马粪海胆多糖的研究
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摘要
多糖是自然界含量最为丰富的生物大分子,具有多种特殊的生物活性,如抗肿瘤、降血糖、降血脂、抗氧化、抗病毒和抗辐射等。海胆是一种低等的海洋无脊椎动物,属于棘皮动物门海胆纲。海胆黄为名贵的海产珍品,具有极高的营养价值和药用价值。本研究所选用的马粪海胆(Hemicentrotus pulcherrimus)是我国主要经济海胆之一,国内外关于海胆活性成分的研究取得了一定进展,但关于马粪海胆的海胆壳棘和海胆内容物活性多糖的研究尚未见报道。我们分别从海胆壳棘和海胆内容物中分离纯化得到均一多糖组分XB-1和XB-3,通过采用理化性质分析,化学方法和波谱技术结构鉴定等手段,证实XB-1和XB-3均为首次从马粪海胆中分离得到的两种结构新颖的硫酸多糖,并均具有一定的抗肿瘤活性和免疫活性。
本论文共分五部分,分别是海胆壳棘和海胆内容物多糖的提取工艺、海胆壳棘和海胆内容物活性多糖组分的分离纯化、海胆壳棘多糖XB-1和海胆内容物多糖XB-3的结构解析、海胆多糖XB-1和XB-3的抗肿瘤活性和免疫活性。
取得的成果如下：
1.海胆壳棘和海胆内容物多糖的提取工艺研究
海胆壳棘和海胆内容物经脱脂、热水煮沸、浓缩、沉淀、去蛋白、去核酸、醇析、常规干燥得海胆壳棘和海胆内容物粗多糖,采用MTT法筛选抗肿瘤活性,采用正交实验测定多糖的最佳提取工艺。结果表明海胆壳棘粗多糖的最佳提取条件为15倍水,90℃提取5h,提取4次,产率为0.01349。海胆内容物粗多糖的提取最佳条件为10倍水,90℃,5小时,提取4次,产率为0.10845。
海胆壳棘粗多糖剂量为5.0μg/mL时对人肺腺癌A549细胞的抑制率达到47.9%；对乳腺癌MCF-7细胞的抑制率达到38.1%；对宫颈癌Hela细胞无明显抑制作用。海胆内容物粗多糖剂量为5.0μg/mL时,对人肺腺癌A549细胞的抑制作用达到47.2%；对宫颈癌Hela细胞的生长也有较好的抑制作用,对乳腺癌MCF-7细胞无明显抑制作用。
2.海胆壳棘和海胆内容物活性多糖组分的分离纯化研究
海胆壳棘粗多糖和海胆内容物粗多糖经热水溶解,乙醇分级醇析以及凝胶过滤柱层析纯化得海胆壳棘多糖XB-1和海胆内容物多糖XB-3。经高效液相色谱(HPLC)和比旋光度鉴定,表明XB-1和XB-3为均一多糖组分；经高效液相色谱和凝胶过滤柱层析测定其相对分子质量为1.90×106Da和2.0×106Da；经旋光度(C 0.1,H20)测定,XB-1和XB-3的比旋光度分别为+105°和+210°,经苯酚-硫酸法测定XB-1和XB-3总糖含量分别为99.1%和99.3%。
3.海胆壳棘多糖XB-1和海胆内容物多糖XB-3的化学结构研究
通过糖组分分析,高碘酸氧化,Smith降解,糖苷键酶降解分析,脱硫前后硫酸基测定,脱硫前后甲基化分析,红外光谱分析,1H-NMR谱分析和原子力显微镜分析等综合实验,研究了海胆多糖和XB-1和XB-3的结构。糖组成分析结果表明,海胆壳棘多糖XB-1为硫酸岩藻聚糖；海胆内容物多糖XB-3为硫酸葡萄半乳聚糖,其中葡萄糖：半乳糖=2：5；高碘酸氧化、Smith降解及甲基化分析表明,XB-1含有1→2糖苷键和非还原末端1→,2/3的0-4上连有硫酸基；XB-3含有1→6、1→2,6糖苷键和非还原末端1→,2/3的0-4和0-2上连有硫酸基；IR结果表明XB-1和XB-3均由α-D-吡喃糖组成；酶降解实验表明XB-1的重复结构单元中含1→2糖苷键,糖残基为α构型。
综合各种实验结果,海胆壳棘多糖XB-1和海胆内容物多糖XB-3的结构单元如下：
4.海胆壳棘多糖XB-1和海胆内容物多糖XB-3的抗肿瘤活性研究
MTT法测定XB-1和XB-3体外对肿瘤细胞的影响。结果表明：
海胆壳棘多糖XB-1对人肺腺癌A549、乳腺癌MCF-7细胞和宫颈癌Hela细胞的生长均有一定的抑制作用,XB-1剂量为50μg/mL时对人肺腺癌A549、乳腺癌MCF-7和宫颈癌Hela细胞的抑制率分别达到63.2%、18.32%和42.08%。三种癌细胞对XB-1的剂量依赖性较大,且海胆壳棘多糖对A549细胞的抑制效果最强。
