两株海洋放线菌发酵液活性成分研究
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摘要
本论文对两株初筛分别具有互动抗真菌活性和HCT-116细胞毒活性的海洋放线菌No. Ms252、No.172221的发酵液进行活性跟踪分离和结构鉴定。利用硅胶柱开放柱色谱、ODS开放柱色谱、制备型HPLC等多种色谱方法,从活性流分中分离得到15个化合物,通过现代波谱学手段(MS、1D及2D-NMR)鉴定了它们的结构,分别为：3,3-二(3′-吲哚基)-1,2丙二醇(化合物1)、星孢菌素苷(化合物2)、5-9H-吡啶并[3,4-b]吲哚基-2-呋喃甲醇(化合物3)、1-[5-(羟基甲基)-2-呋喃基]-9H-吡啶并[3,4-b]吲哚基-3-羧酸(化合物4)、麦角甾-3β，5α,6β-三醇(化合物5)、麦角甾-3β,5α,6β-三醇-βD-葡萄糖苷(化合物6)、β-胡萝卜苷(化合物7)、环(脯-亮)二肽(化合物8)、环(脯-缬)二肽(化合物9)、脯氨酸(化合物10)、十六烷酸(化合物11)、N-苯甲基氨基甲酸(化合物12)、1,3-丙二醇苯乙酸酯(化合物13)、大豆素(化合物14)、腺嘌呤核苷(化合物15)。其中,化合物1～4为吲哚类生物碱,化合物5～7为甾体类化合物,化合物8～9为放线菌常见的代谢产物环二肽类化合物。化合物1、3-4和6为首次从放线菌中分离得到,化合物12～13为新天然产物。通过合作单位对所分离的单体进行活性评价,发现化合物1～4表现出一定抗真菌活性,但无显著互动活性；化合物5～11对HCT-116细胞毒活性较弱。
This thesis has studied on the bioactive-guided isolation and structural determination of active constituents in fermentation broth from two strains of marine actinomycetes No. Ms252 and No.172221 that respectively showed antifungal effect and synergistic antifungal effect and cytotoxic activity for HCT-116 in early screening. Fifteen compounds were isolated from the active fractions by silica gel column, ODS open column chromatography, preparative HPLC and other chromatographic techniques. Their structures were identified as 3,3-bis(3'-indolyl)-1,2-propanediol (1), staurosporine aglycon (2),5-(9H-pyrido[3,4-b]indol-1-yl)-2-furanmethanol (3), 1-[5-(hydroxymethyl)-2-furanyl]-9H-Pyrido[3,4-b]inddole-3-carboxylic acid (4), methylcholestane-3β,5α,6β-triol (5), ergostane-β-D-glucopyranoside (6),β-daucosterol (7), cyclo-(Pro-Leu) (8), cyclo-(Pro-Val) (9), proline (10), palmitic acid (11), N-(phenylmethyl)-carbamic acid(12),1,3-propanediyl benzeneacetic acid ester (13), daidzein (14) and adenosine (15) by modern spectroscopic methods (MS, 1D and 2D-NMR). Among them, compounds 1～4 are indole alkaloids, compounds 5～7 are steroids, compounds 8～9 are cyclic dipeptides usually seen in the microorganism metabolites, whereas 1,3～4 and 6 was isolated from actinomycete for the first time. Compounds 12～13 are new natural products. Then these compounds were evaluated on the activity through co-operations. We found that compounds 1～4 showed certain degree antifungal activity but no significant synergistic effect, compounds 5～15 showed weak cytotoxic activity for HCT-116.
