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Production of saponin in fermentation process of Sanchi (Panax notoginseng) and biotransformation of saponin byBacillus subtilis

Production of saponin in fermentation process of Sanchi (Panax notoginseng) and biotransformation... Annals of Microbiology, 56 (2) 151-153 (2006) Production of saponin in fermentation process of Sanchi (Panax notoginseng) and biotransformation of saponin by Bacillus subtilis 1 2 1 1 Guo Hong LI , Yue Mao SHEN , Yajun LIU , Ke Qin ZHANG * 1 2 Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091; The State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China Received 6 September 2005 / Accepted 27 January 2006 Abstract - An antitumor compound ginsenoside Rh was produced during the fermentation process of Sanchi (Panax notoginseng) by Bacillus subtilis. Saponins ginsenosides Rh and Re were transformed by B. subtilis and were produced two main transformed products. The transformed product of ginsenoside Rh was determined to be 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-pro- topanaxatriol, and the transformed product of ginsenoside Re had 162 atomic mass units (amu) greater than the substrate. Compared with the substrates, the transformed products had one more glucosyl moiety linked respectively, which indicated that glycosylation reac- tion occurred in the transformation process. Key words: fermentation, Sanchi, biotransformation, saponin, Bacillus subtilis, glycosylation. INTRODUCTION two key metabolites, one of which is a new compound. Up to now, there is no report about the microbial fermentation of Traditional Chinese Medicine (TCM) as an integral part of Chi- Sanchi and the glycosylation reaction in the transformation nese culture has been studied for the effective utilization all process of saponins. The difference of saponins between orig- along. The fermentation research of TCM started since 80’s inal and fermented Sanchi as well as the glycosylation reac- last century, but only aimed at fermentation of officinal fungi tion in the transformation process of saponin by Bacillus sub- (Wu et al., 2001). Microorganisms have strong ability of tilis will be discussed in this paper, by which we hope to provide transforming substances and the physiological activities of a new approach to exploit TCM effectively. some TCM’s substances have been changed by the trans- formation of intestinal microbes (Zhao et al., 1998), so the microbial fermentation of TCM’s substances may produce new MATERIALS AND METHODS compounds or change the content of active components. There is no report on the componential change between Microbial strain and Sanchi. The strain of Bacillus subtilis TCM’s substances and their fermented products. was purchased from China General Microbiological Culture Sanchi (Panax notoginseng) is a famous traditional herb, Collection Center (CGMCC No. 1318) and was conserved in which has been clinically tested to have the abilities of pro- nutrient agar medium. moting blood circulation, preventing the formation of blood Sanchi was purchased from medicinal materials market clots (anti-thrombosis property), dissolving blood clots, of Yunnan Province, People’s Republic of China. Ginsenosides enhancing the removal of cellular breakdown products and Rh and Re were prepared from Sanchi with method other debris from the blood circulation. Saponins as the major described by Zhou et al. (1981). components in Sanchi have been proved to have diversiform biological activities (Jiang and Qian, 1995; Li and Chu, 1999). Fermentation