褐藻中酸性多糖的结构分析及神经保护活性研究
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摘要
褐藻中的多糖包含褐藻胶，褐藻多糖硫酸酯和海带淀粉等。褐藻多糖硫酸酯是一类结构复杂的硫酸化杂多糖。本文主要对海带和羊栖菜中的褐藻多糖硫酸酯中的硫酸化杂多糖，硫酸化半乳岩藻聚糖和硫酸化岩藻聚糖（三者统一称为酸性多糖）进行结构表征。探讨褐藻多糖硫酸酯的分子量，岩藻糖和半乳糖摩尔比与其抗凝血活性的构效关系。研究褐藻多糖硫酸酯中发挥神经保护的活性组分，通过马尾藻属多糖进一步确认硫酸化杂多糖为神经保护的活性组分。同时，研究了神经保护与抗氧化之间的关系。主要得到以下结论：
1.通过对F0.5，F1和F2组分的结构研究，推断粗褐藻多糖硫酸酯具有如下结构单元：
①硫酸化的岩藻聚糖：主链以3-连接岩藻糖组成，少量含有4-连接岩藻糖，且硫酸化位置主要为C2或者C4位，同时在岩藻糖的C2位有部分葡萄糖醛酸基化；
②甘露葡萄糖醛酸聚糖：主链以2-连接甘露糖和4-连接葡萄糖醛酸交替连接组成，同时在2-连接甘露糖的C6位有部分硫酸化和C3有部分岩藻糖基化；
③葡萄糖醛酸聚糖：主链以3-连接葡萄糖醛酸组成，其C4位有岩藻糖基支链；
④硫酸化半乳聚糖：以6-连接半乳糖为主链，且硫酸化位置为C4位；
⑤木聚糖：以4-连接木糖为主链。
同时，我们发现分级组分（F0.5，F1和F2）各有相似性和差异性，主要表现在以下几方面：
（Ⅰ）在单糖组分方面，F0.5含有岩藻糖和半乳糖之外的其他单糖组分（甘露糖，葡萄糖醛酸和葡萄糖等），F1主要以岩藻糖和半乳糖为主，伴随有少量的其他单糖组分，F2主要以岩藻糖为主。
（Ⅱ）在硫酸根方面，F0.5，F1和F2的硫酸根含量依次增加。
（Ⅲ）在分子量方面，F0.5，F1和F2的分子量依次增加。
（Ⅳ）但是从结构单元来讲，F0.5，F1和F2均具有硫酸化的岩藻聚糖，F0.5和F1均具有甘露葡萄糖醛酸聚糖，葡萄糖醛酸聚糖和木聚糖等。
2.羊栖菜来源的褐藻多糖硫酸酯与海带来源的褐藻多糖硫酸酯的差异性：两者有共同的结构特征单元（岩藻聚糖，甘露葡萄糖醛酸聚糖，半乳聚糖和木聚糖等），但是其主链上的硫酸化的方式以及支链的类别不同，最为不同的是羊栖菜中的硫酸化半乳岩藻聚糖是以岩藻聚糖（岩藻寡糖）和半乳聚糖（半乳寡糖）交替连接的主链。此外，羊栖菜中含有相当量的海带淀粉。
3.分子量和半乳糖与岩藻糖摩尔比对抗凝血活性的影响：通过阴离子交换色谱法和过氧化氢降解法，制备了七种不同分子量和四种不同半乳糖与岩藻糖摩尔比的样品。通过抗凝血活性测定，推断样品的分子量和半乳糖与岩藻糖的摩尔比影响抗凝血活性。且在APTT和TT实验中，抗凝血活性还与样品浓度有关。
4.褐藻多糖硫酸酯及其分级组分的神经保护活性及神经保护活性与抗氧化的关系：通过对海带来源的褐藻多糖硫酸酯的分级组分，降解分级组分以及二次分级组分进行神经保护活性测定，推测神经保护活性的物质主要为杂多糖，含有相对较少的岩藻糖含量（少于20%）和硫酸根含量（少于25%），高的糖醛酸含量（多于10%）。为了验证此假设，对马尾藻属多糖进行神经保护活性测定，发现部分马尾藻属多糖没有活性，同时没有活性的马尾藻多糖缺少糖醛酸含量较高的组分，因此对所有马尾藻多糖通过阴离子交换色谱法进行分级，然而失望的是，所有分级组分均无活性，因此只能暂时推测神经保护的活性需要各组分的协同作用。最后，基于本实验的数据，我们推测去除羟自由基活性，去除DPPH自由基活性和还原能力与神经保护活性有关，但是去除超氧自由基活性与神经保护活性无关。
Polysaccharides from brown seaweeds contained mainly algin, fucoidan andlaminaran. Fucoidan was a family of complex sulfated heteropolysaccharides. Thisstudy focused on the structural elucidation of the fucoidan from Saccharina japonica,including sulfated heteropolysaccharides, sulfated galactofucan and sulfated fucan,which are named together as acidic polysaccharides. In addition, the structure of thefucoidan from Sargassum fusiforme (Hizikia fusiforme) was investigated. Then therelationships between molecular weight, the molar ratio of galactose (Gal) to fucose(Fuc) and anticoagulant activity were also discussed. Finally, sulfatedheteropolysaccharides contributed to the neuroprotective activity, which wasconfirmed by the results of fucoidan and its fractions from S. japonica and Sargassumspecies. The main results were as follows:
1. According to the results of F0.5, F1and F2, it was concluded that the structuralfeatures of fucoidan from S. japonica were as follows:
(1) Sulfated fucan: a backbone of (1→3)-linked Fucp majorly and (1→4)-linkedFucp minorly, sulfated at C2or C4and branched partly at C2with glucuronic acid(GlcA);
(2) Sulfated fucoglucuronomannan: a backbone of alternating2-linked mannose(Man) and4-linked GlcAp, sulfated partly at C6of Man and branched partly at C3with Fuc;
