中国科学院海洋研究所机构知识库

抗生素的出现，在很大程度上提高了人类的存活率，但随着抗生素的滥用，使得很多致病菌产生耐药性，对人类的健康以及经济的发展造成了不可逆的影响。目前，临床上亟需新型抗生素的发现，而海洋作为活性分子的储存宝库，拥有无限潜力。海洋微生物能够产生结构丰富、活性多样的初级和次级代谢产物，是天然药物的重要来源。本论文详细阐述了两株海洋真菌Talaromyces scorteus AS-242以及Trichoderma longibrachiatum EN-586产生的次级代谢产物类型及其抗菌活性。 

菌株T. scorteus AS-242是从麦哲伦海山角海葵内部的新鲜组织中分离出的一株踝节菌属真菌。综合利用减压硅胶柱层析、反相硅胶柱层析、正相硅胶柱层析、凝胶柱层析、薄层层析、高效液相色谱、重结晶等多种分离纯化方法，结合现代波谱学技术（如1D/2D NMR，HR-MS，IR，UV等）从其大米培养基发酵产物中共分离鉴定36个单体化合物（TS 1-TS 36），发现10个新化合物，结构类型包括2个环七肽（TS 1-TS 2），2个酸酐类化合物（TS 3-TS 4），4个二萜类衍生物（TS 6-TS 8和TS10），1个羊毛甾烷型三萜化合物（TS 17）以及1个缩酚酮酸酯类化合物（TS 25）。此外，从凹顶藻内生真菌长柄木霉Trichoderma longibrachiatum EN-586 的大米培养基发酵产物中共分离鉴定4个化合物，其中3个为新型糖苷类化合物（TM 1-TM 3）。

对分离获得的单体化合物进行了抗菌活性评价，包括3株人类致病菌、8株水产致病菌以及12株植物致病菌。测试结果表明，多个化合物对供试菌株具有不同程度的抑制活性，其中环多肽类化合物TS 1和TS 2对穗状弯孢菌具有显著的抑制作用，MIC值分别为2和1 µg/mL；酸酐类化合物TS 3-TS 5对层生镰刀菌表现出显著的抑制活性，其MIC值分别为1、1和2 µg/mL，其中化合物TS 3对蓝莓尖孢菌和胶孢炭疽病菌的MIC值也均为1 µg/mL；二萜类化合物TS 6-TS 16表现出较广谱的抑菌作用，MIC值介于1-64 µg/mL；此外，azaphilone类化合物TS 20-TS 24对蓝莓尖孢菌和柑橘炭疽病菌的MIC值介于4-32 µg/mL。

本文通过对两株海洋真菌的次级代谢产物进行系统研究，分离鉴定40个单体化合物，包括13个结构新颖的化合物，通过对23种致病菌的抗菌活性筛选与评价，发现一系列具有显著抑制活性的先导化合物，并且对构效关系进行了探讨。本论文研究成果在海洋药物，特别是对抗菌活性化合物的发现提供了物质基础和理论依据。

The emergence of antibiotics has greatly improved the survival rate of human beings. However, with the abuse of antibiotics, many pathogenic bacteria obtained the abilities of drug resistance, causing irreversible impacts on human health and economic development. At present, the advent of new antibiotics is urgently needed in clinical practice, and the ocean as a repository of bioactive molecules has boundless potential. Marine microorganisms can produce primary and secondary metabolites with complicated structures and diverse bioactivities, therefore they are one of dominant sources of natural medicine. In the thesis, the structure types and antimicrobial activities of secondary metabolites produced by two marine fungi Talaromyces scorteus AS-242 and Trichoderma longibrachiatum EN-586 were elucidated in detail.

The fungal strain T. scorteus AS-242 was obtained from the inner fresh tissue of a deep sea Cerianthus sp. sea anemone collected from the Strait of Magellan. The condensed organic crude extract of rice medium which AS-242 cultured was isolated with vacuum silica gel column chromatography, reverse silica gel column chromatography, normal silica gel column chromatography, sephadex column chromatography, thin layer chromatography, high performance liquid chromatography, recrystallization and other separation and purification methods, we also combined modern spectroscopic techniques (such as 1D/2D NMR, HR-MS, IR, UV and et al) to identify 36 monomer compounds (TS 1-TS 36). Among them, ten compounds as novel structural natural products were isolated, the types of chemical structures included 2 cycloheptapeptides (TS 1-TS 2), 2 anhydrides (TS 3-TS 4), and 4 diterpenoids (TS 6-TS 8 and TS 10), 1 lanosterane triterpenoid compound (TS 17) and one acetyl ketonate compound (TS 25). In addition, 3 novel glycosides (TM 1-TM 3) were isolated and identified from rice medium fermentation products of the endophytic fungus T. longibrachiatum EN-586, which was obtained from Laurencia obtusa.

