Structure And Anticoagulant Activity Analysis Of Acidic Polysaccharides From Patinopecten Yessoensis Viscera

Scallop(Patinopecten yessoensis)is mainly produced in our country,such as Liaoning and Shandong provinces.The more scallop processing industries increase,the more by-products are produced resulting in environmental pollution and waste of resources.Therefore,the rational and efficient use of this resource has become a problem to be solved.In this paper,viscera which are the by-products of scallop processing were treated by enzymolysis,alcohol precipitation and cetylpyridinium chloride precipitation to afford the crude polysaccharide.The DEAE anion exchange column was used to obtain five fractions,namely SVP1,SVP2,SVP3,SVP4 and SVP5.The major fraction SVP2 was further purified by CL-6B gel chromatography to obtain two homogeneous polysaccharide fractions,named SVP2-1 and SVP2-2.Their protein contents were 5.5%and 3.7%,repectively,as measured by Folin-phenol reagent method.The uronic acid contents of SVP2-1 and SVP2-2 were determined as 2.2%and 4.3%,repectively,by tetrahydroxybiphenyl method.The gelatin nophelometry method was used to determine sulfated contents which were 8.1%and 13.8%,respectively.The molecular weight of SVP2-1 calculated from the calibration curve of dextran standards by gel chromatography was approximately 630 KDa,while the molecular weight of SVP2-2 was approximately 63 KDa.The results of scanning electron microscopy showed that the surface structure of the two components were flaky and smooth.In order to elucidate the structure of SVP2-1 and SVP2-2,the liquid-phase mass spectrometry,NMR and methylation techniques were applied.Monosaccharide compositions were measured by GC showing that they both have xylose,arabinose,fucose,rhamnose,galactose,mannose,glucose,glucosamine,galactosamine,and glucuronic acid but with different ratios.From the results of disaccharide analysis,the main disaccharide fragments of both SVP2-1 and SVP2-2 included hexose-uronic acid(m/z 687),hexose-hexose(m/z 673),and deoxyhexose-hexose(m/z 657).Comparing with the methylation of the desulfation samples and the native polysaccharide,the linkage pattern and the sulfation position of the SVP2-1 and SVP2-2 can be obtained.From the results,there are many kinds of glycosidic bonds,and the sulfate substitution in both SVP2-1 and SVP2-2 was at the C-4 of Man(1?and?3)Gal(1?.From the partial acid hydrolysis result,the backbone of SVP2-2consists of?6)Man_p(1?3)Gal_p(1?,and Xly or Glc was linked to C-4 position of the?3)Gal_p(1?.Finally,the anticoagulant activities of the two components were analyzed and the anticoagulant activities of SVP2-1 and SVP2-2 were concentration-dependent.SVP2-1 and SVP2-2 can inhibit clotting and thrombin activity through intrinsic and common pathways,and they can inhibit the transformation of fibrinogen into fibrin.It was observed that SVP2-1 showed better anticoagulant ability than SVP2-2 in terms of APTT,FIB and TT.