**Meet the editors**

Dr. Rao completed his M. Sc. and Ph. D. degrees in Food Science from Oregon State University, USA. After being a Full Professor in the Department of Nutritional Sciences, Faculty of Medicine, he currently holds the position of Professor Emeritus at the University of Toronto. He also served as the Director of the Program in Food Safety, Nutrition and Regulatory Affairs, Director of the Collab-

orative Program in Toxicology, Member of the Institute of Environmental Studies, and Undergraduate Coordinator for the Department of Nutritional Sciences. He is a member of several national and international scientific and professional organizations including the International Bifidus Foundation (Japan). He has established a major focus in the area of diet and health with particular emphasis on the role of dietary phytochemicals and intestinal microflora. His research has focused on the role of probiotics and prebiotics in human health and oxidative stress and antioxidants in the causation and prevention of chronic diseases, with particular emphasis on the role of carotenoids and polyphenols. He is one of the pioneering researchers to study the bioavailability, metabolism, mechanisms of action, and biological role of lycopene, a carotenoid antioxidant present in tomatoes and other fruits and vegetables. Dr. Rao served as a member of the Provincial and National Expert Committees in Canada in the areas of nutrition, health, food safety and agriculture. He has a distinguished academic career spanning over 45 years as an educator and a researcher. He serves as a Senior Scientific Consultant to provincial and federal government agencies and food and pharmaceutical industries globally. He is popularly sought by the international media to express his opinions on the subjects of nutrition and health.

Dr. Leticia Rao is an Associate (Adjunct) Professor of Medicine and a full member of the Graduate Faculty, Institute of Medical Sciences, at the University of Toronto. She is also the Director of the Calcium Research Laboratory, Department of Medicine and a Staff Scientist at St Michael's Hospital, Toronto, Ontario. She obtained a Master of Science Degree in Food Science from Oregon

State University and a Ph.D. degree in Biochemistry from the University of Toronto. Her current expertise is in the area of bone cell biology and calcium metabolism, and her long term research goal is to uncover the causes and prevention of osteoporosis by studying bone cells in the laboratory. She has extensive basic research experience on drugs that have been approved for osteoporosis prevention and treatment such as raloxifene and estrogen. She has conducted clinical studies to find out how the fat-soluble carotenoid antioxidant lycopene present predominantly in tomatoes and tomato products and the water-soluble antioxidant polyphenols in colourful fruits

and vegetables and natural food supplement can help in the prevention of postmenopausal osteoporosis. She has presented her research data to a number of symposia and has been invited to give talks on her clinical research on osteoporosis. Her publications in peer reviewed scientific journals are extensive. She co-authored a book entitled "Bone Building Solution" and presently co-editing the book "Polyphenols."

### Contents

#### **Preface XIII**



D. Donno, G.L. Beccaro, A.K. Cerutti, M.G. Mellano and G. Bounous


Chapter 14 **Eleutherine Plicata – Quinones and Antioxidant Activity 323** Luiz Claudio da Silva Malheiros, João Carlos Palazzo de Mello and Wagner Luiz Ramos Barbosa

Chapter 7 **Aspidosperma Terpenoid Alkaloids — Biosynthetic Origin, Chemical Synthesis and Importance 163**

**Section 3 Extraction and Identification of Phytochemicals 185**

Chapter 8 **Bud Extracts as New Phytochemical Source for Herbal**

**Section 4 Effect of Stress on Phytochemical Activation 219**

Chapter 9 **Effects of Abiotic Stress (UV-C) Induced Activation of Phytochemicals on the Postharvest Quality of**

**Section 5 Biological and Health Significance of Phytochemicals 245**

Chapter 10 **Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of Phytochemical Antioxidants Lycopene and Polyphenol-containing Nutritional Supplements 247**

Chapter 11 **Phytochemicals and Cancer – Possible Molecular Targets of**

Chapter 12 **Lead Compounds from Cucurbitaceae for the Treatment**

**Phytochemicals in Cancer Prevention and Therapy 261** Victor P. Bagla, Matlou P. Mokgotho and Leseilane J. Mampuru

Marcos Soto-Hernández, Jorge Cadena Iñiguez, Lourdes C. Arévalo-Galarza, Edelmiro Santiago-Osorio, Itzen Aguiñiga -Sánchez and

**Analytical Fingerprint 187**

**Horticultural Crops 221**

Rohanie Maharaj

L.G. Rao and A.V. Rao

**of Cancer 289**

Lucero del Mar Ruíz-Posadas

Chapter 13 **Green Tea Catechins for Prostate Cancer Chemoprevention 305**

Ganna Chornokur and Nagi B. Kumar

Araújo-Júnior

**VI** Contents

Pedro Gregório Vieira Aquino, Thiago Mendonça de Aquino, Magna Suzana Alexandre-Moreira, Bárbara Viviana de Oliveira Santos, Antônio Euzébio Goulart Santana and João Xavier de

**Preparations — Quality Control and Standardization by**

D. Donno, G.L. Beccaro, A.K. Cerutti, M.G. Mellano and G. Bounous

### Preface

Global dietary recommendations emphasize the consumption of plant-based foods for the prevention and management of chronic diseases. Plants contain many biologically active compounds referred to as 'phytochemicals' or 'functional ingredients'. These compounds play an important role in human health. Prior to establishing the safety and health benefits of these compounds, they must first be isolated, purified, and their physico-chemical prop‐ erties established. Once identified, their mechanisms of actions are studied. They can them be evaluated for their biological activity using *in vitro* and *in vivo* techniques including the use of cell cultures, animal models and clinical studies. Recognizing the importance of the phytochemicals in human health, the contents of this book include all the above aspects. The chapters are arranged in the order from isolation, purification and identification to in vivo and clinical studies, there by covering not only the analytical procedures used but also their nutraceutical and therapeutic properties. This book will be beneficial to researchers, health professionals, government regulatory agencies and industrial personnel. We are confident that the readers of this book which follows two previously published books on the same topic by In Tech Publication will find it highly useful as a standard reference book.

#### **Dr. L. G. Rao**

Associate (Adjunct) Professor and Director Calcium Research Laboratory St. Michael's Hospital University of Toronto

#### **Dr. A. V. Rao**

Professor Emeritus Department of Nutritional Science Faculty of Medicine University of Toronto, Canada

## **Phytochemical Composition**

## **The Phytochemical Constitution of Maltese Medicinal Plants – Propagation, Isolation and Pharmacological Testing**

Everaldo Attard, Henrietta Attard, Antoine Tanti, Jurgen Azzopardi, Mario Sciberras, Victor Pace, Neville Buttigieg, Andrew Mangion Randon, Bernardette Rossi, Marie Josette Parnis, Karin Vella, Michelle Zammit and Anthony Serracino Inglott

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60094

**1. Introduction**

In spite of its small size (31,500 hectares), the Maltese Archipelago hosts a large number of medicinal and aromatic plants that have been utilised medicinally for several centuries. The Maltese Archipelago lies in the middle of the Mediterranean Sea, 35°50' north of the Equator and14°35' eastofGreenwich.The climate is characterizedbyhotdrysummers,mildwetwinters (an average rainfall of 500 mm and temperatures ranging between 13°C in winter and 35°C in summer) and a high relative humidity all the year round. Most of the wild plants thrive in very shallow soil pockets that, in some cases, contribute to the production of phytochemicals as a means of protection against other plants or other organisms. In general, Maltese soils contain a high amount of calcium carbonate (>53%), which is the parentrock material, a high pH (>8) and a high clay content with a good physical structure but lacking organic matter (<4.5 %).

The Maltese flora comprises around 1284 vascular plants 66% originating from the Mediterranean region while the other 34% originating from the cold European and warm subtropicalregions [1]. Out ofthese,there are about 458 medicinaltaxa with approximately 300 originating from the Mediterranean region. The main plant families of medicinal importance are Asteraceae (15%), Lamiaceae (7%), Fabaceae (6%), Umbelliferae (4%) and Rosaceae (4%) amongst others. The biodiversity in medicinalflora is high probably due to severalreasons that include:

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The number of medicinal species is on the decline to the extent that some have already become extinct. This is not mainly attributed to overuse problems but due to various human activities. There were isolated cases where a medicinal plant was under treat due to over-harvesting. One typical example was the seaside squill (*Drimia maritima*) which was over-harvested due to export.

#### **2. Medicinal flora of the Maltese islands**

The pharmacological assessment of the Maltese medicinal flora, contributed to a portion of the research conducted on these species. Intensive research has been conducted in other fields, particularly in the ethnobotanical, agronomic, *in vitro* propagation and phytochemical fields. Phytochemistry plays a very important role in medicinal plant research (figure 1). The quality and safety of these plants depends mainly on their phytochemical constitution. These metab‐ olites determine the categorization of plants; whether a medicine, food supplement or cosmetic. The quality and quantity of these metabolites depends mainly on the growing conditions. This instigated researchers to study different aspects of medicinal plants with phytochemistry as the common aspect. The pharmacological assessment of the Maltese medicinal flora, contributed to a portion of the research conducted on these species. Intensive research has been conducted in other fields, particularly in the ethnobotanical, agronomic, in vitro propagation and phytochemical fields. Phytochemistry plays a very important role in medicinal plant research (figure 1). The quality and safety of these plants depends mainly on their phytochemical constitution. These metabolites determine the categorization of plants; whether a medicine, food supplement or cosmetic. The quality and quantity of these metabolites depends mainly on the growing conditions. This instigated researchers to study different aspects of medicinal plants with phytochemistry as the common aspect.

Figure 1. The importance of phytochemistry in medicinal plant research **Figure 1.** The importance of phytochemistry in medicinal plant research

table 1.

Medicinal plants have been classified either on their phytochemical constitution or else on their pharmacological activities. These plant contain a myriad of metabolite classes and single metabolites. In most cases, more has to be discovered as the information is either unavailable or else still uninvestigated yet. Locally, medicinal plants have been classified on their pharmacological activity. Some would include the following effects: cardiotonic (e.g. squill, oleander), anticancer (e.g. squirting cucumber, borage), immunomodulatory (e.g. squirting cucumber, olive tree), antiflammatory and skin disorders (e.g. marigold, aloe, erica), antihypertensive (e.g. hawthorn), antimicrobial and antifungal (poison ivy, sage, garden basil, Medicinal plants have been classified either on their phytochemical constitution or else on their pharmacological activities. These plant contain a myriad of metabolite classes and single

> sticky fleabane, couch grass, garlic, fig tree, caper plant, pellitory of the Wall), antidiabetic (karela), insect repellents and insecticides (pennyroyal, tree tobacco), antihelmintic (pumpkin), spasmodic and antispasmodic (vervain, henbane), sedative (blue passion flower, orange-flower water, chamomile), kidney stone problems (micromeria), volatile oil (lavander, garden rue, lemon balm, rosemary, laurel, spearmint) and fixed oils (olive tree, castor oil plant). Some of these plants are listed in

> Local ethnobotanical research has contributed towards the discovery of new leads. In such studies, the traditional claims are challenged using scientific methods. Possible conservation strategies were also considered, particularly for endangered species. However, there are limitations since there are no national incentives to conserve these plant species unless cultivated

metabolites. In most cases, more has to be discovered as the information is either unavailable or else still uninvestigated yet. Locally, medicinal plants have been classified on their phar‐ macological activity. Some would include the following effects: cardiotonic (e.g. squill, oleander), anticancer (e.g. squirting cucumber, borage), immunomodulatory (e.g. squirting cucumber, olive tree), antiflammatory and skin disorders (e.g. marigold, aloe, erica), antihy‐ pertensive (e.g. hawthorn), antimicrobial and antifungal (poison ivy, sage, garden basil, sticky fleabane, couch grass, garlic, fig tree, caper plant, pellitory of the Wall), antidiabetic (karela), insect repellents and insecticides (pennyroyal, tree tobacco), antihelmintic (pumpkin), spasmodic and antispasmodic (vervain, henbane), sedative (blue passion flower, orangeflower water, chamomile), kidney stone problems (micromeria), volatile oil (lavander, garden rue, lemon balm, rosemary, laurel, spearmint) and fixed oils (olive tree, castor oil plant). Some of these plants are listed in table 1.

Local ethnobotanical research has contributed towards the discovery of new leads. In such studies, the traditional claims are challenged using scientific methods. Possible conservation strategies were also considered, particularly for endangered species. However, there are limitations since there are no national incentives to conserve these plant species unless cultivated or sold as pot plants. However, there are few plants that are legally bound. A typical example is the carob tree. The grower cannot uproot a carob tree to pursue cultivation needs.


**Table 1.** The Maltese medicinal plants in this study.

#### **2.1.** *Drimia maritima* **(L.) Stearn**

**•** favourable Mediterranean climate **•** availability of fertile calcareous soils

**•** former conquerors of the Maltese Islands

**•** Maltese interest in herbal medicine

to export.

**•** considerable area of uncultivated land (wastelands)

4 Phytochemicals - Isolation, Characterisation and Role in Human Health

**2. Medicinal flora of the Maltese islands**

phytochemistry as the common aspect.

table 1.

phytochemistry as the common aspect.

Figure 1. The importance of phytochemistry in medicinal plant research

**Figure 1.** The importance of phytochemistry in medicinal plant research

The number of medicinal species is on the decline to the extent that some have already become extinct. This is not mainly attributed to overuse problems but due to various human activities. There were isolated cases where a medicinal plant was under treat due to over-harvesting. One typical example was the seaside squill (*Drimia maritima*) which was over-harvested due

The pharmacological assessment of the Maltese medicinal flora, contributed to a portion of the research conducted on these species. Intensive research has been conducted in other fields, particularly in the ethnobotanical, agronomic, *in vitro* propagation and phytochemical fields. Phytochemistry plays a very important role in medicinal plant research (figure 1). The quality and safety of these plants depends mainly on their phytochemical constitution. These metab‐ olites determine the categorization of plants; whether a medicine, food supplement or cosmetic. The quality and quantity of these metabolites depends mainly on the growing conditions. This instigated researchers to study different aspects of medicinal plants with

The pharmacological assessment of the Maltese medicinal flora, contributed to a portion of the research conducted on these species. Intensive research has been conducted in other fields, particularly in the ethnobotanical, agronomic, in vitro propagation and phytochemical fields. Phytochemistry plays a very important role in medicinal plant research (figure 1). The quality and safety of these plants depends mainly on their phytochemical constitution. These metabolites determine the categorization of plants; whether a medicine, food supplement or cosmetic. The quality and quantity of these metabolites depends mainly on the growing conditions. This instigated researchers to study different aspects of medicinal plants with

Medicinal plants have been classified either on their phytochemical constitution or else on their pharmacological activities. These plant contain a myriad of metabolite classes and single metabolites. In most cases, more has to be discovered as the information is either unavailable or else still uninvestigated yet. Locally, medicinal plants have been classified on their pharmacological activity. Some would include the following effects: cardiotonic (e.g. squill, oleander), anticancer (e.g. squirting cucumber, borage), immunomodulatory (e.g. squirting cucumber, olive tree), antiflammatory and skin disorders (e.g. marigold, aloe, erica), antihypertensive (e.g. hawthorn), antimicrobial and antifungal (poison ivy, sage, garden basil, sticky fleabane, couch grass, garlic, fig tree, caper plant, pellitory of the Wall), antidiabetic (karela), insect repellents and insecticides (pennyroyal, tree tobacco), antihelmintic (pumpkin), spasmodic and antispasmodic (vervain, henbane), sedative (blue passion flower, orange-flower water, chamomile), kidney stone problems (micromeria), volatile oil (lavander, garden rue, lemon balm, rosemary, laurel, spearmint) and fixed oils (olive tree, castor oil plant). Some of these plants are listed in

Medicinal plants have been classified either on their phytochemical constitution or else on their pharmacological activities. These plant contain a myriad of metabolite classes and single

> Local ethnobotanical research has contributed towards the discovery of new leads. In such studies, the traditional claims are challenged using scientific methods. Possible conservation strategies were also considered, particularly for endangered species. However, there are limitations since there are no national incentives to conserve these plant species unless cultivated

*Drimia maritima* or *Urginea maritima* is one of the local medicinal plants which was harvested and exported. It is a member of the Asparagaceae family, with cardiac glycosides that reside in the bulb of this plant. It is renowned for its emetic, diuretic, cardiotonic [2], expectorant, rodenticide [3] and anticancer activities. The seaside squill has been extensively studied for its propagation potential. Locally, cultivation studies have been associated with the cardiac glycosidic content while micropropagation has been linked to biomass production.

The main constituents of the seaside squill are the cardiac glycosides and phenolic compounds [4]. It also contains mucilage and calcium oxalate crystals. The squill cardiac glycosides are bufadienolides. In principle, these are similar to triterpenoids having a sugar group and a lactone ring at C17. Scillaren A accounts for about 70% of the total glycosidal content of squill. It contains one unit of rhamnose and one unit of glucose. When scillaren A is hydrolyzed by enzymes, it breaks down to proscillaridin A and D-glucose (Figure 2).

Figure 2. The structure of cardiac glycosides of Drimia maritima (L.) Stearn.: (A) Scillaren A and (B) Proscillaridin A **Figure 2.** The structure of cardiac glycosides of *Drimia maritima* (L.) Stearn.: (A) Scillaren A and (B) Proscillaridin A

These bufadienolides are therefore important cardiotonic, blood pressure stimulating and antitumour agents. The main glycosides with digoxin-like effects are scillaren A and proscillaridin A [7]. Several cultivation parameters were studied for Drimia maritima in relation to dry matter yield and the total glycosidal content [8]. These include methods of propagation, planting at different depths, effects of nitrogen (N) , phosphorus (P) and potassium (K) fertilizers, cultivation in different soil types, age of harvesting and seasonal timing of harvesting. Propagation These glycosides act by binding to the Na+ /K+ ATPase pumps. This occurs due to the presence of the lactone group [5, 6]. These bufadienolides are therefore important cardiotonic, blood pressure stimulating and antitumour agents. The main glycosides with digoxin-like effects are scillaren A and proscillaridin A [7].

These glycosides act by binding to the Na<sup>+</sup>/K<sup>+</sup> ATPase pumps. This occurs due to the presence of the lactone group [5, 6].

by bulb division only takes 10 weeks to produce a seedling as opposed to seed propagation that requires 56 weeks. The type of soil does not contribute to the variation of glycosides in the squill bulb. In fact, Maltese squill grown on four soil types, namely terra soil, xerorendzina soil, carbonate raw and sandy soil exhibited average glycosidal contents of 0.575 % (w/w). Fertiliser studies revealed that the use of different ratios of N, P and K affect the rate of growth but no change in glycosidal content (average of 0.59 % w/w). For the best annual production of dry weight and glycosidal content, it is advisable to harvest squill in the third year after transplanting (table 2) immediately after flowering. The highest glycosidal content is obtained from the roots (Figure 3) Year of harvest Treatment Mean glycosidal content(%) First Control 0.25 Fertiliser-treated 0.26 Second Control 0.66 Fertiliser-treated 0.68 Third Control 0.57 Fertiliser-treated 0.58 Fourth Control 0.58 Fertiliser-treated 0.58 Several cultivation parameters were studied for *Drimia maritima* in relation to dry matter yield and the total glycosidal content [8]. These include methods of propagation, planting at different depths, effects of nitrogen (N), phosphorus (P) and potassium (K) fertilizers, cultivation in different soil types, age of harvesting and seasonal timing of harvesting. Propagation by bulb division only takes 10 weeks to produce a seedling as opposed to seed propagation that requires 56 weeks. The type of soil does not contribute to the variation of glycosides in the squill bulb. In fact, Maltese squill grown on four soil types, namely terra soil, xerorendzina soil, carbonate raw and sandy soil exhibited average glycosidal contents of 0.575 % (w/w). Fertiliser studies revealed that the use of different ratios of N, P and K affect the rate of growth but no change in glycosidal content (average of 0.59 % w/w). For the best annual production of dry weight and glycosidal content, it is advisable to harvest squill in the third year after transplanting (table 2) immediately after flowering. The highest glycosidal content is obtained from the roots (Figure 3)

Table 2. The mean percentage glycosidal content in the squill bulb with year of harvest [8].

Fifth Control 0.38

Sixth Control 0.31

Fertiliser-treated 0.40

Fertiliser-treated 0.34

The Phytochemical Constitution of Maltese Medicinal Plants – Propagation, Isolation and Pharmacological Testing http://dx.doi.org/10.5772/60094 7


**Table 2.** The mean percentage glycosidal content in the squill bulb with year of harvest [8].

**Figure 3.** The mean percentage glycosidal content in different parts of the squill bulb [8].

Figure 3. The mean percentage glycosidal content in different parts of the squill bulb [8]. Micropropagation of squill was carried out by direct and indirect organogenesis. Regeneration was successfully achieved Micropropagation of squill was carried out by direct and indirect organogenesis. Regeneration was successfully achieved using bulb explants by direct organogenesis. Although the process of regeneration was slow, callus cultures maintained in high auxin concentrations (4 mg/l 2,4- D + 2 mg/l NAA) induced root formation, when the plant growth regulators (PGRs) were removed [9].

#### **2.2. Ecballium elaterium (L.) A.Rich.**

in the bulb of this plant. It is renowned for its emetic, diuretic, cardiotonic [2], expectorant, rodenticide [3] and anticancer activities. The seaside squill has been extensively studied for its propagation potential. Locally, cultivation studies have been associated with the cardiac

The main constituents of the seaside squill are the cardiac glycosides and phenolic compounds [4]. It also contains mucilage and calcium oxalate crystals. The squill cardiac glycosides are bufadienolides. In principle, these are similar to triterpenoids having a sugar group and a lactone ring at C17. Scillaren A accounts for about 70% of the total glycosidal content of squill. It contains one unit of rhamnose and one unit of glucose. When scillaren A is hydrolyzed by

Figure 2. The structure of cardiac glycosides of Drimia maritima (L.) Stearn.: (A) Scillaren A and (B) Proscillaridin A

of the lactone group [5, 6]. These bufadienolides are therefore important cardiotonic, blood pressure stimulating and antitumour agents. The main glycosides with digoxin-like effects are

Several cultivation parameters were studied for *Drimia maritima* in relation to dry matter yield and the total glycosidal content [8]. These include methods of propagation, planting at different depths, effects of nitrogen (N), phosphorus (P) and potassium (K) fertilizers, cultivation in different soil types, age of harvesting and seasonal timing of harvesting. Propagation by bulb division only takes 10 weeks to produce a seedling as opposed to seed propagation that requires 56 weeks. The type of soil does not contribute to the variation of glycosides in the squill bulb. In fact, Maltese squill grown on four soil types, namely terra soil, xerorendzina soil, carbonate raw and sandy soil exhibited average glycosidal contents of 0.575 % (w/w). Fertiliser studies revealed that the use of different ratios of N, P and K affect the rate of growth but no change in glycosidal content (average of 0.59 % w/w). For the best annual production of dry weight and glycosidal content, it is advisable to harvest squill in the third year after transplanting (table 2) immediately after flowering. The highest glycosidal content is obtained

**Figure 2.** The structure of cardiac glycosides of *Drimia maritima* (L.) Stearn.: (A) Scillaren A and (B) Proscillaridin A

glycosides with digoxin-like effects are scillaren A and proscillaridin A [7].

First

Fourth

Table 2. The mean percentage glycosidal content in the squill bulb with year of harvest [8].

obtained from the roots (Figure 3)

These glycosides act by binding to the Na+

scillaren A and proscillaridin A [7].

from the roots (Figure 3)

These glycosides act by binding to the Na<sup>+</sup>/K<sup>+</sup> ATPase pumps. This occurs due to the presence of the lactone group [5, 6]. These bufadienolides are therefore important cardiotonic, blood pressure stimulating and antitumour agents. The main

/K+ ATPase pumps. This occurs due to the presence

Several cultivation parameters were studied for Drimia maritima in relation to dry matter yield and the total glycosidal content [8]. These include methods of propagation, planting at different depths, effects of nitrogen (N) , phosphorus (P) and potassium (K) fertilizers, cultivation in different soil types, age of harvesting and seasonal timing of harvesting. Propagation by bulb division only takes 10 weeks to produce a seedling as opposed to seed propagation that requires 56 weeks. The type of soil does not contribute to the variation of glycosides in the squill bulb. In fact, Maltese squill grown on four soil types, namely terra soil, xerorendzina soil, carbonate raw and sandy soil exhibited average glycosidal contents of 0.575 % (w/w). Fertiliser studies revealed that the use of different ratios of N, P and K affect the rate of growth but no change in glycosidal content (average of 0.59 % w/w). For the best annual production of dry weight and glycosidal content, it is advisable to harvest squill in the third year after transplanting (table 2) immediately after flowering. The highest glycosidal content is

Year of harvest Treatment Mean glycosidal

Second Control 0.66

Third Control 0.57

Fifth Control 0.38

Sixth Control 0.31

Control 0.25 Fertiliser-treated 0.26

Fertiliser-treated 0.68

Fertiliser-treated 0.58

Control 0.58 Fertiliser-treated 0.58

Fertiliser-treated 0.40

Fertiliser-treated 0.34

content(%)

glycosidic content while micropropagation has been linked to biomass production.

enzymes, it breaks down to proscillaridin A and D-glucose (Figure 2).

6 Phytochemicals - Isolation, Characterisation and Role in Human Health

using bulb explants by direct organogenesis. Although the process of regeneration was slow, callus cultures maintained in high auxin concentrations (4 mg/l 2,4-D + 2 mg/l NAA) induced root formation, when the plant growth regulators (PGRs) *Ecballium elaterium* (squirting cucumber), a member of the Cucurbitaceae family, is a Mediter‐ ranean medicinal plant in a monotypic genus. In the past, the squirting cucumber was used as

2.2. Ecballium elaterium (L.) A.Rich.

been proven through various scientific investigations.

inhibitor, and Astacus protease inhibitor [18].

Ecballium elaterium (squirting cucumber), a member of the Cucurbitaceae family, is a Mediterranean medicinal plant in a

monotypic genus. In the past, the squirting cucumber was used as a purgative, emetic, for the treatment of jaundice and

oedema. It was also used for the treatment of otitis, hydrophobia and malarial fever. Locally, it was prepared in various

dosage forms such as powders, solutions, semisolid blocks and dried cubes for exportation. It also used to be prepared in the

form of lozenges with gum Arabic. The fresh fruit juice was renowned for several pharmacological effects mainly as

antibilirubinaemic, antihepatotoxic and lacrimation stimulant. The dried juice, also known as the elaterium, was effective as

a laxative, anti-inflammatory, antitumour and as an aflatoxin suppressor [10, 11]. Most of these pharmacological effects have

The main constituents of this plant are the cucurbitacins (Cu), the major ones being CuE and CuB (Figure 4), particularly

present in the fruit juice. Other cucurbitacins include cucurbitacins D, G, H, I, R, L, hexanorcucurbitacin I, 16-deoxy-�<sup>16</sup>-

hexanorcucurbitacin O, anhydro-22-deoxo-3-epi-isocucurbitacin D, and their glycosides [12-14]. The squirting cucumber also

contains sterols, fatty acids, elaterases, tannins [15], complex phenolic compounds and flavonoids [16], amino acids and their

derivatives as well as the Ecballium elaterium protease inhibitors (EEPIs). These EEPIs are obtained from seed extracts and are

effective against at least four different serine proteinases [17]. In fact, these are termed as trypsin inhibitors I, II, and III, also

known as the trypsin isoinhibitors (EETIso), chymotrypsin inhibitor (8 kDa), subtilisin inhibitor (9 kDa), and elastase

Although this plant is abundant in wastelands throughout the Maltese Archipelago, micropropagation was attempted for

two main reasons. These were as a means to study the responses of explants from the squirting cucumber to different plant

growth regulators, and to determine the potential propagation of high-yielding mother plants. In this attempt, seeds were

germinated in Murashige-Skoog (MS) medium. Different concentrations and types of PGRs, mainly auxins and cytokinins,

were added. Subculturing with the different PGRs was performed every 4 weeks and explants were maintained at about 25 ±

1 °C and 3250 ± 250 lx. Once developed, the plantlets were transferred to Jiffy® pots until rooting and then repotted

(compost:peat:perlite, 2:2:1) until flowering [19]. The main four responses of explants were bud multiplication, shoot

Figure 4. The structures of (A) cucurbitacin E and (B) Cucurbitacin B found in Ecballium elaterium (L.) A.Rich.

elongation, callus production and rooting, as illustrated in Figure 5.

were removed [9].

a purgative, emetic, for the treatment of jaundice and oedema. It was also used for the treatment of otitis, hydrophobia and malarial fever. Locally, it was prepared in various dosage forms such as powders, solutions, semisolid blocks and dried cubes for exportation. It also used to be prepared in the form of lozenges with gum Arabic. The fresh fruit juice was renowned for several pharmacological effects mainly as antibilirubinaemic, antihepatotoxic and lacrimation stimulant. The dried juice, also known as the elaterium, was effective as a laxative, antiinflammatory, antitumour and as an aflatoxin suppressor [10, 11]. Most of these pharmaco‐ logical effects have been proven through various scientific investigations. 2.2. Ecballium elaterium (L.) A.Rich. Ecballium elaterium (squirting cucumber), a member of the Cucurbitaceae family, is a Mediterranean medicinal plant in a monotypic genus. In the past, the squirting cucumber was used as a purgative, emetic, for the treatment of jaundice and oedema. It was also used for the treatment of otitis, hydrophobia and malarial fever. Locally, it was prepared in various dosage forms such as powders, solutions, semisolid blocks and dried cubes for exportation. It also used to be prepared in the form of lozenges with gum Arabic. The fresh fruit juice was renowned for several pharmacological effects mainly as antibilirubinaemic, antihepatotoxic and lacrimation stimulant. The dried juice, also known as the elaterium, was effective as

Figure 3. The mean percentage glycosidal content in different parts of the squill bulb [8].

Micropropagation of squill was carried out by direct and indirect organogenesis. Regeneration was successfully achieved using bulb explants by direct organogenesis. Although the process of regeneration was slow, callus cultures maintained in

The main constituents of this plant are the cucurbitacins (Cu), the major ones being CuE and CuB (Figure 4), particularly present in the fruit juice. Other cucurbitacins include cucurbitacins D, G, H, I, R, L, hexanorcucurbitacin I, 16-deoxy-∆16-hexanorcucurbitacin O, anhydro-22 deoxo-3-epi-isocucurbitacin D, and their glycosides [12-14]. The squirting cucumber also contains sterols, fatty acids, elaterases, tannins [15], complex phenolic compounds and flavonoids [16], amino acids and their derivatives as well as the *Ecballium elaterium* protease inhibitors (EEPIs). These EEPIs are obtained from seed extracts and are effective against at least four different serine proteinases [17]. In fact, these are termed as trypsin inhibitors I, II, and III, also known as the trypsin isoinhibitors (EETIso), chymotrypsin inhibitor (8 kDa), subtilisin inhibitor (9 kDa), and elastase inhibitor, and Astacus protease inhibitor [18]. a laxative, anti-inflammatory, antitumour and as an aflatoxin suppressor [10, 11]. Most of these pharmacological effects have been proven through various scientific investigations. The main constituents of this plant are the cucurbitacins (Cu), the major ones being CuE and CuB (Figure 4), particularly present in the fruit juice. Other cucurbitacins include cucurbitacins D, G, H, I, R, L, hexanorcucurbitacin I, 16-deoxy-�<sup>16</sup> hexanorcucurbitacin O, anhydro-22-deoxo-3-epi-isocucurbitacin D, and their glycosides [12-14]. The squirting cucumber also contains sterols, fatty acids, elaterases, tannins [15], complex phenolic compounds and flavonoids [16], amino acids and their derivatives as well as the Ecballium elaterium protease inhibitors (EEPIs). These EEPIs are obtained from seed extracts and are effective against at least four different serine proteinases [17]. In fact, these are termed as trypsin inhibitors I, II, and III, also known as the trypsin isoinhibitors (EETIso), chymotrypsin inhibitor (8 kDa), subtilisin inhibitor (9 kDa), and elastase inhibitor, and Astacus protease inhibitor [18].

Figure 4. The structures of (A) cucurbitacin E and (B) Cucurbitacin B found in Ecballium elaterium (L.) A.Rich. **Figure 4.** The structures of (A) cucurbitacin E and (B) Cucurbitacin B found in *Ecballium elaterium* (L.) A.Rich.

Although this plant is abundant in wastelands throughout the Maltese Archipelago, micropropagation was attempted for two main reasons. These were as a means to study the responses of explants from the squirting cucumber to different plant growth regulators, and to determine the potential propagation of high-yielding mother plants. In this attempt, seeds were germinated in Murashige-Skoog (MS) medium. Different concentrations and types of PGRs, mainly auxins and cytokinins, were added. Subculturing with the different PGRs was performed every 4 weeks and explants were maintained at about 25 ± 1 °C and 3250 ± 250 lx. Once developed, the plantlets were transferred to Jiffy® pots until rooting and then repotted (compost:peat:perlite, 2:2:1) until flowering [19]. The main four responses of explants were bud multiplication, shoot elongation, callus production and rooting, as illustrated in Figure 5. Although this plant is abundant in wastelands throughout the Maltese Archipelago, micro‐ propagation was attempted for two main reasons. These were as a means to study the responses of explants from the squirting cucumber to different plant growth regulators, and to determine the potential propagation of high-yielding mother plants. In this attempt, seeds were germinated in Murashige-Skoog (MS) medium. Different concentrations and types of PGRs, mainly auxins and cytokinins, were added. Subculturing with the different PGRs was performed every 4 weeks and explants were maintained at about 25 ± 1 °C and 3250 ± 250 lx. Once developed, the plantlets were transferred to Jiffy® pots until rooting and then repotted (compost:peat:perlite, 2:2:1) until flowering [19]. The main four responses of explants were bud multiplication, shoot elongation, callus production and rooting, as illustrated in Figure 5.

A regeneration protocol was devised as follows. Briefly, the seeds were germinated on MS medium (8 - 9 weeks). Bud multiplication of node explants was performed on naphthalene‐ acetic acid/6-benzylaminopurine (NAA/BAP) medium (for 2 - 3 subcultures every 4 weeks). high auxin concentrations (4 mg/l 2,4-D + 2 mg/l NAA) induced root formation, when the plant growth regulators (PGRs) The Phytochemical Constitution of Maltese Medicinal Plants – Propagation, Isolation and Pharmacological Testing http://dx.doi.org/10.5772/60094 9

a purgative, emetic, for the treatment of jaundice and oedema. It was also used for the treatment of otitis, hydrophobia and malarial fever. Locally, it was prepared in various dosage forms such as powders, solutions, semisolid blocks and dried cubes for exportation. It also used to be prepared in the form of lozenges with gum Arabic. The fresh fruit juice was renowned for several pharmacological effects mainly as antibilirubinaemic, antihepatotoxic and lacrimation stimulant. The dried juice, also known as the elaterium, was effective as a laxative, antiinflammatory, antitumour and as an aflatoxin suppressor [10, 11]. Most of these pharmaco‐

Figure 3. The mean percentage glycosidal content in different parts of the squill bulb [8].

Micropropagation of squill was carried out by direct and indirect organogenesis. Regeneration was successfully achieved using bulb explants by direct organogenesis. Although the process of regeneration was slow, callus cultures maintained in

Ecballium elaterium (squirting cucumber), a member of the Cucurbitaceae family, is a Mediterranean medicinal plant in a monotypic genus. In the past, the squirting cucumber was used as a purgative, emetic, for the treatment of jaundice and oedema. It was also used for the treatment of otitis, hydrophobia and malarial fever. Locally, it was prepared in various dosage forms such as powders, solutions, semisolid blocks and dried cubes for exportation. It also used to be prepared in the form of lozenges with gum Arabic. The fresh fruit juice was renowned for several pharmacological effects mainly as antibilirubinaemic, antihepatotoxic and lacrimation stimulant. The dried juice, also known as the elaterium, was effective as a laxative, anti-inflammatory, antitumour and as an aflatoxin suppressor [10, 11]. Most of these pharmacological effects have

The main constituents of this plant are the cucurbitacins (Cu), the major ones being CuE and CuB (Figure 4), particularly present in the fruit juice. Other cucurbitacins include cucurbitacins D, G, H, I, R, L, hexanorcucurbitacin I, 16-deoxy-�<sup>16</sup> hexanorcucurbitacin O, anhydro-22-deoxo-3-epi-isocucurbitacin D, and their glycosides [12-14]. The squirting cucumber also contains sterols, fatty acids, elaterases, tannins [15], complex phenolic compounds and flavonoids [16], amino acids and their derivatives as well as the Ecballium elaterium protease inhibitors (EEPIs). These EEPIs are obtained from seed extracts and are effective against at least four different serine proteinases [17]. In fact, these are termed as trypsin inhibitors I, II, and III, also known as the trypsin isoinhibitors (EETIso), chymotrypsin inhibitor (8 kDa), subtilisin inhibitor (9 kDa), and elastase

The main constituents of this plant are the cucurbitacins (Cu), the major ones being CuE and CuB (Figure 4), particularly present in the fruit juice. Other cucurbitacins include cucurbitacins D, G, H, I, R, L, hexanorcucurbitacin I, 16-deoxy-∆16-hexanorcucurbitacin O, anhydro-22 deoxo-3-epi-isocucurbitacin D, and their glycosides [12-14]. The squirting cucumber also contains sterols, fatty acids, elaterases, tannins [15], complex phenolic compounds and flavonoids [16], amino acids and their derivatives as well as the *Ecballium elaterium* protease inhibitors (EEPIs). These EEPIs are obtained from seed extracts and are effective against at least four different serine proteinases [17]. In fact, these are termed as trypsin inhibitors I, II, and III, also known as the trypsin isoinhibitors (EETIso), chymotrypsin inhibitor (8 kDa), subtilisin inhibitor (9 kDa), and elastase inhibitor, and Astacus protease inhibitor [18].

Figure 4. The structures of (A) cucurbitacin E and (B) Cucurbitacin B found in Ecballium elaterium (L.) A.Rich.

elongation, callus production and rooting, as illustrated in Figure 5.

**Figure 4.** The structures of (A) cucurbitacin E and (B) Cucurbitacin B found in *Ecballium elaterium* (L.) A.Rich.

Although this plant is abundant in wastelands throughout the Maltese Archipelago, micro‐ propagation was attempted for two main reasons. These were as a means to study the responses of explants from the squirting cucumber to different plant growth regulators, and to determine the potential propagation of high-yielding mother plants. In this attempt, seeds were germinated in Murashige-Skoog (MS) medium. Different concentrations and types of PGRs, mainly auxins and cytokinins, were added. Subculturing with the different PGRs was performed every 4 weeks and explants were maintained at about 25 ± 1 °C and 3250 ± 250 lx. Once developed, the plantlets were transferred to Jiffy® pots until rooting and then repotted (compost:peat:perlite, 2:2:1) until flowering [19]. The main four responses of explants were bud multiplication, shoot elongation, callus production and rooting, as illustrated in Figure 5. A regeneration protocol was devised as follows. Briefly, the seeds were germinated on MS medium (8 - 9 weeks). Bud multiplication of node explants was performed on naphthalene‐ acetic acid/6-benzylaminopurine (NAA/BAP) medium (for 2 - 3 subcultures every 4 weeks).

logical effects have been proven through various scientific investigations.

been proven through various scientific investigations.

inhibitor, and Astacus protease inhibitor [18].

2.2. Ecballium elaterium (L.) A.Rich.

8 Phytochemicals - Isolation, Characterisation and Role in Human Health

were removed [9].

**Figure 5.** The effects of plant growth regulators on *Ecballium elaterium* explants in tissue culture [20].

shoot elongation was obtained on Gibberellic Acid (GA3) medium (4 weeks), followed by an auxin shock on Indole-3-acetic acid (IAA) medium (1 week) then, treated with rooting hormone powder and finally transfer to Jiffy® pots (3 - 4 weeks). The plants were then repotting and acclimatised for 4 - 5 weeks [19]. The whole process takes between 24 and 35 weeks. Figure 5. The effects of plant growth regulators on Ecballium elaterium explants in tissue culture [20]. A regeneration protocol was devised as follows. Briefly, the seeds were germinated on MS medium (8 - 9 weeks). Bud multiplication of node explants was performed on naphthaleneacetic acid/6-benzylaminopurine (NAA/BAP) medium (for 2 - 3 subcultures every 4 weeks). shoot elongation was obtained on Gibberellic Acid (GA3) medium (4 weeks), followed by an

Although this plant is abundant in wastelands throughout the Maltese Archipelago, micropropagation was attempted for two main reasons. These were as a means to study the responses of explants from the squirting cucumber to different plant growth regulators, and to determine the potential propagation of high-yielding mother plants. In this attempt, seeds were germinated in Murashige-Skoog (MS) medium. Different concentrations and types of PGRs, mainly auxins and cytokinins, were added. Subculturing with the different PGRs was performed every 4 weeks and explants were maintained at about 25 ± 1 °C and 3250 ± 250 lx. Once developed, the plantlets were transferred to Jiffy® pots until rooting and then repotted The *Ecballium elaterium* explants produced a high amount of callus and this led to further studies to determine the production of cucurbitacins in these undifferentiated cells. Callus masses were treated with different PGRs at different concentrations. The best PGR combina‐ tion for biomass accumulation was 2,4-Dichlorophenoxyacetic acid/kinetin (2,4D/Ki) while for metabolite production, the NAA/BAP combinations showed optimum yields [20]. A growthlinked accumulation of metabolites was observed (figure 6). auxin shock on Indole-3-acetic acid (IAA) medium (1 week) then, treated with rooting hormone powder and finally transfer to Jiffy® pots (3 - 4 weeks). The plants were then repotting and acclimatised for 4 - 5 weeks [19]. The whole process takes between 24 and 35 weeks. The Ecballium elaterium explants produced a high amount of callus and this led to further studies to determine the production of cucurbitacins in these undifferentiated cells. Callus masses were treated with different PGRs at different concentrations.

(compost:peat:perlite, 2:2:1) until flowering [19]. The main four responses of explants were bud multiplication, shoot The production of cucurbitacins from cultivated sources, is significantly higher in fruit compared to stems and leaves (figure 7). A drop in ambient temperature results in lower production of cucurbitacins [21]. The best PGR combination for biomass accumulation was 2,4-Dichlorophenoxyacetic acid/kinetin (2,4D/Ki) while for metabolite production, the NAA/BAP combinations showed optimum yields [20]. A growth-linked accumulation of metabolites was observed (figure 6).

> Pharmacological testing has been extensively carried out on this plant. Extracts exhibited a marked effect on prostate cancer cells (IC50= 9.35 nM) and moderate effects on melanoma and breast cancer cells (IC50 = 0.87 and 1.95 μM, respectively) *in vitro*. Negligible cytotoxic effects

**Figure 6.** Growth-linked accumulation of metabolites in *Ecballium elaterium* cultures.

Figure 6. Growth-linked accumulation of metabolites in Ecballium elaterium cultures.

7). A drop in ambient temperature results in lower production of cucurbitacins [21].

Figure 7. The total cucurbitacin content in elaterium produced from Ecballium elaterium fruit, stems and leaves with time and temperature [21]. **Figure 7.** The total cucurbitacin content in elaterium produced from *Ecballium elaterium* fruit, stems and leaves with time and temperature [21].

were observed on normal fibroblasts (IC50 = 93.8 μM) [22]. It was demonstrated that CuE provoked apoptosis in cancer cell lines. This was exhibited by the condensation of chromatin and also DNA fragmentation using gel electrophoresis. CuE was also effective as an immune modulator. Human peripheral T-lymphocytes were freshly isolated and challenged with phytohaemagglutinin (PHA) and *Ecballium elaterium* extracts [23]. Cucurbitacins in the juice Figure 7. The total cucurbitacin content in elaterium produced from Ecballium elaterium fruit, stems and leaves with time and temperature [21]. Pharmacological testing has been extensively carried out on this plant. Extracts exhibited a marked effect on prostate cancer cells (IC50= 9.35 nM) and moderate effects on melanoma and breast cancer cells (IC50 = 0.87 and 1.95 µM, respectively) in vitro. Negligible cytotoxic effects were observed on normal fibroblasts (IC50 = 93.8 µM) [22]. It was demonstrated that CuE provoked apoptosis in cancer cell lines. This was exhibited by the condensation of chromatin and also DNA fragmentation using gel electrophoresis. CuE was also effective as an immune modulator. Human peripheral T-lymphocytes were freshly

Pharmacological testing has been extensively carried out on this plant. Extracts exhibited a marked effect on prostate cancer cells (IC50= 9.35 nM) and moderate effects on melanoma and breast cancer cells (IC50 = 0.87 and 1.95 µM, respectively) in vitro. Negligible cytotoxic effects were observed on normal fibroblasts (IC50 = 93.8 µM) [22]. It was demonstrated that CuE provoked apoptosis in cancer cell lines. This was exhibited by the condensation of chromatin and also DNA fragmentation using gel electrophoresis. CuE was also effective as an immune modulator. Human peripheral T-lymphocytes were freshly extract of *Ecballium elaterium*, also exhibited potential anti-inflammatory, analgesic and antipyretic activities in rodents [10, 24].

#### **2.3. Mentha pulegium L.**

were observed on normal fibroblasts (IC50 = 93.8 μM) [22]. It was demonstrated that CuE provoked apoptosis in cancer cell lines. This was exhibited by the condensation of chromatin and also DNA fragmentation using gel electrophoresis. CuE was also effective as an immune modulator. Human peripheral T-lymphocytes were freshly isolated and challenged with phytohaemagglutinin (PHA) and *Ecballium elaterium* extracts [23]. Cucurbitacins in the juice

**Figure 7.** The total cucurbitacin content in elaterium produced from *Ecballium elaterium* fruit, stems and leaves with

Pharmacological testing has been extensively carried out on this plant. Extracts exhibited a marked effect on prostate cancer cells (IC50= 9.35 nM) and moderate effects on melanoma and breast cancer cells (IC50 = 0.87 and 1.95 µM, respectively) in vitro. Negligible cytotoxic effects were observed on normal fibroblasts (IC50 = 93.8 µM) [22]. It was demonstrated that CuE provoked apoptosis in cancer cell lines. This was exhibited by the condensation of chromatin and also DNA fragmentation using gel electrophoresis. CuE was also effective as an immune modulator. Human peripheral T-lymphocytes were freshly

Figure 7. The total cucurbitacin content in elaterium produced from Ecballium elaterium fruit, stems and leaves with time and temperature

cells (IC50= 9.35 nM) and moderate effects on melanoma and breast cancer cells (IC50 = 0.87 and 1.95 µM, respectively) in vitro.

[21].

time and temperature [21].

[21].

Figure 6. Growth-linked accumulation of metabolites in Ecballium elaterium cultures.

7). A drop in ambient temperature results in lower production of cucurbitacins [21].

**Figure 6.** Growth-linked accumulation of metabolites in *Ecballium elaterium* cultures.

10 Phytochemicals - Isolation, Characterisation and Role in Human Health

Figure 6. Growth-linked accumulation of metabolites in Ecballium elaterium cultures.

7). A drop in ambient temperature results in lower production of cucurbitacins [21].

*Mentha pulegium* L. is a perennial plant, belonging to the Lamiaceae family. During Roman times, the plant was used for several ailments particularly for headaches, flatulence and even as an abortifacient. The name 'pulegium' derived from the Latin word 'pulex' for flea, indicates that in Roman times the plant was used as a flea repellent [25]. Locally, it was well-reputed as a treatment for common cold, as a carminative, emmenagogue but also as an insect repellent [26]. *Mentha pulegium* used to be hung in wardrobes to ward off fleas and placed on windowsills to repel mosquitoes especially during the summer months. The most important extract from this plant is the essential oil, known as the pennyroyal oil. isolated and challenged with phytohaemagglutinin (PHA) and Ecballium elaterium extracts [23]. Cucurbitacins in the juice extract of Ecballium elaterium, also exhibited potential anti-inflammatory, analgesic and antipyretic activities in rodents [10, 24]. 2.3. Mentha pulegium L.

The production of cucurbitacins from cultivated sources, is significantly higher in fruit compared to stems and leaves (figure The production of cucurbitacins from cultivated sources, is significantly higher in fruit compared to stems and leaves (figure In a study by [27], pennyroyal oil contained 38.0% piperitone, 33.0% piperitenone, 4.7% αterpineol and 2.3% pulegone as the major components (Figure 8). The authors concluded that Iranian pennyroyal oil is rich in piperitone/piperitenone. In another study, the pulegone content of Iranian pennyroyal oil ranged between 1.3 – 52.0%, when extracted by supercritical fluid extraction, while hydrodistillation yielded around 37.8% of pulegone. Piperitenone consituted only 6.8% to the extracted essential oil [28]. Similarly, in another study [29], the content of pulegone in Greek pennyroyal oil was in the range of 42.9% and 90.7% attributed to two populations. In other wild populations, the pulegone content did not exceed 35.6%. Such populations were rich in either menthone/isomenthone or in piperitone/piperitenone. In Tunisian pennyroyal oil, 41.8 % of the oil was pulegone [30] while Portuguese pennyroyal oil contained 23.2 % of pulegone [31]. The pennyroyal oil was extracted from wild Maltese populations using hydrodistillation with a yield of 0.73 % [32]. The pulegone content in the oil was 85.8 %, followed by other constituents; (-) limonene (0.984 %), myrcene (0.109 %) and βpinene (0.191 %). This was determined by GC-FID. Mentha pulegium L. is a perennial plant, belonging to the Lamiaceae family. During Roman times, the plant was used for several ailments particularly for headaches, flatulence and even as an abortifacient. The name 'pulegium' derived from the Latin word 'pulex' for flea, indicates that in Roman times the plant was used as a flea repellent [25]. Locally, it was wellreputed as a treatment for common cold, as a carminative, emmenagogue but also as an insect repellent [26]. Mentha pulegium used to be hung in wardrobes to ward off fleas and placed on windowsills to repel mosquitoes especially during the summer months. The most important extract from this plant is the essential oil, known as the pennyroyal oil. In a study by [27], pennyroyal oil contained 38.0% piperitone, 33.0% piperitenone, 4.7% α-terpineol and 2.3% pulegone as the major components (Figure 8). The authors concluded that Iranian pennyroyal oil is rich in piperitone/piperitenone. In another study, the pulegone content of Iranian pennyroyal oil ranged between 1.3 – 52.0%, when extracted by supercritical fluid extraction, while hydrodistillation yielded around 37.8% of pulegone. Piperitenone consituted only 6.8% to the extracted essential oil [28]. Similarly, in another study [29], the content of pulegone in Greek pennyroyal oil was in the range of 42.9% and 90.7% attributed to two populations. In other wild populations, the pulegone content did not exceed 35.6%. Such populations were rich in either menthone/isomenthone or in piperitone/piperitenone. In Tunisian pennyroyal oil, 41.8 % of the oil was pulegone [30] while Portuguese pennyroyal oil contained 23.2 % of pulegone [31]. The pennyroyal oil was extracted from wild Maltese populations using hydrodistillation with a yield of 0.73 % [32]. The pulegone content in the oil was 85.8 %, followed by other constituents; (-) limonene (0.984 %), myrcene (0.109 %) and β-pinene (0.191 %). This was determined by GC-FID.

Pharmacological testing has been extensively carried out on this plant. Extracts exhibited a marked effect on prostate cancer Figure 8. The most abundant monoterpenoids of Mentha pulegium L. essential oil: (A) pulegone, (B) piperitone, (C) piperitenone, (D) αterpineol and (E) menthofuran. **Figure 8.** The most abundant monoterpenoids of *Mentha pulegium* L. essential oil: (A) pulegone, (B) piperitone, (C) pi‐ peritenone, (D) α-terpineol and (E) menthofuran.

Figure 7. The total cucurbitacin content in elaterium produced from Ecballium elaterium fruit, stems and leaves with time and temperature Negligible cytotoxic effects were observed on normal fibroblasts (IC50 = 93.8 µM) [22]. It was demonstrated that CuE provoked apoptosis in cancer cell lines. This was exhibited by the condensation of chromatin and also DNA fragmentation using gel electrophoresis. CuE was also effective as an immune modulator. Human peripheral T-lymphocytes were freshly Apart from its abortifacient activity, pennyroyal oil is also hepatotoxic and causes pulmonary necrosis. Hepatotoxicity is mainly attributed to the conversion of pulegone into its epoxide or menthofuran derivatives [33-35]. Insect repellent activity of pennyroyal was determined by using two setups (Figure 9) with citronella oil and distilled water Apart from its abortifacient activity, pennyroyal oil is also hepatotoxic and causes pulmonary necrosis. Hepatotoxicity is mainly attributed to the conversion of pulegone into its epoxide or menthofuran derivatives [33-35].

used as positive and negative controls, respectively [32]. Setup 1 consisted of a trough with a diameter of 30 cm and a height of 12 cm. Four zones were designated within the trough (Figure 9A). The mosquitoes were introduced inside the container, and the oil sample was then injected by a syringe. Sixteen mosquitoes were observed every two minutes for a period of 20 minutes and their position within the trough was recorded. After the second minute, 75 % of the mosquitoes were found in the compartment furthest from the injection site. A gradient was achieved at this time interval and the mosquitoes moved away from the source. After the tenth minute, this compartmental difference was no longer observed, most probably due to Insect repellent activity of pennyroyal was determined by using two setups (Figure 9) with citronella oil and distilled water used as positive and negative controls, respectively [32]. Setup 1 consisted of a trough with a diameter of 30 cm and a height of 12 cm. Four zones were designated within the trough (Figure 9A). The mosquitoes were introduced inside the container, and the oil sample was then injected by a syringe. Sixteen mosquitoes were observed every two minutes for a period of 20 minutes and their position within the trough was recorded. After the second minute, 75 % of the mosquitoes were found in the compartment furthest from the injection site. A gradient was achieved at this time interval and the mosqui‐ toes moved away from the source. After the tenth minute, this compartmental difference was no longer observed, most probably due to the fact that the oil must have saturated the trough and hence there was no trend in mosquito distribution. Setup 2 consisted of a glass tube with an internal diameter of 2.5 cm and a length of 150 cm. Seven zones were designated within the tube (Figure 9B). Twenty mosquitoes were observed every two minutes for half an hour and their position recorded, following injection of the pennyroyal oil. As with setup 1, there was a statistical difference between zone 1 and zone 7 of the tube, but this difference became negligible with time. Similar results were observed with citronella. In spite of this similarity, GC-FID determination of the citronella oil revealed the presence of geraniol (60.0 %), citronellal (15.0 %) and camphene (> 15.0 %), but no significant pulegone content. With water a more random distribution of mosquitoes was observed [32]. the fact that the oil must have saturated the trough and hence there was no trend in mosquito distribution. Setup 2 consisted of a glass tube with an internal diameter of 2.5 cm and a length of 150 cm. Seven zones were designated within the tube (Figure 9B). Twenty mosquitoes were observed every two minutes for half an hour and their position recorded, following injection of the pennyroyal oil. As with setup 1, there was a statistical difference between zone 1 and zone 7 of the tube, but this difference became negligible with time. Similar results were observed with citronella. In spite of this similarity, GC-FID determination of the citronella oil revealed the presence of geraniol (60.0 %), citronellal (15.0 %) and camphene (> 15.0 %), but

no significant pulegone content. With water a more random distribution of mosquitoes was observed [32].

Figure 9. The experimental setups used to determine the insect repellent properties of pennyroyal oil [32]. **Figure 9.** The experimental setups used to determine the insect repellent properties of pennyroyal oil [32].

inflammatory [41] and antiviral effects [42] and is also used to control Alzheimer's disease [43]

content [36].

2.4. Salvia officinalis L.

Pennyroyal oil exhibited repellent and insecticidal effects. After 90 minutes exposure, none of the mosquitoes were airborne and those that were in contact with the oil were dead. The insect repellent activity was attributed to the high pulegone

Salvia officinalis, more commonly known as garden sage, is a member of the Lamiaceae family. Sage has been renowned for its healing properties since the Ancient Greeks. The Romans inherited the medicinal knowledge on sage and used it to enhance diuresis, menstruation and to stop bleeding of wounds. It was also used to treat pain associated with colds and rheumatism [37]. Scientifically, sage has several medicinal properties, such as, antioxidant [38, 39], antibacterial [40], anti-

Sage contains several metabolites primarily monoterpenoids and sesquiterpenoids, diterpenoids [43], triterpenoids, such as ursolic and oleanolic acid [41, 44], and also flavonoids and phenolic glycosides [45]. The essential oil of Portuguese sage Pennyroyal oil exhibited repellent and insecticidal effects. After 90 minutes exposure, none of the mosquitoes were airborne and those that were in contact with the oil were dead. The insect repellent activity was attributed to the high pulegone content [36].

#### **2.4. Salvia officinalis L.**

Insect repellent activity of pennyroyal was determined by using two setups (Figure 9) with citronella oil and distilled water used as positive and negative controls, respectively [32]. Setup 1 consisted of a trough with a diameter of 30 cm and a height of 12 cm. Four zones were designated within the trough (Figure 9A). The mosquitoes were introduced inside the container, and the oil sample was then injected by a syringe. Sixteen mosquitoes were observed every two minutes for a period of 20 minutes and their position within the trough was recorded. After the second minute, 75 % of the mosquitoes were found in the compartment furthest from the injection site. A gradient was achieved at this time interval and the mosqui‐ toes moved away from the source. After the tenth minute, this compartmental difference was no longer observed, most probably due to the fact that the oil must have saturated the trough and hence there was no trend in mosquito distribution. Setup 2 consisted of a glass tube with an internal diameter of 2.5 cm and a length of 150 cm. Seven zones were designated within the tube (Figure 9B). Twenty mosquitoes were observed every two minutes for half an hour and their position recorded, following injection of the pennyroyal oil. As with setup 1, there was a statistical difference between zone 1 and zone 7 of the tube, but this difference became negligible with time. Similar results were observed with citronella. In spite of this similarity, GC-FID determination of the citronella oil revealed the presence of geraniol (60.0 %), citronellal (15.0 %) and camphene (> 15.0 %), but no significant pulegone content. With water a more

the fact that the oil must have saturated the trough and hence there was no trend in mosquito distribution. Setup 2 consisted of a glass tube with an internal diameter of 2.5 cm and a length of 150 cm. Seven zones were designated within the tube (Figure 9B). Twenty mosquitoes were observed every two minutes for half an hour and their position recorded, following injection of the pennyroyal oil. As with setup 1, there was a statistical difference between zone 1 and zone 7 of the tube, but this difference became negligible with time. Similar results were observed with citronella. In spite of this similarity, GC-FID determination of the citronella oil revealed the presence of geraniol (60.0 %), citronellal (15.0 %) and camphene (> 15.0 %), but

no significant pulegone content. With water a more random distribution of mosquitoes was observed [32].

Figure 9. The experimental setups used to determine the insect repellent properties of pennyroyal oil [32].

**Figure 9.** The experimental setups used to determine the insect repellent properties of pennyroyal oil [32].

inflammatory [41] and antiviral effects [42] and is also used to control Alzheimer's disease [43]

content [36].

2.4. Salvia officinalis L.

Pennyroyal oil exhibited repellent and insecticidal effects. After 90 minutes exposure, none of the mosquitoes were airborne and those that were in contact with the oil were dead. The insect repellent activity was attributed to the high pulegone

Salvia officinalis, more commonly known as garden sage, is a member of the Lamiaceae family. Sage has been renowned for its healing properties since the Ancient Greeks. The Romans inherited the medicinal knowledge on sage and used it to enhance diuresis, menstruation and to stop bleeding of wounds. It was also used to treat pain associated with colds and rheumatism [37]. Scientifically, sage has several medicinal properties, such as, antioxidant [38, 39], antibacterial [40], anti-

Sage contains several metabolites primarily monoterpenoids and sesquiterpenoids, diterpenoids [43], triterpenoids, such as ursolic and oleanolic acid [41, 44], and also flavonoids and phenolic glycosides [45]. The essential oil of Portuguese sage

random distribution of mosquitoes was observed [32].

12 Phytochemicals - Isolation, Characterisation and Role in Human Health

*Salvia officinalis*, more commonly known as garden sage, is a member of the Lamiaceae family. Sage has been renowned for its healing properties since the Ancient Greeks. The Romans inherited the medicinal knowledge on sage and used it to enhance diuresis, menstruation and to stop bleeding of wounds. It was also used to treat pain associated with colds and rheumatism [37]. Scientifically, sage has several medicinal properties, such as, antioxidant [38, 39], anti‐ bacterial [40], anti-inflammatory [41] and antiviral effects [42] and is also used to control Alzheimer's disease [43]

Sage contains several metabolites primarily monoterpenoids and sesquiterpenoids, diterpe‐ noids [43], triterpenoids, such as ursolic and oleanolic acid [41, 44], and also flavonoids and phenolic glycosides [45]. The essential oil of Portuguese sage according to [46] contains αthujone (17.4 %), α-humulene (13.3 %), 1,8-cineole (12.7 %), *E*-caryophyllene (8.5%) and borneol (8.3%) as major constituents. In another study [47], the sage essential oil contained mainly αthujone (29.1 %), camphor (26.3 %), 1,8-cineole (9.3 %), α-humulene (4.4 %) and terpinen-4-ol (4.0%). Similar results were obtained in a local study [48], where the Maltese sage oil was found to contain mainly α-thujone (29.28 %), camphor (26.61 %) and 1,8-cineole (15.53 %) as the major constituents (Figure 10). according to [46] contains α-thujone (17.4 %), α-humulene (13.3 %), 1,8-cineole (12.7 %), E-caryophyllene (8.5%) and borneol (8.3%) as major constituents. In another study [47], the sage essential oil contained mainly α-thujone (29.1 %), camphor (26.3 %), 1,8-cineole (9.3 %), α-humulene (4.4 %) and terpinen-4-ol (4.0%). Similar results were obtained in a local study [48], where the Maltese sage oil was found to contain mainly α-thujone (29.28 %), camphor (26.61 %) and 1,8-cineole (15.53 %) as the major constituents (Figure 10).

Figure 10. The common constituents of Salvia officinalis L. essential oil: (A) thujone, (B) camphor, (C) 1,8-cineole and (D) carnosolic acid. Another significantly important metabolite in sage is carnosolic acid, a bitter abietane diterpenoid derivative with a carboxylic acid structure. This compound possesses antimicrobial, antioxidant, antiviral and anticancer activities [49]. **Figure 10.** The common constituents of *Salvia officinalis* L. essential oil: (A) thujone, (B) camphor, (C) 1,8-cineole and (D) carnosolic acid.

Carnosolic acid was extracted using Soxhlet extraction and petroleum ether as extractant. The extract was dried and dissolved in pyridine/acetic anhydride. The neutral fraction was then chromatographed using silica gel as support [48]. Cultivation studies revealed that sage is best cultivated under shade conditions with irrigation. Propagation is best performed by cuttings every three weeks during spring after the plants have ceased to flower. The recommended planting distance is 30 cm in a row with a cultivation density of 10 plants per m<sup>2</sup>. Plants should be irrigated immediately after planting of cuttings and twice weekly in summer. The monthly harvesting of leaves produced a variable content of essential oil on fresh weight basis with the peak reached during the month of August (2.24 % v/v) and the least during December (0.52 Another significantly important metabolite in sage is carnosolic acid, a bitter abietane diterpe‐ noid derivative with a carboxylic acid structure. This compound possesses antimicrobial, antioxidant, antiviral and anticancer activities [49]. Carnosolic acid was extracted using Soxhlet extraction and petroleum ether as extractant. The extract was dried and dissolved in pyridine/ acetic anhydride. The neutral fraction was then chromatographed using silica gel as support [48].

% v/v) (Figure 11). Cultivation studies revealed that sage is best cultivated under shade conditions with irrigation. Propagation is best performed by cuttings every three weeks during spring after the plants have ceased to flower. The recommended planting distance is 30 cm in a row with a cultivation density of 10 plants per m2 . Plants should be irrigated immediately after planting of cuttings and twice weekly in summer. The monthly harvesting of leaves produced a variable content of essential oil on fresh weight basis with the peak reached during the month of August (2.24 % v/v) and the least during December (0.52 % v/v) (Figure 11).

**Figure 11.** The yield of Maltese sage essential oil throughout the year [48].

sources was negligible [58].

#### **2.5. Verbena officinalis L.**

2.5. Verbena officinalis L. Verbena officinalis, a member of the Verbenaceae family, is also known as vervain. This plant is indigenous to Europe, North Africa and Asia but has been introduced to North America and Australia. Some of the common traditional uses of vervain, worldwide, were in the treatment of respiratory problems such as cough, wheezing and shortness of breath [37], as a purgative, in the treatment of haemorrhoids, eye problems [50], wounds, fever and stomach upsets [51]. In Malta, vervain *Verbena officinalis*, a member of the Verbenaceae family, is also known as vervain. This plant is indigenous to Europe, North Africa and Asia but has been introduced to North America and Australia. Some of the common traditional uses of vervain, worldwide, were in the treatment of respiratory problems such as cough, wheezing and shortness of breath [37], as a purgative, in the treatment of haemorrhoids, eye problems [50], wounds, fever and stomach upsets [51]. In Malta, vervain was used in the treatment of many ailments particularly, carbuncles, boils, wounds, eczema, high blood pressure, diarrhoea, dysentery, cough and arthritis [52].

Figure 11. The yield of Maltese sage essential oil throughout the year [48].

yields an essential oil, with citral, geraniol, limonene and verbenone as main constituents [56]. Other constituents include the flavone derivative artemetin, phenylpropane glycosides verbascoside and eukovoside and the triterpenes ursolic acid, βsitosterol and lupeol [57]. Some of these constituents are highlighted in figure 12. The volume of oil obtained from Maltese

was used in the treatment of many ailments particularly, carbuncles, boils, wounds, eczema, high blood pressure, diarrhoea, dysentery, cough and arthritis [52]. The main constituents of Verbena officinalis are iridoid glycosides, namely verbenalin [53], hastatoside [54] and aucubin [55]. It The main constituents of *Verbena officinalis* are iridoid glycosides, namely verbenalin [53], hastatoside [54] and aucubin [55]. It yields an essential oil, with citral, geraniol, limonene and verbenone as main constituents [56]. Other constituents include the flavone derivative artemetin, phenylpropane glycosides verbascoside and eukovoside and the triterpenes ursolic

acid, β-sitosterol and lupeol [57]. Some of these constituents are highlighted in figure 12. The volume of oil obtained from Maltese sources was negligible [58]. flavone derivative artemetin, phenylpropane glycosides verbascoside and eukovoside and the triterpenes ursolic acid, βsitosterol and lupeol [57]. Some of these constituents are highlighted in figure 12. The volume of oil obtained from Maltese sources was negligible [58].

yields an essential oil, with citral, geraniol, limonene and verbenone as main constituents [56]. Other constituents include the

Verbena officinalis, a member of the Verbenaceae family, is also known as vervain. This plant is indigenous to Europe, North Africa and Asia but has been introduced to North America and Australia. Some of the common traditional uses of vervain, worldwide, were in the treatment of respiratory problems such as cough, wheezing and shortness of breath [37], as a purgative, in the treatment of haemorrhoids, eye problems [50], wounds, fever and stomach upsets [51]. In Malta, vervain was used in the treatment of many ailments particularly, carbuncles, boils, wounds, eczema, high blood pressure, diarrhoea,

Figure 11. The yield of Maltese sage essential oil throughout the year [48].

2.5. Verbena officinalis L.

**Figure 12.** Typical constituents of *Verbena officinalis* L.: (A) verbenalin, (B) artemetin and (C) verbascoside

A hydromethanolic extract of the dried aerial parts of Maltese vervain was obtained by Soxhlet extraction[58].The constitutionofverbenalin wasdeterminedby HPLCusing SupelcosilLC-18 column, acetonitrile/water-phosphoric acid (pH 2) gradient mobile phase with a flow rate of 1.5 ml/min. The content of verbenalin expressed as dry weight of plant material was 2.09 % (w/ w).Previousreports[59]declaredthatcontentsofverbenalinwerelessthan0.1%whenextracted with ether but the content in methanolic extracts varied between 0.12 and 0.50 % [60].

Several pharmacological activities are attributed to vervain, namely, anti-inflammatory [54, 61], neuroprotective [62], antioxidant, antifungal [63], antileukaemic [64] and hepatoprotective [65]. Verbenalin, from Maltese vervain sources, was tested on mammalian intestinal smooth muscle *in vitro* and compared to acetylcholine [58]. Final molar concentrations of acetylcholine (40nM to 10 μM) and verbenalin (21.3 μM to 2.6 mM) were prepared. The smooth muscle was placed in an organ bath with a 30 ml-muscle chamber in freshly prepared Tyrode's solution maintained at 37°C. The muscle was challenged for a period of 30 seconds with the two substances at the stated concentrations (Figure 13). Between additions, the muscle was allowed to achieve baseline activity. The median effective concentration for acetylcholine and verbe‐ nalin were 1.54 μM and 0.32mM, respectively, with acetylcholine being approximately 200 times more potent than verbenalin. In spite of its mild effects, the presence of verbenalin in vervain is not recommended in pregnancy [66].

#### **2.6. Hedera helix L.**

have ceased to flower. The recommended planting distance is 30 cm in a row with a cultivation

and twice weekly in summer. The monthly harvesting of leaves produced a variable content of essential oil on fresh weight basis with the peak reached during the month of August (2.24

% v/v) and the least during December (0.52 % v/v) (Figure 11).

14 Phytochemicals - Isolation, Characterisation and Role in Human Health

. Plants should be irrigated immediately after planting of cuttings

Figure 11. The yield of Maltese sage essential oil throughout the year [48].

*Verbena officinalis*, a member of the Verbenaceae family, is also known as vervain. This plant is indigenous to Europe, North Africa and Asia but has been introduced to North America and Australia. Some of the common traditional uses of vervain, worldwide, were in the treatment of respiratory problems such as cough, wheezing and shortness of breath [37], as a purgative, in the treatment of haemorrhoids, eye problems [50], wounds, fever and stomach upsets [51]. In Malta, vervain was used in the treatment of many ailments particularly, carbuncles, boils,

The main constituents of *Verbena officinalis* are iridoid glycosides, namely verbenalin [53], hastatoside [54] and aucubin [55]. It yields an essential oil, with citral, geraniol, limonene and verbenone as main constituents [56]. Other constituents include the flavone derivative artemetin, phenylpropane glycosides verbascoside and eukovoside and the triterpenes ursolic

> yields an essential oil, with citral, geraniol, limonene and verbenone as main constituents [56]. Other constituents include the flavone derivative artemetin, phenylpropane glycosides verbascoside and eukovoside and the triterpenes ursolic acid, βsitosterol and lupeol [57]. Some of these constituents are highlighted in figure 12. The volume of oil obtained from Maltese

2.5. Verbena officinalis L.

**Figure 11.** The yield of Maltese sage essential oil throughout the year [48].

**2.5. Verbena officinalis L.**

dysentery, cough and arthritis [52].

wounds, eczema, high blood pressure, diarrhoea, dysentery, cough and arthritis [52].

sources was negligible [58].

density of 10 plants per m2

Verbena officinalis, a member of the Verbenaceae family, is also known as vervain. This plant is indigenous to Europe, North Africa and Asia but has been introduced to North America and Australia. Some of the common traditional uses of vervain, worldwide, were in the treatment of respiratory problems such as cough, wheezing and shortness of breath [37], as a purgative, in the treatment of haemorrhoids, eye problems [50], wounds, fever and stomach upsets [51]. In Malta, vervain was used in the treatment of many ailments particularly, carbuncles, boils, wounds, eczema, high blood pressure, diarrhoea, *Hedera helix* L. or common ivy, a member of the Araliaceae family, is indigenous to Europe but its presence has been reported in Asia (as far as Japan), Africa and North America. Records of the use of ivy as a medicinal plant, dates back to the times of Hippocrates. The flowers were used to treat dysentery, earache and headache, while the leaves were usedas an emmenagogue [67). Others claimed it to be effective in the treatment of sunburn, ulcers, tuberculosis, bronchitis, whooping-cough, constipation, wounds and various skin diseases [68-70].

The main constituents of Verbena officinalis are iridoid glycosides, namely verbenalin [53], hastatoside [54] and aucubin [55]. It The main constituents of *Hedera helix* are the saponins, more commonly known as hederasa‐ ponins. This is a group of structurally related triterpenoid glycosides with an oleanane

Figure 12. Typical constituents of Verbena officinalis L.: (A) verbenalin, (B) artemetin and (C) verbascoside

but the content in methanolic extracts varied between 0.12 and 0.50 % [60].

A hydromethanolic extract of the dried aerial parts of Maltese vervain was obtained by Soxhlet extraction [58]. The constitution of verbenalin was determined by HPLC using Supelcosil LC-18 column, acetonitrile/water-phosphoric acid (pH 2) gradient mobile phase with a flow rate of 1.5 ml/min. The content of verbenalin expressed as dry weight of plant material was 2.09 % (w/w). Previous reports [59] declared that contents of verbenalin were less than 0.1 % when extracted with ether

Several pharmacological activities are attributed to vervain, namely, anti-inflammatory [54, 61], neuroprotective [62], antioxidant, antifungal [63], antileukaemic [64] and hepatoprotective [65]. Verbenalin, from Maltese vervain sources, was tested on mammalian intestinal smooth muscle in vitro and compared to acetylcholine [58]. Final molar concentrations of acetylcholine (40nM to 10 µM) and verbenalin (21.3 µM to 2.6 mM) were prepared. The smooth muscle was placed in an organ bath with a 30 ml-muscle chamber in freshly prepared Tyrode's solution maintained at 37°C. The muscle was challenged for a period of 30 seconds with the two substances at the stated concentrations (Figure 13). Between additions, the muscle was allowed to achieve baseline activity. The median effective concentration for acetylcholine and verbenalin were 1.54 µM and 0.32mM, respectively, with acetylcholine being approximately 200 times more potent than verbenalin. In spite

**Figure 13.** The spasmodic response of the smooth intestinal muscle with (a) acetylcholine and (b) verbenalin [58].

backbone (Figure 14). These are divided into mono- and bidesmosides. Monodesmosides include α-hederin and hederagenin 3-O-β-glucoside, while bidesmosides include hederasa‐ ponins С, A, B, D, E, F, G, H and I [71, 72]. 2.6. Hedera helix L.

Figure 13. The spasmodic response of the smooth intestinal muscle with (a) acetylcholine and (b) verbenalin [58].

Figure 14. The main pentacyclic triterpenoids of Hedera helix L. **Figure 14.** The main pentacyclic triterpenoids of *Hedera helix* L.

hederasaponins С, A, B, D, E, F, G, H and I [71, 72].

(table 3).

Microorganism

Table 3. Minimum Inhibitory Concentrations (mg/l) for Hedera extracts [75].

Enterobacter aerogenes

hederasaponin

Another important group is that represented by phenolics (flavonoids, anthocyanins, coumarins and phenolic acids) [71, 73]. The essential oil from ivy stems and leaves contains germacrene D, β-caryophyllene, sabinene, β-pinene, limonene, and αpinene [74]. Hederasaponins, from ivy grown in Malta, were extracted with 70 % ethanol by Soxhlet extraction [75]. Spring, summer, autumn and winter leaves yielded 12.75 %, 11.82 %, 10.74 % and 10.97 % (w/w) of dried extract. The hederosaponin content was determined by HPLC using Supelcosil LC- 18 column, acetonitrile/water-phosphoric acid (0.01 N) gradient mobile phase with a flow rate of 1 ml/min. Hederasaponin C and α-hederin were used as standards. The 70 % ethanolic extract contained 46.7 % hederasaponin C and 6.1 % α-hederin totaling 52.8 %. Purification of the ethanolic extract through an alumina column with methanol as solvent resulted in 62.2 % hederasaponin C and 9.2 % α-hederin. This goes in accordance with other authors [76, 77] who confirmed that hederasaponin C is the main saponin in common ivy. Hedera helix was investigated for its pharmacological potential, by many scientists. Typical reported activities include antiinflammatory [78, 79], antiviral [80], antifungal [81], antibacterial, mucolytic, antispasmodic agent and in vitro bronchodilatory [82, 83]. The ivy leaf extracts, obtained from Maltese sources, and the standards were tested for their antimicrobial activity [75]. The tested organisms were Staphylococcus aureus, Escherichia coli, Enterobacter aerogenes, Klebsiella sp., Serrata sp. and Candida Another important group is that represented by phenolics (flavonoids, anthocyanins, cou‐ marins and phenolic acids) [71, 73]. The essential oil from ivy stems and leaves contains germacrene D, β-caryophyllene, sabinene, β-pinene, limonene, and α-pinene [74]. Hederasa‐ ponins, from ivy grown in Malta, were extracted with 70 % ethanol by Soxhlet extraction [75]. Spring, summer, autumn and winter leaves yielded 12.75 %, 11.82 %, 10.74 % and 10.97 % (w/ w) of dried extract. The hederosaponin content was determined by HPLC using Supelcosil LC-18 column, acetonitrile/water-phosphoric acid (0.01 N) gradient mobile phase with a flow rate of 1 ml/min. Hederasaponin C and α-hederin were used as standards. The 70 % ethanolic extract contained 46.7 % hederasaponin C and 6.1 % α-hederin totaling 52.8 %. Purification of the ethanolic extract through an alumina column with methanol as solvent resulted in 62.2 % hederasaponin C and 9.2 % α-hederin. This goes in accordance with other authors [76, 77] who confirmed that hederasaponin C is the main saponin in common ivy.

albicans. Pure α-hederin was inactive against all organisms presumably due to its poor solubility in water as was reported by [84]. On the other hand, pure hederasaponin C was active against all the tested organisms. It was more active than both ivy extracts against Staphylococcus aureus, Enterobacter aerogenes, Klebsiella sp. and Serrata sp. It was just as effective as the purified ivy extract against Escherichia coli and Candida albicans. The only difference between hederasaponin C and α-hederin is that the former has an extra sugar group. Being a bidesmoside, hederasaponin C is more water soluble. There are no other structural differences that may have contributed to a better antimicrobial activity. In conclusion, the purified ivy extract (62.2 % hederasaponin C) and pure hedersaponin C were more active against Staph. aureus and least active against Candida albicans

C α-hederin Ethanolic

Staph. aureus 0.312 - 1.25 – 2.50 0.625 – 1.25 Escherichia coli 5 - 10 5 - 10

Klebsiella sp. 1.25 - 5 – 10 2.5 – 5 Serrata sp 2.5 - 5 – 10 - Candida albicans 10 - - 10

Minimum Inhibitory Concentrations (mg/l)

2.5 - 5 – 10 5 – 10

extract

Purified ethanolic extract

*Hedera helix* was investigated for its pharmacological potential, by many scientists. Typical reported activities include anti-inflammatory [78, 79], antiviral [80], antifungal [81], antibac‐ terial, mucolytic, antispasmodic agent and *in vitro* bronchodilatory [82, 83].

The ivy leaf extracts, obtained from Maltese sources, and the standards were tested for their antimicrobial activity [75]. The tested organisms were *Staphylococcus aureus*, *Escherichia coli*, *Enterobacter aerogenes*, *Klebsiella* sp., *Serrata* sp. and *Candida albicans*. Pure α-hederin was inactive against all organisms presumably due to its poor solubility in water as was reported by [84]. On the other hand, pure hederasaponin C was active against all the tested organisms. It was more active than both ivy extracts against *Staphylococcus aureus*, *Enterobacter aerogenes*, *Klebsiella* sp. and *Serrata* sp. It was just as effective as the purified ivy extract against *Escherichia coli* and *Candida albicans*. The only difference between hederasaponin C and α-hederin is that the former has an extra sugar group. Being a bidesmoside, hederasaponin C is more water soluble. There are no other structural differences that may have contributed to a better antimicrobial activity. In conclusion, the purified ivy extract (62.2 % hederasaponin C) and pure hedersaponin C were more active against *Staph. aureus* and least active against *Candida albicans* (table 3).


**Table 3.** Minimum Inhibitory Concentrations (mg/l) for *Hedera* extracts [75].

#### **2.7. Crataegus monogyna Jacq.**

backbone (Figure 14). These are divided into mono- and bidesmosides. Monodesmosides include α-hederin and hederagenin 3-O-β-glucoside, while bidesmosides include hederasa‐

**Figure 13.** The spasmodic response of the smooth intestinal muscle with (a) acetylcholine and (b) verbenalin [58].

Figure 13. The spasmodic response of the smooth intestinal muscle with (a) acetylcholine and (b) verbenalin [58].

Figure 12. Typical constituents of Verbena officinalis L.: (A) verbenalin, (B) artemetin and (C) verbascoside

of its mild effects, the presence of verbenalin in vervain is not recommended in pregnancy [66].

but the content in methanolic extracts varied between 0.12 and 0.50 % [60].

A hydromethanolic extract of the dried aerial parts of Maltese vervain was obtained by Soxhlet extraction [58]. The constitution of verbenalin was determined by HPLC using Supelcosil LC-18 column, acetonitrile/water-phosphoric acid (pH 2) gradient mobile phase with a flow rate of 1.5 ml/min. The content of verbenalin expressed as dry weight of plant material was 2.09 % (w/w). Previous reports [59] declared that contents of verbenalin were less than 0.1 % when extracted with ether

Several pharmacological activities are attributed to vervain, namely, anti-inflammatory [54, 61], neuroprotective [62], antioxidant, antifungal [63], antileukaemic [64] and hepatoprotective [65]. Verbenalin, from Maltese vervain sources, was tested on mammalian intestinal smooth muscle in vitro and compared to acetylcholine [58]. Final molar concentrations of acetylcholine (40nM to 10 µM) and verbenalin (21.3 µM to 2.6 mM) were prepared. The smooth muscle was placed in an organ bath with a 30 ml-muscle chamber in freshly prepared Tyrode's solution maintained at 37°C. The muscle was challenged for a period of 30 seconds with the two substances at the stated concentrations (Figure 13). Between additions, the muscle was allowed to achieve baseline activity. The median effective concentration for acetylcholine and verbenalin were 1.54 µM and 0.32mM, respectively, with acetylcholine being approximately 200 times more potent than verbenalin. In spite

Another important group is that represented by phenolics (flavonoids, anthocyanins, coumarins and phenolic acids) [71, 73]. The essential oil from ivy stems and leaves contains germacrene D, β-caryophyllene, sabinene, β-pinene, limonene, and αpinene [74]. Hederasaponins, from ivy grown in Malta, were extracted with 70 % ethanol by Soxhlet extraction [75]. Spring, summer, autumn and winter leaves yielded 12.75 %, 11.82 %, 10.74 % and 10.97 % (w/w) of dried extract. The hederosaponin content was determined by HPLC using Supelcosil LC- 18 column, acetonitrile/water-phosphoric acid (0.01 N) gradient mobile phase with a flow rate of 1 ml/min. Hederasaponin C and α-hederin were used as standards. The 70 % ethanolic extract contained 46.7 % hederasaponin C and 6.1 % α-hederin totaling 52.8 %. Purification of the ethanolic extract through an alumina column with methanol as solvent resulted in 62.2 % hederasaponin C and 9.2 % α-hederin. This goes in

Another important group is that represented by phenolics (flavonoids, anthocyanins, cou‐ marins and phenolic acids) [71, 73]. The essential oil from ivy stems and leaves contains germacrene D, β-caryophyllene, sabinene, β-pinene, limonene, and α-pinene [74]. Hederasa‐ ponins, from ivy grown in Malta, were extracted with 70 % ethanol by Soxhlet extraction [75]. Spring, summer, autumn and winter leaves yielded 12.75 %, 11.82 %, 10.74 % and 10.97 % (w/ w) of dried extract. The hederosaponin content was determined by HPLC using Supelcosil LC-18 column, acetonitrile/water-phosphoric acid (0.01 N) gradient mobile phase with a flow rate of 1 ml/min. Hederasaponin C and α-hederin were used as standards. The 70 % ethanolic extract contained 46.7 % hederasaponin C and 6.1 % α-hederin totaling 52.8 %. Purification of the ethanolic extract through an alumina column with methanol as solvent resulted in 62.2 % hederasaponin C and 9.2 % α-hederin. This goes in accordance with other authors [76, 77] who

Hedera helix was investigated for its pharmacological potential, by many scientists. Typical reported activities include antiinflammatory [78, 79], antiviral [80], antifungal [81], antibacterial, mucolytic, antispasmodic agent and in vitro

The ivy leaf extracts, obtained from Maltese sources, and the standards were tested for their antimicrobial activity [75]. The tested organisms were Staphylococcus aureus, Escherichia coli, Enterobacter aerogenes, Klebsiella sp., Serrata sp. and Candida albicans. Pure α-hederin was inactive against all organisms presumably due to its poor solubility in water as was reported by [84]. On the other hand, pure hederasaponin C was active against all the tested organisms. It was more active than both ivy extracts against Staphylococcus aureus, Enterobacter aerogenes, Klebsiella sp. and Serrata sp. It was just as effective as the purified ivy extract against Escherichia coli and Candida albicans. The only difference between hederasaponin C and α-hederin is that the former has an extra sugar group. Being a bidesmoside, hederasaponin C is more water soluble. There are no other structural differences that may have contributed to a better antimicrobial activity. In conclusion, the purified ivy extract (62.2 % hederasaponin C) and pure hedersaponin C were more active against Staph. aureus and least active against Candida albicans

C α-hederin Ethanolic

Staph. aureus 0.312 - 1.25 – 2.50 0.625 – 1.25 Escherichia coli 5 - 10 5 - 10

Klebsiella sp. 1.25 - 5 – 10 2.5 – 5 Serrata sp 2.5 - 5 – 10 - Candida albicans 10 - - 10

Minimum Inhibitory Concentrations (mg/l)

2.5 - 5 – 10 5 – 10

extract

Purified ethanolic extract

accordance with other authors [76, 77] who confirmed that hederasaponin C is the main saponin in common ivy.

ponins С, A, B, D, E, F, G, H and I [71, 72].

16 Phytochemicals - Isolation, Characterisation and Role in Human Health

2.6. Hedera helix L.

Figure 14. The main pentacyclic triterpenoids of Hedera helix L.

**Figure 14.** The main pentacyclic triterpenoids of *Hedera helix* L.

Microorganism

Table 3. Minimum Inhibitory Concentrations (mg/l) for Hedera extracts [75].

Enterobacter aerogenes

hederasaponin

confirmed that hederasaponin C is the main saponin in common ivy.

bronchodilatory [82, 83].

(table 3).

*Crataegus monogyna* (may, quick or common hawthorn) belongs to the Rosaceae family. Records show that it has been used since the Ancient Roman times. Dioscorides and later Paracelsus reported the effects of the shrub in heart conditions [85]. Mediterranean folk medicine utilized the shrub as an astringent, febrifuge, sedative, in the treatment of diarrhoea, whitlow's, heart disease, high blood pressure and to improve circulation [86].

Hawthorn contains several constituents, most of which are either pharmacologically active or have a nutritional value. Triterpenoids, flavonoids, coumarins and amines are the main groups of compounds that possess a significant activity in the treatment of cardiovascular diseases [87]. The two triterpenoids, abundantly found in hawthorns, are ursolic and oleanolic acids (figure 15). These account for 90 % of the total pentacyclic triterpenoids present in the shrub [88]. The triterpenoids oleanolic, ursolic and crataegolic acids were extracted as a crude mixture with 96 % alcohol [89, 90], as an acid-ether extract [91] and as a tincture of *Crataegus monogyna* [92]. treatment of cardiovascular diseases [87]. The two triterpenoids, abundantly found in hawthorns, are ursolic and oleanolic acids (figure 15). These account for 90 % of the total pentacyclic triterpenoids present in the shrub [88]. The triterpenoids oleanolic, ursolic and crataegolic acids were extracted as a crude mixture with 96 % alcohol [89, 90], as an acid-ether extract [91] and as a tincture of Crataegus monogyna [92].

Crataegus monogyna (may, quick or common hawthorn) belongs to the Rosaceae family. Records show that it has been used since the Ancient Roman times. Dioscorides and later Paracelsus reported the effects of the shrub in heart conditions [85]. Mediterranean folk medicine utilized the shrub as an astringent, febrifuge, sedative, in the treatment of diarrhoea, whitlow's,

Hawthorn contains several constituents, most of which are either pharmacologically active or have a nutritional value. Triterpenoids, flavonoids, coumarins and amines are the main groups of compounds that possess a significant activity in the

The hydroethanolic extract of Crataegus monogyna was studied for its angiotensin-converting enzyme (ACE) inhibitory

values (335.00 µg/ml and 3.61 µM, respectively) in comparison to captopril (46.9 nM). However, these results suggest that the

2.7. Crataegus monogyna Jacq.

Figure 15. Structure of oleanolic acid and derivatives. For oleanolic acid, R1 and R2 are hydrogen atoms. For the triterpenoid glycosides, R<sup>1</sup> and R2 represent different sugar groups. **Figure 15.** Structure of oleanolic acid and derivatives. For oleanolic acid, R1 and R2 are hydrogen atoms. For the triter‐ penoid glycosides, R1 and R2 represent different sugar groups.

Crataegus species are renowned for their flavonoid content [93]. Flavonoids include vitexin, hyperoside [94], rutin, quercetin, luteolin-7-glucoside [95] and apigenin [96]. The most abundant in flowers was the hyperoside [94]. Other flavonoids included catechin, luteolin, epicatechin, quercetrin, quercetrin-3-rhamnogalactoside and luteolin-3',7-diglucoside [87, 97]. Hawthorn contains a large variety of cardiotonic amines in different plants parts especially the leaves and flowers. These include di- and trimethylamine, ethanolamine, ethylamine [87], isoamyl and isobutylamines [92]. Choline and acetylcholine are also present. It contains other minor constituents [98]. Hawthorn extracts have been tested for several pharmacological activities such as antimicrobial, antioxidant [99, 100], peroxysmal tachycardia [101], prevention of cardiac necrosis [102-104], hyperglycaemia [105], atherosclerosis [106] and hypertension [107]. *Crataegus* species are renowned for their flavonoid content [93]. Flavonoids include vitexin, hyperoside [94], rutin, quercetin, luteolin-7-glucoside [95] and apigenin [96]. The most abundant in flowers was the hyperoside [94]. Other flavonoids included catechin, luteolin, epicatechin, quercetrin, quercetrin-3-rhamnogalactoside and luteolin-3',7-diglucoside [87, 97]. Hawthorn contains a large variety of cardiotonic amines in different plants parts especially the leaves and flowers. These include di- and trimethylamine, ethanolamine, ethylamine [87], isoamyl and isobutylamines [92]. Choline and acetylcholine are also present. It contains other minor constituents [98].

activity [108]. The direct interaction of extracts and pure compounds with ACE was performed using a microtiter plate method modified for the ACE detection kit (Sigma, MO) at 430 nm (Figure 16). The crude extract contained triterpenic acids, flavonoids and coumarins. The ACE inhibitory activity of the crude extract and pure oleanolic acid (a triterpenoid) were compared to captropril, the latter used as a control drug. The hydroethanolic extract and oleanolic acid showed higher IC<sup>50</sup> Hawthorn extracts have been tested for several pharmacological activities such as antimicro‐ bial, antioxidant [99, 100], peroxysmal tachycardia [101], prevention of cardiac necrosis [102-104], hyperglycaemia [105], atherosclerosis [106] and hypertension [107].

anti-ACE activity of the hydroethanolic extract from hawthorn is due to oleanolic acid and other triterpenic acids present. The hydroethanolic extract of *Crataegus monogyna* was studied for its angiotensin-convert‐ ing enzyme (ACE) inhibitory activity [108]. The direct interaction of extracts and pure compounds with ACE was performed using a microtiter plate method modified for the ACE detection kit (Sigma, MO) at 430 nm (Figure 16). The crude extract contained triterpenic acids, flavonoids and coumarins. The ACE inhibitory activity of the crude extract and pure oleanolic acid (a triterpenoid) were compared to captropril, the latter used as a control drug. The hydroethanolic extract and oleanolic acid showed higher IC50 values (335.00 μg/ml and 3.61 μM, respectively) in comparison to captopril (46.9 nM). However, these results suggest that the anti-ACE activity of the hydroethanolic extract from hawthorn is due to oleanol‐ ic acid and other triterpenic acids present. This was the first study to suggest that triterpen‐ ic acids contribute to the antihypertensive activity of hawthorn. In previous studies, the ACE inhibitory activity of *C. monogyna* extracts was always attributed to flavonoids and proanthocyanidins.

studies, the ACE inhibitory activity of C. monogyna extracts was always attributed to flavonoids and proanthocyanidins.

rhamnose and arabinose subunits. Other constituents include the essential oil, triterpene alcohols, phenolic acids, tannins, sterols, tocopherols, N-paraffins, pyrethrins, sesquiterpenes and coumarins. Monoterpenes and sesquiterpenes make up the essential oil. However, the latter does not contain sesquiterpene lactones. Moreover, 50 - 60 % of the oil present in the seeds is

made up of calendulic acid, an unsaturated fatty acid having an unusual chemical structure.

Figure 16. The interaction of angiotensin converting enzyme and compounds (such as captopril and oleanolic acid) with the chromophore N- [3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). **Figure 16.** The interaction of angiotensin converting enzyme and compounds (such as captopril and oleanolic acid) with the chromophore N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG).

#### 2.8. Calendula officinalis L. **2.8. Calendula officinalis L.**

The two triterpenoids, abundantly found in hawthorns, are ursolic and oleanolic acids (figure 15). These account for 90 % of the total pentacyclic triterpenoids present in the shrub [88]. The triterpenoids oleanolic, ursolic and crataegolic acids were extracted as a crude mixture with 96 % alcohol [89, 90], as an acid-ether extract [91] and as a tincture of *Crataegus monogyna* [92].

treatment of cardiovascular diseases [87].

and R2 represent different sugar groups.

[102-104], hyperglycaemia [105], atherosclerosis [106] and hypertension [107].

hypertension [107].

are also present. It contains other minor constituents [98].

Hawthorn extracts have been tested for several pharmacological activities such as antimicro‐ bial, antioxidant [99, 100], peroxysmal tachycardia [101], prevention of cardiac necrosis

The hydroethanolic extract of *Crataegus monogyna* was studied for its angiotensin-convert‐ ing enzyme (ACE) inhibitory activity [108]. The direct interaction of extracts and pure compounds with ACE was performed using a microtiter plate method modified for the ACE detection kit (Sigma, MO) at 430 nm (Figure 16). The crude extract contained triterpenic acids, flavonoids and coumarins. The ACE inhibitory activity of the crude extract and pure oleanolic acid (a triterpenoid) were compared to captropril, the latter used as a control drug. The hydroethanolic extract and oleanolic acid showed higher IC50 values (335.00 μg/ml and 3.61 μM, respectively) in comparison to captopril (46.9 nM). However, these results suggest that the anti-ACE activity of the hydroethanolic extract from hawthorn is due to oleanol‐ ic acid and other triterpenic acids present. This was the first study to suggest that triterpen‐ ic acids contribute to the antihypertensive activity of hawthorn. In previous studies, the ACE inhibitory activity of *C. monogyna* extracts was always attributed to flavonoids and

**Figure 15.** Structure of oleanolic acid and derivatives. For oleanolic acid, R1 and R2 are hydrogen atoms. For the triter‐

*Crataegus* species are renowned for their flavonoid content [93]. Flavonoids include vitexin, hyperoside [94], rutin, quercetin, luteolin-7-glucoside [95] and apigenin [96]. The most abundant in flowers was the hyperoside [94]. Other flavonoids included catechin, luteolin, epicatechin, quercetrin, quercetrin-3-rhamnogalactoside and luteolin-3',7-diglucoside [87, 97]. Hawthorn contains a large variety of cardiotonic amines in different plants parts especially the leaves and flowers. These include di- and trimethylamine, ethanolamine, ethylamine [87], isoamyl and isobutylamines [92]. Choline and acetylcholine are also present. It contains other

[92].

penoid glycosides, R1 and R2 represent different sugar groups.

minor constituents [98].

proanthocyanidins.

heart disease, high blood pressure and to improve circulation [86].

Crataegus monogyna (may, quick or common hawthorn) belongs to the Rosaceae family. Records show that it has been used since the Ancient Roman times. Dioscorides and later Paracelsus reported the effects of the shrub in heart conditions [85]. Mediterranean folk medicine utilized the shrub as an astringent, febrifuge, sedative, in the treatment of diarrhoea, whitlow's,

Hawthorn contains several constituents, most of which are either pharmacologically active or have a nutritional value. Triterpenoids, flavonoids, coumarins and amines are the main groups of compounds that possess a significant activity in the

The two triterpenoids, abundantly found in hawthorns, are ursolic and oleanolic acids (figure 15). These account for 90 % of the total pentacyclic triterpenoids present in the shrub [88]. The triterpenoids oleanolic, ursolic and crataegolic acids were extracted as a crude mixture with 96 % alcohol [89, 90], as an acid-ether extract [91] and as a tincture of Crataegus monogyna

Figure 15. Structure of oleanolic acid and derivatives. For oleanolic acid, R1 and R2 are hydrogen atoms. For the triterpenoid glycosides, R<sup>1</sup>

Crataegus species are renowned for their flavonoid content [93]. Flavonoids include vitexin, hyperoside [94], rutin, quercetin, luteolin-7-glucoside [95] and apigenin [96]. The most abundant in flowers was the hyperoside [94]. Other flavonoids included catechin, luteolin, epicatechin, quercetrin, quercetrin-3-rhamnogalactoside and luteolin-3',7-diglucoside [87, 97]. Hawthorn contains a large variety of cardiotonic amines in different plants parts especially the leaves and flowers. These include di- and trimethylamine, ethanolamine, ethylamine [87], isoamyl and isobutylamines [92]. Choline and acetylcholine

Hawthorn extracts have been tested for several pharmacological activities such as antimicrobial, antioxidant [99, 100], peroxysmal tachycardia [101], prevention of cardiac necrosis [102-104], hyperglycaemia [105], atherosclerosis [106] and

The hydroethanolic extract of Crataegus monogyna was studied for its angiotensin-converting enzyme (ACE) inhibitory activity [108]. The direct interaction of extracts and pure compounds with ACE was performed using a microtiter plate method modified for the ACE detection kit (Sigma, MO) at 430 nm (Figure 16). The crude extract contained triterpenic acids, flavonoids and coumarins. The ACE inhibitory activity of the crude extract and pure oleanolic acid (a triterpenoid) were compared to captropril, the latter used as a control drug. The hydroethanolic extract and oleanolic acid showed higher IC<sup>50</sup> values (335.00 µg/ml and 3.61 µM, respectively) in comparison to captopril (46.9 nM). However, these results suggest that the anti-ACE activity of the hydroethanolic extract from hawthorn is due to oleanolic acid and other triterpenic acids present.

