三株尖瓣海莲内生真菌抗肿瘤活性次级代谢产物及其作用机制研究

Mangrove endophytic fungi could produce secondary metabolites with novel structures and significant bioactivities, which has become a hotspot in the studies of marine drugs. The Bruguiera sexangula var. rhynchopetala as a traditional folk meidcinal resource is an endemic mangrove plant in Hainan province. The research group were primary studied on the bioactive metabolites of endophytic fungi isolated from the B. sexangula var. rhynchopetala for the first time. Five human tumor cells including human hepatoma (BEL-7402), TLC and HPLC were used to select the endophytic fungi isolated from B. sexangula var. rhynchopetalai. Three endophytic fungi which could be produce rich secondary metabolites with cytotoxic activity were selected as the preferred target strains. This project intends to foucs on these three target strains to carry out the following work: (1) Under the guidance of the cytotoxic activity, second metabolites were isolated from the fermentation broth of these endophytic fungi. (2) The secondary metabolites with significant cytotoxic activity were determined, and the structure-activity relationship were explored. (3) The action mechanism of active constituents on cell proliferation, cell apoptosis, andwere investigated. Based on these research, one or two drug candidate molecules with novel structure, good activity and selectivity will be screened out, and lay the foundation for creating the independent intellectual property rights of new antitumor agents.

红树内生真菌可产生结构新颖、活性独特的次级代谢产物，已成为目前海洋药物的研究热点。海南特有红树植物尖瓣海莲为民间传统药用资源，本课题组首次对来源于尖瓣海莲的内生真菌的活性次级代谢产物进行初步研究，利用人肝癌细胞 (BEL-7402)等 5 种典型抗肿瘤活性模型，结合 TLC 和 HPLC-DAD 检测筛选尖瓣海莲内生真菌，获得 3 株抗肿瘤活性显著且代谢产物丰富的内生真菌作为优选目标菌株。本项目拟以3株优选目标菌株为研究对象，开展如下工作：（1）以抗肿瘤活性为导向，从发酵产物中分离获得活性次级代谢产物；（2）确定具有显著抗肿瘤活性的化合物，并探讨构效关系；（3）阐明优良活性化合物对肿瘤细胞周期、细胞凋亡和信号传导等的作用机制。通过以上研究，有望优选出1-2个结构新颖、活性强、选择性好的药物候选分子，为研发具有自主知识产权的新型抗肿瘤药物奠定基础。

红树内生真菌次级代谢产物研究，为海洋药物的热点研究领域。本研究从海南特有药用红树植物尖瓣海莲中分离筛选内生真菌，在活性指导下对优选的目标菌株进行发酵培养，将从发酵产物中分离纯化的单体化合物进行抗肿瘤等活性筛选，为创新药物的研发提供候选因子。在尖瓣海莲的不同部位分离内生真菌共106株，采用现代色谱分离方法和波谱鉴定技术，从6株目标菌株的发酵产物中分离并鉴定了146个单体化合物，其中新化合物60个；为海洋微生物库增加内生真菌106株，为海洋微生物化合物库新增化合物样品146个，在本课题组建全了红树内生真菌的系统研究方法（包括内生真菌分离、种属鉴定、发酵、次级代谢产物的纯化、单体化合物的结构鉴定、抗菌活性测试、机制研究、表观遗传修饰等）。通过X-ray单晶衍射测试、ECD量化计算、化学衍生化等方法确定新化合物的绝对构型；活性筛选发现54个活性化合物，其中15个具有抗肿瘤活性、15个具有抗菌活性、11个具有α-葡萄糖苷酶抑制活性、13个具有抗虫或杀虫活性，对1个优良活性化合物进行抗肿瘤作用机制研究。在J. Nat. Prod., Mar. Drugs 等期刊发表论文 12 篇，申请国家发明专利9项（其中2项授权，4项公开）。本研究兼顾了红树资源的保护和开发利用，研究成果对海洋药物的研发提供物质基础和理论依据。