Physicochemical Characteristics And Anticoagulant Activities Of The Polysaccharides From Sea Cucumber Pattalus Mollis

Sea cucumber(Echinodermata,Holothuroidea)is a marine invertebrate.The body wall of sea cucumber contains bioactive acid polysaccharides,including fucosylated glycos-aminoglycan(FG),fucan sulfate(FS)and neutral glycan(NG).Native FG exhibits diverse pharmacological activities,such as intrinsic coagulation FXase(FVIIIa-FIXa-PL-Ca2+ enzy-me complex,FXase)inhibition,contact activation and platelet aggregation etc..Compared with native FQ its depolymerized FG(dFG)has lower molecular weight.The dFG can selectively inhibit FXase,while has no significant effect on contact activation and platelet aggregation.Most FS from echinoderm are structurally regular,consisting of repeating structural unit.The FS also have various pharmacological activities,including anticoagulant activity.Native FG and FS share some common structural feature,they both have abundant characteristic in structure,particularly,the glycosidic linkages and sulfated patterns are diverse.To further investigate the structural diversity of native polysaccharides from sea cucu-mber,the polysaccharides from body wall of Pattalus mollis were studied in this proj ect,including the polysaccharide types,physicochemical properties,structural characteristics Moreover,the anticoagulant activities of PmFG and PmFS,and structure-activity relation-ship were systematically evaluated.These studies will provide valuable data for the development of the novel anticoagulant and antithrombotic drugThe methods and results used in this project are listed as below(1)Extraction,purification and physicochemical properties of polysaccharides from P.mollis:PmFQ PmFS,PmNG-1 and PmNG-2 were extracted and purified by enzymatic-alkaline hydrolysis,ethanol-grading precipitation and strong ion exchange column chromatography.The HPGPC profiles showed that these polysaccharides had good homog-eneity.The physicochemical properties of PmFQ PmFS,PmNG-1 and PmNG-2 were analyzed by studying their molecular weight,monosaccharide composition,sulfate easter content,optical rotation,IR,MS and NMR spectra(2)Structure analysis of PmFG,PmFS and PmNG:PmFG and PmNG were depolym-erized by H2O2 treatment and trifluoroacetic acid,respectively,to prepare dPmFG and dPmNG.Then dPmFG-Ⅰ--Ⅲ were fractioned from the dPmFG by GPC(Gel permeation chromatography,GPC).PmFS-Ⅰ--Ⅲ were fractioned from PmFS by GPCAccording to the 1D\2D NMR spectra of the native(PmFG)and its depolymerized products,the chemical structure of PmFG was deduced.The backbone sequence of PmFG was {-4-D-GlcA-β1,3-D-GalNAc4S6S-β1-},and the branches L-Fuc linked to GlcA via al,3 glycosidic bonds.The structure of native PmFG was {-4-[L-FucR-α1,3]-D-GlcA-β1,3-D-GalNAc4S6S-β1-}n(R was 3S4S,2S4S or 4S with a molar ratio of 2.5:2:1)According to the 1D\2D NMR spectra of PmFS,its structure sequence was {-4-L-Fuc2S-α1,4-[L-FucR-α1,3]-L-Fuc2S-al,4-L-Fuc2S-α1-}n(R represented 4S or 3S with a molar ratio of 4:1),it possessed a sulfated α-Fuc in every trisaccharide unit.Its Mw(～6.12 kDa)was lower than the FS from other sea cucumbers reported previouslyBesides,the two neutral glycans,PmNG-1&-2,were structurally similar to animal glycogen.They consisted of D-Glc residues which linked via a1,4(major)and α1,6(minor,branching)glycosidic bonds(3)Preparation of series oligosaccharides of PmFG:PmFG was depolymerized by glycosidic-selective β-eliminative method.The glycosidic linkages of GalNAc-1,4-GlcA were selectively cleaved,and distinctive △4,5 unsaturated hexuronic acid was formed at the non-reducing end of resulting fragments,which had maximum absorption at λ232nm.A series of oligosaccharides Ⅰ-Ⅳ(tri-,penta-,octa-,hendecasaccharides,respectively)were purified from the depolymerized products(d’PmFG)by GPC.According to the HPGPC and NMR analysis,the structure of I was L-FucR-α1,3-L-△4,5 GlcA-α1,3-D-GalNAc4S6S-ol.And Ⅱ-Ⅳpossessed the structure of L-FucR-α1,3-L-△4,5 GlcA-α1,3-D-GalNAc4S6S-β1,4-{[L-FucR-α1,3-]-D-GlcA-β1,3-D-GalNAc4S6S-β1,14-}n-[L-FucR-α1,3-]-D-GlcA-ol(n=0,1,2;R = 3S4S,2S4S or 4S)(4)Anticoagulant activity and structure-activity relationship study:The results of APTT,PT and TT tests showed that both PmFG and PmFS had significantly APTT prolonging activity,while had no significant effect on PT and TT.PmNG-1&2 had no significant anticoagulant activity.Particularly,PmFG and its depolymerized products displayed potent anti-FXase activity,and their activity decreased with the reduction of the chain length.PmFS and PmFS-Ⅰ--Ⅲ also had potent anti-FXase activity,which was not significantly affected by the Mw.For FG oligosaccharides obtained by β-eliminative depolymerization,the octasaccharide was the the smallest structural fragment with potent FXase inhibition activity.