Structure And Bioactivity Analysis Of Several Acid Polysaccharides From Sea Animal: Searching For Alternatives To Heparin

Heparin, a complex glycosaminoglycan polysaccharide, is widely used as an anticoagulant in a number of settings, including kidney dialysis and acute coronary syndromes for many years.-Recently, certain heparins have been proved to associate with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. These observations indicate that we still need alternatives to heparin, perhaps new polysaccharides with fewer side effects and which would be more appropriate for each clinical condition. There is now more interest in therapeutics prepared from non-mammalian sources, thus avoiding the risk of contamination with pathogenic agents. The sulfated polysaccharides from marine are important nature resources. In this article we focused on analyzing structure and bioactivity of three kinds of sea animal acid polysaccharides, the Fucosylated chondroitin sulfate (FCS) and the sulfated fucan from sea cucumber, as well as a glycosaminoglycans-like non-sulfated polysaccharide from squid ink. The content includes the following five parts:1) Structure determination of the a non-sulfated polysaccharide from squid ink. A non-sulfated polysaccharide was isolated from the ink sac of squid Ommastrephes bartrami after removal of the melanin granules. The carbohydrate sequence of this polysaccharide was assigned by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions produced by partial acid hydrolysis of the polysaccharide. The structural determination was completed by NMR for assignment of anomeric configuration and confirmation of linkage and it was unambiguously identified as a glycosaminoglycan-like polysaccharide containing a glucuronic acid-fucose (GlcA-Fuc) disaccharide repeat in the main chain and a branched N-acetylgalactosamine (GalNAc) at the Fuc position:-[3GlcAβ1-4(GalNAca1-3)Fucal]n-. Partial hydrolysis of the polysaccharide to obtain several oligosaccharide fractions with different numbers of the repeating unit assisted the assignment.In the negative-ion tandem mass spectrometric analysis, the unique 0,2A type fragmentation was important to establish the presence of a 4-linked fucose in the main polysaccharide chain and a GalNAc branch at the Fuc position of the disaccharide repeat.2) Comparison on Structure and anticoagulant activity comparasion of Fucosylated chondroitin sulfates (fCSs) from four kinds of sea cucumbers. the Fucosylated chondroitin sulfates (fCSs) were isolated from four sea cucumbers, Pearsonothuria graeffei (Indo-Pacific), Stichopus tremulus (Western Indian Ocean), Holothuria vagabunda (Norwegian coast), and Isostichopus badionotus (Western Atlantic). The detailed sequences of fCSs, particularly their fucose branches, were characterized and compared.'H-and 13C-NMR of the polysaccharides clearly identified three different sulfation patterns on the branched fucoses,4-O-mono-,2,3-O-di-and 2,4-O-disulfation, variously distributed in the four fCSs. Anticoagulant activities of the four fCSs were assessed and compared.3) Selective mild acid hydrolysis of a novel fucan from sea cucumber with no sulfate loss: preparation and structure analysis using analysis using ESI-MS and NMR, and anticoagulant activity of the oligosaccharides. A novel linear sulfated fucan was isolated from sea cucumber Isostichopus badionotus, no specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. The structural determination of the fucan was assigned by 1D and 2D NMR firstly, and the sequence of this polysaccharide and was further confirmed by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions. The structure of the fucan was finally identified as a regular repeating sequence of [3-a-L-Fucp-(2,4SO4)-(1→3)-a-L-Fucp-(2SO4)-(1→3)-a-L-Fucp-(2SO4)-(1→3)-a-L-Fucp-(1→]. And acid choose a preferential cleavage of the glycosidic linkage formed between the nonsulfated residue (D in Figure 1) and the second 2,4-sulfated unit (unit A), no obvious loose of sulfate group happened. In the negative-ion tandem mass spectrometric analysis, the unique 1,4A ions in squid was important to establish the presence of a 3-linked