Alternative Production of Saponins from Panax ginseng

Transcription

1 Alternative Production of Saponins from Panax ginseng L. Langhansová, P. Maršík and T. Vaněk Institute of Organic Chemistry and Biochemistry, AS CR, Flemingovo nám , Praha 6 Czech Republic Keywords: ginseng, adventitious roots, bioreactor, TIS Abbreviations: IBA indole-3-butric acid, TIS temporary immersion system, SH Schenk and Hildebrandt (1972), MS Murashige and Skoog (1962), NAA naphthalene acetic acid Abstract Different systems of large-scale cultivation of multiple adventitious roots of Panax ginseng C. A. Meyer comparing to common cultivation in Erlenmayer flasks were established. Adventitious roots were isolated from plantlets regenerated from somatic embryos and cultivated separately in liquid media. Formation of adventitious roots was reached on liquid Schenk and Hildebrandt (1972) medium containing 3% sucrose, and 24.6 µm indole-3-butric acid. The highest saponin content of 2,09 mg.g -1 of fresh weight was found in adventitious roots cultivated in Rita system and in Osmotek Life Reactor. The best production of biomass was achieved in standard Erlenmayer flasks placed on rotary shaker, followed by Rita bioreactor. Saponin production in Erlenmayer flasks was considerably lower (0.72 mg.g -1 of fresh weight). The saponin production of roots cultivated in other tested bioreactor systems was not significant. INTRODUCTION Panax ginseng C. A. Meyer (Araliaceae) is very important tonic medicinal plant. Biologically active compound ginsenosides are extracted from roots. Natural sources of wild growing plants in mountain areas, from Nepal to Manchuria, and from eastern Siberia to Korea have been overexploited and therefore are very limited. The current supply of ginseng mainly depends on field cultivation, held primarily in Japan, Korea and north China but it is a long and laborious process. Ginseng plants need 5-7 years prior to harvest and the content of ginsenosides is low. In vitro mass production in large-scale systems seems to be a potentially more efficient alternative for production of ginseng bioactive components. The aim of our previous study was to establish in vitro cultures producing ginsenosides identical to those in native ginseng plants. We found a full range of ginsenosides distributed analogous as in roots of native plants in adventitious roots cultivated in liquid Schenk and Hildebrandt media supplemented with 24.6 µm IBA (Choi et al., 2002). Moreover we found some polyacetylen compounds, which have been reported to have a cytotoxic activity (Newal et al., 1996). Recently we have tested cultivation of adventitious roots in different bioreactor systems and we are adjusting culture conditions to increase yield while preserving the ability of adventitious roots to produce whole spectrum of biologically active compounds. The cultivation was primarily held in 250 ml Erlenmayer flasks on rotary shaker (127 rpm) where the total ginsenoside content varies from 0.25 to 0.53% in dry weight. MATERIALS AND METHODS Adventitious Roots Induction Adventitious roots of Panax ginseng were isolated from plantlets regenerated from somatic embryos and rooted on third-strength MS medium. Separated roots were Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law Eds. C. Franz, Á. Máthé, L.E. Craker and Z.E. Gardner Acta Hort. 678, ISHS

