Medicinal and Aromatic Plant Science and Biotechnology ©2007 Global Science Books Clerodendrum and Healthcare: An Overview - Part II Phytochemistry and Biotechnology Neeta Shrivastava* • Tejas Patel B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabad - 380054, Gujarat, India Corresponding author: * neetashrivastava_perd@yahoo.co.in ABSTRACT The genus Clerodendrum is very widely distributed throughout the world and has more than five hundred species. Many species of this genus have been described in various indigenous systems of medicine and are used in preparation of folklore medicines for the treatment of various life-threatening diseases. From the genus few species are very well studied for their chemical constituents and biological activities, the latter having been covered in our previous review. This review mainly focuses on phytochemistry i.e. isolation, identification and characterization of chemical constituents and biotechnological prospects of the Clerodendrum genus. Some of the species described in the review are Clerodendrum trichotomum, C. bungei, C. chinense, C. colebrookianum, C. inerme, C. phlomidis, C. petasites, C. grayi, C. indicum, C. serratum, C. campbellii, C. calamitosum and C. cyrtophyllum. The major chemical constituents present in this genus were identified as phenolics, flavonoids, terpenes, steroids and oils. Biotechnological aspects have also been discussed in the review. _____________________________________________________________________________________________________________ Keywords: flavonoids, in vitro, phenolics, steroids, terpenes Abbreviations: BA, benzyl adenine; CMV, Cucumber mosaic virus; GC, gas chromatography; HPLC, high performance liquid chromatography; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; IR, infrared; MPLC, medium pressure liquid chromatography; MS, Murashige and Skoog; NAA, -naphthalene acetic acid; NMR, nuclear magnetic resonance; PVY, Potato virus Y; TLC, thin layer chromatography; ToMV, Tomato mosaic tobamovirus; UV, ultraviolet CONTENTS INTRODUCTION...................................................................................................................................................................................... 209 PHYTOCHEMICAL INVESTIGATION OF CLERODENDRUM GENUS.............................................................................................. 210 PHENOLICS.............................................................................................................................................................................................. 210 FLAVONOIDS........................................................................................................................................................................................... 211 TERPENES................................................................................................................................................................................................ 211 STEROIDS................................................................................................................................................................................................. 212 OTHER CHEMICAL CONSTITUENTS .................................................................................................................................................. 214 GENERAL ISOLATION AND EXTRACTION METHOD ...................................................................................................................... 215 IDENTIFICATION OF COMPOUNDS .................................................................................................................................................... 215 BIOTECHNOLOGY AND ITS FUTURE PROSPECTS .......................................................................................................................... 216 SUMMARY ............................................................................................................................................................................................... 217 ACKNOWLEDGEMENTS ....................................................................................................................................................................... 217 REFERENCES........................................................................................................................................................................................... 