Affinity measurement of ligands in Perilla frutescens extract towards α-glucosidase using affinity-based...

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 α-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 α-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 α-glucosidase, and their K_ds were calculated as 44.9 μM and 88.5 μM, 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.