色谱

色谱 ›› 2015, Vol. 33 ›› Issue (5): 530-534.DOI: 10.3724/SP.J.1123.2015.01003

• 研究论文 • 上一篇    下一篇

细胞膜色谱法研究5-羟色胺受体与藁本内酯的亲和作用

杜晖, 周娜, 李静静, 范菲   

  1. 西安市食品药品检验所, 陕西 西安 710054
  • 收稿日期:2015-01-04 发布日期:2015-04-23
  • 通讯作者: 杜晖
  • 基金资助:

    陕西省中医药管理局科研课题(13-ZY005).

A cell membrane chromatography method for investigation of 5-hydroxytryptamine receptor-ligustilide interaction

DU Hui, ZHOU Na, LI Jingjing, FAN Fei   

  1. Xi'an Institute for Food and Drug Control, Xi'an 710054, China
  • Received:2015-01-04 Published:2015-04-23

PDF

152

被引次数

10

摘要：

建立了大鼠纹状体细胞膜色谱前沿分析法,研究5-羟色胺(5-HT)受体5-HT1D与藁本内酯的亲和作用。通过大鼠纹状体组织制备得到色谱固定相,利用酶联免疫吸附剂测定法(ELISA)分别测定硅胶吸附前后细胞膜悬液中5-HT的量,求得细胞膜固定相上5-HT受体含量为每克硅胶(40.5±2.3) pg。利用细胞膜色谱与液相色谱的离线联用,特异性地识别混合对照品中的舒马普坦和藁本内酯;以不同浓度(24.2~242 nmol/L)的5-HT1D受体激动剂舒马普坦为模型药物,连续通过细胞膜色谱柱,记录舒马普坦的突破曲线,测得舒马普坦与受体作用的平衡解离常数(KD)为389 nmol/L;并将舒马普坦通过不同浓度(37.0~370 nmol/L)的藁本内酯饱和后的细胞膜色谱柱,记录色谱柱饱和前后舒马普坦突破曲线的变化,测得藁本内酯与受体作用的KD值为4.21 μmol/L。该方法快速、有效,适用于求解存在竞争结合时药物与受体作用的平衡解离常数。

关键词: 5-羟色胺受体, 藁本内酯, 前沿分析, 舒马普坦, 细胞膜色谱

Abstract:

A rat striatum cell membrane chromatography (CMC) frontal analysis method was developed for the determination of the equilibrium dissociation constants (KD) for 5-hydroxytryptamine (5-HT) receptor 5-HT1D-ligustilide interactions. Rat striatum was used for preparation of the cell membrane stationary phase (CMSP). An enzyme-linked immunosorbent assay (ELISA) was applied to determine the 5-HT level of CMSP before and after the adsorption of cell membrane, and the value was (40.5±2.3) pg per gram of silica. The CMC-offline-HPLC system was applied to specifically recognize the mixed standard solution of sumatriptan and ligustilide. Sumatriptan, a 5-HT1D agonist of 24.2 to 242 nmol/L, was pumped through a CMC column continuously, and the breakthrough curves were recorded. For further competitive studies, the mobile phase that contained ligustilide (37.0-370 nmol/L) was pumped through the column to saturate the binding sites. Afterwards, sumatriptan was propelled towards the column. The breakthrough curves were recorded and compared with those obtained from the column without saturation. KD values obtained using frontal analysis were 389 nmol/L and 4.21 μmol/L for sumatriptan and ligustilide, respectively. The competitive binding study indicated that the CMC method could be a quick and efficient way for determining the KD values in drug-receptor interactions.

Key words: 5-hydroxytryptamine (5-HT) receptor, cell membrane chromatography (CMC), frontal analysis, ligustilide, sumatriptan

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