Studies On Extraction, Separation, Identification And Biological Ablity Of Flavonoids From Fructus Aurantii Immaturus

With the aim of extraction,separation and purification flavonoids from Fructus Aurantii Immaturus,the technological condition of extracting flavonoid glycosides through reflux extraction was studied in this paper.Seven kinds of flavonoids were separated from Fructus Aurantii Immaturus,and the structures of flavonoids were confirmed with RP—HPLC,ESI—MS,1D—,2D—NMR.1.Detecting method of flavonoid glycosides from Fructus Aurantii ImmaturusThe contents of flavonoid glycosides from Fructus Aurantii Immaturus were detected by developed reversed phase high performance liquid chromatography(RP—HPLC).Flavonoid glycosides could be separated on Simadazu ODS(RP—18,5μm,250mm×4.6)with UV detector(280 nm).The mobile phase was:78%water(22%acetonitrile) gradient elution 25 min,then the gradient renturn to 10%water(90% acetonitrile)and remained for 20 min.The flow rate is 0.8 ml/min.This method is exclusively to detect flavonoid glycosides,and the analysis result is more accurate and reliable.With this condition,take LC/MS as the examination method,four kinds of flavonoid glycosides were determined.The study establishes an extraction standard using extraction rate of four kinds of flavonoid glycosides.2.Extraction Process of flavonoid glycosides from Fructus Aurantii ImmaturusCalculating the Percentage of four kinds of flavonoid glycosides as extraction rate,we studied the effects of alcohol concentration,extraction time,the feed-liquid ratio on the extraction rate of flavonoids;and the times of extraction was explored.Considering the cost of extraction and preparation,the test results showed that the optimum parameters are:60% alcohol concent;extracting time,1.5h;the feed-liquid ratio,1:10;and extracted 2 or 3 times.3.Separation,purification and identification of flavonoids from Fructus Aurantii Immaturus.Four kinds of flavonoid glycosides and three kinds of Polymethoxy-flavonoids are Separated and Purified,which are identified as narinrutin, naringin,hesperidin,neohesperidin and tangeretin,nobiletin,4′,5,7,8—Tetramethoxyflavone.This is the first isolation of tangeretin,nobiletin,4′,5,7,8—Tetramethoxyflavone from Fructus Aurantii Immaturus. 4.Biological activity of Polymethoxy-flavonoids from Fructus Aurantii Immaturus.By Anti-tumor biological activity in vitro screening test,we found all of the three kinds of Poly methoxy-flavonoids which are tangeretin,nobiletin,4′,5,7,8—Tetramethoxyflavone have antiproliferative activity to human breast adenocarcinoma Bcap37 cells and human liver cancer SMMC7721 cells.In the three kinds of Polymethoxyflavonoids, tangeretin is stronger to inhibit the Bcap37 cells growth than others and nobiletin is the strongest one to inhibit the SMMC7721 cells growth.