of Sanchi. Sanchi was fermented by B. sub- Some work related with the transformation and metabolism tilis in solid form. The fibres of Sanchi were ground into pow- of Sanchi saponins (Chen et al., 1999; Bae et al., 2000; Lin et der and mixed with water; the mixture (400 g) was put into al., 2001; Dong et al., 2003) has been done to elucidate that flask (1 litre) and sterilized at 121 °C for 40 min. Bacillus sub- the metabolism of ginsenoside Rb begins with cleavage of the tilis (40 ml), cultured in Wort medium, pH 7.0, at 30 °C for -1 terminal sugar moiety, then gradually the other sugars. The 72 h on an orbital shaker at 120 rev min , was inoculated transformation of ginsenoside Rb by Curvularia lunata gives into each flask. After culturing at 30 °C for 10 days, the fer- mented products of Sanchi were dried in oven at 60 °C. Biotransformation procedures. Bacillus subtilis was inoc- * Corresponding author. Phone: + 86 871 5034878; ulated in 250 ml flask containing 100 ml Wort medium at pH Fax: +86 871 5034838; E-mail: Kqzhang111@yahoo.com.cn 7.0. Ginsenoside Rh and ginsenoside Re, dissolved in 1 152 G.H. Li et al. ethanol, were each added into three flasks of Wort medium 9.0, 15.2, H-3), 4.36 (1H, m, H-6), 5.32 (1H, m, H-24), 4.94 inoculated with B. subtilis to make the final concentration of (1H, d, 7.7, H-6-glc-1’), 5.91 (1H, d, 8.0, H-6-glc-1’’); ginsenosides Rh 0.4 mg/ml and ginsenoside Re 0.2 mg/ml. C-NMR (C D N, 100 MHz): 39.4 (C-1), 27.9 (C-2), 89.2 1 5 5 Two controls of equal volume of ethanol added into the (C-3), 40.3 (C-4), 61.5 (C-5), 80.3 (C-6), 45.3 (C-7), 41.2 flasks of Wort medium with and without B. subtilis were pre- (C-8), 50.2 (C-9), 39.7 (C-10), 32.1 (C-11), 71.1 (C-12), pared. All the flasks were cultured at 30 °C for 96 h at 120 48.3 (C-13), 51.7 (C-14), 31.3 (C-15), 26.9 (C-16), 54.8 (C- -1 rev min . 17), 17.4 (C-18), 17.7 (C-19), 73.0 (C-20), 27.1 (C-21), 35.9 (C-22), 23.0 (C-23), 126.4 (C-24), 130.8 (C-25), 25.8 Isolation and identification compounds. The fermenta- (C-26), 17.6 (C-27), 31.7 (C-28), 16.3 (C-29), 16.8 (C-30), tion mixture (2 kg) was extracted three times with 80% 105.8 (C-1’), 75.6 (C-2’), 80.3 (C-3’), 72.0 (C-4’), 78.6 ethanol exhaustively and dissolved in water; then was (C-5’), 62.5 (C-6’), 103.1 (C-1’’), 74.8 (C-2’’), 77.6 (C-3’’), extracted five times with n-butanol to give 61 g of residue. 71.0 (C-4’’), 77.6 (C-5’’), 62.6 (C-6’’). The residue was subjected over silica gel column (chloro- Compound 3: white power, negative FAB-MS m/z: 1108 - - form:methanol:water, 65:35:10, v/v/v), reversed-phase C ([M – H] , 100), 962 ([M – H – 146] , 6), 946 ([M – H – - - (RP-18) column (methanol:water, 65:35, v/v) and Sephadex 162] , 6), 476 ([M – 146 – 3×162] , 11). LH-20 column (methanol) to give compound 1 (16 mg). The three flasks (300 ml) of B. subtilis culture, trans- forming ginsenoside Rh and Re respectively, were filtered. RESULTS AND DISCUSSION The filtrates were extracted four times with n-butanol, 4.5 g residue containing transformational product of ginsenoside According to TLC result, the spot appeared in fermented Rh (TRh) and 1.5 g residue containing transformational Sanchi but not in original Sanchi was selected as isolation product of ginsenoside Re (TRe) were obtained, respective- object, and compound 1 was obtained. From the broths of ly. The fraction TRh was chromatographied on a silica gel col- B. subtilis transforming ginsenoside Rh and Re, compounds umn (12 g) eluting with chloroform and methanol (6:1, v/v) 2 and 3 were isolated as transformational