豨莶草内生真菌抗肿瘤活性次生代谢产物的研究

The ability of produce pharmacologically important natural products previously only known in plant is occasionally also inherent to endophytic fungi. To the best of our knowledge, no study on the endophytic fungi from Siegesbeckia Puescens and also no antitumor activity of its fungi secondary metabolites have been reported. .In our previous work, the effect of S. Puescens on human cervix cancer Hela cell in vitro was studied. Our group isolated the cytotoxic active fungi from S. Puescens based on Hela cell in vitro, with 3 fungi being the best. To study the effects of the fungi extract on gene expression, cDNA microarray which contained 105 genes associated with tumor cell differentiation and proliferation was applied. Further study showed that the tumor cell apoptosis induced by the 3 fungi extracts associated with Wnt-3a and Fz-7, which is the key of Wnt/β-catenin signaling pathway..The objective of this project is to optimize the culture condition and isolat the active compounds from the extract of the 3 fungi with the molecular target of Wnt-3a and Fz-7. The structure of the active ones will be elucidated using one- and two-dimensional NMR techniques. Furthermore, series of derivatives will be prepared through the structural modification in order to investigate the possible structure - activity relationship, thus, the effective anti-tumor leading compounds which target the Wnt/β-catenin signaling pathway might be found.

从药用植物内生真菌次生代谢产物中寻找结构独特、活性显著的化合物已成为国内外研究的热点。本课题组前期分离、筛选了豨莶草(Siegesbeckia Puescens)中具强细胞毒活性的5株内生真菌，用基因芯片检测显示其中的3株真菌发酵提取物能选择性地抑制Wnt/β-catenin信号通路的启动子Wnt-3a和Fz-7，而Wnt/β-catenin信号通路与十几种高发性癌变密切相关。本项目利用内生真菌易培养、易控制的特点，优化内生真菌的发酵条件以富集更多的活性物质，选择发酵产物活性最强的培养条件进行目标菌株的大量发酵培养，在此基础上，以Wnt-3a和Fz-7为靶点，在活性指导下跟踪分离发酵物中诱导肿瘤细胞凋亡的单体成分，解析其结构，并通过结构改造制备系列衍生物探讨可能的结构-活性关系，以期发现作用显著、针对"Wnt/β-catenin信号通路"的抗肿瘤先导化合物。

Wnt/β-catenin信号通路与十几种高发性癌变的发生密切相关，寻找药物分子与该通路作用的靶位，是抗癌创新药物开发的热点。前期研究发现豨莶草(Siegesbeckia Puescens)内生真菌次生代谢产物中存在着调控该通路而抗肿瘤的活性成分。本项目以豨莶草内生真菌F. begoniae、F. sp.和P. lanosum为出发菌株，以下调Wnt-3a和Fz-7活性为考察指标，以“星点设计-效应面法”获得了发酵产物活性最强的培养条件；运用各种色谱分离手段，分离鉴定了活性化合物14个，其中环肽类化合物8个；此外，对豨莶草内生真菌A. brassicicola次生代谢产物进行了研究，鉴定了21个化合物，其中3个fusicoccane型新骨架、7个新化合物；从内生真菌C. capsici 发酵物中分离纯化了6个化合物，其中，化合物colletotrichumine A为新奇的 C16N2型骨架；本项目对所获得的化合物进行了抗肿瘤等方面的活性评价，发现了多种针对Wnt/β-catenin信号通路的抗肿瘤先导化合物，具有进一步研究和开发的价值。