Quantification of Asiatic Acid from Plant Parts of Centella asiatica L. and Enhancement of Its Synthesis through Organic Elicitors in in Vitro

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1 Hort. Environ. Biotechnol. 55(6): DOI /s Research Report ISSN (print) : ISSN (online) : Quantification of Asiatic Acid from Plant Parts of Centella asiatica L. and Enhancement of Its Synthesis through Organic Elicitors in in Vitro Behrooz Mohammadparast 1*, Mousa Rasouli 2, Ali Reza Rustaiee 3, Sannaz Zardari 4, and Veena Agrawal 5 1 Biology Department, Faculty of Science, Malayer University, Malayer, Iran 2 Landscape Engineering Department, Faculty of Agriculture, Malayer University, Malayer, Iran 3 Horticultural Department, Agriculture Research Center, University of Zabol, Zabol, Iran 4 Horticultural Department, Agriculture Faculty, Tabriz University, Tabriz, Iran 5 Botany Department, North Campus, University of Delhi, Delhi , India *Corresponding author: bmp2013@yahoo.com Received November 28, 2013 / Revised June 22, 2014 / Accepted July 2, 2014 C Korean Society for Horticultural Science and Springer 2014 Abstract. Medicinal plants are nearly unlimited source of phytochemicals. Centella asiatica L. is a tropical medicinal plant with a long history of therapeutic uses, dermal disorders, venous insufficiency micro-angiopathy, and anticancer. The present study highlights (i) in vivo evaluation of asiatic acid (as an anticancer compound) content in different plant parts of C. asiatica and (ii) in vitro elicitation of asiatic acid employing organic elicitors. Based on the HPLC analysis, the highest amount of asiatic acid (190.2 µg g -1 fresh wt.) was found in root part in in vivo condition. Quantitative analyses were done to enhance of asiatic acid content by some organic elicitors. Results showed the highest quantity of asiatic acid content (258.3 µg g -1 fw.) among all of the organic elicitors was found at 200 mg L -1 yeast extract in leaf callus cultures. All these observations suggest that exogenous application of organic elicitors could have induced a subset of secondary metabolite biosynthetic genes, which may modulate expression of genes and accumulation of compounds induced by elicitors. Additional key words: glycine, myo-inositol, sucrose, yeast extract Introduction Medicinal plants can provide biologically active molecules for the development of modified derivatives with enhanced activity and reduced toxicity. Various secondary metabolites are useful in plants interaction with the environment of various stress factors. Wide arrays of external stimuli are capable of triggering changes in the plant cell which lead to a cascade of reactions ultimately resulting in the formation and accumulation of secondary metabolites which helps plant to overcome the stress factors. The biotic and abiotic elicitors can result in an enhancement of the secondary metabolite production. The stimuli are perceived by receptors which then result in the activation of the secondary messengers (Zhao et al., 2005). Centella asiatica L. (Apiaceae) is an important medicinal herb indigenous to India that is considered to be of immense medicinal value. The leaves of this species are used in the treatment of syphilis, all types of fever, children s abdominal disorder, elephantiasis, and hydrocele. It also is useful in treating dermatosis, anaemia, diabetes, cough, dysoponea, emaciation, and insanition (Joshi, 2000). Centella asiatica is a profusely branched prostrate herb consisting of active principles such as vallarine, asiaticoside, sitosterol, tannin, oxy-asiaticoside and asiatic acid that are used in pharmaceuticals, health, and body care products. Asiaticoside is used in the treatment of leprosy. Sitosterol and tannin possess antiprotozoal and spasmolytic property. Asiatic acid (AA) is an important anticancerous compound that is considered a good candidate for the therapeutic intervention of human breast and skin cancer (Hsu et al., 2005; Park et al., 2005). Four triterpenes precursors, squalene, farnesyl pyrophosphate (FPP), isopentenyl pyrophosphate (IPP) and leucine, which are involved directly or indirectly in the triterpene biosynthetic pathway, were used to increase the triterpenes production. Squalene was found to be the best precursor in promoting triterpenes production in callus cultures. Squalene treated callus also produced the highest biomass production compared to the other precursors tested (Kiong et al., 2005). Improvement of the AA content through different elicitors