products respec- and further subjected on Sephadex LH-20 eluting with tively. methanol to obtain compound 2 (5 mg). The fraction TRe was According to spectra data compound 1 was determined isolated by silica gel column (12 g) and eluted with chloro- to be ginsenoside Rh (Fig. 1) and, based on the reference form, methanol and water (65:35:10, v/v/v), then further of Baek et al. (1996), it had cytotoxinic effect to some types purified on Sephadex LH-20 eluting with methanol to obtain of tumor cell. There was no report before on the isolation of compound 3 (1 mg). ginsenoside Rh from Sanchi that, in our experiment, was not detected by TLC in the extract of unfermented Sanchi, Structure of the compounds. Optical rotations were meas- so it must be produced during the fermentation process of ured on a JASCO DIP-370 Digital Polarimeter. The IR spec- Sanchi by B. subtilis. tra were measured on a Perkin-Elmer-577 spectropho- Compound 2 was determined to be 3-O-β-D-glucopyra- tometer. The NMR spectra were recorded on Bruker AM-400 nosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol (Fig. and Brucker DRX-500 spectrometers. MS were performed on 2), which was identified as a new compound (Li et al., 2005) a VG AutoSpec-3000 spectrometer. and had additional β-glucopyranosyl linked with C-3 com- Compound 1: white powder, negative FAB-MS m/z: 619 pared with the substrate ginsenoside Rh (Fig. 2). Com- - - 1 ([M – H] , 100), 600 ([M – H – 18] , 20); H-NMR (C D N, pound 3 gave a quasi-molecular ion peek at m/z 1108 ([M 5 5 400 MHz): 0.83 (3H, s, H-19), 1.02 (3H, s, H-18), 1.22 (3H, – H] in the negative FAB-MS, which had 162 atomic mass s, H-28), 1.57 (6H, s, H-26, H-27), 1.62 (3H, s, H-21), 2.03 units (amu) greater than its substrate ginsenoside Re, and (3H, s, H-29), 2.77 (3H, m, H-23), 3.92 (1H, dd, J =J = 8 indicated that glycosylation replacement reaction had also 1 2 Hz, H-2), 4.23 (1H, m, H-6), 4.97 (1H, d, J = 7.7 Hz, H-6- taken place. Compounds 2 and 3 had higher polarity than glc-1’), 4.99 (1H, d, J = 7.4 Hz, H-24), 5.47 (1H, d, J = 7.4 the substrates and were not detected by TLC analysis either Hz, H-22); C-NMR (C D N, 100 MHz): 79.7 (C-3), 78.2 (C- in other controls or in the methanol extracts of bacterial cell. 5 5 6), 30.5 (C-13,), 140.2 (C-20), 123.6 (C-22), 124.0 (C-24), Biotransformation was an efficient way to produce new 106.1 (C-1’), 63.1 (C-6’). structure products, e.g. the metabolism of ginsenoside Rb 22,D Compound 2: white power, [α] 180 (c, 0.1, CH OH), by bacteria gave a few novel compounds, but most results negative FAB-MS m/z: 799 ([M – H] , 100), 637 ([M – H – showed that some substituents were cleaved from sub- - - 162] , 6), 475([M – H – 2×162] 14); IR (KBr) ν 3414, 2958, strates in the transformational process (Chen et al., 1999; –1 1 1633, 1455, 1146, 1077, 1031 cm ; H-NMR (C D N, 400 Bae et al., 2000). Although glycosylation reaction was gen- 5 5 MHz): 1.05 (3H, s, H-19), 1.16 (3H, s, H-18), 4.23 (1H, dd, eral in organism, it was very important in drug research OH OH OH OH Oglc OH OH OH Oglc Oglc Oglc-rha Gingsenoside Rh Gingsenoside Rh Gingsenoside Re FIG. 1 – The hypothetic pathways of producing ginsenoside Rh . 