沙蚕(Perinereis aibuhitensis)硫酸多糖的结构表征及抗凝血活性研究

Structure characterization and anticoagulant activities of a sulfated polysaccharide fromPerinereis aibuhitensis

目的对来源于沙蚕(Perinereis aibuhitensis)的硫酸多糖进行结构表征和抗凝血活性研究。方法采用两步酶解法从沙蚕中提取多糖;利用强阴离子交换色谱和凝胶渗透色谱对粗多糖进行分离纯化;通过离子色谱法、高效液相色谱法(HPLC)、高效凝胶渗透色谱-多角度激光光散射仪(HPGPC-MALLS)联用技术、硫酸软骨素酶ABC酶法分析、核磁共振氢谱(1 H-NMR)等方法,研究纯化多糖的化学组成和结构特征;通过测定APTT、PT和TT评价其体外抗凝血活性。结果从沙蚕中纯化得到了2种硫酸多糖组分PAE1和PAE2,PAE1主要由Gal、Glc、GalN、GlcA及少量Fuc组成;PAE2主链为硫酸软骨素C[→4GlcAβ1→3GalNAc6Sβ1→],支链主要由Fuc、Gal和Glc构成。PAE1和PAE2均可明显延长APTT和PT。结论首次从沙蚕中提取、分离得到2种硫酸多糖,其中PAE2是1种含有Fuc、Gal与Glc支链并具有明显的体外抗凝血活性的结构新颖的类硫酸软骨素,该发现为沙蚕硫酸多糖的开发提供了依据。

Objective To study the structure characteristics and anticoagulant activities of the sulfated polysaccharides fromPerinereis aibuhitensis.Methods The crude polysaccharides fromPerinereis aibuhitensis were prepared by the trypsin and papain digestion,which were further fractionated and purified by anion-exchange and gel-permeation chromatography.The chemical composition and structural characteristic of the purified polysaccharides were determined by ion chromatography,HPLC,HPGPCMALLS,chondroitinase ABC digestion and 1 H-NMR.The anticoagulant activities of the polysaccharides were evaluated by activated partial thromboplastin time（APTT）,prothrombin time（PT）and thrombin time（TT）.Results Two purified polysaccharides,designated as PAE1 and PAE2,were obtained fromPerinereis aibuhitensis.The contents of total sugar,protein,sulfate and molecular weight of PAE1 were 65.21%,14.31%,0.33%and 24.49 kDa,respectively.Monosaccharide composition analysis showed that PAE1 had 43.58%of Gal,32.63%of Glc,8.71%of GalNAc,7.66%of GlcA and2.77% of Fuc.The contents of total sugar,protein,sulfate and molecular weight of PAE2 were53.08%,11.33%,13.46% and 57.79 kDa,respectively.The backbone of PAE2 was chondroitin sulfate C（GlcAβ1→3GalNAc6S）with branched Fuc（35.33%）,Gal（20.9%）and Glc（8.98%）as side chains.In vitro anticoagulant assay indicated that PAE1 and PAE2could prolong APTT and PT,and exhibit anticoagulant activities through endogenous and extrinsic coagulation pathway.Conclusion Two polysaccharides were first isolated fromPerinereis aibuhitensis.The sulfated polysaccharide PAE2 was a structurally unique chondroitin sulfate with Fuc,Gal and Glc as branches,and exhibited remarkable anticoagulant activities in vitro.These data provided a basis for the exploitation of sulfated polysaccharides derived fromPerinereis aibuhitensis.