海胆内容物多糖XB-3对宫颈癌Hela细胞的生长有一定的抑制作用,剂量为50μg/mL时对宫颈癌Hela细胞的抑制率达到35.15%；对乳腺癌MCF-7细胞和咽喉癌hep-2细胞无明显抑制作用。
5.海胆壳棘多糖XB-1和海胆内容物多糖XB-3的免疫活性研究
体外免疫实验表明,海胆壳棘多糖XB-1对脾淋巴细胞有细胞毒作用,在1000～1.95μg/mL范围内,随着XB-1浓度的降低对脾淋巴细胞的细胞毒性减弱；海胆内容物多糖XB-3对脾淋巴细胞无明显的抑制作用；采用MTT法检测海胆多糖对ConA、LPS诱导下T、B淋巴细胞增殖反应的影响,结果显示,在62.5～1.95μg/mL浓度范围内海胆壳棘多糖XB-1对ConA诱导的T淋巴细胞增殖反应无明显影响,在125～1000μg/mL浓度范围内有轻度的抑制作用；但对LPS诱导的B淋巴细胞增殖反应有显著的增强作用。海胆内容物多糖XB-3对ConA诱导的T淋巴细胞增殖反应随着XB-3浓度的降低而明显；在1.95～1000μg/mL浓度范围内对LPS诱导的B淋巴细胞增殖反应有显著的增强作用。
Polysaccharide is the richest biomacromolecules in nature,it has many kinds of significant bioactivities, such as antitumor activity, hypoglycemic effect, antilipemic function, anti-oxidative effect, anti-virus, radioprotection and so on.Sea urchins, a kind of marine invertebrates, are sorted as Phylum Echinodermata, Class Echinoidea. Sea urchin roes or eggs are a kind of favorite seafood for their high nutrition and medicinal values in China. Considerable advances have been made at home and abroad in isolation of natural products from sea urchins. Studies on polysaccharides from Hemicentrotus pulcherrimus are not yet reported.Hemicentrotus pulcherrimus, collected for research, is one of the economic sea urchins in China.This paper reported two novel sulfated polysaccharide named as XB-1%和XB-3 was isolated and fractionated from Hemicentrotus pulcherrimus. Its physic-chemical properities were analyzed, and its structure was determined by chemical analysis and spectrum technology, indicating XB-1和XB-3 was the novel polysaccharide purified from sea urchin for the first time and evaluation of the antitumor activities.
This dissertation consists of five parts, which are Optimization of extraction process of polysaccharide, Purification of the crude polysaccharide, Structure elucidation of XB-1 & XB-3,Antitumor and immunological activity of XB-1 & XB-3.
The results are shown as following.
