马比木内生真菌及其抗肿瘤活性物质的研究

Malignant tumor is one of the primary diseases threatening human being. The huge demand for cancer chemotherapeutic drug is the momentous requirement of economic and social development of the state. It would be one of the important symbols for the advancement of science and teqnology of the state to overcome the cancer. The current chemotherapeutic drugs are however facing low efficacy, side effects and increased multi-resistance from the tumor. The discovery of new antitumor bioactive compounds has become necessary. The natural products from medicinal plants has attracted research focus for a long time and resulted these plants to become endangered. To discover new antitumor bioactive compounds from endophytic fungi of medicinal plants not only can explore new anti-tumor drug source, but also protect these plants and the ecological environment. The endophytic fungi are eukaryotic microorganisms parasitizing in plant tissue, often have same enzyme system and secondary metabolites with their host plants. Nothapodytes pittosporoides is a medicinal plant which has seldomly been studied and contain rich camptothecin. This study aim to use N. pittosporoides as materials to isolate endophytic fungi, screening and purifing anti-tumor bioactive compounds, and identifing the chemical structure of them. In our preliminary studies, we have isolated more than 200 endophytic fungal isolates from N.pittosporoides, and screened a compound with relatively high cytotoxicity against gastric cancer, lung cancer and leukemia via in vitro cytotoxicity model. This study is expected to discover new endophytic fungal strains producing new anti-tumor compounds from N. pittosporoides for the pharmaceutical industry.

恶性肿瘤是威胁人类生命的首要疾病之一。肿瘤化疗药物需求巨大，是我国经济和社会发展的重大需求。攻克癌症是国家科技进步和发展的重要标志之一。目前，化疗药物面临着疗效低、副作用大和肿瘤的多药耐药性增强等问题。发现和开发新药源是新型抗肿瘤药研究的重点。植物源天然产物的研发已导致植物资源匮乏。从植物内生真菌中既能开拓抗肿瘤新药源，又能保护珍稀濒危药用植物和生态环境。植物内生真菌能产寄主植物的次生代谢产物。本项目以研究少且富含抗瘤活性喜树碱及其它活性物的马比木为研究材料，分离内生真菌，进行抗肿瘤活性物质的筛选、分离纯化和化学结构鉴定。前期研究已开展了多种药用植物内生真菌及其抗癌活性物质的研究工作。并已分离得到马比木内生真菌200余株；已筛选到对胃癌、肺癌和白血病具有较高活性的内生真菌源化合物。项目的实施预期将筛选到一批产抗肿瘤活性物的菌株和化合物。

为了解我国马比木植物内生真菌的多样性及其真菌次级代谢产物的抗肿瘤活性，本项目通过采集贵州省贵阳、铜仁、遵义及湖南省吉首、湖北省兴山等地区的健康马比木样本15个，于24小时内对其内生真菌进行分离纯化，对所得菌株ITS rDNA序列进行测定分析后，采用分子系统学和形态学方法进行多样性分析。将纯化的内生真菌采用固体或液体发酵，萃取获得粗提物，筛选抗肿瘤活性菌株，将筛选得到的活性菌株进行大量发酵后，萃取浓缩得到的粗提物进行分离，纯化得到单体化合物，并对单体化合物进行抗肿瘤活性复筛。结果表明，项目共分离得到内生真菌1493株、1223个ITS rDNA序列，系统发育树分析表明其共隶属于4个目、23个科、35个属。从160株内生真菌的粗提物筛选得到具有抗肿瘤活性菌株10株，将活性最好的菌株（TRG1-5）进行大量发酵后分离获得单体化合物14个，其中TRG3、TRG4、TRG10具有良好的抗肿瘤细胞活性。但分离所得的单体量太少，不足以进行动物实验。以上结果表明，马比木内生真菌多样性丰富，部分菌株次级代谢产物具有抗肿瘤活性，从活性最好的菌株TRG1-5中分离得到抗肿瘤细胞活性单体化合物TRG3、TRG4和TRG10。