Data obtained from literature review.

**Source:** Maisuthisakul (2007)

content in the extracts.

content was similar (Fig. 12).

Many plants in Thailand show potential as a source of extracts rich in phenolic constituents and natural antioxidants. Phenolic compounds are the major antioxidants in plants. Moreover, practical aspects relevant to the use of this class of compounds need to be considered including extraction efficiency, availability of sufficient raw material, and toxicity or safety considerations. To utilize these significant sources of natural antioxidants, further characterization of the phenolic composition is needed.

#### **6. Acknowledgement**

This book chapter was supported by a grant from University of the Thai Chamber of Commerce (UTCC). The author also thanks Professor Michael H. Gordon for helpful suggestions.

#### **7. References**

Amakura, Y.; Umino, Y.; Tsuji, S. & Tonogai, Y. (2000). Influence of jam processing on the radical scavenging activity and phenolic content in berries. *Journal of Agricultural and Food Chemistry,* Vol. 48, pp 6292-6297.

Phenolic Constituents and Antioxidant Properties of some Thai Plants 209

Helrich, K. (1990). *Official methods of analysis of the association of official analytical chemist*. 15th ed. Virginia: Association of Official Analytical Chemists. pp. 703, 1048–1049. Herodež, S.S.; Hadolin, M.; Škerget, M. & Knez, Z. (2003). Solvent extraction study of

Ho, C.T. (1992). Phenolic compounds in food. In *Phenolic Compounds in Food and their Effects* 

Huang, H & Ferraro, T. (1992). Phenolic compounds in food and cancer prevention. In: M.J.

Ibáñez E.; Oca, A.; Murga, G.; López-Sebastián, A.; Tabera, J. & Reglero, G. (1999).

Katalinic, V.; Milos, M.; Kulisic, T. & Jukic, M. (2006). Screening of 70 medicinal plant

Chuenarom V.; Kerdchoechuen, O. & Laohakunjit, N. (2010). Antioxidant Capacity and

Kitanov, G. & Assenov. I. (1988). Flavonols and xanthones from *Cratoxylum pruniflorum*  Kurz. (Guttiferae). *Pharmazie*, Vol. 43, Number 12, pp 879-880. (Abstract). Kumar, V.; Brecht, V. & Frahm, A. W. (2004). Conformational analysis of the biflavonoids

Larrauri, J.A.; Ruperez, P. & Saura-Calixto, F. (1997). Effect of drying temperature on the

Lee, J. & Scagel, C.F. (2009). Chicoric acid found in basil (*Ocimum basilicum* L.) leaves. *Food* 

Leong, L.P. & Shui, G. (2002). An investigation of antioxidant capacity of fruits in Singapore

Maisuthisakul, P. (2007). *Evaluation of Antioxidant Potential and Phenolic con stituents in Selected Thai Indigenous Plant Extracts.* Dissertation. Kasetsart University. 241 p. Maisuthisakul, P. & Pongsawatmanit, R. (2005). Effect of sample preparation methods and

plants. *Journal of Agricultural and Food Chemistry*, Vol. 47, pp 1400–1404. Kähkonen M.P.; Hopia, A.I.; Vuorela, H.J.; Rauha, J.P.; Pihlaja, K.; Kujala, T.S. & Heinonen,

*Journal of Agricultural and Food Chemistry,* Vol. 47, pp 3954-3962.

*Science Journal*, Vol 41, Number (3/1)(Suppl.), pp 621-624.

*Medica*, Vol. 70, Number 7, pp 646-651. (Abstract).

*Chemistry*, Vol. 115, pp 650-656.

markets. *Food Chemistry,* Vol. 76, pp 69-75.

*of Agricultural and Food Chemistry,* Vol. 45, pp 1390–1393.

Lindsay, R.C. (1996). *Food additives*. Marcel Dekker Inc., New York. pp. 778–780.

403-410.

275–282.

557.

Washington, DC. pp. 2–7.

Washington, DC. pp. 8–34.

antimicrobial and antioxidant activities. *Journal of ethnopharmacology*, Vol. 72, pp

antioxidants from Balm (*Melissa officinalis* L.) leaves. Food Chemistry, Vol. 80, pp

*on Health I*, ed. C. T. Ho, C. Y. Lee & M-T. Huang. ACS Symposium series 506,

Huang, C. Ho & C.Y. Lee, Editors, *Phenolic Compounds in Food and Their Effects on Health. II. Antioxidants and Cancer Prevention*. American Chemical Society,

Supercritical fluid extraction and fractionation of different preprocessed rosemary

M. (1999). Antioxidant activity of plant extract containing phenolic compounds.

extracts for antioxidant capacity and total phenols. *Food Chemistry*, Vol. 94, pp 550-

Total Phenolics of Plant Extract from Annual Seablite (*Suaeda maritima*). *Agricultural* 

GB2 and a polyhydroxylated flavanone-chromone of *Cratoxylum nerifolium*. *Planta* 

stability of polyphenols and antioxidant activity of red grape pomace peels. *Journal* 

extraction methods and extraction time on yield and antioxidant activity from


Amarowicz, R.; Pegg, R. B.; Rahimi-Moghaddam, P.; Barl, B. & Weil, J. A. (2004). Free-

Amonkar, A. J. ; Nagabhushan, M. ; D'Souza, A. V. & Bhide, S. V. (1986). Hydroxychavicol:

Auddy, B.; Ferreira, M.; Blasina, F.; Lafon, L.; Arredondo, F.; Dajas, F.; Tripathi, P.C.; Seal, T.

Bocco, A.; Cuvelier, M. E.; Richard, H. & Berset, C. (1998). Antioxidant activity and

Bonvehí, J. S.; Torrent, M. S. & Lorente, E. C. (2001). Evaluation of polyphenolic and

Chanwitheesuk, A.; Teerawutgulrag, A. & Rakariyatham, N. (2005). Screening of

Chen, G.; Huo, Y.; Tan, D.X.; Liang, Z.; Zhang, W. & Zhang, Y. (2001). Melatonin in Chinese

Conde, E.; Cadahía, E.; García-Vallejo, M.C. & Fernández de Simón, B. (1998). Polyphenolic

Das, M. C. & Mahato, S. B. (1982). Triterpenoid sapogenols from the leaves of *Careya arborea*: structure of careyagenolide. *Phytochemistry*, Vol. 21, Number 8, pp 2069-2073. Demo, A.; petrakis, C.; Kefalas, P. & Boskou, D. (1998). Nutrient antioxidants in some herbs and Mediteranean plant leaves. *Food Research International*, Vol. 31, pp 351-354. Dillard, C.J. & German, J.B. (2000). Review phytochemicals : nutraceuticals and human

Evans, P. H.; Bowers, W. S. & Funk, E. J. (1984). Identification of fungicidal and nematocidal

Fernández de Simón, B.; Cadahía, E., Conde, E. & García-Vallejo, M.C. (1996). Low

Guha P. (2006). Betel leaf: the neglected green gold of India. *Journal of Hum Ecology*, Vol.19,

Gupta, R. K.; Chakraboty, N. K. & Dutta, T. R. (1975). Crystalline constituents from

Habsah, M.; Amran, M.; Mackeen, M.M.; Lajis,N.H.; Kikuzaki, H.; Nakatani, N.; Rahman,

components in the leaves of Piper Betel (*Piperaceae*). *Journal of Agricultural and Food* 

molecular weight phenolic compounds in spanish oak woods. *Journal of Agricultural* 

*Careya arborea* Roxb. leaves. *Indian Journal of Pharmacy,* Vol. 37, Number 6, pp

A.A.; Gahfar, A. & Ali, A.M. (2000). Screening of *Zingiberaceae* extracts for

the Canadian prairies. *Food Chemistry* Vol.84, pp 551-562.

*Journal of ethnopharmacology*, Vol. 84, Number (2-3), pp 131-8.

*Agricultural and Food Chemistry, Vol.* 49, pp 1848-1853.

Thailand. *Food Chemistry*, Vol. 92, Number 3, pp 491-7.

medicinal herbs. *Life Science*, Vol. 73, Number 1, pp 19-26.

*Agricultural and Food Chemistry,*Vol. 46, Number 8, pp 3166–3171.

health. *Journal of Science, Food and Agriculture*, Vol. 80, pp 1744-1756.

Number 12, pp 1321-1324.

*Food Chemistry*, Vol. 46, pp 2123-2129.

*Chemistry*, Vol. 32, pp1254-1256.

pp 87–93.

161-162.

*and Food Chemistry*, Vol. 44, pp 1507–1511.

radical scavenging capacity and antioxidant activity of selected plant species from

a new phenolic antimutagen from Betel leaf. *Food and Chemical Toxicology,* Vol. 24,

& Mukherjee, B. (2003). Screening of antioxidant activity of three Indian medicinal plants, traditionally used for the management of neurodegenerative diseases.

phenolic composition of citrus peel and seed extracts. *Journal of Agricultural and* 

flavonoid compounds in honey-bee collected pollen produced in spain. *Journal of* 

antioxidants activity and antioxidant compounds of some edible plants of

composition of *Quercus suber* cork from different Spanish provenances, *Journal of* 

antimicrobial and antioxidant activities. *Journal of ethnopharmacology*, Vol. 72, pp 403-410.


Phenolic Constituents and Antioxidant Properties of some Thai Plants 211

Nuutilla, A.M.; kammiovirta, K. & Oksman-Caldentey, K.M. (2002). Comparison of methods

Phomkaivon, N. & Areekul, V. (2009). Screening for antioxidant activity in selected Thai wild plants. *Asian Journal of Food Ago-Industry,* Vol. 2, Number 4, pp 433-440 Pokorny, J. & Korczak, J. (2001). Preparation of natural antioxidants. In J. Pokony, N.

Pokorny, J. 2001. Introduction, In J. Pokorny, N. Yanishlieva & M. Gordon, eds. *Antioxidants in Food, Practical applications.* Woodhead Pulbishing Limited, Cambridge. pp 1-3. Povichit, N.; Phrutivoraponkul, A.; Suttajit, M.; Chaiyasut, C. & Leelapornpisid, P. (2010).

Rice-Evans, C.A.; Miller N. J. & Paganga, G. (1997). Structure-antioxidant activity

Rimando, A. M.; Han, B. H.; Park, J. H. & Cantoria, M. C. (1986). Studies on the constituents

Salisbury, F.B. & Ross, C.W. (1992). *Plant Physiology*. Wadsworth Publishing Co., Belmont,

Shahidi, F. & Naczk, M. (2003). *Phenolics in food and nutraceuticals*. Boca Raton, FL, USA: CRC

Sheabar, F.Z. & Neeman, I. (1988). Separation and concentration of natural antioxidants

Siddhuraju, P.; Mohan, P. S. & Becker, K. (2002). Studies on the antioxidant activity of

Skerget, M.; Kotnik, P.; Hadolin, M.; Hra, A.R.; Simoni, M. & Knez, A. (2005). Phenols,

Stahl, W. & Sies, H. (2003). Antioxidant activity of carotenoids. *Molecular Aspects of Medicine*,

Talapatra, B.; Basak, A. & Talapatra, S. K. (1981). Terpenoids and related compounds;

Weinberg, Z.G.; Akiri, B.; Potoyevski, E. & Kanner, J. (1999). Enhancement of polyphenol

*Indian Chemical Society*, Vol. 58, Number 8, pp 814-815. (abstract).

bark, leaves and fruit pulp. *Food Chemistry*, Vol. 79, pp 61-67.

antioxidant activities. *Food Chemistry*, 89, pp 191–198.

HPLC analysis. Food Chemistry, Vol. 76, pp 519-525.

Publishing Limited, Cambride. pp. 311-330.

Number 4, pp 403-408.

Vol. 20, pp 933-956.

Number 2, pp 93-97.

Vol. 24, pp 345–351.

47, pp 2959-2962.

CA. 682 pp.

Press.

993.

for the hydrolysis of flavonoids and phenolic acid from onion and spinash for

Yanishlieva, M. Gordon, eds. *Antioxidants in food, Practical applications*. Woodhead

Phenolic content and in vitro inhibitory effects on oxidation and protein glycation of some Thai medicinal plants. *Pakistan Journal of Pharmaceutical Science*, Vol. 23,

relationships of flavonoids and phenolic acids. *Free Radical Biology and Medicine,* 

of Philippine Piper Betel leaves. *Archiv of Physical Medicine and Rehabilitation*, Vol. 9,

from the rape of olives. *Journal of American Oil Chemist's Society,* Vol. 65, pp 990–

Indian Laburnum (*Cassia fistula*. L): a preliminary assessment of crude from stem,

proanthocyanidins, flavones and flavonols in some plant materials and their

Careaborin, a new titerpene ester from the leaves of *Careya arborea*. *Journal of the* 

recovery from rosemary (*Rosmarinus officinalis*) and sage (*Salvia officinalis*) by enzyme-assisted ensiling (ENLAC). *Journal of Agricultural and Food Chemistry,* Vol.

Kradonbok (*Careya sphaerica* Roxb.) leaves. *Kasetsart Journal*: *Natural Science*, Vol. 38, Number 5, pp 8-14.


Maisuthisakul, P.; Pongsawatmanit, R. & Gordon, M.H. (2007a). Assessment of phenolic

Maisuthisakul, P.; Pongsawatmanit, R. & Gordon, M.H. (2007). Characterization of the

Maisuthisakul, P.; Pasuk, S. & Ritthiruangdej, P. (2008a). Relationship of antioxidant

Maisuthisakul, P. (2008). Phenolic antioxidants from Betel leaf (*Piper betel* Linn.) extract

Marinova, E. M. & Yanishlieva, N. V. (1997). Antioxidative activity of extracts from

Matsuda, H.; Morikawa, T.; Toguchida, I.; Park, J.Y.; Harima, S. & Yoshikawa, M. (2001).

Miliauskas, G.; Venskutonis, P.R. & van Beek, T.A. (2004). Screening of radical scavenging

Morita, N.; Arisawa, M.; Nagaes, M.; Hsu, H. Y. & Chen, Y. (1977). Studies on the

*fistula* and 8 other species. *Shoyakura Zasshi,* Vol. 31, pp 172-174. (Abstract). Moure, A.; Cruz, J. M.; Franco, D.; Dominguez, J. M.; Sineiro, J.; Dominguez, H. & Núñez,

Munishwar, N. G. (1997). Polarity Index: the Guiding Solvent Parameter for Enzyme

Nagabhushan, M.; Amonkar, A. J.; Nair, U. J.; D'Souza, A. V. & Bhide, S. V. (1989).

Núñez Sellés, A.; Vélez Castro, H. T. & Agüero-Agüero, J. (2002). Isolation and Quantitative

Supplement. *Journal of Agricultural and Food Chemistry,* Vol. 50, pp 762–766. Nutrition Division. Department of Health Ministry of Public Health. (1992). *Nutritive Values* 

*formosum* Dyer). *Food Chemistry*, Vol. 100, pp 1620-1629.

*Composition and Analysis,* Vol. 21, pp 229-240.

*Medicinal Chemistry.* Vol. 9, pp 41–50.

*Mutagenesis*, Vol. 4, Number 3, pp 200-204.

*of Thai Foods*. Thai veterans oganisation. Bankok.

Number 5, pp 8-14.

Number 4, pp 52-64.

pp 245–248.

231-237.

pp 145-171.

284-288.

*Chemistry*, Vol. 100, pp 1409-1418.

Kradonbok (*Careya sphaerica* Roxb.) leaves. *Kasetsart Journal*: *Natural Science*, Vol. 38,

content and free radical-scavenging capacity of some Thai indigenous plants. *Food* 

phytochemicals and antioxidant properties of extracts from Teaw (*Cratoxylum* 

properties and chemical composition of some Thai plants. *Journal of Food* 

obtained with different solvents and extraction time. *UTCC journal*, Vol. 28,

selected species of the family Lamiacea in sunflower oil. *Food Chemistry*, Vol. 58,

Antioxidant constituents from rhubarb: structural requirements of stilbenes for the activity and structures of two new anthraquinone glucosides. *Bioorganic and* 

activity of some medicinal and aromatic plant extracts. *Food Chemistry,* Vol. 85, pp

constituents of formorsan leguminosae. III. Flavonoids from *Leucaena glauca*, *Cassia* 

M. J. (2001). Natural antioxidants from residual sources. *Food Chemistry*, Vol. 72,

Stability in Aqueous-Organic Cosolvent Mixtures. *Biotechology Progress,* Vol. 13, pp

Hydroxy-Chavicol: a New Anti-Nitrosating Phenolic Compound from Betel Leaf.

Analysis of Phenolic Antioxidants, Free Sugars, and Polyols from Mango (*Mangifera indica* L.) Stem Bark Aqueous Decoction Used in Cuba as a Nutritional


**10** 

*Brazil* 

*1Universidade de Franca, 2Universidade de São Paulo,* 

**Lignans: Chemical and Biological Properties** 

Wilson R. Cunha1, Márcio Luis Andrade e Silva1, Rodrigo Cassio Sola

The plant kingdom has formed the basis of folk medicine for thousands of years and nowadays continues to provide an important source to discover new biologically active compounds (Fabricant & Farnsworth, 2001; Gurib-Fakim, 2006; Newman, 2008). The research, development and use of natural products as therapeutic agents, especially those derived from higher plants, have been increasing in recent years (Gurib-Fakim, 2006). Several lead metabolites such as vincristine, vinblastine, taxol and morphine have been isolated from plants, and many of them have been modified to yield better analogues for activity, low toxicity or better solubility. However, despite the success of this drug discovery strategy, only a small percentage of plants have been phytochemically investigated and studied for their

The first step in the search of new plant-based drugs or lead compounds is the isolation of the secondary metabolites. In the past, the natural products researchers were more concerned with establishing the structures and stereochemistry of such compounds but, in recent years, a great number of studies have concentrated efforts on their biological activities (Ambrosio et al., 2006). This multidisciplinary approach was reinforced by the substantial progress observed in the development of novel bioassay methods. As a consequence, a great number of compounds isolated from plants in the past have been "rediscovered" (Ambrosio et al., 2008; Ambrosio et al., 2006; Houghton, 2000; Porto et al.,

Several classes of secondary metabolites are synthesized by plants and, among those, lignans are recognized as a class of natural products with a wide spectrum of important biological activities. **Table 1** summarizes the main biological properties described in the

The term "Lignan" was first introduced by Haworth (1948) to describe a group of dimeric phenylpropanoids where two C6-C3 are attached by its central carbon (C8), as shown in **Figure 1**. More recently, Gotlieb (1978) proposed that micromolecules with two phenylpropanoid units coupled in other manners, like C5-C5´ for example should be named "neolignans" (Umezawa, 2003). According to Gordaliza et al (2004), lignans can be found in more than 60 families of vascular plants and have been isolated from different plant parts,

medicinal potential (Ambrosio et al., 2006; Hostettmann et al., 1997; Soejarto, 1996).

2009a; Porto et al., 2009b; Tirapelli et al., 2008).

literature for lignans.

exudates and resins.

**1. Introduction** 

Veneziani1, Sérgio Ricardo Ambrósio1 and Jairo Kenupp Bastos2


### **Lignans: Chemical and Biological Properties**

Wilson R. Cunha1, Márcio Luis Andrade e Silva1, Rodrigo Cassio Sola Veneziani1, Sérgio Ricardo Ambrósio1 and Jairo Kenupp Bastos2

> *1Universidade de Franca, 2Universidade de São Paulo, Brazil*

#### **1. Introduction**

212 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Witzell, J.; Gref, R. & Nüsholm, T. (2003). Plant part specific and temporal variation in

Zheng, W. & Wang, S.Y. (2001). Antioxidant activity and phenolic compounds in selected herbs. *Journal of Agricultural and Food Chemistry,* Vol. 49, pp 5165-5170. Zhishen, J., Mengcheng, T. & Jianming, W. (1999). The determination of flavonoid contents

*and Ecology*, Vol. 31, pp 115-127.

Vol. 64, pp 555-559.

phenolic compounds of boreal bilberry (*Vaccinium myrtillus*). *Biochemical Systematic* 

in mulberry and their scavenging effects on superoxide radicals. *Food Chemistry*,

The plant kingdom has formed the basis of folk medicine for thousands of years and nowadays continues to provide an important source to discover new biologically active compounds (Fabricant & Farnsworth, 2001; Gurib-Fakim, 2006; Newman, 2008). The research, development and use of natural products as therapeutic agents, especially those derived from higher plants, have been increasing in recent years (Gurib-Fakim, 2006). Several lead metabolites such as vincristine, vinblastine, taxol and morphine have been isolated from plants, and many of them have been modified to yield better analogues for activity, low toxicity or better solubility. However, despite the success of this drug discovery strategy, only a small percentage of plants have been phytochemically investigated and studied for their medicinal potential (Ambrosio et al., 2006; Hostettmann et al., 1997; Soejarto, 1996).

The first step in the search of new plant-based drugs or lead compounds is the isolation of the secondary metabolites. In the past, the natural products researchers were more concerned with establishing the structures and stereochemistry of such compounds but, in recent years, a great number of studies have concentrated efforts on their biological activities (Ambrosio et al., 2006). This multidisciplinary approach was reinforced by the substantial progress observed in the development of novel bioassay methods. As a consequence, a great number of compounds isolated from plants in the past have been "rediscovered" (Ambrosio et al., 2008; Ambrosio et al., 2006; Houghton, 2000; Porto et al., 2009a; Porto et al., 2009b; Tirapelli et al., 2008).

Several classes of secondary metabolites are synthesized by plants and, among those, lignans are recognized as a class of natural products with a wide spectrum of important biological activities. **Table 1** summarizes the main biological properties described in the literature for lignans.

The term "Lignan" was first introduced by Haworth (1948) to describe a group of dimeric phenylpropanoids where two C6-C3 are attached by its central carbon (C8), as shown in **Figure 1**. More recently, Gotlieb (1978) proposed that micromolecules with two phenylpropanoid units coupled in other manners, like C5-C5´ for example should be named "neolignans" (Umezawa, 2003). According to Gordaliza et al (2004), lignans can be found in more than 60 families of vascular plants and have been isolated from different plant parts, exudates and resins.

Lignans: Chemical and Biological Properties 215

Most of the known natural lignans are oxidized at C9 and C9´ and, based upon the way in which oxygen is incorporated into the skeleton and on the cyclization patterns, a wide range of lignans of very different structural types can be formed. Due to this fact, lignans are classified in eight subgroups and (Chang et al., 2005; Suzuki & Umezawa, 2007), among these subgroups, the furan, dibenzylbutane and dibenzocyclooctadiene lignans can be further classified in "lignans with C9 (9´)-oxygen" and "lignans without C9 (9´)-oxygen". **Figure 2** displays the main classes of lignans, as well as their subgroups. It is noteworthy that, despite its structural variation, lignans also display a substantial variation on its enantiomeric composition (Umezawa et al., 1997). In this sense, these metabolites can be found as pure enantiomers and as enantiomeric compositions, including racemates (Macias

As mentioned before, lignins and lignans are both originated from C6-C3 units, thus indicating that these metabolites are biosynthesized through the same pathway in the earlier steps. As seen in **Figure 3**, aromatic aminoacids *L*-phenylalanine and *L*-tyrosine are produced from shikimic acid pathway, and then converted in a series of cinnamic acid

CO2H

CO2H

O2 NADPH

COSCoA

NADPH NADPH NADPH

OH

CO2H

SAM

HSCoA HSCoA

CO2H

OH MeO OMe

> sinapic acid

OH MeO OMe

OH MeO OMe

> sinapyl alcohol

OH

COSCoA

OH MeO OH

ferulic acid OH

OH

OH

coniferyl alcohol

SAM

MeO

MeO

MeO

OH

CO2H

O2 NADPH HO

*p-*coumaric acid

OH

OH

*p-*coumaryl alcohol

Fig. 3. Biosynthesis of hydroxycinamyl alcohol monomers, the precursors of lignans

OH

HSCoA COSCoA

OH

O2 NADPH

et al., 2004).

CO2H NH2

*L*-Phe

**2. Chemical aspects of lignans** 

Cinnamic acid

*L*-Tyr

according to Dewick (2002).

OH

CO2H

CO2H NH2


Table 1. Main biological activities of lignans

Fig. 2. Main subclasses of lignans. Adapted from Suzuki & Umezawa (2007).

Antiviral (Charlton, 1998; Cos et al., 2008; McRae & Towers, 1984;

Anticancer (McRae & Towers, 1984; Pan et al., 2009; Saleem et al.,

antioxidant (Fauré et al., 1990; Pan et al., 2009; Saleem et al., 2005)

1 3 2

With C9(9´)-oxygen

7 8 9

O

Aryltetralin Dibenzocyclooctadienes

9´

<sup>9</sup> <sup>9</sup> <sup>9</sup> <sup>9</sup>

O

O

Arylnaphtalene

Fig. 2. Main subclasses of lignans. Adapted from Suzuki & Umezawa (2007).

Dibenzylbutyrolactol

<sup>9</sup> <sup>9</sup>

OH

9´

O

Lignan structure

1'

7' 8' 9'

O

9´

Furan

2' 3'

5'6'

4'

O

With C9(9´)-oxygen Without C9(9´)-oxygen

Dibenzylbutyrolactones

9

OH O

9´ 9´

O

O

9´

9 9

9´ 9´

Without C9(9´)-oxygen

O

5 6

4

Cancer prevention (Huang et al., 2010; Webb & McCullough, 2005)

Yousefzadi et al., 2010)

2005; Yousefzadi et al., 2010)

Biological activity Reference

Anti-inflammatory (Saleem et al., 2005) antimicrobial (Saleem et al., 2005)

immunosuppressive (Saleem et al., 2005) Hepatoprotective (Negi et al., 2008) Osteoporosis prevention (Habauzit & Horcajada, 2008)

Table 1. Main biological activities of lignans

Phenylpropanoid unit

> OH OH

<sup>9</sup> <sup>9</sup> <sup>9</sup>

9´

Fig. 1. Phenylpropanoid unit and lignan structure

OH OH

Dibenzylbutane

With C9(9´)-oxygen Without C9(9´)-oxygen

9´ 9´

O

9´

O

O

9

Furofuran

O

9´

7 8 9 Most of the known natural lignans are oxidized at C9 and C9´ and, based upon the way in which oxygen is incorporated into the skeleton and on the cyclization patterns, a wide range of lignans of very different structural types can be formed. Due to this fact, lignans are classified in eight subgroups and (Chang et al., 2005; Suzuki & Umezawa, 2007), among these subgroups, the furan, dibenzylbutane and dibenzocyclooctadiene lignans can be further classified in "lignans with C9 (9´)-oxygen" and "lignans without C9 (9´)-oxygen". **Figure 2** displays the main classes of lignans, as well as their subgroups. It is noteworthy that, despite its structural variation, lignans also display a substantial variation on its enantiomeric composition (Umezawa et al., 1997). In this sense, these metabolites can be found as pure enantiomers and as enantiomeric compositions, including racemates (Macias et al., 2004).

#### **2. Chemical aspects of lignans**

As mentioned before, lignins and lignans are both originated from C6-C3 units, thus indicating that these metabolites are biosynthesized through the same pathway in the earlier steps. As seen in **Figure 3**, aromatic aminoacids *L*-phenylalanine and *L*-tyrosine are produced from shikimic acid pathway, and then converted in a series of cinnamic acid

Fig. 3. Biosynthesis of hydroxycinamyl alcohol monomers, the precursors of lignans according to Dewick (2002).

Lignans: Chemical and Biological Properties 217

MeO

HO

OH MeO

OH

OH

HO

Other

O O

MeO

Dibenzylbutyrolactols

MeO

O

O

OMe

Fig. 6. Chemical structures of clinically available podophyllotoxin derivatives.

OH

OH O

Fig. 5. Biosynthesis of dibenzylbutyrolactols and aryltetralin lactones (Canel et al., 2000).

O

OH

O O Yatein

OMe

O O

O O HO

H

S

According to You (2005), podophyllotoxin still can be considered a hot prototype for discovery and development of novel anticancer agents, even in the 21st century. This leading compound has been isolated from the roots of *Podophyllum* species and more recently from other genus, such as *Linum* (Yousefzadi et al., 2010). Due to its importance in anticancer therapy, several biotechnological approaches including the use of cell cultures, biotransformation processes and metabolic engineering techniques to manipulate the

MeO

Etoposide Teniposide Etoposide phosphate

secoisolariciresinol

pinoresinol

O

OMe OH

HO

MeO

O

lariciresinol

HO

Aryltetralin lactones

O O

O O HO

H Me

MeO

O

O

OMe

OH

OH O

OMe OH

O

O

OMe

O

P O HO OH

OH O

OH

Coniferyl alcohol

MeO

HO

MeO

HO

O O

MeO

Podophyllotoxin

MeO

MeO

OH

OH

O

O

OMe

OMe

OH

O OH

D resonance forms

O O Matairesinol

> O O

O O HO

H Me

MeO

HO O

O

Dibenzylbutyrolactollignan

OH

OMe

OH

derivatives. The reduction of these acids via coenzyme A of related esters and aldehydes forms three alcohols (*p*-coumaryl alcohol, coniferyl alcohol and sinalpyl alcohol) that are the main precursors of all lignins and lignans.

The peroxidase induces one-electron oxidation of the phenol group allowing the delocalization of the unpaired electron through resonance forms. In these hydroxycinamyl alcohols, conjugation allows the unpaired electron to be delocalized also into the side chain. After this point, radical pairing of these resonance structures originates reactive dimeric systems susceptible to nucleophilic attack from hydroxyl groups, leading to a wide range of lignans, as shown in **Figure 2**.

Among these subgroups, the biosynthesis of C9 (9´)-oxygen lignans is the most well known. This type of lignan is formed through the enantioseletive dimerization of two coniferyl alcohol monomeric units (D resonance form of coniferyl alcohol radical, **Figure 4**) into pinoresinol via intermolecular 8,8´ oxidative coupling with the aid of dirigent protein (Dewick, 2002; Suzuki & Umezawa, 2007).

Fig. 4. Resonance forms of coniferyl alcohol radical

The following steps involve sequencial stereoselective enzymatic reduction of pinosresinol by pinoresinol/lariciresinol reductase to generate lariciresinol and then secoisolariciresinol by secoisolariciresinol dehydrogenase. The main steps of this biosynthetic proposal are depicted in **Figure 5**. Secoisolariciresinol gives the presumably common precursor of all dibenzylbutyrolactol lignans and, through the formation of matairesinol and yatein, also forms the aryltetralin lignans. These subclasses of lignans includes some important bioactive compounds such as cubebin (**1**) and podophyllotoxin (Canel et al., 2000; de Souza et al., 2005; Gordaliza et al., 2004; Saraiva et al., 2007; Silva et al., 2007; Silva et al., 2009; Srivastava et al., 2005; You, 2005; Yousefzadi et al., 2010).

#### **3. Podophyllotoxin: chemical and biological approaches**

Podophyllotoxin (**Figure 6**), a naturally occurring aryltetralin lignin, is one of the most important compound due to its high toxicity and current use as a local antiviral agent (Yousefzadi et al., 2010). Moreover, this metabolite has been used to obtain structural analogues which are employed as anticancer drugs (Ayres & Loike, 1990; Yousefzadi et al., 2010) and several semi-synthetic podophyllotoxin-related derivatives showed to be topoisomerase II inhibitor, acting as an antimitotic compound (You, 2005; Yousefzadi et al., 2010). **Figure 6** also shows the clinically valuable anticancer agents, etoposide, teniposide and etoposide phosphate, obtained from podophyllotoxin.

The peroxidase induces one-electron oxidation of the phenol group allowing the delocalization of the unpaired electron through resonance forms. In these hydroxycinamyl alcohols, conjugation allows the unpaired electron to be delocalized also into the side chain. After this point, radical pairing of these resonance structures originates reactive dimeric systems susceptible to nucleophilic attack from hydroxyl groups, leading to a wide range of

Among these subgroups, the biosynthesis of C9 (9´)-oxygen lignans is the most well known. This type of lignan is formed through the enantioseletive dimerization of two coniferyl alcohol monomeric units (D resonance form of coniferyl alcohol radical, **Figure 4**) into pinoresinol via intermolecular 8,8´ oxidative coupling with the aid of dirigent protein

O

alcohol A B <sup>C</sup> <sup>D</sup>

The following steps involve sequencial stereoselective enzymatic reduction of pinosresinol by pinoresinol/lariciresinol reductase to generate lariciresinol and then secoisolariciresinol by secoisolariciresinol dehydrogenase. The main steps of this biosynthetic proposal are depicted in **Figure 5**. Secoisolariciresinol gives the presumably common precursor of all dibenzylbutyrolactol lignans and, through the formation of matairesinol and yatein, also forms the aryltetralin lignans. These subclasses of lignans includes some important bioactive compounds such as cubebin (**1**) and podophyllotoxin (Canel et al., 2000; de Souza et al., 2005; Gordaliza et al., 2004; Saraiva et al., 2007; Silva et al., 2007; Silva et al., 2009; Srivastava

Podophyllotoxin (**Figure 6**), a naturally occurring aryltetralin lignin, is one of the most important compound due to its high toxicity and current use as a local antiviral agent (Yousefzadi et al., 2010). Moreover, this metabolite has been used to obtain structural analogues which are employed as anticancer drugs (Ayres & Loike, 1990; Yousefzadi et al., 2010) and several semi-synthetic podophyllotoxin-related derivatives showed to be topoisomerase II inhibitor, acting as an antimitotic compound (You, 2005; Yousefzadi et al., 2010). **Figure 6** also shows the clinically valuable anticancer agents, etoposide, teniposide

OH OH OH OH

O

O

OH

MeO

MeO

MeO

derivatives. The reduction of these acids via coenzyme A of related esters and aldehydes forms three alcohols (*p*-coumaryl alcohol, coniferyl alcohol and sinalpyl alcohol) that are the

main precursors of all lignins and lignans.

(Dewick, 2002; Suzuki & Umezawa, 2007).

OH

et al., 2005; You, 2005; Yousefzadi et al., 2010).

Fig. 4. Resonance forms of coniferyl alcohol radical

Coniferyl

O

**3. Podophyllotoxin: chemical and biological approaches** 

and etoposide phosphate, obtained from podophyllotoxin.

MeO


MeO

lignans, as shown in **Figure 2**.

Fig. 5. Biosynthesis of dibenzylbutyrolactols and aryltetralin lactones (Canel et al., 2000).

Fig. 6. Chemical structures of clinically available podophyllotoxin derivatives.

According to You (2005), podophyllotoxin still can be considered a hot prototype for discovery and development of novel anticancer agents, even in the 21st century. This leading compound has been isolated from the roots of *Podophyllum* species and more recently from other genus, such as *Linum* (Yousefzadi et al., 2010). Due to its importance in anticancer therapy, several biotechnological approaches including the use of cell cultures, biotransformation processes and metabolic engineering techniques to manipulate the

Lignans: Chemical and Biological Properties 219

O

O

Me

2HCl

Me

Fig. 8. Examples of antineoplasic candidates developed from podophyllotoxin chemical

The Chagas' disease, or American trypanosomiasis, is endemic in Central and South America and it is estimated that 16–18 million people are currently infected with the protozoan flagellate *Trypanosoma cruzi* (Molfetta et al., 2005) and more than 100 million are

Since it was discovery in 1909, Chagas´ disease infection has been difficult to control due to its multiple characteristics (de Souza et al., 2005). One of the main causes of these difficulties are to find an efficient compound to combat the aetiologic agent (*T. cruzi*) is directly linked to the morphologic characteristics of its strains, mainly due to the occurrence of various subpopulations of the parasite, leading to a different host tissue´s tropism (de Souza et al.,

Clinical treatment of infected patients is relied on two nitroheterocyclic drugs, the nifrofuran nifurtimox, Lampit®, which production has now been discontinued, and the 2 nitroimidazole benznidazole, Rochagan® (Paulino et al., 2005). Both drugs, if administered during the acute phase of the disease, could cure 50-70% of the patients. However, these

MeO

O

OH

OH

O

O

OMe

O

OMe

ON2 + ON2 +

O

P

O

OH O

MeO

N

O

O O HO

H

Me

O

NO2

O

OMe

OH

NK611 GL331 Azatoxin

HN

O

MeO

Etopophos

O

O

OMe

O

N N

O

OMe

**4. Trypanocidal activity of cubebin and its derivatives** 

OH

TOP-53

exposed to the risk of infection (Takeara et al., 2003).

OH

N O

O

O O HO

H

Me

O

MeO

O

O

skeleton.

2005).

MeO

biosynthetic pathway (**Figure 7)**, have been currently developed and are alternatives for the production of podophyllotoxin.

Fig. 7. Biosynthesis proposal of podophyllotoxin according to Canel et al., 2000.

Despite the fact that etoposide, teniposide and etoposide phosphate are clinically valuable anticancer agents, several adverse effects and drug resistance have been associated with the use of these drugs (You, 2005). In this sense, several studies focusing to prepare novel derivatives and to understand the structure-activity relationship (SAR) of podophyllotoxins have been published (You, 2005). Based on these data a great number of potential drug candidates were synthesized (You, 2005; Yousefzadi et al., 2010). **Figure 8** shows the structures of new antineoplasic candidates developed from podophyllotoxin chemical skeleton.

In order to better explore the biological potential of this class of metabolites, our research group has concentrated efforts to investigate the biological activity of some dibenzylbutyrolactone lignans, mainly cubebin (**Figure 9**, **1**) and its semi-synthetic derivatives. In this sense, the most significant achievements in our investigations are described in the following sections.

biosynthetic pathway (**Figure 7)**, have been currently developed and are alternatives for the

OH

H

OH

O

O H H

HO

MeO

HO

MeO

HO

O H H OMe

(+)-pinoresinol

(+)-lariciresinol

OMe

OH

OH

O

OH MeO

OH

H

production of podophyllotoxin.

H

O

O

O

O

OMe

(-)-podophyllotoxin

OMe

OH

Coniferyl alcohol

MeO

HO

O O

skeleton.

following sections.

MeO

MeO

OMe

OH

OH

OH

O

OH

H

OMe

HO

MeO

(-)-matairesinol (-)-secoisolariciresinol

Fig. 7. Biosynthesis proposal of podophyllotoxin according to Canel et al., 2000.

Despite the fact that etoposide, teniposide and etoposide phosphate are clinically valuable anticancer agents, several adverse effects and drug resistance have been associated with the use of these drugs (You, 2005). In this sense, several studies focusing to prepare novel derivatives and to understand the structure-activity relationship (SAR) of podophyllotoxins have been published (You, 2005). Based on these data a great number of potential drug candidates were synthesized (You, 2005; Yousefzadi et al., 2010). **Figure 8** shows the structures of new antineoplasic candidates developed from podophyllotoxin chemical

In order to better explore the biological potential of this class of metabolites, our research group has concentrated efforts to investigate the biological activity of some dibenzylbutyrolactone lignans, mainly cubebin (**Figure 9**, **1**) and its semi-synthetic derivatives. In this sense, the most significant achievements in our investigations are described in the

OMe

Fig. 8. Examples of antineoplasic candidates developed from podophyllotoxin chemical skeleton.

#### **4. Trypanocidal activity of cubebin and its derivatives**

The Chagas' disease, or American trypanosomiasis, is endemic in Central and South America and it is estimated that 16–18 million people are currently infected with the protozoan flagellate *Trypanosoma cruzi* (Molfetta et al., 2005) and more than 100 million are exposed to the risk of infection (Takeara et al., 2003).

Since it was discovery in 1909, Chagas´ disease infection has been difficult to control due to its multiple characteristics (de Souza et al., 2005). One of the main causes of these difficulties are to find an efficient compound to combat the aetiologic agent (*T. cruzi*) is directly linked to the morphologic characteristics of its strains, mainly due to the occurrence of various subpopulations of the parasite, leading to a different host tissue´s tropism (de Souza et al., 2005).

Clinical treatment of infected patients is relied on two nitroheterocyclic drugs, the nifrofuran nifurtimox, Lampit®, which production has now been discontinued, and the 2 nitroimidazole benznidazole, Rochagan® (Paulino et al., 2005). Both drugs, if administered during the acute phase of the disease, could cure 50-70% of the patients. However, these

Lignans: Chemical and Biological Properties 221

*cubeba* (de Souza et al., 2005). **Table 2** shows the results of the trypanocidal activity evaluation of compounds **2**, **3**, **4**, **5**, **6** and benznidazole, against amastigote forms of Y strain

**Compounds Concentration (μM) X % of lyse (± SD) IC50 (μM)** 0.5 2.0 8.0 32.0 **2** 14.5 ± 1.9 26.0 ± 2.5 29.0 ± 5.2 26.4 ± 1.2 1.5 X 104 **3** 37.0 ± 1.4 38.0 ± 7.0 46.8 ± 6.4 68.8 ± 2.9 5.7 **4** 32.6 ± 2.6 55.4 ± 4.3 50.8 ± 1.0 57.8 ± 8.0 4.7 **5** 47.6 ± 9.5 57.0 ± 1.1 57.6 ± 8.9 63.6 ± 5.2 0.7 **6** 34.6 ± 7.9 48.7 ± 1.4 38.9 ± 2.0 48.5 ± 6.1 95.3 Benznidazole 38.4 ± 3.0 67.0 ± 7.2 69.0 ± 4.0 68.6 ± 1.7 0.8 Table 2. Results of the trypanocidal activity evaluation of compounds **2**, **3**, **4**, **5**, **6** and benznidazole, against amastigote forms of Y strain of *T. cruzi* (de Souza et al., 2005).

The production of compound **2** by substitution of the lactol hydrogen of cubebin by an acetyl group led to a strong reduction of its trypanocidal ctivity, in comparison with all other evaluated compounds belonging to the same group (IC50 = 1.5 X 104 μM; **Table 2**). Furthermore, the comparison of compounds **2** and **3** indicate that the biological activity against the amastigote forms of *T. cruzi* was significantly affected by the nature of the substituting group at position C-9, which played an important role in the reduction of the calculated IC50 value for compound **3** (IC50 = 5.7 μM; **Table 2**). Likewise, cubebin derivative **4**, bearing an amino group at the lactol ring, displayed an activity quite similar to compound **3**. Analysis of the obtained results, displayed in **Table 2**, indicate that compound 5 was the most active, with an IC50 value of 0.7 μM similar to that displayed by benznidazole (IC50 = 0.8 μM), a standard drug used as the positive control. On the other hand, most of the other evaluated compounds displayed much lower activity, with the exception of compounds **3**

In this study, De Souza et al. (2005) also pointed out that hinokinin (**HK**, **5**) is a promising compound to continue examining, since at 0.5 μM it displayed higher activity than benznidazole and at the other assayed concentrations (2.0, 8.0 and 32.0 μM) it showed

In view of higher trypanocidal activity displayed by **HK** (**5**) against free amastigote forms of *T. cruzi* (**Table 2**; (de Souza et al., 2005), this lignan was selected to be assayed against epimastigote and intracellular amastigote forms of *T. cruzi*, both in vitro and *in vivo* assays (Saraiva et al., 2007). The results of the trypanocidal activity against epimastigote and

**Compounds Concentration (μM) X % of lyse (± SD) IC50** 

**HK** 21.79±1.82 99.03±0.15 100.0±0.35 100.0±0.35 100.0±0.64 0.67 **Nifurtimox** 11.78±13.92 49.38±6.71 65.46±5.36 81.54±2.71 97.54±1.80 3.08 **benznidazole** 0 1.23±5.87 26.18±10.71 51.13±5.23 76.09±2.74 30.89 Table 3. Results of the trypanocidal activity evaluation of **HK**, benznidazole and nifurtimox

0.5 2.0 8.0 32.0 128.0 **(μM)** 

intracellular amastigote forms of *T. cruzi* are shown in **Tables 3 and 4**, respectively.

(IC50 = 5.7 μM) and **4** (IC50 = 4.7 μM), which showed significant activity.

against epimastigote forms of CL strain of *T. cruzi* (Saraiva et al., 2007).

of *T. cruzi*.

similar activity.

drugs display limited efficacy in the treatment of the chronic phase of the disease and are quite toxic for the patients (de Souza et al., 2005). Therefore, there is an urgent demand for the discovery and development of novel therapeutic compounds to treat Chagas´ disease

De Souza et al. (2005) (de Souza et al., 2005) have reported the trypanocidal activity of cubebin (**1**) and its semi-synthetic derivatives against free amastigote forms of *T. cruzi*. **Figure 9** also shows the compounds obtained by partial synthesis from cubebin (**1**), as well as the reagents and conditions used in these reactions.

Fig. 9. Reagents and conditions: (a) Ac2O, Py, room temperature, 24 h; (b) NaH, BnBr, THF, room temperature, 24h; (c) EtONa, (CH3)2CH2Cl, EtOH, reflux, 6h; (d) PCC, CH2Cl2, room temperature, 12h; (e) HNO3, 2h, -10° C.

The natural cubebin (**1**), used as the starting compound to obtain the evaluated dibenzylbutyrolactone derivatives, did not display activity against trypomastigote forms of *T. cruzi* (Bastos et al., 1999). Hence, the biological evaluation against amastigote forms was undertaken only for lignans **2**, **3**, **4**, **5** and **6**. Cubebin was selected as starting compound because of its availability, being easily isolated in large amounts from the seeds of *Piper* 

drugs display limited efficacy in the treatment of the chronic phase of the disease and are quite toxic for the patients (de Souza et al., 2005). Therefore, there is an urgent demand for the discovery and development of novel therapeutic compounds to treat Chagas´ disease

De Souza et al. (2005) (de Souza et al., 2005) have reported the trypanocidal activity of cubebin (**1**) and its semi-synthetic derivatives against free amastigote forms of *T. cruzi*. **Figure 9** also shows the compounds obtained by partial synthesis from cubebin (**1**), as well

O

O

O

OR

O

Fig. 9. Reagents and conditions: (a) Ac2O, Py, room temperature, 24 h; (b) NaH, BnBr, THF, room temperature, 24h; (c) EtONa, (CH3)2CH2Cl, EtOH, reflux, 6h; (d) PCC, CH2Cl2, room

The natural cubebin (**1**), used as the starting compound to obtain the evaluated dibenzylbutyrolactone derivatives, did not display activity against trypomastigote forms of *T. cruzi* (Bastos et al., 1999). Hence, the biological evaluation against amastigote forms was undertaken only for lignans **2**, **3**, **4**, **5** and **6**. Cubebin was selected as starting compound because of its availability, being easily isolated in large amounts from the seeds of *Piper* 

O

O

(**d**) (**e**)

O

O

O

O

O

O2N

(**5**) (**6**)

O

O

O

NO2

O

O

O

R = Ac (**2**) R = CH2Ph (**3**)

R = (CH3)2N(CH2)2 (**4**)

as the reagents and conditions used in these reactions.

(**a**)

(**b**)

(**c**)

O

(**1**)

O

O

O

OH

O

temperature, 12h; (e) HNO3, 2h, -10° C.


*cubeba* (de Souza et al., 2005). **Table 2** shows the results of the trypanocidal activity


Table 2. Results of the trypanocidal activity evaluation of compounds **2**, **3**, **4**, **5**, **6** and benznidazole, against amastigote forms of Y strain of *T. cruzi* (de Souza et al., 2005).

The production of compound **2** by substitution of the lactol hydrogen of cubebin by an acetyl group led to a strong reduction of its trypanocidal ctivity, in comparison with all other evaluated compounds belonging to the same group (IC50 = 1.5 X 104 μM; **Table 2**). Furthermore, the comparison of compounds **2** and **3** indicate that the biological activity against the amastigote forms of *T. cruzi* was significantly affected by the nature of the substituting group at position C-9, which played an important role in the reduction of the calculated IC50 value for compound **3** (IC50 = 5.7 μM; **Table 2**). Likewise, cubebin derivative **4**, bearing an amino group at the lactol ring, displayed an activity quite similar to compound **3**.

Analysis of the obtained results, displayed in **Table 2**, indicate that compound 5 was the most active, with an IC50 value of 0.7 μM similar to that displayed by benznidazole (IC50 = 0.8 μM), a standard drug used as the positive control. On the other hand, most of the other evaluated compounds displayed much lower activity, with the exception of compounds **3** (IC50 = 5.7 μM) and **4** (IC50 = 4.7 μM), which showed significant activity.

In this study, De Souza et al. (2005) also pointed out that hinokinin (**HK**, **5**) is a promising compound to continue examining, since at 0.5 μM it displayed higher activity than benznidazole and at the other assayed concentrations (2.0, 8.0 and 32.0 μM) it showed similar activity.

In view of higher trypanocidal activity displayed by **HK** (**5**) against free amastigote forms of *T. cruzi* (**Table 2**; (de Souza et al., 2005), this lignan was selected to be assayed against epimastigote and intracellular amastigote forms of *T. cruzi*, both in vitro and *in vivo* assays (Saraiva et al., 2007). The results of the trypanocidal activity against epimastigote and intracellular amastigote forms of *T. cruzi* are shown in **Tables 3 and 4**, respectively.


Table 3. Results of the trypanocidal activity evaluation of **HK**, benznidazole and nifurtimox against epimastigote forms of CL strain of *T. cruzi* (Saraiva et al., 2007).

Lignans: Chemical and Biological Properties 223

These systems have been extensively utilized for oral and parenteral administration (Saraiva et al., 2010). The physical properties and the Food and Drug Administration approval of poly(lactide-co-glycosides) make them the most extensively studied commercially available

The microparticles can be able to sustain the release of the drug for a considerable period of time, to reduce the required frequency of administration increasing patient compliance, to avoid plasmatic fluctuations, to decrease side effects, and to facilitate dosage administration (Hans & Lowman, 2002). In this sense, our research group prepared **HK**-loaded PLGA microparticles to protect HK of biological interactions and promote its sustained release for treatment of Chagas´ disease. Moreover, the trypanocidal effect of microparticles containing

The **HK**-loaded PLGA microparticles were prepared with success (Saraiva et al., 2010) and presented narrow distribution size and a mean diameter of 0.862 μm, with PDI of 0.072 mm. Scanning electron micrographs of PLGA microparticles obtained showed that **HK** loaded microparticles presented, smooth and spherical surface. Due to their small diameter, the **HK**

The trypanocidal *in vivo* experiments were performed using Female Swiss mice (weigh, 20- 22 g) which were infected intraperitoneally with 2 X 104 trypomastigotes forms of *T. cruzi*. The treatment (20 days) was performed through subcutaneous route and initiated 48 h after

The treatment of infected mice with 40 mg kg-1 of **HK**-loaded microparticles each 2 days was able to provoke significant decrease in parasitemia levels compared with those recorded in untreated controls (P<0.05 at days 12, 14, 16, 19 and 21 post-infection with *T. cruzi*). The treatment with an equivalent amount of empty microparticles (without **HK**) had no effect on the parasitemia compared to untreated controls (Saraiva et al., 2010). Moreover, administration of **HK**-loaded microparticles was able to reduce the number of parasites more than the treatment with 20 mg kg-1 day-1 of **HK** not only in the parasitemic peak, but also in the course of infection (P<0.05 at days 14, 16, 19 and 21 post-infection with *T. cruzi*) (Saraiva et al., 2010). The use of PLGA micropartiles as vehicle for HK delivery can improve HK trypanocidal activity. It may be attributed to the fact that it can protect HK of biological interactions and promote its sustained release, with maintenance of its plasmatic

The **HK**-loaded microparticles developed by our research group can be considerable a promising system for sustained release of **HK** for therapeutic use and could be used in future clinical studies (Saraiva et al., 2010). Also, it is very important to point out that other *in vivo* assays have been developed by our research group in order to evaluate the parasitological cure of infection and the activity of this delivery system coating **HK** against

We have been reporting the significant trypanocidal activity of dibenzylbutyrolactone lignans, mainly the semi-synthetic **HK**. Such results aroused the interest within our group

microparticles obtained are better suited for parenteral delivery (Cegnar et al., 2005).

biodegradable polymers (Birnbaum et al., 2000).

infection, according to Saraiva et al. (2010).

concentration in therapeutic levels (Saraiva et al., 2010).

**6. Influences of stereochemistry on trypanocidal activity of** 

**HK** was evaluated *in vivo*.

other strains of *T. cruzi*.

**dibenzylbutyrolactone lignans** 


Table 4. Results of the trypanocidal activity evaluation of HK, benznidazole and nifurtimox against intracellular amastigote forms of CL strain of *T. cruzi* (Saraiva et al., 2007).

As it can be observed in **Table 3**, **HK** showed a very significant activity against epimastigote forms of *T. cruzi*, displaying IC50 value (0.67 μM) much lower than benznidazole and nifurtimox, used as positive controls (Saraiva et al., 2007). **HK**, also showed to be very active against intracellular amastigote forms, displaying IC50 value of 18.36 μM, which was similar to benznidazole (IC50 = 20.0 μM, **Table 4**).

The *in vivo* assays (Saraiva et al., 2007) were performed using five groups of five BALB/c males, weighing approximately 20 g each. The groups were as follows: group 1, animals without infection; group 2, control infected animals; group 3, animals treated with solvent; group 4, animals treated with benznidazole 40 mg kg-1 day-1; group 5, animals treated with **HK** 40 mg kg-1 day-1. The animals were inoculated with 2 X 104 trypomastigote forms of *T. cruzi* (Y strain). The treatment was initiated 48 h after infection and maintained for 20 days. The animals were treated twice a day with 20 mg kg-1 benznidazole and **HK** orally. The results obtained showed that the treatment with **HK** promoted 70.8% of parasitaemia reduction in the parasitaemic peak, while benznidazole displayed approximately 29.0% of parasite reduction (Saraiva et al., 2007). In addition, **HK** was able to reduce the number of parasites more than benznidazole not only in the parasitaemic peak, but also in all curse of infection (Saraiva et al., 2007).

Moreover, it was observed that the groups treated with **HK** displayed better survival rates than the group treated with benznidazole, with survival until the 22nd and 16th day after the beginning of the infection, respectively (Saraiva et al., 2007). Despite the obtained significant results for the *in vivo* assays, the treatment with **HK** or benznidazole did not cause parasitological cure. Overall, considering the promising results displayed by **HK** against both the epimastigote and amastigote forms of the parasite in the *in vitro* assay, as well as the good result displayed in the *in vivo* assay, this lignan has been considered as a lead compound for the development of new drugs for the treatment of Chagas´disease (Saraiva et al., 2007).

In order to obtain better efficacy of **HK** towards the intracellular forms of the parasite, our research group prepared and investigated the effect of **HK** load poly(D,L-lactide-coglycolide) microparticules.

#### **5. Hinokinin-load poly(d,l-lactide-co-glycolide) microparticles for Chagas´ disease**

The drug delivery system were developed for the purposes of bringing, uptaking, retaining, releasing, activating, localizing, and targeting the drugs at the right timing, period, dose, and place (Ueda & Tabata, 2003). The use of biodegradable polymers, as poly(D,L-lactic-coglycolic acid; PLGA), for the controlled release of therapeutic agents is now well established.

**Compounds Concentration (μM) X % of lyse (± SD) IC50 (μM)** 2.0 8.0 32.0 128.0 **HK** 25.84±1.09 31.92±9.29 61.72±9.17 100.0±0.40 18.36 Nifurtimox 44.6±0.99 63.78±1.25 83.31±0.79 90.63±1.13 3.54 benznidazole 14.33±2.65 35.81±0.65 57.28±1.99 78.75±0.67 20.00 Table 4. Results of the trypanocidal activity evaluation of HK, benznidazole and nifurtimox

As it can be observed in **Table 3**, **HK** showed a very significant activity against epimastigote forms of *T. cruzi*, displaying IC50 value (0.67 μM) much lower than benznidazole and nifurtimox, used as positive controls (Saraiva et al., 2007). **HK**, also showed to be very active against intracellular amastigote forms, displaying IC50 value of 18.36 μM, which was similar

The *in vivo* assays (Saraiva et al., 2007) were performed using five groups of five BALB/c males, weighing approximately 20 g each. The groups were as follows: group 1, animals without infection; group 2, control infected animals; group 3, animals treated with solvent; group 4, animals treated with benznidazole 40 mg kg-1 day-1; group 5, animals treated with **HK** 40 mg kg-1 day-1. The animals were inoculated with 2 X 104 trypomastigote forms of *T. cruzi* (Y strain). The treatment was initiated 48 h after infection and maintained for 20 days. The animals were treated twice a day with 20 mg kg-1 benznidazole and **HK** orally. The results obtained showed that the treatment with **HK** promoted 70.8% of parasitaemia reduction in the parasitaemic peak, while benznidazole displayed approximately 29.0% of parasite reduction (Saraiva et al., 2007). In addition, **HK** was able to reduce the number of parasites more than benznidazole not only in the parasitaemic peak, but also in all curse of

Moreover, it was observed that the groups treated with **HK** displayed better survival rates than the group treated with benznidazole, with survival until the 22nd and 16th day after the beginning of the infection, respectively (Saraiva et al., 2007). Despite the obtained significant results for the *in vivo* assays, the treatment with **HK** or benznidazole did not cause parasitological cure. Overall, considering the promising results displayed by **HK** against both the epimastigote and amastigote forms of the parasite in the *in vitro* assay, as well as the good result displayed in the *in vivo* assay, this lignan has been considered as a lead compound for the development of new drugs for the treatment of Chagas´disease (Saraiva

In order to obtain better efficacy of **HK** towards the intracellular forms of the parasite, our research group prepared and investigated the effect of **HK** load poly(D,L-lactide-co-

The drug delivery system were developed for the purposes of bringing, uptaking, retaining, releasing, activating, localizing, and targeting the drugs at the right timing, period, dose, and place (Ueda & Tabata, 2003). The use of biodegradable polymers, as poly(D,L-lactic-coglycolic acid; PLGA), for the controlled release of therapeutic agents is now well established.

**5. Hinokinin-load poly(d,l-lactide-co-glycolide) microparticles for Chagas´** 

against intracellular amastigote forms of CL strain of *T. cruzi* (Saraiva et al., 2007).

to benznidazole (IC50 = 20.0 μM, **Table 4**).

infection (Saraiva et al., 2007).

glycolide) microparticules.

et al., 2007).

**disease** 

These systems have been extensively utilized for oral and parenteral administration (Saraiva et al., 2010). The physical properties and the Food and Drug Administration approval of poly(lactide-co-glycosides) make them the most extensively studied commercially available biodegradable polymers (Birnbaum et al., 2000).

The microparticles can be able to sustain the release of the drug for a considerable period of time, to reduce the required frequency of administration increasing patient compliance, to avoid plasmatic fluctuations, to decrease side effects, and to facilitate dosage administration (Hans & Lowman, 2002). In this sense, our research group prepared **HK**-loaded PLGA microparticles to protect HK of biological interactions and promote its sustained release for treatment of Chagas´ disease. Moreover, the trypanocidal effect of microparticles containing **HK** was evaluated *in vivo*.

The **HK**-loaded PLGA microparticles were prepared with success (Saraiva et al., 2010) and presented narrow distribution size and a mean diameter of 0.862 μm, with PDI of 0.072 mm. Scanning electron micrographs of PLGA microparticles obtained showed that **HK** loaded microparticles presented, smooth and spherical surface. Due to their small diameter, the **HK** microparticles obtained are better suited for parenteral delivery (Cegnar et al., 2005).

The trypanocidal *in vivo* experiments were performed using Female Swiss mice (weigh, 20- 22 g) which were infected intraperitoneally with 2 X 104 trypomastigotes forms of *T. cruzi*. The treatment (20 days) was performed through subcutaneous route and initiated 48 h after infection, according to Saraiva et al. (2010).

The treatment of infected mice with 40 mg kg-1 of **HK**-loaded microparticles each 2 days was able to provoke significant decrease in parasitemia levels compared with those recorded in untreated controls (P<0.05 at days 12, 14, 16, 19 and 21 post-infection with *T. cruzi*). The treatment with an equivalent amount of empty microparticles (without **HK**) had no effect on the parasitemia compared to untreated controls (Saraiva et al., 2010). Moreover, administration of **HK**-loaded microparticles was able to reduce the number of parasites more than the treatment with 20 mg kg-1 day-1 of **HK** not only in the parasitemic peak, but also in the course of infection (P<0.05 at days 14, 16, 19 and 21 post-infection with *T. cruzi*) (Saraiva et al., 2010). The use of PLGA micropartiles as vehicle for HK delivery can improve HK trypanocidal activity. It may be attributed to the fact that it can protect HK of biological interactions and promote its sustained release, with maintenance of its plasmatic concentration in therapeutic levels (Saraiva et al., 2010).

The **HK**-loaded microparticles developed by our research group can be considerable a promising system for sustained release of **HK** for therapeutic use and could be used in future clinical studies (Saraiva et al., 2010). Also, it is very important to point out that other *in vivo* assays have been developed by our research group in order to evaluate the parasitological cure of infection and the activity of this delivery system coating **HK** against other strains of *T. cruzi*.

#### **6. Influences of stereochemistry on trypanocidal activity of dibenzylbutyrolactone lignans**

We have been reporting the significant trypanocidal activity of dibenzylbutyrolactone lignans, mainly the semi-synthetic **HK**. Such results aroused the interest within our group

Lignans: Chemical and Biological Properties 225

competitive antagonist, which may be confirmed by comparison of the IC50 value of the

In conclusion, this study pointed the importance of the stereochemistry on trypanocidal activity of dibenzylbutyrolactone lignans and brings new perspective in the importance to understand the trypanocidal structure-activity relationship for this class of natural compounds.

**7. Antimicrobial potential of some natural and semi-synthetic lignans against**

The lignans possess a wide spectrum of biological activities, including antimicrobial (Saleem et al., 2005). Considering this fact, our research group also decided to investigate the potential of some natural and semi-synthetic lignans against mycobateria and oral

Tuberculosis is a severe infectious disease caused by mycobacteria belonging to the *Mycobacterium tuberculosis* complex. According to WHO, tubercolosis affects nearly 30% of the world´s population and is responsible for 3 million deaths worldwide each year, mainly in developing countries (Raviglione, 2003). The current chemotherapy of this pathology has been based on the use of combined drug therapy with rifampicin, isonizid, and pyrazinamide. However, the incorrect use and long drug administration, as well as the high cost and countless side-effects have led people to abandon the treatment before being completely cured, leading to resistant bacilli (Timmins & Deretic, 2006). In addition, the existence of drug-resistant tuberculosis reinforces the need to develop new safe and effective antimycobacterial drugs. In this sense, our research group evaluated the antimycobacterial

As shown in **Figure 11**, (-)-cubebin (**1**) was isolated from powdered seeds of *Piper cubeba* and then submitted to various semi-synthetic procedures to obtain hinokinin (**HK**, **5**), (-)-Oacetyl-cubebin (**2**), (-)-O-methyl-cubebin (**7**), (-)-O-(N,N-dimethylamine-ethyl)-cubebin (**4**) and (-)-6,6´-dinitrohinokinin (**6**). All these compounds were assayed *in vitro* by the microdilution technique on a Resazurin microtiter assay (REMA) plate, using a procedure

Cubebin (**1**) did not display any activity against the investigated strains (**Table 6**). **HK** (**5**) was moderately active against *Mycobacterium tuberculosis*, with a MIC value equal to 62.5 µg mL-1. Compound (**2**), whose lactol group is acetylated, displayed activity against *M. tuberculosis* (MIC = 125 µg mL-1) and *M. avium* (MIC = 62.5 µg mL-1). The best result was achieved with compound **7**, whose lactol group is methylated, leading to a MIC value equal to 31.25 µg mL-1 against *M. avium*. The other compounds were not active against any of the studied mycobacteria. In the case of the lactol-containing compounds evaluated here, it seems to be essential that the lactol group is absent, and the substituent of this group should be small, as in the case of **2** and **7**. *M. kansaii* (ATCC 12478) was the most resistant mycobacterium concerning

the evaluated compounds, with MIC values varying between 1000 and 2000 µg mL-1.

To sum up, cubebin and hinokinin semi-synthetic derivatives were prepared and evaluated for their antimycobacterial activity. Some derivatives were active against *M. tuberculosis* and *M. avium*, suggesting that this class of compounds may lead to a new generation of

racemic mixture with that of the **–tM** itself (da Silva et al., 2008).

activity of several lignans obtained from cubebin (Silva et al., 2009).

*Mycobacteria* **and oral pathogens** 

adapted from (Palomino et al., 2002).

antituberculosis agents (Silva et al, 2009).

pathogens (Silva et al., 2007; Silva et al., 2009).

to study the effect of stereochemistry in this biological property. For this purpose, methylpluviatolide, one of the most powerful compounds regarding trypanocidal activity (Bastos et al., 1999) was synthesized in its *trans* and *cis* racemic forms. Thus, allowing us to evaluate the trypanocidal activity not only of a mixture of these two stereoisomers, but also of the pure enantiomers, which were separated by chiral HPLC (da Silva et al., 2008).

*Trans* (tM) and *cis* (cM)racemic forms of methylpluviatolide were prepared by a procedure described by Landais et al. (1991) and Charlton and Chee (1997) (**Figure 10**).

Fig. 10. Reagents and conditions: (a) H2, 4 atm, Pd/C, ETOH, HClO4, 60h, room temperature; (b) THF, AC2O, Et3N, DMAP, 2h, room temperature; (c) DBU, CH2Cl2, 5h, room temperature; (d) H2, 4 atm, Pd/C, ETOH, 60h, room temperature.

The results obtained for the racemic mixture of *trans* and *cis* methylpluviatolide against *T. cruzi* showed that racemic *cis*-stereoisomer (**2**) is inactive, while the racemic *trans*stereoisomer (**1**) display significant trypanocidal activity, with an IC50 of 89.3 μM (da Silva et al., 2008).

On the basis of these results, a separation of the *trans*-stereoisomer from the racemic mixture was undertaken by chiral HPLC using an analytical Chiracel OJ (4.6 x 250 mm) column, aiming to evaluate the trypanocidal activity of each enantiomer separately. The chromatogram gave a well resolved peak separation, allowing the isolation of both enantiomers (da Silva et al., 2008), which were evaluated against trypomastigote forms of the Y strain of *T. cruzi*. **Table 5** shows the results of the trypanocidal activity evaluation of (+)-*trans*- methylpluviatolide (**+tM**) and (-)-*trans*- methylpluviatolide (**-tM**) against trypomastigote forms of the Y strain of *T. cruzi*.


Table 5. Results of the trypanocidal activity evaluation of **+tM** or **-tM** against trypomastigotes forms of the Y strain of *T. cruzi*. (da Silva et al., 2008).

The results show that **+tM** is completely inative, whereas the **–tM** displayed good activity, with an IC50 of 18.7 μM (da Silva et al., 2008). These results indicate that despite being completely inactive, the **+tM** blocks the action of the **–tM** when they are present in a racemic mixture. It should be taken into consideration that the **+tM** might bind to the active sites as a

to study the effect of stereochemistry in this biological property. For this purpose, methylpluviatolide, one of the most powerful compounds regarding trypanocidal activity (Bastos et al., 1999) was synthesized in its *trans* and *cis* racemic forms. Thus, allowing us to evaluate the trypanocidal activity not only of a mixture of these two stereoisomers, but also

*Trans* (tM) and *cis* (cM)racemic forms of methylpluviatolide were prepared by a procedure

<sup>O</sup> OCH3

**(a) (b,c,d)**

OCH3

O O

cM

OCH3

OCH3

O O

of the pure enantiomers, which were separated by chiral HPLC (da Silva et al., 2008).

OH

O O

temperature; (b) THF, AC2O, Et3N, DMAP, 2h, room temperature; (c) DBU, CH2Cl2, 5h,

The results obtained for the racemic mixture of *trans* and *cis* methylpluviatolide against *T. cruzi* showed that racemic *cis*-stereoisomer (**2**) is inactive, while the racemic *trans*stereoisomer (**1**) display significant trypanocidal activity, with an IC50 of 89.3 μM (da Silva et

On the basis of these results, a separation of the *trans*-stereoisomer from the racemic mixture was undertaken by chiral HPLC using an analytical Chiracel OJ (4.6 x 250 mm) column, aiming to evaluate the trypanocidal activity of each enantiomer separately. The chromatogram gave a well resolved peak separation, allowing the isolation of both enantiomers (da Silva et al., 2008), which were evaluated against trypomastigote forms of the Y strain of *T. cruzi*. **Table 5** shows the results of the trypanocidal activity evaluation of (+)-*trans*- methylpluviatolide (**+tM**) and (-)-*trans*- methylpluviatolide (**-tM**) against trypomastigote forms of the Y strain of *T.* 

**Compounds Concentration (μM) X % of lyse (± SD) IC50 (μM)** 8.0 32.0 128.0 **+tM** 5.3±2.5 7.6±3.4 9.9±2.5 1.3 X 106 **-tM** 40.6±3.6 52.3±4.4 79.7±0.0 18.7

The results show that **+tM** is completely inative, whereas the **–tM** displayed good activity, with an IC50 of 18.7 μM (da Silva et al., 2008). These results indicate that despite being completely inactive, the **+tM** blocks the action of the **–tM** when they are present in a racemic mixture. It should be taken into consideration that the **+tM** might bind to the active sites as a

Table 5. Results of the trypanocidal activity evaluation of **+tM** or **-tM** against trypomastigotes forms of the Y strain of *T. cruzi*. (da Silva et al., 2008).

Fig. 10. Reagents and conditions: (a) H2, 4 atm, Pd/C, ETOH, HClO4, 60h, room

room temperature; (d) H2, 4 atm, Pd/C, ETOH, 60h, room temperature.

described by Landais et al. (1991) and Charlton and Chee (1997) (**Figure 10**).

O

O O

al., 2008).

*cruzi*.

tM

OCH3

OCH3

O O competitive antagonist, which may be confirmed by comparison of the IC50 value of the racemic mixture with that of the **–tM** itself (da Silva et al., 2008).

In conclusion, this study pointed the importance of the stereochemistry on trypanocidal activity of dibenzylbutyrolactone lignans and brings new perspective in the importance to understand the trypanocidal structure-activity relationship for this class of natural compounds.

#### **7. Antimicrobial potential of some natural and semi-synthetic lignans against** *Mycobacteria* **and oral pathogens**

The lignans possess a wide spectrum of biological activities, including antimicrobial (Saleem et al., 2005). Considering this fact, our research group also decided to investigate the potential of some natural and semi-synthetic lignans against mycobateria and oral pathogens (Silva et al., 2007; Silva et al., 2009).

Tuberculosis is a severe infectious disease caused by mycobacteria belonging to the *Mycobacterium tuberculosis* complex. According to WHO, tubercolosis affects nearly 30% of the world´s population and is responsible for 3 million deaths worldwide each year, mainly in developing countries (Raviglione, 2003). The current chemotherapy of this pathology has been based on the use of combined drug therapy with rifampicin, isonizid, and pyrazinamide. However, the incorrect use and long drug administration, as well as the high cost and countless side-effects have led people to abandon the treatment before being completely cured, leading to resistant bacilli (Timmins & Deretic, 2006). In addition, the existence of drug-resistant tuberculosis reinforces the need to develop new safe and effective antimycobacterial drugs. In this sense, our research group evaluated the antimycobacterial activity of several lignans obtained from cubebin (Silva et al., 2009).

As shown in **Figure 11**, (-)-cubebin (**1**) was isolated from powdered seeds of *Piper cubeba* and then submitted to various semi-synthetic procedures to obtain hinokinin (**HK**, **5**), (-)-Oacetyl-cubebin (**2**), (-)-O-methyl-cubebin (**7**), (-)-O-(N,N-dimethylamine-ethyl)-cubebin (**4**) and (-)-6,6´-dinitrohinokinin (**6**). All these compounds were assayed *in vitro* by the microdilution technique on a Resazurin microtiter assay (REMA) plate, using a procedure adapted from (Palomino et al., 2002).

Cubebin (**1**) did not display any activity against the investigated strains (**Table 6**). **HK** (**5**) was moderately active against *Mycobacterium tuberculosis*, with a MIC value equal to 62.5 µg mL-1. Compound (**2**), whose lactol group is acetylated, displayed activity against *M. tuberculosis* (MIC = 125 µg mL-1) and *M. avium* (MIC = 62.5 µg mL-1). The best result was achieved with compound **7**, whose lactol group is methylated, leading to a MIC value equal to 31.25 µg mL-1 against *M. avium*. The other compounds were not active against any of the studied mycobacteria. In the case of the lactol-containing compounds evaluated here, it seems to be essential that the lactol group is absent, and the substituent of this group should be small, as in the case of **2** and **7**. *M. kansaii* (ATCC 12478) was the most resistant mycobacterium concerning the evaluated compounds, with MIC values varying between 1000 and 2000 µg mL-1.

To sum up, cubebin and hinokinin semi-synthetic derivatives were prepared and evaluated for their antimycobacterial activity. Some derivatives were active against *M. tuberculosis* and *M. avium*, suggesting that this class of compounds may lead to a new generation of antituberculosis agents (Silva et al, 2009).

Lignans: Chemical and Biological Properties 227

Recently, our research group also investigated the antimicrobial activity of cubebin and related derivatives against oral pathogens, mainly those responsible for caries disease,

Dental plaque is defined as a biofilm consisting of cariogenic bacteria adhered on the tooth surface and plays an important role in the development of dental caries (Chung et al., 2006; Xie et al., 2008), one of the main oral diseases that affect humankind (More et al., 2008; Souza et al., 2010). This destructive infection of the dental hard tissues can progress and if untreated, lead to the death of vital pulp tissue and tooth loss (Allaker & Douglas, 2009). Bacteria from the genus *Streptococci* are commonly isolated from the oral cavity (Hirasawa & Takada, 2002) and have been responsible for this infectious disease. Among them, *Streptococcus mutans* is considered one of the main cariogenic microorganisms, due to its ability to synthesize extracellular polysaccharides from sucrose, mainly waterinsoluble glucan, and initiate plaque formation (Koo et al., 2000). Other aerobic bacteria such as *Enterococcus faecalis*, *Lactobacillus casei*, *Streptococcus mitis*, *S. sanguinis*, *S. sobrinus* and *S. salivarius* are also important in the latter formation of the dental biofilm (Chung et

The mechanical removal of the dental plaque is the most efficient procedure to prevent caries, but the majority of the population does not perform this removal efficiently (Ambrosio et al., 2008). Moreover, dental treatment is often very expensive and not readily accessible, especially in developing countries (More et al., 2008). In this sense, the use of chemicals as a complementary measure is necessary and has demonstrated to be of great value in the prevention of the formation and in the decreasing of the tooth surface biofilm

Extensive efforts have been made toward the search for anticariogenic compounds that can be incorporated into dental products, aiming at complementing the mechanical removal. Several antibiotics, such as ampicillin, chlorhexidine, sanguinarine, metronidazole, phenolic-antiseptics and quaternary ammonium-antiseptics have been used to prevent dental caries. Among these compounds, chlorhexidine is considered a gold standard anticariogenic and has received the approval of the American Dental Association Council on Dental Therapeutics (Ambrosio et al., 2008). However, the regular use of oral care products containing this chemical are often associated with tooth and restoration staining, changes in the taste of food, and a burning sensation at the tip of the tongue (Greenberg et al., 2008; More et al., 2008; Porto et al., 2009b). In addition, chlorhexidine is much less effective in reducing the levels of *Lactobacillus* species, which are strongly related to caries evolution (Ambrosio et al., 2008). All these problems, therefore, denote that finding new, safe and

Thus, our research group tested compounds **1**, **4**, **5**, and **6** (**Figure 11**) and another semisynthetic derivative (O-benzyl cubebin, **8**, **Figure 12**)using the broth microdilution method (Andrews, 2001) against the following microorganisms: *Enterecoccus faecalis* (ATCC 4082), *Streptococcus salivarius* (ATCC 25975), *Streptococcus mitis* (ATCC 49456), *Streptococcus mutans* (ATCC 25275), *Streptococcus sobrinus* (ATCC 33478), *Streptococcus sanguinis* (ATCC 10556) and *Candida albicans* (ATCC 28366) (Silva et al. 2007). **Table 7** displays the minimum

which are intimately related with the dental plaque formation.

al., 2006).

(Furiga et al., 2008).

effective anticariogenic coumpounds is still needed.

inhibitory concentration values obtained for these compounds

Fig. 11. Chemical structures and conditions of the reactions (a) Acetic anhydride, room temperature, 24h. (b) Dimethyllethylammonium chloride, EtONa, dry THF, room temperature, N2 atmosphere, 6h. (c) Methyl iodide, NaH, dry THF, room temperature, 6h. (d) PCC (pyridinium chlorochromate) in dry methylene chloride, 24h, in an ice bath with continuous stirring. (e) HNO3, chloroform, -6oC, 2h.


a Standard antibiotic

Table 6. Minimal inhibitory concentration (MIC) of cubebin (**1**) and its derivatives against *M. tuberculosis*, *M. kansasii*, and *M. avium*.

O

O

c d e

N

O

O

NO2

O

O O

O O

6

O2N

O

O O

*M. tuberculosis M. kansaii M. avium* 

O O

1 HK (5)

O

Fig. 11. Chemical structures and conditions of the reactions (a) Acetic anhydride, room temperature, 24h. (b) Dimethyllethylammonium chloride, EtONa, dry THF, room

500 2000 1000 125 1000 62.5 250 2000 250 62.5 2000 500 1000 2000 1000 250 2000 31.25 **Rifampicina** 0.031 0.015 0.062

Table 6. Minimal inhibitory concentration (MIC) of cubebin (**1**) and its derivatives against *M.* 

temperature, N2 atmosphere, 6h. (c) Methyl iodide, NaH, dry THF, room temperature, 6h. (d) PCC (pyridinium chlorochromate) in dry methylene chloride, 24h, in an ice bath with

O

b

4

**Compound MIC [µg mL-1]** 

OH

a

O

O

O O

O O

O O

2

O O

O O

continuous stirring. (e) HNO3, chloroform, -6oC, 2h.

O O

O

OCH3

O O

7

a Standard antibiotic

*tuberculosis*, *M. kansasii*, and *M. avium*.

O O Recently, our research group also investigated the antimicrobial activity of cubebin and related derivatives against oral pathogens, mainly those responsible for caries disease, which are intimately related with the dental plaque formation.

Dental plaque is defined as a biofilm consisting of cariogenic bacteria adhered on the tooth surface and plays an important role in the development of dental caries (Chung et al., 2006; Xie et al., 2008), one of the main oral diseases that affect humankind (More et al., 2008; Souza et al., 2010). This destructive infection of the dental hard tissues can progress and if untreated, lead to the death of vital pulp tissue and tooth loss (Allaker & Douglas, 2009). Bacteria from the genus *Streptococci* are commonly isolated from the oral cavity (Hirasawa & Takada, 2002) and have been responsible for this infectious disease. Among them, *Streptococcus mutans* is considered one of the main cariogenic microorganisms, due to its ability to synthesize extracellular polysaccharides from sucrose, mainly waterinsoluble glucan, and initiate plaque formation (Koo et al., 2000). Other aerobic bacteria such as *Enterococcus faecalis*, *Lactobacillus casei*, *Streptococcus mitis*, *S. sanguinis*, *S. sobrinus* and *S. salivarius* are also important in the latter formation of the dental biofilm (Chung et al., 2006).

The mechanical removal of the dental plaque is the most efficient procedure to prevent caries, but the majority of the population does not perform this removal efficiently (Ambrosio et al., 2008). Moreover, dental treatment is often very expensive and not readily accessible, especially in developing countries (More et al., 2008). In this sense, the use of chemicals as a complementary measure is necessary and has demonstrated to be of great value in the prevention of the formation and in the decreasing of the tooth surface biofilm (Furiga et al., 2008).

Extensive efforts have been made toward the search for anticariogenic compounds that can be incorporated into dental products, aiming at complementing the mechanical removal. Several antibiotics, such as ampicillin, chlorhexidine, sanguinarine, metronidazole, phenolic-antiseptics and quaternary ammonium-antiseptics have been used to prevent dental caries. Among these compounds, chlorhexidine is considered a gold standard anticariogenic and has received the approval of the American Dental Association Council on Dental Therapeutics (Ambrosio et al., 2008). However, the regular use of oral care products containing this chemical are often associated with tooth and restoration staining, changes in the taste of food, and a burning sensation at the tip of the tongue (Greenberg et al., 2008; More et al., 2008; Porto et al., 2009b). In addition, chlorhexidine is much less effective in reducing the levels of *Lactobacillus* species, which are strongly related to caries evolution (Ambrosio et al., 2008). All these problems, therefore, denote that finding new, safe and effective anticariogenic coumpounds is still needed.

Thus, our research group tested compounds **1**, **4**, **5**, and **6** (**Figure 11**) and another semisynthetic derivative (O-benzyl cubebin, **8**, **Figure 12**)using the broth microdilution method (Andrews, 2001) against the following microorganisms: *Enterecoccus faecalis* (ATCC 4082), *Streptococcus salivarius* (ATCC 25975), *Streptococcus mitis* (ATCC 49456), *Streptococcus mutans* (ATCC 25275), *Streptococcus sobrinus* (ATCC 33478), *Streptococcus sanguinis* (ATCC 10556) and *Candida albicans* (ATCC 28366) (Silva et al. 2007). **Table 7** displays the minimum inhibitory concentration values obtained for these compounds

Lignans: Chemical and Biological Properties 229

Despite of the wide spectrum of biological activities related to lignans, the literature used to emphasize the antioxidant properties and the role of these metabolites in cancer treatment and prevention. (Fauré et al., 1990; McRae & Towers, 1984; Pan et al., 2009; Saleem et al., 2005; Yousefzadi et al., 2010). However, in recent years our research group pointed out the importance of such metabolites, specially cubebin and their semi-synthetic derivatives, as potential antichagasic agents (da Silva et al., 2008; de Souza et al., 2005; Saraiva et al., 2010; Saraiva et al., 2007). The very promising results obtained against *T. cruzi* suggested that further investigations of these lignans against other parasitic diseases should be performed. In this sense, our group is now focusing the evaluation of such compounds against, for example, *Schistossoma mansoni* and *Fasciola hepatica*, as well as the obtainment of new

In addition, our results on the antimicrobial activities of these metabolites also highlighted their potential as new antimicrobial agents (Silva et al., 2007; Silva et al., 2009). In this context, the literature also reports additional experiments with the objective of investigating other features of the antimicrobial activity, such as the time-kill curve experiments based on D'Arrigo et al (2010) and investigations about a possible synergistic effect between the most

Allaker R. P.; C. W. I. Douglas. (2009). Novel anti-microbial therapies for dental plaque-

Ambrosio S. R.; C. R. Tirapelli; F. B. da Costa; A. M. de Oliveira. (2006). Kaurane and

Ambrosio S. R.; N. A. J. C. Furtado; D. C. R. De Oliveira; F. B. Da Costa; C. H. G. Martins; T.

Andrews J. M. (2001). Determination of minimum inhibitory concentrations. *Journal of* 

Ayres D. C.; J. D. Loike. (1990). *Lignans: Chemical, Biological and Clinical Properties* (1),

Bastos J. K.; S. Albuquerque; M. L. Silva. (1999). Evaluation of the trypanocidal activity of

Birnbaum D. T.; J. D. Kosmala; D. B. Henthorn; L. Brannon-Peppas. (2000). Controlled

contractility. *Life Sciences*, 79, 10, (2006), pp. 925-933, 0024-3205

*Journal of Biosciences*, 63c, 5-6, (2008), pp. 326-330, 0939-5075

Cambridge University Press, Cambridge

(1999), pp. 541-544, 0032-0943

375-387, 0168-3659

*Antimicrobial Chemotherapy*, 48 Suppl 1, (2001), pp. 5-16, 0305-7453

related diseases. *International Journal of Antimicrobial Agents*, 33, 1, (2009), pp. 8-13,

pimarane-type diterpenes from the *Viguiera* species inhibit vascular smooth muscle

C. De Carvalho; T. S. Porto; R. C. S. Veneziani. (2008). Antimicrobial activity of kaurane diterpenes against oral pathogens. *Zeitschrift Fur Naturforschung Section C-a* 

lignans isolated from the leaves of *Zanthoxylum naranjillo*. *Planta Medica*, 65, 6,

release of beta-estradiol from PLAGA microparticles: the effect of organic phase solvent on encapsulation and release. *Journal of Controlled Release*, 65, 3, (2000), pp.

effective tested lignans and the current used antimicrobial agents (White et al., 1996).

**8. Future perspectives** 

**9. References** 

0924-8579

cubebin-related semi-synthetic derivatives.

Fig. 12. Structure of O-benzyl cubebin (compound **8**)


a Chlorhexidine

Table 7. Values of minimum inhibitory concentrations (in milimolar) of cubebin and its semi-synthetic derivatives against oral pathogens

The semi-synthetic derivative **6** was the most active one against all the evaluated microorganisms (**Table 7**). Compounds **5** and **6** are lignan-lactones and differ from cubebin by the presence of a carbonyl group at C9 **(Figure 11**). Analysis of the obtained results suggested that the presence of the carbonyl group at C9 with introduction of polar groups in the aromatic rings is beneficial for the antimicrobial activity.

The obtained results for antimicrobial activity are in accordance to those obtained for antiinflammatory and analgesic activities. Compounds possessing a lactone ring bearing two methylendioxyaryl groups display significant anti-inflammatory and analgesic activities, and the introduction of polar groups in the aromatic rings is advantageous for these activities . However, with regard to trypanocidal activity, the introduction of nitro groups at the aromatic rings is harmful for this activity. Besides, the lignan-lactone **HK** (**5**) was the most active compound against *T. cruzi* (de Souza et al., 2005).

#### **8. Future perspectives**

228 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

<sup>O</sup> <sup>O</sup>

O

*S. sanguinis* 

Table 7. Values of minimum inhibitory concentrations (in milimolar) of cubebin and its

The semi-synthetic derivative **6** was the most active one against all the evaluated microorganisms (**Table 7**). Compounds **5** and **6** are lignan-lactones and differ from cubebin by the presence of a carbonyl group at C9 **(Figure 11**). Analysis of the obtained results suggested that the presence of the carbonyl group at C9 with introduction of polar groups in

The obtained results for antimicrobial activity are in accordance to those obtained for antiinflammatory and analgesic activities. Compounds possessing a lactone ring bearing two methylendioxyaryl groups display significant anti-inflammatory and analgesic activities, and the introduction of polar groups in the aromatic rings is advantageous for these activities . However, with regard to trypanocidal activity, the introduction of nitro groups at the aromatic rings is harmful for this activity. Besides, the lignan-lactone **HK** (**5**) was the

0.35 0.25 0.22 0.20 0.32 0.27 0.28 0.31 0.21 0.21 0.19 0.28 0.23 0.23 0.38 0.25 0.25 0.25 0.32 0.28 0.28 0.30 0.20 0.21 0.18 0.27 0.23 0.23 0.31 0.20 0.23 0.18 0.29 0.23 0.28 **CHDa** 5.9x10-3 1.7 x10-3 3.9x10-3 5.9x10-3 5.9x10-3 1.5x10-3 7.9x10-3

Fig. 12. Structure of O-benzyl cubebin (compound **8**)

semi-synthetic derivatives against oral pathogens

the aromatic rings is beneficial for the antimicrobial activity.

most active compound against *T. cruzi* (de Souza et al., 2005).

*S. salivarius* 

*E. faecalis* 

**Compound** 

a Chlorhexidine

O

O O

**Microorganism** 

*S. mitis* 

*S. mutans* 

*S. sobrinus* 

*C. albicans*  Despite of the wide spectrum of biological activities related to lignans, the literature used to emphasize the antioxidant properties and the role of these metabolites in cancer treatment and prevention. (Fauré et al., 1990; McRae & Towers, 1984; Pan et al., 2009; Saleem et al., 2005; Yousefzadi et al., 2010). However, in recent years our research group pointed out the importance of such metabolites, specially cubebin and their semi-synthetic derivatives, as potential antichagasic agents (da Silva et al., 2008; de Souza et al., 2005; Saraiva et al., 2010; Saraiva et al., 2007). The very promising results obtained against *T. cruzi* suggested that further investigations of these lignans against other parasitic diseases should be performed. In this sense, our group is now focusing the evaluation of such compounds against, for example, *Schistossoma mansoni* and *Fasciola hepatica*, as well as the obtainment of new cubebin-related semi-synthetic derivatives.

In addition, our results on the antimicrobial activities of these metabolites also highlighted their potential as new antimicrobial agents (Silva et al., 2007; Silva et al., 2009). In this context, the literature also reports additional experiments with the objective of investigating other features of the antimicrobial activity, such as the time-kill curve experiments based on D'Arrigo et al (2010) and investigations about a possible synergistic effect between the most effective tested lignans and the current used antimicrobial agents (White et al., 1996).

#### **9. References**


Lignans: Chemical and Biological Properties 231

Gordaliza M.; P. A. Garcia; J. M. del Corral; M. A. Castro; M. A. Gomez-Zurita. (2004).

Greenberg M.; M. Dodds; M. Tian. (2008). Naturally occurring phenolic antibacterial

Gurib-Fakim A. (2006). Medicinal plants: traditions of yesterday and drugs of tomorrow.

Habauzit V.; M. N. Horcajada. (2008). Phenolic phytochemicals and bone. *Phytochemistry* 

Hans M. L.; A. M. Lowman. (2002). Biodegradable nanoparticles for drug delivery and

Hirasawa M.; K. Takada. (2002). Susceptibility of *Streptococcus mutans* and *Streptococcus* 

Hostettmann K.; J. L. Wolfender; S. Rodriguez. (1997). Rapid detection and subsequent

Houghton P. J. (2000). Use of small scale bioassays in the discovery of novel drugs from natural sources. *Phytotherapy Research*, 14, 6, (2000), pp. 419-423, 0951-418X Huang W. Y.; Y. Z. Cai; Y. Zhang. (2010). Natural phenolic compounds from medicinal

Koo H.; A. M. Vacca Smith; W. H. Bowen; P. L. Rosalen; J. A. Cury; Y. K. Park. (2000). Effects

Landais Y.; J. P. Robin; A. Lebrun. (1991). Ruthenium dioxide in fluoro acid medium: I. A

Macias F. A.; A. Lopez; R. M. Varela; A. Torres; J. M. G. Molinillo. (2004). Bioactive lignans

McRae D. W.; N. G. H. Towers. (1984). Biological activities of lignans. *Phytochemistry*, 23, 6,

Molfetta F. A.; A. T. Bruni; K. M. Honorio; A. B. da Silva. (2005). A structure-activity

More G.; T. E. Tshikalange; N. Lall; F. Botha; J. J. M. Meyer. (2008). Antimicrobial activity of

*Journal of Medicinal Chemistry*, 40, 4, (2005), pp. 329-338, 0223-5234

targeting. *Current Opinion in Solid State & Materials Science*, 6, (2002), pp. 319-327,

*sobrinus* to cell wall inhibitors and development of a novel selective medium for *S-*

isolation of bioactive constituents of crude plant extracts. *Planta Medica*, 63, 1,

herbs and dietary plants: potential use for cancer prevention. *Nutrition and Cancer*,

of *Apis mellifera* propolis on the activities of streptococcal glucosyltransferases in solution and adsorbed onto saliva-coated hydroxyapatite. *Caries Research*, 34, 5,

new agent in the biaryl oxidative coupling. Application to the synthesis of non phenolic bisbenzocyclooctadiene lignan lactones. *Tetrahedron*, 47, 23, (1991), pp.

from a cultivar of *Helianthus annuus*. *Journal of Agricultural and Food Chemistry*, 52,

relationship study of quinone compounds with trypanocidal activity. *European* 

medicinal plants against oral microorganisms. *Journal of Ethnopharmacology*, 119, 3,

*Molecular aspects of medicine*, 27, 1, (2006), pp. 1-93, 0098-2997

*sobrinus*. *Caries Research*, 36, 3, (2002), pp. 155-160, 0008-6568

*Toxicon*, 44, 4, (2004), pp. 441-459, 0041-0101

*Reviews*, 7, (2008), pp. 313-344, 1568-7767

pp. 11151-11156, 1520-5118

(1997), pp. 2-10, 0032-0943

62, 1, (2010), pp. 1-20, 1532-7914

(2000), pp. 418-426, 0008-6568

21, (2004), pp. 6443-6447, 0021-8561

(1984), pp. 1207-1220, 0031-9422

(2008), pp. 473-477, 0378-8741

3787-3804, 0040-4020

1359-0286

Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives.

compounds show effectiveness against oral bacteria by a quantitative structureactivity relationship study. *Journal of Agricultural and Food Chemistry*, 56, 23, (2008),


Canel C.; R. M. Moraes; F. E. Dayan; D. Ferreira. (2000). Podophyllotoxin. *Phytochemistry*, 54,

Cegnar M.; J. Kristl; J. Kos. (2005). Nanoscale polymer carriers to deliver chemotherapeutic

Chang J.; J. Reiner; J. Xie. (2005). Progress on the chemistry of dibenzocyclooctadiene

Charlton J. L.; G.-L. Chee. (1997). Asymmetric synthesis of lignans using oxazolidinones as

Charlton J. L. (1998). Antiviral activity of lignans. *Journal of Natural Products*, 61, 11, (1998),

Chung J. Y.; J. H. Choo; M. H. Lee; J. Hwang. (2006). Anticariogenic activity of macelignan

Cos P.; L. Maes; A. Vlietinck; L. Pieters. (2008). Plant-derived leading compounds for

D'Arrigo M.; G. Ginestra; G. Mandalari; P. M. Furneri; G. Bisignano. (2010). Synergism and

da Silva R.; J. Saraiva; S. de Albuquerque; C. Curti; P. M. Donate; T. N. C. Bianco; J. K.

de Souza V. A.; R. da Silva; A. C. Pereira; A. Royo Vde; J. Saraiva; M. Montanheiro; G. H. de

Dewick P. M. (2002). *Medicinal Natural Products: a Biosynthetic approach* Willey, Chichester,

Fabricant D. S.; N. R. Farnsworth. (2001). The value of plants used in traditional medicine for

Fauré M.; E. Lissi; R. Torres; L. A. Videla. (1990). Antioxidant activities of lignans and

Furiga A.; A. Lonvaud-Funel; G. Dorignac; C. Badet. (2008). In vitro anti-bacterial

flavonoids. *Phytochemistry*, 29, 12, (1990), pp. 3773-3775, 0031-9422

lignans. *Chemical Reviews*, 105, 12, (2005), pp. 4581-4609, 0009-2665

*Phytomedicine*, 13, 4, (2006), pp. 261-266, 0944-7113

*Letters*, 15, 2, (2005), pp. 303-307, 0960-894X


agents to tumours. *Expert Opinion on Biological Therapy*, 5, 12, (2005), pp. 1557-1569,

chiral auxiliaries. *Canadian Journal of Chemistry*, 75, 8, (1997), pp. 1076-1083, 0008-

isolated from *Myristica fragrans* (nutmeg) against *Streptococcus mutans*.

chemotherapy of human immunodeficiency virus (HIV) infection - an update (1998

postantibiotic effect of tobramycin and *Melaleuca alternifolia* (tea tree) oil against *Staphylococcus aureus* and *Escherichia coli*. *Phytomedicine*, 17, 5, (2010), pp. 317-322,

Bastos; M. L. A. Silva. (2008). Trypanocidal structure-activity relationship for cisand trans-methylpluviatolide. *Phytochemistry*, 69, 9, (2008), pp. 1890-1894, 0031-

Souza; A. A. da Silva Filho; M. D. Grando; P. M. Donate; J. K. Bastos; S. Albuquerque; M. L. e Silva. (2005). Trypanocidal activity of (-)-cubebin derivatives against free amastigote forms of *Trypanosoma cruzi*. *Bioorganic & Medicinal Chemistry* 

drug discovery. *Environmental Health Perspectives*, 109 Suppl 1, (2001), pp. 69-75,

and anti-adherence effects of natural polyphenolic compounds on oral bacteria. *Journal of Applied Microbiology*, 105, 5, (2008), pp. 1470-1476, 1365-

(2000), pp. 115-120, 0031-9422

pp. 1447-1451, 0163-3864

1744-7682

4042

1618-095X

978-0-470-74168-9

0091-6765

2672

9422


Lignans: Chemical and Biological Properties 233

Soejarto D. D. (1996). Biodiversity prospecting and benefit-sharing: perspectives from the field. *Journal of Ethnopharmacology*, 51, 1-3, (1996), pp. 1-15, 0378-8741 Souza A. B.; C. H. G. Martins; M. G. M. Souza; N. A. J. C. Furtado; V. C. G. Heleno; J. P. B.

Srivastava V.; A. S. Negi; J. K. Kumar; M. M. Gupta; S. P. Khanuja. (2005). Plant-based

*Bioorganic & Medicinal Chemistry*, 13, 21, (2005), pp. 5892-5908, 0968-0896 Suzuki S.; T. Umezawa. (2007). Biosynthesis of lignans and norlignans. *Journal of Wood* 

Takeara R.; S. Albuquerque; N. P. Lopes; J. L. Lopes. (2003). Trypanocidal activity of

Timmins G. S.; V. Deretic. (2006). Mechanisms of action of isoniazid. *Molecular Microbiology*,

Tirapelli C. R.; S. R. Ambrosio; F. B. da Costa; A. M. de Oliveira. (2008). Diterpenes: a

Ueda H.; Y. Tabata. (2003). Polyhydroxyalkanonate derivatives in current clinical

Umezawa T.; T. Okunishi; M. Shimada. (1997). Stereochemical diversity in lignan

Umezawa T. (2003). Diversity in lignan biosynthesis. *Phytochemistry Reviews*, 2, 3, (2003), pp.

Webb A. L.; M. L. McCullough. (2005). Dietary lignans: potential role in cancer prevention.

White R. L.; D. S. Burgess; M. Manduru; J. A. Bosso. (1996). Comparison of three

Xie Q.; J. Li; X. Zhou. (2008). Anticaries effect of compounds extracted from *Galla chinensis* in

You Y. (2005). Podophyllotoxin derivatives: current synthetic approaches for new

*Nutrition and Cancer*, 51, 2, (2005), pp. 117-131, 0163-5581

(2009), pp. 779-784, 0939-5075

*Science*, 53, (2007), pp. 273-284, 1435-0211

62, 5, (2006), pp. 1220-1227, 0950-382X

*Drug Discovery*, 3, 1, (2008), pp. 1-8, 1574-8901

(2003), pp. 490-493, 0944-7113

85, (1997), pp. 96-125, 0049-7916

220, 1099-1573

0169-409X

371-390,

4804

459-465, 1399-302X

1381-6128

Da Silva Filho. (2009). Antimycobacterial activity of natural and semi-synthetic lignans. *Zeitschrift Fur Naturforschung Section C-a Journal of Biosciences*, 64, 11-12,

d. Sousa; E. M. P. Rocha; J. K. Bastos; W. R. Cunha; R. C. S. Veneziani; S. R. Ambrósio. (2010). Antimicrobial activity of terpenoids from *Copaifera langsdorffii* Desf. against cariogenic bacteria. *Phytotherapy Research*, 25, n/a, (2010), pp. 215-

anticancer molecules: a chemical and biological profile of some important leads.

*Lychnophora staavioides* Mart. (Vernonieae, Asteraceae). *Phytomedicine*, 10, 6-7,

therapeutic promise for cardiovascular diseases. *Recent Patents on Cardiovascular* 

applications and trials. *Advanced Drug Delivery Reviews*, 55, 4, (2003), pp. 501-518,

biosynthesis. *Wood Research: bulletin of the Wood Research Institute Kyoto University*,

different *in vitro* methods of detecting synergy: time-kill, checkerboard, and E test. *Antimicrobial Agents and Chemotherapy*, 40, 8, (1996), pp. 1914-1918, 0066-

a multispecies biofilm model. *Oral Microbiology and Immunology*, 23, 6, (2008), pp.

anticancer agents. *Current Pharmaceutical Design*, 11, 13, (2005), pp. 1695-1717,


Negi A. S.; J. K. Kumar; S. Luqman; K. Shanker; M. M. Gupta; S. P. Khanuja. (2008).

Newman D. J. (2008). Natural products as leads to potential drugs: An old process or the

Palomino J. C.; A. Martin; M. Camacho; H. Guerra; J. Swings; F. Portaels. (2002). Resazurin

Pan J. Y.; S. L. Chen; M. H. Yang; J. Wu; J. Sinkkonen; K. Zou. (2009). An update on lignans:

Paulino M.; F. Iribarne; M. Dubin; S. Aguilera-Morales; O. Tapia; A. O. Stoppani. (2005). The

Porto T. S.; N. A. J. C. Furtado; V. C. G. Heleno; C. H. G. Martins; F. B. da Costa; M. E.

Porto T. S.; R. Rangel; N. Furtado; T. C. de Carvalho; C. H. G. Martins; R. C. S. Veneziani; F.

Raviglione M. C. (2003). The TB epidemic from 1992 to 2002. *Tuberculosis (Edinb)*, 83, 1-3,

Saleem M.; H. J. Kim; M. S. Ali; Y. S. Lee. (2005). An update on bioactive plant lignans.

Saraiva J.; C. Vega; M. Rolon; R. da Silva; E. S. ML; P. M. Donate; J. K. Bastos; A. Gomez-

Saraiva J.; A. A. Lira; V. R. Esperandim; D. da Silva Ferreira; A. S. Ferraudo; J. K. Bastos; E. S.

Silva M. L.; H. S. Coimbra; A. C. Pereira; V. A. Almeida; T. C. Lima; E. S. Costa; A. H.

Silva M. L.; C. H. Martins; R. Lucarini; D. N. Sato; F. R. Pavanb; N. H. Freitas; L. N. Andrade;

*Natural Product Reports*, 22, 6, (2005), pp. 696-716, 0265-0568

1098-1128

2599, 0022-2623

1292, 1460-4752

8, (2002), pp. 2720-2722, 0066-4804

5, 5, (2005), pp. 499-519, 1389-5575

(2003), pp. 4-14, 1472-9792

795, 0932-0113

*Fitoterapia*, 80, (2009a), pp. 432-436, 0367-326X

*Molecules*, 14, 1, (2009b), pp. 191-199, 1420-3049

*Research*, 106, 3, (2010), pp. 703-708, 1432-1955

*Research*, 21, 5, (2007), pp. 420-422, 0951-418X

Recent advances in plant hepatoprotectives: a chemical and biological profile of some important leads. *Medicinal Research Reviews*, 28, 5, (2008), pp. 746-772,

new hope for drug discovery? *Journal of Medicinal Chemistry*, 51, 9, (2008), pp. 2589-

microtiter assay plate: Simple and inexpensive method for detection of drug resistance in *Mycobacterium tuberculosis*. *Antimicrobial Agents and Chemotherapy*, 46,

natural products and synthesis. *Natural Products Reports*, 26, 10, (2009), pp. 1251-

chemotherapy of chagas' disease: an overview. *Mini-Reviews in Medicinal Chemistry*,

Severiano; A. N. Silva; R. C. S. Veneziani; S. R. Ambrosio. (2009a). Antimicrobial *ent*-pimarane diterpenes from *Viguiera arenaria* against Gram-positive bacteria.

B. Da Costa; A. H. C. Vinholis; W. R. Cunha; V. C. G. Heleno; S. R. Ambrosio. (2009b). Pimarane-type Diterpenes: Antimicrobial Activity against Oral Pathogens.

Barrio; S. de Albuquerque. (2007). In vitro and in vivo activity of lignan lactones derivatives against *Trypanosoma cruzi*. *Parasitology Research*, 100, 4, (2007), pp. 791-

ML; C. M. de Gaitani; S. de Albuquerque; J. M. Marchetti. (2010). (-)-Hinokininloaded poly(D,-lactide-co-glycolide) microparticles for Chagas disease. *Parasitology* 

Vinholis; V. A. Royo; R. Silva; A. A. Filho; W. R. Cunha; N. A. Furtado; C. H. Martins; T. C. Carvalho; J. K. Bastos. (2007). Evaluation of *Piper cubeba* extract, (-) cubebin and its semi-synthetic derivatives against oral pathogens. *Phytotherapy* 

A. C. Pereira; T. N. Bianco; A. H. Vinholis; W. R. Cunha; J. K. Bastos; R. Silva; A. A.

Da Silva Filho. (2009). Antimycobacterial activity of natural and semi-synthetic lignans. *Zeitschrift Fur Naturforschung Section C-a Journal of Biosciences*, 64, 11-12, (2009), pp. 779-784, 0939-5075


**11** 

*1Bulgaria 2Spain* 

**The Genus** *Galanthus***:** 

*1AgroBioInstitute, Sofia,* 

**A Source of Bioactive Compounds** 

*2Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Catalonia,* 

The Amaryllidaceae family is one of the 20 most important alkaloid-containing plant families (Zhong, 2005). It comprises about 1100 perennial bulbous species classified in 85 genera, distributed throughout the tropics and warm temperate regions of the world (Willis, 1988). The specific alkaloids produced by the amaryllidaceous plants have attracted considerable attention due to their interesting pharmacological activities. One of them, galanthamine, is a long acting, selective, reversible and competitive inhibitor of the acetylcholinesterase enzyme (Thomsen *et al.,* 1998), which is marketed as a hydrobromide salt under the name of Razadyne® (formerly Reminyl®) and Nivalin® for the treatment of Alzheimer's disease, poliomyelitis and other neurological diseases (Heinrich and Teoh, 2004). After its discovery in *Galanthus woronowii* by Proskurina and co-authors in 1955 (Proskurina *et al.,* 1955), the pharmacological properties of galanthamine soon attracted the attention of the pharmaceutical industry. It was first produced by Sopharma (Bulgaria) under the name of Nivalin® from *G. nivalis* in the early 1960s, but due to the small plant size and variability of galanthamine content, this species was soon replaced by other plant

The genus *Galanthus* (Snowdrop; Greek *gála* "milk", *ánthos* "flower") comprises about 19 species (World Checklist of Selected Plant Families), and to our knowledge 11 have been investigated for their alkaloid content. Although the genus has only been partially studied, phytochemical work has revealed an exceptional diversity of alkaloid structures, many of them reported for the first time and with still unknown bioactivity. The present article

**2. Geographical distribution, taxonomical aspects and ecology of** *Galanthus*  The genus *Galanthus* L. is distributed around Europe, Asia Minor and the Caucasus region. The limits of its area of distribution are the Pyrenees in the west, the Caucasus and Iran in the east, and Sicily, the Peloponnese and Lebanon in the south. The northern distribution limit cannot be assessed due to human introduction and cultivation (Davis, 1999). Some

provides a brief overview of the phytochemical studies within the genus *Galanthus*.

**1. Introduction** 

sources (Berkov *et al.,* 2009*b*).

Strahil Berkov1, Carles Codina2 and Jaume Bastida2

Yousefzadi M.; M. Sharifi; M. Behmanesh; E. Moyano; M. Bonfill; R. M. Cusido; J. Palazon. (2010). Podophyllotoxin: Current approaches to its biotechnological production and future challenges. *Engineering in Life Sciences*, 10, 4, (2010), pp. 281-292, 1618- 2863

### **The Genus** *Galanthus***: A Source of Bioactive Compounds**

Strahil Berkov1, Carles Codina2 and Jaume Bastida2 *1AgroBioInstitute, Sofia, 2Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Catalonia, 1Bulgaria 2Spain* 

#### **1. Introduction**

234 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Yousefzadi M.; M. Sharifi; M. Behmanesh; E. Moyano; M. Bonfill; R. M. Cusido; J. Palazon.

2863

(2010). Podophyllotoxin: Current approaches to its biotechnological production and future challenges. *Engineering in Life Sciences*, 10, 4, (2010), pp. 281-292, 1618-

> The Amaryllidaceae family is one of the 20 most important alkaloid-containing plant families (Zhong, 2005). It comprises about 1100 perennial bulbous species classified in 85 genera, distributed throughout the tropics and warm temperate regions of the world (Willis, 1988). The specific alkaloids produced by the amaryllidaceous plants have attracted considerable attention due to their interesting pharmacological activities. One of them, galanthamine, is a long acting, selective, reversible and competitive inhibitor of the acetylcholinesterase enzyme (Thomsen *et al.,* 1998), which is marketed as a hydrobromide salt under the name of Razadyne® (formerly Reminyl®) and Nivalin® for the treatment of Alzheimer's disease, poliomyelitis and other neurological diseases (Heinrich and Teoh, 2004). After its discovery in *Galanthus woronowii* by Proskurina and co-authors in 1955 (Proskurina *et al.,* 1955), the pharmacological properties of galanthamine soon attracted the attention of the pharmaceutical industry. It was first produced by Sopharma (Bulgaria) under the name of Nivalin® from *G. nivalis* in the early 1960s, but due to the small plant size and variability of galanthamine content, this species was soon replaced by other plant sources (Berkov *et al.,* 2009*b*).

> The genus *Galanthus* (Snowdrop; Greek *gála* "milk", *ánthos* "flower") comprises about 19 species (World Checklist of Selected Plant Families), and to our knowledge 11 have been investigated for their alkaloid content. Although the genus has only been partially studied, phytochemical work has revealed an exceptional diversity of alkaloid structures, many of them reported for the first time and with still unknown bioactivity. The present article provides a brief overview of the phytochemical studies within the genus *Galanthus*.

#### **2. Geographical distribution, taxonomical aspects and ecology of** *Galanthus*

The genus *Galanthus* L. is distributed around Europe, Asia Minor and the Caucasus region. The limits of its area of distribution are the Pyrenees in the west, the Caucasus and Iran in the east, and Sicily, the Peloponnese and Lebanon in the south. The northern distribution limit cannot be assessed due to human introduction and cultivation (Davis, 1999). Some

The Genus *Galanthus*: A Source of Bioactive Compounds 237

*Galanthus reginae-olgae subsp. vernalis Kamari, Bot. Jahrb. Syst. 103: 116 (1982).* 

20. *Galanthus × allenii* Baker, (*G. alpinus × G. woronowii*) Gard. Chron., III, 9: 298 (1891). 21. *Galanthus × valentinei* Beck, (*G. plicatus × G. nivalis*) Wiener Ill. Gart.-Zeitung 19: 57

some species, like *G. cilicicus*, *G. peshmenii* and *G. reginae-olgae*, flower in autumn.

species (*G. nivalis*, *G, elwesii* and *G. woronowii*) from Turkey.

**3. Biosynthesis and structural types of Amaryllidaceae alkaloids** 

A particular characteristic of the Amaryllidaceae plant family is a consistent presence of an exclusive group of isoquinoline alkaloids, which have been isolated from plants of all the genera of this family. As a result of extensive phytochemical studies, over 500 alkaloids have been isolated from the amaryllidaceous plants (Zhong, 2005). The Amaryllidaceae type alkaloids have been structurally classified into nine main subgroups, namely lycorine, crinine, haemanthamine, narciclasine, galanthamine, tazettine, homolycorine, montanine

The habitats of *Galanthus* species are varied, ranging from undisturbed broad-leaved or coniferous woodlands of, for example oak (*Quercus* spp.), beech (*Fagus orientalis*), maple (*Acer* spp.), pines (*Pinus* spp.), Cilician fir (*Abies cilicia*), and cedar of Lebanon (*Cedrus libani*), woodland edges, river banks, scrub, grassland, amongst large rocks, and pockets of soil on rocks and cliff faces. *G. peshmenii* can sometimes be found only 10 m from the sea-shore on Kastellorhizo, a typical hot and dry Aegean island. In contrast, *G. platyphyllus* is a plant of the subalpine to alpine zone, and occurs mainly at altitudes of 2,000 - 2,700 m in alpine grasslands and meadows above the tree-line and at the edges of high-altitude woodlands (Davis, 1999). Typically, the *Galanthus* species are winter-to-spring flowering plants, but

*G. nivalis* and *G. elwesii* are two of the best known and most frequently cultivated bulbous plants. Their popularity is due to their beauty, longevity and because they flower when little else is in season. A vast number of cultivars and clones are available (Davis, 1999). Huge numbers of wild-collected bulbs are exported annually from Turkey. In the early 1980s onwards this trade increased, with many millions of *G. elwesii* bulbs being exported via the Netherlands. The large numbers of *Galanthus* bulbs coming into commerce caused great concern because it was uncertain whether the collection of bulbs in such high numbers was sustainable. For this reason, *Galanthus* was placed on Appendix II of CITES in 1990. The wild harvesting of *G. elwesii* bulbs is now carefully controlled and monitored, and export quotas are set each year. Some snowdrop species are threatened in their wild habitats, and in most countries it is now illegal to collect bulbs from the wild. Under CITES regulations, international trade in any quantity of *Galanthus*, whether bulbs or plants, live or dead, is illegal without a CITES permit. This applies to hybrids and named cultivars as well as species. CITES does, however, allow a limited trade in wild-collected bulbs of just three

13. *Galanthus platyphyllus* Traub & Moldenke, Herbertia 14: 110 (1948). 14. *Galanthus plicatus* M.Bieb., Fl. Taur.-Caucas., Suppl.: 225 (1819).

16. *Galanthus rizehensis* Stern, Snowdrops & Snowflakes: 37 (1956).

*Galanthus reginae-olgae subsp. reginae-olgae.* 

(1894).

15. *Galanthus reginae-olgae* Orph., Atti Congr. Int. Bot. Firenze 1874: 214 (1876).

17. *Galanthus transcaucasicus* Fomin, Opred. Rast. Kavk. Kryma 1: 281 (1909). 18. *Galanthus trojanus* A.P.Davis & Özhatay, Bot. J. Linn. Soc. 137: 409 (2001). 19. *Galanthus woronowii* Losinsk. in V.L.Komarov (ed.), Fl. URSS 4: 749 (1935).

species are widespread, while others are restricted to small areas. *G. nivalis,* for example, is native to a large area of Europe, stretching from the Pyrenees to Italy, Northern Greece, Ukraine, and European Turkey, while *G. trojanus* is a rare plant in the wild, found in a single location (an area less than 10 km2) in western Turkey (Davis and Ozhatay, 2001). Turkey is the country where most species (14) are geographically concentrated (Ünver, 2007).

All species of *Galanthus* are perennial, herbaceous plants that grow from bulbs. They have two or three linear leaves and an erect, leafless scape. The scape bears a pair of bract-like spathe valves at the top, from which emerges a solitary, bell-shaped white flower, held on a slender pedicel. The flower of *Galanthus* consists of six tepals, the outer three being larger and more convex than the inner series. The inner flower segments are marked with a green, or greenish-yellow, bridge-shaped mark at the tip of each tepal. The ovary is three-celled, ripening into a three-celled capsule. Each whitish seed has a small, fleshy tail (elaiosome) containing substances attractive to ants, which distribute the seeds (Davis, 1999). The genus *Galanthus* is closely related to the genus *Leucojum* L. but its plants can be easily distinguished because *Leucojum* has flowers with six equal tepals, from 2 to 6-7 flowers per scape and several leaves (Meerow and Snijman, 1998).

Species of the genus *Galanthus* L. (Amaryllidaceae) are difficult to distinguish and classify because of a lack of clearly definable morphological characteristics and a high level of variability. The search for other useful systematic information has produced little consensus in the enumeration of the species, divisions within the genus and relationships among their various components (Davis and Barnet, 1997). Besides morphological features, cariological (Kamari, 1981), anatomical (Davis and Barnet, 1997) and DNA (Zonneveld *et al.,* 2003) methods have been used to clarify the taxonomy of the genus.

It is generally accepted that the genus *Galanthus* comprises 19 species, 6 varieties and 2 natural interspecies hybrids (World Cheklist of Selected Plant Families):


species are widespread, while others are restricted to small areas. *G. nivalis,* for example, is native to a large area of Europe, stretching from the Pyrenees to Italy, Northern Greece, Ukraine, and European Turkey, while *G. trojanus* is a rare plant in the wild, found in a single location (an area less than 10 km2) in western Turkey (Davis and Ozhatay, 2001). Turkey is

All species of *Galanthus* are perennial, herbaceous plants that grow from bulbs. They have two or three linear leaves and an erect, leafless scape. The scape bears a pair of bract-like spathe valves at the top, from which emerges a solitary, bell-shaped white flower, held on a slender pedicel. The flower of *Galanthus* consists of six tepals, the outer three being larger and more convex than the inner series. The inner flower segments are marked with a green, or greenish-yellow, bridge-shaped mark at the tip of each tepal. The ovary is three-celled, ripening into a three-celled capsule. Each whitish seed has a small, fleshy tail (elaiosome) containing substances attractive to ants, which distribute the seeds (Davis, 1999). The genus *Galanthus* is closely related to the genus *Leucojum* L. but its plants can be easily distinguished because *Leucojum* has flowers with six equal tepals, from 2 to 6-7 flowers per scape and

Species of the genus *Galanthus* L. (Amaryllidaceae) are difficult to distinguish and classify because of a lack of clearly definable morphological characteristics and a high level of variability. The search for other useful systematic information has produced little consensus in the enumeration of the species, divisions within the genus and relationships among their various components (Davis and Barnet, 1997). Besides morphological features, cariological (Kamari, 1981), anatomical (Davis and Barnet, 1997) and DNA (Zonneveld *et al.,* 2003)

It is generally accepted that the genus *Galanthus* comprises 19 species, 6 varieties and 2

*Galanthus alpinus var. bortkewitschianus (Koss) A.P.Davis, Kew Bull. 51: 750 (1996).*  2. *Galanthus angustifolius* Koss, Bot. Mater. Gerb. Bot. Inst. Komarova Akad. Nauk S.S.S.R.

*Galanthus elwesii var. monostictus P.D.Sell in P.D. Sell & G.Murrell, Fl. Great Britain* 

6. *Galanthus gracilis* Celak., Sitzungsber. Königl. Böhm. Ges. Wiss., Math.-Naturwiss. Cl.

9. *Galanthus krasnovii* Khokhr., Byull. Moskovsk. Obshch. Isp. Prir., Otd. Biol., n.s., 68(4):

8. *Galanthus koenenianus* Lobin, C.D.Brickell & A.P.Davis, Kew Bull. 48: 161 (1993).

10. *Galanthus lagodechianus* Kem.-Nath., Zametki Sist. Geogr. Rast. 13: 6 (1947).

12. *Galanthus peshmenii* A.P.Davis & C.D.Brickell, New Plantsman 1: 17 (1994).

the country where most species (14) are geographically concentrated (Ünver, 2007).

several leaves (Meerow and Snijman, 1998).

*Galanthus alpinus var. alpinus.* 

*Galanthus elwesii var. elwesii* 

*Ireland 5: 363 (1996).* 

11. *Galanthus nivalis* L., Sp. Pl.: 288 (1753).

1891(1): 195 (1891).

140 (1963).

14: 134 (1951).

methods have been used to clarify the taxonomy of the genus.

natural interspecies hybrids (World Cheklist of Selected Plant Families):

1. *Galanthus alpinus* Sosn., Vestn. Tiflissk. Bot. Sada 19: 26 (1911).

3. *Galanthus cilicicus* Baker, Gard. Chron. 1897(1): 214 (1897). 4. *Galanthus elwesii* Hook.f., Bot. Mag. 101: t. 6166 (1875), nom. cons.

5. *Galanthus fosteri* Baker, Gard. Chron., III, 5: 458 (1889).

7. *Galanthus ikariae* Baker, Gard. Chron. 1893(1): 506 (1893).


The habitats of *Galanthus* species are varied, ranging from undisturbed broad-leaved or coniferous woodlands of, for example oak (*Quercus* spp.), beech (*Fagus orientalis*), maple (*Acer* spp.), pines (*Pinus* spp.), Cilician fir (*Abies cilicia*), and cedar of Lebanon (*Cedrus libani*), woodland edges, river banks, scrub, grassland, amongst large rocks, and pockets of soil on rocks and cliff faces. *G. peshmenii* can sometimes be found only 10 m from the sea-shore on Kastellorhizo, a typical hot and dry Aegean island. In contrast, *G. platyphyllus* is a plant of the subalpine to alpine zone, and occurs mainly at altitudes of 2,000 - 2,700 m in alpine grasslands and meadows above the tree-line and at the edges of high-altitude woodlands (Davis, 1999). Typically, the *Galanthus* species are winter-to-spring flowering plants, but some species, like *G. cilicicus*, *G. peshmenii* and *G. reginae-olgae*, flower in autumn.

*G. nivalis* and *G. elwesii* are two of the best known and most frequently cultivated bulbous plants. Their popularity is due to their beauty, longevity and because they flower when little else is in season. A vast number of cultivars and clones are available (Davis, 1999). Huge numbers of wild-collected bulbs are exported annually from Turkey. In the early 1980s onwards this trade increased, with many millions of *G. elwesii* bulbs being exported via the Netherlands. The large numbers of *Galanthus* bulbs coming into commerce caused great concern because it was uncertain whether the collection of bulbs in such high numbers was sustainable. For this reason, *Galanthus* was placed on Appendix II of CITES in 1990. The wild harvesting of *G. elwesii* bulbs is now carefully controlled and monitored, and export quotas are set each year. Some snowdrop species are threatened in their wild habitats, and in most countries it is now illegal to collect bulbs from the wild. Under CITES regulations, international trade in any quantity of *Galanthus*, whether bulbs or plants, live or dead, is illegal without a CITES permit. This applies to hybrids and named cultivars as well as species. CITES does, however, allow a limited trade in wild-collected bulbs of just three species (*G. nivalis*, *G, elwesii* and *G. woronowii*) from Turkey.

#### **3. Biosynthesis and structural types of Amaryllidaceae alkaloids**

A particular characteristic of the Amaryllidaceae plant family is a consistent presence of an exclusive group of isoquinoline alkaloids, which have been isolated from plants of all the genera of this family. As a result of extensive phytochemical studies, over 500 alkaloids have been isolated from the amaryllidaceous plants (Zhong, 2005). The Amaryllidaceae type alkaloids have been structurally classified into nine main subgroups, namely lycorine, crinine, haemanthamine, narciclasine, galanthamine, tazettine, homolycorine, montanine

The Genus *Galanthus*: A Source of Bioactive Compounds 239

The biogenetic pathway of gracilines possibly originates from the 6-hydroxy derivatives of haemanthamine-type species (Noyan *et al.,* 1998), while plicamine-type alkaloids most probably proceed from tazettine-type compounds, considering their structural similarities

The phytochemical studies of the genus *Galanthus* started in the early fifties of the last century. Two of the first alkaloids reported for the genus were galanthine (Proskurina and Ordzhonikidze, 1953) and galanthamine (Proskurina *et al.,* 1955), which were isolated from *G. voronowii*. To the best of our knowledge, eleven species from the genus *Galanthus* have been phytochemically studied to date and ninety alkaloids have been found and classified in

Until recently, the distribution of alkaloids within the genus has been studied by classical phytochemical approaches. The collected biomass is extracted with alcohol, the neutral compounds removed at low pH and the alkaloids fractionated after basification of the extract. Individual alkaloids have been separated by column chromatography, preparative TLC, prep. HPLC, etc., and identified by spectroscopy, mainly 1D and 2D NMR. The GC-MS technique has proved to be very effective for rapid separation and identification of complex mixtures of Amaryllidaceae alkaloids obtained from low mass samples (Kreh *et al.,* 1995). Thus, the assessment of alkaloid distribution at species, populational and individual levels and the detection of new compounds have become much easier and faster (Berkov *et al.,*

An overview of the literature indicates that the genus *Galanthus* is a very rich source of novel compounds. Thirty-seven alkaloids (namely **12**, **22**, **26**, **29**, **34**-**39**, **46-49, 53, 56-58, 62, 67, 69-75, 77-86**) or *ca*. 40% of all identified compounds from the genus have been isolated for the first time from *Galanthus*. What is more, the biochemical evolution of the genus has led to the occurrence of two specific subgroups, namely graciline- and plicamine-type

The most studied species are *G. nivalis* and *G. elwesii*. Due to taxonomical changes over the years, the information on the alkaloids of *G. nivalis* is confusing. Thus, until 1966, only one *Galanthus* species had been recognized in Bulgaria, namely *G. nivalis* L. (Jordanov, 1964). This taxon was subsequently separated into *G. nivalis* L. and *G. elwesii* Hook. (Kozuharov, 1992). At present, it is unclear which plant species the alkaloids isolated in the early sixties from Bulgarian *G. nivalis* can be attributed to (Valkova, 1961; Bubeva-Ivanova and Pavlova, 1965). Kaya *et al.* (2004*b*) have reported five alkaloids for *G. nivalis* L. subsp. *silicicus* (Baker) Guttl.-Tann., a taxon regarded as a synonym of *G. silicicus* Baker by other authors (Davis and Barnett, 1997; Davis, 1999). A recent revelation has substantiated that *G. nivalis* subsp. *cilicicus* is identical to the newly introduced species, G*. trojanus* A. P. Davis and N. Özhatay,

Latvala *et al.,* (1995) isolated 18 alkaloids (6 new) from *G. elwesii* in addition to the already reported flexinine, elwesine, tazettine and haemanthamine (Boit and Ehmke, 1955; Boit and Döpke, 1961). The occurrence of elwesine (**26**) in the genus is particularly interesting. This compound displays a β-configuration of its 5,10b-ethano bridge, which is typical of the South African representatives of the family (Viladomat *et al.,* 1997). Although widely

a plant species endemic to Northwestern Turkey (Davis and Özhatay, 2001).

**4. Distribution of alkaloids in the genus** *Galanthus*

(Ünver *et al.,* 1999*a*).

2007*a*, 2009*c*, 2011).

alkaloids.

11 structural types (Table 1, Fig.2).

and norbelladine (Bastida *et al.,* 2006). In the genus *Galanthus*, however, two new structural subgroups, graciline and plicamine type alkaloids, have been found (Ünver, 2007). The following new subgroups have also been reported: specific augustamine-type structures in *Crinum kirkii* (Machocho *et al.,* 2004), a carboline alkaloid in *Hippeastrum vittatum* (Youssef, 2001), mesembrane (*Sceletium*)-type compounds in *Narcissus pallidulus* and *N. triandrus* (Bastida *et al.,* 2006), and phtalideisoquinoline-, benzyltetrahydroisoquinoline- and aporphine-type alkaloids in *G. trojanus* (Kaya *et al.,* 2004*b,* 2011). Mesembrane-type compounds are typical of the genus *Sceletium* of the Aizoaceae, while phtalideisoquinoline-, benzyltetrahydroisoquinoline- and aporphine-type alkaloids are found in the Papaveraceae, both families being dicotyledonous. Tyramine-type protoalkaloids, which are biosynthesized in Poaceae, Cactaceae, some algae and fungi, have also been found in *Leucojum* and *Galanthus* species (Berkov *et al.,* 2009*a,* 2011).

Amaryllidaceae alkaloids are formed biogenetically by intramolecular oxidative coupling of norbelladines derived from the amino acids L-phenylalanine and L-tyrosine (Bastida *et al.,* 2006). The key intermediate metabolite is *O*-methylnorbelladine. *Ortho-para*´ phenol oxidative coupling of *O*-methylnorbelladine results in the formation of a lycorine-type skeleton, from which homolycorine-type compounds proceed. The galanthamine-type skeleton originates from *para-ortho*´ phenol oxidative coupling. *Para-para*´ phenol oxidative coupling leads to the formation of crinine, haemanthamine, tazettine, narciclasine and montanine structures (Bastida *et al.,* 2006). In the present article, for the structures reported by different authors we have adopted the numbering system according to Bastida *et al.,* (2006, Fig. 1).

Fig. 1. Biosynthetic pathway of *Galanthus* alkaloids with representative compounds.

and norbelladine (Bastida *et al.,* 2006). In the genus *Galanthus*, however, two new structural subgroups, graciline and plicamine type alkaloids, have been found (Ünver, 2007). The following new subgroups have also been reported: specific augustamine-type structures in *Crinum kirkii* (Machocho *et al.,* 2004), a carboline alkaloid in *Hippeastrum vittatum* (Youssef, 2001), mesembrane (*Sceletium*)-type compounds in *Narcissus pallidulus* and *N. triandrus* (Bastida *et al.,* 2006), and phtalideisoquinoline-, benzyltetrahydroisoquinoline- and aporphine-type alkaloids in *G. trojanus* (Kaya *et al.,* 2004*b,* 2011). Mesembrane-type compounds are typical of the genus *Sceletium* of the Aizoaceae, while phtalideisoquinoline-, benzyltetrahydroisoquinoline- and aporphine-type alkaloids are found in the Papaveraceae, both families being dicotyledonous. Tyramine-type protoalkaloids, which are biosynthesized in Poaceae, Cactaceae, some algae and fungi, have also been found in

Amaryllidaceae alkaloids are formed biogenetically by intramolecular oxidative coupling of norbelladines derived from the amino acids L-phenylalanine and L-tyrosine (Bastida *et al.,* 2006). The key intermediate metabolite is *O*-methylnorbelladine. *Ortho-para*´ phenol oxidative coupling of *O*-methylnorbelladine results in the formation of a lycorine-type skeleton, from which homolycorine-type compounds proceed. The galanthamine-type skeleton originates from *para-ortho*´ phenol oxidative coupling. *Para-para*´ phenol oxidative coupling leads to the formation of crinine, haemanthamine, tazettine, narciclasine and montanine structures (Bastida *et al.,* 2006). In the present article, for the structures reported by different authors we have adopted the numbering system according to Bastida *et al.,*

*Leucojum* and *Galanthus* species (Berkov *et al.,* 2009*a,* 2011).

(2006, Fig. 1).

**L-Phe**

HO

N

O O

6 6a 7 8 9 10 10a 10b

H H

11 12

O

O

OH

OH

12 11

O O 10 10a

*para-para´*

7

O O

**Homolycorine Narciclasine Graciline Plicamine**

Fig. 1. Biosynthetic pathway of *Galanthus* alkaloids with representative compounds.

H2N

**Tyramine**

HO

H

H

NMe

O

1´

4´

OH

<sup>H</sup> <sup>N</sup>

O

NMe

O

OMe

MeO

1 2 3 4 4a 12 <sup>11</sup> 10b

OH

4

12

O O

<sup>H</sup> <sup>N</sup>

6 8 6a 9

10b

2

11

O O

OH NMe

12

3

4

<sup>H</sup> <sup>N</sup>

6 8 6a 9

**Galanthamine Haemanthamine Tazettine**

10b

O O

**L-Tyr**

2

<sup>1</sup> <sup>3</sup> 4a

11

O

4a 4

11 12

6 6a 7 8 9 10 10a 10b

1 2 OMe

OH

10 10a 1 3 4a

7 **Crinine**

NMe

OH

NH O

OH

<sup>10</sup> 10a 10b

1' 2' 3' 4' 5'

6'

HO

*para-ortho´*

MeO

HO

MeO

*O***-Methylnorbelladine**

NH

**Norbelladine**

O

4

H

2

OH

H

**Lycorine**

H

OH

*ortho-para´*

HO CHO

**Protocatechuic aldehyde**

HO

6a 7 8 9 10 10a 10b

MeN

<sup>H</sup> MeO

MeO

O O The biogenetic pathway of gracilines possibly originates from the 6-hydroxy derivatives of haemanthamine-type species (Noyan *et al.,* 1998), while plicamine-type alkaloids most probably proceed from tazettine-type compounds, considering their structural similarities (Ünver *et al.,* 1999*a*).

#### **4. Distribution of alkaloids in the genus** *Galanthus*

The phytochemical studies of the genus *Galanthus* started in the early fifties of the last century. Two of the first alkaloids reported for the genus were galanthine (Proskurina and Ordzhonikidze, 1953) and galanthamine (Proskurina *et al.,* 1955), which were isolated from *G. voronowii*. To the best of our knowledge, eleven species from the genus *Galanthus* have been phytochemically studied to date and ninety alkaloids have been found and classified in 11 structural types (Table 1, Fig.2).

Until recently, the distribution of alkaloids within the genus has been studied by classical phytochemical approaches. The collected biomass is extracted with alcohol, the neutral compounds removed at low pH and the alkaloids fractionated after basification of the extract. Individual alkaloids have been separated by column chromatography, preparative TLC, prep. HPLC, etc., and identified by spectroscopy, mainly 1D and 2D NMR. The GC-MS technique has proved to be very effective for rapid separation and identification of complex mixtures of Amaryllidaceae alkaloids obtained from low mass samples (Kreh *et al.,* 1995). Thus, the assessment of alkaloid distribution at species, populational and individual levels and the detection of new compounds have become much easier and faster (Berkov *et al.,* 2007*a*, 2009*c*, 2011).

An overview of the literature indicates that the genus *Galanthus* is a very rich source of novel compounds. Thirty-seven alkaloids (namely **12**, **22**, **26**, **29**, **34**-**39**, **46-49, 53, 56-58, 62, 67, 69-75, 77-86**) or *ca*. 40% of all identified compounds from the genus have been isolated for the first time from *Galanthus*. What is more, the biochemical evolution of the genus has led to the occurrence of two specific subgroups, namely graciline- and plicamine-type alkaloids.

The most studied species are *G. nivalis* and *G. elwesii*. Due to taxonomical changes over the years, the information on the alkaloids of *G. nivalis* is confusing. Thus, until 1966, only one *Galanthus* species had been recognized in Bulgaria, namely *G. nivalis* L. (Jordanov, 1964). This taxon was subsequently separated into *G. nivalis* L. and *G. elwesii* Hook. (Kozuharov, 1992). At present, it is unclear which plant species the alkaloids isolated in the early sixties from Bulgarian *G. nivalis* can be attributed to (Valkova, 1961; Bubeva-Ivanova and Pavlova, 1965). Kaya *et al.* (2004*b*) have reported five alkaloids for *G. nivalis* L. subsp. *silicicus* (Baker) Guttl.-Tann., a taxon regarded as a synonym of *G. silicicus* Baker by other authors (Davis and Barnett, 1997; Davis, 1999). A recent revelation has substantiated that *G. nivalis* subsp. *cilicicus* is identical to the newly introduced species, G*. trojanus* A. P. Davis and N. Özhatay, a plant species endemic to Northwestern Turkey (Davis and Özhatay, 2001).

Latvala *et al.,* (1995) isolated 18 alkaloids (6 new) from *G. elwesii* in addition to the already reported flexinine, elwesine, tazettine and haemanthamine (Boit and Ehmke, 1955; Boit and Döpke, 1961). The occurrence of elwesine (**26**) in the genus is particularly interesting. This compound displays a β-configuration of its 5,10b-ethano bridge, which is typical of the South African representatives of the family (Viladomat *et al.,* 1997). Although widely

The Genus *Galanthus*: A Source of Bioactive Compounds 241

*G. elwesii* 

Tyramine (**1**) +1 +1 *+*<sup>25</sup>

Arolycoricidine (**10**) *+*25 *+*<sup>27</sup> Narciprimine (**11**) *+*<sup>27</sup> 

Vittatine/crinine (**24**) +1,5 +24 +26

11-Hydroxyvittatine (**28**) +2 +10 +26 11-Hydroxyvittatine *N*-oxide (**29**) *+*<sup>25</sup>

8-*O*-Demethylmaritidine (**31**) *+*<sup>25</sup> Narcidine (**32**) *+*<sup>25</sup> Haemanthamine (**33**) +1 +1 *+*<sup>25</sup>

Hamayne (**27**, 3-Epihydroxybulbispermine) +7 +7,9 +4

Galanthamine (**12**) +1,2 +1 +19 +22 +4 +23 +24

*O*-Methylnorbelladine (**5**) *+*<sup>25</sup> 

Methyltyramine (**2**) +1 +1 Hordenine (**3**) +1,2 +1 +10

*N*-feruloyltyramine (**4**) *+*<sup>2</sup>

3-Epigalanthamine (**13**) +1 Narwedine (**14**) +1,2 *N*-Demethylgalanthamine (**15**) +1,2 Lycoramine (**16**) +1 +1 3-Epilycoramine (**17**) +1 +1 Sanguinine (**18**) +2 *N*-Formylnorlgalanthamine (**19**) +1 Leucotamine (**20**) +1,2 *O*-Methylleucotamine (**21**) +2 Nivalidine (**22**) *+*<sup>3</sup> *+*<sup>8</sup>

Buphanisine (**23**) +1,4

Flexinine (**25**) +6 Elwesine (**26**) +6

Maritidine (**30**) +1

11-*O*-(3'-Hydroxybutanoyl)hamayne (**34**) +1,7 +9 3,11-*O*-(3',3''-Dihydroxybutanoyl)hamayne (**35**) +9

hydroxybutanoyl)hamayne (**36**) +9

(**37**) +9

3,3'-*O*-(3',3''-Dihydroxybutanoyl)hamayne (**38**) +7

3,11,3'-*O-*(3', 3'', 3'''- Trihydroxybutanoyl)-hamayne

Ismine (**6**) +1 +1,7 +11 *N*-Formylismine (**7**) +12 Trisphaeridine (**8**) +1 +1 +13 5,6-Dihydrobicolorine (**9**) *+*<sup>13</sup> *+*<sup>15</sup>

*G. nivalis* 

*G. plicatus* 

*G. gracilis* 

*G. woronowii* 

*G. caucasicus* 

*G. ikariae* 

*G. krasnovii* 

*G. reginae-olgae* 

*G. trojanus* 

*G. rizehensis* 

**Compound** 

*I. Tyramine type* 

*II. Norbelladine* 

*III. Narciclasine type* 

*IV. Galanthamine type* 

*V. Haemanthamine type* 

3-*O*-(2''-Butenoyl)-11-*O*-(3'-

accepted that *G. nivalis* was the industrial source of galanthamine (in Bulgaria) during the 1960s (Heinrich and Teoh, 2004), later studies on 32 Bulgarian populations of *G. nivalis* and *G. elwesii* indicate that the distribution of this important compound is limited to a few populations of *G. elwesii*, while just one population of *G. nivalis* has been found to contain galanthamine and only as a minor alkaloid (Sidjimova *et al.,* 2003; Berkov *et al.,* 2011). These studies, however, have also shown a great intra-species diversity of alkaloid synthesis in *G. nivalis* and *G. elwesii.* The populations displayed between 6 and 31 alkaloids in their alkaloid patterns and about 70 compounds have been detected in total. Many of them were left unidentified due to the lack of reference spectra, possibly indicating new structures. This biochemical diversity has led to the isolation of eight more new alkaloids from these wellstudied species, after the collection of plant material from populations proven by GC-MS to be a rich source of unknown compounds (Berkov *et al.,* 2007*a*, 2009*c*). Interestingly, many of the *G. elwesii* populations have accumulated the tyramine-type protoalkaloids as major compounds (up to 99 % of all alkaloids). In addition to the tyramine chemotype, homolycorine, lycorine haemanthamine and galanthamine chemotypes have also been found in the studied populations of *G. elwesii*. A galanthamine chemotype population was also found for *G. nivalis*, but in contrast with *G. elwesii*, this *G. nivalis* population accumulated the 4,4a-dihydrogenated derivatives of galanthamine (**12**), lycoramine (**16)** and its isomer (**17**) (Berkov *et al.,* 2011).

As well as a high level of alkaloid diversity and the existence of different chemotypes among the species populations, *G. elwesii* and *G. nivalis* have also shown some important differences in their alkaloid patterns, at least in the studied Bulgarian populations. A study of sympatric populations, and 32 populations from both species showed that the alkaloid pattern of *G. nivalis* is dominated by compounds coming from a *para–para*´ oxidative coupling of *O*-methylnorbelladine (haemanthamine- and tazettine-type alkaloids, Fig. 1). The conjugated and free lycorine-type alkaloids proceeding from an *ortho–para*´ oxidative coupling were relatively less abundant. Homolycorine-type alkaloids were not detected in this plant species. In contrast to *G. nivalis*, the alkaloid pattern of *G. elwesii* was dominated mainly by compounds coming from *ortho–para*´ oxidative coupling: free lycorine- and homolycorine-type alkaloids. The synthesis of *para–para*´ oxidative products in *G. elwesii* is relatively weak (only haemanthamine- and no tazettine-type compounds, Berkov *et al.,* 2008, 2011). In total, 46 and 38 alkaloids have been identified in *G. elwesii* and *G. nivalis,* respectively.

In a study on sympatric *G. nivalis* and *G. elwesii* populations, it was found that the organs of the plants presented different alkaloid patterns (Berkov *et al.,* 2008). Thus, the predominant alkaloids of *G. nivalis* roots were found to belong to the lycorine and tazettine structural types, bulbs were dominated by tazettine, leaves by lycorine and flowers by haemanthamine-type alkaloids. The predominant alkaloids in *G. elwesii* roots, bulbs and leaves were those of the homolycorine type, whereas the flowers accumulated mainly tyramine-type compounds. To the best of our knowledge, no studies of the dynamics of the alkaloid patterns during ontogenesis have been reported for either of these two species or any other *Galanthus* species. Such studies, however, may contribute to the understanding of the chemoecological role of the alkaloids in the genus *Galanthus* and the Amaryllidaceae as a whole. A remarkably high number of alkaloids conjugated with 3-hydroxybutyryl moieties occur in *G. nivalis*. Co-existence of free and conjugated alkaloids in the plant implies that the latter may have a chemoecological role. Such conjugated alkaloids have rarely been reported for Amaryllidaceae plants.

accepted that *G. nivalis* was the industrial source of galanthamine (in Bulgaria) during the 1960s (Heinrich and Teoh, 2004), later studies on 32 Bulgarian populations of *G. nivalis* and *G. elwesii* indicate that the distribution of this important compound is limited to a few populations of *G. elwesii*, while just one population of *G. nivalis* has been found to contain galanthamine and only as a minor alkaloid (Sidjimova *et al.,* 2003; Berkov *et al.,* 2011). These studies, however, have also shown a great intra-species diversity of alkaloid synthesis in *G. nivalis* and *G. elwesii.* The populations displayed between 6 and 31 alkaloids in their alkaloid patterns and about 70 compounds have been detected in total. Many of them were left unidentified due to the lack of reference spectra, possibly indicating new structures. This biochemical diversity has led to the isolation of eight more new alkaloids from these wellstudied species, after the collection of plant material from populations proven by GC-MS to be a rich source of unknown compounds (Berkov *et al.,* 2007*a*, 2009*c*). Interestingly, many of the *G. elwesii* populations have accumulated the tyramine-type protoalkaloids as major compounds (up to 99 % of all alkaloids). In addition to the tyramine chemotype, homolycorine, lycorine haemanthamine and galanthamine chemotypes have also been found in the studied populations of *G. elwesii*. A galanthamine chemotype population was also found for *G. nivalis*, but in contrast with *G. elwesii*, this *G. nivalis* population accumulated the 4,4a-dihydrogenated derivatives of galanthamine (**12**), lycoramine (**16)** and its isomer (**17**) (Berkov *et al.,* 2011).

As well as a high level of alkaloid diversity and the existence of different chemotypes among the species populations, *G. elwesii* and *G. nivalis* have also shown some important differences in their alkaloid patterns, at least in the studied Bulgarian populations. A study of sympatric populations, and 32 populations from both species showed that the alkaloid pattern of *G. nivalis* is dominated by compounds coming from a *para–para*´ oxidative coupling of *O*-methylnorbelladine (haemanthamine- and tazettine-type alkaloids, Fig. 1). The conjugated and free lycorine-type alkaloids proceeding from an *ortho–para*´ oxidative coupling were relatively less abundant. Homolycorine-type alkaloids were not detected in this plant species. In contrast to *G. nivalis*, the alkaloid pattern of *G. elwesii* was dominated mainly by compounds coming from *ortho–para*´ oxidative coupling: free lycorine- and homolycorine-type alkaloids. The synthesis of *para–para*´ oxidative products in *G. elwesii* is relatively weak (only haemanthamine- and no tazettine-type compounds, Berkov *et al.,* 2008, 2011). In total, 46 and 38 alkaloids have been identified in *G. elwesii* and *G. nivalis,* respectively. In a study on sympatric *G. nivalis* and *G. elwesii* populations, it was found that the organs of the plants presented different alkaloid patterns (Berkov *et al.,* 2008). Thus, the predominant alkaloids of *G. nivalis* roots were found to belong to the lycorine and tazettine structural types, bulbs were dominated by tazettine, leaves by lycorine and flowers by haemanthamine-type alkaloids. The predominant alkaloids in *G. elwesii* roots, bulbs and leaves were those of the homolycorine type, whereas the flowers accumulated mainly tyramine-type compounds. To the best of our knowledge, no studies of the dynamics of the alkaloid patterns during ontogenesis have been reported for either of these two species or any other *Galanthus* species. Such studies, however, may contribute to the understanding of the chemoecological role of the alkaloids in the genus *Galanthus* and the Amaryllidaceae as a whole. A remarkably high number of alkaloids conjugated with 3-hydroxybutyryl moieties occur in *G. nivalis*. Co-existence of free and conjugated alkaloids in the plant implies that the latter may have a chemoecological role. Such conjugated alkaloids have rarely been reported

for Amaryllidaceae plants.


The Genus *Galanthus*: A Source of Bioactive Compounds 243

*G. elwesii* 

Another two phytochemically interesting species from which a number of new alkaloids have been isolated are *G. gracilis* and *G. plicatus*. Phytochemical studies on *G. gracilis* resulted in the isolation of three novel monomeric alkaloids (**78**, **80**, **81**) and a dimeric compound (**82**) bearing a 10b,4a-ethanoiminodibenzo[b,d]pyrane skeleton, which represents a new subgroup of Amaryllidaceae alkaloids named gracilines (Fig. 1, Noyan *et al.,* 1998; Ünver *et al.,* 2001). An unusual pentacyclic dinitrogenous alkaloid, gracilamine (**83**), was also isolated from this species (Ünver and Kaya, 2005). Another new graciline-type alkaloid (**79**, Noyan *et al.,* 1998) has been isolated from *G. plicatus*, together with compounds **84**-**86**  (Ünver et al., 1999*a*, 2001), representing a new subgroup of the Amaryllidaceae alkaloids where the oxygen atom at position 5 of a tazettine molecule is replaced by a nitrogen atom, conjugated with a 4-hydroxyphenethyl moiety. This new subgroup, named after the lead compound plicamine (**84**), was found later in another amaryllidaceous plant, *Cyrtanthus obliquus* (Brine *et al.,* 2002). Apart from plicamines, four new tazettine-type alkaloids (**46**-**49**) and a compound with a nonfused indole ring (**75**) have also been isolated in *G. plicatus* (Ünver *et al.,* 1999*b*, 2003). In total, 17 and 12 alkaloids have been reported for *G. plicatus* and

The other *Galanthus* species are relatively less studied. Four known alkaloids (**12**, **42**, **53**, and **54**), including galanthamine, have been reported for *G. woronovii* (Proskurina *et al.,* 1955; Proskurina and Ordzhonikidze, 1953; Yakovleva, 1963). A new compound, galanthusine (**78**),

Bulbocapnine (**87**) +26 Capnoidine (**88**) +26 Stylopine (**89**) *+*<sup>25</sup> Protopine (**90**) *+*<sup>25</sup> 1) Berkov *et al.,* (2011); 2) Latvala *et al.,* (1995); 3) Bubeva-Ivanova and Pavlova (1965); 4) Sener *et al.,* (1998); 5) Berkov *et al.,* (2008); 6) Wildman, (1968); 7) Berkov *et al.,* (2009c); 8) Kalashnikov (1970); 9) Berkov *et al.,* (2007a); 10) Ünver *et al.,* (2003); 11) Akneri and Günes (1998); 12) Ünver *et al.,* (2001); 13) Ünver *et al.,* (1999a); 14) Kaya *et al.,* (2004*a*); 15) Noyan (1999); 16) Noyan *et al.,* (1998); 17) Ünver *et al.,* (1999b); 18) Ünver and Kaya*,* (2005); 19) Proskurina *et al.,* (1955); 20) Yakovleva (1963); 21) Proskurina Ordzhonikidze (1953); 22) Tsakadze *et al.,* (1979); 23) Asoeva *et al.,* (1968); 24) Conforti *et al.,* (2010); 25)

3,4-Dihydro-3-hydroxygraciline (**80**) +12 3-Epi-3,4-dihydro-3-hydroxygraciline (**81**) +12 Digracine (**82**) +16 Gracilamine (**83**) +18

Plicamine (**84**) +17 Plicane (**85**) +12 Secoplicamine (**86**) +17

Kaya *et al.,* (2011); 26) Kaya *et al.,* (2004*b*); 27) Bozkurt *et al.,* (2010).

Table 1. Alkaloids reported in the genus *Galanthus*

*G. gracilis,* respectively (Table 1)*.* 

*G. nivalis* 

*G. plicatus* 

*G. gracilis* 

*G. woronowii* 

*G. caucasicus* 

*G. ikariae* 

*G. krasnovii* 

*G. reginae-olgae* 

*G. trojanus* 

*G. rizehensis* 

**Compound** 

*X. Plicamine type* 

*XI. Other* 


*G. elwesii* 

Lycorine (**54**) +1,2 +1,7 +14 +14 +21 +22 +24 +26

8-*O*-Methyldihydrosternbergine *N*-oxide (**62**) *+*<sup>25</sup> Dihydrolycorine (**63**) *+*<sup>25</sup> 

Neronine (**76**) +24

8-*O*-Demethylhomolycorine (**65**) +1,2 +15 +15 +22 +4

Tazettine (**42**) +1 +7 +11 +15 +20 +22 +4 +24

11,3'-*O-*(3',3''-Dihydroxybutanoyl)hamayne (**39**) +7

11-Deoxytazettine (**40**) +1 +1 6-*O*-Methylpretazettine (**41**) +1 +1

Criwelline (**43**) +6 Macronine (**44**) +1

Anhydrolycorine (**50**) +1 +1 11,12-Dehydroanhydrolycorine (**51**) +1 +1 Caranine (**52**) +5

Incartine (**55**) +1 +1 2-*O*-(3'-Hydroxybutanoyl)lycorine (**56**) +1,7 +1 2?-*O*-(3'-Hydroxybutanoyl)lycorine isomer (**57**) +5 2-*O*-(3'-Acetoxybutanoyl)lycorine (**58**) +1,9 Ungeremine (**59**) *+*<sup>9</sup> 8-*O*-Demethylvasconine (**60**) *+*<sup>7</sup> Nartazine (**61**) +6

Homolycorine (**64**) +1

 Masonine (**66**) +5 +6 2-Methoxy-8-*O*-demethylhomolycorine (**67**) +1,2 Hippeastrine (**68**) +1 +8 Galwesine (**69**) +1,2 8-*O*-Demethylgalwesine (**70**) +2 8-*O*-Demethyl-10b-hydroxygalwesine (**71**) +2 10b-Hydroxygalwesine (**72**) +2 Galasine (**73**) +2 2α-Hydroxyhomolycorine (**74**) +1

Galanthindole (**75**) +10

Graciline (**78**) +16 11-Acetoxygraciline (**79**) +16

Galanthusine (**77**) *+*<sup>22</sup>

Epimacronine (**45)** +7 +11 +15 3-*O*-Demethyl-3-epimacronine (**46**) +13 3-*O*-Demethylmacronine (**47**) +13 3-*O*-(3´-Hydroxybutanoyl)tazettinol (**48**) +12 Isotazettinol (**49**) *+*<sup>13</sup>

Galanthine (**53**) +1,2 +1 +21 +22

*G. nivalis* 

*G. plicatus* 

*G. gracilis* 

*G. woronowii* 

*G. caucasicus* 

*G. ikariae* 

*G. krasnovii* 

*G. reginae-olgae* 

*G. trojanus* 

*G. rizehensis* 

**Compound** 

*VI. Tazettine type* 

*VII. Lycorine type* 

*VIII. Homolycorine type* 

*IX. Graciline type*


1) Berkov *et al.,* (2011); 2) Latvala *et al.,* (1995); 3) Bubeva-Ivanova and Pavlova (1965); 4) Sener *et al.,* (1998); 5) Berkov *et al.,* (2008); 6) Wildman, (1968); 7) Berkov *et al.,* (2009c); 8) Kalashnikov (1970); 9) Berkov *et al.,* (2007a); 10) Ünver *et al.,* (2003); 11) Akneri and Günes (1998); 12) Ünver *et al.,* (2001); 13) Ünver *et al.,* (1999a); 14) Kaya *et al.,* (2004*a*); 15) Noyan (1999); 16) Noyan *et al.,* (1998); 17) Ünver *et al.,* (1999b); 18) Ünver and Kaya*,* (2005); 19) Proskurina *et al.,* (1955); 20) Yakovleva (1963); 21) Proskurina Ordzhonikidze (1953); 22) Tsakadze *et al.,* (1979); 23) Asoeva *et al.,* (1968); 24) Conforti *et al.,* (2010); 25) Kaya *et al.,* (2011); 26) Kaya *et al.,* (2004*b*); 27) Bozkurt *et al.,* (2010).

Table 1. Alkaloids reported in the genus *Galanthus*

Another two phytochemically interesting species from which a number of new alkaloids have been isolated are *G. gracilis* and *G. plicatus*. Phytochemical studies on *G. gracilis* resulted in the isolation of three novel monomeric alkaloids (**78**, **80**, **81**) and a dimeric compound (**82**) bearing a 10b,4a-ethanoiminodibenzo[b,d]pyrane skeleton, which represents a new subgroup of Amaryllidaceae alkaloids named gracilines (Fig. 1, Noyan *et al.,* 1998; Ünver *et al.,* 2001). An unusual pentacyclic dinitrogenous alkaloid, gracilamine (**83**), was also isolated from this species (Ünver and Kaya, 2005). Another new graciline-type alkaloid (**79**, Noyan *et al.,* 1998) has been isolated from *G. plicatus*, together with compounds **84**-**86**  (Ünver et al., 1999*a*, 2001), representing a new subgroup of the Amaryllidaceae alkaloids where the oxygen atom at position 5 of a tazettine molecule is replaced by a nitrogen atom, conjugated with a 4-hydroxyphenethyl moiety. This new subgroup, named after the lead compound plicamine (**84**), was found later in another amaryllidaceous plant, *Cyrtanthus obliquus* (Brine *et al.,* 2002). Apart from plicamines, four new tazettine-type alkaloids (**46**-**49**) and a compound with a nonfused indole ring (**75**) have also been isolated in *G. plicatus* (Ünver *et al.,* 1999*b*, 2003). In total, 17 and 12 alkaloids have been reported for *G. plicatus* and *G. gracilis,* respectively (Table 1)*.* 

The other *Galanthus* species are relatively less studied. Four known alkaloids (**12**, **42**, **53**, and **54**), including galanthamine, have been reported for *G. woronovii* (Proskurina *et al.,* 1955; Proskurina and Ordzhonikidze, 1953; Yakovleva, 1963). A new compound, galanthusine (**78**),

The Genus *Galanthus*: A Source of Bioactive Compounds 245

R1

R4O <sup>H</sup>

**52** R1=OH, R2=H, R3+R4=CH2 **53** R1=OH, R2=OMe, R3=R4=Me **54** R1=R2=OH, R3+R4=CH2

H

**56** R1=OH, R2=OCOCH2CHOHMe, R3+R4=CH2 **58** R1=OH, R2=OCOCH2CHOAcMe, R3+R4=CH2

R2

N

N

O

R2

O

 R1=Me, R2=H **70** R1=R2=H R1=H, R2=OH R1=Me, R2=OH

H

O

H

**59**

**61** R1=R2=OAc, R3+R4=CH2 **63** R1=R2=OH, R3+R4=CH2

R1

MeN

**75 77**

H

H

H

OMe

O

O

R2

N

O

N

N

**50**

N

**51**

N

+

O

O

**73**

H

**62**

**60**

OH

AcO

MeN

H

H

H

OMe

O

O

OH

OH

MeO

O

O

O

O

HO

MeO

MeO

MeO

MeO

MeN

OH

OMe

R3O

R4O

O

O

R3O

MeO

R1O

OH

MeN

O

O

N

H

O

H

O

 R1=H, R2=R3=Me, R4=H R1=R2=H, R3=Me, R4=H R1=H, R2+R3=CH2,R4=H R1=OMe, R2=H, R3=Me, R4=H R1=OH, R2+R3=CH2, R4=H R1=OH, R2=R3=Me, R4=H R1=OH, R2+R3=CH2, R4=OMe

H

R4

MeN

H

**55**

HO

H

OMe

**48** R1=OCOCH2CHOHMe, R2=H, R3=OH, R4=R5=H

O

R1 R2

R1=OMe, R2=H, R3=HR4=R5=H R1=OMe, R2=H, R3=H, R4=H, R5=OMe R1=OMe, R2=H, R3=OH, R4=R5 R1=H, R2=OMe, R3=OH, R4=R5=H R1=H, R2=OMe, R3=H, R4+R5=O R1=OMe, R2=H, R3=H, R4+R5=O R1=OH, R2=H, R3=H, R4+R5=O R1=H, R2=OH, R3=H, R4+R5=O

R5 R4

**49** R1=H, R2=OH, R3=OH, R4=R5=H

R3

H

NMe

O

R1

MeO

O

O

MeO

R3O

R2O

HO

**5**

O

R4O

HO

MeO

N R2

R1

NMe

NR3

R2

NH

<sup>O</sup> <sup>O</sup>

R1

O

 R1=OH, R2=H, R3=R4=Me R1=H, R2=OH, R3=R4=Me R1+R2=O, R3=R4=Me R1=OH, R2=R3=H, R4=Me R1=OH, R2=H, R3=Me, R4=H R1=OH, R2=H, R3=CHO, R4=Me R1=OCOCH2CHOHMe, R2=H, R3=Me, R4=H R1=OCOCH2CHOHMe, R2=H, R3=R4=Me

O OH

O

**9**

H

**25**

N

R1

<sup>R</sup> R2 <sup>3</sup>

N

**37** R1=H, R2=OCOCH2CH(Me)OCOCH2CHOHMe, R3=OCOCH2CHOHMe, R4+R5=CH2

H

**36** R1=H, R2=OCOCH=CHMe, R3=OCOCH2CHOHMe, R4+R5=CH2

**38** R1=H, R2=OCOCH2CH(Me)OCOCH2CHOHMe, R3=OH, R4+R5=CH2 **39** R1=H, R2=OH, R3=OCOCH2CH(Me)OCOCH2CHOHMe, R4+R5=CH2

OH

N

**1** R1=R2=H **2** R1=H, R2=Me **3** R1=R2=Me **4** R1=H, R2= feruloyl

R1

NMe

R2

OR

O

O

R5O

R4O

 R1=OH, R2=R3=H, R4+R5=CH2(vittatine) R1=H, R2=R3=OH, R4+R5=CH2 R1=OH, R2=H, R3=OH, R4+R5=CH2 R1=OH, R2=R3=H, R4=R5=Me R1=OH, R2=R3=R4=H, R5=Me R1=OMe, R2=H, R3=OH, R4=H, R5=Me R1=OMe, R2=H, R3=OH, R4+R5=CH2 R1=H, R2=OH, R3=OCOCH2CHOHMe, R4+R5=CH2 R1=H, R2=R3=OCOCH2CHOHMe, R4+R5=CH2

N

H

**23** R=Me **24** R=H (crinine)

**16** R1=OH, R2=H **17** R1=H, R2=OH

**8**

MeO

OH

O

O

O

O

MeO

O

O

O

O

R1NR2

R1

OH

<sup>H</sup> <sup>N</sup>

**26**

**22**

NMe

OH

**6** R1=H, R2=Me **7** R1=CHO, R2=Me

<sup>O</sup> NH

R2

O

**10** R1=OH, R2=H **11** R1=R2=OH

**75 77**

The Genus *Galanthus*: A Source of Bioactive Compounds 247

ethano bridges, including vittatine (Kaya *et al.,* 2004*b*), which is the optical isomer of crinine, 11-hydroxyvittatine (Latvala *et al.,* 1995; Kaya *et al.,* 2004*b*); Ünver *et al.,* 2003) and hamayne (Berkov *et al.,* 2007*a*; 2009c). On the other hand, elwesine (**26**, 2,3-dihydrocrinine) and buphanisine (**23**) display a *β*-configuration of the 5,10b-ethano bridge (Wildman, 1968, Capo and Saa, 1989). Recently initiated phytochemical studies on *G. rizehensis* (Bozkurt *et al.,* 2010) have identified two narciclasine-type compounds, arolycoricidine (**10**) and narciprimine (**11**). An interesting example of biochemical convergence is the presence of bulbocapnine (**87**), capnoidine (**88**), stilopine (**89**) and protopine (**90**) in *G. trojanus* (studied as *G. nivalis* subsp. *silicicus* (Baker) Gottlieb-Tannenhain). Two new alkaloids, the *N*-oxides of 9-*O*methyldihydrosternbergine (**62**) and 11-hydroxyvittatine (**29**), were also isolated, along with several known alkaloids **2, 5**, **10, 24, 28, 29, 31-33, 54, 62** and **63** (Kaya *et al.,* 2004*b*; Ünver 2007). Compounds **84-90** are benzyltetrahydroisoquinoline-, aporphine- and phthalide-type isoquinolines, found in dicotyledonous plants of the Fumariaceae and Papaveraceae families

**5. Biological and pharmacological activities of the alkaloid found in** 

Alkaloids are important for the well-being of the producing organism. One of their main functions is to provide a chemical defence against herbivores, predators or microorganisms (Wink, 2008). The biological roles of the numerous alkaloids found in the genus *Galanthus* remain largely unknown and only a few have been studied for their pharmacological

The most studied *Galanthus* alkaloid, galanthamine (**12**), is a long-acting, selective, reversible and competitive inhibitor of acetylcholinesterase (AChE) and an allosteric modulator of the neuronal nicotinic receptor for acetylcholine. AChE is responsible for the degradation of acetylcholine at the neuromuscular junction, in peripheral and central cholinergic synapses. Galanthamine has the ability to cross the blood-brain barrier and to act within the central nervous system (Bastida *et al.,* 2006; Heinrich and Teoh, 2006). Owing to its AChE inhibitory activity, galanthamine is used and marketed under the name of Razadine®, formerly Reminyl®, in the USA, for the treatment of certain stages of Alzheimer's Disease (AD). According to data presented by the Alzheimer's Association in 2007, the prevalence of Alzheimer's disease will quadruple by 2050. Galanthamine hydrobromide has superior pharmacological profiles and higher tolerance as compared to the original AChE inhibitors,

Epigalanthamine (**13**), with a hydroxylgroup at *α*-position, and narwedine (**14**), with a keto group at C3, are also active AChE inhibitors, but about 130-times less than galanthamine (Thomsen *et al.,* 1998). The loss of the methyl group at the *N* atom, as in *N*demethylgalanthamine (**15**), decreases the activity 10-fold. On the other hand, sanguinine (**18**), which has a hydroxylgroup at C9 instead of a methoxyl group, is *ca*. 10 times more active than galanthamine. Hydrogenation of the C4-C4a, as in lycoramine (**16**), results in a complete loss of AChE inhibitory activity (López *et al.,* 2002). It is suggested that in plants AChE inhibitors act as pesticides. The synthetic pesticides such as phosphoorganic

(Kametani and Honda 1985; MacLean, 1985).

physostigmine or tacrine (Grutzendler and Morris, 2001).

compounds are non-reversible AChE inhibitors (Hougton *et al.,* 2006).

*Galanthus*

activities.

**Galanthamine-type** 

Fig. 2. Structures of the alkaloids found in the genus *Galanthus*

has been found in *G. caucasicus*, along with five known alkaloids (**12**, **42**, **53**, **54**, and **65;** Tsakadze *et al.,* 1979). Only galanthamine has been reported for *G. krasnovii* (Asoeva *et al.,* 1968). *G. ikariae* has furnished four known alkaloids (**12**, **27, 42**, and **65;** Sener *et al.,* 1998). A recent GC-MS report on *G. reginae-olgae* resulted in the identification of compounds **12**, **24**, **42**, and **66** (Conforti, *et al.,* 2010)**.** The presence of crinine (with the 5,10b-ethano bridge at the β-position) in this species, as well as in *G. elwesii*, as reported in our earlier GC-MS studies (Berkov *et al.,* 2004), is debatable because the absolute configuration of the 5,10b-ethano bridge cannot be established by GC-MS alone. Later phytochemical studies on *Galanthus* resulted in the isolation of crinane-3-ol derivatives with a *α*-configuration of their 5,10bethano bridges, including vittatine (Kaya *et al.,* 2004*b*), which is the optical isomer of crinine, 11-hydroxyvittatine (Latvala *et al.,* 1995; Kaya *et al.,* 2004*b*); Ünver *et al.,* 2003) and hamayne (Berkov *et al.,* 2007*a*; 2009c). On the other hand, elwesine (**26**, 2,3-dihydrocrinine) and buphanisine (**23**) display a *β*-configuration of the 5,10b-ethano bridge (Wildman, 1968, Capo and Saa, 1989). Recently initiated phytochemical studies on *G. rizehensis* (Bozkurt *et al.,* 2010) have identified two narciclasine-type compounds, arolycoricidine (**10**) and narciprimine (**11**). An interesting example of biochemical convergence is the presence of bulbocapnine (**87**), capnoidine (**88**), stilopine (**89**) and protopine (**90**) in *G. trojanus* (studied as *G. nivalis* subsp. *silicicus* (Baker) Gottlieb-Tannenhain). Two new alkaloids, the *N*-oxides of 9-*O*methyldihydrosternbergine (**62**) and 11-hydroxyvittatine (**29**), were also isolated, along with several known alkaloids **2, 5**, **10, 24, 28, 29, 31-33, 54, 62** and **63** (Kaya *et al.,* 2004*b*; Ünver 2007). Compounds **84-90** are benzyltetrahydroisoquinoline-, aporphine- and phthalide-type isoquinolines, found in dicotyledonous plants of the Fumariaceae and Papaveraceae families (Kametani and Honda 1985; MacLean, 1985).

#### **5. Biological and pharmacological activities of the alkaloid found in**  *Galanthus*

Alkaloids are important for the well-being of the producing organism. One of their main functions is to provide a chemical defence against herbivores, predators or microorganisms (Wink, 2008). The biological roles of the numerous alkaloids found in the genus *Galanthus* remain largely unknown and only a few have been studied for their pharmacological activities.

