Abstract: 【Objective】 The aim of this study was to optimize the extraction process of total flavonoids from Viola yedoensis by response surface methodology,identify the composition of compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS),and study its anti-inflammatory activity. 【Method】 The absorbance at 510 nm was determined by rutin standard,and the standard curve of flavonoids was established with absorbance (Y) as ordinate and sample concentration (X) as abscissa.The powder of Viola yedoensi was sieved through 100 mesh,soaked in ethanol for 12 h,and ultrasonically extracted by ultrasonic instrument.The ratio of Viola yedoensis powder to ethanol (solid-liquid ratio),ultrasonic extraction time (ultrasonic time),and ethanol concentration (ethanol concentration) were used as investigation factors.The extraction process of total flavonoids from Viola yedoensis was optimized by Box-Behnken response surface methodology with comprehensive score of total flavonoids extraction.The total flavonoids extracted from Viola yedoensis were purified by D101 macroporous resin,and the composition of the compounds was identified by LC-MS/MS method.Sixty 6-week-old healthy Kunming mice were randomly divided into blank,model,low dose (150 mg/kg),medium dose (300 mg/kg),high dose (600 mg/kg) and levofloxacin (5 mg/kg) groups.Except for blank group,LPS (1 g/L,50 μL/ mouse) was instilled into the nasal cavity to establish an acute lung injury (ALI) model.After successful modeling,the drug intervention was performed for 1 week (1 time/d,continuous 7 days).Blood was collected 1 h after the last administration,followed by anesthesia,execution,and lung collection.The degree of lung injury was observed by HE staining.The levels of interleukin-1 β (IL-1β),IL-6,IL-10 and tumor necrosis factor-α (TNF-α) in serum of mice were detected by ELISA. 【Result】 When the solid-liquid ratio was 1∶20,the extraction time was 22 min,and the ethanol concentration was 73%,the extraction amount of total flavonoids was the highest,which was 25.134 mg/g.It was identified that the extract contained 37 flavonoids (quercetin,luteolin,apigenin,etc.),a small amount of isoflavones,sugars,coumarins,lignans,monoterpenes,and sesquiterpenes.Pathological sections showed that compared with model group,the bleeding points of lung tissue in each administration group were gradually reduced,the area was reduced,and there were different degrees of recovery.The lung tissue of the high dose and the levofloxacin groups showed slight damage in color and morphology.Compared with model group,the damage degree of lung tissue in the low and medium dose groups was reduced,but there was still a certain degree of edema and some bleeding points.The results of ELISA showed that after administration,the levels of TNF-α,IL-β,IL-6 and IL-10 in the low,medium and high dose groups were lower than those in the model group to varying degrees.Except that the levels of TNF-α and IL-β in the serum of mice in the low dose group were not significantly different from those in the model group,the rest of the administration groups were significantly reduced (P＜0.05).The levels of inflammatory factors in the serum of mice in the high dose group were slightly different from those in the levofloxacin group,which were slightly higher than those in the blank group. 【Conclusion】 The response surface method was used to effectively improve the extraction amount of total flavonoids from Viola yedoensis,and the active components of total flavonoids from Viola yedoensis were identified by LC-MS/MS technology.At the same time,through the model of acute lung injury in mice,it was proved that the total flavonoids of Viola yedoensis could improve the acute lung injury in mice and reduce the levels of IL-1β,IL-6,IL-10 and TNF-α in the blood.The total flavonoids of Viola yedoensis had good anti-inflammatory effect.

Key words: Viola yedoensis; flavonoid; response surface methodology; identification; acute lung injury; anti-inflammatory