基于牡蛎糖原硫酸酯结构的多糖构效关系研究
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摘要
天然多糖具有多种生物活性,包括提高免疫力、抗氧化、抗肿瘤、抗病毒、抗凝血等。经过研究发现许多具备某种特定生理活性的天然多糖,都具有着一定特殊的分子结构。这种特定的分子结构决定着其所具备的生物活性能力。但至今对多糖的结构与活性所对应关系(多糖构效关系)的研究仍然进展缓慢,其主要障碍之一就是缺乏能够作为多糖构效关系研究的模式化合物,这种多聚化合物必须具备单一的可比较的单糖组成、明确且简洁的单糖链接糖苷键型、一致的异头物构型、明确的且取代位置相同的取代基、可有效掌控的分子量大小。找到符合上述条件的模式多糖成为多糖构效关系研究中的无法回避的难点之一。天然多糖结构复杂,组成单糖复杂,连接键型多种多样,取代基种类及取代位置变化多端,不同分子量但具有相同结构的多糖难以获取等等,这一系列的难题使得将某种多糖具备的生理活性与其结构精确对应成为一件难以完成的任务。
本研究从大连湾牡蛎,学名Ostrea talienwhanensis Crosse体内提取出一种多糖物质,经过分离纯化以及结构鉴定后得到一种纯度良好的糖原物质,此糖原经过硫酸化修饰后得到了一种硫酸取代基专一取代在C-6位置的硫酸化糖原。此种糖原具有简单的单糖组成和异头物构型、一致的糖苷键键型、可控的分子长短、一致且位置确定的硫酸基取代,成为极其优良的可以做为多糖构效关系研究的模型化合物。
1、为获取纯度良好的牡蛎糖原,本研究采用多糖含量为评价指标优化了牡蛎糖原提取方法,尽可能的去除糖原所共价连接的蛋白成分。最优提取条件为：以胃蛋白酶酶解提取,料液比1：45,加酶量为底物质量的1.5%,pH2.0,温度37℃,酶解时间2h。酶解提取物再经过乙醇醇沉沉淀、DEAE-52阴离子纤维素柱和Sephadex G-100凝胶柱洗脱分离纯化出一种纯度良好的牡蛎多糖聚合物。
2、对所提取到的多糖物质进行了结构解析。样品通过衍生化经气相色谱和液相色谱方法分别鉴定证明其只含有葡萄糖组成成分,IR扫描结果证明其为多糖类结构物质。样品甲基化后经GC-MS分析发现此提取物具有1-4Glc,1-4,6Glc和1-Glc的链接键型结构,属于典型的糖原特征组成键型,并且还存在有痕量的其他葡萄糖组成键型。且1-4,6Glc与1-4Glc键型在数量上的比例约为1：6.6,说明此糖原结构平均每6.6个葡萄糖存在一次分支。核磁共振谱分析同样证明了此提取物的糖原组成结构。从而证实了该牡蛎提取物为牡蛎糖原,且其分支率较高,平均每6.6个葡萄糖残基中发生一次分支。
3、对此牡蛎糖原进行硫酸化修饰后,经过分离纯化得到3个组分,分别对其进行分子结构鉴定,发现其中组分SOG的结构为硫酸基特异性取代于单糖残基的C-6羟基位置的硫酸化牡蛎糖原,而组分SOG1的结构为硫酸基取代在糖残基的C-2和C-3羟基位置的硫酸化牡蛎糖原。对此两种糖原进行体外促淋巴细胞增殖能力的研究发现SOG的活性要明显强于SOG1,而其分子结构除硫酸基取代位置因素不同外其他因素基本一致,这证明在多糖的构效关系中,硫酸基取代位点对于其促淋巴细胞增殖能力有着重要影响,且硫酸基在C-6位置取代的结构其活性最佳
4、通过对硫酸化牡蛎糖原SOG进行高碘酸氧化降解,得到了一个硫酸基含量达到40.6%,分子量为6.3×106的组分SOGF1。将SOGF1与硫酸化修饰后得到的另外3种硫酸酯糖原SOG、SOG1和SOG2编成一研究对照组,构成了一组具有不同硫酸基含量、不同分子质量但结构相似的硫酸酯糖原组,对其进行活性研究表明：此4种硫酸酯糖原均有促淋巴细胞增殖活性,其能力大小关系为SOG>SOGF1>SOG1>SOG2。对照他们的结构与活性之间关系分析发现,多糖结构对促淋巴细胞增殖活性影响因素的强弱顺序为：多糖结构中硫酸基取代位点>多糖中硫酸基含量>硫酸基多糖分子量。
总之,此多个多糖组分经过结构对应的活性测试可以总结出,多糖中硫酸基的含量与活性间有着线性的平行对应规律,但分子量大小因素并没有非常明确的影响规律。在其他结构因素相同的情况下,C-6位置硫酸基取代对多糖活性的提升具有决定性影响,其次,硫酸酯多糖的活性受结构中硫酸基含量的影响,硫酸基含量越多其活性越强烈。最后,硫酸酯多糖的分子量大小对多糖活性有影响,但无明确的相关关系,只有多糖分子量达到一定值区间时多糖结构才能充分发挥其功能。
Natural polysaccharide has many bioactivities, including immunomudulation, antioxidation, antitumor, antivirus, anticoagulant, etc. Research showed that many natural polysaccharides with specific physiological activity have specific molecular structures. These specific molecular structures determine the bioactivity of the polysaccharides. But up to now, research on the relationship between polysaccharides structure and bioactivity progressed slowly. One of the main obstacles is the lack of suitable mode compounds. Such polymeric compounds must have single monosaccharide composition, clear and definite glycosidic bond type and substituent at same substitution positions, consistent anomer configuration, and their molecular weight can be effectively controlled. It's difficult to obtain such polysaccharides as discribed above and this difficulty cannot be easily overcome in the structure-activity relationship studies.
