天麻提取物有效成分对灰树花发酵的代谢影响及机理研究

In our early research on the national natural science fund project, our groups used Grifola frondosa strain's broth to ferment Rhizoma gastrodiae extract, and the result showed that the prepared extracellular polysaccharides (EPS) of Grifola frondosa had an obvious and positive effect on not only inhibition of A549 lung cancer cell in human body, but also proliferation of spleen-lymphocyte in mice. At the same time, a preliminarily but accurately quantitative and qualitative analysis on ingredients of Rhizoma gastrodiae extract fermented by Grifola frondosa strain also showed that many more great changes had taken place, compared with the unfermented Rhizoma gastrodiae extract and pure fermented broth of Grifola frondosa. Accordingly, based on above researches, this project is to track and study on some effective ingredients of Rhizoma gastrodiae extract, such as p-hydroxybenzaldehyde (HBA), in the fermentation system of Grifola frondosa. A research on the interaction between HBA and Grifola frondosa strain is to be carried out, which will clarify HBA's effects on cell growth and EPS synthesis of Grifola frondosa, and the mechanism of their interaction; Additionally, a method of metabonomics will be applied throughout the fermentation process of Grifola frondosa to further clarify great changes occurring in the interaction between ingredients of Rhizoma gastrodiae extract and Grifola frondosa strain, and their mechanism of mutual transformation. In short, all studies above will provide a powerful theory foundation to develop new kinds of active preparations or compounds by the interaction between medicinal fungi and traditional Chinese medicine (TCM), such as Grifola frondosa's fermentation and transformation of Rhizoma gastrodiae. At the same time, this project will also play a positive role in promoting the research on metabolites of medicinal fungi.

在前期国家基金项目（31060272）研究中，本团队以灰树花菌株液态深层发酵转化天麻提取物，结果所制得的灰树花发酵胞外多糖具有显著地抑制人肺癌A549细胞和促进小鼠脾淋巴细胞增殖的活性效果。我们对灰树花菌发酵后的天麻培养液成分进行定量与定性分析，发现与天麻提取物和纯灰树花发酵液相比，其转化制剂在成分上发生明显的变化。基于以上发现，本项目拟在灰树花液体发酵体系中继续跟踪考察天麻提取物的有效成分（如对羟基苯甲醛等）；继续研究对羟基苯甲醛与灰树花发酵间的相互作用，意在阐明对羟基苯甲醛影响灰树花的细胞生长和胞外多糖的合成机理；以代谢组学的研究方法来进一步阐明灰树花发酵转化天麻的增效机理，以及天麻的有效成分与灰树花发酵间的相互作用变化。以其为利用药用真菌发酵中药，如灰树花发酵转化天麻等来开发新的活性制剂或获得新的活性化合物提供坚实的理论基础。本项目的实施对药用真菌代谢组学的研究将具有积极地推动作用。

中药具有多种活性成分，通过药用真菌发酵转化中药，其中药成分不仅能刺激微生物的菌体细胞生长，同时能参与到微生物的代谢过程之中，增加微生物次级代谢产物（例如胞外多糖）的产量和生物活性。本研究是基于中药提取物应用于微生物发酵过程中，其主要研究目的是制定科学实验方案，进一步探明天麻提取物的有效成分影响灰树花菌体生长和胞外多糖合成机理及其相互作用情况,为实现药用真菌发酵生产与中药生物转化的结合进行中药新药(或活性化合物)的开发提供理论基础。实验数据表明，从发酵动力学、代谢组学（转录组学、蛋白组学）等不同角度分析都表明天麻提取物及其有效成分的加入对灰树花深层发酵具有促进和增效作用。研究结果显示，质量浓度为7 g/L的天麻提取物和0.15g/L的对羟基苯甲醛均对灰树花菌体生长及胞外多糖合成的促进作用效果明显高于单一天麻素和对羟基苯甲醇的实验效果；实验发现，天麻提取物和天麻特征成分主要是通过调整EPS合成酶系酶活力促进灰树花EPS合成；基于大量的转录组测序实验数据支撑，本研究首次通过FPKM值法对灰树花菌丝体进行转录组测序，筛选得到181个差异表达基因，并首次提出天麻醇提物的添加会使糖酵解和丙酮酸代谢途径相关基因表达下调，导致胞外多糖的产量增加；本研究发现，添加天麻提取物会改变灰树花胞外多糖单糖组成的摩尔比，进而影响小鼠巨噬细胞（RAW264.7）活性，从而提高灰树花胞外多糖免疫活性。总之，本课题的研究结论和执行情况符合本项目研究目标和设计要求，最终将为中药新制剂研发开辟新的研究方向和路径，对药用真菌代谢组学研究有一定的推动作用。