银杏树内生真菌代谢产物中活性成分的研究
|
摘要
内生真菌长期生活在植物组织内部，与宿主协同进化，在演化过程中二者形成了互惠共生的关系，在宿主的生长发育和系统演化过程中起重要作用。内生真菌可产生一些重要的治疗人类疾病的药物，对内生真菌次级代谢产物的研究，可以筛选得到一些结构新颖、活性专一的物质。本文以银杏树内生真菌(Endophytic fungus of Ginkgo biloba L., EG)为研究对象，通过对内生真菌胞内产物的研究，定向筛选银杏内酯产生菌。
首先，从原始菌种EG_3出发，在筛选过程中分离得到菌株G_(3-1)、G_(3a)、G_(3b)、G_(AR)、G_(AY)、F_(1-3)和F_3，其中菌株G_(3b)、G_(AR)、G_(AY)、F_(1-3)和F_3经菌种鉴定，被归属为半知菌亚门、丝孢纲、瘤座孢目、瘤座孢科、镰孢菌属(fusarium)。
应用高效液相色谱法和电喷雾质谱法以及液相质谱联用技术，以银杏内酯标准品为对照，对G_3系列菌株的胞内产物进行了筛选和检测。由检测结果初步认定，菌株G_(3b)和F_3的代谢产物中含有银杏内酯A、银杏内酯B和白果内酯。
在菌株F_(1-3)的发酵液中，检测到有抗革兰阳性菌的物质存在，通过对活性成分的追踪，分离得到化合物F1-3A和F1-3B。经过质谱、~1H谱、~(13)C谱以及二维核磁共振波谱的测定和解析，确定化合物F1-3A的分子结构为：
cyclo[Hiv-Me-Val-Hiv-Me-Val-Hiv-Me-Val]；质谱、~1H谱及~(13)C谱的数据与环肽类抗生素Enniatin B的文献值基本一致，认定二者同质。经过质谱测定和解析，化合物F1-3B可能与Enniatin B_1或Enniatin D同质。
There exists symbiosis between Endophytic fungus of plants and their hosts, now that Endophytic fungus live in botanical tissues for a long period of time, and evolve together with their hosts. Endophytic fungus play important roles in the growth and evolution of plants, and many of them can produce some compounds with novel structure and specific bioactivity. In this paper, Endophytic fungus of Ginkgo biloba L.(EG) were studied, from which the ginkgolide-producing strains were screened orientationally, by means of studying endo-cellular metabolites from trial-strains.
First of all, strains G3-1, G3a, G3b, GAR, GAY, F1-3 and F3 were obtained from primitive strain EG3, in the course of screening. Moreover, strains G3b, GAR, GAY, F1-3 and F3 were identified as Deuteromycotina,Hyphomycetes,
Tuberculariales, Tuberculariaceae, fusarium sp.
Besides, the endo-cellular metabolites of the all strains were detected and screening, based on the analysis methods in which samples were compared with authentic Ginkgolides by HPLC and ESI-MS. Thus, it may be concluded that ginkgolide A, ginkgolide B and bilobalide occur in metabolites of strains F3 and G3b.
Meanwhile, the substances against Gram-positive bacteria were discovered in culture broths of F1-3. After further isolation and purification, compounds F1-3A and F1-3B were obtained from active extracts. The chemical structure of F1-3A was determined to cyclo[Hiv-Me-Val-Hiv-Me-Val-Hiv-Me-Val] by several structure analysis methods, such as ESI-MS, 1H-NMR,13C-NMR and 2Dimensional-NMR, etc. F1-3A shared the same NMR and MS spectral data with cyclodepsipeptide Enniatin B reported. F1-3B was regarded as Enniatin B1 or Enniatin D according to the analysis of MS.
