Screening of urate-lowering components in TongFengTangSan using an integrated strategy of affinity ultrafiltration, molecular docking and in vitro validation

• TEG and TRG were new XOD inhibitors. • TRG, TEG and PGG were all URAT1 and GLUT9 inhibitor. • TRG, TEG and PGG exhibited dual urate-lowering effect. • Integration of affinity ultrafiltration-HK-2 cell model to screen urate-lowering components. Hyperuricemia arises from an imbalance between uric acid production and excretion, resulting in elevated systemic uric acid levels. Consequently, therapeutic strategies targeting both production and excretion pathways offer a promising approach for managing hyperuricemia. TongFengTangSan (TFTS) has been frequently utilized as the Tibetan prescription in treating hyperuricemia or gout, and its anti-hyperuricemia effect has been confirmed in our previous study. However, the urate-lowering components still remain unclear. Therefore, an integrative strategy was constructed to screen potential urate-lowering components from TFTS by combination with affinity ultrafiltration, molecular docking and in vitro validation. In the present study, the main active fraction of TFTS with xanthine oxidase inhibitory effect was screened out by xanthine oxidase inhibitory assay. The potential ligands in main active fraction were identified by affinity ultrafiltration experiment combined with enzyme channel blocking technology and explored by ultra-high performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry. Then, the screened ligands were further validated by in vitro xanthine oxidase inhibitory assay and molecular docking technique. Furthermore, the regulation effect on uric acid transporters of xanthine oxidase inhibitors was further evaluated by the over-expression of uric acid transporters in uric acid stimulated human Kidney-2 cell model by Western blot assay. In this study, the 60% ethanol-eluted fraction from the ethanol extract of TFTS was confirmed as the main fraction of TFTS with the best xanthine oxidase inhibitory effect. Three potential xanthine oxidase inhibitors were screened out from the 60% ethanol-eluted fraction, including 1,3,6-trigalloylglucose, 1,2,3,6-tetragalloylglucose and 1,2,3,4,6-pentagalloylglucose. Half maximal inhibitory concentration values of 1,3,6-trigalloylglucose, 1,2,3,6-tetragalloylglucose and 1,2,3,4,6-pentagalloylglucose for xanthine oxidase were 54.46, 15.91, 6.58 µg/mL, respectively. 1,3,6-trigalloylglucose, 1,2,3,6-tetragalloylglucose and 1,2,3,4,6-pentagalloylglucose were identified as mixed-type xanthine oxidase inhibitors. Additionally, these compounds demonstrated inhibitory effects on urate transporters 1 and glucose transporter 9. The combination strategy is suitable for screening active components with dual properties of inhibiting uric acid production and promoting uric acid excretion. 1,3,6-trigalloylglucose, 1,2,3,6-tetragalloylglucose and 1,2,3,4,6-pentagalloylglucose were identified as a potential urate-lowering drug and exhibited dual-target characteristics in uric acid metabolism.