**2. Terpenoids**

Terpenoids are the largest and most diverse family of natural products, ranging in structure from linear to polycyclic molecules and in size from the five-carbon hemiterpenes to natural rubber, comprising thousands of isoprene units. All terpenoids are synthesized through the condensation of isoprene units (C5) and are classified by the number of five-carbon units present in the core structure (Mahmoud et al. 2002). Many flavor and aromatic molecules, such as menthol, linalool, geraniol and caryophyllene are formed by monoterpenes (C10), with two isoprene units, and sesquiterpenes (C15), with three isoprene units. Other bioactive compounds, such as diterpenes (C20), triterpenes (C30) and tetraterpenes (C40) show very special properties and will be also discussed in this chapter.

#### **2.1 Monoterpenes and sesquiterpenes (Plant volatiles)**

Plant volatiles are typically lipophilic liquids with high vapor pressures. Non-conjugated plant volatiles can cross membranes freely and evaporate into the atmosphere when there are no barriers to diffusion. The number of identified volatile chemicals synthesized by various plants exceeds 1000 and is likely to grow as more plants are examined with new methods for detecting and analyzing quantities of volatiles that are often minute (Pichersky et al. 2006; Dudareva et al., 2004). Studying the volatile fraction requires analytical methods and technologies that not only evaluate its composition exhaustively but also monitor variations in its profile and detect trace components characterizing the plant being investigated (Bicchi et al., 2011).

The gas chromatography (GC and GC-MS) is a very powerful analytical tool for the identification of essential oil components. However, GC-MS has its limitations. Isomers usually give very similar mass spectra. This is particularly true for terpenes and even more for sesquiterpenes. Therefore, a favorable match factor between mass spectra is not sufficient for identification (Zellner et al., 2010). Retention indices have been used, together with mass spectrometry, for the proper identification of essential oils composition. Misidentification is not rare, however, either if a non-authentic mass spectra library is used, which means a database built with data from the literature, not from the analysis of real standards, or by misuse of retention indices (Joulain & König, 1998). CC has been applied to solve this problem. After isolation, a NMR analysis can be performed for the unknown or suspect compound, so that the correct mass spectrum and retention index can be recorded.

Despite the advances in analytical methods to evaluate the composition of essential oil, CC remains a powerful technology for separation and characterization of specific compounds of interest. Column chromatography has been largely used and reported for searching and identification of new molecules, sometimes associated with their antimicrobial, antibacterial and antifungal activities. Also, this technique has been successfully used to obtain sufficient amounts of a substance for the investigation of its biological properties and allowing the detection of its olfactory properties. Isolation is also applied to and, very important for volatiles, to evaluate its odor.

or analyzed using various adsorbents and eluents through column chromatography (CC)

Terpenoids are the largest and most diverse family of natural products, ranging in structure from linear to polycyclic molecules and in size from the five-carbon hemiterpenes to natural rubber, comprising thousands of isoprene units. All terpenoids are synthesized through the condensation of isoprene units (C5) and are classified by the number of five-carbon units present in the core structure (Mahmoud et al. 2002). Many flavor and aromatic molecules, such as menthol, linalool, geraniol and caryophyllene are formed by monoterpenes (C10), with two isoprene units, and sesquiterpenes (C15), with three isoprene units. Other bioactive compounds, such as diterpenes (C20), triterpenes (C30) and tetraterpenes (C40) show very

Plant volatiles are typically lipophilic liquids with high vapor pressures. Non-conjugated plant volatiles can cross membranes freely and evaporate into the atmosphere when there are no barriers to diffusion. The number of identified volatile chemicals synthesized by various plants exceeds 1000 and is likely to grow as more plants are examined with new methods for detecting and analyzing quantities of volatiles that are often minute (Pichersky et al. 2006; Dudareva et al., 2004). Studying the volatile fraction requires analytical methods and technologies that not only evaluate its composition exhaustively but also monitor variations in its profile and detect trace components characterizing the plant being

