硫酸软骨素寡糖的制备及其体外抗氧化活性研究
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摘要
硫酸软骨素(Chondroitin Sulfate, CS)为高分子糖胺聚糖的一种,因其较高的生物活性现已应用于临床和医药。但由于CS高分子量、高表观粘度、结构复杂及细胞膜的选择透过性等因素,造成了其生物利用率较低。本文研究了CS的降解方法,对降解产物进行了初步分级及纯化,并研究了低分子量CS的体外抗氧化活性,为糖类药物的筛选与应用提供一定的理论参考。
论文探索了不同降解方法对CS降解产物的影响。结果表明:酸法降解所得低分子CS硫酸基脱除率高达3.97%,并伴随有4,5-不饱和糖醛酸等不饱和结构产生,产物颜色为橘红色。而酶解条件温和,所得产物硫酸基保存完好,产物颜色为乳白色。体外抗氧化实验表明,酸解所得产物DPPH·自由基清除能力及还原能力均优于酶解所得产物。为有效控制CS分子量范围,以下文中均选用酶解的方法。
研究了经初步分级后CS的体外抗氧化活性。采用透析袋透析(分子量为5000)及Sephadex G25凝胶柱对CS酶解产物进行初步分级,得到两种重均分子量分别为4875 Da、1326 Da的低分子量CS。分别研究了CS及两种低分子量CS的体外抗氧化活性,结果表明,随着浓度增大,各组分抗氧化活性随之增大;且相同浓度下分子量越低,活性越强。当浓度为10 mg/mL时,低分子量CS(1326 Da)可清除56.22%的羟自由基,高于高分子量CS(21.54%),且对超氧阴离子自由基的清除率可达52.33%。此浓度下,低分子量CS(4875 Da)的还原能力跟低分子量CS(1326 Da)浓度为2 mg/mL时相当。
建立了CS寡糖的分离纯化方法。首次采用DEAE纤维素52阴离子交换柱与制备凝胶电泳联合的方法实现了对低分子量CS的分离纯化。经分离纯化后得到三种组分,分别采用AKTA superdex peptide及荧光辅助糖电泳对每种组分进行纯度验证,均为单一条带,表明上述分离所得组分单一;采用高效凝胶过滤色谱(HPGFC)对单一组分进行分子量测定,结果表明三种组分分子量分别为521 Da、1024 Da及1527 Da,分别与CS二糖(C14H21NO15SNa2)、四糖(C28H40N2O29S2Na4 )、六糖(C42H59N3O43S3Na6)相吻合。
研究了单一组分CS寡糖的体外抗氧化活性。结果表明,所得三种CS寡糖(分子量分别为521 Da、1024 Da及1527 Da)的超氧阴离子自由基清除能力及还原能力均表现出了一定的量效关系,即随着浓度的增大,三种寡糖的活性随之增大。对于以上两种测定方法,三种寡糖活性无显著性差异。浓度为4 mg/mL时,超氧阴离子自由基清除力达到最大,约为35%,而达到最大还原能力所需浓度较大(>10 mg/mL)。
Chondroitin Sulfate (CS), a kind of glycosaminoglycans, has been used in clinical and medicine because of the high biological activities. But its bioavailability is relatively lower due to its high molecular weight, high apparent viscosity, complex structure and selective permeability of cell membrance. The different degradation methods of CS, preliminary separation, purification methods and antioxidant activities in vitro of CS degradations were studied in this paper. It can provide a theoretical to screen and apply for carbohydrate drugs.
In this paper, different degradation methods of CS were studied. It showed that the sulfate removal rate of acid degradation products reached 3.97%, and some 4, 5-unsaturated uronic acids were generated. The color of products was orange-red. While the enzyme hydrolysis condition was mild, the sulfate of enzymatic degradation products was well-preserved, and the color was milky. The vitro antioxidant experiment showed that DPPH? free radical scavenging capacity and reducing power of acid degradations were superior to the enzyme degradations. In order to effective control the degradation, enzymatic method was used in the following paper.
The antioxidant activities in vitro of different molecular weight CS by preliminary separation were also studied. After dialysis (molecular weight 5000) and Sephadex G25 gel column, two low molecular weight CS were gained, which weight-average molecular weight were 4875 Da and 1326 Da, respectively. The antioxidant experiment in vitro showed that the lower molecular weight was, the stronger the activity was. At 10 mg/mL, the low molecular weight CS (1326 Da) could remove 56.22% of hydroxyl radical, higher than CS (21.54%), and the superoxide anion radical scavenging could reach 52.33%. Reducing power of low molecular weight CS (4875 Da) at 10 mg/mL was similar with that of low molecular weight CS (1326 Da) at 2 mg/mL.
The purification methods of CS oligosaccharides were established. It’s the first time to combine DEAE-52 cellulose chromatography with preparative electrophoresis to separate the CS oligosaccharides. Three chondroitin sulfate oligosaccharides were obtained, which have been confirmed single band by AKTA superdex peptide column and fluorescent assisted carbohydrate electrophoresis. Through HPGFC measured, the molecular weights of these three components were 521 Da, 1024 Da and 1527 Da, corresponding to the disaccharide, tetrasaccharide and hexasaccharide, respectively.
Finally, the antioxidant activities in vitro of three CS oligosaccharides (the molecular weights were 521 Da, 1024 Da and 1527 Da) we got were compared. For superoxide anion radical scavenging capacity and reducing power, these three oligosaccharides all showed a definite dose relationship. As the concentration increased, the activities were greater. The activities in the both detecting methods had no significant difference. When the concentration was 4 mg/mL, the superoxide anion radical scavenging abilities of these three oligosaccharides reached the maximum, about 35%. It required higher concentration to achieve the maximal reducing power (> 10 mg/mL).
