诱导子对药用真菌火木层孔菌次生代谢的调节作用研究

In our previous studies, a series of phelligridins with highly unsaturated novel structure were isolated from the fruiting body of medicinal fungus Phellinus igniarius. Some of the components have antitumor and antioxidant activities. But it is very difficult to continue the further research because the content of phelligridins in the cultures of Phellinus igniarius is deeply low. It was reported that the elicitors can significantly affect the secondary metabolism of the plant. But few studies of the elicitors on the the secondary metabolites of medicinal fungi have been published. Therefore, this project investigated the influence of elicitors on the secondary metabolities by firstly isolating the mycorrhiza-associated fungi and mycorrhiza-bacteria from the fruiting body of Phellinus igniarius as biotic elicitors and the precursors substance (Hispidin and phenylalanine) of phelligridins and inhibitor (sodium benzoate) as abiotic elicitors. The above elicitors were added into the cultures of Phellinus igniarius to screen the optimum conditions which can enhance the productivity of phelligridins and their structural analogues, or other special bioactive components. The optimum co-culture conditions and determination system for elicitors and Phellinus igniarius were established. Then, preparative scale culture of the antitumor active chemicals such as phelligridins was carryied out.And then,the system isolation,elucidation and activity screening of the cultures were operated to lay the base for the biological preparation of the phelligridins with anti-tumor activity.

课题前期从药用真菌火木层孔菌(Phellinus igniarius)的子实体桑黄中得到一系列高度不饱和的新骨架类型化合物- - 桑黄素类成分，具有抗肿瘤及抗氧化活性；而火木层孔菌常规液体培养的代谢产物中桑黄素类成分含量极低，给后续研究带来困难。文献表明诱导子能显著影响植物次生代谢，但对药用真菌次生代谢影响方面的研究较少。因此，本项目选择从野生桑黄子实体中分离得到的菌根伴生真菌和细菌作为生物诱导子，桑黄素类成分可能的前体物质(Hispidin和苯丙氨酸)及抑制剂(苯甲酸钠)作为非生物诱导子，并在火木层孔菌液体培养过程中加入以上诱导子，考察诱导子对次生代谢产物的影响，筛选出提高桑黄素及其结构类似物的含量或产生特殊的生物活性成分的培养条件；建立诱导子与火木层孔菌最佳共培养和检测体系，进行桑黄素类抗肿瘤活性成分的放大培养、系统分离、结构鉴定和活性筛选，为桑黄素类抗肿瘤活性成分的生物制备奠定基础。

本项目从生物诱导到物理化学诱导对火木层孔菌的代谢产物的影响的考察发现：通过高效液相检测和液质连用技术确定桑黄内生真菌SHM-2能够诱导火木层孔菌产生桑黄素G；SHTD-4明显提高火木层孔菌代谢产物中桑黄素类含量；通过亚硝酸诱导得到关键诱导菌株PNCM5-1，其代谢产物中发现关键性成分桑黄素D，并比较了诱导菌株PNCM5-1和原始菌株的显微形态特征和色素类代谢产物累积影响；从硫酸二乙酯诱导得到菌株PIDES-1，比较诱导菌株和原始菌株物化性质和对PC12细胞抗氧化作用的影响，发现诱导菌株在释放色素类成分和抗氧化活性方面明显优于原始菌株；对诱导菌株PNCM5-1的代谢产物进行分离得到43个单体化合物，其中三个新化合物；通过抑菌和抗肿瘤活性筛选发现：菌株SHM-2诱导火木层孔菌的代谢液对肝癌HEPG-2和胰腺癌PANC-1细胞株具有抑制活性。