2.7. Crataegus monogyna Jacq.

Calendula officinalis, more commonly known as the pot marigold, belongs to the Asteraceae family. The use of pot marigold for therapeutic purposes has been recognised since the time of St. Hildeguard (1098-1197), who described in her work Causae et Curae and Physica the curative properties of ringula [109]. Calendula officinalis was being used internally during the twelfth century for digestive disturbances and also as an antidote against man and animal intoxication. It was also used externally for the treatment of impetigous eczema. Hundred years later (1193-1280), Albert the Great utilized the Calendula which he called Sponsa Solis, against animal bites and also to alleviate hepatic pain and pain of the spleen. This plant can also be seen in the herbals of the Renaissance. Leonard Fuchs stated that if the plant was to be boiled and held in the mouth for some time it relieved dental pain. Later during the sixteenth Century, Mattioli (1500-1577) attributed the therapeutic properties of the pot marigold in the constriction of the heart and palpitations as a consequence of menstrual fluid retention. According to this author, the water of the marigold has sudatory properties. He was also the first physician to recommend the herb for its therapeutic use against cancer and accordingly called it Herba Cancri. Mattioli's recommendations of Calendula officinalis as a remedy against cancer were fully approved by Osiander and Hufeland [109]. The pharmacist, J. W. Weinmann (1683 - 1741), in his work "Phytantoza iconographica" recommended the aqueous marigold extract for the alleviation of red and inflamed eyes and also used the plant in the treatment of goitre. Pharmacologically-active classes of compounds, in the marigold, include the terpenoids including the carotenoids, flavonoids, coumarins and polysaccharides [110-112]. The saponosides are particularly abundant in the plant. There are also numerous triterpenoid alcohols which are derived from tarassene, lupene, oleanene and ursene. These are present as free or *Calendula officinalis*, more commonly known as the pot marigold, belongs to the Asteraceae family. The use of pot marigold for therapeutic purposes has been recognised since the time of St. Hildeguard (1098-1197), who described in her work Causae et Curae and Physica the curative properties of ringula [109]. *Calendula officinalis* was being used internally during the twelfth century for digestive disturbances and also as an antidote against man and animal intoxication. It was also used externally for the treatment of impetigous eczema. Hundred years later (1193-1280), Albert the Great utilized the *Calendula* which he called Sponsa Solis, against animal bites and also to alleviate hepatic pain and pain of the spleen. This plant can also be seen in the herbals of the Renaissance. Leonard Fuchs stated that if the plant was to be boiled and held in the mouth for some time it relieved dental pain. Later during the sixteenth Century, Mattioli (1500-1577) attributed the therapeutic properties of the pot marigold in the constriction of the heart and palpitations as a consequence of menstrual fluid retention. According to this author, the water of the marigold has sudatory properties. He was also the first physician to recommend the herb for its therapeutic use against cancer and accordingly called it Herba Cancri. Mattioli's recommendations of *Calendula officinalis* as a remedy against cancer were fully approved by Osiander and Hufeland [109]. The pharmacist, J. W. Weinmann (1683 - 1741), in his work "Phytantoza iconographica" recommended the aqueous marigold extract for the alleviation of red and inflamed eyes and also used the plant in the treatment of goitre.

esterified as monols, diols and triols. The content of monoesters of the triterpenoid diols is between 2 and 45 %, of which 1.85 % is made up of faradiol esters. The most common triterpenoid is oleanolic acid (Figure 15). The colour of the flowers is determined by the amount of carotenoids which can vary from 1.5 to 3 %. The orange flowers are made up mainly of carotenes particularly lycopene whereas the yellow flowers contain mainly xanthophylls [113]. The heterosides of quercetin and isorhamnetin (flavonoids) are present in the dry Calendula drug [114]. Their content varies between 0.25 and 0.88 %. The Calendula drug contains 14.75 % of polysaccharides (PS), which are soluble in water. The three main ones are PS I (molecular weight of 15,000), PS II (molecular weight of 25,000) and PS III (molecular weight of 35,000). These are made up of galactose, Pharmacologically-active classes of compounds, in the marigold, include the terpenoids including the carotenoids, flavonoids, coumarins and polysaccharides [110-112]. The sapono‐ sides are particularly abundant in the plant. There are also numerous triterpenoid alcohols which are derived from tarassene, lupene, oleanene and ursene. These are present as free or esterified as monols, diols and triols. The content of monoesters of the triterpenoid diols is between 2 and 45 %, of which 1.85 % is made up of faradiol esters. The most common triter‐

penoid is oleanolic acid (Figure 15). The colour of the flowers is determined by the amount of carotenoids which can vary from 1.5 to 3 %. The orange flowers are made up mainly of carotenes particularly lycopene whereas the yellow flowers contain mainly xanthophylls [113]. The heterosides of quercetin and isorhamnetin (flavonoids) are present in the dry *Calendula* drug [114]. Their content varies between 0.25 and 0.88 %. The *Calendula* drug contains 14.75 % of polysaccharides (PS), which are soluble in water. The three main ones are PS I (molecular weight of 15,000), PS II (molecular weight of 25,000) and PS III (molecular weight of 35,000). These are made up of galactose, rhamnose and arabinose subunits. Other constituents include the essential oil, triterpene alcohols, phenolic acids, tannins, sterols, tocopherols, N-paraffins, pyrethrins, sesquiterpenes and coumarins. Monoterpenes and sesquiterpenes make up the essential oil. However, the latter does not contain sesquiterpene lactones. Moreover, 50 - 60 % of the oil present in the seeds is made up of calendulic acid, an unsaturated fatty acid having an unusual chemical structure.

Flowerheads of *Calendula officinalis* were extracted with methanol and following concentration, the extract was hydrolysed with 0.5 M hydrochloric acid. The mixture was centrifuged and the residue was dissolved in chloroform. This was then dried and subjected to column chromatography (silica gel; mobile phase - petroleum ether:dichloroethylene:acetic acid 50:50:0.7). The collected fractions were analysed by melting point determination, Infrared and Ultraviolet spectroscopy. The content of oleanolic acid extracted from the dried flowerheads was 0.13% (w/w) [115].

The marigold has been investigated for its anti-microbial, anti-inflammatory [116, 117], antitumour [110] activities, effects on the cardiovascular and nervous systems [118, 119] as well as oestrogenic [120], hypolipidaemic [121], anti-ulcer [122] and spermicidal properties [123].

The antimicrobial activity was conducted for oleanolic acid against a number of organisms [115]. Due to the insoluble nature of oleanolic acid in water, it was incorporated in the nutrient agar for bacterial strains and in Sabouraud's dextrose agar for fungi. In fact, [124] stated that the anti-bacterial agent was soluble in alcohol but not in water. According to the results obtained after 24 hours in the study performed, oleanolic acid was active against Gram-positive organisms (*Strep. faecalis, Strep. viridans* and *Staph. aureus*) except *Staph. albus*. However, for *Staph. albus*, there was slight inhibition which resulted in hazy growth. On the other hand, it was inactive against Gram-negative strains. Although for *Morganella* species and *Pseudomonas aeruginosa*, there was some degree of inhibition, the plate which contained the ethanol instead of oleanolic acid showed the same degree of inhibition. Hence, the anti-bacterial activity in these two cases might be attributable to the presence of ethanol. Oleanolic acid did not show any activity against *Candida albicans*.

The topical in vivo effects of oleanolic acid (2.5 %) on inflamed bites induced by mosquitoes (*Culex pipiens*) was studied [115]. The positive and negative controls included indomethacin (2.5 %), hydrocortisone (1 %) and petroleum jelly. The topical anti-inflammatory activity of oleanolic acid, over a 24-hour period, was comparable to that of hydrocortisone, after being applied at 8 hourly periods. However, when compared to indomethacin, oleanolic acid was found to be less effective (P<0.01). In accordance with the study conducted by [125], oleanolic acid was found to have similar effects to those of hydrocortisone. However, other studies relate oleanolic acid to non-steroidal anti-inflammatory agents, like indomethacin [126].

#### **2.9. Melissa officinalis L.**

penoid is oleanolic acid (Figure 15). The colour of the flowers is determined by the amount of carotenoids which can vary from 1.5 to 3 %. The orange flowers are made up mainly of carotenes particularly lycopene whereas the yellow flowers contain mainly xanthophylls [113]. The heterosides of quercetin and isorhamnetin (flavonoids) are present in the dry *Calendula* drug [114]. Their content varies between 0.25 and 0.88 %. The *Calendula* drug contains 14.75 % of polysaccharides (PS), which are soluble in water. The three main ones are PS I (molecular weight of 15,000), PS II (molecular weight of 25,000) and PS III (molecular weight of 35,000). These are made up of galactose, rhamnose and arabinose subunits. Other constituents include the essential oil, triterpene alcohols, phenolic acids, tannins, sterols, tocopherols, N-paraffins, pyrethrins, sesquiterpenes and coumarins. Monoterpenes and sesquiterpenes make up the essential oil. However, the latter does not contain sesquiterpene lactones. Moreover, 50 - 60 % of the oil present in the seeds is made up of calendulic acid, an unsaturated fatty acid having

Flowerheads of *Calendula officinalis* were extracted with methanol and following concentration, the extract was hydrolysed with 0.5 M hydrochloric acid. The mixture was centrifuged and the residue was dissolved in chloroform. This was then dried and subjected to column chromatography (silica gel; mobile phase - petroleum ether:dichloroethylene:acetic acid 50:50:0.7). The collected fractions were analysed by melting point determination, Infrared and Ultraviolet spectroscopy. The content of oleanolic acid extracted from the dried flowerheads

The marigold has been investigated for its anti-microbial, anti-inflammatory [116, 117], antitumour [110] activities, effects on the cardiovascular and nervous systems [118, 119] as well as oestrogenic [120], hypolipidaemic [121], anti-ulcer [122] and spermicidal properties [123]. The antimicrobial activity was conducted for oleanolic acid against a number of organisms [115]. Due to the insoluble nature of oleanolic acid in water, it was incorporated in the nutrient agar for bacterial strains and in Sabouraud's dextrose agar for fungi. In fact, [124] stated that the anti-bacterial agent was soluble in alcohol but not in water. According to the results obtained after 24 hours in the study performed, oleanolic acid was active against Gram-positive organisms (*Strep. faecalis, Strep. viridans* and *Staph. aureus*) except *Staph. albus*. However, for *Staph. albus*, there was slight inhibition which resulted in hazy growth. On the other hand, it was inactive against Gram-negative strains. Although for *Morganella* species and *Pseudomonas aeruginosa*, there was some degree of inhibition, the plate which contained the ethanol instead of oleanolic acid showed the same degree of inhibition. Hence, the anti-bacterial activity in these two cases might be attributable to the presence of ethanol. Oleanolic acid did not show

The topical in vivo effects of oleanolic acid (2.5 %) on inflamed bites induced by mosquitoes (*Culex pipiens*) was studied [115]. The positive and negative controls included indomethacin (2.5 %), hydrocortisone (1 %) and petroleum jelly. The topical anti-inflammatory activity of oleanolic acid, over a 24-hour period, was comparable to that of hydrocortisone, after being applied at 8 hourly periods. However, when compared to indomethacin, oleanolic acid was found to be less effective (P<0.01). In accordance with the study conducted by [125], oleanolic acid was found to have similar effects to those of hydrocortisone. However, other studies relate

oleanolic acid to non-steroidal anti-inflammatory agents, like indomethacin [126].

an unusual chemical structure.

20 Phytochemicals - Isolation, Characterisation and Role in Human Health

was 0.13% (w/w) [115].

any activity against *Candida albicans*.

*Melissa officinalis* L. is a member of the Lamiaceae family. It is also known as lemon balm or simply as balm. The Latin name ''Melissa'' (balm) refers to the Greek word 'melitos', that is honey. It is believed that the plant attracts honey bees. The plant is found mainly in the Mediterranean region and eastwards to Asia and Siberia. Balm is renowned for its effects on the nervous system and is used to treat nervous agitation, insomnia, hysteria, melancholia, migraine, headache, toothache, earache and nerve pains. It is also useful for gastrointestinal problems such as gastric complaints and lower abdominal pain [127, 128].

Lemon balm contains a volatile oil [129], flavonoids (cynaroside, rhamnocitrin, isoquercitrin, cosmosin), phenolic acids (carnosic acid and rosmarinic acid), and triterpene acids (particu‐ larly ursolic and oleanolic acid) [130]. The study by [131] focused mainly on the cultivation parameters that affect the quantity and quality of the lemon balm oil. The oil yield was 0.1 % (v/w) with cis-citral and trans-citral as the major constituents (figure 17).

Figure 17. The main constituents of Melissa officinalis essential oil: (A) citronellal, (B) geranial (trans-citral), (C) neral (cis-citral), (D) transcaryophyllene **Figure 17.** The main constituents of *Melissa officinalis* essential oil: (A) citronellal, (B) geranial (trans-citral), (C) neral (cis-citral), (D) trans-caryophyllene

Seeds were procured from four sources: Maltese (Argotti Gardens), Swiss (Basel Botanic Gardens), German A (Botanischer Garten der Martin-Luther-Universität) and German B (Botanischer Garten der RWTH). The planting distance was of 20 cm in a row with a distance of 50-60 cm between rows. The cultivation density was of 10 – 12 plants per m<sup>2</sup>. The plants were irrigated immediately after transplanting and then once every fortnight in winter but twice weekly in summer. Plots were divided into two: half treated with fertiliser (NPK Mg (12+12+17+2) + Trace elements) while the other half left untreated, as a control. The leaves were harvested in May and subjected to steam distillation extraction and GC-MS analysis. Table 4 illustrates the results obtained in this study. Sample Citronellal Nerol Geranial Neral Caryophyllene Maltese w/o Fertiliser 0.00 0.00 37.11 47.39 1.02 Fertiliser 0.52 0.00 36.82 47.74 1.26 Seeds were procured from four sources: Maltese (Argotti Gardens), Swiss (Basel Botanic Gardens), German A (Botanischer Garten der Martin-Luther-Universität) and German B (Botanischer Garten der RWTH). The planting distance was of 20 cm in a row with a distance of 50-60 cm between rows. The cultivation density was of 10 – 12 plants per m2 . The plants were irrigated immediately after transplanting and then once every fortnight in winter but twice weekly in summer. Plots were divided into two: half treated with fertiliser (NPK Mg (12+12+17+2) + Trace elements) while the other half left untreated, as a control. The leaves were harvested in May and subjected to steam distillation extraction and GC-MS analysis. Table 4 illustrates the results obtained in this study.

Swiss w/o Fertiliser 0.55 0.00 36.08 48.92 2.11 Fertiliser 1.31 0.55 30.73 45.13 2.67 German A w/o Fertiliser 1.24 0.57 30.96 45.79 2.62 Fertiliser 1.25 0.71 32.11 47.23 1.84 German B w/o Fertiliser 1.65 0.56 31.39 47.63 2.13 Fertiliser 1.31 0.74 33.42 49.19 1.94 Table 4. The composition of essential oils obtained from lemon balm of different seed origins [131]. In most cases, the use of fertilizer improved content of the two main terpenoids, geranial and neral. This goes in accordance with [132], stating that nitrogen fertilisers increased the yield of these constituents. In some cases citronellal also showed significant increases with fertilizer application. In another study, the oil yield was found to vary between 0.16 and 0.25% [133]. With farmyard manure, the content of neral (28.23%) and geranial (39.86%) was higher than with other treatments. Oil yield was also significantly affected by planting spacing and nutrient amendments.

In most cases, the use of fertilizer improved content of the two main terpenoids, geranial and neral. This goes in accordance with [132], stating that nitrogen fertilisers increased the yield of these constituents. In some cases citronellal also showed significant increases with fertilizer application. In another study, the oil yield was found to vary between 0.16 and 0.25% [133]. With farmyard manure, the content of neral (28.23%) and geranial (39.86%) was higher than with other treatments. Oil

Olea europea L. is a typical Mediterranean plant within the Oleaceae family with culinary and medicinal virtues. The typical extract from this plant is the fixed oil obtained from the fruit. In the ancient world, by 2000 BC the olive tree was already in

yield was also significantly affected by planting spacing and nutrient amendments.

2.10. Olea europea L.


**Table 4.** The composition of essential oils obtained from lemon balm of different seed origins [131].

#### **2.10. Olea europea L.**

*Olea europea* L. is a typical Mediterranean plant within the Oleaceae family with culinary and medicinal virtues. The typical extract from this plant is the fixed oil obtained from the fruit. In the ancient world, by 2000 BC the olive tree was already in cultivation. Olive and olive oil was used and traded by the Egyptians, Phoenicians, Greeks and Romans. Today, a large number of olive varieties are recognised internationally as table olives and olives for oil production. Extracts from the olive tree were used in the treatment of hypertension, hyperglycaemia, hyperacidity [134], constipation, for treatment of wounds, sunburn and muscle aches [135, 52] amongst others.

The bioactive phenolic compounds present in the olive fruit include phenolic acids, phenolic alcohols, flavonoids and secoiridoids. The main phenolic acids are cinnamic, syringic, pcoumaric, vanillic, caffeic, 3,4-dihydroxyphenylacetic and protocatechuic acid [136]. Phenolic alcohols include 3,4-dihydroxyphenylethanol (hydroxytyrosol) and p-hydroxyphenylethanol (tyrosol) [137-139]. Flavonoids include taxifolin, apigenin, luteolin and lignans represented by pinoresinol and its metabolites [140]. However, an important class of metabolites found in the leaves and fruit of *Olea*, is that of the secoiridoid glycosides. These include oleuropein (figure 18), demethyloleuropein, oleuropein aglycone and elenolic acid [141-144].

Oleuropein was extracted from Maltese olives as follows [145].The leaves were defatted with petroleum ether and then extracted with 50% ethanol for 6-8 hours. The dried extract was then treated with water and sodium chloride was added until saturation was achieved. Chloroform was added and the aqueous extract was collected. Ethylacetate was added to the aqueous extract and following partitioning, the ethylacetate extract was collected. The extract was then subjected to dryness in order to obtain a yellow crystalline substance. Oleuropein in the olive leaf ethanolic extract amounts to 20.6 %, as mentioned by [146], with a content varying from 20 to 25% (w/w) total dry weight.

*Olea europea* was tested for its antimicrobial [147, 148], antiviral [149], antioxidant [146, 150], antihypertensive, antiatherosclerotic [151, 152] and antidiabetic [153] activities amongst

aqueous extract and following partitioning, the ethylacetate extract was collected. The extract was then subjected to dryness in order to obtain a yellow crystalline substance. Oleuropein in the olive leaf ethanolic extract amounts to 20.6 %, as mentioned by [146], with a content varying from 20 to 25% (w/w) total dry weight. The Phytochemical Constitution of Maltese Medicinal Plants – Propagation, Isolation and Pharmacological Testing http://dx.doi.org/10.5772/60094 23

sunburn and muscle aches [135, 52] amongst others.

acid [141-144].

cultivation. Olive and olive oil was used and traded by the Egyptians, Phoenicians, Greeks and Romans. Today, a large number of olive varieties are recognised internationally as table olives and olives for oil production. Extracts from the olive tree were used in the treatment of hypertension, hyperglycaemia, hyperacidity [134], constipation, for treatment of wounds,

The bioactive phenolic compounds present in the olive fruit include phenolic acids, phenolic alcohols, flavonoids and secoiridoids. The main phenolic acids are cinnamic, syringic, p-coumaric, vanillic, caffeic, 3,4-dihydroxyphenylacetic and protocatechuic acid [136]. Phenolic alcohols include 3,4-dihydroxyphenylethanol (hydroxytyrosol) and phydroxyphenylethanol (tyrosol) [137-139]. Flavonoids include taxifolin, apigenin, luteolin and lignans represented by pinoresinol and its metabolites [140]. However, an important class of metabolites found in the leaves and fruit of Olea, is that of the secoiridoid glycosides. These include oleuropein (figure 18), demethyloleuropein, oleuropein aglycone and elenolic

Oleuropein was extracted from Maltese olives as follows [145].The leaves were defatted with petroleum ether and then extracted with 50% ethanol for 6-8 hours. The dried extract was then treated with water and sodium chloride was added until saturation was achieved. Chloroform was added and the aqueous extract was collected. Ethylacetate was added to the

**Figure 18.** Structures of polyphenolic compounds from olive oil: (A) hydroxytyrosol and (B) oleuropein glucoside.

Figure 18. Structures of polyphenolic compounds from olive oil: (A) hydroxytyrosol and (B) oleuropein glucoside.

others. Maltese olive leaf extract was studied for its immunomodulatory activity [145]. Human peripheral blood lymphocytes were isolated and cultured on RPMI medium. Oleuropein (540 – 0.054 μg/ml) was tested alongside phytohaemagglutinin (m-form, Gibco BRL, UK - 1 – 0.0001 % as positive control and *Olea* extracts to final concentrations ranging from 540 – 0.054 μg/ml (oleuropein content). The cells were studied for their survival, death and morphological characteristics using WST-1 assay, LDH (Boehringer-Mannheim, Germany) and the Papani‐ colau staining procedure, respectively. Oleuropein possesses three α,β -moieties; two α, β unsaturated keto systems at the 3,4-dihydroxyphenyl part and one α, β -unsaturated aldehyde system on the secoiridoid part, which are important for the non-toxic but stimulatory activity on lymphocytes. From the results obtained, oleuropein was more effective when it formed part of the extract (SC50, < 0.054 μg/ml) than when used in its pure form (SC50, 0.146 μg/ml). Olea europea was tested for its antimicrobial [147, 148], antiviral [149], antioxidant [146, 150], antihypertensive, antiatherosclerotic [151, 152] and antidiabetic [153] activities amongst others. Maltese olive leaf extract was studied for its immunomodulatory activity [145]. Human peripheral blood lymphocytes were isolated and cultured on RPMI medium. Oleuropein (540 – 0.054 µg/ml) was tested alongside phytohaemagglutinin (m-form, Gibco BRL, UK - 1 – 0.0001 % as positive control and Olea extracts to final concentrations ranging from 540 – 0.054 µg/ml (oleuropein content). The cells were studied for their survival, death and morphological characteristics using WST-1 assay, LDH (Boehringer-Mannheim, Germany) and the Papanicolau staining procedure, respectively. Oleuropein possesses three α,β -moieties; two α, β -unsaturated keto systems at the 3,4-dihydroxyphenyl part and one α, β -unsaturated aldehyde system on the secoiridoid part, which are important for the non-toxic but stimulatory activity on lymphocytes. From the results obtained, oleuropein was more effective when it formed part of the extract (SC50, < 0.054 µg/ml) than when used in its pure form (SC50, 0.146 µg/ml). 2.11. Urtica dubia Forsk.

#### **2.11. Urtica dubia Forsk.**

**2.10. Olea europea L.**

Maltese

Swiss

German A

German B

52] amongst others.

20 to 25% (w/w) total dry weight.

*Olea europea* L. is a typical Mediterranean plant within the Oleaceae family with culinary and medicinal virtues. The typical extract from this plant is the fixed oil obtained from the fruit. In the ancient world, by 2000 BC the olive tree was already in cultivation. Olive and olive oil was used and traded by the Egyptians, Phoenicians, Greeks and Romans. Today, a large number of olive varieties are recognised internationally as table olives and olives for oil production. Extracts from the olive tree were used in the treatment of hypertension, hyperglycaemia, hyperacidity [134], constipation, for treatment of wounds, sunburn and muscle aches [135,

**Sample Citronellal Nerol Geranial Neral Caryophyllene**

22 Phytochemicals - Isolation, Characterisation and Role in Human Health

w/o Fertiliser 0.00 0.00 37.11 47.39 1.02 Fertiliser 0.52 0.00 36.82 47.74 1.26

w/o Fertiliser 0.55 0.00 36.08 48.92 2.11 Fertiliser 1.31 0.55 30.73 45.13 2.67

w/o Fertiliser 1.24 0.57 30.96 45.79 2.62 Fertiliser 1.25 0.71 32.11 47.23 1.84

w/o Fertiliser 1.65 0.56 31.39 47.63 2.13 Fertiliser 1.31 0.74 33.42 49.19 1.94

**Table 4.** The composition of essential oils obtained from lemon balm of different seed origins [131].

The bioactive phenolic compounds present in the olive fruit include phenolic acids, phenolic alcohols, flavonoids and secoiridoids. The main phenolic acids are cinnamic, syringic, pcoumaric, vanillic, caffeic, 3,4-dihydroxyphenylacetic and protocatechuic acid [136]. Phenolic alcohols include 3,4-dihydroxyphenylethanol (hydroxytyrosol) and p-hydroxyphenylethanol (tyrosol) [137-139]. Flavonoids include taxifolin, apigenin, luteolin and lignans represented by pinoresinol and its metabolites [140]. However, an important class of metabolites found in the leaves and fruit of *Olea*, is that of the secoiridoid glycosides. These include oleuropein (figure

Oleuropein was extracted from Maltese olives as follows [145].The leaves were defatted with petroleum ether and then extracted with 50% ethanol for 6-8 hours. The dried extract was then treated with water and sodium chloride was added until saturation was achieved. Chloroform was added and the aqueous extract was collected. Ethylacetate was added to the aqueous extract and following partitioning, the ethylacetate extract was collected. The extract was then subjected to dryness in order to obtain a yellow crystalline substance. Oleuropein in the olive leaf ethanolic extract amounts to 20.6 %, as mentioned by [146], with a content varying from

*Olea europea* was tested for its antimicrobial [147, 148], antiviral [149], antioxidant [146, 150], antihypertensive, antiatherosclerotic [151, 152] and antidiabetic [153] activities amongst

18), demethyloleuropein, oleuropein aglycone and elenolic acid [141-144].

*Urtica dubia* Forsk., stinging nettle, is a member of the Urticaceae family. The *Urtica* species are common weeds found growing wild throughout the temperate zones of both hemispheres worldwide. These species are renowned for their stinging sensation when touched. Since Ancient Greek times, stinging nettle was used as a medical treatment for septic wounds, nosebleeds and as an emmenagogue [154]. They were later used as diuretics and laxatives, in the treatment of asthma, pleurisy, dog bites, tinea and mouth ulcers [155]. In Malta, *Urtica dubia* was used in the treatment of pneumonia, chilblains, as a metabolic stimulant, to improve blood circulation and as a diuretic [156].

Stinging nettle contains bioactive amines such as 5-hydroxytryptamine; flavonoids such as quercetin, kaempferol and their glycosides; coumarins such as scopoletin; organic acids such as caffeic acid and chlorogenic acid; fatty acids such as erucic acid, α-linolenic acid and linoleic acid; an essential oil; carotenoids such as lutein, β-carotene, neoxanthin, violaxanthin and lycopene; agglutinins such as *Urtica dioica* agglutinin (Figure 19); and phytosterols such as βamyrin, stigmasterol, oleanolic acid and β-sitosterol [157-163]. The isolation of *Urtica dubia* agglutinin (UDuA) was based on a procedure described by [164] with some modification [165]. Briefly, the fresh plant materials (rhizomes, leaves and stems) were homogenised with 0.1N HCl (200 g/l) and allowed for 24 h shaking. The filtrate was passed through series of extractions with 2N NaOH and (NH4)2SO4 solutions. The final agglutinin purified extract was washed with phosphate buffer saline (PBS) which was used as the medium for the bioassays. Phyto‐ haemagglutinin (PHA, Invitrogen) was prepared likewise in PBS. The content of UDuA in the rhizomes, leaves and stems was 0.49 %, 0.65 % and 0.16 %, respectively. the medium for the bioassays. Phytohaemagglutinin (PHA, Invitrogen) was prepared likewise in PBS. The content of UDuA in the rhizomes, leaves and stems was 0.49 %, 0.65 % and 0.16 %, respectively.

Urtica dubia Forsk., stinging nettle, is a member of the Urticaceae family. The Urtica species are common weeds found

growing wild throughout the temperate zones of both hemispheres worldwide. These species are renowned for their

stinging sensation when touched. Since Ancient Greek times, stinging nettle was used as a medical treatment for septic

wounds, nosebleeds and as an emmenagogue [154]. They were later used as diuretics and laxatives, in the treatment of

asthma, pleurisy, dog bites, tinea and mouth ulcers [155]. In Malta, Urtica dubia was used in the treatment of pneumonia,

Stinging nettle contains bioactive amines such as 5-hydroxytryptamine; flavonoids such as quercetin, kaempferol and their

glycosides; coumarins such as scopoletin; organic acids such as caffeic acid and chlorogenic acid; fatty acids such as erucic

acid, α-linolenic acid and linoleic acid; an essential oil; carotenoids such as lutein, β-carotene, neoxanthin, violaxanthin and

lycopene; agglutinins such as Urtica dioica agglutinin (Figure 19); and phytosterols such as β-amyrin, stigmasterol, oleanolic

acid and β-sitosterol [157-163]. The isolation of Urtica dubia agglutinin (UDuA) was based on a procedure described by [164]

with some modification [165]. Briefly, the fresh plant materials (rhizomes, leaves and stems) were homogenised with 0.1N

(NH4)2SO4 solutions. The final agglutinin purified extract was washed with phosphate buffer saline (PBS) which was used as

Capparis spinosa L. is a member of the Capparaceae family, also known as the caper plant. Today it is renowned for its

culinary uses, particularly in the Mediterranean region. When stored in brine, the intensive and slightly pungent taste of the

capers is preserved. Capers were used since prehistoric times, although it is believed that other Capparis species were actually

utilised rather than Capparis spinosa [171]. In the past, the root bark and leaves were used as aperient, tonic, diuretic and

expectorant while the flowers were used as anthelmintic, emmenagogue, analgesic, antimicrobial, antifertility, anti-

inflammatory, hepatoprotective, antihyperglycemic, immuno-stimulant and in the treatment of anaemia, diabetes, heart

chilblains, as a metabolic stimulant, to improve blood circulation and as a diuretic [156].

**Figure 19.** The crystal structure of *Urtica dioica* agglutinin isolectin I [166].

Figure 19. The crystal structure of Urtica dioica agglutinin isolectin I [166]. The stinging nettle possesses several pharmacological activities, namely antioxidant, antimicrobial, antiulcer and analgesic activities [167], anti-inflammatory effects [168] and cardiovascular effects [169]. The UDuA extracts from the Maltese Urtica dubia were tested for haemagglutination activity on human red blood cells (RBCs) [165]. Briefly, a 1% suspension of RBCs was prepared and 100 µl aliquots were tested with different concentrations of UDuA and PHA. The agglutination was quantified by lysing the precipitated agglutinated cells and read spectrophotometrically at a wavelength of 405 nm at 20, 40, The stinging nettle possesses several pharmacological activities, namely antioxidant, antimi‐ crobial, antiulcer and analgesic activities [167], anti-inflammatory effects [168] and cardiovas‐ cular effects [169]. The UDuA extracts from the Maltese *Urtica dubia* were tested for haemagglutination activity on human red blood cells (RBCs) [165]. Briefly, a 1% suspension of RBCs was prepared and 100 μl aliquots were tested with different concentrations of UDuA and PHA. The agglutination was quantified by lysing the precipitated agglutinated cells and read spectrophotometrically at a wavelength of 405 nm at 20, 40, 60 and 80 minutes. Over the 80-min period, the best results were obtained after 60 min, as was observed by [170] for the snowdrop lectin. Extracts from all three plant parts exhibited superior haemagglutination activity (AgA) to the standard PHA lectin (AgA - 3.996 ± 0.259). The highest activity was exhibited by the stems, followed by roots and leaves (AgA - 4.824 ± 0.301, 4.693 ± 0.368 and 4.594 ± 0.417, respectively at 1% concentrations).

#### 60 and 80 minutes. Over the 80-min period, the best results were obtained after 60 min, as was observed by [170] for the **2.12. Capparis spinosa L.**

snowdrop lectin. Extracts from all three plant parts exhibited superior haemagglutination activity (AgA) to the standard PHA lectin (AgA - 3.996 ± 0.259). The highest activity was exhibited by the stems, followed by roots and leaves (AgA - 4.824 ± 0.301, 4.693 ± 0.368 and 4.594 ± 0.417, respectively at 1% concentrations). *Capparis spinosa* L. is a member of the Capparaceae family, also known as the caper plant. Today it is renowned for its culinary uses, particularly in the Mediterranean region. When stored in brine, the intensive and slightly pungent taste of the capers is preserved. Capers were used since prehistoric times, although it is believed that other *Capparis* species were actually utilised rather than *Capparis spinosa* [171]. In the past, the root bark and leaves were used as aperient,

2.12. Capparis spinosa L.

the medium for the bioassays. Phytohaemagglutinin (PHA, Invitrogen) was prepared likewise in PBS. The content of UDuA in the rhizomes, leaves and stems was 0.49 %, 0.65 % and 0.16 %, respectively. tonic, diuretic and expectorant while the flowers were used as anthelmintic, emmenagogue, analgesic, antimicrobial, antifertility, anti-inflammatory, hepatoprotective, antihyperglyce‐ mic, immuno-stimulant and in the treatment of anaemia, diabetes, heart problems, amongst other uses [172]. In Malta, caper extracts were used as diuretics, in the treatment of skin rashes and pain associated with gout [135].

Urtica dubia Forsk., stinging nettle, is a member of the Urticaceae family. The Urtica species are common weeds found

growing wild throughout the temperate zones of both hemispheres worldwide. These species are renowned for their

stinging sensation when touched. Since Ancient Greek times, stinging nettle was used as a medical treatment for septic

wounds, nosebleeds and as an emmenagogue [154]. They were later used as diuretics and laxatives, in the treatment of

asthma, pleurisy, dog bites, tinea and mouth ulcers [155]. In Malta, Urtica dubia was used in the treatment of pneumonia,

Stinging nettle contains bioactive amines such as 5-hydroxytryptamine; flavonoids such as quercetin, kaempferol and their

glycosides; coumarins such as scopoletin; organic acids such as caffeic acid and chlorogenic acid; fatty acids such as erucic

acid, α-linolenic acid and linoleic acid; an essential oil; carotenoids such as lutein, β-carotene, neoxanthin, violaxanthin and

lycopene; agglutinins such as Urtica dioica agglutinin (Figure 19); and phytosterols such as β-amyrin, stigmasterol, oleanolic

acid and β-sitosterol [157-163]. The isolation of Urtica dubia agglutinin (UDuA) was based on a procedure described by [164]

with some modification [165]. Briefly, the fresh plant materials (rhizomes, leaves and stems) were homogenised with 0.1N

(NH4)2SO4 solutions. The final agglutinin purified extract was washed with phosphate buffer saline (PBS) which was used as

Capparis spinosa L. is a member of the Capparaceae family, also known as the caper plant. Today it is renowned for its

culinary uses, particularly in the Mediterranean region. When stored in brine, the intensive and slightly pungent taste of the

capers is preserved. Capers were used since prehistoric times, although it is believed that other Capparis species were actually

utilised rather than Capparis spinosa [171]. In the past, the root bark and leaves were used as aperient, tonic, diuretic and

expectorant while the flowers were used as anthelmintic, emmenagogue, analgesic, antimicrobial, antifertility, anti-

inflammatory, hepatoprotective, antihyperglycemic, immuno-stimulant and in the treatment of anaemia, diabetes, heart

chilblains, as a metabolic stimulant, to improve blood circulation and as a diuretic [156].

Figure 19. The crystal structure of Urtica dioica agglutinin isolectin I [166].

HCl (200 g/l) and allowed for 24 h shaking. The filtrate was passed through series of extractions with 2N NaOH and (NH4)2SO4 solutions. The final agglutinin purified extract was washed with phosphate buffer saline (PBS) which was used as the medium for the bioassays. Phyto‐ haemagglutinin (PHA, Invitrogen) was prepared likewise in PBS. The content of UDuA in the

2.12. Capparis spinosa L.

*Capparis spinosa* L. is a member of the Capparaceae family, also known as the caper plant. Today it is renowned for its culinary uses, particularly in the Mediterranean region. When stored in brine, the intensive and slightly pungent taste of the capers is preserved. Capers were used since prehistoric times, although it is believed that other *Capparis* species were actually utilised rather than *Capparis spinosa* [171]. In the past, the root bark and leaves were used as aperient,

The stinging nettle possesses several pharmacological activities, namely antioxidant, antimi‐ crobial, antiulcer and analgesic activities [167], anti-inflammatory effects [168] and cardiovas‐ cular effects [169]. The UDuA extracts from the Maltese *Urtica dubia* were tested for haemagglutination activity on human red blood cells (RBCs) [165]. Briefly, a 1% suspension of RBCs was prepared and 100 μl aliquots were tested with different concentrations of UDuA and PHA. The agglutination was quantified by lysing the precipitated agglutinated cells and read spectrophotometrically at a wavelength of 405 nm at 20, 40, 60 and 80 minutes. Over the 80-min period, the best results were obtained after 60 min, as was observed by [170] for the snowdrop lectin. Extracts from all three plant parts exhibited superior haemagglutination activity (AgA) to the standard PHA lectin (AgA - 3.996 ± 0.259). The highest activity was exhibited by the stems, followed by roots and leaves (AgA - 4.824 ± 0.301, 4.693 ± 0.368 and

rhizomes, leaves and stems was 0.49 %, 0.65 % and 0.16 %, respectively.

24 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Figure 19.** The crystal structure of *Urtica dioica* agglutinin isolectin I [166].

4.594 ± 0.417, respectively at 1% concentrations).

**2.12. Capparis spinosa L.**

According to [173], the capers contain 79% moisture, 1.6% ash, 5.8% protein, 1.6% fat and 5.4% raw fibre. It contains several minerals such as, Ca (871 ppm), Mg (636 ppm), K (542 mg/100mL), Na (226 ppm), Fe (13 ppm) and P (21 mg/100g). Other valuable constituents include the flavonoids such as rutin, kaempferol and its glycosides; alkaloids (Figure 20) such as cadabi‐ cine [174], capparisine A, capparisine B, capparisine C; 2-(5-hydroxymethyl-2-formylpyrrol-1 yl) propionic acid lactone and N-(3′-maleimidy1)-5-hydroxymethyl-2-pyrrole formaldehyde [175]. Other constituents include aldehydes, esters, sesquiterpenes, monoterpenes and sulphur compounds with methyl-isothiocyanate as the main constituent [176], carotenoids with lutein as the main constituent [177], sterols such as β-sitosterol, campesterol, stigmasterol, 5 avenasterol, cholesterol and campestanol [178], and a lectin (*Capparis spinosa* lectin) [179]. problems, amongst other uses [172]. In Malta, caper extracts were used as diuretics, in the treatment of skin rashes and pain associated with gout [135]. According to [173], the capers contain 79% moisture, 1.6% ash, 5.8% protein, 1.6% fat and 5.4% raw fibre. It contains several minerals such as, Ca (871 ppm), Mg (636 ppm), K (542 mg/100mL), Na (226 ppm), Fe (13 ppm) and P (21 mg/100g). Other valuable constituents include the flavonoids such as rutin, kaempferol and its glycosides; alkaloids (Figure 20) such as cadabicine [174], capparisine A, capparisine B, capparisine C; 2-(5-hydroxymethyl-2-formylpyrrol-1-yl) propionic acid lactone and N-(3′-maleimidy1)-5-hydroxymethyl-2-pyrrole formaldehyde [175]. Other constituents include aldehydes, esters, sesquiterpenes, monoterpenes and sulphur compounds with methyl-isothiocyanate as the main constituent [176],

carotenoids with lutein as the main constituent [177], sterols such as β-sitosterol, campesterol, stigmasterol, 5-avenasterol,

The stinging nettle possesses several pharmacological activities, namely antioxidant, antimicrobial, antiulcer and analgesic Figure 20. Typical Capparis spinosa L. alkaloids: (A) capparisine and (B) cadabicine. **Figure 20.** Typical *Capparis spinosa* L. alkaloids: (A) capparisine and (B) cadabicine.

cholesterol and campestanol [178], and a lectin (Capparis spinosa lectin) [179].

activities [167], anti-inflammatory effects [168] and cardiovascular effects [169]. The UDuA extracts from the Maltese Urtica dubia were tested for haemagglutination activity on human red blood cells (RBCs) [165]. Briefly, a 1% suspension of RBCs was prepared and 100 µl aliquots were tested with different concentrations of UDuA and PHA. The agglutination was quantified by lysing the precipitated agglutinated cells and read spectrophotometrically at a wavelength of 405 nm at 20, 40, 60 and 80 minutes. Over the 80-min period, the best results were obtained after 60 min, as was observed by [170] for the The xanthoproteic test for proteins [181], Fehling's test for carbohydrates, Sudan IV test for fats and lipids [182], Dragendorff's test for alkaloids [183], triphenyltetrazolium test for terpenoids and the acidified vanillin test for flavonoids [184] were carried out on the extracts. The petroleum ether extract (0.020 % w/w plant material) contained fats and lipids, the aqueous/methanol extract (2.401 % w/w plant material) contained proteins and terpenoids, the methanol extract (1.398 % w/w plant material) contained alkaloids, while the aqueous extract (3.015 % w/w plant material) contained carbohydrates and terpenoids. The caper plant was tested for several pharmacological activities such as antiviral [185], anti-arthritic [186], anti-oxidant [187], hypolipidaemic [188], antihyperglycaemic [189], chondrocyte protective [190], antiallergic, antihistaminic [191], antifungal [192], anti-Leishmania [193] and antimicrobial [194]. The Brine shrimp test was conducted for the extracts derived from the Maltese caper plants [180]. Briefly, Artemia salina eggs were hatched and challenged with various concentrations of the extracts ranging between 0.0001 and 1 % as 1 in 10 dilutions. After 24 hour the number of dead larvae (nauplii) was determined. The aqueous extract exhibited the lowest LC50 (0.014%) compared to the methanol (0.0475%) and the Metabolites from the Maltese caper plant were obtained by extracting the plant material with four different solvents [180]. The xanthoproteic test for proteins [181], Fehling's test for carbohydrates, Sudan IV test for fats and lipids [182], Dragendorff's test for alkaloids [183], triphenyltetrazolium test for terpenoids and the acidified vanillin test for flavonoids [184] were carried out on the extracts. The petroleum ether extract (0.020 % w/w plant material) contained fats and lipids, the aqueous/methanol extract (2.401 % w/w plant material) contained proteins and terpenoids, the methanol extract (1.398 % w/w plant material) contained alkaloids, while the aqueous extract (3.015 % w/w plant material) contained carbohydrates and terpenoids.

Metabolites from the Maltese caper plant were obtained by extracting the plant material with four different solvents [180].

snowdrop lectin. Extracts from all three plant parts exhibited superior haemagglutination activity (AgA) to the standard PHA lectin (AgA - 3.996 ± 0.259). The highest activity was exhibited by the stems, followed by roots and leaves (AgA - 4.824 ± 0.301, 4.693 ± 0.368 and 4.594 ± 0.417, respectively at 1% concentrations). aqueous/methanol (0.08%) extracts. The chloroform extract did not reach a 50% lethal effect and therefore the LC50 could not be determined. According to [195] the methanol, aqueous and aqueous/methanol extracts were all active as their LC50 was below the 0.1% threshold. 2.13. Ephedra fragilis Desf. The caper plant was tested for several pharmacological activities such as antiviral [185], antiarthritic [186], anti-oxidant [187], hypolipidaemic [188], antihyperglycaemic [189], chondro‐ cyte protective [190], antiallergic, antihistaminic [191], antifungal [192], anti-Leishmania [193] and antimicrobial [194]. The Brine shrimp test was conducted for the extracts derived from the Maltese caper plants [180]. Briefly, Artemia salina eggs were hatched and challenged with

various concentrations of the extracts ranging between 0.0001 and 1 % as 1 in 10 dilutions. After 24 hour the number of dead larvae (nauplii) was determined. The aqueous extract exhibited the lowest LC50 (0.014%) compared to the methanol (0.0475%) and the aqueous/ methanol (0.08%) extracts. The chloroform extract did not reach a 50% lethal effect and therefore the LC50 could not be determined. According to [195] the methanol, aqueous and aqueous/methanol extracts were all active as their LC50 was below the 0.1% threshold.

#### **2.13. Ephedra fragilis Desf.**

*Ephedra fragilis* Desf., a member of the Ephedraceae family, is also known as Mormon tea. *Ephedra* has been listed amongst the most important herbs used by Ancient Chinese civilisa‐ tions. It was known as Ma Huang and was used to treat coughs, colds, headache and fever. It was later used by the Chinese to treat asthma [196] and acute nephritis [197]. This plant contains alkaloids [198], amino acids, proteins [199], tannins and fatty acids [200]. The volatile oil of *Ephedra fragilis* contains (E)-phytol (10.1%), pentacosane (5.2%), 6,10,14-trimethyl-2-pentade‐ canone (5.3%), cis-thujopsene (3.5%), and α-terpineol (3.0%) as the major components [201]. Flavonoids, minerals, and vitamins are also present. The principle alkaloid present in this plant is ephedrine [198] (Figure 21). Ephedra fragilis Desf., a member of the Ephedraceae family, is also known as Mormon tea. Ephedra has been listed amongst the most important herbs used by Ancient Chinese civilisations. It was known as Ma Huang and was used to treat coughs, colds, headache and fever. It was later used by the Chinese to treat asthma [196] and acute nephritis [197]. This plant contains alkaloids [198], amino acids, proteins [199], tannins and fatty acids [200]. The volatile oil of Ephedra fragilis contains (E)-phytol (10.1%), pentacosane (5.2%), 6,10,14-trimethyl-2-pentadecanone (5.3%), cis-thujopsene (3.5%), and α-terpineol (3.0%) as the major components [201]. Flavonoids, minerals, and vitamins are also present. The principle alkaloid present in this plant is ephedrine [198] (Figure 21).

Figure 21. Ephedrine, pseudoephedrine and methylephedrine **Figure 21.** Ephedrine, pseudoephedrine and methylephedrine

Aerial parts of local cultivated Ephedra fragilis specimen were dried in an oven at 30ºC for 48 h, pulverized and dispersed in distilled water for 25 min. After thorough mixing for 30 min at 30ºC (twice), the filtrate was treated with sodium carbonate (15 g). An equal volume of benzene was added and then acidifed and treated with acidifed water. After neutralisation to a pH of 7, the precipitate was oven dried. The alkaloidal content in the different plant parts was determined; 1.8675 % (w/w) in flowers, 0.6234 % in seeds, 0.5198 % in pods and seeds, 0.1389 % in dried pods and 0.0547 % in branches [202]. Clinically, ephedra has been tested for its anti-hypertensive [203], bronchodilator [204], decongestant [205], diuretic [206] and immune booster [207]. The immunomodulatory response of ephedrine and the Ephedra extract were studied on human peripheral lymphocytes [202]. Cell viability, cytotoxicity and morphological characteristics were recorded for the test substances and phytohaemagglutinin (PHA), a mitogen known to stimulate cell division of T-lymphocytes. Over the 96-hour treatment, ephedrine and Ephedra extracts exhibited high cell viability (> 97% viability) and blastogenesis when compared to Aerial parts of local cultivated *Ephedra fragilis* specimen were dried in an oven at 30ºC for 48 h, pulverized and dispersed in distilled water for 25 min. After thorough mixing for 30 min at 30ºC (twice), the filtrate was treated with sodium carbonate (15 g). An equal volume of benzene was added and then acidifed and treated with acidifed water. After neutralisation to a pH of 7, the precipitate was oven dried. The alkaloidal content in the different plant parts was determined; 1.8675 % (w/w) in flowers, 0.6234 % in seeds, 0.5198 % in pods and seeds, 0.1389 % in dried pods and 0.0547 % in branches [202].

the untreated control. The control cells measured 6-10 µm, while treated cells measured 20-40 µm in diameter. The ephedrine present in Ephedra extracts exhibited a direct effect on lymphocytes in vitro. 2.14. Nicotiana glauca RC Graham Nicotiana glauca RC Graham belongs to the Solanaceae family and is known as tree tobacco. This was native to South America but is now naturalized in North America, the Mediterranean, and Africa. Since, this plant was considered as poisonous [208], it has been rarely used in tradition. The more toxic counterpart, Nicotiana tabacum was used for several conditions particularly to expel leeches [209], against snakebite [210] and scabies [211]. Tree tobacco contains pyridine alkaloids [212], as for other Nicotiana species. The major pyridine alkaloids are nicotine and anabasine (figure 22). Nicotine predominated in Nicotiana tabacum [213] and Nicotiana rustica [214] whereas anabasine predominates in Nicotiana glauca [214, 215]. Clinically, ephedra has been tested for its anti-hypertensive [203], bronchodilator [204], decongestant [205], diuretic [206] and immune booster [207]. The immunomodulatory response of ephedrine and the *Ephedra* extract were studied on human peripheral lymphocytes [202]. Cell viability, cytotoxicity and morphological characteristics were recorded for the test substances and phytohaemagglutinin (PHA), a mitogen known to stimulate cell division of Tlymphocytes. Over the 96-hour treatment, ephedrine and *Ephedra* extracts exhibited high cell viability (> 97% viability) and blastogenesis when compared to the untreated control. The control cells measured 6-10 μm, while treated cells measured 20-40 μm in diameter. The ephedrine present in *Ephedra* extracts exhibited a direct effect on lymphocytes *in vitro*.

#### **2.14. Nicotiana glauca RC Graham**

various concentrations of the extracts ranging between 0.0001 and 1 % as 1 in 10 dilutions. After 24 hour the number of dead larvae (nauplii) was determined. The aqueous extract exhibited the lowest LC50 (0.014%) compared to the methanol (0.0475%) and the aqueous/ methanol (0.08%) extracts. The chloroform extract did not reach a 50% lethal effect and therefore the LC50 could not be determined. According to [195] the methanol, aqueous and aqueous/methanol extracts were all active as their LC50 was below the 0.1% threshold.

*Ephedra fragilis* Desf., a member of the Ephedraceae family, is also known as Mormon tea. *Ephedra* has been listed amongst the most important herbs used by Ancient Chinese civilisa‐ tions. It was known as Ma Huang and was used to treat coughs, colds, headache and fever. It was later used by the Chinese to treat asthma [196] and acute nephritis [197]. This plant contains alkaloids [198], amino acids, proteins [199], tannins and fatty acids [200]. The volatile oil of *Ephedra fragilis* contains (E)-phytol (10.1%), pentacosane (5.2%), 6,10,14-trimethyl-2-pentade‐ canone (5.3%), cis-thujopsene (3.5%), and α-terpineol (3.0%) as the major components [201]. Flavonoids, minerals, and vitamins are also present. The principle alkaloid present in this plant

Ephedra fragilis Desf., a member of the Ephedraceae family, is also known as Mormon tea. Ephedra has been listed amongst the most important herbs used by Ancient Chinese civilisations. It was known as Ma Huang and was used to treat coughs, colds, headache and fever. It was later used by the Chinese to treat asthma [196] and acute nephritis [197]. This plant contains alkaloids [198], amino acids, proteins [199], tannins and fatty acids [200]. The volatile oil of Ephedra fragilis contains (E)-phytol (10.1%), pentacosane (5.2%), 6,10,14-trimethyl-2-pentadecanone (5.3%), cis-thujopsene (3.5%), and α-terpineol (3.0%) as the major components [201]. Flavonoids, minerals, and vitamins are also present. The principle alkaloid present in this plant is

Aerial parts of local cultivated Ephedra fragilis specimen were dried in an oven at 30ºC for 48 h, pulverized and dispersed in distilled water for 25 min. After thorough mixing for 30 min at 30ºC (twice), the filtrate was treated with sodium carbonate (15 g). An equal volume of benzene was added and then acidifed and treated with acidifed water. After neutralisation to a pH of 7, the precipitate was oven dried. The alkaloidal content in the different plant parts was determined; 1.8675 % (w/w) in

Aerial parts of local cultivated *Ephedra fragilis* specimen were dried in an oven at 30ºC for 48 h, pulverized and dispersed in distilled water for 25 min. After thorough mixing for 30 min at 30ºC (twice), the filtrate was treated with sodium carbonate (15 g). An equal volume of benzene was added and then acidifed and treated with acidifed water. After neutralisation to a pH of 7, the precipitate was oven dried. The alkaloidal content in the different plant parts was determined; 1.8675 % (w/w) in flowers, 0.6234 % in seeds, 0.5198 % in pods and seeds, 0.1389

Clinically, ephedra has been tested for its anti-hypertensive [203], bronchodilator [204], decongestant [205], diuretic [206] and immune booster [207]. The immunomodulatory response of ephedrine and the Ephedra extract were studied on human peripheral lymphocytes [202]. Cell viability, cytotoxicity and morphological characteristics were recorded for the test substances and phytohaemagglutinin (PHA), a mitogen known to stimulate cell division of T-lymphocytes. Over the 96-hour treatment, ephedrine and Ephedra extracts exhibited high cell viability (> 97% viability) and blastogenesis when compared to the untreated control. The control cells measured 6-10 µm, while treated cells measured 20-40 µm in diameter. The ephedrine

Nicotiana glauca RC Graham belongs to the Solanaceae family and is known as tree tobacco. This was native to South America but is now naturalized in North America, the Mediterranean, and Africa. Since, this plant was considered as poisonous [208], it has been rarely used in tradition. The more toxic counterpart, Nicotiana tabacum was used for several conditions

Clinically, ephedra has been tested for its anti-hypertensive [203], bronchodilator [204], decongestant [205], diuretic [206] and immune booster [207]. The immunomodulatory response of ephedrine and the *Ephedra* extract were studied on human peripheral lymphocytes [202]. Cell viability, cytotoxicity and morphological characteristics were recorded for the test substances and phytohaemagglutinin (PHA), a mitogen known to stimulate cell division of Tlymphocytes. Over the 96-hour treatment, ephedrine and *Ephedra* extracts exhibited high cell viability (> 97% viability) and blastogenesis when compared to the untreated control. The control cells measured 6-10 μm, while treated cells measured 20-40 μm in diameter. The ephedrine present in *Ephedra* extracts exhibited a direct effect on lymphocytes *in vitro*.

Tree tobacco contains pyridine alkaloids [212], as for other Nicotiana species. The major pyridine alkaloids are nicotine and anabasine (figure 22). Nicotine predominated in Nicotiana tabacum [213] and Nicotiana rustica [214] whereas anabasine

flowers, 0.6234 % in seeds, 0.5198 % in pods and seeds, 0.1389 % in dried pods and 0.0547 % in branches [202].

**2.13. Ephedra fragilis Desf.**

26 Phytochemicals - Isolation, Characterisation and Role in Human Health

is ephedrine [198] (Figure 21).

Figure 21. Ephedrine, pseudoephedrine and methylephedrine

**Figure 21.** Ephedrine, pseudoephedrine and methylephedrine

% in dried pods and 0.0547 % in branches [202].

2.14. Nicotiana glauca RC Graham

predominates in Nicotiana glauca [214, 215].

present in Ephedra extracts exhibited a direct effect on lymphocytes in vitro.

particularly to expel leeches [209], against snakebite [210] and scabies [211].

ephedrine [198] (Figure 21).

*Nicotiana glauca* RC Graham belongs to the *Solanaceae* family and is known as tree tobacco. This was native to South America but is now naturalized in North America, the Mediterranean, and Africa. Since, this plant was considered as poisonous [208], it has been rarely used in tradition. The more toxic counterpart, *Nicotiana tabacum* was used for several conditions particularly to expel leeches [209], against snakebite [210] and scabies [211].

Tree tobacco contains pyridine alkaloids [212], as for other *Nicotiana* species. The major pyridine alkaloids are nicotine and anabasine (figure 22). Nicotine predominated in *Nicotiana tabacum* [213] and *Nicotiana* rustica [214] whereas anabasine predominates in *Nicotiana glauca* [214, 215].

Figure 22. (A) Nicotine, (B) Nornicotine and (C) Anabasine, the main pyridine alkaloids of Nicotiana species. **Figure 22.** (A) Nicotine, (B) Nornicotine and (C) Anabasine, the main pyridine alkaloids of *Nicotiana* species.

The leaves of Maltese Nicotiana glauca were dried and extracted with 200ml of 0.5% sodium hydroxide [216]. After volume reduction, chloroform was added to extract the alkaloids in this organic phase. This phase was then treated with acidified water (0.05M hydrochloric acid) and then neutralised with ammonia solution to a pH of 7. The presence of alkaloids was tested at each step using the Dragendorff's reagent [217] and the anabasine content was determined by HPLC. A Shimadzu LC-10A HPLC (Shimadzu, Kyoto, Japan) using a C18 MicroBondapak column, 250 x 4.6mm, 10mm was used. The mobile phase consisted of 40 % methanol containing 0.2 % phosphoric acid buffered to pH 7.25 with triethylamine [218]. The anabasine standard was used for calibration and for the determination of anabasine in Nicotiana extracts. In the Maltese study, the anabasine content (0.258 ± 0.0042 %) concords very closely with the results obtained in a study in Arizona (0.233 ± 0.0061 % anabasine) [219]. In another HPLC determination, the anabasine content of Nicotiana glauca plants in California, was 0.143 % [220]. The nicotine and anabasine have been widely used as pesticides. Nicotine is a powerful insecticide towards aphids [221] and larvae of lepidopterous pests [222]. Anabasine and nicotine exert their insecticidal effect by interacting with nicotinic acetylcholine receptors [222, 223]. Anabasine and Nicotiana glauca extracts were tested for their effects against Pieris rapae larvae [216]. The paralysis of the larvae was an indicator of activity. Standard anabasine produced an effect on Pieris rapae larvae (EC50 - 0.572 mg/larva or 0.286 %) which was higher to that provoked by the extract (EC50 - 1.202 mg/larva or 0.601 %). It is possible that alongside anabasine there may be other metabolites that interfered with anabasine hence reducing the response of the caterpillars to anabasine. 3. Conclusion and further directions The leaves of Maltese *Nicotiana glauca* were dried and extracted with 200ml of 0.5% sodium hydroxide [216]. After volume reduction, chloroform was added to extract the alkaloids in this organic phase. This phase was then treated with acidified water (0.05M hydrochloric acid) and then neutralised with ammonia solution to a pH of 7. The presence of alkaloids was tested at each step using the Dragendorff's reagent [217] and the anabasine content was determined by HPLC. A Shimadzu LC-10A HPLC (Shimadzu, Kyoto, Japan) using a C18 MicroBondapak column, 250 x 4.6mm, 10mm was used. The mobile phase consisted of 40 % methanol con‐ taining 0.2 % phosphoric acid buffered to pH 7.25 with triethylamine [218]. The anabasine standard was used for calibration and for the determination of anabasine in Nicotiana extracts. In the Maltese study, the anabasine content (0.258 ± 0.0042 %) concords very closely with the results obtained in a study in Arizona (0.233 ± 0.0061 % anabasine) [219]. In another HPLC determination, the anabasine content of *Nicotiana glauca* plants in California, was 0.143 % [220].

The studies on the fourteen Maltese medicinal plants, presented herein, demonstrate a wide array of experimental work that is all associated with phytochemical research. This is a very small fraction of the Maltese medicinal flora, but in terms of research, this represents a diversity of research protocols that may be adopted for medicinal plant research. In some cases, phytochemical analysis is the end-point of the research whereas in others, phytochemical analysis leads on to further studies, including pharmacological testing. References [1] Attard E. Status of Medicinal and Aromatic Plants in Malta. In: Baricevic, D., Bernath, J., Maggioni, L. and Lipman E. (eds.) Report of a Working Group on Medicinal and Aromatic Plants, First Meeting, 12-14 September 2002, Gozd Martuljek, Slovenia. Rome: International Plant Genetic Resources Institute; 2004. [2] Kropp B, Krenn L, Draxler M, Hoyer A, Terkola R, Vallaster P, Robien W. Bufadienolides from Urginea maritima from Egypt. Phytochemistry 1996;42:513-522. [3] Dewick PM. Medicinal natural products. A biosynthetic approach. New York: John Wiley & Sons Inc; 1997. The nicotine and anabasine have been widely used as pesticides. Nicotine is a powerful insecticide towards aphids [221] and larvae of lepidopterous pests [222]. Anabasine and nicotine exert their insecticidal effect by interacting with nicotinic acetylcholine receptors [222, 223]. Anabasine and *Nicotiana glauca* extracts were tested for their effects against *Pieris rapae* larvae [216]. The paralysis of the larvae was an indicator of activity. Standard anabasine produced an effect on *Pieris rapae* larvae (EC50 - 0.572 mg/larva or 0.286 %) which was higher to that provoked by the extract (EC50 - 1.202 mg/larva or 0.601 %). It is possible that alongside anabasine there may be other metabolites that interfered with anabasine hence reducing the response of the caterpillars to anabasine.

#### **3. Conclusion and further directions**

The studies on the fourteen Maltese medicinal plants, presented herein, demonstrate a wide array of experimental work that is all associated with phytochemical research. This is a very small fraction of the Maltese medicinal flora, but in terms of research, this represents a diversity of research protocols that may be adopted for medicinal plant research. In some cases, phytochemical analysis is the end-point of the research whereas in others, phytochemical analysis leads on to further studies, including pharmacological testing.

#### **Author details**

Everaldo Attard1\*, Henrietta Attard1 , Antoine Tanti2 , Jurgen Azzopardi2 , Mario Sciberras2 , Victor Pace2 , Neville Buttigieg2 , Andrew Mangion Randon2 , Bernardette Rossi1 , Marie Josette Parnis1 , Karin Vella2 , Michelle Zammit2 and Anthony Serracino Inglott2

\*Address all correspondence to: everaldo.attard@um.edu.mt

1 University of Malta, Institute of Earth Systems, Division of Rural Sciences and Food Systems, Msida, Malta

2 University of Malta, Faculty of Medicine and Surgery, Department of Pharmacy, Msida, Malta

#### **References**


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**3. Conclusion and further directions**

28 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Author details**

Marie Josette Parnis1

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Victor Pace2

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Everaldo Attard1\*, Henrietta Attard1

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\*Address all correspondence to: everaldo.attard@um.edu.mt

The studies on the fourteen Maltese medicinal plants, presented herein, demonstrate a wide array of experimental work that is all associated with phytochemical research. This is a very small fraction of the Maltese medicinal flora, but in terms of research, this represents a diversity of research protocols that may be adopted for medicinal plant research. In some cases, phytochemical analysis is the end-point of the research whereas in others, phytochemical

, Antoine Tanti2

, Michelle Zammit2

, Andrew Mangion Randon2

1 University of Malta, Institute of Earth Systems, Division of Rural Sciences and Food

2 University of Malta, Faculty of Medicine and Surgery, Department of Pharmacy, Msida,

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60422

#### **1. Introduction**

Cancer is an uncontrolled proliferation of anaplastic cells, which tend to invade surrounding tissues and metastasize to other tissues and organs. A hundred years ago, cancer was not a common disease, but today, it is one of the leading causes of death worldwide, andone of three persons is having a lifetime risk of developing cancer. The incidence of cancer has risen alarmingly in the last few decades: in 2008, 12.7 million people were diagnosed with cancer globally, and about 7.6 million people died of it [1]; in 2012, 14.1 million cases of cancer were diagnosed, with 8.2 million deaths; in 2030, more than 21 million new cancer cases are estimated and 13 million deaths are expected. Considering the above data, it confirms the hypothesis that cancer seems to be a civilization disease. The major type of cancer diagnosed among females is breast cancer, accounting for 23% of the total cancer cases; in males, 17% of the total cancer cases are caused by lung cancer [2]. More than 60% of all cancer deaths occur in low- and middle-income countries, many of which lack the medical resources and health systems to support the disease burden [3]. However, cancer mortality surprisingly increased in U.S. population as well, and cancer metastasis attributes to approximately 90% of cancerrelated deaths [4].

After years of intense biomedical research on understanding the mechanisms of tumor genesis and biology of cancer, usual cancer treatment still consists in surgery, radiation, and chemo‐ therapy, each having its own limitations: surgery and radiation therapy could be effective especially for the primary tumor; chemotherapy with serious side effects associated with severe toxicity to normal cells is commonly used for the whole-body treatment of recurrent disease. Considerable research activity is devoted to the discovery of more potent treatments, while minimizing their toxic side effects and to discover selective drugs that can kill malignant tumor cells without affecting normal cells [5, 6].

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Recently, there is a greater global interest in nonsynthetic, natural medicines derived from natural sources due to better tolerance and minimum adverse drug reactions as compared to synthetic medicines. Many cancer patients prefer complementary and alternative medicine; herbal medicines were by far the most commonly used group of treatments because they are believed by the general public to be safe, cause less side effects, and less likely to cause dependency [7].

When we are considering natural antitumor compounds, generally we refer to secondary metabolites. Secondary metabolites are the products of metabolism not essential for normal growth, development, or reproduction of an organism. Many of them have proved invaluable as antibacterial or antifungal agents, anticancer drugs, cholesterol-lowering agents, immuno‐ suppressant, antiparasitic agents, herbicides, diagnostics, and tools for research [8]. Nowa‐ days, the increased interest in the obtaining of considerable amounts of secondary metabolites has led to intensive research in the field of cell cultures technology. A lot of efforts have been put into plant cell, tissue, and organ culture as an alternative method to whole plant cultivation for the production of pharmacologically important plant secondary metabolites [9].

Getting plant metabolites is not the only problem; the delivery of biologically active substances in the human body is also subjected to innovative research such as nanotechnol‐ ogy that has been used for tumor diagnosis and to design and development of targeted drug delivery [10-11].

#### **2. What is cancer?**

Cancer is ultimately the result of cells that uncontrollably grow and do not die. Normal cells in the body follow an orderly path of growth, division, and death. There are three major types of cell death:


Cancer results from a multistage carcinogenesis process that involves three distinguishable but closely connected stages: initiation (normal cell → transformed or initiated cell), promotion (initiated cell → preneoplastic cell), and progression (preneoplastic cell → neoplastic cell). The malignant tumor has the abilities to invade surrounding tissues (metastasis), migrate around the body in the blood or the lymphatic system, and set up secondary foci of cancerous growths at distant sites. Metastasis is responsible for 90% of the deaths caused by cancer [1].

Every living cell in the human body has the potential to become cancerous. A generic name is usually given to a group of cancers, depending on the type of cells of their origin, which include carcinoma, sarcoma, leukemia, lymphoma, and myeloma.


When cancer begins, it invariably produces no symptoms. Signs and symptoms only appear as the mass continues to grow or ulcerates. General symptoms may include unintentional weight loss, fever, being excessively tired, and changes to the skin. The symptoms of metastatic cancers depend on the location of the tumor and can include enlarged lymph nodes, enlarged liver or enlarged spleen, which can be felt in the abdomen, pain or fracture of affected bones, and neurological symptoms [16].

#### **3. What causes cancer?**

Recently, there is a greater global interest in nonsynthetic, natural medicines derived from natural sources due to better tolerance and minimum adverse drug reactions as compared to synthetic medicines. Many cancer patients prefer complementary and alternative medicine; herbal medicines were by far the most commonly used group of treatments because they are believed by the general public to be safe, cause less side effects, and less likely to cause

When we are considering natural antitumor compounds, generally we refer to secondary metabolites. Secondary metabolites are the products of metabolism not essential for normal growth, development, or reproduction of an organism. Many of them have proved invaluable as antibacterial or antifungal agents, anticancer drugs, cholesterol-lowering agents, immuno‐ suppressant, antiparasitic agents, herbicides, diagnostics, and tools for research [8]. Nowa‐ days, the increased interest in the obtaining of considerable amounts of secondary metabolites has led to intensive research in the field of cell cultures technology. A lot of efforts have been put into plant cell, tissue, and organ culture as an alternative method to whole plant cultivation

for the production of pharmacologically important plant secondary metabolites [9].

Getting plant metabolites is not the only problem; the delivery of biologically active substances in the human body is also subjected to innovative research such as nanotechnol‐ ogy that has been used for tumor diagnosis and to design and development of targeted

Cancer is ultimately the result of cells that uncontrollably grow and do not die. Normal cells in the body follow an orderly path of growth, division, and death. There are three major types

**a.** Apoptosis—a naturally occurring programmed and targeted cause of cellular death that may occur in multicellular organisms. Biochemical events lead to characteristic cell

**b.** Autophagy—the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. Autophagy

**c.** Necrosis—a form of cell injury that results in the premature death of cells in living tissue by autolysis, caused by factors external to the cell or tissue, such as infection, toxins, or trauma that result in the unregulated digestion of cell components. Necrosis is almost

Cancer results from a multistage carcinogenesis process that involves three distinguishable but closely connected stages: initiation (normal cell → transformed or initiated cell), promotion (initiated cell → preneoplastic cell), and progression (preneoplastic cell → neoplastic cell). The malignant tumor has the abilities to invade surrounding tissues (metastasis), migrate around

allows the degradation and recycling of cellular components [13].

always detrimental to the organism and can be fatal [14].

dependency [7].

46 Phytochemicals - Isolation, Characterisation and Role in Human Health

drug delivery [10-11].

**2. What is cancer?**

changes (morphology) and death [12].

of cell death:

Cancer results from a mutation in the chromosomal DNA of a normal cell under the action of carcinogens. The mutated cell proliferates indiscriminately (pathological mitosis), usually forming a mass known as neoplasm or malignant tumor. Cancer can be triggered by both internal factors (nutritional imbalance in the diet, aging, immune conditions, hormones, and mutations occurring in metabolism) and external factors (tobacco, alcohol, chemicals, infec‐ tious agents, and radiation). These causal factors may act together or in sequence to initiate or promote the development of cancer [1, 2, 15, 17].

#### **3.1. Carcinogens**

A carcinogen is any substance, radionuclide, or radiation that is an agent directly involved in causing cancer. Carcinogens include a wide range of possibilities: chemical and physical pollution, unhealthy lifestyle, stress, aging, viruses, bacteria, heredity, etc.

#### *3.1.1. Chemical carcinogens*

Most of the human cancers are caused by certain chemical agents.


#### *3.1.2. Physical carcinogens*

Ionizing radiation emitted by radioactive substances ruptures the DNA strands, leading to mutations in the genes. Ultraviolet radiation induces skin cancer. The electromagnetic fields generated by electrical appliances, power lines, and cell phones emit 30-100 times higher electromagnetic fields than the maximum permissible limits. Studies have revealed that prolonged exposure to electromagnetic fields causes mutations in the genes [15].

#### *3.1.3. Biological carcinogens*

Certain viruses are suspected to cause cancer in human: for example, the Epstein-Barr virus is linked to Burkitt's lymphoma; testicular tumors and leiomyoma in children; viruses of hepatitis B and hepatitis C are known to enhance risk of the hepatocellular carcinoma; human papilloma virus is a major risk factor of the cervical and of anal cancers [15].

#### *3.1.4. Lifestyle*

The World Cancer Research Fund and the American Institute for Cancer Research assess that 30-40% of all cancers could be prevented by appropriate diet and physical activity to avoid overweight and obesity. Many lines of evidence demonstrate that a diet based on abundant and various foods of plant origin protects against epithelial cancers, particularly those of the gastrointestinal tract [2].

#### *3.1.5. Age factor*

**•** *Tabacco*. Among the dangerous factors leading to cancer, cigarette smoking (also second‐ hand tobacco smoking) is essential. Smoke contains several carcinogenic pyrolytic products that bind to DNA and cause many genetic mutations. Tobacco smoke generates more than 2000 chemical compounds, and more than 45 are known or suspected as chemical carcino‐

**•** *Polluted environment* is another major source of chemical carcinogens. Industrial toxic chemicals and heavy metals having carcinogenic activity may enter and get accumulated in

**•** *Contaminated food*. The food containing residues of pesticides, insecticides, and herbicides is one of the three major causes of cancer. Most of the food additives including preservatives (butylated hydroxytoluene), sweeteners (saccharin and cyclamates), and synthetic colorants

**•** *Chlorine*, used to purify drinking water, may produce certain carcinogenic compounds in the treated water, including chloroform and trichloroethylene, which have carcinogenic

**•** *Reactive oxygen species* (ROS—superoxide, hydrogen peroxide, hydroxyl radical) are also major causes of DNA, protein, and lipid damages, which lead to cancer or aging [19].

Ionizing radiation emitted by radioactive substances ruptures the DNA strands, leading to mutations in the genes. Ultraviolet radiation induces skin cancer. The electromagnetic fields generated by electrical appliances, power lines, and cell phones emit 30-100 times higher electromagnetic fields than the maximum permissible limits. Studies have revealed that

Certain viruses are suspected to cause cancer in human: for example, the Epstein-Barr virus is linked to Burkitt's lymphoma; testicular tumors and leiomyoma in children; viruses of hepatitis B and hepatitis C are known to enhance risk of the hepatocellular carcinoma; human

The World Cancer Research Fund and the American Institute for Cancer Research assess that 30-40% of all cancers could be prevented by appropriate diet and physical activity to avoid overweight and obesity. Many lines of evidence demonstrate that a diet based on abundant and various foods of plant origin protects against epithelial cancers, particularly those of the

prolonged exposure to electromagnetic fields causes mutations in the genes [15].

papilloma virus is a major risk factor of the cervical and of anal cancers [15].

gens; nicotine is one of the major cancer promoters [18].

48 Phytochemicals - Isolation, Characterisation and Role in Human Health

the human body and damage DNA [2].

have carcinogenic activity [15].

activity [15].

*3.1.2. Physical carcinogens*

*3.1.3. Biological carcinogens*

gastrointestinal tract [2].

*3.1.4. Lifestyle*

The incidence of cancer is three times higher in women as compared to men during 30 to 50 years of age, whereas men have a greater risk of cancer as compared to women during 60 to 80 years of age. Cancer is generally considered as the disease of middle-aged and elderly people, but some cancers are known to affect the children [15].

#### *3.1.6. Genes (hereditary factor)*

Extensive evidence indicates that only 5-10% of cancers are genetically determined. It is believed that members of those families, who are predisposed to a particular cancer, have one or more activated oncogenes in their genome; therefore, fewer additional mutations are required in such persons to develop the cancer. Genetic mutations are commonly seen in the breast and the ovarian cancers, especially in cancers occurring below 30 years [15].

#### **4. How do natural remedies work?**

The plants act on several fronts in the fight against cancer, such as nourishing the body with minerals, vitamins, enzymes, and micronutrients; increasing the immune system of the body; inducing antioxidant action and protecting the body from oxidative stress; enhancing detox‐ ification functions of the body; alkalizing the body fluids; inhibiting cancer-activating en‐ zymes; promoting production of protective enzymes; stimulating DNA repair mechanism; and modulating the activity of specific hormones and enzymes to inhibit growth of cancer.

#### **4.1. Immunomodulatory properties of plant**

The immune system is a complex defense network that protects the host from disease and has a large impact on antitumor resistance. Most of the anticancer drugs currently used in chemotherapy are cytotoxic to normal cells, leading a low immune function of organism and can result in development of the tumor, and difficulty of tumor patients' recovery [20].

A major research interest has focused on the immunomodulatory properties of plant-derived medicines. Several studies have reported that many flavonoids, such as quercetin, catechins, resveratrol, green tea polyphenols, grape seed proanthocyanidins, silymarin, and curcumin, have anti-inflammatory and immunomodulatory properties [2].

#### **4.2. Chemoprevention**

Chemoprevention is defined as the use of natural or synthetic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. Many chemopreventive agents are phytochemicals (secondary metabolites) and nonnutritive plant chemicals that have protective or disease-preventive properties [2, 21]. Several commonly used herbs such as coriander, cumin, garlic, ginger, mint, oregano, and turmeric have been identified by the National Cancer Institute as possessing cancer-preventive properties.

#### **4.3. Impact on cancer induction: antimutagenesis**

During the initiation phase, natural chemopreventive agents can inhibit the absorption of a carcinogen into the organism through the antioxidant activity, prevent inflammatory-induced damage or enhance neutralization, and remove carcinogens through metabolic enzymes [3].

#### **4.4. Anti-inflammatory activity**

Long-term inflammation leads to the development of dysplasia [22]. Many phenolic substan‐ ces, including phenolic acids (e.g., gallic, ellagic, caffeic, chlorogenic), flavonoids (e.g., genistein, kaempferol, quercetin, daidzein, isorhamnetin, naringenin), anthocyanins (e.g., pelargonidin), and catechins, have potent anti-inflammatory properties, affecting different stages of the inflammation. Flavonoid aglycones have been shown to exert higher activity than adequate glycosides [2].

#### **4.5. Antioxidant properties**

Oxidative stress has been proven to be one of the main factors that lead to the formation of cancer. Oxidative stress is defined as a discrepancy between production of free radicals and reactive metabolites, so-called reactive oxygen species [ROS, e.g., hydrogen peroxide (H2O2), superoxide anion (O⋅<sup>2</sup> − ), and hydroxyl radical (⋅OH)], and their eradication by defending mechanisms, referred to as antioxidants. Excessive oxidative stress in the body for extended periods of time activates inflammatory pathways, which cause the transformation of normal cells into cancer cells, support the survival of cancer cells, and finally lead to cancer cell proliferation [23].

Many herbs are known to contain large amounts of phenolic antioxidants having the capacities to quench lipid peroxidation, prevent DNA oxidative damage, and scavenge reactive oxygen species (ROS) [24].

#### **4.6. Pro-oxidative activity**

The pro-oxidant behavior of phenolics is observed in the presence of transition metal ions such as copper or iron. Many studies suggest that the antitumor activity of some polyphenols (e.g., resveratrol, gallic acid, delphinidin, baicalin, quercetin, epicatechin, and epigallocatechin-3 gallate) is also a consequence of their pro-oxidant properties. It seems that while the antioxi‐ dant activity lowers the risk of cancer induction by protecting normal cells from oxidative injury, pro-oxidative properties of polyphenolics are more relevant for apoptosis induction and destruction of existing tumor cells [2].

#### **4.7. Modulation of activity of xenobiotic-metabolizing enzymes**

A carcinogen is a xenobiotic, and generally, biotransformation involves modification, conju‐ gation, and excretion and is strictly involved in the detoxification of carcinogens. Many plantderived phenolic compounds such as flavonoids, including kaempferol, daidzenin, genistein, diosmetin, and theaflavin, have been reported to affect the activity of metabolizing enzymes leading to faster carcinogen detoxification [2].

#### **4.8. Changing multidrug resistance**

**4.3. Impact on cancer induction: antimutagenesis**

50 Phytochemicals - Isolation, Characterisation and Role in Human Health

**4.4. Anti-inflammatory activity**

adequate glycosides [2].

superoxide anion (O⋅<sup>2</sup>

proliferation [23].

species (ROS) [24].

**4.6. Pro-oxidative activity**

and destruction of existing tumor cells [2].

**4.7. Modulation of activity of xenobiotic-metabolizing enzymes**

−

**4.5. Antioxidant properties**

During the initiation phase, natural chemopreventive agents can inhibit the absorption of a carcinogen into the organism through the antioxidant activity, prevent inflammatory-induced damage or enhance neutralization, and remove carcinogens through metabolic enzymes [3].

Long-term inflammation leads to the development of dysplasia [22]. Many phenolic substan‐ ces, including phenolic acids (e.g., gallic, ellagic, caffeic, chlorogenic), flavonoids (e.g., genistein, kaempferol, quercetin, daidzein, isorhamnetin, naringenin), anthocyanins (e.g., pelargonidin), and catechins, have potent anti-inflammatory properties, affecting different stages of the inflammation. Flavonoid aglycones have been shown to exert higher activity than

Oxidative stress has been proven to be one of the main factors that lead to the formation of cancer. Oxidative stress is defined as a discrepancy between production of free radicals and reactive metabolites, so-called reactive oxygen species [ROS, e.g., hydrogen peroxide (H2O2),

mechanisms, referred to as antioxidants. Excessive oxidative stress in the body for extended periods of time activates inflammatory pathways, which cause the transformation of normal cells into cancer cells, support the survival of cancer cells, and finally lead to cancer cell

Many herbs are known to contain large amounts of phenolic antioxidants having the capacities to quench lipid peroxidation, prevent DNA oxidative damage, and scavenge reactive oxygen

The pro-oxidant behavior of phenolics is observed in the presence of transition metal ions such as copper or iron. Many studies suggest that the antitumor activity of some polyphenols (e.g., resveratrol, gallic acid, delphinidin, baicalin, quercetin, epicatechin, and epigallocatechin-3 gallate) is also a consequence of their pro-oxidant properties. It seems that while the antioxi‐ dant activity lowers the risk of cancer induction by protecting normal cells from oxidative injury, pro-oxidative properties of polyphenolics are more relevant for apoptosis induction

A carcinogen is a xenobiotic, and generally, biotransformation involves modification, conju‐ gation, and excretion and is strictly involved in the detoxification of carcinogens. Many plantderived phenolic compounds such as flavonoids, including kaempferol, daidzenin, genistein,

), and hydroxyl radical (⋅OH)], and their eradication by defending

Multidrug resistance is a significant challenge in the treatment of infectious diseases and cancer. The antitumor treatments currently in use often fail at some stage of the sickness because many types of cancers develop resistance to chemotherapeutic drugs. Some plants can change the resistance of tumor cells to antitumor drugs [25].

#### **4.9. Suppression of angiogenesis**

Angiogenesis is the process of formation of new vessels (vascularization) from the preexisting microvascular network. This is as an essential step in tumor dissemination and formation of metastases. Many plant-derived phenolics have been reported to restrain angiogenesis and, consequently, cancer invasion and metastasis [2].

#### **4.10. Apoptosis induction**

The activation of apoptosis in preneoplastic cells is one of the crucial mechanisms of cancer chemoprevention. Polyphenolic compounds have been shown to induce selective promotion of apoptosis in cancerous or precancerous cells, by affecting different cellular mechanisms [2].

#### **5. Which are the phytochemicals with antitumor activity?**

Plant secondary metabolites and their semisynthetic derivatives are playing today an impor‐ tant role in anticancer drug therapy. In this chapter, the main classes of natural compounds are presented, which have been identified to contain substances with antitumor activity.

#### **5.1. Terpenes**

Terpenes are a large and diverse class of organic compounds, often strong-smelling, produced by a variety of plants, particularly conifers. Many terpenes are aromatic hydrocarbons and thus may have had a protective function. The difference between terpenes and terpenoids is that terpenes are hydrocarbons, whereas terpenoids contain additional functional groups. Terpenes are derived biosynthetically from units of isoprene, which has the molecular formula C5H8. The basic molecular formulae of terpenes are multiples of (C5H8)*n*, where *n* is the number of linked isoprene units.

**• Diterpenes are** composed of four isoprene units and have the molecular formula C20H32. Taxanes are diterpenes produced by the plants of the genus *Taxus* and are widely used as chemotherapy agents although present difficulties in formulation as medicines because they are poorly soluble in water. The principal mechanism of action of the taxane class of drugs is inhibition of the process of mitosis [26]. Taxane agents include taxol (or paclitaxel) and docetaxel (Figure 1).

tumor cells to antitumor drugs [25]. **4.9. Suppression of angiogenesis** 

**4.10. Apoptosis induction** 

**5.1. Terpenes** 

consequently, cancer invasion and metastasis [2].

Multidrug resistance is a significant challenge in the treatment of infectious diseases and cancer. The antitumor treatments currently in use often fail at some stage of the sickness because many types of cancers develop resistance to chemotherapeutic drugs. Some plants can change the resistance of

Angiogenesis is the process of formation of new vessels (vascularization) from the preexisting microvascular network. This is as an essential step in tumor dissemination and formation of metastases. Many plant-derived phenolics have been reported to restrain angiogenesis and,

The activation of apoptosis in preneoplastic cells is one of the crucial mechanisms of cancer chemoprevention. Polyphenolic compounds have been shown to induce selective promotion of

Plant secondary metabolites and their semisynthetic derivatives are playing today an important role in anticancer drug therapy. In this chapter, the main classes of natural compounds are presented,

Terpenes are a large and diverse class of organic compounds, often strong-smelling, produced by a variety of plants, particularly conifers. Many terpenes are aromatic hydrocarbons and thus may have had a protective function. The difference between terpenes and terpenoids is that terpenes are hydrocarbons, whereas terpenoids contain additional functional groups. Terpenes are derived biosynthetically from units of isoprene, which has the molecular formula C5H8. The basic molecular formulae of terpenes are multiples of (C5H8)*n*, where *n* is the number of linked isoprene units. **Diterpenes are** composed of four isoprene units and have the molecular formula C20H32. Taxanes are diterpenes produced by the plants of the genus *Taxus* and are widely used as chemotherapy agents

The principal mechanism of action of the taxane class of drugs is inhibition of the process of mitosis

apoptosis in cancerous or precancerous cells, by affecting different cellular mechanisms [2].

**5. Which are the phytochemicals with antitumor activity?** 

which have been identified to contain substances with antitumor activity.

[26]. Taxane agents include taxol (or paclitaxel) and docetaxel (Figure 1).

**Figure 1.** Taxol (or paclitaxel, left) and docetaxel (right). Taxol, originally isolated in 1971 from the bark of Pacific Yew, *Taxus brevifolia*, is a strong **Figure 1.** Taxol (or paclitaxel, left) and docetaxel (right).

anticancer drug approved by the U.S. FDA to treat a variety of tumors, including breast, ovarian, and AIDS-related Kaposi's sarcoma, among others. Among the conifers apart from the genus *Taxus*, only two other species have been reportedly claimed to produce taxanes: *T. brevifolia* and *Taxus baccata*  or the European yew. **Triterpenes and triterpenoids**  Triterpenes consist of *six isoprene* units and have the molecular formula C30H48. Triterpenes are widespread in nature and are highly abundant in medicinal plants, especially in the leaves, bark, Taxol, originally isolated in 1971 from the bark of Pacific Yew, *Taxus brevifolia*, is a strong anticancer drug approved by the U.S. FDA to treat a variety of tumors, including breast, ovarian, and AIDS-related Kaposi's sarcoma, among others. Among the conifers apart from the genus *Taxus*, only two other species have been reportedly claimed to produce taxanes: *T. brevifolia* and *Taxus baccata* or the European yew.

#### fruits, and seeds of the herbs. The pentacyclic triterpenes can be classified into lupane, oleanane, or **• Triterpenes and triterpenoids**

5 ursane groups. Triterpenoids are structurally diverse organic compounds, more than 20,000 known Triterpenes consist of *six isoprene* units and have the molecular formula C30H48. Triterpenes are widespread in nature and are highly abundant in medicinal plants, especially in the leaves, bark, fruits, and seeds of the herbs. The pentacyclic triterpenes can be classified into lupane, oleanane, or ursane groups. Triterpenoids are structurally diverse organic compounds, more than 20,000 known members, widespread in nature. Several triterpenoids, such as ursolic and oleanolic acid, betulinic acid, and lupeol, possess antioxidative and anti-inflammatory, antidiabetic properties, and have been suggested to be potentially promising anticancer agents. Triterpenoids exist in free form or combined with sugar into glycosides—the triterpenoid saponins. In anticancer mechanisms, triterpenoids are multitargeted agents that induce apoptosis, inhibit cell proliferation, suppress angiogenesis, cause mitochondrial dysfunction, and modulate genes and proteins [27].


Phytochemicals in Antitumor Herbs and Herbal Formulas http://dx.doi.org/10.5772/60422 53

**Figure 2.** β-Boswellic acid.

5

**Figure 1.** Taxol (or paclitaxel, left) and docetaxel (right). Taxol, originally isolated in 1971 from the bark of Pacific Yew, *Taxus brevifolia*, is a strong anticancer drug approved by the U.S. FDA to treat a variety of tumors, including breast, ovarian, and AIDS-related Kaposi's sarcoma, among others. Among the conifers apart from the genus *Taxus*, only two other species have been reportedly claimed to produce taxanes: *T. brevifolia* and *Taxus baccata* 

Taxol, originally isolated in 1971 from the bark of Pacific Yew, *Taxus brevifolia*, is a strong anticancer drug approved by the U.S. FDA to treat a variety of tumors, including breast, ovarian, and AIDS-related Kaposi's sarcoma, among others. Among the conifers apart from the genus *Taxus*, only two other species have been reportedly claimed to produce taxanes: *T.*

Triterpenes consist of *six isoprene* units and have the molecular formula C30H48. Triterpenes are widespread in nature and are highly abundant in medicinal plants, especially in the leaves, bark, fruits, and seeds of the herbs. The pentacyclic triterpenes can be classified into lupane, oleanane, or ursane groups. Triterpenoids are structurally diverse organic compounds, more than 20,000 known members, widespread in nature. Several triterpenoids, such as ursolic and oleanolic acid, betulinic acid, and lupeol, possess antioxidative and anti-inflammatory, antidiabetic properties, and have been suggested to be potentially promising anticancer agents. Triterpenoids exist in free form or combined with sugar into glycosides—the triterpenoid saponins. In anticancer mechanisms, triterpenoids are multitargeted agents that induce apoptosis, inhibit cell proliferation, suppress angiogenesis, cause mitochondrial dysfunction,

**• Boswellic acid** (Figure 2), a triterpene isolated from Boswellia serrata (native in India and Pakistan), has been found to possess potent anti-inflammatory and anticancer activity. Boswellic acid have been used to treat Crohn disease, ulcerative colitis, bronchial asthma, endotoxin-induced hepatitis, and arthritis. Boswellic acid is a mixture of four major pentacyclic triterpene acids. Beta-boswellic acid (Figure 2), keto-beta-boswellic acid, and acetyl-keto-beta-boswellic acid have been indicated in apoptosis of cancer cells, in particu‐ lar, brain tumors and cells affected by leukemia or colon cancer, melanoma, hepatoma, and

**• Oleanolic acid** (Figure 3) is a triterpenoid widely found in several dietary and medicinal plants. Oleanolic acid is abundant in ginseng root and in olive plant (*Olea europaea*), which is the primary commercial source for the compound. Oleanolic acid can be easily obtained in high yield from olive pulp remaining after crushing of the olive fruit and also from olive

Triterpenes consist of *six isoprene* units and have the molecular formula C30H48. Triterpenes are widespread in nature and are highly abundant in medicinal plants, especially in the leaves, bark, fruits, and seeds of the herbs. The pentacyclic triterpenes can be classified into lupane, oleanane, or ursane groups. Triterpenoids are structurally diverse organic compounds, more than 20,000 known

Multidrug resistance is a significant challenge in the treatment of infectious diseases and cancer. The antitumor treatments currently in use often fail at some stage of the sickness because many types of cancers develop resistance to chemotherapeutic drugs. Some plants can change the resistance of

Angiogenesis is the process of formation of new vessels (vascularization) from the preexisting microvascular network. This is as an essential step in tumor dissemination and formation of metastases. Many plant-derived phenolics have been reported to restrain angiogenesis and,

The activation of apoptosis in preneoplastic cells is one of the crucial mechanisms of cancer chemoprevention. Polyphenolic compounds have been shown to induce selective promotion of

Plant secondary metabolites and their semisynthetic derivatives are playing today an important role in anticancer drug therapy. In this chapter, the main classes of natural compounds are presented,

Terpenes are a large and diverse class of organic compounds, often strong-smelling, produced by a variety of plants, particularly conifers. Many terpenes are aromatic hydrocarbons and thus may have had a protective function. The difference between terpenes and terpenoids is that terpenes are hydrocarbons, whereas terpenoids contain additional functional groups. Terpenes are derived biosynthetically from units of isoprene, which has the molecular formula C5H8. The basic molecular formulae of terpenes are multiples of (C5H8)*n*, where *n* is the number of linked isoprene units. **Diterpenes are** composed of four isoprene units and have the molecular formula C20H32. Taxanes are diterpenes produced by the plants of the genus *Taxus* and are widely used as chemotherapy agents although present difficulties in formulation as medicines because they are poorly soluble in water. The principal mechanism of action of the taxane class of drugs is inhibition of the process of mitosis

apoptosis in cancerous or precancerous cells, by affecting different cellular mechanisms [2].

**5. Which are the phytochemicals with antitumor activity?** 

which have been identified to contain substances with antitumor activity.

[26]. Taxane agents include taxol (or paclitaxel) and docetaxel (Figure 1).

tumor cells to antitumor drugs [25]. **4.9. Suppression of angiogenesis** 

**4.10. Apoptosis induction** 

**5.1. Terpenes** 

or the European yew.

**• Triterpenes and triterpenoids**

and modulate genes and proteins [27].

prostate cancer [28].

leaves.

**Figure 1.** Taxol (or paclitaxel, left) and docetaxel (right).

52 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Triterpenes and triterpenoids** 

*brevifolia* and *Taxus baccata* or the European yew.

consequently, cancer invasion and metastasis [2].

**Figure 3.** Structure of oleanolic, ursolic acid and betulinic acid.

Oleanolic acid exists in nature as free acid but also serves as an aglycone of triterpenoid saponins, linked with one or more sugar moieties to form glycosides. Effects of oleanolic acid on cancer cells have been demonstrated in chemoprevention and therapy of breast, colorectal, non-small cell lung, epithelial ovarian, pancreatic, prostate cancer, and melanoma. Oleanolic acid inhibited the proliferation of cancer cells and induced apoptosis via ROS-mediated mitochondrial mechanism [29].


pharmacological properties, many derivatives were synthesized and evaluated for cytotoxic activity [30-33].

#### **• Triterpenic saponins**

Saponins are a group of naturally occurring plant glycosides, characterized by their strong foam-forming properties in aqueous solution. Originally named for soapwort plants (*Sapona‐ ria* spp.), saponins consist of a hydrophobic aglycone linked to a hydrophilic carbohydrate. The presence of saponins has been reported in more than 100 families of plants, out of which at least 150 kinds of natural saponins have been found to possess significant anticancer properties. Saponins are common in a variety of higher plants and usually found in roots, tubers, leaves, blooms, or seeds. The amphipathic nature of the class gives them activity as surfactants that can be used to enhance penetration of macromolecules such as proteins through cell membranes. Saponins have also been used as adjuvant in vaccines [34]. Based on the carbon skeletons, saponins were classified into triterpenes and steroids. Their glycone parts were mostly oligosaccharides, arranged either in a linear or branched fashion, attached to hydroxyl groups through an acetal linkage. The considerable variety of aglycones, carbohy‐ drates, and their different attachments results in many different saponins. Modern research found that saponins have antitumor effect on many cancer cells at least by cell cycle arrest and apoptosis. Meanwhile, saponins in combination with conventional tumor treatment strategies result in improved therapeutic success [35].


#### **5.2. Phenols and polyphenols**

Phenolic phytochemicals represent the largest category of phytochemicals widespread in different fruits and vegetables and one of the major groups of secondary metabolites in plants. Numerous phenolic compounds have been reported to demonstrate selective activity, destroying cancer cells without damaging normal cells. Many clinical trials have been made on cancer prevention of breast, colon, gastric, reproductive, head and neck, and prostate cancers by using plant polyphenols such as epigallocatechin-3-gallate, curcumin, resveratrol, genistein, and quercetin [2, 36].

Most of the studies with plant polyphenols showed that cancer-preventing mechanisms include antioxidant activity (the strength of this property is influenced by the number and location of hydroxyl groups, the size and shape of molecules, and steric properties), radical scavenging activity, inactivation of carcinogenic substances, antiproliferation, cell cycle arrest, induction of apoptosis and differentiation, inhibition of angiogenesis, modulation of tumor suppression genes, and others [36].

Plant polyphenols are divided into two major groups (Table 1)—flavonoids (chalcones, flavanols, flavones, flavanones, isoflavones, and anthocyanins) and nonflavonoids (phenolic acids, e.g., caffeic acid, gallic acid; stilbenes, e.g., trans-resveratrol, tannins, lignins) [21]. Flavonoids and phenolic acids account for the majority (60% and 30%, respectively) of total dietary polyphenols. The average daily intake of flavonoids alone is 1-2 g [2].


**Table 1.** Main classes of plant polyphenols

pharmacological properties, many derivatives were synthesized and evaluated for cytotoxic

Saponins are a group of naturally occurring plant glycosides, characterized by their strong foam-forming properties in aqueous solution. Originally named for soapwort plants (*Sapona‐ ria* spp.), saponins consist of a hydrophobic aglycone linked to a hydrophilic carbohydrate. The presence of saponins has been reported in more than 100 families of plants, out of which at least 150 kinds of natural saponins have been found to possess significant anticancer properties. Saponins are common in a variety of higher plants and usually found in roots, tubers, leaves, blooms, or seeds. The amphipathic nature of the class gives them activity as surfactants that can be used to enhance penetration of macromolecules such as proteins through cell membranes. Saponins have also been used as adjuvant in vaccines [34]. Based on the carbon skeletons, saponins were classified into triterpenes and steroids. Their glycone parts were mostly oligosaccharides, arranged either in a linear or branched fashion, attached to hydroxyl groups through an acetal linkage. The considerable variety of aglycones, carbohy‐ drates, and their different attachments results in many different saponins. Modern research found that saponins have antitumor effect on many cancer cells at least by cell cycle arrest and apoptosis. Meanwhile, saponins in combination with conventional tumor treatment strategies

**• Oleananes** are the most common saponins in nature. Their antitumor effect worked through various pathways, such as anticancer, antimetastasis, immunostimulation, chemopreven‐ tion, etc. Some of the most studied triterpene saponins are soybean saponin, saikosaponins,

**• Cycloartane saponins** could be used as chemotherapeutic agent in the treatment of tumors. For example, total Astragalus saponins possess antitumor properties in human colon cancer cells. In addition, Astragalus saponins could be used as an adjuvant in combination with other orthodox chemotherapeutic drugs to reduce the side effects of the latter compounds

Phenolic phytochemicals represent the largest category of phytochemicals widespread in different fruits and vegetables and one of the major groups of secondary metabolites in plants. Numerous phenolic compounds have been reported to demonstrate selective activity, destroying cancer cells without damaging normal cells. Many clinical trials have been made on cancer prevention of breast, colon, gastric, reproductive, head and neck, and prostate cancers by using plant polyphenols such as epigallocatechin-3-gallate, curcumin, resveratrol,

Most of the studies with plant polyphenols showed that cancer-preventing mechanisms include antioxidant activity (the strength of this property is influenced by the number and location of hydroxyl groups, the size and shape of molecules, and steric properties), radical scavenging activity, inactivation of carcinogenic substances, antiproliferation, cell cycle arrest,

activity [30-33]. **• Triterpenic saponins**

result in improved therapeutic success [35].

54 Phytochemicals - Isolation, Characterisation and Role in Human Health

avicins, and tubeimoside [35].

**5.2. Phenols and polyphenols**

genistein, and quercetin [2, 36].

[35].

**Table 2.** Chemical structure of some plant polyphenols with antitumor activity

Flavonoids commonly share the same generic structure, the flavan nucleus consisting of two aromatic rings linked by a pyran ring. Differences in the location of the right phenolic ring to pyran ring make it possible to distinguish between flavonoids (2-phenylbenzopyrans) and isoflavonoids (3-phenylbenzopyrans). 2-Phenylbenzopyran group may be further divided into 3-hydroxyflavonoids (flavonols, flavanols, and anthocyanidins) and flavonoids without substituent at C3 (flavanones and flavones). Flavones differ from flavanones by a C2-C3 double bond [19].

