Isolation,Characterization And Bioactivity Of Heparinoids From Swimming Bladder

As one of the by-products of aquatic product processing,swimming bladder is rich in active substances such as collagen,peptide and mucopolysaccharide,which make it a nourishing treasure for medicine and food.In traditional Chinese medicine,it was known for the functions of tonifying kidney and replenishing essence,dispersing phlegm,swelling and so on.However,its effects and efficacy factors are unknown.In the present study,a heparinoid isolated from swim bladder was structurally characterized and its anticoagulant and anti-inflammatory activities were determined to provide a reference for elucidating the structural composition and structure-activity relationship of the swim bladder heparinoid,and lay the foundation for development and utilization of swim bladder.(1)Establishment and optimization of extracting technology of fish swim bladder heparin: the heparinoids were extracted from swim bladder by enzyme hydrolysis.Then,response surface analysis was adopted to examine the effects of protease consumption,extraction time and temperature on the extraction of heparin-like compounds.The concentration of protease from Bacillus licheniformis was 5.4 mg/mL,extraction temperature was 50?,and extraction time was 20 h.Under these conditions,the predicted extraction yield by mathematical model was 1.79±0.05%,and the standard error was3.24%.(2)Isolation,purification and chemical composition analysis of heparinoids from swim bladder: the heparin-like compounds from swim bladder were isolated by anion exchange resin adsorption,gradient elution of sodium chloride solution,and purified by alcohol precipitation.Response surface analysis was adopted to examine effects of sodium chloride solution concentration,pH and temperature on heparin-like compound adsorption rate.The results show that the concentration of sodium chloride solution was 6.0 mg/mL,pH was 8.0,and the temperature was 50?.Four fractions eluted with sodium chloride solutions of increasing concentration were obtained,F1,F2,F3 and F4.F4 has the highest yield of(2.22±0.02)%.The results of chemical composition analysis showed that the content of heparinoid,uronic acid,hexosamine and sulphate increased gradually and the protein content decreased from F1 to F4.In cellulose acetate membrane electrophoresis test,F1 presented a single band with the lowest mobility;F2 and F3 presented four bands;F4 presented a single band with the highest mobility similar to chondroitin sulfate.F4 which has the highest acquisition rate and the closest chemical properties to heparin,was selected to further structural identification.(3)Structural identification of heparinoids from swim bladder: the structure of F4 were determined by UV spectrum,high performance gel chromatography,pre-column derivatization and high performance liquid chromatography,fourier infrared spectroscopy,enzyme-cleavage MS/MS and nuclear magnetic resonance imaging.The results showed that F4 has higher purity of(85.79±0.63)% heparinoids content,with a molecular weight of 115844±1401 u.F4 is composed of glucuronic acid and N-acetylgalactosamine with a small amount of aldoluronic acid and galactose and has the characteristic absorption peaks of carboxyl,acetyl amino and sulfate groups.It was determined that the heparinoid from fish swim bladder was chondroitin sulfate-A composed of repeated disaccharide units of[?4GlcUA?1?3Gal NAc(4S)?1?].(4)Anticoagulant and anti-inflammatory activities of different molecular weight heparinoids from swim bladder: degradation of F4 with hyaluronidase produced two low-molecular-weight heparinoids: DP1 and DP2,with the average molecular weights of6089±33 u and 4963±37 u,respectively.The in vitro anticoagulant activity of different-molecular-weights swim bladder heparinoids was evaluated by measuring activated partial thromboplastin time,prothrombin time and prothrombin time.In vitro anti-inflammatory activity was evaluated by LPS-stimulated RAW264.7 macrophages inflammation model.The results showed that,compared with heparin sodium,heparinoids from swim bladder showed negligible in vitro anticoagulant activity,and the lower the molecular weight,the more weak the effect play;the heparinoids can also inhibit the secretion of NO and reduce the level of ROS in LPS-stimulated RAW264.7 macrophages inflammation model,meanwhile the effect was in a dose dependent manner,and the lower the molecular weight,the more significant the effect play.Further analysis shows that,DP2 inhibited the LPS-induced inflammatory in RAW264.7 macrophages by down-regulation of inflammatory mediators,such as NO,ROS,IL-6,IL-1? and IL-10.