【作者】 毕淑峰；

【导师】 谭仁祥；

【作者基本信息】 南京大学 ， 植物学， 2011， 博士

【摘要】 自1940年Waksman从放线菌中发现了一个具有划时代意义的天然产物——链霉素之后,放线菌作为一类明星微生物,经历了半个世纪的发展黄金期,从中分离出大量抗生素,使人类的生存质量大大提高,放线菌对人类做出了巨大的贡献。进入二十一世纪后,人们更多地把目光投向海洋放线菌和特殊生境下的陆生放线菌及其产生的生物活性物质,并不断发现结构新颖的生物活性产物,使放线菌天然产物的研究焕发出新的活力。本研究从活性出发,并结合谱图知识,从海鱼和白蚁Odontotermes formosanus (Shiraki)的体内(主要是肠道)筛选出二株放线菌,编号分别为fang-1、 BY-4。对这二株放线菌进行了一定量的液体发酵,用大孔树脂XAD-7对发酵液进行提取,利用硅胶、凝胶(Sephadex LH20)、HPLC等技术对发酵产物进行分离,共分到34个化合物；运用各种波谱学方法(IR, MS,1H-NMR,13C-NMR, HMQC, COSY, HMBC, NOESY和CD等)及X-ray单晶衍射技术对分离到的化合物进行结构鉴定,31个化合物鉴定出结构,3个化合物未鉴定出结构；最后对分离到的部分化合物进行抗菌和细胞毒活性筛选。具体过程和结果如下：从放线菌fang-1的发酵液(约50L)得到粗浸膏4.2g,其中乙酸乙酯段为2.2g,用硅胶柱层析、凝胶层析(Sephadex LH-20)和HPLC等分离方法对乙酸乙酯段进行分离,总共分离到16个化合物,运用MS、一维NMR等波谱方法并查阅文献资料鉴定了它们的结构,均为已知化合物,其中14个化合物为环二肽,另2个化合物是小分子化合物。从放线菌BY-4发酵液(约50L)得到粗浸膏5.8g,其中乙酸乙酯段为2.7g,用硅胶柱层析、凝胶层析(Sephadex LH-20)和HPLC等分离方法对乙酸乙酯段进行粗分和细分,最终共分到18个化合物,综合运用各种波谱方法(IR、MS、一维、二维NMR、UV、CD等)及X-ray单晶衍射技术鉴定15个化合物的结构,其中化合物22、23、24、25、26、27、28、29、30为首次发现的新化合物,另外还培养出2个大环内酯化合物(17和21)的单晶。抗菌研究结果表明,化合物17、18、22、25、28、30具有一定的抗菌活性,其中化合物28对6个菌株有一定的抑制活性。化合物26对幽门螺旋杆菌的抑制作用较好。体外抗肿瘤细胞研究结果表明,化合物19有一定抗肿瘤活性,化合物19对人胃癌细胞SGC7901的IC50为15.51μ/mL、对人肝癌细胞SMMC7721的IC50为23.47μg/mL。另外,本论文对放线菌次生代谢产物的研究做了简单的综述和介绍。更多还原

【Abstract】 Since Waksman found a landmark natural product, streptomycin, in1940, actinomycetes have been one class of star microorganisms developed for half a century with a high speed. The quality of human lives has been largely improved due to many antibiotics from actinomycetes. The actinomycetes has great contribution to human. After the entry of twenty-first century, indigenous marine actinomycetes and terrestrial actinomycetes in special environment along with their bioactive metabolites have been research highlight and more bioactive metabolites with new structures are being discovered, which brings much vitality to the study on the nature product of actinomycetes.In this research, two strains of symbiotic actinomycetes, which were screened by bioactive and spectral analysis, were isolated from the marine fish and termite bodies (mainly from the guts) and the two strains of actinomycetes were respectively named fang-1and BY-4. The two strains of actinomycetes were fermented and the broth was extracted with XAD-7resin.34compounds were separated and purified from the extraction on the basis of spectral experiments such as IR, MS,’H-NMR,13C-NMR, HMQC, COSY, HMBC, NOESY, CD, and single-crystal X-ray diffraction analysis. The structure of31compounds was characterized and the structure of3compounds was not identified. Some of the compounds were studied on antimicrobial and antitumor activity. The results were described in details below.A crude extract (4.2g) were obtained from the fermentation broth (50L) of fang-1.The EtOAc extraction (2.2g) of the crude extract was performed through Silica gel column chromatography, Gel(Sephadex LH-20) column chromatography, HPLC and other chromatographic methods, and then16compounds were separated and purified. By a combination of spectroscopic methods such as MS,1D NMR and related data, these compounds were unambiguously characterized. All the compounds were known.14of them were cyclic peptides and the rest were small molecules’ compounds.A crude extract (5.8g) were obtained from the fermentation broth (50L) of BY-4. The EtOAc extraction (2.2g) of the crude extract was performed through Silica gel column chromatography, Gel (Sephadex LH-20) column chromatography, HPLC and other chromatographic methods, and then18compounds were separated and purified. Using different spectral approaches such as IR, MS,1D NMR,2D NMR, UV, CD, and single-crystal X-ray diffraction analysis, the structures of15compounds were elucidated, the single crystal of compound17,21had been obtained. Compound22,23,24,25,26,27,28,29,30were novel compounds, which were never reported. The antimicrobial tests were shown that compound17,18,22,25,28,30had some activities, and compound28had some activities against six strains of bacteria or fungi. Compound26had activities against H. pylori. The antitumor tests showed that compound19had some activities against human gastric cancer cell SGC7901with IC5015.51μg/mL-1and against human liver cancer cell SMMC7721with IC50=23.47μg/mL.Furthermore, studies on nature products from actinomycetes were reviewed briefly.更多还原