两株海洋内生真菌次级代谢产物及其生物活性的研究

发布时间：2018-01-15 20:32

本文关键词：两株海洋内生真菌次级代谢产物及其生物活性的研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：海洋真菌是是天然产物研究的热点之一,从中发现了许多结构新颖的化合物。其代谢产物包括生物碱、萜类、甾体类、蒽醌类等,表现出较为显著的抗菌、抗氧化、抗肿瘤等活性,具有广阔的药用开发前景,对创新药物研究具有重要意义。本研究主要是针对海洋内生真菌次级代谢产物的研究,分离内生真菌,通过生物活性与化学成分分析确定活性菌株,发酵目标菌株后分离其次级代谢产物,确定单体化合物的结构并检测其抗菌活性。1.海洋内生真菌的分离鉴定以山东省威海市国际海水浴场潮间带动植物为研究对象,共分离得到35株海洋内生真菌菌株,使用三种不同的培养基小批量发酵菌株,乙酸乙酯提取其次级代谢产物。对这35株真菌进行生物活性筛选,通过DPPH法进行抗氧化活性筛选。同时对真菌的次级代谢产物进行高效液相(HPLC)、薄层层析(TLC)分析,含量测定,确定化合物含量多且结构丰富的菌株,作为次级代谢产物分离的样品。2.杂色曲霉PT-20次级代谢产物分离鉴定及抗菌活性研究改良的PDP培养基发酵PT-20菌株,乙酸乙酯提取其次级代谢产物。通过薄层层析、硅胶柱层析、反相硅胶柱层析、SephadexLH-20凝胶柱色谱和重结晶等方法对PT-20次级代谢产物分别进行分离纯化,利用波谱学方法共鉴定出14个化合物结构,包括苯并呋喃衍生物1个、占吨酮类2个、蒽醌类4个、二酮哌嗪3个和甾体类化合物4个。通过滤纸片扩散法检测分离单体化合物的抗菌活性,指示菌株为枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌,其中化合物3具有抗菌活性,并通过微量肉汤稀释法测定活性化合物3对枯草芽孢杆菌的最低抑菌浓度(MIC)为31.1μg/mL。DPPH法测定化合物的抗氧化活性显示化合物8、9、10具有较高的抗氧化活性,其对DPPH的清除率为89.95%、73.75%和88.89%。3.杂色曲霉1403-08次级代谢产物分离鉴定及抗菌活性研究改良的PDP培养基菌株发酵菌株1403-08,共发酵15 L,乙酸乙酯提取其菌丝体与发酵液,SephadexLH-20凝胶柱色谱、正相硅胶柱层析、反相硅胶柱层析、薄层层析等方法分离,波谱学方法鉴定化合物结构,共鉴定出单体化合物6个,为占吨酮类2个和蒽醌类化合物4个。滤纸片法检测了化合物对枯草芽孢杆菌、金黄色葡萄球菌、大肠杆菌的抗菌活性,均未有抗菌活性。DPPH法测定化合物的抗氧化活性,显示化合物不具有抗氧化活性。本文对真菌进行筛选,发酵得到次级代谢产物进行分离,鉴定化合物的结构,发现了一系列蒽醌、蒽酮、甾体、二酮哌嗪类化合物。上述实验研究为海洋天然产物化学中活性菌株以及药物先导化合物的发掘提供了支持,为化合物的结构多样性提供了研究思路。
[Abstract]:Marine fungi are one of the hotspots in the research of natural products, and many novel compounds have been found. Their metabolites include alkaloids, terpenes, steroids, anthraquinones and so on. Antioxidation, anti-tumor and other activities have a broad prospect of pharmaceutical development, which is of great significance for the study of innovative drugs. This study is mainly aimed at the secondary metabolites of marine endophytic fungi, isolation of endophytic fungi. The bioactivity and chemical composition analysis were used to determine the active strain, and the secondary metabolites were isolated after fermentation of the target strain. The isolation and identification of marine endophytic fungi took the intertidal flora and fauna of Weihai City, Shandong Province as the research object. A total of 35 strains of marine endophytic fungi were isolated. Three kinds of small batch fermentation strains were used in different culture medium. Ethyl acetate was used to extract the secondary metabolites. The bioactivity of 35 strains of fungi was screened. The antioxidant activity was screened by DPPH. The secondary metabolites of fungi were analyzed by HPLC and TLC, and the content of the secondary metabolites was determined. The strains with abundant compounds and structures were identified. Isolation and Identification of Secondary metabolites from Aspergillus heterochroma PT-20 and study on Antibacterial activity; fermentation of PT-20 strain on improved PDP medium. The secondary metabolites were extracted from ethyl acetate by thin layer chromatography, silica gel column chromatography and reverse phase silica gel column chromatography. The secondary metabolites of PT-20 were separated and purified by SephadexLH-20 gel column chromatography and recrystallization, and 14 compounds were identified by spectroscopic method. Including 1 benzofuran derivative, 2 tons of ketones, 4 anthraquinones, 3 diketo piperazine and 4 steroids. The antibacterial activity of isolated monomers was detected by filter paper diffusion method. The indicated strains were Bacillus subtilis, Escherichia coli and Staphylococcus aureus, in which compound 3 had antibacterial activity. The minimum inhibitory concentration of active compound 3 against Bacillus subtilis was determined by broth dilution method. The antioxidant activity of the compounds was determined by DPPH method for 31.1 渭 g / mL DPPH. The antioxidation activity of 9N10 was higher, and the clearance rate of DPPH was 89.95%. Isolation and Identification of Secondary metabolites of Aspergillus heterochroma 1403-08 and its Antibacterial activity; fermentation strain 1403-08 of improved PDP medium. The mycelium was separated from fermentation broth by Sephadex LH-20 gel column chromatography, normal phase silica gel column chromatography, reverse silica gel column chromatography and thin layer chromatography. The structure of the compounds was identified by spectroscopic method. Six monomers were identified, including 2 tons of ketones and 4 compounds of anthraquinones. Filter paper method was used to detect the compounds against Bacillus subtilis and Staphylococcus aureus. The antimicrobial activity of Escherichia coli was not determined by .DPPH method, which showed that the compound had no antioxidation activity. The secondary metabolites were isolated and identified by fermentation. A series of anthraquinones, anthrone and steroids were found. The above experimental studies have provided support for the discovery of active strains and drug leading compounds in the chemistry of marine natural products, and provided the research ideas for the structural diversity of the compounds.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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