1.Optimization of extraction process of polysaccharide
The active crude polysaccharides were obtained by acetone treatment, hot water extraction, removing protein and nucleic acid and ethanol precipitation.According to the orthogonal experiment and antitumor activities assay, the optimum conditions of polysaccharide of shellspines extract were 90℃,five hours, four times and 15mL H2O per gram shellspines. the optimum conditions of polysaccharide of eggs extract were 90℃,five hours, four times and 10mL H2O per gram eggs.
Antitumor activities of crude polysaccharides in vitro were evaluated against human cancer cell A549,MCF-7,Hela by MTT method.The growth inhibitory rate of shellspines was 47.9%,38.1% repectively at the dose of 5.0 ug/mL.It has hardly no inhibition effect to Hela cells.The growth inhibitory rate of eggs was 47.2% at the dose of 5.0ug/mL.It has a certain inhibition effect to Hela and has no inhibition effect to MCF-7 cells.
2.Isolation and purification of the active polysaccharide
Novel sulfated polysaccharide named as XB-1 & XB-3 was isolated from the crude polysaccharides by hot water extraction and gel-permeation. The homogeneity of XB-1 & XB-3 was verified by HPLC,sepharose CL--4B gel chromatography.The polysaccharide XB-1 & XB-3 has a weight-average molecular weight of about 1.90×106 Da和2.0×106 Da by HPLC in reference to standard T-series Dextran. The specific rotation of XB-1 & XB-3 is +105°,+210°(c0.1,H2O).The total sugar content of XB-1 & XB-3 was determined to be 99.1% and 99.3% by phenol-sulfuric acid mthod.
3.Structure elucidation of XB-1 and XB-3
The structure of XB-1 and XB-3 has been determined by G.C, Periodate oxidation, Smith degradation, Methylation analysis, IR,1H-NMR, enzyme hydrolysis, SO4 determination and atomic force microscopy etc experiments. The composition was analysed by GC.Results showed that XB-1 was composed of sulfated Fucans. XB-3 was composed of the monosaccharide of galactose and glucose, and the molar ratio is 5:2.The results of periodate oxidation, Smith degradation and fully Methylated showed that XB-1 was composed of(1→2) Fucose and (1→), and 2/3 of 0-4 is S04. XB-3 showed that(1→6) galactose is main chain and(1→4) glucose is side chain,0-4 and 0-2 link of SO42-.The FT-IR spectrum showed that XB-1 and XB-3 had the hydroxyl stretching vibration of the polysaccharide and that the pyranose residues. Its sulfur content is determined by barium sulfide turbidity analysis.Sulfur content of XB-1 and XB-3 was 57.7%.enzyme hydrolysis showed that(1→2)Fucose is the main chain of XB-1.
On the basis of the above-mentioned results,it can be concluded that XB-1 and XB-3 is composed of a repeating unit having the possible structure as shown:
4.Antitumor of polysaccharide XB-1 and XB-3
Antitumor activities of polysaccharides in vitro were evaluated against human cancer cell A549, MCF-7, Hela by a MTT method.
The XB-1 has certain inhibition in A549,MCF-7 cells and Hela cell growth, and showed strongly dependent on the dose.The growth inhibitory rate of XB-1 was 63.2%,18.32 %,42.08% repectively at the dose of 50 ug/mL.
The XB-3 has a certain inhibition in Hela cell growth, The growth inhibitory rate was 35.15% at the dose of 50 ug/mL. It has no inhibition effect to MCF-7 and hep-2 cells.
