| Litopenaeus vannamei is one of the extremely important aquatic products in China.Shrimp head is the main by-product of shrimp processing process,It is rich in compounds such as protein,chitin,astaxanthin,glycosaminoglycan,etc.However,the current high-value development and utilization of shrimp head is insufficient,and most of them are still abandoned except for a certain amount used as feed and fertilizer.Our preliminary research found that shrimp head was rich in heparinoid compounds.Heparinoid compounds are a class of compounds that are similar to heparin in structure and function.Common heparinoid compounds include CS,DS,HS,etc.which have antithrombotic and antitumor activities.Its structure was characterized by being mainly composed of D-Glc A,L-Ido A,N-Glc N,N-Gal Ac,Gal and other components.In this study,shrimp head was used as raw material to prepare heparinoid compounds,and their structures were characterized.Their antithrombotic activities were evaluated by anticoagulation,fibrinolysis,platelet formation and platelet aggregation experiments,with the expect to provide a new way for the high-value utilization of shrimp heads and provide a reference for the development and utilization of antithrombotic functional factors and heparinoid compounds.The major research outcomes and results are as follows:(1)Preparation and physicochemical properties of heparinoids from shrimp headLitopenaeus vannamei shrimp heads are used as raw materials,the heparinoids from shrimp heads were extracted,separated and purified by enzymatic hydrolysis,anion exchange chromatography,and alcohol precipitation,and the physical and chemical properties of the obtained components were analyzed.Then,the purified heparinoids were identified by cellulose acetate membrane electrophoresis,and the thermal stability of the purified heparinoids were analyzed by DSC.The results showed that three heparinoids F,F1 and F2 were obtained from shrimp head with the yield of 213.53 mg/kg,347.00 mg/kg and 91.67 mg/kg respectively.The glycosaminoglycan content of component F was 48.00%,sulfate group content was0.45%,and potency was 18.90 IU/mg,further analysis was not performed.The contents of glycosaminoglycans measured in components F1 and F2 were all above83.40%,the protein mass fraction was below 1.00%,the uronic acid content was33.56% and 21.94%,the sulfate group content was 9.39% and 12.05%,and the titers were 118.29 IU/mg and 109.72 IU/mg,respectively.The results of electrophoresis showed that component F1 mainly contained chondroitin sulfate and a small amount of dermatan sulfate,and component F2 mainly contained heparin.Thermal stability analysis found that the thermal decomposition temperature of F1 and F2 at about240°C,the thermal stability is better.F1 and F2 were selected for detailed structural research in the next step.(2)Structural characterization of heparinoid from shrimp headsThe structure of the heparinoids components F1 and F2 from shrimp heads was identified,the molecular mass was detected by HPGPC,FTIR was used to analyze the functional groups,the monosaccharide composition was analyzed by PMP derivatization-high performance liquid chromatography,the triple helix structure was analyzed by Congo red reagent,the micro morphology was visualized by scanning electron microscope,and enzymatic cleavage-chromatography was used to determine its disaccharide composition.The results showed that the molecular masses of F1 and F2 were 7.80 k Da and 19.80 k Da,respectively.They do not had no triple helix structure;with hah carboxyl,acetyl,sulfate acid and other groups;and the monosaccharide composition showed that the heparinoids F1 and F2 were composed of D-Glc A,L-Ido A,N-Glc N,N-Gal NAc,Gal and Fuc in different proportions.The disaccharide composition results indicate that F1 is composed of 13.18% trisulfated disaccharide,43.20% disulfate disaccharide,11.94% monosulfate disaccharide and10.93% non-sulfated disaccharide;F2 is composed of 12.36% trisulfate It was composed of disaccharides,38.9% disulfate disaccharides,21.05% monosulfate disaccharides and 7.97% non-sulfated disaccharides;the degree of sulfation of F2 is higher than that of F1.(3)Evaluation of anticoagulant and fibrinolytic activity of heparinoids from shrimp headsThe in vitro anticoagulant activity of heparinoids F1 and F2 from shrimp heads was preliminarily evaluated by measuring the three anticoagulant indicators APTT,PT,and TT.Chromogenic substrate method to determine its anti-factor Xa and anti-factor ?a activities,the thrombolytic activity in vitro was explored by clot lysis method,fibrin plate method was used evaluated the fibrinolytic activity in vitro,and the bleeding side effects were evaluated by bleeding time of mice with tail cutting.Results indicate that heparinoids F1 and F2 from shrimp heads had moderate anticoagulant activity,which was about 1/4?1/5 of standard heparin sodium.They exert anticoagulant effect through APTT pathway and TT pathway together,and the anticoagulant activity of F2 was better than that of F1 in vitro.The anticoagulant mechanism of heparinoids F1 and F2 from shrimp heads was partly consistent with that of traditional heparin,which mainly relies on activating AT-? to make more FXa and F? a combine with AT-? to inactivate it.The qualitative and quantitative results of fibrinolytic activity show that heparinoids F1 and F2 from shrimp heads have good fibrinolytic ability,and their fibrinolytic activities are 6.67±0.17 U/mg and5.52±0.54 U/mg,respectively,which was about 0.5-0.7 times that of heparin.The fibrinolytic ability of F1 was better than that of F2 in vitro.Heparanoids F1 and F2 from shrimp heads have good thrombolytic ability in vitro,and the dissolution rates of clots at 10 mg/m L are 66.11% and 64.73%.In addition,the results of bleeding time of tail docking in mice showed that heparinoids F1 and F2 from shrimp heads prolonged tail bleeding time of mice by about 0.7 times that of unfractionated heparin,with less bleeding side effects and better safety.(4)Effects of heparinoids from shrimp heads on thrombosis and platelet aggregationThrough building a model of carrageenan-induced thrombosis in the tail of mice,determining the ratio of black tail formation and the determination of related indicators of mouse plasma fibrinolytic function,preliminary exploration of the effect of heparinoids F1 and F2 from shrimp heads on thrombosis and its action pathway.Micro turbidimetric method was used to explore the effects of heparinoids F1 and F2 from shrimp heads on the platelet aggregation induced by ADP,AA and COLL in rabbits in vitro.The finding showed that the medium and high dose of heparinoids F1 and F2 could significantly reduce the length of black tail in mice,and the effect of F1 was better than that of F2.Heparinoids F1 and F2 could significantly increase the content of t-PA and FDP in mice plasma,effectively inhibit the release of PAI-1,promote the volatilization of fibrinolytic function,and interfere with the formation of thrombus.However,F1 and F2 had no significant effect on the rabbit platelet aggregation induced by ADP,AA and COLL in vitro. |
PDF Full Text Request