Natural polysaccharides are of complicated in structure, complex monosaccharide composition, varied connecting type, substituent types and replace position and so on. In addition, polysaccharide with different molecular weight and same structure are hardly to obtain. These problems made the structure-activity relationship research a hard task.
In this study, polysaccharide was extracted and purified from oyster Ostrea talienwhanensis Crosse. After structure identification, the polysaccharide was proved to be a kind of well purified glycogen from oyster. The oyster glycogen was then sulfated, and the substitution patterns were precisely determined to be C-6in the sulfated oyster glycogen. This kind of glycogen, with simple monosaccharide composition and anomer configuration, consistent glycoside type, ascertain molecular length, consistent and determined position of the sulfuric base, thus can be used as an excellent model compound in the study of structure-activity relationship.
In order to obtain purified oyster glycogen, the polysaccharide content was used as an indix to optimize the extraction method to remove the conjugated pretein. The optimum extraction conditions were determined:the optimize extraction enzyme is pepsin, the ratio of solid material to liquid is1:45, enzyme percentage is1.5%(m/m, substrate), pH2.0, temperature as37℃, enzymolysis time is2h. Ethanol (v/v) was then added into the enzymatic hydrolysate to obtain the crude oyster polysaccharide. The crude oyster polysaccharide was then purified by a series of column chromatography, including dimethylaminoethyl negion exchange column (DEAE-cellulose-52,2.0cm×40cm) and Sephadex G-100column (1.6cm×70cm). The major fraction was collected, concentrated, desalted by dialysis and then vacuum freeze-dried to obtain the homogeneous polysaccharide.
After derivatization, the structure of oyster polysaccharide polymer was analyzed by gas chromatography and high performance liquid chromatography (HPLC). Results showed that the oyster polysaccharide consisted solely of glucose. IR scan results proved the obtained polymer was polysaccharide. The fully methylated products were analyzed by GC-MS and were affirmed to be a kind of glycogen because of its typical glycogen linkage,1-4Glc,1-4,6Glc and1-Glc. Meanwhile, there exist trace other glycogen linkage type. The ratio of1,4-linkage to1,4,6-linkage was6.6, which indicated that the oyster glycogen chain branched for6.6glucose residues averagely. And it was further confirmed by NMR analysis that the oyster polysaccharide was a kind of glycogen and the oyster glycogen chain branched for6.6glucose residues-averagely.
The oyster glycogen was chemically sulfated and further purified. Three fractions of sulfated oyster glycogen were collected, named as SOG, SOG1, and SOG2. SOG was sulfate substituted at C-6position. SOG1was sulfate substituted at C-2and C-3positions. SOG showed better activity in promoting spleen lymphocyte proliferation than SOG1. SOG and SOG1shared similar structure properties, except the sulfate position. It's proved that the position of sulfate substitution played an important role in promoting lymphocyte proliferation ability. The polysaccharide with C-6substitution demonstrated the most significant activity.
After periodate oxidation, SOGF1was obtained by gel chromatography of the degradation products of SOG. The content of sulfate group in SOGF1was40.6%. Molecular weight of SOGF1was determined as6.3×106. Thus SOGF1, SOG, SOG1and SOG2, with different sulfate group content, different molecular weight, but similar structure, could be used as model polysaccharides to research the structure-activity relationship. Activity studies showed that all the above four kinds of glycogens could promote lymphocyte proliferation with an ascending series of SOG>SOGF1>SOG1>SOG2.
Analysis showed that different structural properties of the polysaccharides played different role in promoting lymphocyte proliferation, with an ascending series of sulfate positon>sulfate content> molecular weight.
