Simultaneous screening and isolation of activated constituents from Puerariae Flos by ultrafiltration with liquid chromatography and mass spectrometry combined with high-speed counter-current chromatography.

Abstract

Puerariae Flos is the flower of Puerariae Radix, which is a common Chinese herb containing numerous isoflavones in all parts of the flower. Standard methods for screening and isolating isoflavones are typically labor intensive and time consuming. In this study, a new assay based on ultrafiltration with liquid chromatography and mass spectrometry was developed for the rapid screening and identification of ligands for α-glucosidase, xanthine oxidase and lactate dehydrogenase in the extract of Puerariae Flos. Three isoflavones were identified as α-glucosidase inhibitors, three isoflavones were identified as lactate dehydrogenase inhibitors, and no specific binding ligands were identified for xanthine oxidase in the extract. Subsequently, specific binding ligands, puerarin, genistin, and tectorigenin (purities were 90, 60, 99, and 91.73%, respectively), were separated by high-speed counter-current chromatography. The partition coefficient values of the target compounds and resolutions of peaks were employed as indicators and the solvent system and mobile phase flow rate were optimized for two-stage separation. An optimized two-phase solvent system comprised of ethyl acetate/ethanol/water (4:0.5:3, v/v/v) was successfully used to isolate the three compounds from Puerariae Flos. The monomer compounds isolated, collected, and purified by high-speed counter-current chromatography were analyzed by high-performance liquid chromatography, resulting in the isolation of three targeted compounds. The chemical structures of all three targeted compounds were individually identified by ultra high performance liquid chromatography with high-resolution mass spectrometry. The results demonstrate that ultrafiltration with liquid chromatography and mass spectrometry combined with high-speed counter-current chromatography is not only a powerful tool for screening and isolating α-glucosidase and lactate dehydrogenase inhibitors in complex samples, but also a useful platform for identifying bioactive compounds for preventing and treating diabetes and stroke.