217 _____________________________________________________________________________________________________________ INTRODUCTION Most of the earliest pharmaceuticals used were plant materials and they were used to treat diseases even before history was written (Houghton and Raman 1998). Documentation of the use of natural substances for medicinal purposes can be found as far back as 78 A.D., when Dioscorides wrote De Materia Medica, where he described thousands of medicinal plants. It included descriptions of many medicinal plants that remain important in modern medicine till today, not because they are continuously used for crude drug preparations, but because they serve as the source of important pure chemicals that have become mainstays of modern therapy (Ebadi 2007). The study and identification of chemical constituents present in plants is termed ‘phytochemistry’. Before the 18th century progress in the field of phytochemistry was very slow and very few compounds such as starch, camphor, etc., were known. But the major thrust came in the 19th century, when ‘nicotine’, the first alReceived: 31 August, 2007. Accepted: 1 November, 2007. kaloid, was isolated. In the 20th century with the isolation of many more compounds it gained more importance (Evans 2002). The main reason for interest in biologically active natural compounds was exemplified by changes that have occurred in the Western society with regard to pharmaceuticals during the last quarter of the 20th century (Ebadi 2007). So in the 20th century a major emphasis was given to the isolation, identification and elucidation of biosynthetic pathways of the isolated compounds. These studies were possible because of the use of various separation and identification techniques developed during this era (Harbone 1984; Mann et al. 1994; Kaufman et al. 1999). All the chemical constituents found in plants are not biologically active molecules e.g. carbohydrates, protein, fats, etc. They are produced by plants for their own normal functioning and growth; these chemical constituents are termed primary metabolites. But there are certain compounds which are produced by plants mainly for their defence mechanism during adverse environmental conditions Invited Review Medicinal and Aromatic Plant Science and Biotechnology 1(2), 209-223 ©2007 Global Science Books A B Figs. 1A and 1B: Flowering twigs of C. inerme. A B Fig. 2 C. phlomidis in wild. Vegetative (A) and flowering (B) stages. used as folk and traditional medicines in various parts of the world such as India, China, Korea, Japan, Thailand, Africa, etc. Various Clerodendrum species are reported to be used for remedial purpose in inflammatory disorders, diabetes, cancers, malaria, fever, etc. The traditional or ethnomedical claims of the species have also been evaluated. The biological activities of these species described in ancient literature have been reported to be associated with the chemical constituents present in the species (Shrivastava and Patel 2007). The major groups of chemical constituents present in the Clerodendrum genus are phenolics, flavonoids, terpenoids and steroids. or pathogen attack, and these compounds are termed secondary metabolites and they have biological importance. These compounds are also termed biologically active compounds. These secondary metabolites contribute towards the therapeutic value of plants and when isolated from plants form not only valuable drugs but also valuable lead molecules. These lead molecules can be further modified chemically for designing synthetic molecules responsible for having better or similar biological activity as their natural counterparts. The chemical constituents found in plants can broadly be grouped on the basis of their functional group. The major groups are phenolics, flavonoids, terpenoids, steroids, alkaloids, oils, etc. In this review the chemical constituents of the genus Clerodendrum are discussed in detail with reference to these groups. Biological activities of these chemical groups and individual compounds have been discussed in detail in our previous review on this genus (Shrivastava and Patel 2007). Figs. 1 and 2 represent two species of the genus, C. inerme and C. phlomidis in the vegetative and reproductive stage. PHENOLICS Phenolics constitute the largest group in plant secondary metabolites. In the Clerodendrum genus many phenolic compounds have been reported to be isolated from various species. The phenolic compounds in general and in the genus Clerodendrum are found in both free as well as bound to sugar moieties (Harbone 1984; Mann et al. 1994). On the basis of their structure phenolic compounds are further subgrouped into phenols, phenolic acids, phenyl propanoids, flavonoids, etc. As flavonoids represent a major constituent in this genus it will be dealt with separately. The various phenolic compounds isolated from the genus are listed in Table 1. All the major phenolic compounds which have been isolated from various species of Clerodendrum genus are given in Fig. 3A-C. Some of the phenolic compounds isolated were directly correlated with biologically activities such as antioxidant, antimicrobial, antiproliferative, antihypertensive