广东海带岩藻多糖的分离纯化、结构鉴定及生物活性研究
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摘要
海带岩藻多糖，又叫岩藻聚糖,是富含硫酸根的L-岩藻糖，研究发现海带岩藻多糖具有抗病毒、抗凝血、抑制肿瘤以及调节免疫功能等多种作用。因此弄清它的结构和功能关系，有助于开发应用这类功能性多糖。
由于岩藻多糖结构十分复杂，它随产地、品种、季节、组织部位及处理方法的不同而显示出较大的差异，这为研究带来了很大的麻烦，往往使研究结果之间存在一定的差异性。
目的:对广东海域海带岩藻多糖的提取和纯化工艺、抗氧化活性进行研究，以期为广东海域海带岩藻多糖的药物及功能性食品开发提供科学依据和参考。
方法：采用稀酸和微波辅助提取工艺，设计正交实验优选提取条件；分别用EDTA、硅藻土去除岩藻多糖中的金属离子；用凝胶柱层析、高效液相色谱法、比旋光度法对岩藻粗多糖（LJF）分离纯化后的主要组分LJF-2的纯度进行鉴定；紫外光谱、红外光谱、XRD、刚果红试验对LJF-2的结构进行表征；研究了LJF和LJF-2的体外抗氧化活性、还原能力及对H_2O_2诱导血红细胞氧化溶血的抑制作用。
结果：稀酸提取的最佳工艺条件为: pH为2、提取温度为60℃、提取时间为5h,此条件下多糖得率为4.85%。微波辅助最佳提取工艺条件为：微波作用20min，微波功率560W，提取次数为4次，此条件下多糖得率为5.46%。EDTA对金属离子Cd、Fe的去除率分别为71.78%和66.78%，明显高于相同浓度下硅藻土的去除率。用凝胶柱层析、高效液相色谱、比旋光度法对LJF-2纯度进行鉴定,结果表明LJF-2为单一组分；紫外光谱表明LJF-2不含蛋白质、多肽，红外光谱显示LJF-2具有多糖的特征吸收峰，含有硫酸根及糖醛酸,是一种硫酸化的杂多糖；XRD表明LJF-2几乎不含结晶，刚果红试验显示LJF-2在水溶液中的空间构象无规则卷曲状。体外抗氧化活性结果显示且在一定浓度范围内LJF和LJF-2对自由基清除效果与多糖浓度呈正相关。当多糖浓度为6.0g/L时，LJF-2对DPPH、·OH、超氧阴离子自由基的清除率分别为79.30%、89.66%、79.61%，低于同浓度下LJF对自由基的清除能力；当多糖浓度为6.0g/L时，LJF-2的吸光值为0.812，低于LJF的吸光值0.840，表明LJF的还原能力要高于LJF-2。血红细胞氧化溶血的抑制实验显示当多糖浓度相同时，LJF对H_2O_2诱导血红细胞氧化溶血的抑制作用要强于LJF-2，这与其抗氧化性结果一致。
研究表明，相对于稀酸提取，微波辅助提取海带岩藻多糖，提取率更高，提取所得粗多糖比纯化后的岩藻多糖具有的更强的抗氧化活性。本研究为后续对岩藻多糖组分纯化，功能发现提供技术基础。
Laminaries japonica fucoidin, also known as Fucoidan, consists a number ofL-fucoses with sulphate radicals. Fucoidan has many bioactivities, such as anti-virus,anticoagulant, killing tumor cells and adjusting immune function. Laminaria japonicafucoidin is a sulphated polysaccharide, and its structure is very complex and differentfrom each other with its origin, growing in various seasons, and different tissues, andtreatment methods, which leads to a lot of troubles in puzzling results from differentgroups.
Objective: We seeked the conditions of extraction and purification, and testedantioxidant activity of fucoidan from Laminaria japonica in Southern China sea. Inorder to provide primary dates on developing medicament and food based on fucoidanextracted from Laminaria japonica in Southern China sea.
Methods: Diluted hydyochloric acid and microwave-assisted extractiontechnology were used to investigate the extraction technology of the fucoidan fromLaminarie japonica. The orthogonal test was practised in seeking the optimalextraction conditions. The effect of competitive chelators, EDTA and diatomaceousearth on depriving heavy metals binding with fucoidan was checked. The purificationof main component in crude fucoidan(LJF)﹑LJF-2was proved by chromatography ofSephadexG-200﹑HPLC and polarimetry respectively. The structure of LJF-2wascharacterized with UV﹑FT-IR﹑XRD and Congo red test. Antioxidant activity andthe reducing ability and protective effects of LJF and LJF-2on H_2O_2inducinghemolysis of erythrocytes were measured by the in vitro system.