The gas chromatography (GC and GC-MS) is a very powerful analytical tool for the identification of essential oil components. However, GC-MS has its limitations. Isomers usually give very similar mass spectra. This is particularly true for terpenes and even more for sesquiterpenes. Therefore, a favorable match factor between mass spectra is not sufficient for identification (Zellner et al., 2010). Retention indices have been used, together with mass spectrometry, for the proper identification of essential oils composition. Misidentification is not rare, however, either if a non-authentic mass spectra library is used, which means a database built with data from the literature, not from the analysis of real standards, or by misuse of retention indices (Joulain & König, 1998). CC has been applied to solve this problem. After isolation, a NMR analysis can be performed for the unknown or suspect compound, so that the correct mass spectrum and retention index can be recorded. Despite the advances in analytical methods to evaluate the composition of essential oil, CC remains a powerful technology for separation and characterization of specific compounds of interest. Column chromatography has been largely used and reported for searching and identification of new molecules, sometimes associated with their antimicrobial, antibacterial and antifungal activities. Also, this technique has been successfully used to obtain sufficient amounts of a substance for the investigation of its biological properties and allowing the detection of its olfactory properties. Isolation is also applied to and, very important for

and thin layer chromatography (TLC).

investigated (Bicchi et al., 2011).

volatiles, to evaluate its odor.

special properties and will be also discussed in this chapter.

**2.1 Monoterpenes and sesquiterpenes (Plant volatiles)** 

**2. Terpenoids** 

#### **2.1.1 Compound identification**

The major constituents present in the *volatile oils* of different aromatic species may also be isolated or fractionated by silica gel on preparative TLC or CC. For example, the essential oil of *Tanacetum chiliophyllum* was subjected to silica gel CC, using a solvent mixture of nhexane and ethyl acetate, to isolate one of its major components, dihydro-α-cyclogeranyl hexanoate (Salamci et al, 2007).

Djabou et al. (2010) have characterized the essential oils of *Teucrium massiliense* L. from Corsican and Sardinian islands, using a combination of capillary GC/retention indices, GC/MS and 13CNMR spectroscopy after fractionation on CC. A mixture of all Corsican oil samples was submitted to flash chromatography [FC; silica gel, elution with *n*-pentane, then with diethyl ether]. The polar fraction was separated on silica gel and 14 fractions were eluted with a mixture of *n*-pentane and diethyl ether of increasing polarity to give the major components: 6-methyl-3-heptyl acetate, 3-octyl acetate, isobutyl isovalerate, germacrene D and linalool. Successive CC revealed the unknown compound 6-methyl-3-heptyl acetate.

Chemical investigations on the essential oil of *Lippia integrifolia* performed by CC (silica gel using *n*-hexane with increasing amounts of diethyl ether), followed by HPLC, GC–MS, 1H and 13C-NMR spectroscopy led to the identification of 78 components. A new sesquiterpenic alcohol, *trans*-africanan-1α-ol, was identified (Coronel et al. 2006).

Sutour et al. (2008) studied the essential oil of *Mentha suaveolens* ssp*. insularis* (Req.) Greuter, finding pulegone to be the major constituent. However, the second most abundant component of the essential oil remained unknown, being isolated by repeated chromatography on silica gel and submitted to a full set of NMR experiments, and identified as *cis-cis-p-*menthenolide. Fractions were eluted with a gradient of solvents of increasing polarity (pentane:diethyl ether 100:0–0:100).

The essential oil from the leaves of *Piper hispidinervum*, a native species from the Amazon, is very rich in safrole (Maia et al., 1987). During an extensive agronomical study with this species, a population produced an essential oil rich in a different phenylpropanoid (up to 78%). The mass spectrum was identical to that of myristicin (Figure 1). However, a difference of near 30 units in retention index between myristicin and the phenylpropanoid was strong evidence that it was another compound, probably a myristicin isomer. No retention indices were available for the two possible isomers, croweacin and sarisan. For identification, the unknown compound was isolated by CC on silica gel and eluted with hexane-ethyl acetate mixtures. After 1H and 13CNMR studies, it was identified as sarisan (Bizzo et al., 2001).

Fig. 1. Possible isomers of myristicin from the oil of a population of *Piper hispidinervum*; sarisan was the actual compound present.