#### **Galanthamine-type**

246 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

N

O

**84**

OMe

NMe **<sup>78</sup>**R1=H **<sup>79</sup>**R1=OAc **80** R1=OH, R2=H **81** R1=H, R2=OH

O

NMe R2

R1

O

OH

Me

Me

H

O

O

O

O

O HO

MeO

OH

**86 87**

O

Fig. 2. Structures of the alkaloids found in the genus *Galanthus*

O

NMe

O

NMe

H

O

O

has been found in *G. caucasicus*, along with five known alkaloids (**12**, **42**, **53**, **54**, and **65;** Tsakadze *et al.,* 1979). Only galanthamine has been reported for *G. krasnovii* (Asoeva *et al.,* 1968). *G. ikariae* has furnished four known alkaloids (**12**, **27, 42**, and **65;** Sener *et al.,* 1998). A recent GC-MS report on *G. reginae-olgae* resulted in the identification of compounds **12**, **24**, **42**, and **66** (Conforti, *et al.,* 2010)**.** The presence of crinine (with the 5,10b-ethano bridge at the β-position) in this species, as well as in *G. elwesii*, as reported in our earlier GC-MS studies (Berkov *et al.,* 2004), is debatable because the absolute configuration of the 5,10b-ethano bridge cannot be established by GC-MS alone. Later phytochemical studies on *Galanthus* resulted in the isolation of crinane-3-ol derivatives with a *α*-configuration of their 5,10b-

O

O

O

OH

O

O

**90**

N

**85**

NMe

<sup>H</sup> <sup>O</sup> <sup>O</sup>

H

NMe

**88**

O

O

O

O

OMe

**82**

O

<sup>O</sup> <sup>O</sup>

H

O

O

O

NMe

NMe

H

H O

H

H

H

O

O

O

HN

O

NMe

R1

N

<sup>O</sup> <sup>N</sup>

H

**89**

OMe

**83**

H

N

CHO

Me

H

NMe

OEt <sup>O</sup>

O

O

O

O

O

The most studied *Galanthus* alkaloid, galanthamine (**12**), is a long-acting, selective, reversible and competitive inhibitor of acetylcholinesterase (AChE) and an allosteric modulator of the neuronal nicotinic receptor for acetylcholine. AChE is responsible for the degradation of acetylcholine at the neuromuscular junction, in peripheral and central cholinergic synapses. Galanthamine has the ability to cross the blood-brain barrier and to act within the central nervous system (Bastida *et al.,* 2006; Heinrich and Teoh, 2006). Owing to its AChE inhibitory activity, galanthamine is used and marketed under the name of Razadine®, formerly Reminyl®, in the USA, for the treatment of certain stages of Alzheimer's Disease (AD). According to data presented by the Alzheimer's Association in 2007, the prevalence of Alzheimer's disease will quadruple by 2050. Galanthamine hydrobromide has superior pharmacological profiles and higher tolerance as compared to the original AChE inhibitors, physostigmine or tacrine (Grutzendler and Morris, 2001).

Epigalanthamine (**13**), with a hydroxylgroup at *α*-position, and narwedine (**14**), with a keto group at C3, are also active AChE inhibitors, but about 130-times less than galanthamine (Thomsen *et al.,* 1998). The loss of the methyl group at the *N* atom, as in *N*demethylgalanthamine (**15**), decreases the activity 10-fold. On the other hand, sanguinine (**18**), which has a hydroxylgroup at C9 instead of a methoxyl group, is *ca*. 10 times more active than galanthamine. Hydrogenation of the C4-C4a, as in lycoramine (**16**), results in a complete loss of AChE inhibitory activity (López *et al.,* 2002). It is suggested that in plants AChE inhibitors act as pesticides. The synthetic pesticides such as phosphoorganic compounds are non-reversible AChE inhibitors (Hougton *et al.,* 2006).

The Genus *Galanthus*: A Source of Bioactive Compounds 249

as an anti-inflammatory agent (Citoglu *et al.,* 1998). Lycorine has also been shown to have insect antifeedant activity (Evidente *et al.,* 1986). As a potential chemotherapeutic drug, this compound has been studied as an antiproliferative agent against a number of cancer cell lines (Likhitwitayawuid *et al.,* 1993). The *in vitro* mode of action in a HL-60 leukemia cell line model is associated with suppressing tumor cell growth and reducing cell survival via cell cycle arrest and induction of apoptosis (Liu *et al.,* 2004). Further investigation showed that it is able to decrease tumor cell growth and increase survival rates with no observable adverse effects in treated animals (Liu *et al.,* 2007), thus being a good candidate for a therapeutic

Anhydrolycorine (**50**), in contrast to caranine (**52**), has shown a higher ability to inhibit ascorbic acid synthesis than lycorine (Evidente *et al.,* 1986). Analgesic and hypotensive effects have been reported for caranine and galanthine (**53**), the latter also being active against *Tripanosoma brucei rhodesiense* and *Plasmodium falciparum*. Some lycorine-type compounds such as caranine and ungeremine (**59**) have shown acetylcholinesterase inhibitory activity (Bastida *et al.,* 2006). Incartine was found to be cytotoxic and to weakly

Cytotoxic activity has been demonstrated for homolycorine (**64**), 8-*O*-demethylhomolycorine (**65**), and hippeastrine (**68**). Homolycorine has shown high antiretroviral activity, while hippeastrine is active against *Herpes simplex* type 1. Homolycorine and 8-*O*demethylhomolycorine have a hypotensive effect on normotensive rats. In addition, hippeastrine shows antifungal activity against *Candida albicans* and also possesses a weak

The bioactivity of the plicamine- and graciline-type alkaloids is largely unknown. Bulbocapline (**87**) and protopine (**90**) have been shown to act as inhibitors of acetylcholinesterase (Kim et al., 1999; Adsersen *et al.,* 2007) and dopamine biosynthesis (Shin *et al.,* 1998). Stylopine (**89**) suppresses the NO and PGE2 production in macrophages by

Although only some of the species of this phytochemically interesting genus have been studied, it has yielded a considerable number of new structures. Moreover, the high level of intraspecies diversity indicates that new compounds can be expected from already studied taxons. Only a few of the new alkaloids have been screened for their bio- and pharmacological activities, probably due to the small amounts isolated. Consequently, their

Adsersen, A.; Kjølbye, A.; Dall, O.; Jäger, A.K. (2007). Acetylcholinesterase and

Alzheimer's Association (2010). Alzheimer's disease facts and figures Alzheimer's &

butyrylcholinesterase inhibitory compounds from *Corydalis cava* Schweigg. & Kort.

agent against leukaemia (Liu *et al.,* 2009).

inhibit AChE (Berkov *et al.,* 2007).

insect antifeedant activity (Bastida *et al.,* 2006).

inhibiting iNOS and COX-2 expression (Jang et al., 2004).

*Journal of Ethnopharmacology* 113, 79-82

Dementia 6, 158–194

synthesis or *in silico* studies will facilitate further bioactivity assessment.

**Homolycorine-type** 

**6. Conclusions** 

**7. References** 

#### **Tyramine-type**

Compounds **1**-**4** can be attributed to the group of the phenolic amines that impact the hypothalamic-pituitary-adrenal axis (Vera-Avila *et al.,* 1996) due to their structural similarity to adrenaline (epinefrine). The consequent release of adrenocorticotropic hormone and cortisol results in sympathomimetic action with toxic effects in animals (Clement *et al.,*  1998). Hordenine (**3**) possesses diuretic, disinfectant and antihypotensive properties, and acts as a feeding repellent against grasshoppers (Dictionary of Natural Products).

#### **Narciclasine-type**

Trisphaeridine (**8**) has a high retroviral activity but a low therapeutic index. Ismine (**6**) shows a significant hypotensive effect on rats and cytotoxicity against Molt 4 lymphoid and LMTK fibroblastic cell lines (Bastida *et al.,* 2006). A recent study revealed that arolycoricidine (**10**) and narciprimine (**11**) were considerably effective in DNA topoisomerase reactions in a dosedependent manner. Topoisomerase-interfering ability of these alkaloids partially correlated with cytostatic assays, using HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells (Bozkurt *et al.,* 2010). Arolycoricidine showed inhibitory activity against African trypanosomes, (*Trypanosoma brucei rhodesiense)* at micromolar levels (Kaya *et al.,* 2011).

#### **Haemanthamine type**

Haemanthamine **(33)** has been shown to be a potent inducer of apoptosis in tumour cells at micromolar concentrations (McNulty *et al.,* 2007). This compound also possesses antimalarial activity against strains of chloroquine-sensitive *Plasmodium falciparum,* hypotensive effects and antiretroviral activity (Bastida *et al.,* 2006; Kaya *et al.,* 2011). Vittatine (**24**) and maritidine (**30)**  have shown cytotoxic activity against HT29 colon adenocarcinoma, lung carcinoma and RXF393 renal cell carcinoma (Bastida *et al.,* 2006; Silva *et al.,* 2008). Antibacterial activity against Gram-positive *Staphylococcus aureus* and Gram-negative *E. coli* have been reported for vittatine (**24**) and 11-hydroxyvittatine (**28**) (Kornienko and Evidente, 2008). Data about the bioactivity of recently isolated compounds **34**-**39** is still lacking.

#### **Tazettine-type**

Moderate cytotoxic activity has been reported for tazettine (**42**), and epimacronine (**45**) (Weniger *et al.,* 1995). Tazettine, however, is an isolation artefact of chemically labile pretazettine, which is indeed present in plants. This compound has shown remarkable cytotoxicity against a number of tumor cell lines, being therapeutically effective against advanced Rauscher leucemia, Ehrlich ascites carcinoma, spontaneous AKR lymphocytic leukaemia, and Lewis lung carcinoma (Bastida *et al.,* 2006).

#### **Lycorine-type**

Lycorine (**54**), one of the most frequently occurring alkaloids in Amaryllidaceae plants, possesses a vast array of biological properties. It has been reported as a potent inhibitor of ascorbic acid synthesis, cell growth and division and organogenesis in higher plants, algae, and yeasts, inhibiting the cell cycle during the interphase (Bastida *et al.,* 2006). Additionally, lycorine exhibits antiviral (against poliovirus, vaccine smallpox virus and SARS-associated coronavirus), antifungal (*Saccharomyces cerevisiae, Candida albicans*), and anti-protozoan (*Trypanosoma brucei*) activities (McNulty *et al.,* 2009), and is more potent than indomethacin as an anti-inflammatory agent (Citoglu *et al.,* 1998). Lycorine has also been shown to have insect antifeedant activity (Evidente *et al.,* 1986). As a potential chemotherapeutic drug, this compound has been studied as an antiproliferative agent against a number of cancer cell lines (Likhitwitayawuid *et al.,* 1993). The *in vitro* mode of action in a HL-60 leukemia cell line model is associated with suppressing tumor cell growth and reducing cell survival via cell cycle arrest and induction of apoptosis (Liu *et al.,* 2004). Further investigation showed that it is able to decrease tumor cell growth and increase survival rates with no observable adverse effects in treated animals (Liu *et al.,* 2007), thus being a good candidate for a therapeutic agent against leukaemia (Liu *et al.,* 2009).

Anhydrolycorine (**50**), in contrast to caranine (**52**), has shown a higher ability to inhibit ascorbic acid synthesis than lycorine (Evidente *et al.,* 1986). Analgesic and hypotensive effects have been reported for caranine and galanthine (**53**), the latter also being active against *Tripanosoma brucei rhodesiense* and *Plasmodium falciparum*. Some lycorine-type compounds such as caranine and ungeremine (**59**) have shown acetylcholinesterase inhibitory activity (Bastida *et al.,* 2006). Incartine was found to be cytotoxic and to weakly inhibit AChE (Berkov *et al.,* 2007).

#### **Homolycorine-type**

248 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Compounds **1**-**4** can be attributed to the group of the phenolic amines that impact the hypothalamic-pituitary-adrenal axis (Vera-Avila *et al.,* 1996) due to their structural similarity to adrenaline (epinefrine). The consequent release of adrenocorticotropic hormone and cortisol results in sympathomimetic action with toxic effects in animals (Clement *et al.,*  1998). Hordenine (**3**) possesses diuretic, disinfectant and antihypotensive properties, and

Trisphaeridine (**8**) has a high retroviral activity but a low therapeutic index. Ismine (**6**) shows a significant hypotensive effect on rats and cytotoxicity against Molt 4 lymphoid and LMTK fibroblastic cell lines (Bastida *et al.,* 2006). A recent study revealed that arolycoricidine (**10**) and narciprimine (**11**) were considerably effective in DNA topoisomerase reactions in a dosedependent manner. Topoisomerase-interfering ability of these alkaloids partially correlated with cytostatic assays, using HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells (Bozkurt *et al.,* 2010). Arolycoricidine showed inhibitory activity against African trypanosomes, (*Trypanosoma brucei rhodesiense)* at

Haemanthamine **(33)** has been shown to be a potent inducer of apoptosis in tumour cells at micromolar concentrations (McNulty *et al.,* 2007). This compound also possesses antimalarial activity against strains of chloroquine-sensitive *Plasmodium falciparum,* hypotensive effects and antiretroviral activity (Bastida *et al.,* 2006; Kaya *et al.,* 2011). Vittatine (**24**) and maritidine (**30)**  have shown cytotoxic activity against HT29 colon adenocarcinoma, lung carcinoma and RXF393 renal cell carcinoma (Bastida *et al.,* 2006; Silva *et al.,* 2008). Antibacterial activity against Gram-positive *Staphylococcus aureus* and Gram-negative *E. coli* have been reported for vittatine (**24**) and 11-hydroxyvittatine (**28**) (Kornienko and Evidente, 2008). Data about the bioactivity of

Moderate cytotoxic activity has been reported for tazettine (**42**), and epimacronine (**45**) (Weniger *et al.,* 1995). Tazettine, however, is an isolation artefact of chemically labile pretazettine, which is indeed present in plants. This compound has shown remarkable cytotoxicity against a number of tumor cell lines, being therapeutically effective against advanced Rauscher leucemia, Ehrlich ascites carcinoma, spontaneous AKR lymphocytic

Lycorine (**54**), one of the most frequently occurring alkaloids in Amaryllidaceae plants, possesses a vast array of biological properties. It has been reported as a potent inhibitor of ascorbic acid synthesis, cell growth and division and organogenesis in higher plants, algae, and yeasts, inhibiting the cell cycle during the interphase (Bastida *et al.,* 2006). Additionally, lycorine exhibits antiviral (against poliovirus, vaccine smallpox virus and SARS-associated coronavirus), antifungal (*Saccharomyces cerevisiae, Candida albicans*), and anti-protozoan (*Trypanosoma brucei*) activities (McNulty *et al.,* 2009), and is more potent than indomethacin

acts as a feeding repellent against grasshoppers (Dictionary of Natural Products).

**Tyramine-type** 

**Narciclasine-type** 

micromolar levels (Kaya *et al.,* 2011).

recently isolated compounds **34**-**39** is still lacking.

leukaemia, and Lewis lung carcinoma (Bastida *et al.,* 2006).

**Haemanthamine type** 

**Tazettine-type** 

**Lycorine-type** 

Cytotoxic activity has been demonstrated for homolycorine (**64**), 8-*O*-demethylhomolycorine (**65**), and hippeastrine (**68**). Homolycorine has shown high antiretroviral activity, while hippeastrine is active against *Herpes simplex* type 1. Homolycorine and 8-*O*demethylhomolycorine have a hypotensive effect on normotensive rats. In addition, hippeastrine shows antifungal activity against *Candida albicans* and also possesses a weak insect antifeedant activity (Bastida *et al.,* 2006).

The bioactivity of the plicamine- and graciline-type alkaloids is largely unknown. Bulbocapline (**87**) and protopine (**90**) have been shown to act as inhibitors of acetylcholinesterase (Kim et al., 1999; Adsersen *et al.,* 2007) and dopamine biosynthesis (Shin *et al.,* 1998). Stylopine (**89**) suppresses the NO and PGE2 production in macrophages by inhibiting iNOS and COX-2 expression (Jang et al., 2004).

#### **6. Conclusions**

Although only some of the species of this phytochemically interesting genus have been studied, it has yielded a considerable number of new structures. Moreover, the high level of intraspecies diversity indicates that new compounds can be expected from already studied taxons. Only a few of the new alkaloids have been screened for their bio- and pharmacological activities, probably due to the small amounts isolated. Consequently, their synthesis or *in silico* studies will facilitate further bioactivity assessment.

#### **7. References**


The Genus *Galanthus*: A Source of Bioactive Compounds 251

Clement, B.; Goff, C.; Forbes, D. (1998). Toxic amines and alkaloids from *Acacia rigidula*.

Conforti, F.; Loizzo, M.; Marrelli, M.; Menichini, F.; Statti, G.; Uzunov, D.; Menichini, F.

Davis, A.; Barnett, J. (1997). The leaf anatomy of the genus *Galanthus* L. (Amaryllidaceae J.

Davis, A.P. (1999). The genus *Galanthus*. In: Mathew, B. (Ed.) A Botanical Magazine

Davis, A.P.; Özhatay, N. (2001). *Galanthus trojanus*: a new species of *Galanthus*

Evidente, A.; Arrigoni, O.; Luso, R.; Calabrese, G.; Randazzo, G. (1986). Further experiments

Grutzendler J.; Morris J.C. (2001). Cholinesterase Inhibitors for Alzheimer's Disease, *Drugs*

Heinrich, M.; Teoh, H.L. (2004). Galanthamine from snowdrop-the development of a

Houghton, P.; Ren, Y.; Howes, M.-J. 2006. Acetylcholinesterase inhibitors from plants and

Jordanov, D. (1964). Genus *Galanthus* L. In: Jordanov, D. (Ed.), Flora of the People's Republic

Kaya, G.; Fillik, A.; Hisil, Y.; Ünver, N. (2004a). High pressure liquid chromatographic

Kaya, I.; Ünver, N.; Gözler, B.; Bastida, J. (2004b). (-)-Capnoidine and (+)-bulbocapnine from

Kametani, T.; Honda, T. (1985). Aporphine alkaloids. In: *The alkaloids - chemistry and pharmacology,* Brossi, A.R. (Ed.), Vol. 24, Academic Press Inc., Orlando, pp 153–251 Kim, S.R.; Hwang, S.Y.; Jang, Y.P.; Park, M.J.; Markelonis, G.J.; Oh, T.H.; Kim, Y.C. (1999).

Kornienko, A.; Evidente, A. (2008). Chemistry, biology and medicinal potential of

narciclasine and its congeners. *Chemical Reviews* 108, 1982–2014

Kaya, G.; Sarkaya B.; Onur, M.; Unver, N.; Viladomat F.; Codina, C.; Bastida, J.; Lauinger,

analysis of lycorine in four *Galanthus* species grown in Turkey. *Turkish Journal of* 

an Amaryllidaceae species, *Galanthus nivalis* subsp. *cilicicus*. *Biochemical Systematics* 

Kaiser, M.; Tasdemir D. (2011). Antiprotozoal Alkaloids from *Galanthus trojanus.* 

Protopine from C*orydalis ternata* has anticholinesterase and antiamnesic activities.

, I.;

Kalashnikov, I. (1970). Alkaloids from *Galanthus nivalis*. *Khimija Prirodnix Coedinenii* 6, 380. Kamari, G. (1981). A biosystematic study of the genus *Galanthus* L. in Greece, part II

St.-Hil.). *Botanical Journal of the Linnean Society* 123, 333–352

Monograph, Timber Press Inc. Oregon, pp 15–17, 140–155

(2010). Quantitative determination of Amaryllidaceae alkaloids from *Galanthus reginae-olgae* subsp. *vernalis* and *in vitro* activities relevant for neurodegenerative

(Amaryllidaceae) from northwestern Turkey. *Botanical Journal of the Linnean Society*

on structure-activity relationships among lycorine alkaloids. *Phytochemistry* 25,

modern drug against Alzheimer's disease from local Caucasian knowledge. *Journal* 

*Phytochemistry* 38, 266-279

137, 409–412

2739–2743

61, 41-52

diseases. *Pharmaceutical Biology* 48, 2-9

Dictionary of Natural Products, v17.1, Taylor & Francis Group, http://dnp.chemnetbase.com (accessed on 16.05.2009).

*of Ethnopharmacology* 92, 147–162

fungi. *Natural Products Reports* 23, 181-199.

(Cytology). *Botanika Chronika* 1, 60–98

*Pharmaceutical Sciences.* 1, 105-114

*Phytochemistry Letters* 4, 301-305.

*and Ecology* 32, 1059-1062

*Planta Medica* 65, 218-21

of Bulgaria, Vol. 2 Izdatelstvo na BAN, Sofia, pp. 318–319


Akneri, G.; Gunes, H.S. (1998) *Galanthus plicatus* ssp. *byzantinus* as a new source for some

Asoeva, E.; Murabeva, D.; Molodozhnikov, M.; Rabinovich, I. (1968). *Galanthus krasnovii* – A

Bastida, J.; Lavilla, R.; Viladomat, F. (2006). Chemical and biological aspects of *Narcissus* 

Berkov, S; Sidjimova, B.; Popov, S.; Evstatieva, L. (2004). Intraspecies variability in alkaloid

Berkov, S.; Bastida, J.; Viladomat, F.; Codina, C. (2007a). Alkaloids from *Galanthus nivalis*.

Berkov, S.; Bastida, J.; Cilpa-Reyes, R.; Viladomat, F.; Codina, C. (2007b). Revized RMN data for incartine: a bioactive compound from *Galanthus elwesii*. *Molecules* 12, 1430-1435 Berkov, S.; Bastida, J.; Sidjimova.; B.; Viladomat, F.; Codina, C. (2008). Phytochemical

Berkov, S.; Pavlov, A.; Georgiev, V.; Bastida, J.; Burrus, M.; Ilieva, M.; Codina, C. (2009a).

Berkov, S.; Georgieva, L.; Kondakova, V.; Atanassov, A.; Viladomat, F.; Bastida, J.; Codina,

Berkov, S.; Cuadrado, M.; Osorio, E.; Viladomat, F.; Codina, C.; Bastida, J. (2009c). Three

Berkov, S.; Bastida, J.; Sidjimova, B.; Viladomat, F.; Codina, C. (2011). Alkaloid diversity in *Galanthus elwesii* and *Galanthus nivalis. Chemistry and Biodiversity* 8, 115-130 Boit, H. -G.; Ehmke, H. (1955). Alkaloide von *Sprekelia formosissima*, *Galanthus elwesii*,

Boit, H, -G.; Döpke, W. (1961). Alkaloide aus *Haemanthus, Zephrantes-, Galanthus*- und

Bozkurt, B.; Zencir,S.; Ünver, N.; Kaya, G.; Onur, M.; Zupko, I; Topcu, Z; (2010). Biological

Brine, N.; Campbell, W.; Bastida, J.; Herrera M.; Viladomat, F.; Codina, C.; Smith, P. (2002) A dinitrogenous alkaloid from *Cyrtanthus obliquus*. *Phytochemistry* 61, 443–447 Bubeva-Ivanova, L.; Pavlova, H. (1965). Varhu alkaloidite na *Galanthus nivalis* L. var. *gracilis* (Celak). VIII Saobshtenie. Amaryllidaceae alkaloidi. *Farmacia* 15, 103-105 Capo, M.; Saa, J.M. (1989). Alkaloids from *Leucojum aestivum* sub. *pulchellum*

Citoglu, G.; Tanker, M.; Gumusel, B. (1998). Antiinfl ammatory effects of lycorine and

aspects. *Biotechnology and Biotechnological Equipment.* 23, 1170-1176

alkaloids. In *The Alkaloids*, Vol. 63, Cordell, G.A. (Ed.), Elsevier Scientific,

differentiation of *Galanthus nivalis* and *Galanthus elwesii:* a case study. *Biochemical* 

*Leucojum aestivum in vitro* cultures: variation in the alkaloid patterns. *Natural* 

C. (2009b). Plant sources of galanthamine: phytochemical and biotechnological

new alkaloids from *Galanthus nivalis* and *Galanthus elwesii. Planta Medica* 75, 1351-

*Zephyranthes candida* and *Crinum powellii* (VIII. Mitteil. Über Amaryllidaceen

activity of arolycoricidine and narciprimine on tumor cell killing and topoisomerase enzyme activities. Abstract Book of International Postgraduate Student Meeting on Pharmaceutical Sciences, p. 90, Ismir, Turkey, June 24-27, 2010

(Amaryllidaceae). *Anales de química. Serie C: Química Orgánica y Bioquímica* 85, 119-

Amaryllidaceae alkaloids. *J Fac Pharm Gazi* 15, 99–106

Amsterdam, pp. 87–179

*Phytochemistry* 68*,* 1791-1798

*Systematics and. Ecolology* 36, 638-645

*Product Communications 4, 359-364*

Alkaloide), *Chemische Beichte* 88, 1590-1594

*Crinum*-Arten, *Die Naturwissenschaften* 48, 406-407

haemanthidine. *Phytotherapy Research 12,* 205-206

1355

121

source for obtaining galanthamine. *Farmacia (Moskow)* 17, 47-49

metabolism in *Galanthus elwesii*. *Phytochemistry* 65, 579-586


The Genus *Galanthus*: A Source of Bioactive Compounds 253

Sener, B.; Koyuncu, M.; Bingöl, F.; Muhtar, F. (1998). Production of bioactive compounds

Shin, J.S.; Kim, K.T.; Lee, M.K. (1998). Inhibitory effects of bulbocapnine on dopamine

Sidjimova, B.; Berkov, S.; Popov, S.; Evstatieva, L. (2003). Galanthamine distribution in

Silva, A.; de Andrade J.; Machado K.; Rocha, A.B.;, Apel, M.A.; Sobral, M.E.G.;

Thomsen, T.; Bickel, U.; Fischer, J.; Kewitz, H. (1998). Stereoselectivity of cholinesterase

Tsakadze, D.; Kadirov, K.; Kiparenko, T.; Abdusamatov, A. (1979). New alkaloid from

Ünver, N.; Noyan, S.; Gözler, T.; Önür, M.; Gözler, B.; Hesse, M. (1999a). Three new tazettine

Ünver, N.; Gözler, T.; Walch, N.; Gözler, B.; Hesse, M. (1999b). Two novel dinitrogenous

Ünver, N.; Noyan, S.; Gözler, B.; Gözler, T.; Werner, C.; Hesse, M. (2001). Four new

Ünver, N.; Kaya, G.; Werner, C.; Verpoorte, R.; Gozler,B. (2003). Galanthindole:a new indole alkaloid from *Galanthus plicatus ssp. byzantus*. *Planta Medica* 69, 869-871 Ünver, N.; Kaya, G. (2005). An unusual pentacyclic dinitrogenous alkaloid from *Galanthus* 

Ünver, N. (2007). New skeletons and new concepts in Amaryllidaceae alkaloids.

Valkova, A. (1961). Varhu dokazvaneto i opredeljaneto na alkaloidite na *Galanthus nivalis* L.

Vera-Avila, H.; Forbes, T.; Randel, R. (1996). Plant phenolic amines: Potential effects on

Viladomat, F.; Bastida, J.; Codina, C.; Nair, J.; Campbell, W. (1997). Alkaloids of the South

Weniger, B.; Italiano, L.; Beck, P.; Bastida, J.; Bergoñón, S.; Codina, C.; Lobstein, A. Anton, R. (1995). Cytotoxic Activity of Amaryllidaceae Alkaloids. *Planta Medica* 61, 77-79 Wildman, W.C. (1968). *The Alkaloids Chemistry and Pharmacology*, Vol. 11, Manske, R.H.F.;

Willis, J.C. (1988). Amaryllidaceae. In: Shaw, A.H.K. (Ed.), A Dictionary of the Flowering

Holmes, H.L. (Eds.), New York, Academic Press Inc., pp. 307-405.

Plants & Ferns, 8th edn. Cambridge University Press, Cambridge

sympathoadrenal medullary, hypothalamic-pituitary-adrenal, and hypothalamicpituitary-gonadal function in ruminants. *Domestic Animal Endocrinology 13, 2*85-296

African Amaryllidaceae. *Recent Research and Developments in Phytochemistry* 1, 131-

Henriques, A.T.; Zuanazzi, J.A.S. (2008). Screening for cytotoxic activity of extracts and isolated alkaloids from bulbs of *Hippeastrum vittatum*. *Phytomedicine* 15, 882–885

inhibition by galanthamine and tolerance in humans. *European Journal of Clinical* 

*Galanthus caucasicus*. *Izvestija Akademii Nauk Gruzinskoi SSR*, *Serija Chimicheskaja* 5,

type alkaloids from *Galanthus gracilis* and *Galanthus plicatus* subsp. *byzanthus*. *Planta* 

alkaloids from *Galanthus plicatus* subsp. *byzanthus* (Amaryllidacea). *Phytochemistry*

Amaryllidaceae alkaloids from *Galanthus gracilis* and *Galanthus plicatus* subsp.

from Turkish geophytes. *Pure and Applied Chemistry* 70, 2131

biosynthesis in PC12 cells. *Neuroscience Letters* 244, 161-164

Bulgarian *Galanthus* species. *Pharmazie* 58, 936-937

*Pharmacology* 39, 603-605

*Medica* 65, 347-350

*byzantinus*. *Heterocycles* 55, 641-652

*Phytochemical Reviews* 6, 125–135

*gracilis*. *Turkish Journal of Chemistry* 29, 547-533

var. *gracilis* i *Leucojum aestivum*, *Farmatsia* 11, 17–22

50, 1255-1261

171

191-192


Kozuharov, S. (1992). Field Guide to the Vascular Plants in Bulgaria. Naouka & Izkustvo,

Kreh, M.; Matusch, R.; Witte, L. (1995). Capillary gas chromatography–mass spectrometry of

Latvala, A.; Önür, M.; Gözler, T.; Linden, A.; Kivçak, B.; Hesse, M. (1995). Alkaloids of

Liu, J.; Hu, W.X.; He, L.F.; Li, Y.; Ye, M. (2004). Effects of lycorine on HL-60 cells via arresting cell cycle and inducing apoptosis, *FEBS Letters* 578, 245–250 Liu, J.; Li, Y.; Tang, L.J.; Zhang, G.P.; Hu, W.X. (2007). Treatment of lycorine on SCID mice model with human APL cells, *Biomedicine and. Pharmacother*apy 61, 229–234 Liu, X.; Jiang, J.; Jiao, X.; Wu Y.; Lin, J.; Cai, Y. (2009) Lycorine induces apoptosis and downregulation of Mcl-1 in human leukemia cells. *Cancer Letters* 274 16–24. Likhitwitayawuid, K.; Angerhofer, C.K.; Chai, H.; Pezzuto, J.M.; Cordell, GA.. (1993).

López, S.; Bastida, J.; Viladomat, F.; Codina, C. (2002). Acetylcholinesterase inhibitory

Jang, S.; Kim, B.; Lee, W.-Y.; An, S.; Choi, H.;... Jeon, B.; Chung, H-T.; Rho, J.-R.; Kim, Y.-J.;

Machocho, A.K.; Bastida, J.; Codina, C.; Viladomat, F.; Brun, R.; and Chhabra, S.C. (2004). Augustamine type alkaloids from *Crinum kirkii*, *Phytochemistry* 65, 3143-3149 McNulty, J.; Nair, J.; Codina, C.; Bastida, J.; Pandey, S.; Gerasimoff, J.; Griffin C*.* (2007).

McNulty, J.; Nair, J.; Bastida, J.; Pandey, S.; Griffin, C. (2009). Structure-activity studies on

MacLean D.B. (1985). Phtalideisoquinoline alkaloids and related compounds. In: *The* 

Meerow, A.V.; Snijman, D.A. (1998). Amaryllidaceae, In: The Families and Genera of

Noyan, S.; Rentsch, G.; Önür, M.; Gözler, T.; Gözler, B.; Hesse, M. (1998). The Gracilines: a novel subgroup of the Amaryllidaceae alkaloids. *Heterocycles* 48, 1777-1791 Noyan, S. (1999) Isolation and structural elucidation studies on the alkaloids of *G. gracilis*

Proskurina, N.; Ordzhonikidze, S. (1953). Alkaloids of *Galanthus woronovii*. Structure of

Proskurina, N.; Yakovleva, A.; Ordzhonikidze, S. (1955). Alkaloids of *Galanthus woronovii* III.

Structure of galanthamine. *Zhurnal Obshchei Khimii* 25, 1035-1039

galanthine. *Dokladi Akademii Nauk SSSR*. 90, 565-567

Cytotoxic and antimalarial alkaloids from the bulbs of *Crinum amabile*. *Journal of* 

activity of some Amaryllidaceae alkaloids and *Narcissus* extracts. *Life Sciences* 71,

Chai, K.-Y. (2004). Stylopine from *Chelidonium majus* inhibits LPS-induced inflammatory mediators in RAW 264.7 cells. *Archives of Pharmacal Research* 27, 923-

Selective apoptosis-inducing activity of *Crinum*-type Amaryllidaceae alkaloids.

the lycorine pharmacophore: A potent inducer of apoptosis in human leukemia

*alkaloids - chemistry and pharmacology,* Brossi, A.R. (Ed.), Vol. 24, Academic Press

Vascular Plants, Vol. 3, Kubitzki, K. (Ed.), Springer-Verlag, Berlin Heiderlberg, pp

Célak (Amaryllidaceae) growing wildly in Mount Nif, Kemalpasa, Izmir.

Amaryllidaceae alkaloids. *Phytochemistry* 38, 773–776

*Galanthus elwesii*. *Phytochemistry* 39, 1229-1249

*Natural Products* 56, 1331-1338

*Phytochemistry* 68, 1068-1074

Inc., Orlando, pp. 253–286

Dissertation, Ege University

cells. *Phytochemistry* 70, 913–919

2521–2529

929

83-110

Sofia


**12** 

*Egypt* 

**Silymarin, Natural Flavonolignans** 

Plants are a valuable source of pharmaceuticals, food ingredients, agrochemicals, insecticides, flavors and pigments. These compounds are called secondary metabolites. These are compounds with a restricted occurrence in taxonomic groups that are not essential for an organism to live but play a role in the interaction of the organism with its environment, ensuring the survival of the organism in its ecosystem (Verpoorte and Alfermann, 2000).

Milk thistle or St. Mary's thistle [*Silybum marianum* (L.) Gaertn. (Syn. *Cardus marianum*) Asteraceae] is an annual or biennial herb. The plant is native to the Mediterranean and North African regions (Boulos, 2000). It grows wild throughout Europe, North Africa, Americas and Australia (Hamid et al., 1983). The plant reaches to heights 10 feet. It has a stem of 20-150 cm high, erect, ridged and branched in the upper part. A distinguishing characteristic of milk thistle is the white patches found along the veins of the dark green leaves (Fig. 1). The broad leaves are deeply lobed, 50 cm long and 25 cm wide. The leaf margins are yellow and tipped with woody spines (3-12 mm long). The leaves are alternate and clasping to the stem. Each stem ends with solitary composite flower heads, about 2 inches in diameter, consisting of purple disc florets. The flower heads of milk thistle differ from other thistles by the presence of leathery bracts that are also tipped with stiff spines. The fruits (Fig. 2) are hard skimmed achenes, 6-8 mm long flat, smooth and shiny dark brown in color. The fruits yield 1.5-3% of an isomeric mixture of flavonolignans collectively known as silymarin (Morazzoni and Bombardelli, 1995). Silymarin accumulates mainly in

The principal components of silymarin are silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B and silydianin (Fig. 3). The first six compounds exist as equimolar mixtures as trans diastereoisomers. These diastereomers have very similar 1H and 13C NMR spectra and have no characteristic signals for facile identification of the individual isomers (Lee and Liu, 2003). A number of other chemically related compounds have been found in the fruits including dehydrosilybin, desoxysilychristin, desoxysilydianin, silandrin, silybinome, silyhermin and neosilymermin. The common feature of these

the external cover of the fruits of *S*. *marianum* (Madrid and Corchete, 2010).

**2. Chemistry of flavonolignans** 

**1. Introduction** 

 **from Milk Thistle** 

*Faculty of Pharmacy, University of Beni-Sueif,* 

Sameh AbouZid


## **Silymarin, Natural Flavonolignans from Milk Thistle**

Sameh AbouZid *Faculty of Pharmacy, University of Beni-Sueif, Egypt* 

#### **1. Introduction**

254 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Wink, M. (2008). Ecological Roles of Alkaloids, In: *Modern Alkaloids*, Fattorusso, E.;

http://apps.kew.org/wcsp/qsearch.do;jsessionid=56A58C50E62834507137259ECD

Yakovlea, A. (1963). Alkaloids of *Galanthus woronovii*. VI Isolation of tazettine. *Zhurnal* 

Youssef, D.T. (2001). Alkaloids of the flowers of *Hippeastrum vittatum*. *Journal of Natural* 

Zhong, J. (2005). Amaryllidaceae and *Sceletium* alkaloids. *Natural Products Reports* 22, 111–

Zonneveld, B.; Grimshaw, J.; Davis, A. (2003). The systematic value of the nuclear DNA

content in *Galanthus*. Plant Systematics and Evolution 241, 89–102

World Cheklist of Selected Plant Families, Kew Garden, (accessed May, 2011)

pp. 3-52

D7B0E0

126

*Obshchei Khimii* 33, 1691-1693

*Products* 64, 839-41.

Taglialatella-Scafati, O., (Eds.),Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Plants are a valuable source of pharmaceuticals, food ingredients, agrochemicals, insecticides, flavors and pigments. These compounds are called secondary metabolites. These are compounds with a restricted occurrence in taxonomic groups that are not essential for an organism to live but play a role in the interaction of the organism with its environment, ensuring the survival of the organism in its ecosystem (Verpoorte and Alfermann, 2000).

Milk thistle or St. Mary's thistle [*Silybum marianum* (L.) Gaertn. (Syn. *Cardus marianum*) Asteraceae] is an annual or biennial herb. The plant is native to the Mediterranean and North African regions (Boulos, 2000). It grows wild throughout Europe, North Africa, Americas and Australia (Hamid et al., 1983). The plant reaches to heights 10 feet. It has a stem of 20-150 cm high, erect, ridged and branched in the upper part. A distinguishing characteristic of milk thistle is the white patches found along the veins of the dark green leaves (Fig. 1). The broad leaves are deeply lobed, 50 cm long and 25 cm wide. The leaf margins are yellow and tipped with woody spines (3-12 mm long). The leaves are alternate and clasping to the stem. Each stem ends with solitary composite flower heads, about 2 inches in diameter, consisting of purple disc florets. The flower heads of milk thistle differ from other thistles by the presence of leathery bracts that are also tipped with stiff spines. The fruits (Fig. 2) are hard skimmed achenes, 6-8 mm long flat, smooth and shiny dark brown in color. The fruits yield 1.5-3% of an isomeric mixture of flavonolignans collectively known as silymarin (Morazzoni and Bombardelli, 1995). Silymarin accumulates mainly in the external cover of the fruits of *S*. *marianum* (Madrid and Corchete, 2010).

#### **2. Chemistry of flavonolignans**

The principal components of silymarin are silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B and silydianin (Fig. 3). The first six compounds exist as equimolar mixtures as trans diastereoisomers. These diastereomers have very similar 1H and 13C NMR spectra and have no characteristic signals for facile identification of the individual isomers (Lee and Liu, 2003). A number of other chemically related compounds have been found in the fruits including dehydrosilybin, desoxysilychristin, desoxysilydianin, silandrin, silybinome, silyhermin and neosilymermin. The common feature of these

Silymarin, Natural Flavonolignans from Milk Thistle 257

silybin has been identified in 1975 using a degradative method (Lee and Liu, 2003). The first trials to synthesize silybin suffered from the problem of giving a product which is a mixture of regioisomers, silybin and isosilybin (57:43). Regioselective synthesis of diastereomeric silybin in 63% overall yield was achieved by synthesizing a key intermediate which was coupled with 2,4,6-trimethoxyacetophenone to form a chalcone intermediate. Epoxidation,

deprotection and acidic cyclization were followed (Tanaka et al., 1985).

**O CH2OH**

**OCH3 OH**

**OCH3 OH**

**O**

**O**

**O**

**OH**

**OH**

**Silybin B**

**O**

**O**

**O**

**OH**

**OH**

**HO**

**HO**

**O OH**

**O**

**OCH3**

**OH**

**HO**

**OH**

**Isosilybin B**

**OH**

**OH**

**Silychristin B**

**O O**

**O**

**O**

**O CH2OH**

**CH2OH**

**CH2OH OCH3**

**OCH3 OH**

**OH**

**OCH3 OH**

**O**

**O**

**O**

**O**

**O**

**OH**

**OH**

**HO**

**HO**

**OH**

**Silybin A**

**O**

**O**

**OH**

**HO**

**O**

**O O**

**CH2OH**

**OH**

**Isosilybin A**

**OH**

**OH**

**Silychristin A**

Fig. 3. Chemical structures of silymarin components.

**OH**

**O**

**O**

**OH**

**HO**

**OH**

**Silydianin**

**CH2OH OCH3**

Fig. 1. Milk thistle with white patches along the veins of dark green leaves.

Fig. 2. *Silybum marianum* fruits.

compounds is a flavonolignan skeleton (C25H22O10, mol wt 482). Basically, flavonolignan nucleus consists of the dihydroflavanol taxifolin linked to coniferyl alcohol moiety through an oxeran ring. The oxeran ring is responsible for the biological activity of silymarin, and opening of this ring results in loss of activity. Only silybins and isosilybins contain the 1,4 dioxane ring system in their structure. Silybin and isosilybin have the same trans conformation of C-2, C-3 and C-7', C-8'. Silybin is considered the major and most active component in silymarin (Ligeret et al., 2008; Kim et al., 2009). The chemical structure of

Fig. 1. Milk thistle with white patches along the veins of dark green leaves.

compounds is a flavonolignan skeleton (C25H22O10, mol wt 482). Basically, flavonolignan nucleus consists of the dihydroflavanol taxifolin linked to coniferyl alcohol moiety through an oxeran ring. The oxeran ring is responsible for the biological activity of silymarin, and opening of this ring results in loss of activity. Only silybins and isosilybins contain the 1,4 dioxane ring system in their structure. Silybin and isosilybin have the same trans conformation of C-2, C-3 and C-7', C-8'. Silybin is considered the major and most active component in silymarin (Ligeret et al., 2008; Kim et al., 2009). The chemical structure of

Fig. 2. *Silybum marianum* fruits.

silybin has been identified in 1975 using a degradative method (Lee and Liu, 2003). The first trials to synthesize silybin suffered from the problem of giving a product which is a mixture of regioisomers, silybin and isosilybin (57:43). Regioselective synthesis of diastereomeric silybin in 63% overall yield was achieved by synthesizing a key intermediate which was coupled with 2,4,6-trimethoxyacetophenone to form a chalcone intermediate. Epoxidation, deprotection and acidic cyclization were followed (Tanaka et al., 1985).

**Silybin A**

**Isosilybin A**

**Silybin B**

**Isosilybin B**

**Silydianin**

Fig. 3. Chemical structures of silymarin components.

Silymarin, Natural Flavonolignans from Milk Thistle 259

separation of all compounds allowed the purity control of each peak, plotting of UV spectra, useful for the peak identification and a more correct quantification. However, time consumption, the need for pre-purification step and availability of pure reference compounds are the main disadvantages of HPLC. Analysis of silymarin components by HPLC on RP-18 in our laboratory only showed separation of the two diastereomers of

Capillary zone electrophoresis has been proposed as a method for separation and determination of silymarin components (Kvasnička et al., 2003). Repeatability, accuracy, linearity and limit of detection were evaluated. The method was comparable to HPLC results. Shorter analysis time and better resolution of silydianin and silychristin from sample constituents were the main advantages of this method. High Performance Capillary Electrophoresis (HPCE) was used for determination of silymarin in the extract of *S*. *marianum* using borate buffer solution at pH 9. At this pH the flavonolignans having many phenolic groups in their structure were negatively charged (Quaglia et al., 1999). In these conditions isosilybin co-eluted together with silybin. Adding 12 mM dimethyl β–cyclodextrins solution to

silybin. However, the two peaks were not base-line resolved (Fig. 4).

the running buffer, the separation of silybin from isosilybin was obtained.

Fig. 5. Analysis of silymarin components by HPLC on RP18 (analysis was carried out in

Ultra-Performance Liquid Chromatography (UPLC) offer many advantages over traditional HPLC for separation and quantification of multicomponent analytes such as silymarin

**3.5 Ultra performance liquid chromatography analysis** 

**3.4 Capillary electrophoresis analysis** 

author laboratory).

#### **3. Analysis of flavonolignans**

Extract obtained from the fruits of *S*. *marianum* is available worldwide in the pharmaceutical market as antihepatotoxic drug under a variety of brand names. There are many products that contain silymarin either as a single component or in a mixture with other active constituents. The extract contains about 80% wt/wt of flavonolignans. Due to its poor water solubility and thus low bioavailability, silymarin is complexed with phosphatidylcholin, βcyclodextrin or even given as glycosides, which have better water solubility and higher activity. A method for extraction of silymarin from plants on an industrial level has been reported (Madaus et al. 1983). In this method large part of the fruit oil is removed by cold pressing, the compressed mass is broken up, the pressed residue is extracted with ethyl acetate and the ethyl acetate extract is evaporated and processed. There is a need to have a selective and accurate analytical method for qualitative and quantitative determination of silymarin flavonolignan components during standardization of the extract. This is expressed as silymarin percentage and it corresponds to the sum of silybins, isosilybins, and silychristins and silydianin concentrations. It is important that the analytical method characterizes and quantifies each component in silymarin.

#### **3.1 Thin layer chromatography analysis**

Flavonolignans were analyzed by Thin Layer Chromatography (TLC) (Wagner et al., 2009). Chloroform-acetone-formic acid (75:16.5:8.5) was used as a solvent system and detection was done using natural products-polyethylene glycol reagent. Silymarin is characterized in UV-365 nm by two intense green-blue fluorescent zones of silybin/isosilybin (Rf = 0.6), silychristin (Rf = 0.35) and an orange zone of taxifolin (Rf = 0.4).

#### **3.2 UV-visible spectrophotometry analysis**

UV-visible spectrophotometry was proposed for the quantitative determination of flavonolignans (Famacopea Ufficiale Italiana, 1985). This spectrophotometric method is time consuming, shows a non-satisfactory repeatability and measures total and not individual flavonoligans. A fast, simple and sensitive spectrophotometric method for determination of silymarin in pure form and in pharmaceutical formulations was reported. This method was based on oxidation with potassium permanganate at pH 7. The reaction was followed spectrophotometrically by measuring the decrease in the absorbance at 530 nm (Rahman et al., 2004).

#### **3.3 High performance liquid chromatography analysis**

High Performance Liquid Chromatography (HPLC) was proposed as a method for determination of silymarin (Quaglia et al., 1999). Two reversed stationary phases, RP-18 and RP-8, were compared for resolution of all considered flavonolignans. The RP-18 stationary phase showed good separation among silybin and isosilybin, while silydianin and silychristin were not baseline resolved. The increase in water concentration in the mobile phase allowed the separation of two distereomers of silybin. RP-8 stationary phase, a more polar phase, improved the resolution of peaks related to all flavonolignans but did not allow the resolution of the two silybin diastereomers. Among the advantages of this method are precision, sensitivity, ability to measure individual constituents in a mixture, the good

Extract obtained from the fruits of *S*. *marianum* is available worldwide in the pharmaceutical market as antihepatotoxic drug under a variety of brand names. There are many products that contain silymarin either as a single component or in a mixture with other active constituents. The extract contains about 80% wt/wt of flavonolignans. Due to its poor water solubility and thus low bioavailability, silymarin is complexed with phosphatidylcholin, βcyclodextrin or even given as glycosides, which have better water solubility and higher activity. A method for extraction of silymarin from plants on an industrial level has been reported (Madaus et al. 1983). In this method large part of the fruit oil is removed by cold pressing, the compressed mass is broken up, the pressed residue is extracted with ethyl acetate and the ethyl acetate extract is evaporated and processed. There is a need to have a selective and accurate analytical method for qualitative and quantitative determination of silymarin flavonolignan components during standardization of the extract. This is expressed as silymarin percentage and it corresponds to the sum of silybins, isosilybins, and silychristins and silydianin concentrations. It is important that the analytical method

Flavonolignans were analyzed by Thin Layer Chromatography (TLC) (Wagner et al., 2009). Chloroform-acetone-formic acid (75:16.5:8.5) was used as a solvent system and detection was done using natural products-polyethylene glycol reagent. Silymarin is characterized in UV-365 nm by two intense green-blue fluorescent zones of silybin/isosilybin (Rf = 0.6),

UV-visible spectrophotometry was proposed for the quantitative determination of flavonolignans (Famacopea Ufficiale Italiana, 1985). This spectrophotometric method is time consuming, shows a non-satisfactory repeatability and measures total and not individual flavonoligans. A fast, simple and sensitive spectrophotometric method for determination of silymarin in pure form and in pharmaceutical formulations was reported. This method was based on oxidation with potassium permanganate at pH 7. The reaction was followed spectrophotometrically by measuring the decrease in the absorbance at 530 nm (Rahman et

High Performance Liquid Chromatography (HPLC) was proposed as a method for determination of silymarin (Quaglia et al., 1999). Two reversed stationary phases, RP-18 and RP-8, were compared for resolution of all considered flavonolignans. The RP-18 stationary phase showed good separation among silybin and isosilybin, while silydianin and silychristin were not baseline resolved. The increase in water concentration in the mobile phase allowed the separation of two distereomers of silybin. RP-8 stationary phase, a more polar phase, improved the resolution of peaks related to all flavonolignans but did not allow the resolution of the two silybin diastereomers. Among the advantages of this method are precision, sensitivity, ability to measure individual constituents in a mixture, the good

**3. Analysis of flavonolignans** 

characterizes and quantifies each component in silymarin.

silychristin (Rf = 0.35) and an orange zone of taxifolin (Rf = 0.4).

**3.3 High performance liquid chromatography analysis** 

**3.1 Thin layer chromatography analysis** 

**3.2 UV-visible spectrophotometry analysis** 

al., 2004).

separation of all compounds allowed the purity control of each peak, plotting of UV spectra, useful for the peak identification and a more correct quantification. However, time consumption, the need for pre-purification step and availability of pure reference compounds are the main disadvantages of HPLC. Analysis of silymarin components by HPLC on RP-18 in our laboratory only showed separation of the two diastereomers of silybin. However, the two peaks were not base-line resolved (Fig. 4).

#### **3.4 Capillary electrophoresis analysis**

Capillary zone electrophoresis has been proposed as a method for separation and determination of silymarin components (Kvasnička et al., 2003). Repeatability, accuracy, linearity and limit of detection were evaluated. The method was comparable to HPLC results. Shorter analysis time and better resolution of silydianin and silychristin from sample constituents were the main advantages of this method. High Performance Capillary Electrophoresis (HPCE) was used for determination of silymarin in the extract of *S*. *marianum* using borate buffer solution at pH 9. At this pH the flavonolignans having many phenolic groups in their structure were negatively charged (Quaglia et al., 1999). In these conditions isosilybin co-eluted together with silybin. Adding 12 mM dimethyl β–cyclodextrins solution to the running buffer, the separation of silybin from isosilybin was obtained.

Fig. 5. Analysis of silymarin components by HPLC on RP18 (analysis was carried out in author laboratory).

#### **3.5 Ultra performance liquid chromatography analysis**

Ultra-Performance Liquid Chromatography (UPLC) offer many advantages over traditional HPLC for separation and quantification of multicomponent analytes such as silymarin

Silymarin, Natural Flavonolignans from Milk Thistle 261

**OH**


Radical coupling

**OH**

**O**

**O**

**O**

**OH**

**OH**

**O**

**O CH2OH**

**OCH3**

**OH**

**OCH3**

**O**

**..**

**HO**

Silymarin has been used for centuries to treat liver, spleen and gall bladder disorders (Shaker et al., 2010). It is known to possess hepatoprotective, antioxidant (Morazzoni and Bombardelli, 1995), anticancer (Zi et al., 1997), anti-inflammatory (De La Puerta, 1996) and anti-diabetic (Maghrani et al., 2004) properties. As a hepatoprotective agent, silymarin is used for oral treatment of toxic liver damage and for the therapy of chronic inflammatory

**OH**

**OCH3**

Coniferyl alcohol (phenylpropanoid)

**.**

**OH**

**O**

**OCH3**

**OH**

**OH**

**<sup>O</sup> OH**

**O .**

**OH**

**OH**

**OH**

Taxifolin (flavonoid)

**O**

**O**

**HO**

**OH**

**HO**

**O**

**O**

liver diseases (Flora et al., 1998).

**OH**

**HO**

**OH**

**5. Biological activity of flavonolignans** 

**O**

**O CH2OH**

**OH**

**O**

**O**

**OCH3**

**OH**

Fig. 5. Proposed biosynthetic pathway to silybin in *Silybum marianum*.

**OH**

**OH**

**O**

**HO**

components. Among these advantages are short analysis time, maintaining the resolution and increasing peak capacity and sensitivity. Complete separation of the seven major active flavonolignans of silymarin by UPLC RP18 column was reported (Wang et al., 2010). In this study, the use of electrospray ionization tandem mass spectrometry allowed to obtain detailed analysis of fragmentation and distinguish between the seven flavonolignans for online identification. Advantages and disadvantages of different methods for quantitative analysis of flavonolignan components in silymarin are summarized in table 1.


Table 1. Advantages and disadvantages of different methods for quantitative analysis of flavonolignan components in silymarin.

#### **4. Biosynthesis of flavonolignans in** *Silybum marianum*

Flavonolignans are formed by combination of flavonoid and lignan structures. This occurs by oxidative coupling processes between a flavonoid and a phenylpropanoid, usually coniferyl alcohol (Dewich, 2002). Oxidative coupling occurs between free radical generated from the flavanol taxifolin and the free radical generated from coniferyl alcohol. This would lead to an adduct formation. This adduct could cyclize by attachment of the phenol nucleophile on to the quinine methide generated from coniferyl alcohol (Figure 5). The product in this case would be silybin. The fact that silybin exists in *S*. *marianum* in a mixture of two diastereomers reveals that the radical coupling reaction is not stereospecific. This is also true for isosilybin and silychristin. The latter flavonolignan originate from a mesomer of the taxifolin-derived free radical. Silydianin has a more complex structure and is formed by intramolecular cyclization of the coupling product. This is followed by hemiketal formation.

components. Among these advantages are short analysis time, maintaining the resolution and increasing peak capacity and sensitivity. Complete separation of the seven major active flavonolignans of silymarin by UPLC RP18 column was reported (Wang et al., 2010). In this study, the use of electrospray ionization tandem mass spectrometry allowed to obtain detailed analysis of fragmentation and distinguish between the seven flavonolignans for online identification. Advantages and disadvantages of different methods for quantitative

Individual flavonolignans are

 Not all flavonolignans are separated from each other

 Needs pre-purification step Pure reference compounds are

Needs pre-purification step

 Needs pre-purification step Needs calibration curve

not quantified

Time consuming

needed

Expensive

analysis of flavonolignan components in silymarin are summarized in table 1.

**Method Advantages Disadvantages** 

Individual flavonolignans are

 Less solvent consumption Individual flavonolignans are quantified including diastereomers

 Less solvent consumption Increased resolution

Increased peak capacity and

Table 1. Advantages and disadvantages of different methods for quantitative analysis of

Flavonolignans are formed by combination of flavonoid and lignan structures. This occurs by oxidative coupling processes between a flavonoid and a phenylpropanoid, usually coniferyl alcohol (Dewich, 2002). Oxidative coupling occurs between free radical generated from the flavanol taxifolin and the free radical generated from coniferyl alcohol. This would lead to an adduct formation. This adduct could cyclize by attachment of the phenol nucleophile on to the quinine methide generated from coniferyl alcohol (Figure 5). The product in this case would be silybin. The fact that silybin exists in *S*. *marianum* in a mixture of two diastereomers reveals that the radical coupling reaction is not stereospecific. This is also true for isosilybin and silychristin. The latter flavonolignan originate from a mesomer of the taxifolin-derived free radical. Silydianin has a more complex structure and is formed by intramolecular cyclization of the coupling product.

sensitivity

**4. Biosynthesis of flavonolignans in** *Silybum marianum*

Spectrophotometric Fast and simple

HPCE Shorter analysis

UPLC Short analysis time

flavonolignan components in silymarin.

This is followed by hemiketal formation.

HPLC Precise and sensitive

Sensitive

quantified Peak identification Purity control

Fig. 5. Proposed biosynthetic pathway to silybin in *Silybum marianum*.

#### **5. Biological activity of flavonolignans**

**HO**

Silymarin has been used for centuries to treat liver, spleen and gall bladder disorders (Shaker et al., 2010). It is known to possess hepatoprotective, antioxidant (Morazzoni and Bombardelli, 1995), anticancer (Zi et al., 1997), anti-inflammatory (De La Puerta, 1996) and anti-diabetic (Maghrani et al., 2004) properties. As a hepatoprotective agent, silymarin is used for oral treatment of toxic liver damage and for the therapy of chronic inflammatory liver diseases (Flora et al., 1998).

Silymarin, Natural Flavonolignans from Milk Thistle 263

Activity of silymarin was examined against bronchial anaphylaxis and against postanaphylactic, propranolol- or platelet activating factor-induced hyperreactivity in guineapigs (Breschi et al., 2002). Silymarin pretreatment reduced the bronchospasm induced by antigen-challenge in sensitized animals. This protective effect was due to indirect mechanism that reduces airway responsiveness to histamine, and consequently the immediate anaphylactic response. Therefore, silymarin can be used as protective agent in

Several studies have reported the immunostimulatory actions of silymarin (Wilarusmee et al., 2002). The effect of treatment with silymarin was studied on glutathione level and proliferation of peripheral blood mononuclear cells of β–thalassemia major patients (Alidoost et al., 2006). In vitro treatment with 10 g/ml silymarin restored glutathione levels

Many patients cannot tolerate the side effects of pharmaceutical agents available for treatment of obsessive-compulsive disorder, do not respond properly to the treatment or the medications lose their effectiveness after a period of treatment. An 8-week pilot doubleblind randomized clinical trial on 35 adult patients was conducted to compare the efficacy of the extract of *S*. *marianum* with fluoxetine in the treatment of obsessive-compulsive disorder (Sayyah et al., 2010). The results showed that the extract of *S*. *marianum* has positive effects on obsession and compulsion starting from the fifth week. There were no any serious side

*S*. *marianum* fruits have been traditionally used by nursing mothers for stimulating milk production (Newall et al., 1996). It was demonstrated that milk thistle increases lactation (Carotenuto and Di Pierro, 2005). The mechanism that led to the increase in lactation has been studied by measuring the concentration of circulating prolactin in female rats treated with silymarin (Capasso et al., 2009). It was shown that silymarin is able to produce a significant increase in circulating prolactin levels after oral administration. The levels of prolactin remains elevated for up to 66 days after silymarin discontinuation. Fig. 6 shows a

An average daily dose of silymarin (420 mg/day for 41 months) was found to be non-toxic, relative to placebo, in clinical trials (Tamayo and Diamond, 2007). Drug-drug interaction and liver toxicity by interference with co-drugs by induction or inhibition of cytochrome-P450 is a major concern for the use of silymarin (Izzo and Ernst, 2009). Studies were performed to investigate the potential for hepatotoxicity, cytochrome-P450 isoenzymes induction and inhibition on dry extract from *S. marianum*, as contained in HEPAR-PASC® film-coated tablets (Doehmer et al., 2011). The results indicated that interference or

summary of the wide range of biological activities attributed to silymain.

and enhanced cellular proliferation. This was explained by its antioxidant activity.

**5.4 Effect on asthma** 

the management of asthmatic disorders.

**5.6 Treatment of obsessive-compulsive disorder** 

effects accompanying *S. marianum* extract administration.

**5.5 Immunostimulatory activity** 

**5.7 Hyperprolactinemic effect** 

**5.8 Toxicity of silymarin** 

#### **5.1 Hepatoprotective activity**

Silymarin is one of the most investigated plant extracts with known mechanisms of action for oral treatment of toxic liver damage (Hiroshi et al., 1984). Silymarin is used as a protective treatment in acute and chronic liver diseases (Flora *et al.*, 1998). Silymarin supports the liver cells through multifactor action including binding to cell membrane to suppress toxin penetration into the hepatic cells, increasing superoxide dismutase activity (Feher and Vereckei, 1991), increasing glutathione tissue level (Pietrangelo *et al*., 1995), inhibition of lipid peroxidation (Bosisio *et al*., 1992; Carini *et al*., 1992) and enhancing hepatocyte protein synthesis (Takahara et al., 1986). The hepatoprotective activity of silymarin can be explained based on antioxidant properties due to the phenolic nature of flavonolignans. It also acts through stimulating liver cells regeneration and cell membrane stabilization to prevent hepatotoxic agents from entering hepatocytes (Fraschini et al., 2002). Recently it has been shown that flavonolignans inhibit leucotriene production; this inhibition explains their anti-inflammatory and antifibrotic activity (Dehmlow et al., 1996).

#### **5.2 Anticancer activity of silymarin**

Silymarin is also beneficial for reducing the chances for developing certain cancers (Deep et al., 2007; Zhao et al., 1999). The molecular targets of silymarin for cancer prevention have been studied (Ramasamy and Agrawal, 2008). Silymarin interfere with the expressions of cell cycle regulators and proteins involved in apoptosis to modulate the imbalance between cell survival and apoptosis. Sy-Cordero et al., 2010, isolated four key flavonolignan diastereoisomers (silybin A, silybin B, isosilybin A and isosilybin B) from *S*. *marianum* in gram scale. These compounds and other two related analogues, present in extremely minute quantities, were evaluated for antiproliferative/cytotoxic activity against human prostate cancer cell lines. Isosilybin B showed the most potent activity (Deep et al., 2007; Deep et al., 2008a; Deep et al., 2008b). The isolation of six isomers afforded a preliminary analysis of structure-activity relationship toward prostate cancer prevention. The results suggested that an *ortho* relationship for the hydroxyl and methoxy substituents in silybin A, silybin B, isosilybin A and isosilybin B was more favorable than the *meta* relationship for the same substituents in the minor flavonolignans. Silymarin suppressed UVA-induced oxidative stress that can induce skin damage (Svobodová et al., 2007). Therefore, topical application of silymarin can be a useful strategy for protecting against skin cancer.

#### **5.3 Anti-inflammatory activity**

Silymarin seems to possess anti-inflammatory properties by acting through different mechanisms such as its antioxidant action, membrane-stabilizing effect and inhibition of the production or release of inflammatory mediators such as arachidonic acid metabolites (Breschi et al., 2002). Gastric anti-ulcer activity of silymarin has been reported (Alarcon et al., 1992). This action was attributed to the inhibition of enzymatic peroxidation in the lipoxygenase pathway and free radical scavenging activity (Bauman et al., 1980). Silymarin exhibited significant anti-inflammatory and antiarthritic activities in the papaya latex induced model of inflammation and mycobacterial adjuvant induced arthritis in rats (Gupta et al., 2000). This action is mediated through inhibition of 5-lipoxygenase.

#### **5.4 Effect on asthma**

262 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Silymarin is one of the most investigated plant extracts with known mechanisms of action for oral treatment of toxic liver damage (Hiroshi et al., 1984). Silymarin is used as a protective treatment in acute and chronic liver diseases (Flora *et al.*, 1998). Silymarin supports the liver cells through multifactor action including binding to cell membrane to suppress toxin penetration into the hepatic cells, increasing superoxide dismutase activity (Feher and Vereckei, 1991), increasing glutathione tissue level (Pietrangelo *et al*., 1995), inhibition of lipid peroxidation (Bosisio *et al*., 1992; Carini *et al*., 1992) and enhancing hepatocyte protein synthesis (Takahara et al., 1986). The hepatoprotective activity of silymarin can be explained based on antioxidant properties due to the phenolic nature of flavonolignans. It also acts through stimulating liver cells regeneration and cell membrane stabilization to prevent hepatotoxic agents from entering hepatocytes (Fraschini et al., 2002). Recently it has been shown that flavonolignans inhibit leucotriene production; this inhibition explains their anti-inflammatory and antifibrotic activity (Dehmlow et al.,

Silymarin is also beneficial for reducing the chances for developing certain cancers (Deep et al., 2007; Zhao et al., 1999). The molecular targets of silymarin for cancer prevention have been studied (Ramasamy and Agrawal, 2008). Silymarin interfere with the expressions of cell cycle regulators and proteins involved in apoptosis to modulate the imbalance between cell survival and apoptosis. Sy-Cordero et al., 2010, isolated four key flavonolignan diastereoisomers (silybin A, silybin B, isosilybin A and isosilybin B) from *S*. *marianum* in gram scale. These compounds and other two related analogues, present in extremely minute quantities, were evaluated for antiproliferative/cytotoxic activity against human prostate cancer cell lines. Isosilybin B showed the most potent activity (Deep et al., 2007; Deep et al., 2008a; Deep et al., 2008b). The isolation of six isomers afforded a preliminary analysis of structure-activity relationship toward prostate cancer prevention. The results suggested that an *ortho* relationship for the hydroxyl and methoxy substituents in silybin A, silybin B, isosilybin A and isosilybin B was more favorable than the *meta* relationship for the same substituents in the minor flavonolignans. Silymarin suppressed UVA-induced oxidative stress that can induce skin damage (Svobodová et al., 2007). Therefore, topical application of

Silymarin seems to possess anti-inflammatory properties by acting through different mechanisms such as its antioxidant action, membrane-stabilizing effect and inhibition of the production or release of inflammatory mediators such as arachidonic acid metabolites (Breschi et al., 2002). Gastric anti-ulcer activity of silymarin has been reported (Alarcon et al., 1992). This action was attributed to the inhibition of enzymatic peroxidation in the lipoxygenase pathway and free radical scavenging activity (Bauman et al., 1980). Silymarin exhibited significant anti-inflammatory and antiarthritic activities in the papaya latex induced model of inflammation and mycobacterial adjuvant induced arthritis in rats (Gupta

silymarin can be a useful strategy for protecting against skin cancer.

et al., 2000). This action is mediated through inhibition of 5-lipoxygenase.

**5.1 Hepatoprotective activity** 

**5.2 Anticancer activity of silymarin** 

**5.3 Anti-inflammatory activity** 

1996).

Activity of silymarin was examined against bronchial anaphylaxis and against postanaphylactic, propranolol- or platelet activating factor-induced hyperreactivity in guineapigs (Breschi et al., 2002). Silymarin pretreatment reduced the bronchospasm induced by antigen-challenge in sensitized animals. This protective effect was due to indirect mechanism that reduces airway responsiveness to histamine, and consequently the immediate anaphylactic response. Therefore, silymarin can be used as protective agent in the management of asthmatic disorders.

#### **5.5 Immunostimulatory activity**

Several studies have reported the immunostimulatory actions of silymarin (Wilarusmee et al., 2002). The effect of treatment with silymarin was studied on glutathione level and proliferation of peripheral blood mononuclear cells of β–thalassemia major patients (Alidoost et al., 2006). In vitro treatment with 10 g/ml silymarin restored glutathione levels and enhanced cellular proliferation. This was explained by its antioxidant activity.

#### **5.6 Treatment of obsessive-compulsive disorder**

Many patients cannot tolerate the side effects of pharmaceutical agents available for treatment of obsessive-compulsive disorder, do not respond properly to the treatment or the medications lose their effectiveness after a period of treatment. An 8-week pilot doubleblind randomized clinical trial on 35 adult patients was conducted to compare the efficacy of the extract of *S*. *marianum* with fluoxetine in the treatment of obsessive-compulsive disorder (Sayyah et al., 2010). The results showed that the extract of *S*. *marianum* has positive effects on obsession and compulsion starting from the fifth week. There were no any serious side effects accompanying *S. marianum* extract administration.

#### **5.7 Hyperprolactinemic effect**

*S*. *marianum* fruits have been traditionally used by nursing mothers for stimulating milk production (Newall et al., 1996). It was demonstrated that milk thistle increases lactation (Carotenuto and Di Pierro, 2005). The mechanism that led to the increase in lactation has been studied by measuring the concentration of circulating prolactin in female rats treated with silymarin (Capasso et al., 2009). It was shown that silymarin is able to produce a significant increase in circulating prolactin levels after oral administration. The levels of prolactin remains elevated for up to 66 days after silymarin discontinuation. Fig. 6 shows a summary of the wide range of biological activities attributed to silymain.

#### **5.8 Toxicity of silymarin**

An average daily dose of silymarin (420 mg/day for 41 months) was found to be non-toxic, relative to placebo, in clinical trials (Tamayo and Diamond, 2007). Drug-drug interaction and liver toxicity by interference with co-drugs by induction or inhibition of cytochrome-P450 is a major concern for the use of silymarin (Izzo and Ernst, 2009). Studies were performed to investigate the potential for hepatotoxicity, cytochrome-P450 isoenzymes induction and inhibition on dry extract from *S. marianum*, as contained in HEPAR-PASC® film-coated tablets (Doehmer et al., 2011). The results indicated that interference or

Silymarin, Natural Flavonolignans from Milk Thistle 265

*In vitro* cultured cells of *S. marianum* may offer an alternative and renewable source for this valuable natural product. However, the yield of silymarin was very low or sometimes not detectable in undifferentiated cultured cells (Becker and Schrall, 1977). In order to obtain silymarin in concentrations high enough for commercial manufacturing, many approaches have been made to stimulate the productivity of silymarin in cultured cells of *S. marianum*. These approaches compromise changes in the media composition (Cacho et al., 1999), treatment with elicitors such as yeast extract and methyl jasmonate (Sánchez-Sampedro et al., 2005a), addition of precursor (Tůmová et al., 2006) and morphological differentiation. Such approaches for improving silymarin production by manipulating plant cell cultures may also help in studying signal transduction pathways, cloning biosynthetic genes,

studying metabolic flux and regulation of silymarin production (Zhao et al., 2005).

Fig. 7. Callus of *Silybum marianum* developed on explants.

Becker and Schrall, (1977) cultured cotyledon explants on MS media using different growth hormones for establishment of cell suspension culture. Typical flavonolignans of *S. marianum* were not detected. This was possible after feeding coniferyl alcohol and taxifolin

**6.1 Cell culture** 

hepatotoxicity of the dry extract from *S. marianum* at the recommended maximum daily dose equivalent to 210 mg silybin is unlikely and is to be considered safe.

Fig. 6. Biological activities of silymarin.

#### **6. Tissue culture studies**

Plant tissue culture can be a potential source for important secondary metabolites (Misawa, 1994). This is based mainly on using plant cultures in a similar manner to microbial fermentation for factory-type production of pharmaceuticals and food additives. This technology has some advantages over conventional agricultural methods: production is independent of variation in crop quality or failure, yield of secondary metabolites would be constant and geared to demand, there is no difficulty in applying good manufacturing practice to the early stages of production, production would be possible anywhere under strictly controlled conditions, independent of political problems, free from risk of contamination with pesticides, herbicides or fertilizers and new methods of production can be patented (AbouZid et al., 2008). Cell suspension culture and hairy root culture were established from *S. marianum*. The former is established from callus tissue that developed on injured plant surface as a result of wounding or exogenous hormones (Fig. 7). The latter represent an approach to increase the yield of flavonolignans using morphologically differentiated/organized cultures.

#### **6.1 Cell culture**

264 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

hepatotoxicity of the dry extract from *S. marianum* at the recommended maximum daily

**Hepatoprotective** 

**Silymarin Biological Activities** 

**Anticancer** 

**Antiinflammatory** 

Plant tissue culture can be a potential source for important secondary metabolites (Misawa, 1994). This is based mainly on using plant cultures in a similar manner to microbial fermentation for factory-type production of pharmaceuticals and food additives. This technology has some advantages over conventional agricultural methods: production is independent of variation in crop quality or failure, yield of secondary metabolites would be constant and geared to demand, there is no difficulty in applying good manufacturing practice to the early stages of production, production would be possible anywhere under strictly controlled conditions, independent of political problems, free from risk of contamination with pesticides, herbicides or fertilizers and new methods of production can be patented (AbouZid et al., 2008). Cell suspension culture and hairy root culture were established from *S. marianum*. The former is established from callus tissue that developed on injured plant surface as a result of wounding or exogenous hormones (Fig. 7). The latter represent an approach to increase the yield of flavonolignans using morphologically

**stimulant Anti-asthma** 

dose equivalent to 210 mg silybin is unlikely and is to be considered safe.

Fig. 6. Biological activities of silymarin.

**Immuno-**

**Hyperprolactinemia** 

**6. Tissue culture studies** 

**Against obsessivecompulsive disorder** 

differentiated/organized cultures.

*In vitro* cultured cells of *S. marianum* may offer an alternative and renewable source for this valuable natural product. However, the yield of silymarin was very low or sometimes not detectable in undifferentiated cultured cells (Becker and Schrall, 1977). In order to obtain silymarin in concentrations high enough for commercial manufacturing, many approaches have been made to stimulate the productivity of silymarin in cultured cells of *S. marianum*. These approaches compromise changes in the media composition (Cacho et al., 1999), treatment with elicitors such as yeast extract and methyl jasmonate (Sánchez-Sampedro et al., 2005a), addition of precursor (Tůmová et al., 2006) and morphological differentiation. Such approaches for improving silymarin production by manipulating plant cell cultures may also help in studying signal transduction pathways, cloning biosynthetic genes, studying metabolic flux and regulation of silymarin production (Zhao et al., 2005).

Fig. 7. Callus of *Silybum marianum* developed on explants.

Becker and Schrall, (1977) cultured cotyledon explants on MS media using different growth hormones for establishment of cell suspension culture. Typical flavonolignans of *S. marianum* were not detected. This was possible after feeding coniferyl alcohol and taxifolin

Silymarin, Natural Flavonolignans from Milk Thistle 267

Fig. 8. Root culture of *Silybum marianum* growing in Murashige and Skoog medium.

flavonolignans (Hasanloo et al., 2009).

valuable compounds in high yield will facilitate such studies.

**7. Future directions** 

lipoxygenase to allow for the production of jasmonate. It was concluded that jasmonate signaling is an integral part of the yeast extract signal transduction for the production of

Plant tissue culture studies have contributed to our understanding of biosynthesis and regulation of silymarin in *S*. *marianum*. Using elicitation technology may offer an effective approach to improve silymarin production for industrial purpose. However, the possible signaling pathway that may be involved in accumulation of silymarin is still unknown. Understanding the basic components of this pathway is mandatory before these biotechnological methods can replace field crops as the basic source of pharmaceutical raw material. Establishment of plant tissue culture systems able to produce these biologically

to cell suspension cultures (Schrall and Becker 1977). Feeding the culture medium with precursor of coniferyl alcohol offered enhancement of silydianin production but other components of silymarin were not influenced (Tůmová et al., 2006). Cacho et al. (1999) reported that callus and cell cultures of *S*. *marianum* could produce silymarin but to a lesser extent than that accumulates in the fruits. They also reported that elimination of calcium ion positively affected silymarin production. This point was further confirmed by Sánchez-Sampedro et al. (2005a), who also reported that silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. These results suggest that inhibition of external and internal calcium fluxes play a significant role in flavonolignans metabolism in *S. marianum* cell cultures. Sánchez-Sampedro et al. (2005b) reported that yeast extract and methyl jasmonate elicited the production of silymarin. Elicitation is one of the most effective approaches to enhance the yield of secondary metabolites in *in vitro* cultures (Namdeo, 2007). It has been shown that elicitors can affect level of secondary metabolites in medicinal plants by modulating the rates of biosynthesis, accumulation, and/or vacuolar transit, turnover and degradation (Barz et al., 1990). Jasmonic acid and its methyl ester are known to be involved in the plant defense response through altering the gene expression. The mechanism by which jasmonate induces gene expression was studied in *Catharanthus roseus* (van der Fits and Memelink, 2000). In this plant species induction occurs through an ORCA3 transcription factor with a conserved jasmonate–response domain. The use of methyl jasmonate as an elicitor has an advantage of being only one compound of welldefined chemical structure. The effect of elicitation with picloram, jasmonic acid and light on silymarin production was reported (Hasanloo et al., 2008). The greatest silymarin content (0.41 mg/g DW) was obtained with 3 mg/l picloram and 2 mg/l jasmonic acid in the dark after 28 days. The sequence of the signaling processes leading to stimulation of flavonolignan production by methyl jasmonate is not well-known. Madrid and Corchete, 2010, studied the possible involvement of a phospholipase D-mediated lipid signaling in the elicitation of flavonolignans. It was reported that methyl jasmonate increased the activity of phospholipase D. Mastoparan, a phospholipase D activity stimulator, caused a substantial increase in silymarin production. Phosphatidic acid, a product of phospholipase D activity, promoted silymarin accumulation. N-butanol which inhibits phospholipase D activity prevented silymarin elicitation by methyl jasmonate or mastoparan.

#### **6.2 Root culture**

Production of flavonolignans from root cultures (Fig. 8) of *S*. *marianum* was reported before (Alikaridis et al ., 2000). Silybin (1.79 x 10-3 % DW) and silychristin (0.81 x 10-3 % DW) were the major flavonolignans produced by the established root cultures. In the referred study hairy root cultures of *S*. *marianum* were established. Hairy root cultures are the roots obtained by genetic transformation of plant tissues with the pathogenic soil bacterium *Agrobacterium rhizogenes.* These roots can then be cultured on hormone-free media and have three main advantages: genetic and biochemical stability, cultivation without addition of growth regulators and ability to give high final biomasses from low inocula.

Salicylic acid was effective in increasing the flavonolignan content 2.42 times in hairy root cultures of *S. marianum* higher than control cultures (Khalili et al., 2009). Yeast extract stimulated flavonolignan production in hairy root cultures two-fold higher than the control cultures. Moreover, it was reported that yeast extract treatment induced the activity of

to cell suspension cultures (Schrall and Becker 1977). Feeding the culture medium with precursor of coniferyl alcohol offered enhancement of silydianin production but other components of silymarin were not influenced (Tůmová et al., 2006). Cacho et al. (1999) reported that callus and cell cultures of *S*. *marianum* could produce silymarin but to a lesser extent than that accumulates in the fruits. They also reported that elimination of calcium ion positively affected silymarin production. This point was further confirmed by Sánchez-Sampedro et al. (2005a), who also reported that silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. These results suggest that inhibition of external and internal calcium fluxes play a significant role in flavonolignans metabolism in *S. marianum* cell cultures. Sánchez-Sampedro et al. (2005b) reported that yeast extract and methyl jasmonate elicited the production of silymarin. Elicitation is one of the most effective approaches to enhance the yield of secondary metabolites in *in vitro* cultures (Namdeo, 2007). It has been shown that elicitors can affect level of secondary metabolites in medicinal plants by modulating the rates of biosynthesis, accumulation, and/or vacuolar transit, turnover and degradation (Barz et al., 1990). Jasmonic acid and its methyl ester are known to be involved in the plant defense response through altering the gene expression. The mechanism by which jasmonate induces gene expression was studied in *Catharanthus roseus* (van der Fits and Memelink, 2000). In this plant species induction occurs through an ORCA3 transcription factor with a conserved jasmonate–response domain. The use of methyl jasmonate as an elicitor has an advantage of being only one compound of welldefined chemical structure. The effect of elicitation with picloram, jasmonic acid and light on silymarin production was reported (Hasanloo et al., 2008). The greatest silymarin content (0.41 mg/g DW) was obtained with 3 mg/l picloram and 2 mg/l jasmonic acid in the dark after 28 days. The sequence of the signaling processes leading to stimulation of flavonolignan production by methyl jasmonate is not well-known. Madrid and Corchete, 2010, studied the possible involvement of a phospholipase D-mediated lipid signaling in the elicitation of flavonolignans. It was reported that methyl jasmonate increased the activity of phospholipase D. Mastoparan, a phospholipase D activity stimulator, caused a substantial increase in silymarin production. Phosphatidic acid, a product of phospholipase D activity, promoted silymarin accumulation. N-butanol which inhibits phospholipase D activity

prevented silymarin elicitation by methyl jasmonate or mastoparan.

growth regulators and ability to give high final biomasses from low inocula.

Production of flavonolignans from root cultures (Fig. 8) of *S*. *marianum* was reported before (Alikaridis et al ., 2000). Silybin (1.79 x 10-3 % DW) and silychristin (0.81 x 10-3 % DW) were the major flavonolignans produced by the established root cultures. In the referred study hairy root cultures of *S*. *marianum* were established. Hairy root cultures are the roots obtained by genetic transformation of plant tissues with the pathogenic soil bacterium *Agrobacterium rhizogenes.* These roots can then be cultured on hormone-free media and have three main advantages: genetic and biochemical stability, cultivation without addition of

Salicylic acid was effective in increasing the flavonolignan content 2.42 times in hairy root cultures of *S. marianum* higher than control cultures (Khalili et al., 2009). Yeast extract stimulated flavonolignan production in hairy root cultures two-fold higher than the control cultures. Moreover, it was reported that yeast extract treatment induced the activity of

**6.2 Root culture** 

Fig. 8. Root culture of *Silybum marianum* growing in Murashige and Skoog medium.

lipoxygenase to allow for the production of jasmonate. It was concluded that jasmonate signaling is an integral part of the yeast extract signal transduction for the production of flavonolignans (Hasanloo et al., 2009).

#### **7. Future directions**

Plant tissue culture studies have contributed to our understanding of biosynthesis and regulation of silymarin in *S*. *marianum*. Using elicitation technology may offer an effective approach to improve silymarin production for industrial purpose. However, the possible signaling pathway that may be involved in accumulation of silymarin is still unknown. Understanding the basic components of this pathway is mandatory before these biotechnological methods can replace field crops as the basic source of pharmaceutical raw material. Establishment of plant tissue culture systems able to produce these biologically valuable compounds in high yield will facilitate such studies.

Silymarin, Natural Flavonolignans from Milk Thistle 269

Breschi, M.C., Martinotti, E., Apostoliti, F., Nieri, P. (2002) Protective effect of silymarin in

Cacho, M., Moran, M., Corchete, P., Fernandez-Tarrago, J. (1999) Influence of medium

Capasso, R., Aviello, G., Capasso, F., Savino, F., Isso, A.A., Lembo, F., Borrelli, F. (2009)

Carini, R., Comoglio, A., Albano, E., Poli, G. (1992) Lipid peroxidation and irreversible

Carotenuto, D., Di Pierro, F. (2005) Studio sulla tollerabilità ed efficacia dela silimarina BIO-

De La Puerta, R. (1996) Effect of silymarin on different acute inflammation models and in leukocyte migration. *Journal of Pharmacy and Pharmacology* 48, 9, 968-970. Deep, G., Oberlies, N.H., Kroll, D.J., Agarwal, R. (2007) Isosilybin B and isosilybin A inhibit

Deep, G., Oberlies, N.H., Kroll, D.J., Agarwal, R. (2008) Isosilybin B causes androgen

Deep, G., Oberlies, N.H., Kroll, D.J., Agarwal, R. (2008) Identifying the differential effects of

Dehmlow, C., Erhard, J., De Groot, H. (1996) Inhibition of Kupffer cell functions as an

Dewick, P.M. (2002) Medicinal natural products. A Biosynthetic Approach, John Wiley &

Doehmer, J., Weiss, G., McGregor, G.P., Appel, K. (2011) Assessment of a dry extract from

Famacopea Ufficiale Italiana, ed. IX, vol. II 1673, Istituto Poligraficao e Zecca cello Stato-

Flora, K., Hahn, M., Rosen, H., Benner, K. (1998) Milk thistle (*Silybum marianum*) for the therapy of liver disease. *American Journal of Gastroenterology* 93, 139-143. Fraschini, F., Demartini, G., Esposti, D. (2002) Pharmacology of silymarin. *Clinical Drug* 

Gupta, O.P., Sing, S., Bani, S., Sharma, N., Malhotra, S., Gupta, B.D., Banerjee, S.K., Handa,

Hamid, S., Sabir, A., Khan, S., Aziz, P. (1983) Experimental cultivation of *Silybum marianum*

through inhibition of 5-lipoxygenase. *Phytomedicine* 7, 21-24.

S.S. (2000) Antiinflammatory and antiarithritic activities of silymarin acting

and chemical composition of its oil. *Pakistan Journal of Scientific and Industrial* 

hperprolactinemia in intact female rats. *Phytomedicine* 16, 839-844.

complex IdB 1016. *Biochemical Pharmacology* 43, 10, 2111-2115.

prostate cancer cells. *International Journal of Cancer* 123, 41-50.

*European Journal of Pharmacology* 437, 91-95.

*marianum* (L.) Gaertn. *Plant Science* 144, 63-68.

and 22Rv1 cells. *Carcinogenesis* 28, 1533-1542.

mediated pathway. *Oncogene* 27, 3986-3998.

activities. *Toxicology in Vitro* 25, 1, 21-27.

Sons, Ltd, Chichester, UK.

*Investigation* 22(1), 51-65.

*Research* 26, 244-246.

Roma, 1985.

antigen challenge- and histamin-induced brochoconstriction in in vivo guinea-pigs.

composition on the accumulation of flavonolignans in cultured cells of *Silybum* 

Silymarin BIO-C®, and extract from *Silybum marianum* fuits, induces

damage in the rat hepatocyte model: Protection by the silybin-phospholipid

C® (Piừlatte®) micronizzata come galattagogo. *Acta Neonatology Pediatrics* 4, 393-400.

growth, induce G1 arrest and cause apoptosis in human prostate cancer LNCaP

receptor degradation in human prostate carcinoma cells via P13K-Akt-Mdm2-

silymarin constituents on cell growth and cell cycle regulatory molecules in human

explanation for the hepatoprotective properties of silybinin. *Hepatology* 23, 749-754.

milk thistle (*Silybum marianum*) for interference with human liver cytochrome-P450

#### **8. Conclusion**

Milk thistle is an annual or biennial herb native to the Mediterranean and North African regions. The fruits of the plant contain an isomeric mixture of flavonolignans collectively known as silymarin. Basically, flavonolignan nucleus consists of the dihydroflavanol taxifolin linked to coniferyl alcohol moiety through an oxeran ring. Little is known about the coupling of coniferyl alcohol to taxifolin. Silymarin is widely used as a hepatoprotective agent for oral treatment of toxic liver damage and for the therapy of chronic inflammatory liver diseases. The hepatoprotective activity of silymarin is based on antioxidant properties, stimulating liver cells regeneration and cell membrane stabilization to prevent hepatotoxic agents from entering hepatocytes. It has been shown that flavonolignans exhibit wide range of biological activity including anticancer, anti-inflammatory, hyperprolactinemic properties. Various methods have been developed for analysis of the content and composition of main silymarin components in plant material and pharmaceuticals. Among these methods are thin layer chromatography, spectrophotometric, high performance liquid chromatography, capillary zone electrophoresis and ultra performance liquid chromatography. *In vitro* cultured cells of *S. marianum* may offer an alternative and renewable source for this valuable natural product. Flavonolignans production in cell and root cultures of *S. marianum* has been reported. Many approaches have been used to increase the yield of flavonolignans in *S*. *marianum* tissue culture including change in media composition, addition of precursors and elicitation.

#### **9. References**


Milk thistle is an annual or biennial herb native to the Mediterranean and North African regions. The fruits of the plant contain an isomeric mixture of flavonolignans collectively known as silymarin. Basically, flavonolignan nucleus consists of the dihydroflavanol taxifolin linked to coniferyl alcohol moiety through an oxeran ring. Little is known about the coupling of coniferyl alcohol to taxifolin. Silymarin is widely used as a hepatoprotective agent for oral treatment of toxic liver damage and for the therapy of chronic inflammatory liver diseases. The hepatoprotective activity of silymarin is based on antioxidant properties, stimulating liver cells regeneration and cell membrane stabilization to prevent hepatotoxic agents from entering hepatocytes. It has been shown that flavonolignans exhibit wide range of biological activity including anticancer, anti-inflammatory, hyperprolactinemic properties. Various methods have been developed for analysis of the content and composition of main silymarin components in plant material and pharmaceuticals. Among these methods are thin layer chromatography, spectrophotometric, high performance liquid chromatography, capillary zone electrophoresis and ultra performance liquid chromatography. *In vitro* cultured cells of *S. marianum* may offer an alternative and renewable source for this valuable natural product. Flavonolignans production in cell and root cultures of *S. marianum* has been reported. Many approaches have been used to increase the yield of flavonolignans in *S*. *marianum* tissue culture including change in media composition, addition of precursors and elicitation.

AbouZid, S., Nasib, A., Khan, S., Qureshi, J., Choudhary, M.I. (2010) Withaferin A

Alarcon, C., Martin, M.J., Marhuenda, E. (1992) Gastric anti-ulcer activity of silymarin, a lipoxygenase inhibitor, in rats. *Journal of Pharmacy and Pharmacology* 44, 929-931. Alidoost, F., Gharagozloo, M., Bagherpour, B., Jafarian, A., Sajjadi, S.E., Hourfar, H.,

Alikaridis, F., Papadakis, D., Pantelia, K., Kephalas, T. (2000) Flavonolignan production

Barz, W.A., Beimen, B., Drae, U., Jaques, C., Sue, O.E., Upmeier, B. (1990) Turnover and

Becker, H., Schrall, R. (1977) Tissue and suspension cultures of *Silybum marianum*: the

Bosisio, E., Benelli, C., Pirola, O. (1992) Effect of the flavolignans of *Silybum marianum* L. on

Boulos, L. (2000) *Flora of Egypt*, (1st edition), Al Hadara Publishing Inc., Cairo, Egypt.

*Research in Natural Products* 2(5), 23-27.

*International Journal of Immunopharmacology* 6, 1305-1310.

scavenging properties. *Achieves of Pharmacology* 313, 330-337.

production by root cultures of *Withania coagulans*. *International Journal of Applied* 

Moayedi, B. (2006) Effects of silymarin on the proliferation and glutathione levels of peripheral blood mononuclear cells from β–thalassemia major patients.

from *Silybum marianum* transformed and untransformed root cultures. *Fitoterapia*,

storage of secondary products in cell cultures. In: Charlwood BV, Rhodes MJC, eds. Secondary products from plant tissue culture. Oxford: Clarendon Press, 79-102. Baumann, J., Wurm, G., Von Bruhhansen, F. (1980) Prostaglandin relation to their oxygen-

formation of flavanolignans by flavanoids and coniferyl alcohol. *Planta Medica*,

lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes.

**8. Conclusion** 

**9. References** 

71(4), 379-384.

32(1), 27-32.

*Pharmacology Research* 25, 147-154.


Silymarin, Natural Flavonolignans from Milk Thistle 271

Rahman, N., Khan, N.A., Azmi, S.N.H. (2004) Kinetic spectrophotometric method for the

Ramasamy, K., Agrawal, R. (2008) Multitargeted therapy of cancer by silymarin. *Cancer* 

Pietrangelo, A., Borella, F., Casalgrandi, G. (1995) Antioxidant activity of silybin *in vivo* during long-term iron overload in rats. *Gastroenterology* 109, 1941-1949. Sánchez-Sampedro, M.A., Fernández-Tárrago, J., Corchete, P. (2005a) Yeast extract and

Sánchez-Sampedro, M.A., Fernández-Tárrago, J., Corchete, P. (2005b) Some common signal

Sayyah, M., Boostani, H., Pakseresht, S., Malayeri, A. (2010) Comparison of *Silybum marianum*

Shah, J., Klessig, D.F. (1999) Salicylic acid: signal perception and transduction. In Libbenga,

Shaker, E., Mahmoud, H., Mnaa, S. (2010) Silymarin, the antioxidant component and

Skehan, P., Storeng, R. (1990) New colorimetric cytotoxicity assay for anti cancer drug

Svobodová, A., Zdařilová, A., Walterová, D., Vostálová, J. (2007) Flavonolignans from

Takahara, E., Ohta, S., Hirobe, M. (1986) Stimulatory effects of silibinin on the DNA

Tamayo, C., Diamond, S. (2007) Review of clinical trials evaluating safety and efficacy of milk thistle (*Silybum marianum* [L.] Gaertn.). *Integrated Cancer Therapy* 6, 146-157. Tanaka, H., Shibata, M., Ohira, K., Ito, K. (1985) Total synthesis of (±)-silybin, an antihepatotoxic flavanolignan. *Chemical and Pharmaceutical Bulletin* 33, 1419-1423. Tůmová, L., Řimáková, J., Tůma, J., Dušek, J. (2006) *Silybum marianum in vitro*-flavonolignan

van der Fits, L., Memelink, J. (2000) ORCA3, a jasmonate responsive transcriptional regulator of plant primary and secondary metabolism. *Science* 289, 295–297. Verpoorte, R., Alfermann, A.W. (2000) Metabolic Engineering of Plant Secondary

*Progress in Neuro-Psychopharmacology & Biological Psychiatry* 34, 362-365. Schrall, R., Becker, H. (1977) Produktion von catechinen und oligomeren

*Cratжgus oxyacantha* und *Ginkgo biloba*. *Planta Medica* 32, 297-307.

screening. *Journal of National Cancer Institute* 82, 1107-1112.

keratinocytes. *Journal of Dermatological Science* 48, 213-224.

production. *Plant Soil and Environment* 52(10), 454–458.

Metabolism. Kluwer Academic Publishers, The Netherlands.

Hormones. Elsevier, Oxford, 513–541.

*marianum* (L.) Gaertn. *Journal of Plant Physiology* 162(10), 1177-1182.

permanganate as antioxidant. *Pharmazie* 59, 112- 116.

*Biomedical Analysis* 19, 435-442.

*Letters* 269, 352-362.

1466-1473.

803-806.

541.

liquid chromatography and capillary electrophoresis. *Journal of Pharmaceutical and* 

determination of silymarin in pharmaceutical formulations using potassium

methyl jasmonate-induced silymarin production in cell cultures of *Silybum* 

transduction events are not necessary for the elicitor-induced accumulation of silymarin in cell cultures of *Silybum marianum. Journal of Plant Physiology* 165(14),

(L.) Gaertn. with fluoxetine in the treatment of Obsessive–Compulsive Disorder.

proanthocyanidinen in callus- und suspensionskulturen von *Cratжgus monogyna*,

K., Hall. M., Hooykaas, P.J.J., editors. Biochemistry and Molecular Biology of Plant

*Silybum marianum* extracts prevents liver damage. *Food and Chemical Toxicology* 48,

*Silybum marianum* moderate UVA-induced oxidative damage to HaCaT

synthesis in partially hepatectomized rate livers. *Biochemical Pharmacology* 35, 538-


Hasanloo, T., Kavari-Nejad, R.A., Majidi, E., Shams, Ardakani, M.R. (2008) Flavonolignan

Hiroshi, H., Yoshinobu, K., Wagner, H., Manfred, F. (1984) antihepatotoxic actions of flavonolignans from *Silybum marianum* fruits. *Planta Medica* 51, 248-250. Kim, S., Choi, J.H., Lim H.I., Lee, S., Kim, W.W., Kim J.S., Kim J., Choe, J., Yang, J., Nam, S.J.,

Klassen, C.D., Plaa, G.L. (1969) Comparison of the biochemical alteration elicited in liver of

Kvasnička, F., Biĩba, B., Ševčík, Voldřich, M., Krátká, J. (2003) Analysis of the active

Izzo, A.A., Ernst, E. (2009) Interactions between herbal medicines and prescribed drugs: and

Lee, D.Y.-W., Liu, Y. (2003) Molecular structure and stereochemistry of silybin A, silybin B,

Lee, S.K., Mbwambo, Z.H.Y., Chung, H., Luyengi, L., Gamez, E.J.C., Mehta, R.G., Kinghorn,

Madrid, E., Corchete, P. (2010) Silymarin secretion and its elicitation by methyl jasmonate in

Maghrani, M., Zeggwagh, N.A., Lemhadri, A., EI Amraoui, M., Michael, J.B., Eddouks, M.

Misawa, M. (1994) Plant tissue culture: an alternative for production of useful metabolites.

Morazzoni, P., Bombardelli, E. (1995) *Silybum marianum* (*Cardus marianum*). *Fitoterapia* 66, 3-

Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with

Namdeo, A.G. (2007) Plant cell elicitation for production of secondary metabolites: A

Newall, C.A., Anderson, L.A., Phillipson, J.D. (1996) Herbal Medicines: A Guide for Health-

Quaglia, M.G., Bossù, E., Donati, E., Mazzanti, G., Brandt, A. (1999) Determination of

silymarin in the extract from the dried *silybum marianum* fruits by high performance

products. *Combinatorial Chemistry and High Throughput Screening* 1, 35-46. Ligeret, H., Brault, A., Vallerand, D., Haddad, Y., Haddad, P.S. (2008) Antioxidant and

components of silymarin. *Journal of Chromatography A* 990, 239-245.

46(12), 876-882.

18(8), 2019- 2027.

cancer cells. *Phytomedicine* 16, 573-580.

*of Natural Products* 66, 1171-1174.

*Ethnopharmacology* 91, 309-316.

42.

updated systematic review. *Drugs* 69, 1777-1798.

liver injury*. Journal of Ethnopharmacology* 115, 507-514.

tobacco tissue culture. *Physiologia Plantarum* 15, 473-497.

Care Professionals. Pharmaceutical Press, London pp. 46-47.

acid. *Journal of Experimental Botany* 61(3), 747-754.

Madaus, R., Gorler, K., Molls, W. (1983) US patent 4,368,195.

FAO Agricultural Services Bulletin 108.

review. *Pharmacognosy Review* 1, 69-79.

Production in Cell Suspension Culture of *Silybum marianum*. *Pharmaceutical Biology*

Lee, J.E. (2009) Silibinin prevents TPA-induced MMP-9 expression and VEGF secrestion by inactivation of the Raf/MEK/ERK pathway in MCF-7 human breast

rats treated with carbon tetra chloride and chloroform. *Biochemical Pharmacology*

isosilybin A, and isosilybin B, isolated from *Silybum marianum* (Milk thistle). *Journal* 

A.D., Pezzuto, J.M. (1998) Evaluation of the antioxidant potential of natural

mitochondrial protective effects of silibinin in cold preservation-warm reperfusion

cell cultures of *Silybum marianum* is mediated by phospholipase D-phosphatidic

(2004) Study of the hypoglycaemic activity of *Fraxinus excelsior* and *Silybum marianum* in an animal model of type 1 diabetes mellitus. *Journal of*  liquid chromatography and capillary electrophoresis. *Journal of Pharmaceutical and Biomedical Analysis* 19, 435-442.


**13** 

*USA* 

**Phytocannabinoids** 

*University of Mississippi,* 

Afeef S. Husni and Stephen J. Cutler

What is marijuana? Marijuana, also known as *Cannabis*, is defined as any preparation of the *Cannabis sativa* plant used to exploit psychoactive effects whether it is recreational or medicinal. According to the 2004 World Drug Report, 3.7% of the population 15-64 years of age consumed marijuana from 2001-2003 (World Drug Report, 2004). The use of marijuana is associated with numerous pharmacological effects; most, but not all may attributed to tetrahydrocannabinol (THC). The combination of THC and other compounds from *Cannabis sativa* may all exhibit specific pharmacological effects. These isolates from *Cannabis* are

Cannabinoids are a chemical class of C21 terpenophenolic compounds that represent a group of compounds found in *Cannabis sativa* (Mechoulam & Gaoni, 1967). Phytocannabinoids are the naturally occurring cannabinoids from *Cannabis* sp (Pate, 1999). It is now known that at least 85 cannabinoids have been derived from *Cannabis sativa* (El-Alfy et al., 2010). It is also

In order to gain a better understanding of the pharmacological effects of the phytocannabinoids, human and rodent receptors are used to evaluate binding affinity of these compounds to two cannabinoid receptors that have been reported in literature, CB1 and CB2. CB1 receptors are located mainly in the brain, while CB2 receptors are primarily

known that some of these compounds are of medical importance in today's society.

**1. Introduction** 

known as cannabinoids (ElSohly, 2010).


## **Phytocannabinoids**

Afeef S. Husni and Stephen J. Cutler *University of Mississippi, USA* 

#### **1. Introduction**

272 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Wagner, H., Baldt, S., Rickl, V. (2009) Plant Drug Analysis: A Thin Layer Chromatography

Wang, K., Zhang, H., Shen, L., Du, Q., Li, J. (2010) Rapid separation and characterization of

Wilasrusmee, C., Kittur, S., Shah, G., Siddiqui, J., Bruch, D., Wilasrusmee, S., Kittur, D.S.

Zhao, B., Wolf, D.M., Agrawal, R. (1999) Inhibition of human carcinoma cell growth and

Zhao, J., Davis, L.C.T., Verpoorte R. (2005) Elicitor signal transduction leading to production

Zi, X., Mukhtar, H., Agarwal R. (1997) Novel cancer chemopreventative effects of a

of plant secondary metabolites. *Journal of Biotechnology* 23, 283-333.

active flavonolignans of *Silybum marianum* by ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry. *Journal of* 

(2002) Immunostimulatory effect of *Silybum marianum* (milk thistle) extract. *Medical* 

DNA synthesis by silybinin, an active constituent of milk thistle: comparison with

flavonoid constituent silymarin: inhibition of mRNA expression of an endogenous tumor promoter TNF alpha. *Biochemical and Biophysical Research Communications*

Atlas. Second Edition, Springer, Germany.

*Science Monitor 8, BR439-434.*

239, 334–339.

silymarin. *Cancer Letters* 147, 77-84.

*Pharmaceutical and Biomedical Analysis* 53, 1053-1057.

What is marijuana? Marijuana, also known as *Cannabis*, is defined as any preparation of the *Cannabis sativa* plant used to exploit psychoactive effects whether it is recreational or medicinal. According to the 2004 World Drug Report, 3.7% of the population 15-64 years of age consumed marijuana from 2001-2003 (World Drug Report, 2004). The use of marijuana is associated with numerous pharmacological effects; most, but not all may attributed to tetrahydrocannabinol (THC). The combination of THC and other compounds from *Cannabis sativa* may all exhibit specific pharmacological effects. These isolates from *Cannabis* are known as cannabinoids (ElSohly, 2010).

Cannabinoids are a chemical class of C21 terpenophenolic compounds that represent a group of compounds found in *Cannabis sativa* (Mechoulam & Gaoni, 1967). Phytocannabinoids are the naturally occurring cannabinoids from *Cannabis* sp (Pate, 1999). It is now known that at least 85 cannabinoids have been derived from *Cannabis sativa* (El-Alfy et al., 2010). It is also known that some of these compounds are of medical importance in today's society.

In order to gain a better understanding of the pharmacological effects of the phytocannabinoids, human and rodent receptors are used to evaluate binding affinity of these compounds to two cannabinoid receptors that have been reported in literature, CB1 and CB2. CB1 receptors are located mainly in the brain, while CB2 receptors are primarily

Phytocannabinoids 275

Endogenous cannabinoids, or endocannabinoids, are substances produced in the body that activate the cannabinoid receptors. Generally, neurotransmitters are released presynaptically and activate the receptors on a postsynaptic cell. However, unlike most neurotransmitters, the endocannabinoids work in a reverse fashion. Endocannabinoids use retrograde signaling to achieve cannabinoid receptor activation. This means that the ligands are being produced postsynaptically, but acting presynaptically (Lambert, 2009). Another critical point in understanding the function of the endocannabinoids is that the endocannabinoid system can produce endocannabinoids "on demand" in response to an increase in intracellular calcium

Shortly after the cloning of the cannabinoid receptors, researchers began searching for endogenous ligands that activate these receptors. The first endocannabinoid discovered was anandamide (Figure 2) in 1992 (Devane et al., 1992). Several years after the discovery of anandamide the second endogenous ligand, 2-arachadonoyl-glycerol (2-AG, Figure 3), was discovered (Sugiura et al., 2006). Anandamide and 2-AG act as a partial agonist and full agonist, respectively, at the CB1 and CB2 receptors. Although the structure of anandamide differs significantly from THC, both of these ligands have similar pharmacological profiles (Grotenhermen, 2002). Understanding the mechanism of how cannabinoids produce their

Although the physiological roles of the endocannabinoids are not fully defined, several pharmacological functions have been described. Studies suggest that these endogenous ligands may aid in pain relief, enhancement of appetite, blood pressure lowering during

shock, embryonic development, and blocking of working memory (ElSohly, 2010).

effects is in part because of the discovery of the endocannabinoid system.

**3. Endocannabinoids** 

levels (Sugiura et al., 2006).

Fig. 2. Chemical structure of anandamide.

Fig. 3. Chemical structure of 2-AG.

peripheral and found on mature B cells and macrophages within the tonsils and spleen (Raymon & Walls, 2010). When activated, the CB1 receptors exhibit the psychoactive effects caused by *Cannabis* use. Since CB1 receptors are not present in the medulla oblongata, part of the brain stem responsible for respiratory and cardiovascular functions, there is not a risk of overdose resulting in respiratory depression or cardiovascular failure that may be seen with abuse of other drugs, such as the opioids. CB2 receptors are said to be responsible for antiinflammatory effects.

#### **2. Cannabinoid receptor function**

Cannabinoid receptors are G-protein coupled receptors (Figure 1), which are a large family of seven member transmembrane receptors that act in a second messenger fashion. When cannabinoid receptors are activated, they inhibit the enzyme adenylate cyclase. Adenylate cyclase is responsible for breaking ATP to form cyclic AMP (cAMP). When a ligand binds to the extracellular surface of cannabinoid receptors, it causes a conformational change of the receptor. This change activates the second messenger by exchanging guanosine diphosphate (GDP) for guanosine triphosphate (GTP). Then, the Gprotein's alpha subunit separates from the beta/gamma subunit to cause intracellular proteins to function properly. In CB1 and CB2 receptors, cAMP acts as the second messenger. When these receptors are activated, cAMP levels decrease within the cell. Therefore, the result of activating cannabinoid receptors leads to a decrease in cAMP levels, and in turn leads to an inhibition of function.

Fig. 1. Example of a G-Protein Coupled Receptor.

peripheral and found on mature B cells and macrophages within the tonsils and spleen (Raymon & Walls, 2010). When activated, the CB1 receptors exhibit the psychoactive effects caused by *Cannabis* use. Since CB1 receptors are not present in the medulla oblongata, part of the brain stem responsible for respiratory and cardiovascular functions, there is not a risk of overdose resulting in respiratory depression or cardiovascular failure that may be seen with abuse of other drugs, such as the opioids. CB2 receptors are said to be responsible for anti-

Cannabinoid receptors are G-protein coupled receptors (Figure 1), which are a large family of seven member transmembrane receptors that act in a second messenger fashion. When cannabinoid receptors are activated, they inhibit the enzyme adenylate cyclase. Adenylate cyclase is responsible for breaking ATP to form cyclic AMP (cAMP). When a ligand binds to the extracellular surface of cannabinoid receptors, it causes a conformational change of the receptor. This change activates the second messenger by exchanging guanosine diphosphate (GDP) for guanosine triphosphate (GTP). Then, the Gprotein's alpha subunit separates from the beta/gamma subunit to cause intracellular proteins to function properly. In CB1 and CB2 receptors, cAMP acts as the second messenger. When these receptors are activated, cAMP levels decrease within the cell. Therefore, the result of activating cannabinoid receptors leads to a decrease in cAMP

inflammatory effects.

**2. Cannabinoid receptor function** 

levels, and in turn leads to an inhibition of function.

Fig. 1. Example of a G-Protein Coupled Receptor.

#### **3. Endocannabinoids**

Endogenous cannabinoids, or endocannabinoids, are substances produced in the body that activate the cannabinoid receptors. Generally, neurotransmitters are released presynaptically and activate the receptors on a postsynaptic cell. However, unlike most neurotransmitters, the endocannabinoids work in a reverse fashion. Endocannabinoids use retrograde signaling to achieve cannabinoid receptor activation. This means that the ligands are being produced postsynaptically, but acting presynaptically (Lambert, 2009). Another critical point in understanding the function of the endocannabinoids is that the endocannabinoid system can produce endocannabinoids "on demand" in response to an increase in intracellular calcium levels (Sugiura et al., 2006).

Shortly after the cloning of the cannabinoid receptors, researchers began searching for endogenous ligands that activate these receptors. The first endocannabinoid discovered was anandamide (Figure 2) in 1992 (Devane et al., 1992). Several years after the discovery of anandamide the second endogenous ligand, 2-arachadonoyl-glycerol (2-AG, Figure 3), was discovered (Sugiura et al., 2006). Anandamide and 2-AG act as a partial agonist and full agonist, respectively, at the CB1 and CB2 receptors. Although the structure of anandamide differs significantly from THC, both of these ligands have similar pharmacological profiles (Grotenhermen, 2002). Understanding the mechanism of how cannabinoids produce their effects is in part because of the discovery of the endocannabinoid system.

Fig. 2. Chemical structure of anandamide.

Although the physiological roles of the endocannabinoids are not fully defined, several pharmacological functions have been described. Studies suggest that these endogenous ligands may aid in pain relief, enhancement of appetite, blood pressure lowering during shock, embryonic development, and blocking of working memory (ElSohly, 2010).

Fig. 3. Chemical structure of 2-AG.

Phytocannabinoids 277

Tetrahydrocannabinol and cannabidiol are the two most discussed phytocannabinoids, but not the only ones known. ElSohly and co-investigators have divided the phytocannabinoids into ten subclasses: 1) Cannabigerol type – propyl side chains and monomethyl ether derivatives 2) Cannabichromene type – analogs present in the C-5 position 3) Cannabidiol type – analogs varying from C-1 to C-5 positions 4) Delta-9-tetrahydrocannabinol type – double bond in the C-9 position; responsible for psychoactive effects 5) Delta-8 tetrahydrocannabinol type – double bond in the C-8 position; thermodynamically more stable than delta-9-THC , however, 20% less active 6) Cannabicyclol type – five atom ring and C-1 bridge 7) Cannabielsoin type – artifacts formed from CBD 8) Cannabinol and Cannabinodiol types – A ring aromatization 9) Cannabitriol type – additional hydroxyl substitution 10) Miscellaneous types – ex: furano ring, carbonyl function, tetrahydroxy

Another phytocannabinoid that shows a significant amount of importance is cannabinol (CBN, Figure 6); it is a metabolite of tetrahydrocannabinol. It was the first cannabinoid identified from *Cannabis sativa*. (Wood et al., 1896). Along with THC, cannabinol is also a psychoactive component of *Cannabis* due to its interaction with CB1 receptors. Compared to

THC, it acts a weak agonist at both the CB1 and CB2 receptors.

Fig. 5. Chemical structure of cannabidiol.

substitution (ElSohly, 2010).

Fig. 6. Chemical structure of cannabinol.

#### **4. Phytocannabinoids**

The first cannabinoid identified was cannabigerol, and its precursor cannabigeric acid was shown to be the cannabinoid formed in the plant as well as endogenously (Yamauchi, 1975). Today, the most discussed phytocannabinoid is delta-9-tetrahydrocannabinol. In 1964, Gaoni and Mechoulam isolated and elucidated the chemical structure of THC from the leaves of *Cannabis sativa* (Mechoulam & Gaoni, 1964). THC is pharmacologically and toxicologically the best studied constituent of *Cannabis*, responsible for most of the psychoactive effects of natural *Cannabis* preparations (Grotenhermen, 2002). THC and cannabidiol (CBD) are the two most common naturally occurring cannabinoids.

As mentioned earlier, THC (Figure 4) is the main component of *Cannabis* responsible for the psychoactive effects. Other than *Cannabis* being abused to achieve a state of euphoria, it is now being used medicinally to aid in acquired immunodeficiency syndrome (AIDS) patients with wasting syndrome and for pain management, nausea, and vomiting associated with patients receiving cancer chemotherapy. Since THC is responsible for the psychoactive effects of *Cannabis*, people have learned how to genetically increase the concentration of THC within each plant to produce a stronger "high." Since 1980, the concentration of THC within marijuana has increased from less than 1.5% to approximately 20% (ElSohly et al., 2000). THC acts a partial agonist at the CB1 and CB2 receptors, but functions via interaction with the CB1 receptor.

Fig. 4. Chemical structure of delta-9-THC.

The second major constituent of *Cannabis*, cannabidiol (CBD, Figure 5), is responsible for the anti-inflammatory effects due to its interactions with the human CB2 receptor. CBD was first isolated in 1940 (Adams et al., 1940); however, it was not until 1963 that Mechoulam and Shvo elucidated its correct structure (Mechoulam & Shvo, 1963). At the human CB2 receptor, CBD's mechanism of action shows inverse agonism activity (Pertwee et al., 2007). In 1995, Benet and colleagues show that cannabidiol is not only responsible for anti-inflammatory effects, but may also aid in reducing unpleasant side effects from THC, including reduced anxiety (Benet et al., 1995). They found that CBD inhibits cytochrome P450 3A11, which causes THC to change into its more potent metabolite 11-hydroxy-THC (Gallily et al., 2002).

The first cannabinoid identified was cannabigerol, and its precursor cannabigeric acid was shown to be the cannabinoid formed in the plant as well as endogenously (Yamauchi, 1975). Today, the most discussed phytocannabinoid is delta-9-tetrahydrocannabinol. In 1964, Gaoni and Mechoulam isolated and elucidated the chemical structure of THC from the leaves of *Cannabis sativa* (Mechoulam & Gaoni, 1964). THC is pharmacologically and toxicologically the best studied constituent of *Cannabis*, responsible for most of the psychoactive effects of natural *Cannabis* preparations (Grotenhermen, 2002). THC and

As mentioned earlier, THC (Figure 4) is the main component of *Cannabis* responsible for the psychoactive effects. Other than *Cannabis* being abused to achieve a state of euphoria, it is now being used medicinally to aid in acquired immunodeficiency syndrome (AIDS) patients with wasting syndrome and for pain management, nausea, and vomiting associated with patients receiving cancer chemotherapy. Since THC is responsible for the psychoactive effects of *Cannabis*, people have learned how to genetically increase the concentration of THC within each plant to produce a stronger "high." Since 1980, the concentration of THC within marijuana has increased from less than 1.5% to approximately 20% (ElSohly et al., 2000). THC acts a partial agonist at the CB1 and CB2 receptors, but functions via interaction

The second major constituent of *Cannabis*, cannabidiol (CBD, Figure 5), is responsible for the anti-inflammatory effects due to its interactions with the human CB2 receptor. CBD was first isolated in 1940 (Adams et al., 1940); however, it was not until 1963 that Mechoulam and Shvo elucidated its correct structure (Mechoulam & Shvo, 1963). At the human CB2 receptor, CBD's mechanism of action shows inverse agonism activity (Pertwee et al., 2007). In 1995, Benet and colleagues show that cannabidiol is not only responsible for anti-inflammatory effects, but may also aid in reducing unpleasant side effects from THC, including reduced anxiety (Benet et al., 1995). They found that CBD inhibits cytochrome P450 3A11, which causes THC to change into its more potent metabolite 11-hydroxy-THC (Gallily et al.,

cannabidiol (CBD) are the two most common naturally occurring cannabinoids.

**4. Phytocannabinoids** 

with the CB1 receptor.

2002).

Fig. 4. Chemical structure of delta-9-THC.

Fig. 5. Chemical structure of cannabidiol.

Tetrahydrocannabinol and cannabidiol are the two most discussed phytocannabinoids, but not the only ones known. ElSohly and co-investigators have divided the phytocannabinoids into ten subclasses: 1) Cannabigerol type – propyl side chains and monomethyl ether derivatives 2) Cannabichromene type – analogs present in the C-5 position 3) Cannabidiol type – analogs varying from C-1 to C-5 positions 4) Delta-9-tetrahydrocannabinol type – double bond in the C-9 position; responsible for psychoactive effects 5) Delta-8 tetrahydrocannabinol type – double bond in the C-8 position; thermodynamically more stable than delta-9-THC , however, 20% less active 6) Cannabicyclol type – five atom ring and C-1 bridge 7) Cannabielsoin type – artifacts formed from CBD 8) Cannabinol and Cannabinodiol types – A ring aromatization 9) Cannabitriol type – additional hydroxyl substitution 10) Miscellaneous types – ex: furano ring, carbonyl function, tetrahydroxy substitution (ElSohly, 2010).

Another phytocannabinoid that shows a significant amount of importance is cannabinol (CBN, Figure 6); it is a metabolite of tetrahydrocannabinol. It was the first cannabinoid identified from *Cannabis sativa*. (Wood et al., 1896). Along with THC, cannabinol is also a psychoactive component of *Cannabis* due to its interaction with CB1 receptors. Compared to THC, it acts a weak agonist at both the CB1 and CB2 receptors.

Fig. 6. Chemical structure of cannabinol.

Phytocannabinoids 279

amount of labeled ligand bound to the receptor will be low resulting in high binding affinity of the test compound. A compound showing strong binding affinity for either of the

A functional assay determines whether the compound is acting as an agonist, antagonist, or inverse agonist. As opposed to the binding assay, an in vitro functional assay is not based upon competitive binding, but rather "tracking" the amounts of guanosine triphosphate (GTP). When the membrane is not stimulated, there is a pool of guanosine diphosphate (GDP) associated with it. Upon stimulation, this pool of GDP is converted into GTP. To monitor this response, 35S labeled GTP is added to the assay to bind to the receptors. Therefore, an increase in GTP is directly proportional to stimulation of the receptor by labeled ligand. An agonist compound is indicated by an increase in GTP. Delta-9-THC has a functional Ki of approximately 300nM, which means it is acting as a partial agonist, yet is still responsible for the psychoactive effects associated with *Cannabis* (Figure 8). To detect an antagonist, the compound must be tested in the presence of a known agonist at that specific receptor. The antagonist blocks the ability of the agonist to fully stimulate the receptor, thus

Fig. 8. Functional assay activity of delta-9-THC at Cannabinoid Receptor 1 and Cannabinoid

Cannabinoids that show promising activity in the functional assay, whether acting as an agonist or antagonist, may be tested in vivo using the tetrad assay in mice. In the late 1980s, Little and his colleagues began testing rodents treated with cannabinoids in this tetrad assay. The term tetrad describes a series of four different tests to help evaluate the biological effects of a compound: 1) Locomotor activity 2) Catalepsy 3) Hypothermia and 4) Analgesia. The locomotor activity test allows a researcher to determine if the rodent is acting "lazy." The rodent is placed in a box with perpendicular gridlines, which are beams of light. The test determines the amount of times the beams are broken in an allotted time period, an increase in the number of times broken correlates with a decrease in locomotor activity. To determine if the drug causes cataleptic effects, a rodent is placed on a bar elevated off the ground surface. If

cannabinoid receptors, warrants testing to determine the functional activity.

resulting in a right shift of the agonist EC50.

Receptor 2.

**6.** *In Vivo* **bioassays** 

Extracts that have been isolated from marijuana may be tested to see if they have affinity for each of the CB1- or CB2- type receptors. THC remains the best phytocannabinoid in terms of affinity for the cannabinoid receptors with a binding Ki of 14nM (Figure 7). Most of the compounds isolated from *Cannabis* show a sufficient amount of binding activity at both of the cannabinoid receptors. However, not all compounds isolated show interactions with either CB1 or CB2. For instance, even though cannabidiol is a major constituent of *Cannabis*  and shows pharmacological effects, it has little or no activity for CB1 or CB2 receptors (Mechoulam & Rodriguez, 2007). To determine binding affinity and functional activity, in vitro assays are performed.

Fig. 7. Binding affinity of delta-9-THC at Cannabinoid Receptor 1 and Cannabinoid Receptor 2.

#### **5.** *In vitro* **bioassays**

In order to have success with in vitro assays, cultured cells containing the specific receptors must be developed. At the University of Mississippi HEK293 cells have been transfected with full length human CB1 and human CB2 DNA via electroporation. Once "shocked," the cells open and accept the human CB1 and CB2 cDNA with a linked specific antibiotic resistant plasmid. Since not all cells will receive the DNA, a selection process using the specific antibiotic is added to the cultured cells in order to kill off cells without the cDNA. After an allotted time period for growth, a single cell is selected and clonal colonies are grown in cell culture. The replication of a single cell containing either CB1 or CB2 DNA allows researchers to guarantee the over expression of cannabinoid receptors on the cell membrane. With this, mass subculture followed by "scraping" of the cells leads to the membrane with the receptors. Once the protein concentration is determined this membrane may be used for in vitro assays.

Phytocannabinoids may be tested for their binding affinity toward each of the cannabinoid receptors. A competitive binding assay is done to determine the binding affinity of each compound. The competition is between the chosen phytocannabinoid and a labeled ligand, such as 3H- CP-55, 940. It is known that the labeled ligand will tightly bind to each of the cannabinoid receptors; therefore, if a test compound shows affinity for the receptors, the

Extracts that have been isolated from marijuana may be tested to see if they have affinity for each of the CB1- or CB2- type receptors. THC remains the best phytocannabinoid in terms of affinity for the cannabinoid receptors with a binding Ki of 14nM (Figure 7). Most of the compounds isolated from *Cannabis* show a sufficient amount of binding activity at both of the cannabinoid receptors. However, not all compounds isolated show interactions with either CB1 or CB2. For instance, even though cannabidiol is a major constituent of *Cannabis*  and shows pharmacological effects, it has little or no activity for CB1 or CB2 receptors (Mechoulam & Rodriguez, 2007). To determine binding affinity and functional activity, in

Fig. 7. Binding affinity of delta-9-THC at Cannabinoid Receptor 1 and Cannabinoid Receptor

In order to have success with in vitro assays, cultured cells containing the specific receptors must be developed. At the University of Mississippi HEK293 cells have been transfected with full length human CB1 and human CB2 DNA via electroporation. Once "shocked," the cells open and accept the human CB1 and CB2 cDNA with a linked specific antibiotic resistant plasmid. Since not all cells will receive the DNA, a selection process using the specific antibiotic is added to the cultured cells in order to kill off cells without the cDNA. After an allotted time period for growth, a single cell is selected and clonal colonies are grown in cell culture. The replication of a single cell containing either CB1 or CB2 DNA allows researchers to guarantee the over expression of cannabinoid receptors on the cell membrane. With this, mass subculture followed by "scraping" of the cells leads to the membrane with the receptors. Once the protein concentration is determined this membrane may be used for in vitro assays.

Phytocannabinoids may be tested for their binding affinity toward each of the cannabinoid receptors. A competitive binding assay is done to determine the binding affinity of each compound. The competition is between the chosen phytocannabinoid and a labeled ligand, such as 3H- CP-55, 940. It is known that the labeled ligand will tightly bind to each of the cannabinoid receptors; therefore, if a test compound shows affinity for the receptors, the

vitro assays are performed.

2.

**5.** *In vitro* **bioassays** 

amount of labeled ligand bound to the receptor will be low resulting in high binding affinity of the test compound. A compound showing strong binding affinity for either of the cannabinoid receptors, warrants testing to determine the functional activity.

A functional assay determines whether the compound is acting as an agonist, antagonist, or inverse agonist. As opposed to the binding assay, an in vitro functional assay is not based upon competitive binding, but rather "tracking" the amounts of guanosine triphosphate (GTP). When the membrane is not stimulated, there is a pool of guanosine diphosphate (GDP) associated with it. Upon stimulation, this pool of GDP is converted into GTP. To monitor this response, 35S labeled GTP is added to the assay to bind to the receptors. Therefore, an increase in GTP is directly proportional to stimulation of the receptor by labeled ligand. An agonist compound is indicated by an increase in GTP. Delta-9-THC has a functional Ki of approximately 300nM, which means it is acting as a partial agonist, yet is still responsible for the psychoactive effects associated with *Cannabis* (Figure 8). To detect an antagonist, the compound must be tested in the presence of a known agonist at that specific receptor. The antagonist blocks the ability of the agonist to fully stimulate the receptor, thus resulting in a right shift of the agonist EC50.

Fig. 8. Functional assay activity of delta-9-THC at Cannabinoid Receptor 1 and Cannabinoid Receptor 2.

#### **6.** *In Vivo* **bioassays**

Cannabinoids that show promising activity in the functional assay, whether acting as an agonist or antagonist, may be tested in vivo using the tetrad assay in mice. In the late 1980s, Little and his colleagues began testing rodents treated with cannabinoids in this tetrad assay. The term tetrad describes a series of four different tests to help evaluate the biological effects of a compound: 1) Locomotor activity 2) Catalepsy 3) Hypothermia and 4) Analgesia. The locomotor activity test allows a researcher to determine if the rodent is acting "lazy." The rodent is placed in a box with perpendicular gridlines, which are beams of light. The test determines the amount of times the beams are broken in an allotted time period, an increase in the number of times broken correlates with a decrease in locomotor activity. To determine if the drug causes cataleptic effects, a rodent is placed on a bar elevated off the ground surface. If

Phytocannabinoids 281

mentioned, CBD has shown to reduce anxiety and other unpleasant side effects caused by ingestion of pure THC (Zuardi et al., 1982). The preference of whole *Cannabis* over synthetic formulations of THC is due to the lack of extra side effects associated with the whole *Cannabis*. This opens the door for scientists to study what is actually causing all of the side effects associated with synthetic THC. This also shows that some of the compounds associated with *Cannabis sativa* may be working synergistically to alleviate unwanted effects from THC when used alone (McPartland & Russo, 2001). So, the ultimate goal in cannabinoid drug development would be to mimic the non-psychotropic effects associated with CB1, mimic the beneficial effects associated with CB2, and not deal with the negative

Depression may be described as a mood disorder associated with feeling down, sad, angry, or lost that interferes with everyday life. The most commonly associated drug categories for the treatment of depression include monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), selective-serotonin reuptake inhibitors (SSRIs), and serotoninnorepinephrine reuptake inhibitors (SNRIs). A new field of research involving *Cannabis* may be the link to the treatment of depression. However, studies show conflicting data as to whether *cannabis* is beneficial (Grinsponn & Balkar, 1998) or detrimental for the treatment of depression (Bovassa, 2001). Due to the conflicting results of these studies, Witkin switched the focus to the role of the endocannabinoid system and the treatment of depression from exogenously administered cannabinoids (Witkin et al., 2005). Since 2005, it has been concluded that the endocannabinoid system does play a role in the treatment of depression,

New research has found that a common characteristic of *Cannabis*, mood elevation, may be the link to the treatment of depression. A study published by El-Alfy and co-investigators in 2010 describes the antidepressant effects associated with administration of phytocannabinoids. The objective of this study was to isolate the major cannabinoids from *Cannabis* and evaluate the antidepressant effects using the mouse forced swim test (FST), followed by the tail suspension test (TST). Typically in mice, when cannabinoids are administered they exert hypothermia and catalepsy, which means that a psychoactive state is being achieved. For these depression studies, only low dosages of these phytocannabinoids were administered so that the test subjects did not demonstrate psychoactive effects. The cannabinoids isolated and tested were cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC), cannabidiol (CBD), delta-8-

Fig. 10. Chemical structures of Nabilone (left) and Dronabinol (right).

side effects associated with marijuana or synthetic THC.

**8. Phytocannabinoids and depression** 

but differs from minor depression to major depression.

THC, and delta-9-THC (THC) (Figure 12).

the rodent remains immobile, it is considered cataleptic. Hypothermia, also know as a rectal temperature assay, is simply a measure of the rodents rectal temperature after the drug has been administered. For the last part of the tetrad assay, there are two different methods of testing for analgesic effects. One method is the hot plate (Figure 9) assay. In this assay, a rodent is placed upon a hot plate and the time it takes for the rodent to react, usually a small jump, is recorded. The second method is known as the tail-flick assay. In this assay, the rodent is immobilized and a high temperature beam of light is sporadically placed on the tail. If the rodent feels pain, it will move its tail either left or right (Little, 1988).

Fig. 9. Analgesic portion of tetrad assay: hot plate test.

#### **7. Medicinal uses of marijuana**

According the United Nations, *Cannabis* "is the most widely used illicit substance in the world" (World Drug Report, 2010). There are people who use Cannabis medicinally, and there are others who abuse *Cannabis* in order get "high," or obtain a state of euphoria. Those who use marijuana regularly for medicinal purposes use strict, smaller amounts to control the strength and duration of the "high." However, those who abuse marijuana attempt to smoke or ingest as much as necessary to achieve their own personal state of euphoria. This abuse negatively affects the people who do need *Cannabis* to help with side effects of chemotherapy and AIDS. *Cannabis* is not only used to help those suffering from cancer chemotherapy and AIDS, but it also lowers intraocular eye pressure for those with glaucoma, acts a pain reliever, and more recently has been found to help with symptoms of multiple sclerosis and depression. Therefore, researchers are attempting to formulate a synthetic cannabinoid that resembles the compounds isolated from *Cannabis*, but do not exploit psychotropic properties.

The goal of research in this area is to synthesize a cannabinoid-like compound that warrants a high affinity for either CB1 or CB2 receptors, or both, and can help patients without causing some of the unwanted side effects of marijuana, such as the psychotropic effects associated with CB1. With this said, studies show that *Cannabis* users have fewer psychological side effects than those users administering synthetic THC. There are two synthetic cannabinoid products available on the market in the United States, Nabilone and Dronabinol (Figure 10). Some of these side effects from synthetic cannabinoids include dysphoria, depersonalization, anxiety, and paranoia (Grinsponn & Bakalar, 1997). As previously

the rodent remains immobile, it is considered cataleptic. Hypothermia, also know as a rectal temperature assay, is simply a measure of the rodents rectal temperature after the drug has been administered. For the last part of the tetrad assay, there are two different methods of testing for analgesic effects. One method is the hot plate (Figure 9) assay. In this assay, a rodent is placed upon a hot plate and the time it takes for the rodent to react, usually a small jump, is recorded. The second method is known as the tail-flick assay. In this assay, the rodent is immobilized and a high temperature beam of light is sporadically placed on the tail. If the

According the United Nations, *Cannabis* "is the most widely used illicit substance in the world" (World Drug Report, 2010). There are people who use Cannabis medicinally, and there are others who abuse *Cannabis* in order get "high," or obtain a state of euphoria. Those who use marijuana regularly for medicinal purposes use strict, smaller amounts to control the strength and duration of the "high." However, those who abuse marijuana attempt to smoke or ingest as much as necessary to achieve their own personal state of euphoria. This abuse negatively affects the people who do need *Cannabis* to help with side effects of chemotherapy and AIDS. *Cannabis* is not only used to help those suffering from cancer chemotherapy and AIDS, but it also lowers intraocular eye pressure for those with glaucoma, acts a pain reliever, and more recently has been found to help with symptoms of multiple sclerosis and depression. Therefore, researchers are attempting to formulate a synthetic cannabinoid that resembles the compounds isolated from *Cannabis*, but do not

The goal of research in this area is to synthesize a cannabinoid-like compound that warrants a high affinity for either CB1 or CB2 receptors, or both, and can help patients without causing some of the unwanted side effects of marijuana, such as the psychotropic effects associated with CB1. With this said, studies show that *Cannabis* users have fewer psychological side effects than those users administering synthetic THC. There are two synthetic cannabinoid products available on the market in the United States, Nabilone and Dronabinol (Figure 10). Some of these side effects from synthetic cannabinoids include dysphoria, depersonalization, anxiety, and paranoia (Grinsponn & Bakalar, 1997). As previously

rodent feels pain, it will move its tail either left or right (Little, 1988).

Fig. 9. Analgesic portion of tetrad assay: hot plate test.

**7. Medicinal uses of marijuana** 

exploit psychotropic properties.

mentioned, CBD has shown to reduce anxiety and other unpleasant side effects caused by ingestion of pure THC (Zuardi et al., 1982). The preference of whole *Cannabis* over synthetic formulations of THC is due to the lack of extra side effects associated with the whole *Cannabis*. This opens the door for scientists to study what is actually causing all of the side effects associated with synthetic THC. This also shows that some of the compounds associated with *Cannabis sativa* may be working synergistically to alleviate unwanted effects from THC when used alone (McPartland & Russo, 2001). So, the ultimate goal in cannabinoid drug development would be to mimic the non-psychotropic effects associated with CB1, mimic the beneficial effects associated with CB2, and not deal with the negative side effects associated with marijuana or synthetic THC.

Fig. 10. Chemical structures of Nabilone (left) and Dronabinol (right).

#### **8. Phytocannabinoids and depression**

Depression may be described as a mood disorder associated with feeling down, sad, angry, or lost that interferes with everyday life. The most commonly associated drug categories for the treatment of depression include monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), selective-serotonin reuptake inhibitors (SSRIs), and serotoninnorepinephrine reuptake inhibitors (SNRIs). A new field of research involving *Cannabis* may be the link to the treatment of depression. However, studies show conflicting data as to whether *cannabis* is beneficial (Grinsponn & Balkar, 1998) or detrimental for the treatment of depression (Bovassa, 2001). Due to the conflicting results of these studies, Witkin switched the focus to the role of the endocannabinoid system and the treatment of depression from exogenously administered cannabinoids (Witkin et al., 2005). Since 2005, it has been concluded that the endocannabinoid system does play a role in the treatment of depression, but differs from minor depression to major depression.

New research has found that a common characteristic of *Cannabis*, mood elevation, may be the link to the treatment of depression. A study published by El-Alfy and co-investigators in 2010 describes the antidepressant effects associated with administration of phytocannabinoids. The objective of this study was to isolate the major cannabinoids from *Cannabis* and evaluate the antidepressant effects using the mouse forced swim test (FST), followed by the tail suspension test (TST). Typically in mice, when cannabinoids are administered they exert hypothermia and catalepsy, which means that a psychoactive state is being achieved. For these depression studies, only low dosages of these phytocannabinoids were administered so that the test subjects did not demonstrate psychoactive effects. The cannabinoids isolated and tested were cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC), cannabidiol (CBD), delta-8- THC, and delta-9-THC (THC) (Figure 12).

Phytocannabinoids 283

To assess that hypothermia and catalepsy were not achieved, the tetrad assay was completed after administration of each cannabinoid. Out of the six cannabinoids tested, only delta-8-THC and delta-9-THC showed a U-shaped dose response in the forced swim test. With this, only delta-9-THC showed significant antidepressant-like effects. Administration of the non-psychoactive components revealed that CBC and CBD displayed antidepressantlike effects in the forced swim test. However, a high dose of CBD was used to display these

Fig. 13. Effects of each phytocannabinoid on immobility time in the mouse forced swim test

To further confirm these tests, delta-9-THC and CBC were evaluated in the tail suspension test. Between these two phytocannabinoids, only delta-9-THC continued to exhibit these antidepressant-like effects at low doses. Therefore, the results of this study show that delta-9-THC and other phytocannabinoids administered exogenously do indeed aid with the

Patients suffering from AIDS are now becoming the main target for the therapeutic use of *Cannabis*. Those with AIDS tend to lose their desire to eat regularly throughout the day. When this occurs, the patient becomes weak, agitated, tired, and anorexic; this occurrence in known as Wasting Syndrome. Research shows that at least 90% of patients who smoked marijuana had the desire to eat immediately after use (Haines & Green, 1970). With the use of *Cannabis* as a therapeutic drug to stimulate appetite, the suffering patients may be able to eat on a regular basis throughout the day, thus improving their quality of life. Several studies have shown that the use of marijuana does increase appetite, which also increases energy in daily life routines. In a study conducted by Mattes and colleagues, the appetite stimulating effects of cannabinoids, specifically THC, were examined. A major focus in this study, for a means of clarification from previous research, was the route of administration of THC. The four

antidepressant-like effects.

(El-Alfy et al., 2010).

treatment of depression (El-Alfy et al., 2010).

**9. Phytocannabinoids and appetite stimulation** 

Fig. 11. Chemical structure of the tricyclic antidepressant, Amitriptyline.

Fig. 12. The six phytocannabinoids tested for antidepressant-like effects (El-Alfy et al., 2010).

Fig. 11. Chemical structure of the tricyclic antidepressant, Amitriptyline.

Fig. 12. The six phytocannabinoids tested for antidepressant-like effects (El-Alfy et al., 2010).

To assess that hypothermia and catalepsy were not achieved, the tetrad assay was completed after administration of each cannabinoid. Out of the six cannabinoids tested, only delta-8-THC and delta-9-THC showed a U-shaped dose response in the forced swim test. With this, only delta-9-THC showed significant antidepressant-like effects. Administration of the non-psychoactive components revealed that CBC and CBD displayed antidepressantlike effects in the forced swim test. However, a high dose of CBD was used to display these antidepressant-like effects.

Fig. 13. Effects of each phytocannabinoid on immobility time in the mouse forced swim test (El-Alfy et al., 2010).

To further confirm these tests, delta-9-THC and CBC were evaluated in the tail suspension test. Between these two phytocannabinoids, only delta-9-THC continued to exhibit these antidepressant-like effects at low doses. Therefore, the results of this study show that delta-9-THC and other phytocannabinoids administered exogenously do indeed aid with the treatment of depression (El-Alfy et al., 2010).

#### **9. Phytocannabinoids and appetite stimulation**

Patients suffering from AIDS are now becoming the main target for the therapeutic use of *Cannabis*. Those with AIDS tend to lose their desire to eat regularly throughout the day. When this occurs, the patient becomes weak, agitated, tired, and anorexic; this occurrence in known as Wasting Syndrome. Research shows that at least 90% of patients who smoked marijuana had the desire to eat immediately after use (Haines & Green, 1970). With the use of *Cannabis* as a therapeutic drug to stimulate appetite, the suffering patients may be able to eat on a regular basis throughout the day, thus improving their quality of life. Several studies have shown that the use of marijuana does increase appetite, which also increases energy in daily life routines.

In a study conducted by Mattes and colleagues, the appetite stimulating effects of cannabinoids, specifically THC, were examined. A major focus in this study, for a means of clarification from previous research, was the route of administration of THC. The four

Phytocannabinoids 285

the most widely used illegal drug, along with one of the most widely studied plants. There

It is possible that the cannabinoid system has several other receptors that may explain the mechanism of action of compounds that exhibit cannabinoid-like effects when there is little or no affinity for CB1 or CB2. GPR55 and GPR119, both G-protein coupled receptors, are said to be novel cannabinoid receptors. All cannabinoid receptor antagonists appear to act as inverse agonists instead of neutral antagonists. There are few ligands starting to appear in literature as being neutral antagonists. Interest in this area could is important to help develop pharmacological tools to aid in finding neutral antagonists. These findings may possess unknown therapeutic advantages over receptor antagonists that act as inverse

It is now known that phytocannabinoids interact with the CB1 and CB2 receptors, and that the human body consists of an endocannabinoid system that activates these two receptors. However, what these receptors look like remains a mystery. A general structure-activity relationship has been determined for the cannabinoids, but there is no limitation to synthesizing new compounds that will interact strongly with these receptors. In *vitro* and in *vivo* bioassays play a crucial role in determining the affinities and functions of compounds associated with the CB1 and CB2 receptors. The information determined from these bioassays will continue to help develop novel therapeutic drugs that potentially have

Adams, R., Hunt, M., & Clark, J. (1940). Structure of cannabidiol, a product isolated from the marihuana extract of Minnesota wild hemp. I. *J. Am. Chem. Society.* 62, 196-199. Bornheim, L., Kim, K., Li, J., Perotti, B., & Benet, L. (1995). Effect of cannabidiol pretreatment

Bovassa, G. (2001). Cannabis abuse as a risk factor for depressive symptoms. *American* 

Devane, W., Hanus, L., Breur, A., Pertwee, R., Stevenson, L., Griffin, G., Gibson, D.,

ElSohly, M., Ross, S., Mehmedic, Z., Arafat, R., Yi, B., & Banahan, B. (2000). Potency trends

ElSohly, M. (2010). *Marijuana and the Cannabinoids*. Humana Press. ISBN: 978-1-61737-581-

Grinsponn, L. & Bakalar, J. (1997). *Marihuana, the forbidden medicine*, revised edition. New

Grinsponn, L. & Balkar, J. (1998). The use of cannabis as a mood stabilizer in bipolar disorder:

on the kinetics of Tetrahydrocannabinol metabolites in mouse brain. *Drug Metab.* 

Mandelbaum, A., Etinger, A., & Mechoulam, R. (1992). Isolation and Structure of a Brain Constituent That Binds to the Cannabinoid Receptor. *Science*. 258, 1946-1949. El-Alfy, A., Ivey, K., Robinson, K., Ahmed, S., Radwan, M., Slade, D., Khan, I., ElSohly, M.,

& Ross, S. (2010). Antidepressant-like effect of [Delta]9-tetrahydrocannabinol and other cannabinoids isolated from Cannabis sativa L. *Pharmacology Biochemistry and* 

of Delta-9-THC and other cannabinoids in confiscated marijuana from 1980-1997. *J.* 

anectodotal evidence and the need for clinical research. *Journal Psychoactive Drugs*. 30,

pharmacological effects related to *Cannabis* without the deleterious side effects.

are still many questions to be answered within the *Cannabis* field of study.

agonists (Pertwee, 2005).

**11. References** 

*Dispos.* 23, 825-831.

*Forens. Sci.* 45, 24-30.

1.Totowa, New Jersey.

171-177.

*Journal Psychiatry*. 158: 2033-2037.

*Behavior*, 95, 4, June 2010, 434-442, ISSN 0091-3057.

Gaoni, Y. & Mechoulam, R. (1964). *J. Am. Chem. Soc.* 86, 1646.

Haven, CT: Yale University Press.

Fig. 14. Effects of THC and CBC on immobility time in the mouse tail suspension test (El-Alfy et al., 2010).

different ways in which THC was administered includes oral, inhaled, sublingual, and suppository. There are high levels of variability in determining if THC does actually stimulate appetite. Factors such as environment, age, gender, tolerance, dosage, and social influences play a role in the effect of THC on appetite. During one study, the suppository route of administration resulted in the highest energy intake when compared to oral, sublingual, and inhaled administration of THC (Figure 15).

Fig. 15. Mean data from patients dosed orally and via suppository over a 72 hour time period (Mattes et al., 1994).

There is no single outcome on the effect of THC on appetite stimulation no matter the form of administration. The results vary from having no effect to the possibility of having major food cravings. In some circumstances, not only did the food cravings become increased, but during a meal the food seemed to also have an increased taste of delightfulness. The conclusion of this study indicates that THC as an appetite stimulant produces its highest effects on healthy, adult individuals who use low dosage amounts (Mattes et al., 1994).

#### **10. Future directions**

The growing population is becoming more aware of *Cannabis* as a medicinal plant, and not only a recreational drug. The first *Cannabis* publications date back to the early 1940's in which there was only one publication from 1940-1949. Today, when a search is performed there are over 7,000 journal articles that discuss anything associated with the words *Cannabis*, cannabinoids, or endocannabinoids. Over the last 50 years, marijuana has become

 Fig. 14. Effects of THC and CBC on immobility time in the mouse tail suspension test (El-

different ways in which THC was administered includes oral, inhaled, sublingual, and suppository. There are high levels of variability in determining if THC does actually stimulate appetite. Factors such as environment, age, gender, tolerance, dosage, and social influences play a role in the effect of THC on appetite. During one study, the suppository route of administration resulted in the highest energy intake when compared to oral,

Fig. 15. Mean data from patients dosed orally and via suppository over a 72 hour time

There is no single outcome on the effect of THC on appetite stimulation no matter the form of administration. The results vary from having no effect to the possibility of having major food cravings. In some circumstances, not only did the food cravings become increased, but during a meal the food seemed to also have an increased taste of delightfulness. The conclusion of this study indicates that THC as an appetite stimulant produces its highest effects on healthy, adult individuals who use low dosage amounts (Mattes et al., 1994).

The growing population is becoming more aware of *Cannabis* as a medicinal plant, and not only a recreational drug. The first *Cannabis* publications date back to the early 1940's in which there was only one publication from 1940-1949. Today, when a search is performed there are over 7,000 journal articles that discuss anything associated with the words *Cannabis*, cannabinoids, or endocannabinoids. Over the last 50 years, marijuana has become

sublingual, and inhaled administration of THC (Figure 15).

Alfy et al., 2010).

period (Mattes et al., 1994).

**10. Future directions** 

the most widely used illegal drug, along with one of the most widely studied plants. There are still many questions to be answered within the *Cannabis* field of study.

It is possible that the cannabinoid system has several other receptors that may explain the mechanism of action of compounds that exhibit cannabinoid-like effects when there is little or no affinity for CB1 or CB2. GPR55 and GPR119, both G-protein coupled receptors, are said to be novel cannabinoid receptors. All cannabinoid receptor antagonists appear to act as inverse agonists instead of neutral antagonists. There are few ligands starting to appear in literature as being neutral antagonists. Interest in this area could is important to help develop pharmacological tools to aid in finding neutral antagonists. These findings may possess unknown therapeutic advantages over receptor antagonists that act as inverse agonists (Pertwee, 2005).

It is now known that phytocannabinoids interact with the CB1 and CB2 receptors, and that the human body consists of an endocannabinoid system that activates these two receptors. However, what these receptors look like remains a mystery. A general structure-activity relationship has been determined for the cannabinoids, but there is no limitation to synthesizing new compounds that will interact strongly with these receptors. In *vitro* and in *vivo* bioassays play a crucial role in determining the affinities and functions of compounds associated with the CB1 and CB2 receptors. The information determined from these bioassays will continue to help develop novel therapeutic drugs that potentially have pharmacological effects related to *Cannabis* without the deleterious side effects.

#### **11. References**


**14** 

*Malaysia* 

**Alkaloids and Anthraquinones** 

Nor Hadiani Ismail, Asmah Alias and Che Puteh Osman

The flora of Malaysia is one of the richest flora in the world due to the constantly warm and uniformly humid climate. Malaysia is listed as 12th most diverse nation (Abd Aziz, 2003) in the world and mainly covered by tropical rainsforests. Tropical rainforests cover only 12% of earth's land area; however they constitute about 50% to 90% of world species. At least 25% of all modern drugs originate from rainforests even though only less than 1% of world's tropical rainforest plant species have been evaluated for pharmacological properties (Kong*, et al.*, 2003). The huge diversity of Malaysian flora with about 12 000 species of flowering plants offers huge chemical diversities for numerous biological targets. Malaysian flora is a rich source of numerous class of natural compounds such as alkaloids, anthraquinones and phenolic compounds. Plants are usually investigated based on their ethnobotanical use. The phytochemical study of several well-known plants in folklore medicine such as *Eurycoma longifolia, Labisia pumila*, *Andrographis paniculata*, *Morinda citrifolia* and *Phyllanthus niruri* yielded many bioactive phytochemicals. This review describes our work on the alkaloids of *Fissistigma latifolium* and *Meiogyne virgata* from family Annonaceae and anthraquinones of

Annonaceae, known as *Mempisang* in Malaysia (Kamarudin, 1988) is a family of flowering plants consisiting of trees, shrubs or woody lianas. This family is the largest family in the Magnoliales consisting of more than 130 genera with about 2300 to 2500 species. Plants of the family Annonaceae are well known as source of a variety of alkaloids (Cordell, 1981). Many alkaloids have important physiological effects on human and exhibit marked pharmacological activity which is useful as medicine. For examples, atropine is used widely as an antidote to cholinesterase inhibitors such as physostigmine. Morphine and codeine are narcotic analgesics and antitusive agent while caffeine, which occurs in coffee, tea and cocoa is a central nervous system stimulant. Caffeine is also used as cardiac and respiratory stimulant andbesides as an antidote to barbiturate and morphine poisoning (Parker, 1997). The first report on phytochemical studies of alkaloids from Malaysian Annonaceae plants was on the leaves of *Desmos dasymachalus* which has led to the isolation of new 7-

**1. Introduction** 

*Renellia* and *Morinda* from Rubiaceae family.

**2. The family** *Annonaceae* **as source of alkaloids** 

hydroxyaporphine, dasymachaline (Chan & Toh, 1985).

 **from Malaysian Flora** 

*Universiti Teknologi MARA,* 


### **Alkaloids and Anthraquinones from Malaysian Flora**

Nor Hadiani Ismail, Asmah Alias and Che Puteh Osman *Universiti Teknologi MARA, Malaysia* 

#### **1. Introduction**

286 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Grotenhermen, F. (2002). Effects of Cannabis and the cannabinoids, in Cannabis and

Haines, L. & Green, W. (1970). Marijuana use patterns. *British Journal of Addiction*. 65, 347-362. Lambert, D. (2009). *Cannanbinoids in Nature and Medicine*. Wiley-Verlag Helvetica Chimica

Little, P., Compton, D., Johnson, M., Melvin, L., & Martin, B. (1988). Pharmacology and

Mattes, R., Engelman, K., Shaw, L., & ElSohly, M. (1994). Cannabinoids and appetite

McPartland, J. & Russo, E. (2001). Cannabis and cannabis extracts: greater than the sum of

Mechoulam, R. & Shvo, Y. (1963) Hasish-I. The structure of cannabidiol. *Tetrahedron* 19,

Mechoulam, R. & Gaoni, Y. (1967). Recent advances in the chemistry of hashish. *Fortschr.* 

Mechoulam, R., Parker, L., & Gallily, R. (2002) Cannabidiol: an overview of some

Mechoulam, R. & Peters, M. (2007). Cannabidiol-Recent advances. *Chemistry and Biochemistry*

Pate, D. (1999) Anandamide structure-activity relationships and mechanisms of action on

Pertwee, R. (2005). Pharmacological actions of cannabinoids. *Handbook of Experimental* 

Raymon, L. & Walls, H. (2010). Marijuana and the Cannabinoids. *Pharmacology of Cannabinoids.* 

Shoyama, Y., Yagi, M., Nishioka, I., & Yamauchi, T. (1975). Biosynthesis of cannabinoid

Sugiura, T., Kishimoto, S., Oka, S., & Gokoh, M. (2006). Biochemistry, pharmacology and

Thomas, A., Baillie, G., Phillips, A., Razdan, R., Ross, R., Pertwee, R. (2007) *British Journal of* 

Witkin, J., Tzavara, E., Davis, R., Li, X., & Nomikos, G. (2005). A therapeutic role for

Yamauchi, T., Shoyama, Y., Yagi, M., & Nishioka, I. (1975). Biosynthesis of cannabinoid

Zuardi, A., Morais, S., Guimaraes, F., & Mechoulam, R. (1995). Antipsychotic effect of

*2004 World Drug Report*, United Nations, Office of Drugs and Crime. Oxford University

*2010 World Drug Report*, United Nations, Office of Drugs and Crime. Oxford University

Wood, T., Spivey, W., & Easterfield, T. (1896). *Journal of the Chemical Society.* 69, 539.

intraocular pressure in the normotensive rabbit model. *Doctoral dissertation.* Kuopio

physiology of 2-arachadonoylglycerol, and endogenous cannabinoid receptor

cannabinoid CB1 receptor antagonists in major depressive disorders. *Trends* 

stimulation. *Pharamcology, Biochemistry, and Behavior*. 49, 187-195

pharmacological aspects. *J. Clinical Pharmacology*. 42, 11S-19S.

F. and Russo, E., pages 55-65, Haworth Press, New York.

Acta. ISBN: 978-3-906390-56-7. Zurich, Switzerland.

*Exp. Ther.* 247, 1046-1051.

2073-2078.

4, 8, 1678-1692.

Page 97-143.

their parts? *J. Cann. Therap.* 1, 103-132.

*Chem. Org. Naturst*, 25, 175-213.

University Publications, Kuopio.

acids. *Phytochemistry,* 14, 2189-2192.

ligand. *Prog. Lipid Res.* 45, 405-446.

acids. *Phytochemistry*. 14, 10, 2189-2192.

Press, Oxford, United Kingdom.

Press, Oxford, United Kingdom.

cannabidiol. *J. Clinical Psychiatry.* 56, 485-486.

*Pharmacology*. 168: 1-51.

*Pharmacology.* 150, 613.

*Pharmacol. Sci.* 26, 609-617.

Cannabinoids, In: *Pharmacology, Toxicology, and Therapeutic Potential,* Grotenhermen,

stereoselectivity of structurally novel cannabinoids in mice. *Journal of Pharmacol.* 

The flora of Malaysia is one of the richest flora in the world due to the constantly warm and uniformly humid climate. Malaysia is listed as 12th most diverse nation (Abd Aziz, 2003) in the world and mainly covered by tropical rainsforests. Tropical rainforests cover only 12% of earth's land area; however they constitute about 50% to 90% of world species. At least 25% of all modern drugs originate from rainforests even though only less than 1% of world's tropical rainforest plant species have been evaluated for pharmacological properties (Kong*, et al.*, 2003). The huge diversity of Malaysian flora with about 12 000 species of flowering plants offers huge chemical diversities for numerous biological targets. Malaysian flora is a rich source of numerous class of natural compounds such as alkaloids, anthraquinones and phenolic compounds. Plants are usually investigated based on their ethnobotanical use. The phytochemical study of several well-known plants in folklore medicine such as *Eurycoma longifolia, Labisia pumila*, *Andrographis paniculata*, *Morinda citrifolia* and *Phyllanthus niruri* yielded many bioactive phytochemicals. This review describes our work on the alkaloids of *Fissistigma latifolium* and *Meiogyne virgata* from family Annonaceae and anthraquinones of *Renellia* and *Morinda* from Rubiaceae family.

#### **2. The family** *Annonaceae* **as source of alkaloids**

Annonaceae, known as *Mempisang* in Malaysia (Kamarudin, 1988) is a family of flowering plants consisiting of trees, shrubs or woody lianas. This family is the largest family in the Magnoliales consisting of more than 130 genera with about 2300 to 2500 species. Plants of the family Annonaceae are well known as source of a variety of alkaloids (Cordell, 1981). Many alkaloids have important physiological effects on human and exhibit marked pharmacological activity which is useful as medicine. For examples, atropine is used widely as an antidote to cholinesterase inhibitors such as physostigmine. Morphine and codeine are narcotic analgesics and antitusive agent while caffeine, which occurs in coffee, tea and cocoa is a central nervous system stimulant. Caffeine is also used as cardiac and respiratory stimulant andbesides as an antidote to barbiturate and morphine poisoning (Parker, 1997). The first report on phytochemical studies of alkaloids from Malaysian Annonaceae plants was on the leaves of *Desmos dasymachalus* which has led to the isolation of new 7 hydroxyaporphine, dasymachaline (Chan & Toh, 1985).

Alkaloids and Anthraquinones from Malaysian Flora 289

Previous studies on *F. fulgens* and *F. manubriatum* have resulted in the isolation of aporphine, oxoaporphine and protoberberine alkaloids. Similarly, the studies on alkaloids from *Fissistigma latifolium* led to the isolation of a new aporphine alkaloid, (-)-*N*methylguattescidine **1** (Alias, *et al*., 2010). This alkaloid, together with eight known alkaloids, namely liriodenine **2**, lanuginosine **3**, (-)-asimilobine **4**, dimethyltryptamine **5**, (-) remerine **6**, (-)-anonaine **7**, columbamine **8** and lysicamine **9**, were obtained from the methanol extract of the bark of the plant*.* The new compound was characterized by analysis of spectroscopic methods such as NMR (Nuclear Magnetic Resonance), IR (Infrared) and

(-)-*N*-Methylguattescidine **1** exhibited a molecular formula of C19H17O4N based on the HRESIMS spectrum (positive mode), which showed a pseudomolecular ion at *m/z* 324.3581 [M+H]+ (calcd. 324.3595). The UV spectrum showed an absorption band at 310 nm, suggesting the compound was an aporphine alkaloid with substitutions at position 1 and 2. The IR spectrum indicated the presence of C-H aromatic at 3056, C-O at 1266 and OH at 3409 cm-1, respectively. The absorption of methyl group appeared at 2945 and 2833. The 13C-NMR spectrum showed presence of 19 carbons. The signal at δ 198.0 ppm confirmed the presence of the carbonyl group, while the signal at δ 153.1 ppm is evidence for the oxygenated aromatic carbon. The DEPT spectrum revealed three methylene carbons at δ 26.9 ppm, 41.4 ppm and 96.9 ppm. Signal at δ 96.9 ppm is indicative of a methylenedioxy carbon. This is consistent with two doublets at δ 5.99 ppm (*J* = 1.2 Hz) and δ 6.07 ppm (*J* = 1.2 Hz) in the 1H-NMR spectrum for the protons of methylenedioxy group which is typically located at positions 1 and 2. The characteristic ABD aromatic signals of H-11, H-10 and H-8 of aporphine alkaloid were observed at δ 8.24 ppm (*d, J =* 8.7 Hz), δ 7.13 ppm (*dd, J =* 8.7, 2.7 Hz) and δ 7.39 ppm (*d, J =* 2.7 Hz), respectively. The 1H-NMR spectrum also exhibited an *N*methyl signal at δ 2.34 ppm and another methyl group attached to C-6a gave a singlet at δ 1.52 ppm. The assignment of this methyl group at the 6a position is confirmed through its HMBC correlation with C-6a at δ 62.7 ppm, C-1b at δ 118.3 ppm and C-7 at δ 198.0 ppm. HMQC spectrum shows two cross peaks at δ 26.9 ppm (C-4) axis, represented the correlations of C-4 to H-4 (δ 2.55 ppm) and H-4' (δ 3.00 ppm). At δ 41.4 ppm (C-5) axis, two

Fig. 1. *Fissistigma latifolium* 

GC-MS (Gas-Chromatography-Mass Spectrometry).

The phytochemical investigation of Malaysian Annoaceous plants for their alkoloidal content continue to flourish. Phytochemical survey of the flora of the Peninsula Malaysia and Sabah, with systematic screening for alkaloids resulted in reports on chemical constituents of several plants from Annonaceae illustrating great interest in this field (Teo, *et al*., 1990). Lavault *et al.,* (1981) analysed the alkaloid content of three Annonaceae plants; *Disepalum pulchrum*, *Polyalthia macropoda* and *Polyalthia stenopetala* which led to the isolation of several isoquinoline compounds. Isolation of two new 7,7′-bisdehydroaporphine alkaloids; 7,7′-bisdehydro-*O*-methylisopiline and 7-dehydronornuciferine-7′-dehydro-*O*methylisopiline from bark of *Polyalthia bullata* was reported by Connolly *et al*., (1996). Kam (1999) reviewed the alkaloids derived from Malaysian flora in a book entitled chemical and biological approach of alkaloids.

In Malaysia, eight species of *Fissistigma* are known. They are *F. mobiforme, F. cylindrium, F. fulgens, F. kingii, F. lanuginosum, F. latifolium, F. munubriatum* and *F. kinabaluensis* (Nik Idris *et al*., 1994). Not much has been reported on the phytochemical studies of *Fissistigma* species. The studies on the alkaloids from *Fissistigma fulgens* have led to the isolation of aporphine, oxoaporphine and protoberberine alkaloids. Liriodenine, anonaine, argentinine, discretamine and kikemanine were found from this species (Awang, *et al*., 2000). The phytochemical work on alkaloidal composition of the Malaysian *Fissistigma manubriatum* by Saaid and Awang (2005) yielded two oxoaporphines, lanuginosine and liriodenine together with two tetrahydroprotoberberines, tetrahydropalmatine and discretine. We studied the alkaloids of *Fissistigma latifolium* and reported the isolation of nine alkaloids including a new aporphine compound (Alias *et al*., 2010).

*Meiogyne cylindrocarpa*, *Meiogyne monosperma* and *Meiogyne virgata* are the only three *Meiogyne* species found in Malaysia. Only *Meiogyne virgata* was studied by Tadic *et al.* (1987). The sample collected from Mount Kinabalu, Sabah was reported to contain azafluorene alkaloid, kinabaline, together with liriodenine, cleistopholine and other aporphine alkaloids. Our work on *Meiogyne virgata* from Hulu Terengganu yielded nine alkaloids from aporphine, oxoaporphines and azaanthracene groups.

#### **2.1 Alkaloids of** *Fissistigma latifolium* **and** *Meiogyne virgata*

Since the last three decades, a large number of alkaloidal compounds have been isolated from some Annonaceae species. Tertiary and quaternary isoquinoline and quinoline alkaloids are pharmacologically important compounds commonly found in Annonaceae plants. Continuing our interest on this family of plants, we pursued phytochemical investigation on *Fissistigma latifolium* and *Meiogyne virgata.* 

#### **2.1.1 Alkaloids of** *Fissistigma latifolium*

*Fissistigma latifolium* (Dunal) Merr. from the genus *Fissistigma* is a climbing shrub found in lowland forest of Malaysia, Sumatra, Borneo and Philippines (Verdout, 1976). The genus *Fissistigma* (Annonaceae) consists of about 80 species and is widely distributed in Asia and Australia (Sinclair, 1955). Several species of the genus *Fissistigma* have been used in Southeast Asia as traditional medicines (Perry, 1980). They have been used for muscular atrophy, hepatomegaly and hepatosplenomegaly (Kan, 1979). In Malaysia, the medicinal uses of *Fissistigma* species was briefly mentioned by Burkill as the treatment for childbirth, malaria, wounds, ulcer and rheumatism (Kamarudin, 1988).

The phytochemical investigation of Malaysian Annoaceous plants for their alkoloidal content continue to flourish. Phytochemical survey of the flora of the Peninsula Malaysia and Sabah, with systematic screening for alkaloids resulted in reports on chemical constituents of several plants from Annonaceae illustrating great interest in this field (Teo, *et al*., 1990). Lavault *et al.,* (1981) analysed the alkaloid content of three Annonaceae plants; *Disepalum pulchrum*, *Polyalthia macropoda* and *Polyalthia stenopetala* which led to the isolation of several isoquinoline compounds. Isolation of two new 7,7′-bisdehydroaporphine alkaloids; 7,7′-bisdehydro-*O*-methylisopiline and 7-dehydronornuciferine-7′-dehydro-*O*methylisopiline from bark of *Polyalthia bullata* was reported by Connolly *et al*., (1996). Kam (1999) reviewed the alkaloids derived from Malaysian flora in a book entitled chemical and

In Malaysia, eight species of *Fissistigma* are known. They are *F. mobiforme, F. cylindrium, F. fulgens, F. kingii, F. lanuginosum, F. latifolium, F. munubriatum* and *F. kinabaluensis* (Nik Idris *et al*., 1994). Not much has been reported on the phytochemical studies of *Fissistigma* species. The studies on the alkaloids from *Fissistigma fulgens* have led to the isolation of aporphine, oxoaporphine and protoberberine alkaloids. Liriodenine, anonaine, argentinine, discretamine and kikemanine were found from this species (Awang, *et al*., 2000). The phytochemical work on alkaloidal composition of the Malaysian *Fissistigma manubriatum* by Saaid and Awang (2005) yielded two oxoaporphines, lanuginosine and liriodenine together with two tetrahydroprotoberberines, tetrahydropalmatine and discretine. We studied the alkaloids of *Fissistigma latifolium* and reported the isolation of nine alkaloids including a new

*Meiogyne cylindrocarpa*, *Meiogyne monosperma* and *Meiogyne virgata* are the only three *Meiogyne* species found in Malaysia. Only *Meiogyne virgata* was studied by Tadic *et al.* (1987). The sample collected from Mount Kinabalu, Sabah was reported to contain azafluorene alkaloid, kinabaline, together with liriodenine, cleistopholine and other aporphine alkaloids. Our work on *Meiogyne virgata* from Hulu Terengganu yielded nine alkaloids from

Since the last three decades, a large number of alkaloidal compounds have been isolated from some Annonaceae species. Tertiary and quaternary isoquinoline and quinoline alkaloids are pharmacologically important compounds commonly found in Annonaceae plants. Continuing our interest on this family of plants, we pursued phytochemical

*Fissistigma latifolium* (Dunal) Merr. from the genus *Fissistigma* is a climbing shrub found in lowland forest of Malaysia, Sumatra, Borneo and Philippines (Verdout, 1976). The genus *Fissistigma* (Annonaceae) consists of about 80 species and is widely distributed in Asia and Australia (Sinclair, 1955). Several species of the genus *Fissistigma* have been used in Southeast Asia as traditional medicines (Perry, 1980). They have been used for muscular atrophy, hepatomegaly and hepatosplenomegaly (Kan, 1979). In Malaysia, the medicinal uses of *Fissistigma* species was briefly mentioned by Burkill as the treatment for childbirth,

biological approach of alkaloids.

aporphine compound (Alias *et al*., 2010).

aporphine, oxoaporphines and azaanthracene groups.

**2.1 Alkaloids of** *Fissistigma latifolium* **and** *Meiogyne virgata*

investigation on *Fissistigma latifolium* and *Meiogyne virgata.* 

malaria, wounds, ulcer and rheumatism (Kamarudin, 1988).

**2.1.1 Alkaloids of** *Fissistigma latifolium* 

#### Fig. 1. *Fissistigma latifolium*

Previous studies on *F. fulgens* and *F. manubriatum* have resulted in the isolation of aporphine, oxoaporphine and protoberberine alkaloids. Similarly, the studies on alkaloids from *Fissistigma latifolium* led to the isolation of a new aporphine alkaloid, (-)-*N*methylguattescidine **1** (Alias, *et al*., 2010). This alkaloid, together with eight known alkaloids, namely liriodenine **2**, lanuginosine **3**, (-)-asimilobine **4**, dimethyltryptamine **5**, (-) remerine **6**, (-)-anonaine **7**, columbamine **8** and lysicamine **9**, were obtained from the methanol extract of the bark of the plant*.* The new compound was characterized by analysis of spectroscopic methods such as NMR (Nuclear Magnetic Resonance), IR (Infrared) and GC-MS (Gas-Chromatography-Mass Spectrometry).

