基于血小板P2Y12受体色谱技术探究典型活血化瘀中药药效物质基础

Screening, identification and evaluation of the effective substances in traditional Chinese medicines (TCMs) are the crucial problems in the modernization of TCMs, and there is an urgent need to establish screening systems which are suitable for the identification of active components in TCMs. In this study, aim at typical TCMs for promoting circulation and dissipating stasis like Salvia miltiorrhiza, safflower and Ligusticum Chuanxiong, we focus on platelet P2Y12 receptor, which is the key target of anti-thrombosis drugs. The receptor could be obtained with the affinity chromatography, and immobilized on the surface of functional grapheme oxide-macroporous silica gel uniformly and covalently with the mode of moderate chemical coupled reaction. The stationary phase would be characterized with the bond quantity of receptor, bioactivity and retention mechanism and the online-coupling system of platelet P2Y12 receptor chromatography and SPE/NMR/Q-TOF-MS would be established. This system would be applied in the quick screening, separation and identification of active components which could react with platelet P2Y12 receptor in the TCMs mentioned before, and the results, supplemented with platelet aggregation tests, may serve as the support of methodology for the discovery of active compounds and clarification of the material basis of TCMs which could promote blood circulation and dissipate blood stasis,and it may also serve as a new model for quick screening of active compounds from TCMs.

中药药效物质基础的发现、确定与评价是制约中药现代化进程的瓶颈，建立中药复杂体系活性成分（群）的筛选方法是亟待解决的关键问题。本研究针对丹参、红花、川芎等典型活血化瘀类中药，以抗血栓药物发挥药效的主要靶点- - 血小板P2Y12受体为例，通过亲和层析法从血小板中分离纯化该受体，以温和的化学偶联方式将其通过共价键均匀地固载于氧化石墨烯功能化硅胶表面，对固定相受体键合量、生物活性和保留特性进行表征，并构建血小板受体色谱/SPE/NMR/Q-TOF-MS联用体系，对上述活血化瘀中药中与P2Y12受体有特异性作用的活性成分进行快速筛选、分离、鉴定并辅以抗血小板聚集药理活性确证，为抗血小板活性成分的发现及阐明活血化瘀中药的药效物质基础提供方法学支撑，为中药复杂体系有效成分的快速筛选提供新的模式。

中药药效物质基础的确定是制约中药现代化进程的瓶颈，建立针对中药复杂体系活性成分（群）的筛选方法是亟待解决的关键问题。本项目通过建立昆虫杆状病毒表达系统并结合N2+柱纯化从而获得高纯度P2Y12受体蛋白；采用高级氧化技术中的Photo-Sono协同H2O2技术，通过“氧化切割”方式成功制备了石墨烯量子点（GQDs）并对其进行表征，证明该方法制备的GQDs尺寸均匀且光电化学性质佳；以EDCI和NHS作为偶联剂，牛血清白蛋白BSA作为工具蛋白，建立了基于石墨烯量子点固定化蛋白质的新方法；通过前沿色谱法研究小分子药物与石墨烯量子点固定化蛋白的相互作用，表明该方法能分离具相同母核结构的六种配体，证明GQDs可稳定固载蛋白制备蛋白色谱柱用于分离分析并应用于研究药物与蛋白相互作用；基于以上方法，成功构建了基于石墨烯量子点-P2Y12R复合固定相的受体色谱药物筛选模型，并进行了活血化瘀中药活性成分及系列君使化合物的活性筛选，确定了丹参醇提液中与P2Y12R具有相互作用的3个活性成分、水提液中的10个活性成分、丹红注射液中的7个活性成分，并筛选出君使化合物DBZ1及DBZ2的抗血小板聚集活性，并辅以ADP诱导的血小板聚集实验验证其活性，部分阐释了活血化瘀中药的药效物质基础。研究工作共发表论文5篇，其中SCI论文4篇。本项目的完成为抗血小板活性成分的发现及阐明活血化瘀中药的药效物质基础提供方法学支撑，为中药复杂体系有效成分的快速筛选提供新的模式。