Secondary Metabolites 135

methyl-*p*-anisaldehydes, trimethoxybenzene, 5-hydroxy-2-methyl-4*H*-pyran-4-one, and lilac

Shimoda et al. (1995) reported that a column extraction method was applied to the separation of volatile compounds from infusions of green tea and black tea, wherein their aromas degrade during distillation even under reduced pressure, and from sake, wherein

Linalool is a very frequent terpenoid in essential oils, usually responsible for the scent of a specific plant, such as mint, basil, and other plant species. In order to evaluate the effect on humans of inhalation of linalool enantiomers (Figure 4), repeated flash chromatography was used to isolate R-(-)-linalool from lavender essential oil, while (S)-(+)-linalool was obtained

The Australian finger lime (*Citrus australasica*), an endemic Australian species, is an unique citrus fruits in terms of their size, shape and aroma. The fractionation of finger lime peel oil by chromatography on silica gel (with a gradient of pentane:ether) was able to identify a high number of terpenyl esters enriched in a medium polar fraction. This included citronellyl and geranyl esters, commonly found in lime peel oils, but also several less common (*Z*)-3-hexenyl esters and bornyl esters. In finger limes these compounds, together with isomenthone, menthol, carvone, *cis*-3-hexenol and methyl salicylate, may also

L-menthyl-L-lactate [(2*S*, 1′*R*, 2′*S*, 5′*R*)-2-isopropoyl-5-methylcyclohexyl 2-hydroxypropanoate, LM-LL] is widely used as a cooling compound in mint flavors, fruit flavors, oral care products, confections and beverages. L-menthyl-L-lactate was identified in dementholized cornmint oil from India by comparing mass spectrum and retention time with a reference sample on two columns. Separation was achieved by increasing polarity of the hexane–methyl-tert-butyl-ether (MTBE) gradient on a flash chromatography column packed with silica gel. It is worth noting that L-menthyl-L-lactate was identified in *M. arvensis* oils from India but not in *M. piperita* oils from the USA. Model experiments proved that LM-LL is formed during water vapor distillation of mint leaves in the presence of lactic acid. Lactic acid is spontaneously formed when mint herb is stored for some days in a humid environment. A prolonged storage of damp mint herb may increase formation of lactic acid and thus give rise to the formation of LM-LL during distillation. The presence of lactic acid will be influenced by the agricultural practice employed (Gassenmeier, 2006).

contribute to the characteristic fresh green aroma of the fruits (Delort et al., 2009).

aldehydes seemed to contribute to Haze honey's aroma (Shimoda et al., 1996).

ethyl alcohol disturbs a quantitative separation of volatile compounds.

from coriander oil and commercial linalool (Sugawara et al. 2000).

Fig. 4. Linalool enantiomers.

Evaluating the essential oils from the leaves of 40 individuals of *Croton cajucara* Benth. from a germplasm bank in Manaus (State of Amazonas, Brazil), Quadros et al. (2011) observed some plants, instead of linalool, produced an oil rich in a hydroxylated sesquiterpene. The mass spectrum was very similar to that of 5-hydroxycalamenene. After the last edition of Adams' reference book (Adams, 2007), it was verified that the retention index of the sesquiterpenic alcohol did not correspond to that of 5-hydroxycalamenene. The oil was chromatographed over silica gel with hexane and hexane-ethyl acetate mixtures. The fraction containing the compound of interest was re-chromatographed on a silica gel preparative TLC plate with hexane-ethyl acetate (90:10). The purified compound was extracted from the silica with chloroform. Two clear singlets in the aromatic hydrogens at 6.56 and 6.94 ppm instead of a doublet pointed to a 7-hydroxy-substituted structure (Figure 2). Chemical shifts were in good agreement with published data for *cis*-7-hydroxycalamene.