论文探索了不同降解方法对CS降解产物的影响。结果表明:酸法降解所得低分子CS硫酸基脱除率高达3.97%,并伴随有4,5-不饱和糖醛酸等不饱和结构产生,产物颜色为橘红色。而酶解条件温和,所得产物硫酸基保存完好,产物颜色为乳白色。体外抗氧化实验表明,酸解所得产物DPPH·自由基清除能力及还原能力均优于酶解所得产物。为有效控制CS分子量范围,以下文中均选用酶解的方法。
研究了经初步分级后CS的体外抗氧化活性。采用透析袋透析(分子量为5000)及Sephadex G25凝胶柱对CS酶解产物进行初步分级,得到两种重均分子量分别为4875 Da、1326 Da的低分子量CS。分别研究了CS及两种低分子量CS的体外抗氧化活性,结果表明,随着浓度增大,各组分抗氧化活性随之增大;且相同浓度下分子量越低,活性越强。当浓度为10 mg/mL时,低分子量CS(1326 Da)可清除56.22%的羟自由基,高于高分子量CS(21.54%),且对超氧阴离子自由基的清除率可达52.33%。此浓度下,低分子量CS(4875 Da)的还原能力跟低分子量CS(1326 Da)浓度为2 mg/mL时相当。
建立了CS寡糖的分离纯化方法。首次采用DEAE纤维素52阴离子交换柱与制备凝胶电泳联合的方法实现了对低分子量CS的分离纯化。经分离纯化后得到三种组分,分别采用AKTA superdex peptide及荧光辅助糖电泳对每种组分进行纯度验证,均为单一条带,表明上述分离所得组分单一;采用高效凝胶过滤色谱(HPGFC)对单一组分进行分子量测定,结果表明三种组分分子量分别为521 Da、1024 Da及1527 Da,分别与CS二糖(C14H21NO15SNa2)、四糖(C28H40N2O29S2Na4 )、六糖(C42H59N3O43S3Na6)相吻合。
研究了单一组分CS寡糖的体外抗氧化活性。结果表明,所得三种CS寡糖(分子量分别为521 Da、1024 Da及1527 Da)的超氧阴离子自由基清除能力及还原能力均表现出了一定的量效关系,即随着浓度的增大,三种寡糖的活性随之增大。对于以上两种测定方法,三种寡糖活性无显著性差异。浓度为4 mg/mL时,超氧阴离子自由基清除力达到最大,约为35%,而达到最大还原能力所需浓度较大(>10 mg/mL)。
Chondroitin Sulfate (CS), a kind of glycosaminoglycans, has been used in clinical and medicine because of the high biological activities. But its bioavailability is relatively lower due to its high molecular weight, high apparent viscosity, complex structure and selective permeability of cell membrance. The different degradation methods of CS, preliminary separation, purification methods and antioxidant activities in vitro of CS degradations were studied in this paper. It can provide a theoretical to screen and apply for carbohydrate drugs.
In this paper, different degradation methods of CS were studied. It showed that the sulfate removal rate of acid degradation products reached 3.97%, and some 4, 5-unsaturated uronic acids were generated. The color of products was orange-red. While the enzyme hydrolysis condition was mild, the sulfate of enzymatic degradation products was well-preserved, and the color was milky. The vitro antioxidant experiment showed that DPPH? free radical scavenging capacity and reducing power of acid degradations were superior to the enzyme degradations. In order to effective control the degradation, enzymatic method was used in the following paper.
The antioxidant activities in vitro of different molecular weight CS by preliminary separation were also studied. After dialysis (molecular weight 5000) and Sephadex G25 gel column, two low molecular weight CS were gained, which weight-average molecular weight were 4875 Da and 1326 Da, respectively. The antioxidant experiment in vitro showed that the lower molecular weight was, the stronger the activity was. At 10 mg/mL, the low molecular weight CS (1326 Da) could remove 56.22% of hydroxyl radical, higher than CS (21.54%), and the superoxide anion radical scavenging could reach 52.33%. Reducing power of low molecular weight CS (4875 Da) at 10 mg/mL was similar with that of low molecular weight CS (1326 Da) at 2 mg/mL.
The purification methods of CS oligosaccharides were established. It’s the first time to combine DEAE-52 cellulose chromatography with preparative electrophoresis to separate the CS oligosaccharides. Three chondroitin sulfate oligosaccharides were obtained, which have been confirmed single band by AKTA superdex peptide column and fluorescent assisted carbohydrate electrophoresis. Through HPGFC measured, the molecular weights of these three components were 521 Da, 1024 Da and 1527 Da, corresponding to the disaccharide, tetrasaccharide and hexasaccharide, respectively.
Finally, the antioxidant activities in vitro of three CS oligosaccharides (the molecular weights were 521 Da, 1024 Da and 1527 Da) we got were compared. For superoxide anion radical scavenging capacity and reducing power, these three oligosaccharides all showed a definite dose relationship. As the concentration increased, the activities were greater. The activities in the both detecting methods had no significant difference. When the concentration was 4 mg/mL, the superoxide anion radical scavenging abilities of these three oligosaccharides reached the maximum, about 35%. It required higher concentration to achieve the maximal reducing power (> 10 mg/mL).
引文
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