5.Immunological activity of polysaccharide XB-1 and XB-3
By means of MTT, The immuno-modulating assay in vitro showed that XB-3 has no obvious inhibition effect to spleen lymphocyte. Within the range of 1000～1.95μg/mL, With the decreasing concentration of XB-1,the positive effect is weaker gradually.Effect of XB-3 to the propagation of T, B lymphocyte induced by ConA or LPS.XB-1 didn't co-stimulate spleen lymphocyte proliferation with ConA at 62.5～1.95μg/mL,XB-1 co-stimulated spleen lymphocyte proliferation with LPS(10μg/mL) at the dosage of 125～1000μg/mL remarkably. The effect of XB-3 to the propagation of T lymphocyte induced by ConA is more pronounced with decreasing concentration. Within the concentration of 1.95～1000μg/mL, XB-3 has a pronounced increasing effect to the propagation of B lymphocyte induced by LPS.
本论文共分五部分,分别是海胆壳棘和海胆内容物多糖的提取工艺、海胆壳棘和海胆内容物活性多糖组分的分离纯化、海胆壳棘多糖XB-1和海胆内容物多糖XB-3的结构解析、海胆多糖XB-1和XB-3的抗肿瘤活性和免疫活性。
取得的成果如下：
1.海胆壳棘和海胆内容物多糖的提取工艺研究
海胆壳棘和海胆内容物经脱脂、热水煮沸、浓缩、沉淀、去蛋白、去核酸、醇析、常规干燥得海胆壳棘和海胆内容物粗多糖,采用MTT法筛选抗肿瘤活性,采用正交实验测定多糖的最佳提取工艺。结果表明海胆壳棘粗多糖的最佳提取条件为15倍水,90℃提取5h,提取4次,产率为0.01349。海胆内容物粗多糖的提取最佳条件为10倍水,90℃,5小时,提取4次,产率为0.10845。
海胆壳棘粗多糖剂量为5.0μg/mL时对人肺腺癌A549细胞的抑制率达到47.9%；对乳腺癌MCF-7细胞的抑制率达到38.1%；对宫颈癌Hela细胞无明显抑制作用。海胆内容物粗多糖剂量为5.0μg/mL时,对人肺腺癌A549细胞的抑制作用达到47.2%；对宫颈癌Hela细胞的生长也有较好的抑制作用,对乳腺癌MCF-7细胞无明显抑制作用。
2.海胆壳棘和海胆内容物活性多糖组分的分离纯化研究
海胆壳棘粗多糖和海胆内容物粗多糖经热水溶解,乙醇分级醇析以及凝胶过滤柱层析纯化得海胆壳棘多糖XB-1和海胆内容物多糖XB-3。经高效液相色谱(HPLC)和比旋光度鉴定,表明XB-1和XB-3为均一多糖组分；经高效液相色谱和凝胶过滤柱层析测定其相对分子质量为1.90×106Da和2.0×106Da；经旋光度(C 0.1,H20)测定,XB-1和XB-3的比旋光度分别为+105°和+210°,经苯酚-硫酸法测定XB-1和XB-3总糖含量分别为99.1%和99.3%。
3.海胆壳棘多糖XB-1和海胆内容物多糖XB-3的化学结构研究
通过糖组分分析,高碘酸氧化,Smith降解,糖苷键酶降解分析,脱硫前后硫酸基测定,脱硫前后甲基化分析,红外光谱分析,1H-NMR谱分析和原子力显微镜分析等综合实验,研究了海胆多糖和XB-1和XB-3的结构。糖组成分析结果表明,海胆壳棘多糖XB-1为硫酸岩藻聚糖；海胆内容物多糖XB-3为硫酸葡萄半乳聚糖,其中葡萄糖：半乳糖=2：5；高碘酸氧化、Smith降解及甲基化分析表明,XB-1含有1→2糖苷键和非还原末端1→,2/3的0-4上连有硫酸基；XB-3含有1→6、1→2,6糖苷键和非还原末端1→,2/3的0-4和0-2上连有硫酸基；IR结果表明XB-1和XB-3均由α-D-吡喃糖组成；酶降解实验表明XB-1的重复结构单元中含1→2糖苷键,糖残基为α构型。
综合各种实验结果,海胆壳棘多糖XB-1和海胆内容物多糖XB-3的结构单元如下：
4.海胆壳棘多糖XB-1和海胆内容物多糖XB-3的抗肿瘤活性研究
MTT法测定XB-1和XB-3体外对肿瘤细胞的影响。结果表明：
海胆壳棘多糖XB-1对人肺腺癌A549、乳腺癌MCF-7细胞和宫颈癌Hela细胞的生长均有一定的抑制作用,XB-1剂量为50μg/mL时对人肺腺癌A549、乳腺癌MCF-7和宫颈癌Hela细胞的抑制率分别达到63.2%、18.32%和42.08%。三种癌细胞对XB-1的剂量依赖性较大,且海胆壳棘多糖对A549细胞的抑制效果最强。
海胆内容物多糖XB-3对宫颈癌Hela细胞的生长有一定的抑制作用,剂量为50μg/mL时对宫颈癌Hela细胞的抑制率达到35.15%；对乳腺癌MCF-7细胞和咽喉癌hep-2细胞无明显抑制作用。