4 Ann. Microbiol., 56 (2), 151-153 (2006) 153 OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH Gingsenoside Rh Compound 2 FIG. 2 – The structures of ginsenoside Rh and its transformed product compound 2. because glycosylation could change polarity of substrate grateful to Mr. Y.N. He and Ms. H.L. Liang in the Kunming and modulate the metabolism and distribution of active com- Institute of Botany, Chinese Academy of Sciences, for meas- pounds by enhancing the hepatocyte uptake. uring NMR and MS data, respectively. Sanchi (Panax notoginseng), a famous traditional herb in Southeast Asia, has been widely used, but the Sanchi saponins are not reported on possessing obvious antitumor REFERENCES activity. Based on our study, an antitumor compound gin- Bae E.A., Park S.Y., Kim D.H. (2000). Constitutive β-glucosidases senoside Rh was produced in the fermentation process of hydrolyzing ginsenoside Rb and Rb from human intestinal 1 2 Sanchi by B. subtilis, which proved that microbe could change bacterial. Biol. Pharm. Bull., 23: 1481-1485. the components of TCM’s substances and resulted in the Baek N.I., Kim D.S., Lee Y.H., Park J.D., Lee C.B., Kim S.I. (1996). change of drug effect. In order to investigate the origin of Ginsenoside Rh , a genuine dammarane glycoside from Kore- ginsenoside Rh in fermented Sanchi, ginsenosides Rh and an red gingseng. Planta Med., 62: 86-87. 4 1 Re that had the similar structure with ginsenoside Rh were 4 Chen X., Zhou Q.L., Wang B.X. (1999). The metabolism of gin- selected as the objects to study the biotransformation of senoside Rb by intestinal bacterial. Acta Pharmacol. Sin., 49: 442-446. saponins by B. subtilis. The pathways of producing gin- senoside Rh were assumed to be (i) losing hydroxy from gin- Dong A.L., Ye M., Guo H.Z., Zheng J.H., Guo D.A. (2003). Micro- bial transformation of ginsenoside Rb by Rhizopus stolonifer senoside Rh or (ii) losing sugar moiety from ginsenoside Re and Curvularia lunata. Biotechnol. Lett., 25: 339-344. in the transformation process (Fig. 1). But our experiment Jiang K.Y., Qian Z.N. (1995). Effects of Panax notoginseng saponins results showed that glycosylation reaction occurred in the cul- on posthypoxic cell damage of neurons in vitro. Acta Phar- ture of B. subtilis after feeding singular compound ginseno- macol. Sin., 16: 399-402. side Rh and Re respectively which was contrary to the Li S.H., Chu Y. (1999). Anti-inflammatory effects of total saponins hypothesis, so we could not logically explain the origin of gin- of Panax notoginseng. Acta Pharmacol. Sin., 20: 551-554. senoside Rh in fermented Sanchi. Li G.H., Shen Y.M., Zhang K.Q. (2005). A new saponin trans- These results indicate that the fermentation process of formed from ginsenoside Rh by Bacillus subtilis. Chinese TCM’s substances by microbes is so complex; however, it Chem. Lett., 16 (3): 359-361. might provide diversiform fermented products for screening Lin M.C., Wang K.C., Lee S.S. (2001). Transformation of ginseno- active compounds. From our experiments it is obvious that sides Rg and Rb , and crude Sanchi saponins by human 1 1 the components and drug effect of TCM’s substances may be intestinal microflora. J. Chin. Chem. Soc., 48: 113-120. changed after fermentation by microbe and it is feasible to Wu B.X., Niu J.J., Sun X.L., Dong L.S. (2001). Technology of tra- ditional Chinese medicine’s fermentation pharmacy. Shan- utilize the microbial fermentation of TCM’s substances in dong Journal of Traditional Chinese Medicine, 20 (3): 179-180. the discovery of leads and improvement the drug action of Zhao R., Xing Z.T., Li X.G., Cheng K.D., Yang X.W. (1998). Chem- TCM. ical modification of natural rug by intestine bacteria. Journal of Jilin Agricultural University, 20 (2): 103-110. Acknowledgments Zhou J., Wu M.Z., Taniyasu S. (1981). Dammarane-Saponins of This work was supported by the National Natural Science sanqi-ginseng, roots of Panax notoginseng: structures of new Foundation (20362010) and Department of Science and saponins, notoginsenosides–R1 and –R2, and identification of Technology of Yunnan Province (2001ZCBFB01C), and we are ginsenoside-Rg2 and Rh1. Chem. Pharm. Bull., 29: 2844-2850. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Production of saponin in fermentation process of Sanchi (Panax notoginseng) and biotransformation of saponin byBacillus subtilis