Fig. 2. Hydroxylated sesquiterpenes from *Croton cajucara.*

Sometimes, despite all careful efforts and extensive study, wrong structure assignment is published. For example, a new triquinane sesquiterpenic alcohol was isolated from the essential oil of *Anemia tomentosa* var. *anthriscifolia* by CC on silicagel with a hexane-ethyl acetate gradient. A new structural formula, namely (−)-*epi*-presilphiperfolan-1-ol, was initially proposed, after extensive 1D- and 2D-NMR analyses, as well as by GC–MS, chiral bidimensional GC, dehydration reactions, and a comparative (GIAO/DFT) theoretical study of the 13C NMR chemical shifts (Pinto et al., 2009a). Further examination by X-ray diffraction and vibrational circular dichroism studies, however, led to its reassignment to (−)-9-*epi*presilphiperfolan-1-ol (Figure 3), also a new compound. Its absolute configuration was established as 1S,4S,7R,8R,9S (Joseph-Nathan et al., 2010).

Fig. 3. Isomers of (−)-presilphiperfolan-1-ol: the 9-*epi*-isomer was isolated from *Anemia tomentosa* var. *anthriscifolia* essential oil.

#### **2.1.2 Olfactory analysis**

Aroma concentrate separated from an aqueous solution of Haze honey by adsorptive CC had stronger aroma intensity than that separated by other methods. Fractions separated by preparative GC were sniffed to evaluate the sensory importance of the volatile compounds of Haze honey. Benzeneacetaldehyde, linalool, phenethyl alcohol, *p*-cresol, *p*-anisaldehyde,

Evaluating the essential oils from the leaves of 40 individuals of *Croton cajucara* Benth. from a germplasm bank in Manaus (State of Amazonas, Brazil), Quadros et al. (2011) observed some plants, instead of linalool, produced an oil rich in a hydroxylated sesquiterpene. The mass spectrum was very similar to that of 5-hydroxycalamenene. After the last edition of Adams' reference book (Adams, 2007), it was verified that the retention index of the sesquiterpenic alcohol did not correspond to that of 5-hydroxycalamenene. The oil was chromatographed over silica gel with hexane and hexane-ethyl acetate mixtures. The fraction containing the compound of interest was re-chromatographed on a silica gel preparative TLC plate with hexane-ethyl acetate (90:10). The purified compound was extracted from the silica with chloroform. Two clear singlets in the aromatic hydrogens at 6.56 and 6.94 ppm instead of a doublet pointed to a 7-hydroxy-substituted structure (Figure 2). Chemical shifts were in good agreement with published data for *cis*-7-hydroxycalamene.

Sometimes, despite all careful efforts and extensive study, wrong structure assignment is published. For example, a new triquinane sesquiterpenic alcohol was isolated from the essential oil of *Anemia tomentosa* var. *anthriscifolia* by CC on silicagel with a hexane-ethyl acetate gradient. A new structural formula, namely (−)-*epi*-presilphiperfolan-1-ol, was initially proposed, after extensive 1D- and 2D-NMR analyses, as well as by GC–MS, chiral bidimensional GC, dehydration reactions, and a comparative (GIAO/DFT) theoretical study of the 13C NMR chemical shifts (Pinto et al., 2009a). Further examination by X-ray diffraction and vibrational circular dichroism studies, however, led to its reassignment to (−)-9-*epi*presilphiperfolan-1-ol (Figure 3), also a new compound. Its absolute configuration was

Fig. 3. Isomers of (−)-presilphiperfolan-1-ol: the 9-*epi*-isomer was isolated from *Anemia* 

Aroma concentrate separated from an aqueous solution of Haze honey by adsorptive CC had stronger aroma intensity than that separated by other methods. Fractions separated by preparative GC were sniffed to evaluate the sensory importance of the volatile compounds of Haze honey. Benzeneacetaldehyde, linalool, phenethyl alcohol, *p*-cresol, *p*-anisaldehyde,

Fig. 2. Hydroxylated sesquiterpenes from *Croton cajucara.*

established as 1S,4S,7R,8R,9S (Joseph-Nathan et al., 2010).

*tomentosa* var. *anthriscifolia* essential oil.

**2.1.2 Olfactory analysis** 

methyl-*p*-anisaldehydes, trimethoxybenzene, 5-hydroxy-2-methyl-4*H*-pyran-4-one, and lilac aldehydes seemed to contribute to Haze honey's aroma (Shimoda et al., 1996).

