人参内生菌Burkholderia sp. GE 17-7制备人参皂苷Rg3的研究
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摘要
许多研究表明人参皂苷是人参中起主要药效活性的物质,尤其是人参稀有皂苷比人参主皂苷在抗癌、抗心脑血管病、抗糖尿病等方面具有更好的疗效。利用从17年生野山参中分离、筛选获得一株人参内生真菌Burkholderia sp. GE 17-7生物转化人参主皂苷。通过薄层色谱法、高效液相色谱法等方法对人参主皂苷(Rb1、Rb2、Rc、Rd、Re和Rg1)的转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定。同时,对转化路径进行了分析。结果表明人参内生真菌Burkholderia sp. GE 17-7能够特异性水解原人参二醇型皂苷C-20位糖基,对其内部糖基不具有水解特性。经鉴定其转化产物为人参稀有皂苷Rg3。转化路径为人参皂苷Rb1→人参皂苷Rd→人参稀有皂苷Rg3。人参内生真菌Burkholderia sp. GE 17-7能够特异性制备人参稀有皂苷Rg3,为工业制备人参稀有皂苷提供了新的微生物资源。
Many investigational studies have shown that ginsenosides are the principal active constituents of ginseng in antioxidant, antineoplastic, anti-inflammatory and biomodulatory processes. In particular, minor ginsenoside monomers, such as F2, Rh2, Rg3 and compound K(CK), have greater antitumor activity than the major ginsenoside monomers Rb1, Rb2, Rc, Rd, Re and Rg1. An endophytic bacterium GE 17-7 strain isolated from ginseng roots for 17 years, belonging to the genus Burkholderia, had been used in converting minor ginsenosides. The major ginsenoside monomers Rb1, Rb2, Rc, Rd, Re and Rg1 were used by strain GE 17-7. Biotransformation products were separated and purified through thin-layer chromatography(TLC) and high performance liquid chromatography(HPLC) analysis and identified by spectral analysis and physical constants. The results indicated that Burkholderia sp. GE 17-7 had specific hydrolytic activity for the outer glucose at the C-20 position in protopanaxadiol-type ginsenosides without hydrolysis of the inner glucose. The final metabolite was ginsenoside Rg3 by nuclear magnetic resonance(NMR) analysis. This suggests that the hydrolytic pathway of ginsenoside Rb1 by strain GE 17-7 was ginsenoside Rb1→ginsenoside Rd→ginsenoside Rg3. We have successfully found a specificity ginsenoside Rg3-producing endophytic bacterium GE 17-7 from ginseng.
Many investigational studies have shown that ginsenosides are the principal active constituents of ginseng in antioxidant, antineoplastic, anti-inflammatory and biomodulatory processes. In particular, minor ginsenoside monomers, such as F2, Rh2, Rg3 and compound K(CK), have greater antitumor activity than the major ginsenoside monomers Rb1, Rb2, Rc, Rd, Re and Rg1. An endophytic bacterium GE 17-7 strain isolated from ginseng roots for 17 years, belonging to the genus Burkholderia, had been used in converting minor ginsenosides. The major ginsenoside monomers Rb1, Rb2, Rc, Rd, Re and Rg1 were used by strain GE 17-7. Biotransformation products were separated and purified through thin-layer chromatography(TLC) and high performance liquid chromatography(HPLC) analysis and identified by spectral analysis and physical constants. The results indicated that Burkholderia sp. GE 17-7 had specific hydrolytic activity for the outer glucose at the C-20 position in protopanaxadiol-type ginsenosides without hydrolysis of the inner glucose. The final metabolite was ginsenoside Rg3 by nuclear magnetic resonance(NMR) analysis. This suggests that the hydrolytic pathway of ginsenoside Rb1 by strain GE 17-7 was ginsenoside Rb1→ginsenoside Rd→ginsenoside Rg3. We have successfully found a specificity ginsenoside Rg3-producing endophytic bacterium GE 17-7 from ginseng.