5.海胆壳棘多糖XB-1和海胆内容物多糖XB-3的免疫活性研究
体外免疫实验表明,海胆壳棘多糖XB-1对脾淋巴细胞有细胞毒作用,在1000～1.95μg/mL范围内,随着XB-1浓度的降低对脾淋巴细胞的细胞毒性减弱；海胆内容物多糖XB-3对脾淋巴细胞无明显的抑制作用；采用MTT法检测海胆多糖对ConA、LPS诱导下T、B淋巴细胞增殖反应的影响,结果显示,在62.5～1.95μg/mL浓度范围内海胆壳棘多糖XB-1对ConA诱导的T淋巴细胞增殖反应无明显影响,在125～1000μg/mL浓度范围内有轻度的抑制作用；但对LPS诱导的B淋巴细胞增殖反应有显著的增强作用。海胆内容物多糖XB-3对ConA诱导的T淋巴细胞增殖反应随着XB-3浓度的降低而明显；在1.95～1000μg/mL浓度范围内对LPS诱导的B淋巴细胞增殖反应有显著的增强作用。
Polysaccharide is the richest biomacromolecules in nature,it has many kinds of significant bioactivities, such as antitumor activity, hypoglycemic effect, antilipemic function, anti-oxidative effect, anti-virus, radioprotection and so on.Sea urchins, a kind of marine invertebrates, are sorted as Phylum Echinodermata, Class Echinoidea. Sea urchin roes or eggs are a kind of favorite seafood for their high nutrition and medicinal values in China. Considerable advances have been made at home and abroad in isolation of natural products from sea urchins. Studies on polysaccharides from Hemicentrotus pulcherrimus are not yet reported.Hemicentrotus pulcherrimus, collected for research, is one of the economic sea urchins in China.This paper reported two novel sulfated polysaccharide named as XB-1%和XB-3 was isolated and fractionated from Hemicentrotus pulcherrimus. Its physic-chemical properities were analyzed, and its structure was determined by chemical analysis and spectrum technology, indicating XB-1和XB-3 was the novel polysaccharide purified from sea urchin for the first time and evaluation of the antitumor activities.
This dissertation consists of five parts, which are Optimization of extraction process of polysaccharide, Purification of the crude polysaccharide, Structure elucidation of XB-1 & XB-3,Antitumor and immunological activity of XB-1 & XB-3.
The results are shown as following.
1.Optimization of extraction process of polysaccharide
The active crude polysaccharides were obtained by acetone treatment, hot water extraction, removing protein and nucleic acid and ethanol precipitation.According to the orthogonal experiment and antitumor activities assay, the optimum conditions of polysaccharide of shellspines extract were 90℃,five hours, four times and 15mL H2O per gram shellspines. the optimum conditions of polysaccharide of eggs extract were 90℃,five hours, four times and 10mL H2O per gram eggs.
