受体毛细管电泳对赤芍中扩血管活性成分的筛选研究

Screening of the active components is very important for modernization of traditional Chinese medicine. Relied on the interaction between protein and drugs, novel screening methods of active ingredients from traditional Chinese medicine could be established based on capillary electrophoresis (CE). CE has its inherent characteristics such as high separation selectivity, small sample size requirement, high speed of analysis, high efficiency, and excellent mass sensitivity. One of the attractive main features of capillary electrophoresis is the capability to simultaneously separate a wide variety of analytes which is benefit to analysis of the complicated system of traditional Chinese medicine. Drug target of cardiovascular disease, beta2 - adrenergic receptor (beta2-AR), was chosen as the target protein in the work..First, beta2-AR was oriented bonded to the inner surface of capillary by covalent bond to improve its stability and biological activity. Second, the capillary containing oriented immobilized selectors was used as an affinity ligand to capture specific analyte, i.e., to screen active components of cardiovascular diseases from traditional Chinese medicine by capillary electrophoresis. The advances of oriented immobilization technology of receptor in interactions study between drug and receptor were evalued. The binding constants, binding sites and other parameters were all used to characterize the selective interactions between receptor and drugs. The specific selectivity of beta2-AR in drug screening was investigated by interaction studying between beta2-AR and propranolol hydrochloride and pueraria lobata by capillary electrophoresis. Third, the method was applied to screen active components of extending artery from traditional Chinese medicine paeonia obovata. .The works are very helpful to establishment of high throughput screening methods of active components screening of traditional Chinese medicine by capillary electrophoresis. Furthermore, it is helpful to develop new active ingredients screening techniques of traditional Chinese medicine which based on the receptor and capillary electrophoresis.

中药活性成分的筛选是实现中药现代化的关键。根据蛋白与药物相互作用原理，毛细管电泳为建立中药活性成分筛选新方法提供了可能。本研究以心血管疾病类药物靶点beta2-肾上腺素受体（beta2-adrenceptor，beta2-AR）作为靶标蛋白，将其以共价键定向键合在毛细管内表面，以提高受体的稳定性和生物活性。阐明受体的定向固定化技术在研究药物与受体相互作用中的先进性。采用结合常数、结合位点等多参数表征受体对药物的选择性相互作用，以beta2-AR阳性药为例验证定向键合受体毛细管电泳在药物筛选中的特异性。以赤芍为研究对象，对其扩血管活性成分进行筛选，最终建立以受体毛细管电泳为核心的中药抗心血管疾病活性成分筛选新方法，并结合在线质谱联用技术对活性成分进行结构鉴定。该项目的实施，可为中药活性成分的毛细管电泳高通量筛选研究提供新思路，为建立基于受体毛细管电泳为核心的中药活性成分筛选研究提供新方法。

中药活性成分的筛选是实现中药现代化的关键。根据蛋白与药物相互作用原理，毛细管电泳为建立中药活性成分筛选新方法提供了可能。本研究以心血管疾病类药物靶点beta2-肾上腺素受体（beta2-adrenceptor，β2-AR）作为靶标蛋白，将其以共价键定向键合在毛细管内表面，以提高受体的稳定性和生物活性。.对赤芍用70%乙醇进行提取，得B15-27等系列组分。分别采用非定向和定向键合法将β2-AR固定在毛细管内表面：以γ-氨丙基三甲氧基硅烷（γ-aminopropyltrimethoxyl silane, APTS）和甲基丙烯酸缩水甘油脂（Glycidyl methacrylate, GMA）为间隔臂，制备毛细管内壁的非定向固定化涂层；以γ-氨丙基三甲氧基硅烷（APTS）为间隔臂，与β2-AR的高碘酸（H5IO6）定向氧化产物反应，制备定向固定化毛细管涂层柱。其中非定向方法的键合率为5.64%，定向键合的键合率为28.40%。将药物在未涂层毛细管柱、定向及非定向β2-AR开管毛细管电色谱柱中进行电泳分离，通过归一化容量因子（KRCE）值大小评价药物与受体作用强弱，KRCE值越大，相互作用力越强。实验结果表明，药物在非定向与定向β2-AR开管毛细管电色谱柱上的迁移时间较未涂层柱长，表明药物与β2-AR确实发生了相互作用；依据计算所得KRCE值可知，肾上腺素类药物与其作用活性强弱顺序为盐酸肾上腺素>重酒石酸去甲肾上腺素>盐酸普萘洛尔；赤芍各提取物中B18-19-②与β2-AR相互作用活性最强，可能成为β2-AR的阳性药物；通过改变受体通入时间控制涂层长度，根据涂层长度与药物淌度变化的关系，获得药物与β2-AR的结合常数。实验结果显示，所得定向β2-AR受体开管毛细管电色谱柱的稳定性、重现性良好；得盐酸肾上腺素、重酒石酸去甲肾上腺素、盐酸普萘洛尔与β2-AR蛋白的结合常数分别为2.5346×104L/mol、2.4998×104L/mol 、1.7179×104L/mol，表明三种药物与β2-AR相互作用强弱顺序为盐酸肾上腺素重酒石酸去甲肾上腺素盐酸普萘洛尔，与文献报道一致。.本研究可为中药活性成分的毛细管电泳高通量筛选研究提供新思路，为建立基于受体毛细管电泳为核心的中药活性成分筛选研究提供新方法。