三峡库区土壤来源真菌SXW-05的分离鉴定及其活性产物的初步研究

发布时间：2018-05-16 06:17

本文选题：三峡库区土壤 + Aspergillus　；参考：《西南大学》2011年硕士论文

【摘要】：研究背景：近年来,随着各种抗生素的广泛应用,病原微生物的耐药问题日趋严重,寻找新型有效抗生素刻不容缓；抗肿瘤药物虽种类繁多,但其毒副作用、高昂成本也迫使人们去寻找新型有效抗肿瘤药物,因此国内外科研工作者正从天然产物中积极探索、寻找活性先导化合物。真菌特有的遗传背景和生物合成途径,使其次级代谢产物极为丰富,成为筛选新型抗菌药物的和抗肿瘤活性先导化合物的重要来源。自第一个广谱抗生素—青霉素问世以来,从真菌中寻找具有生物活性、结构新颖的次级代谢产物的研究长期受到生物医药工作者的重视。三峡库区地处四川盆地与长江中下游平原的交界处,巫山地处三峡库区腹心,属于亚热带季风湿润气候,雨量充沛,独特的喀斯特地貌和明显的立体气候特征使巫山地区土壤蕴藏着丰富的微生物资源,各种微生物可能因环境的特殊而代谢多种活性次级代谢产物,成为新药筛选中重要的先导化合物来源。研究目的：本文从三峡库区巫山土壤样品中分离培养真菌,经过抗菌活性、抗癌活性筛选后,选择活性抗菌谱广、活性较强的菌株作为研究对象,对其次级代谢产物进行初步研究,以获得活性产物,寻找先导化合物,丰富三峡库区天然药用先导化合物的来源。研究方法：本文对采集的土壤样品进行处理后,利用平板稀释法进行真菌的分离培养；对所得到的真菌菌株采用牛津杯双层平板法和MTT法,以金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、白色念珠菌(Candida albicans)、藤黄八叠球菌(Sarcina lutea)、枯草芽孢杆菌(Bacillus subtilis)为受试细菌,结肠癌细胞SW480为受试细胞,进行抗菌活性和抗癌活性筛选；对菌株SXW-05在三种鉴定培养基(PDA培养基、查氏培养基、马丁培养基)上的菌落培养特征、显微形态特征的观察及18S rDNA序列测定和系统发育分析,结合真菌鉴定手册(魏景超)综合判断,确定该菌株的种属地位；以PDA液体培养基中25℃,210r/min发酵8天后所得产物用乙酸乙酯充分萃取,浓缩成浸膏,采用用薄层层析、柱层析、高效液相色谱、重结晶等分离方法结合活性追踪对其活性产物进行分离纯化；利用质谱、紫外、核磁共振等波谱手段对所得活性单体进行结构鉴定；采用MTT法对单体进行抗肿瘤活性评价,以宫颈癌Hela、结肠癌细胞SW480为受试细胞,测定该化合物对这两种癌细胞的增殖抑制率,对活性单体抗肿瘤活性的实现初步评价。研究结果：本文从三峡库区巫山土壤样品中共分离获得形态各异的真菌20株。经活性筛选后发现一株发酵产物对金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、白色念珠菌(Candida albicans)、藤黄八叠球菌(Sarcina lutea)均有明显抑制作用,并且对结肠癌细胞SW480表现出抑制活性的菌株,将其作为为最终研究的目标菌株,并命名为SXW-05。经过菌落形态、显微形态观察,菌种鉴定后,确定该菌株与土曲霉Aspergillus terreus同源性最高,为98%,并且与Aspergillus terreus FJ878634.1处于同一分支,亲缘关系最近。对菌株SXW-05进行菌落观察时,发现在PDA固体平板上生长情况最为良好,因此最终确定该菌侏的发酵条件为：PDA液体培养基中25℃,210r/min发酵8天,经过乙酸乙酯充分萃取后得到浸膏5kg,将其分离纯化后最终得到一个有抗肿瘤活性单体FB44,对其结构鉴定后确定该化合物为4,5-二羟基-3-丙烯基-1-环戊烯基-1-酮,与文献报道的terrein结构一致。对单体FB44进行抗肿瘤活性评价,以宫颈癌Hela、结肠癌细胞SW480、肺癌细胞A549为受试细胞,测定化合物FB44分别对两种癌细胞的增值抑制率,测定其IC5o值分别为0.03mM、0.23mM、9μM,表现出良好的抗肿瘤活性。结论：本文最终从三峡库区土壤分离到具有抗肿瘤活性的真菌Aspergillus terreusSXW-05,并得到其主要代谢产物之一化合物4,5-二羟基-3-丙烯基-2-环戊烯基-1-酮,为后续研究该化合物其他生理活性以及活性机理奠定了基础,丰富了三峡库区天然药用先导化合物的来源。
[Abstract]:Research background: in recent years, with the wide application of various antibiotics, the drug resistance of pathogenic microbes is becoming more and more serious. It is urgent to find new effective antibiotics. Although there are many kinds of antitumor drugs, their toxic side effects and high cost also force people to find new effective antitumor drugs. Therefore, researchers at home and abroad are in the right place. The natural products are actively exploring and searching for active precursor compounds. The specific genetic background and biosynthesis pathway of fungi are very rich in secondary metabolites and become an important source of screening new antiseptic drugs and antitumor precursor compounds. Since the first broad-spectrum antibiotic - penicillin came out, it has been found from fungi. Biopharmaceutical workers have attached great importance to the research of secondary metabolites with biological activity and novel structure.
The Three Gorges Reservoir area is located at the junction of the Sichuan basin and the plain of the middle and lower reaches of the Yangtze River. Wushan is located in the heart of the Three Gorges Reservoir area. It belongs to the subtropical monsoon humid climate. The rainfall is abundant, the unique Karst landforms and obvious three-dimensional climatic characteristics make the soil of Wushan rich in microbial resources. All kinds of microorganisms may be replaced by the special environment. Thanks to many active secondary metabolites, it has become an important lead compound source in new drug screening.
The purpose of this study is to isolate and culture fungi from the soil samples of Wushan in the Three Gorges Reservoir area. After screening the antibacterial activity and anti-cancer activity, this paper selects the active antibacterial strain as the research object and studies its secondary metabolites, in order to obtain the active products, find the precursor compounds and enrich the natural medicine of the Three Gorges Reservoir area. The source of the pilot compound.
