Extraction,Purification,Structural Analysis And Antioxidant Activity Of Polysaccharide From Rana Dybowskii Skin

Rana skin contains many biological activity ingredients with special molecular structures and different bioactive functions. As byproduct of Oviductus Ranae, rana chensinensis skin has a great value in use and potential development prospects. To our knowledge no systematic investigations concerning extraction, purification, structure analysis and bioactive of polysaccharide from rana chensinensis have been published previously, which makes the rich and precious resources cannot be made the best and is the major roadblocks in their development for pharmaceutical fields and promoting health. The technique of extraction, purification, structure properties and antioxidant activity in vitro of polysaccharide from Rana dybowskii skin were studied with modern scientific methods systematacially. Current works aimed at developing and utilizing rana chensinensis comprehensively in our country, especially providing foundation works for the Rana dybowskii skin resources development and utilization. Experiment design and results are listed in the following:(1) Study on extraction of polysaccharide from Rana dybowskii skin. With the polysaccharide yield as index, technological conditions of traditional water extraction, enzymatic extraction and ultrasonic-enzymatic synergistic extraction were optimized. The yields of polysaccharide were1.357%,1.638%and2.286%respectively. The results showed that ultrasonic-enzymatic synergistic method could improve polysaccharide yield significantly.(2) Study on deproteinization of polysaccharide from Rana dybowskii skin. TCA method was compared with Sevage method, evaluated by clearance ratio of protein and loss ratio of polysaccharide. The results showed that the best deproteinizing method was Sevage method. After six times deproteinization, clearance ratio of protein was82.3%and loss ratio of polysaccharide was only34%.(3) Study on purification process of polysaccharide from Rana dybowskii skin. Two components RDP Ⅰ and RDPⅡ were gained by DEAE-52ion-exchange column chromatography after deproteinization and dialysis. The high peak component-RDP Ⅱ was collected and a single symmetrical peak by Sephadex G-100gel filtration chromatography was gained. Purity identification results showed that RDP Ⅱ was a uniform component by UV scanning and HPGPC chromatogram.(4) Study on physicochemical and structure properties of polysaccharide from Rana dybowskii skin. Solubility test, Phcnol-sulfuric acid reaction, Fcling test and Ninhydrin test were utilized to confirm that RDP Ⅱ was typical polysaccharide compound and no residues of protein and small molecular oligosaccharides. Its relative molecular mass was12.5kDal by using HPGPC. Infra-red spectrometry showed that RDP Ⅱ had typical absorption peaks of polysaccharides and it was pyranose with alpha glycosidic bond connection; GC-MS result indicated that RDPⅡ was composed of glucose, galactose and mannose, and the moore percentage of them was88.61:2.16:1.35.’H-NMR and13C-NMR spectra proofed that RDP II was typical heterozygous sugar chains, mainly composed of glucose and trace amounts of galactose and mannose. Its monosaccharide had a configuration. These results added to the evidences that have been confirmed by using IR and GC-MS.(5) Study on antioxidant activity in vitro of polysaccharide from Rana dybowskii skin. With the clearance rate of superoxide radicals, hydroxyl radicals, DPPH free radicals, total reductive ability and total antioxidant capacity as indexes, the results indicated that the polysaccharide from Rana dybowskii skin had antioxidant activity. The clearance rate of O2-was nearly50%while the concentration of RDP II was0.3mg-mL-1; the clearance rate of DPPH-was nearly100%while the concentration of RDP II was5mg·mL-1. The three kinds of free radicals clearance rate, total reductive ability and total antioxidant capacity were increased with increasing concentration of polysaccharide and it showed dose-effect relationship in certain concentration range.