Abstract:

A rat striatum cell membrane chromatography (CMC) frontal analysis method was developed for the determination of the equilibrium dissociation constants (K_D) for 5-hydroxytryptamine (5-HT) receptor 5-HT_1D-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-HT_1D 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. K_D 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 K_D values in drug-receptor interactions.

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