#### *5.2.1. Flavonoids as anticancer agents*

**(+) - Catechin**

56 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Epigallocatechin gallate**

**(-) - Epicatechin**

**Apigenin**

**Luteolin Wogonin**

**Baicalein Quercetin**

**Myricetin Kaempferol**

**Genistein Daidzein**

#### **• Chalcones as anticancer agents**

Chalcone (Table 2) is an aromatic ketone that forms the central core for a variety of important biological compounds, which are known collectively as chalcones or chalconoid. They are a group of plant-derived polyphenolic compounds belonging to the flavonoids family even if the chroman ring is not yet formed (they are precursors of flavonoid structure). Some of the most significant chalcones identified from plants include flavokawin, butein, xanthoangelol, 4-hydroxyderricin, cardamonin, 2′,4′-dihydroxychalcone, and naringenin chalcone (phlore‐ tin). These chalcones have been linked with immunomodulation, antibacterial, antiviral, antiinflammatory, antioxidant, and anticancer activities [36].

#### **• Anthocyanins as anticancer agents**

Anthocyanins (also named anthocyans; Table 2) are water-soluble vacuolar pigments that may appear red, purple, or blue depending on the pH. Anthocyanins occur in all tissues of higher plants, including leaves, stems, roots, flowers, and fruits. Dietary intake of anthocyanins has been estimated at up to 200 mg/day, which is higher than other flavonoids. More than 550 different anthocyanins are discovered until now. Anthocyanins are derived from anthocyani‐ dins by adding pendant sugars and are mostly 3-glucosides of the anthocyanidins. From various studies, it is found that some plants or their parts containing anthocyanins have anticancer property, and their analogues may be helpful in synthesizing newer effective anticancer agents in future. The number of hydroxyl groups and presence of sugar moiety is crucial for the specific modulatory actions of anthocyanins. Numerous *in vitro* and *in vivo* studies showed that anthocyanins can affect basic cell functions related to cancer development. They may inhibit the formation and growth of tumors by induction of cell cycle arrest and apoptosis, thus eliminating damaged cells or tumor cells [36].

#### **• Flavanones as anticancer agents**

The consumption of citrus fruits and juices has been widely investigated for its possible role in the prevention of cardiovascular disease and cancer. These beneficial effects are mainly attributed to flavanones, the typical polyphenols of citrus species. Major flava‐ nones in plant species include hesperetin, naringenin, eriodictyol, isosakuranetin, and their respective glycosides. Hesperetin and its derivatives are characteristic flavanones of sweet orange, tangelo, lemon, and lime, while naringenin and its derivatives are those of grapefruit and sour orange. The major citrus flavanones can be effective in fighting carcinogenesis by minimizing DNA damage (protecting effect of naringenin against UV-induced damage of DNA), tumor development, and proliferation [19].

Naringenin (Table 2) is found in high concentrations in citrus fruit while low concentrations are also found in tomatoes and their products. Naringenin can be found as aglycone and/or as glycosides, such as naringin (naringenin-7-neohesperidoside) and narirutin (naringenin-7 rutinoside). Naringenin was successfully investigated for its cell antiproliferation effect on colon cancer cell line. In a comparative study, flavanones showed a significant antiproliferative activity against lung, colon, breast, prostate, and melanoma cancerous cell lines [19].

Hesperetin (Table 2) and its glycosides are also mainly present in citrus fruit. The aglycone is less dominant in nature than the glycosides. The most widely distributed glycosides of hesperetin are hesperidin (hesperetin-7-rutinoside) and neohesperidin (hesperetin-7-neohes‐ peridoside), which are conjugates with rutinose and neohesperidose, respectively [19]. Recent study shows that hesperetin exhibits a potential anticancer activity against human cervical cancer cell lines *in vitro* through the reduction in cell viability and the induction of apoptosis. Altogether, these data sustain our contention that hesperetin has anticancer properties and merits further investigation as a potential therapeutic agent [37].

#### **• Flavanols as anticancer agents**

Flavan-3-ols (flavanols) possess two benzene rings (called the A- and B-rings) and a dihydro‐ pyran heterocycle (the C-ring) with a hydroxyl group on carbon 3 and have been shown to have the ability to scavenge free radicals, reduce the rate of LDL oxidation, inhibit lipid peroxidation, modify enzymes that activate or detoxify, and participate in the modulation of the immune response in several biological systems. The hydroxylation pattern of the B-ring appears to have a critical influence on their activities, particularly in the inhibition of protein kinase, which can induce antiproliferate activities [38]. **Catechins** belong to flavan-3-ols and there are two chiral centers on the molecule on carbons 2 and 3 (they are four diastereoisomers). Two of the isomers are in *trans* configuration and are called catechin, and the other two are in *cis* configuration and are called epicatechin. The most common catechin isomer is the (+) catechin (Table 2). The most common epicatechin isomer is (**-**)-epicatechin (Table 2). Catechins and epicatechins are found in cocoa (108 mg/100 g), prune juice (25 mg/100 ml) and broad bean pod (16 mg/100 g), açaí oil (67 mg/kg), argan oil, barley grain, peaches, green tea, and vinegar [39-42].

**• Anthocyanins as anticancer agents**

58 Phytochemicals - Isolation, Characterisation and Role in Human Health

**• Flavanones as anticancer agents**

apoptosis, thus eliminating damaged cells or tumor cells [36].

DNA), tumor development, and proliferation [19].

Anthocyanins (also named anthocyans; Table 2) are water-soluble vacuolar pigments that may appear red, purple, or blue depending on the pH. Anthocyanins occur in all tissues of higher plants, including leaves, stems, roots, flowers, and fruits. Dietary intake of anthocyanins has been estimated at up to 200 mg/day, which is higher than other flavonoids. More than 550 different anthocyanins are discovered until now. Anthocyanins are derived from anthocyani‐ dins by adding pendant sugars and are mostly 3-glucosides of the anthocyanidins. From various studies, it is found that some plants or their parts containing anthocyanins have anticancer property, and their analogues may be helpful in synthesizing newer effective anticancer agents in future. The number of hydroxyl groups and presence of sugar moiety is crucial for the specific modulatory actions of anthocyanins. Numerous *in vitro* and *in vivo* studies showed that anthocyanins can affect basic cell functions related to cancer development. They may inhibit the formation and growth of tumors by induction of cell cycle arrest and

The consumption of citrus fruits and juices has been widely investigated for its possible role in the prevention of cardiovascular disease and cancer. These beneficial effects are mainly attributed to flavanones, the typical polyphenols of citrus species. Major flava‐ nones in plant species include hesperetin, naringenin, eriodictyol, isosakuranetin, and their respective glycosides. Hesperetin and its derivatives are characteristic flavanones of sweet orange, tangelo, lemon, and lime, while naringenin and its derivatives are those of grapefruit and sour orange. The major citrus flavanones can be effective in fighting carcinogenesis by minimizing DNA damage (protecting effect of naringenin against UV-induced damage of

Naringenin (Table 2) is found in high concentrations in citrus fruit while low concentrations are also found in tomatoes and their products. Naringenin can be found as aglycone and/or as glycosides, such as naringin (naringenin-7-neohesperidoside) and narirutin (naringenin-7 rutinoside). Naringenin was successfully investigated for its cell antiproliferation effect on colon cancer cell line. In a comparative study, flavanones showed a significant antiproliferative

Hesperetin (Table 2) and its glycosides are also mainly present in citrus fruit. The aglycone is less dominant in nature than the glycosides. The most widely distributed glycosides of hesperetin are hesperidin (hesperetin-7-rutinoside) and neohesperidin (hesperetin-7-neohes‐ peridoside), which are conjugates with rutinose and neohesperidose, respectively [19]. Recent study shows that hesperetin exhibits a potential anticancer activity against human cervical cancer cell lines *in vitro* through the reduction in cell viability and the induction of apoptosis. Altogether, these data sustain our contention that hesperetin has anticancer properties and

Flavan-3-ols (flavanols) possess two benzene rings (called the A- and B-rings) and a dihydro‐ pyran heterocycle (the C-ring) with a hydroxyl group on carbon 3 and have been shown to

activity against lung, colon, breast, prostate, and melanoma cancerous cell lines [19].

merits further investigation as a potential therapeutic agent [37].

**• Flavanols as anticancer agents**

The results of a recent study show that catechin present in the extract of *Ligaria cuneifolia,* a hemiparasite species that belongs to Argentine flora, can reduce proliferation and induces apoptosis of lymphoma cell line [38]. The recent data show that catechins also affect the molecular mechanisms involved in angiogenesis, regulation of cell death and multidrug resistance in cancer. Catechins present antioxidant activity by scavenging free radicals, chelating redox active transition-metal ions, inhibiting redox active transcription factors, inhibiting pro-oxidant enzymes, and inducing antioxidant enzymes [43].

Tea (*Camellia sinensis*), one of the most popular beverages in the world, is manufactured as black (78%), green (20%), or oolong tea (2%) [43]. Catechins are the major group of bioactive compounds found in green tea, meaning about 25% of the dry weight of fresh tea leaf [44], which contains characteristic polyphenolic compounds, such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC). The chemopreventive properties of this beverage are mostly connected with EGCG that EGCG and includes induction of apoptosis, promotion of cell growth arrest, inhibition of cell proliferation and transformation, inhibition of angiogenesis, and metastasis [2]. Tea polyphenols inhibited in experimental studies the growth of lung, mammary, and stom‐ ach human cancer cell lines [45].

Also, red wine containing higher levels of catechin showed significant reducing powers, thereby confirming the antioxidant potential of red wine due to catechin content. It is obvious that drinking green tea or red wine helps in improving the general well-being in humans.

Epigallocatechin gallate (EGCG, Table 2), also known as epigallocatechin 3-gallate, is the ester of epigallocatechin and gallic acid. EGCG has been shown to inhibit the growth of different human hepatoma cell lines at concentrations of 50-100 μg/ml and increased tumor necrosis [21]. Various clinical studies have revealed that treatment by EGCG inhibits tumor incidence and multiplicity in different organ sites such as liver, stomach, skin, lung, mammary gland, and colon. Preclinical research data in recent studies show promising results, and EGCG has great potential in cancer prevention because of its safety, low cost, and bioavailability [36].

Theaflavins are antioxidant polyphenols that are formed from the condensation of flavan-3 ols in tea leaves during the enzymatic oxidation (fermentation) of black tea [46]. Theaflavin-3gallate, theaflavin-3′-gallate, and theaflavin-3-3′-digallate are the principle of theaflavins. Previous research studies led in mice showed beneficial inhibitory effects of topically applied black tea theaflavins (e.g., theaflavin-3,3′-digallate) providing a good promise for chemopre‐ vention (e.g., decreased occurrence of melanoma and nonmelanomas such as squamous cell carcinoma and basal cell carcinoma) [47].

#### **• Flavones as anticancer agents**

Apigenin (Table 2) is a flavonoid present in various fruits, vegetables, and spices like onions, parsley, oranges, thyme, celery, and sweet red pepper. It possesses several chemopreventive properties, including induction of apoptosis, inhibition of cancer cells proliferation, and involvement in angiogenesis [2].

Luteolin (Table 2) is most often found in leaves, but it is also seen in celery, parsley, broccoli, onion, carrots, peppers, cabbages, apples, thyme, dandelion, chamomile tea, carrots, green pepper, olive oil, peppermint, rosemary, oranges, and oregano. The chemopreventive mech‐ anism of action of luteolin includes induction of apoptosis, inhibition of proliferation of cancer cells, and suppression of angiogenesis and metastasis. Luteolin has been reported to inhibit growth of tumors in human skin, hepatoma, and ovarian cancer cells; in lung and breast cancer, luteolin has been shown to inhibit the invasion of cancer cells [2]. Among 68 plant polyphenols examined for their inhibitory ability against hepatocellular carcinoma cells, luteolin was one of the most potent [21].

Wogonin, baicalein (Table 2), and baicalin (baicalein 7-*O*-glucuronide), all naturally occurring flavonoid extracted from *Scutellaria baicalensis Georgi* (*Lamiaceae*), were reported to have potential antitumor effects in several studies. These phytochemicals are not only cytostatic but also cytotoxic to various human tumor cell lines *in vitro* and inhibit tumor growth *in vivo*. Numerous studies demonstrated that wogonin had a cytostatic effect due to the apoptotic cell death on various tumor cell lines (such as sarcoma, hepatoma cells, breast cancer, and nasopharyngeal carcinoma cells) and antimetastatic ability as well [48, 49].

#### **• Flavonols**

Quercetin (Table 2) is the most abundant flavonoid in foods. It is present in different fruits (apples, berries, and grapes), vegetables (onions, broccoli), as well as in tea and red wine, mostly in glycosylated forms. The daily intake of quercetin has been estimated at 30 mg. Quercetin is probably the most studied flavonoids, and antioxidant properties are known as outstanding. This flavonol was found to have antiproliferative activity in many situations, such as adenomatous polyposis [36] and breast cancer [2].

Myricetin (Table 2) is the next flavonol occurring in fruits, vegetables, or red wine exerting anticancer activity. In lung cancer, this compound has been observed to block invasion and migration of human lung adenocarcinoma cells. According to the study with human leukemia cell line, myricetin induced apoptosis in these cells [2].

Kaempferol (Table 2) is another flavonol present in tea, broccoli, grapefruit, brussels sprouts, apples, and other plants. This compound exerts antioxidant, anti-inflammatory, and anticancer effects on several types of cancer. Kaempferol has been reported to inhibit the invasion of the human invasive breast carcinoma cell line, angiogenesis, and human ovarian cancer cells. The available data indicate that kaempferol could be a potential chemopreventive agent against various cancers such as lung, colon, prostate, liver, pancreas, and skin [2].

#### **• Isoflavones as anticancer agents**

gallate, theaflavin-3′-gallate, and theaflavin-3-3′-digallate are the principle of theaflavins. Previous research studies led in mice showed beneficial inhibitory effects of topically applied black tea theaflavins (e.g., theaflavin-3,3′-digallate) providing a good promise for chemopre‐ vention (e.g., decreased occurrence of melanoma and nonmelanomas such as squamous cell

Apigenin (Table 2) is a flavonoid present in various fruits, vegetables, and spices like onions, parsley, oranges, thyme, celery, and sweet red pepper. It possesses several chemopreventive properties, including induction of apoptosis, inhibition of cancer cells proliferation, and

Luteolin (Table 2) is most often found in leaves, but it is also seen in celery, parsley, broccoli, onion, carrots, peppers, cabbages, apples, thyme, dandelion, chamomile tea, carrots, green pepper, olive oil, peppermint, rosemary, oranges, and oregano. The chemopreventive mech‐ anism of action of luteolin includes induction of apoptosis, inhibition of proliferation of cancer cells, and suppression of angiogenesis and metastasis. Luteolin has been reported to inhibit growth of tumors in human skin, hepatoma, and ovarian cancer cells; in lung and breast cancer, luteolin has been shown to inhibit the invasion of cancer cells [2]. Among 68 plant polyphenols examined for their inhibitory ability against hepatocellular carcinoma cells, luteolin was one

Wogonin, baicalein (Table 2), and baicalin (baicalein 7-*O*-glucuronide), all naturally occurring flavonoid extracted from *Scutellaria baicalensis Georgi* (*Lamiaceae*), were reported to have potential antitumor effects in several studies. These phytochemicals are not only cytostatic but also cytotoxic to various human tumor cell lines *in vitro* and inhibit tumor growth *in vivo*. Numerous studies demonstrated that wogonin had a cytostatic effect due to the apoptotic cell death on various tumor cell lines (such as sarcoma, hepatoma cells, breast cancer, and

Quercetin (Table 2) is the most abundant flavonoid in foods. It is present in different fruits (apples, berries, and grapes), vegetables (onions, broccoli), as well as in tea and red wine, mostly in glycosylated forms. The daily intake of quercetin has been estimated at 30 mg. Quercetin is probably the most studied flavonoids, and antioxidant properties are known as outstanding. This flavonol was found to have antiproliferative activity in many situations, such

Myricetin (Table 2) is the next flavonol occurring in fruits, vegetables, or red wine exerting anticancer activity. In lung cancer, this compound has been observed to block invasion and migration of human lung adenocarcinoma cells. According to the study with human leukemia

Kaempferol (Table 2) is another flavonol present in tea, broccoli, grapefruit, brussels sprouts, apples, and other plants. This compound exerts antioxidant, anti-inflammatory, and anticancer effects on several types of cancer. Kaempferol has been reported to inhibit the invasion of the

nasopharyngeal carcinoma cells) and antimetastatic ability as well [48, 49].

as adenomatous polyposis [36] and breast cancer [2].

cell line, myricetin induced apoptosis in these cells [2].

carcinoma and basal cell carcinoma) [47].

60 Phytochemicals - Isolation, Characterisation and Role in Human Health

**• Flavones as anticancer agents**

involvement in angiogenesis [2].

of the most potent [21].

**• Flavonols**

Isoflavone (3-phenyl-4*H*-1-benzopyr-4-one) differs from flavone in location of the phenyl group. Most members of the *Fabaceae* family contain significant quantities of isoflavones. Genistein and daidzein (Table 2), the most prevalent compounds of isoflavonoid class, were found in various legumes including soybean, green bean, alfalfa sprout, mung bean sprout, cowpea, kudzu root, and red clover sprout. Due to the estrogen-like structure of isoflavones found in soybeans and the known role of estrogens in breast carcinogenesis, most soy research has focused on the hormonal activity of these compounds [50]. Some studies showed that daidzein can inhibit hepatoma cell growth and induce apoptosis [21]. Regarding genistein, the major isoflavone in soy, laboratory studies have demonstrated different mechanisms of action, including antiproliferative activity and induction of apoptosis in both animal and human cell lines. In addition, genistein possesses antioxidant activity and the ability to scavenge free radicals, protecting the body against oxidative DNA damage. The chemopreventive activity of genistein has been investigated mainly against breast and prostate cancer.

#### **• Influence of glycosilation in flavonoid family**

The dietary flavonoids in nature almost all exist as their O/C-glycosides, such as glucoside, galactoside, rhamnoside, arabinoside, and rutinoside. The most abundant flavonoid glyco‐ sides in plants are flavone and flavonol O-glycosides. The glycosidation are found mainly as their 3 or 7 O-glycosides, although the 5, 8, and 4′ O-glycosides were also reported in some cases. Regarding the antioxidant and antitumoral properties, it was illustrated that the flavonoid aglycones showed higher anticancer potential than their glycosides in cell level; e.g., rutin did not inhibit cell proliferation of any of the cancer cell lines tested; C/O-glycosylation of apigenin significantly weakened the inhibition of cancer cells; genistein and daidzein glycosides exhibited no noticeable activity on human breast carcinoma cell. It was demon‐ strated that cellulase can remarkably transform baicalin and wogonoside to their aglycones (baicalein and wogonin) with enhance of antiproliferative effects [51]. Generally, glycosylation reduced the antiproliferative activity in flavonoid classes, and a C2-C3 double bond seems important for the antiproliferative activity of flavonoids, and indeed flavones are typically more potent that flavanones [19].

#### *5.2.2. Nonflavonoids as anticancer agents*

#### **• Stilbenoids**

Stilbenoids are hydroxylated derivatives of stilbene with a C6-C2-C6 structure. They are secondary metabolites occurring naturally in various families of plants; grapes and related products are considered the most important dietary sources of these substances. Transresveratrol (3,4′,5-trihydroxystilbene, Table 2), a hydroxylated stilbene, is today a well-known chemopreventive substance for cardiovascular diseases, antiaging, antiviral, and several malignant neoplasms [36]. High amounts of trans-resveratrol can be found in grapes and grape skins used for wine production, raspberries, mulberries, blueberries cranberries, peanuts, and certain types of pine [21]. All anticancer data obtained on preclinical animal studies showed that resveratrol affects all three discrete stages of carcinogenesis (initiation, promotion, and progression) by modulating signal transduction pathways that control cell division and growth, apoptosis, inflammation, angiogenesis, and metastasis [36]. A synthetic stilbene derivative, tamoxifen, is currently used for the treatment of several types of breast cancer in women, and as a hormone treatment for male breast cancer [52].

#### **• Caffeic acid**

Caffeic acid (Table 2) is a hydroxycinnamic acid found in most plants, including coffee beans, nuts, berries, and grains. Several studies have investigated the antitumor potential and hepatoprotective activity of the caffeic acid derivative, which have been identified in particular in honey and propolis [21]. Data showed that caffeic acid phenethyl ester (CAPE), a component of propolis, induces cell cycle arrest and has antiproliferation effect on glioma cells *in vitro* and *in vivo*. In addition, CAPE inhibited the metastasis of glioma cells [53].

Caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE), a natural polyphenol from *Sarcandra glabra*, has potent *in vitro* anticancer activity through multiple targets. This compound significantly decreased tumor growth in hepatoma and sarcoma tumor-bearing mice, and also significantly inhibited ascites development. CADPE did not show any toxicity *in vivo* and anticancer efficacies were equivalent to those of 5-fluorouracil and cyclophosphamide (drugs in cancer treatment). The tested 59 human cancer cell lines from leukemia and nine different solid tumors, including colon cancer, gastric cancer, breast cancer, CNS cancer, ovarian cancer, melanoma, lung cancer, renal cancer, and prostate cancer, are sensitive to CADPE that suppresses tumor growth and angiogenesis [54].

#### **• Protocatechuic acid**

Protocatechuic acid (Table 2), a dihydroxybenzoic acid widely distributed in the plant kingdom, is the major metabolite of antioxidant polyphenols found in green tea. Protocate‐ chuic acid has antioxidant and anti-inflammatory activity and has been shown to inhibit HepG2 (human liver carcinoma cell line) cell growth and induce apoptosis [21].

#### **• Curcuminoids**

A curcuminoid is a linear diarylheptanoid, with molecules such as curcumin (Table 2) or derivatives of curcumin with different chemical groups that confers increase solubility and make them suitable for drug formulation. Curcumin is the principal curcuminoid of the spice turmeric, which is a member of the ginger family. It has been traditionally used for centuries for treating numerous diseases. Over the past few years, a number of studies uncovered several pharmacological properties of curcumin. It has been shown that curcumin inhibited HepG2 cell growth by inducing apoptosis [21]. Recent studies showed substantial evidence that curcumin inhibited proliferation, migration, invasion and metastasis, and induced apoptosis via modulating multiple signaling pathways in head and neck cancer [36].

#### **• Gallic acid**

malignant neoplasms [36]. High amounts of trans-resveratrol can be found in grapes and grape skins used for wine production, raspberries, mulberries, blueberries cranberries, peanuts, and certain types of pine [21]. All anticancer data obtained on preclinical animal studies showed that resveratrol affects all three discrete stages of carcinogenesis (initiation, promotion, and progression) by modulating signal transduction pathways that control cell division and growth, apoptosis, inflammation, angiogenesis, and metastasis [36]. A synthetic stilbene derivative, tamoxifen, is currently used for the treatment of several types of breast cancer in

Caffeic acid (Table 2) is a hydroxycinnamic acid found in most plants, including coffee beans, nuts, berries, and grains. Several studies have investigated the antitumor potential and hepatoprotective activity of the caffeic acid derivative, which have been identified in particular in honey and propolis [21]. Data showed that caffeic acid phenethyl ester (CAPE), a component of propolis, induces cell cycle arrest and has antiproliferation effect on glioma cells *in vitro* and

Caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE), a natural polyphenol from *Sarcandra glabra*, has potent *in vitro* anticancer activity through multiple targets. This compound significantly decreased tumor growth in hepatoma and sarcoma tumor-bearing mice, and also significantly inhibited ascites development. CADPE did not show any toxicity *in vivo* and anticancer efficacies were equivalent to those of 5-fluorouracil and cyclophosphamide (drugs in cancer treatment). The tested 59 human cancer cell lines from leukemia and nine different solid tumors, including colon cancer, gastric cancer, breast cancer, CNS cancer, ovarian cancer, melanoma, lung cancer, renal cancer, and prostate cancer, are sensitive to CADPE that

Protocatechuic acid (Table 2), a dihydroxybenzoic acid widely distributed in the plant kingdom, is the major metabolite of antioxidant polyphenols found in green tea. Protocate‐ chuic acid has antioxidant and anti-inflammatory activity and has been shown to inhibit

A curcuminoid is a linear diarylheptanoid, with molecules such as curcumin (Table 2) or derivatives of curcumin with different chemical groups that confers increase solubility and make them suitable for drug formulation. Curcumin is the principal curcuminoid of the spice turmeric, which is a member of the ginger family. It has been traditionally used for centuries for treating numerous diseases. Over the past few years, a number of studies uncovered several pharmacological properties of curcumin. It has been shown that curcumin inhibited HepG2 cell growth by inducing apoptosis [21]. Recent studies showed substantial evidence that curcumin inhibited proliferation, migration, invasion and metastasis, and induced apoptosis

HepG2 (human liver carcinoma cell line) cell growth and induce apoptosis [21].

via modulating multiple signaling pathways in head and neck cancer [36].

women, and as a hormone treatment for male breast cancer [52].

62 Phytochemicals - Isolation, Characterisation and Role in Human Health

*in vivo*. In addition, CAPE inhibited the metastasis of glioma cells [53].

suppresses tumor growth and angiogenesis [54].

**• Protocatechuic acid**

**• Curcuminoids**

**• Caffeic acid**

Gallic acid (Table 2) is a hydroxybenzoic acid occurring mostly in certain red fruits, black radish, onions, and also fresh tea leaves containing up to 4.5 g/kg. Gallic acid has been shown to exhibit biological activity, including anticancer and chemopreventive potential, capacity to induce apoptosis in human leukemia. Because gallic acid exhibits anti-invasive and antime‐ tastatic activities in various cancer cells, it might be a potential preventive and therapeutic agent against gastric cancer metastasis [2].

#### **• Ellagic acid**

Ellagic acid (Table 2) is a dimeric derivative of gallic acid occurring mostly in fruits and berries (such as raspberries, strawberries, blackberries, and pomegranates) and a primary constituent of ellagitannins. The data demonstrate that ellagic acid inhibits carcinogenesis, induces apoptosis in pancreatic and leukemia cancer cells, and also can delay the tumor latency and significantly reduce the estrogen-induced mammary tumors in rats. As a consequence, ellagic acid as well as gallic acid may be considered in a promising new class of cancer chemopre‐ ventive and therapeutic agents [2, 55].

#### **• Tannins**

Tannin (also known as tannoid) is an astringent, bitter plant polyphenolic compound that binds to and precipitates proteins and various other organic compounds, including amino acids and alkaloids. The tannin compounds are widely distributed in many species of plants, where they play a role in protection from predation, and perhaps also as pesticides, and in plant growth regulation. They showed antimutagenic activity without any obvious toxicity. Pentagalloylglucose and geraniin were among the most active tannins. Geraniin was isolated from the dry leaf of *Geranium thunbergii*. The plant is an official medicine in the Japanese Pharmacopoeia and is used for the treatment of diarrhea and for controlling intestinal function [45]. The grape (*Vitis vinifera*) skin mostly contains the highest amount of condensed tannins, alongside monomeric flavanols and flavonols, phenolic acids, and resveratrol. The grape skin extract induced prostate tumor cell line apoptosis, and the extract from pomace has a signifi‐ cant antiproliferative effect on human colon adenocarcinoma cells [56].

#### **5.3. Polysaccharides**

Polysaccharides are biopolymers with linear or branched side chains, composed of monosac‐ charides linked together through glycosidic bonds. Considering that the repeating units in the polymer backbone are often six-carbon monosaccharides, the general formula can be repre‐ sented as (C6H10O5)*n*, where 40 ≤ *n* ≤ 3000. Natural polysaccharides can be obtained from various organisms, such as plants, algae, microorganisms, and animals, and exist in a variety of chemical compositions, molecular weights, and structures [1]. Most polysaccharides derived from higher plants are relatively nontoxic and do not cause significant side effects; thus, plant polysaccharides are ideal candidates for therapeutics with immunomodulatory and antitumor effects and low toxicity [20]. Natural polysaccharides isolated from herbal plants have been shown to possess bioactivities. For example, the immune-stimulatory properties of *Aloe vera* glucomannan have been confirmed and used for treatment of immune-related diseases [23].

The type of linkage between saccharide units—the glycosidic linkage—seems to be important in immunomodulatory and anticancer activities. The literature also demonstrates that most of antitumor polysaccharides contain B-1,3-glucans, β-1,6-glucans, and α-1,3-glucans [23]. Although today is investigating especially polysaccharides of bacterial, algae, and fungi origins, plants have an important as sources of bioactive polymers especially because they are nontoxic. Here are some examples in the following lines:


**•** *Astragalus membranaceus* is commonly used in Chinese herbalism, where it is considered to be one of the 50 fundamental herbs. The plant, used especially for treatment of the kidneys and also to avoid senility, has been shown to be effective in immune enhancement and in the treatment of diabetes, viral infections, and cancers as well. The polysaccharides isolated from the radix of *A. membranaceus* are an active anticancer component. Structural analysis indicated that the polysaccharides were mainly composed of α-(1 → 4)-glucan with α-(1 → 6)-linked branches or α-(1 → 3)-glucan with side-chains containing arabinose and xylose [1].

shown to possess bioactivities. For example, the immune-stimulatory properties of *Aloe vera* glucomannan have been confirmed and used for treatment of immune-related diseases [23].

The type of linkage between saccharide units—the glycosidic linkage—seems to be important in immunomodulatory and anticancer activities. The literature also demonstrates that most of antitumor polysaccharides contain B-1,3-glucans, β-1,6-glucans, and α-1,3-glucans [23]. Although today is investigating especially polysaccharides of bacterial, algae, and fungi origins, plants have an important as sources of bioactive polymers especially because they are

**•** The genus *Actinidia* (*Actinidiaceae*) consists of over 58 species widely distributed in the Asian continent (China, Taiwan, Korea, Japan, southeast Siberia, and south of Indochina). Some *Actinidia* species, such as *A. macrosperma*, are the important in traditional medicine being used as health foods and medical products. In temperate climate zones, roots of *Actinidia eriantha Benth* (a commonly liana plant) have been used for gastric carcinoma, nasophar‐ yngeal carcinoma, breast carcinoma, and hepatitis in traditional Chinese medicine. The water extracts of this drug possess antitumor and immunopotentiating activities. Four polysaccharides were isolated and purified from the roots of *A. eriantha*, and the chemical composition of these polysaccharides could affect their antitumor and immunomodulatory

**•** *Rhizoma arisaematis* comes from the rhizome of *Pinellia pedatisecta Schott*, which has bitter, warm, pungent, and toxic properties. It has be recorded in Chinese Pharmacopoeia as a traditional Chinese medicine, displaying sedative, stomachic, analgesic, anticoagulant, anti-inflammatory, antiemetic, and antitumor activities. The pharmacognostical study identifies the active components of *R. arisaematis* to be beta-sitosterol, total alkaloids, guanosine, gama-aminobutyric acid, dipeptides, and recently a water soluble polysacchar‐ ide composed of rhamnose, fucose, arabinose, mannose, galactose and glucose, with molar ratios of 0.4:0.5:0.3:0.6:0.9:5.3. This polysaccharide significantly inhibits the growth of tumor in animal experiments; immunomodulation might be the mechanism of the

**•** *Rhodiola rosea L.* belongs to the family Crassullaceae that grows in the Arctic and in the mountainous regions of Europe, Asia, and North America. The rhizome and roots has been widely used for a long time in Russian and Chinese folk medicine to increase human physical and mental performance, longevity, and resistance to high-altitude sickness and to treat fatigue, anemia, cancer, bacterial infection, impotence, nervous system disorders, and cardiovascular diseases. Phytochemical studies of *R. rosea* have revealed the presence of glycosides, flavonoids, essential oils, fats, waxes, sterols, organic acids, tannins, proteins, and polysaccharides. A homogeneous polysaccharide (composed of glucose, galactose, manose, and rhamnose with a relative molar ratio of 4.2:2.4:1.6:1.0) from *R. rosea* was tested for its immunomodulation and anticancer activity *in vitro* and *in vivo* experiments on sarcoma cells. The results showed that this polysaccharide could be used as a novel

promising immunotherapeutic agent in cancer treatment [58].

nontoxic. Here are some examples in the following lines:

64 Phytochemicals - Isolation, Characterisation and Role in Human Health

activity [20].

antitumor activity [57].


polysaccharides, one of the main components of tea extracts, have been demonstrated to have immunological, antiradiation, antioxidant, anticancer, and hypoglycemic effects [63].

Although they are not derived from plants, we must remember some polysaccharides that have been demonstrated to have powerful antitumor activity: chitin, the most abundant renewable natural resource after cellulose, a homopolymer of *N*-acetyl-d-glucosamine, is active on bladder human cancer cells and colon carcinoma [64]; a water-soluble polysaccharide from *Inonotus obliquus* has immunomodulatory and antitumor activity [65]; the polysacchar‐ ides extracted from *Pleurotus eryngii* have been demonstrated to have multiple functions, such as antitumor, antioxidant, hepatoprotective activity, and enhance immunity [66]; gamacarrageenan sulfated polysaccharides extracted from marine red alga, with different molecular weight, has antitumor and immunostimulating activities [67].

#### **5.4. Alkaloids**

Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. have been demonstrated to have multiple functions, such as antitumor, antioxidant, hepatoprotective activity, and enhance immunity [66]; gama-carrageenan sulfated polysaccharides extracted from

**•** Camptothecin (Figure 4) is a naturally occurring pyridoindole (quinoline) alkaloid isolated from the seeds of Chinese plant Camptotheca acuminata and Mappia foetida. Camptothecin and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors including colorectal, breast, lung, and ovarian cancers. Derivatives of camp‐ tothecin such as 18-OH-camptothecin, 11-OH-camptothecin, and 10-OH-camptothecin have been found to possess a strong antileukemic activity [3, 68]. marine red alga, with different molecular weight, has antitumor and immunostimulating activities [67]. **5.4. Alkaloids**  Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. Camptothecin (Figure 4) is a naturally occurring pyridoindole (quinoline) alkaloid isolated from the seeds of Chinese plant *Camptotheca acuminata* and *Mappia foetida.* Camptothecin and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors including

The primary limitations of camptothecin are its extremely low water solubility, and the hydrolysis of the active lactone ring to the inactive carboxylate, which reduces the drug efficacy and can lead to side effects. To overcome these stability and solubility problems of campto‐ thecin, several new approaches have been investigated such as using drug delivery technolo‐ gies, e.g., incorporation in liposomes, polymer micelles, microemulsions, and microspheres [69]. Specifically, camptothecin nanocrystals were prepared with a sonication-precipitation method with promising results [68]. colorectal, breast, lung, and ovarian cancers. Derivatives of camptothecin such as 18-OHcamptothecin, 11-OH-camptothecin, and 10-OH-camptothecin have been found to possess a strong antileukemic activity [3, 68]. The primary limitations of camptothecin are its extremely low water solubility, and the hydrolysis of the active lactone ring to the inactive carboxylate, which reduces the drug efficacy and can lead to side effects. To overcome these stability and solubility problems of camptothecin, several new approaches have been investigated such as using drug delivery technologies, e.g., incorporation in liposomes, polymer micelles, microemulsions, and microspheres [69]. Specifically, camptothecin

nanocrystals were prepared with a sonication–precipitation method with promising results [68].

Figure 4. Camptothecin (left) and harmine (right). Harmine (Figure 4), the most representative naturally occurring beta-carboline alkaloid, was **Figure 4.** Camptothecin (left) and harmine (right).

synthesized by modification of β-carboline nucleus [70].

**5.5. Peptides, proteins, and lectins** 

gland cancer and ovarian neoplasm [71].

originally isolated from *Peganum harmala*, which is being widely used as a traditional herbal drug in the Middle East and North Africa. In Northwest China, the extracts of the seeds of *P. harmala* have been used for hundreds of years to treat the gastrointestinal cancers and malaria. Harmine was reported to exhibit a diverse range of pharmacological properties such as hallucinogenic, antitumor, antiviral, and antiparasitic activities. In order to search for novel leading compounds with better **•** Harmine (Figure 4), the most representative naturally occurring beta-carboline alkaloid, was originally isolated from *Peganum harmala*, which is being widely used as a traditional herbal drug in the Middle East and North Africa. In Northwest China, the extracts of the seeds of

antitumor activities and less neurotoxicities, a series of harmine derivatives were designed and

 Peptides are short chains of amino acids linked by peptide bonds. They are naturally, abundantly occurring biological molecules. Many plants are an enormously rich source of peptides with potential antitumor effect, e.g., *Violaceae*, *Rubiaceae*, and *Cucurbitaceae* families. The mechanism of anticancer peptides action consists of inhibition of tumor angiogenesis, induction of tumor apoptosis, induction of tumor necrosis, immunomodulation. Studies have shown that peptides from plants exhibit marked inhibitory effects on the proliferation of various tumor cell lines, such as murine leukemia, rat osteoblastlike sarcoma, human nasopharyngeal carcinoma, lung, liver, and mammary

17

Studies have shown that most peptides isolated from plants are cyclic peptides, so-called cyclopeptides (which usually consist of less than 14 amino acid residues with no disulfide bond). More than 450 cyclopeptides have been discovered in higher plants. They exhibit more potent biological activities, possibly due to the stable configuration provided by their cyclic structure. Some *P. harmala* have been used for hundreds of years to treat the gastrointestinal cancers and malaria. Harmine was reported to exhibit a diverse range of pharmacological properties such as hallucinogenic, antitumor, antiviral, and antiparasitic activities. In order to search for novel leading compounds with better antitumor activities and less neurotoxicities, a series of harmine derivatives were designed and synthesized by modification of β-carboline nucleus [70].

#### **5.5. Peptides, proteins, and lectins**

polysaccharides, one of the main components of tea extracts, have been demonstrated to have immunological, antiradiation, antioxidant, anticancer, and hypoglycemic effects [63].

Although they are not derived from plants, we must remember some polysaccharides that have been demonstrated to have powerful antitumor activity: chitin, the most abundant renewable natural resource after cellulose, a homopolymer of *N*-acetyl-d-glucosamine, is active on bladder human cancer cells and colon carcinoma [64]; a water-soluble polysaccharide from *Inonotus obliquus* has immunomodulatory and antitumor activity [65]; the polysacchar‐ ides extracted from *Pleurotus eryngii* have been demonstrated to have multiple functions, such as antitumor, antioxidant, hepatoprotective activity, and enhance immunity [66]; gamacarrageenan sulfated polysaccharides extracted from marine red alga, with different molecular

Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic

have been demonstrated to have multiple functions, such as antitumor, antioxidant, hepatoprotective activity, and enhance immunity [66]; gama-carrageenan sulfated polysaccharides extracted from marine red alga, with different molecular weight, has antitumor and immunostimulating activities

Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic

The primary limitations of camptothecin are its extremely low water solubility, and the hydrolysis of the active lactone ring to the inactive carboxylate, which reduces the drug efficacy and can lead to side effects. To overcome these stability and solubility problems of camptothecin, several new approaches have been investigated such as using drug delivery technologies, e.g., incorporation in liposomes, polymer micelles, microemulsions, and microspheres [69]. Specifically, camptothecin nanocrystals were prepared with a sonication–precipitation method with promising results [68].

Figure 4. Camptothecin (left) and harmine (right).

 Harmine (Figure 4), the most representative naturally occurring beta-carboline alkaloid, was originally isolated from *Peganum harmala*, which is being widely used as a traditional herbal drug in the Middle East and North Africa. In Northwest China, the extracts of the seeds of *P. harmala* have been used for hundreds of years to treat the gastrointestinal cancers and malaria. Harmine was reported to exhibit a diverse range of pharmacological properties such as hallucinogenic, antitumor, antiviral, and antiparasitic activities. In order to search for novel leading compounds with better antitumor activities and less neurotoxicities, a series of harmine derivatives were designed and

**•** Harmine (Figure 4), the most representative naturally occurring beta-carboline alkaloid, was originally isolated from *Peganum harmala*, which is being widely used as a traditional herbal drug in the Middle East and North Africa. In Northwest China, the extracts of the seeds of

 Peptides are short chains of amino acids linked by peptide bonds. They are naturally, abundantly occurring biological molecules. Many plants are an enormously rich source of peptides with potential antitumor effect, e.g., *Violaceae*, *Rubiaceae*, and *Cucurbitaceae* families. The mechanism of anticancer peptides action consists of inhibition of tumor angiogenesis, induction of tumor apoptosis, induction of tumor necrosis, immunomodulation. Studies have shown that peptides from plants exhibit marked inhibitory effects on the proliferation of various tumor cell lines, such as murine leukemia, rat osteoblastlike sarcoma, human nasopharyngeal carcinoma, lung, liver, and mammary

**•** Camptothecin (Figure 4) is a naturally occurring pyridoindole (quinoline) alkaloid isolated from the seeds of Chinese plant Camptotheca acuminata and Mappia foetida. Camptothecin and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors including colorectal, breast, lung, and ovarian cancers. Derivatives of camp‐ tothecin such as 18-OH-camptothecin, 11-OH-camptothecin, and 10-OH-camptothecin have

The primary limitations of camptothecin are its extremely low water solubility, and the hydrolysis of the active lactone ring to the inactive carboxylate, which reduces the drug efficacy and can lead to side effects. To overcome these stability and solubility problems of campto‐ thecin, several new approaches have been investigated such as using drug delivery technolo‐ gies, e.g., incorporation in liposomes, polymer micelles, microemulsions, and microspheres [69]. Specifically, camptothecin nanocrystals were prepared with a sonication-precipitation

 Camptothecin (Figure 4) is a naturally occurring pyridoindole (quinoline) alkaloid isolated from the seeds of Chinese plant *Camptotheca acuminata* and *Mappia foetida.* Camptothecin and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors including colorectal, breast, lung, and ovarian cancers. Derivatives of camptothecin such as 18-OHcamptothecin, 11-OH-camptothecin, and 10-OH-camptothecin have been found to possess a strong

17

Studies have shown that most peptides isolated from plants are cyclic peptides, so-called cyclopeptides (which usually consist of less than 14 amino acid residues with no disulfide bond). More than 450 cyclopeptides have been discovered in higher plants. They exhibit more potent biological activities, possibly due to the stable configuration provided by their cyclic structure. Some

weight, has antitumor and immunostimulating activities [67].

66 Phytochemicals - Isolation, Characterisation and Role in Human Health

been found to possess a strong antileukemic activity [3, 68].

synthesized by modification of β-carboline nucleus [70].

**5.5. Peptides, proteins, and lectins** 

gland cancer and ovarian neoplasm [71].

**5.4. Alkaloids**

nitrogen atoms.

[67].

method with promising results [68].

**Figure 4.** Camptothecin (left) and harmine (right).

antileukemic activity [3, 68].

**5.4. Alkaloids** 

nitrogen atoms.


However, plant lectins attracted increasing attention from cancer biologists due to their possible antitumor properties. A large-scale study in colorectal cancer patients and a control group showed some beneficial effects of consuming plant lectins, but the pathways remain unclear [73].

*Astragalus membranaceus* lectin displays antiproliferative properties toward human leukemia cells *in vitro*. Mistletoe lectin, which is used as an adjuvant in cancer therapy, is known to activate caspases, enzymes involved in the self destruction of cells [74].

*Concanavalin A*, a member of the legume lectin family with a mannose/glucose-binding specificity, was reported to induce apoptosis in human melanoma cells and demonstrated potent therapeutic effect in liver cancer [75].

Recently, some antitumoral lectins have been discovered and researched, such as mistletoe (*V. album*) lectins, rice (*Oryza sativa)* bran agglutinin with remarkable antitumour activities, wheat (*Triticum* spp.) germ agglutinin, a typical chitin-binding lectin with strong inhibitory effects on the growth of the pancreatic tumour cells, and garlic (*Allium sativum L*) lectin, isolated from garlic which induced apoptosis at a low concentration [76]. *Phaseolus vulgaris* lectin can also induce apoptotic cell death toward various types of cancers; more interestingly, induces autophagic cell death in hepatocarcinoma [77].

#### **5.6. Quinones**

Quinones are secondary metabolites generally having a hexacyclic di-ketone system derived from the oxidation of hydroquinones, and isolated principally from plants. Naturally occur‐ ring quinones are widely distributed and include benzoquinones, naphthoquinones, anthra‐ quinones, and polyquinones. Quinones exhibit numerous biological activities, such as neurological, antibacterial, antioxidant, antitumor, and anti-HIV activities that have been proven to be related to the redox properties of their carbonyl functions. Quinones, in general, and naphthoquinones, in particular, are well known for antibacterial, antifungal, and antitu‐ moral activities.


Plumbagin has been shown to have activity against breast, prostate, ovarian, pancreatic, lung, liver, renal, cervical, and skin cancer, in addition to having activity against myeloma and leukemia. It has been shown to inhibit cell proliferation and cell invasion and effectively induces apoptosis and causes cell cycle arrest [80].

**•** Emodin (Figure 5), isolated from *Rheum emodi*, a Himalayan rhubarb, is also produced by many species of fungi, including *Aspergillus* fungi. The pharmacological studies have demonstrated that emodin when isolated from rhubarb exhibits anticancer effects on several human cancers, including human pancreatic cancer [81].

**Figure 5.** Chemical structures of quinones with antitumoral activity.

#### **5.7. Isothiocyanates**

*Astragalus membranaceus* lectin displays antiproliferative properties toward human leukemia cells *in vitro*. Mistletoe lectin, which is used as an adjuvant in cancer therapy, is known to

*Concanavalin A*, a member of the legume lectin family with a mannose/glucose-binding specificity, was reported to induce apoptosis in human melanoma cells and demonstrated

Recently, some antitumoral lectins have been discovered and researched, such as mistletoe (*V. album*) lectins, rice (*Oryza sativa)* bran agglutinin with remarkable antitumour activities, wheat (*Triticum* spp.) germ agglutinin, a typical chitin-binding lectin with strong inhibitory effects on the growth of the pancreatic tumour cells, and garlic (*Allium sativum L*) lectin, isolated from garlic which induced apoptosis at a low concentration [76]. *Phaseolus vulgaris* lectin can also induce apoptotic cell death toward various types of cancers; more interestingly, induces

Quinones are secondary metabolites generally having a hexacyclic di-ketone system derived from the oxidation of hydroquinones, and isolated principally from plants. Naturally occur‐ ring quinones are widely distributed and include benzoquinones, naphthoquinones, anthra‐ quinones, and polyquinones. Quinones exhibit numerous biological activities, such as neurological, antibacterial, antioxidant, antitumor, and anti-HIV activities that have been proven to be related to the redox properties of their carbonyl functions. Quinones, in general, and naphthoquinones, in particular, are well known for antibacterial, antifungal, and antitu‐

**•** Lapachol (Figure 5), the most studied substance of natural quinone class, is the main constituent in Pau D'Arco (a natural remedy) and has been extracted from the bark of South American tree, *Tabebuia impetiginosa*. Lapachol possesses strong biological activity against liver, kidney, breast, prostate, cervix cancer, and leukemia. Unfortunately the toxic effect

**•** Plumbagin (Figure 5) is a naphthoquinone compound that displayed antiproliferative activity on a panel of 60 cancer cell lines. However, plumbagin, identified in the Cameroo‐ nian plants *Diospyros crassiflora* and *Diospyros canaliculata,* was suggested as a promising

Plumbagin has been shown to have activity against breast, prostate, ovarian, pancreatic, lung, liver, renal, cervical, and skin cancer, in addition to having activity against myeloma and leukemia. It has been shown to inhibit cell proliferation and cell invasion and effectively

**•** Emodin (Figure 5), isolated from *Rheum emodi*, a Himalayan rhubarb, is also produced by many species of fungi, including *Aspergillus* fungi. The pharmacological studies have demonstrated that emodin when isolated from rhubarb exhibits anticancer effects on several

activate caspases, enzymes involved in the self destruction of cells [74].

potent therapeutic effect in liver cancer [75].

68 Phytochemicals - Isolation, Characterisation and Role in Human Health

autophagic cell death in hepatocarcinoma [77].

**5.6. Quinones**

moral activities.

limits the use of lapachol [78].

anticancer lead drug [79].

induces apoptosis and causes cell cycle arrest [80].

human cancers, including human pancreatic cancer [81].

Cruciferous vegetables are a rich source of glucosinolates (natural chemicals most likely contribute to plant defense against pests and diseases), which are converted to isothiocyanates. Evidence supports that consumption of cruciferous vegetables has substantial chemopreven‐ tive activity against various human malignancies, including pancreatic cancer. Benzyl isothiocyanate (Figure 6), an agent that is present in cruciferous vegetables such as watercress, cabbage, cauliflower, mustard, and horseradish, is widely consumed as part of a routine diet. Benzyl isothiocyanate is quite effective in suppressing pancreatic tumor growth by inhibiting various key signaling pathways [82].

**Figure 6.** Benzyl isothiocyanate (R=benzyl).

#### **6. Herbs and herbal formulas with antitumor activity**

#### **6.1. Aloe vera**

Aloe vera is one of the most impressive herbs, called by many people "a miracle." The plant works on multiple plans purifying, strengthening, and healing the body. In a single plant, aloe vera offers potent, natural medicine that nourishes the body with minerals, vitamins, enzymes, and saccharides; alkalizes the body, helping to balance overly acidic dietary habits; prevents and treats Candida infections; protects the body from oxidative stress; boosts the oxygenation of your blood; reduces inflammation; purifies the blood, repairs "sludge blood"; halts the growth of cancer tumors; stops colon cancer, heals the intestines and lubricates the digestive tract [83].

Scientific research shows strong immunomodulatory and antitumor properties for aloe vera polysaccharides. Acemannan (Figure 7) is the name given to the major carbohydrate fraction obtained from the gel of the aloe vera leaf. This compound has several important therapeutic properties including acceleration of wound healing, immune stimulation, and antiviral and anticancer effects. The research results suggest that acemannan accelerates the destruction of cancer tumors, improves survival time and results in far better recovery from toxic cancer treatments [83, 84]. Acemannan is currently being used for treatment and clinical management of fibrosarcoma in dogs and cats. Administration of acemannan has been shown to increase tumor necrosis and prolonged host survival [85].

**Figure 7.** Acemannan structure

Also, *Aloe vera* amplifies the antioxidant effects of vitamins C and vitamin E probably due to its effect on enhancing blood quality and allowing the blood to more effectively transport oxygen and nutrients to the body's cells [83].

#### **6.2. Catharanthus roseus or Vinca rosea**

This contains vinca alkaloids, which were the first phytoconstituents ever used to treat cancer. Intense work on *C. roseus*, a folklore hypoglycemic drug, led to isolation of more than 70 dimeric indole alkaloids, which include vinblastine, vincristine, alstonine, ajmalicine, and reserpine. Vinca alkaloids execute anticancer effect by arresting division of the cancerous cells. Vinblastine is used in the treatment of Hodgkin's disease, non-Hodgkin's lymphoma, and cancers of the kidney and the testis. Vincristine is usually given in combination with other anticancer agents to treat acute lymphocytic leukemia, Wilm's tumour, neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma, lymphoma, and cancers of the breast, lung, bladder, and the cervix [15].

#### **6.3. Garlic (***Allium sativum***)**

and saccharides; alkalizes the body, helping to balance overly acidic dietary habits; prevents and treats Candida infections; protects the body from oxidative stress; boosts the oxygenation of your blood; reduces inflammation; purifies the blood, repairs "sludge blood"; halts the growth of cancer tumors; stops colon cancer, heals the intestines and lubricates the digestive

Scientific research shows strong immunomodulatory and antitumor properties for aloe vera polysaccharides. Acemannan (Figure 7) is the name given to the major carbohydrate fraction obtained from the gel of the aloe vera leaf. This compound has several important therapeutic properties including acceleration of wound healing, immune stimulation, and antiviral and anticancer effects. The research results suggest that acemannan accelerates the destruction of cancer tumors, improves survival time and results in far better recovery from toxic cancer treatments [83, 84]. Acemannan is currently being used for treatment and clinical management of fibrosarcoma in dogs and cats. Administration of acemannan has been shown to increase

Also, *Aloe vera* amplifies the antioxidant effects of vitamins C and vitamin E probably due to its effect on enhancing blood quality and allowing the blood to more effectively transport

This contains vinca alkaloids, which were the first phytoconstituents ever used to treat cancer. Intense work on *C. roseus*, a folklore hypoglycemic drug, led to isolation of more than 70 dimeric indole alkaloids, which include vinblastine, vincristine, alstonine, ajmalicine, and reserpine. Vinca alkaloids execute anticancer effect by arresting division of the cancerous cells. Vinblastine is used in the treatment of Hodgkin's disease, non-Hodgkin's lymphoma, and cancers of the kidney and the testis. Vincristine is usually given in combination with other anticancer agents to treat acute lymphocytic leukemia, Wilm's tumour, neuroblastoma,

tract [83].

tumor necrosis and prolonged host survival [85].

70 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Figure 7.** Acemannan structure

oxygen and nutrients to the body's cells [83].

**6.2. Catharanthus roseus or Vinca rosea**

Garlic has been used for thousands of years to treat various diseases. Garlic contains approx‐ imately 33 sulfur compounds (aliin, allicin, ajoene, allylpropyl disulfide, diallyl trisulfide, sallylcysteine, vinyldithiines, S-allylmercaptocystein, and others), several enzymes (allinase, peroxidases, myrosinase, and others), 17 amino acids (arginine and others), and minerals (selenium, germanium, tellurium, and other trace minerals). Garlic has shown significant therapeutic effect in stomach, colorectal, and breast cancer in humans. Biological effects of garlic are attributed to its characteristic organ sulfur compounds [86]. The constitutive compounds of garlic can selectively inhibit tumor proliferation by a number of factors, e.g., controlling DNA repair mechanisms, chromosomal stability, and cell cycle regulation. Garlic constituents can suppress carcinogen formation, carcinogen bioactivation, and tumor prolif‐ eration [87, 88].

#### **6.4. Parsley (***Petroselinum crispum***)**

Parsley has a variety of nutrients that protect against developing cancer. It is an excellent source of vitamin C and a good source of beta-carotene, folic acid, vitamin K, flavonoids, and volatile oil components—including myristicin, limonene, eugenol, and alpha-thujene. Parsley's volatile oils—particularly myristicin—have been shown to inhibit tumor formation in animal studies, and particularly tumor formation in the lungs. The flavonoids include apiin, apigenin, crisoeriol, and luteolin [89]. Luteolin have been shown to function as antioxidants that combine with ROS and help prevent oxygen-based damage to cells. Apigenin research studies have associated it with a decreased risk of pancreatic cancer, leukemia, cervical, and ovarian cancer. Apigenin has also been shown to interfere with cancer cell proliferation, exhibiting strong antitumor properties [90].

#### **6.5. Chaparal (***Larrea divaricata Cav***.—Zygophyllaceae)**

This contains five species of evergreen shrubs that are native to the Southwestern United States, and it is a herb derived from the common desert shrubs *Larrea tridentata* and *Larrea divaricata*. It is a plant widely used in popular medicine to treat tumors, infections, and inflammatory diseases. Many studies have shown that chaparral possesses antioxidant properties and exhibits cytotoxic properties in a variety of cell types. The principal ingredient in chaparral is nordihydroguaiaretic acid that is an anticancer agent and a potent antioxidant [91]. This herb also has anti-inflammatory, analgesic, expectorant, emetic, and diuretic properties. Chaparral chelates remove heavy metals in the body and offer protection against the harmful effects of radiation, sun exposure, and the formation of tumors and cancer cells. The lignans found in chaparral that are very similar to estrogen; they are isolated from the flowering tops of *Larrea tridentata* and are effective against human breast cancer, human colon cancer, and human melanoma cell lines [92].

#### **6.6. Essiac**

Essiac is an herbal formula that was originally known to an American tribe and was renamed Essiac in 1920 by a Canadian nurse, Rene Caisse. Essiac is a tea prepared from a mixture of herbs: burdock root (*Arctium lappa*), sheep sorrel (*Rumex acetosella*), slippery elm bark (*Ulmus rubra*), and turkey rhubarb (*Rheum officinale*), and it has been used in alternative medicine for over 50 years. The phytochemicals found in these ingredients are antioxidants protecting cells against oxidative damage manifest interference with DNA replication and have antibacterial effects. Essiac enhances activity of the immune cells and reduces toxic side effects of chemo‐ therapy and radiotherapy. Essiac has been used in the treatment of malignant melanoma, lymphoma, and cancers of the pancreas, breast, ovary, esophagus, bladder, bile duct, and bone. Despite a lack of clinical studies reporting efficacy, 72% of the patients taking Essiac reported a positive opinion of the product [93].

#### **6.7. Hoxsey herbal formula**

Hoxsey herbal formula was discovered incidentally in by Elder Hoxsey, when one of his horses suffering from cancer on the leg, got cured after grazing certain herbs. Later, his grandson Harry Hoxsey tried these herbs on the human cancer. The cancer treatment practiced by Harry M. Hoxsey is one of the longest-lived unconventional therapies of this century. Hoxsey herbal formula contains *Arctium lappa*, *Berberis vulgaris*, *Glycyrrhiza glabra*, *Larrea tridentata*, *Picramnia antidesma*, *Rhamnus purshianus*, *Stillingia sylvatica*, *Trifolium pratense*, and *Xanthoxylum ameri‐ canum*. Hoxsey herbal formula has been used to treat malignant melanoma, lymphoma and cancer of the skin [94].

#### **6.8. Iscaddor**

Iscaddor is a fermented extract of *Viscum album* (mistletoe) that was discovered by the Austrian scientist, Rudolf Steiner, and has been used in the treatment of cancer by European physicians since 1920s. Mistletoe extracts are complex multicomponent mixtures, containing various biologically active substances such as glycoproteins, in particular the mistletoe lectins, polypeptides, amino acids, and oligo- and polysaccharides. More than 20 prospective clinical trials using mistletoe extracts in patients with various malignancies have been reported, and in most of these studies, it was concluded that mistletoe extracts had therapeutic benefit in terms of response rate, overall survival, quality of life, and reduce side effects [95].

#### **7. Conclusion**

Conventional chemotherapy, radiotherapy, and surgical treatments of cancer mainly focus on mass cell killing without high specificity and often cause severe side effects and toxicities. Because of these reasons and high mortality rate associated with cancer, many cancer patients seek alternative methods of treatment and herbal medicines have a vital role to play in the prevention and treatment of cancer.

Over the past decade, there has been an increasing demand of drug development against cancer; at the same time there is considerable scientific and commercial interest in the con‐ tinuing discovery of new anticancer agents from natural product sources, especially herbs. Already a great number of modern drugs in clinical use, having the ability to control cancer cells, are of natural product origins; with advanced knowledge and modern device in isolation and structure elucidation techniques and in molecular science, a more efficient research for finding new plant remedies is possible. There are an impressive number of higher plants, and it is estimated that only 10% of all plants have been examined for their chemical composition and only about 100,000 types of chemical structures have been identified. Thus, there is still a lot to be done in the search for novel natural chemopreventive compounds.

Chemically speaking, it can notice a large structural diversity in herbs: alkaloids, polyphenols, tannins, fatty acids, terpenes, polysaccharides, etc. We can say that nature has cures for serious diseases, in every place all around the globe; most of them are waiting to be discovered and used for the good of people.

Until now, the scientific researchers have investigated mainly particular herbs well known in worldwide alternative medicine ; far less have been investigated the mixtures of plants, e.g., some recognized herbs formulations (e.g., Essiac, Hoxsey, Iscador) that proved to be effective on hundreds of patients. The synergisms arising among the biologically active compounds are waiting to be demonstrated.

In scientific terms, no magic overall formula has been found so far to be applicable to all types of cancer. However, there are enough people who had a longer life or even healed with plants; therefore, medical and herbal research must continue.

#### **Author details**

**6.6. Essiac**

a positive opinion of the product [93].

72 Phytochemicals - Isolation, Characterisation and Role in Human Health

**6.7. Hoxsey herbal formula**

cancer of the skin [94].

**6.8. Iscaddor**

**7. Conclusion**

prevention and treatment of cancer.

Essiac is an herbal formula that was originally known to an American tribe and was renamed Essiac in 1920 by a Canadian nurse, Rene Caisse. Essiac is a tea prepared from a mixture of herbs: burdock root (*Arctium lappa*), sheep sorrel (*Rumex acetosella*), slippery elm bark (*Ulmus rubra*), and turkey rhubarb (*Rheum officinale*), and it has been used in alternative medicine for over 50 years. The phytochemicals found in these ingredients are antioxidants protecting cells against oxidative damage manifest interference with DNA replication and have antibacterial effects. Essiac enhances activity of the immune cells and reduces toxic side effects of chemo‐ therapy and radiotherapy. Essiac has been used in the treatment of malignant melanoma, lymphoma, and cancers of the pancreas, breast, ovary, esophagus, bladder, bile duct, and bone. Despite a lack of clinical studies reporting efficacy, 72% of the patients taking Essiac reported

Hoxsey herbal formula was discovered incidentally in by Elder Hoxsey, when one of his horses suffering from cancer on the leg, got cured after grazing certain herbs. Later, his grandson Harry Hoxsey tried these herbs on the human cancer. The cancer treatment practiced by Harry M. Hoxsey is one of the longest-lived unconventional therapies of this century. Hoxsey herbal formula contains *Arctium lappa*, *Berberis vulgaris*, *Glycyrrhiza glabra*, *Larrea tridentata*, *Picramnia antidesma*, *Rhamnus purshianus*, *Stillingia sylvatica*, *Trifolium pratense*, and *Xanthoxylum ameri‐ canum*. Hoxsey herbal formula has been used to treat malignant melanoma, lymphoma and

Iscaddor is a fermented extract of *Viscum album* (mistletoe) that was discovered by the Austrian scientist, Rudolf Steiner, and has been used in the treatment of cancer by European physicians since 1920s. Mistletoe extracts are complex multicomponent mixtures, containing various biologically active substances such as glycoproteins, in particular the mistletoe lectins, polypeptides, amino acids, and oligo- and polysaccharides. More than 20 prospective clinical trials using mistletoe extracts in patients with various malignancies have been reported, and in most of these studies, it was concluded that mistletoe extracts had therapeutic benefit in

Conventional chemotherapy, radiotherapy, and surgical treatments of cancer mainly focus on mass cell killing without high specificity and often cause severe side effects and toxicities. Because of these reasons and high mortality rate associated with cancer, many cancer patients seek alternative methods of treatment and herbal medicines have a vital role to play in the

terms of response rate, overall survival, quality of life, and reduce side effects [95].

Mariana Albulescu1,2

Address all correspondence to: Malbulescu@yahoo.com

1 Faculty of Chemistry, Biology, Geography, Department of Biology-Chemistry, West Uni‐ versity of Timişoara, Timişoara, Romania

2 Advanced Environmental Research Laboratories, West University of Timişoara, Timişoara, Romania

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### **Advances in Studies of** *Vernonanthura patens* **(Kunth) H. Rob. Growing in Ecuador**

P.I. Manzano, M. Miranda, M.F. Quijano and L. Monzote

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59866

#### **1. Introduction**

[83] Adams M. Aloe Vera Miracle. Truth Publishing International Ltd.; 2007. pp. 1-16, http://www.naturalnews.com/specialreports/aloeveramiracle.pdf (accessed 20 Sep‐

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an Journal of Integrative Medicine 2012; 3: 55-62.

Asteracea family (Asteraceae) includes more than 23,000 species and is the group of Angio‐ sperms with the richest biodiversity existing in the world. *Vernonia* (*Vernonanthura*) is the most representative gene among 1000 species [1 - 4].

Various chemical components have been reported for the genus: sesquiterpene lactones of glaucolide type, hirsutinolide, vernomargolide, eudesmanolide, cardinanolide, and elephan‐ topus, reported for most species, with some exceptions [5-11]. Moreover, diterpenes derived from ent-kaurano and kaurano, pentacyclic triterpenoids from oleanane and ursane, phytos‐ terols, and flavonoids have been isolated and identified [12-15], while the antimicrobial and molluscicidal pharmacological activities have been the most investigated [15-17].

Within the genus *Vernonia*, *Vernonanthura patens* has been one of the less studied species. It is a shrub that grows wild in Ecuador and is widely used by people in rural areas, who attribute various medicinal properties among them: analgesic, anti-inflammatory, anticancer, and antileishmanial. However, medicinal use, studies on the chemical composition, and pharma‐ cological activities until 2010 were very few. The first information collected on the chemical composition of the species reported the absence of sesquiterpene lactones [18], which consti‐ tuted a chemical marker of gender. Nevertheless, in 1986, the aerial parts were studied, isolated and 10 lactones of a species were identified, existing contradictions on the composition [19]. Jakupovic and Schmedia-Hirschmann, With respect to biological activity, less information exists. The first, was the effect demonstrated by a methanol extract of the leaves against *Artemia salina*, the inertness to the potato disc tumor produced by the introduction of *Agrobacterium tumefaciens*, and the lack of cytotoxicity against cell lines V79 [20]. The antimalarial activity of

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

aqueous extract of leaves has been described and a mean inhibitory concentration (IC50) of 38.7 ± 10.1 μg/ml against *Plasmodium falciparum* has been reported [21], *whereas* others authors have reported that an aqueous extract of leaves, got antileishmanial activity against strains of *Leishmania amazonensis* with an IC50 >100 μg/ml, being these the only reports found for the species [22].

#### **1.1. Studies in the species** *V. patens* **in the Ecuadorian coast**

In an earlier chapter of this book, has been published the results for the fractionation of a methanol extract of the species, the study of fractions as antifungal against strains of *Fusarium oxysporum* and *Penicilium notatum*, and the chemical characterization by gas chromatography/ mass spectrometry of the fraction that showed that activity. The hexane fraction presented antifungal activity against these strains, and 33 compounds were possible to identify. Three pentacyclic triterpenoids (lupeol, epilupeol, and lupeol acetate), presented as major compo‐ nents, were also isolated and characterized for fractions[23]

Unlike other species of *Vernonanthura* and the own *V. patens* living in other regions of South America, the one that grows in Ecuador presents as major components pentacyclic triterpe‐ noids and no sesquiterpene lactones. These results forced to perform a genetic characterization of the genes from the plastid rbcL and matK of leaf chloroplast DNA fraction, and it confirmed that the species was *V. patens* [24, 25]

This research group has continued with studies to verify the identity of the species, obtaining the identification of 53 compounds from leaves of a methanolic extract within the terpene compounds are highlighted (oxygenated sesquiterpenoids and triterpenoids), aliphatic hydrocarbons, fatty acids, and their methyl and ethyl esters and sugars [26, 27].

In others studies, two types of waxes of leaves and fat fractions of stems and flowers were isolated and were identified 29 fatty acids and 8 triterpenoids as components of the lipid fractions of these organs, which had not been previously reported (Tables 1 and 2) [28]. Figure 1 presents some of the chemical structures identified in this species.

Regarding biological studies, the antileishmanial evaluation of ethanolic extract of the leaves and stems was performed, proving the traditional use of the leaves of the species as antileish‐ manial. The ethanol extract of stems was highly toxic. Leaves extract showed a higher selectivity index than pentamidine, used as reference drug [29].



aqueous extract of leaves has been described and a mean inhibitory concentration (IC50) of 38.7 ± 10.1 μg/ml against *Plasmodium falciparum* has been reported [21], *whereas* others authors have reported that an aqueous extract of leaves, got antileishmanial activity against strains of *Leishmania amazonensis* with an IC50 >100 μg/ml, being these the only reports found for the

In an earlier chapter of this book, has been published the results for the fractionation of a methanol extract of the species, the study of fractions as antifungal against strains of *Fusarium oxysporum* and *Penicilium notatum*, and the chemical characterization by gas chromatography/ mass spectrometry of the fraction that showed that activity. The hexane fraction presented antifungal activity against these strains, and 33 compounds were possible to identify. Three pentacyclic triterpenoids (lupeol, epilupeol, and lupeol acetate), presented as major compo‐

Unlike other species of *Vernonanthura* and the own *V. patens* living in other regions of South America, the one that grows in Ecuador presents as major components pentacyclic triterpe‐ noids and no sesquiterpene lactones. These results forced to perform a genetic characterization of the genes from the plastid rbcL and matK of leaf chloroplast DNA fraction, and it confirmed

This research group has continued with studies to verify the identity of the species, obtaining the identification of 53 compounds from leaves of a methanolic extract within the terpene compounds are highlighted (oxygenated sesquiterpenoids and triterpenoids), aliphatic

In others studies, two types of waxes of leaves and fat fractions of stems and flowers were isolated and were identified 29 fatty acids and 8 triterpenoids as components of the lipid fractions of these organs, which had not been previously reported (Tables 1 and 2) [28]. Figure

Regarding biological studies, the antileishmanial evaluation of ethanolic extract of the leaves and stems was performed, proving the traditional use of the leaves of the species as antileish‐ manial. The ethanol extract of stems was highly toxic. Leaves extract showed a higher

 Nonanedioic acid dimethyl ester 0.37 - 0.01 - Tetradecanoic acid 0.31 0.26 0.03 0.23 Pentadecanoic acid 0.31 0.24 0.06 0.15 Acid, 9-hexadecenoic (ISOM) 0.33 0.45 0.04 -

**Percent relative abundance HSCB HSCN TSC FSC**

hydrocarbons, fatty acids, and their methyl and ethyl esters and sugars [26, 27].

1 presents some of the chemical structures identified in this species.

selectivity index than pentamidine, used as reference drug [29].

**Compounds**

**1.1. Studies in the species** *V. patens* **in the Ecuadorian coast**

82 Phytochemicals - Isolation, Characterisation and Role in Human Health

nents, were also isolated and characterized for fractions[23]

that the species was *V. patens* [24, 25]

species [22].

HSCB = saponifiable white wax fraction leaves; HSCN = saponifiable orange wax fraction leaves; TSC = saponifiable wax fraction stems, FSC = saponifiable wax fraction flowers.

**Table 1.** Compounds identified in the fatty acid fractions of leaves, stems and flowers of *Vernonanthura patens*

All these studies have contributed to knowledge of the species growing in the Ecuadorian coast and serve as reference for other studies.


HICB and HICN, unsaponifiable leaves fractions; TIC, unsaponifiable stems fractions; FIC, unsaponifiable flowers fractions

+

**Table 2.** Compounds identified in the unsaponifiable fractions *Vernonanthura patens*

**Figure 1.** Some compounds isolated from *Vernonanthura patens.*

#### **2. Materials and methods**

Leaves, flowers, and stems of the species in the phenological stage of flowering were used and collected around the Biotechnology Research Center of Ecuador located at Km. 30.5 via Perimeter province of Guayas, Ecuador. A sample of the plant material was taken for botanical identification, which was botanized at the National Herbarium of Ecuador (QCNE), Quito, with the key CIBE37a.

#### **2.1. Extraction**

**Pico Compound**

84 Phytochemicals - Isolation, Characterisation and Role in Human Health

HO

**PALMITATO DE LUPEOL**

**2. Materials and methods**

C15H31 C-O

O

HO

**GLUTINOL**

**Relative abundance % HICB HICN TIC FIC**

+

 β-Amyrin 2.45 15.52 28.5 19.8 α-Amyrin - - - 66.83 Lupeol 97.5 88.48 - 0.49 α-Amyrin + lupeol - - 55.59 - Glutinol - - 2.70 - Taraxasterol acetate - - 1.93 - Taraxasterol - - - 7.9 Neoganmacer 22(29)-en-3.ol - - - 4.9

HICB and HICN, unsaponifiable leaves fractions; TIC, unsaponifiable stems fractions; FIC, unsaponifiable flowers fractions

**EPILUPEOL OH 3**a

CH3

CH2CH3

**CAMPESTEROL**

**SITOSTEROL**

**LUPEOL OH 3**b

HO

HO

HO

Leaves, flowers, and stems of the species in the phenological stage of flowering were used and collected around the Biotechnology Research Center of Ecuador located at Km. 30.5 via Perimeter province of Guayas, Ecuador. A sample of the plant material was taken for botanical

+

**Table 2.** Compounds identified in the unsaponifiable fractions *Vernonanthura patens*

**ACETATO DE LUPEOL**

N

O

**ACETATO DE TARAXASTEROL**

N

**LUPANINA**

CH3COO

CH2CH3

> H3CCOO

**ESTIGMASTEROL**

**Figure 1.** Some compounds isolated from *Vernonanthura patens.*

The extraction from the aerial parts of the species (leaves, flowers, and stems) was performed in water and ethanol by triplicate in an ultrasonic bath VWR of 35 KHz power [30]. In all cases, 10 g of sample was extracted in the solvent (water or ethanol), in the following time intervals: 5, 15, 30, 45, and 60 min.

#### **2.2. Determination of antioxidant activity**

The determination of antioxidant activity was performed based on the stability of 1,1 diphenyl-2-picrylhydrazyl (DPPH) radical to alcoholic and aqueous extracts obtained from the aerial parts. A total of 800 μl of 0.1 N DPPH and 200 μl of the extracts were taken, and the absorbance was measured at 517 nm after 30 min using 200 μl of ethanol and 800 μl of DPPH as control. Determinations were performed in triplicate.

#### **2.3. Determination of total polyphenols**

The total polyphenol content was measured by the Folin-Ciocalteu method in a spectropho‐ tometer (BioTek), at 760 nm using gallic acid monohydrate (CAS 149-91-7) as patron of the calibration curve. Results were expressed as milligrams of gallic acid per gram of sample (mg GA/g sample). A total of 250 μl of sample and 350 μl of Folin-Ciocalteu 1 N were mixed; 5 min after, 350 μl of 20% of sodium carbonate was added. After the 90-min incubation at room temperature, the absorbance was measured at 760 nm. Determinations were performed in triplicate.

#### **2.4. Chromatographic profile of the extracts by HPLC**

Chromatographic profiles by high-resolution liquid chromatography (HPLC) were performed in all extracts obtained at different extraction times to determine whether the extraction time produces chemical changes in the extracts. HPLC analysis was performed with Perkin Elmer Series 2000 HPLC with TotalCrom Software operating system. The phenolic compounds were detected at 280 nm with a flow rate of 1 ml/min. C18 column was used at a temperature of 30°C. Separations were carried out in a pumping system by varying the proportion of 2.5% (v/ v) acetic acid in water (mobile phase A) and 70% methanol in water (mobile phase B). The solvent gradient elution program was as follows: 10% to 26% B (v/v) in 10 min, to 70% B at 20 min, and finally to 90% B at 25 to 31 min. The injection volume for all samples was 10μL.

#### **2.5. Statistical analysis**

A factorial design 2 × 3 × 5 was used involving the categorical factors: extraction solvent (water and alcohol), plant organ (leaves, flowers, and stems), and extraction time (5, 15, 30, 45, and 60 min), with antioxidant activity and total polyphenol content as response variables.

#### **2.6. Antileishmanial activity**

This test was performed with aqueous extracts obtained as described.

#### *2.6.1. Aqueous extract*

Extraction was carried out by decoction of plant materials in proportion of 10% in water during 20 min. The aqueous extract was evaporated to 87ºC and 400 mmHg. Finally, the concentrated extract was lyophilized to 120 × 10-3 mbar and 47ºC below zero.

#### *2.6.2. Reference drug*

Pentamidine (Richet, Buenos Aires, Argentina) diluted in sterile distilled water was used for *L. amazonensis*. *L. amazonensis* (MHOM/77BR/LTB0016) was kindly provided by the Depart‐ ment of Immunology, Oswaldo Cruz Foundation (FIOCRUZ), Brazil. Parasites were routinely isolated from mouse lesions and maintained as promastigotes at 26ºC in Schneider's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (HFBS) (Sigma, St. Louis, MO, USA), 100 mg of streptomycin/ml, and 100 U penicillin/ml. The parasites were not used after the tenth passage.

#### *2.6.3. Antileishmanial activity*

The activity of the extracts against intracellular amastigotes was evaluated as described previously. The peritoneal macrophages were harvested and plated at 106/ml in 24-well Lab-Tek (Costarâ, USA) and incubated at 37ºC under an atmosphere of 5% CO2 for 2 h. Nonadherent cells were removed by washing with prewarmed phosphate-buffered saline (PBS). Stationaryphase *L. amazonensis* promastigotes were added at a 4:1 parasite/macrophage ratio, and the cultures were incubated for further 4 h. The cell monolayers were washed three times with prewarmed PBS to remove free parasites. Then 999 ml of RPMI completed medium and 1 ml of the different products dissolved in DMSO were added in duplicate for further 48 h. The cultures were then fixed in absolute methanol, stained with Giemsa, and examined under a light microscope. The number of intracellular amastigotes was determined by counting the amastigotes in 100 macrophages per each sample. In addition, the percentage of infected macrophages was calculated. The results were expressed as percent of reduction of the infection rate in comparison to that of the controls, where the infection rates were obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages [31]. The IC50 value was determined from the linear regression of concentration– response curves.

The IC50 of the extracts for the viability of mouse peritoneal macrophages was determined. Twenty-two macrophages were collected from peritoneal cavities of normal BALB/c mice in ice-cold RPMI 1640 medium (Sigma, St. Louis, Mo, USA) supplemented with antibiotics and seeded at 30,000 cells/well. The cells were incubated for 2 h at 37ºC in 5% CO2. Nonadherent cells were removed by washing with PBS, and then 1 μl of product solution was added to 200 μl medium containing 10% HFBS and antibiotics. Macrophages treated with 1 μl DMSO were included as controls. The cytotoxicity was determined using the colorimetric assay with 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). MTT solutions were prepared at 5 mg/ml in saline solution, filtered, and sterilized at the moment of use, and 15 μl was added to each well. After incubation for an additional 3 h, the formazan crystals were dissolved by addition of 100 μl DMSO. The optical density was determined using an EMS Reader MF Version 2.4-0, at a test wavelength of 560 nm and a reference wavelength of 630 nm [32]. The IC50 was obtained by fitting a sigmoidal Emax model to dose–response curves. Selectivity indexes were calculated by dividing the IC50 for perito‐ neal macrophage of BALB/c mice by the IC50 for *L. amazonensis* amastigotes. The IC50 of the extracts for the viability of mouse peritoneal macrophages was determined [31].

#### **3. Results and discussions**

**2.6. Antileishmanial activity**

*2.6.1. Aqueous extract*

*2.6.2. Reference drug*

were not used after the tenth passage.

*2.6.3. Antileishmanial activity*

response curves.

This test was performed with aqueous extracts obtained as described.

86 Phytochemicals - Isolation, Characterisation and Role in Human Health

extract was lyophilized to 120 × 10-3 mbar and 47ºC below zero.

Extraction was carried out by decoction of plant materials in proportion of 10% in water during 20 min. The aqueous extract was evaporated to 87ºC and 400 mmHg. Finally, the concentrated

Pentamidine (Richet, Buenos Aires, Argentina) diluted in sterile distilled water was used for *L. amazonensis*. *L. amazonensis* (MHOM/77BR/LTB0016) was kindly provided by the Depart‐ ment of Immunology, Oswaldo Cruz Foundation (FIOCRUZ), Brazil. Parasites were routinely isolated from mouse lesions and maintained as promastigotes at 26ºC in Schneider's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (HFBS) (Sigma, St. Louis, MO, USA), 100 mg of streptomycin/ml, and 100 U penicillin/ml. The parasites

The activity of the extracts against intracellular amastigotes was evaluated as described previously. The peritoneal macrophages were harvested and plated at 106/ml in 24-well Lab-Tek (Costarâ, USA) and incubated at 37ºC under an atmosphere of 5% CO2 for 2 h. Nonadherent cells were removed by washing with prewarmed phosphate-buffered saline (PBS). Stationaryphase *L. amazonensis* promastigotes were added at a 4:1 parasite/macrophage ratio, and the cultures were incubated for further 4 h. The cell monolayers were washed three times with prewarmed PBS to remove free parasites. Then 999 ml of RPMI completed medium and 1 ml of the different products dissolved in DMSO were added in duplicate for further 48 h. The cultures were then fixed in absolute methanol, stained with Giemsa, and examined under a light microscope. The number of intracellular amastigotes was determined by counting the amastigotes in 100 macrophages per each sample. In addition, the percentage of infected macrophages was calculated. The results were expressed as percent of reduction of the infection rate in comparison to that of the controls, where the infection rates were obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages [31]. The IC50 value was determined from the linear regression of concentration–

The IC50 of the extracts for the viability of mouse peritoneal macrophages was determined. Twenty-two macrophages were collected from peritoneal cavities of normal BALB/c mice in ice-cold RPMI 1640 medium (Sigma, St. Louis, Mo, USA) supplemented with antibiotics and seeded at 30,000 cells/well. The cells were incubated for 2 h at 37ºC in 5% CO2. Nonadherent cells were removed by washing with PBS, and then 1 μl of product solution was added to 200 μl medium containing 10% HFBS and antibiotics. Macrophages treated with 1 μl DMSO were included as controls. The cytotoxicity was determined using the colorimetric assay with 3-[4,5-

#### **3.1. Determination of antioxidant activity**

The percentage of antioxidant activity by the method of DPPH of the aqueous and the alcoholic extracts of different plant organs, which were obtained at different times of ultrasonic extraction, were determined and differences in time of extraction were observed.

The highest percentage of antioxidant activity was obtained by employing an extraction time of 5 min for the aqueous extracts of leaves and flowers. However, the highest percentage of activity was obtained at 15 min of extraction for the aqueous extract of stems.

Alcoholic extracts of leaves and stems showed no activity at any time of extraction; in contrast, a greater activity at 45 and 60 min of extraction was observed in flowers of *V. patens* with no differences of significance between these times (*p* > 0.05) (Table 3, Figure 2).


Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not significantly different (*p* > 0.05).

**Table 3.** Antioxidant activity (%) of aerials parts of *Vernonanthura patens*

There is only one antecedent of antioxidant activity for the *Vernonia* genre, for the species *Vernonia tweedieana*, with no previous studies for *V. patens* [33].

**Aerial part of the plant** 

**Flowers**

Table 3. Antioxidant activity (%) of aerials parts of *Vernonanthura patens*

**Leaves** 80.98 ± 1.7 Aa 48.36 ± 6.69 Ba 51.68 ± 3.14 Ba 54.16 ± 4.95 Ba 44.72 ± 4.33 Ba

**Flowers** 82.85 ± 3.79 Aa 73.85 ± 5.83 ABb 62.63 ± 2.69 BCb 68.05 ± 8.77 BCa 56.26 ± 5.38 Cb

**Stems** 35.10 ± 9.18 Ab 78.76 ± 6.09 Bb 59.14 ± 5.65 Cab 61.35 ± 2.84 Ca 54.60 ± 2.97 Cb

**(ethanol)** 34.24 ± 6.36 Ab 28.07 ± 3.29 Ac 69.34 ± 3.09 Bb 79.42 ± 3.29 BCb 80.82 ± 2.60 Cc

**Extraction time 5 min 15 min 30 min 45 min 60 min** 

**Figure 2.** Antioxidant activity of aerials parts of *Vernonanthura patens* according to extraction time.

#### **3.2. Determination of total polyphenols**

The part of the plant, the extraction time, and the solvent employed have influence in the content of polyphenols. There is only one antecedent of antioxidant activity for the *Vernonia* genre, for the species *Vernonia* 

Figure 2. Antioxidant activity of aerials parts of *Vernonanthura patens* according to extraction time.

No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the spectrophotometric determination of polyphenols in the extract. *tweedieana*, with no previous studies for *V. patens* [33]. **3.2 Determination of total polyphenols**  The part of the plant, the extraction time, and the solvent employed have influence in the content of

When water is used as solvent, there is a direct relationship between the extraction time and the concentration of polyphenols; that is, the longer the extraction, the higher the concentration independent of the plant organ used (Figure 3). polyphenols. No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the

In all cases, the highest percentage of total polyphenols was obtained at 45 min of aqueous extraction without significant differences (*p* > 0.05) with 60 min of extraction. At this time of extraction (45 min), the leaves of the species exhibited the highest percentage of polyphenols, followed by flowers. Stems exhibited the lowest percentage of polyphenol (Table 4). spectrophotometric determination of polyphenols in the extract.

> There are several reports regarding the presence of phenolic compounds in the different plant organs for the *Vernonanthura* (*Vernonia*) genre. Flavonoids have been designated as constituents of gender [34]. Flavonoids, tannins, and phenolics are reported for leaves of *V. cinerea* [35], whereas others authors have reported the presence of flavonoids [14]. For flowers of the same species, flavonoids, tannins, and phenolic compounds have been identifiedas well [35]. Flavonoids and tannins has been detected for leaves of *Vernonia amigdalina*, [36, 37].

The part of the plant, the extraction time, and the solvent employed have influence in the content of polyphenols.

Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not

There is only one antecedent of antioxidant activity for the Vernonia genre, for the species Vernonia tweedieana, with no

No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the

When water is used as solvent, there is a direct relationship between the extraction time and the concentration of

Figure 2. Antioxidant activity of aerials parts of Vernonanthura patens according to extraction time.

Table 3. Antioxidant activity (%) of aerials parts of Vernonanthura patens

significantly different (p > 0.05).

previous studies for V. patens [33].

3).

3.2. Determination of total polyphenols

spectrophotometric determination of polyphenols in the extract.

**Figure 3.** Polyphenol concentration *vs* time for different plant organs.

Figure 3. Polyphenol concentration vs time for different plant organs.

**3.2. Determination of total polyphenols**

88 Phytochemicals - Isolation, Characterisation and Role in Human Health

independent of the plant organ used (Figure 3).

spectrophotometric determination of polyphenols in the extract.

content of polyphenols.

**3.2 Determination of total polyphenols** 

**Antioxidant**

**Activity**

**%**

*tweedieana*, with no previous studies for *V. patens* [33].

extract.

polyphenols.

not significantly different (*p* > 0.05).

**Aerial part of the plant** 

**Flowers**

The part of the plant, the extraction time, and the solvent employed have influence in the

ethanol

5 min 15 min 30 min 45min 60 min

**Extraction time**

There is only one antecedent of antioxidant activity for the *Vernonia* genre, for the species *Vernonia* 

Figure 2. Antioxidant activity of aerials parts of *Vernonanthura patens* according to extraction time.

Leaves Flowers Stems Flowers

**Aerial Part of** *V. patens*

**Figure 2.** Antioxidant activity of aerials parts of *Vernonanthura patens* according to extraction time.

Table 3. Antioxidant activity (%) of aerials parts of *Vernonanthura patens*

**Leaves** 80.98 ± 1.7 Aa 48.36 ± 6.69 Ba 51.68 ± 3.14 Ba 54.16 ± 4.95 Ba 44.72 ± 4.33 Ba

**Flowers** 82.85 ± 3.79 Aa 73.85 ± 5.83 ABb 62.63 ± 2.69 BCb 68.05 ± 8.77 BCa 56.26 ± 5.38 Cb

**Stems** 35.10 ± 9.18 Ab 78.76 ± 6.09 Bb 59.14 ± 5.65 Cab 61.35 ± 2.84 Ca 54.60 ± 2.97 Cb

**(ethanol)** 34.24 ± 6.36 Ab 28.07 ± 3.29 Ac 69.34 ± 3.09 Bb 79.42 ± 3.29 BCb 80.82 ± 2.60 Cc

Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are

**Extraction time 5 min 15 min 30 min 45 min 60 min** 

No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the spectrophotometric determination of polyphenols in the

When water is used as solvent, there is a direct relationship between the extraction time and the concentration of polyphenols; that is, the longer the extraction, the higher the concentration

The part of the plant, the extraction time, and the solvent employed have influence in the content of

No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the

> In all cases, the highest percentage of total polyphenols was obtained at 45 min of aqueous extraction without significant differences (*p* > 0.05) with 60 min of extraction. At this time of extraction (45 min), the leaves of the species exhibited the highest percentage of polyphenols,

> There are several reports regarding the presence of phenolic compounds in the different plant organs for the *Vernonanthura* (*Vernonia*) genre. Flavonoids have been designated as constituents of gender [34]. Flavonoids, tannins, and phenolics are reported for leaves of *V. cinerea* [35], whereas others authors have reported the presence of flavonoids [14]. For flowers of the same species, flavonoids, tannins, and phenolic compounds have been identifiedas well [35].

followed by flowers. Stems exhibited the lowest percentage of polyphenol (Table 4).

Flavonoids and tannins has been detected for leaves of *Vernonia amigdalina*, [36, 37].


the plant 5 min 15 min 30 min 45 min 60 min Leaves 6.07 ± 1.7 Aa 5.57 ± 0.22 Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not significantly different (*p* > 0.05)

Aa 8.53 ± 1.2 Aa 10.95 ± 0.91 Ba 11.46 ± 1.94 Ba 1.41 ± 0.17 **Table 4.** Total polyphenol content (mg GA/g) of aerial parts of *Vernonanthura patens* using water as solvent.

Flowers 1.12 ± 0.12 Ab Ab 2.55 ± 0.67 Ab 5.74 ± 0.06 Bb 6.85 ± 0.96 Bb Stems 0.98 ± 0.06 Ac 1.09 ± 0.14 Ac 1.47 ± 0.22 ABc 2.06 ± 0.48 BCc 2.99 ± 0.49 Cc Phenolic compounds, tannins, and flavonoids among other compounds in the leaves, stems, and flowers of the species *V. patens* of Ecuadorian coast have been reported [38]. This may explain the total polyphenol content found in different plant organs studied, although in not a very high proportion.

#### **3.3. Chromatographic profile of the extracts by HPLC**

Considering the fact that the extraction method using ultrasound sometimes causes changes in the chemical composition of the extracts, a chromatographic profile was performed to the aqueous extracts obtained at 5 and 45 min of extraction.

Some changes in the chromatographic profile of the aqueous extract of leaves at 45 min are observed (Figure 4), especially in the baseline, although the highest chromatographic peak intensity observed at about 56.9 min has no variation.

Chromatographic profile changes are minor in the aqueous extract of the flowers; a major chromatographic peak around 56.6 min is also seen (Figure 5).

Nevertheless, the aqueous extract of the stems suffered no change in the chromatographic profile at different extraction times analyzed (5, 15, and 45 min). The retention time of the major peak was found at 56.9 min (Figure 6).

Only the study for alcoholic extracts of flowers was done because it was the only one that showed antioxidant activity. Minimal changes were observed in the chromatographic profile of 5 and 45 min extraction, which may be due to the transmission of the ultrasonic waves in the ethanol solvent, which are lower than when water is used [38, 39]. A chromatographic peak around 57 min was also observed in this extract (Figure 7).

The results lead us to believe that in all plant organs of the species obtained with water and the alcoholic extract of the flowers, there is a major component or a mixture thereof, which eluted at a similar retention time by HPLC, in the studied conditions, which may or may not be responsible for the antioxidant activity found for these compounds. However, it is impor‐ tant to note that no correlation between the polyphenol content of the extracts and the antioxidant activity was found. In all cases, the higher the percentage of antioxidant activity, the lower the percentage of total polyphenolics (Figure 8).

These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or more compounds are influential in this activity.

**Figure 4.** HPLC chromatographic profile of the aqueous extract of the leaves of *Vernonanthura patens* obtained with 5 and 45 min of ultrasound extraction

Chromatographic profile changes are minor in the aqueous extract of the flowers; a major

Nevertheless, the aqueous extract of the stems suffered no change in the chromatographic profile at different extraction times analyzed (5, 15, and 45 min). The retention time of the major

Only the study for alcoholic extracts of flowers was done because it was the only one that showed antioxidant activity. Minimal changes were observed in the chromatographic profile of 5 and 45 min extraction, which may be due to the transmission of the ultrasonic waves in the ethanol solvent, which are lower than when water is used [38, 39]. A chromatographic peak

The results lead us to believe that in all plant organs of the species obtained with water and the alcoholic extract of the flowers, there is a major component or a mixture thereof, which eluted at a similar retention time by HPLC, in the studied conditions, which may or may not be responsible for the antioxidant activity found for these compounds. However, it is impor‐ tant to note that no correlation between the polyphenol content of the extracts and the antioxidant activity was found. In all cases, the higher the percentage of antioxidant activity,

These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or more compounds are influential in

**Figure 4.** HPLC chromatographic profile of the aqueous extract of the leaves of *Vernonanthura patens* obtained with 5

chromatographic peak around 56.6 min is also seen (Figure 5).

90 Phytochemicals - Isolation, Characterisation and Role in Human Health

around 57 min was also observed in this extract (Figure 7).

the lower the percentage of total polyphenolics (Figure 8).

peak was found at 56.9 min (Figure 6).

this activity.

and 45 min of ultrasound extraction

**Figure 5.** HPLC chromatographic profile of the aqueous extract of the flowers of *Vernonanthura patens* obtained with 5 and 45 min of ultrasound extraction

**Figure 6.** HPLC chromatographic profile of the aqueous extract of the stems of *Vernonanthura patens* obtained with 5, 15, and 45 min of ultrasound extraction

**Figure 7.** HPLC chromatographic profile of the ethanolic extract of the flowers of *Vernonanthura patens* obtained with 5 and 45 min of ultrasound extraction However, it is important to note that no correlation between the polyphenol content of the extracts and the antioxidant activity was found. In all cases, the higher the percentage of antioxidant activity, the lower the percentage of total

#### These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of **3.4. Evaluation of antileishmanial activity**

3.4. Evaluation of antileishmanial activity

polyphenolics (Figure 8).

polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or more compounds are influential in this activity. Aqueous extracts of leaves and stems showed activity and selectivity against *L. amazonensis* (Table 5). The activity and selectivity present in the aqueous extract of the stems showed a

Figure 8. Total polyphenol content vs antioxidant activity of the leaves, flowers, and stems of Vernonanthura patens

Aqueous extracts of leaves and stems showed activity and selectivity against L. amazonensis (Table 5). The activity and selectivity present in the aqueous extract of the stems showed a different behavior to that reported for the ethanol extract different behavior to that reported for the ethanol extract of this plant organ [28]. The evalu‐ ation was reported as not detectable because it caused a minimal toxicity in the concentrations tested, destroying the cell monolayer.

The results obtained for the aqueous extract support the traditional use of the species and are highly relevant as these extracts would enhance the usefulness of a possible low-cost source for the development of an effective herbal drug for the treatment against Leishmania.


However, it is important to note that no correlation between the polyphenol content of the extracts and the antioxidant **Table 5.** Antileishmanial activity and cytotoxicity of aqueous extracts of *Vernonanthura patens* against *L. amazonensis*

> IC50, half maximal inhibitory concentration, is expressed as the concentration of extract (mg/ml) that inhibits 50% of the parasite growth. CC50, median cytotoxic concentration, is expressed as the concentration of extract (mg/ml) causing 50% of parasite mortality. *X*¯(*S*), mean (standard deviation). SI, selectivity index: CC50 macrophages / IC50 Leishmania. SI ≤ 5 more selective for the cell, SI ≥5 more selective for parasite.

#### **4. Conclusion**

**Figure 7.** HPLC chromatographic profile of the ethanolic extract of the flowers of *Vernonanthura patens* obtained with 5

R² = 0.239

R² = 0.588

Figure 8. Total polyphenol content vs antioxidant activity of the leaves, flowers, and stems of Vernonanthura patens

Aqueous extracts of leaves and stems showed activity and selectivity against L. amazonensis (Table 5). The activity and selectivity present in the aqueous extract of the stems showed a different behavior to that reported for the ethanol extract

activity was found. In all cases, the higher the percentage of antioxidant activity, the lower the percentage of total

Leaves Flowers Stems

Linear (Leaves) Linear (Flowers) Linear (Stems)

and 45 min of ultrasound extraction

0

2

4

6

Total polyphenols content (mgEGA/g)

8

10

12

polyphenolics (Figure 8).

more compounds are influential in this activity.

**3.4. Evaluation of antileishmanial activity**

3.4. Evaluation of antileishmanial activity

R² = 6E-05

20 40 60 80 100

Antioxidant Activity %

**Figure 8.** Total polyphenol content *vs* antioxidant activity of the leaves, flowers, and stems of *Vernonanthura patens*

Aqueous extracts of leaves and stems showed activity and selectivity against *L. amazonensis* (Table 5). The activity and selectivity present in the aqueous extract of the stems showed a

92 Phytochemicals - Isolation, Characterisation and Role in Human Health

The extraction time ultrasound influences the concentration of polyphenolic compounds in all organs tested, when water is used as solvent. Ethanol extracts are unable to determine the concentration of phenolic compounds due to the possible interference of colored compounds extracted with this solvent. Aqueous extracts of the leaves and flowers and the alcoholic extract of flowers showed higher antioxidant activity than the aqueous extracts of the stems. Antiox‐ idant activity in the alcoholic extracts of leaves and stems was not observed.

The HPLC chromatographic profiles of all extracts tested showed a majority chromatographic peak between 56 and 57 min, which could correspond to a mixture of compounds responsible for the antioxidant activity found.

No correlation between antioxidant activity and polyphenol content was found, so presumably they are not solely responsible for the antioxidant activity found.

These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or Aqueous extracts of the leaves and stems of the species *V. patens* showed higher activity and selectivity than the alcoholic extracts, against *L. amazonensis*, corroborating the traditional use of the species.

#### **5. Future directions**

The results obtained in the chemical and biological study of the species *V. patens* show its potential as antimicrobial, antioxidant, and particularly antileishmanial, antiparasitic diseases of high incidence in Ecuador and other countries. However, inclusion in the therapeutic still requires further studies among which may be mentioned toxicology to demonstrate the safety of their preparations and as a development of a suitable dosage form to the pathology to be treated, aspects that should motivate future research of our work group.

### **Acknowledgements**

This study was supported by grants from SENESCYT, Prometheus program, and ESPOL (Ecuador).

#### **Author details**

P.I. Manzano1\*, M. Miranda2 , M.F. Quijano2 and L. Monzote3

\*Address all correspondence to: pmanzano@espol.edu.ec

1 Escuela Superior Politécnica del Litoral, Facultad de Ciencias Naturales y Matemáticas (ESPOL-FCNM), Guayaquil, Ecuador

2 Escuela Superior Politécnica del Litoral, Centro de Investigaciones Biotecnológicas del Ecuador (ESPOL-CIBE), Guayaquil, Ecuador

3 Institute of Tropical Medicine Pedro Kouri, Havana, Cuba

#### **References**


[4] Bremer K, Anderber A. (1994). *Asteraceae:* Cladistics & Classification*.* Universidad de Michigan, Timber Press. ISBN-10: 0881922757. ISBN-13: 978-0881922752.

**5. Future directions**

94 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Acknowledgements**

(Ecuador).

**Author details**

**References**

P.I. Manzano1\*, M. Miranda2

(ESPOL-FCNM), Guayaquil, Ecuador

Ecuador (ESPOL-CIBE), Guayaquil, Ecuador

The results obtained in the chemical and biological study of the species *V. patens* show its potential as antimicrobial, antioxidant, and particularly antileishmanial, antiparasitic diseases of high incidence in Ecuador and other countries. However, inclusion in the therapeutic still requires further studies among which may be mentioned toxicology to demonstrate the safety of their preparations and as a development of a suitable dosage form to the pathology to be

This study was supported by grants from SENESCYT, Prometheus program, and ESPOL

1 Escuela Superior Politécnica del Litoral, Facultad de Ciencias Naturales y Matemáticas

2 Escuela Superior Politécnica del Litoral, Centro de Investigaciones Biotecnológicas del

[1] Rodríguez A. (2005). *Asteraceae*. In: Hammel BE, Zamora N, Grayum MH (eds.). Plant Manual of Costa Rica. Missouri Bot. Gard. Press, St. Louis & Inst. Nac. de Biodiversi‐

[2] Stevens PF. (2006). «Asteraceae». Angiosperm Phylogeny Website. Version 7, Mayo.