2 Hort. Environ. Biotechnol. 55(6): such as organic elicitors not attempted yet. Prior to this study, the AA had been extracted from whole plant of C. asiatica (Bonfill et al., 2006; Gunther and Wagner, 1996; Schaneberg et al., 2003; Singh and Rasatogi, 1969). The present study reports the evaluation of the AA content in (i) in vivo different plant parts of C. asiatica and (ii) in vitro elicitation of AA content by employing organic elicitors. Materials and Methods Plant Materials The leaf explants of C. asiatica were taken from seed beds of Botanical Garden, Department of Botany, University of Delhi. These explants were washed thoroughly under running tap water for 20 min and treated with 1% bavistin (w/v) for 10 min with constant vigorous shaking on rotary table top shaker at 150 rpm, to provide better surface contact with the fungicide. Those were rewashed under running tap water to remove any traces of bavistin. After pouring out excess of water, the explants were sterilized with 0.1% (w/v) aqueous mercuric chloride solution for 2 min. They were given 4 or 5 time washings with sterilized distilled water. Culture Media Culture media MS and B5 were used. Analytical grade (AR) salts (Qualigens or Glaxo Fine Chemicals, Mumbai, India) were used to prepare the stock solutions. The basal medium was supplemented with various growth regulators such as N6-benzyladenine (BA), a-naphthalene acetic acid (NAA), kinetin (Sigma Aldrich, St. Louis, MO, USA), organic compounds (casein hydrolyzate, glycine, myo-inositol, glutamine, proline, yeast extract in the range of 1, 5, 25, 50, 100, 200, and 300 mg L -1 and sucrose: 1.5, 3, 4.5, 6, 7.5, and 9%). As a source of carbon, 3% (w/v) was used. The media were gelled with 0.8% agar (Qualigens Fine Chemicals, Mumbai, India) and the ph of media was adjusted to 5.8 using 0.1 N NaOH or HCl before autoclaving. Approximately, 20 ml media was dispensed in each cm test tubes plugged with non-absorbent cotton wrapped in muslin cloth and was autoclaved at 1.06 kg cm -2 at 121 C for 15 min. Sample Preparation and Estimation of Asiatic Acid The method used for triterpenes extraction was a slight modification from that of Inamdar et al. (1996). Centella asiatica samples were oven-dried at 50 C. The dried sample was grounded with mortar and pestle and 1 g was extracted with 20 ml of absolute analytical grade methanol thrice and kept for one week at room temperature under dark condition. The samples were then filtered and the filtrate was evaporated to dryness using rotary evaporator to obtain a dark brown crude extract. The dried crude extract was dissolved in 1.0 ml of absolute HPLC grade methanol. The extracts were finally filtered through a 0.45 µm filter prior to running a sample on an HPLC system. The HPLC system (4A Shimadzu, Kyoto, Japan) was equipped with a UV detector was operated under the following parameters: column, C 18 ; column packing, Zorbex ODS; solvent, methanol; injection volume, 20 µl; flow rate, 0.5 ml min -1 ; detection, UV 210 nm for the AA (A2612, Sigma-Aldrich 3050 Spruce, St. Louis, MO, USA) Cultures of Leaf Explants The leaf explants were incubated in continuous light ( µw cm -2 ) using cool day light fluorescent incandescent tubes (40 W, Philips, Kolkata, India). The cultures were maintained in a culture room at the temperature of 25 ± 2 C and 55 ± 10% relative humidity. The explants were subcultured after every day interval on the same fresh medium. Observations were recorded at an interval of seven days. The final data were recorded after 30 days of inoculation. Statistical Analysis The evaluation of the AA content from various callus samples of C. asiatica was performed with four replicates each and the data obtained were analyzed statistically. The statistical analyses were performed by ANOVA using SPSS. The differences between means were tested for significance by Duncan s multiple range test at p Results Quantitative Analysis of Asiatic Acid from Different Plant Parts A comparative study of AA estimation of different parts had revealed that the maximum quantity of AA was present in root callus (190.2 µg g -1 fresh wt.) (Fig. 1). Fig. 2 shows HPLC chromatograms of C. asiatica samples, in calli of root, rhizome, leaves and petiole. Since callus derived from leaf proved greatest in terms of AA content and had good response callus formation, it was further used for enhancement of the AA content using various elicitors. Effect of Various Organic Elicitors on Asiatic Acid Synthesis The AA yield was elicitor specific and concentration dependent. The highest quantity of the AA was found (258.3 µg g -1 fresh wt.) at 200 mg L -1 of yeast extract which was also highest in comparison to organic elicitor that had been studied (Table 1). With the increase in concentration of yeast extract from 1 to 200 mg L -1, AA content increased gradually. However, at concentrations higher than 200 mg L -1 its quantity decreased. With the gradual increase in concentration