引文
[1]LI Q, LI B, DONG C Y, et al. 20(S)-Ginsenoside Rh2 suppresses proliferation and migration of hepatocellular carcinoma cells by targeting EZH2 to regulate CDKN2A-2B gene cluster transcription [J]. European Journal of Pharmacology, 2017, 815(1): 173-180.
[2]LI P, ZHOU X Y, QU D, et al. Preliminary study on fabrication, characterization and synergistic anti-lung cancer effects of self-assembled micelles of covalently conjugated celastrol-polyethylene glycol-ginsenoside Rh2 [J]. Drug Delivery, 2017, 24(1): 834-845.
[3]KIM W Y, KIM J M, HAN S B, et al. Steaming of ginseng at high temperature enhances biological activity [J]. Journal of Natural Products, 2000, 63(12): 1702-1704.
[4]CHO S H, CHUNG K S, CHOI J H, et al. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells [J]. BMC Cancer, 2009, 9(1): 449.
[5]TONG-LIN WU T, TONG Y C, CHEN I H, et al. Induction of apoptosis in prostate cancer by ginsenoside Rh2 [J]. Oncotarget, 2018, 9(13): 11109-11118.
[6]SHI X, YANG J, WEI G. Ginsenoside 20(S)-Rh2 exerts anti-cancer activity through the Akt/GSK3β signaling pathway in human cervical cancer cells [J]. Molecular Medicine Reports, 2018, 17(3): 4811-4816.
[7]YANG L, ZHANG Z, HOU J, et al. Targeted delivery of ginsenoside compound K using TPGS/PEG-PCL mixed micelles for effective treatment of lung cancer [J]. International Journal of Nanomedicine, 2017, 12:7653-7667.
[8]尹天翔, 王燕燕. 人参皂苷Rg3对人肝癌细胞增殖、迁移、黏附和凋亡的影响及其作用机制[J]. 基础医学与临床, 2015, 35(3): 303-307.
[9]张味娜, 徐逸, 俞敏. 人参皂苷Rg3温敏纳米粒制备及对肝癌细胞抑制作用研究[J]. 实用药物与临床, 2017, 20(11): 1231-1235.
[10]YUAN Z G, JIANG H, ZHU X H, et al. Ginsenoside Rg3 promotes cytotoxicity of Paclitaxel through inhibiting NF-κB signaling and regulating Bax/Bcl-2 expression on triple-negative breast cancer[J]. Biomedicine & Pharmacotherapy, 2017, 89(1): 227-232.
[11]ZHOU Y, ZHENG X, LU J, et al. Ginsenoside 20(S)-Rg3 inhibits the warburg effect via modulating DNMT3A/MiR-532-3p/HK2 pathway in ovarian cancer cells [J]. Celluar Physiolgy and Biochemistry, 2018, 45(6): 2548-2559.
[12]TANG M, WANG W, CHENG L, et al. The inhibitory effects of 20(R)-ginsenoside Rg3 on the proliferation, angiogenesis, and collagen synthesis of hypertrophic scar derived fibroblasts in vitro[J]. Iranian Journal of Basic Medical Sciences, 2018, 21(3): 309-317.
[13]LIU C, GONG Q, CHEN T, et al. Treatment with 20(S)-ginsenoside Rg3 reverses multidrug resistance in A549/DDP xenograft tumors [J]. Oncology Letters, 2018, 15(4): 4376-4382.
[14]STROBELL G, DAISY B. Bioprospecting for microbial endophytes and their natural products [J]. Microbiology and Molecular Biology Reviews, 2003, 67(4): 491-502.
[15]边军昌, 冯永辉, 杨静, 等. 中药内生菌的研究进展[J]. 光明中医, 2010, 25(1): 164-165.
[16]HALLMANN J, HALLMANN Q A, MAHAFFEE W F, et al. Bacterial endophytes in agricultural crops [J]. Canadian Journal of Microbiology, 1997, 43(10): 895-914.