This thesis has studied on the bioactive-guided isolation and structural determination of active constituents in fermentation broth from two strains of marine actinomycetes No. Ms252 and No.172221 that respectively showed antifungal effect and synergistic antifungal effect and cytotoxic activity for HCT-116 in early screening. Fifteen compounds were isolated from the active fractions by silica gel column, ODS open column chromatography, preparative HPLC and other chromatographic techniques. Their structures were identified as 3,3-bis(3'-indolyl)-1,2-propanediol (1), staurosporine aglycon (2),5-(9H-pyrido[3,4-b]indol-1-yl)-2-furanmethanol (3), 1-[5-(hydroxymethyl)-2-furanyl]-9H-Pyrido[3,4-b]inddole-3-carboxylic acid (4), methylcholestane-3β,5α,6β-triol (5), ergostane-β-D-glucopyranoside (6),β-daucosterol (7), cyclo-(Pro-Leu) (8), cyclo-(Pro-Val) (9), proline (10), palmitic acid (11), N-(phenylmethyl)-carbamic acid(12),1,3-propanediyl benzeneacetic acid ester (13), daidzein (14) and adenosine (15) by modern spectroscopic methods (MS, 1D and 2D-NMR). Among them, compounds 1～4 are indole alkaloids, compounds 5～7 are steroids, compounds 8～9 are cyclic dipeptides usually seen in the microorganism metabolites, whereas 1,3～4 and 6 was isolated from actinomycete for the first time. Compounds 12～13 are new natural products. Then these compounds were evaluated on the activity through co-operations. We found that compounds 1～4 showed certain degree antifungal activity but no significant synergistic effect, compounds 5～15 showed weak cytotoxic activity for HCT-116.
引文
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[28]Rui L, Tian Z, Dh L, et. al, Two indolocarbazole alkaloids with apoptosis activity from a marine-derived actinomycete Z2039-2[J]. Arch. Pharm. Res.,2007,30 (3):270-274
[29]唐刚华,王世真,吴淑琴,等,川芎哚的有机合成研究[J].中国药学杂志,1998,33(8)：492-494
[30]董泽军,王飞,刘吉开,等,点柄乳牛肝菌子实体中抗HIV-1活性成分[J].中草药,2007,38(3)：337-339
[31]Pruna LB, Henriqes RD, Hunnerck S, et. al, Chemical studies of Cuban gorgonians, Part 10:Di-and tri-hydroxysteroids from Plexaurella grisea[J]. Pharmazie,1984,39(2):117-120.
[32]Junichi K, Yoshiteru I, Naoki N, et. al. Studies on the constituents of crude drug "Fritillariae Bulus" Ⅵ. on the sterol, organic acid, and nucleoside of fresh bulus of Fritillariae thunbergii Mro. and crude drug "Bai-mo"[J]. Yakugaku Zasshi.1982,102(11):1016-1020
[33]张庆文,叶文才,车镇涛,等,安徽银莲花的化学成分研究[J].中国中药杂志,2001,26(9):612-615
[34]Yan PS, Song Y, Sakuno E, et al. Cyclo(L-leucyl-L-prolyl) produced by Achromobacter xylosoxidans inhibits aflatoxin production by Aspergillus parasiticus.[J], Environ. Microbiol. 2004.70 (12):7466-7473.
[35]Gamini S. Jayatilake, Maureen P. Bill J. et. al. Metabolites from an Antarctic sponge-associated bacterium Pseudomonas aeruginosa. [J]. J. Nat. Prod.1996,59(3),293-296
[36]于德泉,杨峻山.分析化学手册[M].第2版,第7分册.北京：化学工业出版社,1999：495-496
[37]汤海峰,易炳华,姚新生,等.褐藻果叶马尾藻化学成分的研究[J].中国海洋药物杂志,2002,90(60)：11-15
[38]Ralph N, Elona A, Richard M, et. al. Efficient Cs2CO3-promoted solution and solid phase synthesis of carbonates and carbamates in the presence of TBAI [J]. Tetrohedron,2002,58: 3329-3347
[39]Stefano R, Ricardo T. Binding of acetylcholine and quaternary ammonium cations to macrocyclic and acyclic "Phane" esters. Evaluation of the cation-(?) primary interaction through adaptive aromatic hosts [J]. J.Am. Chem. Soc,1998,120:12443-12445
[40]Masaki K, Yoshimoto O, Introduction of stress metabolite formation in suspension cultures of Vigna Angu-laris[J]. Phytochemistry,1983,22:1257-1261.
[41]杨顺丽,刘锡葵.竹叶菜中的核苷类化学成分[J].中国天然药物,2003,1(4)：196-198
[42]刘新泳,赵全芹,李朝武,等,川芎咔啉碱的合成及其对血管内皮细胞损伤的保护作用[J].山东大学学报(医学版),2003,41(5)：485-488