Research methods: after processing the collected soil samples, the fungi were separated and cultivated by plate dilution method. The obtained fungal strains were used in Oxford cup double deck plate method and MTT method, with Staphylococcus aureus (Staphylococcus aureus), Escherichia coli, Candida albicans (Candida albicans), and Garcinia. Sarcina lutea (Sarcina lutea) and Bacillus subtilis (Bacillus subtilis) were tested bacteria, and colon cancer cell SW480 was tested as tested cells to conduct antimicrobial activity and screening of anticancer activity. The micromorphological characteristics of strain SXW-05 in the culture medium (PDA medium, chagfish medium, Martin medium) and microscopic morphological characteristics were observed and observed. 18S rDNA sequence determination and phylogenetic analysis, combined with the comprehensive judgment of the fungal identification manual (Wei Jingchao), determine the species status of the strain; extract the products from PDA liquid medium at 25 and 210r/min after 8 days, and concentrate them into the extract by ethyl acetate, and use thin layer chromatography, column chromatography, high performance liquid chromatography, recrystallization and other separation. Methods the active products were isolated and purified by combined activity tracing. The structure of the active monomers was identified by mass spectrometry, ultraviolet and nuclear magnetic resonance spectroscopy. The anti-tumor activity of the monomers was evaluated by MTT. The Hela of cervical cancer and SW480 of colon cancer cells were tested, and the increase of the compound to these two kinds of cancer cells was determined. Inhibition rate of colonization and preliminary evaluation of the anti-tumor activity of active monomer.
Results: 20 strains of different fungi were isolated from the Wushan soil samples in the Three Gorges Reservoir area. After active screening, a strain of Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Candida albicans (Candida albicans), and eight Staph (Sarcina lutea) were found to be obvious after screening. The inhibitory activity, and the strain that showed inhibitory activity to the colon cancer cell SW480, was used as the target strain for the final study. The strain was named as SXW-05. through colony morphology, microscopic observation, and after identification, it was identified that the strain was the highest homology to Aspergillus Aspergillus terreus, 98%, and Aspergillus terreus FJ878634.1 In the same branch, close relatives recently. When the strain SXW-05 was observed in the colony, it was found that the growth of the PDA solid plate was the best. Therefore, the fermentation conditions of the bacteria were determined as follows: 25 C in the PDA liquid medium, 8 days for the fermentation of the bacteria, and the extract 5kg after the full extraction of ethyl acetate. An anti-tumor active monomer FB44 was obtained. After identification of its structure, the compound was identified as 4,5- two hydroxyl -3- propyl -1- cyclopentenyl -1- ketone, consistent with the terrein structure reported in the literature. The anti-tumor activity of the monomer FB44 was evaluated, the cervical cancer Hela, the colon cancer cell SW480, and the lung cancer cell A549 were tested, and the compound FB44 was tested for FB44. The inhibition rates of two kinds of cancer cells were measured respectively, and their IC5o values were 0.03mM, 0.23mM and 9 M respectively, showing a good antitumor activity.
Conclusion: in this paper, Aspergillus terreusSXW-05 with antitumor activity was isolated from the soil of the Three Gorges Reservoir area, and 4,5- two hydroxyl -3- propene -2- cyclopentenyl -1- ketone, one of its main metabolites, was obtained. It laid the foundation for further study of other physiological activities and active mechanism of the compound, enriching the three Gorges Reservoir area. Sources of natural medicinal precursor compounds.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R379

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