Results showed that there was a proportional relation betrelationship between sulfate content and lymphocyte proliferation activity. But molecular weight did not demonstrate any effect on the bioactivity.
Results showed that C-6position sulfate substitution has decisive influence on the bioactivity of polysaccharide if other structural properties were similar. The content of sulfuric acid played the second role, more sulfate content, stronger bioactivity. Finally, molecular weight of sulfate polysaccharides also could influence the bioactivity, but there is no clear correlation between them. Perhaps the molecular weight might play some role when reaches a certain value.
本研究从大连湾牡蛎,学名Ostrea talienwhanensis Crosse体内提取出一种多糖物质,经过分离纯化以及结构鉴定后得到一种纯度良好的糖原物质,此糖原经过硫酸化修饰后得到了一种硫酸取代基专一取代在C-6位置的硫酸化糖原。此种糖原具有简单的单糖组成和异头物构型、一致的糖苷键键型、可控的分子长短、一致且位置确定的硫酸基取代,成为极其优良的可以做为多糖构效关系研究的模型化合物。
1、为获取纯度良好的牡蛎糖原,本研究采用多糖含量为评价指标优化了牡蛎糖原提取方法,尽可能的去除糖原所共价连接的蛋白成分。最优提取条件为：以胃蛋白酶酶解提取,料液比1：45,加酶量为底物质量的1.5%,pH2.0,温度37℃,酶解时间2h。酶解提取物再经过乙醇醇沉沉淀、DEAE-52阴离子纤维素柱和Sephadex G-100凝胶柱洗脱分离纯化出一种纯度良好的牡蛎多糖聚合物。
2、对所提取到的多糖物质进行了结构解析。样品通过衍生化经气相色谱和液相色谱方法分别鉴定证明其只含有葡萄糖组成成分,IR扫描结果证明其为多糖类结构物质。样品甲基化后经GC-MS分析发现此提取物具有1-4Glc,1-4,6Glc和1-Glc的链接键型结构,属于典型的糖原特征组成键型,并且还存在有痕量的其他葡萄糖组成键型。且1-4,6Glc与1-4Glc键型在数量上的比例约为1：6.6,说明此糖原结构平均每6.6个葡萄糖存在一次分支。核磁共振谱分析同样证明了此提取物的糖原组成结构。从而证实了该牡蛎提取物为牡蛎糖原,且其分支率较高,平均每6.6个葡萄糖残基中发生一次分支。
3、对此牡蛎糖原进行硫酸化修饰后,经过分离纯化得到3个组分,分别对其进行分子结构鉴定,发现其中组分SOG的结构为硫酸基特异性取代于单糖残基的C-6羟基位置的硫酸化牡蛎糖原,而组分SOG1的结构为硫酸基取代在糖残基的C-2和C-3羟基位置的硫酸化牡蛎糖原。对此两种糖原进行体外促淋巴细胞增殖能力的研究发现SOG的活性要明显强于SOG1,而其分子结构除硫酸基取代位置因素不同外其他因素基本一致,这证明在多糖的构效关系中,硫酸基取代位点对于其促淋巴细胞增殖能力有着重要影响,且硫酸基在C-6位置取代的结构其活性最佳
4、通过对硫酸化牡蛎糖原SOG进行高碘酸氧化降解,得到了一个硫酸基含量达到40.6%,分子量为6.3×106的组分SOGF1。将SOGF1与硫酸化修饰后得到的另外3种硫酸酯糖原SOG、SOG1和SOG2编成一研究对照组,构成了一组具有不同硫酸基含量、不同分子质量但结构相似的硫酸酯糖原组,对其进行活性研究表明：此4种硫酸酯糖原均有促淋巴细胞增殖活性,其能力大小关系为SOG>SOGF1>SOG1>SOG2。对照他们的结构与活性之间关系分析发现,多糖结构对促淋巴细胞增殖活性影响因素的强弱顺序为：多糖结构中硫酸基取代位点>多糖中硫酸基含量>硫酸基多糖分子量。
总之,此多个多糖组分经过结构对应的活性测试可以总结出,多糖中硫酸基的含量与活性间有着线性的平行对应规律,但分子量大小因素并没有非常明确的影响规律。在其他结构因素相同的情况下,C-6位置硫酸基取代对多糖活性的提升具有决定性影响,其次,硫酸酯多糖的活性受结构中硫酸基含量的影响,硫酸基含量越多其活性越强烈。最后,硫酸酯多糖的分子量大小对多糖活性有影响,但无明确的相关关系,只有多糖分子量达到一定值区间时多糖结构才能充分发挥其功能。
Natural polysaccharide has many bioactivities, including immunomudulation, antioxidation, antitumor, antivirus, anticoagulant, etc. Research showed that many natural polysaccharides with specific physiological activity have specific molecular structures. These specific molecular structures determine the bioactivity of the polysaccharides. But up to now, research on the relationship between polysaccharides structure and bioactivity progressed slowly. One of the main obstacles is the lack of suitable mode compounds. Such polymeric compounds must have single monosaccharide composition, clear and definite glycosidic bond type and substituent at same substitution positions, consistent anomer configuration, and their molecular weight can be effectively controlled. It's difficult to obtain such polysaccharides as discribed above and this difficulty cannot be easily overcome in the structure-activity relationship studies.
