Plant Cell Tiss Organ Cult (2008) 94:9199 DOI 10.

1007/s11240-008-9391-z

ORIGINAL PAPER

Characterisation of two phenotypes of Centella asiatica in Southern Africa through the composition of four triterpenoids in callus, cell suspensions and leaves
Jacinda T. James Riaan Meyer Ian A. Dubery

Received: 18 March 2008 / Accepted: 30 April 2008 / Published online: 16 May 2008 Springer Science+Business Media B.V. 2008

Abstract Two morphologically distinct phenotypes of Centella asiatica (Type-1 and Type-2) in South Africa were compared in relation to the levels of triterpenoid saponins with the aim of assessing their potential for biotechnological manipulation of triterpenoid synthesis. The metabolites investigated included madecassoside and asiaticoside and their sapogenins madecassicand asiatic acid; produced in cultured undifferentiated cells (cell suspensions and calli) and leaves. Weight determination in plant cell suspensions and the accumulation of secondary metabolites after 16 days for Type-1 and 20 days for Type-2 were investigated since these secondary metabolites accumulate during the period that follows the active growth phase. The four triterpenoids of interest were analysed and quantied by HPLC in crude ethanolic extracts. A difference in bioactive triterpenoids was exhibited that was tissue specic and varied between the two phenotypes. The triterpenoids from leaf tissue were more easily quantiable in each phenotype than in the case of the undifferentiated cells (callus and cell suspensions), which had lower, but still quantiable, levels of these targeted secondary metabolites. Leaves contained the highest triterpenoid levels (ranging from 1.8 to 5% dry weight for the triterpenoid acids and their glycosides,
J. T. James R. Meyer I. A. Dubery (&) Department of Biochemistry, University of Johannesburg, PO Box 524, Auckland Park 2006, South Africa e-mail: idubery@uj.ac.za

respectively), with the free acids occurring in a ratio of approximately 1:2.5 in relation to the glycoside content. Keywords Apiaceae Asiatic acid Asiaticoside Glycosides Madecassic acid Madecassoside Umbelliferae

Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid AS Anisaldehyde-sulphuric acid BAP 6-Benzylaminopurine HPLC High performance liquid chromatography Rf Retardation factor Rt Retention time

Introduction Saponins are a vast group of glycosides, widely distributed in higher plants. A number of different plant species synthesize triterpenoid saponins as part of normal growth and development with the most predominant group being pentacyclic triterpene derivatives and their sapogenins (Haralampidis et al. 2002). Their surface-active properties are what distinguish these amphiphilic compounds from other glycosides (Sparg et al. 2004). The triterpenoid structure (aglycone) is hydrophobic and contains a hydrophilic sugar chain (glycone) and these

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characteristics are responsible for the biological activity of saponins (Singelton et al. 2000). The sugar(s) are attached to the aglycone and varies both in type and number. The substances of therapeutic interest are the saponins containing triterpene acids and their sugar esters (Sparg et al. 2004). Triterpenoid saponins are synthesised via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons. The triterpenoid backbone then undergoes various modications (oxidation, substitution and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyl transferases and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is still largely uncharacterised, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies (Haralampidis et al. 2002). Centella compromises some 45 species belongings to the plant family Apiaceae which includes the medicinally important C. asiatica. This slender, weakly aromatic, small creeping perennial herbaceous plant is an umbellifer which has many common names including Gotu Kota and Indian Pennywort (Matsuda et al. 2001). It has been utilised for centuries in Ayurvedic medicine to alleviate symptoms of anxiety (Wijeweera et al. 2006) and to promote broblast proliferation and collagen synthesis (Maquart et al. 1999). Centella terpenoids include asiaticoside, centelloside, madecassoside, brahmoside, brahminoside, thankuniside, sceffoleoside, centellose, and asiatic, brahmic, centellic and madecasic acids (Aziz et al. 2007). Depending on the origin of the Centella plant material, these saponins can account for between 1 and 8% of the constituents (Brinkhaus et al. 2000). It was reported that C. asiatica extracts contained three bio-active triterpenoids, namely asiatic acid, madecassic acid and asiaticoside, which have healing properties. The most abundant triterpenoid saponin, asiaticoside, has antibacterial, fungicidal and cell proliferative activities which have been shown to aid in the treatment of wounds (Shukla et al. 1999), ulcers, various skin