首先，从原始菌种EG_3出发，在筛选过程中分离得到菌株G_(3-1)、G_(3a)、G_(3b)、G_(AR)、G_(AY)、F_(1-3)和F_3，其中菌株G_(3b)、G_(AR)、G_(AY)、F_(1-3)和F_3经菌种鉴定，被归属为半知菌亚门、丝孢纲、瘤座孢目、瘤座孢科、镰孢菌属(fusarium)。
应用高效液相色谱法和电喷雾质谱法以及液相质谱联用技术，以银杏内酯标准品为对照，对G_3系列菌株的胞内产物进行了筛选和检测。由检测结果初步认定，菌株G_(3b)和F_3的代谢产物中含有银杏内酯A、银杏内酯B和白果内酯。
在菌株F_(1-3)的发酵液中，检测到有抗革兰阳性菌的物质存在，通过对活性成分的追踪，分离得到化合物F1-3A和F1-3B。经过质谱、~1H谱、~(13)C谱以及二维核磁共振波谱的测定和解析，确定化合物F1-3A的分子结构为：
cyclo[Hiv-Me-Val-Hiv-Me-Val-Hiv-Me-Val]；质谱、~1H谱及~(13)C谱的数据与环肽类抗生素Enniatin B的文献值基本一致，认定二者同质。经过质谱测定和解析，化合物F1-3B可能与Enniatin B_1或Enniatin D同质。
There exists symbiosis between Endophytic fungus of plants and their hosts, now that Endophytic fungus live in botanical tissues for a long period of time, and evolve together with their hosts. Endophytic fungus play important roles in the growth and evolution of plants, and many of them can produce some compounds with novel structure and specific bioactivity. In this paper, Endophytic fungus of Ginkgo biloba L.(EG) were studied, from which the ginkgolide-producing strains were screened orientationally, by means of studying endo-cellular metabolites from trial-strains.
First of all, strains G3-1, G3a, G3b, GAR, GAY, F1-3 and F3 were obtained from primitive strain EG3, in the course of screening. Moreover, strains G3b, GAR, GAY, F1-3 and F3 were identified as Deuteromycotina,Hyphomycetes,
Tuberculariales, Tuberculariaceae, fusarium sp.
Besides, the endo-cellular metabolites of the all strains were detected and screening, based on the analysis methods in which samples were compared with authentic Ginkgolides by HPLC and ESI-MS. Thus, it may be concluded that ginkgolide A, ginkgolide B and bilobalide occur in metabolites of strains F3 and G3b.
Meanwhile, the substances against Gram-positive bacteria were discovered in culture broths of F1-3. After further isolation and purification, compounds F1-3A and F1-3B were obtained from active extracts. The chemical structure of F1-3A was determined to cyclo[Hiv-Me-Val-Hiv-Me-Val-Hiv-Me-Val] by several structure analysis methods, such as ESI-MS, 1H-NMR,13C-NMR and 2Dimensional-NMR, etc. F1-3A shared the same NMR and MS spectral data with cyclodepsipeptide Enniatin B reported. F1-3B was regarded as Enniatin B1 or Enniatin D according to the analysis of MS.
引文
[1] 郭良栋．内生真菌研究进展．菌物系统，2001，20(1)：148-152
[2] 李桂玲，王建锋．植物内生真菌抗肿瘤活性菌株的筛选．菌物系统，2001，20(3)：387-391
[3] 钱天秀，杨世林，徐丽珍．银杏研究现状．国外医药(植物药分册)，1997，12 (4)：157-161
[4] 刘媛文．银杏药理作用的研究进展．时珍国药研究，1997，8(6)：564-565
[5] 唐于平，楼风昌等．银杏外种皮的化学成分及药理作用．药学进展，2000，24 (3)：152-155
[6] Yeu Su, Chang-Ming San, Hao-Hsuan Chuang, et al. Studies on the cytotoxic mechanisms of ginkgetin in a human ovarian adenocarcinoma cell. Naunyn-Schiemdeberg's Archives of pharmacology. 2000, 362 (1): 82-90
[7] Cheon, Bong Sun, Kim,Young Ha. Effects of Prenylated Flavonoids and Biflavonoids on Lipopolysaccharide-Induced Nitric Oxide production from the Mouse Macropharge Cell line RAW 264.7. Planta medica, 2000,66(7): 596-600.
[8] 游松，姚新生，陈英杰．银杏的化学及药理研究进展．沈阳药学院学报，1988，5(2)：142-147
[9] Ines Grsel, GerhardReuter. Analysis of 6-Hydroxkynurenic Acid in Ginkgo biloba and Ginkgo Preparations. Planta Med, 1998 (64): 566-570.
