药用植物蕲艾内生真菌代谢产物分子多样性与抗海洋病害菌活性研究

Aquaculture is a mega-industry in China. With the expansion of the scale and intensive degree of aquaculture, the incidence of diseases and the degree of impacts are increasing very quickly, and there is urgent need to discover new bioactive substances to against the marine pathogens. It is likely easier to find active substances for anti-marine pathogens from the species that genetically far from marine organisms. Qiai (Artemisia argyi), growing in Qichun County of Hubei Province, is a famous Chinese herbs with long history for the treatment of various diseases. In the preliminary screening we found that this mugwort is rich in endophytic fungi, with two species (QA-3 and QA-8) belonging to the genus Trichoderma have prolific metabolites and strong activity against marine pathogens, and some polyketides having ketal substructures with potent activity against auqticpathogens and some diterpenoids with rare skeleton have been identified from the fungal strain QA-3. Further works revealed that these two strains (QA-3 and QA-8) could produce various metabolites in different culturing conditions. Based on these work, the current project will focus on mining the molecular diversity of the secondary metabolites of these two strains by using of a kind of cultivation on environment-stressed culture conditions. By constructing a research mode with combination of Qiai-endophyte and stress-induced biological activity, and by employing modern chromatographic and spectral methods, a sensitive and efficient separation/purification and identification system to obtain various metabolites with new structure and strong activity will be approached. The molecular diversity of the metabolites and structure–activity relationships will be disclosed. The project will provide scientific basis for the sustainable utilization of endophytic resources in Chinese medicinal plants.

中国是水产养殖大国，随着养殖规模的扩大和集约化程度的提高，病害的发生频率和影响程度越来越大，迫切需要研发新的抗海洋病害菌活性物质。从亲缘关系较远的物种中更有可能发现作用显著的活性分子。蕲艾是湖北蕲春著名道地药材，我们前期研究发现其富含内生真菌，经筛选发现两种木霉属内生真菌QA-3和QA-8的代谢产物丰富且具有显著的抗海洋病害菌活性，进一步研究发现在不同培养条件下QA-3和QA-8能产生不同类型的代谢产物,已从中发现活性显著的含缩酮结构特征的聚酮和结构新颖的二萜类化合物。拟在此基础上采取环境胁迫等手段深入挖掘上述内生真菌代谢产物的分子多样性，构建蕲艾-内生真菌-（人工诱导）抗菌活性相结合的研究模式，建立蕲艾内生真菌代谢产物高效灵敏的分离检测体系，发现结构新颖、活性显著的代谢产物，探讨蕲艾内生真菌次生代谢的分子多样性与活性并总结、获得构效关系规律，为我国药用植物内生真菌资源的利用提供科学依据。

本项目主要开展了药用植物蕲艾内生真菌拟康宁木霉QA-3（Trichoderma koningiopsis QA-3）和绿木霉QA-8（Trichoderma virens QA-8）的代谢产物研究和抗水产病害菌活性研究。通过优化培养和规模发酵，采用现代色谱分离技术从菌株QA-3和QA-8的发酵产物中分离纯化单体化合物81个，综合运用多种波谱技术、X-射线晶体衍射技术、ECD计算等方法鉴定了所有化合物的分子结构，发现新化合物38个（出新率46.9%），其中聚酮类新化合物15个、杜松烷类倍半萜新化合物12个、胡萝卜烷类倍半萜新化合物8个。对所有新化合物和部分已知化合物开展了抗水产病害菌活性筛选，发现18个化合物对多种水产病害菌如迟缓爱德华氏菌(Edwardsiella tarda)和鳗弧菌(Vibrio anguillarum)等有较强抑制活性，其中强活性化合物10个，MIC值在 1-2 µg/mL以下，优于阳性对照或与阳性对照相当。对蕲艾及其内生真菌挥发性成分快速分析的样品前处理新方法开展了研究探索。项目实施期间在Journal of Natural Products, Bioorganic Chemistry等期刊发表SCI论文4篇、会议论文1篇；获发明专利授权1项、实用新型专利授权2项；培养毕业硕士3人，协助指导培养博士1人、博士后1人。研究结果丰富了天然产物的分子多样性，为蕲艾内生真菌的有效利用提供了科学依据。