diseases, vein insufciency, tuberculosis and in the treatment of mental disorders (Mathur et al. 2000). Recent studies have led to the isolation of other triterpenoids with healing abilities, namely terminolic acid, madecassoside and asiaticoside-B (Shaneberg et al. 2003). Most plant-derived pharmacologically active compounds have complex structures, making chemical synthesis an economically uncompetitive option. Plant cell culture has been used in attempts to increase the production of bio-active secondary metabolites of pharmaceutical interest (Gaines 2004). A particular important potential benet is the ability to manipulate and improve the production of desired compounds within the plant cell through manipulation of cultured cells by elicitors and plant hormones. Two phenotypes of C. asiatica which will be referred to as Type-1 (a reniform (broad and round) leaf shape with crenate leaf margins) and Type 2 (a cardate leaf with sinuated leaf margins) (Fig. 1), are found in South Africa. A comparison and evaluation of the triterpenoid content (asiatic acid, madecassic acid and their glycosides) in callus, cell suspensions and leaves of these phenotypes are reported. The reported data will contribute to the establishment of knowledge about the triterpenoid saponin composition of C. asiatica found in Southern Africa in comparison to other geographical areas, and lays a foundation for future studies on the manipulation of the phytochemical composition of C. asiatica.

Fig. 1 Two phenotypes of South African C. asiatica (left): a broad and round leaf shape with crenate leaf margins (Type-1) and (right) a cardate leaf with sinuated leaf margins (Type-2)

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Materials and methods Plant material Commercially cultivated C. asiatica was obtained from a local nursery and designated Type-1. The morphologically wild relative, Type-2, (previously known as swamp Centella or C. cordifolia) was collected from a marshy area in the Gauteng province, South Africa. Voucher specimens of both Type-1 and Type-2 were deposited in the herbarium of the Botany Department, University of Johannesburg, South Africa. Preparation of callus and cell cultures Explants from stems of Type-1 and Type-2 was cultivated and maintained in agar solidied Murashige and Skoog (MS) medium with vitamins (50 mg nicotinic acid, 50 mg thiamine HCl, 10 mg pyridoxine HCl and 10 g myo-inositol per 100 ml, Highveld Biologicals, South Africa) supplemented with 1 lM 2,4-D, 1 lM BAP, 30 g l-1 sucrose and 1 g l-1 casein hydrolysate (Bouhouche et al. 1998). To obtain callus proliferation, 1 g of callus was transferred aseptically to solid MS salt solution as described above. The cultures were kept in a culture room with 18/6 h light/dark cycle and the temperature was regulated at 23C for 21 days. In addition, callus was transferred to sterile liquid medium, to initiate cell suspensions. These were incubated on an orbital shaker in the dark at 23C. Every 7 days the homogenous cell suspension was subcultured in a 1:1 (v/v) ratio with fresh MS medium. Determination of dry/wet weight A series of Erlenmeyer asks, each containing 40 ml of culture medium were all individually inoculated with 10 ml of an established cell suspension and allowed to grow in the dark at 23C. The growth was terminated on alternate days by ltering the cell suspension through a 0.45 lm nylon membrane. The wet weight of the cells were determined and these cells were either dried in pre-weighed polypropylene tubes (Merck) at 65C for 48 h to determine the dry weight, or added to the required amount of ethanol for metabolite extraction as described below.

