Ligand fishing based on bioaffinity ultrafiltration for screening xanthine oxidase inhibitors from citrus plants

Citrus plants are valuable medicinal plants with abundant flavonoids content in the parts of fruits and peels, which exhibit potential hypouricemic effect. In the present study, a ligand fishing assay was performed based on bio-affinity ultrafiltration for rapidly screening and identifying xanthine oxidase inhibitors from citrus plants. Under the optimal experimental conditions, five potential ligands were fished out when xanthine oxidase acted as the targeted protein. Subsequently, the chemical structures of all five compounds were identified by quadrupole time-of-flight mass spectrometry. Among them, hesperidin and naringin were confirmed as high-efficiency xanthine oxidase inhibitors. The half maximal inhibitory concentration values of hesperidin and naringin were 0.15 and 1.82 μM, respectively. Compared with the clinical antigout drug, allopurinol (half maximal inhibitory concentration = 8.03 μM), lower half maximal inhibitory concentration values indicated higher enzyme inhibitory activity. The Lineweaver-Burk plots indicated that the two compounds inhibited xanthine oxidase in a noncompetitive manner. The results demonstrate that the bioaffinity ultrafiltration method is a powerful tool for screening out xanthine oxidase inhibitors from natural products.