[10] Hideji ltokawa, Nobuo Totsuka, Keisuke Nakahara, et al. Antitumor Principles from Ginkgo biloba L. Chem.Pharm.Bull., 1987, 35:3016-3020
[11] O.Sticher. Quality of Ginkgo Preparations. Planta Med, 1993, 59:2-10
[12] S.Furukawa. Scientific Papers of the Institute of Physical and Chemical Research (Japan), 1932, 27:19
[13] M.Maruyama, A.Terahara, Y. Itagaki, et al. The Ginkgolides. Ⅰ.Isolation and Characterization of the various groups. Tetrahedron Letters, 1967(4):299-302
[14] M.Maruyama, A.Terahara, Y. Itagaki, et al. The Ginkgolides. Ⅱ.Derivation of partial Structures. Tetrahedron Letters, 1967(4): 303-308
[15] 游松，姚新生，崔承斌等．银杏叶的活性成分的研究．沈阳药学院学报，1988，5(7)：216
[16] E.J.Corey, Myung-chol Kang, Manoj C.Desai, et al. Total synthesis of (±) -Ginkgolide B. J.Am. Chem. Soc., 1988, 110:649-651
[17] E.J.Corey, Arun K. Ghosh. Total synthesis of Ginkgolide A. Tetrahedron lett., 1988, 29:3201-3204
[18] Carrier D J, et al. Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture. Plant Cell Rep, 1990, 8(11): 635-638
[19] Laurain D, Van Beek T A, et al. Production of ginkgolide and bilobalide in transformed and gametophyte derived cell cultures of Ginkgo biloba. Phytochemistry, 1997, 46 (1): 127
[20] Braquet Pierre G, Etienne A, et al. Involvement of platelet activating factor in respiratory anaphylaxis, demonstrated by PAF-acether inhibitor BN52021. Lancet, 1985, 1 (8444): 1501
[21] 杨铭．药物研究中的分子识别．北京：北京医科大学中国协和医科大学联合出版社，1999，138-145
[22] 王敬勉，廖德胜，张永洪．银杏萜内酯的提制及检测．食品科学，1997，18(12)：46-49
[23] 熊宗贵．发酵工艺原理．北京：中国医药科技出版社，1995，1—11
[24] 林恩·马古利斯．生物共生的行星．上海：上海科学技术出版社，1999，84-88
[25] Leuchtmann A, Clay K. Isozyme variation in the fungus Atkinsonella hypoxylon within and among populations of its
host grasses. Can J Bot, 1989,67:2600-2607
[26] HemmerliUA, Brindle U E,Petrini O, et al. Differentiation of isolates of Discula umbrinella from beech, chestnut and oak using RAPD marks. Mol Plant-Microbe Interactions, 1992, 5:479-483
[27] 颜方贵，秦杰．赤霉素A4、A7的研究进展．微生物学通报，1994，21 (3)：163-167
[28] 王建锋，吕华鹰，苏文金．植物内生真菌产紫杉醇的研究．微生物学通报，2000，27(1)：58-60
[29] 周与良，邢来君．真菌学．北京：高等教育出版社，1989，128-131，194-197，448
[30] Carrier D. J., Van Beek T. A., et al.Distribution of Ginkgolides and Terpeniod Biosynthetic Activity in Ginkgo Biloba.1998,48(1): 89-92
[31] 王伟，朱平等．植物赤霉素生物合成和信号传导的分子生物学．植物学通报，2002，19(2)：137-149
[32] 黄新，邱德有等．紫杉醇生物合成途径中相关酶的研究进展．中国生物工程杂志，2002，22(5)：27-33
[33] Andrea Stierle, Gary Strobel, Donald Stierle. Taxol and Taxane Production by Taxomyces androanae, an Endophytic Fungus of Pacific Yew. SCIENCE,1993,260:214-216.
[34] Gary Strobel, Xianshu Yang, Joe Sear, et al. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiology, 1996,142:435-440.