(-)-*N*-Methylguattescidine **1** exhibited a molecular formula of C19H17O4N based on the HRESIMS spectrum (positive mode), which showed a pseudomolecular ion at *m/z* 324.3581 [M+H]+ (calcd. 324.3595). The UV spectrum showed an absorption band at 310 nm, suggesting the compound was an aporphine alkaloid with substitutions at position 1 and 2. The IR spectrum indicated the presence of C-H aromatic at 3056, C-O at 1266 and OH at 3409 cm-1, respectively. The absorption of methyl group appeared at 2945 and 2833. The 13C-NMR spectrum showed presence of 19 carbons. The signal at δ 198.0 ppm confirmed the presence of the carbonyl group, while the signal at δ 153.1 ppm is evidence for the oxygenated aromatic carbon. The DEPT spectrum revealed three methylene carbons at δ 26.9 ppm, 41.4 ppm and 96.9 ppm. Signal at δ 96.9 ppm is indicative of a methylenedioxy carbon. This is consistent with two doublets at δ 5.99 ppm (*J* = 1.2 Hz) and δ 6.07 ppm (*J* = 1.2 Hz) in the 1H-NMR spectrum for the protons of methylenedioxy group which is typically located at positions 1 and 2. The characteristic ABD aromatic signals of H-11, H-10 and H-8 of aporphine alkaloid were observed at δ 8.24 ppm (*d, J =* 8.7 Hz), δ 7.13 ppm (*dd, J =* 8.7, 2.7 Hz) and δ 7.39 ppm (*d, J =* 2.7 Hz), respectively. The 1H-NMR spectrum also exhibited an *N*methyl signal at δ 2.34 ppm and another methyl group attached to C-6a gave a singlet at δ 1.52 ppm. The assignment of this methyl group at the 6a position is confirmed through its HMBC correlation with C-6a at δ 62.7 ppm, C-1b at δ 118.3 ppm and C-7 at δ 198.0 ppm. HMQC spectrum shows two cross peaks at δ 26.9 ppm (C-4) axis, represented the correlations of C-4 to H-4 (δ 2.55 ppm) and H-4' (δ 3.00 ppm). At δ 41.4 ppm (C-5) axis, two

Alkaloids and Anthraquinones from Malaysian Flora 291

Asimilobine (**4**), brownish amorphous; MS m/z : 267, C17H17O2N; UV λmax nm EtOH : 274, 308; IR υmax cm-1 : 3390, 1675, 1600, 1225; 1H NMR (CDCl3, 300MHz) δ ppm : 8.30 (1H, d, *J* = 7.8 Hz, H-11), 7.36 – 7.25 (3H, m, H-8, H-9, H-10), 6.73 (1H, s, H-3), 3.92 (1H, m, H-6a), 3.50 (1H, m, H-5'), 3.08 (1H, d, H-4'), 3.04 (1H, d, H-5), 2.99 (1H, m, H7), 2.85 (1H, m, H7), 2.74 (1H, d, H-4), 3.61 (3H, s, OCH3), 2.00 (1H, s, N-H); 13C NMR (CDCl3, 75MHz) δ ppm : 148.6 (C-2), 143.0 (C-1), 135.6 (C-7a), 131.7 (C-11a), 129.4 (C-16), 128.1 (C-3a), 127.7 (C-8), 127.4 (C-10), 127.3 (C-9), 127.2 (C-11), 125.5 (C-1a), 114.6 (C-3), 53.4 (C-

Dimethyltryptamine (**5**), reddish amorphous; MS m/z : 188, C12H16N2; UV λmax nm EtOH : 240, 252; IR υmax cm-1 : 3945, 3055, 2305, 1634, 1422, 1265, 1046, 896; 1H NMR (CDCl3, 300MHz) δ ppm : 7.60 (1H, d, *Jo* = 5.7 Hz, H-7), 7.38 (1H, d, *Jo* = 7.1 Hz, H-4), 7.20 (1H, td, *Jo* = 6.9Hz; *Jm*0.9 Hz, H-5), 7.12 (1H, td, *Jo* = 6.9 Hz; *Jm* = 0.9 Hz, H-6), 3.03 (2H, m, H-8), 2.80 (2H, m, H-9), 8.28 (1H, brs, N-H), 2.49 (6H, s, 2(CH3); 13CNMR (CDCl3, 75MHz) δ ppm : 136.0 (C-7), 127.0 (C-3a), 122.0 (C-6), 121.7 (C-2), 119.2 (C-5), 118.7 (C-4), 59.4 (C-9), 44 (C-3), 22.9 (C-8), 44.9

Remerine (**6**), yellow amorphous; MS m/z : 279, C18H17O2N; UV λmax nm EtOH : 234, 264; IR υmax cm-1 : 1401, 1361, 1053, 942; 1H NMR (CDCl3, 300MHz) δ ppm : 8.09 (1H, d, *Jo* = 7.5 Hz, H-11), 7.34 – 7.24 (3H, m, H-8, H-9, H-10), 6.59 (1H, s, H-3), 4.000 (1H, m, H-6a), 3.4 (1H, m, H-5'), 3.10 (1H, m, H-4'), 3.00 (1H, m, H-5), 2.90 (1H, m, H-7'), 2.80 (2H, m, H-7, H-4), 6.11 (1H, d, *Jm* = 1.2 Hz, CH-O), 5.96 (1H, d, *Jo* = 1.2 Hz, CH-O), 2.62 (3H, s, CH3); 13C NMR (CDCl3, 75MHz) δppm : 146.7 (C-2), 142.8 (C-1), 136.3 (C-7a), 128.1 (C-8), 128.0 (C-1b), 127.6 (C-9), 127.0 (C-10), 127.0 (C-11), 125.4 (C-3a), 126.5 (C-1a), 126.0 (C-11a), 125.4 (C-3a), 62.4 (C-6a), 53.3 (C-6a), 43 (C-5), 36.9 (C-

Anonaine (**7**)**,** yellow amorphous; MS m/z : 265, C17H13O2N; UV λmax nm EtOH : 234, 272, 315; IR υmax cm-1 : 1040, 945; 1H NMR (CDCl3, 300MHz) δ ppm : 8.09 (1H, d, *Jo* = 7.5 Hz, H=11), 7.36 – 7.19 (3H, m, H-8, H-9, H-10), 6.6 (1H, s, H-3), 4.04 (1H, dd, H-6a), 3.48 (1H, m, H-5'), 3.1 (1H, m, H-4'), 3.07 (1H, m, H-5), 3.02 (1H, m, H-7'), 6.12 (1H, d, *Jm* = 1.5, CH – O), 5.97 (1H, d, *Jm* = 1.5, CH – O); 13C NMR (CDCl3, 75MHz) δ ppm : 147.0 (C-2), 143.0 (C-1), 135.4 (C-7a), 131.4 (C-11a), 129.0 (C-1b), 128.0 (C-3a), 127.8 (C-8), 127.7 (C-9), 127.0 (C-10), 126.1 (C-11), 116.3 (C-1a), 53.6 (C-6a), 43.6 (C-5), 37.4 (C-7), 29.6 (C-4), 100.6

7), 28 (C-4), 100.7 (O – CH2 – O), 39.0 (CH3).

6a), 42.8 (C-5), 36.7 (C-7), 28.2 (C-4), 60.4 (OCH3).

**(4)**

HO

H3CO

NH

O

**(5)**

<sup>N</sup> <sup>N</sup>

N

CH3

NH

**(6)**

O

O

O

O

**(7)**

(2CH3).

(O –CH2– O).

cross peaks showed the correlations between C-5 and H-5 (δ 2.99 ppm) and H-5' (δ 3.01 ppm). The quaternary carbon signals were assigned based on HMBC experiment. C-1a at δ 108.9 ppm, C-7a at δ 126.0 ppm and C-9 at δ 153.1 ppm were assigned based on their correlations with H-11 at δ 8.24 ppm, while C-1b at δ 118.3 ppm and C-2 at δ 143.2 ppm showed correlations with H-3 at δ 6.54 ppm. (-)-*N*-methylguattescidine, is a rare 6a-methylated-7-oxoaporphine alkaloid, having only been previously reported by Reynald *et al*. in 1982. Presented below are structures and spectroscopic data of the isolated compounds.

(-)-N-Methylguattescidine (**1**). yellow amorphous solid; [α] <sup>30</sup>*<sup>D</sup>* : -20º (c = 0.1 mg mL-1, CHCl3); MS *m/z*: 324.1242, C19H17O4N; UV λmax nm EtOH: 235, 310; IR υmax cm-1: 3409, 1710, 1266; 1H NMR (CDCl3, 300 MHz) δ ppm : 8.24 (1H, d, *J* = 8.7 Hz, H-11), 7.39 (1H, d, *J* = 2.7 Hz, H-8), 7.13 (1H, dd, *Jo* = 8.7 Hz; *Jm* = 2.7 Hz, H-10), 6.54 (1H, s, H-3), 6.07 (1H, d, *J* = 1.2 Hz, H-2), 5.99 (1H, d, *J* = 1.2 Hz, H-1), 3.52 (1H, m, H-11a), 3.01 (1H, m, H-5), 3.00 (1H, m, H-4), 2.99 (1H, m, H-5'), 2.55 (1H, m, H-4'); 13C NMR (CDCl3, 75 MHz) δppm : 153.1 (C-9), 143.2 (C-2), 138.8 (C-1), 126.0 (C-7a), 125.3 (C-3a), 123.1 (C-11a), 122.7 (C-11), 122.2 (C-10), 118.3 (C-1b), 110.3 (C-8), 108.9 (C-1a), 103.9 (C-3), 96.9 (O-CH2-O), 62.7 (C-6a), 41.4 (C-5), 34.1 (N-CH3), 26.9 (C-4), 25.0 (CH3).

Liriodenine (**2**), yellow needles; MS m/z : 275, C17H9O3N; UV λmax nm EtOH : 215, 246, 268, 395, 412; IR υmax cm-1 : 3054, 1726, 1421, 1265; 1H NMR (CDCl3, 300 MHz) δ ppm : 8.9 (1H, d, *J* = 5.1 Hz, H-5), 8.66 (1H, dd, *Jo* = 7.2 Hz; *Jm* = 1.2 Hz, H-11), 8.59 (1H, dd, *Jo* = 7.8 Hz; *Jm* = 1.2Hz, H-8), 7.79 (1H, d, *J* = 5.1 Hz, H-4), 7.76 (1H, td, *Jo* = 7.8 Hz; 7.2 Hz; *Jm* = 1.5 Hz, H-10), 7.59 (1H, td, *Jo* = 7.8 Hz;7.2; *Jm* = 1.2 Hz, H-9), 7.16 (1H, s, H-3), 6.40 (2H, s, O-CH2-O); 13C NMR (CDCl3, 75 MHz) δppm : 151.7 (C-2), 147.9 (C-1), 146 (C-6a), 145.4 (C-3a), 144.9 (C-5), 135.7 (C-1a), 133.9 (C-10), 132.9 (C-7a), 131.3 (C-11a), 128.8(C-8), 128.6 (C-9), 127.4 (C-11), 124.2 (C-4), 108.2 (C-1b), 103.3 (C-3), 102.4 (O-CH2-O), 182.4 (C-7).

Lanuginosine (**3**), yellow needles; MS m/z : 305, C18H11O4N; UV λmax nm EtOH : 246, 271, 315, 258, 283, 334; IR υmax cm-1 : 3055, 2987, 2306, 1712, 1635, 1363, 1265, 1046, 896; 1H NMR (CDCl3, 300MHz) δ ppm : 8.85 (1H, d, *J* = 5.4 Hz, H-5), 8.58 (1H, d, *Jo* = 9.0 Hz, H-11), 8.04 (1H, d, *J* = 3 Hz, H-8), 7.79 (1H, d, *J* = 5.4 Hz, H-4), 7.32 (1H, dd, *Jo* = 9.0 Hz; *Jm* = 3 Hz, H-10), 7.17 (1H, s, H-3), 6.47 (2H, s, O – CH2 – O); 13C NMR (CDCl3, 75MHz) δ ppm : 158.0 (C-9), 151.0 (C-2), 146.0 (C-1), 144.9 (C-5), 144.0 (C-6a), 136.0 (C-3a), 133.0 (C-7a), 131.9 (C-1b), 129.1 (C-11), 126.2 (C-11a), 124.3 (C-4), 122.6 (C-10), 110.2 (C-8), 109.0 (C-1a), 102.3 (C-3), 55.8 (OCH3), 102.5 (O – CH2 – O), 182.0 (C-7).

cross peaks showed the correlations between C-5 and H-5 (δ 2.99 ppm) and H-5' (δ 3.01 ppm). The quaternary carbon signals were assigned based on HMBC experiment. C-1a at δ 108.9 ppm, C-7a at δ 126.0 ppm and C-9 at δ 153.1 ppm were assigned based on their correlations with H-11 at δ 8.24 ppm, while C-1b at δ 118.3 ppm and C-2 at δ 143.2 ppm showed correlations with H-3 at δ 6.54 ppm. (-)-*N*-methylguattescidine, is a rare 6a-methylated-7-oxoaporphine alkaloid, having only been previously reported by Reynald *et al*. in 1982. Presented

> (-)-N-Methylguattescidine (**1**). yellow amorphous solid; [α] <sup>30</sup>*<sup>D</sup>* : -20º (c = 0.1 mg mL-1, CHCl3); MS *m/z*: 324.1242, C19H17O4N; UV λmax nm EtOH: 235, 310; IR υmax cm-1: 3409, 1710, 1266; 1H NMR (CDCl3, 300 MHz) δ ppm : 8.24 (1H, d, *J* = 8.7 Hz, H-11), 7.39 (1H, d, *J* = 2.7 Hz, H-8), 7.13 (1H, dd, *Jo* = 8.7 Hz; *Jm* = 2.7 Hz, H-10), 6.54 (1H, s, H-3), 6.07 (1H, d, *J* = 1.2 Hz, H-2), 5.99 (1H, d, *J* = 1.2 Hz, H-1), 3.52 (1H, m, H-11a), 3.01 (1H, m, H-5), 3.00 (1H, m, H-4), 2.99 (1H, m, H-5'), 2.55 (1H, m, H-4'); 13C NMR (CDCl3, 75 MHz) δppm : 153.1 (C-9), 143.2 (C-2), 138.8 (C-1), 126.0 (C-7a), 125.3 (C-3a), 123.1 (C-11a), 122.7 (C-11), 122.2 (C-10), 118.3 (C-1b), 110.3 (C-8), 108.9 (C-1a), 103.9 (C-3), 96.9 (O-CH2-O), 62.7 (C-6a), 41.4 (C-5), 34.1 (N-CH3), 26.9 (C-

> Liriodenine (**2**), yellow needles; MS m/z : 275, C17H9O3N; UV λmax nm EtOH : 215, 246, 268, 395, 412; IR υmax cm-1 : 3054, 1726, 1421, 1265; 1H NMR (CDCl3, 300 MHz) δ ppm : 8.9 (1H, d, *J* = 5.1 Hz, H-5), 8.66 (1H, dd, *Jo* = 7.2 Hz; *Jm* = 1.2 Hz, H-11), 8.59 (1H, dd, *Jo* = 7.8 Hz; *Jm* = 1.2Hz, H-8), 7.79 (1H, d, *J* = 5.1 Hz, H-4), 7.76 (1H, td, *Jo* = 7.8 Hz; 7.2 Hz; *Jm* = 1.5 Hz, H-10), 7.59 (1H, td, *Jo* = 7.8 Hz;7.2; *Jm* = 1.2 Hz, H-9), 7.16 (1H, s, H-3), 6.40 (2H, s, O-CH2-O); 13C NMR (CDCl3, 75 MHz) δppm : 151.7 (C-2), 147.9 (C-1), 146 (C-6a), 145.4 (C-3a), 144.9 (C-5), 135.7 (C-1a), 133.9 (C-10), 132.9 (C-7a), 131.3 (C-11a), 128.8(C-8), 128.6 (C-9), 127.4 (C-11), 124.2 (C-4), 108.2 (C-1b), 103.3 (C-

> Lanuginosine (**3**), yellow needles; MS m/z : 305, C18H11O4N; UV λmax nm EtOH : 246, 271, 315, 258, 283, 334; IR υmax cm-1 : 3055, 2987, 2306, 1712, 1635, 1363, 1265, 1046, 896; 1H NMR (CDCl3, 300MHz) δ ppm : 8.85 (1H, d, *J* = 5.4 Hz, H-5), 8.58 (1H, d, *Jo* = 9.0 Hz, H-11), 8.04 (1H, d, *J* = 3 Hz, H-8), 7.79 (1H, d, *J* = 5.4 Hz, H-4), 7.32 (1H, dd, *Jo* = 9.0 Hz; *Jm* = 3 Hz, H-10), 7.17 (1H, s, H-3), 6.47 (2H, s, O – CH2 – O); 13C NMR (CDCl3, 75MHz) δ ppm : 158.0 (C-9), 151.0 (C-2), 146.0 (C-1), 144.9 (C-5), 144.0 (C-6a), 136.0 (C-3a), 133.0 (C-7a), 131.9 (C-1b), 129.1 (C-11), 126.2 (C-11a), 124.3 (C-4), 122.6 (C-10), 110.2 (C-8), 109.0 (C-1a), 102.3 (C-3), 55.8 (OCH3), 102.5 (O – CH2 – O),

below are structures and spectroscopic data of the isolated compounds.

4), 25.0 (CH3).

182.0 (C-7).

3), 102.4 (O-CH2-O), 182.4 (C-7).

**(1)**

OH

O

O

O

O

O

O

N

O

N

O

N

O

**(2)** 

**(3)**

OCH3

Asimilobine (**4**), brownish amorphous; MS m/z : 267, C17H17O2N; UV λmax nm EtOH : 274, 308; IR υmax cm-1 : 3390, 1675, 1600, 1225; 1H NMR (CDCl3, 300MHz) δ ppm : 8.30 (1H, d, *J* = 7.8 Hz, H-11), 7.36 – 7.25 (3H, m, H-8, H-9, H-10), 6.73 (1H, s, H-3), 3.92 (1H, m, H-6a), 3.50 (1H, m, H-5'), 3.08 (1H, d, H-4'), 3.04 (1H, d, H-5), 2.99 (1H, m, H7), 2.85 (1H, m, H7), 2.74 (1H, d, H-4), 3.61 (3H, s, OCH3), 2.00 (1H, s, N-H); 13C NMR (CDCl3, 75MHz) δ ppm : 148.6 (C-2), 143.0 (C-1), 135.6 (C-7a), 131.7 (C-11a), 129.4 (C-16), 128.1 (C-3a), 127.7 (C-8), 127.4 (C-10), 127.3 (C-9), 127.2 (C-11), 125.5 (C-1a), 114.6 (C-3), 53.4 (C-6a), 42.8 (C-5), 36.7 (C-7), 28.2 (C-4), 60.4 (OCH3).

Dimethyltryptamine (**5**), reddish amorphous; MS m/z : 188, C12H16N2; UV λmax nm EtOH : 240, 252; IR υmax cm-1 : 3945, 3055, 2305, 1634, 1422, 1265, 1046, 896; 1H NMR (CDCl3, 300MHz) δ ppm : 7.60 (1H, d, *Jo* = 5.7 Hz, H-7), 7.38 (1H, d, *Jo* = 7.1 Hz, H-4), 7.20 (1H, td, *Jo* = 6.9Hz; *Jm*0.9 Hz, H-5), 7.12 (1H, td, *Jo* = 6.9 Hz; *Jm* = 0.9 Hz, H-6), 3.03 (2H, m, H-8), 2.80 (2H, m, H-9), 8.28 (1H, brs, N-H), 2.49 (6H, s, 2(CH3); 13CNMR (CDCl3, 75MHz) δ ppm : 136.0 (C-7), 127.0 (C-3a), 122.0 (C-6), 121.7 (C-2), 119.2 (C-5), 118.7 (C-4), 59.4 (C-9), 44 (C-3), 22.9 (C-8), 44.9 (2CH3).

Remerine (**6**), yellow amorphous; MS m/z : 279, C18H17O2N; UV λmax nm EtOH : 234, 264; IR υmax cm-1 : 1401, 1361, 1053, 942; 1H NMR (CDCl3, 300MHz) δ ppm : 8.09 (1H, d, *Jo* = 7.5 Hz, H-11), 7.34 – 7.24 (3H, m, H-8, H-9, H-10), 6.59 (1H, s, H-3), 4.000 (1H, m, H-6a), 3.4 (1H, m, H-5'), 3.10 (1H, m, H-4'), 3.00 (1H, m, H-5), 2.90 (1H, m, H-7'), 2.80 (2H, m, H-7, H-4), 6.11 (1H, d, *Jm* = 1.2 Hz, CH-O), 5.96 (1H, d, *Jo* = 1.2 Hz, CH-O), 2.62 (3H, s, CH3); 13C NMR (CDCl3, 75MHz) δppm : 146.7 (C-2), 142.8 (C-1), 136.3 (C-7a), 128.1 (C-8), 128.0 (C-1b), 127.6 (C-9), 127.0 (C-10), 127.0 (C-11), 125.4 (C-3a), 126.5 (C-1a), 126.0 (C-11a), 125.4 (C-3a), 62.4 (C-6a), 53.3 (C-6a), 43 (C-5), 36.9 (C-7), 28 (C-4), 100.7 (O – CH2 – O), 39.0 (CH3).

Anonaine (**7**)**,** yellow amorphous; MS m/z : 265, C17H13O2N; UV λmax nm EtOH : 234, 272, 315; IR υmax cm-1 : 1040, 945; 1H NMR (CDCl3, 300MHz) δ ppm : 8.09 (1H, d, *Jo* = 7.5 Hz, H=11), 7.36 – 7.19 (3H, m, H-8, H-9, H-10), 6.6 (1H, s, H-3), 4.04 (1H, dd, H-6a), 3.48 (1H, m, H-5'), 3.1 (1H, m, H-4'), 3.07 (1H, m, H-5), 3.02 (1H, m, H-7'), 6.12 (1H, d, *Jm* = 1.5, CH – O), 5.97 (1H, d, *Jm* = 1.5, CH – O); 13C NMR (CDCl3, 75MHz) δ ppm : 147.0 (C-2), 143.0 (C-1), 135.4 (C-7a), 131.4 (C-11a), 129.0 (C-1b), 128.0 (C-3a), 127.8 (C-8), 127.7 (C-9), 127.0 (C-10), 126.1 (C-11), 116.3 (C-1a), 53.6 (C-6a), 43.6 (C-5), 37.4 (C-7), 29.6 (C-4), 100.6 (O –CH2– O).

Alkaloids and Anthraquinones from Malaysian Flora 293

Most of oxoaporphine and aporphine alkaloids showed IR spectra typified by the 7-oxo group with absorption band in the 1635-1660 cm-1 region. The UV spectra data for these type of compounds are quite characteristic for the skeletal type. There is indication that they may also be diagnostic for a particular oxygenation pattern. For example, 1, 2-methylenedioxy

υmax cm-1 : 1040, 945

Nornuciferine (**10**), Colourless crystalline solid; MS m/z : 281 (M+); UV λmax nm EtOH : 234, 272, 315; IR

1H NMR (CDCl3, 300 MHz) δ ppm : 8.39 (1H, *d,*J= 7.8 Hz, H-11), 7.33-7.20 (3H, *m,* H-8, H-9, H-10), 6.65 (1H, *s*, H-3), 3.98 (1H, *dd*, *J*= 13.4; 5.2 Hz, H-6a), 3.41 (1H, *dd*, *J*= 12.3; 6.3 Hz, H-5'), 3.90 (3H, *s*, OMe-2), 3.68 (3H, *s*, OMe-1), 3.08 (1H, *dd*, *J*=13.2 Hz, H-4), 3.04 (1H, *td*, *J*= 12.3; 5.1 Hz, H-5), 2.85 (1H, *dd*, *J*= 13.4 ; 5.2 Hz, H-7'), 2.68 (1H, *dd*, *J*= 13.2; 6.0 Hz, H-4'), 2.64 (1H, *t*, *J*= 13.4 Hz, H-7).; 13C NMR (CDCl3, 125 MHz) δ ppm : 152.3 (C-2), 145.2 (C-1), 135.0 (C-7a), 132.1 (C-1b), 132.1 (C-11a), 131.2 (C-3a), 128.4 (C-8), 127.8 (C-10), 127.4 (C-9), 127.1 (C-11), 126.6 (C-1a), 111.8 (C-3), 60.3 (OMe-1), 55.9 (OMe-2), 53.6 (C-6a), 43.0 (C-5), 37.2 (C-7), 29.7 (C-4).

Norushinsunine (**11**), Colourless crystalline solid; MS m/z : 281; UV λmax nm EtOH : 217, 247, 252, 259, 273, 319; IR υmax cm-1 : 3488, 3355, 1574, 1215; 1H NMR (CDCl3, 300MHz) δ ppm : 8.16 (1H, dd, *J* = 7.2;1.2 Hz, H=11), 7.45 (1H, td, *J* = 8.7;1.2 Hz, H-10 ), 7.40 (1H, dd, *J* = 8.1;0.9 Hz, H-8), 7.34 (1H, td, *J* = 7.2;1.2 Hz, H-9), 6.59 (1H, s, H-3), 6.11 (1H, d, *J* =1.5 Hz, O – CH2 – O), 5.95 (1H, d, *J* = 1.2 Hz, O – CH2 – O), 4.61 (1H, d, *J* = 3.0 Hz, H-7 ), 4.06 (1H, d, *J* = 3.3 Hz, CH – O). 3.37 (1H, ddd, *J* = 5.0;3.9;1.2 Hz, H-4', 2.68 (1H,dd, J=16.2;3.9 Hz, H-4); 13C NMR (CDCl3, 75MHz) δ ppm : 147.1 (C-1), 142.6 (C-2), 135.6 (C-1a), 130.3 (C-7a), 129.4 (C-9), 129.1 (C-3a), 123.6 (C-1b), 115.6 (C-11a), 108.4 (O – CH2 – O), 71.0 (C-7), 57.2 (C-6a), 43.1 (C-5), 29.2 (C-4).

Cleistopholine (**12**), yellow glassy solid; MS m/z : 281 (M+); UV λmax nm EtOH : 234, 272, 315; IR υmax cm-1 : 1040, 945; 1H NMR (CDCl3, 400 MHz) δ ppm : 8.95 (1H, *d,J*= 4.8 Hz, H-2), 8.31 (1H, *dd,J*= 8.5;2.2 Hz, H-5), 8.21 (1H, *dd,J*= 8.5;2.2 Hz, H-8), 7.79 (1H, m*,* H-6), 7.79 (1H, m, H-7), 2.89 (1H, s, CH3); 13C NMR (CDCl3, 100.6 MHz) δ ppm : 184.7 (C-9), 181.9 (C-10), 153.4 (C-2), 151.6 (C-4), 150.1 (C-9a), 134.6 (C-7), 134.2 (C-6), 132.6 (C-10a), 131.2 (C-3), 129.1 (C-4a), 127.4 (C-5),

127.2 (C-8), 22.8 (CH3).

(**10)** 

H3CO

H3CO

O

NH

(**11)** 

O R1

N

R2

(**12)** 

O

N

O CH3

Table 2.

Table 1.

#### **2.1.1 Alkaloids of** *Meiogyne virgata*

*Meiogyne virgata* is a rainforest tree grows in Peninsular Malaysia, Borneo, Java and Sumatera. The genus *Meiogyne* (Annonaceae) consists of about 24 species and widely distributed in Indo-china, Thailand, Peninsular Malaysia, Sumatra, Java, Borneo and the Philippines. There is no formal report on the traditional uses of *Meiogyne virgata* in Malaysia. However, being an alkaloid rich species, it could be useful medicinally.

We have conducted phytochemical work on *Meiogyne virgata*. Six of the aporphine alkaloids in *Fissistigma latifolium* were also found in *Meiogyne virgata* collected from the Peninsular Malaysia. Isolation and purification of alkaloids from the bark of *Meiogyne virgata* afforded nine alkaloids; four oxoaporphines, liriodenine **2**, lanuginosine **3**, asimilobine **4** and lysicamine **9**; four aporphines, anonaine **7**, remerine **6**, nornuciferine **10** and norushinsunine **11**; and one azaanthracene alkaloid, cleistopholine **12**.

Most of the compounds are yellowish or colorless hygroscopic liquid at room temperature while impure samples will appear brownish. They have low solubility in water but dissolve well in methanol, chloroform, acetone, dichloromethane and other common organic solvent. They are also soluble in dilute acid as the protonated derivative. The melting point of these thype of compounds in range 100-300 oC.

58.0 (OCH3), 57.0 (OCH3).

Columbamine (**8**), red amorphous solid; MS m/z : 338, C20H20O4N; UV λmax nm EtOH : 206, 225, 265, 345; IR υmax cm-1 : 3390, 1600; 1H NMR (CDCl3, 300MHz) δ ppm : 9.00 (1H, s, H-8), 8.08 (1H, s, H-13), 7.65 (1H, d, *Jo* = 9.0 Hz, H-11), 7.61 (1H, d, *Jo* = 8.7 Hz, H-12), 7.27 (1H, s, H-1), 6.79 (1H, s, H-4), 4.68 (2H, t, *Jo* = 6.6 Hz; 6.3 Hz, H-6), 3.18 (2H, t, *Jo* = 6.0 Hz, H-5), 4.02 (3H, OCH3), 3.99 (3H, OCH3), 3.92 (3H, OCH3). ; 13C NMR (CDCl3, 75MHz) δ ppm : 163.0 (C-10), 151.0 (C-3), 150.0 (C-4a), 149.0 (C-2), 142.0 (C-9), 140.0 (C-8), 139.0 (C-11), 132.0 (C-14), 129.0 (C-12a), 126.0 (C-1a), 123.3 (C-12), 120.0 (C-13), 119.0 (C-8a), 111.0 (C-4), 108.0 (C-1), 56.0 (C-6), 27.0 (C-5), 60.0 (OCH3),

Lysicamine (**9**), yellow amorphous; MS m/z : 291, C18H13O3N; UV λmax nm EtOH : 214, 250, 255, 261, 319; IR υmax cm-1 : 1675, 1600, 1225; 1H NMR (CDCl3, 300MHz) δ ppm : 9.10 (1H, d, *Jo* = 5.1 Hz, H-11), 8.70 (1H, d, *Jo* = 6.9 Hz, H-5) 8.48 (1H, dd, *Jo* = 7.5 Hz; *Jm* = 1.5 Hz, H-8), 7.76 (1H, td, *Jo* = 7.2 Hz; *Jm* = 1.5 Hz, H-10), 7.7 (1H, d, *Jo* = 6.9 Hz, H-4), 7.55 (1H, td, *Jo* = 7.2 Hz; *Jm* = 1.2 Hz, H-9), 7.24 (1H, s, H-3), 4.05 (3H, s, OCH3), 3.97 (3H, s, OCH3); 13C NMR (CDCl3, 75MHz) δppm : 156.7 (C-6a), 152.0 (C-2), 145.2 (C-1), 139.0 (C-5), 135.3 (C-3a), 134.7 (C-11a), 132.0 (C-7a), 130.9 (C-10), 125.7 (C-9), 122.0 (C-1b), 119.6 (C-1a),

108.7 (C-3), 65.1 (OCH3), 56.8 (OCH3), 182.5 (C=O).

*Meiogyne virgata* is a rainforest tree grows in Peninsular Malaysia, Borneo, Java and Sumatera. The genus *Meiogyne* (Annonaceae) consists of about 24 species and widely distributed in Indo-china, Thailand, Peninsular Malaysia, Sumatra, Java, Borneo and the Philippines. There is no formal report on the traditional uses of *Meiogyne virgata* in Malaysia.

We have conducted phytochemical work on *Meiogyne virgata*. Six of the aporphine alkaloids in *Fissistigma latifolium* were also found in *Meiogyne virgata* collected from the Peninsular Malaysia. Isolation and purification of alkaloids from the bark of *Meiogyne virgata* afforded nine alkaloids; four oxoaporphines, liriodenine **2**, lanuginosine **3**, asimilobine **4** and lysicamine **9**; four aporphines, anonaine **7**, remerine **6**, nornuciferine **10** and norushinsunine

Most of the compounds are yellowish or colorless hygroscopic liquid at room temperature while impure samples will appear brownish. They have low solubility in water but dissolve well in methanol, chloroform, acetone, dichloromethane and other common organic solvent. They are also soluble in dilute acid as the protonated derivative. The melting point of these

However, being an alkaloid rich species, it could be useful medicinally.

**11**; and one azaanthracene alkaloid, cleistopholine **12**.

thype of compounds in range 100-300 oC.

**(8)**

OCH3

OCH3

N

O

H3CO

HO

**(9)**

**2.1.1 Alkaloids of** *Meiogyne virgata* 

Table 1.

H3CO

H3CO

Most of oxoaporphine and aporphine alkaloids showed IR spectra typified by the 7-oxo group with absorption band in the 1635-1660 cm-1 region. The UV spectra data for these type of compounds are quite characteristic for the skeletal type. There is indication that they may also be diagnostic for a particular oxygenation pattern. For example, 1, 2-methylenedioxy

Nornuciferine (**10**), Colourless crystalline solid; MS m/z : 281 (M+); UV λmax nm EtOH : 234, 272, 315; IR υmax cm-1 : 1040, 945

1H NMR (CDCl3, 300 MHz) δ ppm : 8.39 (1H, *d,*J= 7.8 Hz, H-11), 7.33-7.20 (3H, *m,* H-8, H-9, H-10), 6.65 (1H, *s*, H-3), 3.98 (1H, *dd*, *J*= 13.4; 5.2 Hz, H-6a), 3.41 (1H, *dd*, *J*= 12.3; 6.3 Hz, H-5'), 3.90 (3H, *s*, OMe-2), 3.68 (3H, *s*, OMe-1), 3.08 (1H, *dd*, *J*=13.2 Hz, H-4), 3.04 (1H, *td*, *J*= 12.3; 5.1 Hz, H-5), 2.85 (1H, *dd*, *J*= 13.4 ; 5.2 Hz, H-7'), 2.68 (1H, *dd*, *J*= 13.2; 6.0 Hz, H-4'), 2.64 (1H, *t*, *J*= 13.4 Hz, H-7).; 13C NMR (CDCl3, 125 MHz) δ ppm : 152.3 (C-2), 145.2 (C-1), 135.0 (C-7a), 132.1 (C-1b), 132.1 (C-11a), 131.2 (C-3a), 128.4 (C-8), 127.8 (C-10), 127.4 (C-9), 127.1 (C-11), 126.6 (C-1a), 111.8 (C-3), 60.3 (OMe-1), 55.9 (OMe-2), 53.6 (C-6a), 43.0 (C-5), 37.2 (C-7), 29.7 (C-4).

Norushinsunine (**11**), Colourless crystalline solid; MS m/z : 281; UV λmax nm EtOH : 217, 247, 252, 259, 273, 319; IR υmax cm-1 : 3488, 3355, 1574, 1215; 1H NMR (CDCl3, 300MHz) δ ppm : 8.16 (1H, dd, *J* = 7.2;1.2 Hz, H=11), 7.45 (1H, td, *J* = 8.7;1.2 Hz, H-10 ), 7.40 (1H, dd, *J* = 8.1;0.9 Hz, H-8), 7.34 (1H, td, *J* = 7.2;1.2 Hz, H-9), 6.59 (1H, s, H-3), 6.11 (1H, d, *J* =1.5 Hz, O – CH2 – O), 5.95 (1H, d, *J* = 1.2 Hz, O – CH2 – O), 4.61 (1H, d, *J* = 3.0 Hz, H-7 ), 4.06 (1H, d, *J* = 3.3 Hz, CH – O). 3.37 (1H, ddd, *J* = 5.0;3.9;1.2 Hz, H-4', 2.68 (1H,dd, J=16.2;3.9 Hz, H-4); 13C NMR (CDCl3, 75MHz) δ ppm : 147.1 (C-1), 142.6 (C-2), 135.6 (C-1a), 130.3 (C-7a), 129.4 (C-9), 129.1 (C-3a), 123.6 (C-1b), 115.6 (C-11a), 108.4 (O – CH2 – O), 71.0 (C-7), 57.2 (C-6a), 43.1 (C-5), 29.2 (C-4).

Cleistopholine (**12**), yellow glassy solid; MS m/z : 281 (M+); UV λmax nm EtOH : 234, 272, 315; IR υmax cm-1 : 1040, 945; 1H NMR (CDCl3, 400 MHz) δ ppm : 8.95 (1H, *d,J*= 4.8 Hz, H-2), 8.31 (1H, *dd,J*= 8.5;2.2 Hz, H-5), 8.21 (1H, *dd,J*= 8.5;2.2 Hz, H-8), 7.79 (1H, m*,* H-6), 7.79 (1H, m, H-7), 2.89 (1H, s, CH3); 13C NMR (CDCl3, 100.6 MHz) δ ppm : 184.7 (C-9), 181.9 (C-10), 153.4 (C-2), 151.6 (C-4), 150.1 (C-9a), 134.6 (C-7), 134.2 (C-6), 132.6 (C-10a), 131.2 (C-3), 129.1 (C-4a), 127.4 (C-5), 127.2 (C-8), 22.8 (CH3).

Table 2.

Alkaloids and Anthraquinones from Malaysian Flora 295

Anthraquinones of the Malaysian Rubiaceae are generally of the *Rubia* type. Rings A and B of the anthraquinone skeleton are biosynthetically derived from chorismic acid and *α*ketoglutarate *via* o-succinylbenzoic acid, whereas ring C is formed from isopentenyl diphosphate *via* the terpenoid pathway (Han*, et al.*, 2001). Chorismate is first converted to isochorismate, and then to *o*-succinoylbenzoic acid (OSB) in the presence of ketoglutarate and thiamine diphosphate. OSB is activated at the aliphatic carboxyl group to produce an OSB-CoA ester. It is the ring closure of OSB-CoA which results in the formation of 1,4-dihydroxy-2-naphthoic acid (DHNA) leading to ring A and B. The prenylation of DHNA at C-3, leads to naphthoquinol or naphthoquinone. The ring C formation is a consequence of the cyclization *via* C-C bond between the aromatic ring of the naphthoquinone and an isoprene unit, isopentenyl diphosphate (IPP) or 3,3-

Of the anthraquinone from Malaysian Rubiaceeae are substituted only on ring C while the remaining are substituted on both ring A and ring C. Anthraquinones from genus *Morinda* are typically substituted at C-1, C-2, and C-5, C-6 or C-7, C-8 and C-1, C-2 and C-3

Glucose

I. Shikimate Pathway II. Mevalonate Pathway

COOH

COOH

Tricarboxylic acid

+ O

CO2 Phosphoenol Pyruvate

COOHCOOH

+

O

O

Fig. 2. Biosynthetic Pathway of Anthraquinones

COOH

o-succinyl benzoic acid(OSB)

SCoA

<sup>O</sup> OH

CoA ester of OSB 1,4-dihydroxy-2-

COOH

<sup>3</sup> <sup>+</sup>

naphtoic acid 3,3-dimethylallyl

O

O A B C

OPP C

OH

HO O COOH Mevalonic acid

Acetyl Coa Pyruvate Glyceraldehyde-3-phospahate

+

OH OP

2-C-methyl-D-erythritol 4-P

CO2

Thiamine diphosphate

OH HO

3-hydroxy-3-glutaryl CoA 1-Deoxy-D-xylulose-5-P

II. MVA Pathway IV. MEP Pathway

OPP IPP isomerase

diphosphate

A B OH

dimethylallyl diphosphate (DMAPP).

Phosphoenol Pyruvate + Eryhtrose-4-Phosphate

Shikimic acid

Chorismic acid

OH O COOH

Thiamine diphosphate

HOOC

derivative in compound **2** gives increase to a bathochromic shift in the 235-250 nm bands on comparison with the corresponding compound **9.** The addition of acid will gives a substantial bathochromic shift of the longest-wavelength band. In oxoaporphine and aporphine, position 1 and 2 are constantly oxygenated. It is frequent to find further oxygen substituent at C-9, C-10 and C-11 and occasionally at C-8. Other than that, H-4 and H-5 will give a characteristic AB system with doublet of doublet at about 7.6 ppm and 8.7 ppm with a coupling constant about 5.4 Hz. The small *J* value is due to the adjacent of electronegative nitrogen atom. The methylenedioxyl group gives singlet peak at about 6.0 ppm due to the inductive effect cause by existence of the neighboring C-7 carbonyl. The C-11 proton usually the most deshielded and the C-3 protons always appeared at a higher field then the aromatic hydrogen (Cordell, 1981). Presented below are structures and spectroscopic data of the isolated compounds.

#### **3. The family of Rubiaceae as source of anthraquinones**

Rubiaceae is among the largest flowering plants family comprising of 450 genera and 13,000 species. In Malaysia, 70 genera and 555 species of Rubiaceous plants were reported (Wong, 1989). Most Rubiaceous plants are shrubs or small trees and infrequently herbs (Hutchinson, 1973). Rubiaceous plants are distributed worldwide but they are mainly tropical. They are easily recognized at family level by decussate, entire leaves, presence of stipules, actinomorph flowers and inferior ovary.

Rubiaceous plants are known to accumulate substantial amount of anthraquinones particularly in the roots (Han*, et al.*, 2001). Anthraquinones containing plants are used traditionally for various ailments and health complaints such as diarrhea, loss of appetite, fever, wounds and cancer. The plant extracts are used in form of poultice, lotion and decoction from various plant parts. *Morinda*, *Hedyotis*, *Primatomeris* and *Rennellia* are among anthraquinone containing-genera that are widely used in Malaysian traditional medicine (Ismail*, et al.*, 1997; Jasril*, et al.*, 2003; Ahmad*, et al.*, 2005; Lajis*, et al.*, 2006; Osman*, et al.*, 2010).

*Morinda* comprises of approximately 80 species, distributed worldwide in tropical areas. It is considered to be highly nutritious plant and is used as traditional medicine. In Malaysia *M. citrifolia* and *M. elliptica* are widely used. The roots of *M. elliptica* are used to treat jaundice and gastric complaints and the leaves are used to treat flatulence and fever. *Prismatomeris* and *Hedyotis* species on the other hand are recorded in various traditional medicine systems such as Traditional Chinese Medicine. Several well-known *Prismatomeris species* used in folk medicine in Malaysia are *P. glabra* and *P. malayana*. *P. glabra* is claimed to be aphrodisiac and widely used in the east coast of Malaysia. *P. Malayana* contained the anthraquinones, rubiadin and rubiadin-1-methyl ether (Lee, 1969). *Hedyotis* plants are generally consumed as tonic or febrifuge for treatment of diarrhea and dysentery (Lajis, et al., 2006). Several species of *Hedyotis* native to Malaysia are *H.capitellata*, *H. Herbaceae*, *H.dichtoma*, *H. diffusa* and *H. verticillata*. Besides anthraquinones, *Hedyotis* also contain β-cabolline alkaloids, flavonoids and triterpenes. *Rennellia* is another small genus of Rubiaceae family. Consists of shrubs and small trees, the plants may be found in lowland tropical rainforest of Peninsular Malaysia and Sumatra. *R. elliptica*, is used for general health improvements and dubbed as Malaysian Ginseng most likely due to the appearance of its yellow roots.

derivative in compound **2** gives increase to a bathochromic shift in the 235-250 nm bands on comparison with the corresponding compound **9.** The addition of acid will gives a substantial bathochromic shift of the longest-wavelength band. In oxoaporphine and aporphine, position 1 and 2 are constantly oxygenated. It is frequent to find further oxygen substituent at C-9, C-10 and C-11 and occasionally at C-8. Other than that, H-4 and H-5 will give a characteristic AB system with doublet of doublet at about 7.6 ppm and 8.7 ppm with a coupling constant about 5.4 Hz. The small *J* value is due to the adjacent of electronegative nitrogen atom. The methylenedioxyl group gives singlet peak at about 6.0 ppm due to the inductive effect cause by existence of the neighboring C-7 carbonyl. The C-11 proton usually the most deshielded and the C-3 protons always appeared at a higher field then the aromatic hydrogen (Cordell, 1981). Presented below are structures and spectroscopic data of the

Rubiaceae is among the largest flowering plants family comprising of 450 genera and 13,000 species. In Malaysia, 70 genera and 555 species of Rubiaceous plants were reported (Wong, 1989). Most Rubiaceous plants are shrubs or small trees and infrequently herbs (Hutchinson, 1973). Rubiaceous plants are distributed worldwide but they are mainly tropical. They are easily recognized at family level by decussate, entire leaves, presence of stipules,

Rubiaceous plants are known to accumulate substantial amount of anthraquinones particularly in the roots (Han*, et al.*, 2001). Anthraquinones containing plants are used traditionally for various ailments and health complaints such as diarrhea, loss of appetite, fever, wounds and cancer. The plant extracts are used in form of poultice, lotion and decoction from various plant parts. *Morinda*, *Hedyotis*, *Primatomeris* and *Rennellia* are among anthraquinone containing-genera that are widely used in Malaysian traditional medicine (Ismail*, et al.*, 1997; Jasril*, et al.*, 2003; Ahmad*, et al.*, 2005; Lajis*, et al.*, 2006; Osman*, et al.*,

*Morinda* comprises of approximately 80 species, distributed worldwide in tropical areas. It is considered to be highly nutritious plant and is used as traditional medicine. In Malaysia *M. citrifolia* and *M. elliptica* are widely used. The roots of *M. elliptica* are used to treat jaundice and gastric complaints and the leaves are used to treat flatulence and fever. *Prismatomeris* and *Hedyotis* species on the other hand are recorded in various traditional medicine systems such as Traditional Chinese Medicine. Several well-known *Prismatomeris species* used in folk medicine in Malaysia are *P. glabra* and *P. malayana*. *P. glabra* is claimed to be aphrodisiac and widely used in the east coast of Malaysia. *P. Malayana* contained the anthraquinones, rubiadin and rubiadin-1-methyl ether (Lee, 1969). *Hedyotis* plants are generally consumed as tonic or febrifuge for treatment of diarrhea and dysentery (Lajis, et al., 2006). Several species of *Hedyotis* native to Malaysia are *H.capitellata*, *H. Herbaceae*, *H.dichtoma*, *H. diffusa* and *H. verticillata*. Besides anthraquinones, *Hedyotis* also contain β-cabolline alkaloids, flavonoids and triterpenes. *Rennellia* is another small genus of Rubiaceae family. Consists of shrubs and small trees, the plants may be found in lowland tropical rainforest of Peninsular Malaysia and Sumatra. *R. elliptica*, is used for general health improvements and dubbed as Malaysian

**3. The family of Rubiaceae as source of anthraquinones** 

Ginseng most likely due to the appearance of its yellow roots.

isolated compounds.

2010).

actinomorph flowers and inferior ovary.

Anthraquinones of the Malaysian Rubiaceae are generally of the *Rubia* type. Rings A and B of the anthraquinone skeleton are biosynthetically derived from chorismic acid and *α*ketoglutarate *via* o-succinylbenzoic acid, whereas ring C is formed from isopentenyl diphosphate *via* the terpenoid pathway (Han*, et al.*, 2001). Chorismate is first converted to isochorismate, and then to *o*-succinoylbenzoic acid (OSB) in the presence of ketoglutarate and thiamine diphosphate. OSB is activated at the aliphatic carboxyl group to produce an OSB-CoA ester. It is the ring closure of OSB-CoA which results in the formation of 1,4-dihydroxy-2-naphthoic acid (DHNA) leading to ring A and B. The prenylation of DHNA at C-3, leads to naphthoquinol or naphthoquinone. The ring C formation is a consequence of the cyclization *via* C-C bond between the aromatic ring of the naphthoquinone and an isoprene unit, isopentenyl diphosphate (IPP) or 3,3 dimethylallyl diphosphate (DMAPP).

Of the anthraquinone from Malaysian Rubiaceeae are substituted only on ring C while the remaining are substituted on both ring A and ring C. Anthraquinones from genus *Morinda* are typically substituted at C-1, C-2, and C-5, C-6 or C-7, C-8 and C-1, C-2 and C-3

Fig. 2. Biosynthetic Pathway of Anthraquinones

Alkaloids and Anthraquinones from Malaysian Flora 297

*R. elliptica* is used for general health improvements and dubbed as Malaysian Ginseng may be due to the appearance of its yellow roots. Its medicinal uses were documented as treatment of body aches, after-birth tonic and aphrodisiac (Mat Salleh & Latiff, 2002). The root extract of *R. elliptica* was reported to be antimalarial (Osman*, et al.*, 2010) and antioxidant (Ahmad*, et al.*, 2010). Further study is warranted to investigate the antimalarial

Phytochemical studies of the roots of *R. elliptica* Korth. resulted a new anthraquinone 1,2 dimethoxy-6-methyl-9,10-anthraquinone **18**, along with ten known ones. The known anthraquinones were nordamnacanthal **13**, 2-formyl-3-hydroxy-9,10-anthraquinone **14**, damnacanthal **15,** 1-hydroxy-2-methoxy-6-methyl-9,10-anthraquinone **16**, lucidin-*ω*-methyl ether **17**, 3-hydroxy-2-methoxy-6-methyl-9,10-anthraquinone **19**, rubiadin 2**0**, 3-hydroxy-2 methyl-9,10-anthraquinone **21**, rubiadin-1-methyl ether **22** and 3-hydroxy-2-hydroxymethyl-

Anthraquinone **18**, 1,2-dimethoxy-6-methyl-9,10-anthraquinone, isolated for the first time as bright yellow amorphous solid. The HREIMS of **18** displayed a [M H]+ peak at 283.0968 [calc 283.3067] suggesting a molecular formula of C17H14O4. The absorption maxima in the UV spectrum were observed at 373, 341 and 257 nm, indicative of an anthraquinone moiety. The IR spectrum did not show presence of chelated carbonyl and hydroxyl groups. The *sp*<sup>2</sup> C-H stretch for the aromatic ring was observed at 3,081 cm-1. With the exception of the sharp singlet in the downfield region for the hydrogen-bonded hydroxyl group, the 1H NMR spectrum resembles that of compound **16**, suggesting a similar substitution pattern. Splitting pattern of the five aromatic proton signals suggested substitutions on both rings. Two overlapping doublets centered at δH 8.17 are due to H-8 (*d*, *J* = 7.8 Hz) and H-4 (*d, J =* 8.7 Hz), the *peri*-hydrogens. A doublet at δH 7.28 (*J* = 8.7 Hz) is due to H-3, meanwhile H-7 gave another doublet of doublet at δH 7.58 (*J*o = 7.8 Hz, *J*m = 1.7 Hz). These assignments were confirmed by their respective correlations in the COSY spectrum. H-5 resonated as a singlet at 8.06 ppm. In addition, two sharp singlets at δH 2.53 (3H, *s*) and 4.02 (6H, *s*) due to a methyl and two methoxy groups, respectively, were also observed. The location of the methoxy groups were established at C-1 and C-2 of ring C based on its NOE correlation with H-3. Thus, the only possible location for the methyl substituent is at C-6. This assignment was confirmed through NOE correlations of the methyl group with H-5 and H-7. The placement of methyl group at C-6 was further confirmed by HMBC experiment

potential of roots of *R. elliptca*.

Fig. 4. Rennellia elliptica Korth

9,10-anthraquinone **23**.

meanwhile anthraquinones from *Hedyotis* are differed by rare substitution at C-1, C-2 and C-4. Anthraquinones from *Hedyotis* displayed wide structural variation. *H. capitellata* contains furanoanthraquinones (Ahmad*, et al.*, 2005) and *H. dichotoma* was reported to contain both 9,10- and 1,4-anthraquinone (Hamzah & Lajis, 1998). Genus *Rennellia* is closely related to *Morinda* and anthraquinones reported from *R. elliptica* are similar to those from genus *Morinda* (Osman *et al.*, 2010). One particular difference is the occurrence of anthraquinone with methyl substitution at C-6 which is characteristic to this plant.

Fig. 3. Basic Skeleton of Anthraquinones

There are several characteristic spectroscopic data that distinguished anthraquinones from other types of compounds. In mass spectra, the major fragmentations are due to two consecutive loss of carbonyls, [M-CO]+ and [M-2CO]+. In the IR spectra, the unchelated carbonyl only viewed as one sharp stretching band at 1670 cm-1 due to symmetrical character of 9,10-anthraquinone (Derksen*, et al.*, 2002). Anthraquinones substituted with hydroxyl at *peri* position displayed two carbonyl absorption bands at about 1670 cm-1 and 1630 cm-1. Anthraquinones give several chracteristic UV absorptions at 265-280 nm and 285- 290 nm due to electron transfers bonds of benzoid chromophore and at 430-437 nm due local excitation of quinoid carbonyls. The location hydroxyl substituent can be distinguished by observing the absorption maxima in UV spectra. Addition of dilute sodium hydroxide solution caused bathchromic shift of absorption maxima. The shift is useful in distinguishing substitution pattern of polyhydroxyanthraquinones. Proton NMR spectra of 9,10-anthraquinones shows typical A2B2 substitution pattern of *ortho*-substituted aromatic ring. An unsubstituted anthraquinone ring can be easily distinguished by the presence of at least two sets of multiplets at ca. δH 8.10 and ca. δH 7.20 in the aromatic region. Anthraquinones substituted at both rings A and C will give several doublets in the aromatic region. The two carbonyl groups in the molecule can be easily distinguished if hydroxyl substituents present in *para* position. Hydroxyl groups adjacent to carbonyl can be seen as sharp singlets much downfield at δH 12-14 due to strong intramolecular hydrogen bonding to the adjacent carbonyl. The presence of hydroxyl adjacent to carbonyl cause significant shift of carbonyl carbon resonance to downfield region at 186-189 ppm.

#### **3.1 Anthraquinones of** *Rennellia elliptica* **Korth.**

*R. elliptica* Korth. was also previously known as *R. elongata* (King & Gamble) Ridl. It is a shrub of about 2 m tall. This shrub can be found in lowland to hill forest to c. 500m above sea level. *R. elliptica* Korth. is widely distributed from Southern Myanmar to West Malaysia.

meanwhile anthraquinones from *Hedyotis* are differed by rare substitution at C-1, C-2 and C-4. Anthraquinones from *Hedyotis* displayed wide structural variation. *H. capitellata* contains furanoanthraquinones (Ahmad*, et al.*, 2005) and *H. dichotoma* was reported to contain both 9,10- and 1,4-anthraquinone (Hamzah & Lajis, 1998). Genus *Rennellia* is closely related to *Morinda* and anthraquinones reported from *R. elliptica* are similar to those from genus *Morinda* (Osman *et al.*, 2010). One particular difference is the occurrence of anthraquinone

with methyl substitution at C-6 which is characteristic to this plant.

8

5

shift of carbonyl carbon resonance to downfield region at 186-189 ppm.

*R. elliptica* Korth. was also previously known as *R. elongata* (King & Gamble) Ridl. It is a shrub of about 2 m tall. This shrub can be found in lowland to hill forest to c. 500m above sea level. *R. elliptica* Korth. is widely distributed from Southern Myanmar to West Malaysia.

**3.1 Anthraquinones of** *Rennellia elliptica* **Korth.** 

11

10

O

There are several characteristic spectroscopic data that distinguished anthraquinones from other types of compounds. In mass spectra, the major fragmentations are due to two consecutive loss of carbonyls, [M-CO]+ and [M-2CO]+. In the IR spectra, the unchelated carbonyl only viewed as one sharp stretching band at 1670 cm-1 due to symmetrical character of 9,10-anthraquinone (Derksen*, et al.*, 2002). Anthraquinones substituted with hydroxyl at *peri* position displayed two carbonyl absorption bands at about 1670 cm-1 and 1630 cm-1. Anthraquinones give several chracteristic UV absorptions at 265-280 nm and 285- 290 nm due to electron transfers bonds of benzoid chromophore and at 430-437 nm due local excitation of quinoid carbonyls. The location hydroxyl substituent can be distinguished by observing the absorption maxima in UV spectra. Addition of dilute sodium hydroxide solution caused bathchromic shift of absorption maxima. The shift is useful in distinguishing substitution pattern of polyhydroxyanthraquinones. Proton NMR spectra of 9,10-anthraquinones shows typical A2B2 substitution pattern of *ortho*-substituted aromatic ring. An unsubstituted anthraquinone ring can be easily distinguished by the presence of at least two sets of multiplets at ca. δH 8.10 and ca. δH 7.20 in the aromatic region. Anthraquinones substituted at both rings A and C will give several doublets in the aromatic region. The two carbonyl groups in the molecule can be easily distinguished if hydroxyl substituents present in *para* position. Hydroxyl groups adjacent to carbonyl can be seen as sharp singlets much downfield at δH 12-14 due to strong intramolecular hydrogen bonding to the adjacent carbonyl. The presence of hydroxyl adjacent to carbonyl cause significant

O

14

4

1

3

2

13 9

12

7

6

Fig. 3. Basic Skeleton of Anthraquinones

*R. elliptica* is used for general health improvements and dubbed as Malaysian Ginseng may be due to the appearance of its yellow roots. Its medicinal uses were documented as treatment of body aches, after-birth tonic and aphrodisiac (Mat Salleh & Latiff, 2002). The root extract of *R. elliptica* was reported to be antimalarial (Osman*, et al.*, 2010) and antioxidant (Ahmad*, et al.*, 2010). Further study is warranted to investigate the antimalarial potential of roots of *R. elliptca*.

Fig. 4. Rennellia elliptica Korth

Phytochemical studies of the roots of *R. elliptica* Korth. resulted a new anthraquinone 1,2 dimethoxy-6-methyl-9,10-anthraquinone **18**, along with ten known ones. The known anthraquinones were nordamnacanthal **13**, 2-formyl-3-hydroxy-9,10-anthraquinone **14**, damnacanthal **15,** 1-hydroxy-2-methoxy-6-methyl-9,10-anthraquinone **16**, lucidin-*ω*-methyl ether **17**, 3-hydroxy-2-methoxy-6-methyl-9,10-anthraquinone **19**, rubiadin 2**0**, 3-hydroxy-2 methyl-9,10-anthraquinone **21**, rubiadin-1-methyl ether **22** and 3-hydroxy-2-hydroxymethyl-9,10-anthraquinone **23**.

Anthraquinone **18**, 1,2-dimethoxy-6-methyl-9,10-anthraquinone, isolated for the first time as bright yellow amorphous solid. The HREIMS of **18** displayed a [M H]+ peak at 283.0968 [calc 283.3067] suggesting a molecular formula of C17H14O4. The absorption maxima in the UV spectrum were observed at 373, 341 and 257 nm, indicative of an anthraquinone moiety. The IR spectrum did not show presence of chelated carbonyl and hydroxyl groups. The *sp*<sup>2</sup> C-H stretch for the aromatic ring was observed at 3,081 cm-1. With the exception of the sharp singlet in the downfield region for the hydrogen-bonded hydroxyl group, the 1H NMR spectrum resembles that of compound **16**, suggesting a similar substitution pattern. Splitting pattern of the five aromatic proton signals suggested substitutions on both rings. Two overlapping doublets centered at δH 8.17 are due to H-8 (*d*, *J* = 7.8 Hz) and H-4 (*d, J =* 8.7 Hz), the *peri*-hydrogens. A doublet at δH 7.28 (*J* = 8.7 Hz) is due to H-3, meanwhile H-7 gave another doublet of doublet at δH 7.58 (*J*o = 7.8 Hz, *J*m = 1.7 Hz). These assignments were confirmed by their respective correlations in the COSY spectrum. H-5 resonated as a singlet at 8.06 ppm. In addition, two sharp singlets at δH 2.53 (3H, *s*) and 4.02 (6H, *s*) due to a methyl and two methoxy groups, respectively, were also observed. The location of the methoxy groups were established at C-1 and C-2 of ring C based on its NOE correlation with H-3. Thus, the only possible location for the methyl substituent is at C-6. This assignment was confirmed through NOE correlations of the methyl group with H-5 and H-7. The placement of methyl group at C-6 was further confirmed by HMBC experiment

O

CHO

OH

O

Nordamnacanthal. (**13**) Orange crystals. Mps 216-219 ° [lit. 220 ͦ C (Me2CO) Chang (1984)]. UVλmax EtOH nm: 421, 295, 259. UVλmax EtOH/ -OH nm: 512, 357, 283. IR νmax (KBr) cm-1 :3460, 1646, 1627, 1382. MS m/z 268 [M+], 240, 212, 184, 138. 1H NMR (CDCl3, 300MHz): 14.05 (1H, s, 1-OH), 12.70 (1H, s, 3-OH), 10.52 (1H, s, 2-CHO), 8.30 (2H, m, H-5, H-8), 7.88 (2H, m, H-6, H-7), 7.36 (1H, s, H-4). 13C NMR (CDCl3, 75.5 MHz): 193.9 (2- CHO), 186.8 (C=O, C-9), 181.4 (C=O, C-10), 169.2 (C-OH,C-1), 168. 1 (C-OH, C-3), 139.1 (C-2), 134.8 (C-7), 134.7 (C-6), 133.3 (C-14), 133.2 (C-13), 127. 8 (C-8), 127.0 (H-5), 112.1 (C-14), 109.4 (C-4), 109.1 (C-13)

Alkaloids and Anthraquinones from Malaysian Flora 299

Lucidin-*ω*-methyl ether (**17**). Yellow crystals. Mps 175- 179°C [lit 170 ͦ C, Dictionary of Natural Products (1995); 163-166 ͦ C, Leistner (1975)]. UVλmax EtOH nm: 412, 280, 24. UVλmax EtOH/ -OH nm: 491, 314, 242 IR νmax (KBr) cm-1: 3428, 2927, 1668, 162. MS m/z: 284 [M+], 263, 241, 213, 185. 1H NMR (CDCl3, 300MHz): 13.29 (1H, s, 1-OH), 9.39 (1H, s br, 3-OH), 8.27 (2H, m, H-5, H-8), 7.80 (2H, m, H-6, H-7), 7.32 (1H, s, H-4), 4.90 (2H, s, 2-**CH2**OCH3), 3.59 (3H, s, 2-CH2O**CH3**). 13C NMR (CDCl3, 75.5 MHz): 186.9 (C=O, C-9), 182.2 (C=O, C-10), 164.1 (C-1), 161.7 (C-3), 134.1 (C-11), 134.1 (C-12), 133.6 (C-6), 133.5 (C-7), 126.8 (C-2), 114.4 (C-4), 109.7 (C-8), 109.7 (C-13), 109.6 (C-5), 109.6 (C-14), 68.9 (2-**CH2**OCH3), 59.3 (2-CH2OCH3)

1,2-Dimethoxy-6-methyl-9,10-anthraquinone (**18**). Bright yellow crystals. Mps 193-196 °C. UVλmax EtOH nm: 373, 341, 257, 222. UVλmax EtOH/ -OH nm: 373, 342, 257, 222. IR νmax (KBr) cm-1:1666, 1601, 1327, 1267MS m/z: 282 [M+], 253, 221, 194, 165, 139. 1H NMR (CDCl3, 300MHz): 8.17 (2H, dd, *J*=8.7, 7.8, H-4, H-8), 8.06 (1H, s, H-5), 7.58 (1H, d, *J*=7.8, H-7), 7.28 (1H, d, *J*=8.7, H-3), 4.02 (6H, s, 1- OCH3, 2-OCH3), 2.53 (3H, s, 6-CH3).13C NMR (CDCl3, 75.5 MHz): 182.7 (C=O, C-9), 182.7 (C=O, C-10), 159.1 (C-1), 149.6 (C-2), 144.6 (C-6), 134.8 (C-7), 132. 9 (C-11), 132.9 (C-12), 127.5 (C-14), 127.4 (C-13), 127.1 (C-8), 126.9 (C-5), 125.2 (C-4), 115.9 (C-3), 61.3 (1-OCH3), 56.3 (2-OCH3),

2-Hydroxy-3-methoxy-6-methyl-9,10-anthraquinone (**19**) Light yellow amorphous solid. Mp 210-215 °C. UVλmax EtOH nm: 393, 286, 244. UVλmax EtOH/ -OH nm: 509, 316, 250. IR νmax (KBr) cm-1: 3203, 2927, 2869, 1666, 1265. MS m/z: 268 (M+], 239, 207, 169. 1H NMR (CDCl3, 300MHz): 8.18 (1H, d, *J*=8.1, H-8), 8.08 (1H, s, H-5), 7.79 (1H, s, H-1), 7.76 (1H, s, H-4),7.57 (1H, d, *J*=8.1, H-7), 6.23 (1H, s br, 2-OH), 4.11 (3H, s, 3-OCH3), 2.54 (3H, s, 6-CH3). 13C NMR (CDCl3, 75.5 MHz): 182.4 (C=O), 162.8(C-OH, C-2), 151.4 (C-OCH3, C-3), 144.9, 134.5, 133.6, 127.4, 127.2, 112.6, 108.3, 56.6 (3-OCH3),

O

OH

**(17)** 

O

O

O

O

**(19)** 

H3C OCH3

**(18)** 

H3C

CH2OCH3

OH

OCH3

OCH3

OH

21.8 (6-CH3)

21.9 (6-CH3)

O

3-Formyl-2-hydroxy-9,10-anthraquinone (**14**). Bright orange needle crystals. Mps 212-214 °C [259-260 ͦ C, Rath et al.( 1995)]. UVλmax EtOH nm: 380, 277, 246. UVλmax EtOH/ -OH nm: 466, 392, 310, 254. IR νmax (KBr) cm-1: 3467, 1655, 1657, 1564. MS m/z 252 [M+], 229, 206, 167, 139. 1H NMR (CDCl3, 300MHz): 11.45 (1H, s, 3-OH), 10.17 (1H, s, 2-CHO), 8.68 (1H, s, H-4), 8.35 (2H, m, H-5, H-8), 7.88 (2H, m, H-6, H-7), 7.86(1H, s, H-1) .13C NMR (CDCl3, 75.5 MHz): 196.8 (2-CHO), 181.0 (C=O, C-9, C-10), 165.3 (C-OH, C-3), 139.1, 134.8 (C-4), 134.8, 133.3, 127.6, 127.5, 127.3, 126.1, 124.5, 123.4, 116.5 (C-1)

O O OCH3 CHO OH **(15)** 

**(14)** 

Damnacanthal (**15**). Yellow crystals. Mps 208-211°C [ lit. 218-218.5 ͦ C (Me2CO) Chang (1984)]. UVλmax EtOH nm: 381, 284, 250, 213. UVλmax EtOH/ -OH nm: 460, 379, 315, 262, 250. IR νmax (KBr) cm-1: 3437, 1644, 1561. MS m/z: 282 [M+], 254, 225, 196. 1H NMR (CDCl3, 300MHz): 12.29 (1H, s, 3-OH), 10.49 (1H, s, 2-CHO), 8.25 (2H, m, H-5, H-8), 7.84 (2H, m, H-6, H-7), 7.68 (1H, s, H-4), 4.14 (3H, s, 1- OCH3)

1-Hydroxy-2-methoxy-6-methyl-9,10-anthraquinone (**16**). Red needle crystals. Mps 220-221 °C. UVλmax EtOH nm: 421, 278, 262, 231. UVλmax EtOH/ -OH nm: 505, 314, 258. IR νmax (KBr) cm-1: 3467, 1653, 1637. MS m/z: 268 [M+], 239, 197, 169,139, 115. 1H NMR (CDCl3, 300MHz): 13.20 (1H, s, 1-OH), 8.23 (1H, d, *J*=8.1, H-8), 8.12 (1H, s, H-5), 7.89 (1H, d, *J*=8.4, H-4), 7.61 (1H, d, *J*=8.1, H-7), 7.19 (1H, d, *J*=8.4, H-3), 4.04 (3H, s, 2-OCH3), 2.56 (3H, s, 6- CH3). 13C NMR (CDCl3, 75.5 MHz): 189.1(C=O, C-9), 181.8 (C=O, C-10), 154.0 (C-OH, C-1), 152.7 (C-OCH3, C2), 146.2 (C-6), 134.6 (C-7), 134.0 (C-11), 131.10 (C-12), 127.8 (C-5), 127.1 (C-8), 125.5 (C-14), 121.0 (C-4), 116.1 (C-13), 115.6 (C-3), 56.4 (2-OCH3), 22.0 (6-CH3)

109.4 (C-4), 109.1 (C-13)

Nordamnacanthal. (**13**) Orange crystals. Mps 216-219 ° [lit. 220 ͦ C (Me2CO) Chang (1984)]. UVλmax EtOH nm: 421, 295, 259. UVλmax EtOH/ -OH nm: 512, 357, 283. IR νmax (KBr) cm-1 :3460, 1646, 1627, 1382. MS m/z 268 [M+], 240, 212, 184, 138. 1H NMR (CDCl3, 300MHz): 14.05 (1H, s, 1-OH), 12.70 (1H, s, 3-OH), 10.52 (1H, s, 2-CHO), 8.30 (2H, m, H-5, H-8), 7.88 (2H, m, H-6, H-7), 7.36 (1H, s, H-4). 13C NMR (CDCl3, 75.5 MHz): 193.9 (2- CHO), 186.8 (C=O, C-9), 181.4 (C=O, C-10), 169.2 (C-OH,C-1), 168. 1 (C-OH, C-3), 139.1 (C-2), 134.8 (C-7), 134.7 (C-6), 133.3 (C-14), 133.2 (C-13), 127. 8 (C-8), 127.0 (H-5), 112.1 (C-14),

3-Formyl-2-hydroxy-9,10-anthraquinone (**14**). Bright orange needle crystals. Mps 212-214 °C [259-260 ͦ C, Rath et al.( 1995)]. UVλmax EtOH nm: 380, 277, 246. UVλmax EtOH/ -OH nm: 466, 392, 310, 254. IR νmax (KBr) cm-1: 3467, 1655, 1657, 1564. MS m/z 252 [M+], 229, 206, 167, 139. 1H NMR (CDCl3, 300MHz): 11.45 (1H, s, 3-OH), 10.17 (1H, s, 2-CHO), 8.68 (1H, s, H-4), 8.35 (2H, m, H-5, H-8), 7.88 (2H, m, H-6, H-7), 7.86(1H, s, H-1) .13C NMR (CDCl3, 75.5 MHz): 196.8 (2-CHO), 181.0 (C=O, C-9, C-10), 165.3 (C-OH, C-3), 139.1, 134.8 (C-4), 134.8, 133.3,

127.6, 127.5, 127.3, 126.1, 124.5, 123.4, 116.5 (C-1)

Damnacanthal (**15**). Yellow crystals. Mps 208-211°C [ lit. 218-218.5 ͦ C (Me2CO) Chang (1984)]. UVλmax EtOH nm: 381, 284, 250, 213. UVλmax EtOH/ -OH nm: 460, 379, 315, 262, 250. IR νmax (KBr) cm-1: 3437, 1644, 1561. MS m/z: 282 [M+], 254, 225, 196. 1H NMR (CDCl3, 300MHz): 12.29 (1H, s, 3-OH), 10.49 (1H, s, 2-CHO), 8.25 (2H, m, H-5, H-8), 7.84 (2H, m, H-6, H-7), 7.68 (1H, s, H-4), 4.14 (3H, s, 1-

1-Hydroxy-2-methoxy-6-methyl-9,10-anthraquinone (**16**). Red needle crystals. Mps 220-221 °C. UVλmax EtOH nm: 421, 278, 262, 231. UVλmax EtOH/ -OH nm: 505, 314, 258. IR νmax (KBr) cm-1: 3467, 1653, 1637. MS m/z: 268 [M+], 239, 197, 169,139, 115. 1H NMR (CDCl3, 300MHz): 13.20 (1H, s, 1-OH), 8.23 (1H, d, *J*=8.1, H-8), 8.12 (1H, s, H-5), 7.89 (1H, d, *J*=8.4, H-4), 7.61 (1H, d, *J*=8.1, H-7), 7.19 (1H, d, *J*=8.4, H-3), 4.04 (3H, s, 2-OCH3), 2.56 (3H, s, 6- CH3). 13C NMR (CDCl3, 75.5 MHz): 189.1(C=O, C-9), 181.8 (C=O, C-10), 154.0 (C-OH, C-1), 152.7 (C-OCH3, C2), 146.2 (C-6), 134.6 (C-7), 134.0 (C-11), 131.10 (C-12), 127.8 (C-5), 127.1 (C-8), 125.5 (C-14), 121.0 (C-4), 116.1 (C-

13), 115.6 (C-3), 56.4 (2-OCH3), 22.0 (6-CH3)

O

OH

CHO

OH

CHO

OH

CHO

OH

OCH3)

O

O

O

O

O

**(15)** 

O

OH

OCH3

O

**(16)** 

H3C

**(14)** 

OCH3

**(13)** 

Lucidin-*ω*-methyl ether (**17**). Yellow crystals. Mps 175- 179°C [lit 170 ͦ C, Dictionary of Natural Products (1995); 163-166 ͦ C, Leistner (1975)]. UVλmax EtOH nm: 412, 280, 24. UVλmax EtOH/ -OH nm: 491, 314, 242 IR νmax (KBr) cm-1: 3428, 2927, 1668, 162. MS m/z: 284 [M+], 263, 241, 213, 185. 1H NMR (CDCl3, 300MHz): 13.29 (1H, s, 1-OH), 9.39 (1H, s br, 3-OH), 8.27 (2H, m, H-5, H-8), 7.80 (2H, m, H-6, H-7), 7.32 (1H, s, H-4), 4.90 (2H, s, 2-**CH2**OCH3), 3.59 (3H, s, 2-CH2O**CH3**). 13C NMR (CDCl3, 75.5 MHz): 186.9 (C=O, C-9), 182.2 (C=O, C-10), 164.1 (C-1), 161.7 (C-3), 134.1 (C-11), 134.1 (C-12), 133.6 (C-6), 133.5 (C-7), 126.8 (C-2), 114.4 (C-4), 109.7 (C-8), 109.7 (C-13), 109.6 (C-5), 109.6 (C-14), 68.9 (2-**CH2**OCH3), 59.3 (2-CH2OCH3)

1,2-Dimethoxy-6-methyl-9,10-anthraquinone (**18**). Bright yellow crystals. Mps 193-196 °C. UVλmax EtOH nm: 373, 341, 257, 222. UVλmax EtOH/ -OH nm: 373, 342, 257, 222. IR νmax (KBr) cm-1:1666, 1601, 1327, 1267MS m/z: 282 [M+], 253, 221, 194, 165, 139. 1H NMR (CDCl3, 300MHz): 8.17 (2H, dd, *J*=8.7, 7.8, H-4, H-8), 8.06 (1H, s, H-5), 7.58 (1H, d, *J*=7.8, H-7), 7.28 (1H, d, *J*=8.7, H-3), 4.02 (6H, s, 1- OCH3, 2-OCH3), 2.53 (3H, s, 6-CH3).13C NMR (CDCl3, 75.5 MHz): 182.7 (C=O, C-9), 182.7 (C=O, C-10), 159.1 (C-1), 149.6 (C-2), 144.6 (C-6), 134.8 (C-7), 132. 9 (C-11), 132.9 (C-12), 127.5 (C-14), 127.4 (C-13), 127.1 (C-8), 126.9 (C-5), 125.2 (C-4), 115.9 (C-3), 61.3 (1-OCH3), 56.3 (2-OCH3), 21.8 (6-CH3)

2-Hydroxy-3-methoxy-6-methyl-9,10-anthraquinone (**19**) Light yellow amorphous solid. Mp 210-215 °C. UVλmax EtOH nm: 393, 286, 244. UVλmax EtOH/ -OH nm: 509, 316, 250. IR νmax (KBr) cm-1: 3203, 2927, 2869, 1666, 1265. MS m/z: 268 (M+], 239, 207, 169. 1H NMR (CDCl3, 300MHz): 8.18 (1H, d, *J*=8.1, H-8), 8.08 (1H, s, H-5), 7.79 (1H, s, H-1), 7.76 (1H, s, H-4),7.57 (1H, d, *J*=8.1, H-7), 6.23 (1H, s br, 2-OH), 4.11 (3H, s, 3-OCH3), 2.54 (3H, s, 6-CH3). 13C NMR (CDCl3, 75.5 MHz): 182.4 (C=O), 162.8(C-OH, C-2), 151.4 (C-OCH3, C-3), 144.9, 134.5, 133.6, 127.4, 127.2, 112.6, 108.3, 56.6 (3-OCH3), 21.9 (6-CH3)

**(21)** 

O

Rubiadin (**20**). Yellow crystals. Mps 250-258 °C [lit. 280- 283 ͦ C, Leistner (1975)]. UVλmax EtOH nm: 413, 279. UVλmax EtOH/ -OH nm: 496, 314, 241 IR νmax (KBr) cm-1: 3436, 1653, 1626. MS m/z: 254 [M+], 226, 197, 152, 115. 1H NMR (Acetone-d6, 300MHz): 13.20 (1H, s, 1-OH), 8.31 (1H, m, H-8), 8.23 (1H, m, H-5), 7.92 (2H, m, H-6, H-7), 7.38 (1H, s, H), 2.20 (3H, s, 2-CH3). 13C NMR (Acetone-d6, 75.5 MHz): 186.9 (C=O,C9), 181.8 (C=O, C-10), 163.2 (C-OH, C-1), 162.4 (C-OH, C-3), 134.3, 134.2, 133.5, 133.5, 132.4, 126.8, 126.5, 117.9, 107.17, 7.3

Alkaloids and Anthraquinones from Malaysian Flora 301

which showed a 3*J* correlation with H-7. The methine carbons (C-3, C-4, C-5, C-7 and C-8) were assigned through HMQC correlations while the quaternary carbons (C-1, C-2, C-6, C-11, C-12, C-13 and C-14) were assigned based on careful analysis of HMBC spectrum. Both carbonyl carbons in this compound resonated very closely to each other with only 0.01 ppm difference at δC 182.70 and 182.71, which further confirmed the unchelated nature of the carbonyls. Presented below are structures and spectroscopic data of the isolated

*Morinda elliptica* or locally known as 'mengkudu kecil' is a shrub or small tree and it is very common in wild state of Malay Peninsula and northwards Burma (Burkill, 1966). It can be seen growing wild in newly developed areas, bushes and lowland secondary forest throughout the peninsula. *M. elliptica* is very common and always available and mostly used by the Malays for medicinal purposes. Traditionally, different parts of the plant are used in various ways for a number of health problems and ailments. The leaves may be added to rice for loss of appetite and taken for headache, cholera, diarrhea and wounds. Sometimes a lotion is made and used for hemorrhoid and applied upon body after childbirth (Burkill, 1966). The extracts and anthraquinones isolated from *M. elliptica* were reported to possess wide spectrum of biological activities such as antioxidant (Ismail*, et al.*, 2002; Jasril*, et al.*,

Five anthraquinones in roots of *M. elliptica* which are nordamnacanthal **13**, damnacanthal **15**, lucidin-ω-methyl ether **17,** rubiadin **20** and rubiadin-l-methyl ether **22** are the same constituents found in *R. elliptica*. The others are 1-hydroxy-2-methylanthraquinone **23**, soranjidiol **25**, morindone **26**, morindone-5- methyl ether **27** and alizarin-1-methyl ether **28.** In addition, 2-formyl-1-hydroxyanthraquinone **24** was reported as a new naturally occuring anthraquinone from roots of *M. elliptica*. HR-MS of **24** showed molecular ion peak at 252.0414 consistent with molecular formula of C15H14O4. A bathchromic shift (407 to 531 nm) upon adding NaOH suggested the presence of OH at C-1 of the anthraquinone skeleton. The presence of hydroxyl group was evident from the broad stretching band observed at 3448 cm-1. Two sharp stretching vibrations due to chelated and unchelated carbonyls were observed at 1638 and 1676 cm-1, respectively. In the proton NMR, the signal for chelated hydroxyl group is at δH 13.26. The splitting pattern of 1H NMR suggest substitution pattern

compounds.

Fig. 5. *Morinda elliptica*

**3.2 Anthraquinones of** *Morinda elliptica* 

2003), antimicrobial, anti-HIV and anticancer (Ali*, et al.*, 2000).

3-Hydroxy-2-methyl-9,10-anthraquinone (**21**). Yellow crystals. Mps 138- 142 ͦ C. UVλmax EtOH nm: 379, 329, 274, 245, 239. UVλmax EtOH/ -OH nm: 496, 314, 246 . IR νmax (KBr) cm-1: 3436, 1663, 651. MS m/z 238 [M+], 238, 210, 181, 152, 105. 1H NMR (Acetone-d6, 300MHz): 8.23 (2H, m, H-5, H-8), 8.05 (1H, s, H-1), 7.89 (2H, m, H-6, H-7), 7.67 (1H, s, H-4), 2.39 (3H, s, 2-CH3). 13C NMR (Acetone-d6, 75.5 MHz): 182.6 (C=O, C-10), 181.5 (C=O, C-9), 161.0 (C-OH, C-3), 134.1 (C-2), 133.8 (C-14), 133.7 (C-7), 133.6 (C-6), 132.2 (C-13), 130.1 (C-1),126.6 (C-5), 126.5 (C-8), 111.4 (C-4), 15.6 (2-CH3)

Rubiadin-1-methyl ether (**22**). Light yellow crystal. Mps 302-304 ͦ C [282-284, Briggs (1976); 300 ͦ C, Roberts (1977)]. UVλmax EtOH nm: 354, 332, 279. UVλmax EtOH/ - OH nm: 440, 314, 246. IR νmax (KBr) cm-1: 3437, 2913, 2847, 1668, 1651. MS m/z : 268, 239, 207, 181. 1H NMR (Acetone-d6, 300MHz): 9.50 (1H, s, br, 3-OH), 8.20 (2H, m, H-5, H-8), 7.87 (2H, m, H-6, H-7), 7.63 (1H, s, H-4), 3.90 (3H, s, 1-OCH3), 2.27 (3H, s, 2-CH3). 13C NMR (Acetoned6, 75.5 MHz): 182.7 (C=O), 180.4 (C=O), 161.2 (C-OH), 140.6, 134.4, 134.2, 133.0, 132.6, 126.8, 126.0, 60.4 (OCH3), 8.4 (CH3)

3-Hydroxy-2-hydroxymethyl-9,10-anthraquinone (**23**). Light yellow solid . UVλmax EtOH nm: 374, 274, 238. UVλmax EtOH/ -OH nm: 481, 311, 246 IR νmax (KBr) cm-1: 3468, 1628. 1H NMR (Acetone-d6, 300MHz): 8.38 (1H, s, H-4), 8.24 (2H, m, H-5, H-8), 7.89 (2H, m, H-6, H-7), 7.63 (1H, s, H-1), 4.803 (2H,s, 2-CH2OH). 13C NMR (Acetoned6, 75.5 MHz): 182.9 (C=O, C-9), 181.7 (C=O, C-10), 160.1 (C-OH, C-3), 136. 1 (C-2), 134.2 (C-14), 134.1 (C-11), 133.8 (C-6), 133.7 (C-7), 133.6 (C-12), 126.7 (C-4), 126.6 (C-5), 125.9 (C-13), 125.6 (C-8), 111.5 (C-1), 59.0 (2-**CH2**OH)

Table 3.

132.4, 126.8, 126.5, 117.9, 107.17, 7.3

126.5 (C-8), 111.4 (C-4), 15.6 (2-CH3)

Rubiadin (**20**). Yellow crystals. Mps 250-258 °C [lit. 280- 283 ͦ C, Leistner (1975)]. UVλmax EtOH nm: 413, 279. UVλmax EtOH/ -OH nm: 496, 314, 241 IR νmax (KBr) cm-1: 3436, 1653, 1626. MS m/z: 254 [M+], 226, 197, 152, 115. 1H NMR (Acetone-d6, 300MHz): 13.20 (1H, s, 1-OH), 8.31 (1H, m, H-8), 8.23 (1H, m, H-5), 7.92 (2H, m, H-6, H-7), 7.38 (1H, s, H), 2.20 (3H, s, 2-CH3). 13C NMR (Acetone-d6, 75.5 MHz): 186.9 (C=O,C9), 181.8 (C=O, C-10), 163.2 (C-OH, C-1), 162.4 (C-OH, C-3), 134.3, 134.2, 133.5, 133.5,

3-Hydroxy-2-methyl-9,10-anthraquinone (**21**). Yellow crystals. Mps 138- 142 ͦ C. UVλmax EtOH nm: 379, 329, 274, 245, 239. UVλmax EtOH/ -OH nm: 496, 314, 246 . IR νmax (KBr) cm-1: 3436, 1663, 651. MS m/z 238 [M+], 238, 210, 181, 152, 105. 1H NMR (Acetone-d6, 300MHz): 8.23 (2H, m, H-5, H-8), 8.05 (1H, s, H-1), 7.89 (2H, m, H-6, H-7), 7.67 (1H, s, H-4), 2.39 (3H, s, 2-CH3). 13C NMR (Acetone-d6, 75.5 MHz): 182.6 (C=O, C-10), 181.5 (C=O, C-9), 161.0 (C-OH, C-3), 134.1 (C-2), 133.8 (C-14), 133.7 (C-7), 133.6 (C-6), 132.2 (C-13), 130.1 (C-1),126.6 (C-5),

Rubiadin-1-methyl ether (**22**). Light yellow crystal. Mps 302-304 ͦ C [282-284, Briggs (1976); 300 ͦ C, Roberts (1977)]. UVλmax EtOH nm: 354, 332, 279. UVλmax EtOH/ - OH nm: 440, 314, 246. IR νmax (KBr) cm-1: 3437, 2913, 2847, 1668, 1651. MS m/z : 268, 239, 207, 181. 1H NMR (Acetone-d6, 300MHz): 9.50 (1H, s, br, 3-OH), 8.20 (2H, m, H-5, H-8), 7.87 (2H, m, H-6, H-7), 7.63 (1H, s, H-4), 3.90 (3H, s, 1-OCH3), 2.27 (3H, s, 2-CH3). 13C NMR (Acetoned6, 75.5 MHz): 182.7 (C=O), 180.4 (C=O), 161.2 (C-OH), 140.6, 134.4, 134.2, 133.0, 132.6, 126.8, 126.0, 60.4 (OCH3),

3-Hydroxy-2-hydroxymethyl-9,10-anthraquinone (**23**). Light yellow solid . UVλmax EtOH nm: 374, 274, 238. UVλmax EtOH/ -OH nm: 481, 311, 246 IR νmax (KBr) cm-1: 3468, 1628. 1H NMR (Acetone-d6, 300MHz): 8.38 (1H, s, H-4), 8.24 (2H, m, H-5, H-8), 7.89 (2H, m, H-6, H-7), 7.63 (1H, s, H-1), 4.803 (2H,s, 2-CH2OH). 13C NMR (Acetoned6, 75.5 MHz): 182.9 (C=O, C-9), 181.7 (C=O, C-10), 160.1 (C-OH, C-3), 136. 1 (C-2), 134.2 (C-14), 134.1 (C-11), 133.8 (C-6), 133.7 (C-7), 133.6 (C-12), 126.7 (C-4), 126.6 (C-5), 125.9 (C-13), 125.6 (C-8), 111.5 (C-1), 59.0 (2-**CH2**OH)

O

OH

CH3

OH

CH3

OH

OCH3

CH3

OH

CH2OH

OH

8.4 (CH3)

O

O

O

O

O

O

O

Table 3.

**(23)** 

**(22)** 

**(21)** 

**(20)** 

which showed a 3*J* correlation with H-7. The methine carbons (C-3, C-4, C-5, C-7 and C-8) were assigned through HMQC correlations while the quaternary carbons (C-1, C-2, C-6, C-11, C-12, C-13 and C-14) were assigned based on careful analysis of HMBC spectrum. Both carbonyl carbons in this compound resonated very closely to each other with only 0.01 ppm difference at δC 182.70 and 182.71, which further confirmed the unchelated nature of the carbonyls. Presented below are structures and spectroscopic data of the isolated compounds.

#### **3.2 Anthraquinones of** *Morinda elliptica*

*Morinda elliptica* or locally known as 'mengkudu kecil' is a shrub or small tree and it is very common in wild state of Malay Peninsula and northwards Burma (Burkill, 1966). It can be seen growing wild in newly developed areas, bushes and lowland secondary forest throughout the peninsula. *M. elliptica* is very common and always available and mostly used by the Malays for medicinal purposes. Traditionally, different parts of the plant are used in various ways for a number of health problems and ailments. The leaves may be added to rice for loss of appetite and taken for headache, cholera, diarrhea and wounds. Sometimes a lotion is made and used for hemorrhoid and applied upon body after childbirth (Burkill, 1966). The extracts and anthraquinones isolated from *M. elliptica* were reported to possess wide spectrum of biological activities such as antioxidant (Ismail*, et al.*, 2002; Jasril*, et al.*, 2003), antimicrobial, anti-HIV and anticancer (Ali*, et al.*, 2000).

Fig. 5. *Morinda elliptica*

Five anthraquinones in roots of *M. elliptica* which are nordamnacanthal **13**, damnacanthal **15**, lucidin-ω-methyl ether **17,** rubiadin **20** and rubiadin-l-methyl ether **22** are the same constituents found in *R. elliptica*. The others are 1-hydroxy-2-methylanthraquinone **23**, soranjidiol **25**, morindone **26**, morindone-5- methyl ether **27** and alizarin-1-methyl ether **28.** In addition, 2-formyl-1-hydroxyanthraquinone **24** was reported as a new naturally occuring anthraquinone from roots of *M. elliptica*. HR-MS of **24** showed molecular ion peak at 252.0414 consistent with molecular formula of C15H14O4. A bathchromic shift (407 to 531 nm) upon adding NaOH suggested the presence of OH at C-1 of the anthraquinone skeleton. The presence of hydroxyl group was evident from the broad stretching band observed at 3448 cm-1. Two sharp stretching vibrations due to chelated and unchelated carbonyls were observed at 1638 and 1676 cm-1, respectively. In the proton NMR, the signal for chelated hydroxyl group is at δH 13.26. The splitting pattern of 1H NMR suggest substitution pattern

Alkaloids and Anthraquinones from Malaysian Flora 303

62.3 (OCH3)

on ring C only. H-3 and H-4 appeared as doublets at δH 8.23 and 7.89 respectively. A formyl group (δH 10.63) is attached to C-2. HMBC correlations of C-10 with H-3 and H-5 confirmed the assignment of the protons at their respective positions and supported by their respective COSY correlations. 13C NMR showed fifteen carbons peaks as expected. One of the chelated carbonyl carbon was further downfield at δC 188.9 (C-9), confirming the chelated nature of this carbonyl. The assignment of carbons were accomplished using FGHMQC and FGHMBC experiment. Presented below are structures and spectroscopic data of the isolated compounds.

The phytochemical study on *Fissistigma latifolium* and *Meiogyne virgata* (Annonaceae) yielded twelve alkaloids; (-)-*N*-methylguattescidine **1**, liriodenine **2**, lanuginosine **3**, (-)-asimilobine **4**, dimethyltryptamine **5**, (-)-remerine **6**, (-)-anonaine **7**, columbamine **8**, lysicamine **9**, nornuciferine **10**, norushinsunine **11** and cleistopholine **12**. Tryptamine alkaloids have never been reported from *Fissistigma* species, whereas (-)-*N*-methylguattescidine **1** represents a rare finding of a naturally occurring 6a-methylated-7-oxo-aporphine alkaloid. Alkaloids **3**, **6**,

*Rennellia* and *Morinda* are often confused with each other due to their similar traditional usage. Both plants are traditionally used for fever, postpartum and body ache treatment. Our phytochemical study on roots extract of *R. elliptica* showed significant similarities of major anthraquinones with those found in Morinda species. The major constituents of *R. elliptica*, nordamnacanthal, damnacanthal, rubiadian, rubiadin methyl ether and lucidin-ω-

Universiti Teknologi MARA, Universiti of Malaya, Ministry of Higher Education for Research grants and Ministry of Science, Technology and Innovation Malaysia for scholarship awarded

O

OCH3

OH

**9** and **10** have never been reported from *Meiogyne* species.

methyl ether are also present in *M. elliptica* and *M. citrifolia*.

O

**4. Conclusion** 

**5. Acknowledgment** 

to Alias,A.

**(28)** 

(1H, s, 6-OH), 4.03 (3H, s, OCH3), 2.37 (3H, s, CH3). 13C NMR (CDCl3, 125 MHz): 187.8 (C=O), 182.0 (C=O), 160.6 (C-OH), 155.9, 146.8, 136.9, 134.5, 132.3, 127.1, 125.9, 125.5,

Alizarin-1-methyl ether. Yellow-orange crystals (**28**). Mp 164 [lit 178-179 ͦ C, Chang & Lee (1984)]. UVλmax EtOH nm: 313, 378, 485. UVλmax EtOH/ -OH nm: 315, 333, 493. IR νmax (KBr) cm-1: 3443 (OH), 2926, 1671 (C=O unchelated), 1589 (C=C) aromatic. MS m/z 254 (M+), 236, 208, 183. 1H NMR (DMSO-d6, 500MHz): 8.28 (2H, m, H-5, H-8), 8.15 (1H, d, J= 8.55 Hz, H-4), 7.78 (2H, m, H-7, H-6), 7.37 (1H, d, J = 8.54 Hz, H-3), 6.70 (1H, s, 2-OH), 4.04 (3H, s, OCH3). 13C NMR (DMSO-d6 125 MHz): 182.7 (C=O), 182.1 (C=O), 155.5, 146.6, 131.4, 133.9, 132.9, 127.5, 127.1, 126.8, 125.8, 125.6, 120.2,

112.0, 118.9, 114.7, 62.3 (OCH3), 16.1 (CH3)

2-Formyl-1-hydroxy-9,10-anthraquinone (**24**). Mps 183-185 ͦ C [lit. 259-260 ͦ C, Rath et al. (1995)]. UVλmax EtOH nm: 229, 278, 331, 407. UVλmax EtOH/ -OH nm: 229, 280, 308, 531. IR νmax (KBr) cm-1: 3448 (OH), 1696 (aldehyde), 1676 (C=O unchelated), 1638 (C=O chelated), 1592 (C=C aromatic). MS m/z 252 (M+), 2224, 196, 168. 1H NMR (CDCl3, 500MHz): 13.26 (1H,s, 1-OH), 10.63 (1H, s, CHO), 8.35 (1H, m, H-8), 8.32 (1H, m, H-5), 8.23 (1H, d, J= 8.0 Hz, H-3), 7.89 (1H, d, J= 8.0 Hz, H-4), 7.88 (2H, m, H-6, H-7). 13C NMR(CDCl3, 125 MHz): 164.5 (C-1), 128.4 (C-2), 135.4 (C-3), 118.7 (C-4), 127.7 (C-5), 134.7 (C-6), 135.3 (C-7), 127.2 (C-8), 188.9 (C-9), 181.8 (C-10), 117.4 (C-11), 137.2 (C-12), 134.8 (C-13), 133.3 (C-14) and 188.0 (C-15)

Soranjidiol. Yellow-orange neddles (**25**) Mps 276-273 ͦ C [lit. 271-272 ͦ C, Adesogan (1973)]. UVλmax EtOH nm: 265, 409. UVλmax EtOH/ -OH nm: 308, 489. IR νmax (KBr) cm-1: 3401 (OH), 1667 (C=O unchelated), 1635 (C=O chelated), 1593 (C=C aromatic). MS m/z 254 (M+), 226, 197, 115. 1H NMR (DMSO-d6, 500 MHz): 13.10 (1H,s, 1-OH), 11.21 (1H, s, 6- OH), 7.63 (1H, d, J= 7.57 Hz, H-3), 7.57 (1H, d, J=7.57 Hz, H-4), 7.25 (1H, dd, J7,8 = 8.55 Hz, J7,5 = 2.69 Hz, H-7), 7.45 (1H, d, J= 2.69 Hz, H-5), 2.27 (3H, s, CH3). 13C NMR (DMSO-d6, 125 MHz): 187.6 (C=O), 181.8 (C=O), 163.8 (C-OH), 160.0 (C-OH), 136.9, 135.6, 134.2, 131.1, 129.8, 124.5, 121.4, 118.6, 114.7, 112.5, 15.8 (CH3)

Morindone (**26**). Orange needles. Mps 240-241 ͦ C (CHCl3) [lit. 248-249.5 ͦ C, Leistner (1975)]. UVλmax EtOH nm: 260, 299, 448. UVλmax EtOH/ -OH nm: 260, 302, 338, 558. IR νmax (KBr) cm-1: 3462 (OH), 1628 (C=O chelated). MS m/z 270 (M+), 242, 135.1H NMR (CDCl3, 500MHz): 13.21 (1H, s, 1- OH), 12.95 (1H, s, 5-OH), 7.85 (1H, d- J=8.2 Hz, H-8), 7.75 (1H, d, J= 7.7 Hz, H-4), 7.52 (1H, d, J=7.6Hz, H-3), 7.26 (1H, d, J=8.2 Hz, H-7), 6.32 (1H, s, 6-OH), 2.39 (3H, s, CH3). 13C NMR (CDCl3, 125 MHz): 186.6 (C=O),179.9 (C=O), 170.4 (C-OH), 169.8 (C-OH), 161.4 (C-OH), 151.3, 149.4, 136.6, 130.9, 121.3, 119.8, 118.9, 115.3, 16.3 (CH3)

Morindone-5-methyl ether (**27**). Orange cystals. Mp 232 ͦ C [lit. 223 ͦ C, Chang & Lee (1984)]. UVλmax EtOH nm: 410, 497. UVλmax EtOH/ -OH nm: 314, 388, 498. IR νmax (KBr) cm-1: 3389 (OH), 2926, 1672 (C=O unchelated), 1630 (C=O chelated), 1581 (C=C aromatic). MS m/z 284 (M+), 266, 238, 197. 1H NMR (CDCl3, 500MHz): 13.02 (1H, s, 1-OH),8.14 (1H, d, J=8.55 Hz, H-8), 7.70 (1H, d, J= 8.06 Hz, H-4), 7.51 (1H, d, J=7.81 Hz, H-3), 7.35 (1H, d, J=8.54 Hz, H-7), 6.73

2-Formyl-1-hydroxy-9,10-anthraquinone (**24**). Mps 183-185 ͦ C [lit. 259-260 ͦ C, Rath et al. (1995)]. UVλmax EtOH nm: 229, 278, 331, 407. UVλmax EtOH/ -OH nm: 229, 280, 308, 531. IR νmax (KBr) cm-1: 3448 (OH), 1696 (aldehyde), 1676 (C=O unchelated), 1638 (C=O chelated), 1592 (C=C aromatic). MS m/z 252 (M+), 2224, 196, 168. 1H NMR (CDCl3, 500MHz): 13.26 (1H,s, 1-OH), 10.63 (1H, s, CHO), 8.35 (1H, m, H-8), 8.32 (1H, m, H-5), 8.23 (1H, d, J= 8.0 Hz, H-3), 7.89 (1H, d, J= 8.0 Hz, H-4), 7.88 (2H, m, H-6, H-7). 13C NMR(CDCl3, 125 MHz): 164.5 (C-1), 128.4 (C-2), 135.4 (C-3), 118.7 (C-4), 127.7 (C-5), 134.7 (C-6), 135.3 (C-7), 127.2 (C-8), 188.9 (C-9), 181.8 (C-10), 117.4 (C-11), 137.2 (C-12),

Soranjidiol. Yellow-orange neddles (**25**) Mps 276-273 ͦ C [lit. 271-272 ͦ C, Adesogan (1973)]. UVλmax EtOH nm: 265, 409. UVλmax EtOH/ -OH nm: 308, 489. IR νmax (KBr) cm-1: 3401 (OH), 1667 (C=O unchelated), 1635 (C=O chelated), 1593 (C=C aromatic). MS m/z 254 (M+), 226, 197, 115. 1H NMR (DMSO-d6, 500 MHz): 13.10 (1H,s, 1-OH), 11.21 (1H, s, 6- OH), 7.63 (1H, d, J= 7.57 Hz, H-3), 7.57 (1H, d, J=7.57 Hz, H-4), 7.25 (1H, dd, J7,8 = 8.55 Hz, J7,5 = 2.69 Hz, H-7), 7.45 (1H, d, J= 2.69 Hz, H-5), 2.27 (3H, s, CH3). 13C NMR (DMSO-d6, 125 MHz): 187.6 (C=O), 181.8 (C=O), 163.8 (C-OH), 160.0 (C-OH), 136.9, 135.6, 134.2, 131.1, 129.8, 124.5,

Morindone (**26**). Orange needles. Mps 240-241 ͦ C (CHCl3) [lit. 248-249.5 ͦ C, Leistner (1975)]. UVλmax EtOH nm: 260, 299, 448. UVλmax EtOH/ -OH nm: 260, 302, 338, 558. IR νmax (KBr) cm-1: 3462 (OH), 1628 (C=O chelated). MS m/z 270 (M+), 242, 135.1H NMR (CDCl3, 500MHz): 13.21 (1H, s, 1- OH), 12.95 (1H, s, 5-OH), 7.85 (1H, d- J=8.2 Hz, H-8), 7.75 (1H, d, J= 7.7 Hz, H-4), 7.52 (1H, d, J=7.6Hz, H-3), 7.26 (1H, d, J=8.2 Hz, H-7), 6.32 (1H, s, 6-OH), 2.39 (3H, s, CH3). 13C NMR (CDCl3, 125 MHz): 186.6 (C=O),179.9 (C=O), 170.4 (C-OH), 169.8 (C-OH), 161.4 (C-OH), 151.3, 149.4, 136.6,

Morindone-5-methyl ether (**27**). Orange cystals. Mp 232 ͦ C [lit. 223 ͦ C, Chang & Lee (1984)]. UVλmax EtOH nm: 410, 497. UVλmax EtOH/ -OH nm: 314, 388, 498. IR νmax (KBr) cm-1: 3389 (OH), 2926, 1672 (C=O unchelated), 1630 (C=O chelated), 1581 (C=C aromatic). MS m/z 284 (M+), 266, 238, 197. 1H NMR (CDCl3, 500MHz): 13.02 (1H, s, 1-OH),8.14 (1H, d, J=8.55 Hz, H-8), 7.70 (1H, d, J= 8.06 Hz, H-4), 7.51 (1H, d, J=7.81 Hz, H-3), 7.35 (1H, d, J=8.54 Hz, H-7), 6.73

134.8 (C-13), 133.3 (C-14) and 188.0 (C-15)

121.4, 118.6, 114.7, 112.5, 15.8 (CH3)

130.9, 121.3, 119.8, 118.9, 115.3, 16.3 (CH3)

O

OH

CHO

O

**(24)** 

O

OH

OH

OH

OH

CH3

CH3

O

**(25)**

O

O

**(26)** 

O

O

**(27)**

OCH3

OH

HO

HO

HO

(1H, s, 6-OH), 4.03 (3H, s, OCH3), 2.37 (3H, s, CH3). 13C NMR (CDCl3, 125 MHz): 187.8 (C=O), 182.0 (C=O), 160.6 (C-OH), 155.9, 146.8, 136.9, 134.5, 132.3, 127.1, 125.9, 125.5, 112.0, 118.9, 114.7, 62.3 (OCH3), 16.1 (CH3)

Alizarin-1-methyl ether. Yellow-orange crystals (**28**). Mp 164 [lit 178-179 ͦ C, Chang & Lee (1984)]. UVλmax EtOH nm: 313, 378, 485. UVλmax EtOH/ -OH nm: 315, 333, 493. IR νmax (KBr) cm-1: 3443 (OH), 2926, 1671 (C=O unchelated), 1589 (C=C) aromatic. MS m/z 254 (M+), 236, 208, 183. 1H NMR (DMSO-d6, 500MHz): 8.28 (2H, m, H-5, H-8), 8.15 (1H, d, J= 8.55 Hz, H-4), 7.78 (2H, m, H-7, H-6), 7.37 (1H, d, J = 8.54 Hz, H-3), 6.70 (1H, s, 2-OH), 4.04 (3H, s, OCH3). 13C NMR (DMSO-d6 125 MHz): 182.7 (C=O), 182.1 (C=O), 155.5, 146.6, 131.4, 133.9, 132.9, 127.5, 127.1, 126.8, 125.8, 125.6, 120.2, 62.3 (OCH3)

on ring C only. H-3 and H-4 appeared as doublets at δH 8.23 and 7.89 respectively. A formyl group (δH 10.63) is attached to C-2. HMBC correlations of C-10 with H-3 and H-5 confirmed the assignment of the protons at their respective positions and supported by their respective COSY correlations. 13C NMR showed fifteen carbons peaks as expected. One of the chelated carbonyl carbon was further downfield at δC 188.9 (C-9), confirming the chelated nature of this carbonyl. The assignment of carbons were accomplished using FGHMQC and FGHMBC experiment. Presented below are structures and spectroscopic data of the isolated compounds.

#### **4. Conclusion**

The phytochemical study on *Fissistigma latifolium* and *Meiogyne virgata* (Annonaceae) yielded twelve alkaloids; (-)-*N*-methylguattescidine **1**, liriodenine **2**, lanuginosine **3**, (-)-asimilobine **4**, dimethyltryptamine **5**, (-)-remerine **6**, (-)-anonaine **7**, columbamine **8**, lysicamine **9**, nornuciferine **10**, norushinsunine **11** and cleistopholine **12**. Tryptamine alkaloids have never been reported from *Fissistigma* species, whereas (-)-*N*-methylguattescidine **1** represents a rare finding of a naturally occurring 6a-methylated-7-oxo-aporphine alkaloid. Alkaloids **3**, **6**, **9** and **10** have never been reported from *Meiogyne* species.

*Rennellia* and *Morinda* are often confused with each other due to their similar traditional usage. Both plants are traditionally used for fever, postpartum and body ache treatment. Our phytochemical study on roots extract of *R. elliptica* showed significant similarities of major anthraquinones with those found in Morinda species. The major constituents of *R. elliptica*, nordamnacanthal, damnacanthal, rubiadian, rubiadin methyl ether and lucidin-ωmethyl ether are also present in *M. elliptica* and *M. citrifolia*.

#### **5. Acknowledgment**

Universiti Teknologi MARA, Universiti of Malaya, Ministry of Higher Education for Research grants and Ministry of Science, Technology and Innovation Malaysia for scholarship awarded to Alias,A.

Alkaloids and Anthraquinones from Malaysian Flora 305

Kan, W. S. (1979). In *Pharmaceutical Botany*; National Research Institute of Chinese Medicine:

Kam, T. S. (1999). Alkaloids from Malaysian Flora, in Alkaloids: Chemical and Biological

Kamaruddin, M. S. (1998). Proceedings Malaysian Traditional Medicine, 10-11 June 1998,

Kong, J.-M., Goh, N.-K., & Chia, T.-F. (2003). Recent Advances in Traditional Plant Drugs

Lajis, N. H., Ahmad, R., & Atta-ur, R. (2006). Phytochemical studies and pharmacological

Lavault, M., Cabalion, P. and Bruneton, J. (1981). Alkaloids of Uncaria guianensis. *Planta* 

Lee, H. H. (1969). Colouring matters from Prismatomeris malayana. *Phytochemistry, 8*(2),

Leistner,E.,1975. Isolation,identifica tion and biosynthesis of anthraquinones in cell suspension cultures of Morinda citrifolia. Planta Med. (Suppl.) 214–224. Mat Salleh, K., & Latiff, A. (2002). *Tumbuhan Ubatan Malaysia*: Universiti Kebangsaan

Nik Idris, Y., Lim, S. Y., Zaemah, J. and Ikram, M. S. (1994). *Alkaloid daripada Batang* 

Osman, C. P., Ismail, N. H., Ahmad, R., Ahmat, N., Awang, K., & Jaafar, F. M. (2010).

Rath, G., Ndonzao, M., & Hostettmann, K. (1995). Antifungal Anthraquinones from *Morinda* 

Roberts, J. L., Rutledge, P. S., and Trebilcock, M. J. (1977). Experiments Directed Towards the

Saaid M., and Awang, K. (2005) Alkaloids of *Fissistigma manubriatum*. Malaysian *Journal of* 

Sinclair, J., (1955) A revision of the Malayan Annonaceae. *The Gardens' Bulletin Singapore,* 14,

Tadic, D., Cassels, B. K., Leboeuf, M. and Cavé, A. (1987). Kinabaline and the aporphinoid biogenesis azaanthracene and azafluorene alkaloids. *Phytochemistry*, 26, 537–541. Teo, L.E., Pachiaper, G., Chan, K.C., Hadi, H.A., Weber, J.F., Deverre, J.R., David, B. &

screening and plant chemical studies. *J. Ethnopharmacol.* 28(1) : 63-101. Verdout, B. (1976). *Annonaceae, Flora of Tropical East Africa*; Crown Agents for Oversea

Government and Administrations: London, UK, pp. 101–102.

*Fissistigma latifolium (Annonaceae) dan Potensinya Sebagai Dadah Anti-Leukaemia*,

Anthraquinones with Antiplasmodial Activity from the Roots of Rennellia elliptica

Synthesis of Anthracyclinones. I Synthesis of 2-Formylmethoxyanthraquinones

Sevenet, T. (1990). A new phytochemical survey of Malaysia V. Preliminary

*Biotechnology, 11*(1), 3-7.

Universiti Malaya, Kuala Lumpur, 80.

and Orchids. *Acta Pharmacologica Sinica, 24*(1), 17-21.

Malaysia & Kem, Sains, Teknologi dan Alam Sekitar.

Korth. (Rubiaceae). *Molecules, 15*(10), 7218-7226. Parker, Sybil P. (1997). *Chemistry; Dictionaries*. McGraw-Hill (New York). Perry, L. M. (1980). *Medicinal Plants of Southeast Asia.* MIT: Cambridge, 19.

Laporan Teknik FSFG 4 : 199-204

*lucida*. *Int J. Pharmacogn., 33*, 107-114.

*Aust. J. Chem.,* , 30, 1553.

*Science*. 24 (1), 41-45.

149-69.

*Chemistry* (Vol. Volume 33, Part 13, pp. 1057-1090): Elsevier.

Taiwan, 268.

285-435.

*Med*. 42, 50.

501-503.

Suspension Culture of *Morinda elliptica*. *Asia Pacific Journal of Molecular Biology and* 

Perspectives, S. W. Pelletier (Ed.), Pergamon, Amsterdam, Volume 14, Chapter 2,

activities of plants in genus Hedyotis/oldenlandia *Studies in Natural Products* 

#### **6. References**


Abd Aziz, R. (2003). *Siri Syarahan Perdana Professor*. Skudai: Universiti Teknologi

Adesogan, E.K. (1973). Anthraquinones and anthraquinols from *Morinda lucida*. The biological significance of oruwal and oruwalol, *Tetrahedron* 29 (1973), p. 4099. Ahmad, R., Mahbob, E. N. M., Noor, Z. M., Ismail, N. H., Lajis, N. H., & Shaari, K. (2010).

Ahmad, R., Shaari, K., Lajis, N. H., Hamzah, A. S., Ismail, N. H., & Kitajima, M. (2005). Anthraquinones from *Hedyotis capitellata*. *Phytochemistry, 66*(10), 1141-1147. Ali, A. M., Ismail, N. H., Mackeen, M. M., Yazan, L. S., Mohamed, S. M., Ho, A. S. H., et al.

Awang, K.; Hamid, A.; Hadi, A. (2000). Protoberberine Alkaloids From *Fissistigma fulgens*

Briggs, L.H., Beachen, J.F., Cambie, R.C., Dudman, N.P.B., Steggles, A.W. & Rutledge, P.S.

Burkill, I. H. (1966). *A Dictionary of the Economic Product of the Malay Peninsular*. Volumes I &

Chan, K. C. & Toh, H. T. (1985). A new aporphinoid from *Desmos dasymachallus*. In:

Chang, P. and Lee, K., Cytotoxic antileukemic anthraquinones from *Morinda parvifolia*.

Connolly, J. D., Haque, M. D. E., Kadir, A. A. (1996): Two 7,7-bisdehydroaporphine

Cordell, G.A. (1981). *Introduction to Alkaloid: A Biogenetic Approach;* John Wiley & Sons: New

Derksen, G. C. H., Van Beek, T. A., & Atta ur, R. (2002). Rubia tinctorum L *Studies in Natural* 

Hamzah, A. S., & Lajis, N. H. (1998). Chemical Constituents of Hedyotis herbaceae. *ARBEC,* 

Han, Y.-S., der Heiden, R. V., & Verpoorte, R. (2001). Biosynthesis of Anthraquinones in Cell Cultures of the Rubiaceae. *Plant Cell, Tissue and Organ Culture, 67*, 201-220. Hutchinson, J. (1973). *The Families of Flowering Plants* (3rd ed.): Oxford University Press. Ismail, N. H., Ali, A. M., Aimi, N., Kitajima, M., Takayama, H., & Lajis, N. H. (1997). Anthraquinones from *Morinda elliptica*. *Phytochemistry, 45*(8), 1723-1725. Ismail, N. H., Mohamad, H., Mohidin, A., & Lajis, N. H. (2002). Antioxidant Activity of Anthraquinones from *Morinda elliptica*. *Natural Product Science, 8*(2), 48-51. Jasril, Lajis, N. H., Lim, Y. M., Abdullah, M. A., Sukari, M. A., & Ali, A. M. (2003). Antitumor

Promoting and Antioxidant Activities of Anthraquinones Isolated from the Cell

*Fissistigma latifolium* Dunal Merr. *Molecules*, 15, 4583-4588.

II. Ministry of Agriculture and Cooperatives, Kuala Lumpur.

alkaloids from *Polyalthia bullata*. *Phytochemistry* 43: 295–297.

*Products Chemistry* (Vol. Volume 26, Part 7, pp. 629-684): Elsevier.

Merr. (Annonaceae). *Malaysian J. Sci.*, *19*, 41–44.

species. *J. Chem. Soc. Perkin Trans. I*, 1789–1792

*Phytochemistry*, 23, 1733-1736. (1984).

Evaluation of antioxidant potential of medicinal plants from Malaysian Rubiaceae (subfamily Rubioideae). *African Journal of Biotechnology, 9*(46), 7948-

(2000). Antiviral, Cytotoxic and Antimicrobial Activities of Anthraquinones Isolated from the Root of *Morinda elliptica*. *Pharmaceutical Biology, 38*, 298-301. Alias, A., Hazni, H., Mohd Jaafar, F., Awang, K. and Ismail, N. H. (2010). Alkaloids from

(1976) Chemistry of *Coprosma* genus. Part XIV. Constituents of five New Zealand

*Proceedings of the 2nd Meeting of the Natural Products Group*, edited by Said, I.M. & Zakaria, Z. 17-20. Jabatan Kimia, Fakulti Fizis dan Gunaan, Universiti Kebangsaan

**6. References** 

Malaysia.

7954.

Malaysia, Bangi.

York, NY, USA, 6–19.

*Article II*, 1-6.

Suspension Culture of *Morinda elliptica*. *Asia Pacific Journal of Molecular Biology and Biotechnology, 11*(1), 3-7.


**Phytochemistry of some Brazilian Plants** 

Cinara V. da Silva, Fernanda M. Borges and Eudes S. Velozo

Since time immemorial man has used various parts of plants in the treatment and prevention of many ailments, including sexual impotence (Ayyanar & Ignacimuthu, 2009 as cited in Chah et al., 2006). Ancient people knew about herbal and animal aphrodisiacs, used in combinations like potions to mystical rites to infertility, to increase sexual performance,

One of the first mentions of aphrodisiacs is in the Egyptian papyruses from 2300 to 1700 B.C. In the papyrus of Ebers, mandragora, garlic, onion and blue lotus were found as plants with

The tomb of Tutankhamon contain a gold plated shrine decorated with a bas-relief of a pharaoh holding a blue lotus and two mandragoras in his left hand, since the Egyptians

Hindu poems dating from 2000 to 1000 B.C. and the Kama Sutra had already reported to the use of some products to enhance the sex (Zanolari, 2003). The traditional Chinese Medicine uses with aphrodisiac purpose, among others, ginseng, Chinese chive and parts of animals

On this basis, the legendary love potions, such as Spanish fly, glandular products from musk deer and civet cats, varieties of natural oats (*Avena sativa*), ginseng, belladonna, and erotic foods like fish and oysters, are known aphrodisiacs (Drewes et al., 2003 as cited in

The word aphrodisiac has its origin in Greek Mythology, most precisely from the goddess of love, Aphrodite. It has been used to define the products applied with proposal of increasing desire and drive associated with sexual instinct. Besides they have represented a passion of man, since historically, in all cultures, the sexual potency is considered as a significant part of the male ego and the anxiety and humiliation is frequently associated with a declining

An aphrodisiac includes any food or drug that arouses the sexual instinct, induces venereal desire and increases pleasure and performance. There are two main types of aphrodisiacs: psychophysiological stimuli (visual, tactile, olfactory and aural) preparations and internal

preparations (food, alcoholic drinks and love potion) (Malviya et al., 2011).

**1. Introduction**

desire and pleasure (Malviya et al., 2011).

believed in sexual life after death (Bertol et al., 2004).

sexual ability (Malviya et al., 2011; Zanolari, 2003).

for example: dogs, rhino, bear and tiger penis and testicles (Still, 2003).

aphrodisiac activity (Zanolari, 2003).

Choudhary & Ur-Rahman, 1997).

**with Aphrodisiac Activity** 

*Federal University of Bahia,* 

*Brazil* 

Wong, K. M. (1989). Rubiaceae (from the genus Rubia). In F. S. P. Ng (Ed.), *Tree Flora of Malaya; A Manual for Foresters* (Vol. 4, pp. 324-337, 404-405): Longman Malaysia. **15** 

## **Phytochemistry of some Brazilian Plants with Aphrodisiac Activity**

Cinara V. da Silva, Fernanda M. Borges and Eudes S. Velozo *Federal University of Bahia, Brazil* 

#### **1. Introduction**

306 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Wong, K. M. (1989). Rubiaceae (from the genus Rubia). In F. S. P. Ng (Ed.), *Tree Flora of* 

Since time immemorial man has used various parts of plants in the treatment and prevention of many ailments, including sexual impotence (Ayyanar & Ignacimuthu, 2009 as cited in Chah et al., 2006). Ancient people knew about herbal and animal aphrodisiacs, used in combinations like potions to mystical rites to infertility, to increase sexual performance, desire and pleasure (Malviya et al., 2011).

One of the first mentions of aphrodisiacs is in the Egyptian papyruses from 2300 to 1700 B.C. In the papyrus of Ebers, mandragora, garlic, onion and blue lotus were found as plants with aphrodisiac activity (Zanolari, 2003).

The tomb of Tutankhamon contain a gold plated shrine decorated with a bas-relief of a pharaoh holding a blue lotus and two mandragoras in his left hand, since the Egyptians believed in sexual life after death (Bertol et al., 2004).

Hindu poems dating from 2000 to 1000 B.C. and the Kama Sutra had already reported to the use of some products to enhance the sex (Zanolari, 2003). The traditional Chinese Medicine uses with aphrodisiac purpose, among others, ginseng, Chinese chive and parts of animals for example: dogs, rhino, bear and tiger penis and testicles (Still, 2003).

On this basis, the legendary love potions, such as Spanish fly, glandular products from musk deer and civet cats, varieties of natural oats (*Avena sativa*), ginseng, belladonna, and erotic foods like fish and oysters, are known aphrodisiacs (Drewes et al., 2003 as cited in Choudhary & Ur-Rahman, 1997).

The word aphrodisiac has its origin in Greek Mythology, most precisely from the goddess of love, Aphrodite. It has been used to define the products applied with proposal of increasing desire and drive associated with sexual instinct. Besides they have represented a passion of man, since historically, in all cultures, the sexual potency is considered as a significant part of the male ego and the anxiety and humiliation is frequently associated with a declining sexual ability (Malviya et al., 2011; Zanolari, 2003).

An aphrodisiac includes any food or drug that arouses the sexual instinct, induces venereal desire and increases pleasure and performance. There are two main types of aphrodisiacs: psychophysiological stimuli (visual, tactile, olfactory and aural) preparations and internal preparations (food, alcoholic drinks and love potion) (Malviya et al., 2011).

Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 309

leads to relaxation of smooth muscle. The erection ceases after a while because cGMP is hydrolysed by phosphodiesterase enzime into inactive GMP. Five types of phosphodiesterases are known to cause hydrolysis in cGMP. In the penis, phosphodiesterase is type V. Thus, a drug that inhibits the phosphodiesterase type V (cGMP-specific) should accelerate the action

The treatment with a psychotherapeutic approach is indicated to patients with psychological disorders. To patients with physical disorders, current treatments include oral

Some oral medications are available and well-established for ED treatment, among of them, two natural products: Cantharidin (Spanish fly) and Yohimbine, besides synthetic selective inhibitors, such as sildenafil (Viagra®), vardenafil (Levitra®), tadalafil (Cialis®), lodenafil (Helleva®) and udenafil (Zydena®) (see fig.2). The PDE-5 inhibitors have shown efficacy compared to placebo, in addition to present similar form of action and side effects like headache, flushing, dyspepsia and nasal congestion (Matheus et al., 2009; Wang et al., 2008). The cantharidin is a lactone found in Spanish flies (also called Cantharides), beetles that have been cited in most of Asian and European Pharmacopoeias and have been used in dried form in internal preparations to impotence. Cantharides acts causing irritation of the urethra with vascular congestion, and inflammation of the erectile tissue. The Spanish flies

Yohimbine is an indole alkaloid with a 2-adrenergic blocking activity. It comes from the bark of the African tree *Corynanthe yohimbe*, its first isolation was in the early 1930s and remained on the African market until 1973 like a drug marketed Aphrodex. Renewed

medication, intracavernosal injection, vacuum pumps and penile prosthesis.

are fallen into disuse due to their toxic effects (Zanolari, 2003).

of nitric oxide and cGMP in erection (Drewes et al., 2003).

Fig. 1. Penis anatomy diagram

**2.1 Male dysfunction therapies** 

Currently, the increase in life expectancy of human beings has increased the demand for substances capable of improving quality of this longevity. Among these are products that enhance sexual performance, treat impotence or erectile dysfunction.

Brazil is the country with around 55,000 species of higher plants about a quarter of all known and greatest biodiversity in the world (Velozo et al., 2002). Many of these plants are used in folk medicine to aphrodisiac purposes in the form of teas, mixed with alcohol and other beverages. Some of them are belonging to the families like Anacardiaceae, Fabaceae, Sapindaceae, Amarantaceae, Amaryllidaceae, Aristolochiaceae, Bignoniaceae, Erythroxylaceae, Oleaceae, Asteraceae, Sapindaceae, Annonaceae and Dilleniaceae.

Several phytochemical studies, with species from these families above cited, have enabled the isolation of secondary metabolites possibly related to its pharmacological activity, such as alkaloids, flavonoids and saponins.

This chapter is a review on the chemical composition of Brazilian plants most used by the population for aphrodisiac purpose, searching rationalization between the chemical structure and biological activity (SAR).

#### **2. Erectile dysfunction and aphrodisiac products**

Erectile dysfunction (ED) is experienced at least some of the time by the most of men who have reached 45 years of age, and it is projected to affect 322 million men worldwide by 2025. This prevalence is high in men of all ages but increases greatly in the elderly (Seftel et al., 2002).

Sexual dysfunction, erectile dysfunction or male impotence is characterized by the inability to develop or maintain an erection of the penis and can be caused by psychological disorders like anxiety, stress and depression, physical disorders like chronic diseases: diabetes and hypertension; hormonal problems or sedentary life-style, alcohol and smoking abuses (Malviya et al., 2011; Sumalatha et al., 2010).

Drugs play a significant role in the pathogenesis of ED, altering hormonal or vascular mechanics needed for erection. Alterations in penile vessels can be observed in the elderly and in particular, lack of androgens may lead to a reduction of smooth muscle cells content in the penis and an increase in the caliber of vascular spaces (Vignera et al., 2011 as cited in Galiano et al., 2010).

An erection is a hemodynamic balance between inflow and outflow of blood within two chambers named corpus cavernosum and it starts with sensory and mental stimulation. There is a relaxation of the smooth muscles and arterioles which allows blood supply to flow in the sinusoidal space. The increased flow of blood, compress venules between sinusoids and the tunica albuginea of the corpus cavernosum. The lack of the distension of tunica albuginea results in venous occlusion, which increases the intracavernosal pressure, generating and sustaining a full erection (Zanolari, 2003).

The erection ends when the muscles of penis contract, opening outflow channels. The relaxation of cavernous smooth muscle is mediated by Nitric Oxide (NO) via cyclic guanosine monophosphate (cGMP). After sexual stimulation, nitric oxide is released by nerve endings and endothelial cells. Nitric oxide (NO) stimulates GMP cyclase to produce cGMP, which

Currently, the increase in life expectancy of human beings has increased the demand for substances capable of improving quality of this longevity. Among these are products that

Brazil is the country with around 55,000 species of higher plants about a quarter of all known and greatest biodiversity in the world (Velozo et al., 2002). Many of these plants are used in folk medicine to aphrodisiac purposes in the form of teas, mixed with alcohol and other beverages. Some of them are belonging to the families like Anacardiaceae, Fabaceae, Sapindaceae, Amarantaceae, Amaryllidaceae, Aristolochiaceae, Bignoniaceae,

Several phytochemical studies, with species from these families above cited, have enabled the isolation of secondary metabolites possibly related to its pharmacological activity, such

This chapter is a review on the chemical composition of Brazilian plants most used by the population for aphrodisiac purpose, searching rationalization between the chemical

Erectile dysfunction (ED) is experienced at least some of the time by the most of men who have reached 45 years of age, and it is projected to affect 322 million men worldwide by 2025. This prevalence is high in men of all ages but increases greatly in the elderly (Seftel et

Sexual dysfunction, erectile dysfunction or male impotence is characterized by the inability to develop or maintain an erection of the penis and can be caused by psychological disorders like anxiety, stress and depression, physical disorders like chronic diseases: diabetes and hypertension; hormonal problems or sedentary life-style, alcohol and smoking

Drugs play a significant role in the pathogenesis of ED, altering hormonal or vascular mechanics needed for erection. Alterations in penile vessels can be observed in the elderly and in particular, lack of androgens may lead to a reduction of smooth muscle cells content in the penis and an increase in the caliber of vascular spaces (Vignera et al., 2011 as cited in

An erection is a hemodynamic balance between inflow and outflow of blood within two chambers named corpus cavernosum and it starts with sensory and mental stimulation. There is a relaxation of the smooth muscles and arterioles which allows blood supply to flow in the sinusoidal space. The increased flow of blood, compress venules between sinusoids and the tunica albuginea of the corpus cavernosum. The lack of the distension of tunica albuginea results in venous occlusion, which increases the intracavernosal pressure,

The erection ends when the muscles of penis contract, opening outflow channels. The relaxation of cavernous smooth muscle is mediated by Nitric Oxide (NO) via cyclic guanosine monophosphate (cGMP). After sexual stimulation, nitric oxide is released by nerve endings and endothelial cells. Nitric oxide (NO) stimulates GMP cyclase to produce cGMP, which

Erythroxylaceae, Oleaceae, Asteraceae, Sapindaceae, Annonaceae and Dilleniaceae.

enhance sexual performance, treat impotence or erectile dysfunction.

as alkaloids, flavonoids and saponins.

structure and biological activity (SAR).

al., 2002).

Galiano et al., 2010).

**2. Erectile dysfunction and aphrodisiac products** 

abuses (Malviya et al., 2011; Sumalatha et al., 2010).

generating and sustaining a full erection (Zanolari, 2003).

leads to relaxation of smooth muscle. The erection ceases after a while because cGMP is hydrolysed by phosphodiesterase enzime into inactive GMP. Five types of phosphodiesterases are known to cause hydrolysis in cGMP. In the penis, phosphodiesterase is type V. Thus, a drug that inhibits the phosphodiesterase type V (cGMP-specific) should accelerate the action of nitric oxide and cGMP in erection (Drewes et al., 2003).

Fig. 1. Penis anatomy diagram

#### **2.1 Male dysfunction therapies**

The treatment with a psychotherapeutic approach is indicated to patients with psychological disorders. To patients with physical disorders, current treatments include oral medication, intracavernosal injection, vacuum pumps and penile prosthesis.

Some oral medications are available and well-established for ED treatment, among of them, two natural products: Cantharidin (Spanish fly) and Yohimbine, besides synthetic selective inhibitors, such as sildenafil (Viagra®), vardenafil (Levitra®), tadalafil (Cialis®), lodenafil (Helleva®) and udenafil (Zydena®) (see fig.2). The PDE-5 inhibitors have shown efficacy compared to placebo, in addition to present similar form of action and side effects like headache, flushing, dyspepsia and nasal congestion (Matheus et al., 2009; Wang et al., 2008).

The cantharidin is a lactone found in Spanish flies (also called Cantharides), beetles that have been cited in most of Asian and European Pharmacopoeias and have been used in dried form in internal preparations to impotence. Cantharides acts causing irritation of the urethra with vascular congestion, and inflammation of the erectile tissue. The Spanish flies are fallen into disuse due to their toxic effects (Zanolari, 2003).

Yohimbine is an indole alkaloid with a 2-adrenergic blocking activity. It comes from the bark of the African tree *Corynanthe yohimbe*, its first isolation was in the early 1930s and remained on the African market until 1973 like a drug marketed Aphrodex. Renewed

Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 311

There are many herbal drugs that have been used by men with ED with varying degrees of success. Most potent aphrodisiacs herbal are available and have few side effects (Malviya et

Some of the genera and species listed in this work in *in vitro* tests showed satisfactory answers to such an aphrodisiac effect like *Turnera diffusa* (Estrada-Reyes et al., 2009), *Pfaffia paniculata* (Arletti et al., 1999), *Passiflora* (Patel et al. 2009), *Mucuna pruriens* (Suresh et al., 2009), *Mimosa pudica* (Pande & Pathak, 2009), *Mimosa tenuiflora* (Souza et al., 2008), *Achyrocline satureioides*  (Hnatyszyn et al, 2004; Simões et al., 1986) and *Anemopaegma arvense* (Chieregatto, 2005).

The effects of the Brazilian herbal medicine Catuama® and each of its plant constituents (*Paullinia cupana, Trichilia catigua, Zingiber officinalis and Ptychopetalum olacoides*) were investigated on rabbit corpus cavernosum. Catuama® induced relaxations, but P*. cupana* was the most effective, increased the cAMP levels by 200% indicating that it is the main

*Achyrocline satureioides* (Asteraceae) Inflorescence Macela do campo

*Aristolochia cymbifera* (Aristolochiaceae) Stem Cipó mil homens *Arrabidaea chica* **(**Bignoniaceae) Leaves Cipó cruz

*Davilla rugosa* (Dilleniaceae) Stem , Leaves Cipó caboclo *Erythroxylum viceniifolium* (Erythroxylaceae) Stem bark Catuaba *Hippeastrum psittacinum* (Amaryllidaceae) Bulbs Alho-bravo

*Mimosa pudica* (Fabaceae) Stem bark Dormideira *Mimosa tenuiflora* (Fabaceae) Stem bark Jurema preta *Mucuna pruriensis* (Fabaceae) Seeds Pó-de-mico

*Nymphaea ampla* (Nymphaeaceae) Whole plant Ninfa branca

*Pfaffia paniculata* (Amarantaceae) Roots Ginseng brasileiro *Ptychopetalum olacoides* (Oleaceae) Bark Marapuama *Schinus terebinthifolius* (Anarcadiaceae) Bark Aroeira vermelha

*Paulinia cupana* (Sapindaceae) Seeds Guaraná

*Trichilia catigua* (Meliaceae) Bark , Leaves Catuaba *Turnera diffusa* (Turneraceae) Leaves Damiana

*Artocarpus integrifolia* (Moraceae) Seeds Jaca

Specie (Family) Part used Popular Name

Pseudo-fruit

Roots

Macela

Carajiru

Alho-do-mato Açucena-do-campo

Mucuna preta

Leaves Maracujá

Catuaba verdadeira Marapuama Alecrim do campo

Caju

extract responsible for the relaxing effect (Antunes et al., 2001).

*Anacardium Ocidentale (*Anacardiaceae) Nut

*Passiflora sp.* (*P. edulis, P. alata and P. caerulea)*

Table 1. Main Brazilian species with aphrodisiac activity

(Passifloraceae)

*Anemopaegma arvense* (Bignoniaceae) Stem bark

al., 2011).

interest in yohimbine for ED has prompted several new investigative trials; however, there are indications of side-effects such as hypertension, anxiety, manic symptoms and interactions with used medications (Drewes et al., 2003).

Some natural products act like non-selective PDE inhibitors as the methylxanthines caffeine and theophylline, but others show similar effects to PDE-5 inhibitors, for example: flavonoids and derivatives (quercetin from *Allium cepa*, pyrano-isoflavones from *Eriosema kraussianum* - Kraussianone 1 and 2); alkaloids (Neferin from *Nelumbo nucifera*, Berberine from *Berberis aristata*, Papaverine from *Papaver somniferum* – used in association with Prostaglandin-E1 to injections intracavernosal), saponins (Steroidal saponins from *Allium tuberosum*), coumarins (Osthole from *Angelica pubescens*) and terpenes (Forskolin from *Coleus forskohlii*) (Drewes et al., 2003; Guohua et al., 2009; Rahimi et al., 2009; Sumalatha et al., 2010; Zanolari, 2003).

Fig. 2. Selective inhibitors of PDE-5

Fig. 3. Examples of natural products with aphrodisiac effect

#### **2.2 Chemical of some Brazilian aphrodisiacs species and rationalization between structure and activity**

The success of PDE-5 inhibitors, particularly of Viagra, the first inhibitor that has been marketed, the aging of the population and the quest for improved quality of life led to the search for new drugs with fewer side effects. As sources of research, plants used as aphrodisiacs have turned to folk medicine in whole world.

interest in yohimbine for ED has prompted several new investigative trials; however, there are indications of side-effects such as hypertension, anxiety, manic symptoms and

Some natural products act like non-selective PDE inhibitors as the methylxanthines caffeine and theophylline, but others show similar effects to PDE-5 inhibitors, for example: flavonoids and derivatives (quercetin from *Allium cepa*, pyrano-isoflavones from *Eriosema kraussianum* - Kraussianone 1 and 2); alkaloids (Neferin from *Nelumbo nucifera*, Berberine from *Berberis aristata*, Papaverine from *Papaver somniferum* – used in association with Prostaglandin-E1 to injections intracavernosal), saponins (Steroidal saponins from *Allium tuberosum*), coumarins (Osthole from *Angelica pubescens*) and terpenes (Forskolin from *Coleus forskohlii*) (Drewes et al., 2003; Guohua et al., 2009; Rahimi et al., 2009; Sumalatha et al., 2010;

interactions with used medications (Drewes et al., 2003).

Zanolari, 2003).

EtO

O2S <sup>N</sup>

N

O O

O

OH O <sup>O</sup> OH

Cantharidin

O O

**structure and activity** 

Sildenafil

O

N

N <sup>N</sup> NH O

Fig. 2. Selective inhibitors of PDE-5

MeO

MeO

EtO

O2S <sup>N</sup>

N

N

Vardenafil

Papaverine

Kraussianone 1 Kraussianone 2

Fig. 3. Examples of natural products with aphrodisiac effect

aphrodisiacs have turned to folk medicine in whole world.

N N <sup>N</sup> NH O

O

O2S NH

OMe

OMe

OH O

N

<sup>N</sup> Udenafil

OH O <sup>O</sup> OH

**2.2 Chemical of some Brazilian aphrodisiacs species and rationalization between** 

The success of PDE-5 inhibitors, particularly of Viagra, the first inhibitor that has been marketed, the aging of the population and the quest for improved quality of life led to the search for new drugs with fewer side effects. As sources of research, plants used as

N H

Yohimbine

N <sup>N</sup> NH O

EtO

O2S <sup>N</sup>

N

OH MeO O

> O O

N

Lodenafil OH

N

N <sup>N</sup> NH O

> N H

N+

Forskolin

OH

OH

O

OMe Berberine

OMe

<sup>O</sup> <sup>O</sup> O

N

O

O O Tadalafil

O

N

There are many herbal drugs that have been used by men with ED with varying degrees of success. Most potent aphrodisiacs herbal are available and have few side effects (Malviya et al., 2011).

Some of the genera and species listed in this work in *in vitro* tests showed satisfactory answers to such an aphrodisiac effect like *Turnera diffusa* (Estrada-Reyes et al., 2009), *Pfaffia paniculata* (Arletti et al., 1999), *Passiflora* (Patel et al. 2009), *Mucuna pruriens* (Suresh et al., 2009), *Mimosa pudica* (Pande & Pathak, 2009), *Mimosa tenuiflora* (Souza et al., 2008), *Achyrocline satureioides*  (Hnatyszyn et al, 2004; Simões et al., 1986) and *Anemopaegma arvense* (Chieregatto, 2005).

The effects of the Brazilian herbal medicine Catuama® and each of its plant constituents (*Paullinia cupana, Trichilia catigua, Zingiber officinalis and Ptychopetalum olacoides*) were investigated on rabbit corpus cavernosum. Catuama® induced relaxations, but P*. cupana* was the most effective, increased the cAMP levels by 200% indicating that it is the main extract responsible for the relaxing effect (Antunes et al., 2001).


Table 1. Main Brazilian species with aphrodisiac activity

Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 313

OH

OH

OH O

O

Flavan

Chalcone

O

O

Flavanone

O

OH

O Dihydrochalcone

O

O

Dihydroflavonol

O

Isoflavone

OH

OH

COOH

C15H31

O

5' <sup>7</sup>

2' 2

4' 3'

OH

O

Flavone

OH

O

Fig. 5. Basic Structures of Flavonoids

OH

Catechin

OH

OH

Fig. 6. Phenolic substances

OH OH

<sup>O</sup><sup>+</sup> OH

OH

Anthocyanidin

OH

R

OH

R1

O

OH OH

Studies conducted by Ko and colleagues (2004) in flavonoids as inhibitors of PDE have suggested that C-4' and C-5' hydroxyl groups is not important for PDE-5 inhibition. The replacement of the hydroxyl by a methoxyl did not alter its inhibitory effect and it deletion resulted in no effect on PDE-5 inhibition. However, the C-7 hydroxyl group is very important for PDE-5 inhibition. C-7-glucoside showed no inhibition of the enzyme, being possible that the bulky glycosyl residues may hinder its binding to active site. Also, the C-3-

The luteolin showed more potent than other flavonoids, indicating that the presence of a double bond between C-2 and C-3 is important for PDE-5 inhibition. Between a flavon and an isoflavone, it may be easier for isoflavones than flavones to bind to the moiety of PDE-5. The removal of the C-5 hydroxyl group promoted the loss of inhibition of PDE, proposing

Caffeic Acid Chlorogenic Acid Anacardic Acid

O

OH

hydroxyl group of flavonols seems difficult the binding with the PDE-5.

that the hydroxyl group is vital for PDE-5 inhibition (Ko et al., 2004).

OH

O

O OH OH

OH

5 6

O

O

Flavonol

OH

#### **2.2.1 Aphrodisiacs chemical classes**

The classes of substances discussed were those with proven aphrodisiac activity or with this possible action. The compounds were separated in three main groups, according to structures similarities: flavonoids and others phenolics compounds; alkaloids, xanthins and others amines; and saponins.

#### **2.2.1.1 Flavonoids and other phenolic compounds**

Flavonoids are polyphenols with a diphenylpropane core. According to the chemical and biosynthetic routes, flavonoids are separated into different classes: chalcones, flavonols, flavones, dihydroflavonoids, anthocyanidins, isoflavones, aurones, pterocarpanes, neoflavonoids, bioflavonoids and are presents in all flowering plants.

The major classes are flavones, flavonols, anthocyanins, isoflavones and the flavan-3-ol derivatives (catechin and tannins) (Miean & Mohamed, 2001).

The flavonoids are widely distributed in gymnosperms and angiosperms with therapeutic potential because of their antioxidant, anti-inflammatory, hepatoprotective, cardio protective, antiulcer, anticancer, antimutagenic, antispasmodic, anti-allergic and antiviral activities, besides to show inhibit xanthine oxidase, protein kinase C and PDE (Rahimi et al., 2009; Ko et al., 2004).

Miean & Mohamed (2001) studied 62 tropical species to presence of flavonoids and observed that flavonol quercetin and derivatives, mainly quercetin glycosides, had major occurrence, however glycosides of kaempferol, luteolin and apigenin were also present. In fruits contained almost exclusively quercetin glycosides.

In plants surveyed, in addition to flavonoids, other phenols were found such as caffeic and chlorogenic acid in *Achyrocline satureioides* (Desmarchelier et al., 2000) and chlorogenic acid in *Trichilia catigua* (Lagos, 2006), besides anacardic acid in *Anacardium ocidentale* (Kubo et al., 1994).

Fig. 4. Biosynthetic relationship among classes of flavonoids (Barron & Ibrahim, 1996)

The classes of substances discussed were those with proven aphrodisiac activity or with this possible action. The compounds were separated in three main groups, according to structures similarities: flavonoids and others phenolics compounds; alkaloids, xanthins and others

Flavonoids are polyphenols with a diphenylpropane core. According to the chemical and biosynthetic routes, flavonoids are separated into different classes: chalcones, flavonols, flavones, dihydroflavonoids, anthocyanidins, isoflavones, aurones, pterocarpanes,

The major classes are flavones, flavonols, anthocyanins, isoflavones and the flavan-3-ol

The flavonoids are widely distributed in gymnosperms and angiosperms with therapeutic potential because of their antioxidant, anti-inflammatory, hepatoprotective, cardio protective, antiulcer, anticancer, antimutagenic, antispasmodic, anti-allergic and antiviral activities, besides to show inhibit xanthine oxidase, protein kinase C and PDE (Rahimi et al.,

Miean & Mohamed (2001) studied 62 tropical species to presence of flavonoids and observed that flavonol quercetin and derivatives, mainly quercetin glycosides, had major occurrence, however glycosides of kaempferol, luteolin and apigenin were also present. In

In plants surveyed, in addition to flavonoids, other phenols were found such as caffeic and chlorogenic acid in *Achyrocline satureioides* (Desmarchelier et al., 2000) and chlorogenic acid in *Trichilia catigua* (Lagos, 2006), besides anacardic acid in *Anacardium ocidentale* (Kubo et al.,

1,3-Diphenylpropane

Chalcone Dihydrochalcone

Flavan Flavanone Flavone

Flavan 3-ol & -3,4-diol

Fig. 4. Biosynthetic relationship among classes of flavonoids (Barron & Ibrahim, 1996)

Dihydroflavonol Flavonol

**2.2.1 Aphrodisiacs chemical classes**

**2.2.1.1 Flavonoids and other phenolic compounds** 

neoflavonoids, bioflavonoids and are presents in all flowering plants.

derivatives (catechin and tannins) (Miean & Mohamed, 2001).

fruits contained almost exclusively quercetin glycosides.

amines; and saponins.

2009; Ko et al., 2004).

p-Hydroxycinnamoyl CoA

(3) Malonyl CoA

1994).

Fig. 5. Basic Structures of Flavonoids

Fig. 6. Phenolic substances

Studies conducted by Ko and colleagues (2004) in flavonoids as inhibitors of PDE have suggested that C-4' and C-5' hydroxyl groups is not important for PDE-5 inhibition. The replacement of the hydroxyl by a methoxyl did not alter its inhibitory effect and it deletion resulted in no effect on PDE-5 inhibition. However, the C-7 hydroxyl group is very important for PDE-5 inhibition. C-7-glucoside showed no inhibition of the enzyme, being possible that the bulky glycosyl residues may hinder its binding to active site. Also, the C-3 hydroxyl group of flavonols seems difficult the binding with the PDE-5.

The luteolin showed more potent than other flavonoids, indicating that the presence of a double bond between C-2 and C-3 is important for PDE-5 inhibition. Between a flavon and an isoflavone, it may be easier for isoflavones than flavones to bind to the moiety of PDE-5. The removal of the C-5 hydroxyl group promoted the loss of inhibition of PDE, proposing that the hydroxyl group is vital for PDE-5 inhibition (Ko et al., 2004).

Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 315

OH

Narigenin 4'-O-methyltaxifolin Quercetin (David et al, 2006)

(Souza et al., 2008)

O

OH O

Glu

O

Apigenin and luteolin derivatives (*P. edulis*) (Ferreres et al., 2007) Flavonoids above (*P. alata*)(Doyama et al, 2005)

> Quercetin, myricetin, Kaempferol and derivatives (Ceruks et al., 2007; Johann et al., 2010)

OH O

Isovitexin

Glu OH

OH

OH OH

(Sapindaceae) Epicathechins, Cathechins (Ushirobira et al., 2007)

(Meliaceae) Chlorogenic acid, catechin and epicatechin (Lagos, 2006)

Vitexin Scoparin

OH

O OH

MeO

OH O

R= O Me - Kukulkan A R= OH - Kukulkan B

O

O

OH OH

OH O

Isoorientin

O Chrysin (*P. Caeruleae*) Dhwan et al., 2002)

OH

OH

Glu OH

OH

OH OMe

OMe OH

R

**Specie (Family) Flavonoids and phenols** 

OH O

R1 OR2

R= H; R1= H; R2= Me - 6-Dimethoxy-4'-O-methylcapilarisin

O

OH Glu

OH O

O

OH O

Orientin

Glu

OH

OH

(Nymphaeaceae) Quercetin derivatives (glycosides) (Marquina et al., 2005)

OH O

(Dilleniaceae) OH <sup>O</sup>

(Fabaceae) OH

R= O Me; R1= OH; R2= Me - Tenuiflorin A

= OMe; R2= H - Tenuiflorin B R= H; R1= OH; R2= Me - Tenuiflorin C R= H; R1= H; R2= H - 6-Dimethoxycapilarisin

OH O O

OH O

R

R= R1

*Davilla rugosa*

*Mimosa tenuiflora*

*Nymphaea ampla*

*Passiflora sp.* (Passifloraceae)

*Paulinia cupana*

*Trichilia catigua* 

*Schinus terebinthifolius* (Anarcadiaceae)


OH

Luteolin Quercetin

3-O-Metil Quercetin And Caffeic, chlorogenic and isochlorogenic acids (Desmarchelier et al., 2000).

O

Myricetin And Quercetin, Anacardic Acids and derivatives (Kubo et al., 1994; Miean & Mohamed, 2001).

O

OH OH

OH

Catuabine A Cinchonain Ia R = Cinchonain IIa

(Tabanca et al.,2007)

O

O

OMe

MeO O

OH

O

OH OH

OH

O

OH

OH OH

OH

OH

OH OH

OCH3

OH OH

O

O

OH

OH O

O

OH OH

OH OH O

OH

OH

O

O

OH OH

> OH OH

> > OH

Carajuruflavone (Takemura et al., 1995)

OMe O 4'-hydroxi-3,7-dimethoxiflavone (Barbosa et al., 2008)

OH

OH O

OH O

Kaempferol (Barbosa et al., 2008)

OH

OH

O

R= KandelinAl OH OH

O

OH O

Acacetin (Zorn et al., 2001)

OH OH

R

OH OH

OH OH

OH

**Specie (Family) Flavonoids and phenols** 

OH OH

O

O OH

OH

*(*Anacardiaceae) <sup>O</sup>

O

OH

O O

OH

OH <sup>O</sup>

OH OMe O

OH

O+

OR R1

Vicenin-A (Barbosa et al., 2008)

R1= OH; R= H - 6,7,3',4'-tetrahydroxy-5-methoxyflavylium R1= H; R= H - 6,7,4'-trihydroxy-5- methoxyflavylium

OCH3

O

OH H OH O

OH OH

OH OH

R1= H; R = CH3 - Carajurin

(Zorn et al., 2001)

OH OH

OH

O

OH

OH

OH

(Asteraceae) <sup>O</sup>

*Achyrocline satureioides*

*Anacardium Ocidentale* 

*Anemopaegma arvense* 

(Bignoniaceae)

*Arrabidaea chica* **(**Bignoniaceae)


Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 317

MeO

OH OH

MeO Magnoflorine

<sup>N</sup> <sup>O</sup> O

(Wu et al.,2005)

O O

OMe OMe

O O

OH MeO OMe

N

Catuabin H Catuabin I Vaccinine A Vaccinine B And derivatives (Zanolari, 2003)

O O

OH

OMe OH

N

OH

OH

Hippeastrine Galanthamine Montanine

Lycorine Lycosinine Pretazettine

(Jin, 2011; Pagliosa et al., 2010)

N

Catuabin A Catuabin B Catuabin C Catuabin D

MeO

<sup>N</sup> <sup>O</sup>

<sup>O</sup> <sup>N</sup>

Catuabin F

O O N

O

O N

O O N+

<sup>N</sup> <sup>O</sup> O N

> O O NH

> > <sup>N</sup> OH

OH

N

N

OH

O O

MeO MeO O O

OH

O

O

N

OH

OH

N

OH

Catuabin G

<sup>N</sup> <sup>O</sup> O N H

O O

O O N

**Specie (Family) Alkaloids, xanthines and others amines** 

<sup>N</sup> <sup>O</sup>

O O

OMe OMe

O O N

OH

O

<sup>O</sup> <sup>O</sup> OH

MeO

O

<sup>N</sup> OH

N

OMe

N

OH

MeO

<sup>N</sup> <sup>O</sup>

<sup>N</sup> <sup>O</sup> O N

Catuabin E

<sup>O</sup> <sup>N</sup>

N O

*Aristolochia cymbifera*

(Aristolochiaceae)

*Erythroxylum viceniifolium* (Erythroxylaceae)

*Hippeastrum psittacinum* (Amaryllidaceae)


Table 2. Aphrodisiacs plants, their flavonoids and phenols

#### **2.2.1.2 Alkaloids, xanthines and others amines**

In broad sense, the alkaloids are natural nitrogen-containing secondary metabolites mostly derived from amino acids and found in about 20% of flowering plants. They are not limited to plants but also occur in marine organisms, insects, microorganisms and some animals (Rahimi et al., 2009).

Until 2005, 150,000 compounds were known and 14% these have been alkaloids. They are special interesting due to the heterogeneity of the group and the great bioactive potential particularly as inhibitors of PDE (Silva, 2006). Many of them have been used as a basis for design and development of new and more selective drugs with reduced side effects.

The methylxanthines are purine bases and have structural similarity with the cAMP and cGMP, therefore bind competitively to the sites of the various PDEs. They are considered non-selective inhibitors, such as caffeine found in *Paullinia cupana* seeds, theobromine and adenine from *Ptychopetalum olacoides*, which validate its aphrodisiac effect.

Introducing achiral cyclopenthyl and hexylamines moiety in xanthines analogues enhanced inhibitory activity. The ethyl group at the N-1 and N-3 positions showed the highest effect in PDE-5 (Wang et al., 2002).

Aporphines alkaloids act as dopamine agonists, due to their structural similarity. They improve central pro-erectile mechanisms by binding to receptors in the paraventricular nucleus of the hypothalamus. In clinical trials, apomorphine was found to be effective in patients with ED of various aetiologies and levels of severity, albeit with substantially less efficacy than any of the PDE-5 inhibitors (Seftel, 2002).

Other plants that seem to act this way are: *Mimosa tenuiflora, Mimosa pudica and Mucuna pruriens*, but they need more studies to investigation their aphrodisiac activities.

While many β-carbolines have effect as a selective inhibitor of PDE-5, the alkaloids of *Passiflora* seems to have effect as serotonin uptake inhibitors and therefore act with antidepressants. Recently, harmine and numerous related β-carboline derivatives were found as potent and specific inhibitors of cyclin-dependent kinases (CDKs), and the structure activity relationships (SARs) analysis demonstrated that the degree of aromaticity

O

OH O Chrysoeriol

In broad sense, the alkaloids are natural nitrogen-containing secondary metabolites mostly derived from amino acids and found in about 20% of flowering plants. They are not limited to plants but also occur in marine organisms, insects, microorganisms and some animals

Until 2005, 150,000 compounds were known and 14% these have been alkaloids. They are special interesting due to the heterogeneity of the group and the great bioactive potential particularly as inhibitors of PDE (Silva, 2006). Many of them have been used as a basis for

The methylxanthines are purine bases and have structural similarity with the cAMP and cGMP, therefore bind competitively to the sites of the various PDEs. They are considered non-selective inhibitors, such as caffeine found in *Paullinia cupana* seeds, theobromine and

Introducing achiral cyclopenthyl and hexylamines moiety in xanthines analogues enhanced inhibitory activity. The ethyl group at the N-1 and N-3 positions showed the highest effect

Aporphines alkaloids act as dopamine agonists, due to their structural similarity. They improve central pro-erectile mechanisms by binding to receptors in the paraventricular nucleus of the hypothalamus. In clinical trials, apomorphine was found to be effective in patients with ED of various aetiologies and levels of severity, albeit with substantially less

Other plants that seem to act this way are: *Mimosa tenuiflora, Mimosa pudica and Mucuna* 

While many β-carbolines have effect as a selective inhibitor of PDE-5, the alkaloids of *Passiflora* seems to have effect as serotonin uptake inhibitors and therefore act with antidepressants. Recently, harmine and numerous related β-carboline derivatives were found as potent and specific inhibitors of cyclin-dependent kinases (CDKs), and the structure activity relationships (SARs) analysis demonstrated that the degree of aromaticity

*pruriens*, but they need more studies to investigation their aphrodisiac activities.

design and development of new and more selective drugs with reduced side effects.

adenine from *Ptychopetalum olacoides*, which validate its aphrodisiac effect.

efficacy than any of the PDE-5 inhibitors (Seftel, 2002).

OH

OMe OH

Luteolin, apigenin, quercetin, orientin and vitexin derivatives (Zhao et al., 2007)

OH

O

OH

OH O

Echinacin

GlucO O O

**Specie (Family) Flavonoids and phenols** 

OMe OH OMe

OH O

OH O

Tricin

Table 2. Aphrodisiacs plants, their flavonoids and phenols

**2.2.1.2 Alkaloids, xanthines and others amines** 

*Turnera diffusa* (Turneraceae)

(Rahimi et al., 2009).

in PDE-5 (Wang et al., 2002).


Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 319

N

Caffeine (Ushirobira et al., 2007)

O

And caffeine, muirapuamine (Montrucchio, 2005)

O

O

<sup>N</sup> <sup>N</sup>

N

NH <sup>N</sup>

Theobromine

O

N

**Specie (Family) Alkaloids, xanthines and others amines** 

N <sup>N</sup> <sup>N</sup> H

Adenine

Table 3. Aphrodisiacs plants and their alkaloids, xanthines and others amines

seem to have the same action in the transport of dopamine (Singh, 2000).

NH2

N

of the tricyclic ring and the positioning of substituents were crucial for inhibitory activity. In addition, N-2-furoyl and N-2- pyrimidinyl β-carbolines were found to strongly inhibit

Tropane alkaloids present in *Erytroxylum* species have a structure similar to cocaine and

Saponins are a vast group of non-nitrogenous compounds, in general glycosides of steroids or polycyclic terpenes and widely distributed in higher plants. Their surfactant properties are what distinguish these compounds from others. They are soluble in water and form

They have a diverse range of biological activities including hemolytic, hepatoprotective,

Saponins are high molecular weight substances and occur in complex mixtures due to the concomitant presence of structures with varying number of sugars or because of the presence of various aglycones. As a result of structural complexity, isolation and structural elucidation of these compounds can be very difficult and has developed only recently

Although some saponins inhibit PDE-5, like those present in *Allium tuberosum*, those found in plants studied did not have any reports for this activity (Guohua et al., 2009; Rahimi et al, 2009), except *Pfaffia paniculata* (Brazilian ginseng) presented saponins as the main active components due to its similarity with those saponins from *Panax ginseng,* known as ginsenosides (Rates & Gosmann, 2002). The ginsenosides are adaptogens substances or anti-

The term adaptogen, or resistogen, as it is called to classify a group of substances that can improve nonspecific resistance of body after being exposed to various stressing factors,

colloidal solutions that foam upon shaking (Schenkel et al., 2007; Sparg et al. 2004).

antimutagenic, antiviral, antileishmanial and antiinflammatory (Rahimi et al., 2009).

stress agents, but their action mechanisms are not clear (Schenkel et al., 2007).

(Oleaceae) <sup>N</sup>

activity against phosphodiesterases (PDEs) (Cao et al., 2007).

*Paulinia cupana* (Sapindaceae)

*Ptychopetalum olacoides*

**2.2.1.3 Saponins** 

(Schenkel et al., 2007).



Table 3. Aphrodisiacs plants and their alkaloids, xanthines and others amines

of the tricyclic ring and the positioning of substituents were crucial for inhibitory activity. In addition, N-2-furoyl and N-2- pyrimidinyl β-carbolines were found to strongly inhibit activity against phosphodiesterases (PDEs) (Cao et al., 2007).

Tropane alkaloids present in *Erytroxylum* species have a structure similar to cocaine and seem to have the same action in the transport of dopamine (Singh, 2000).

#### **2.2.1.3 Saponins**

318 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

O

NH COOH

> N H

OH

4) R=R1 = Me

(Misra & Wagner, 2004; Siddhuraju &Becker, 2001)

(Ingale & Hivrale, 2010)

OH R R1

N

O

OH OH

L-dopa

N

(Souza et al., 2008)

COOH NH2

NH

OH R R1

COOH

(Muthumani et al., 2010; Ueda & Yamamura, 1999a, 1999b)

OH

Mimosiine Pheniletylamine

COOH

NH2

OH

N

OH OH

Yuremamine

OH

COOH NH2

OH

OH

L-dopa

N H

<sup>N</sup> <sup>R</sup>

R=OH - Harmalol R=OCH3 - Harmalin N(CH3)2

OH OH

OH

OH NH2

OH

**Specie (Family) Alkaloids, xanthines and others amines** 

OH OH <sup>N</sup> <sup>N</sup>

NH2 OH NH2

Mimopudine

NH OH COOH

NH2

OH

N,N-dimethyltryptamine 5 Hydroxytryptamine

OH COOH

R R1

N H

<sup>N</sup> <sup>R</sup>

R=H- Harman R=OH - Harmol R=OCH3 - Harmin

=Me

4) R=R1 = Me

R R1

N

O

OH

1) R=R1 = H 2) R=H; R1 =Me

3) R=R1 = Me

OH

(Fabaceae) NH

N H

OH

1) R=R1 = H 2) R=H; R1

3) R=R1 = Me

*Mimosa pudica* (Fabaceae)

*Mimosa tenuiflora* (Fabaceae)

*Mucuna pruriensis*

*Passiflora sp.* (Passifloraceae) Saponins are a vast group of non-nitrogenous compounds, in general glycosides of steroids or polycyclic terpenes and widely distributed in higher plants. Their surfactant properties are what distinguish these compounds from others. They are soluble in water and form colloidal solutions that foam upon shaking (Schenkel et al., 2007; Sparg et al. 2004).

They have a diverse range of biological activities including hemolytic, hepatoprotective, antimutagenic, antiviral, antileishmanial and antiinflammatory (Rahimi et al., 2009).

Saponins are high molecular weight substances and occur in complex mixtures due to the concomitant presence of structures with varying number of sugars or because of the presence of various aglycones. As a result of structural complexity, isolation and structural elucidation of these compounds can be very difficult and has developed only recently (Schenkel et al., 2007).

Although some saponins inhibit PDE-5, like those present in *Allium tuberosum*, those found in plants studied did not have any reports for this activity (Guohua et al., 2009; Rahimi et al, 2009), except *Pfaffia paniculata* (Brazilian ginseng) presented saponins as the main active components due to its similarity with those saponins from *Panax ginseng,* known as ginsenosides (Rates & Gosmann, 2002). The ginsenosides are adaptogens substances or antistress agents, but their action mechanisms are not clear (Schenkel et al., 2007).

The term adaptogen, or resistogen, as it is called to classify a group of substances that can improve nonspecific resistance of body after being exposed to various stressing factors,

Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 321

Pharmacological studies of *P. paniculata* extracts indicate that they might act mainly by increasing central noradrenergic and dopaminergic tone, and possibly (indirectly)

It is possible to speculate that the activity is related to the distance between the groups at C-3

Despite the search promoted by pharmaceutical companies for analogues of sildenafil, the use and interest in herbal products based on folk and traditional medicine is growing globally, aiming to increase access to treatment for erectile dysfunction and to reduce the

The investigation of classes of metabolites present in plants can indicate a possible rationalization of relations between the structure - aphrodisiac activity of substances, contributing to the development and generation of new drugs more effective and secure

Antunes, E.; Gordo, W. M.; Oliveira, J. F. de; Teixeira, C.E. ; Hyslop, S. & Nucci, G. de.

Arletti, R.; Benelli, A; Cavazzuti, E.; Scarpetta, G. & Bertolini, A. (1999). Stimulating property

Barbosa, W. L. R.; Pinto, L. Do N.; Quignard, E.; Vieira, J.M. dos S. ; Silva Jr., J.O.C. &

*Psychopharmacology*, Vol. 143, No. 1, n.d., pp. 15-19. ISSN: 0033-3158. Ayyanar, M. & Ignacimuthu, S. (2009). Herbal medicines for wound healing among tribal

No. 4, (October/December 2008), pp. 544-548. ISSN: 0102-695X.

(2001). The relaxation of isolated rabbit corpus cavernosum by the herbal medicine Catuama® and its constituents. *Phytotherapy Research*, Vol. 15, n.d., pp. 416-421.

of *Turnera diffusa* and *Pfaffia paniculata* extracts on the sexual-behavior of male rats.

people in Southern India: Ethnobotanical and Scientific evidences*. International Journal Of Applied Research in Natural Products*, Vol. 2, No. 3, n.d., pp. 29-42. ISSN:

Albuquerque, S. (2008). *Arrabidaea chica* (HBK) Verlot: phytochemical approach, antifungal and trypanocidal activities. *Revista Brasileira de Farmacognosia*, Vol. 18,

COOR2

R1O

OR2

OH O

OH

and groups at C-17 and the architecture of the molecule must be important.

OH R3O

OR2 R1O R1O

Fig. 7. Saponins basic strutures from *Panax Ginseng* (Jia & Zhao, 2009)

adverse effects and costs, improving the quality of life.

oxytocinergic transmission (Arletti et al., 1999).

10 5

11 12 14 13

**3. Conclusion** 

**4. References** 

18

<sup>17</sup> <sup>16</sup> <sup>19</sup> <sup>20</sup> <sup>22</sup> <sup>21</sup>

30

15

derivatives from regional floras.

ISSN: 1099-1573.

1940-6223.

23 24 25 26

27

28 29

promoting a state of adaptation to the exceptional situation. Some plants like *Pfaffia paniculata, Paulinia cupana, Turnera diffusa, Anemopaegma arvense, Ptychopetalum olacoides* and *Trichilia catigua* are considered adaptogens (Mendes, 2011).

Table 4. Saponins and derivatives found in some Brazilian plants

Pharmacological studies of *P. paniculata* extracts indicate that they might act mainly by increasing central noradrenergic and dopaminergic tone, and possibly (indirectly) oxytocinergic transmission (Arletti et al., 1999).

It is possible to speculate that the activity is related to the distance between the groups at C-3 and groups at C-17 and the architecture of the molecule must be important.

Fig. 7. Saponins basic strutures from *Panax Ginseng* (Jia & Zhao, 2009)

#### **3. Conclusion**

320 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

promoting a state of adaptation to the exceptional situation. Some plants like *Pfaffia paniculata, Paulinia cupana, Turnera diffusa, Anemopaegma arvense, Ptychopetalum olacoides* and

O

OH

O O

OH OH OH

OH

OH OH OH

O

O

O

CO2R1

= OH

HOH2C

COOH

O R

Mimoside A: R1 = R2= H

<sup>O</sup> OH

Mimoside B: R1 = R2= H

(Souza et al., 2008)

RO

(*P. alata*) (Doyama et al., 2005)

R= -D-glucoronic acid(2-1)--D-xilose (Rates & Gosmann, 2002)

Quadrangoloside (R= gentibiose) 3-sophorosil oleanolic acid (R=sophorose)

Mimoside C:R1 = R2

<sup>O</sup> OH

OH OH

OH OH

R2

OH O

O

O O

OH

O O

COOH

RO

R= H - Pfaffic acid

OH

H OH O

OH

*Trichilia catigua* are considered adaptogens (Mendes, 2011).

RO

Table 4. Saponins and derivatives found in some Brazilian plants

*Mimosa tenuiflora* (Fabaceae)

*Passiflora sp.* (Passifloraceae)

*Pfaffia paniculata* (Amarantaceae)

**Specie (Family) Saponins** 

Despite the search promoted by pharmaceutical companies for analogues of sildenafil, the use and interest in herbal products based on folk and traditional medicine is growing globally, aiming to increase access to treatment for erectile dysfunction and to reduce the adverse effects and costs, improving the quality of life.

The investigation of classes of metabolites present in plants can indicate a possible rationalization of relations between the structure - aphrodisiac activity of substances, contributing to the development and generation of new drugs more effective and secure derivatives from regional floras.

#### **4. References**


Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 323

Ingale, A. G. & Hivrale, A. U. (2010). Pharmacological studies of *Passiflora sp*. and their

Jia, L. & Zhao, Y. (2009). Current Evaluation of the Millennium Phytomedicine-Ginseng (I):

Jin, Z. (2011). *Amaryllidaceae* and *Sceletium* alkaloids. *Natural Product Reports*, Vol. 28, No. 6,

Johann, S.; Sá, N. P.; Lima, L. A. R. S.; Cisalpino, P. S.; Cota, B. B.; Alves, T. M.A.; Siqueira, E.

Ko, W-C.; Shih, C-M.; Lai, Y-H.; Chen, J-H. & Huang, H-L. (2004). Inhibitory effects of

Kubo, I.; Kinst-Hori, I. & Yokokawa, Y. (1994). Tyrosinase inhibitors from *Anacardium* 

Lagos, J. B. (2006). *Estudo comparativo da composição química das folhas e cascas da Trichilia* 

Malviya, N.; Jain, S.; Gupta, V. B. & Vyas, S. (2011). Recent studies on aphrodisiac herbs for

Marquina, S.; Bonilla-Barbosa, J. & Alvarez, L. (2005). Comparative phytochemical analysis

Matheus, W. E.; Fregonesi, A. & Ferreira, U. (2009). Disfunção erétil. *Revista Brasileira de Medicina*, Vol. 66, No. 12, (October 2009), pp. 85-89. ISSN: 0034-7264. Mendes, F. R. (2011). Tonic, fortifier and aphrodisiac: adaptogens in the Brazilian folk

Miean, K. H. & Mohamed, S. (2001). Flavonoid (Myrcetin, Quercetin, Kaempferol, Luteolin,

Misra, L. & Wagner, H. (2004). Alkaloidal constituents of *Mucuna pruriens* seeds. *Phytochemistry*, Vol. 65, No. 18, n.d., pp. 2565-2567. ISSN: 0031-9422. Montrucchio, D. P.; Miguel, O. G.; Miguel, M. D.; Monache, F. D. & Carvalho, J. L. S.

Muthumani, P.; Meera, R.; Devi, P.; Koduri, L.V.S.K. ; Manavarthi, S. & Badmanaban, R.

*Chemistry*, Vol. 49, n.d., pp. 3106-3112. ISSN: 0021-8561.

pp. 417-426. ISSN: 1996-0824.

n.d., pp. 1126-42. ISSN : 1460-4752.

No. 1, n.d., pp. 09-30. ISSN: 1476-0711.

Vol. 68, No. 1, n.d., pp. 3-8. ISSN 0001-6837.

ISSN: 0006-2952.

ISSN: 0031-9422.

0163-3864.

Paraná.

695X.

1518-5192.

bioactive compounds. *African Journal of Plant Science*, Vol. 4, No. 10, (October 2010),

Etymology, Pharmacognosy, Phytochemistry, Market and Regulations. *Current Medicinal Chemistry*, Vol. 16, No. 19, (January 2010), pp. 2475-2484. ISSN: 0929-8673.

P. & Zani, C. L. (2010). Antifungal activity of schinol and a new biphenyl compound isolated from *Schinus terebinthifolius* against the pathogenic fungus *Paracoccidioides brasiliensis*. *Annals of Clinical Microbiology and Antimicrobials*, Vol. 9,

flavonoids on phosphodiesterase isozymes from guinea pig and their structureactivity relationships. *Biochemical Pharmacology*, Vol. 68, No. 10, n.d., pp. 2087-2094.

*Occidentale* fruits. *Journal of Natural Products*, Vol. 57, No. 4, n.d., pp. 545-551. ISSN:

*catigua A. Juss., Meliaceae*. Thesis of Master Degrees. Universidade Federal da

the management of male sexual dysfunction - a review. *Acta Poloniae Pharmaceutica*,

of four Mexican *Nymphaea* species. *Phytochemistry*, Vol. 66, No. 8, n.d., pp. 921-927.

medicine. *Revista Brasileira de Farmacognosia*, Vol. 21, No. 3, in print. ISSN: 0102-

and Apigenin) content of edible tropical plants. *Journal of Agricultural and Food* 

Componentes Químicos e Atividade Antimicrobiana de *Ptychopetalum Olacoides* Bentham. *Visão Acadêmica*, Vol. 6, No. 2, (July-December 2005), pp. 48-52. ISSN:

(2010). Phytochemical investigation and enzyme inhibitory activity of *Mimosa* 


Barron, D.& Ibrahim, R. K. (1996). Isoprenylated flavonoids—a survey. *Phytochemistry*, Vol.

