铁皮石斛多糖体外抗氧化活性的研究
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摘要
石斛为我国传统中药材,其药用价值为人们所共识。经研究发现,多糖是石斛属植物的主要活性成分。本文以石斛之上品-铁皮石斛-为主要研究对象,对铁皮石斛总多糖(DSP)进行了提取和分级分离,测定了铁皮石斛总多糖及分级多糖的分子量,研究了铁皮石斛及分级多糖抗氧化活性。为石斛的生产与使用提供了依据,并且为铁皮石斛多糖构效关系的深入研究奠定了基础。获得的主要结果如下:
1铁皮石斛总多糖的分离及其分子量的测定
采用分步醇沉法对铁皮石斛总多糖进行分离,分别得到40%醇沉多糖(DSP3),70%醇沉多糖(DSP2)和90%醇沉多糖(DSP1)。
用凝胶层析法测定了铁皮石斛总多糖及分级多糖分子量,得到DSP、DSP1、DSP2、DSP3的分子量范围分别为1.57×1 0~4-15.7×10~4Da、1.48×10~4 Da、3.53×10~4-7.44×10~4 Da、11.51×10~4-15.7×10~4 Da。
2铁皮石斛总多糖及分级多糖抗氧化活性的研究
本文分别采用清除活性氧和DPPH自由基体系,金属离子螯合体系、还原力体系、Fe~(2+)-V_C诱导的小鼠肝匀浆脂质过氧化体系、ABTS法测总抗氧化能力体系、抑制H_2O_2诱导红细胞氧化溶血能力体系和对羟基自由基介导的DNA损伤的保护体系,利用分光光度法和琼脂糖凝胶电泳法分别对铁皮石斛多糖DSP、DSP1、DSP2、DSP3的体外抗氧化活性进行测定。结果表明,除在清除O_2~-·自由基体系中4种多糖无抗氧化作用外,在其它体系中均表现出较强的抗氧化能力,并具有显著的量效关系,但抗氧化体系不同,其抗氧化活性差异显著。DSP1对DPPH·的清除作用、还原力、对Fe~(2+)-V_C诱导的小鼠肝匀浆脂质过氧化抑制作用、ABTS总抗氧化能力和抑制H_2O_2诱导红细胞氧化溶血作用效果最佳;DSP对·OH的清除率和金属螯合力最大;DSP3对H_2O_2和NO的清除作用的清除率最大;4种多糖均可显著抑制羟基自由基介导的DNA氧化断裂。
综上所述:4种铁皮石斛多糖均具有不同程度的抗氧化活性,表明,铁皮石斛多糖抗氧化活性大小与其分子量有关,还可能与铁皮石斛多糖的结构有关,其机制有待更深一步探讨。
Dendrobium species as traditional Chinese herbs has been used for centuries. In many studies, polysaccharides from Dendrobii plants were found to be the main active components. In this paper, the total polysaccharide (DSP) was obtained from Dendrobium candidum, which was the top grade of Dendrobii, and then separated by ethanol to get different polysaccharide fractions. DSP and different polysaccharide fractions were determined to make sure the molecular weights and antioxidant activities, which would provid the basis for the application of Dendrobium candidum in medicine and food. The main results are as follows:
1. Fractionation of DSP and determination of molecular weights
DSP was dissolved in deionised water and then continuously deposited by ethanol with final concentrations of 40%, 70% and 90% to obtain different polysaccharides fractions of DSP3, DSP2 and DSP1.
Molecular weights of DSP, DSP1, DSP2 and DSP3, determined by gel chromatography method, were in the range of 1.57×10~4-15.7×10~4 Da, 1.48×10~4 Da, 3.53×10~4-7.44×10~4 Da and 11.5×10~4-15.7×10~4 Da, respectively.
2. Antioxidant activities of DSP and different polysaccharide fractions
The antioxidant activities of DSP and different polysaccharide fractions were evaluated by O_2~-·, DPPH, chelating metal ions, anti-lipid peroxidation induced by Fe~(2+)-V_C, ABTS, anti-erythrocyte hemolysis induced by H_2O_2 and DNA damage induced by·OH. Results showed that different polysaccharide fractions had strong antioxidant activities in dose-dependent manner in all assays except O_2~-·. DSP1 exhibited strongest antioxidant activities among polysaccharide fractions in DPPH-scavenging activity, reducing power, anti-lipid peroxidation induced by Fe~(2+)-V_C, ABTS-scavenging activity and anti-erythrocyte hemolysis induced by H_2O_2. DSP had better antioxidant activities than others in·OH-scavenging activity and chelating metal ions. DSP3 showed better scavenging activities on H_2O_2 radical and NO radical than others. All the polysaccharide fractions showed excellent protection effect on DNA damage induced by·OH.