[35] 颜玉贞，谢培山，钱浩良等。银杏叶提取物及制剂萜类内酯薄层色谱荧光扫描定量测定研究．中国中药杂志，1997，22(3)：159-162
[36] T.A.Vanbeek, H.A.Scheeren, et al. Determination of ginkgolides and bilobalide in Ginkgo biloba leaves and phytopharmaceuticals, J. Chromatography, 1991, 543:375-387
[37] 毛忠贵．生物工业下游技术．北京：中国轻工业出版社，1999，98-106
[38] Weings Klaus, Hepp Wichael, Jaggy Hermam. Chemistry of
ginkgolide Ⅱ,Isolation and structural elucidation Liebigs. Ann. Chem., 1987 (6): 521
[39] 陈惠芳译，提取银杏叶的新工艺．国外药讯，1992(4)：35
[40] Hoon Huh, et al. Ontogenic Aspects of Ginkgolide Production in Ginkgo biloba. PlantaMed, 1993, 59:232
[41] 姚渭溪．银杏叶中活性成分的提取工艺、测定及其进展．中草药，1995，24 (3)：157-159
[42] 彭柏林．阿维菌素生产工艺的研究：[学位论文]．沈阳：沈阳药科大学，2001．
[43] 陈维军，谢笔钧，胡慰望．分光光度法快速测定银杏叶中萜内酯含量的研究．中草药，1998，(29)增刊：45-46
[44] 毕殿洲．药剂学(第四版)，北京：人民卫生出版社，1999，324-325
[45] 白秀峰．新抗生素的筛选(选修课教材)．沈阳：沈阳药科大学，1998，24-38
[46] Gordon W L. Distribution and prevalence of Fusarium moniliforme sheld. Gibberella jujikuroi (saw.) Wr. Producing substances with Gibberellin-like biological properties. Nature 1960, 186:698-704
[47] Eisenberg W V, Keenan G L. Microscopic-crystallograic properties of crystalline sodium penicillin G[J].J Am Pharm Assoc Sci Ed, 1974, 36 (2): 294
[48] 洪丽娜，唐信东，缪昌城，等．镰刀菌4-11产生环孢菌素Ⅱ．提取、分离纯化和鉴别[J]．抗生素．1984，9(1)：5
[49] 于德泉，杨俊山．分析化学手册(第七分册)．北京：化学工业出版社，1999：123、909
[50] Zhong Jiang, Marc-OlivierBarret, Kenneth G. Boyd, et al.JM47, a cyclic tetrapeptide HC-toxin analogue from a marine Fusarium S P. Phytochemistry, 2002,60. 33-38
[51] Gumann E, et al. Enniatin, a new antibiotic active against Mycobacteria. Experientia, 1947,9:202-203
[52] Deol B.S., Ridley D.D., et al. Isolation of cyclodepsipepyides from plant pathogenic fungi. Australian Journal of Chemistry, 1978,31: 1397-1399
[53] Hiroshi Tomoda, Hiroyuki Nishida, et al. New Cyclodepsipeptides, Enniatins D、E and F produced by Fusarium sp. FO-1305.J.Antibiotics, 1992,45: 1207-1215
[54] Tawnya C. McKee, Heidi R. Bokesch, Jinping L. McCormick, et al. Isolation and Characterization of New Anti-HIV and Cytotoxic leads from Plants, Marine, and Microbial Organism. J. NaturalProducts,1997,60 (5):431-438
[55] http: //home.nicifcrf. gov/mtddp/compounds/692895.html
[56] 白秀峰．生物药物分析．北京：中国医药科技出版社，2002，473-477
[57] Pierluigi Mauri, Barbara Migliazza, Piergiorgio Pietta. Liquid Chromatography/Electrospay Mass Spectrometry of Bioactive Terpenoids in Ginkgo biloba L. J. Mass. Spectrum, 1999, 34: 1361-1367
[58] 杨义方，夏野鹰．银杏叶及其制剂中萜内酯含量测定方法研究进展．国外医药·植物药分册，2000，15(2)：61-66
[59] Bernice Steinke, Bernd Müller, Hildebert Wagner. Biologische Standardisierungs methode für Ginkgo-Extrate. Planta Med,1993, 59:155-160
[2] 李桂玲，王建锋．植物内生真菌抗肿瘤活性菌株的筛选．菌物系统，2001，20(3)：387-391
[3] 钱天秀，杨世林，徐丽珍．银杏研究现状．国外医药(植物药分册)，1997，12 (4)：157-161
[4] 刘媛文．银杏药理作用的研究进展．时珍国药研究，1997，8(6)：564-565
[5] 唐于平，楼风昌等．银杏外种皮的化学成分及药理作用．药学进展，2000，24 (3)：152-155
[6] Yeu Su, Chang-Ming San, Hao-Hsuan Chuang, et al. Studies on the cytotoxic mechanisms of ginkgetin in a human ovarian adenocarcinoma cell. Naunyn-Schiemdeberg's Archives of pharmacology. 2000, 362 (1): 82-90
[7] Cheon, Bong Sun, Kim,Young Ha. Effects of Prenylated Flavonoids and Biflavonoids on Lipopolysaccharide-Induced Nitric Oxide production from the Mouse Macropharge Cell line RAW 264.7. Planta medica, 2000,66(7): 596-600.
