In this study,the relaxant activity on uterine smooth muscle of the extracts and chemical components from Cyathulae Radix was investigated.The uterine contraction model in vitro was used to evaluate the uterine smooth muscle relaxant activity of the 70% EtOH extract and its EtOAc and n-BuOH fractions of Cyathulae Radix.Silica gel column chromatography,preparative thin layer chromatography,and semi-preparative high performance liquid chromatography were used to isolate the compounds from the best active portion,then the structures of isolated compounds were determined by various modern spectroscopic techniques.Finally,the relaxant effects of these compounds on uterine smooth muscle was screened.The results showed that the EtOAc fraction (0.4 mg/mL) could significantly inhibited the contractile tension and contractile activity inhibition on rat uterine smooth muscle and exhibited the best activity,with inhibition rates at (67.09±5.37)% and (79.27±2.49)% .In addition,under the cumulative dosing,the maximum tension inhibition rate was (77.45±12.12)% ,and the EC₅₀ was 0.10±0.01 mg/mL.Nine compounds were isolated from the EtOAc fraction of Cyathulae Radix and were identified as irilin A (1),irilin B (2),2′-hydroxy-5,6,7-trimethoxyisoflavone (3),betavulgarin (4),scoparone (5),N-cis-feruloyl-3-methoxytyramine (6),N-trans-feruloylmethoxytyramine (7),N-cis-fruloyltyramine (8),and N-trans-feruloyltyramine (9).Among them,compounds 2,3,6,7,8 and 9 were isolated from this plant for the first time.Compared with control,compounds 1-3,5-9 inhibited the contractile tension and activity on uterine smooth muscle at 50 μmol/L(P < 0.05 or P < 0.01),among which compound 2 had the best activity,with an tension inhibition rate of (36.16±2.75)% and the activity inhibition rate of (47.03±5.84)% at 50 μmol/L.In this study,the relaxant activity on the uterine smooth muscle of Cyathulae Radix extract and its chemical components were found,which provided a scientific basis for clarifying the material basis of Cyathulae Radix in the treatment of dysmenorrhea.