Ethanolic extracts of C. asiatica tissues After the required growth period was achieved, the cell suspensions of Type-1 and Type-2 were ltered through a 0.45 lm nylon membrane and cells were added to absolute ethanol (Saarchem, South Africa) in a 1:3 (w/v) ratio. Extracts of callus were prepared similarly by transfer to absolute ethanol. The suspension were homogenised for 10 min and centrifuged at 2,200 9 g for 20 min. The supernatants were collected and vacuum dried to remove the excess solvent. Leaves of C. asiatica (Type-1 and Type-2) were weighed, cut into strips and placed in absolute ethanol (1:12 (w/v)) to extract secondary metabolites. This was placed on a magnetic stirrer for 24 h and then centrifuged at 2,200 9 g for 20 min. The supernatant was decanted and concentrated in 10 times with a rotary evaporator at 45C under vacuum. The extracts were then used for analysis. TLC analysis of triterpene saponins Concentrated crude extracts of callus, cell suspensions and/or leaves were reconstituted in a minimal amount of ethanol and applied to Silica gel 60 F254 TLC-plates (20 9 20 cm, Merck, Germany). These were developed in a chloroform, glacial acetic acid, methanol and dH2O (60:32:12:8 (v/v/v/v)) developing solution. Detection of triterpenoids was achieved by spraying with anisaldehyde-sulphuric acid (AS) reagent (Kraemer et al. 2002). The TLC plates were sprayed with 10 ml of the spray reagent and then heated at 95C for 10 min. Authentic standards of asiatic acid, madacassic acid and their respective glycosides (Extrasynthase, France) were also chromatographed on the plates to calculate corresponding Rf values. Violet spots develop with a density proportional to the total saponin content present. This approach, described by Gurnkel and Rao (2002) was referred to as direct densitometry, which allows for the rapid analysis of many samples. Quantitive analysis using HPLC Chromatography was carried out on a Shimadzu 10AVP system consisting of a dual set of 10AT solvent delivery modules, a 10 AD Shimadzu autosampler and a Shimadzu SPD-M VP diode array

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detector. Data were collected and analyzed using the Shimadzu CLASS VP software supplied. The eluate was monitored continuously from 200 to 600 nm. Column temperature was maintained at 25C. A reverse-phase C18 Zorbax (250 9 4.6 mm) column coupled to a Phenomenex SecurityGuard guard column was used. Acetonitrile and water (Sigma, St. Louis, USA) were used as the mobile phase; the column was eluted with a gradient from 20% acetonitrile in water to 100% acetonitrile over 35 min, maintaining acetonitrile for a further 10 min. Flow rates of 1 ml min-1 were used. Quantication was carried out by injection of the four standards, the peak areas of the standards were determined at the wavelength providing maximum absorbance using the Shimadzu CLASS VP software. Concentrations from 1 to 250 lg ml-1 were injected in order to construct response versus concentration standard curves for the linearity range and regression equation. All the samples and standards were ltered through a 0.2 lm Millex-HV-lter and then put into a 2 ml SRI vial. Samples of 25 ll were injected for analysis. Under these conditions, the obtained retention times (Rt) for madecassoside, asiaticoside, madecassic acid and asiatic acid were 6.54, 8.94, 14.34 and 16.27 min, respectively. Identication of the metabolites in extracts was based on Rt values and UV absorbance spectra. Further verication of the four compounds was done by spiking with authentic standards to conrm retention time and spectral properties. Metabolite proling using densitometric analysis Zones (by calculated Rf-values) on the TLC plate which correlating to authentic standards commercially available were analysed using Quantity-One software (BioRad).

A
Wet weight (g)

18 15 12 9 6 3 0 1 4 7 10 13 16 19 22

0.6

0.4 0.3 0.2 0.1 0

Time (days)

B
Wet weight (g)

15 12 9

0.4 0.3 0.2

6 3 0 1 4 7 10 13 16 19 22 0.1 0

Time (days)

Fig. 2 The growth curves ( wet and - - - dry mass) of C. asiatica Type-1 (a) and Type-2 (b) in liquid medium, indicative of when the stationary phase is achieved ( ) and the production of secondary metabolites is predominant