Shimoda et al. (1995) reported that a column extraction method was applied to the separation of volatile compounds from infusions of green tea and black tea, wherein their aromas degrade during distillation even under reduced pressure, and from sake, wherein ethyl alcohol disturbs a quantitative separation of volatile compounds.

Linalool is a very frequent terpenoid in essential oils, usually responsible for the scent of a specific plant, such as mint, basil, and other plant species. In order to evaluate the effect on humans of inhalation of linalool enantiomers (Figure 4), repeated flash chromatography was used to isolate R-(-)-linalool from lavender essential oil, while (S)-(+)-linalool was obtained from coriander oil and commercial linalool (Sugawara et al. 2000).

Fig. 4. Linalool enantiomers.

The Australian finger lime (*Citrus australasica*), an endemic Australian species, is an unique citrus fruits in terms of their size, shape and aroma. The fractionation of finger lime peel oil by chromatography on silica gel (with a gradient of pentane:ether) was able to identify a high number of terpenyl esters enriched in a medium polar fraction. This included citronellyl and geranyl esters, commonly found in lime peel oils, but also several less common (*Z*)-3-hexenyl esters and bornyl esters. In finger limes these compounds, together with isomenthone, menthol, carvone, *cis*-3-hexenol and methyl salicylate, may also contribute to the characteristic fresh green aroma of the fruits (Delort et al., 2009).

L-menthyl-L-lactate [(2*S*, 1′*R*, 2′*S*, 5′*R*)-2-isopropoyl-5-methylcyclohexyl 2-hydroxypropanoate, LM-LL] is widely used as a cooling compound in mint flavors, fruit flavors, oral care products, confections and beverages. L-menthyl-L-lactate was identified in dementholized cornmint oil from India by comparing mass spectrum and retention time with a reference sample on two columns. Separation was achieved by increasing polarity of the hexane–methyl-tert-butyl-ether (MTBE) gradient on a flash chromatography column packed with silica gel. It is worth noting that L-menthyl-L-lactate was identified in *M. arvensis* oils from India but not in *M. piperita* oils from the USA. Model experiments proved that LM-LL is formed during water vapor distillation of mint leaves in the presence of lactic acid. Lactic acid is spontaneously formed when mint herb is stored for some days in a humid environment. A prolonged storage of damp mint herb may increase formation of lactic acid and thus give rise to the formation of LM-LL during distillation. The presence of lactic acid will be influenced by the agricultural practice employed (Gassenmeier, 2006).

Secondary Metabolites 137

and *Carum carvii* and from leaves of *Ocimum basilicum,* were fractionated by silica gel over CC and tested in the laboratory for volatile toxicity against three stored rice pests. Fractions, where combinations of products occurred with or without other minor compounds, were

The main component of the essential oil of *Anemia tomentosa* var. *anthriscifolia,* (−)-9-*epi*presilphiperfolan-1-ol (Figure 3) was isolated by CC over silica gel, eluted with hexane and hexane-ethyl acetate (95:5), then tested against *Mycobacterium tuberculosis* (H37Rv) and *M. smegmatis*. A minimum inhibitory concentration of 120 µg/ml was recorded for *M.* 

The diterpene compounds arise from geranylgeranyl diphosphate, and present 20 carbon units in their basic skeletal type. One of the simplest and most important of the diterpenes is phytol (Figure 5), a reduced form of geranylgeraniol, which forms the lipophilic side-chain

Cyclization reactions of geranylgeranyl diphosphate led to many structural types of diterpenoids, presenting a large range of polarity nature, from apolar hydrocarbons such as cembrene (Villanueva and Setzer, 2010), a 14-membered ring, to fully oxidized skeleton of virescenoside (Figure 5), isolated from marine fungus *Acremonium striatisporum* (Ebel, 2010).

phytol

OH

O

H

H OH

O

OH

virescenoside W

OH

OH OH

OH

OH

Sesterterpenes (C25) may be the least common group of terpenoids. This class of compounds arises from geranylfarnesyl diphosphate (Figure 6), which by cyclization can give rise to various skeletal types, presenting different oxidation levels and several biological activities.

often more toxic than any one compound alone (Lopez et al, 2008).