[17]KIM S S, SEONG S, KIM S Y. Synergistic effect of ginsenoside Rg3 with verapamil on the modulation of multidrug resistance in human acute myeloid leukemia cells [J]. Oncology Letters, 2014, 7(4): 1265-1269.
[18]CUI L, WU S Q, ZHAO C A, et al. Microbial conversion of major ginsenosides in ginseng total saponins by Platycodon grandiflorm endophytes [J]. Journal of Ginseng Research, 2015, 40(4): 366-374.
[19]PARK Y H, LEE S G, AHN D J, et al. Diversity of fungal endophytes in various tissues of Panax ginseng Meyer cultivated in Korea [J]. Journal of Ginseng Research, 2012, 36(2): 211-217.
[20]PARK Y H, KIM Y C, PARK S U, et al. Age-dependent distribution of fungal endophytes in Panax ginseng roots cultivated in Korea [J]. Journal of Ginseng Research, 2012, 36(3): 327-333.
[21]WU H, YANG H Y, YOU X L, et al. Isolation and characterization of saponin-producing fungal endophytes from Aralia elata in Northeast China [J]. International Journal of Molecular Sciences, 2012, 13(12): 16255-16266.
[22]WU H, YANG H Y, YOU X L, et al. Diversity of endophytic fungi from roots of Panax ginseng and their saponin yield capacities[J]. Springer Plus, 2013, 2(1): 107.
[23]CHO K M, HONG S Y, LEE S M, et al. Endophytic bacterial communities in ginseng and their antifungal activity against pathogens [J]. Microbial Ecology, 2007, 54(2): 343-351.
[24]姜云, 尹望, 陈长卿, 等. 人参内生菌的分离及拮抗菌株的筛选[J]. 吉林农业大学学报, 2012, 34(5): 517-521.
[25]曲红光, 靳慧娟, 马丽伟, 等. 人参内生细菌的分离及其体外抗肿瘤活性分析[J]. 中国妇幼保健, 2014, 29(12): 1926-1928.
[26]林星辰, 王泽, 牛林飞, 等. 人参内生菌B69菌株抑菌活性物质产生条件的优化[J]. 人参研究, 2018, 1(1): 6-11.
[27]崔磊, 金英今, 尹成日. 党参内生菌转化人参根总皂苷为稀有人参皂苷F2和C-K的研究[J]. 延边大学学报, 2015, 41(1):79-84.
[28]郭从亮, 杨晓艳, 陈子明, 等. 一株植物内生菌Coniochaeta sp.对三七总皂苷中人参皂苷Rb1的特异性转化含量测定[J]. 中药材, 2016, 5(39): 1075-1078.
[29]FU Y, YIN Z H, YIN C R. Biotransformation of ginsenoside Rb1 to ginsenoside Rg3 by endophytic bacterium Burkholderia sp. GE 17-7 isolated from Panax ginseng [J]. Journal of Applied Microbiology, 2017, 122(6): 1579-1585.
[30]LI P Y, LIU J P, LU D. Standard NMR spectrum of ginsenosides [M]. Beijing: Chemical Industry Press, 2012.
[31]王毅, 蒋艳, 王本祥, 等. 人参皂苷Rg3及其肠内菌代谢产物Rh1对小鼠免疫细胞功能的影响[J]. 药学学报, 2002, 37(12): 927-929.
[32]ZENG X S, ZHOU X S, LUO F C, et al. Comparative analysis of the neuroprotective effects of ginsenosides Rg1 and Rb1 extracted from Panax notoginseng against cerebral ischemia [J]. Canadian Journal of Physiology and Pharmacology, 2014, 92(2): 102-108.
[33]CHRISTENSEN L P. Ginsenosides chemistry, biosynthesis, analysis, and potential health effects [J]. Advances in Food and Nutrition Research, 2009, 55(1): 1-99.
[34]张虹, 付雯雯, 徐华丽, 等. 20(S)-原人参二醇、人参皂苷Rh2及人参皂苷Rg3抗肿瘤作用的对比[J]. 中国老年学杂志, 2014, 34(17): 4911-4913.