and anticancer activities (Shrivastava and Patel 2007). PHYTOCHEMICAL INVESTIGATION OF CLERODENDRUM GENUS Genus Clerodendrum [family: Lamiaceae (Verbenaceae)] was reported for the first time in 1753 (Hsiao and Lin 1995; Steane et al. 1999; Shrivastava and Patel 2007). This genus has more than 500 species and is very widely distributed throughout the world and comprises from herbs to small trees (Moldenke 1985; Rueda 1993). Few species of the genus like C. indicum, C. phlomidis, C. serratum, C. trichotomum, C. chinense, C. petasites, etc. are being extensively 210 Phytochemistry and biotechnology of Clerodendrum genus. Shrivastava and Patel Table 1 Phenolic compounds isolated from genus Clerodendrum. Species Compound C. aculeatum Cistanoside D, acteoside C. bungei Anisic acid, vanillic acid, maltol, acteoside, leucosceptoside A, isoacteoside, jinoside C. calmitosum Pheophorbide related compounds C. cryptophyllum Pheophorbide related compounds C. fragrans Acteoside, leucosceptoside A, isoacteoside, methyl and ethyl esters of caffeic acid, jinoside C. grayi Lucumin, prunasin C. inerme (3-methoxy-4-hydroxyl phenyl) ethyl-O-2, 3-diacetyl-Į-Lrhanopyranosyl-(1-3)-4-O-(E)-fernloyl-ȕ-D-glucopyranoside, verbascoside, isoverbascoside, Neolignans (I-III) C. indicum Cleroindicin A-F C. infortunatum Acteoside, fumaric acid, methyl and ethyl esters of caffeic acid C. myricoides Myricoide, acteoside C. trichotomum Kusagenin, indolizino[8,7-ȕ] indole 5-carboxylic acids, acteoside, acteoside isomer, leucosceptoside A, martynoside, isomartynoside, isoacteoside, jinoside, trichotomoside Part Whole plant Whole plant Reference Garnier et al. 1989 Zhou et al. 1982; Li et al. 2005 Whole plant Whole plant Whole plant Cheng et al. 2001 Cheng et al. 2001 Gao et al. 2003 Whole plant Whole plant Miller et al. 2006 Spencer and Flippen-Anderson 1981; Nan et al. 2005 Aerial parts Whole plant, flower Root Whole plant Tain et al. 1997 Sinha et al. 1980, 1982 Cooper et al. 1980 Tayoda et al. 1982; Sukurai and Kato 1983; Kim et al. 2001; Nagao et al. 2001; Kang et al. 2003; Chae et al. 2004, 2005, 2006; Lee et al. 2006 binding activities (Shrivastava and Patel 2007). Isolation of flavonoids is carried out based on the polarity of the compounds. Less polar flavonoids are extracted with non-polar solvents such as chloroform, dichloromethane, diethyl ether or ethyl acetate, while polar flavonoids which are mainly glycosides are extracted with alcohols or mixture of alcohol and water e.g. the flavonoid hispidulin was extracted from alcoholic extract by partitioning with ethyl acetate/methanol/water. The organic phase obtained was again dissolved in ethanol and the insoluble fraction was fractionated with counter current chromatography in solvent system (chloroform/methanol/n-propanol/water) to obtain pure hispidulin (Hazekamp et al. 2001). Another flavonoid cleroflavone was isolated from leaves by extracting them with petroleum ether and then the extract was chromatographed which yielded pure cleroflavone (Ganapaty and Rao 1990). 7-hydroxyflavone, 7-hydroxyflavonone, naringin-4-O-Į-glucopyranoside and chalcone glucoside were isolated from flowers of C. phlomidis by extracting it in hexane and methanol, the hexane and methanolic extract were chormatographed which yielded these flavonoids (Anam 1997). Structures of isolated flavonoids are given in Fig. 4. The general procedure for isolation of phenolic compounds depends on the type of phenolic compound present i.e. whether it is present in glycosidic form or free form. For the extraction of phenolic moieties from its glycosides, the glycosides are first hydrolyzed; usually hydrolysis is carried out either with acid or alkali to break the glycosidic bond. The phenolic moieties are then extracted in non-polar solvents such as ethers. Extraction of free phenolic compounds is carried out by extracting the plant material with polar solvents. The extract obtained is then concentrated and the required compound is separated by various separation techniques such as preparative thin layer chromatography, column chromatography, HPLC and other techniques. Isolation of acteoside from flowers of C. infortunatum was carried out by extracting the material with alcohol after defatting it. The alcoholic extract was then successively extracted with various non-polar solvents like petroleum ether, n-hexane and diethyl ether and subjected to column chromatography which finally yielded acteoside (Sinha et al. 1982). Phenyl propanoid glycosides were isolated from stems of C. trichotomum by extracting the material with methanol and the methanolic fraction was further partitioned with solvents such as dichloromethane, ethyl acetate and n-butanol. From these ethyl acetate fraction was chromatographed which yielded acteoside, leucosceptoside A, martynoside, acteoside