Results: The optimal process condition for diluted hydyochloric acid extraction ofthe fucoidan from Laminaria japonica was pH value being4and treating time being5h，and the extraction temperature being60℃, and the extracting ratio of crudefucoidan was4.85%. For microwave-assisted extraction was extraction time being20min, microwave power with560W and extraction times being4times. And theextracting ratio was5.46%. The removal ratios of Cd, and Fe ions by EDTA were71.78%and66.78%respectively, which was significantly higher than those by diatomaceous earth. The purity of LJF-2was identified by chromatography ofSephadexG-200, HPLC and polarimetry respectively. Results showed that LJF-2waspurific without proteins, peptides and other polysaccharides. The FT-IR result provedthat LJF-2had a characteristic absorption peak of polysaccharide containing sulfateand uronic acid. The result of XRD showed that LJF-2was non crystal. The Congored test showed the space conformation of LJF-2in aqueous solution was non-rulecurly instead of triple helix as polysaccharides usually do. The results of antioxidantsshowed that LJF and LJF-2had certain abilities of scavenging free radicals. Theirscavenging effects showed positive correlation with their concentrations. When theconcentration of polysaccharide was6.0g/L, the scavenging rates of LJF-2on DPPH﹑hydroxyl radicals and superoxide anion radicals were79.30%﹑89.66%﹑79.61%respectively. They were all lower than the scavenging rates of LJF with the sameconcentration. When the concentration of polysaccharide was6.0g/L, the absorbancevalue was0.812, and lower than the same concentrations of the absorbance value ofLJF-2, which ment that the reducing ability of LJF was stronger than that of LJF-2.The protective effects of LJF and LJF-2on H_2O_2induced hemolysis of erythrocytesshowed that LJF was stronger than LJF-2, which was consistent with their antioxidantactivities.
In conclusion, the extraction rate of microwave-assisted of fucoidan was thehighest in all extraction processes. The antioxidant activity of crude fucoidan wasstronger than the purified fucoidan. These demonstates present a sophisticated processto find out relations of structures and functions of other components from fucoidan.
由于岩藻多糖结构十分复杂，它随产地、品种、季节、组织部位及处理方法的不同而显示出较大的差异，这为研究带来了很大的麻烦，往往使研究结果之间存在一定的差异性。
目的:对广东海域海带岩藻多糖的提取和纯化工艺、抗氧化活性进行研究，以期为广东海域海带岩藻多糖的药物及功能性食品开发提供科学依据和参考。
方法：采用稀酸和微波辅助提取工艺，设计正交实验优选提取条件；分别用EDTA、硅藻土去除岩藻多糖中的金属离子；用凝胶柱层析、高效液相色谱法、比旋光度法对岩藻粗多糖（LJF）分离纯化后的主要组分LJF-2的纯度进行鉴定；紫外光谱、红外光谱、XRD、刚果红试验对LJF-2的结构进行表征；研究了LJF和LJF-2的体外抗氧化活性、还原能力及对H_2O_2诱导血红细胞氧化溶血的抑制作用。
结果：稀酸提取的最佳工艺条件为: pH为2、提取温度为60℃、提取时间为5h,此条件下多糖得率为4.85%。微波辅助最佳提取工艺条件为：微波作用20min，微波功率560W，提取次数为4次，此条件下多糖得率为5.46%。EDTA对金属离子Cd、Fe的去除率分别为71.78%和66.78%，明显高于相同浓度下硅藻土的去除率。用凝胶柱层析、高效液相色谱、比旋光度法对LJF-2纯度进行鉴定,结果表明LJF-2为单一组分；紫外光谱表明LJF-2不含蛋白质、多肽，红外光谱显示LJF-2具有多糖的特征吸收峰，含有硫酸根及糖醛酸,是一种硫酸化的杂多糖；XRD表明LJF-2几乎不含结晶，刚果红试验显示LJF-2在水溶液中的空间构象无规则卷曲状。体外抗氧化活性结果显示且在一定浓度范围内LJF和LJF-2对自由基清除效果与多糖浓度呈正相关。当多糖浓度为6.0g/L时，LJF-2对DPPH、·OH、超氧阴离子自由基的清除率分别为79.30%、89.66%、79.61%，低于同浓度下LJF对自由基的清除能力；当多糖浓度为6.0g/L时，LJF-2的吸光值为0.812，低于LJF的吸光值0.840，表明LJF的还原能力要高于LJF-2。血红细胞氧化溶血的抑制实验显示当多糖浓度相同时，LJF对H_2O_2诱导血红细胞氧化溶血的抑制作用要强于LJF-2，这与其抗氧化性结果一致。
研究表明，相对于稀酸提取，微波辅助提取海带岩藻多糖，提取率更高，提取所得粗多糖比纯化后的岩藻多糖具有的更强的抗氧化活性。本研究为后续对岩藻多糖组分纯化，功能发现提供技术基础。
Laminaries japonica fucoidin, also known as Fucoidan, consists a number ofL-fucoses with sulphate radicals. Fucoidan has many bioactivities, such as anti-virus,anticoagulant, killing tumor cells and adjusting immune function. Laminaria japonicafucoidin is a sulphated polysaccharide, and its structure is very complex and differentfrom each other with its origin, growing in various seasons, and different tissues, andtreatment methods, which leads to a lot of troubles in puzzling results from differentgroups.
