无梗五加果抗血小板和抗血栓药效物质基础及作用机制研究

Thrombotic disease is a common cardio-cerebrovascular disease, which is a serious threat to human health. Therefore, discovering new resources of traditional Chinese medicine possessing the effect of the prevention and control of cardiovascular diseases has important scientific significance and broad application prospects. In a wide range of applications, Acanthopanax sessiliflorus fruits showed nontoxicity and food therapy effect of improving cardiovascular system function. On the basis of identification of the effective fraction of Acanthopanax sessiliflorus fruits and and its antiplatelet and antithrombotic activity, this project planned to separate the effective fraction by chromatographic separation stratagy such as ODS and Sephadex. The phenolic acids and triterpenoid saponins from the separation were prepared for systematic chemical separation. The chemical structure of monomeric compound from the separation was elucidated by the means of spectroscopy, assisted by the pharmacodynamics research to identify active constituents and effective components of Acanthopanax sessiliflorus fruits. On that basis, this project investigated the antiplatelet and antithrombotic activity of effective fractions, effective constituents and effective components, the three levels of pharmacodynamic basis, including the effect on the blood rheological properties, platelet function (adhesion, aggregation and release reaction), thrombosis and blood coagulation system. Through the determination of released particles contents by adhesion and aggregation of platelets stimulated by the outward factors and metabolites produced by platelet activation process, the mechanisms of antiplatelet and antithrombotic actions of the three levels of pharmacodynamic basis are clarified and the relationship of effective fractions, effective constituents and effective components were investigated. This project would deepen the cognition of the scientific meaning of efficacy of Acanthopanax sessiliflorus fruits, provide important scientific basis for developing new antithrombotic drugs with good curative effects and independent intellectual property rights, and provide new ideas for researching other antithrombotic traditional Chinese medicine.

血栓是一种常见的心脑血管疾病，挖掘具有防治心脑血管疾病疗效并且副作用较小的中药新资源存在着重要的科学意义和广阔的应用前景。无梗五加果在其广泛的应用中显示出无毒性和良好的抗血栓食疗效果。在已确定其其抗血小板及抗血栓活性及有效部位的基础上，本项目拟对其有效部位进行拆分，将得到的酚酸和三萜皂苷类成分进行系统的化学分离，并结合药效学研究确定无梗五加果的有效组分和有效成分。在此基础上，研究三个层次药效物质基础的抗血小板和抗血栓药理活性，包括对血液流变学特性、血小板功能、血栓形成和凝血系统的影响。通过测定血小板接受外界刺激，发生粘附、聚集的基础上所释放的颗粒内容物以及血小板激活过程中所产生的代谢物，来阐明其抗血小板和抗血栓的作用机制，探讨有效部位/有效组分/有效成分-功效-作用机制的相关性。本项目为研制出疗效好、具有自主知识产权的抗血栓新药提供重要科学依据，为其他抗血栓中药提供新的思路。

本项目在构建体外体内、外抗血小板聚集药效筛选药理模型基础上，发现了五加科植物无梗五加果抗血小板聚集的有效部位和有效组分，并采用大孔吸附树脂和聚酰胺等柱色谱实现了有效组分的拆分。发现无梗五加果70%乙醇提取物经大孔吸附树脂梯度洗脱后的30%洗脱层为抗血小板聚集的有效部位；其中三萜皂苷类及酚酸类组分为有效组分；以对血液流变性、血栓形成、血小板功能、抗凝血功能的影响为研究对象，对无梗五加果有效部位和有效组分的抗血小板聚集药效学进行研究，发现无梗五加果30%乙醇组和有效组分II组均能明显降低血瘀大鼠全血黏度，通过降低红细胞刚性指数IR和降低血沉及血沉方程K，增强红细胞的变形性，降低红细胞聚集性，从而表现出一定的改善血液循环作用；有效部位和有效组分均显著降低血栓重量，抑制血栓形成；有效部位和有效组分以及三萜类化合物22α-hydroxychiisanogenin，chiisanogenin有抗血小板聚集作用；无梗五加果对凝血系统作用不强，主要影响凝血过程第三阶段。对有效部位进行系统细致的化学分离，分离鉴定了42化学成分。并采用RP-HPLC法建立了7个有机酸类成分的质量控制方法，建立的方法简单灵敏、重现性好，并用该方法对不同产地的无梗五加药材进行了含量测定。在此基础上，对活性成分三萜皂苷的大鼠体内药代动力学进行了研究。采用UPLC-ESI-MS/MS法对给予无梗五加果提取物后大鼠体内4种三萜皂苷的多组分药动学进行了研究。这些结果都可以为无梗五加果在体内的药理学，毒理学，药物代谢研究奠定基础。