isomer, and isomartynoside (Kang et al. 2003; Chae et al. 2005). Another phenolic compound trichotomoside was also isolated from stems of C. trichotomum by extracting the material in methanol and was further partitioned with solvents like dichloromethane, hexane, butanol and ethyl acetate. The dichlormethane fraction was further subjected to column chromatography the fractions obtained were further separated and purified by MPLC which yielded trichotomoside (Chae et al. 2006). Cleroindicins from C. indicum were isolated by extracting the aerial parts in alcohol, which was further defatted with petroleum ether and residue obtained was chromatographed to obtain cleroindicins (Tain et al. 1997). TERPENES Many terpenoids have been reported from this genus. Broadly terpenes are grouped on the basis of isoprene units present into hemiterpenoid (C5), monoterpenoid (C10), sesquiterpenoid (C15), diterpenoid (C20), sesterterpenoid (C25), triterpenoid (C30) and carotenoid (C40). Terpenoids are generally found to be bound to sugar moieties by a glycoside linkage. Usually they are present as glycosides in their ȕ-Dglucosidic form (Harbone 1984; Mann et al. 1994). Terpenes isolated and identified from Clerodendron genus are listed in Table 3 and some of the terpenes had weak CNS activity, strong molluscicidal and fungitoxic activities (Shrivastava and Patel 2007). Structures of isolated terpenoids from the genus are shown in Fig. 5A-C. Isolation of terpenoids is generally carried out with nonpolar and polar solvents. Triterpenoid Mi-saponin was isolated from roots of C. wildii by first extracting it with chloroform followed by methanolic, water and butanol extraction. The butanol extract obtained was then chromatographed to get pure Mi-saponin A. Inerminosides were isolated from leaves of C. inerme by extracting the leaves in methanol which was further partitioned with petroleum ether, diethyl ether and butanol. The butanol fraction was chromatographed which yielded inerminosides (Calis et al. 1994a). Megastigmane iridoid glycosides were also isolated by extracting the aerial parts of C. inerme with methanol FLAVONOIDS Flavonoids are one of the major groups present in Clerodendrum genus possessing promising biological activities. Flavonoids found in this genus are in both free and bound form. These flavonoids are further sub-grouped into catechins, leucoanthocyanidins, flavanones, flavanonols, flavones, anthocyanidins, flavonols, chalcones, aurones and isoflavones (Harbone 1984; Mann et al. 1994). Various flavonoids isolated from the Clerodendrum genus are mentioned in Table 2. These isolated flavonoids possess potent antioxidant, antimicrobial, antiasthmatic, antitumor and CNS211 Medicinal and Aromatic Plant Science and Biotechnology 1(2), 209-223 ©2007 Global Science Books O O H 2CO OCH 2 H 2CO H 3CO H 3CO H 2CO OCH 2 OCH3 H 2CO H3CO Neolignan I H 3CO OCH3 Neolignan II HOOC CH3 O H 3CO CH 3 OCH2 CH 3 COOH CH3 H 3CO H3CO H 3CO OCH2 HO H 3C CH 3 Neolignan III Serratagenic acid O CH 2OH O HO O O O OH HO H 3C O OH HO HO OH OH Acteoside HO OH O HO O O O H 3C HO OH OH OCH3 O HO HO Jionoside D Fig. 3A Phenolic compounds of Clerodendrum genus. and this methanolic extract was defatted with diethyl ether and the aqueous fraction was chromatographed which yielded iridoid glycosides (Kanchanapoom et al. 2001). Iridiod glucosides were also isolated from C. incisum by extracting the aerial parts with methanol and the methanolic extract was further chromatographed to get iridoid glucosides (Stenzel et al. 1986). and they are termed phytosterols. ȕ-sitosterol was reported to be isolated from various species of Clerodendrum genus such as C. inerme, C. fragrans, C. colebrookianum, C. paniculatum, C. tomentosum, C. bungei, C. phlomidis and C. infortunatum (Chirva et al. 1980; Sinha et al. 1980; Singh and Singhi 1981; Hsu et al. 1983; Pinto and Nes 1985; Att-Ur-Rehman et al. 1997; Yang et al. 2002; Gao et al. 2003). (24S)-ethylcholestra-5, 22,25-triene-3ȕ-ol was reported in C. inerme, C. paniculatum and C. fragrans (Singh and Singhi 1981; Singh and Prakash 1983; Hsu et al. 1983) and 24ȕ-ethylcholesta-5,22E, 25(27)-trien-3ȕ-ol was isolated from C. splendens (Pinto et al. 1985). Other steroids such as clerosterol and deucosterol were isolated from petroleum ether extracts of C. fragrans (Singh and Singhi 1981). Ȗ-sitosterol was reported from C. STEROIDS Steroids are terpenes based on the cyclopentane perhydroxy phenanthrene ring, but they are considered separately because of their chemical, biological and medicinal importance. Steroids are found in nature in free as well as in glycosidic form. There are many steroids reported from plants 212 Phytochemistry and biotechnology of Clerodendrum genus. Shrivastava and Patel H2C CH 3 H2C CH3 H 3C NH O CH 3 H3C CH 3 HO H HOOC HN N H3C CH 2OOCH3 