蛇孢假壳素类化合物的发酵优化及制备方法研究
发布时间：2019-01-18 10:10
【摘要】：特殊环境来源的微生物具有独特的适应机制和代谢途径,从而代谢产生结构新颖和活性多样的次级代谢产物,成为先导化合物的重要来源。代谢产物丰富,活性突出的“天才菌株”受到科学界的广泛关注,是极具潜力的药物开发资源。海南文昌红树林木榄根泥来源的特境微生物Aspergillus ustus 094102代谢产生的Ophiobolin O在前期药理活性研究中发现可以逆转耐阿霉素乳腺癌MCF-7细胞(MCF/ADR)对阿霉素的抗药性,但由于Ophiobolin O产量较小,影响了对该化合物的进一步研究。为解决药源供应问题,我们开展了蛇孢假壳素类化合物的发酵优化和制备方法的研究工作。同时为寻找活性先导化合物,对该菌产生的蛇孢假壳素类化合物和南海海绵样品中获得的1株放线菌的次生代谢产物进行研究。本论文主要包括以下内容：1.研究了特境微生物Aspergillus ustus 094102代谢产生的蛇孢假壳素类化合物的定性定量检测、发酵优化、萃取与制备方法。2.采用薄层色谱、正反相硅胶柱色谱、凝胶柱色谱和高效液相等多种分离纯化色谱方法从焦曲霉Aspergillus ustus 094102分离出17个化合物,从放线菌71号中分离到3个化合物,共得到20个化合物,利用IR、UV、MS、NMR等波谱学方法和X-射线单晶衍射及化学反应的方法确定了这些化合物的化学结构,其中4个为新化合物。蛇孢假壳素类化合物14个(1-14)、3个补身烷型倍半萜类化合物(15～17)、2个环肽类化合物(18～19)和1个吲哚咔唑类化合物(20)。3.运用生物活性筛选模型,对菌株Aspergillus ustus 094102和放线菌71号中分离出的化合物进行活性测试。采用MTT法评价化合物的抗肿瘤活性。结果表明：化合物4-5和7-11对胰腺癌G3K细胞及乳腺癌MD-MAB-231、MCF-7和MCF/3K细胞抑制活性较强。化合物18有强效的对人白血病细胞株K562的抑制活性。4.对非曲霉和青霉来源的海洋真菌新天然产物进行总结,总结了从20世纪50年代头孢菌素类抗生素发现开始至2014年8月共1123个化合物的来源、结构和活性。综上所述,本文对产蛇孢假壳素类化合物的特境微生物Aspergillus ustus094102进行发酵优化,建立蛇孢假壳素类化合物定性和定量分析的方法及萃取分离制备方法,为此类化合物的成药性研究中的药源问题的解决提供了参考思路。对其衍生物进行分离鉴定,分离得到14个蛇孢假壳素类化合物,其中4个为新化合物。此外还分离得到3个补身烷型倍半萜类化合物,并在另一株放线菌中分离得到3个化合物。活性评价显示蛇孢假壳素类化合物和棘霉素有很好的细胞毒活性,这为抗肿瘤药物的研究提供了先导化合物。对1123个非曲霉和青霉来源的海洋真菌新天然产物的来源、结构和活性的总结,为海洋真菌资源的开发提供了重要参考。
[Abstract]:Microbes from special environmental sources have unique adaptation mechanisms and metabolic pathways, which lead to the production of novel secondary metabolites with novel structure and diverse activities, thus becoming an important source of lead compounds. "Genius strains", which have abundant metabolites and outstanding activity, are widely concerned by the scientific community and are potential resources for drug development. Ophiobolin O produced by metabolism of Aspergillus ustus 094102, a special microorganism from mangrove root mud in Wenchang, Hainan Province, was found to reverse the resistance of adriamycin-resistant breast cancer MCF-7 cells (MCF/ADR) to adriamycin. However, the production of Ophiobolin O was small, which affected the further study of the compound. In order to solve the problem of drug supply, we have studied the fermentation optimization and preparation method of the compound. In order to search for active lead compounds, the secondary metabolites of Actinomyces actinomycetes from the spongy samples of the South China Sea were studied. This thesis mainly includes the following contents: 1. The qualitative and quantitative detection, fermentation optimization, extraction and preparation methods of serpentine compounds produced by metabolism of Aspergillus ustus 094102, a special microorganism, were studied. 2. Seventeen compounds were isolated from Aspergillus ustus 094102 by TLC, silica gel column chromatography, gel column chromatography and high performance liquid chromatography. Three compounds were isolated from actinomycetes 71. A total of 20 compounds were obtained. The chemical structures of these compounds were determined by means of IR,UV,MS,NMR and X-ray single crystal diffraction and chemical reaction methods, four of which were new compounds. There are 14 compounds (1-14), 3 sesquiterpenes (1517), 2 cyclic peptides (1819) and 1 indole carbazole (20). The bioactivity of the compounds isolated from strain Aspergillus ustus 094102 and actinomycetes 71 was tested by bioactivity screening model. The anti-tumor activity of the compound was evaluated by MTT method. The results showed that compounds 4-5 and 7-11 had strong inhibitory activities on G3K cells of pancreatic cancer and MD-MAB-231,MCF-7 and MCF/3K cells of breast cancer. Compound 18 has strong inhibitory activity on human leukemia cell line K562. 4. The new natural products of marine fungi from non-aspergillus and penicillium were summarized. From the discovery of cephalosporins in 1950s to August 2014, the origin, structure and activity of 1123 compounds were summarized. To sum up, this paper optimized the fermentation of Aspergillus ustus094102, a special microorganism, which produced the compounds, and established the methods of qualitative and quantitative analysis of the compounds and the preparation method of extraction and separation. It provides a reference for solving the problem of drug source in the study of the drug properties of this kind of compounds. The derivatives were isolated and identified, and 14 compounds were isolated, 4 of which were new compounds. In addition, three sesquiterpenoids were isolated and three compounds were isolated from another actinomycetes. The activity evaluation showed that the pseudo-chitosan compounds and spiramycin had very good cytotoxic activity, which provided the leading compounds for the study of antitumor drugs. The sources, structures and activities of 1123 new natural products of marine fungi from non-Aspergillus and penicillium were summarized, which provided an important reference for the exploitation of marine fungi resources.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R915