fucose in the main polysaccharide chain.4) Preparation of the fucosylated chondrotin sulfate oligosaccharides from sea cucumberⅠ. badionotus using peroxide oxidation and the anticoagulant activity of the oligosaccharides. The fucose branches are reported to be the key factor on the keeping the bioactivity of FCS and it is liable to lose in acid condition. The mechanism of the acid hydrolysis and the peroxide were investigated.Acid hydrolysis brought on loss of fucose branches and sulfate group in the backbone GalNAc, which will resulted in loss of anticoagulant activity. Using a peroxide strategy mediated by the copper ions, we prepared oligosaccharides with different degree of polymerization from a new fCS fromⅠ. badionotus, the free radical mainly attacked on the GlcA in the backbone and have no obvious effect on the sulfated fucose branch and sulfation pattern of the backbone GalNAc. ESI-MS/MS and NMR were used to analyze the structure of these highly sulfated oligosaccharides. The structure of fucosylated chondrotin sulfate were determination to be:-[3GalNAcβ(4,6S)1-4GlcAβ(Fuca(2,4S)1-3) 1]n-.5) Anticoagulant and anti-thrombosis activity analysis of sea cucumber chondroitin sufalte The results indicated that the difference in their anticoagulant activities can be attributed to the difference in sulfation pattern of the fucose branch of the chondroitin sulfate, and 2,4-O-disulfation is important for anticoagulant activity. Their influence on anti-thrombin and anti-FXa activities mediated by antithrombinⅢand heparin cofactorⅡwere further evaluated. The fucosylated chondroitin sulfate inhibits thrombin by mainly heparin cofactorⅡ, whereas the sulfated fucan inhibits both thrombin and FXa activities mediated by antithrombinⅢ. Those SC-CHS with 2,4-SO4 have better effect than the 4-SO4 fucose branch. As a result of the complex anticoagulant mechanism, the invertebrate polysaccharides differ in their effects on experimental thrombosis in vitro indicated those with 2,4-SO4 sulfated fucose branches also have a better effect, but both of the SC-CHS have better effect than the sulfated fucan FUC-Ib.Further bioactivity research on the oligosaccharides from geltration and HPLC-SAX shows a both the oligosaccharides D1-D8 and D4Ⅱ, D7I prolong APTT, but have no effect on PT and TT. Further evaluating their influence on anti-thrombin activities mediated by antithrombinⅢand heparin cofactorⅡindicated the oligosaccharides D1 and D4II have the effect of on inhibition of thrombin mediated by heparin cofactorⅡ, and D4Ⅱthe heptasacchride with 10 sulfates is the minimum unit to have an anticoagulant activity, the structure of the oligosaccharide were:GalNAcOOHβ(4,6S)1-4GlcAβ(Fuca(2,4S)1-3)1-3GalNAcβ(4,6S)-4GlcAβ(Fuca(2,4S)1-3) 1-3GalNAcβ(4,6S). This is the first research on structure-activity relationship about sea cucumber FCSs in an oligosaccharides level.6) Sulfation of squid ink polysaccharides and its effect on anticoagulant and tumor metastasis. The squid ink polysaccharides (SIP) and tributylammonium salt of SIP were firstly sulfated with pyridine-sulphur-trioxide complex in DMSO sulfated. Structure characterization of sulfated SIPs using NMR indicated that sulfation mainly occurred at the 4,6-postion of GalNAc. Anticoagulant activity assay shows it has no obvious effect on TT, but prolong PT and APTT, which indicated the anticoagulant effects multi-coagulation processes. By evaluating their influence on anti-thrombin and anti-FXa activities mediated by antithrombinⅢand heparin cofactorⅡ, the sulfate SIP TBA-1 obviously inhibited the activity of FIIa and FXa. The effects of the sulfated SIP (TBA-1) on tumor cell growth, invasion, and migration were examined in vitro, and its antiangiogenesis effect was measured in vivo using the chick embryo chorioallantoic membrane (CAM) assay. TBA-1 did not have any obvious effects on the proliferation of HepG2 tumor cells, but induced a dose-dependent suppression of cell invasion and migration in HepG2. Moreover, TBA-1 significantly inhibited angiogenesis in a CAM model. Thus, our results indicate that TBA-1 is a potential candidate compound for the prevention of tumor metastasis.