(3) Fucoglucuronan:3-linked GlcA, branched at C4with Fuc;
(4) Sulfated galactan:6-linked Galp, sulfated at C4;
(5) Xylan:4-linked xylose (Xyl).
In addition, after compared with the results of F0.5, F1and F2, it was concludedthat the similarities and differences among F0.5, F1and F2were as follows:Firstly, F0.5contained Fuc, Gal, Man and Glc, et al., F1consisted majorly of Fuc and Gal,and F2was composed of Fuc on the aspects of monosaccharides. Secondly, the sulfatecontents and molecular weight of F0.5, F1and F2increased. Thirdly, F0.5, F1and F2all had a backbone of (1→3)-linked Fucp, and F0.5and F2had glucuronomannan,glucuronan and Xylan et al.
2. The similarities and differences of fucoidans from S. japonica and S. fusiforme:Both fucoidans contained the same structure units (fucan, glucuronomannan, galactanand xylan et al.). Except the differences of the sulfation pattern and branched residues,the major difference was sulfated galactaofucan from S. fusiforme, which had abackbone of either alternating Gal and Fuc sulfated at C2or alternating (Gal)nand(Fuc)nsulfated at C2. Moreover, fucoidan from S. fusiforme had substantial amount oflaminaran.
3. The relationships between molecular weight, the molar ratio of galactose (Gal) tofucose (Fuc) and anticoagulant activity: Seven fucoidans that differed with theaverage molecular weight and four fucoidan that differed with the average molecularweight and the molar ratio of Gal to Fuc were obtained by anion exchangechromatography and degradation with hydrogen peroxide and ascorbic acid. It wasconcluded that both the average molecular weigh and the molar ratio of Gal to Fucaffected the anticoagulant activity. Moreover, this activity exhibited dose dependencein the APTT and TT assays.
4. The neuroprotective activity of fucoidan and its fraction and the relationshipbetween neuroprotective activity and antioxidant activity: After the neuroprotectiveactivities of fucoidan and its fractions from S. japonica were determined, it wasconcluded that sulfated heteropolysaccharide, which contained Fuc (less than20%),sulfate (less than25%) and uronic acid (UA)(more than10%), contributed to theneuroprotective activity. In addition, the neuroprotective activities of fucoidan fromSargassum species were also determined. It was indicated that fraction that containedhigh UA was the principal difference between the active and non-active fucoidans byanion exchange chromatography. However, all fractions did not correlate withneuroprotective effect, suggesting that the neuroprotective effect depended on a variety of factors. Finally, the hydroxyl-radical scavenging effect, the DPPH-redicalscavenging effect and reducing power, not superoxide-radical scavenging effect werecorrelated with the neuroprotective activities based on the data.