COOCH 3 N NH HN N CH 3 CH3 H3C N NH H 3C CH 3 CH3 H3C N HN N H2C CH 3 O COOCH 3 O COOCH 3 HOOC HOOC I III II Pheophorbide related compounds O HOH2C HO O O O OH O OH HO H 3C HO O OH OH OH Verbascoside OCH3 OCH 3 H N HO O N OH OH HO OCOCH 3 O O O O O OCOCH 3 O Indolizino [8,7-b] indole 5-carboxlic acid OH H3C H 3CO OH Trichotomoside Fig. 3B: Phenolic compounds of Clerodendrum genus. cyrtophyllum (Wu 1980) and C. serratum (Banerjee et al. 1969; Anynomous 2005), while 24S-stigmata-5,22,25-dine3ȕ-ol, 22E, 24S-stigmata 5,22,25-trine-3ȕ-ol were isolated from hydroalcoholic extract of C. mandarinorum root bark, aerial parts of C. inerme and C. campbellii (Bolger et al. 1970a, 1970b; Zhu et al. 1996; Pandey et al. 2003). Also steroids such as clerosterol, taraxerol were reported from C. colebrookianum, C. paniculatum, and C. tomentosum species (Joshi et al. 1979; Chirva and Garg 1980; Goswami et al. 1996). New steroids colebrin A-E and colebroside were also isolated from aerial parts of C. colebrookianum (Yang et al. 2000a, 2000b). Cholestanol was also isolated from stem of C. phlomidis (Akihisa et al. 1989). Steroids such as taraxerol, glochidone, glochidonol, glochidiol were isolated from C. bungei (Gao et al. 2003). Some of the major steroids isolated have been shown in Fig. 6A and 6B. Steroids are extracted by various solvents, steroidal gly- cosides are extracted in polar solvents while free steroids are extracted with non-polar solvents. To obtain steroidal aglycan from steroidal glycoside, the glycoside is hydrolyzed and then extracted in non-polar solvents. Ȗ-sitosterol was isolated from C. serratum roots by extracting it in petroleum ether which yielded a dark yellow residue which was further chromatographed to get Ȗ-sitosterol. 4Į-methyl sterols were also isolated from C. inerme by extracting the aerial parts with methanol. Then the methanolic fraction was concentrated and to it dimethyl ketone was added, the soluble fraction was further extracted with alkaline ethanol and then further extracted it with diethyl ether. The ether fraction was chromatographed which yielded 4Į-methyl sterols (Akihisa et al. 1990). Other steroidal compounds like clerosterol, E-sitosterol were isolated from C. colebrookianum by extracting the leaves with hexane and subjecting the hexane fraction to column chromatography (Singh et al. 213 Medicinal and Aromatic Plant Science and Biotechnology 1(2), 209-223 ©2007 Global Science Books CN CH3 OH HO O OH OH HO O O O H 3C O OH H3C C(CH 2)15CH 3 O CH 3 (R)-lucumin HO HO CH3 CH2 H H O O OH OH CN HO O OH O Colebrin D OH CH3 H 3C OH O (R)-prunasin H3C C(CH2) 15CH3 O CH3 HO HO CH3 CH2 H H O O H OH Colebrin C Fig. 3C: Phenolic compounds of Clerodendrum genus. Table 2 Flavonoids isolated from the genus Clerodendrum. Species Compound C. fragrans Kaempferol C. indicum Hispidulin, scutellarein, scutellarein-7-O-ȕ-D-glucuronide C. inerme C. infortunatum C. mandarinorum C. nerrifolium C. petasites C. phlomidis C. siphonenthus C. tomentosum C. trichotomum Part Leaves Flowers Apigenin, acacetin, cosmosin, luteolin, cynaroside, salvigenin, 5-hydroxy-4-7-dimethoxy-6-flavone, 5-hydroxy-4-7dimethoxy flavone, 4-methylscutellarein Apigenin, acacetin and methyl esters of acacetin-7-Oglucuronide, cabruvin, quercetin, scutellarein, scutellarein-7O-ȕ-D-glucuronide, Hispidulin Cirsimartin, cirsimartin 4-glucoside, quercetin-3-methyl ether Cleroflavone Hispidulin Apigenin, pectolinerngenin, chalconeglucoside, 2-4-4trihydroxy-6-methylchalcone, 7-hydroxyflavanone, an its ȕ-Dglucoside, naringin-4-O-Į-glucopyranoside Pectolinerngenin 5-hydroxy-4-7-dimethoxy flavone Apigenin Aerial parts, stem, leaves Reference Gao et al. 2003 Sankara Subramanian and Ramachandran Nair 1973; Gunasegaran et al. 1993 Vendatham et al. 1977; Achari et al. 1990; Raha et al. 1991; El-Shamy et al. 1996 Flowers, roots Sankara Subramanian and Ramachandran Nair AG 1973; Sinha et al. 1981; Roy et al. 1996 Roots Leaves Flowers Flowers, leaves, whole plant Zhu et al. 1996 Ganapaty and Rao 1990 Hazekamp et al. 2001 Seth et al. 1982; Roy et al. 1994, 1995; Roy and Pandey 1995; Anam 1997, 1999 Flowers Stems Whole plant Pal et al. 1989 Chirva and Garg 1980 Min et al. 2005 from C. serratum roots (Garg and Verma 2006). A cyclic hexapeptide cleromyrine I (Ala-Gly-Pro-Ile-Val-Phe) was isolated from C. myricoides by chiral chromatography (Bashwira et al. 1989) and two new spermidine alkaloids, myricoidine and dihydromyricoidine were also reported from C. myricoides (Bashwira and Hootele 1988); also other spermidine alkaloids buchnerine and N'-(Z)-p-methoxycinnamoylbuchnerine were isolated from leaves of C. buchneri (Lumba and Hootele 1993). Lectins and two pigments trichotomine and trichotomine G1 were also isolated from fruits and leaves of C. trichotomum (Iwadare et al. 1974; Kitagaki-Ogawa et al. 1986). Glycoproteins CIP-29 and CIP-34 were isolated from C. inerme were reported to be responsible for inducing systemic resistance against tobacco mosaic virus in Nicotina tabacum (Prasad et al. 1995; Olivier et al. 1996), another protein identified as Crip-31 was also isolated from the same species and it was also showing systemic viral resistance against Cucumber mosaic virus (CMV), Tomato mosaic tobamovirus (ToMV) and Potato virus Y (PVY) in Nicotiana tabacum (Praveen et al. 2001) (Fig. 7). 