Study On The Isolation, Purification And Hypoglycemic Activities Of Polysaccharides From The Stem Of Acanthopanax Trifoliatus

Acanthopanax Trifoliatus(Linn.) Merr. is a kind of shrub of Araliaceae family. The study on the extraction, isolation and hypoglycemic activities of polysaccharides from the stem of A. Trifoliatus was conducted in this thesis, which made a foundation for further research on natural hypoglycemic substances and also provided evidences for development of A. Trifoliatus. The main research contents and results are as follows:1. The content of polysaccharides and proteins in A. Trifoliatus was determined by phenol-sulfuric acid and coomassie brilliant blue method, respectively. According to the results of methodology study, the linear range, precision, repeatability, stability and recoveries of determination methods were good.The crude A. Trifoliatus polysaccharide(ATP) was prepared by the water-extraction and alcohol precipitation method. Polysaccharides and proteins content of ATP were 28.5% and 1.1% respectively.2. Deproteinization technology of polysaccharides from A. Trifoliatus was conducted according to Sevag method. After extracting with Sevag for 4 times, the protein removal rate and the retention rate of polysaccharides were 78.21% and 84.57% repectively. The decolorization effect between hydrogen peroxide and different types of macroporous resin were compared, the polysaccharides retention rate and decolorization rate were both considered. Ultimately, HPD-600 resin was selected as the decolorizing agent. On the basis of single-factor tests, orthogonal design was used for the optimization of decolorization process of HPD-600 macroporous resin. The optimal decolorization process: p H value of polysaccharide solution was 4; the volume of solution and resin content ratio of 30:1(m L/g); the adsorption of 4 h at room temperature. The retention rate and decolorization of polysaccharides was 80.09% and 87.91%, respectively. After deproteinization and decolorization, the purified ATP was obtained.3. Purified ATP was isolated by DEAE-52 cellulose chromatography, and neutral polysaccharides and acidic polysaccharides were obtained. Neutral polysaccharide was named as ATP1, while acidic polysaccharides were named as ATP2 and ATP3 respectively. Analysis of the monosaccharide composition of the neutral and acidic polysaccharides was performed by precolumn derivatization high performance liquid chromatography(HPLC). The result showed that ATP1 polysaccharide was consisted of mannose(Man), rhamnose(Rha), glucose(Glc), galactose(Gla), xylose(Xyl) and arabinose(Ara). ATP2 polysaccharide was consisted of Man, Rha, glucuronic acid(Glc A), Glc, Gla, Xyl and Ara. ATP3 polysaccharide was consisted of Rha, Glu A, Glu, Gla, Xyl and Ara.ATP1 was further isolated by Sephadex G50 column chromatography, and a single symmetrical component was obtained, which was named GATP1. Through the analysis of high performance gel permeation chromatography(HPGPC), the result showed that GATP1 was homogeneous polysaccharide. The peak distribution width of GATP1 was 1.02. The number average molecular weight was 2270. The weight average molecular weight was 2310. Analysis of the monosaccharide composition of GATP1 was performed by precolumn derivatization gas chromatography(GC), and GATP1 was mainly composed of Rha, Man, Glc, Gla and inositol, with the ratio of 0.63:0.56:5.52:0.62:1.00.4. Anti-hyperglycemic effects of ATP and decolored polysaccharide were studied on the Streptozotocin(STZ) induced diabetic mice. Weight change, fasting blood glucose were used to evaluate the hypoglycemic activities of ATP and decolored polysaccharide. In addition, oral glucose tolerance, hepatic glycogen content, muscle glycogen content and HE staining of kidney and spleen were used to evaluate the activities of ATP. ATP and decolored polysaccharide could decrease blood glucose of STZ induced diabetic mice significantly. The hypoglycemic actitiy of ATP and decolored polysaccharide showed a dosage-effect relationship. The hypoglycemic effect of middle and high dosage of ATP and decolored polysaccharide were equal to that of metformin. With repeated gastric perfusion for 2 weeks, the inhibition rate of ATP and decolored polysaccharide high-dose group were 32.02% and 43.04%, respectively.The results showed decolored polysaccharide had enhanced effect on reducing blood glucose than ATP. ATP could improve glucose tolerance and increase the hepatic glycogen and muscle glycogen cotent of mice. HE staining results showed that ATP had therapeutic effect on mouse islet and could enhance immune function on mouse spleen.