[8] 游松，姚新生，陈英杰．银杏的化学及药理研究进展．沈阳药学院学报，1988，5(2)：142-147
[9] Ines Grsel, GerhardReuter. Analysis of 6-Hydroxkynurenic Acid in Ginkgo biloba and Ginkgo Preparations. Planta Med, 1998 (64): 566-570.
[10] Hideji ltokawa, Nobuo Totsuka, Keisuke Nakahara, et al. Antitumor Principles from Ginkgo biloba L. Chem.Pharm.Bull., 1987, 35:3016-3020
[11] O.Sticher. Quality of Ginkgo Preparations. Planta Med, 1993, 59:2-10
[12] S.Furukawa. Scientific Papers of the Institute of Physical and Chemical Research (Japan), 1932, 27:19
[13] M.Maruyama, A.Terahara, Y. Itagaki, et al. The Ginkgolides. Ⅰ.Isolation and Characterization of the various groups. Tetrahedron Letters, 1967(4):299-302
[14] M.Maruyama, A.Terahara, Y. Itagaki, et al. The Ginkgolides. Ⅱ.Derivation of partial Structures. Tetrahedron Letters, 1967(4): 303-308
[15] 游松，姚新生，崔承斌等．银杏叶的活性成分的研究．沈阳药学院学报，1988，5(7)：216
[16] E.J.Corey, Myung-chol Kang, Manoj C.Desai, et al. Total synthesis of (±) -Ginkgolide B. J.Am. Chem. Soc., 1988, 110:649-651
[17] E.J.Corey, Arun K. Ghosh. Total synthesis of Ginkgolide A. Tetrahedron lett., 1988, 29:3201-3204
[18] Carrier D J, et al. Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture. Plant Cell Rep, 1990, 8(11): 635-638
[19] Laurain D, Van Beek T A, et al. Production of ginkgolide and bilobalide in transformed and gametophyte derived cell cultures of Ginkgo biloba. Phytochemistry, 1997, 46 (1): 127
[20] Braquet Pierre G, Etienne A, et al. Involvement of platelet activating factor in respiratory anaphylaxis, demonstrated by PAF-acether inhibitor BN52021. Lancet, 1985, 1 (8444): 1501
[21] 杨铭．药物研究中的分子识别．北京：北京医科大学中国协和医科大学联合出版社，1999，138-145
[22] 王敬勉，廖德胜，张永洪．银杏萜内酯的提制及检测．食品科学，1997，18(12)：46-49
[23] 熊宗贵．发酵工艺原理．北京：中国医药科技出版社，1995，1—11
[24] 林恩·马古利斯．生物共生的行星．上海：上海科学技术出版社，1999，84-88
[25] Leuchtmann A, Clay K. Isozyme variation in the fungus Atkinsonella hypoxylon within and among populations of its
host grasses. Can J Bot, 1989,67:2600-2607
[26] HemmerliUA, Brindle U E,Petrini O, et al. Differentiation of isolates of Discula umbrinella from beech, chestnut and oak using RAPD marks. Mol Plant-Microbe Interactions, 1992, 5:479-483
[27] 颜方贵，秦杰．赤霉素A4、A7的研究进展．微生物学通报，1994，21 (3)：163-167
[28] 王建锋，吕华鹰，苏文金．植物内生真菌产紫杉醇的研究．微生物学通报，2000，27(1)：58-60
[29] 周与良，邢来君．真菌学．北京：高等教育出版社，1989，128-131，194-197，448
[30] Carrier D. J., Van Beek T. A., et al.Distribution of Ginkgolides and Terpeniod Biosynthetic Activity in Ginkgo Biloba.1998,48(1): 89-92
[31] 王伟，朱平等．植物赤霉素生物合成和信号传导的分子生物学．植物学通报，2002，19(2)：137-149
[32] 黄新，邱德有等．紫杉醇生物合成途径中相关酶的研究进展．中国生物工程杂志，2002，22(5)：27-33
[33] Andrea Stierle, Gary Strobel, Donald Stierle. Taxol and Taxane Production by Taxomyces androanae, an Endophytic Fungus of Pacific Yew. SCIENCE,1993,260:214-216.
