九州虫草-黄芪双向固体发酵菌质抗非小细胞肺癌药效物质基础及分子机制研究

Bi-directional solid fermentation technology has made great progress, but there is no report about the bi-directional solid fermentation of Cordyceps kyushuensis and Chinese medicine at home and abroad. The applicant selected traditional Chinese medicine which is proved by clinical experiments have definite anticancer efficacy as drug matrix added Cordyceps kyushuensis culture for bidirectional solid fermentation medium in non-small cell lung cancer as the object, screening of active fermentation quality, found that Cordyceps kyushuensis- Astragalus fungus anti cancer activity was significantly enhanced. The application study for Cordyceps kyushuensis-Astragalus solid co fermentation bacteria, using the metabonomics method, carry out fungus extract analysis and comparative study of polarity, analyzed the variation of species, the number of active components of the fermentation process under the action of two-way, to explore the biosynthetic pathway and key enzymes of fermentation products may be the key matter. The application project of non-small cell lung cancer as the research object, the antitumor constituents of non cytotoxic focus on fermentation of fungus, trying to explore the molecular mechanism of fermentation medium induced apoptosis and autophagy of tumor cells, and has good effect for the development of anticancer drugs and fungi provide a theoretical basis for the experiment. It lays the foundation for the practical application of "two-way solid fermentation" based on biotransformation technology in Chinese medicine and the promotion of the innovation and development of holistic medicine and traditional Chinese medicine.

双向固体发酵技术取得了很多可喜的进展，但尚未见国内外有关九州虫草真菌与中药双向固体发酵的报道。申请者前期实验选取多味经临床实验证明有确切抗癌疗效的中药作为药性基质加入九州虫草培养基中进行双向固体发酵，以非小细胞肺癌为对象，筛选活性发酵菌质，发现九州虫草-黄芪菌质抗癌活性显著增强。申请课题拟针对九州虫草-黄芪固体共发酵菌质，采用代谢组学方法，开展菌质极性提取物成分分析和对比研究，分析双向作用下发酵过程中活性成分种类、数量的变化规律，探讨菌质关键发酵产物可能的生物合成途径及其关键作用酶。申请课题选择非小细胞肺癌作为研究对象，着眼于共发酵菌质的非细胞毒抗肿瘤活性成分，力图探究发酵菌质诱导肿瘤细胞凋亡和自噬的分子机理，为开发具有良好功效的抗癌真菌类药物提供理论与实验依据。为基于生物转化技术的“双向固体发酵”在中药学的实际应用和促进整体医学及中医药创新发展奠定基础。

课题将黄芪作为药性基质加入九州虫草培养基中进行双向固体发酵。随发酵时间的增加，虫草素和腺苷的含量先增加后降低，且在发酵第30天时含量最高，分别为17.31mg/g和0.94mg/g，是大米培养基（未添加黄芪）的8.6倍和2.0倍。黄酮类物质含量逐渐下降，皂苷类物质含量是先增加，并且在发酵第17d左右达到最高，之后均呈下降趋势。通过转录组测序分析，在基因水平上对发酵过程中活性物质含量变化动态进行深入研究。发现九州虫草的菌丝生长期，调节疏水蛋白合成的基因表达量高于其他三个时期，有利于菌丝生长。在转色期，与嘌呤代谢相关的基因表达高于其他三个时期，因此合成较多的腺苷和虫草素。在原基生长期，大量合成脂质、芳香族氨基酸等物质，同时细胞内的信息传递增加，促进九州虫草原基的形成，并促使子实体的发生。在子实体时期，核酸代谢、核糖体形成以及碳代谢加速，细胞大量增殖形成子实体和孢子。对发酵菌质提取分离，进行抗非小细胞肺癌活性筛选，发现乙酸乙酯部位作用最为明显，且随着浓度的增加，细胞抑制作用增强，有明显的剂量依赖关系。使用硅胶柱色谱等方法对乙酸乙酯部位进行了分离纯化，得到虫草素等21个单体化合物，并进行了光谱鉴定。采用MTT法对分离得到的单体化合物进行抗A549细胞活性研究，发现5种化合物具有较强的抑制A549肺癌细胞增殖活性，尤以虫草素效果最好，IC50值为2.27µg/mg。对虫草素处理后的H460和H1299细胞进行了流式细胞术和Western blot分析。流式结果表明，使用2009M虫草素处理24h后，1-1460和H1299的凋亡率可分别达到29.39%和36.55%；Western blot结果显示，虫草素可明显增强Caspase8、Caspase9、Caspase3和PARP的切割水平，并呈时间和浓度依赖效应，表明在人类NSCLC细胞中，虫草素可诱导Caspase途径依赖的细胞凋亡。实验结果为开发具有良好功效的抗癌菌物药物提供了理论与实验依据。