基于中药炮制与复方配伍理论结合的四逆散“疏肝解郁”作用机制研究

Chinese medicine processing and Chinese medicinal formula compatibility are two outstanding characteristics in the clinical applications of Chinese medicine. However, current studies most often focus on compatibility mechanism or processing mechanism alone with very few combining them together organically, and the reports discussing Chinese medicine processing mechanism in Chinese medicinal formula have been rarely seen. As a result, the selection of the processed products of Chinese medicine in Chinese medicinal formula has to rely on the experience of clinicians without sufficient basis of scientific theories. Sinisan has been regarded as an effective anti-depression prescription according to the traditional Chinese medicine theories of six channels and depression. However, the employment of processed Bupleuri Radix and processed Paeoniae Radix Alba in Sinisan is quite confused, which largely affects its curative effects in clinic. In order to solve this embarrassing situation, we creationally put forward three progressive levels - single herb, herb pair, and complicated Chinese herbal formula - based on the representative drug Bupleuri Radix, Bupleuri Radix - Paeoniae Radix Alba herb-pair, and Sinisan, and adopted the research idea of “chemical material basis - endogenous metabolite - physiological and biochemical index”. Starting from the overall evaluation of the in vitro and in vivo and the molecular biological level, systematical investigations were carried out and compared on the changes of chemical constituents, pharmacological effects, and pharmacokinetics of Bupleuri Radix and Paeoniae Radix Alba, compatibility, and Sinisan before and after processing to clarify pharmacodynamic material basis and herein reveal scientific connotation of processing compatibility. We are looking forward to seek out the common mechanism of processing compatibility so as to provide the scientific theory basis for safe clinical applications of Chinese medicine and rational drug processing in Chinese herbal formula through the research of this project.

中药炮制与复方配伍是中医临床用药的两大特色。目前已有的研究大多单一专注于配伍机制或炮制机理，很少将二者有机结合起来，涉及在复方中探讨中药炮制作用机制的报道更是罕见，这直接导致复方中中药炮制品种的选择缺乏科学理论依据而只能依赖于临床医师的经验。根据六经学说和郁症学说，四逆散为中医治疗抑郁症良方，但方中柴胡和白芍的炮制品使用极为混乱，严重影响其疗效。鉴于此，本项目立足于单味药、药对、复杂复方三个层次，以代表性药物柴胡、柴胡-白芍药对、四逆散为例，采用“化学物质基础-内源性代谢产物-生理生化指标”的研究思路，从体内外整体评价和分子生物水平入手，系统研究比较三个层次下柴胡和白芍炮制前后及配伍和组方中化学成分、药效作用和体内动态过程的变化，阐明其药效物质基础，揭示炮制配伍的科学内涵。通过本项目的研究，旨在探索炮制配伍研究的共性规律，为指导中医临床安全用药及复方中饮片合理炮制提供科学理论依据。

本课题立足于“单味药-药对-复方”三种不同的药物组成形式，以代表性方剂四逆散为例，从体内外整体评价和分子生物学水平入手，系统考察了柴胡和白芍醋炙前后及配伍和组方中化学成分、药理作用和体内动态过程的变化。通过UHPLC-Q-TOF/MS方法并结合多元统计分析策略，共鉴定出柴胡和白芍醋炙前后的“单味药、药对及配伍组方四逆散”中122种成分。结果表明，中药炮制及配伍会使中药化学成分发生改变。通过体内外药效实验系统考察了炮制与配伍对柴胡和白芍抗抑郁作用的影响。结果表明，含醋炙柴胡和醋炙白芍的四逆散抗抑郁效果更为显著，且与单味药和药对之间存在显著性差异。采用快速、灵敏的UHPLC-QqQ-MS/MS方法同时定量评价四逆散中“君臣佐使”药的14种主要成分在抑郁大鼠血浆中的含量，并应用于柴胡和白芍醋炙前后的“单味药-药对-复方”三种不同药物组成形式的整体药动学比较研究。结果显示，在柴胡和白芍醋炙前后的“单味药-药对-复方”三种不同药物组成形式中，部分柴胡皂苷及白芍总苷类成分的Cmax、AUC0-24、AUC0-∞、t1/2和MRT0-24均存在显著差异。同时，含醋炙柴胡和醋炙白芍的四逆散组中的甘草苷、甘草酸、甘草素、橙皮苷及橙皮素的AUC0-24、AUC0-∞和Cmax均显著高于四逆散组（P < 0.05，P < 0.01）。通过代谢组学技术，筛选出16种与抑郁症发病相关的潜在生物标志物，并结合代谢通路数据库，探讨了炮制与配伍前后四逆散抗抑郁作用机制的差异。采用Western Blot及PCR技术研究四逆散对BDNF-ERK-CREB通路的调控。结果表明，四逆散及含醋炙柴胡和醋炙白芍的四逆散的抗抑郁作用可能是通过调控BDNF-ERK-CREB通路实现的，且含醋炙柴胡和醋炙白芍的四逆散的上调作用更强。