Results and discussion Primary metabolism is associated with the log or exponential phase of a culture during where the sole products of metabolism are either essential for growth or are the by-products of energy-yielding metabolism (Prescott et al. 1999). Ideophase refers to the period in a batch culture in which secondary metabolites are synthesised in preference to primary metabolites; it

generally corresponds to the stationary phase and the end of the log phase. Growth curves of C. asiatica cells were constructed in order to determine the active and stationary phases of growth as is illustrated in Fig. 2. In some cases the production of secondary metabolites does not show a positive correlation with the maximal growth rate of the culture. It was ascertained that Type-1 reaches the stationary phase at day 13 of growth in suspension under the conditions described, whereas Type-2 was able to sustain a longer latent and growth phase up to day 19. Under the described experimental culture conditions, cell suspensions of C. asiatica Type-1 exhibited a lag phase from day 13, followed by an active exponential growth phase to day 12. The stationary phase was achieved after 1314 days. Similar ndings were reported by Bouhouche et al. (1998) who reported a latent phase between 0 and 3 days and an exponential growth phase from 3 to 12 days and a stationary phase from 12 to 14 days under the same conditions. Nath and Buragohain (2005) found an initial lag phase up to day 10 of incubation for C. asiatica cell suspensions followed by a steep rise in growth rate until the third week. In contrast, C. asiatica Type-2 was able to sustain a longer growth cycle. There is a latent phase from 1 to

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Dry weight (g)

Dry weight (g)

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11 days in culture before an exponential growth phase is encountered for 7 days; the stationary phase is only achieved after day 19. The highest concentrations of the targeted triterpenoids in cell suspensions detected by TLC with the AS reagent were seen on day 16 for Type-1 and day 20 for Type-2 once the stationary phase has been attained. Media ltered off from the cell cultures were analysed under the same conditions as for extracts from callus and cell suspensions. None of the investigated triterpenes could be detected (results not shown), indicating that the metabolites are not secreted to a signicant extent and therefore that harvesting of the triterpenoid saponins from the culture medium is not an option. These observations may reect a competition for metabolites utilized in primary metabolism with those pathways leading to the formation of secondary products. One approach used to regulate metabolic pathways favouring the production of specic secondary metabolites has been to add precursors to the culture medium, though it is not known if this option has been investigated for C. asiatica cells. Another is the manipulation of metabolic ux through specic pathways in response to elicitors and signal molecules of plant defense responses. The instability of cell cultures for the continued production of secondary products poses another problem; some cell lines loose the ability to synthesize the desired compound after prolonged culture. The relationship between cell differentiation and tissue organisation and the

biosynthesis of secondary compounds is also obscure. Plant secondary metabolites are normally synthesised by specialised cells, often at distinct stages of plant development and certain compounds are not synthesised if cells remain undifferentiated as in cell suspensions (Kim et al. 2002b). The distribution between mRNA transcripts, enzymes and biosynthetic products within and between cells is an important component of regulation for secondary plant metabolic processes. Many metabolic pathways are compartmentalised, enabling the separation of incompatible or competing reactions, and concentrating enzymes and metabolites (Samanani and Facchini 2006). Biotechnological attempts to overproduce the quantities of asiaticoside through cell or tissue culture have encountered limited success (Kim et al. 2002a). Callus is undifferentiated tissue which has the ability to develop into any plant organ whether it is a root, shoot or leaf, under the correct growth hormone concentrations. This might be the reason behind the presence of all the four triterpenes in the callus samples, albeit in low concentrations (Table 1). The detection of asiaticoside and madecassoside in callus contrasts with the ndings of Kim et al. (2004) who failed to detect asiaticoside in undifferentiated cells of Korean C. asiatica, but is supported by Nath and Buragohain (2005) who stated that callus and cell suspensions of Indian origin did, in fact, synthesise asiaticoside. All four of the triterpenes under consideration were found in leaves of both Type-1 and Type-2

