Affinity selection-based two-dimensional chromatography coupled with high-performance liquid chromatography-mass spectrometry for discovering xanthine oxidase inhibitors from Radix Salviae Miltiorrhizae

Xanthine oxidase (XOD) is a key oxidative enzyme to the pathogenesis of hyperuricemia and certain diseases induced by excessive reactive oxygen species. XOD inhibitors could provide an important therapeutic approach to treat such diseases. A new method using affinity selection-based two-dimensional chromatography coupled with liquid chromatography-mass spectrometry was developed for the online screening of potential XOD inhibitors from Radix Salviae Miltiorrhizae. Based on our previous study, the two-dimensional, turbulent-flow chromatography (TFC) was changed to a mixed-mode anion-exchange/reversed-phase column and one reversed-phase column. The developed method was validated to be selective and sensitive for screening XOD-binding compounds, especially weak acidic ones, in the extracts. Three salvianolic acids were screened from the Radix Salviae Miltiorrhizae extract via the developed method. The XOD inhibitory activities of salvianolic acid C and salvianolic acid A were confirmed, and their inhibitory modes were measured. Salvianolic acid C exhibited potent XOD inhibitory activity with an IC50 of 9.07 μM. This work demonstrated that the developed online, two-dimensional TFC/LC-MS method was effective in discovering the binding affinity of new compounds from natural extracts for target proteins, even at low concentrations.