Research On Extraction-separation And Antiviral Activity Of Echinacea Water-soluble Polysaccharides And Acyl Tartaric Acid Based On CPE Method

Objective: To research the extraction and purification technology of soluble Echinacea polysaccharide,and for analyze monosaccharide composition;To research the separation and purification route of Echinacea coffee acyl tartaric acid;According to Echinacea antiviral effect in vitro,to filtrate its sensitive strains;According to the efficacy experiment in vitro,to filtrate its antiviral active ingredients.Methods: Use the single factor investigation method and the orthogonal experiment method to optimize the preparation process conditions of Echinacea polysaccharide by water extract-alcohol precipitation method,and to determine the content of crude polysaccharide in the selected traditional chinese medicinal materials;Use DEAE-52 cellulose column to do research on the separation and purification of Echinacea polysaccharide;Use HPLC first derivative method to determine the monosaccharide composition of Echinacea polysaccharide;Use Macroporous resin column,Preparation of liquid phase and Analysis of liquid phase to do research on the purification of Echinacea coffee acyl tartaric acid.Adopt CPE inhibition assay to observe Echinacea polysaccharide inhibiting effect on RSV,EV-71,Cox-B5 in Monkey embryonic kidney cells,Human rhabdomyosarcoma cells and human laryngeal epithelial cells;Observe the inhibition effect of various fractions isolated from DEAE-52 cellulose column on RSV,and observe the inhibition effect of phenolic acids in MA104 cells on RSV;By Determining median poisoning concentration(TC50),median effective concentration(EC50),and calculating therapeutic index(TI),to filtrate the optimal antiviral species of Echinacea,and research the mechanism of action.Results:Optimized the best extraction technology conditions of Echinacea polysaccharide: solid-liquid ratio is 1:20,ultrasonic extraction under 60 ? for 50 minutes,precipitated with three times of ethyl alcohol.Monosaccharide composition of Polysaccharide contains mannose,glucuronic acid,rhamnose,glucose,xylose and arabinose.Pharmacodynamics experient results showed that Echinacea polysaccharide has good inhibition effect on RSV,EV-71 in vitro experiment,but it has no effect on Cox-B5.The antiviral research in which various separation of DEAE is resistance to RSV in vitro,only the composition eluted in 0.3 mol/LNa Cl and 0.5 mol/LNa Cl has effect on RSV,whose poison suppression index are respectively 64 and 256.Single coffee acyl tartaric acid,chlorogenic acid,caffeic acid of Echinacea phenolic acids all have no effect on RSV.Conclusion: Researched and established the optimum process conditions of Echinacea polysaccharide with ultrasonic extraction method,the method has the advantages of high efficiency,energy saving;Established Echinacea polysaccharide separation method and composition analysis method for determination of Echinacea polysaccharide;Since early only acid in chicory was studied,not study of other organic acids,therefore,represented by a single coffee acyl tartaric acid,the establishment of a single coffee acyl tartaric acid purification method.The pharmacokinetics study proved that represented by a single coffee acyl tartaric acid for respiratory syncytial virus no effect of organic acids and Echinacea polysaccharide has a certain therapeutic effect on RSV.