[3] Jeffrey C. (2007). Compositae: Introduction with Key to Tribes. 7 in Families and Genera of Vascular Plants. Flowering Plants, Eudicots, *Asterales*. Springer-Verlag,

and L. Monzote3

treated, aspects that should motivate future research of our work group.

, M.F. Quijano2

\*Address all correspondence to: pmanzano@espol.edu.ec

3 Institute of Tropical Medicine Pedro Kouri, Havana, Cuba

dad, Santo Domingo de Heredia, Costa Rica.

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[28] Manzano Santana PI, Miranda Martínez M, de Oca Porto RM, Orellana León T, Abreu Payrol J, Peralta García EL. (2013c). Chemical study of lipid compounds of the leaves, stems and flowers *Vernonanthura patens* (Kunth) H. Rob. (Asteraceae). Cuban Journal of Medicinal Plants, 18(4).

[17] Borkosky S, Ponce S, Gabriela Juárez G, González M, Bardón A. (2009). Molluscicidal sesquiterpene lactones from species of the tribe Vernonieae (Compositae). Chemistry

[18] Mabry TJ, Abdel-Baset Z, Padolina WG. (1975). Systematic implications of flavo‐ noides and sesquiterpene lactones in species of *Vernonia*. Biochemical Systematics

[19] Jakupovic J, Schmedia-Hirschmann G. (1986). Hirsutinolides, glaucolides and sesqui‐

[20] Gupta M, Monge A. (1986). Screening of Panamanian medicinal plants for brine shrimp toxicity, crown gall tumor inhibition, cytotoxicity and DNA intercalation. In‐

[21] Fournet A, Barrios A. (1994). Leishmanicidal and trypanocidal activities of Bolivian

[22] Valadeau C, Pabon A, Deharo E, Albán-Castillo J, Estévez Y, Lores F, Rojas R, Gam‐ boa D, Sauvain M, Castillo D, Bourdy G. (2009). Medicinal plants from the Yanesha (Peru): evaluation of the leishmanicidal and antimalarial activity of selected extracts.

[23] Manzano Santana PI, Silva Osorio M, Sterner O, Peralta Garcìa EL. (2012). Phyto‐ chemical Studies of Fractions and Compounds Present in *Vernonanthura patens* with Antifungal Bioactivity and Potential as Antineoplastic, Phytochemicals—A Global Perspective of Their Role in Nutrition and Health, Dr Venketeshwer Rao (Ed.). ISBN:

[24] Santos E, Manzano P, Navarrete O, Hidalgo L, Miranda M, Peralta E. (2012a). *Verno‐ nanthura patens* maturase K (matK) gene, partial cds; chloroplast. National Center for Biotechnology Information. GenBank: JX276535.1. http://www.ncbi.nlm.nih.gov/

[25] Santos E, Manzano P, Navarrete O, Hidalgo L, Miranda M, Peralta E. (2012b). *Verno‐ nanthura patens* ribulosa-1,5-bifosfato carboxilasa/oxigenasa subunidad grande (rbcL) de genes, cds parcial; cloroplasto National Center for Biotechnology Information. GenBank: JX276536.1. http://www.ncbi.nlm.nih.gov/nuccore/JX276535.1; http://

[26] Manzano Santana P, Miranda Martínez M, Abreu Payrol J, Silva M, Olov S, Peralta E. (2013a). Pentacyclic triterpenoids with antimicrobial activity from the leaves of *Ver‐ nonanthura patens* (Asteraceae). Emirates Journal of Food and Agriculture, 25(7). doi:

[27] Manzano Santana PI, Orellana León T, Miranda Martínez M, Abreu Payrol J, Ruíz O, Peralta García EL. (2013b). Some pharmacognostic parameters of *Vernonanthura pat‐ ens* (Kunth) H. Rob. (Asteraceae) endemic to Ecuador. Cuban Journal of Medicinal

terpene lactones in species of *Vernonia*. Phytochemistry, 25(2): 145–158.

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Plants, 18(1).


### **Phytochemical Profile of Honey**

Aldina Kesić, Nadira Ibrišimović-Mehmedinović and Almir Šestan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60087

#### **1. Introduction**

During the process of cellular respiration in living organisms some reactive oxygen species (ROS) are constantly forming, sometimes as a result of exogenous sources such as pollution, radiation and ionizing radiation and drugs [1, 2, 3, 4]. They can harm vitally important structures, such as cell membranes, destroying deoxyribonucleic acid (DNA) that is an essential core component of every cell and damaging the respiratory enzymes and genetic material, thus creating the preconditions for the emergence of degenerative and malignant diseases [5, 6, 7, 8].

Production of prooxidant species, especially ROS, is in equilibrium with the antioxidant protection of the organism under normal conditions.

Increased production of pro-oxidants and / or reduced antioxidant protection of the organism can lead to tissue damage and disease. This situation is called oxidative stress, which is the cause or contributing factor in the pathology of many diseases [9].

So, if there is a genetic predisposition or exposure to external factors such as cigarette smoke, sun light, pollution, etc., the balance of prooxidants / antioxidants may be impaired (Figure 1).

Oxidative stress, induced mainly by ROS is now recognized as a major cause of several human health disorders (Figure1) [10, 11]. Antioxidants of both endogenous and exogenous sources have the ability to donate electrons and are effective in mitigating the damaging effects of ROS on cellular components [12]. Honey is a complex mixture of more than seventy different compounds present in varying proportions [13,14]. Many of these phytochemicals such as polyphenols and flavonoids present in honey have antioxidant properties [15,16, 17,18,19]. In view of the importance of the antioxidant phytochemicals present in honey, we undertook a

**Figure 1.** ROS: Occurrence and major consequences on the human body.

study to characterize the photochemical profiles of several honey samples from different botanical and geographical origins.

#### **2. Methods and materials**

The study included 60 samples of honey from different botanical and geographical origin. Samples of honey were collected in cooperation with the Union of Beekeepers of Tuzla Canton, Bosnia and Herzegovina. Information about the botanical and geographical origin of the samples was collected from the producers themselves. The analysis of total antioxidants in the samples analyzed, and an analysis of the content of polyphenols were made [20, 21, 22]. Then, statistical analysis of the data was performed and examined the correlation between the total antioxidant capacity of honey and polyphenol content. The total content of antioxidants in the samples was determined by Ferric Reducing Antioxidant Power (FRAP) method [23, 24, 25]. Total polyphenols in the samples were determined using the Folin-Ciocalteu (FC) method [26].

#### **2.1. Determination of total antioxidant capacity of honey using the FRAP method**

In order to determine the antioxidant capacity, a lot of methods based on different mechanisms of antioxidant defensce systems were developed, such as the removal or inhibition of free radicals or chelating metal ions, which would otherwise lead to the formation of free radicals.

The method used in our study is an indirect FRAP method. The FRAP is a method that was described in 1996 by Benzie and Strain, but in 1999 was further modified [25, 27]. This is a simple, quick test method based on the reduction of iron from ferric Fe3+ into the ferrous Fe2+ form in the presence of antioxidants, where at low pH level, a deep- blue colored complex of ferro tripyridyltriazine is developed, which has an absorption maximum at 593 nm (Figure 2.). The reaction is not specific. The results are expressed as the μmol Fe2+ equivalent of (Fe) / mL sample.

**Figure 2.** Ferrum reduction reaction - 2,4,6-tripyridyl-s-triazine (TPTZ).

Analysis results were determined by measuring absorbance of standard solutions, which have been incorporated in the calibration line.

The concentration of antioxidants in the measured sample on the basis of the obtained value is calculated according to the formula:

$$X = \frac{Y - 0.0243}{0.0011}$$

where:

study to characterize the photochemical profiles of several honey samples from different

The study included 60 samples of honey from different botanical and geographical origin. Samples of honey were collected in cooperation with the Union of Beekeepers of Tuzla Canton, Bosnia and Herzegovina. Information about the botanical and geographical origin of the samples was collected from the producers themselves. The analysis of total antioxidants in the samples analyzed, and an analysis of the content of polyphenols were made [20, 21, 22]. Then, statistical analysis of the data was performed and examined the correlation between the total antioxidant capacity of honey and polyphenol content. The total content of antioxidants in the samples was determined by Ferric Reducing Antioxidant Power (FRAP) method [23, 24, 25]. Total polyphenols in the samples were determined using the Folin-Ciocalteu (FC) method [26].

botanical and geographical origins.

**Figure 1.** ROS: Occurrence and major consequences on the human body.

100 Phytochemicals - Isolation, Characterisation and Role in Human Health

**2. Methods and materials**

X – antioxidant concentration

Y – sample average absorbance

The results are expressed in μmol FeII/L 10 % of honey solution.

#### **3. Determination of total polyphenols using the FC method**

The content of total polyphenols in the honey samples was determined using the FC method. The FC method is one of the oldest indirect methods that is sensitive to phenolic and poly‐ phenolic compounds. This method has been standardized and widely used in the determina‐ tion of polyphenols.

### **4. Results and discussion**

The components of honey, with antioxidant properties, are phenolic acids and flavonoids, enzymes, ascorbic acid, organic acids, amino acids, proteins and some micro biogenic elements [28, 29]. Honey characterization helps us to understand its antioxidant characteristics, thereby, its use as natural foodstuff, i.e., as sources of antioxidant human nutrition.

Our results have shown that antioxidant activity of honey from Bosnia and Herzegovina is in the range of 4.7 μMFeII/L to 1606.54 μMFeII/L.

According to Jerkovic and co-workers´ research, antioxidant activity of honey from the Croatian territory was from 101.5 μMFe to 955.9 μMFe [30].

With the analysis of total antioxidant activity along with the concentration of polyphenols, we came to the conclusion that there has been a significant correlation between these two parameters.

This conclusion is confirmed by research scientist Al-Mamary et-al. [31], who showed that antioxidant activity of several types of honey originating from different countries depends on the concentration of phenolic groups.

In addition, researches and other antioxidant activities of honey that support this assertion were also included [32, 33, 34, 35].

Our results are also in correlation with the results of the Slovenian scientists [26] who, with their studies, confirmed the polyphenol content of 4.48 mg/100 g in locust honey and 24.14 mg/ 100g in forest honey. Italian scientists [35] confirmed the contents of polyphenols in honey in the value of 3 mg/100g to 17.5 mg/100g. These results show slightly lower values of polyphe‐ nols in Italian honey samples compared to the honey from the territory of Bosnia and Herze‐ govina.

A high correlation coefficient between the antioxidant activity of honey and total polyphenol content has also been confirmed in our studies (Figure 3):

With statistical analysis of the given results, we got Pearson's correlation coefficient that is equal to 0.957. This means that 95.7% of honey samples with a stronger antioxidant activity have higher polyphenol content.

The chemical composition of honey largely depends on botanical origin as an important factor. According to the botanical origin, analyzed honey samples are divided into several groups:

**Figure 3.** Correlation between total antioxidant activity of honey and polyphenol content

**3. Determination of total polyphenols using the FC method**

its use as natural foodstuff, i.e., as sources of antioxidant human nutrition.

tion of polyphenols.

parameters.

govina.

**4. Results and discussion**

the range of 4.7 μMFeII/L to 1606.54 μMFeII/L.

102 Phytochemicals - Isolation, Characterisation and Role in Human Health

the concentration of phenolic groups.

were also included [32, 33, 34, 35].

have higher polyphenol content.

Croatian territory was from 101.5 μMFe to 955.9 μMFe [30].

content has also been confirmed in our studies (Figure 3):

The content of total polyphenols in the honey samples was determined using the FC method. The FC method is one of the oldest indirect methods that is sensitive to phenolic and poly‐ phenolic compounds. This method has been standardized and widely used in the determina‐

The components of honey, with antioxidant properties, are phenolic acids and flavonoids, enzymes, ascorbic acid, organic acids, amino acids, proteins and some micro biogenic elements [28, 29]. Honey characterization helps us to understand its antioxidant characteristics, thereby,

Our results have shown that antioxidant activity of honey from Bosnia and Herzegovina is in

According to Jerkovic and co-workers´ research, antioxidant activity of honey from the

With the analysis of total antioxidant activity along with the concentration of polyphenols, we came to the conclusion that there has been a significant correlation between these two

This conclusion is confirmed by research scientist Al-Mamary et-al. [31], who showed that antioxidant activity of several types of honey originating from different countries depends on

In addition, researches and other antioxidant activities of honey that support this assertion

Our results are also in correlation with the results of the Slovenian scientists [26] who, with their studies, confirmed the polyphenol content of 4.48 mg/100 g in locust honey and 24.14 mg/ 100g in forest honey. Italian scientists [35] confirmed the contents of polyphenols in honey in the value of 3 mg/100g to 17.5 mg/100g. These results show slightly lower values of polyphe‐ nols in Italian honey samples compared to the honey from the territory of Bosnia and Herze‐

A high correlation coefficient between the antioxidant activity of honey and total polyphenol

With statistical analysis of the given results, we got Pearson's correlation coefficient that is equal to 0.957. This means that 95.7% of honey samples with a stronger antioxidant activity

The chemical composition of honey largely depends on botanical origin as an important factor. According to the botanical origin, analyzed honey samples are divided into several groups: meadow, acacia, forest, mixed, lime, chestnut honey, mountain honey, hawthorn, heather, sage and rosemary. Different antioxidant activity is shown in honey samples of different botanical origin. Antioxidant activity of honey samples divided according to botanical origin grows according to the following sequence: hawthorn <acacia <lime <meadow <chestnut <mixed <sage <ling <forest <mountain <rosemary. This relationship is shown in Figure 4:

**Figure 4.** Total antioxidant activity of honey samples of different botanical origin

Rosemary honey has got the highest antioxidant activity. Because of its relatively high content of antioxidants and low content of essential oil and chlorophyll, rosemary is the best source of natural antioxidants. Plants such as rosemary, sage, oregano, thyme, pepper and green tea, contain active substances with strong antioxidant activities. Rosemary extracts have a high concentration of active substances carnosol acid, carnosol, methyl carnosol and rosemary acid. Besides carnosol acid and its derivatives which are the most powerful natural antioxidants, rosemary extract also contains ursolic and oleanolic acid (in its chemical composition triter‐ penoids).

Rosemary honey showed the highest antioxidant activity since rosemary is a significant source of natural antioxidants. Although the data showed [36] that hawthorn is a significant source of antioxidants, in our analysis, hawthorn honey showed a lower antioxidant activity com‐ pared to other analyzed honey samples. Since our study determined the total antioxidant activity of aqueous solutions of honey, there is a possibility that hawthorn contains more antioxidants, that are not soluble in water.

If we divide the analyzed honey samples according to their geographical origin, we can say that the samples from the middle Bosnia are of the richest sources of antioxidants, followed by specimens from North-eastern Bosnia, and slightly weaker source of antioxidants are samples of honey from the north and west of Bosnia and Herzegovina (Figure 5).

**Figure 5.** Antioxidant activity of honey samples from different geographic origin

The content of polyphenols in honey is affected by the botanical origin of honey besides having a significant impact on the overall antioxidant activity (Figure 6).

**Figure 6.** Polyphenol content in honey of different botanical origin.

Rosemary honey has got the highest antioxidant activity. Because of its relatively high content of antioxidants and low content of essential oil and chlorophyll, rosemary is the best source of natural antioxidants. Plants such as rosemary, sage, oregano, thyme, pepper and green tea, contain active substances with strong antioxidant activities. Rosemary extracts have a high concentration of active substances carnosol acid, carnosol, methyl carnosol and rosemary acid. Besides carnosol acid and its derivatives which are the most powerful natural antioxidants, rosemary extract also contains ursolic and oleanolic acid (in its chemical composition triter‐

Rosemary honey showed the highest antioxidant activity since rosemary is a significant source of natural antioxidants. Although the data showed [36] that hawthorn is a significant source of antioxidants, in our analysis, hawthorn honey showed a lower antioxidant activity com‐ pared to other analyzed honey samples. Since our study determined the total antioxidant activity of aqueous solutions of honey, there is a possibility that hawthorn contains more

If we divide the analyzed honey samples according to their geographical origin, we can say that the samples from the middle Bosnia are of the richest sources of antioxidants, followed by specimens from North-eastern Bosnia, and slightly weaker source of antioxidants are

samples of honey from the north and west of Bosnia and Herzegovina (Figure 5).

**Figure 5.** Antioxidant activity of honey samples from different geographic origin

a significant impact on the overall antioxidant activity (Figure 6).

The content of polyphenols in honey is affected by the botanical origin of honey besides having

penoids).

antioxidants, that are not soluble in water.

104 Phytochemicals - Isolation, Characterisation and Role in Human Health

According to the results of the study, the polyphenol content is increased according to the following order: acacia <lime <chestnut <meadow <mixed <winter savory <sage <forest <rosemary <hawthorn <mountain.

By analyzing these results we can say that the botanical origin significantly affects the polyphenol content and the total antioxidant activity of honey samples. Influence of polyphe‐ nol content to total antioxidant activity is shown in Figure 7:

**Figure 7.** Polyphenol influence on antioxidant activity of honey samples of different botanical origin.

Most samples showed a correlation between polyphenol content and total antioxidant activity.

Exceptions are the samples of honey from the hawthorn where this correlation does not exist. Polyphenols also exhibit antioxidant activity in the presence of copper ions and prooxidant action. So, it can be assumed that the honey samples contain higher amounts of this micro biogenic element, as it affects the overall antioxidant activity.

#### **5. Conclusion**

Honey, as a natural food product, is known to be a significant source of antioxidants in the human body. The total antioxidant activity of honey is the result of its complex chemical composition and the complex interactions between different substances. There is a high degree of correlation among the total antioxidant activity and polyphenol content in honey. The chemical composition of honey is largely dependent on its botanical origin. The antioxidant activity of honey samples from different botanical origin is different. Nectarian honey samples are lower than forest honey as a source of antioxidants and have a lower content of polyphe‐ nols.

Influence of geographical origin of honey can be linked to the influence of botanical origin. Different plant cover is developed at different geographical locations and, therefore, under different climatic conditions. Therefore, the phytochemical profile present in honey, to a large extent, is a picture of the botanical and geographical origin of honey. The samples of honey from central Bosnia are the richest sources of antioxidants, followed by specimens from northeastern Bosnia - a slightly weaker source of antioxidants are samples of honey from the northern and western parts of Bosnia and Herzegovina.

#### **Author details**

Aldina Kesić\* , Nadira Ibrišimović-Mehmedinović and Almir Šestan

\*Address all correspondence to: aldina.kesic@untz.ba

University in Tuzla, Faculty of Science, Department of Chemistry, Bosnia and Herzegovina

#### **References**

[1] Ames B N, Shigenaga M, Hagen T. Oxidants, antioxidants, and the degenerative dis‐ eases of aging. Proc. Natl. Acad. Sci. USA. 1993; 90: 7915-7922.

[2] Gutteride JMC, Halliwell B. Free radicals and antioxidants in aging and disease; fact of fantasy. In Antioxidants in nutrition, heath, and disease. Oxford, UK: Oxford Uni‐ versity Press. 1994; 111-135.

Most samples showed a correlation between polyphenol content and total antioxidant activity.

Exceptions are the samples of honey from the hawthorn where this correlation does not exist. Polyphenols also exhibit antioxidant activity in the presence of copper ions and prooxidant action. So, it can be assumed that the honey samples contain higher amounts of this micro

Honey, as a natural food product, is known to be a significant source of antioxidants in the human body. The total antioxidant activity of honey is the result of its complex chemical composition and the complex interactions between different substances. There is a high degree of correlation among the total antioxidant activity and polyphenol content in honey. The chemical composition of honey is largely dependent on its botanical origin. The antioxidant activity of honey samples from different botanical origin is different. Nectarian honey samples are lower than forest honey as a source of antioxidants and have a lower content of polyphe‐

Influence of geographical origin of honey can be linked to the influence of botanical origin. Different plant cover is developed at different geographical locations and, therefore, under different climatic conditions. Therefore, the phytochemical profile present in honey, to a large extent, is a picture of the botanical and geographical origin of honey. The samples of honey from central Bosnia are the richest sources of antioxidants, followed by specimens from northeastern Bosnia - a slightly weaker source of antioxidants are samples of honey from the

, Nadira Ibrišimović-Mehmedinović and Almir Šestan

eases of aging. Proc. Natl. Acad. Sci. USA. 1993; 90: 7915-7922.

University in Tuzla, Faculty of Science, Department of Chemistry, Bosnia and Herzegovina

[1] Ames B N, Shigenaga M, Hagen T. Oxidants, antioxidants, and the degenerative dis‐

biogenic element, as it affects the overall antioxidant activity.

106 Phytochemicals - Isolation, Characterisation and Role in Human Health

northern and western parts of Bosnia and Herzegovina.

\*Address all correspondence to: aldina.kesic@untz.ba

**5. Conclusion**

**Author details**

Aldina Kesić\*

**References**

nols.


[33] Busserolles J, Gueux E, Rock E, Mazur A, Rayssiguier Y. Substitutinghoney for re‐ fined carbohydrates protects rats from hypertriglyceridemicand prooxidative effects of fructose. J Agric Food Chem. 2002; 50: 5870–5877.

[18] Beretta G, Granata P, Ferrero M, Orioli M, Facino RM. Standardization of antioxidant properties of honey by a combination of spectrophotometric/fluorimetric assays and

[20] Gryglewski RJ, Korbut R, Robak J. On the mechanism of antithrombotic action of fla‐

[21] Frankel E. Nutritional benefits of flavonoids. International conference on food fac‐ tors: Chemistry and cancer prevention, Hamamatsu, Japan. Abstracts. 1995; C 6-2. [22] Husain SR, Cillard J, Cillard P. Hydroxyl radical scavenging activity of flavonoids,

[23] Foti M, Piatelli M, Baratta MT, Ruberto G. Flavonoids, coumarins and cinnamic acids as antioxidants in a micellar system. Structure-activity relationship. J. Agric. Food

[24] Bors W, Heller W, Michel C, Saran M. Flavonoids as antioxidants: determination of

[25] Halliwel B, Aeschbach R, Loliger J, Aruoma Ol. The characterization of antioxidants.

[26] Jasna B, Urška D, Mojca J, Terezija G. "Evaluation of the phenolic content, antioxi‐ dant activity and colour of Slovenian honey" Analytical, Nutritional and Clinical

[27] Vraćar LJ. Priručnik za kontrolu svježeg i prerađenog voća, povrća i pećurki i osvje‐

[28] Šenk N. Vrednotenje medu: zveza med barvo medu in antioksidativno aktivnostjo.

[29] Andrade P, Ferreres F, Amaral MT. Analysis of honey phenolic acidsby HPLC, its application to honey botanical characterization. J. Liq. Chrom. Rel. Technol. 1997;

[30] Igor J, Zvonimir M, Oak (Quercus frainetto Ten.) Honeydew Honey—Approach to Screening of Volatile Organic Composition and Antioxidant Capacity (DPPH and

[31] Socha R, Juszczak L, Pietrzyk S, Fortuna T. Antioxidant activity and phenolic compo‐

[32] Al-Mamary M, Al-Meery A, Al-Habori M. Antioxidant activities and total phenolic

of different types of honey. Nutrition Research, 2002; 22, 9: 1041-1047

FRAP Assay, Molecules), 2010; 15: 3744-3756. doi:10.3390/molecules15053744

radical-scavenging efficiencies, Methods Enzymol. 1990; 186: 343–355.

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Phytochemistry. 1987; 26: 2489–2491.

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Food Chem Toxicol 1995; 33: 601-17

Methods Food Chemistry. 2007; 2,105: 822-828

žavajućih bezalkoholnih pića. Novi Sad. 2001;

Univerzitet u Ljubljani, Biotehnički fakultet. 2006; 8-19.

sition of herbhoneys. Food Chemistry. 2009; 113, 2: 568-574

Chem. 1996; 44: 497-501.

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[19] Anonymous. Flavonoids in honey. 2004; http://www.chemsoc.org/


**Specific Bioactive Phytochemicals**

### **Coumarins — An Important Class of Phytochemicals**

Maria João Matos, Lourdes Santana, Eugenio Uriarte, Orlando A. Abreu, Enrique Molina and Estela Guardado Yordi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59982

#### **1. Introduction**

Phytochemicals are chemical compounds that occur naturally in the plant kingdom. Some are responsible for the organoleptic properties of the natural sources in which they are present. The term is generally used to refer to those chemicals that may have biological significance, for example carotenoids, flavonoids, coumarins, or chromones, but not all are established as essential nutrients. There may be as many as 4,000 different phytochemicals having potential activity against several diseases such as cancer and metabolic or degenerative diseases.

Among them, coumarins are a family of benzopyrones (1,2-benzopyrones or 2H-1-benzopyr‐ an-2-ones) widely distributed in the nature. They represent an important family of naturally occurring and/or synthetic oxygen-containing heterocycles, bearing a typical benzopyrone framework (Figure 1) [1].

**Figure 1.** Chemical structure of coumarin and the IUPAC numeration of this scaffold.

The name coumarin comes from a French term for the Tonka bean, *coumarou*, seeds of *Dipteryx odorata* (*Coumarouna odorata*) (*Fabaceae*/*Leguminosae*), one of the sources from which coumarin was first isolated as a natural product in 1820. It has a sweet odor, easy to be recognized as the

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

scent of new-mown hay; because of that, coumarin has been used in perfumes since 1882. It is presumed to be produced by plants as a chemical defense to discourage predation [2, 3].

Coumarinic compounds are a class of lactones structurally constructed by a benzene ring fused to α-pyrone ring, and essentially possess - conjugated system with rich electron and good charge-transport properties [4, 5]. The simplicity and versatility of the coumarin scaffold make it an interesting starting-point for a wide range of applications [6-8]. There are coumarins as perfumes, cosmetics, and industrial additives. Some of its derivatives have been used as aroma enhancers in tobaccos and certain alcoholic drinks [9, 10]. But their most relevant role is described in natural products, organic chemistry, and medicinal chemistry [11, 12]. The extraction, synthesis, and evaluation of coumarins have become an extremely attractive and rapidly developing topic [13, 14]. Moreover, a lot of coumarin compounds as medicinal candidates for drugs with strong pharmacological activity, low toxicity and side effects, fewer drug resistance, high bioavailability, broad spectrum, better curative effects, etc., to treat various types of diseases are being actively studied [15]. Several efforts have been made mainly in developing coumarin-based anticoagulant, antioxidant [16], antimicrobial (anti-viral, antifungal, and anti-parasitic) [10, 17], anticancer [18-20], anti-diabetic, analgesic, antineurodegenerative, and anti-inflammatory agents [10, 21]. Moreover, the unique and versatile oxygen-containing heterocyclic structure makes coumarin compounds occupy an important place in medicinal chemistry [22, 23]. In addition, studies have been done regarding coumarins as bioactive agents [24], as well as supramolecular medicinal drugs, diagnostic agents and pathologic probes, and biological stains [25]. Particularly, the large - conjugated system in the coumarinic ring, with electron-rich and charge-transport properties, is important in the interaction of this scaffold with molecules and ions. Coumarin-based ion receptors, fluorescent probes, and biological stains are growing quickly and have extensive applications to monitor timely enzyme activity, complex biological events, as well as accurate pharmacological and pharmacokinetic properties in living cells [26, 27].

Coumarin was first synthesized in 1868, and it was used in the pharmaceutical industry as a precursor in the synthesis of a number of synthetic anticoagulant pharmaceuticals, starting with dicoumarol (removed from the current therapy) [28]. So far, some interesting coumarinbased anticoagulant drugs have extensively been used in clinics [29]. Coumarins are a type of vitamin K antagonists [30]. The most notable ones are warfarin, acenocumarol, and phenpro‐ coumon, currently in use in several countries [31, 32]. Warfarin is employed more frequently than acenocoumarol because of its longer half-life (36 h), theoretically providing more stable anticoagulation and avoiding factor VII fluctuations that potentially occur during acenocou‐ marol treatment (half-life 10 h) [33]. Nowadays, some coumarins proved to be enzymatic inhibitory agents [monoamine oxidase (MAO) inhibitors, acetylcholinesterase (AChE) inhibitors, and butyrylcholinesterase (BuChE) inhibitors] with great potential in neurodege‐ nerative diseases (ND) [34-38]. These studies represent an important tendency in the coumar‐ in's chemistry and biological evaluation [39-41].

Therefore, the coumarin ring is prevalently applied to construct several functional molecules in the medicinal field. A great deal of work has been done directed towards the separation and purification of naturally occurring biological coumarins from a variety of plants, animals, and microorganisms, as well as towards the artificial synthesis of coumarin compounds with novel structures and properties [42]. Coumarin compounds as medicinal drugs have been increas‐ ingly attracting special interest due to their underlying outstanding contributions in the prevention and treatment of diseases, and the related researches and developments have become an extremely attractive highlighted area.

In this context, an overview of the role of coumarins as important phytochemicals and their interesting applications will be presented and discussed. The origin, natural sources, biosyn‐ thesis, and applications are going to be presented in this chapter.

### **2. Natural occurring coumarins**

scent of new-mown hay; because of that, coumarin has been used in perfumes since 1882. It is presumed to be produced by plants as a chemical defense to discourage predation [2, 3].

Coumarinic compounds are a class of lactones structurally constructed by a benzene ring fused to α-pyrone ring, and essentially possess - conjugated system with rich electron and good charge-transport properties [4, 5]. The simplicity and versatility of the coumarin scaffold make it an interesting starting-point for a wide range of applications [6-8]. There are coumarins as perfumes, cosmetics, and industrial additives. Some of its derivatives have been used as aroma enhancers in tobaccos and certain alcoholic drinks [9, 10]. But their most relevant role is described in natural products, organic chemistry, and medicinal chemistry [11, 12]. The extraction, synthesis, and evaluation of coumarins have become an extremely attractive and rapidly developing topic [13, 14]. Moreover, a lot of coumarin compounds as medicinal candidates for drugs with strong pharmacological activity, low toxicity and side effects, fewer drug resistance, high bioavailability, broad spectrum, better curative effects, etc., to treat various types of diseases are being actively studied [15]. Several efforts have been made mainly in developing coumarin-based anticoagulant, antioxidant [16], antimicrobial (anti-viral, antifungal, and anti-parasitic) [10, 17], anticancer [18-20], anti-diabetic, analgesic, antineurodegenerative, and anti-inflammatory agents [10, 21]. Moreover, the unique and versatile oxygen-containing heterocyclic structure makes coumarin compounds occupy an important place in medicinal chemistry [22, 23]. In addition, studies have been done regarding coumarins as bioactive agents [24], as well as supramolecular medicinal drugs, diagnostic agents and pathologic probes, and biological stains [25]. Particularly, the large - conjugated system in the coumarinic ring, with electron-rich and charge-transport properties, is important in the interaction of this scaffold with molecules and ions. Coumarin-based ion receptors, fluorescent probes, and biological stains are growing quickly and have extensive applications to monitor timely enzyme activity, complex biological events, as well as accurate pharmacological and

Coumarin was first synthesized in 1868, and it was used in the pharmaceutical industry as a precursor in the synthesis of a number of synthetic anticoagulant pharmaceuticals, starting with dicoumarol (removed from the current therapy) [28]. So far, some interesting coumarinbased anticoagulant drugs have extensively been used in clinics [29]. Coumarins are a type of vitamin K antagonists [30]. The most notable ones are warfarin, acenocumarol, and phenpro‐ coumon, currently in use in several countries [31, 32]. Warfarin is employed more frequently than acenocoumarol because of its longer half-life (36 h), theoretically providing more stable anticoagulation and avoiding factor VII fluctuations that potentially occur during acenocou‐ marol treatment (half-life 10 h) [33]. Nowadays, some coumarins proved to be enzymatic inhibitory agents [monoamine oxidase (MAO) inhibitors, acetylcholinesterase (AChE) inhibitors, and butyrylcholinesterase (BuChE) inhibitors] with great potential in neurodege‐ nerative diseases (ND) [34-38]. These studies represent an important tendency in the coumar‐

Therefore, the coumarin ring is prevalently applied to construct several functional molecules in the medicinal field. A great deal of work has been done directed towards the separation and purification of naturally occurring biological coumarins from a variety of plants, animals, and

pharmacokinetic properties in living cells [26, 27].

114 Phytochemicals - Isolation, Characterisation and Role in Human Health

in's chemistry and biological evaluation [39-41].

Coumarin (Figure 1) and its derivatives are an important group of natural compounds widely distributed in the natural kingdom [43]. They can be found in the integument of seeds, fruits, flowers, roots, leaves, and stems, although the largest concentration is generally in fruits and flowers [44]. Originally, coumarin was isolated from the seed of *D. odorata*. Coumarins are secondary metabolites of higher plants, few microorganisms (bacteria and fungi), and sponges [45]. The function of this type of end product of secondary metabolism is related to defense mechanisms against herbivores and attacks by microorganisms. These compounds are biosynthesized from phenylalanine via the shikimic acid [46]. Natural coumarins are generally unsaturated lactones and comprise another class of compounds C6C3. Almost all the natural coumarins have an oxygenated substituent at position 7 [47], either free as in hydroxylated umbelliferone, or combined (methyl, sugars, etc.) in other derivatives. Structurally, they are considered derivatives of the *ortho*-hydroxy-cinnamic acid.

There are different classifications for the coumarin derivatives. Generally, they can be chem‐ ically classified according to the most common cores: simple coumarins, complex coumarins, and various coumarins. More complex coumarins are generally fused with other heterocycles [3]. Therefore, they can be classified as: simple coumarins, furocoumarins, dihydrofurocou‐ marins, pyranocoumarins (linear and angular), phenylcoumarins, and biscoumarins [1]. As said before, hundreds of coumarins have been identified in natural sources, especially plants [48, 49]. Major coumarin constituents isolated from plants include: simple hydroxycoumarins, furocoumarins and isofurocoumarins, pyranocoumarins, biscoumarins, and dihydroisocou‐ marins (Figure 2) [1].

Coumarins have been isolated from hundreds of plants species distributed in more than 40 different families. There were isolated more than different 1300 coumarins, well distributed in *Angiospermae*, *Monocotyledoneae* and *Dicotyledoneae* families. Orders with occurrence numbers > 100 are *Araliales*, *Rutales*, *Asterales*, *Fabales*, *Oleales*, *Urticales*, and *Thymelaeales*. Families with occurrence numbers > 100 are *Apiaceae* (*Umbelliferae*), *Rutaceae*, *Asteraceae* (*Compositae*), *Fabaceae* (*Leguminosae*), *Oleaceae*, *Moraceae*, and *Thymelaeaceae*, respectively (Figure 3) [50]. The best known and researched coumarins in the field of phytochemistry, pharmacology, medicinal chemistry, and the food science can be found in these families. Therefore, these are the coumarins that are going to be further addressed in the next sections of this chapter.

**Figure 2.** Principal types of coumarins isolated from plants.

**Figure 3.** Number of coumarins presented in seven different families of plants[50].

Coumarins usually are in the free state in plants as they are polar structures, and many of them can sublimate. They might also be found in the form of glycosides, including psoralen corerelated structures [44]. They are characterized by UV light absorption, resulting in a very characteristic blue fluorescence; they are also very photosensitive as they can be altered by natural light [44]. These features are used in the isolation and analysis, as well as in unusual therapies such as photochemotherapy and the industry of chemical sensors [51, 52].

#### **3. Biosynthesis of coumarins**

Simple coumarins are biogenetically derived from shikimic acid, via cinnamic acid. The specificity of the process is the C-2 hydroxylation, producing a break (*β*-oxidation) of the side chain (i.e. *Salix* spp.), or chain isomerization and subsequent lactonization, generating the umbelliferone. Figure 4 explains the entire process [46, 53].

**Figure 4.** Biosynthesis of simple coumarins.

**Figure 3.** Number of coumarins presented in seven different families of plants[50].

**Figure 2.** Principal types of coumarins isolated from plants.

116 Phytochemicals - Isolation, Characterisation and Role in Human Health

Coumarins usually are in the free state in plants as they are polar structures, and many of them can sublimate. They might also be found in the form of glycosides, including psoralen corerelated structures [44]. They are characterized by UV light absorption, resulting in a very characteristic blue fluorescence; they are also very photosensitive as they can be altered by natural light [44]. These features are used in the isolation and analysis, as well as in unusual

therapies such as photochemotherapy and the industry of chemical sensors [51, 52].

Pyrano and furocoumarins (Figure 2) are also biogenetically derived from shikimic acid. These coumarins could be divided in two groups—lineal and angular—depending on the position where the isopentenyl pyrophosphate is condensed to further cyclize and form the heterocycle. The biosynthesis of these complex coumarins could also be the result of the cyclization of a simple coumarin previously prenylated [53].

Among the coumarins classified as "various" is the dicoumarol, which is formed by bacterial fermentation of Yellow Sweet Clover, and was isolated for the first time from decomposed leaves of *Melilotus albus* (*Fabaceae*/*Leguminosae*).

An approximation for the dicumarol biogenesis is the hydroxylation of the 4-position of the coumarin, that then captures a molecule of formaldehyde and is condensed with another molecule of 4-hydroxycoumarin, and finally enolize the keto group forming the dicumarol [46].

From a chemotaxonomic approach, Ribeiro & Kaplan (2002) evidenced that the diversity and structural complexity of the coumarins constitute an example of higher plant evolution. Simple coumarins are the most common in all angiosperms, especially in *Oleaceae* and *Asteraceae*, and their occurrence is of 100% and 98, 68%, respectively [50]. The second most prevalent coumar‐ ins are furocoumarins and pyranocoumarins. Coumarin in some families are high (*Thymelaea‐ ceae*, *Rutaceae*, *Apiaceae*, *Fabaceae*, and *Moraceae*). In the case of well-diversified structural types in *Apiaceae* and *Rutaceae*, coumarins are considered as chemotaxonomic markers [50].

*Apiaceae* is the major source of coumarins (Figure 3) and one of the more diverse, containing five different types of coumarin derivatives (simple coumarins, lineal furocoumarins, angular furocoumarins, lineal pyranocoumarins, and angular pyranocoumarins) [50, 54]. *Rutaceae* is also highlighted in both occurrence and diversification. Generally, the division *Angiospermae* is preferably rich in simple coumarins, followed by the furo and pyranocoumarins [50].

#### **4. Coumarins in medicinal plants**

A large number of valuable species used commonly as medicinal plants, aromatic plants, and edible plants for human and animal feeding belongs to coumarin-rich plant families. Among them are species with well-documented biological activity, in which coumarins are part of the active principles. Table 1 shows a selection of plants of these families (first listed seven families with number of occurrence > 100) and some other families with species of particular pharma‐ cological interest on chronic diseases. Coumarins presenting great pharmacological interest have been isolated in different geographical regions from other botanical families. Also shown are the coumarin compounds having species and their yield (if available).

Most of these plants are well known by people and scientists as part of herbal medicine repertories in Europe, Asia, or the Americas [55-58]. From the list, several coumarin-containing species or genera have also ethnomedical records in Cuba and the Caribbean Basin [59, 60]. Among of plant included are species with a great historical record of ethnomedicinal uses, and are present in *traditional medicine systems*: Ayurveda Medicine, Traditional Chinese Medicine and Unani Medicine, or in other recent cultures. Also, renowned species used on conventional therapeutics and modern herbal medicine are included, ie. *Aesculus hippocastanum* (Horsechestnut), *Passiflora incarnata* (Passion Flower), *Lawsonia inermis* (Henna), *Hypericum perfora‐ tum* (Saint John Wort), *Tilia cordata* (Lime Tree) and *Uncaria tomentosa* (Cat's Claw).

Coumarins are also present in several species belonging to different botanical families, which are widespread in the northeastern region of Brazil [61]. Some of them are reported in folk medicine as traditional remedies drugs for the treatment of respiratory diseases [55]. Many pharmacological activities have been ascribed to coumarins such as anticlotting, hypotensive, antimicrobial, anti-inflammatory, and antitumor activities [61].

Recent studies and review manuscripts regarding the coumarin scaffold describe the huge variety of biological activities that may be present in the natural coumarins [8, 18, 62-64]. Venugopala et al. (2013) presented several coumarins displaying activities such as antiinflammatory, anticoagulant, antibacterial, antifungal, antiviral, anticancer, anti-hypertensive, antitubercular, anticonvulsant, anti-adipogenic, Cytochrome P450 inhibiting, anti-hypergly‐ cemic, antioxidant, and neuroprotective. Several recent reviews summarize and highlight advances in the application of coumarins, especially concerning their antioxidant and anti‐ cancer properties [62-70].From *Calophyllum* spp., it is remarkable the antiviral activity of calanolides and other related pyranocoumarins on Epstein-Barr virus and HIV [56]. As active compound of molluscicidal effects on *Biomphalaria glabrata* of *C. brasiliense* extracts were determined (-) mammea A/BB, also found in *C. Verticillatum* [71].

It is the great structural diversity of coumarinic compounds that allows for their several applications, and also allows for the high interest of these derivatives as phytochemicals. The pharmacological and biochemical properties and therapeutic applications of simple coumarins depend upon the pattern of substitution [68].


**4. Coumarins in medicinal plants**

118 Phytochemicals - Isolation, Characterisation and Role in Human Health

A large number of valuable species used commonly as medicinal plants, aromatic plants, and edible plants for human and animal feeding belongs to coumarin-rich plant families. Among them are species with well-documented biological activity, in which coumarins are part of the active principles. Table 1 shows a selection of plants of these families (first listed seven families with number of occurrence > 100) and some other families with species of particular pharma‐ cological interest on chronic diseases. Coumarins presenting great pharmacological interest have been isolated in different geographical regions from other botanical families. Also shown

Most of these plants are well known by people and scientists as part of herbal medicine repertories in Europe, Asia, or the Americas [55-58]. From the list, several coumarin-containing species or genera have also ethnomedical records in Cuba and the Caribbean Basin [59, 60]. Among of plant included are species with a great historical record of ethnomedicinal uses, and are present in *traditional medicine systems*: Ayurveda Medicine, Traditional Chinese Medicine and Unani Medicine, or in other recent cultures. Also, renowned species used on conventional therapeutics and modern herbal medicine are included, ie. *Aesculus hippocastanum* (Horsechestnut), *Passiflora incarnata* (Passion Flower), *Lawsonia inermis* (Henna), *Hypericum perfora‐*

*tum* (Saint John Wort), *Tilia cordata* (Lime Tree) and *Uncaria tomentosa* (Cat's Claw).

antimicrobial, anti-inflammatory, and antitumor activities [61].

determined (-) mammea A/BB, also found in *C. Verticillatum* [71].

depend upon the pattern of substitution [68].

Coumarins are also present in several species belonging to different botanical families, which are widespread in the northeastern region of Brazil [61]. Some of them are reported in folk medicine as traditional remedies drugs for the treatment of respiratory diseases [55]. Many pharmacological activities have been ascribed to coumarins such as anticlotting, hypotensive,

Recent studies and review manuscripts regarding the coumarin scaffold describe the huge variety of biological activities that may be present in the natural coumarins [8, 18, 62-64]. Venugopala et al. (2013) presented several coumarins displaying activities such as antiinflammatory, anticoagulant, antibacterial, antifungal, antiviral, anticancer, anti-hypertensive, antitubercular, anticonvulsant, anti-adipogenic, Cytochrome P450 inhibiting, anti-hypergly‐ cemic, antioxidant, and neuroprotective. Several recent reviews summarize and highlight advances in the application of coumarins, especially concerning their antioxidant and anti‐ cancer properties [62-70].From *Calophyllum* spp., it is remarkable the antiviral activity of calanolides and other related pyranocoumarins on Epstein-Barr virus and HIV [56]. As active compound of molluscicidal effects on *Biomphalaria glabrata* of *C. brasiliense* extracts were

It is the great structural diversity of coumarinic compounds that allows for their several applications, and also allows for the high interest of these derivatives as phytochemicals. The pharmacological and biochemical properties and therapeutic applications of simple coumarins

are the coumarin compounds having species and their yield (if available).



**Family-specie (vernacular name)**

*Aegle marmelos* (Bael fruit)

*Citrus aurantium* (Bitter

*Murraya paniculata* (*M. exotica*)(Orange Jessamine,

*Tetradium daniellii* (*Euodia*

*Toddalia aculeata* (*T. asiatica*)

*Zanthoxilum americanum* (Northern Prickly Ash)

*Ageratum conyzoides*

*Conyza sumatrensis* (Fleabane)

Snakeroot)

*Chamaemelum nobile* (Roman

*Eupatorium triplinerve* (White

Chinese box)

*daniellii*)

Orange tree)

**Coumarin Use\* Reference**

bergapten, bergaptol, citropten M, F [58]

Coumarins M [74]

Coumarins M [79, 80]

Osthol M [56]

Coumarins M [84]

M, F [75-77]

M, F [58]

M [57]

Sesquiterpenic coumarin ethers, diterpenic coumarin ethers, triterpenic coumarin ethers, sesterterpenic coumarin ethers, auraptene,

Volatile Coumarins (0.09%): aurapteno, auraptenol, bergapteno, bergaptol, escoparona,

*C. sinensis* (Orange tree) Herniarin, scopoletin M, F [60] *Melicope spp.* Coumarins, chromones, dichromones M [56]

*Paramygnya monophylla* Poncitrin, nordentatin M [56] *Stauracanthus perforates* Coumarins M [78]

(Orange climber) Ulopterol <sup>M</sup> [74, 81]

Xanthyletin, xanthoxyletin, alloxanthoxyletin, 8-

*Z. syncarpum* Coumarins M [82]

*Achillea millefolium* (Yarrow) Coumarins (0.35% ) M [58]

(Mexican ageratum) 1-2 benzopirone <sup>M</sup> [83]

*Arnica montana* (Arnica) Scopoletin, umbelliferone M, F [57, 58]

Chamomile) Scopoletin-7-glucoside M, F [57, 58]

*Cichorium* intybus (Chicory) Coumarins M, F [73]

*Asteraceae/Compositae*

(3,3-dimethylallyl)-alloxanthoxyletin.

epoxyauraptene, marmin.

citropteno.

120 Phytochemicals - Isolation, Characterisation and Role in Human Health

*C. limonum* (Lemon tree) Escopoletin, umbelliferone, bergamotin,



**Family-specie (vernacular name)**

*Eleutherococcus senticosus* (Eleutherococcus)

*Herniaria glabra* (Rupture

*Vivurnum prunifolium* (American black haw)

*Calophyllum brasiliense* (Guanandi, Ocuje)

*Calophyllum inophyllum* (Borneo mahogany)

*Calophyllum lanigerum* var

*Radicula armoracia* (Horseradish root) **Coumarin Use\* Reference**

fucosides, daphnetin, - [94, 95]

methoxy-7-hydroxycoumarin - [96]

Coumarins M [58]

Aesculetin M, F [57, 58]

brasimarins A, B, and C [71, 98]

Coumarins [56]

M [58]

*Achanthaceae*

*Araliaceae*

*Brassicacae/Cruciferae*

*Caryophylacae*

*Caprifoliaceae*

*Clusiaceae/Guttiferae*

*D. odora* (Winter daphne) daphnetin - [93]

*Justicia pectoralis* (Tilo) Coumarin, umbelliferone M [97]

wort) (0.1-0.4%) umbelliferone, herniarin <sup>M</sup> [58]

Scopoletin (7-hidroxy-6-methoxicoumarin),

volatile Coumarins, (-) mammea A/BB,

*C. cerasiferum* (-) calanolide B M [56] *C. cordato-oblongum* Coumarins M [56]

*austrocoriaceum* (+)- calanolide A <sup>M</sup> [56] *C. teysmannii* var *inophylloide* (-) calanolide B, sonlattrolide M [56] *C. verticillatum* mammea A/BB [71]

*Connarus monocarpus* Bergenin1.5% M [56]

*Connaraceae*

*Cupresaceae*

scopolin, sculetin

*D. oleoides* dimeric coumarin glycoside, trimeric coumarin

122 Phytochemicals - Isolation, Characterisation and Role in Human Health

*D. pedunculata* 3-[(3-hydroxy-4-ethylpropanpicatephenyl)oxy]-6-


**Table 1.** Medicinal and food plant uses of some species from major coumarin-containing families.

#### **5. Natural coumarins, non-nutrients presented in the food**

Phytochemicals are defined as bioactive non-nutrient plant compounds presented in fruits, vegetables, grains, and other food plants that have been linked to reducing the risk of major chronic diseases. It is estimated that > 5,000 individual phytochemicals have been identi‐ fied in fruits, vegetables, and grains, but a large percentage still remain unknown and need to be identified before we can fully understand the health benefits of phytochemicals in whole foods [101].

Phenolics are compounds possessing one or more aromatic rings with one or more hydroxy groups, and generally are categorized as phenolic acids, flavonoids, stilbenes, coumarins, and tannins [102]. Phenolics are the products of secondary metabolism in plants, providing essential functions in the reproduction and the growth of the plants, acting as defense mechanisms against pathogens, parasites, and predators, as well as contributing to the color of plants [103]. In addition to their roles in plants, phenolic compounds in our diet may provide health benefits associated with reduced risk of chronic diseases. Among the 11 common fruits consumed in the United States, cranberry has the highest total phenolic content, followed by apple, red grape, strawberry, pineapple, banana, peach, lemon, orange, pear, and grapefruit [104]. Some of these fruits as important antioxidant and antiproliferative activities [104]. Among the 10 common vegetables consumed in the United States, broccoli possesses the highest total phenolic content, followed by spinach, yellow onion, red pepper, carrot, cabbage, potato, lettuce, celery, and cucumber [105]. Some of these vegetables proved to display interesting antioxidant and antiproliferative activities [105]. It is estimated that flavonoids account for approximately two thirds of the phenolics in our diet and the remaining one third are from phenolic acids [106].

**Family-specie (vernacular name)**

124 Phytochemicals - Isolation, Characterisation and Role in Human Health

*Passiflora incarnate* (Passion

*Plantago major* (Large

*Galium odoratum* (*Asperula*

*Uncaria tomentosa* (Cat's

\* M: Medicine, F: Food.

whole foods [101].

Claw)

Plantain)

**Coumarin Use\* Reference**

*Peumus boldus* (Boldus) Coumarins 125 ppm M [84]

Flower) Scopoletin, umbelliferone M, F [57, 58]

*Zea mays* (Corn) Coumarins: 2,000 ppm F [84]

*Zizyphus jujube* (Jujube) Coumarins: 3,000 ppm M, F [84]

*odorata*) (Woodruff) Coumarins: 13,000 ppm <sup>M</sup> [84]

*Tilia cordata* (Lime tree) Fraxosides, sculosides M [58]

*Urtica dioica* (Nettle) Scopoletin M, F [57, 58]

**Table 1.** Medicinal and food plant uses of some species from major coumarin-containing families.

**5. Natural coumarins, non-nutrients presented in the food**

*Passifloraceae*

*Plantaginaceae*

*Poaceae (Graminae)*

*Rhamnaceae*

*Rubiaceae*

*Tiliaceae*

*Urticaceae*

Phytochemicals are defined as bioactive non-nutrient plant compounds presented in fruits, vegetables, grains, and other food plants that have been linked to reducing the risk of major chronic diseases. It is estimated that > 5,000 individual phytochemicals have been identi‐ fied in fruits, vegetables, and grains, but a large percentage still remain unknown and need to be identified before we can fully understand the health benefits of phytochemicals in

Phenolics are compounds possessing one or more aromatic rings with one or more hydroxy groups, and generally are categorized as phenolic acids, flavonoids, stilbenes, coumarins, and tannins [102]. Phenolics are the products of secondary metabolism in plants, providing

Esculetin M [58]

Coumarins M [100]

Epidemiological studies have consistently shown that a high dietary intake of fruits and vegetables as well as whole grains is strongly associated with reduced risk of developing chronic diseases, such as cancer and cardiovascular disease [107-109]. Even if it is not so described in the bibliographic sources, most of the food plants, spice plants, and culinary herbs used regionally or worldwide are coumarin-containing plants, thus its effect on health cannot be ignored. For example, the potentially health-promoting role of popular vegetables and spices proved to be derived from *Apiaceae* [110]. Additionally, the above vegetables and spices also contain several bioactive phytochemicals such as flavonoids (quercetin, rutin) and coumarins (bergapten, isopimpinellin, xanthotoxin), which are reported to have curative, preventive, or nutritive value [84]. The above coumarins have also been found to inhibit multiplication of bacteria, fungi, and viruses [111] and demonstrated anti-allergy [112], antiinflammation [113], and immunosuppression activities [114].

Table 1 also shows the importance of a number of families containing coumarins in human nutrition. Among other species of interest, the *Apiaceae* family is a prominent food source of coumarins: carrots, celery, parsley, coriander, cumin, fennel, and aniseed are present in the culinary practice around the world and in the food industry (fixative) [110]. *Rutaceae* also proved to contain a great number of coumarins with nutritional and economic interest, particularly the c*itrus* and some other fruits such as Bael [115]. Besides fruits and vegetables, olive oil and beverages such as coffee, wine, and black and green tea are also important dietary sources of coumarins [73].

It is also known that essential oils derived from some plants also contain coumarin derivatives and are used as flavoring in foods. Some essential oils such as Chinese cinnamon oil [116], cinnamon bark oil [117], and lavender oil [118] have important amounts of coumarins. Coumarin's aroma has been described as sweet, aromatic, creamy vanilla bean odor with nutlike tones that are heavy, but not sharp or brilliant [119]. A major source in alcoholic beverages is *Hierochloe odorata*, which is used to flavor a special kind of vodka, produced mainly in Eastern Europe [120].

According to Lake (1999), the main source of coumarin in human diet is the cinnamon. Cinnamon comes from the dried bark of *Cinnamomum verum* and *C. cassia/C. aromaticum*, and is considered a spice. Cinnamon is widely used in various cultures in preparation of desserts, cakes, candy, etc., to decorate and some flavoring dishes. It is also used in some places as a beverage or tea. It is also an ingredient in many curries and other dishes Eastern. Intake levels [tolerable daily intake (TDI)] of coumarin derivatives are 0.1 mg/kg bw [121]. For food and beverages in general, the maximum permissible level is 2 mg/kg [122.

It has been estimated that human exposure to coumarins diet is approximately 0.02 mg/kg/day (Lake, 1999). The theoretical maximum daily intake (TAMDI) of coumarin via food was estimated to be 4.085 mg/day (0.07 mg/kg bw/day) [123].

Evidence has suggested that coumarin is not a genotoxic agent [121, 124]. The International Agency for Research on Cancer [125] has classified coumarin as belonging to group 3 (''not classifiable as to its carcinogenicity in humans"). No epidemiological data relevant to the carcinogenicity of coumarin were available and there was only limited evidence in experi‐ mental animals for the carcinogenicity of coumarin [125].

The field of food science is of great interest to develop research related to consumer safety and also those designed to elucidate the potentially health-promoting capacity and biological activity of bioactive components that are part of so-called functional foods. However, the beneficial role of these phytocompounds when in synergy on the original food matrix or when isolated (nutraceutics or food supplement) is currently a hot topic [126].

Due to the structural diversity and versatility of applications of coumarins, not only in food sciences (including diet supplements), it is necessary to continue research related to the safety, and also its bioavailability, interactions with other dietary compounds, and therapeutic and environmental components. It is also important to amplify omics techniques, including epigenetic studies.

Different environmental insults can influence epigenetics and nutrition is one of the major factors that contribute to epigenetic regulation of diseases. Particularly, non-communicable diseases phenotypes can be determined by the role of prenatal or early-age nutrition and epimutations can have transgenerational effects, while some "epi-nutrients" and food products can also stabilize the genome [127, 128]. Therefore, the role of food-based "epi-bioactive" compounds has become an emerging field. Nutri-epigenomics is part of this new era too, since this approach on research has been carried out on chronic or degenerative diseases [129-133].

Besides micronutrients (folate, selenium, retinoic acid, and vitamins D and E) effects on epigenome, investigations on dietary phytochemicals has been carried out to determine their ability to reverse adverse epigenetic marks, mainly in cancer, for instance: polyphenols (resveratrol, curcumin, catechin, ellagitannin); genistein and soy isoflavones; sulfur-contain‐ ing compounds (sulforaphane, phenylethyl isothiocyanat, phenylhexyl isothiocyanate, diallyldisulfide, allyl mercaptan) from *Allium* spp. (*Alliaceae*); and cruciferous (*Brassicaceae*) vegetables [129, 133].

Histone modifications are one of the major epigenetic mechanisms and its acetylation is mediated by the interplay of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The class III HDACs, called sirtuins (SIRT), has been shown to deacetylate the transcription factor p53. Given the regulatory functions of p53 in cell metabolism, inhibition of SIRT1 might contribute to inhibition of glycolysis and inhibit cell proliferation and the apoptosis [129, 133, 134].

is considered a spice. Cinnamon is widely used in various cultures in preparation of desserts, cakes, candy, etc., to decorate and some flavoring dishes. It is also used in some places as a beverage or tea. It is also an ingredient in many curries and other dishes Eastern. Intake levels [tolerable daily intake (TDI)] of coumarin derivatives are 0.1 mg/kg bw [121]. For food and

It has been estimated that human exposure to coumarins diet is approximately 0.02 mg/kg/day (Lake, 1999). The theoretical maximum daily intake (TAMDI) of coumarin via food

Evidence has suggested that coumarin is not a genotoxic agent [121, 124]. The International Agency for Research on Cancer [125] has classified coumarin as belonging to group 3 (''not classifiable as to its carcinogenicity in humans"). No epidemiological data relevant to the carcinogenicity of coumarin were available and there was only limited evidence in experi‐

The field of food science is of great interest to develop research related to consumer safety and also those designed to elucidate the potentially health-promoting capacity and biological activity of bioactive components that are part of so-called functional foods. However, the beneficial role of these phytocompounds when in synergy on the original food matrix or when

Due to the structural diversity and versatility of applications of coumarins, not only in food sciences (including diet supplements), it is necessary to continue research related to the safety, and also its bioavailability, interactions with other dietary compounds, and therapeutic and environmental components. It is also important to amplify omics techniques, including

Different environmental insults can influence epigenetics and nutrition is one of the major factors that contribute to epigenetic regulation of diseases. Particularly, non-communicable diseases phenotypes can be determined by the role of prenatal or early-age nutrition and epimutations can have transgenerational effects, while some "epi-nutrients" and food products can also stabilize the genome [127, 128]. Therefore, the role of food-based "epi-bioactive" compounds has become an emerging field. Nutri-epigenomics is part of this new era too, since this approach on research has been carried out on chronic or degenerative diseases [129-133].

Besides micronutrients (folate, selenium, retinoic acid, and vitamins D and E) effects on epigenome, investigations on dietary phytochemicals has been carried out to determine their ability to reverse adverse epigenetic marks, mainly in cancer, for instance: polyphenols (resveratrol, curcumin, catechin, ellagitannin); genistein and soy isoflavones; sulfur-contain‐ ing compounds (sulforaphane, phenylethyl isothiocyanat, phenylhexyl isothiocyanate, diallyldisulfide, allyl mercaptan) from *Allium* spp. (*Alliaceae*); and cruciferous (*Brassicaceae*)

Histone modifications are one of the major epigenetic mechanisms and its acetylation is mediated by the interplay of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The class III HDACs, called sirtuins (SIRT), has been shown to deacetylate the

beverages in general, the maximum permissible level is 2 mg/kg [122.

isolated (nutraceutics or food supplement) is currently a hot topic [126].

was estimated to be 4.085 mg/day (0.07 mg/kg bw/day) [123].

126 Phytochemicals - Isolation, Characterisation and Role in Human Health

mental animals for the carcinogenicity of coumarin [125].

epigenetic studies.

vegetables [129, 133].

Dihydrocoumarin (DHC), which is found in *Melilotus officinalis* (*Fabaceae*) (sweet clover) or synthetized, is commonly added as flavoring agent to food and cosmetics. This compound was studied by Olaharski et al. (2005), on different assays to evaluate it as an "epi-bioactive" agent and resulted that DHC inhibited the deacetylase activities of yeast SIRT2p and human SIRT1. DHC exposure in the human TK6 lymphoblastoid cell line also caused concentration-depend‐ ent increases in p53 acetylation, cytotoxicity, and flow cytometric analysis, demonstrating that DHC increased apoptosis more than 3-fold over controls [135].

Authors also stated that these findings on DHC could be potentially worrisome, since SIRT1 inhibition may lead to epigenetic alterations, as well as possible stem cell depletion and early tissue senescence, a phenotype associated with senescence and aging. However, on the possibility of deleterious human exposition to epigenetic toxicants that inhibit SIRT deacety‐ lases, this effect can be desirable in cancer treatment mediated chemopreventive potential epigenetic mechanism [36, 129].

#### **6. Extraction techniques and identification of coumarins**

There have been a variety of methods described for extraction of coumarins. Generally, coumarins extraction can be performed either on dry or fresh material, with solvents of different polarities, depending on the type of structure. Some coumarins are sparingly soluble in apolar solvents and often they can be crystallized directly by cooling or concentrating the solvent.

Miranda and Cuéllar (2001), in their book entitled "Farmacognosia y Productos Naturales ", raised the possibility of following four variants using the Soxhlet method:


**•** The dried and ground plant material is extracted with acetone continuously in a Soxhlet apparatus, for at least 3 days, and the extract is concentrated to dryness giving a residue that contains the coumarins.

Purification and separation of coumarins contained in various extracts could be performed by using chromatographic columns, using as a carrier aluminum oxide and as solvent the eluotropic series: benzene-hexane (1:2.5); benzene; chloroform; chloroform-acetone, in proportions of a linear gradient to pure acetone [44]. For recognition of the described structures some trials were described, within which there are:


The last test is commonly used for phytochemical screening that is initially performed in plant research. Carrying on this approach, Payo et al., (1996), from 39 Cuban species screened, detected that 51.2% were positive for coumarin test.

Other extraction methods used in coumarins are: microwave, sonication, and supercritical fluid extraction (SFE) [136], these tests also propose capillary electrophoresis for natural products isolation.

Therefore, on the isolation and analysis of coumarins diverse methods have been used: chromatography (paper chromatography, thin layer chromatography, gas chromatography, and high-performance liquid chromatography), titrimetric, and spectrophotometric (colori‐ metric and polarographic) methods [1].

Due to coumarin-characteristic chromophore groups and its strong UV absorption at around 300 nm, it is routinely possible to be detected by feasible methods such as ultraviolet–visible spectroscopy (UV–vis) [137]. UV–vis detector is used in high performance liquid chromatog‐ raphy (HPLC) and also other hyphenated techniques are employed to characterize and quantify natural products such as Liquid Chromatography (LC)–Photo Diode Array Detector (PDA), coupling of Mass Spectrometry to LC–[137], or Ultra Performance Liquid Chromatog‐ raphy coupled with Mass Spectrometry (UPLC-MS) [138].

A simple spectroscopic technique [35] and HPLC [85] were employed to determine coumarins in the Brazilian medicinal plant "guaco", *Mikania glomerata* (*Asteraceae*). Coumarin HPLC detection is also used in Cuba to standardize a sedative herbal medicine based on *Justicia pectoralis* (*Acathanceae*) [97, 139].

#### **7. Conclusion**

**•** The dried and ground plant material is extracted with acetone continuously in a Soxhlet apparatus, for at least 3 days, and the extract is concentrated to dryness giving a residue

Purification and separation of coumarins contained in various extracts could be performed by using chromatographic columns, using as a carrier aluminum oxide and as solvent the eluotropic series: benzene-hexane (1:2.5); benzene; chloroform; chloroform-acetone, in proportions of a linear gradient to pure acetone [44]. For recognition of the described structures

**•** Those that recognize coumarin's phenolic substitutions where Emerson's Reagent is used,

**•** The presence of lactone groups can be observed leading to changes of pH in the medium. When coumarins are dissolved in ethanol, solutions change the color when acidified (yellow

**•** The furan ring can be recognized by using the Erlich test. The extract is treated with a solution of dimethylamino-benzaldehyde (5% ethanol), and then acidified by bubbling

The last test is commonly used for phytochemical screening that is initially performed in plant research. Carrying on this approach, Payo et al., (1996), from 39 Cuban species screened,

Other extraction methods used in coumarins are: microwave, sonication, and supercritical fluid extraction (SFE) [136], these tests also propose capillary electrophoresis for natural

Therefore, on the isolation and analysis of coumarins diverse methods have been used: chromatography (paper chromatography, thin layer chromatography, gas chromatography, and high-performance liquid chromatography), titrimetric, and spectrophotometric (colori‐

Due to coumarin-characteristic chromophore groups and its strong UV absorption at around 300 nm, it is routinely possible to be detected by feasible methods such as ultraviolet–visible spectroscopy (UV–vis) [137]. UV–vis detector is used in high performance liquid chromatog‐ raphy (HPLC) and also other hyphenated techniques are employed to characterize and quantify natural products such as Liquid Chromatography (LC)–Photo Diode Array Detector (PDA), coupling of Mass Spectrometry to LC–[137], or Ultra Performance Liquid Chromatog‐

A simple spectroscopic technique [35] and HPLC [85] were employed to determine coumarins in the Brazilian medicinal plant "guaco", *Mikania glomerata* (*Asteraceae*). Coumarin HPLC detection is also used in Cuba to standardize a sedative herbal medicine based on *Justicia*

gaseous hydrochloric acid. The orange color indicates a positive test.

that contains the coumarins.

developing color.

color disappears).

products isolation.

some trials were described, within which there are:

128 Phytochemicals - Isolation, Characterisation and Role in Human Health

detected that 51.2% were positive for coumarin test.

raphy coupled with Mass Spectrometry (UPLC-MS) [138].

metric and polarographic) methods [1].

*pectoralis* (*Acathanceae*) [97, 139].

Coumarins have been increasingly attracting special interest as phytochemicals due to their underlying outstanding contributions in the prevention and treatment of diseases. Coumarins represent a diverse class of phytochemicals that are ubiquitous in the human diet. Some of the medicinal usages of extracts of plants containing coumarins have been proven in experimental models, which suggested that the extracts possess various pharmacological actions. Several related researches and developments make coumarins an extremely attractive scaffold. The role of coumarins as important phytochemicals and their interesting applications were presented and discussed in this book chapter. The origin, natural sources, biosynthesis, and applications were also described.

#### **Acknowledgements**

The authors thank the partial financial support of University of Santiago de Compostela, University of Camagüey Ignacio Agramonte Loynaz and Galician Plan of research, innovation and growth 2011-2015 (Plan I2C, ED481B 2014/086-0)

#### **Author details**

Maria João Matos1 , Lourdes Santana1 , Eugenio Uriarte1 , Orlando A. Abreu2 , Enrique Molina1,2 and Estela Guardado Yordi1,2\*

\*Address all correspondence to: estela.guardado@reduc.edu.cu

1 Faculty of Pharmacy, University of Santiago de Compostela, Spain

2 Faculty of Chemistry, University of Camaguey Ignacio Agramonte Loynaz, Cuba

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## **Quinolines, Isoquinolines, Angustureine, and Congeneric Alkaloids — Occurrence, Chemistry, and Biological Activity**

Gaspar Diaz, Izabel Luzia Miranda and Marisa Alves Nogueira Diaz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59819

### **1. Introduction**

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The alkaloids are a family of compounds widely found in nature. Therefore, they They are nitrogenous secondary metabolites heterocyclic derivatives of amino acids or by the transa‐ mination process, which confers basic character [1].

The term alkaloid, linguistically derived from the Arabic *al*-*quali* (ash plants), is used to designate pharmacologically active nitrogen compounds found predominantly in Angio‐ sperms [2]. Their distribution is not uniform in that division. It is estimated that approximately 40% of all plant families have at least one plant species that contains alkaloids. They are easily found in Fabaceaes and Solanaceaes but are rare in Gymnosperms and Pteridophytes such as ferns and monocots [3]. Although most occur in plants, alkaloids have also been isolated from algae, insects, marine and land animals, microorganisms, and fungi [4].

Since the dawn of civilization, there are reports of the use of plant extracts containing alkaloids for various purposes. The death of Socrates in 399 BC, for example, was attributed to the consumption of the extract of *Conium maculatum* containing the alkaloid coniine. There are also reports that during the last century BC, Cleopatra employed extracts of *Hyoscyamus muticus* containing the alkaloid atropine to dilate the pupils to look more attractive. It is known that medieval European women employed extract from *Atropa belladonna* with the same goal. Moreover, 25% of contemporary medicines are plant-derived. For example, morphine is extracted from the poppy. It is a narcotic drug used as analgesic and marketed since 1827 by Merck [2].

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Several alkaloids (papaverine, morphine, and cocaine) can cause different stimuli in the nervous system of an animal; a common phenomenon in ecological relationships may constitute, for example, a defense mechanism by plants against herbivores [1].

The importance of alkaloids in the development of medicine is unquestionable since several advances made in the battle against diseases like malaria, leukemia, cancer, and neurodege‐ nerative diseases would not have occurred without the use of these substances [1].

This chapter focuses on the occurrence occurring, chemistry, and biological activity of the quinoline and isoquinoline alkaloids, including the angustureine and congeneric alkaloids.

#### **2. Quinoline and isoquinoline alkaloids**

Among the various classes of alkaloid compounds, this section highlights the quinoline and isoquinoline alkaloids. They were originally obtained from natural sources, whose remarkable biological activities and relatively simple structures have attracted great interest in the scientific community, especially researchers involved in the chemistry of natural products. However, these compounds have also attracted the interest of synthetic organic chemists due to the need to obtain increased amounts aimed at additional biological research, as well as in developing efficient synthetic routes for these alkaloids and their derivatives, whose chemical and biological properties could become greatly enhanced by the design of new structures from these modifications.

#### **2.1. Methods of extraction of quinoline and isoquinoline alkaloids**

The general method for extracting and isolating alkaloids from plants consists of an acid-base extraction. The dried and pulverized plant is extracted with organic solvents or with acidified water. When the extraction is carried out with organic solvents immiscible with water, the plant sample is alkalized prior to being extracted. For this, the sample is wrapped in filter paper, as a cartridge, humidified with dilute basic solution, e.g., ammonium hydroxide, and extracted under heating using Soxhlet apparatus with organic solvent such as ethyl ether, chloroform, or dichloromethane. After extraction, the solvent volume is reduced by half in rotary evaporator under reduced pressure. The remaining volume of the solvent containing the alkaloid residue is transferred to a separatory funnel and extracted with aqueous phos‐ phoric acid solution (pH 1 to 2) or hydrochloric acid. The wetting of the plant sample with basic solution allows the alkaloids are released from organic acids, thus facilitating its extraction by organic solvents. Meanwhile, when using an acidified solution, alkaloids tend to form salts with these strong acids, facilitating their removal from the two-phase system, water-solvent for the separation of these into the aqueous phase [5].

Another extraction technique uses polar solvent such as ethanol, for example. However, if a plant contains a high amount of lipids, a preextraction with a nonpolar solvent such as hexanes or petroleum ether is necessary for removing these lipids. Next, the organic solvent is concen‐ trated under reduced pressure and the residue solubilized in water [6]. As most alkaloids are basic or are found in salt form, aqueous solutions of phosphoric acid (pH 1 to 2) or hydrochloric acid are used for their removal. Neutral amide alkaloids, such as colchicine and piperine (Figure 1), remain in the organic phase when treated with an organic solvent (usually ethyl acetate) during the extraction process, while most of the other alkaloids may be extracted using an organic solvent only after neutralization of the aqueous phase with a base [4].

**Figure 1.** Structure of the neutral amide alkaloids colchicine and piperine.

Several alkaloids (papaverine, morphine, and cocaine) can cause different stimuli in the nervous system of an animal; a common phenomenon in ecological relationships may

The importance of alkaloids in the development of medicine is unquestionable since several advances made in the battle against diseases like malaria, leukemia, cancer, and neurodege‐

This chapter focuses on the occurrence occurring, chemistry, and biological activity of the quinoline and isoquinoline alkaloids, including the angustureine and congeneric alkaloids.

Among the various classes of alkaloid compounds, this section highlights the quinoline and isoquinoline alkaloids. They were originally obtained from natural sources, whose remarkable biological activities and relatively simple structures have attracted great interest in the scientific community, especially researchers involved in the chemistry of natural products. However, these compounds have also attracted the interest of synthetic organic chemists due to the need to obtain increased amounts aimed at additional biological research, as well as in developing efficient synthetic routes for these alkaloids and their derivatives, whose chemical and biological properties could become greatly enhanced by the design of new structures from

The general method for extracting and isolating alkaloids from plants consists of an acid-base extraction. The dried and pulverized plant is extracted with organic solvents or with acidified water. When the extraction is carried out with organic solvents immiscible with water, the plant sample is alkalized prior to being extracted. For this, the sample is wrapped in filter paper, as a cartridge, humidified with dilute basic solution, e.g., ammonium hydroxide, and extracted under heating using Soxhlet apparatus with organic solvent such as ethyl ether, chloroform, or dichloromethane. After extraction, the solvent volume is reduced by half in rotary evaporator under reduced pressure. The remaining volume of the solvent containing the alkaloid residue is transferred to a separatory funnel and extracted with aqueous phos‐ phoric acid solution (pH 1 to 2) or hydrochloric acid. The wetting of the plant sample with basic solution allows the alkaloids are released from organic acids, thus facilitating its extraction by organic solvents. Meanwhile, when using an acidified solution, alkaloids tend to form salts with these strong acids, facilitating their removal from the two-phase system,

Another extraction technique uses polar solvent such as ethanol, for example. However, if a plant contains a high amount of lipids, a preextraction with a nonpolar solvent such as hexanes or petroleum ether is necessary for removing these lipids. Next, the organic solvent is concen‐ trated under reduced pressure and the residue solubilized in water [6]. As most alkaloids are

constitute, for example, a defense mechanism by plants against herbivores [1].

nerative diseases would not have occurred without the use of these substances [1].

**2. Quinoline and isoquinoline alkaloids**

142 Phytochemicals - Isolation, Characterisation and Role in Human Health

**2.1. Methods of extraction of quinoline and isoquinoline alkaloids**

water-solvent for the separation of these into the aqueous phase [5].

these modifications.

Distillation methods are rarely employed for isolating alkaloids. This is possible only in cases of low molecular weight alkaloids like coniine and sparteine (Figure 2) [4].

**Figure 2.** Structure of the alkaloids coniine and sparteine.

Generally, the crude extract is purified by column chromatography (CC), employing silica gel or alumina as the stationary phase and a mixture of appropriate solvents as the mobile phase, following an appropriate choice made via thin layer chromatography (TLC). Often, alkaloids still need additional purification by recrystallization, employing solvent systems such as ethanol/water, methanol/chloroform, or acetone/methanol [4].

While these techniques are successful to extract and to isolate most alkaloids, for those highly water soluble and, therefore, partially or totally insoluble in organic solvents, these methods are not appropriates [6].

Although generally higher solubility in aqueous solutions involves greater bioactivities [6], the employment of more suitable methods for the extraction of highly water-soluble alkaloids is very useful.

In the case of quaternary alkaloids, the residual aqueous solution should be tested with a reagent suitable for the detection of alkaloids (Dragendorff reagent, for example) after completion of conventional partition methods. If it is positive, one should employ meth‐ ods such as direct crystallization, precipitation as insoluble salts, extraction with polar solvent water-immiscible, or formation of pseudobases to extract the alkaloids from the aqueous phase [6].

Alkaloids *N*-oxides can be isolated by indirect methods or chemical derivatization, through a reaction of reduction to the corresponding tertiary basis and extracted using the conventional method, and subsequently regenerated by an oxidation reaction, using oxidizing agents such as peroxide hydrogen or *m*-chloroperbenzoic acid. In some cases, the *N*-oxides containing higher aliphatic chains can be almost completely extracted by increasing the basicity of the aqueous phase, but for those who have high levels of hydroxylation, it is infeasible [6].

The methods employed to extract most common polyhydroxylated alkaloids are the direct extraction and ion exchange chromatography [6].

After extraction of alkaloids, a quantitative analysis that may be performed by traditional methods such as simple weighing or base titration after dilution under acidic conditions is recommended. Other widely used techniques for the quantification of alkaloids are by employing the HPLC apparatus with UV detection and by testing the refractive index depending on the structure of the isolated compound(s) [5].

#### **2.2. Occurrence in nature of quinoline and isoquinoline alkaloids**

The quinoline and isoquinoline alkaloids were initially extracted from coal tar in 1834 and 1835, respectively. Quinoline, which has high boiling temperature, is commonly employed in organic synthesis as a solvent. Isoquinoline has a low melting temperature and both have moderate basicity (p*K*a = 4.9 and 5.1, respectively) [7].

Although the vast majority of quinoline and isoquinoline alkaloids derived from flowered plants, they can also be isolated from animals and microorganisms, some representative examples are given in Table 1 [7].


Quinolines, Isoquinolines, Angustureine, and Congeneric Alkaloids — Occurrence, Chemistry, and Biological Activity http://dx.doi.org/10.5772/59819 145

solvent water-immiscible, or formation of pseudobases to extract the alkaloids from the

Alkaloids *N*-oxides can be isolated by indirect methods or chemical derivatization, through a reaction of reduction to the corresponding tertiary basis and extracted using the conventional method, and subsequently regenerated by an oxidation reaction, using oxidizing agents such as peroxide hydrogen or *m*-chloroperbenzoic acid. In some cases, the *N*-oxides containing higher aliphatic chains can be almost completely extracted by increasing the basicity of the aqueous phase, but for those who have high levels of hydroxylation, it is infeasible [6].

The methods employed to extract most common polyhydroxylated alkaloids are the direct

After extraction of alkaloids, a quantitative analysis that may be performed by traditional methods such as simple weighing or base titration after dilution under acidic conditions is recommended. Other widely used techniques for the quantification of alkaloids are by employing the HPLC apparatus with UV detection and by testing the refractive index

The quinoline and isoquinoline alkaloids were initially extracted from coal tar in 1834 and 1835, respectively. Quinoline, which has high boiling temperature, is commonly employed in organic synthesis as a solvent. Isoquinoline has a low melting temperature and both have

Although the vast majority of quinoline and isoquinoline alkaloids derived from flowered plants, they can also be isolated from animals and microorganisms, some representative

**Structure Usual name Source Biological activity**

Argemonine *Argemone* sp. -

Berlambine *Thalictrum foliolosum* -

Antiprotozoal Antimalarial Antibacterial Antidiarrheal

Topoisomerase inhibitor (anticancer agent)

Berberine Family of *Berberidaceae*

Camptothecine *Camptotheca acuminata*

aqueous phase [6].

extraction and ion exchange chromatography [6].

144 Phytochemicals - Isolation, Characterisation and Role in Human Health

depending on the structure of the isolated compound(s) [5].

moderate basicity (p*K*a = 4.9 and 5.1, respectively) [7].

examples are given in Table 1 [7].

H3CO H3CO

NCH3

N

OCH3 O

N N

<sup>N</sup> H3CO OCH3

H3CO

OCH3 OCH3

O O

O O

O O HO H3C

**2.2. Occurrence in nature of quinoline and isoquinoline alkaloids**


N


Quinolines, Isoquinolines, Angustureine, and Congeneric Alkaloids — Occurrence, Chemistry, and Biological Activity http://dx.doi.org/10.5772/59819 147

**Structure Usual name Source Biological activity**

H3CO Jamtine *Cocculus hirsutus* Anti-hyperglycemic

HO CH3 Lephocereine *Lephocereus schotti* -

<sup>H</sup> <sup>H</sup> Lycorine *Lycoris radiata* hypotensive

Lavendamycin *Streptomyces levendulae* Antitumor antibiotic

Maritidine - -

Morphine *Opium* Analgesic

Nandinine *Nandina domestica* -

α1 Adrenergic and serotonin antagonist 5-HT2A

Topoisomerase inhibitor (anticancer agent)

Nantenine *Nandina domestica*

Nitidine *Zanthoxylum nitidum*

*N*-norlaudanosine - -

Palmitine *Jateorhiza palmate* -

N H H3CO2C

146 Phytochemicals - Isolation, Characterisation and Role in Human Health

N HN CH3 CO2H

> N CH3 CH3

N

OH

CH3

O O

> O O

N

H

N

N

H CH3

N

NH <sup>H</sup>

N

OCH3 OCH3

> OCH3 OCH3

H3CO CH3

OH HO

H3CO

H2N N O

H3CO

O O

H3CO H3CO

> H3CO O H N

> > HO

H3CO OH

> H3CO H3CO

> > O O

H3CO

H3CO H3CO

H3CO H3CO O


O

CH3

CH3


**Table 1.** Quinoline and isoquinoline alkaloids: natural sources and biological activities.

#### **2.3. Biosynthetic origin of quinoline and isoquinoline alkaloids**

The most One of the systems used for the classification of alkaloids is based on the kind of nitrogen heterocycle in the structure and its botanical origin (plant family which is extracted); thus, the name given to an alkaloid derived from the genus or species of organism produced, including the term "ine." Another commonly used system takes into account its precursor amino acid. Thus, alkaloid derivatives of tryptophan and tyrosine are biosynthetically classified as quinoline and isoquinoline, respectively (Figure 3) [3].

Thus, quinoline alkaloids are derived from 3-hydroxyanthranilic acid, a metabolite formed from tryptophan through a sequence of enzymatic reactions (Figure 4). The condensation between 3-hydroxyanthranilic acid and malonyl-SCoA, followed by cyclization, produces quinoline alkaloid (Figure 4) [8-10].

Quinolines, Isoquinolines, Angustureine, and Congeneric Alkaloids — Occurrence, Chemistry, and Biological Activity http://dx.doi.org/10.5772/59819 149

**Figure 3.** Structure of quinoline and isoquinoline alkaloids.

**Structure Usual name Source Biological activity**

Skimmianine *Skimmia japoinica*

Stepholidine *Stephania intermedia*

<sup>H</sup> Thalictricavine *Corydalis tuberosa* -

The most One of the systems used for the classification of alkaloids is based on the kind of nitrogen heterocycle in the structure and its botanical origin (plant family which is extracted); thus, the name given to an alkaloid derived from the genus or species of organism produced, including the term "ine." Another commonly used system takes into account its precursor amino acid. Thus, alkaloid derivatives of tryptophan and tyrosine are biosynthetically

Thus, quinoline alkaloids are derived from 3-hydroxyanthranilic acid, a metabolite formed from tryptophan through a sequence of enzymatic reactions (Figure 4). The condensation between 3-hydroxyanthranilic acid and malonyl-SCoA, followed by cyclization, produces

Sanguinarine *Sanguinaria* Antibacterial

Streptonigrin *Streptomyces flocculus* Antitumor antibiotic

Tetrahydropa-lmitine *Corydalis family* Analgesic

*Chododendron tomentosum*

Sedative Anticonvulsant

D1 receptor agonist D2 receptor antagonist

> Neuromuscular blocking agent

<sup>H</sup> <sup>N</sup> <sup>O</sup> 3CO OCH3

N

N

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

N

N

O OH

H3CO

CH3

OCH3 OCH3

O O

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

<sup>H</sup> CH3 3C Tubocurarin

**Table 1.** Quinoline and isoquinoline alkaloids: natural sources and biological activities.

**2.3. Biosynthetic origin of quinoline and isoquinoline alkaloids**

classified as quinoline and isoquinoline, respectively (Figure 3) [3].

H2N CO2H H2N CH3 OH OCH3 OCH3

H3C

O O

H3CO HO

H3CO

H3CO H3CO

> H3CO OCH3

> > N

CH3

OCH3 OH O

quinoline alkaloid (Figure 4) [8-10].

O

O

OCH3

O O

148 Phytochemicals - Isolation, Characterisation and Role in Human Health

OCH3 OH

**Figure 4.** Biosynthesis of quinoline alkaloids.

In the case of isoquinoline systems, the biosynthesis proceeds from the cyclization of the Schiff base formed between the dopamine and an aliphatic aldehyde [8]. Dopamine is obtained from the hydroxylation and decarboxylation of tyrosine. A second intermediate, *p*-hydroxyphenyl acetaldehyde, can also be formed by transamination, decarboxylation, and hydroxylation of tyrosine. The condensation of these intermediates followed by a sequence of steps (cyclization, hydroxylation, and methylation) produces the (*S*)-reticuline, a biosynthetic intermediate of all isoquinoline alkaloids (Figure 5) [11].

#### **2.4. Biological activities of quinoline and isoquinoline alkaloids**

In addition to the biological activities shown in Table 1, many quinoline alkaloids and their analogues represent the most important drugs currently used to combat malaria, with chloroquine, amodiaquine, piperaquine, primaquine, quinine, and mefloquine (Figure 6)

**Figure 5.** Biosynthesis of isoquinoline alkaloids.

being the most representative drugs of this group [12]. Even today, many studies are still being conducted to increase the efficiency of quinoline derivatives such as AZT-chloro-quinoline, quinine-dihydroartemisinin, and MEFAS, a salt derived from mefloquine and artesunate [13] (Figure 7).

**Figure 6.** Structure of some quinoline alkaloids and their analogues.

Quinolines, Isoquinolines, Angustureine, and Congeneric Alkaloids — Occurrence, Chemistry, and Biological Activity http://dx.doi.org/10.5772/59819 151

**Figure 7.** Structure of other quinoline derivatives.

being the most representative drugs of this group [12]. Even today, many studies are still being conducted to increase the efficiency of quinoline derivatives such as AZT-chloro-quinoline, quinine-dihydroartemisinin, and MEFAS, a salt derived from mefloquine and artesunate [13]

HO

CO2H Dopamine

descarboxylase HO

H3CO HO

HO

tyrosine descarboxylase or DOPA descarboxylase phenol oxidase

150 Phytochemicals - Isolation, Characterisation and Role in Human Health

transaminase,

*N*-methylcoclaurine NCH 3'-hydroxylase <sup>3</sup>

3'-hydroxy-*N*-methylcoclaurine 4'-*O*-methyltransferase

NCH3

H

HO NH2

O H

norcoclaurine

coclaurine *<sup>N</sup>*-methyltransferase NCH3

synthase NH HO HO

HO

norcoclaurine 6-*O*-methyltransferase

> H3CO HO

> > HO

H

NH

H

(*S*)-norcoclaurine

*p*-hydroxyphenyl acetaldehyde

H

OH

N CH3 CH3

quinine

N

HO <sup>N</sup> H

H

Cl N

N

piperaquine

N

CF3

H

HO <sup>N</sup> H

mefloquine

CF3

N 3 N

N

N Cl

(Figure 7).

H3 C N H3 C

> Cl chloroquine

N

H3 CO

NH2

H

L-tyrosine

H3CO HO

> HO HO

H3CO HO

H3CO HO

(*S*)-reticuline

**Figure 5.** Biosynthesis of isoquinoline alkaloids.

N

HN CH3

primaquine

**Figure 6.** Structure of some quinoline alkaloids and their analogues.

Cl N

NH2

HN

amodiaquine

H3 CO

NH CH3

On the other hand, the quinoline nucleus has also demonstrated a critical role in the develop‐ ment of new anticancer drugs. Some of its derivatives showed excellent results on various types of cancer cells, through different mechanisms of action. Recently, three protein kinase inhibitors (Bosutinib, Lenvatinib, and Cabozantinib) and an inhibitor of farnesyltransferase (Tipifarnib) (Figure 8), considered as potential anticancer agents, entered into phase of clinical trials [14].

**Figure 8.** Structure of bosutinib, lenvatinib, cabozantinib, and tipifarnib quinoline nucleus.

Knowing that functionalized quinolines have also shown high anti-carcinogenic potential, in addition to acting as anti-angiogenic agents, and inhibitors of telomerase in various human tumor cells, Muñoz and coworkers [15] selected the DM8 and DM12 tetrahydroquinolines (Figure 9) for testing cytotoxic activity. They observed that individually these compounds significantly inhibited cell growth of breast cancer, and when tested concomitantly with other two anticancer drugs, they work synergistically to increase their cytotoxic activities. The results obtained for the DM12 quinoline made this compound a promising candidate as a new adjunctive anticancer agent.

**Figure 9.** Structure of the DM8 and DM12 tetrahydroquinoline alkaloids.

Furthermore, Villemagne and Okamura [16] obtained promising results with other quinoline derivatives used as selective ligands for Tau proteins, associated with brain damage, which represent a risk factor for diseases such as Alzheimer's, which allowed the investigation of the causes, diagnosis, and treatment of neurodegenerative diseases, encephalopathy, and trau‐ matic brain injury.

Biological activities of hundreds of other substituted quinolines have been reported, many of which are promising in terms of their potential as pharmaceutical agents. 2-Methyl-5-hy‐ droxy-1,2,3,4-tetrahydroquinoline exhibits analgesic activity with a potency one-eighth that of morphine [17]. 1,2,3,4-Tetrahydroquinoline-4-carboxylic acid is used in tissue-irrigating solutions [18]. A wide array of other biological activities have been reported, including *inter alia* inhibition of (H+ /K+ )-ATPase [19], blood serum monoamine oxidase [20], angiotensin I converting enzyme [21], lipoxygenase [22], lipid peroxidation [23], bone resorption [24], leukotriene synthesis [25, 26], and bacterial dihydrofolate reductase [27]. Relevance to many other indications has also been noted [28-54].

In addition to the numerous utilities in the pharmacological area, studies are being performed in order to investigate the potential activities of these alkaloids as agrochemical agents. Sanguinarine and chelerythrine (Figure 10), for example, have shown high activity against fungi and phytopathogenic bacteria *in vitro*, with sanguinarine being the most effective against *Rhizoctonia solani* fungus, which has a wide range of hosts, which causes disease in most plants grown in the world [55].

**Figure 10.** Structure of the sanguinarine and chelerythrine agrochemical agents.

### **3. Tetrahydroquinoline alkaloids angustureine, galipeine, galipinine, and cuspareine**

H N NH2

152 Phytochemicals - Isolation, Characterisation and Role in Human Health

OH DM8

**Figure 9.** Structure of the DM8 and DM12 tetrahydroquinoline alkaloids.

matic brain injury.

*alia* inhibition of (H+

grown in the world [55].

O

O

/K+

other indications has also been noted [28-54].

N

**Figure 10.** Structure of the sanguinarine and chelerythrine agrochemical agents.

H3C

CH3

H N NO2

O2N

OH DM12

)-ATPase [19], blood serum monoamine oxidase [20], angiotensin I

CH3

OCH3

OCH3

Furthermore, Villemagne and Okamura [16] obtained promising results with other quinoline derivatives used as selective ligands for Tau proteins, associated with brain damage, which represent a risk factor for diseases such as Alzheimer's, which allowed the investigation of the causes, diagnosis, and treatment of neurodegenerative diseases, encephalopathy, and trau‐

Biological activities of hundreds of other substituted quinolines have been reported, many of which are promising in terms of their potential as pharmaceutical agents. 2-Methyl-5-hy‐ droxy-1,2,3,4-tetrahydroquinoline exhibits analgesic activity with a potency one-eighth that of morphine [17]. 1,2,3,4-Tetrahydroquinoline-4-carboxylic acid is used in tissue-irrigating solutions [18]. A wide array of other biological activities have been reported, including *inter*

converting enzyme [21], lipoxygenase [22], lipid peroxidation [23], bone resorption [24], leukotriene synthesis [25, 26], and bacterial dihydrofolate reductase [27]. Relevance to many

In addition to the numerous utilities in the pharmacological area, studies are being performed in order to investigate the potential activities of these alkaloids as agrochemical agents. Sanguinarine and chelerythrine (Figure 10), for example, have shown high activity against fungi and phytopathogenic bacteria *in vitro*, with sanguinarine being the most effective against *Rhizoctonia solani* fungus, which has a wide range of hosts, which causes disease in most plants

O

N

H3C

OCH3

OCH3

O

sanguinarine chelerythrine

O

O

Among the simplest natural alkaloids that contain the 1,2,3,4-tetrahydroquinoline core are angustureine and its congeners, galipeine, cuspareine, and galipinine (Figure 11). The struc‐ tures are more narrowly described as 2-alkyl-1-methyl-1,2,3,4-tetrahydroquinolines. They are chiral by virtue of the stereogenic center at position 2. There is an excellent review of the chemistry and synthesis of 1,2,3,4-tetrahydroquinolines by Diaz and Dudley [56].

**Figure 11.** Structure of the 1,2,3,4-tetrahydroquinolin alkaloids angustureine, galipeine, cuspareine, and galipinine.

In 1999, Jacquemond-Collet and coworkers [57] isolated and characterized these four tetrahy‐ droquinoline alkaloids (Figure 11) from the extract of the bark of *Galipea officinalis*, commonly known as "angostura" [58]. The genus *Galipea* comprises about 20 species that are found predominantly in the Northern of South America. *G. officinalis* is a shrub known by rural communities in the Venezuelan mountains for their pharmacological activities, and its extract is used in the control of dyspepsia, dysentery, and chronic diarrhea, in addition to presenting antimalarial, cytotoxic [59], molluscicidal activities, and antimicrobial properties, inhibiting the growth of *Mycobacterium tuberculosis*, the etiological agent of tuberculosis [60].

In later trials, galipinine and galipeine exhibited promising biological activities *in vitro* such as antimalarial (IC50: 0.24-6.12 and 0.33-13.78 mM, respectively) for the protozoa species *Plasmodium falciparum*, one of the species causing malaria that is already resistant to chloro‐ quine, one of the main drugs used to combat the disease [61].

Due to their structural simplicities associated with the promising pharmacological activities, these alkaloids, especially angustureine, have attracted the attention of the synthetic organic community. For this alkaloid alone, more than 24 different syntheses have already been described [62-85]. Some recent examples of the syntheses of these alkaloids are described below.

Diaz *et al*. [83] described the enantioselective synthesis of both alkaloids (*R*)-(-)- and (*S*)-(+) angustureine with overall yields of 80% and 44%, respectively, and excellent enantiomeric excesses (95% and 96%, respectively), starting from the (*S*)-β-amino ester and (*R*)-sodium carboxylate, prepared following enzymatic resolution of the β-amino ester racemate [86, 87].

Foubelo *et al*. [85] described a second-generation synthesis of (-)-angustureine by way of 2 allyl-tetrahydroquinoline. A further sequence of three steps from this intermediate provided angustureine in 36% overall yield. However, 2-allyl-tetrahydroquinolines can serve as common precursors to various 2-substituted tetrahydroquinoline alkaloids by making use of the cross-metathesis reaction with the Hoveyda-Grubbs reagent (second-generation rutheni‐ um catalyst) to achieve the appropriate side-chain homologation events.

In turn, Lam *et al*. [88] synthesized analogues of tetrahydroquinoline alkaloids employing asymmetric hydrogenation catalyzed by iridium. These compounds were tested *in vitro* for antitumor activities against lung cancer, breast cancer, hepatocellular carcinoma, and sarcoma cancer and have achieved remarkable results, as well as *in vivo*, using mice as animal models receiving hepatocellular tumor grafts, being that 1,2,3,4-tetrahydroquin-8-ol (Figure 12) revealed the highest toxicity to human cancer cells.

1,2,3,4-tetrahydroquin-8-ol

**Figure 12.** Structure of 1,2,3,4-tetrahydroquin-8-ol anticancer agent.

#### **4. Concluding remarks**

This chapter is an illustration of the fascinating world of chemistry of quinoline and isoqui‐ noline alkaloids. The range of pharmacological and agrochemical activities, among others, associated with their biosynthetic routes and various natural sources demonstrates the creative universe of the nature of these alkaloids. This universe of compounds arouses interest to organic chemists in general, stimulated by the simplicity and its varied forms of exploitation throughout constant search for the discovery of novel pharmacological agents targeting the treatment of several diseases that affect society.

#### **5. Future directions**

In this chapter, we can verify the importance of quinoline and isoquinoline alkaloids due to their potent biological activities demonstrated by *in vitro* and *in vivo* assays, as well as agrochemical agents reported throughout this chapter. Based on these facts, this family of compounds is presented as an inexhaustible source for research groups working with natural and synthetic products for the pharmaceutical area. In this context, our group intends to continue the study of these alkaloids with the synthesis of novel quinoline and isoquinoline derivatives, aimed at the discovery of new pharmacological agents.

### **Acknowledgements**

Diaz *et al*. [83] described the enantioselective synthesis of both alkaloids (*R*)-(-)- and (*S*)-(+) angustureine with overall yields of 80% and 44%, respectively, and excellent enantiomeric excesses (95% and 96%, respectively), starting from the (*S*)-β-amino ester and (*R*)-sodium carboxylate, prepared following enzymatic resolution of the β-amino ester racemate [86, 87]. Foubelo *et al*. [85] described a second-generation synthesis of (-)-angustureine by way of 2 allyl-tetrahydroquinoline. A further sequence of three steps from this intermediate provided angustureine in 36% overall yield. However, 2-allyl-tetrahydroquinolines can serve as common precursors to various 2-substituted tetrahydroquinoline alkaloids by making use of the cross-metathesis reaction with the Hoveyda-Grubbs reagent (second-generation rutheni‐

In turn, Lam *et al*. [88] synthesized analogues of tetrahydroquinoline alkaloids employing asymmetric hydrogenation catalyzed by iridium. These compounds were tested *in vitro* for antitumor activities against lung cancer, breast cancer, hepatocellular carcinoma, and sarcoma cancer and have achieved remarkable results, as well as *in vivo*, using mice as animal models receiving hepatocellular tumor grafts, being that 1,2,3,4-tetrahydroquin-8-ol (Figure 12)

> N H

OH 1,2,3,4-tetrahydroquin-8-ol

This chapter is an illustration of the fascinating world of chemistry of quinoline and isoqui‐ noline alkaloids. The range of pharmacological and agrochemical activities, among others, associated with their biosynthetic routes and various natural sources demonstrates the creative universe of the nature of these alkaloids. This universe of compounds arouses interest to organic chemists in general, stimulated by the simplicity and its varied forms of exploitation throughout constant search for the discovery of novel pharmacological agents targeting the

In this chapter, we can verify the importance of quinoline and isoquinoline alkaloids due to their potent biological activities demonstrated by *in vitro* and *in vivo* assays, as well as

um catalyst) to achieve the appropriate side-chain homologation events.

revealed the highest toxicity to human cancer cells.

154 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Figure 12.** Structure of 1,2,3,4-tetrahydroquin-8-ol anticancer agent.

treatment of several diseases that affect society.

**4. Concluding remarks**

**5. Future directions**

The authors are grateful to CAPES and FAPEMIG for their financial support.

