银杏叶制剂等效成分群的发现与质量控制新模式研究

Traditional Chinese Medicines (TCMs) have long been postulated as a natural template of 'multidrug' therapy in clinical practice. It is one of the most characteristic and advantageous filed for potential independent innovation in China. The current quality standard for ginkgo herbal products (GHP) represents a high level for natural products. The exact composition of effective components, however, remains elusive due to the lack of adequate methodology. There are still some problems to be solved, such as the concentration ranges of flavonoid glycosides and terpene lactones, the proportions of the chemical constituents, and the rationality of the transcription factors between flavonoid glycosides and aglycones in quantitative analysis. In this project, we propose that there are bioactive equivalent combinatorial components (BECCs) that can represent the efficacy of GHP. The project is aimed at developing a novel quality control strategy for GHP through exploring the BECCs. To test the complexity of TCMs consisting of a mixture of constituents, multi-disciplinary methods and collaboration among chemists, biologists and computer scientists will be performed. Differing from traditional screening methods that separate GHP into many single compounds and test them one at a time, this work focuses on the whole GHP and its chemical compositions in their original proportions. The main research content of this work includes: 1) Characterizing complex constituents, especially original flavonoid glycosides and trace terpene lactones, in GHP and their metabolites in vivo with highly sensitive UPLC-QTOF MS methods. 2) Discovering BECCs from GHP and evaluating the efficacies of BECCs and original GHP by in vitro and in vivo bioassays using a bioactive equivalence oriented feedback screening strategy. 3) Proposing a novel quality control strategy for GHP based on chemical markers of BECCs. This project will make it possible to establish an international advanced technology for quality control of herbal medicines. The main characteristic of this project is that the multicomponent integrative effects are always considered in the whole screening and quality control process.

中药与天然药物为已公认的多成分复杂体系，具有多成分多靶点整合作用特点，现今主流的通过几个活性成分或指标成分的质量控制模式难以体现其整体疗效。本项目以中医药整体观为指导,以代表中药与天然药物质量控制世界先进水平的银杏叶制剂为研究载体，以等效成分群为切入点（即中药制剂中化学组成明确、结构清楚、比例关系确定且能够代表原制剂药效的一类化学成分群），开展中药质量控制模式创新研究，即在银杏叶制剂全成分、原有比例和临床起效浓度背景下，①全面解析银杏叶制剂的物质基础与体内过程，尤其关注原型黄酮苷和微量内酯类；②建立等效成分群反馈筛选策略，应用化学成分实时捕集整合系统，结合细胞、整体动物模型的药效评价及统计分析，解码能够表征银杏叶制剂的等效成分群，明确成分群的组成、比例关系及波动范围；③针对等效成分群，建立普适性、具有中药与天然药物特色的质量控制新模式。本研究对中药质量标准引领世界具有示范作用。

本项目在中医药整体观指导下，以银杏叶提取物为研究载体，进一步发展了“代谢处置-网络调控”的中药等效成分群发现新方法。首先，从化学成分出发，对银杏中的相关成分进行定向分离和富集，制备包括稀有内酯K、L在内的银杏相关化学对照品；建立银杏叶提取物化学成分体内外的表征方法，从银杏叶提取物中鉴定化合物61个，并对其中44个进行了定量；采用MCAO模型小鼠，对银杏叶提取物在病理状态下的血脑分布进行了研究，结果显示，内酯类和黄酮苷类成分在脑部和外周均有分布，黄酮苷类则代谢较快，内酯类含量较高代谢较慢；有机酸类在脑部和外周少量分布；黄酮苷元和花青素只在外周有少量分布。进一步建立了超临界流体-质谱分析方法，阐释了银杏内酯体内水解产物的药代动力学特征。在明确银杏叶提取物中各成分代谢规律的基础上，构建“代谢处置-网络调控”导向的等效筛选策略，从404个相关蛋白中，筛选出银杏叶提取物与缺血性脑卒中高度相关的9条通路，并依据主效性排序，发现潜在的等效成分群。经过药理学验证，确证由12个化合物构成的组合可作为银杏叶提取物治疗脑卒中的等效成分群，该组合包括8个黄酮、2个内酯、1个有机酸和1个花青素，约占银杏叶提取物总含量的16.5%。在此基础上，以等效成分群为质控指标，构建与药效关联的质量控制方法，为保证药物临床疗效稳定奠定基础。研究相关内容已发表SCI论文7篇，申请发明专利3项（授权2项），应邀作国际会议大会报告2次；培养博士研究生1名，硕士研究生3名。