本文编号：2410603
[Abstract]:Microbes from special environmental sources have unique adaptation mechanisms and metabolic pathways, which lead to the production of novel secondary metabolites with novel structure and diverse activities, thus becoming an important source of lead compounds. "Genius strains", which have abundant metabolites and outstanding activity, are widely concerned by the scientific community and are potential resources for drug development. Ophiobolin O produced by metabolism of Aspergillus ustus 094102, a special microorganism from mangrove root mud in Wenchang, Hainan Province, was found to reverse the resistance of adriamycin-resistant breast cancer MCF-7 cells (MCF/ADR) to adriamycin. However, the production of Ophiobolin O was small, which affected the further study of the compound. In order to solve the problem of drug supply, we have studied the fermentation optimization and preparation method of the compound. In order to search for active lead compounds, the secondary metabolites of Actinomyces actinomycetes from the spongy samples of the South China Sea were studied. This thesis mainly includes the following contents: 1. The qualitative and quantitative detection, fermentation optimization, extraction and preparation methods of serpentine compounds produced by metabolism of Aspergillus ustus 094102, a special microorganism, were studied. 2. Seventeen compounds were isolated from Aspergillus ustus 094102 by TLC, silica gel column chromatography, gel column chromatography and high performance liquid chromatography. Three compounds were isolated from actinomycetes 71. A total of 20 compounds were obtained. The chemical structures of these compounds were determined by means of IR,UV,MS,NMR and X-ray single crystal diffraction and chemical reaction methods, four of which were new compounds. There are 14 compounds (1-14), 3 sesquiterpenes (1517), 2 cyclic peptides (1819) and 1 indole carbazole (20). The bioactivity of the compounds isolated from strain Aspergillus ustus 094102 and actinomycetes 71 was tested by bioactivity screening model. The anti-tumor activity of the compound was evaluated by MTT method. The results showed that compounds 4-5 and 7-11 had strong inhibitory activities on G3K cells of pancreatic cancer and MD-MAB-231,MCF-7 and MCF/3K cells of breast cancer. Compound 18 has strong inhibitory activity on human leukemia cell line K562. 4. The new natural products of marine fungi from non-aspergillus and penicillium were summarized. From the discovery of cephalosporins in 1950s to August 2014, the origin, structure and activity of 1123 compounds were summarized. To sum up, this paper optimized the fermentation of Aspergillus ustus094102, a special microorganism, which produced the compounds, and established the methods of qualitative and quantitative analysis of the compounds and the preparation method of extraction and separation. It provides a reference for solving the problem of drug source in the study of the drug properties of this kind of compounds. The derivatives were isolated and identified, and 14 compounds were isolated, 4 of which were new compounds. In addition, three sesquiterpenoids were isolated and three compounds were isolated from another actinomycetes. The activity evaluation showed that the pseudo-chitosan compounds and spiramycin had very good cytotoxic activity, which provided the leading compounds for the study of antitumor drugs. The sources, structures and activities of 1123 new natural products of marine fungi from non-Aspergillus and penicillium were summarized, which provided an important reference for the exploitation of marine fungi resources.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R915
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1 朱统汉;蛇孢假壳素类化合物的发酵优化及制备方法研究[D];中国海洋大学;2015年
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