[35]朱晓丽. 人参皂苷药理作用研究进展[J]. 中国药物经济学, 2017, 12(12): 152-154.
[2]LI P, ZHOU X Y, QU D, et al. Preliminary study on fabrication, characterization and synergistic anti-lung cancer effects of self-assembled micelles of covalently conjugated celastrol-polyethylene glycol-ginsenoside Rh2 [J]. Drug Delivery, 2017, 24(1): 834-845.
[3]KIM W Y, KIM J M, HAN S B, et al. Steaming of ginseng at high temperature enhances biological activity [J]. Journal of Natural Products, 2000, 63(12): 1702-1704.
[4]CHO S H, CHUNG K S, CHOI J H, et al. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells [J]. BMC Cancer, 2009, 9(1): 449.
[5]TONG-LIN WU T, TONG Y C, CHEN I H, et al. Induction of apoptosis in prostate cancer by ginsenoside Rh2 [J]. Oncotarget, 2018, 9(13): 11109-11118.
[6]SHI X, YANG J, WEI G. Ginsenoside 20(S)-Rh2 exerts anti-cancer activity through the Akt/GSK3β signaling pathway in human cervical cancer cells [J]. Molecular Medicine Reports, 2018, 17(3): 4811-4816.
[7]YANG L, ZHANG Z, HOU J, et al. Targeted delivery of ginsenoside compound K using TPGS/PEG-PCL mixed micelles for effective treatment of lung cancer [J]. International Journal of Nanomedicine, 2017, 12:7653-7667.
[8]尹天翔, 王燕燕. 人参皂苷Rg3对人肝癌细胞增殖、迁移、黏附和凋亡的影响及其作用机制[J]. 基础医学与临床, 2015, 35(3): 303-307.
[9]张味娜, 徐逸, 俞敏. 人参皂苷Rg3温敏纳米粒制备及对肝癌细胞抑制作用研究[J]. 实用药物与临床, 2017, 20(11): 1231-1235.
[10]YUAN Z G, JIANG H, ZHU X H, et al. Ginsenoside Rg3 promotes cytotoxicity of Paclitaxel through inhibiting NF-κB signaling and regulating Bax/Bcl-2 expression on triple-negative breast cancer[J]. Biomedicine & Pharmacotherapy, 2017, 89(1): 227-232.
[11]ZHOU Y, ZHENG X, LU J, et al. Ginsenoside 20(S)-Rg3 inhibits the warburg effect via modulating DNMT3A/MiR-532-3p/HK2 pathway in ovarian cancer cells [J]. Celluar Physiolgy and Biochemistry, 2018, 45(6): 2548-2559.
[12]TANG M, WANG W, CHENG L, et al. The inhibitory effects of 20(R)-ginsenoside Rg3 on the proliferation, angiogenesis, and collagen synthesis of hypertrophic scar derived fibroblasts in vitro[J]. Iranian Journal of Basic Medical Sciences, 2018, 21(3): 309-317.
[13]LIU C, GONG Q, CHEN T, et al. Treatment with 20(S)-ginsenoside Rg3 reverses multidrug resistance in A549/DDP xenograft tumors [J]. Oncology Letters, 2018, 15(4): 4376-4382.
[14]STROBELL G, DAISY B. Bioprospecting for microbial endophytes and their natural products [J]. Microbiology and Molecular Biology Reviews, 2003, 67(4): 491-502.
[15]边军昌, 冯永辉, 杨静, 等. 中药内生菌的研究进展[J]. 光明中医, 2010, 25(1): 164-165.
[16]HALLMANN J, HALLMANN Q A, MAHAFFEE W F, et al. Bacterial endophytes in agricultural crops [J]. Canadian Journal of Microbiology, 1997, 43(10): 895-914.
[17]KIM S S, SEONG S, KIM S Y. Synergistic effect of ginsenoside Rg3 with verapamil on the modulation of multidrug resistance in human acute myeloid leukemia cells [J]. Oncology Letters, 2014, 7(4): 1265-1269.