5-O-ethylcleroindicin and bungein A were isolated by 1995). OTHER CHEMICAL CONSTITUENTS Many other chemical constituents are also reported from the genus which include volatile constituents such as 5-O-ethylcleroindicin D, cleroindicin (A, C, E and F), linalool, benzyl acetate, benzyl benzoate, benzaldehyde and octen-3-ol which have been isolated from C. bungei, C. canescens, C. cyrtophyllum, C. inerme and C. philippinum, C. buchholzii (Yang et al. 2002; Yu 2004; Nyegue et al. 2005; Wong and Tan 2005). Inactive wax bungein A was also isolated from aerial parts of C. bungei (Yang et al. 2002). Amino acids such as lysine, arginine, serine, proline, threonine, glutamic acid; sugars like galactose, glucose and fructose and pentadecanoic acid-E-D-glucoside were also isolated from C. inerme (Desai and Baxi 1991; Pandey et al. 2006). Palmitic, oleic and linoleic acids were extracted from seeds of C. infortunatum (Siddiqui et al. 1973). 2-methyleicosa 2,9diene,10,11,32-trimethyltetratriacontanol, pentatriacontane, palmitic acid were isolated from the leaves of C. colebrookianum (Singh et al. 1995). D-manitol was also isolated 214 Phytochemistry and biotechnology of Clerodendrum genus. Shrivastava and Patel Table 3 Terpenes isolated from the genus Clerodendrum. Species Compound C. chinense Monomelittoside, melittoside, harpagide, 5-O-ȕ-glucopyranosylharpagide, 8-O-acetylharpagide C. colebrookianum Triacatane, clerodin, clerodendrin A C. incisum 8-O-foliamenthoyleuphroside, 2’-O,8-Odifoliameuthoyleuphroside, plantarenaloside, euphroside C. inerme Į and ȕ-amyrin, royleanone dehydroroyleanone, caryoptin, 3epicaryoptin, 14,15-dihydro-15ȕ-methoxy-3-epicaryoptin, clerodermic acid, glutinol, gramisterol, Iridoids such as (inerminoside A-1, B, C, D), clerodendrins (A-H), clerodendrin B acetate, monomelittoside, inermes A, B, sammangaoside A-C, betulin, clerodermic acid C. mandarinorum Friedelanone, lupeol, betulinic acid C. neriifolium (-)-Hardwickic acid C. paniculatum Triacatane, clerodin, clerodendrin A, 3ȕ-acetyloleanolic acid, 3ȕacetyloleanolic aldehyde, glutinol C. phlomidis Triacatane, clerodin, clerodendrin A C. serratum Queretaroic acid, serratagenic acid C. siphonenthus Unicinatone C. thomsonae Monomelittoside, melittoside, harpagide, 5-O-ȕ-glucopyranosylharpagide, 8-O-acetylharpagide, aucubin, reptoside, ajugoside, 8O-acetylmioporoside C. trichotomum Clerodendrins (A-H) C. ugandenese Ugandoside C. uncinatum Unicinatone C. wildii Mi-saponin A extracting the aerial parts of C. bungei in methanol and further defatting it with petroleum ether. The residue obtained was partitioned with ethyl acetate and n-butanol successsively. The ethyl acetate fraction was chromatographed which yielded the two compounds, 5-O-ethylcleroindicin and bungein A (Yang et al. 2002). Isolation of spermidine alkaloids from C. buchneri leaves was carried out by extracting the leaves with methanol. Methanolic fraction was filtered and the filtrate obtained was acidified with dilute acid and then neutralized, this neutralized fraction was further extracted with chloroform. The chloroform extract was concentrated and the residue was distributed between chloroform and aqueous citric acid. It was further basified with alkali and then extracted with chloroform which yielded crude alkaloidal fraction. This fraction upon chromatography yielded two spermidine alkaloids (Lumbu and Hootele 1993). For isolation of sugars and amino acids, first the material was defatted and then the remaining residue was extracted with hydro-alcoholic mixture. The filtrate thus obtained was concentrated and acidified with dilute acid and then extracted with non-polar solvents like ether. The aqueous acidic fraction remained after separation was further extracted with ethyl acetate for removal of flavonoids. The aqueous fraction then obtained was neutralized and subjected to column chromatography which yielded sugars and amino acids. Volatile constituents from fresh plant material were reported to be extracted by steam distillation (Houghton and Raman 1998). Part Aerial parts Reference Kanchanapoom et al. 2005 Whole plant Whole plant Joshi et al. 1979 Stenzel et al. 1989 Leaves, aerial parts Roots Leaves Leaves Abdul-Alim 1971; Singh and Prakash 1983; Achari et al. 1990, 1992; Akihisa et al. 1990; Rao et al. 1993; Calis et al. 1994a, 