Study On The Separation,Extraction And In Vitro Activity From Puerariae Radix,Puerariae Flos And Soybean

In this study,high-speed counter-current chromatography(HSCCC)and semi-preparative high-performance liquid chromatography(SPHPLC)were used to isolate the active compounds from the extracts of Puerariae radix,Puerariae flos and soybean,ultrafiltration combined with PC12 cells model were developed to screen the anti-lactate dehydrogenase compounds.Puerariae radix is the dry root of pueraria lobata widely used in traditional Chinese medicine as well as in food.This study adopted the reflux technology to research the extraction process of Puerariae radix by the use of the single factor experiment integrated with orthogonal experiment to optimize it.And the optimal extraction methods were as follows: 80% of ethanol concentration and by a ratio of one to twenty with extracting twice and 120 mins each time to obtain the total extract of Pueraria isoflavones.The extraction rate of daidzein was 1.263 mg/g.Lactate dehydrogenase(LDH)was used as the target enzyme to confirm the inhibitory activity by virtue of the ultrafiltration technique as the screening method for enzyme inhibitor.And,HSCCC purification with a binary phase system made up of petroleum ether/ ethyl acetate/ methanol/ water(5.0:5.0:4.0:5.0,v/v/v/v)separate daidzein into a solution with a purity of 99.2 %.Puerariae flos is the dried flower of Pueraria lobata Ohwi or P.thomsonii Benth.Isoflavone compounds was extracted by reflux technology,the optimal parameters was tested by orthogonal test.And the optimal extraction methods are as follows: 70% of ethanol concentration and by a ratio of one to fifteen with extracting third and120 mins each time to obtain the total extract of Puerariae flos isoflavones.The extraction rate of tectoridin was 5.601 mg/g.The use of LDH as the target enzyme is to confirm the inhibitory activity by means of ultrafiltration technique as a screening method for selecting enzyme inhibitor and to test the cell viability by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.There are three kinds of compounds separated by the application of SPHPLC.And the purity of these three compounds is tectorigenin-7-O-xylosylglucosid(90.1%),tectoridin(93.4%)and tectorigenin(95.5%).Therefore,the experiment further proves that the total extract of Puerariae flos can protect the MPP+-induced cell death and apoptosis.Soybean is a leguminous herb plant attributed to fabaceae.This study applied the reflux technology to extract the soybean isoflavone and used the LDH as the target enzyme to evaluate the activity of anti-stroke of it by the employment of ultrafiltration technique as the selection method of enzyme inhibitor.And the result showed that six kinds of compounds from Soybean were LDH inhibitors.The inhibition rates were as follows: acetyldaidzin(48.36%),malonylgenistin(47.14%),daidzin(41.92%),glycitin(39.13%),genistin(28.41%)and acetylcitin(24.21%).MTT test indicates that the total extract of soybean have certain protective effects against neuronal cell injuries.