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冈村凹顶藻内生真菌单培养和共培养次级代谢产物的化学多样性及其生物活性研究
李洪雷
学位类型博士
导师王斌贵
2017-05-22
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词内生真菌 单培养 共培养 次级代谢产物 生物活性
摘要天然产物是新药研发的重要资源,海洋真菌具有十分多样和特殊的生存环境,代谢途径独特,是天然产物研究不可或缺的一部分。而另一方面,在天然产物研究中,已知化合物的重复分离和新化合物发现几率的降低成为一个普遍性问题,针对这一问题,微生物共培养策略在天然产物研究中逐渐受到重视,在海洋真菌次级代谢产物研究中的应用逐渐增多,是发掘海洋真菌次级代谢潜力的有效途径。
本论文通过对冈村凹顶藻内生真菌的分离和筛选,综合分析菌株代谢产物生物量、生物活性以及TLC和HPLC分析等指标,选择了Talaromyces islandicus EN-501,Cladosporium cladosporioides EN-399和Aspergillus terreus EN-539三株真菌进行单培养代谢产物研究,选择开展了C. cladosporioides EN-399与Bacillus subtilis MA-288共培养、A. terreus EN-539与Paecilomyces lilacinus EN-531共培养探索并对其共培养代谢产物进行了研究。综合利用多种色谱方法和波谱技术分离鉴定了66个化合物,包括17个新化合物,结构类型包括还原型蒽醌类、二苯甲酮类、氧杂蒽酮类、枝孢菌素类、混源萜类和土曲霉酮类化合物等。对新化合物进行了DPPH和ABTS自由基清除活性、抗菌活性和抗肿瘤活性测试,发现二苯甲酮类和氧杂蒽酮类化合物具有显著的DPPH和ABTS自由基清除活性,枝孢菌素类化合物抗肿瘤活性显著,具有潜在的应用价值。
T. islandicus EN-501单培养代谢产物中分离鉴定了12个化合物,其中9个新化合物,包括5个新的还原型蒽醌类化合物(HT 1*HT 5*),2个新的二苯甲酮类化合物(HT 6*HT 7*)和2个新的氧杂蒽酮化合物(HT 8*HT 9*);从C. cladosporioides EN-399单培养代谢产物中分离鉴定了7个化合物,包括5个新的枝孢菌素类化合物(RST 1*RST 4*RST 6*),通过TDDFT-ECD和OR计算确定了枝孢菌素类化合物的绝对构型,并发现所有已报道的枝孢菌素类化合物C-4'绝对构型应修正为(S);从A. terreus EN-539单培养代谢产物中分离鉴定了12个化合物,包括混源萜类、丁内酯类和生物碱类等化合物;从C. cladosporioides EN-399与B. subtilis MA-288共培养代谢产物中分离鉴定了17个化合物,包括5个小分子吲哚类化合物(HW 1HW 5),该类化合物在真菌EN-399单独培养时被少量检测到,共培养增加了化合物的表达量;从A. terreus EN-539与P. lilacinus EN-531共培养代谢产物中分离鉴定了21个化合物,其中3个新化合物,包括1个新的混源萜类化合物AP 1*和2个新的土曲霉酮类化合物AP 6*AP 7*,混源萜类化合物AP 1*AP 2以及土曲霉酮类化合物AP 6*AP 8为真菌共培养诱导产生,在两株真菌单独培养时未检测到。
对新化合物进行了DPPH和ABTS自由基清除活性、抗菌活性和抗肿瘤活性测试。菌株EN-501代谢产物中,化合物HT 1*9*对DPPH和ABTS自由基均具有较强的清除活性,其IC50值为8.3−52 µM;化合物HT 1*5*对金黄色葡萄球菌、大肠杆菌和爱德华氏菌具有抑制活性,MIC值为2.0−64 μg/ml,化合物HT 6*HT 8*HT 9*对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌、溶藻弧菌和副溶血弧菌具有较强的抑制活性,MIC值为4.0−64 μg/ml;菌株EN-399代谢产物中,化合物RST 1*RST 6*对大肠杆菌、藤黄微球菌和哈氏弧菌具有较强的抑制活性,MIC值为4.0−128 μg/mL,化合物RST 3*对A549,Huh7和LM3肿瘤细胞株具有显著抑制活性,IC50值分别为5.0,1.0和4.1 μM,化合物RST 5*对H446肿瘤细胞株具有显著抑制活性,IC50值分别为4.0 μM;菌株EN531与EN539共培养代谢产物中,化合物AP 2对藤黄微球菌具有一定的抑制活性,MIC值为32 µg/mL,化合物AP 6*AP 9对迟缓爱德华氏菌,金黄色葡萄球菌,白菜黑斑病菌,大葱紫斑病菌,苹果轮纹病菌具有不同程度的抑制活性,MIC值为8.0−64 µg/mL。
本文开展了微生物单培养与共培养次级代谢产物的研究,获得了分子多样性丰富的代谢产物,增加了凹顶藻内生真菌次级代谢产物的分子多样性,证明了共培养策略是挖掘真菌次级代谢潜力的有效方法,丰富了海洋真菌次级代谢产物的研究内容,为海洋药物的开发和利用提供了一定的实验和理论基础。
其他摘要Natural products are a historical resources of new drugs or pharmaceutical lead compounds. Marine fungi are an indispensable part of natural product research, offering various structures with potent bioactivities, due to their unique and complicated habitats. Nevertheless, rare occurrences of the novel compounds and chemical redundancy are a chronic problem in the microbial secondary metabolites chemistry. As an available way for activation of silent genes and induction of new compounds, fungal co-culture strategy has attracted more and more attentions in recent years.
