Preparation Standard Of Pueraria Lobata Extract And Study On Quality Control Of Pueraria Lobata Compound And Its Anti-atherosclerosis Effect

Today,cardiovascular disease is the first major cause of death and the prevalence rate and mortality is still in a rising stage.Therefore,it is urgent to develop or to optimize existing drugs so as to obtain curative effect medicines for prevention and treatment of cardiovascular and cerebrovascular diseases.Traditional Chinese medicine(TCM)P.lobata and its compound is commonly used in clinical medicine for the prevention and treatment of cardiovascular and cerebrovascular diseases,Isoflavones are the Active components in P.lobata,This study was to establish P.lobata fingerprint or chemical bar code based on multiple isoflavones and a method for quality control.The method could be used to optimize the preparation of P.lobata extract in order to ensure its quality and efficacy.Danshen and Sanqi(Panax notoginseng)are also applied for cardiovascular diseases with clear constituent.The three herbs in combination often appear in formula used for cardiovascular disease,In order to control the quality of the three herbs combination,first established each drug's fingerprint and presented them in the compound's chromatogram,then constructed simultaneous qualitative and quantitative detection of rapid resolution liquid chromatography(RRLC)analysis method on multiple components.The new method can distinguish ingredients originating from each herb and make it possible to control the quality of TCM formula.Then we assessed the anti-athrosclerosis effect of different proportion of three extracts with clear constituents and filtered out the one with significant effect.1.Study on preparation of P.lobata extractResponse surface methods(RSM)were used to study the the extraction process of P.lobata and concentration of ethanol(0-100%),liquid material ratio(8-12 times)and extraction time(1-3h)were investigated.Combining the optimization results of RSM and the actual situation of manufacture,the extract conditons were confirmed to oncentration of ethanol(65%),liquid material ratio(11 times)and extraction time 2 hours for two times and extraction temperature 80oC.The process and the effects of P.lobata isoflavones purified by macroporous resin were studied.Macroporous resin selection,sampling volume,sampling rate,dilution ratio,ratio of diameter to height,rinsing wate and ethanol concentration and dosage used for eluting were considered.AB-8 was chosen to purify P.lobata isoflavones because it had the best comprehensive performance.Sampling volume for raw material: resin was 1:2(m/m);diameter height ratio 1:6;sampling concentration 0.5 g raw drug/ml solution;sampling rate 3BV/h;after sampled,rinsed the column with 8BV purified water,then eluted with 7BV 70% ethanol,collecting the eluent,dried it.2.RRLC used in P.lobata and its comound quality analysis2.1 Establishment of RRLC QAMS(quantitative analysis of multicomponents by single maker)and characteristic chromatogram for P.lobata six isoflavones(3'-hydroxypuerarin,puerarin,puerarin 6''-O-xyloside,3'-methoxypuerarin,mirificin and daidzin)were chosen as markers to control the quality of P.lobata.Relative correction factor(RCF)between puerarin and the other ingredients were 1.253,1.422,1.297,1.332 and 1.030,respectively.The results calculated by QAMS were consistent with the convenient method.38 batches P.lobata,6 batches P.thomsonii and 7 batches P.lobata extract were detected by this method.One batch contains no isoflavones at all.PCA analysis and statistic were performed base on the normalization data of 6 isoflavones peak area.PCA analysis could eaily discriminate P.lobata and P.thomsonii.It can be easily to decide which sample is better just based on its position on PC1,because the samples' value of PC1 have positive correlation with their content of total isoflavones.By comparing with the ratio of different compounds' peak area in all tested samples,it showed that the ratio of all those examined were consistent between P.lobata and its extracts,but the ratio of 3'-hydroxypuerarin/puerarin,3'-methoxy puerarin/puerarin and mirificin/puerarin in P.lobata was more than 0.1 while in P.thomsonii it was less than 0.1;3'-methoxy puerarin/daidzin and mirificin/ daidzin in P.lobata was more than 0.6,but in P.thomsonii it was opposite.Similarity evaluation system of traditional Chinese medicine chromatographic fingerprint 2004 Edition was used to evaluate a total of 46 batches of samples analysis,including P.lobata,its extract and P.lobata standard herb.There were 9 common peaks.The similarity calculation results showed RRLC patterns of 46 batches of samples and the control fingerprint similarity calculation results were all above 0.96,indicating that each batch of herbs or extracts and the control fingerprint has good consistency.2.2 Establishment of multiple component analysis for P.lobata,Salvia miltiorrhiza and Panax ginseng combinaton and its applicationTen ingredients,that 3'-hydroxypuerarin,puerarin,3'-methoxypuerarin,mirificin and daidzin originating from Pueraria lobata,salvionolic acid B,cryptotanshinone and tanshinone IIA from Salvia miltiorrhiza,and ginsenoside Rg1,ginsenoside Rb1 from Panax notoginseng.Eluted out first(0-6.5min)were components of P.lobata isoflavones;middle segment(6.5-14min)were P.notoginseng total saponin and salvianolic acid B;tanshinones were in the last segment(14-25min),All the components and their proportional relationship of the three Chinese herbal medicines obtained a good presentation in the chromatogram.The method was applied for the determination of components in three herbs' extracts,identification of Panax genus(P.notoginseng,P.ginseng and P.quinquefolium)plant and preliminary quality evaluation of TCM preparations which contain P.lobata,S.miltiorrhiza and P.ginseng.3 Study on anti-atherosclerosis(AS)effect on LDL-R(-/-)hamster of P.lobata,S.miltiorrhiza and P.ginseng combinationThe AS effects of different proportions of three herb extracts combination were investigated.Seventy LDL-R(-/-)hamsters were divided equally into 7 groups.Blood was bleed from eyes at the zero week(before feeding drugs),fourth week,eighth week and twelfth week and the levels of total cholesterol and total glyceride in serum were detected.The drugs' alleviating AS lesions was evaluated by its effect on the whole aorta and the inwall of aorta.The existing results showed that when the amount of three herbs in the ratio of S.miltiorrhiza: P.lobata: P.notoginseng(7:3:9),it could significantly reduce TC and TG in hamsters' serum and ease AS lesions comparing with the model group.