Bertol, E.; Fineschi, V.; Karch, S. B.; Mari, F. & Riezzo, I. (2004). *Nymphaea* cults in ancient

Ceruks, M.; Romoff, P.; Fávero, O. A. & Lago, J. H. G. (2007). Constituíntes fenólicos polares

Chieregatto, L. C. (2005). *Efeito do tratamento crônico com extratos de Heteropterys aphrodisiaca* 

David, J. M.; Souza, J. C.; Guedes, M. L. S. & David, J. P.(2006). Estudo fitoquímico de *Davilla* 

Desmarchelier, C.; Ciccia, G. & Coussio, J. (2000). Recent advances in the search for

Dhawan, K.; Kumar, S. & Sharma, A. (2002). Beneficial Effects of Chrysin and Benzoflavone

Doyama, J. T.; Rodrigues, H. G.; Novelli, E. L. B.; Cereda, E. & Vilegas, W. (2005). Chemical

Drewes, S. E.; George, J. & Khan, F. (2003). Recent findings on natural products with erectile-

Estrada-Reyes, R.; Ortiz-López, P.; Gutiérrez-Ortíz, J. & Martínez-Mota, L. (2009). *Turnera* 

Guohua, H.; Yanhua, L.; Rengang, M.; Dongzhi, W.; Zhengzhi, M. & Hua, Z. 2009.

Hnatyszyn, O.; Moscatelli, V.; Garcia, J.; Rondina, R ; Costa, M. ; Arranz, C.; Balaszczuk, A. ; Ferraro, G. & Coussio, J.D. (2003). Argentinian plant extracts with relaxant effect on the

*Ethnopharmacology*, Vol. 122, n.d., pp. 579-582. ISSN: 0378-8741.

Thesis of Master Degrees. Universidade Federal de Viçosa.

(January/March 2006), pp. 105-108. ISSN: 0102-695X.

Egypt and the New World: a lesson in empirical pharmacology. *Journal of the Royal Society of Medicine*, Vol. 97, No. 2, (February 2004), pp. 84-85. ISSN: 0141-0768. Cao, R.; Peng, W.; Wang, Z. & Xu, A. (2007). β-Carboline alkaloids: biochemical and

pharmacological functions. *Current Medicinal Chemistry*, Vol. 14, No. 4, n.d., pp. 479-

de *Schinus Terebinthifolius* Raddi (Anacardiaceae). *Química Nova*, Vol. 30, No. 3, n.d.,

*O. Mach. e Anemopaegma Arvense (Vell.) Stellf. no testículo de ratos wistar adultos*.

*rugosa*: flavonóides e terpenóides. *Revista Brasileira de Farmacognosia*, v. 16, No. 1,

antioxidant activity in South American plants. In : *Studies in Natural Products Chemistry*. Rahman, A. Vol. 22, n.d., pp. 343-367. Elsevier. ISBN: 9780444531810.

on Virility in 2-Year-Old Male Rats. *Journal Of Medicinal Food*, Vol. 5, No. 1, n.d., pp.

investigation and effects of the tea of *Passiflora alata* on biochemical parameters in rats. *Journal of Ethnopharmacology,* Vol. 96, No. 3, n.d., pp. 371-374. ISSN: 0378-8741.

dysfunction activity. *Phytochemistry*, Vol. 62, No. 7, n.d., pp. 1019-1025. ISSN: 0031-

*diffusa* Wild (Turneraceae) recovers sexual behavior in sexually exhausted males. *Journal of Ethnopharmacology*, Vol. 123, No. 3, n.d., pp. 423-429. ISSN: 0378-8741. Ferreres, F.; Sousa, C.; Valentão, P.; Andrade, P. B. ; Seabra, R. M. & Gil-Izquierdo, A. (2007).

New C-deoxyhexosyl flavones and antioxidant properties of *Passiflora edulis* leaf extract. *Journal of Agricultural and Food Chemistry*, Vol. 55, No. 25, (November 2007),

Aphrodisiacs properties of *Allium tuberosum* seeds extract. *Journal of* 

smooth muscle of the corpus cavernosum of guinea pig. *Phytomedicine: International Journal of Phytotherapy and Phytopharmacology*, Vol. 10, No. 8, n.d., pp. 669-674.

43, No. 5, n.d., pp. 921-982. ISSN: 0031-9422.

500. ISSN: 0929-8673.

pp. 597-599. ISSN: 0100-4042.

Amsterdam, Netherlands.

43-48. ISSN: 1096-620X.

pp. 10187-10193. ISSN: 0021-8561

ISSNČ 0944-7113.

9422.


Phytochemistry of some Brazilian Plants with Aphrodisiac Activity 325

Still, J. (2003). Use of animal products in traditional Chinese medicine: environmental

Sumalatha, K. & Kumar, A. (2010). Review on natural aphrodisiac potentials to treat sexual

Suresh, S.; Prithiviraj, E. & Prakash, S. (2009). Dose-and time-dependent effects of ethanolic

Takemura, O. (1995). A flavone from leaves of *Arrabidaea chica* f. cuprea. *Phytochemistry*, Vol.

Ueda, M. & Yamamura, S. (1999a). Leaf-opening substance of *Mimosa pudica* L.; chemical

Ueda, M. & Yamamura, S. (1999b) Leaf-closing Substance of *Mimosa pudica* L.; Chemical

Ushirobira, T. M. A.; Yamaguti, E.; Uemura, L. M.; Nakamura, C.V. ; Dias Filho, B.P. &

Velozo, E. da S.; Barreto, M. M.; Jesus, E. L. de & Silva, C. V. da. (2002). A Etnofarmacologia

Vignera, S. L.; Condorelli, R.; Vicari, E.; Agata, R. D. & Calogero, A. E.(2011). Physical

Wang, H.; Ye, M.; Robinson, H.; Francis, S. H. & Ke, H. (2008). Conformational Variations of

Wu, T-S.; Damu, A. G.; Su, C-R. & Kuo, P-C. (2005). Chemical constituents and

*Andrology*, Vol. 32, No. 3, (May/June 2011), pp. 1-17, ISSN: 0196-3635. Wang, Y.; Chackalamannil, S.; Hu, Z.; Boyle, C. D. ; Lankin, C. M. ; Xia, Y. ; Xu, R. ;

38, No. 5, n.d., pp. 1299-1300. ISSN: 0031-9422.

(January 1999), pp. 353-356. ISSN: 0040-4039

(April 1999), pp. 2981-2984. ISSN: 0040-4039

26, No. 1, n.d., pp. 5-9. ISSN: 0326-2383.

n.d., pp. 3149-3152. ISSN: 0960-894X

No. 1, n.d., pp. 104-110. ISSN: 0026-895X.

*Journal of Ethnopharmacology*, Vol. 122, n.d., pp. 497-501. ISSN: 0378-8741. Tabanca, N.; Pawar, R. S.; Ferreira, D.; Marais, J.P.J. ; Khan, S. I. ; Vaishali, J. ; Wedge, D. E. &

pp. 118-122. ISSN: 0965-2299.

6-14. ISSN: 0976 – 0342.

ISSN: 0032-0943.

– BA, Brazil.

Netherlands.

impact and health hazards. *Complementary Therapies in Medicine*, Vol. 11, No. 2, n.d.,

dysfunction. *International Journal of Pharmacy & Therapeutics*, Vol. 1, No. 1, n.d., pp.

extracts of *Mucuna pruriens* Linn. seed on sexual behaviour of normal male rats.

Khan, I. A. (2007). Flavan-3-ol-Phenylpropanoid Conjugates from *Anemopaegma arvense* and their antioxidant activities. *Planta Medica*, Vol. 73, n.d., pp. 1107-1111,

studies on the other leaf movement of mimosa. *Tetrahedron Letters*, v. 40, No. 2,

Studies on Another Leaf-movement of Mimosa II. *Tetrahedron Letters*, v. 40, No. 15,

Palazzo de Mello, J.C. (2007). Chemical and Microbiological Study of Extract from Seeds of Guaraná (*Paullinia cupana* var. *sorbilis*). *Acta Farmacéutica Bonaerense*, Vol.

dos terreiros nagôs-baianos. In : *O Mundo das folhas*. Serra, O.; Velozo, E. da S. ; Bandeira, F. ; Pacheco, L. pp.143-175. UEFS/EDUFBA. ISBN: 8573950862, Salvador

activity and erectile dysfunction in middle-aged men : a brief review. *Journal of* 

Asberom, T. ; Pissarnitski, D. ; Stamford, A. W. ; Greenlee, W. J.; Skell, J. ; Kurowski, S. ; Vemulapalli, S. ; Palamanda, J. ; Chintala, M. ; Wu, P.; Myers, J. & Wang, P. (2002). Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors. *Bioorganic & Medicinal Chemistry Letters*, Vol. 12, No. 21,

Both Phosphodiesterase-5 and Inhibitors Provide the Structural Basis for the Physiological Effects of Vardenafil and Sildenafil. *Molecular Pharmacology*, Vol. 73,

pharmacology of *Aristolochia* species. In : *Studies in Natural Products Chemistry*. Rahman, A. Vol. 32, n.d., pp. 855-1018. Elsevier. ISBN: 9780444531810. Amsterdam,

*pudica* Linn. *Journal of Chemical and Pharmaceutical Research*, Vol. 2, No. 5, n.d., pp. 108-114. ISSN: 0975-7384


Pagliosa, L.B.; Monteiro, S. C.; Silva, K. B.; de Andrade, J. P. ; Dutilh, J. Bastida, J. ;

Pande, M. & Pathak, A. (2009). Aphrodisiac Activity of Roots of *Mimosa pudica* Linn.

Rahimi, R.; Ghiasi, S.; Azimi, H.; Fakhari, S. & Abdollahi, M. (2009). A review of the herbal

Rates, S. M. K. & Gosmann, G. Gênero *Pfaffia*: aspectos químicos, farmacológicos e

Schenkel, E. P.; Gosmann, G. & Athayde, M. L. (2007). Saponinas. In : *Farmacognosia: da* 

Seftel, A. D. (2002). Challenges in oral therapy for erectile dysfunction. *Journal of Andrology*, Vol. 23, No. 6, (November/December 2002), pp. 729-736. ISSN: 0196-3635. Siddhuraju, P. & Becker, K. (2001). Rapid reversed-phase high performance liquid

Silva, C. V. da. (2006) *Alcalóides benzofenantridínicos e outros metabólitos do caule e frutos de* 

Simões, C. M. O.; Rech, N. & Lapa, A. J. (1986). Investigação farmacológica do extrato

51, No. 5, (September-October 2008), pp. 937-947. ISSN: 1516-8913.

Sparg, S. G.; Light, M. E. & Staden, J. Van. (2004). Biological activities and distribution of

*Reviews,* Vol. 3, No. 5, n.d., pp. 186-192. ISSN: 0973-7847.

No. 2, (November 2009), pp. 123-129. ISSN: 1043-4666.

12, No. 2, (July-December 2002), pp. 85-93. ISSN: 0102-695X.

Grande do Sul. ISBN : 9788570259271, Porto Alegre-RS, Brazil.

108-114. ISSN: 0975-7384

698-701. ISSN: 0944-7113.

0308-8146.

ISSN: 0378-8741.

Bahia.

*pudica* Linn. *Journal of Chemical and Pharmaceutical Research*, Vol. 2, No. 5, n.d., pp.

Cammarota, M & Zuanazzi, J. A. S. (2010). Effect of isoquinoline alkaloids from two *Hippeastrum* species on in vitro acetylcholinesterase activity*. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology*, Vol. 17, No. 8-9, n.d., pp.

ethanolic extract in mice. *International Journal of Sciences and Nanotechnology Pharmaceutical*, Vol. 2, No. 1, (April-June 2009), pp. 477-486. ISSN: 0974-3278 . Patel, S. S.; Verma, N. K. & Gauthaman, K. (2009). *Passiflora Incarnata* Linn: A Review on

Morphology, Phytochemistry and Pharmacological Aspects. *Pharmacognosy* 

phosphodiesterase inhibitors; future perspective of new drugs. *Cytokine*, Vol. 49,

implicações para o seu emprego terapêutico. *Revista Brasileira de Farmacognosia*, Vol.

*planta ao medicamento*. Simões, C.M.O. ; Schenkel, E. P.; Gosmann, G. Mello, J. C. P.; Mentz, L.A & Petrovick, P.R. pp. 711-740. UFRGS : Universidade Federal do Rio

chromatographic method for the quantification of L-Dopa tetrahydroisoquinoline compounds from *Mucuna* beans. *Food Chemistry*, Vol. 72, n.d., pp. 389-394. ISSN:

*Zanthoxylum tingoassuiba St. Hil.* Thesis of Master Degrees. Universidade Federal da

aquoso de folhas/caules de *Achyrocline satureioides* (Lam.) Dc., Compositae (Marcela). *Caderno de Farmácia*, Vol. 2, No. 1, n.d., pp. 37-54. ISSN: 0102-6593. Singh, S. (2000). Chemistry, design, and structure-activity relationship of cocaine antagonists. *Chemical Reviews*, Vol. 100, No. 3, n.d., pp. 925-1024. ISSN: 0009-2665. Souza, R. S. O. D.; Albuquerque, U. P. D.; Monteiro, J. M. & Amorim, E. L. C. D. (2008).

Jurema-Preta (*Mimosa tenuiflora* [Willd.] Poir.): a review of its traditional use, phytochemistry and pharmacology. *Brazilian Archives of Biology and Technology*, Vol.

plant saponins. *Journal of Ethnopharmacology*, Vol. 94, No. 2-3, n.d., pp. 219-243.


**16** 

*Brazil* 

**A Phytochemical and Ethnopharmacological** 

João X. de Araújo-Júnior, Mariana S.G. de Oliveira, Pedro G.V. Aquino,

Considered in acient times as a connection to the divine, the use of this medicinal plant is as old as human civilization itself. Whole nations dominated its secrets, often associated with magic and religious rites, searching in nature's resources to improve life conditions, and

In 1978, the World Health Organization (WHO) recognized folk medicine and its beneficial effects to health, during the *Alma Ata* conference, which published in 1985 that approximatly 80% of the global population, resorted to traditional medicine as their primary health treatment (Herbarium, 2008). Medicinal plants have been used as a means of curing or preventing diseases, now called phytotherapy, in all regions of the world, with regional variations due to the influence of cultural characteristics of the population, as well as its

Since the nineteenth century, humanity discovered the endless and diverse therapeutic arsenal present in medicinal plants, due to the discovery of active substances that in their natural state or after chemical transformation showed biological activity, and often already

According to Yamada (1998) it is necessary to carry out more studies and to propagate medicinal plant utilization as a way to diminish the costs of public health programs since the utilization of these plants may constitute a very useful therapeutic value due their efficacy coupled with low operating costs and the relative ease of obtaining the plants

According to Brazilian legislation, a new herbal medicine can be introduced to the market in two forms: as a finished product – industrially produced, or as an official product – manufactured in pharmacies. Both forms should ensure quality, safety and efficacy of the herbal medicines supplied to the consumer. On the other hand, medicinal plants sold at popular markets or obtained directly from farmers at an informal market, have no guarantee provided by law, especially with regards to safety and efficacy (Herbarium, 2008). However we cannot rule out the cultural importance that popular knowledge inputs, being

confirmed by popular use and/or proven scientifically (Miguel & Miguel, 2004).

**1. Introduction** 

(Matos, 1994).

increase chances of survival (Herbarium, 2008).

flora, soil and climate (Lewinsohn, 2003).

transmitted from generation to generation.

**Review of the Genus** *Erythrina*

*Universidade Federal de Alagoas* 

Magna S. Alexandre-Moreira and Antônio E.G. Sant'Ana


## **A Phytochemical and Ethnopharmacological Review of the Genus** *Erythrina*

João X. de Araújo-Júnior, Mariana S.G. de Oliveira, Pedro G.V. Aquino, Magna S. Alexandre-Moreira and Antônio E.G. Sant'Ana *Universidade Federal de Alagoas Brazil* 

#### **1. Introduction**

326 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Zanolari, B. (2003). *Natural Aphrodisiacs. Studies of comercially-available herbal recipes, and* 

Zhao, J.; Pawar, R. S.; Ali, Z.; Khan, I. A. (2007). Phytochemical investigation of *Turnera diffusa*. *Journal of Natural Products*. Vol. 70, No. 2, n.d., pp. 289-292. ISSN 0163-3864.

Zorn, B.; García-Piñeres, A. J.; Castro, V.; Murillo, R. ; Mora, G. & Merfort, I. (2001). 3-

Doctor Degrees. University of Lausanne.

831-835. ISSN: 0031-9422.

*phytochemical investigation of Erythroxylum vacciniifolium Mart. from Brazil*. Thesis of

Desoxyanthocyanidins from *Arrabidaea chica*. *Phytochemistry*, Vol. 56, No. 8, n.d., pp.

Considered in acient times as a connection to the divine, the use of this medicinal plant is as old as human civilization itself. Whole nations dominated its secrets, often associated with magic and religious rites, searching in nature's resources to improve life conditions, and increase chances of survival (Herbarium, 2008).

In 1978, the World Health Organization (WHO) recognized folk medicine and its beneficial effects to health, during the *Alma Ata* conference, which published in 1985 that approximatly 80% of the global population, resorted to traditional medicine as their primary health treatment (Herbarium, 2008). Medicinal plants have been used as a means of curing or preventing diseases, now called phytotherapy, in all regions of the world, with regional variations due to the influence of cultural characteristics of the population, as well as its flora, soil and climate (Lewinsohn, 2003).

Since the nineteenth century, humanity discovered the endless and diverse therapeutic arsenal present in medicinal plants, due to the discovery of active substances that in their natural state or after chemical transformation showed biological activity, and often already confirmed by popular use and/or proven scientifically (Miguel & Miguel, 2004).

According to Yamada (1998) it is necessary to carry out more studies and to propagate medicinal plant utilization as a way to diminish the costs of public health programs since the utilization of these plants may constitute a very useful therapeutic value due their efficacy coupled with low operating costs and the relative ease of obtaining the plants (Matos, 1994).

According to Brazilian legislation, a new herbal medicine can be introduced to the market in two forms: as a finished product – industrially produced, or as an official product – manufactured in pharmacies. Both forms should ensure quality, safety and efficacy of the herbal medicines supplied to the consumer. On the other hand, medicinal plants sold at popular markets or obtained directly from farmers at an informal market, have no guarantee provided by law, especially with regards to safety and efficacy (Herbarium, 2008). However we cannot rule out the cultural importance that popular knowledge inputs, being transmitted from generation to generation.

A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 329

Studies have demonstrated the presence of analgesic and anti-inflammatory effects in extracts obtained from *E. senegalensis*, *E. velutina* and *E. mulungu* (Vasconcelos et al., 2003). In folk medicine, various species are utilized as a tranquilizer, against insomnia and to treat

We conducted a literature review using the database SciFinder Scholar®, and from the results obtained, we prepared two tables of data showing the correlation between popular use and the plant part utilized, as well as the form of utilization (Table 1), and the biological activities of the extracts obtained from *Erythrina* species (Table 2). Due to the large amount of data for phytochemicals isolated from the *Erythrina* species, we organized them in a

**Species Locality Reference** 

Kenya Ichimaru et al. (1996) Kamat et al. (1981)

(1998)

Tanzania Chhabra et al. (1984)

Uganda Kamusiime et al. (1996)

Rwanda Chagnon (1984)

(1995)

Zimbabwe Ndamba et al. (1994)

India Selvanayahgam et al. (1994)

East Africa Kokwaro (1976)

Mexico Hastings (1990)

Dominguez & Alcorn (1985)

Boily & Van Puyvelde (1986)

Maikere-Faniyo et al. (1989) Vlietinck et al.

Moriyasu et al.

*Erythrina abyssinica* 

*abyssinica* 

*abyssinica* 

*abyssinica* 

*abyssinica* 

*abyssinica* 

*abyssinica* 

*Erythrina americana* 

**Use and Administration**

Unspecified, external

Unspecified Decoction, oral *Erythrina* 

Unspecified, oral Unspecified, oral Unspecified, oral Unspecified, oral

Unspecified, oral *Erythrina* 

Unspecified, oral *Erythrina* 

Unspecified, oral Unspecified,oral Unspecified

inflammation (Garcia-Mateos et al., 2001).

**Uses Part Utilized Kind of Extract/ Way of** 

Colic Roots Decoction, oral *Erythrina* 

Syphilis Flowers Infusion, oral *Erythrina* 

Poison antidote Roots Unspecified *Erythrina* 

Bark Roots Roots Bark

Leaves

Stalk Stalk

**3.1 Bibliographic review** 

simplified table (Table 3).

Trachoma Malaria Syphilis Elephantiasis

Fever Leprosy

tract

Dysentery Gonorrhea Hepatitis

Schistosomiasis of the urinary

Contraception Parturition Malaria Insomnia

Anthelmintic Green bark

stem

Bark Bark Whole plant Flowers

The WHO strategy on traditional medicine for the period of 2002-2005 has brought as one of its objectives, the strengthening of traditional remedies by placing them in the National Health Systems through policies and programs determined by their respective governments. The National Policy on Integrative and Complementary Practices of the Brazilian Unified Health System (SUS, *Sistema Único de Saúde*) (2006), for example, fulfills these requests by proposing the inclusion of medicinal plants, phytotherapy, homeopathy, traditional Chinese medicine, acupuncture, hydrotherapy and crenotherapy as therapeutic options for the SUS. Another example is the Brazilian National Policy on Medicinal Plants and Herbal Medicines, which includes as one of its guidelines the promotion and recognition of popular practices in the use of herbal and home remedies. Therefore, a strategy that can be used to meet this demand proposed by the federal government is to conduct a survey of plants used by communities in order to strengthen with the establishment a list of Medicinal Plants of Interest to SUS (RENISUS), which aims to give priority to the naturally occurring species of regions or to those easily cultivated. In this context, the Brazilian Ministry of Health released the RENISUS list, containing 71 species of medicinal plants for therapeutic use (http://portal.saude.gov.br/portal/ arquivos/pdf/RENISUS.pdf).

#### **2. The Fabaceae family**

Also known as a sub-family of Leguminosae, the Fabaceae family is one of the largest botanical families and widely distributed around the world, spread out over temperate, tropical and cold regions. Thus family is composed of 32 tribes, whose genera are chemically represented by a variety of flavonoid skeletons, notably pterocarpans and isoflavones. There are about 650 genera comprising about 18,000 species (Polhil & Raven, 1981). The genus *Erythrina* is represented by about 290 species (Cronquist, 1981; http://www.tropicos.org/Name/40005932). The Fabaceae family produces valuable medicinal drugs, ornamental species, fodders plants, oil producing plants, inseticides and species with various other functions (Salinas, 1992).

#### **3. The** *Erythrina* **Genus**

The genus *Erythrina* is one among several genera from the Fabaceae family. The origin of the name *Erythrina* comes from the Greek word "erythros" which means red, alluding to the bright red flowers of the trees of the genus (Krukoff & Barneby, 1974). Over 130 species of "coral tree" belong to the genus *Erythrina*, which has been widely studied and are distributed in tropical and subtropical regions of the world. In South America, these species are present in Argentina, Bolivia, Paraguay, French Guiana, Colombia and Peru (Hickey & King, 1981). In Brazil the genus is spread throughout all of the Brazilian biomes, like the Atlantic forest, *cerrado*, Amazon rainforest and Brazilian northeast *caatinga* (Corrêa, 1984). In Brazil, there are eight species found: *E. mulungu, E. velutina, E. cista-galli, E. poeppigiana, E. fusca, E. falcata, E. speciosa* and *E. verna* (Lourenzi, 1992).

Phytochemical analysis has demonstrated the presence of terpenes in plants from the *Erythrina* genus (Serragiotto et al., 1981; Nkengfack et al., 1997), that are also recognized as bioactive alkaloid-rich plants (Ghosal et al., 1971; Barakat et al., 1977) and flavonoids, especially, isoflavones, pterocarpanes, flavanones and isoflavanones (Chacha et al., 2005). Some of these flavonoids have demonstrated a wide variety of biological activities (Table 2).

Studies have demonstrated the presence of analgesic and anti-inflammatory effects in extracts obtained from *E. senegalensis*, *E. velutina* and *E. mulungu* (Vasconcelos et al., 2003). In folk medicine, various species are utilized as a tranquilizer, against insomnia and to treat inflammation (Garcia-Mateos et al., 2001).

#### **3.1 Bibliographic review**

328 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

The WHO strategy on traditional medicine for the period of 2002-2005 has brought as one of its objectives, the strengthening of traditional remedies by placing them in the National Health Systems through policies and programs determined by their respective governments. The National Policy on Integrative and Complementary Practices of the Brazilian Unified Health System (SUS, *Sistema Único de Saúde*) (2006), for example, fulfills these requests by proposing the inclusion of medicinal plants, phytotherapy, homeopathy, traditional Chinese medicine, acupuncture, hydrotherapy and crenotherapy as therapeutic options for the SUS. Another example is the Brazilian National Policy on Medicinal Plants and Herbal Medicines, which includes as one of its guidelines the promotion and recognition of popular practices in the use of herbal and home remedies. Therefore, a strategy that can be used to meet this demand proposed by the federal government is to conduct a survey of plants used by communities in order to strengthen with the establishment a list of Medicinal Plants of Interest to SUS (RENISUS), which aims to give priority to the naturally occurring species of regions or to those easily cultivated. In this context, the Brazilian Ministry of Health released the RENISUS list, containing 71 species of medicinal plants for therapeutic use

Also known as a sub-family of Leguminosae, the Fabaceae family is one of the largest botanical families and widely distributed around the world, spread out over temperate, tropical and cold regions. Thus family is composed of 32 tribes, whose genera are chemically represented by a variety of flavonoid skeletons, notably pterocarpans and isoflavones. There are about 650 genera comprising about 18,000 species (Polhil & Raven, 1981). The genus *Erythrina* is represented by about 290 species (Cronquist, 1981; http://www.tropicos.org/Name/40005932). The Fabaceae family produces valuable medicinal drugs, ornamental species, fodders plants, oil producing plants, inseticides and

The genus *Erythrina* is one among several genera from the Fabaceae family. The origin of the name *Erythrina* comes from the Greek word "erythros" which means red, alluding to the bright red flowers of the trees of the genus (Krukoff & Barneby, 1974). Over 130 species of "coral tree" belong to the genus *Erythrina*, which has been widely studied and are distributed in tropical and subtropical regions of the world. In South America, these species are present in Argentina, Bolivia, Paraguay, French Guiana, Colombia and Peru (Hickey & King, 1981). In Brazil the genus is spread throughout all of the Brazilian biomes, like the Atlantic forest, *cerrado*, Amazon rainforest and Brazilian northeast *caatinga* (Corrêa, 1984). In Brazil, there are eight species found: *E. mulungu, E. velutina, E. cista-galli, E. poeppigiana, E.* 

Phytochemical analysis has demonstrated the presence of terpenes in plants from the *Erythrina* genus (Serragiotto et al., 1981; Nkengfack et al., 1997), that are also recognized as bioactive alkaloid-rich plants (Ghosal et al., 1971; Barakat et al., 1977) and flavonoids, especially, isoflavones, pterocarpanes, flavanones and isoflavanones (Chacha et al., 2005). Some of these flavonoids have demonstrated a wide variety of biological activities (Table 2).

(http://portal.saude.gov.br/portal/ arquivos/pdf/RENISUS.pdf).

species with various other functions (Salinas, 1992).

*fusca, E. falcata, E. speciosa* and *E. verna* (Lourenzi, 1992).

**2. The Fabaceae family** 

**3. The** *Erythrina* **Genus** 

We conducted a literature review using the database SciFinder Scholar®, and from the results obtained, we prepared two tables of data showing the correlation between popular use and the plant part utilized, as well as the form of utilization (Table 1), and the biological activities of the extracts obtained from *Erythrina* species (Table 2). Due to the large amount of data for phytochemicals isolated from the *Erythrina* species, we organized them in a simplified table (Table 3).


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 331

**Species Locality Reference** 

Argentina Perez &

Anesini (1994) Bandoni et al.

(1976)

Brazil Simões et al. (1999)

Argentina Filipoy (1994)

Mexico Hastings (1990)

Mexico Zamora-

Peru Duke (1994)

Thailand Wasuwat

Indonesia Widianto

Peru Jovel et

(1967)

(1980)

al.(1996) Duke (1994)

Diaz (1977) Pennington (1973) Bye (1986)

Martinez & Pola (1992) Hastings (1990)

*Erythrina crista-galli* 

*Erythrina crista-galli* 

*Erythrina dominguezii*

*Erythrina flabelliformis*

*Erythrina folkersii* 

*Erythrina fusca* 

*fusca* 

*Erythrina fusca* 

*Erythrina glauca* 

**Use and Administration**

Unspecified, external Unspecified, oral Unspecified, external

Suspension in water,

Infusion, oral Unspecified, oral Unspecified, oral Unspecified, oral Unspecified, oral Unspecified, oral

Decocction, oral Unspecified, oral Unspecified

Infusion, external Decoction, external Decoction, external Decoction, oral

Unspecified Unspecified

Infusion, oral Infusion, oral Infusion, oral

Unspecified, oral *Erythrina* 

oral

Decoction, oral Decoction, oral Decoction, oral Unspecified, external Unspecified, oral Unspecified, external

**Uses Part Utilized Kind of Extract/ Way of** 

Bark Bark Bark Leaves Stalk Stalk

Stalk+leaves Stalk+leaves Stalk+leaves

Bark Bark

Leaves Seeds Seeds Seeds Seeds Seeds

Bark

Bark Bark Bark Flowers

Bark and leaves

Bark and leaves Bark and

Seeds Seeds

Whole plant Seeds

Urinary Tract Infection Respiratory Tract Infection Diarrhea Anti-

hemorrhoids Narcotic Antiseptic

Antimicrobial Throat infections Astringent in wound healing

Swelling Healing

Diarrhea Toothache Erotic dreams

Toxic Purgative Contraceptive

Inflammation of uterus Appendicitis Diuretic

Migraine Infected wounds Fungal dermatosis Antitussive

Anti-

Itch

inflammatory

Skin infections

Headache Narcotic Kidney


**Species Locality Reference** 

Nepal Manandhar (1995)

India Rao (1981)

Guatemala Giron et al. (1991) Caceres et al. (1987)

India Selvanayahgam et al. (1994)

Mexico Hastings (1990)

Morton (1994)

Ayensu (1994)

Bhattarai (1991)

*Erythrina arborescens*

*arborescens*

*Erythrina berteroana* 

*berteroana* 

*Erythrina berteroana* 

*Erythrina berteroana* 

*berteroana* 

*Erythrina corallodendron*

*coralloides* 

Central America

Panama Duke &

Antilles Ayensu (1978)

Mexico Hastings (1990)

**Use and Administration**

Unspecified, external Unspecified, oral Unspecified, external Unspecified, external

Unspecified, oral Unspecified, External

Decoction, oral Juice of leaves, aural

Infusion, oral

Poison antidote Bark Infusion, oral *Erythrina* 

Unspecified Unspecified, oral

Decoction, oral Decoction, oral Decoction, oral Unspecified, oral Decoction, Unspecified

Infusion, oral Unspecified, oral Unspecified, oral

Unspecified Unspecified, oral

Measles Seeds Unspecified, external *Erythrina* 

Unspecified Unspecified, oral *Erythrina* 

Leaves Unspecified, external *Erythrina* 

Unspecified, external Unspecified, external Unspecified, external Unspecified, external

**Uses Part Utilized Kind of Extract/ Way of** 

Flowers Fruits

Leaves Leaves Leaves Seeds

Bark Leaves

Bark Leaves Leaves Leaves

Leaves

Branches Whole plant

Flowers Flowers Flowers Seeds Unspecified

Levaes and flowers Flowers Flowers

Bark Leaves

Hypnotic Inflammation of the arms, legs, hair and

eyes. Abscesses Insect bites Ulcers Curare-like effect

Anthelmintic Earache

Pork skin disease

Snakebite Abscesses Boils Infections of skin and mucous Dermatitis and inflammation

Fish poison Female diseases Sedative Bleeding Dysentery Poison Narcotic

Sedative Bleeding Dysentery

Female diseases

Antiasthmatic Expel placenta


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 333

**Species Locality Reference** 

Solomon Islands

Adaman Islands

New Guinea

Nigeria Etkin (1997)

Thailand Anderson

(1986)

Mexico Hastings (1990)

India Chopra (1933)

East Indias Burkill (1966)

Peru Desmarcheilier

Argentina Desmarcheilier

India Pushpangadan

Rotuma Mc Clatchey (1996)

Brazil Sarragiotto et al. (1981)

India Das (1955)

et al. (1997) Desmarcheilier et al. (1996)

et al. (1996)

Awasth (1991)

& Atal (1984) John (1984)

Holdsworth (1984)

Mokkhasmit et al. (1971)

Blackwood (1935)

Dominguez & Alcorn (1985)

*senegalensis*

*Erythrina species* 

*species* 

*Erythrina standleyana*

*Erythrina stricta* 

*subumbrans*

*ulei* 

*ulei* 

*variegata* 

*Erythrina variegata* 

*variegata* 

*Erythrina variegata* 

*Erythrina variegata* 

*variegata* 

**Use and Administration**

Unspecified, oral Unspecified, oral

Decoction, external Unspecified, oral

**Uses Part Utilized Kind of Extract/ Way of** 

Antimalarial Roots Unspecified *Erythrina* 

Analgesic Leaves Unspecified, oral *Erythrina* 

Menorrhagia Leaves Unspecified, oral *Erythrina* 

Antiseptic Bark Unspecified, external *Erythrina* 

Antiseptic Stem Bark Unspecified, external *Erythrina* 

Antipyretic Bark Decoction, oral *Erythrina* 

Swelling Bark Unspecified, external *Erythrina* 

Infusion, oral Unspecified, oral Unspecified, external

Unspecified, oral Unspecified

Unspecified, oral Juice, oral

Infusion, oral Infusion, oral Infusion, oral

Unspecified, oral Unspecified, oral Unspecified, oral

Leaves Juice, oral *Erythrina* 

Twigs and leaves Twigs and leaves

Bark Leaves

Bark and leaves Roots Roots

Bark Bark

Bark Bark

Bark Bark Bark

Flowers Flowers Flowers

Table 1. Popular uses of *Erythrina* species

Ulcers Venereal diseases

Broken bones Antipyretic

Parturition induction Toothache Nosebleed

Epilepsy Leprosy

Epilepsy Stomach ache

Amenorrhea Conception Dysmenorrhea

Antipyretic Sedative Antiasthmatic

Induce menstruation


*herbacea* 

*humeana* 

*Erythrina indica* 

*lanata* 

*mildbraedii*

*mulungu* 

*Erythrina sacleuxii* 

*Erythrina senegalensis*

*Erythrina senegalensis*

**Species Locality Reference** 

South Africa

Mexico Hastings (1990)

India Khan et al. (1994) John (1984) Chopra & Ghosh (1935) Pushpangadan & Atal (1984)

Mexico Hastings (1990)

Guinea Vasileva (1969)

Brazil Brandão (1985)

Guinea Vasileva (1969)

Senegal Le Grand &

al.(1995)

Wondergem (1987) Le Grand (1989)

Tanzania Gessler et

Pillay et al. (2001)

**Use and Administration**

Seeds Unspecified, oral *Erythrina* 

Unspecified, oral Unspecified, oral Unspecified, external Unspecified, oral Unspecified, ophthalmic Unspecified, oral Juice, oral With milk, oral Unspecified, oral Unspecified, oral Unspecified, oral Unspecified, oral

Tuberculosis Bark Infusion, oral *Erythrina* 

Poison Whole plant Unspecified *Erythrina* 

Aphrodisiac Bark Unspecified, oral *Erythrina* 

Antipyretic Bark Decoction, oral *Erythrina* 

oral

Decoction/infusion,

Unspecified, oral Unspecified, oral

Unspecified, oral,

Unspecified, oral Unspecified, oral Unspecified, oral Unspecified, external

external

Infusion, oral Decocction, oral

**Uses Part Utilized Kind of Extract/ Way of** 

leaves Bark and leaves Bark and leaves Unspecified

Bark Bark and leaves Bark Bark Bark Bark Bark Bark+roots Leaves Leaves Leaves Leaves

inflammation Purgative Antimalarial

Rats and dogs poison

Antipyretic Anthelmintic Astringent Expectorant Eye drops Antibilious Stomach upset Menstrual regulator Aphrodisiac Laxative Diuretic Stimulation of

milk production

Antimalarial Leaves an

Postpartum (women) Treatment of female sterility

Serious injury Yellow fever Bronchial diseases Eye disorders Injuries

roots

Bark Bark+leaves

Bark Bark Bark Bark Twigs and leaves


Table 1. Popular uses of *Erythrina* species

A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 335

Kenya Tachibana et al (1993)

> Kloos et al (1987) Kamat et al. (1981)

Taniguchi et al.

Yenesew et al. (2003a)

Maikere-faniyo et al. (1989) Vlietinck et al.

(1978)

(1998)

Rwanda Chagnon (1984)

(1995)

East Africa Taniguchi &

Unspecified Dominguez &

Mexico Garin-Aguilar et al. (2000)

India Dhar et al. (1968)

Guatemala Caceres et al. (1987)

Kubo (1993)

Alcorn (1985)

Sudan Omer et al.

**Species Part of the Plant Biological Activities Location Reference** 

Mitogenic activity Cell Culture Molluscicidal

(*Biomphalaria pfeifferi*) Anti-bacterial Anti-bacterial (Grampositive species, *Escherichia coli*)

Anti-yeast (*Saccharomyces* 

*aeruginosa*, *Bacillus subtilis*  e *Staphylococcus aureus*)

Uterine relaxing and

Muscle Relaxing and

Periferic muscle relaxing

Anti-bacterial (*Salmonella* 

*dysenteriae*, *Shigella boyd*,

Stem Bark Anti-inflammatory Cameroon Talla et al. (2003)

*cerevisiae*) Antimalárico

Bark Anti-bacterial (*Escherichia coli*, *Pseudomonas* 

stimulant

stimulant

and stimulant Toxic effect in rats Antidiarrheal

*typhi*, *Shigella flexnerishigella* 

*Shigella sonnei*) Antiviral

Anti-fungal

inhibition Molluscicidal

Seeds Central Nervous System depressor

> Hypotensive Cytotoxic Antispasmodic Uterine stimulant

Anti-bacterial

*Erythrina* Leaves+Twigs Cytotoxic Panamá Chapuis et al.

Root bark Anti-bacterial

Bark Plant germination

Leaves Anti-yeast

*Erythrina abyssinica* 

*Erythrina abyssinica* 

*Erythrina abyssinica* 

*Erythrina abyssinica* 

*Erythrina addisoniae* 

*Erythrina americana* 

*Erythrina americana* 

*Erythrina arborescens* 

*Erythrina berteroana*  Bark

Leaves

Roots Roots Root Bark

Root Bark

Leaves

Trunk

Leaves Leaves, stem

Stem Stem

#### **3.1.1 Ethnopharmacological data**

Plants of the *Eryhtrina* genus are utilized for a wide array of human diseases (Table 1). With regards the parts of the plants that are utilized, the most used is the bark, being 40.8% of the total of citations, as shown in Graphic 1.

Graphic 1. Parts of the plants utilized in folk medicine.

#### **3.1.2 Biological activity data**

Analysis of the biological activity data (Table 2) shows the wide variety of biological activity of plants from the *Erythrina* genus, and shows too that most of this corroborates with popular knowledge and uses.

It is noteworthy to point out that most of these activities, mainly the antibacterial and analgesic properties, confirm the different popular applications of extracts obtained from plants of this genus. We would like also to draw attention to the fact that in the Brazilian market there is the availability of a phytotherapeutic product from *Erythrina mulungu* widely used for anxiolytic purposes and as a sedative, activities confirmed by popular knowledge, but that, to our knowledge, have not yet been confirmed in pharmacological tests, showing that, despite the wide array of available data related to plants of this genus, there is still a need for more research about some of them.

It is important to note that some of the activities shown in the biological tests were not cited in the ethnopharmacological studies, which indicates yet another importance for plants of the *Erythrina* genus, which have the potential to provide new compounds for the development of drugs for the treatment of diseases such as cancer, diabetes and hypertension.

#### **3.1.3 Phytochemical data**

The phytochemical data (Table 3) analysis allowed for the verificationof a predominance of alkaloids and flavonoids in the *Erythrina* genus. It is important to note that alkaloids are recognized as markers for plants of this genus in addition to showing a wide array of biological activities, and being important candidates in the development of new drugs.

Plants of the *Eryhtrina* genus are utilized for a wide array of human diseases (Table 1). With regards the parts of the plants that are utilized, the most used is the bark, being 40.8% of the

Analysis of the biological activity data (Table 2) shows the wide variety of biological activity of plants from the *Erythrina* genus, and shows too that most of this corroborates with

It is noteworthy to point out that most of these activities, mainly the antibacterial and analgesic properties, confirm the different popular applications of extracts obtained from plants of this genus. We would like also to draw attention to the fact that in the Brazilian market there is the availability of a phytotherapeutic product from *Erythrina mulungu* widely used for anxiolytic purposes and as a sedative, activities confirmed by popular knowledge, but that, to our knowledge, have not yet been confirmed in pharmacological tests, showing that, despite the wide array of available data related to plants of this genus,

It is important to note that some of the activities shown in the biological tests were not cited in the ethnopharmacological studies, which indicates yet another importance for plants of the *Erythrina* genus, which have the potential to provide new compounds for the development of drugs for the treatment of diseases such as cancer, diabetes and

The phytochemical data (Table 3) analysis allowed for the verificationof a predominance of alkaloids and flavonoids in the *Erythrina* genus. It is important to note that alkaloids are recognized as markers for plants of this genus in addition to showing a wide array of biological activities, and being important candidates in the development of new drugs.

**3.1.1 Ethnopharmacological data** 

total of citations, as shown in Graphic 1.

Graphic 1. Parts of the plants utilized in folk medicine.

there is still a need for more research about some of them.

**3.1.2 Biological activity data** 

popular knowledge and uses.

hypertension.

**3.1.3 Phytochemical data** 


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 337

Thailand Unakul (1950)

Indonesia Widianto et al. (1980)

South Africa

South Africa

South Africa (1997)

Egypt Ross et al. (1980)

Sri Lanka Ratnasooriya &

India Singh &

Nigeria Waffo et al. (2000)

(2001)

Pillay et al. (2001)

Pillay et al. (2001) Rabe & Van Staden (1997) Motsei et al. (2003)

(1997)

Cameroon Njamen et al. (2007)

Unspecified Suffness et al (1988)

Nigeria Mitscher et al. (1988)

Egypt Ross et al. (1980)

Pillay et al. (2001)

Dharmasiri (1999)

Chatterjee (1979)

Nkengfack et al.

**Species Part of the Plant Biological Activities Location Reference**  *flabelliformis* (1977)

Uterine stimulant

*Erythrina glauca* Bark Antiviral Guatemala Mc Kee et al.

COX1 inhibitor

Anti-bacterial

lymphocyte blastogenesis

Anti-mycobacterial Anti-bacterial Cytotoxic

Anti-bacterial

Anti-bacterial

Anti-bacterial Anti-yeast

Antidiabetic Rises seric LDL

Toxic effect Cytotoxic

Anti-bacterial

Root Antiviral Tanzania Mc Kee et al.

Bone formation stimulant

Bark, leaves COX1 inhibitor

Stem bark Estrogenic

Whole plant Anti-tumoral

Root Anti-mycobacterial

*Erythrina indica* Seeds Immunosuppressor India Singh (1979)

depressor

Diuretic

depressor

*Erythrina fusca* Leaves Hypotensive

*Erythrina indica* Leaves Anti-fungal

Stem bark

*Erythrina indica* Seeds Anti-fungal

*Erythrina latissima* Bark, leaves COX1 inhibitor

*Erythrina indica* Root bark

*Erythrina humeana* 

*Erythrina lysistemon* 

*Erythrina lysistemon* 

*Erythrina lysistemon* 

*Erythrina mildbraedii* 

*Erythrina mildbraedii* 

*Erythrina fusca* Seeds Central Nervous System

*Erythrina indica* Leaves Central Nervous System

*Erythrina indica* Unspecified Stimulant and inhibitor of

Bark, leaves Anti-bacterial


Stem Pherormone Puerto Rico Keiser et al.

Leaves+stem Cancer induction USA Caldwell &

Seeds Trypsin inhibition Israel Joubert & Sharon

Flowers Anti-mutagenic Unspecified Ishii et al. (1984)

Leaves + stem Animal repellent Germany Wink (1984)

Seeds Trypsin inhibition Uruguay Joubert & Sharon

Root bark Anti-bacterial Cameroon Nkengfack et al.

Whole plant Molluscicidal Puerto Rico Medina&

Maillard et al.

Tanaka (1994) Iinuma et al. (1994)

Brewer (1983)

Woodbury (1979)

Perez & Anesini

Pillay et al. (2001)

(1996)

(1985)

Argentina Mino et al. (2002)

(1994)

(1996)

Egypti Ross et al. (1980)

Brazil Simoes et al. (1999)

Bolivia Mitscher et al. (1984)

(1988)

(1985)

(1995)

(1993)

Mitscher et al.

(1987)

(1975)

Okinawa Iinuma &

South Africa

Antiphagocytic Greece Yannitsaros

Anti-phagocytic Greece Yannitsaros

**Species Part of the Plant Biological Activities Location Reference**  *berteroana* Root bark Anti-fungal (1988)

> Anti-bacterial Anticoagulant

Anti-bacterial

Analgesic

Anti-bacterial

Anti-bacterial Anti-mycobacterial

*Erythrina excelsa* Root bark Anti-bacterial/anti-fungal East Africa Taniguchi et al.

*Erythrina* Seeds Larvicidal Unspecified Janzen et al.

Antiviral

Anti-inflammatory Anti-bacterial Anti-fungal

Root bark Anti-fungal

*Erythrina caffra* Bark, leaves COX1 inhibitor

Dry fruit + leaves + stem

Aerial parts

Fresh fruit + leaves + stem

Root and stem

bark

Leaves Anti-fungal

Leaves + stem Cytotoxic

Bark

*Erythrina berteroana* 

*hybrid* 

*Erythrina breviflora* 

*Erythrina corallodendron* 

*Erythrina corallodendron* 

*Erythrina coromandelianum* 

*galli* 

*galli* 

*galli* 

*galli* 

*galli* 

*galli* 

*galli* 

*galli* 

*Erythrina eriotricha* 

*Erythrina crista-*

*Erythrina crista-*

*Erythrina crista-*

*Erythrina crista-*

*Erythrina crista-*

*Erythrina crista-*

*Erythrina crista-*

*Erythrina crista-*

*Erythrina bidwillii* 


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 339

Unspecified Dominguez &

India Bhakuni et al. (1988)

India Dhar et al. (1968)

Thailand Silpasuwon (1979)

India Aswal et al. (1984)

Peru Desmarcheilier

India Chauhan et al. (1989)

(1984)

(1975)

et al. (1996) Desmarcheilier et al. (1997)

Bhale et al. (1979) Tripathi & Rizvi

Prabhu & John

Joshi et al. (1981)

Alcorn (1985)

Dhar et al. (1968)

**Species Part of the Plant Biological Activities Location Reference** 

Spasmolytic Hypotermic Diuretic Anticonvulsant Analgesic Antiviral Anti-fungal Anti-yeast Anti-protozoan Toxicity evaluation

Cytotoxic

Hypotensive Anti-spermatogenic Anti-androgen Anti-gonadotropin Anti tumoral Toxicity evaluation Hypoglicemic Cytotoxic Antispasmodic

Anti-bacterial Anti-fungal

Uterine stimulant Anti tumoral Abortive effect Toxicity evaluation

"DNA linker" Antioxidant

Inhibition of plant germination and growing

Juvenile hormone activity

Anti-bacterial Anti-fungal

Bark Anti gastric ulcer Japan Muto et al. (1994)

Aerial parts Fetal anti-implantation

Inhibition of plant germination

Bark Molluscicidal

*Erythrina standleyana* 

*Erythrina stricta* Stem

*Erythrina suberosa* Leaves

*Erythrina suberosa* Leaves, seed

*Erythrina subumbrans* 

*Erythrina variegata* 

*Erythrina variegata* 

oil

Leaves

Stem bark

*Erythrina ulei* Bark Anti-crustacean

Bark, leaves

Seeds oil

Stem


Unspecified Cytotoxic Colombia De Cerain et al.

(1996)

India Aswal et al. (1984)

Unspecified Pezzuto et al. (1991)

Tanzania Gessler et al. (1994) Gessler et al. (1995)

Nigeria Saidu et al.

(2000) Etkin (1997) Ajaiyeoba et al.

(2004)

(1991)

(1988)

Cameroon Biyiti et al. (1988)

(1994)

(1993)

(1949)

(1975)

(1990)

Bolivia Fournet et al. (1994)

al. (1971)

Senegal Le Grand &

Bissau

Hussain & Deeni

Okunji & Iwu

Wondergem (1988)

Silva et al. (1997)

Nkengfack et al.

Benedicta et al.

Nkeh et al. (1996)

**Species Part of the Plant Biological Activities Location Reference** 

Roots Fetal anti-implantation Anti-tumoral Uterine stimulant

Abortive

Cytotoxic

Antimalarial Cytotoxic

Antimalarial Analgesic

Anti-fungal

Anti-yeast Anti-bacterial Anti-fungal

Antispasmodic Spasmolytic

*Erythrina species* Bark Anti-bacterial China Gaw & Wang

*Erythrina species* Leaves Pherormone Puerto Rico Keiser et al.

*Erythrina species* Leaves Anti tumoral Indonesia Itokawa et al.

*Erythrina species* Leaves Antipyretic Thailand Mokkhasmit et

Anti-trypanossomiasis

Raiz Antiviral Guinea-

Skeletal muscle relaxing

Anti-inflammatory Anti-bacterial Molluscicidal

Twigs "DNA linker"

Bark Anti-bacterial

Toxicity evaluation

*Erythrina poeppigiana* 

*Erythrina resupinata* 

*Erythrina rubrinervia* 

*Erythrina senegalensis* 

*Erythrina senegalensis* 

*Erythrina senegalensis* 

*Erythrina sigmoidea* 

*Erythrina sacleuxii* Leavess, root

bark

Bark, root, stem bark

Flowers

Bark

Bark, root bark

*Erythrina species* Stem bark Anti-leishmaniasis

Stem bark


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 341

**Classes of Compounds Occurrence Percentage**  Alkaloids 461 41.57 Coumarins 1 0.09 Steroids 29 2.62 Flavonoids 330 29.76 Lipids 32 2.88 Proteins 112 10.10 Triterpenes 31 2.80 Other compounds 113 10.19 Total 1109 100

Table 3. Occurrence of the different classes of compounds in the *Erythrina* genus

**N**

**N**

Erysotramidine

**O**

This review showed that *Erythrina* species are commonly utilized for numerous diseases and that many ethnopharmacological studies have been performed in order to confirm the activities attributed to these species. Moreover, several classes of substances have been

Despite the large amount of available data, some of the plants of this genus remain to be studied. An example is *Erythrina mulungu*, largely used in Brazil, yet a significant number of studies regarding its pharmacological properties and chemical composition were unable to

isolated from the *Erythrina* genus, mainly alkaloids (41.57%) and flavonoids (29.76%).

**OH**

**N**

**N**

**OH**

**O**

**O**

**O**

Erythartine

**O**

Erysotrine

**O**

**O**

**O**

Erytharbine

**O**

**O**

**O**

**O**

Fig. 1. Common alkaloids found in the *Erythrina* genus.

nitrogen atom.

**4. Conclusion** 

Some important alkaloids that are distributed within plants from the *Erythrina* genus are erytharbine, erythartine, erysotramidine and erysotrine, shown in figure 1. It is noteworthy that a characteristic feature of these alkaloids is the spiro structure in the rings bearing the

**O**

**O**


Table 2. Biological activity of *Erythrina* extracts.


Table 3. Occurrence of the different classes of compounds in the *Erythrina* genus

Some important alkaloids that are distributed within plants from the *Erythrina* genus are erytharbine, erythartine, erysotramidine and erysotrine, shown in figure 1. It is noteworthy that a characteristic feature of these alkaloids is the spiro structure in the rings bearing the nitrogen atom.

Fig. 1. Common alkaloids found in the *Erythrina* genus.

#### **4. Conclusion**

340 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Prostaglandin synthesis

Samoa Hegde et al. (1997)

Thailand Avirutnant &

Vietnam Nguyen et al. (1991) Nguyen et al. (1992)

Taiwan Yanfg et al.

Dunstan et al.

Cox et al. (1989)

Pongpan (1983)

Telikepalli et al.

Dhar et al. (1968)

(1997)

(2007)

(1987)

(1990)

(1971)

Brazil Marchioro et al. (2005) Barros et al. (1970) Pinheiro de Sousa & Rouquayrol (1974)

Australia Rogers et al. (2001)

South Africa (2002)

Pillay et al. (2001)

India Dhar et al. (1968)

Central Nervous System

Fresh flowers Anxiolytic Brazil Flausino et al.

Leaves Anti tumoral Philippines Masilungan et al.

**Species Part of the Plant Biological Activities Location Reference** 

inhibitor

inhibidor

Anti-bacterial

Anti-inflammatory Skeletal muscle relaxing Barbiturates potentiator

pyruvate-transaminase

Antispasmodic Cytotoxic

Anti-yeast Anti-bacterial Anti-mycobacterial

Cytotoxic Antispasmodic

Analgesic

Bark Inhibition of platelet

aggregation Serotonin release inhibition

*Erythrina vogelii* Root bark Anti-fungal Ivory Coast Queiroz et al.

COX1 Inhibitor

Anti-inflammatory Uterine stimulant Molluscicidal

Toxicity evaluation

effects Spasmolytic

Roots Inhibitor of glutamate-

Flowers Anti-yeast

Phospholipase A2

*Erythrina variegata* 

*Erythrina variegata* 

*Erythrina variegata* 

*Erythrina variegata* 

*Erythrina variegata* 

*Erythrina variegate var. orientalis* 

*Erythrina variegate var. orientalis* 

*Erythrina vespertilio* 

*Erythrina velutina* Leaves

Bark Stem bark

Leaves Unspecified

Leaves

Roots

Stem bark

Stem bark Trunk bark

*Erythrina zeyheri* Leaves Anti-bacterial

Table 2. Biological activity of *Erythrina* extracts.

This review showed that *Erythrina* species are commonly utilized for numerous diseases and that many ethnopharmacological studies have been performed in order to confirm the activities attributed to these species. Moreover, several classes of substances have been isolated from the *Erythrina* genus, mainly alkaloids (41.57%) and flavonoids (29.76%).

Despite the large amount of available data, some of the plants of this genus remain to be studied. An example is *Erythrina mulungu*, largely used in Brazil, yet a significant number of studies regarding its pharmacological properties and chemical composition were unable to

A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 343

Biyiti, L., Pesando, D., & Puiseux-Dao, S. (1988). Antimicrobial activity of two flavanones

Blackwood, B. (1935). Both sides of buka passage, Clarendon Press, ISBN 0404159079,

Boily, Y. & Van Puyvelde, L. (1986). Screening of medicinal plants of Rwanda (central

Brandão, M., Botelho, M., & Krettli, E. (1985). Antimalarial experimental chemotherapy

Burkill, I. H. (1966). Dictionary of the economic products of the Malay Peninsula. Ministry of

Bye, J. R. (1986). Medicinal plants of the Sierra Madre: comparative study of Tarahumara

Caceres, A., Giron, L. M., Alvarado, S. R., & Torres, M. F. (1987). Screening of antimicrobial

Caldwell, M. E. & Brewer, W. R. (1983). Plants with potential to enhance significant tumor growth. Cancer Research, Vol.43, No.12, pp. 5775-5777, ISSN 0008-5472 Chacha, M., Bojase-Moleta, G., & Majinda, R. R. T. (2005). Antimicrobial and radical

Chagnon, M. (1984). General pharmacologic inventory of medicinal plants of Rwanda. Journal of Ethnopharmacology, Vol.12, No.3, pp. 239-251, ISSN 0378-8741 Chapuis, J. C., Sordat, B., & Hostettmann, K. (1988). Screening for cytotoxic activity of plants

Chauhan, J. S. (1989). Screening of higher plants for specific herbicidal principle active

Chhabra, S. C., Uiso, F. C., & Mshiu, E. N. (1984). Phytochemical screening of tanzanian

Chopra, R. N. (1933). Indigenous drugs of india. Their medical and economic aspects, The

Chopra, R. N. & Ghosh, S. (1935). Some common indigenous remedies. Indian Medical

Corrêa, M. P. (1984). Dicionário das plantas úteis do Brasil e das exóticas cultivadas, Vol. 5,

Cox, P. A., Sperry, L. B., Tuominen, M., & Bohlin, L. (1989). Pharmacological activity of the

Samoan ethnopharmacopoeia. Economic Botany, Vol.43, No.4, pp. 487-497, ISSN

agriculture and cooperatives, Volume I, Kuala Lumpur, Malaysia

No.2, pp. 126-128, ISSN 0032-0943

Oxford, United Kingdom

1-13, ISSN 0378-8741

237, ISSN 0378-8741

273-284, ISSN 0378-8741

0378-8741

0013-0001

Vol.66, pp. 99-104, ISSN 0031-9422

Vol.27, No.10, pp. 877-884, ISSN 0019-5189

Art Press, ASIN B003E2YJ6W, Calcutta, India

Ministério da Agricultura, Rio de Janeiro, Brazil

Record, Vol.55, p. 77, ISSN 0019-5898

0029

0013-0001

isolated from the cameroonian plant *Erythrina sigmoidea*. Planta Medica, Vol.54,

Africa) for antimicrobial activity. Journal of Ethnopharmacology, Vol.16, No.1, pp.

using natural products. Ciência e Cultura, Vol.37, No.7, pp. 1152-1163, ISSN 1980-

and Mexican market plants. Economic Botany, Vol.40, No.1, pp. 103-124, ISSN

activity of plants popularly used in Guatemala for the treatment of dermatomucosal diseases. Journal of Ethnopharmacology, Vol.20, No.3, pp. 223-

scavenging flavonoids from the stem wood of *Erythrina latissima*. Phytochemistry,

used in traditional medicine. Journal of Ethnopharmacology, Vol.23, No.2/3, pp.

against dodder, *Cuscuta reflexa* roxb. Indian Journal of Experimental Biology,

medicinal plants. Journal of Ethnopharmacology, Vol.11, No.2, pp. 157-179, ISSN

be found. A recent contribution to the knowledge about this plant is given by our group, regarding the anti-inflammatory and antinociceptive activities of a hydroalcoholic extract obtained from *E. mulungu* (Oliveira et al., *in press*).

#### **5. Acknowledgments**

The authors aknowledge the Brazilian National Research Council (CNPq), FAPEAL and CAPES for their financial support in the form of grants and fellowship awards.

#### **6. References**


be found. A recent contribution to the knowledge about this plant is given by our group, regarding the anti-inflammatory and antinociceptive activities of a hydroalcoholic extract

The authors aknowledge the Brazilian National Research Council (CNPq), FAPEAL and

Ajaiyeoba, E., Ashidi, J., Abiodun, O., Okpako, L., Ogbole, O., Akinboye, D., Falade, C.,

Anderson, E. F. (1986). Ethnobotany of hill tribes of northern Thailand. II. Lahu medicinal

Aswal, B. S., Bhakuni, D. S., Goel, A. K., Kar, K., Mehrotra, B. N., & Mukherjee, K. C. (1984).

Avirutnant, W. & Pongpan, A. (1983). The antimicrobial activity of some thai flowers and

Awasth, A. K. (1991). Ethnobotanical studies on the negrito islanders of andaman islands,

Ayensu, E. S. (1978). Medicinal plants of the West Indies, Unpublished Manuscript, p. 110 Bandoni, A. L., Mendiondo, M. E., Rondina, R. V. D., & Coussio, J. D. (1976). Survey of

Barakat, I., Jackson, A. H., & Abdulla, M. I. (1977). Further studies of *Erythrina* alkaloids.

Barros, G. S. G., Matos, F. J. A., Vieira, J. E. V., Sousa, M. P., & Medeiros, M. C. (1970).

Benedicta, N. N., Kamanyi, & A. Bopelet, M. (1993). Anticholinergic effects of the methanol

Bhakuni, D. S., Goel, A. K., Jain, S., Mehrotra, B. N., Patnaik, G. K., & Prakash, V. (1988).

Phytotherapy Research, Vol.7, No.2, pp. 120-123, ISSN 0951-418X

Experimental Biology, Vol.26, No.11, pp. 883RY-904, ISSN 0019-5189 Bhale, B., Jain, P. K., & Bokadia, M. M. (1979). The *in vitro* antimicrobial activity of the fixed oil of *Erythrina indica*. Indian Drugs Pharm Ind, Vol.14, No.3, pp. 39-40 Bhattarai, N. K. (1991). Folk herbal medicines of Makawanpur district, Nepal. International Journal of Pharmacognosy, Vol.29, No.4, pp. 284-295, ISSN 0925-1618

Lloydia, Vol.40, No.5, pp. 471-475, ISSN 0024-5461

Pharmacology, Vol.22, p. 116, ISSN 0022-3573

plants. Economic Botany, Vol.40, No.4, pp. 442-450, ISSN 0013-0001

Experimental Biology, Vol.22, No.6, pp. 312-332, ISSN 0019-5189

Bolaji, O., Gbotosho, G., Falade, M., Itiola, O., Houghton, P.,Wright, C., & Oduola, A. (2004). Antimalarial ethnobotany: in vitro antiplasmodial activity of seven plants indentified in the Nigerian middle belt. Pharmaceutical Biology, Vol.42, No.8, pp.

Screening of indian plants for biological activity: part X. Indian Journal of

plants. Mahidol University Journal of Pharmaceutical Sciences, Vol.10, No.3, pp.

India - the great andamanese. Economic Botany, Vol.45, No.2, pp. 274-280, ISSN

Argentine medicinal plants. Economic Botany, Vol.30, pp. 161-185, ISSN 0013-

Pharmacological screening of some brazilian plants. Journal of Pharmacy and

stembark extract of *Erythrina sigmoidea* on isolated rat ileal preparations.

Screening of indian plants for biological activity: part XIII. Indian Journal of

CAPES for their financial support in the form of grants and fellowship awards.

obtained from *E. mulungu* (Oliveira et al., *in press*).

588-591, ISSN 1744-5116

81-86, ISSN 0125-1570

0013-0001

0001

**5. Acknowledgments** 

**6. References** 


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 345

Gaw, H. Z. & Wang, H. P. (1949). Survey of chinese drugs for presence of antibacterial

Gessler, M. C., Nkunyak, M. H. H., Mwasumbi, L. B., Heinrich, M., & Tanner, M. (1994).

Gessler, M. C., Tanner, M., Chollet, J., Nkunya, N. H. H., & Heinrich, M. (1995). Tanzanian

Ghosal, S., Singh, S., & Bhattacharya, S. K. (1971). Alkaloids of Mucuna pruriens, Chemistry

Giron, L. M., Freire, V., Alonzo, A., & Caceres, A. (1991). Ethnobotanical survey of the

Hastings, R. B. (1990). Medicinal legumes of mexico: Fabaceae, Papilionoidea, part one.

Hegde, V. R., Dai, P., Patel, M. G., Puar, M. S., Das, P., Pai, J., Bryant, R., & Cox, P. A. (1997).

Herbarium (2008). Introdução à fitoterapia: utilizando adequadamente as plantas

Hickey, M. & King, C. (1981). 100 families of flowering plants, Cambridge University Press,

Holdsworth, D. (1984). Phytomedicine of the Madang province, Papua New Guinea part I.

Hussain, H. S. & Deeni, Y. Y. (1991). Plants in kano ethomedicine; screening for

Ichimaru, M., Moriyasu, M., Nishiyama, Y., Kato, A., Mathenge, S. G., Juma, F. D., &

Iinuma, M. & Tanaka, T. (1994). Isoflavanone derivatives with antibacterial activity from

Iinuma, M., Okawa,Y., Tanaka, T., Kobayashi, Y., & Miyauchi, K. I. (1994). Phenolic

Ishii, R., Yoshikawa, K., Minakata, H., Komura, H., & Kada, T. (1984). Specificities of bio-

Heterocycles, Vol.39, No.2, pp. 687-692, ISSN 1881-0942

and Pharmacology, Planta Medica, Vol.19, p. 279, ISSN 0032-0943

Economic Botany, Vol.44, No.3, pp. 336-348, ISSN 0013-0001

Natural Products, Vol.60, No.6, pp. 537-539, ISSN 0163-3864

ISBN 0521337003, 9780521337007, Cambridge, United Kingdom

substances. Science, Vol.110, pp. 11-12, ISSN 1095-9203

No.1, pp. 65-77, ISSN 0001-706X

pp. 504-508, ISSN 0951-418X

119, ISSN 0167-7314

Koho-1994. 6,312,983.

1520-6025

Vol.34, No.2/3, pp. 173-187, ISSN 0378-8741

medicinais, Colombo: Herbarium Lab. Bot. Ltda

Vol.29, No.1, pp. 51-56, ISSN 0976-8858

No.10, pp. 2587-2591, ISSN 0002-136

0378-8741

behavior in rats. Journal of Ethnopharmacology, Vol.69, No.2, pp. 189-196, ISSN

Screening tanzanian medicinal plants for antimalarial activity. Acta Tropica, Vol.56,

medicinal plants used traditionally for the treatment of malaria: in vivo antimalarial and *in vitro* cytotoxic activities. Phytotherapy Research, Vol.9, No.7,

medicinal flora used by the caribs of Guatemala. Journal of Ethnopharmacology,

Phospholipase A 2 inhibitors from an *Erythrina* species from Samoa. Journal of

Karkar island. International Journal of Crude Drug Research, Vol.22, No.3, pp. 111-

antimicrobial activity and alkaloids. International Journal of Pharmacognosy,

Nganga, J. N. (1996). Structural elucidation of new flavanones isolated from *Erythrina abyssinica*. Journal of Natural Products, Vol.59, No.12, pp. 1113-1116, ISSN

*Erythrina bidwilli* and antibacterial agents for mouth. Patent-Japan Kokai Tokkyo

compounds in *Erythrina bidwillii* and their activity against oral microbial organisms.

antimutagens in plant kingdom. Agricultural and Biological Chemistry, Vol.48,


Cronquist, A. (1981). An integrated system of classification of flowering plants, Columbia

Das, S. K. (1955). Medicinal, economic and useful plants of India, West Bengal, ISBN

De Cerain A. L. (1996). Cytotoxic activities of colombian plant extracts on chinense

Desmarchelier, C., Gurni, A., Ciccia, G., & Giulietti, A. M. (1996). Ritual and medicinal

Desmarchelier C., Repetto, M., Coussio, J., Llesuy, S., & Ciccia, G. (1997). Total reactive

Dhar, M. L., Dhar, M. M., Dhawan, B. N., Mehrotra, B. N., & Ray, C. (1968). Screening of

Diaz, J. L. (1977). Ethnopharmacology of sacred psychoactive plants used by the Indians of

Dominguez, X. A. & Alcorn, J. B. (1985). Screening of medicinal plants used by huastec

Duke, J. A. (1994). Amazonian ethnobotanical dictionary, CRC Press, ISBN 0849336643,

Dunstan, C. A., Noreen, Y., Serrano, G., Cox, P. A., Perera, P., & Bohlin, L. (1997). Evaluation

Etkin, N. L. (1997). Antimalarial plants used by Hausa in northern Nigeria. Tropical Doctor,

Filipoy, A. (1994). Medicinal plants of the pilaga of central chaco. Journal of

Flausino, O., Santos, L. S.,Verli, H., Pereira, A. M., Bolzani, S., & Nunes-De-Souza, R. L.

Fournet, A. Barrios, A. A., & Munoz, V. (1994). Leishmanicidal and trypanocidal activities of

García-Mateos, R., Soto-Hernández, M., & Vibrans, H. (2001). *Erythrina americana* Miller

Garin-Aguilar, M. E., Luna, J. E. R., Soto-Hernandez, M., Del Toro, G. V., & Vazquez, M.

Ethnopharmacology, Vol.44, No.3, pp. 181-193, ISSN 0378-8741

of Natural Products, Vol.70, pp. 48-53, ISSN 0163-3864

Ethnopharmacology, Vol.52, No.1, pp. 45-51, ISSN 0378-8741

Pharmacognosy, Vol.35, No.4, pp. 288-296, ISSN 1388-0209

Vol.6, pp. 232-247, ISSN 0019-5189

Vol.27, No.1, pp. 12-16, ISSN 0049-4755

Vol.55, pp. 391–400, ISSN 0013-0001

America

0951-418X

ISSN:0362-1642

USA

8741

139-156, ISSN 0378-8741

37, ISSN 0378-8741

B00117DIX2, India

University Press, ISBN 0231038801, 9780231038805, New York, United States of

hamster lung fibroblasts. Phytotherapy Research, Vol.10, No.5, pp. 431-432, ISSN

plants of the ese'ejas of the amazonian rainforest (Madre de Dios, Peru). Journal of

antioxidant potential (TRAP) and total antioxidant reactivity (TAR) of medicinal plants used in southwest Amazonas (Bolivia and Peru). International Journal of

indian plants for biological activity: part I. Indian Journal of Experimental Biology,

Mexico. Annual Review of Pharmacology and Toxicology, Vol.17, pp. 647-75,

mayans of northeastern Mexico. Journal of Ethnopharmacology, Vol.13, No.2, pp.

of some Samoan and Peruvian medicinal plants by prostaglandin biosynthesis and rat ear edema assays. Journal of Ethnopharmacology, Vol.57, pp. 35-56, ISSN 0378-

(2007). Anxiolytic effects of erythrinian alkaloids from *Erythrina mulungu*. Journal

bolivian medicinal plants. Journal of Ethnopharmacology, Vol.41, No.1/2, pp. 19-

('Colorín'; Fabaceae), a versatile resource from Mexico: a review. Economic Botany,

M. (2000). Effect of crude extracts of *Erythrina americana* Mill. on aggressive

behavior in rats. Journal of Ethnopharmacology, Vol.69, No.2, pp. 189-196, ISSN 0378-8741


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 347

Lewinsohn, R. (2003). Três Epidemias. Lições do Passado, p. 81-85, Editora Unicamp, ISBN

Lourenzi, H. (1992). Árvores brasileiras: manual de identificação e cultivo de plantas,

Maikere-Faniyo, R., Van Puyvelde, L., Mutwewingabo, A., & Habiyaremye, F. X. (1989).

Maillard, M., Gupta, M. P., & Hostettmann, K. (1987). A new antifungal prenylated

Manandhar, N. P. (1995). Medicinal Folk-Lore about the plants used as anthelmintic agents

Marchioro, M., Blank, M. D. F. A., Mourao, R. H. V., & Antoniolli, A. R. (2005). Anti-

Masilungan, V. A., Vadlamudi, S., & Goldin, A. (1971). Screening of philippine medicinal

Matos, F. J. (1994). Farmácias vivas: sistema de utilização de plantas medicinais projetado

Mc Clatchey, W. (1996). The ethnopharmacopoeia of Rotuma. Journal of

Mc Kee T. C., Bokesch, H. R., Mc Cormick, J. L., Rashid, A., Spielvogel, D., Gustafson, K. R.,

Medina, F. R. & Woodbury, R. Terrestrial plants molluscicidal to lymnaeid hosts of

Miguel, M. D. & Miguel, O. G. (2004). Desenvolvimento de fitoterápicos, 2nd Ed., Robe

Mino, J., Gorzalczany, S., Moscatelli, V., Ferraro, G., Acevedo, C., & Hnatyszyn, O. (2002).

Mitscher,L. A., Ward, J. A., Drake, S., & Rao, G. S. (1984). Antimicrobial agents from

Mitscher, L. A., Okwute, S. K., Gollapudi, S. R., Drake, S., & Avona, E. (1988). Antimicrobial

Farmaceutica Bonaerense, Vol.21, No.2, pp. 93-98, ISSN 0326-2383

Ethnopharmacology, Vol.50, No.3, pp. 147-156, ISSN 0378-8741

Ethnopharmacology, Vol. 26, No.2, pp. 101-109, ISSN 0378-8741

in Nepal. Fitoterapia, Vol.66, No. 2, pp. 149-155, ISSN 0367-326X

8741

0032-0943

Brazil

0163-3864

1881-0942

3449-3452, ISSN 0031-9422

8526805827, Campinas, Brazil

Plantarum, ISBN 9788586714320, São Paulo, Brazil

Vol.76, No.7-8, pp. 637-642, ISSN 0367-326X

Part 2, Vol.2, pp. 135-140, ISSN 0069-0120

Rico, Vol.63, pp. 366-376, ISSN 0041-994X

Editorial, ISBN 9788586652196, São Paulo, Brazil

43 species. Journal of Ethnopharmacology, Vol.25, No.3, pp. 315-338, ISSN 0378-

Study of rwandese medicinal plants used in the treatment of diarrhea. Journal of

flavanone from *Erythrina berteroana*. Planta Medica, Vol.53, No.6, pp. 563-564, ISSN

nociceptive activity of the aqueous extract of *Erythrina velutina* leaves. Fitoterapia,

plants for anticancer agents using CCNSC protocols. Cancer Chemother Reports

para pequenas comunidades, 2nd Ed, EUFC, ISBN 10 8572820086, Fortaleza,

Alavanja, M. M., Cardelina I. I. J. H., & Boyd, M. R. (1997). Isolation and characterization of new anti-HIV and cytotoxic leads from plants, marine, and microbial organisms. Journal of Natural Products, Vol.60, No.5, pp. 431-438, ISSN

*Fasciliasis hepatica* in Puerto Rico. Journal of Agriculture of the University of Puerto

Actividad antinociceptiva y antiinflammatoria de *Erythrina crista-galli*. Acta

higher plants. Erycristagalin, a new pterocarpene from the roots of the bolivian coral tree, *Erythrina crista-galli*. Heterocycles, Vol.22, No.8, pp. 1673-1675, ISSN

pterocarpans of nigerian *Erythrina mildbraedii*. Phytochemistry, Vol.27, No.11, pp.


Itokawa, H., Furukawa, H., & Tanaka, H. (1990). Screening test for antitumor activity of

Janzen,D. H., Juster, H. B., & Bell, E. A. (1977). Toxicity of secondary compounds to the seed-

John, D. (1984). One hundred useful raw drugs of the kani tribes of trivandrum forest

Joshi, R., Jain, N. K., & Garg, B. D. (1981). Antimicrobial activity of the oil and its

Joubert, F. J. & Sharon, N. (1985). Proteinase inhibitors from Erythrina corallodendron and

Jovel, E. M., Cabanillas, J., & Towers, G. H. H. (1996).An ethnobotanical study of the

Kamat, V. S., Chuo, F. Y., Kubo, I., & Nakanishi, K. (1981). Antimicrobial agents from an east

Kamusiime, H., Pedersen, A. T., Andersen, O. M., & Kiremire, B. (1996). Kaempferol 3-o-(2 o-beta-d-glucopyranosyl-6-o-alpha-l-rhamnopyranosyl-beta-d-glucopyranoside) from the african plant *Erythrina abyssinica*. International Journal of Pharmacognosy,

Keiser, I., Harris, E. J., Miyashita, D. H., Jacobson, M., & Perdue, R. E. (1975). Attraction of

mediterranean fruit flies. Lloydia, Vol.38, No.2, pp. 141-152, ISSN 0024-5461

Krukoff, B. A. & Barneby, R. C. (1974). Conspectus of species of the genus *Erythrina*. Lloydia,

Le Grand, A. & Wondergem, P. A. (1987). Antiinfective phytotherapy of the savannah

Le Grand, A. & Wondergem, P. A. (1988). Anti-infectious phytotherapies of the tree-

Le Grand, A. (1989). Anti-infectious phytotherapy of the tree-savannah, Senegal (western

Ethnopharmacology, Vol.22, No.1, pp. 25-31, ISSN 0378-8741

Ethnopharmacology, Vol.53, pp. 149-156, ISSN 0378-8741

Vol.34, No.5, pp. 370-373, ISSN 0975-4873

Bureau, ISBN 9789966846846 Nairobi, Kenya

Vol.37, No.3, pp. 332-459, ISSN 0024-5461

Vol.21, No.2, pp. 109-125, ISSN 0378-8741

Shoyakugaku Zasshi. Vol.44, No.1, pp. 58-62, ISSN 0037-4377

pp. 223-227, ISSN 0031-9422

pp. 17-39, ISSN 0167-7314

9422

ISSN 1881-0942

Vol.18, p. 411, ISSN 0019-462X

crude drugs (III). Studies on antitumor activity of indonesian medicinal plants.

eating larvae of the bruchid beetle *callosobruchus maculatus*. Phytochemistry, Vol.16,

division, Kerala, India. International Journal of Crude Drug Research, Vol.22, No.1,

unsaponifiable matter from the seeds of *Erythrina suberosa* roxb. Indian Drugs,

*Erythrina cristagalli* seeds. Phytochemistry, Vol.24, No.6, pp. 1169-1179, ISSN 0031-

traditonal medicine of the mestizo people of suni mirano, Loreto, Peru. Journal of

african medicinal plant *Erythrina abyssinica*. Heterocycles, Vol.15, pp. 1163-1170,

ethyl ether extracts of 232 botanicals to oriental fruit flies, melon flies, and

Khan, M. A., Khan, T., & Ahmad, Z. (1994). Barks used as source of medicine in madhya pradesh, India. Fitoterapia, Vol.65, No.5, pp. 444-446, ISSN 0367-326X Kloos, H., Thiongo, F. W., Ouma, J. H., & Butterworth, A. E. (1987). Preliminary evaluation

of some wild and cultivated plants for snail control in machakos district, Kenya. Journal of Tropical Medicine & Hygiene, Vol.90, No.4, pp. 197-204, ISSN 0022-5304 Kokwaro, J. O. (1976). Medicinal Plants of East Africa, East Africa Literature

forests of Senegal (east Africa). I. An inventory. Journal of Ethnopharmacology,

savannah of senegal (west-africa). II. Antimicrobial activity of 33 species. Journal of

Africa) III: a review of the phytochemical substances and anti-microbial activity of

43 species. Journal of Ethnopharmacology, Vol.25, No.3, pp. 315-338, ISSN 0378- 8741


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 349

Nkengfack, A. E., Azebaze, A. G. B., Waffo, A. K., Fomum, Z. T., Meyer, M., & Van Heerden,

Okunji, C. O. & Iwu, M. M. (1988). Control of schistosomiasis using nigerian medicinal

Oliveira, M. S. G., Aquino, A. B., Silva, D. L., Aquino, P. G. V., Santos, M. S., Porfírio, A. P.

Omer, M. E. A., Al Magboul, A. Z., & El Egami, A. A. (1998). Sudanese plants used in

Pennington, C. W. (1973). Medicinal plants utilized by the pima montanes of chihuahua.

Pérez, C. & Anesini C. (1994). In vitro antibacterial activity of Argentine folk medicinal

Pezzuto, J. M., Che, C. T., Mc Pherson, D. D., Zhu, J. P., Topcu, G., Erdelmeier, C. A. J., &

Pillay C. C. N., Jager, A. K., Mulholland, D. A., & Van Staden, J. (2001). Cyclooxygenase

Pinheiro de Sousa, M. & Rouquayrol, M. Z. (1974). Molluscicidal activity of plants from

Polhill, R. M. & Raven P. H. (E.d.) (1981). Advances in legume systematic I and II, Kew

Prabhu, V. K. K. & John, M. (1975). Juvenomimetic activity in some plants. Univ Kerala

Pushpangadan, P. & Atal, C. K. (1984). Ethno-medico-botanical investigations in kerala i.

Queiroz, E. F., Atindehou, K. K., Terreaux, C., Antus, S., & Hostettmann, K. (2002).

Rabe, T. & Van Staden, J. (1997). Antibacterial activity of south african plants used for

Rao, R. R. (1981). Ethnobotany of Meghalaya: medicinal plants used by khasi and garo

Ethnopharmacology, Vol.74, No.3, pp. 231-237, ISSN 0378-8741

Ethnopharmacology, Vol.11, No.1, pp. 59-77, ISSN 0378-8741

tribes. Economic Botany, Vol.35, No.1, pp. 4-9, ISSN 0013-0001

Publishing, ISBN 9780855212247, United Kingdom

Products, Vol.65, No.3, pp. 403-406, ISSN 0163-3864

America Indigena, Vol.33, pp. 213-232, ISSN 0185-1179

No.7, pp. 1113-1120, ISSN 0031-9422

No.4, pp. 246-252, ISSN 0167-7314

Farmacognosia, ISSN 0102-695X

No.6, pp. 542-545, ISSN 0367-326X

pp. 1522-1530, ISSN 0163-3864

46, ISSN 0378-8741

389-394

4754

8741

F. R. (2001). Cytotoxic isoflavones from *Erythrina indica*. Phytochemistry, Vol.58,

plants as molluscicides. International Journal of Crude Drug Research, Vol.26,

R., Sant'Ana, A. E. G., Santos, B. V. O. Alexandre-Moreira, M. S., & Araújo-Júnior, J. X. (*Article in Press*) Antinociceptive and anti-inflammatory activity of hydroalcoholic extracts and phases from *Erythrina mulungu*. Revista Brasileira de

folkloric medicine: screening for antibacterial activity. Part IX. Fitoterapia, Vol.69,

plants against *Salmonella typhi*. Journal of Ethnopharmacology, Vol.44, No.1, pp. 41-

Cordell, G. A. (1991). DNA as an affinity probe useful in the detection and isolation of biologically active natural products. Journal of Natural Products, Vol.54, No.6,

inhibiting and anti-bacterial activities of South African *Erythrina* species. Journal of

northeast Brazil. Revista Brasileira de Pesquisa Medica e Biológica, Vol.7, No.4, pp.

Dept Zoology Trivandrum Kerala India. Experientia, Vol. 31, p. 913, ISSN 0014-

Some primitive tribals of western ghats and their herbal medicine. Journal of

Prenylated isoflavonoids from the root bark of *Erythrina vogelii*. Journal of Natural

medicinal purposes. Journal of Ethnopharmacology, Vol.56, pp. 81-87, ISSN 0378-


Politica\_Nacional\_de\_Praticas\_Integrativas\_e\_Complementares\_SUS.pdf


Mokkhasmit, M., Ngarmwathana, W., Sawasdimongkol, K., & Permphiphat, U. (1971).

Moriyasu, M., Ichimaru, M., Nishiyama, Y., Kato, A., Mathenge, S. G., Juma, F. D., &

Morton, J. F. (1994). Pito (*Erythrina berteroana*) and chipilin (*Crotalaria longirostrata*),

Motsei, M. L., Lindsey, K. L., Van Staden, J., & Jager, A. K. (2003). Screening of traditionally

of Ethnopharmacology, Vol.76, No.2/3, pp. 235-241, ISSN 0378-8741 Muto, Y., Ichikawa, H., Kitagawa, O., Kumagai, K., Watanabe, M., Ogawa, E., Seiki, M.,

activity. Yakugaku Zasshi, Vol.114, No.2, pp. 980-994, ISSN 1347-5231 National Policy on Integrative and Complementary Practices (2006). Avaiable at:

Politica\_Nacional\_de\_Praticas\_Integrativas\_e\_Complementares\_SUS.pdf Ndamba, J., Nyazema, N., Makaza, N., Anderson, C., & Kaondera, K. C. (1994). Traditional

Journal of Ethnopharmacology, Vol.42, No.2, pp. 125-132, ISSN 0378-8741 Nguyen, V. T., Pham, T. K., Pho, D. T., & Do, C. H. (1991). The pharmacological action of

Nguyen, V. T., Pham, T. K., Pho, D. T., & Do, C. H. (1992). The anti-inflammatory effect of

Njamen, D., Nde, C. B. M., Fomum, Z. T., & Mbanya, J. C. (2007). Preventive effects of an

Nkengfack, A. E., Vouffo, T. W., Formum, Z. T., Meyer, M., Bergendorff, O., & Sterner, O.

Nkengfack, A. E., Vardamides, J. C., Fomum, Z. T., & Meyer, M. (1995). Prenylated

Nkengfack, A. E., Vouffo, W., Vardamides, J. C., Kouam, J., Fomum, Z. T., Meyer, M., &

Phytochemistry, Vol.36, No.4, pp. 1047-1051, ISSN 0031-9422

http://www.telessaudebrasil.org.br/lildbi/docsonline/3/1/113-

Association of Thailand, Vol.54, No.7, pp. 490-504, ISSN 0125-2208

Products, Vol.61, No.2, pp. 185-188, ISSN 1520-6025

No.2, pp. 130-138, ISSN 0013-0001

Vol.6, pp. 13-17, ISSN 0258-6967

444-446, ISSN 0951-418X

ISSN 0031-9422

Duoc Hoc, Vol.1, pp. 25-27, ISSN 0258-6967

Vol.46, No.3, pp. 573-578, ISSN 0031-9422

Pharmacological evaluation of Thai medicinal plants. Journal of the Medical

Nganga, J. N. (1998). Minor flavanones from *Erythrina abyssinica*. Journal of Natural

(Fabaceae), two soporific vegetables of central america. Economic Botany, Vol.48,

used south african plants for antifungal activity aganist Candida albicans. Journal

Shirataki, Y., Yokoe, I., & Komatsu, M. (1994). Studies on antiulcer agents. I. The effects of various methanol and aqueous extracts of crude drugs on antiucler

herbal remedies used for the treatment of urinary schistosomiasis in Zimbabwe.

total alkaloids extracted from *Erythrina orientalis* (L.) Murr. Tap Chi Duoc Hoc,

the total alkaloids extracted from the leaves of Erythrina orientalis Murr. Tap Chi

extract of *Erythrina lysistemon* (Fabaceae) on some menopausal problems: studies on the rat. Journal of Complementary and Integrative Medicine, Vol.4, No.1, pp. 1-17 Nkeh, B., Kamany, A., Bopelet, M., Ayafor, J. F., & Mbfor, J. T. (1996). Inhibition of

histamine-induced contraction of rat ileum by promethazine and the methanol stembark extract of *Erythrina sigmoidea*. Phytotherapy Research, Vol.10, No.5, pp.

(1994). Prenylated isoflavanone from the roots of *Erythrina sigmoidea*.

isoflavanone from *Erythrina eriotricha*. Phytochemistry, Vol.40, No.6, pp. 1803-1808,

Sterner, O. (1997). Phenolic metabolites from *Erythrina* species. Phytochemistry,


A Phytochemical and Ethnopharmacological Review of the Genus *Erythrina* 351

Taniguchi, M. & Kubo, I. (1993). Ethnobotanical drug discovery based on medicine men's

Telikepalli, H., Gollapudi, S. R., Keshavarz-Shokri, A., Velazquez, L., Sandmann, R. A.,

Tripathi, A. K. & Rizvi, S. M. A. (1984).Antifeedant activity of indigenous plants against

Unakul, S. (1950). Pharmacological studies. 2. Study of the leaves of *Erythrina fusca* lour.

Vasconcelos, S. M. M., Oliveira, G. R., Carvalho, M. M., Rodrigues, A. C. P., Silveira, E. R.,

Vlietinck, A. J., Van Hoof, L., Totte, J., Lasure, A., Vanden Berghe, D., Rwangabo, P. C., &

Waffo, A. K., Azebaze, G. A., Nkengfack, A. E., Fomum, Z. T., Meyer, M., Bodo, B., & Van

Wasuwat, S. (1967). A list of thai medicinal plants, ASRCT, Bangkok report project. 17

Widianto, M. B., Padmawinata, K., & Suhalim, H. (1980). An evaluation of the sedative effect

Wink, M. (1984). Chemical Defense of lupins. Mollusc-repellent properties of quinolizidine

Yamada, C. S. B. (1998). Fitoterapia sua história e importância. Revista Racine, Vol.43, pp.

Yanfg, L. L., Yen, K. Y., Kiso, Y., & Kikino, H. (1987). Antihepatotoxic actions of formosan

Yannitsaros, A. (1996). Screening for antiphage activity of plants growing in greece.

Yenesew, A., Derses, S., Irungu, B., Midiwo, J. O., Waters, N. C., Liyala, P., Akala, H.,

Fitoterapia, Vol.67, No.3, pp. 205-214, ISSN 0367-326X

Vol.69, No.7, pp. 658-661, ISSN 0032-0943

Research report, A.S.R.C.T., Nº.1 on Research Project, Vol. 17, p. 22

Siriraj Hospital Gazette, Vol.2, No.4, pp. 177-189, ISSN 0125-152X

Vasileva, B. (1969). Plantes medicinales de Guinee. Univ Moscow, Russia

Vol.29, No.6, pp. 2005-2007, ISSN 0031-9422

3864

3891

9422

5075

8741

ISSN 1347-5215

pp. 31-47, ISSN 0378-8741

50-51, ISSN 1807-166X

Spices, p. 147, Bangkok, Thailand, 1980

trials in the african savanna: screening of east african plants for antimicrobial activity II. Journal of Natural Products, Vol. 56, No.9, pp. 1539-1546, ISSN 0163-

Veliz, E. A., Rao, K. V. J., Madhavi, A. S., & Mitscher, L. A. (1990). Isoflavonoids and a cinnamyl phenol from root extracts of *Erythrina variegata*. Phytochemistry,

diacrisia obliqua walker. Curr Science, Vol.54, No.13, pp. 630-631, ISSN 0011-

Fonteles, M. M. F., Sousa, F. C. L., & Viana, G. S. B. (2003). Antinociceptive activities of the hydroalcoholic extracts from *Erythrina velutina* and *Erythrina mulungu* in mice. Biological and Pharmaceutical Bulletin, Vol. 26, pp. 946-949,

Mvukiyumwami, J. (1995). Screening of hundred rwandese medicinal plants for antimicrobial and antiviral properties. Journal of Ethnopharmacology, Vol.46, No.1,

Heerden, F. R. Indicanines B and C, two isoflavonoid derivatives from the root bark of *Erythrina indica*. Phytochemistry, Vol.53, No.8, pp. 981-985, ISSN 0031-

of the seeds of *Erythrina fusca* lour, 4th Asian Symposium on Medicinal Plants and

alkaloids. Zeitschrift für Naturforschung C., Vol.39, No.6, pp. 553-558, ISSN 0939-

plant drugs. Journal of Ethnopharmacology, Vol.19, No.1, pp. 103-110, ISSN 0378-

Heydenreich, M., & Peter, M. G. (2003a). Flavonoids and isoflavonoids with antiplasmodial activites from the root bark of *Erythrina abyssinica*. Planta Medica,


Ratnasooriya, W. D. & Dharmasiri, M. G. (1999). Aqueous extract of sri lankan *Erythrina*

Rogers, K. L., Grice, I. D., & Griffiths, L. R. (2001). Modulation of in vitro platelet 5-ht release

Ross, S. A., Megalla, S. E., Bishay, D. W., & Awad, A. H. (1980). Studies for determining

Saidu, K., Onah, J., Orisadipe, A., Olusola, A., Wambebe, C., & Gamaniel, K. (2000).

Selvanayahgam, Z. E., Gnanevendhan, S. G., Balakrishna, K., & Rao, R. B. (1994). Antisnake

Serragiotto, M. H., Leitão Filho, H., & Marsaioli, A. (1981). Erysotrine-N-oxide and

Silpasuwon, S. (1979). Studies of the effects of some medicinal plants on growth of some

Silva, O., Barbosa, S., Diniz, A., Valdeira, M. L., & Gomes, E. (1997). Plant extracts

Simões, C. M. O., Falkenberg, M., Auler Mentz, L., Schenkel, E. P., Amoros, M., & Girre, L.

Singh, L. M. & Chatterjee, S. (1979). Effect of amoora rohituka on in vitro blastogenesis of

Suffness, M., Abbott, B., Statz, D. W., Wonilowicz, E., & Spjut, R. (1988). The utility of

Tachibana, Y., Kato, A., Nishiyama, Y., Kawanishi, K., Tobe, H., Juma, F. D., Ogeto, J. O., &

Talla, E., Njamen, D., Mbafor, J. T., Fomum, Z. T., Kamanyi, A., Mbanya, J. C., Giner, R. M.,

Taniguchi, M., Chapya, A., Kubo, I., & Nakanishi, K. (1978). Screening of east African plants

activity. Fitoterapia, Vol.51, pp. 303-308, ISSN 0367-326X

of Chemistry, Vol.59, No.18, pp. 2771-2775, ISSN 1480-3291

Phytomedicine, Vol.6, No.3, pp. 205-214, ISSN 0975-0185

Planta Medica, Vol.59, No.4, pp. 354-358, ISSN 0032-0943

Products, Vol.66, No.6, pp. 891-893, ISSN 0163-3864

311-313, ISSN 0367-326X

No.1/2, pp. 275-280, ISSN 0378-8741

Plants, Vol.2, No.4, pp. 45-100, ISSN 1540-3580

ISSN 0024-3205

2522

0976-8858

0951-418X

Vol.14, No.1, pp. 45-48

2913, ISSN 1347-5223

*indica* leaves had sedative but not analgesic activity. Fitoterapia, Vol.70, No.3, pp.

by species of *Erythrina* and *Cymbopogon*. Life Sciences, Vol.69, No.15, pp. 1817-1829,

antibiotic substances in some egyptian plants. Part I. Screening for antimicrobial

Antiplasmodial, anaglesic, and anti-inflammatory activities of the aqueous extract of the stem bark of *Erythrina senegalensis*. Journal of Ethnopharmacology, Vol.71,

venom botanicals from ethnomedicine. Journal of Herbs, Spices and Medicinal

erythartine-N-oxide, two novel alkaloids from *Erythrina mulungu*. Canadian Journal

bacteria in the family enterobacteriaceae. Ms.Thesis Res Chiangmai University, p.

antiviral activity against herpes simplex virus type 1 and african swine fever virus. International Journal of Pharmacognosy, Vol.35, No.1, pp. 12-16, ISSN

(1999). Antiviral activity of south Brazilian medicinal plant extracts.

lymphocytes. Journal of Research in Indian Medicine, Yoga and Homeopathy,

p388 leukemia compared to b16 melanoma and colon carcinoma 38 for in vivo screening of plant extracts. Phytotherapy Research, Vol.2, No.2, pp. 89-97, ISSN

Mathenge, S. G. (1993). Mitogenic activities in african traditional herbal medicines.

Recio, M. C., Manez, S., & Rios, J. L. (2003). Warangalone, the isoflavonoid antiinflammatory principle of *Erythrina addisoniae* stem bark. Journal of Natural

for antimicrobial activity. Chemical and Pharmaceutical Bulletin, Vol.26, pp. 2910-


**17** 

*Brazil* 

**Phytochemistry, Pharmacology** 

**and Agronomy of Medicinal Plants:** 

Luzia Kalyne A. M. Leal and Glauce Socorro B. Viana

*Amburana cearensis***, an Interdisciplinary Study** 

Plants are an important source of biologically active substances, therefore they have been used for medicinal purposes, since ancient times. Plant materials are used as home remedies, in over-the-counter drug products, dietary supplements and as raw material for obtention of phytochemicals. The use of medicinal plants is usually based on traditional knowledge, from which their therapeutic properties are oftenly ratified in pharmacological

Nowadays, a considerable amount of prescribed drug is still originated from botanical sources and they are associated with several pharmacological activities, such as morphine (**I**) (analgesic), scopolamine (**II**) atropine (**III**) (anticholinergics), galantamine (**IV**) (Alzheimer's disease), quinine (**V**) (antimalarial), paclitaxel (**VI**), vincristine (**VII**) and vinblastine (**VIII**) (anticancer drugs), as well as with digitalis glycosides (**IX**) (heart failure) (Fig. 1). The versatility of biological actions can be attributed to the huge amount and wide variety of secondary metabolites in plant organisms, belonging to several chemical classes as

The large consumption of herbal drugs, in spite of the efficiency of synthetic drugs, is due to the belief that natural products are not toxic and/or have fewer side effects, the preference/need for alternative therapies, and their associated lower costs. In developing countries, herbal medicine is the main form of health care. In Brazil, where there is one of greatest biodiversity of plants in the world, pharmaceutical assistance programs, such as "Living Pharmacies", have a prominent role in spreading the rational use of medicinal plants mainly for poor people, under recognition by World Health Organization (WHO). Furthermore, herbal medicines also represent a significant pharmaceutical market share in

On the flip side, herbal drugs are discredited by most of the health related professionals, owing to a lack of scientific research supporting its efficacy and safety. In general, physicians feel insecure in prescribing herbal medicines, as most of them do not undergo through clinical trials, phytochemical analysis, and their active principles not being

alkaloids, coumarins, flavonoids, tannins, terpenoids, xanthones, etc.

some industrialized countries like Germany.

**1. Introduction** 

studies.

Kirley M. Canuto, Edilberto R. Silveira, Antonio Marcos E. Bezerra,

*Empresa Brasileira de Pesquisa Agropecuária, Universidade Federal do Ceará,* 

Zamora-Martinez, M. C. & Pola, C. N. P. (1992). Medicinal plants used in some rural populations of Oaxaca, Puebla and Veracruz, Mexico. Journal of Ethnopharmacology, Vol. 35, No.3, pp. 229-257, ISSN 0378-8741

```
http://portal.saude.gov.br/portal/arquivos/pdf/RENISUS.pdf
```
http://www.tropicos.org/Name/40005932

### **Phytochemistry, Pharmacology and Agronomy of Medicinal Plants:**  *Amburana cearensis***, an Interdisciplinary Study**

Kirley M. Canuto, Edilberto R. Silveira, Antonio Marcos E. Bezerra, Luzia Kalyne A. M. Leal and Glauce Socorro B. Viana *Empresa Brasileira de Pesquisa Agropecuária, Universidade Federal do Ceará, Brazil* 

#### **1. Introduction**

352 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Zamora-Martinez, M. C. & Pola, C. N. P. (1992). Medicinal plants used in some rural

Ethnopharmacology, Vol. 35, No.3, pp. 229-257, ISSN 0378-8741

http://portal.saude.gov.br/portal/arquivos/pdf/RENISUS.pdf

http://www.tropicos.org/Name/40005932

populations of Oaxaca, Puebla and Veracruz, Mexico. Journal of

Plants are an important source of biologically active substances, therefore they have been used for medicinal purposes, since ancient times. Plant materials are used as home remedies, in over-the-counter drug products, dietary supplements and as raw material for obtention of phytochemicals. The use of medicinal plants is usually based on traditional knowledge, from which their therapeutic properties are oftenly ratified in pharmacological studies.

Nowadays, a considerable amount of prescribed drug is still originated from botanical sources and they are associated with several pharmacological activities, such as morphine (**I**) (analgesic), scopolamine (**II**) atropine (**III**) (anticholinergics), galantamine (**IV**) (Alzheimer's disease), quinine (**V**) (antimalarial), paclitaxel (**VI**), vincristine (**VII**) and vinblastine (**VIII**) (anticancer drugs), as well as with digitalis glycosides (**IX**) (heart failure) (Fig. 1). The versatility of biological actions can be attributed to the huge amount and wide variety of secondary metabolites in plant organisms, belonging to several chemical classes as alkaloids, coumarins, flavonoids, tannins, terpenoids, xanthones, etc.

The large consumption of herbal drugs, in spite of the efficiency of synthetic drugs, is due to the belief that natural products are not toxic and/or have fewer side effects, the preference/need for alternative therapies, and their associated lower costs. In developing countries, herbal medicine is the main form of health care. In Brazil, where there is one of greatest biodiversity of plants in the world, pharmaceutical assistance programs, such as "Living Pharmacies", have a prominent role in spreading the rational use of medicinal plants mainly for poor people, under recognition by World Health Organization (WHO). Furthermore, herbal medicines also represent a significant pharmaceutical market share in some industrialized countries like Germany.

On the flip side, herbal drugs are discredited by most of the health related professionals, owing to a lack of scientific research supporting its efficacy and safety. In general, physicians feel insecure in prescribing herbal medicines, as most of them do not undergo through clinical trials, phytochemical analysis, and their active principles not being

Phytochemistry, Pharmacology

not considered to be herbal medicines. (Calixto, 2000).

derived from plant origins (Sahoo et al., 2010)

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 355

under different forms: oral tablets, capsules, gel caps, syrup, extracts and infusions. In general, combinations with chemically defined active substances or isolated constituents, are

The information about the therapeutical properties and usage of medicinal plants are commonly based on the empirical knowledge of ancient people, which was passed over several generations and originated the traditional medicine systems, utilized all over the world (Traditional Chinese Medicine, Ayurvedic system, Western and African Herbalisms). An estimated quantity of 50 000 plant species are used for medicinal purposes, from which the stand out species of the following families, such as Apocynaceae, Araliaceae, Apiaceae, Asclepiadaceae, Canellaceae, Clusiaceae and Menispermaceae (Schippmann et al., 2002). From the total of 252 drugs in the WHO's essential medicine list, 11% are exclusively

Herbal drugs are consumed by three-quarters of the world's population in the treatment of mainly chronic diseases, particularly headache, rheumatological disorders and asthma (Inamdar et al., 2010). In the developing countries, the population relies basically on medicinal plants for primary health care, since modern medicine is expensive and not easily accessible. However, the consumption of herbal drugs is also large in developed countries. Phytotherapy is popular in many countries of Western Europe (Germany, France, Italy, etc.), since people believe that either herbal drugs are devoid of side-effects or seek a healthier life style. Americans usually buy herbal products as a dietary supplement in the United States, aiming at preventing aging and diseases like cancer, as well as diabetes (Calixto, 2000).

Herbal drugs have some features which distinguish themselves considerably from synthetic drugs. Herbal medicines are always formed from a complex mixture of chemical compounds (eg. *Scutellaria baicalensis* has over 2000 components), and they may be constituted by many plants, therefore herbal drugs show an ample therapeutic usage. It is quite common to find a medicinal plant with several therapeutic properties (Sahoo et al., 2010; Calixto, 2000). The combination of either many plants, containing diverse bioactive substances or a pool of structural analogs, can produce a synergistic action that results in a stronger effect, therefore, permitting a reduction of dosage, which implies in lower risks of intoxication and undesirable side effects. As some diseases (e.g. AIDS or various types of cancer) possess a multi-causal etiology and a complex pathophysiology, a medical treatment may be more effective through well-chosen drug combinations than a single drug. Ginkgolides A and B, isolated from *Ginkgo biloba*, duly demonstrated a greater effect on the thrombocyte aggregation inhibition, when used as a mixture as opposed to what would be

On the other hand, plant-based products do not possess a well-defined chemical composition, due partially to chemical complexity stated above. Hence, the active principles of herbal drugs are frequently unknown, in addition to their standardization and quality control, being hardly achieved (Calixto, 2000) owing to mainly chemical variability in raw material. Secondary metabolites are the bioactive components from herbal drugs and their contents are strongly influenced by several factors: genetic (genotypes, chemotypes), physiologic (circadian rhythm, phenology, age), environmental (climate, sunlight exposure, water availability, soil, agronomic conditions) and manufacturing conditions (harvesting,

expected from the sum of the two compounds separately (Wagner, 2011).

storage and processing) (Tab. 1), (Sahoo et al., 2010; Gobbo-Beto & Lopes, 2007).

determined. Therefore, herbal medicines do not have a defined dosage, information on the chemical composition and warnings about possible risks. Additionally, the poor quality control of herbal drugs, which are subject to adulteration and intrinsic factors related to used raw material, do produce variables and inconsistent effects. Furthermore, most herbal drugs are produced from wild source, limiting the production at industrial level and putting the species used under threat of extinction.

Due to these aforesaid limitations, disadvantages and drawbacks of herbal medicine, we would like to present an updated review of chemical, pharmacological and agronomic studies of *Amburana cearensis* as a well succeeded example of a scientific research on wild plants and a model of a sustainable economic utilization of medicinal plants.

Fig. 1. Chemical structures of plant-derived drugs

#### **2. Herbal drugs and phytopharmaceuticals**

According to the WHO definition, herbal drugs are preparations containing plant parts (leaves, roots, seeds, stem bark, etc.) or whole plant materials in the crude or processed form, as active ingredients, besides some excipients. Herbal preparations can be found

determined. Therefore, herbal medicines do not have a defined dosage, information on the chemical composition and warnings about possible risks. Additionally, the poor quality control of herbal drugs, which are subject to adulteration and intrinsic factors related to used raw material, do produce variables and inconsistent effects. Furthermore, most herbal drugs are produced from wild source, limiting the production at industrial level and putting

Due to these aforesaid limitations, disadvantages and drawbacks of herbal medicine, we would like to present an updated review of chemical, pharmacological and agronomic studies of *Amburana cearensis* as a well succeeded example of a scientific research on wild

H N H

O

**VI**

HO

NH

O

**II III**

O

O

HO

O

O

O

O

OH

HO O O

O

O

H H

OH

O

O

OH H

According to the WHO definition, herbal drugs are preparations containing plant parts (leaves, roots, seeds, stem bark, etc.) or whole plant materials in the crude or processed form, as active ingredients, besides some excipients. Herbal preparations can be found

O OH HO

N

H H

O

O

O OH OCH3

<sup>H</sup> <sup>3</sup>

**IX**

plants and a model of a sustainable economic utilization of medicinal plants.

O

O

N

**V**

H

N

H H

O

HO **<sup>I</sup>**

HO

N

H

OH

<sup>H</sup> <sup>H</sup>

HO

<sup>N</sup> H3CO

R

O

HO

the species used under threat of extinction.

N

N

H

H3CO

N H

H3CO O

N

O

HO

**VII. R= CHO VIIII. R= CH3**

**IV**

H3CO

O

H HO

HO

H

Fig. 1. Chemical structures of plant-derived drugs

**2. Herbal drugs and phytopharmaceuticals** 

under different forms: oral tablets, capsules, gel caps, syrup, extracts and infusions. In general, combinations with chemically defined active substances or isolated constituents, are not considered to be herbal medicines. (Calixto, 2000).

The information about the therapeutical properties and usage of medicinal plants are commonly based on the empirical knowledge of ancient people, which was passed over several generations and originated the traditional medicine systems, utilized all over the world (Traditional Chinese Medicine, Ayurvedic system, Western and African Herbalisms). An estimated quantity of 50 000 plant species are used for medicinal purposes, from which the stand out species of the following families, such as Apocynaceae, Araliaceae, Apiaceae, Asclepiadaceae, Canellaceae, Clusiaceae and Menispermaceae (Schippmann et al., 2002). From the total of 252 drugs in the WHO's essential medicine list, 11% are exclusively derived from plant origins (Sahoo et al., 2010)

Herbal drugs are consumed by three-quarters of the world's population in the treatment of mainly chronic diseases, particularly headache, rheumatological disorders and asthma (Inamdar et al., 2010). In the developing countries, the population relies basically on medicinal plants for primary health care, since modern medicine is expensive and not easily accessible. However, the consumption of herbal drugs is also large in developed countries. Phytotherapy is popular in many countries of Western Europe (Germany, France, Italy, etc.), since people believe that either herbal drugs are devoid of side-effects or seek a healthier life style. Americans usually buy herbal products as a dietary supplement in the United States, aiming at preventing aging and diseases like cancer, as well as diabetes (Calixto, 2000).

Herbal drugs have some features which distinguish themselves considerably from synthetic drugs. Herbal medicines are always formed from a complex mixture of chemical compounds (eg. *Scutellaria baicalensis* has over 2000 components), and they may be constituted by many plants, therefore herbal drugs show an ample therapeutic usage. It is quite common to find a medicinal plant with several therapeutic properties (Sahoo et al., 2010; Calixto, 2000). The combination of either many plants, containing diverse bioactive substances or a pool of structural analogs, can produce a synergistic action that results in a stronger effect, therefore, permitting a reduction of dosage, which implies in lower risks of intoxication and undesirable side effects. As some diseases (e.g. AIDS or various types of cancer) possess a multi-causal etiology and a complex pathophysiology, a medical treatment may be more effective through well-chosen drug combinations than a single drug. Ginkgolides A and B, isolated from *Ginkgo biloba*, duly demonstrated a greater effect on the thrombocyte aggregation inhibition, when used as a mixture as opposed to what would be expected from the sum of the two compounds separately (Wagner, 2011).

On the other hand, plant-based products do not possess a well-defined chemical composition, due partially to chemical complexity stated above. Hence, the active principles of herbal drugs are frequently unknown, in addition to their standardization and quality control, being hardly achieved (Calixto, 2000) owing to mainly chemical variability in raw material. Secondary metabolites are the bioactive components from herbal drugs and their contents are strongly influenced by several factors: genetic (genotypes, chemotypes), physiologic (circadian rhythm, phenology, age), environmental (climate, sunlight exposure, water availability, soil, agronomic conditions) and manufacturing conditions (harvesting, storage and processing) (Tab. 1), (Sahoo et al., 2010; Gobbo-Beto & Lopes, 2007).

Phytochemistry, Pharmacology

to thousands of kilograms per annum. (McChesney, 2007)

*serpentina* and *Pterocarpus santalinus* (Schippmann et al., 2002).

been obtained by cultivation for a long time (Canter et al., 2005).

**4. Cultivation of medicinal plants** 

**3. Medicinal plants threatened by extinction** 

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 357

The interest in herbal drugs is continuously growing and they account for a significant share in the pharmaceutical market. The global herbal pharmaceutical industry (including drugs from herbal precursors and registered herbal medicines) invoices approximately US\$ 50 billion/year (2008). In 2006, the best selling herbal products were: Ginseng (>U\$ 1 billion global sales), Ginkgo (U\$ 1 billion), Noni (U\$ 1 billion), Saw Palmetto (U\$ 600 millions) Echinacea (U\$ 500 millions), Valerian (U\$ 450 millions), and Green Tea (U \$ 450 millions) (Gruenwald, 2008- Entrepreneur). The United States, China, Japan, Germany, South Korea and India, are the largest market. Medicinal plants have also been utilized as source of phytochemicals for the pharmaceutical, cosmetic and agrochemical industries. The most successful examples are paclitaxel (an anticancer drug from *Taxus baccata*), artemisinin (an antimalarial agent from *Artemisia annua*), vincristine/vinblastine (anticancer substances from *Catharanthus roseus*). The Pharmaceutical industry is interested in phytochemicals, however, the availability of quantities of pure chemical substances is normally a limiting factor, since the market demand for phytochemicals, usually reaches a scale from hundreds

The increasing demand for medicinal plants has endangered several species, since the main source of herbal drugs is the wild plant and the amount required from plant materials invariably exceeds the supply available from its natural source. Although the Convention on Biological Diversity (CBD), held in 1992, has established as goals, the conservation of biological diversity, the sustainable use of its components as well as the fair and equitable sharing of the benefits from the usage of genetic resources, it is still estimated that slightly more than 4000 medicinal plant species are under threat of extinction. The Convention on International Trade of Endangered Species of Wild Fauna and Flora (CITES), being the principal tool for monitoring or restricting the international trade of species threatened by over-exploitation, has published a biannual list of medicinal species like: *Taxus wallichiana*, *Panax quinquefolius*, *Dioscorea deltoidea*, *Hydrastis canadensis*, *Prunus africana* , *Rauvolfia* 

The overexploitation of a certain wild medicinal plant and consequent depletion of its raw material affect inevitably the economic feasibility of any phytopharmaceutical business in medium or long-term, since the production cost tends to be higher and the product supply become discontinuous. Furthermore, the extractivism provokes loss of genetic diversity, becoming the remaining plant population more vulnerable to diseases/pests and diminishing the variability of genotypes with features of interest such as yielding, bioactive substance content and resistance to biotic and abiotic factors (Rao et al., 2004). Hence, agencies concerned with conservation policies are recommending that wild species be brought into cultivation systems in order to assure the economic and environmental sustainability of herbal medicines trade (Schippmann et al., 2002). *Ginkgo biloba* and *Hypericum perforatum* are some of the topselling medicinal plants, however they are not endangered, because their plant materials have

The cultivation of medicinal plants is advocated as a means for meeting current and future demands for large quantities of herbal drugs, but also as a way to relieve the pressure of


Table 1. Effects on the production of secondary metabolites

Furthermore, most herbal drugs are utilized and commercialized without having a proven efficacy and safety through well-controlled double-blind clinical and toxicological trials, as pharmaceuticals are usually tested prior to being marketed. The safety and efficacy of herbal drugs are supported by their long historical use. Nevertheless, it is known that various herbal drugs fail, after testing in clinical trials and there are numerous reports on intoxication cases associated with their consumption. The WHO database has over sixteen thousand suspected case reports, related to intoxication by herbal drugs. The most frequent adverse reactions are hypertension, hepatitis, convulsions, thrombocytopenia and allergic reactions. Cardiovascular problems with the use of ephedra, hepatotoxicity caused by the consumption of kava-kava and comfrey, as well as licorice-related water retention, are some side effects claimed by the pharmacovigilance authorities. In addition to intrinsic factors mentioned above, the herbal drugs efficacy and safety, may also be seriously affected due to botanical misidentification or intentional usage of fake plants, contamination with pesticide residue, toxic heavy metals, pathogens and mycotoxins, as well as adulterants added to increase potency (synthetic substances) or the weight of herbal products in order to reduce costs (Sahoo et al., 2010; Calixto, 2000).

For the purpose of overcoming or mitigating the aforesaid inconvenient issues, WHO has developed a series of technical guidelines and documents in relation to the safety and the quality assurance of medicinal plants and herbal drugs preparations, such as "Quality Control Methods for Medicinal Plant Materials" (a collection of recommended test procedures for assessing the identity, purity and content of medicinal plant materials), "Guidelines on good agricultural and collection practices for medicinal plants", as well as "WHO guidelines for assessing quality of herbal medicines with reference to contaminants and residues". In turn, pharmaceutical laboratories have been investing in the enhancement of the quality for herbal products, aiming at the approval by governmental regulatory agencies, as a strategy to offer more reliable products, therefore conquering the confidence of health care professionals and consumers (Sahoo et al., 2010).

The interest in herbal drugs is continuously growing and they account for a significant share in the pharmaceutical market. The global herbal pharmaceutical industry (including drugs from herbal precursors and registered herbal medicines) invoices approximately US\$ 50 billion/year (2008). In 2006, the best selling herbal products were: Ginseng (>U\$ 1 billion global sales), Ginkgo (U\$ 1 billion), Noni (U\$ 1 billion), Saw Palmetto (U\$ 600 millions) Echinacea (U\$ 500 millions), Valerian (U\$ 450 millions), and Green Tea (U \$ 450 millions) (Gruenwald, 2008- Entrepreneur). The United States, China, Japan, Germany, South Korea and India, are the largest market. Medicinal plants have also been utilized as source of phytochemicals for the pharmaceutical, cosmetic and agrochemical industries. The most successful examples are paclitaxel (an anticancer drug from *Taxus baccata*), artemisinin (an antimalarial agent from *Artemisia annua*), vincristine/vinblastine (anticancer substances from *Catharanthus roseus*). The Pharmaceutical industry is interested in phytochemicals, however, the availability of quantities of pure chemical substances is normally a limiting factor, since the market demand for phytochemicals, usually reaches a scale from hundreds to thousands of kilograms per annum. (McChesney, 2007)

### **3. Medicinal plants threatened by extinction**

356 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Seasonality *Hypericum perforatum* Hypericin 300x content larger in summer than

Age *Papaver somniferum* Morphine 6x content larger on the 75th day

isoorientin

Temperature *Nicotiana tabacum* Scopolamine [Scopolamine] 4x larger after

Hypericin

Furthermore, most herbal drugs are utilized and commercialized without having a proven efficacy and safety through well-controlled double-blind clinical and toxicological trials, as pharmaceuticals are usually tested prior to being marketed. The safety and efficacy of herbal drugs are supported by their long historical use. Nevertheless, it is known that various herbal drugs fail, after testing in clinical trials and there are numerous reports on intoxication cases associated with their consumption. The WHO database has over sixteen thousand suspected case reports, related to intoxication by herbal drugs. The most frequent adverse reactions are hypertension, hepatitis, convulsions, thrombocytopenia and allergic reactions. Cardiovascular problems with the use of ephedra, hepatotoxicity caused by the consumption of kava-kava and comfrey, as well as licorice-related water retention, are some side effects claimed by the pharmacovigilance authorities. In addition to intrinsic factors mentioned above, the herbal drugs efficacy and safety, may also be seriously affected due to botanical misidentification or intentional usage of fake plants, contamination with pesticide residue, toxic heavy metals, pathogens and mycotoxins, as well as adulterants added to increase potency (synthetic substances) or the weight of herbal products in order to reduce

For the purpose of overcoming or mitigating the aforesaid inconvenient issues, WHO has developed a series of technical guidelines and documents in relation to the safety and the quality assurance of medicinal plants and herbal drugs preparations, such as "Quality Control Methods for Medicinal Plant Materials" (a collection of recommended test procedures for assessing the identity, purity and content of medicinal plant materials), "Guidelines on good agricultural and collection practices for medicinal plants", as well as "WHO guidelines for assessing quality of herbal medicines with reference to contaminants and residues". In turn, pharmaceutical laboratories have been investing in the enhancement of the quality for herbal products, aiming at the approval by governmental regulatory agencies, as a strategy to offer more reliable products, therefore conquering the confidence

*H. perforatum* Hyperforin/

Table 1. Effects on the production of secondary metabolites

of health care professionals and consumers (Sahoo et al., 2010).

costs (Sahoo et al., 2010; Calixto, 2000).

Phenology *Gentiana lutea* Mangiferin/

**Plant Compound (s) Result** 

*Ocimum gratissimum* Eugenol 98 % at 17h, but it is 11 % at

winter. (Southwell & Bourke, 2001)

after germination than on the 50th day (Williams & Ellis, 1989)

Before flowering- [Mangiferin] ; during flowering- [Isoorientin]

midday (Silva et al. 1999)

(Menković et al., 2000)

freezing (Koeppe et al, 1970)

Under water stress: 2x [Hyperforin] and [Hypericin]

(Zobayed et al, 2007)

**Evaluated Effect** 

Harvesting time

Water availability

> The increasing demand for medicinal plants has endangered several species, since the main source of herbal drugs is the wild plant and the amount required from plant materials invariably exceeds the supply available from its natural source. Although the Convention on Biological Diversity (CBD), held in 1992, has established as goals, the conservation of biological diversity, the sustainable use of its components as well as the fair and equitable sharing of the benefits from the usage of genetic resources, it is still estimated that slightly more than 4000 medicinal plant species are under threat of extinction. The Convention on International Trade of Endangered Species of Wild Fauna and Flora (CITES), being the principal tool for monitoring or restricting the international trade of species threatened by over-exploitation, has published a biannual list of medicinal species like: *Taxus wallichiana*, *Panax quinquefolius*, *Dioscorea deltoidea*, *Hydrastis canadensis*, *Prunus africana* , *Rauvolfia serpentina* and *Pterocarpus santalinus* (Schippmann et al., 2002).

> The overexploitation of a certain wild medicinal plant and consequent depletion of its raw material affect inevitably the economic feasibility of any phytopharmaceutical business in medium or long-term, since the production cost tends to be higher and the product supply become discontinuous. Furthermore, the extractivism provokes loss of genetic diversity, becoming the remaining plant population more vulnerable to diseases/pests and diminishing the variability of genotypes with features of interest such as yielding, bioactive substance content and resistance to biotic and abiotic factors (Rao et al., 2004). Hence, agencies concerned with conservation policies are recommending that wild species be brought into cultivation systems in order to assure the economic and environmental sustainability of herbal medicines trade (Schippmann et al., 2002). *Ginkgo biloba* and *Hypericum perforatum* are some of the topselling medicinal plants, however they are not endangered, because their plant materials have been obtained by cultivation for a long time (Canter et al., 2005).

#### **4. Cultivation of medicinal plants**

The cultivation of medicinal plants is advocated as a means for meeting current and future demands for large quantities of herbal drugs, but also as a way to relieve the pressure of

Phytochemistry, Pharmacology

respectively. (Canter et al., 2005)

and private laboratories (Fig. 3).

**5. Amburana cearensis** 

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 359

However, the cultivation of medicinal plant in agroforestry system can be a good alternative for more viable and environmentally sustainable farming. In China, ginseng (*Panax ginseng*) and other medicinal plants are grown in pine (*Pinus* spp.), *Paulownia tomentosa* and spruce (*Picea* spp.) forests; besides some medicinal herbs are often planted with bamboo (*Bambusa*  spp.). In New Zealand, American ginseng showed better growth under *Pinus radiate.* The shade offered by forest species seems to favor the growth of medicinal plants. Likewise, quinine yields of *Cinchona ledgeriana* increase when it is protected by shade of other species, such as *Crotalaria anagyroides* and *Tephrosia candida*. In India, some medicinal plants that have also been successfully intercropped with fuel wood trees (e.g., *Acacia auriculiformis* and *Eucalyptus tereticornis*) and coconut. Intercropping gives some income to farmers during the

Application of traditional and biotechnological plant-breeding techniques can become the cultivation of medicinal plants a trade more attractive (eg. increasing the yielding) as well as it can improve features of the plant that affect the efficacy and safety of a herbal drug (eg. levels of bioactive compounds or presence of potentially toxic substances). *Mentha* spp (mints) have been engineered to modify essential oil production and to enhance the resistance of the plant to fungal infection and abiotic stresses. Genetic engineering allowed the enhancement of scopolamine and artemisinin in *Atropa belladonna* and *Artemisia annua*,

*A. cearensis* (Fabaceae) is a native tree from "Caatinga" (a kind of vegetation found in the Brazilian semi-arid region), where it is popularly known as "cumaru" or "imburana-decheiro" (Fig. 2). Because of these said popular names, *A. cearensis* is usually misidentified as *Dipteryx odorata* (Fabaceae) and *Commiphora leptophloeos* (Burseraceae). *A. cearensis* occurs widely in South America (from Peru to Argentina), along with another species of this taxon, *Amburana acreana*, which is found chiefly in the southwestern region of the Amazon Forest. *A. cearensis* can reach 15 m of height and 50 cm of diameter, but it is characterized by white flowers and dark pods containing only one seed each, besides its stem bark possessing reddish stains and a vanilla-like aroma of coumarin (**1**). At the early stage of development (seedlings), *A. cearensis* displays a hypertrophied and subterraneous tube-like structure, called xylopodium, which acts as a storage of water and nutrients, therefore it is considered

Given the various applications, *A. cearensis* has a great commercial importance in Northeastern region of Brazil. Its wood is used in the carpentry for the manufacturing of furniture, doors and crates, owing to its recognized durability, whereas the seeds are used as flavoring and insect repellents. The wood powder from it can be added to alcoholic beverage barrels for accelerating the aging process of sugar cane distilled spirits (cachaça) (Aquino et al, 2005). The seeds and stem bark are traditionally utilized for treating respiratory diseases, such as influenza, asthma and bronchitis due to anti-inflammatory, analgesic and bronchodilator properties. As far as folk medicine is concerned, *A. cearensis* is consumed as a homemade medication called "lambedô (a sugary drink), however in an industrial scale, the syrup is a pharmaceutical form, which is produced by the government

period when the main trees have not started production. (Rao et al., 2004).

an adaptive strategy for arid habitats (Lima, 1989; Cunha & Ferreira, 2003).

harvesting on wild populations (Schippmann et al., 2002). In China, one of the largest markets of herbal medicine, 380,000 ha of lands are utilized for farming of medicinal plants.

Medicinal plants are also cultivated for supplying phytochemicals. Bristol-Myers Squibb developed a system of production based upon isolation of a precursor of Taxol from the leaves or needles of cultivated *Taxus baccata* or *T. wallichiana* that provide the hundreds of kilograms of Taxol required per year for the treatment of cancer patients. (McChesney, 2007)

From the perspective of the market, domestication and cultivation provide a number of advantages over wild harvest for production of herbal drugs: (1) reliable botanical identification; (2) uniform and high quality raw material. As wild plants are dependent on many factors that cannot be controlled and the irregularity of supply is a common feature, the cultivation assures a steady source of raw material; (3) price and volume between farmer and pharmaceutical companies can be more easily negotiable, since the production forecast is more precise; (4) genetic breeding and biotechnology tools can lead to the development of plant materials with agronomically and commercially desirable features, permitting to optimize yield and to meet regulations as well as consumer preferences, respectively; (5) cultivated material can be easily certified as "organic product" (Schippmann et al., 2002; Canter et al, 2005).

Cultivated plants account for 60-90 % in terms of amount of plant material employed by Herbal medicines companies, but the number of wild species still is larger. Although the cultivation is apparently more advantageous than wild harvesting, only 130-140 species are cultivated in Europe, while just 20 out of 400 medicinal plants marketed in India are grown in field. Likewise, amongst 1000 plants more commonly used with medicinal purposes in China, only 100-250 species are sourced from cultivation. There are some reasons that can explain this low utilization of cultivated plants:

(1) Belief of that wild specimens are more potent than cultivated plants. Chinese believe that the physical appearance of wild roots to the human body symbolizes vitality and this feature is crucial for the potency of the ginseng roots, nevertheless cultivated roots do not exhibit this characteristic shape. Furthermore, some scientific studies support partly this hypothesis saying that secondary metabolites, the main responsible for therapeutic properties of herbal medicines, are biosynthesized by plants under particular conditions of stress and competition in their natural environments. Hence, perhaps the secondary metabolites would not be so expressed in monoculture conditions, therefore the active ingredient levels can be much lower in cultivated plant.

(2) Domestication of wild plant is not always technically possible. Many species are difficult to cultivate because of certain biological features or ecological requirements (slow growth rate, special soil requirements, low germination rates, susceptibility to pests, etc.).

(3) Economical feasibility. Domestication requires a long time of agronomical studies and high financial investment for the plantation. Generally, production costs through cultivation are higher than wild harvesting, thus few species can be marketed at a high sufficient price to make cultivation profitable, for instance *Garcinia afzelii, Panax quinquefolius, Saussurea costus* and *Warburgia salutaris.* Hence, many endangered medicinal plants only will bring into cultivation, if exists governmental incentive (Schippmann et al., 2002).

However, the cultivation of medicinal plant in agroforestry system can be a good alternative for more viable and environmentally sustainable farming. In China, ginseng (*Panax ginseng*) and other medicinal plants are grown in pine (*Pinus* spp.), *Paulownia tomentosa* and spruce (*Picea* spp.) forests; besides some medicinal herbs are often planted with bamboo (*Bambusa*  spp.). In New Zealand, American ginseng showed better growth under *Pinus radiate.* The shade offered by forest species seems to favor the growth of medicinal plants. Likewise, quinine yields of *Cinchona ledgeriana* increase when it is protected by shade of other species, such as *Crotalaria anagyroides* and *Tephrosia candida*. In India, some medicinal plants that have also been successfully intercropped with fuel wood trees (e.g., *Acacia auriculiformis* and *Eucalyptus tereticornis*) and coconut. Intercropping gives some income to farmers during the period when the main trees have not started production. (Rao et al., 2004).

Application of traditional and biotechnological plant-breeding techniques can become the cultivation of medicinal plants a trade more attractive (eg. increasing the yielding) as well as it can improve features of the plant that affect the efficacy and safety of a herbal drug (eg. levels of bioactive compounds or presence of potentially toxic substances). *Mentha* spp (mints) have been engineered to modify essential oil production and to enhance the resistance of the plant to fungal infection and abiotic stresses. Genetic engineering allowed the enhancement of scopolamine and artemisinin in *Atropa belladonna* and *Artemisia annua*, respectively. (Canter et al., 2005)

#### **5. Amburana cearensis**

358 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

harvesting on wild populations (Schippmann et al., 2002). In China, one of the largest markets of herbal medicine, 380,000 ha of lands are utilized for farming of medicinal plants. Medicinal plants are also cultivated for supplying phytochemicals. Bristol-Myers Squibb developed a system of production based upon isolation of a precursor of Taxol from the leaves or needles of cultivated *Taxus baccata* or *T. wallichiana* that provide the hundreds of kilograms of Taxol required per year for the treatment of cancer patients. (McChesney,

From the perspective of the market, domestication and cultivation provide a number of advantages over wild harvest for production of herbal drugs: (1) reliable botanical identification; (2) uniform and high quality raw material. As wild plants are dependent on many factors that cannot be controlled and the irregularity of supply is a common feature, the cultivation assures a steady source of raw material; (3) price and volume between farmer and pharmaceutical companies can be more easily negotiable, since the production forecast is more precise; (4) genetic breeding and biotechnology tools can lead to the development of plant materials with agronomically and commercially desirable features, permitting to optimize yield and to meet regulations as well as consumer preferences, respectively; (5) cultivated material can be easily certified as "organic product" (Schippmann et al., 2002;

Cultivated plants account for 60-90 % in terms of amount of plant material employed by Herbal medicines companies, but the number of wild species still is larger. Although the cultivation is apparently more advantageous than wild harvesting, only 130-140 species are cultivated in Europe, while just 20 out of 400 medicinal plants marketed in India are grown in field. Likewise, amongst 1000 plants more commonly used with medicinal purposes in China, only 100-250 species are sourced from cultivation. There are some reasons that can

(1) Belief of that wild specimens are more potent than cultivated plants. Chinese believe that the physical appearance of wild roots to the human body symbolizes vitality and this feature is crucial for the potency of the ginseng roots, nevertheless cultivated roots do not exhibit this characteristic shape. Furthermore, some scientific studies support partly this hypothesis saying that secondary metabolites, the main responsible for therapeutic properties of herbal medicines, are biosynthesized by plants under particular conditions of stress and competition in their natural environments. Hence, perhaps the secondary metabolites would not be so expressed in monoculture conditions, therefore the active

(2) Domestication of wild plant is not always technically possible. Many species are difficult to cultivate because of certain biological features or ecological requirements (slow growth

(3) Economical feasibility. Domestication requires a long time of agronomical studies and high financial investment for the plantation. Generally, production costs through cultivation are higher than wild harvesting, thus few species can be marketed at a high sufficient price to make cultivation profitable, for instance *Garcinia afzelii, Panax quinquefolius, Saussurea costus* and *Warburgia salutaris.* Hence, many endangered medicinal plants only will bring

rate, special soil requirements, low germination rates, susceptibility to pests, etc.).

into cultivation, if exists governmental incentive (Schippmann et al., 2002).

2007)

Canter et al, 2005).

explain this low utilization of cultivated plants:

ingredient levels can be much lower in cultivated plant.

*A. cearensis* (Fabaceae) is a native tree from "Caatinga" (a kind of vegetation found in the Brazilian semi-arid region), where it is popularly known as "cumaru" or "imburana-decheiro" (Fig. 2). Because of these said popular names, *A. cearensis* is usually misidentified as *Dipteryx odorata* (Fabaceae) and *Commiphora leptophloeos* (Burseraceae). *A. cearensis* occurs widely in South America (from Peru to Argentina), along with another species of this taxon, *Amburana acreana*, which is found chiefly in the southwestern region of the Amazon Forest. *A. cearensis* can reach 15 m of height and 50 cm of diameter, but it is characterized by white flowers and dark pods containing only one seed each, besides its stem bark possessing reddish stains and a vanilla-like aroma of coumarin (**1**). At the early stage of development (seedlings), *A. cearensis* displays a hypertrophied and subterraneous tube-like structure, called xylopodium, which acts as a storage of water and nutrients, therefore it is considered an adaptive strategy for arid habitats (Lima, 1989; Cunha & Ferreira, 2003).

Given the various applications, *A. cearensis* has a great commercial importance in Northeastern region of Brazil. Its wood is used in the carpentry for the manufacturing of furniture, doors and crates, owing to its recognized durability, whereas the seeds are used as flavoring and insect repellents. The wood powder from it can be added to alcoholic beverage barrels for accelerating the aging process of sugar cane distilled spirits (cachaça) (Aquino et al, 2005). The seeds and stem bark are traditionally utilized for treating respiratory diseases, such as influenza, asthma and bronchitis due to anti-inflammatory, analgesic and bronchodilator properties. As far as folk medicine is concerned, *A. cearensis* is consumed as a homemade medication called "lambedô (a sugary drink), however in an industrial scale, the syrup is a pharmaceutical form, which is produced by the government and private laboratories (Fig. 3).

Phytochemistry, Pharmacology

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 361

The agronomical study of *A. cearensis* was carried out with seedlings obtained by seed germination. Each plot consisted of six regularly spaced rows of 20 cm, whose sowing density was 50 seeds/row. The seedlings were transplanted to four garden beds (1.2m×10 m), fertilized prior to an organic fertilizer (2.8 kg m-2), containing each of them 20 young

Eight harvestings were performed monthly, starting on the 2nd month until the 9th month after the sowing. The plants harvested were evaluated with the following parameters: fresh plant weight, plant height, xylopodium diameter, root size, ethanol extract yield from the aerial part and xylopodium (Fig. 4). The fresh biomass production of *A. cearensis* seedlings increased almost eight-fold, during the 2nd through 9th month after the sowing. With reference to ethanol extract yield, there was a tendency of decrease for the extract weigh/xylopodium weight ratio over a period of time, while an oscillatory behavior was observed for yield of the ethanol extract from its aerial part, achieving a plateau on the 3rd

Fig. 3. Syrups made from the trunk bark of *Amburana cearensis*.

Fig. 4. Seedlings of *A. cearensis* harvested in 8th month of growth

**5.1 Agronomical study of** *A. cearensis*

and 7th month (Leal et al. 2011)

plants.

Fig. 2. A wild specimen of *Amburana cearensis* in its natural habitat.

The medicinal use of *A. cearensis* is based on scientific studies, which demonstrated that this plant possesses therapeutic properties that justify its recommendation for the treatment of respiratory illnesses. Preclinical tests demonstrated bronchodilator, analgesic and antiinflammatory activities for the hydro-alcohol extract from the stem bark of the *A. cearensis*, which also showed to be free of toxicity in therapeutic doses. The chemical composition of the stem bark and seeds from it, consists basically of coumarin, flavonoids, phenol acids and phenol glucosides. Some of them were tested individually and showed pharmacological activities similar to the extract, hence they were considered the active principles of the *A. cearensis*.

However, the intense commercial use of *A. cearensis* has led to the threat of extinction for this specie. In order to ensure the conservation and the economic utilization of *A. cearensis*, we proposed the replacement of its stem bark of a wild adult plant for a young specimen, cultivated under controlled agronomic parameters. In an interdisciplinary study, ethanol extracts of cultivated plants were compared to the extracts of this wild plant through preclinical trials and phytochemical analysis.

Fig. 3. Syrups made from the trunk bark of *Amburana cearensis*.

#### **5.1 Agronomical study of** *A. cearensis*

360 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Fig. 2. A wild specimen of *Amburana cearensis* in its natural habitat.

preclinical trials and phytochemical analysis.

*cearensis*.

The medicinal use of *A. cearensis* is based on scientific studies, which demonstrated that this plant possesses therapeutic properties that justify its recommendation for the treatment of respiratory illnesses. Preclinical tests demonstrated bronchodilator, analgesic and antiinflammatory activities for the hydro-alcohol extract from the stem bark of the *A. cearensis*, which also showed to be free of toxicity in therapeutic doses. The chemical composition of the stem bark and seeds from it, consists basically of coumarin, flavonoids, phenol acids and phenol glucosides. Some of them were tested individually and showed pharmacological activities similar to the extract, hence they were considered the active principles of the *A.* 

However, the intense commercial use of *A. cearensis* has led to the threat of extinction for this specie. In order to ensure the conservation and the economic utilization of *A. cearensis*, we proposed the replacement of its stem bark of a wild adult plant for a young specimen, cultivated under controlled agronomic parameters. In an interdisciplinary study, ethanol extracts of cultivated plants were compared to the extracts of this wild plant through The agronomical study of *A. cearensis* was carried out with seedlings obtained by seed germination. Each plot consisted of six regularly spaced rows of 20 cm, whose sowing density was 50 seeds/row. The seedlings were transplanted to four garden beds (1.2m×10 m), fertilized prior to an organic fertilizer (2.8 kg m-2), containing each of them 20 young plants.

Fig. 4. Seedlings of *A. cearensis* harvested in 8th month of growth

Eight harvestings were performed monthly, starting on the 2nd month until the 9th month after the sowing. The plants harvested were evaluated with the following parameters: fresh plant weight, plant height, xylopodium diameter, root size, ethanol extract yield from the aerial part and xylopodium (Fig. 4). The fresh biomass production of *A. cearensis* seedlings increased almost eight-fold, during the 2nd through 9th month after the sowing. With reference to ethanol extract yield, there was a tendency of decrease for the extract weigh/xylopodium weight ratio over a period of time, while an oscillatory behavior was observed for yield of the ethanol extract from its aerial part, achieving a plateau on the 3rd and 7th month (Leal et al. 2011)

Phytochemistry, Pharmacology

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 363

Fig. 5. Chemical structures of constituents isolated from wild *A. cearensis*.

#### **5.2 Phytochemistry of** *A. cearensis*

#### **5.2.1 Wild plant**

While the pharmacological research about *A. cearensis* advanced, it was necessary to have a better understanding of the chemistry in this specie, previously limited to coumarin and a few phenol compounds, in order to discover its active principles. Bastos (1983), in her master thesis, found coumarin (**1**) as the most abundant component and described the isolation of isokaempferide (**2**), methyl 3,4-dimethoxy-cinnamate (**3**), afrormosin (**4**), 7 hidroxy-8,4'-dimethoxy-isoflavone (**5**), 24-methylenecycloartanol (**6**), -sitosterol (**7**) and 6 hidroxy-coumarin (**8**). Additionally, the seeds presented a high oil content (23 %), which is composed of triglycerides of the following fatty acids: oleic acid (53 %), palmitic acid (19 %), stearic acid (8 %) and linoleic acid (7 %). Bravo et al. (1999) isolated amburosides A [(4-*O*-- D-glucopyranosylbenzyl) protocatechuate] (**9**) and B [(4-*O*--D-glucopyranosylbenzyl) vanillate] (**10**) from the ethyl acetate extract of the trunk bark, utilizing just silica gel preparative Thin-Layer Chromatography (TLC) (Fig. 5)

Canuto & Silveira (2006) carried out a phytochemical investigation of the ethanol extract from the stem bark. The ethanol extract was partitioned with water and ethyl acetate. The aqueous phase showed to be very rich in sucrose (**11**), whereas the organic phase was dried and submitted to successive chromatographic columns on silica gel and dextran gels. These chromatographic separations led to the isolation of coumarin (**1**), two phenol acids [vanillic acid (**12**) and protocatechuic acid (**13**)], five flavonoids [isokaempferide (**2**), afrormosin (**4**), kaempferol (**14**), quercetin (**15**) and 4'-methoxy-fisetin (**16**)], amburoside A (**9**) and a mixture of -sitosterol and stigmasterol glycosides (**17-18**). Later on, this same methodology was applied to isolate an isoflavone formononetin (**19**) and a novel coumarin 6-coumaryl protocatechuate (**20**) (Canuto et al., 2010) (Fig. 5). Continuing with the phytochemical study of *A. cearensis*, the seeds revealed high presence of phenol glucosides. Liquid-liquid partitioning from the ethanol extracts followed by chromatography on Sephadex LH-20 and a reversed-phase HPLC chromatography of the ethyl acetate fraction, resulted in the isolation of six new amburosides (C-H). 4-*O*--D-(6´´-*O*-galloylglucopyranosyl)-benzyl protocatechuate (**21**), 4-*O*--D-(6´´-*O*-acetylglucopyranosyl)-benzyl protocatechuate (**22**), 4- *O*--D-(6´´-*O*-protocatechuoylglucopyranosyl)-benzyl protocatechuate (**23**), 4-*O*--D-(6´´-*O*feruloylglucopyranosyl)-benzyl protocatechuate (**24**), 4-*O*--D-(6´´-*O*vanilloylglucopyranosyl)-benzyl protocatechuate (**25**) and 4-*O*--D-(6´´-*O*sinapoylglucopyranosyl)-benzyl protocatechuate (**26**). Additionally, amburoside A (**9**), isokaempferide (**2**), vanillic acid (**12**), 6-hydroxycoumarin (**8**) and (*E*)-*o*-coumaric acid (**27**) were isolated from this same extract. (Canuto et al., 2010) (Fig. 5).

The isolation of 6-coumaryl protocatechuate (**20**) (trunk bark) and 6-hydroxycoumarin (**8**) (seeds) from *A. cearensis* presents an intriguing finding for biosynthesis of coumarins, since monoxygenated-coumarins are preferentially substituted at C-7 position (umbelliferone and its derivatives), according to biogenetic rules. This substitution pattern is due to the usual precursor of coumarins, *p*-coumaric acid, which is biosynthentized by the shikimate pathway from either tyrosine-deamination or *p*-hydroxylation of cinnamic acid (Dewick, 2002). Nevertheless, despite a large occurrence of simple coumarins oxygenated at the C-7 position, 6-hydroxycoumarin (**8**) was also found in some species like *Bidens parviflora*  (Asteraceae), *Paeonia suffruticosa* (Paeoniaceae) and *Hydrangea chinensis* (Hydrangeaceae) (Tommasi et al., 1992; Wu et al., 2002; Khalil et al., 2003)

While the pharmacological research about *A. cearensis* advanced, it was necessary to have a better understanding of the chemistry in this specie, previously limited to coumarin and a few phenol compounds, in order to discover its active principles. Bastos (1983), in her master thesis, found coumarin (**1**) as the most abundant component and described the isolation of isokaempferide (**2**), methyl 3,4-dimethoxy-cinnamate (**3**), afrormosin (**4**), 7 hidroxy-8,4'-dimethoxy-isoflavone (**5**), 24-methylenecycloartanol (**6**), -sitosterol (**7**) and 6 hidroxy-coumarin (**8**). Additionally, the seeds presented a high oil content (23 %), which is composed of triglycerides of the following fatty acids: oleic acid (53 %), palmitic acid (19 %), stearic acid (8 %) and linoleic acid (7 %). Bravo et al. (1999) isolated amburosides A [(4-*O*-- D-glucopyranosylbenzyl) protocatechuate] (**9**) and B [(4-*O*--D-glucopyranosylbenzyl) vanillate] (**10**) from the ethyl acetate extract of the trunk bark, utilizing just silica gel

Canuto & Silveira (2006) carried out a phytochemical investigation of the ethanol extract from the stem bark. The ethanol extract was partitioned with water and ethyl acetate. The aqueous phase showed to be very rich in sucrose (**11**), whereas the organic phase was dried and submitted to successive chromatographic columns on silica gel and dextran gels. These chromatographic separations led to the isolation of coumarin (**1**), two phenol acids [vanillic acid (**12**) and protocatechuic acid (**13**)], five flavonoids [isokaempferide (**2**), afrormosin (**4**), kaempferol (**14**), quercetin (**15**) and 4'-methoxy-fisetin (**16**)], amburoside A (**9**) and a mixture of -sitosterol and stigmasterol glycosides (**17-18**). Later on, this same methodology was applied to isolate an isoflavone formononetin (**19**) and a novel coumarin 6-coumaryl protocatechuate (**20**) (Canuto et al., 2010) (Fig. 5). Continuing with the phytochemical study of *A. cearensis*, the seeds revealed high presence of phenol glucosides. Liquid-liquid partitioning from the ethanol extracts followed by chromatography on Sephadex LH-20 and a reversed-phase HPLC chromatography of the ethyl acetate fraction, resulted in the isolation of six new amburosides (C-H). 4-*O*--D-(6´´-*O*-galloylglucopyranosyl)-benzyl protocatechuate (**21**), 4-*O*--D-(6´´-*O*-acetylglucopyranosyl)-benzyl protocatechuate (**22**), 4- *O*--D-(6´´-*O*-protocatechuoylglucopyranosyl)-benzyl protocatechuate (**23**), 4-*O*--D-(6´´-*O*feruloylglucopyranosyl)-benzyl protocatechuate (**24**), 4-*O*--D-(6´´-*O*vanilloylglucopyranosyl)-benzyl protocatechuate (**25**) and 4-*O*--D-(6´´-*O*sinapoylglucopyranosyl)-benzyl protocatechuate (**26**). Additionally, amburoside A (**9**), isokaempferide (**2**), vanillic acid (**12**), 6-hydroxycoumarin (**8**) and (*E*)-*o*-coumaric acid (**27**)

The isolation of 6-coumaryl protocatechuate (**20**) (trunk bark) and 6-hydroxycoumarin (**8**) (seeds) from *A. cearensis* presents an intriguing finding for biosynthesis of coumarins, since monoxygenated-coumarins are preferentially substituted at C-7 position (umbelliferone and its derivatives), according to biogenetic rules. This substitution pattern is due to the usual precursor of coumarins, *p*-coumaric acid, which is biosynthentized by the shikimate pathway from either tyrosine-deamination or *p*-hydroxylation of cinnamic acid (Dewick, 2002). Nevertheless, despite a large occurrence of simple coumarins oxygenated at the C-7 position, 6-hydroxycoumarin (**8**) was also found in some species like *Bidens parviflora*  (Asteraceae), *Paeonia suffruticosa* (Paeoniaceae) and *Hydrangea chinensis* (Hydrangeaceae)

**5.2 Phytochemistry of** *A. cearensis*

preparative Thin-Layer Chromatography (TLC) (Fig. 5)

were isolated from this same extract. (Canuto et al., 2010) (Fig. 5).

(Tommasi et al., 1992; Wu et al., 2002; Khalil et al., 2003)

**5.2.1 Wild plant** 

Fig. 5. Chemical structures of constituents isolated from wild *A. cearensis*.

Phytochemistry, Pharmacology

(*Z*)-Coumaric acid

p-Hidroxi-benzoic

**Substances Retention** 

three-fold lower than in the latter.

**time (min.)** 

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 365

glucoside 3,85 - + + + + - - - -

acid 4,99 + + + + + - - - - Protocatechuic acid 7,36 + + - + + + + + + Vanillic acid 10,87 + + + + + + + + + Coumarin 13,98 + + + + + + + + + Amburoside A 14,65 + + - + + - + + + Ayapin 14,85 - + + + - + + + + Amburoside B 17,17 + - + + + + + + +

Table 2. Distribution of some constituents in *A. cearensis* (**+**, presence; **-** ,not detected)

Industrial Quality: linearity, selectivity, accuracy, precision as well as the limit of detection and the limit of quantification. As can be noticed on Table 3, amburoside A (**9**) was the most abundant component in EETB, followed by coumarin (**1**), protocatechuic acid (**13**) and vanillic acid (**12**). However, coumarin (**1**) was the only component detected in EES. Among cultivated plants extracts, vanillic acid (**12**) was the principal component in 3 out of 4 periods analyzed through EEW, while coumarin (**1**) appeared as the major compound in the 7th month. Protocatechuic acid (**13**) and amburoside A (**9**) were below the limit of quantification in all extracts, except in the 9th month, whereby amburoside A (**9**) had a considerable content (Leal et al., 2011). In EEAP, vanillic acid (**12**) was the main constituent evaluated in all seasons, reaching the highest concentration in the 7th month (8520 mg/100g ext). On the other hand, coumarin (**1**) was the major component in xylopodium (4th month: 3760 mg/100g ext), except in the last month, when amburoside A (**9**) was the most abundant (680 mg/100g ext). Protocatechuic acid (**13**) presented measurable levels only in the EEAP of 4 months (360 mg/100g ext). Coumarin (**1**) and vanillic acid (**12**) were found preferentially in the aerial part, while amburoside A (**9**) was present mainly in the xylopodium. In comparison with wild plants, EEX of 9 months was the only cultivated plant extract which had amburoside A as the major component like EETB, even so at a concentration being

EEAP and EEX extracts harvested in the 7th month of growth were submitted to partition H2O/EtOAc, yielding aqueous and ethyl acetate fractions from each extract. Isokaempferide (**2**), amburoside B (**10**), vanillic acid (**12**), *p*-hidroxy-benzoic acid (**38**) and the coumarin ayapin (**36**) were isolated from the ethyl acetate fraction derived from EEAP after being chromatographed on silica and dextran gels, while the aqueous fraction of EEAP yielded (*E*)-melilotoside (**35**) and amburoside B (**10**), again, through C18 solid-phase extraction (SPE) and C18 HPLC. On the flip side, adsorption and exclusion chromatography of the ethyl acetate fraction derived of EEX, afforded the isolation of amburoside A (**9**) and protocatechuic acid (**13**), whereas (*Z*)-melilotoside (**37**) was isolated by Sephadex LH-20 followed with the purification through C18 HPLC (Fig. 6). (*E/Z*)- melilotoside (**35** and **37**) are trivial names for *o*-coumaric acid glucoside in allusion to the genus *Melilotus*, where these compounds were firstly identified. Interestingly, the *E* and *Z*-melilotosides were found in different parts of the *A. cearensis*: (*E*)-stereoisomer exclusively in the aerial part, while (Z)-

**Xylopodium (month)** 

**2 4 7 9 2 4 7 9** 

**Aerial Part (month)** 

**Trunk Bark** 

Recently, Bandeira et al (2011) studied a resin exuded from the trunk of *A. cearensis* and found a flavonoid-rich material. The resin ethanol extract was partitioned with water and organic solvents, yielding an ethyl acetate fraction, which was chromatographed on silica gel. From this chromatography, a chloroform fraction was separated by a Sephadex LH-20, resulting in the isolation of a novel compound 3',4'-dimethoxy-1'-(7-methoxy-4-oxo-4Hcromen-3-yl)-benzo-2',5'-quinone (**28**), along with six known compounds:, 7,8,3',4' tetramethoxyisoflavone (**29**), 3',4'-dimethoxy-7-hydroxyisoflavone (**30**) and 6,7,4' trimethoxy-3'-hydroxyisoflavone (**31**), 4,2',4'-trihydroxychalcone (**32**), 4,2',4'-trihydroxy-3 methoxychalcone (**33**), 3,4,5-trimethoxycinnamaldehyde (**34**).

#### **5.2.2 Cultivated plant**

In order to seek a sustainable alternative for an economic utilization of *A. cearensis*, our research became focused on this *A. cearensis* cultivated plant. The chemical study of the cultivated *A. cearensis* was divided into two parts: (1) a Nuclear Magnetic Resonance (NMR) and the HPLC profiling of ethanol extracts obtained from the aerial part (EEAP) and xylopodium (EEX) of specimens cultivated according to the growing conditions described above; (2) A refined phytochemical analysis of EEAP and EEX extracts produced from specimens in 7 months of growth, where was chosen with basis on pharmacological results.

NMR profiling was performed with extracts of specimens from the 2nd through the 9th month of growth. 1H NMR spectra, recorded in deuterated dimethylsulfoxide, revealed that the extracts from specimens with 2, 4, 7 and 9 months of growth, presented significantly different profiles, requiring further analysis. Hence, these extracts were duly analyzed comparatively with the wild plant extract by Photodiode Array detector (PDA)-HPLC profiling, utilizing constituents previously isolated from the wild *A. cearensis* as an analytical standard. The separations were performed on a C18 analytical column and the mobile phase was a gradient composed of H2O (pH 3, H3PO4-Et3N)/MeOH. The run time was 40 min and the chromatograms were observed at 254 nm. The qualitative analysis consisted of identification from analytical standards in the chromatograms of ethanol extracts, which were derived from the trunk bark (wild plant), xylopodium and the aerial part (cultivated plant) by retention time and UV spectra. Only 8 out of the 13 standards injected were detected in the samples. Coumarin (**1**) and vanillic acid (**12**) were the only substances present in all extracts of the *A. cearensis.* Amburoside B (**10**) and protocatechuic acid (**13**) were found in all extracts, except in the xylopodium extracts from specimens harvested in the 2nd and 4th month of growth, respectively. (*E*)-*o*-coumaric acid (**27**) and its glucoside (**35**), along with isokaempferide (**2**), afrormorsin (**4**) and kaempferol (**14**) were not detected in any extracts. Ayapin (**36**) and (*Z*)-*o*-coumaric acid glucoside (**37**) were found only in cultivated plants (Table 2).

A quantitative analysis was carried out for four major constituents of the *A. cearensis* [coumarin (**1**), amburoside A (**9**), vanillic acid (**12**), protocatechuic acid (**13**)] in ethanol extracts from trunk bark (EETB) and seeds (EES) of the said wild plant, as well as the whole plant (EEWP), xylopodium (EEX), as well as the aerial part (EEAP) of cultivated specimens in the four following selected months (2, 4, 7 and 9), accounting for 10 samples. The HPLC method was developed in chromatographic condition similar to the once described above and validated according to analytical parameters, defined by the Brazilian Health Surveillance Agency and the Brazilian Institute of Metrology, Standardization and


Recently, Bandeira et al (2011) studied a resin exuded from the trunk of *A. cearensis* and found a flavonoid-rich material. The resin ethanol extract was partitioned with water and organic solvents, yielding an ethyl acetate fraction, which was chromatographed on silica gel. From this chromatography, a chloroform fraction was separated by a Sephadex LH-20, resulting in the isolation of a novel compound 3',4'-dimethoxy-1'-(7-methoxy-4-oxo-4Hcromen-3-yl)-benzo-2',5'-quinone (**28**), along with six known compounds:, 7,8,3',4' tetramethoxyisoflavone (**29**), 3',4'-dimethoxy-7-hydroxyisoflavone (**30**) and 6,7,4' trimethoxy-3'-hydroxyisoflavone (**31**), 4,2',4'-trihydroxychalcone (**32**), 4,2',4'-trihydroxy-3-

In order to seek a sustainable alternative for an economic utilization of *A. cearensis*, our research became focused on this *A. cearensis* cultivated plant. The chemical study of the cultivated *A. cearensis* was divided into two parts: (1) a Nuclear Magnetic Resonance (NMR) and the HPLC profiling of ethanol extracts obtained from the aerial part (EEAP) and xylopodium (EEX) of specimens cultivated according to the growing conditions described above; (2) A refined phytochemical analysis of EEAP and EEX extracts produced from specimens in 7 months of growth, where was chosen with basis on pharmacological results. NMR profiling was performed with extracts of specimens from the 2nd through the 9th month of growth. 1H NMR spectra, recorded in deuterated dimethylsulfoxide, revealed that the extracts from specimens with 2, 4, 7 and 9 months of growth, presented significantly different profiles, requiring further analysis. Hence, these extracts were duly analyzed comparatively with the wild plant extract by Photodiode Array detector (PDA)-HPLC profiling, utilizing constituents previously isolated from the wild *A. cearensis* as an analytical standard. The separations were performed on a C18 analytical column and the mobile phase was a gradient composed of H2O (pH 3, H3PO4-Et3N)/MeOH. The run time was 40 min and the chromatograms were observed at 254 nm. The qualitative analysis consisted of identification from analytical standards in the chromatograms of ethanol extracts, which were derived from the trunk bark (wild plant), xylopodium and the aerial part (cultivated plant) by retention time and UV spectra. Only 8 out of the 13 standards injected were detected in the samples. Coumarin (**1**) and vanillic acid (**12**) were the only substances present in all extracts of the *A. cearensis.* Amburoside B (**10**) and protocatechuic acid (**13**) were found in all extracts, except in the xylopodium extracts from specimens harvested in the 2nd and 4th month of growth, respectively. (*E*)-*o*-coumaric acid (**27**) and its glucoside (**35**), along with isokaempferide (**2**), afrormorsin (**4**) and kaempferol (**14**) were not detected in any extracts. Ayapin (**36**) and (*Z*)-*o*-coumaric acid glucoside (**37**) were found only in cultivated

A quantitative analysis was carried out for four major constituents of the *A. cearensis* [coumarin (**1**), amburoside A (**9**), vanillic acid (**12**), protocatechuic acid (**13**)] in ethanol extracts from trunk bark (EETB) and seeds (EES) of the said wild plant, as well as the whole plant (EEWP), xylopodium (EEX), as well as the aerial part (EEAP) of cultivated specimens in the four following selected months (2, 4, 7 and 9), accounting for 10 samples. The HPLC method was developed in chromatographic condition similar to the once described above and validated according to analytical parameters, defined by the Brazilian Health Surveillance Agency and the Brazilian Institute of Metrology, Standardization and

methoxychalcone (**33**), 3,4,5-trimethoxycinnamaldehyde (**34**).

**5.2.2 Cultivated plant** 

plants (Table 2).

Table 2. Distribution of some constituents in *A. cearensis* (**+**, presence; **-** ,not detected)

Industrial Quality: linearity, selectivity, accuracy, precision as well as the limit of detection and the limit of quantification. As can be noticed on Table 3, amburoside A (**9**) was the most abundant component in EETB, followed by coumarin (**1**), protocatechuic acid (**13**) and vanillic acid (**12**). However, coumarin (**1**) was the only component detected in EES. Among cultivated plants extracts, vanillic acid (**12**) was the principal component in 3 out of 4 periods analyzed through EEW, while coumarin (**1**) appeared as the major compound in the 7th month. Protocatechuic acid (**13**) and amburoside A (**9**) were below the limit of quantification in all extracts, except in the 9th month, whereby amburoside A (**9**) had a considerable content (Leal et al., 2011). In EEAP, vanillic acid (**12**) was the main constituent evaluated in all seasons, reaching the highest concentration in the 7th month (8520 mg/100g ext). On the other hand, coumarin (**1**) was the major component in xylopodium (4th month: 3760 mg/100g ext), except in the last month, when amburoside A (**9**) was the most abundant (680 mg/100g ext). Protocatechuic acid (**13**) presented measurable levels only in the EEAP of 4 months (360 mg/100g ext). Coumarin (**1**) and vanillic acid (**12**) were found preferentially in the aerial part, while amburoside A (**9**) was present mainly in the xylopodium. In comparison with wild plants, EEX of 9 months was the only cultivated plant extract which had amburoside A as the major component like EETB, even so at a concentration being three-fold lower than in the latter.

EEAP and EEX extracts harvested in the 7th month of growth were submitted to partition H2O/EtOAc, yielding aqueous and ethyl acetate fractions from each extract. Isokaempferide (**2**), amburoside B (**10**), vanillic acid (**12**), *p*-hidroxy-benzoic acid (**38**) and the coumarin ayapin (**36**) were isolated from the ethyl acetate fraction derived from EEAP after being chromatographed on silica and dextran gels, while the aqueous fraction of EEAP yielded (*E*)-melilotoside (**35**) and amburoside B (**10**), again, through C18 solid-phase extraction (SPE) and C18 HPLC. On the flip side, adsorption and exclusion chromatography of the ethyl acetate fraction derived of EEX, afforded the isolation of amburoside A (**9**) and protocatechuic acid (**13**), whereas (*Z*)-melilotoside (**37**) was isolated by Sephadex LH-20 followed with the purification through C18 HPLC (Fig. 6). (*E/Z*)- melilotoside (**35** and **37**) are trivial names for *o*-coumaric acid glucoside in allusion to the genus *Melilotus*, where these compounds were firstly identified. Interestingly, the *E* and *Z*-melilotosides were found in different parts of the *A. cearensis*: (*E*)-stereoisomer exclusively in the aerial part, while (Z)-

Phytochemistry, Pharmacology

coumarin (**1**) (Dewick et al., 2002).

**5.3 Pharmacology of** *A. cearensis*

2001; 2003ab; 2005; 2006ab; 2008).

and 2D NMR (COSY, HSQC, HMBC and NOESY).

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 367

stereoisomer was present only in xylopodium. In *Melilotus alba* (a legume), the melilotosides are considered the precursors of coumarin, being one of the major constituents of *A. cearensis*. (*E*)-melilotoside (**35**) exposed to UV radiation (sunlight) may be converted to the less stable stereoisomer (*Z*), which do undergo enzyme-catalyzed lactonization to yield

As part of our effort for finding out which substances are responsible by medicinal properties of *A. cearensis*, isokaempferide (**2**), afrormosin (**4**), amburoside A (**9**), vanillic acid (**12**) and protocatechuic acid (**13**) obtained from this work were assayed in diverse pharmacological tests, which will be discussed briefly. The chemical structures of the new compounds were elucidated by means of spectroscopic techniques such as IR, HRMS, 1D

The literature reports several toxicological and pharmacological studies carried out with the extracts and substances isolated from wild and cultivated *A. cearensis.* The focus of them is on the anti-inflammatory, antioxidant, smooth muscle relaxant, antinociceptive, neuroprotector and platelet antiaggregant effects (Leal, 1995; 2006; Leal et al., 1997; 2000;

A toxicological study carried out with the hydroalcoholic extract (HAE) from the trunk bark of the *A. cearensis* administered to rats by the oral route did not show any toxic effects (Leal et al., 2003). Further studies demonstrated that the HAE administered to rats daily for 50 days did not interfere with the pregnancy rate and development during the 1st as well as the 2nd generation of animals (Leal et al., 2003a, Leal et al., 2006a). The cytotoxicity of isokaempferide (**2**), kaempferol (**14**), amburoside A (**9**) and protocatechuic acid (**13**) from the *A. cearensis,* were evaluated on tumor cell lines and on the sea urchin egg development, as well as their lytic properties on mouse erythrocytes. The results showed that isokaempferide (**2**) and kaempferol (**14**), but not amburoside A (**9**) and protocatechuic acid (**13**), inhibited the sea urchin egg development, as well as tumor cell lines. However, only protocatechuic acid

Previous studies (Leal et al., 1997; 2000) reported the antinociceptive, antiedematogenic and smooth muscle relaxant properties of HAE, coumarin (**1**), and the flavonoid fraction, from wild *A. cearensis*. The antiedematogenic activity was manifested in inflammatory process dependents on polimorphonuclear cells, while the antinociceptive effect of coumarin (**1**) and HAE seems to occur by a mechanism at least in part dependent on the opioid system. Nevertheless, the nitridergic system has also an important role in the coumarin nociception. Additional studies about the pharmacological potential of the HAE, coumarin (**1**) and the flavonoid fraction emphasized the anti-inflammatory potential of these species, which

Like other medicinal plants containing coumarin (**1**) such as *Justicia pectoralis, Pterodon polygaliflorus, Hybanthus ipecacuanha* and *Eclipta alba*, *A. cearensis,* also has a relaxing activity on isolated guinea pig tracheal muscles (Leal et al., 2000). Confirming this as particular effect, it was recently (Leal et al., 2006) demonstrated the relaxant action of the isokaempferide (**2**). The relaxation of the guinea-pig isolated trachea, induced by isokaempferide (**2**), was a direct and an epithelium-independent phenomenon, resulting

seems to be related to the presence of coumarin (**1**) in the plant (Leal et al., 2003).

(**13**) induced lysis on mouse erythrocytes (Costa-Lotufo et al., 2003).


Table 3. Concentrations of four major compounds of *A.cearensis* in different extracts.

Fig. 6. Chemical structures of constituents isolated from cultivated *A. cearensis*.

stereoisomer was present only in xylopodium. In *Melilotus alba* (a legume), the melilotosides are considered the precursors of coumarin, being one of the major constituents of *A. cearensis*. (*E*)-melilotoside (**35**) exposed to UV radiation (sunlight) may be converted to the less stable stereoisomer (*Z*), which do undergo enzyme-catalyzed lactonization to yield coumarin (**1**) (Dewick et al., 2002).

As part of our effort for finding out which substances are responsible by medicinal properties of *A. cearensis*, isokaempferide (**2**), afrormosin (**4**), amburoside A (**9**), vanillic acid (**12**) and protocatechuic acid (**13**) obtained from this work were assayed in diverse pharmacological tests, which will be discussed briefly. The chemical structures of the new compounds were elucidated by means of spectroscopic techniques such as IR, HRMS, 1D and 2D NMR (COSY, HSQC, HMBC and NOESY).

#### **5.3 Pharmacology of** *A. cearensis*

366 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Seeds ND ND 23520 *(3,9)* ND

2 months ND 1520 *(1,3)* 1020 *(5,7)* ND 4 months ND 2680 *(13,4)* 2000 *(7,0)* ND 7 months ND 3440 *(4,3)* 4060 *(6,5)* ND 9 months ND 1520 *(6,1)* 660 *(1,8)* 400 *(5,7)*

2 months ND 4780 *(10,6)* 1540 *(3,0)* ND 4 months 360 (3,3) 6120 *(4,8)* 1660 *(2,0)* 260 *(8,7)* 7 months ND 8520 *(1,0)* 6060 *(7,9)* ND 9 months ND 3460 *(6,3)* 1500 *(3,3)* ND

2 months ND 780 (5,3) 1320 *(1,0)* 380 *(5,3)* 4 months ND 760 (7,5) 3760 *(0,9)* ND 7 months ND 1380 (2,9) 2500 *(4,2)* 300 *(10,4)* 9 months ND 540 (9,8) 420 *(10,8)* 680 *(9,4)*

OCH3

**2**

OH

OH

OH OH

OH

OH

OH CO2H

OH

O

O

**35.** <sup>7</sup> *trans 37***.** <sup>7</sup> *cis*

R

**36**

**12**. R= OCH3 **13.** R= OH **38.** R= H

O O

Table 3. Concentrations of four major compounds of *A.cearensis* in different extracts.

O

HO

O

OH

O O HO

**9.** R= H **10.** R= CH3

Fig. 6. Chemical structures of constituents isolated from cultivated *A. cearensis*.

<sup>O</sup> <sup>O</sup> HO

CH=CHCO <sup>2</sup>H

**Protocatechuic acid Vanillic acid Coumarin Amburoside** 

*(6,3)* <sup>200</sup>*(5,6)* 1340 *(6,8)* <sup>2180</sup>

*(5,5)*

**Extracts Concentration (mg/100g extract)–** *CV* **(%)** 

Wild Plant

**Cultivated Plant**  *Whole* 

*Aerial Part* 

*Xylopodium* 

O O

**1**

O

O

RO

HO

Trunk bark <sup>320</sup>

The literature reports several toxicological and pharmacological studies carried out with the extracts and substances isolated from wild and cultivated *A. cearensis.* The focus of them is on the anti-inflammatory, antioxidant, smooth muscle relaxant, antinociceptive, neuroprotector and platelet antiaggregant effects (Leal, 1995; 2006; Leal et al., 1997; 2000; 2001; 2003ab; 2005; 2006ab; 2008).

A toxicological study carried out with the hydroalcoholic extract (HAE) from the trunk bark of the *A. cearensis* administered to rats by the oral route did not show any toxic effects (Leal et al., 2003). Further studies demonstrated that the HAE administered to rats daily for 50 days did not interfere with the pregnancy rate and development during the 1st as well as the 2nd generation of animals (Leal et al., 2003a, Leal et al., 2006a). The cytotoxicity of isokaempferide (**2**), kaempferol (**14**), amburoside A (**9**) and protocatechuic acid (**13**) from the *A. cearensis,* were evaluated on tumor cell lines and on the sea urchin egg development, as well as their lytic properties on mouse erythrocytes. The results showed that isokaempferide (**2**) and kaempferol (**14**), but not amburoside A (**9**) and protocatechuic acid (**13**), inhibited the sea urchin egg development, as well as tumor cell lines. However, only protocatechuic acid (**13**) induced lysis on mouse erythrocytes (Costa-Lotufo et al., 2003).

Previous studies (Leal et al., 1997; 2000) reported the antinociceptive, antiedematogenic and smooth muscle relaxant properties of HAE, coumarin (**1**), and the flavonoid fraction, from wild *A. cearensis*. The antiedematogenic activity was manifested in inflammatory process dependents on polimorphonuclear cells, while the antinociceptive effect of coumarin (**1**) and HAE seems to occur by a mechanism at least in part dependent on the opioid system. Nevertheless, the nitridergic system has also an important role in the coumarin nociception. Additional studies about the pharmacological potential of the HAE, coumarin (**1**) and the flavonoid fraction emphasized the anti-inflammatory potential of these species, which seems to be related to the presence of coumarin (**1**) in the plant (Leal et al., 2003).