3 580 Behrooz Mohammadparast, Mousa Rasouli, Ali Reza Rustaiee, Sannaz Zardari, and Veena Agrawal Fig. 1. Asiatic acid contents from different plant parts of C. asiatica. of myo-inositol from 1 to 25 mg L -1 there was a gradual increase in the AA content and the optimum response in terms of AA content (208 µg g -1 fresh wt.) was detected at 25 mg L -1 myo-inositol. With the gradual increase in concentration of casein hydrolysate from 1 to 100 mg L -1, there was a gradual increase in the AA content in comparison to control (183.3 µg g -1 fresh wt.) and the highest value (231.7 µg g -1 fresh wt.) was detected at 100 mg L -1 casein hydrolysate. Addition of sucrose in the medium enhanced the quantity of the AA content significantly. Sucrose at 9% induced the maximum response (225.6 µg g -1 fresh wt.). Addition of proline did not affect in elevating of AA content significantly. A maximum of µg g -1 fresh wt. of AA was observed at 1 mg L -1 of proline (Table 2). Beyond this level, a gradual decline in the AA content was observed. The maximum quantity of AA content ( µg g -1 fresh wt.) was detected at 50 mg L -1 of glycine. At concentrations higher than 50 mg L -1 of glycine, the AA content decreased. With the gradual increase in concentration of glutamine from 1 to 100 mg L -1 there was a gradual increase in the AA content and the greatest quantity of AA (241.6 µg g -1 fresh wt.) was found at 100 mg L -1 glutamine. Discussion The yeast extract significantly increased the intracellular content of AA in comparison to the control. These results could be attributed to the content of some cations like Zn, Ca, and Co, present in yeast extract which could act as abiotic elicitors (Suzuki et al., 1985). As the yeast extract was composed of a variety of compounds, apart from amino acids, vitamins, and minerals, it was also possible that the elicitation effects might be due to other components that was still not identified. Similar to our results, an enhancement in metabolite content using yeast extract was also reported in Orthosiphon aristatus, Salvia miltiorrhiza (Sumaryono et Fig. 2. HPLC determination of asiatic acid from different callus derived plant parts of C. asiatica: A, root; B, rhizome; C, petiole; and D, leaf. al., 1991; Yan et al., 2006; Zhang et al., 2004). The effect of

4 Hort. Environ. Biotechnol. 55(6): Table 1. Effect of different elicitors on asiatic acid content in C. asiatica leaf callus cultures. Treatment Asiatic acid content Amino acid Concentration (μg g -1 fresh wt.) Control f z Yeast extract (mg L -1 ) g e d c b a a Myo-inositol (mg L -1 ) c b a b c c c Casein hydrolysate (mg L -1 ) e c d b a c e Sucrose (%) e d c c b a z Results for the asiatic acid content through HPLC for different elicitors used are the mean of four replicates. Letters on the columns indicate significant differences according to Duncan s multiple range test at p yeast extract might be due to its role in increasing the activity of key enzyme of triterpenes pathway that catalyzed the production of secondary metabolites with triterpenes skeleton (Seidel et al., 2002). Akin to this, amino acid also enhanced the AA content in C. asiatica callus cultures and this might be due to production of some plant hormones under the influence of amino acids. Many authors have described the biochemical basis of synthesis of other secondary metabolites in different plants (Perassolo et al., 2007). Some amino acids were incorporated into intermediate compounds of glycolysis and TCA cycle after deamination (Bonner, 1972). Acetyl-CoA was a precursor Table 2. Effect of different amino acids on asiatic acid content in C. asiatica leaf callus cultures. Treatment Asiatic acid content Amino acid Concentration (μg g -1 fresh wt.) Control f z Proline (mg L -1 ) a b d c c e f Glycine (mg L -1 ) e d c a a b b Glutamine (mg L -1 ) e d b b a c c z Results for the asiatic acid content through HPLC for different elicitors used are the mean of four replicates. Letters on the columns indicate significant differences according to Duncan s multiple range test at p of secondary metabolites and is converted into isopentyl pyrophosphate which was a precursor of terpenoids. Lower levels inositol was found to be optimum dose in C. asiatica, this might be due to the role of inositol in signal transduction pathway (Jian et al., 2005). It was able to increase Ca 2+ concentration by associating with a membrane protein called IP3-gated channel or IP3 receptor. Inositol of 1, 4, 5 triphosphate must bind to sites on the cytosolic side of the membrane protein to open the channel and release Ca 2+ (Berg et al., 2001). Phosphatidyl inositol-4-monophosphate and phosphatidylinositol-4, 5-bisphosphate increased the activity of ATPase associated with plasma membranes isolated from both sunflower hypocotyls and carrot suspension culture cells (Memon et al., 1989). Our data suggested that activation of the inositol phospholipid kinases could be a critical step in signal transduction in plants. Casein improved three essential nutrients, Ca 2+, phosphate, and amino acid. The numerous instances showed, phosphorylation dephosphorylation cycles regulated the activity of