1994b; Att-Ur-Rehman et al. 1997; Kanchanapoom et al. 2001; Kumari et al. 2003; Pandey et al. 2005 Zhu et al. 1996 Ganapaty and Rao 1990 Joshi et al. 1979; Hsu et al. 1983 Whole plant Whole plant Roots Aerial parts Joshi et al. 1979 Rangaswami et al. 1969 Doraz et al. 2004 Gabriele and Rimpler 1981 Whole plant Leaves Roots Roots Kawai et al. 1998 Gabriele and Rimpler 1983 Doraz et al. 2004 Toyota 1990 late chemical constituents the method mainly employed is based on fractionation by solvents of varying polarity. The fractions obtained after extraction with various solvents are then subjected to different separation techniques like precipitation techniques, TLC, paper chromatography, column chromatography, HPLC, GC, etc. to yield pure compounds (Fig. 8). The procedure of isolation can be modified depending on the substance that is under investigation (Harbone 1984; Mann et al. 1994; Houghton and Raman 1998). IDENTIFICATION OF COMPOUNDS Once the compound is isolated, it is necessary to identify it. The compound identification is done by determining the properties of the compound such as melting point, boiling point, purity, solubility and Rf value of the compounds. For characterization of the compounds various analytical techniques such as ultra violet (UV) spectroscopy, infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy (MS) and X-ray crystallography are used. In the case of ultraviolet spectroscopy the underline principle is the amount of light absorbed by the compound. A spectrum of the compound is recorded against solvent blank in which it is dissolved. The compound will absorb maximum amount of light at a specific wavelength which is termed as absorbance maxima. Such spectral measurements help in checking the purity of the isolated compound. While in infrared spectroscopy the type of chemical group present in the compound can be identified on the basis of bending and stretching vibration property of the compound. IR spectroscopy helps in structural elucidation and identification of the compound. In NMR spectroscopy the principle is based on the spinning property of the active nucleus so it will also have a magnetic moment and an angular momentum. The ratio of these two properties (magnetic moment and angular momentum) is utilized as a characteristic property of a compound for its identification. Molecular weight of the compound is determined by mass spectroscopy where it determines the molecular weight based on the mass to charge ratio of the particles present in the compound. So by using these techniques compound are identified and its structures are elucidated. The data obtained then can be compared with the authentic standards materials and confirmed. In case where authentic samples are not available the above data are exploited to identify and cha- GENERAL ISOLATION AND EXTRACTION METHOD Ideally, the plant material to be used is collected fresh at the right stage of growth and then dried under the shade or in oven at 40-45°C, the dried plant material is used for the extraction. Drying of plant material should be carried out under control conditions to prevent the changes occurring in its constituents due to drying. In the case of volatile constituents, fresh plant material is used because drying leads to degradation/loss of volatile constituents from the material. Another criterion is that plant material should be free from any type of contamination before it is used for isolation studies because contamination can also lead to loss or degradation of chemical constituents present. Prior to extraction the plant material should be authenticated. To investigate/iso215 Medicinal and Aromatic Plant Science and Biotechnology 1(2), 209-223 ©2007 Global Science Books OH HO HO O O O OH OH O HO Apigenin 7-hydroxy f lavanone O H3CO OH O Hispudilin O HO OH O OH O O OH 2'-4-4'-trihydroxy-6'-methyl chalcone O 5-hydroxy-4'-7-Dimethoxy f lavone OH OCH3 HO HO O O HO H 3C OH H3CO O O Scutellarein Clerof lavone OH CH2OH O O OH OCH3 HO O OH O O O OH H 3CO OH O OH O CH 3 O OH OH Ninargin Pectolinarigenin Fig. 4 Flavonoids of Clerodendrum genus. dendrum genus. Direct shoot regeneration from leaf explants of C. inerme was reported by Baburaj et al. (2000) on MS medium supplemented with BA alone at 4 mg/l. The regenerated shoots were rooted in MS medium supplemented with IAA (2 mg/l). In our laboratory we have reported micropropagation of C. inerme using axillary buds. Axillary buds were multiplied using BA at 16 μM with 3% sucrose. Rooting of the regenerated shoots was observed in basal MS medium without plant growth regulators. The phytochemical profile of in vitro plants was found to be similar to that of in vivo plants (Kothari et al. 2006). Adventitious shoot regeneration in MS medium supplemented with BA (5 mg/l), NAA and IBA (0.5 mg/l of each), was reported in