Antitumor activities of crude polysaccharides in vitro were evaluated against human cancer cell A549,MCF-7,Hela by MTT method.The growth inhibitory rate of shellspines was 47.9%,38.1% repectively at the dose of 5.0 ug/mL.It has hardly no inhibition effect to Hela cells.The growth inhibitory rate of eggs was 47.2% at the dose of 5.0ug/mL.It has a certain inhibition effect to Hela and has no inhibition effect to MCF-7 cells.
2.Isolation and purification of the active polysaccharide
Novel sulfated polysaccharide named as XB-1 & XB-3 was isolated from the crude polysaccharides by hot water extraction and gel-permeation. The homogeneity of XB-1 & XB-3 was verified by HPLC,sepharose CL--4B gel chromatography.The polysaccharide XB-1 & XB-3 has a weight-average molecular weight of about 1.90×106 Da和2.0×106 Da by HPLC in reference to standard T-series Dextran. The specific rotation of XB-1 & XB-3 is +105°,+210°(c0.1,H2O).The total sugar content of XB-1 & XB-3 was determined to be 99.1% and 99.3% by phenol-sulfuric acid mthod.
3.Structure elucidation of XB-1 and XB-3
The structure of XB-1 and XB-3 has been determined by G.C, Periodate oxidation, Smith degradation, Methylation analysis, IR,1H-NMR, enzyme hydrolysis, SO4 determination and atomic force microscopy etc experiments. The composition was analysed by GC.Results showed that XB-1 was composed of sulfated Fucans. XB-3 was composed of the monosaccharide of galactose and glucose, and the molar ratio is 5:2.The results of periodate oxidation, Smith degradation and fully Methylated showed that XB-1 was composed of(1→2) Fucose and (1→), and 2/3 of 0-4 is S04. XB-3 showed that(1→6) galactose is main chain and(1→4) glucose is side chain,0-4 and 0-2 link of SO42-.The FT-IR spectrum showed that XB-1 and XB-3 had the hydroxyl stretching vibration of the polysaccharide and that the pyranose residues. Its sulfur content is determined by barium sulfide turbidity analysis.Sulfur content of XB-1 and XB-3 was 57.7%.enzyme hydrolysis showed that(1→2)Fucose is the main chain of XB-1.
On the basis of the above-mentioned results,it can be concluded that XB-1 and XB-3 is composed of a repeating unit having the possible structure as shown:
4.Antitumor of polysaccharide XB-1 and XB-3
Antitumor activities of polysaccharides in vitro were evaluated against human cancer cell A549, MCF-7, Hela by a MTT method.
The XB-1 has certain inhibition in A549,MCF-7 cells and Hela cell growth, and showed strongly dependent on the dose.The growth inhibitory rate of XB-1 was 63.2%,18.32 %,42.08% repectively at the dose of 50 ug/mL.
The XB-3 has a certain inhibition in Hela cell growth, The growth inhibitory rate was 35.15% at the dose of 50 ug/mL. It has no inhibition effect to MCF-7 and hep-2 cells.
5.Immunological activity of polysaccharide XB-1 and XB-3
By means of MTT, The immuno-modulating assay in vitro showed that XB-3 has no obvious inhibition effect to spleen lymphocyte. Within the range of 1000～1.95μg/mL, With the decreasing concentration of XB-1,the positive effect is weaker gradually.Effect of XB-3 to the propagation of T, B lymphocyte induced by ConA or LPS.XB-1 didn't co-stimulate spleen lymphocyte proliferation with ConA at 62.5～1.95μg/mL,XB-1 co-stimulated spleen lymphocyte proliferation with LPS(10μg/mL) at the dosage of 125～1000μg/mL remarkably. The effect of XB-3 to the propagation of T lymphocyte induced by ConA is more pronounced with decreasing concentration. Within the concentration of 1.95～1000μg/mL, XB-3 has a pronounced increasing effect to the propagation of B lymphocyte induced by LPS.
引文
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