蓬莪术醋制增强行气破血功效的炮制原理研究

In clinical practice of traditional Chinese medicine (TCM), Curcuma phaeocaulis Val. (Peng Ezhu) is recommended as a vital medicine of blood-breaking and disorder-eliminating, with qi-moving and blood-breaking being the main efficacy of it. Modern research shows that platelet aggregation is regarded as one of the important parts of Blood Stasis. And among all the possible pathways of platelet aggregation process, MAPK signal pathway is shown as a major one. It was found by our former researches that the efficacy of Qi-moving and Blood-breaking of Peng Ezhu could be enhanced after processed by vinegar. In order to verify the mechanism of the efficacy-enhancement of Peng Ezhu, the hypothesis is presented: the vinegar products of Peng Ezhu can strengthen the regulatory role of the MAPK signal pathway, thus inhibit the platelet aggregation involved by platelet activating factor (PAF) and prevent the formation of Blood Stasis, then reach to enhancement of the efficacy of Qi-moving and Blood-breaking..Base on the established efficacy research platform of zebrafish and start with the entry point of the regulatory effect of the vinegar products of Peng Ezhu which influences the platelet aggregation through the MAPK signal pathway, the main goal is to build the chromatography-activity model of Peng Ezhu and its vinegar products, then, to confirm the active components regulating MAPK pathway by in situ hybridization and to establish its dose-effect relationship through the application of reverse transcription polymerase chain reaction. Analyzing from three levels, “characteristics of the states of matter, molecular mechanism, relationship between property and efficacy”, the final objectives are to investigate into the key effecting step of how the vinegar products of Peng Ezhu regulate MAPK signal pathway and inhibit platelet aggregation. Thus, the scientific connotation, why Peng Ezhu should be processed by vinegar, was embodied.

蓬莪术被中医临床誉为破血消癥要药，其主要功效是行气破血。现代研究表明血小板聚集是“瘀血”形成的重要环节，MAPK信号通路是血小板聚集过程的主要通路之一。前期研究显示蓬莪术醋制后行气破血功效增强，为探明其醋制增效的炮制机理，提出假说：蓬莪术醋制后增强对MAPK信号通路的调控作用，阻断血小板活化因子PAF激活血小板聚集环节，防止“瘀血”形成，达到增强行气破血功效的作用。. 基于已建立的斑马鱼模式生物药物研究平台，以醋制蓬莪术调控MAPK信号通路抗血小板聚集为切入点，构建蓬莪术醋制前后抗血小板聚集的谱-效模型，以整体原位杂交技术明确其调控MAPK通路的活性成分，应用逆转录聚合酶链式反应技术建立其量效关系，从“物质基础-分子机制-性效关联”三个层次探索醋制蓬莪术调控MAPK通路以抗血小板聚集的关键环节，阐明蓬莪术醋制增强行气破血功效的炮制原理，丰富蓬莪术醋制的科学内涵。

蓬莪术是川产道地药材的代表性品种之一，醋制历史悠久，炮制始载于南北朝的《雷公炮炙论》。醋制后主入肝经血分，既能增强“破积消坚，去积聚癖块”之效，又可缓其峻猛药性，并成为《中国药典》2015年版一部莪术项下收载的炮制品。中医临床以醋莪术最为常用，而其炮制机理尚不完全清楚。本课题在多年研究蓬莪术的基础上，借助成功申报国家自然科学青年基金项目的科研平台，经过课题组成员两年的艰苦攻关，通过研究蓬莪术醋制前后化学成分的变化差异、蓬莪术醋制前后毒性作用的变化、活性成分的网络靶标等，进一步阐释蓬莪术醋制机理，证明传统醋制理论的科学性。.开展蓬莪术醋制前后化学成分（挥发油类成分、微量元素）的对比研究，发现了炮制前后蓬莪术物质基础的改变。醋制增效与挥发油类化合物种类和含量的变化密切相关。采用GC-MS技术并应用自动质谱退卷积定性系统(AMDIS) 和保留指数进行比较分析，醋制后新增γ-芹子烯和荜澄茄油烯等成分，萜品油烯、δ-榄香烯、吉马烯和γ-荜澄茄烯等成分消失。不同产地醋莪术挥发油中主要特征性成分β-榄香烯、吉马酮、新莪术二酮含量均增加，而莪术醇含量降低。用ICP-MS法测定不同产地莪术醋制前后微量元素含量的变化。提示炮制不仅改变了元素的含量，而且可能改变了元素的存在形态，经炮制后Zn、Ti 、B、Ni等微量必需元素的含量有所升高，Cu、V、Co的含量有所降低； Li 、As、Cd、Pb等潜在有毒元素的含量有所下降。.采用CCK-8法、Edu细胞增殖检测、qPCR荧光实时定量技术研究蓬莪术醋制前后抗肿瘤作用及其机制，表明炮制之后的药材对肿瘤细胞毒活性具有降低的趋势，能调控基因的表达水平，从分子层面阐释了炮制理论的科学性。首次运用模式生物斑马鱼胚胎、荧光血管斑马鱼（flk-GLP）研究蓬莪术的炮制机理，醋莪术对斑马鱼胚胎致畸、急性毒性及心脏毒性有减小趋势，并有明显抑制血管生成作用。进一步揭示蓬莪术孕妇禁用的科学内涵，阐释炮制前后蓬莪术“所增之效”及“所减之毒”，阐明蓬莪术醋制增强行气破血功效的炮制原理，取得了一定成果。已发表的相关创新性科研论文共有8篇，研究成果参加国内学术会议及交流3次，培养掌握相关研究知识和实验技能的学生7名。丰富了蓬莪术醋制的炮制内涵，为蓬莪术及其炮制品的合理应用打下坚实的基础，为今后川产道地药材蓬莪术的临床合理应用及综合开发利用提供科学依据。