三株大西洋深海真菌抗流感活性成分研究

Influenza virus are major human pathogens that cause the most deaths in respiratory diseases. However, there are no ideal anti-influenza drugs in clinic because of the high drug-resistant influenza virus. Thus, it is urgent to discover new anti-influenza drugs. Currently, to explore anti-influenza bioactive compounds from deep-sea fungi is a hot spot for discovery anti-influenza drugs. Previously, eighty six fungi were isolated from deep-sea sulphide ore deposits of the Atlantic. The cultured extracts of the eighty six fungi were subjected to anti-H1N1 bioassay and further to UPLC-DAD analysis. As a result, three fungi of Aspergillus sp.XT20、Graphostroma sp.XW42 and Acremonium sp.XW19 were found with potent bioactivities and diverse chemical components. Therefore, they were selected in this project for further investigation. In brief, fermentation optimization and large-scale cultivation will be carried out according to bioactivity and chemical analysis of the culture extracts. Then, anti-influenza bioactive compounds will be obtained from crude extracts using bio-guided isolation and their structures will be determined mainly by detailed analysis of NMR data and HRESIMS spectroscopic data. Finally, all compounds will be evaluated for their anti-influenza virus activities and structure-activity relationships will be preliminary discussed. This project will give the first report on chemical constitutions of the fungal genus Graphostroma. In addition to, it will be expected to provide drug precursors and model structures for the discovery and development marine-derived anti-influenza drugs.

流感是历史上死亡人数最多的呼吸道传染病，目前临床上尚无理想的抗流感药物，因此新型抗流感药物的研发迫在眉睫。从深海真菌中寻找结构奇特、机制新颖的活性成分是当前抗流感药物研发的重要方向。前期申请人对大西洋深海热液区硫化物来源的86株真菌进行了流感活性测试及UPLC-DAD化学分析，发现Aspergillus sp.XT20、Graphostroma sp.XW42和Acremonium sp.XW19等3株真菌代谢产物丰富且活性较好。因此本项目拟以该3株深海真菌为研究对象，以活性和化学为评价指标进行发酵条件优化及大规模培养；进而以活性为导向，采用现代色谱分离方法从发酵产物中分离抗流感活性成分；然后利用NMR等理化数据鉴定结构并对其进行构效关系探讨。通过本项目的研究，可填补Graphostroma属真菌化学研究空白，并获得一批抗流感活性化合物，有望为新型抗流感药物的研发提供海洋来源的先导化合物。

深海真菌由于其独特的生存环境以及产生丰富次级代谢产物的能力，近几年成为天然产物研究的热点领域。本项目以前期筛选获得的具有抗流感病毒活性的深海来源真菌为研究对象，通过培养条件优化、扩大规模发酵、次级代谢产物的分离纯化以及结构鉴定、活性评价，从中寻找抗流感病毒活性成分。基于此，我们对三株深海来源真菌Graphostroma sp. MCCC 3A00421、Aμspergillus versicolor MCCC 3A00324、Botryotinia fuckeliana 3A00494进行了系统的化学成分研究，共分离鉴定219个化合物，其中新化合物146个。对单体化合物进行抗流感病毒活性测试，结果发现来自A. versicolor MCCC 3A00324中的Asperterpene A2和 Asperterpene A4具有显著的抑制H1N1病毒活性，其IC50 值分别为13.5和18.2 M。为了拓展其生物活性，我们对其分别进行了抗炎、抗过敏以及抗肿瘤活性筛选，发现来自Graphostroma sp. MCCC 3A00421中的化合物6和17具有较强抗炎活性，其IC50值分别为17和14 μM，显著强于阳性对照aminoguanidine (IC50为23 μM)。化合物34具有较强的抗过敏反应，其IC50值为13.5 μM（氯雷他定，IC50 = 91.6 μM），机制研究发现通过抑制组胺的释放发挥抗过敏活性。来自B. fuckeliana 3A00494的化合物14具有对T24肿瘤细胞具有较强的抑制活性，其IC50值为2.5 μM。通过免疫印迹实验发现14通过DNA损伤应答来发挥抗肿瘤作用。该化合物目前正在与恩城制药公司合作研究开发抗肿瘤药物。此外，本项目还拓展系统研究了另外三株深海来源真菌P. chrysogenum MCCC 3A00292、P. granulatum MCCC 3A00475、P. commune MCCC 3A00940化学成分。从这三株深海来源真菌中共分离鉴定45个化合物，其中新化合物11个。通过活性筛选发现部分单体化合物具有较强的抗肿瘤活性。本项目的实施为从深海真菌来源的抗流感药物研发提供模式结构或药物前体。同时本项目拓展研究发现了一系列单体化合物具有显著的生物学活性，有望成为抗炎、抗过敏以及抗肿瘤先导化合物。