Comparsion Of Physicochemical Characteristics And Anticoagulant Activities Of Sulfated Polysaccharides From Four Species Of Sea Cucumbers

Fucosylated glycosaminoglycan (FGAG) is a glycosaminoglycan derivative with a special structure, which is found only in echinoderm so far. FGAG has strong potent anticoagulant activity, and its main pharmacological target of anticoagulant activity is intrinsic-tenase (f.Xase). Meanwhile, fucan sulfate(FS)has certain anticoagulant activity. So it has a good academic value and an application potentiality.In this study, the pured polysaccharides were isolated from the body wall of four species of sea cucumbers Holothuria edulis, Holothurica nobilis, Apostichopus japonicus and Stichopus variegates by alkaline hydrolysis, enzymatic digestion, salting-out, ethanol precipitation, gel filtration and ion-exchange chromatography. The polysaccharides were four FSs and four FGAGs named HE-1, HN-1, AJ-1, SV-1, HE-2, HN-2, AJ-2and SV-2, respectively.Physicochemical property analysis of HE-1, HN-1, AJ-1and SV-1showed that they had the fundamental property of acid mucopolysaccharides. Firstly, all the weight-average molecular masses (Mw) of four FSs were more than419,900D (419,900-615.500D), and their molecular weight distributions (Mw/Mn) were from1.19to1.86. Secondly, all the FSs were laevorotatory substances, and their optical rotations ranged from-193.4°to-165.3°. Thirdly, intrinsic viscosities of the four FSs were greater than208.6mL/g (208.6～605.6mL/g). The Huggins constants (KH) were greater than0, but Kraemer constants (KK) were less than0. Fourthly, as the composition analysis of the polysaccharides shown, these four kinds of FSs contained fucose as the only sugar with content of sulfate ester. In this paper, the mass percentages of sulfate radical were similar among different Holothuria of Holothuriae, and they remained a tiny discrepancy from different genera of Stichopodidae. The later family was bigger than the former family. Fifthly, infrared spectras (IRs) of the four FSs were in coincidence with the analysis of monosaccharide compositions. Nuclear magnetic resonance (NMR) analysis showed that there were some differences existed in these fundamental structures of four FSs. Finally, as the biological activities shown, all four FSs possessed certain anticoagulant activities, which were influenced by molecular weight and content of sulfate radical.The physicochemical properties of HE-2, HN-2, AJ-2and SV-2showed that the Mw of four FGAGs were less than51,090(51,090～62,456D), and molecular weight distributions (Mw/Mn) of these FGAGs were from1.19to1.27. The specific optical rotations of the FGAGs are levorotatory, and the values varied from-37.9°to-56.2°. The specific optical rotations were similar among those of same family, and the values of specific optical rotations from Stichopodidae were more than those from Holothuriae in this paper. The intrinsic viscosities of four FGAGs were less than42.7mL/g (24.6-42.7mL/g, KH<0, Kk<0). Analysis of monosaccharide compositions showed that four types of FGAGs were composed of three monosaccharides, namely glucuronic acid (GlcA). acetylgalactosamine (GalNAc) and L-fucose. In this paper, The mass percentages of sulfate radical were similar among those of Holothuria of Holothuriae, and the mass percentages were also close from different genera of Stichopodidae. The mass percentages of sulfate radical from Stichopodidae were more than the Holothuriae. The IRs were consistent with the analysis of monosaccharide compositions. As shown in NMR spectra, four types of FGAGs had an accordant basic structure, while the sulfation patterns and number were significantly different. The contents of2,4-di-O-sulfated fucose(L-Fuc2S4S) of SV-2, AJ-2, HE-2and HN-2were68%,45%,18%and0%, respectively. The anticoagulant activities of four FGAGs showed that anticoagulant activity of SV-2was the highest, and that of HN-2was least. The doubling activated partial thromboplastin time of AJ-2,SV-2, HE-2and HN-2were116U/mg,101U/mg,89U/mg,59U/mg, respectively. While the Mw of SV-2was more than the AJ-2. So the content of L-Fuc2S4S and molecular sizes result in the difference in anticoagulant activities of the FGAGs.Comparing the above results, the different type of polysaccharides had obviously different physicochemical characteristics in the same sea cucumber, while the same type of polysacchrides had similar physicochemical properties from different sea cucumber. The Mws of FSs were7.4times more than those of FGAGs. Four FGAGs were made of three kinds of monosaccharide, while the FSs containing.only L-fucose sulfate. The sulfate radical contents of the FGAGs and FSs were greater than20%. All the FSs and FGAGs were laevorotatory substances, and the optical rotation values of FSs were3times of those of FGAGs; however, the intrinsic viscosities of four FGAGs were less than those of the FSs. A good linear relationship was observed between log Mw and log [η] for eight different Mw of the sulfated polysaccharide solutions, and the Mark-Houwink-Sakurada equation, log[η]=1.181ogMw-5.00, was obtained by a linear regression (R2=0.97). Thus, the MHS parameters, K and a were given as K=1.00×10-5mL/g and a=1.18, respectively. In this paper, the FGAGs had stronger anticoagulant activities than those of the FSs, and the anticoagulant activities of Stichopodidae polysaccharides were stronger than those from the Holothuriae. So we could draw a conclusion that the anticoagulant activities of sulfated polysaccharides are likely related to the source of polysaccharides, monosaccharide composition, molecular masses and sulfate content.