### **Author details**

Gaspar Diaz1\*, Izabel Luzia Miranda2 and Marisa Alves Nogueira Diaz3

\*Address all correspondence to: gaspardm@qui.ufmg.br

1 Departament of Chemistry, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

2 Departament of Chemistry, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil

3 Departament of Biochemistry and Molecular Biology, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil

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## **Aspidosperma Terpenoid Alkaloids — Biosynthetic Origin, Chemical Synthesis and Importance**

Pedro Gregório Vieira Aquino, Thiago Mendonça de Aquino, Magna Suzana Alexandre-Moreira, Bárbara Viviana de Oliveira Santos, Antônio Euzébio Goulart Santana and João Xavier de Araújo-Júnior

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59758

### **1. Introduction**

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cine 2013;20(2) 166-171.

Since a long time ago health sciences and natural products have been linked by the use of remedies and poisons, and nowadays there is little doubt that humans used natural drugs long before the emergence of written history [1,2]. The Ebers Papyrus dating from about 1600 BC, is one of the oldest medical treatise, which documents natural product-derived drugs used by the Sumerians and Akkadians in the 3rd century BC. Today there is information on medicinal plants dating back over about 500 years, as documented in herbaria. Laboratory studies of medicinal natural products started only about 200 years ago, with the isolation of of morphine, an alkaloid, from opium (*Papaver* spp.) [2,3].

*Aspisoperma* genus (Apocynaceae) species are trees of a great diversity of sizes that grow in different habitats and are distributed mainly among the Americas; In Brazil about 50 species of this genus have been catalogued [4–6]. There are several reports in the literature concerning the folk utilization of plants of this genus, as in treatment of malaria, dysentery, appendicitis, wounds, fever, dyspnea, asthma, scabies, stomachache, cough, constipation, boils, rheuma‐ tism, leishmaniasis, toothache, urinary tract inflammation and dermatitis. However several studies show that some plants of the genus are not recommended for pregnant women because of their potential abortifacient and teratogenic effects [7–28].

Given the diversity of popular uses of plants of the genus *Aspidosperma* as well as the pre‐ dominance of terpenoid-alkaloids production in this genus and the importance of these substances for organic synthesis, medicinal chemistry and for knowledge of the biosynthetic

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

pathways used by plants to produce them, we propose to review the literature concerning the aspidosperma-type terpenoid-alkaloids chemical synthesis and their biological potential.

#### **2. Biosynthesis of Aspidosperma terpenoid alkaloids**

The isolation of alkaloids from species of *Aspidosperma* trees and their structural elucidation give rise to theories that attempt to explain their biosynthetic origin. In the field of indole-type alkaloids, one of the earliest theories to explain its biosynthesis arose in 1933, proposing that this type of alkaloid has origin in the reaction between tryptophan, phenylalanine and glicine (although at the time the proposed structures do not represent exactly the known reality today) [29]. Revisions of this theory lead to a new biosynthetic route, proposing that the indole-type alkaloids are derived from shikimic and prephenic acids and their interactions with *seco*prephenate-formadehyde units and aromatic aminoacids as tryptamine and tryptophan (figure 1) [30,31]. As Aspidosperma type alkaloids were isolated theories that tried to explain their biosynthetic origin began to emerge, which, at this moment, were based on the chemical synthesis of such alkaloids, as done by several research groups [32].

**Figure 1.** Early propositions for indole alkaloid precursors.

Early work on proof of terpenoid alkaloid biosynthesis were based on administration of deuterium-labelled precursors of alkaloids to the plants tested and analysis of the metabolites produced to confirm a proposed biosynthetic way. The earliest proposition for the biosynthesis of Aspidosperma terpenoid alkaloids was the synthesis *via* mevalonate pathway, demonstrat‐ ed in 1966 by the administration of 1-[²H2]-geraniol and 2-¹4 C-geraniol to *Vinca rosea* and mass spectrometry detection of labeled vindoline, what allowed the proposition of the biosynthetic route showed in figure 2 [33,34]. This biosynthetic way was refined two years later with the demonstration that administration of 14C-labelled loganin (produced by the administration of 2-¹4 C-geraniol to *Meyanthes trifoliata*) to *V. rosea* and *Rawfolia serpentina* allowed the isolation of 14C-labeled catharantine, serpentine, ajmalicine, vindoline and perivine [35], whose biosyn‐ thetic mechanism was detailed elsewhere [36–47].

**Figure 2.** Proposal to Aspidosperma terpenoid alkaloid biosynthesis (adapted from [1-2]).

#### **3. Chemical synthesis of Aspidosperma terpenoid alkaloids**

Since the structure elucidation of the first isolated Aspidosperma alkaloids, various alterna‐ tives and techniques have emerged, due mainly the great structural complexity of this family of alkaloids. One of the earliest syntheses of an Aspidosperma alkaloid was published by a group from Havard University in 1959, which obtained the recently-isolated alkaloid ellipti‐ cine from condensation of indole with 3-acetylpyridine followed by reduction and pyrolisis, as shown in figure 3 [48].

**Figure 3.** First synthesis of ellipticine.

pathways used by plants to produce them, we propose to review the literature concerning the aspidosperma-type terpenoid-alkaloids chemical synthesis and their biological potential.

The isolation of alkaloids from species of *Aspidosperma* trees and their structural elucidation give rise to theories that attempt to explain their biosynthetic origin. In the field of indole-type alkaloids, one of the earliest theories to explain its biosynthesis arose in 1933, proposing that this type of alkaloid has origin in the reaction between tryptophan, phenylalanine and glicine (although at the time the proposed structures do not represent exactly the known reality today) [29]. Revisions of this theory lead to a new biosynthetic route, proposing that the indole-type alkaloids are derived from shikimic and prephenic acids and their interactions with *seco*prephenate-formadehyde units and aromatic aminoacids as tryptamine and tryptophan (figure 1) [30,31]. As Aspidosperma type alkaloids were isolated theories that tried to explain their biosynthetic origin began to emerge, which, at this moment, were based on the chemical

O O

Early work on proof of terpenoid alkaloid biosynthesis were based on administration of deuterium-labelled precursors of alkaloids to the plants tested and analysis of the metabolites produced to confirm a proposed biosynthetic way. The earliest proposition for the biosynthesis of Aspidosperma terpenoid alkaloids was the synthesis *via* mevalonate pathway, demonstrat‐

spectrometry detection of labeled vindoline, what allowed the proposition of the biosynthetic route showed in figure 2 [33,34]. This biosynthetic way was refined two years later with the demonstration that administration of 14C-labelled loganin (produced by the administration of

C-geraniol to *Meyanthes trifoliata*) to *V. rosea* and *Rawfolia serpentina* allowed the isolation of 14C-labeled catharantine, serpentine, ajmalicine, vindoline and perivine [35], whose biosyn‐

O HO

O

O OH

NH2

NH2

OH

O

N H

C-geraniol to *Vinca rosea* and mass

N H

*seco*-prephenate-formadehyde Tryptamine Tryptophan

**2. Biosynthesis of Aspidosperma terpenoid alkaloids**

164 Phytochemicals - Isolation, Characterisation and Role in Human Health

synthesis of such alkaloids, as done by several research groups [32].

HO

2-¹4

OH

Shikimic acid

O OH

OH

OH

**Figure 1.** Early propositions for indole alkaloid precursors.

Prephenic acid

O

O

OH

ed in 1966 by the administration of 1-[²H2]-geraniol and 2-¹4

thetic mechanism was detailed elsewhere [36–47].

HO

Many years later, a new synthesis of aspidosperma-type skeleton was published, in a very simple way using four steps (figure 4) [49]. Another example is the synthesis of quebrachamine, one way published in 1966, and another three ways, one of them based on alkylation of cyclic enamines, other starting with 1,3-propanediol and another based on the cleavage of a thioketal group (figure 5) [50–53].

**Figure 4.** Synthesis of 3-Methylaspidospermidine (adapted from [49]).

**Figure 5.** Total synthesis of quebrachamine.

Despite the many synthetic routes described for obtaining quebrachamine, none was obtained with enantiomeric purity until the problem was addressed in 1980, with the development of a synthetic route to (+)-quebrachamine using L-glutamic acid as a chiral template (figure 6) [54].

(+)-Quebrachamine

**Figure 6.** Enantioselective total synthesis of quebrachamine

Enamines were also utilized in the synthesis of aspidospermine, as showed in 1971 by a group from Rice University (figure 7) and other groups [55,56].

**Figure 7.** Synthesis of aspidospermine.

O O

OO

4 steps

166 Phytochemicals - Isolation, Characterisation and Role in Human Health

N

HN

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

2 steps

O O

O

**Figure 5.** Total synthesis of quebrachamine.

NH2

N H

(+)-Quebrachamine

**Figure 6.** Enantioselective total synthesis of quebrachamine

N

O

N

O S S

> O O

H HO

> N H

N

H

N H

O

3 steps

Despite the many synthetic routes described for obtaining quebrachamine, none was obtained with enantiomeric purity until the problem was addressed in 1980, with the development of a synthetic route to (+)-quebrachamine using L-glutamic acid as a chiral template (figure 6) [54].

O O

O O O NH

4 steps

4 steps

N H 2 steps

N H

O O

N H N O

N H NH2

H

HO

N MeSO

O O O

N

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

O O

NH2

HN

N H

N O

N O

O OH

2 steps

N H N

Quebrachamine

N H

2 steps

2 steps

OHO

N

O

HO OH

6 steps

O O O O O

OO

O O

O

HO

O

In 1978 was published a work that introduced a conceptually new approach to the synthesis of Aspidosperma-type alkaloids, the photocyclization-rearrangement or heteroatom directed photoarylation of anilinocyclohexanones, exemplified by the synthesis of the indolines A and B shown in figure 8 [57], this concept being expanded many years later, with the demonstration of different techniques of photo-induced reactions [58–60].

**Figure 8.** Photoarylation in the synthesis of Aspidosperma-type substructures.

Given the biosynthetic route proposed by Wenkert [30], a group from Yale University developed a synthetic route for obtaining the alkaloid minovine in a biogenetically modeled way (figure 9), refined many years later by the same group [61,62].

**Figure 9.** Synthesis of minovine.

Based on the fact that the Aspidosperma alkaloids share common structural features, a group from the Chinese Academy of Synthesis developed a strategy to aspidophytine enantioselec‐ tive and stereo-controlled synthesis that could be applied to the synthesis of several other alkaloids of this family by simply varying the initial aniline (figure 10) [63].

**Figure 10.** Aspidophytine synthesis (adapted from [63]).

Another powerful technique for the Aspidosperma alkaloids skeleton is the utilization of aza-Cope rearrangements, utilized for the first time in 1981 for the stereoselective synthesis of 9aarylhydrolilolidines precursors of vindoline (figure 11) and later expanded to other alkaloids [64–66].

**Figure 11.** Application of aza-Cope rearrangement (adapted from [64]).

Based on the premise that Heck reaction is a powerful method for the construction of quater‐ nary carbon centers, researchers from Kyoto University decided to apply this methodology to the entantioselective synthesis of (-)-epieburnamonine (figure 12) [67].

Aspidosperma Terpenoid Alkaloids — Biosynthetic Origin, Chemical Synthesis and Importance http://dx.doi.org/10.5772/59758 169

**Figure 12.** Utilization of Heck reaction on the construction of intermediates in terpenoid alkaloids synthesis (adapted from [67]).

Exploiting the possibilities of C-H bond functionalization on the pyrrole ring, a group from Cambridge University recently proposed the total synthesis of rhanizilam-type alkaloids as precursors to Aspidosperma-type alkaloids, as shown in figure 13 [68].

**Figure 13.** Metal-catalyzed C-H bond functionalization on terpenoid alkaloid synthesis (adapted from [68]).

#### **3.1. Aspidosperma alkaloid precursors**

N

O

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

O

EtO EtO <sup>O</sup>

O

**Figure 10.** Aspidophytine synthesis (adapted from [63]).

**Figure 11.** Application of aza-Cope rearrangement (adapted from [64]).

O

PhS

[64–66].

O Si

Cl

O OH

**Figure 9.** Synthesis of minovine.

2 steps

168 Phytochemicals - Isolation, Characterisation and Role in Human Health

N O

alkaloids of this family by simply varying the initial aniline (figure 10) [63].

MeO

NH2 OMe

> N H

OMe

MeO

Another powerful technique for the Aspidosperma alkaloids skeleton is the utilization of aza-Cope rearrangements, utilized for the first time in 1981 for the stereoselective synthesis of 9aarylhydrolilolidines precursors of vindoline (figure 11) and later expanded to other alkaloids

O

the entantioselective synthesis of (-)-epieburnamonine (figure 12) [67].

Cl O

*2 steps*

Based on the premise that Heck reaction is a powerful method for the construction of quater‐ nary carbon centers, researchers from Kyoto University decided to apply this methodology to

N

O *4 steps*

Aspidophytine

OMe MeO O

O

N O H O

N

Based on the fact that the Aspidosperma alkaloids share common structural features, a group from the Chinese Academy of Synthesis developed a strategy to aspidophytine enantioselec‐ tive and stereo-controlled synthesis that could be applied to the synthesis of several other

N

O O 2 steps

EtO <sup>N</sup>

N

Minovine

H

N MeO H

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

HO N

MeO <sup>O</sup>

MeO

MeO

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

O

*8 steps*

EtO

O O

N

N

Another field of great interest is in synthesis of precursors which can serve to the achieve greater structural diversity from common structures. Various approaches have been utilized in this field, such as ketone annelation of endocyclic enamines [69] (figure 14) and the utiliza‐ tion of photochemistry with the one pot synthesis of a 9-membered ring system that could be applied not only in the synthesis of Aspidosperma-type alkaloids, but also Strychnos, Schiz‐ ozygane and Eburnamine-types, as shown in figure 15 [70].

**Figure 14.** Ketone annelation of endocyclic enamines on the synthesis of alkaloids.

**Figure 15.** Photochemistry on the synthesis of alkaloids.

Another approach relied on the conversion of *para*-substituted anisoles into 4,4-dissubstitued cyclohexenones via cyclohexadiene-Fe(CO)3 complexes, to obtain the tetrasubstituted carbon of Aspidosperma-type alkaloids, as demonstrated by the synthesis shown in figure 16 [71]. The iron complexes were also utilized in the synthesis of limaspermine derivatives, as shown in figure 17 [72]. Iron [73] and others metals were also utilized in Aspidosperma alkaloids synthesis, such as rhodium [74–77], copper [75,78], ruthenium and molybdenum [79], titanium [80] and palladium [81,82].

**Figure 16.** Synthesis of alkaloids with functionalised C(20) substituents via diene-Fe(CO)3 complex (adapted from [71]).

Another precursor of Aspidosperma type alkaloids was synthesized in 1978, from azocetones or iminomalonates via acid-catalysed and Birch reduction reactions (figure 18) [83].

Aspidosperma Terpenoid Alkaloids — Biosynthetic Origin, Chemical Synthesis and Importance http://dx.doi.org/10.5772/59758 171

**Figure 17.** Utilization of organoiron chemistry in limaspermine synthesis.

**Figure 18.** Synthesis of synthons for Aspidosperma alkaloids synthesis (adapted from [83]).

#### **3.2. Novel strategies**

applied not only in the synthesis of Aspidosperma-type alkaloids, but also Strychnos, Schiz‐

N H

N

Strychnos Eburnamine

Another approach relied on the conversion of *para*-substituted anisoles into 4,4-dissubstitued cyclohexenones via cyclohexadiene-Fe(CO)3 complexes, to obtain the tetrasubstituted carbon of Aspidosperma-type alkaloids, as demonstrated by the synthesis shown in figure 16 [71]. The iron complexes were also utilized in the synthesis of limaspermine derivatives, as shown in figure 17 [72]. Iron [73] and others metals were also utilized in Aspidosperma alkaloids synthesis, such as rhodium [74–77], copper [75,78], ruthenium and molybdenum [79], titanium

> O O

**Figure 16.** Synthesis of alkaloids with functionalised C(20) substituents via diene-Fe(CO)3 complex (adapted from [71]).

Another precursor of Aspidosperma type alkaloids was synthesized in 1978, from azocetones

or iminomalonates via acid-catalysed and Birch reduction reactions (figure 18) [83].

O

N

N O

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

N

HN HN

O

O

N

Aspidosperma Schizozygane

N

N O

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

O O

O

ozygane and Eburnamine-types, as shown in figure 15 [70].

170 Phytochemicals - Isolation, Characterisation and Role in Human Health

HN

O

**Figure 14.** Ketone annelation of endocyclic enamines on the synthesis of alkaloids.

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

N H

**Figure 15.** Photochemistry on the synthesis of alkaloids.

O

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

O O

N

O

O

Fe(CO)3

[80] and palladium [81,82].

*4 steps*

O

O O

Fe(CO)3

N

O

O

N

O

NH2

N

*hv*

One of the main concerns of chemists worldwide is the development of more efficient and "green" procedures in organic synthesis procedures. Among the procedures developed we can cite the so-called domino synthesis, where several bonds are formed in sequence, without isolation of intermediates, addition of reagents or changes in reaction conditions, so that the subsequent reaction result as a consequence of the functionality formed in the previous step [84]. One example of domino synthesis application to Aspidosperma alkaloids synthesis was recently published, where the alkaloids (-)-aspidospermidine, (-)-tabersonine and (-)-vinca‐ difformine were synthesized in an asymmetric domino Michael/Mannich/N-alkylation sequence, as shown in figure 19 [85].

The majority of synthetic strategies employed to obtain natural products are based on the construction of a single target skeleton, in contrast with the strategy utilized by plants, where

**Figure 19.** Domino Michael/Mannich/N-alkylation sequence to Aspidosperma alkaloids synthesis (adapted from [85]).

divergent molecular cyclizations of a polyunsaturated common intermediate produce different scaffolds, as recently demonstrated in two different papers, by the synthesis of different Aspidosperma alkaloids[81] and diverse indole alkaloids skeletons [86] from a common intermediate in a biogenetically-inspired way, as shown in figure 20 [86].

**Figure 20.** Synthesis of indole alkaloids in a biogenetically-inspired way (adapted from [86]).

#### **4. Biological importance of Aspidosperma terpenoid alkaloids**

One of the research interests of our group is the isolation of alkaloids from *Aspidosperma* species with pharmacological potential. From a chemotaxonomic point of view, alkaloids are sub‐ stances of great potential in malaria treatment [87,88]. In this perspective, we decided to study the alkaloids produced by *A*. *pyrifolium*, resulting in the isolation of the alkaloids 15-deme‐ thoxypyrifoline, aspidofractinine and *N*-formylaspidofractinine [89]. We have identified in *A. pyrifolium* insecticidal [90], antibacterial [91] and hypotensive activities [92]. Another plant studied by our group was *A. tomentosum*, which showed great anti-hipertensive [93,94], antinociceptive, anti-inflammatory and analgesic [95–98] and *A. macrocarpum*, which showed anti-hypertensive activity in spontaneously hypertensive mice [99].

divergent molecular cyclizations of a polyunsaturated common intermediate produce different scaffolds, as recently demonstrated in two different papers, by the synthesis of different Aspidosperma alkaloids[81] and diverse indole alkaloids skeletons [86] from a

**Figure 19.** Domino Michael/Mannich/N-alkylation sequence to Aspidosperma alkaloids synthesis (adapted from [85]).

common intermediate in a biogenetically-inspired way, as shown in figure 20 [86].

N PhO2S

> H *t*-Bu

N O Et

Li OMe <sup>S</sup> <sup>O</sup>

*M annich Reaction*

PhO2S

N

S <sup>O</sup> *<sup>t</sup>*-Bu Br

> Et CO2Me

N

N

CO2Me CO2Me

N

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

N

O O

R

R

N

N

O R'

N

N

Et

R' O

N

R

H O O

N H

Iboga

N H N H

CO2Me

One of the research interests of our group is the isolation of alkaloids from *Aspidosperma* species with pharmacological potential. From a chemotaxonomic point of view, alkaloids are sub‐ stances of great potential in malaria treatment [87,88]. In this perspective, we decided to study the alkaloids produced by *A*. *pyrifolium*, resulting in the isolation of the alkaloids 15-deme‐

Andraginine

NH2

N SO2Ph

N S *t*-Bu O Li

172 Phytochemicals - Isolation, Characterisation and Role in Human Health

CO2Me Et *M ichael Reaction*

HN

MeO2C

N

<sup>H</sup> CO2Me

Aspidosperma

**Figure 20.** Synthesis of indole alkaloids in a biogenetically-inspired way (adapted from [86]).

**4. Biological importance of Aspidosperma terpenoid alkaloids**

N H tryptamine

N

H2N <sup>S</sup> O *t*-Bu O

SO2Ph

Some species have been the subject of research in order to identify its pharmacological properties and other biological activities. In vitro assay with aqueous extracts of the aerial parts and roots of A*. pachypterum* against *Staphylococcus aureus* and the Human Immunodeficiency Virus (HIV), respectively, showed that this species exhibited a moderate activity [100,101]. The methanolic extract of the aerial parts of *A. ramiflorum* was active in vitro against gram-negative bacterium *Escherichia coli* [102] and against the fungus *Cryptococcus neoformans* (causing opportunistic infections in humans) [103] while the methanol extract of the stem bark of the same species was found to be moderately active against gram-positive bacteria and inactive against gram-negatives ones [104]. Studying tailings from the processing of hardwoods in Paraná (Brazil), it was found that the methanol extract of the wood of the plant identified as Peroba pink (*Aspidosperma* sp.) had a composition rich in phenols and alkaloids as well as strong activity against gram-negative bacteria *Proteus mirabilis* [105]. In two trials conducted with various plant species, among them five from *Aspidosperma* genre, it was observed that the ethanol extract of the stem bark of *A. excelsum*, *A. megalocarpon*, *A. oblongum* and *A. marcgra‐ vianum* were active against gram-positive bacteria *Bacillus subtilis* and that the same extracts and also the ethanol extract of the stem bark of *A. album* were active against gram-positive *S. aureus* [106,107]. In a study of Peruvian plants, it was reported that the extract of the bark of *A. rigidum* showed antibacterial activity against *B. subtilis* [108].

Another reported activity for species was the anti-Leishmania, where in vitro assay for *Leishmania amazonensis* promastigotes ahead and *L. braziliensis*, the fraction rich in alkaloids obtained from the stem bark proved to be active, with the highest activity observed against the first species [109]. Yet in order to find alternatives for the treatment of neglected diseases, the methanol extract of the bark of *A. megalocarpon* was tested against the D2 and F32 *Plasmo‐ dium falciparum* strains, being active [110]. The dichloromethane extract of the roots of *A. tomentosum* was active front *P. falciparum* (strain FcB1/Colombia) with a selectivity index of 67.5 compared with the activity front NIH-3T3 cells. In relation to substances with antifungal properties, it was seen that the ethanol extract of the stem of *A. polyneuron* was capable of inhibiting *Cladosporium herbarum* (pathogen of plants) [111].

In order to find alternatives for the treatment of cancer, the dichloromethane extract of the aerial parts of *A. tomentosum* was capable of inhibiting the proliferation of cell lines MCF-7 (breast cancer), UACC62 (melanoma), NCIADR (breast cancer phenotype with resistance to multiple drugs and NCl460 (lung cancer), and we observed that the activity was concentrated in fractions rich in terpenes and species of high polarity [112].

In vivo assay of the ethanol extract of the stem of *A. nitidum* showed significant anti-inflam‐ matory activity when evaluated in the trial of edema induced by carrageenan in mice. Prospecting for sources of antioxidant compounds, the hot aqueous extract of *A. quebracho-* *blanco* was tested for oxidation power / ferric reduction, showing a low activity and is therefore not considered as potential producer of antioxidant compounds [113].

It was observed that administration of a fraction rich in alkaloids obtained from the root bark of *A. ulei* exerted pro-erectile effect in rats and suggested a mechanism of action via blocking presynaptic α2-adrenergic receptors, the activation of the dopaminergic system and release of nitric oxide [114]. When the same fraction was tested in corpus cavernosum penis obtained from rabbit, its ability to cause relaxation was observed and the proposed mechanism blocking the influx of calcium into the cells [115]. In assay using α-adrenergic receptors isolated from human penis, it was shown that the crude extract and four fractions obtained from the bark of *A. quebracho-blanco* were able to block them, and the magnitude of interaction directly propor‐ tional to the content of the alkaloid yohimbine [116].

Despite reports of low toxicity associated with the use of plants of the genus *Aspidosperma* [109,110,117–119], some studies show a contrary position regarding the species *A. pyrifolium* [89,120]. In a study of the species *A. pyrifolium* cases of abortion in goats were reported due to ingestion of parts of the plants and when the ethanol extract of the leaves was administered to pregnant rats reduced fetal weight and maternal toxicity was observed, as well as hemolysis and toxicity test the front microcrustacean *Artemia salina* [120]. In a toxicity study with the microcrustacean *A. franciscana* with several species found in the Brazilian Amazon, among them seven species of *Aspidosperma*, it was reported that the bark extracts of *A. marcgravia‐ num*, *A. vargasii*, *A. nitidum* and *A. sprucenaum* led to mortality of 100, 94, 70 and 65% of the crustaceans, whilst extracts from the bark of *A. desmanthum*, *A. sandwithianum* and *A. shulte‐ sii* led to a mortality rate of 6, 0 and 0% crustaceans, showing the potential toxicity of some species gender [121]. In another test with brine shrimp, both the dichloromethane extract and the methanol extract of the bark of *A. excelsum* showed toxicity [14].

#### **5. Conclusion**

The present literature review shows the importance of the study of Aspidosperma type alkaloids due to the widespread usage of plants that produce these substances in folk medicine and the great array of potential biomedical applications that these substances exhibit. Beyond this it is clear the importance of developments in synthetic organic chemistry to obtain these substances without the necessity of extraction from natural sources.

#### **Acknowledgements**

We would like to thank the financial support given by CNPq, CAPES, FINEP and FAPEAL agencies.

### **Author details**

*blanco* was tested for oxidation power / ferric reduction, showing a low activity and is therefore

It was observed that administration of a fraction rich in alkaloids obtained from the root bark of *A. ulei* exerted pro-erectile effect in rats and suggested a mechanism of action via blocking presynaptic α2-adrenergic receptors, the activation of the dopaminergic system and release of nitric oxide [114]. When the same fraction was tested in corpus cavernosum penis obtained from rabbit, its ability to cause relaxation was observed and the proposed mechanism blocking the influx of calcium into the cells [115]. In assay using α-adrenergic receptors isolated from human penis, it was shown that the crude extract and four fractions obtained from the bark of *A. quebracho-blanco* were able to block them, and the magnitude of interaction directly propor‐

Despite reports of low toxicity associated with the use of plants of the genus *Aspidosperma* [109,110,117–119], some studies show a contrary position regarding the species *A. pyrifolium* [89,120]. In a study of the species *A. pyrifolium* cases of abortion in goats were reported due to ingestion of parts of the plants and when the ethanol extract of the leaves was administered to pregnant rats reduced fetal weight and maternal toxicity was observed, as well as hemolysis and toxicity test the front microcrustacean *Artemia salina* [120]. In a toxicity study with the microcrustacean *A. franciscana* with several species found in the Brazilian Amazon, among them seven species of *Aspidosperma*, it was reported that the bark extracts of *A. marcgravia‐ num*, *A. vargasii*, *A. nitidum* and *A. sprucenaum* led to mortality of 100, 94, 70 and 65% of the crustaceans, whilst extracts from the bark of *A. desmanthum*, *A. sandwithianum* and *A. shulte‐ sii* led to a mortality rate of 6, 0 and 0% crustaceans, showing the potential toxicity of some species gender [121]. In another test with brine shrimp, both the dichloromethane extract and

The present literature review shows the importance of the study of Aspidosperma type alkaloids due to the widespread usage of plants that produce these substances in folk medicine and the great array of potential biomedical applications that these substances exhibit. Beyond this it is clear the importance of developments in synthetic organic chemistry to obtain these

We would like to thank the financial support given by CNPq, CAPES, FINEP and FAPEAL

not considered as potential producer of antioxidant compounds [113].

the methanol extract of the bark of *A. excelsum* showed toxicity [14].

substances without the necessity of extraction from natural sources.

**5. Conclusion**

**Acknowledgements**

agencies.

tional to the content of the alkaloid yohimbine [116].

174 Phytochemicals - Isolation, Characterisation and Role in Human Health

Pedro Gregório Vieira Aquino1 , Thiago Mendonça de Aquino1 , Magna Suzana Alexandre-Moreira2 , Bárbara Viviana de Oliveira Santos3 , Antônio Euzébio Goulart Santana1 and João Xavier de Araújo-Júnior1\*

\*Address all correspondence to: jotaaraujo2004@gmail.com

1 Institute of Chemistry and Biotechnology, Federal University of Alagoas, Maceió, Brazil

2 Institute of Biological and Health Sciences, Federal University of Alagoas, Maceió, Brazil

3 Pharmaceutical Sciences Department, Federal University of Paraíba, João Pessoa, Brazil

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## **Bud Extracts as New Phytochemical Source for Herbal Preparations — Quality Control and Standardization by Analytical Fingerprint**

D. Donno, G.L. Beccaro, A.K. Cerutti, M.G. Mellano and G. Bounous

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59759

**1. Introduction**

#### **1.1. General, economic, and social notes**

#### *1.1.1. Historical background of the gemmotherapy method*

The use of buds for therapeutic purposes dates back many centuries. Ancient Indian medicine (Ayurveda) used, and still uses, plant buds for therapeutic purposes. In Western Europe, Galen (second century A.D.) prepared Acopon, one of the most renowned vulnerary balsams of that time, by soaking poplar buds in olive oil for three months. Nicolas De Myrepse, a medieval Greek physician, revived this idea and formulated the famous "Unguentum Populeum."

Buds and sprouts were identified with the persistence of the life cycle, which renewed itself each spring [1, 2]. Paracelsus realized that the different parts of a single plant had distinct properties [3].

In ancient times, physicians, botanists, and naturalists were interested in defining plants and their derivative properties. According to Theophrastus, herb quality was dependent on a series of factors, including plant age, collection method, used plant part, and geographical origin, as well as preparation methods and storage conditions of the final extracts. In the first century AD, Plinio il Vecchio warned his readers about the herbal preparation quality in relation to possible substitutes, pollutants, deterioration due to age, pests, or inadequate storage. Two centuries later, Galen emphasized the importance of distinguishing good quality products on the basis of sensory tests, drug potency, and geographical origin. During the Renaissance,

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

scientists and scholars also performed medicinal plant quality control through studies and experiments on fresh plants. Safety, quality, and efficacy, therefore, have always been the key factors distinguishing a therapeutic plant-based product, and over time they have become increasingly complex and important criteria.

Pol Henry was the first researcher who systematically studied bud-preparations. He is therefore considered to be the true "founding father" of gemmotherapy in its current form. He was the first to have the idea of using extracts derived from meristematic tissues rather than adult plant parts for human therapy and proposed a therapeutic method based on analogical protocols. He conducted a series of clinical trials on humans and animals to establish the psychopharmacological effects of, initially, some twenty or so gemmotherapy remedies. The results of his initial works were published in the "Archives Homeopathiques de Normandie" in 1959. Since then, many scientific publications have confirmed the validity of this therapeutic method [8, 9].

Gemmotherapy is a therapeutic method belonging to the field of Biotherapy, which, based on the analogical–biochemical principles of biological drainage, uses hydroglyceroalcoholic solutions of macerated fresh plant extracts in the first decimal dilution for therapeutic purposes [1, 6]. These extracts consist of meristematic tissues, such as buds, sprouts, young stems, rootlets, catkins, the inner root cortex, the young branch cortex, sap, seeds, and other meris‐ tematic plant tissues in the growth phase [10]. To express the concept at a taxonomic level, it would be more correct to describe the method as "meristemotherapy," since the used plant tissues are of meristematic origin.

Gemmotherapy/meristemotherapy is therefore a medical method that uses extracts of fresh plant tissues in the growth phase. Sprouts, buds, and other meristematic plant tissues retain all the anabolic faculties of the primitive plant cell, which is capable of developing all the potential no longer found in the adult plant. This therapeutic plant medicine belongs to "renewed phytotherapy," a branch of the biotherapeutic medicines classed between allopathic medicine and homeopathy. It was relatively unknown until a few years ago, but it is now growing considerably, in Italy and elsewhere [13]. The reason for this popularity lies in the fact that bud-preparations are easy to administer: they are sold ready-for-use and simply need to be diluted in water.

#### *1.1.2. New interest and current diffusion of phytotherapy*

Phytotherapy has thus developed from knowledge that was handed down over the course of millennia and has been confirmed, modified, or disproved through many pharmacological studies and clinical trials (Figure 1). It has therefore become a fully fledged medical discipline, since the knowledge gleaned from folk medicine has since been subjected to methodical scientific assessment in order to provide evidence of its efficacy [1, 15].

Progressively deteriorating environmental quality, pollution, excessive reliance on technolo‐ gy, continuous innovation of products and synthetic drugs perceived as alien to the organism, the indiscriminate use of many chemicals, and the change in the concept of health, which is no longer seen merely as the absence of disease but as a general sense of well-being, have Bud Extracts as New Phytochemical Source for Herbal Preparations — Quality Control and Standardization... http://dx.doi.org/10.5772/59759 189

**Figure 1.** Methods for pharmacological validation of the popular use of medicinal plants [12].

scientists and scholars also performed medicinal plant quality control through studies and experiments on fresh plants. Safety, quality, and efficacy, therefore, have always been the key factors distinguishing a therapeutic plant-based product, and over time they have become

Pol Henry was the first researcher who systematically studied bud-preparations. He is therefore considered to be the true "founding father" of gemmotherapy in its current form. He was the first to have the idea of using extracts derived from meristematic tissues rather than adult plant parts for human therapy and proposed a therapeutic method based on analogical protocols. He conducted a series of clinical trials on humans and animals to establish the psychopharmacological effects of, initially, some twenty or so gemmotherapy remedies. The results of his initial works were published in the "Archives Homeopathiques de Normandie" in 1959. Since then, many scientific publications have confirmed the validity of this therapeutic

Gemmotherapy is a therapeutic method belonging to the field of Biotherapy, which, based on the analogical–biochemical principles of biological drainage, uses hydroglyceroalcoholic solutions of macerated fresh plant extracts in the first decimal dilution for therapeutic purposes [1, 6]. These extracts consist of meristematic tissues, such as buds, sprouts, young stems, rootlets, catkins, the inner root cortex, the young branch cortex, sap, seeds, and other meris‐ tematic plant tissues in the growth phase [10]. To express the concept at a taxonomic level, it would be more correct to describe the method as "meristemotherapy," since the used plant

Gemmotherapy/meristemotherapy is therefore a medical method that uses extracts of fresh plant tissues in the growth phase. Sprouts, buds, and other meristematic plant tissues retain all the anabolic faculties of the primitive plant cell, which is capable of developing all the potential no longer found in the adult plant. This therapeutic plant medicine belongs to "renewed phytotherapy," a branch of the biotherapeutic medicines classed between allopathic medicine and homeopathy. It was relatively unknown until a few years ago, but it is now growing considerably, in Italy and elsewhere [13]. The reason for this popularity lies in the fact that bud-preparations are easy to administer: they are sold ready-for-use and simply need

Phytotherapy has thus developed from knowledge that was handed down over the course of millennia and has been confirmed, modified, or disproved through many pharmacological studies and clinical trials (Figure 1). It has therefore become a fully fledged medical discipline, since the knowledge gleaned from folk medicine has since been subjected to methodical

Progressively deteriorating environmental quality, pollution, excessive reliance on technolo‐ gy, continuous innovation of products and synthetic drugs perceived as alien to the organism, the indiscriminate use of many chemicals, and the change in the concept of health, which is no longer seen merely as the absence of disease but as a general sense of well-being, have

increasingly complex and important criteria.

188 Phytochemicals - Isolation, Characterisation and Role in Human Health

method [8, 9].

tissues are of meristematic origin.

to be diluted in water.

*1.1.2. New interest and current diffusion of phytotherapy*

scientific assessment in order to provide evidence of its efficacy [1, 15].

driven "modern people" to "return to nature." The use of phytotherapy has considerably increased in recent years, thanks to important studies confirming the medicinal properties of plant extracts and the continuous growth in scientific knowledge of their positive effects on humans and animals.

However, the use of plants for therapeutic purposes should be assessed by both the treating physician as well as the patient; the bioactive substances found in plants and their derivatives can also cause unwanted side effects, allergic reactions, or interactions with other plants or medicines [17]. For some diseases, moreover, no "miracle plants" are currently available, and so official medicine is the only option. They can, however, replace or complement synthetic drugs for several functional and somatic disorders. For example, many laxatives, painkillers, and tranquilizers can be replaced by a milder treatment based on medicinal plants [18].

The recent ISTAT multipurpose survey "Health conditions and the use of health services," conducted in 2005, has a section on the use of nonconventional therapies, which are also referred to as alternative, complementary, or traditional (at times ambiguously). The survey was carried out on a sample of approximately 60,000 households and showed that 13.9% of the Italian population (7.9 million) used nonconventional medicine during the three years preceding the survey, particularly homeopathy (7%), manual therapies (6.4%), and phyto‐ therapy (3.7%). There was also a clear prevalence in the north of the country (North-east Italy 21.9%, North-west Italy 17.9%, Central Italy 13.6%, Southern Italy 5.4% and the Italian islands 7%). The users were mostly women (15.8% compared to 11.2% of men) between thirty-five and forty-four years of age.

The inclination to use nonconventional treatment methods increases with the level of educa‐ tion: 18.7% of people with a degree or diploma, 13.5% of people with a middle-school diploma, and 9.2% of those with primary school certificates use these methods. Executives, entrepre‐ neurs, professionals, and office workers, in particular, most often used alternative therapies during the period covered by the survey [19]. According to the ISTAT survey, 71.3% of the patients stated that they chose nonconventional medicine because they considered it less toxic than conventional medical treatments.

General public opinion regarding UMs remained divided. However, 48.8% considered them to be effective and 51.2% regarded them as useless. Men were found to be more critical than women in regard to the usefulness of these methods. Most (73.5%) of the people interviewed in 2005 combined both homeopathy and phytotherapy with standard medicines. Specifically, 44.2% of them tried homeopathy and phytotherapy but mainly relied on conventional medical therapies, whereas 29.3% stated that they had combined conventional medical treatments with alternative therapies, relying mainly on the latter; 17% of the questioned people used homeo‐ pathy and phytotherapy exclusively, without combining them with other conventional medical treatments.

More recently, Eurispes indicated that 14.5% of Italians used nonconventional medicines in 2011, a decrease of 4% from 2010. Homeopathy remains the preferred therapy (70.6%), followed by phytotherapy (39.2%), osteopathy (21.5%), acupuncture (21%), and chiropractic (17.2%). There is a greater tendency toward regular use of alternative therapies in other European countries: 49% and 46% of the population in France and Germany, respectively, used it regularly, along with 35%, 31%, and 25% of the population in the United Kingdom, Belgium, and the countries of Northern Europe, respectively [20].

There are many reasons for the increasing interest in and availability of nonconventional medicines. Many patients try them because they have not benefited from traditional medical remedies or because they caused side effects. Moreover, the basic UM doctrines are holistic in nature, i.e., they consider the patient in his or her psychophysical entirety. A particular doctor– patient relationship is thereby established, based on the premise that a specific disorder may originate from a multiplicity of factors [3, 21]. The approach to the problem is therefore not confined to the treatment of a particular symptom, but extends to an analysis of the patient's personal and family background.

The growing popularity of these alternative medicine practices is also reflected in the efforts over the last twenty years by the World Health Organization (WHO), and, more recently, by the National Center for Complementary and Alternative Medicine (NCCAM) of the National Institute of Health (USA) to establish guidelines for methods to research and evaluate the effectiveness of nonconventional medicine [1, 22].

#### **1.2. Bud-preparations**

the Italian population (7.9 million) used nonconventional medicine during the three years preceding the survey, particularly homeopathy (7%), manual therapies (6.4%), and phyto‐ therapy (3.7%). There was also a clear prevalence in the north of the country (North-east Italy 21.9%, North-west Italy 17.9%, Central Italy 13.6%, Southern Italy 5.4% and the Italian islands 7%). The users were mostly women (15.8% compared to 11.2% of men) between thirty-five and

The inclination to use nonconventional treatment methods increases with the level of educa‐ tion: 18.7% of people with a degree or diploma, 13.5% of people with a middle-school diploma, and 9.2% of those with primary school certificates use these methods. Executives, entrepre‐ neurs, professionals, and office workers, in particular, most often used alternative therapies during the period covered by the survey [19]. According to the ISTAT survey, 71.3% of the patients stated that they chose nonconventional medicine because they considered it less toxic

General public opinion regarding UMs remained divided. However, 48.8% considered them to be effective and 51.2% regarded them as useless. Men were found to be more critical than women in regard to the usefulness of these methods. Most (73.5%) of the people interviewed in 2005 combined both homeopathy and phytotherapy with standard medicines. Specifically, 44.2% of them tried homeopathy and phytotherapy but mainly relied on conventional medical therapies, whereas 29.3% stated that they had combined conventional medical treatments with alternative therapies, relying mainly on the latter; 17% of the questioned people used homeo‐ pathy and phytotherapy exclusively, without combining them with other conventional

More recently, Eurispes indicated that 14.5% of Italians used nonconventional medicines in 2011, a decrease of 4% from 2010. Homeopathy remains the preferred therapy (70.6%), followed by phytotherapy (39.2%), osteopathy (21.5%), acupuncture (21%), and chiropractic (17.2%). There is a greater tendency toward regular use of alternative therapies in other European countries: 49% and 46% of the population in France and Germany, respectively, used it regularly, along with 35%, 31%, and 25% of the population in the United Kingdom, Belgium,

There are many reasons for the increasing interest in and availability of nonconventional medicines. Many patients try them because they have not benefited from traditional medical remedies or because they caused side effects. Moreover, the basic UM doctrines are holistic in nature, i.e., they consider the patient in his or her psychophysical entirety. A particular doctor– patient relationship is thereby established, based on the premise that a specific disorder may originate from a multiplicity of factors [3, 21]. The approach to the problem is therefore not confined to the treatment of a particular symptom, but extends to an analysis of the patient's

The growing popularity of these alternative medicine practices is also reflected in the efforts over the last twenty years by the World Health Organization (WHO), and, more recently, by the National Center for Complementary and Alternative Medicine (NCCAM) of the National

forty-four years of age.

medical treatments.

than conventional medical treatments.

190 Phytochemicals - Isolation, Characterisation and Role in Human Health

and the countries of Northern Europe, respectively [20].

personal and family background.

#### *1.2.1. Buds and others meristematic tissues*

Research has shown that, in addition to cytoplasmic substances, meristematic tissue cells also contain plant hormones (auxins, gibberellins, cytokinins, abscisic acid, and ethylene), en‐ zymes, aminoacids, trace elements, vitamins, flavonoids [23, 24], and nucleic acids (DNA and RNA), many bioactive compounds are often found only in trace amounts in adult plants [9, 25]. The effectiveness of bud-preparations is due to the combination of all these substances (called "botanicals"), which constitute the bud-phytocomplex.

Plants preserve meristematic cells in their meristematic tissue. The meristem biochemical profile is not comparable to the adult plant organ profile, because plant growth imposes a specialization aimed at specific biological roles [11, 26]. Meristematic tissues are composed of groupings of meristematic cells: the specialized adult tissue cells are subsequently differenti‐ ated from these cells. Buds are particular organs produced by the plant to protect these tissues during the cold season: new plant parts will emerge at the end of this unfavorable period from these tissues [25].

The following parts are used in gemmotherapy: buds, sprouts, rootlets, root cortex, young branch cortex, and seeds. Buds and sprouts are the most common tissues used for the budpreparation production [8, 27].

The buds spend the winter in a sleeping state, which begins with a period marked by decreas‐ ing temperatures or a progressive reduction of the photoperiod. The end of dormancy is determined by the exposure of the buds to relatively low temperatures for a certain number of hours (chilling requirement) and by a decrease in the level of abscisic acid, with an increase in cytokinin and gibberellin content. Indeed, in preparation for winter, abscisic acid is produced in terminal buds: this slows plant growth and directs leaf primordia to develop scales to protect the dormant buds during the cold season. It also inhibits the division of cells in the vascular cambium, adjusting to cold conditions in the winter by suspending primary and secondary growth. Gibberellins and cytokinins are also involved in the natural process of breaking dormancy and cell differentiation [25].

The breaking of dormancy is also associated with an increase in organic acid content and respiration in buds. Organic acid content was inversely related to carbohydrate content in developing buds [28].

Moreover, in the early stages of development, the plant tissues contain specific bioactive compounds: later, these molecules are subjected to metabolic transformations, as the removal/ adding of hydroxyl groups, diversifying, for example, the polyphenolic patrimony; in this case, recent histochemical tests revealed the presence of three types of polyphenolic compounds in buds, differing in what their intracellular localization is concerned: granular polyphenols, vacuolar polyphenols, and droplike polyphenols. Finally, the sugar moieties are diversified in mature organs: in buds, the preferred sugar for glycosylation is galactose, while, later, the glycosylation preferentially occurs with rhamnose [27].

#### *1.2.2. Preparation protocols*

The official procedure for bud-preparation production is detailed in the monograph "Home‐ opathic preparations" published in the 8th edition of the *French Pharmacopoeia* and subsequent editions [29].

This book describes the different stages of the extraction and preparation process:

**Collection**. Meristematic tissues should be collected in their balsamic period, which generally coincides with late winter–early spring, as the greatest concentration of bioactive compounds occurs at this time.

**Cleaning**. The newly harvested fresh parts are cleaned thoroughly to remove any foreign parts or soil residues.

**Measurement of humidity level and dry weight**. Samples are placed in a stove at 105°C to dehydrate for few days in order to reach a changeless weight, defined as the dehydrated weight. This operation allows the quantity of ethanol and glycerol to be calculated for the subsequent maceration step.

**Grinding**. The clean portion of fresh plant material is manually fragmented, using a mortar, for example, in the case of large buds. This operation facilitates extraction by the solvent.

**Maceration**. The fresh plant material is left to macerate for 21 days in a mixture of 95% ethanol and glycerol in a 1:1 ratio: the solvent quantities are calculated in order to obtain a 1:20 glycerine macerate, so that the final product is 20 times the weight of the raw material in the dry state. The maceration must be performed immediately after harvesting, to prevent the fresh tissue undergoing enzymatic and oxidative degradation.

**Decanting, filtration, and pressing**. Once the maceration is finished, the mixture is decanted and then filtered. The filter is pressed using a manual press. The product obtained from the pressing is added to the filtrate, which is then left to settle for another 48 hours. Finally, it is filtered once more, obtaining the mother preparation.

**Dilution**. The macerate is diluted to the first Hahnemann decimal (DH 1): one part of the mother preparation is diluted with nine parts of a mixture containing 50 parts (by weight) of glycerol, 30 parts of ethanol, and 20 parts of water. The mixture is then stirred for optimal mixing of the preparation. The alcohol content normally attained is 38°.

**Checks**. The final product is checked to assess and determine the smell and taste (by sensory analysis), color (by spectrophotometry), and alcohol content (e.g., by gas chromatography). A further test is useful to detect the presence of any contaminants, such as molds, yeasts, methanol, or 2-propanol.

#### *1.2.3. Relationship between drugs/bud-preparations and phytochemicals*

mature organs: in buds, the preferred sugar for glycosylation is galactose, while, later, the

The official procedure for bud-preparation production is detailed in the monograph "Home‐ opathic preparations" published in the 8th edition of the *French Pharmacopoeia* and subsequent

**Collection**. Meristematic tissues should be collected in their balsamic period, which generally coincides with late winter–early spring, as the greatest concentration of bioactive compounds

**Cleaning**. The newly harvested fresh parts are cleaned thoroughly to remove any foreign parts

**Measurement of humidity level and dry weight**. Samples are placed in a stove at 105°C to dehydrate for few days in order to reach a changeless weight, defined as the dehydrated weight. This operation allows the quantity of ethanol and glycerol to be calculated for the

**Grinding**. The clean portion of fresh plant material is manually fragmented, using a mortar, for example, in the case of large buds. This operation facilitates extraction by the solvent.

**Maceration**. The fresh plant material is left to macerate for 21 days in a mixture of 95% ethanol and glycerol in a 1:1 ratio: the solvent quantities are calculated in order to obtain a 1:20 glycerine macerate, so that the final product is 20 times the weight of the raw material in the dry state. The maceration must be performed immediately after harvesting, to prevent the fresh tissue

**Decanting, filtration, and pressing**. Once the maceration is finished, the mixture is decanted and then filtered. The filter is pressed using a manual press. The product obtained from the pressing is added to the filtrate, which is then left to settle for another 48 hours. Finally, it is

**Dilution**. The macerate is diluted to the first Hahnemann decimal (DH 1): one part of the mother preparation is diluted with nine parts of a mixture containing 50 parts (by weight) of glycerol, 30 parts of ethanol, and 20 parts of water. The mixture is then stirred for optimal

**Checks**. The final product is checked to assess and determine the smell and taste (by sensory analysis), color (by spectrophotometry), and alcohol content (e.g., by gas chromatography). A further test is useful to detect the presence of any contaminants, such as molds, yeasts,

This book describes the different stages of the extraction and preparation process:

glycosylation preferentially occurs with rhamnose [27].

192 Phytochemicals - Isolation, Characterisation and Role in Human Health

*1.2.2. Preparation protocols*

editions [29].

occurs at this time.

or soil residues.

subsequent maceration step.

methanol, or 2-propanol.

undergoing enzymatic and oxidative degradation.

filtered once more, obtaining the mother preparation.

mixing of the preparation. The alcohol content normally attained is 38°.

The principle of pure and pharmacologically active compounds was and remains the goal of pharmaceutical-chemical research in this century. Plants have been considered a very impor‐ tant source of active molecules for direct extraction or as a basis for the synthesis of molecules with specific pharmacological properties. Chemists have identified some of these many compounds and extracted them from plants, and physicians have studied their action mech‐ anisms and therapeutic effects. This has allowed the identification not only of bioactive compound classes, such as flavonoids, saponins, terpenes, but also of some single molecules (caffeic acid, ferulic acid, chlorogenic acid, quercetin, and many more). Pharmaceutical chemistry has thus begun to reproduce their fundamental structures, sometimes modifying them to obtain products with therapeutic effects of equal, if not greater, effect.

Conventional drugs contain a single active compound (represented by molecules by synthesis or by plant extraction), which has the purpose of interacting with a specific tissue-target in order to produce a fast and effective result. They are therefore suitable for acute diseases or in deteriorating chronic conditions [21, 33]. Phytotherapy treatments, like synthetic drugs, act on the organism by chemical substances, which have a pharmacological effect [6]. Due to the large quantity of bioactive compounds found in phytotherapeutic products, many of which act synergistically, there is a preference to attribute the pharmacological effect to the "phytocom‐ plex," rather than to any single active compound, as in the case of standard medicine.

The phytocomplex consists of a combination of different substances, both active principles and other plant components, which contribute to the overall therapeutic effect. The combination of individual components contained in the phytocomplex is able to release a synergistic action as a whole [21] and to reduce the risk of addiction and toxicity, producing a more complete and less drastic pharmacological effect [9] than that of one or a few of its components taken separately.

The main compounds with phytotherapeutic action are the plant secondary metabolites, i.e., compounds without a direct function in regard to growth and development. Unlike the primary metabolites, such as chlorophyll, aminoacids, nucleotides, or simple carbohydrates, they do not have roles in the processes of assimilation, respiration, transport, and differentia‐ tion. Although they are widely distributed throughout the plant kingdom, secondary metab‐ olites are normally found in a specific plant species or in a group of related species and are only localized in specific tissues and organs or in particular stages of development [6, 34].

#### **1.3. Main critical points**

#### *1.3.1. Final product and supply chain*

The current literature on bud-preparations shows a discrepancy regarding the preparation protocols. The *French Pharmacopoeia* requires the bioactive compounds to be extracted in a mixture consisting of ethanol and glycerol in equal parts. It also requires the "mother solution" to be diluted with a mixture of water, alcohol, and glycerol in a 1:10 ratio (one part of base macerated in nine parts of this mixture).

According to some experts, however, if the base preparation is diluted in a ratio of 1.5:10 (a more concentrated product), the bud-preparation will provide a higher number of clinical benefits [8]. Other herbal companies propose yet another maceration method, divided into two steps. During the first step, which lasts for a week, the plant material is macerated in ethanol alone. In the second step, after seven days, a solution of water (revitalized through a particular process) and glycerol in equal parts is added. In other cases, the bud-preparation is prepared by macerating the plant material (100 g) in a 1 L mixture with different extraction solvents, in a 1:2 ratio, while stirring vigorously. After a month, the macerate is filtered and the solvent removed with a rotary evaporator [24].

Another critical point concerns the botanical nomenclature showed in the bud-preparation package. It is not uncommon to find inaccuracies regarding the species names, as in lemon and chestnut herbal products. The latter, for example, is named as *Castanea vesca*, and this scientific name was replaced many years ago by *Castanea sativa*. In the European market, most of the plant material (80%) for herbal preparations is harvested in the wild, but also when harvested from specialized cultivations herbal products are only generally labeled with the species name without any regard to genotype (Figure 2).

**Figure 2.** No cultivar information is reported on the bud-preparation packages.

No plants are specifically grown for the phytotherapeutic market, and there is a lack of basic knowledge, i.e., researches on the potential qualitative and quantitative differences between different genotypes of a species.

Finally, the bud-preparation regulatory classifications are ambiguous (Figure 3): they have been associated with homeopathic products by the *French Pharmacopoeia*, based on their dilution method, but they can also now be associated with the food supplement category.

**Figure 3.** Bud-preparation regulatory classification is still contradictory and ambiguous.

#### *1.3.2. Analytical techniques*

According to some experts, however, if the base preparation is diluted in a ratio of 1.5:10 (a more concentrated product), the bud-preparation will provide a higher number of clinical benefits [8]. Other herbal companies propose yet another maceration method, divided into two steps. During the first step, which lasts for a week, the plant material is macerated in ethanol alone. In the second step, after seven days, a solution of water (revitalized through a particular process) and glycerol in equal parts is added. In other cases, the bud-preparation is prepared by macerating the plant material (100 g) in a 1 L mixture with different extraction solvents, in a 1:2 ratio, while stirring vigorously. After a month, the macerate is filtered and the solvent

Another critical point concerns the botanical nomenclature showed in the bud-preparation package. It is not uncommon to find inaccuracies regarding the species names, as in lemon and chestnut herbal products. The latter, for example, is named as *Castanea vesca*, and this scientific name was replaced many years ago by *Castanea sativa*. In the European market, most of the plant material (80%) for herbal preparations is harvested in the wild, but also when harvested from specialized cultivations herbal products are only generally labeled with the species name

removed with a rotary evaporator [24].

194 Phytochemicals - Isolation, Characterisation and Role in Human Health

without any regard to genotype (Figure 2).

**Figure 2.** No cultivar information is reported on the bud-preparation packages.

different genotypes of a species.

No plants are specifically grown for the phytotherapeutic market, and there is a lack of basic knowledge, i.e., researches on the potential qualitative and quantitative differences between Compared to other phytotherapic products, bud-preparations are currently affected by critical aspects, due to the total lack of analytical studies and comparative references to other research experiences at national and international levels [38].

An important issue concerns the analysis methods. Technological innovation in the analytical instruments has allowed increasingly sophisticated qualitative and quantitative assessments of the plant extract chemical composition. Chromatographic techniques are mainly used to analyze herbal preparations with some analytical problems related to the complexity of the initial matrix and the nature of the extraction solvents, because glycerol does not allow for the application of certain commonly used analytical techniques. Thin Layer Chromatography (TLC), for example, which is normally used to analyze mother tinctures, cannot be applied to bud-preparations, because the glycerol does not allow the analyte migration to the chromato‐ graphic plate. With paper chromatography and TLC, very small samples could be analyzed, and the resolution and reproducibility could be improved. However, quantitation is still inadequate and the resolution of similar compounds is difficult.

Chromatography includes a range of different instrumental techniques, from the simplest to the most sophisticated (Table 1).


**Table 1.** The major chromatographic methods used in phytochemistry [40].

Their common feature is a separation achieved through the flowing of a mobile phase, the eluent, containing the analytes to be separated, over a fixed phase, the adsorbent, which exerts a selective action on the compounds. Chromatography offers very powerful separation ability, such that the complex chemical components in herbal extracts can be separated into many relatively simple subfractions. Furthermore, the recent approaches of applying hyphenated chromatography and spectrometry such as high-performance liquid chromatography–diode array detection (HPLC–DAD), gas chromatography–mass spectroscopy (GC–MS), capillary electrophoresis–diode array detection (CE-DAD), HPLC–MS, and HPLC–NMR, can provide additional spectral information. This is very helpful for the qualitative analysis and for the online structural elucidation.

More specifically, High-Performance Liquid Chromatography is currently the most widely used analytical method, combined with different detectors (diode arrays, mass spectrometry, and fluorescence). All the compounds in herbal preparations can be analyzed by HPLC because this popular analytical method is easy and not limited by compound volatility and stability.

bud-preparations, because the glycerol does not allow the analyte migration to the chromato‐ graphic plate. With paper chromatography and TLC, very small samples could be analyzed, and the resolution and reproducibility could be improved. However, quantitation is still

Chromatography includes a range of different instrumental techniques, from the simplest to

**Chromatographic Method Separation Mechanism**

Paper (Adsorption), partition

Ion-pair chromatography Ion pair formation, ion interaction

Liquid–solid chromatography Adsorption

Gas liquid chromatography (GLC) Adsorption, partition Thin-layer chromatography (TLC) Adsorption, partition

High-performance liquid chromatography (HPLC) Adsorption, partition Ultra-performance liquid chromatography (UPLC) Adsorption, partition Supercritical fluid chromatography (SFC) Adsorption, partition Liquid–liquid chromatography (LLC) Partition Countercurrent chromatography (CCC) Partition Ion-exchange chromatography (IEC) Ion exchange Capillary electrophoresis Charge

Hydrophobic interaction chromatography (HIC) (Adsorption), partition Size exclusion chromatography (SEC) Size of analyte

Their common feature is a separation achieved through the flowing of a mobile phase, the eluent, containing the analytes to be separated, over a fixed phase, the adsorbent, which exerts a selective action on the compounds. Chromatography offers very powerful separation ability, such that the complex chemical components in herbal extracts can be separated into many relatively simple subfractions. Furthermore, the recent approaches of applying hyphenated chromatography and spectrometry such as high-performance liquid chromatography–diode array detection (HPLC–DAD), gas chromatography–mass spectroscopy (GC–MS), capillary electrophoresis–diode array detection (CE-DAD), HPLC–MS, and HPLC–NMR, can provide additional spectral information. This is very helpful for the qualitative analysis and for the on-

More specifically, High-Performance Liquid Chromatography is currently the most widely used analytical method, combined with different detectors (diode arrays, mass spectrometry,

Affinity chromatography Biological affinity

inadequate and the resolution of similar compounds is difficult.

196 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Table 1.** The major chromatographic methods used in phytochemistry [40].

line structural elucidation.

the most sophisticated (Table 1).

It is a chemical–physical separation method: different molecules distribute themselves, according to determined relationships, between two separate and distinct phases, one fixed into a column and the other mobile. In this case, there is a real molecules separation, while in other analytical methods, as spectrophotometry, the single compounds remain united and the separation consists in highlighting the different analyte interaction with the radiation. In the analysis of herbal products, reversed-phase (RP) HPLC columns are the most used stationary phases for compound separation. Different factors are involved in chromatographic separation as mobile phase chemical composition, pH of solvents, pump pressure, flow rate, and selected wavelengths [44].

Finally, the implementation of fast and reliable methodologies for analysis based on GC–MS, HPLC–UV–MS, etc., made possible the identification and quantification of known compounds in plants and other organisms [16].

#### **2. Phytochemical composition and quality of gemmotherapy products**

Gemmotherapy can provide effective therapy only through the availability of suitable plant extracts that are as uniform as possible. A great deal of information is necessary to ensure the herbal preparation's safety and quality (Table 2).



**Table 2.** Information relating to herbal product identification, characterization, and standardization [50].

#### **2.1. Genotype and bud phenological stage**

The bud-preparation quality begins in the field through the species identification before bud collecting. Species identification should be done by botanists or agronomists, as the diagnostic features typically used in botanical identification (leaves, flowers, etc.) are not usually present in the bud-harvesting season. However, some dichotomous keys allow trees to be identified in winter, through bud color and shape analysis [8]. Identification is an important step in the wild plant collection, even if it is often overlooked by the herbal companies: in the European market only 20% of the plants for herbal products are collected in germplasm repositories. Moreover, an individual plant species may have different cultivars, with different genetic characteristics, chemical compositions, and health effects on final product efficacy and consumer's health [52].

The buds are picked up in the early spring, when they emerge from winter sleeping. This time is known as the "balsamic period," a specific phenological stage in which the meristems have high concentrations of bioactive compounds [8]. The balsamic period, specific to each plant part, depends on the plant vegetative cycle; it can also vary because of different sampling sites and years. Tree species development is also influenced by natural factors (endogenous or exogenous), as well as by human factors. These factors can influence the plant organ chemical composition (and the respective herbal products).

#### **2.2. Pedoclimatic conditions and agrotechnique**

The herbal preparation quality is not only dependent on intrinsic characteristics (genetic factors), but is also closely linked to the collecting-area pedoclimatic conditions. Indeed, plants growing in more suitable areas have greater productive potential in terms of product quantity and quality [56]. In the case of plant material for bud-preparation production, knowledge of environmental characteristics, such as the best pedoclimatic conditions for the plant develop‐ ment, can help choose the best raw material to be used for bud-preparations [3, 12].

Each species requires its own best soil and climate, in terms of altitude, latitude, mean temperature, average annual rainfall, light availability, and physico-chemical soil properties. Climatic conditions show a direct effect on plant physiological processes and phenology, such as growth, flowering, and fruit ripening, thus they can also affect the availability of essential metabolites for the active compound biosynthesis [56, 57]. A plant can almost completely lose the ability to synthesize specific bioactive molecules outside of its own habitat [25]. The use of advanced agrotechniques together with a dedicated genetic improvement allow tree species to fit better with the cultivation site and can also improve the herbal product final quality [57].

#### **2.3. Extraction methods**

**Process applied to starting material** 1. Steps in preparation (e.g., separation, extraction processes, solvents)

**Botanical preparation** 1. Standardization criteria (markers: active constituents, other relevant components; plant extract ratio)

6. Storage conditions

**containing the botanical preparation)** 2. Fate on industrial processing and/or domestic preparation for consumption

**Table 2.** Information relating to herbal product identification, characterization, and standardization [50].

The bud-preparation quality begins in the field through the species identification before bud collecting. Species identification should be done by botanists or agronomists, as the diagnostic features typically used in botanical identification (leaves, flowers, etc.) are not usually present in the bud-harvesting season. However, some dichotomous keys allow trees to be identified in winter, through bud color and shape analysis [8]. Identification is an important step in the wild plant collection, even if it is often overlooked by the herbal companies: in the European market only 20% of the plants for herbal products are collected in germplasm repositories. Moreover, an individual plant species may have different cultivars, with different genetic characteristics, chemical compositions, and health effects on final product efficacy and

The buds are picked up in the early spring, when they emerge from winter sleeping. This time is known as the "balsamic period," a specific phenological stage in which the meristems have high concentrations of bioactive compounds [8]. The balsamic period, specific to each plant part, depends on the plant vegetative cycle; it can also vary because of different sampling sites and years. Tree species development is also influenced by natural factors (endogenous or exogenous), as well as by human factors. These factors can influence the plant organ chemical

The herbal preparation quality is not only dependent on intrinsic characteristics (genetic factors), but is also closely linked to the collecting-area pedoclimatic conditions. Indeed, plants

**End product (food or supplement** 1. Fate in food or formulated product

198 Phytochemicals - Isolation, Characterisation and Role in Human Health

**2.1. Genotype and bud phenological stage**

composition (and the respective herbal products).

**2.2. Pedoclimatic conditions and agrotechnique**

consumer's health [52].

2. Specifications: levels and range for markers

3. Specific precautions addressing, e.g., light/temperature sensitivity,

3. Physicochemical properties of relevant constituents; stability 4. Purity criteria by chain control or analysis; microbiological, mycotoxins, pesticides, environmental contaminants 5. Nature and level of excipients; formulation methodology

2. Methods used

oxidation

However, the herbal preparation quality is also determined by the following processing and storage procedures. The extraction technique is an important processing step that requires specific chemical and analytical knowledge. The extraction method and the used solvents show a high influence on the extract releasing and bioactivity. Each plant organ, due to the different chemical composition, has its own specific extraction method. The extraction method and its effectiveness depend on the solvent nature, which is linked to the different chemical compound polarities [24, 35], and to the extraction time and temperature [58].

Due to bud-preparation variability and complexity, it is very difficult to control their product quality: the key factors in achieving this objective are determination of the chemical compo‐ sition and standardization. The definition of an analytic fingerprint allows for qualitative and quantitative phytocomplex component evaluation: for this reason, botanical fingerprints become an effective tool for quality control [44]. This quantity should not be below a minimum level; otherwise the product will not have the proper therapeutic effect.

### **3. Analysis of phytochemicals in herbal products**

#### **3.1. Analytical methods**

Identification and quality control of the herbal material can be performed through macroscopic and microscopic techniques and the use of marker compounds [41]: a marker compound is a chemical constituent of a botanical raw material, drug substance, or drug product that is used for identification and/or quality control purposes, especially when the active constituents are not known or identified. The active compounds are responsible for the intended pharmaco‐ logical activity or synergistic therapeutic effects [35]. It is important to assess the phytochemical composition of herbal medicines in order to study pharmacological and clinical properties (bioactivity and possible side effects) of these products and their bioactive constituents. [44].

Sample preparation is the most important step in the development of analytical methods for the analysis of botanicals and herbal preparations. The basic operation included steps, as prewashing, drying of plant materials or freeze drying, grinding to obtain a homogeneous sample and often improving the kinetics of analyte extraction: the official extraction method, detailed in the *French Pharmacopoeia*, should be chosen in order to produce commercial preparations.

After bioactive compound extraction, in order to evaluate the entire pattern, chromatography combined with a suitable detection technique offers a powerful tool for separating the individual compounds and developing a characteristic profile of the sample, called a finger‐ print. In particular, High-Performance Liquid Chromatography (HPLC) is the most popular analytical technique for analyzing herbal products: HPLC is an easy to operate, fully auto‐ matable technique with high resolution, selectivity, and sensitivity. One of the main advan‐ tages of HPLC is the possibility to couple the technique to different detectors: combining a chromatographic separation system on-line with a spectroscopic detector has become the most important approach for the identification and/or confirmation of the identity of target and unknown chemical compounds in herbal preparations. For most (trace-level) analytical problems in the research field of herbal medicines, the combination of column liquid chroma‐ tography with a UV–vis or a mass spectrometer (HPLC–DAD, LC–MS, respectively) becomes the preferred approach for the analysis of herbal medicines. HPLC coupled with UV photo‐ diode array detection (DAD) allow the running of a chromatographic separation with simultaneous detection at different wavelengths. The UV spectra of bud-products give useful information on the type of constituents: indeed, new instruments allow the recording of UV spectra of compounds, which can be performed automatically when screening for known constituents [40]. The data obtained from hyphenated instruments are the so-called two-way data (one way for chromatogram and the other way for spectrum), which provide much more information than the classic one-way chromatography in order to obtain a complete chemical fingerprint. If hyphenated chromatography is further combined with chemometric ap‐ proaches, clear pictures might be developed for chromatographic fingerprints obtained.

#### **3.2. Statistical data analysis**

After selecting an appropriate technique to analyze the bud-preparation samples, an adequate method should be developed, optimized, and validated with a multivariate statistical ap‐ proach, as the Design of Experiments (DoE) or experimental design. In the multivariate approaches, a predefined number of experiments are performed according to a well-designed experimental setup in order to examine several factors simultaneously. The factors having the most effect on the outcome of a chromatographic analytical method are determined from screening designs: these factors included, amongst others, mobile phase composition, buffer concentration and pH, detection wavelength, column temperature, injection volume, sample concentration, and column type.

Due to the complexity of herbal medicines, and in particular bud-preparations, it is necessary to consider many factors for their fingerprint evaluation: some minor differences between strongly related genotypes may not be observed, but can largely affect the health of the patient. Classification of, and discrimination between herbal species is an important strategy in the quality control of herbal products [71]: the quality of bud-preparations is closely related to the chemical constituents and their concentration, and might vary slightly according to differences in climate, cultivation conditions, harvest time, drying, and storage [42]. To analyze the large number of generated data, a variety of analytical methods prove to be useful and versatile tools for the extraction, visualization, and interpretation of the information. Pattern recognition methods as Principal Component Analysis (PCA) allow better visualizing of the information that is included in the fingerprints. Exploratory data analysis is easier if represented as a multivariate data table as a low-dimensional plane. The original variables are transformed into latent variables summarizing the systemic patterns of variation between the samples. For this reason, PCA is often applied to chemical data expressed by bud-preparation fingerprinting.

### **4. Product quality**

#### **4.1. Safety**

Sample preparation is the most important step in the development of analytical methods for the analysis of botanicals and herbal preparations. The basic operation included steps, as prewashing, drying of plant materials or freeze drying, grinding to obtain a homogeneous sample and often improving the kinetics of analyte extraction: the official extraction method, detailed in the *French Pharmacopoeia*, should be chosen in order to produce commercial

After bioactive compound extraction, in order to evaluate the entire pattern, chromatography combined with a suitable detection technique offers a powerful tool for separating the individual compounds and developing a characteristic profile of the sample, called a finger‐ print. In particular, High-Performance Liquid Chromatography (HPLC) is the most popular analytical technique for analyzing herbal products: HPLC is an easy to operate, fully auto‐ matable technique with high resolution, selectivity, and sensitivity. One of the main advan‐ tages of HPLC is the possibility to couple the technique to different detectors: combining a chromatographic separation system on-line with a spectroscopic detector has become the most important approach for the identification and/or confirmation of the identity of target and unknown chemical compounds in herbal preparations. For most (trace-level) analytical problems in the research field of herbal medicines, the combination of column liquid chroma‐ tography with a UV–vis or a mass spectrometer (HPLC–DAD, LC–MS, respectively) becomes the preferred approach for the analysis of herbal medicines. HPLC coupled with UV photo‐ diode array detection (DAD) allow the running of a chromatographic separation with simultaneous detection at different wavelengths. The UV spectra of bud-products give useful information on the type of constituents: indeed, new instruments allow the recording of UV spectra of compounds, which can be performed automatically when screening for known constituents [40]. The data obtained from hyphenated instruments are the so-called two-way data (one way for chromatogram and the other way for spectrum), which provide much more information than the classic one-way chromatography in order to obtain a complete chemical fingerprint. If hyphenated chromatography is further combined with chemometric ap‐ proaches, clear pictures might be developed for chromatographic fingerprints obtained.

After selecting an appropriate technique to analyze the bud-preparation samples, an adequate method should be developed, optimized, and validated with a multivariate statistical ap‐ proach, as the Design of Experiments (DoE) or experimental design. In the multivariate approaches, a predefined number of experiments are performed according to a well-designed experimental setup in order to examine several factors simultaneously. The factors having the most effect on the outcome of a chromatographic analytical method are determined from screening designs: these factors included, amongst others, mobile phase composition, buffer concentration and pH, detection wavelength, column temperature, injection volume, sample

Due to the complexity of herbal medicines, and in particular bud-preparations, it is necessary to consider many factors for their fingerprint evaluation: some minor differences between strongly related genotypes may not be observed, but can largely affect the health of the patient.

preparations.

200 Phytochemicals - Isolation, Characterisation and Role in Human Health

**3.2. Statistical data analysis**

concentration, and column type.

Although botanicals have played a role in the marketing of health products to reduce age effects, there is an increased interest today due to their perceived health benefits. Not only do consumers increasingly take charge of their health, but the scientific information and under‐ standing of the beneficial health effects of bioactive substances in food, functional foods, and food supplements have improved. Increasing use of these products has also led to concerns about their actual safety.

The safety assessment of botanicals and botanical preparations in food and food supplement is complicated by, amongst others, the variability of composition and it should at least involve:


An increasing number of botanical products are used as food ingredients or supplements and these are a commercially important part of the health and food market. Botanical products may have different formulations, from whole foods to pharmaceutical-like preparations in unit dose form, as tablets, capsules, or drops [75].

The regulatory position with regard to food supplements is unclear (food or medicine?), and the increasing number of products in the food market raises concern about the safety assurance of these products, as many cases of intoxication with the use of botanical products are reported [50]. In some cases, these have resulted from contamination with other plant species. In addition, different parts of the plant source may have widely different concen‐ trations of toxic constituents, e.g., pyrrolizidine alkaloids [76], and climatic and agronom‐ ic differences may lead to great variability in composition [53]. Since compositional variability of bud-products may occur, it is important that the safety-evaluated botanical material is representative of the commercial material and has adequate specifications of identity and purity. Also, identification of the plant material, details of all the applied processes and compositional standards for the finished product are pivotal. Data should be based on a sufficient number of batches to permit the assessment of the natural variabili‐ ty under different agronomic conditions and establishment of appropriate specifications, and sampling plans should take the variability into account [33].

For some botanical products, good epidemiological and clinical human studies addressing both efficacy and safety may be available. However, if a herbal extract was to be used as a food supplement, the safety evaluation may be focused on the effects of the fractionation process, i.e., nature and concentrations of bioactive compounds relative to the traditional source and likely effects on bioavailability and intake [20].

Hazard identification (adverse effects and adverse effect nature) and hazard characterization (human-relevant dose levels) are necessary to determine the nature and level of risk associated with a particular level of herbal preparation exposure (intake) [75]. The risk assessment/safety evaluation relates to the material as consumed, thus data applicable to the finished products are of major importance. Therefore, detailed analytical fingerprint and other data are required to give assurance that the tested material provides relevant information, bearing in mind the possibility of matrix effects, e.g., on bioavailability, absorption, and pharmacokinetic behavior [60]. There is need of adequate chemical information related to all the phenological stages and to all the potential biological properties of the botanical material, also evaluating the possible botanical interactions with pharmaceuticals, aspects not normally considered for food additives [50].

#### **4.2. Standardization**

Scientists have been proceeding in a systematic way in order to validate the claims of many medicinal plant species. For some symptoms and ailments, this may be fairly easy to prove, but in more complex health conditions, the situation becomes a bit complicated. Nonetheless, medicinal plant extracts are showing a great deal of promise, even in instances of complexity of illnesses.

Millions of dollars are spent each year on herbal products that are marketed as food supple‐ ments, but in reality very few know, chemically, what they are purchasing or using. Very often the dosage varies from the different brands of the same herbal product. The chemistry and efficacy of many of these plants are relatively unknown and there is a chance of toxicity or overdose until the secondary compounds are known and understood [74, 77]. There is a tendency in the United States and in Europe to regulate and license this market, and this has led to greater and more effective use of these important medicinal plants. There is also general agreement that chemical standardization is the way forward in order for herbal remedies to be prescribed to patients who seek to be treated with medicinal plants [2].

Standardization is a method for assuring a minimum level of bioactive ingredients in the extracts and is becoming increasingly important as a means of ensuring a consistent supply of high-quality phyto-pharmaceutical products. It can be defined as the establishment of reproducible pharmaceutical quality by comparing a product with established reference substances, and by defining minimum amounts of one or several compounds or groups of compounds [35]. In general, in the field of phyto-medicines, standardization is only applied to extracts and not to diluted products. Standards for bioactive molecules to be used in medicinal products may be found in monographs and/or pharmacopeias. A pharmacopeia is a collection of quality standards for medicines and their components. In order to obtain marketing authorization for a medical product, the ingredients or the medicinal product must generally comply with a pharmacopeial standard. Thus, pharmacopeial standard may provide guidance on acceptable purity criteria for that ingredient.

It is accepted that concentration or dosages are very important because herbal medicines (in common with conventional medicines) contain biologically active substances that may produce nontrivial side effects when taken in excessive doses. Very low doses, on the other hand, may have no therapeutic value. Moreover, plant material is often highly variable, so that a minimum concentration or a concentration range is often used rather than an exact level. An upper limit is necessary with highly active or potentially harmful ingredients, as most plants have a wide therapeutic window (e.g., a toxic compound is considerably higher than the therapeutic dose). In the case of compounds with a narrow therapeutic window, chemical entities are favored, as opposed to extracts. Standardization also allows comparison of the clinical effectiveness, pharmacological effects and side effects of a series of products (e.g., against a placebo) [5]. Standardized products provide more security and increase the level of trust people have in herbal drugs.

Different kinds of extracts should be considered depending on standardization:


Standardization by blending different batches of a herbal drug before extraction, or by mixing different lots of herbal drug preparation, is acceptable but adjustment using excipients is not acceptable [22, 77].

#### **4.3. Quality control**

species. In addition, different parts of the plant source may have widely different concen‐ trations of toxic constituents, e.g., pyrrolizidine alkaloids [76], and climatic and agronom‐ ic differences may lead to great variability in composition [53]. Since compositional variability of bud-products may occur, it is important that the safety-evaluated botanical material is representative of the commercial material and has adequate specifications of identity and purity. Also, identification of the plant material, details of all the applied processes and compositional standards for the finished product are pivotal. Data should be based on a sufficient number of batches to permit the assessment of the natural variabili‐ ty under different agronomic conditions and establishment of appropriate specifications,

For some botanical products, good epidemiological and clinical human studies addressing both efficacy and safety may be available. However, if a herbal extract was to be used as a food supplement, the safety evaluation may be focused on the effects of the fractionation process, i.e., nature and concentrations of bioactive compounds relative to the traditional source and

Hazard identification (adverse effects and adverse effect nature) and hazard characterization (human-relevant dose levels) are necessary to determine the nature and level of risk associated with a particular level of herbal preparation exposure (intake) [75]. The risk assessment/safety evaluation relates to the material as consumed, thus data applicable to the finished products are of major importance. Therefore, detailed analytical fingerprint and other data are required to give assurance that the tested material provides relevant information, bearing in mind the possibility of matrix effects, e.g., on bioavailability, absorption, and pharmacokinetic behavior [60]. There is need of adequate chemical information related to all the phenological stages and to all the potential biological properties of the botanical material, also evaluating the possible botanical interactions with pharmaceuticals, aspects not normally considered for food

Scientists have been proceeding in a systematic way in order to validate the claims of many medicinal plant species. For some symptoms and ailments, this may be fairly easy to prove, but in more complex health conditions, the situation becomes a bit complicated. Nonetheless, medicinal plant extracts are showing a great deal of promise, even in instances of complexity

Millions of dollars are spent each year on herbal products that are marketed as food supple‐ ments, but in reality very few know, chemically, what they are purchasing or using. Very often the dosage varies from the different brands of the same herbal product. The chemistry and efficacy of many of these plants are relatively unknown and there is a chance of toxicity or overdose until the secondary compounds are known and understood [74, 77]. There is a tendency in the United States and in Europe to regulate and license this market, and this has led to greater and more effective use of these important medicinal plants. There is also general agreement that chemical standardization is the way forward in order for herbal remedies to

be prescribed to patients who seek to be treated with medicinal plants [2].

and sampling plans should take the variability into account [33].

likely effects on bioavailability and intake [20].

202 Phytochemicals - Isolation, Characterisation and Role in Human Health

additives [50].

of illnesses.

**4.2. Standardization**

Quality control for natural medicine begins in the field and ends with a safe and effective product being delivered to the patient, followed by post-marketing pharmacovigilance: the evaluation of genetic, agronomic, and environmental parameters is the first critical step in all the quality control supply chain. It is an evolutionary process. The World Health Organization has recognized this, and developed a series of guidelines to assist nations to develop their strategies for the quality control of herbal medicines.

Phytochemistry has an important and pivotal role in the implementation of each of the quality control steps. Each step requires knowing what is in the plant in terms of bioactive compounds, knowing what is happening to those constituents during the processing of the plant, and assuring appropriate analysis methods when materials are being tested biologically, and eventually delivered to the patient in a finished product [33].

There are serious concerns being expressed all over the world with respect to contamination and adulteration of traditional medicines. Contaminants may include various pesticides, herbicides, insecticides, heavy metals (arsenic, cadmium, lead, and mercury), microbial species, radiation, etc., and adulterants may include other (perhaps cheaper) plant materials with similar biological effects or synthetic drugs [5]. Microbial contamination can be control‐ led. It is a fact that natural materials harbor a large number of spores and other microorganisms. However, on the contrary, the maximum number or microorganisms allowed is regulated in most pharmacopeia [4].

Quality control is also very important to control side effects (plant extract toxicity). Botanical secondary compounds are not benign molecules. Ecologically speaking, these have evolved as chemical defenses that can repel, stun, poison, or kill other species. It would be easy on anybody's behalf to think that every plant extract is necessarily safe for human consumption. Nonetheless, it would be difficult for an inexpert consumer to distinguish between effective medicines from deadly poisons. Over and above the dosage factor, it is adulteration either accidentally or intentionally.

It is not a simple exercise of applying modern technologies to quality control of the products that have been in constant use for centuries. New technologies for botanical authentication, phytochemical analysis, and biological determination are employed for the development of new strategies to improve the quality control of medicinal plants [4]. Microscopy is less important here as opposed to phytochemical methods. In the case of the herbal extracts, Thin-Layer Chromatography is only feasible for some components, as the flavonoids, while the presence of vital minor compounds may be masked and may not be very visible. It is in this particular instance that the complementarity of analytical methods as HPLC are of paramount importance for analyzing both the lead and the minor compounds, as polyphenols and vitamins.

The use of chromatographic fingerprinting demonstrates to be an effective and efficient tool for the bud-extract quality control of the available herbal products. The complex relationship between the chromatographic fingerprints and efficacy of the herbal medicines could also be one of the most important aspects for the herbal medicine quality control. Chemical finger‐ prints might be linked to biological assays to provide assurance of efficacy and consistency. In general, the research work on this aspect is far from sufficient to meet the needed criteria: the studies on the analytical fingerprint of herbal preparations in relation to their efficacy are an important task for quality control of these products. At the same time, it is urgent to evaluate the possible contaminations in phytotherapic medicines (e.g., pesticides, microbial agents, heavy metals, and toxins).

#### **4.4. Phytochemical fingerprint**

the quality control supply chain. It is an evolutionary process. The World Health Organization has recognized this, and developed a series of guidelines to assist nations to develop their

Phytochemistry has an important and pivotal role in the implementation of each of the quality control steps. Each step requires knowing what is in the plant in terms of bioactive compounds, knowing what is happening to those constituents during the processing of the plant, and assuring appropriate analysis methods when materials are being tested biologically, and

There are serious concerns being expressed all over the world with respect to contamination and adulteration of traditional medicines. Contaminants may include various pesticides, herbicides, insecticides, heavy metals (arsenic, cadmium, lead, and mercury), microbial species, radiation, etc., and adulterants may include other (perhaps cheaper) plant materials with similar biological effects or synthetic drugs [5]. Microbial contamination can be control‐ led. It is a fact that natural materials harbor a large number of spores and other microorganisms. However, on the contrary, the maximum number or microorganisms allowed is regulated in

Quality control is also very important to control side effects (plant extract toxicity). Botanical secondary compounds are not benign molecules. Ecologically speaking, these have evolved as chemical defenses that can repel, stun, poison, or kill other species. It would be easy on anybody's behalf to think that every plant extract is necessarily safe for human consumption. Nonetheless, it would be difficult for an inexpert consumer to distinguish between effective medicines from deadly poisons. Over and above the dosage factor, it is adulteration either

It is not a simple exercise of applying modern technologies to quality control of the products that have been in constant use for centuries. New technologies for botanical authentication, phytochemical analysis, and biological determination are employed for the development of new strategies to improve the quality control of medicinal plants [4]. Microscopy is less important here as opposed to phytochemical methods. In the case of the herbal extracts, Thin-Layer Chromatography is only feasible for some components, as the flavonoids, while the presence of vital minor compounds may be masked and may not be very visible. It is in this particular instance that the complementarity of analytical methods as HPLC are of paramount importance for analyzing both the lead and the minor compounds, as polyphenols and

The use of chromatographic fingerprinting demonstrates to be an effective and efficient tool for the bud-extract quality control of the available herbal products. The complex relationship between the chromatographic fingerprints and efficacy of the herbal medicines could also be one of the most important aspects for the herbal medicine quality control. Chemical finger‐ prints might be linked to biological assays to provide assurance of efficacy and consistency. In general, the research work on this aspect is far from sufficient to meet the needed criteria: the studies on the analytical fingerprint of herbal preparations in relation to their efficacy are an important task for quality control of these products. At the same time, it is urgent to evaluate

strategies for the quality control of herbal medicines.

204 Phytochemicals - Isolation, Characterisation and Role in Human Health

most pharmacopeia [4].

accidentally or intentionally.

vitamins.

eventually delivered to the patient in a finished product [33].

Few data are available on natural product quality (intended as safety and efficacy) because adequate and validated methods and health care policies for herbal medicine evaluation are still lacking. Currently, only one or two biomarkers were considered for the evaluation of herbal product quality and authenticity, and for the assessment of the quantitative chemical composition of a herbal preparations and in assessing the quantitative herbal composition of a herbal product [81, 44]. This kind of determination, however, does not give a complete picture of a herbal product because multiple constituents are usually responsible for its therapeutic effects: it is necessary a chemical fingerprint be obtained by several analytical techniques.

By definition, a chromatographic fingerprint of a herbal medicine is a chromatographic pattern of the extract of the most common pharmacologically active compounds. With the help of obtained chromatographic fingerprints, the authentication and identification of herbal medicines can be accurately conducted even if the amount and/or concentration of the chemically characteristic constituents are not exactly the same for different samples of this preparation: the chromatographic fingerprints could demonstrate both the "sameness" and "differences" between various samples successfully.

A herbal preparation may contain several natural compounds and, for this reason, a combi‐ nation of different herbs could create interactions with many of these constituents during the extraction phases: therefore, a chromatographic fingerprint may present a complete represen‐ tation of all the chemical molecules of a natural medicine.

The chromatographic technique allows obtaining a relatively complete profile of budpreparations, the chromatographic fingerprint, representing the so-called phytocomplex. Obtaining a good chromatographic fingerprint representing the herbal preparation phyto‐ complex depended on several analytical factors, such as the extracting methods, the measure‐ ment instruments and the measurement conditions, according to other studies [66]. In particular, it is necessary to have a good extracting method, in order to obtain an informative fingerprint of herbal products with almost all the bioactive compounds to represent the integrity of the bud-preparations. Moreover, a chromatogram with good separation and a representative concentration profile of the bioactive components detected by a useful detector, as diode array detector, is also required.

A botanical fingerprint with all of its peaks just completely separated should be featured by maximal information content, and further separation cannot provide any more information and becomes unnecessary. It is a multivariate system, since in general it embraces most of the phytochemical constituents of herbal products [62]. Once different analytical fingerprints are obtained from the same herbal product or from different preparations, first of all it is important to evaluate any similarities and/or differences between them, also considering possible variation sources and their effects (e.g., retention time shift) from one chromatographic pattern to another. Moreover, the concentration profiles changes greatly for the herbal medicines depending on the different producing places and/or harvest seasons and wrong results would have been obtained by simply seeking the optimal correlation coefficient between the chro‐ matograms.

The construction of chromatographic fingerprints aims at evaluating the bud-product quality. Even if one or two markers or bioactive compounds are currently employed for evaluating the quality and authenticity of a herbal medicine, this determination does not give a complete picture of a herbal product. The fundamental reason of herbal medicine quality control is based on the concept of total herbal phytocomplex in order to identify the real herbal medicine and the false one [41]. If some fingerprints from different extraction methods and/or from different herbal medicines are considered, it is possible to see the same phytochemical constituents or to understand their bioactivities and possible side effects: it is very important for the quality assessment of different samples to determine the presence or absence of interested components among the different chromatographic fingerprints.

#### **4.5. Case study: Phytochemicals in Ribes nigrum bud-preparations**

*Ribes nigrum* L. (Family: *Grossulariaceae*) is commonly used as herbal medicine and it is popularized for its alleged tonic effect and possible curative and restorative properties; for this reason, blackcurrant bud-preparations, derived from embryonic fresh plant tissues, are important therapeutic remedies, prescribed in hepatic, respiratory, circulatory, and inflam‐ matory disorders, but data on their chemical composition are lacking as, to date, phytochemical studies have principally been performed on barks, roots and root exudates, leaves, fruit, and seeds. The most important industrial product of *Ribes nigrum* is fruit; however, due to their particular chemical composition and excellent flavor, leaves and buds are also used in some applications as a raw material for the herbal and cosmetic industries: many people use the buds as medicinal preparation for their anti-inflammatory activity and antidermal diseases (eczema and psoriasis) [86].

By nature herbal preparations are complex matrices, comprising a multitude of compounds that are prone to variation due to environmental factors and manufacturing conditions. The current practice of identifying herbal extracts is by measuring the concentration of the main botanicals; their concentrations are used to characterize the herbal preparations and finger‐ printing is recommended by the main pharmacopeias as a potential and reliable strategy for the quality control of complex mixtures.

The aim of this research was to perform an analytical study of *Ribes nigrum* bud-preparations, in order to identify and quantify the main bioactive compounds, obtaining a specific chemical fingerprint to evaluate the single class contribution to herbal preparation phytocomplex. The same analyses were performed using a High-Performance Liquid Chromatograph-Diode Array Detector (HPLC-DAD). In March 2013, samples of *Ribes nigrum* L. buds were picked up in a germplasm repository in San Secondo di Pinerolo, Turin Province (Italy): two different varieties (Rozenthal and Tenah) were sampled in different phenological stages (bud sleeping, bud break, first leaves), in order to test the genotype/sampling time effect on the chemical composition of the final product. Buds were used fresh to produce herbal preparations.

The protocol of bud-preparations is detailed in the monograph "Homeopathic preparations," quoted in the *French Pharmacopoeia*, 8th edition, 1965 [29]. Bioactive compounds were extracted through a cold maceration process for 21 days, in a solution of ethanol (95%) and glycerol, followed by a first filtration, a manual pressing and, after two days of decanting, a second filtration. Macerated samples were then stored at N.A., at 4°C and 95% R.H. Different chro‐ matographic methods were used to analyze the macerated samples, two for polyphenols and one for terpenic compounds, organic acids, and vitamins, respectively. Mineral elements were also considered.

depending on the different producing places and/or harvest seasons and wrong results would have been obtained by simply seeking the optimal correlation coefficient between the chro‐

The construction of chromatographic fingerprints aims at evaluating the bud-product quality. Even if one or two markers or bioactive compounds are currently employed for evaluating the quality and authenticity of a herbal medicine, this determination does not give a complete picture of a herbal product. The fundamental reason of herbal medicine quality control is based on the concept of total herbal phytocomplex in order to identify the real herbal medicine and the false one [41]. If some fingerprints from different extraction methods and/or from different herbal medicines are considered, it is possible to see the same phytochemical constituents or to understand their bioactivities and possible side effects: it is very important for the quality assessment of different samples to determine the presence or absence of interested components

*Ribes nigrum* L. (Family: *Grossulariaceae*) is commonly used as herbal medicine and it is popularized for its alleged tonic effect and possible curative and restorative properties; for this reason, blackcurrant bud-preparations, derived from embryonic fresh plant tissues, are important therapeutic remedies, prescribed in hepatic, respiratory, circulatory, and inflam‐ matory disorders, but data on their chemical composition are lacking as, to date, phytochemical studies have principally been performed on barks, roots and root exudates, leaves, fruit, and seeds. The most important industrial product of *Ribes nigrum* is fruit; however, due to their particular chemical composition and excellent flavor, leaves and buds are also used in some applications as a raw material for the herbal and cosmetic industries: many people use the buds as medicinal preparation for their anti-inflammatory activity and antidermal diseases

By nature herbal preparations are complex matrices, comprising a multitude of compounds that are prone to variation due to environmental factors and manufacturing conditions. The current practice of identifying herbal extracts is by measuring the concentration of the main botanicals; their concentrations are used to characterize the herbal preparations and finger‐ printing is recommended by the main pharmacopeias as a potential and reliable strategy for

The aim of this research was to perform an analytical study of *Ribes nigrum* bud-preparations, in order to identify and quantify the main bioactive compounds, obtaining a specific chemical fingerprint to evaluate the single class contribution to herbal preparation phytocomplex. The same analyses were performed using a High-Performance Liquid Chromatograph-Diode Array Detector (HPLC-DAD). In March 2013, samples of *Ribes nigrum* L. buds were picked up in a germplasm repository in San Secondo di Pinerolo, Turin Province (Italy): two different varieties (Rozenthal and Tenah) were sampled in different phenological stages (bud sleeping, bud break, first leaves), in order to test the genotype/sampling time effect on the chemical composition of the final product. Buds were used fresh to produce herbal preparations.

matograms.

among the different chromatographic fingerprints.

206 Phytochemicals - Isolation, Characterisation and Role in Human Health

(eczema and psoriasis) [86].

the quality control of complex mixtures.

**4.5. Case study: Phytochemicals in Ribes nigrum bud-preparations**

The content of total bioactive compounds in the evaluated bud-preparations is reported in Figure 4. Statistically significant differences were observed among the analyzed samples depending on genotypes and bud phenological stages.

**Figure 4.** Total bioactive compound content in Ribes nigrum bud-preparations.

Ribes nigrum was identified as a rich source of anti-inflammatory and antioxidant compounds: the observed analytical fingerprint demonstrated that these bud-preparations represent a rich source of organic acids, terpenic and polyphenolic compounds, especially catechins and phenolic acids (Table 3).

Chemical fingerprint showed the prevalence of organic acids and monoterpenes in chemical composition of all the analyzed samples (mean values were considered): the most important class was organic acids (75.57%), followed by monoterpenes (18.45%), polyphenolic com‐ pounds (4.94%), vitamins (0.95%), and mineral elements (0.09%) (Figure 5).


**Table 3.** Information relating to botanical fingerprint of Ribes nigrum bud-preparations.

Analytical fingerprinting could be an important tool to study the assessment of chemical composition and bioactivities of plant-derived products, helping in finding new sources of natural health-promoting compounds: this study allowed to develop an effective tool for quality control through the botanical fingerprinting of bud-preparations.

Bud Extracts as New Phytochemical Source for Herbal Preparations — Quality Control and Standardization... http://dx.doi.org/10.5772/59759 209

**Figure 5.** Ribes nigrum bud-preparation phytocomplex.

#### **5. Conclusion**

**Bioactive Compounds mg/100 gFW**

*ellagic acid* 34.270

*chlorogenic acid* 20.354 *coumaric acid* 7.645 *ferulic acid* 87.175

*epicatechin* 63.501

*isoquercitrin* 1.801 *quercetin* 68.921 *quercitrin* 34.066 *rutin* n.d.

*iron* 0.165 *magnesium* 0.697 *phosphorus* 1.626 *potassium* 4.694

*phellandrene* 163.176 *sabinene* 27.607 *γ-terpinene* 57.633 *terpinolene* 1194.037

*malic acid* 2189.727 *oxalic acid* 57.434 *quinic acid* 3259.735 *succinic acid* 481.857 *tartaric acid* 391.420

*B2* 0.002 *C* 80.747

**Benzoic Acids** *gallic acid* 46.018

208 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Cinnamic acids** *caffeic acid* 1.757

**Catechins** *catechin* 53.026

**Flavonols** *hyperoside* n.d.

**Mineral elements** *calcium* 0.701

**Monoterpenes** *limonene* 121.583

**Organic acids** *citric acid* 28.063

**Vitamins** *B1* 0.015

Analytical fingerprinting could be an important tool to study the assessment of chemical composition and bioactivities of plant-derived products, helping in finding new sources of natural health-promoting compounds: this study allowed to develop an effective tool for

**Table 3.** Information relating to botanical fingerprint of Ribes nigrum bud-preparations.

quality control through the botanical fingerprinting of bud-preparations.

#### **5.1. Herbal preparation supply chain**

The creation of specific horticultural models for bud-production could be an interesting opportunity to obtain a high standardized raw material for herbal preparations. If natural products are to be produced for utilization in the pharmaceutical industry, there will be certain criteria that a system of production must meet. First, it clearly must be economical: if the drug costs hundreds to thousands of dollars per dose, there is no viable product [39].

The production system must be sustainable and reliable because the plant part has to be harvested directly from its source (thus endangering the survival of the species), and the natural compound extraction may not be economically viable [8]. A source of bioactive compounds must be available to meet different medical needs, but today's society requires that production of natural products to be environmentally safe and nonenvironmentally impacting. Alternative methods for herbal preparation production include cultivation of the target species, synthesis, and biotech production. These approaches closely follow the original phytochemical study in order to assess the quality and yield of the production and the purity of the final product, but to be economical, these methods must also include fast analytical techniques.

To meet these criteria and establish a viable production system, all the steps of production of a natural product must be systematically evaluated. First, a source of bioactive substances must be identified: cultivars of the same species must be considered in order to choose the highest and the most stable concentration of the natural compounds. Once this source has been identified, an uninterruptible and stable supply of plant material must be secured. This means that an agronomic system for biomass production must be developed in order to allow the full expression of the genetic capability of the cultivar.

Climate and soil should fit with the species requirements. The impact of fertilization and irrigation on the biomass production and its chemical constituents must be understood and, generally, the economic growth and cultivation of the raw material must be explored [53]. In the future, a plant production of secondary metabolites may be controlled with different growth regulators according to Good Agriculture Practices (G.A.P.) used in this agronomic production [90].

The successful production of biomass should include the development of appropriate harvest techniques. Topics to consider are the determination of the best bioactive compound concen‐ tration in the plant material during the growing season and the handling of freshly harvested biomass to retain bioactive content: the choice of the best phenological stage during budharvesting period is a fundamental step to obtain high values of bioactive compounds in budproducts. Moreover, mechanization of the harvest process must be addressed in order to maintain an economic system of production. Harvested biomass should require an economic processing facility in order to process biomass for the plant material isolation over an entire calendar year, not just immediately after harvesting. Therefore, technology should be devel‐ oped to stabilize the biomass so that it retains bioactive content during storage before its ultimate processing: this usually involves developing an appropriate drying process. For this reason, analytical fingerprint could be also an important tool to evaluate the bioactive compound stability during product storage.

Finally, once the biomass processing is initiated, the extraction–purification system should be economical and efficient in its recovery of the bioactive compounds from the biomass. Moreover, it should be safe in its operation, and the generation of waste products should be minimized so that there is no deleterious environmental impact from the material processing.

