Isolation,Characterization And Antithrombotic Activity Of Heparin From Common Shellfish In South China Sea

Heparin is a glycosaminoglycan composed of uronic acid(iduronic acid or glucuronic acid)and glucosamine and its derivatives(acetylated,sulfated).It has a high content of sulfate groups and carboxyl groups.It is the biological macromolecule with the highest negative charge density known.Because of its strong anticoagulant effect,it is widely used in clinical practice as an anticoagulant.Traditional heparin comes from pigs,cattle,and sheep.Due to its strong anticoagulant effect,side effects such as bleeding effects will inevitably occur.Some scholars have proposed that heparin is extracted from marine organisms.Marine source heparin has the characteristics of mitigating anticoagulant effects and low bleeding side effects.Therefore,in this paper,heparin is extracted from various marine organisms,and the differences are compared.The raw materials that can replace traditional heparin are screened,the structure is analyzed,and the bleeding effect and antithrombotic activity are evaluated.It provides a reference for the structure-activity relationship of heparin and lays the foundation for the comprehensive utilization of marine biological resources.The main research contents and results of this topic are as follows:(1)Extraction and comparison of heparin from different marine organisms.Eight types of marine crude heparin have the basic structural characteristics of heparin.With different raw materials,the physical and chemical properties of crude heparin are also inconsistent.Heparin content,sulfate content and heparin titer were used as the main reference indexes to screen heparin raw materials from marine organisms.Among them,the titer size is:Mactra antiquata>Meretrix lusoria>Tapes dorsatus>Sinonovacula constricta>Tegillarca granosa> Panopea abrupta >Argopectenirradias > Ruditapes philippinarum.The titers of Mactra antiquata and Meretrix lusoria were 69.1 U/mg and 44.6 U/mg,respectively.The heparin content is in order of Mactra antiquata > Tegillarca granosa > Sinonovacula constricta >Meretrix lusoria >Tapes dorsatus >Argopectenirradias>Panopea abrupta >Ruditapes philippinarum.Among them,the heparin content of Mactra antiquata and Meretrix lusoria was 239.2 μg/mg and 214.8 μg/mg,respectively.The content of sulfuric acid in the order of Meretrix lusoria>Mactra antiquata>Tegillarca granosa>Sinonovacula constricta>Argopectenirradias>Tapes dorsatus>Panopea abrupta>Ruditapes philippinarum.The content of sulfuric acid group in Mactra antiquata and Meretrix lusoria was 6.2% and 7.7%respectively.Therefore,Mactra antiquata and Meretrix lusoria heparin were used as research objects for subsequent experiments.(2)Isolation,Purification and Physicochemical Properties of heparin from Mactra antiquata and Meretrix lusoria.Macroporous anion resin was used for separation and purification,and three components(G1,G2,G2)and(M1,M2,M3)were separated from Mactra antiquata and Meretrix lusoria,respectively.Among them,G2 and M2 all performed well.The extraction rates of G2 and M2 were 1.17 mg/g and 0.53 mg/g(dry weight);The titers of of G2 and M2 were 149.6 U/mg and122 U/mg;the heparin content of G2 and M2 were 88.1% and 61.4%;the glycosaminoglycan content of G2 and M2 were 92.8% and 69.8%;the uronic acid content of G2 and M2 were:31.2% and 18.4%.the aminohexose content of G2 and M2 were: 36.8% and 20.7%;the sulfate content of G2 and M2 were: 21.4% and 14.8%;the protein content of G2 and M2 were: 1.4%and 5.8%.Both ultraviolet spectrum and cellulose acetate electrophoresis showed that G2 and M2 were of high purity,and both were characterized as heparin.(3)Structural characterization of heparin from Mactra antiquata and Meretrix lusoria.The molecular weights of G2 and M2 measured by HPGPC were 30.99 k Da and 22.58 k Da,respectively.Congo red experiments and DSC analysis showed that neither G2 nor M2 had a triple-stranded spiral structure,and their structures were thermally stable.Through functional group analysis,monosaccharide composition analysis,disaccharide composition analysis,and nuclear magnetic resonance analysis,the sugar chain structures of G2 and M2 were similar to heparin.The main disaccharide repeat units of G2 are: →4)-α-Ido A2S-(1→4)-α-Glc NS3S6S-(1 →(12.03%)、→4)-α-Ido A2S-(1 →4)-α-Glc NS6S-(1 →(25.81%)、→4)-β-Glc A-(1 →4)-α-Glc NS6S-(1 →(33.32%)、→4)-α-Ido A2S-(1→4)-α-Glc NS-(1→(26.53%)、→4)-β-Glc A-(1 →4)-α-Glc NS-(1→(9.88%)and a small amount→4)-β-Glc A-(1 →4)-α-Glc NAc(1→(1.63%)。 The tetrasaccharide structural unit of G2 are: →4)-α-Ido A2S-(1 →4)-α-Glc NS3S6S-(1 →4)-β-Glc A-(1→4)-α-Glc NS6S(or Glc NAC)-(1→.and →4)-α-Ido A2S-(1 →4)-α-Glc NS6S-(1→4)-β-Glc A-(1 →4)-α-Glc NS6S(or Glc NAC)-(1→.The main disaccharide repeat units of M2 are: →4)-α-Ido A2S-(1 →4)-α-Glc NS6S-(1 →(31.19%)、→4)-β-Glc A-(1 →4)-α-Glc NAc(1→(23.21%)、→4)-β-Glc A-(1 →4)-α-Glc NS(1→(13.87%),→4)-α-Ido A2S-(1 →4)-α-Glc NS(1→(8.95%),→4)-β-Glc A-(1 →4)-α-Glc NAc6S(1→(7.39%)and→4)-β-Glc A-(1 →4)-α-Glc NS6S(1→(7.63%)。(4)Antithrombotic activity of heparin from Mactra antiquata and Meretrix lusoria|G2 and M2 both have good anticoagulant activity.Among them,G2 exerts anticoagulant effect mainly through exogenous coagulation pathway,endogenous coagulation pathway and blocking the formation of fibrin;M2 exerts its anticoagulant effect mainly through exogenous coagulation pathway and endogenous coagulation pathway.The fibrinolytic activities of G2 and M2 measured by agarose fibrin plate method were 1.96 ± 0.11 U/mg and 1.41 ± 0.10U/mg,respectively.G2 can promote fibrinolytic activity in vivo by promoting the release of t-PA and u-PA,while inhibiting the release of PAI-1;M2 can exert fibrinolytic activity by promoting the release of t-PA and u-PA.In addition,mouse tail-break experiments have shown that G2 and M2 have lower bleeding side effects than traditional heparin.