狭叶南五味子内生真菌的代谢产物和功能研究

More and more attention was paid to endophytic fungi which is a kind of resource looking for new bioactive substances and alternative medicinal plant,and has a potential application in synthetic drug biotransformed. Recently, series of terpenoids from medicinal plants of Schisandraceae which have important physiological activities and novel structures aroused people`s new interest. We have found the endophytic fungi which showed the antitumor activities and were fermented on the cultural media mainly consistent of the stems or the chemical component nigranoic acid of Kadsura angustifolia in our previous study. We also have found that nigranoic acid can be biodegraded to micranoic acid, a trace component from the host plant and biotransformed to new triterpenoids which increased the degree of oxidation by the endophytic fungi from Kadsura angustifolia.These indicated that the biosynthesis of terpenoids from the Schisandraceae family may be associated with the presence and involvement of the endophytic fungi. Therefore, this project is to select 3-5 endophytic fungi from Kadsura angustifolia which are capable of biotansforming nigranoic acid and biosynthesizing the host plant characteristic terpenoids or other novel antitumor metabolites through the high-througput screening, to ferment them respectively on the culture media which contain the host plant material/nigranoic acid or not, to isolate and identify their secondary metabolites from their pure cultures and co-cultures of these strains, to analyze the metabolic difference and relationship between the endophytic fungi and host plant, to explore the effect on the host plant secondary metabolism of the endophytic fungi, to design experiments to verify the metabolic relationship between the endophytic fungi and host plant, and to clarify the function of the endophytic fungi during the biosynthesis process of the host plant characteristic terpenoids. Finally, the project will also provide many novel metabolites for measuring their antitumor activities, and establish the foundation for biosynthesizing terpenoids from the medicinal plants of Schisandraceae and their endophytic fungi more effectively.

植物内生真菌是一类寻找新生物活性物质和替代药用植物的资源，且在生物转化合成药物中应用潜力大，越来越受重视。近年从五味子科植物中发现系列结构新奇高活性萜类化合物，引起人们新的兴趣。前期发现部分内生真菌具有抗癌活性、能发酵狭叶南五味子茎及其主要成分三萜酸nigranoic acid，三萜酸能被内生真菌降解为宿主微量成分micranoic acid或转化为氧化度增加的新三萜，说明五味子萜类的生成可能与内生真菌存在和参与有关。因而项目拟通过筛选实验，优选3-5株能转化三萜酸、抗癌活性稳定、可能产生新结构或宿主化学成分的内生真菌进行次生代谢化学研究，分离和鉴定菌株纯发酵、与三萜酸及与五味子材料共发酵代谢物，分析代谢物和宿主化学成分的异同，探讨菌株对五味子萜类成分生成的影响并实验验证，阐明菌株对五味子萜类成分生成的作用，为药物筛选提供丰富的材料，为调控五味子和内生真菌合成萜类化合物寻找依据和形成方法。

本项目从狭叶南五味子中分到次生代谢多样的426株内生真菌；发现40株菌能明显代谢转化宿主成分，鉴定其隶属11属；发现16株抗癌活性较稳内生菌；优选Penicillium ochrochloron SWUKD4.1850、Eupenicillium brefeldianum SWUKD2.0300和Umbelopsis dimorpha SWUKD3.1410进行了发酵和次生代谢研究，不重复分离鉴定代谢物94个，其中41个为内生菌源五味子三萜或内生菌转化宿主三萜修饰物，22个新化合物；完成了15-17、56-61和nigranoic acid的抗癌活性测试，发现15-17对HepG2和HL-60有微弱细胞毒活性，56-61比nigranoic acid对HepG2生物活性更强，构效研究表明氧化度提高是主要原因；分析了内生菌与宿主特征活性成分生成相关性及其作用，检测到U. dimorpha SWUKD3.1410和Trichoderma harzianum SWUKD3.1601纯发酵均产宿主成分nigranoic acid，发现U. dimorpha SWUKD3.1410和P. ochrochloron SWUKD4.1850能增加宿主代谢物多样性和丰富度，探讨了所获高氧化度三萜的生物合成途径；发现内生菌转化宿主成分时宿主植物环境起重要作用。.发现优势菌群青霉属和正青霉属等均大量生产对外源植物病原菌和内生菌具有明显抑菌作用的brefeldin A。此外，发现狭叶南五味子内生菌多数属公认的植物病原菌，包括大丽轮枝杆菌。为探讨大丽轮枝杆菌在宿主中的作用，总结了该菌与宿主相互作用研究，分析了该菌微菌核形成的转录组，发现Vdpf蛋白通过调控cAMP和G蛋白介导的信号通路调控该菌的表型和致病力。.项目执行期间，从其它材料（包括内生菌、植物Schisandra sphenanthera和Myricaria laxiflora）中也不重复分离获得次生代谢物33个，其中新化合物11个。.项目开展和完成证实内生菌具有合成宿主特征活性成分能力，能提高宿主代谢物多样性和丰富度，在宿主成分生成中起重要作用，为科学调控五味子和内生菌生物合成萜类化合物找到材料，从内生菌角度阐释药材品质及其地道性的形成做了有益的探索，对将来中药材的生产实践也具有重要的指导或借鉴意义。