#### **5.2. Directions for further studies**

Research into medicinal plants and the search for plant-derived preparations require a multidisciplinary approach with integrated projects, financial and technical support, and a very carefully planned strategy [12]. The research field of herbal medicine quality control is really an interdisciplinary research: it needs a crossover of chemistry, agricultural science, pharmacology, medicine, and even statistics to provide a platform for the quality control of herbal products and further discovery of the novel therapeutics composed of multiple chemical compounds [44]. The aims should consider demands in terms of public health, preservation of Biodiversity and the technical qualification of each laboratory or research group involved ; advances in technology and knowledge of natural products should be viewed not merely from the perspective of drug development, but also as a special tool for the understanding of biological phenomenon in order to contribute to the well-being of humanity [4]. Chemical analysis of extracts from plant material could play a central role in the development and modernization of natural medicines, botanicals and herbal preparations.

In order to evaluate the entire substances pattern of complex bud-products, chromatography offers a powerful tool for separating the individual compounds and hereby creates a specific fingerprint profile. Later on, when the analyses are performed, data handling methods should be used to extract the useful information residing within the enormous amounts of generated data. The use of an experimental design for the selection of the most influential factors and their optimization is important.

Once the analytical method is established, the herbal fingerprints can be recorded. Although major improvements in the quality control of herbal medicines have been made, proper fingerprint analysis does not only include an adequate choice of the analytical technique [40]. For this reason, more attention has been paid to the optimization of the extraction and separation procedures using appropriate experimental designs, even if an adequate prepro‐ cessing of the fingerprints and a critical validation of the results obtained by the data handling techniques are too often neglected [41].

Compounds as the macromolecules (e.g., polysaccharides), a major part of the bioactive compounds in herbal species, are often not considered in herbal fingerprinting, and should be taken into account during future development of quality control criteria. For this, several innovative methods based on the interaction with biological systems and dialysis methods are at the point of breakthrough and could allow scientists to approach quality control issues from other angles, reducing the complexity of the matrix and focusing on the directly related biologically active compounds. Accordingly, the biological activity of herbal samples can be integrated in the fingerprint development, focusing on the relevant biological information.

Just as for pharmaceutical products, a well-validated method can help in monitoring the stability of the botanicals and herbal preparations over a period of time: the fingerprint could be also applied in this direction. It is likely that the combination of chemical standardization with biological assay could provide further knowledge about therapeutic effects of the medicinal plants [37].

Finally, the use of validated methods in the chemical standardization of botanicals and herbal preparations could enhance the quality of the products, assist in pharmacological studies, perform credible clinical trials and propel the move toward evidence-based medicine.

#### **Author details**

To meet these criteria and establish a viable production system, all the steps of production of a natural product must be systematically evaluated. First, a source of bioactive substances must be identified: cultivars of the same species must be considered in order to choose the highest and the most stable concentration of the natural compounds. Once this source has been identified, an uninterruptible and stable supply of plant material must be secured. This means that an agronomic system for biomass production must be developed in order to allow the full

Climate and soil should fit with the species requirements. The impact of fertilization and irrigation on the biomass production and its chemical constituents must be understood and, generally, the economic growth and cultivation of the raw material must be explored [53]. In the future, a plant production of secondary metabolites may be controlled with different growth regulators according to Good Agriculture Practices (G.A.P.) used in this agronomic

The successful production of biomass should include the development of appropriate harvest techniques. Topics to consider are the determination of the best bioactive compound concen‐ tration in the plant material during the growing season and the handling of freshly harvested biomass to retain bioactive content: the choice of the best phenological stage during budharvesting period is a fundamental step to obtain high values of bioactive compounds in budproducts. Moreover, mechanization of the harvest process must be addressed in order to maintain an economic system of production. Harvested biomass should require an economic processing facility in order to process biomass for the plant material isolation over an entire calendar year, not just immediately after harvesting. Therefore, technology should be devel‐ oped to stabilize the biomass so that it retains bioactive content during storage before its ultimate processing: this usually involves developing an appropriate drying process. For this reason, analytical fingerprint could be also an important tool to evaluate the bioactive

Finally, once the biomass processing is initiated, the extraction–purification system should be economical and efficient in its recovery of the bioactive compounds from the biomass. Moreover, it should be safe in its operation, and the generation of waste products should be minimized so that there is no deleterious environmental impact from the material processing.

Research into medicinal plants and the search for plant-derived preparations require a multidisciplinary approach with integrated projects, financial and technical support, and a very carefully planned strategy [12]. The research field of herbal medicine quality control is really an interdisciplinary research: it needs a crossover of chemistry, agricultural science, pharmacology, medicine, and even statistics to provide a platform for the quality control of herbal products and further discovery of the novel therapeutics composed of multiple chemical compounds [44]. The aims should consider demands in terms of public health, preservation of Biodiversity and the technical qualification of each laboratory or research group involved ; advances in technology and knowledge of natural products should be viewed not merely from the perspective of drug development, but also as a special tool for the understanding of

expression of the genetic capability of the cultivar.

210 Phytochemicals - Isolation, Characterisation and Role in Human Health

compound stability during product storage.

**5.2. Directions for further studies**

production [90].

D. Donno\* , G.L. Beccaro, A.K. Cerutti, M.G. Mellano and G. Bounous

\*Address all correspondence to: dario.donno@unito.it

Department of Agricultural, Forest and Food Sciences, University of Turin, Grugliasco (TO), Italy

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## **Effects of Abiotic Stress (UV-C) Induced Activation of Phytochemicals on the Postharvest Quality of Horticultural Crops**

Rohanie Maharaj

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60050

#### **1. Introduction**

Phytochemicals are bioactive non-nutrient plant components that have gained considerable attention as photoprotective agents in providing certain health benefits and are well known for their powerful antioxidant and free radical scavenging potential [1-2, 8]. Phytochemicals found in plant-based foods (fruits, vegetables, legumes, nuts, cereals, and grains) belong to several classes according to their chemical structures and physiological functions and include polyphenols, flavonoids, isoflavonoids, phytoalexins, phenols, anthocyanidins, nitrogen compounds (polyamines), chlorophyll derivatives, beta carotene (pro-vitamin A), and other carotenoids, ascorbic acid (vitamin C), folic acid, and α-tocopherol (vitamin E). Figure 1 illustrates the classification of dietary phytochemicals, which are widely distributed with different structures at the tissue, cellular, and sub-cellular levels. While it has been reported that there are over 4,000 phytochemicals, the most studied are the phenolics (largest category of phytochemicals) and carotenoids [3, 5], whereas some phytochemicals are distributed only among limited taxonomic groups. For example, glucosinolates are only found in the crucifer‐ ous vegetables crops, whereas the occurrence of sulfides is restricted to the Liliaceae. Addi‐ tionally, each fruit and vegetable species has a distinct profile of phytochemicals, which is also within a special phytochemical group [4]. Many phytochemicals, particularly the pigment molecules, are often concentrated in the outer layers of the various plant tissues [5]. Some sources of phytochemicals include lycopene from tomatoes, isoflavones from soy, β-carotene from carrots, and anthocyanins from blueberries and grapes. The phytochemicals lutein and zeaxanthin are carotenoids found in spinach, kale, and turnip greens. Another group of phytochemicals called allyl sulfides are found in garlic and onions. http://www.cancer.org/ treatment/treatmentsandsideeffects/complementaryandalternativemedicine/herbsvitamin‐ sandminerals/phytochemicals

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Classification of dietary phytochemicals (Adapted from [3]).

The beneficial effects of phytochemicals are mediated through several mechanisms that include enzyme stimulation, interference with DNA replication, anti-bacterial effect, and physical action. Enhancing the health benefits of fresh produce through physical methods could therefore add value and create new opportunities for growers and processors by tapping into health-oriented markets [12, 57]. Abiotic stresses (negative impact of non-living environ‐ mental factors on growth and productivity of crops) are significant determinants of quality and nutritional value of crops along the food value chain. While abiotic stress is essentially unavoidable, simple modifications to postharvest handling systems may result in significant reduction in stress exposure, which will positively impact on phytochemical content and storage and/or shelf-life extension [7, 10]. There is a need to provide technologies that can ensure the delivery of high-quality products with high levels of the desired phytonutrients [6, 11-12, 15]. There is now considerable literature on the use of low dose UV-C radiation to control postharvest pathogens, delay postharvest senescence, and improve shelf-life of many horti‐ cultural crops. It also has potential for commercial use as a surface treatment in the food industry, particularly for whole and fresh-cut produce. However, the use of UV-C radiation as an elicitor of phytochemicals which enhances additional health benefits and mechanisms related to improvement in shelf-life or increase in the content of phytonutrients are still not well understood. The objective of this chapter will focus on a review of recent studies of UV-C abiotic stress on the production and activation of phytochemicals and its effect on the quality of crops.

### **2. UV radiation and its interactions with biological systems**

UV radiation is generally divided into three classes: UV-C (200-280 nm), UV-B (280-320 nm), and UV-A (315-400 nm) [21]. Each band can induce significantly different biological effects in plant systems. UV-C wavelengths (highly energetic) result in high levels of damage very quickly [22]; however, such radiations are effectively absorbed by ozone and atmospheric gases and are not present in sunlight at the earth's surface. UV radiation below 320 nm is actinic, which means it causes photochemical reactions [21]. Such reactions occur as a result of the absorption of photons by chromophores (chemical grouping of molecules) at particular wavelengths.

#### **2.1. Direct and indirect effects of UV radiation**

**Figure 1.** Classification of dietary phytochemicals (Adapted from [3]).

222 Phytochemicals - Isolation, Characterisation and Role in Human Health

of crops.

The beneficial effects of phytochemicals are mediated through several mechanisms that include enzyme stimulation, interference with DNA replication, anti-bacterial effect, and physical action. Enhancing the health benefits of fresh produce through physical methods could therefore add value and create new opportunities for growers and processors by tapping into health-oriented markets [12, 57]. Abiotic stresses (negative impact of non-living environ‐ mental factors on growth and productivity of crops) are significant determinants of quality and nutritional value of crops along the food value chain. While abiotic stress is essentially unavoidable, simple modifications to postharvest handling systems may result in significant reduction in stress exposure, which will positively impact on phytochemical content and storage and/or shelf-life extension [7, 10]. There is a need to provide technologies that can ensure the delivery of high-quality products with high levels of the desired phytonutrients [6, 11-12, 15]. There is now considerable literature on the use of low dose UV-C radiation to control postharvest pathogens, delay postharvest senescence, and improve shelf-life of many horti‐ cultural crops. It also has potential for commercial use as a surface treatment in the food industry, particularly for whole and fresh-cut produce. However, the use of UV-C radiation as an elicitor of phytochemicals which enhances additional health benefits and mechanisms related to improvement in shelf-life or increase in the content of phytonutrients are still not well understood. The objective of this chapter will focus on a review of recent studies of UV-C abiotic stress on the production and activation of phytochemicals and its effect on the quality

The destructive action of UV radiation results from both direct absorption of photons by DNA and indirect mechanisms involving excitation of photosensitizers and the generation of reactive oxygen species (ROS). Nucleic acids, proteins, lipids, indole acetic acids, flavorproteins, and phytochromes are molecules that contain conjugated double bonds and absorb energy in the UV region. They have key roles in plant cell function and structure and any alterations of these compounds due to UV radiation might be expected to cause physiological alterations in crops. Plants may differ in resistance to ROS depending upon the efficiency of the plant defense systems that involve DNA repair systems and quenching systems involving either a suppression of ROS production or scavenging of ROS, which have already been produced with enzymatic and non-enzymatic scavenging pathways. This can affect the secondary metabolism of fresh produce and increase the synthesis of phytochemicals or reduce the synthesis of undesirable compounds [4, 12, 16]. The biological consequences of free radical reactions leading to cellular dysfunction and cell death are summarized in Figure 2.

#### **3. Postharvest abiotic stress and the production of phytochemicals**

Harvested crops can be potentially exposed to various abiotic stresses that impact on quality in the food value chain. These include qualitative and quantitative losses including sensorial, microbial, and nutritional losses [7]. Wide variation in resistance to stress injury exists in higher plants and the stress tolerance threshold are determined by plant species, the developmental stage of the plant, type of stressor, exposure time, as well as other factors such as the plant stress-coping mechanisms. Abiotic stresses play a major role in determining the distribution of plant species across different types of environments. Many breeding programs impose abiotic stresses on the plants in a very quantitative way to develop stress tolerance that will allow crops to adapt to climate change [10]. However, it is not clear whether breeding in the field will also extend stress resistance characteristics in the postharvest phase including shelflife extension and phytonutrient quality of crops.

Various abiotic stresses (salinity, water stress, drought, heavy metals, wind, air pollution, altered gas composition, light, temperature extremes, and exposure to ultraviolet or gamma

**Figure 2.** Schematic representation of the photobiological effects of UV-C radiation in plants [26].

radiation) are known to have deleterious effects on plant tissues as a result of the production of ROS, causing progressive oxidative damage, which can damage biomolecules and may affect biochemical pathways, leading to cell death (as shown in Figure 2) and cause substantial crop losses worldwide [13, 16, 18-19]. Postharvest abiotic stressors can lead to numerous quality problems in fruits and vegetables, including scald, core and flesh browning of fruits, sweetening, pitting, water-soaked appearance, abnormal ripening, russeting, tissue softening, and loss of nutrient constituents [10]. Hence an understanding of the effects of field abiotic stresses on postharvest stress susceptibility will become more important since postharvest stresses limit the storage and shelf-life potential of crops. Characterization of mutants and transgenic plants with altered expression of antioxidants is also a potentially powerful approach to understanding the functioning of the antioxidant system and its role in protecting plants against stress [6, 13]. The use of genetically modified crops has its biases as in most cases it is considered as potential biological hazards that create an ecological imbalance [6, 9].

As a secondary response, some postharvest abiotic stress treatments could induce some mechanisms that affect the metabolic activity of the treated produce, such as triggering antioxidants. Studies have shown that plants with higher levels of antioxidants, whether constitutive or induced, showed a greater resistance to different types of environmental stresses [13]. Plants possibly protect themselves against increased UV irradiation by an increased synthesis of pigments in the epidermis [23]. However, the mechanisms of cellular oxidative stress are complex, and other biochemical and physiological mechanisms may act in concert under UV stress. Figure 3 illustrates possible mechanisms for using controlled postharvest abiotic stresses to enhance the phytochemical content of crops [7].

Effects of Abiotic Stress (UV-C) Induced Activation of Phytochemicals on the Postharvest Quality of Horticultural Crops http://dx.doi.org/10.5772/60050 225

**Figure 3.** Effect of postharvest abiotic stress on the production of phytochemicals (adapted from [7]).

#### **4. UV-C as a postharvest treatment**

radiation) are known to have deleterious effects on plant tissues as a result of the production of ROS, causing progressive oxidative damage, which can damage biomolecules and may affect biochemical pathways, leading to cell death (as shown in Figure 2) and cause substantial crop losses worldwide [13, 16, 18-19]. Postharvest abiotic stressors can lead to numerous quality problems in fruits and vegetables, including scald, core and flesh browning of fruits, sweetening, pitting, water-soaked appearance, abnormal ripening, russeting, tissue softening, and loss of nutrient constituents [10]. Hence an understanding of the effects of field abiotic stresses on postharvest stress susceptibility will become more important since postharvest stresses limit the storage and shelf-life potential of crops. Characterization of mutants and transgenic plants with altered expression of antioxidants is also a potentially powerful approach to understanding the functioning of the antioxidant system and its role in protecting plants against stress [6, 13]. The use of genetically modified crops has its biases as in most cases it is considered as potential biological hazards that create an ecological imbalance [6, 9].

Exposure to Radiation

Excitation of Molecules

Molecular Changes

Physiological and Biochemical Effects

Direct Action Indirect Action

Reversible Irreversible

Amelioration Death

**Figure 2.** Schematic representation of the photobiological effects of UV-C radiation in plants [26].

224 Phytochemicals - Isolation, Characterisation and Role in Human Health

As a secondary response, some postharvest abiotic stress treatments could induce some mechanisms that affect the metabolic activity of the treated produce, such as triggering antioxidants. Studies have shown that plants with higher levels of antioxidants, whether constitutive or induced, showed a greater resistance to different types of environmental stresses [13]. Plants possibly protect themselves against increased UV irradiation by an increased synthesis of pigments in the epidermis [23]. However, the mechanisms of cellular oxidative stress are complex, and other biochemical and physiological mechanisms may act in concert under UV stress. Figure 3 illustrates possible mechanisms for using controlled

postharvest abiotic stresses to enhance the phytochemical content of crops [7].

Non-ionizing artificial UV-C irradiation is a postharvest treatment that can be adjunct to refrigeration for delaying postharvest ripening, senescence, and decay in different fruit and vegetable species. UV-C radiation is mainly used as a surface treatment because it penetrates only 5-30 microns of the tissue and is beneficial for keeping the integrity and freshness of fruits and vegetables [27, 65]. It has been extensively used for many years in the disinfection of equipment, glassware, and air by food and medical industries. Exposure to abiotic UV-C radiation stress is well known to have deleterious effects on plant tissues however low levels may stimulate beneficial responses of plants, a phenomenon known as Hormesis [14, 28]. The use of UV-C hormesis to improve the postharvest quality of fresh fruits and vegetables has been the subject of numerous research activities during the last two decades, with some early applications in the eighties on the control of postharvest pathogens. UV-C doses above the optimal level can lead to detrimental color development and poor appearance of fruits and vegetables, thus lowering aesthetic value and lead to poor marketability of such treated produce [15]. Turtoi [27] and Ribeiro [11] conducted recent reviews on the effects of UV-C primarily on decontamination and disease control in fresh fruits and vegetables. UV-C irradiation also holds considerable promise as a non-chemical treatment to delay ripening and senescence and to improve shelf-life of fresh fruits and vegetables and the associated health and therapeutic benefits with their consumption [11, 28]. In a more recent study, UV-C hormesis was induced at the pre-harvest stage, which has commercial implications particularly for crops that are easily damaged during postharvest treatment [20]. UV-C treated crops, with the ability to scavenge and/or control the level of cellular ROS with the consequential activation of phytochemicals may be useful to improve postharvest storage life and enhance nutritional quality and health benefits. However, the reported biochemical and molecular mechanisms of such effects are complex and quite diverse and depend on several factors such as differences in crops sensitivity, maturity levels, UV-C doses, equipment, and environmental conditions as previously noted.

#### **4.1. UV-C technology**

UV-C irradiation can be applied using low and medium pressure mercury vapor discharge lamps with a peak emission at 254 nm inside an enclosed chamber that is kept closed during irradiation. The lamps are suspended directly over the sample in the chamber at a fixed distance from the sample and radiation doses are varied using different exposure times. The intensity of the light is affected by the distance the source is from the sample. Manual rotation is generally employed to the treated commodity to ensure irradiation uniformity. The use of devices to rotate the crops to ensure radiation uniformity is noted in [20]. The dose is calculated from the product of exposure time and irradiance, as measured by portable handheld digital radiometers. The irradiance, sometimes called intensity, has the preferred units of mWcm-2 while the UV dose has the preferred units of kJm-2 [21]. UV dose may be calculated from the following formula:

#### D = I (T)

where D = dosage, I = applied intensity, and T = time of exposure.

There are several advantages in the use of UV-C technology such as:


The beneficial effects from UV-C radiation result from the use of very low UV doses ranging from 0.125 kJm−2 to 9 kJm−2 and the time scale for the induction of such effects is generally measured over hours (h) or even days [11, 15, 17]. The design of UV-C equipment and technology on a commercial scale for fresh fruits and vegetables and fresh-cut food industry poses a challenge with respect to ensuring affordable technology and simultaneously protect‐ ing operators from harmful effects of UV-C rays while effectively treating fruit tissue to ensure significant and consistent microbial load reduction and activation of beneficial phytochemi‐ cals.

#### **5. UV-C radiation and its effects on phytochemicals**

Plants possess a variety of phytochemicals to protect against the adventitious production of ROS caused by specific postharvest elicitor treatments [4, 13]. Such phytochemicals include the enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reduca‐ tase (DHAR), glutathione reductase (GR), polyphenol oxidases (PPO), guaiacol peroxidase (GPX), as well as water and fat-soluble antioxidants (ascorbic acid, thiol containing com‐ pounds, tocopherols, carotenes) and general antioxidants such as phenolic compounds. Nonenzymic phytochemicals play a significant role in protecting the cell from oxidative stress that occurs when there is a disturbance between the production of free radicals and antioxidant defenses [13, 18, 24]. A summary of some changes in enzymic and non-enzymic phytochemi‐ cals in postharvest UV-C treatment of some crops are illustrated below.

#### **5.1. Enzymic phytochemcials**

been the subject of numerous research activities during the last two decades, with some early applications in the eighties on the control of postharvest pathogens. UV-C doses above the optimal level can lead to detrimental color development and poor appearance of fruits and vegetables, thus lowering aesthetic value and lead to poor marketability of such treated produce [15]. Turtoi [27] and Ribeiro [11] conducted recent reviews on the effects of UV-C primarily on decontamination and disease control in fresh fruits and vegetables. UV-C irradiation also holds considerable promise as a non-chemical treatment to delay ripening and senescence and to improve shelf-life of fresh fruits and vegetables and the associated health and therapeutic benefits with their consumption [11, 28]. In a more recent study, UV-C hormesis was induced at the pre-harvest stage, which has commercial implications particularly for crops that are easily damaged during postharvest treatment [20]. UV-C treated crops, with the ability to scavenge and/or control the level of cellular ROS with the consequential activation of phytochemicals may be useful to improve postharvest storage life and enhance nutritional quality and health benefits. However, the reported biochemical and molecular mechanisms of such effects are complex and quite diverse and depend on several factors such as differences in crops sensitivity, maturity levels, UV-C doses, equipment, and environmental conditions

226 Phytochemicals - Isolation, Characterisation and Role in Human Health

UV-C irradiation can be applied using low and medium pressure mercury vapor discharge lamps with a peak emission at 254 nm inside an enclosed chamber that is kept closed during irradiation. The lamps are suspended directly over the sample in the chamber at a fixed distance from the sample and radiation doses are varied using different exposure times. The intensity of the light is affected by the distance the source is from the sample. Manual rotation is generally employed to the treated commodity to ensure irradiation uniformity. The use of devices to rotate the crops to ensure radiation uniformity is noted in [20]. The dose is calculated from the product of exposure time and irradiance, as measured by portable handheld digital radiometers. The irradiance, sometimes called intensity, has the preferred units of mWcm-2 while the UV dose has the preferred units of kJm-2 [21]. UV dose may be calculated from the

where D = dosage, I = applied intensity, and T = time of exposure.

**•** it does not involve complex expensive equipment,

**•** it generally lacks regulatory restrictions.

There are several advantages in the use of UV-C technology such as: **•** it leaves no residues after treatment, including moisture residues,

**•** it is simpler and more economical to use than ionizing radiation, and

The beneficial effects from UV-C radiation result from the use of very low UV doses ranging from 0.125 kJm−2 to 9 kJm−2 and the time scale for the induction of such effects is generally

as previously noted.

**4.1. UV-C technology**

following formula:

D = I (T)

#### *5.1.1. Superoxide Dismutase (SOD), Glutathione Reductase (GR), Catalase (CAT)*

SOD is a ubiquitous defensive enzyme against superoxide damage to aerobic organisms. SOD catalyzes the dismutation of the one-electron reduced form of oxygen (O2⋅-) or superoxide radical resulting in the production of the less toxic hydrogen peroxide and oxygen: 2 O2⋅- + 2H + → H2O2 + O2 [18, 25]. The H2O2 formed by the action of SOD is itself a strong oxidant and is toxic to cells. It can then be converted to oxygen and water in consecutive reactions with ascorbate and glutathione or with CAT in plant tissues [20]. SOD activity and enzymes of the H2O2 scavenging pathway are induced by diverse environmental stresses. High SOD activity has been reported to increase in plants exposed to various stresses [18]. GR is a key enzyme in the glutathione-ascorbate cycle and can regenerate reduced glutathione from its oxidized form. Together with glutathione, it is an important component of the ROS scavenging system in plant cells. GR has its main function as a detoxifier of peroxide from the cells. Peroxide decomposes to form highly reactive free radicals which can damage proteins, DNA and lipids. CAT a high capacity but low affinity enzyme destroys H2O2 and promotes the redox reac‐ tion:-2H2O2→2H2O + O2. This enzyme has been reported to show a general decline in activity with increasing illumination, with degradation exceeding the capacity for repair [13]. In one study, activities of SOD, GR, and CAT were more than 2-fold higher during the first 1 h after UV-C treatment of peanut seedling [38]. Overall, the general trend is that UV-C stressed crops show an initial increase in enzyme activity followed by a decrease in the antioxidant enzymes. Table 1 summarizes the changes in SOD, GR, and CAT activities with postharvest UV-C treatment of some crops.


**Table 1.** Summary of changes in SOD, GR, and CAT activities with postharvest UV-C treatment of crops.

#### **5.2. Non-enzymic phytochemicals**

#### *5.2.1. Phytoalexins*

Table 1 summarizes the changes in SOD, GR, and CAT activities with postharvest UV-C

**Crop UV-C conditions Results Source**

to control fruit during later storage.

SOD activity in both control and UVC-treated fruit decreased with storage time, with a slight increase at the end of storage, but UV-C treated fruit maintained higher SOD activity throughout storage. UV-C treatment led to significantly higher activities of SOD, CAT, POD, APX, and GR compared

UV-C illumination at 6.6 kJm−2 enhanced the activities of enzymes such as CAT, SOD, GPX, and APX in yellow bell pepper during storage when compared to control fruit.

Activities of SOD, GR, and CAT in Yali pear fruit were significantly enhanced with UV-C dose compared to control

Total SOD activity decreased in both control and UV-C treated strawberries, but after 15 days of storage all UVtreatments showed higher SOD levels that controls. UVtreated fruits at 2.15 kJm−2 dose (5 min) had the highest GR activity. GR activity increased during the first 10 days of storage and then declined after 15 days of storage.

SOD activity increased at day 5 but then declined during storage. Extracts from all UV-C treated fruits had higher SOD activities compared to control fruits during storage. UV-C doses at 0.5 kJm−2 and 0.75 kJm−2 enhanced SOD and CAT activities in fruit after 5 days but levels declined after.

SOD levels were lowest at day 0 and increased during the pre-climacteric phase attaining a maximum level by day 14 for control and day 21 for UV-C treated fruits (both doses) prior to declining in the post-climacteric phase. SOD levels were generally lower for UV-C treated fruit than controls.

No significant differences were found in SOD activity of *damiana* leaves between UV-C treatments and control plants. [37]

The general trend in CAT activity was a decline from initial values however the 1.6 kJm−2 and 4.8 kJm−2 treatments kept the initial activity of this enzyme throughout shelf-life.

fruit even up to the end of the storage period.

[30]

[32]

[33]

[34]

[35]

[36]

[39]

treatment of some crops.

0.02 kJm−2, 0.03 kJm−2 and 0.04 kJm−2 and storage at 5°C and 25°C for 7 days and

228 Phytochemicals - Isolation, Characterisation and Role in Human Health

2.2 kJm−2, 4.4 kJm−2, and 6.6 kJm−2 and storageat 12 ± 1°C for 15 days

5 kJm−2 and storage at

0.43 kJm−2, 2.15 kJm−2, and 4.3 kJm−2 and storage at 10°C for 15

0.25, 0.5 kJm−2 and 0.75 kJm−2 and storage at 10°C for 15

3.7 kJm−2 and 24.4 kJm−2 and storage at 16°C for 28 days

0.38 mWcm-2; 5, 10, and 20 min day-1 and storage for 10

1.6, kJm−2, 2.8 kJm−2, 4.8 kJm−2, and 7.2 kJm −2 and storage at 5°C

**Table 1.** Summary of changes in SOD, GR, and CAT activities with postharvest UV-C treatment of crops.

21 days

20°C for 42 days

days

days

days

for 11 days

Banana (cv. Cavendish)

Yellow Bell Pepper

Pear (cv. Yali)

Strawberry

Strawberry

Tomato (cv. Capello)

plant)

*Turnera diffusa* Willd (*Damiana* medicinal

Fresh-cut watermelon

Resistance to infection by pathogen or abiotic stress is correlated with the induction of plant defense mechanisms and this is manifested through the stimulation of anti-microbial com‐ pounds such as phytoalexins. Allixin was the first compound isolated from garlic as a phytoalexin and a possible compound for cancer prevention. Trans-resveratrol produced by a large number of plants has a wide range of beneficial biological properties including being attributed with a relatively low incidence of cardiovascular disease and associated with reduced cancer risk [8]. A more detailed review of the stilbene resveratrol in grape and grape products irradiated with UV-C can be found in [8] and in peanuts [38, 43]. Resveratrol was present in substantial amounts (1.2-2.6 μg/g FW) in leaves, roots, and shells, but very little (0.05-0.06 μg/g FW) was found in developing seeds and seed coats of field-grown peanuts. Accumulation of resveratrol in leaves increased over 200-fold in response to UV light, over 20 fold in response to paraquat, and between 2- and 9-fold in response to wounding, H2O2, salicylic acid (SA), jasmonic acid, and ethephon, 24 h after treatment. Changes in resveratrol content were correlated with levels of resveratrol synthase (RS) mRNA, indicating a tran‐ scriptional control of resveratrol synthase activity [40]. These gene changes underline the biochemical and physiological changes induced by UV-C such as increased defense ability, delayed softening, better maintenance of nutritional and sensory qualities and extension of shelf-life [30-31]. Table 2 summarizes the changes in phytoalexin content with postharvest UV-C treatment of some crops.


**Table 2.** Summary of changes in phytoalexin content with postharvest UV-C treatment of crops.

#### *5.2.2. Phenolics and flavonoids*

The largest category of phytochemicals and most widely distributed secondary products in plants are the phenolics. They exist in higher plants in many different forms including hydroxybenzoic derivatives, cinnamates, flavonoids (flavonols, flavones, flavanols, flava‐ nones, isoflavones, proanthocyanidins), lignans, and stilbenes, and affect quality characteris‐ tics of plants such as appearance, flavor, and health-promoting properties. Besides their role as antioxidants, phenolic compounds also possess antimicrobial properties and are involved in disease resistance by contributing to the healing of wounds by lignification of cell walls around wounded sites [44]. The accumulation of phenolic and flavonoid compounds may act as a protective filter against excessive UV radiation [28, 45-48]. Phenolics also serve as substrates for browning reactions. The enzymes phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), polyphenol oxidases (PPO; EC 1.14.18.1) and peroxidises (POD; EC 1.11.1.7) are the main enzymes responsible for phenolic degradation that often leads to quality loss in crops [44]. Skin discoloration or external browning is one effect of UV-C treatment and is dose dependent with the effect being more pronounced as the dose is increased and has been reported in several crops notably tomato, strawberries, peaches, and Star Ruby grapefruit [28]. Table 3 summarizes the changes in Phenolics content with postharvest UV-C treatment of some crops. Time course measurements of the effects of UV-C have shown that the strongest responses of fruit to UV-C treatment occurred instantly after the illumination and the effects diminished with time.


Effects of Abiotic Stress (UV-C) Induced Activation of Phytochemicals on the Postharvest Quality of Horticultural Crops http://dx.doi.org/10.5772/60050 231

*5.2.2. Phenolics and flavonoids*

230 Phytochemicals - Isolation, Characterisation and Role in Human Health

diminished with time.

1.5 kJm−2 and storage in field conditions after 3 and 5 days

0.02 kJm−2, 0.03 kJm−2, and 0.04 kJm−2 and storage at 5 and 25°C for 7 and 21 days

2.2 kJm−2, 4.4 kJm−2, and 6.6 kJm−2 and storage at 12 ± 1◦Cfor

5 kJm−2 and storage at

3.7 kJm−2 and 24.4 kJm−2 and storage at 16°C for 28 days

15 days

20°C for 42 days

*Spilanthes acmella* (toothache plant)

Yellow Bell Pepper

Pear (cv. Yali)

Tomato (cv. Capello)

Banana (cv. Cavendish)

The largest category of phytochemicals and most widely distributed secondary products in plants are the phenolics. They exist in higher plants in many different forms including hydroxybenzoic derivatives, cinnamates, flavonoids (flavonols, flavones, flavanols, flava‐ nones, isoflavones, proanthocyanidins), lignans, and stilbenes, and affect quality characteris‐ tics of plants such as appearance, flavor, and health-promoting properties. Besides their role as antioxidants, phenolic compounds also possess antimicrobial properties and are involved in disease resistance by contributing to the healing of wounds by lignification of cell walls around wounded sites [44]. The accumulation of phenolic and flavonoid compounds may act as a protective filter against excessive UV radiation [28, 45-48]. Phenolics also serve as substrates for browning reactions. The enzymes phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), polyphenol oxidases (PPO; EC 1.14.18.1) and peroxidises (POD; EC 1.11.1.7) are the main enzymes responsible for phenolic degradation that often leads to quality loss in crops [44]. Skin discoloration or external browning is one effect of UV-C treatment and is dose dependent with the effect being more pronounced as the dose is increased and has been reported in several crops notably tomato, strawberries, peaches, and Star Ruby grapefruit [28]. Table 3 summarizes the changes in Phenolics content with postharvest UV-C treatment of some crops. Time course measurements of the effects of UV-C have shown that the strongest responses of fruit to UV-C treatment occurred instantly after the illumination and the effects

**Crop UV-C conditions Results Source**

UV-C illumination at 6.6 kJ/m2

content.

storage.

Anthocyanin and flavonoid concentration increased with UV-C treatment. [29]

UV-C doses reduced both the incidence of CI and its severity compared with controls in banana fruit peel. UV-C treatment activated PAL and resulted in higher levels of total phenolic compounds in comparison with untreated controls.

Enzyme activities of PAL and β-1,3-glucanase were induced to high levels by UV-C treated pears and thought to be responsible for the reduction in postharvest decay.

A 4.5- and 4.9-fold increase in total phenols with UV doses of 3.7 kJm−2 and 24.4 kJm−2, respectively, when compared to controls on day 14 was observed. Levels of total phenols were higher with UV-treated fruits than controls at the end of the

enhanced total flavonoid

[30]

[32]

[33]

[36]


**Table 3.** Summary of changes in phenolics and flavonoids content with postharvest UV-C treatment of crops.

*5.2.3. Photosynthetic pigments, antioxidants carotenoids, lycopene and vitamins α-tocopherol and ascorbic acid*

#### *5.2.3.1. Chlorophyll*

During chlorophyll degradation, chlorophyll *a* is transformed to chlorophyllide *a* through the action of the chlorophyllase enzyme [52]. Chlorophyllide *a* is then acted by the enzyme Mgdechelatase removing Mg2+ from the molecule and forming pheophorbide *a*, consequently the green color is lost. In general, the chloroplast is the first organelle to show injury response with UV radiation and reduction in the chlorophyll contents may be due to inhibition of biosynthesis or due to degradation of chlorophyll and their precursors [29]. Table 4 summarizes the changes in chlorophyll content with postharvest UV-C treatment of some crops. Photosynthetic pigments such as chlorophyll *a* and *b* as well as total chlorophyll contents were considerably reduced in UV-C treated crops [29, 37]. In these studies, chlorophyll *a* and chlorophyll *b* were more sensitive to UV-C radiation. The reduction of chlorophyll content is thought to have a negative effect on plant photosynthetic efficiency due to UV-C radiation being too severe. Previous studies have demonstrated the inhibitory effect of UV-C light on chlorophyll breakdown and on the activities of Mg-dechelatase, chlorophyllase and chlorophyll degrading peroxidase in crops [53-55].



**Table 4.** Summary of changes in chlorophyll content with postharvest UV-C treatment of crops.

#### *5.2.3.2. Carotenoids and lycopene*

*5.2.3. Photosynthetic pigments, antioxidants carotenoids, lycopene and vitamins α-tocopherol and*

During chlorophyll degradation, chlorophyll *a* is transformed to chlorophyllide *a* through the action of the chlorophyllase enzyme [52]. Chlorophyllide *a* is then acted by the enzyme Mgdechelatase removing Mg2+ from the molecule and forming pheophorbide *a*, consequently the green color is lost. In general, the chloroplast is the first organelle to show injury response with UV radiation and reduction in the chlorophyll contents may be due to inhibition of biosynthesis or due to degradation of chlorophyll and their precursors [29]. Table 4 summarizes the changes in chlorophyll content with postharvest UV-C treatment of some crops. Photosynthetic pigments such as chlorophyll *a* and *b* as well as total chlorophyll contents were considerably reduced in UV-C treated crops [29, 37]. In these studies, chlorophyll *a* and chlorophyll *b* were more sensitive to UV-C radiation. The reduction of chlorophyll content is thought to have a negative effect on plant photosynthetic efficiency due to UV-C radiation being too severe. Previous studies have demonstrated the inhibitory effect of UV-C light on chlorophyll breakdown and on the activities of Mg-dechelatase, chlorophyllase and chlorophyll degrading

**Crop UV-C conditions Results Source**

Chlorophyll *a*, chlorophyll *b*,and total chlorophyll contents were considerably reduced with UV-C treatment. [29]

Chlorophyll *a* content significantly decreased in plants exposed to UV-C radiation for 5 and 20 min day-1 while chlorophyll *b* content significantly decreased with UV-C 20

All UV treatments delayed yellowing and chlorophyll degradation. UV-C dose of 10 kJm−2 reduced the degradation of both chlorophyll *a* and *b* in broccoli florets and this was correlated to a reduced activity of chlorophyllase and chlorophyll peroxidase in treated florets, which maintained a greener color than the controls. UV-C treated broccoli also maintained lower Mg-dechelatase level than controls.

UV-C doses of 3.6 kJm−2 and 5.4 kJm−2 delayedleaf yellowing and chlorophyll loss depicted as higher chlorophyll contents and lower activity of chlorophyllase, Mg-dechelatase and chlorophyll-degrading peroxidase as compared to the other

[37]

[53]

[54]

min day-1 dose relative to control plants.

*ascorbic acid*

*5.2.3.1. Chlorophyll*

peroxidase in crops [53-55].

1.5 kJm−2 and storage in field conditions after 3 and 5 days

232 Phytochemicals - Isolation, Characterisation and Role in Human Health

0.38 mWcm-2; 5 min day-1, 10 min day-1, and 20 min day-1 exposure and storage

4 kJm−2, 7 kJm−2, 10 kJm−2, or 14 kJm−2 and storage at 20°C

1.8 kJm−2, 3.6 kJm−2, 5.4 kJm−2, and 7.2 kJm −2 and storage at 20°C

treatments.

for 10 days

for 6 days

for 8 days

*Spilanthes acmella* (toothache plant)

*Turnera diffusa* Willd (*Damiana* medicinal

Broccoli heads (cv. de

*Brassica oleracea* var.

plant)

Cicco)

*alboglaba* (Chinese kale) Carotenoids (β-carotene) or pro-vitamin A are quenching agents that facilitate the return of singlet oxygen to its ground state. Table 5 summarizes changes in Total Carotenoids Content (TCC) and Lycopene content with postharvest UV-C treatment of some crops. In some cases, decreases in β-carotene as a result of UV-C treatment, are due most likely to the phenomenon of photobleaching [37, 45]. These observations appear to be at variance with those of other authors where carotenoid levels increased in medicinal plants [29], tomato [55], and minimally processed carrots [61] with UV-C treatment. The increase in total carotenoids may be part of the antioxidant system where carotenes are involved in protection of the chloroplast against photooxidation.



**Table 5.** Summary of changes in total carotenoids content (TCC) and lycopene with postharvest UV-C treatment of crops.

#### *5.2.3.3. α-tocopherol (Vitamin E)*

Alpha-tocopherol is a major lipid-soluble antioxidant that breaks the chain of free radical reactions of polyunsaturated fatty acids (PUFAs) in cell membranes. Alpha-tocopherol protects membrane PUFAs from ROS and free radical damage by reacting with lipid radicals produced in the lipid peroxidation chain reaction [16]. Levels of tocopherol in plants are as a result of synthesis, recycling, and degradation. Changes in α-tocopherol levels during plant responses to environmental stress are characterized by two phases; in the first phase there is an increase in tocopherol synthesis that was followed by a second phase of net tocopherol loss [24]. Endogenous α-tocopherol levels are also severely affected by the extent of its degradation and recycling under stress. As stress is more severe and the amounts of ROS in chloroplasts increase, α-tocopherol levels tend to decrease. Irreversible degradation of α-tocopherol may also occur when α-tocopheroxyl radicals, which result from the scavenging of lipid peroxyl radical by α-tocopherol, are not recycled back by ascorbate. Table 6 summarizes the changes

in α-Tocopherol (Vitamin E) content with postharvest UV-C treatment of some crops. While it has been reported that α-tocopherol levels increase in photosynthetic plant tissues in response to a variety of abiotic stresses, results from a study with UV-C irradiated tomato exocarp demonstrated declining levels and do not support the hypothesis that antioxidants such as α-tocopherol are stimulated for plant defense against oxidative stress caused by UV-C radiation [36]. On the other hand, the increased vitamin E content in medicinal *Damiana* plants exposed to UV-C radiation may provide protection against the oxidative damage induced by UV-C radiation [37].


**Table 6.** Summary of changes in α-Tocopherol (vitamin E) content with postharvest UV-C treatment of crops.

#### *5.2.3.4. Ascorbic acid (Vitamin C)*

**Crop UV-C conditions Results Source**

treated for 10 mins and 5 mins.

throughout storage.

**Table 5.** Summary of changes in total carotenoids content (TCC) and lycopene with postharvest UV-C treatment of

Alpha-tocopherol is a major lipid-soluble antioxidant that breaks the chain of free radical reactions of polyunsaturated fatty acids (PUFAs) in cell membranes. Alpha-tocopherol protects membrane PUFAs from ROS and free radical damage by reacting with lipid radicals produced in the lipid peroxidation chain reaction [16]. Levels of tocopherol in plants are as a result of synthesis, recycling, and degradation. Changes in α-tocopherol levels during plant responses to environmental stress are characterized by two phases; in the first phase there is an increase in tocopherol synthesis that was followed by a second phase of net tocopherol loss [24]. Endogenous α-tocopherol levels are also severely affected by the extent of its degradation and recycling under stress. As stress is more severe and the amounts of ROS in chloroplasts increase, α-tocopherol levels tend to decrease. Irreversible degradation of α-tocopherol may also occur when α-tocopheroxyl radicals, which result from the scavenging of lipid peroxyl radical by α-tocopherol, are not recycled back by ascorbate. Table 6 summarizes the changes

dose.

over those of control. *E*-lycopene decreased with 12.2 kJ/m2

β-carotene content decreased with storage in all treatments including controls with the major reduction observed in fruits

There was a significant decline in lycopene accumulation with time for both UV-doses. UV-3.7 kJm−2 exhibited significantly higher levels of TCC compared to control fruits

Lycopene content was enhanced by UV-C treatment but the concentration of β-carotene was not affected by UV-C. [56]

UV treatment significantly reduced the lycopene content. [57]

A 64% loss in TCC in UV-C treated samples compared to controls was noted just after processing. However, UV samples exhibited a consistent increase in TCC levels with levels reaching 3-fold higher at day 7 than at day 0.

[48]

[55]

[61]

storage at RT for 8

234 Phytochemicals - Isolation, Characterisation and Role in Human Health

1 min day-1, 3 min day-1, 5 min day-1, and 10 min day-1 exposure and storage at 5°C for 15

3.7 kJm−2 and 24.4 kJm−2 and storage at 16°C for 28-35 days

13.7 kJm−2 and storage at 12°C-14°C for 21

3.7 kJm−2 and storage at 13°C for

0.78-0.36 kJm−2 and storage at 5°C for 10

days

days

days

30 days

days

*5.2.3.3. α-tocopherol (Vitamin E)*

Fresh-cut Mango (cv. Tommy Atkins)

Mature Green Tomato

Mature Green (Breaker

Mature Green Tomato

Fresh-cut Carrots (cv. Nantes)

crops.

(cv. Capello)

stage)Tomato

Vitamin C content, considered to be a nutritional quality index for fruits and vegetables occurs as L-ascorbic acid and dehydroascorbic acid (oxidised form of ascorbic acid). Ascorbate is an electron donor and this property explains its function as an antioxidant or reducing agent and is easily destroyed by oxidation, exposure to light or high temperatures. Ascorbic acid scavenges free radicals in the water soluble compartment of the cell and may also regenerate α-tocopherol (vitamin E), an important lipid-phase antioxidant [16]. Table 7 summarizes the changes in ascorbic acid (vitamin C) content with postharvest UV-C treatment of some crops. In most cases, UV-C treatment decreased the vitamin C content of fruits or did not affect ascorbic acid levels.



**Table 7.** Summary of changes in ascorbic acid (vitamin C) content with postharvest UV-C treatment of crops.

#### *5.2.4. Glutathione*

Thiol (-SH) groups and other functions readily donate hydrogen atoms and are scavengers of hydroxyl radicals. The tri-peptide Glutathione (γ‐glutamylcysteinylglycine) is a major free non-protein thiol compound found in plant tissues. In non-stressed cells, 90% of glutathione is mainly present in its reduced form (GSH) while the oxidized GSSG levels are lower. Upon oxidative stress, the glutathione redox status may shift to a more oxidized form, ROOH +2GSH →ROH +GSSG+H2O, due to increased GSH oxidation and/or decreased GSSG reduction. Such a shift in the glutathione status in plants has been reported upon exposure to a variety of environmental factors associated with oxidative stresses [62]. The ratio of reduced GSH to oxidized GSSG within the cells can be used to measure cellular toxicity. Table 8 summarizes the changes in glutathione content with postharvest UV-C treatment of some crops. Although glutathione has been used as an oxidative stress indicator in plants, there is conflicting evidence about glutathione responses under oxidative stress conditions. While some studies have found an increase in glutathione synthesis in response to oxidative stress, others have found no increase in GSH by environmental stresses.

Effects of Abiotic Stress (UV-C) Induced Activation of Phytochemicals on the Postharvest Quality of Horticultural Crops http://dx.doi.org/10.5772/60050 237


**Table 8.** Summary of changes in glutathione content with postharvest UV-C treatment of crops

#### *5.2.5. Polyamines(PAs)*

**Crop UV-C conditions Results Source**

fruits irradiated for 10 min.

in fruit juices.

**Table 7.** Summary of changes in ascorbic acid (vitamin C) content with postharvest UV-C treatment of crops.

Thiol (-SH) groups and other functions readily donate hydrogen atoms and are scavengers of hydroxyl radicals. The tri-peptide Glutathione (γ‐glutamylcysteinylglycine) is a major free non-protein thiol compound found in plant tissues. In non-stressed cells, 90% of glutathione is mainly present in its reduced form (GSH) while the oxidized GSSG levels are lower. Upon oxidative stress, the glutathione redox status may shift to a more oxidized form, ROOH +2GSH →ROH +GSSG+H2O, due to increased GSH oxidation and/or decreased GSSG reduction. Such a shift in the glutathione status in plants has been reported upon exposure to a variety of environmental factors associated with oxidative stresses [62]. The ratio of reduced GSH to oxidized GSSG within the cells can be used to measure cellular toxicity. Table 8 summarizes the changes in glutathione content with postharvest UV-C treatment of some crops. Although glutathione has been used as an oxidative stress indicator in plants, there is conflicting evidence about glutathione responses under oxidative stress conditions. While some studies have found an increase in glutathione synthesis in response to oxidative stress, others have found no

UV treatment decreased ascorbic acid levels. [47]

A reduction in total ascorbic acid content with UV treatment was observed. The lowest values for ascorbic acid occurred in

Apart from day 1 where UV treated mushrooms had lower ascorbic acid content than controls, during storage, levels increased to a maximum at day 14 and remained stable until day 21 for both control and UV-treated mushrooms.

Ascorbate oxidase activity exhibited a decline during storage for both control and UV-C treated fruit, however the amount of this enzyme was lower in UV-C-treated fruit and could

The antioxidant capacity of UV-C-treated fruit did not change during storage, but showed a slight increase in the control. [59]

UV treatment decreased ascorbic acid levels. The longer the exposure to UV, the higher the losses of ascorbic acid content

correlate to decreases in levels of ascorbic acid.

[48]

[50]

[58]

[60]

Fresh-cut pineapple (cv. Honey), banana (cv. Pisang mas) and Thai seedless guava

Fresh-cut Mango (cv. Tommy Atkins)

*Button* mushrooms

Tomato (cv. Trust)

Fresh-cut mature green

bell pepper

Fresh fruit juices

*5.2.4. Glutathione*

10 min day-1, 20 min day-1, and 30 min day-1 exposure and storage at RT

236 Phytochemicals - Isolation, Characterisation and Role in Human Health

1 min day-1, 3 min day-1, 5 min day-1, and 10 min day-1 exposure and storage at 5°C for 15 days

0.225 kJm−2, 0.45 kJm −2, and 0.90 kJm−2 and storage at 4°C for 21

3.7 kJm−2 at and storage at 16°C for 25

3 kJm−2, 10 kJm−2, and 20 kJm−2 and storage at 10°C for 8 days

Various time intervals of exposure

to UV-C

increase in GSH by environmental stresses.

days

days

Polyamines (PAs) are implicated in a variety of regulatory processes ranging from regulation of growth and cell division, regulating the activity of ribonucleotides and proteinase to inhibition of ethylene (C2H4) production and senescence. The anti-senescent activity of PAs may also be related to their ability to be effective free radical scavengers, as well as stabilizing DNA and membranes by their positively charged cations associating with negative charges on nucleic acids and phospholipids [26]. Changes in plant PA metabolism occur in response to a variety of abiotic stresses and have been shown to enhance the ability of plants to resist environmental stresses. However, the physiological significance of elevated PA levels in abiotic stress responses is still unclear in terms of whether such a response is as a result of stress-induced injury or a protective response to abiotic stress [64]. Table 9 summarizes the changes in PA content with postharvest UV-C treatment of some crops.



**Table 9.** Summary of changes in polyamine (PA) content with postharvest UV-C treatment of crops.

#### **6. Conclusions**

Studies on the benefit of phytochemicals continue to be of interest, as researchers have been exploring alternative medicine in the management of common lifestyle diseases and preven‐ tative actions, as well as the promotion of good health and nutrition. Abiotic stresses are significant determinants of quality and nutritional value of crops during their life cycle. Notwithstanding, climate change has created additional environmental variables that may influence postharvest stress susceptibility of crops. While breeding programs are underway for many crops to develop stress resistance that will allow them to adapt to climate change, it is not clear that breeding for stress resistance in the field will also extend to stress resistance characteristics of harvested crops particularly fresh fruits and vegetables and fresh-cut produce. Proper postharvest management of crops can positively influence susceptibility to abiotic stress. It is important to understand the various response networks to abiotic stresses encountered in the field and in the postharvest continuum to better evaluate the benefits that may yield from such stresses. Controlled stresses may be used as tools by the fresh produce and food processing industries to obtain enhanced phytochemical or health promoting components of fresh-cut or whole fresh produce and for growers interested in finding alternative uses for their crops. In the context of the different postharvest handling treatments, the potential of UV-C irradiation has enormous possibilities as a non-chemical treatment to sanitize and reduce microbial loads, delay ripening, and improve shelf-life of fresh fruits and vegetables. Further, the elicitation of health promoting phytochemicals with UV-C is an indication that such an approach may be of benefit to growers, processors, and consumers in enhancing sensory characteristics of fresh fruits and vegetables. The beneficial action of UV-C is thought to be as a result of the activation of multiple defense systems involving secondary stress metabolites such as phytoalexins and enzymic and non-enzymic antioxidants. The intensification of natural defense mechanisms of higher plant so that they can defend them‐ selves against infection or adverse stresses is one alternative approach to controlling post‐ harvest losses. Depending on the experimental conditions including UV-C dose, commodity, maturity stage, storage, and environmental conditions administered, such bioactive com‐ pounds have been found to be variable, in some instances may decrease or increase, and this change may impact quality of the crops. In many studies, the strongest responses to UV-C treatment occurred almost immediately after the illumination with some beneficial effects of crops occurring after UV-C treatment with the effects decreasing with time. There is need for further research on the molecular and biochemical mechanisms by which these beneficial responses are derived in UV-C treated fresh fruits and vegetables. This is in order to help guide the development of approaches to reliably confer health and nutritional benefits. The potential application of UV-C is novel and relevant particularly for the fresh fruit and vegetable and fresh-cut trade industries.

#### **Author details**

**Crop UV-C conditions Results Source**

Studies on the benefit of phytochemicals continue to be of interest, as researchers have been exploring alternative medicine in the management of common lifestyle diseases and preven‐ tative actions, as well as the promotion of good health and nutrition. Abiotic stresses are significant determinants of quality and nutritional value of crops during their life cycle. Notwithstanding, climate change has created additional environmental variables that may influence postharvest stress susceptibility of crops. While breeding programs are underway for many crops to develop stress resistance that will allow them to adapt to climate change, it is not clear that breeding for stress resistance in the field will also extend to stress resistance characteristics of harvested crops particularly fresh fruits and vegetables and fresh-cut produce. Proper postharvest management of crops can positively influence susceptibility to abiotic stress. It is important to understand the various response networks to abiotic stresses encountered in the field and in the postharvest continuum to better evaluate the benefits that may yield from such stresses. Controlled stresses may be used as tools by the fresh produce and food processing industries to obtain enhanced phytochemical or health promoting components of fresh-cut or whole fresh produce and for growers interested in finding alternative uses for their crops. In the context of the different postharvest handling treatments, the potential of UV-C irradiation has enormous possibilities as a non-chemical treatment to sanitize and reduce microbial loads, delay ripening, and improve shelf-life of fresh fruits and vegetables. Further, the elicitation of health promoting phytochemicals with UV-C is an indication that such an approach may be of benefit to growers, processors, and consumers in enhancing sensory characteristics of fresh fruits and vegetables. The beneficial action of UV-C is thought to be as a result of the activation of multiple defense systems involving secondary stress metabolites such as phytoalexins and enzymic and non-enzymic antioxidants. The intensification of natural defense mechanisms of higher plant so that they can defend them‐ selves against infection or adverse stresses is one alternative approach to controlling post‐ harvest losses. Depending on the experimental conditions including UV-C dose, commodity, maturity stage, storage, and environmental conditions administered, such bioactive com‐ pounds have been found to be variable, in some instances may decrease or increase, and this change may impact quality of the crops. In many studies, the strongest responses to UV-C treatment occurred almost immediately after the illumination with some beneficial effects of crops occurring after UV-C treatment with the effects decreasing with time. There is need for further research on the molecular and biochemical mechanisms by which these beneficial responses are derived in UV-C treated fresh fruits and vegetables. This is in order to help guide the development of approaches to reliably confer health and nutritional benefits. The potential

**Table 9.** Summary of changes in polyamine (PA) content with postharvest UV-C treatment of crops.

damage as a result of UV-C irradiation.

Endogenous free, conjugated, and bound PAs (Spm, Spd, and Put) in leaves of young pea plants reduced membrane

[68]

Intact Pea Plants 0.1 kJm−2 and

**6. Conclusions**

0.3 kJm−2 and storage

238 Phytochemicals - Isolation, Characterisation and Role in Human Health

Rohanie Maharaj

Address all correspondence to: rohanie.maharaj@utt.edu.tt

Biosciences, Agriculture and Food Technologies (BAFT), ECIAF Campus, The University of Trinidad and Tobago, Caroni North Bank Road, Centeno, Piarco, W.I., Trinidad & Tobago

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**Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of Phytochemical Antioxidants Lycopene and Polyphenol-containing Nutritional Supplements**

L.G. Rao and A.V. Rao

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60446

### **1. Introduction**

Osteoporosis is a systemic disease that is characterized by low bone mass and deterioration of the microarchitecture of bone, resulting in an increased risk of fracture in postmenopausal women and men over 50 years old. Several factors have been identified that contribute to the risk of osteoporosis, and oxidative stress has now emerged as one of the most important life style risk factor associated with loss of bone mass. Phytochemicals as antioxidants have been shown to counteract the deleterious effects of oxidative stress in the risk of osteoporosis. This review will include an overview on osteoporosis, the deleterious effects of oxidative stress and the beneficial effects of phytochemical antioxidants, with emphasis on the results of our clinical studies on the phytochemical lycopene and polyphenols present in nutritional supplements.

#### **2. Osteoporosis – An overview**

Osteoporosis is a metabolic bone disease known as "the silent thief" because the gradual loss of bone associated with this disease usually occurs over the years, and there are usually no noticeable symptoms until the bones are so fragile that a fracture occurs [1]. Osteoporosis is "a major public health threat" that is projected to results to 8.1 million fractures (78 % women, 22 % men) during the period between 2010 and 2050 [2]. Approximately 1 in 2 women and 1 in 5 men older than 50 years will eventually experience osteoporotic fractures [3]. The condition costs our healthcare system \$18 billion per year [4]. Newer findings on all aspects of osteopo‐ rosis have increased exponentially. The more importantly ones are discovering an ever

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

increasing number of risk factors including oxidative stress, opening up new knowledge on the involvement of the bone forming cells osteoblasts and the bone resorbing cells osteoclasts in the development of osteoporosis, the introduction and improvement of more sensitive diagnostic instruments, and finding new drugs and the nutritional alternatives for the prevention and treatment of osteoporosis. Advances in the knowledge on osteoporosis is not without pitfalls. Hormone Replacement Therapy (HRT), once a first line of treatment for osteoporosis has been discontinued due to side effects [5]. It is becoming more evident that the drugs known as bisphosphonates, although effective in stopping the resoption of bone and preventing osteoporosis in women, are associated with a number of side effects [6, 7]. Because of this, a number of women are now resorting to other modes of treatment, including that from natural food components. Our laboratory has carried out studies on the use of phytochemical antioxidants such as lycopene and polyphenols present in nutritional supplements as possible alternatives and/or complementary to drugs in the treatment and prevention of osteoporosis. This chapter will include an overview on osteoporosis, oxidative stress as a risk factor in the development of osteoporosis and a review of studies on the use of antioxidants in counteract‐ ing oxidative stress in the prevention of osteoporosis. These topics should put our research in perspective and offer a rationale to our study approaches. Finally we will highlight our pioneering clinical studies on the lipid-soluble phytochemical antioxidant lycopene and the water-soluble antioxidant polyphenols present in a nutritional supplement in the prevention of risk for osteoporosis in postmenopausal women.

#### **2.1. Risk factors for osteoporosis**

Some of the risk factors for osteoporosis [8, 9] are presented in Table 1 [10]. The risk factors that are of interest in our studies are the oxidative stress-generating factors, including nutrition deficiency, low antioxidant status, smoking, alcohol intake, excessive sports activity and caffeine intake.


**Table 1.** Risk Factors for Osteoporosis

#### **2.2. Prevention and treatment of osteoporosis**

increasing number of risk factors including oxidative stress, opening up new knowledge on the involvement of the bone forming cells osteoblasts and the bone resorbing cells osteoclasts in the development of osteoporosis, the introduction and improvement of more sensitive diagnostic instruments, and finding new drugs and the nutritional alternatives for the prevention and treatment of osteoporosis. Advances in the knowledge on osteoporosis is not without pitfalls. Hormone Replacement Therapy (HRT), once a first line of treatment for osteoporosis has been discontinued due to side effects [5]. It is becoming more evident that the drugs known as bisphosphonates, although effective in stopping the resoption of bone and preventing osteoporosis in women, are associated with a number of side effects [6, 7]. Because of this, a number of women are now resorting to other modes of treatment, including that from natural food components. Our laboratory has carried out studies on the use of phytochemical antioxidants such as lycopene and polyphenols present in nutritional supplements as possible alternatives and/or complementary to drugs in the treatment and prevention of osteoporosis. This chapter will include an overview on osteoporosis, oxidative stress as a risk factor in the development of osteoporosis and a review of studies on the use of antioxidants in counteract‐ ing oxidative stress in the prevention of osteoporosis. These topics should put our research in perspective and offer a rationale to our study approaches. Finally we will highlight our pioneering clinical studies on the lipid-soluble phytochemical antioxidant lycopene and the water-soluble antioxidant polyphenols present in a nutritional supplement in the prevention

Some of the risk factors for osteoporosis [8, 9] are presented in Table 1 [10]. The risk factors that are of interest in our studies are the oxidative stress-generating factors, including nutrition deficiency, low antioxidant status, smoking, alcohol intake, excessive sports activity and

Chronic inactivity

Low body weight

Medication used Oxidative stress-related

Smoking

Factors:

Low lifetime calcium intake

High Alcohol intake Low antioxidant status Nutrition deficiency Excessive sports activity Excessive caffeine intake

**Unmodifiable Modifiable**

of risk for osteoporosis in postmenopausal women.

248 Phytochemicals - Isolation, Characterisation and Role in Human Health

**2.1. Risk factors for osteoporosis**

**Table 1.** Risk Factors for Osteoporosis

caffeine intake.

Race Sex Age Genetics Body size Family History Previous Fractures Until 10 years ago, the first line of treatment for women who have gone through menopause and were diagnosed with osteoporosis was hormone replacement therapy (HRT). However, results of the Women's Health Initiative (WHI) revealed that women taking HRT had higher risks for breast cancer, cardiovascular events, blood clots, cognitive decline, and more [5]. This treatment for osteoporosis has since been discontinued and is prescribed only for a short period of time to alleviate hot flashes in menopausal women [11]. The current treatments which inhibit bone resorption that are approved by the Food and Drug Administration (FDA) include a number of bisphosphonates under specific trademarks [12]. Some are taken daily while others are formulated for weekly, monthly or intermittent oral use [13, 14]. The newer bisphophonates are injectables such as Zoledronate and Ibandronate [14]. Other drugs available include calcitonin; Raloxifene (Evista), the Selective Estrogen Receptor Modulator (SERM) and strontium renalate [15]. Parathyroid hormone, PTH1-34 or teriparatide (Forteo), is the only anabolic agent currently approved for use by the FDA [16, 17]. The new class of osteoporosis drug now approved for use is a human monoclonal antibody (Denosumab) which bind to RANKL, imitating the effects of OPG and acting as an inhibitor of RANKL [18]. Other drugs are still being tested clinically for osteoporotic treatment and prevention [16].

None of the drugs are without side effects. Side effects that emerged in clinical trials include acute phase response with iv treatment or high-dose oral therapy and esophageal irritation with oral administration. Osteonecrosis of the jaw, musculoskeletal complaints, and atypical fractures are some uncommon side effects that have been noted with wide clinical use of bisphosphonate. The number of these events are small, and a clear cause-and-effect relation‐ ship between events and bisphosphonate treatment has not been established. Accumulation of Bisphosphonates in the bone create a reservoir leading to continued release from bone for months or years and provide some residual anti-fracture reduction long after treatment is stopped [19]. As a result, there is a recommendation for a drug holiday after 5 –10 yr of treatment with bisphosphonate [7, 19]. The length of the holiday is based on previous duration of treatment, BMD status and fracture risk. Studies with alendronate and risedronate showed that if treatment is stopped after 3–5 yr, there is at least 1–2 yr persisting anti-fracture efficacy. The consensus from expert panels [7] for those who are not on holiday is not to stop the use of drug since the side effects are often rare, and that the benefits outweigh the side effects. In the balance, most individuals who have osteoporosis are much better taking an osteoporosis medication [6].

#### **2.3. Alternative approach to prevention and treatment of osteoporosis**

Diet is now recognized as an important life-style factor in the management of bone health [20]. Given that many nutrients have been identified as being beneficial to bone health [21, 22], there is strong scientific support for the potential benefits of incorporating therapeutic nutritional interventions with contemporary pharmaceutical treatments [23]. As a result of the possible adverse side effects of HRT [5] and the ever increasing reports on the side effects of bisphosph‐ onates that are prescribed for the management of postmenopausal osteoporosis [19], comple‐ mentary and alternative medicine (CAM) is in demand as an alternative for the prevention and treatment of osteoporosis [24]. CAM is the term for medical practices, services and products that are not a part of standard care. Some of the approaches include exercise, acupuncture, diet, herbs rich in polyphenols and nutritional supplements including calcium, zinc, magnesium boron and other vitamins and minerals [24]. Recent dietary guidelines for the prevention of chronic diseases have recommended an increase in the consumption of fruits and vegetables worldwide [25] that are good sources of dietary antioxidants [26]. The beneficial effects of antioxidants in bone health and osteoporosis are demonstrated epidemiologically and through clinical intervention. As will be reviewed in this chapter, our clinical studies on lycopene treatment and nutritional supplements containing polyphenols and other nutritional components showed positive results on bone health.

#### **3. Oxidative stress and antioxidants – An overview**

Oxidative stress is caused by reactive oxygen species (ROS) which are the main by-products formed in the cells of aerobic organisms that can initiate autocatalytic reactions in such a way that the target molecules get converted into free radicals causing a chain of damage [27]. There is ample evidence to show that oxidative stress induces an increase in the rate of bone loss and is therefore a risk factor for osteoporosis. Epidemiological evidence in humans and studies in animals indicate that aging and the associated increase in reactive oxygen species (ROS) are responsible for bone loss [28]. Oxidative stress is associated with the activity and function of both the osteoblasts and osteoclasts cells, the two major bone cells involved in the pathogenesis of osteoporosis [29, 30].

Under normal physiological conditions, the cells can fight free radical attack or oxidative stress by promoting antioxidant defenses. A number of endogenous defense mechanisms are present in the body, including the metal chelating proteins and the endogenous antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) [31]. Exoge‐ nous antioxidants come from dietary sources present in fruits and vegetables containing several phytonutrient antioxidants two of which are the potent antioxidant lipid-soluble lycopene and the water-soluble antioxidant polyphenols [32]. In cases where the exogenous antioxidants or antioxidants from diet fail to prevent oxidative damage, the repair antioxidants come into play which include DNA repair enzymes, protease, lipase, and transferase [33]. When antioxidants loses its fight with oxidative stress, diseases associated with oxidative stress develop, which include cardiovascular disease, cancer, diabetes, neurological diseases and osteoporosis [26].

#### **3.1. Lycopene, a lipid-soluble phytochemical antioxidant**

The role of lycopene in the prevention of human diseases is supported by a number of evidence and previously reviewed [34, 35]. Since then, there have been several epidemiological as well as clinical intervention studies showing the relationship between lycopene intake and the prevention of cancers at other sites, as well as coronary heart disease, diabetes, hypertension, macular degenerative disease, neurodegenerative disease and male infertility [26]. The role of lycopene in bone health has so far been based on its potent antioxidant properties, the wellknown role of oxidative stress in bone health, the limited studies on the effects of lycopene in bone cells in culture [34, 35] and the results of epidemiological studies [36, 37]. To date our clinical intervention studies at St. Michael's Hospital on the role of lycopene and elucidation of its mechanism in lowering the risk for osteoporosis in postmenopausal women (aged 50 to 60 years) are so far the only clinical studies reported in the literature.

#### **3.2. Polyphenols, the water-soluble antioxidant**

Polyphenols are a class of water-soluble molecules naturally found in plants [38]. It is estimated that there are 10,000 different phytonutrients (phyto, meaning from plants). The health benefits associated with fruits, vegetables, tea, red wine, and Mediterranean diets are probably linked to the polyphenol antioxidants [21, 39, 40]. The polyphenols of interest in our study are a mixture of flavonoids such as quercetin, apigenin, kaempferol and luteolin present in the supplement greens+TM [41]. greens+TM in combination with another supplement, bone build‐ erTM, were used in our study on osteoblasts cells and in clinical intervention studies on the prevention of risk of osteoporosis in postmenopausal women as will be reviewed below.

#### **4. Studies on the antioxidants polyphenols and lycopene**

#### **4.1. Studies on lycopene**

and treatment of osteoporosis [24]. CAM is the term for medical practices, services and products that are not a part of standard care. Some of the approaches include exercise, acupuncture, diet, herbs rich in polyphenols and nutritional supplements including calcium, zinc, magnesium boron and other vitamins and minerals [24]. Recent dietary guidelines for the prevention of chronic diseases have recommended an increase in the consumption of fruits and vegetables worldwide [25] that are good sources of dietary antioxidants [26]. The beneficial effects of antioxidants in bone health and osteoporosis are demonstrated epidemiologically and through clinical intervention. As will be reviewed in this chapter, our clinical studies on lycopene treatment and nutritional supplements containing polyphenols and other nutritional

Oxidative stress is caused by reactive oxygen species (ROS) which are the main by-products formed in the cells of aerobic organisms that can initiate autocatalytic reactions in such a way that the target molecules get converted into free radicals causing a chain of damage [27]. There is ample evidence to show that oxidative stress induces an increase in the rate of bone loss and is therefore a risk factor for osteoporosis. Epidemiological evidence in humans and studies in animals indicate that aging and the associated increase in reactive oxygen species (ROS) are responsible for bone loss [28]. Oxidative stress is associated with the activity and function of both the osteoblasts and osteoclasts cells, the two major bone cells involved in the pathogenesis

Under normal physiological conditions, the cells can fight free radical attack or oxidative stress by promoting antioxidant defenses. A number of endogenous defense mechanisms are present in the body, including the metal chelating proteins and the endogenous antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) [31]. Exoge‐ nous antioxidants come from dietary sources present in fruits and vegetables containing several phytonutrient antioxidants two of which are the potent antioxidant lipid-soluble lycopene and the water-soluble antioxidant polyphenols [32]. In cases where the exogenous antioxidants or antioxidants from diet fail to prevent oxidative damage, the repair antioxidants come into play which include DNA repair enzymes, protease, lipase, and transferase [33]. When antioxidants loses its fight with oxidative stress, diseases associated with oxidative stress develop, which include cardiovascular disease, cancer, diabetes, neurological diseases and

The role of lycopene in the prevention of human diseases is supported by a number of evidence and previously reviewed [34, 35]. Since then, there have been several epidemiological as well as clinical intervention studies showing the relationship between lycopene intake and the prevention of cancers at other sites, as well as coronary heart disease, diabetes, hypertension, macular degenerative disease, neurodegenerative disease and male infertility [26]. The role of

components showed positive results on bone health.

250 Phytochemicals - Isolation, Characterisation and Role in Human Health

of osteoporosis [29, 30].

osteoporosis [26].

**3. Oxidative stress and antioxidants – An overview**

**3.1. Lycopene, a lipid-soluble phytochemical antioxidant**

The direct role of lycopene in osteoblasts and osteoclasts, the cells involved in the pathogenesis of osteoporosis, is now being unraveled. This involvement is further supported by both epidemiological and clinical intervention with lycopene in postmenopausal women who are at risk of osteoporosis [29, 30].

An epidemiological study to determine the beneficial role of lycopene in the prevention of risk for osteoporosis was carried out by Rao et al [36]. In a cross-sectional study, 33 postmenopausal women aged 50–60 years participants were recruited and asked to provide seven-day dietary records and blood samples for analyses of total antioxidant capacity; oxidative stress param‐ eters including lipid peroxidation and protein oxidation; and bone turnover markers including bone resorption marker NTX and bone formation marker. Their results showed that the estimated dietary lycopene had a significant and direct correlation with serum lycopene, suggesting that lycopene from the diet is bioavailable. Their conclusion that the higher serum lycopene was associated with a low NTx (*p*<0.005) and lower protein oxidation (*p*<0.05) supports the antioxidative properties of lycopene involvement in its mechanisms of action in bone [36].

The overall conclusions that can be derived from the cross-sectional study is that lycopene has a role in the prevention of risk for osteoporosis. Further clinical studies described below support this conclusion.

Mackinnon et al [42] studied whether the elevated dose obtained through lycopene supple‐ mentation compared to intakes typically obtained from the usual daily diet was more beneficial in reducing bone turnover markers. Serum lycopene, bone turnover markers and oxidative stress parameter data were compared between postmenopausal women who were supple‐ mented with lycopene and those who obtained both a low and high intake lycopene from daily food diet. Results showed that women who consumed lycopene supplement had significantly lower TBARS values than participants who obtained a low intake or high intake lycopene through their usual daily diets. These differences in TBARS value may be attributed to a significantly higher concentration of serum 5-*cis* in lycopene-supplemented participants compared to participants who obtained their lycopene from low or high usual daily diet. This suggests that it is the 5-*cis* isomer, with the most potent antioxidant capacity which, at higher concentrations, decreases bone turnover markers due to its ability to provide the greatest protection against oxidative stress. It also appears to show that supplementation with lycopene may be necessary in spite of the daily intake of lycopene [42].

Another study was carried out to determine the effects of a lycopene-restricted diet on oxidative stress parameters and bone turnover markers in postmenopausal women [43]. Results showed that restricting the participants from consuming lycopene-containing prod‐ ucts resulted in significant decreases in serum lycopene, α-/β-carotene and lutein/zeaxanthin, with the overall change in the serum carotenoids being lower than that seen for lycopene. All configurations of lycopene (all trans, 5-cis- and other cis lycopene) were found to be decreased and the antioxidant enzymes SOD and CAT were also significantly depressed after lycopene restriction. These changes were accompanied by a significant increase in the bone resorption marker NTx. The important conclusion from this study is the possibility that the significant increase in the bone resorption marker NTx could lead to a long-term decrease in BMD and increased fracture risk as was observed by Brown et al. [44]; longer restriction period may be detrimental to a group of postmenopausal women who were already at high risk for osteo‐ porosis. Therefore, shorter wash-out periods of no lycopene consumption is all that is needed in clinical trials examining the effects of lycopene on bone health [43].

A clinical fully randomized controlled intervention study was next carried out by Mackin‐ non et al [45] to investigate directly the effects of lycopene supplementation on decreas‐ ing the risk for osteoporosis. Lycopene supplements include lycopene capsules, tomato juice with normal amount of lycopene, tomato juice with high amount of lycopene. They have shown that after the 4-month duration, the LYCOPENE-supplemented group had a significant increase in total antioxidant capacity, decrease in oxidative stress parameters protein oxidation as shown by increase in thiol values and lipid peroxidation as shown by TBARS which correlated to a decrease in NTx; all changes were significantly different from the PLACEBO group. These findings suggest that lycopene obtained in the form of tomato juice or capsule exerted equivalent antioxidant potency in reducing the risk of osteoporo‐ sis in postmenopausal women [45].

Mackinnon et al studied whether polymorphism plays a role in the development of osteopo‐ rosis [46]. To do this, Mackinnon et al. studied the role of 172T→A or 584A→G polymorphisms of the paraoxonase 1 (PON 1) in modulating the effects of serum lycopene on antioxidant capacity, oxidative stress parameters and bone turnover markers, and in women between the ages of 25-70 years). Their results showed that the PON1 polymorphism modified the associ‐ ation between lycopene and NTx and BAP, an interaction that may also moderate the risk of osteoporosis [46].

In another study, they showed that there was a significant interaction between PON1 genotype and change in TBARS (p<0.05) suggesting that supplementation with lycopene resulted in decreased lipid peroxidation, which interacted with the PON1 genotype to decrease bone resorption markers in postmenopausal women. These results provided a mechanistic evidence of how intervention with lycopene may act to decrease lipid peroxidation and thus the risk of osteoporosis in postmenopausal women [45, 47].

In conclusion, the demand for the use of other natural food components in the management of postmenopausal osteoporosis has increased due to reports on the adverse side effects of the conventional therapy (eg, HRT and bisphosphonates). The studies reviewed above revealed evidence that antioxidants such as lycopene can counteract the damaging effects of oxidative stress brought about by ROS that lead to the development of osteoporosis. The results of studies reviewed here indicate that lycopene maybe useful either as a dietary alternative to drug therapy or as a complement to the drugs presently approved for used by women at risk of osteoporosis.

#### **4.2. Studies on polyphenols**

Mackinnon et al [42] studied whether the elevated dose obtained through lycopene supple‐ mentation compared to intakes typically obtained from the usual daily diet was more beneficial in reducing bone turnover markers. Serum lycopene, bone turnover markers and oxidative stress parameter data were compared between postmenopausal women who were supple‐ mented with lycopene and those who obtained both a low and high intake lycopene from daily food diet. Results showed that women who consumed lycopene supplement had significantly lower TBARS values than participants who obtained a low intake or high intake lycopene through their usual daily diets. These differences in TBARS value may be attributed to a significantly higher concentration of serum 5-*cis* in lycopene-supplemented participants compared to participants who obtained their lycopene from low or high usual daily diet. This suggests that it is the 5-*cis* isomer, with the most potent antioxidant capacity which, at higher concentrations, decreases bone turnover markers due to its ability to provide the greatest protection against oxidative stress. It also appears to show that supplementation with lycopene

Another study was carried out to determine the effects of a lycopene-restricted diet on oxidative stress parameters and bone turnover markers in postmenopausal women [43]. Results showed that restricting the participants from consuming lycopene-containing prod‐ ucts resulted in significant decreases in serum lycopene, α-/β-carotene and lutein/zeaxanthin, with the overall change in the serum carotenoids being lower than that seen for lycopene. All configurations of lycopene (all trans, 5-cis- and other cis lycopene) were found to be decreased and the antioxidant enzymes SOD and CAT were also significantly depressed after lycopene restriction. These changes were accompanied by a significant increase in the bone resorption marker NTx. The important conclusion from this study is the possibility that the significant increase in the bone resorption marker NTx could lead to a long-term decrease in BMD and increased fracture risk as was observed by Brown et al. [44]; longer restriction period may be detrimental to a group of postmenopausal women who were already at high risk for osteo‐ porosis. Therefore, shorter wash-out periods of no lycopene consumption is all that is needed

A clinical fully randomized controlled intervention study was next carried out by Mackin‐ non et al [45] to investigate directly the effects of lycopene supplementation on decreas‐ ing the risk for osteoporosis. Lycopene supplements include lycopene capsules, tomato juice with normal amount of lycopene, tomato juice with high amount of lycopene. They have shown that after the 4-month duration, the LYCOPENE-supplemented group had a significant increase in total antioxidant capacity, decrease in oxidative stress parameters protein oxidation as shown by increase in thiol values and lipid peroxidation as shown by TBARS which correlated to a decrease in NTx; all changes were significantly different from the PLACEBO group. These findings suggest that lycopene obtained in the form of tomato juice or capsule exerted equivalent antioxidant potency in reducing the risk of osteoporo‐

Mackinnon et al studied whether polymorphism plays a role in the development of osteopo‐ rosis [46]. To do this, Mackinnon et al. studied the role of 172T→A or 584A→G polymorphisms

may be necessary in spite of the daily intake of lycopene [42].

252 Phytochemicals - Isolation, Characterisation and Role in Human Health

in clinical trials examining the effects of lycopene on bone health [43].

sis in postmenopausal women [45].

It is well known that polyphenols have a role in the prevention of chronic diseases such as cancers, diabetes, cardiovascular diseases, neurodegenerative diseases, and osteoporosis. Interest on polyphenols and bone health has increased in the last 10 years [48-51]. The anabolic role of phytonutrients and especially polyphenols in bone was reviewed by Horcajada [49], the mechanisms of action of polyphenol in osteoblast function and its interaction with osteoclasts was reviewed by Trzeciakiewicz [50] and the beneficial effects of green tea polyphenols has been reviewed [52, 53].

Currently, most of the research on polyphenols and their effects have emerged from *in vitro* and *in vivo* animal studies with only a few clinical studies available. In our recent review, we have included tables listing all the studies on polyphenols *in vitro* bone cell culture and the epidemiologic studies on the protective effects of polyphenol consumption against osteopo‐ rosis [54].

Combinations of polyphenols have also been studied. One such source is the nutritional supplement greens+TM, a blend of several herbal and botanical products containing a substan‐ tial amount of polyphenols including quercetin, apigenin and luteolin [41] which act as antioxidants and therefore should be able to counteract oxidative stress. Thus, Rao et al [55] have shown that greens+*TM*, is more effective in stimulating osteoblasts to form more bone nodules in a dose-dependant manner than epicatechin, the main polyphenol found in green tea. We have further shown that this stimulatory effect is accompanied by decreases in the reactive oxygen species H2O2 [56].

Two additional nutritional supplements have since been formulated which may prove to be good for bone health. These are the bone builderTM and the greens+bone builderTM ; the latter is the original greens+TM product that has been supplemented with the bone builderTM formula which contains several compounds including vitamins, minerals, and antioxidants. These various components have been separately shown to have some beneficial effect on bone [57]. Using the human osteoblast SaOS-2 cells, Rao et al showed that similarly to the greens+TM, the water-soluble bone-builderTM extract had a significant dose-dependent stimulatory effect on bone nodules formation [58]. It was additionally shown that similarly to the greens+TM, the watersoluble bone-builderTM extract had a significant dose-dependent stimulatory effect on bone nodules formation [58]. In a later study, they have shown 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 [59]*.* This led Rao et al to believe that synergistic effects of greens +*TM* and bone builder*TM* may have a beneficial effect on osteoporosis. A product called greens +bone builderTM is available commercially [57].

A clinical study was then carried out to evaluate the effect of the nutritional supplement greens + bone builder*TM* for 8 weeks on the risk for osteoporosis in postmenopausal women compared to placebo control [60, 61]. Results have shown that there was an increase in total antioxidant capacity, 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 [60] suggesting that the nutritional supplement may have a beneficial effect on bone health by mitigating the effects of oxidative stress. In order to test whether the antioxidant properties of greens+bone build‐ erTM can prevent the risk of osteoporosis in postmenopausal women. Kang et al [60] also measured the serum bone turnover markers, C-terminal telopeptide of type I collagen (CTX) as indicator of bone resorption, and procollagen type I N-terminal propeptide (PINP) as indicator of bone formation and determined their correlations with the serum antioxidant capacity, and the oxidative stress parameters lipid peroxidation, protein oxidation. Statistical analysis showed that at 8 weeks, the greens +bone builderTM supplement group significantly decreased the bone resorption marker CTX, while the Placebo group showed no significant changes. The supplement group was also significantly different from that of the Placebo group in all parameters measured. This decrease CTX correlated to the increase in their serum total antioxidant capacity and decreases in oxidative parameters protein oxidation lipid peroxida‐ tion [61]. These results suggest that a daily supplementation with polyphenols and micronu‐ trients may be important in reducing oxidative damage by reducing bone resorption, thereby reducing the risk of osteoporosis in postmenopausal women [60, 61].

In summary, studies reported in the literature on the role of polyphenols in bone health have exploded in the last 10 years, but most of the reports involved in vitro studies in osteoclasts and osteoblasts, animal studies and epidemiologicai studies. There is little doubt from the excellent studies reported that oxidative stress is one of the primary culprits responsible for the pathogenesis of osteoporosis via its role in osteoclastic resoption and the detrimental effects on the bone-forming osteoblasts. To date, only four clinical intervention studies have been reported, including ours. It is easy to see why it is very difficult to evaluate the role of polyphenols since, as we learned from this review, there are at least 8,000 different polyphenols identified to date, and each one probably having different effects on humans. Additionally, polyphenols are present in food with other constituents that may also be beneficial to bone health. In our clinical study, we combined the effects of a combination of polyphenols present in the nutritional supplement from greens+TM with the nutritional components present in bone builderTM such as minerals, vitamins and other nutrients. It is possible that the effects of greens +bone builderTM in increasing total antioxidant capacity, decreasing the oxidative stress markers protein oxidation and lipid peroxidation which correlated to the decrease in bone turnover marker for bone resorption is a result of the combined effects of the different polyphenols it contained with those of the other nutritional components present in the bone builderTM. It remained for future studies to zero in on specific component that is responsible for its beneficial effect.

In conclusion, we showed that oxidative stress due to ROS that are shown to cause the development of osteoporosis may be prevented by supplementation with the antioxidants lycopene and polyphenols. Results of in vitro studies in osteoblasts and osteoclasts, animal intervention studies, epidemiological studies and clinical intervention studies on lycopene and polyphenols are evidence for their potential use as alternative or complementary agent with other established drugs approved for the prevention or treatment of osteoporosis in postme‐ nopausal women.

#### **Acknowledgements**

tea. We have further shown that this stimulatory effect is accompanied by decreases in the

Two additional nutritional supplements have since been formulated which may prove to be good for bone health. These are the bone builderTM and the greens+bone builderTM ; the latter is the original greens+TM product that has been supplemented with the bone builderTM formula which contains several compounds including vitamins, minerals, and antioxidants. These various components have been separately shown to have some beneficial effect on bone [57]. Using the human osteoblast SaOS-2 cells, Rao et al showed that similarly to the greens+TM, the water-soluble bone-builderTM extract had a significant dose-dependent stimulatory effect on bone nodules formation [58]. It was additionally shown that similarly to the greens+TM, the watersoluble bone-builderTM extract had a significant dose-dependent stimulatory effect on bone nodules formation [58]. In a later study, they have shown 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 [59]*.* This led Rao et al to believe that synergistic effects of greens +*TM* and bone builder*TM* may have a beneficial effect on osteoporosis. A product called greens

A clinical study was then carried out to evaluate the effect of the nutritional supplement greens + bone builder*TM* for 8 weeks on the risk for osteoporosis in postmenopausal women compared to placebo control [60, 61]. Results have shown that there was an increase in total antioxidant capacity, 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 [60] suggesting that the nutritional supplement may have a beneficial effect on bone health by mitigating the effects of oxidative stress. In order to test whether the antioxidant properties of greens+bone build‐ erTM can prevent the risk of osteoporosis in postmenopausal women. Kang et al [60] also measured the serum bone turnover markers, C-terminal telopeptide of type I collagen (CTX) as indicator of bone resorption, and procollagen type I N-terminal propeptide (PINP) as indicator of bone formation and determined their correlations with the serum antioxidant capacity, and the oxidative stress parameters lipid peroxidation, protein oxidation. Statistical analysis showed that at 8 weeks, the greens +bone builderTM supplement group significantly decreased the bone resorption marker CTX, while the Placebo group showed no significant changes. The supplement group was also significantly different from that of the Placebo group in all parameters measured. This decrease CTX correlated to the increase in their serum total antioxidant capacity and decreases in oxidative parameters protein oxidation lipid peroxida‐ tion [61]. These results suggest that a daily supplementation with polyphenols and micronu‐ trients may be important in reducing oxidative damage by reducing bone resorption, thereby

reducing the risk of osteoporosis in postmenopausal women [60, 61].

In summary, studies reported in the literature on the role of polyphenols in bone health have exploded in the last 10 years, but most of the reports involved in vitro studies in osteoclasts and osteoblasts, animal studies and epidemiologicai studies. There is little doubt from the excellent studies reported that oxidative stress is one of the primary culprits responsible for

reactive oxygen species H2O2 [56].

254 Phytochemicals - Isolation, Characterisation and Role in Human Health

+bone builderTM is available commercially [57].

Funding for this research into Oxidative Stress, Antioxidants and Bone Health is shared by H.J. Heinz Co (Canada), Kagome Co. (Japan), LycoRed Natural Product Industries, Ltd. (Israel), Genuine Health Ltd (Canada), Millenium Biologix Inc. (Canada), and matched by the Canadian Institutes of Health Research (CIHR). We sincerely thanked the valuable contribu‐ tions to this research by the following students/graduate students and staff at the Calcium Research Laboratory, Department of Medicine at St Michael's Hospital and the University of Toronto and Department of Nutritional Sciences, University of Toronto: Dr. Bala Balachan‐ dran, Jaclyn Beca, Dawn Snyder, Loren Chan, Honglei Shen, Salva Sadeghi, Ayesha Quireshi, Dr. Erin Mackinnon and Nancy Kang. Their contributions were based on their experimental data, written reports published/in press manuscripts/theses. We would also like to thank to Dr. R.G. Josse for providing us with his medical expertise as well as allowing us access to his list of patients we were able to recruit. Special thanks to Dr. H. Vandenberghe for carrying out the CTX assay and for her valuable suggestions.

### **Author details**

L.G. Rao1\* and A.V. Rao2

\*Address all correspondence to: leticia.rao@utoronto.ca

1 Department of Medicine, St Michael's Hospital and University of Toronto, Canada

2 Department of Nutritional Sciences, University of Toronto, Canada

Parts of this chapter are © [2013] IEEE. Reprinted, with permission, from Rao LG and Rao AV Oxidative stress and antioxidants in the risk of osteoporosis - Role of the antioxidants lycopene and polyphenols. Chapter 5, pages 117-161. In: Topics in Osteoporosis. Ed. M. Vades-Flores.Intech Publishing. 2013.

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### **Phytochemicals and Cancer – Possible Molecular Targets of Phytochemicals in Cancer Prevention and Therapy**

Victor P. Bagla, Matlou P. Mokgotho and Leseilane J. Mampuru

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59873

#### **1. Introduction**

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ing; December 10-13, 2010; Singapore2010.

Phytochemicals and their synthetic derivatives have, over the decades, attracted huge attention and made significant contribution in modern drug discovery programs for their relevance in leveraging the severity or cure of several human diseases, including cancer. These natural products and their derivatives thereof have demonstrated immense pharmacological and biological properties. Although the molecular mechanisms of action of a majority of these phytochemicals are yet to be elucidated, cumulative evidence and the continued generation of new scientific data on their health benefits in disease prevention and cure have accrued over the years. Recent advancement in molecular biology, high throughput screening, biomarker identifications, target selection and genomic approaches have enabled researchers to under‐ stand salient interactions of natural products or their derivatives with cancer cells.

Most phytochemicals exhibit their pharmacologic effects in nature through a multi-targeted approach; a property that is highly desirable since therapy for carcinomas invariably involves dysregulation of multiple genes and associated cell-signalling pathways at various stages of initiation, progression and metastasis. On the other hand, in cancer initiation and progression, acquired genetic alterations, microenvironment-mediated epigenetic (heritable changes in gene activity and expression that occur without alteration in DNA sequences and are suffi‐ ciently powerful to regulate the dynamics of gene expression) perturbations have primarily been considered to play an important role in neoplastic development [9]. Genetic factors which control epigenetic modifications have been extensively documented [53].

One of the most widely studied phytochemical with anticancer properties is curcumin. Indeed, curcumin together with a number of related chemically-defined derivatives have been used

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

extensively in the treatment of a number of malignant growths, such as breast cancer (Figure 1). The rhizome of the plant *Curcuma longa* L., commonly known as turmeric, has been used for centuries as a spice and colouring agent. The dry rhizome of turmeric contains curcumin, the main bioactive component. Curcumin displays a diverse range of molecular targets, supporting the concept that it acts upon numerous biochemical and molecular cascades (Figure 2). Although the precise mode of action of this compound is yet to be fully elucidated, studies have shown that the chemopreventive action of curcumin might be due to its ability to induce apoptosis by several pathways. Curcumin physically binds to as many as 33 different proteins, including thioredoxin reductase, cyclooxygenase-2, (COX-2), protein kinase C, 5-lipoxygenase (5-LOX), and tubulin. Various molecular targets modulated by this agent include transcription factors, growth factors and their receptors, cytokines, enzymes, and genes regulating cell proliferation, and apoptosis. Since phytochemicals exhibit their therapeutic effect through multi-mechanism of action, research into the mechanism of action of curcumin in cancer has demonstrated its relevance in various biochemical pathways. The modulation of anti-apoptotic and survival pathways by curcumin as a strategy to induce apoptosis in cancer cells is well documented as well as its ability to inhibit proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell-signalling proteins [29, 48]. A list of some of apoptotic and growth inhibitory pathways activated by curcumin in tumour cells has been well documented [47].

Several reports indicate conflicting evidence on the sensitivity of different cancer cells to the effect of the same phytochemical content of extracts in exhibiting their anti-proliferative effect. This review will therefore focus on recent research developments in anticancer therapy using curcumin, as a representative plant-derived compound, against breast, lung, colorectal, cervical and prostate cancers, generated between 2008 and 2014.

#### **2. Curcumin and breast cancer**

Tumour progression is characterised by a mass formed by multiple populations of cells with mechanisms capable of inhibiting apoptosis, while promoting survival pathways and the invasion of healthy tissues through the blood and lymphatic circulation. The manifestation of breast cancer can be determined by the expression of receptors to oestrogen (ER) and to progesterone (PR) and of Her2 (c-erbB2, Her2/neu). Triple-negative breast cancer (TNBC) lacks the expression of the oestrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/EGFR2) and is an aggressive breast cancer phenotype with a poor prognosis. The p27, a CIP/KIP member, is a cyclin-dependent kinase inhibitor that causes G1 arrest by inhibiting G1 cyclin-CDK activities and the reduction of p27 is initiated by enhanced ubiquitin-mediated degradation, in which the Her2/Grb2/MAPK pathway has been implicated in the decrease of p27 stability. The effect of curcumin has been reported to stabilise p27 levels, a lack of which is associated with poor prognosis in breast cancer. In order to investigate whether this effect is mediated through changes in the S-phase kinase-associated protein 2 (Skp2) or Her2 expression, Sun and co-workers determined the inhibitory effect of curcumin on Skp2-mediated p27 ubiquitination in Her2/Skp2-overexpressing cancer cell lines (MDA-MB-231/Her2 cells) [57]. Their findings revealed that curcumin represses cell prolifer‐ ation, induces G1 arrest at a low dosage, and triggers apoptosis at a higher dosage and blocks cell migration in MDA-MB-231/Her2 cells. Curcumin at low dose was also shown to increases p27 and decreases Skp2, Her2, Cyclin E, CDK kinases in a time- and dose-dependent manner, a finding that is suggestive that p27, Skp2 and Her2 may be involved in the curcumin-induced growth inhibition in MDA-MB-231/Her2 cells. On the contrary, higher doses of curcumin produce a dose-dependent apoptotic death in MDA-MB-231/Her2 cells, an event that was observed to be related to cleaved forms of PARP and caspase-3 [57]. [16] on the other hand observed that since the F-box protein S-phase kinase-associated protein 2 (Skp2), which acts as an oncogene through targeting p27 for degradation, is overexpressed in many different human cancers; and that since curcumin induces p27 expression and growth arrest through the inhibition of Skp2 in MDA-MB-231 cells, a therapeutic strategy that could be designed to reduce Skp2 may play a central role in the treatment of ER/HER2 negative breast cancers. In another study, curcumin has been shown to exhibit an inhibitory effect on the proliferation of MDA-MB-231 cells and induced G2/M arrest in a dose-dependent manner. The study further demonstrated curcumin to increase the protein expression levels of p21 and Bax and decreased the levels of p53 and Bcl-2, a finding that suggests that one molecular mechanism by which curcumin inhibits the proliferation of MDA-MB-231 cells could be either through the upregulation of p21 expression for apoptosis to occur or through the up-regulation of the Baxto-Bcl-2 ratio [8].

extensively in the treatment of a number of malignant growths, such as breast cancer (Figure 1). The rhizome of the plant *Curcuma longa* L., commonly known as turmeric, has been used for centuries as a spice and colouring agent. The dry rhizome of turmeric contains curcumin, the main bioactive component. Curcumin displays a diverse range of molecular targets, supporting the concept that it acts upon numerous biochemical and molecular cascades (Figure 2). Although the precise mode of action of this compound is yet to be fully elucidated, studies have shown that the chemopreventive action of curcumin might be due to its ability to induce apoptosis by several pathways. Curcumin physically binds to as many as 33 different proteins, including thioredoxin reductase, cyclooxygenase-2, (COX-2), protein kinase C, 5-lipoxygenase (5-LOX), and tubulin. Various molecular targets modulated by this agent include transcription factors, growth factors and their receptors, cytokines, enzymes, and genes regulating cell proliferation, and apoptosis. Since phytochemicals exhibit their therapeutic effect through multi-mechanism of action, research into the mechanism of action of curcumin in cancer has demonstrated its relevance in various biochemical pathways. The modulation of anti-apoptotic and survival pathways by curcumin as a strategy to induce apoptosis in cancer cells is well documented as well as its ability to inhibit proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell-signalling proteins [29, 48]. A list of some of apoptotic and growth inhibitory pathways activated by curcumin in tumour cells has

262 Phytochemicals - Isolation, Characterisation and Role in Human Health

Several reports indicate conflicting evidence on the sensitivity of different cancer cells to the effect of the same phytochemical content of extracts in exhibiting their anti-proliferative effect. This review will therefore focus on recent research developments in anticancer therapy using curcumin, as a representative plant-derived compound, against breast, lung, colorectal,

Tumour progression is characterised by a mass formed by multiple populations of cells with mechanisms capable of inhibiting apoptosis, while promoting survival pathways and the invasion of healthy tissues through the blood and lymphatic circulation. The manifestation of breast cancer can be determined by the expression of receptors to oestrogen (ER) and to progesterone (PR) and of Her2 (c-erbB2, Her2/neu). Triple-negative breast cancer (TNBC) lacks the expression of the oestrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/EGFR2) and is an aggressive breast cancer phenotype with a poor prognosis. The p27, a CIP/KIP member, is a cyclin-dependent kinase inhibitor that causes G1 arrest by inhibiting G1 cyclin-CDK activities and the reduction of p27 is initiated by enhanced ubiquitin-mediated degradation, in which the Her2/Grb2/MAPK pathway has been implicated in the decrease of p27 stability. The effect of curcumin has been reported to stabilise p27 levels, a lack of which is associated with poor prognosis in breast cancer. In order to investigate whether this effect is mediated through changes in the S-phase kinase-associated protein 2 (Skp2) or Her2 expression, Sun and co-workers determined the inhibitory effect of curcumin on Skp2-mediated p27 ubiquitination in Her2/Skp2-overexpressing cancer cell lines

cervical and prostate cancers, generated between 2008 and 2014.

been well documented [47].

**2. Curcumin and breast cancer**

The effect of curcumin in inducing paraptosis in malignant breast cancer cell lines, including MDA-MB-435S, MDA-MB-231, and Hs578T cells has also been demonstrated. Apoptosis was demonstrated to be promoted by vacuolation that results from swelling and fusion of mito‐ chondria and/or the endoplasmic reticulum (ER) of the cell. The importance of protein synthesis in the process was tested by the use of cycloheximide. Cycloheximide was shown to block curcumin-induced vacuolation and subsequent cell death. AIP-1/Alix protein levels, an inhibitor protein of paraptosis, remained increasingly down-regulated in curcumin-treated malignant breast cancer cells while their overexpression decreased curcumin-induced cell death. ERK2 and JNK activation were shown to be associated with curcumin-induced cell death. It was also shown that mitochondrial superoxide acts as a critical early signal in curcumin-induced paraptosis, whereas proteasomal dysfunction was mainly responsible for the paraptotic changes associated with oestrogen receptor (ER) dilation [69]. Other authours have focused on the testing of the potency of curcumin analogues in comparison with curcumin. In one such study, an *ortho*-hydroxy substituted analogue of curcumin (BDMC-A) was analysed for its cytotoxicity. The analogue inhibited MCF-7 cells at a dose equivalent to that of curcumin. Further analysis of the apoptotic mechanism of the analogue, in comparison with curcumin, demonstrated that the analogue exerted more potent effect on the modulation of selective apoptotic markers of the intrinsic pathway: p53, Bcl-2, Bax, cytochrome c, Apaf-1, caspases-9, -3, PARP and those of the extrinsic pathway: FasL, caspase-8, as compared to curcumin. mRNA expression studies for Bcl-2/Bax also buttressed the efficacy of the analogue. An *in silico* molecular docking study with PI3K revealed that the docking of the analogue was more potent compared to curcumin. Increased apoptotic induction by the analogue was also demonstrated using different techniques in which characteristic apoptotic features such as nuclear fragmentation and chromatin condensation were exhibited [39].

