宽礁膜（Monostroma latissium）多糖的分离、结构和抗凝血活性研究
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摘要
宽礁膜(Monostroma latissium)隶属于绿藻门,绿藻纲,礁膜科,礁膜属。多糖是宽礁膜的有效成分。本文以宽礁膜为原料,对宽礁膜多糖的提取分离、化学特征、结构和抗凝血活性进行了研究。
论文第一部分,以宽礁膜为原料,依次采用冷水、热水和碱液提取,分别得到三个多糖组分CE、HE和AE,运用化学、气相色谱和高效液相色谱等方法分别对其化学组成进行了分析比较。结果表明:所得多糖总得率为40.41%,三种多糖都为含硫酸根的水溶性多糖,硫酸根含量分别为21.33%、24.92%和25.34%,所含单糖主要为鼠李糖,另外还含有少量的葡萄糖和木糖,糖醛酸种类为葡萄糖醛酸。
论文第二部分,在对各多糖组分基本理化性质分析测定的基础上,以HE为研究对象,通过各种色谱分离纯化出3个纯品多糖HEA、HEB和HEC,并采用化学和现代波谱学技术对HEA、HEB和HEC的化学组成和结构进行了研究。结果表明,HEA分子量较大(725.4kD), HEB和HEC分子量相近(54.8 kD和68.8 kD),三种多糖的单糖组成相似,其中HEC硫酸含量最高;HEA和HEB主要含有1→3、1→2连接的鼠李糖,硫酸基位于1→2连接鼠李糖的C-4位或C-3位上。HEB主链是由1,2-Rha和少量的1,3-Rha连接组成的硫酸化鼠李聚糖,并带有少量分支;HEA是由各种键型交替相连的结构复杂的一种硫酸化鼠李聚糖。
论文第三部分,探讨了氧化降解和酸降解方法制备低分子量宽礁膜多糖片段的工艺,在此基础上采用凝胶色谱等技术,制备获得了不同分子量的多糖片段,并通过活化部分凝血活酶时间、凝血酶原时间和凝血酶时间法分析测定不同分子量的多糖片段的抗凝血活性。结果表明,本文所采用的H2O2降解方法对多糖的基本结构没有影响和破坏,而H2SO4降解方法对多糖的基本结构的影响也很小。通过这两种方法可以有效地制备获得一系列不同分子量的多糖片段。抗凝血活性研究表明,宽礁膜多糖主要抑制内源性凝血途径和/或共有凝血途径以及抑制凝血酶活性或纤维蛋白原转化为纤维蛋白,而对外源性凝血途径没有影响。而且,具有较高分子量的多糖片段具有与母核宽礁膜多糖相似的抗凝血活性,但随着多糖片段分子量的进一步降低,其抗凝血活性下降。结果揭示,分子量大小对宽礁膜多糖的抗凝血活性有重要作用,宽礁膜多糖抑制凝血酶的作用要求其糖链具有一定的长度。
论文第四部分,采用酸降解方法对宽礁膜多糖进行降解,通过凝胶色谱技术分离得到7个寡糖组分,并采用甲基化分析、红外光谱、电喷雾质谱和电喷雾电离-碰撞诱导解离二级质谱等技术对寡糖的结构进行研究。结果表明,所得寡糖组分主要由2-7糖组成,其主要结构单元为:由1→3连接鼠李糖组成的直链结构,硫酸根的位置位于非还原端,可能在C-2或C-4位上;葡萄糖醛酸与鼠李糖组成直链结构,且位于非还原端。
本研究的创新点在于:采用各种色谱分离技术、化学和先进的仪器分析技术,首次对宽礁膜多糖进行了系统的研究,揭示了宽礁膜多糖的化学特征和结构。首次利用氧化降解、酸降解方法和凝胶色谱等技术,制备宽礁膜多糖的不同分子量的多糖片段,并初步揭示了宽礁膜多糖抗凝血活性的作用机制和构效关系。
多糖是宽礁膜的有效成分,对其开展结构和活性的研究十分重要和必要。本研究为宽礁膜多糖的研究提供了理论数据,为开发利用宽礁膜奠定了基础,对促进“海洋糖库”的建设,发展具有我国特色的海洋新药都有重要意义。
Monostroma latissium is a solt of green algae plant in ocean. It grows widely in the brackish water area in the upper part of the intertidal zone. Monostroma latissium exhibits many biological activities such as anticoagulant, antiviral, antioxidant activities and radioprotective effect. It has been confirmed that the main active substance in Monostroma latissium is soluble sulfated polysaccharide.