[18]CUI L, WU S Q, ZHAO C A, et al. Microbial conversion of major ginsenosides in ginseng total saponins by Platycodon grandiflorm endophytes [J]. Journal of Ginseng Research, 2015, 40(4): 366-374.
[19]PARK Y H, LEE S G, AHN D J, et al. Diversity of fungal endophytes in various tissues of Panax ginseng Meyer cultivated in Korea [J]. Journal of Ginseng Research, 2012, 36(2): 211-217.
[20]PARK Y H, KIM Y C, PARK S U, et al. Age-dependent distribution of fungal endophytes in Panax ginseng roots cultivated in Korea [J]. Journal of Ginseng Research, 2012, 36(3): 327-333.
[21]WU H, YANG H Y, YOU X L, et al. Isolation and characterization of saponin-producing fungal endophytes from Aralia elata in Northeast China [J]. International Journal of Molecular Sciences, 2012, 13(12): 16255-16266.
[22]WU H, YANG H Y, YOU X L, et al. Diversity of endophytic fungi from roots of Panax ginseng and their saponin yield capacities[J]. Springer Plus, 2013, 2(1): 107.
[23]CHO K M, HONG S Y, LEE S M, et al. Endophytic bacterial communities in ginseng and their antifungal activity against pathogens [J]. Microbial Ecology, 2007, 54(2): 343-351.
[24]姜云, 尹望, 陈长卿, 等. 人参内生菌的分离及拮抗菌株的筛选[J]. 吉林农业大学学报, 2012, 34(5): 517-521.
[25]曲红光, 靳慧娟, 马丽伟, 等. 人参内生细菌的分离及其体外抗肿瘤活性分析[J]. 中国妇幼保健, 2014, 29(12): 1926-1928.
[26]林星辰, 王泽, 牛林飞, 等. 人参内生菌B69菌株抑菌活性物质产生条件的优化[J]. 人参研究, 2018, 1(1): 6-11.
[27]崔磊, 金英今, 尹成日. 党参内生菌转化人参根总皂苷为稀有人参皂苷F2和C-K的研究[J]. 延边大学学报, 2015, 41(1):79-84.
[28]郭从亮, 杨晓艳, 陈子明, 等. 一株植物内生菌Coniochaeta sp.对三七总皂苷中人参皂苷Rb1的特异性转化含量测定[J]. 中药材, 2016, 5(39): 1075-1078.
[29]FU Y, YIN Z H, YIN C R. Biotransformation of ginsenoside Rb1 to ginsenoside Rg3 by endophytic bacterium Burkholderia sp. GE 17-7 isolated from Panax ginseng [J]. Journal of Applied Microbiology, 2017, 122(6): 1579-1585.
[30]LI P Y, LIU J P, LU D. Standard NMR spectrum of ginsenosides [M]. Beijing: Chemical Industry Press, 2012.
[31]王毅, 蒋艳, 王本祥, 等. 人参皂苷Rg3及其肠内菌代谢产物Rh1对小鼠免疫细胞功能的影响[J]. 药学学报, 2002, 37(12): 927-929.
[32]ZENG X S, ZHOU X S, LUO F C, et al. Comparative analysis of the neuroprotective effects of ginsenosides Rg1 and Rb1 extracted from Panax notoginseng against cerebral ischemia [J]. Canadian Journal of Physiology and Pharmacology, 2014, 92(2): 102-108.
[33]CHRISTENSEN L P. Ginsenosides chemistry, biosynthesis, analysis, and potential health effects [J]. Advances in Food and Nutrition Research, 2009, 55(1): 1-99.
[34]张虹, 付雯雯, 徐华丽, 等. 20(S)-原人参二醇、人参皂苷Rh2及人参皂苷Rg3抗肿瘤作用的对比[J]. 中国老年学杂志, 2014, 34(17): 4911-4913.
[35]朱晓丽. 人参皂苷药理作用研究进展[J]. 中国药物经济学, 2017, 12(12): 152-154.