肠浒苔多糖结构及生物活性研究
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摘要
本论文采用蒸馏水和稀碱溶液依次提取肠浒苔水溶性多糖,通过不同方法对两种粗多糖进行分级,研究多糖的化学结构、抗肿瘤和免疫活性,并探讨多糖的结构与生物活性的关系。
用95%的乙醇回流后,用蒸馏水以浸提比为1:20提取多糖,常规干燥后得到肠浒苔水溶性粗多糖WE;残渣洗净、干燥后,用0.5 M NaOH室温下提取,盐酸中和,离心,常规干燥后得碱提水溶性粗多糖AE。WE经冻融分级、脱蛋白、离子交换层析和分子筛凝胶层析纯化后得到中性糖WEA和酸性糖WEB;AE经冻融分级、脱蛋白、超滤分级和分子筛凝胶层析后得到DAEA(过膜部分)和DAEB(膜上部分)。HPLC检测WEA、WEB、DAEA和DAEB均为单一峰,平均分子量分别为72.03kDa、60.12kDa、18.21kDa和46.80kDa。
GC分析、间羟联苯法及离子色谱检测表明(摩尔百分含量,以下同):WE中含有鼠李糖(58.00%)、葡萄糖(20.25%)、木糖(13.54%)、半乳糖(2.27%)、甘露糖(2.26%)和葡萄糖醛酸(3.43%);WEA含有葡萄糖(55.24%)、鼠李糖(23.76%)、木糖(17.11%)、甘露糖(2.21%)及半乳糖(1.67%);WEB含有鼠李糖(68.81%)、木糖(9.40%)、半乳糖(4.89%)和葡萄糖醛酸(16.89%),硫酸根19.98%。AE含有鼠李糖(15.93%)、甘露糖(18.04%)、半乳糖(13.09%)、葡萄糖(11.96%)阿拉伯糖(10.94%)、核糖(8.89%)、木糖(5.29%)和葡萄糖醛酸(15.86%);DAEA含有葡萄糖(33.69%)、鼠李糖(11.44%)、半乳糖(9.64%)、甘露糖(7.92%)、木糖(2.79%)、阿拉伯糖(7.58%)和葡萄糖醛酸(29.65%);DAEB含有鼠李糖(69.20%)、木糖(12.90%)、葡萄糖(8.20%)、半乳糖(7.40%)及葡萄糖醛酸(2.30%),硫酸根9.6%。
对肠浒苔多糖各级分免疫活性研究表明:WE、WEA、WEB、AE和DAEB能够增强淋巴细胞的增殖和腹腔巨噬细胞的吞噬作用,促进巨噬细胞分泌NO,增强诱导型一氧化氮合酶(iNOS)活性,促进肿瘤坏死因子-α(TNF-α)的分泌。其中水提多糖中WEB的免疫活性优于WEA和WE;碱提多糖中DAEB的免疫活性优于AE,而DAEA活性不明显。
对肠浒苔多糖各级分进行体外抗肿瘤实验研究表明:WE、WEA、WEB、AE、DAEA及DAEB对S 180、Hela、HepG2细胞均没有抑制作用。
根据上述结果,选择WEA、WEB和DAEB进行抗肿瘤实验研究。结果表明:WEA、WEB和DAEB均能抑制小鼠体内移植性肿瘤S180的生长。WEB抗肿瘤活性优于WEA和DAEB,WEB在400 mg/kg时抑瘤率最高,为74.57%。并且WEA、WEB和DAEB均能增加荷瘤小鼠的相对脾重和相对胸腺重,增加血清中TNF-α的含量。
采用部分酸水解、高碘酸氧化、Smith降解、甲基化及IR、GC、GC-MS、13C-NMR等方法对WEB和DAEB进行结构分析。WEBD和WEBD-R甲基化结果比较表明WEB中Rha以(1→4)键型为主,还有(1→2),(1→2,4)、(1→4)、和(1→3)连接键型;Xyl和Gal的连接键型均为(1→),同时含有(1→4)-GlcA及少量(1→)-GlcA;该糖中每5个(1→4)-Rha中就有一个分支,分支点在O-2。通过WEB和WEBD的13C-NMR结果比较发现,WEB中硫酸根位于鼠李糖的O-3。DAEB甲基化分析结果表明DAEB由(1→)、(1→4)、(1→2,4)-Rha;(1→)、(1→2,3)、(1→3)-Xyl;(1→4)-Glc及(1→3)-Gal组成;每6个(1→4)-Rha中就有一个分支,分支点在O-2。另外,通过对DAEB和DAEB-D的甲基化结果比较表明,大部分硫酸根位于(1→4)-Rha的O-3,还有一少部分硫酸根位于(1→3)-Xyl的O-2位。
最后,本论文检测了硫酸根离子对WEB免疫活性的影响。结果表明,WEBD对淋巴细胞转化的促进作用,以及对巨噬细胞的激活作用明显减弱。可见,硫酸根与酸性多糖WEB的免疫活性密切相关。
The polysaccharides were isolated sequently from Enteromorpha intestinalis by hot water, diluted alkali solution. Purification, immunostimulation and antitumor activities and the structure of these polysaccharides were studied in this paper. The relationship between the structure and activities of the polysaccharides was also discussed.