1.通过对F0.5，F1和F2组分的结构研究，推断粗褐藻多糖硫酸酯具有如下结构单元：
①硫酸化的岩藻聚糖：主链以3-连接岩藻糖组成，少量含有4-连接岩藻糖，且硫酸化位置主要为C2或者C4位，同时在岩藻糖的C2位有部分葡萄糖醛酸基化；
②甘露葡萄糖醛酸聚糖：主链以2-连接甘露糖和4-连接葡萄糖醛酸交替连接组成，同时在2-连接甘露糖的C6位有部分硫酸化和C3有部分岩藻糖基化；
③葡萄糖醛酸聚糖：主链以3-连接葡萄糖醛酸组成，其C4位有岩藻糖基支链；
④硫酸化半乳聚糖：以6-连接半乳糖为主链，且硫酸化位置为C4位；
⑤木聚糖：以4-连接木糖为主链。
同时，我们发现分级组分（F0.5，F1和F2）各有相似性和差异性，主要表现在以下几方面：
（Ⅰ）在单糖组分方面，F0.5含有岩藻糖和半乳糖之外的其他单糖组分（甘露糖，葡萄糖醛酸和葡萄糖等），F1主要以岩藻糖和半乳糖为主，伴随有少量的其他单糖组分，F2主要以岩藻糖为主。
（Ⅱ）在硫酸根方面，F0.5，F1和F2的硫酸根含量依次增加。
（Ⅲ）在分子量方面，F0.5，F1和F2的分子量依次增加。
（Ⅳ）但是从结构单元来讲，F0.5，F1和F2均具有硫酸化的岩藻聚糖，F0.5和F1均具有甘露葡萄糖醛酸聚糖，葡萄糖醛酸聚糖和木聚糖等。
2.羊栖菜来源的褐藻多糖硫酸酯与海带来源的褐藻多糖硫酸酯的差异性：两者有共同的结构特征单元（岩藻聚糖，甘露葡萄糖醛酸聚糖，半乳聚糖和木聚糖等），但是其主链上的硫酸化的方式以及支链的类别不同，最为不同的是羊栖菜中的硫酸化半乳岩藻聚糖是以岩藻聚糖（岩藻寡糖）和半乳聚糖（半乳寡糖）交替连接的主链。此外，羊栖菜中含有相当量的海带淀粉。
3.分子量和半乳糖与岩藻糖摩尔比对抗凝血活性的影响：通过阴离子交换色谱法和过氧化氢降解法，制备了七种不同分子量和四种不同半乳糖与岩藻糖摩尔比的样品。通过抗凝血活性测定，推断样品的分子量和半乳糖与岩藻糖的摩尔比影响抗凝血活性。且在APTT和TT实验中，抗凝血活性还与样品浓度有关。
4.褐藻多糖硫酸酯及其分级组分的神经保护活性及神经保护活性与抗氧化的关系：通过对海带来源的褐藻多糖硫酸酯的分级组分，降解分级组分以及二次分级组分进行神经保护活性测定，推测神经保护活性的物质主要为杂多糖，含有相对较少的岩藻糖含量（少于20%）和硫酸根含量（少于25%），高的糖醛酸含量（多于10%）。为了验证此假设，对马尾藻属多糖进行神经保护活性测定，发现部分马尾藻属多糖没有活性，同时没有活性的马尾藻多糖缺少糖醛酸含量较高的组分，因此对所有马尾藻多糖通过阴离子交换色谱法进行分级，然而失望的是，所有分级组分均无活性，因此只能暂时推测神经保护的活性需要各组分的协同作用。最后，基于本实验的数据，我们推测去除羟自由基活性，去除DPPH自由基活性和还原能力与神经保护活性有关，但是去除超氧自由基活性与神经保护活性无关。
Polysaccharides from brown seaweeds contained mainly algin, fucoidan andlaminaran. Fucoidan was a family of complex sulfated heteropolysaccharides. Thisstudy focused on the structural elucidation of the fucoidan from Saccharina japonica,including sulfated heteropolysaccharides, sulfated galactofucan and sulfated fucan,which are named together as acidic polysaccharides. In addition, the structure of thefucoidan from Sargassum fusiforme (Hizikia fusiforme) was investigated. Then therelationships between molecular weight, the molar ratio of galactose (Gal) to fucose(Fuc) and anticoagulant activity were also discussed. Finally, sulfatedheteropolysaccharides contributed to the neuroprotective activity, which wasconfirmed by the results of fucoidan and its fractions from S. japonica and Sargassumspecies. The main results were as follows:
1. According to the results of F0.5, F1and F2, it was concluded that the structuralfeatures of fucoidan from S. japonica were as follows:
(1) Sulfated fucan: a backbone of (1→3)-linked Fucp majorly and (1→4)-linkedFucp minorly, sulfated at C2or C4and branched partly at C2with glucuronic acid(GlcA);
(2) Sulfated fucoglucuronomannan: a backbone of alternating2-linked mannose(Man) and4-linked GlcAp, sulfated partly at C6of Man and branched partly at C3with Fuc;
(3) Fucoglucuronan:3-linked GlcA, branched at C4with Fuc;
(4) Sulfated galactan:6-linked Galp, sulfated at C4;
(5) Xylan:4-linked xylose (Xyl).