[34] Gary Strobel, Xianshu Yang, Joe Sear, et al. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiology, 1996,142:435-440.
[35] 颜玉贞，谢培山，钱浩良等。银杏叶提取物及制剂萜类内酯薄层色谱荧光扫描定量测定研究．中国中药杂志，1997，22(3)：159-162
[36] T.A.Vanbeek, H.A.Scheeren, et al. Determination of ginkgolides and bilobalide in Ginkgo biloba leaves and phytopharmaceuticals, J. Chromatography, 1991, 543:375-387
[37] 毛忠贵．生物工业下游技术．北京：中国轻工业出版社，1999，98-106
[38] Weings Klaus, Hepp Wichael, Jaggy Hermam. Chemistry of
ginkgolide Ⅱ,Isolation and structural elucidation Liebigs. Ann. Chem., 1987 (6): 521
[39] 陈惠芳译，提取银杏叶的新工艺．国外药讯，1992(4)：35
[40] Hoon Huh, et al. Ontogenic Aspects of Ginkgolide Production in Ginkgo biloba. PlantaMed, 1993, 59:232
[41] 姚渭溪．银杏叶中活性成分的提取工艺、测定及其进展．中草药，1995，24 (3)：157-159
[42] 彭柏林．阿维菌素生产工艺的研究：[学位论文]．沈阳：沈阳药科大学，2001．
[43] 陈维军，谢笔钧，胡慰望．分光光度法快速测定银杏叶中萜内酯含量的研究．中草药，1998，(29)增刊：45-46
[44] 毕殿洲．药剂学(第四版)，北京：人民卫生出版社，1999，324-325
[45] 白秀峰．新抗生素的筛选(选修课教材)．沈阳：沈阳药科大学，1998，24-38
[46] Gordon W L. Distribution and prevalence of Fusarium moniliforme sheld. Gibberella jujikuroi (saw.) Wr. Producing substances with Gibberellin-like biological properties. Nature 1960, 186:698-704
[47] Eisenberg W V, Keenan G L. Microscopic-crystallograic properties of crystalline sodium penicillin G[J].J Am Pharm Assoc Sci Ed, 1974, 36 (2): 294
[48] 洪丽娜，唐信东，缪昌城，等．镰刀菌4-11产生环孢菌素Ⅱ．提取、分离纯化和鉴别[J]．抗生素．1984，9(1)：5
[49] 于德泉，杨俊山．分析化学手册(第七分册)．北京：化学工业出版社，1999：123、909
[50] Zhong Jiang, Marc-OlivierBarret, Kenneth G. Boyd, et al.JM47, a cyclic tetrapeptide HC-toxin analogue from a marine Fusarium S P. Phytochemistry, 2002,60. 33-38
[51] Gumann E, et al. Enniatin, a new antibiotic active against Mycobacteria. Experientia, 1947,9:202-203
[52] Deol B.S., Ridley D.D., et al. Isolation of cyclodepsipepyides from plant pathogenic fungi. Australian Journal of Chemistry, 1978,31: 1397-1399
[53] Hiroshi Tomoda, Hiroyuki Nishida, et al. New Cyclodepsipeptides, Enniatins D、E and F produced by Fusarium sp. FO-1305.J.Antibiotics, 1992,45: 1207-1215
[54] Tawnya C. McKee, Heidi R. Bokesch, Jinping L. McCormick, et al. Isolation and Characterization of New Anti-HIV and Cytotoxic leads from Plants, Marine, and Microbial Organism. J. NaturalProducts,1997,60 (5):431-438
[55] http: //home.nicifcrf. gov/mtddp/compounds/692895.html
[56] 白秀峰．生物药物分析．北京：中国医药科技出版社，2002，473-477
[57] Pierluigi Mauri, Barbara Migliazza, Piergiorgio Pietta. Liquid Chromatography/Electrospay Mass Spectrometry of Bioactive Terpenoids in Ginkgo biloba L. J. Mass. Spectrum, 1999, 34: 1361-1367
[58] 杨义方，夏野鹰．银杏叶及其制剂中萜内酯含量测定方法研究进展．国外医药·植物药分册，2000，15(2)：61-66
[59] Bernice Steinke, Bernd Müller, Hildebert Wagner. Biologische Standardisierungs methode für Ginkgo-Extrate. Planta Med,1993, 59:155-160