On the other hand, a major metabolite of curcumin tetrahydrocurcumin (THC) has been investigated for its efficacy and associated mechanism of action in MCF-7 cells. The metabolite was shown to exhibit significant cell growth inhibition by inducing MCF-7 cells to undergo mitochondrial apoptosis and G2/M arrest, while co-treatment of cells with THC and p38 MAPK inhibitor was observed to effectively reverse the dissipation in mitochondrial membrane potential, and was also shown to block THC-mediated Bax up-regulation, Bcl-2 downregulation, caspase-3 activation as well as p21 up-regulation. This finding thus highlights the role of p38 MAPK in THC-induced mediated apoptosis and G2/M arrest, and its relevance, following the biotransformation of curcumin *in vivo* in the treatment of breast cancer [20].

Maspin is a serine protease inhibitor, which suppresses tumour growth and metastasis *in vivo* and tumour cell motility and invasion *in vitro*. In another study, curcumin was shown to up-regulate the expression of miR-15a and miR-16 and down-regulate the expression levels of Bcl-2 in MCF-7 treated cells, while silencing of miR-15a and miR-16 expression by specific inhibitors was shown to restore the expression of Bcl-2 levels. It was concluded from that study that curcumin can reduce the expression of Bcl-2 by up-regulating the expression of miR-15a and miR-16 in MCF-7 cells [44, 66].

Chronic inflammation is considered a major risk factor in the development and metastatic progression of cancers, while obesity on the other hand increases the risk of breast cancer in postmenopausal women. In obese individuals, there are increased levels of growth factors including insulin and insulin-like growth factors (IGFs). High insulin levels lead to an increase in the secretion of oestrogen, by binding to the circulating sex hormone binding globulin (SHBG). Consequently, the increased oestrogen-mediated downstream signalling favours breast cancer development.

Recent reports have shown that curcumin inhibits the expression of the pro-inflammatory cytokines CXCL-1 and CXCL-2, thereby enhancing the diminished formation of breast cancer metastasis [28]. In one study, the authours analysed the correlation between the effects of curcumin on miRNA expression using microarray miRNA expression analyses. Their findings revealed curcumin to modulate the expression of a series of miRNAs, including miR181b, in metastatic breast cancer cells while miR181b was observed to down-modulate CXCL-1 and CXCL-2 through a direct binding to their 3′-UTR [28]. [2] have also demonstrated that reduction of CXCL-1 and CXCL-2 messenger RNA levels is NFĸB dependent and requires intact IĸBα expression. Furthermore, the silencing of CXCL-1 and CXCL-2 was observed to result in down-regulation of several metastasis-promoting genes among which was the cytokine receptor CXCR4. The ability of curcuminoids to prevent transforming growth factor (TGF-β) induction of parathyroid hormone-related protein (PTHrP) and to reduce osteolytic bone destruction by blockade of Smad signalling in breast cancer cells has also been investi‐ gated [63]. To further understand the underlying mechanism, the effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide PTHrP and on lytic breast cancer bone metastasis were evaluated in the study. Curcumin was shown to inhibit TGF-β-stimu‐ lated PTHrP secretion in MDA-MB-231 human breast cancer cells independent of effects on cell growth inhibition *in vitro*. The effect on TGF-β signalling, reveal decreases in phospho-Smad2/3 and Ets-1 protein levels with no effect on p-38 MAPK-mediated TGF-β signalling [63].

demonstrated using different techniques in which characteristic apoptotic features such as

On the other hand, a major metabolite of curcumin tetrahydrocurcumin (THC) has been investigated for its efficacy and associated mechanism of action in MCF-7 cells. The metabolite was shown to exhibit significant cell growth inhibition by inducing MCF-7 cells to undergo mitochondrial apoptosis and G2/M arrest, while co-treatment of cells with THC and p38 MAPK inhibitor was observed to effectively reverse the dissipation in mitochondrial membrane potential, and was also shown to block THC-mediated Bax up-regulation, Bcl-2 downregulation, caspase-3 activation as well as p21 up-regulation. This finding thus highlights the role of p38 MAPK in THC-induced mediated apoptosis and G2/M arrest, and its relevance, following the biotransformation of curcumin *in vivo* in the treatment of breast cancer [20]. Maspin is a serine protease inhibitor, which suppresses tumour growth and metastasis *in vivo* and tumour cell motility and invasion *in vitro*. In another study, curcumin was shown to up-regulate the expression of miR-15a and miR-16 and down-regulate the expression levels of Bcl-2 in MCF-7 treated cells, while silencing of miR-15a and miR-16 expression by specific inhibitors was shown to restore the expression of Bcl-2 levels. It was concluded from that study that curcumin can reduce the expression of Bcl-2 by up-regulating the expression of miR-15a

Chronic inflammation is considered a major risk factor in the development and metastatic progression of cancers, while obesity on the other hand increases the risk of breast cancer in postmenopausal women. In obese individuals, there are increased levels of growth factors including insulin and insulin-like growth factors (IGFs). High insulin levels lead to an increase in the secretion of oestrogen, by binding to the circulating sex hormone binding globulin (SHBG). Consequently, the increased oestrogen-mediated downstream signalling favours

Recent reports have shown that curcumin inhibits the expression of the pro-inflammatory cytokines CXCL-1 and CXCL-2, thereby enhancing the diminished formation of breast cancer metastasis [28]. In one study, the authours analysed the correlation between the effects of curcumin on miRNA expression using microarray miRNA expression analyses. Their findings revealed curcumin to modulate the expression of a series of miRNAs, including miR181b, in metastatic breast cancer cells while miR181b was observed to down-modulate CXCL-1 and CXCL-2 through a direct binding to their 3′-UTR [28]. [2] have also demonstrated that reduction of CXCL-1 and CXCL-2 messenger RNA levels is NFĸB dependent and requires intact IĸBα expression. Furthermore, the silencing of CXCL-1 and CXCL-2 was observed to result in down-regulation of several metastasis-promoting genes among which was the cytokine receptor CXCR4. The ability of curcuminoids to prevent transforming growth factor (TGF-β) induction of parathyroid hormone-related protein (PTHrP) and to reduce osteolytic bone destruction by blockade of Smad signalling in breast cancer cells has also been investi‐ gated [63]. To further understand the underlying mechanism, the effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide PTHrP and on lytic breast cancer bone metastasis were evaluated in the study. Curcumin was shown to inhibit TGF-β-stimu‐ lated PTHrP secretion in MDA-MB-231 human breast cancer cells independent of effects on

nuclear fragmentation and chromatin condensation were exhibited [39].

264 Phytochemicals - Isolation, Characterisation and Role in Human Health

and miR-16 in MCF-7 cells [44, 66].

breast cancer development.

On the other hand, the mechanism of action of EF24, a novel curcumin analogue, in comparison with curcumin has been evaluated on MDA-MB231 breast cancer cells. EF24 and/or curcumin were shown to inhibit HIF-1α protein levels and, the subsequent inhibition of HIF transcrip‐ tional activity. The induction of HIF inhibition was demonstrated to occur in a VHL-dependent but proteasome-independent manner. While curcumin was seen to inhibit HIF-1α gene transcription, EF24 on the other hand exerted its activity by inhibiting HIF-1α post-transcrip‐ tion. EF24 was also shown to induce microtubule stabilisation in cells, although it had no stabilising effect on tubulin polymerisation in an *in vitro* assay using purified bovine brain tubulin, a finding that suggests that EF24-induced cytoskeletal disruption in cells may be related to an upstream signalling event rather than the direct binding of EF24 direct to tubulin [59]. In triple negative breast cancer cells however, curcumin was shown to induce DNA damage in association with phosphorylation, increased expression, and cytoplasmic retention of the BRCA1 protein and was shown to promote apoptosis and prevents anchorage-inde‐ pendent growth and migration of the cells [49].

TNF-related apoptosis inducing ligand (TRAIL) has also shown promising anti-cancer therapeutic activity and natural compound such as curcumin could potentially sensitise resistant cancer cells to TRAIL. Although significant percentage of primary tumours resistant to TRAIL-induced apoptosis remains an obstacle to the extensive use of TRAIL-based monotherapies, the combination of TRAIL with curcumin treatment has been investigated in an effort to induce apoptosis in TRAIL-resistant breast cancer cells. Findings revealed the combination to synergistically induce apoptosis in three TRAIL- resistant breast cancer cells due to the effect of curcumin on the expression and activation of TRAIL-associated cell death proteins to be related to differential effects of curcumin on the expression of Mcl-1 and activities of ERK and Akt. Although curcumin-induced production of reactive oxygen species was not observed to affect total expression of DR5 in this study, it was shown to enhance mobilisation of DR5 to the plasma membrane as well as induce the down-regulation of IAP proteins [43].

Another pathway by which curcumin has been shown to induce apoptosis in various malig‐ nant cancer cell lines is the induction of apoptosis through the PI3K/Akt signalling pathway. Protein kinase B (PKB) (Akt) is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. When active, Akt regulates cell survival and proliferation in addition to inhibition of apoptosis. At apoptotic concentration, curcumin has been shown to induce Akt phosphorylation, complemented by an increase in phosphorylation of glycogen synthase kinase 3-β (GSK3β), a pro-growth signalling molecule. In the study, the combination of curcumin with a PI3K inhibitor (LY290042) was shown to exhibit synergistic effect in inducing apoptosis while the inhibitor, on the other hand, was shown to attenuate curcumininduced Akt phosphorylation and activation of GSK3β [27]. Other mechanisms have shown curcumin-treated MDA-MB-435 human breast cancer cells to accumulate in the G1 phase of the cell cycle, accompanied by the suppression of the expression of Enhancer of Zeste Homolog 2 (EZH2) gene via the stimulation of three major members of the mitogen-activated protein kinase (MAPK) pathway: c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 kinase [15].

Recently, a new type of non-apoptotic cell death, termed paraptosis has been reported to be induced by insulin-like growth factor-1 receptor, epidermal growth factor, and TAJ/TROY, a member of the tumour necrosis factor (TNF) receptor superfamily. Some authours have demonstrated the ability of curcumin to induce paraptosis in malignant breast cancer cell lines, including MDA-MB-435S, MDA-MB-231, and Hs578T cells, by promoting vacuolation consequent of the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). Inhibition of protein synthesis was demonstrated to block curcumin-induced vacuolation and subsequent cell death, a finding that underscores the relevance of protein synthesis in the process of paraptosis. The levels of AIP-1/Alix protein, a known paraptosis inhibitor protein complex, were progressively down-regulated in malignant breast cancer cells exposed to curcumin, and AIP-1/Alix overexpression was shown to attenuate curcumin-induced cell death, while ERK2 and JNK activation were positively associated with curcumin-induced cell death [69].

The different mechanisms through which curcumin inhibits cancer cell functions such as cell growth, survival and motility, continue to be widely explored. In one study, the effect of curcumin on the function of integrin α6β4, a laminin adhesion receptor that plays an important role in the invasion and migration of cancer cells, was assessed. The study revealed curcumin to considerably reduce α6β4-dependent breast cancer cell motility and invasion in a concen‐ tration-dependent manner, without affecting apoptosis in MDA-MB-435/ β4β4-integrin transfectants and MDA-MB-231 breast cancer cell lines. Curcumin was also shown to selec‐ tively reduce the basal phosphorylation of β4 integrin (Y1494), which is essential in mediating α6β4-dependent phosphatidylinositol 3-kinase activation and cell motility as well as the blocking of α6β4-dependent Akt activation and expression of the cell motility-promoting factor ENPP2 in MDA-MB-435/β4 cell line [24]. The control of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) activity in recent years has also come to great significance. Matrix metalloproteinases play an important role in remodelling the extracellular matrix and their activities are regulated by tissue inhibitor of metalloproteinases (TIMPs) family. To investigate the role of curcumin in regulating cell metastasis, the effect of curcumin on metastatic MMPs and anti-metastatic TIMPs genes on MDA breast cancer cells has been assessed and was shown at high concentration to up-regulate TIMP-1, -2, -3 and -4 genes after 48 hours of treatment, accompanied by down-regulation of MMP-2 and MMP-9 gene expression levels in a concentration- and time-dependent manner [13]. This finding highlights the role of curcumin in regulating cell metastasis by inhibition of MMP-2 and MMP-9 and the up-regulation of TIMP-1 and TIMP-4 gene expression in breast cancer cells. In another study, the authours tested the comparative effect of the major component of turmeric (curcumin, demethoxycurcumin, *bis*demethoxycurcumin) in the modulation of MMP-3 activity and its secretion in MDA-MB-231 breast cancer cells. Analysis of MMP-3 expression by casein zymography exhibited high expression in MDA-MB-231 invasive breast carcinoma cells, but not in MCF-7 non-invasive breast cancer cells. In the ELISA assays however, MMP-3 levels were shown to be significantly decreased in all curcuminoid treat‐ ments while in using zymography, exposure to non-toxic doses of curcuminoid compounds except curcumin, was shown to reduce MMP-3 levels [3].

kinase (MAPK) pathway: c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated

Recently, a new type of non-apoptotic cell death, termed paraptosis has been reported to be induced by insulin-like growth factor-1 receptor, epidermal growth factor, and TAJ/TROY, a member of the tumour necrosis factor (TNF) receptor superfamily. Some authours have demonstrated the ability of curcumin to induce paraptosis in malignant breast cancer cell lines, including MDA-MB-435S, MDA-MB-231, and Hs578T cells, by promoting vacuolation consequent of the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). Inhibition of protein synthesis was demonstrated to block curcumin-induced vacuolation and subsequent cell death, a finding that underscores the relevance of protein synthesis in the process of paraptosis. The levels of AIP-1/Alix protein, a known paraptosis inhibitor protein complex, were progressively down-regulated in malignant breast cancer cells exposed to curcumin, and AIP-1/Alix overexpression was shown to attenuate curcumin-induced cell death, while ERK2 and JNK activation were positively associated with curcumin-induced cell

The different mechanisms through which curcumin inhibits cancer cell functions such as cell growth, survival and motility, continue to be widely explored. In one study, the effect of curcumin on the function of integrin α6β4, a laminin adhesion receptor that plays an important role in the invasion and migration of cancer cells, was assessed. The study revealed curcumin to considerably reduce α6β4-dependent breast cancer cell motility and invasion in a concen‐ tration-dependent manner, without affecting apoptosis in MDA-MB-435/ β4β4-integrin transfectants and MDA-MB-231 breast cancer cell lines. Curcumin was also shown to selec‐ tively reduce the basal phosphorylation of β4 integrin (Y1494), which is essential in mediating α6β4-dependent phosphatidylinositol 3-kinase activation and cell motility as well as the blocking of α6β4-dependent Akt activation and expression of the cell motility-promoting factor ENPP2 in MDA-MB-435/β4 cell line [24]. The control of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) activity in recent years has also come to great significance. Matrix metalloproteinases play an important role in remodelling the extracellular matrix and their activities are regulated by tissue inhibitor of metalloproteinases (TIMPs) family. To investigate the role of curcumin in regulating cell metastasis, the effect of curcumin on metastatic MMPs and anti-metastatic TIMPs genes on MDA breast cancer cells has been assessed and was shown at high concentration to up-regulate TIMP-1, -2, -3 and -4 genes after 48 hours of treatment, accompanied by down-regulation of MMP-2 and MMP-9 gene expression levels in a concentration- and time-dependent manner [13]. This finding highlights the role of curcumin in regulating cell metastasis by inhibition of MMP-2 and MMP-9 and the up-regulation of TIMP-1 and TIMP-4 gene expression in breast cancer cells. In another study, the authours tested the comparative effect of the major component of turmeric (curcumin, demethoxycurcumin, *bis*demethoxycurcumin) in the modulation of MMP-3 activity and its secretion in MDA-MB-231 breast cancer cells. Analysis of MMP-3 expression by casein zymography exhibited high expression in MDA-MB-231 invasive breast carcinoma cells, but not in MCF-7 non-invasive breast cancer cells. In the ELISA assays however, MMP-3 levels were shown to be significantly decreased in all curcuminoid treat‐

kinase (ERK), and p38 kinase [15].

266 Phytochemicals - Isolation, Characterisation and Role in Human Health

death [69].

Other studies have shown that demethoxycurcumin (DMC) inhibits the adhesion, migration and invasion of MDA-MB-231 human breast cancer cells, by decreasing levels of extracellular matrix (ECM) degradation-associated proteins including matrix metalloproteinase-9 (MMP-9), membrane type-1 matrix metalloproteinase (MT1-MMP), urokinase plasminogen activator (uPA) and uPA receptor (uPAR), while those of uPA inhibitor (PAI-1) have been shown to be up-regulated. Demethoxycurcumin was also shown to reduce the expression of intercellular adhesion molecule-1 (ICAM-1) and chemokine receptor 4, (CXCR4), which is involved in the modulation of the tumour metastasis process. The authours also demonstrated that DMC treatment inhibits DNA binding activity of nuclear factor-kappa B (NF-κB), which is known to mediate the expression of MMPs, uPA, uPAR, ICAM-1, and CXCR4, a finding that is suggestive that the mechanism of DMC mediated anti-invasive activity may involve the modulation of the expression of invasion-associated proteins, possibly by targeting NF-κB in MDA-MB-231 cells [68]. Similarly, the effect of curcumin on NF-κB, cell cycle regulatory proteins and matrix metalloproteinases (MMPs) in two breast cancer cell lines (MDA-MB-231 and BT-483) were evaluated. It was shown that Curcumin exhibited its anti-proliferation effect on MDA-MB-231 and BT-483 cells in a time- and dose-dependent manner, while the expression of cyclin D1 in MDA-MB-231 and the expression of CDK4 in BT-483 were shown to decline. MMP1 mRNA expression in BT-483 and MDA-MB-231 significantly decreased in curcumin treatment group when compared with untreated control group [35]; a finding that further buttresses the notion of the involvement of the regulation of the NF-kB inducing gene by curcumin in breast cancer cell proliferation and invasion. The effect of curcumin (diferuloyl‐ methane) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression, cell invasion and the molecular mechanisms involved in MCF-7 cells invasion has also been reported. Curcumin was shown to inhibit TPA-induced MMP-9 expression and cell invasion through suppressing NF-κB and AP-1 activation. It was also revealed to repress TPA-induced phosphorylation of p38 and JNK, and inhibits TPA-induced translocation of PKCα from the cytosol to the membrane, suggesting that curcumin-mediated inhibition of TPA-induced matrix metalloproteinase (MMP)-9 expression and cell invasion is associated with the sup‐ pression of the PKCα, MAPK and NF-κB/AP-1 pathway in MCF-7 cells [26]. In another study to evaluate the effects of curcumin on matrixmetalloproteinase-9 (MMP-9) and invasion ability induced by transforming growth factor-β1 (TGF-β1) in MDA-MB-231 cells, it was shown that low doses of curcumin had no obvious toxicity on cells while a change in concentration resulted in a concentration-dependent reduction in cell invasion provoked by TGF-β1. Curcumin was also shown to markedly inhibit TGF-β1-regulated MMP-9 and activation of Smad2, ERK1/2 and p38 in a dose- and time-dependent manner, a mechanism that maybe associated with TGFβ/Smad and TGF-β/ERK signalling [38].

The effect of low concentrations of curcumin has also been tested on patient-derived primary breast cancer-associated fibroblasts (CAF) cells. Cancer-associated fibroblasts actively partic‐ ipate in tumour growth, invasion, and metastasis. This involves many chemokines, growth factors, and matrix metalloproteinases (MMPs), which transmit the message in both directions, thus allowing cooperative crosstalk between cancer cells and their stroma. Recent reports show that curcumin treatment up-regulates p16INK4A and other tumour suppressor proteins and inactivates the JAK2/STAT3 pathway which results in the reduction level of alpha-smooth muscle actin (α-SMA) and the migration/invasion abilities of CAF cells. Curcumin was also demonstrated to further suppress the expression/secretion of stromal cell-derived factor-1 (SDF-1), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), MMP-9, and transforming growth factor-β, thereby impeding their paracrine procarcinogenic potential. Intriguingly, these effects were sustained even after curcumin withdrawal and cell splitting. Curcuminrelated senescence in this study was shown to be p16INK4A-dependent and occurred with no associated inflammatory secretory phenotype and that curcumin can trigger DNA damageindependent and safe senescence in stromal fibroblasts [14].

The efficacy of curcumin in blocking *Recepteur d'Origine Nantais* (RON) tyrosine kinase– mediated invasion of breast cancer cells has also been analysed. Curcumin-mediated inhibition of RON expression has been shown to result in the blockade of RON ligand, MSP-induced invasion of breast cancer cells and reduced RON expression by distorting p65 protein expres‐ sion and transcriptional activity. The treatment of MDA-MB-231 cells with pyrrolidine dithiocarbamate, an inhibitor of p65, or small interfering RNA knockdown of p65, leads to the blockage of RON gene expression and MSP-mediated invasion of MDA-MB-231 cells which further highlights the role of curcumin in blocking RON tyrosine kinase-mediated invasion of carcinoma cells [42]. Other studies have focused on the role of curcumin in preventing or delaying the progression of cancer by disruption of epithelial-mesenchymal transition (EMT), a key event in cancer cell invasion and metastasis. The authours showed that curcumin inhibits LPS-induced morphological changes, decreased the expression of LPS-induced markers of EMT such as vimentin, and increased the expression of E-cadherin and as a consequence, the inhibition of motility and invasiveness of MCF-7 and MDA-MB-231 breast cancer cell lines *in vitro*, mediated through the inactivation of NF-κB-Snail signalling pathways [17].

Of equal importance is the frequent association of obesity with breast cancer, an association that is possibly mediated by adipokines. Visfatin, an adipokine, has recently been shown to be related to the development and progression of breast cancer. Hence the consideration that its down-regulation may be a novel strategy for breast cancer therapy has been explored. To investigate this, the effect of curcumin on visfatin gene expression and the characterisation of its functional role in breast cancer have been assessed. It was found that the mRNA and protein levels of visfatin were down-regulated by curcumin in MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cells, along with decreased activity of constitutive NF-κB which high‐ lights the effect of curcumin to down-regulate visfatin gene expression in human breast cancer cells by a mechanism that is, at least in part, NF-κB dependent [26]. On the other hand obesity has also been shown to results in change in the expression profiles of several adipokines and cytokines including leptin, adiponectin, IL-6, TNF-α and IL-1β. Increased levels of leptin and decreased adiponectin secretions are directly associated with breast cancer development while increased levels of pro-inflammatory cytokines within the tumour microenvironment promote tumour development. The cumulative evidence of different adipokine- and cytokine-mediated molecular signalling pathways involved in obesity-associated breast cancer have been documented [22].

thus allowing cooperative crosstalk between cancer cells and their stroma. Recent reports show that curcumin treatment up-regulates p16INK4A and other tumour suppressor proteins and inactivates the JAK2/STAT3 pathway which results in the reduction level of alpha-smooth muscle actin (α-SMA) and the migration/invasion abilities of CAF cells. Curcumin was also demonstrated to further suppress the expression/secretion of stromal cell-derived factor-1 (SDF-1), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), MMP-9, and transforming growth factor-β, thereby impeding their paracrine procarcinogenic potential. Intriguingly, these effects were sustained even after curcumin withdrawal and cell splitting. Curcuminrelated senescence in this study was shown to be p16INK4A-dependent and occurred with no associated inflammatory secretory phenotype and that curcumin can trigger DNA damage-

The efficacy of curcumin in blocking *Recepteur d'Origine Nantais* (RON) tyrosine kinase– mediated invasion of breast cancer cells has also been analysed. Curcumin-mediated inhibition of RON expression has been shown to result in the blockade of RON ligand, MSP-induced invasion of breast cancer cells and reduced RON expression by distorting p65 protein expres‐ sion and transcriptional activity. The treatment of MDA-MB-231 cells with pyrrolidine dithiocarbamate, an inhibitor of p65, or small interfering RNA knockdown of p65, leads to the blockage of RON gene expression and MSP-mediated invasion of MDA-MB-231 cells which further highlights the role of curcumin in blocking RON tyrosine kinase-mediated invasion of carcinoma cells [42]. Other studies have focused on the role of curcumin in preventing or delaying the progression of cancer by disruption of epithelial-mesenchymal transition (EMT), a key event in cancer cell invasion and metastasis. The authours showed that curcumin inhibits LPS-induced morphological changes, decreased the expression of LPS-induced markers of EMT such as vimentin, and increased the expression of E-cadherin and as a consequence, the inhibition of motility and invasiveness of MCF-7 and MDA-MB-231 breast cancer cell lines *in*

*vitro*, mediated through the inactivation of NF-κB-Snail signalling pathways [17].

Of equal importance is the frequent association of obesity with breast cancer, an association that is possibly mediated by adipokines. Visfatin, an adipokine, has recently been shown to be related to the development and progression of breast cancer. Hence the consideration that its down-regulation may be a novel strategy for breast cancer therapy has been explored. To investigate this, the effect of curcumin on visfatin gene expression and the characterisation of its functional role in breast cancer have been assessed. It was found that the mRNA and protein levels of visfatin were down-regulated by curcumin in MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cells, along with decreased activity of constitutive NF-κB which high‐ lights the effect of curcumin to down-regulate visfatin gene expression in human breast cancer cells by a mechanism that is, at least in part, NF-κB dependent [26]. On the other hand obesity has also been shown to results in change in the expression profiles of several adipokines and cytokines including leptin, adiponectin, IL-6, TNF-α and IL-1β. Increased levels of leptin and decreased adiponectin secretions are directly associated with breast cancer development while increased levels of pro-inflammatory cytokines within the tumour microenvironment promote tumour development. The cumulative evidence of different adipokine- and cytokine-mediated

independent and safe senescence in stromal fibroblasts [14].

268 Phytochemicals - Isolation, Characterisation and Role in Human Health

Furthermore, the development and progression of malignant tumours depends on the formation of new blood vessels inside the tumour through a process termed angiogenesis. It is a vital process that ensues during cancer progression, and depends on the expression and activation of various angiogenic molecules, cytokines, growth factors, kinases and transcrip‐ tion factors. It had been previously demonstrated that the chemokine-like ECM-associated protein osteopontin (OPN) ignites the angiogenic switch by up-regulating the expression of vascular endothelial growth factor (VEGF) in a human breast cancer model. In this study [4], the authours demonstrated that curcumin (diferuloylmethane) abolishes OPN-induced VEGF expression and controls OPN-induced VEGF-dependent breast tumour angiogenesis *in vivo*. It was also observed that curcumin, in combination with anti-VEGF or anti-neuropilin (NRP)-1 antibody, was able to boost anti-angiogenic activity when compared to curcumin alone.

Furthermore, the over-expression of Flap endonuclease 1 (Fen1), a DNA repair-specific nuclease, has been implicated in the development of breast cancer. Nrf-2 is a leading regulator of cellular antioxidant defence systems and its inhibition of proliferation of breast cancer cells through its-mediated down-regulation of Fen1 expression by curcumin has been reported. Curcumin has been demonstrated to inhibit Fen1-dependent proliferation of MCF-7 cells, significantly induce Nrf-2 protein expression and inhibit Fen1 protein expression. It has also been shown to down-regulate Fen1 gene expression in an Nrf-2-dependent manner, as well as causing Nrf-2 translocation from the cytoplasm to the nucleus and to decrease Fen1 promoter activity by decreasing the recruitment of Nrf-2 to the Fen1 promoter [5]. While the abnormal activation of the Wnt/β-catenin signalling pathway and subsequent up-regulation of β-catenin driven downstream targets c-Myc and cyclin D1 is said to be associated with development of breast cancer in another study, the possibility that the efficacy of curcumin in the inhibition of cell proliferation and induction of apoptosis occur through modulation of β-catenin pathway in human breast cancer cells have also been suggested [45].

In order to help circumvent the problem associated with the low bioavailability of curcumin, the activities of analogues of curcumin have been tested in comparison with curcumin. In one study, an analogue of curcumin, GO-Y030, was tested for its efficacy in human breast MDA-MB-231 cell line. Both compounds were shown to reduce cell viability and induce apoptosis, although GO-Y030 was substantially more potent. It was also demonstrated that GO-Y030 was capable of interfering with STAT3 by inhibiting its phosphorylation and transcriptional activity, whereas comparable dosages of curcumin had little or no effect [18]. STAT3 is a persistently activated transcription factor in many cancer types. With regard to STAT3 phosphorylation, another curcumin analogue, FLLL12 was found to be a more potent inhibitor than the other, FLLL11. The reduction of phosphorylation of STAT3 was observed to correlate with the induction of apoptosis (determined by cleavage of PARP and caspase-3) [32]. Similarly, another synthetic curcumin analogue (hydrazinocurcumin) was shown to be more effective than curcumin in inhibiting STAT3 phosphorylation and down-regulation of an array of STAT3 downstream targets which contribute to suppression of cell proliferation, loss of colony formation, depression of cell migration and invasion as well as induction of cell apoptosis [61]. Inhibition of IkB kinase-nuclear factor-kB signalling pathway by 3,5-*bis*(2 flurobenzylidene)piperidin-4-one (EF24), a novel monoketone analogue of curcumin has also been demonstrated. EF24 has been shown to potently suppress the NF-ĸB signalling pathway through direct action on IĸB kinase (IKK) [21].

Hypoxia-inducible factors (HIFs) are transcription factors that play a central role in the adaptation and response to low oxygen levels in metazoan cells. Curcumin has been attributed with tumour growth inhibiting effects, possibly mediated by promoting hypoxia-inducible factor (HIF) degradation and also as exhibiting properties of an iron chelator, suggesting its potential of inhibiting HIF-α prolyl hydroxylase (PHD) activity. In order to clarify the divergent action of curcumin, researchers studied the concentration- and time-dependent effects of curcumin on HIF-α and -β protein levels and activity in hepatoma and breast carcinoma cell cultures under normoxic and hypoxic conditions. HIF-1α was shown to accumulate in normoxia after the application of higher doses of curcumin. The effect of curcumin was shown to lower HIF-1α and HIF-2α protein levels in hypoxia. HIF-1β (ARNT; arylhydrocarbon nuclear translocator) protein levels and HIF transcriptional activity were also reduced in normoxia and hypoxia after 4 h and 24 h of exposure. Furthermore, curcumin treatment was shown to negatively impact on clonogenic cell survival of Hep3B hepatoma and MCF-7 breast carcinoma cells. Effects of curcumin on cell growth and survival factor expres‐ sion was suggested to be of potential benefit in the treatment of cancer without a direct radiosensitising influence of the drug on these cells [56].

The effects of curcumin on triple-negative breast cancer (TNBC) cells and the possible molecular mechanisms have been evaluated in MDA-MB-231 cells [57]. The authours exam‐ ined the anti-proliferative effect of curcumin, its ability to induce apoptosis and the expression levels of extracellular regulated protein kinase (ERK1/2), pERK1/2, EGFR and pEGFR and concluded that the inhibition of the epidermal growth factor receptor (EGFR) signalling pathway is the likely underlying molecular mechanism of curcumin action in these cells. Since the functional interaction between integrin α6β4 and growth factor receptors has been implicated in key signalling pathways important for cancer cell function, the functional interaction between α6β4 and the epidermal growth factor receptor (EGFR) has also been examined. Findings revealed that curcumin is able to disrupt the functional and physical interactions between α6β4 and EGFR, as well as block α6β4/EGFR-dependent functions of carcinoma cells expressing the signalling competent form of α6β4. It has also been established that curcumin inhibits EGF-dependent mobilisation of α6β4 from hemi-desmosomes to the leading edges of migrating cells such as lammelipodia and filopodia, and thereby preventing α6β4 distribution to lipid rafts where functional interactions between α6β4 and EGFR occur. This finding highlights a novel paradigm in which curcumin inhibits α6β4 signalling and functions by altering intracellular localisation of α6β4, preventing its association with signal‐ ling receptors such as EGFR [55].

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apoptosis [61]. Inhibition of IkB kinase-nuclear factor-kB signalling pathway by 3,5-*bis*(2 flurobenzylidene)piperidin-4-one (EF24), a novel monoketone analogue of curcumin has also been demonstrated. EF24 has been shown to potently suppress the NF-ĸB signalling pathway

Hypoxia-inducible factors (HIFs) are transcription factors that play a central role in the adaptation and response to low oxygen levels in metazoan cells. Curcumin has been attributed with tumour growth inhibiting effects, possibly mediated by promoting hypoxia-inducible factor (HIF) degradation and also as exhibiting properties of an iron chelator, suggesting its potential of inhibiting HIF-α prolyl hydroxylase (PHD) activity. In order to clarify the divergent action of curcumin, researchers studied the concentration- and time-dependent effects of curcumin on HIF-α and -β protein levels and activity in hepatoma and breast carcinoma cell cultures under normoxic and hypoxic conditions. HIF-1α was shown to accumulate in normoxia after the application of higher doses of curcumin. The effect of curcumin was shown to lower HIF-1α and HIF-2α protein levels in hypoxia. HIF-1β (ARNT; arylhydrocarbon nuclear translocator) protein levels and HIF transcriptional activity were also reduced in normoxia and hypoxia after 4 h and 24 h of exposure. Furthermore, curcumin treatment was shown to negatively impact on clonogenic cell survival of Hep3B hepatoma and MCF-7 breast carcinoma cells. Effects of curcumin on cell growth and survival factor expres‐ sion was suggested to be of potential benefit in the treatment of cancer without a direct radio-

The effects of curcumin on triple-negative breast cancer (TNBC) cells and the possible molecular mechanisms have been evaluated in MDA-MB-231 cells [57]. The authours exam‐ ined the anti-proliferative effect of curcumin, its ability to induce apoptosis and the expression levels of extracellular regulated protein kinase (ERK1/2), pERK1/2, EGFR and pEGFR and concluded that the inhibition of the epidermal growth factor receptor (EGFR) signalling pathway is the likely underlying molecular mechanism of curcumin action in these cells. Since the functional interaction between integrin α6β4 and growth factor receptors has been implicated in key signalling pathways important for cancer cell function, the functional interaction between α6β4 and the epidermal growth factor receptor (EGFR) has also been examined. Findings revealed that curcumin is able to disrupt the functional and physical interactions between α6β4 and EGFR, as well as block α6β4/EGFR-dependent functions of carcinoma cells expressing the signalling competent form of α6β4. It has also been established that curcumin inhibits EGF-dependent mobilisation of α6β4 from hemi-desmosomes to the leading edges of migrating cells such as lammelipodia and filopodia, and thereby preventing α6β4 distribution to lipid rafts where functional interactions between α6β4 and EGFR occur. This finding highlights a novel paradigm in which curcumin inhibits α6β4 signalling and functions by altering intracellular localisation of α6β4, preventing its association with signal‐

through direct action on IĸB kinase (IKK) [21].

270 Phytochemicals - Isolation, Characterisation and Role in Human Health

sensitising influence of the drug on these cells [56].

ling receptors such as EGFR [55].

2,6‐*bis*(3,4‐dimethoxybenzylidene)‐cyclohexanone

3 Figure 1: Chemical structure of curcumin and related derivatives/analogues widely used in the treatment of various forms of cancers. **Figure 1.** Chemical structure of curcumin and related derivatives/analogues widely used in the treatment of various forms of cancers.

The use of curcumin as potential candidate in the treatment for HER-2-overexpressed breast cancer has also been reported [30]. HER-2 is an important oncoprotein that is overexpressed in about 15–25% of breast cancers. In one particular study, cell growth, cell cycle change, the anti-mobility effect, signal transduction, and xenograft volume analysis between groups treated with herceptin and/or curcumin were examined. Curcumin was shown to effect a decrease in cell growth of MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr breast cancer cell lines while phosphorylation of Akt, MAPK, and expression of NF-κB were shown to be reduced in BT-474 cells, but not in SK-BR-3-hr cells, after treatment with herceptin. Following treatment with curcumin, the HER-2 oncoprotein, phosphorylation of Akt, MAPK and expression of NF-κB were shown to decrease in both BT-474 and SK-BR-3-hr cells [30]. Evidence indicates that curcumin reverses chemo-resistance and sensitises cancer cells to chemotherapy and targeted therapy in breast cancer. Studies have therefore been undertaken to explore curcumin's potential anti-proliferation effects and resistance reversal in antioestrogen-resistant breast cancer cell line MCF-7/LCC2 and MCF-7/LCC9. The effect of curcumin treatment revealed anti-proliferative and pro-apoptotic activities and induction of cell cycle arrest at G2/M interphase. Moreover, the combination of curcumin and tamoxifen exhibited a synergistic survival inhibition in MCF-7/LCC2 and MCF-7/LCC9 cells. It was also revealed that curcumin targets multiple signals that are involved in growth maintenance and resistance acquisition in endocrine resistant cells. In the cell types used, curcumin was shown to suppress the expression of pro-growth and anti-apoptosis molecules, induce inactivation of NF-κB, Src and Akt/mTOR pathways and down-regulates the key epigenetic modifier EZH2 [19]. The chemosensitisation of breast cancer cell by curcumin to 5-fluorouracil (5-FU) has also been demonstrated. 5-Fluorouracil is an antimetabolite which achieves its therapeutic efficacy through inhibition of the enzyme thymidylate synthase (TS), essential for the synthesis and repair of DNA. Prolonged exposure to 5-FU induces TS overexpression, which leads to 5-FU resistance in cancer cells. Curcumin was demonstrated to sensitise the breast cancer cells to 5- FU through TS-dependent down-regulation of NF-κB. Silencing of TS was shown to suppress 5-FU-induced NF-κB activation, whereas inactivation of NF-κB was not shown to affect 5-FUinduced TS up-regulation, a finding that indicates that TS is upstream of NF-κB and is responsible for the regulation of the activation of NF-κB in 5-FU-induced signalling pathway. Although Akt/PI3 kinase and mitogen-activated protein kinase pathways were activated by 5-FU and down-regulated by curcumin, they were not shown to play a role in regulating the synergism in the study [60].

#### **3. Curcumin and lung cancer**

The molecular antitumour mechanism of a new 4-arylidene curcumin analogue (T63) has recently been reported to significantly inhibit the proliferation of A549 and H460 human lung cell lines via induction of G0/G1 cell cycle arrest and apoptosis. The study implicated reactive oxygen species (ROS)-activated FOXO3a cascade to be responsible to playing a central role in T63-induced cell proliferation inhibition. Enhancement of ROS production by T63 was shown to induce FOXO3a expression and nuclear translocation through activation of p38MAPK and inhibition of AKT, with subsequent elevation of the expression of FOXO3a target genes, including p21, p27, and Bim, as well as increasing the levels of activated caspase-3 and decreased levels of cyclin D1. N-acetylcysteine, an antioxidant, was shown to noticeably block the above effects, while small interfering RNA-mediated knockdown of FOXO3a significantly decreased T63-induced cell cycle arrest and apoptosis [34]. Evident in another study, was the ability of curcumin to cause DNA damage and endoplasmic reticulum (ER) stress and mitochondrial-dependent-induced apoptosis through the activation of caspase-3 at a treat‐ ment concentration of 30 μM in human lung cancer A549 cells. In contrast, lower concentra‐ tions of 5–10 μM curcumin showed no significant apoptotic inducing effect but rather induced G2/M-phase arrest in A549 cells. It was also shown to increase intracellular oxidative stress, indicators of ER stress, Ca2+ levels and the mitochondrial membrane potential in A549 cells thereby highlighting the role of curcumin in the activation of pathways involved in inducing G2/M-phase arrest and apoptosis [33].

anti-mobility effect, signal transduction, and xenograft volume analysis between groups treated with herceptin and/or curcumin were examined. Curcumin was shown to effect a decrease in cell growth of MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr breast cancer cell lines while phosphorylation of Akt, MAPK, and expression of NF-κB were shown to be reduced in BT-474 cells, but not in SK-BR-3-hr cells, after treatment with herceptin. Following treatment with curcumin, the HER-2 oncoprotein, phosphorylation of Akt, MAPK and expression of NF-κB were shown to decrease in both BT-474 and SK-BR-3-hr cells [30]. Evidence indicates that curcumin reverses chemo-resistance and sensitises cancer cells to chemotherapy and targeted therapy in breast cancer. Studies have therefore been undertaken to explore curcumin's potential anti-proliferation effects and resistance reversal in antioestrogen-resistant breast cancer cell line MCF-7/LCC2 and MCF-7/LCC9. The effect of curcumin treatment revealed anti-proliferative and pro-apoptotic activities and induction of cell cycle arrest at G2/M interphase. Moreover, the combination of curcumin and tamoxifen exhibited a synergistic survival inhibition in MCF-7/LCC2 and MCF-7/LCC9 cells. It was also revealed that curcumin targets multiple signals that are involved in growth maintenance and resistance acquisition in endocrine resistant cells. In the cell types used, curcumin was shown to suppress the expression of pro-growth and anti-apoptosis molecules, induce inactivation of NF-κB, Src and Akt/mTOR pathways and down-regulates the key epigenetic modifier EZH2 [19]. The chemosensitisation of breast cancer cell by curcumin to 5-fluorouracil (5-FU) has also been demonstrated. 5-Fluorouracil is an antimetabolite which achieves its therapeutic efficacy through inhibition of the enzyme thymidylate synthase (TS), essential for the synthesis and repair of DNA. Prolonged exposure to 5-FU induces TS overexpression, which leads to 5-FU resistance in cancer cells. Curcumin was demonstrated to sensitise the breast cancer cells to 5- FU through TS-dependent down-regulation of NF-κB. Silencing of TS was shown to suppress 5-FU-induced NF-κB activation, whereas inactivation of NF-κB was not shown to affect 5-FUinduced TS up-regulation, a finding that indicates that TS is upstream of NF-κB and is responsible for the regulation of the activation of NF-κB in 5-FU-induced signalling pathway. Although Akt/PI3 kinase and mitogen-activated protein kinase pathways were activated by 5-FU and down-regulated by curcumin, they were not shown to play a role in regulating the

272 Phytochemicals - Isolation, Characterisation and Role in Human Health

The molecular antitumour mechanism of a new 4-arylidene curcumin analogue (T63) has recently been reported to significantly inhibit the proliferation of A549 and H460 human lung cell lines via induction of G0/G1 cell cycle arrest and apoptosis. The study implicated reactive oxygen species (ROS)-activated FOXO3a cascade to be responsible to playing a central role in T63-induced cell proliferation inhibition. Enhancement of ROS production by T63 was shown to induce FOXO3a expression and nuclear translocation through activation of p38MAPK and inhibition of AKT, with subsequent elevation of the expression of FOXO3a target genes, including p21, p27, and Bim, as well as increasing the levels of activated caspase-3 and decreased levels of cyclin D1. N-acetylcysteine, an antioxidant, was shown to noticeably block

synergism in the study [60].

**3. Curcumin and lung cancer**

In another effort to finding novel putative intervention sites as chemo-protective and chemo‐ therapeutic target for curcumin in squamous cell lung carcinoma, Sen and co-authours, demonstrated that curcumin induces apoptosis in these cells, while microarray analysis revealed about 34 and 31 genes to be up- and down-regulated, respectively, following curcumin treatment. Likewise, growth arrest and DNA damage genes, GADD45a and peroxiredoxin-I was also shown to be up-regulated more than 2-folds (Sen *et al.,* 2008).

The effect of curcumin in non-small cell lung cancer (NSCLC), the leading cause of cancerrelated mortality, has also been evaluated. High expression of Rad51, a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, plays a key role in chemo- or radio-resistant carcinomas and thus HR represents a novel target for cancer therapy. Studies to evaluate the effect of curcumin in enhancing the effect of mitomycin C (MMC), a DNA inter-strand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression have been reported. Findings revealed curcumin treatment of non-small lung cancer (NSCLC) cell lines (A549 and H1975) was capable of suppressing MMC-induced MKK1/2–ERK1/2 signal activation and Rad51 protein expression. On the other hand, enhance‐ ment of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) was shown to increase Rad51 protein levels in curcumin and MMC co-treated human lung cells. The synergistic cytotoxic effect induced by curcumin–MMC treatment was established to be decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. On the contrary, the MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through down-regulation of ERK1/2 activation and Rad51 expression, while depletion of endogenous Rad51 expression by siRad51 RNA transfection was demon‐ strated to significantly enhance MMC and/or curcumin-induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected the lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. It was thus concluded that Rad51 inhibition may be an additional mechanism of action for enhancing the chemosensitisation of MMC by curcumin in NSCLC [27].

In a similar study, the effect of curcumin treatment on the expression of nuclear factor κBrelated proteins *in vitro* and *in vivo* and on growth and metastasis in an intra-lung tumour mouse model alone or in combination with gemcitabine or cisplatin has been assessed. Western blot analyses showed that the expressions of IkB, nuclear p65, cyclooxygenase-2 (COX-2) and p-ERK1/2 were down-regulated by curcumin *in vitro*. In *in vivo*, curcumin was shown to potentiate gemcitabine- or cisplatin-mediated antitumour effects and was also capable of reducing COX-2 expression in subcutaneous tumours with a decrease in weight of intra-lung tumours accompanied by a significant survival rate increase. The effect of curcumin in the inhibition of COX-2, p65 expression and ERK1/2 activity in NSCLC cells was observed to be associated with decreased survival and increased induction of apoptosis [32].

The anti-metastasis effects and mechanism of curcumin action in lung cancer have also been elucidated. Rac1 is an important small Rho GTPases family protein and has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metasta‐ sis. In order to investigate its role, [5] examined the influence of curcumin on *in vitro* invasive‐ ness of human lung cancer cells and the expression pattern of Rac1. Their findings revealed that curcumin at 10 μM was capable of slightly reducing the proliferation of 801D lung cancer cells with a pronounced inhibitory effect on epidermal growth factor or transforming growth factor β1-induced lung cancer cell migration and invasion. The suppression of invasiveness correlat‐ ed with the inhibition of Rac1/PAK1 signalling pathways and matrix metalloproteinases (MMP) -2 and -9 protein expression when curcumin treatment was combined with the methods of Rac1 gene silencing and overexpression in lung cancer cells. It was also revealed by laser confocal microscopic analysis that Rac1-regulated actin cytoskeleton rearrangement may be involved in anti-invasion effect of curcumin on lung cancer cell. The authours concluded that low-toxic levels of curcumin could efficiently inhibit migration and invasion of lung cancer cells through inhibition of Rac1/PAK1 signalling pathway and MMP-2 and MMP-9 expression [5].

The effect of curcumin as a chemosensitiser in lung cancer has also been examined on HIF-1α in cisplatin (DDP) sensitive A549 and resistant A549/DDP cell lines. Findings revealed HIF-1α in A549/DDP cells to be overexpressed at both mRNA and protein levels together with a poor response to DDP. It was also shown that HIF-1α abnormality contributes to DDP resistance in A549/DDP lung cancer cells while combined curcumin and DDP treatment was observed to markedly inhibited A549/DDP cells proliferation, reversed DDP resistance and triggered apoptotic death by promoting HIF-1α degradation and activation of caspase-3, respectively. The expression of HIF-1α-dependent P-gp was also observed to decrease in response to curcumin in a dose-dependent manner; a finding that highlights the drug resistant reversing effect of curcumin in lung cancer cells by inhibiting HIF-1α expression and activation of caspase-3 [67].

A novel inflammation-related mechanism for curcumin-induced inhibition of lung tumour growth has also been reported. Neutrophil elastase, an important regulator of inflammatory processes has been found to directly triggered tumour cell proliferation in human lung adenocarcinoma A549 cells. Alpha1-antitrypsin synthesised by tumour cells is a natural inhibitor of neutrophil elastase and curcumin has been shown to counter the decrease of α1 antitrypsin induced by neutrophil elastase by prompting the promoter activity of α1-antitryp‐ sin, thereby promoting its expression in A549 cells. The inhibition of neutrophil elastaseinduced proliferation was shown to be dependent on the PI3K/Akt pathway. Knockdown of α1-antitrypsin by siRNA was demonstrated to further enhance the tumour cell proliferation induced by neutrophil elastase and significantly blocked the anti-proliferative effect of curcumin against neutrophil elastase. In *in vivo*, curcumin was also observed to remarkably inhibit the primary tumour growth of Lewis lung carcinoma (LLC) in C57BL/6 mice. The authours also demonstrated that curcumin up-regulates the level of α1-antitrypsin in primary tumour tissue by promoting its local expression, while the protein level of neutrophil elastase in tumour tissue was observed to decrease in mice treated with curcumin. This finding further highlights the roles of neutrophil elastase and α1-antitrypsin in modulating lung tumour proliferation in inflammatory microenvironment and the effect of curcumin in inhibiting neutrophil elastase-induced tumour proliferation via the up-regulation of α1-antitrypsin expression *in vitro* and *in vivo* [64].

#### **4. Curcumin and colorectal cancer**

potentiate gemcitabine- or cisplatin-mediated antitumour effects and was also capable of reducing COX-2 expression in subcutaneous tumours with a decrease in weight of intra-lung tumours accompanied by a significant survival rate increase. The effect of curcumin in the inhibition of COX-2, p65 expression and ERK1/2 activity in NSCLC cells was observed to be

The anti-metastasis effects and mechanism of curcumin action in lung cancer have also been elucidated. Rac1 is an important small Rho GTPases family protein and has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metasta‐ sis. In order to investigate its role, [5] examined the influence of curcumin on *in vitro* invasive‐ ness of human lung cancer cells and the expression pattern of Rac1. Their findings revealed that curcumin at 10 μM was capable of slightly reducing the proliferation of 801D lung cancer cells with a pronounced inhibitory effect on epidermal growth factor or transforming growth factor β1-induced lung cancer cell migration and invasion. The suppression of invasiveness correlat‐ ed with the inhibition of Rac1/PAK1 signalling pathways and matrix metalloproteinases (MMP) -2 and -9 protein expression when curcumin treatment was combined with the methods of Rac1 gene silencing and overexpression in lung cancer cells. It was also revealed by laser confocal microscopic analysis that Rac1-regulated actin cytoskeleton rearrangement may be involved in anti-invasion effect of curcumin on lung cancer cell. The authours concluded that low-toxic levels of curcumin could efficiently inhibit migration and invasion of lung cancer cells through

associated with decreased survival and increased induction of apoptosis [32].

274 Phytochemicals - Isolation, Characterisation and Role in Human Health

inhibition of Rac1/PAK1 signalling pathway and MMP-2 and MMP-9 expression [5].

of caspase-3 [67].

The effect of curcumin as a chemosensitiser in lung cancer has also been examined on HIF-1α in cisplatin (DDP) sensitive A549 and resistant A549/DDP cell lines. Findings revealed HIF-1α in A549/DDP cells to be overexpressed at both mRNA and protein levels together with a poor response to DDP. It was also shown that HIF-1α abnormality contributes to DDP resistance in A549/DDP lung cancer cells while combined curcumin and DDP treatment was observed to markedly inhibited A549/DDP cells proliferation, reversed DDP resistance and triggered apoptotic death by promoting HIF-1α degradation and activation of caspase-3, respectively. The expression of HIF-1α-dependent P-gp was also observed to decrease in response to curcumin in a dose-dependent manner; a finding that highlights the drug resistant reversing effect of curcumin in lung cancer cells by inhibiting HIF-1α expression and activation

A novel inflammation-related mechanism for curcumin-induced inhibition of lung tumour growth has also been reported. Neutrophil elastase, an important regulator of inflammatory processes has been found to directly triggered tumour cell proliferation in human lung adenocarcinoma A549 cells. Alpha1-antitrypsin synthesised by tumour cells is a natural inhibitor of neutrophil elastase and curcumin has been shown to counter the decrease of α1 antitrypsin induced by neutrophil elastase by prompting the promoter activity of α1-antitryp‐ sin, thereby promoting its expression in A549 cells. The inhibition of neutrophil elastaseinduced proliferation was shown to be dependent on the PI3K/Akt pathway. Knockdown of α1-antitrypsin by siRNA was demonstrated to further enhance the tumour cell proliferation induced by neutrophil elastase and significantly blocked the anti-proliferative effect of curcumin against neutrophil elastase. In *in vivo*, curcumin was also observed to remarkably Insulin resistance and obesity are associated with increased colorectal cancer (CRC) risk and high reoccurrence rates. The effect of dietary compounds in reducing insulin-induced cell proliferation in normal and metastatic colon epithelial cells has been demonstrated. Murine colon epithelial cells (YAMC) and adenocarcinoma cells (MC38) were treated with docosa‐ hexaenoic acid (DHA) or curcumin alone, followed by the combination of co-treatments of the diet-derived compound and insulin. Insulin-stimulated MAPK and MEK phosphorylation was shown to be inhibited by DHA and curcumin in MC38 cancer cells, suggesting that curcumin and DHA are capable of blocking insulin-induced colon cancer cell proliferation *in vitro* via a MEK-mediated mechanism [12]. Other indicators implicate non-steroidal antiinflammatory drugs (NSAIDs), particularly the highly selective COX-2 inhibitors in the prevention of colon cancer. As such, some authours have demonstrated the effects of diclofe‐ nac, a preferential COX-2 inhibitor, and curcumin in inducing apoptosis in colon cancer cells. Both diclofenac and curcumin were shown to lower COX-2 activity and PGE-2 levels while the expression of IκBα was shown to be higher, with a lowered IKK activity suggesting that these agents may suppress the transfer of NF-κB to the nucleus and its pro-inflammatory gene transcription. Both drugs were also shown to down-regulate the level of pro-inflammatory cytokines, TNF-α, IL-1β and IL-2, through the inhibition of NF-κB and subsequent induction of apoptosis, thus confirming the regulatory role of NF-κB in the process [46]. Similarly, targeting nutraceuticals to tumours can enhance their effectiveness. Nanoparticles encapsu‐ lating curcumin have been shown to be more effective than the free curcumin, eventually inhibiting NF-κB regulated transcription and angiogenesis [41].

In a study, [50] exposed human colorectal cancer cells to clinically relevant doses of gamma rays, in an attempt to elucidate the mechanism of their radio-resistance. The authours charac‐ terised NF-κB activation as a mechanism of inducible radio-resistance in colorectal cancer. Curcumin was shown to inhibit the proliferation and the post-irradiation clonogenic survival of multiple colorectal cancer cell lines. Radiation was observed to stimulate NF-κB activity in a dose- and time-dependent manner, whereas curcumin was shown to suppress this radiationinduced NF-κB activation via inhibition of radiation-induced phosphorylation and degrada‐ tion of inhibitor of κB alpha, inhibition of κB kinase activity, and the inhibition of Akt phosphorylation. Curcumin was also shown to suppress NF-κB–regulated gene products (Bcl-2, Bcl-xL, inhibitor of apoptosis protein-2, COX-2, and cyclin D1). The authours concluded that transient inducible NF-κB activation provides a pro-survival response to radiation that may account for development of radio-resistance and that curcumin blocks this signalling pathway and further potentiates the antitumour effects of radiation therapy.

The induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment, demonstrating a functional link between senescence and autophagy in curcumintreated cells, has been reported. Activation of SA-β-galactosidase activity following curcumin treatment has been observed in p53+/+ and p53−/− cells, although the later was less sensitive to the pro-senescent activity. The authours also demonstrated the up-regulation of p53 and p21 proteins in p53+/+ HCT116 cells, while p53-independent induction of p21 was observed in p53−/− HCT116 cell. Senescence of HCT116 cells was shown to be accompanied by autoph‐ agy that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, puncture staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi, was observed to decrease the number of senescent cells induced by curcumin, but was not shown to lead to increased cell death [40].

#### **5. Curcumin and cervical cancer**

Human papillomavirus (HPV) infections remain a leading cause of mortality worldwide and cervical cancer is associated with infection with high risk human papillomaviruses (HPVs). Cervical cancer is the second leading cause of cancer death for women in the world. The effect of low concentration of curcumin on human cervical cancer cell line (HeLa) has been shown to mediate decrease in the cell number and viability, and increase in apoptotic events and superoxide level. Treatment of cells with curcumin was revealed to be toxic even at concen‐ trations as low as 1 μM even though no genotoxic effect was observed in these cells. Since argyrophilic nucleolar protein (AgNOR protein) expression is elevated in malignant cells compared to normal cells, the effect of curcumin-associated changes in size (area) and number of silver deposits were also evaluated. Curcumin was shown to induce decreased AgNOR protein pools, mediated possibly by global DNA hypermethylation observed after low concentration of curcumin treatment [31].

Curcumin is an anti-inflammatory agent that is known to have anti-COX-2 activity. One way of preventing and treating cervical cancer is by targeting COX-2. In order to evaluate the effect of curcumin in cervical cancer, the expression of COX-2 and its precursors have been examined by immunohistochemistry. The inhibitory effect of curcumin on cervical cancer cells was determined via 2-dimensional gel electrophoresis, data analysis, and ingenuity pathway analysis. The authours observed no significant differences in the expression of COX-2 in squamous cell carcinoma, and carcinoma *in situ*, although that was not the case in the expres‐ sion of COX-2 in adenocarcinoma in comparison to normal and squamous cell carcinoma tissues. However, proteins associated with cancer and the cell cycle were shown to be signif‐ icantly altered in cultured cells [37].

Since cervical cancer is associated with infection with high risk human papillomaviruses (HPVs), the molecular mechanism of curcumin induced apoptosis in HPV-positive cervical cancer HeLa, SiHa and CaSki cells have also been evaluated. Curcumin was shown to cause distinct inhibition of human telomerase reverse transcriptase (hTERT), the catalytic core of telomerase thereby reducing proliferation of cancer cells. Findings in the study revealed that curcumin-mediated apoptosis in these cells may be associated with the up-regulation of proapoptotic Bax, AIF, release of cytochrome c and down regulation of anti-apoptotic Bcl-2 and Bcl-xL, accompanied by an increase in caspase-3 and -9 activities. As such, the effect of curcumin as an anti-inflammatory and anti-proliferative agent in these cells was shown to be associated with down regulation of COX-2, iNOS and cyclin D1 at varying extents [54]. Recently, in an attempt to develop a curcumin–based therapy for cervical cancer, the effect of curcumin on four human papillomavirus HPV (+) cervical cancer cell lines and normal fibroblasts has been assessed. Curcumin treatment was shown to selectively eliminate a variety of HPV (+) cervical cancers cells (HeLa, ME-180, SiHa, and SW756), suppress the transforming antigen E6, dramatically inhibits the expression of the pro-cancer protein epidermal growth factor receptor (EGFR), and concomitantly induced p53 levels. In addition, vacurin, a colloidal solution of curcumin which is incorporated in clinically used amphipathic vaginal cream was shown to eliminate apposed HeLa cells while suppressing the expression of EGFR [11].

#### **6. Curcumin and prostate cancer**

that transient inducible NF-κB activation provides a pro-survival response to radiation that may account for development of radio-resistance and that curcumin blocks this signalling

The induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment, demonstrating a functional link between senescence and autophagy in curcumintreated cells, has been reported. Activation of SA-β-galactosidase activity following curcumin treatment has been observed in p53+/+ and p53−/− cells, although the later was less sensitive to the pro-senescent activity. The authours also demonstrated the up-regulation of p53 and p21 proteins in p53+/+ HCT116 cells, while p53-independent induction of p21 was observed in p53−/− HCT116 cell. Senescence of HCT116 cells was shown to be accompanied by autoph‐ agy that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, puncture staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi, was observed to decrease the number of senescent cells induced by curcumin, but

Human papillomavirus (HPV) infections remain a leading cause of mortality worldwide and cervical cancer is associated with infection with high risk human papillomaviruses (HPVs). Cervical cancer is the second leading cause of cancer death for women in the world. The effect of low concentration of curcumin on human cervical cancer cell line (HeLa) has been shown to mediate decrease in the cell number and viability, and increase in apoptotic events and superoxide level. Treatment of cells with curcumin was revealed to be toxic even at concen‐ trations as low as 1 μM even though no genotoxic effect was observed in these cells. Since argyrophilic nucleolar protein (AgNOR protein) expression is elevated in malignant cells compared to normal cells, the effect of curcumin-associated changes in size (area) and number of silver deposits were also evaluated. Curcumin was shown to induce decreased AgNOR protein pools, mediated possibly by global DNA hypermethylation observed after low

Curcumin is an anti-inflammatory agent that is known to have anti-COX-2 activity. One way of preventing and treating cervical cancer is by targeting COX-2. In order to evaluate the effect of curcumin in cervical cancer, the expression of COX-2 and its precursors have been examined by immunohistochemistry. The inhibitory effect of curcumin on cervical cancer cells was determined via 2-dimensional gel electrophoresis, data analysis, and ingenuity pathway analysis. The authours observed no significant differences in the expression of COX-2 in squamous cell carcinoma, and carcinoma *in situ*, although that was not the case in the expres‐ sion of COX-2 in adenocarcinoma in comparison to normal and squamous cell carcinoma tissues. However, proteins associated with cancer and the cell cycle were shown to be signif‐

pathway and further potentiates the antitumour effects of radiation therapy.

was not shown to lead to increased cell death [40].

276 Phytochemicals - Isolation, Characterisation and Role in Human Health

**5. Curcumin and cervical cancer**

concentration of curcumin treatment [31].

icantly altered in cultured cells [37].

Emerging evidence suggests that chronic inflammation is a major risk factor for the develop‐ ment and metastatic progression of prostate cancer. In evaluating the effect of curcumin on prostate carcinoma growth, apoptosis and metastasis, curcumin was shown to inhibit the translocation of NFκB to the nucleus through the inhibition of the IκB-kinase (IKKβ), leading to stabilisation of the inhibitor of NFκB, IκBα, in PC-3 prostate carcinoma cells. Inhibition of NFκB activity was demonstrated to reduce expression of CXCL-1 and -2 and abolished the autocrine/paracrine loop that links the two chemokines to NFκB. When used in combination with the synthetic IKKβ inhibitor, SC-541, no additive or synergistic effect was observed while treatment of cells with curcumin and siRNA-based knockdown of CXCL-1 and -2 was shown to induce apoptosis, inhibit proliferation and down-regulate several important metastasispromoting factors like COX-2, SPARC and EFEMP. In an orthotopic mouse model of hema‐ togenous metastasis, treatment with curcumin inhibited statistically significant formation of lung metastasis. The authours concluded that chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells by maintaining a positive pro-inflammatory and prometastatic feedback loop between NFκB and CXCL-1/-2, while reduced metastasis formation *in vivo* can be achieved by the disruption of this feedback loop by curcumin-induced inhibition of the NFκB signalling [23].

On the other hand, protein kinase D1 (PKD1), a multifunctional kinase that is highly expressed in normal prostate and decreased expression levels, has been associated with the progression of prostate cancer. Curcumin has been found to activate PKD1, with subsequent changes in β-catenin signalling by hindering nuclear β-catenin transcription activity and increasing the levels of membrane β-catenin in prostate cancer cells. Modulation of these cellular events by curcumin is shown to correlate with decreased cell proliferation, colony formation, cell motility and enhanced cell-cell aggregation in prostate cancer cells. It has also been demonstrated that inhibition of cell motility is mediated by decreasing the levels of active cofilin, a downstream target of PKD1. The potent anti-cancer effect of curcumin *in vitro* in the study was shown to correlate well with those in prostate cancer xenograft mouse model, thus highlighting a novel molecular mechanism of curcumin action via the activation of PKD1 in prostate cancer cells [58]. The effect of curcumin in suppressing prostate cancer cell invasion, tumour growth, and metastasis has also been assessed. Curcumin was shown to be capable of suppressing epider‐ mal growth factor (EGF)-stimulated and heregulin-stimulated PC-3 cell invasion, as well as androgen-induced LNCaP cell invasion. Treatment of cells with curcumin was also shown to significantly result in reduced matrix metalloproteinase 9 activities and down-regulation of cellular matriptase, a membrane-anchored serine protease with oncogenic roles in tumour formation and invasion. It was also demonstrated that curcumin inhibits the induction effects of androgens and EGF on matriptase activation, as well as the reduction of the activated levels of matriptase after its overexpression. The reduction of activated matriptase in cells by curcumin was also observed to be partly due to its effect on promoting the shedding of matriptase into an extracellular environment without altering matriptase gene expression. In addition, curcumin was also shown to significantly suppress the invasive ability of prostate cancer cells induced by matriptase overexpression. The data from the study indicate that curcumin exhibits a suppressive effect on prostate cancer cell invasion, tumour growth, and metastasis, in part via the down-regulation of matriptase function [7]. In another study, the anticancer activity of curcumin and genistein combination in human prostate cancer (PC3) cell line with respect to their anti-angiogenic effect has been examined. The combination of both compounds was shown to decrease cell viability, induce apoptosis and cell cycle arrest at G0 phase in a dose- and time-dependent manner. In order to understand the anti-angiogenic effect of the combination, the authours determined the expression of ARNT and HIF-1α protein levels which were shown to significant decline when compared to the control group and their respective monotherapy-treated groups [1].

The transcriptional activity of the androgen receptor (AR) is modulated by interaction with co-regulators, one of which is β-catenin. The effect of curcumin in inhibiting AR expression has also been elucidated through its role in mediating Wnt/β-catenin signalling pathway with regard to AR/β-catenin interactions. Curcumin induced a significant inhibition of AR expres‐ sion in a dose-dependent manner as well as its suppression of β-catenin in the nuclear and cytoplasmic extracts and whole cell lysates. Phosphorylation of Akt and glycogen synthase kinase-3β was shown to be attenuated, while phosphorylated β-catenin was increased after curcumin treatment. Cyclin D1 and c-myc, the target genes of the β-catenin/T-cell factor transcriptional complex, were also shown to be decreased; a finding that highlights the effect of curcumin in modulating the Wnt/β-catenin signalling pathway and may thus play a significant role in mediating inhibitory effects on LNCaP prostate cancer cells [10]. The antitumour activity of curcumin against androgen-independent prostate cancer cells *in vitro* and the possible mechanism of action have also been investigated. The results showed curcumin to effectively inhibit the proliferation of PC-3 cells *in vitro* with cell cycle arrest at the G2/M interphase. The percentage of apoptotic cells was shown to be significantly higher in curcumintreated groups than in control group and curcumin was shown to selectively inhibit the activities of NF-κB and AP-1 signalling pathways in PC-3 cells significantly [36].

levels of membrane β-catenin in prostate cancer cells. Modulation of these cellular events by curcumin is shown to correlate with decreased cell proliferation, colony formation, cell motility and enhanced cell-cell aggregation in prostate cancer cells. It has also been demonstrated that inhibition of cell motility is mediated by decreasing the levels of active cofilin, a downstream target of PKD1. The potent anti-cancer effect of curcumin *in vitro* in the study was shown to correlate well with those in prostate cancer xenograft mouse model, thus highlighting a novel molecular mechanism of curcumin action via the activation of PKD1 in prostate cancer cells [58]. The effect of curcumin in suppressing prostate cancer cell invasion, tumour growth, and metastasis has also been assessed. Curcumin was shown to be capable of suppressing epider‐ mal growth factor (EGF)-stimulated and heregulin-stimulated PC-3 cell invasion, as well as androgen-induced LNCaP cell invasion. Treatment of cells with curcumin was also shown to significantly result in reduced matrix metalloproteinase 9 activities and down-regulation of cellular matriptase, a membrane-anchored serine protease with oncogenic roles in tumour formation and invasion. It was also demonstrated that curcumin inhibits the induction effects of androgens and EGF on matriptase activation, as well as the reduction of the activated levels of matriptase after its overexpression. The reduction of activated matriptase in cells by curcumin was also observed to be partly due to its effect on promoting the shedding of matriptase into an extracellular environment without altering matriptase gene expression. In addition, curcumin was also shown to significantly suppress the invasive ability of prostate cancer cells induced by matriptase overexpression. The data from the study indicate that curcumin exhibits a suppressive effect on prostate cancer cell invasion, tumour growth, and metastasis, in part via the down-regulation of matriptase function [7]. In another study, the anticancer activity of curcumin and genistein combination in human prostate cancer (PC3) cell line with respect to their anti-angiogenic effect has been examined. The combination of both compounds was shown to decrease cell viability, induce apoptosis and cell cycle arrest at G0 phase in a dose- and time-dependent manner. In order to understand the anti-angiogenic effect of the combination, the authours determined the expression of ARNT and HIF-1α protein levels which were shown to significant decline when compared to the control group and their

The transcriptional activity of the androgen receptor (AR) is modulated by interaction with co-regulators, one of which is β-catenin. The effect of curcumin in inhibiting AR expression has also been elucidated through its role in mediating Wnt/β-catenin signalling pathway with regard to AR/β-catenin interactions. Curcumin induced a significant inhibition of AR expres‐ sion in a dose-dependent manner as well as its suppression of β-catenin in the nuclear and cytoplasmic extracts and whole cell lysates. Phosphorylation of Akt and glycogen synthase kinase-3β was shown to be attenuated, while phosphorylated β-catenin was increased after curcumin treatment. Cyclin D1 and c-myc, the target genes of the β-catenin/T-cell factor transcriptional complex, were also shown to be decreased; a finding that highlights the effect of curcumin in modulating the Wnt/β-catenin signalling pathway and may thus play a significant role in mediating inhibitory effects on LNCaP prostate cancer cells [10]. The antitumour activity of curcumin against androgen-independent prostate cancer cells *in vitro* and the possible mechanism of action have also been investigated. The results showed curcumin to effectively inhibit the proliferation of PC-3 cells *in vitro* with cell cycle arrest at the G2/M

respective monotherapy-treated groups [1].

278 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Figure 2.** Molecular targets of curcumin and/or its chemically-related analogues and possible mechanisms of action in various types of malignant growths.

The effect of six cyclohexanone analogues of curcumin (Figure 1) has also been investigated for their effects on growth and apoptosis, by evaluating the ability of these compounds to inhibit NF-κB activity in PC-3 human prostate cancer cells. Five out of the six curcumin analogues were shown to have stronger inhibitory effects compared to curcumin on the growth of these cells. They also showed stronger stimulatory effects on apoptosis in PC-3 cells than curcumin, with a more potent inhibition of NF-κB activity than curcumin which correlates well with effects on growth inhibition and apoptosis stimulation in PC-3 cells [62]. Furthermore, the therapeutic potential of a novel poly(lactic-co-glycolic acid)-curcumin nanoparticles (PLGA-Curcumin NPs) for prostate cancer treatment has also been assessed. Findings revealed PLGA-Curcumin NPs to efficiently internalise in prostate cancer cells and release biologically active curcumin in cytosolic compartment of cells for effective therapeutic activity. It was also shown that PLGA-Curcumin NPs can effectively inhibit the proliferation and colony formation ability of prostate cancer cells than free curcumin. PLGA-Curcumin NPs showed superior tumour regression compared to curcumin in xenograft mice. It was also revealed that PLGA-Curcumin NPs inhibit nuclear β-catenin and androgen receptor (AR) expression in cells and in tumour xenograft tissues. Furthermore, it was shown to suppress STAT3 and Akt phos‐ phorylation resulting in apoptosis via inhibition of key anti-apoptotic proteins, Mcl-1, Bcl-xL and caused induction of PARP cleavage [65].).

#### **7. Conclusion**

The limiting factor associated with curcumin is its poor solubility in water and likewise when soluble, it is extremely sensitive at physiological pH. However, the potential of curcumin, its derivatives and/or metabolites on cancer cells has been recognised and demonstrated in various cancer cells and its varying mechanisms of action elucidated depending on the tumour cell type. Coupled with this, the pleiotropic property of the curcumin molecule enables it to target the DNA, RNA and enzymes (proteins) within cells thereby eliciting sequential and/or simultaneous therapeutic effects. It is therefore pertinent to strive to refine the properties of curcumin through targeted delivery, tissue distribution and bioavailability in tumour cells in the presence of an adjuvant. These strategies can be achieved through the design and devel‐ opment of nanoparticles, self-assemblies, nanogels, liposome and other formulations that possess characteristics tailored according to specific requirements in order to efficiently harness therapeutic potential of curcumin in the treatment of a variety of malignant growths.

#### **Acknowledgements**

This work was made possible by grants from the Medical Research Council (South Africa) awarded to LJM, National Research Foundation, NRF (Thuthuka) awarded to MPM and the University of Limpopo.

#### **Author details**

Victor P. Bagla, Matlou P. Mokgotho and Leseilane J. Mampuru\*

\*Address all correspondence to: Leseilane.mampuru@ul.ac.za

Department of Biochemistry, Microbiology and Biotechnology, School of Molecular and Life Sciences, Faculty of Science and Agriculture, University of Limpopo, Sovenga, South Africa

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**7. Conclusion**

280 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Acknowledgements**

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**Author details**

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The limiting factor associated with curcumin is its poor solubility in water and likewise when soluble, it is extremely sensitive at physiological pH. However, the potential of curcumin, its derivatives and/or metabolites on cancer cells has been recognised and demonstrated in various cancer cells and its varying mechanisms of action elucidated depending on the tumour cell type. Coupled with this, the pleiotropic property of the curcumin molecule enables it to target the DNA, RNA and enzymes (proteins) within cells thereby eliciting sequential and/or simultaneous therapeutic effects. It is therefore pertinent to strive to refine the properties of curcumin through targeted delivery, tissue distribution and bioavailability in tumour cells in the presence of an adjuvant. These strategies can be achieved through the design and devel‐ opment of nanoparticles, self-assemblies, nanogels, liposome and other formulations that possess characteristics tailored according to specific requirements in order to efficiently harness therapeutic potential of curcumin in the treatment of a variety of malignant growths.

This work was made possible by grants from the Medical Research Council (South Africa) awarded to LJM, National Research Foundation, NRF (Thuthuka) awarded to MPM and the

Department of Biochemistry, Microbiology and Biotechnology, School of Molecular and Life Sciences, Faculty of Science and Agriculture, University of Limpopo, Sovenga, South Africa

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*apy* 10: 883–891.

187: 393–400.

22–30.

1189-1195.

279-284.

*nal of Natural Products* 76(3): 316–321.


### **Lead Compounds from Cucurbitaceae for the Treatment of Cancer**

Marcos Soto-Hernández, Jorge Cadena Iñiguez, Lourdes C. Arévalo-Galarza, Edelmiro Santiago-Osorio, Itzen Aguiñiga -Sánchez and Lucero del Mar Ruíz-Posadas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60048

### **1. Introduction**

#### **1.1. Phytochemicals in the cucurbitaceae**

The seeds of several species are a rich source of proteins, lipids, unsaturated fatty acids (table 1), phytosterols, vitamin E, and some minerals such as Mn, Zn, Cu, and carotenoids. The flesh of the fruits also contains some important compounds, such as flavonoids, that show antioxi‐ dant activity.

Other phytochemicals in the cucurbits are scarce, but polysaccharides is important to mention. These compounds, bound to proteins, are often considered as key active compounds in some species particularly with regards to diabetes. Alkaloids and saponins have been described in *Momordica* and *Sechium*, and also polyamines and galactolipids. In *Sechium*, the presence of proteins with ribosome-inhibiting activities has been described.


© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Data obtained from the US Department of Agriculture, Agricultural Research Service (2001) USDA Nutrient Database for Standard Reference, Release 14. Nutrient Data Laboratory Home (www.nat.usda.gov/fnic/foodcomp).

**Table 1.** Nutrient composition of Cucurbits in 100-g edible raw portion

#### **2. Cucurbitacins**

The cucurbitacins, as characteristic compounds of many species of the cucurbits, are tetracyclic triterpenes arising from a rearrangement of the protostane cation. They are unsaturated and polyfunctional oxygenated compounds and occur most often as glycosides. They are particu‐ larly toxic substances, the bitterness and cytotoxicity being the contributing factors for this toxicity [1].

They are divided into twelve groups, from cucurbitacin A to cucurbitacin T. The cucurbita‐ cin I, B, D, E, and L the most used *in vivo* and *in vitro* studies and differ by the acetyla‐ tion of groups OH or by the presence of double bond (Figure 1) that increase the lipophilic and toxic properties [2, 3].

**Figure 1.** The chemical structure of major cucurbitacins

**Crop Water (%) Calories**

290 Phytochemicals - Isolation, Characterisation and Role in Human Health

*Winter squash*

**(kcal)**

Acorn 80 40 0.8 0.1 10.4 1.5

*Summer squash* 94 20 1.2 0.2 404 1.9 Cucumber 96 13 0.7 0.1 2.8 0.8 *Watermelon* 92 32 0.6 0.4 7.2 0.5 *Bitter gourd (momordica)* 94 17 1.0 0.2 3.7 2.8 *Wax gourd (Benincasa)* 96 13 0.4 0.2 3.0 2.9

*Chayote (Sechium)* 94 19 0.8 0.1 4.5 1.7 *Pumpkin and squash, seeds* 7 541 24.5 45.9 17.8 3.9

for Standard Reference, Release 14. Nutrient Data Laboratory Home (www.nat.usda.gov/fnic/foodcomp).

Data obtained from the US Department of Agriculture, Agricultural Research Service (2001) USDA Nutrient Database

The cucurbitacins, as characteristic compounds of many species of the cucurbits, are tetracyclic triterpenes arising from a rearrangement of the protostane cation. They are unsaturated and polyfunctional oxygenated compounds and occur most often as glycosides. They are particu‐ larly toxic substances, the bitterness and cytotoxicity being the contributing factors for this

They are divided into twelve groups, from cucurbitacin A to cucurbitacin T. The cucurbita‐ cin I, B, D, E, and L the most used *in vivo* and *in vitro* studies and differ by the acetyla‐ tion of groups OH or by the presence of double bond (Figure 1) that increase the lipophilic

Butternut 86 45 1.0 0.1 11.7 Hubbard 88 40 2.0 0.5 8.7 Spaghetti 92 31 0.6 0.6 6.9

*Luffa gourd (Luffa)* 94 20 1.2 0.2 4.4 *Calabash gourd (Lagenaria)* 96 14 0.6 0.02 3.4

*Watermelon, seeds* 5 557 28.3 47.4 15.3

**Table 1.** Nutrient composition of Cucurbits in 100-g edible raw portion

**2. Cucurbitacins**

and toxic properties [2, 3].

toxicity [1].

**Protein (g) Fat (g) Carbohydrate (g) Fiber (g)**

#### **3. Physical properties and solubility of cucurbitacins**

Some of the cucurbitacins are crystalline solids, but others are gums or semisolids and their structure contribute to their scarce solubility in water and this factor is a challenge in pursuing their biological activity. There are some studies [4, 5, 6] that tried to search how to deliver the active principle without this problem, such as the use of polymeric micelles for nanoscale drug delivery. The amphiphilic block copolymers has the capacity to accommodate several types of molecules for drug delivery and the poly(ethylene oxide)-block-poly(ε-caprolactone) called PEO-b-PCL, which is a biocompatible copolymer successfully used for the solubilization of compounds of poor solubility in water, is suitable for solubilization and controlled delivery of cucurbitacin I and B [6].

### **4. Bioactivity of cucurbitacins**

Besides their cytotoxic and anticancer activity, they also show other pharmacological effects *in vivo* as in *in vitro*, for example, as purgative, anti-inflammatory, cough, flu, fever, or antifertility. They also show other functions as plant growth regulators and antifeeding agents in insects. A combination of cucurbitacins B + E glucosides showed a notorious antioxidant activity and it is believed that their beneficial properties are due to their ability to interact with reactive oxygen species [10]. It is know that nitric oxide has various functions, but its uncon‐ trolled production can be toxic in many pathological conditions, such as the inflammatory tissue damaged, and in this connection some members of the *Cucurbitaceae* family have been tested as potential inhibitory agents for nitric oxide production [11]. From *Hemsleya pengxia‐ nensis*, Dihydrocurbitacin F-25-O-acetate was isolated as one of the main components and it was shown that this compound plays a main role as an anti-infection agent [12].

It was shown that at the molecular level, the cucurbitacins have a role in the inhibition of the JAK/STAT3 pathway that is a major contributor in oncogenesis and five cucurbitacins were tested, A, B, E, I, and Q, and found that the last one inhibit the activation of STAT3 but not JAK2, instead cucurbitacin A inhibit JAK2 but not STAT3 and the cucurbitacins B,E, and I inhibit the activation of both. Furthermore, cucurbitacin Q but not A induces apoptosis and inhibits human tumor growth in mice [13]. To validate these interesting results, another model was described in the Sézay syndrome(Sz), which is an aggressive lymphoma/leukemia of the skin, that used cucurbitacin I to inhibit the JAK/STA3 pathway [16] and it was demonstrated that inhibition of STAT3 using cucurbitacin I induced apoptosis of the cells and it was mentioned that this inhibitor of STAT3 is a potent therapeutic agent for Sz.

Cucurbitacin B and R isolated from *Cayaponia tayuya* induced dramatic changes in the cytos‐ keleton, inhibiting proliferation and induced apoptosis of isogenic colon cancer cell lines HCT116 and Hke-3 [14]. The cucurbitacin glucosides extracted from *Citrillus colocynthis* leaves effects on human breast cancer cell growth and it is suggested that this kind of glucosides exhibit pleitropic effects on cells causing both cell cycle arrest and apoptosis [13].

Also, the cucurbitacin E has demonstrated some interesting results because it has emerged in one empirical screening strategy to define agents with potent growth inhibitory activity. It was demonstrated that one early effect of this cucurbitacin is the rearrangement of the actin and the vimentin cytoskeleton. The growth inhibitory actions of a series of cucurbitacins correlate with these effects and that the actin and vermectin cytoskeleton are potential targets for this kind of compounds [17].

In another model with cucurbitacin B isolated from *Trichosanthes cucurmerina*, it was demon‐ strated that this compound has a cytotoxic effect on breast cancer cell lines SKBR-3 and MCF-7 and that growth inhibition was attributed to G2/M phase arrest and apoptosis and also that the cucurbitacin B mediates its effect by inhibiting the translocation of β-catenin and galectin-3 proteins to the nucleus rather than by disrupting the β-catenin/galectin-3/TCF-4 complex formation. This means that cucurbitacin B targeting of the Wnt signaling pathway could be an innovative approach for breast cancer treatment [19].

It was observed that when an antitumor agent, such as doxorubicin (DOX), is combined with some natural products such as capsaicin derivatives, gingerol, ferulic acid, or cucurbitacin E, it has a notorious effect on tumor cells. The first ones did not change DOX permeability in the tumor cells but instead cucurbitacin E significantly promoted DOX influx into the tumour cells and maintained its levels in the tumour cells [18].

In another model, the induction of cancer cell-specific apoptosis via activation of TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) signaling has become an important focus of cancer research and in this sense it was found that cucurbitacins B and D were among the sensitizers of cancer cells to TRAIL-mediated apoptosis in a high-throughput screen [32]. It was found that sensitization by these cucurbitacins is rapid and persistent, making them potentially useful reagents for developing increased understanding of the sequence of molecular events that can lead to TRAIL sensitization and subsequent apoptosis.

The model of proteasome is another approach to understand the molecular mechanism of chemotherapeutic agents. It was found that the proteasome is an abundant catalytic complex present in both nucleus and cytoplasm of eukaryotic cells. Proteasome-mediated degradation plays an essential role in the regulation of most intracellular proteins such as NF-κB and recently proteasome inhibitors have been used as a new anticancer therapy. In this sense it was found that cucurbitacin D induced apoptosis through suppression of proteasome activity both in vivo and in vitro, making this compound a promising candidate for clinical applications in the treatment of T-cell leukemia [20].

#### **5. Response of cancer cells to cucurbitacin exposure**

**4. Bioactivity of cucurbitacins**

292 Phytochemicals - Isolation, Characterisation and Role in Human Health

kind of compounds [17].

innovative approach for breast cancer treatment [19].

Besides their cytotoxic and anticancer activity, they also show other pharmacological effects *in vivo* as in *in vitro*, for example, as purgative, anti-inflammatory, cough, flu, fever, or antifertility. They also show other functions as plant growth regulators and antifeeding agents in insects. A combination of cucurbitacins B + E glucosides showed a notorious antioxidant activity and it is believed that their beneficial properties are due to their ability to interact with reactive oxygen species [10]. It is know that nitric oxide has various functions, but its uncon‐ trolled production can be toxic in many pathological conditions, such as the inflammatory tissue damaged, and in this connection some members of the *Cucurbitaceae* family have been tested as potential inhibitory agents for nitric oxide production [11]. From *Hemsleya pengxia‐ nensis*, Dihydrocurbitacin F-25-O-acetate was isolated as one of the main components and it

It was shown that at the molecular level, the cucurbitacins have a role in the inhibition of the JAK/STAT3 pathway that is a major contributor in oncogenesis and five cucurbitacins were tested, A, B, E, I, and Q, and found that the last one inhibit the activation of STAT3 but not JAK2, instead cucurbitacin A inhibit JAK2 but not STAT3 and the cucurbitacins B,E, and I inhibit the activation of both. Furthermore, cucurbitacin Q but not A induces apoptosis and inhibits human tumor growth in mice [13]. To validate these interesting results, another model was described in the Sézay syndrome(Sz), which is an aggressive lymphoma/leukemia of the skin, that used cucurbitacin I to inhibit the JAK/STA3 pathway [16] and it was demonstrated that inhibition of STAT3 using cucurbitacin I induced apoptosis of the cells and it was

Cucurbitacin B and R isolated from *Cayaponia tayuya* induced dramatic changes in the cytos‐ keleton, inhibiting proliferation and induced apoptosis of isogenic colon cancer cell lines HCT116 and Hke-3 [14]. The cucurbitacin glucosides extracted from *Citrillus colocynthis* leaves effects on human breast cancer cell growth and it is suggested that this kind of glucosides

Also, the cucurbitacin E has demonstrated some interesting results because it has emerged in one empirical screening strategy to define agents with potent growth inhibitory activity. It was demonstrated that one early effect of this cucurbitacin is the rearrangement of the actin and the vimentin cytoskeleton. The growth inhibitory actions of a series of cucurbitacins correlate with these effects and that the actin and vermectin cytoskeleton are potential targets for this

In another model with cucurbitacin B isolated from *Trichosanthes cucurmerina*, it was demon‐ strated that this compound has a cytotoxic effect on breast cancer cell lines SKBR-3 and MCF-7 and that growth inhibition was attributed to G2/M phase arrest and apoptosis and also that the cucurbitacin B mediates its effect by inhibiting the translocation of β-catenin and galectin-3 proteins to the nucleus rather than by disrupting the β-catenin/galectin-3/TCF-4 complex formation. This means that cucurbitacin B targeting of the Wnt signaling pathway could be an

was shown that this compound plays a main role as an anti-infection agent [12].

mentioned that this inhibitor of STAT3 is a potent therapeutic agent for Sz.

exhibit pleitropic effects on cells causing both cell cycle arrest and apoptosis [13].

Like other plant-derived substances, cucurbitacins induce toxicity in different cancer cell lines with several morphological and physiological changes (Table 2). Drastic changes in cell shape, such as rounding, swelling, pinocytic blebbing, submembranous inclusions, and blisters, are observed within a couple of hours. Some of the morphological changes could be explained by the dysregulation of cytoskeleton homeostasis by cucurbitacins. Duncan et al. reported a dramatic increase in F-actin to G-actin ratio and abnormal reorganization of the vimentin network by cucurbitacin E in human prostate cancer cell lines [17, 28]. Studies with cucurbi‐ tacin B also showed the aggregation of F-actin in various human cancer cell lines [29, 30, 31], implying the disruption of the dissociation process of F-actin by an unknown mechanism. However, unlike vinca alkaloids and taxanes, there is no clear evidence that cucurbitacin affects the microtubule network.

Multinucleation is another common morphological change that was consistently reported in human cancer cell cultures exposed to cucurbitacin for more than 24 h. According to Duncan et al., multinucleation implies that cucurbitacin blocks cytokinesis, but not karyokinesis [17]. This is in conjunction with the observation that actin (which is involved in cytokinesis) is disrupted, whereas microtubules (which are involved in karyonesis) are not.

Multinucleation can result from the disruption of the cell cycle. Many reports showed that cucurbitacins induced cell cycle arrest, mostly in the G2/M phase [31, 32, 33, 39], but S-phase

arrest in HL-60 and U937 human leukaemia cell lines was also reported [11]. G2/M arrest happens in the early period of cucurbitacin exposure and results in apoptotic death of the tumor cells [39]. Tannin-Spitz et al. showed that G2/M arrest occurred in breast cancer cell lines (MCF-7 and MDA-MB-231) exposed to cucurbitacin B/E glucosides by the inhibition of cyclindependent kinase (cdk) p34 CDC2 and cyclin B1, both in expression level and activation status [33]. G2/M has shown arrest by upregulation of cdk inhibitor p21WAFI, and by downregulation of cyclin A and cyclin E in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2) exposed to cucurbitacin B [32].

#### **6. Cucurbitacins and their molecular mechanism of action**

By what molecular mechanism do cucurbitacins achieve cell cycle arrest, apoptosis, and growth suppression of cancer cells? There are several oncogenic signaling pathways that are commonly involved in cancer cell proliferation and survival. The JAK-STAT pathway, the Akt-PKB pathway, and the MAPK pathway are important in cancer cells and are also targets of the cucurbitacin family.

The JAK-STAT pathway induces Janus-kinases (JACKs) and signal transducers and activators of transcription (STATs), and regulates cytokine and growth factor signals (Figure 2). In many cancer cells, constitutive activation of STAT3 and STAT5 has been known to play important roles in tumorigenesis [34]. After the initial finding by Blaskovich et al. that cucurbitacin I (JSI-124) is a dual inhibitor of STAT3 and JACK [16], many studies confirmed that cucurbitacin I is a powerful JAK-STAT inhibitor by blocking the tyrosine phosphorylation of STAT3 and JAK2 in various human cancers [33, 35, 36, 37, 38, 39]. However, cucurbitacin I did not affect other oncogenic signaling pathways, such as the Akt-PKB or MAPK/ERK pathways [35].

Furthermore, it has been discovered that cucurbitacin B inhibits the tyrosine phosphorylation of STAT3, STAT5, and JAK2 in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2) *in vitro* and in Panc-1 xenografts *in vivo* [32]. Inhibition of the JACK-STAT pathway affected various downstream targets involved in progrowth signaling (e.g., c-myc, cyclins, surviving) and apoptosis (e.g., p53, Bcl-xL, Bcl-2) [34, 36]. Therefore G2/M arrest and apoptosis in pancreatic cancer cells exposed to cucurbitacin B could be explained as a result of inhibition of the JAK-STAT pathway and was confirmed by the downregulation of p21WAFI, cyclin A, and cyclin E, and upregulation of Bcl-xL. Like cucurbitacin I, cucurbitacin B did not inhibit other progrowth signaling pathways, such as the Akt/PKB and MAPK/ERK pathways in pancreatic cancer cells [32]. Interestingly, Sun et al. found that cucurbitacin A, which only differs with cucurbitacin B by its C-11 hydroxyl group, lost its activity as a STAT3 inhibitor while maintaining its activity as a JACK2 inhibitor [36], showing that the inhibition of STAT3 and JAK2 may follow different molecular mechanisms.

The anti-cancer mechanism of cucurbitacin in breast cancer cells is still not clear. Tannin-Spitz et al. exposed breast cancer cell lines (MCF-7 and MDA-MB-231) to cucurbitacin B/E glucosides and found that cucurbitacins increased the tyrosine phosphorylation of STAT3, unlike other cancers [33]. Considering the activated JAK-STAT pathway in breast cancer [34], this result was contradictory. The authors hypothesized that concurrent inactivation of PBK in a cell typespecific manner might explain this unique regulation, which requires further research.

arrest in HL-60 and U937 human leukaemia cell lines was also reported [11]. G2/M arrest happens in the early period of cucurbitacin exposure and results in apoptotic death of the tumor cells [39]. Tannin-Spitz et al. showed that G2/M arrest occurred in breast cancer cell lines (MCF-7 and MDA-MB-231) exposed to cucurbitacin B/E glucosides by the inhibition of cyclindependent kinase (cdk) p34 CDC2 and cyclin B1, both in expression level and activation status [33]. G2/M has shown arrest by upregulation of cdk inhibitor p21WAFI, and by downregulation of cyclin A and cyclin E in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2) exposed to

By what molecular mechanism do cucurbitacins achieve cell cycle arrest, apoptosis, and growth suppression of cancer cells? There are several oncogenic signaling pathways that are commonly involved in cancer cell proliferation and survival. The JAK-STAT pathway, the Akt-PKB pathway, and the MAPK pathway are important in cancer cells and are also targets of the

The JAK-STAT pathway induces Janus-kinases (JACKs) and signal transducers and activators of transcription (STATs), and regulates cytokine and growth factor signals (Figure 2). In many cancer cells, constitutive activation of STAT3 and STAT5 has been known to play important roles in tumorigenesis [34]. After the initial finding by Blaskovich et al. that cucurbitacin I (JSI-124) is a dual inhibitor of STAT3 and JACK [16], many studies confirmed that cucurbitacin I is a powerful JAK-STAT inhibitor by blocking the tyrosine phosphorylation of STAT3 and JAK2 in various human cancers [33, 35, 36, 37, 38, 39]. However, cucurbitacin I did not affect other oncogenic signaling pathways, such as the Akt-PKB or MAPK/ERK pathways [35].

Furthermore, it has been discovered that cucurbitacin B inhibits the tyrosine phosphorylation of STAT3, STAT5, and JAK2 in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2) *in vitro* and in Panc-1 xenografts *in vivo* [32]. Inhibition of the JACK-STAT pathway affected various downstream targets involved in progrowth signaling (e.g., c-myc, cyclins, surviving) and apoptosis (e.g., p53, Bcl-xL, Bcl-2) [34, 36]. Therefore G2/M arrest and apoptosis in pancreatic cancer cells exposed to cucurbitacin B could be explained as a result of inhibition of the JAK-STAT pathway and was confirmed by the downregulation of p21WAFI, cyclin A, and cyclin E, and upregulation of Bcl-xL. Like cucurbitacin I, cucurbitacin B did not inhibit other progrowth signaling pathways, such as the Akt/PKB and MAPK/ERK pathways in pancreatic cancer cells [32]. Interestingly, Sun et al. found that cucurbitacin A, which only differs with cucurbitacin B by its C-11 hydroxyl group, lost its activity as a STAT3 inhibitor while maintaining its activity as a JACK2 inhibitor [36], showing that the inhibition of STAT3 and JAK2 may follow different

The anti-cancer mechanism of cucurbitacin in breast cancer cells is still not clear. Tannin-Spitz et al. exposed breast cancer cell lines (MCF-7 and MDA-MB-231) to cucurbitacin B/E glucosides and found that cucurbitacins increased the tyrosine phosphorylation of STAT3, unlike other cancers [33]. Considering the activated JAK-STAT pathway in breast cancer [34], this result

**6. Cucurbitacins and their molecular mechanism of action**

294 Phytochemicals - Isolation, Characterisation and Role in Human Health

cucurbitacin B [32].

cucurbitacin family.

molecular mechanisms.

Considering the important role of STAT3 during inflammation [40], it is not surprising that part of the anti-cancer activity of cucurbitacins is linked to their anti-inflammatory activity. Chronic inflammation can make individuals predisposed to many types of cancer [40]. This seems to affect both cancer cells and normal macrophages through different mechanisms. In cancer cells, cucurbitacins work as STAT3 inhibitors, and make cells more susceptible to the attack of reactive oxygen species (ROS) and free radicals during inflammation [43]. In normal macrophages, however, cucurbitacins work as inhibitors of the IKK/NF-κB pathway rather than inhibitors of STAT3 [42, 43]. Inhibition of the IKK/NF-kB pathway by cucurbitacins results in the inhibition of key inflammatory enzymes, such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), whose overproduction contributes to tumorigenesis [41, 42, 43]. However, it is still not clear how cucurbitacins can selectively choose their target pathway depending on cell type.

Interestingly, when Escandell et al. used two human colon cancer cell lines without activated STAT3 (HCT116 and Hke-3), 23,24-dihydrocucurbitacin B and cucurbitacin R still suppressed tumour growth at a significant level [44]. Furthermore, the presence of active kRas in HCT116 cells showed more protection from apoptosis than Hke-3 cells, which do not have active kRas [25]. Since kRas is upstream of ERK, the result implies the effect of cucurbitacins on the MAPK pathway.

Indeed, the MAPK signaling cascade was another target that cucurbitacins acted upon. It has been shown that cucurbitacin B affects the MAPK pathway in glioblastoma (GBM) multiforme cells *in vitro* [31]. Cucurbitacin B-treated GBM cells showed an increased level of phosphory‐ lated p38, phosphorylated JNK,and phosphorylated c-Jun, as well as a decreased level of phosphorylated ERK at the same time. Upregulation of JNK may induce apoptosis in GBM cells, and downregulation of p38 and ERK may block cytokine signaling. Chan et al. also showed that both phosphotyrosine-STAT3 and phospho-ERK were suppressed in the K562 leukemia cell line [45]. Increased JNK phosphorylation by cucurbitacin D in the Hep3B human hepatocellular carcinoma cell line was also reported [46].

The Akt-PKB pathway mediates signals from receptor tyrosine kinases (RTHKs) and integrins. Currently, no cucurbitacins are known to inhibit the phosphorylation of Akt. Although Tannin-Spintz et al. showed the downregulation of PKB in breast cancer cell lines [33], further research is required to confirm the effect of cucurbitacin on the Akt-PKB pathway.

#### **7. Synergistic effect of cucurbitacins with chemotherapeutic agents**

Despite its excellent anti-cancer activity, clinical use of cucurbitacin has challenges to over‐ come, such as low therapeutic index and nonspecific toxicity. One of the solutions to these problems would be the use of cucurbitacins in combination, not only to enhance the efficacy of the treatment, but also to avoid the build-up of resistance in cancer cells. Moreover, some drug combinations show strong synergism that helps to achieve the same therapeutic effect with a lower dose, and hence less toxicity. Encouragingly, some reports have shown that cucurbitacins show a synergistic effect with chemotherapeutic agents that are already estab‐ lished in the treatment of human cancers.

Saduka et al. showed that cucurbitacin E promotes cellular accumulation of doxorubicin, both by facilitating influx to and by preventing efflux from the tumour cells, implying synergistic effects of the two drugs [47]. Another study by Ramalhete et al. using cucurbitacin derivatives from *Momordica balsamica* confirmed statically the synergistic effect of some cucurbitacin derivatives and doxorubicin using the fractional inhibitory concentration index (FIX) [48]. Recently, the synergistic effect of cucurbitacin B with gemcitabine in pancreatic cancer was discovered [32]. Using an isobologram and combination index (CI) method, it was shown that in a certain concentration range, cucurbitacin B and gemcitabine showed a CI value less than 0.9, which showed synergism between two drugs *in vitro*.

Strikingly, cucurbitacin B induced no apparent toxicity *in vivo* [32]. As a single agent at high dose (1.0 mg/kg), cucurbitacin B induced a slight body weight loss after the first treatment, but no further toxicity was observed during the seven weeks of treatment. Any signs of toxicity after treatment were searched for using various immunohistochemistry stainings on major organs such as the liver, spleen, and kidney, as well as a blood and serum chemistry test. However, no signs of toxicity by cucurbitacin B were found. Considering the high dose of cucurbitacin B near LD50 value (1.1 mg/kg) [26], the result was remarkable.

