刺梨多糖分离纯化、理化性质及生物活性研究
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摘要
本论文对刺梨汁中多糖成分进行分离纯化,并对其理化性质及生物活性进行研究,主要内容如下:
1.刺梨多糖的分离纯化及理化性质研究
刺梨汁经浓缩、乙醇沉淀、脱蛋白、脱色、DEAE-纤维素柱和Sepharose CL-6B柱层析,分离纯化出RRTP-1～RRTP-8共8个组分。其中RRTP-1和RRTP-3组分经凝胶柱层析法和聚丙烯酰胺凝胶电泳法鉴定,证实为分子量分布均一组分。红外光谱显示其具有多糖特征吸收峰。凝胶渗透色谱法测定,RRTP-1和RRTP-3分子量分别为3200、52000。气相色谱分析RRTP-1由鼠李糖、阿拉伯糖、未知糖、木糖、甘露糖、半乳糖、葡萄糖等中性单糖组成,中性单糖摩尔比为1.00:16.75:13.37:5.86:11.49:22.73:7.80;RRTP-3由鼠李糖、阿拉伯糖、未知糖、甘露糖、半乳糖、葡萄糖等中性单糖组成,摩尔比为1.00:5.47:1.31:3.02:15.88:1.58。RRTP-1和RRTP-3糖醛酸含量分别为12.7%,16%。
2.刺梨多糖的生物活性
刺梨粗多糖RRTP及纯组分RRTP-1对羟基自由基体系、超氧阴离子自由基体系、二苯代苦味肼基自由基均有不同程度的清除作用,呈现一定的量效关系,且粗多糖对该三种自由基的清除效果均大于同浓度时的纯组分。
中、高剂量刺梨粗多糖能使衰老小鼠血清、肝脏、脑组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活力显著提高,丙二醛(MDA)含量均显著降低,并呈一定的量效关系,表明刺梨粗多糖能提高衰老小鼠体内抗氧化能力。
加入RRTP-1的实验组神经干细胞损伤有所减轻,高浓度实验组细胞死亡率和乳酸脱氢酶漏出率较低。显示刺梨多糖RRTP-1对神经干细胞硫代硫酸钠损伤和谷氨酸损伤有保护作用。
多数刺梨多糖组分能刺激PC12细胞产生神经纤维样突起,具有神经营养活性。但在本试验剂量范围内,刺梨多糖纯组分RRTP-1和RRTP-3与产生的活性细胞率未表现出量效关系。
In this paper,polysaccharides were extracted and isolated from Rosa roxburghii Tratt juice,and their physicochemical characteristics and bioactivities were studied.The main contents are as follows:
1.Isolation,purification and physicochemical characteristics of Rosa roxburghii polysaccharides.
RRTP-1～RRTP-8 were obtained by concentration,precipitation with ethanol, deprotein and decoloufing,DEAE-cellulose column and Sepharose CL-6B column chromatography from the Rosa roxburghii Tratt juice.RRTP-1 and RRTP-3 were confirmed to be homogeneous composition by gel chromatography and polyacrylamide gel electrophoresis,and the IR spectrum showed typical characteristic absorption peak of polysaccharides.The molecular weight of RRTP-1 and RRTP-3 were measured to be 3200 and 52000 respectively by gel permeation chromatography.By GC analysis, RRTP-1 was consisted of rhamnose,arabinose,unknown sugar,xylose,mannose, galactose,the molar ratio of neutromonosaccharide were 1.00:16.75:13.37:5.86:11.49: 22.73:7.80;RRTP-3 was consisted of rhamnose,arabinose,unknown sugar,mannose, galactose,glucose,with the molar ratio of neutromonosaccharide 1.00:5.47:1.31:3.02: 15.88:1.58.The uronic acid content of RRTP-1 and RRTP-3 were 12.7%and 16% respectively.
2.The bioactivities of Rosa roxburghii Tratt polysaccharides
Crude and pure polysaccharides had different scavenging effect on hydroxy free radical,superoxide anion free radical and DPPH free radical,and they had dose-effect relationship.Forthemore,the scavenging effects of the crude polysaccharide on the three kinds of free radical were better than those of pure fraction on the same concentration.
Middle and high dosage of polysaccharides can increase the activity of SOD,CAT in plasma,liver and brain in aged mice significantly,while the content of MDA were decreased,and it had dose-effect relationship.It suggested that Rosa roxburghii Tratt polysaccharides can enhance the antioxide capability of aged mice.
The neural stem cells damage of test group adding RRTP-1 were alleviated,the cells death rate and LDH leakage of high concentration test group were significantly lower than those of Na_2S_2O_3 and glutamic acid group.It suggested that RRTP-1 has protective effect on neural stem cells damage caused by Na_2S_2O_3 and glutamic acid.
Most of polysaccharide fractions can induce differentiation of PC12 cell and had neural nutrition activity.While in our dosage range,pure polysaccharide fractions RRTP-1 and RRTP-3 show no dose-effect relationship on positive rates of PC12 cell.
