Preparation And Analysis Of Panax Notoginseng Saponins Knock-out Extracts

Objective:The main effective substance of both panax notoginsengs and ginsengs is dammarane ginsenoside,which can be mainly divided into two types——protopanaxadiol type and protopanaxatriol type——according to the structure of sapogenin.The proportion of protopanaxatriol saponin in total saponins of panax ginsengs is relatively higher,while protopanaxadiol type accounts for a larger portion in total saponins of panax notoginsengs.In addition,total saponins of panax notoginsengs also contain protopanaxatriol saponin that is specially involved in panax notoginsengs,that is,notoginsenoside R1.As we all know,there is a great difference in the traditional application methods of panax notoginsengs and ginsengs.The material bases determine the difference in efficacy.Hence,whether the difference in efficacy between panax notoginsengs and ginsengs is determined by the peculiar saponin or the component group in total saponins of panax notoginsengs is worth being explored in depth.Therefore,this study has used component knockout techniques to knock out the peculiar saponin,notoginsenoside R1,or change of PPD/PPT from total saponins of panax notoginsengs so as to prepare the knock-out extracts of total saponins of panax notoginsengs,thereby providing material guarantee for subsequent investigation of the role of notoginsenoside R1 and protopanaxadiol saponin played in the efficacy of panax notoginsengs,and accumulating data on the advantages and disadvantages of the two knockout methods applied in the study.Methods:Experiment 1:Preparation of high-purity total saponins of panax notoginsengs by resin coupling technologyOn the basis of the preparation of crude Panax notoginseng saponins by D101 macroporous resin,using anion and cation exchange resins can further purify the crude saponins of Panax notoginseng to obtain high-purity Panax notoginseng saponins and using UV spectrophotometry and vanillin colorimetry to evaluate its purity.Experiment 2:Quantitative analysis of total saponins of panax notoginsengs by UPLC-CAD post-column compensation combining an internal reference methodUse UPLC-CAD column post-compensation and internal reference materials to quantify the content of each monomer saponin in Panax notoginseng saponins,to evaluate the purity of Panax notoginseng saponins and to calculate the ratio of the content of protopanaxadiol saponins and protopanaxatriol saponins,the ratio of the content of Rb1 saponins and Rg1 saponins.Experiment 3:Extraction of notoginsenoside R1 from total saponins of panax notoginsengs through liquid polymerizationWeigh the appropriate amount of Panax notoginseng saponins and dissolve in 10%methanol.The liquid-phase elution procedures are determined on the basis of the component,notoginsenoside R1,to be knocked out.The mobile phase consisted of water and acetonitrile.Through pressure-reducing collection below 60° C,the target component is collected according to the retention time and the solution without the target component is also collected at the same time,so as to obtain the desired samples.UPLC-CAD post-column compensation is used to further analyze the knock-out situation of notoginsenoside R1 as well as the variation of the ratio of protopanaxadiol and protopanaxatriol before and after it is knocked out.Experiment 4:Knocking out the notoginsenoside R1 from total saponins of panax notoginsengs by immunoaffinity column chromatographyNotoginsenoside R1 and BSA are coupled by sodium periodate oxidation,forming notoginsenoside R1-BSA antigens,which then are used to immunize rabbits so that to achieve notoginsenoside R1 antibodies.After removing the ammonium sulfate precipitation,the antibodies in the impurities could be extracted.Afterwards,immunoaffinity columns are prepared by using these antibodies,and then through immunoaffinity column chromatography,notoginsenoside R1 in total saponins of panax notoginsengs could be knocked out.The collected samples are tested by UPLC-CAD post-column compensation method so as to observe the knock-out situation of notoginsenoside R1 and the variation of the ratio of protopanaxadiol and protopanaxatriol after it is knocked out.Experiment 5:Change of PPD/PPT from total saponins of panax notoginsengs by macroporous resin column chromatographyD101 macroporous adsorption resin was preprocessed.The total saponins of Panax notoginsengs were dissolved in about 100 ml of pure water,and the solution was then passed through the columns for about 3 times repeatedly.Ethanol was divided into three groups in concentration-30%and 80%,40%and 80%,and 50%and 80%.Firstly,the solution was eluted in the ethanol of low concentration of each group,and the collected liquid was eluent 1.Then,the eluent 1 was eluted in the ethanol of higher concentration of each group,and the liquid was collected as eluent 2.The eluents were dried at 60° C respectively to obtain six samples in total.After then,the amount of notoginsenoside was detected by UPLC-CAD again.Through comparing the ratios of protopanaxadiol and protopanaxatriol under different elution conditions,a reasonable scheme for the change of PPD/PPT could be figured out.Results:Experiment l:The combination of resins of D101,D201 and D113 in the test showed excellent purification effect.According to vanillin colorimetry,the estimated contents of total saponins reached approximately 107%respectively.Therefore,the study indicated that the separation by resin combination is a viable purification method for neutral and natural polar components.Experiment 2:UPLC-CAD post-column compensation method can successfully separate the main components of high-purity Panax notoginseng saponins and the peak shape is relatively clear.The internal reference method is used to measure the total sum of the monomer saponins up to 92%while the ratio of protopanaxadiol saponin and protopanaxatriol saponin content is about 0.54,the ratio of Rbl and Rgl is 0.37.Experiment 3:The preparation of liquid phase method can successfully knock out notoginsenoside R1 from Panax notoginseng saponins,and the ratio of protopanaxadiol saponin to protopanaxatriol saponin after knock-out is 0.70,the ratio of Rbl and Rgl after knock-out is 1.82.Experiment 4:The immunoaffinity method can partially knock out notoginsenoside R1,and the ratio of protopanaxadiol saponin to protopanaxatriol saponin after knock-out is 0.53,the ratio of Rbl and Rgl after knock-out is 0.33.Experiment 5:The ratio of protopanaxadiol saponin to protopanaxatriol saponin in the six different elution conditions is compared.The 3 0%ethanol eluate has the lowest ratio of PPD/PPT,0.26,Rbl/Rgl is 0.20.The 80%ethanol eluate after 50%ethanol washing had the PPD/PPT ratio of 2.5,highest among the six groups,Rbl/Rgl is 2.38.Conclusions:1.The combination of anion and cation exchange resins can be used to produce high-purity total saponins of panax notoginsengs.Concentration of individual components of the total saponins can be quantitatively analyzed through the integration of the UPLC-CAD post-column compensation and the internal reference method.2.Both the chromatographic knock-out and the antibody knock-out methods can remove notoginsenoside R1 from total saponins of panax notoginsengs.However,comparing to antibody knock-out,chromatographic knock-out is more efficient and easy to perform.Chromatographic knock-out removes notoginsenoside R1 more thoroughly,does not substantially change the protopanaxadiol saponin to protopanaxatriol saponin ratio,and has better reproducibility.The performance of antibody knock-out highly depends on experimentalconditions which is difficult to control.Although it can partially knock out notoginsenoside R1,the method introducessignificant changes to the protopanaxadiol saponin to protopanaxatriol saponin ratio.3.Macroporous resin column chromatography can be used to change the ratio of protopanaxadiol saponin to protopanaxatriol saponin significantly.