First, the air dried algae was extracted with 95% ethanol at 60°C for 2 h to remove pigment and micromolecular substance. Then the residues were extracted with distilled water at 90°C for 2 h, the solid-liquid ratio was 1:20, the progress was repeated twice. After combination, concentration and lyophillization, we obtained the crude polysaccharide WE. The water unextractable solid was washed, dried and extracted with 0.5 M NaOH solution which contained 0.3% (w/w) KBH4 at room temperature for 2 h, and the green slurry was filtered through line cloth. The suspension was neutralized with hydrochloric acid (1 M) and filtered. The supernatant containing water-soluble polysaccharide was dialyzed, concentrated, lyophilized and then dried, yielding the crude polysaccharide AE.
The water-extracted polysaccharide WE was fractionated by frozen-thawed, deproteinated by Sevag method, and purified by anion-exchange chromatography on a column of DEAE-Sepharose CL-6B and gel filtration chromatography on a column of Sepharose CL-6B. Then two fractions were obtained and named as WEA (the neutral polysaccharide) and WEB ( the acid polysaccharide), respectively. WEA and WEB appeared as single symmetrical peak on HPLC, and the average molecular weights were 72.03kDa and 60.12kDa which were caculated through calculation from dextran standard. The alkali-extracted polysaccharide AE was fractionated by frozen-thawed, deproteinated by Sevag method, and purified ultrafiltration and gel filtration chromatography on a column of Sepharose CL-6B. Then two fractions were obtained and named as DAEA and DAEB, respectively. DAEA and DAEB appeared as single symmetrical peak on HPLC, and the average molecular weights were 18.21kDa and 46.80kDa.
GC analysis showed that WE was composed of Rha (58.00%), Glc (20.50%), Xyl (13.54%), Gal (2.27%), Man (2.26%), and small content of glucuronic acid (3.43%); WEA was composed of Glc (55.24%), Rha (23.76%), Xyl (17.11%), Man (2.21%) and Gal (1.67%); the acid fraction WEB was composed of Rha (68.81%), Xyl (9.40%), Gal (4.89%) and Glucuronic acid (16.89%), the sulfate content of WEB was 19.98%. AE was composed of Rha (15.93%), Man (18.04%), Gal (13.09%), Glc (11.96%), Ara (10.94%), Rib (8.89%), Xyl (5.29%), and glucuronic acid (15.86%); DAEA consisted of Glc (33.69%), Rha (11.44%), Gal (9.64%), Man (7.92%), Ara (7.58%) and Xyl (2.79%); DAEB consisted of Rha (69.20%), Xyl (12.90%), Glc (8.20%), Gal (7.40%) and small content of glucuronic acid (2.30%), the sulfate content of WEB was 9.6%.
