血小板电色谱法筛选典型活血化瘀中药活性成分研究

Anti-platelet aggregation is one of the main mechanisms for the anti-coagulation effect of HuoXueHuaYu traditional Chinese medicines (TCMs), development of platelet based open tubular capillary electrochromatography can be used for screening of platelet active compounds from HuoXueHuaYu TCMs. Based on the theory of cell membrane chromatography and capillary electrocrhomatography,to develop a novel drug screening method platelet based open tubular capillary electrochromatography (P-OT-CEC), which has affinity electrophoretic and cell membrane chromatographic dual properties. Combined with pharmacological, receptorotical and chromatographic theories, the adaptability, stability and sepecificity of the developed method will be intensively investigated by using positive and negative drugs. Then the developed method will be applied for the screening of platelet active compounds from Ligusticum chuanxiong,Salvia miltiorrhiza, Angelica sinensis, Caulis Spatholobi and Herba Leonuri. Furthermore, in vitro platelet aggregation test will be done to confirm the platelet activity of the compounds. The binding constants for the screened out compounds will be determined by peak shift assay, and the relationship between the binding constants and their activities will be evaluated.The present study develop a effective, specific and rapid method for screening of anti-platelet aggregative compounds from TCMs, which further develop the affinity caplillary electrophoresis and broadening the application of new drug screening method in the field of TCMs research.

抗血小板聚集是中药抗凝血作用的重要机制之一，创立以血小板为固定相的开管毛细管电色谱法能筛选活血化瘀中药中的血小板作用活性成分。本项目以细胞膜色谱和毛细管电色谱为理论基础，建立一种具有亲和毛细管电泳及细胞膜色谱双重作用机制的血小板-开管毛细管电色谱（P-OT-CEC）药物筛选新方法。结合药理学、受体学及色谱理论基础，采用阳性药及阴性药考察所建立方法的适应性、稳定性及特异性等。建立的P-OT-CEC 法应用于筛选川芎、丹参、当归、鸡血藤及益母草等中药的血小板作用活性成分，并进行体外抗凝血活性测试加以验证。同时采用峰漂移法测定活性成分与血小板的结合常数，评价结合常数与活性的相关性。通过本项目的研究建立一种高效、特异、快速的中药抗血小板聚集活性成分筛选新方法，进一步发展了亲和毛细管电泳，并拓展了药物筛选新方法在中药研究领域的应用。

本研究首先应用所建立的体外抗血小板聚集实验方法，系统、全面的评价和比较了31个传统活血化瘀中药和14个日常食用食物不同提取物的抗血小板聚集作用，由结果得出活性强的中药及食物。然后采用血小板亲和萃取法分别筛选延胡索（活性强的代表性中药）和柠檬（活性强的代表性食物）中的抗血小板聚集活性成分，结果从延胡索和柠檬中分别筛选得到5个和6个活性成分。进一步研究延胡索的抗血小板作用机制，考察了延胡索提取物对血小板的cGMP、P-选择素、5-HT和Ca2+释放的影响，结果显示延胡索显著的抗血小板聚集作用可能与升高cGMP表达及抑制Ca2+浓度、P-选择素和5-HT的释放有密切联系；同时采用双向电泳（2-DE）结合MDLAI-TOF-MS/MS技术分析中药延胡索粗提物作用于血小板后引起的蛋白质组变化，并构建血小板的蛋白质组图谱和数据库，推测出延胡索抗血小板聚集的两条可能作用途径。此外，创新性地提出并建立了一种测定血小板聚集率的毛细管电泳法，可在线评价药物的抗血小板聚集作用。最后成功建立了一种血小板涂覆开管毛细管电色谱方法（OT-ACEC）并用于评价小分子与血小板的亲和作用。通过本项目的研究，取得了一系列的研究成果，成功搭建了抗血小板聚集活性成分筛选、评价及作用机理研究的平台，为进一步的深入及拓展研究奠定扎实的基础。