海带多糖的分离纯化、结构鉴定及生物活性研究
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摘要
海带是一种分布较广、营养价值较高的食用藻类。海带多糖有很好的生物活性,对人类健康很有益处。文献中对海带多糖的研究大多集中在硫酸化的岩藻聚糖上,对海带多糖的系统分级,特别是对褐藻胶的分离提取、结构鉴定和活性研究报道较少。为了探讨海带多糖活性和机理,更好地应用海带及其多糖,本文对海带多糖的分离、纯化,及各级分的结构特征与生物活性进行了系统的研究。
海带经过水提、醇沉后得到两种多糖,20%醇沉得到的沉淀LFA和60%醇沉得到的沉淀LFF。LFA再经过酸分级得到两种多糖,分别是PH为2.85时的沉淀LFAG和上清LFAM,LFAG和LFAM分别经Sepharose CL-6B凝胶过滤层析制备得到多糖LFAGS和LFAMS;LFF通过DEAE-Cellulose离子交换层析和Sepharose CL-6B凝胶过滤层析制备后得到多糖LFF-1S。本论文对这三种纯化后的多糖分别进行了理化性质的研究和结构分析,其结果如下:
LFAGS为白色粉末,微溶于水,分子量约为42 KDa,由GulA、ManA和GalA三种单糖组成,以1, 4-β-GulAp-1, 4-β-ManAp为主链,每四个ManA连有一个GulA,无分支,主链中有微量的β-1, 4-GalAp。LFAMS为白色粉末,难溶于水,分子量约为12 KDa,由GulA、ManA和GalA三种单糖组成,以1, 4-β-GulAp-1,4 -β-ManAp的重复单位为主链,ManAp的3位有分支,连有β-1, 4-GulAp糖残基或寡糖残基,支链末端为ManA。LFF-1S为白色粉末,易溶于水,单糖组成为GulA、ManA、Man、GluA、Gal和Fuc,分子量约为14 KDa。
实验结果表明海带多糖LFAGS、LFAMS和LFF-1S对淋巴细胞有明显的调节作用。无ConA和LPS诱导时,LFAMS和LFF-1S可显著地增强小鼠脾混合淋巴细胞的增殖;在ConA和LPS诱导下,三种多糖级分可以显著的刺激ConA和LPS诱导T淋巴细胞和B淋巴细胞增殖,表明三种级分多糖对ConA和LPS诱导的T细胞和B细胞有明显激活作用。当浓度大于100μg/ml时,三种多糖均能极显著地(p < 0.01)增强巨噬细胞的吞噬能力;此外,三种多糖也能增强巨噬细胞产生NO的能力,并且呈现出一定的浓度依赖性;海带多糖LFAGS具有抑制结肠癌细胞生长的作用,而LFAMS和LFF-1S对结肠癌细胞的生长没有明显影响。细胞迁移实验结果表明这三种多糖对L929细胞的迁移没有影响。
Laminaria japonica is a very widespread edible algae. Laminarin has good bioactivities which are beneficial to people’s health. Most studies concerning the L. japonica focus on the sulfated fucosan, the systematic fractionation of L. japonica and in particular, its algin separation, structure identification, and examination of the activity are neglected. This thesis studied on the algin separation, structure identification, and examination of the activity of L. japonica.
The polysaccharide fractions from L. japonica were isolated and purified. First, three polysaccharides, LFAG, LFAM and LFF, were obtained by fractionation with ethanol precipitation and dilute acid. LFAG and LFAM were further purified by Sepharose CL-6B to produce LFAGS and LFAMS, respectively. LFF was first separated with DEAE-Cellulose, then it was fractionated on a Sepharose CL-6B column to give LFF-1S. The physical and chemical properties of these three L. japonica polysaccharides were analyzed, and the major structural features of them were elucidated using 13C-nuclear magnetic resonance spectrometer (13C-NMR). The results are as follows:
LFAGS is white powder and its solubility in water is very low. Sugar composition analysis by high performance liquid chromatography (HPLC) indicated that LFAGS consists of GulA, ManA and GalA. The 13C-NMR results showed that the backbone of LFAGS was in the linkage form of 1, 4-β-GulAp-1, 4-β-ManAp, it is composed of four ManA attaching one GulA units in the order of a heteropolymeric sequences, and LFAGS has no side chain, the molecular weight is 42 KDa. LFAMS is also a kind of white powder which is difficult to dissolve in water. The sugar composition of LFAMS is GulA, ManA and GalA, too. While the backbone of LFAMS is composed of repeated units 1, 4-β-GulAp-1, 4-β-ManAp, there isβ-1, 4 -GulAp attached to other sugar residues side-chain in the C-3 position of ManAp, moreover, sugar composition of the end of the side chain is ManA. The molecular weight of LFAGS is 12 KDa. LFF-1S is white powder and easily soluble in water, the molecular weight is 14 KDa, and its sugar composition is GulA, ManA, Man, GluA, Gal and Fuc.
The results indicate that the L. japonica polysaccharides LFAGS, LFAMS and LFF-1S can regulate the activity of lymphocyte obviously. When there is no ConA and LPS induction, LFAMS and LFF-1S can enhance the proliferative response of mouse lymphocyte markedly; all of LFAGS, LFAMS and LFF-1S can enormously strengthen the lymphocyte proliferative ablility under ConA and LPS induction, this manifests that these three polysaccharides has obvious activating effect on T cell and B cell. The effect of these three compounds reinforcing the phagocytosis of macrophage was obviously significant (p < 0.01) when concentrations were larger than 100μg/ml; the three polysaccharide can also enhance the ability for producing NO of macrophage, and the effect was concentration-dependent. In addition, the experimental results also indicated that LFAGS could inhibit the growth of colon carcinoma cell, while LFAMS and LFF-1S have little influence on colon carcinoma cell growth. The three compounds have no influence on migration of L929 cell.
