自然药学观指导的含药肝孵育液法用于麻杏石甘汤平喘较优药物体系解析初探

The Natural Pharmacy is an important guiding ideology for the research of ideal drug of TCM, core of the theory is to association analysis and find network system’s characteristics library of optimal drug system about TCM-Components-effects. In vitro pharmacological plays an important role in drug screening, study on cellular and molecular pharmacology. However, the serum pharmacological/pharmaceutical chemistry was poor reproducibility due to the interference of endogenous components, and the in vivo pharmacokinetic characteristics caused low content components, thus it is difficult to accurately grasp the relationship between the three. Although the intestinal absorption liquid containing medicine composition can solve the problem of serum, but lack of metabolic process. Based on the above considerations, we further propose a new method, the method of liver incubated buffer containing medicine. That is prepare liquid containing medicine composition in vitro pharmacokinetic process and use it at in vitro pharmacological study, the method can directly echo PK-PD process. In this study, formula of Maxingshigan Decoction which’s designed by mixture homogeneous design was studied as the object, the method of liver incubated buffer containing medicine, technology of HCS and RT-PCR, Pearson analysis were used to construct antiasthmatic effective matrix from multi-level. Technology of URLC-QTOF/MS and partial least squares were used to construct component matrix of antiasthmatic. Principal component analysis and correlation analysis were used to find each layer local area network system, thus, to reveal the essence and law of the compatibility of the optimal drug system. Through the study, we hope to explore a new method in vitro pharmacological research more suitable for TCM, deepen the understanding of the essence and connotation of the TCM drug system, and provide demonstration for the research of the ideal drug.

自然药学观是指导中药理想药物研究的重要学说，核心是关联分析发现最佳药物体系的中药-成分-效应表征库。体外药理研究在药物活性筛选、细胞分子药理研究起重要作用，但血清药理因内源性成分干扰而重现性差，且体内药代特征而成分含量低，难以准确地把握三者间关系；含药肠吸收液无代谢过程故缺乏活性代谢物。基于上述分析，我们提出“含药肝孵育液”的新方法：制备经离体肠吸收并肝细胞孵育的含药体系用于体外药理研究，可解决上述问题并将PK-PD直接呼应起来。本研究以麻杏石甘汤混料均匀设计配方为对象，采用含药肝孵育液法、Pearson分析，多水平构建疗喘的效应矩阵；联合URLC-QTOF/MS检测、偏最小二乘回归分析，构建疗喘的成分矩阵；经关联分析两矩阵间关系、发现各局域网络体系表征库，初步揭示较优药物体系的配伍规律。通过研究探寻更适合中药体外药理研究的方法，以深化认识中药药物体系的本质及内涵，为理想药物研究提供示范。

体外实验是药理学研究不可或缺的部分，目前中药常用的含药血清法、含药脑脊液法和含药肠吸收液法等各有其优缺点。含药肠吸收液法虽作为含药血清法的一种替代，但其因无代谢过程故缺乏体内可能的活性代谢物，据此，课题组提出了一种新方法--含药肝孵育液法，以期为更科学的中药体外药理研究方法的探索提供借鉴。 . 本项目拟在自然药学观的指导下，以麻杏石甘汤混料均匀设计配方的水煎液和含药肝孵育液为对象，液质联用检测、PLSR分析，初步探明其药效物质基础；从整体（水煎液）、组织和细胞水平（含药肝孵育液）探讨复方减轻气道上皮损伤、对EGFR/PI3K 信号通路的影响，初步阐释其作用机理；经关联分析，探讨配伍-成分-效应的联动变化关系，初步分析各局域网络体系表征库、揭示最优药物体系配伍的本质与规律。. 本项目建立了复方四药味共136种成分的目标数据库及53种成分的有效化合物库；建立了水煎液、含药肝孵育液、含药血清的RRLC-QTOF/MS检测、PLSR分析方法，得出了复方8种、含药肝孵育液（无异甘草素）和含药血清（无甲基麻黄碱）各7种主要差异性成分；含药肝孵育液有成分含量较高、稳定性更好的优点。复方有良好的平喘作用，该效应与其延长引喘潜伏期/松驰支气管平滑肌，调节上皮细胞EGFR表达、减少炎症因子释放，从而减轻气道上皮细胞损伤等密切相关；含药肝孵育液对上皮细胞损伤模型PI3K和AKT蛋白表达的作用趋势与含药血清一致，但结果重现性更好。分析了上述差异性成分与各效应指标的交互关系，其中甘草素对各效应指标的影响最为明显，而麻黄碱次之应是麻黄的主要有效成分；复方延长引喘潜伏期的最优配比为N11（麻：杏：甘：石=22.9：1.6：28.3：47.2），其对多数效应指标影响的活性比率均较高而并非最高。. 本研究初步验证了含药肝孵育液可作为更可靠的体外药理学研究方法用于中药非神经系统的各水平研究，初步探明了麻杏石甘汤疗喘的物质基础、EGFR/ PI3K作用机理及“配伍-成分-效应”之间的变化，深化了对该复方的现代阐释。