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四株深海真菌次级代谢产物化学多样性及生物活性研究
迟路坪
Subtype博士
Thesis Advisor王斌贵
2020-05-22
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword深海真菌 海洋天然产物 次级代谢产物 生物活性
Abstract

天然产物因其新颖的化学结构和多样的生物活性在新药研发中被赋予独特的地位。深海(>1000 m)通常具有高压、无光等环境特点,局部具有冷泉、热液等极端环境,深海微生物具有与深海环境相适应的独特遗传背景和代谢途径,蕴含丰富的次级代谢产物资源,具有独特的生物功能。

本论文从不同海域和水深的深海沉积物及动物样品中分离获得137株真菌,经小试培养筛选及培养条件优化,结合化学和生物学指标以及文献调研,最终选取4株潜力菌株(Epicoccum nigrum SD-388Aspergillus insuetus SD-512A. penicillioides SD-311以及Trichoderma longibrachiatum SD-529)进行规模发酵,综合运用各种色谱手段(CCTLCHPLC等),结合波谱分析(NMRMSUVECD等)及X-射线晶体学技术,从其发酵产物中共分离鉴定化合物结构91个,包括21个新化合物,1个新天然产物,并对部分化合物进行了抗菌、抗病毒和抗肿瘤等活性评价。

从深海真菌E. nigrum SD-388的发酵产物中共分离鉴定32个化合物(EN 1EN 32),包括11个新化合物(EN 1*–EN 10*)和1个新天然产物(EN 11#),结构类型主要为二酮哌嗪类。通过RDCRCSA、手性拆分、ECD计算等方法确定了新化合物的相对和绝对构型,并培养获得了二酮哌嗪类化合物的晶体结构。抗肿瘤和抗菌活性测试结果显示,化合物EN 5*EN 28具有显著抑制Huh 7.5肿瘤细胞生长的活性,IC50值分别为9.54.9 μM;化合物EN 5*EN 289株病原细菌表现出中等抑制活性;对映异构体(±)-EN 6*中具有2R构型的(–)-EN 6*8株病原细菌表现出中等抑制活性;EN 8*EN 9*具有中等抑菌活性。从深海冷泉真菌A. insuetus SD-512的发酵产物中分离鉴定34个化合物(AI 1AI 34),包括9个新化合物(AI 1*–AI 3*AI 9*–AI 13*AI 34*,结构类型涵盖萜类、苯酚类、糖苷类、环肽类等。通过ECD计算以及糖的水解和衍生确定新化合物的绝对构型,并培养获得了二倍半萜类化合物的晶体结构。抗菌活性测试结果表明,差向异构体AI 2*AI 3*因构型不同表现出活性差异,其中具有6S构型的AI 3*8株致病细菌表现出中等抑制活性;化合物AI 9*AI 11*因取代基不同引起活性差异,活性强度由大到小依次为乙酰基取代、双键、羟基取代;化合物AI 12*AI 34*具有中等抑菌活性。从深海真菌A. penicillioides SD-311的发酵产物中分离鉴定19个化合物(AP 1AP 19),包括1个新化合物(AP 1*),结构类型为甾体类和生物碱类等。通过培养得到AP 1*的晶体结构,确定了新化合物的绝对构型。抗菌和抗病毒活性测试结果显示,化合物AP 1*具有微弱的抑菌活性;化合物AP 8具有H1N1流感病毒抑制活性。从深海冷泉真菌T. longibrachiatum SD-529的发酵产物中分离鉴定6个化合物(TL 1TL 6),结构类型包括倍半萜类、甾体类、甘油酯类等。

本论文对4株深海真菌的发酵条件进行了优化,并对真菌次级代谢产物进行了系统的分离鉴定与活性测试,丰富了深海真菌天然产物的研究内容,为深海微生物资源的开发利用提供了研究基础。

Other Abstract

Structurally unique and biologically active natural products have historically served as a rich source for drug discovery. The special environmental characteristics in deep-sea (>1000 m), such as high pressure, darkness, and extreme living conditions like cold seep and hydrothermal area, has played a major role in the evolution of microbial diversity. Strikingly, deep-sea-derived microorganisms survive under extreme environment, leading to prolific metabolisms and special biological diversity.

In this paper, 137 strains of fungi were isolated from the deep-sea sediments and benthos samples obtained in different sea areas and water depths. After screening and optimization of culture conditions, in conjunction with chemical and biological indicators, as well as pre-literature searching, four potential strains (Epicoccum nigrum SD-388, Aspergillus insuetus SD-512, A. penicillioides SD-311, and Trichoderma longibrachiatum SD-529) were finally selected for further chemical investigations. As a result, 91 compounds were isolated and identified, containing 21 new compounds and one new natural product. Besides, their antimicrobial, antiviral and cytotoxic activities were evaluated.