Li, Guo; Shen, Yue; Liu, Yajun; Zhang, Ke
Annals of Microbiology , Volume 56 (2) – Nov 20, 2009
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Springer Journals
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Copyright © 2006 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
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10.1007/BF03174997
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Abstract

Annals of Microbiology, 56 (2) 151-153 (2006) Production of saponin in fermentation process of Sanchi (Panax notoginseng) and biotransformation of saponin by Bacillus subtilis 1 2 1 1 Guo Hong LI , Yue Mao SHEN , Yajun LIU , Ke Qin ZHANG * 1 2 Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091; The State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China Received 6 September 2005 / Accepted 27 January 2006 Abstract - An antitumor compound ginsenoside Rh was produced during the fermentation process of Sanchi (Panax notoginseng) by Bacillus subtilis. Saponins ginsenosides Rh and Re were transformed by B. subtilis and were produced two main transformed products. The transformed product of ginsenoside Rh was determined to be 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-pro- topanaxatriol, and the transformed product of ginsenoside Re had 162 atomic mass units (amu) greater than the substrate. Compared with the substrates, the transformed products had one more glucosyl moiety linked respectively, which indicated that glycosylation reac- tion occurred in the transformation process. Key words: fermentation, Sanchi, biotransformation, saponin, Bacillus subtilis, glycosylation. INTRODUCTION two key metabolites, one of which is a new compound. Up to now, there is no report about the microbial fermentation of Traditional Chinese Medicine (TCM) as an integral part of Chi- Sanchi and the glycosylation reaction in the transformation nese culture has been studied for the effective utilization all process of saponins. The difference of saponins between orig- along. The fermentation research of TCM started since 80’s inal and fermented Sanchi as well as the glycosylation reac- last century, but only aimed at fermentation of officinal fungi tion in the transformation process of saponin by Bacillus sub- (Wu et al., 2001). Microorganisms have strong ability of tilis will be discussed in this paper, by which we hope to provide transforming substances and the physiological activities of a new approach to exploit TCM effectively. some TCM’s substances have been changed by the trans- formation of intestinal microbes (Zhao et al., 1998), so the microbial fermentation of TCM’s substances may produce new MATERIALS AND METHODS compounds or change the content of active components. There is no report on the componential change between Microbial strain and Sanchi. The strain of Bacillus subtilis TCM’s substances and their fermented products. was purchased from China General Microbiological Culture Sanchi (Panax notoginseng) is a famous traditional herb, Collection Center (CGMCC No. 1318) and was conserved in which has been clinically tested to have the abilities of pro- nutrient agar