In vitro xanthine oxidase inhibitory properties of Flos Sophorae Immaturus and potential mechanisms

Flos Sophorae Immaturus (FSI) possessed potential xanthine oxidase (XO) inhibitory activity as a uric acid-lowing natural product. The present work identified and quantified the free and bound polyphenols of FSI by UPLC-QTOF-MS. Then determined the primary polyphenols with XO inhibitory effect and clarified their potential mechanisms by omission experiment, interaction assay, inhibition type, and fluorescence measurements. The results revealed that nine polyphenols were detected in the free polyphenol extract and ten polyphenols were detected in the bound polyphenol extract. Meanwhile, seven polyphenols were identified as XO inhibitors, including quercetin, kaempferol, isorhamnetin, rutin, hyperoside, protocatechuic acid, and quercitrin with the IC50 values of 0.03, 0.11, 0.07, 5.62, 11.48, 22.13, and 367.82 mg/mL, but their inhibition stability was lower than 24 h. Although the content of quercetin (18.87 mg/g) was not the highest, it played a crucial role to the XO inhibitory effect of FSI. Furthermore, kaempferol and isorhamnetin alone revealed the sub-additive effect with quercetin, while the combination of other polyphenols with quercetin generated the interference or antagonism effects. Quercetin, isorhamnetin, and kaempferol were mixed-type and competitive inhibitors, which significantly quenched the fluorescence intensity of XO. Moreover, the binding processes of quercetin-XO, kaempferol-XO, and isorhamnetin-XO were spontaneous and endothermic, and the hydrophobic interaction was the key driving force. In general, quercetin, kaempferol, and isorhamnetin in FSI can be used as potential XO inhibitors.