WE, WEA, WEB, AE, DAEA and DAEB was cultivated with lymphocyte cells or peritoneal macrophages. Among the six polysaccharides, WEA, WEB and DAEB could induce lymphoctes proliferation, increase the production of TNF-αin macrophages, and dose-dependently stimulate macrophages to produce NO through the up-regulation of inducible NO synthase (iNOS) activity. The activity of WEB was better than that of WEA and WE. After desulfation, the immunostimulation activities of the polysaccharide WEB was strongly decreased, or even completely disappeared. It was indicated the sulfate group was indispensable for immunostimulation of WEB. Among the alkali-extracted polysaccharides, DAEB showed the better immunostimulation activity.
At the dose of 100, 200, and 400mg/kg, WEA, WEB and DAEB could inhibit tumor growth in S180 tumor-bearing mice, WEB had better activity than WEA and DAEB, the maximal tumor inhibition rate was 74.70% at the does of 400 mg/kg. Meanwhile, they increased in the relative spleen and thymus weight and expression of tumor necrosis factor-alpha (TNF-α) in serum.
However, WE, WEA, WEB, AE, DAEA and DAEB at the concentration up to 800μg/ml did not affect the growth of S180, Hela and HepG2 tumor cells. These results indicated that the tumor inhibitory activity of the three fractions was not due to their cytotoxicities on tumor cells, but may be related to the stimulation of immune system, which has a great significance in therapeutics of the tumor growth.
On the basis of the methylation analysis applied for native polysaccharide, desulfated polysaccharide and desulfated-reduced polysaccharide, partial acid hydrolysis, periodate oxidation and 13C NMR analysis suggested that WEB was mainly contained (1→4)-linked-rhamnose, and other linkages as (1→2), (1→2, 4) and (1→3)-rhamnose, and with a branch at the O-2 position every five (1→4)-linked-rhamnose residues. It also had nonreducing terminal Xyl and Gal; amount of (1→4)-linked-GlcA, and small content of (1→)-linked-GlcA. The O-3 of (1→4)-linked-rhamnose was the sites of sulfation. DAEB was branched and contained principally terminal, (1→4), (1→2,4)-linked-rhamnose; terminal, (1→3)-linked-xylose, (1→4)-linked-glucose, and (1→3)-linked-galactose. Desulfaction of DAEB demonstrated that the O-3 of rhamnose and O-2 of xylose were the sites of sulfation.
用95%的乙醇回流后,用蒸馏水以浸提比为1:20提取多糖,常规干燥后得到肠浒苔水溶性粗多糖WE;残渣洗净、干燥后,用0.5 M NaOH室温下提取,盐酸中和,离心,常规干燥后得碱提水溶性粗多糖AE。WE经冻融分级、脱蛋白、离子交换层析和分子筛凝胶层析纯化后得到中性糖WEA和酸性糖WEB;AE经冻融分级、脱蛋白、超滤分级和分子筛凝胶层析后得到DAEA(过膜部分)和DAEB(膜上部分)。HPLC检测WEA、WEB、DAEA和DAEB均为单一峰,平均分子量分别为72.03kDa、60.12kDa、18.21kDa和46.80kDa。