As Raikhlin-Eisenkraft et al. pointed out, many factors can affect the toxicity of cucurbitacins [25]. The bioreactivity of a compound can vary greatly depending on the presence of other compounds and the microenvironment *in vivo*. The discrepancy between the results and previous case reports can be partly explained by the extent of purity of cucurbitacins. Rapid advances in purification technology over the decades may have eliminated the other impurities coeluted with cucurbitacins from the plant source, which may be the true cause of toxicity in the past. Changes in the type of solvents for elution and dilution can also play a role. Defective immune system in athymic nude mice can be another possibility. For this reason, it was suggested that cucurbitacin toxicity in humans needs to be re-studied and should not be the reason to rule it out as a potential anti-cancer drug.



drug combinations show strong synergism that helps to achieve the same therapeutic effect with a lower dose, and hence less toxicity. Encouragingly, some reports have shown that cucurbitacins show a synergistic effect with chemotherapeutic agents that are already estab‐

Saduka et al. showed that cucurbitacin E promotes cellular accumulation of doxorubicin, both by facilitating influx to and by preventing efflux from the tumour cells, implying synergistic effects of the two drugs [47]. Another study by Ramalhete et al. using cucurbitacin derivatives from *Momordica balsamica* confirmed statically the synergistic effect of some cucurbitacin derivatives and doxorubicin using the fractional inhibitory concentration index (FIX) [48]. Recently, the synergistic effect of cucurbitacin B with gemcitabine in pancreatic cancer was discovered [32]. Using an isobologram and combination index (CI) method, it was shown that in a certain concentration range, cucurbitacin B and gemcitabine showed a CI value less than

Strikingly, cucurbitacin B induced no apparent toxicity *in vivo* [32]. As a single agent at high dose (1.0 mg/kg), cucurbitacin B induced a slight body weight loss after the first treatment, but no further toxicity was observed during the seven weeks of treatment. Any signs of toxicity after treatment were searched for using various immunohistochemistry stainings on major organs such as the liver, spleen, and kidney, as well as a blood and serum chemistry test. However, no signs of toxicity by cucurbitacin B were found. Considering the high dose of

As Raikhlin-Eisenkraft et al. pointed out, many factors can affect the toxicity of cucurbitacins [25]. The bioreactivity of a compound can vary greatly depending on the presence of other compounds and the microenvironment *in vivo*. The discrepancy between the results and previous case reports can be partly explained by the extent of purity of cucurbitacins. Rapid advances in purification technology over the decades may have eliminated the other impurities coeluted with cucurbitacins from the plant source, which may be the true cause of toxicity in the past. Changes in the type of solvents for elution and dilution can also play a role. Defective immune system in athymic nude mice can be another possibility. For this reason, it was suggested that cucurbitacin toxicity in humans needs to be re-studied and should not be the

Lung: A549 cell lines

Leukemia and lymphoma: HL60, U937, THP1, NB4, K562, BALL1, Reh, RCH, LY4, Daudi, D901, SP49, Jeko1 and NCEB1. Hepatocellular: Hep-2. Breast: SKBR2, MCF-7, T47D and MDA-B435. Lung: A549, SK

cucurbitacin B near LD50 value (1.1 mg/kg) [26], the result was remarkable.

**Cucurbitacin Plant Source Effectiveness on cancer cell lines**

lished in the treatment of human cancers.

296 Phytochemicals - Isolation, Characterisation and Role in Human Health

0.9, which showed synergism between two drugs *in vitro*.

reason to rule it out as a potential anti-cancer drug.

Cucurbitacin A *Trichosanthes cucumerina* (snake gourd)

(snakegourd)

*Wibrandia ebracteata.*

squash).

*Cucurbita andreana* (buttercup

Cucurbitacin B *Trichosanthes cucumerina*

**Table 2.** Cucurbitacin compounds from different plant species and their bioactivity on cancer cells.

**Figure 2.** Mechanism of activation of Janus kinases and signal transducer and activator transcription (JAK/STAT) path‐ way. Upon activator cytokine binding to its receptor on cell surface (e.g.,IL-6 receptor), JAK/STAT pathway is activat‐ ed (left) leading to sequential cell response. Inhibition signalling process by cucurbitacins is indicate on JAK or STAT STAT signalling.

#### **8. Conclusions**

This review has highlighted the interest or importance of some phytochemicals present in members of the *Cucurbitaceae* family, a group of plants used for a long time for many purposes such as ornamental, food, and medicinal. It is in this last area where their phytochemicals constituents have been pointed out as responsible for their medicinal properties. Flavonoids, carotenoids, sterols, oils, vitamins, and minerals are some of these phytochemicals, but is the terpenoid fraction known as cucurbitacins that has received special attention. This is because such compound has been associated as a potential cytotoxic compounds. More than ten years ago, a large number of cucurbitacins have been isolated and the use or applications for the treatment of different types of cancer was described. The review also described details about the mechanisms of how these phytochemicals act at the cellular level, but it seems that they are not specifically bound protein targets forming thioethers bonds through a Michael-type addition. This would allow these compounds to be conjugated with a broad array of potential proteins targets, many of which would be inhibited or disrupted as a consequence. As a result, their value as chemical biology probes is still limited and must be confirmed by independent means. The different findings of the reports reviewed also indicated that the binding mode of cucurbitacins to protein targets means that optimization for selectivity would be unlikely to work, which would make it very difficult to reduce toxicities or improve the therapeutic window for future clinical applications. However, alternative ways that their potential therapeutic utility could be improved in the future is through targeted delivery to tumour cells, for example, through antibody-conjugation or incorporation in nanoparticles.

#### **Author details**

Marcos Soto-Hernández1\*, Jorge Cadena Iñiguez2 , Lourdes C. Arévalo-Galarza3 , Edelmiro Santiago-Osorio4 , Itzen Aguiñiga -Sánchez4 and Lucero del Mar Ruíz-Posadas1

\*Address all correspondence to: msoto@colpos.mx

1 Colegio de Postgraduados Campus Montecillo, Botany Department Texcoco, Estado de México, México

2 Colegio de Postgraduados Campus San Luis Potosí SLP, México

3 Colegio de Postgraduados Campus Montecillo, Postharvest Physiology Department, Tex‐ coco, Edo. de México, México

4 Facultad de Estudios Superiores Zaragoza, National Autonomos University of Mexico, Mexico City, Mexico

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**Figure 2.** Mechanism of activation of Janus kinases and signal transducer and activator transcription (JAK/STAT) path‐ way. Upon activator cytokine binding to its receptor on cell surface (e.g.,IL-6 receptor), JAK/STAT pathway is activat‐ ed (left) leading to sequential cell response. Inhibition signalling process by cucurbitacins is indicate on JAK or STAT

This review has highlighted the interest or importance of some phytochemicals present in members of the *Cucurbitaceae* family, a group of plants used for a long time for many purposes such as ornamental, food, and medicinal. It is in this last area where their phytochemicals constituents have been pointed out as responsible for their medicinal properties. Flavonoids, carotenoids, sterols, oils, vitamins, and minerals are some of these phytochemicals, but is the terpenoid fraction known as cucurbitacins that has received special attention. This is because such compound has been associated as a potential cytotoxic compounds. More than ten years ago, a large number of cucurbitacins have been isolated and the use or applications for the treatment of different types of cancer was described. The review also described details about the mechanisms of how these phytochemicals act at the cellular level, but it seems that they are not specifically bound protein targets forming thioethers bonds through a Michael-type addition. This would allow these compounds to be conjugated with a broad array of potential proteins targets, many of which would be inhibited or disrupted as a consequence. As a result, their value as chemical biology probes is still limited and must be confirmed by independent means. The different findings of the reports reviewed also indicated that the binding mode of

STAT signalling.

**8. Conclusions**

298 Phytochemicals - Isolation, Characterisation and Role in Human Health


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### **Green Tea Catechins for Prostate Cancer Chemoprevention**

Ganna Chornokur and Nagi B. Kumar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59872

#### **1. Introduction**

#### **1.1. Prostate cancer: Opportunity for prevention**

Prostate cancer (PCa) is the most commonly diagnosed cancer and second most common cause of cancer deaths in American men. The American Cancer Society estimates that there will be 233,000 new cases of prostate cancer in the United States (US) in 2014, and 29,480 men will die from this disease. [1] The initiation and progression of PCa involves a complex series of events. During progression, genetic changes and loss of cellular control are observed as cell pheno‐ types change from normal to dysplasia (prostatic intraepithelial neoplasia or PIN), severe dysplasia (high-grade PIN or HGPIN), atypical small acinar proliferation (ASAP) or focal glandular prostatic atypia (FGA), [2, 3] clinically localized and finally to metastatic disease. [2, 4 - 7] The frequency of HGPIN as well as latent PCa is evenly distributed, suggesting that external factors such as diet, physical activity, and other lifestyle factors are important in the transformation from these early stages into more aggressive, clinical cancer. [2, 4 - 9] Although early screening and detection has been used historically as strategies for PCa prevention, these recommendations have been a subject of much debate in recent years. Although screening using serum prostate-specific antigen (PSA) has not been shown to significantly reduce either PCa-specific or overall mortality, it has been linked to the substantial overtreatment of clinically insignificant, potentially indolent tumors. [10 - 12] With questions about value of prostate-specific antigen (PSA)-based screening, [10 - 12] few prevention options are available for asymptomatic men who are at high risk for PCa. To address the urgent need for alternative preventive strategies, hormonal agents including the 5-alpha-reductase inhibitors finasteride and dutasteride, which block the conversion of testosterone to dihydrotestosterone, have been evaluated for PCA chemoprevention. [13 - 15] Although a reduction in PCa incidence of 23%–

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

25% was observed with these agents, [13, 15] concerns about higher incidence of aggressive cancers and toxicities have limited their clinical adoption, establishing the need to identify alternative chemoprevention agents [14] with a more favorable safety profile. These features of prostate cancer, namely, high prevalence, long latency, significant mortality and morbidity, availability of HGPIN and ASAP as intermediate predictive stages of progression, and urgent need for alternative strategies for prevention other than screening, provide the most promise and best opportunity for evaluating agents for chemoprevention.

#### **2. Cancer chemoprevention**

Chemoprevention refers to the inhibition of preinvasive and invasive cancer and its progres‐ sion or treatments of identifiable precancers. [16, 17] Successful chemopreventive interven‐ tions require a thorough evidence-based understanding of the mechanism and hallmarks of carcinogenesis. Novel technologies, methods, and approaches in genomics, metabolomics, and proteomics have enabled this field of research. In order for chemopreventive efforts to be successful, it is imperative to employ these innovative methods and approaches to develop pharmacologic agents (including botanicals/biologicals) to reverse or halt the process of carcinogenesis. Chemopreventive agents include antipromotion and antiprogression com‐ pounds that could prevent or stop the survival, growth, and dissemination/metastasis of cells that are already committed to become malignant. [16, 17]

#### **3. Botanicals as agents for cancer chemoprevention**

Although several targeted "smart" drugs have emerged over the past decade, it is clear that diseases like cancer have an etiology based on perturbations of multiple signaling pathways. Thus, targeting multiple pathways may represent a more effective approach to cancer control. [18 - 20] In addition, the monotargeted "smart" drugs are associated with high cost and produce numerous side effects. These drawbacks of monotargeted drugs underscore the importance for the development of multitargeted, innocuous, inexpensive, and readily available botanicals for the prevention of cancer. [20] Botanicals have been shown to influence multiple biochemical and molecular cascades that inhibit mutagenesis and proliferation, induce apoptosis, and suppress the formation and growth of human cancers, modulating several hallmarks of carcinogenesis, with a significantly superior safety profile than most agents evaluated to date. Multiple botanicals have been identified and appear promising for PCa chemoprevention. [31]

The objective of this chapter is to review and summarize the most current literature focusing on GTC for prostate cancer chemoprevention based on evidence from laboratory, *in vitro*, and *in vivo* studies and clinical chemoprevention trials.

#### **4. Green tea catechins and prostate cancer chemoprevention**

25% was observed with these agents, [13, 15] concerns about higher incidence of aggressive cancers and toxicities have limited their clinical adoption, establishing the need to identify alternative chemoprevention agents [14] with a more favorable safety profile. These features of prostate cancer, namely, high prevalence, long latency, significant mortality and morbidity, availability of HGPIN and ASAP as intermediate predictive stages of progression, and urgent need for alternative strategies for prevention other than screening, provide the most promise

Chemoprevention refers to the inhibition of preinvasive and invasive cancer and its progres‐ sion or treatments of identifiable precancers. [16, 17] Successful chemopreventive interven‐ tions require a thorough evidence-based understanding of the mechanism and hallmarks of carcinogenesis. Novel technologies, methods, and approaches in genomics, metabolomics, and proteomics have enabled this field of research. In order for chemopreventive efforts to be successful, it is imperative to employ these innovative methods and approaches to develop pharmacologic agents (including botanicals/biologicals) to reverse or halt the process of carcinogenesis. Chemopreventive agents include antipromotion and antiprogression com‐ pounds that could prevent or stop the survival, growth, and dissemination/metastasis of cells

Although several targeted "smart" drugs have emerged over the past decade, it is clear that diseases like cancer have an etiology based on perturbations of multiple signaling pathways. Thus, targeting multiple pathways may represent a more effective approach to cancer control. [18 - 20] In addition, the monotargeted "smart" drugs are associated with high cost and produce numerous side effects. These drawbacks of monotargeted drugs underscore the importance for the development of multitargeted, innocuous, inexpensive, and readily available botanicals for the prevention of cancer. [20] Botanicals have been shown to influence multiple biochemical and molecular cascades that inhibit mutagenesis and proliferation, induce apoptosis, and suppress the formation and growth of human cancers, modulating several hallmarks of carcinogenesis, with a significantly superior safety profile than most agents evaluated to date. Multiple botanicals have been identified and appear promising for

The objective of this chapter is to review and summarize the most current literature focusing on GTC for prostate cancer chemoprevention based on evidence from laboratory, *in vitro*, and

and best opportunity for evaluating agents for chemoprevention.

306 Phytochemicals - Isolation, Characterisation and Role in Human Health

that are already committed to become malignant. [16, 17]

**3. Botanicals as agents for cancer chemoprevention**

**2. Cancer chemoprevention**

PCa chemoprevention. [31]

*in vivo* studies and clinical chemoprevention trials.

#### **4.1. Epidemiological evidence of the role of GTC and prostate cancer prevention**

Several reviews summarizing epidemiological evidence have suggested a protective effect of tea consumption against human cancers of the breast, cervix, colon and rectum, gallbladder, liver, lung, nasopharynx, pancreas, prostate, stomach, ovary, and uterus. [32 - 35] Twenty percent of green tea is consumed in Asian countries such as China, Japan, and Korea, where populations drinking green tea consistently demonstrate lower PCa risks. Epidemiological studies, however, have been mixed, potentially due to confounding factors such as variations in tea consumption(salted, hot), dietary, tobacco and alcohol use, genetic differences, and recall bias. [36, 50] The risk for PCa increases in Asians who migrate to the US if original dietary habits are abandoned. There is epidemiological evidence that Asian men consuming green tea have lower PCa risk, potentially due to exposure over their lifetime that prevents transforma‐ tion or progression to later stages of prostate carcinogenesis. The conflicting epidemiological results have been attributed to confounding factors that include consumption of salted or very hot tea, geographical location, use of tobacco and alcohol, other dietary differences, and lack of standardization of quantities and compositions of the tea products consumed. In a case– control study conducted in China during 2001–2002, prostate cancer risk declined with increasing frequency, duration, and quantity of green tea consumption [OR = 0.28 (95% CI = 0.17−0.47)], showing a dose–response relationship. [39] Four to five times more men die of CaP in the US than that in Japan. [36] It also appears that the onset of CaP occurs later in life and/or CaPs grow more slowly in Japanese populations compared to Western populations, which may be attributed to the consumption of GTC and soy products. Thus, the potential preventive properties of GTP in prostate cancer, as demonstrated by evidence from epide‐ miological studies although limited, appear promising.

#### **4.2. Preclinical evidence of the role of GTC and prostate cancer prevention**

Several published preclinical studies using green tea, green tea leaves, green tea extracts, GTP mixtures, green tea catechin (GTC) mixtures, and the individual catechins have demonstrated chemopreventive efficacy in several cancers. [51 - 57] Using the TRAMP mice model, Gupta et al. [51] were able to demonstrate that oral infusion of GTP extract at a human achievable dose (equivalent to six cups of green tea per day) significantly inhibits CaP development and increases survival in these mice. When 0.1% GTP (weight/volume) was provided as the sole source of drinking water to transgenic adenocarcinoma mouse prostate (TRAMP) mice 8–32 weeks of age, the result was (i) substantial reduction in the tumor incidence, tumor burden, and delay in metastasis as assessed by magnetic resonance imaging (MRI); (ii) substantial reduction in prostate (by 64%) and genitourinary (GU) (by 72%) weight; (iii) substantially decreased serum insulin-like growth factor-I (IGF-I) and restored to normal insulin-like growth factor binding protein-3 levels (IGFBP-3); and (iv) marked decrease in the expression of proliferating cell nuclear antigen (PCNA) in the prostate compared with control (water-fed) TRAMP mice. Furthermore, GTP consumption caused significant apoptosis of CaP cells, which possibly resulted in reduced dissemination of cancer cells, thereby causing the inhibition of CaP development, progression, and metastasis of CaP to distant organ sites. However, in another similar animal model, epigallocatechin gallate (EGCG) only slightly reduced these levels. [52] These observations may be attributed to the pharmacokinetic properties of EGCG, which has relatively low oral bioavailability. [52] These inconsistencies could also be explained by differential doses, different methods of infusion, duration of intervention, and inadequate timing of castration necessary to observe changes in markers of progression and the antioxi‐ dant property of EGCG. Oral administration of GTPs (*vs.* pure EGCG) at 500 mg/kg/day in drinking water to TRAMP mice is expected to cause a higher systemic exposure compared to gavage and may explain the protective effects observed by Gupta et al. [51] compared with Suttie et al. [52] Earlier preclinical trials [54, 57, 107] have observed decreasing effectiveness of GTC with advancing stage of PCa using a TRAMP mouse model. We investigated the safety and efficacy of PolyE administered with the goal to reduce the progression of CaP in TRAMP mice which is an established model of CaP. [56] In this study, 119 TRAMP and 119 C57BL/6J mice were treated orally with one of three doses of PolyE (200, 500, and 1000 mg/kg/day) in drinking water ad libitum, replicating human achievable doses. Safety and efficacy assess‐ ments in our study were performed when mice were 12, 22, and 32 weeks old. The number and the size of tumors in the PolyE group were significantly decreased compared with the control group (water-fed). In water-fed 32-week-old control TRAMP mice, prostate carcinoma metastasis to distant sites was observed in 100% of mice (8/8), compared with 13% of mice (2/16) treated with high-dose PolyE during the same period. Furthermore, PolyE treatment significantly inhibited metastasis in TRAMP mice in a dose-dependent manner (*P* = 0.0003). Long-term (32 weeks) treatment with PolyE was safe and well tolerated with no evidence of toxicity in C57BL/6J mice. PolyE similar to other GTC formulations has been observed to be effective chemopreventive agents in preventing the progression of prostate cancer in TRAMP mice with no toxicity in these mouse models. Our findings provide additional preclinical evidence for the safety and chemopreventive effect of PolyE in preventing metastatic progres‐ sion of prostate cancer. [56] Preclinical studies, including our recent work with PolyE, have consistently demonstrated chemopreventive efficacy in PCa, significantly delaying primary tumor incidence and tumor burden, with reduction in markers of proliferation and potent and selective *in vitro* and *in vivo* proapoptotic activity on PCa cells.

#### **4.3. Potential mechanism by which GTC modulates prostate carcinogenesis:**

A body of research evidence indicates that tea and tea compounds reduce the growth of several human cancer cell lines *in vitro*, including stomach, lung, prostate, colon, leukemia, oral tumor, liver, breast, and cervix, as well as HPV-immortalized cervical epithelial cells. Among the constituents of GTCs, laboratory studies have identified EGCG as the most potent chemopre‐ ventive agent that affects a number of molecular processes including induction of apoptosis and inhibition of tumor growth and angiogenesis with no significant effect on benign controls. [58 - 62] EGCG has been reported to affect several cancer-related proteins, including p27, Bcl-2 or Bcr-Abl oncoproteins, Bax, matrix metalloproteinases (MMP-2 and MMP-9), [63] androgen receptor, EGF receptor, activator protein 1 (AP1), and some cell cycle regulators. [64 - 66]

In their recent review of signaling pathways that are affected by green tea polyphenols, Khan et al. [67] reported that various pathways mediated via mitogen-activated protein kinases (MAPK) signaling were affected, including extracellular-signal-related kinases (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2/3 or SAPKs), p38 MAPK, ERK3/4, ERK5, and ERK7/8. Ultimately, MMP-2 and MMP-9 expressions were affected by EGCG via inhibition of phosphorylation of ERK1/2 and p38 pathways. Since MMP-9 and MMP-2 are known biomarkers of invasion and metastasis, the inhibition of their expression leads to inhibition of prostate cancer spread. Insulin-like growth factors (IGF) exert multiple effects on glucose, fat, and protein metabolism and play important roles in regulating cell proliferation, differentiation, apoptosis, and transformation. PI3K/Akt and Ras/MAPK pathways can be activated via IGF-signaling, supporting cell proliferation. GTCs are known to suppress cellular signaling via the IGF-axis, thus affecting cell proliferation. Cyclooxygenase (COX) is a ratelimiting enzyme in the prostaglandin biosynthesis. COX-2 can be activated via NF-κB signaling pathway, leading to inflammation and cell proliferation. EGCG was shown to inhibit COX-2 expression at both the mRNA and the protein levels in the prostate, thus slowing down the cancer growth. Finally, EGCG have a notable effect on cell cycle arrest. Gupta et al. [68] have earlier reported that treatment of CaP cell lines LNCaP and DU145 with EGCG caused cell cycle disruptions and reduced proliferation of cells due to down-regulation of various cyclins as well as altered binding to CDKs. Treatment of human CaP cell lines LNCaP and PC-3 with GTP caused the inhibition of class I HDAC activity, leading to cell cycle arrest and induction of apoptosis.

CaP development, progression, and metastasis of CaP to distant organ sites. However, in another similar animal model, epigallocatechin gallate (EGCG) only slightly reduced these levels. [52] These observations may be attributed to the pharmacokinetic properties of EGCG, which has relatively low oral bioavailability. [52] These inconsistencies could also be explained by differential doses, different methods of infusion, duration of intervention, and inadequate timing of castration necessary to observe changes in markers of progression and the antioxi‐ dant property of EGCG. Oral administration of GTPs (*vs.* pure EGCG) at 500 mg/kg/day in drinking water to TRAMP mice is expected to cause a higher systemic exposure compared to gavage and may explain the protective effects observed by Gupta et al. [51] compared with Suttie et al. [52] Earlier preclinical trials [54, 57, 107] have observed decreasing effectiveness of GTC with advancing stage of PCa using a TRAMP mouse model. We investigated the safety and efficacy of PolyE administered with the goal to reduce the progression of CaP in TRAMP mice which is an established model of CaP. [56] In this study, 119 TRAMP and 119 C57BL/6J mice were treated orally with one of three doses of PolyE (200, 500, and 1000 mg/kg/day) in drinking water ad libitum, replicating human achievable doses. Safety and efficacy assess‐ ments in our study were performed when mice were 12, 22, and 32 weeks old. The number and the size of tumors in the PolyE group were significantly decreased compared with the control group (water-fed). In water-fed 32-week-old control TRAMP mice, prostate carcinoma metastasis to distant sites was observed in 100% of mice (8/8), compared with 13% of mice (2/16) treated with high-dose PolyE during the same period. Furthermore, PolyE treatment significantly inhibited metastasis in TRAMP mice in a dose-dependent manner (*P* = 0.0003). Long-term (32 weeks) treatment with PolyE was safe and well tolerated with no evidence of toxicity in C57BL/6J mice. PolyE similar to other GTC formulations has been observed to be effective chemopreventive agents in preventing the progression of prostate cancer in TRAMP mice with no toxicity in these mouse models. Our findings provide additional preclinical evidence for the safety and chemopreventive effect of PolyE in preventing metastatic progres‐ sion of prostate cancer. [56] Preclinical studies, including our recent work with PolyE, have consistently demonstrated chemopreventive efficacy in PCa, significantly delaying primary tumor incidence and tumor burden, with reduction in markers of proliferation and potent and

308 Phytochemicals - Isolation, Characterisation and Role in Human Health

selective *in vitro* and *in vivo* proapoptotic activity on PCa cells.

**4.3. Potential mechanism by which GTC modulates prostate carcinogenesis:**

A body of research evidence indicates that tea and tea compounds reduce the growth of several human cancer cell lines *in vitro*, including stomach, lung, prostate, colon, leukemia, oral tumor, liver, breast, and cervix, as well as HPV-immortalized cervical epithelial cells. Among the constituents of GTCs, laboratory studies have identified EGCG as the most potent chemopre‐ ventive agent that affects a number of molecular processes including induction of apoptosis and inhibition of tumor growth and angiogenesis with no significant effect on benign controls. [58 - 62] EGCG has been reported to affect several cancer-related proteins, including p27, Bcl-2 or Bcr-Abl oncoproteins, Bax, matrix metalloproteinases (MMP-2 and MMP-9), [63] androgen receptor, EGF receptor, activator protein 1 (AP1), and some cell cycle regulators. [64 - 66]

Yang et al. [69] have recently reported that phenolic groups of EGCG can directly bind to multiple molecules due to their hydrogen bonds. The molecules of interest include B-cell CLL/ lymphoma 2 protein (BCL2), which has antiapoptotic properties, leading to induction of apoptosis. Some other EGCG binding targets include glucose-regulated protein 78 kDa, vimentin, IGF-1R, and peptidylcis/trans isomerase. Via this mechanism, EGCG can modulate the formation of reactive oxygen species (ROS) as well as affect other molecular pathways described above.

Rizzi et al. [70] reported that Poly E induced cell cycle arrest at the G0/G1 checkpoint for PNT1a prostate cell lines (modeling early stage disease) and G2/M for PC3 cells (modeling late stage disease). The authors showed that in the model of an early stage disease, autophagy was the first mechanism to activate in response to endoplasmic reticulum stress (ERS). After that stage, which lasted approximately 12 h, activation of caspases occurred, indicating anoikis cell death. In the model of an aggressive CaP, Poly E induced severe ERS which eventually led to GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. These results may aid in the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

Hagen et al. [71] reported that EGCG induced apoptosis in PC3 cells, via the caspase 9 dependent mechanism. Furthermore, EGCG, both alone and in combination with cisplatin, promoted the expression of the proapoptotic splice isoform of caspase 9, and it modifies the alternative splicing of caspase 9, favoring the proapoptotic isoform. The latter finding suggests that EGCG may affect the alternative splicing of cancer-associated genes *in vivo*.

Connors et al. [72] have previously proposed a mechanistic model according to which GTCs antitumor action in prostate cancer acts through proteasome. GTC-induced inhibition of chymotrypsin-like activity of the proteasome results in the accumulation proteasome targets p21, p27, Bax, and IκBα. The accumulation of cell cycle regulators p21 and p27 result in G1 cell cycle arrest, whereas the accumulation of the proapoptotic protein, Bax, contributes to cell apoptosis. The oncogenic transcription factor, NFκB, is down-regulated, presumably by the elevation of IκBα, its intrinsic inhibitor. This results in the reduced expression of NFκB target genes, antiapoptotic, Bcl-xL and Bcl-2, cell cycle regulators, cyclin D and cyclin E, and metastasis-related genes, VEGF, angiopoietin 1/2, MMPs, and uPA. Reductions in cyclins D and E, Bcl-2, and Bcl-xL further drive the processes of cell cycle arrest, decreased cell prolif‐ eration, and apoptosis, respectively. Additionally, the reduction in metastasis-related genes inhibits tumor cell invasion and metastasis.

Based on their studies of GTP in cell culture systems, Adhami et al. [63] were able to demon‐ strate that EGCG in GTP induces apoptosis, cell growth inhibition, and cyclin kinase inhibitor WAF-1/p21-mediated cell cycle dysregulation.Using cDNA microarrays, they also observed that the EGCG treatment of LNCaP cells results in the induction of genes that functionally exhibit growth-inhibitory effects and repression of genes that belong to the G-protein signaling network. When oral feeding of GTP was the sole source of drinking fluid for TRAMP mice, a significant inhibition of VEGF, MMP-2, and MMP-9 was demonstrated, suggesting antimeta‐ static and antiangiogenic properties of GTP that may affect CaP progression. [63] Bettuzzi et al. have validated these findings, showing that oral administration of GTP to TRAMP mice reduced CaP onset from 100% to 20% [94] mediated by induction of clusterin (CLU) expression, which is potently up-regulated during prostate gland involution [95] but down-regulated in human CaP specimens [73, 74] and exerts antiproliferative [75] and proapoptotic activity [76 - 79] in PNT1a and PC-3 cells.

EGCG potently and selectively inhibits the proteasome activity in intact human CaP cells and consequently accumulates IkBα and p27 proteins, leading to growth arrest (Figure 1). [59 - 61] ECCG has been shown to decrease NF-κB DNA binding and abrogates NF-κB activation by DNA-damaging agents. Our group also reported that EGCG was able to inhibit NF-κB activation through stabilization of IκBα. [58 - 61] NF-κB is a eukaryotic transcription factor involved in the regulation of COX-2 and many other genes. [79] The constitutive activation of NF-κB has been reported in many tumors, [80, 81] associating it with progression of epithelial cells, including prostate, toward malignancy. This inhibition of proteasome activity by EGCG occurred at or near physiological concentrations *in vitro* (IC50 = 0.1−0.2 mM) and *in vivo* (1−10 mM) at the concentrations found in the serum of green tea drinkers. [59 - 61] The ubiquitin/ proteasome system plays an important role in the degradation of cellular proteins. This proteolytic system includes two distinct steps: ubiquitination and degradation. [82, 83] The 26S proteasome is a high molecular weight (2.4MDa), ATP-dependent, multiprotease complex. [84 - 86] This complex, which exists in both the cytoplasm and nucleus, is composed of a catalytic subunit (20S) and regulatory subunits. [87] The 20S catalytic subunit contains at least three enzymatic activities, chymotrypsin-like (prefers Tyr or Phe at the P1 site), trypsin-like (prefers Lys or Arg), and glutamyl peptidyl hydrolytic-like (prefers Asp or Glu). [88] The inhibition of only the chymotrypsin-like activity has been tightly associated with apoptosis induction. Most substrates that are degraded by the proteasome are first covalently modified with ubiquitin (a 76 amino acid polypeptide). [82] Many substrates of the proteasome are proteins that are involved in the regulation of cell cycle progression and apoptosis. [86] For example, the proteasome degrades regulators of cyclin-dependent kinases (CDK) such as cyclins and the CDK inhibitors p21waf and p27kip as well as the CDK phosphatases CDC25 A, B, and C. The proteasome also degrades IκBα, an important inhibitor of the tumor survival factor NF-κB. Many physical (i.e., radiation), chemical (cancer chemotherapeutic agents), viral, and biological (cytokines and growth factors) agents induce phosphorylation, ubiquitination, and subsequent degradation of IκBα by the proteasome, freeing up NF-κB to translocate to the nucleus and to modulate genes involved in proliferation, invasion, and tumor survival. [89, 90] For example, NF-κB up-regulates the antiapoptotic protein Bcl2 and down-regulates the proapoptotic protease caspase 8. Therefore, by inhibiting the proteasome, IκBα will accumu‐ late, which will inhibit NF-κB from promoting tumor survival. The proteasome is also responsible for degrading the tumor suppressor p53. Many tumor cells inactivate p53 by overexpressing an E3 ligase called mdm2, which binds p53 and ubiquitinates it for degradation by the proteasome. [82, 83] In human tumors, which overexpress mdm2, the inhibition of the proteasome is predicted to induce tumor cell apoptosis by accumulating p53. CEP1612, a dipeptidyl proteasome inhibitor, was able to rapidly induce apoptosis in all the human cancer cell lines tested, including breast, prostate, leukemia, lung, bone, brain, and head and neck, but not in human normal fibroblasts and normal breast cells. [85, 91] They also reported that proteasome inhibition was sufficient to overcome apoptotic protection by Bcl-2 or Bcr-Abl oncoprotein. [91] Recently, it has been reported that proteasome inhibition accumulates Bax (but not Bcl-2) protein in mitochondria, resulting in increased ratio of Bax/Bcl-2, associated with cytochrome *c* release and apoptosis induction. [92] It has also been reported that during TNF-α-induced apoptosis, Bcl-2, but not Bax, protein is degraded through ubiquitin/protea‐ some-dependent pathway, [93] which also increased the Bax/Bcl-2 ratio. Therefore, selectively degrading one or more Bcl-2 family proteins by the proteasome should change the ratio of proto antiapoptotic proteins, which might contribute to the apoptotic commitment.

alternative splicing of caspase 9, favoring the proapoptotic isoform. The latter finding suggests

Connors et al. [72] have previously proposed a mechanistic model according to which GTCs antitumor action in prostate cancer acts through proteasome. GTC-induced inhibition of chymotrypsin-like activity of the proteasome results in the accumulation proteasome targets p21, p27, Bax, and IκBα. The accumulation of cell cycle regulators p21 and p27 result in G1 cell cycle arrest, whereas the accumulation of the proapoptotic protein, Bax, contributes to cell apoptosis. The oncogenic transcription factor, NFκB, is down-regulated, presumably by the elevation of IκBα, its intrinsic inhibitor. This results in the reduced expression of NFκB target genes, antiapoptotic, Bcl-xL and Bcl-2, cell cycle regulators, cyclin D and cyclin E, and metastasis-related genes, VEGF, angiopoietin 1/2, MMPs, and uPA. Reductions in cyclins D and E, Bcl-2, and Bcl-xL further drive the processes of cell cycle arrest, decreased cell prolif‐ eration, and apoptosis, respectively. Additionally, the reduction in metastasis-related genes

Based on their studies of GTP in cell culture systems, Adhami et al. [63] were able to demon‐ strate that EGCG in GTP induces apoptosis, cell growth inhibition, and cyclin kinase inhibitor WAF-1/p21-mediated cell cycle dysregulation.Using cDNA microarrays, they also observed that the EGCG treatment of LNCaP cells results in the induction of genes that functionally exhibit growth-inhibitory effects and repression of genes that belong to the G-protein signaling network. When oral feeding of GTP was the sole source of drinking fluid for TRAMP mice, a significant inhibition of VEGF, MMP-2, and MMP-9 was demonstrated, suggesting antimeta‐ static and antiangiogenic properties of GTP that may affect CaP progression. [63] Bettuzzi et al. have validated these findings, showing that oral administration of GTP to TRAMP mice reduced CaP onset from 100% to 20% [94] mediated by induction of clusterin (CLU) expression, which is potently up-regulated during prostate gland involution [95] but down-regulated in human CaP specimens [73, 74] and exerts antiproliferative [75] and proapoptotic activity [76

EGCG potently and selectively inhibits the proteasome activity in intact human CaP cells and consequently accumulates IkBα and p27 proteins, leading to growth arrest (Figure 1). [59 - 61] ECCG has been shown to decrease NF-κB DNA binding and abrogates NF-κB activation by DNA-damaging agents. Our group also reported that EGCG was able to inhibit NF-κB activation through stabilization of IκBα. [58 - 61] NF-κB is a eukaryotic transcription factor involved in the regulation of COX-2 and many other genes. [79] The constitutive activation of NF-κB has been reported in many tumors, [80, 81] associating it with progression of epithelial cells, including prostate, toward malignancy. This inhibition of proteasome activity by EGCG occurred at or near physiological concentrations *in vitro* (IC50 = 0.1−0.2 mM) and *in vivo* (1−10 mM) at the concentrations found in the serum of green tea drinkers. [59 - 61] The ubiquitin/ proteasome system plays an important role in the degradation of cellular proteins. This proteolytic system includes two distinct steps: ubiquitination and degradation. [82, 83] The 26S proteasome is a high molecular weight (2.4MDa), ATP-dependent, multiprotease complex. [84 - 86] This complex, which exists in both the cytoplasm and nucleus, is composed of a catalytic subunit (20S) and regulatory subunits. [87] The 20S catalytic subunit contains at least

that EGCG may affect the alternative splicing of cancer-associated genes *in vivo*.

inhibits tumor cell invasion and metastasis.

310 Phytochemicals - Isolation, Characterisation and Role in Human Health


In summary, there is increasing experimental evidence that GTCs slow down prostate carcinogenesis via an umbrella of mechanisms and cellular pathways that work in concert to affect multiple hallmarks of cancer. The main pathways include proteasome inhibition, cell cycle arrest, inhibition of cell proliferation, induction of apoptosis, suppression of growth and invasion, and inhibition of metastasis. Further research should elucidate the exact sequence of cellular events induced by GTCs that lead to suppression of prostate malignancy.

#### **4.4. Clinical experience: green tea polyphenols and prostate cancer**

Several phase I studies, sponsored by the National Cancer Institute's Division of Cancer Prevention (NCI, DCP) and others, comparing the pharmacokinetics and safety of oral green tea, polyphenon E, and EGCG, have been completed and published. [96 - 99, 100] By conduct‐

**Figure 1.** Modeling relative to the primary mechanism of Polyphenon E in Prostate Cancer

ing a phase I trial of oral green tea extract in adult patients with solid tumors, Pisters et al. [98] reported that a safe dose of green tea extract was equivalent to 7–8 Japanese cups (120 ml) of green tea three times daily for 6 months. They concluded that the side effects (neurological and gastrointestinal effects) of the green tea extract preparation were caffeine related, and not from EGCG*.* The average cup of green tea contains 10–50 mg of caffeine; therefore, overcon‐ sumption may have a stimulatory effect in some individuals. Thus, although green tea is practical, nontoxic, and has the potential to be developed for chemoprevention, one has to consume 8–10 cups to obtain these benefits and the side effects due to caffeine are a concern.

In a single-dose study sponsored by NCI, DCP, each subject received two single doses containing 200, 400, 600, or 800 mg EGCG provided by each of two different formulations (polyphenon E or a purified EGCG preparation) separated by a 2-week washout period. [96] Plasma EGCG levels increased with dose and formulation had no effect on EGCG pharmaco‐ kinetics. Although little EGCG circulated in conjugated form, EGC and EC were highly conjugated. In a completed multidose study, healthy subjects with sun-sensitive skin received 800 mg EGCG alone or as polyphenon E in one or divided daily doses for 4 weeks. [101] Adverse effects were predominantly mild gastrointestinal complaints. Plasma levels of EGCG were significantly higher in the 400 mg qd versus 400 mg bid dose group for both formulations. Plasma antioxidant levels and UV light-induced minimum erythema dose (MED) were unaffected by any treatment. In the third completed phase I study, the effect of fasting on pharmacokinetics was examined in healthy adults taking 400, 800, or 1200 mg EGCG as polyphenon E. Plasma levels of free EGCG were dramatically higher when taking polyphenon E in the fasting state compared to the fed state for all three dose levels; the average *C*max increased more than 3.6-fold, and the average AUC increased more than 2.3-fold when taking polyphenon E on an empty stomach. In a study to test if the oral bioavailability of green tea catechins can be enhanced when consumed in the absence of food, Chow et al. [97] observed that greater bioavailability of free catechins can be achieved by taking the polyphenon E capsules on an empty stomach after an overnight fast thus optimizing the biological effects of tea catechins. Recent studies including individual case reports have indicated several grade 1–2 AEs, including case studies of liver toxicities and rectal bleeding (personal communication from DCP), all of which indicate the need for continued monitoring of safety in well-designed clinical trials.

Bettuzzi et al. [102 - 103] completed a randomized clinical trial to assess safety and efficacy of GTCs for chemoprevention of CaP in men diagnosed with HGPIN. [73] Purity and content of GTCs preparations were assessed by HPLC (EGC 5.5%, EC 12.24%, EGCG 51.88%, ECG 6.12%, total GTCs 75.7%, caffeine <1%). After 1-year intervention with GTC tablets containing 600 mg EGCG/day, only one tumor was diagnosed among the 30 GTC-treated men (incidence: about 3%), while nine cancers were found among the 30 placebo-treated men (incidence: 30%). After 9 months of treatment, subjects taking GTC had a 17% decrease in total PSA values; otherwise, PSA values did not change significantly between the two arms of the study, probably because of high individual differences. No significant side effects were documented. As a secondary observation, they found that the administration of GTCs at this dose was also effective at reducing LUTS. However, other studies intervening in men with localized prostate cancer in the presurgical phase (from biopsy to prostatectomy) with GTC failed to observe chemopre‐ ventive benefit [104 - 106] observed in trials targeting earlier stages of prostate carcinogenesis (HGPIN). These early trials indicate that GTC may not exert a meaningful effect once diag‐ nosed with prostate cancer and, consistent with earlier preclinical trials, [54, 104] have observed decreasing effectiveness of GTC with advancing stage of PCa using a TRAMP mouse model. In summary, evidence from phase I/II studies has demonstrated bioavailability and tolerance to GTC at doses ranging from 200 to 1200 mg per day and for a duration of 1 year with observation of chemoprevention effects in the early stages (HGPIN) of prostate carcino‐ genesis and not in the later stages. With few options available for the chemoprevention of early precursors of PCa, GTC shows promise to be further evaluated as chemoprevention agents targeting high-risk cohorts with HGPIN.

#### **5. Conclusions and future directions**

ing a phase I trial of oral green tea extract in adult patients with solid tumors, Pisters et al. [98] reported that a safe dose of green tea extract was equivalent to 7–8 Japanese cups (120 ml) of green tea three times daily for 6 months. They concluded that the side effects (neurological and gastrointestinal effects) of the green tea extract preparation were caffeine related, and not from EGCG*.* The average cup of green tea contains 10–50 mg of caffeine; therefore, overcon‐ sumption may have a stimulatory effect in some individuals. Thus, although green tea is practical, nontoxic, and has the potential to be developed for chemoprevention, one has to consume 8–10 cups to obtain these benefits and the side effects due to caffeine are a concern.

**Figure 1.** Modeling relative to the primary mechanism of Polyphenon E in Prostate Cancer

312 Phytochemicals - Isolation, Characterisation and Role in Human Health

In a single-dose study sponsored by NCI, DCP, each subject received two single doses containing 200, 400, 600, or 800 mg EGCG provided by each of two different formulations (polyphenon E or a purified EGCG preparation) separated by a 2-week washout period. [96] Plasma EGCG levels increased with dose and formulation had no effect on EGCG pharmaco‐ kinetics. Although little EGCG circulated in conjugated form, EGC and EC were highly conjugated. In a completed multidose study, healthy subjects with sun-sensitive skin received 800 mg EGCG alone or as polyphenon E in one or divided daily doses for 4 weeks. [101] Adverse effects were predominantly mild gastrointestinal complaints. Plasma levels of EGCG were significantly higher in the 400 mg qd versus 400 mg bid dose group for both formulations. Plasma antioxidant levels and UV light-induced minimum erythema dose (MED) were unaffected by any treatment. In the third completed phase I study, the effect of fasting on pharmacokinetics was examined in healthy adults taking 400, 800, or 1200 mg EGCG as polyphenon E. Plasma levels of free EGCG were dramatically higher when taking polyphenon E in the fasting state compared to the fed state for all three dose levels; the average *C*max increased more than 3.6-fold, and the average AUC increased more than 2.3-fold when taking polyphenon E on an empty stomach. In a study to test if the oral bioavailability of green tea

GTCs are promising agents for the chemoprevention of prostate cancer. Early observations indicate that GTCs at human achievable doses are most effective against early stage prostate carcinogenesis in preclinical and clinical trials and are attractive chemopreventive agents with a favorable safety profile. There is an urgent need to continue to identify chemopreventive agents to reduce the burden of cancer. However, it is just as critical to learn from the experience of past chemoprevention trials [108 - 115] that underscore the need for systematically charac‐ terizing these agents for cancer chemoprevention and defining their efficacy, safety, and mechanism of action using preclinical and early-phase work before undertaking phase III trials and ultimately translating the findings to clinical practice.

#### **Author details**

Ganna Chornokur and Nagi B. Kumar\*

\*Address all correspondence to: nagi.kumar@moffitt.org

Department of Cancer Epidemiology, Moffitt Cancer Center and Research Institute, Tampa, FL, USA

#### **References**


[11] Ilic D, O'Connor D, Green S, Wilt TJ. Screening for prostate cancer: an updated Co‐ chrane systematic review. BJU Int. 2011;107(6):882–91.

**Author details**

FL, USA

**References**

Ganna Chornokur and Nagi B. Kumar\*

\*Address all correspondence to: nagi.kumar@moffitt.org

314 Phytochemicals - Isolation, Characterisation and Role in Human Health

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### *Eleutherine Plicata* **– Quinones and Antioxidant Activity**

Luiz Claudio da Silva Malheiros, João Carlos Palazzo de Mello and Wagner Luiz Ramos Barbosa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59865

#### **1. Introduction**

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[112] Bertagnolli MM, Eagle CJ, Zauber AG, et al. Celecoxib for the prevention of sporadic

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322 Phytochemicals - Isolation, Characterisation and Role in Human Health

Natl Cancer Inst. 1998;90(18):1371–88.

Iridaceae is a family of petalloid monocots with 77 genera and more than 1,630 species, which, despite worldwide distribution, has a remarkable concentration in the southern continents. This family has its major center of irradiation in Southern Africa and in Brazil, it is represented by 14 genera and 110 species [1].

*Eleutherine plicata* Herb. is an Iridaceae popularly known in the Amazonian region as maru‐ pazinho, marupari, palmerinha, and marupá-pirranga, where it occurs in the form of a clump, with red bulbs like an onion (Figure 1). Its leaves are entire, pleated, and simple; the flowers are colored in white to pink; and the red bulbs are widely used in Brazilian folk phytotherapy, especially in the Amazonian region [2].

The ethnoguided surveys conducted by Barbosa et al. (2009) [3] in the city of Igarapé-Miri and by Jardim et al. [4] in Santa Barbara, both in the Brazilian State Pará, revealed the use of Marupazinho (*Eleutherine plicata* Herb.) to treat diarrhea caused by amoeba and that the people prepare a tea from the bulb of the plant, which is drunk before the meal.

The aqueous extract (AE) of the dried bulbs contains isoeleutherine, which appears as a peak of 99.14% purity in the LC-DAD chromatographic analyses. A buffered aqueous solution containing 2.5 mg of dried AE/mL showed antiamoebic activity, a complete inhibition of the *Entamoeba histolytica* trophozoites growth, in 24 hours [5]. It is remarkable that this solution is less concentrated than the tea (about 3.5 mg/mL), both prepared according to popular knowledge, but normalized according to the Brazilian Pharmacopoeia [6], being indeed sufficient to promote the total annihilation of the tested trophozoites.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** *Eleutherine plicata* Herb. (herbaceousborneo.blogspot.com)

The experimental characterization of the antiamoebic activity of the decoction prepared with bulbs of *E. plicata* contributes to validate its alleged popular use. The detection of isoeleutherine in the analyzed decoction can explain partially the reported antiamoebic activity, which can be attributed to the pro-oxidant activity of the substance [7].

*Eleutherine bulbosa* (Mill.) Urb., an accepted name for *E. plicata,* according to Tropicos®1 , is also used by traditional Zulu healers as anti-diarrheal [8] and yields eleutherinone (1), eleutherine (2), and isoleutherine (3), three quinones that show strong antifungal activity [9] Figure 2). Quinones show various pharmacological activities including bactericidal, fungicidal, and antiprotozoal, noting that they cause oxidative stress by inducing the endogenous formation of reactive oxygen species [7]. Quinones are oxygenated aromatic derivatives, characterized by the presence of a diketocyclohexa-1,4-diene residue (para-quinones) or alternatively by a diketocyclohexa-1,3-diene grouping (ortho-quinones) [10]. derivatives, characterized by the presence of a diketocyclohexa-1,4-diene residue (para-quinones) or alternatively by a diketocyclohexa-1,3-diene grouping (ortho-quinones) [10](BRUNETON, 2001).

1 – Eleutherinone 2 – Eleutherine (3‐S isomer) 3 – Isoeleutherine (3‐R isomer)

ˉ), which in the presence of superoxide dismutase is converted into H2O2.

inside the cell. Although H2O2 is not a free radical, it's a very reactive

Figure 2. Chemical constituents isolated from *E. bulbosa* (Mill.) Urb. **Figure 2.** Chemical constituents isolated from *E. bulbosa* (Mill.) Urb.

•

superoxide anion radical ([O2]

Reaction) generates [HO]•

3.1.1 REAGENTS

3.1.2 EQUIPMENTS

**3 MATERIALS AND METHODS**  3.1 MATERIALS AND EQUIPMENTS

and silica gel for column chromatography.

[O2] •

Naphthoquinones act under enzymatic influence, accepting an electron to form semiquinone anion radical under catalysis of reduced nicotinamide-adenine dinucleotide 3'-phosphate

Naphthoquinones act under enzymatic influence, accepting an electron to form semiquinone

phosphate (NADPH) cytochrome-P-450-reductase, NADPH cytochrome-b5-reductase, and NADPH ubiquinone-oxidoreductase. The semiquinone anion radical then reduces molecular oxygen to

ˉ under catalysis of transition metals (Fenton Reaction) or reacting with H2O2 (Haber-Weiss

 Acetone, ethyl acetate, acetonitrile, chloroform, deuterated chloroform, ethanol, hexane, potassium hydroxide, methanol, dimethylsulfoxide (DMSO), silica gel for thin layer chromatography,

 Stainless steel knives mill (Tecnal®, model TCL-650); ultraviolet (UV) visible (VIS) spectrophotometer spectrum SP 2000; UV chamber 254 and 365nm; analytical balance GEHAKA BK 600; high-performance liquid chromatography system LaChrom7000 Merck-HITACHI® with diode

substance and can also promote the oxidation of certain biomolecules [7](SILVA et al., 2003).

anion radical under catalysis of reduced nicotinamide-adenine dinucleotide 3'- 1 Tropicos.org. Missouri Botanical Garden. 28 Oct 2014 <http://www.tropicos.org>

(NADPH) cytochrome-P-450-reductase, NADPH cytochrome-b5-reductase, and NADPH ubiquinone-oxidoreductase. The semiquinone anion radical then reduces molecular oxygen to superoxide anion radical ([O2] ⋅ ¯), which in the presence of superoxide dismutase is con‐ verted into H2O2. [O2] ⋅ ¯ under catalysis of transition metals (Fenton Reaction) or reacting with H2O2 (Haber-Weiss Reaction) generates [HO]<sup>⋅</sup> inside the cell. Although H2O2 is not a free radical, it's a very reactive substance and can also promote the oxidation of certain biomole‐ cules [7].

#### **2. Materials and methods**

#### **2.1. Materials and equipments**

#### *2.1.1. Reagents*

The experimental characterization of the antiamoebic activity of the decoction prepared with bulbs of *E. plicata* contributes to validate its alleged popular use. The detection of isoeleutherine in the analyzed decoction can explain partially the reported antiamoebic activity, which can

used by traditional Zulu healers as anti-diarrheal [8] and yields eleutherinone (1), eleutherine (2), and isoleutherine (3), three quinones that show strong antifungal activity [9] Figure 2). Quinones show various pharmacological activities including bactericidal, fungicidal, and antiprotozoal, noting that they cause oxidative stress by inducing the endogenous formation of reactive oxygen species [7]. Quinones are oxygenated aromatic derivatives, characterized by the presence of a diketocyclohexa-1,4-diene residue (para-quinones) or alternatively by a

1 – Eleutherinone 2 – Eleutherine (3‐S isomer) 3 – Isoeleutherine (3‐R isomer)

Naphthoquinones act under enzymatic influence, accepting an electron to form semiquinone

Naphthoquinones act under enzymatic influence, accepting an electron to form semiquinone anion radical under catalysis of reduced nicotinamide-adenine dinucleotide 3'-phosphate

ˉ under catalysis of transition metals (Fenton Reaction) or reacting with H2O2 (Haber-Weiss

 Acetone, ethyl acetate, acetonitrile, chloroform, deuterated chloroform, ethanol, hexane, potassium hydroxide, methanol, dimethylsulfoxide (DMSO), silica gel for thin layer chromatography,

 Stainless steel knives mill (Tecnal®, model TCL-650); ultraviolet (UV) visible (VIS) spectrophotometer spectrum SP 2000; UV chamber 254 and 365nm; analytical balance GEHAKA BK 600; high-performance liquid chromatography system LaChrom7000 Merck-HITACHI® with diode

substance and can also promote the oxidation of certain biomolecules [7](SILVA et al., 2003).

ˉ), which in the presence of superoxide dismutase is converted into H2O2.

inside the cell. Although H2O2 is not a free radical, it's a very reactive

anion radical under catalysis of reduced nicotinamide-adenine dinucleotide 3' phosphate (NADPH) cytochrome-P-450-reductase, NADPH cytochrome-b5-reductase, and NADPH ubiquinone-oxidoreductase. The semiquinone anion radical then reduces molecular oxygen to

Figure 2. Chemical constituents isolated from *E. bulbosa* (Mill.) Urb.

*Eleutherine bulbosa* (Mill.) Urb., an accepted name for *E. plicata,* according to Tropicos®1

derivatives, characterized by the presence of a diketocyclohexa-1,4-diene residue (para-quinones) or alternatively by a diketocyclohexa-1,3-diene grouping (ortho-quinones) [10](BRUNETON, 2001).

be attributed to the pro-oxidant activity of the substance [7].

**Figure 1.** *Eleutherine plicata* Herb. (herbaceousborneo.blogspot.com)

324 Phytochemicals - Isolation, Characterisation and Role in Human Health

diketocyclohexa-1,3-diene grouping (ortho-quinones) [10].

**Figure 2.** Chemical constituents isolated from *E. bulbosa* (Mill.) Urb.

1 Tropicos.org. Missouri Botanical Garden. 28 Oct 2014 <http://www.tropicos.org>

•

superoxide anion radical ([O2]

Reaction) generates [HO]•

3.1.1 REAGENTS

3.1.2 EQUIPMENTS

**3 MATERIALS AND METHODS**  3.1 MATERIALS AND EQUIPMENTS

and silica gel for column chromatography.

[O2] • Acetone, ethyl acetate, acetonitrile, chloroform, deuterated chloroform, ethanol, hexane, potassium hydroxide, methanol, dimethylsulfoxide (DMSO), silica gel for thin layer chroma‐ tography, and silica gel for column chromatography.

#### *2.1.2. Equipments*

Stainless steel knives mill (Tecnal®, model TCL-650); ultraviolet (UV) visible (VIS) spectro‐ photometer spectrum SP 2000; UV chamber 254 and 365nm; analytical balance GEHAKA BK 600; high-performance liquid chromatography system LaChrom7000 Merck-HITACHI® with diode array detector (DAD) and Agilent LiChrospher100 (250 mm × 4.6 mm) column; nuclear magnetic resonance (NMR) spectrometer Plus 300 MHz Variant.

#### **2.2. Methods**

, is also

#### *2.2.1. Collection and identification of botanical material*

The plant material purchased at the Ver-O-Peso Market, Belem, Pará State, Brazil was collected in October 2007 in the same region. The botanical identification occurred by comparison of the prepared exsiccata (Figure 3) to a voucher deposited at the Herbarium of the Emilio Goeldi Museum registered under the number 105432 .

#### *2.2.2. Processing of plant material and extraction*

Approximately 5 kg of fresh bulbs were sliced and after washing, aeration and selection, dehydrated at room temperature for 2 days. Drying was completed under forced hot air circulation at about 40° C. The dried material was ground in a Wiley knives mill to yield 1.20 kg of herbal drug.

<sup>2</sup> The authors acknowledge Prof.Dr.Mario Augusto Gonçalves Jardim by the characterization of the plant material.

**Figure 3.** Voucher specimen of *E. plicata* Herb. prepared for botanical identification.

An ethanol extract (EE) was obtained by successive macerations, using 500 g of the herbal drug and Ethanol 96o GL, until total drug exhaustion. Thereafter, the solvent was removed under reduced pressure in a rotary evaporator.

#### *2.2.3. Fractionation of ethanol extract*

About 30 g EE were suspended in 500 mL of methanol/water (1:1) and partitioned with Hexane (4 × 100mL) – HF (Hexane Fraction); Chloroform (5 × 100mL) – CF (Chloroform Fraction) and Ethyl Acetate (5 × 100mL) – EAF (Ethyl Acetate Fraction), leaving a residual hydromethanolic solution – RF. Solvents were removed under reduced pressure in rotary evaporator, for water, a lyophilizer was employed.

#### *2.2.4. Phytochemical screening*

Chemical tests were performed on EE, HF, CF, EAF, and RF based on the Guide for Phyto‐ chemical Analysis of Plant Extracts [11] in order to verify the presence of 18 classes of secondary metabolites.

#### *2.2.5. Thin Layer Chromatography (TLC) analyses*

TLC analyses aiming to corroborate the phytochemical results on EE, HF, CF, EAF, and RF were performed employing silica gel as stationary phase and as eluents hexane/acetone (80:20), chloroform/methanol (90:10), chloroform/methanol/water (70:25:05), chloroform/acetone (99:03) and (99:01), thus the corresponding chromatographic profiles were defined. The obtained chromatograms were observed under visible and ultraviolet light at 254 nm and 365 nm and then sprayed with KOH 10% in methanol to make possible the detection of quinones. The chromatographic profiles were obtained by determining the retention factor (Rf) and describing the color of each chemical constituent observed as a zone in the chromatograms.

#### *2.2.6. Isolation of substances*

Using 2.0 g of lyophilized CF, the first chromatographic separation (A) was performed on a 32–63 μm silica gel column (40 × 2.0 cm) as stationary phase, and chloroform/acetone (99.5:0.5) as mobile phase. The fractions were pooled according their TLC profiles, providing the following samples: A1 = 83 mg (corresponding to the substance **1**); A2 = 380 mg; A3 = 71mg; and A4 = 156 mg.

Sample A2 was chromatographed on a 32–63 μm silica gel column (35 × 1.5 cm), eluted with chloroform/acetone (99.5:0.5) and monitored by TLC, yielding the following fractions: B1 = 175 mg, B2 = 12 mg, B3= 53 mg, and B4 = 74 mg.

Using the same stationary phase in a 20 × 1.5 cm column and chloroform/acetone (99.3:0.7) as eluent, B1 was separated by column chromatography, yielding the following fractions after TLC monitoring: C1 = 8 mg, C2 = 28 mg, C3 = 22 mg, C4=69 mg, and C5 = 15 mg. About 65 mg of C4 were subjected to preparative TLC using normal phase silica gel on a standard chroma‐ toplate (20 × 20 cm), eluted with chloroform/acetone (99:01) to obtain **2**.

#### *2.2.7. Characterization of 1 and 2*

An ethanol extract (EE) was obtained by successive macerations, using 500 g of the herbal drug

About 30 g EE were suspended in 500 mL of methanol/water (1:1) and partitioned with Hexane (4 × 100mL) – HF (Hexane Fraction); Chloroform (5 × 100mL) – CF (Chloroform Fraction) and Ethyl Acetate (5 × 100mL) – EAF (Ethyl Acetate Fraction), leaving a residual hydromethanolic solution – RF. Solvents were removed under reduced pressure in rotary evaporator, for water,

Chemical tests were performed on EE, HF, CF, EAF, and RF based on the Guide for Phyto‐ chemical Analysis of Plant Extracts [11] in order to verify the presence of 18 classes of secondary

TLC analyses aiming to corroborate the phytochemical results on EE, HF, CF, EAF, and RF were performed employing silica gel as stationary phase and as eluents hexane/acetone (80:20), chloroform/methanol (90:10), chloroform/methanol/water (70:25:05), chloroform/acetone (99:03) and (99:01), thus the corresponding chromatographic profiles were defined. The obtained chromatograms were observed under visible and ultraviolet light at 254 nm and 365 nm and then sprayed with KOH 10% in methanol to make possible the detection of quinones.

GL, until total drug exhaustion. Thereafter, the solvent was removed under

and Ethanol 96o

reduced pressure in a rotary evaporator.

326 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Figure 3.** Voucher specimen of *E. plicata* Herb. prepared for botanical identification.

*2.2.5. Thin Layer Chromatography (TLC) analyses*

*2.2.3. Fractionation of ethanol extract*

a lyophilizer was employed.

*2.2.4. Phytochemical screening*

metabolites.

About 20 mg of **1** and **2** were dissolved in deuterated chloroform (CDCl3) to be analyzed in a Variant brand Plus NMR Spectrometer. The characterization of these substances was achieved by comparison of their 1 H- (300 MHz) and 13C- NMR (75MHz) spectra to those obtained from the same naphthoquinones isolated from other species of *Eleutherine* and reported in the scientific literature.

#### *2.2.8. LC profile of EE, CF, 1, and 2*

The LC-DAD profile was recorded using a LaChrom 7000 Merck HITACHI® chromatograph hyphenated to a DAD equipped with a LiChrospher100 Agilent column (250 × 4.6 mm). The mobile phase consisted of ultrapure water and acetonitrile (ACN) as described in Table 1 and was pumped at 1 mL/min. The oven temperature was 26°C (± 1°C) and the detection occurred between 200 nm and 500 nm, this method was adapted from Paramapojn et al. (2008) [12].


**Table 1.** Gradient composition (in %) of the eluent used to analyze EE, CF, **1,** and **2**.

Aliquots of 50 μL were applied at the following concentrations: **1**, 500 μg/mL; **2,** 1 mg/mL; CF, 2,500 μg/mL; and crude EE, 1 mL.

#### *2.2.9. Antioxidant activity of EE, 1, and 2*

The antioxidant capacity of EE, Ep1, and Ep2 was evaluated using the free radical 2,2 diphenyl-1-picrylhydrazyl ([DPPH]⋅+) and as reference the substance butylhydroxytoluene (BHT). The reaction was accompanied by color change and the activity is monitored by the decrease in absorbance of the mixture at 517 nm relative to the solvent as blank [13].

### **3. Results and discussion**

#### **3.1. Botanical identification**

The characterization of an exsiccata containing herborized plant material of *Eleutherine plicata* Herb. confirmed the identity of the investigated herbal drug. According to Tropicos®, *Eleutherine bulbosa* is an accepted name for *E. plicata*.

#### **3.2. Extraction and fractionation**

The ethanol extract, EE, weighed 63 g, from which *circa* 30g provided four fractions by solid/ liquid partition: HF = 2.103 g, 7%; CF = 3,224 g, 11%; EAF = 5,551 g, 18%; and RF = 12.494 g, 41%. The process generates a loss in mass of about 25%, partially due to the solubility of the constituents of the extract in the employed solvents and the partition coefficient of them; and to the evaporation of volatile substances inherent to the methods used to obtain and concen‐ trate the fractions.

#### **3.3. Phytochemical screening**

The positive results of the phytochemical approach of EE and fractions considering the presence of 18 classes of secondary metabolites are shown in Table 2.

The metabolites were detected in fractions according to the polarity of the solvents used in the fractionation, such as steroids and triterpenes, azulenes, anthraquinones and naphthoqui‐ nones in HF and CF, which are solvents and metabolites of low polarity. Moreover, saponins, tannins and phenols, and coumarin derivatives present in EAF and RF show middle to high polarities. Reducing sugars, detected only in EE, are metabolites of very high polarity, which in liquid-liquid partition do not migrate to organic layer. saponin, coumarin derivatives, and tannins and phenols were not detected in EE, probably due to their concentration in the crude extract or the occurrence of interference on the reagent used.

#### **3.4. Thin Layer Chromatography (TLC) analyses**

TLC analyses are used to define the chromatographic profile of extracts and fractions, thereby contributing to the quality control of herbal drugs and their derivatives. In present case,


coumarin derivatives present in EAF and RF show middle to high polarities. Reducing sugars, detected **Table 2.** Metabolic classes detected in derivatives of *E*. *plicata*.

Aliquots of 50 μL were applied at the following concentrations: **1**, 500 μg/mL; **2,** 1 mg/mL; CF,

The antioxidant capacity of EE, Ep1, and Ep2 was evaluated using the free radical 2,2 diphenyl-1-picrylhydrazyl ([DPPH]⋅+) and as reference the substance butylhydroxytoluene (BHT). The reaction was accompanied by color change and the activity is monitored by the

The characterization of an exsiccata containing herborized plant material of *Eleutherine plicata* Herb. confirmed the identity of the investigated herbal drug. According to Tropicos®,

The ethanol extract, EE, weighed 63 g, from which *circa* 30g provided four fractions by solid/ liquid partition: HF = 2.103 g, 7%; CF = 3,224 g, 11%; EAF = 5,551 g, 18%; and RF = 12.494 g, 41%. The process generates a loss in mass of about 25%, partially due to the solubility of the constituents of the extract in the employed solvents and the partition coefficient of them; and to the evaporation of volatile substances inherent to the methods used to obtain and concen‐

The positive results of the phytochemical approach of EE and fractions considering the

The metabolites were detected in fractions according to the polarity of the solvents used in the fractionation, such as steroids and triterpenes, azulenes, anthraquinones and naphthoqui‐ nones in HF and CF, which are solvents and metabolites of low polarity. Moreover, saponins, tannins and phenols, and coumarin derivatives present in EAF and RF show middle to high polarities. Reducing sugars, detected only in EE, are metabolites of very high polarity, which in liquid-liquid partition do not migrate to organic layer. saponin, coumarin derivatives, and tannins and phenols were not detected in EE, probably due to their concentration in the crude

TLC analyses are used to define the chromatographic profile of extracts and fractions, thereby contributing to the quality control of herbal drugs and their derivatives. In present case,

presence of 18 classes of secondary metabolites are shown in Table 2.

extract or the occurrence of interference on the reagent used.

**3.4. Thin Layer Chromatography (TLC) analyses**

decrease in absorbance of the mixture at 517 nm relative to the solvent as blank [13].

2,500 μg/mL; and crude EE, 1 mL.

**3. Results and discussion**

**3.2. Extraction and fractionation**

**3.3. Phytochemical screening**

trate the fractions.

*Eleutherine bulbosa* is an accepted name for *E. plicata*.

**3.1. Botanical identification**

*2.2.9. Antioxidant activity of EE, 1, and 2*

328 Phytochemicals - Isolation, Characterisation and Role in Human Health

different chromatographic systems were tested to obtain the chromatographic profile of EE, HF, and CF, wherein normal phase silica gel and chloroform/acetone (99:1) produced chro‐ matograms with very good resolution. organic layer. saponin, coumarin derivatives, and tannins and phenols were not detected in EE, probably due to their concentration in the crude extract or the occurrence of interference on the reagent used. 4.4 THIN LAYER CHROMATOGRAPHY (TLC) ANALYSES

only in EE, are metabolites of very high polarity, which in liquid-liquid partition do not migrate to

TLC analyses are used to define the chromatographic profile of extracts and fractions, thereby

Figure 4A shows three colored yellow zones with Rfs 0.25, 0.31, and 0.44, respectively, present in EE as well in the fractions HF and CF, and pink colored areas with Rf 0.5 and in 0.62 HF and CF. When the same chromatogram is observed under UV light at 254 nm (Figure 4B), three absorption zones in EE and fractions can be observed with Rfs 0.25, 0.31 and 0.44, respectively, and in HF, one bluish zone by Rf 0.62. contributing to the quality control of herbal drugs and their derivatives. In present case, different chromatographic systems were tested to obtain the chromatographic profile of EE, HF, and CF, wherein normal phase silica gel and chloroform/acetone (99:1) produced chromatograms with very good resolution. Figure 4A shows three colored yellow zones with Rfs 0.25, 0.31, and 0.44, respectively, present in EE as well in the fractions HF and CF, and pink colored areas with Rf 0.5 and in 0.62 HF and CF.

This chromatogram also shows, in EE and fractions, brown colored areas with Rfs 0.25, 0.31, and 0.44, respectively, indicating the presence of naphthoquinones after treatment with KOH 10% in methanol (Figure 4C), which is a reagent to detect quinones [14]. Additionally, three rose colored spots with Rfs 0.5, 0.62, and 0.87, respectively, can be seen in HF. When the same chromatogram is observed under UV light at 254 nm (Figure 4B), three absorption zones in EE and fractions can be observed with Rfs 0.25, 0.31 and 0.44, respectively, and in HF, one bluish zone by Rf 0.62. This chromatogram also shows, in EE and fractions, brown colored areas with Rfs 0.25, 0.31, and 0.44, respectively, indicating the presence of naphthoquinones after treatment with KOH 10% in

> methanol (Figure 4C), which is a reagent to detect quinones [14](WAGNER, BLADT, 2001). Additionally, three rose colored spots with Rfs 0.5, 0.62, and 0.87, respectively, can be seen in HF.

**Figure 4.** Chromatograms on silica gel eluted with chloroform/acetone 99:1; A – observed under visible light; B – under UV 254nm; C – visible light, after KOH 10%.

#### **3.5. Isolation of major chemical constituents**

Sample A1 (83 mg) appears as an isolated chemical substance when analyzed by TLC; it was named **1** and can be observed in HF fraction with Rf 0.62 (Figure 4A, B, C). From C-4, a sample reacting like a naphthoquinone could be purified by preparative TLC yielding 51 mg of a substance that was named **2** that in the TLC analyses showed Rf 0.44 and can also be observed in Figure 4A, B, C.

#### **3.6. Structural characterization of isolated substances**

#### *3.6.1. Isoeleutherol*

The 1 H-NMR spectral data of **1** listed in Table 3 shows characteristic signals of aromatic hydrogen at positions C-4, C-6, C-7, and C-8. The aromatic hydrogen H-4 appears as a singlet (δ = 7.860 ppm), H-6 hydrogen (δ = 7.545 ppm) appears coupled with H-7 (δ = 7.399 ppm) making a doublet. Hydrogen H-7 (δ = 7.399 ppm) couples with H-6 (δ = 7,545 ppm) and H-8 (δ = 6.940ppm), appearing as an overlaid double doublet or false triplet. H-8 hydrogen (δ = 6.940 ppm) appears coupled with H-7 (δ = 7.399) as a doublet.

The signal observed as a singlet (δ = 4.108 ppm) refers to the hydrogen atoms of the methoxy group attached to the aromatic ring. There is also a signal of a methyl group (δ = 1.736 ppm), which is attached to C-1 of the furan ring, coupling with H-1 (δ = 5.718ppm), thus appearing as a doublet.

The hydrogen at C-1 (δ = 5.718ppm) couples with the hydrogen atoms of the methyl group at the same position, generating a quartet. Finally, a phenolic hydrogen appears as a singlet at δ = 9.644.


**Table 3.** Data of 1 H-NMR analysis of Ep1 compared to authentic isoeleutherol.

The 13C-NMR spectral data of **1** listed in Table 4 shows the presence of 14 carbon atoms. The signals with δ = 19.34 and δ = 56.77 are characteristic of methyl carbon atoms of C-11 and C-10, respectively, and the signal at δ = 76.80 is characteristic of carbon C-1. The signals at δ = 126.79, δ = 123.82, δ = 116.67, and δ = 106.44 correspond to the carbons C-4, C-5, C-6 and C-7, respec‐ tively. The carbon C-3 of furan ring that is double bonded to an oxygen atom shows a signal at δ = 170.69 and the resonance of non-substituted aromatic carbon atoms such as C-4a and C-8a appears at δ = 137.37 and δ = 117.66. The resonance at δ = 156.42 refers to C-8 where a methoxy group is attached, while the signal at δ = 149.20 corresponds to C-9 bonded to the hydroxyl group. Finally, the carbon atoms C-3a, C-9a of the furan residue condensed with an aromatic ring resonate at δ = 126.04 and δ = 128.07, respectively.

**3.5. Isolation of major chemical constituents**

330 Phytochemicals - Isolation, Characterisation and Role in Human Health

**3.6. Structural characterization of isolated substances**

6.940 ppm) appears coupled with H-7 (δ = 7.399) as a doublet.

in Figure 4A, B, C.

*3.6.1. Isoeleutherol*

as a doublet.

**HYDROGEN Ep1 δ(ppm)**

\*From: HARA et al., 1997[15]

**Table 3.** Data of 1

= 9.644.

The 1

Sample A1 (83 mg) appears as an isolated chemical substance when analyzed by TLC; it was named **1** and can be observed in HF fraction with Rf 0.62 (Figure 4A, B, C). From C-4, a sample reacting like a naphthoquinone could be purified by preparative TLC yielding 51 mg of a substance that was named **2** that in the TLC analyses showed Rf 0.44 and can also be observed

H-NMR spectral data of **1** listed in Table 3 shows characteristic signals of aromatic hydrogen at positions C-4, C-6, C-7, and C-8. The aromatic hydrogen H-4 appears as a singlet (δ = 7.860 ppm), H-6 hydrogen (δ = 7.545 ppm) appears coupled with H-7 (δ = 7.399 ppm) making a doublet. Hydrogen H-7 (δ = 7.399 ppm) couples with H-6 (δ = 7,545 ppm) and H-8 (δ = 6.940ppm), appearing as an overlaid double doublet or false triplet. H-8 hydrogen (δ =

The signal observed as a singlet (δ = 4.108 ppm) refers to the hydrogen atoms of the methoxy group attached to the aromatic ring. There is also a signal of a methyl group (δ = 1.736 ppm), which is attached to C-1 of the furan ring, coupling with H-1 (δ = 5.718ppm), thus appearing

The hydrogen at C-1 (δ = 5.718ppm) couples with the hydrogen atoms of the methyl group at the same position, generating a quartet. Finally, a phenolic hydrogen appears as a singlet at δ

> **\* ISOELEUTHEROL δ(ppm)**

1-ME 1.736 1.73 d 6.5

H-1 5.718 5.70 dd 6.5 H-5 6.927 6.93 d 7.7 H-6 7.399 7.39 t 7.7 H-7 7.545 7.54 t 7.7

8-OME 4.108 4.11 s

H-4 7.863 7.84 s 9-OH 9.644 9.63 s

H-NMR analysis of Ep1 compared to authentic isoeleutherol.

**MULTIPLICITY J (Hz)**


**Table 4.** Data of 13C-NMR analysis of Ep1 compared to authentic isoeleutherol.

The very close correspondence of the 1 H- and 13C-NMR spectral data of **1** to those found in the literature (Tables 3 and 4) allows to infer that the isolated substance is isoeleutherol (Figure 5), which has been isolated from *Eleutherine americana* Merr. et Heyne by Hara et al. (1997) [15] being this the first report of its occurrence in *E*. *plicata* Herb.

Hara et al. (1997) [15] found that isoeleutherol did not inhibit the enzyme topoisomerase II DNA dependent, but significantly hinders the HIV replication in H9 lymphocytes.

used as a chemical marker of *E. plicata* and its derivatives.

Figure 5. Chemical structure of isoeleutherol. **Figure 5.** Chemical structure of isoeleutherol.

Since isoeleutherol seems to be very stable and the major chemical constituent in EE, it could be used as a chemical marker of *E. plicata* and its derivatives. Hara et al. (1997)[15] found that isoeleutherol did not inhibit the enzyme topoisomerase II DNA dependent, but significantly hinders the HIV replication in H9 lymphocytes.

#### *3.6.2. Isoeleutherine* Since isoeleutherol seems to be very stable and the major chemical constituent in EE, it could be

Substance **2** was analyzed by 1 H-NMR and its structure was characterized by comparison of the obtained spectral data to those reported in the literature. Table 5 shows the resonance values of H-6, H-7, and H-8 from the aromatic ring δ = 7.73, δ = 7.64, and δ = 7.27, respectively, with identical coupling constant *J*6H-7H=*J*7H-8H=6.7Hz. The signal of H-6 appears as a doublet since it couples with H-7, which in turn appears as a false triplet due the coupling with H-6 and H-8. 4.6.2 ISOELEUTHERINE Substance **2** was analyzed by <sup>1</sup> H-NMR and its structure was characterized by comparison of the obtained spectral data to those reported in the literature. Table 5 shows the resonance values of H-6, H-7, and H-8 from the aromatic ring δ = 7.73, δ = 7.64, and δ = 7.27, respectively, with identical coupling

The hydrogen atoms of the methyl group attached to C-1 appear as a doublet at δ = 1:54 (*J*H1- CH3=6.7Hz) and that one bonded to C-3 at δ = 1:33 (*J*H3-CH3=6.1Hz). constant *J*6H-7H=*J*7H-8H=6.7Hz. The signal of H-6 appears as a doublet since it couples with H-7, which in turn appears as a false triplet due the coupling with H-6 and H-8.


**Table 5.** Data of 1 H-NMR analysis of Ep2 compared to authentic isoeleutherine.

The spectral data listed in Table 5 when compared with that from the literature permit to deduce that **2** corresponds to isoeleutherine (Figure 6), a naphthoquinone already isolated from *Eleutherine bulbosa* Mill [9] and from *E. americana* Merr. by Hara et al. (1997) [15]. This is the first report of the occurrence of isoeleutherine in *E. plicata*.

The spectral data listed in Table 5 when compared with that from the literature permit to deduce

that **2** corresponds to isoeleutherine (Figure 6), a naphthoquinone already isolated from *Eleutherine* 

*bulbosa* Mill [9](ALVES et al., 2003) and from *E. americana* Merr. by Hara et al. (1997)[15]. This is

**Figure 6.** Chemical structure of isoeleutherine. The LC-DAD profile of EE and CF was obtained using the method developed by

Since isoeleutherol seems to be very stable and the major chemical constituent in EE, it could

Hara et al. (1997)[15] found that isoeleutherol did not inhibit the enzyme topoisomerase II DNA

1 2

10

Since isoeleutherol seems to be very stable and the major chemical constituent in EE, it could be

Figure 5. Chemical structure of isoeleutherol.

3a 3

9a

the obtained spectral data to those reported in the literature. Table 5 shows the resonance values of H-6, H-7, and H-8 from the aromatic ring δ = 7.73, δ = 7.64, and δ = 7.27, respectively, with identical coupling constant *J*6H-7H=*J*7H-8H=6.7Hz. The signal of H-6 appears as a doublet since it couples with H-7, which in turn appears as a false triplet due the coupling with H-6

The hydrogen atoms of the methyl group attached to C-1 appear as a doublet at δ = 1:54 (*J*H1-

**\* ISOELEUTHERINE δ(ppm)**

The hydrogen atoms of the methyl group attached to C-1 appear as a doublet at δ = 1:54 (*J*H1-

\* ISOELEUTHERINE

1-H 5.00 5.01 q 6.7 1-ME 1.53 1.53 d 6.7

3-ME 1.33 1.34 d 6.1 4-αH 2.69 2.68 dd 3.5–19.0

H-NMR analysis of Ep2 compared to authentic isoeleutherine.

δ(ppm) MULTIP. J (Hz)

obtained spectral data to those reported in the literature. Table 5 shows the resonance values of H-6, H-7, and H-8 from the aromatic ring δ = 7.73, δ = 7.64, and δ = 7.27, respectively, with identical coupling constant *J*6H-7H=*J*7H-8H=6.7Hz. The signal of H-6 appears as a doublet since it couples with H-7, which in

1-H 5.00 5.01 q 6.7 1-ME 1.53 1.53 d 6.7

3-ME 1.33 1.34 d 6.1 4-αH 2.69 2.68 dd 3.5–19.0 4-βH 2.23 2.23 dd 11.0–19.0 6-H 7.73 7.74 d 6.7 7-H 7.64 7.64 t 6.7 8-H 7.27 7.27 d 6.7

3-H 3.95 3.96 m

3-H 3.95 3.96 m

9-OME 4.00 4.00 s

H-NMR analysis of Ep2 compared to authentic isoeleutherine.

H-NMR and its structure was characterized by comparison of

**R=αME**

H-NMR and its structure was characterized by comparison of the

**MULTIP. J (Hz)**

be used as a chemical marker of *E. plicata* and its derivatives.

6 7

332 Phytochemicals - Isolation, Characterisation and Role in Human Health

8

11

used as a chemical marker of *E. plicata* and its derivatives.

dependent, but significantly hinders the HIV replication in H9 lymphocytes.

4 4a 5

8a <sup>9</sup>

CH3=6.7Hz) and that one bonded to C-3 at δ = 1:33 (*J*H3-CH3=6.1Hz).

turn appears as a false triplet due the coupling with H-6 and H-8.

CH3=6.7Hz) and that one bonded to C-3 at δ = 1:33 (*J*H3-CH3=6.1Hz).

*3.6.2. Isoeleutherine*

and H-8.

Substance **2** was analyzed by 1

4.6.2 ISOELEUTHERINE

**Figure 5.** Chemical structure of isoeleutherol.

Substance **2** was analyzed by <sup>1</sup>

**HYDROGÊN Ep2 δ(ppm)**

Table 5. Data of 1

HYDROGÊN Ep2 δ(ppm)

\*From: HARA et al., 1997[15]

**Table 5.** Data of 1

#### **3.7. Chromatographic profile by LC-DAD** Paramapojn et al. [12](2008) with modifications and the best chromatograms were registered at

the first report of the occurrence of isoeleutherine in *E. plicata*.

The LC-DAD profile of EE and CF was obtained using the method developed by Paramapojn et al. (2008) [12] with modifications and the best chromatograms were registered at 250 nm. EE profile shows two peaks of high intensity with retention time – Rt = 18.93 min and 20.83 min, with areas of 45,854,675 and 60,180,902 and a purity of 99.97% and 99.72%, respectively (Figure 7). 250 nm. EE profile shows two peaks of high intensity with retention time – Rt = 18.93 min and 20.83 min, with areas of 45,854,675 and 60,180,902 and a purity of 99.97% and 99.72%, respectively (Figure 7).

Figure 7. LC-DAD profile of EE registered at 250 nm. **Figure 7.** LC-DAD profile of EE registered at 250 nm.

CF chromatogram shows two peaks with high intensity at 19.12 min (Peak 1) and 21.18 min (Peak 2), with areas of 7,813,739 and 1,900,571 and purity of 98.29% and 99.75%, respectively (Figure 8).

The isolated isoeleutherol was also analyzed by LC-DAD under the same conditions as EE and CF, generating a peak at 21.71 min with 3,641,711 area and purity of 99.92% (Figure 9).

0,59 0,93 1,041,36

0,0

0,4 0,5

0,6

0,7

0,5

1,0

Absorbance (AU)

1,5

2,0

1,71 1,97

4,85 6,00 8,03 8,409,55 10,8012,40

6,61 Peak1

2), with areas of7,813,739and1,900,571and purity of98.29% and99.75%, respectively(Figure 8).

14,19 15,01 17,39

Figure 7.LC-DADprofileofEEregistered at 250nm.

0 5 10 15 20 25 30 35 40 45 Retention Time (min)

26,40 26,99 28,40 29,04 30,72 33,07

Peak 2

Peak 2

18,93 20,85

CF chromatogram shows twopeakswith high intensityat19.12min (Peak 1)and21.18 min (Peak

21,12

The isolated isoeleutherolwas also analyzed byLC-DADunder the same conditionsas EEandCF, Figure 8. LC-DAD profile of the chloroform fraction registered at 250 nm. **Figure 8.** LC-DAD profile of the chloroform fraction registered at 250 nm.

generating a peak at21.71minwith 3,641,711areaand purity of99.92% (Figure 9).

Figure 9.HPLCchromatogramofisoeleutherolat a wavelength of250nm. **Figure 9.** HPLC chromatogram of isoeleutherol at a wavelength of 250 nm.

registered.

A

bs

or

ba

nc

e

The same procedure was adopted to analyze the obtained isoeleutherine, producing the LC-DAD profile, showed in Figure 10, where a peak at 18.13min with area 24,727,851 and purity of 99.14% is registered. Figure 9. HPLC chromatogram of isoeleutherol at a wavelength of 250 nm. The same procedure was adopted to analyze the obtained isoeleutherine, producing the LC-DAD profile, showed in Figure 10, where a peak at 18.13min with area 24,727,851 and purity of 99.14% is

> The reverse survey feature in the library of the chromatograph shows correlations of 97.40% and 99.89% between the UV spectrum of the peak 01 in EE (Figure 7) and in CF (Figure 8) and that of isoeleutherine. Similarly, the peak 02 in EE (Figure 7) and in CF (Figure 8) showed a correlation of 99.84% and 99.98%, respectively, between the UV spectra of both the peaks and that of isoeleutherol. 18,1 3

> > Figure 10. LC-DAD profile of isoeleutherine registered at 250 nm.

32,96

Figure 9. HPLC chromatogram of isoeleutherol at a wavelength of 250 nm.

15,95

The same procedure was adopted to analyze the obtained isoeleutherine, producing the LC-DAD

**0 5 10 15 20 25 30 35 40 45 Retention Time (min)**

The isolated isoeleutherol was also analyzed by LC-DAD under the same conditions as EE and

21,71

CF, generating a peak at 21.71 min with 3,641,711 area and purity of 99.92% (Figure 9).

Figure 10. LC-DAD profile of isoeleutherine registered at 250 nm. **Figure 10.** LC-DAD profile of isoeleutherine registered at 250 nm.

**0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4**

**Absorbance (AU)**

Figure 7.LC-DADprofileofEEregistered at 250nm.

0 5 10 15 20 25 30 35 40 45 Retention Time (min)

26,40 26,99 28,40 29,04 30,72 33,07

Peak 2

registered.

Peak 2

Peak 2

18,93 20,85

CF chromatogram shows twopeakswith high intensityat19.12min (Peak 1)and21.18 min (Peak

21,12

2), with areas of7,813,739and1,900,571and purity of98.29% and99.75%, respectively(Figure 8).

14,19 15,01 17,39

0,59 0,93 1,041,36

0,91

0,0 0,1

**0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7**

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4

of 99.14% is registered.

that of isoeleutherol.

Absorbance (AU)

registered.

A

bs

or

ba

nc

e

0,2

0,3

0,4 0,5

Absorbance (AU)

**Absorbance (AU)**

0,6

0,7

1,81 2,13

> 0,91 1,81 2,13

6,56

334 Phytochemicals - Isolation, Characterisation and Role in Human Health

6,56 7,20 8,699,68 9,92 11,20 11,92 12,29 13,73 14,43 15,41 16,00 17,55

**Figure 8.** LC-DAD profile of the chloroform fraction registered at 250 nm.

7,20

8,69

9,68 9,92 11,20

11,92 12,29

Peak 1

13,73

Peak 1

14,43

15,41

16,00

17,55

19,12 19,12

21,12

0,0

0,5

1,0

Absorbance (AU)

1,5

2,0

1,71 1,97

4,85 6,00 8,03 8,409,55 10,8012,40

6,61 Peak1

Figure 8.LC-DAD profileof thechloroformfractionregistered at 250nm.

**0 5 10 15 20 25 30 35 40 45 Retention Time (min)**

Figure 8. LC-DAD profile of the chloroform fraction registered at 250 nm.

21,71

The isolated isoeleutherol was also analyzed by LC-DAD under the same conditions as EE and

32,96

0 5 10 15 20 25 30 35 40 45 Retention Time (min)

Figure 9.HPLCchromatogramofisoeleutherolat a wavelength of250nm.

The same procedure was adopted to analyze the obtained isoeleutherine, producing the LC-DAD

The same procedure was adopted to analyze the obtained isoeleutherine, producing the LC-DAD profile, showed in Figure 10, where a peak at 18.13min with area 24,727,851 and purity

Figure 9. HPLC chromatogram of isoeleutherol at a wavelength of 250 nm.

The reverse survey feature in the library of the chromatograph shows correlations of 97.40% and 99.89% between the UV spectrum of the peak 01 in EE (Figure 7) and in CF (Figure 8) and that of isoeleutherine. Similarly, the peak 02 in EE (Figure 7) and in CF (Figure 8) showed a correlation of 99.84% and 99.98%, respectively, between the UV spectra of both the peaks and

profile, showed in Figure 10, where a peak at 18.13min with area 24,727,851 and purity of 99.14% is

Figure 10. LC-DAD profile of isoeleutherine registered at 250 nm.

0 5 10 15 20 25 30 35 40 45 Retention Time (min)

15,95

generating a peak at21.71minwith 3,641,711areaand purity of99.92% (Figure 9).

CF, generating a peak at 21.71 min with 3,641,711 area and purity of 99.92% (Figure 9).

**Figure 9.** HPLC chromatogram of isoeleutherol at a wavelength of 250 nm.

18,1

3

#### **3.8. Antioxidant activity of EE, isoeleutherol, and isoeleutherine**

Figure 11 and Table 6 show the evaluation of the antioxidant activity of EE, isoeleutherol, and isoeleutherine on DPPH in comparison to the results obtained for BHT used as standard. The antioxidant activity of isoeleutherol (Ep1) appears in concentrations up from 4 μg/mL; for EE, 5 μg/mL; and isoeleutherine (Ep2), 6 μg/mL; while BHT showed activity in concentrations above 1 μg/mL. Figure 11 and Table 6 show the evaluation of the antioxidant activity of EE, isoeleutherol, and isoeleutherine on DPPH in comparison to the results obtained for BHT used as standard. The antioxidant activity of isoeleutherol (Ep1) appears in concentrations up from 4 μg/mL; for EE, 5 μg/mL; and

isoeleutherine (Ep2), 6 μg/mL; while BHT showed activity in concentrations above 1 μg/mL.

Figure 11. Evaluation of antioxidant activity of EE, and isoeleuterol, and isoeleutherine against DPPH. **Figure 11.** Evaluation of antioxidant activity of EE, and isoeleuterol, and isoeleutherine against DPPH.

about 5× lower.

 Among the tested samples, isoeleutherol showed the best antioxidant activity considering its Inhibition Concentration value (Table 6), followed by EE and isoeleutherine. However, when the IC50 values of isoeleutherol and BHT are compared, stay clear that the antioxidant activity of isoeleutherol is

Table 6. Determination of IC50 of EE, isoeleutherol, isoeleutherine and BHT.

The antioxidant activity of isoeleutherol, higher than that observed for EE and isoelutherine, may

be attributed to the hydroxyl group at C-9; this phenolic residue may act as free radical scavenger and

**SAMPLE IC50** BHT 17.83 EE 94.72

ISOELEUTHEROL 84.63 ISOELEUTHERINE 281.04 Among the tested samples, isoeleutherol showed the best antioxidant activity considering its Inhibition Concentration value (Table 6), followed by EE and isoeleutherine. However, when the IC50 values of isoeleutherol and BHT are compared, stay clear that the antioxidant activity of isoeleutherol is about 5× lower.


**Table 6.** Determination of IC50 of EE, isoeleutherol, isoeleutherine and BHT.

The antioxidant activity of isoeleutherol, higher than that observed for EE and isoelutherine, may be attributed to the hydroxyl group at C-9; this phenolic residue may act as free radical scavenger and sometimes as chelating agent of metal ion with effective action, mainly in preventing lipid oxidation, acting both on the initiation step as on the propagation step of this oxidative process.

Table 6 shows that EE has a higher IC50 than isoeleutherol, but lower than that of isoeleutherine. This fact can find explanation in the presence of tannins in EE, which are polyphenols that have adequate chemical structure for the capture of free radicals, contributing to an effective antioxidant capacity of the sample. It is noteworthy to mention that the presence of naphtho‐ quinones in EE may antagonize the activity of tannins since these secondary metabolites can induce oxidative stress or show pro-oxidant capacity, leading EE to present a very low antioxidant activity. Indeed, isoeleutherine presents a negligible antioxidant activity as expected from its structural characteristics, but its occurrence in aqueous extract may be a reason for the antiamoebic activity detected in a previous work [5] and described for *E. bulbosa*, a synonym of *E. plicata* according to TROPICOS®3, 30 years before today [16].

#### **4. Conclusions**

Isoeleutherol and isoeleutherine described before in other *Eleutherine* species were isolated from the chloroform fraction of EE and characterized by 1 H- and 13C-NMR. Their HPLC analyses allow confirmation that both are present in the herbal drug, dried bulbs of *E. plicata*. Isoeleutherol seems to be the major chemical constituent in EE and thus can be indicated as a chemical marker for the quality control of this plant species and its derivatives. The occurrence of isoeleutherine in aqueous antiamoebic extract indicates this substance to be used in the quality control of the extract and derivatives, mainly if the substance can be linked to the

<sup>3</sup> tropicos.org an information system of Missouri Botanical Garden's electronic databases, Access: 21 Sep. 2014.

reported activity. The isolation and characterization of isoeleutherol and isoeleutherine, which show well-described pharmacological activities, justify the potential of *E. plicata* Herb. to originate a phytomedicine to face neglected diseases, such as Amoeba infection, and probably explain the reason why this plant finds a wide popular use in Amazonian countries.

#### **Author details**

Among the tested samples, isoeleutherol showed the best antioxidant activity considering its Inhibition Concentration value (Table 6), followed by EE and isoeleutherine. However, when the IC50 values of isoeleutherol and BHT are compared, stay clear that the antioxidant activity

> **SAMPLE IC50** BHT 17.83 EE 94.72

ISOELEUTHEROL 84.63 ISOELEUTHERINE 281.04

The antioxidant activity of isoeleutherol, higher than that observed for EE and isoelutherine, may be attributed to the hydroxyl group at C-9; this phenolic residue may act as free radical scavenger and sometimes as chelating agent of metal ion with effective action, mainly in preventing lipid oxidation, acting both on the initiation step as on the propagation step of this

Table 6 shows that EE has a higher IC50 than isoeleutherol, but lower than that of isoeleutherine. This fact can find explanation in the presence of tannins in EE, which are polyphenols that have adequate chemical structure for the capture of free radicals, contributing to an effective antioxidant capacity of the sample. It is noteworthy to mention that the presence of naphtho‐ quinones in EE may antagonize the activity of tannins since these secondary metabolites can induce oxidative stress or show pro-oxidant capacity, leading EE to present a very low antioxidant activity. Indeed, isoeleutherine presents a negligible antioxidant activity as expected from its structural characteristics, but its occurrence in aqueous extract may be a reason for the antiamoebic activity detected in a previous work [5] and described for *E.*

*bulbosa*, a synonym of *E. plicata* according to TROPICOS®3, 30 years before today [16].

Isoeleutherol and isoeleutherine described before in other *Eleutherine* species were isolated

analyses allow confirmation that both are present in the herbal drug, dried bulbs of *E. plicata*. Isoeleutherol seems to be the major chemical constituent in EE and thus can be indicated as a chemical marker for the quality control of this plant species and its derivatives. The occurrence of isoeleutherine in aqueous antiamoebic extract indicates this substance to be used in the quality control of the extract and derivatives, mainly if the substance can be linked to the

3 tropicos.org an information system of Missouri Botanical Garden's electronic databases, Access: 21 Sep. 2014.

H- and 13C-NMR. Their HPLC

of isoeleutherol is about 5× lower.

336 Phytochemicals - Isolation, Characterisation and Role in Human Health

oxidative process.

**4. Conclusions**

**Table 6.** Determination of IC50 of EE, isoeleutherol, isoeleutherine and BHT.

from the chloroform fraction of EE and characterized by 1

Luiz Claudio da Silva Malheiros1 , João Carlos Palazzo de Mello2 and Wagner Luiz Ramos Barbosa1\*

\*Address all correspondence to: barbosa@ufpa.br

1 Faculty of Pharmaceutical Sciences, Health Sciences Institute, Pará Federal University; Be‐ lém, Pará, Brazil

2 Maringá State University, Health Sciences Center, Department of Pharmacy and Pharma‐ cology; Maringá, Paraná, Brazil

#### **References**


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[8] Lin, J.; Puckree, T.; Mvelase, T.P. Anti-diarrhoeal evaluation of some medicinal plants used by Zulu traditional healer. Journal of Ethnopharmacology. 79, 1, 53, 2002.

[9] Alves, T.M.A.; Kloos, H.; Zani, C.L. Eleutherinone, a novel fungitox naphthoquinone from *Eleutherine bulbosa* (Iridaceae). Mem. Inst. Oswaldo Cruz, Rio de Janeiro, 98, 5,

[10] Bruneton, J. Farmacognosia. Fitoquímica e plantas medicinales. 2nd Ed. Zaragoza:

[11] Barbosa, W.L.R. Manual para análise fitoquímica e cromatográfica de extratos vege‐ tais. Belém: UFPA, 2001. Available in http://www2.ufpa.br/rcientifica/didaticos\_cien‐

[12] Paramapojn, S.; Ganzera, M.; Gritsanapan, W.; Stuppner, H. Analysis of naphthoqui‐ none derivatives in the Asian medicinal plant *Eleutherine americana* by RP-HPLC and LC-MS. Journal of Pharmaceutical and Biomedical Analysis, 47, 990-993, 2008. Avail‐

[13] Blois, M.S. Antioxidant determinations by the use of a stable free radical. Nature, 181, 1199-1200, 1958. Available in: www.nature.com/nature/journal/v181/n4617/abs/

[14] Wagner, H; Bladt, S. Plant drug analysis: A thin layer chromatography atlas. 2nd Ed.

[15] Hara, H.; Maruyama, N.; Yamashita, S.; Hayashi, Y.; Lee, K.; Bastow, K.F.; Marumo‐ to, R.; Imakura, M. Elecanacin, a novel new naphthoquinone from the bulb of *Eleu‐ therine americana* Merr. Et Heyne. Chemical Pharmaceutical Bulletin, 45, 10, 1714-1716, 1997. Available in: www.journalarchive.jst.go.jp Accessed: 12 Sep. 2008.

[16] Vickers, W. T. & Plowman, T. *Useful plants* of the Siona and *Secoya*. *Indians of Eastern Ecuador*. Fieldiana. 15:1-63. 1984. Available online on Scholar Google. Access: 21 Sep.

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Nova. 26, 3. São Paulo. May/June, 2003.

338 Phytochemicals - Isolation, Characterisation and Role in Human Health

ACRIBIA S.A. 2001, 405-420.

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Springer, 275-279, 2001.

2014.

### *Edited by A. Venket Rao and Leticia G. Rao*

Global dietary recommendations emphasize the consumption of plant-based foods for the prevention and management of chronic diseases. Plants contain many biologically active compounds referred to as phytochemicals or functional ingredients. These compounds play an important role in human health. Prior to establishing the safety and health benefits of these compounds, they must first be isolated, purified, and their physico-chemical properties established. Once identified, their mechanisms of actions are studied. The chapters are arranged in the order from isolation, purification and identification to in vivo and clinical studies, there by covering not only the analytical procedures used but also their nutraceutical and therapeutic properties.

Phytochemicals - Isolation, Characterisation and Role in Human Health

Phytochemicals

Isolation, Characterisation and Role

in Human Health

*Edited by A. Venket Rao and Leticia G. Rao*

Photo by ajijchan / iStock