三株海洋木霉次生代谢产物及生物活性研究

	三株海洋木霉次生代谢产物及生物活性研究
	梁小蕊
学位类型	博士
导师	季乃云
	2016-05-17
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋天然产物与药物化学
关键词	海洋木霉木霉次生代谢产物化学结构生物活性
摘要	海洋来源木霉属真菌的次生代谢产物以其新颖的结构、显著的生物活性成为农药或医药先导化合物的重要来源。本文选取3株海洋来源的木霉属真菌为研究对象，包括棘孢木霉Trichoderma asperellum dl34、桔绿木霉T. citrinoviride cf27和哈茨木霉T. harzianum R5，采用不同的培养基进行筛选，以化学多样性和生物活性为指标，选择合适的培养基进行规模发酵，对其次生代谢产物进行分离纯化、结构鉴定以及活性评价。采用硅胶柱层析、反相硅胶柱层析、凝胶Sephadex LH-20柱层析、制备薄层层析、半制备高效液相色谱及重结晶等分离纯化方法，综合运用各种现代波谱学技术（1D/2D NMR、MS、IR和ECD等），并结合量子化学计算，从上述三株海洋来源木霉属真菌的发酵产物中共分离鉴定了55个化合物，其中4个为新化合物（2个新骨架结构）。从棘孢木霉dl34的发酵粗提物中分离鉴定了18个化合物（TA1～TA18），结构类型包括1个新的二萜化合物（TA1），1个已知二萜化合物（TA2），10个甾体类化合物（TA3~TA12），2个二酮哌嗪类化合物（TA14和TA15），1个核苷（TA16）和3个聚酮类化合物（TA13、TA17和TA18）。从桔绿木霉cf27的发酵粗提物中分离鉴定23个化合物（TC1~TC23），结构类型包括1个新的二萜类化合物（TC1），1个新的降二萜化合物（TC2），5个三萜化合物（TC3~TC7），1个倍半萜化合物（TC8），4个甾体类化合物（TC9~TC12），8个聚酮类化合物（TC13~TC20），2个生物碱类化合物（TC21和TC22）。从哈茨木霉R5的发酵粗提物中分离鉴定14个化合物（TH1~TH14），结构类型包括1个新的萜类化合物（TH1）、6个甾体化合物（TH2~TH7）、2个聚酮类化合物（TH8和TH9）、2个生物碱类化合物（TH10和TH11）和2个核苷（TH13和TH14）。对分离鉴定的部分化合物进行了活性测试，发现若干化合物具有较好的抑菌、卤虫毒性和微藻抑制活性。其中化合物TC2对金黄色葡萄球菌（Staphyloccocus aureus）有明显抑制效果，最小抑菌浓度MIC值为12.4 μg/mL；化合物TA1、TA2、TC2和TH1对卤虫表现出明显的致死活性，半数致死浓度LC₅₀值分别为12.0、38.2、65.6 和68.6 μg/mL；化合物TC2对海洋卡盾藻、赤潮异弯藻和东海原甲藻表现出一定的抑制活性，浓度为100 μg/mL时的抑制率分别为37.2、23.4和14.1%；化合物TC1和TC13对东海原甲藻表现出良好的抑制活性，在100 μg/mL浓度下致死率分别达到54.1%和36.7%。本文对三株海洋来源木霉属真菌次生代谢产物及其生物活性进行了系统研究，丰富了海洋真菌来源的次生代谢产物的结构多样性，为海洋木霉属真菌的开发利用提供了一定的科学依据。
其他摘要	Due to the unique structures and potent bioactivities, natural products of marine-derived Trichoderma origin continued to be the rich resources of new leading compounds and functional agents. In this dissertation, three marine-derived Trichoderma fungi (Trichoderma asperellum dl34、T. citrinoviride cf27 and T. harzianum R5) were screened under different media. The fermentation extracts were evaluated on the basis of thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and bioassay. As a result, suitable media were selected for further investigation of chemical constituents. Most of the isolates were preliminarily screened for their bioactivities. The compounds were isolated by repeated column chromatography on silica gel, reversed-phase silica gel, and Sephadex LH-20, preparative TLC, and semi-preparative HPLC as well as recrystallization. Their structures were elucidated by means of spectroscopic methods including 1D/2D-NMR, MS, IR, ECD and by quantum chemical calculation methods. A total of 55 compounds including 4 new ones were isolated and identified. From the cultures of T. asperellum dl34, 18 compounds were isolated and identified, including one new diterpenoid (TA1), one known diterpenoid (TA2), ten known steroids (TA3~TA12), two diketopiperazines (TA14 and TA15), one nucleotide (TA16) and three polyketides (TA13, TA17 and TA18). 23 compounds were isolated and identified from the cultures of T. citrinoviride cf27, including one new diterpenoid (TC1), one new norditerpenoid (TC2), five known triterpenoids (TC3~TC7), one sesquiterpene (TC8), four steroids (TC9~TC12), eight polyketides (TC13~TC20), and two alkaloids (TC21 and TC22). 14 compounds including one new terpenoid (TH1), six known sterols (TH2~TH7), two known polyketides (TH8 and TH9), two alkaloids (TH10 and TH11), and two nucleotides (TH13 and TH14) were isolated and identified from the cultures of T. harzianum R5. Most of the isolated compounds were screened for biological activities, including inhibition against bacteria, phytopathogenic fungi, marine zooplankton, and marine phytoplankton, and some of them showed significant activities. The results showed that TC2 inhibited the growth of Staphyloccocus aureus with an MIC value of 12.4 μg/mL; TA1、TA2、TC2 and TH1 were toxic to Artemia salina with LC₅₀ value of 12.0、38.2、65.6 and 68.6 μg/mL; TC2 exhibited 37.2、23.4 and 14.1% inhibition of Chattonella marina, Heterosigma akashiwo, and Prorocentrum donghaiense at 100 μg/mL, respectively; TC1 and TC13 exhibited 54.1 and 36.7% inhibition of P. donghaiense at 100 μg/mL, respectively. In this dissertation, secondary metabolites of three marine-derived Trichoderma fungi were thoroughly studied. It enriched the chemical diversity of marine natural products and may provide important proof to the utilization and exploration of marine Trichoderma-derived natural products.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/17531
专题	中国科学院烟台海岸带研究所知识产出_学位论文
作者单位	中国科学院烟台海岸带研究所
第一作者单位	中国科学院烟台海岸带研究所
推荐引用方式 GB/T 7714	梁小蕊. 三株海洋木霉次生代谢产物及生物活性研究[D]. 北京. 中国科学院大学,2016.

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