In this paper, the polysaccharide from Monostroma latissium was extracted by cool water, boiled water and alkali liquid successively. Three kinds of polysaccharides were obtained and named as CE, HE and AE, respectively. The chemical characteristics of the polysaccharides were analyzed by chemical method, GC and HPLC. The results showed that the total yield of the polysaccharides was 40.41% and the three polysaccharides were soluble sulfated polysaccharides with the different sulfate content. The sulfate contents of polysaccharides are 21.33%, 24.92% and 25.34%, respectively. Three sulfated polysaccharides were found to contain mainly rhamnose with small amounts of glucose and xylose and trace amounts of glactose and mannose. The uronic acid is consisted of glucuronic in the sulfated polysaccharide.
Based on the physicochemical analysis, three pure fractions HEA, HEB and HEC were obtained from HE using ion-exchange chromatography and low-pressure gel permeation chromatography. The chemical characteristics and primary structures of the pure fractions were investigated. The result showed that HEA was a polysaccharide with high molecular size (725.4kD). The molecular weights of HEB and HEC (54.8 kD, 68.8 kD) are similar. The monosaccharide compositions of the three polysaccharides are similar. The backbone of HEA and HEB were consisted of 1,3-Rha, 1,2-Rha and the sulfate groups were substituted at C-4 or at C-3 in 1,2-Rha. While HEB was a kind of sulfated rhamnan consisting of 1,2-Rha with little branch, HEA was a complicated sulfated rhamnan consisting of 1,2-Rha, 1,3- Rha, 1,2,4-Rha and 1,2,3,4-Rha.
The sulfated polysaccharide from Monostroma latissimum was hydrolyzed into smaller fragments with different molecular weights by H2O2 or H2SO4 degradation. The polysaccharide fragments with different molecular weights were further fractionated by gel permeation chromatography. Molecular weight of the polysaccharide fragments prepared by H2O2 were 216.4kDa, 123.7kDa, 61.9kDa, 26.0kDa and 10.6kDa, respectively; the molecular weight of polysaccharide fragments prepared by H2SO4 degradation were assayed as follows: 156.8 kDa, 61.7 kDa, 46.1 kDa, and 15.7 kDa. Moreover, chemical and instrumental analysis indicated that chemical components and structure of the sulfated polysaccharide fragments are similar to that of the parent sulfated polysaccharide. The results showed that the basic structures of polysaccharides had not been destroyed by H2O2. H2SO4 degradation had little effect on the basic structure of the sulfated polysaccharide. A series polysaccharide fragments with different molecular weights can be prepared effectively by the two ways.
Anticoagulant activities of the parent sulfated polysaccharide and its fragments were investigated by studying the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using human plasma. The study on anticoagulant activity showed that the sulfated polysaccharide isolated from Monostroma latissimum inhibited both the intrinsic and or common pathways of coagulation and thrombin activity or conversion of fibrinogen to fibrin. The sulfated polysaccharide fragments with higher molecular weight had slightly higher anticoagulant activities than their parent sulfated polysaccharide at the same concentration. The sulfated polysaccharide fragment with a lower molecular weight had a lower anticoagulant activity. These results suggest that the molecular size has a profound effect on the anticoagulant activity of the sulfated polysaccharides from Monostroma latissimum, and plays an important role in anticoagulant action. The sulfated polysaccharides from Monostroma latissimum require longer chains to achieve complete thrombin inhibition.
In the last part of the paper, the oligosaccharides from the parent sulfated polysaccharide were prepared by H2SO4 method. Seven oligosaccharide products were obtained by gel chromatography. The structures of the oligosaccharides were characterized by methylation analysis, IR, ESI-MS and ESI-CID-MS/MS. The results showed that the oligosaccharide products are mainly consist of disaccharide to heptasaccharide. The oligosaccharide was straight line consisting of 1,3-Rha and the sulfate groups were substituted at C-2 or C-4 in 1,3-Rha.