5 582 Behrooz Mohammadparast, Mousa Rasouli, Ali Reza Rustaiee, Sannaz Zardari, and Veena Agrawal many enzymes and regulatory proteins (Nelson and Cox, 2001). In the present study, our data revealed that lower concentrations of casein hydrolysate proved to be effective for enhancing the amount of AA in C. asiatica cultures, 100 mg L -1 CH was found to be optimum dose for cultures. In this study, all the levels of sucrose improved the AA content in C. asiatica cultures. However, sucrose at 1.5% and 3% induced lower amount of AA in C. asiatica callus cultures. Despite this, the response obtained was dose dependent. The improvement of AA perhaps related to the role of the sucrose acted as an ATP generation compound that was needed for various biological reactions (Berg et al., 2001). Literature Cited Berg, J., T. John, and L. Stryer Biochemistry. Freeman WH and Co., New York. Bonfill, M., S. Mangas, R.M. Cusidü, L. Osuna, M.T. Piñol, and J. Palazün Identification of triterpenoid compounds of Centella asiatica by thin-layer chromatography and mass spectrometry. Biomed. Chromatogr. 20: Bonner, J The isoprenoids, p In: J. Bonner and J.E. Varner (eds.). Plant biochemistry. Academic Press, New York. Gunther. B. and H. Wagner Quantitative determination of triterpenes in extracts and phytoparations of Centella asiatica (L.) Urban. Phytomedicine 3: Hsu, Y.L., P.L. Kuo, L.T. Lin, and C.C. Lin Asiatic acid, a triterpene, induces apoptosis and cell cycle arrest through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells. J. Pharmacol. Exp. Ther. 313: Inamdar, P.K., R.D. Yeole, A.B. Ghogare, and N.J. Souza Determination of biologically active constituents in Centella asiatica. J. Chromatogr. A 742: Jian, Z., L.C. Davis, and R. Verpoorte Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol. Adv. 23: Joshi, S.G Medicinal plants. Oxford & IBH Publishing Co., Pvt, Ltd., New Delhi, India. Kiong, A.L.P., M. Mahmood, N.M. Fadzillah, and S.K. Daud Effects of precursor supplementation on the production of triterpenes by Centella asiatica callus cultures. Pak. J. Biol. Sci. 8: Memon, A.R., Q.Y. Chen, and W.F. Boss Inositol phospholipids activate plasma membrane ATPase in plants. Biochem. Biophys. Res. Commun. 162: Nelson, D.L. and M.M. Cox Lehninger s principles of biochemistry. Freeman WH and Co., New York. p Park, B.C., K.O. Bosire, E.S. Lee, Y.S. Lee, and J.A. Kim Asiatic acid induces apoptosis in SK-MEL-2 human melanoma cells. Cancer Lett. 218: Perassolo, M., C. Quevedo, V. Busto, F. Ianone, A.M. Giulietti, and J. Rodriguez Talou Enhance of anthraquinone production by effect of proline and aminoindan-2-phosphonic acid in Rubia tinctorum suspension cultures. Enzyme Microb. Technol. 41: Schaneberg, B.T., J.R. Mikell, E. Bedir, and I.A. Khan An improved HPLC method for quantitative determination of six triterpenes in Centella asiatica extracts and commercial products. Pharmazie 58: Seidel, V., J. Windhövel, G. Eaton, A.W. Alfermann, R.R. Arroo, M. Medarde, M. Petersen, and J.G. Woolley Biosynthesis of podophyllotoxin in Linum album cell cultures. Planta. 215: Singh, B. and R.P. Rastogi A re-investigation of the triterpenes of Centella asiatica. Phytochemistry 8: Sumaryono, W., P. Proksch, T. Hartmann, M. Nimtz, and V. Wray Induction of rosmarinic acid accumulation in cell suspension cultures of Orthosiphon aristatus after treatment with yeast extract. Phytochemistry 30: Suzuki, T., H. Mori, T. Yamame, and S. Shimuzu Automatic supplementation of minerals in fed-batch culture to high cell mass concentration. Biotech. Bioeng. 27: Yan, Q., Z.D. Hu, and J.Y. Wu Synergistic effects of biotic and abiotic elicitors on the production of tanshinones in Salvia miltiorrhiza hairy root culture. China. J. Chin. Mater. Med. 31: Zhang, C., Q. Yan, W.K. Cheuk, and J. Wu Enhancement of tanshinone production in Salvia miltiorrhiza hairy root culture by Ag+ elicitation and nutrient feeding. Planta Med. 70: Zhao, J., L.C. Davis, and R. Verpoorte Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol. Adv. 23: