Study On The Extraction, Structure And Biological Activity Of Polysaccharides(SWP) From Sarcopyramis Nepalensis Wall

Objective:Extract and isolate polysaccharides from Sarcopyramis nepalensis Wall,seeking optimum conditions of extraction.Study on the purification of polysaccharides,the structure of polysaccharides,and biological activity in vivo and vitro.To elucidate new pharmacological action and new medicinal value,and furnish the theoretical basis for developing and exploiting new drug from Sarcopyramis nepalensis Wall.Methods:(1) Classical hot water extracting and ethanol precipitating method was employed to isolate polysaccharides and the content of total sugar was determined by phenol-sulfuric acid method.Single factor tests were done to analyze the influence of each factor including temperature,extraction time,ratio of material to water,extraction times,and the volume of ethanol. Then the optimum conditions for the extraction were obtained by using orthogonal design L₉3⁴．(2) After the crude SWP was extracted with hot water and precipitated with ethanol,protein and pigment were also removed.DEAE-cellulose chromatography was used to separate crude SWP,further purification was carried on by SephadexG-200 chromatography.Finally,it's homogeneity was proved by HPLC.(3) The chemical composition of SWPâ…¡was analyzed by using TLC,gas chromatography,Uv spectrum,IR spectrum,NMR spectrum.The monosaccharide and linkage of SWPâ…¡were investigated through chemical analyzation.(4) Antioxidant activity of polysaccharides in vitro are determined by cell culturing,liver injury which was induced by carbon retrachloride model,were carried out to examine SWP's anti-oxidation,hepatoprotective,immunostimulating activity.ResuIts:(l)The results of single factor tests and orthogonal experiments demonstrated that the optimum SWP extraction conditions are as follows:solid-liquid ratio is 1:60,temperature is 100℃,extraction time is 2．5 hours,the extraction times is 3 and precipitated with four times of alcohol.Under this condition,the content of total sugar is 15．9%.(2) The crude polysaccharide was fractionated by column chromatography DEAE-52,Sephadex G-200,and SWPâ… ,SWPâ…¡were obtained.It was proved to be homo-geneous polysaccharide on molecular weight.(3) IR and NMR spectroscopy analysis revealed the main chain of SWPâ…¡mostly linked byβ-glycosidic bond,rarely linked byα-glycosidic bond;IR, UV and element analysis showed that N and S is absent,it's not a aminosugar.The results of GC and TLC analysis showed the monosaccharide are Rha,Glc and Gal,with the molar ratio of 11．6:16．4:28．6．The average molecular weight of SWPâ…¡is 848,180．(4) The results of the vitro-anti-oxidation suggested that SWP had good anti-oxidation action,with the dosage of 3．0 mg/mL the inhibitory rate on hydroxyl free radical was 39．3%,each dosage could inhibite superoxide anion,with the dosage of 4．0 mg/mL (SWPâ…¡) the inhibitory rate on the restraining auto-oxididation of erythrocytes was 87．3%,with the dosage of 1．4 mg/mL the inhibitory rate on lipidperoxidation was 50%.We found that SWPâ…¡and crude SWP could increase proliferation index of splenic lymphocyte.SWP could significantly augment the percentage of activated macrophage, phagocytic activity,the amount of NO.Each dose group of SWP could significantly increase the activies of IgG,IgM,T-AOC,SOD ,and can enhance the weight of liver and spleen compared with control group. Conclusion:By using single factor tests and orthogonal design,we got the optimum conditions for extraction of SWP.Using this method,the extraction yield is high,the reaction conditions are gentle,easy to operate,apt to industrialization.The homogeneous heterosaccharide SWP,obtained by the optimized extraction and purification method,showed a good anti-oxidation and hepatoprotective.activities and also had a strong immunity activities.