【作者】 嵇国利；

【导师】 于广利；

【作者基本信息】 中国海洋大学 ， 药物化学， 2010， 硕士

【摘要】 本文以刺松藻(Codium fragile (Suringar) Hariot)为原料,经85%乙醇脱脂后采用室温水、90℃热水和70℃2%Na2CO3水溶液提取,分别得到了3种粗多糖CFC、CFH和CFA,其得率分别为7.0%,8.1%和0.9%。运用高效凝胶渗透色谱(HPGPC)、高效离子色谱(HPIC)及傅里叶变换红外光谱(FTIR)等方法对其分子量、单糖组成和结构特征进行了分析。结果表明,CFC、CFH和CFA均含有不同含量的半乳糖(Gal)、阿拉伯糖(Arb)、葡萄糖(Glc)、甘露糖(Man)和木糖(Xyl),其摩尔比分别为3.65：2.71：1.00：0.85：0.55和1.10：1.01：1.00: 0.92：0.21及0.51：0.34：1.00：0.61：0.05。CFC硫酸化程度最高,CFH次之,CFA硫酸化程度最低,其硫酸基含量分别为23.4%,17.5%和9.7%。在上述理化性质分析基础上,采用离子交换色谱分离技术,从CFC和CFH中纯化得到了组分CFCP1～P6和CFHP1～P5共11种多糖。对CFCP4和CFCP6进一步采用Sepharose 6B-FF柱层析纯化得到分子量均一的多糖组分。结果表明,CFCP4和CFCP6分子量分别为29 kD和56 kD,硫酸基含量分别为21.0%和32.4%,单糖组成分别以Gal和Ara为主。通过核磁共振波谱(NMR)和脱硫前后甲基化分析表明,CFCP4是硫酸半乳聚糖,以β1,3连接为主链(60%),β1,6链接为支链(40%)；链内硫酸基位于C4位,分支末端硫酸基位于C6位；分支末端C3和C4位-OH与丙酮酸环化形成五元环。CFCP6是硫酸阿拉伯聚糖,以β1,3连接为主链,30%的阿拉伯糖在C2-OH处有分支,硫酸基位于阿拉伯糖的C4位和C2位,分子中含有少量半乳糖。为了确定其细微结构,纯化的CFCP4和CFCP6分别经氢型强阳离子交换树脂和自由基降解获得寡糖混合物,再经BioGel-P4柱分离法,从中获得了14种寡糖,并通过电喷雾碰撞诱导串联质谱法(ESI-CID-MS/MS)对CFCP4中8种硫酸半乳寡糖和CFCP6中6种硫酸阿拉伯寡糖进行了序列分析,确定其结构序列分别是：Galp6Sβ1→6Galp、Galpβ1→6Galpβ1→3Galp4S、Galp6Sβ1→6Galpβ1→6Galpβ1→6Galp、Galp6Sβ1→3Galpβ1→3 Galpβ1→3Galpβ1→6Galp Galp6Sβ1→6Galp4Sβ1→6Galp、Galp6Sβ1→6Galpβ1→6Galp4Sβ1→6Galp Galp6Sβ1→6Galpβ1→6Galpβ1→6Galpβ1→6Galp4S、3,4PyrGalp6Sβ1→6Galp以及Arbpβ1→3Arbp4S、Arbp4Sβ1→3Arbpβ1→3Arbp、Arbp4Sβ1→3Arbp4S、Arbp4Sβ1→3Arbp2Sβ1→3)Arbp、Arbp4Sβ1→3Arbp2Sβ1→3Arbpβ1→3Arbp、Arbp4Sβ1→3Arbpβ1→3Arbp2Sβ1→3Arbpβ1→2Arbp。这些寡糖结构进一步证明CFCP4是β1,6-链接为支链的硫酸半乳聚糖,CFCP6是β1,3-链接为主链的硫酸化阿拉伯聚糖。这些结构特殊的寡糖不仅丰富了海洋寡糖库数据,也为进一步从事糖生物芯片研究,探讨其与蛋白之间的相互作用提供了基础。在上述结构研究基础上,以APTT、TT、PT为评价指标,对三种粗多糖CFC、CFH和CFA以及三种纯化多糖CFCP1、CFCP4和CFCP6的抗凝血活性进行了评价。通过与肝素对照的结果揭示,CFC及其纯化组分CFCP1抗凝活性较强,而CFCP6的抗凝活性较弱,说明刺松藻多糖的抗凝活性除了与结构有关外,还与分子量有关,分子量越高,抗凝活性越强。CFCP1主要通过抑制内源性凝血途径和共同凝血途径起作用,而对外源性凝血途径影响较弱。该结果为开发海洋抗凝血药物提供了基础。更多还原