The active substance in Monostroma latissimum is polysaccharide. It is important and neccssary to research the structure and activity of the polysaccharides. This research provided the theory information for the sulfated polysaccharide, settled the foundation for utilizing the sulfated polysaccharide from Monostroma latissimum and develop the establishing the“saccharide-bank”.
论文第一部分,以宽礁膜为原料,依次采用冷水、热水和碱液提取,分别得到三个多糖组分CE、HE和AE,运用化学、气相色谱和高效液相色谱等方法分别对其化学组成进行了分析比较。结果表明:所得多糖总得率为40.41%,三种多糖都为含硫酸根的水溶性多糖,硫酸根含量分别为21.33%、24.92%和25.34%,所含单糖主要为鼠李糖,另外还含有少量的葡萄糖和木糖,糖醛酸种类为葡萄糖醛酸。
论文第二部分,在对各多糖组分基本理化性质分析测定的基础上,以HE为研究对象,通过各种色谱分离纯化出3个纯品多糖HEA、HEB和HEC,并采用化学和现代波谱学技术对HEA、HEB和HEC的化学组成和结构进行了研究。结果表明,HEA分子量较大(725.4kD), HEB和HEC分子量相近(54.8 kD和68.8 kD),三种多糖的单糖组成相似,其中HEC硫酸含量最高;HEA和HEB主要含有1→3、1→2连接的鼠李糖,硫酸基位于1→2连接鼠李糖的C-4位或C-3位上。HEB主链是由1,2-Rha和少量的1,3-Rha连接组成的硫酸化鼠李聚糖,并带有少量分支;HEA是由各种键型交替相连的结构复杂的一种硫酸化鼠李聚糖。
论文第三部分,探讨了氧化降解和酸降解方法制备低分子量宽礁膜多糖片段的工艺,在此基础上采用凝胶色谱等技术,制备获得了不同分子量的多糖片段,并通过活化部分凝血活酶时间、凝血酶原时间和凝血酶时间法分析测定不同分子量的多糖片段的抗凝血活性。结果表明,本文所采用的H2O2降解方法对多糖的基本结构没有影响和破坏,而H2SO4降解方法对多糖的基本结构的影响也很小。通过这两种方法可以有效地制备获得一系列不同分子量的多糖片段。抗凝血活性研究表明,宽礁膜多糖主要抑制内源性凝血途径和/或共有凝血途径以及抑制凝血酶活性或纤维蛋白原转化为纤维蛋白,而对外源性凝血途径没有影响。而且,具有较高分子量的多糖片段具有与母核宽礁膜多糖相似的抗凝血活性,但随着多糖片段分子量的进一步降低,其抗凝血活性下降。结果揭示,分子量大小对宽礁膜多糖的抗凝血活性有重要作用,宽礁膜多糖抑制凝血酶的作用要求其糖链具有一定的长度。
论文第四部分,采用酸降解方法对宽礁膜多糖进行降解,通过凝胶色谱技术分离得到7个寡糖组分,并采用甲基化分析、红外光谱、电喷雾质谱和电喷雾电离-碰撞诱导解离二级质谱等技术对寡糖的结构进行研究。结果表明,所得寡糖组分主要由2-7糖组成,其主要结构单元为:由1→3连接鼠李糖组成的直链结构,硫酸根的位置位于非还原端,可能在C-2或C-4位上;葡萄糖醛酸与鼠李糖组成直链结构,且位于非还原端。
本研究的创新点在于:采用各种色谱分离技术、化学和先进的仪器分析技术,首次对宽礁膜多糖进行了系统的研究,揭示了宽礁膜多糖的化学特征和结构。首次利用氧化降解、酸降解方法和凝胶色谱等技术,制备宽礁膜多糖的不同分子量的多糖片段,并初步揭示了宽礁膜多糖抗凝血活性的作用机制和构效关系。
多糖是宽礁膜的有效成分,对其开展结构和活性的研究十分重要和必要。本研究为宽礁膜多糖的研究提供了理论数据,为开发利用宽礁膜奠定了基础,对促进“海洋糖库”的建设,发展具有我国特色的海洋新药都有重要意义。
Monostroma latissium is a solt of green algae plant in ocean. It grows widely in the brackish water area in the upper part of the intertidal zone. Monostroma latissium exhibits many biological activities such as anticoagulant, antiviral, antioxidant activities and radioprotective effect. It has been confirmed that the main active substance in Monostroma latissium is soluble sulfated polysaccharide.