Objective: We seeked the conditions of extraction and purification, and testedantioxidant activity of fucoidan from Laminaria japonica in Southern China sea. Inorder to provide primary dates on developing medicament and food based on fucoidanextracted from Laminaria japonica in Southern China sea.
Methods: Diluted hydyochloric acid and microwave-assisted extractiontechnology were used to investigate the extraction technology of the fucoidan fromLaminarie japonica. The orthogonal test was practised in seeking the optimalextraction conditions. The effect of competitive chelators, EDTA and diatomaceousearth on depriving heavy metals binding with fucoidan was checked. The purificationof main component in crude fucoidan(LJF)﹑LJF-2was proved by chromatography ofSephadexG-200﹑HPLC and polarimetry respectively. The structure of LJF-2wascharacterized with UV﹑FT-IR﹑XRD and Congo red test. Antioxidant activity andthe reducing ability and protective effects of LJF and LJF-2on H_2O_2inducinghemolysis of erythrocytes were measured by the in vitro system.
Results: The optimal process condition for diluted hydyochloric acid extraction ofthe fucoidan from Laminaria japonica was pH value being4and treating time being5h，and the extraction temperature being60℃, and the extracting ratio of crudefucoidan was4.85%. For microwave-assisted extraction was extraction time being20min, microwave power with560W and extraction times being4times. And theextracting ratio was5.46%. The removal ratios of Cd, and Fe ions by EDTA were71.78%and66.78%respectively, which was significantly higher than those by diatomaceous earth. The purity of LJF-2was identified by chromatography ofSephadexG-200, HPLC and polarimetry respectively. Results showed that LJF-2waspurific without proteins, peptides and other polysaccharides. The FT-IR result provedthat LJF-2had a characteristic absorption peak of polysaccharide containing sulfateand uronic acid. The result of XRD showed that LJF-2was non crystal. The Congored test showed the space conformation of LJF-2in aqueous solution was non-rulecurly instead of triple helix as polysaccharides usually do. The results of antioxidantsshowed that LJF and LJF-2had certain abilities of scavenging free radicals. Theirscavenging effects showed positive correlation with their concentrations. When theconcentration of polysaccharide was6.0g/L, the scavenging rates of LJF-2on DPPH﹑hydroxyl radicals and superoxide anion radicals were79.30%﹑89.66%﹑79.61%respectively. They were all lower than the scavenging rates of LJF with the sameconcentration. When the concentration of polysaccharide was6.0g/L, the absorbancevalue was0.812, and lower than the same concentrations of the absorbance value ofLJF-2, which ment that the reducing ability of LJF was stronger than that of LJF-2.The protective effects of LJF and LJF-2on H_2O_2induced hemolysis of erythrocytesshowed that LJF was stronger than LJF-2, which was consistent with their antioxidantactivities.
In conclusion, the extraction rate of microwave-assisted of fucoidan was thehighest in all extraction processes. The antioxidant activity of crude fucoidan wasstronger than the purified fucoidan. These demonstates present a sophisticated processto find out relations of structures and functions of other components from fucoidan.
引文
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