Like other medicinal plants containing coumarin (**1**) such as *Justicia pectoralis, Pterodon polygaliflorus, Hybanthus ipecacuanha* and *Eclipta alba*, *A. cearensis,* also has a relaxing activity on isolated guinea pig tracheal muscles (Leal et al., 2000). Confirming this as particular effect, it was recently (Leal et al., 2006) demonstrated the relaxant action of the isokaempferide (**2**). The relaxation of the guinea-pig isolated trachea, induced by isokaempferide (**2**), was a direct and an epithelium-independent phenomenon, resulting

Phytochemistry, Pharmacology

standard drug (Fig. 7).

by our laboratory.

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 369

administered to rats, was shown to significantly inhibit the carrageenan, but not the dextran-induced edema. It also reduced the accumulation of PMN into the peritoneal cavity of rats and this effect was comparable to that observed with dexamethasone, used as a

Vanillic acid (**12**) is a benzoic acid derivative that is used as a flavoring agent. It is an intermediate in the production of vanillin from ferulic acid (Prince et al., 2011; Kim et al., 2011). Previous studies have shown antifilarial, antibacterial, antioxidant, hepatoprotective and anti-inflammatory (Kim et al., 2011; Prince et al., 2011; Itoh et al., 2009) effects of the vanillic acid. This compound exerts its anti-inflammatory effect by suppressing the production of prostaglandin E2, nitric oxide and cytokines. Furthermore, it also suppressed the activation of nuclear-factor-kappa B and caspase (Kim et al., 2011; Itoh et al., 2009). These findings confirm the anti-inflammatory activity of vanillic acid (**12**) as demonstrated

Fig. 7. Antinociceptive and anti-inflammatory effects of ethanolic extracts (EtOHE) and

A growing body of evidence suggests that the extract and chemical constituents from the wild *A. cearensis* have pharmacological properties which justify at least in part its traditional use in the treatment of asthma. Among others, the anti-inflammatory activity is possible due

1 I : effects of EtOHE (cultivated, (4, 7 and 9 months(m) or wild plants: 200 mg/kg, p.o.), VA (50 mg/kg, p.o.) or morphine (MP, 5 mg/kg, s.c.) on the formalin-induced nociception in mice (6-18 animals/group). II, III and IV: anti-inflammatory effects of EtOHE and VA on the carrageenan (Cg)-

vanillic acid (VA) from *Amburana cearensis* in rodents.1

induced mice paw edema and Cg-inuced rat peritonitis.

from several intracellular actions through a common pathway e.g., the opening of Ca2+ and ATP-sensitive K+ channels.

Previous studies (Leal et al., 2003; Leal, 2006) showed that the anti-inflammatory activity of HAE, coumarin (**1**), isokaempferide (**2**) and amburoside A (**9**) from *A. cearensis,* seems to occur by an inhibitory action on the release of inflammatory mediators, and/or alternatively by interfering with a certain phase of the neutrophil migration into the inflammatory focus. Other data (Leal et al., 2008) corroborated this hypothesis showing that both the isokaempferide (**2**) and amburoside A (**9**) exert their anti-inflammatory activities mainly by inhibiting the lipopolysaccharide-induced release of TNF-α, although the involvement of other inflammatory mediators cannot be excluded. Furthermore, inhibitions of some biological functions of neutrophils, namely, accumulation of cells and activity of hydrolytic enzymes, as myeloperoxidase, may also play a role.

Amburoside A (**9**) showed a hepatoprotective property in the CCl4-induced liver toxicity model in rats. This effect may be due to its capacity to modulate the oxidative stress, especially by reducing of the lipid peroxidation, as well as by a significant restoration to normal levels of the catalase activity and GSH contents as observed in CCl4-intoxicated rats after the amburoside A (**9**) treatment (Leal et al., 2008).

The large-scale usage and demand for the wild *A. cearensis,* as a medicinal plant by communities in the Northeastern of Brazil, governmental programs of phytotherapy as well as the pharmaceutical industry, are contributing to decrease availability on these species, presently considered as endangered ones. In this sense, our laboratory has conducted comparative studies on the pharmacological profile of the ethanolic extract (EtOHE) or vanillic acid (**12**) from the wild and cultivated *A. cear*ensis, by evaluating their antinociceptive and antiedematogenic activities in several experimental models, such as the formalin test, carrageenan or dextran-induced edema and carragenan-induced neutrophil migration into the rat peritoneal cavity (Leal et al., 2010).

The acute treatment with both the EtOHE prepared from all parts of the cultivated *A. cearensis* (4, 7 or 9 months) or the wild *A. cearensis,* present antinociceptive and antiinflammatory activities (Fig. 7). In addition, vanillic acid (**12**), which together with coumarin (**1**) are the major compounds present in cultivated *A. cearensis*, also showed an antinociceptive activity by inhibiting both phases of the formalin test in mice, and this effect was partially blocked by naloxone. Thus, the data suggest that antinociceptive effect of vanillic acid (**12**) occur by a mechanism at least in part dependent on the opioid system (Leal et al., 2010).

Coumarin (**1**) has been found in several Brazilian medicinal plants including *J. pectoralis*, *M. glomerata* and *A. cearensis*. It has been reported that the antinociceptive and the antiinflammatory activities of these species seems to be related at least in part to the presence of coumarin (**1**) (Leal et al., 1997; 2003; Leal et al., 1997; Lino et al., 1997; Leal et al., 2000; Freitas et al., 2008). The biological effects of coumarin (**1**) include antibacterial, antiviral, antiedematogenic, antioxidant, lipoxygenase inhibition, lipid peroxidation inhibition, and scavenging of superoxide hydroxyl radicals (Hoult & Paya, 1996; Chang et al., 1996; Casley-Smith et al., 1993; Rajarajeswari & Pari, 2011).

Recently (Leal et al., 2010), it was also determined that the anti-inflammatory effect of vanillic acid (**12**) is isolated from the cultivated *A. cearensis.* This compound orally

from several intracellular actions through a common pathway e.g., the opening of Ca2+ and

Previous studies (Leal et al., 2003; Leal, 2006) showed that the anti-inflammatory activity of HAE, coumarin (**1**), isokaempferide (**2**) and amburoside A (**9**) from *A. cearensis,* seems to occur by an inhibitory action on the release of inflammatory mediators, and/or alternatively by interfering with a certain phase of the neutrophil migration into the inflammatory focus. Other data (Leal et al., 2008) corroborated this hypothesis showing that both the isokaempferide (**2**) and amburoside A (**9**) exert their anti-inflammatory activities mainly by inhibiting the lipopolysaccharide-induced release of TNF-α, although the involvement of other inflammatory mediators cannot be excluded. Furthermore, inhibitions of some biological functions of neutrophils, namely, accumulation of cells and activity of hydrolytic

Amburoside A (**9**) showed a hepatoprotective property in the CCl4-induced liver toxicity model in rats. This effect may be due to its capacity to modulate the oxidative stress, especially by reducing of the lipid peroxidation, as well as by a significant restoration to normal levels of the catalase activity and GSH contents as observed in CCl4-intoxicated rats

The large-scale usage and demand for the wild *A. cearensis,* as a medicinal plant by communities in the Northeastern of Brazil, governmental programs of phytotherapy as well as the pharmaceutical industry, are contributing to decrease availability on these species, presently considered as endangered ones. In this sense, our laboratory has conducted comparative studies on the pharmacological profile of the ethanolic extract (EtOHE) or vanillic acid (**12**) from the wild and cultivated *A. cear*ensis, by evaluating their antinociceptive and antiedematogenic activities in several experimental models, such as the formalin test, carrageenan or dextran-induced edema and carragenan-induced neutrophil

The acute treatment with both the EtOHE prepared from all parts of the cultivated *A. cearensis* (4, 7 or 9 months) or the wild *A. cearensis,* present antinociceptive and antiinflammatory activities (Fig. 7). In addition, vanillic acid (**12**), which together with coumarin (**1**) are the major compounds present in cultivated *A. cearensis*, also showed an antinociceptive activity by inhibiting both phases of the formalin test in mice, and this effect was partially blocked by naloxone. Thus, the data suggest that antinociceptive effect of vanillic acid (**12**) occur by a mechanism at least in part dependent on the opioid system (Leal

Coumarin (**1**) has been found in several Brazilian medicinal plants including *J. pectoralis*, *M. glomerata* and *A. cearensis*. It has been reported that the antinociceptive and the antiinflammatory activities of these species seems to be related at least in part to the presence of coumarin (**1**) (Leal et al., 1997; 2003; Leal et al., 1997; Lino et al., 1997; Leal et al., 2000; Freitas et al., 2008). The biological effects of coumarin (**1**) include antibacterial, antiviral, antiedematogenic, antioxidant, lipoxygenase inhibition, lipid peroxidation inhibition, and scavenging of superoxide hydroxyl radicals (Hoult & Paya, 1996; Chang et al., 1996; Casley-

Recently (Leal et al., 2010), it was also determined that the anti-inflammatory effect of vanillic acid (**12**) is isolated from the cultivated *A. cearensis.* This compound orally

ATP-sensitive K+ channels.

et al., 2010).

enzymes, as myeloperoxidase, may also play a role.

after the amburoside A (**9**) treatment (Leal et al., 2008).

migration into the rat peritoneal cavity (Leal et al., 2010).

Smith et al., 1993; Rajarajeswari & Pari, 2011).

administered to rats, was shown to significantly inhibit the carrageenan, but not the dextran-induced edema. It also reduced the accumulation of PMN into the peritoneal cavity of rats and this effect was comparable to that observed with dexamethasone, used as a standard drug (Fig. 7).

Vanillic acid (**12**) is a benzoic acid derivative that is used as a flavoring agent. It is an intermediate in the production of vanillin from ferulic acid (Prince et al., 2011; Kim et al., 2011). Previous studies have shown antifilarial, antibacterial, antioxidant, hepatoprotective and anti-inflammatory (Kim et al., 2011; Prince et al., 2011; Itoh et al., 2009) effects of the vanillic acid. This compound exerts its anti-inflammatory effect by suppressing the production of prostaglandin E2, nitric oxide and cytokines. Furthermore, it also suppressed the activation of nuclear-factor-kappa B and caspase (Kim et al., 2011; Itoh et al., 2009). These findings confirm the anti-inflammatory activity of vanillic acid (**12**) as demonstrated by our laboratory.

Fig. 7. Antinociceptive and anti-inflammatory effects of ethanolic extracts (EtOHE) and vanillic acid (VA) from *Amburana cearensis* in rodents.1

A growing body of evidence suggests that the extract and chemical constituents from the wild *A. cearensis* have pharmacological properties which justify at least in part its traditional use in the treatment of asthma. Among others, the anti-inflammatory activity is possible due

<sup>1</sup> I : effects of EtOHE (cultivated, (4, 7 and 9 months(m) or wild plants: 200 mg/kg, p.o.), VA (50 mg/kg, p.o.) or morphine (MP, 5 mg/kg, s.c.) on the formalin-induced nociception in mice (6-18 animals/group). II, III and IV: anti-inflammatory effects of EtOHE and VA on the carrageenan (Cg) induced mice paw edema and Cg-inuced rat peritonitis.

Phytochemistry, Pharmacology

180-185.

666.

1158–1163

Sons, New York, 515 p.

*Phytochemistry* Vol. 50: 71-74.

*Society* Vol. 21: 1746-1753.

*Biological Research.* Vol. 33: 179-189.

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 371

Bastos, C.R.V. (1983). *Contribuição ao conhecimento químico de Torresea cearensis* (Fr. All.)

Bravo, J.A., Sauvain, M., Gimenez, A., Muñoz, V., Callapa, J., Le Men-Olivier, L., Massiot,

Calixto, J.B. (2000). Efficacy, safety, quality control, marketing and regulatory guidelines for

Canter, P.H., Thomas, H. & Ernst, E. (2005). Bringing medicinal plants into cultivation:

Canuto, K.M., Lima, M.A.S. & Silveira, E.R., (2010). Amburosides C-H and 6-*O*-

Canuto, K.M., Silveira, E.R. & Bezerra, A.M.E., (2010). Estudo Fitoquímico de espécimens

Canuto, K.M. & Silveira, E.R., (2006). Chemical constituents of trunk bark of *Amburana* 

Carvalho, P.E.R. (1994). *Espécies florestais brasileiras: recomendações silviculturais, potencialidades* 

Casley-Smith, J.R., Morgan, R.G. & Piller, N.B. (1993). Treatment of lymphedema of the arms

Chang, W.S., Lin, C.C., Chuang, S.C. & Chiang, H.C. (1996). Superoxide anion scavenging effect of coumarins. *The American Journal of Chinese Medicine* Vol. 24: 11-17 Costa-Lotufo, L.V., Jimenez, P. C., Wilke, D.V., Leal, L.K.A.M., Cunha, G.M.A., Silveira, E.R.,

Cunha, M.C.L. Ferreira, R.A. (2003). Aspectos morfológicos da semente e do

Dewick, P.M. (2002). *Medicinal Natural Products: a biosynthetic approach*, 2nd ed., John Wiley &

Freitas, T.P., Silveira, P.C., Rocha, L.G., Rezin, G.T., Rocha, J., Citadini-Zanette, V., Romao, P.

Gobbo-Neto, L., Lopes, N.P. (2007). Medicinal plants: factors of influence on the content of

Gruenwald, J. (2008). The global herbs & botanicals market; Herbs and botanicals are

http://www.entrepreneur.com/tradejournals/article/181916708\_1.html

A.C. Smith. *Zeitschrift fur Naturforschung C* Vol. 58c: p. 675-680.

Papilionoideae. Revista Brasileira de Sementes Vol. 25: 89-96.

exposure. *Journal of Medicinal Food* Vol. 11: 761-766.

secondary metabolites. *Quimica Nova,* Vol. 30: 374-381, 2007

*cearensis* A.C. Smith., *Quimica Nova* Vol. 29: 1241-1243.

*e uso da madeira,* EMBRAPA, Brasília, pp.

G., & Lavaud, C. (1999). Bioactive phenolic glycosides from *Amburana cearensis*,

herbal medicines (phytotherapeutic agents), *Brazilian Journal of Medical and* 

opportunities and challenges for biotechnology, *TRENDS in Biotechnology* Vol. 23:

protocatechuoyl coumarin from *Amburana cearensis, Journal of the Brazilian Chemical* 

cultivados de cumaru (*Amburana cearensis* A. C. Smith), *Quimica Nova* Vol. 33: 662-

and legs with 5,6-benzo-[]pyrone. The *New England Journal of Medicine* Vol. 329:

Canuto, K.M., Viana, G.S.B., Moraes, M.E.A., Moraes, M.O. & Pessoa, C., (2003). Antiproliferative effects of several compounds isolated from *Amburana cearensis*

desenvolvimento da planta jovem de *Amburana cearensis* A.C. Smith- Leguminosae

T., Dal-Pizzol, F., Pinho, R.A., Andrade, V.M. & Streck, E.L. (2008). Effects of *Mikania glomerata* Spreng. and *Mikania laevigata* Schultz Bip. ex Baker (Asteraceae) extracts on pulmonary inflammation and oxidative stress caused by acute coal dust

currently showing the most potential in functional foods and cosmetics. *Entrepreneur* URL:

(Master Thesis), Universidade Federal do Ceará, Fortaleza, pp.

to their capacity to modular several responses, especially those related to oxidative stress, the production of inflammatory mediators, and the accumulation and/or activation of inflammatory cells as neutrophils.

The preliminary pharmacological study of the cultivated *A. cearensis* (Leal et al., 2010) showed that both cultivated and wild plants have antinociceptive and anti-inflammatory activities in rodents. Coumarin (**1**) and vanillic acid (**12**) are possibly responsible for the pharmacological activities of the cultivated *A. cearensis* extracts, however the pharmacological importance of other chemical constituents present in the cultivated species cannot be ruled out.

#### **6. Conclusions**

The interdisciplinary study of the *A. cearensis* revealed that its ethanol extracts from cultivated and wild sources have similar phytochemical profiles, as consequence, both extracts possess similar pharmacological activities. Hence, these findings support the idea of the utilization of cultivated plants for the manufacturing of herbal drugs preparations by pharmaceutical laboratories, favoring the uniform and constant supply of high quality raw material, as well as the conservation of the wild specimens in the original biome. Indeed, this research indicates promising prospects for the rational use of the *A. cearensis,* however, it is still needed to be advanced in some issues concerning with agronomical, phytochemical and pharmacological knowledge of these species. The influence of some agronomic parameters (plant spacing, shading or sunlight exposure, water supply, etc) on the chemical composition will be performed. Chemical markers or a metabolomic approach will be developed in order to evaluate the influence of the aforementioned agronomic parameters on chemical composition. Pharmacological testing with other types of inflammation experimental models and clinical trials will be carried out aiming to elucidate the mechanisms of action of the *A. cearensis* active principles as well as to evaluate the efficacy in human beings. Additionally, an economic analysis should be performed in order to evaluate the economic feasibility in the production of *A. cearensis* herbal drug preparations from cultivated source*.* 

#### **7. Acknowledgements**

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by Banco do Nordeste do Brasil (BNB).

#### **8. References**


to their capacity to modular several responses, especially those related to oxidative stress, the production of inflammatory mediators, and the accumulation and/or activation of

The preliminary pharmacological study of the cultivated *A. cearensis* (Leal et al., 2010) showed that both cultivated and wild plants have antinociceptive and anti-inflammatory activities in rodents. Coumarin (**1**) and vanillic acid (**12**) are possibly responsible for the pharmacological activities of the cultivated *A. cearensis* extracts, however the pharmacological importance of other chemical constituents present in the cultivated species

The interdisciplinary study of the *A. cearensis* revealed that its ethanol extracts from cultivated and wild sources have similar phytochemical profiles, as consequence, both extracts possess similar pharmacological activities. Hence, these findings support the idea of the utilization of cultivated plants for the manufacturing of herbal drugs preparations by pharmaceutical laboratories, favoring the uniform and constant supply of high quality raw material, as well as the conservation of the wild specimens in the original biome. Indeed, this research indicates promising prospects for the rational use of the *A. cearensis,* however, it is still needed to be advanced in some issues concerning with agronomical, phytochemical and pharmacological knowledge of these species. The influence of some agronomic parameters (plant spacing, shading or sunlight exposure, water supply, etc) on the chemical composition will be performed. Chemical markers or a metabolomic approach will be developed in order to evaluate the influence of the aforementioned agronomic parameters on chemical composition. Pharmacological testing with other types of inflammation experimental models and clinical trials will be carried out aiming to elucidate the mechanisms of action of the *A. cearensis* active principles as well as to evaluate the efficacy in human beings. Additionally, an economic analysis should be performed in order to evaluate the economic feasibility in the production of *A. cearensis* herbal drug preparations from

This work was supported by Conselho Nacional de Desenvolvimento Científico e

Aquino, F.W.B., Rodrigues, S., Nascimento, R.F. & Casimiro, A.R.S. (2005). Phenolic

Bandeira, P.N., Farias, S.S., Lemos, T.L.G., Braz-Filho, R., Santos, H.S., Albuquerque,

compounds in imburana (*Amburana cearensis*) powder extracts, *European Food* 

M.R.J.R. & Costa, S.M.O. (2011). New isoflavone derivative and other flavonoids from the resin of *Amburana cearensis*, *Journal of the Brazilian Chemical Society* Vol. 22:

Tecnológico (CNPq) and by Banco do Nordeste do Brasil (BNB).

*Research and Technology* Vol. 221: 739-745.

inflammatory cells as neutrophils.

cannot be ruled out.

**6. Conclusions**

cultivated source*.* 

**8. References** 

**7. Acknowledgements**

372-375.


http://www.entrepreneur.com/tradejournals/article/181916708\_1.html

Phytochemistry, Pharmacology

p.

58–66.

21.

*Toxicology* Vol. 25: 1-7.

*Fitoterapia* Vol. 82: 462–471.

*Fitoterapia* Vol. 63: 470.

and Agronomy of Medicinal Plants: *Amburana cearensis*, an Interdisciplinary Study 373

Leal, L.K.A.M; Oliveira, F.G.; Fontenele, J.B.; Ferreira, M.A.D. & Viana, G.S.B. (2003b).

Leal, L.K.A.M., Ferreira, A.A.G. & Viana G.S.B. (2000). Antinociceptive, anti-inflammatory

Leal, L.K.A.M, Matos, M.E.; Matos, F.J.A., Ribeiro, R.A., Ferreira, F.V. & Viana, G.S.B. (1997).

Lino, C.S., Taveira, M.L., Viana, G.S.B. & Matos, F.J.A. (1997). Analgesic and

Menković, N., Šavikin-Fodulović, K. & Savin, K. (2000). Chemical composition and seasonal

Prince, P.S.M., Dhanasekar, K. & Rajakumar, S. (2011). Preventive effects of vanillic acid on

Rajarajeswari, N & Pari, L. (2011). Antioxidant Role of Coumarin on Streptozotocin–

Rao, M.R., Palada, M.C. & Becker, B.N. (2004). Medicinal and aromatic plants in agroforestry

Sahoo, N., Manchikanti, P. & Dey, S. (2010). Herbal drugs: Standards and regulation

Schippmann, U., Leaman, D.J. & Cunnningham, A.B. (2002). *Impact of cultivation and* 

Silva, M.G.V., Craveiro, A.A.; Matos, F.J.A., Machado, M.I.L. & Alencar, J.W. (1999).

Southwell, I.A. & Bourke, C.A. (2001). Seasonal variation in hypericin content of *Hypericum* 

Tommasi, N., Feo, V., Pizza, C. & Zhou, Z.I. (1992). Constituents of *Bidens parviflora*.

Wagner, H. (2011). Synergy research: Approaching a new generation of

Williams, R.D. & Ellis, B.E. (1989). Age and tissue distribution of alkaloids in *Papaver* 

*perforatum* L. (St. John's Wort), *Phytochemistry* Vol. 56: 437-.441

coumarin and umbelliferone. *Phytotherapy Research* Vol. 11: 211-215. McChesney, J.D., Venkataraman, S.K., & Henri, J.T. (2007). Plant natural products: Back to

the future or into extinction? *Phytochemistry* Vol. 68: 2015–2022

coumarin from *Torresea cearensis* Fr. All. *Phytomedicine* Vol. 4: p. 221-227. Lima, D.A. (1989) *Plantas das caatingas*, Academia Brasileira de Ciências Rio de Janeiro, 243

comparative study. *Journal of Ethnopharmacology* Vol. 70: 151–9.

Smith. *Phytotherapy Research* Vol. 17: 335-340.

*Pharmaceutical Biology* Vol. 41: p. 308-314.

flowers, *Planta Medica* Vol. 66: 178-180

systems, *Agroforestry Systems* Vol. 61: 107–122.

*gratissimum* leaves, *Fitoterapia* Vol. 70: 32-34.

phytopharmaceuticals. *Fitoterapia* Vol. 82: 34–37.

*somniferum, Phytochemistry* Vol. 28: 2085-2088.

hydroalcoholic extract and chemical constituents from *Amburana cearensis* A. C.

Toxicological study of hydroalcoholic extract from *Amburana cearensis* in rats.

and bronchodilador activities of Brazilian medicinal plants containing coumarin: a

Antinociceptive and antiedematogenic effects of the hydroalcoholic extract and

antiinflammatory activities of *Justicia pectoralis* Jacq and its main constituents:

variations in the amount of secondary compounds in *Gentiana lutea* leaves and

lipids, Bax, Bcl-2 and myocardial infarct size on isoproterenol-induced myocardial infarcted rats: a biochemical and in vitro study, *Cardiovascular Toxicology* Vol. 11:

Nicotinamide-Induced Type 2 Diabetic Rats, *Journal of Biochemical and Molecular* 

*gathering of medicinal plants on biodiversity: global trends and issues.* FAO, Rome, pp. 1–

Chemical variation during daytime of constituents of the essential oil of *Ocimum* 


Guedes, R.S., Alves, E.U., Gonçalves, E.P., Viana, J.S., Moura, M.F. & Costa, E.G. (2010).

Guedes, R.S., Alves, E.U., Gonçalves, E.P., Viana, J.S., França, P.R.C. & Santos, S.S. (2010b)

Hoult, J.R.S. and Paya, M. (1996). Pharmacological and biochemical actions of simple

Itoh, A., Isoda, K., Kondoh, M., Kawase, M., Kobayashi, M., Tamesada, M. & Yagi, K., (2009).

induced liver injury. *Biological & Pharmaceutical Bulletin* Vol. 32: 1215–1219. Khalil, A.T., Chang, F.R., Lee, Y. H., Chen, C.Y., Liaw, C.C., Ramesh, P., Yuan, S.S.F., & Wu,

Kim, M.C., Kim, S.J., Kim, D.S., Jeon, Y.D., Park, S.J., Lee, H.S., Um, J.Y., & Hong, S.H.

Koeppe, D.E., Rohrbaugh, L.M., Rice, E.L. & Wender, S.H. (1970). Effect of age and chilling

Leal, L.K.A.M., Pierdoná, T.M., Góes, J.G.S., Fonsêca, K.S., Canuto, K.M., Silveira, E.R.,

cultivated *Amburana cearensis* A.C. Smith. *Phytomedicine* Vol. 18: 230-233. Leal, L.K.A.M., Canuto, K.M., Costa, K.C.S., Nobre-Júnior, H.V., Vasconcelos, S.M., Silveira,

neutrophils. *Basic & Clinical Pharmacology & Toxicology* Vol. 104: 198. Leal, L.K.A.M., Fonseca, F.N., Pereira, F.A., Canuto, K.M., Felipe, C.F.B., Fontenele, J. B.,

hepatotoxicity in rats. *Planta Medica* Vol. 74: 497.

induced neurotoxicity. *Neuroscience Letters* Vol. 388, 86-90.

*Semina: Ciências Agrárias* Vol. 31: 331-342.

850.

27: 713-722.

*Research* Vol. 31: 15-20.

*Sciences* Vol. 79: 98-104.

*and Immunotoxicology* Vol. 33:1-8

*Physiologia Plantarum* Vol. 23: 258-266.

Emergence and vigor of *Amburana cearensis* (Allemão) A.C. Smith seedling in function of the sowing position and depth, *Semina: Ciências Agrárias* Vol. 31: 843-

.Physiological quality of *Amburana cearensis* (Allemão) A.C. Smith seeds stored,

coumarins: natural products with therapeutic potential. *General Pharmacology* Vol.

Hepatoprotective effect of syringic acid and vanillic acid on concanavalin a-

Y.C. (2003). Chemical constituents from the *Hydrangea chinensis*. *Archives Pharmacal* 

(2011). Vanillic acid inhibits inflammatory mediators by suppressing NF-κB in lipopolysaccharide-stimulated mouse peritoneal macrophages *Immunopharmacology* 

temperature on the concentration of scopolin and caffeoylquinic acids in tobacco,

Bezerra, A.M.E., Viana, G. S. B. (2011). A comparative chemical and pharmacological study of standardized extracts and vanillic acid from wild and

E.R., Ferreira, M.V.P., Fontenele, J.B., Andrade, G. M., Viana, G. S. B. (2009). Effects of amburoside A and isokaempferide, polyphenols from *Amburana cearensis*, on rodent inflammatory processes and myeloperoxidase activity in human

Pitombeira, M.V., Silveira, E.R., Viana, G.S.B., (2008). Protective effects of amburoside A, a phenol glucoside from *Amburana cearensis*, against CCl4-induced

Viana, G.S.B., (2006). Mechanisms underlying the relaxation induced by isokaempferide from *Amburana cearensis* in the guinea-pig isolated trachea. *Life* 

Silveira, E.R., Canuto, K.M., Viana, G.S.B., (2005). Amburoside A, a glucoside from *Amburana cearensis*, protects mesencephalic cells against 6-hydroxydopamine-

G.S.B., (2003a). Anti-inflammatory and smooth muscle relaxant activities of the

Leal, L.K.A.M., Costa, M.F., Pitombeira, M., Barroso, V.M., Silveira, E.R., Canuto, K.M.,

Leal, L.K.A.M., Nobre, H.V., Cunha, G.M.A., Moraes, M.O., Pessoa, C., Oliveira, R.A.,

Leal, L.K.A.M., Nechio, M., Silveira, E.R., Canuto, K.M., Fontenele, J.B., Ribeiro, R.A., Viana,

hydroalcoholic extract and chemical constituents from *Amburana cearensis* A. C. Smith. *Phytotherapy Research* Vol. 17: 335-340.


**18** 

*Beirut, Lebanon* 

**General Introduction** 

 **on Family Asteracea** 

*Beirut Arab University,* 

*Dept. of Pharmacognosy, Faculty of Pharmacy,* 

Maha Aboul Ela, Abdalla El-Lakany and Mohamad Ali Hijazi

Asteraceae is the largest family of the plant kingdom, very abundant and also a diverse one. The Asteraceae plants are the most widely distributed of all the families (Porter, C.L. (1969); Evans W. (1989); Hutchinson, J.(1973); Core, E. L. (1955) of the angiosperms. It includes about 1400 genera and over 25000 species (Harborne , J. B., Turner, B.L. (1984); Aboul Ela,

Asteraceae has characteristic taxonomical characters (Muschler, R. (1912). Members of the family are generally herbs of annul or perennial habits and some tropical forms occur as shrubs. Flowers are grouped in heads known as capitula, surrounded by involucres. It is of two kinds of florets; tubular or disc florets with tubular corolla and mostly hermaphrodite,

Genus Matricaria comprises plants with various secondary metabolites of different chemical nature recorded mainly in *Matricaria chamomilla.*German chamomile flowers contain 0.24- to 2% volatile oil which is blue in color. Chamomile also contains up to 8% flavone glycosides and flavonol; up to 10 percent mucilage polysaccharides; up to 0.3 percent choline; and approximately 0.1 percent coumarines. The tannin level in chamomile is less than one

Following is a review of the chemical compounds that have been isolated previously from

**Name Source Structure References**  a) Azulene derivatives

M. A.,(1991), forming approximately 10% of the flowering plants.

and ligulate or ray floret, with starp like corolla and mostly female.

**2. Chemistry of genus Matricaria** 

percent. (Alternative Medicine Review (2008))

genus Matricaria (Tables 1, 2, 3, and 4).

**2.1 Volatile oil** 

**1. Introduction** 


## **General Introduction on Family Asteracea**

Maha Aboul Ela, Abdalla El-Lakany and Mohamad Ali Hijazi

*Dept. of Pharmacognosy, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon* 

#### **1. Introduction**

374 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Wu, S.; Ma, Y., Luo, X., Hao, X. & Wu, D. (2002). Studies on chemical constituents in root

Zobayed, S.M.A., Afreen, F., Kozai, T. (2007). Phytochemical and physiological changes in

the leaves of St. John's wort plants under a water stress condition. *Environmental* 

back of *Paeonia suffruticosa*. *Zhongcaoyao*, Vol. 33: 679-680.

*and Experimental Botany* Vol. 59: 109–116

Asteraceae is the largest family of the plant kingdom, very abundant and also a diverse one. The Asteraceae plants are the most widely distributed of all the families (Porter, C.L. (1969); Evans W. (1989); Hutchinson, J.(1973); Core, E. L. (1955) of the angiosperms. It includes about 1400 genera and over 25000 species (Harborne , J. B., Turner, B.L. (1984); Aboul Ela, M. A.,(1991), forming approximately 10% of the flowering plants.

Asteraceae has characteristic taxonomical characters (Muschler, R. (1912). Members of the family are generally herbs of annul or perennial habits and some tropical forms occur as shrubs. Flowers are grouped in heads known as capitula, surrounded by involucres. It is of two kinds of florets; tubular or disc florets with tubular corolla and mostly hermaphrodite, and ligulate or ray floret, with starp like corolla and mostly female.

#### **2. Chemistry of genus Matricaria**

Genus Matricaria comprises plants with various secondary metabolites of different chemical nature recorded mainly in *Matricaria chamomilla.*German chamomile flowers contain 0.24- to 2% volatile oil which is blue in color. Chamomile also contains up to 8% flavone glycosides and flavonol; up to 10 percent mucilage polysaccharides; up to 0.3 percent choline; and approximately 0.1 percent coumarines. The tannin level in chamomile is less than one percent. (Alternative Medicine Review (2008))

Following is a review of the chemical compounds that have been isolated previously from genus Matricaria (Tables 1, 2, 3, and 4).

#### **2.1 Volatile oil**


General Introduction on Family Asteracea 377

OR1

4

*M. aurea* H Ac H

OR

1

4

5

6

2

3

*M. aurea* Ac Ahmed A. Ahmed, et

HO OH

7

10

11

*M. aurea* 

8

9

5

6

OR3

R1 R2 R3 Ahmed A. Ahmed,

H H H (1999)

*M. aurea* Ac H H Ahmed A. Ahmed, et

Maha A. Abou Elela

al.(1993)

Ahmed A. Ahmed, Maha A. Abou Elela (1999)

R Ahmed A. Ahmed, et

al.(1993)

H al.(1993)

<sup>1</sup> OR2

2

3

HO OH

7

10

11

*M. aurea* 

8

9

(1R\*,2R\*,3R\*,6R\*,7R\*)1,2 ,3,6,7-pentahydroxybisabolol-10(11)-ene

(1R\*,2R\*,3R\*,6R\*,7R\*)1,2 ,3,6,7-tetrahydroxy-1 acetoxy-bisabolol-10(11)-ene

(1R\*,2R\*,3R\*,6R\*,7R\*)1,2 ,3,6,7-tetrahydroxy-2 acetoxy-bisabolol-10(11)-ene

(1R\*,6R\*,7R\*)1,6,7 trihydroxy-bisabolol-2,10- diene

(1R\*,6R\*,7R\*)1,6,7 trihydroxy-1 acetoxybisabolol-2,10 diene


HOOC

O

HO

H

H3C

O

H3C

O

b) Sesquiterpenes i) Oxygenated sesquiterpenes

O

O

OCOCH3

Alternative Medicine Review 2008, Ness, A.,Metzger, J. W., Shmidt, P. C. (1996)

Stahl, E. (1954)

Motl, O. ,Repcak, M. (1979), Motl, O. ,Repcak, M. ,Ubik, K. (1983)

Alternative Medicine Review (2008), Sorm, F.,Nowak, J., Herout, V.(1953), Cekan, Z., Herout, V., Sorm, F.,(1957)

OCOCH3 Alternative Medicine

Review (2008)

Chamazulene *M. chamomilla* 

Carboxylic acid *M. chamomilla* 

Chamavioline *M. chamomilla* 

Matricarin *M. chamomilla* 

Ligulate and tubular floret only of *M. chamomilla*

Chamazulene

Matricin (proazulene)


General Introduction on Family Asteracea 379

Alternative Medicine Review (2008), Motl, O., et al(1977)

Reichling, J., et al (1983)

Anne ORAV, Tiiu KAILAS, and Kaire IVASK (2001)

Alternative Medicine Review (2008), Lemberovics, E. (1979)

O

HO

ii) Unsaturated sesquiterpenes

*Trans* --farnesene *M. chamomilla* Lemberovics, E. (1979)

Spathulenol *M. chamomilla* 

Caryophyllene epoxide *M. chamomilla*

β-bisabolene *M. chamomilla* 

*Trans*-- farnesene *M. chamomilla* 


OH

OH

OH

OH

O

O

Alternative Medicine Review (2008), Sorm, F.,Zaoral M.,Herout, V.(1951)

Alternative Medicine Review 2008, Sampath , V., et al (1969)

Alternative Medicine Review (2008), Sampath ,V.,Sabata, et al ,(1969)

Schilcher, H., et al (1976)

Hölzl, J., Demuth, G.(1973)

(-)--bisabolol *M. chamomilla* 

(-)--bisabolol oxide A *M. chamomilla* <sup>O</sup>

(-)--bisabolol oxide B *M. chamomilla* <sup>O</sup>

(-)--bisabolol oxide C *M. chamomilla* <sup>O</sup>

*M. chamomilla* growing in turkey



General Introduction on Family Asteracea 381

Calamemene *M. chamomilla* Motl, O., Repcak,

c) Monoterpenes

α-Terpinene *M. chamomilla* A.Pizard, et al.(2006)

In the root oil of *M. chamomilla*

α-pinene *M. chamomilla* 

β-caryophyllene

M.(1979)

Reichling, J., et al (1983)

Anne ORAV, Tiiu KAILAS, and Kaire IVASK (2001), A.Pizard, et al.(2006)


β-selinene *M. chamomilla* A.Pizard, et al.(2006)

Germacrene A *M. chamomilla* A.Pizard, et al.(2006)

Bicyclo germacrene *M. chamomilla* A.Pizard, et al.(2006)


Anne ORAV, Tiiu KAILAS, and Kaire IVASK (2001) A.Pizard, et al.(2006)

Alternative Medicine Review (2008), Anne ORAV, Tiiu KAILAS, and Kaire IVASK (2001)

M.(1979)

Germacrene D *M. chamomilla* 

Cadinene *M. chamomilla* 

General Introduction on Family Asteracea 383

O

R3'

6'

3'

Alternative Medicine Review (2008), Bohlmann, F., et al ,(1961), Bohlmann, F., Zdero, C. (1982)

Ahmed A. Ahmed, Maha A. Abou Elela (1999)

R4'

Powe, F., Browning, H. Jr. (1914), Sorm, P.,et al.,(1952), Kunde, R., Isaac, O.(1979), Carle, R. and Isaac, O. (1985)

Carle, R. and Isaac, O. (1985)

Reichling, J., et al., (1979)

4'

5'

H

HO OH

*M. chamomilla* <sup>O</sup>

*M. aurea* <sup>O</sup>

a) Flavone aglycon and glycosides isolated from species *Chamomilla*

O

2

1'

2'

3 4

i) Flavone aglycon

Luteolin flowers OH OH OH OH Kunde, R., Isaac, O.(1979),

Chrysoseriol flowers OH OH OCH3 OH Carle, R. and Isaac, O. (1985) ,

OH OH OH

1

O

**Name Source R5 R6 R7 R3' R4' Ref.** 

Table 1. Volatile components isolated from Matricaria species

R5

8

5

Trans (E)-enyne dicycloether *trans*-2-[hexadiyne)- (2,4)-ylidene]-1,6 dioxaspiro-[4,4]-nonene

(3S\*,4S\*,5R\*)-(E)-3,4 dihydroxy-2-(hexa-2,4 diynyliden)-1,6 dioxaspiro-(4,5) decane

R7

R6

Apigenin

6

Ligulate florets flowers

7

**2.2 Flavonoids** 

Table 1. Volatile components isolated from Matricaria species

#### **2.2 Flavonoids**

382 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Myrcene *M. chamomilla* Stransky, K., et al.,

CH2OH

O

Spiroethers

*M. chamomilla* <sup>O</sup>

Sabinene *M. chamomilla* 

Gerianol *M. chamomilla* 

Cis (Z)-enyne dicycloether *cis*-2-[hexadiyne)- (2,4) ylidene]-1,6-dioxaspiro- [4,4]-nonene)

(1981)

Anne ORAV, Tiiu KAILAS, and Kaire IVASK (2001) , A. Pizard, et al.(2006)

Stransky, K., et al., (1981)

<sup>H</sup> Alternative Medicine

Review (2008), Bohlmann, F., Zdero, C. (1982), Bohlmann, F., et al ,(1961)

General Introduction on Family Asteracea 385

i) Flavonol aglycones

Kunde, R., Isaac, O.(1979), Carle, R. and Isaac, O. (1985) , Ahmed A. Ahmed, Maha A. Abou Elela (1999)

Kunde, R., Isaac, O.(1979), Carle, R. and Isaac, O. (1985), Greger, H. (1975)

Carle, R. and Isaac, O. (1985), Exner, J., et al., (1981), Hänsel, R., Rimpler, H., Walther, K. (1966)

Carle, R. and Isaac, O. (1985) , Hänsel, R., Rimpler, H., Walther, K. (1966)

Kunde, R., Isaac, O.(1979), Carle, R. and Isaac, O. (1985), Exner, J., et al., (1981), Hänsel, R., Rimpler, H., Walther, K. (1966)

Carle, R. and Isaac, O. (1985), Exner, J., et al., (1981), Hänsel, R., Rimpler, H., Walther, K. (1966)

Kunde, R., Isaac, O.(1979), Lang, W., Schwandt, K. (1957) , Horhammer, L.,

OH OH OCH3 OH OH OH

OH OH OH OH OH

OH OH OCH3 OCH3 OH OH

ii) Flavonol glycosides

flower OH OH OCH3 OCH3 OH

floret OH OH OGlu OH OH

flower OCH3 OH OCH3 OCH3 OH OH

in Flowers OCH3 OH OCH3 OCH3 OCH3 OH

**Name Source R3 R5 R6 R7 R3' R4' Ref.** 

Lutuletin

Quercetin

Chrysosplenit

Eupatoletin

Quercetin-7 glucoside (Quercimeritri n)

Eupalitin Chamomile

Chrysosplenol Chamomile

Tubular floret flowes

Leaves Tubular floret flowers

Chamomile flower Ligulate florets

Tubular


ii) Flavone glycosides

Kunde, R., Isaac, O.(1979), Elkiey, M. A.,et al., (1963), Greger, H. (1975)

Kunde, R., Isaac, O.(1979), Lang, W., Schwandt, K. (1957), Hörhammer, L., Wagner, H., Salfner, B. (1963)

Kunde, R., Isaac, O.(1979), Redaelli, C., Formentini, L., Santaniello, E. (1979)

Kunde, R., Isaac, O.(1979), Wagner, H., Kirmayer, W. (1957)

R4'

4'

5'

OH OGlu OH OH

7-glucoside Leaves OH OGlu OCH3 OH Greger, H. (1975)

ac.

OH

R3'

6'

3'

Apio. OH

2

3

1'

R3

2'

b) Flavonol aglycones and glycosides isolated from *Chamomilla* species

O

1

O

4

R5

8

5

floret OH OGlu OH

Luteolin-7 glucoside

Luteolin-4'-

Chrysoseriol-

Apigenin-7 glucoside (Apigetrin)

Apigenin-7- (6''-Oacetyl) glucoside

Apigenin-7- (6''-Oapiosyl) glucoside (apiin)

Egyptian chamomile floret (leaves) Ligulate florets

Ligulate

Ligulate

Ligulate

R7

R6

6

7

glucoside OH OH OH OGlu

florets OH OGlu-

florets OH OGlu-


General Introduction on Family Asteracea 387

COOH OCH3 OH OCH3

COOH H OH OCH3

Anisic acid *M. chamomilla* COOH H OCH3 H Reichling, J., et al.,

Caffeic acid *M. chamomilla* CH2 CH2COOH OH OH H Reichling, J., et al.,

Table 3. Coumarines and other polyphenolic compounds isolated from genus Matricaria

rhamnnose (Janecke, H., Weiser, W. (1964))( Janecke, H., Weiser, W.(1965)).

protein, similar to pectin), and arabino-3, 6-galactane glycoproteins.

lysine, DL- threonine, DL-serine, and L-glutaminic acid. - Tannin level is less than 1% (Alternative Medicine Review (2008).

chamomile constituents (Schilcher, H.,(1987)).

Chamomile contains up to 10% mucilage polysaccharides (Alternative Medicine Review (2008)).The main chain of the polysaccharide consists of -1-> 4 connected D-galacturone acids (Carle, R. and Isaac, O.,(1985)). In addition to xylose, arabinose, galactose, glucose,

Recently, three polysaccharides were isolated and showed remarkable antiphlogistic activity against mouse ear edema induced by crotone oil (Füller, E., (1992)) as fructane (74.3% fructose and 3.4% glucose, similar to inulin), rhamnogalacturonane (28% uronic acid, 3.2%



**3. Some reported pharmacological activity of the chemical constituents of** 

Several pharmacological actions have been assigned for German chamomile, based primarily on *in vitro* and animal studies. Such actions include antibacterial, antifungal, antiinflammatory, antispasmodic, anti-ulcer, antiviral, carminative, and sedative effects (Alternative Medicine Review 2008). It is important to mention that therapeutic effectiveness is mainly due to the combined pharmacological and biochemical effects of several

Reichling, J., et al., (1979)

Reichling, J., et al., (1979)

(1979)

(1979)

**Name Source R1 R2 R3 R4 Ref.** 

Synergic acid

Vanillic acid

extract.

**Matricaria** 

Ligulate and tubular floret of *M. chamomilla*

Ligulate and tubular floret of *M. chamomilla*

**2.4 Miscellaneous substances** 


Table 2. Flavone and flavonol aglycon and glycosides isolated from species *Chamomilla*

#### **2.3 Coumarines and other polyphenolic compounds**


Table 2. Flavone and flavonol aglycon and glycosides isolated from species *Chamomilla*

a) Coumarines

5 4

R1O O O

Isoscopoletin *M. chamomilla* CH3 H Kotov, A. G., et al., (1991) Esculetin *M. chamomilla* H OH Kotov, A. G., et al., (1991) Scopoletin *M. chamomilla* H OCH3 Kotov, A. G., et al., (1991) b) Phenyl carboxylic acid

R3

R1

8

**Name Source R1 R2 Ref.** 

1

of *M. chamomilla* CH3 H Schilcher, H. (1985)

of *M. chamomilla* H H Schilcher, H. (1985)

R4

2

3

Rham OH OH OH OH Elkiey, M. A.,et al.,

e flower OGal OH OH OH OH Elkiey, M. A.,et al.,

Quercetin-3 rutinoside

Quercetin-3 galactoside Chamomil e flower

Chamomil

OGlu-

**2.3 Coumarines and other polyphenolic compounds** 

R2

Herniarin Ligulate and Tubular floret

Umbelliferone Ligulate and Tubular floret

6

R2

7

Wagner, H., Salfner, B. (1963)

(1963)

(1963)


Table 3. Coumarines and other polyphenolic compounds isolated from genus Matricaria

#### **2.4 Miscellaneous substances**

Chamomile contains up to 10% mucilage polysaccharides (Alternative Medicine Review (2008)).The main chain of the polysaccharide consists of -1-> 4 connected D-galacturone acids (Carle, R. and Isaac, O.,(1985)). In addition to xylose, arabinose, galactose, glucose, rhamnnose (Janecke, H., Weiser, W. (1964))( Janecke, H., Weiser, W.(1965)).

Recently, three polysaccharides were isolated and showed remarkable antiphlogistic activity against mouse ear edema induced by crotone oil (Füller, E., (1992)) as fructane (74.3% fructose and 3.4% glucose, similar to inulin), rhamnogalacturonane (28% uronic acid, 3.2% protein, similar to pectin), and arabino-3, 6-galactane glycoproteins.


#### **3. Some reported pharmacological activity of the chemical constituents of Matricaria**

Several pharmacological actions have been assigned for German chamomile, based primarily on *in vitro* and animal studies. Such actions include antibacterial, antifungal, antiinflammatory, antispasmodic, anti-ulcer, antiviral, carminative, and sedative effects (Alternative Medicine Review 2008). It is important to mention that therapeutic effectiveness is mainly due to the combined pharmacological and biochemical effects of several chamomile constituents (Schilcher, H.,(1987)).

General Introduction on Family Asteracea 389

Both flavonoids and essential oil contribute to the musculotropic antispasmodic effect of chamomile. Apigenin, alpha-bisabolol, and the cis-spiroethers appear to provide the most



about 1 mg of papaverine as for musculotropic effect. (Della Loggia, R. 1985) - Other flavonoids contribute to the smooth muscle relaxation but to lesser degree. They can be classified in descending activity as follows: apigenin, quercetin, luteolin, kaempferol, luteolin-7-glucoside, and apigenin-7-glucoside. (Hörhammer, L.,et al.,

Preliminary *in vitro* studies on the antimicrobial activity of chamomile have yielded





inhibiting effect on adhesion of this microorganism of phospholipid — lecithin. - Turi M. *et al.* in 1997 showed that chamomile extract inhibited the growth of poliovirus and herpes virus while chamomile esters and lactones demonstrated activity against

*Mycobacterium tuberculosis* and *Mycobacterium avium*.

some fungicidal activity against *Candida albicans*

significant antispasmodic effects. (Alternative Medicine Review (2008).

**3.3 Antispasmodic effect** 

inhibition.

(1963)

promising results.

**3.4 Antimicrobial effect** 

*Escherichia coli*.

#### **3.1 Apoptotic effect against cancerous cell**


#### **3.2 Sedative and anxiolytic effect**


#### **3.3 Antispasmodic effect**

388 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health








flowers inhibited [3H]-flunitrazepam binding in the bovine cerebral cortex.

significant prolongation of sleeping time induced by barbiturates in mice.

**3.1 Apoptotic effect against cancerous cell** 

needed (Barton, H. 1959).

**3.2 Sedative and anxiolytic effect** 

hypnotic and anxiolytic activity in animals.

cells or in animals was absent. (Elena Darra, et al., (2008))

Cancer Lett. (2000)) and (Srivastava JK, Gupta S. (2007))

responsible for these effects, (Deendayal patel et al. (2007))

Both flavonoids and essential oil contribute to the musculotropic antispasmodic effect of chamomile. Apigenin, alpha-bisabolol, and the cis-spiroethers appear to provide the most significant antispasmodic effects. (Alternative Medicine Review (2008).


#### **3.4 Antimicrobial effect**

Preliminary *in vitro* studies on the antimicrobial activity of chamomile have yielded promising results.


General Introduction on Family Asteracea 391

Yoshinari et al. in 2008 showed that the spiroethers of German chamomile inhibited production of aflatoxin G1 AFG1 by *Aspergillus parasiticus* with inhibitory concentration 50% (IC50) values of 2.8 and 20.8 mM respectively. This is through inhibiton of cytochrome P450 monooxygenase CYPA and without inhibiting fungal growth. In addition, it also inhibited production of 3-acetyldeoxynivalenol 3-ADON by *Fusarium graminearum* by inhibiting TRI4. The inhibitory activity of the (E)-spiroether isomer was much stronger than that of the (Z) spiroether in both cases. Inhibition of TRI4 by the spiroethers showed that TRI4 may be a




**3.7 Other Pharmacological actions** 

**3.7.1 Inhibition of Aflatoxin G1 production** 

good target for inhibiting biosynthesis of trichothecene mycotoxins.

**3.7.2 Protective effect on diabetic complications** 

ATSUSHI KATO,et al ., (2008)

electron scavenging (Cristina Lado, et al.,(2004)).

**3.7.4 Inhibition of morphine dependence** 

morphine withdrawal syndrome.

**3.7.3 Antioxidant effect** 

*Streptococcus faecalis,* and *Pseudomonas aeruginosa* and inhibits the growth of strains of *Bacterium phlei* that were resistant against standard anti-infectives (Szabo-Szalontai, M., et al., (1976) and (Szalontai, M.,et al., (1975). Bisabolol, together with enyne dicycloethers, also showed fungistatic activity against *Candida albicans, Trichophytone menthagrophytes,* and *Trichophytone rubrum* at a concentration of 100µg/ml. Chamazulene also had this fungistatic activity, but at higher concentrations (Szalontai.,M., Verzar-petri, G., Florian, E. (1977).

#### **3.5 Anti inflammatory effect**


#### **3.6 Anti ulcerative effect**

Torrado S, et al., in 1995 reported that significant protective effect against gastric toxicity of 200 mg/kg acetylsalicylic acid where achieved after oral administration of chamomile oil to rats at doses ranging from 0.8-80 mg/kg bisabolol. Moreover, *in vitro* studies revealed that alpha-bisabolol inhibited gastric ulcer formation induced by indomethacin, ethanol, or stress, Szelneyi I, Isaac O., thiemer K. (1979)





Torrado S, et al., in 1995 reported that significant protective effect against gastric toxicity of 200 mg/kg acetylsalicylic acid where achieved after oral administration of chamomile oil to rats at doses ranging from 0.8-80 mg/kg bisabolol. Moreover, *in vitro* studies revealed that alpha-bisabolol inhibited gastric ulcer formation induced by indomethacin, ethanol, or

activity which is similar to non-steroidal anti inflammatory drugs.

stress, or alcohol (Szelenyi J., Isaac, O., Thiemer, K., (1979))

myricetin > apigenin-7-glucoside > rutin.

stress, Szelneyi I, Isaac O., thiemer K. (1979)

(Szalontai.,M., Verzar-petri, G., Florian, E. (1977).

**3.5 Anti inflammatory effect** 

et al. (1959).

**3.6 Anti ulcerative effect** 

*Streptococcus faecalis,* and *Pseudomonas aeruginosa* and inhibits the growth of strains of *Bacterium phlei* that were resistant against standard anti-infectives (Szabo-Szalontai, M., et al., (1976) and (Szalontai, M.,et al., (1975). Bisabolol, together with enyne dicycloethers, also showed fungistatic activity against *Candida albicans, Trichophytone menthagrophytes,* and *Trichophytone rubrum* at a concentration of 100µg/ml. Chamazulene also had this fungistatic activity, but at higher concentrations

#### **3.7 Other Pharmacological actions**

#### **3.7.1 Inhibition of Aflatoxin G1 production**

Yoshinari et al. in 2008 showed that the spiroethers of German chamomile inhibited production of aflatoxin G1 AFG1 by *Aspergillus parasiticus* with inhibitory concentration 50% (IC50) values of 2.8 and 20.8 mM respectively. This is through inhibiton of cytochrome P450 monooxygenase CYPA and without inhibiting fungal growth. In addition, it also inhibited production of 3-acetyldeoxynivalenol 3-ADON by *Fusarium graminearum* by inhibiting TRI4. The inhibitory activity of the (E)-spiroether isomer was much stronger than that of the (Z) spiroether in both cases. Inhibition of TRI4 by the spiroethers showed that TRI4 may be a good target for inhibiting biosynthesis of trichothecene mycotoxins.

#### **3.7.2 Protective effect on diabetic complications**


#### **3.7.3 Antioxidant effect**


#### **3.7.4 Inhibition of morphine dependence**


General Introduction on Family Asteracea 393






In an open case study to examine the cardiac effects of two cups of chamomile tea on patients undergoing cardiac catheterization, Gould L. et al. observed that 10 of 12 patients in the study achieved deep sleep within 10 minutes of drinking the tea, Gould L, et al. (1973). The patients had a small but significant increase in mean brachial artery pressure. No other


the weeping wound area and increased drying compared to the placebo group.

and mastitis and in rare cases secondarily healing episiotomies.

superior activity to bufexamac and fluocortin butyl ester.

concentrated form for swabbing inflammatory lesions of the mucosa.

are excised; superficial ones heal without proteolytic ferments.

**4.3 Dermatologyical effect** 

dermatitis.

**4.4 Sleep enhancement** 

**4.5 Radiation therapy** 

significant hemodynamic changes were observed.

colpitis senilis, and about the improvement of the healing of wounds after surgical operations carried out by laser in gynecology after taking a chamomile (hip) bath. - Carle et al. in 1987, and according to reports of various gynecological hospitals, showed that chamomile extract is a suitable remedy for the treatment of bartholinitis, vulvitis,

#### **3.7.5 Tachykinin receptor antagonist**


#### **4. Clinical indications of** *Matricaria chamomilla*

German chamomile is a well-known and widely used herb in different parts of the world. Few well designed, randomized, double-blind; placebo-controlled studies are available to fully assess its therapeutic benefit. (Alternative Medicine Review 2008)

#### **4.1 Gastrointestinal effect**


#### **4.2 General anti-inflammatory effect**



German chamomile is a well-known and widely used herb in different parts of the world. Few well designed, randomized, double-blind; placebo-controlled studies are available to


experienced colic relief compared to 26 percent in the placebo group (p<0.01). - Schmid et al. in 1975 showed that chamomile extract is successfully applied in pediatrics due to its carminative and spasmolytic effect with diseases of the gastrointestinal tract and the effect as such is said to set in immediately after taking the preparation. The internal administration of chamomile tea or preparations from chamomile extracts is appropriate in different gastric troubles that can be classed under



term of "dyspepsia," as recorded by Weiss in 1987.

**4.2 General anti-inflammatory effect** 

chamomile bath and inhalation.

**3.7.5 Tachykinin receptor antagonist** 

**4.1 Gastrointestinal effect** 

**4. Clinical indications of** *Matricaria chamomilla*

fully assess its therapeutic benefit. (Alternative Medicine Review 2008)

colpitis senilis, and about the improvement of the healing of wounds after surgical operations carried out by laser in gynecology after taking a chamomile (hip) bath.


#### **4.3 Dermatologyical effect**


#### **4.4 Sleep enhancement**

In an open case study to examine the cardiac effects of two cups of chamomile tea on patients undergoing cardiac catheterization, Gould L. et al. observed that 10 of 12 patients in the study achieved deep sleep within 10 minutes of drinking the tea, Gould L, et al. (1973). The patients had a small but significant increase in mean brachial artery pressure. No other significant hemodynamic changes were observed.

#### **4.5 Radiation therapy**


General Introduction on Family Asteracea 395

(10-40cm in height, with erect, branching stems **.**the capitulum (to 1.5cm in diameter) comprises 12-20 white ligulate orets surrounding a conical hollow receptacle on which

A lot of studies have been conducted on *Matricaria chamomilla* all over the world where many important biologically active compounds have been separated and identified. However, very few studies are available for *Matricaria aurea* world wide. Nowadays, researches are focusing on exploring the pharmacological profile of compounds from natural origin, where promising results aroused. Challenges remain in finding ways to benefit from these biologically important compounds in treating human health problems.

[1] Porter, C.L. "Taxonomy of flowering plants", Eurasia Publishing House (Pvt.) Ltd., Ram

[2] Evans, W. "Pharmacognosy"; 13th Edition, Bailleire Tinadall., London, Philladelphis,

[3] Hutchinson, J. "The families of flowering plants", 2nd Edition, Oxford University Press,

[5] Harborne, J. B.; Turner, B.L. "Plant Chemosystematics", Academic Press, London, 113

[6] Aboul Ela, M. A; "A Thesis of Doctor of Philosophy degree in Pharmaceutical sciences"; Faculty of Pharmacy, Alexandria University, Alexandria, Egypt 4 (1991). [7] Muschler, R. "A manual Flora of Egypt", Berlin, Freid Laender and sohn Karlstrase,

[9] Ness, A., Metzger, J. W., Schmidt, P. C. (1996) Pharm. Acta Helvet., 71, 265-271. 83. Piesse, S. (1863) Comptes Rend. hebdom. Séances Acad. Sciences, 57, 1016.

[4] Core, E.L. "Plant taxonomy", Engle Cliffs, N.J. Prentice-Hall inc., 423 (1955).

numerous yellow tubular (disk) orets are inserted (Bruneton J. (1995))

Fig. 2. Photograph of *Matricaria chamomilla.*

**6. Conclusions and recommendations** 

Nagar, New Delhi, India, 410 (1969)

Ely House, London, 482 (1973).

Toronto, Sydney and Tokyo, 226 (1989).

[8] Alternative Medicine Review Volume 13, Number 1 2008

**7. References** 

(1984).

Volume II (1912).

were divided into two groups. One group of 66 patients (20 undergoing radiation therapies, 46 undergoing chemotherapy) participated in prophylactic oral care with the mouthwash. The remaining 32 patients underwent chemotherapy and were treated therapeutically after mucositis had developed. Of the 20 patients undergoing radiation, only one developed high-grade (grade 3) mucositis in the final week of treatment, 65 percent developed intermediate grade mucositis, and 30 percent developed low-grade mucositis. Of the 46 patients concurrently receiving chemotherapy and the mouthwash, 36 remained free of any clinically significant mucositis. Of the 32 patients with existing mucositis, all noted immediate relief from mouth discomfort, and within seven days almost all patients had no clinical sign of mucositis.


#### **4.6 Other uses**


#### **5. Photograph of the two matricaria specie**

Fig. 1. Photograph of *Matricaria aurea* 




alleviating effect in cases of inflammatory and painful esophageal diseases.

almost all patients had no clinical sign of mucositis.

**5. Photograph of the two matricaria specie** 

Fig. 1. Photograph of *Matricaria aurea* 

**4.6 Other uses** 

cream to the placebo for its rapid absorption and stainlessness.

were divided into two groups. One group of 66 patients (20 undergoing radiation therapies, 46 undergoing chemotherapy) participated in prophylactic oral care with the mouthwash. The remaining 32 patients underwent chemotherapy and were treated therapeutically after mucositis had developed. Of the 20 patients undergoing radiation, only one developed high-grade (grade 3) mucositis in the final week of treatment, 65 percent developed intermediate grade mucositis, and 30 percent developed low-grade mucositis. Of the 46 patients concurrently receiving chemotherapy and the mouthwash, 36 remained free of any clinically significant mucositis. Of the 32 patients with existing mucositis, all noted immediate relief from mouth discomfort, and within seven days

Fig. 2. Photograph of *Matricaria chamomilla.*

(10-40cm in height, with erect, branching stems **.**the capitulum (to 1.5cm in diameter) comprises 12-20 white ligulate orets surrounding a conical hollow receptacle on which numerous yellow tubular (disk) orets are inserted (Bruneton J. (1995))

#### **6. Conclusions and recommendations**

A lot of studies have been conducted on *Matricaria chamomilla* all over the world where many important biologically active compounds have been separated and identified. However, very few studies are available for *Matricaria aurea* world wide. Nowadays, researches are focusing on exploring the pharmacological profile of compounds from natural origin, where promising results aroused. Challenges remain in finding ways to benefit from these biologically important compounds in treating human health problems.

#### **7. References**


General Introduction on Family Asteracea 397

[39] Power, F., Browning, H. Jr. (1914) J. Chem. Soc., London, 105, 2280, in Becker, H.,

[44] Elkiey, M. A., Darwish, M., Mustafa, M. A. (1963) Fac. Pharm. Cairo Univ., 2, 107, ref. in

[53] Schilcher, H. (1985) Zur Biologie von Matricaria chamomilla, syn. "Chamomilla recutita

[54] Kotov, A. G., Khvorost, P. P., Komissarenko, N. F. Khimiya Prirodnykh Soedinenii

[57] Schilcher, H. (1987) Die Kamille — Handbuch für Arzte, Apotheker und andere Naturwissenschaftler. Wissenschaftl Verlagsgesellschaft, Stuttgart, Germany.

[62] Streibel, M. (1980) Presentation, DFG Conference in Kiel, ref. in: Seifen, Öle, Wachse,

[63] Schilcher, H. (1987) Die Kamille — Handbuch für Ärzte, Apotheker und andere Wissenschaftler, Wissenschaftliche Verlagsgesellschaft, Stuttgart, Germany. [64] Elena Darra , Safwat Abdel-Azeim , Anna Manara , Kazuo Shoji , Jean-Didier Mare´chal

[65] J.H. Joo, G. Liao, J.B. Collins, S.F. Grissom, A.M. Jetten, Cancer Res. 67 (2007) 7929–7936.

[67] Srivastava JK, Gupta S. Antiproliferative and apoptotic effects of chamomile extract in various human cancer cells. J. Agric. Food Chem. (2007) 55:9470- 9478. [68] Deendayal Patel, Sanjeev Shukla and Sanjay Gupta, "Apigenin and cancer

Bid". 476 (2008) 113–123 Archives of Biochemistry and Biophysics

[66] Adany, Cancer Lett. 79 (1994) 175–179. Rioja, FEBS Lett. 467 (2000) 291–295.

,Sofia Mariotto , Elisabetta Cavalieri , Luigi Perbellini , Cosimo Pizza ,David Perahia , Massimo Crimi , Hisanori Suzuki , "Insight into the apoptosis-inducing action of a-bisabolol towards malignant tumor cells: Involvement of lipid rafts and

chemoprevention: Progress, potential and promise". International Journal of

(L.) Raus- chert," Research report 1968-1981, I Pharmakognosie and Phytochemie of

Reichling, J.(1981) Dtsch. Apoth. Ztg, 121, 1285.

[46] Lang, W., Schwandt, K. (1957) Dtsch. Apoth. Ztg., 97, 149.

[55] Janecke, H., Weiser, W. (1964) Planta Med., 12, 528. [56] Janecke, H., Weiser, W. (1965) Pharmazie, 20, 580.

[58] Füller, E. (1992) Dissertation, University of Regensburg.

[60] Bayer, J., Katona, K., Tardos, L. (1958) Naturwiss., 45, 629.

[61] Schilcher, H. (1970) Planta Med., 18, 101-113.

Oncology 30: 233-245, 2007 [69] Barton, H. (1959) Acta Biol. Med. Gem. 2, 555.

[59] Bayer, J., Katona, K., Tardos, L. (1958) Acta Pharm. Hung., 28, 164.

[48] Tschirsch, K., Hölzl, J. (1992) PZ-Wissenschaft, 137, (5) 208–214.

[50] Wagner, H., Kirmayer, W. (1957) Naturwissenschaften, 44, 307.

[41] Kunde, R., Isaac, O. (1979) Planta Med., 37, 124.

[45] Greger, H. (1975) Plant. Syst. Evol., 124, 35.

the FU, Berlin.

(1991), 853

106, 503.

[40] Sorm, P., Zekan, Z., Herout, V., Raskova, H. (1952) Chem. Listy, 46, 308.

[42] Carle, R. and Isaac, O. (1985) Dtsch. Apoth. Ztg., 125 Nr. 43/Suppl. 1, 2–8. [43] Reichling, J., Becker, H., Exner, J., Dräger, P. D. (1979) Pharmaz. Ztg. 124, 1998.

Becker, H., Reichling, J. (1981) Dtsch. Apoth. Ztg, 121, 1285.

[47] Hörhammer, L., Wagner, H., Salfner, B. (1963) Arzneim. Forsch., 13, 33.

[49] Redaelli, C., Formentini, L., Santaniello, E. (1979) Herba Hung., 18, 323.

[51] Exner, J., Reichling, J., Cole, T. H., Becker, H. (1981) Planta Med., 41, 198. [52] Hänsel, R., Rimpler, H., Walther, K. (1966) Naturwissenschaften, 53, 19.


[15] Cekan, Z., Herout, V., Sorm, F. (1954) Collect Czechoslov. Chem. Commun., 19, 798. [16] Cekan, Z., Herout, V., Sorm, F. (1957) Collect Czechoslov. Chem. Commun., 22, 1921. [17] Ahmed A. Ahmed, Maha A. Abou Elela, "Highly oxygenated bisabolenes and acetylene

[18] Ahmed A. Ahmed, J. Jakupovic, Maha A. Abou Elela, Ahmed A. seif El-Din and Nadia

[19] Sorm, F., Zaoral, M., Herout, V. (1951) Collect Czechoslov. Chem. Commun., 16, 626-

[20] Sampath, V., Trivedi, G. K., Paknikar, S. K., Bhattacharyya, S. C. (1969) Indian J. Chem.,

[21] Sampath, V., Trivedi, G. K., Paknikar, S. K., Sabata, B. K., Bhattacharyya, S. C. (1969)

[22] Schilcher, H., Novotny, L., Ubik, K., Motl, O., Herout, V. (1976) Arch. Pharm., 309, 189.

[25] Anne ORAV, Tiiu KAILAS, and Kaire IVASK, "Volatile Constituents of Matricaria recutita L. f". Proc. Estonianrom Estonia" Acad. Sci. Chem., 2001, 50, 1, 39-45

[28] A.Pizard, H. Alyari, M.R. Shakiba , S. Zehtab-Salmasi and A. Mohammadi, "Essential

[30] Kumar, S., Das, M., Singh, A., Ram, G., Mallavarapu, G. R., Ramesh, S. (2001) J. Med.

[31] Bohlmann, F., Herbst, P., Arndt, Ch., Schönowski, U., Gleinig, H. (1961) Chem. Ber., 94,

[33] F.Bohman and H. Kapteyn (1967): Die Polyine aus Chrysanthemum carintum. Chemical

[34] F.Bohman and H. Kapteyn (1967): Die Polyine aus Chrysanthemum carintum. Chemical

[36] W. Donald Macrae and G. H. Tower (1984): Biological activities of lignans.

[37] R. silverstein and G. Bassler (1986): spectroscopic identification of Organic compounds.

[38] F. Bouhlman, W. Kramp Gupta, R. King and H. Robinson (1981): Four guaianolides and

Different Irrigation Regimes. Journal of Agronomy 5 (3): 451-455, 2006 [29] Stransky, K., Streibel, M., Ubik, K., Kohoutova, J., Novotny, L. (1981) Fette, Seifen,

Oil content and composition of German Chamomile (Matricaria chamomilla L.) at

[24] Motl, O., Felklova, M., Lukes, V., Jasikova, M. (1977) Arch. Pharm., 310, 210.

[26] Reichling, J., Bisson, W., Becker, H., Schilling, G. (1983) Z. Naturforsch., 38 c, 159.

S. Hussein, (1993)" Two Bisabolanes from Matricaria aurea". Natural product

from Matricaria aurea". Phytochemistry 51 (1999) 551-554

[10] Stahl, E. (1954) Chem. Ber., 87, 202, 205, 1626. [11] Motl, O., Repcak, M. (1979) Planta Med., 36, 272.

letters 3(4): 277-281

Indian J. Chem., 7, 1060

[27] Lemberovics, E. (1979) Sci. Pharm., 47, 330.

Arom. PlantSciences, 23, 617–623.

[32] Bohlmann, F., Zdero, C. (1982) Phytochemistry, 21, 2543-9.

[35] Yamazaki, H., Miyakado, T., Mabry, T. J. (1982) J. Nat. Prod., 45, 508.

2nd Ed. John Wiley & Sons. Inc., New York, London, Sydney

other constituents from three Kaunia species. Phytochemistry

Anstrichmittel, 83, 347.

Berichte, 100, 1927

Berichte, 100, 1927

Phytochemistry, 23, 1207

[23] Hölzl, J., Demuth, G. (1973) Dtsch. Apoth. Ztg., 113, 671.

638.

7, 100

3193.

[12] Motl, O., Repcak, M., Ubik, K. (1983) Arch. Pharm., 316, 908. [13] So rm, F., Nowak, J., Herout, V. (1953) Chem. Listy, 47, 1097. [14] Cekan, Z., Herout, V., Sorm, F. (1954) Chem. Listy, 48, 1071.


General Introduction on Family Asteracea 399

[97] Kato, L., Gözsy, B. zit., Tur, W., Joss, B. (1959) Azulen im Lichte der medizinischen

[102] Della Loggia, R., Tubaro, A., Zilli, C. (1984) 32nd Annual Congress for Medicinal Plant

[103] Della Loggia, R.; Tubaro, A., Dri, P., Zilli, C., Del Negro, P. (1986) Plant Flavonoids in

[105] Torrado S, Torrado S, Agis A, et al. Effect of dissolution profile and (-)-alpha-bisabolol on the gastrotoxicity of acetylsalicylic acid. Pharmazie 1995;50:141-143. [106] Szelenyi I, Isaac O, Thiemer K. Pharmacological experiments with compounds of

[107] Tomoya Yoshinari, Atsushi Yaguchi, Naoko Takahashi-Ando, Makoto Kimura, Haruo

[108] Atsushi Kato, Yuka Minoshima, Jo Yamamoto, Isao Adachi, Alison A Watson, and

[109] Cristina Lado, Ma´ ria Then, Ilona Varga, E´ va Szo ke, and Kla´ra Szentmiha´ lyi,

[110] Adel Gomaa,, Tahia Hashem, Mahmoud Mohamed, and Esraa Ashry, "*Matricaria* 

[112] De la Motte S, Bose-O'Reilly S, Heinisch M, Harrison F. Double-blind comparison of an

[113] Weizman Z, Alkrinawi S, Goldfarb D, Bitran C. Efficacy of herbal tea preparation in

[114] Schmid, F. (1975) in Demling, L., Nasemann, T. (Eds.), Erfahrungstherapie — späte

Biology and Medicine — Biochemical, Pharmacological and Structure-Activity

chamomile. III. Experimental studies of the ulcerprotective effect of chamomile

Takahashi, Takashi Nakajima, Yoshiko Sugita-Konishi, Hiromichi Nagasawa & Shohei Sakuda" Spiroethers ofGerman chamomile inhibit production ofa£atoxinG1 and trichothecenemycotoxin by inhibiting cytochromeP450 monooxygenases involved in their biosynthesis". FEMS Microbiol. let. 2008 Jul;284(2):184-90. E-pub

Robert J. Nash, "Protective Effects of Dietary Chamomile Tea on Diabetic

"Antioxidant Property of Volatile Oils Determined by the Ferric Reducing Ability".

*chamomilla* Extract Inhibits Both Development of Morphine Dependence and Expression of Abstinence Syndrome in Rats". J. Pharmacol. Sci 92, 50 – 55 (2003) [111] Atsushi Yamamoto, Ko Nakamura, Kazuhito Furukawa, Yukari Konishi, Takashi

Ogino, Kunihiko Higashiura, Hisashi Yago, Kaoru Okamoto, and Masanori Osuka, "A New Nonpeptide Tachykinin NK1 Receptor Antagonist Isolated from the Plants

apple pectin-chamomile extracts preparation with placebo in children with

Stadtler, R., Isaac, O. (1973) Arzneim.-Forsch. 23, 756. [98] Heubner, W., Grabe, E, (1933) Arch. Exp. Pathol. Pharmakol. 171, 329.

[100] Ammon, H. P. T., Sabieraj, J., Kaul, R. (1996) Dtsch. Apoth. Ztg. 136, 1821 [101] Baumann, J., Wurm, G., Bruchhausen, F. (1980) Arch. Pharm. 313, 330.

[104] Wurm, G., Baumann, J., Geres, V. (1982) Dtsch. Apoth. Ztg. 122, 2062.

Complications". J. Agric. Food Chem. 2008, 56, 8206–8211

of Compositae". Chem. Pharm. Bull. 50(1) 47—52 (2002)

diarrhea. Arzneimittel forschung 1997; 47:1247-1249.

Rechtfertigung, Verlag G. Braun, Karlsruhe, Germany

infantile colic. J Pediatr 1993;122:650-652.

[115] Weiss, R. F. (1987) Kneipp-Blätter, 1, 4.

[99] Pommer, Ch. (1942) Arch. Exp. Pathol. Pharmakol. 199, 74.

Relationships, Alan R. Liss, Inc., pp. 481–484

(author's transl). Planta Med 1979; 35:218 227.

Z. Naturforsch. 59c, 354D358 (2004)

Research, Antwerp, Abstracts L.16.

2008 May 19

Weltliteratur, Flyer of the company Th. Geyer KG, Stuttgart, ref. in Thiemer, K.,


[70] Kazuaki Shimoniya, Toshio inoue, Yoshiaki Utsu, Shin Tokunaga, Takayoshi Masuoka,

[71] Avallone R., Zanoli P., Puia G., Kleinschnitz M., Schreier P., Baraldi M., Biochem.

[72] Viola H, Wasowski C 16. , Levi de Stein M, et al. Apigenin, a component of *Matricaria* 

[75] Della Loggia, R.; Tubaro, A., Dri, P., Zilli, C., Del Negro, P. (1986) Plant Flavonoids in

[77] Achterrath-Tuckermann, U., Kunde, R., Flaskamp, E., Isaac, O., Thiemer, K. (1980)

[79] Janku, J. (1981) Paper at 2nd Physiolog. Conf. Königgrätz, ref. in Becker, H., Reichling, J.

[82] Annuk H, Hirmo S, Turi E, et al. Effect on cell surface hydrophobicity and susceptibility

[83] Shikov, A. N., Pozharitskaya, O. N., Makarov, V. G. et al. (1999) Method of allocation of

[84] Turi M, Turi E, Koljalg S, Mikelsaar M. Influence of aqueous extracts of medicinal plants

[86] Szabo-Szalontai, M., Verzár-Petri, G. (1976) 24. Jahres versammlung d. Ges. f.

[87] Szalontai, M., Verzár-Petri, G., Florián, E., Gimpel, F. (1975) Dtsch. Apoth. Ztg. 115, 912. [88] Szalontai, M., Verzár-Petri, G., Florián, E., Gimpel, F. (1975) Pharmaz. Ztg. 120, 982. [89] Szalontai, M., Verzár-Petri, G., Florián, E. (1976) Acta Pharm.-Hung. 46, 232. [90] Szalontai, M., Verzár-Petri, G., Florián, E. (1977) Parfümerie und Kosmetik 58, 121. [91] Janmejai K. Srivastava, Mitali Pandey, Sanjay Gupta, "Chamomile, a novel and selective COX-2 inhibitor with anti-inflammatory activity". Life Sciences 85 (2009) 663–669

[96] Uda, T. (1960) Nippon Yak. Zasshi 56, 1151; ref. in Chem. Abstr. 50, 4058 (1962).

of Helicobacter pylori to medicinal plant extracts. FEMS Microbiol Lett 1999;172:41-

biologically active substances from plant material. Patent Ru 214 1336 from Nov. 2,

on surface hydrophobicity of Escherichia coli strains of different origin. APMIS

[73] Gould L., Reddy C. V. R., Gomprecht R. F., J. Clin. Pharmacol., 13, 475—479 (1973).

[74] Della Loggia R., Tubaro A., Redaelli C., Riv. Neurol., 51, 297—310 (1981).

Relationships, Alan R. Liss, Inc., pp. 481–484 [76] Carle, R., Gomaa, K. (1992) Drugs of Today 28, 559.

[80] Della Loggia, R. (1985) Dtsch. Apoth. Ztg. 125, Suppl. I, 9.

[85] Berry M. The chamomiles. Pharm. J 1995; 254:191-193.

Arzneipflanzen forsch., Munich, Germany.

[92] Szelenyi, J., Isaac, O., Thiemer, K. (1979) Planta Med. 35, 218.

[94] Zierz, P., Kiessling, W. (1953) Dtsch. Med. Wschr. 78, 1166. [95] Zierz, P., Lehmann, A., Craemer, R. (1957) Hautarzt 8, 552.

[93] Büchi, O. (1959) Arch. Int. Pharmacodyn. 123, 140.

[81] Hörhammer, L., Wagner, H., Salfner, B. (1963) Arzneim.-Forsch. 13, 33.

Pharmacol. 59, 1387—1394 (2000).

Planta Med. 1995; 61:213-216.

Planta Med. 39, 38–50.

45.

1999.

1997; 105:956-962.

[78] Hava M., Janku J. (1957) Rev. Czech. Med. 3, 130

(1981) Dtsch. Apoth. Ztg. 121, 1285.

Asae Ohmori, and Chiaki Kamei, "Hypnotic Activities of Chamomile and Passiflora Extracts inSleep-Disturbed Rats". Biol. Pharm. Bull. 28(5) 808—810 (2005)

*recutita* flowers, is a central benzodiazepine receptors-ligand with anxiolytic effects.

Biology and Medicine — Biochemical, Pharmacological and Structure-Activity


**19** 

*1Indonesia 2Malaysia* 

**Bioavailability of Phytochemicals** 

Phytochemicals are increasingly accepted as health promoting, maintaining, and repairing agents in cells, tissues, or the whole human body. Phytochemicals are compounds obtained from plants that exert particular health effects; generally, they are not necessarily basic nutrients (minerals, vitamins, carbohydrates, proteins or lipids), medicines or toxins. The phytochemicals that are frequently associated with human health are phenolics, carotenoids, organic acids, and several miscellaneous bioactive compounds such as saponin and sterols. The contributions of phytochemicals in public health cover various issues world-widely and thus it is seen by researchers, industries, general society, and policy makers as a new tool to manage public health. Ironically, the roles of phytochemicals in health are poorly understood, which warrant the needs for validation as well as scientific database on safety issues and mechanisms of the functions. Even though various genetic-base studies propose mechanisms and health interventions of phytochemicals (Noe et al., 2004), many findings are inconclusive. Hence, the emerging health potentials of phytochemicals are inconclusive; and internationally it has been the reason for new policies/regulations in food trading. This is partly due to limited understanding on phytochemical bioavailability by which the health benefits depend on. Moreover, transport mechanisms for phytochemicals delivery into the target sites, phytochemical metabolisms by the human body, and biomarkers exerting the health benefits are also poorly understood. These complexities call for a new framework on how and to what extent dietary phytochemicals should be recommended in order to reach

In the human body, bioavailability is defined as substances obtained from ingested materials that reach circulatory system for further delivery into designated tissues so that the beneficial compounds are biologically available for exerting healthy functions. The normal routes of dietary phytochemicals thus include ingestion, digestions, and transport across gastrointestinal epithelium prior to circulatory vessels. The epithelium in the gastrointestinal tract is a polarized enterocyte cell having two different sides facing luminal hollow (Apical side) and blood capillaries (Basolateral side) where each side is equipped with different transport facilities and barriers. The epithelial cells are critical for bioavailability of target compounds either as entrance gates or as metabolizing machines which release different compounds from the parent molecules. These make further complexing bioavailability routes because the metabolisms and transport processes are also

**1. Introduction** 

biologically-safe active dosages.

*2Swinburne University of Technology, Sarawak Campus,* 

Indah Epriliati1 and Irine R. Ginjom2 *1Widya Mandala Catholic University,* 


### **Bioavailability of Phytochemicals**

#### Indah Epriliati1 and Irine R. Ginjom2

*1Widya Mandala Catholic University, 2Swinburne University of Technology, Sarawak Campus, 1Indonesia 2Malaysia* 

#### **1. Introduction**

400 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

[116] Schilcher, H. (1999) Phytotherapie in der Kinderheilkunde, 3rd ed., Wissenschaftliche

[117] Nasemann, T., Patzelt-Wenczler, R. (1991) Kamillosan im Spiegel der Literatur, pmi-

[119] Stechele, U. (1979) Expert report from a pediatric practice. Ref. in Nasemann, T.,

[120] Aertgeerts P., Albring M., Klaschka F. et al. Comparative testing of Kamillosan cream

[121] Born, W.: Personal communication to company Homburg (letter of August 6, 1980), ref.

[122] Contzen, H. (1975) in Demling, L., Nasemann, T. (Eds.), Erfahrungs therapie — späte

[123] Glowania HJ, Raulin C, Swoboda M. Effect of chamomile on wound healing – a clinical

[124] Gould L, Reddy CV, Gomprecht RF. Cardiac effectsof chamomile tea. J. Clin.

[125] Fidler P, Loprinzi CL, O'Fallon JR, et al. Prospective evaluation of a chamomile

[126] Carl W, Emrich LS. Management of oral mucositis during local radiation and systemic chemotherapy: a study of 98 patients. J Prosthet. Dent. 1991;66:361- 369. [127] Maiche AG, Grohn P, Maki-Hokkonen H. Effect of chamomile cream and almond ointment on acute radiation skin reaction. Acta Oncol 1991; 30:395-396.

[130] Bruneton J. Pharmacognosy, phytochemistry, medicinal plants. Paris, Lavoisier, 1995.

mouthwash for prevention of 5-FU-induced oral mucositis. Cancer 1996;77:522-

Patzelt-Wenczler, R. (Eds.) Kamillosan im Spiegel der Literatur, pmi-Verlag

and steroidal (0.25% hydrocortisone, 0.75% fluocortin butyl ester) and nonsteroidal (5% bufexamac) dermatologic agents in maintenance therapy of

in T. Nasemann, R. Patzelt-Wenczler (Eds.), Kamillosan im Spiegel der Literatur ,

Verlagsgesellschaft mbH, Stuttgart, Germany.

eczematous diseases. Z. Hautkr 1985;60:270-277.

Rechtfertigung; Verlag G. Braun, Karlsruhe, Germany.

double blind study. Z Hautkr 1987;62:1262,1267-1271.

[128] Blumenberg, E.-W., Hoefer-Janker, H. (1972) Radiologie, 12, 209.

[118] Carle, R., Isaac, O. (1987) Zschr.-f. Phytoth ., 8 , 67.

pmi-Verlag Frankfurt/ Main (1991).

Pharmacol. 1973;13:475 479.

[129] Hinz, D. (1995) Therapiewoche,8, 478.

525.

Verlag Frankfurt/ Main.

Frankfurt/Main (1991).

Phytochemicals are increasingly accepted as health promoting, maintaining, and repairing agents in cells, tissues, or the whole human body. Phytochemicals are compounds obtained from plants that exert particular health effects; generally, they are not necessarily basic nutrients (minerals, vitamins, carbohydrates, proteins or lipids), medicines or toxins. The phytochemicals that are frequently associated with human health are phenolics, carotenoids, organic acids, and several miscellaneous bioactive compounds such as saponin and sterols. The contributions of phytochemicals in public health cover various issues world-widely and thus it is seen by researchers, industries, general society, and policy makers as a new tool to manage public health. Ironically, the roles of phytochemicals in health are poorly understood, which warrant the needs for validation as well as scientific database on safety issues and mechanisms of the functions. Even though various genetic-base studies propose mechanisms and health interventions of phytochemicals (Noe et al., 2004), many findings are inconclusive. Hence, the emerging health potentials of phytochemicals are inconclusive; and internationally it has been the reason for new policies/regulations in food trading. This is partly due to limited understanding on phytochemical bioavailability by which the health benefits depend on. Moreover, transport mechanisms for phytochemicals delivery into the target sites, phytochemical metabolisms by the human body, and biomarkers exerting the health benefits are also poorly understood. These complexities call for a new framework on how and to what extent dietary phytochemicals should be recommended in order to reach biologically-safe active dosages.

In the human body, bioavailability is defined as substances obtained from ingested materials that reach circulatory system for further delivery into designated tissues so that the beneficial compounds are biologically available for exerting healthy functions. The normal routes of dietary phytochemicals thus include ingestion, digestions, and transport across gastrointestinal epithelium prior to circulatory vessels. The epithelium in the gastrointestinal tract is a polarized enterocyte cell having two different sides facing luminal hollow (Apical side) and blood capillaries (Basolateral side) where each side is equipped with different transport facilities and barriers. The epithelial cells are critical for bioavailability of target compounds either as entrance gates or as metabolizing machines which release different compounds from the parent molecules. These make further complexing bioavailability routes because the metabolisms and transport processes are also

Bioavailability of Phytochemicals 403

sites Infant Adults Elderly Female Male

Improper chewing, may be with incomplete salivary enzymes

Acid & pepsin digestion

Complete tissues and enzymes

Lossing Bifido bacteria

Table 1. Summary of digestion characteristics of infant, adult and elderly, female and male

Fig. 1. Principles of human digestion system adapted and modified from Johnson (2001)

Cell wall materials significantly modulate digestion of plant foods. Nunan et al. (1998) state that in grape berry during its development of the berry fruits the Na2CO3-soluble fraction increases before veraison but decreases as the berries softened. It implies that the Na2CO3 soluble fraction is the cell wall component which is responsible for firmness and strength. Epriliati (2008) observe that ripe mango, tomato, and papaya behave differently when Na2CO3 is added into *in vitro* digestion model mimicking small intestine where not all

Chewing Chewing

Acid & pepsin digestion

Lossing Bifido bacteria

Hormonal related digestive secretion , disturbed by reproductive organ cycles

Acid & pepsin digestion

Hormonal related digestive secretion, undisturbed by reproductive cycles

Lossing Bifido bacteria

Chewing, complete salivary enzymes

Acid & pepsin digestion

Complete tissues and enzymes

Lossing Bifido bacteria

Digestion

Improper chewing,

enzymes

digestion

Predominant Bifido bacteria

**2.4 Effects of digestion on phytochemicals** 

Gastric Acid & pepsin

intestine Immature system

imperfect salivary

Mouth

Small

Large intestine

involved in the orchestrated physiological regulations maintaining homeostasis states of the human body. However, bioavailability of phytochemicals by which the health benefits depend on are not well understood; consequently, it is difficult to be measured.

The difficulties in studies of bioavailability are mainly due to the complexities involved in the biological system, i.e. (a) variation in food materials and the human subjects or surrogate models which are not always representative; (b) complex interactions amongst huge chemicals/food components during postharvest, storage, processing, digestion, and absorption that may alter health benefits; and (c) mechanism pathways. In this paper, fundamental aspects of phytochemical bioavailability are reviewed.

#### **2. Digestibility of phytochemicals**

It is known that major phytochemicals are located inside vacuoles of plant cells; and several phenolics form complexes with fibres in the cell wall. These natural existences make the phytochemicals poorly accessed by enzymes or hardly released out from the plant matrices during digestion. Most cell wall materials are indigestible by human enzymic systems. Moreover, it is also poorly permeable for important molecules such as phytochemicals. Therefore, digestibility of the phytochemicals is of great interest, in particular, to reveal how the phytochemicals can affect human health and fight or prevent diseases if the phytochemicals are strongly contained in the food matrices.

#### **2.1 Digestion: principles of human gastrointestinal tract**

The digestion compartments in human consist of mouth, gastric, small intestine, and colon (Figure 1). Each has slightly different digestion performances depending on age and gender as listed in Table 1. In the gastrointestinal tract, net nutriome1 is released as a result of orchestrated secretions, enzymic activities, and physical-mechanical actions of peristaltic movements. The nutriomes will diffuse out from the food particles to chyme solutions. The levels of nutriome in this stage are called availability or accessibility of the components. However, bio-/chemical degradations of the molecules can take place. Hence, digestibility will also provide metabolites/derivatives. Nevertheless, availability and accessibility parameters can only account for intact molecules but not the metabolites.

Architecture and material of the plant tissues is generally unfavourable for activities of enzymic system in the human gastrointestinal tract. As a consequence, limited cell contents of the ingested food materials are released into chyme solution in the gastrointestinal tract. Natural pores and plasmodesmatas may not play predominant roles in diffusion of the nutriome. Nevertheless, according to Stolle-Smits et al. (2009), natural matrices of tomato, mango, apple, and kiwi undergo galactan solubilisation during ripening stage; thus the release of nutriome can be altered. However, processing and chemical compositions of the food matrices themselves may change physicochemical environments of the chyme for nutriome mass transfer. The most recent finding indicates that ingested foods are necessarily designed such that the diffusion of the nutriome favours nutriome absorption by epithelial cells; even for phenolics, it requires lipid-complex called phytosome (Kidd & Head, 2005) to penetrate gut lining and to enter the circulatory system.

<sup>1</sup> Nutriome is a term referring to all beneficial food components

involved in the orchestrated physiological regulations maintaining homeostasis states of the human body. However, bioavailability of phytochemicals by which the health benefits

The difficulties in studies of bioavailability are mainly due to the complexities involved in the biological system, i.e. (a) variation in food materials and the human subjects or surrogate models which are not always representative; (b) complex interactions amongst huge chemicals/food components during postharvest, storage, processing, digestion, and absorption that may alter health benefits; and (c) mechanism pathways. In this paper,

It is known that major phytochemicals are located inside vacuoles of plant cells; and several phenolics form complexes with fibres in the cell wall. These natural existences make the phytochemicals poorly accessed by enzymes or hardly released out from the plant matrices during digestion. Most cell wall materials are indigestible by human enzymic systems. Moreover, it is also poorly permeable for important molecules such as phytochemicals. Therefore, digestibility of the phytochemicals is of great interest, in particular, to reveal how the phytochemicals can affect human health and fight or prevent diseases if the

The digestion compartments in human consist of mouth, gastric, small intestine, and colon (Figure 1). Each has slightly different digestion performances depending on age and gender as listed in Table 1. In the gastrointestinal tract, net nutriome1 is released as a result of orchestrated secretions, enzymic activities, and physical-mechanical actions of peristaltic movements. The nutriomes will diffuse out from the food particles to chyme solutions. The levels of nutriome in this stage are called availability or accessibility of the components. However, bio-/chemical degradations of the molecules can take place. Hence, digestibility will also provide metabolites/derivatives. Nevertheless, availability and accessibility

Architecture and material of the plant tissues is generally unfavourable for activities of enzymic system in the human gastrointestinal tract. As a consequence, limited cell contents of the ingested food materials are released into chyme solution in the gastrointestinal tract. Natural pores and plasmodesmatas may not play predominant roles in diffusion of the nutriome. Nevertheless, according to Stolle-Smits et al. (2009), natural matrices of tomato, mango, apple, and kiwi undergo galactan solubilisation during ripening stage; thus the release of nutriome can be altered. However, processing and chemical compositions of the food matrices themselves may change physicochemical environments of the chyme for nutriome mass transfer. The most recent finding indicates that ingested foods are necessarily designed such that the diffusion of the nutriome favours nutriome absorption by epithelial cells; even for phenolics, it requires lipid-complex called phytosome (Kidd &

depend on are not well understood; consequently, it is difficult to be measured.

fundamental aspects of phytochemical bioavailability are reviewed.

phytochemicals are strongly contained in the food matrices.

**2.1 Digestion: principles of human gastrointestinal tract** 

parameters can only account for intact molecules but not the metabolites.

Head, 2005) to penetrate gut lining and to enter the circulatory system.

1 Nutriome is a term referring to all beneficial food components

**2. Digestibility of phytochemicals** 


Table 1. Summary of digestion characteristics of infant, adult and elderly, female and male

Fig. 1. Principles of human digestion system adapted and modified from Johnson (2001)

#### **2.4 Effects of digestion on phytochemicals**

Cell wall materials significantly modulate digestion of plant foods. Nunan et al. (1998) state that in grape berry during its development of the berry fruits the Na2CO3-soluble fraction increases before veraison but decreases as the berries softened. It implies that the Na2CO3 soluble fraction is the cell wall component which is responsible for firmness and strength. Epriliati (2008) observe that ripe mango, tomato, and papaya behave differently when Na2CO3 is added into *in vitro* digestion model mimicking small intestine where not all

Bioavailability of Phytochemicals 405

pH as detected by Ginjom (2009). Similarly, (+)-catechin is stable in *in vitro* digestion up to pH 7.4 at 37 C for 8 h different from that of (-)-catechin (Friedman & Jurgen, 2000 and Donovan et al., 2006). Overall, phenolics in wine do not undergo significant changes during

In red wine, anthocyanidin is important component of phenolics. Anthocyanin availability is reduced by 32% after pancreatic digestion compared to that of gastric digestion and undigested sample (Ginjom, 2009). Pancreatic environment, however, decreases monomeric anthocyanin more severely (58.75%) than polymeric anthocyanins (17.72%). Pancreatic condition in the intestine modifies molecular structures of peonidin-3-glucoside, malvidin-3 glucoside, and malvidin glucoside pyruvate as indicated by the changes in their retention time during HPLC/UPLC analyses, although it is unclear why this can happen. However, during *in vitro* digestion, Ginjom (2009) speculates that the losses of monomer are related to their polymerization during the pancreatic digestion. Although non-anthocyanins are insignificantly affected by gastric digestion, pancreatic digestion severely reduces them by ca 88% (equivalent to 22% of total phenolics in red wine). On the other hand, flavan– catechin is speculated to polymerize with anthocyanins or tannin forming precipitates during pancreatic digestion; consequently, they either being eliminated during sample

Phenolics in tomato products are released into digest solutions more during *in vitro* gastric digestion than during pancreatic digestion and the highest release is from tomato juice (Epriliati, 2008). The main phenolics in tomato are caffeic, catechin, rutin, chlorogenic acid, and coumaric acids. More phenolics are obtained from tomato juice than those from dried and fresh tomato indicating the natural barrier of cell wall has been eliminated. Noticeable changes of phenolic compounds due to processing and digestion are found but the new compounds are not able to be identified. Rutin and catechin are consistently found in fresh, juiced, and dried products. Meanwhile, no *p*-coumaric is found in fresh product whereas *p*coumaric gradually appears in juiced and in dried products. In contrast, chlorogenic acid is present in fresh products but it gradually disappears in juiced and dried products. This could be caused the different extractability due to different matrices of the products or by chemical changes due to processing and digestion environments (Epriliati, 2008). Gastric digestion does not affect phenolic compounds. However, the phenolic levels are significantly reduced in consecutive gastric-intestinal digestion. Apparently, tomato pectin neither gels nor traps phenolic compounds at lower pH. Altering pH from gastric to

Similarly, there are different phenolics released from mango during *in vitro* digestion. The phenomena consistently support the possibilities of impermeable pectin where more phenolics are released in a consecutive gastric-intestinal digestion when aggregated boli can be broken down with the addition of Na2CO3/NaHCO3 (Epriliati, 2008). Recently, phenolics in gastrointestinal tract markedly behave in a similar way to that of carotenoids incorporated in chylomicrons, thus, all emulsified phytochemical compounds are called phytosome3 (Kidd & Head, 2005). Therefore, the presence of pancreatic juices and bile extract improve phenolics release during consecutive gastric-intestinal digestions of mango.

gastric digestion.

preparation or disposed in aqueous fractions.

intestine may obstruct the molecular phenolic stability.

3 Phytosome is a term for vehicles in which phytochemical compounds are bound

aggregated boli from human *in vivo* chewing can be broken down. The diverse resistances of plant cell wall material amongst plant species during digestion may be partly due to Na2CO3 soluble fractions. It is more likely that mango and tomato have different levels of Na2CO3 soluble fractions which result in diverse *in vitro* digestion effects compared to papaya. Furthermore, processing will affect the way nutriome being released. Meanwhile, heating of the filtered fresh-juices (tomato, mango, and papaya) results in formation of clumpy substances (Epriliati, 2008). Similar clumps are also found as remnant of pectin gel used for taste masking agent of paracetamol and ambroxol (Miyazaki et al., 2005). These imply that consumption of fresh and processed various fruits, rich in pectin, can yield a wide range of phytochemical bioavailability depending on their cell wall material compositions. Furthermore, pasteurisation may render phytochemical release from the clumpy substances.

**Phenolics.** There are two main routes for digestion of dietary phenolics; i.e. digestion along the gastrointestinal tract and digestion inside the enterocytes. This can happen because hydrolase enzymes, i.e. lactase phlorizin hydrolase are available in intestinal lumen, brush border, and enterocyte (Williamson, 2004). Metabolisms that take place along the gastrointestinal tract are mainly aiming at deglycosilation of glycoside form of dietary phenolics. This deglycosilation is also carried out by microbiota in the colon.

Inside the enterocyte, dietary phenolic glucuronidation of the aglycone form are catalyzed by UGT2. Meanwhile, the glycone forms are hydrolyzed and conjugated. The conjugated forms from both glycone and aglycone dietary phenolics are either effluxed into intestinal lumen or translocated into the portal blood vessel. The circulated conjugates of dietary phenolics in plasma can be absorbed by liver and hepatocytes will metabolize them further. For instance, the hepatocyte converts flavonoids into glucuronidated and sulphated forms, which are polar rendering to dissolve in water easier and then be excreted in urine or bile. The pivotal roles of liver indicate that these conjugations are apparently one of physiological needs in the body, for example for bile synthesis in mammalians.

All compounds in wine, which are free from cell wall materials, show clearer responses during gastrointestinal digestion. Flavonol and proanthocyanidin interact with protein in the salivary secretion. However, catechin interacts stronger than epicatechin indicating that molecular characteristics play an important role in this interaction (de Freitas & Mateus 2001). Flavonols and proanthocyanidins remain intact but they may also be broken down when pH is sufficiently low in the stomach. Phenolics stability is strongly affected by pH as studied by Ginjom (2009). For example, syringic and *p-*coumaric acids are stable at pH 2-9 for 24 h. Generally, pH higher than 7.4 is unfavourable for phenolics and the effects of high pH are worsened by lengthy exposures. The number of -OH groups in benzene ring of simple phenolics can also be critical clues for phenolic stability. High pH results in unstable quinones which are oxidized further into diketones and other degradation products. In contrast, the stability of polyphenols such as quercetin, malvidin-3-glucoside, and resveratrol which have more than one benzene ring does not solely depend on their -OH groups. Quercetin is unstable during gastric and pancreatic digestions because quercetin is easily degraded at high pH, yet it is stable at pH 2 and pH 5.5 (Ginjom, 2009). In contrast, *trans*-resveratrol is stable at pH 1-7. Catechin isomers also show different stability at high

<sup>2</sup> UDP glucuronosyltransferase

aggregated boli from human *in vivo* chewing can be broken down. The diverse resistances of plant cell wall material amongst plant species during digestion may be partly due to Na2CO3 soluble fractions. It is more likely that mango and tomato have different levels of Na2CO3 soluble fractions which result in diverse *in vitro* digestion effects compared to papaya. Furthermore, processing will affect the way nutriome being released. Meanwhile, heating of the filtered fresh-juices (tomato, mango, and papaya) results in formation of clumpy substances (Epriliati, 2008). Similar clumps are also found as remnant of pectin gel used for taste masking agent of paracetamol and ambroxol (Miyazaki et al., 2005). These imply that consumption of fresh and processed various fruits, rich in pectin, can yield a wide range of phytochemical bioavailability depending on their cell wall material compositions. Furthermore, pasteurisation may render phytochemical release from the

**Phenolics.** There are two main routes for digestion of dietary phenolics; i.e. digestion along the gastrointestinal tract and digestion inside the enterocytes. This can happen because hydrolase enzymes, i.e. lactase phlorizin hydrolase are available in intestinal lumen, brush border, and enterocyte (Williamson, 2004). Metabolisms that take place along the gastrointestinal tract are mainly aiming at deglycosilation of glycoside form of dietary

Inside the enterocyte, dietary phenolic glucuronidation of the aglycone form are catalyzed by UGT2. Meanwhile, the glycone forms are hydrolyzed and conjugated. The conjugated forms from both glycone and aglycone dietary phenolics are either effluxed into intestinal lumen or translocated into the portal blood vessel. The circulated conjugates of dietary phenolics in plasma can be absorbed by liver and hepatocytes will metabolize them further. For instance, the hepatocyte converts flavonoids into glucuronidated and sulphated forms, which are polar rendering to dissolve in water easier and then be excreted in urine or bile. The pivotal roles of liver indicate that these conjugations are apparently one of physiological

All compounds in wine, which are free from cell wall materials, show clearer responses during gastrointestinal digestion. Flavonol and proanthocyanidin interact with protein in the salivary secretion. However, catechin interacts stronger than epicatechin indicating that molecular characteristics play an important role in this interaction (de Freitas & Mateus 2001). Flavonols and proanthocyanidins remain intact but they may also be broken down when pH is sufficiently low in the stomach. Phenolics stability is strongly affected by pH as studied by Ginjom (2009). For example, syringic and *p-*coumaric acids are stable at pH 2-9 for 24 h. Generally, pH higher than 7.4 is unfavourable for phenolics and the effects of high pH are worsened by lengthy exposures. The number of -OH groups in benzene ring of simple phenolics can also be critical clues for phenolic stability. High pH results in unstable quinones which are oxidized further into diketones and other degradation products. In contrast, the stability of polyphenols such as quercetin, malvidin-3-glucoside, and resveratrol which have more than one benzene ring does not solely depend on their -OH groups. Quercetin is unstable during gastric and pancreatic digestions because quercetin is easily degraded at high pH, yet it is stable at pH 2 and pH 5.5 (Ginjom, 2009). In contrast, *trans*-resveratrol is stable at pH 1-7. Catechin isomers also show different stability at high

phenolics. This deglycosilation is also carried out by microbiota in the colon.

needs in the body, for example for bile synthesis in mammalians.

clumpy substances.

2 UDP glucuronosyltransferase

pH as detected by Ginjom (2009). Similarly, (+)-catechin is stable in *in vitro* digestion up to pH 7.4 at 37 C for 8 h different from that of (-)-catechin (Friedman & Jurgen, 2000 and Donovan et al., 2006). Overall, phenolics in wine do not undergo significant changes during gastric digestion.

In red wine, anthocyanidin is important component of phenolics. Anthocyanin availability is reduced by 32% after pancreatic digestion compared to that of gastric digestion and undigested sample (Ginjom, 2009). Pancreatic environment, however, decreases monomeric anthocyanin more severely (58.75%) than polymeric anthocyanins (17.72%). Pancreatic condition in the intestine modifies molecular structures of peonidin-3-glucoside, malvidin-3 glucoside, and malvidin glucoside pyruvate as indicated by the changes in their retention time during HPLC/UPLC analyses, although it is unclear why this can happen. However, during *in vitro* digestion, Ginjom (2009) speculates that the losses of monomer are related to their polymerization during the pancreatic digestion. Although non-anthocyanins are insignificantly affected by gastric digestion, pancreatic digestion severely reduces them by ca 88% (equivalent to 22% of total phenolics in red wine). On the other hand, flavan– catechin is speculated to polymerize with anthocyanins or tannin forming precipitates during pancreatic digestion; consequently, they either being eliminated during sample preparation or disposed in aqueous fractions.

Phenolics in tomato products are released into digest solutions more during *in vitro* gastric digestion than during pancreatic digestion and the highest release is from tomato juice (Epriliati, 2008). The main phenolics in tomato are caffeic, catechin, rutin, chlorogenic acid, and coumaric acids. More phenolics are obtained from tomato juice than those from dried and fresh tomato indicating the natural barrier of cell wall has been eliminated. Noticeable changes of phenolic compounds due to processing and digestion are found but the new compounds are not able to be identified. Rutin and catechin are consistently found in fresh, juiced, and dried products. Meanwhile, no *p*-coumaric is found in fresh product whereas *p*coumaric gradually appears in juiced and in dried products. In contrast, chlorogenic acid is present in fresh products but it gradually disappears in juiced and dried products. This could be caused the different extractability due to different matrices of the products or by chemical changes due to processing and digestion environments (Epriliati, 2008). Gastric digestion does not affect phenolic compounds. However, the phenolic levels are significantly reduced in consecutive gastric-intestinal digestion. Apparently, tomato pectin neither gels nor traps phenolic compounds at lower pH. Altering pH from gastric to intestine may obstruct the molecular phenolic stability.

Similarly, there are different phenolics released from mango during *in vitro* digestion. The phenomena consistently support the possibilities of impermeable pectin where more phenolics are released in a consecutive gastric-intestinal digestion when aggregated boli can be broken down with the addition of Na2CO3/NaHCO3 (Epriliati, 2008). Recently, phenolics in gastrointestinal tract markedly behave in a similar way to that of carotenoids incorporated in chylomicrons, thus, all emulsified phytochemical compounds are called phytosome3 (Kidd & Head, 2005). Therefore, the presence of pancreatic juices and bile extract improve phenolics release during consecutive gastric-intestinal digestions of mango.

<sup>3</sup> Phytosome is a term for vehicles in which phytochemical compounds are bound

Bioavailability of Phytochemicals 407

Fig. 2. Absorption tissue: epithelial cell (left) and intestinal brush border (right)

the coarse particles are not abrasive towards the epithelial cells.

**3.2 Transport mechanisms** 

4 Sodium dependent glucose transporter

Other barrier in intestine is mucus. Most absorptive tissues comprise of epithelial cells which protect the human body from hazardous components in ingested foods. The epithelial cells are critical gate for human body. Due to its critical roles, the epithelial cells along gastrointestinal tract are covered with mucus secreted by goblet cells making an unstirred water layers so that

Epithelial cell membrane is an important part of transport facility. It controls and selectively takes up molecules required for living or treats hazardous molecules. The fate of its work is not well understood despite studies finding many facilities and signaling processes available for regulating transport molecules. The transport modes include passive and active mechanisms. Passive transport is transcellular or paracellular transports and cynocitosis. Active transports are characterized by the use of protein transporters: channels/pump, binding protein transport, and formation of vehicles that is mainly emulsion system incorporating oil soluble compounds, such as chylomicrons. The transporter is able to promote transmembrane movement without hydrolyzing ATP (Johnson, 2001). They are categorized as uniporter (single compound moving down along the electrochemical gradients) and symporter (two molecules at the same time moving in one direction) or antiporter (two molecules at the same time moving in opposite directions). Several transporters act as cellular efflux port for flavonoids: *P-*glycoproteins, multidrug resistance associated proteins, and breast cancer resistance protein (Johnson, 2001). They generally have loose substrate specificity and also involve in regulating non-nutritional compounds. Several findings point out glucose transporters which allow quercetin glucoside to be absorbed intact besides its aglycone forms. They are SGLT14 (SLC5A15),

**Carotenoids.** About 50% of extractable carotenoids dominated by lycopene and -carotene in tomato, mango, and papaya products are released to digest solution in a non-lipidic digestion model (Epriliati, 2008). The release of carotenoids increases significantly in intestinal digestion where bile extract and pancreatic secretions exist. Consecutive gastricintestinal digestions do not help with higher release of carotenoids. This is more likely due to insufficient emulsifier-water ratios to provide emulsification of carotenoids which are fat soluble. It is concluded that mango, tomato, and papaya carotenoids are released better in intestinal digestion where the model is without addition of oil (Epriliati, 2008).

**Organic acids.** Pectin content in tomato hinders organic acid release thus the total organic acids in *in vitro* gastric digest solution is lower than that of consecutive gastric-pancreatic digestion. This is evidenced by the changes in pH from highly acidic gastric pH to higher small intestinal pH (~6), that causes disaggregation of boli during *in vitro* digestion. For all types of mango samples, organic acid including ascorbic acid (Vitamin C) is released better during gastric digestion. Apparently, the pectinous materials in mango do not trap organic acids (Epriliati, 2008).

#### **3. Absorption of phytochemicals**

Currently, there is no well-established molecular form of absorbed substances in the gastrointestinal tract, i.e. whether they are absorbed intact or as metabolites. On the other hand, it is well known that lifestyle, behavior, diets, and basal metabolism of the subjects are more important affecting factors than age, gender, body weight, and plasma volume (Manners et al., 2003) in bioavailability determination. Therefore, standardized experimental conditions controlling such critical factors of absorption *in vivo* and *in vitro* is a must despite individual human variability.

#### **3.1 Absorptive tissue structures**

The main absorptive tissue is the small intestine. In human 81% of the total intestinal lengths is by small intestine and 19% is large intestine. The stretched length of jejunum is around 30.78% of the intestinal lengths. The transit time along human small and large intestine is 3-4 h and 2-4 d, respectively (Vermeulen, 2009). Principles of the intestinal absorptive structures are depicted in Figure 2. A single enterocyte has microvilli and each microvillus has glycocalyx. Such structure considerably increases contact surface areas with luminal contents. Each microvillus also contains a complex structure providing various facilities for uptake/influx and efflux molecules, signalling ports, cytoplasm, and lipid matrix. The glycocalyx and microvilli are the areas where the human body depends on for collecting nutriomes but rejecting hazardous compounds including microbes.

Each enterocyte attaches onto adjacent cells through tight, adherence and gap junctions. Cellular transport from intestinal lumen to portal blood vessel occurs in two ways: paracellular and transcellular. The paracellular entrances for hazards are tightly controlled by those three types of junctions. Molecular weight cut off limits the hazardous substances crossing through both enterocyte lining cells and tight junction. The enterocytes collect compounds that reach apical side. The compounds then traverse into basolateral side where they end up in capillary vessel for circulation into the whole human body.

**Carotenoids.** About 50% of extractable carotenoids dominated by lycopene and -carotene in tomato, mango, and papaya products are released to digest solution in a non-lipidic digestion model (Epriliati, 2008). The release of carotenoids increases significantly in intestinal digestion where bile extract and pancreatic secretions exist. Consecutive gastricintestinal digestions do not help with higher release of carotenoids. This is more likely due to insufficient emulsifier-water ratios to provide emulsification of carotenoids which are fat soluble. It is concluded that mango, tomato, and papaya carotenoids are released better in

**Organic acids.** Pectin content in tomato hinders organic acid release thus the total organic acids in *in vitro* gastric digest solution is lower than that of consecutive gastric-pancreatic digestion. This is evidenced by the changes in pH from highly acidic gastric pH to higher small intestinal pH (~6), that causes disaggregation of boli during *in vitro* digestion. For all types of mango samples, organic acid including ascorbic acid (Vitamin C) is released better during gastric digestion. Apparently, the pectinous materials in mango do not trap organic

Currently, there is no well-established molecular form of absorbed substances in the gastrointestinal tract, i.e. whether they are absorbed intact or as metabolites. On the other hand, it is well known that lifestyle, behavior, diets, and basal metabolism of the subjects are more important affecting factors than age, gender, body weight, and plasma volume (Manners et al., 2003) in bioavailability determination. Therefore, standardized experimental conditions controlling such critical factors of absorption *in vivo* and *in vitro* is a must despite

The main absorptive tissue is the small intestine. In human 81% of the total intestinal lengths is by small intestine and 19% is large intestine. The stretched length of jejunum is around 30.78% of the intestinal lengths. The transit time along human small and large intestine is 3-4 h and 2-4 d, respectively (Vermeulen, 2009). Principles of the intestinal absorptive structures are depicted in Figure 2. A single enterocyte has microvilli and each microvillus has glycocalyx. Such structure considerably increases contact surface areas with luminal contents. Each microvillus also contains a complex structure providing various facilities for uptake/influx and efflux molecules, signalling ports, cytoplasm, and lipid matrix. The glycocalyx and microvilli are the areas where the human body depends on for collecting

Each enterocyte attaches onto adjacent cells through tight, adherence and gap junctions. Cellular transport from intestinal lumen to portal blood vessel occurs in two ways: paracellular and transcellular. The paracellular entrances for hazards are tightly controlled by those three types of junctions. Molecular weight cut off limits the hazardous substances crossing through both enterocyte lining cells and tight junction. The enterocytes collect compounds that reach apical side. The compounds then traverse into basolateral side where

nutriomes but rejecting hazardous compounds including microbes.

they end up in capillary vessel for circulation into the whole human body.

intestinal digestion where the model is without addition of oil (Epriliati, 2008).

acids (Epriliati, 2008).

**3. Absorption of phytochemicals** 

individual human variability.

**3.1 Absorptive tissue structures** 

Fig. 2. Absorption tissue: epithelial cell (left) and intestinal brush border (right)

Other barrier in intestine is mucus. Most absorptive tissues comprise of epithelial cells which protect the human body from hazardous components in ingested foods. The epithelial cells are critical gate for human body. Due to its critical roles, the epithelial cells along gastrointestinal tract are covered with mucus secreted by goblet cells making an unstirred water layers so that the coarse particles are not abrasive towards the epithelial cells.

#### **3.2 Transport mechanisms**

Epithelial cell membrane is an important part of transport facility. It controls and selectively takes up molecules required for living or treats hazardous molecules. The fate of its work is not well understood despite studies finding many facilities and signaling processes available for regulating transport molecules. The transport modes include passive and active mechanisms. Passive transport is transcellular or paracellular transports and cynocitosis. Active transports are characterized by the use of protein transporters: channels/pump, binding protein transport, and formation of vehicles that is mainly emulsion system incorporating oil soluble compounds, such as chylomicrons. The transporter is able to promote transmembrane movement without hydrolyzing ATP (Johnson, 2001). They are categorized as uniporter (single compound moving down along the electrochemical gradients) and symporter (two molecules at the same time moving in one direction) or antiporter (two molecules at the same time moving in opposite directions).

Several transporters act as cellular efflux port for flavonoids: *P-*glycoproteins, multidrug resistance associated proteins, and breast cancer resistance protein (Johnson, 2001). They generally have loose substrate specificity and also involve in regulating non-nutritional compounds. Several findings point out glucose transporters which allow quercetin glucoside to be absorbed intact besides its aglycone forms. They are SGLT14 (SLC5A15),

<sup>4</sup> Sodium dependent glucose transporter

Bioavailability of Phytochemicals 409

components of diets immediately after diffusing out from the food particles. The emulsion acts as vehicles moving along the intestinal lumen. Contacting with the epithelial brush border and unstirred water layer on the top of the epithelial lining cells, rearrangement of vehicle emulsion take place which eventually releases the lipid soluble compounds into the cells. These compounds then traverse across the epithelium cells and end up in the lymph circulation. Nevertheless, many studies show losses material balances during transport across the epithelial lining gut. Moreover, the proportion of traversing compounds which are both water soluble and lipid soluble nutriomes that survive intact entering the circulatory system is not well understood. Similarly, proportion of metabolized nutriome

**Phenolics.** Many studies support evidences that aglycone polyphenols are not only absorbed in the small intestine but also in the large intestine after microbial digestions. The

In the human small intestine and stomach, 95% of caffeic acid is absorbed while 62% of its ester form (called chlorogenic acid) is reduced. All are absorbed intact, except chlorogenic acid which mostly enters the human body from colon. Proanthocyanidins are pH sensitive thus it is likely to be broken down in stomach so that they may be readily absorbable. Meanwhile, catechin and epicatechin is poorly absorbed in the small intestine (≤20%) in a dose dependent manner. However, enterocytes can act differently; for instance, in intestinal jejunum it metabolizes flavanols into glucuronidated conjugates whereas in ileum it translocates flavanols intact. In the large bowel, most microflora metabolize flavonols and proanthocyanidins; for example, catechin metabolites include (-)-5[3'4'5'-trihydroxyphenyl]- -valerolactone; (-)-5[3'4'-dihydroxy phenyl]--valerolactone; 3-hydroxyphenylpropionic

With a new bilitranslocase transport mechanism it is likely that the determinations of bioavailability of phytochemicals are necessarily being revised. pH and temperature are necessarily taken into account in order to avoid underestimation/overestimation regarding its stability. Several issues include absorption of quercetin and anthocyanin, glycone and aglycone forms, and conjugation/glucuronidation of phytochemicals as well as the presence of alcohol. Quercetin absorption varies from one food source to another. Its absorption from wine is enhanced by alcohol presence. Resident time of quercetin expressed as half-life clearance is 11-28 h (Manach et al., 2005). A very low level of intact anthocyanins is found in plasma after administration of anthocyanins. Resveratrol is absorbed well in the small intestine and being glucuronidated. Consumption of red wine would provide a good level of resveratrol bioavailability can be questioned whether this is because of alcohol presence. Flavonoid is one of the group molecules with molecular weights >500 Da and has bioavailability level of <1%. Such molecules are unlikely to be transported through passive diffusion pathways. Further study found that influx membrane transporters cannot recognize flavonoid (signalling) whereas the efflux transporters do. Consequently, potential of flavonoids to be expelled is higher than that of influxed into the cells (Johnson, 2001).

In determination of phytochemical bioavailability, researchers should not limit their detection for ingested molecular forms only based on reported presence in the diets. It has been proven that at plasma levels many phytochemicals have been conserved by digestion and by hepatic activity. Fitting the mass balance of ingested phytochemical is challenging.

used up by the epithelial cells as energy source is unclear.

steps may involve hydrolysis of sugar moiety by intestinal enzymes.

acid; 3-hydroxybenzoic acid; or 3-hydroxyhippuric acid (Ginjom, 2009).

GLUT26 (SLC2A2), MCT7, OAT8, and OATP9. However, results from the *in vitro* cell culturebased experiments are contradictive. Recently, bilitranslocase transport was introduced (Passamonti et al., 2009), that suggests the existence of a uniporter for flavonoids which is assumed to be an analogue of phthalien due to their similar molecular structures. The bilitranslocase is distributed in goblet and parietal cells in gastric, in apical jejunum of rat intestine, and in basolateral site of proximal tubular cell in kidney. However, further research is required for better understanding.

Briefly, bilitranslocase description indicates that target molecules interact with bilitranslocase through hydrogen bonds (hydrophilic properties of the active site); thus, nonionic inhibitors would not interact with it electrostatically. However, a negative charge is found to play an important role for electrogenic movement along the translocation pathway. These are observed through structural analysis (Passamonti et al., 2009). Similarly, the competitiveness of the target compounds can be explained by characteristics of C4 in C-ring flavon building block where the target molecules are inactively competitive if the sugar moiety is in nonplanar position; otherwise, the molecules will be actively competitive. Taking an example of quercetin-3-glucoside, the C4 carbonyl forces 3-glucosyl moiety is perpendicular to the plane of flavonol aglycone resulting in a non-planar molecule. In contrast, its best competitor cyanidin-3-glucoside has a co-planar sugar moiety to the aglycone. Similarly, comparison of myricetin and delphinidin behave noncompetitively and competitively, respectively. Consequently, noncompetitor and competitor can be in one target molecule if its molecular structure has quinoidal, anionic tautomer, and neutral phenolics. They simultaneously can bind both the noncompetitive and competitive sites of bilitranslocase. Inconclusive role of bilitranslocation is compounded further by noninhibitor responses of other flavonoids, for instance flavonol (+)-catechin and isoflavones─genistin, genistein, daidzin, daidzein, and puerarin (Passamonti et al., 2009).

Bilitranslocase sheds a light for phenolics bioavailability and transport studies. The most striking relevance is that phenolics bioavailabilty is not delivered to blood circulation; instead, it is delivered through lymphatic system. This corrects understanding of hydrophobic nature of phytochemicals. The presence of bilitranslocase also clarifies disappearance of flavonoids in apical side but no basolateral level obtained in Ginjom (2009) and Epriliati (2008), despite GLUT2, MRP10, organic cation, and amino acid/peptide transporters are available in basolateral domain. This specificity is promising for explaining the diverse bioavailability studies of phytochemicals.

#### **3.3 Phytochemical absorption**

There are two groups of nutriome: water soluble and less polar-solvent soluble. The water soluble components diffuse out from the food particles into chyme, traverse across the epithelial lining cells along the brush borders, and enter the portal blood circulation. On the other hand, the lipid soluble nutriome will be emulsified by bile salts and lipidic

<sup>5</sup> Solute carrier

<sup>6</sup> Glucose transporter

<sup>7</sup> Monocarboxylate transporter

<sup>8</sup> Organic anion transporter

<sup>9</sup> Organic anion transporting polypeptide

<sup>10</sup> Multidrug resistance-associated protein

GLUT26 (SLC2A2), MCT7, OAT8, and OATP9. However, results from the *in vitro* cell culturebased experiments are contradictive. Recently, bilitranslocase transport was introduced (Passamonti et al., 2009), that suggests the existence of a uniporter for flavonoids which is assumed to be an analogue of phthalien due to their similar molecular structures. The bilitranslocase is distributed in goblet and parietal cells in gastric, in apical jejunum of rat intestine, and in basolateral site of proximal tubular cell in kidney. However, further

Briefly, bilitranslocase description indicates that target molecules interact with bilitranslocase through hydrogen bonds (hydrophilic properties of the active site); thus, nonionic inhibitors would not interact with it electrostatically. However, a negative charge is found to play an important role for electrogenic movement along the translocation pathway. These are observed through structural analysis (Passamonti et al., 2009). Similarly, the competitiveness of the target compounds can be explained by characteristics of C4 in C-ring flavon building block where the target molecules are inactively competitive if the sugar moiety is in nonplanar position; otherwise, the molecules will be actively competitive. Taking an example of quercetin-3-glucoside, the C4 carbonyl forces 3-glucosyl moiety is perpendicular to the plane of flavonol aglycone resulting in a non-planar molecule. In contrast, its best competitor cyanidin-3-glucoside has a co-planar sugar moiety to the aglycone. Similarly, comparison of myricetin and delphinidin behave noncompetitively and competitively, respectively. Consequently, noncompetitor and competitor can be in one target molecule if its molecular structure has quinoidal, anionic tautomer, and neutral phenolics. They simultaneously can bind both the noncompetitive and competitive sites of bilitranslocase. Inconclusive role of bilitranslocation is compounded further by noninhibitor responses of other flavonoids, for instance flavonol (+)-catechin and isoflavones─genistin, genistein, daidzin, daidzein, and

Bilitranslocase sheds a light for phenolics bioavailability and transport studies. The most striking relevance is that phenolics bioavailabilty is not delivered to blood circulation; instead, it is delivered through lymphatic system. This corrects understanding of hydrophobic nature of phytochemicals. The presence of bilitranslocase also clarifies disappearance of flavonoids in apical side but no basolateral level obtained in Ginjom (2009) and Epriliati (2008), despite GLUT2, MRP10, organic cation, and amino acid/peptide transporters are available in basolateral domain. This specificity is promising for explaining the diverse bioavailability

There are two groups of nutriome: water soluble and less polar-solvent soluble. The water soluble components diffuse out from the food particles into chyme, traverse across the epithelial lining cells along the brush borders, and enter the portal blood circulation. On the other hand, the lipid soluble nutriome will be emulsified by bile salts and lipidic

research is required for better understanding.

puerarin (Passamonti et al., 2009).

studies of phytochemicals.

7 Monocarboxylate transporter 8 Organic anion transporter

9 Organic anion transporting polypeptide 10 Multidrug resistance-associated protein

5 Solute carrier 6 Glucose transporter

**3.3 Phytochemical absorption** 

components of diets immediately after diffusing out from the food particles. The emulsion acts as vehicles moving along the intestinal lumen. Contacting with the epithelial brush border and unstirred water layer on the top of the epithelial lining cells, rearrangement of vehicle emulsion take place which eventually releases the lipid soluble compounds into the cells. These compounds then traverse across the epithelium cells and end up in the lymph circulation. Nevertheless, many studies show losses material balances during transport across the epithelial lining gut. Moreover, the proportion of traversing compounds which are both water soluble and lipid soluble nutriomes that survive intact entering the circulatory system is not well understood. Similarly, proportion of metabolized nutriome used up by the epithelial cells as energy source is unclear.

**Phenolics.** Many studies support evidences that aglycone polyphenols are not only absorbed in the small intestine but also in the large intestine after microbial digestions. The steps may involve hydrolysis of sugar moiety by intestinal enzymes.

In the human small intestine and stomach, 95% of caffeic acid is absorbed while 62% of its ester form (called chlorogenic acid) is reduced. All are absorbed intact, except chlorogenic acid which mostly enters the human body from colon. Proanthocyanidins are pH sensitive thus it is likely to be broken down in stomach so that they may be readily absorbable. Meanwhile, catechin and epicatechin is poorly absorbed in the small intestine (≤20%) in a dose dependent manner. However, enterocytes can act differently; for instance, in intestinal jejunum it metabolizes flavanols into glucuronidated conjugates whereas in ileum it translocates flavanols intact. In the large bowel, most microflora metabolize flavonols and proanthocyanidins; for example, catechin metabolites include (-)-5[3'4'5'-trihydroxyphenyl]- -valerolactone; (-)-5[3'4'-dihydroxy phenyl]--valerolactone; 3-hydroxyphenylpropionic acid; 3-hydroxybenzoic acid; or 3-hydroxyhippuric acid (Ginjom, 2009).

With a new bilitranslocase transport mechanism it is likely that the determinations of bioavailability of phytochemicals are necessarily being revised. pH and temperature are necessarily taken into account in order to avoid underestimation/overestimation regarding its stability. Several issues include absorption of quercetin and anthocyanin, glycone and aglycone forms, and conjugation/glucuronidation of phytochemicals as well as the presence of alcohol. Quercetin absorption varies from one food source to another. Its absorption from wine is enhanced by alcohol presence. Resident time of quercetin expressed as half-life clearance is 11-28 h (Manach et al., 2005). A very low level of intact anthocyanins is found in plasma after administration of anthocyanins. Resveratrol is absorbed well in the small intestine and being glucuronidated. Consumption of red wine would provide a good level of resveratrol bioavailability can be questioned whether this is because of alcohol presence.

Flavonoid is one of the group molecules with molecular weights >500 Da and has bioavailability level of <1%. Such molecules are unlikely to be transported through passive diffusion pathways. Further study found that influx membrane transporters cannot recognize flavonoid (signalling) whereas the efflux transporters do. Consequently, potential of flavonoids to be expelled is higher than that of influxed into the cells (Johnson, 2001).

In determination of phytochemical bioavailability, researchers should not limit their detection for ingested molecular forms only based on reported presence in the diets. It has been proven that at plasma levels many phytochemicals have been conserved by digestion and by hepatic activity. Fitting the mass balance of ingested phytochemical is challenging.

Bioavailability of Phytochemicals 411

epimer limonin at C17. It is clear that the human body does not necessarily control levels of plasma limonin and its absorption in the gut whereas limonin glucoside enters blood plasma through GLUT pathways, but it is necessarily hydrolyzed and lactonized. If it is absorbed through GLUT pathways without being metabolized, it should enter blood plasma at the same rate with sugars. The problem is that variation of individuals cannot be ignored since by the time it shows accumulation or decrease of detected limonin levels. The consequence of this accumulation is also not understood. Overall, limonin aglycone form is apparently safer than that of limonin glucosides; therefore, the high level of limonin glucoside form is controlled. Based on transit time of chyme in the gut, 6 h will be long enough to bring the chyme completely passing the small intestine. Therefore, lower level of ingestion results in limonin absorption after microbial glucoside hydrolisis in bowel. These

**Interactions involve in various phytochemicals and nutrient transports.** Since phytochemical are generally reactive molecules they can interact with various compounds in the chyme and this will affect phytochemical bioavailability and vice versa. Phytochemicals that interact with vitamin E include lignans, curcumin, anthocyanins, phenolic acid and catechin, as well as cereal alkylresorcinol (Frank, 2004). Interaction of vitamin E and plant lignans significantly increases vitamin E bioavailability as much as 900% in plasma level; 1,350% in liver; and 1,556% in lung using rat model. On the other hand, using human and rat model tocopherol--hydrolase activity is effectively inhibited by sesamin11. Sesamin also reduces degradation of -tocopherol and urinary secretion so that it increases -tocopherol level in plasma. However, not all lignans show similar effects. For instance, sesamin or flaxseed lignan secoisolariciresinol diglucoside, either its monomer or oligomers decrease tocopherol by 50%. Experiment using rat model indicates that flaxseed lignan decreases and tocopherol availability in a dose dependent manner. However, it presence increases lipid peroxidation. The majority of flaxseed lignan is converted into

In contrast, the effect of curcumin studied using rat model on tocopherol bioavailability is less apparent when compared to flaxseed or sesame lignans where it is only detected in lung. In fact, curcumin is absorbed, metabolized, and secreted as glucuronidated metabolites. Similarly, the effect of anthocyanins on tocopherol bioavailability is neglected. Using the same rat model, it is found that caffeic acid increases -tocopherol in the liver and it is also converted into its metabolites 5-caffeoylquinic acid which in turn increases the levels of -tocopherol in lung. However, when ingested as 5-caffeoylquinic acid, it is metabolized into caffeic acid and quinic acid; and caffeic acid is absorbed and found in plasma both in human and rat models. In contrast, ferulic acid is found to form complexes with albumin in blood plasma and LDL; hence, it does not affect tocopherol bioavailability. Interestingly, (+)-catechin and (-)-catechin isomers similarly improve -tocopherol bioavailability in plasma and liver (Frank, 2004). There is a slight difference regarding their effects on -tocopherol where 2R,3R-isomer(-)-epicatechin enhances -tocopherol bioavailability whereas 2R,3R-(+)-catechin has no effect on it. The differences between and tocopherol is estimated due to (i) antioxidant activity of catechin isomers on a tocopherol and (ii) different effects of the isomers on cytochrome P450 enzymes such as

speculations remain to be elucidated.

11 Lignan exists in sesamin

mammalian lignan allowing them to be absorbed (Frank, 2004).

For instance, total metabolites in plasma levels are found reaching 4 mmol/L when intake is 50 mg aglycone equivalent whereas urinary excretion levels are 0.3-43% of the ingested doses, depending on polyphenol types. Flavonol such as quercetin in broccoli is rarely found as free quercetin. Human who consume 21-100 mg/d of quercetin show exclusive form of methyl, sulphate, or glucuronic acid conjugates totally amounted to maximum 1-5 mol/L aglycone equivalent (Moreno et al., 2006). However, several phytochemicals are found intact, especially those which are absorbed easily. The ranks of phytochemical absorption is gallic acid and isoflavones > catechins and flavanones, quercetin glucoside > proanthocyanidins, galloylated tea catechins, and anthocyanins (Moreno et al., 2006).

**Carotenoids.** Carotenoids of mango, tomato, and papaya in caco-2 absorption model are not detected (Epriliati, 2008) in spite of *in vivo* data indicates that carotenoid plasma level increase after consumption of carotenoid-rich foods. Processing altered matrices of ingested food system and more likely degraded carotenoids which caused variation in bioavailability of carotenoids. A comparative study of organic and inorganic carrot found that apparently organic farming practices do not affect bioavailability of carotenoids in carrot consumption. Ingestion of total carotenoids of 24.3±1.4 mg organic carrot and 23.2±2.5 mg inorganic carrot results in 700 nmol/L -carotene and 350 nmol/L -carotene, and 150 nmol/L lutein after 2 weeks interventions (Stracke et al., 2009).

**Organic acids.** Organic acid provides organic anion important for metal binding and counteracting acidosis as well as preventing chronic diseases (Sabboh et al., 2011). Particular organic acids are apparently absorbed into plasma. Most organic acids in tomato, mango, and papaya products are absorbed in *in vitro* caco-2 model but they are not found in the basolateral sides (Epriliati, 2008). On the other hand, citric acid and oxalic from banana and sweet potato are consistently found to be absorbed and translocated into basolateral sides in *in vitro* caco-2 model (Sabboh et al., 2011). The absorbed organic acids are much lower compared to the original levels in food materials, thus, the retained organic acids in particles may be useful for controlling pH in colonic fermentation because selection of microbes in the large bowel is important.

**Miscellaneous.** Phytosterol could be absorbed at very low level using the same transport facilities for cholesterol due to structural similarities. It needs emulsion vehicle to diffuse in the aqueous lumen system, crossing the lipid membrane, and, finally, entering circulatory system. This requires evaluation because absorption is closely connected to which mechanisms are involved in health function, which is still debatable (Kang et al., 2010).

Triterpenoids citrus limonin glucoside is one of metabolites in citrus plant. Generally, it is water soluble; yet few aglycone forms of liminoids are insoluble. According to Manners et al. (2003) liminoid metabolites are found in human after ingestion of citrus juice containing limonin glucoside which may undergo epimerization from limonin glucoside to epilimonin (m/z 471.2). This may be from reaction pathways of hydrolization of glucoside moiety followed by lactonization. Although low level of limonin is ingested, it is eventually available in plasma after 6 h (Manners et al., 2003). During the first 3 h the higher ingestion level of limonin results in more significant changes in plasma epilimonin levels, regardless of age and gender. However, after 6 h, all volunteers show increased levels of plasma epilimonins at any ingestion levels of 0.25 g/200 mL–2 g/200 mL that is equivalent to 7 glasses of natural juices. The authors conclude that ingestion of limonin glucoside produces speculations remain to be elucidated.

410 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

For instance, total metabolites in plasma levels are found reaching 4 mmol/L when intake is 50 mg aglycone equivalent whereas urinary excretion levels are 0.3-43% of the ingested doses, depending on polyphenol types. Flavonol such as quercetin in broccoli is rarely found as free quercetin. Human who consume 21-100 mg/d of quercetin show exclusive form of methyl, sulphate, or glucuronic acid conjugates totally amounted to maximum 1-5 mol/L aglycone equivalent (Moreno et al., 2006). However, several phytochemicals are found intact, especially those which are absorbed easily. The ranks of phytochemical absorption is gallic acid and isoflavones > catechins and flavanones, quercetin glucoside >

proanthocyanidins, galloylated tea catechins, and anthocyanins (Moreno et al., 2006).

weeks interventions (Stracke et al., 2009).

the large bowel is important.

**Carotenoids.** Carotenoids of mango, tomato, and papaya in caco-2 absorption model are not detected (Epriliati, 2008) in spite of *in vivo* data indicates that carotenoid plasma level increase after consumption of carotenoid-rich foods. Processing altered matrices of ingested food system and more likely degraded carotenoids which caused variation in bioavailability of carotenoids. A comparative study of organic and inorganic carrot found that apparently organic farming practices do not affect bioavailability of carotenoids in carrot consumption. Ingestion of total carotenoids of 24.3±1.4 mg organic carrot and 23.2±2.5 mg inorganic carrot results in 700 nmol/L -carotene and 350 nmol/L -carotene, and 150 nmol/L lutein after 2

**Organic acids.** Organic acid provides organic anion important for metal binding and counteracting acidosis as well as preventing chronic diseases (Sabboh et al., 2011). Particular organic acids are apparently absorbed into plasma. Most organic acids in tomato, mango, and papaya products are absorbed in *in vitro* caco-2 model but they are not found in the basolateral sides (Epriliati, 2008). On the other hand, citric acid and oxalic from banana and sweet potato are consistently found to be absorbed and translocated into basolateral sides in *in vitro* caco-2 model (Sabboh et al., 2011). The absorbed organic acids are much lower compared to the original levels in food materials, thus, the retained organic acids in particles may be useful for controlling pH in colonic fermentation because selection of microbes in

**Miscellaneous.** Phytosterol could be absorbed at very low level using the same transport facilities for cholesterol due to structural similarities. It needs emulsion vehicle to diffuse in the aqueous lumen system, crossing the lipid membrane, and, finally, entering circulatory system. This requires evaluation because absorption is closely connected to which mechanisms are involved in health function, which is still debatable (Kang et al., 2010).

Triterpenoids citrus limonin glucoside is one of metabolites in citrus plant. Generally, it is water soluble; yet few aglycone forms of liminoids are insoluble. According to Manners et al. (2003) liminoid metabolites are found in human after ingestion of citrus juice containing limonin glucoside which may undergo epimerization from limonin glucoside to epilimonin (m/z 471.2). This may be from reaction pathways of hydrolization of glucoside moiety followed by lactonization. Although low level of limonin is ingested, it is eventually available in plasma after 6 h (Manners et al., 2003). During the first 3 h the higher ingestion level of limonin results in more significant changes in plasma epilimonin levels, regardless of age and gender. However, after 6 h, all volunteers show increased levels of plasma epilimonins at any ingestion levels of 0.25 g/200 mL–2 g/200 mL that is equivalent to 7 glasses of natural juices. The authors conclude that ingestion of limonin glucoside produces epimer limonin at C17. It is clear that the human body does not necessarily control levels of plasma limonin and its absorption in the gut whereas limonin glucoside enters blood plasma through GLUT pathways, but it is necessarily hydrolyzed and lactonized. If it is absorbed through GLUT pathways without being metabolized, it should enter blood plasma at the same rate with sugars. The problem is that variation of individuals cannot be ignored since by the time it shows accumulation or decrease of detected limonin levels. The consequence of this accumulation is also not understood. Overall, limonin aglycone form is apparently safer than that of limonin glucosides; therefore, the high level of limonin glucoside form is controlled. Based on transit time of chyme in the gut, 6 h will be long enough to bring the chyme completely passing the small intestine. Therefore, lower level of ingestion results in limonin absorption after microbial glucoside hydrolisis in bowel. These

**Interactions involve in various phytochemicals and nutrient transports.** Since phytochemical are generally reactive molecules they can interact with various compounds in the chyme and this will affect phytochemical bioavailability and vice versa. Phytochemicals that interact with vitamin E include lignans, curcumin, anthocyanins, phenolic acid and catechin, as well as cereal alkylresorcinol (Frank, 2004). Interaction of vitamin E and plant lignans significantly increases vitamin E bioavailability as much as 900% in plasma level; 1,350% in liver; and 1,556% in lung using rat model. On the other hand, using human and rat model tocopherol--hydrolase activity is effectively inhibited by sesamin11. Sesamin also reduces degradation of -tocopherol and urinary secretion so that it increases -tocopherol level in plasma. However, not all lignans show similar effects. For instance, sesamin or flaxseed lignan secoisolariciresinol diglucoside, either its monomer or oligomers decrease tocopherol by 50%. Experiment using rat model indicates that flaxseed lignan decreases and tocopherol availability in a dose dependent manner. However, it presence increases lipid peroxidation. The majority of flaxseed lignan is converted into mammalian lignan allowing them to be absorbed (Frank, 2004).

In contrast, the effect of curcumin studied using rat model on tocopherol bioavailability is less apparent when compared to flaxseed or sesame lignans where it is only detected in lung. In fact, curcumin is absorbed, metabolized, and secreted as glucuronidated metabolites. Similarly, the effect of anthocyanins on tocopherol bioavailability is neglected. Using the same rat model, it is found that caffeic acid increases -tocopherol in the liver and it is also converted into its metabolites 5-caffeoylquinic acid which in turn increases the levels of -tocopherol in lung. However, when ingested as 5-caffeoylquinic acid, it is metabolized into caffeic acid and quinic acid; and caffeic acid is absorbed and found in plasma both in human and rat models. In contrast, ferulic acid is found to form complexes with albumin in blood plasma and LDL; hence, it does not affect tocopherol bioavailability. Interestingly, (+)-catechin and (-)-catechin isomers similarly improve -tocopherol bioavailability in plasma and liver (Frank, 2004). There is a slight difference regarding their effects on -tocopherol where 2R,3R-isomer(-)-epicatechin enhances -tocopherol bioavailability whereas 2R,3R-(+)-catechin has no effect on it. The differences between and tocopherol is estimated due to (i) antioxidant activity of catechin isomers on a tocopherol and (ii) different effects of the isomers on cytochrome P450 enzymes such as

<sup>11</sup> Lignan exists in sesamin

Bioavailability of Phytochemicals 413

properties of the target molecules. Molecular forms of phenolics such as glycone or aglycone definitely make diverse variations on bioavailability levels. In addition to these factors, individual gastrointestinal tract of the human also affects bioavailability. Gastrointestinal pH, level of secretions, microbiota, and age have been established as crucial factors affecting digestion and absorption of phytochemicals. Equally, the role of interactions amongst food components and their interactions with gastrointestinal secretions contribute significant

Tannin-protein interactions occur starting from mouth and food systems. The interaction depends on size, conformation, and charges of proteins; molecular size, flexibility, and water solubility of phenolics; and environmental conditions such as pH. Proteins with higher molecular weights or loose conformational structures or rich in proline/hydrophobic amino acids, increase its potential to be precipitated by tannin. On the other hand, flavonols (three orthohydroxyl groups on the B-ring) has higher affinity to protein compared to those with two orthohydroxyl groups. Similarly, the affinity increases with increasing galloylation degrees. The order of flavonols affinity is (-)-epigallocatechin gallate >(-)-gallocatechin gallate >(-)-epicatechin gallate >(-)epigallocatechin or (-)-epicatechin or (+)-catechin (Ginjom, 2009). Interestingly, tannin also plays pivotal roles in its capability to act as health

Effects of *in vitro* digestion on wine phytochemicals are significant during pancreatic digestion step, especially for nonpolar compounds. Therefore, water solubility level is crucial in generating an appropriate *in vitro* digestion model. In contrast, acid does not

*In vitro* model for absorption using a monolayer cell culture can help bioavailability determinations with human surrogates; however, the results should be carefully considered. More importantly, the results cannot be liberately generalized for human system biology. Yun et al. (2004) propose a constant to equalize *in vitro* measurement using caco-2 monolayer with human *in vivo* measurement for iron. Furthermore, there are critical factors in utilizing such *in vitro* model for a bioavailability study that should be carefully considered. For instance, the original composition of digest containing bile salts decreases TEER (transepithelial electrical resistance) indicating serious detrimental effect on the cell monolayer integrity. In addition, alcohol content in wine also affects the monolayer integrity so that alcohol removal is required although alcohol enhances phenolic absorption. This is unrealistic wine samples. Furthermore, the delicate properties of the monolayers may result from lacking of mucus/unstirred water layer protecting the epithelium. The development of

an appropriate and standardized *in vitro* model needed to be persued continuously.

Kinetics study of phytochemicals is scarce. Several experiments are reviewed below to

**Quercetin.** Quercetin is more likely to be absorbed quickly in the human gut after ingestion, e.g. quercetin-3-glucoside from blackcurrant juice is 4 h or pure quercetin glucoside capsule is ca 30 min. Quercetin-3-rutinoside takes longer time to reach peak plasma levels compared

effects in determining bioavailability of phytochemicals.

**4.1** *In vitro* **model of digestion and transport** 

**4.2 Kinetics of phytochemical bioavailability** 

understand phytochemical kinetics after ingestion.

significantly affect the phytocemical components in wine.

protective antioxidant.

CYP1A1, CYP1A2, CYP2B1, AND CYP3A4 as well as CYP4F2. Alkylresorcinols in outer layer of wheat and rye is also absorbed and metabolized. Its presence improves -tocopherol in liver and lung but not -tocopherol observed in rat. The various effects on tocopherol isomers are unclear although molecular differences of alkylresorcinol and tocopherol is known.

Addition of citric acid affects iron uptake. In reverse, citrate reduction improves iron bioavailability (Glahn et al., 1998). Iron bioavailability is also influenced by purple and brown pigments in rice; apparently, the pigment behaves similarly to tannin, phenolic, anthocyanin, or phytic acid (Glahn et al., 2002).

Interactions amongst carotenoids (Kostic et al., 1995; van den Berg, 1999) show that carotene inhibits lutein uptake. These interactions perhaps also occurred at the micelle formation and transport levels, or their combination (van het Hof et al., 2000). Similarly, carotene shows competitive inhibition to lycopene transport (Johnson, 1998). Meanwhile, carotenoids can interact with proteins and pectin decreasing absorption the carotenoids (Williams, 1998). Moreover, the cathecol structure in the ring of flavonols and 2,6-di-*tert*butyl-4-mehtylphenol inhibits the dioxygenase enzyme and conversion of carotene (Nagao et al., 2000; Nagao, 2004). On the other hand, metabolites of bio-oxidation may act as pro-oxidants in the body (Nagao, 2004). Konishi found that tea phenolics inhibit other dietary phenolics (Konishi et al., 2003).

Among several fruits and vegetables, papaya and tomato consumption are found to be benefecial in hypolipidemic diet components, with similar mechanisms observed during *in vivo* experiments using rats (Kumar et al., 1997). Here, soluble and insoluble fibers can bind bile acids, thus influencing micelle formation and absorption of lipophilic substances by the brush border. Lignin and guar gum are apparently better bile binders than cellulose, which is relatively inert. Interaction also occurs between fiber and intestinal mucin, which probably alters absorption and nutrient diffusion from bulk lumen content (Vahouny & Cassidy, 1985). Moreover, fiber bound health promoters include lycopene in tomato peel (Awad et al., 2002) and antioxidants in mango peel (Larrauri et al., 1996), where the antioxidants found in mango peel, pulp and seed include gallotannins (Berardini et al., 2004). Consumption of fiber-rich food products can reduce minerals and vitamin (Schneeman & Gallaher, 1985). Generally, those authors agree that pectin and cellulose play important roles, especially in reducing the activity of digestive enzymes, or hormones such as insulin (Schneeman & Gallaher, 1985; Vahouny & Cassidy, 1985).

#### **4. Kinetics simulation of phytochemical bioavailability**

Kinetics is a study observing changes of the phytochemicals after ingestion including elimination period. To understand kinetics of phytochemicals after ingestion, kinetics simulation is frequently carried out. The limitations of simulations should be acknowledged in interpreting the results. Moreover, bioavailability closely relates to absorption and metabolism, yet there are limited understanding of bioavailability markers. Furthermore, the markers need validating, i.e. the molecular forms selected as bioavailability markers are necessarily those which actually cause health effects.

Affecting factors of phenolic bioavailability include matrix of food sources, processing condition during food preparations, chemical compositions, and molecular physicochemical

CYP1A1, CYP1A2, CYP2B1, AND CYP3A4 as well as CYP4F2. Alkylresorcinols in outer layer of wheat and rye is also absorbed and metabolized. Its presence improves -tocopherol in liver and lung but not -tocopherol observed in rat. The various effects on tocopherol isomers are unclear although molecular differences of alkylresorcinol and tocopherol is

Addition of citric acid affects iron uptake. In reverse, citrate reduction improves iron bioavailability (Glahn et al., 1998). Iron bioavailability is also influenced by purple and brown pigments in rice; apparently, the pigment behaves similarly to tannin, phenolic,

Interactions amongst carotenoids (Kostic et al., 1995; van den Berg, 1999) show that carotene inhibits lutein uptake. These interactions perhaps also occurred at the micelle formation and transport levels, or their combination (van het Hof et al., 2000). Similarly, carotene shows competitive inhibition to lycopene transport (Johnson, 1998). Meanwhile, carotenoids can interact with proteins and pectin decreasing absorption the carotenoids (Williams, 1998). Moreover, the cathecol structure in the ring of flavonols and 2,6-di-*tert*butyl-4-mehtylphenol inhibits the dioxygenase enzyme and conversion of carotene (Nagao et al., 2000; Nagao, 2004). On the other hand, metabolites of bio-oxidation may act as pro-oxidants in the body (Nagao, 2004). Konishi found that tea phenolics inhibit other

Among several fruits and vegetables, papaya and tomato consumption are found to be benefecial in hypolipidemic diet components, with similar mechanisms observed during *in vivo* experiments using rats (Kumar et al., 1997). Here, soluble and insoluble fibers can bind bile acids, thus influencing micelle formation and absorption of lipophilic substances by the brush border. Lignin and guar gum are apparently better bile binders than cellulose, which is relatively inert. Interaction also occurs between fiber and intestinal mucin, which probably alters absorption and nutrient diffusion from bulk lumen content (Vahouny & Cassidy, 1985). Moreover, fiber bound health promoters include lycopene in tomato peel (Awad et al., 2002) and antioxidants in mango peel (Larrauri et al., 1996), where the antioxidants found in mango peel, pulp and seed include gallotannins (Berardini et al., 2004). Consumption of fiber-rich food products can reduce minerals and vitamin (Schneeman & Gallaher, 1985). Generally, those authors agree that pectin and cellulose play important roles, especially in reducing the activity of digestive enzymes, or hormones such

Kinetics is a study observing changes of the phytochemicals after ingestion including elimination period. To understand kinetics of phytochemicals after ingestion, kinetics simulation is frequently carried out. The limitations of simulations should be acknowledged in interpreting the results. Moreover, bioavailability closely relates to absorption and metabolism, yet there are limited understanding of bioavailability markers. Furthermore, the markers need validating, i.e. the molecular forms selected as bioavailability markers are

Affecting factors of phenolic bioavailability include matrix of food sources, processing condition during food preparations, chemical compositions, and molecular physicochemical

as insulin (Schneeman & Gallaher, 1985; Vahouny & Cassidy, 1985).

**4. Kinetics simulation of phytochemical bioavailability** 

necessarily those which actually cause health effects.

known.

anthocyanin, or phytic acid (Glahn et al., 2002).

dietary phenolics (Konishi et al., 2003).

properties of the target molecules. Molecular forms of phenolics such as glycone or aglycone definitely make diverse variations on bioavailability levels. In addition to these factors, individual gastrointestinal tract of the human also affects bioavailability. Gastrointestinal pH, level of secretions, microbiota, and age have been established as crucial factors affecting digestion and absorption of phytochemicals. Equally, the role of interactions amongst food components and their interactions with gastrointestinal secretions contribute significant effects in determining bioavailability of phytochemicals.

Tannin-protein interactions occur starting from mouth and food systems. The interaction depends on size, conformation, and charges of proteins; molecular size, flexibility, and water solubility of phenolics; and environmental conditions such as pH. Proteins with higher molecular weights or loose conformational structures or rich in proline/hydrophobic amino acids, increase its potential to be precipitated by tannin. On the other hand, flavonols (three orthohydroxyl groups on the B-ring) has higher affinity to protein compared to those with two orthohydroxyl groups. Similarly, the affinity increases with increasing galloylation degrees. The order of flavonols affinity is (-)-epigallocatechin gallate >(-)-gallocatechin gallate >(-)-epicatechin gallate >(-)epigallocatechin or (-)-epicatechin or (+)-catechin (Ginjom, 2009). Interestingly, tannin also plays pivotal roles in its capability to act as health protective antioxidant.

#### **4.1** *In vitro* **model of digestion and transport**

Effects of *in vitro* digestion on wine phytochemicals are significant during pancreatic digestion step, especially for nonpolar compounds. Therefore, water solubility level is crucial in generating an appropriate *in vitro* digestion model. In contrast, acid does not significantly affect the phytocemical components in wine.

*In vitro* model for absorption using a monolayer cell culture can help bioavailability determinations with human surrogates; however, the results should be carefully considered. More importantly, the results cannot be liberately generalized for human system biology. Yun et al. (2004) propose a constant to equalize *in vitro* measurement using caco-2 monolayer with human *in vivo* measurement for iron. Furthermore, there are critical factors in utilizing such *in vitro* model for a bioavailability study that should be carefully considered. For instance, the original composition of digest containing bile salts decreases TEER (transepithelial electrical resistance) indicating serious detrimental effect on the cell monolayer integrity. In addition, alcohol content in wine also affects the monolayer integrity so that alcohol removal is required although alcohol enhances phenolic absorption. This is unrealistic wine samples. Furthermore, the delicate properties of the monolayers may result from lacking of mucus/unstirred water layer protecting the epithelium. The development of an appropriate and standardized *in vitro* model needed to be persued continuously.

#### **4.2 Kinetics of phytochemical bioavailability**

Kinetics study of phytochemicals is scarce. Several experiments are reviewed below to understand phytochemical kinetics after ingestion.

**Quercetin.** Quercetin is more likely to be absorbed quickly in the human gut after ingestion, e.g. quercetin-3-glucoside from blackcurrant juice is 4 h or pure quercetin glucoside capsule is ca 30 min. Quercetin-3-rutinoside takes longer time to reach peak plasma levels compared

Bioavailability of Phytochemicals 415

conventional nutrition believes that phytic acid and phytate is not traversed across lipid

**Sulfur compounds**. Bioavailability of isothiocyanates is better than glucosinolate in the human gut. In spite of different cruciferous origins and types, isothiocyanate is always found in plasma and its metabolites in urine is consistently found as dicarbamate or mercapturic acid. It is important to note that not all glucosinolates behave similarly. Generally, heated or cooked glucosinolate is less bioavailable (1.8-43%) than raw (8.2-113); and it is quickly absorbed in the gut and quickly excreted in urine (24 h). The exceptions are from pak choi (butenyl and pentenyl isothiocyanates, 8%), garden cress (benzyl isothiocyanate, 14%), and water cress (phenylethyl isothicyanate, 50%) compared to 100% isothiocyanate. Critical factors of the study remain: (i) individual variations (different microflora in the bowel, metabolism, and chewing ability), (ii) natural cruciferous matrices so that strongly entrapped glycosinolate in the cells will be hardly released during chewing,

Vegetable consumption during lunchtime shows a general lag phase for excretion of mercapturic acid at 4 h after ingestion (Vermeulen, 2009). The sulfocompounds (isothiocyanate) in the body is conjugated. Raw vegetable consumption results in a fast excretion whereas cooked vegetable has longer resident time of conjugated form. Elimination for half-life of the compound is 2-4 h, which is longer than that of other study (1.8 h) (Ye et al.,

Food source of sulfur compounds is a determinant factor in absorption in addition to processing and physiological conditions. Sulforaphane content in raw and cooked broccoli is 9.92 and 61.4 mole/kg, respectively; and 37 and 3.4 % of them are recovered in urine in the form of sulforaphane mercapturic acids. On the other hand, 54% of benzyl isothicyanate from garden cress is found in urine but phenylethyl isothicyanate from watercress after chewing is 47%. When cooked watercress is administered, only 1.2-7.3% of glucosinolates is recovered; this is much lower than sulforaphane (17.2-77.7%). Monitoring dithiocarbamate in urine shows 12% recovery from boiled broccoli sprout. The recovery increased to 80% when the boiled broccoli sprout is treated with myrosinase. About 68% of allyl isothicyanate from mustard is excreted in urine as mercapturic acid while sinigrin is present at 15% and 37% from cooked and raw cabbage12, respectively (Vermeulen, 2009). Generally, the routes of metabolisms in the human body vary depending on the target molecules and food sources. Glucoraphanin and sulforaphane from cooked and raw broccoli peak for maximum 6 and 1.6 h, respectively (Vermeulen, 2009). The half-life clearance of sulforaphane in the human body from the aforementioned vegetables is 4.6 and 3.8 h, respectively. These are different from half-life time of mercapturic acid which is 2.4 and 2.6 h, respectively. Further investigations using human subjects show inconclusive results that particular polymorphism S-glutathione transferase (GST M1, T1 and P1) and N-acetyl transferase (NAT2) gene affect the variations (Vermeulen, 2009). It is important to view these

ka: intercept and slope with a residual method; k: natural log of plasma amounts plotted against time; ke: natural log of absolute excreted amounts vs time; area under curve (concentration vs time) extrapolated using trapezoid method; lag phase is determined from empiric curve obtained; base line is

2002), with excretion rate of 0.18-0.33 h-1 by assuming the first order reaction.

and (iii) types of glucosinolates (Vermeulen, 2009).

phenomena under the holistic affecting factors.

12 Data is calculated from : First order kinetics

not used

bilayer.

to the two previously mentioned, i.e. after 5-10 h. Short- and long- term studies show kinetics absorption of quercetin is quick and easy; and there are no interactions with other food components. Moreover, bioavailable quercetin can be obtained from normal diet regardless of whether it contains the berries or not. Therefore, it is proposed that fasting quercetin bioavailability is used as a biomarker of high fruit and vegetable intakes for all plant based foods (Erlund et al., 2006).

**Soyasaponin**. Soyasaponin has a very low bioavailability when investigated using *in vivo* experiments involving animals and human (Kang et al., 2010). However, it is also found that possible metabolites of soyasaponin are detected in *in vitro* and *in vivo* studies, although it is found several days after ingestion (Kang et al., 2010). The metabolites include soyasapogenol B, which is secreted into faeces in human *in vivo* experiments. However, the metabolism is more likely due to microbiota in the colon which is supported by *in vitro* data using fresh faecal microbiota. *In vitro* data show sequential metabolism of sapogenin by the microbiota as follows: soyasaponin I after 48 h incubation at 37 C, and it is converted into soyasaponin III after 24 h and disappeared at 48 h where the predicted final metabolite is soyasapogenol B. These sequential metabolisms take place through sugar hydrolysis which results in the formation of more hydrophobic metabolites and smaller molecules (Kang et al., 2010).

**Lignan**. Low lignan bioavailability is recovered in plasma in human after ingestion of lignans (Kang et al., 2010). It is interesting that lignan is easily absorbed into plasma after ingestion. The available information is for secoisolariciresinol diglucoside and its aglycone secoisolariciresinol and matairesinol. Based on the studies, at least 40% of ingested lignans are metabolized by intestinal bacteria and these metabolites can be detected in the plasma. Metabolites of lignans appearing in the human plasma after ingestion follows the sequences: (i) at 14.8±5.1 h enterodiol is maximally found in plasma, (ii) at 19.7±6.2 h enterolactone is maximally bioavailable, and (iii) at 8-10 h enterolignans is bioavailable. Resident time of lignan metabolites in plasma is 20.6±5.9 h for enterodiol and 35.8±10.6 h for enterolactone, respectively (Kang et al., 2010).

**Phytosterol**. Low phytosterol bioavailability is observed in human plasma after ingestion. Definite small amount (0.6-7.5%) of phytosterol is transported through gut epithelial cells *in vivo*. Chemically, phytosterol is similar to cholesterol; yet cholesterol is absorbed at much higher levels than phytosterol. This is because of side chain differences, i.e. ethyl/methyl group in C24 which increases hydrophobicity but reduces absorption; and the presence of <sup>5</sup> double bond. The similarity of absorption mechanism of phytosterol and cholesterol is that (i) they need to be in emulsion system and (ii) to be facilitated by Niemann-Pick C1 like 1 (NPC1L1) protein. Surprisingly, it is just recently acknowledged that many of bioactive compounds need to be in emulsion system to make them more bioavailable (Kang et al., 2010).

**Phytate**. Phytate bioavailability is low in human plasma levels after ingestion. Plasma myo- [inositol-2-H3(N)]hexakisphosphate in human after ingestion is dose-dependent and it only reaches 3-5 times higher than that of diet poor in myo-[inositol-2-H3(N)]hexakisphosphate. Further study using rat found that absorbed phytate is quickly distributed into tissues such as brain, kidneys, liver, and bone in its original dietary molecular forms. The highest level is in brain reaching 10 times compared to average of tissues (Kang et al., 2010). This is beyond

to the two previously mentioned, i.e. after 5-10 h. Short- and long- term studies show kinetics absorption of quercetin is quick and easy; and there are no interactions with other food components. Moreover, bioavailable quercetin can be obtained from normal diet regardless of whether it contains the berries or not. Therefore, it is proposed that fasting quercetin bioavailability is used as a biomarker of high fruit and vegetable intakes for all

**Soyasaponin**. Soyasaponin has a very low bioavailability when investigated using *in vivo* experiments involving animals and human (Kang et al., 2010). However, it is also found that possible metabolites of soyasaponin are detected in *in vitro* and *in vivo* studies, although it is found several days after ingestion (Kang et al., 2010). The metabolites include soyasapogenol B, which is secreted into faeces in human *in vivo* experiments. However, the metabolism is more likely due to microbiota in the colon which is supported by *in vitro* data using fresh faecal microbiota. *In vitro* data show sequential metabolism of sapogenin by the microbiota as follows: soyasaponin I after 48 h incubation at 37 C, and it is converted into soyasaponin III after 24 h and disappeared at 48 h where the predicted final metabolite is soyasapogenol B. These sequential metabolisms take place through sugar hydrolysis which results in the formation of more hydrophobic metabolites and smaller molecules (Kang et

**Lignan**. Low lignan bioavailability is recovered in plasma in human after ingestion of lignans (Kang et al., 2010). It is interesting that lignan is easily absorbed into plasma after ingestion. The available information is for secoisolariciresinol diglucoside and its aglycone secoisolariciresinol and matairesinol. Based on the studies, at least 40% of ingested lignans are metabolized by intestinal bacteria and these metabolites can be detected in the plasma. Metabolites of lignans appearing in the human plasma after ingestion follows the sequences: (i) at 14.8±5.1 h enterodiol is maximally found in plasma, (ii) at 19.7±6.2 h enterolactone is maximally bioavailable, and (iii) at 8-10 h enterolignans is bioavailable. Resident time of lignan metabolites in plasma is 20.6±5.9 h for enterodiol and 35.8±10.6 h for enterolactone,

**Phytosterol**. Low phytosterol bioavailability is observed in human plasma after ingestion. Definite small amount (0.6-7.5%) of phytosterol is transported through gut epithelial cells *in vivo*. Chemically, phytosterol is similar to cholesterol; yet cholesterol is absorbed at much higher levels than phytosterol. This is because of side chain differences, i.e. ethyl/methyl group in C24 which increases hydrophobicity but reduces absorption; and the presence of <sup>5</sup> double bond. The similarity of absorption mechanism of phytosterol and cholesterol is that (i) they need to be in emulsion system and (ii) to be facilitated by Niemann-Pick C1 like 1 (NPC1L1) protein. Surprisingly, it is just recently acknowledged that many of bioactive compounds need to be in emulsion system to make them more bioavailable (Kang et al.,

**Phytate**. Phytate bioavailability is low in human plasma levels after ingestion. Plasma myo- [inositol-2-H3(N)]hexakisphosphate in human after ingestion is dose-dependent and it only reaches 3-5 times higher than that of diet poor in myo-[inositol-2-H3(N)]hexakisphosphate. Further study using rat found that absorbed phytate is quickly distributed into tissues such as brain, kidneys, liver, and bone in its original dietary molecular forms. The highest level is in brain reaching 10 times compared to average of tissues (Kang et al., 2010). This is beyond

plant based foods (Erlund et al., 2006).

respectively (Kang et al., 2010).

al., 2010).

2010).

conventional nutrition believes that phytic acid and phytate is not traversed across lipid bilayer.

**Sulfur compounds**. Bioavailability of isothiocyanates is better than glucosinolate in the human gut. In spite of different cruciferous origins and types, isothiocyanate is always found in plasma and its metabolites in urine is consistently found as dicarbamate or mercapturic acid. It is important to note that not all glucosinolates behave similarly. Generally, heated or cooked glucosinolate is less bioavailable (1.8-43%) than raw (8.2-113); and it is quickly absorbed in the gut and quickly excreted in urine (24 h). The exceptions are from pak choi (butenyl and pentenyl isothiocyanates, 8%), garden cress (benzyl isothiocyanate, 14%), and water cress (phenylethyl isothicyanate, 50%) compared to 100% isothiocyanate. Critical factors of the study remain: (i) individual variations (different microflora in the bowel, metabolism, and chewing ability), (ii) natural cruciferous matrices so that strongly entrapped glycosinolate in the cells will be hardly released during chewing, and (iii) types of glucosinolates (Vermeulen, 2009).

Vegetable consumption during lunchtime shows a general lag phase for excretion of mercapturic acid at 4 h after ingestion (Vermeulen, 2009). The sulfocompounds (isothiocyanate) in the body is conjugated. Raw vegetable consumption results in a fast excretion whereas cooked vegetable has longer resident time of conjugated form. Elimination for half-life of the compound is 2-4 h, which is longer than that of other study (1.8 h) (Ye et al., 2002), with excretion rate of 0.18-0.33 h-1 by assuming the first order reaction.

Food source of sulfur compounds is a determinant factor in absorption in addition to processing and physiological conditions. Sulforaphane content in raw and cooked broccoli is 9.92 and 61.4 mole/kg, respectively; and 37 and 3.4 % of them are recovered in urine in the form of sulforaphane mercapturic acids. On the other hand, 54% of benzyl isothicyanate from garden cress is found in urine but phenylethyl isothicyanate from watercress after chewing is 47%. When cooked watercress is administered, only 1.2-7.3% of glucosinolates is recovered; this is much lower than sulforaphane (17.2-77.7%). Monitoring dithiocarbamate in urine shows 12% recovery from boiled broccoli sprout. The recovery increased to 80% when the boiled broccoli sprout is treated with myrosinase. About 68% of allyl isothicyanate from mustard is excreted in urine as mercapturic acid while sinigrin is present at 15% and 37% from cooked and raw cabbage12, respectively (Vermeulen, 2009). Generally, the routes of metabolisms in the human body vary depending on the target molecules and food sources. Glucoraphanin and sulforaphane from cooked and raw broccoli peak for maximum 6 and 1.6 h, respectively (Vermeulen, 2009). The half-life clearance of sulforaphane in the human body from the aforementioned vegetables is 4.6 and 3.8 h, respectively. These are different from half-life time of mercapturic acid which is 2.4 and 2.6 h, respectively. Further investigations using human subjects show inconclusive results that particular polymorphism S-glutathione transferase (GST M1, T1 and P1) and N-acetyl transferase (NAT2) gene affect the variations (Vermeulen, 2009). It is important to view these phenomena under the holistic affecting factors.

<sup>12</sup> Data is calculated from : First order kinetics

ka: intercept and slope with a residual method; k: natural log of plasma amounts plotted against time; ke: natural log of absolute excreted amounts vs time; area under curve (concentration vs time) extrapolated using trapezoid method; lag phase is determined from empiric curve obtained; base line is not used

Bioavailability of Phytochemicals 417

Fig. 3. Bioassay of caffeine using simulated transit method (top panel: model A; using 2 transwell-inserts), static apical solution method (middle panel: model B; using 4 transwellinserts), and static apical and basolateral solution procedures for 22 h (bottom panel: model

13 Model A: apical side is replenished every 30 min, Model B: basolateral side is refreshed every 30 min,

C; using 2 transwell-inserts)13

Model C: both apical and basolateral are not replenished for 22 h

**Phenolics**. Generally, the least absorbed polyphenols are proanthocyanidins, galloylated tea catechins, and anthocyanins (Epriliati, 2008 and Ginjom, 2009).

