Study On Extraction,Isolation,Structure And Bioactivity Of Polysaccharide From Rana Chensinensis Skin

Objective: To optimize the extraction process of polysaccharide from Rana dybowskii Guenther skin by water extraction,to separate and purify polysaccharide from Rana dybowskii Guenther skin by chromatographic column and to analyze the structure of polysaccharide from Rana dybowskii Guenther skin by IR,HPLC and NMR to investigate anti-aging activity,providing experimental basis for developing and utilizing Rana dybowskii Guenther.Methods:(1)Using Rana dybowskii Guenther skin as raw material,extracted with hot water,optimized by single factor investigation and orthogonal test,to determine the best process conditions for polysaccharide extraction,and measure the polysaccharide yield.(2)The crude polysaccharide DGP was obtained by extraction and alcohol precipitation,the free protein was removed by the Sevage method to obtain the DGPI,and the bound protein and free protein were removed by the enzymatic hydrolysis combined with the sevage method to obtain a higher-purity polysaccharide DGPII.DGPⅠa and DGPⅡa were separated by Gel chromatography;their purity was verified by high performance gel permeation chromatography,the type of glycopeptide bond was determined by β-elimination reaction,and making preliminary analysis of the monosaccharide composition and its special groups of DGPⅠa and DGPⅡa determined by HPLC,IR and NMR.(3)The aging model was established by intraperitoneal injection of D-galactose.The changes of body weight in each group were observed.Morris water maze test was used to detect the learning and memory ability of mice in each group,and the pathological morphology of the brain tissue of the mice in each group was detected by HE staining Changes in mouse liver,serum tissue SOD,T-AOC,CAT,GSH-PX activity and MDA content were measured to explore the effect of Rana dybowskii Guenther polysaccharide on aging mice.Results:(1)The optimal process conditions were determined: the extraction temperature of 100 °C for 3 hrs,with the ratio of liquid of 1:40,and the polysaccharide yield of 1.9782%;(2)The β-elimination reaction was used to determine the polysaccharide DGPⅠa of Rana dybowskii Guenther skin is an O-glycopeptide bond type;the monosaccharide composition analysis shows that both DGPⅠa and DGPⅡa are composed of Man,Glc A,Gal N,Gal,Glc and Fuc;IR results show that DGPⅠa and DGPⅡa have the characteristic group of glycan,and the glycosidic bond are all pyran type;NMR analysis of DGPⅡa confirms that it has the characteristics of glycosaminoglycan,and the result is consistent with IR.(3)In vivo experiments showed that compared with the normal group,the model group had pathological damage to the hippocampus;and within the dose range of100-400 mg/Kg,the aging mice could improve the weight loss of the mice to varying degrees;improve spatial learning and memory ability;and repair the pathological damage in the hippocampus of aging mice and in a dose-dependent manner.Conclusion: The optimal extraction conditions were determined by single factor investigation method and orthogonal test analysis,and the Rana dybowskii Guenther polysaccharide DGPⅡa was preliminarily determined as glycosaminoglycan by IR,HPLC and NMR techniques,and the anti-aging activity in mice was determined.The polysaccharide from Rana dybowskii Guenther skin can improve the characterization of D-galactose-induced aging,which providing a new experimental basis for developing anti-aging drugs.