medium. moting blood circulation, preventing the formation of blood Sanchi was purchased from medicinal materials market clots (anti-thrombosis property), dissolving blood clots, of Yunnan Province, People’s Republic of China. Ginsenosides enhancing the removal of cellular breakdown products and Rh and Re were prepared from Sanchi with method other debris from the blood circulation. Saponins as the major described by Zhou et al. (1981). components in Sanchi have been proved to have diversiform biological activities (Jiang and Qian, 1995; Li and Chu, 1999). Fermentation of Sanchi. Sanchi was fermented by B. sub- Some work related with the transformation and metabolism tilis in solid form. The fibres of Sanchi were ground into pow- of Sanchi saponins (Chen et al., 1999; Bae et al., 2000; Lin et der and mixed with water; the mixture (400 g) was put into al., 2001; Dong et al., 2003) has been done to elucidate that flask (1 litre) and sterilized at 121 °C for 40 min. Bacillus sub- the metabolism of ginsenoside Rb begins with cleavage of the tilis (40 ml), cultured in Wort medium, pH 7.0, at 30 °C for -1 terminal sugar moiety, then gradually the other sugars. The 72 h on an orbital shaker at 120 rev min , was inoculated transformation of ginsenoside Rb by Curvularia lunata gives into each flask. After culturing at 30 °C for 10 days, the fer- mented products of Sanchi were dried in oven at 60 °C. Biotransformation procedures. Bacillus subtilis was inoc- * Corresponding author. Phone: + 86 871 5034878; ulated in 250 ml flask containing 100 ml Wort medium at pH Fax: +86 871 5034838; E-mail: Kqzhang111@yahoo.com.cn 7.0. Ginsenoside Rh and ginsenoside Re, dissolved in 1 152 G.H. Li et al. ethanol, were each added into three flasks of Wort medium 9.0, 15.2, H-3), 4.36 (1H, m, H-6), 5.32 (1H, m, H-24), 4.94 inoculated with B. subtilis to make the final concentration of (1H, d, 7.7, H-6-glc-1’), 5.91 (1H, d, 8.0, H-6-glc-1’’); ginsenosides Rh 0.4 mg/ml and ginsenoside Re 0.2 mg/ml. C-NMR (C D N, 100 MHz): 39.4 (C-1), 27.9 (C-2), 89.2 1 5 5 Two controls of equal volume of ethanol added into the (C-3), 40.3 (C-4), 61.5 (C-5), 80.3 (C-6), 45.3 (C-7), 41.2 flasks of Wort medium with and without B. subtilis were pre- (C-8), 50.2 (C-9), 39.7 (C-10), 32.1 (C-11), 71.1 (C-12), pared. All the flasks were cultured at 30 °C for 96 h at 120 48.3 (C-13), 51.7 (C-14), 31.3 (C-15), 26.9 (C-16), 54.8 (C- -1 rev min . 17), 17.4 (C-18), 17.7 (C-19), 73.0 (C-20), 27.1 (C-21), 35.9 (C-22), 23.0 (C-23), 126.4 (C-24), 130.8 (C-25), 25.8 Isolation and identification compounds. The fermenta- (C-26), 17.6 (C-27), 31.7 (C-28), 16.3 (C-29), 16.8 (C-30), tion mixture (2 kg) was extracted three times with 80% 105.8 (C-1’), 75.6 (C-2’), 80.3 (C-3’), 72.0 (C-4’), 78.6 ethanol exhaustively and dissolved in water; then was (C-5’), 62.5 (C-6’), 103.1 (C-1’’), 74.8 (C-2’’), 77.6 (C-3’’), extracted five times with n-butanol to give 61 g of residue. 71.0 (C-4’’), 77.6 (C-5’’), 62.6 (C-6’’). The residue was subjected over silica gel column (chloro- Compound 3: white power, negative FAB-MS m/z: 1108 - - form:methanol:water, 65:35:10, v/v/v), reversed-phase C ([M – H] , 100), 962 ([M – H – 146] , 6), 946 ([M – H – - - (RP-18) column (methanol:water, 65:35, v/v) and Sephadex 162] , 6), 476 ([M – 146 – 3×162] , 11). LH-20 column (methanol) to give compound 1 (16 mg). The three flasks (300 ml) of B. subtilis culture, trans- forming ginsenoside Rh and Re respectively, were filtered. RESULTS AND DISCUSSION The filtrates were extracted four times with n-butanol, 4.5 g