Natural polysaccharides are of complicated in structure, complex monosaccharide composition, varied connecting type, substituent types and replace position and so on. In addition, polysaccharide with different molecular weight and same structure are hardly to obtain. These problems made the structure-activity relationship research a hard task.
In this study, polysaccharide was extracted and purified from oyster Ostrea talienwhanensis Crosse. After structure identification, the polysaccharide was proved to be a kind of well purified glycogen from oyster. The oyster glycogen was then sulfated, and the substitution patterns were precisely determined to be C-6in the sulfated oyster glycogen. This kind of glycogen, with simple monosaccharide composition and anomer configuration, consistent glycoside type, ascertain molecular length, consistent and determined position of the sulfuric base, thus can be used as an excellent model compound in the study of structure-activity relationship.
In order to obtain purified oyster glycogen, the polysaccharide content was used as an indix to optimize the extraction method to remove the conjugated pretein. The optimum extraction conditions were determined:the optimize extraction enzyme is pepsin, the ratio of solid material to liquid is1:45, enzyme percentage is1.5%(m/m, substrate), pH2.0, temperature as37℃, enzymolysis time is2h. Ethanol (v/v) was then added into the enzymatic hydrolysate to obtain the crude oyster polysaccharide. The crude oyster polysaccharide was then purified by a series of column chromatography, including dimethylaminoethyl negion exchange column (DEAE-cellulose-52,2.0cm×40cm) and Sephadex G-100column (1.6cm×70cm). The major fraction was collected, concentrated, desalted by dialysis and then vacuum freeze-dried to obtain the homogeneous polysaccharide.
After derivatization, the structure of oyster polysaccharide polymer was analyzed by gas chromatography and high performance liquid chromatography (HPLC). Results showed that the oyster polysaccharide consisted solely of glucose. IR scan results proved the obtained polymer was polysaccharide. The fully methylated products were analyzed by GC-MS and were affirmed to be a kind of glycogen because of its typical glycogen linkage,1-4Glc,1-4,6Glc and1-Glc. Meanwhile, there exist trace other glycogen linkage type. The ratio of1,4-linkage to1,4,6-linkage was6.6, which indicated that the oyster glycogen chain branched for6.6glucose residues averagely. And it was further confirmed by NMR analysis that the oyster polysaccharide was a kind of glycogen and the oyster glycogen chain branched for6.6glucose residues-averagely.
The oyster glycogen was chemically sulfated and further purified. Three fractions of sulfated oyster glycogen were collected, named as SOG, SOG1, and SOG2. SOG was sulfate substituted at C-6position. SOG1was sulfate substituted at C-2and C-3positions. SOG showed better activity in promoting spleen lymphocyte proliferation than SOG1. SOG and SOG1shared similar structure properties, except the sulfate position. It's proved that the position of sulfate substitution played an important role in promoting lymphocyte proliferation ability. The polysaccharide with C-6substitution demonstrated the most significant activity.
After periodate oxidation, SOGF1was obtained by gel chromatography of the degradation products of SOG. The content of sulfate group in SOGF1was40.6%. Molecular weight of SOGF1was determined as6.3×106. Thus SOGF1, SOG, SOG1and SOG2, with different sulfate group content, different molecular weight, but similar structure, could be used as model polysaccharides to research the structure-activity relationship. Activity studies showed that all the above four kinds of glycogens could promote lymphocyte proliferation with an ascending series of SOG>SOGF1>SOG1>SOG2.
Analysis showed that different structural properties of the polysaccharides played different role in promoting lymphocyte proliferation, with an ascending series of sulfate positon>sulfate content> molecular weight.
Results showed that there was a proportional relation betrelationship between sulfate content and lymphocyte proliferation activity. But molecular weight did not demonstrate any effect on the bioactivity.
Results showed that C-6position sulfate substitution has decisive influence on the bioactivity of polysaccharide if other structural properties were similar. The content of sulfuric acid played the second role, more sulfate content, stronger bioactivity. Finally, molecular weight of sulfate polysaccharides also could influence the bioactivity, but there is no clear correlation between them. Perhaps the molecular weight might play some role when reaches a certain value.
引文
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