In‐depth analysis of the xanthine oxidase inhibitors of Cicer arietinum L.‐based receptor–ligand affinity coupled with complex chromatography

Cicer arietinum L. is the choice of health food for people with diabetes, hypertension, and hyperlipidemia. As an essential source of high-nutrition legumes, it is also an important source of dietary isoflavones.In order to improve the preparation efficiency of natural plants, a rapid biological activity screening and preparation of xanthine oxidase inhibitors from C. arietinum L. was established.Xanthine oxidase (XOD) inhibitors were rapidly screened using ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) based on receptor-ligand affinity. The change in XOD activity was evaluated by enzymatic reaction kinetics measurement. The potential bioactive compounds were verified through molecular docking. In addition, the biological activity of ligands screened was separated and purified by complex chromatography. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy.Three active ingredients, namely daidzin, daidzein, calycosin with XOD binding affinities were identified and isolated from the raw plant materials via semi-preparative high-performance liquid chromatography (HPLC), 0-60 min, 5-50% B and countercurrent chromatography (CCC) (ethyl acetate/acetic acid/water [5:0.8:10, v/v/v]).This study will help to elucidate the mechanisms of action of natural plants of interest at the molecular level and could also provide more opportunities for the discovery and development of new nutritional value from other natural resources.