Affinity measurement of ligands in Perilla frutescens extract towards alpha-glucosidase using affinity-based ultrafiltration-high-performance liquid chromatography

摘要

It is a time-consuming and challenging task for affinity measurement of drug lead compounds from a plant extract because of its chemical complexity. In this research, a strategy of ultrafiltration-high-performance liquid chromatography (HPLC) was developed to directly measure dissociation constant (K-d) of compounds from natural product extract to target protein, and the K-d measurement of alpha-glucosidase ligands from the ethyl acetate fraction of Perilla frutescens (L.) Britt. (PFEA) was performed. The recovery value, binding degree, and signal-to-noise ratio of alpha-glucosidase ligands from PFEA were first determined according to the ultrafiltration-HPLC results; the K-d values were then calculated using proposed equilibrium. Finally, oleanolic acid (4) and apigenin (8) from PFEA were determined as the high affinity ligands for alpha-glucosidase, and their K(d)s were calculated as 44.9 mu M and 88.5 mu M, respectively, which agreed with the isothermal titration calorimetry analysis, kinetic analysis, and computer simulation of molecular docking. These results suggested that the proposed strategy is a simple and convenient method for the direct K-d determination of compounds from natural product extract without using any internal calibrants or internal standards.