32 compounds (EN 1EN 32), including eleven new compounds (EN 1*–EN 10*) and one new natural product (EN 11#), were isolated and identified from the deep-sea-derived fungus E. nigrum SD-388. The relative and absolute configurations of new compounds were determined by RDC and RCSA, chiral HPLC, ECD calculation, as well as X-ray crystallography. Bioactivity assays showed that compounds EN 5* and EN 28 exhibited potent cytotoxic activity against Huh7.5 liver tumor cells with IC50 values of 9.5 and 4.9 μM, respectively, which were comparable to that of the positive control. Besides, EN 5* and EN 28 showed moderate inhibitory effect against nine strains of pathogenic bacteria. Moreover, (-)-EN 6* inhibited eight strains of pathogenic bacteria, indicating 2R configuration may be essential for the activity. Meanwhile, EN 8* and EN 9* displayed moderate antimicrobial activity. 34 compounds (AI 1AI 34), containing nine new compounds (AI 1*–AI 3*AI 9*–AI 13*AI 34*), were isolated and elucidated from the cold-seep-derived fungus A. insuetus SD-512. The absolute configurations of new compounds were determined by ECD calculation, acid hydrolysis and derivation and X-ray crystallography. Antibacterial assays showed that AI 3* inhibited eight pathogenic bacteria, implying 6S configuration may be important for the activity. Furthermore, caused by the substituents, AI 9*AI 11* displayed differences in anti-microbial activity, while the decreasing trend of the activity should be deduced as acetyl substitution > double bond > hydroxy-substitution. In addition, AI 12* and AI 34* exhibited moderate antibacterial activity. Nineteen compounds (AP 1AP 19), covering one new compound (AP 1*), were isolated and clarified from the deep-sea-derived fungus A. penicillioides SD-311. The absolute configuration of AP 1* was established by X-ray crystallography. Bioactivity assays showed that AP 1* displayed weak antibacterial activity. Meanwhile, AP 8 inhibited H1N1 influenza virus at the concentration of 30 μM, with the inhibition ratio of 61.7%. Six compounds (TL 1TL 6) were isolated and identified from the cold-seep-derived fungus T. longibrachiatum SD-529.

In summary, this study provided the cultural condition optimization, systematical isolation and identification of the secondary metabolites, and bioactivity assays for the metabolites of four deep-sea-derived fungal strains, which enriched the structural types of natural products from the deep-sea-derived fungi and offered foundation for further exploitation and utilization of deep-sea microbial resources.

Subject Area海洋科学
MOST Discipline Catalogue理学
Pages184
Language中文
Table of Contents

第一章 绪论... 1

1.1 引言... 1

1.2 深海曲霉属真菌次级代谢产物研究进展... 3

1.3 深海青霉属真菌次级代谢产物研究进展... 13

1.4 其他深海真菌次级代谢产物研究进展... 16

1.5 小结... 20

第二章 Epicoccum nigum SD-388次级代谢产物研究... 23

2.1 菌株SD-388化合物的结构解析... 23

2.1.1 新化合物的结构解析... 23

2.1.2 已知化合物的结构解析... 46

2.1.3 部分新化合物的晶体数据... 59

2.1.4 新化合物的物理性质... 61

2.2 生物活性评价... 63

2.2.1 抑菌活性... 63

2.2.3 肿瘤细胞毒活性... 64

2.3 实验部分... 65

2.3.1 实验仪器与试剂... 65

2.3.2 菌株SD-388的分离、鉴定与保藏... 66

2.3.3 菌株SD-388培养条件优化与规模发酵... 67

2.3.4 菌株SD-388发酵粗提物的分离与纯化... 68

第三章 Aspergillus insuetus SD-512次级代谢产物研究... 71

3.1 菌株SD-512化合物的结构解析... 71

3.1.1 新化合物的结构解析... 71

3.1.2 已知化合物的结构解析... 89

3.1.3 部分化合物的晶体数据... 102

3.1.4 部分新化合物的物理性质... 103

3.2 生物活性评价... 104

3.2.1 抑菌活性... 105

3.3 实验部分... 105

3.3.1 实验仪器与试剂... 105

3.3.2 菌株SD-512的分离、鉴定与保藏... 105

3.3.3 菌株SD-512培养条件优化与规模发酵... 106

3.3.4 菌株SD-512发酵粗提物的分离与纯化... 107

第四章 Aspergillus penicillioides SD-311次级代谢产物研究... 109

4.1 菌株SD-311化合物的结构解析... 109

4.1.1 新化合物的结构解析... 109

4.1.2 已知化合物的结构解析... 111

4.1.3 新化合物的晶体数据... 120

4.1.4 新化合物的物理性质... 120

4.2 生物活性评价... 121

4.2.1 抑菌活性... 121

4.2.2 抗病毒活性... 121

4.3 实验部分... 121

4.3.1 实验仪器与试剂... 121

4.3.2 菌株SD-311的分离、鉴定和保藏... 121

4.3.3 菌株SD-311培养条件优化与规模发酵... 122

4.3.4 菌株SD-311发酵粗提物的分离与纯化... 123

第五章 Trichoderma longibrachiatum SD-529次级代谢产物研究... 125

5.1 菌株SD-529化合物的结构解析... 125

5.1.1 已知化合物的结构解析... 125

5.2 实验部分... 128

5.2.1 实验仪器与试剂... 128

5.2.2 菌株SD-529的分离、鉴定和保藏... 128

5.2.3 菌株SD-529培养条件优化与规模发酵... 129

5.2.4 菌株SD-529发酵粗提物的分离与纯化... 130

第六章 结论与创新点... 131

6.1 ... 131

6.2 创新点... 132

参考文献... 133

附录... 145

部分化合物NMR图谱... 145

致谢... 157

作者简历... 159

攻读学位期间已发表和待发表的学术论文... 161

 

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/164689
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
迟路坪. 四株深海真菌次级代谢产物化学多样性及生物活性研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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