虫生真菌Hypocrea sp.新颖代谢产物的挖掘、评价与生物合成途径研究

Entomogenous fungi are a valuable source of bioactive natural products due to the unique environment they reside and their special relationships with the insects. In our previous study, we have identified an antitumor agent hypocrolide A from the crude extract of Hypocrea sp., an entomogenous fungus isolated from the Septobasidium-infected insect, Serrataspis sp. Hypocrolide A is the first hybrid metabolite originates from botrydiol and coumarin, possessing the unique 5/6/6/5/6/6 skeleton (Org. Lett. 2013). However, the HPLC-MS fingerprint of the crude extract showed the presence of other minor analogues that could not be identified due to sample limitations. In addition, the key hypothetical biosynthetic precursors were not isolated in the current work and proposed biosynthetic pathway of hypocrolide A could not be verified. The purpose of this study is to identify the minor analogues of hypocrolide A and the possible biosynthetic precursors through optimization of the fermentation condition and chemical investigations, evaluate their antitumor effects, and explore the mechanism of action for the identified antitumor compounds. Through HPLC-MS analysis and comparison of the fingerprints for the extracts of the insect Serrataspis sp. obtained before and after infection by Septobasidium sp., to explore the origin of coumarin, and to verify the proposed biosynthetic pathway for hypocrolide A based on the structures of the isolated analogues and plausible precursors. The results of this study will provide valuable information and serve as the basis for the discovery of novel bioactive metabolites and lead compounds, and shed light on biosynthesis of the complicated natural products from Hypocrea sp.

独特的生存环境与物种间互作关系，使虫生真菌产生新活性化合物的几率远高于普通真菌资源。我们前期从隔担菌侵染的盾蚧中分离了真菌Hypocrea sp.，获得了具有独特5/6/6/5/6/6新骨架的抗肿瘤分子hypocrolide A，并推测了可能的生物合成途径；该化合物为首个botrydiol-香豆素杂合分子。对粗提物的指纹分析发现尚有微量同系物有待于深入挖掘，而且推测的香豆素类前体的存在与生物合成途径也有待于证实。本研究拟通过优化发酵条件并应用高效微量分离技术，挖掘发酵产物中其它微量同系物及其前体，评价抑瘤活性并探索目标分子的作用机制；分析隔担菌侵染前后盾蚧提取物的化学指纹，探讨香豆素的可能来源；初步明确该类新骨架的生物合成途径。本研究的实施，将为新结构活性化合物与先导分子的发现、复杂天然产物的生物合成途径研究奠定基础，并为真菌−昆虫互作关系的研究与特境真菌资源的有效利用提供借鉴。

独特的生存环境与物种间互作关系，使虫生真菌产生新活性化合物的几率远高于普通真菌资源。从隔担菌侵染盾蚧中分离的相关真菌由于与宿主拟定的互利共生关系，引起我们的关注。前期从隔担菌侵染的盾蚧中分离了真菌Hypocrea sp.，获得了具有独特新骨架的抗肿瘤分子hypocrolide A，并推测了可能的生物合成途径。对粗提物的指纹分析发现尚有微量同系物有待于深入挖掘，而且推测的香豆素类前体的存在与生物合成途径也有待于证实。本研究通过多次条件摸索完成了hypocrolide A产生菌的放大发酵和hypocrolide A的大量制备；对多次放大发酵提取物进行分离，获得40余个不同类型次级代谢产物，其中10个新化合物，1个新天然产物；从产生菌中获得了botryane系列化合物生物合成前体化合物，推测完善了其可能的生物合成途径。对新化合物、新天然产物及1个单端孢霉烯类化合物（30）进行了体外活性评价，单端孢霉烯类化合物对多种肿瘤细胞均显示显著的细胞毒活性，是Hypocrea sp.中主要的细胞毒活性成分，其能特异性干扰（抑制）细胞NFκB通路，最终导致细胞死亡相关的NFκB通路激活；化合物30诱发的细胞毒性还伴随线粒体膜电位和细胞核膜通透性的改变以及细胞核损伤反应；细胞毒表征分析表明，HepG2细胞较Caco2细胞对化合物30的敏感性更高，推测该化合物可能存在代谢活化作用。相关化合物的作用机制研究仍在进行中。研究结果为新结构天然化合物的发现、复杂天然产物的生物合成途径研究奠定了基础，并为真菌−昆虫互作关系的研究与特境真菌资源的有效利用提供了借鉴。