GC分析、间羟联苯法及离子色谱检测表明(摩尔百分含量,以下同):WE中含有鼠李糖(58.00%)、葡萄糖(20.25%)、木糖(13.54%)、半乳糖(2.27%)、甘露糖(2.26%)和葡萄糖醛酸(3.43%);WEA含有葡萄糖(55.24%)、鼠李糖(23.76%)、木糖(17.11%)、甘露糖(2.21%)及半乳糖(1.67%);WEB含有鼠李糖(68.81%)、木糖(9.40%)、半乳糖(4.89%)和葡萄糖醛酸(16.89%),硫酸根19.98%。AE含有鼠李糖(15.93%)、甘露糖(18.04%)、半乳糖(13.09%)、葡萄糖(11.96%)阿拉伯糖(10.94%)、核糖(8.89%)、木糖(5.29%)和葡萄糖醛酸(15.86%);DAEA含有葡萄糖(33.69%)、鼠李糖(11.44%)、半乳糖(9.64%)、甘露糖(7.92%)、木糖(2.79%)、阿拉伯糖(7.58%)和葡萄糖醛酸(29.65%);DAEB含有鼠李糖(69.20%)、木糖(12.90%)、葡萄糖(8.20%)、半乳糖(7.40%)及葡萄糖醛酸(2.30%),硫酸根9.6%。
对肠浒苔多糖各级分免疫活性研究表明:WE、WEA、WEB、AE和DAEB能够增强淋巴细胞的增殖和腹腔巨噬细胞的吞噬作用,促进巨噬细胞分泌NO,增强诱导型一氧化氮合酶(iNOS)活性,促进肿瘤坏死因子-α(TNF-α)的分泌。其中水提多糖中WEB的免疫活性优于WEA和WE;碱提多糖中DAEB的免疫活性优于AE,而DAEA活性不明显。
对肠浒苔多糖各级分进行体外抗肿瘤实验研究表明:WE、WEA、WEB、AE、DAEA及DAEB对S 180、Hela、HepG2细胞均没有抑制作用。
根据上述结果,选择WEA、WEB和DAEB进行抗肿瘤实验研究。结果表明:WEA、WEB和DAEB均能抑制小鼠体内移植性肿瘤S180的生长。WEB抗肿瘤活性优于WEA和DAEB,WEB在400 mg/kg时抑瘤率最高,为74.57%。并且WEA、WEB和DAEB均能增加荷瘤小鼠的相对脾重和相对胸腺重,增加血清中TNF-α的含量。
采用部分酸水解、高碘酸氧化、Smith降解、甲基化及IR、GC、GC-MS、13C-NMR等方法对WEB和DAEB进行结构分析。WEBD和WEBD-R甲基化结果比较表明WEB中Rha以(1→4)键型为主,还有(1→2),(1→2,4)、(1→4)、和(1→3)连接键型;Xyl和Gal的连接键型均为(1→),同时含有(1→4)-GlcA及少量(1→)-GlcA;该糖中每5个(1→4)-Rha中就有一个分支,分支点在O-2。通过WEB和WEBD的13C-NMR结果比较发现,WEB中硫酸根位于鼠李糖的O-3。DAEB甲基化分析结果表明DAEB由(1→)、(1→4)、(1→2,4)-Rha;(1→)、(1→2,3)、(1→3)-Xyl;(1→4)-Glc及(1→3)-Gal组成;每6个(1→4)-Rha中就有一个分支,分支点在O-2。另外,通过对DAEB和DAEB-D的甲基化结果比较表明,大部分硫酸根位于(1→4)-Rha的O-3,还有一少部分硫酸根位于(1→3)-Xyl的O-2位。
最后,本论文检测了硫酸根离子对WEB免疫活性的影响。结果表明,WEBD对淋巴细胞转化的促进作用,以及对巨噬细胞的激活作用明显减弱。可见,硫酸根与酸性多糖WEB的免疫活性密切相关。
The polysaccharides were isolated sequently from Enteromorpha intestinalis by hot water, diluted alkali solution. Purification, immunostimulation and antitumor activities and the structure of these polysaccharides were studied in this paper. The relationship between the structure and activities of the polysaccharides was also discussed.
First, the air dried algae was extracted with 95% ethanol at 60°C for 2 h to remove pigment and micromolecular substance. Then the residues were extracted with distilled water at 90°C for 2 h, the solid-liquid ratio was 1:20, the progress was repeated twice. After combination, concentration and lyophillization, we obtained the crude polysaccharide WE. The water unextractable solid was washed, dried and extracted with 0.5 M NaOH solution which contained 0.3% (w/w) KBH4 at room temperature for 2 h, and the green slurry was filtered through line cloth. The suspension was neutralized with hydrochloric acid (1 M) and filtered. The supernatant containing water-soluble polysaccharide was dialyzed, concentrated, lyophilized and then dried, yielding the crude polysaccharide AE.