**Caffeine.** Using pharmacological principles, absorption simulations of pure compound in intestine is mimicked by caco-2 monolayers. During the simulated transit method (model A), unchanged caffeine was transported across epithelial cells (Figure 3). This indicates that caffeine is directly transported to the basolateral compartment without damaging the tight junctions. This transport is selectively occurring in the apical to basolateral direction over the bioassay time (240 min). The apical caffeine levels from simulation of transit method even after 120 min (Figure 3, top panel) are higher than that of semi dynamic apical solution method (B model) (Figure 3, middle panel). Caffeine was transported by the enterocytes in the apical to basolateral direction apparently without an equilibrium state being generated. Uptake of caffeine was rapid and basolateral secretion possibly required a certain amount of caffeine intracellularly is retained. When a high gradient concentration was maintained, continuous basolateral secretion of caffeine took place at a constant rate. As a result, the final level of basolateral caffeine was higher than the apical levels, even when it was subjected to a 22 h bioassay (C model) (Figure 3, bottom panel). The transport mechanism of caffeine may be a simple passive diffusion. However, another study shows caffeine can also be transported by the transcellular route (Mao, 2007). In addition, caffeine is found interacts with glucose uptake sensitivity (Pizziol, 1998).

**Catechin.** The simulation transit method of catechin indicates that it is retained in the apical compartment at about one-third of the initial amount (86.8 nmol) and remains at about the same level throughout the experiment (Figure 4, top panel). However, basolateral compartment analysis did not indicate equal amount of translocated catechin. In contrast, most basolateral samples contain very little catechin. In the static apical solution method (Figures 4, middle and bottom panels), apical catechin was reduced to 39 nmol after 22 h, but there were no indication of transported catechin in the basolateral compartment. In the present study, there may have been some metabolism of catechin based on apical losses which require further study to identify possible metabolites of catechin.

**Lycopene.** Lycopene is neither transported (Figure 5) nor chemically changed during bioassay using all three bioassay methods for all time periods. Its hydrophobicity and unfavorable molecular geometry apparently prevents lycopene from passing through monolayers via either paracellular or transcellular routes. This was confirmed by the decrease of TEER values for all monolayers, which is not accompanied by lycopene translocation into the basolateral compartment from the apical solutions. In the present study, the apical lycopene do not show disappearance in the transit model (Figure 5, top panel). Instead, lycopene shows apical accumulations with renewal solutions. Similar results are obtained from the semi dynamic model (Figure 5, middle panel) and confirmed in the 22 h static model (Figure 5, bottom panel). Lycopene absorption has been shown to be affected by the presence of other carotenoids, the lipid status, and plasma antioxidant capacity (Bohm & Bitsch, 1999). However, another study found that lycopene plasma levels after consumption of cherry tomatoes are insignificantly different from the plasma base line (Bugianesi et al., 2004). Further absorption from micelles has been shown to be slow (e.g. lycopene absorbed by LNCaP and Hs888Lu cells took approximately 10 h; Xu et al., 1999). This suggests that epithelial cells may have specific mechanisms that are not micelle dependent.

416 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

**Phenolics**. Generally, the least absorbed polyphenols are proanthocyanidins, galloylated tea

**Caffeine.** Using pharmacological principles, absorption simulations of pure compound in intestine is mimicked by caco-2 monolayers. During the simulated transit method (model A), unchanged caffeine was transported across epithelial cells (Figure 3). This indicates that caffeine is directly transported to the basolateral compartment without damaging the tight junctions. This transport is selectively occurring in the apical to basolateral direction over the bioassay time (240 min). The apical caffeine levels from simulation of transit method even after 120 min (Figure 3, top panel) are higher than that of semi dynamic apical solution method (B model) (Figure 3, middle panel). Caffeine was transported by the enterocytes in the apical to basolateral direction apparently without an equilibrium state being generated. Uptake of caffeine was rapid and basolateral secretion possibly required a certain amount of caffeine intracellularly is retained. When a high gradient concentration was maintained, continuous basolateral secretion of caffeine took place at a constant rate. As a result, the final level of basolateral caffeine was higher than the apical levels, even when it was subjected to a 22 h bioassay (C model) (Figure 3, bottom panel). The transport mechanism of caffeine may be a simple passive diffusion. However, another study shows caffeine can also be transported by the transcellular route (Mao, 2007). In addition, caffeine is found interacts

**Catechin.** The simulation transit method of catechin indicates that it is retained in the apical compartment at about one-third of the initial amount (86.8 nmol) and remains at about the same level throughout the experiment (Figure 4, top panel). However, basolateral compartment analysis did not indicate equal amount of translocated catechin. In contrast, most basolateral samples contain very little catechin. In the static apical solution method (Figures 4, middle and bottom panels), apical catechin was reduced to 39 nmol after 22 h, but there were no indication of transported catechin in the basolateral compartment. In the present study, there may have been some metabolism of catechin based on apical losses

**Lycopene.** Lycopene is neither transported (Figure 5) nor chemically changed during bioassay using all three bioassay methods for all time periods. Its hydrophobicity and unfavorable molecular geometry apparently prevents lycopene from passing through monolayers via either paracellular or transcellular routes. This was confirmed by the decrease of TEER values for all monolayers, which is not accompanied by lycopene translocation into the basolateral compartment from the apical solutions. In the present study, the apical lycopene do not show disappearance in the transit model (Figure 5, top panel). Instead, lycopene shows apical accumulations with renewal solutions. Similar results are obtained from the semi dynamic model (Figure 5, middle panel) and confirmed in the 22 h static model (Figure 5, bottom panel). Lycopene absorption has been shown to be affected by the presence of other carotenoids, the lipid status, and plasma antioxidant capacity (Bohm & Bitsch, 1999). However, another study found that lycopene plasma levels after consumption of cherry tomatoes are insignificantly different from the plasma base line (Bugianesi et al., 2004). Further absorption from micelles has been shown to be slow (e.g. lycopene absorbed by LNCaP and Hs888Lu cells took approximately 10 h; Xu et al., 1999). This suggests that epithelial cells may have specific

which require further study to identify possible metabolites of catechin.

catechins, and anthocyanins (Epriliati, 2008 and Ginjom, 2009).

with glucose uptake sensitivity (Pizziol, 1998).

mechanisms that are not micelle dependent.

Fig. 3. Bioassay of caffeine using simulated transit method (top panel: model A; using 2 transwell-inserts), static apical solution method (middle panel: model B; using 4 transwellinserts), and static apical and basolateral solution procedures for 22 h (bottom panel: model C; using 2 transwell-inserts)13

<sup>13</sup> Model A: apical side is replenished every 30 min, Model B: basolateral side is refreshed every 30 min, Model C: both apical and basolateral are not replenished for 22 h

Bioavailability of Phytochemicals 419

**carotene.** There are always reductions of apical levels but not necessarily accompanied by release into the basolateral side (Figure 6). Meanwhile, -carotene completely disappears in the 22 h static model, both from the apical and basolateral sides although TEER values drops from 0.497 to 0.125 kΩ.cm2.-carotene may diffuse better than lycopene, as indicated by the -carotene apical disappearances; however, neither is translocated. This may be related to intrinsic solubility, as -carotene is more soluble than lycopene in the mixed aqueous/organic solvents. In the semi dynamic model after 120 min, apical -carotene

Fig. 5. Apical lycopene bioassay; **a** transit model (model A), **b** basolateral renewals (model

B), **c** static model (model C) in buffer-0.5% DMSO

decreases and in the static model after 22 h, -carotene disappears completely.

Fig. 4. (+) Catechin transport using simulation of transit chyme (top panel: model A; using 2 transwell inserts), static apical solution methods (middle panel: model B; using 4 transwell inserts), and static apical and basolateral solution after 22 h (bottom panel: model C; using 4 transwell inserts)

Fig. 4. (+) Catechin transport using simulation of transit chyme (top panel: model A; using 2 transwell inserts), static apical solution methods (middle panel: model B; using 4 transwell inserts), and static apical and basolateral solution after 22 h (bottom panel: model C; using 4

transwell inserts)

**carotene.** There are always reductions of apical levels but not necessarily accompanied by release into the basolateral side (Figure 6). Meanwhile, -carotene completely disappears in the 22 h static model, both from the apical and basolateral sides although TEER values drops from 0.497 to 0.125 kΩ.cm2.-carotene may diffuse better than lycopene, as indicated by the -carotene apical disappearances; however, neither is translocated. This may be related to intrinsic solubility, as -carotene is more soluble than lycopene in the mixed aqueous/organic solvents. In the semi dynamic model after 120 min, apical -carotene decreases and in the static model after 22 h, -carotene disappears completely.

Fig. 5. Apical lycopene bioassay; **a** transit model (model A), **b** basolateral renewals (model B), **c** static model (model C) in buffer-0.5% DMSO

Bioavailability of Phytochemicals 421

25-500 ppm

 100 ppm; 24 h 25-75 M; 12 h

50 M; 24 h

1, 2.5, and 5 mM; 12 h

IC50 at 1.26 mM; 96 h

IC50 at 1.32 mM; 96 h

 IC50 at 4.18 mM; 96 h 0.25-5 mM; 6 d 0.5-4 mM; 24 h 0.25-2 mM; 24 h

than 4-6 weeks; drinking diluted water extract of fresh potato (Tan & Rahardja, 2010)

 1-2 times a day; drinking mature seed extract made from 20 g powder in a ½ cup

 Drinking extract of a 6 g of seed and flesh powder 2-3 times a day; drinking young luffa juice sweetened

once a day; consuming 10 g

of hot water

with sugar

of boiled leaves regularly 3 times a day; consuming 10-15 pieces of

13 mmol.L; 12 h

**Phytochemicals Effects Dosages**  Suppress HTC 15 cell proliferation

B16F10 cells

cells

cells

231cells

Soyasapogenol A and B (Kang et al., 2010)

Soyasaponin – soyasapogenol B monoglucuronide mixture (Kang et

Phytate (Kang et

al., 2010)

al., 2010)

Phenolics in colourful potatoes

Phytochemicals from luffa [*Luffa cylindrica* Roem.; *Luffa Aegyptica* Mill.; *L. Cattupincina* Ser.; *L. Pentandra* Roxb.]

Phytochemicals from Indian champor weed [*Pluchea indica* (L.)  Induce macroautophagy Decrease migratory

ability/increase adhesion of

 Decrease expression of TNF-a and TNF II in Caco-2 cells Inhibit proliferation of HT 29

Inhibit growth of MCF-7/Adr

Inhibit growth of MDA-MB

 Inhibit growth of MCF-7 cells Inhibit growth of HepG2 cells Inhibit growth of LNCaP cells Inhibit growth of DU145 cells

To treat intestinal inflammation

To improve breast milk

For fever and emetic sweat

For body odour removal

production To treat asthma

removal

Enhance adhesion of MCF7 cells

Suppress HT 29 cell growth 6-50 ppm; 72 h

Suppress HT 29 cell growth 50 ppm; 72 h

Treating gastric ulcer 2-3 times a day for no more

Fig. 6. Apical -Carotene in HBSS-25 mM HEPES contained 0.5 % DMSO: **a** transit chyme (model A), **b** static apical solution (model B)

#### **4.3 Dosages**

Establishing the most suitable dosages for an optimal health benefit of a phytochemical is not an easy task. As an antioxidant, phytochemicals are generally required in small doses due to its ability to become pro-oxidant. Based on its traditional usage, the doses are commonly determined from folklores, thus the key compounds mostly responsible for their health functions and their mechanisms remain to be explored through epidemiological studies. Table 3 lists what doses studied *in vitro* and folklores.


Fig. 6. Apical -Carotene in HBSS-25 mM HEPES contained 0.5 % DMSO: **a** transit chyme

Establishing the most suitable dosages for an optimal health benefit of a phytochemical is not an easy task. As an antioxidant, phytochemicals are generally required in small doses due to its ability to become pro-oxidant. Based on its traditional usage, the doses are commonly determined from folklores, thus the key compounds mostly responsible for their health functions and their mechanisms remain to be explored through epidemiological

> 100-300 l/mL; 24 h 150, 300, 600 ppm; 72 h IC50 at 30 g/mL; 48 h

25-75 M; 48 h

(model A), **b** static apical solution (model B)

studies. Table 3 lists what doses studied *in vitro* and folklores.

**Phytochemicals Effects Dosages** 

glioblastoma cells

 Decrease HT cell growth Inhibit AFB1-DNA adduct formation in HepG2 liver cells Induce apoptosis in SNB 19

Inhibits metastasis HT-1080 cells

**4.3 Dosages** 

Soya saponin (Kang et al., 2010)


Bioavailability of Phytochemicals 423

concentrated twice twice a day; consuming 60 g

 1-2 times a week for 8 weeks; applying a mixture of ground boiled-black nightshade fruits at the suffering tissues 3 times twice a week; drinking a ½ glasses of decoction of 30 g ground black nightshade fruits and *Celosia cristata* flowers in a 3 glasses of water, and concentrated (Dalimartha,

 once a day for 14 days for adult; given infusion liquid of 1 waxy gourd fruit as big as a palm hand, added by 10 pieces of anises/fennels, a ±1cm length of *Alixa stellata*, and a tea spoon of honey; (Kementerian Lingkungan

 Consuming 100-150 g boiled or juiced waxy guard (Wijayakusuma, 2008)

 twice a day; drinking a ½ glasses of decoction of 15 g lemon basil grass in a 2 glass of water for 15 minutes

of boiled shrub 3 times a day; chewing around 15 black nightshade

fruits

2008)

Hidup, 2011)

(Hariana, 2006)

**Phytochemicals Effects Dosages** 

For xeropthalia

For pektay

For cervical erosion

Curing hemorrhoid

To treat diabetes

 To ease people suffering from early ejaculation, late

and for removing fever

menstruation, breast milk and gas cleanser in the human body,

Table 2. Resume of dosages used in studies regarding phytochemicals health effects and in

14 The information of Indonesian medicinal folklores is obtained through a collaboration project between Korean Food Research Institution and Bogor Agricultural University, Indonesia in 2011.

For eczema or dermatitis

Miller, *Solanum nodiflorum* Jacq, *Solanum ningrum*  auct non L.]

Phytochemicals from Waxy gourd [*Benincasa hispida*  (Thunb) Cogn., *B. cerifera savi, Cucurbita hispida*  Thunb*. Lagenaria dasystemon* Miq]

Phytochemicals from Lemon basil

[*Ocimum americanum, O. citriodorum, O. africanum, O. canum Sims, O. brachiatum Blume*]

folklores14.


raw or steamed leaves with

(Dalimartha, 2005; Hariana,

 2-3 times a day; consuming soup made from 250 g watercress and pig bone, added with sufficient salt Consuming soup made from 60 g of watercress and sugar

consuming soup of boiled

 Consuming soup made from 250 g of watercress and palm sugar (Muchlisah & Hening,

drinking extract of 3 bilimbi fruits in 3 glasses of water, concentrated 3 times 3 times a day; applying a mixture of 6-8 ground bilimbi fruits, a ½ tea spoon of salt, a ¼ glasses of water

 Consuming 3-5 pieces of crushed leaves mixed with

soft rice (porridge)

several times a day;

once every three days;

 Applying a mixture of 25 pieces of bilimbi leaves, 10 clove, and 15 pepper, ground finely, added with a small amount of vinegar, at suffering body/tissues Chewing 5 pieces of bilimbi fruits with a little salt and at the suffering teeth (Hariana,

 twice a day; drinking a ½ glasses of extract of 30 g of black nightshade fruits with *Hedyotis diffusa* grass, and *Phyllantus urinearia* in a 3

glasses of water,

watercress

2009)

onto acne

2006).

rice

2006).

**Phytochemicals Effects Dosages** 

For tuberculosis

For skin irritation

For hypertension

For muscle pain

For teeth cavities

For urethra infection

For acne

For urinary problems

 For relieving gastrointestinal disorders in children

For inflamed lung and coughing

Less.]

Phytochemicals from Watercress [*Nasturtium officinale* R. Brown*, N. officinale* W.T. Aiton*, N. nasturtiumaquaticum* (L) H. Karst*., Radicula nasturtium Cav.,*]

Phytochemicals from bilimbi [*Averrhoa bilimbi* 

Phytochemicals from Glossy nightshade, Black nightshade [*Solanum americanum* 

Linn]


Table 2. Resume of dosages used in studies regarding phytochemicals health effects and in folklores14.

 14 The information of Indonesian medicinal folklores is obtained through a collaboration project between Korean Food Research Institution and Bogor Agricultural University, Indonesia in 2011.

Bioavailability of Phytochemicals 425

allowance establishment, there is a need for dosage allowance for each bioactive. Similarly, when recommended allowance has been established, food chain supply needs to provide necessary quantity of the phytochemical sources for people. Such data are currently unavailable, and thus, a database and information system for it needs to be established.

Phytochemicals bioavailability is strongly dependent on cell wall compositions of the food matrices they originate from, structural chemistry of the phytochemicals, history of processing, as well as individual human gastrointestinal system. Determination of phytochemical bioavailability is increasingly developed using both *in vitro* and *in vivo* approaches*,* and yet the results are still inconclusive. The main challenge is to develop an *in vitro* model that can represent human *in vivo* condition for practical uses. On the other hand, many aspects of bioavailability is not well understood, prompting further research and database for recommended dosages and consequently per capita phytochemical demands for public health management. Currently, folklores are the main sources of public health management using phytochemicals and database remains to be pursued for better scientific base of

Berardini, N.; Carle, R. & Schieber, A. (2004). Characterization of gallotannins and

Awad, H.M.; Boersma, M.G.; Boeren, S.; van Bladeren, P.J.; Vervoort, J. & Rietjens, I.M.

Bohm, V. & Bitsch, R. (1999). Intestinal absorption of lycopene from different matrices and

Bugianesi, R.; Salucci, M.; Leonardi, C.; Ferracane, R.; Catasta, G.; Azzini, E. & Maiani, G.

de Freitas, V. & Mateus, N. (2001). Structural features of procyanidin interactions with

Epriliati, I. Nutriomic analysis of fresh and processed fruits through the development of an

Erlund, I.; Freese, R.; Marniemi, J.; Hakala, P. & Alfthan, G. (2006). Bioavailability of Quercetin from Berries and The Diet. *Nutr Cancer*, 54, 1, (2006), pp. 13–17

human plasma. *Eur J Nutr,* 38, (1999), pp. 118 - 125.

Dalimartha, S. (2005). *Tanaman Obat di Lingkungan Sekitar*. Puspa Swara, Depok Dalimartha S. (2008). *Atlas Tumbuhan Obat Indonesia*. Jilid 5. Pustaka Bunda, Depok

salivary proteins. *J Agric Food Chem.,* 49, (2001), pp. 940-945.

benzophenone derivatives from mango (*Mangifera indica* L. cv. 'Tommy Atkins') peels, pulp and kernels by high-performance liquid chromatography electrospray ionization mass spectrometry. *Rapid Communications in Mass Spectrometry,* 18,

(2002). The regioselectivity of glutathione adduct formation with flavonoid quinone/quinone methides is pH-dependent. *Chem Res Toxicol,* 15, (2002), pp. 343 -

interactions to other carotenoids, the lipid status, and the antioxidant capacity of

(2004). Effect of domestic cooking on human bioavailability of naringenin, chlorogenic acid, lycopene and beta-carotene in cherry tomatoes. *Eur J Nutr [NLM -* 

*in-vitro* model of human digestive system, PhD dissertation, The University of

**6. Conclusion** 

folklores practices.

**7. References** 

351.

Queensland.

(2004), pp. 2208 - 2216.

*MEDLINE],* 43, (2004), pp. 360.

#### **5. Recommended daily allowance**

#### **5.1 Recommended daily allowance**

Recommended uses and maximum limits of uses in modern public health management are limited. The ancient uses are based on folklores and old documents. This information should be followed up with proper scientific investigations and documentations. Even for broccoli which is extensively studied, the recommended daily intake has not been officially established. US national cholesterol education program recommends adult subject to consume 2 g of phytosterol/d for optimally lowering LDL-C and coronary heart disease risks by 10% (Kang et al., 2010). The mechanism of this is still vague but it is known that phytosterol/phytostanol does not necessarily present simultaneously with cholesterol to control cholesterol absorption.

#### **5.2 Public health management**

There is limited information on detailed diet prescription aiming at treating a particular disease, except those recorded in ancient medications. Dieticians usually arrange diets for patients not aiming for disease treatments but to meet certain nutritional requirements to improve their stamina or immune system to combat their physiological problems. Mechanism for phytochemical health benefits have been studied extensively. Current understanding shows that public health would take benefits from diet management for prevention and maintaining public health instead of treating it. Many research results found scientific base of phytochemicals. For example, liminoids has increasingly proven positive health effects including induction of glutathione S-transferase, inhibiting cancers growth, and lowering cholesterols (Kang et al., 2010), yet officially, this still has not been established for recommended daily allowance. On the other hand, information from ancient medicinal prescriptions as listed in Table 3 is mostly in the form of decoction of the phytochemical sources and the boiled water is drunk. Interesting research area is to establish whether such preparation preserve biological functions of the phytochemicals or, instead, the methods modify molecular form of the phytochemicals that is a much safer and/or more bioactive than its original forms.

#### **5.3 Phytochemicals incorporation in diets**

Phytochemicals are commonly consumed as supplements either in capsules, tablets, or powders. The incorporation of such ingredients in food products may or may not face problems of stability, especially at its extraction step and formulation and food processing in which heating is one of predominant aspects for generating food palatability. Most conventional food preparations are of high risks on phytochemical instability. Attempt to improve food technology remains inconclusive. Health effect study indicates that enriched ground beef with soy phytosterol reduces total cholesterol, LDL-C, and TC/HDL cholesterol by 9.3, 14.6, and 9.1%, respectively (Vermeulen, 2009). Such attempts require standardization for establishment of functional food regulations.

#### **5.4 Phytochemical demands per capita**

In order to maintain health where phytochemicals are involved, a daily recommended allowance similar to other nutrients is required. Therefore, prior to daily recommended allowance establishment, there is a need for dosage allowance for each bioactive. Similarly, when recommended allowance has been established, food chain supply needs to provide necessary quantity of the phytochemical sources for people. Such data are currently unavailable, and thus, a database and information system for it needs to be established.

#### **6. Conclusion**

424 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Recommended uses and maximum limits of uses in modern public health management are limited. The ancient uses are based on folklores and old documents. This information should be followed up with proper scientific investigations and documentations. Even for broccoli which is extensively studied, the recommended daily intake has not been officially established. US national cholesterol education program recommends adult subject to consume 2 g of phytosterol/d for optimally lowering LDL-C and coronary heart disease risks by 10% (Kang et al., 2010). The mechanism of this is still vague but it is known that phytosterol/phytostanol does not necessarily present simultaneously with cholesterol to

There is limited information on detailed diet prescription aiming at treating a particular disease, except those recorded in ancient medications. Dieticians usually arrange diets for patients not aiming for disease treatments but to meet certain nutritional requirements to improve their stamina or immune system to combat their physiological problems. Mechanism for phytochemical health benefits have been studied extensively. Current understanding shows that public health would take benefits from diet management for prevention and maintaining public health instead of treating it. Many research results found scientific base of phytochemicals. For example, liminoids has increasingly proven positive health effects including induction of glutathione S-transferase, inhibiting cancers growth, and lowering cholesterols (Kang et al., 2010), yet officially, this still has not been established for recommended daily allowance. On the other hand, information from ancient medicinal prescriptions as listed in Table 3 is mostly in the form of decoction of the phytochemical sources and the boiled water is drunk. Interesting research area is to establish whether such preparation preserve biological functions of the phytochemicals or, instead, the methods modify molecular form of the phytochemicals that is a much safer and/or more bioactive

Phytochemicals are commonly consumed as supplements either in capsules, tablets, or powders. The incorporation of such ingredients in food products may or may not face problems of stability, especially at its extraction step and formulation and food processing in which heating is one of predominant aspects for generating food palatability. Most conventional food preparations are of high risks on phytochemical instability. Attempt to improve food technology remains inconclusive. Health effect study indicates that enriched ground beef with soy phytosterol reduces total cholesterol, LDL-C, and TC/HDL cholesterol by 9.3, 14.6, and 9.1%, respectively (Vermeulen, 2009). Such attempts require standardization

In order to maintain health where phytochemicals are involved, a daily recommended allowance similar to other nutrients is required. Therefore, prior to daily recommended

**5. Recommended daily allowance 5.1 Recommended daily allowance** 

control cholesterol absorption.

**5.2 Public health management** 

than its original forms.

**5.3 Phytochemicals incorporation in diets** 

for establishment of functional food regulations.

**5.4 Phytochemical demands per capita** 

Phytochemicals bioavailability is strongly dependent on cell wall compositions of the food matrices they originate from, structural chemistry of the phytochemicals, history of processing, as well as individual human gastrointestinal system. Determination of phytochemical bioavailability is increasingly developed using both *in vitro* and *in vivo* approaches*,* and yet the results are still inconclusive. The main challenge is to develop an *in vitro* model that can represent human *in vivo* condition for practical uses. On the other hand, many aspects of bioavailability is not well understood, prompting further research and database for recommended dosages and consequently per capita phytochemical demands for public health management. Currently, folklores are the main sources of public health management using phytochemicals and database remains to be pursued for better scientific base of folklores practices.

#### **7. References**


Bioavailability of Phytochemicals 427

Mao, X.; Chai, Y. & Lin, Y-F. (2007). Dual regulation of ATP-sensitive potassium channel by

Miyazaki, S., Kubo, W., Itoh, K., Konno, Y., Fujiwara, M., Dairaku, M., Togashi, M., Mikami,

Moreno, D.A.; Carvajal, M.; L´opez-Berenguer, C. & Garc´a-Viguera, C. (2006). Chemical

Muchlisah, F. & Hening, S. (2009). *Sayur dan bumbu dapur berkhasiat obat*. Penebar Swadaya,

Nagao, A.; Maeda, M.; Lim, B. P.; Kobayashi, H. & Terao, J. (2000). Inhibition of [beta]-

Nagao, A. 2004. Oxidative conversion of carotenoids to retinoids and other products. *The* 

Noe, V.; Penuelas, S.; Lamuela-Raventos, R.M.; Permanyer, J.; Ciudad, C.J. & Izquerdo-

Nunan, K.J.; Sims, I.M.; Bacic, A.; Robinson, S.P. & Fincher, G.B. (1998). Changes in cell wall

Passamonti, S.; Terdoslavich, M.; Franca, R.; Vanzo, A.; Tramer, F.; Braidot, E.; Petrussa, E. &

Pizziol, A.; Tikhonoff, V.; Paleaeri, C.D.; Russo, E.; Mazza, A.; Ginocchio, G.; Onesta, C.;

tolerance: a placebo-controlled study. *Eur J Clin Nutr*, 52, (1998), pp. 846 - 9. Sabboh-Jourdan, H.; Valla, F.; Epriliati, I. & Gidley, M.J. (2011). Organic acid bioavailability

Schneeman, B. O. & Gallaher, D. (1985). Effects of dietry fiber on digestive enzyme activity

Stracke, B.A.; Ru¨fer, C.E.; Bub, A.; Briviba, K.; Seifert, S.; Kunz, C. & Watzl, B.

Stolle-Smits, T.; Beekhuizen, J.G.; Kok, M.T.C.; Pijnenburg, M.; Recourt, K.; Derksen, J. &

Vahouny, G.V. & Cassidy, M.M. (1985). Dietary fibres and absorption of nutrients. *Proc Soc* 

during development. *Plant Physiology*, 121, (October 1999), pp. 363–372, Tan, H.T. & Rahardja, K. (2010). *Obat-obat sederhana untuk gangguan sehari-hari*. PT Gramedia

van den Berg, H. (1999). Carotenoid interactions. *Nutrition Reviews,* 57, 1 (1999), pp.

Eur J Nutr., 50, 1, (2011), pp. 31-40, DOI: 10.1007/s00394-010-0112-0

expresión in human Caco-2 cells. J Nutr., 134, (2004), pp. 2509-2516

R. and Attwood, D. 2005. The effect of taste masking agents on in situ gelling pectin formulations for oral sustained delivery of paracetamol and ambroxol. *International* 

and biological characterisation of nutraceutical compounds of Broccoli. *J Pharmaceu* 

carotene-15,15'-dioxygenase activity by dietary flavonoids. *The Journal of Nutritional* 

Pulido, M. (2004). Epicatechin and cocoa polyphenolic extract modulate gene

composition during ripening of grape berries. *Plant Physiol*., 118, (1998). pp. 783–

Vianello, A. (2009). Bioavailability of flavonoids: A Review of their membrane transport and the function of bilitranslocase in animal and plant organisms. *Current* 

Pavan, L.; Casiglia, E. & Dessina, A.C. (1998). Effects of caffeine on glucose

from banana and sweet potato using an *in vitro* digestion and Caco-2 cell model.

and bile acids in the small intestine. *Proc Soc Exp Biol Med,* 180, (1985), pp. 409 - 414.

Bioavailability and nutritional effects of carotenoids from organically and conventionally produced carrots in healthy men. *Br J Nutr*., 101, (2009), pp. 1664–

Voragen, A.G.J. (1999). Changes in cell wall polysaccharides of green bean pods

caffeine. *Am J Physiol Cell Physiol*, 292, (2007), pp. C2239 - 58.

*Journal of Pharmaceutics,* 297, 38 – 49

*Biomed Anal*, 41, (2006), pp. 1508–1522

*Biochemistry,* 11, (2004), pp. 348 - 355.

*Journal of Nutrition,* 134, (2004), PP. 237S.

*Drug Metabolism*, 10, (2009). pp. 369-394

Jakarta

792

1672

Pustaka Utama, Jakarta

*Exp Biol Med,* 180, (1985), pp. 432 - 446.


Frank, J. (2004). *Dietary Phenolic Compounds and Vitamin E Bioavailability–Model studies in rats* 

Friedman, M. & Jurgens, H. S. (2000). Effect of pH on the stability of plant phenolic

Ginjom, I.R.H. Health aspects of wine antioxidants: composition and *in vitro* bioavailability.

Glahn, R.P.; Cheng, Z.; Welch, R.M. & Gregorio, G.B. (2002). Comparison of iron

Glahn, R.P.; Lai, C.; Hsu, J. & Thompson, J.F. (1998). Decreased citrate improves iron

Johnson, E.J. (1998). Human studies on bioavailability and plasma response of lycopene.

Johnson, L.R. (2001). *Gastrointestinal Physiology*. 6th ed., Mosby, ISBN 0-323-01239-6 St. Louis Kang, J.; Badger, T.M.; Ronis, M.J.J.; & Wu, X. (2010). Non-isoflavone Phytochemicals in Soy and Their Health Effects. *J. Agric. Food Chem*. 58, (2010), pp. 8119–8133 Kidd, P. & Head, K. (2005). A review of the bioavailability and clinical efficacy of milk thistle

Konishi, Y.; Kobayashi, S. & Shimizu, M. (2003). Tea polyhenols inhibit the transport of

in separate or combined oral doses. *Am J Clin Nutr,* 62, (1995), pp. 604 - 610. Kementerian Lingkungan Hidup. (2011). Potensi sumberdaya genetik tanaman: Bligu

Kumar, G. P.; Sudheesh, S.; Ushakumari, B.; Valsa, A. K.; Vijayakumar, S.; Sandhya, C. &

Larrauri, J.A.; Goni, I.; MartinCarron, N.; Ruperez, P. & SauraCalixto, F. (1996).

Manach, C.; Williamson, G.; Morand, C.; Scalbert, A. & Remesy, C. (2005). Bioavailability

Manners, G.D.; Jacob, R.A.; Breksa III, A.P.; Schoch, T.K. & Hasegawa, S. (2003).

*American Journal of Clinical Nutrition,* 81, (2005), pp. 230S-242.

of Food Science, Swedish University Agricultural Science

PhD dissertation. The University of Queensland.

*Proc Soc Exp Biol Med,* 218, (1998), pp. 115 - 120.

*Rev*., 10, 3, (2005), pp. 193-203

*and Agriculture,* 71, (1996), pp. 515 - 519.

2011]

103 - 107.

4156-4161

compounds. *J Agric Food Chemistry,* 48, (2000). pp. 2101-2110.

cell culture model. *J Agric Food Chem.,* 50, (2002), pp. 3586 - 3591.

culture model. *The Journal of Nutrition,* 128, (1998), pp. 257. Hariana, A. (2006). *Tanaman Obat dan Khasiatnya*. Seri1. Penebar Swadaya, Jakarta

*and humans*. Doctoral dissertation. ISSN 1401-6249, ISBN 91-576-6453-6. Department

biaovailability from 15 rice genotypes: studies using an *in vitro* digestion/caco-2

availability from infant formula: Application of an *in vitro* digestion/Caco-2 cell

phytosome: A silybin-phosphatidylcholine complex (Silipos®). *Alternative Medicine* 

dietary phenolic acids mediated by the monocarboxyclic acid transporters (MCT) in intestinal caco-2 cell monolayers. *J Agric Food Chem.,* 51, (2003), pp. 7296 - 7302. Kostic, D.; White, W. & Olson, J. (1995). Intestinal absorption, serum clearance, and

interactions between lutein and beta-carotene when administered to human adults

(*Benincasa hispida (Thunb.) Cogn*). http://bk.menlh.go.id/sdg/ [Accessed June 5,

Vijayalakshmi, N.R. (1997). A comparative study on the hypolipidemic activity of eleven different pectins. *Journal of Food Science and Technology Mysore,* 34, (1997), pp.

Measurement of health-promoting properties in fruit dietary fibres: Antioxidant capacity, fermentability and glucose retardation index. *Journal of the Science of Food* 

and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies.

Bioavailability of Citrus Limonoids in Humans. *J. Agric. Food Chem*., 51, (2003), pp.


**20** 

*Brasil* 

*Ximenia americana***: Chemistry, Pharmacology** 

*Programa de Pós-Graduação em Química Universidade Federal do Ceará, Fortaleza - Ceará* 

The use of plants as medicinal agents to the treat of many diseases has been investigated for a long time since the antique civilizations. Several plants are used in traditional medicine against inflammatory diseases as well as various types of tumors on the base the potential of their chemical constituents. Although many compounds are extremely toxic, when we have the relation between the toxicity of a compound and its chemical pattern of substitution that can result in a more in-depth understanding of these compounds (Atta-ur-Rahman, 2005). Today, even after more than 200 years, the chemistry of natural products remains a challenge and an important field of research in several science areas (chemistry, biology, medicine, agronomy, botany and pharmacy). The reasons for it's large use are the considerable pharmacological potential observed in natural products, in the great development in the process of detection, isolation, purification and, especially, the advances in spectrometric techniques [infrared (IR), mass spectrometry (MS) and nuclear magnetic resonance (NMR 1H and 13C) for structural elucidation of new and complex compounds. These advances were outstanding in both NMR and MS spectrometry. The NMR allows the complete 1H and 13C NMR spectral assignments (chemical shifts and coupling constants) which serve to build a data base to support computer assisted structure elucidation. These data are also useful in the fuller understanding of the correlations between molecular conformation and biological activity of natural substances with biological importance (Loganathan *et al.*, 1990). Mass spectrometry has a huge application in chemistry, biochemistry, medicine, pharmacology, agriculture and food science. Although the mass spectrometric ionization techniques EI (electron impact) and CI (chemical ionization) required the analyte molecules to be present in the gas phase and were thus suitable only for volatile compounds, the development of several desorption ionization methods [FD (field desorption), FABMS (fast atom bombardment), ESIMS (electrospray), MALDI-MS (matrix assisted laser desorption ionization)] allowed the hight-precision mass spectrometric

The genus Ximenia belongs to the Olacaceae and comprises about 8 species (Brasileiro et al., 2008): Ximenia roiigi, Ximenia aegyptiaca, Ximenia parviflora, Ximenia coriaceae, Ximenia aculeata, Ximenia caffra, Ximenia americana and Ximenia aegyptica. X. caffra stands out for

**1. Introduction** 

analysis of different classes of biomolecules.

**and Biological Properties, a Review** 

Francisco José Queiroz Monte1, Telma Leda Gomes de Lemos1, Mônica Regina Silva de Araújo2 and Edilane de Sousa Gomes1

*Depatamento de Química, Universidade Federal do Piauí, Teresina - Piauí,* 


### *Ximenia americana***: Chemistry, Pharmacology and Biological Properties, a Review**

Francisco José Queiroz Monte1, Telma Leda Gomes de Lemos1, Mônica Regina Silva de Araújo2 and Edilane de Sousa Gomes1 *Programa de Pós-Graduação em Química Universidade Federal do Ceará, Fortaleza - Ceará Depatamento de Química, Universidade Federal do Piauí, Teresina - Piauí, Brasil* 

#### **1. Introduction**

428 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

van het Hof, K.H.; West, C.E.; Weststrate, J.A. & Hautvast, J.G.A.J. (2000). Dietary factors

Vermeulen, M. (2009). Isothiocyanates from cruciferous vegetables: Kinetics, biomarkers and

Williams, A.W. (1998). Factors influencing the uptake and absorption of carotenoids. *Proc* 

Williamson, G. (2004). Common features in the pathways of absorption and metabolism of

Xu, X.; Wang, Y.; Constantinou, A.; Stacewicz-Sapuntzakis, M.; Bowen, P. & van Breemen,

Ye, L.X.; Dinkova-Kostova, A.T.; Wade, K.L.; Zhang, Y.S.; Shapiro, T.A. & Talalay, P. (2002).

Yun, S.M.; Habicht, J.P.; Miller, D.D. & Glahn, R. P. (2004). An *in vitro* digestion/Caco-2 cell

RANDOLPH, R. K. (Eds.) *Phytochemicals.* Boca Raton, CRC Press.

lycopene to cells in culture. *Lipids,* 34, (1999), pp. 1031 - 1036.

humans. *Clinica Chimica Acta*, 316, 1-2,. (2002), pp. 43-53.

Wijayakusuma, H. (2008). *Bebas Diabetes ala Hembing*. Puspa Swara, Jakarta

*Soc Exp Biol Med,* 218, (1998), pp.106 - 108.

503 - 506.

90-8585-312-1

that affect the bioavailability of carotenoids. *The Journal of Nutrition,* 130, (2000), pp.

effects. Thesis. Wageningen University, Wageningen, The Netherlands ISBN 978-

flavonoids. IN MESKIN, M. S., BIDLACK, W. R., DAVIES, A. J., LEWIS, D. S. &

R. (1999). Solubilization and stabilization of carotenoids using micelles: Delivery of

Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in

culture system accurately predicts the effects of ascorbic acid and polyphenolic compounds on iron bioavailability in humans. *J Nutr.,* 134, (2004), pp. 2717 - 2721.

The use of plants as medicinal agents to the treat of many diseases has been investigated for a long time since the antique civilizations. Several plants are used in traditional medicine against inflammatory diseases as well as various types of tumors on the base the potential of their chemical constituents. Although many compounds are extremely toxic, when we have the relation between the toxicity of a compound and its chemical pattern of substitution that can result in a more in-depth understanding of these compounds (Atta-ur-Rahman, 2005). Today, even after more than 200 years, the chemistry of natural products remains a challenge and an important field of research in several science areas (chemistry, biology, medicine, agronomy, botany and pharmacy). The reasons for it's large use are the considerable pharmacological potential observed in natural products, in the great development in the process of detection, isolation, purification and, especially, the advances in spectrometric techniques [infrared (IR), mass spectrometry (MS) and nuclear magnetic resonance (NMR 1H and 13C) for structural elucidation of new and complex compounds. These advances were outstanding in both NMR and MS spectrometry. The NMR allows the complete 1H and 13C NMR spectral assignments (chemical shifts and coupling constants) which serve to build a data base to support computer assisted structure elucidation. These data are also useful in the fuller understanding of the correlations between molecular conformation and biological activity of natural substances with biological importance (Loganathan *et al.*, 1990). Mass spectrometry has a huge application in chemistry, biochemistry, medicine, pharmacology, agriculture and food science. Although the mass spectrometric ionization techniques EI (electron impact) and CI (chemical ionization) required the analyte molecules to be present in the gas phase and were thus suitable only for volatile compounds, the development of several desorption ionization methods [FD (field desorption), FABMS (fast atom bombardment), ESIMS (electrospray), MALDI-MS (matrix assisted laser desorption ionization)] allowed the hight-precision mass spectrometric analysis of different classes of biomolecules.

The genus Ximenia belongs to the Olacaceae and comprises about 8 species (Brasileiro et al., 2008): Ximenia roiigi, Ximenia aegyptiaca, Ximenia parviflora, Ximenia coriaceae, Ximenia aculeata, Ximenia caffra, Ximenia americana and Ximenia aegyptica. X. caffra stands out for

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 431

The present review compiles the published chemical and pharmacological information on the species *X. americana* and update important data reported in the last ten years in the

To evaluate the scientific basis for the use of numerous plants species used to treat diseases of infectious origin, crude extracts of these plants were investigated. The antimicrobial activity of the extracts of the various parts of the investigated plants such as roots, leaves, seeds, stem barks and fruits, appears to be due to the presence of secondary metabolites such polyphenols, triterpenes, sterols, saponins, tannins, alkaloids, glycosides and polysaccharides (Geyid *et al.*, 2005; James *et al.*, 2007; Maikai *et al.*,2009; Ogunleye *et al.*,

*X. americana* is a plant used in traditional medicine for the treatment of malaria, leproutic ulcers and infectious diseases of mixed origin by natives in Ethiopia, Guinea, Sudan and in the Northern part of Nigeria (Geyid *et al.*, 2005; James *et al.*, 2007; Magassouba *et al.*, 2007;

The crude extracts of *X. americana* show antimicrobial and antifungal activities. The crude aqueous, methanolic, ethanolic, butanolic and chloroform extracts from different parts (leaves, root, stem and stem bark) of the plant were subjected to phytochemical screening and from the test carried out, it was observed that the secondary metabolites contained were saponins, flavonoids, tannins, terpenoids, sterols, quinones, alkaloids, cyanogenetic glycosides, cardiac glycosides and carbohydrates in the form of sugars and soluble starch. The results of phytochemical screening of various parts solvent extracts of *X. americana* are

The MeOH extract from leaves of *X. americana* inhibited or retarded growth of *Neisseria gonorrhea* organism at dilution as low as 250 µg/ml. This same extract showed antifungal effect against *Candida albicans* and *Cryptococus neoformans* in concentration of 4000 µg/ml. Chemical screening conducted on the extract showed the presence of several secondary metabolites as tannins, sterols, terpenoids, flavonoids and saponins (Geyid *et al.*, 2005). The antimicrobial activities of ethanol extract of the leaves were evaluated against six common bacterial isolates (*Pseudomonas aeruginosa*, *Proteus vulgaris*, *Bacillus subtilis*, *Escherichia coli*, *Staphylococus aureus* and *Candida albicans*) and was active against all of them. The highest degree of activity was for *P. aeruginosa* (inhi bition zone: 20 mm), followed by *B. subtilis* and *C. albicans* (inhibition zone: 10 mm). Activity of the organic extract of the plant was comparable to that of commercially available penicillin disc (2 µg) which was more active against *P. aeruginosa* but less effective against *S. aureus.* The results of phytochemical analysis indicated the presence of saponins, flavonoids, tannins and cyanogenetic glycosides. Alkaloids and anthraquinones were not present (Ogunleye *et al.*, 2003). The root, stem bark and leaves aqueous and methanolic extracts of *X. americana* were tested against five bacteria and they inhibited the growth of *Staphylococus aureus* and *Klebsiella pneumoniae* while *Shigella flexineri* was inhibited by only methanolic leaves, aqueous bark and aqueous leaves extracts. *Salmonella typhi* and *Escherichia coli* were not affected by these extracts. The

scientific literature.

2003).

**2. Biological activity** 

presented in Table 1.

**2.1 Antimicrobial and antifungal activities** 

Maikai *et al.*, 2009; Ogunleye *et al.*, 2003; Omer & Elnima, 2003).

being used in Tanzania for the treatment of irregular menstruation, rheumatism and cancer (Chhabra & Viso, 1990) and, in Limpopo Province, South Africa, for treatment diarrhea (Mathabe, 2006). However, X. americana Linn. is the most common, being native to Australia and Asia where is commonly known as Yellow Plum or Sea Lemon. It is found mainly in tropical regions (Africa, India, New Zealand, Central America and south America), specially Africa and Brazil. The plant is characterized as a small tree spinose 3-4 feet tall, gray or reddish bark, with leaves small, simple, alternate, of bright green color and with a strong smell of almonds. The flowers are yellowish-white, curved and aromatic. Fruit are yellow-orange, aromatic, measuring 1.5 to 2.0 cm in diameter, surrounding a single seed and have a pleasant plum-like flavor (Matos, 2007). In Asia, the young leaves are consumed as a vegetable, however, the leaves also contain cyanide and need to be thoroughly cooked, and should not be eaten in large amounts.

*X.* americana, commonly called "ameixa do mato", "ameixa de espinho" and "ameixa da Bahia", is widely distributed in northeast Brazil. A tea obtained from its barks has been used in popular medicine as cicatrizing, adstringent and as an agent against excessive menstruation. As a powder, it treats stomach ulcers and the seeds are purgative (Braga, 1976; Pio-Correia, 1984). This specimen has been recently examined (Araújo *et al.*, 2008,2009) and the stem ethanolic extract afforded steroids (stigmasterol and sitosterol), triterpenoids (betulinic acid, oleanolic acid, 28-O-(-D-glucopyranosyl) oleanolic acid, 3-oxo-oleanolic acid, 3β–hydroxycicloart-24(*E*)-ene-26-oic acid and sesquiterpenoids (furanoic and widdrane type). A large number of sesquiterpenes are constituents of essential oils of higher plants and seem to intervene in the pharmacological properties attributed to these volatile fractions (Bruneton, 1999). It has been clarified that the biological activities of the liverworths are due to terpenoids and lipophilic aromatic compounds (Atta-ur-Rahman, 1988). Steroids and triterpenes with therapeutic interest and manufacturing employment, are a group of secondary metabolites of outstanding importance (Bruneton, 1999). Considerable recent work strongly indicates the great potential of the triterpenoids as source of use medicinal (Mahato *et al.*, 1992).

Investigations in the past 10 years showed that the constituents of *X. americana* have shown several biological activities such as, antimicrobial, antifungal, anticancer, antineoplastic, antitrypanosomal, antirheumatic, antioxidant, analgesic, moluscicide, pesticidal, also having hepatic and heamatological effects.

In general, the compounds found in *X. americana* were saponins, glicosydes, flavonoids, tannins, phenolics, alkaloids, quinones and terpenoids types. In addition, the plant is potentially rich in fatty acids and glycerides and the seeds contain derivatives cyanide. The identified compounds did not demonstrate a representative pattern of each class. For example, the sesquiterpene were furanoic and widdrane while, triterpenes exhibited oleanane and cycloartane skeletal type. Concerning the fatty acids, in addition to common C16, C18 and C22, a distinctive feature is the presence of acetylenic, as well as, very long chain fatty acids.

We can see, from all the information summarized above, that work on plants of the genus Ximenia is justified, particularly *Ximenia americana* species, where systematic study is still not satisfactory, specially, relative to specific biological activity of their chemical constituents.

The present review compiles the published chemical and pharmacological information on the species *X. americana* and update important data reported in the last ten years in the scientific literature.

### **2. Biological activity**

430 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

being used in Tanzania for the treatment of irregular menstruation, rheumatism and cancer (Chhabra & Viso, 1990) and, in Limpopo Province, South Africa, for treatment diarrhea (Mathabe, 2006). However, X. americana Linn. is the most common, being native to Australia and Asia where is commonly known as Yellow Plum or Sea Lemon. It is found mainly in tropical regions (Africa, India, New Zealand, Central America and south America), specially Africa and Brazil. The plant is characterized as a small tree spinose 3-4 feet tall, gray or reddish bark, with leaves small, simple, alternate, of bright green color and with a strong smell of almonds. The flowers are yellowish-white, curved and aromatic. Fruit are yellow-orange, aromatic, measuring 1.5 to 2.0 cm in diameter, surrounding a single seed and have a pleasant plum-like flavor (Matos, 2007). In Asia, the young leaves are consumed as a vegetable, however, the leaves also contain cyanide and need to be thoroughly cooked,

*X.* americana, commonly called "ameixa do mato", "ameixa de espinho" and "ameixa da Bahia", is widely distributed in northeast Brazil. A tea obtained from its barks has been used in popular medicine as cicatrizing, adstringent and as an agent against excessive menstruation. As a powder, it treats stomach ulcers and the seeds are purgative (Braga, 1976; Pio-Correia, 1984). This specimen has been recently examined (Araújo *et al.*, 2008,2009) and the stem ethanolic extract afforded steroids (stigmasterol and sitosterol), triterpenoids (betulinic acid, oleanolic acid, 28-O-(-D-glucopyranosyl) oleanolic acid, 3-oxo-oleanolic acid, 3β–hydroxycicloart-24(*E*)-ene-26-oic acid and sesquiterpenoids (furanoic and widdrane type). A large number of sesquiterpenes are constituents of essential oils of higher plants and seem to intervene in the pharmacological properties attributed to these volatile fractions (Bruneton, 1999). It has been clarified that the biological activities of the liverworths are due to terpenoids and lipophilic aromatic compounds (Atta-ur-Rahman, 1988). Steroids and triterpenes with therapeutic interest and manufacturing employment, are a group of secondary metabolites of outstanding importance (Bruneton, 1999). Considerable recent work strongly indicates the great potential of the triterpenoids as source of use medicinal

Investigations in the past 10 years showed that the constituents of *X. americana* have shown several biological activities such as, antimicrobial, antifungal, anticancer, antineoplastic, antitrypanosomal, antirheumatic, antioxidant, analgesic, moluscicide, pesticidal, also

In general, the compounds found in *X. americana* were saponins, glicosydes, flavonoids, tannins, phenolics, alkaloids, quinones and terpenoids types. In addition, the plant is potentially rich in fatty acids and glycerides and the seeds contain derivatives cyanide. The identified compounds did not demonstrate a representative pattern of each class. For example, the sesquiterpene were furanoic and widdrane while, triterpenes exhibited oleanane and cycloartane skeletal type. Concerning the fatty acids, in addition to common C16, C18 and C22, a distinctive feature is the presence of acetylenic, as well as, very long

We can see, from all the information summarized above, that work on plants of the genus Ximenia is justified, particularly *Ximenia americana* species, where systematic study is still not satisfactory, specially, relative to specific biological activity of their chemical

and should not be eaten in large amounts.

having hepatic and heamatological effects.

(Mahato *et al.*, 1992).

chain fatty acids.

constituents.

#### **2.1 Antimicrobial and antifungal activities**

To evaluate the scientific basis for the use of numerous plants species used to treat diseases of infectious origin, crude extracts of these plants were investigated. The antimicrobial activity of the extracts of the various parts of the investigated plants such as roots, leaves, seeds, stem barks and fruits, appears to be due to the presence of secondary metabolites such polyphenols, triterpenes, sterols, saponins, tannins, alkaloids, glycosides and polysaccharides (Geyid *et al.*, 2005; James *et al.*, 2007; Maikai *et al.*,2009; Ogunleye *et al.*, 2003).

*X. americana* is a plant used in traditional medicine for the treatment of malaria, leproutic ulcers and infectious diseases of mixed origin by natives in Ethiopia, Guinea, Sudan and in the Northern part of Nigeria (Geyid *et al.*, 2005; James *et al.*, 2007; Magassouba *et al.*, 2007; Maikai *et al.*, 2009; Ogunleye *et al.*, 2003; Omer & Elnima, 2003).

The crude extracts of *X. americana* show antimicrobial and antifungal activities. The crude aqueous, methanolic, ethanolic, butanolic and chloroform extracts from different parts (leaves, root, stem and stem bark) of the plant were subjected to phytochemical screening and from the test carried out, it was observed that the secondary metabolites contained were saponins, flavonoids, tannins, terpenoids, sterols, quinones, alkaloids, cyanogenetic glycosides, cardiac glycosides and carbohydrates in the form of sugars and soluble starch. The results of phytochemical screening of various parts solvent extracts of *X. americana* are presented in Table 1.

The MeOH extract from leaves of *X. americana* inhibited or retarded growth of *Neisseria gonorrhea* organism at dilution as low as 250 µg/ml. This same extract showed antifungal effect against *Candida albicans* and *Cryptococus neoformans* in concentration of 4000 µg/ml. Chemical screening conducted on the extract showed the presence of several secondary metabolites as tannins, sterols, terpenoids, flavonoids and saponins (Geyid *et al.*, 2005). The antimicrobial activities of ethanol extract of the leaves were evaluated against six common bacterial isolates (*Pseudomonas aeruginosa*, *Proteus vulgaris*, *Bacillus subtilis*, *Escherichia coli*, *Staphylococus aureus* and *Candida albicans*) and was active against all of them. The highest degree of activity was for *P. aeruginosa* (inhi bition zone: 20 mm), followed by *B. subtilis* and *C. albicans* (inhibition zone: 10 mm). Activity of the organic extract of the plant was comparable to that of commercially available penicillin disc (2 µg) which was more active against *P. aeruginosa* but less effective against *S. aureus.* The results of phytochemical analysis indicated the presence of saponins, flavonoids, tannins and cyanogenetic glycosides. Alkaloids and anthraquinones were not present (Ogunleye *et al.*, 2003). The root, stem bark and leaves aqueous and methanolic extracts of *X. americana* were tested against five bacteria and they inhibited the growth of *Staphylococus aureus* and *Klebsiella pneumoniae* while *Shigella flexineri* was inhibited by only methanolic leaves, aqueous bark and aqueous leaves extracts. *Salmonella typhi* and *Escherichia coli* were not affected by these extracts. The

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 433

glycosides, saponins, tannins and flavonoids while alkaloids were absent in all the extracts. It was concluded that the extracts of methanolic roots, stem bark and leaves have bacteridal activities over the concentration of 2,5x104 - 1,25x104 gmL-1 and that the presence of carbohydrates, glycosides, flavonoids and tannins in the diferent extracts are responsible for their antibacterial activity. The antimicrobial properties of the bark, leave, root and stem extracts of *Ximenia americana* were screened against *Bacillus subtilis*, *Staphyllococus aureus*, *Escherishia coli* and *Pseudomonas aeruginosa* (Table 2) using the cup-plate agar diffusion method

system % Yield Inhibition zone (mm) MIC (mg/ml)

CHCl3 1.1 13 12 11 15 N.D N.D N.D N.D MeOH 21.1 23 30 19 22 0.31 0.62 19.79 19.79 H2O 8.9 18 18 16 14 0.40 1.62 3.24 1.62

CHCl3 10.7 13 14 - 12 N.D N.D N.D N.D MeOH 26.6 23 22 - 25 1.55 0.77 9997 12.45 H2O 5.0 17 19 16 22 0.59 1.19 >25.5 19.11

CHCl3 2.2 15 13 12 13 N.D N.D N.D N.D MeOH 3.7 15 21 19 15 3.27 6.54 >34.88 >34.48 H2O 5.7 13 13 - - 2.68 10.74 28.65 28.65

CHCl3 2.7 - 11 11 - N.D N.D N.D N.D MeOH 11.8 20 25 - 24 >72.75 3.41 >72.75 >72.75 H2O 2.7 17 17 13 13 5.12 5.12 >13.65 >13.65

B.s, *Bacillus subtilis;* S.a, *Staphyllococus aureus*; E.c, *Escherichia coli*; Ps.a, *Pseudomonas aeruginosa;* 

concentration of extracts 100 mg/ml, 0.1 ml/cup; inhibition zones are the mean of three replicates. MIC,

The methanolic extract was the most active one. The aqueous extract also exhibited high activity which justifies its traditional use. *Staphyllococus aureus* was the most susceptible bacterium among the tested organisms. The table 3 show the antibacterial activity of

Several other studies to determine the presence of antimicrobial activity in crude extracts of *Ximenia americana* were performed (Magassouba *et al.*, 2007; Maikai *et al.*, 2009). In all, the various extracts were found to have broad spectrum effect against standard organisms (*Escherichia coli*, *Pseudomonas aeruginosa, Staphylococus aureus, Proteus vulgaris, Candida albicans, Bacillus subtilis, Salmonella typhi* and *Shigella flexineri*) and supports the traditional

In general, the antimicrobial activity of extracts of the various parts of the plants appears to be due to presence of secondary metabolites. In some experiments, was remarked that the

Table 2. Antibacterial activity of *Ximenia americana* extracts against standard organisms.

B.s S.a E.c Ps.a B.s S.a E.c Ps.a

and the minimum inhibitory concentration by agar dilution method (Omer *et al*., 2003).

Part used Solvent

minimum inhibitory concentration; N.D, not detected.

*Ximenia Americana* against the pharmaceuticals patterns.

usage of this plant as remedy in treatement of microbial infections.

Bark

Leaves

Root

Stem

(Placed on the table 2)


+: present; -: absent; Ref.: references

Some extracts showed the presence of carbohydrates in the form of sugars and soluble starch (James *et al.*, 2007 & Ogunleye *et al.*, 2003); few extracts showed also the presence of cyanogenetic glycosides (Ogunleye *et al.*, 2003). Quinones are of the anthraquinone type; terpenes are sesquiterpenes and triterpenes type (Araújo *et al.*, 2008, 2009).

Table 1. Phytochemical screening of stem bark, leaves, root and stem extracts of *X. Americana.* (Placed on the table 1)

Minimum Inhibitory Concentration (MIC) was only evident for the methanolic extracts at 1.25x104 µgmL-1 (1:4) against *Staphylococus aureus* while the Minimum Bactericidal Concentration (MBC) of the extracts was obtained at 2.50x10-4µg mL-1 (1:2) (James *et al.*, 2007). From the results, inhibitory activity of extracts (methanolic root) was more pronounced on *Klebsiella pneumonia* whereas it shows no activity against *Escherichia coli*, *Salmonella typhi* and *Shigella flexineri*. The methanolic root extract showed highly significant (p<0.05) activity on *Klebsiella pneumonia* when compared with leaf extracts and methanolic bark extract. The phytochemical constituents present in the extracts were carbohydrates in the form of sugars and soluble starch (except for aqueous and leaves extracts), cardiac

Flavonoids

Leaves MeOH + + - + - - - Geyid *et al.*, 2005

Leaves H2O + - - + + - + - Ogunleye *et al.*, EtOH + - - + + - + - 2003

Leaves H2O + - - + + - + + James *et al.*, 2007 MeOH + - - + + - + -

Root H2O + - - + + - + +

BuOH + - + + + + + -

H2O + - + + + - - +

Root CHCl3 Omer & Elnima, MeOH + + 2003)

Stem EtOH + + Araújo *et al.*,

Some extracts showed the presence of carbohydrates in the form of sugars and soluble starch (James *et al.*, 2007 & Ogunleye *et al.*, 2003); few extracts showed also the presence of cyanogenetic glycosides (Ogunleye *et al.*, 2003). Quinones are of the anthraquinone type; terpenes are sesquiterpenes and

Minimum Inhibitory Concentration (MIC) was only evident for the methanolic extracts at 1.25x104 µgmL-1 (1:4) against *Staphylococus aureus* while the Minimum Bactericidal Concentration (MBC) of the extracts was obtained at 2.50x10-4µg mL-1 (1:2) (James *et al.*, 2007). From the results, inhibitory activity of extracts (methanolic root) was more pronounced on *Klebsiella pneumonia* whereas it shows no activity against *Escherichia coli*, *Salmonella typhi* and *Shigella flexineri*. The methanolic root extract showed highly significant (p<0.05) activity on *Klebsiella pneumonia* when compared with leaf extracts and methanolic bark extract. The phytochemical constituents present in the extracts were carbohydrates in the form of sugars and soluble starch (except for aqueous and leaves extracts), cardiac

Table 1. Phytochemical screening of stem bark, leaves, root and stem extracts of *X.* 

Alcaloids

MeOH + - + + + + + + Maikai *et al.*, 2009

Class of Compounds Ref.

Glycosids

Quinones

2008,2009

Cardiac

Plant part Solvent

Stem

Stem bark

MeOH

+: present; -: absent; Ref.: references

triterpenes type (Araújo *et al.*, 2008, 2009).

*Americana.* (Placed on the table 1)

Tannins

Steroids

Terpenes

bark H2O + - - + + - + + MeOH + - - + + - + +

Saponins

glycosides, saponins, tannins and flavonoids while alkaloids were absent in all the extracts. It was concluded that the extracts of methanolic roots, stem bark and leaves have bacteridal activities over the concentration of 2,5x104 - 1,25x104 gmL-1 and that the presence of carbohydrates, glycosides, flavonoids and tannins in the diferent extracts are responsible for their antibacterial activity. The antimicrobial properties of the bark, leave, root and stem extracts of *Ximenia americana* were screened against *Bacillus subtilis*, *Staphyllococus aureus*, *Escherishia coli* and *Pseudomonas aeruginosa* (Table 2) using the cup-plate agar diffusion method and the minimum inhibitory concentration by agar dilution method (Omer *et al*., 2003).


B.s, *Bacillus subtilis;* S.a, *Staphyllococus aureus*; E.c, *Escherichia coli*; Ps.a, *Pseudomonas aeruginosa;*  concentration of extracts 100 mg/ml, 0.1 ml/cup; inhibition zones are the mean of three replicates. MIC, minimum inhibitory concentration; N.D, not detected.

Table 2. Antibacterial activity of *Ximenia americana* extracts against standard organisms. (Placed on the table 2)

The methanolic extract was the most active one. The aqueous extract also exhibited high activity which justifies its traditional use. *Staphyllococus aureus* was the most susceptible bacterium among the tested organisms. The table 3 show the antibacterial activity of *Ximenia Americana* against the pharmaceuticals patterns.

Several other studies to determine the presence of antimicrobial activity in crude extracts of *Ximenia americana* were performed (Magassouba *et al.*, 2007; Maikai *et al.*, 2009). In all, the various extracts were found to have broad spectrum effect against standard organisms (*Escherichia coli*, *Pseudomonas aeruginosa, Staphylococus aureus, Proteus vulgaris, Candida albicans, Bacillus subtilis, Salmonella typhi* and *Shigella flexineri*) and supports the traditional usage of this plant as remedy in treatement of microbial infections.

In general, the antimicrobial activity of extracts of the various parts of the plants appears to be due to presence of secondary metabolites. In some experiments, was remarked that the

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 435

*et al.*, 2007; Maikai *et al.*, 2009; Ogunleye *et al.*, 2003). Although it is difficult to speculate on the mechanism of action of the constituents of the extracts on the basis of studies conducted to date, the antimicrobial activity of these extracts is due, no doubt, the presence of these secondary metabolites. In the case of extracts of *Ximenia americana,* probably, due the presence of tannins, flavonoids, triterpenes/steroids, saponins or cyanogenetic glycosides. In summary, the results justified the use of *X. americana* as having antibacterial properties and support its use as agent in new drugs for therapy of infectious diseases caused by pathogens.

Olecaceous seed oils are a rich source of acetylenic lipids and unsaturated fatty acids (Badami & Patil, 1981 & Sptizer *et al.*, 1997). Acetylenic metabolites show some biological activities including, insecticidal activity (Jacbson, 1971). *X. americana* was recorded to contain octadec-11-en-9-ynoic acid, named xymeninic acid as well as icosenoic-triacontenoic acids, all of which belong to the -9 series (Rezanka, & Sigler, 2007). Bioactivity-driven fractionation of the CHCl3 extract of the root of *X. americana* using the Brine Shrimp Lethality Test (BST) and hatchability test with *Clavigralla tomentosicollis* eggs yielded two fractions (F006, soluble in petroleum ether and F005, soluble in 10% H2O in MeOH) as the most actives (F005, BST LC50 78 (129-48) µg/mL and F006, BST LC%50 76(121-49) µg/mL) (Fatope *et al.*, 2000). A combination of F005 and F006 was submitted to hatchability test (inhibition of hatching = 68 % of control) and successive BST-dircted fractionation on silica gel column and preparative TLC yielded oleanene palmitates (**1**), β-sitosterol (**2**) and C18

COOH

HO **<sup>1</sup> <sup>2</sup>**

The substance **4** suppressed the hatchability of *C. tomentosicollis* eggs at 92 % of control when tested at 4 x 10 4 µg/mL (correcting for unhatched eggs in the control using Abbott's

% control = [(% unhatched of treated group - % unhatched of untreated group)/ (100 - % unhatched of untreated group)] x 100 These acetylenic fatty acids show characteristic spectrometric data. The 13C NMR spectrum of **3** displayed absorptions diagnostic of acetylenic carbons at C 80.4 (C) and 80.1 (C) and of carboxylic carbon at C 189.1 (C), in agreement with its IR spectrum which exhibited bands at 2200 and at 1713 cm-1, characteristic of acetylenic and acid groups, respectively. Compound **3** had molecular formulaC18H32O2, as established by HREI-MS (*m*/*z* 280.2378 for [M+]) in combination with its 1H and 13C NMR spectra. From analysis spectral data compound **3** was thus established as octadeca-5-ynoic acid (tariric acid). Compound **4** had a

**2.2 Pesticidal activity** 

acetylenic fatty acids (**3** and **4**) as yellow oils.

O

O ( )14

formula):


Interpretation of sensitivity test results: Gram(+) bacteria\*; Gram(-) bacteria \*\*;

>18 mm (M.DIZ)= sensitive; >16 mm (M.DIZ)=sensitive;

14-18 mm (M.DIZ)= intermediate; 13-16 mm (M.DIZ)= intermediate;

<14 mm (M.DIZ)= resistant; and < 13mm (M.DIZ)= resistant.

Table 3. The activity of *Ximenia Americana* against the clinical isolates. (Placed on the table 3)

plants which accumulate polyphenols, tannins and unsaturated sterols/terpenes showed to inhibit or significantly retard growth of eight of the ten test organisms; the species, which constitute polyphenols and unsaturated sterols/terpenes; and polyphenols, tannins, unsaturated sterols/terpenes, saponins and glycosides inhibited six organisms each while, those with polyphenols, tannins, unsaturated sterols/terpenes, saponins; and alkaloids and unsaturated sterols/terpenes inhibited growth of five bacterial strains each (Geyid *et al.*, 2005). Cyanogenetic glycosides are reported to possess antimicrobial activity (Finnermore *et al.*, 1988). Tannins have been traditionally used for protection of inflamed surfaces of the mouth and treatment of catarrh, wounds, haemorrhoids and diarrhea and as antidote in heavy metal poisoning. They have the ability to inactivate microbial adhesions, enzymes, cell envelope transport proteins and also complex with polysaccharide (Maikai *et al.*, 2009; Scalbert, 1991; Ya *et al.*, 1988). Flavonoids are naturally occurring phenols, which posses numerous biological activities including anti-inflamatory, antiallegic, antibacterial, antifungal and vasoprotective effects and, also have been reported to complex with extracellular and soluble proteins and to complex with bacterial cell walls (Dixon *et al.*, 1983; Geyid *et al.*, 2005; Hostettman *et al.*, 1995; James *et al.*, 2007; Maikai *et al.*, 2009; Ogunleye *et al.*, 2003). Terpenoids have also been reported to be active against bacteria, the mechanism of action involve membrane disruption by the lipophilic compounds (Geyid *et al.*, 2005; James *et al.*, 2007; Maikai *et al.*, 2009; Ogunleye *et al.*, 2003). Although it is difficult to speculate on the mechanism of action of the constituents of the extracts on the basis of studies conducted to date, the antimicrobial activity of these extracts is due, no doubt, the presence of these secondary metabolites. In the case of extracts of *Ximenia americana,* probably, due the presence of tannins, flavonoids, triterpenes/steroids, saponins or cyanogenetic glycosides.

In summary, the results justified the use of *X. americana* as having antibacterial properties and support its use as agent in new drugs for therapy of infectious diseases caused by pathogens.

#### **2.2 Pesticidal activity**

434 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

MDIZ B.s S.a E.c Ps.a

40 14 25 - - 20 13 22 - - 10 - 19 - - 5 - 18 - -

40 - 37 - - 20 - 33 - - 10 - 28 - - 5 - 24 - -

40 - 29 - - 20 - 27 - - 10 - 22 - - 5 - 18 - -

40 24 18 25 22 20 22 16 17 15 10 17 14 16 12 5 15 13 11 -

Reference drugs

Ampicillin

Benzyl penicillin

Cloxacillin

Gentamicin

Concentration /ml

Interpretation of sensitivity test results: Gram(+) bacteria\*; Gram(-) bacteria \*\*;

Table 3. The activity of *Ximenia Americana* against the clinical isolates. (Placed on the table 3)

plants which accumulate polyphenols, tannins and unsaturated sterols/terpenes showed to inhibit or significantly retard growth of eight of the ten test organisms; the species, which constitute polyphenols and unsaturated sterols/terpenes; and polyphenols, tannins, unsaturated sterols/terpenes, saponins and glycosides inhibited six organisms each while, those with polyphenols, tannins, unsaturated sterols/terpenes, saponins; and alkaloids and unsaturated sterols/terpenes inhibited growth of five bacterial strains each (Geyid *et al.*, 2005). Cyanogenetic glycosides are reported to possess antimicrobial activity (Finnermore *et al.*, 1988). Tannins have been traditionally used for protection of inflamed surfaces of the mouth and treatment of catarrh, wounds, haemorrhoids and diarrhea and as antidote in heavy metal poisoning. They have the ability to inactivate microbial adhesions, enzymes, cell envelope transport proteins and also complex with polysaccharide (Maikai *et al.*, 2009; Scalbert, 1991; Ya *et al.*, 1988). Flavonoids are naturally occurring phenols, which posses numerous biological activities including anti-inflamatory, antiallegic, antibacterial, antifungal and vasoprotective effects and, also have been reported to complex with extracellular and soluble proteins and to complex with bacterial cell walls (Dixon *et al.*, 1983; Geyid *et al.*, 2005; Hostettman *et al.*, 1995; James *et al.*, 2007; Maikai *et al.*, 2009; Ogunleye *et al.*, 2003). Terpenoids have also been reported to be active against bacteria, the mechanism of action involve membrane disruption by the lipophilic compounds (Geyid *et al.*, 2005; James

14-18 mm (M.DIZ)= intermediate; 13-16 mm (M.DIZ)= intermediate; <14 mm (M.DIZ)= resistant; and < 13mm (M.DIZ)= resistant.

>18 mm (M.DIZ)= sensitive; >16 mm (M.DIZ)=sensitive;

Olecaceous seed oils are a rich source of acetylenic lipids and unsaturated fatty acids (Badami & Patil, 1981 & Sptizer *et al.*, 1997). Acetylenic metabolites show some biological activities including, insecticidal activity (Jacbson, 1971). *X. americana* was recorded to contain octadec-11-en-9-ynoic acid, named xymeninic acid as well as icosenoic-triacontenoic acids, all of which belong to the -9 series (Rezanka, & Sigler, 2007). Bioactivity-driven fractionation of the CHCl3 extract of the root of *X. americana* using the Brine Shrimp Lethality Test (BST) and hatchability test with *Clavigralla tomentosicollis* eggs yielded two fractions (F006, soluble in petroleum ether and F005, soluble in 10% H2O in MeOH) as the most actives (F005, BST LC50 78 (129-48) µg/mL and F006, BST LC%50 76(121-49) µg/mL) (Fatope *et al.*, 2000). A combination of F005 and F006 was submitted to hatchability test (inhibition of hatching = 68 % of control) and successive BST-dircted fractionation on silica gel column and preparative TLC yielded oleanene palmitates (**1**), β-sitosterol (**2**) and C18 acetylenic fatty acids (**3** and **4**) as yellow oils.

The substance **4** suppressed the hatchability of *C. tomentosicollis* eggs at 92 % of control when tested at 4 x 10 4 µg/mL (correcting for unhatched eggs in the control using Abbott's formula):

% control = [(% unhatched of treated group - % unhatched of untreated group)/ (100 - % unhatched of untreated group)] x 100

These acetylenic fatty acids show characteristic spectrometric data. The 13C NMR spectrum of **3** displayed absorptions diagnostic of acetylenic carbons at C 80.4 (C) and 80.1 (C) and of carboxylic carbon at C 189.1 (C), in agreement with its IR spectrum which exhibited bands at 2200 and at 1713 cm-1, characteristic of acetylenic and acid groups, respectively. Compound **3** had molecular formulaC18H32O2, as established by HREI-MS (*m*/*z* 280.2378 for [M+]) in combination with its 1H and 13C NMR spectra. From analysis spectral data compound **3** was thus established as octadeca-5-ynoic acid (tariric acid). Compound **4** had a

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 437

*americana* in doses containing 10 to 100 mg/kg P.C, inhibits contractions of the abdomen with analgesic effects comparable to those of phenylbutazone. In fact, at doses of 100 mg / kg P.C, phenylbutazone causes an inhibition of pain in 45.2±2%. The percentage of inhibition by extract of *X. ameriacana* is 61.1±% in the same concentration. These properties are probably due to the presence of flavonoids and saponins, detected in the extract (Soro *et al.*, 2009). The analgesic activity of the methanol extract of *X. americana* leaf was investigated in chemical models of nociception in mice. The extract at doses of 200, 400 and 600 mg/kg i.p. produced an inhibition of 54.13, 63.74, and 66.4% respectively, of the abdominal writhes induced by acetic acid in mice. In the formalin test, the administration of 200, 400 and 600 mg/kg i.p. had no effects in the first phase (0 to 5 min) but produced a dose dependent analgesic effect on the second phase (15 to 40 min) with inhibitions of the licking time of 29.3, 47.8 and 59.8%, respectively. These observations suggested that methanol extract of *X.* 

The bark of stem of *X. americana* has been used in West Africa for the treatment of pain and fever. To verify this second property, the treatment of rats in hyperthermia with *Ximenia americana* stem bark aqueous and with beer yeast was compared to those obtained with lysine acetylsalicylate (Aspegic). The study showed an antipyretic action of the extract. Moreover, the toxicological study of the stem extract indicated a LD50 of 237.5 mg/kg P.C according to the classification of Diezi this plant is relatively toxic. The experiments show that the properties of *X. americana* could due to the presence of saponosides, as show by screening tests performed in this study. These results justified the use of *X. americana* in

The in vitro antitrypanosomal activity of methanolic and aqueous extracts of stem bark of *Ximenia americana* was evaluated on Trypanosoma congolense. Blood obtained from a high infected mice with *T. congolense* (10(7) was incubated with methanolic and aqueous extracts at 20, 10 and 5 mg/ml and Diminal(R) (diminazene aceturate) at 200, 100 and 50 µg/ml in a 96 micro plate. The results revealed that methanol and aqueous extracts had activity at 20 and 40 mg/ml however, the methanolic extracts were more active than aqueous extracts at 10 and 5 mg/ml. Phytochemical screening of the methanolic and aqueous extracts of the bark showed that they both had flavonoids, anthraquinones, saponins, terpenes and tannins. The aqueous and methanolic extracts appears to show some potential activity against *T.* 

Plants have been show to provide a useful source of natural products that are effective in the treatment of human neoplastic diseases. Information recorded from ancient civilizations has demonstrated the use of plants in search of treatment for various types of cancer (Hartwell, 1967-1971). An analysis of plant materials that had been studied at the National Cancer Institute (NCI), USA for discovering new anticancer drugs showed that if ethnopharmacological information had been used, the yield of plants harboring antineoplastic activity would have been significantly increased (Spjut & Perdue, 1976). The

*americana* leaf possesses analgesic activity (Siddaiah *et al.*, 2009).

traditional cure of fever treatment (Soro *et al.*, 2009).

**2.4 Antipyretic activity** 

**2.5 Antitrypanosomal activity** 

*congolense* (Maikai *et al.*, 2008).

**2.6 Anticancer activity** 

mol wt 6 mass units less than that of **3** with molecular formula C18H26O2 as revealed by HREI-MS (*m*/*z* 274.2021 for [M+]) in combination with its 1H and 13CNMR spectra. The 13C NMR spectrum of **4** displayed absorptions diagnostic of acetylenic carbons at C 83.4 (C) and 74.1 (C) and of carboxylic carbon at 179.3 (C), in agreement with its IR spectrum which exhibited bands at 2232 and 1702 cm-1, characteristic of acetylenic and acid groups, respectively. The 13C RMN spectrum also exhibited six resonance at C 148.2 (CH), 140.9 (CH), 136.9 (CH), 129.8 (CH), 109.3 3(CH) and 108.6 (CH), revealing the presence of three double bonds. From a detailed spectral analysis considering, especially, the multiplicity of signals and coupling constants in the 1H NMR spectrum, as well as the presence of diagnostic peaks in the mass spectrum, compound **4** was thus established as 10*Z*,14*E,*16*E*-octadeca - 10,14,16-triene-12-ynoic acid, a ene-ene-yneene acetylenic fatty acid (Fatope *et al.*, 2000).

$$\text{CH}\_3\text{---}(\text{CH}\_2)\_{11}\text{C}\underline{\text{m}}\text{C}\text{---}(\text{CH}\_2)\_3\text{CO}\_2\text{H} \qquad \text{CH}\_3\text{CH=C}\_{16}\text{H}\text{CH=C}\_{14}\text{HC}\underline{\text{m}}\text{C}\_{12}\text{CH=C}\_{10}\text{H}(\text{CH}\_2)\_6\text{CO}\_2\text{H}$$

$$\text{3}$$

In addition, *Ximenia* seed oil have been found to contain fatty acids with more than 22 carbon atoms (very long fatty acids) which are found only rarely in nature. Using liquid chromatography in combination with mass spectrometry was founded that *Ximenia* oil to contain fatty acids with chain length C34 and C36 (Rezanka & Sigler, 2007). Effectively, two very long chain unsaturated fatty acids C40 and C35 (**5** and **6**) were isolated (Saeed & Bashier, 2010) from *X. americana* seeds and fruits, respectively. The mass spectrum of the major component (**5**) showed a molecular ion at m/z 604 corresponding to the molecular formula C40H76O3. The IR spectrum of **5** showed a broad absorption band at 3600-3200 cm-1 (OH) and the presence of strong absorption at 1742 cm-1 attributed to ester group. The base peak appeared at m/z 55 (C4H7+) due to allylic bond cleavage and peaks at m/z 479 and 151 furnished from fragmentation in C28-C29 and C26-C27, respectively. In addition, the peaks at m/z 31, 59, 73 and 74 (McLafferty rearrangement) were compatible with unit CH3OCO(CH2)3-. The compound **5** was identified as methyl-14,14-dimethyl-18 hydroxyheptatracont-27,35-dienoate. The mass spectrum of **6** showed a molecular ion at 578, corresponding to the molecular formula C35H62O6. The IR spectrum showed bands at 3500, 1731 and 1645 cm-1 corresponding to OH, C=O and C=C groups, respectively. The base peak appeared at m/z 73 (C3H5O2+) which is characteristic for the methyl ester, reinforced by additional peaks at m/z 31, 59 and 74 (McLafferty rearrangement). An peak at m/z 479 was due to M-C5H7O2 and one at m/z 339 is due to the cleavage C13-C14 while, those at m/z 126 and 265 were due to C7H10O2 and M-C17H28O2, respectively. The compound **6** was identified as dimethyl-5-Methyl-28,29-dihydroxydotriacont-3,14,26-triendioate.

### [CH3OCO(CH2)12C(CH3)2(CH2)3CHOH(CH2)8CH=CH(CH2)6CH=CHCH3] **5**

#### [CH3OCOCH2)CH=CHCH3(CH2)8CH=CH(CH2)10CH=CH(CHOH)2(CH2)2COOCH3]

#### **6**

#### **2.3 Analgesic activity**

The aqueous extract of stem bark of *X. american* has analgesic properties that justify its use popular in countries such as Tanzania, Senegal, Zimbawe and Nigeria. The extract of *X.*  *americana* in doses containing 10 to 100 mg/kg P.C, inhibits contractions of the abdomen with analgesic effects comparable to those of phenylbutazone. In fact, at doses of 100 mg / kg P.C, phenylbutazone causes an inhibition of pain in 45.2±2%. The percentage of inhibition by extract of *X. ameriacana* is 61.1±% in the same concentration. These properties are probably due to the presence of flavonoids and saponins, detected in the extract (Soro *et al.*, 2009). The analgesic activity of the methanol extract of *X. americana* leaf was investigated in chemical models of nociception in mice. The extract at doses of 200, 400 and 600 mg/kg i.p. produced an inhibition of 54.13, 63.74, and 66.4% respectively, of the abdominal writhes induced by acetic acid in mice. In the formalin test, the administration of 200, 400 and 600 mg/kg i.p. had no effects in the first phase (0 to 5 min) but produced a dose dependent analgesic effect on the second phase (15 to 40 min) with inhibitions of the licking time of 29.3, 47.8 and 59.8%, respectively. These observations suggested that methanol extract of *X. americana* leaf possesses analgesic activity (Siddaiah *et al.*, 2009).

#### **2.4 Antipyretic activity**

436 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

mol wt 6 mass units less than that of **3** with molecular formula C18H26O2 as revealed by HREI-MS (*m*/*z* 274.2021 for [M+]) in combination with its 1H and 13CNMR spectra. The 13C NMR spectrum of **4** displayed absorptions diagnostic of acetylenic carbons at C 83.4 (C) and 74.1 (C) and of carboxylic carbon at 179.3 (C), in agreement with its IR spectrum which exhibited bands at 2232 and 1702 cm-1, characteristic of acetylenic and acid groups, respectively. The 13C RMN spectrum also exhibited six resonance at C 148.2 (CH), 140.9 (CH), 136.9 (CH), 129.8 (CH), 109.3 3(CH) and 108.6 (CH), revealing the presence of three double bonds. From a detailed spectral analysis considering, especially, the multiplicity of signals and coupling constants in the 1H NMR spectrum, as well as the presence of diagnostic peaks in the mass spectrum, compound **4** was thus established as 10*Z*,14*E,*16*E*-octadeca - 10,14,16-triene-12-ynoic acid, a ene-ene-yneene acetylenic fatty acid (Fatope *et al.*, 2000).

CH3 (CH2)11C C (CH2)3CO2H CH3CH C16HCH C14HC C12CH C10H(CH2)8CO2H

In addition, *Ximenia* seed oil have been found to contain fatty acids with more than 22 carbon atoms (very long fatty acids) which are found only rarely in nature. Using liquid chromatography in combination with mass spectrometry was founded that *Ximenia* oil to contain fatty acids with chain length C34 and C36 (Rezanka & Sigler, 2007). Effectively, two very long chain unsaturated fatty acids C40 and C35 (**5** and **6**) were isolated (Saeed & Bashier, 2010) from *X. americana* seeds and fruits, respectively. The mass spectrum of the major component (**5**) showed a molecular ion at m/z 604 corresponding to the molecular formula C40H76O3. The IR spectrum of **5** showed a broad absorption band at 3600-3200 cm-1 (OH) and the presence of strong absorption at 1742 cm-1 attributed to ester group. The base peak appeared at m/z 55 (C4H7+) due to allylic bond cleavage and peaks at m/z 479 and 151 furnished from fragmentation in C28-C29 and C26-C27, respectively. In addition, the peaks at m/z 31, 59, 73 and 74 (McLafferty rearrangement) were compatible with unit CH3OCO(CH2)3-. The compound **5** was identified as methyl-14,14-dimethyl-18 hydroxyheptatracont-27,35-dienoate. The mass spectrum of **6** showed a molecular ion at 578, corresponding to the molecular formula C35H62O6. The IR spectrum showed bands at 3500, 1731 and 1645 cm-1 corresponding to OH, C=O and C=C groups, respectively. The base peak appeared at m/z 73 (C3H5O2+) which is characteristic for the methyl ester, reinforced by additional peaks at m/z 31, 59 and 74 (McLafferty rearrangement). An peak at m/z 479 was due to M-C5H7O2 and one at m/z 339 is due to the cleavage C13-C14 while, those at m/z 126 and 265 were due to C7H10O2 and M-C17H28O2, respectively. The compound **6** was identified

> [CH3OCO(CH2)12C(CH3)2(CH2)3CHOH(CH2)8CH=CH(CH2)6CH=CHCH3] **5**

[CH3OCOCH2)CH=CHCH3(CH2)8CH=CH(CH2)10CH=CH(CHOH)2(CH2)2COOCH3] **6**

The aqueous extract of stem bark of *X. american* has analgesic properties that justify its use popular in countries such as Tanzania, Senegal, Zimbawe and Nigeria. The extract of *X.* 

 **3 4** 

as dimethyl-5-Methyl-28,29-dihydroxydotriacont-3,14,26-triendioate.

**2.3 Analgesic activity** 

The bark of stem of *X. americana* has been used in West Africa for the treatment of pain and fever. To verify this second property, the treatment of rats in hyperthermia with *Ximenia americana* stem bark aqueous and with beer yeast was compared to those obtained with lysine acetylsalicylate (Aspegic). The study showed an antipyretic action of the extract. Moreover, the toxicological study of the stem extract indicated a LD50 of 237.5 mg/kg P.C according to the classification of Diezi this plant is relatively toxic. The experiments show that the properties of *X. americana* could due to the presence of saponosides, as show by screening tests performed in this study. These results justified the use of *X. americana* in traditional cure of fever treatment (Soro *et al.*, 2009).

#### **2.5 Antitrypanosomal activity**

The in vitro antitrypanosomal activity of methanolic and aqueous extracts of stem bark of *Ximenia americana* was evaluated on Trypanosoma congolense. Blood obtained from a high infected mice with *T. congolense* (10(7) was incubated with methanolic and aqueous extracts at 20, 10 and 5 mg/ml and Diminal(R) (diminazene aceturate) at 200, 100 and 50 µg/ml in a 96 micro plate. The results revealed that methanol and aqueous extracts had activity at 20 and 40 mg/ml however, the methanolic extracts were more active than aqueous extracts at 10 and 5 mg/ml. Phytochemical screening of the methanolic and aqueous extracts of the bark showed that they both had flavonoids, anthraquinones, saponins, terpenes and tannins. The aqueous and methanolic extracts appears to show some potential activity against *T. congolense* (Maikai *et al.*, 2008).

#### **2.6 Anticancer activity**

Plants have been show to provide a useful source of natural products that are effective in the treatment of human neoplastic diseases. Information recorded from ancient civilizations has demonstrated the use of plants in search of treatment for various types of cancer (Hartwell, 1967-1971). An analysis of plant materials that had been studied at the National Cancer Institute (NCI), USA for discovering new anticancer drugs showed that if ethnopharmacological information had been used, the yield of plants harboring antineoplastic activity would have been significantly increased (Spjut & Perdue, 1976). The

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 439

The antineoplastic activity in vitro of various extracts from *Ximenia americana*, plant used in African traditional medicine for the treating cancer, was investigated (Voss *et al.*, 2006, 2006). The most active, aqueous extract was subjected to a detailed investigation in a panel of 17 tumor cell lines (Table 4) originating from human (16 lines) and rat (1 line), showing a averageI C50 of 49 mg raw powder/ml medium. The majority of cell lines (11 out of 17) were classified as sensitive (the sensitivity varied from 1.7 mg/ml in MCF7 breast cancer cells to 170 mg/ml in AR230 chronic-myeloid leukemia cells) and three of these (MCF7 breast cancer, BV173 CML and CC531 rat colon carcinoma) showed a particularly high sensitivity, with ratios lower than 0.1 of the average IC50. The *in vivo* antitumor activity was determined in the CC531 coloretal rat model and significant anticancer activity was found following

A comparison of the antineoplastic activity of the extract with three clinically used agents is given in Table 5. The cytotoxicity profiles of four cell lines are illustrated by the respective IC10, IC50 and IC90 values, as well as by the corresponding IC90 to IC10 ratio, describing the slop of the concentration-effect curve. Most prominently, the ranking in sensitivity differed between the extract and the positive controls. In variance to the extract, which resulted in the lowest IC50 and IC90/IC10 ratio in MCF7 cells, miltefosine and cisplatinum caused the lowest IC50 and IC90/IC10 ratio in HEp2 cells. Similar to the extract, the lowest IC50 following gemcitabine exposure was seen in NCF7 cellls. However, this agent differed from all the others by its lack in effecting 90% growth inhibition, were the HEp2 cells; notably, the cells were most resistant to the agent. In contrast, SAOS2 cells were found to best most resistant

Cell line Treatment IC50 IC50 IC90 IC90/IC10 MCF7 Extract (µg/ml) 0.6 1.8 10 16.7 Miltefosine (µM) 6.5 40 80 12.3 Cisplatinum (µg/ml) 0.22 2.2 10 45 Gemcitabine (µM) 0.001 0.012 >100 >105 U87-MG Extract (µg/ml) 1.0 9.0 100 100 Miltefosine (µM) 4.7 27 70 14.9 Cisplatinum (µg/ml) 0.12 1.6 18 150 Gemcitabine (µM) 0.002 0.014 >100 >5x104 HEp2 Extract (µg/ml) 5.0 21 100 20 Miltefosine (µM) 1.2 2,8 8.0 6.7 Cisplatinum (µg/ml) 0.09 0.4 1.4 15.6 Gemcitabine (µM) 0.2 0.47 17 85 SAOS2 Extract (µg/ml) 20 66 1000 50 Miltefosine (µM) 5.0 40 120 24 Cisplatinum (µg/ml) 0.11 3.1 10 91 Gemcitabine (µM) 0.007 0.034 >100 >104

Table 5. Cytotoxicity profiles of the extract and three standard antineoplastic agents in a

peroral administration, indicating a 95% reduced activity.

to the extract as well as to miltefosine and cisplatinum.

subpanel of the cell lines

list of natural products stored for study as more effective drugs for the treatment of human cancers (NCI) were generated by searching for specific structural types (Steven & Russel, 1993). However, the presence of some large class cannot be ruled out. Examples of anticancer agents developed from higher plants are the antileukemic bis-indole alkaloids vinblastine and vincristine from the *Catharantus roseus* (Apocynaceae); diterpene taxol, used to treat breast cancer, lung cancer, and ovarian cancer and also used to treat AIDS-related (Kaposi's sarcoma) from *Taxus breviflora* (Taxaceae); pyrrolo[3,4,b]-quinoline alkaloid camptothecin (antileukemic) from *Camptotheca acuminate* (Nyssaceae) and pyridocarbazole alkaloid elipticine (antitumor) contained in *Ochrosia elliptica* (Apocynaceae). A large number of other active natural products with toxicity to cells in culture (Walker carcinosarcoma 256, mouse L-1210 leukemia, Ehrlich ascite tumor, sarcoma 180 and mouse P-388 leukemia cell lines) have been detected (Geran *et al.*, 1972 & Lee *et al.*, 1988).


aInhibitory concentration 10 (concentration inhibiting the cell growth by 10%), as accessed by MTT assay; bInhibitory concentration 50 (concentration inhibiting the cell growth by 50%), as accessed by MTT assay. cInhibitory concentration 90 (concentration inhibiting the cell growth by 90%), as accessed by MTT assay. dRatio of IC90 and IC10 values.

Table 4. Antiproliferative activity of an aqueous extract from *X. americana* in 16 human and one rodent tumor cell lines and in 4 immortalized non-tumor cell lines.

list of natural products stored for study as more effective drugs for the treatment of human cancers (NCI) were generated by searching for specific structural types (Steven & Russel, 1993). However, the presence of some large class cannot be ruled out. Examples of anticancer agents developed from higher plants are the antileukemic bis-indole alkaloids vinblastine and vincristine from the *Catharantus roseus* (Apocynaceae); diterpene taxol, used to treat breast cancer, lung cancer, and ovarian cancer and also used to treat AIDS-related (Kaposi's sarcoma) from *Taxus breviflora* (Taxaceae); pyrrolo[3,4,b]-quinoline alkaloid camptothecin (antileukemic) from *Camptotheca acuminate* (Nyssaceae) and pyridocarbazole alkaloid elipticine (antitumor) contained in *Ochrosia elliptica* (Apocynaceae). A large number of other active natural products with toxicity to cells in culture (Walker carcinosarcoma 256, mouse L-1210 leukemia, Ehrlich ascite tumor, sarcoma 180 and mouse P-388 leukemia cell

Cell line

MCF7 0.6 1.7 10 16.7 BV173 0.4 1.8 7.0 17.5 CC531 0.8 3.3 12 15.0 U87-MG 1.0 9.0 100 100 K562 5.0 11 180 36 SKW-3 3.1 20 700 226 HEp2 5.0 21 100 20 NC1-H460 4.0 21 150 38 PC3 3.5 26 >1000 >300 MDA-MB231 5.0 33 100 20 HT29 8.0 40 350 44 U333 7.0 65 300 43 SAOS2 20 66 1000 50 LAMA84 10 90 600 60 HL60 30 90 1000 33 CML-T1 2.5 160 1000 400 AR230 17 170 700 41 Non tumor cell lines MCF10 35 >100 >100 >2.0 MDCK 12 27 60 5.0 N1H/3T3 2 33 >100 >50 PNT-2 2 20 >100 >50 aInhibitory concentration 10 (concentration inhibiting the cell growth by 10%), as accessed by MTT assay; bInhibitory concentration 50 (concentration inhibiting the cell growth by 50%), as accessed by MTT assay. cInhibitory concentration 90 (concentration inhibiting the cell growth by 90%), as accessed by MTT assay.

Table 4. Antiproliferative activity of an aqueous extract from *X. americana* in 16 human and

one rodent tumor cell lines and in 4 immortalized non-tumor cell lines.

 (ug/ml medium)

IC90 c

 (ug/ml medium)

IC90/IC10

medium)

d

IC50 b

lines) have been detected (Geran *et al.*, 1972 & Lee *et al.*, 1988).

a (ug/ml medium)

*Tumor cell lines* IC10

dRatio of IC90 and IC10 values.

The antineoplastic activity in vitro of various extracts from *Ximenia americana*, plant used in African traditional medicine for the treating cancer, was investigated (Voss *et al.*, 2006, 2006). The most active, aqueous extract was subjected to a detailed investigation in a panel of 17 tumor cell lines (Table 4) originating from human (16 lines) and rat (1 line), showing a averageI C50 of 49 mg raw powder/ml medium. The majority of cell lines (11 out of 17) were classified as sensitive (the sensitivity varied from 1.7 mg/ml in MCF7 breast cancer cells to 170 mg/ml in AR230 chronic-myeloid leukemia cells) and three of these (MCF7 breast cancer, BV173 CML and CC531 rat colon carcinoma) showed a particularly high sensitivity, with ratios lower than 0.1 of the average IC50. The *in vivo* antitumor activity was determined in the CC531 coloretal rat model and significant anticancer activity was found following peroral administration, indicating a 95% reduced activity.

A comparison of the antineoplastic activity of the extract with three clinically used agents is given in Table 5. The cytotoxicity profiles of four cell lines are illustrated by the respective IC10, IC50 and IC90 values, as well as by the corresponding IC90 to IC10 ratio, describing the slop of the concentration-effect curve. Most prominently, the ranking in sensitivity differed between the extract and the positive controls. In variance to the extract, which resulted in the lowest IC50 and IC90/IC10 ratio in MCF7 cells, miltefosine and cisplatinum caused the lowest IC50 and IC90/IC10 ratio in HEp2 cells. Similar to the extract, the lowest IC50 following gemcitabine exposure was seen in NCF7 cellls. However, this agent differed from all the others by its lack in effecting 90% growth inhibition, were the HEp2 cells; notably, the cells were most resistant to the agent. In contrast, SAOS2 cells were found to best most resistant to the extract as well as to miltefosine and cisplatinum.


Table 5. Cytotoxicity profiles of the extract and three standard antineoplastic agents in a subpanel of the cell lines

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 441

The compounds **7** and **8** were recently isolated and their structures were elucidated on the basis of spectral analysis (IV, MS and NMR) and the complete assignment of the 1H and 13C NMR signals were achieved by 1D(1H, 13C and DEPT) and 2D (1H - 1H COSY, 1H - 13C HMQC, 1H-13C HMBC and 1H - 1H NOESY) NMR experiments. The sesquiterpene **7**, isolated

combination with its 1H and 13C NMR spectra. The 1H and 13C NMR spectra combined with distortionless enhancement by polarization transfer (DEPT) technique exhibited signals that allowed characterize the three isoprene units (C-1, C-2, C-3, C-4 and C-13; C-8, C-9, C-10, C-11 and C-12; C-5, C-6, C-7, C-14 and C-15) of **7**. Thus, the 13C NMR spectra exhibited signals for six sp2 carbons [olefinic bond: C-2 (C 128.9), C-3 (C 141.7) and furan ring: C-9 (C 127.9), C-10 (C 147.1), C-11 (C 144.4), C-12 (C 108.9)], two carbonyl [conjugated ketone, C-8 (<sup>C</sup> 195.3) and conjugated carboxylic acid, C-1 (C 173.1)]), three methylene [C-4 (C 41.2), C-6 (<sup>C</sup> 36.5) and C-7 (C 35.9), three methyl [C-13 (C 12.4), C-14 (C 25.9) C-15 (C 25.9) and one quaternary carbon [C-5 (C 34.3)]. One conjugated ketone (C 195.3) was also evident from the absorption at 1682 cm-1 in the IR spectrum. In the HMBC spectrum, obvious long-range connectivities between the methylene group 2H-7 (H 2.71, dd, 7.9, 6.0 Hz) and C-8 (<sup>C</sup> 195.57) and between the methylene group 2H-4 (H 2.20, d, 7.7 Hz) and C-5 (C 34.56) allowed the assembly of the molecule and show it to consist of a furanoid sesquiterpene. Others diagnostic 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC correlations permitted to assign all the hydrogen and carbon atoms. The sesquiterpene, **8** isolated as a white solid, has

13C NMR spectra. The 1H and 13C NMR spectra combined with distortionless enhancement by polarization transfer (DEPT) technique exhibited signals that allowed characterize the three isoprene units (C-1, C-2, C-3, C-11 and C-12; C-4, C-5, C-6, C-13 and C-14; C-7, C-8, C-9, C-10 and C-15) of **8**. The 13C NMR spectra exhibited signals for four sp2 carbons [three substituted, C-8 (C 132.34) and C-9 (C 145.01) and disubstituted, C-1 (C 154.71) and C-12 (C 111.63) bonds; one conjugated carboxylic acid, C-15 (C 173.71), besides signals to five methylene, two methyne, one quaternary and two methyl carbons. The possibility of himachalano type structure was eliminated based on the interpretation of spin-spin interactions revealed by 1H-1H COSY spectrum, which clearly showed the presence of cross peaks corresponding to the couplings of two atoms of hydrogen 2H-6 [H 1.68 (m) and 1.50 (m)] with H-5 hydrogen [H 1.81 (m)] and with the two hydrogen atoms 2H-7 (H 2.45 and 2.35) besides interaction of H-5 (H 1.81) with H-11 (H 2.50, q). This sequence does not appear in the skeleton type himachalano. The *trans* configuration fusion ring was supported by correlations observed in NOESY NMR spectrum, that exhibited the presence of nOes indicating that the hydrogens 3H-13 (H 1.01, s), H-5 (<sup>H</sup> 1.81) and H-3ax (<sup>H</sup> 1.58, t, 10.8 Hz) are oriented on the same side (á) of the molecule, while the hydrogens 3H-14 has the same orientation (â) that the hydrogens H-11 (<sup>H</sup> 2.50, q), H-6ax (<sup>H</sup> 1.50) and H-3eq (<sup>H</sup> 1.74, dd, 10.8 , 8.9). Others diagnostic 1H-1H COSY, 1H 13C C HMQC and 1H 13C 13C HMBC

The stem bark MeOH extract of *X. americana* as well as several others plant species used by the Maasai pastoralis of East Africa showed antiviral effect against measles virus *in vitro* by

264) in

234) in combination with its 1H and

as a white powder, has molecular formulaC15H20O4 deduced from its EIMS (M+**.**

molecular formulaC15H22O2 deduced from its EIMS (M+**.**

correlations permitted to assign all the hydrogen and carbon atoms.

**2.7 Others activities 2.7.1 Antiviral effect** 

In order to define the substance class of the active component(s) (Voss *et al.*, 2006) experiments were carried out on physicochemical properties. In the process, lipids and lipophilic plant secondary metabolites could be excluded, since the biological activity was only extracted by strongly polar solvents. Large amounts of tannins were identified in the aqueous extract. However, extracts prepared in methanol or 70% acetone, both solvents known to efficiently extract tannins from plant materials, had only a low (methanol) or no (70% acetone) cytotoxic activity. Molecules smaller than 10 kDa were excluded by ultrafiltration. Out of the known class of plant cell macromolecules, DNA and RNA were not found in the aqueous extract and digestion experiments with DNase or RNase had not effect biological activity. However, proteins and polysaccharides were shown to be present in the aqueous extracts and could not be further separated by physicochemical methods. Digestion experiments with trypsin and proteinase K hinted at a protein being responsible for the cytotoxic activity.

A well-defined family of cytotoxic plant proteins is that of the type II ribosome-inactiving proteins (RIPs). These proteins with molecular weight of about 60 kDa, consist of two polypeptide chain, termed A- and B- chain, with an MW of about 30 kDa each, being held together by disulphide bridge. Cumulative evidences (cytotoxic effects, MW, two-chain structure of the proteins in the affinity-purified fraction and one mass-spectrometrically sequenced tryptic peptides) strongly suggests that the active components of the plant material are so far unknown proteins belonging to the type II RIP family.

By a combination of preextraction, extraction, ion exchange and affinity chromatography, a mixture of two cytotoxic proteins was isolated. The eluted peptides were analyzed by electrospray ionization mass spectrometry (MS/MS). The MS/MS mass spectrum is a method in which a first analyzer isolates a precursor ion which then undergoes a fragmentation yielding a product ions and neutral fragments. A second spectrometer analyzes the product ions. MS/MS applications are plentiful in the study of fragmentation mechanisms, observation of ion-molecule reactions, applications to high-selectivity and high-sensitivity analysis and determination of elementary compositions. Thus, it is a rapid selective analysis method for the components of a complex mixture and macromolecules in biological fluids. The homology of the translated protein sequence from isolated peptides to known type II RIP precursor protein sequence demonstrates that the new protein termed "riproximin" is a so far unknown member of this class. In conclusion, from biological activity of each of the two proteins as well as from MS/MS sequence analysis, showing the presence of two B-chain and two A-chain in the mixture, the *X. americana* extract analyzed contains a mixture of two new proteins, riproximin, belongs to the family of type II ribosome-inactivating proteins.

Two sesquiterpenes (**7** and **8**) isolated from the EtOH extract of the stems of *X. americana* did not inhibit the growth of HL-60 (human leukemia), HTC-8 (human colon) and MDA-MB-435 (human breast cancer) cell lines.

The compounds **7** and **8** were recently isolated and their structures were elucidated on the basis of spectral analysis (IV, MS and NMR) and the complete assignment of the 1H and 13C NMR signals were achieved by 1D(1H, 13C and DEPT) and 2D (1H - 1H COSY, 1H - 13C HMQC, 1H-13C HMBC and 1H - 1H NOESY) NMR experiments. The sesquiterpene **7**, isolated as a white powder, has molecular formulaC15H20O4 deduced from its EIMS (M+**.** 264) in combination with its 1H and 13C NMR spectra. The 1H and 13C NMR spectra combined with distortionless enhancement by polarization transfer (DEPT) technique exhibited signals that allowed characterize the three isoprene units (C-1, C-2, C-3, C-4 and C-13; C-8, C-9, C-10, C-11 and C-12; C-5, C-6, C-7, C-14 and C-15) of **7**. Thus, the 13C NMR spectra exhibited signals for six sp2 carbons [olefinic bond: C-2 (C 128.9), C-3 (C 141.7) and furan ring: C-9 (C 127.9), C-10 (C 147.1), C-11 (C 144.4), C-12 (C 108.9)], two carbonyl [conjugated ketone, C-8 (<sup>C</sup> 195.3) and conjugated carboxylic acid, C-1 (C 173.1)]), three methylene [C-4 (C 41.2), C-6 (<sup>C</sup> 36.5) and C-7 (C 35.9), three methyl [C-13 (C 12.4), C-14 (C 25.9) C-15 (C 25.9) and one quaternary carbon [C-5 (C 34.3)]. One conjugated ketone (C 195.3) was also evident from the absorption at 1682 cm-1 in the IR spectrum. In the HMBC spectrum, obvious long-range connectivities between the methylene group 2H-7 (H 2.71, dd, 7.9, 6.0 Hz) and C-8 (<sup>C</sup> 195.57) and between the methylene group 2H-4 (H 2.20, d, 7.7 Hz) and C-5 (C 34.56) allowed the assembly of the molecule and show it to consist of a furanoid sesquiterpene. Others diagnostic 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC correlations permitted to assign all the hydrogen and carbon atoms. The sesquiterpene, **8** isolated as a white solid, has molecular formulaC15H22O2 deduced from its EIMS (M+**.** 234) in combination with its 1H and 13C NMR spectra. The 1H and 13C NMR spectra combined with distortionless enhancement by polarization transfer (DEPT) technique exhibited signals that allowed characterize the three isoprene units (C-1, C-2, C-3, C-11 and C-12; C-4, C-5, C-6, C-13 and C-14; C-7, C-8, C-9, C-10 and C-15) of **8**. The 13C NMR spectra exhibited signals for four sp2 carbons [three substituted, C-8 (C 132.34) and C-9 (C 145.01) and disubstituted, C-1 (C 154.71) and C-12 (C 111.63) bonds; one conjugated carboxylic acid, C-15 (C 173.71), besides signals to five methylene, two methyne, one quaternary and two methyl carbons. The possibility of himachalano type structure was eliminated based on the interpretation of spin-spin interactions revealed by 1H-1H COSY spectrum, which clearly showed the presence of cross peaks corresponding to the couplings of two atoms of hydrogen 2H-6 [H 1.68 (m) and 1.50 (m)] with H-5 hydrogen [H 1.81 (m)] and with the two hydrogen atoms 2H-7 (H 2.45 and 2.35) besides interaction of H-5 (H 1.81) with H-11 (H 2.50, q). This sequence does not appear in the skeleton type himachalano. The *trans* configuration fusion ring was supported by correlations observed in NOESY NMR spectrum, that exhibited the presence of nOes indicating that the hydrogens 3H-13 (H 1.01, s), H-5 (<sup>H</sup> 1.81) and H-3ax (<sup>H</sup> 1.58, t, 10.8 Hz) are oriented on the same side (á) of the molecule, while the hydrogens 3H-14 has the same orientation (â) that the hydrogens H-11 (<sup>H</sup> 2.50, q), H-6ax (<sup>H</sup> 1.50) and H-3eq (<sup>H</sup> 1.74, dd, 10.8 , 8.9). Others diagnostic 1H-1H COSY, 1H 13C C HMQC and 1H 13C 13C HMBC correlations permitted to assign all the hydrogen and carbon atoms.

#### **2.7 Others activities**

440 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

In order to define the substance class of the active component(s) (Voss *et al.*, 2006) experiments were carried out on physicochemical properties. In the process, lipids and lipophilic plant secondary metabolites could be excluded, since the biological activity was only extracted by strongly polar solvents. Large amounts of tannins were identified in the aqueous extract. However, extracts prepared in methanol or 70% acetone, both solvents known to efficiently extract tannins from plant materials, had only a low (methanol) or no (70% acetone) cytotoxic activity. Molecules smaller than 10 kDa were excluded by ultrafiltration. Out of the known class of plant cell macromolecules, DNA and RNA were not found in the aqueous extract and digestion experiments with DNase or RNase had not effect biological activity. However, proteins and polysaccharides were shown to be present in the aqueous extracts and could not be further separated by physicochemical methods. Digestion experiments with trypsin and

A well-defined family of cytotoxic plant proteins is that of the type II ribosome-inactiving proteins (RIPs). These proteins with molecular weight of about 60 kDa, consist of two polypeptide chain, termed A- and B- chain, with an MW of about 30 kDa each, being held together by disulphide bridge. Cumulative evidences (cytotoxic effects, MW, two-chain structure of the proteins in the affinity-purified fraction and one mass-spectrometrically sequenced tryptic peptides) strongly suggests that the active components of the plant

By a combination of preextraction, extraction, ion exchange and affinity chromatography, a mixture of two cytotoxic proteins was isolated. The eluted peptides were analyzed by electrospray ionization mass spectrometry (MS/MS). The MS/MS mass spectrum is a method in which a first analyzer isolates a precursor ion which then undergoes a fragmentation yielding a product ions and neutral fragments. A second spectrometer analyzes the product ions. MS/MS applications are plentiful in the study of fragmentation mechanisms, observation of ion-molecule reactions, applications to high-selectivity and high-sensitivity analysis and determination of elementary compositions. Thus, it is a rapid selective analysis method for the components of a complex mixture and macromolecules in biological fluids. The homology of the translated protein sequence from isolated peptides to known type II RIP precursor protein sequence demonstrates that the new protein termed "riproximin" is a so far unknown member of this class. In conclusion, from biological activity of each of the two proteins as well as from MS/MS sequence analysis, showing the presence of two B-chain and two A-chain in the mixture, the *X. americana* extract analyzed contains a mixture of two new proteins, riproximin,

Two sesquiterpenes (**7** and **8**) isolated from the EtOH extract of the stems of *X. americana* did not inhibit the growth of HL-60 (human leukemia), HTC-8 (human colon) and MDA-MB-435

CO2H

1

<sup>2</sup> <sup>3</sup>

15

4 5 6

OH

H

12

14 13

11

2 3 4 5 <sup>6</sup> <sup>7</sup> 8

<sup>15</sup> <sup>1</sup>

10 9

H

**8**

O

proteinase K hinted at a protein being responsible for the cytotoxic activity.

material are so far unknown proteins belonging to the type II RIP family.

belongs to the family of type II ribosome-inactivating proteins.

(human breast cancer) cell lines.

O

<sup>7</sup> <sup>8</sup> <sup>9</sup>

12 13 14

**7**

O

11

10

#### **2.7.1 Antiviral effect**

The stem bark MeOH extract of *X. americana* as well as several others plant species used by the Maasai pastoralis of East Africa showed antiviral effect against measles virus *in vitro* by

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 443

Besides the substances mentioned in the text of this chapter, several other originated from

OH

O

**<sup>13</sup> <sup>14</sup> <sup>15</sup>**

OH

COOH

**O**

**19**

O

**<sup>11</sup> <sup>12</sup>**

OH

OH

OH

11

**17** O

COOH

**3. Others constituents isolated from** *X. americana*

*Ximenia americana* were isolated.

O

**10**

OH

11

**18**

**16**

**Isoprenoids** 

**Fatty acids** 

**Triterpenes** 

HO

plaque reduction neutralization assay. Potentially active constituents from extracts of all the plants include polyphenols, alkaloids, tannins, sterols, terpenes, saponins and glycosides, between others (Parker *et al.*, 2007).

#### **2.7.2 Hepatic and heamatological effects**

A study (James *et al.*, 2008) was conducted from the leaves, stem bark and root aqueous extract of *X. americana* with albino rats. The results of this work shows that the extracts significantly (P<0.05) increasing the level of serum alanine transaminase (ALT) and aspartate transaminase (AST), results indicative of hepatocellular damage. The result also shows that the root has the ability to impair albumin synthesis as observed by the decrease of level of serum albumin. The weight of the animal showed a significant (P<0.05) reduction on administering the leaves extract as compared to the control and the others extracts. This reduction might be due to poor intake and utilization of food by the animals in the leaves extract group. The significantly (P<0.05) higher content of hydrogen cyanide, saponins, and oxalates in the root extracts indicates that the root extracts may be more toxic. Hydrogen cyanide is known to cause gastrointestinal inflammation and inhibition of cellular respiration. Saponins are known to have haemolytic properties and the ability to reduce body cholesterol by preventing its reabsorption. The high saponin content in the root may lead to gastroenteritis manifested by diarrhea. Oxalates have been known to cause irreversible oxalate nefrosis when ingested in large doses. Thus, there is need to isolate the specific component(s) responsible for the toxicity in the root extract in order to standardized the preparation for maximum therapeutic benefit.

#### **2.7.3 Toxicity**

The stem bark of *X. americana* was evaluated for its phytochemical constituents and acute toxicity effect on the Swiss albino mice (Maikai *et al.*, 2008). The results from the extracts administered intraperitoneally/orally at doses of 10, 100 and 1000 mg/kg body weight revealed no death with doses up 5000 mg/kg body weight. Post mortem, hematological and histopathological examination did not show any significant (P<0.05) weight changes. Phytochemical screening of the aqueous extract stem bark revealed the presence of cardiac glycosides, flavonoids, saponins and tannins. The results suggested that the aqueous extract is not acutely toxic to the mice.

#### **2.7.4 Food composition and cosmetic use**

Glyceride blends containing ximenynic acid (**9**) (found in *X. americana*) are useful for the preparation of food compositions or food supplements, including margarine, chocolate, ice cream, mayonnaises, cheese, dry soups, drinks, cereal bars and sauces and snack bars. The blend provides a composition providing health benefits consisting of insulin resistance, or related disorders such as diabetes, delaying the onset of symptoms related to development of Alzheimer's disease, improving memory function, lowering blood lipid levels, anticancer effects or skin antiageing effects (Koenen *et al.*, 2004). Food *X. americana* flowers are a replacement for orange blossoms with similar fragrance and soothing cosmetic properties (Paolo, 1979).

> CH3(CH2)5CH C C(CH2)7CO2H E

#### **3. Others constituents isolated from** *X. americana*

Besides the substances mentioned in the text of this chapter, several other originated from *Ximenia americana* were isolated.

#### **Isoprenoids**

442 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

plaque reduction neutralization assay. Potentially active constituents from extracts of all the plants include polyphenols, alkaloids, tannins, sterols, terpenes, saponins and glycosides,

A study (James *et al.*, 2008) was conducted from the leaves, stem bark and root aqueous extract of *X. americana* with albino rats. The results of this work shows that the extracts significantly (P<0.05) increasing the level of serum alanine transaminase (ALT) and aspartate transaminase (AST), results indicative of hepatocellular damage. The result also shows that the root has the ability to impair albumin synthesis as observed by the decrease of level of serum albumin. The weight of the animal showed a significant (P<0.05) reduction on administering the leaves extract as compared to the control and the others extracts. This reduction might be due to poor intake and utilization of food by the animals in the leaves extract group. The significantly (P<0.05) higher content of hydrogen cyanide, saponins, and oxalates in the root extracts indicates that the root extracts may be more toxic. Hydrogen cyanide is known to cause gastrointestinal inflammation and inhibition of cellular respiration. Saponins are known to have haemolytic properties and the ability to reduce body cholesterol by preventing its reabsorption. The high saponin content in the root may lead to gastroenteritis manifested by diarrhea. Oxalates have been known to cause irreversible oxalate nefrosis when ingested in large doses. Thus, there is need to isolate the specific component(s) responsible for the toxicity in the root extract in order to standardized the preparation for maximum therapeutic benefit.

The stem bark of *X. americana* was evaluated for its phytochemical constituents and acute toxicity effect on the Swiss albino mice (Maikai *et al.*, 2008). The results from the extracts administered intraperitoneally/orally at doses of 10, 100 and 1000 mg/kg body weight revealed no death with doses up 5000 mg/kg body weight. Post mortem, hematological and histopathological examination did not show any significant (P<0.05) weight changes. Phytochemical screening of the aqueous extract stem bark revealed the presence of cardiac glycosides, flavonoids, saponins and tannins. The results suggested that the aqueous extract

Glyceride blends containing ximenynic acid (**9**) (found in *X. americana*) are useful for the preparation of food compositions or food supplements, including margarine, chocolate, ice cream, mayonnaises, cheese, dry soups, drinks, cereal bars and sauces and snack bars. The blend provides a composition providing health benefits consisting of insulin resistance, or related disorders such as diabetes, delaying the onset of symptoms related to development of Alzheimer's disease, improving memory function, lowering blood lipid levels, anticancer effects or skin antiageing effects (Koenen *et al.*, 2004). Food *X. americana* flowers are a replacement for

orange blossoms with similar fragrance and soothing cosmetic properties (Paolo, 1979).

CH3(CH2)5CH C C(CH2)7CO2H

**9**

E

between others (Parker *et al.*, 2007).

**2.7.3 Toxicity** 

is not acutely toxic to the mice.

**2.7.4 Food composition and cosmetic use** 

**2.7.2 Hepatic and heamatological effects** 

OH

**Fatty acids** 

**Triterpenes** 

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 445

HO

**26**

From an extensive literature review was observed that the *Ximenia americana* is widely used as a popular alternative remedy in certain regions of some countries of the Africa (Guinea, Ethiopia, Nigeria, Sudan) and in the Brazil. The plant, used by their crude extracts, especially, aqueous and methanolic, showed several biological activities such as antimicrobial, antifungal, anticancer, antitrypanosomal, antirheumatic, antioxidant, analgesic, moluscicide, pesticidal, antipyretic, antifugal, among others. There are several papers in the literature confirming these activities. The crude extracts consist of complex mixture of compounds called secondary metabolites produced by plants, which include, mainly, flavonoids, saponins, alkaloids, quinones, terpenoids, phenols, glycosides and

Many plants have a prolonged and uneventful use that may serve as indirect evidence to their efficacy. However, in the absence of objective proof of efficacy and without the knowledge of the constituents responsible for the physiological actions, the validity of the remedies is questionable and its use restricted. It generally was observed that the more the constituents in a given species, the more diverse the micro-organisms it acts upon. The difference of activity appears to be directly related to the qualitative and/or quantitative diversity of the compounds that are being accumulated by the plants investigated.

O

O

CH2OH

**25**

**Steroids** 

O

sterols.

**24**

HO HO

HO

**4. Summary/conclusion/future directions** 

O

O

HO

HO

OH OH

HO **22**

O

CO

O

OH

CO2H

OH

OH

COOH

O

**20**

OH

O

HO

**21**

O

O

O

OH

O

O

O

CO2H

OH

<sup>H</sup> **23**

CH3

HOCH2 O

OH

OH OH

OH

HO

#### **4. Summary/conclusion/future directions**

From an extensive literature review was observed that the *Ximenia americana* is widely used as a popular alternative remedy in certain regions of some countries of the Africa (Guinea, Ethiopia, Nigeria, Sudan) and in the Brazil. The plant, used by their crude extracts, especially, aqueous and methanolic, showed several biological activities such as antimicrobial, antifungal, anticancer, antitrypanosomal, antirheumatic, antioxidant, analgesic, moluscicide, pesticidal, antipyretic, antifugal, among others. There are several papers in the literature confirming these activities. The crude extracts consist of complex mixture of compounds called secondary metabolites produced by plants, which include, mainly, flavonoids, saponins, alkaloids, quinones, terpenoids, phenols, glycosides and sterols.

Many plants have a prolonged and uneventful use that may serve as indirect evidence to their efficacy. However, in the absence of objective proof of efficacy and without the knowledge of the constituents responsible for the physiological actions, the validity of the remedies is questionable and its use restricted. It generally was observed that the more the constituents in a given species, the more diverse the micro-organisms it acts upon. The difference of activity appears to be directly related to the qualitative and/or quantitative diversity of the compounds that are being accumulated by the plants investigated.

*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 447

Atta-ur-Rahman (Elsevier) (2005). *Studies in Natural Products, Bioactive Natural Products (Part L)*, *Vol. 32,* Atta-ur-Rahamn, Karachi, Pakistan , ISBN 9780444521712. Badami, R. C. & Patil, K. B. (1981). Structure and Ocurrence of Unusual Fatty Acids in Minor Seed Oils. *Progress in l Lipid Research*, Vol. 19, pp. 119-153, ISSN 01637827. Braga, R. (3ª Ed). (1976). *Plantas do Nordeste, especialmente do Ceará*, Escola Superior de

Brasileiro, M. T.; Egito, M. A. & Lima, J. R.; Randau, K. P.; Pereira G. C.; Neto, P. J. R.

Bruneton, J. (3a Ed) (1999). *Pharmacognosie, phytochimie, plantes médicinales*, Tec & Doc Ed.,

Chhabra S. C.; Viso, F. C., (1990). A Survey of the Medicinal Plants Eastern Tanzania for

Dixon, R. A.; Dey, P. M. & Lamb, C. J. (1983). Phytoalexins: enzymology and molecular

Fatope, M. O.; Adoum, O. A. & Takeda, Y. (2000). C18 Acetylenic Fatty Acids of *Ximenia* 

Finnermore, H. J. M. Cooper, M. B. Stanlet, J. H. Cobcroff & L. J. Harris, (1988). Journal of the

Geran, R. T.; Greenberg, M. N.; MacDonald, A. M.; Schumacher, A. M. & Abbot, B. J. (1972).

Geyid, A.; Abebe, D.; Debella, A.; Makonnen, Z.; Aberra, F.; Teka, F.; Kebede, T.; Urga, K.;

Hartwell, J. L. (1967; 1968; 1969; 1970; 1971). Plants used against cancer. *Loydia*, Vol. 30 p.

Hostettman, K.; Marston, A. J.; Wolfender, L & Miallard, M. (1995). *Screening for flavonoids* 

Jacbson, M. (1971). *Naturally Occurring Insecticides*, M. Crosby. D. G. Eds.: Dekker, New

James, D. B.; Abu, E. A.; Wurochekke, A. U. & Orgi, G. N. (2007). Phytochemical and

James, D. B.; Owolabi, A. O.; Ibiyeye, H.; Magaji, J. & Ikugiyi, Y. A. (2008). Assessment of the

(2008). *Ximenia americana* L: botânica, química e farmacologia no interesse da tecnologia farmacêutica. *Revista Brasileira Farmacognosia*, 89, 2, pp. 164-167, ISSN

Alkaloids, Flavonoids, Saponins and Tannins. *Fitoterapia,* Vol. 61, No. 4, pp. 307-

*americana* with Potential Pesticidal Activity. *Journal of Agricultural and Food* 

Protocols for screening chemical agents and natural products against animal tumors and other biological systems. *Cancer Chemotherapy Reports,* Vol. 3. p. 1, ISSN

Yersaw, K.; Biza, T.; Mariam, B. H. & Guta, M. (2005). Screening of medicinal plants of Ethiopia for their anti-microbial properties and chemical profiles. *Journal of* 

379; Vol. 31, p. 71; Vol. 32, p. 71, 153, 247; Vol. 33, p. 98, 288; Vol. 34, p. 103, 204, 310,

*and related compounds in medicinal plants by LC-UV-MS and subsequent isolation of* 

Antimicrobial Investigation of the Aqueous and Methanolic Extracts of *Ximenia americana*. Journal of *Medical Science*, Vol. 7, No. 2, (15th february 2007), pp. 284-288,

hepatic effects , heamatological effect and some phytochemical constituents of

Agricultura, Mossoró, Brasil.

0370-372X.

00690112.

386.

York, U.S.A.

ISSN 20721625.

Angers, France.

316, ISSN 2367326X.

bilogy. *Advance Enzymology*, Vol. 55, pp. 1-69.

*Chemistry*, Vol. 48, pp. 1872-1874, ISSN 00218561.

Indian chemical Society, Vol. 57, pp. 162-169 ISSN 0019-4522.

*Ethnopharmacology*, Vol. 97, pp. 421-427, ISSN 0378-8741.

*bioctive compounds,* Akademiiai, Kiaho, *Budapest, Hungry.* 

However, detailed studies on the toxicity of extracts revealed through phytochemical screening showed that many constituents chemicals can affect the animal positively or negatively as a result of prolong usage. Thus, was founded that tannins and anthraquinones are thought to have both proxidant and antioxidant effects on the body. While the antioxidant protects, the proxidant damage the tissues and organs. Also, was observed that the presence of tannins and other compounds interferes with absorption of nutrients such proteins and minerals resulting in weight loss. The extracts contained the presence of saponins has been reported to produce free radicals and hydrogen peroxide during its oxidation to semiquinone in the body, is thought to damage the cells of the body. The results of several studies conducted so far have produced a scientific basis that can justify the use of *Ximenia americana* in medicine. As we see the many works on *X. americana* show its effectiveness in treating various diseases. In all studies, were highlighted the participation and importance of secondary metabolites produced by them. However, there are still many details to be clarified. As mentioned above, in general, it was observed that the more the constituents in a given species, the more diverse the micro-organisms it acts upon. Moreover, the activity of plant extracts seems to be related to quality and quantity of metabolites present, possibly due to the possibility of synergism while, different types of metabolites appear to be related to specific biologic actions. In this context it is important to point out that the norisoprenoid isophorane (**10**), shown to be carcinogenic agent (Mevy *et al.*, 2006), was identified in the leaves of *X. americana*, which would conflict with its use in treating cancer. The last report about compounds isolated from *X. americana* up to date were the sesquiternes **7** and **8**, triterpenoids **18**-**22** and steroids **24**-**26,** all from ethanol extract of stems (Araújo *et al.*, 2008, 2009). Some of them have not yet been exhaustively investigated from the point of view of biological activity.

Future studies should be performed using chromatographic methods such as HPLC (high performance liquid chromatography) and LC-MS (Liquid chromatography coupled to mass spectroscopy) to obtain the chromatographic profile of the chemical composition of the extracts. Then carry out guided study (biological activity) in order to isolate and identify the pure constituents. Finally, as reported, many compounds may exhibit both carcinogenic and anticarcinogenic effects but it is not excluded that the occurrence of compounds other than volatile constituents may act in the anticarcinogenic process. Consequently, these results encourage further investigations to extracts and identify the active chemical compounds responsible for the specific biological activity in order to standardized the plant preparation for maximum therapeutic benefit.

#### **5. References**


However, detailed studies on the toxicity of extracts revealed through phytochemical screening showed that many constituents chemicals can affect the animal positively or negatively as a result of prolong usage. Thus, was founded that tannins and anthraquinones are thought to have both proxidant and antioxidant effects on the body. While the antioxidant protects, the proxidant damage the tissues and organs. Also, was observed that the presence of tannins and other compounds interferes with absorption of nutrients such proteins and minerals resulting in weight loss. The extracts contained the presence of saponins has been reported to produce free radicals and hydrogen peroxide during its oxidation to semiquinone in the body, is thought to damage the cells of the body. The results of several studies conducted so far have produced a scientific basis that can justify the use of *Ximenia americana* in medicine. As we see the many works on *X. americana* show its effectiveness in treating various diseases. In all studies, were highlighted the participation and importance of secondary metabolites produced by them. However, there are still many details to be clarified. As mentioned above, in general, it was observed that the more the constituents in a given species, the more diverse the micro-organisms it acts upon. Moreover, the activity of plant extracts seems to be related to quality and quantity of metabolites present, possibly due to the possibility of synergism while, different types of metabolites appear to be related to specific biologic actions. In this context it is important to point out that the norisoprenoid isophorane (**10**), shown to be carcinogenic agent (Mevy *et al.*, 2006), was identified in the leaves of *X. americana*, which would conflict with its use in treating cancer. The last report about compounds isolated from *X. americana* up to date were the sesquiternes **7** and **8**, triterpenoids **18**-**22** and steroids **24**-**26,** all from ethanol extract of stems (Araújo *et al.*, 2008, 2009). Some of them have not yet been

exhaustively investigated from the point of view of biological activity.

for maximum therapeutic benefit.

Elsevier, New York, U.S.A.

**5. References** 

Future studies should be performed using chromatographic methods such as HPLC (high performance liquid chromatography) and LC-MS (Liquid chromatography coupled to mass spectroscopy) to obtain the chromatographic profile of the chemical composition of the extracts. Then carry out guided study (biological activity) in order to isolate and identify the pure constituents. Finally, as reported, many compounds may exhibit both carcinogenic and anticarcinogenic effects but it is not excluded that the occurrence of compounds other than volatile constituents may act in the anticarcinogenic process. Consequently, these results encourage further investigations to extracts and identify the active chemical compounds responsible for the specific biological activity in order to standardized the plant preparation

Araújo, M. R. S.; Assunção, J. C. C., Dantas, I. N. F., Costa-Lotufo, L. V. & Monte, F. J. Q.

Araújo, M. R. S.; Monte, F. J. Q. & Braz-Filho, R. (2009). A New Sesquiterpene from *Ximenia americana* Linn. *Helvetica Chimica Acta*, Vol. 92, pp. 127-129, ISSN 0018-019X. Atta-ur-Rahman. (1988). Studies *in Natural Products Chemistry*, *Structure Elucidation*, Vol.32,

*Communications*, Vol. 3, No. 6, pp. 857-860, ISSN 1934-578X

(2008). Chemical Constituents of *Ximenia americana. Natural Products* 


*Ximenia americana*: Chemistry, Pharmacology and Biological Properties, a Review 449

Parker, M. E.; Chabot, S.; Ward, B. J. & Johns, T. (2007). Traditional dietary additives of the

Pio-Correia, M. (1984). *Dicionário de Plantas Úteis do Brasil e das Éxoticas Cultivadas,* Imprensa

Rezanka, T & Sigler, K. (2007). Identification of very long chain unsaturated fatty acids from

Scalbert, A. (1991). Antimicrobial properties of tannins. *Phytochemist*y, Vol. 30, pp. 3875-3883,

Siddaiah, M.; Jayavcera, K. N.; Mallikarjuna, R. P.; Ravindra, R. K.; Yasodha, K. Y. &

Soro, T. Y.; Traore, F. ; Datte, J. Y. & Nene-Bi, A. S. (2009). Antipyretic activity of aqueous

Soro, T. Y.; Traore, F. & Sakande, J. (2009). Activité analgésique de l' extrait aqueux de

Spjut, R. W. & Perdue Jr., R. E. (1976). Plant folklore: a tool for predicting sources of

Sptizer, V.; Tomberg, W. & Aichholz, R. (1997). Analysis of Seed Oil of *Heisteria silvanii*

Steven, M. C. & Russel, J. M. (1993). *Bioactive Natural Products,* CRC Press, ISBN 0-8493-4372-

Tassou, C. C.; Drosinos, E. H. &. Nychas, G. J. E. (1995). Effects of essential oils from mint

Taylor, R. S.L.; Edet, F. Manandhar, N. P. & Towers, G. H. N. (1996). Antimicrobial activities

Voss, C.; Eyol, E. & Berger, M. R. (2006). Identification of potent anticancer activity in

Voss, C.; Eyol, E.; Frank, M.; von der Lieth, Claus-W & Berger, M. R. (2006). Identification

*and Applied Pharmacology,* Vol. 211, pp. 177-178, ISSN 0041-008X.

antitumor activity ?. *Cancer Treatiment Reports*, Vol. 60, pp. 979-985.

mass spectroscopy. *Phytochemistry*,Vol. 68, pp. 925-934, ISSN 00319422. Saeed, A. E. M. & Bashier, R. S. M. (2010). Physico-chemical analysis of *Ximenia americana* L*.* 

114, pp. 146-152, ISSN 0378-8741.

Nacional, Rio de Janeiro, Brasil.

No. 1, pp. 23-25, ISSN 0973-9874.

21412502.

8597.

ISSN 00319422.

377, ISSN 16310691.

1189-1200, ISSN 00244201.

0, Boca Raton, U. S. A.

102, ISSN 0378-8741.

00218847.

Maasai are antiviral against the measles virus. *Journal of Ethnopharmacology*, Vol.

*Ximenia* oil by atmospheric pressure chemical ionization liquid chromatography-

oil and structure elucidation of some chemical constituents of its seed oil and fruit pulp. *Journal of Pharmacognosy and Phytotherapy*, Vol. 2, No. 4, pp. 49-55. ISSN

Narender, R. G. (2009). Phytochemical screening and analgesic activity of methanolic extract of *Ximenia americana. Journal of Pharmacy and Chemistry*, Vol. 3,

extract of *Ximenia americana*. *Phytoterapie,* Vol. 7, No. 6, pp. 297-303, ISSN 1624-

*Ximenia americana* (Linné) (Olacaceae). *Comptes Rendus Biologies*, Vol. 332, pp. 371-

(Olacaceae) – A rich Source of Novel C18 Acetylenic Fatty Acid. *Lipids*, Vol. 32, pp.

(*Mentha piperita*) on S*almonella enteitidis* and *Listeria monocytogenes* in model Food systems at 4° and 10°C. *Journal of Applied Bacteriology*, Vol. 78, pp. 593-600, ISSN

of southern Nepalase medicinal plants. *Journal of Ethnopharmacology,* Vol. 50, pp. 97-

*Ximenia americana* aqueous extracts used by African traditional medicine. *Toxicology* 

and characterization of riproximin, a new type II ribosome-inactivating protein

*Ximenia Americana* (Leaves, stem and root) extracts. *African Journal of Biotechnology*, Vol. 7, No. 23, (December, 2008), pp. 4274-4278, ISSN 1684-5315.


Koenen, C.; Schmid, U.; Rogers, J.; Peilow, A.; Bosley, J.; Eggink, M. & Stam, W. (2004).

Magassouba, F. B.; Diallo, A.; Kouyaté, M.; Mara, F.; Mara, O.; Bangoura, O.; Camara, A.;

Mahato, S. B.; Nandy, A. K. & Roy, G. (1992). Triterpenoids. *Phytochemistry*, Vol. 31, pp.

Maikai, V. A.; Kobo, P. I. & Adaudi, A. O. (2008). Acute toxicity studies of aqueous stem

Maikai, V. A.; Maikai, B. V. & Kobo, P. I. (2009). Antimicrobial Properties of Stem Bark

Maikai, V. A.; Nok, J. A.; Adaudi, A. O. & Alawa, C. B. I. (2008). In vitro antitrypanosomal

Mathabe, M. C.; Nikova, R. V.; Lall, N. & Nyazema, N. Z. (2006). Antibacterial activities of

Mevy, J-P.; Bessiere, J-M.; Greff, S.; Zombre, G. & Viano, J. (2006). Composition of the

Ogunleye, D. S.; Ibitoye & Trop,S. F. (2003). Studies of antimicrobial activity and chemical

Omer, M. E. F. A. & Elnima, E. I. (2003). Antimicrobial activiy of *Ximenia americana*.

Paolo, R. (1979). Cosmetic use of the oil and flowers of *Ximenia americana*. *Rivista Italiana* 

Africa. *Journal of Ethnopharmacology*, 105, pp. 286-293, ISSN 0378-8741. Matos, F. J. A. (2007). *Plantas medicinais: guia de seleção e emprego de plantas usadas em fitoterapia* 

*no Nordeste do Brasil*, Imprensa Universitária, Fortaleza, Brasil.

bark extract of *Ximenia Americana. African Journal of Biotechnology*, Vol. 7, No. 10,

Extracts of *Ximenia americana*. *Journal of Agricultural Science*., Vol.1, No. 2,

activity of aqueous and methanolic crude extracts of stem bark of *Ximenia americana*  on *Trypanosoma congolense. Journal of Medicinal Plants*, Vol. 2, No. 3, pp. 55-58, ISSN

medicinal plants used for the treatment of diarrhoea in Limpopo province, South

volatile oil from leaves of *Ximenia americana* L. *Biochemical Systematics and Ecology*,

constituents of *Ximenia americana*. *Journal of Pharmmaceutical Research*, Vol. 2, No. 2,

Vol. 7, No. 23, (December, 2008), pp. 4274-4278, ISSN 1684-5315.

*Innovations Index*, patent No. EP1402787-A1, (June 2004), U.S.A., 4p. Loganathan, D.; Trivedi, G. K. & Chary, K. V. R. (1990). A Two Dimensional NMR Strategy

ISSN 1097-458X.

ISSN 0378-8741.

16840240.

2199-2249, ISSN 0031-9422.

(May, 2008), pp. 1600-1603, ISSN 1684-5315.

(December 2009), pp. 30-34. ISSN 00218596.

Vol. 34, pp. 549-553, ISSN 0305-1978.

(December 2003), pp. 239-241, ISSN 00223549.

*Fitoterapia*, Vol. 74, pp. 122-126, ISSN 0367326X.

*Essenze*, Vol. 61, No. 5, pp. 190-193, ISSN 0391-4658.

*Ximenia Americana* (Leaves, stem and root) extracts. *African Journal of Biotechnology*,

Blend used in preparing, food composition, e. g. margarine, comprises ximenynic acid originating from natural source and fatty acids or glycerides. *Derwent* 

for the Complete 1H Chemical Shift Assignment of Extended Proton Spin Systems in Triterpenoids. *Magnetic Resonance in Chemistry*, 28, 11, (July 1990), pp. 925-930,

Traoré, S.; Diallo, A. K.; Zaoro, M.; Lamah, K.; Diallo, S.; Camara, G.; Kéita, A.; Camara, M. K.; Barry, R.; Kéita, S.; Oularé, K.; Barry, M. S.; Donzo, M.; Camara, K.; Toté, K.;. Vanden Berghe, D.; Totté, J.; Pieters, L.; Vlietinck, A. J. & Baldé, A. M. (2007). Ethnobotanical survey and antibacterial activity of some plants used in Guinean traditional medicine. *Journal of Ethnopharmacology*, Vol. 114, pp. 44-53,


**21** 

*South Korea* 

**Phytochemicals and Their Pharmacological** 

Botanical medicines have been applied for the treatment of various human diseases with thousands of years of history all over the world. In some Asian and African countries, 80 % of population depends on traditional medicine in primary health care. On the other hand, in many developed countries, 70 % to 80 % of the population has used some forms of alternative or complementary medicine. The long tradition of using plants for medicine, supplemented by pharmaceutical research, has resulted in many plant-based Western medicines. Traditional medicine has provided Western medicine with over 40 % of all pharmaceuticals (Samuelsson & Bohlin, 2004). In the past decades, therefore, research has

*Acanthopanax* species (Araliaceae) are widely distributed in Asia, Malaysia, Polynesia, Europe, North Africa and the America. There are about 40 species of *Acanthopanax* to be found in over the world. *Acanthopanax* species have traditionally been used as a tonic and sedative as well as in the treatment of rheumatism, and diabetes. *A. koreanum* Nakai is an indigenous plant prevalently distributed throughout South Korea. It is deciduous shrub with upright to slightly arching stems, small, fresh green, trilobed to palmately divided leaves and several axillary as well as terminal round clusters of decorative, bluish black berries in late summer and autumn. Extensive investigation of chemical components in *A. koreanum* has been reported by many researchers in the worldwide. Several types of compounds have been isolated from this plant. Major active constituents are reported as lupanes and their glycosides, diterpenes, monoterpenes, lignans, phenylpropanoids, flavonoids from whole parts of *A. koreanum.* Of these, lupane triterpenes were reported as major components of leaves and *ent*-kauranes are main components of the roots of *A. koreanum.* They showed significant biological effects by several bioassay systems such as 1) anti-inflammatory activities: inhibit lipopolysaccharide (LPS)-stimulated TNF-α, IL-6, and IL-12 p40 productions in bone marrow-derived dendritic cells, decrease the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, and reduce iNOS and COX-2 mRNA in a dose-dependent pattern, 2) anticancer, and 3) antiosteoporosis by effects on the differentiation of osteoblastic MC3T3-E1 cells. The desired target of this chapter is to introduce explanations of structures and pharmacological activities of novel compounds, which have been isolated and identified from *A. koreanum* since 1985. Those studies have reported and focused on bioactivities of unambiguous

been focused on scientific evaluation of traditional drugs of plant origin.

**1. Introduction** 

**Aspects of** *Acanthopanax koreanum*

Young Ho Kim, Jeong Ah Kim and Nguyen Xuan Nhiem

*Chungnam National University,* 

with antineoplastic activity from *Ximenia americana. Toxicology and Applied Pharmacology,* Vol. 20, pp. 334-345, ISSN 0041008X.

Ya, C.; Gaffney, S. H.; Lilley, T. H. & Haslam. E. (R. W. Heminway and J. J. Karchesy Ed). (1988). *Carbohydrate-polyphenol complexation,* Plenum Press, New York, U.S.A.

### **Phytochemicals and Their Pharmacological Aspects of** *Acanthopanax koreanum*

Young Ho Kim, Jeong Ah Kim and Nguyen Xuan Nhiem *Chungnam National University, South Korea* 

#### **1. Introduction**

450 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Ya, C.; Gaffney, S. H.; Lilley, T. H. & Haslam. E. (R. W. Heminway and J. J. Karchesy Ed). (1988). *Carbohydrate-polyphenol complexation,* Plenum Press, New York, U.S.A.

*Pharmacology,* Vol. 20, pp. 334-345, ISSN 0041008X.

with antineoplastic activity from *Ximenia americana. Toxicology and Applied* 

Botanical medicines have been applied for the treatment of various human diseases with thousands of years of history all over the world. In some Asian and African countries, 80 % of population depends on traditional medicine in primary health care. On the other hand, in many developed countries, 70 % to 80 % of the population has used some forms of alternative or complementary medicine. The long tradition of using plants for medicine, supplemented by pharmaceutical research, has resulted in many plant-based Western medicines. Traditional medicine has provided Western medicine with over 40 % of all pharmaceuticals (Samuelsson & Bohlin, 2004). In the past decades, therefore, research has been focused on scientific evaluation of traditional drugs of plant origin.

*Acanthopanax* species (Araliaceae) are widely distributed in Asia, Malaysia, Polynesia, Europe, North Africa and the America. There are about 40 species of *Acanthopanax* to be found in over the world. *Acanthopanax* species have traditionally been used as a tonic and sedative as well as in the treatment of rheumatism, and diabetes. *A. koreanum* Nakai is an indigenous plant prevalently distributed throughout South Korea. It is deciduous shrub with upright to slightly arching stems, small, fresh green, trilobed to palmately divided leaves and several axillary as well as terminal round clusters of decorative, bluish black berries in late summer and autumn. Extensive investigation of chemical components in *A. koreanum* has been reported by many researchers in the worldwide. Several types of compounds have been isolated from this plant. Major active constituents are reported as lupanes and their glycosides, diterpenes, monoterpenes, lignans, phenylpropanoids, flavonoids from whole parts of *A. koreanum.* Of these, lupane triterpenes were reported as major components of leaves and *ent*-kauranes are main components of the roots of *A. koreanum.* They showed significant biological effects by several bioassay systems such as 1) anti-inflammatory activities: inhibit lipopolysaccharide (LPS)-stimulated TNF-α, IL-6, and IL-12 p40 productions in bone marrow-derived dendritic cells, decrease the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, and reduce iNOS and COX-2 mRNA in a dose-dependent pattern, 2) anticancer, and 3) antiosteoporosis by effects on the differentiation of osteoblastic MC3T3-E1 cells. The desired target of this chapter is to introduce explanations of structures and pharmacological activities of novel compounds, which have been isolated and identified from *A. koreanum* since 1985. Those studies have reported and focused on bioactivities of unambiguous

Phytochemicals and Their Pharmacological Aspects of *Acanthopanax koreanum* 453

Name Parts R1 R2 R3 Reference

steamed

steamed

steamed

30-norlupane-28-oic acid (**2**) leaves CHO OH =O (Park et al., 2010)

en-23-al-28-oic acid (**3**) leaves CHO OH CH2 (Park et al., 2010)

Fig. 1. Structures of main lupane-type triterpenes isolated from *Acanthopanax koreanum*

Fig. 2. 3-*O*-β-D-glucopyranosyl 3α,11α-dihydroxylup-20(29)-en-28-oic acid (**7**) and 3αhydroxylup-20(29)-en-23,28-oic acid 28-*O*-[β-D-Glucopyranosyl-(1→6)-β-D-Glucopyranosyl]

roots CH3 OH CH2 (Cai et al., 2004b)

leaves COOH H CH(OCH3)2 (Kim et al., 2010)

leaves COOH H CH2 (Kim et al., 2010)

leaves CH2OH OH CH2 (Kim et al., 2010)

Impressic acid (**1**) leaves

3α,11α-Dihydroxy-20,23-dioxo-

3α,11α-Dihydroxy-lup-20(29)-

3α-Hydroxylup-20(29)-en-23,28-

3α,11α,23-Trihydroxy-lup-20(29)-en-28-oic acid (**6**)

ester (**8**) (Kim et al., 2010)

(20R)-3α-Hydroxy-29 dimethoxylupan-23,28-dioic

acid (**4**)

dioic acid (**5**)

compounds from *A. koreanum*, therefore we discuss new pharmacological findings on these compounds.

The depth and breadth of research involving this plant has been organized into easily accessible and comparable information. Using Chemical Abstracts, Scifinder Scholar, and BIOSIS databases, relevant research papers were selected by based on pertinence and specificity to ethnopharmacology and phytochemistry, as well as readability. This collection was then carefully reviewed, extracted, and corroborated with available characterization data from other sources.

#### **2. Phytochemistry and pharmacology of** *A. koreanum*

#### **2.1 Lupane aglycones**

Impressic acid (**1**) was isolated for the first time from *Schefflera impressa* by (Srivastava, 1992) and it was found in the roots (Cai et al., 2004b) and the leaves (Kim et al., 2010) of *A. koreaum*. Impressic acid exhibited potently NFAT inhibitory activity with IC50 value of 12.6 μM. In the studies of (Kim et al., 2010), impressic acid (**1**) and (20*R*)-3α-hydroxy-29 dimethoxylupan-23,28-dioic acid (**4**) showed significantly anti-inflammatory activities by inhibiting TNF-α, IL-6, and IL-12 p40 productions in bone marrow-derived dendritic cells with LPS-stimulated. Furthermore, impressic acid was found to inhibit TNF-α-induced NFκB activity by inhibiting the induction of COX-2 and iNOS in HepG2 cells. Impressic acid also up-regulated the transcriptional activity of PPAR by elevating the expression of PPARγ1, PPARγ2, and SREBF-2, and by suppressing the expression of Insig-2 (Kim et al., 2011). One new norlupane, 3α,11α-dihydroxy-20,23-dioxo-30-norlupane-28-oic acid (**2**) as well as two known lupane aglycones, impressic acid (**1**), 3α,11α-dihydroxy-lup-20(29)-en-23 al-28-oic acid (**3**) were isolated and determined by (Park et al., 2010). They were evaluated for the differentiation of osteoblastic MC3T3-E1 cells. Among of them, compound **1**  significantly increased osteoblastic cell growth and differentiation as assessed by MTT assay and collagen content. Compound **2** significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of osteoblastic cell differentiation as assessed by the alkaline phosphatase activity (Park et al., 2010). Other compounds, 3α,11α-dihydroxy-lup-20(29)-en-23-al-28-oic acid, 3α-hydroxylup-20(29)-en-23,28-dioic acid (**5**), and 3α,11α,23 trihydroxy-lup-20(29)-en-28-oic acid (**6**) were also isolated from steamed leaves (Kim et al., 2010). However, they showed weak anti-inflammatory activity. 3α-Hydroxylup-20(29)-en-23,28-dioic acid (**5**) possessed broader antiviral activity against respiratory syncytial, influenza (H1N1), coxsackie B3, and herpes simplex virus type 1 viruses with IC50 values of 6.2, 25.0, 12.5, and 18.8 μg/mL, respectively (Li et al., 2007).

#### **2.2 Lupane-triterpene glycosides**

Up to date, eighteen lupane-type triterpene glycosides have been isolated from this plant and almost of them from the leaves of *A. koreanum.* They are main saponin components of the leaves of *A. koreanum.* The first lupane triterpene glycoside, acantrifoside A (**1**) was isolated from both *A. koreanum* and *A. trifoliatus* in a year of 1998 by (Yook et al., 1998). And then, two new saponins, acankoreoside A (**10**) and acankoreoside B (**11**) were isolated from the leaves of this plant (Chang et al., 1998). Our group reported seven new lupane-type triterpene glycosides, acankoreosides I-O. Their biological activities were evaluated for

compounds from *A. koreanum*, therefore we discuss new pharmacological findings on these

The depth and breadth of research involving this plant has been organized into easily accessible and comparable information. Using Chemical Abstracts, Scifinder Scholar, and BIOSIS databases, relevant research papers were selected by based on pertinence and specificity to ethnopharmacology and phytochemistry, as well as readability. This collection was then carefully reviewed, extracted, and corroborated with available characterization

Impressic acid (**1**) was isolated for the first time from *Schefflera impressa* by (Srivastava, 1992) and it was found in the roots (Cai et al., 2004b) and the leaves (Kim et al., 2010) of *A. koreaum*. Impressic acid exhibited potently NFAT inhibitory activity with IC50 value of 12.6 μM. In the studies of (Kim et al., 2010), impressic acid (**1**) and (20*R*)-3α-hydroxy-29 dimethoxylupan-23,28-dioic acid (**4**) showed significantly anti-inflammatory activities by inhibiting TNF-α, IL-6, and IL-12 p40 productions in bone marrow-derived dendritic cells with LPS-stimulated. Furthermore, impressic acid was found to inhibit TNF-α-induced NFκB activity by inhibiting the induction of COX-2 and iNOS in HepG2 cells. Impressic acid also up-regulated the transcriptional activity of PPAR by elevating the expression of PPARγ1, PPARγ2, and SREBF-2, and by suppressing the expression of Insig-2 (Kim et al., 2011). One new norlupane, 3α,11α-dihydroxy-20,23-dioxo-30-norlupane-28-oic acid (**2**) as well as two known lupane aglycones, impressic acid (**1**), 3α,11α-dihydroxy-lup-20(29)-en-23 al-28-oic acid (**3**) were isolated and determined by (Park et al., 2010). They were evaluated for the differentiation of osteoblastic MC3T3-E1 cells. Among of them, compound **1**  significantly increased osteoblastic cell growth and differentiation as assessed by MTT assay and collagen content. Compound **2** significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of osteoblastic cell differentiation as assessed by the alkaline phosphatase activity (Park et al., 2010). Other compounds, 3α,11α-dihydroxy-lup-20(29)-en-23-al-28-oic acid, 3α-hydroxylup-20(29)-en-23,28-dioic acid (**5**), and 3α,11α,23 trihydroxy-lup-20(29)-en-28-oic acid (**6**) were also isolated from steamed leaves (Kim et al., 2010). However, they showed weak anti-inflammatory activity. 3α-Hydroxylup-20(29)-en-23,28-dioic acid (**5**) possessed broader antiviral activity against respiratory syncytial, influenza (H1N1), coxsackie B3, and herpes simplex virus type 1 viruses with IC50 values of

Up to date, eighteen lupane-type triterpene glycosides have been isolated from this plant and almost of them from the leaves of *A. koreanum.* They are main saponin components of the leaves of *A. koreanum.* The first lupane triterpene glycoside, acantrifoside A (**1**) was isolated from both *A. koreanum* and *A. trifoliatus* in a year of 1998 by (Yook et al., 1998). And then, two new saponins, acankoreoside A (**10**) and acankoreoside B (**11**) were isolated from the leaves of this plant (Chang et al., 1998). Our group reported seven new lupane-type triterpene glycosides, acankoreosides I-O. Their biological activities were evaluated for

**2. Phytochemistry and pharmacology of** *A. koreanum* 

6.2, 25.0, 12.5, and 18.8 μg/mL, respectively (Li et al., 2007).

**2.2 Lupane-triterpene glycosides** 

compounds.

data from other sources.

**2.1 Lupane aglycones** 


Fig. 1. Structures of main lupane-type triterpenes isolated from *Acanthopanax koreanum*

Fig. 2. 3-*O*-β-D-glucopyranosyl 3α,11α-dihydroxylup-20(29)-en-28-oic acid (**7**) and 3αhydroxylup-20(29)-en-23,28-oic acid 28-*O*-[β-D-Glucopyranosyl-(1→6)-β-D-Glucopyranosyl] ester (**8**) (Kim et al., 2010)

Phytochemicals and Their Pharmacological Aspects of *Acanthopanax koreanum* 455

Name Part R Reference

HO

3

24

HO

OH

Name Part R1 R2 R3 R4 Reference Acankoreoside E (**20**) leaves COOH H H CHO (Park et al., 2005) Acankoreoside H (**21**) leaves CHO H H COOH (Choi et al., 2008) Acankoreoside J (**22**) leaves COOH H =O - (Nhiem et al., 2010a) Acankoreoside K (**23**) leaves COOH H OH Me (Nhiem et al., 2010a) Acankoreoside L (**24**) leaves COOH H OH CH2OH (Nhiem et al., 2010a) Acankoreoside O (**25**) leaves COOH OH H CH3 (Nhiem et al., 2010b) Fig. 3. Structures of lupane-type triterpene glycosides from *A*. *koreanum* (continued)

O

HO

HO Me

Acankoreoside C (**18**) leaves H (Chang et al., 1999) Acankoreoside N (**19**) leaves OH (Nhiem et al., 2010b)

R1

O

OH OHO

HO

H

R3

R4

R2

19

HO HO O

O

OH

28

O

O

cytotoxic activities including A549 (lung), HL-60 (acute promyelocytic leukemia), MCF-7 (breast), U937 (leukemia) cancer cell lines; immune enhancement activity (INF-γ and IL-2 release in spleen cells); anti-inflammatory (inhibitory TNF-α, IL-6, and IL-12 p40 productions in bone marrow-derived dendritic cells with LPS-stimulated, and RAW 264.7). Searching for anticancer activities from natural compounds, several acankoreosides showed significantly cytotoxic activities in various cancer cell lines (A549, HL-60, MCF-7, and U937). The effects of three new lupane glycosides, acankoreosides F-H (**13**, **14**, and **21**) on the LPS-induced production of nitric oxide and prostaglandin E2 were evaluated in RAW 264.7 macrophages. Among of them, acankoreoside F (**13**) showed the most potent inhibitory PGE2 (59.0 %) and NO (42.0 %) production at concentration of 200.0 μM. Furthermore, eleven lupane triterpene glycosides from *A. koreanum,* including three new compounds acankoreoside M-O (**16**, **24**, and **25**) were evaluated for Con A-induced splenolytic production of IL-2 and IFN-γ. The results indicated that acankoreosides A (**10**), D (**12**), L (**24**), and acantrifoside A (**9**)


Fig. 3. Structures of lupane-type triterpene glycosides from *A*. *koreanum*

cytotoxic activities including A549 (lung), HL-60 (acute promyelocytic leukemia), MCF-7 (breast), U937 (leukemia) cancer cell lines; immune enhancement activity (INF-γ and IL-2 release in spleen cells); anti-inflammatory (inhibitory TNF-α, IL-6, and IL-12 p40 productions in bone marrow-derived dendritic cells with LPS-stimulated, and RAW 264.7). Searching for anticancer activities from natural compounds, several acankoreosides showed significantly cytotoxic activities in various cancer cell lines (A549, HL-60, MCF-7, and U937). The effects of three new lupane glycosides, acankoreosides F-H (**13**, **14**, and **21**) on the LPS-induced production of nitric oxide and prostaglandin E2 were evaluated in RAW 264.7 macrophages. Among of them, acankoreoside F (**13**) showed the most potent inhibitory PGE2 (59.0 %) and NO (42.0 %) production at concentration of 200.0 μM. Furthermore, eleven lupane triterpene glycosides from *A. koreanum,* including three new compounds acankoreoside M-O (**16**, **24**, and **25**) were evaluated for Con A-induced splenolytic production of IL-2 and IFN-γ. The results indicated that acankoreosides A (**10**), D (**12**), L (**24**), and acantrifoside A (**9**)

Names Parts R1 R2 R3 Ref.

3-Epibetulinic acid 28-*O*-

Acantrifoside A (**9**) leaves CH3 OH H (Yook et al., 1998) Acankoreoside A (**10**) leaves, roots COOH H H (Chang et al., 1998)

Acankoreoside B (**11**) leaves CH2OH OH H (Chang et al., 1998) Acankoreoside D (**12**) leaves CHO OH H (Chang et al., 1999) Acankoreoside F (**13**) leaves COOH H OH (Choi et al., 2008) Acankoreoside G (**14**) leaves CHO H OH (Choi et al., 2008) Acankoreoside I (**15**) leaves CHO OH OH (Nhiem et al., 2009) Acankoreoside M (**16**) leaves COOH OH OH (Nhiem et al., 2010b)

glc-glc-rha (**17**) leaves, roots CH3 H H (Cai et al., 2004b)

Fig. 3. Structures of lupane-type triterpene glycosides from *A*. *koreanum*

(Cai et al., 2004b)


Fig. 3. Structures of lupane-type triterpene glycosides from *A*. *koreanum* (continued)

Phytochemicals and Their Pharmacological Aspects of *Acanthopanax koreanum* 457

The hepatoprotective effects of acanthoic acid were evaluated in a D-galactosamine/ lipopolysaccharide-induced fulminant hepatic failure mouse model. The effects were likely associated with a significant decrease in serum TNF-α levels, which are correlated not only with those of alanine aminotransferase and aspartate aminotransferase but also with the reduced number of apoptotic hepatocytes in the liver as confirmed using the terminal deoxynucleotidyl transferase-mediated (dUTP) nick end-labeling method and DNA fragmentation assay (Nan et al., 2008). The protective effect of acanthoic acid was investigated in acetaminophen-induced hepatic toxicity. These results indicated that acanthoic acid protected liver tissue from oxidative stress elicites by acetaminopheninduced liver damage (Wu et al., 2010). Acanthoic acid markedly suppressed the protein expression of TNF-α, COX-2, NF-κB and chymase as well as the mRNA expression of TNF-α

Isopimara-9(11),15-dien-19-ol (**29**) roots (Chung & Kim, 1986) Acanthokoreoic acid A (**30**) roots (Cai et al., 2003a)

7β-Hydroxy-*ent*-pimara-8(14),15-dien-19-oic acid (**31**) roots (Cai et al., 2004a) Sumogaside (**32**) roots (Cai et al., 2004a)

In study of (Cai et al., 2003a), a new compound, acanthokoreoside acid A (**30**) as well as acanthoic acid (**27**), (-)-pimara-9(11),15-dien-19-ol (**26**), and sumogaside (**32**) were isolated from CH2Cl2 fraction of *A. koreanum* roots. They were evaluated for inhibitory activity on IL-8 secretion in TNF-α-stimulated HT-29 and TNF-α secretion in trypsin-stimulated HMC-1. In the TNF- α-stimulated HT-29, acanthoic acid and sumogaside significantly inhibited IL-8 secretion at concentrations of 1, 10, and 100 μM and at concentrations of 10 and 100 μM,

Fig. 4. Structures of pimarane-type diterpenes from *A*. *koreanum* (continued)

and COX-2 (Kang et al., 2010).

respectively.

significantly increased both IL-2 and IFN-γ. The structure-activity relationship of these compounds was also discussed. Moreover, lupane aglycones and lupane glycosides were assayed for LPS-stimulated pro-inflammatory cytokine production. These results suggested lupane aglycone inhibited pro-inflammatory cytokine production stronger than lupane glycosides (Kim et al., 2010). This was further confirmed by the study of (Cai et al., 2004b).

#### **2.3 Pimarane-type diterpenes**

A number of pimarane-type diterpenes have been isolated and associated with significant biological activity. There are seven pimarane-type diterpenes from *A. koreanum*. All of them were isolated from the roots. Acanthoic acid was presented in roots and leaves of this plant, and was one of compounds having potent anti-inflammatory activity. Acanthoic acid, a pimarane diterpene ((-)-pimara-9(11),15-dien-19-oic acid), was isolated for the first time from *A. koreanum* in a year of 1988 by (Kim et al., 1988b) and was proved with high content of this plant. Acanthoic acid has widely exhibited of biological activities. In study of (Kang et al., 1996), acanthoic acid has potent anti-inflammatory effects by reducing the production of proinflammatory cytokines such as IL-1 and TNF-α. It was also effective in supressing experimental silicosis and cirrhosis. Furthermore, acanthoic acid was found to suppress TNF-α gene expression (Kang et al., 1998) and TNF-α-induced IL-8 production in a dosedependent manner. Acanthoic acid also inhibited TNF-α-induced MAPKs activation, IκB degradation, NF-κB nuclear translocation, and NF-κB/DNA binding activity (Kim et al., 2004). Furthermore, acanthoic acid significantly inhibited production of both TNF-α and tryptase in trypsin-stimulated human leukemic mast cell-1 at concentrations of 10 and 100 μg/mL with a dose-dependent manner. Acanthoic acid inhibited ERK phosphorylation and NF-κB activation induced by trypsin treatment without blocking of trypsin activity even though 100 μg/mL. These results suggested that acanthoic acid may inhibit the production of inflammatory mediators through inhibition of ERK phosphorylation and NF-κB activation pathway in human mast cells (Kang et al., 2006).


Fig. 4. Structures of pimarane-type diterpenes from *A*. *koreanum*

significantly increased both IL-2 and IFN-γ. The structure-activity relationship of these compounds was also discussed. Moreover, lupane aglycones and lupane glycosides were assayed for LPS-stimulated pro-inflammatory cytokine production. These results suggested lupane aglycone inhibited pro-inflammatory cytokine production stronger than lupane glycosides (Kim et al., 2010). This was further confirmed by the study of (Cai et al., 2004b).

A number of pimarane-type diterpenes have been isolated and associated with significant biological activity. There are seven pimarane-type diterpenes from *A. koreanum*. All of them were isolated from the roots. Acanthoic acid was presented in roots and leaves of this plant, and was one of compounds having potent anti-inflammatory activity. Acanthoic acid, a pimarane diterpene ((-)-pimara-9(11),15-dien-19-oic acid), was isolated for the first time from *A. koreanum* in a year of 1988 by (Kim et al., 1988b) and was proved with high content of this plant. Acanthoic acid has widely exhibited of biological activities. In study of (Kang et al., 1996), acanthoic acid has potent anti-inflammatory effects by reducing the production of proinflammatory cytokines such as IL-1 and TNF-α. It was also effective in supressing experimental silicosis and cirrhosis. Furthermore, acanthoic acid was found to suppress TNF-α gene expression (Kang et al., 1998) and TNF-α-induced IL-8 production in a dosedependent manner. Acanthoic acid also inhibited TNF-α-induced MAPKs activation, IκB degradation, NF-κB nuclear translocation, and NF-κB/DNA binding activity (Kim et al., 2004). Furthermore, acanthoic acid significantly inhibited production of both TNF-α and tryptase in trypsin-stimulated human leukemic mast cell-1 at concentrations of 10 and 100 μg/mL with a dose-dependent manner. Acanthoic acid inhibited ERK phosphorylation and NF-κB activation induced by trypsin treatment without blocking of trypsin activity even though 100 μg/mL. These results suggested that acanthoic acid may inhibit the production of inflammatory mediators through inhibition of ERK phosphorylation and NF-κB

Name Part R Reference

(-)-Pimara-9(11),15-dien-19-ol (**26**) root barks CH2OH (Kim et al., 1988b) Acanthoic acid (**27**) roots, leaves COOH (Kim et al., 1988b) (-)-Pimara-9(11),15-dien-19-ol 19-acetate (**28**) root barks CH2OAc (Kim et al., 1988b) (-)-Pimara-9(11),15-diene (**29**) root barks CH3 (Kim et al., 1988b)

**2.3 Pimarane-type diterpenes** 

activation pathway in human mast cells (Kang et al., 2006).

Fig. 4. Structures of pimarane-type diterpenes from *A*. *koreanum*

The hepatoprotective effects of acanthoic acid were evaluated in a D-galactosamine/ lipopolysaccharide-induced fulminant hepatic failure mouse model. The effects were likely associated with a significant decrease in serum TNF-α levels, which are correlated not only with those of alanine aminotransferase and aspartate aminotransferase but also with the reduced number of apoptotic hepatocytes in the liver as confirmed using the terminal deoxynucleotidyl transferase-mediated (dUTP) nick end-labeling method and DNA fragmentation assay (Nan et al., 2008). The protective effect of acanthoic acid was investigated in acetaminophen-induced hepatic toxicity. These results indicated that acanthoic acid protected liver tissue from oxidative stress elicites by acetaminopheninduced liver damage (Wu et al., 2010). Acanthoic acid markedly suppressed the protein expression of TNF-α, COX-2, NF-κB and chymase as well as the mRNA expression of TNF-α and COX-2 (Kang et al., 2010).

Fig. 4. Structures of pimarane-type diterpenes from *A*. *koreanum* (continued)

In study of (Cai et al., 2003a), a new compound, acanthokoreoside acid A (**30**) as well as acanthoic acid (**27**), (-)-pimara-9(11),15-dien-19-ol (**26**), and sumogaside (**32**) were isolated from CH2Cl2 fraction of *A. koreanum* roots. They were evaluated for inhibitory activity on IL-8 secretion in TNF-α-stimulated HT-29 and TNF-α secretion in trypsin-stimulated HMC-1. In the TNF- α-stimulated HT-29, acanthoic acid and sumogaside significantly inhibited IL-8 secretion at concentrations of 1, 10, and 100 μM and at concentrations of 10 and 100 μM, respectively.

Phytochemicals and Their Pharmacological Aspects of *Acanthopanax koreanum* 459

Two lignans were found from the roots of *A. koreanum*. Those were acanthoside D (**39**) (Hahn et al., 1985) and ariensin (**40**) (Kim et al., 1988a). Beside these lignans, the first

**2.5 Other class compounds** 

Fig. 6. Structures of compounds isolated from *A*. *Koreanum* 

#### **2.4** *ent***-Kaurane-type diterpenes**

*ent*-Kaurane, a tetracyclic diterpene, has been proven to excert various biological activities like cytotoxicity, anti-inflammation, and so on. From the roots of *A. koreanum*, (Kim et al., 1988b) and (Cai et al., 2003b) isolated six *ent*-kaurane-type diterpenes, including *ent*-kaur-16-en-19-oic acid (**33**), 16α-hydroxy-*ent*-kauran-19-oic acid (**34**), paniculoside IV (**35**), 16αH,17 isovaleryloxy-*ent*-kauran-19-oic acid (**36**), 16α-hydroxy-17-isovaleryloxy-*ent*-kauran-19-oic acid (**37**), and 16α,17-dihydroxy-*ent*-kauran-19-oic acid (**38**). (Cai et al., 2003b) evaluated five *ent*kauranes for TNF-α secretion from HMC-1, a trypsin-stimulated human leukemic mast cell line. The results indicated that 16αH,17-isovaleryloxy-*ent*-kauran-19-oic acid (**36**) exhibited potently an inhibitory activity with IC50 value of 16.2 μM. Furthermore, these compounds were assayed for their inhibitory effect against NFAR transcription factor and 16α-hydroxy-17 isovaleryloxy-*ent*-kauran-19-oic acid (**37**) was found to significantly inhibit NFAT transcription factor with IC50 of 6.7 μM (Cai et al., 2004a). The authors also found that remain compounds containing a hydroxyl group at C-16 or a glycoside at C-4 showed no activity.