residue containing transformational product of ginsenoside According to TLC result, the spot appeared in fermented Rh (TRh) and 1.5 g residue containing transformational Sanchi but not in original Sanchi was selected as isolation product of ginsenoside Re (TRe) were obtained, respective- object, and compound 1 was obtained. From the broths of ly. The fraction TRh was chromatographied on a silica gel col- B. subtilis transforming ginsenoside Rh and Re, compounds umn (12 g) eluting with chloroform and methanol (6:1, v/v) 2 and 3 were isolated as transformational products respec- and further subjected on Sephadex LH-20 eluting with tively. methanol to obtain compound 2 (5 mg). The fraction TRe was According to spectra data compound 1 was determined isolated by silica gel column (12 g) and eluted with chloro- to be ginsenoside Rh (Fig. 1) and, based on the reference form, methanol and water (65:35:10, v/v/v), then further of Baek et al. (1996), it had cytotoxinic effect to some types purified on Sephadex LH-20 eluting with methanol to obtain of tumor cell. There was no report before on the isolation of compound 3 (1 mg). ginsenoside Rh from Sanchi that, in our experiment, was not detected by TLC in the extract of unfermented Sanchi, Structure of the compounds. Optical rotations were meas- so it must be produced during the fermentation process of ured on a JASCO DIP-370 Digital Polarimeter. The IR spec- Sanchi by B. subtilis. tra were measured on a Perkin-Elmer-577 spectropho- Compound 2 was determined to be 3-O-β-D-glucopyra- tometer. The NMR spectra were recorded on Bruker AM-400 nosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol (Fig. and Brucker DRX-500 spectrometers. MS were performed on 2), which was identified as a new compound (Li et al., 2005) a VG AutoSpec-3000 spectrometer. and had additional β-glucopyranosyl linked with C-3 com- Compound 1: white powder, negative FAB-MS m/z: 619 pared with the substrate ginsenoside Rh (Fig. 2). Com- - - 1 ([M – H] , 100), 600 ([M – H – 18] , 20); H-NMR (C D N, pound 3 gave a quasi-molecular ion peek at m/z 1108 ([M 5 5 400 MHz): 0.83 (3H, s, H-19), 1.02 (3H, s, H-18), 1.22 (3H, – H] in the negative FAB-MS, which had 162 atomic mass s, H-28), 1.57 (6H, s, H-26, H-27), 1.62 (3H, s, H-21), 2.03 units (amu) greater than its substrate ginsenoside Re, and (3H, s, H-29), 2.77 (3H, m, H-23), 3.92 (1H, dd, J =J = 8 indicated that glycosylation replacement reaction had also 1 2 Hz, H-2), 4.23 (1H, m, H-6), 4.97 (1H, d, J = 7.7 Hz, H-6- taken place. Compounds 2 and 3 had higher polarity than glc-1’), 4.99 (1H, d, J = 7.4 Hz, H-24), 5.47 (1H, d, J = 7.4 the substrates and were not detected by TLC analysis either Hz, H-22); C-NMR (C D N, 100 MHz): 79.7 (C-3), 78.2 (C- in other controls or in the methanol extracts of bacterial cell. 5 5 6), 30.5 (C-13,), 140.2 (C-20), 123.6 (C-22), 124.0 (C-24), Biotransformation was an efficient way to produce new 106.1 (C-1’), 63.1 (C-6’). structure products, e.g. the metabolism of ginsenoside Rb 22,D Compound 2: white power, [α] 180 (c, 0.1, CH OH), by bacteria gave a few novel compounds, but most results negative FAB-MS m/z: 799 ([M – H] , 100), 637 ([M – H – showed that some substituents were cleaved from sub- - - 162] , 6), 475([M – H – 2×162] 14); IR (KBr) ν 3414, 2958, strates in the transformational process (Chen et al., 1999; –1 1 1633, 1455, 1146, 1077, 1031 cm ; H-NMR (C D N, 400 Bae et al., 2000). Although glycosylation reaction was gen- 5 5 MHz): 1.05 (3H, s, H-19), 1.16 (3H, s, H-18), 4.23 (1H, dd, eral in organism, it was very important in drug research OH OH OH OH Oglc OH OH OH Oglc Oglc Oglc-rha Gingsenoside Rh Gingsenoside Rh Gingsenoside Re FIG. 1 – The hypothetic pathways of producing ginsenoside Rh . 