Cell membrane chromatography with zonal elution for characterization of seven alkaloids binding to α_1A adrenoreceptor

A high expression α_1A adrenoreceptor (α_1AAR) cell membrane chromatography (CMC) combined with zonal elution method was developed to study the affinity interaction between the three kinds of alkaloid in Lotus Plumule and four kinds of alkaloid in Uncaria rhynchophylla with the α_1AAR. HEK293 α_1AAR cells were used for preparation of the CMC stationary phase. All measurements were performed with diode array detector at 37°C. In the self- and direct-competitive displacement assay, tamsulosin was used as competitor to evaluate the effects of the competitors concentration on the retention factor and the seven kinds of alkaloid, respectively. The competition behavior between tamsulosin and the seven alkaloids were also been examined. The results indicated that tamsulosin and other seven alkaloids had the same affinity sites on α_1AAR. The association equilibrium constants (KA) values obtained were (2.99 ± 0.41) × 10⁵ L/mol for tamsulosin, (6.26 ± 1.22) × 10⁵ L/mol for liensinine, (7.59802 ± 1.81918) × 10⁵ L/mol for neferine, (8.45 ± 1.49) × 10⁵ L/mol for isoliensinine, and (6.79 ± 1.11) × 10⁵ L/mol for isorhynchophylline, (4.68 ± 1.71) × 10⁵ L/mol for rhynchophylline, (4.03 ± 0.97) × 10⁵ L/mol for isocorynoxeine, (5.69 ± 1.13) × 10⁴ L/mol for corynoxeine at pH 7.4 and 37°C, respectively. The method applied in this study for drug-receptor affinity interaction studies is not limited to these alkaloids or α_1AAR, but also can be extended to other drugs and receptors.