药用菌蝉花化学成分及其免疫抑制活性研究

In previous studies, only T lymphocytes were thought to play important roles in the pathogenesis of autoimmune diseases and organ transplantation rejection. Recent studies have shown that B lymphocytes also play important roles in the pathogenesis of autoimmune diseases and organ transplantation rejection. The immunosuppressive drugs acting on T cells have dominated the market for a long time, however, some of them were reported to have serious side effects. To develop new types of immunosuppressive drugs with specific inhibition on B lymphocyte proliferation have broad market prospects. Fungal natural products are one of the important sources of lead compounds. The Traditional Chinese Medicine cicada flowers is an insect-fungus complex formed by the fungus Isaria cicadae infesting on the cicada larvae. During the formation of cicada flowers, fungi must produce immunosuppressive substances to fight against the insect immune system, so as to achieve the goal of successful infesting. Inspired by this process, a preliminary study on the liquid cultures of I. cicadae led to the isolation of five novel tricyclic macrolactones harboring an unusual enol-ether moiety, of which three selectively inhibited the proliferation of B cells. Based on these findings, this project aims to further study the secondary metabolites of the commercial materials of cicada flowers and three fermentation culture conditions of I. cicadae strains (rice, liquid and liquid with epigenetic modification), and then to find more molecules that selectively inhibit on the proliferation of B cells or T/B cells, which will lay a foundation for the research and development of new immunosuppressive agents.

过去认为仅T淋巴细胞在自身免疫性疾病的发病和器官移植后的排异反应中发挥重要作用，然而近年来研究表明B淋巴细胞在其中也扮演重要角色。作用于T细胞免疫抑制药物一直占据着主导地位，但具有严重副反应。开发新型的特异性抑制B细胞增殖的免疫抑制药物具有广阔的市场前景。真菌天然产物是药用先导化合物的重要来源之一。传统中药蝉花是由真菌蝉棒束孢感染蝉幼虫时形成的虫菌复合体。蝉花形成过程中，真菌必定产生具有免疫抑制活性的物质对抗昆虫免疫系统，达到成功入侵的目的。以此为启示，本项目前期从蝉花菌种的小试液体发酵中分离鉴定了5个含有罕见的“烯醚”片段的三环系大环内酯类化合物，其中3个能选择性抑制B细胞增殖。以此为基础，本项目拟深入研究蝉花药材和其菌种三种发酵培养方式（固体、液体和表观遗传修饰）的次生代谢产物多样性，从中寻找更多选择性抑制B细胞或同时抑制T、B细胞增殖的活性分子，为新型免疫抑制剂的研究和开发打下基础。

传统中药蝉花是由真菌蝉棒束孢感染蝉幼虫时形成的虫菌复合体。蝉花形成过程中，真菌必定产生活性的物质干扰宿主实现成功入侵。本项目以此为启示，系统性地研究了四种蝉花药用资源，包括液体、固体、醋酸钠为表观遗传修饰剂处理的发酵提取物、市售药材等的化学成分，共分离鉴定了化合物52个，包括新化合物16个（新骨架类型12个）。化合物的类型涉及聚酮、环二肽类、环缩酚肽类、酚苷、甾醇类、聚酮-非核糖体肽类、脂肪酸和非核糖体肽类等。在分离得到的化合物中，化合物1-6、8-11具有罕见的10/5-5环系骨架和烯醚官能团，化合物7具有罕见的10/7/8环系，化合物44具有稀有的10-6/5骨架，化合物45具有稀有的烯醚官能团和螺环结构。通过X-射线单晶衍射确定了化合物1和2的绝对构型。从生物合成角度考虑，这类结构蕴含一种全新的聚酮的多聚化后修饰机制，即某未知的酶催化了二分子虫草真菌特有的C10聚酮化合物通过Michael加成反应形成2-4位连接的二聚体。以家蚕为模型的拒食活性筛选研究发现，化合物1和2具有显著的拒食活性，其EC50值分别为65.4和57.0 μg/cm2。阳性对照药EC50为25.2 μg/cm2。然而，cephalosporolide E是化合物1和2的生源前体单体，其拒食活性却不明显。因此，本项目的开展首次对蝉花的次生代谢产物有了全面的认识，有利于蝉花药用资源的进一步开发和利用。本研究还解答了两个科学问题，一是天然产物的多聚现象确实有利于提高其生物活性，二是虫生真菌入侵昆虫宿主的机制还可能是通过分泌特定的次生代谢产物作用于昆虫宿主，使其食欲减弱而成功实现入侵。化合物1和2具备进一步研究的价值，一是对它们进行生物合成研究，有助于科学界认识一种全新的聚酮的生物合成机制，二是结合合成生物学手段实现其量产，可以进一步研究其作为杀虫绿色农药的潜力。