药用植物内生真菌抗菌活性次生代谢产物研究

Endophytes are those microorganisms that inhabit interior of plants especially leaves, stems, roots show no apparent harm to host. Endophytic fungi from medicinal plants could be an outstanding source of antimicrobial secondary metabolites. There are abundant medicinal plant resources in the northwest of China.We have found that endophytes could be isolated from different antimicrobial plants belong to the different families and classes and grow under different ecological and geographical conditions in this region..Establishing the crude extract library of medicinal plants, fermentation broth and mycelium of endophytes, collecting HPLC-MS analysis results and screening antimicrobial data of the extract are premise and basis for carrying this project forward. When the potent fungus was confirmed by screening activity and testing chromatograph, the species will be identified according to their morphology, culture characteristics, and 16S rDNA or 18S rDNA sequences. With the purpose of finding out the optimum fermentation conditions of active strain which could be used to increase the biomass and the yield of active substances,optimum carbon and nitrogen source, initial pH, medium loading quantity, inoculation amount, rotary rate and fermentation temperature will be researched. Under the optimal conditions,the strains will be cultured using a large number of fermentation flasks.Bioassay-guided isolation of the extract of potent strains by TLC, column chromatography and HPLC, the single chemical entities with antimicrobial activities will be purified. The structures of those compounds will be elucidated by NMR, MS, UV, IR, X-ray, optical rotation (OR), circular dichroism (CD) techniques, computational data of OR and CD and chemical structure conversion. In order to increase the water/fat-solubility, absorption and therapeutic effect of bioactive compounds, the structural modification will be carried out with hydroxyl, amino, glycosyl, and PEG functional group conversions. The strucutre and activity relationship, toxicological, and pharmacological will also be studied. .It is beneficial to the research and development of endophytes from medicinal plants in the northwest of china, the protection of rare and endangered medicinal plant resources, and the maintenance of ecological balance. It is to lay a good foundation for filling the resource library of antimicrobial endophytes and founding new bioactive compounds.

内生菌广泛分布于药用植物中，可产生多种多样的次生代谢产物。因此本项目拟对西北地区丰富的药用植物内生菌进行研究，选择具有抗菌活性的药用植物组织作为内生菌分离材料，从所分离的内生菌中，筛选其提取物的抗菌活性，建立由植物提取物、内生菌发酵物组成的样品库和HPLC-MS及活性筛选数据库。对筛选出的广谱抗菌内生菌进行种属鉴定和发酵优化培养。通过活性跟踪分离得到单体化合物，运用光谱、波谱、单晶衍射技术、计算方法和结构转化等鉴定结构。采用大分子聚合物修饰等新方法进行结构修饰，研究单体化合物的活性及作用机制，探讨此类化合物的构效关系。通过本研究为创制新型抗菌药物奠定基础，促进西北地区药用植物内生菌资源的开发利用和珍稀濒危药用植物资源的保护。

细菌耐药性是21世纪全球关注的热点，它对人类生命健康所构成的威胁不亚于艾滋病、癌症和心血管疾病，因此低毒高效的新型抗生素日益受到人们的关注，植物内生菌尤其是药用植物内生菌的研究给这个问题的解决带来了光明。本课题组采集了80种生长于西北地区的药用植物，对其进行了内生真菌的分离纯化工作，得到445株内生真菌。在研究中亦采集了7个植物根系土壤、9个昆虫样品和一个气体样品，获得了87株真菌。对大部分真菌使用土豆、查氏、YES以及优化培养基进行液体恒温摇床发酵，21天后分别用甲醇超声提取菌丝体、大孔树脂吸附发酵液，甲醇洗脱获得发酵液和菌丝体提取物，对这些提取物进行了HPLC-MS、NMR及抗菌活性筛选，获得最优菌株，对筛选出的9株广谱抗菌内生菌进行了18s RNA测序种属鉴定和发酵优化培养。通过活性跟踪从9株真菌中分离得到160个单体化合物，运用光谱、波谱、单晶衍射、计算方法和结构转化等技术鉴定了它们的结构。另外进行了一种药用植物甘肃丹参次生代谢产物研究，获得32个单体化合物。发现的单体化合物中有16个新结构化合物，6个新骨架化合物。以上研究丰富了天然产物化合物库，同时新结构和新骨架化合物的发现为合成化学家提供了新的目标分子。对获得的大部分化合物进行了抑制枯草芽孢杆菌、巨大芽孢杆菌、大肠杆菌、产气荚膜杆菌、四联球菌以及耐甲氧西林金黄色葡萄球菌等革兰氏阳性及阴性菌的活性筛选，链霉素、四环素和氨苄西林等常用抗菌药品为阳性对照，获得了大量的活性优于阳性对照的化合物，这类化合物有望开发为抗菌药物。对部分结构进行了修饰研究，得到了一些修饰产物，探讨了所得单体化合物的构效关系。另外从来源于艾草的内生真菌中发现了与宿主植物结构类似的倍半萜类化合物，该菌株有望开发为药品，代替艾草入药，可节省药用植物资源，解决自然资源不足等问题。通过本研究为创制新型抗菌药物奠定了一定的物质基础，同时促进了西北地区药用植物内生菌资源的开发利用和珍稀濒危药用植物资源的保护。