C. aculeatum. The shoots were rooted in MS medium con- racterize the isolated compounds (Harbone 1984; Houghton and Raman 1998; Anonymous 1 2004). BIOTECHNOLOGY AND ITS FUTURE PROSPECTS In the recent past, there has been a resurgence of interest in herbal medicines, which has disturbed the equation of demand and supply. To deal with these demands search of a potential alternative method for supply of good quality raw material has become a prime importance. In the last few decades biotechnological methods, specifically the plant tissue culture system, has emerged as a potential alternative source of high quality plant material. However, very little work has been reported on tissue culture aspects of Clero216 Phytochemistry and biotechnology of Clerodendrum genus. Shrivastava and Patel H 2C H H3C CH3 CH 3 O H O CH3 COOH CH 3 H HO Clerodermic acid H 3C H CH3 CH2OH COOH CH 3 CH 3 HO H3C H CH3 CH3 Oleanolic acid Betulin H 3C CH 3 CH3 CH3 H2C CH3 H H H H CH 3 CH3 CH 3 CH 3 CH3 H HO CH 3 CH 3 H 3C Friedelin H CH 3 O H Betulinic acid OCH3 CH 3 CH3 AcO O O O OAc CH2OAc 14,15-dihydro-15-hydroxy-3-epicaryoptin H CH 3 CH3 OH O HO AcO HO O OH O CH 3 O H COOH O OH HO OAc CH2OAc 14,15-dihydro-15E -methoxy-3-epicaryoptin O HO HOH2C HO HO H CH3 O O OH 5-O-E -glucopyranosyl-harpagide Fig. 5A: Terpenes of Clerodendrum genus. taining 0.5 mg/l of NAA and IBA (Srivastava et al. 2004). Multiple shoot regeneration was observed in C. colebrookianum with six different cytokinins. Optimum shoot induction was observed with the medium containing BA (Mao et al. 1995). From the above reports, it is clear that only certain aspects of biotechnology have been worked out in a few species of the genus. Extensive research has to be done in this field of biotechnology, especially in the area of molecular taxonomy because the genus shows much diversity and a clear pedigree of the genus is not yet known. yet unfolded. The need of the hour is to explore the potential of various species of this widely distributed and available genus to fight against many diseases. New transgenic varieties could be created as efficient green production lines for pharmaceuticals by using genetic engineering and tissue culture for multiplying and conserving the species, which are difficult to regenerate by conventional methods and to save them from extinction. ACKNOWLEDGEMENTS SUMMARY The authors wish to thank Dr. H. Srinivasa for his help in preparing the manuscript and Dr. Ritesh P. Vaidya (taxonomist) for his help in identification of two Clerodendrum species. 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African Journal of Bio- Medicinal and Aromatic Plant Science and Biotechnology 1(2), 209-223 ©2007 Global Science Books C 2H 5 H3C C 2H 5 CH3 CH 3 H 3C CH 3 CH3 CH 3 CH 3 CH3 CH 3 H HO HO J -Sitosterol E -Sitosterol H3C H3C CH3 CH 3 CH 3 CH 3 CH3 CH 3 CH3 CH3 CH 3 HO HO H Campesterol Cholestanol H 3C CH 3 CH3 CH3 C 2H 5 CH 3 CH 3 H3C CH 3 HO CH 2 CH3 CH 3 CH 3 24-ethyl cholesterol HO Clerosterol CH 3 H 3C H 3C CH 3 H 3C CH3 CH 2 CH 2 H H HO H HO CH 3 H3C CH3 CH 3 OH OH COOH Colebrin B Colebrin A Fig. 6A: Steroids of Clerodendrum genus. technology 5, 415-418 Kumari GNK, Balachandran J, Aravind S, Ganesh MR (2003) Antifeedant and growth inhibitory effects of some neoclerodane diterpenoids isolated from Clerodendron species (Verbenaceae) on Earias vitella and Sodoptera litura. 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Shrivastava and Patel CH 3 CH 3 H3C O H3C C(CH 2)15CH 3 O HO H 3C O HO CH 2 H H CH 3 O H OH H 3C C(CH2) 15CH3 Colebrin C CH3 H 3C O O H3C HO O HO CH 2 H H O OH OH Colebrin D CH 3 CH 3 H 3C O H3C C(CH 2)15CH 3 O H3C HO O HO CH 2 H H 3C OH CH 3 O H OH CH 2 H3C CH 3 CH3 Colebrin E HO 4D -methylsterol, 4D -methyl- 24E-ethyl5D -cholesta-14, 25-dien-3E -ol H 3C CH 3 H CH3 CH 3 H3C CH 3 CH3 HO 24E -ethylcholesta-5, 9(11), 22E-trien-3E -ol Fig. 6B: Steroids of Clerodendrum genus. from Clerodendrum inerme. Indian Journal of Chemistry 34B, 2161-2163 Pandey R, Verma RK, Singh SC, Gupta MM (2003) 4Į-methyl-24ȕ-ethyl5Į-cholesta-14,25-doeme-3ȕ-ol and 24ȕ-ethylcholesta-5 9(11)22e-trien-3-ȕol, sterols from Clerodendrum inerme. Phytochemistry 63, 415-420 Pinto WJ, Nes WR (1985) 24ȕ-ethylsterols, n-alkanes and n-alkanols of Clerodendrum splendens. 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Planta Medica 62, 393-396 Material Extract the material with alcohol : water Marc Filtrate Extract the marc with ethyl acetate Partition the filtrate with chloroform Marc Filtrate (POLYSACCHARIDES) (FAT, WAXES) Chloroform fraction Aqueous fraction (TERPENOIDS, PHENOLICS) Basify the aqueous fraction and extract it with chloroform : methanol Chloroform : methanol Aqueous basic fraction (ALKALOIDS) (QUATERNARY ALKALOIDS) Fig. 8 General schematic diagram for isolation of chemical constituents. Based on Harbone 1984. 223