蛇孢假殼素類化合物的發(fā)酵優(yōu)化及制備方法研究
發(fā)布時間：2019-01-18 10:10
【摘要】：特殊環(huán)境來源的微生物具有獨特的適應機制和代謝途徑,從而代謝產(chǎn)生結(jié)構(gòu)新穎和活性多樣的次級代謝產(chǎn)物,成為先導化合物的重要來源。代謝產(chǎn)物豐富,活性突出的“天才菌株”受到科學界的廣泛關注,是極具潛力的藥物開發(fā)資源。海南文昌紅樹林木欖根泥來源的特境微生物Aspergillus ustus 094102代謝產(chǎn)生的Ophiobolin O在前期藥理活性研究中發(fā)現(xiàn)可以逆轉(zhuǎn)耐阿霉素乳腺癌MCF-7細胞(MCF/ADR)對阿霉素的抗藥性,但由于Ophiobolin O產(chǎn)量較小,影響了對該化合物的進一步研究。為解決藥源供應問題,我們開展了蛇孢假殼素類化合物的發(fā)酵優(yōu)化和制備方法的研究工作。同時為尋找活性先導化合物,對該菌產(chǎn)生的蛇孢假殼素類化合物和南海海綿樣品中獲得的1株放線菌的次生代謝產(chǎn)物進行研究。本論文主要包括以下內(nèi)容：1.研究了特境微生物Aspergillus ustus 094102代謝產(chǎn)生的蛇孢假殼素類化合物的定性定量檢測、發(fā)酵優(yōu)化、萃取與制備方法。2.采用薄層色譜、正反相硅膠柱色譜、凝膠柱色譜和高效液相等多種分離純化色譜方法從焦曲霉Aspergillus ustus 094102分離出17個化合物,從放線菌71號中分離到3個化合物,共得到20個化合物,利用IR、UV、MS、NMR等波譜學方法和X-射線單晶衍射及化學反應的方法確定了這些化合物的化學結(jié)構(gòu),其中4個為新化合物。蛇孢假殼素類化合物14個(1-14)、3個補身烷型倍半萜類化合物(15～17)、2個環(huán)肽類化合物(18～19)和1個吲哚咔唑類化合物(20)。3.運用生物活性篩選模型,對菌株Aspergillus ustus 094102和放線菌71號中分離出的化合物進行活性測試。采用MTT法評價化合物的抗腫瘤活性。結(jié)果表明：化合物4-5和7-11對胰腺癌G3K細胞及乳腺癌MD-MAB-231、MCF-7和MCF/3K細胞抑制活性較強�；衔�18有強效的對人白血病細胞株K562的抑制活性。4.對非曲霉和青霉來源的海洋真菌新天然產(chǎn)物進行總結(jié),總結(jié)了從20世紀50年代頭孢菌素類抗生素發(fā)現(xiàn)開始至2014年8月共1123個化合物的來源、結(jié)構(gòu)和活性。綜上所述,本文對產(chǎn)蛇孢假殼素類化合物的特境微生物Aspergillus ustus094102進行發(fā)酵優(yōu)化,建立蛇孢假殼素類化合物定性和定量分析的方法及萃取分離制備方法,為此類化合物的成藥性研究中的藥源問題的解決提供了參考思路。對其衍生物進行分離鑒定,分離得到14個蛇孢假殼素類化合物,其中4個為新化合物。此外還分離得到3個補身烷型倍半萜類化合物,并在另一株放線菌中分離得到3個化合物�；钚栽u價顯示蛇孢假殼素類化合物和棘霉素有很好的細胞毒活性,這為抗腫瘤藥物的研究提供了先導化合物。對1123個非曲霉和青霉來源的海洋真菌新天然產(chǎn)物的來源、結(jié)構(gòu)和活性的總結(jié),為海洋真菌資源的開發(fā)提供了重要參考。
[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.
【學位授予單位】：中國海洋大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R915
本文編號：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.
【學位授予單位】：中國海洋大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R915
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1 朱統(tǒng)漢;蛇孢假殼素類化合物的發(fā)酵優(yōu)化及制備方法研究[D];中國海洋大學;2015年
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