Table 1 Quantitative determination by HPLC of asiatic acid, madecassic acid and their glycosides, asiaticoside and madecassoside, in ethanolic extracts from C. asiatica Type-1 and Type-2 from cultured cells (suspensions and callus) and leaves Sample C. asiatica Type-1 Cell suspensions Callus Leaves C. asiatica Type-2 Cell suspensions Callus Leaves Authentic standards Rf-value (TLC) Rt-value (HPLC) (min) Asiatic acid 0.16 0.032a 0.19 0.016a 1.89 0.080a 0.14 0.024a 0.24 0.021 1.79 0.102 0.95 16.27
a a

Madecassic acid 0.28 0.036a 0.24 0.013a 1.97 0.007a 0.15 0.012a 0.19 0.010 1.88 0.070 0.86 14.34
a a

Asiaticoside 1.38 0.020a 2.46 0.092a 5.23 0.025a 1.23 0.015a 2.84 0.012 4.52 0.138 0.39 8.94
a a

Madecassoside 1.67 0.012a 2.35 0.098a 4.76 1.342a 1.31 0.018a 1.98 0.056a 4.28 0.124a 0.32 6.54

Cells suspensions at day 16 (Type-1) and 20 (Type-2), respectively, were used for analyses. Quantication and identication were as described under Materials and methods (values presented are expressed as % dry weight and standard deviationa n = 5, calculated to be in the magnitude of 0.1000.032). The values presented in this table are an average of at least 5 individual experiments carried out. Authentic standards were used to set up standard curves for quantication purposes. R2 values of 0.97, 0.98, 0.99 and 0.99 were obtained for the regression lines for asiatic acid, madecassic acid, asiaticoside and madecassoside, respectively

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C. asiatica and this nding thus supports the claimed medicinal value of this herb. This point is indirectly conrmed by the fact that it is the leaves of C. asiatica that are used as material for various medicinal products and health foods (Brinkhaus et al. 2000). Analysis of callus, cell suspensions and leaf tissue by means of TLC showed differences in the asiatic acid, madecassic acid and their glycoside content for both phenotypes. These ndings were veried by HPLC analysis which allowed the quantication of the four targeted metabolites. HPLC offers the advantages of speed, sensitivity and the capability to analyse without derivatization. However, two major difculties of saponin characterisation is the lack of a chromophore and available standards. The fact that the compounds do not contain chromophores limits the detection and types of solvents which can be utilised in the HPLC method as these may absorb in the UV region. The use of acetonitrile/water on reverse-phase columns at a detection wavelength of 205 nm made the qualitative and quantitative determination of the triterpenes of C. asiatica and its pharmaceutical preparations nther and Wagner 1996; Inamdar et al. possible (Gu 1996; Schaneberg et al. 2003). By means of these parameters and our own modications, our solvent of choice was ethanol for extraction and an eluent consisting of a linear gradient of acetonitrile and water for HPLC in order to effectively separate all four compounds in a single run. Chromatograms of plant extracts which demonstrated the optimum separation and ideal evaluation of individual constituents in the plant extract is shown in Fig. 3. The detection limit by HPLC for the four standards was determined to be 1.5 lg ml-1 for madecassoside and asiaticoside with a slightly higher value of 1.8 lg ml-1 for madecassic acid and asiatic acid (the limit of detection (LOD) was calculated using peak height to three times the baseline noise). This is somewhat higher to the detection limit of 1 lg ml-1 of each standard obtained by TLC. Standard curves exhibited linearity of the above mentioned concentration ranges with R2-values of 0.970.99. Extraction efciency was studied by adding known concentrations of pure standards to plant extracts to be studied. The extraction efciency (n = 6) for madecassoside was 98.3% (relative standard deviation (R.S.D) of 0.35%), asiaticoside 98.7% (R.S.D. of 0.45%), madecassic acid 96.4% (R.S.D. of 0.67%) and asiatic acid