4 Ann. Microbiol., 56 (2), 151-153 (2006) 153 OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH Gingsenoside Rh Compound 2 FIG. 2 – The structures of ginsenoside Rh and its transformed product compound 2. because glycosylation could change polarity of substrate grateful to Mr. Y.N. He and Ms. H.L. Liang in the Kunming and modulate the metabolism and distribution of active com- Institute of Botany, Chinese Academy of Sciences, for meas- pounds by enhancing the hepatocyte uptake. uring NMR and MS data, respectively. Sanchi (Panax notoginseng), a famous traditional herb in Southeast Asia, has been widely used, but the Sanchi saponins are not reported on possessing obvious antitumor REFERENCES activity. Based on our study, an antitumor compound gin- Bae E.A., Park S.Y., Kim D.H. (2000). Constitutive β-glucosidases senoside Rh was produced in the fermentation process of hydrolyzing ginsenoside Rb and Rb from human intestinal 1 2 Sanchi by B. subtilis, which proved that microbe could change bacterial. Biol. Pharm. Bull., 23: 1481-1485. the components of TCM’s substances and resulted in the Baek N.I., Kim D.S., Lee Y.H., Park J.D., Lee C.B., Kim S.I. (1996). change of drug effect. In order to investigate the origin of Ginsenoside Rh , a genuine dammarane glycoside from Kore- ginsenoside Rh in fermented Sanchi, ginsenosides Rh and an red gingseng. Planta Med., 62: 86-87. 4 1 Re that had the similar structure with ginsenoside Rh were 4 Chen X., Zhou Q.L., Wang B.X. (1999). The metabolism of gin- selected as the objects to study the biotransformation of senoside Rb by intestinal bacterial. Acta Pharmacol. Sin., 49: 442-446. saponins by B. subtilis. The pathways of producing gin- senoside Rh were assumed to be (i) losing hydroxy from gin- Dong A.L., Ye M., Guo H.Z., Zheng J.H., Guo D.A. (2003). Micro- bial transformation of ginsenoside Rb by Rhizopus stolonifer senoside Rh or (ii) losing sugar moiety from ginsenoside Re and Curvularia lunata. Biotechnol. Lett., 25: 339-344. in the transformation process (Fig. 1). But our experiment Jiang K.Y., Qian Z.N. (1995). Effects of Panax notoginseng saponins results showed that glycosylation reaction occurred in the cul- on posthypoxic cell damage of neurons in vitro. Acta Phar- ture of B. subtilis after feeding singular compound ginseno- macol. Sin., 16: 399-402. side Rh and Re respectively which was contrary to the Li S.H., Chu Y. (1999). 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Shan- utilize the microbial fermentation of TCM’s substances in dong Journal of Traditional Chinese Medicine, 20 (3): 179-180. the discovery of leads and improvement the drug action of Zhao R., Xing Z.T., Li X.G., Cheng K.D., Yang X.W. (1998). Chem- TCM. ical modification of natural rug by intestine bacteria. Journal of Jilin Agricultural University, 20 (2): 103-110. Acknowledgments Zhou J., Wu M.Z., Taniyasu S. (1981). Dammarane-Saponins of This work was supported by the National Natural Science sanqi-ginseng, roots of Panax notoginseng: structures of new Foundation (20362010) and Department of Science and saponins, notoginsenosides–R1 and –R2, and identification of Technology of Yunnan Province (2001ZCBFB01C), and we are ginsenoside-Rg2 and Rh1. Chem. Pharm. Bull., 29: 2844-2850.

Journal

Annals of Microbiology – Springer Journals

Published: Nov 20, 2009

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