The water-extracted polysaccharide WE was fractionated by frozen-thawed, deproteinated by Sevag method, and purified by anion-exchange chromatography on a column of DEAE-Sepharose CL-6B and gel filtration chromatography on a column of Sepharose CL-6B. Then two fractions were obtained and named as WEA (the neutral polysaccharide) and WEB ( the acid polysaccharide), respectively. WEA and WEB appeared as single symmetrical peak on HPLC, and the average molecular weights were 72.03kDa and 60.12kDa which were caculated through calculation from dextran standard. The alkali-extracted polysaccharide AE was fractionated by frozen-thawed, deproteinated by Sevag method, and purified ultrafiltration and gel filtration chromatography on a column of Sepharose CL-6B. Then two fractions were obtained and named as DAEA and DAEB, respectively. DAEA and DAEB appeared as single symmetrical peak on HPLC, and the average molecular weights were 18.21kDa and 46.80kDa.
GC analysis showed that WE was composed of Rha (58.00%), Glc (20.50%), Xyl (13.54%), Gal (2.27%), Man (2.26%), and small content of glucuronic acid (3.43%); WEA was composed of Glc (55.24%), Rha (23.76%), Xyl (17.11%), Man (2.21%) and Gal (1.67%); the acid fraction WEB was composed of Rha (68.81%), Xyl (9.40%), Gal (4.89%) and Glucuronic acid (16.89%), the sulfate content of WEB was 19.98%. AE was composed of Rha (15.93%), Man (18.04%), Gal (13.09%), Glc (11.96%), Ara (10.94%), Rib (8.89%), Xyl (5.29%), and glucuronic acid (15.86%); DAEA consisted of Glc (33.69%), Rha (11.44%), Gal (9.64%), Man (7.92%), Ara (7.58%) and Xyl (2.79%); DAEB consisted of Rha (69.20%), Xyl (12.90%), Glc (8.20%), Gal (7.40%) and small content of glucuronic acid (2.30%), the sulfate content of WEB was 9.6%.
WE, WEA, WEB, AE, DAEA and DAEB was cultivated with lymphocyte cells or peritoneal macrophages. Among the six polysaccharides, WEA, WEB and DAEB could induce lymphoctes proliferation, increase the production of TNF-αin macrophages, and dose-dependently stimulate macrophages to produce NO through the up-regulation of inducible NO synthase (iNOS) activity. The activity of WEB was better than that of WEA and WE. After desulfation, the immunostimulation activities of the polysaccharide WEB was strongly decreased, or even completely disappeared. It was indicated the sulfate group was indispensable for immunostimulation of WEB. Among the alkali-extracted polysaccharides, DAEB showed the better immunostimulation activity.
At the dose of 100, 200, and 400mg/kg, WEA, WEB and DAEB could inhibit tumor growth in S180 tumor-bearing mice, WEB had better activity than WEA and DAEB, the maximal tumor inhibition rate was 74.70% at the does of 400 mg/kg. Meanwhile, they increased in the relative spleen and thymus weight and expression of tumor necrosis factor-alpha (TNF-α) in serum.
However, WE, WEA, WEB, AE, DAEA and DAEB at the concentration up to 800μg/ml did not affect the growth of S180, Hela and HepG2 tumor cells. These results indicated that the tumor inhibitory activity of the three fractions was not due to their cytotoxicities on tumor cells, but may be related to the stimulation of immune system, which has a great significance in therapeutics of the tumor growth.
On the basis of the methylation analysis applied for native polysaccharide, desulfated polysaccharide and desulfated-reduced polysaccharide, partial acid hydrolysis, periodate oxidation and 13C NMR analysis suggested that WEB was mainly contained (1→4)-linked-rhamnose, and other linkages as (1→2), (1→2, 4) and (1→3)-rhamnose, and with a branch at the O-2 position every five (1→4)-linked-rhamnose residues. It also had nonreducing terminal Xyl and Gal; amount of (1→4)-linked-GlcA, and small content of (1→)-linked-GlcA. The O-3 of (1→4)-linked-rhamnose was the sites of sulfation. DAEB was branched and contained principally terminal, (1→4), (1→2,4)-linked-rhamnose; terminal, (1→3)-linked-xylose, (1→4)-linked-glucose, and (1→3)-linked-galactose. Desulfaction of DAEB demonstrated that the O-3 of rhamnose and O-2 of xylose were the sites of sulfation.
引文
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