Objective To evaluate the inhibitory effect of Gnaphalium affine extracts on xanthine oxidase(XO) activity in vitro and to analyze the mechanism of this effect. Methods In this in vitro study, Kinetic measurements were performed in 4 different inhibitor concentrations and 5 different xanthine concentrations(60, 100, 200, 300, 400 μmol/L). Dixon and Lineweaver-Burk plot analysis were used to determine Ki values and the inhibition mode for the compounds isolated from Gnaphalium affine extract. Results Four potent xanthine oxidase inhibitors were found in 95% ethanolic(v/v) Gnaphalium affine extract. Among them, the f lavone Eupatilin exhibited the strongest inhibitory effect on XO with a inhibition constant(Ki) of 0.37 μmol/L, lower than the Ki of allopurinol(4.56 mol/L), a known synthetic XO inhibitor. Apigenin(Ki of 0.56 μmol/L, a proportion of 0.0053‰ in Gnaphalium affine), luteolin(Ki of 2.63 μmol/L, 0.0032‰ in Gnaphalium affine) and 5-hydroxy-6,7,3’,4’-tetramethoxyflavone(Ki of 3.15 μmol/L, 0.0043‰ in Gnaphalium affine) also contributed to the inhibitory effect of Gnaphalium affine extract on XO activity. Conclusions These results suggest that the use of Gnaphalium affine in the treatment of gout could be attributed to its inhibitory effect on XO. This study provides a rational basis for the traditional use of Gnaphalium affine against gout. Objective To evaluate the inhibitory effect of Gnaphalium affine extracts on xanthine oxidase (XO) activity in vitro and to analyze the mechanism of this effect. Methods In this in vitro study, Kinetic measurements were performed in 4 different inhibitor concentrations and 5 different xanthine concentrations ( 60, 100, 200, 300, 400 μmol / L). Dixon and Lineweaver-Burk plot analysis were used to determine Ki values ​​and the inhibition mode for the compounds isolated from Gnaphalium affine extract. Results Four potent xanthine oxidase inhibitors were found in 95 % ethanolic (v / v) Gnaphalium affine extract. Among them, the f lavone Eupatilin exhibits the strongest inhibitory effect on XO with a inhibition constant (Ki) of 0.37 μmol / L, lower than the Ki of allopurinol (4.56 mol / L) , a known synthetic XO inhibitor. Apigenin (Ki of 0.56 μmol / L, a proportion of 0.0053 ‰ in Gnaphalium affine), luteolin (Ki of 2.63 μmol / L, 0.0032 ‰ in Gnaphalium affine) and 5-hydroxy- 3 ’, 4’-tetramethoxyflavone (Ki of 3.15 μmol / L, 0.0043 ‰ in Gnaphalium affine) also contributed to the inhibitory effect of Gnaphalium affine extract on XO activity. Conclusions These results suggest that the use of Gnaphalium affine in the treatment of gout could be attributed to its inhibitory effect on XO This study provides a rational basis for the traditional use of Gnaphalium affine against gout.