【Abstract】 In this paper, the green alga Codium fragile (Suringar) Hariot, was used as starting material, the alga was firstly delipided with 85% ethanol, then extracted with room temperature water,90℃hot water and 2% sidium bicarbonate water solution at 70℃, three types of crude polysaccharides, which named as CFC, CFH and CFA, were acquired. The yields of CFC, CFH and CFA were 7.0%,8.1% and 0.9%, respectively. Their relative molecular weight (Mw), monosaccharide composition and structural features were determined with high performance gel permeation chromatography (HPGPC), high performance ion chromatography (HPIC) and Fourier transform infrared spectroscopy (FTIR), respectively. The results showed that CFC, CFH and CFA were composed of different contents of Gal, Ara, Glc, Man and Xyl, with the molar ratio of 3.65:2.71:1.00:0.85:0.55,1.10:1.01:1.00:0.92:0.21 and 0:51:0.34:1.00:0.61:0.05. Their sulfate contents were different, CFC have the highest sulfate content, then was CFH and CFA, of which were 23.4%,17.5% and 9.7%, respectively.Based on the physicochemical analysis of CFC and CFH, six kinds of polysaccharides (CFCP1～P6) and five kinds of polysaccharides (CFHP1～P5) were respectively separated from CFC and CFH by using Q-Sepharose FF anion-exchange chromatography. Two kinds of molecular uniform pure fractions CFCP4 and CFCP6 were further acquired from CFC with Sepharose 6B Fast Flow gel permeation chromatography. The Mw of CFCP4 and CFCP6 were 29 and 56 kD, separately; the sulfate content were 21.0% and 32.4%, respectively. CFCP4 and CFCP6 was mainly composed by galactose and arabinose, separately. Based on the NMR and methylation analysis to CFCP4 and CFCP6 and their desulfated, counterpart, CFCP4 and CFCP6 were determined as sulfated galactan and sulfated arabinan. The backbone of CFCP4 wasβ(1→3)-linked galactose (60%), and the branched chain wasβ(1→6)-linked galactose residues (40%). The sulfate groups were located at C4-OH and C6-OH, and pyruvic acid was linked at C3-OH and C4-OH of terminal galactose residues. The backbone of the CFCP6 was consists ofβ(1→3)-arabinose residues, and the branch points were found at C2-OH (30%), and the sulfate located at C4-OH and C2-OH.To determine their fine structures, the purified polysaccharide CFCP4 and CFCP6 were degraded with the methods of hydrogen form strong-cation exchange resin and free radical degradation, the oligosaccharides mixture was separated on BioGel-P4 column,14 kinds of oliogosaccharides were acquired. The sequence and structure of eight galactooligosaccharides and six arabinooligosaccharides were identified with electrospray ionization tandem mass spectrometry (ESI-CID-MS/MS). Their structures were as following:Galp6Sβ1→6Galp, Galpβ1→6Galpβ1→3Galp4S, Galp6Sβ1→6Galpβ1→6Galpβ1→6Galp, Galp6Sβ1→3Galpβ1→3 Galpβ1→3Galpβ1→6 Galp, Galp6Sβ1→6Galp4Sβ1→6Galp, Galp6Sβ1→6Galpβ1→6Galp4Sβ1→6 Galp,Galp6Sβ1→6Galpβ1→6Galpβ1→6Galpβ1→6Galp4S,3,4PyrGalp6Sβ1→6Galp; and Arbpβ1→3Arbp4S, Arbp4Sβ1→3Arbpβ1→3Arbp, Arbp4Sβ1→3Arbp4S, Arbp4Sβ1→3Arbp2Sβ1→3)Arbp,Arbp4Sβ1→3Arbp2Sβ1→3Arbpβ1→3Arbp,and Arbp4Sβ1→3Arbpβ1→3Arbp2Sβ1→3Arbpβ1→2Arbp.All these unique oligosaccharides offered useful message for construction of marine carbohydrate library. More importantly, they provide the foundation for the preparation of the oligosaccharide-chip and the investigation on the oligosaccharide-protein Interaction.Based on the structure analysis, the anticoagulant activities of three crude polysaccharides (CFC, CFH and CFA) and three purified polysaccharides (CFCP1, CFCP4 and CFCP6) were evaluated by assays of the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT). The results showed that CFC and CFCP1 showed high anticoagulant activities with heparin as positive control, CFCP6, which have similar physicochemical properties to CFCP1, showed lower anticoagulant activities. Apart from the structural features, the major differences of anticoagulant activities between these polysaccharides were associated with relative molecular weight, the higher Mw showed the stronger anticoagulant. The mechanism of anticoagulant of CFCP1 was due to intrinsic and common pathway, but not extrinsic pathway. The results provided theoretical foundation to marine drugs development.更多还原