Extraction And Purification Of Polysaccharides In Rabdosia Rubescens And The Primary Study Of The Immunocompetence

Objective:This research is to fine a good technique of extracting,isolating and purifying polysaccharides from Rabdosia rubescens,to study their physic-chemical properties,polysaccharides content and monosaccharide constituents,and to study their immunological activity.Method:1.Screening for optimum extraction process by studying the effect of water-extracted process,cellulose-enzyme-extracted process,pectase-enzyme -extracted process and combined-enzyme-extracted process on the dry weight of crude polysaccharides.By the loss rate of polysaccharides and removal rate of protein to study the effect of trichloroaceticacid-normal-Butanol,trichloracetic acid method and Sevag method,Sevag method,trichloracetic acid on removing protein from polysaccharides.Evaluating decolorizing effect of Hydrogen Peroxide and active carbon on the polysaccharides from Rabdosia rubescens by the loss rate of polysaccharides and the transmittance of clarified sugar solution.DEAE-Sepharose Fast Flow(Cl~-) weak polar anion-exchange chromatography and stepwise ethanol precipitation were used to isolate and purify the polysaccharides from Rabdosia rubescens respectively.2.The purified polysaccharides was identified byα-naphthol, anthrone,phenol,Fehling reagent,iodine-postassium iodide.To select determination by comparing phenol-sulfuric acid and anthrone-sulfuric acid and select the potimizal reaction conditions by reaction time,reaction temperature and reference control. Barium sulfate turbidity was used for determining the content of sulfate group in polysaccharides from Rabdosia rubescens.UV spectrophotometer,paper chromatography and sephadex column chromatography were used to analyze the purity of separate segment RRPS-Ⅱ-Ⅰand TLC method and HPLC were used to analyze the monosaccharide composition of purified polysaccharides RRPS and separate segment RRPS-Ⅱ-Ⅰ.To study the chemical structure of separate segments obtained from Rabdosia rubescens by infrared spectrum andβ-elimination reaction. Automatic polarimeter was used to determine the optical rotation of RRPS-Ⅱ-Ⅰ.3. MTT method was used to evaluate the immune activity.Result:1.The optimum conditions of extraction were as follows:30 times water based on the weight of Rabdosia rubescens,extracted by thermal reflux twice, alcohol precipitation concentration was 80%,the method of trichloracetic acid, repeating two times was the best for removing proteins,its concentration was 10%,1.5%activated carbon was used to remove impurity,DEAE-Sepharose Fast Flow(Cl~-) weak polar anion-exchange chromatography and stepwise ethanol precipitation were used to isolate and purify the polysaccharides from Rabdosia rubescens respectively.Five components were obtained from DEAE-Sepharose Fast Flow(Cl~-) weak polar anion-exchange chromatography and the mainly component was RRPS-B and RRPS-D.four polysaccharides were obtained through alcohol precipitation,namely RRPS-ⅠRRPS-ⅡRRPS-ⅢRRPS-Ⅳ.2.The reaction conditions of sulfuric acid anthrone colorimetric were 0.2%anthrone-sulfuric acid,water bath heating 15 min,ice cooling 40 min,its RSD was 0.8%.The color of separate segment RRPS-Ⅱ-Ⅰwas hazel powder and was supposed to one kind of proteinose.It dissolves in water easily,and its polysaccharide content was 89.1%, protein content was 7.8%,optical rotation was—412.16°.And it was connected by N-type glycopeptides linkage.Infrared spectrum showed that the structure of monosaccharide in RRPS-Ⅱ-Ⅰ,which was connected byα- glycosidic bond, belonged to pyran model.3.Polysaccharides from Rabdosia rubescens showed immunological activity,both RRPS-Ⅱand RRPS-Ⅱ-Ⅰcan accelerate the proliferation of sleenlymphocytes of mice during 5～100μg/ml.100μg/ml RRPS-Ⅱand 5μg/ml,50μg/ml RRPS-Ⅱ-Ⅰboth can reinforce the lymphocyte proliferation induced by ConA.Conclusion:Extraction,isolation and purification method of polysaccharides from Rabdosia rubescens were confirmed and its determination also had been optimized. Polysaccharides from Rabdosia rubescens show immunological activity.Their immunomodulatory effect and related mechanism were to be researched further.