發(fā)布時(shí)間：2018-01-15 20:32

  本文關(guān)鍵詞：兩株海洋內(nèi)生真菌次級(jí)代謝產(chǎn)物及其生物活性的研究　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 海洋真菌 雜色曲霉 次級(jí)代謝產(chǎn)物 結(jié)構(gòu)鑒定 抗菌活性

【摘要】：海洋真菌是是天然產(chǎn)物研究的熱點(diǎn)之一,從中發(fā)現(xiàn)了許多結(jié)構(gòu)新穎的化合物。其代謝產(chǎn)物包括生物堿、萜類、甾體類、蒽醌類等,表現(xiàn)出較為顯著的抗菌、抗氧化、抗腫瘤等活性,具有廣闊的藥用開發(fā)前景,對(duì)創(chuàng)新藥物研究具有重要意義。本研究主要是針對(duì)海洋內(nèi)生真菌次級(jí)代謝產(chǎn)物的研究,分離內(nèi)生真菌,通過(guò)生物活性與化學(xué)成分分析確定活性菌株,發(fā)酵目標(biāo)菌株后分離其次級(jí)代謝產(chǎn)物,確定單體化合物的結(jié)構(gòu)并檢測(cè)其抗菌活性。1.海洋內(nèi)生真菌的分離鑒定以山東省威海市國(guó)際海水浴場(chǎng)潮間帶動(dòng)植物為研究對(duì)象,共分離得到35株海洋內(nèi)生真菌菌株,使用三種不同的培養(yǎng)基小批量發(fā)酵菌株,乙酸乙酯提取其次級(jí)代謝產(chǎn)物。對(duì)這35株真菌進(jìn)行生物活性篩選,通過(guò)DPPH法進(jìn)行抗氧化活性篩選。同時(shí)對(duì)真菌的次級(jí)代謝產(chǎn)物進(jìn)行高效液相(HPLC)、薄層層析(TLC)分析,含量測(cè)定,確定化合物含量多且結(jié)構(gòu)豐富的菌株,作為次級(jí)代謝產(chǎn)物分離的樣品。2.雜色曲霉PT-20次級(jí)代謝產(chǎn)物分離鑒定及抗菌活性研究改良的PDP培養(yǎng)基發(fā)酵PT-20菌株,乙酸乙酯提取其次級(jí)代謝產(chǎn)物。通過(guò)薄層層析、硅膠柱層析、反相硅膠柱層析、SephadexLH-20凝膠柱色譜和重結(jié)晶等方法對(duì)PT-20次級(jí)代謝產(chǎn)物分別進(jìn)行分離純化,利用波譜學(xué)方法共鑒定出14個(gè)化合物結(jié)構(gòu),包括苯并呋喃衍生物1個(gè)、占噸酮類2個(gè)、蒽醌類4個(gè)、二酮哌嗪3個(gè)和甾體類化合物4個(gè)。通過(guò)濾紙片擴(kuò)散法檢測(cè)分離單體化合物的抗菌活性,指示菌株為枯草芽孢桿菌、大腸桿菌、金黃色葡萄球菌,其中化合物3具有抗菌活性,并通過(guò)微量肉湯稀釋法測(cè)定活性化合物3對(duì)枯草芽孢桿菌的最低抑菌濃度(MIC)為31.1μg/mL。DPPH法測(cè)定化合物的抗氧化活性顯示化合物8、9、10具有較高的抗氧化活性,其對(duì)DPPH的清除率為89.95%、73.75%和88.89%。3.雜色曲霉1403-08次級(jí)代謝產(chǎn)物分離鑒定及抗菌活性研究改良的PDP培養(yǎng)基菌株發(fā)酵菌株1403-08,共發(fā)酵15 L,乙酸乙酯提取其菌絲體與發(fā)酵液,SephadexLH-20凝膠柱色譜、正相硅膠柱層析、反相硅膠柱層析、薄層層析等方法分離,波譜學(xué)方法鑒定化合物結(jié)構(gòu),共鑒定出單體化合物6個(gè),為占噸酮類2個(gè)和蒽醌類化合物4個(gè)。濾紙片法檢測(cè)了化合物對(duì)枯草芽孢桿菌、金黃色葡萄球菌、大腸桿菌的抗菌活性,均未有抗菌活性。DPPH法測(cè)定化合物的抗氧化活性,顯示化合物不具有抗氧化活性。本文對(duì)真菌進(jìn)行篩選,發(fā)酵得到次級(jí)代謝產(chǎn)物進(jìn)行分離,鑒定化合物的結(jié)構(gòu),發(fā)現(xiàn)了一系列蒽醌、蒽酮、甾體、二酮哌嗪類化合物。上述實(shí)驗(yàn)研究為海洋天然產(chǎn)物化學(xué)中活性菌株以及藥物先導(dǎo)化合物的發(fā)掘提供了支持,為化合物的結(jié)構(gòu)多樣性提供了研究思路。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R915

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本文編號(hào)：1429961