In continuation of our efforts to identify new bioactive secondary metabolites from marine-derived fungi, chemical investigations were performed on three strains of marine red alga Laurencia okamurai-derived fungi Talaromyces islandicus EN-501, Cladosporium cladosporioides EN-399, and Aspergillus terreus EN-539 in single-strain culture. Moreover, we investigated the secondary metabolites from the dual-strain culture of C. cladosporioides EN-399 and the mangrove-derived bacterial Bacillus subtilis MA-288, as well as the dual-strain culture of Paecilomyces lilacinus EN-531 and A. terreus EN-539. As a result, 66 compounds were isolated and identified from the above culture extracts, of which 17 were new compounds.
Twelve compounds, including five new hydroanthraquinones, two new diphenylketones and two new xanthones, were isolated and elucidated from the fungus T. islandicus EN-501. Seven compounds, including five new cladosporols, were isolated and identified from the fungus C. cladosporioides EN-399. Twelve compounds were isolated and identified from the fungus A. terreus EN-539. Seventeen compounds were isolated and identified from the dual culture of C. cladosporioides EN-399 and B. subtilis MA-288, including five small molecule indoles which could be detected by the trace in the single culture of the fungus C. cladosporioides EN-399. Twenty-one compounds were isolated and identified from the dual culture of P. lilacinus EN-531 and A. terreus EN-539, including one new meroterpenoid and two new terreins which could not be detected in the single culture of both fungi.
The new compouds were evaluated for their DPPH and ABTS scavenging, antimicrobial and cytotoxic activities. Compounds HT 1*9* showed significant DPPH and ABTS scavenging activities with IC50 values ranging from 8.3 to 52 µM and moderate antibacterial activities with MIC values ranging from 2 to 64 µg/mL. Compounds RST 1*RST 6* exhibited inhibitory activities against Escherichia coli, Micrococcus luteus, and Vibrio harveyi, with MIC values ranging from 4 to 128 µg/mL. In adddition, compounds RST 2*, RST 3*, and RST 6* displayed cytotoxic activities against most of the tested cell lines: A549, H446, Hela, Huh7, L02, LM3, MCF-7, and SW1990, with IC50 values ranging from 1.0 to 20.0 μM. Compound AP 2 showed inhibitory activity against M. luteus with MIC value of 32 µg/mL and compounds AP 6*AP 9 showed activities against Alternaria brassicae, Alternaria porri, Edwardsiella tarda, Physalospora piricolav, Staphylococcus aureus, with MIC values ranging from 8 to 64 µg/mL.
语种英语
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136601
专题实验海洋生物学重点实验室
作者单位中国科学院海洋研究所
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李洪雷. 冈村凹顶藻内生真菌单培养和共培养次级代谢产物的化学多样性及其生物活性研究[D]. 北京. 中国科学院大学,2017.
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