In addition, after compared with the results of F0.5, F1and F2, it was concludedthat the similarities and differences among F0.5, F1and F2were as follows:Firstly, F0.5contained Fuc, Gal, Man and Glc, et al., F1consisted majorly of Fuc and Gal,and F2was composed of Fuc on the aspects of monosaccharides. Secondly, the sulfatecontents and molecular weight of F0.5, F1and F2increased. Thirdly, F0.5, F1and F2all had a backbone of (1→3)-linked Fucp, and F0.5and F2had glucuronomannan,glucuronan and Xylan et al.
2. The similarities and differences of fucoidans from S. japonica and S. fusiforme:Both fucoidans contained the same structure units (fucan, glucuronomannan, galactanand xylan et al.). Except the differences of the sulfation pattern and branched residues,the major difference was sulfated galactaofucan from S. fusiforme, which had abackbone of either alternating Gal and Fuc sulfated at C2or alternating (Gal)nand(Fuc)nsulfated at C2. Moreover, fucoidan from S. fusiforme had substantial amount oflaminaran.
3. The relationships between molecular weight, the molar ratio of galactose (Gal) tofucose (Fuc) and anticoagulant activity: Seven fucoidans that differed with theaverage molecular weight and four fucoidan that differed with the average molecularweight and the molar ratio of Gal to Fuc were obtained by anion exchangechromatography and degradation with hydrogen peroxide and ascorbic acid. It wasconcluded that both the average molecular weigh and the molar ratio of Gal to Fucaffected the anticoagulant activity. Moreover, this activity exhibited dose dependencein the APTT and TT assays.
4. The neuroprotective activity of fucoidan and its fraction and the relationshipbetween neuroprotective activity and antioxidant activity: After the neuroprotectiveactivities of fucoidan and its fractions from S. japonica were determined, it wasconcluded that sulfated heteropolysaccharide, which contained Fuc (less than20%),sulfate (less than25%) and uronic acid (UA)(more than10%), contributed to theneuroprotective activity. In addition, the neuroprotective activities of fucoidan fromSargassum species were also determined. It was indicated that fraction that containedhigh UA was the principal difference between the active and non-active fucoidans byanion exchange chromatography. However, all fractions did not correlate withneuroprotective effect, suggesting that the neuroprotective effect depended on a variety of factors. Finally, the hydroxyl-radical scavenging effect, the DPPH-redicalscavenging effect and reducing power, not superoxide-radical scavenging effect werecorrelated with the neuroprotective activities based on the data.
引文
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