95.8 % (R.S.D. of 0.52%). The retention times of the four metabolites are given in Table 1. HPLC quantication indicated that leaves contained higher levels of triterpenoids than the undifferentiated cultured cells for both types, with Type-1 (the commercial species) having a higher triterpenoid content generally. In addition, all four the targeted metabolites were found in the undifferentiated cultures cells, with calli exhibiting the higher concentrations compared to cell suspensions (Fig. 3, Table 1). Secondary metabolite production may require interaction between roots and leaves with metabolic precursors generated in roots and passing to aerial parts of plants for bioconversion in leaves (Giri and Naraseu 2000). The biosynthesis of major secondary metabolites is often either tissue or organ specic (Aziz et al. 2007), as also indicated by Kim et al. in the case of C. asiatica terpenoid saponins. In contrast to our data, they could not detect asiaticoside in undifferentiated cells. Their results have shown that asiaticoside biosynthesis is concentrated in the leaves (0.41.4% dry weight) and that the level of asiaticoside content is quite low in the roots of whole plants (Kim et al. 2002a, 2004, 2005, 2007). Differences between varieties in medicinal plants of the same species (chemotypes) are common and variation in secondary metabolites has been observed with identical phenotypes and growth conditions, depending on plant origin (Aziz et al. 2007). Significant differences in active constituents have also been observed between samples of C. asiatica originating from different countries, possibly as a result of genomic diversity (Das and Mallick 1991). Gupta et al. (1999) reported variable asiaticoside content in ve lines of C. asiatica from India. Similarly, Rouillard-Guellec et al. (1997) investigated the secondary metabolites in India and Madagascar, and reported that plants from the latter contained the highest level of asiaticoside. The distribution of asiaticoside and madecassoside throughout the plant was organ specic with leaves of both lines containing the higher content of these compounds. In a study of C. asiatica from Madagascar, Randriamampionona et al. (2007) reported asiaticoside content of between 2.6 and 6.42% dry weight. The authors achieved in vitro propagation of C. asiatica in a hormone-free media but these in vitro plants displayed lower asiaticoside content. Aziz et al. (2007) reported two phenotypes of C. asiatica exhibiting differences in

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Plant Cell Tiss Organ Cult (2008) 94:9199 Fig. 3 Reverse phaseHPLC chromatograms representing characteristic proles from ethanolic extracts of Centella asiatica leaves (a), callus (b) and cell suspensions (c); (Type-1 (upper trace) and Type-2 (lower trace). HPLC conditions are as described in Materials and methods. A chromatogram of the four standards (25 ng of each): madecassoside (1), asiaticoside (2), madecassic acid (3) and asiatic acid (4) are displayed for comparative purposes. All chromatograms were normalized to the second largest peak. The y-axes were offset with 250 mAbs units and the x-axes for Type I (upper trace) offset by 2 min, in order to facilitate visual comparison. The peaks indicative of the target triterpenoid are designated by an asterisk

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terpenoid content that were tissue specic and varied between glasshouse-grown and tissue-derived material. Triterpenoid saponin content was highest in leaves (asiaticoside and madecassoside concentrations of 0.70.9 and 1.11.6% dry weight were, respectively, reported), and roots contained the lowest content of asiaticoside. In their study, asiaticoside and madecassoside were undetectable in transformed roots and undifferentiated callus. Most studies on the quantication of terpenoid saponins from C. asiatica only report on the concentration of asiaticoside. The individual identication

and quantication of the two main acids, asiatic acid and madecassic acid, as well as their glycosides, asiaticoside and madecassoside in C. asiatica extracts can be accomplished by our adapted HPLC protocol. Our data indicates that (i) the total triterpenoid saponin content of the two South African phenotypes is generally comparable to that reported from India, Korea and Madagascar, (ii) all four targeted metabolited were found to occur together (as end products of a shared, initial biosynthetic pathway), even in cell suspensions and calli, (iii) the two phenotypes we studied were found to vary in triterpenoid