海带经过水提、醇沉后得到两种多糖,20%醇沉得到的沉淀LFA和60%醇沉得到的沉淀LFF。LFA再经过酸分级得到两种多糖,分别是PH为2.85时的沉淀LFAG和上清LFAM,LFAG和LFAM分别经Sepharose CL-6B凝胶过滤层析制备得到多糖LFAGS和LFAMS;LFF通过DEAE-Cellulose离子交换层析和Sepharose CL-6B凝胶过滤层析制备后得到多糖LFF-1S。本论文对这三种纯化后的多糖分别进行了理化性质的研究和结构分析,其结果如下:
LFAGS为白色粉末,微溶于水,分子量约为42 KDa,由GulA、ManA和GalA三种单糖组成,以1, 4-β-GulAp-1, 4-β-ManAp为主链,每四个ManA连有一个GulA,无分支,主链中有微量的β-1, 4-GalAp。LFAMS为白色粉末,难溶于水,分子量约为12 KDa,由GulA、ManA和GalA三种单糖组成,以1, 4-β-GulAp-1,4 -β-ManAp的重复单位为主链,ManAp的3位有分支,连有β-1, 4-GulAp糖残基或寡糖残基,支链末端为ManA。LFF-1S为白色粉末,易溶于水,单糖组成为GulA、ManA、Man、GluA、Gal和Fuc,分子量约为14 KDa。
实验结果表明海带多糖LFAGS、LFAMS和LFF-1S对淋巴细胞有明显的调节作用。无ConA和LPS诱导时,LFAMS和LFF-1S可显著地增强小鼠脾混合淋巴细胞的增殖;在ConA和LPS诱导下,三种多糖级分可以显著的刺激ConA和LPS诱导T淋巴细胞和B淋巴细胞增殖,表明三种级分多糖对ConA和LPS诱导的T细胞和B细胞有明显激活作用。当浓度大于100μg/ml时,三种多糖均能极显著地(p < 0.01)增强巨噬细胞的吞噬能力;此外,三种多糖也能增强巨噬细胞产生NO的能力,并且呈现出一定的浓度依赖性;海带多糖LFAGS具有抑制结肠癌细胞生长的作用,而LFAMS和LFF-1S对结肠癌细胞的生长没有明显影响。细胞迁移实验结果表明这三种多糖对L929细胞的迁移没有影响。
Laminaria japonica is a very widespread edible algae. Laminarin has good bioactivities which are beneficial to people’s health. Most studies concerning the L. japonica focus on the sulfated fucosan, the systematic fractionation of L. japonica and in particular, its algin separation, structure identification, and examination of the activity are neglected. This thesis studied on the algin separation, structure identification, and examination of the activity of L. japonica.
The polysaccharide fractions from L. japonica were isolated and purified. First, three polysaccharides, LFAG, LFAM and LFF, were obtained by fractionation with ethanol precipitation and dilute acid. LFAG and LFAM were further purified by Sepharose CL-6B to produce LFAGS and LFAMS, respectively. LFF was first separated with DEAE-Cellulose, then it was fractionated on a Sepharose CL-6B column to give LFF-1S. The physical and chemical properties of these three L. japonica polysaccharides were analyzed, and the major structural features of them were elucidated using 13C-nuclear magnetic resonance spectrometer (13C-NMR). The results are as follows:
LFAGS is white powder and its solubility in water is very low. Sugar composition analysis by high performance liquid chromatography (HPLC) indicated that LFAGS consists of GulA, ManA and GalA. The 13C-NMR results showed that the backbone of LFAGS was in the linkage form of 1, 4-β-GulAp-1, 4-β-ManAp, it is composed of four ManA attaching one GulA units in the order of a heteropolymeric sequences, and LFAGS has no side chain, the molecular weight is 42 KDa. LFAMS is also a kind of white powder which is difficult to dissolve in water. The sugar composition of LFAMS is GulA, ManA and GalA, too. While the backbone of LFAMS is composed of repeated units 1, 4-β-GulAp-1, 4-β-ManAp, there isβ-1, 4 -GulAp attached to other sugar residues side-chain in the C-3 position of ManAp, moreover, sugar composition of the end of the side chain is ManA. The molecular weight of LFAGS is 12 KDa. LFF-1S is white powder and easily soluble in water, the molecular weight is 14 KDa, and its sugar composition is GulA, ManA, Man, GluA, Gal and Fuc.
The results indicate that the L. japonica polysaccharides LFAGS, LFAMS and LFF-1S can regulate the activity of lymphocyte obviously. When there is no ConA and LPS induction, LFAMS and LFF-1S can enhance the proliferative response of mouse lymphocyte markedly; all of LFAGS, LFAMS and LFF-1S can enormously strengthen the lymphocyte proliferative ablility under ConA and LPS induction, this manifests that these three polysaccharides has obvious activating effect on T cell and B cell. The effect of these three compounds reinforcing the phagocytosis of macrophage was obviously significant (p < 0.01) when concentrations were larger than 100μg/ml; the three polysaccharide can also enhance the ability for producing NO of macrophage, and the effect was concentration-dependent. In addition, the experimental results also indicated that LFAGS could inhibit the growth of colon carcinoma cell, while LFAMS and LFF-1S have little influence on colon carcinoma cell growth. The three compounds have no influence on migration of L929 cell.
引文
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