冬虫夏草定殖真菌中抗肿瘤与免疫抑制活性次级代谢产物研究

Cancer, transplant rejection and autoimmune diseases are a serious hazard to human health, there are still no effective treatments for these diseases, and new drugs are urgently needed. The Chinese caterpillar fungus (Ophiocordyceps sinensis ≡ Cordyceps sinensis; anamorph: Hirsutella sinensis) is a famous fungal traditional Chinese medicine with antitumor and immunomodulatory effects, and has been widely used as a tonic and/or medicine for hundreds of years in the Orient. However, due to its growing popularity, the natural fungus has been over-harvested to the extent that it is an endangered species. In its life span, many fungal species colonize its fruiting body and larvae, and we named them Cordyceps-colonizing fungi because of their ill-defined relationships with the host. This phenomenon led to postulation whether the claimed medical benefits of O. sinensis originated from itself or the metabolites produced by its colonizing fungi. Based on ecological considerations, we initiated chemical investigations of the fungal colonizers associated with O. sinensis collected from Sichuan and Tibet. Our initial work has resulted in the discovery of a variety of new fungal metabolites; many of them possess unique structural features and significant bioactivities. This proposed project will screen secondary metabolite library prepared from Cordyceps-colonizing fungi, and obtain antitumor and immunosuppressive components by application of the LC-UV and LC-MSn technology, antitumor and immunosuppressive screening models. In the mean time, by using secondary assays to validate the activity of obtained pure compounds and further explore the mechanism for their antitumor and immunosuppressive effects. The results will expand the source for compounds with antitumor and immunosuppressive properties, and provide lead compounds for antitumor and immunosuppressive drugs.

癌症、器官移植排斥及自身免疫性疾病都严重危害人类的健康，目前仍缺少有效的治疗药物，针对这些重大疾病的新药创制变得日益紧迫。冬虫夏草是我国青藏高原特有的名贵药材，具有抗肿瘤与免疫调节等功效，其资源稀少，价格昂贵，限制了它的广泛使用。冬虫夏草的子实体和虫体中存在大量的其它真菌，被称为"冬虫夏草定殖真菌"。定殖真菌与寄主具有相似的生存环境及适应机制，极可能产生与寄主类似的次级代谢产物作为一种化学防御机制。前期我们从该类真菌中发现了50多个结构新颖的活性次级代谢产物，本项目拟以定殖真菌为材料，通过多培养基发酵建立其代谢产物样品库，采用LC-UV和LC-MSn联用技术以及抗肿瘤与免疫抑制筛选模型，应用微量分离技术高效发现新结构活性化合物，并初步探索它们的作用机制。本研究将有助于拓展抗肿瘤及免疫抑制活性物质的来源，为抗肿瘤及免疫抑制药物的研发提供先导化合物。

冬虫夏草定殖及相关真菌的活性化学成分研究越来越成为热点研究。本项目选择冬虫夏草定殖及周围土壤菌株进行小样发酵，获得了320个代谢产物样品以及相应的HPLC分析的图谱库。根据活性筛选结果及指纹图谱分析，选择了6株真菌进行放大发酵，然后系统地进行化学分离纯化，共获得了73个化合物，其中34个为新结构，它们的结构类型包括聚酮类、萜类、肽类、细胞松弛素及糖苷类。对所分离化合物进行针对不同肿瘤细胞株的细胞毒活性与免疫抑制活性，以及抗氧化活性及评价。结果表明：化合物3对人宫颈癌细胞HeLa、人乳腺癌细胞MCF-7和人肝癌细胞HepG2具有较强的细胞毒活性(IC50分别为11.0、14.7、11.0 µM)；化合物6和7对人白血病细胞K562具有较强的细胞毒活性(IC50: 12.1, 13.0 μM)；化合物15和16对K562细胞具有弱的细胞毒活性(IC50: 53.7, 93.0 μg/mL)。Terpestacin被认为是一个从真菌中发现的，在抗肿瘤和AIDS的药物研发中极具潜力的先导分子，在我们的研究中发现了一株分离自冬虫夏草子实体，能高产terpestacin的多孔木霉Tolypocladium inflatum (编号: P086)，10g的该菌株大米发酵粗提取能获得50mg的terpestacin，研究还发现该化合物能抑制小鼠巨噬细胞释放NO的活性(IC50 = 10.3 μM)；其对巨噬细胞具有较弱的细胞毒活性(IC50 = 85.0 μM)。.上述研究结果发表SCI论文3篇，申请专利1项。