The antibacterial and antifungal activities of the metabolites produced by T. scorteus AS-242 and T. longibrachiatum EN-586 were evaluated by tested strains including three strains of human pathogenic bacteria, eight strains of aquatic pathogenic bacteria, as well as twelve agricultural pathogenic fungi. The results showed that many compounds possessed different degrees of inhibitory activities against the tested strains. Among the assayed compounds, the cyclic polypeptides TS 1 and TS 2 had a significant inhibitory effect on Curvularia spicifera with MIC values of 2 and 1 µg/mL, respectively; anhydrides compounds TS 3-TS 5 generally showed potent inhibitory activity against Fusarium proliferatum, with MIC values of 1, 1 and 2 µg/mL, severally. Besides, the MIC values of TS 3 against F. oxysporum and Colletotrichum gloeosporioides were 1 µg/mL; diterpenoids compounds TS 6-TS 16 also generally exhibited a broad spectrum of antimicrobial activities, with MIC values ranging from 1 to 64 µg/mL; azaphilone analogues TS 20-TS 24 owned inhibitory effects on F. oxysporum and C. gloeosporioides Penz with MIC values ranging from 4 to 32 µg/mL.

 In this thesis, the systematic researches for the secondary metabolites of two marine fungi were performed. Eventually, forty monomer compounds were isolated and indentified, including thirteen novel natural compounds. There are a series of lead compounds which had potent antimicrobial activities were identified by screening and evaluating 23 pathogenic microorganisms, furthermore, the structure-activity relationship was discussed in this thesis. The research results provided material basis and scientific reference for the study of marine drugs, particularly for antibiotic compounds.

目  录

第1章  绪  论

1.1  引言

1.2  海洋动物来源的抗菌物质

1.2.1  软体动物

1.2.2  多孔动物

1.2.3  棘皮动物

1.2.4  刺胞动物

1.2.5  苔藓动物

1.2.6  脊索动物

1.2.7  节肢动物

1.3  海洋植物来源的抗菌物质

1.3.1  大型藻类

1.3.2  微藻

1.3.3  海草

1.4  海洋真菌来源的抗菌物质

1.4.1  海洋植物共生真菌

1.4.2  海洋动物共生真菌

1.4.3  沉积物来源的真菌

1.4.4  海水来源的真菌

1.5  海洋细菌来源的抗菌物质

1.6  小结

第2章  海洋真菌AS-242次级代谢产物及抗菌活性评价

2.1  单体化合物结构鉴定

2.1.1  新化合物结构鉴定

2.1.2  已知化合物结构鉴定

2.2  实验部分

2.2.1  实验仪器

2.2.2  材料与试剂

2.2.3  真菌Talaromyces scorteus AS-242菌种信息

2.2.4  化合物物理参数及波谱数据

2.2.5  单体化合物X-射线单晶衍射结果

2.3  单体化合物抗菌活性评价

2.3.1  抗细菌活性测试结果

2.3.2  抗真菌活性测试结果

第3章  海洋真菌EN-586次级代谢产物及抗菌活性评价

3.1  单体化合物结构鉴定

3.1.1  新化合物结构鉴定

3.1.2  已知化合物结构鉴定

3.2  实验部分

3.2.1  实验仪器、试剂与材料

3.2.2  真菌Trichoderma longibrachiatum EN-586菌株信息

3.2.3  化合物物理参数及波谱数据

3.2.4  单体化合物X-射线单晶衍射结果

3.3  单体化合物抗菌活性测试

3.3.1  抗菌活性结果

第4章 结语与创新点

4.1  结语

4.2  创新点

参考文献

附录：部分化合物谱图

致  谢

作者简历及攻读学位期间发表的学术论文与研究成果