2 transferred in liquid MS medium supplemented with 1 µm NAA and cultivation was held in 500 ml Erlenmayer flasks on rotary shaker (125 rpm). Formation of adventitious roots was reached on liquid SH media supplemented with 24.6 µm IBA (Choi et al., 2000). Bioreactor Cultures Adventitious roots cultures were cultivated in different bioreactor systems: 1. Erlenmayer flasks with volume of 0,5 L control cultivation system 2. Temporary Immersion System bioreactor RITA (Fig. 1) 3. MAFE 0,5 L bioreactor (Fig. 2) 4. The Aplikon Bioreactor (Aplikon, Netherlands) with total volume of 3 L 5. Simple conical airlift bioreactor made from 1 L separation funnel (Fig. 3) 6. Life Reactor System (Osmotek, Israel) with total volume of 5 L (Fig. 4) Detection of Ginsenosides by HPLC Adventitious roots were homogenized and extracted with methanol (7 ml/1 g fresh weight) for 5 days at room temperature. The sample was then filtered, and evaporated to dryness under vacuum. The residue of extract was redissolved in destil water and partitioned with dietyl-ether, and twice in n-buoh saturated with water. The n-buoh layer was concentrated in vacuo to obtain crude saponin fraction (Sanada, 1974). The n- BuOH soluble fraction was analyzed by HPLC for identification and quantification of ginsenosides Rb 1, Rb 2, Rc, Rd, Re, Rf and Rg 1. HPLC analyses were performed on the system consisting of two high pressure pumps (DeltaChrom, SDS 020 a SDS 030) with a mixer (SunChrom GmbH) and PDA detector (JASCO, MD 1510); the stainless steel column (250 x 4 mm) packed by reverse phase Si-C18, 7 µm (Biospher); flow-rate 1 ml/min. The injection volume was set up at 20 µl in the autosampler (TSP, AS300). Eluents: (A) 15% acetonitrile and water, (B) 100% acetonitrile; Gradient elution profile: 0-40 min, 0-35% B; min, 35% B. The peaks were monitored by UV detection at 203 nm (Soldati and Sticher, 1980; Pietta et al., 1986; Petersen and Palmqvist, 1990). Each ginsenoside was identified by comparison of retention time and UV spectra with authentic ginsenosides purchased from Carl Roth GmbH & Co., Germany. Ginsenoside content was expressed in mg.g -1 of fresh weight. Presence of ginsenosides was additionally confirmed by LC-MS. RESULTS AND DISCUSSION We are testing these large-scale systems for biomass production of adventitious roots. The best production of biomass was achieved in standard Erlenmayer flasks placed on rotary shaker, followed by RITA bioreactor (Fig. 5). Growth value in evaporation funnel bioreactor (simple airlift bioreactor) and Osmotek Life Reactor was very similar. In both, airlift bioreactor and Osmotek, the cultivation conditions are similar in system of aeration. Air is sparged below the root flowing afloat the bioreactor funnel or bag. The proportion of particular ginsenosides compared with native material is almost identical after cultivation in all bioreactor systems with exception of Applikon bioreactor where we observed inhibition of the protopanaxadiol group and in ½ L Mafe bioreactor the biosynthesis of the protopanaxadiol group was significantly stimulated. Total saponin content in the root of nature Panax ginseng is commonly in the range of 1-3% dry weight (Bruneton, 1995). The highest content of 2.1 mg.g -1 fresh weight of ginsenosides (Rb 1, Rb 2, Rc, Rd, Re, Rf and Rg 1 ) in adventitious roots was achieved in RITA TIS and in Osmotek Life Reactor (Fig. 6). However, in order to reach effective production we have to combine two factors, which is the saponin content and biomass growth. In conclusion, here we found root culture in the RITA TIS optimal for ginsenoside production regarding a high biomass growth. On the other hand Osmotek Life Reactor is of larger volume and easier to handle. 46

3 ACKNOWLEDGEMENTS This work was supported by 521/02/P064, COST and Z projects. Literature Cited Bruneton, J Pharmacognosy, Phytochemistry, Medicinal Plants. Lavoisier Publishing. 915p. Choi, S.M., Son, S.H., Yun, S.R., Kwon, O.W., Seon, J.H. and Paek, K.Y Pilotscale culture of adventitious roots of ginseng in a bioreactor system. Plant cell, tissue and organ culture 62(3): Sanada, S Studies on the Saponins of Ginseng. I Structures of Ginsenoside - Ro,- Rb 1, - Rb 2, - Rc and - Rd. Chem. Pharm. Bull. 22(2): Newall, C.A., Anderson, L.A. and Phillipson, J.D Herbal Medicines. A Guide for Health-care Professionals. The Pharmaceutical Press, London. 296p. Schenk, R.U. and Hildebrandt, A.C Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50: Soldati, F. and Sticher, O HPLC Separation and Quantitative Determination of Ginsenosides from Panax ginseng, Panax quinquefolium and from Ginseng Drug Preparations. Planta Medica 38: Petersen, T.G. and Palmqvist, B Utilizing column selectivity in developing a highperformance liquid chromatographic method for ginsenoside assay. J. Chromatography 504: Pietta, P. and Rava, P.M Improved high-performance liquid chromatographic method for the analysis of ginsenosides in Panax ginseng extracts and products. J. Chromatography 356:

4 Figures Fig. 1. RITA, Temporary immersion system. Fig. 2. MAFE, 0,5 L bioreactor. 48

5 Fig. 3. Simple conical airlift bioreactor made from separation funnel. Fig. 4. Life Reactor System (Osmotek). 49

6 3.0 6 Biomass growth (g/100ml) Inoculum per 100 ml Biomass grow th per 100 ml Grow th value Growth value 0.0 Erlen. flasks RITA MAFE Applikon Airlift Osmotek Bioreactor system 0 Fig. 5. Growth adventitious roots in different bioreactor systems after 2 months of cultivation. Values are expressed in dry weight. Saponin content (mg/g fw) Erlen. Flasks RITA MAFE Applicon Airlift Osmotek Bioreactor system Fig. 6. Total ginsenoside production. 50