Fig. 5. Structures of *ent*-kaurane-type diterpenes from *A*. *koreanum*

#### **2.5 Other class compounds**

458 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

*ent*-Kaurane, a tetracyclic diterpene, has been proven to excert various biological activities like cytotoxicity, anti-inflammation, and so on. From the roots of *A. koreanum*, (Kim et al., 1988b) and (Cai et al., 2003b) isolated six *ent*-kaurane-type diterpenes, including *ent*-kaur-16-en-19-oic acid (**33**), 16α-hydroxy-*ent*-kauran-19-oic acid (**34**), paniculoside IV (**35**), 16αH,17 isovaleryloxy-*ent*-kauran-19-oic acid (**36**), 16α-hydroxy-17-isovaleryloxy-*ent*-kauran-19-oic acid (**37**), and 16α,17-dihydroxy-*ent*-kauran-19-oic acid (**38**). (Cai et al., 2003b) evaluated five *ent*kauranes for TNF-α secretion from HMC-1, a trypsin-stimulated human leukemic mast cell line. The results indicated that 16αH,17-isovaleryloxy-*ent*-kauran-19-oic acid (**36**) exhibited potently an inhibitory activity with IC50 value of 16.2 μM. Furthermore, these compounds were assayed for their inhibitory effect against NFAR transcription factor and 16α-hydroxy-17 isovaleryloxy-*ent*-kauran-19-oic acid (**37**) was found to significantly inhibit NFAT transcription factor with IC50 of 6.7 μM (Cai et al., 2004a). The authors also found that remain compounds

COOH

**36**

**37**

**38**

Name Part Reference

COOH

COOH

*ent*-Kaur-16-en-19-oic acid (**33**) roots (Kim et al., 1988b) α-Hydroxy-ent-kauran-19-oic acid (**34**) roots (Cai et al., 2003b) Paniculoside IV (**35**) roots (Cai et al., 2003b) αH,17-Isovaleryloxy-*ent*-kauran-19-oic acid (**36**) roots (Cai et al., 2003b) α-Hydroxy-17-isovaleryloxy-*ent*-kauran-19-oic acid (**37**) roots (Cai et al., 2003b) α,17-Dihydroxy-*ent*-kauran-19-oic acid (**38**) roots (Kim et al., 1988b)

H

OH

OH OH

O Me

O Me

O Me

Me O

containing a hydroxyl group at C-16 or a glycoside at C-4 showed no activity.

OH

OH OH

Fig. 5. Structures of *ent*-kaurane-type diterpenes from *A*. *koreanum*

**2.4** *ent***-Kaurane-type diterpenes** 

COOH

**33**

**34**

**35**

COOH

COOGlc

Two lignans were found from the roots of *A. koreanum*. Those were acanthoside D (**39**) (Hahn et al., 1985) and ariensin (**40**) (Kim et al., 1988a). Beside these lignans, the first

Fig. 6. Structures of compounds isolated from *A*. *Koreanum* 

Phytochemicals and Their Pharmacological Aspects of *Acanthopanax koreanum* 461

**Pos. 13 14 15 16 17 18 19 20 21 22 23 24 25**  1 33.2 33.1 35.9 35.5 36.5 36.1 36.7 33.3 33.1 34.0 34.2 33.9 34.5 2 26.1 26.7 27.0 26.4 26.6 21.8 19.2 26.1 26.7 26.1 26.1 26.1 26.1 3 72.8 73.0 73.8 73.8 73.5 81.3 82.4 72.7 73.0 73.4 73.3 73.6 73.3 4 51.8 52.5 53.7 53.0 40.3 37.8 38.5 51.7 52.5 52.2 52.1 52.3 52.1 5 45.4 44.0 44.5 45.4 49.9 50.5 51.0 45.6 44.0 46.6 46.9 46.2 47.4 6 21.7 20.9 22.0 22.6 19.2 18.4 22.2 21.7 21.1 22.2 22.2 22.3 22.2 7 34.5 34.1 36.2 36.1 36.3 35.4 36.4 34.6 34.1 35.1 35.5 35.3 35.5 8 41.8 41.8 41.6 43.5 43.4 42.6 43.5 41.7 41.8 42.5 43.0 42.6 43.1 9 51.0 50.6 56.3 56.7 50.2 55.8 56.3 50.6 50.2 51.9 52.0 51.7 52.0 10 37.4 36.9 38.2 40.0 38.3 39.6 40.4 37.4 36.9 38.2 38.1 38.1 38.2 11 21.0 21.0 71.1 71.1 24.2 69.7 71.2 20.9 20.8 22.0 22.4 22.0 22.2 12 27.1 27.0 38.3 39.4 26.5 38.1 39.5 26.9 26.9 28.5 29.8 28.3 26.1 13 38.3 38.3 39.5 38.2 38.2 37.3 38.3 38.2 38.2 38.6 39.5 39.4 39.1 14 42.8 42.8 44.3 44.3 43.8 42.9 44.0 43.0 43.0 43.6 44.5 44.0 44.3 15 30.2 30.1 30.8 30.8 31.6 30.0 30.9 30.0 30.0 30.9 31.2 30.9 30.9 16 32.2 32.1 32.8 32.9 32.8 32.2 32.9 32.0 32.1 32.4 33.1 32.9 34.5 17 57.0 56.9 57.9 57.9 57.9 56.8 58.0 57.0 57.3 58.0 60.1 58.0 59.4 18 50.2 50.2 50.9 50.9 50.1 49.4 51.0 48.5 48.9 50.8 49.5 49.7 50.4 19 43.2 43.2 43.7 43.7 48.2 47.1 43.8 37.3 40.6 52.8 45.7 39.3 85.4 20 156.5 156.5 155.9 155.9 151.3 150.4 156.0 50.1 42.1 215.4 76.1 39.5 36.8 21 32.7 32.7 33.5 33.6 30.6 30.8 33.6 24.6 25.0 29.3 28.8 24.38 34.5 22 36.8 36.7 37.3 37.4 37.5 36.7 37.4 37.4 37.3 37.6 37.5 38.3 37.2 23 181.2 209.8 211.2 180.6 29.5 29.8 29.8 181.8 209.9 183.4 184.2 181.8 185.2 24 18.2 14.6 15.2 18.2 23.0 23.0 23.3 18.3 14.6 18.3 18.4 18.2 18.6 25 16.8 16.4 17.2 17.5 17.8 16.8 17.3 16.8 16.3 17.2 17.4 17.2 17.4 26 16.7 16.5 18.3 18.1 17.0 17.6 18.0 16.6 16.5 16.9 17.2 17.0 17.1 27 14.9 14.8 15.1 15.1 14.9 14.7 15.2 14.7 14.9 15.2 15.6 15.1 15.5 28 175.1 175.0 176.3 176.3 176.2 174.8 176.4 175.0 174.9 176.2 177.0 176.4 176.7 29 106.1 106.1 107.7 107.7 110.8 110.1 107.6 204.6 180.0 29.9 71.2 8.9 21.9 30 64.3 64.3 65.5 65.5 19.6 19.6 65.6 7.0 10.0 19.6 110.3 27.0 Solv. *a a b b b a b a a b b b b* 

a recorded in pyridine-*d5*, b recorded in methanol-*d4*.

2005); **21**: (Choi et al., 2008); **22**, **23**, **24**: (Nhiem et al., 2010a).

Table 1. 13C-NMR data of lupane aglycone moieties (continued)

Note: NMR data were obtained from **13**, **14**, **21**: (Choi et al., 2008); **15**: (Nhiem et al., 2009) (Cai et al., 2004b), **16**, **19**, **25**: (Nhiem et al., 2010b); **17**: (Cai et al., 2004b); **18**: (Chang et al., 1999), **20**: (Park et al.,


a recorded in pyridine-*d5*, b recorded in methanol-*d4*.

Note: NMR data were obtained from **1:** (Srivastava, 1992); **2**, **3** (Park et al., 2010); **4**, **8**: (Kim et al., 2010); **5**, **6**, **10**, **11**: (Chang et al., 1998); **7**, **12:** (Chang et al., 1999)

Table 1. 13C-NMR data of lupane aglycone moieties

Phytochemicals and Their Pharmacological Aspects of *Acanthopanax koreanum* 461

a recorded in pyridine-*d5*, b recorded in methanol-*d4*.

**5**, **6**, **10**, **11**: (Chang et al., 1998); **7**, **12:** (Chang et al., 1999) Table 1. 13C-NMR data of lupane aglycone moieties

Note: NMR data were obtained from **1:** (Srivastava, 1992); **2**, **3** (Park et al., 2010); **4**, **8**: (Kim et al., 2010);

**Pos. 1 2 3 4 5 6 7 8 9 10 11 12**  1 35.8 35.7 35.1 32.8 32.6 35.9 36.2 33.1 36.2 33.1 35.9 34.9 2 26.8 26.9 25.5 26.0 25.9 27.1 21.9 26.2 26.9 26.2 27.1 26.8 3 74.8 73.7 75.8 72.8 72.7 75.9 81.5 71.5 75.3 73.0 75.7 72.7 4 38.4 53.5 37.7 51.8 51.7 41.2 37.9 51.9 38.5 52.8 41.1 52.6 5 49.4 44.4 48.8 44.7 44.8 43.9 50.6 45.3 49.6 45.0 43.8 43.9 6 19.2 21.8 19.3 21.6 21.0 18.3 18.4 21.7 18.6 21.8 18.3 21.0 7 36.0 35.7 35.4 23.8 34.5 35.6 35.7 34.6 35.7 34.6 35.4 35.2 8 42.7 43.2 42.2 41.6 41.5 42.8 42.7 41.8 42.8 42.8 42.7 42.4 9 55.9 56.2 55.6 50.7 50.8 56.2 55.9 51.0 56.2 51.2 55.6 55.6 10 39.8 39.6 39.1 38.2 37.2 39.6 39.7 37.4 39.9 37.6 39.6 38.7 11 69.8 70.7 70.4 21.1 21.5 69.9 69.9 20.9 69.8 21.2 69.8 69.4 12 38.4 39.4 37.7 27.7 25.8 38.4 38.3 26.0 38.3 26.0 38.3 37.8 13 37.5 37.5 37.2 28.6 38.4 37.6 37.7 38.4 37.4 38.4 37.4 37.0 14 42.9 44.1 42.6 43.0 42.7 42.8 42.9 42.9 43.0 43.0 42.9 43.0 15 29.9 30.6 29.5 30.2 30.0 30.1 30.2 30.1 30.0 30.1 30.0 29.6 16 32.8 32.7 32.0 34.7 32.8 32.8 32.9 32.2 32.3 32.4 32.2 31.9 17 56.5 57.1 56.2 56.7 56.4 56.5 56.6 57.0 56.9 57.7 56.9 56.6 18 47.5 50.0 46.6 49.1 49.5 49.4 49.4 49.8 49.5 49.7 49.4 49.1 19 49.4 52.5 48.6 43.7 47.5 47.5 47.5 47.4 47.2 47.5 47.1 46.8 20 151.0 215.0 149.7 37.6 151.1 150.8 150.9 150.9 150.4 151.0 150.4 150.1 21 31.0 29.4 30.5 24.6 30.9 31.2 31.3 30.8 30.9 30.9 30.9 30.6 22 37.5 37.7 36.8 37.2 37.3 37.4 37.4 36.9 36.8 37.2 36.7 36.4 23 29.6 210.9 28.7 178.9 179.6 71.9 29.9 181.3 29.8 179.0 71.9 209.7 24 22.7 15.1 22.2 17.8 17.7 18.3 23.0 18.1 22.9 18.0 18.3 14.6 25 18.0 17.2 17.3 16.6 16.5 17.0 16.8 16.8 16.9 16.8 17.1 16.5 26 17.4 17.8 16.2 16.6 16.5 17.7 17.6 16.7 17.7 16.6 17.7 17.4 27 14.4 15.0 14.6 14.5 14.6 14.8 14.8 14.8 14.8 14.8 14.8 14.4 28 179.2 179.4 181.5 179.4 178.6 178.8 178.9 175.0 175.0 175.9 175.0 174.6 29 110.0 30.0 110.2 107.8 109.7 110.0 110.1 110.0 110.2 110.0 110.0 109.9 30 19.2 17.9 16.4 19.2 19.5 19.6 19.4 19.5 19.3 19.5 19.2 Solv. *a a b a a a a a a a a a* 


a recorded in pyridine-*d5*, b recorded in methanol-*d4*.

Note: NMR data were obtained from **13**, **14**, **21**: (Choi et al., 2008); **15**: (Nhiem et al., 2009) (Cai et al., 2004b), **16**, **19**, **25**: (Nhiem et al., 2010b); **17**: (Cai et al., 2004b); **18**: (Chang et al., 1999), **20**: (Park et al., 2005); **21**: (Choi et al., 2008); **22**, **23**, **24**: (Nhiem et al., 2010a).

Table 1. 13C-NMR data of lupane aglycone moieties (continued)

Phytochemicals and Their Pharmacological Aspects of *Acanthopanax koreanum* 463

3. When 23-methyl group was replaced with aldehydic group, chemical shifts of C-23 and C-4 moved to down field from 28.0-28.8 to 209.0-210.0, 37.5-39.5 to 54.9-56.3 ppm, respectively. When 23-methy group was replaced with carboxylic group, chemical shifts of C-23 and C-4 changed from 28.5 to 178.0, 37.8 to 53.0 respectively, and when 23 methyl group was replaced with CH2OH, chemical shift of C-23 had a large change

4. When 30-methyl group was replaced with CH2OH, the chemical shifts of C-20 and C-30 downshifted from 151.0 to 156.5, from 19.5 to 64.5 ppm, respectively; chemical shifts of

This chapter is intended to serve as a reference tool for people in all fields of ethnopharmacology and natural products chemistry. The pharmacological studies on *A. koreanum* indicated the immense potential possibility of this plant in the treatment of conditions such as inflammation, rheumatism, diabetes, cardiovascular, and virus. However, the diverse pharmacological activities of solvent extracts and phytochemicals of *A. koreanum* have only been tested in *in vitro* assay using laboratory animals, and obtained too unclearly and ambiguously for the case of human beings to be conducted on enough. However, these gaps in the studies demand to be bridged in order to exploit medicinal potential of the entire plant of *A. koreanum*. It is still clear that *A. koreanum* is massively and widespreadly consumed, and also contiuously studied expecting clinical treatment of various diseases for the future in Korea as well as in the world. From these viewpoints, impressic acid and acanthoic acid, major components of *A. koreanum* are good candidates for further studies in clinical trials, and the development of products derived from *A. koreanum*  can be an important part of our biodiversity to respect and sustain for coming generation.

C-19 and C-29 upshifted from 47.5 to 43.0, from 110.0 to 106.0 ppm, respectively. 5. When hydroxyl group was at C-11, chemical shift of C-11 downshifted from 21.1 to 71.0

from 28.0 to 71.5 ppm; chemical shift of C-4 had small change about 2.0ppm.

ppm. Furthermore, configuration of hydroxyl group at this position is α.

Fact sheet No 134: Traditional medicine. World Health Organization. December 2008.

Vol.51, No.5, pp. 605-607, ISSN 0009-2363

Vol.26, No.9, pp. 731-734, ISSN 0253-6269

Cai, X. F.; Shen, G.; Dat, N. T.; Kang, O. H.; Kim, J. A.; Lee, Y. M.; Lee, J. J., & Kim, Y. H.

Cai, X. F.; Shen, G.; Dat, N. T.; Kang, O. H.; Lee, Y. M.; Lee, J. J., & Kim, Y. H. (2003b).

Cai, X. F.; Lee, I. S.; Dat, N. T.; Shen, G., & Kim, Y. H. (2004a). Diterpenoids with inhibitory

Cai, X. F.; Lee, I. S.; Shen, G.; Dat, N. T.; Lee, J. J., & Kim, Y. H. (2004b). Triterpenoids from

Archives of Pharmacal Research, Vol.27, No.8, pp. 825-828, ISSN 0253-6269

Phytotherapy Research, Vol.18, No.8, pp. 677-680, ISSN 0951-418X

(2003a). Inhibitory effect of TNF-α and IL-8 secretion by pimarane-type diterpenoids from *Acanthopanax koreanum*. Chemical & Pharmaceutical Bulletin,

Inhibitory effect of kaurane type diterpenoids from *Acanthopanax koreanum* on TNFα secretion from trypsin-stimulated HMC-1 cells. Archives of Pharmacal Research,

activity against NFAT transcription factor from *Acanthopanax koreanum*

*Acanthopanax koreanum* root and their inhibitory activities on NFAT transcription.

**4. Conclusion** 

**5. References**

phenylpropanoid, syrinoside (**44**) were isolated from the roots of *A. koreanum* by (Hahn et al., 1985) and then was ariensin (**43**) (Kim et al., 1988a). In study antioxidant activity of chemical components from the leaves of this plant, (Nhiem et al., 2011) isolated one new phenylpropanoid named acanthopanic acid and one known 1,2-O-dicaffeoylcyclopenta-3 ol. These compounds showed significantly antioxidant activity by the intracellular ROS radical scavenging DCF-DA assay with IC50 values of 3.8 and 2.9 μM, respectively. Until now, only rutin (**45**), a quercetin glycoside was isolated from this plant with large amount. Rutin is used in many countries as medication for blood vessel protection and are ingredients of numerous multivitamin preparations and herbal remedies. Rutin has various biological activities that are beneficial to human health such as antioxidant effect (Nhiem et al., 2011), protective effect against hepatotoxicity, and anti-inflammatory effect. On the other hand, from the leaves of *A. koreanum*, two monoterpenoids, 4S)-α-terpineol O-β-D glucopyranoside (**46**) (Nhiem et al., 2011) and betulabuside B (**47**) (Park et al., 2010) were isolated. From fruits, citric, maleic succinic, malonic, furmaric, and malic acid were isolated (Shin & Kim, 1985).

#### **3. NMR data of lupane aglycones**

Lupane triterpenes are a class of the most compounds isolated from the leaves and roots of *A. koreanum*, which were determined that this type of compounds are main chemical components of this plant.

Structure of lupanes were elucidated with 1H-NMR, 13C-NMR, DEPT (distortionless enhancement by polarization transfer), COSY (1H-1H shift correlation spectroscopy), TOCSY (total correlation spectroscopy), HMBC (heteronuclear multiple bond correlation), HSQC (heteronuclear single quantum coherence), NOESY (nuclear overhauser enhancement spectroscopy, and ROESY (rotating frame overhause effect spectroscopy). Proton coupling networks of sugar moieties were indicated with 1H-NMR, COSY, HMBC and HSQC. Herein, we suggest statistical results of 13C-NMR data of lupane-type triterpene aglycones and their derivatives in comparison with data of references (Table 1).

Observed the isolated compounds from *A. koreanum,* we found that there are four main classes including lupane triterpenoids, pimarane diterpenoids, *ent*-kaurane diterpenoids, and lignans. Among of them, lupane triterpenes were isolated as numerous of compounds with high yield. These lupanes often contain hydroxyl group at C-3, carboxyl at C-28. In some compounds, hydroxyl, aldehydic, carboxylic groups were at C-11, C-23, and C-30, glycoside was at C-28 and rarely at C-3.

From Table 1, we summarized all 13C-NMR characteristics of lupane aglycones as follows:


### **4. Conclusion**

462 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

phenylpropanoid, syrinoside (**44**) were isolated from the roots of *A. koreanum* by (Hahn et al., 1985) and then was ariensin (**43**) (Kim et al., 1988a). In study antioxidant activity of chemical components from the leaves of this plant, (Nhiem et al., 2011) isolated one new phenylpropanoid named acanthopanic acid and one known 1,2-O-dicaffeoylcyclopenta-3 ol. These compounds showed significantly antioxidant activity by the intracellular ROS radical scavenging DCF-DA assay with IC50 values of 3.8 and 2.9 μM, respectively. Until now, only rutin (**45**), a quercetin glycoside was isolated from this plant with large amount. Rutin is used in many countries as medication for blood vessel protection and are ingredients of numerous multivitamin preparations and herbal remedies. Rutin has various biological activities that are beneficial to human health such as antioxidant effect (Nhiem et al., 2011), protective effect against hepatotoxicity, and anti-inflammatory effect. On the other hand, from the leaves of *A. koreanum*, two monoterpenoids, 4S)-α-terpineol O-β-D glucopyranoside (**46**) (Nhiem et al., 2011) and betulabuside B (**47**) (Park et al., 2010) were isolated. From fruits, citric, maleic succinic, malonic, furmaric, and malic acid were

Lupane triterpenes are a class of the most compounds isolated from the leaves and roots of *A. koreanum*, which were determined that this type of compounds are main chemical

Structure of lupanes were elucidated with 1H-NMR, 13C-NMR, DEPT (distortionless enhancement by polarization transfer), COSY (1H-1H shift correlation spectroscopy), TOCSY (total correlation spectroscopy), HMBC (heteronuclear multiple bond correlation), HSQC (heteronuclear single quantum coherence), NOESY (nuclear overhauser enhancement spectroscopy, and ROESY (rotating frame overhause effect spectroscopy). Proton coupling networks of sugar moieties were indicated with 1H-NMR, COSY, HMBC and HSQC. Herein, we suggest statistical results of 13C-NMR data of lupane-type triterpene aglycones and their

Observed the isolated compounds from *A. koreanum,* we found that there are four main classes including lupane triterpenoids, pimarane diterpenoids, *ent*-kaurane diterpenoids, and lignans. Among of them, lupane triterpenes were isolated as numerous of compounds with high yield. These lupanes often contain hydroxyl group at C-3, carboxyl at C-28. In some compounds, hydroxyl, aldehydic, carboxylic groups were at C-11, C-23, and C-30,

From Table 1, we summarized all 13C-NMR characteristics of lupane aglycones as

1. When hydroxyl group at C-3, chemical shift of C-3 was about 73.0 ppm and configuration of hydroxyl group at C-3 is α orientation. When glycosidation is at C-3,

2. Free carboxylic group at C-28 were confirmed by chemical shift about 178.0~180.0 ppm. When sugar moiety was at C-28, chemical shift of C-28 is 174.6~176.3 ppm, decreased

chemical shift of C-3 moved to down field with δC of 81.0 ppm.

isolated (Shin & Kim, 1985).

components of this plant.

**3. NMR data of lupane aglycones** 

glycoside was at C-28 and rarely at C-3.

about 2.5-3.8 ppm.

follows:

derivatives in comparison with data of references (Table 1).

This chapter is intended to serve as a reference tool for people in all fields of ethnopharmacology and natural products chemistry. The pharmacological studies on *A. koreanum* indicated the immense potential possibility of this plant in the treatment of conditions such as inflammation, rheumatism, diabetes, cardiovascular, and virus. However, the diverse pharmacological activities of solvent extracts and phytochemicals of *A. koreanum* have only been tested in *in vitro* assay using laboratory animals, and obtained too unclearly and ambiguously for the case of human beings to be conducted on enough. However, these gaps in the studies demand to be bridged in order to exploit medicinal potential of the entire plant of *A. koreanum*. It is still clear that *A. koreanum* is massively and widespreadly consumed, and also contiuously studied expecting clinical treatment of various diseases for the future in Korea as well as in the world. From these viewpoints, impressic acid and acanthoic acid, major components of *A. koreanum* are good candidates for further studies in clinical trials, and the development of products derived from *A. koreanum*  can be an important part of our biodiversity to respect and sustain for coming generation.

#### **5. References**

Fact sheet No 134: Traditional medicine. World Health Organization. December 2008.


Phytochemicals and Their Pharmacological Aspects of *Acanthopanax koreanum* 465

Kim, Y. H.; Chung, B. S., & Sankawa, U. (1988b). Pimaradiene diterpenes from *Acanthopanax koreanum*. Journal of Natural Products, Vol.51, No.6, pp. 1080-1083, ISSN 163-3864 Li, Y.; Jiang, R.; Ooi, L. S. M.; But, P. P. H., &Ooi, V. E. C. (2007). Antiviral triterpenoids from

Nan, J. X.; Jin, X. J.; Lian, L. H.; Cai, X. F.; Jiang, Y. Z.; Jin, H. R., & Lee, J. J. (2008). A

Nhiem, N. X.; Kiem, P. V.; Minh, C. V.; Ha, D. T.; Tai, B. H.; Yen, P. H.; Tung, N. H.; Hyun, J.

Nhiem, N. X.; Kiem, P. V.; Minh, C. V.; Tai, B. H.; Tung, N. H.; Ha, D. T.; Soung, K. S.; Kim,

Nhiem, N. X.; Kim, K. C.; Kim, A.-D.; Hyun, J. W.; Kang, H. K.; Kiem, P. V.; Minh, C. V.;

Park, S. H.; Nhiem, N. X.; Kiem, P. V.; Choi, E. M.; Kim, J. A., & Kim, Y. H. (2010). A new

Park, S. Y.; Choi, H. S.; Yook, C. S., & Nohara, T. (2005). A new lupane glycoside from the

Samuelsson, G. & Bohlin, L. (2004). Drugs of natural origin: A textbook of pharmacognosy. 5 Ed.; Swedish Pharmaceutical Press, ISBN 9197431842, Stockkholm, Sweden Shin, E. T. & Kim, C. S. (1985). Composition of fatty acid and organic acid in Acanthopanax. Han'guk Sikp'um Kwahakhoechi, Vol.17, No.5, pp. 403-405, ISSN 0367-6293 Srivastava, S. K. (1992). A new triterpenic acid from *Schefflera impressa*. Journal of Natural

Wu, Y. L.; Jiang, Y. Z.; Jin, X. J.; Lan, L. H.; Piao, J. Y.; Wan, Y.; Jin, H. R.; Lee, J. J., & Nan, J.

X. (2010). Acanthoic acid, a diterpene in *Acanthopanax koreanum*, protects acetaminophen-induced hapatic toxicity in mice. Phytomedicine, Vol.17, No.6, pp.

Natural Products Research Vol.13, No.1, pp. 56-61, ISSN 1028-6020

466-470, ISSN 0951-418X

0960-894X

No.9, pp. 986-989, ISSN 0009-2363

Vol.76, No.2, pp. 189-194, ISSN 0032-0943

Vol.33, No.1, pp. 75-80, ISSN 0253-6269

Products, Vol.55, No.3, pp. 298-302, ISSN 0163-3864

pp. 97-99, ISSN 0009-2363

475-479, ISSN 0944-7113

the medicinal plant *Schefflera heptaphylla*. Phytotherapy Research, Vol.21, No.5, pp.

diterpenoid acanthoic acid from *Acanthopanax koreanum* protects against Dgalactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice. Biological & Pharmaceutical Bulletin, Vol.31, No.4, pp. 738-742, ISSN 0918-6158 Nhiem, N. X.; Tung, N. H.; Kiem, P. V.; Minh, C. V.; Ding, Y.; Hyun, J. H.; Kang, H. K., &

Kim, Y. H. (2009). Lupane triterpene glycosides from leave of *Acanthopanax koreanum* and their cytotoxic activity. Chemical & Pharmaceutical Bulletin, Vol.57,

H.; Kang, H. K., & Kim, Y. H. (2010a). Lupane-type triterpene glycosides from the leaves of *Acanthopanax koreanum* and their in vitro cytotoxicity. Planta Medica,

J. H.; Ahn, J. Y.; Lee, Y. M., & Kim, Y. H. (2010b). Structure-activity relationship of lupane-triterpene glycosides from *Acanthopanax koreanum* on spleen lymphocyte IL-2 and INF-γ. Bioorganic & Medicinal Chemistry Letters, No.20, pp. 4927-4931, ISSN

Thu, V. K.; Tai, B. H.; Kim, J. A., & Kim, Y. H. (2011). Phenylpropanoids from the leaves of *Acanthopanax koreanum* and their antioxidant activity. Journal of Asian

norlupane triterpene from the leaves of *Acanthopanax koreanum* increases the differentiation of osteoblastic MC3T3-e1 cells. Archives of Pharmacal Research,

leaves of *Acanthopanax koreanum*. Chemical & Pharmaceutical Bulletin, Vol.51, No.1,


Chang, S. Y.; Yook, C. S., & Nohara, T. (1998). Two new lupane-triterpene glycosides from

Chang, S. Y.; Yook, C. S., & Nohara, T. (1999). Lupane-triterpene glycosides from leaves of

Choi, H. S.; Kim, H. J.; Nam, S. G.; Kim, I. S.; Lee, K. T.; Yook, C. S., & Lee, Y. S. (2008).

Hahn, D.-R.; Kim, C. J., & Kim, J. H. (1985). A study on the chemical constituents of

Kang, H. S.; Kim, Y. H.; Lee, C. S.; Lee, J. J.; Choi, I., & Pyun, K. H. (1996). Suppression of

Kang, H. S.; Song, H. K.; Lee, J. J.; Pyun, K. H., & Choi, I. (1998). Effects of acanthoic acid on

Kang, O.-H.; Choi, Y. A.; Park, H. J.; Kang, C. S.; Song, B. S.; Choi, S. C.; Nah, Y. H.; Yun, K.

Kang, O. H.; Kim, D. K.; Cai, X. F.; Kim, Y. H., & Lee, Y. M. (2010). Attenuation of

Kim, J. A.; Yang, S. Y.; Koo, J.-E.; Koh, Y. S., & Kim, Y. H. (2010). Lupane-type triterpenoids

Kim, J. A.; Yang, S. Y.; Song, S. B., & Kim, Y. H. (2011). Effects of impressic acid from

Kim, Y. H.; Chung, B. S.; Ko, Y. S., & Han, H. J. (1988a). Studies on the chemical constituents

Research, Vol.34, No.8, pp. 1347-1351, ISSN 0253-6269

Archives of Pharmacal Research, Vol.33, No.1, pp. 87-93, ISSN 0253-6269 Kim, J. A.; Kim, D. K.; Tae, J.; Kang, O. H.; Choi, Y. A.; Choi, S. C.; Kim, T. H.; Nah, Y. H.;

Pharmaceutical Bulletin, Vol.56, No.11, pp. 1613–1616, ISSN 0009-2363 Chung, B. S. & Kim, Y. H. (1986). Studies on the constituents of *Acanthopanax koreanum*.

Saengyak Hakhoechi, Vol.17, No.1, pp. 62-66, ISSN 0253-3073

Vol.29, No.6, pp. 357-361, ISSN 0513-4234

Vol.170, No.2, pp. 212-221, ISSN 0008-8749

Vol.7, No.4, pp. 257-259, ISSN 0962-9351

No.3, pp. 326-331, ISSN 0378-8741

pp. 163-165, ISSN 0009-2363

9422

8981

6707, ISSN 0960-894X

159-162, ISSN 0253-6269

leaves of *Acanthopanax koreanum*. Chemical & Pharmaceutical Bulletin, Vol.46, No.1,

*Acanthopanax koreanum*. Phytochemistry, Vol.50, No.8, pp. 1369-1374, ISSN 0031-

Lupane glycosides from the leaves of *Acanthopanax koreanum*. Chemical &

*Acanthopanax koreanum* Nakai and its pharmacological activities. Yakhak Hoechi

interleukin-1 and tumor necrosis factor-. production by acanthoic acid, (-)-pimara-9(11),15-dien-19-oic acid, and its antifibrotic effects in vivo. Cellular Immunology,

TNF-a gene expression and haptoglobin synthesis. Mediators of Inflammation,

J.; Cai, X. F.; Kim, Y. H.; Bae, K., & Lee, Y. M. (2006). Inhibition of trypsin-induced mast cell activation by acanthoic acid. Journal of Ethnopharmacology, Vol.105,

experimental murine colitis by acanthoic acid from *Acanthopanax koreanum*.

Choi, S. J.; Kim, Y. H.; Bae, K. H., & Lee, Y. M. (2004). Acanthoic acid inhibits IL-8 production via MAPKs and NF-kappaB in a TNF-a-stimulated human intestinal epithelial cell line. Clinica Chimica Acta, Vol. 342, No.1-2, pp. 193-202, ISSN 0009-

from the steamed leaves of *Acanthopanax koreanum* and their inhibitory effects on the LPS-stimulated pro-inflammatory cytokine production in bone marrow-derived dendritic cells. Bioorganic & Medicinal Chemistry Letters, Vol.20, No.22, pp. 6703-

*Acanthopanax koreanum* on NFκB and PPARγ activities. Archives of Pharmacal

of *Acanthopanax koreanum*. (II). Archives of Pharmacal Research, Vol.11, No.2, pp.


**22** 

*Canada* 

**A Review** 

L.G. Rao1, N. Kang2 and A.V. Rao2

**Polyphenol Antioxidants and Bone Health:** 

*2Department of Nutritional Sciences, Faculty of Medicine, University of Toronto,* 

Osteoporosis is a skeletal disease characterized by bone loss and structural deterioration of the bone tissue, leading to an increase in bone fragility and susceptibility to fractures, most frequently in the hip, wrist and spine (Sendur *et al.,* 2009). Bone loss is associated with such factors as age, menopause in women, smoking, alcohol excess, calcium and vitamin D deficiency, low weight and muscle mass, anticonvulsant and corticosteroid use as well as certain co-morbid conditions such as rheumatoid arthritis (Javaid *et al.,* 2008). Worldwide, it has been estimated that fractures caused by osteoporosis account for approximately one in three among women and approximately one in five among men over the age of 50. Although the mechanisms underlying osteoporosis are not fully understood, there is evidence suggesting that oxidative stress caused by reactive oxygen species (ROS) is associated with its pathogenesis (Sahnoun *et al.,* 1997; Basu *et al.,* 2001; Rao *et al.,* 2007).

Oxidative stress is a condition that can be characterized by an imbalance of pro-oxidants and antioxidants with the scale being tipped towards an excess of pro-oxidants, creating abnormally high concentrations of ROS. ROS are a family of highly reactive, oxygencontaining molecules and free radicals, including hydroxyl (OH**·**–) and superoxide radicals (O2**·**–), hydrogen peroxide (H2O2), singlet oxygen, and lipid peroxides (Juránek and Bezek, 2005). Several recent studies reported the impact of oxidative stress on osteoclast differentiation as well as on its function resulting to an increase in bone resorption (Garrett *et al.,* 1990; Bax *et al.,* 1992; Mody *et al.,* 2001; Lean, 2003). Furthermore, recent *in vitro* studies have shown the important detrimental role of ROS on osteoblast activity (Park *et al.,* 2005; Bai *et al.,* 2004; Bai *et al.,* 2005). In addition to *in vitro* and animal models, there is also increasing clinical evidence that oxidative stress might be involved in the pathogenesis of osteoporosis

Antioxidants are known to mitigate the damaging effects of oxidative stress on cells. Epidemiological evidence has indicated a link between dietary intake of antioxidants and bone health. Fruits and vegetables are important sources of antioxidant phytochemicals that have been shown to play an important role in bone metabolism. Higher consumption of fruits and vegetables has been correlated with a reduction in the risk for the development of osteoporosis. (Arikan *et al.,* 2011; Prentice *et al.*, 2006; Macdonald *et al.,* 2004; Macdonald *et al.,* 2008; Palacios

(Melhus *et al.,* 1999; Sontakke & Tare., 2002; Basu *et al.,* 2001; Maggio *et al.,* 2003).

*et al.,* 2006; Tucker *et al.,* 1999; Lister *et al.,* 2007; New*,* 2003; Trzeciakiewicz *et al.,* 2009).

**1. Introduction** 

*1Department of Medicine, St. Michael's Hospital, University of Toronto,* 

Yook, C. S.; Kim, I. H.; Hahn, D. R.; Nohara, T., & Chang, S. Y. (1998). A lupane-triterpene glycoside from leaves of two Acanthopanax. Phytochemistry, Vol.49, No.3, pp. 839- 843, ISSN 0031-9422

### **Polyphenol Antioxidants and Bone Health: A Review**

L.G. Rao1, N. Kang2 and A.V. Rao2 *1Department of Medicine, St. Michael's Hospital, University of Toronto, 2Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Canada* 

#### **1. Introduction**

466 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Yook, C. S.; Kim, I. H.; Hahn, D. R.; Nohara, T., & Chang, S. Y. (1998). A lupane-triterpene

843, ISSN 0031-9422

glycoside from leaves of two Acanthopanax. Phytochemistry, Vol.49, No.3, pp. 839-

Osteoporosis is a skeletal disease characterized by bone loss and structural deterioration of the bone tissue, leading to an increase in bone fragility and susceptibility to fractures, most frequently in the hip, wrist and spine (Sendur *et al.,* 2009). Bone loss is associated with such factors as age, menopause in women, smoking, alcohol excess, calcium and vitamin D deficiency, low weight and muscle mass, anticonvulsant and corticosteroid use as well as certain co-morbid conditions such as rheumatoid arthritis (Javaid *et al.,* 2008). Worldwide, it has been estimated that fractures caused by osteoporosis account for approximately one in three among women and approximately one in five among men over the age of 50. Although the mechanisms underlying osteoporosis are not fully understood, there is evidence suggesting that oxidative stress caused by reactive oxygen species (ROS) is associated with its pathogenesis (Sahnoun *et al.,* 1997; Basu *et al.,* 2001; Rao *et al.,* 2007).

Oxidative stress is a condition that can be characterized by an imbalance of pro-oxidants and antioxidants with the scale being tipped towards an excess of pro-oxidants, creating abnormally high concentrations of ROS. ROS are a family of highly reactive, oxygencontaining molecules and free radicals, including hydroxyl (OH**·**–) and superoxide radicals (O2**·**–), hydrogen peroxide (H2O2), singlet oxygen, and lipid peroxides (Juránek and Bezek, 2005). Several recent studies reported the impact of oxidative stress on osteoclast differentiation as well as on its function resulting to an increase in bone resorption (Garrett *et al.,* 1990; Bax *et al.,* 1992; Mody *et al.,* 2001; Lean, 2003). Furthermore, recent *in vitro* studies have shown the important detrimental role of ROS on osteoblast activity (Park *et al.,* 2005; Bai *et al.,* 2004; Bai *et al.,* 2005). In addition to *in vitro* and animal models, there is also increasing clinical evidence that oxidative stress might be involved in the pathogenesis of osteoporosis (Melhus *et al.,* 1999; Sontakke & Tare., 2002; Basu *et al.,* 2001; Maggio *et al.,* 2003).

Antioxidants are known to mitigate the damaging effects of oxidative stress on cells. Epidemiological evidence has indicated a link between dietary intake of antioxidants and bone health. Fruits and vegetables are important sources of antioxidant phytochemicals that have been shown to play an important role in bone metabolism. Higher consumption of fruits and vegetables has been correlated with a reduction in the risk for the development of osteoporosis. (Arikan *et al.,* 2011; Prentice *et al.*, 2006; Macdonald *et al.,* 2004; Macdonald *et al.,* 2008; Palacios *et al.,* 2006; Tucker *et al.,* 1999; Lister *et al.,* 2007; New*,* 2003; Trzeciakiewicz *et al.,* 2009).

Polyphenol Antioxidants and Bone Health: A Review 469

Table 1. The different categories of polyphenols, their chemical structures and sources

categories of polyphenols, their chemical structures and sources are shown in Table 1.

osteoporosis and present results of studies undertaken in our laboratory.

**2. Oxidative stress, antioxidants and osteoporosis** 

Of particular interest among the antioxidant phytochemicals present in fruits and vegetables are the polyphenols. Polyphenols can be sub classified as non-flavonoids and flavonoids. Ellagic acid and stilbenes are among the major non-flavonoid polyphenols. Included in the flavonoid polyphenols are the anthocyanins, catechins, flavones, flavonols and isoflavones. The different

Numerous studies have shown the health-promoting properties of polyphenols, providing additional mechanisms through which they promote skeletal health by reducing resorption caused by high oxidative stress (Trzeciakiewicz *et al.,* 2009; Tucker, 2009; Hunter *et al.,* 2008). The antioxidant properties of polyphenols have been widely studied and reported in the literature (Liu *et al.*, 2005; Miyamoto *et al.*,1998; Rassi *et al.*, 2002; Viereck *et al.*, 2002; Ward *et al.*, 2001; Shen *et al.*, 2011; Rao *et al.*, 2007). They strongly support the role of polyphenols in the delayed onset or reduction in the progression of osteoporosis. The protective effects of polyphenols against diseases, including osteoporosis, have generated new expectations for improvements in health. This review will focus mainly on the role of polyphenols in

Oxidative stress occurs when the production of free radicals through a number of cellular events exceeds the ability of the cell's antioxidant defense to eliminate these oxidants (Baek *et al.,* 2010). These free radicals have the ability to change the integrity of, and thus, damage several biomolecules, such as DNA, proteins and lipids (Baek *et al.,* 2010). There is increasing evidence that oxidative stress is responsible for the pathophysiology of the aging process and may also be involved in the pathogenesis of atherosclerosis, neurodegenerative diseases, cancer, and diabetes. Recently, ROS were shown to be responsible for the development of osteoporosis (Sahnoun *et al.,* 1997; Basu *et al.,* 2001; Rao *et al.,* 2007; Altindag *et al.*, 2008; Becker, 2006; Feng & McDonald, 2011). Several *in vitro* and animal studies have shown that oxidative stress diminishes the level of bone formation by reducing the differentiation and survival of osteoblasts (Baek *et al.,* 2010). Furthermore, it has been reported that ROS activate osteoclasts and thus, enhance bone resorption (Baek *et al.,* 2010). The presence of ROS in osteoclasts was also demonstrated by Rao *et al.* in 2003 Recent evidences from a few clinical studies have also revealed that ROS and/or antioxidant systems might play a role in the pathogenesis of bone loss (Rao *et al.,* 2007; Mackinnon *et al.,*

A number of studies have shown that antioxidants have a fundamental role in preventing postmenopausal osteoporosis. For instance, estrogens, whose antioxidant activity is essential in protecting women of reproductive age from cardiovascular disease, stimulate osteoblastic activity through specific receptors, thus favouring bone growth (Banfi *et al.,* 2008). Antioxidant

deficiency has been shown to have adverse effect on bone mass (Maggio *et al.* 2003).

Resveratrol gapes skins, red

Secoisolaiciresinol flaxseeds

wine

Stilbenes

Lignans

2010; Abdollahi *et al.,* 2005).


acids Gallic acid

Anthocyanidins Cyanidin

Flavonols Quercetin

Isoflavones Genistein

acids Caffeic acid coffee beans

**Common** 

Catechins

Silybin

Apigenin

Tangeritin

Luteolin

Proanthocyanidins

**Flavonoid Food Examples** 

gallnuts, sumac, witch hazel, tea leaves, oak bark,

berries, purple cabbage, beets, grape seed extract, and red

white, green and black teas

chocolate, fruits and vegetables, red wine, onion, apple skin

blessed milk thistle

chamomile, celery, parsley

tangerine and other citrus peels

celery, thyme, green pepers,

soy, alfalfa sprouts, red clover, chickpeas, peanuts, other legumes.

red and yellow onions, tea, wine, apples, cranberries, buckwheat, beans

wine

Theaflavins black teas

Hesperidin citrus fruits Narigenin citrus fruits

**Category Subclass Structure** 

Hydroxycinnamic

Hydroxybenzoic

Flavanols

Flavanones

Flavones

Phenolic acids

Flavonoids


Table 1. The different categories of polyphenols, their chemical structures and sources

Of particular interest among the antioxidant phytochemicals present in fruits and vegetables are the polyphenols. Polyphenols can be sub classified as non-flavonoids and flavonoids. Ellagic acid and stilbenes are among the major non-flavonoid polyphenols. Included in the flavonoid polyphenols are the anthocyanins, catechins, flavones, flavonols and isoflavones. The different categories of polyphenols, their chemical structures and sources are shown in Table 1.

Numerous studies have shown the health-promoting properties of polyphenols, providing additional mechanisms through which they promote skeletal health by reducing resorption caused by high oxidative stress (Trzeciakiewicz *et al.,* 2009; Tucker, 2009; Hunter *et al.,* 2008). The antioxidant properties of polyphenols have been widely studied and reported in the literature (Liu *et al.*, 2005; Miyamoto *et al.*,1998; Rassi *et al.*, 2002; Viereck *et al.*, 2002; Ward *et al.*, 2001; Shen *et al.*, 2011; Rao *et al.*, 2007). They strongly support the role of polyphenols in the delayed onset or reduction in the progression of osteoporosis. The protective effects of polyphenols against diseases, including osteoporosis, have generated new expectations for improvements in health. This review will focus mainly on the role of polyphenols in osteoporosis and present results of studies undertaken in our laboratory.

#### **2. Oxidative stress, antioxidants and osteoporosis**

Oxidative stress occurs when the production of free radicals through a number of cellular events exceeds the ability of the cell's antioxidant defense to eliminate these oxidants (Baek *et al.,* 2010). These free radicals have the ability to change the integrity of, and thus, damage several biomolecules, such as DNA, proteins and lipids (Baek *et al.,* 2010). There is increasing evidence that oxidative stress is responsible for the pathophysiology of the aging process and may also be involved in the pathogenesis of atherosclerosis, neurodegenerative diseases, cancer, and diabetes. Recently, ROS were shown to be responsible for the development of osteoporosis (Sahnoun *et al.,* 1997; Basu *et al.,* 2001; Rao *et al.,* 2007; Altindag *et al.*, 2008; Becker, 2006; Feng & McDonald, 2011). Several *in vitro* and animal studies have shown that oxidative stress diminishes the level of bone formation by reducing the differentiation and survival of osteoblasts (Baek *et al.,* 2010). Furthermore, it has been reported that ROS activate osteoclasts and thus, enhance bone resorption (Baek *et al.,* 2010). The presence of ROS in osteoclasts was also demonstrated by Rao *et al.* in 2003 Recent evidences from a few clinical studies have also revealed that ROS and/or antioxidant systems might play a role in the pathogenesis of bone loss (Rao *et al.,* 2007; Mackinnon *et al.,* 2010; Abdollahi *et al.,* 2005).

A number of studies have shown that antioxidants have a fundamental role in preventing postmenopausal osteoporosis. For instance, estrogens, whose antioxidant activity is essential in protecting women of reproductive age from cardiovascular disease, stimulate osteoblastic activity through specific receptors, thus favouring bone growth (Banfi *et al.,* 2008). Antioxidant deficiency has been shown to have adverse effect on bone mass (Maggio *et al.* 2003).

Polyphenol Antioxidants and Bone Health: A Review 471

**polyphenol Model Main findings** 

stem cells (BMSCs)

were prepared by removing from the femora and tibiae of

peripheral blood monocytic cells (PBMC)

young female piglets

osteoblasts (hOB)

osteoblasts (hFOB), and osteosarcoma cells (MG-63)

Myeloma cell lines U266 and OPM-2

6wk old mice

Daidzein MC3T3-E1 cells increase alkaline phosphatase activity

ICR mice

Choi (2011) Kaempferol MC3T3-E1 cells induced the activation of PI3K

KS483 ↑ nodule formation

HOS58 & SaOS-2 ↑ mineralization of bone cell

Dose-specific (1–100 μg/ml) of the naringin solution may enhance the proliferation and osteogenic differentiation of human BMSCs

growth of MC3T3-E1 cells and caused a significant elevation of alkaline phosphatase (ALP) activity and collagen content in the cells

luteolin decreased differentiation of both bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, inhibited the bone resorptive activity of

differentiated osteoclasts.

(phosphoinositide 3-kinase), Akt (protein kinase B), and CREB (cAMPresponse element-binding protein). This may prevent or reduce degerneration of osteoblasts

Quercetin (0.1–10 mM) decreased osteoclastogenesis in a dose dependent manner in both models with significant effects observed at low concentrations,

inhibits development of osteoclasts from cultures of porcine bone marrow and reduces bone resorption

up-regulated OPG production 2–6-fold in a time- and dose-dependent manner,

Inhibits osteoclast differentiation by suppressing the activation of MAPKs (p38 MAPK, ERK and JNK)

piceatannol increased BMP-2 synthesis, induced osteoblasts maturation and

Resveratrol analogues showed an up to 5,000-fold increased potency to inhibit osteoclast differentiation and promoted osteoblast maturation compared to

from 1 to 5 mM

neutralizing RANKL

differentiation

resveratrol.

**Polyphenol** 

**Phenolic Acids** 

**Class Reference** 

Ayoub et al., (2009)

**Flavonoids** Zhang et al. (2009)

Kim et al. (2011)

Wattel et al. (2004)

**Isoflavones** Sugimoto &

Rassi et al. (2002)

Viereck et al. (2002)

**Lignans** Hasegawa et al. (2010)

**Stilbenes** Chang et al. (2006)

Kupisiewicz

et al. (2010)

Table 2. Polyphenols- *In vitro* studies

Yamaguchi (2000)

Papoutsi et al., (2008)

**Principal** 

(25ug/ml)

Ellagic acid (10- 100nM)

3-methoxyellagic acid

Naringin bone mesenchymal

Luteolin Bone marrow cells

Quercetin RAW 264.7 cells,

Daidzein osteoclasts from

Genistein mature human

Honokiol bone marrow cells of

Piceatannol immortalized fetal

Modified resveratrol analogues

Choi (2007) Apigenin MC3T3-E1 cells Apigenin (0.01 mM) increased the

Antioxidant enzymes are regarded as the markers of antioxidant defense mechanism against bone resorption. Several studies have investigated the relationship between antioxidant enzymes such as glutathione peroxidase (GPx) and catalase (CAT) and osteoporosis (MacKinnon *et al.,* 2011; Hahn *et al.,* 2008*;* Maggio *et al., 2003;* Sontakke & Tare*, 2002*).

Recently, many dietary antioxidant nutrients have also been reported to decrease the oxidative stress that takes part in bone-resorptive processes (Rao *et al.*, 2007; Weber, 2001; Peters & Martini, 2010; Macdonald *et al.,* 2004). In addition to the antioxidant enzymes and nutrients, studies have also been directed towards the role of antioxidant phytochemicals such as the carotenoids in osteoporosis which will not be covered here, but has previously been reviewed (Rao & Rao, 2007; Sahni *et al., 2009;* Tucker, *2009*).

Fig. 1. The role of oxidative stress in osteoporosis and how/where antioxidants play a role in mitigating ROS

#### **3. Natural phytochemical antioxidants**

Within the last decade, there has been an increased interest on polyphenols as a result of the *in vitro* evidence demonstrating that they may have numerous benefits to human health, mainly due to their antioxidative and free radical quenching properties (Hendrich, 2006; Lotito & Frei 2006; Heinonen, 2007; Stevenson & Hurst 2007; Aron & Kennedy 2008; Lopez-Lazaro, 2009; Saura-Calixto *et al.* 2007). It is therefore hypothesized that polyphenols may aid in the prevention of aging-associated diseases, particularly cardiovascular diseases, cancers, and osteoporosis.

Polyphenolic compounds are the products of the secondary metabolism of plant and are an essential part of human diet (Goldberg, 2003; Stevenson & Hurst 2007; D'Archivio *et al.*, 2007; Saura-Calixto *et al.* 2007). To date, more than 8,000 polyphenols that have one common structural feature have been identified, a phenol, which is an aromatic ring possessing at least one hydroxyl substituent (Hendrich, 2006; Scalbert & Williamson, 2000; Harborne, 1993). The main classes of polyphenols include phenolic acids, flavonoids, stilbene, and lignans (Spencer *et al.*, 2008; D'Archivio *et al.*, 2007). Figure 1 illustrates the different groups of polyphenols, the chemical structures and food sources. Their total dietary intake can range up to 1 gram/day, which is considerably higher than that of all other classes of phytochemicals (Velioglu *et al.*, 1998). There is much evidence demonstrating that polyphenols improve the status of different oxidative stress biomarkers. However, there is uncertainty regarding both the relevance of these biomarkers as predictors of disease risk and the appropriateness of the different methods used.

Antioxidant enzymes are regarded as the markers of antioxidant defense mechanism against bone resorption. Several studies have investigated the relationship between antioxidant enzymes such as glutathione peroxidase (GPx) and catalase (CAT) and osteoporosis

Recently, many dietary antioxidant nutrients have also been reported to decrease the oxidative stress that takes part in bone-resorptive processes (Rao *et al.*, 2007; Weber, 2001; Peters & Martini, 2010; Macdonald *et al.,* 2004). In addition to the antioxidant enzymes and nutrients, studies have also been directed towards the role of antioxidant phytochemicals such as the carotenoids in osteoporosis which will not be covered here, but has previously

Fig. 1. The role of oxidative stress in osteoporosis and how/where antioxidants play a role

Within the last decade, there has been an increased interest on polyphenols as a result of the *in vitro* evidence demonstrating that they may have numerous benefits to human health, mainly due to their antioxidative and free radical quenching properties (Hendrich, 2006; Lotito & Frei 2006; Heinonen, 2007; Stevenson & Hurst 2007; Aron & Kennedy 2008; Lopez-Lazaro, 2009; Saura-Calixto *et al.* 2007). It is therefore hypothesized that polyphenols may aid in the prevention of aging-associated diseases, particularly cardiovascular diseases,

Polyphenolic compounds are the products of the secondary metabolism of plant and are an essential part of human diet (Goldberg, 2003; Stevenson & Hurst 2007; D'Archivio *et al.*, 2007; Saura-Calixto *et al.* 2007). To date, more than 8,000 polyphenols that have one common structural feature have been identified, a phenol, which is an aromatic ring possessing at least one hydroxyl substituent (Hendrich, 2006; Scalbert & Williamson, 2000; Harborne, 1993). The main classes of polyphenols include phenolic acids, flavonoids, stilbene, and lignans (Spencer *et al.*, 2008; D'Archivio *et al.*, 2007). Figure 1 illustrates the different groups of polyphenols, the chemical structures and food sources. Their total dietary intake can range up to 1 gram/day, which is considerably higher than that of all other classes of phytochemicals (Velioglu *et al.*, 1998). There is much evidence demonstrating that polyphenols improve the status of different oxidative stress biomarkers. However, there is uncertainty regarding both the relevance of these biomarkers as predictors of disease risk

(MacKinnon *et al.,* 2011; Hahn *et al.,* 2008*;* Maggio *et al., 2003;* Sontakke & Tare*, 2002*).

been reviewed (Rao & Rao, 2007; Sahni *et al., 2009;* Tucker, *2009*).

in mitigating ROS

cancers, and osteoporosis.

**3. Natural phytochemical antioxidants** 

and the appropriateness of the different methods used.


Table 2. Polyphenols- *In vitro* studies

Polyphenol Antioxidants and Bone Health: A Review 473

(-)Epigallocatechin

(-)Epigallocatechin

(-)Epigallocatechin

gallate

Black tea extract Theaflavin Bilaterally

40 female CD rats

50 OVX 0.5%

oophorecto

concentratio n of GTP in drinking water

2.5% aqueous

gallate

OVX rat 0.1% or 0.5%

0.5% concentratio n of GTP in drinking water

concentratio n of GTP in drinking water

gallate

calcium loss while significantly decreasing trabecular separation. No significant effects of treatment on serum or urine measures of bone

GTP supplementation increased urinary epigallocatechin and epicatechin concentrations, femur BMD, decreased urinary 8-hydroxy-2′ deoxyguanosine and urinary calcium levels; no effect on serum estradiol

GTP supplementation increased urinary epigallocatechin and epicatechin concentrations and showed higher values for femur BMC, BMD and serum OC, but lower values for serum TRAP, urinary 8-OHdG and spleen mRNA expression of TNF-α and COX-2 levels.

GTP supplementation resulted in increased serum osteocalcin concentrations, bone mineral density, and trabecular volume, number, and strength of femur; increased trabecular volume and thickness and bone formation in both the proximal tibia and periosteal tibial shaft

BTE increase serum estradiol level

turnover.

polyphenol (equivalent to 7.5% DP powder); (5) 2% FOS+7.5% DP juice; (6) 2% FOS+7.5% DP puree; (7) 2% FOS+7.5% DP pulp skins; (8) 2% FOS+7.5% raisin; (9) 2% FOS+7.5% fig; (10) 2% FOS+7.5% date; (11) 2% FOS+7.5% blueberry; (12) 2% FOS+0.25% HMB; and (13) 0.25% HMB.

Green tea polyphenols (GTP)

Green tea polyphenols (GTP)

Green tea polyphenols (GTP)

Shen et al. (2008)

Shen et al. (2010)

Shen et al. (2011)

Das et al. (2005)

#### **4. Polyphenols and osteoporosis**

There has been an increase interest in the field of bone health and nutrients, and within the last decade, it has been well recognized that some polyphenols, whether ingested as supplements or with food, do in fact improve bone health status. Currently, most of the research on polyphenols and their effects has emerged from *in vitro* and *in vivo* studies with only a few clinical studies available. Compounds present in fruits and vegetables influence bone health as shown with *in vitro* osteoblast cell culture. On the other hand, epidemiologic studies tend to have mixed results with regards to the protective effects of polyphenol consumption against osteoporosis. Tables 2, 3, and 4 illustrate some of the recent *in vitro, in vivo* and clinical studies that have been reported in the literature, respectively.


There has been an increase interest in the field of bone health and nutrients, and within the last decade, it has been well recognized that some polyphenols, whether ingested as supplements or with food, do in fact improve bone health status. Currently, most of the research on polyphenols and their effects has emerged from *in vitro* and *in vivo* studies with only a few clinical studies available. Compounds present in fruits and vegetables influence bone health as shown with *in vitro* osteoblast cell culture. On the other hand, epidemiologic studies tend to have mixed results with regards to the protective effects of polyphenol consumption against osteoporosis. Tables 2, 3, and 4 illustrate some of the recent *in vitro, in* 

*vivo* and clinical studies that have been reported in the literature, respectively.

Chen (2010) Blueberries Phenolic acid

**Principal** 

mixture

coumaric, chlorogenic, clohexanecarbox ylic acid

coumaric, chlorogenic acid

phenolic acids and flavonols

Curcumin Wistar

Ferulic,caffeic, *p-*

Caffeic, *p-*

Blueberries Variety of

**polyphenol Model** 

Sprague-Dawley rats

Wistar Cmd:(WI)W U rats

Cmd:(WI)W U rats

Wistar Cmd:(WI)W U rats

**Dose per** 

10 mg/kg p.o.

10 mg/kg, po

10 mg/kg p.o.

Variety OVX rat diet of 5% FOS + 7.5%

OVX rat 5% w/w Ovx resulted in loss of

**day Main findings** 

Increase serum

osteoblast progenitors, increased osteoblast differentiation, reduced osteoclastogenesis, increase bone mass

caffeic acid worsened bone mechanical properties

no sig. improvement of bone mineralizasation or mechanical properties

caffeic acid ↓ bone mass, p-coumaric acid ↑ bone mass/body mass ratio and bone mineral mass/body mass ratio in long bones

whole-body, tibial, femoral, and 4th lumbar BMD by approximately 6%. Blueberry treatment was able to prevent the loss of whole-body BMD and had an intermediary effect on prevention of tibial and femoral BMD

dried plum was most effective in reversing both right femur and fourth lumbar BMD and fourth lumbar

**4. Polyphenols and osteoporosis** 

**Class Reference Substance given** 

**Polyphenol** 

**Phenolic Acids** 

Zych et al. (2010)

Folwarczna

Folwarczna

**Flavonoids** Devareddy

Arjmandi et al. (2010)

(1) 2%

Fructooligosacchari des (FOS); 5% FOS+7.5% DP; 2% FOS+5% DP; 2% FOS+2% DP

et al. (2010)

et al. (2009)

et al. (2008)


Polyphenol Antioxidants and Bone Health: A Review 475

isopentenylnarin

Lignans 56 OVX/6J

specificpathogenfree (SPF) female mice

Silymarin Silymarin OVX rats 50 mg/kg protected trabecula

20 Sprague-Dawley male rats

C57BL/6NI A mice

*trans-Resveratrol* Resveratrol OVX rat 0.7 mg/kg epiphysis BMD and

293 μmol SDG/kg

100 mg/kg or 400 mg/kg

Miyamoto et al (1998)

**Lignans** Xiao et al. (2011)

El-Shitany et al. (2010)

Ward et al. (2001)

**Stilbenes** Pearson et

Liu et al.

al. (2008)

(2005)

Table 3. Polyphenols- *In vivo* Studies

8-

nin

Sambucus williamsii HANCE

(SWH)

isopentenylnaringe

8-

Flaxseed Secoisolariciresin

Resveratrol Resveratrol Male

ol diglucoside

genin

different mechanism of

naringenin prevented decrease in BMD and bone turnover markers

SWC significantly restored bone mineral density and improved bone size and bone content in femur and

thickness, decreased serum levels of ALP and increased serum levels of both calcium and phosphorus

exposure to a diet with flaxseed during lactation through to early adolescence can reduce bone strength, but lignan does is not the mediator, no sig. change in BMD and BMC those fed flaxseed

Both diets improved distal trabecular tissue mineral density (TMD) and bone volume to total volume ratio over the entire femur compared to control

bone calcium content was significantly greater with resveratrol treatment than that in the OVX group, no differences in femoral midpoint BMD

estrogen

tibia

OVX rats 30 mg/day 8-isopentenyl

17boestradiol (3·2 mg/kg), SWH (60% ethanol crude extract; 1·0 g/kg), SWA (water eluate; 0·570 g/kg), SWB (30% ethanol eluate; 0·128 g/kg) or SWC (50 and 95% ethanol eluates; 0·189 g/kg)


Hesperidin & αglucosylhesperid

luteolin Luteolin OVX mice 5 and 20

Rubus coreanus Anthocyanin OVX rats 100 & 200

Soy protein Genistein 72 OVX rats 1462 mg/kg

Rutin Rutin OVX rats 2.5 g/kg Rutin prevented

Soybean Glycitein 24 OVX rats 6.25 g/kg soybean isoflavone

in

Chiba et al. (2003)

Park et al. (2008)

Kim et al. (2011)

Do et al.

Horcajada-

**Isoflavones** Arjmandi et al. (1998)

Lee et al.

(2004)

(2008)

Molteni et al. (2000)

hesperidin & αglucosylhesperidin mized rats BTE at a

OVX mice 0.5 g/100 g

apigenin Apigenin OVX rats 10 mg/kg apigenin increased the

single dose of 1 ml /100 g body weight

hesperidin, 0.7 g/100 g αglucosylhes peridin

mg/kg

mg/kg

genistein, 25.1 mg/kg daidzin, 11.3 mg/kg daidzein

hesperidin or αglucosylhesperidin restored BMD caused

mineral content and density of the trabecular bone at the neck of the left femur, decreased body weight

luteolin increased bone mineral density and bone mineral content of trabecular and cortical bones in the femur as compared to those of OVX controls

RCM increased femur trabecular bone area in a dose-dependent manner in ovariectomized rats, increased osteoblast differentiation and osteoclast apoptosis.

decrease in both total and distal metaphyseal femoral mineral density by slowing down resorption and increasing osteoblastic activity caused by OVX,

no effect on BMC

appear to prevent bone loss in femur and lumber vertebrae via a

and dietary consumption

by OVX, αglucosylhesperidin significantly prevented loss of trabecular bone volume and trabecular thickness in the femoral distal metaphysis


Table 3. Polyphenols- *In vivo* Studies

Polyphenol Antioxidants and Bone Health: A Review 477

There have been several results suggesting that the combination of polyphenolic compounds found naturally in fruits and vegetables may reduce the risk of osteoporosis via increasing bone mineral density (Wu *et al.*, 2002; Morton *et al.*, 2001; Melhus *et al.*, 1999; Leveille *et al.*, 1997; Singh, 1992). In 1992, Singh was able to show that polyphenols afford protection against oxidative stress-induced bone damage during strenuous exercise. Similarly, Melhus was able

to show its counteractive effect of polyphenols among smokers (Melhus *et al.*, 1999).

**author's laboratory** 

(p<0.05)

in Sa0S-2 cells (p< 0.05).

**5. Research results on the role of polyphenols in osteoporosis from the** 

Previous *in vitro* results from our laboratory have shown that a supplement rich in a variety of polyphenols commercially known as greens+*TM*, is more effective in stimulating

Fig. 2. Dose dependent effect of greens+*TM* (g+) and epicatechin (EC) compared to vehicle.

Fig. 3. Time and dose-dependent effects of bone builder*TM* on mineralized bone nodule area


Table 4. Polyphenols - Clinical Studies

Scottish women

postmenopausal women

postmenopausal women

postmenopausal women >65 years old

199 menopausal women

50 men, 50 postmenopausal women

placebo: 0 mg isoflavones + 500 mg calcium, middose:40 mg isoflavones+ 500 mg calcium, high-dose:80 mg isoflavones + 500 mg calcium

25 g protein and 60 mg isoflavones

18 g soy protein and 105 mg isoflavone tablets

**polyphenol Model Dose per day Main findings** 

flavanones were

negatively associated with bone-resorption markers, association between energyadjusted total flavonoid intakes and BMD at the femoreal neck and lumbar spine, annual percent change in BMD was associated with intakes of procyanidins and catechins

no effect on BMD in all groups, effect of soy isoflavones on BMC at the total hip and trochanter was less strong in women in early menopause or in those with higher body weight, nonsignificantin BMC in those with a high level of dietary calcium intake

Whole body and lumbar BMD and BMC significantly decreased, and BSAP and osteocalcin increasedin control and soy groups

no differences in

BMD

no effect on BMD

 no sig. change in BMD

**Principal** 

None Catechin perimenopausal

Daizein 203

Soy protein Daizein 87

Isoflavones 131

Soy protein + isoflavone tablets

Flaxseed Secoisolaricir esinol diglucoside

Flaxseed Secoisolaricir esinol diglucoside

**Polyphenol** 

**Class Reference** 

**Flavonoids** Hardcastle

**Isoflavones** Chen et al.

Arjmandi et

Kenny et al. (2009)

**Lignans** Cornish et

Dodin et al. (2005)

al. (2009)

Table 4. Polyphenols - Clinical Studies

al. (2005)

(2004)

Soy isoflavone

et al. (2011)

**Substance given** 

There have been several results suggesting that the combination of polyphenolic compounds found naturally in fruits and vegetables may reduce the risk of osteoporosis via increasing bone mineral density (Wu *et al.*, 2002; Morton *et al.*, 2001; Melhus *et al.*, 1999; Leveille *et al.*, 1997; Singh, 1992). In 1992, Singh was able to show that polyphenols afford protection against oxidative stress-induced bone damage during strenuous exercise. Similarly, Melhus was able to show its counteractive effect of polyphenols among smokers (Melhus *et al.*, 1999).

#### **5. Research results on the role of polyphenols in osteoporosis from the author's laboratory**

Previous *in vitro* results from our laboratory have shown that a supplement rich in a variety of polyphenols commercially known as greens+*TM*, is more effective in stimulating

Fig. 2. Dose dependent effect of greens+*TM* (g+) and epicatechin (EC) compared to vehicle. (p<0.05)

Fig. 3. Time and dose-dependent effects of bone builder*TM* on mineralized bone nodule area in Sa0S-2 cells (p< 0.05).

Polyphenol Antioxidants and Bone Health: A Review 479

Fig. 6. Change in TBARS over 4 and 8-weeks of nutritional intervention with g+bbTM

Fig. 7. Change in protein oxidation over 4 and 8-weeks of nutritional intervention with

Although epidemiologic studies are practical for the evaluation of human health effects on the physiologic concentrations of polyphenols, reliable data on polyphenol contents of foods are limited. This review has shown that polyphenols or polyphenol-rich diets can provide significant protection or treatment for the development and progression of osteoporosis. Keeping in mind that many nutrients are co-dependent, and they may interact among themselves and others. The complexity of these interactions may possibly be the reason why many studies show controversial or inconsistent results regarding the effects of a single nutrient or groups of nutrients in bone health. Based on current knowledge, polyphenols offer a platform for the prevention of many human chronic diseases involved with oxidative

To value the actual significance of food phenolics, it is necessary to investigate not only their bioavailability, but also their mechanisms of action and their possible synergism with other constituents either in the diet or within the human body, as well as the polyphenolic content and composition of foods. We have attained this goal by studying the nutritional supplement greens+*TM,* which is rich in polyphenols and their interactions with minerals, vitamins and nutrients that were present in the nutritional supplement

compared to placebo (p<0.001).

g+bbTM compared to placebo (p<0.05).

stress, including osteoporosis.

bone builder*TM*.

**6. Conclusions** 

osteoblasts to form more bone nhodules in a dose-dependant manner than epicatechin, the main polyphenol found in green tea (Fig. 2). Our laboratory also studied the effects of a second supplement, bone builder*TM*, which is rich in minerals, vitamins and nutrients. Similarly to the greens+*TM*, the water-soluble bone-builder extract had a significant dosedependent stimulatory effect on bone nodules formation (Fig. 3). Figure 4 shows that when the two supplements, greens+*TM* and bone builder*TM*, were tested as combination, the effects were six times more effective than either one alone*.* This led us to believe that synergistic effects of greens+*TM* and bone builder*TM* may have a beneficial effect on osteoporosis. We then conducted a clinical evaluation of this nutritional supplement greens+ bone builder*TM* Results have shown that there was an increase in total antioxidant capacity after 8 weeks of treatment compared to placebo (Fig 4). as well as a decrease in both lipid and protein oxidation over a 4 and 8-weeks of intervention with greens+ bone builder*TM* compared to placebo (Fig. 6 & 7). This suggests that the nutritional supplement may have a beneficial effect on bone health by mitigating the effects of oxidative stress.

Fig. 4. Dose Dependent Effect of greens + (g+) with and without 0.5 mg/ml of bone builder (bb) on the area of mineralized bone nodules in osteoblasts Sa0S-2 Cells. \* p<0.0005, \*\*p<0.005; \*\*\*p<0.05; # p<0.0001; ## p<0.001; ### p<0.01; significance differences were found when treatment with g+ plus 0.5 mg/ml bb was compared to treatment with g+ alone as follows: a\b< 0.0001; bp< 0.005

Fig. 5. The effect of nutritional intervention with g+bbTM compared to placebo on serum total antioxidant capacity (p<0.05).

osteoblasts to form more bone nhodules in a dose-dependant manner than epicatechin, the main polyphenol found in green tea (Fig. 2). Our laboratory also studied the effects of a second supplement, bone builder*TM*, which is rich in minerals, vitamins and nutrients. Similarly to the greens+*TM*, the water-soluble bone-builder extract had a significant dosedependent stimulatory effect on bone nodules formation (Fig. 3). Figure 4 shows that when the two supplements, greens+*TM* and bone builder*TM*, were tested as combination, the effects were six times more effective than either one alone*.* This led us to believe that synergistic effects of greens+*TM* and bone builder*TM* may have a beneficial effect on osteoporosis. We then conducted a clinical evaluation of this nutritional supplement greens+ bone builder*TM* Results have shown that there was an increase in total antioxidant capacity after 8 weeks of treatment compared to placebo (Fig 4). as well as a decrease in both lipid and protein oxidation over a 4 and 8-weeks of intervention with greens+ bone builder*TM* compared to placebo (Fig. 6 & 7). This suggests that the nutritional supplement may have a beneficial

Fig. 4. Dose Dependent Effect of greens + (g+) with and without 0.5 mg/ml of bone builder

(bb) on the area of mineralized bone nodules in osteoblasts Sa0S-2 Cells. \* p<0.0005, \*\*p<0.005; \*\*\*p<0.05; # p<0.0001; ## p<0.001; ### p<0.01; significance differences were found when treatment with g+ plus 0.5 mg/ml bb was compared to treatment with g+ alone

Fig. 5. The effect of nutritional intervention with g+bbTM compared to placebo on serum

effect on bone health by mitigating the effects of oxidative stress.

as follows: a\b< 0.0001; bp< 0.005

total antioxidant capacity (p<0.05).

Fig. 6. Change in TBARS over 4 and 8-weeks of nutritional intervention with g+bbTM compared to placebo (p<0.001).

Fig. 7. Change in protein oxidation over 4 and 8-weeks of nutritional intervention with g+bbTM compared to placebo (p<0.05).

#### **6. Conclusions**

Although epidemiologic studies are practical for the evaluation of human health effects on the physiologic concentrations of polyphenols, reliable data on polyphenol contents of foods are limited. This review has shown that polyphenols or polyphenol-rich diets can provide significant protection or treatment for the development and progression of osteoporosis. Keeping in mind that many nutrients are co-dependent, and they may interact among themselves and others. The complexity of these interactions may possibly be the reason why many studies show controversial or inconsistent results regarding the effects of a single nutrient or groups of nutrients in bone health. Based on current knowledge, polyphenols offer a platform for the prevention of many human chronic diseases involved with oxidative stress, including osteoporosis.

To value the actual significance of food phenolics, it is necessary to investigate not only their bioavailability, but also their mechanisms of action and their possible synergism with other constituents either in the diet or within the human body, as well as the polyphenolic content and composition of foods. We have attained this goal by studying the nutritional supplement greens+*TM,* which is rich in polyphenols and their interactions with minerals, vitamins and nutrients that were present in the nutritional supplement bone builder*TM*.

Polyphenol Antioxidants and Bone Health: A Review 481

Chang, J. K., Hsu, Y. L., Teng, I. C., & Kuo, P. L. (2006). Piceatannol stimulates osteoblast

Chen, J. R., Lazarenko, O. P., Wu, X., Kang, J., Blackburn, M. L., Shankar, K., et al. (2010).

Chiba, H., Uehara, M., Wu, J., Wang, X., Masuyama, R., Suzuki, K., et al. (2003). Hesperidin,

Choi, E. M. (2007). Apigenin increases osteoblastic differentiation and inhibits tumor

Choi, E. M. (2011). Kaempferol protects MC3T3-E1 cells through antioxidant effect and

Cornish, S. M., Chilibeck, P. D., Paus-Jennsen, L., Biem, H. J., Khozani, T., Senanayake, V., et

D'Archivio, M., Filesi, C., Di Benedetto, R., Gargiulo, R., Giovannini, C., & Masella, R. (2007).

Das, A. S., Das, D., Mukherjee, M., Mukherjee, S., & Mitra, C. (2005). Phytoestrogenic effects

Devareddy, L., Hooshmand, S., Collins, J. K., Lucas, E. A., Chai, S. C., & Arjmandi, B. H.

Do, S. H., Lee, J. W., Jeong, W. I., Chung, J. Y., Park, S. J., Hong, I. H., et al. (2008). Bone-

Dodin, S., Lemay, A., Jacques, H., Legare, F., Forest, J. C., & Masse, B. (2005). The effects of

El-Shitany, N. A., Hegazy, S., & El-Desoky, K. (2010). Evidences for antiosteoporotic and

norvegicus) model of osteoporosis. *Life Sciences, 77*(24), 3049-3057.

osteoclasts. *Menopause (New York, N.Y.), 15*(4 Pt 1), 676-683.

ovariectomized mice. *The Journal of Nutrition, 133*(6), 1892-1897.

osteoblastic MC3T3-E1 cells. *Die Pharmazie, 62*(3), 216-220.

production. *European Journal of Pharmacology, 551*(1-3), 1-9.

*Menopause (New York, N.Y.), 11*(3), 246-254.

1805.

699.

1390-1397.

*Metabolisme, 34*(2), 89-98.

*Sanita, 43*(4), 348-361.

differentiation that may be mediated by increased bone morphogenetic protein-2

Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/betacatenin canonical wnt signaling. *Journal of Bone and Mineral Research : The Official Journal of the American Society for Bone and Mineral Research, 25*(11), 2399-2411. Chen, Y. M., Ho, S. C., Lam, S. S., Ho, S. S., & Woo, J. L. (2004). Beneficial effect of soy

isoflavones on bone mineral content was modified by years since menopause, body weight, and calcium intake: A double-blind, randomized, controlled trial.

a citrus flavonoid, inhibits bone loss and decreases serum and hepatic lipids in

necrosis factor-alpha-induced production of interleukin-6 and nitric oxide in

regulation of mitochondrial function. *Food and Chemical Toxicology : An International Journal Published for the British Industrial Biological Research Association, 49*(8), 1800-

al. (2009). A randomized controlled trial of the effects of flaxseed lignan complex on metabolic syndrome composite score and bone mineral in older adults. *Applied Physiology, Nutrition, and Metabolism = Physiologie Appliquee, Nutrition Et* 

Polyphenols, dietary sources and bioavailability. *Annali Dell'Istituto Superiore Di* 

of black tea extract (camellia sinensis) in an oophorectomized rat (rattus

(2008). Blueberry prevents bone loss in ovariectomized rat model of postmenopausal osteoporosis. *The Journal of Nutritional Biochemistry, 19*(10), 694-

protecting effect of rubus coreanus by dual regulation of osteoblasts and

flaxseed dietary supplement on lipid profile, bone mineral density, and symptoms in menopausal women: A randomized, double-blind, wheat germ placebocontrolled clinical trial. *The Journal of Clinical Endocrinology and Metabolism, 90*(3),

selective estrogen receptor modulator activity of silymarin compared with

#### **7. References**


Abdollahi, M., Larijani, B., Rahimi, R. & Salari, P. (2005). Role of oxidative stress in

Arikan, D. C., Coskun, A., Ozer, A., Kilinc, M., Atalay, F., & Arikan, T. (2011). Plasma

Arjmandi, B. H., Birnbaum, R., Goyal, N. V., Getlinger, M. J., Juma, S., Alekel, L., et al.

Arjmandi, B. H., Getlinger, M. J., Goyal, N. V., Alekel, L., Hasler, C. M., Juma, S., et al.

Arjmandi, B. H., Johnson, C. D., Campbell, S. C., Hooshmand, S., Chai, S. C., & Akhter, M. P.

Arjmandi, B. H., Lucas, E. A., Khalil, D. A., Devareddy, L., Smith, B. J., McDonald, J., et al.

Aron, P. M., & Kennedy, J. A. (2008). Flavan-3-ols: Nature, occurrence and biological

Ayoub, N. A., Hussein, S. A., Hashim, A. N., Hegazi, N. M., Linscheid, M., Harms, M., et al.

Baek, K. H., Oh, K. W., Lee, W. Y., Lee, S. S., Kim, M. K., Kwon, H. S., et al. (2010).

Banfi, G., Iorio, E. L., & Corsi, M. M. (2008). Oxidative stress, free radicals and bone

Basu, S., Michaelsson, K., Olofsson, H., Johansson, S., & Melhus, H. (2001). Association

Bax, B. E., Alam, A. S. M. T., Banerji, B., Bax, C. M. R., Bevis, P. J. R., Stevens, C. R., et al.

*Biochemical and Biophysical Research Communications, 183*(3), 1153-1158. Becker, C. (2006). Pathophysiology and clinical manifestations of osteoporosis. *Clinical* 

selenium, zinc, copper and lipid levels in postmenopausal turkish women and their relation with osteoporosis. *Biological Trace Element Research, 144*(1-3), 407-417. Altindag, O., Erel, O., Soran, N., Celik, H., & Selek, S. (2008). Total oxidative/anti-oxidative

status and relation to bone mineral density in osteoporosis. *Rheumatol Int, 28*(4),

(1998). Bone-sparing effect of soy protein in ovarian hormone-deficient rats is related to its isoflavone content. *The American Journal of Clinical Nutrition, 68*(6

(1998). Role of soy protein with normal or reduced isoflavone content in reversing bone loss induced by ovarian hormone deficiency in rats. *The American Journal of* 

(2010). Combining fructooligosaccharide and dried plum has the greatest effect on restoring bone mineral density among select functional foods and bioactive

(2005). One year soy protein supplementation has positive effects on bone formation markers but not bone density in postmenopausal women. *Nutrition* 

(2009). Bone mineralization enhancing activity of a methoxyellagic acid glucoside

Association of oxidative stress with postmenopausal osteoporosis and the effects of hydrogen peroxide on osteoclast formation in human bone marrow cell cultures.

remodeling. *Clinical Chemistry and Laboratory Medicine : CCLM / FESCC, 46*(11),

between oxidative stress and bone mineral density. *Biochem Biophys Res Commun,* 

(1992). Stimulation of osteoclastic bone resorption by hydrogen peroxide.

**7. References** 

317-321.

*Journal, 4*, 8.

1550-1555.

*288*(1), 275-279.

*Cornerstone, 8*(1), 19-27.

Suppl), 1364S-1368S.

osteoporosis. *Therapy,* 2(5), 787-796.

*Clinical Nutrition, 68*(6 Suppl), 1358S-1363S.

*Calcified Tissue International, 87*(3), 226-235.

compounds. *Journal of Medicinal Food, 13*(2), 312-319.

activity. *Molecular Nutrition & Food Research, 52*(1), 79-104.

from a feijoa sellowiana leaf extract. *Die Pharmazie, 64*(2), 137-141.


Polyphenol Antioxidants and Bone Health: A Review 483

Kim, T. H., Jung, J. W., Ha, B. G., Hong, J. M., Park, E. K., Kim, H. J., et al. (2011). The effects

Kupisiewicz, K., Boissy, P., Abdallah, B. M., Hansen, F. D., Erben, R. G., Savouret, J. F., et al.

Lee, Y. B., Lee, H. J., Kim, K. S., Lee, J. Y., Nam, S. Y., Cheon, S. H., et al. (2004). Evaluation of

Leveille, S. G., LaCroix, A. Z., Koepsell, T. D., Beresford, S. A., Van Belle, G., & Buchner, D.

Lister, C., Skinner, M., & Hunter, D. (2007). Fruits, vegetables and their phytochemicals for

Liu, Z. P., Li, W. X., Yu, B., Huang, J., Sun, J., Huo, J. S., et al. (2005). Effects of trans-

Lopez-Lazaro, M. (2009). Distribution and biological activities of the flavonoid luteolin. *Mini* 

Lotito, S. B., & Frei, B. (2006). Consumption of flavonoid-rich foods and increased plasma

Maggio, D., Barabani, M., Pierandrei, M., Polidori, M. C., Catani, M., Mecocci, P., et al.

Results of a cross-sectional study. *J Clin Endocrinol Metab, 88*(4), 1523-1527. Macdonald, H. M., Black, A. J., Aucott, L., Duthie, G., Duthie, S., Sandison, R., et al. (2008).

Macdonald, H. M., New, S. A., Golden, M. H., Campbell, M. K., & Reid, D. M. (2004).

Mackinnon, E. S., Rao, A. V., & Rao, L. G. (2011). Dietary restriction of lycopene for a period

Melhus, H., Michaelsson, K., Holmberg, L., Wolk, A., & Ljunghall, S. (1999). Smoking, antioxidant vitamins, and the risk of hip fracture. *J Bone Miner Res, 14*(1), 129-135.

trial. *The American Journal of Clinical Nutrition, 88*(2), 465-474.

promoters of osteoblasts. *Calcified Tissue International, 87*(5), 437-449. Lean, J. M., Davies, J. T., Fuller, K., Jagger, C. J., Kirstein, B., Partington, G. A., et al. (2003). A

bone loss. *The Journal of Nutritional Biochemistry, 22*(1), 8-15.

*Bioscience, Biotechnology, and Biochemistry, 68*(5), 1040-1045.

bone and joint health. *Curr Top Nutraceut Res, 5*, 67–82.

model. *Journal of Medicinal Food, 8*(1), 14-19.

*Reviews in Medicinal Chemistry, 9*(1), 31-59.

*Radical Biology & Medicine, 41*(12), 1727-1746.

*90*(1), 234-242.

*112*(6), 915-923.

485.

165.

*15*(2), 133-138.

women: A randomized controlled trial. *The American Journal of Clinical Nutrition,* 

of luteolin on osteoclast differentiation, function in vitro and ovariectomy-induced

(2010). Potential of resveratrol analogues as antagonists of osteoclasts and

crucial role for thiol antioxidants in estrogen-deficiency bone loss. *J Clin Invest,* 

the preventive effect of isoflavone extract on bone loss in ovariectomized rats.

M. (1997). Dietary vitamin C and bone mineral density in postmenopausal women in washington state, USA. *Journal of Epidemiology and Community Health, 51*(5), 479-

resveratrol from polygonum cuspidatum on bone loss using the ovariectomized rat

antioxidant capacity in humans: Cause, consequence, or epiphenomenon? *Free* 

(2003). Marked decrease in plasma antioxidants in aged osteoporotic women:

Effect of potassium citrate supplementation or increased fruit and vegetable intake on bone metabolism in healthy postmenopausal women: A randomized controlled

Nutritional associations with bone loss during the menopausal transition: Evidence of a beneficial effect of calcium, alcohol, and fruit and vegetable nutrients and of a detrimental effect of fatty acids. *The American Journal of Clinical Nutrition, 79*(1), 155-

of one month resulted in significantly increased biomarkers of oxidative stress and bone resorption in postmenopausal women. *The Journal of Nutrition, Health & Aging,* 

ethinylestradiol in ovariectomized rats. *Phytomedicine : International Journal of Phytotherapy and Phytopharmacology, 17*(2), 116-125.


Feng, X., & McDonald, J. M. (2011). Disorders of bone remodeling. *Annual Review of* 

Folwarczna, J., Zych, M., & Trzeciak, H. I. (2010). Effects of curcumin on the skeletal system

Folwarczna, J., Zych, M., Burczyk, J., Trzeciak, H., & Trzeciak, H. I. (2009). Effects of natural

Garrett, I. R., Boyce, B. F., Oreffo, R. O., Bonewald, L., Poser, J., & Mundy, G. R. (1990).

Goldberg, G. (2003). *Plants: Diet and health: The report of the british nutrition foundation task force*. Oxford, UK.: Blackwell Science for the British Nutrition Foundation. Hahn, M., Conterato, G. M. M., Frizzo, C. P., Augusti, P. R., da Silva, J. C. N., Unfer, T. C., et

enzymes in postmenopausal women. *Clinical Biochemistry, 41*(1-2), 69-74. Harborne, J. B. (1993). *The flavonoids: Advances in research since 1986*. London: Chapman and

Hardcastle, A. C., Aucott, L., Reid, D. M., & Macdonald, H. M. (2011). Associations between

Hasegawa, S., Yonezawa, T., Ahn, J. Y., Cha, B. Y., Teruya, T., Takami, M., et al. (2010).

Heinonen, M. (2007). Antioxidant activity and antimicrobial effect of berry phenolics--a finnish perspective. *Molecular Nutrition & Food Research, 51*(6), 684-691. Hendrich, A. B. (2006). Flavonoid-membrane interactions: Possible consequences for

Horcajada-Molteni, M. N., Crespy, V., Coxam, V., Davicco, M. J., Remesy, C., & Barlet, J. P.

Hunter, D. C., Skinner, M. A., & Lister, C. E. (2008). Impact of phytochemicals on

Javaid, M. K., & Holt, R. I. (2008). Understanding osteoporosis. *Journal of Psychopharmacology* 

Juranek, I., & Bezek, S. (2005). Controversy of free radical hypothesis: Reactive oxygen

Kenny, A. M., Mangano, K. M., Abourizk, R. H., Bruno, R. S., Anamani, D. E., Kleppinger,

*Phytotherapy and Phytopharmacology, 17*(2), 116-125.

in rats. *Pharmacological Reports : PR, 62*(5), 900-909.

in vitro and in vivo. *J Clin Invest, 85*(3), 632-639.

*Pathology, 6*, 121-145.

1567-1572.

Hall.

*Research, 26*(5), 941-947.

*Research, 15*(11), 2251-2258.

*(Oxford, England), 22*(2 Suppl), 38-45.

*27*(1), 27-40.

*24*(4), 390-392.

*24*(3), 263-278.

*Pharmaceutical Bulletin, 33*(3), 487-492.

ethinylestradiol in ovariectomized rats. *Phytomedicine : International Journal of* 

phenolic acids on the skeletal system of ovariectomized rats. *Planta Medica, 75*(15),

Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone

al. (2008). Effects of bone disease and calcium supplementation on antioxidant

dietary flavonoid intakes and bone health in a scottish population. *Journal of Bone and Mineral Research : The Official Journal of the American Society for Bone and Mineral* 

Honokiol inhibits osteoclast differentiation and function in vitro. *Biological &* 

biological effects of some polyphenolic compounds. *Acta Pharmacologica Sinica,* 

(2000). Rutin inhibits ovariectomy-induced osteopenia in rats. *Journal of Bone and Mineral Research : The Official Journal of the American Society for Bone and Mineral* 

maintaining bone and joint health. *Nutrition (Burbank, Los Angeles County, Calif.),* 

species--cause or consequence of tissue injury? *General Physiology and Biophysics,* 

A., et al. (2009). Soy proteins and isoflavones affect bone mineral density in older

women: A randomized controlled trial. *The American Journal of Clinical Nutrition, 90*(1), 234-242.


Polyphenol Antioxidants and Bone Health: A Review 485

Saura-Calixto, F., Serrano, J., & Goñi, I. (2007). Intake and bioaccessibility of total

Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of polyphenols. *The* 

Sendur, O. F., Turan, Y., Tastaban, E., & Serter, M. (2009). Antioxidant status in patients with

Shen, C. L., Cao, J. J., Dagda, R. Y., Tenner, T. E.,Jr, Chyu, M. C., & Yeh, J. K. (2011).

Supplementation with green tea polyphenols improves bone microstructure and quality in aged, orchidectomized rats. *Calcified Tissue International, 88*(6), 455-463. Shen, C. L., Wang, P., Guerrieri, J., Yeh, J. K., & Wang, J. S. (2008). Protective effect of green

tea polyphenols on bone loss in middle-aged female rats. *Osteoporosis International : A Journal Established as Result of Cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA, 19*(7), 979-990. Shen, C. L., Yeh, J. K., Cao, J. J., Chyu, M. C., & Wang, J. S. (2011). Green tea and bone health:

Evidence from laboratory studies. *Pharmacological Research : The Official Journal of the* 

polyphenols mitigate bone loss of female rats in a chronic inflammation-induced

of the intake of dietary polyphenols: Strengths, limitations and application in

Shen, C. L., Yeh, J. K., Cao, J. J., Tatum, O. L., Dagda, R. Y., & Wang, J. S. (2010). Green tea

Sontakke, A. N., & Tare, R. S. (2002). A duality in the roles of reactive oxygen species with

Spencer, J. P., Abd El Mohsen, M. M., Minihane, A. M., & Mathers, J. C. (2008). Biomarkers

Stevenson, D. E., & Hurst, R. D. (2007). Polyphenolic phytochemicals – just antioxidants or

Sugimoto, E., & Yamaguchi, M. (2000). Stimulatory effect of daidzein in osteoblastic MC3T3-

Trzeciakiewicz, A. (2009). When nutrition interacts with osteoblast function: molecular

Tucker, K.L. (2009). Osteoporosis Prevention and Nutrition. *Current Osteoporosis Reports*,

Tucker, K. L., Hannan, M. T., Chen, H., Cupples, L. A., Wilson, P. W., & Kiel, D. P. (1999).

Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and total

Viereck, V., Grundker, C., Blaschke, S., Siggelkow, H., Emons, G., & Hofbauer, L. C. (2002).

trabecular osteoblasts. *Journal of Cellular Biochemistry, 84*(4), 725-735.

Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women. *The American Journal of Clinical* 

phenolics in selected fruits, vegetables, and grain products. *Journal of Agricultural* 

Phytoestrogen genistein stimulates the production of osteoprotegerin by human

bone loss model. *The Journal of Nutritional Biochemistry, 21*(10), 968-974. Singh, V. N. (1992). A current perspective on nutrition and exercise. *The Journal of Nutrition,* 

respect to bone metabolism. *Clin Chim Acta, 318*(1-2), 145-148.

nutrition research. *The British Journal of Nutrition, 99*(1), 12-22.

mechanisms of polyphenols. *Nutrition Research Reviews, 22*, 68-81.

much more? *Cellular & Molecular Life Sciences, 64*, 2900-16.

E1 cells. *Biochemical Pharmacology, 59*(5), 471-475.

polyphenols in a whole diet. *Food Chemistry, 101*, 492–501.

osteoporosis: A controlled study. *Joint Bone Spine, 76*(5), 514-518.

*Journal of Nutrition, 130*(8S Suppl), 2073S-85S.

*Italian Pharmacological Society, 64*(2), 155-161.

*122*(3 Suppl), 760-765.

7(4), 111.

*Nutrition, 69*(4), 727-736.

*Food & Chemistry, 46*, 4113–4117.


Miyamoto, M., Matsushita, Y., Kiyokawa, A., Fukuda, C., Iijima, Y., Sugano, M., et al. (1998).

Mody, N., Parhami, F., Sarafian, T. A., & Demer, L. L. (2001). Oxidative stress modulates

Morton, D. J., Barrett-Connor, E. L., & Schneider, D. L. (2001). Vitamin C supplement use

New, S. A. (2003). Intake of fruit and vegetables: Implications for bone health. *The* 

Palacios, C. (2006). The role of nutrients in bone health, from A to Z. *Critical Reviews in Food* 

Papoutsi, Z., Kassi, E., Chinou, I., Halabalaki, M., Skaltsounis, L. A., & Moutsatsou, P. (2008).

Park, J. A., Ha, S. K., Kang, T. H., Oh, M. S., Cho, M. H., Lee, S. Y., et al. (2008). Protective

Pearson, K. J., Baur, J. A., Lewis, K. N., Peshkin, L., Price, N. L., Labinskyy, N., et al. (2008).

Prentice, A., Schoenmakers, I., Laskey, MA., de Bono, S., Ginty, F. & Goldberg, GR. (2006). Nutrition and bone growth and development. *Proc Nutr Soc 65*, 348–360. Rao, A. V., & Rao, L. G. (2007). Carotenoids and human health. *Pharmacological Research,* 

Rao, L. G., Krishnadev, N., Banasikowska, K., & Rao, A. V. (2003). Lycopene I--effect on

Rao, L. G., Mackinnon, E. S., Josse, R. G., Murray, T. M., Strauss, A., & Rao, A. V. (2007).

Rassi, C. M., Lieberherr, M., Chaumaz, G., Pointillart, A., & Cournot, G. (2002). Down-

Sahni, S., Hannan, M. T., Blumberg, J., Cupples, L. A., Kiel, D. P., & Tucker, K. L. (2009).

Sahnoun, Z., Jamoussi, K., & Zeghal, KM. (1997). Free radicals and antioxidants: human

osteoclasts: Lycopene inhibits basal and parathyroid hormone-stimulated osteoclast formation and mineral resorption mediated by reactive oxygen species in

Lycopene consumption decreases oxidative stress and bone resorption markers in

regulation of osteoclast differentiation by daidzein via caspase 3. *Journal of Bone and Mineral Research : The Official Journal of the American Society for Bone and Mineral* 

Inverse association of carotenoid intakes with 4-y change in bone mineral density in elderly men and women: The framingham osteoporosis study. *The American* 

the cell line KS483. *The British Journal of Nutrition, 99*(4), 715-722.

osteoporosis. *Arq Bras Endocrinol Metab, 54*(2), 179-185.

rat bone marrow cultures. *J Med Food, 6*(2), 69-78.

*Journal of Clinical Nutrition, 89*(1), 416-424.

postmenopausal women. *Osteoporos Int, 18*(1), 109-115.

*Proceedings of the Nutrition Society, 62*(4), 889-899.

*Science and Nutrition, 46*(8), 621-628.

519.

140.

1217-1223.

*55*(3), 207-216.

*Research, 17*(4), 630-638.

*Medicine, 31*(4), 509-519.

Prenylflavonoids: A new class of non-steroidal phytoestrogen (part 2). estrogenic effects of 8-isopentenylnaringenin on bone metabolism. *Planta Medica, 64*(6), 516-

osteoblastic differentiation of vascular and bone cells. *Free Radical Biology &* 

and bone mineral density in postmenopausal women. *J Bone Miner Res, 16*(1), 135-

Walnut extract (juglans regia L.) and its component ellagic acid exhibit antiinflammatory activity in human aorta endothelial cells and osteoblastic activity in

effect of apigenin on ovariectomy-induced bone loss in rats. *Life Sciences, 82*(25-26),

Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending life span. *Cell Metabolism, 8*(2), 157-168. Peters, B. S., & Martini, L. A. (2010). Nutritional aspects of the prevention and treatment of


**23** 

*1,3México 2Spain* 

**The Pentacyclic Triterpenes , -amyrins:** 

**A Review of Sources and Biological Activities** 

Liliana Hernández Vázquez1, Javier Palazon2 and Arturo Navarro-Ocaña3,\* *1Universidad Autónoma Metropolitana, Unidad Xochimilco, Depto. Sistemas Biológicos, 2Laboratorio de Fisiología Vegetal, Facultat of Farmacia, Universitat de Barcelona, 3Departamento de Alimentos y Biotecnología, Facultad de Química "E" – UNAM,* 

Pentacyclic triterpenes are ubiquitously distributed throughout the plant kingdom, in a free form as aglycones or in combined forms, and have long been known to have a number of biological effects. The compounds-amyrin and -amyrin are commonly found in medicinal plants and oleo-resin obtained by bark incision of several species of *Bursera or Protium* of the Burseraceae family. Both *in vitro* and in *vivo* studies have shown that -

In light of the considerable interest recently generated in the chemistry and pharmacological properties of amyrins and their analogs, we have undertaken this review in an effort to summarize the available literature on these promising bioactive natural products. The review will detail the recent studies on the chemistry and bioactivity of , -amyrins, which is presented in the following sections: the isolation and distribution of -amyrin and -amyrin, giving a brief introduction to amyrins as natural products and the methods used in their isolation; the biological activities of amyrins, examining the biological properties associated

The chemical structure of -amyrin (3-hydroxy-urs-12-en-3-ol) is shown in Fig. 1. The chemical formula of -amyrin is C30H50O, its melting point is 184-186 0C (Sirat, et al., 2010), and it presents an MS ion Peak at m/z 426 (M+) (Dias et al., 2011). The infra-red spectrum of amyrin is IR umax (KBr) cm-1: 3450, 2895 and 2895. The chemical structure of -amyrin (3 hydroxy-olean-12-en-3-ol) is also depicted in (Fig. 1) and its formula is C30H50O. The infra-red spectrum of -amyrin shows the presence of a hydroxyl function and the olefinic moiety at a spectrum of 3360 and 1650 cm-1 and MS studies of -amyrin confirm a parent ion peak at m/z 426 (M+) (Dias et al., 2011), other work of HR-EI-MS m/z: 426.2975 (calcd. for C30H50O, 426.3861) (Jabeen et al., 2011). The melting point of -amyrin is 189-191 ºC (Lin et al., 2011).

with these compounds with a focus on their potential chemotherapeutic applications.

**1. Introduction** 

**2.1.1 Structure** 

amyrin also has important biological functions.

**2.1 Chemical structure, detection, analysis and sources** 


### **The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities**

Liliana Hernández Vázquez1, Javier Palazon2 and Arturo Navarro-Ocaña3,\* *1Universidad Autónoma Metropolitana, Unidad Xochimilco, Depto. Sistemas Biológicos, 2Laboratorio de Fisiología Vegetal, Facultat of Farmacia, Universitat de Barcelona, 3Departamento de Alimentos y Biotecnología, Facultad de Química "E" – UNAM, 1,3México* 

*2Spain* 

#### **1. Introduction**

486 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Ward, W. E., Yuan, Y. V., Cheung, A. M., & Thompson, L. U. (2001). Exposure to flaxseed

Wattel, A., Kamel, S., Prouillet, C., Petit, J. P., Lorget, F., Offord, E., et al. (2004). Flavonoid

Wu, C. H., Yang, Y. C., Yao, W. J., Lu, F. H., Wu, J. S., & Chang, C. J. (2002). Epidemiological

Xiao, H. H., Dai, Y., Wan, H. Y., Wong, M. S., & Yao, X. S. (2011). Bone-protective effects of

Zhang, P., Dai, K. R., Yan, S. G., Yan, W. Q., Zhang, C., Chen, D. Q., et al. (2009). Effects of

mesenchymal stem cell. *European Journal of Pharmacology, 607*(1-3), 1-5. Zych, M., Folwarczna, J., Pytlik, M., Sliwinski, L., Golden, M. A., Burczyk, J., et al. (2010).

*Journal of Toxicology and Environmental Health.Part A, 63*(1), 53-65.

Weber, P. (2001). Vitamin K and bone health. *Nutrition, 17*(10), 880-887.

*Internal Medicine, 162*(9), 1001-1006.

*Journal of Nutrition,* 1-8.

*Planta Medica, 76*(5), 407-411.

285-295.

and its purified lignan reduces bone strength in young but not older male rats.

quercetin decreases osteoclastic differentiation induced by RANKL via a mechanism involving NF kappa B and AP-1. *Journal of Cellular Biochemistry, 92*(2),

evidence of increased bone mineral density in habitual tea drinkers. *Archives of* 

bioactive fractions and ingredients in sambucus williamsii HANCE. *The British* 

naringin on the proliferation and osteogenic differentiation of human bone

Administration of caffeic acid worsened bone mechanical properties in female rats.

Pentacyclic triterpenes are ubiquitously distributed throughout the plant kingdom, in a free form as aglycones or in combined forms, and have long been known to have a number of biological effects. The compounds-amyrin and -amyrin are commonly found in medicinal plants and oleo-resin obtained by bark incision of several species of *Bursera or Protium* of the Burseraceae family. Both *in vitro* and in *vivo* studies have shown that amyrin also has important biological functions.

In light of the considerable interest recently generated in the chemistry and pharmacological properties of amyrins and their analogs, we have undertaken this review in an effort to summarize the available literature on these promising bioactive natural products. The review will detail the recent studies on the chemistry and bioactivity of , -amyrins, which is presented in the following sections: the isolation and distribution of -amyrin and -amyrin, giving a brief introduction to amyrins as natural products and the methods used in their isolation; the biological activities of amyrins, examining the biological properties associated with these compounds with a focus on their potential chemotherapeutic applications.

#### **2.1 Chemical structure, detection, analysis and sources**

#### **2.1.1 Structure**

The chemical structure of -amyrin (3-hydroxy-urs-12-en-3-ol) is shown in Fig. 1. The chemical formula of -amyrin is C30H50O, its melting point is 184-186 0C (Sirat, et al., 2010), and it presents an MS ion Peak at m/z 426 (M+) (Dias et al., 2011). The infra-red spectrum of amyrin is IR umax (KBr) cm-1: 3450, 2895 and 2895. The chemical structure of -amyrin (3 hydroxy-olean-12-en-3-ol) is also depicted in (Fig. 1) and its formula is C30H50O. The infra-red spectrum of -amyrin shows the presence of a hydroxyl function and the olefinic moiety at a spectrum of 3360 and 1650 cm-1 and MS studies of -amyrin confirm a parent ion peak at m/z 426 (M+) (Dias et al., 2011), other work of HR-EI-MS m/z: 426.2975 (calcd. for C30H50O, 426.3861) (Jabeen et al., 2011). The melting point of -amyrin is 189-191 ºC (Lin et al., 2011).

The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 489

38.7 38.7 28.7 27.2 3.16 (*dd*, *J* = 5.1; 11.2) 79.6 3.15 (*dd*, *J* = 4.4; 10.8) 79.3 38.7 38.5 0.67 (*d*, *J* = 11.6) 55.1 0.68 (*d*, *J* = 11.0) 55.1 18.4 18.6 32.2 32.4 40.7 39.8 47.7 47.6 36.6 36.9 23.3 23.6 5.06 (*t*, *J* = 3.2) 124.4 5.12 (*t*, *J* = 3.2) 121.7 139.5 145.2 42.0 41.7 1.94 (*td*, *J* = 4.5; 13.5 H) 27.2 1.89 (*td*, *J* = 4.0; 14.0 H) 26.2 1.76 (*td*, *J* = 5.0; 13.5 H) 26.6 1.70 (*td*, *J* = 4.3; 13.5 H) 26.1 33.7 32.6 59.0 47.2 39.6 1.93 (*dd*, *J* = 4.0; 13.7 H) 46.8 39.6 31.0 31.2 34.7 1.85 (*dt*, *J* = 3.0; 7.0) 41.5 1.80*m* 37.1 0.93*s* 28.1 0.77*s* 28.0 0.74*s* 15.6 0.90*s* 15.4 0.73*s* 15.6 0.73*s* 15.4 0.89*s* 16.8 0.93*s* 16.8 1.01*s* 23.2 1.19*s* 25.9 0.94*s* 28.1 1.07*s* 28.4 0.85 (*d*, *J* = 6.0) 17.4 0.87*s* 33.8 0.73 (*d*, *J* = 7.0) 21.4 0.80*s* 23.7

1H 13C 1H 13C

**Position -amyrin -amyrin** 

Table 1. The 1H and 13C-NMR Spectral Data of - and -amyrin

employing silica gel columns or liquid-liquid partition is also necessary.


Fig. 1. Estructure of amyrins

NMR methods have indisputably become the single most important spectroscopic techniques for the identification and structure elucidation of amyrins. Several ID and 2D NMR methods are now commonly used for the characterization of pentaclyclic triterpenes. These methods incluye 1H and 13C-NMR, APT, DEPT, COSY, HMQC, HMBC and TOCSY. The 1H and 13C-NMR assignments of - -amyrin are presented in Table 1, (Dias et al., 2011).

#### **2.1.2 Detection**

Amyrins are found in various plants and plant materials such as leaves, bark, wood, and resins. This material has to be pre-treated prior to isolation of the target compounds. First, the plant material is usually dried, then ground into a power and sieved. Second, extractions are carried out with dichloromethane, chloroformo, *n*-hexane, and methanol. The samples can be subjected to alkaline hydrolysis, derivatization and separation by thin layer chromatography, and the resulting material can be directly subjected to analysis. Gas chromatography (CG) and high performance thin layer chromatography (HPTLC) techniques are the most commonly employed methods to quantitate -, -amyrin in plants.

TLC provided an easy and rapid way to study plant extract profiles and partially identify compounds. The first step for the identification of -amyrin, -amyrin and 3-*epi*-lupeol was to compare RF values of reference standards with those of sample extracts. TLC on silica gel revealed that -amyrin on tracks 6 and 15, -amyrin on track 14 and the -, -amyrin mixture on track 16, as well as two standards, all had the same RF (Fig. 2). The -amyrin band was observed as brown, while the -amyrin band appeared violet, as did the band for the -, -amyrin mixture. TLC analysis revealed the presence of -amyrin, -amyrin and 3 *epi*-lupeol by a comparison of the position and color of the triterpene spots with those of authentic compounds (Fig. 2). The bands of - and -amyrin or their mixture were observed in all commercial resin tracks 1-5 and medicinal plant tracks 8-13, while 3-*epi*-lupeol track 7 was detected only in the commercial Mexican Copal resins tracks 1-4. Attempts were made to separate the -, -amyrin mixture, which had appeared homogenous on TLC, but without success. These results showed that TLC can be used as a simple method for a preliminary analysis of these triterpenes in extracts of commercial resins and plants, but cannot be employed for the analysis of the -, -amyrin mixture. (Hernández-Vázquez et al., 2010)


The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 489

NMR methods have indisputably become the single most important spectroscopic techniques for the identification and structure elucidation of amyrins. Several ID and 2D NMR methods are now commonly used for the characterization of pentaclyclic triterpenes. These methods incluye 1H and 13C-NMR, APT, DEPT, COSY, HMQC, HMBC and TOCSY. The 1H and 13C-NMR assignments of - -amyrin are presented in Table 1, (Dias et al., 2011).

Amyrins are found in various plants and plant materials such as leaves, bark, wood, and resins. This material has to be pre-treated prior to isolation of the target compounds. First, the plant material is usually dried, then ground into a power and sieved. Second, extractions are carried out with dichloromethane, chloroformo, *n*-hexane, and methanol. The samples can be subjected to alkaline hydrolysis, derivatization and separation by thin layer chromatography, and the resulting material can be directly subjected to analysis. Gas chromatography (CG) and high performance thin layer chromatography (HPTLC) techniques are the most commonly

TLC provided an easy and rapid way to study plant extract profiles and partially identify compounds. The first step for the identification of -amyrin, -amyrin and 3-*epi*-lupeol was to compare RF values of reference standards with those of sample extracts. TLC on silica gel revealed that -amyrin on tracks 6 and 15, -amyrin on track 14 and the -, -amyrin mixture on track 16, as well as two standards, all had the same RF (Fig. 2). The -amyrin band was observed as brown, while the -amyrin band appeared violet, as did the band for the -, -amyrin mixture. TLC analysis revealed the presence of -amyrin, -amyrin and 3 *epi*-lupeol by a comparison of the position and color of the triterpene spots with those of authentic compounds (Fig. 2). The bands of - and -amyrin or their mixture were observed in all commercial resin tracks 1-5 and medicinal plant tracks 8-13, while 3-*epi*-lupeol track 7 was detected only in the commercial Mexican Copal resins tracks 1-4. Attempts were made to separate the -, -amyrin mixture, which had appeared homogenous on TLC, but without success. These results showed that TLC can be used as a simple method for a preliminary analysis of these triterpenes in extracts of commercial resins and plants, but cannot be employed for the analysis of the -, -amyrin mixture. (Hernández-Vázquez et al., 2010)

Fig. 1. Estructure of amyrins

employed methods to quantitate -, -amyrin in plants.

**2.1.2 Detection** 

Table 1. The 1H and 13C-NMR Spectral Data of - and -amyrin


The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 491

analyse hexane extracts from seven oleoresins of *Protium* species. High concentrations of and -amyrin were identified in *P. strumosum* (64%) and *P. tenuifolium* (66.7%) (Silva et al., 2009). Finally, the analytical performances of three atmospheric-pressure sources, electrospray (ESI), atmospheric-pressure chemical ionization (APCI) and atmosphericpressure photoionization (APPI), were evaluated for the analysis of pentacyclic triterpenes in liquid chromatography-mass spectrometry (LC-MS) (Zarrouk et al., 2010). The developed LC-MS method was used to characterize pentacyclic triterpenes in tree plant extracts. The main component of birch bark was betulin and the extracts of Okume resin exhibited high amounts of - and -amyrin (Rhourri-Frih et al., 2008). Other technique used to quantitate and determine amyrins is Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). HPLC was used for analysis of some isomeric plant triterpenoids (-amyrin and amyrin -amyrin, lupeol, lupenon, lupeol acetate, cycloartenol acetate, ursolic acid oleanolic acid and two sterols) (Martelanc et al., 2009), other studies was for analysis of medicinal plants and Mexican Copal resins (Hernández-Vázquez et al., 2010) and resin obtained from


Plants reported since 2008 to possess -amyrin, -amyrin and a , -amyrin mixture in minor amounts (detected and isolated) are listed here. -Amyrin has been isolated from the resin of *Boswellia carterii* Birdw (Wang et al., 2011), detected in stemwood and bark from *Populus x euramericana* (Xu et al., 2010), isolated (65 mg/kg) from the *n*-hexane extract of the leaves of *Melastoma malabathricum* L (Sirat et al., 2010), identified in the methanol extract of the stem bark of *Poncirus trifoliate* (Feng et al., 2010), isolated (1 mg/kg) from the methanol extract of the stem bark of the African tree *Antiaris Africana* Engler (Vouffo et al., 2010), detected in seed oil of Saskatoon berries (*Amelanchier alnifolia* Nutt.) (Bakowska-Barczak et al., 2009), isolated (23 mg/kg) from the methanol extract of stem bark and leaves of *Ficus pandurata* Hance (Ramadan et al., 2009), dried rhizomes of *Nelumbo nucifera* (Chaudhuri et

species of the genus *Protium* (Burseraceae) (Dias et al., 2011).

**2.1.4 Sources of -, -amyrins** 

other triterpenes (Silva et al., 2009).

al., 2009), and detected in bread wheat (Nurmi et al., 2008).

Fig. 2. TLC plate. Tracks: 1=MCT; 2=MCS; 3=MCN; 4=MCP; 5=MER; 6 and 15=-amyrin; 7=3-*epi*-lupeol; 8=Dandelion; 9=Olive; 10=Cancerina; 11=Nance wastes; 12=Bearberry; 13=Pot marigold; 14=-amyrin; 15=-amyrin and 16=mixture -, -amyrin.

#### **2.1.3 Analysis**

Gas chromatography (CG) is applied to determine the concentration of -, -amyrin and -, -amyrin mixtures. The chemical composition of the essential oil of Lemon Catnip (*Nepeta cataria* L. var. citriodora Balbis) was determined by CG-MS, and -amyrin was detected in the hydrodistilled volatile of Lemon Catnip (Wesolowska et al., 2011). -amyrin has also been determined in the kernel fats of the shea tree (*Vitellaria paradoxa*; Sapotaceae) of sub-Saharan countries (Akihisa et al., 2010). CG of the apolar extract from *Clusia Minor* L. leaves led to the identification of 25 compounds, lupeol and -amyrin being the most abundant triterpenoids (Mangas-Marin et al., 2008). CG-MS fingerprints for cerumen from the stingless bee *Tetragonula carbonaria* in South East Queensland, Australia, showed trace quantities of TMS ethers of -amyrins (Massaro et al., 2010). Studies on the constituents of yellow Cuban propolis by CG-MS revealed the presence of large amounts of triterpenic alcohols including -amyrin, (Márquez-Hernández et al., 2010). Solid-phase extraction and GC-MS were developed to separate and enrich only sterols from unsaponifiables of vegetable, hazelnut and olive oils, detecting sterols, lupeol and -, -amyrin (Azadmard-Damirchi et al., 2010). Epicuticular and intracuticular waxes from both adaxial and abaxial surfaces of *Kalanchoe daigremontiana* leaves (Hamet et Perr. De la bathie) were analyzed by CG. All wax mixtures were found to contain triterpenoids and fatty acids, the triterpenoid fraction containing small amounts of -amyrin (van Maarseveen & Jetter., 2009). Fatty acid, phytosterol, and polyamine conjugate profiles of corn edible oils were analyzed by GC-MS and HPLC, and a few minor sterols and -amyrin were identified and quantified using GC-FID (Moreau et al., 2009). Fatty acids, phytosterols and tocopherols of Milk thistle (*Silybum marianum*) seeds were determined in four varieties grown in Ardebil-Iran. In this study using TLC-GC, dimethylsterols were predominant followed by cycloartenol and -amyrin, (Fathi-Achachlouei, Azadmard-Damirchi., 2009). CG proved an effective method for quantitative measurement of the -sitosterol content of white mulberry (*Morus alba*) leaves and bark without derivatization. Sterols, lupeol and , -amyrin were identified in leaves and bark by GC-FID analysis (Böszörményi et al., 2009). GC and CG-MS were used to analyse hexane extracts from seven oleoresins of *Protium* species. High concentrations of and -amyrin were identified in *P. strumosum* (64%) and *P. tenuifolium* (66.7%) (Silva et al., 2009). Finally, the analytical performances of three atmospheric-pressure sources, electrospray (ESI), atmospheric-pressure chemical ionization (APCI) and atmosphericpressure photoionization (APPI), were evaluated for the analysis of pentacyclic triterpenes

in liquid chromatography-mass spectrometry (LC-MS) (Zarrouk et al., 2010). The developed LC-MS method was used to characterize pentacyclic triterpenes in tree plant extracts. The main component of birch bark was betulin and the extracts of Okume resin exhibited high amounts of - and -amyrin (Rhourri-Frih et al., 2008). Other technique used to quantitate and determine amyrins is Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). HPLC was used for analysis of some isomeric plant triterpenoids (-amyrin and amyrin -amyrin, lupeol, lupenon, lupeol acetate, cycloartenol acetate, ursolic acid oleanolic acid and two sterols) (Martelanc et al., 2009), other studies was for analysis of medicinal plants and Mexican Copal resins (Hernández-Vázquez et al., 2010) and resin obtained from species of the genus *Protium* (Burseraceae) (Dias et al., 2011).

#### **2.1.4 Sources of -, -amyrins**

490 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Fig. 2. TLC plate. Tracks: 1=MCT; 2=MCS; 3=MCN; 4=MCP; 5=MER; 6 and 15=-amyrin; 7=3-*epi*-lupeol; 8=Dandelion; 9=Olive; 10=Cancerina; 11=Nance wastes; 12=Bearberry;

Gas chromatography (CG) is applied to determine the concentration of -, -amyrin and -, -amyrin mixtures. The chemical composition of the essential oil of Lemon Catnip (*Nepeta cataria* L. var. citriodora Balbis) was determined by CG-MS, and -amyrin was detected in the hydrodistilled volatile of Lemon Catnip (Wesolowska et al., 2011). -amyrin has also been determined in the kernel fats of the shea tree (*Vitellaria paradoxa*; Sapotaceae) of sub-Saharan countries (Akihisa et al., 2010). CG of the apolar extract from *Clusia Minor* L. leaves led to the identification of 25 compounds, lupeol and -amyrin being the most abundant triterpenoids (Mangas-Marin et al., 2008). CG-MS fingerprints for cerumen from the stingless bee *Tetragonula carbonaria* in South East Queensland, Australia, showed trace quantities of TMS ethers of -amyrins (Massaro et al., 2010). Studies on the constituents of yellow Cuban propolis by CG-MS revealed the presence of large amounts of triterpenic alcohols including -amyrin, (Márquez-Hernández et al., 2010). Solid-phase extraction and GC-MS were developed to separate and enrich only sterols from unsaponifiables of vegetable, hazelnut and olive oils, detecting sterols, lupeol and -, -amyrin (Azadmard-Damirchi et al., 2010). Epicuticular and intracuticular waxes from both adaxial and abaxial surfaces of *Kalanchoe daigremontiana* leaves (Hamet et Perr. De la bathie) were analyzed by CG. All wax mixtures were found to contain triterpenoids and fatty acids, the triterpenoid fraction containing small amounts of -amyrin (van Maarseveen & Jetter., 2009). Fatty acid, phytosterol, and polyamine conjugate profiles of corn edible oils were analyzed by GC-MS and HPLC, and a few minor sterols and -amyrin were identified and quantified using GC-FID (Moreau et al., 2009). Fatty acids, phytosterols and tocopherols of Milk thistle (*Silybum marianum*) seeds were determined in four varieties grown in Ardebil-Iran. In this study using TLC-GC, dimethylsterols were predominant followed by cycloartenol and -amyrin, (Fathi-Achachlouei, Azadmard-Damirchi., 2009). CG proved an effective method for quantitative measurement of the -sitosterol content of white mulberry (*Morus alba*) leaves and bark without derivatization. Sterols, lupeol and , -amyrin were identified in leaves and bark by GC-FID analysis (Böszörményi et al., 2009). GC and CG-MS were used to

13=Pot marigold; 14=-amyrin; 15=-amyrin and 16=mixture -, -amyrin.

**2.1.3 Analysis** 


Plants reported since 2008 to possess -amyrin, -amyrin and a , -amyrin mixture in minor amounts (detected and isolated) are listed here. -Amyrin has been isolated from the resin of *Boswellia carterii* Birdw (Wang et al., 2011), detected in stemwood and bark from *Populus x euramericana* (Xu et al., 2010), isolated (65 mg/kg) from the *n*-hexane extract of the leaves of *Melastoma malabathricum* L (Sirat et al., 2010), identified in the methanol extract of the stem bark of *Poncirus trifoliate* (Feng et al., 2010), isolated (1 mg/kg) from the methanol extract of the stem bark of the African tree *Antiaris Africana* Engler (Vouffo et al., 2010), detected in seed oil of Saskatoon berries (*Amelanchier alnifolia* Nutt.) (Bakowska-Barczak et al., 2009), isolated (23 mg/kg) from the methanol extract of stem bark and leaves of *Ficus pandurata* Hance (Ramadan et al., 2009), dried rhizomes of *Nelumbo nucifera* (Chaudhuri et al., 2009), and detected in bread wheat (Nurmi et al., 2008).

The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 493

amyrins are not readily visible on TLC plates UV ( = 254 and 365 nm) but are easily detected following exposure to iodine vapors, anisaldehyde-H2SO4 or vanillin-H2SO4 spray

Fig. 3. Sources of amyrins; a) Copal Piedra, b) White Copal, c) Bursera bark, d) Propolis

, -amyrins have been shown to exhibit various pharmacological actitivies *in vitro* and *in vivo* conditions against various health-related conditions, including conditions such as

The antimicrobial properties of *n*-hexane and methanol extracts of *Bombax malabaricum* flowers were examined against different bacterial, fungal and yeast strains. The methanol extract was highly active against *Staphylococcus aureus*, *Bacillus subtilis*, *Stretoccocus faecalis*, *Neisseria gonorrehea*, *Pseudomonas aeruginosa* and *Candida albicans*, whereas the *n*-hexane extract displayed moderate-to-weak activities against the same test microorganisms. An *n*-hexane extract afforded sterols including -amyrin (El-Hagrassi et al., 2011). A bioassay-guided fractionation of *n*-hexane extracts of *Bursera simaruba* (L) Sarg. leaves resulted in the isolation and identification of five sterols and -amyrin. Additionally, *n*-hexane extracts have displayed anti-inflammatory activity on adjuvant-carrageenan-induced inflammation in rats (Carretero et al., 2008). -Amyrin and other compounds have been proposed as possible biomarkers for

**2.2 An overview of pharmacological activities of , -amyrins** 

inflammation, microbial, fungal, and viral infections and cancer cells.

the fungal resistance of grape-vine leaves (*Vitis vinifera*) (Batovska et al., 2008).

**2.2.1 Anti-microbial and anti-fungal** 

reagents.


Table 2. List of selected materials containing -, -amyrin and /-amyrins


An- and -amyrin mixture has been detected in the following plants: *n*-hexane and chloroform extracts of the epicuticular wax layer of *Mandevilla guanabarica* and *Mandevilla moricandiana*, (Cordeiro et al., 2011), an ethanolic extract of roots of *Salacia amplifolia*, (Wang et al., 2011), and chloroform extracts of Blue Honeysuckle (*Lonicera caerulea* L.) (Palíkova et al., 2008).

A multitude of extraction and isolation schemes have been used for the procurement of amyrin, -amyrin and an / amyrin mixture. Typically, dry material (resins, leafs and stem barks) is extracted with hexane or another non-polar solvent, (Fig. 3), and the resulting extract is directly subjected to column or thin layer chromatography. An alternative procedure is sequential fractionation by silica gel columns using various solvents. The

Mexican copal 5g/Kg Hernandez-Vazquez

*Cassia obtusifolia* 0.14g/kg Sob et al., 2010

*Nelumbo nucifera* Gaertn 3g/kg (Xu. et al., 2011) *Amphipterygium adstringens* 2.4g/kg Rosas-Acevedo et al.,

*Protium sp* 3.1g/kg 1.7g/Kg Dias et al., 2011 *Eucalyptus globulus* 0.3g/kg Domingues et al., 2010 *Ficus carica* 1.2g/Kg Olivera et al 2010 *Ficus cordata* 0.2g/Kg Kuete et al., 2008 *Byrsonima crassa* Niedenzu (IK) 1.3g/kg (Higuchi et al., 2008. *Byrsonima crassifolia* (Nance) 9g/kg Hernández-Vázquez

*Byrsonima fagifolia* 2.3g/Kg Higuchi et al., 2008 *Pouteria gardnerii* (Mart & Miq) X Silva et al., 2009


An- and -amyrin mixture has been detected in the following plants: *n*-hexane and chloroform extracts of the epicuticular wax layer of *Mandevilla guanabarica* and *Mandevilla moricandiana*, (Cordeiro et al., 2011), an ethanolic extract of roots of *Salacia amplifolia*, (Wang et al., 2011), and chloroform extracts of Blue Honeysuckle (*Lonicera caerulea* L.) (Palíkova et

A multitude of extraction and isolation schemes have been used for the procurement of amyrin, -amyrin and an / amyrin mixture. Typically, dry material (resins, leafs and stem barks) is extracted with hexane or another non-polar solvent, (Fig. 3), and the resulting extract is directly subjected to column or thin layer chromatography. An alternative procedure is sequential fractionation by silica gel columns using various solvents. The

Table 2. List of selected materials containing -, -amyrin and /-amyrins

**amyrin Ref** 

Manguro, et al., 2009.

2011

et al., 2010

et al., 2010

**Plant -amyrin -amyrin -**

*Commiphora holtziana* (syn. *Commiphora erythraea*)

al., 2008).

amyrins are not readily visible on TLC plates UV ( = 254 and 365 nm) but are easily detected following exposure to iodine vapors, anisaldehyde-H2SO4 or vanillin-H2SO4 spray reagents.

Fig. 3. Sources of amyrins; a) Copal Piedra, b) White Copal, c) Bursera bark, d) Propolis

#### **2.2 An overview of pharmacological activities of , -amyrins**

, -amyrins have been shown to exhibit various pharmacological actitivies *in vitro* and *in vivo* conditions against various health-related conditions, including conditions such as inflammation, microbial, fungal, and viral infections and cancer cells.

#### **2.2.1 Anti-microbial and anti-fungal**

The antimicrobial properties of *n*-hexane and methanol extracts of *Bombax malabaricum* flowers were examined against different bacterial, fungal and yeast strains. The methanol extract was highly active against *Staphylococcus aureus*, *Bacillus subtilis*, *Stretoccocus faecalis*, *Neisseria gonorrehea*, *Pseudomonas aeruginosa* and *Candida albicans*, whereas the *n*-hexane extract displayed moderate-to-weak activities against the same test microorganisms. An *n*-hexane extract afforded sterols including -amyrin (El-Hagrassi et al., 2011). A bioassay-guided fractionation of *n*-hexane extracts of *Bursera simaruba* (L) Sarg. leaves resulted in the isolation and identification of five sterols and -amyrin. Additionally, *n*-hexane extracts have displayed anti-inflammatory activity on adjuvant-carrageenan-induced inflammation in rats (Carretero et al., 2008). -Amyrin and other compounds have been proposed as possible biomarkers for the fungal resistance of grape-vine leaves (*Vitis vinifera*) (Batovska et al., 2008).

The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 495

*Ligustrum* spp. was measured by HPLC, the highest content of -amyrin, betulinic acid and lupeol being found in LP. This work suggested that these three triterpenoids are responsible

A recent report describes that the roots of *Calotropis gigantea* (Linn.) R.Br, traditionally used in India to treat asthma, possess anti-lipoxygenase activity, it was found that intraperitoneal administration of indomethacin did not block edema formation, but edema was inhibited by montelukast and methanolic extracts of *C. gigantea* roots. This result indicates that the extract from *C. gigantea* was responsible for the inhibition of the lipoxygenase pathway in the arachidonate metabolism. Therefore, it can be concluded that *C*. *gigantea* may have a similar mechanism of action as dexamethasone as well as antioxidant and anti-lipoxygenase

Aqueous and organic extracts of *Acacia visco* Lor. Ap Griseb (Fabaceae) were tested for antiinflammatory activity in experimental rat models. The extracts revealed an antiinflammatory effect against carrageenan-induced oedema, phospholipaseA-induced oedema, and cotton pellet-induced granuloma without any acute toxic effects. Among the class of compounds characterized from *A. visco* leaves, the triterpenoids lupeol, α-amyrin and β-amyrin may be mainly responsible for these anti-anflammatory properties (Padernera et al., 2010). α-, β-Amyrin ameliorates L-arginine-induced acute pancreatitis in rats. It has been demonstrated that the crude resin of *Protium heptaphyllum* (March.) has an - and amyrin ratio of 63:37. The mixture of both compounds and methylprednisolone treatments significantly (P < 0.05) attenuated the L-arginine-induced increases in pancreatic wet weight/body weight ratio, and decreased the serum levels of amylase and lipase, and TNFα and IL-6, in comparison with the vehicle control. Also, pancreatic levels of MPO activity, TBARS, and nitrate/nitrite were significantly lower. The conclusion of this study is that , amyrin has the potential to combat acute pancreatitis by acting as an anti-inflammatory and

Another study has shown the systemic preventive or therapeutic anti-inflammatory action of the triterpenes - and -amyrin in TNBS-induced colitis in mice. It was found that , amyrin is as efficacious as dexamethasone in reversing the macroscopic and microscopic outcomes of TNBS-induced colitis, including the restoration of cytokine balance. Furthermore, the results also indicate that inhibition of NF- and CREB activation is certainly the main mechanism through which these triterpenes exert their anti-inflammatory action (Vitor et al., 2009). Another report demonstrated for the first time that , -amyrin isolated from *Protium heptaphyllum* modulates acute periodontal inflammation in rats by reducing neutrophil infiltration, oxidative stress and the production of proinflammatory cytokine TNF-a, and suggests that these triterpenes might be useful as a therapeutic agent for the treatment of gingivitis and to retard the progression of periodontitis (Holanda-Pinto


effects, possibly due to the presence of -amyrin and -amyrin (Bulani et al., 2011).

for the anti-inflammatory potency of LP (Wu et al., 2011).

antioxidant agent (Melo et al., 2010).

**2.2.3 Other pharmacological activities** 

et al., 2008).


In a recent study on the leaves of *Siraitia grosvenorii*, -amyrin and other bioactive compounds were obtained, and their activities against the growth of oral bacterial species *Streptococcus mutans*, *Actinobacillus actinomycetemcomitans*, and *Fusobacterium nucleatum* and the yeast *C. albicans* were evaluated *in vitro*. -amyrin only exhibited a slight inhibition of *Streptococcus mutans* and *Fusobacterium nucleatum* (Zheng et al., 2011). Bioassay-guided fractionation of the methanol extract of the stem bark of *Klainedoxa gabonensis* Pierre ex Engl. (Irvingiaceae) afforded 12 compounds: four flavonoids and eight (including -amyrin) triterpenes. Antimicrobial activities in the triterpenoids ranged from low to non-existent (Wansi et al., 2010). In this study, the *in vitro* antibacterial activity of the methanolic extract and isolated compounds from the bark of *Byrsonima Crassifolia* against twelve bacteria and the yeast *C. albicans* was investigated. Eight known compounds, -amyrin, betulin, betulinic and oleanolic acid, quercetin, epicatechin, gallic acid and-sitosterol, were isolated and evaluated for their antimicrobial activity. Bacterial growth was inhibited by -amyrin, olenolic and gallic acid at concentrations ranging from 64 to 1088 g.mL-1 (Rivero-Cruz et al., 2008).

#### **2.2.2 Anti-inflammatory activity**

Hexane extracts of *Bursera simaruba* (L.) Sarg. leaves display an anti-inflammatory effect on adjuvant-carrageenan-induced inflammation in rats. In order to isolate and identify the active compounds of the hexane extract, we performed a preliminary phytochemical study and a bioassay-directed fractionation using the carrageenan-induced paw oedema test in mice. From the nine fractions (A–I) obtained, A and E showed the strongest antiinflammatory activity, comparable to that of the reference drug phenylbutazone. Sterols and-amyrin have been isolated and characterized from these fractions, the evidence suggesting that these bioactive compounds may play a key role in the anti-inflammatory effects of *B*. *Simaruba* extracts (Carretero et al., 2008).

*Ligustrum* (privet) plants are used by Chinese physicians to prevent and cure hepatitis and chronic bronchitis. Three common *Ligustrum* plant spp., namely *Ligustrum lucidum* Ait. (LL), *L. pricei* Hayata (LP) and *L. sinensis* Lour. (LS) were collected to assess their analgesic/antiinflammatory effects on chemical-induced nociception and carrageenan-induced inflammation in rodents. The methanol extracts from *Ligustrum* plant leaves effectively inhibited nociceptive responses induced by 1% acetic acid and 1% formalin. LP and LL reduced the edema induced by 1% carrageenan. The most potent *Ligustrum* plant was LP, which also reduced abdominal Evans blue extravasations caused by lipopolysaccharide, lipoteichoic acid, autocrines and sodium nitroprusside. The triterpenoid content of the three


In a recent study on the leaves of *Siraitia grosvenorii*, -amyrin and other bioactive compounds were obtained, and their activities against the growth of oral bacterial species *Streptococcus mutans*, *Actinobacillus actinomycetemcomitans*, and *Fusobacterium nucleatum* and the yeast *C. albicans* were evaluated *in vitro*. -amyrin only exhibited a slight inhibition of *Streptococcus mutans* and *Fusobacterium nucleatum* (Zheng et al., 2011). Bioassay-guided fractionation of the methanol extract of the stem bark of *Klainedoxa gabonensis* Pierre ex Engl. (Irvingiaceae) afforded 12 compounds: four flavonoids and eight (including -amyrin) triterpenes. Antimicrobial activities in the triterpenoids ranged from low to non-existent (Wansi et al., 2010). In this study, the *in vitro* antibacterial activity of the methanolic extract and isolated compounds from the bark of *Byrsonima Crassifolia* against twelve bacteria and the yeast *C. albicans* was investigated. Eight known compounds, -amyrin, betulin, betulinic and oleanolic acid, quercetin, epicatechin, gallic acid and-sitosterol, were isolated and evaluated for their antimicrobial activity. Bacterial growth was inhibited by -amyrin, olenolic and gallic acid at concentrations ranging from 64 to 1088 g.mL-1 (Rivero-Cruz et

Hexane extracts of *Bursera simaruba* (L.) Sarg. leaves display an anti-inflammatory effect on adjuvant-carrageenan-induced inflammation in rats. In order to isolate and identify the active compounds of the hexane extract, we performed a preliminary phytochemical study and a bioassay-directed fractionation using the carrageenan-induced paw oedema test in mice. From the nine fractions (A–I) obtained, A and E showed the strongest antiinflammatory activity, comparable to that of the reference drug phenylbutazone. Sterols and-amyrin have been isolated and characterized from these fractions, the evidence suggesting that these bioactive compounds may play a key role in the anti-inflammatory

*Ligustrum* (privet) plants are used by Chinese physicians to prevent and cure hepatitis and chronic bronchitis. Three common *Ligustrum* plant spp., namely *Ligustrum lucidum* Ait. (LL), *L. pricei* Hayata (LP) and *L. sinensis* Lour. (LS) were collected to assess their analgesic/antiinflammatory effects on chemical-induced nociception and carrageenan-induced inflammation in rodents. The methanol extracts from *Ligustrum* plant leaves effectively inhibited nociceptive responses induced by 1% acetic acid and 1% formalin. LP and LL reduced the edema induced by 1% carrageenan. The most potent *Ligustrum* plant was LP, which also reduced abdominal Evans blue extravasations caused by lipopolysaccharide, lipoteichoic acid, autocrines and sodium nitroprusside. The triterpenoid content of the three

mg mL-1 (Jabeen et al., 2011).

**2.2.2 Anti-inflammatory activity** 

effects of *B*. *Simaruba* extracts (Carretero et al., 2008).

al., 2008).

*Ligustrum* spp. was measured by HPLC, the highest content of -amyrin, betulinic acid and lupeol being found in LP. This work suggested that these three triterpenoids are responsible for the anti-inflammatory potency of LP (Wu et al., 2011).

A recent report describes that the roots of *Calotropis gigantea* (Linn.) R.Br, traditionally used in India to treat asthma, possess anti-lipoxygenase activity, it was found that intraperitoneal administration of indomethacin did not block edema formation, but edema was inhibited by montelukast and methanolic extracts of *C. gigantea* roots. This result indicates that the extract from *C. gigantea* was responsible for the inhibition of the lipoxygenase pathway in the arachidonate metabolism. Therefore, it can be concluded that *C*. *gigantea* may have a similar mechanism of action as dexamethasone as well as antioxidant and anti-lipoxygenase effects, possibly due to the presence of -amyrin and -amyrin (Bulani et al., 2011).

Aqueous and organic extracts of *Acacia visco* Lor. Ap Griseb (Fabaceae) were tested for antiinflammatory activity in experimental rat models. The extracts revealed an antiinflammatory effect against carrageenan-induced oedema, phospholipaseA-induced oedema, and cotton pellet-induced granuloma without any acute toxic effects. Among the class of compounds characterized from *A. visco* leaves, the triterpenoids lupeol, α-amyrin and β-amyrin may be mainly responsible for these anti-anflammatory properties (Padernera et al., 2010). α-, β-Amyrin ameliorates L-arginine-induced acute pancreatitis in rats. It has been demonstrated that the crude resin of *Protium heptaphyllum* (March.) has an - and amyrin ratio of 63:37. The mixture of both compounds and methylprednisolone treatments significantly (P < 0.05) attenuated the L-arginine-induced increases in pancreatic wet weight/body weight ratio, and decreased the serum levels of amylase and lipase, and TNFα and IL-6, in comparison with the vehicle control. Also, pancreatic levels of MPO activity, TBARS, and nitrate/nitrite were significantly lower. The conclusion of this study is that , amyrin has the potential to combat acute pancreatitis by acting as an anti-inflammatory and antioxidant agent (Melo et al., 2010).

Another study has shown the systemic preventive or therapeutic anti-inflammatory action of the triterpenes - and -amyrin in TNBS-induced colitis in mice. It was found that , amyrin is as efficacious as dexamethasone in reversing the macroscopic and microscopic outcomes of TNBS-induced colitis, including the restoration of cytokine balance. Furthermore, the results also indicate that inhibition of NF- and CREB activation is certainly the main mechanism through which these triterpenes exert their anti-inflammatory action (Vitor et al., 2009). Another report demonstrated for the first time that , -amyrin isolated from *Protium heptaphyllum* modulates acute periodontal inflammation in rats by reducing neutrophil infiltration, oxidative stress and the production of proinflammatory cytokine TNF-a, and suggests that these triterpenes might be useful as a therapeutic agent for the treatment of gingivitis and to retard the progression of periodontitis (Holanda-Pinto et al., 2008).

#### **2.2.3 Other pharmacological activities**


The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 497

pathways of other biologically active compounds such as avenacine, centellosides, glycyrrhizin or ginsenosides. The development of biotransformation systems to convert amyrins into these or other compounds would open new ways for using - and -amyrins as a source of bioactive plant secondary metabolites more scarcely distributed in the plant kingdom. In this context, the bioconversion of -amyrin into centellosides in *Centella asiatica*

We thank the PAPIIT-UNAM, IN223611 and CONACYT, CB2009 IN 129061, for financial

Akihisa, T., Kojima, N., Katoh, N., Ichimura, Y., Suzuki, H., Fukatsu, M., Maranz, S. &

Bakowska-Barczak, A. M., Schieber, A. & Kalodziejczyk, P. ( 2009). Charaterization of

Batovska, D. I., Todorova, I. T., Nedelcheva, D. V., Parushev, S. P., Atanassov, A. J.,

Börzörményi, A., Szarka, S., Héthelyi É., Gyurján, I., László, M., Simándi, B., Szoke. É. &

Carretero, M. E., López-Pérez, J. L., Abad, M. J., Bermejo, P., Tillet, S., Israel. A. & Noguera-

Chaudhuri, P. C. & Deepika, S. (2009). A new lipid and other constituents from the rhizomes

Ching, J., Chua, T., Chin, L., Lau, A., Pang, Y., Jaya, J. M., Tan, C. & Koh, H. (2010). -

Masters, E. (2010). Triterpene alcohol and fatty composition of sea nuts from seven African countries, *Journal of the Oleo Science*, 59 2(7): 351-360. ISSN: 1347-3352 Azadmard-Damirchi, S., Nemati, M., Hesari, H., Ansarin, M. & Fathi-Achalouei, B. (2010)

Rapid separating and enrichment of 4.4´-dimethylsterols of vegetable oils by solidphase extraction, *Journal of American Oil Chemical Society*, 87: 1155-1159. ISSN: 003-

Saskatoon berry (*Amelanchier alnifolia* Nutt.) seed oil, *Journal of Agricultural and Food* 

Hvarleva, T. D., Djakova, G. J., bankova, V. S. (2008). Preliminary study on biomarkers for the fangal resitence in *Vitis vinifera* leaves, *Journal of Plant Physiology*,

Lemberkovics, É. (2009) Triterpenes in traditional an d supercritical fluids extracts of *Morus alba* and stem bark, *Acta Chromatographica*, 4: 659-669. ISSN 1231-2522 Bulani, V., Biyani, K., Kale, R., Joshi, U., Charhate, K., Kumar, D. & Pagore, R. (2011).

Inhibitory effect of *Calotropis gigantea* extract on ovalbumin-induced airway inflammation and Arachidonic acid induced inflammation in a murine model of asthma, *International Journal of Current Biological and Medical Science* 1(2): 19-25. ISSN

P, B. (2008). Preliminary study of the anti-inflammatory activity of hexane extract and fraction from *Bursera simaruba* (Linneo) Sarg. (Burseraceae) leaves, *Journal of* 

of *Nelumbo nucifera*, *Journal of Asian Natural Products Research*, 11(7): 583-587. ISSN:

Amyrin from *Ardisia elliptica* Thunb. Is more potent than aspirin in inhibiting

cell cultures has been recently reported (Hernandez-Vazquez et al., 2010).

*Chemistry*, 57: 5401-5406. ISSN:1520-5118

*Ethnopharmacology*, 116: 11-15. ISSN: 0378-8741

165: 791-795. ISSN: 0176-1617

**4. Acknowledgement** 

support.

**5. References** 

021X

2231-6256

1477-2213

cohulupone and garcinielliptone were isolated from the pericarp, heartwood and seed of *Garcinia subelliptica*, respectively, and the three compounds showed an inhibitory effect on xanthine oxidase. Treatment of NTUB1, a human bladder cancer cell, with -amyrin or amyrin in cotreatment with cisplatin for 24 h resulted in a reduced viability of cells. This work suggested that -amyrin exhibited weak cytotoxic activities against NTUB1 cells (Lin et al., 2011). The antiproliferative effects of *n*-hexane, chloroform and aqueous methanol extracts prepared from the whole plant of *Centaurea arenaria* M.B. ex Willd. were investigated against cervix adenocarcinoma (HeLa), breast adenocarcinoma (MCF7) and skin epidermoid carcinoma (A431) cells, using the MTT assay. Only the flavonoids and lignans showed moderate activity against these cell lines and -amyrin was inactive (Csapi et al., 2010). From the ethyl acetate fraction of the stem bark of *Camellia japonica*, three new triterpenoids, 3--O-acetyl-16b-hydroxy-12-oxoolean, 3-O-acetyl-16-hydroxy-11-oxoolean-12-ene, and 3--O-acetyl-16-hydroxyolean-12-ene, along with seven known compounds, 3- -hydroxy-1-oxofriedelan, friedelin, 3--friedelanol, canophyllol, 3-oxofriedelan-1(2)-ene, amyrin, camellenodiol, and camelledionol, were isolated. Their structures were established on the basis of spectroscopic analysis and chemical evidence. The isolated compounds were tested *in vitro* for their cytotoxic activities against the A549, LLC, HL-60 and MCF-7 cancer cell lines. Among them, -amyrin exhibited weak cytotoxicity against A549 and HL-60 cancer cell lines with IC (50) values of 46.2 and 38.6 M, respectively (Thao et al., 2010). Another report showed that the methanol extract obtained from soxhlet extraction of leaves of *Ardisia elliptica* Thunberg (Myrsinaceae) contained - and -amyrin, determined by GS-MS. The leaf extract inhibited platelet aggregation with an IC50 value of 167 g/mL, using bioassay guided fractionation. -Amyrin was isolated and purified showing an IC50 value of 4.5 g/mL, while that of aspirin was found to be 11 g/mL, indicating that -amyrin is more potent that aspirin in inhibiting collagen-induced platelet aggregation (Ching et al., 2010). Two triterpenes, -amyrin and 12-oleanene 3, 21-diol, were isolated as a mixture from the chloroform soluble fraction of an ethanolic extract of *Duranta repens* (Verbanaceae) stem. The mixture was highly effective against the larvae of *Culex quinquefasciatus* Say (Diptera: Culicidae) as a mosquitocide. *C*. *quinquefasciatus* is a potential vector of *Wuchereria bancrofti* (Filarioidae), the causative agent of human lymphatic filariasis (Nikkon et al., 2010). One study has examined the potential trypanocidal activity of different plant species growing in the Brazilian Cerrado, after *in vitro* screening of 20 extracts obtained from 10 plants. The phytochemical analysis of the most active extracts (hexane extracts) allowed the identification of -amyrin, -amyrin, lupeol and other triterpenes and sterols. The results showed that pure amyrins are inactive whereas the *n*-hexane leaf extract of *Tibouchina stenocarpa* cogn. Melastomataceae was active. The trypanocidal activity of the extract may be due to the presence of other compounds (Cunha et al., 2009).

#### **3. Conclusion**


#### **4. Acknowledgement**

We thank the PAPIIT-UNAM, IN223611 and CONACYT, CB2009 IN 129061, for financial support.

#### **5. References**

496 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

cohulupone and garcinielliptone were isolated from the pericarp, heartwood and seed of *Garcinia subelliptica*, respectively, and the three compounds showed an inhibitory effect on xanthine oxidase. Treatment of NTUB1, a human bladder cancer cell, with -amyrin or amyrin in cotreatment with cisplatin for 24 h resulted in a reduced viability of cells. This work suggested that -amyrin exhibited weak cytotoxic activities against NTUB1 cells (Lin et al., 2011). The antiproliferative effects of *n*-hexane, chloroform and aqueous methanol extracts prepared from the whole plant of *Centaurea arenaria* M.B. ex Willd. were investigated against cervix adenocarcinoma (HeLa), breast adenocarcinoma (MCF7) and skin epidermoid carcinoma (A431) cells, using the MTT assay. Only the flavonoids and lignans showed moderate activity against these cell lines and -amyrin was inactive (Csapi et al., 2010). From the ethyl acetate fraction of the stem bark of *Camellia japonica*, three new triterpenoids, 3--O-acetyl-16b-hydroxy-12-oxoolean, 3-O-acetyl-16-hydroxy-11-oxoolean-12-ene, and 3--O-acetyl-16-hydroxyolean-12-ene, along with seven known compounds, 3- -hydroxy-1-oxofriedelan, friedelin, 3--friedelanol, canophyllol, 3-oxofriedelan-1(2)-ene, amyrin, camellenodiol, and camelledionol, were isolated. Their structures were established on the basis of spectroscopic analysis and chemical evidence. The isolated compounds were tested *in vitro* for their cytotoxic activities against the A549, LLC, HL-60 and MCF-7 cancer cell lines. Among them, -amyrin exhibited weak cytotoxicity against A549 and HL-60 cancer cell lines with IC (50) values of 46.2 and 38.6 M, respectively (Thao et al., 2010). Another report showed that the methanol extract obtained from soxhlet extraction of leaves of *Ardisia elliptica* Thunberg (Myrsinaceae) contained - and -amyrin, determined by GS-MS. The leaf extract inhibited platelet aggregation with an IC50 value of 167 g/mL, using bioassay guided fractionation. -Amyrin was isolated and purified showing an IC50 value of 4.5 g/mL, while that of aspirin was found to be 11 g/mL, indicating that -amyrin is more potent that aspirin in inhibiting collagen-induced platelet aggregation (Ching et al., 2010). Two triterpenes, -amyrin and 12-oleanene 3, 21-diol, were isolated as a mixture from the chloroform soluble fraction of an ethanolic extract of *Duranta repens* (Verbanaceae) stem. The mixture was highly effective against the larvae of *Culex quinquefasciatus* Say (Diptera: Culicidae) as a mosquitocide. *C*. *quinquefasciatus* is a potential vector of *Wuchereria bancrofti* (Filarioidae), the causative agent of human lymphatic filariasis (Nikkon et al., 2010). One study has examined the potential trypanocidal activity of different plant species growing in the Brazilian Cerrado, after *in vitro* screening of 20 extracts obtained from 10 plants. The phytochemical analysis of the most active extracts (hexane extracts) allowed the identification of -amyrin, -amyrin, lupeol and other triterpenes and sterols. The results showed that pure amyrins are inactive whereas the *n*-hexane leaf extract of *Tibouchina stenocarpa* cogn. Melastomataceae was active. The trypanocidal activity of the extract may be

due to the presence of other compounds (Cunha et al., 2009).


**3. Conclusion** 


The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 499

Holanda Pinto, S. A., Pinto, L. M. S., Cunha, G. M. A., Chaves, M. H., Santos, F. A. & Rao, V.

Jabeen, K., Javaid, A., Ahmad, E. & Athar, M. (2011). Antifungal compounds from *Melia* 

Jalali, H. T., Ebrahimian, Z. J., Evtuguin, D. V. & Neto, C. P. (2011). Chemical composition of

Kuete, V., Ngameni, B., Simo, C. C. F., Tankeu, R. K., Ngadjui, B. T., Meyer, J. J. M., Lall, N.

Lin, K., Huang, A., Tu, H., Lee, L., Wu, C., Hour, T., Yang., S., Pu., Y & Lin, C. (2011).

Mangas-Marín, R., Montes de Oca-Porto, R., Bello-Alarcón, A., Nival Vázquez-Lavín, A.

Marquez-Hernandez, I., Cuesta-Rubio, O., Campo-Fernández, M., Rosado-Perez, A., Montes

*Journal of Agricultural and Food Chemistry*, 58: 4725-4730. ISSN: 1520-5118 Martelanc, M., Vovk, I. & Simonovska, B. (2009) Separation and identification of some

*Alternative Medicine*, 2011: 1-5. ISSN: 1741-4288

*Natural Products Research*, 25(3): 264-276. ISSN: 1029-2349

48-52. ISSN: 0925-4692

ISSN: 0926-6690

ISSN: 0378-8741

ISSN: 1480-3291

7530

329-337. ISSN: 1432-1904

1520-5118

Compounds with antitubercular activity, *Evidence-Based Complementary and* 

S. (2008). Anti-inflammatory effect of α, β-Amyrin, a pentacyclic triterpene from *Protium heptaphyllum* in rat model of acute periodontitis, *Inflammopharmacology*, 16:

*azederach* leaves for management of *Ascochyta rabiei*, the cause of chickpea blight,

oleo-gum-resin from *Ferula gummosa*, *Industrial Crops and Products*, 33: 549-553.

& Kuiate, J. R. (2008). Antimicrobial activity of the crude extracts and compounds from *Ficus chlamydocarpa* and *Ficus cordata*, *Journal of ethnopharmacology*, 120: 17-24.

Xanthine oxidase inhibitory triterpenoid and phloroglucinol from guttiferaceous plants inhibit growth and induced apoptosis in human NTB1 cells through a ROSdependent mechanism, *Journal of Agricultural and Food Chemistry*, 59: 407-414. ISSN:

(2008) Caracterización por cromatografía de Gases/Espectrofotometría de Masas del Extracto Apolar de las hojas de *Clusia minor* L, *Latin American Journal of Pharmacy (formerly Acta Farmaceutica Bonaerense)*, 27(5): 747-51. ISSN 0326-2383 Manguro, L. O. A., Opiyo, S. A., Herdtweck, E. & Lemmen, P. (2009) Triterpenes of

*Commiphora holtziana* oleo-gum resin, *Canadian Journal of Chemistry*, 87: 1173-1179.

de Oca-Porto, R., Piccinelli, A.L. & Rastrelli, L. (2010) Studies on the constituents of yellow Cuban propolis: GC-MS Determination of Triterpenoids and Flavonoids*,* 

common isomeric plant triterpenoids by thin-layer and high-performance liquid chromatography, *Journal of Chromatography A*., 1216: 6662-6670. ISSN: 0021-9673 Martins, A., Vasas, A., Schelz, Z. S., Viveiros, M., Molnár, J., Hohmann, J. & Amaral, L.

(2010).Constituents of *Carpobrotus edulis* Inhibit P-glycoprotein of MDR1 transfected mouse lymphoma cells, *Anticancer Research*, 30: 829-835. ISSN: 1791-

Antibacterial properties of compounds isolated from *Carpobrotus edulis,* 

singles bees (*Tretragonula carbonariua*): gas chromatography-mass spectrometry fingersprint and potential anti-inflammatory properties, *Naturwissenschaften*, 98 (4)

Martins, A., Vasas, A., Viveiros, M., Molnár, J., Hohmann, J. & Amaral, L. (2011).

*International Journal of Antimicrobial Agents*, 37: 438-444. ISSN 0924-8579 Massaro, F. C., Brooks ,P. R., Wallace, H. M. & Russell, F. D. (2011). Cerumen of Australian

collegen-induced platelet aggregation, *Indian Journal of Experimental Biology*, 48: 275- 279. ISSN 0975-1009


Cordeiro, S. Z., Simas, N. K., Arruda, R. C. O. & Sato, A. (2011). Composition of epicuticular

Cunha, W.R., Dos Santos, F. M., Peixoto, J. A., Veneziani, C. S., Crotti, A. E. M., Siva, M. L.

Dias, M. M., Hamerski, L. & Pinto, A. (2011). Separacao semipreparative de e -amyrina

Domingues, R. M. A., Sousa, G. D. A., Freire, C. S. R., Silvestre, A. J. D. & Pascoal-Neto, C.

El-Hagrassi, A. M., Ali, M. M., Osman, A. F. & Shaaban, M. (2011). Phytochemical

Falodun, A., Chaudhry, A. M. A. & Choudhary, I. M. (2009). Phytotoxic and chemical

Fathi-Achachlouei, B. & Azadmard-Damirchi, S. (2009) Milk thistle seed oil constituents

Feng, T., Wang, R., Cai, X., Zheng, T. & Luo, X. (2010). Anti-human immunodeficiency

He, X. & Lui, R. L. (2008). Phytochemicals of apple peels: isolation, structure, elucidation,

Hernandez-Vázquez, L., Mangas, S., Palazón, J. & Navarro-Ocaña, A. (2010) Valuable

Hernandez-Vázquez, L., Bonfil, M., Moyano, E., Cusido, R. M., Navarro-Ocaña, A. &

Higuchi, C. T., Pavan, F. R., Leite, Sannomiya, M., Vilegas, W., Leite, S. R. A., Sacramento, L.

Higuchi, C. T., Sannomiya, F. R., Pavan, F. R., M., Leite, S.R. A., Sato, D. N., Sacramento, L.

*Centella asiatica*, *Biotechnology Letters*, 32(2): 94-104. ISSN: 0141-5492

*Phytotherapia Research*, 24: 1664-1669. ISSN 1099-1573

*Pharmaceutical Biology*, 47(8): 744-749. ISSN 1744-5116

*Research*, 25(2): 141-151. ISSN: 1029-2349

*Bulletin*, 58(7): 971-975. ISSN: 0009-2363

*Chemistry*, 56: 9905-9910. ISSN: ISSN 1520-5118

*Quimica Nova*, 31(7): 1719-1721. ISSN: 1678-7064

*Industrial Crops and Products*, 31: 476-480. ISSN: 0926-6690

279. ISSN 0975-1009

7064

0926-6690

13-17. ISSN: 1819-3471

643-649. ISSN: 003-021X

collegen-induced platelet aggregation, *Indian Journal of Experimental Biology*, 48: 275-

wax layer of two species of *Mandevilla* (Apocynoideae, Apocynaceae) from Rio de Janeiro, Brazil, *Biochemical Systematics and Ecology*, 39: 198-202. ISSN 0305-1978 Csapi, B., Zsuzsanna, H., Zupkó, I., Berényi, A., Forgo, P., Szabó, P & Hohmann, J. (2010).

Bioactivity-guided isolation of antiproliferative compounds from *Centaurea arenaria*,

A., Filho, A. A. S., Albuquerque, S., Turatti, I. C. C & Bastos, J. K. (2009). Screeing of plantas extracts from the Brazilian Cerrado for their *in vitro* trypanocidal activity,

por cromatografia líquida de alta eficiencia, *Qumica Nova*, 34(4): 704-706. ISSN 1678-

(2010). *Eucalyptus globulus* biomass residues from pulping industry as a source of high value triterpenic compounds, *Industrial Crops and Product*, 31: 65-70. ISSN:

investigation and biological studies of *Bombax malabaricum* flowers, *Natural Products* 

investigations of a Nigerian medicinal plant, *Research Journal of Phytochemistry*, 3(1):

from different varieties grown in Iran, *Journal of American Oil Chemical Society*, 86:

virus-1 constituents of the Bark of *Poncirus trifoliata*, *Chemical Pharmaceutical* 

and their antiproliferative and antioxidant activities, *Journal of Agricultural and Food* 

medicinal plants and resins: Comercial phytochemicals whith bioactive properties,

Palazón, J. (2010), Conversion of -amyrininto centellosides by plant cell cultures of

V. & Sato, D. N. (2008). Triterpenes and antitubercular activity of *Byrsonima crassa,* 

V. S. Vilegas, W. & Leite, C. Q. F. (2008). *Byrsonima fagifolia* Niedenzu Apolar

Compounds with antitubercular activity, *Evidence-Based Complementary and Alternative Medicine*, 2011: 1-5. ISSN: 1741-4288


The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 501

Rhourri-Frih, B., Chaimbault, P., Claude,B., Lamy, C., André, P. & Lafosse, M. (2008).

Rivero-Cruz, F., Sánchez-Nieto, S., Benítez, G., Casimiro, X., Ibarra-Alvarado, C., Rojas-

Rosas-Acevedo, H., Terrazas, T., González-Trujano M. E., Guzmán, Y. & Soto-Hernández,

Silva, A. M., Simeoni, L. A. & Silveira, D. (2009) Genus Pouteria: Chemistry and biological activity, *Brazilian Journal of Pharmacognosy*, 19(2A): 501-509. ISSN: 0102-695X Silva, J. R. A, Zoghbi, M. G. B., Pinto. A., Godoy, R. L. O. and Amaral, A.C. F. (2009)

Simo, C. C. F., Kouam, S. F., Poumale, H. P., Simo, I. K., Ngadjui, B. T., Green, I. R. & Krohn,

Sirat, H. M., Susanti, D., Ahmad, F., Takayama, H. & Kitajima, M. (2010). Amides, triterpene

Sob, S. V. T., K. Wabbo, H. K., Tchinda, A. T., Tane, P., Ngadju, B. T. & Ye, Y. (2010)

Szewczyk, K., Komsta, L. & Skalska-Kaminska, A. (2009) Densitometric HPTLC method for

Thao, N. T. P., Hung, T. M., Lee, M. K., Kim, J. C., Min, B. S. & Bae, K. (2010). Triterpenoids

Tlili, N., El Guizani, T., Nasri, N., Khaldi, A. & Triki, S. (2011). Protein, lipid, aliphatic and

Uchida, H., Ohyama, K., Suzuki, M., Yamashita, H., Muranaka, T. & Ohyama, K. (2010).

Van Maarseveen, C. & Jetter, R. (2009). Composition of the epicuticular and intracuticular

Vitor, C. E., Figueiredo, C. P., Hara, D. B., Bento, A. F., Mazzuco, T. L & Calixto, J. B. (2009)

*Biochemical Systematics and Ecology*, 38:342-345. ISSN: 0305-1978

and APPI, *Journal of Mass Spectrometry*, 44: 71-80. ISSN: 1387-3806

*Journal of Ethnopharmacology*, 134: 67-73. ISSN: 0378-8741

*Essential Oil Research*, 21(4): 305-308. ISSN: 1041-2905

*Medicine*, 64: 492-495. ISSN 1861-0293

58(1): 121- 124. ISSN: 0009-2363

*Chemical Society* 88: 265-270. ISSN: 0003-021X

*Biotechnology*, 27: 105-109. ISSN: 1342-4580

*Phytochemistry*, 70: 899-906. ISSN: 0031-9422

*of Pharmacology*, 157: 1034-1044. ISSN: 1476-5381

5943

0305-1978

4173

Analysis of pentacyclic triterpenes by LC-M. A comparative study between APCL

Molina, A. & Rivero-Cruz, B. (2009). Antibacterial compounds isolated from *Byrsonima crassifolia*, *Revista Latinoamericana de Química*, 37(2): 155-163. ISSN: 0370-

M. (2011) Anti-ulcer activity of *Cyrtocarpa procera* analogous to that of *Amphipterygium adstringens*, both assayed on the experimental gastric injury in rats,

Analysis of the hexane extracts from seven oleoresins of *Protium* species, *Journal of* 

K. (2008) Benjaminamide: A new ceramide and other compounds from the twigs of *Ficus benjamina* (Moraceae), *Biochemical Systematics and Ecology,* 36: 238-243. ISSN:

and flavonoids from the leaves of *Melastoma malabathricum* L., *Journal of Natural* 

Anthraquinones, sterols, triterpeniods and xanthones from *Cassia obtusifolia*,

analysis of triterpenoids in the leaves of *Jovibarba sobolifera* (Sims.) Opiz (Hen and Chickens houseleek), *Journal of Planar Chromatography*, 22(85): 367-369. ISSN: 0993-

from *Camellia japonica* and Their Cytotoxic Activity*, Chemical Pharmaceutical Bulletin*,

triterpenic alcohol content of caper seeds "*Capparis spinosa", Journal American Oil* 

Triterpenoid levels are reduced during Euphorbia *tirucalli* L. callus formation, *Plant* 

wax layers on *Kalanchoe daigremontiana* (Hamet et Perr. de la Bathie) leaves,

Therapeutic action and underlying mechanisms of a combination of two pentacyclic triterpenes, α- and β-amyrin, in a mouse model of colitis, *British Journal* 


Mbouangouere, R. N., Tane, P., Choudhary, M- P., Djemgou, P. C., Ngadjui, B. T. &

Moreau, R.A., Lampi, A. M. & Hicks, K.B. (2009) Fatty Acid, Phytosterol and Polyamine

Nikkon, F., Salam, K. A., Yeasmin, T., Mosaddik, A., Khondkar P. & Haque, M. E. (2010).

Nurmi, T., Nyström, L., Edelmann, M., Lampi, A. & Pironen, V. (2008). Phtosterols in wheat

Oliveira, A. P., Silva, L. R., Andrade, P. B., Valentão, P., Silva, B. M., Goncalves, R, F.,

Palíková, I., Heinrich, J., Bednár, P., Marhol, P., Kren, V., Cvak, L., Valentová, C., Ruzicka,

Parveen, M., Ghalib, R. M., Mehdi, S. H., Mattu, R. U. H. & Ali, M. (2009). A novel

Parveen, M., Khanam, Z., Ali, M. & Rahman, S.Z. (2010). A novel lupene-type triterpenic

Parveena, M., Basudan, O. A., Mushfiq, M. & Ghalib, R. M. (2008). A new benzofuranic acid

Pedernera, A. M., Guardia, T., Calderón, C. E. G., Rotelli, A. E., de la Rocha, N. E., Saad, J. R.,

Ragasa, C. Y-, Lapina, M. C., Lee, J. J., Mandia, E. H. & Rideout, J. A. (2008). Secondary

Ramadan, M., Ahmad, A. S., Nafady, A. M. & Mansour, A. I. (2009). Chemical composition

*Journal of Saudi Chemical Society*, 13: 287-290. ISSN: 1319-6103

*africana*, *Research Journal of Phytochemistry*, 2(1): 27-34. ISSN: 1819-3471 Melo, C. M., Carvalho, K. M. M. B., Neves, J. C. S., Morais, T. C., Rao, V. S., Santos, F. A.,

*Gastroenterology*, 16 (34): 4272-4280. ISSN: 1007-9327

48(3): 264-268. ISSN: 1744-5116

167-176. ISSN: 1029-2349

*Chemistry*, 56: 9710-9715. ISSN: 1520-5118

021X

1520-5118

5118

6419

0925-4692

ISSN: 1478-6419

23(13): 1218-1230. ISSN: 1029-2349

Ngamga, D. (2008). Pipthadenol A-C and -glucosidase inhibitor from *Piptadenia* 

Brito, G. A. B. & Chaves, M. H. (2010). α,β-amyrin, a natural triterpenoid ameliorates L-arginne-induced acute pancreatitis in rats, *World Journal of* 

Conjugates Profiles of Edible Oils Extracted from Corn Germn, Corn Fiber, and Corn Kernels. *Journal of American Oil Chemical Society*, 86(12): 1209-1214. ISSN: 003-

Mosquitocidal triterpenes from the stem of *Duranta repens*, *Pharmaceutical Biology*,

genotypes in the HEALTHGRAIN diversity screen, *Journal of Agricultural and Food* 

Pereira, J. A. & Pinho, P. G., (2010) Further Insight into the látex metabolite profile of *Ficus carica*, *Journal of Agricultural and Food Chemistry*, 58: 10855-10863. ISSN:

F., Holá, V., Kolár, M., Simánek, V. & Ulrichová, J. (2008). Constituents and antimicrobial properties of blue honeysucke: A novel source for phenolic antioxidants, *Journal of Agricultural and Food Chemistry*, 56: 11883-11889. ISSN: 1520-

antimicrobial triterpenic acid from the leaves of *Ficus benjamina* (var. *comosa*),

glucoside from the leaves of *Clerodendrum inerme*, *Natural Product Research*, 24(2):

from the leaves of *Rhus alata*, *Natural Product Research*, 22(5): 371-382. ISSN: 1478-

López Verrilli, M. A., Aseff, S. G. & Pelzer, L.E. (2010). Anti-inflammatory effect of *Acacia visco* extracts in animal models, *Inflammopharmacology*, 18: 253-260. ISSN:

metabolites from *Tectona philippinensis*, *Natural Product Research*, 22(9): 820-824.

of the stem bark and leaves of *Ficus pandurata* Hance, *Natural Product Research*,


**24** 

**Phytochemical Studies of Fractions** 

**Patens with Antifungal Bioactivity** 

Patricia Isabel Manzano Santana1, Mario Silva Osorio2,

*2Laboratorio de Productos Naturales de la Facultad de Ciencias Naturales y Oceanográficas de la Universidad de Concepción,* 

*Centro de Investigaciones Biotecnológicas del Ecuador, CIBE-ESPOL,* 

**and Potential as Antineoplastic** 

Olov Sterner3 and Esther Lilia Peralta Garcìa1

*3Centre for Analysis and Synthesis, Lund University,* 

*1Laboratorio Bioproductos,* 

*1Ecuador 2Chile 3Sweden* 

**and Compounds Present in Vernonanthura** 

Phytochemical research is closely related to the needs of finding new and effective pharmaceuticals. Searching for plant substances that are capable forbeing used to develop new therapeutic drugs against catastrophic recognized illnesses such as cancer, diabetes and AIDS is one of the main topics that researchers around the world have been focusing.

The wonderful plant diversity of South America and more specifically from the Amazon region has around 30-50% of the worlds biodiversity therafor it is an important source for this type of study. Beside the significant undiscovered resources from these regions, ancestral knowledge of indigenous peoples is another relevant and complementary source for biodiscovery programs. Traditional healers guard centuries of accumulated knowledge about natural medicinal resources of this region. These ancient "physicians" hold the key to discovering new drugs that could benefit millions of people around the world. The Amazon forest has contributed dozens of substances to western medicine. Among the best known are the "curare"; a key component of modern anesthetics and quinine, the first contribution of

Study of new plant species and the structural elucidation of its bioactive molecules are the most important aims of phytochemical research which is in constant technological development.

1 Fundación Icaro. La medicina tradicional de los pueblos indígenas amazónicos: Descubriendo la

icaros.org/index.php/component/content/article/8-descubriendo-la-amazonia-europea

Amazonía europea. Disponible en el sitio: http://www.fundacion-

**1. Introduction** 

"natural medicine" to treat malaria1.


## **Phytochemical Studies of Fractions and Compounds Present in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic**

Patricia Isabel Manzano Santana1, Mario Silva Osorio2, Olov Sterner3 and Esther Lilia Peralta Garcìa1 *1Laboratorio Bioproductos, Centro de Investigaciones Biotecnológicas del Ecuador, CIBE-ESPOL, 2Laboratorio de Productos Naturales de la Facultad de Ciencias Naturales y Oceanográficas de la Universidad de Concepción, 3Centre for Analysis and Synthesis, Lund University, 1Ecuador 2Chile 3Sweden* 

#### **1. Introduction**

502 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Vouffo, B., Dongo, E., Facey, P., Thorn, A., Sheldrick, G., Maier, A., Fiebig, H. & Laatsch, H.

Wang, F., Li, Z., Cui, H., Hua, H., Jing, Y. & Liang, M. (2011). Two new triterpenoids from

Wang, X., Li, C., Shi, Y. & Di, D. (2009). Two new secoiridoid glycosides from the leaves of

Wang, Y., Chen, W., Wu, Z., Xi, Z., Chen, W., Zhao, G., Li, X. & Sun, L. (2011). Chemical

Wansi, J. P., Chiozem, D. D., Tcho, A. T., Toze, F. A. A., Devkota, K. P., Ndjakou, B. L.,

Wesolowska, A., Jadczak, D. & Grzeszczuk, M. (2011). GC-MS Analysis of lemon catnip

Wu, C., Hseu, Y., Lien, J., Lin, L., Lin, Y. & Ching, H. (2011). Triterpenoid contents and anti-

Xu, C., Qin, M., Fu, Y., Liu, N., Hemming, J., Holmbom, B. & Willförd, S. (2010). Lipophilic

Xu, X., Dong, J., Mu, X. & Sun, L. (2011). Supercritical CO2 extraction of oil, carotenoids,

Zarrouk, W., Carrasco-Pancorbo, A., Segura-Carretero, A., Fernandez-Gutierrez, A. &

*Wood Chemistry and Technology*, 30: 105-117. ISSN: 1532-2319

*Bioproducts Processing*, 89(1): 47-52. ISSN: 0960-3085

1717-1723. ISSN: 0032-0943

197. ISSN: 1477-2213

1477-2213

ISSN: 0305-1978

ISSN: 1744-5116

ISSN: 1029-2349

169-180. ISSN: 0231-2522

*Molecules*, 16: 1-15. ISSN: 1420-3049

(2010). Antiarol cinnamate and africanoside, a cinnamoyl triterpene and a hydroperoxy-cardenolide from the stem bark of *Antiaris africana*, *Planta Medica*, 76:

the resin of *Boswellia carterii*, *Journal of Asian Natural Products Research*, 13(3): 193-

*Olea europaea* L., *Journal of Asian Natural Products Research*, 11(11): 940-944. ISSN:

consituents from *Salacia amplifolia*, *Biochemical Systematics and Ecology*, 39: 205-208.

Wandji, J. & Sewald, N. (2010). Antimicrobial and antioxidante effects of phenolic constituents from *Klainedoxa gabonensis*, *Pharmaceutical Biology*, 48(10): 1124-1129.

(*Nepeta cataria* L. var. *citriodora* Balbis) essential oil, *Acta Chromatographica*, 23(1):

Inflammatory properties of the methanol extracts of *Ligustrum* species leaves,

extractives in *Populos x euramericana* "Guariento" stemwood and bark, *Journal of* 

squalene and sterols from lotus (*Nelumbo nucifera Gaertn*) bee pollen, *Food and* 

Zarrouk, M. (2010) Exploratory characterization of the unsaponifiable fraction of Tunisdian Virgin Olive Oils by a global approach with HPLC-APCL MS/MS analysis, *Journal of Agricultural and Food Chemistry*, 58: 6418-6426. ISSN: 1520-5118 Zheng, Y., Huang, W., Yoo, J., Ebersole, J. L. & Huang, C. B. (2011). Antibacterial

compounds from *Siraitia grasvenorii* leaves, *Natural Products Research*, 25(9): 890-897.

Phytochemical research is closely related to the needs of finding new and effective pharmaceuticals. Searching for plant substances that are capable forbeing used to develop new therapeutic drugs against catastrophic recognized illnesses such as cancer, diabetes and AIDS is one of the main topics that researchers around the world have been focusing.

The wonderful plant diversity of South America and more specifically from the Amazon region has around 30-50% of the worlds biodiversity therafor it is an important source for this type of study. Beside the significant undiscovered resources from these regions, ancestral knowledge of indigenous peoples is another relevant and complementary source for biodiscovery programs. Traditional healers guard centuries of accumulated knowledge about natural medicinal resources of this region. These ancient "physicians" hold the key to discovering new drugs that could benefit millions of people around the world. The Amazon forest has contributed dozens of substances to western medicine. Among the best known are the "curare"; a key component of modern anesthetics and quinine, the first contribution of "natural medicine" to treat malaria1.

Study of new plant species and the structural elucidation of its bioactive molecules are the most important aims of phytochemical research which is in constant technological development.

<sup>1</sup> Fundación Icaro. La medicina tradicional de los pueblos indígenas amazónicos: Descubriendo la Amazonía europea. Disponible en el sitio: http://www.fundacion-

icaros.org/index.php/component/content/article/8-descubriendo-la-amazonia-europea

Phytochemical Studies of Fractions and Compounds Present

Genus *Vernonanthura* 

Number 415138

Quinindé, Bilsa, Viche, Esmeraldas, Muisne and Salina Prov. Esmeralda

**2.1.3 Geographical distribution** 

Table 1. List of vernacular names assigned to *V. patens* 

*V. micradenia DC. V. monsonensis Hieron V. pacchensis Benth V. salamana Gleason V. suaveolens Kunt V. treberbaneri Hieron* 

**2.1.1 Taxonomy** 

**2.1.2 Vernacular names** 

countries it is grown.

Colombia

Ecuador

Garden2.

in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic 505

Taxon *Vernonanthura patens* (Kunth) H. Rob

Synonyms *Vernonia patens* Kunth (basionym)

Verified name 02-Jun-2008 by systematic botanists of ARS.

Table 1 presents a list of vernacular names that are assigned to *V. patens* according to the

**Country/Location Vernacular name References** 

Valle del Cauca Pebetero Terreros *et al*, proyecto

Tulua, Valle del Cauca- Yasmiande, varejón Blair, 2005

Costa Rica Cusuco Chavarría *et al*., 1998

Chilca

El Salvador Chalatenango Sukunang PROMABOS a, 2006 Guatemala Xuqunán Xuquinái PROMABOS, 2006 Panamá Salvia blanca, Sanalego Diéguez et al, 2006

*V. patens* is native from America and can be found in Belize, Costa Rica, Brazil, Venezuela, Panama, Bolivia, Mexico and Ecuador according to the data reported by Missouri Botanical

2 Tropicos.org. Missouri Botanical Garden. 23 Jun 2011.http://www.tropicos.org/Name/2740044.

Prov. Loja and El Oro Laritaco Tobías, 1996 Prov. Guayas Chilco Blanco León, 2006

Last update: 02-Jun-2008 (ARS-GRIN, 2009)

Tuete, tuete blanco Rodríguez, 2005

ECOFONDO-ACDI 2004-2009

REMACH, 2004

Family *Asteraceae* (alt. Compositae)

Place of publication Phytologia 73:72, 1992

Initial phytochemical screening and further isolation, purification and identification of molecules structure have made a major breakthrough with the development of new methods of chromatography and spectroscopy. The establishment of new and more effective bioassays is also one of the essential aspects that support biodiscovery programs today.

This chapter contains the main results on the phytochemical study of *Vernonanthura patens* leaves which according to ancestral knowledge, have been used to treat different diseases in humans.

#### **2. Botanical classification, general characteristics and ethnobotanical knowledge on** *Vernonanthura patens*

*Vernonanthura patens* is a wild plant broadly distributed throughout America. It grows from 0 to 2200 meters above sea level in the Ecuadorian coastal region. Folk medicine uses its leaves cooked to combat malaria, postpartum treatment and for healing infected wounds of animals by washing with a plant mixture which includes *V. patens* leaves (Blair, 2005).

It is also used against headaches, to clean and heal wounds (Kvist *et al.,* 2006); treatment of leishmaniasis (Gachet *et al*., 2010); preparation of antivenom (Tene *et al*., 2007) and as a poultice of leaves to combat athlete's foot (Valadeau *et al*., 2009). Its usefulness for treating certain types of cancer has also been referred by indigenous healers. There is however there are few chemical studies about this species

#### **2.1** *Vernonanthura patens* **(Kunth) H. Rob. botanical classification and general characteristics**

Species *V. patens* belongs to the *Asteraceae* family, quoting 60 synonyms and one basionym (*Vernonia patens* Kunth) (ARS-GRIN, 2009). Referred to *Vernonia patens* HBK in the list of lignocellulose species investigated in Ecuador, it is a source of raw material for pulping and papermaking (Acuña, 2000). It is also commercially important in the beekeeping industry, and is ranked as one of the most important honeybee plants from Tundo, Olmedo and Loja (Camacho, 2001) for its excellent production and availability of nectar and pollen (Ramirez *et al*., 2001).

In the Ecuadorian province of Zamora it is one of four ecologically important species belonging to the typical families of disturbed forests that are been regenerated (Camacho, 2001; REMACH, 2004). It is now registered as representative tree species of secondary forests in Ecuadorian coastal zone (Aguirre, 2001).

The species has the following synonyms (Blair, 2005):

*Cacalia patens (Kunth), Kuntze C. aschenborniana (Schauer) Kuntze C. baccharoides (Kunth) Kuntze C. haenkeana (DC.) Kuntze C. lanceolaris (DC.) Kuntze C. suaveolens (H.B.K.) Kuntze Vernonanthura patens (Kunth) H. Rob Vernonia ascherbotniana Schauer V. lanceolaris D.C.* 

Initial phytochemical screening and further isolation, purification and identification of molecules structure have made a major breakthrough with the development of new methods of chromatography and spectroscopy. The establishment of new and more effective bioassays is also one of the essential aspects that support biodiscovery programs today.

This chapter contains the main results on the phytochemical study of *Vernonanthura patens* leaves which according to ancestral knowledge, have been used to treat different diseases in

*Vernonanthura patens* is a wild plant broadly distributed throughout America. It grows from 0 to 2200 meters above sea level in the Ecuadorian coastal region. Folk medicine uses its leaves cooked to combat malaria, postpartum treatment and for healing infected wounds of animals by washing with a plant mixture which includes *V. patens* leaves (Blair, 2005).

It is also used against headaches, to clean and heal wounds (Kvist *et al.,* 2006); treatment of leishmaniasis (Gachet *et al*., 2010); preparation of antivenom (Tene *et al*., 2007) and as a poultice of leaves to combat athlete's foot (Valadeau *et al*., 2009). Its usefulness for treating certain types of cancer has also been referred by indigenous healers. There is however there

Species *V. patens* belongs to the *Asteraceae* family, quoting 60 synonyms and one basionym (*Vernonia patens* Kunth) (ARS-GRIN, 2009). Referred to *Vernonia patens* HBK in the list of lignocellulose species investigated in Ecuador, it is a source of raw material for pulping and papermaking (Acuña, 2000). It is also commercially important in the beekeeping industry, and is ranked as one of the most important honeybee plants from Tundo, Olmedo and Loja (Camacho, 2001) for its excellent production and availability of nectar and pollen (Ramirez

In the Ecuadorian province of Zamora it is one of four ecologically important species belonging to the typical families of disturbed forests that are been regenerated (Camacho, 2001; REMACH, 2004). It is now registered as representative tree species of secondary

**2. Botanical classification, general characteristics and ethnobotanical** 

**2.1** *Vernonanthura patens* **(Kunth) H. Rob. botanical classification and general** 

humans.

**characteristics** 

*et al*., 2001).

**knowledge on** *Vernonanthura patens* 

are few chemical studies about this species

forests in Ecuadorian coastal zone (Aguirre, 2001). The species has the following synonyms (Blair, 2005):

*Cacalia patens (Kunth), Kuntze C. aschenborniana (Schauer) Kuntze C. baccharoides (Kunth) Kuntze C. haenkeana (DC.) Kuntze C. lanceolaris (DC.) Kuntze C. suaveolens (H.B.K.) Kuntze Vernonanthura patens (Kunth) H. Rob Vernonia ascherbotniana Schauer* 

*V. lanceolaris D.C.* 


#### **2.1.1 Taxonomy**


#### **2.1.2 Vernacular names**

Table 1 presents a list of vernacular names that are assigned to *V. patens* according to the countries it is grown.


Table 1. List of vernacular names assigned to *V. patens* 

#### **2.1.3 Geographical distribution**

*V. patens* is native from America and can be found in Belize, Costa Rica, Brazil, Venezuela, Panama, Bolivia, Mexico and Ecuador according to the data reported by Missouri Botanical Garden2.

<sup>2</sup> Tropicos.org. Missouri Botanical Garden. 23 Jun 2011.http://www.tropicos.org/Name/2740044.

Phytochemical Studies of Fractions and Compounds Present

labor and to purge (Blair, 2005).

**3. Phytochemical screening** 

(+) or absence (-) of the color reactions.

certain Asian countries.

2007).

2001).

**2.3 Biological and chemical activity** 

poultices to combat athlete's foot is referred by Valadeau *et al.*, (2009).

in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic 507

Gacheta *et al*., (2010) informed its usefulness for leishmanianis treatment; Tene *et al* (2007) indicating its use in the preparation of antivenon and the use of "laritaco" leaves in

Different uses of *V. patens* have been registered in other South American countries. In the Bolivian community of Tacama, the juice of the plant stem is applied against conjunctivitis (Tacana, 1999) and in Colombia the watery brews of the aerial parts mixed with "panela"4, white wine and rosemary are used against malaria. It is also used to relieve pain due to

There are very few biological and chemical studies of the specie *V. patens*. The only results published so far refer to the antimalarial activity against *Plasmodium falciparum*, Itg2 strain (Blair, 2005) ,anti-*Leishmania* activity (Valadeau *et al.,* 2009) of the leaves of this species and no antiprotozoal activity against different strains of Leishmania (Fournet, 1994). On the chemical composition of the species, reports lack of sesquiterpene lactones and sesquiterpenes present in the aerial parts (Mabry, 1975; Jakupovic, 1986). There are some references on genus *Vernonanthura* that show the presence of diterpenes compounds (Portillo *et al.,* 2005; Valadeau *et al.,* 2009), flavonoids (Borkosky *et al*., 2009; Mendonça *et al.,* 2009), triterpenes (Tolstikova *et al.,* 2006, Gallo *et al.,* 2009), saponins (Borkosky *et al.,* 2009) and sesquiterpene lactones. In addition, different biological activities have been described assuming that certain chemical groups could be responsible for the therapeutic properties attributed to species of this genus (Pollora *et al.,* 2003, 2004; Portillo *et al.,* 2005; Bardon *et al.,*

These were the main factors that led to the Laboratorio Bioproductos Centro de Investigaciones Biotecnológicas del Ecuador to undertake a chemical-pharmacological study of *Vernonanthura patens* leaves from plants growing in Ecuadorian areas. Such investigations

As an initial step of thephytochemical screening research allows to determine qualitatively the main groups of chemical constituents present in a plant. This screening can guide the subsequent extraction and / or fractionation of extracts for the isolation of groups of interest. The phytochemical screening routine is performed by extraction with suitable solvents of increasing polarity and the application of color reactions (Miranda & Cuellar,

These reactions are characterized by their selectivity to types or groups of compounds, their simplicity, short time consuming and capacity to detect small amount of compounds using a minimum requirement of laboratory equipment. The results are recorded by the presence

4 "Panela" is a unrefined sugarcane product obtained from the boiling and evaporation of sugarcane juice. It contains sucrose and fructose and is a typical product of Latin America, but can be finding in

are part of the Biodiscovery Program developed by this center.

#### **2.1.4 Habitat**

*V.patens* grows wild in the inter-Andean forest located in the south of Ecuador; its maximum height is 3-6 meters and its altitudinal distribution is between 0 and 2000 meters above sea level (Tobías, 1996; León, 2006). This species has been identified in the vegetal community of dry forests at the south-west of Ecuador3.

This species is sometimes grown or kept in farms after its spontaneous appearance. Generally it can be found near the forest trail and on the edge of the rivers. Flowering and fruiting occurs between May and October.

#### **2.1.5 Botanical information**

*V. patens* (Figure 1), is a small branched shrub, growins up to six meters high with furrowed stems and ferruginous trichomes. Alternate leaves are petiolate, narrowly lanceolate, petiole tomentose with ferruginous trichomes, 4-11mm long; the leaves are entirely or weakly serrate, rounded base with a sharp or acuminate apex leaves are 7-15 cm long and 1.3 - 1.2 cm wide, the adaxial surface is bright and the abaxial is pubescent or puberulent, subcoriaceous, penninerved. Inflorescence is paniculate, terminal, extended branched with the endings scorpioid, provided with leaves and bracts, capitates sessile and very shortly pedicellate, with numerous bell-shaped flowers, 8 mm long, 4-5 sets bracts imbricated, tomentose and of dark brown color, corolla glabrous, about 5 mm long, weakly pubescent achenes, pappus hairs-layered irregular shaped edges that are about 7 mm long. A detailed description of the botanical characteristics of this species has been published by Blair (2005).

Fig. 1. *Vernonanthura patens* (laritaco). It grows wild in different Ecuadorian areas belonging to the provinces of Loja, El Oro, Guayas, Manabí and Los Ríos**.** 

#### **2.2 Ethnomedical information**

In Ecuador the inhabitants of the south-west of Loja and the Marcabelí region of El Oro province recognize both its healing power and analgesic action. They use the leaves of *V. patens* to wash wounds and to relieve headaches. It is also employed as anti-inflammatory to soothe coughs and against certain types of cancers. In addition, a veterinary practice is described as it can heal infected wounds by washing with a mixture of plants that includes leaves from this species (Blair, 2005)**.** Other interesting uses have been also reported.

 3 http://www.darwinnet.org/index.php?option=com\_content&view=article&id=153%3Aarticuloscientificos-y-reportes-&catid=25%3Acontenido&Itemid=1

Gacheta *et al*., (2010) informed its usefulness for leishmanianis treatment; Tene *et al* (2007) indicating its use in the preparation of antivenon and the use of "laritaco" leaves in poultices to combat athlete's foot is referred by Valadeau *et al.*, (2009).

Different uses of *V. patens* have been registered in other South American countries. In the Bolivian community of Tacama, the juice of the plant stem is applied against conjunctivitis (Tacana, 1999) and in Colombia the watery brews of the aerial parts mixed with "panela"4, white wine and rosemary are used against malaria. It is also used to relieve pain due to labor and to purge (Blair, 2005).

#### **2.3 Biological and chemical activity**

506 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

*V.patens* grows wild in the inter-Andean forest located in the south of Ecuador; its maximum height is 3-6 meters and its altitudinal distribution is between 0 and 2000 meters above sea level (Tobías, 1996; León, 2006). This species has been identified in the vegetal community of

This species is sometimes grown or kept in farms after its spontaneous appearance. Generally it can be found near the forest trail and on the edge of the rivers. Flowering and

*V. patens* (Figure 1), is a small branched shrub, growins up to six meters high with furrowed stems and ferruginous trichomes. Alternate leaves are petiolate, narrowly lanceolate, petiole tomentose with ferruginous trichomes, 4-11mm long; the leaves are entirely or weakly serrate, rounded base with a sharp or acuminate apex leaves are 7-15 cm long and 1.3 - 1.2 cm wide, the adaxial surface is bright and the abaxial is pubescent or puberulent, subcoriaceous, penninerved. Inflorescence is paniculate, terminal, extended branched with the endings scorpioid, provided with leaves and bracts, capitates sessile and very shortly pedicellate, with numerous bell-shaped flowers, 8 mm long, 4-5 sets bracts imbricated, tomentose and of dark brown color, corolla glabrous, about 5 mm long, weakly pubescent achenes, pappus hairs-layered irregular shaped edges that are about 7 mm long. A detailed description of the botanical characteristics of this species has been published by Blair (2005).

Fig. 1. *Vernonanthura patens* (laritaco). It grows wild in different Ecuadorian areas belonging

In Ecuador the inhabitants of the south-west of Loja and the Marcabelí region of El Oro province recognize both its healing power and analgesic action. They use the leaves of *V. patens* to wash wounds and to relieve headaches. It is also employed as anti-inflammatory to soothe coughs and against certain types of cancers. In addition, a veterinary practice is described as it can heal infected wounds by washing with a mixture of plants that includes leaves from this species (Blair, 2005)**.** Other interesting uses have been also reported.

3 http://www.darwinnet.org/index.php?option=com\_content&view=article&id=153%3Aarticulos-

to the provinces of Loja, El Oro, Guayas, Manabí and Los Ríos**.** 

cientificos-y-reportes-&catid=25%3Acontenido&Itemid=1

**2.1.4 Habitat** 

dry forests at the south-west of Ecuador3.

fruiting occurs between May and October.

**2.1.5 Botanical information** 

**2.2 Ethnomedical information** 

There are very few biological and chemical studies of the specie *V. patens*. The only results published so far refer to the antimalarial activity against *Plasmodium falciparum*, Itg2 strain (Blair, 2005) ,anti-*Leishmania* activity (Valadeau *et al.,* 2009) of the leaves of this species and no antiprotozoal activity against different strains of Leishmania (Fournet, 1994). On the chemical composition of the species, reports lack of sesquiterpene lactones and sesquiterpenes present in the aerial parts (Mabry, 1975; Jakupovic, 1986). There are some references on genus *Vernonanthura* that show the presence of diterpenes compounds (Portillo *et al.,* 2005; Valadeau *et al.,* 2009), flavonoids (Borkosky *et al*., 2009; Mendonça *et al.,* 2009), triterpenes (Tolstikova *et al.,* 2006, Gallo *et al.,* 2009), saponins (Borkosky *et al.,* 2009) and sesquiterpene lactones. In addition, different biological activities have been described assuming that certain chemical groups could be responsible for the therapeutic properties attributed to species of this genus (Pollora *et al.,* 2003, 2004; Portillo *et al.,* 2005; Bardon *et al.,* 2007).

These were the main factors that led to the Laboratorio Bioproductos Centro de Investigaciones Biotecnológicas del Ecuador to undertake a chemical-pharmacological study of *Vernonanthura patens* leaves from plants growing in Ecuadorian areas. Such investigations are part of the Biodiscovery Program developed by this center.

#### **3. Phytochemical screening**

As an initial step of thephytochemical screening research allows to determine qualitatively the main groups of chemical constituents present in a plant. This screening can guide the subsequent extraction and / or fractionation of extracts for the isolation of groups of interest. The phytochemical screening routine is performed by extraction with suitable solvents of increasing polarity and the application of color reactions (Miranda & Cuellar, 2001).

These reactions are characterized by their selectivity to types or groups of compounds, their simplicity, short time consuming and capacity to detect small amount of compounds using a minimum requirement of laboratory equipment. The results are recorded by the presence (+) or absence (-) of the color reactions.

<sup>4 &</sup>quot;Panela" is a unrefined sugarcane product obtained from the boiling and evaporation of sugarcane juice. It contains sucrose and fructose and is a typical product of Latin America, but can be finding in certain Asian countries.

Phytochemical Studies of Fractions and Compounds Present

using solvents of different polarity.

2010).

kept in polyethylene bags of low density at 24 C.

**4. Plant extracts, fractions and compounds** 

in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic 509

Fig. 3. Chemical reactions carried out in each type of V*. patens'* leaf extracts obtained from

consent was obtained and authorized by the corresponding agencies of the government. The fieldwork and data collection were conducted in accordance with the institutional, national and international principles and guidelines for using and conserving plant biodiversity.

For conducting the phytochemical screening, extraction and fractionation, leaves samples were dried using an automatic dryer (45 °C, 8 hours) and then pulverized in a blender and screened. The fraction that remained in the sieve of 2 mm in diameter was collected and

The result of phytochemical screening is presented in Table 2. This reveals moderate to low concentration of essentials oils, alkaloids, reducing compounds, phenols, tannins, flavonoids, quinones, saponins, triterpenes and steroids. Some of these chemical compounds have been associated to antibacterial, antifungal, antiprotozoal and citotoxicity properties and thus have a potential therapeutic use (Nweze *et al.,* 2004; Reuben *et al.,* 2008; Vital *et al*.,

The dry plant material (67 g of leaves of *V. patens*) was subjected to successive extractions with HPLC grade methanol by maceration in a closed container and in the absence of light. The extraction time was eight days and was conducted until total depletion of plant

material; agitator and a rotary evaporator were used for solvent recovery.

The general outline of steps followed for performing the phytochemical screening of *V. patens'* leaves is presented in Figure 2, while the analysis of the extracts obtained at different polarities is schematically shown in Figure 3. This methodology has been referred previously (Miranda & Cuellar, 2000; Manzano *et al.*, 2009).

Fig. 2. General procedure used for performing the phytochemical screening of *V. patens* leaves.

The plant material of adult leaves of *Vernonanthura patens* (laritaco) were used from plants at the vegetative state which were growing in the citadels "July 25", "Imbabura" and "June 24" and all belonget to the Canton Marcabelí, province El Oro, Ecuador. Leaves were collected at early morning at different dates during the months of December to February in 2009 and 2010.

Botanical identification was performed and voucher specimens of the herbs were prepared and deposited at the National Herbarium of Ecuador (QCNE) and a duplicated sample (CIBE37) was kept as herbal witness in the laboratory of the CIBE-ESPOL Bioproducts. Prior

The general outline of steps followed for performing the phytochemical screening of *V. patens'* leaves is presented in Figure 2, while the analysis of the extracts obtained at different polarities is schematically shown in Figure 3. This methodology has been referred

Fig. 2. General procedure used for performing the phytochemical screening of *V. patens*

The plant material of adult leaves of *Vernonanthura patens* (laritaco) were used from plants at the vegetative state which were growing in the citadels "July 25", "Imbabura" and "June 24" and all belonget to the Canton Marcabelí, province El Oro, Ecuador. Leaves were collected at early morning at different dates during the months of December to February in 2009 and

Botanical identification was performed and voucher specimens of the herbs were prepared and deposited at the National Herbarium of Ecuador (QCNE) and a duplicated sample (CIBE37) was kept as herbal witness in the laboratory of the CIBE-ESPOL Bioproducts. Prior

leaves.

2010.

previously (Miranda & Cuellar, 2000; Manzano *et al.*, 2009).

Fig. 3. Chemical reactions carried out in each type of V*. patens'* leaf extracts obtained from using solvents of different polarity.

consent was obtained and authorized by the corresponding agencies of the government. The fieldwork and data collection were conducted in accordance with the institutional, national and international principles and guidelines for using and conserving plant biodiversity.

For conducting the phytochemical screening, extraction and fractionation, leaves samples were dried using an automatic dryer (45 °C, 8 hours) and then pulverized in a blender and screened. The fraction that remained in the sieve of 2 mm in diameter was collected and kept in polyethylene bags of low density at 24 C.

The result of phytochemical screening is presented in Table 2. This reveals moderate to low concentration of essentials oils, alkaloids, reducing compounds, phenols, tannins, flavonoids, quinones, saponins, triterpenes and steroids. Some of these chemical compounds have been associated to antibacterial, antifungal, antiprotozoal and citotoxicity properties and thus have a potential therapeutic use (Nweze *et al.,* 2004; Reuben *et al.,* 2008; Vital *et al*., 2010).

#### **4. Plant extracts, fractions and compounds**

The dry plant material (67 g of leaves of *V. patens*) was subjected to successive extractions with HPLC grade methanol by maceration in a closed container and in the absence of light. The extraction time was eight days and was conducted until total depletion of plant material; agitator and a rotary evaporator were used for solvent recovery.

Phytochemical Studies of Fractions and Compounds Present

Fraction 2 Hex/EtoAc: 90:10

Fig. 6. Isolated fractions from methanol extract of *V. patens* 

Fraction 1 Hexane

**5. Bioassays** 

in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic 511

Fraction 3 Hex/EtoAc: 80:20

 Compound 1 Compound 2 Compound 3 Fig. 7. Chromatographic plate (TLC) showing the three pure compounds isolated from *V.* 

Assays for screening the bioactivity of natural products has had an impressive history of development and is one of the keys for discovering new natural bioactive compounds.

In this study, a qualitative preliminary evaluation of the antifungal capacity of fractions and pure compounds isolated were conducted in order to select the most active. Those selected

The diffusion method (Avello *et al.,* 2009) in potato dextrose agar (PDA) was used to determine the antifungal activity of fractions and pure compounds isolated from *V. patens* leaves at 100 and 200 µg mL-1. Dilutions were made with dimethylsulfoxide (DMSO) 10%.

*patens*. Pure compounds were isolated from Fr 2 Hex / EtOAc 90:10 (1370mg).

were re-evaluated to quantify their ability to inhibit fungal growth.

Fraction 4 Hex/EtoAc: 30:70

Fraction 5 EtoAc 100%

Fraction 6 EtoAc/MeO H 70:30


Table 2. Chemical groups detected in *V. patens* leaves through the phytochemical screening.

The extract was evaporated to dryness, yielding 7g (10.44%) of methanol extract. The methanol residue was subjected to fractionation by successive column chromatography (CC) packed with activated silica from 60 to 200 mesh; elution was performed with solvents of increasing polarity using mixtures of hexane and ethyl acetate (10, 9:1 , 8:2, 3:7, 10) (Table 3). The extracts were analyzed by thin layer chromatography (TLC) on 60 F254 silica gel cromatofolios (Merck) with fluorescent indicator and a solvent system hexane / ethyl acetate (9:1). Plates were observed under UV light at 254 and 366 nm wavelengths.


Table 3. Solvents and proportions used in the chromatographic column fractionation of *V. patens*.

Six fractions were obtained (Figure 6): Fr 1 hexane (79mg), Fr 2 Hex / EtOAc 90:10 (1370mg), Fr 3 Hex / EtOAc 80:20 (0.60 mg), Fr 4 Hex / EtOAc 30:70 (0.41mg), Fr 5 EtOAc (0.21 mg), fraction 6 EtOAc / MeOH 70:30 (1760m g) and three pure compounds of the EtOAc fraction 10 and 20% (Figure 7): 57 mg of the compound [1] , 20 mg of the compound [2] and 90 mg of the compound [3].

The isolated fractions with different solvents from methanol extract of leaves of *V. patens* by column chromatography, have not been referred to this species, resulting in a high mass in the hexane fraction (79mg) compared with other extracted fractions. Nevertheless, methanol, ethyl acetate and hexane extracts from other plant species had showed a relevant antimicrobial activity (Ramya *et al.,* 2008).

Fraction 1 Hexane Fraction 2 Hex/EtoAc: 90:10 Fraction 3 Hex/EtoAc: 80:20 Fraction 4 Hex/EtoAc: 30:70 Fraction 5 EtoAc 100% Fraction 6 EtoAc/MeO H 70:30

Fig. 6. Isolated fractions from methanol extract of *V. patens* 

Compound 1 Compound 2 Compound 3

Fig. 7. Chromatographic plate (TLC) showing the three pure compounds isolated from *V. patens*. Pure compounds were isolated from Fr 2 Hex / EtOAc 90:10 (1370mg).

#### **5. Bioassays**

510 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Ether Alcoholic Aqueous

**Chemical groups Essays Extracts**

Essential oils, fatty compounds Sudan + Alkaloids Dragendorff Mayer + + Aminoacids Ninhidrine - - Antocianidine Antocianidine - - Cardiotonic Kedde - - Reducing compounds Fehling - + Phenols and tannins Ferric chloride + + Flavonoids Shinoda - + Lactones Baljet - - Mucilages Mucilages - - Bitter principles Bitter principles - - Quinones Börntrager + - Resins Resins - - Saponins Foam + + Triterpenes and steroids Lieberman-Buchard + + - Table 2. Chemical groups detected in *V. patens* leaves through the phytochemical screening.

The extract was evaporated to dryness, yielding 7g (10.44%) of methanol extract. The methanol residue was subjected to fractionation by successive column chromatography (CC) packed with activated silica from 60 to 200 mesh; elution was performed with solvents of increasing polarity using mixtures of hexane and ethyl acetate (10, 9:1 , 8:2, 3:7, 10) (Table 3). The extracts were analyzed by thin layer chromatography (TLC) on 60 F254 silica gel cromatofolios (Merck) with fluorescent indicator and a solvent system hexane / ethyl

**Solvent Proportion (%)**

acetate (9:1). Plates were observed under UV light at 254 and 366 nm wavelengths.

Hexane 100 Hexane/ethyl acetate 90:10 Hexane/ethyl acetate 80:20 Hexane/ethyl acetate 30:70 Ethyl acetate 100 Ethyl acetate/methanol 70:30

Table 3. Solvents and proportions used in the chromatographic column fractionation of *V.* 

Six fractions were obtained (Figure 6): Fr 1 hexane (79mg), Fr 2 Hex / EtOAc 90:10 (1370mg), Fr 3 Hex / EtOAc 80:20 (0.60 mg), Fr 4 Hex / EtOAc 30:70 (0.41mg), Fr 5 EtOAc (0.21 mg), fraction 6 EtOAc / MeOH 70:30 (1760m g) and three pure compounds of the EtOAc fraction 10 and 20% (Figure 7): 57 mg of the compound [1] , 20 mg of the compound [2] and 90 mg of

The isolated fractions with different solvents from methanol extract of leaves of *V. patens* by column chromatography, have not been referred to this species, resulting in a high mass in the hexane fraction (79mg) compared with other extracted fractions. Nevertheless, methanol, ethyl acetate and hexane extracts from other plant species had showed a relevant

*patens*.

the compound [3].

antimicrobial activity (Ramya *et al.,* 2008).

Assays for screening the bioactivity of natural products has had an impressive history of development and is one of the keys for discovering new natural bioactive compounds.

In this study, a qualitative preliminary evaluation of the antifungal capacity of fractions and pure compounds isolated were conducted in order to select the most active. Those selected were re-evaluated to quantify their ability to inhibit fungal growth.

The diffusion method (Avello *et al.,* 2009) in potato dextrose agar (PDA) was used to determine the antifungal activity of fractions and pure compounds isolated from *V. patens* leaves at 100 and 200 µg mL-1. Dilutions were made with dimethylsulfoxide (DMSO) 10%.

Phytochemical Studies of Fractions and Compounds Present

**6.2 Structural identification of isolated compounds** 

(compound 2) and Epi Lupeol (compound 3) (Figure 9).

standard Bruker XWIN-NMR software (rev. 010101).

for *V. patens*.

diseases by reducing cholesterol and triglycerides (Garcia *et al.,* 2010).

in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic 513

and antimicrobial activities, in addition to its beneficial effect for preventing cardiovascular

Fig. 8. Analytical gas chromatogram of the hexane fraction of *Vernonanthura patens*.

For this reason, it is possible to hypothesis that antifungal activity of *V. patens* against *F. oxysporum* and *P. notatum* which has been determined could be directed related to the squalene presence despite not being the main component of the fraction tested. The remaining compounds, individually or collectively, could also be involved in the bioactivity demonstrated. The results described here have not been reported previously

The structures of the three compounds isolated from the hexane soluble fraction by column chromatography were identified by their spectroscopic patterns as compared with references. These pure compounds were identified as Lupeol (compound 1), Acetyl Lupeol

Spectroscopy was performed in the Laboratory of Organic Chemistry at the University of Lund. 1H NMR (500 MHz) and 13C NMR (125 MHz) were recorded at room temperature with a Bruker DRX500 spectrometer with an inverse multinuclear 5 mm probe head equipped with a shielded gradient coil. The spectra were recorded in CDCl3, and the solvent signals (7.26 and 77.0 ppm, respectively) were used as reference. The chemical shifts (δ) are given in ppm, and the coupling constants (*J*) in Hz. COSY, HMQC and HMBC experiments were recorded with gradient enhancements using sine shaped gradient pulses. For the 2D heteronuclear correlation spectroscopy the refocusing delays were optimized for 1*J*CH=145 Hz and n*J*CH=10 Hz. The raw data were transformed and the spectra were evaluated with the

Strains of *Fusarium oxysporum* and *Penicillium notatum*, isolated from infected *Pinus radiata* and *Citrus sinense* fruits and maintained in the Collection of Fungi at University of Concepcion were used.

Holes of 5 mm Ø were made in the agar with a sterile cork borer and filled with 20 µL of each concentration of fractions and pure compounds. DMSO 10% was used as negative control in each plate. A disc (5 mm Ø) of already grown fungus was placed in the center of Petri dishes and incubated at 22 °C. Evaluations were made during two weeks.

Experimental design was completely randomized and each assay was performed in triplicate. Descriptive statistics of the experimental data was made in order to represent and point out its most important features.

Most relevant antifungal activity was observed in fraction 1 (100% hexane) and pure compounds 1 and 3 at the both concentrations tested.

The hexane fraction inhibited the growth of both fungal species tested. Highest inhibition exerted against *Penicillium notatum* (80.2%) and *Fusarium oxysporum* (81.5%) occurred when using 200 g mL-1 of this fraction. Statistical differences (P≤0,05) with negative controls indicated that DMSO did not influence the results of biological evaluation.

Pure compounds showed selective inhibition properties and a certain concentration dependence in its antifungal activity. Compound 1 showed a rate of inhibition of 50 and 90% (100 and 200 µg mL1 respectively) against *Penicillium notatum* while compound 3 was capable to inhibit 80 and 100% of the *Fusarium oxysporum* growth for each assayed concentrations.

Screening for antifungal activity of fractions and pure compounds of *V. patens* has been conducted for the first time. The potential of these results is relevant.

#### **6. Structural identification and quantitative analysis of the fractions and isolated compounds**

#### **6.1 Chemical characterization of the fraction with antifungal activity**

The isolated fraction with antifungal activity were analyzed for structural identification by gas chromatography-mass spectrometry (GC-MS) using an Agilent 7890A gas chromatograph with an Agilent 5975 detector (Avondale, PA.USA) equipped with a column HP-5MS of 5m long (0.25 mm in diameter and 0.25 cm inside diameter). Helium was used as the carrier gas; the analytical conditions were: initial temperature: 100 ° C (increasing 8 ° C per minute to a final temperature of 250 º C); inlet temperature and mass detector: 250 oC and 300 °C respectively. The mass detector was used in scan mode ("scan") with a range of 100 to 400 amu.

According to this technique and the analytical conditions described, this chromatogram was obtained and is as shown in Figure 8.

Using the library computer and taking into consideration those compounds that exceeded the 90% of confidence, structures of 33 components could be assigned (Table 4).

The compounds identified are mostly hydrocarbons, a logical result given the solvent used. There was a relative abundance of possible bicyclical sesquiterpenos (peaks 1-5) and of the acyclic triterpeno squalene (peak 30). For the sesquiterpenos exist antecedents of antimicrobial activity (Gregori *et al .,* 2005) and for the escualeno reports of activity antioxidant, antitumor

Strains of *Fusarium oxysporum* and *Penicillium notatum*, isolated from infected *Pinus radiata* and *Citrus sinense* fruits and maintained in the Collection of Fungi at University of

Holes of 5 mm Ø were made in the agar with a sterile cork borer and filled with 20 µL of each concentration of fractions and pure compounds. DMSO 10% was used as negative control in each plate. A disc (5 mm Ø) of already grown fungus was placed in the center of

Experimental design was completely randomized and each assay was performed in triplicate. Descriptive statistics of the experimental data was made in order to represent and

Most relevant antifungal activity was observed in fraction 1 (100% hexane) and pure

The hexane fraction inhibited the growth of both fungal species tested. Highest inhibition exerted against *Penicillium notatum* (80.2%) and *Fusarium oxysporum* (81.5%) occurred when using 200 g mL-1 of this fraction. Statistical differences (P≤0,05) with negative controls

Pure compounds showed selective inhibition properties and a certain concentration dependence in its antifungal activity. Compound 1 showed a rate of inhibition of 50 and 90% (100 and 200 µg mL1 respectively) against *Penicillium notatum* while compound 3 was capable to inhibit 80 and 100% of the *Fusarium oxysporum* growth for each assayed concentrations.

Screening for antifungal activity of fractions and pure compounds of *V. patens* has been

The isolated fraction with antifungal activity were analyzed for structural identification by gas chromatography-mass spectrometry (GC-MS) using an Agilent 7890A gas chromatograph with an Agilent 5975 detector (Avondale, PA.USA) equipped with a column HP-5MS of 5m long (0.25 mm in diameter and 0.25 cm inside diameter). Helium was used as the carrier gas; the analytical conditions were: initial temperature: 100 ° C (increasing 8 ° C per minute to a final temperature of 250 º C); inlet temperature and mass detector: 250 oC and 300 °C respectively. The mass detector was used in scan mode ("scan") with a range of 100 to 400 amu. According to this technique and the analytical conditions described, this chromatogram was

Using the library computer and taking into consideration those compounds that exceeded

The compounds identified are mostly hydrocarbons, a logical result given the solvent used. There was a relative abundance of possible bicyclical sesquiterpenos (peaks 1-5) and of the acyclic triterpeno squalene (peak 30). For the sesquiterpenos exist antecedents of antimicrobial activity (Gregori *et al .,* 2005) and for the escualeno reports of activity antioxidant, antitumor

the 90% of confidence, structures of 33 components could be assigned (Table 4).

**6. Structural identification and quantitative analysis of the fractions and** 

Petri dishes and incubated at 22 °C. Evaluations were made during two weeks.

indicated that DMSO did not influence the results of biological evaluation.

conducted for the first time. The potential of these results is relevant.

**6.1 Chemical characterization of the fraction with antifungal activity** 

Concepcion were used.

**isolated compounds** 

obtained and is as shown in Figure 8.

point out its most important features.

compounds 1 and 3 at the both concentrations tested.

and antimicrobial activities, in addition to its beneficial effect for preventing cardiovascular diseases by reducing cholesterol and triglycerides (Garcia *et al.,* 2010).

Fig. 8. Analytical gas chromatogram of the hexane fraction of *Vernonanthura patens*.

For this reason, it is possible to hypothesis that antifungal activity of *V. patens* against *F. oxysporum* and *P. notatum* which has been determined could be directed related to the squalene presence despite not being the main component of the fraction tested. The remaining compounds, individually or collectively, could also be involved in the bioactivity demonstrated. The results described here have not been reported previously for *V. patens*.

#### **6.2 Structural identification of isolated compounds**

The structures of the three compounds isolated from the hexane soluble fraction by column chromatography were identified by their spectroscopic patterns as compared with references. These pure compounds were identified as Lupeol (compound 1), Acetyl Lupeol (compound 2) and Epi Lupeol (compound 3) (Figure 9).

Spectroscopy was performed in the Laboratory of Organic Chemistry at the University of Lund. 1H NMR (500 MHz) and 13C NMR (125 MHz) were recorded at room temperature with a Bruker DRX500 spectrometer with an inverse multinuclear 5 mm probe head equipped with a shielded gradient coil. The spectra were recorded in CDCl3, and the solvent signals (7.26 and 77.0 ppm, respectively) were used as reference. The chemical shifts (δ) are given in ppm, and the coupling constants (*J*) in Hz. COSY, HMQC and HMBC experiments were recorded with gradient enhancements using sine shaped gradient pulses. For the 2D heteronuclear correlation spectroscopy the refocusing delays were optimized for 1*J*CH=145 Hz and n*J*CH=10 Hz. The raw data were transformed and the spectra were evaluated with the standard Bruker XWIN-NMR software (rev. 010101).

Phytochemical Studies of Fractions and Compounds Present

Fig. 9. Structure of compounds identified in *V. patens*.

completely inhibited the *Fusarium oxysporum* growth.

**7. Concluding remarks** 

program of CIBE.

**9. References** 

**8. Acknowledgements**

in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic 515

Lupeol Acetyl Lupeol Epi Lupeol

Phytochemical screening of *V. patens* has showed the presence of essentials oils, alkaloids, reducing compounds, phenols, tannins, flavonoids, quinones, saponins, triterpenes and steroids, of which some have been previously associated to important biological activities.

Fractions and pure compounds of this species were screened for the first time for antifungal activity. Hexane fraction and two pure compounds further identified as Lupeol and Epilupeol, were active against two important fungal pathogens at high rate (80-100%). Hexane fraction reduced the growth of *Fusarium oxysporum* in 80% and Epilupeol

Thirty-three chemical compounds in the hexane fraction from *V. patens* leaves were determined, Of which must are hydrocarbons. Antifungal activity of this fraction can be related to presence of squalene and/or combined activity of others identified compounds. Further research must be done for determining specific bioactivity of identified compounds. Chemical structures of three isolated compounds were elucidated, corresponding to Lupeol, Acetyl Lupeol and Epi Lupeol. These compounds are recognized for their significant and

Results of this study show that *V. patens* can be considered as important potential candidate for further chemical and biological researches and justify its inclusion in the biodiscovery

Acuña O. (2000). Valoración de las características físico químicas de especies ligno-

celulósicas y subproductos agroindustriales en la obtención de pulpa y elaboración

diverse biological activities, including antimicrobial and antineoplastic actions.

This study was supported by grants from SENESCYT and ESPOL (Ecuador)

1 3 5 7

H HO

23 24

25

10

30

28

22

27

26

11

29

The results that are shown in this chapter are unpublished and have not been previously registered for the species *V. patens*. Even though, the elucidated structures of the pure compounds have been found in other vegetal species, and recognize their diverse biological activity which includes antineoplastic action against certain types of cancer (Gallo & Sarachine, 2009).


Table 4. Identified compounds in hexane fraction of *V. patens* with antifungal activity.

Fig. 9. Structure of compounds identified in *V. patens*.

### **7. Concluding remarks**

514 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

The results that are shown in this chapter are unpublished and have not been previously registered for the species *V. patens*. Even though, the elucidated structures of the pure compounds have been found in other vegetal species, and recognize their diverse biological activity which includes antineoplastic action against certain types of cancer (Gallo &

2 8.678 Napthalene, 1, 2, 3, 4, 4a, 5, 6, 8a-octahydro-7-methyl-4-methylene-1-(1 methylethyl) - (1 ., 4a. , 8a. ). (bicyclic sesquiterpene) 3 9.156 Naphthalene, 1, 2, 4a, 5, 6, 8a-hexahydro-4, 7-dimethyl-1-(1-methylethyl)

4 9.234 Naphthalene, 1, 2, 3, 5, 6, 8a-hexahydro-4, 7-dimethyl-1-(1-methylethyl)

5 9.426 Naphthalene, 1, 2, 4a, 5, 6, 8a-hexahydro-4, 7-dimethyl-1-(1-methylethyl) -

[1S-(1. , 4a. ., 8a. )] (bicyclic sesquiterpene)

Table 4. Identified compounds in hexane fraction of *V. patens* with antifungal activity.

**retention Name** 

1 8.435 -caryophyllene (sesquiterpene)

6 9.950 2 - tetradecene (E) - 7 10.090 Hexadecane

11 11.646 Tritetracontano 12 11.937 Heptadecane, 3-methyl-13 12.191 3 - octadecane, (E) - 14 12.295 Heptadecane

16 13.976 Octadecane 17 14.199 (E) -3 - eicosane, 18 14.272 Eicosane 19 15.180 Heneicosano

21 16.031 Docosenoic

29 20.841 Nonadecane 30 21.157 Squalene 31 21.738 Eicosane

15 12.357 4-methyl-heptadecane,

20 15.527 Octadeciloxy –2-Ethanol

22 16.088 2 - Bromo dodecane 23 16.399 1 - bromo-octadecane 24 16.944 1-iodo-Hexadecane 25 17.769 Tetracosanoic

26 18.563 11-decyl-tetracosanoic 27 19.325 1-chloro-Heptadecosano, 28 20.057 5,14-dibutyl-octadecane

32 22.791 9-octyl-Heptadecane 33 24.104 Hentriacontane

(bicyclic sesquiterpene)

(bicyclic sesquiterpene)

8 10.583 2, 6, 10, - trimethyl-pentadecane, 9 11.153 2,6,11-trimetil-dodecano, 10 11.226 2,6,11-trimethyl-dodecane,

Sarachine, 2009).

**Peak Time** 

Phytochemical screening of *V. patens* has showed the presence of essentials oils, alkaloids, reducing compounds, phenols, tannins, flavonoids, quinones, saponins, triterpenes and steroids, of which some have been previously associated to important biological activities.

Fractions and pure compounds of this species were screened for the first time for antifungal activity. Hexane fraction and two pure compounds further identified as Lupeol and Epilupeol, were active against two important fungal pathogens at high rate (80-100%). Hexane fraction reduced the growth of *Fusarium oxysporum* in 80% and Epilupeol completely inhibited the *Fusarium oxysporum* growth.

Thirty-three chemical compounds in the hexane fraction from *V. patens* leaves were determined, Of which must are hydrocarbons. Antifungal activity of this fraction can be related to presence of squalene and/or combined activity of others identified compounds. Further research must be done for determining specific bioactivity of identified compounds.

Chemical structures of three isolated compounds were elucidated, corresponding to Lupeol, Acetyl Lupeol and Epi Lupeol. These compounds are recognized for their significant and diverse biological activities, including antimicrobial and antineoplastic actions.

Results of this study show that *V. patens* can be considered as important potential candidate for further chemical and biological researches and justify its inclusion in the biodiscovery program of CIBE.

#### **8. Acknowledgements**

This study was supported by grants from SENESCYT and ESPOL (Ecuador)

#### **9. References**

Acuña O. (2000). Valoración de las características físico químicas de especies lignocelulósicas y subproductos agroindustriales en la obtención de pulpa y elaboración

Phytochemical Studies of Fractions and Compounds Present

66o.pdf

2005. 39(2).

96.

in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic 517

http://www.globalsciencebooks.info/JournalsSup/images/0906/IJBPS\_3(SI1)46-

García Luján, Martínez A., Ortega J. & Castro F. (2010). *Componentes químicos y su relación con* 

Gregori Valdés Susana. *Estructura y actividad de los antifúngicos*. Revista Cubana de Farmacia

Jakupovic, J., Schmedia-Hirschmann, G. *Hirsutinolides, glaucolides and sesquiterpene lactones in* 

Kvist L. P., Aguirre Z. & Sánchez O. (2006). *Bosques montanos bajos occidentales en Ecuador y* 

 http://www.beisa.dk/Publications/BEISA%20Book%20pdfer/Capitulo%2013.pdf León M. R. & Guiracocha F. G. (2006). Tesis Ing. Agro. *Diversidad vegetal asociada a cacaotales de dos zonas agroecológicas en la región litoral del Ecuador*. 144 pp., ESPOL, Ecuador. Mabry, T.J., Abdel-Baset, Z. *Systematic implications of flavonoids and sesquiterpene lactones in* 

Manzano, P., Miranda, M., Orellana-Manzano A., García, G., Gutiérrez, Y. & Orellana, T.

 http://scielo.sld.cu/scielo.php?pid=S1028-47962009000300007&script=sci\_arttext Mendonça C., Gonçalves-Esteves V., Esteves R., & Nunes A. (2009). *Palynotaxonomy of* 

Miranda M. & Cuéllar A. (2001). *Farmacognosia y productos naturales*, Editorial Félix Varela,

Miranda M. & Cuellar A. (2000). *Manual de Prácticas de Laboratorio. Farmacognosia y Productos* 

Nweze, E.T. Okafor, J.I. & Njoku, O. (2004). *Antimicrobial Activities of Methanolic extract of* 

Pollora G. C., Bardón A., Catalan C., Gedris E. & Herz W. (2003) *Elephantopus-type* 

Pollora G. C ., Bardón A., Catalán C., Griffin C. l. & Herz W. (2004). Elephantopus-type

Portillo A., Vila R., Freixaa B., Ferrob E., Parellac T., Casanovad J. & Cañiguerala S. (2005).

Promabos (2006). *Proyecto de manejo de abejas y del bosque.* Flora of la palma (Chalatenango, El

*Medicinal Practice*. J. Bio. Res. *Biotechnol*. Vol. 2 (1): pp. 34-46.

http://cat.inist.fr/?aModele=afficheN&cpsidt=14685237

http://cat.inist.fr/?aModele=afficheN&cpsidt=15798184

http://www.bio.uu.nl/promabos/flora/alt\_index.html

Ethnopharmacology Vol. 97(1): pp. 49-52.

*species of Vernonia.* Biochem. Phytochemistry. 1986. 25:145-158.

de San Andrés, La Paz pp. 205-223. Avaliable from

*species of Vernonia*. Biochem. Sist. Ecol. 1975. 2:185.

medicinales. Vol. 14(3), pp: 45-53. Available in

Brasil. Bot. Vol. 32(4): pp. 647-662.

Ciudad de La Habana, pp. 207-222.

Habana, p. 44-49.

625. Avaliable from

Salvador), Avaliable from

*las actividades biológicas de algunos extractos vegetales*. Química Viva*,* Vol. : 9(2) pp. 86-

*sus plantas útiles.* Botánica Económica de los Andes Centrales Universidad Mayor

(2009) *Efecto antiinflamatorio y composición química del aceite de ramas de Bursera graveolens Triana & Planch. (palo santo) de Ecuador*. Revista cubana de plantas

*vernonanthura H. Rob. (Vernonieae, Asteraceae) Species from southeast Brazil.* Revista

*Naturales. Instituto de Farmacia y Alimentos*. Editorial Félix Varel, Ciudad de La

*Trumeguineesis (Schumm and Thorn) and Morinda lucinda Benth used in Nigerian Herb.* 

*sesquiterpene lactones from a Vernonanthura species, Vernonanthura nebularum Biochemical Systematics and Ecology*, Voumen 31(4) : pp. 397-405. Avaliable from

Sesquiterpene Lactones from a Second *Vernonanthura* species, *Vernonanthura lipeoensis*, *Biochemical Systematics and ecology.* Elsevier, Amsterdam. Vol. 32: pp. 619-

*Antifungal sesquiterpene from the root of Vernonanthura tweedieana.* Journal of

de papel. *Primer encuentro Nacional de productores y artesanos de fibras naturales*. Memorias técnicas, Ibarra-Ecuador. Avaliable from

http://biblioteca.espe.edu.ec/upload/Memorias\_Tecnicas.pdf.

Aguirre N. (2001). ECOPAR: *Sistemas forestales en la costa del Ecuador*: una propuesta para la zona de amortiguamiento de la reserva Mache – Chindul. University of Amsterdam. Programa FACE de Forestación del Ecuador S.A. Quito, Ecuador. Available from

 http://www.rncalliance.org/WebRoot/rncalliance/Shops/rncalliance/4C15/9487 /9F8B/02A1/3D0A/C0A8/D218/9663/Aguirre\_et\_al\_2001\_SFCosta2001.pdf


http://www.pancanal.com/cich/documentos/indice-integridad-biologica.pdf


Aguirre N. (2001). ECOPAR: *Sistemas forestales en la costa del Ecuador*: una propuesta para la

 http://www.rncalliance.org/WebRoot/rncalliance/Shops/rncalliance/4C15/9487 /9F8B/02A1/3D0A/C0A8/D218/9663/Aguirre\_et\_al\_2001\_SFCosta2001.pdf Avello M., Valdivia R., Sanzana R., Mondaca, M., Mennickent S., Aeschlimann V., Bittner

Bardón A., Borkoskya S., Ybarra M., Montanaroa S. & Cartagena E. (2007). Bioactive plants

Borkosky S., Ponce S., Gabriela Juárez G., González M. & Bardón A. (2009). Molusquicida

Blair S. (2005). *Plantas antimaláricas de Tumaco*: Costa Pacífica Colombiana; Vol. 1(347) pp. 84-

http://books.google.com.ec/books?id=8a7CKa3yXr0C&pg=PA84&lpg=PA84&dq

Camacho A. (2001). *Potencialidad melífera y polinífera de dos zonas de vida de la provincia de Loja*.

Chavarría F., Masís A., Pérez D., Espinoza R. & Guadamuz A. (1998). Species Page of

Diéguez M., Luque D., Domínguez I., Somoza A., Ortega G., Tejada I., Veces A., Gallardo

Fournet, A., Barrios, A.A. *Leishmamanicidal and trypanocidal activities of Bolivian medicinal* 

Gacheta M., Salazar J., Kaiserc M., Brunc R., Navarrete H., Muñoz R., Bauer R. & Schühlya

*de Agua Región Oriental de la Cuenca del Canal*, pp. 51. Avaliable from http://www.pancanal.com/cich/documentos/indice-integridad-biologica.pdf ECOPAR (2001). *Sistemas forestales en la costa del Ecuador: una propuesta para la zona de* 

from Argentina and Bolivia. *Fitoterapia* Vol. 78(3), pp. 227-231.

*Chemistry & Biodiversity*, Vol. 6(4), pp. 513–519. Avaliable from http://onlinelibrary.wiley.com/doi/10.1002/cbdv.200800156/citedby

Investigación y el Desarrollo de la Botánica, Avaliable from

87. Universidad de Antioquia. Avaliable from

=botanica++vernonanthura+patens&source

http://joethejuggler.com/Funbotanica/Boletin9.html

FACE de Forestación del Ecuador S.A. Quito.

*plants*. Journal of Ethnopharmacology. 1994. 41:19-37

Guanacaste, Costa Rica. Avaliable from

http://www.acguanacaste.ac.cr

Avaliable from

Memorias técnicas, Ibarra-Ecuador. Avaliable from http://biblioteca.espe.edu.ec/upload/Memorias\_Tecnicas.pdf.

Available from

Aromáticas, Vol. 8 (6), pp. 479-486.

de papel. *Primer encuentro Nacional de productores y artesanos de fibras naturales*.

zona de amortiguamiento de la reserva Mache – Chindul. University of Amsterdam. Programa FACE de Forestación del Ecuador S.A. Quito, Ecuador.

M., Becerra J. (2009). Extractos antioxidantes y antimicrobianos de *Aristotelia chilensis* y *Ugni molinae* y sus aplicaciones como preservantes en productos cosméticos . Boletín Latinoamericano y del Caribe de Plantas Medicinales y

Sesquiterpene Lactones from Species of the Tribe Vernonieae (Compositae).

Editores: Pablo Lozano y Zhofre Aguirre. Fundación Ecuatoriana para la

*Vernonia patens* (Asteraceae). *Species* Home Pages, Área de Conservación

M., Araúz Y. & Núñez E. (2006). Convenio de Cooperación ANAM – ACP. Monitoreo de la Cuenca Hidrográfica del Canal de Panamá. *Componente de Calidad* 

*amortiguamiento de la reserva Mache* – Chindul. University of Amsterdam. Programa

W. (2010). *Assessment of anti-protozoal activity of plants traditionally used in Ecuador in the treatment of leishmaniasis*. Journal of Ethnopharmacology, pp. 128, 184–197. Gallo M. & Sarachine M. (2009). *Biological Activities of Lupeol*. International Journal of

Biomedical and pharmaceutical Sciences. Global Sciences Books. pp. 46-62.

 http://www.globalsciencebooks.info/JournalsSup/images/0906/IJBPS\_3(SI1)46- 66o.pdf


http://www.beisa.dk/Publications/BEISA%20Book%20pdfer/Capitulo%2013.pdf


http://scielo.sld.cu/scielo.php?pid=S1028-47962009000300007&script=sci\_arttext


http://cat.inist.fr/?aModele=afficheN&cpsidt=15798184


http://www.bio.uu.nl/promabos/flora/alt\_index.html

**25** 

*Poland* 

**The Inhibitory Effect of Natural** 

Renata Mikstacka1, Zbigniew Dutkiewicz1, Stanisław Sobiak1 and Wanda Baer-Dubowska2

 **Stilbenes and Their Analogues on Catalytic** 

*1Departament of Chemical Technology of Drugs, Poznań University of Medical Sciences 2Department of Pharmaceutical Biochemistry, Poznań University of Medical Sciences* 

In the last decade, increasing interest in the role of nutrition in disease prevention has been observed. The World Health Organization (WHO) reported that one-third of all cancer deaths could be prevented, and that diet plays a key role in prevention (Bode & Dong, 2009). The term *chemoprevention* introduced and developed by Sporn (2005) and Wattenberg (1985) refers in general to multi-targeted pharmacological and nutritional intervention with the use of naturally occurring or chemically synthesized compounds. For this purpose, dietary phytochemicals believed to be safe for human use seem to be very promising. The importance of natural chemopreventive agents relies on their non-toxicity when given in small amounts for longer periods of time. Moreover, using a combination of phytochemicals

Cancer cell growth arises through a complex multistep process by which cancer cells acquire characteristics of unlimited proliferation potential, lack of response to growth signals, and resistance to cell death. Thus, preventive/therapeutic action of phytochemicals may be directed towards numerous molecular targets that are proteins involved in procarcinogen metabolism, cell transformation and proliferation, and signaling pathways leading to apoptosis of damaged or transformed cells (William et al., 2009). Targeting enzymes of the P450 superfamily may provide one of the strategies for enhancing the efficacy of

Mechanistic studies of natural compounds are of great value regarding their characteristics of bioactivity, efficacy, selectivity and potential adverse side effects. Targeted inhibition of metabolic activation of carcinogens and induction of detoxifying enzymes has been considered a fundamental strategy for blocking the early stage of carcinogenesis. For example, inhibition of CYP1 enzymes was one test in the battery of assays employed in

**1. Introduction**

provides synergistic or additive preventive effects.

chemopreventive and therapeutic agents (Swanson et al., 2010).

**Activity of Cytochromes P450 Family 1** 

**in Comparison with Other Phenols** 

**– Structure and Activity Relationship** 

Promabos. (2006). *Proyecto de Manejo de Abejas y Bosques. Árboles melíferos para reforestar*. Xuqunán o Xuquinái, Avaliable from

http://www.bio.uu.nl/promabos/arbolesmeliferos/pdf\_files/Xuqun%E1n.pdf

Ramírez J., Camacho A., Merino B. & Ureña J. (2001). *Recursos florales y origen botánico de las mieles de las abejas (Apis mellifera, L.) en las provincias de Loja y Zamora Chinchipe.* Área agropecuaria y de recursos renovables. Universidad Nacional de Loja. Revista informática, Avaliable from

http://issuu.com/miltonric/docs/recursosflorales#comments.


http://www.darwinnet.org/docs/PlanManejoMacheChindul.pdf.


http://darnis.inbio.ac.cr/FMPro?-DB=UBIpub.fp3&-lay=WebAll&-

Format=/ubi/detail.html&-Op=bw&id=6713&-Find

Tacana. (1999). *Conozcan nuestros árboles, nuestras hierbas.* Editores UMSA - ClPTA – IRD. La Paz, pp. 27. Avaliable from http://horizon.documentation.ird.fr/exl-doc/pleins\_textes/divers10-

04/010018852.pdf


 http://proyecto.ecofondo.org.co/index2.php?option=com\_docman&task=doc\_vie w&gid=46&Itemid=36.


http://www.springerlink.com/content/e754t15r231l4117/


### **The Inhibitory Effect of Natural Stilbenes and Their Analogues on Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols – Structure and Activity Relationship**

Renata Mikstacka1, Zbigniew Dutkiewicz1,

Stanisław Sobiak1 and Wanda Baer-Dubowska2 *1Departament of Chemical Technology of Drugs, Poznań University of Medical Sciences 2Department of Pharmaceutical Biochemistry, Poznań University of Medical Sciences Poland* 

#### **1. Introduction**

518 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Promabos. (2006). *Proyecto de Manejo de Abejas y Bosques. Árboles melíferos para reforestar*.

Ramya S., Kalayansundaram M., Kalaivani T. & Jayakumararaj, R. (2008). *Phytochemical* 

REMACH, (2004). *Plan de Manejo y Gestión participativa de la reserva ecológica Manche-Chindul*,

Reuben, K.D., Abdulrahman F.I., Akan J.C., Usman H., Sodipo O.A. & Egwu G.O. (2008).

Rodriguez A. (2005). *Vernonia patens Kunth*. INBIO. Instituto Nacional de Biodiversidad.

Tacana. (1999). *Conozcan nuestros árboles, nuestras hierbas.* Editores UMSA - ClPTA – IRD. La

Tene V., Malangón O., Vita P., Vidari G., Armijos Ch. & Zaragoza T. (2007). *An ethnobotanical* 

Terreros G. & Adriana M. (2009). *Recuperación del conocimiento ancestral en manejo de plantas* 

http://proyecto.ecofondo.org.co/index2.php?option=com\_docman&task=doc\_vie

Tolstikova T. G., SorokingI. V., Tolstikov G. A. Tolstikov A. G. & Flekhter O. B. (2006).

Valadeau C., Pabon A., Deharo E., Albán–Castillo J., Estévez Y., Lores F., Rojas R., Gamboa

Vital, P.G., Velasco Jr, R.N., Demigillo, J.M. & Rivera, W.L. (2010). *Antimicrobial activity,* 

*extracts*. Journal of Medicinal Plants Research, Vol. 4(1): pp. 58-63.

*survey of medicinal plants used in Loja and Zamora-Chinchipe, Ecuador*. Journal of

*con fines medicinales en comunidades étnicas el norte del Cauca y en la vereda la colonia (Yotoco)*. Proyecto Regional ECOFONDO-ACDI, Valle-Norte del Cauca, 2004-2009.

*Biological Activity and Pharmacological Prospects of Lupane Terpenoids: I. Natural Lupane Derivatives*. Russian Journal of Bioorganic Chemistry Vol. 32(1): pp. 37–49.

D., Sauvain M., Castillo D. & Bourdy G. (2009). Medicinal plants from the Yanesha (Peru): *Evaluation of the leishmanicidal and antimalarial activity of selected extracts.* 

*cytotoxicity and phytochemical screening of Ficus septica Burm and Sterculia foetida L. leaf* 

*mieles de las abejas (Apis mellifera, L.) en las provincias de Loja y Zamora Chinchipe.* Área agropecuaria y de recursos renovables. Universidad Nacional de Loja. Revista

*Screening and Antibacterial Activity of Leaf Extracts of Pterocarpusmarsupium Roxb.* 

*Phytochemical Screening and In Vitro Antimicrobial Investigation of the Methanolic Extract of Croton Zambesicus Muell ARG. Stem Bark.* European Journal of Scientific

 http://www.bio.uu.nl/promabos/arbolesmeliferos/pdf\_files/Xuqun%E1n.pdf Ramírez J., Camacho A., Merino B. & Ureña J. (2001). *Recursos florales y origen botánico de las* 

http://issuu.com/miltonric/docs/recursosflorales#comments.

*(Fabaceae).* Ethnobotanical Leaflets Vol. 12: pp. 1029-34.

http://www.darwinnet.org/docs/PlanManejoMacheChindul.pdf.

 http://darnis.inbio.ac.cr/FMPro?-DB=UBIpub.fp3&-lay=WebAll&- Format=/ubi/detail.html&-Op=bw&id=6713&-Find

http://horizon.documentation.ird.fr/exl-doc/pleins\_textes/divers10-

Tobías. (1996). EC076. *Cañón del río Catamayo. Darwinnet*, Avaliable from http://www.darwinnet.org/docs/Ibas\_RT/EC076.pdf.

http://www.springerlink.com/content/e754t15r231l4117/

*Journal of Ethnopharmacology* pp. 123, 413–422.

Xuqunán o Xuquinái, Avaliable from

informática, Avaliable from

Research, Vol. 23(1) : pp. 134-140

Paz, pp. 27. Avaliable from

Ethnopharmacology Vol. 111: pp. 63–81.

Avaliable from

Avaliable from

04/010018852.pdf

Avaliable from

Avaliable from

w&gid=46&Itemid=36.

In the last decade, increasing interest in the role of nutrition in disease prevention has been observed. The World Health Organization (WHO) reported that one-third of all cancer deaths could be prevented, and that diet plays a key role in prevention (Bode & Dong, 2009). The term *chemoprevention* introduced and developed by Sporn (2005) and Wattenberg (1985) refers in general to multi-targeted pharmacological and nutritional intervention with the use of naturally occurring or chemically synthesized compounds. For this purpose, dietary phytochemicals believed to be safe for human use seem to be very promising. The importance of natural chemopreventive agents relies on their non-toxicity when given in small amounts for longer periods of time. Moreover, using a combination of phytochemicals provides synergistic or additive preventive effects.

Cancer cell growth arises through a complex multistep process by which cancer cells acquire characteristics of unlimited proliferation potential, lack of response to growth signals, and resistance to cell death. Thus, preventive/therapeutic action of phytochemicals may be directed towards numerous molecular targets that are proteins involved in procarcinogen metabolism, cell transformation and proliferation, and signaling pathways leading to apoptosis of damaged or transformed cells (William et al., 2009). Targeting enzymes of the P450 superfamily may provide one of the strategies for enhancing the efficacy of chemopreventive and therapeutic agents (Swanson et al., 2010).

Mechanistic studies of natural compounds are of great value regarding their characteristics of bioactivity, efficacy, selectivity and potential adverse side effects. Targeted inhibition of metabolic activation of carcinogens and induction of detoxifying enzymes has been considered a fundamental strategy for blocking the early stage of carcinogenesis. For example, inhibition of CYP1 enzymes was one test in the battery of assays employed in

The Inhibitory Effect of Natural Stilbenes and Their Analogues on

(Lamb et al, 2007).

chemotherapy (Bruno & Njar, 2007).

**effective chemopreventive approach** 

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 521

amines. Additionally, CYP1B1 metabolizes 17-estradiol (E2) to 4-hydroxyestradiol (4-OH-E2), which is further oxidized by peroxidase to estradiol-3,4-quinone to form a quinone-DNA adducts responsible for estrogen-related carcinogenesis (Liehr et al., 1996). This pathway of metabolism is extensively studied with respect to polymorphism of CYP1 enzymes and its association with carcinogenic metabolite formation (Kisselev et al., 2005). All members of the human CYP1 family are expressed in extrahepatic tissues. However, CYP1A2 is the only constitutive form of liver enzyme, and as such takes part in metabolism of xenobiotics, including numerous drugs (caffeine, theophylline, methadone, verapamil, propranolol, warfarin, tamoxifen). On the other hand, it is worth mentioning that microbial CYPs are considered as drug targets and may be used as biocatalysts in drug biosynthesis

In humans, CYP1B1 is overexpressed in tumor cells, and this has important implications for tumor development and progression (Castro et al., 2008). It was found that CYP1B1 knockout mice were highly resistant to 7,12-dimethylbenz[a]anthracene induced tumor formation (Gonzalez, 2002). Thus, regulators of the expression and catalytic activity of family 1 cytochromes appear to play an important role in cancer chemoprevention by blocking the initial stages of tumorigenesis. With respect to cancer chemotherapy, CYP1A1 and CYP1B1 have the ability to metabolize cytostatics, diminishing their toxic effect on cancer cells (McFadyen & Murray, 2001). Considering this, the inhibition of CYP1B1, an enzyme up-regulated in many cancers, would be a strategy to prevent the loss of cytostatics effectiveness. On the other hand, the development of anticancer prodrugs specifically activated by CYP1B1 to cytotoxic compounds might be a promising novel strategy in cancer

**3. Mechanism of the expression of CYP1 genes – AHR as a target for** 

preventing this undesirable effect might be a chemopreventive strategy.

Members of the CYP1 family are under the transcriptional control of the aryl hydrocarbon receptor (AHR) localized in cytosol that is activated by polyhalogenated aromatic hydrocarbons, among them 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR agonists are well known environmental pollutants. As a result of activation AHR translocates into the nucleus and forms a dimer with ARNT (aryl hydrocarbon nuclear translocator). The AHR/ARNT complex is characterized by a high affinity to specific DNA recognition sites termed DREs (dioxin response elements) or AHREs (aryl hydrocarbon response element) which upregulate a battery of target genes, including those involved in metabolism of chemical carcinogens such as CYP1A1, CYP1A2 and CYP1B1 (Fig. 2). In this way, agonists induce the expression of xenobiotic metabolizing enzymes (XMEs) that activate procarcinogens to genotoxic forms. Thus, the treatment with AHR antagonists by

There are phytochemicals that possess the ability to block agonist interaction with the ligand-binding site of the AHR and agonist induction of the AHR-signaling pathways. In that respect, resveratrol is the best recognized stilbene derivative. Moreover, it is one of the best-characterized chemopreventive phytochemicals (Goswami and Das, 2009). It occurs mainly in small fruits like berries and grapes, peanuts and red wine. Its chemopreventive properties found in studies on animals *in vivo* were described for the first time by Jang and

screening of potential cancer chemopreventive agents (Gerhauser et al., 2003). Variable dietary exposure to phytochemicals may contribute to some of the inter-individual variation in the pharmacokinetics and pharmacological responses that are observed for drugs such as phenacetin, caffeine, and theophylline, which are substrates for CYP1A2 (Rendic & Di Carlo, 1997). Further research is needed to determine the extent to which the effect of dietary exposure may be modified by genetic polymorphism of xenobiotic metabolizing enzymes.

Phenolics are a diverse group of aromatic compounds broadly distributed in plants. Among this group, stilbenoids are compounds displaying multiple activities of interest with regard to cancer prevention and therapy, and their anticancer properties have been proven in various animal models (Szekeres et al., 2010). In this review, we summarize the results of studies on inhibitory activity of *trans*-resveratrol (3,4',5-trimethoxy-*trans*-stilbene), the best recognized *trans*-stilbene (Figure 1), and its natural and synthetic analogues toward expression and activity of CYPs responsible for procarcinogen activation. We discuss the role of cytochrome family 1 inhibitors in cancer chemoprevention and chemotherapy. Additionally, we compare their effect with other natural phenols occurring in plant foods in relatively high amount and exerting significant bioactivity. Finally, we analyze the use of computational methods for biomolecular docking in structure and activity relationship studies of CYP1 inhibitors.

#### **2. Potential strategies targeting CYPs for cancer therapy and prevention**

One of the strategies of cancer chemoprevention is directed at drug-metabolizing enzymes such as cytochromes P450 (CYPs), a superfamily which metabolizes a wide spectrum of endogenous and exogenous substrates. Cytochrome P450 family 1 comprises three important isoforms: CYP1A1, CYP1A2 and CYP1B1 that catalyze the activation of procarcinogens such as polycyclic aromatic hydrocarbons, and aromatic and heterocyclic

screening of potential cancer chemopreventive agents (Gerhauser et al., 2003). Variable dietary exposure to phytochemicals may contribute to some of the inter-individual variation in the pharmacokinetics and pharmacological responses that are observed for drugs such as phenacetin, caffeine, and theophylline, which are substrates for CYP1A2 (Rendic & Di Carlo, 1997). Further research is needed to determine the extent to which the effect of dietary exposure may be modified by genetic polymorphism of xenobiotic metabolizing enzymes. Phenolics are a diverse group of aromatic compounds broadly distributed in plants. Among this group, stilbenoids are compounds displaying multiple activities of interest with regard to cancer prevention and therapy, and their anticancer properties have been proven in various animal models (Szekeres et al., 2010). In this review, we summarize the results of studies on inhibitory activity of *trans*-resveratrol (3,4',5-trimethoxy-*trans*-stilbene), the best recognized *trans*-stilbene (Figure 1), and its natural and synthetic analogues toward expression and activity of CYPs responsible for procarcinogen activation. We discuss the role of cytochrome family 1 inhibitors in cancer chemoprevention and chemotherapy. Additionally, we compare their effect with other natural phenols occurring in plant foods in relatively high amount and exerting significant bioactivity. Finally, we analyze the use of computational methods for biomolecular docking in structure and activity relationship

OR2

Fig. 1. Structure of *trans*-resveratrol and its natural analogues

 R1 R2 R3 R4 1. *trans-*Resveratrol H H H H 2. Piceatannol H H OH H 3. Rhapontigenin H H OH CH3 4. Desoxyrhapontigenin H H H CH3 5. Pinostilbene H CH3 H H 6. Pterostilbene CH3 CH3 H H

**2. Potential strategies targeting CYPs for cancer therapy and prevention** 

One of the strategies of cancer chemoprevention is directed at drug-metabolizing enzymes such as cytochromes P450 (CYPs), a superfamily which metabolizes a wide spectrum of endogenous and exogenous substrates. Cytochrome P450 family 1 comprises three important isoforms: CYP1A1, CYP1A2 and CYP1B1 that catalyze the activation of procarcinogens such as polycyclic aromatic hydrocarbons, and aromatic and heterocyclic

5

R1O <sup>3</sup>

OR4

R3

3'

4'

studies of CYP1 inhibitors.

amines. Additionally, CYP1B1 metabolizes 17-estradiol (E2) to 4-hydroxyestradiol (4-OH-E2), which is further oxidized by peroxidase to estradiol-3,4-quinone to form a quinone-DNA adducts responsible for estrogen-related carcinogenesis (Liehr et al., 1996). This pathway of metabolism is extensively studied with respect to polymorphism of CYP1 enzymes and its association with carcinogenic metabolite formation (Kisselev et al., 2005).

All members of the human CYP1 family are expressed in extrahepatic tissues. However, CYP1A2 is the only constitutive form of liver enzyme, and as such takes part in metabolism of xenobiotics, including numerous drugs (caffeine, theophylline, methadone, verapamil, propranolol, warfarin, tamoxifen). On the other hand, it is worth mentioning that microbial CYPs are considered as drug targets and may be used as biocatalysts in drug biosynthesis (Lamb et al, 2007).

In humans, CYP1B1 is overexpressed in tumor cells, and this has important implications for tumor development and progression (Castro et al., 2008). It was found that CYP1B1 knockout mice were highly resistant to 7,12-dimethylbenz[a]anthracene induced tumor formation (Gonzalez, 2002). Thus, regulators of the expression and catalytic activity of family 1 cytochromes appear to play an important role in cancer chemoprevention by blocking the initial stages of tumorigenesis. With respect to cancer chemotherapy, CYP1A1 and CYP1B1 have the ability to metabolize cytostatics, diminishing their toxic effect on cancer cells (McFadyen & Murray, 2001). Considering this, the inhibition of CYP1B1, an enzyme up-regulated in many cancers, would be a strategy to prevent the loss of cytostatics effectiveness. On the other hand, the development of anticancer prodrugs specifically activated by CYP1B1 to cytotoxic compounds might be a promising novel strategy in cancer chemotherapy (Bruno & Njar, 2007).

#### **3. Mechanism of the expression of CYP1 genes – AHR as a target for effective chemopreventive approach**

Members of the CYP1 family are under the transcriptional control of the aryl hydrocarbon receptor (AHR) localized in cytosol that is activated by polyhalogenated aromatic hydrocarbons, among them 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR agonists are well known environmental pollutants. As a result of activation AHR translocates into the nucleus and forms a dimer with ARNT (aryl hydrocarbon nuclear translocator). The AHR/ARNT complex is characterized by a high affinity to specific DNA recognition sites termed DREs (dioxin response elements) or AHREs (aryl hydrocarbon response element) which upregulate a battery of target genes, including those involved in metabolism of chemical carcinogens such as CYP1A1, CYP1A2 and CYP1B1 (Fig. 2). In this way, agonists induce the expression of xenobiotic metabolizing enzymes (XMEs) that activate procarcinogens to genotoxic forms. Thus, the treatment with AHR antagonists by preventing this undesirable effect might be a chemopreventive strategy.

There are phytochemicals that possess the ability to block agonist interaction with the ligand-binding site of the AHR and agonist induction of the AHR-signaling pathways. In that respect, resveratrol is the best recognized stilbene derivative. Moreover, it is one of the best-characterized chemopreventive phytochemicals (Goswami and Das, 2009). It occurs mainly in small fruits like berries and grapes, peanuts and red wine. Its chemopreventive properties found in studies on animals *in vivo* were described for the first time by Jang and

The Inhibitory Effect of Natural Stilbenes and Their Analogues on

BPDE-DNA adduct formation in the lungs of mice (Revel et al., 2003).

Acetone 0.2 ml 65.3 4.6 5,6-Benzoflavone 8 M 336 17.2 Protocatechuic acid 8 M 75.3 2.1

Chlorogenic acid 8 M 71.7 3.2

*trans*-Resveratrol 8 M 18.9 2.6

Summarising, resveratrol inhibits AHR-dependent transcription by preventing AHR/ARNT binding to the AHRE. The activity of preventing the conversion of ligand-bound cytosolic AHR into its nuclear DNA-binding form and/or the interaction between the AHR and the transcription initiation complex at the CYP1A1 gene promoter may be an important part of the chemopreventive activity of resveratrol. However, the action of resveratrol is not specific because this natural stilbene as a phytoestrogen is also a potent ER (estrogen receptor) agonist. Recently, experiments on human breast cancer cells revealed that the estrogenic properties of resveratrol and its influence on the ER expression are independent of its ability to inhibit the expression of genes controlled by AHR (MacPherson & Matthews, 2010). New stilbene derivatives of resveratrol that were synthesized appeared to be selective for AHR and devoid of affinity for ER. Among the *trans*-stilbenes synthesized, all displayed a significantly higher affinity than resveratrol for AHR. Substitution of 3- and/or 5-hydroxy groups with chlorine atoms coupled with replacement of 4'-hydroxy with chlorine or a methoxy group yielded selective TCDD antagonists with high affinity for the AHR that was much higher than resveratrol. Interestingly, one of the studied compounds, 3-hydroxy-5 chloro-4'-trifluoromethyl-*trans*-stilbene, was a selective AHR agonist exerting extremely high-affinity to AHR with a Ki of 0.2 nM. None of the compounds studied showed any detectable affinity for the ER that should eliminate estrogen-related risks, such as the

In the Table 2 we summarized the studies on the effects of resveratrol and its derivatives on AHR related expression of CYP1 enzymes. However, the results of *in vivo* experiments on animals are highly dependent on the dose of a studied compound, as well as the duration and manner of its administration. Further, the effect of a studied substance may also be tissue-dependent. The expression of CYP1A1 and CYP1A2-related monooxygenases in hepatic subcellular preparations from resveratrol treated male mice did not differ from the control; while in pulmonary subcellular preparations significantly lower expression of

Table 1. Effect of phenolic compounds on mouse epidermal AHH activity

increased risk of ER-related cancers (de Medina et al. 2005).

CYP1A1/2 –dependent enzymes was observed (Canistro et al., 2009).

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 523

*vivo* experiments on phytochemicals with four different structures, where only resveratrol given topically on mouse epidermis inhibited aryl hydrocarbon hydroxylase (AHH) activity in a dose dependent manner (Table 1) (Szaefer et al., 2004). Moreover, resveratrol has been shown to prevent genotoxicity of B[a]P by inhibiting B[a]P-induced CYP1A1 expression and

**Treatment Dose Activity [pmol/min/mg** 

16 M 83.4 6.4

16 M 83.9 2.8

16 M 0.08 0.01

**protein]** 

Fig. 2. AHR signaling pathway; L – ligand; Hsp90 – heat shock protein 90; ARNT – aryl hydrocarbon nuclear receptor; AHRE – aryl hydrocarbon response element. AHR/ARNT complexes bind to the DNA recognition sequence AHRE located in regulatory regions of phase I (CYP1A1, CYP1A2, CYP1B1) and phase II drug metabolizing enzymes.

coworkers (Jang et al., 1997). Chen and collaborators have reported that resveratrol strongly inhibited TCDD-induced AHR binding activity in human mammary epithelial (MCF-1-A) cells (Chen et al., 2004). The inhibition of CYP1A1 expression by resveratrol was observed in rat primary hepatocytes (Andrieux et al., 2004). In human HepG2 hepatoma cells, resveratrol inhibited the increase in CYP1A1 mRNA caused by TCDD in a concentrationdependent manner. The induction of transcription of an aryl hydrocarbon-responsive reporter vector containing the CYP1A1 promoter by TCDD was likewise inhibited by resveratrol. Resveratrol also inhibited the constitutive level of CYP1A1 mRNA and reporter vector transcription in human hepatoma HepG2 cells (Ciolino et al., 1998). Resveratrol was also effective in inhibiting CYP1A1 transcription induced by the aryl hydrocarbon dimethylbenz[a]anthracene in human mammary carcinoma MCF-7 cells and B[a]P-treated HepG2 cells (Ciolino et al., 1999). These data demonstrate that resveratrol inhibits aryl hydrocarbon-induced CYP1A activity *in vitro* by directly inhibiting CYP1A1/1A2 enzyme activity, and by inhibiting the signal transduction pathway that up-regulates the expression of carcinogen activating enzymes. The antagonistic action of resveratrol was supported by *in* 

**Hsp 90**

**TRANSLOCATION**

Fig. 2. AHR signaling pathway; L – ligand; Hsp90 – heat shock protein 90; ARNT – aryl hydrocarbon nuclear receptor; AHRE – aryl hydrocarbon response element. AHR/ARNT complexes bind to the DNA recognition sequence AHRE located in regulatory regions of

coworkers (Jang et al., 1997). Chen and collaborators have reported that resveratrol strongly inhibited TCDD-induced AHR binding activity in human mammary epithelial (MCF-1-A) cells (Chen et al., 2004). The inhibition of CYP1A1 expression by resveratrol was observed in rat primary hepatocytes (Andrieux et al., 2004). In human HepG2 hepatoma cells, resveratrol inhibited the increase in CYP1A1 mRNA caused by TCDD in a concentrationdependent manner. The induction of transcription of an aryl hydrocarbon-responsive reporter vector containing the CYP1A1 promoter by TCDD was likewise inhibited by resveratrol. Resveratrol also inhibited the constitutive level of CYP1A1 mRNA and reporter vector transcription in human hepatoma HepG2 cells (Ciolino et al., 1998). Resveratrol was also effective in inhibiting CYP1A1 transcription induced by the aryl hydrocarbon dimethylbenz[a]anthracene in human mammary carcinoma MCF-7 cells and B[a]P-treated HepG2 cells (Ciolino et al., 1999). These data demonstrate that resveratrol inhibits aryl hydrocarbon-induced CYP1A activity *in vitro* by directly inhibiting CYP1A1/1A2 enzyme activity, and by inhibiting the signal transduction pathway that up-regulates the expression of carcinogen activating enzymes. The antagonistic action of resveratrol was supported by *in* 

phase I (CYP1A1, CYP1A2, CYP1B1) and phase II drug metabolizing enzymes.

**L L**

**AHR**

*CYTOSOL*

**AHR ARNT**

**L**

*NUCLEUS*

**AHRE**

**CYP1A1 CYP1A2 CYP1B1**

**PROTEINS**

**OTHER GENE PRODUCTS**

**TRANSLATION**

**AHR Hsp 90 Hsp 90**

**L**

**LIGAND BINDING**

**L**

*cell membrane*

**AHR Hsp 90 Hsp 90**

*vivo* experiments on phytochemicals with four different structures, where only resveratrol given topically on mouse epidermis inhibited aryl hydrocarbon hydroxylase (AHH) activity in a dose dependent manner (Table 1) (Szaefer et al., 2004). Moreover, resveratrol has been shown to prevent genotoxicity of B[a]P by inhibiting B[a]P-induced CYP1A1 expression and BPDE-DNA adduct formation in the lungs of mice (Revel et al., 2003).


Table 1. Effect of phenolic compounds on mouse epidermal AHH activity

Summarising, resveratrol inhibits AHR-dependent transcription by preventing AHR/ARNT binding to the AHRE. The activity of preventing the conversion of ligand-bound cytosolic AHR into its nuclear DNA-binding form and/or the interaction between the AHR and the transcription initiation complex at the CYP1A1 gene promoter may be an important part of the chemopreventive activity of resveratrol. However, the action of resveratrol is not specific because this natural stilbene as a phytoestrogen is also a potent ER (estrogen receptor) agonist. Recently, experiments on human breast cancer cells revealed that the estrogenic properties of resveratrol and its influence on the ER expression are independent of its ability to inhibit the expression of genes controlled by AHR (MacPherson & Matthews, 2010). New stilbene derivatives of resveratrol that were synthesized appeared to be selective for AHR and devoid of affinity for ER. Among the *trans*-stilbenes synthesized, all displayed a significantly higher affinity than resveratrol for AHR. Substitution of 3- and/or 5-hydroxy groups with chlorine atoms coupled with replacement of 4'-hydroxy with chlorine or a methoxy group yielded selective TCDD antagonists with high affinity for the AHR that was much higher than resveratrol. Interestingly, one of the studied compounds, 3-hydroxy-5 chloro-4'-trifluoromethyl-*trans*-stilbene, was a selective AHR agonist exerting extremely high-affinity to AHR with a Ki of 0.2 nM. None of the compounds studied showed any detectable affinity for the ER that should eliminate estrogen-related risks, such as the increased risk of ER-related cancers (de Medina et al. 2005).

In the Table 2 we summarized the studies on the effects of resveratrol and its derivatives on AHR related expression of CYP1 enzymes. However, the results of *in vivo* experiments on animals are highly dependent on the dose of a studied compound, as well as the duration and manner of its administration. Further, the effect of a studied substance may also be tissue-dependent. The expression of CYP1A1 and CYP1A2-related monooxygenases in hepatic subcellular preparations from resveratrol treated male mice did not differ from the control; while in pulmonary subcellular preparations significantly lower expression of CYP1A1/2 –dependent enzymes was observed (Canistro et al., 2009).

The Inhibitory Effect of Natural Stilbenes and Their Analogues on

mechanism-based manner (Chang et al, 2001).

**4.2 Natural resveratrol analogues**

stilbenes are summarized in Table 3.

**4.1** *Trans***-resveratrol**

**4. Inhibitory effect of stilbene derivatives on CYP1A enzymes** 

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 525

The studies of the inhibitory effect of phytochemicals on cytochrome P450 dependent enzymes are mainly conducted with the use of *in vitro* techniques on cDNA-expressed enzymes. Recombinant biscistronic supersomes express particular CYP activity and cytochrome c reductase activity. It was reported that resveratrol inhibited human recombinant P450 1A1 activity in a competitive manner (Chun et al., 1999), but the IC50 value (the concentration that causes 50% inhibition of enzyme activity) of 23 M was much higher than the IC50 value of 1.4 M obtained for CYP1B1 inhibition (Chang et al., 2000). Interestingly, resveratrol inactivated human recombinant CYP1A2 indirectly in a

Mechanism-based inhibition was not observed in rat liver microsomes; EROD (7 ethoxyresorufin-O-deethylase) activity as an indicator of both CYP1A1 and CYP1A2 was inhibited by resveratrol and piceatannol (3,3',4,5'-tetrahydroxy-*trans*-stilbene) with Ki value of 0.4 M for both compounds and a mixed type of inhibition (Chang et al., 2007). It was found that resveratrol is metabolized to piceatannol in the reaction of hydroxylation catalyzed by CYP1A2 (Piver et al. 2004) and CYP1B1 (Potter et al. 2002). Poor bioavailability of resveratrol caused by its fast metabolism to glucuronides and sulphates limits the use of this stilbene as a potent chemopreventive / chemotherapeutic agent (Walle et al., 2004). To explain the bioactivity of resveratrol, its accumulation to active levels in target organs or

During the last decade, other naturally occurring stilbenoid compounds with potential health benefit were found and examined. Piceatannol and pterostilbene (3,5-dimethoxy-4' hydroxy-*trans-*stilbene) occur mainly in grapes and blueberries, with their amount depending on plant variety (Rimando et al., 2004). Pterostilbene that was shown to have cancer chemopreventive activity similar to resveratrol (Rimando et al., 2002) occurs also in some medicinal plants used in traditional medicine. Beneficial bioactivity of natural resveratrol analogues have been demonstrated in numerous *in vitro* experiments and in preclinical animal models (Rimando and Suh, 2008). Resveratrol analogues exert multiple bioactivities involved in cancer chemoprevention; for example, they are efficient inhibitors of family 1 cytochromes. The inhibitory action of natural stilbenes appears to be highly selective depending on the cytochrome isoform. Moreover, the extent of CYP inhibition changes according to the stilbene structure; the types and positioning of functional groups linked to the stilbene scaffold significantly influence inhibitory activity of stilbene derivatives. Rhapontigenin (3,5,3'-trihydroxy-4'-methoxystilbene) was found to be a very selective and potent inactivator of CYP1A1 activity with IC50 value 0.4 M and Ki value of 0.09 M (Chun et al., 2001a). Pinostilbene (3,4'-dihydroxy-5-methoxy-*trans*-stilbene), pterostilbene and desoxyrhapontigenin (3,5-dihydroxy-4'-methoxy-*trans*-stilbene) were more efficient inhibitors of CYP1A1 and CYPA2 in comparison to the parent compound, while they inhibited CYP1B1 to the same extent as resveratrol (Guengerich et al., 2003; Mikstacka et al., 2006, 2007). The data on the inhibition of CYP1 enzymes by natural

synergistic / additive effects with other food components are taken into account.


Table 2. AHR as a molecular target for chemopreventive action of resveratrol and its derivatives

#### **4. Inhibitory effect of stilbene derivatives on CYP1A enzymes**

#### **4.1** *Trans***-resveratrol**

524 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

resveratrol 47DRE reporter cell line

resveratrol TCDD-treated MCF-10A cells

resveratrol B[a]P-treated HepG2

resveratrol TCDD treated human

resveratrol lung tissue from BP-

line

HepG2

cells T-47D

cells T-47D

resveratrol TCDD-induced

resveratrol TCDD-induced

piceatannol TCDD-induced

Table 2. AHR as a molecular target for chemopreventive action of resveratrol and its

resveratrol rat primary

resveratrol and 24 other stilbenes

cells and DMBAtreated MCF-7 cells

HepG2 cells

treated mice

hepatocytes

47DRE reporter cell

human breast cancer cell line MCF-7, and human hepatocellular carcinoma cell line,

human breast cancer

human breast cancer

Casper et al., 1999

Chen et al, 2004

Ciolino et al., 1999

Ciolino et al., 1998

Revel et al., 2003

Andrieux et al.,

de Medina et al.,

Beedanagari et al.,

MacPherson and Matthews, 2010

MacPherson and Matthews, 2010

2004

2005

2009

Effect Compound Experimental Model References

AHR translocation AHRE transactivation

AHR DNA binding , expression and activity of

Expression and activity of

constitutive level of CYP1A1 mRNA and reporter vector

CYP 1A1/1B1

CYP1A1/1A2

transcription

formation

resveratrol

CYP1A1 expression BPDE-DNA adduct

CYP1A1 expression by

AHR binding ability of resveratrol and its derivatives

Expression of human CYP1A1 and CYP1B1 Recruitment of the AHR complex and RNA polymerase II to the regulatory regions

AHR-dependent

and CYP1B1

expression

derivatives

transcription of CYP1A1

Recruitment of AHR and ARNT to CYP1A1 and CYP1B1 enhancer regions

CYP1A1 and CYP1B1

CYP1A1 expression Induction of transcription of AHR reporter vector containing the CYP1A1 promoter by TCDD

The studies of the inhibitory effect of phytochemicals on cytochrome P450 dependent enzymes are mainly conducted with the use of *in vitro* techniques on cDNA-expressed enzymes. Recombinant biscistronic supersomes express particular CYP activity and cytochrome c reductase activity. It was reported that resveratrol inhibited human recombinant P450 1A1 activity in a competitive manner (Chun et al., 1999), but the IC50 value (the concentration that causes 50% inhibition of enzyme activity) of 23 M was much higher than the IC50 value of 1.4 M obtained for CYP1B1 inhibition (Chang et al., 2000). Interestingly, resveratrol inactivated human recombinant CYP1A2 indirectly in a mechanism-based manner (Chang et al, 2001).

Mechanism-based inhibition was not observed in rat liver microsomes; EROD (7 ethoxyresorufin-O-deethylase) activity as an indicator of both CYP1A1 and CYP1A2 was inhibited by resveratrol and piceatannol (3,3',4,5'-tetrahydroxy-*trans*-stilbene) with Ki value of 0.4 M for both compounds and a mixed type of inhibition (Chang et al., 2007). It was found that resveratrol is metabolized to piceatannol in the reaction of hydroxylation catalyzed by CYP1A2 (Piver et al. 2004) and CYP1B1 (Potter et al. 2002). Poor bioavailability of resveratrol caused by its fast metabolism to glucuronides and sulphates limits the use of this stilbene as a potent chemopreventive / chemotherapeutic agent (Walle et al., 2004). To explain the bioactivity of resveratrol, its accumulation to active levels in target organs or synergistic / additive effects with other food components are taken into account.

#### **4.2 Natural resveratrol analogues**

During the last decade, other naturally occurring stilbenoid compounds with potential health benefit were found and examined. Piceatannol and pterostilbene (3,5-dimethoxy-4' hydroxy-*trans-*stilbene) occur mainly in grapes and blueberries, with their amount depending on plant variety (Rimando et al., 2004). Pterostilbene that was shown to have cancer chemopreventive activity similar to resveratrol (Rimando et al., 2002) occurs also in some medicinal plants used in traditional medicine. Beneficial bioactivity of natural resveratrol analogues have been demonstrated in numerous *in vitro* experiments and in preclinical animal models (Rimando and Suh, 2008). Resveratrol analogues exert multiple bioactivities involved in cancer chemoprevention; for example, they are efficient inhibitors of family 1 cytochromes. The inhibitory action of natural stilbenes appears to be highly selective depending on the cytochrome isoform. Moreover, the extent of CYP inhibition changes according to the stilbene structure; the types and positioning of functional groups linked to the stilbene scaffold significantly influence inhibitory activity of stilbene derivatives. Rhapontigenin (3,5,3'-trihydroxy-4'-methoxystilbene) was found to be a very selective and potent inactivator of CYP1A1 activity with IC50 value 0.4 M and Ki value of 0.09 M (Chun et al., 2001a). Pinostilbene (3,4'-dihydroxy-5-methoxy-*trans*-stilbene), pterostilbene and desoxyrhapontigenin (3,5-dihydroxy-4'-methoxy-*trans*-stilbene) were more efficient inhibitors of CYP1A1 and CYPA2 in comparison to the parent compound, while they inhibited CYP1B1 to the same extent as resveratrol (Guengerich et al., 2003; Mikstacka et al., 2006, 2007). The data on the inhibition of CYP1 enzymes by natural stilbenes are summarized in Table 3.

The Inhibitory Effect of Natural Stilbenes and Their Analogues on

with recognized CYP1A1/2 inhibitory properties (Rodeiro et al., 2009).

CYP1A1 (IC50 < 0.1 µM) and CYP1A2 (IC50 < 0.3 µM).

stilbenes comprise: flavonoids, isothiocyanates, coumarin and its derivatives.

Numerous natural phenols demonstrate inhibitory activity toward CYP1 enzymes. Phytochemicals that exert inhibitory effects on CYP1A enzymes comparable to natural

**Flavonoids** represent a large class of phenolic phytochemicals. They are ubiquitously present in plant-derived foods and are important microcomponents of the human diet. Humans ingest approximately 0.6-1 g of these bioactive compounds daily (Kuhnau, 1976). The effects of flavonoids on CYP1 activities have been explored since the early nineties, including the effects of flavone and five hydroxylated derivatives on the methoxyresorufin O-demethylase activity catalyzed by human recombinant CYP1A1 and CYP1A2 (Zhai et al., 1998). The authors found galangin (3,5,7-trihydroxyflavone) as the most potent inhibitor of CYP1A2 with Ki value of 8 nM. It should be mentioned that no stilbene derivative with a comparable inhibitory potency toward CYP1A2 was found. Furthermore, galangin showed almost 5-fold selectivity for CYP1A2 over CYP1A1; while, 7-hydroxyflavone exhibited 6-fold greater selectivity for CYP1A1 over CYP1A2. The other hydroxylated flavone derivatives: 3 hydroxy; 5-hydroxy; 7-hydroxy- and 3,7-dihydroxyflavone were also potent inhibitors of

In experiments with the use of human recombinant CYPs, seven flavonoids (myricetin, apigenin, kaempferol, quercetin, amentoflavone, quercitrin, and rutin) occurring in St. John's Wort were tested. They were found to be slightly more selective for CYP1B1 activity compared to CYP1A1. Apigenin and amentoflavone were competitive inhibitors of CYP1B1, while quercetin showed a mixed type of inhibition. The most potent CYP1B1 inhibitor was apigenin with Ki of 60 nM. The same authors investigated CYP1 inhibition in cell system. Myricetin, apigenin, kaempferol and quercetin inhibited TCDD-induced EROD activity in

**4.4 Other natural phenols**

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 527

The influence of other phenolic phytochemicals on CYP1 activities is worth presenting in the context of possible additive or synergistic effects of the micro-components of human diet. The properties of plant extracts rich in numerous bioactive substances are particularly interesting in terms of herb-drug interaction, which could be a subject of independent review. At the beginning of the last decade, Piver and collaborators (2003) discovered that non-volatile components of red wine or various Cognac beverages exert stronger inhibitory effect on CYP1A1, CYP1A2, and CYP1B1 than resveratrol and its dimer ε-viniferin. Another extract, prepared from the most widely used herbal medicine *Ginkgo biloba,* was tested for its ability to inhibit the major human cytochrome P450 enzymes (Gaudineau et al., 2004). It was demonstrated that the flavonoidic fraction of standardized extract inhibits human CYP1A2 and other cytochromes (CYP2C9, CYP2E1, and CYP3A4), wheras its terpenoidic fraction significantly inhibits only CYP2C9. *In vivo* CYP1A2 induction was observed as a result of herbal dietary supplementation (Rye et al., 2003). Effects of Cuban and Mexican herbal extracts used in traditional medicine (obtained from *Heliopsis longipes*, *Mangifera indica* L. and *Thalassia testudinum*) on CYP1A1/2 and other cytochromes involved in drug metabolism of CYP3A4 and CYP2D6 were studied with the use of human liver microsomes and compared with the pure constituents isolated from the extracts of affinin (an alkamide isolated from the *H. longipes* extract), N-*iso*-butyl-decanamide, and mangiferin. The extracts significantly inhibited CYP1A1/2 activities, which reflects the high content of flavonoids

#### **4.3 Resveratrol methyl ethers and other synthetic stilbenes**

In the last decade, new stilbene derivatives have been designed and synthesized in order to find more potent chemopreventive agents (Szekeres et al., 2010). The additional aim of this approach was to find resveratrol derivatives demonstrating better bioavailability in comparison to the parent compound. The bioactivity of resveratrol analogues could be altered due to the presence and positioning of methoxy groups on the basic resveratrol backbone that prevent the conjugation reaction with sulphuric and glucuronic acids. Synthesized derivatives are tested with regard to their inhibitory activity toward CYP 1 enzymes in order to find more efficient and selective inhibitors. A series of *trans*-stilbene derivatives containing a 3,5-dimethoxyphenyl moiety were prepared and evaluated on human recombinant CYP1A, CYP1A2 and CYP1B1 to find a potent and selective CYP1B1 inhibitor. It was shown that substitution at the 2-position of the stilbene skeleton plays a very important role in discriminating between CYP1A1/2 and CYP1B1. Chun and his group found 3,5,2',4'-tetramethoxy-*trans*-stilbene as a new selective and very potent inhibitor of human CYP1B1 (Chun et al., 2001b). Among the whole series of compounds tested, 3,5,2',4' tetramethoxy-*trans*-stilbene exerted the most potent inhibitory activity toward CYP1B1 with an IC50 value of 2 nM. 2-[2-(3,5-dimethoxyphenyl)vinyl]tiophene showed comparable inhibitory activities, but its selectivity toward CYP 1B1 was lower (Kim et al. 2002).

Another series of stilbenes with 4-methylthiophenyl moiety were synthesized and their inhibitory potency toward human recombinant CYPs: CYP1A1, CYP1A2 and CYP1B1 was evaluated. Among compounds tested, 2-methoxy-4'-methylthio-*trans*-stilbene and 3 methoxy-4'-methylthio*-trans*-stilbene demonstrated the most potent and selective inhibitory effect on CYP1A1 and CYP1B1 activities (Mikstacka et al, 2008).


a Chang et al., 2001; b Chun et al., 2001; c in mouse liver microsomes (Mikstacka et al., 2006); n.d. not determined

Table 3. Effect of natural *trans*-stilbenes on human recombinant CYP1A1, CYP1A2 and CYP1B1 activities

#### **4.4 Other natural phenols**

526 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

In the last decade, new stilbene derivatives have been designed and synthesized in order to find more potent chemopreventive agents (Szekeres et al., 2010). The additional aim of this approach was to find resveratrol derivatives demonstrating better bioavailability in comparison to the parent compound. The bioactivity of resveratrol analogues could be altered due to the presence and positioning of methoxy groups on the basic resveratrol backbone that prevent the conjugation reaction with sulphuric and glucuronic acids. Synthesized derivatives are tested with regard to their inhibitory activity toward CYP 1 enzymes in order to find more efficient and selective inhibitors. A series of *trans*-stilbene derivatives containing a 3,5-dimethoxyphenyl moiety were prepared and evaluated on human recombinant CYP1A, CYP1A2 and CYP1B1 to find a potent and selective CYP1B1 inhibitor. It was shown that substitution at the 2-position of the stilbene skeleton plays a very important role in discriminating between CYP1A1/2 and CYP1B1. Chun and his group found 3,5,2',4'-tetramethoxy-*trans*-stilbene as a new selective and very potent inhibitor of human CYP1B1 (Chun et al., 2001b). Among the whole series of compounds tested, 3,5,2',4' tetramethoxy-*trans*-stilbene exerted the most potent inhibitory activity toward CYP1B1 with an IC50 value of 2 nM. 2-[2-(3,5-dimethoxyphenyl)vinyl]tiophene showed comparable

inhibitory activities, but its selectivity toward CYP 1B1 was lower (Kim et al. 2002).

Compound CYP1A1 CYP1A2 CYP1B1

Piceatannol 3.01 competitive 9.67c mixed type 0.27 competitive

Desoxyrhapontigenin 0.16 competitive 1.04 mixed type 2.06 competitive

Pinostilbene 0.13 mixed type 0.94 mixed type 0.90 competitive

Pterostilbene 0.57 competitive 0.39c mixed type 0.91 competitive

a Chang et al., 2001; b Chun et al., 2001; c in mouse liver microsomes (Mikstacka et al., 2006); n.d. not

Table 3. Effect of natural *trans*-stilbenes on human recombinant CYP1A1, CYP1A2 and

competitive

*Ki*  [M]

5.33c

160 (IC50) n.d.

Mode of inhibition

*Ki* [M]

mixed type 0.75a mixed type

9 (IC50) n.d.

Mode of inhibition

Mode of inhibition

effect on CYP1A1 and CYP1B1 activities (Mikstacka et al, 2008).

*Ki* [M]

Resveratrol 1.2a mixed type 15.5a

0.4 (IC50)

Rhapontigenin 0.21b

determined

CYP1B1 activities

Another series of stilbenes with 4-methylthiophenyl moiety were synthesized and their inhibitory potency toward human recombinant CYPs: CYP1A1, CYP1A2 and CYP1B1 was evaluated. Among compounds tested, 2-methoxy-4'-methylthio-*trans*-stilbene and 3 methoxy-4'-methylthio*-trans*-stilbene demonstrated the most potent and selective inhibitory

**4.3 Resveratrol methyl ethers and other synthetic stilbenes**

The influence of other phenolic phytochemicals on CYP1 activities is worth presenting in the context of possible additive or synergistic effects of the micro-components of human diet. The properties of plant extracts rich in numerous bioactive substances are particularly interesting in terms of herb-drug interaction, which could be a subject of independent review. At the beginning of the last decade, Piver and collaborators (2003) discovered that non-volatile components of red wine or various Cognac beverages exert stronger inhibitory effect on CYP1A1, CYP1A2, and CYP1B1 than resveratrol and its dimer ε-viniferin. Another extract, prepared from the most widely used herbal medicine *Ginkgo biloba,* was tested for its ability to inhibit the major human cytochrome P450 enzymes (Gaudineau et al., 2004). It was demonstrated that the flavonoidic fraction of standardized extract inhibits human CYP1A2 and other cytochromes (CYP2C9, CYP2E1, and CYP3A4), wheras its terpenoidic fraction significantly inhibits only CYP2C9. *In vivo* CYP1A2 induction was observed as a result of herbal dietary supplementation (Rye et al., 2003). Effects of Cuban and Mexican herbal extracts used in traditional medicine (obtained from *Heliopsis longipes*, *Mangifera indica* L. and *Thalassia testudinum*) on CYP1A1/2 and other cytochromes involved in drug metabolism of CYP3A4 and CYP2D6 were studied with the use of human liver microsomes and compared with the pure constituents isolated from the extracts of affinin (an alkamide isolated from the *H. longipes* extract), N-*iso*-butyl-decanamide, and mangiferin. The extracts significantly inhibited CYP1A1/2 activities, which reflects the high content of flavonoids with recognized CYP1A1/2 inhibitory properties (Rodeiro et al., 2009).

Numerous natural phenols demonstrate inhibitory activity toward CYP1 enzymes. Phytochemicals that exert inhibitory effects on CYP1A enzymes comparable to natural stilbenes comprise: flavonoids, isothiocyanates, coumarin and its derivatives.

**Flavonoids** represent a large class of phenolic phytochemicals. They are ubiquitously present in plant-derived foods and are important microcomponents of the human diet. Humans ingest approximately 0.6-1 g of these bioactive compounds daily (Kuhnau, 1976). The effects of flavonoids on CYP1 activities have been explored since the early nineties, including the effects of flavone and five hydroxylated derivatives on the methoxyresorufin O-demethylase activity catalyzed by human recombinant CYP1A1 and CYP1A2 (Zhai et al., 1998). The authors found galangin (3,5,7-trihydroxyflavone) as the most potent inhibitor of CYP1A2 with Ki value of 8 nM. It should be mentioned that no stilbene derivative with a comparable inhibitory potency toward CYP1A2 was found. Furthermore, galangin showed almost 5-fold selectivity for CYP1A2 over CYP1A1; while, 7-hydroxyflavone exhibited 6-fold greater selectivity for CYP1A1 over CYP1A2. The other hydroxylated flavone derivatives: 3 hydroxy; 5-hydroxy; 7-hydroxy- and 3,7-dihydroxyflavone were also potent inhibitors of CYP1A1 (IC50 < 0.1 µM) and CYP1A2 (IC50 < 0.3 µM).

In experiments with the use of human recombinant CYPs, seven flavonoids (myricetin, apigenin, kaempferol, quercetin, amentoflavone, quercitrin, and rutin) occurring in St. John's Wort were tested. They were found to be slightly more selective for CYP1B1 activity compared to CYP1A1. Apigenin and amentoflavone were competitive inhibitors of CYP1B1, while quercetin showed a mixed type of inhibition. The most potent CYP1B1 inhibitor was apigenin with Ki of 60 nM. The same authors investigated CYP1 inhibition in cell system. Myricetin, apigenin, kaempferol and quercetin inhibited TCDD-induced EROD activity in

The Inhibitory Effect of Natural Stilbenes and Their Analogues on

Opong et al., 2008).

2003).

protein and ligand.

0.37 M, respectively (Badal et al., 2011).

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 529

**Curcumin** is a natural plant food additive obtained from turmeric used in spices and traditional Indian medicine. Its chemopreventive anticancer potential is well documented (Aggarwal et al., 2003). It belongs to hydroxycinnamic acid derivatives observed ubiquitously in plants. Earlier reports on the inhibition of rat liver microsomal CYPs by curcumin showed that curcumin is a strong inhibitor of CYP1A enzymes and CYP2B as well (Oetari et al., 1996; Thapliyal and Maru, 2001, 2003). However, these data were not confirmed in studies with human recombinant cytochrome P450s, where curcumin appeared to be a moderate inhibitor of CYP1A2 with IC50 value 40 M (Appiah-Opong et al., 2007). Appiah-Opong and coworkers synthesized curcumin derivatives that exhibited about 10- to 40-fold greater potency towards inhibition of CYP1A2 than curcumin itself (Appiah-

Other natural phenols studied more recently with respect to CYP1 inhibition include **phytocannabinoids,** constituents of marijuana, and **chromene amides** from *Amyris plumieri*, a plant grown in the Caribbean, Central America and Venezuela used in folk medicine. Three major constituents in marijuana; 9-tetrahydrocannabinol, cannabidiol and cannabinol inhibited activities of human recombinant CYP1s: CYP1A1, CYP1A2 and CYP1B1 in a competitive manner (Yamaori et al., 2010). One of the amides (chromene acetamide) tested appeared to inhibit potently CYP1A1 activity *in vitro* with IC50 and Ki values 1.547 M and

Interestingly, in the studies on different natural phenols Schwarz and Roots demonstrated that the inhibitory effect depends not only on the structure of the inhibitor, but also the substrate of the reaction catalyzed by CYPs used in the assay. They found flavonoids like myricetin, apigenin, quercetin, and kaempferol, as well as tea polyphenol (-)epigallo catechin gallate, strongly inhibited the formation of benzo(a)pyrene diolepoxide, the ultimate carcinogenic product of benzo(a)pyrene activation. Furthermore, resveratrol, an inhibitor of CYP1A1-catalyzed ethoxyresorufin deethylation, exhibited only slightly inhibitory effect on CYP1A1-mediated epoxidation of 7,8-diol-B(a)P (Schwarz & Roots,

**5. Docking studies – The new approach to CYPs-phytochemical interaction**  Mechanistic studies of the inhibitory effect of stilbenes on enzyme activities are mainly conducted *in vitro* with the use of human recombinant cytochromes. However, the affinity of compounds to cytochromes may be determined by computational analysis of inhibitor/substrate docking in the enzyme active site. Molecular modeling is presumed to be helpful in predicting inhibitory potential of CYP regulators by characteristics of ligandenzyme interactions. We review *in silico* research on elucidating the mechanism of inhibitory action of phytochemicals by analysis of structure and activity relationship. Potential phytochemical candidates can be selected by *in silico* virtual screening, based on natural compound libraries (www.bioscreening.com). When active chemicals are selected, they may be "docked" into the target protein by using available programs, enabling detailed proteinligand interactions to be obtained and the best fit of a candidate compound to be identified. The main objective of molecular docking is to determine the binding interactions between

intact 22Rv1 human prostate cancer cells. Because flavonoids were added 30 minutes prior to the EROD assay, the inhibition did not reflect down regulation of CYP1 mRNA or protein level (Chaudhary et al., 2006). The influence of flavonoid constituents of St. John's Wort were also studied by Schwarz's group. They demonstrated the differentiated inhibition of CYP1A1-catalyzed estradiol 2-hydroxylation according to CYP1A1 genotype. The variant CYP1A1.2 (Ile462Val) was significantly inhibited by quercetin, hypericin and pseudohypericin (naphthodiantrones), with IC50 values for 2-hydroxylation being more than two times lower than the wild-type enzyme. Additionally, the wild-type enzyme was efficiently inhibited by kaempferol, myricetin and resveratrol (Schwarz et al., 2011).

The synthesis of structures differentiated by type and positions of substituents leads to a continuation of structure and activity relationship (SAR) studies. Recently, Takemura and coworkers (2010) evaluated the structure–property relationship of 18 major flavonoids on inhibiting enzymatic activity of CYP1A1, 1A2 and 1B1 by using an ethoxyresorufin Odeethylation assay. Flavones and flavonols indicated relatively strong inhibitory effects on CYP1s compared with flavanone that does not have the double bond between Cpositions 2 and 3 on the C-ring. Flavonoids used in this study selectively inhibited CYP1B1 activity.

Special attention is paid to methoxy derivatives of flavone, which have inhibitory potency exceeding that of the parent compound (Walle & Walle, 2007). In particular, methoxy types of flavones and flavonols such as chrysoeriol and isorhamnetin showed strong and selective inhibition against CYP1B1 (Takemura et al., 2010). The most potent inhibitors of CYP1 catalyzed ethoxyresorufin O-deethylation were the methoxylated flavones acacetin, diosmetin, eupatorin and the dihydroxylated flavone chrysin, indicating that the 4'-OCH3 group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition (Androutsopoulos et al., 2011). It was observed that high metabolic turnover of methoxylated flavonoids may result in enhanced antiproliferative activity. Several flavonoid metabolites produced in reactions catalyzed by CYP1A1 or CYP1B1 have been shown to inhibit cancer cell cycle progression. The authors observed CYP1A1-catalyzed biotransformation of acacetin to luteolin, apigenin and scutellarein. The chemopreventive ability of these metabolites was previously established. Generally, it is suggested that dietary flavonoids exhibit three distinct modes of action with CYP1 enzymes: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1 enzymes.

**Coumarin (1,2-benzopyrone) and its derivatives** occur naturally in several plant families. They are components of essential oils, and are often used as fragrance ingredients in human diet. Their effect on CYP1 activities have been studied since the early nineties. The naturally occurring coumarins: bergamotin, coriandrin, isoimperatorin, imperatorin, ostruthin are potent inhibitors of the metabolic activation of benzo(a)pyrene and dimethylbenzanthracene in the cell culture model system of mouse epidermis (Cai et al., 1997). In experiments *in vitro*, mechanism-based inactivation of hepatic EROD activity by natural coumarin coriandrin was observed (Cai et al., 1996). These results demonstrate that certain coumarins to which humans are exposed in their diet are bioactivated by CYP1A1 to reactive intermediates that subsequently form covalent adducts with the apoprotein, effectively destroying enzyme activity.

intact 22Rv1 human prostate cancer cells. Because flavonoids were added 30 minutes prior to the EROD assay, the inhibition did not reflect down regulation of CYP1 mRNA or protein level (Chaudhary et al., 2006). The influence of flavonoid constituents of St. John's Wort were also studied by Schwarz's group. They demonstrated the differentiated inhibition of CYP1A1-catalyzed estradiol 2-hydroxylation according to CYP1A1 genotype. The variant CYP1A1.2 (Ile462Val) was significantly inhibited by quercetin, hypericin and pseudohypericin (naphthodiantrones), with IC50 values for 2-hydroxylation being more than two times lower than the wild-type enzyme. Additionally, the wild-type enzyme was

efficiently inhibited by kaempferol, myricetin and resveratrol (Schwarz et al., 2011).

CYP1B1 activity.

enzymes.

destroying enzyme activity.

The synthesis of structures differentiated by type and positions of substituents leads to a continuation of structure and activity relationship (SAR) studies. Recently, Takemura and coworkers (2010) evaluated the structure–property relationship of 18 major flavonoids on inhibiting enzymatic activity of CYP1A1, 1A2 and 1B1 by using an ethoxyresorufin Odeethylation assay. Flavones and flavonols indicated relatively strong inhibitory effects on CYP1s compared with flavanone that does not have the double bond between Cpositions 2 and 3 on the C-ring. Flavonoids used in this study selectively inhibited

Special attention is paid to methoxy derivatives of flavone, which have inhibitory potency exceeding that of the parent compound (Walle & Walle, 2007). In particular, methoxy types of flavones and flavonols such as chrysoeriol and isorhamnetin showed strong and selective inhibition against CYP1B1 (Takemura et al., 2010). The most potent inhibitors of CYP1 catalyzed ethoxyresorufin O-deethylation were the methoxylated flavones acacetin, diosmetin, eupatorin and the dihydroxylated flavone chrysin, indicating that the 4'-OCH3 group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition (Androutsopoulos et al., 2011). It was observed that high metabolic turnover of methoxylated flavonoids may result in enhanced antiproliferative activity. Several flavonoid metabolites produced in reactions catalyzed by CYP1A1 or CYP1B1 have been shown to inhibit cancer cell cycle progression. The authors observed CYP1A1-catalyzed biotransformation of acacetin to luteolin, apigenin and scutellarein. The chemopreventive ability of these metabolites was previously established. Generally, it is suggested that dietary flavonoids exhibit three distinct modes of action with CYP1 enzymes: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1

**Coumarin (1,2-benzopyrone) and its derivatives** occur naturally in several plant families. They are components of essential oils, and are often used as fragrance ingredients in human diet. Their effect on CYP1 activities have been studied since the early nineties. The naturally occurring coumarins: bergamotin, coriandrin, isoimperatorin, imperatorin, ostruthin are potent inhibitors of the metabolic activation of benzo(a)pyrene and dimethylbenzanthracene in the cell culture model system of mouse epidermis (Cai et al., 1997). In experiments *in vitro*, mechanism-based inactivation of hepatic EROD activity by natural coumarin coriandrin was observed (Cai et al., 1996). These results demonstrate that certain coumarins to which humans are exposed in their diet are bioactivated by CYP1A1 to reactive intermediates that subsequently form covalent adducts with the apoprotein, effectively **Curcumin** is a natural plant food additive obtained from turmeric used in spices and traditional Indian medicine. Its chemopreventive anticancer potential is well documented (Aggarwal et al., 2003). It belongs to hydroxycinnamic acid derivatives observed ubiquitously in plants. Earlier reports on the inhibition of rat liver microsomal CYPs by curcumin showed that curcumin is a strong inhibitor of CYP1A enzymes and CYP2B as well (Oetari et al., 1996; Thapliyal and Maru, 2001, 2003). However, these data were not confirmed in studies with human recombinant cytochrome P450s, where curcumin appeared to be a moderate inhibitor of CYP1A2 with IC50 value 40 M (Appiah-Opong et al., 2007). Appiah-Opong and coworkers synthesized curcumin derivatives that exhibited about 10- to 40-fold greater potency towards inhibition of CYP1A2 than curcumin itself (Appiah-Opong et al., 2008).

Other natural phenols studied more recently with respect to CYP1 inhibition include **phytocannabinoids,** constituents of marijuana, and **chromene amides** from *Amyris plumieri*, a plant grown in the Caribbean, Central America and Venezuela used in folk medicine. Three major constituents in marijuana; 9-tetrahydrocannabinol, cannabidiol and cannabinol inhibited activities of human recombinant CYP1s: CYP1A1, CYP1A2 and CYP1B1 in a competitive manner (Yamaori et al., 2010). One of the amides (chromene acetamide) tested appeared to inhibit potently CYP1A1 activity *in vitro* with IC50 and Ki values 1.547 M and 0.37 M, respectively (Badal et al., 2011).

Interestingly, in the studies on different natural phenols Schwarz and Roots demonstrated that the inhibitory effect depends not only on the structure of the inhibitor, but also the substrate of the reaction catalyzed by CYPs used in the assay. They found flavonoids like myricetin, apigenin, quercetin, and kaempferol, as well as tea polyphenol (-)epigallo catechin gallate, strongly inhibited the formation of benzo(a)pyrene diolepoxide, the ultimate carcinogenic product of benzo(a)pyrene activation. Furthermore, resveratrol, an inhibitor of CYP1A1-catalyzed ethoxyresorufin deethylation, exhibited only slightly inhibitory effect on CYP1A1-mediated epoxidation of 7,8-diol-B(a)P (Schwarz & Roots, 2003).

#### **5. Docking studies – The new approach to CYPs-phytochemical interaction**

Mechanistic studies of the inhibitory effect of stilbenes on enzyme activities are mainly conducted *in vitro* with the use of human recombinant cytochromes. However, the affinity of compounds to cytochromes may be determined by computational analysis of inhibitor/substrate docking in the enzyme active site. Molecular modeling is presumed to be helpful in predicting inhibitory potential of CYP regulators by characteristics of ligandenzyme interactions. We review *in silico* research on elucidating the mechanism of inhibitory action of phytochemicals by analysis of structure and activity relationship. Potential phytochemical candidates can be selected by *in silico* virtual screening, based on natural compound libraries (www.bioscreening.com). When active chemicals are selected, they may be "docked" into the target protein by using available programs, enabling detailed proteinligand interactions to be obtained and the best fit of a candidate compound to be identified. The main objective of molecular docking is to determine the binding interactions between protein and ligand.

The Inhibitory Effect of Natural Stilbenes and Their Analogues on

CYP1A1 (Androutsopoulos et al., 2011).

mode B, or Asn265 and Asn228 in mode A (Fig. 3d and 3e).

ligand to the binding site.

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 531

active site where it may cause a direct effect on substrate binding (Cho et al., 2003). Further studies with the use of molecular docking were aimed at methoxyflavonoids with a 2–3 double bond, which exerted strong inhibitory effect on CYP1 activities, particularly CYP1B1 (Takemura et al., 2010). The authors observed that the binding specificity of methoxyflavonoids is based on the interactions between the methoxy groups and specific CYP1s residues. For example, chrysoeriol and isorhamnetin fit well into the active site of CYP1B1, but do not fit into the active site of CYP1A2 and 1A1 because of steric collisions between the methoxy substituent of these methoxyflavonoids and Ser122 in CYP1A1 and Thr124 in CYP1A2. Androutsopoulos's group described molecular docking of several flavonoids with regard to their metabolism and inhibitory activity. The simulated binding orientation of the compounds tested was in accordance with the study of Takemura and coworkers (2010). Diosmetin and eupatorin are predicted to be oriented with ring-B over the prosthetic group so that 4'-methoxy group is at ~4.5 Ǻ from the heme iron. The less substituted chrysin and acacetin also were shown to bind CYP1A1 with ring-B over the ironheme group. However, a lower number of interactions were found within the active site of

To better characterize stilbenes as ligands of CYPs, we performed molecular docking by simulation of resveratrol and pterostilbene binding in active sites of CYP1A2 and CYP1B. Resveratrol and pterostilbene molecules were docked into the cavities of CYP1A2 (PDB code: 2hi4) and CYP1B1 (PDB code: 3pm0) with the use of the CDOCKER procedure implemented in Accelrys Discovery Studio 2.5.5. CDOCKER uses a CHARMm-based molecular dynamics (MD) scheme to dock ligands into a receptor binding site. For assigning receptor and ligand atom partial charges, we applied the charging rules used in the MMFF94 forcefield. Docked poses were scored by the negative value of CDOCKER energy for the –CDOCKER\_ENERGY function, which include interaction energy and internal ligand energy: the higher positive value of –CDOCKER\_ENERGY, the stronger affinity of a

Our docking experiment showed that in the CYP1A2 active site, all possible poses of resveratrol can be grouped into two sets. This observation indicated that two binding modes are possible for resveratrol molecule. In mode A, represented by the pose with highest score, a resveratrol molecule is directed with 4'-OH group toward a heme (Fig. 3a). In mode B, the second ring with 3-OH and 5-OH substituents is situated in the vicinity of a prosthetic group (Fig. 3b). In both orientations, resveratrol binding is stabilized by – stacking interactions, with phenyl ring of Phe226 (mode A), and with Phe226 and Phe260 (mode B). Contrary to resveratrol, a pterostilbene molecule was docked in the CYP1A2 active site only in one orientation with 4'-OH group directed toward a heme (Fig. 3c). Pterostilbene binding was stabilized by – interaction with an aromatic ring of Phe226 . For a resveratrol molecule docked in the active site of CYP1B1, we also distinguished two binding modes. In contrast to CYP1A2, the highest scored pose corresponded to binding mode B. In both orientations (A and B), resveratrol was stabilized by two – interactions between both of its rings and a phenyl ring of Phe231, and additionally by two hydrogen bonds with Asn265 and Asp333 in

Similar to interaction with CYP1A2, a pterostilbene molecule represented only one type of orientation in the CYP1B1 cavity (Fig. 2f). The binding conformation with 4'-OH group close to a heme was stabilized by two – stacking interactions with Phe231. In the case of

Computational procedures of molecular modeling have been employed since the nineties. Studies of Lewis and coworkers (1997) on CYP1 family enzymes structure and ligand docking in enzyme cavities have been a great contribution to the development of this field. Lewis formulated the general characteristic of CYP1 ligands as planar and polar polycyclic molecules. Substituents linked to the polycyclic hydrocarbon core influence the ligand binding responsible for molecular interactions: hydrogen bonds; - stacking; and hydrophobic interactions. The effect of structural modification on the inhibitory selectivity of phytochemical derivatives on CYP1A1, CYP1A2, and CYP1B1 help to elucidate which interactions determine the inhibitory ability of the compounds. There are similarities between the active sites of CYP1A2 and CYP1A1 which are in accordance with the overlapping substrate specificities of the two enzymes. However, the CYP1A1 substrates are generally of higher lipophilicity than those of CYP1A2. The reason lies in the more hydrophobic character of the CYP1A1 active site region (including the access channel) in comparison to CYP1A2 active site (Lewis et al., 1999). The differences in the structure of enzyme binding sites may determine the metabolism pathways of a substrate. With the use of computational docking the mechanism of E2 2-hydroxylation and 4-hydroxylation catalyzed by CYP1A1/2 and CYP1B1, respectively, were elucidated. CYP1A1 and CYP1A2 produced 2-OH-E2 and 4-OH-E2 in a ratio of 10 : 1; whereas CYP1B1 produces 2-OH-E2 and 4-OH-E2 in a ratio of 1 : 3 (Lee et al., 2003). The docking study suggests that CYP1A1 and CYP1A2 generate 2-OH-E2 rather than 4-OH-E2, and that CYP1B1 generates both 2-OH-E2 and 4-OH-E2. Particular amino acids residues for each CYP were identified as playing an important role in estradiol recognition (Itoh et al., 2010).

Several groups of phytochemicals were tested for affinity to active sites of CYP1 members. The first studied compounds were rutaecarpine derivatives. An alkaloid rutaecarpine preferentially inhibited CYP1A2 activity with IC50 value of 22 nM. However, 1-methoxyrutaecarpine and 1,2 dimethoxyrutaecarpine were the most selective CYP1A2 inhibitors. Molecular modeling showed a good fitting of rutaecarpine and the active site of CYP1A2. Two hydrogen bonds between the keto- and N14-groups of rutaecarpine and the Thr208 and Thr473 residues of CYP1A2, respectively, were visualized with molecular modeling procedures. The C-ring moiety of rutaecarpine formed - stacking interaction with the aromatic ring of Phe205 residue (Don et al., 2003).

Coumarin was shown to be a substrate of human CYPs, specifically: CYP1A1 and CYP1A2. Molecular modeling led to recognition and localization of the amino acid residues which interact with coumarin molecules resulting in the orientation of coumarin with 3,4 bond directly above the heme moiety. Coumarin 3,4-epoxide is produced and then rearranged to hydroxyphenylacetaldehyde, which can be further metabolized to toxic products. In the CYP1A1 active site, Ser113 forms a hydrogen bond with coumarin, while Phe205 and Phe358 are responsible for aromatic - stacking. In CYP1A2, Thr113 forms hydrogen bonds with coumarin, and Phe205 is responsible for - stacking (Lewis et al., 2006). However the different key residues take part in the interactions with coumarin, they determine the same site of metabolism, and in consequence, the pathway of coumarin metabolism is the same for both CYP1A1 and CYP1A2 .

7,8-benzoflavone (-naphthoflavone) is a prototype flavonoid which has been used to examine the mechanism of action on P450 enzymes. Molecular modeling studies revealed that 7,8-naphthoflavone is positioned in a hydrophobic cavity of CYP1A2 next to the

Computational procedures of molecular modeling have been employed since the nineties. Studies of Lewis and coworkers (1997) on CYP1 family enzymes structure and ligand docking in enzyme cavities have been a great contribution to the development of this field. Lewis formulated the general characteristic of CYP1 ligands as planar and polar polycyclic molecules. Substituents linked to the polycyclic hydrocarbon core influence the ligand binding responsible for molecular interactions: hydrogen bonds; - stacking; and hydrophobic interactions. The effect of structural modification on the inhibitory selectivity of phytochemical derivatives on CYP1A1, CYP1A2, and CYP1B1 help to elucidate which interactions determine the inhibitory ability of the compounds. There are similarities between the active sites of CYP1A2 and CYP1A1 which are in accordance with the overlapping substrate specificities of the two enzymes. However, the CYP1A1 substrates are generally of higher lipophilicity than those of CYP1A2. The reason lies in the more hydrophobic character of the CYP1A1 active site region (including the access channel) in comparison to CYP1A2 active site (Lewis et al., 1999). The differences in the structure of enzyme binding sites may determine the metabolism pathways of a substrate. With the use of computational docking the mechanism of E2 2-hydroxylation and 4-hydroxylation catalyzed by CYP1A1/2 and CYP1B1, respectively, were elucidated. CYP1A1 and CYP1A2 produced 2-OH-E2 and 4-OH-E2 in a ratio of 10 : 1; whereas CYP1B1 produces 2-OH-E2 and 4-OH-E2 in a ratio of 1 : 3 (Lee et al., 2003). The docking study suggests that CYP1A1 and CYP1A2 generate 2-OH-E2 rather than 4-OH-E2, and that CYP1B1 generates both 2-OH-E2 and 4-OH-E2. Particular amino acids residues for each CYP were identified as playing an

Several groups of phytochemicals were tested for affinity to active sites of CYP1 members. The first studied compounds were rutaecarpine derivatives. An alkaloid rutaecarpine preferentially inhibited CYP1A2 activity with IC50 value of 22 nM. However, 1-methoxyrutaecarpine and 1,2 dimethoxyrutaecarpine were the most selective CYP1A2 inhibitors. Molecular modeling showed a good fitting of rutaecarpine and the active site of CYP1A2. Two hydrogen bonds between the keto- and N14-groups of rutaecarpine and the Thr208 and Thr473 residues of CYP1A2, respectively, were visualized with molecular modeling procedures. The C-ring moiety of rutaecarpine formed - stacking interaction with the aromatic ring of Phe205

Coumarin was shown to be a substrate of human CYPs, specifically: CYP1A1 and CYP1A2. Molecular modeling led to recognition and localization of the amino acid residues which interact with coumarin molecules resulting in the orientation of coumarin with 3,4 bond directly above the heme moiety. Coumarin 3,4-epoxide is produced and then rearranged to hydroxyphenylacetaldehyde, which can be further metabolized to toxic products. In the CYP1A1 active site, Ser113 forms a hydrogen bond with coumarin, while Phe205 and Phe358 are responsible for aromatic - stacking. In CYP1A2, Thr113 forms hydrogen bonds with coumarin, and Phe205 is responsible for - stacking (Lewis et al., 2006). However the different key residues take part in the interactions with coumarin, they determine the same site of metabolism, and in consequence, the pathway of coumarin metabolism is the same

7,8-benzoflavone (-naphthoflavone) is a prototype flavonoid which has been used to examine the mechanism of action on P450 enzymes. Molecular modeling studies revealed that 7,8-naphthoflavone is positioned in a hydrophobic cavity of CYP1A2 next to the

important role in estradiol recognition (Itoh et al., 2010).

residue (Don et al., 2003).

for both CYP1A1 and CYP1A2 .

active site where it may cause a direct effect on substrate binding (Cho et al., 2003). Further studies with the use of molecular docking were aimed at methoxyflavonoids with a 2–3 double bond, which exerted strong inhibitory effect on CYP1 activities, particularly CYP1B1 (Takemura et al., 2010). The authors observed that the binding specificity of methoxyflavonoids is based on the interactions between the methoxy groups and specific CYP1s residues. For example, chrysoeriol and isorhamnetin fit well into the active site of CYP1B1, but do not fit into the active site of CYP1A2 and 1A1 because of steric collisions between the methoxy substituent of these methoxyflavonoids and Ser122 in CYP1A1 and Thr124 in CYP1A2. Androutsopoulos's group described molecular docking of several flavonoids with regard to their metabolism and inhibitory activity. The simulated binding orientation of the compounds tested was in accordance with the study of Takemura and coworkers (2010). Diosmetin and eupatorin are predicted to be oriented with ring-B over the prosthetic group so that 4'-methoxy group is at ~4.5 Ǻ from the heme iron. The less substituted chrysin and acacetin also were shown to bind CYP1A1 with ring-B over the ironheme group. However, a lower number of interactions were found within the active site of CYP1A1 (Androutsopoulos et al., 2011).

To better characterize stilbenes as ligands of CYPs, we performed molecular docking by simulation of resveratrol and pterostilbene binding in active sites of CYP1A2 and CYP1B. Resveratrol and pterostilbene molecules were docked into the cavities of CYP1A2 (PDB code: 2hi4) and CYP1B1 (PDB code: 3pm0) with the use of the CDOCKER procedure implemented in Accelrys Discovery Studio 2.5.5. CDOCKER uses a CHARMm-based molecular dynamics (MD) scheme to dock ligands into a receptor binding site. For assigning receptor and ligand atom partial charges, we applied the charging rules used in the MMFF94 forcefield. Docked poses were scored by the negative value of CDOCKER energy for the –CDOCKER\_ENERGY function, which include interaction energy and internal ligand energy: the higher positive value of –CDOCKER\_ENERGY, the stronger affinity of a ligand to the binding site.

Our docking experiment showed that in the CYP1A2 active site, all possible poses of resveratrol can be grouped into two sets. This observation indicated that two binding modes are possible for resveratrol molecule. In mode A, represented by the pose with highest score, a resveratrol molecule is directed with 4'-OH group toward a heme (Fig. 3a). In mode B, the second ring with 3-OH and 5-OH substituents is situated in the vicinity of a prosthetic group (Fig. 3b). In both orientations, resveratrol binding is stabilized by – stacking interactions, with phenyl ring of Phe226 (mode A), and with Phe226 and Phe260 (mode B). Contrary to resveratrol, a pterostilbene molecule was docked in the CYP1A2 active site only in one orientation with 4'-OH group directed toward a heme (Fig. 3c). Pterostilbene binding was stabilized by – interaction with an aromatic ring of Phe226 . For a resveratrol molecule docked in the active site of CYP1B1, we also distinguished two binding modes. In contrast to CYP1A2, the highest scored pose corresponded to binding mode B. In both orientations (A and B), resveratrol was stabilized by two – interactions between both of its rings and a phenyl ring of Phe231, and additionally by two hydrogen bonds with Asn265 and Asp333 in mode B, or Asn265 and Asn228 in mode A (Fig. 3d and 3e).

Similar to interaction with CYP1A2, a pterostilbene molecule represented only one type of orientation in the CYP1B1 cavity (Fig. 2f). The binding conformation with 4'-OH group close to a heme was stabilized by two – stacking interactions with Phe231. In the case of

The Inhibitory Effect of Natural Stilbenes and Their Analogues on

**6. Conclusion** 

**7. References** 

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 533

pterostilbene, which is a dimethoxy analogue of resveratrol, it is suggested that hydrophobic

In studies of *trans*-resveratrol metabolism by human microsomal CYP1B1 enzyme (Potter et al., 2002), the authors observed formation of two metabolites, M1 and M2. The major metabolite M2 has been identied as piceatannol (3,4,3',5'-tetrahydroxystilbene), while 3,4,5,4'-tetrahydroxystilbene was proposed as the M1 product. More recent work (Piver et al., 2004) provided evidence that CYP1A2 is also engaged in the metabolism of *trans*resveratrol to piceatannol and tetrahydroxystilbene M1. Our studies confirmed the possibility of two pathways of metabolism on the grounds of molecular docking analysis.

The finding of high affinity ligands among natural compounds for each of the CYP1 family enzymes will help to reveal more about enzyme specificity, providing a starting point for more extensive studies and improved predictive capabilities. Particularly, a selective inhibition against CYP1B1 that influences the chemopreventive properties of phytochemicals for E2 related breast cancer seems to be promising. There is a need for better characterization of potential chemopreventive/therapeutic agents in order to understand their abilities and limits to influencing numerous pathways leading to cancer development. Novel classes of anti-cancer drugs including those of plant origin are being developed that can target both drug-metabolizing enzymes and disease modifying pathways. Recently, interest in the combinatory effect of different phytochemicals is growing, with respect to the multi-targeted action of numerous components of a food matrix. Wenzel and co-workers found that metabolism of resveratrol present in beverages such as wine or grape juice is inhibited by other polyphenols due to competitive reactions with Phase -II enzymes, resulting in an increased concentration of the free form (Wenzel et al., 2005). It is suggested that an efficient chemoprevention strategy lies in the use of combinations of several chemopreventive and/or therapeutic agents which may exert multi-targeted action. In conclusion, the search for potent and selective CYP1A inhibitors appears to hold promise

and should be continued with the use of novel computational techniques.

2003), pp. 363-398, ISSN 0250-7005

ISSN 0026-895X

Aggarwal, B.B.; Kumar, A. & Bharti, A.C. (2003). Anticancer potential of curcumin:

Andrieux, L.; Langouet, S.; Fautrel, A.; Ezan, F.; Krauser, J.A.; Savouret, J.F.; Guengerich,

Androutsopoulos, V.P.; Papakyriakou, A.; Vourloumis, D. & Spandidos, D.A. (2011).

*Bioorg. Med. Chem.,* Vol. 19, No. 9, (May 2011), pp. 2842-2849, ISSN 0968-0896 Appiah-Opong, R.; Commandeur, J.N.M.; van Vugt, B. & Vermeulen, N.P.E. (2007). Inhibition of

products. *Toxicology*, Vol. 235, No. 1-2, (June 2007), pp. 83-91, ISSN 0300-483X

preclinical and clinical studies. *Anticancer Res*., Vol. 23, No. 1A, (January-February

F.P.; Baffet, G. & Guillouzo, A. (2004). Aryl hydrocarbon receptor activation and cytochrome P450 1A induction by the mitogen-activated protein kinase inhibitor UO126 in hepatocytes. *Mol. Pharmacol*., Vol. 65, No 4, (April 2004), pp. 934-943,

Comparative CYP1A1 and CYP1B1 substrate and inhibitor profile of dietary flavonoids.

human recombinant cytochrome P450s by curcumin and curcumin decomposition

interactions might play a key role determining and stabilizing its docking orientation.

Fig. 3. Putative binding modes of resveratrol and pterostilbene in active sites of CYP1A2 (a – c) and CYP1B1 (d – f) with key residues involved in – stacking interactions and hydrogen bonds represented by solid blue lines and dashed blue lines, respectively. Heme molecule is at the bottom. CYP1A2 active site in complex with: (a) resveratrol in binding mode A, (b) resveratrol in mode B, (c) pterostilbene. CYP1B1 active site in complex with: (d) resveratrol in binding mode B, (e) resveratrol in mode A, (f) pterostilbene.

pterostilbene, which is a dimethoxy analogue of resveratrol, it is suggested that hydrophobic interactions might play a key role determining and stabilizing its docking orientation.

In studies of *trans*-resveratrol metabolism by human microsomal CYP1B1 enzyme (Potter et al., 2002), the authors observed formation of two metabolites, M1 and M2. The major metabolite M2 has been identied as piceatannol (3,4,3',5'-tetrahydroxystilbene), while 3,4,5,4'-tetrahydroxystilbene was proposed as the M1 product. More recent work (Piver et al., 2004) provided evidence that CYP1A2 is also engaged in the metabolism of *trans*resveratrol to piceatannol and tetrahydroxystilbene M1. Our studies confirmed the possibility of two pathways of metabolism on the grounds of molecular docking analysis.

#### **6. Conclusion**

532 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Fig. 3. Putative binding modes of resveratrol and pterostilbene in active sites of CYP1A2 (a – c) and CYP1B1 (d – f) with key residues involved in – stacking interactions and hydrogen bonds represented by solid blue lines and dashed blue lines, respectively. Heme molecule is at the bottom. CYP1A2 active site in complex with: (a) resveratrol in binding mode A, (b) resveratrol in mode B, (c) pterostilbene. CYP1B1 active site in complex with: (d) resveratrol

in binding mode B, (e) resveratrol in mode A, (f) pterostilbene.

The finding of high affinity ligands among natural compounds for each of the CYP1 family enzymes will help to reveal more about enzyme specificity, providing a starting point for more extensive studies and improved predictive capabilities. Particularly, a selective inhibition against CYP1B1 that influences the chemopreventive properties of phytochemicals for E2 related breast cancer seems to be promising. There is a need for better characterization of potential chemopreventive/therapeutic agents in order to understand their abilities and limits to influencing numerous pathways leading to cancer development. Novel classes of anti-cancer drugs including those of plant origin are being developed that can target both drug-metabolizing enzymes and disease modifying pathways. Recently, interest in the combinatory effect of different phytochemicals is growing, with respect to the multi-targeted action of numerous components of a food matrix. Wenzel and co-workers found that metabolism of resveratrol present in beverages such as wine or grape juice is inhibited by other polyphenols due to competitive reactions with Phase -II enzymes, resulting in an increased concentration of the free form (Wenzel et al., 2005). It is suggested that an efficient chemoprevention strategy lies in the use of combinations of several chemopreventive and/or therapeutic agents which may exert multi-targeted action. In conclusion, the search for potent and selective CYP1A inhibitors appears to hold promise and should be continued with the use of novel computational techniques.

#### **7. References**


The Inhibitory Effect of Natural Stilbenes and Their Analogues on

(August 2999), pp. 20-24, ISSN 0006-291X

(April 2001), pp. 389-393, ISSN 0090-9556

2003), pp. 2535-2538, ISSN 0960-894X

March 2003), pp. 163-172, ISSN 0027-5107

No. 1, (October 2009), pp. 1-6, ISSN 0304-3835

(February-March), pp. 173-182, ISSN 0027-5107

205, ISSN 0300-483X

ISSN 1520-4804

2005-2013, ISSN 0143-3334

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 535

Chaudhary, A. & Willett, K.L. (2006). Inhibition of human cytochrome CYP1 enzymes by

Chen, Z.H.; Hurh, Y.J.; Na, H.K.; Kim, J.H.; Chun, Y.J.; Kim, D.H.; Kang, K.S.; Cho, M.H. &

Cho, U.S.; Park, E.Y.; Dong, M.S.; Park, B.S.; Kim, K. & Kim, K.H. (2003). Tight-binding

Chun, Y.J.; Kim, M.Y. & Guengerich, F.P. (1999). Resveratrol is a selective human

Chun, Y.J.; Ryu, S.Y.; Jeong, T.C. & Kim, M.Y. (2001a). Mechanism-based inhibition of

Chun, Y.J.; Kim, S.; Kim, D.; Lee, S.K. & Guengerich, F.P. (2001b). A new selective and potent

Ciolino, H.P. & Yeh, G.C. (1999). Inhibition of aryl hydrocarbon-induced cytochrome P-450

Ciolino, H.P.; Daschner, P.J. & Yeh, G.C. (1998). Resveratrol inhibits transcription of

Don, M.-J.; Lewis, D.F.V.; Wang, S.-Y.; Tsai, M.-W. & Ueng, Y.F. (2003). Effect of structural

Gaudineau, C.; Beckerman, R.; Welbourn, S. & Auclair, K. (2004). Inhibition of human P450

*Res. Commun*., Vol. 318, No. 4, (June 2004), pp. 1072-1078, ISSN 0006-291X Gerhauser, C.; Klimo, K.; Heiss, E.; Neumann, I.; Gamal-Eldeen, A.; Knauft, J.; Liu, G.Y.;

Gonzalez, F.J. (2002).Transgenic models in xenobiotic metabolism and toxicology. *Toxicology*,

Goswami, S.K. & Das, D.K. (2009). Resveratrol and chemoprevention. *Cancer Lett*., Vol. 284,

Guengerich, F.P.; Chun, Y-J.; Kim, D.; Gillam, E.M.J. & Shimada, T. (2003). Cytochrome P450

Vol. 181-182 (December 2002), pp. 237-239, ISSN 0300-483X

*Res*., Vol. 58, No. 24, (December 1998), pp. 5707-5712, ISSN 0008-5472 de Medina, P.; Casper, R.; Savouret, J.-F. & Poirot, M. (2005). Synthesis and biological

*Res*., Vol. 61, No. 22, (November 2001), pp. 8164-8170, ISSN 0008-5472

56, No. 4, (October 1999), pp. 760-767, ISSN 0026-895X

*Acta*, Vol. 1648, No. 1-2, (May 2003), pp. 195-202, ISSN 0006-3002

flavonoids of St. John's wort. *Toxicology*, Vol. 217, No. 2-3, (January 2006), pp. 194-

Surh, Y.J. (2004). Resveratrol inhibits TCDD-induced expression of CYP1A1 and CYP1B1 and catechol estrogen-mediated oxidative DNA damage in cultured human mammary epithelial cells. *Carcinogenesis*, Vol. 25, No. 10, (October 2004), pp.

inhibition by –naphthoflavone of human cytochrome P450 1A2. *Biochim. Biophys.* 

cytochrome P450 1A1 inhibitor. *Biochem. Biophys. Res. Commun.*, Vol. 262, No. 1,

human cytochrome P450 1A1 by rhapontigenin. *Drug Metab. Dispos*., Vol. 29, No. 4,

inhibitor of human cytochrome P450 1B1 and its application to antimutagenesis. *Cancer* 

1A1 enzyme activity and CYP1A1 expression by resveratrol. *Mol. Pharmacol*., Vol.

CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor. *Cancer* 

properties of new stilbene derivatives of resveratrol as new selective aryl hydrocarbon modulators. *J. Med. Chem*., Vol. 48, No. 1, (January 2005), pp. 287-291,

modification on the inhibitory selectivity of rutaecarpine derivatives on human CYP1A1, CYP1A2, and CYP1B1. *Bioorg. Med. Chem. Lett.*, Vol. 13, No.15, (August

enzymes by multiple constituents of the Ginkgo biloba extract. *Biochem. Biophys.* 

Sitthimonchai, S. & Frank, N. (2003) Mechanism-based in vitro screening of potential cancer chemopreventive agents. *Mutat. Res.,* Vol. 523-524, (February-

1B1: a target for inhibition in anticarcinogenesis strategies. *Mutat. Res*., Vol. 523-524,


Appiah-Opong, R.; de Esch, I.; Commandeur, J.N.; Andarini M. & Vermeulen, N.P. (2008).

Badal, S.; Williams, S.A.; Huang, G.; Francis, S.; Vendantam, P.; Dunbar, O.; Jacobs, H.;

Beedanagari, S.R.; Bebenek, I.; Bui, P. & Hankinson, O. (2009). Resveratrol inhibits dioxin-

Bode, A.M. & Dong, Z. (2009). Cancer prevention research - then and now. *Nature Rev.* 

Bruno, R.D. & Njar, V.C.O. (2007). Targeting cytochrome P450 enzymes: A new approach in

Cai, Y.; Baer-Dubowska, W.; Ashwood-Smith, M.J.; Ceska, O.; Tachibana, S. & DiGiovanni, J.

Cai, Y.; Baer-Dubowska, W.; Ashwood-Smith, M. & DiGiovanni, J. (1997). Inhibitory effects

*Carcinogenesis*, Vol. 18, No. 8 (August 1997), pp. 215–222, ISSN 0143-3334 Canistro, D.; Bonamassa, B.; Pozzetti, L.; Sapone, A.; Abdel-Rahman, S.Z.; Biagi, G.L. &

Casper, R.F.; Quesne, M.; Rogers, I.M.; Shirota, T.; Jolivel, A.; Milgrom, E. & Savouret, J.F.

Castro, D.J.; Baird, W.M.; Pereira, C.B.; Giovanini, J.; Lohr, C.V.; Fischer, K.A.; Yu, Z.;

Chang, T.K.H.; Lee, W.B. & Ko, H.H. (2000). Trans-resveratrol modulates the catalytic activity

Chang, T.K.H.; Chen, J. & Lee, W.B. (2001). Differential inhibition and inactivation of human

Chang, T.K.H.; Chen, J. & Yu, C.-T. (2007). In vitro inhibition of rat CYP1A1 and CYP1A2 by

*Cancer Prev. Res. (Phila),* Vol. 1, No. 2, pp. 128-134, ISSN 1940-6207

*Fitoterapia*, Vol. 82, No. 2, (March 2011), 230-236, ISSN 0367-326X

*Cancer*, Vol. 9, No. 7 (June 2009), pp. 508-516, ISSN 1474-175X

(August 2008), pp. 1621-1631, ISSN 0223-5234

67, ISSN 1096-6080

pp. 5047-5060, ISSN 0968-0896

454-461, ISSN 0278-6915

ISSN 0022-3565

1996), pp. 729-736, ISSN 0893-228X

(October 1999), pp. 784-790, ISSN 0026-895X

(January 2007), pp. 13-16, ISSN 1872-3128

Structure-activity relationships for the inhibition of recombinant human cytochromes P450 by curcumin analogues. *Eur. J. Med. Chem*., Vol. 43, No. 8,

Tzeng, T.J.; Gangemi, J. & Delgoda, R. (2011). Cytochrome P450 1 enzyme inhibition and anticancer potential of chromene amides from *Amyris plumieri*.

induced expression of human CYP1A1 and CYP1B1 by inhibiting recruitment of the aryl hydrocarbon receptor complex and RNA polymerase II to the regulatory regions of the corresponding genes. *Toxicol. Sci.,* Vol. 110, No. 1, (July 2009), pp. 61-

anti-cancer drug development. *Bioorg. Med. Chem*., Vol. 15, No. 15 (August 2007),

(1996). Mechanism-based inactivation of hepatic ethoxyresorufin O-dealkylation activity by naturally occurring coumarins. *Chem. Res. Toxicol*., Vol. 9, No. 4, (June

of naturally occurring coumarins on the metabolic activation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured mouse keratinocytes.

Paolini, M., (2009). Alteration of xenobiotic metabolizing enzymes by resveratrol in liver and lung of CD1 mice. *Food Chem. Toxicol*. Vol. 47, No. 2, (February 2009), pp.

(1999). Resveratrol has antagonist activity on the aryl hydrocarbon receptor: implications for prevention of dioxin toxicity. *Mol. Pharmacol*. Vol. 56, No. 4,

Gonzalez, F.J.; Krueger, S.K. & Williams, D.E. (2008). Fetal mouse Cyp1b1 and transplacental carcinogenesis from maternal exposure to dibenzo(a,1)pyrene.

and mRNA expression of the procarcinogens-activating human cytochrome P450 1B1. *Can. J. Physiol. Pharmacol.*, Vol. 78, No. 11 (November 2011), pp. 874-881, ISSN 0008-4212

CYP1 enzymes by trans-resveratrol: evidence for mechanism-based inactivation of CYP1A2. *J. Pharmacol. Exp. Ther*., Vol. 299, No. 3, (December 2001), pp. 874-882,

piceatannol, a hydroxylated metabolite of trans-resveratrol. *Drug Metab. Lett.,* 1, 1,


The Inhibitory Effect of Natural Stilbenes and Their Analogues on

(July 2003), pp. 1199-1213, ISSN 0024-3205

773-783, ISSN 0006-2952

ISSN 0032-0943

pp. 279-282, ISSN 0951-418X

(June 2003), pp. 861-866, ISSN 0278-6915

52, No. S1, (June 2008), pp. S77-S83, ISSN 1613-4125

Catalytic Activity of Cytochromes P450 Family 1 in Comparison with Other Phenols… 537

Mikstacka, R.; Baer-Dubowska, W.; Wieczorek, M. & Sobiak, S. (2008). Thiomethylstilbenes

Oetari, S.; Sudibyo, M.; Commandeur, J.N.M.; Samhoedi, R. & Vermeulen, N.P.E. (1996).

Piver, B.; Fer, M.; Vitrac, X.; Merillon, J.-M.; Dreano, Y.; Berthou, F. & Lucas, D. (2004).

Potter, G.A.; Patterson, L.H.; Wanogho, E.; Perry, P.J.; Butler, P.C.; Iljaz, T.; Ruparelia, K.C.; Lamb,

Rendic, S. & Di Carlo, F.J. (1997). Human cytochromes P450 enzymes: a status report

Revel, A.; Raanani, H.; Younglai, E.; Xu, J.; Rogers, I.; Han, R.; Savouret, J.F. & Casper, R.F.

Rimando, A.M.; Cuendet, M.; Desmarchelier, C.; Mehta, R.G.; Pezzuto, J.M. & Duke, O.D.

Rimando, A.M.; Kalt, W.; Magee, J.B.; Dewey, J. & Ballington, J.R. (2004). Resveratrol,

Rimando, A.M. & Suh, N. (2008). Biological/chemopreventive activity of stilbenes and their

Rodeiro, I.; Donato, M.T.; Jimenez, N.; Garrido, G.; Molina-Torres, J.; Menendez, R.; Castell,

Ryu, S.-D. & Chung, W.-G. (2003). Induction of the procarcinogen-activating CYP1A2 by a

Schwarz, D. & Roots, I. (2003). In vitro assessment of inhibition by natural polyphenols of

*Commun.*, Vol. 303, No. 3, (April 2003), pp. 902-907, ISSN 0006-291X Schwarz, D.; Kisselev, P.; Schunck, W.-H. & Roots, I. (2011). Inhibition of 17-estradiol

Vol. 29, No. 1-2, (February-May 1997), pp. 413-580, ISSN 0360-2532

23, No. 4, (July-August 2003), pp. 255-261, ISSN 0260-437X

(June 2002), pp. 3453-3457, ISSN 0021-8561

No. 15, (July 2004), pp. 4713-4719, ISSN 0021-8561

as inhibitors of CYP1A1, CYP1A2 and CYP1B1 activities. *Mol. Nutr. Food Res*., Vol.

Effects of curcumin on cytochrome P450 and glutathione-S-transferase activities in rat liver. *Biochem. Pharmacol*., Vol. 51, No. 1, (January 1996), pp. 39-45, ISSN 0006-2952 Piver, B.; Berthou, F.; Dreano, Y. & Lucas, D. (2003). Differential inhibition of human

cytochrome P450 enzymes by ε-viniferin, the dimer of resveratrol: comparison with resveratrol and polyphenols from alcoholized beverages. *Life Sci*., Vol. 73, No. 9,

Involvment of cytochrome P450 1A2 in the biotransformation of trans-resveratrol in human liver microsomes. *Biochem. Pharmacol.*, Vol. 68, No. 4, (August 2004), pp.

J.H.; Farmer, P.B.; Stanley, L.A. & Burke, M.D. (2002). The cancer preventive agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYP1B1. *Br. J. Cancer*, Vol. 86, No. 5, (March 2002), pp. 774-778, ISSN 0007-0920

summarizing their reactions, substrates, inducers, and inhibitors. *Drug Metab. Rev*.,

(2003). Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects lung from DNA damage and apoptosis caused by benzo[a]pyrene. *J. Appl. Toxicol*., Vol.

(2002). Cancer chemopreventive and antioxidant activities of pterostilbene, a naturally occurring analogue of resveratrol. *J. Agric. Food Chem., Vol.* 50, No. 12,

pterostilbene, and piceatannol in Vaccinium berries. *J. Agric. Food Chem*., Vol. 52,

effect on colon cancer. *Planta Med*., Vol. 74, No. 13, (October 2008), pp. 1635-1643,

J.V. & Gomez-Lechon, M.J. (2009). Inhibition of human P450 enzymes by natural extracts used in traditional medicine. *Phytother. Res*., Vol. 23, No. 2, (February 2009),

herbal dietary supplement in rats and humans. *Food Chem. Toxicol*., Vol. 41, No. 6,

metabolic activation of procarcinogens by human CYP1A1. *Biochem. Biophys. Res.* 

activation by CYP1A1: genotype- and regioselective inhibition by St. John's Wort


Itoh, T.; Takemura, H.; Shimoi, K. & Yamamoto, K. (2010). A 3D model of CYP1B1 explains

Jang, M.; Cai, L.; Udeani, G.O.; Slowing, K.V.; Thomas, C.F.; Beecher, C.W.W.; Fong, H.H.S.;

Kisselev, P.; Schunck, W.H.; Roots, I. & Schwarz, D. (2005). Association of CYP1A1

Lamb, D.C.; Waterman, M.R.; Kelly, S.L. & Guengerich, F.P. (2007). Cytochromes P450 and

Lee, A.J.; Cai, M.X.; Thomas, P.E.; Conney, A.H. & Zhu, B.T. (2003). Characterization of the

Lewis, D.F.V. (1997). Quantitative structure – activity relationships in substrates, inducers

Lewis, D.F.V.; Lake, B.G.; George, S.G.; Dickins, M.; Eddershaw, P.J.; Tarbit, M.H.; Beresford,

Liehr, J.G. & Ricci, M.J. (1996). 4-Hydroxylation of estrogens as marker of human mammary

MacPherson, L. & Matthews, J. (2010). Inhibition of aryl hydrocarbon receptor-dependent

Mc Fadyen, M.C. & Murray, G.I. (2005). Cytochrome P450 1B1: a novel anticancer therapeutic target. *Future Oncol*. 1, No. 2, (April 2005), pp. 259-263, ISSN 1479-6694 Mikstacka, R.; Przybylska, D.; Rimando, A.M. & Baer-Dubowska, W. (2007). Inhibition of human

*Mol. Nutr. Food Res*., Vol. 51, No. 5, (May 2007), pp. 517-524, ISSN 1613-4125 Mikstacka, R.; Rimando, A.M.; Szalaty, K.; Stasik, K. & Baer-Dubowska, W. (2006). Effect of

*Cancer Res*., Vol. 65, No. 7, (April 2005), pp. 2972-2978, ISSN 0008-5472 Kuhnau, J. (1976). A class of semiessential food components: their role in human nutrition.

*World Rev. Nutr. Diet*, Vol. 24, pp. 117-191, ISSN 0084-2230

(August 2003), pp. 3382-3398, ISSN 0013-7227

(August 1997), pp. 589-650, ISSN 0360-2532

2010), pp. 1173-1178, ISSN 1549-9596

160-164, ISSN 1520-4804

ISSN 0958-1669

ISSN 0887-2333

ISSN 0027-8424

2010), pp. 119-129, ISSN 0304-3835

the dominant 4-hydroxylation of estradiol. *J.Chem. Inf. Model.*, Vol. 50, No. 6, (June

Farnsworth, N.R.; Kinghorn, A.D.; Mehta, R.G.; Moon, R.C. & Pezzuto, J.M. (1997). Cancer chemopreventive activity of resveratrol, a natural product derived from grapes, *Science*, Vol. 275, No. 5297, (January 1997), pp. 218-220, ISSN 0036-8075 Kim, S.; Ko, H.; Park, J.E.; Jung, S.; Lee, S.K. & Chun, Y.J. (2002). Design, synthesis and

discovery of novel trans-stilbene analogues as potent and selective human cytochrome P450 1B1 inhibitors. *J. Med. Chem.,* Vol. 45, No. 1, (January 2002), pp.

polymorphism with differential metabolic activation of 17--estradiol and estrone.

drug discovery. *Curr. Opin. Biotech*., Vol. 18, No. 6, (December 2007), pp. 504-512,

oxidative metabolites of 17-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms. *Endocrinology*, Vol. 144, No. 8,

and inhibitors of cytochrome P450 (CYP1). Drug Metab. Rev., Vol. 29, No. 3,

A.P.; Goldfarb, P.S. & Guegerich, F.P. (1999). Molecular modeling of CYP1 family enzymes CYP1A1, CYP1A2, CYP1A6 and CYP1B1 based on sequence homology with CYP102. *Toxicology*, Vol. 139, No. 1-2, (November 1999), pp. 53-79, ISSN 0300-483X Lewis, D.F.V.; Ito, Y. & Lake, B.G. (2006). Metabolism of coumarin by human P450s: A

molecular modeling study. *Toxicol. in vitro*, Vol. 20, No 2, (March 2006), pp. 256-264,

tumors. *Proc. Natl. Acad. Sci. U.S.A*. , Vol. 93, No. 8, (April 1996), pp. 3294-3296,

transcription by resveratrol or kaempferol is independent of estrogen receptor expression in human breast cancer cells. *Cancer Lett*., Vol. 299, No. 2, (December

recombinant cytochromes P450 CYP1A1 and CYP1B1 by trans-resveratrol metyl ethers.

natural analogues of trans-resveratrol on cytochromem P450 1A2 and 2E1 catalytic activities. *Xenobiotica*, Vol. 36, No. 4, (April 2006), pp. 269-285, ISSN 0049-8254


and natural polyphenols. *Biochim. Biophys. Acta*, Vol. 1814, No. 1, (January 2011), pp. 168-174, ISSN 0006-3002


Sporn, M.B. & Liby, K.T. (2005) Cancer chemoprevention: scientific promise, clinical

Swanson, H.I.; Njar, W.C.O.; Yu, Z.; Castro, D.J.; Gonzalez, F.J.; Williams, D.E.; Huang, Y.;

Szekeres, T.; Fritzer-Szekeres, M.; Saiko, P. & Jager, W. (2010). Resveratrol and resveratrol

Takemura, H.; Itoh, T.; Yamamoto, K.; Sakakibara, H. & Shimoi, K. (2010). Selective

Thapliyal, R. & Maru, G.B. (2001). Inhibition of cytochrome P450 isozymes by curcumins in vitro and in vivo. *Food Chem. Toxicol.*, Vol. 39, No. 6, (June 2001), pp. 541-547, ISSN 0278-6915 Walle, T.; Hsieh, F.; DeLegge, M.H.; Oatis, J.E.,Jr. & Walle, U.K. (2004). High absorption but

Walle, U.K. & Walle, T. (2007). Novel methoxylated flavone inhibitors of cytochrome P450

Wattenberg, L.W. (1985). Chemoprevention of cancer. *Cancer Res*., Vol. 45, No. 1, (January

Wenzel, E.; Soldo, T.; Erbersdobler, H. & Somoza, V. (2005). Bioactivity and metabolism of

William, W.N., Jr.; Heymach, J.V.; Kim, E.S. & Lippman, S.M. (2009). Molecular targets for

Yamaori, S.; Kushihara, M.; Yamamoto, I. & Watanabe, K. (2010). Characterization of major

Zhai, S.; Dai, R.; Friedman, F.K. & Vestal, R.E. (1998). Comparative inhibition of human

Vol. 18, No. 17, (September 2010), pp. 6310-6315, ISSN 0968-0896

No. 12, (December 2004), pp. 1377-1382, ISSN 0090-9556

pp. 168-174, ISSN 0006-3002

pp. 70-77, ISSN 0163-5581

1042-1048, ISSN 0724-8741

2007), pp. 857-862, ISSN 0022-3573

2010), pp. 1691-1698, ISSN 0006-2952

(October 1998), pp. 989-992, ISSN 0090-9556

No. 5, (May 2005), pp. 482-494, ISSN 1613-4125

1985), pp. 1-8, ISSN 0008-5472

213-225, ISSN 1474-1776

525, ISSN 1743-4254

and natural polyphenols. *Biochim. Biophys. Acta*, Vol. 1814, No. 1, (January 2011),

uncertainty. *Nature Clinical Practise Oncology*, Vol. 2, No. 10, (October 2005), pp. 518-

Kong, A.-N. T.; Doloff, J.C.; Ma, J.; Waxman, D.J. & Scott, E.E. (2010). Targeting drug-metabolizing enzymes for effective chemoprevention and chemotherapy. *Drug Metab. Dispos.*, Vol. 38, No. 4, (April 2010), pp. 539-544, ISSN 0090-9556 Szaefer, H.; Cichocki, M.; Brauze, D. & Baer-Dubowska, W. (2004). Alteration in phase I and

II enzyme activities and polycyclic aromatic hydrocarbon-DNA adduct formation by plant phenolics in mouse epidermis. *Nutr. Cancer* Vol. 48, No. 1, (January 2004),

analogues – structure-activity relationship. *Pharm. Res*., 27, 6, (March 2010), pp.

inhibition of methoxyflavonoids on human CYP1B1 activity. *Bioorg. Med. Chem*.,

very low bioavailability of oral resveratrol in humans. *Drug Metab. Dispos*., Vol. 32,

1B1 in SCC9 human oral cancer cells. *J. Pharm. Pharmacol*., Vol. 59, No. 6, (June

trans-resveratrol orally administered to Wistar rats. *Mol. Nutr. Food Res*., Vol. 49,

cancer chemoprevention. *Nat. Rev. Drug Discov*., Vol. 8, No. 3, (March 2009), pp.

phytocannabinoids, cannabidiol and cannabinol, as isoform selective and potent inhibitiors of human CYP1 enzymes. *Biochem. Pharmacol.*, Vol. 79, No. 11, (June

cytochromes P450 1A1 and 1A2 by flavonoids. *Drug Metab. Dispos*., Vol. 26, No. 10,

### *Edited by Venketeshwer Rao*

Phytochemicals are biologically active compounds present in plants used for food and medicine. A great deal of interest has been generated recently in the isolation, characterization and biological activity of these phytochemicals. This book is in response to the need for more current and global scope of phytochemicals. It contains chapters written by internationally recognized authors. The topics covered in the book range from their occurrence, chemical and physical characteristics, analytical procedures, biological activity, safety and industrial applications. The book has been planned to meet the needs of the researchers, health professionals, government regulatory agencies and industries. This book will serve as a standard reference book in this important and fast growing area of phytochemicals, human nutrition and health.

Photo by Vonschonertagen / iStock

Phytochemicals - A Global Perspective of Their Role in Nutrition and Health

Phytochemicals

A Global Perspective of Their Role in

Nutrition and Health

*Edited by Venketeshwer Rao*