*tuberculosis* (Pinto et al., 2009b).

**2.2 Diterpenes and sesterterpenes** 

of the chlorophylls (Vetter and Schröder, 2011).

Fig. 5. Diterpenes phytol, cembrene and virescenoside W.

cembrene

Gas chromatography-olfactometry (GC–O), gas chromatography–mass spectrometry (GC– MS) and preparative CC were used to identify the key odorants present in laboratoryextracted clementine oil from Spain. Almost 50 odorants were identified using GC–O, many of which were unsaturated aldehydes with high odor spectrum values. - and -sinensal, *trans*-4,5-epoxy-(*E*)-2-decanal, (*E*,*Z*)-2,6-dodecadienal and linalool were found to dominate clementine oil aroma. Enrichment of the oxygenates using preparative CC provided further identification of a total of 50 aldehydes, which were originally present in the oil at concentrations not high enough to produce a response using GC–O (Chisholm et al., 2003).

#### **2.1.3 Biological activities**

Human enteropathogens (*Campylobacter jejuni*, *Campylobacter coli*, and *Campylobacter lari* strains) were inhibited by the essential oil of wild *Daucus carota* L. DCEO. Major components of the essential oil responsible for the antibacterial activity were isolated on CC and identified as (*E*)-methyl-isoeugenol and elemicin (Rossi et al., 2007).

Confertifolin (6,6,9a-trimethyl-4,5,5a,6,7,8,9,9a-octahydronaphtho[1,2-c] furan-3 (1*H*)-one), isolated from the essential oil of *Polygonum hydropiper* L. (Polygonaceae) leaves using CC, showed activity both against bacteria and fungi. The oil was chromatographed on a column packed with silica gel and eluted with hexane-ethyl acetate gradient solvent system with increasing polarity, yielding 117 fractions (Duraipandiyan et al., 2010).

The essential oil of *Daucus crinitus* Desf. was submitted to flash chromatography (silica gel, using petroleum ether with increasing amounts of diethyl ether as eluent), and fractions were analyzed by GC-MS. Those containing the isochavicol esters were gathered for the preparation of isochavicol. The antibacterial and antifungal activities of the whole essential oil, of these two esters, and of isochavicol itself, were investigated against a wide range of bacteria and fungi. Additionally, their antimalarial and antiradical properties were also evaluated, showing an interesting antiplasmodial activity of isochavicol (Lanfranchi et al., 2010).

Patchouli oil obtained from *Pogostemon cablin* (Blanco) Benth and its main constituent, patchouli alcohol, were tested for their repellency and toxicity against Formosan subterranean termites (*Coptotermes formosanus* Shiraki). Both were found to be toxic and repellent. The fraction containing patchouli alcohol was purified by silica CC, and its purity was determined by GC-MS. Unusual tissue destruction was noted inside the exoskeleton of the termite after patchouli alcohol was topically applied to the dorsum (Zhu et al., 2003).

A successful breeding program of *Cannabis sativa* cultivars with a low content of the hallucinogenous D-9-tetrahydrocannabinol (THC) is a prerequisite to avoid drug abuse of hemp. Four TLC systems for analyzing cannabinoids were compared, being able to identify the absence of THC in the hemp essential oils from five commercial fiber varieties. The system using HPTLC, RP-18 and acetonitrile-water (9:1) achieved the best separation of hallucinogenous and non-hallucinogenous cannabinoids (Novak et al., 2001).

Nine fractions of essential oil extracted from *Thymus praecox* were separated by silica gel CC and the fractions were demonstrated to have antioxidant activity (Ozen et al, 2011). The essential oil of *Coriandrum sativum* was submitted to silica gel on dry-CC, resulting in six fractions. Essential oil and its fractions presented antimicrobial potential against Candida yeast infections (Begnami et al, 2010). Essential oils, distilled from seeds of *Coriander sativum*

Gas chromatography-olfactometry (GC–O), gas chromatography–mass spectrometry (GC– MS) and preparative CC were used to identify the key odorants present in laboratoryextracted clementine oil from Spain. Almost 50 odorants were identified using GC–O, many of which were unsaturated aldehydes with high odor spectrum values. - and -sinensal, *trans*-4,5-epoxy-(*E*)-2-decanal, (*E*,*Z*)-2,6-dodecadienal and linalool were found to dominate clementine oil aroma. Enrichment of the oxygenates using preparative CC provided further identification of a total of 50 aldehydes, which were originally present in the oil at concentrations not high enough to produce a response using GC–O (Chisholm et al., 2003).