1.刺梨多糖的分离纯化及理化性质研究
刺梨汁经浓缩、乙醇沉淀、脱蛋白、脱色、DEAE-纤维素柱和Sepharose CL-6B柱层析,分离纯化出RRTP-1～RRTP-8共8个组分。其中RRTP-1和RRTP-3组分经凝胶柱层析法和聚丙烯酰胺凝胶电泳法鉴定,证实为分子量分布均一组分。红外光谱显示其具有多糖特征吸收峰。凝胶渗透色谱法测定,RRTP-1和RRTP-3分子量分别为3200、52000。气相色谱分析RRTP-1由鼠李糖、阿拉伯糖、未知糖、木糖、甘露糖、半乳糖、葡萄糖等中性单糖组成,中性单糖摩尔比为1.00:16.75:13.37:5.86:11.49:22.73:7.80;RRTP-3由鼠李糖、阿拉伯糖、未知糖、甘露糖、半乳糖、葡萄糖等中性单糖组成,摩尔比为1.00:5.47:1.31:3.02:15.88:1.58。RRTP-1和RRTP-3糖醛酸含量分别为12.7%,16%。
2.刺梨多糖的生物活性
刺梨粗多糖RRTP及纯组分RRTP-1对羟基自由基体系、超氧阴离子自由基体系、二苯代苦味肼基自由基均有不同程度的清除作用,呈现一定的量效关系,且粗多糖对该三种自由基的清除效果均大于同浓度时的纯组分。
中、高剂量刺梨粗多糖能使衰老小鼠血清、肝脏、脑组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活力显著提高,丙二醛(MDA)含量均显著降低,并呈一定的量效关系,表明刺梨粗多糖能提高衰老小鼠体内抗氧化能力。
加入RRTP-1的实验组神经干细胞损伤有所减轻,高浓度实验组细胞死亡率和乳酸脱氢酶漏出率较低。显示刺梨多糖RRTP-1对神经干细胞硫代硫酸钠损伤和谷氨酸损伤有保护作用。
多数刺梨多糖组分能刺激PC12细胞产生神经纤维样突起,具有神经营养活性。但在本试验剂量范围内,刺梨多糖纯组分RRTP-1和RRTP-3与产生的活性细胞率未表现出量效关系。
In this paper,polysaccharides were extracted and isolated from Rosa roxburghii Tratt juice,and their physicochemical characteristics and bioactivities were studied.The main contents are as follows:
1.Isolation,purification and physicochemical characteristics of Rosa roxburghii polysaccharides.
RRTP-1～RRTP-8 were obtained by concentration,precipitation with ethanol, deprotein and decoloufing,DEAE-cellulose column and Sepharose CL-6B column chromatography from the Rosa roxburghii Tratt juice.RRTP-1 and RRTP-3 were confirmed to be homogeneous composition by gel chromatography and polyacrylamide gel electrophoresis,and the IR spectrum showed typical characteristic absorption peak of polysaccharides.The molecular weight of RRTP-1 and RRTP-3 were measured to be 3200 and 52000 respectively by gel permeation chromatography.By GC analysis, RRTP-1 was consisted of rhamnose,arabinose,unknown sugar,xylose,mannose, galactose,the molar ratio of neutromonosaccharide were 1.00:16.75:13.37:5.86:11.49: 22.73:7.80;RRTP-3 was consisted of rhamnose,arabinose,unknown sugar,mannose, galactose,glucose,with the molar ratio of neutromonosaccharide 1.00:5.47:1.31:3.02: 15.88:1.58.The uronic acid content of RRTP-1 and RRTP-3 were 12.7%and 16% respectively.
2.The bioactivities of Rosa roxburghii Tratt polysaccharides
Crude and pure polysaccharides had different scavenging effect on hydroxy free radical,superoxide anion free radical and DPPH free radical,and they had dose-effect relationship.Forthemore,the scavenging effects of the crude polysaccharide on the three kinds of free radical were better than those of pure fraction on the same concentration.
Middle and high dosage of polysaccharides can increase the activity of SOD,CAT in plasma,liver and brain in aged mice significantly,while the content of MDA were decreased,and it had dose-effect relationship.It suggested that Rosa roxburghii Tratt polysaccharides can enhance the antioxide capability of aged mice.
The neural stem cells damage of test group adding RRTP-1 were alleviated,the cells death rate and LDH leakage of high concentration test group were significantly lower than those of Na_2S_2O_3 and glutamic acid group.It suggested that RRTP-1 has protective effect on neural stem cells damage caused by Na_2S_2O_3 and glutamic acid.
Most of polysaccharide fractions can induce differentiation of PC12 cell and had neural nutrition activity.While in our dosage range,pure polysaccharide fractions RRTP-1 and RRTP-3 show no dose-effect relationship on positive rates of PC12 cell.
引文
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