Study On Extraction And Isolation Of Active Ingredients From Trollius Altaicus And Its Preliminary Anti-inflammatory Activity

Trollius altaicus（Ranunculaceae）is a commonly used to treat various inflammatory diseases Altai region of Xinjiang in China,such as upper respiratory infection and tonsillitis.Flavonoid C-glycosides is the main characterical compound of this plant.In this paper,enzyme-assisted ultrasonic extraction technique of total flavonoids from T.altaicus（TATFE）and Orientin 2"-O-β-L galactosides（OGA）were studied by single factor experiment combined with response surface optimization,and furtherly optimized their purification process of macroporous resin by single factor experiment combined with response surface optimization by orthogonal experimental design.Moreover,anti-inflammory activities of TATFEE and OGA were evaluated by lipopolysaccharide（LPS）induced RAW246.7 cell model.The main findings are briefly described as follows:（1）An HPLC method for the determination of OGA and Orientin was established,and the contents of OGA and Orientin in several trollius were determined.The results showed that the content of OGA in T.altaicus is higher,the highest content can reach25.07?0.39 mg/g;The content of orientin is highest in the Trollius from Chengde Hebei,with the content of 16.97?0.20 mg/g.（2）Ultrasonic enzyme-assisted extraction method was used to study extraction process of TATFE and OGA.The optimal process condition was:p H=4,material-liquid ratio 1:40,ultrasonic power 90W,enzyme addition amount 1.4%;ethanol concentration 47.0%,extraction time 38.0 min and extraction temperature 39.0℃.Under this condition,extraction rate of TATFE was more than17.90?0.33%%,the relative error from the theoretical value is 0.26%,extraction rate of OGA was reached to 2.79?0.02%,The relative error from the theoretical value is 0.2%.（3）Through macroporous resin adsorption and desorption experiments,D101resin selected from six resins of D101,AB-8,NKA-9,NKA,H1020,and X-5 has good enrichment performance for Trollius OGA（static adsorption Amount of12.7mg/g resin）,and easy to desorb（desorption rate is 97.8%）.The results of the dynamic desorption experiment showed that the maximum loading volume was 6.3mg/g,the loading mass concentration was 0.2790 mg/m L,the loading rate was 8BV/h,the amount of impurity removal water was 8 BV,and the eluent dosage was 6BV The eluent is 30%ethanol,and the elution flow rate is 4 BV/h.The TATFEE of the T.altaicus in the resulting flavonoid content is above 58.34?1.30%,and the OGA content is above 21.62?0.47%.The TATFEE was further purified by MCI gel CHP20p chromatogra Phy to obtain OGA monomer compounds with a purity greater than95.14%.（4）TATFEE and its component OGA had no toxic effect on RAW264.7 cells at the test dose range.Compared with LPS-stimulated mouse macrophage RAW 264.7cell inflammation model group,TATFEE and OGA can significantly inhibit the release of NO,inhibit the secretion of inflammatory cytokines TNF-α,IL-6,and show Dose-dependent relationship.The IC₅₀ of TATFEE for COX-1 and COX-2 enzymes were 103.66μg/m L and 6.94μg/m L;the IC₅₀ of OGA to COX-1 and COX-2 was122.9μg/m L and 5.01μg/m L.The results show that T.altaicus and its active ingredient OGA have significant anti-inflammatory activities.