Studies On The Extraction, Purification, Structure And Anticoagulant Activity Of Dermatan Sulfate From Lophius Litulon Skin

In this paper,the extraction,purification,structure and anticoagulant activity of dermatan sulfate(DS)from the skin of Lophius litulon was investigated.The study is to provide a theoretical basis for utilization of Lophius litulon skin and development of DS.The method of enzymatic degradation had been used to extract the DS from Lophius litulon skin.The condition of extraction was optimized using orthogonal design and response surface techniques.The optimum conditions for DS extraction was a solvent-biomass ratio of 26 mE/g,at 76℃for 19h using 25mg enzyme(papain:pancreas=1:2,W/W)per gram of tissue.After centrifugation,3 volumes of ethanol were added to the supernatant,the precipitate was freeze-dried to afford crude DS.TCA method,Sevag method and CPC method were used to purify the crude DS from Lophius litulon skin.Both Sevag and CPC method can effectively purify DS,while TCA method can not.Sevag method is easy to perform,so it is better than CPC method.The DS from Lophius litulon skin was verified to be mono-fraction by HPGPC and cellulose acetate electrophoretic method,its purity is 99.15%.Chemical methods were used to anlysis the basic constituents of DS. It showed that the DS from Lophius litulon skin contains hexosamine (35.75±1.70%),uronic acid(29.02±2.90%)and SO₄^-(18.83±0.60%),and the rate of OSO₃^-:COO^- is 1.02±0.14:1.The SO₄^- content was most in the DS from Lophius litulon skin compared with the DS from pig skin, chicken skin and bloody noun skin.Chemical composition of the DS from Lophius litulon skin is different from others.The structures of the DS from Lophius litulon skin were demermined by GPC,GC,CLC,MS and IR techniques.The molecular weight of the DS was 1.59×10⁹,it is consisted of xylose,galactose,glucuronic acid, iduronic acid and N-acetyl-D-galactosamine,and their mole ratio is 0.07: 0.04:0.07:1.12:1.19.Xylose and galatose are the few monosaccharides between polysaccharide chains and core protein.The DS from Lophius litulon skin can be well digested by chondroitinase ABC at 37℃for 4 hours.Polysaccharide chains of the DS consists of a prevailing disaccharide unit IdUA-[1→3]-GalNAc-4S and some IdUA-[1→3]-GalNAc-6S and GlcUA-[1→3]-GalNAc,their mole ratio is 1.71:0.01:0.01.DS has a characteristic IR spectrum as glycosaminogly,its monosaccharide is pyranose.The anticoagulant activity of the DS from Lophius litulon skin was invested in terms of effection of DS on coagulation time(CT)and prothrombin time(PT)with heparinum as control.The results showed that the DS has significant effect on CT and has no effect on PT which indicating that the DS mainly effect on intrinsic pathway.The anticoagulant activity of heparinum is better than the DS from Lophius litulon skin by possessing significant effect on both CT and PT.The anticoagulant activities of the DS were related to their differences in concentration.The anticoagulant activity of DS is worse than heparinum; however it has potential utilization because of its relatively low side effect of hemorrhage.