Human enteropathogens (*Campylobacter jejuni*, *Campylobacter coli*, and *Campylobacter lari* strains) were inhibited by the essential oil of wild *Daucus carota* L. DCEO. Major components of the essential oil responsible for the antibacterial activity were isolated on CC and

Confertifolin (6,6,9a-trimethyl-4,5,5a,6,7,8,9,9a-octahydronaphtho[1,2-c] furan-3 (1*H*)-one), isolated from the essential oil of *Polygonum hydropiper* L. (Polygonaceae) leaves using CC, showed activity both against bacteria and fungi. The oil was chromatographed on a column packed with silica gel and eluted with hexane-ethyl acetate gradient solvent system with

The essential oil of *Daucus crinitus* Desf. was submitted to flash chromatography (silica gel, using petroleum ether with increasing amounts of diethyl ether as eluent), and fractions were analyzed by GC-MS. Those containing the isochavicol esters were gathered for the preparation of isochavicol. The antibacterial and antifungal activities of the whole essential oil, of these two esters, and of isochavicol itself, were investigated against a wide range of bacteria and fungi. Additionally, their antimalarial and antiradical properties were also evaluated, showing an

Patchouli oil obtained from *Pogostemon cablin* (Blanco) Benth and its main constituent, patchouli alcohol, were tested for their repellency and toxicity against Formosan subterranean termites (*Coptotermes formosanus* Shiraki). Both were found to be toxic and repellent. The fraction containing patchouli alcohol was purified by silica CC, and its purity was determined by GC-MS. Unusual tissue destruction was noted inside the exoskeleton of the termite after patchouli alcohol was topically applied to the dorsum (Zhu et al., 2003).

A successful breeding program of *Cannabis sativa* cultivars with a low content of the hallucinogenous D-9-tetrahydrocannabinol (THC) is a prerequisite to avoid drug abuse of hemp. Four TLC systems for analyzing cannabinoids were compared, being able to identify the absence of THC in the hemp essential oils from five commercial fiber varieties. The system using HPTLC, RP-18 and acetonitrile-water (9:1) achieved the best separation of

Nine fractions of essential oil extracted from *Thymus praecox* were separated by silica gel CC and the fractions were demonstrated to have antioxidant activity (Ozen et al, 2011). The essential oil of *Coriandrum sativum* was submitted to silica gel on dry-CC, resulting in six fractions. Essential oil and its fractions presented antimicrobial potential against Candida yeast infections (Begnami et al, 2010). Essential oils, distilled from seeds of *Coriander sativum*

identified as (*E*)-methyl-isoeugenol and elemicin (Rossi et al., 2007).

increasing polarity, yielding 117 fractions (Duraipandiyan et al., 2010).

interesting antiplasmodial activity of isochavicol (Lanfranchi et al., 2010).

hallucinogenous and non-hallucinogenous cannabinoids (Novak et al., 2001).

**2.1.3 Biological activities** 

and *Carum carvii* and from leaves of *Ocimum basilicum,* were fractionated by silica gel over CC and tested in the laboratory for volatile toxicity against three stored rice pests. Fractions, where combinations of products occurred with or without other minor compounds, were often more toxic than any one compound alone (Lopez et al, 2008).

The main component of the essential oil of *Anemia tomentosa* var. *anthriscifolia,* (−)-9-*epi*presilphiperfolan-1-ol (Figure 3) was isolated by CC over silica gel, eluted with hexane and hexane-ethyl acetate (95:5), then tested against *Mycobacterium tuberculosis* (H37Rv) and *M. smegmatis*. A minimum inhibitory concentration of 120 µg/ml was recorded for *M. tuberculosis* (Pinto et al., 2009b).