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Plant Cell Tiss Organ Cult (2008) 94:9199 Th (ed) Advances in biochemical engineering/biotechnology, vol 75. Springer-Verlag, Berlin, Heidelberg, pp 3249 Inamdar PK, Yeole RD, Ghogare AB, de Souza NJ (1996) Determination of biologically active constituents in Centella asiatica. J Chromatogr 742:127130 Kim OT, Kim MY, Hong MH, Ahn JC, Oh MH, Hwang B (2002a) Production of triterpene glycosides from whole plant cultures of Centella asiatica (L.) Urban. Korean J Plant Biotechnol 29:275279 Kim Y, Wyslouzil BE, Weathers PJ (2002b) Secondary metabolism of hairy root cultures in bioreactors. In vitro Cell Dev Biol Plant: J Tissue Cult Assoc 38:110 Kim OT, Kim MY, Hong MH, Ahn JC, Hwang B (2004) Stimulation of asiaticoside accumulation in the whole plant cultures of Centella asiatica (L.) Urban by elicitors. Plant Cell Rep 23:339344 Kim OT, Kim MY, Huh SM, Bai DG, Ahn JC, Hwang B (2005) Cloning of a cDNA probably encoding oxidosqualene cyclise associated with asiaticoside biosynthesis from Centella asiatica (L.) Urban. Plant Cell Rep 24:304311 Kim OT, Kyong-Hwan B, Shin Y-S, Lee M-J, Jung S-J, Hyun D-Y, Kim Y-C, Seong N-S, Cha S-W, Hwang B (2007) Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate. Plant Cell Rep 26:19411949 Kraemer KH, Schenkel EP, Verpoorte R (2002) Ilex paraguariensis cell suspension culture characterisation and response against ethanol. Plant Cell Tissue Organ Cult 68:257263 Maquart FX, Chastang F, Simeon A, Birembaut P, Gillery P, Wegrowski Y (1999) Triterpenes from Centella asiatica stimulate extracellular matrix accumulation in rat experimental wounds. Eur J Dermatol 9:289296 Mathur S, Verma RK, Gupta MM, Ram M, Sharma S, Kumar S (2000) Screening of genetic resources for the medicinalvegetable plant Centella asiatica for herb and asiaticoside yields under shaded and full light conditions. J Horticult Sci Biotechnol 75:551554 Matsuda H, Morikawa T, Ueda H, Yokhikawa M (2001) Medicinal foodstuffs XXVII. Saponin constituents of Gotu Kola (2): structures of new ursane- and oleananetype triterpenene oligoglycerides, Centellasaponins B, C, and D, from Centella asiatica cultivated in Sri Lanka. Chem Pharm Bull 49:13681371 Nath S, Buragohain AK (2005) Establishment of callus and cell suspension cultures of Centella asiatica. Biol Plant 49:411413 Prescott LM, Harley JP, Klein D (1999) Microbiology, 4th edn. McGraw-Hill, Boston, p 938 Randriamampionona D, Diallo B, Rakotoniriana F, Rabemanantsoa C, Cheuk K, Corbisier A-M, Mahillion J, Ratsimamanga S, Jaziri MEJ (2007) Comparative analysis of active constituents in Centella asiatica samples from Madagascar: application for ex situ conservation and clonal propagation. Fitoterapia 78:482489 Rouillard-Guellec F, Robin JR, Ratsimamanga AR, Ratsimamanga S, Rasaoanaivo R (1997) Comparative study of Centella asiatica of Madagascar origin and Indian origin. te Botanique Acta Botanica Gallica: Bulletin de la Socie de France 144:489493

composition and concentration and that (iv) the four metabolites occur in leaf tissue with the free acids to glycosides in a ratio of approximately 1:2.5 (Table 1). The fourth point differs from the ndings of Randriamampionona et al. (2007) who reported free acid: glycoside ratios ranging from 1:5 to 1:30 for leaf tissue of C. asiatica from Madagascar. The differences in the triterpenoid saponin composition and content of various C. asiatica chemotypes can perhaps be attributed to genetic variation in the oxidosqualene cyclase and other genes involved in the biosynthesis (Haralampidis et al. 2002; Kim et al. 2007), as well as the presence and activity of enzymes involved in the attachment of the sugar residues to the aglycones. Metabolic pathways for these triterpenoids should therefore be further investigated and the ux through these pathways elucidated to obtain a better understanding of the biochemical conversions that will allow the manipulation and exploitation of secondary product synthesis in C. asiatica.

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