基于“菌-质”相应的杜仲药材品质评价及高品质形成机理研究

Eucommia bark is one of the unique characteristic medicine resources in China. Due to the shortage of the wild resourses and the variable quality evaluation standards on it. Eucommia bark is ragged in quality as a medicine material. It’s contribute to define the best genuine producing areas and promote the quality of the traditional Chinese medicine (TCM) by establishing the quality evaluation pattern and the identification method of genuine medicinal materials. The formation of the TCM with high quality are closely related to the endophytes in medical plants. Our preliminary study showed a large correlation between endophytic fungi and effective ingredients in Eucommia bark. Thus we put forward a hypothesis on “Endophyte-Quality Correspondence”(EQC), which we could use to characterize high quality characters by molecular markers of specific endophytes. This project intends to distinguish the high-quality ones from the others by determine and compare the Q-markers of Eucommia bark from different habitats. Then further to seek the distribution features of endophytes in high-quality Eucommia bark by analyze the diversity of endophytic bacteria and fungi in Eucommia bark with high throughput sequencing technology. Find the corresponding relation between specific endophyte and quality and explore a new pattern of quality evaluation based on EQC. Clarify the formation mechanism of high-quality Cortex Eucommiae through confirming the vital role endophyte play in this process. The results will be of value to the quality improvement of Cortex Eucommiae, and will make a difference to use the resourses sustainably and more efficiently.The new technique system established will lend some support to the quality evaluation of TCM, and expand way of thinking on identification of genuine medicinal materials.

杜仲皮为我国独有特色资源，野生资源匮乏，道地性界限模糊，药材品质参差不齐。建立杜仲药材品质评价及道地性辨识方法，有助于明确杜仲道地产区，提升药材品质。高品质药材形成与内生菌关系密切。申请人前期研究发现，杜仲药材有效成分与其干皮中内生菌密切相关，提出“菌-质”相应的假说，以杜仲特征内生菌分子标记表征杜仲药材品质。本项目拟通过不同产地杜仲药材质量标志物的确定及比较，筛选高品质杜仲药材；通过高通量测序技术分析不同产地杜仲鲜干皮中内生细菌及真菌种群多样性，挖掘内生菌在高品质杜仲中的分布特征，与质量标志物进行相关性分析，寻找“菌-质”相应关系，探索一种基于内生菌的中药材品质评价体系，明确特征菌在杜仲品质形成中的作用，初步阐明杜仲高品质道地药材形成机制。研究成果有助于提升杜仲药材品质，对杜仲资源的高效可持续利用具有重要意义。建立的技术体系可为中药材品质评价提供支撑，为道地药材评价和辨识提供新思路。

本研究探索建立一种基于内生菌的杜仲药材的品质评价及道地药材辨识方法，围绕药用植物杜仲“菌-质”相应关系，寻找特征内生菌分子标记表征杜仲药材品质开展工作。收集不同产地、不同季节杜仲皮和叶样品，从传统药性、药效、网络药理、化学成分体内代谢等现代药理作用，结合皮和叶不同入药部位的化学成分特征，确定了杜仲质量的化学标志物。基于质量标志物对4个产地的92份杜仲皮样品和70份杜仲叶样品进行了有效成分含量测定，不同产地杜仲皮药材桃叶珊瑚苷、京尼平苷酸的含量有显著性差异。采用高通量测序分析手段，基于细菌16s RNA和真菌ITS，对不同产地、不同季节杜仲皮和杜仲叶中内生细菌和内生真菌多样性进行分析，揭示了杜仲内生菌的产地特征、季节分布特征和不同组织部位定殖差异。发现陕西和贵州产地的杜仲皮内生菌群落组成较为相似。各产地共有菌属占比较小，不同产地杜仲皮内生菌群体组成差异明显。通过LEfSe比较分析，筛选出20个属特征细菌、14个属特征真菌作为不同产地杜仲皮特征内生菌。15个属特征细菌和3个属内生真菌作为不同产地杜仲叶特征内生菌。杜仲皮和杜仲叶内生菌组成分析发现，3个属内生细菌和7个属内生真菌可作为杜仲皮的特征内生菌，8个属的内生真菌可作为杜仲叶的特征内生菌。杜仲皮内生菌群与有效成分含量相关性分析表明，从丰度前20的内生菌中筛选出14个属的内生细菌、10个属的内生真菌与杜仲皮有效成分含量显著相关。结合产地和组织特异性、有效成分相关性，共同筛选出11个属的内生细菌和7个属内生真菌作为杜仲皮“菌-质”相应分子标记。已发表论文1篇，在投SCI论文2篇，授权发明专利1项，待出版专著1部。作学术报告1次，培养学生2名。本研究从内生菌角度建立中药品质评价及道地药材辨识方法，为中药材的品质评价提供了新思路。