In this paper, the polysaccharide from Monostroma latissium was extracted by cool water, boiled water and alkali liquid successively. Three kinds of polysaccharides were obtained and named as CE, HE and AE, respectively. The chemical characteristics of the polysaccharides were analyzed by chemical method, GC and HPLC. The results showed that the total yield of the polysaccharides was 40.41% and the three polysaccharides were soluble sulfated polysaccharides with the different sulfate content. The sulfate contents of polysaccharides are 21.33%, 24.92% and 25.34%, respectively. Three sulfated polysaccharides were found to contain mainly rhamnose with small amounts of glucose and xylose and trace amounts of glactose and mannose. The uronic acid is consisted of glucuronic in the sulfated polysaccharide.
Based on the physicochemical analysis, three pure fractions HEA, HEB and HEC were obtained from HE using ion-exchange chromatography and low-pressure gel permeation chromatography. The chemical characteristics and primary structures of the pure fractions were investigated. The result showed that HEA was a polysaccharide with high molecular size (725.4kD). The molecular weights of HEB and HEC (54.8 kD, 68.8 kD) are similar. The monosaccharide compositions of the three polysaccharides are similar. The backbone of HEA and HEB were consisted of 1,3-Rha, 1,2-Rha and the sulfate groups were substituted at C-4 or at C-3 in 1,2-Rha. While HEB was a kind of sulfated rhamnan consisting of 1,2-Rha with little branch, HEA was a complicated sulfated rhamnan consisting of 1,2-Rha, 1,3- Rha, 1,2,4-Rha and 1,2,3,4-Rha.
The sulfated polysaccharide from Monostroma latissimum was hydrolyzed into smaller fragments with different molecular weights by H2O2 or H2SO4 degradation. The polysaccharide fragments with different molecular weights were further fractionated by gel permeation chromatography. Molecular weight of the polysaccharide fragments prepared by H2O2 were 216.4kDa, 123.7kDa, 61.9kDa, 26.0kDa and 10.6kDa, respectively; the molecular weight of polysaccharide fragments prepared by H2SO4 degradation were assayed as follows: 156.8 kDa, 61.7 kDa, 46.1 kDa, and 15.7 kDa. Moreover, chemical and instrumental analysis indicated that chemical components and structure of the sulfated polysaccharide fragments are similar to that of the parent sulfated polysaccharide. The results showed that the basic structures of polysaccharides had not been destroyed by H2O2. H2SO4 degradation had little effect on the basic structure of the sulfated polysaccharide. A series polysaccharide fragments with different molecular weights can be prepared effectively by the two ways.
Anticoagulant activities of the parent sulfated polysaccharide and its fragments were investigated by studying the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using human plasma. The study on anticoagulant activity showed that the sulfated polysaccharide isolated from Monostroma latissimum inhibited both the intrinsic and or common pathways of coagulation and thrombin activity or conversion of fibrinogen to fibrin. The sulfated polysaccharide fragments with higher molecular weight had slightly higher anticoagulant activities than their parent sulfated polysaccharide at the same concentration. The sulfated polysaccharide fragment with a lower molecular weight had a lower anticoagulant activity. These results suggest that the molecular size has a profound effect on the anticoagulant activity of the sulfated polysaccharides from Monostroma latissimum, and plays an important role in anticoagulant action. The sulfated polysaccharides from Monostroma latissimum require longer chains to achieve complete thrombin inhibition.
In the last part of the paper, the oligosaccharides from the parent sulfated polysaccharide were prepared by H2SO4 method. Seven oligosaccharide products were obtained by gel chromatography. The structures of the oligosaccharides were characterized by methylation analysis, IR, ESI-MS and ESI-CID-MS/MS. The results showed that the oligosaccharide products are mainly consist of disaccharide to heptasaccharide. The oligosaccharide was straight line consisting of 1,3-Rha and the sulfate groups were substituted at C-2 or C-4 in 1,3-Rha.
The active substance in Monostroma latissimum is polysaccharide. It is important and neccssary to research the structure and activity of the polysaccharides. This research provided the theory information for the sulfated polysaccharide, settled the foundation for utilizing the sulfated polysaccharide from Monostroma latissimum and develop the establishing the“saccharide-bank”.
引文
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