In conclusion, 4 polysaccharide fractions of D. candidum exhibited different antioxidant activities, which were possibly related with molecular weight and the construction of polysaccharides.
1铁皮石斛总多糖的分离及其分子量的测定
采用分步醇沉法对铁皮石斛总多糖进行分离,分别得到40%醇沉多糖(DSP3),70%醇沉多糖(DSP2)和90%醇沉多糖(DSP1)。
用凝胶层析法测定了铁皮石斛总多糖及分级多糖分子量,得到DSP、DSP1、DSP2、DSP3的分子量范围分别为1.57×1 0~4-15.7×10~4Da、1.48×10~4 Da、3.53×10~4-7.44×10~4 Da、11.51×10~4-15.7×10~4 Da。
2铁皮石斛总多糖及分级多糖抗氧化活性的研究
本文分别采用清除活性氧和DPPH自由基体系,金属离子螯合体系、还原力体系、Fe~(2+)-V_C诱导的小鼠肝匀浆脂质过氧化体系、ABTS法测总抗氧化能力体系、抑制H_2O_2诱导红细胞氧化溶血能力体系和对羟基自由基介导的DNA损伤的保护体系,利用分光光度法和琼脂糖凝胶电泳法分别对铁皮石斛多糖DSP、DSP1、DSP2、DSP3的体外抗氧化活性进行测定。结果表明,除在清除O_2~-·自由基体系中4种多糖无抗氧化作用外,在其它体系中均表现出较强的抗氧化能力,并具有显著的量效关系,但抗氧化体系不同,其抗氧化活性差异显著。DSP1对DPPH·的清除作用、还原力、对Fe~(2+)-V_C诱导的小鼠肝匀浆脂质过氧化抑制作用、ABTS总抗氧化能力和抑制H_2O_2诱导红细胞氧化溶血作用效果最佳;DSP对·OH的清除率和金属螯合力最大;DSP3对H_2O_2和NO的清除作用的清除率最大;4种多糖均可显著抑制羟基自由基介导的DNA氧化断裂。
综上所述:4种铁皮石斛多糖均具有不同程度的抗氧化活性,表明,铁皮石斛多糖抗氧化活性大小与其分子量有关,还可能与铁皮石斛多糖的结构有关,其机制有待更深一步探讨。
Dendrobium species as traditional Chinese herbs has been used for centuries. In many studies, polysaccharides from Dendrobii plants were found to be the main active components. In this paper, the total polysaccharide (DSP) was obtained from Dendrobium candidum, which was the top grade of Dendrobii, and then separated by ethanol to get different polysaccharide fractions. DSP and different polysaccharide fractions were determined to make sure the molecular weights and antioxidant activities, which would provid the basis for the application of Dendrobium candidum in medicine and food. The main results are as follows:
1. Fractionation of DSP and determination of molecular weights
DSP was dissolved in deionised water and then continuously deposited by ethanol with final concentrations of 40%, 70% and 90% to obtain different polysaccharides fractions of DSP3, DSP2 and DSP1.
Molecular weights of DSP, DSP1, DSP2 and DSP3, determined by gel chromatography method, were in the range of 1.57×10~4-15.7×10~4 Da, 1.48×10~4 Da, 3.53×10~4-7.44×10~4 Da and 11.5×10~4-15.7×10~4 Da, respectively.
2. Antioxidant activities of DSP and different polysaccharide fractions
The antioxidant activities of DSP and different polysaccharide fractions were evaluated by O_2~-·, DPPH, chelating metal ions, anti-lipid peroxidation induced by Fe~(2+)-V_C, ABTS, anti-erythrocyte hemolysis induced by H_2O_2 and DNA damage induced by·OH. Results showed that different polysaccharide fractions had strong antioxidant activities in dose-dependent manner in all assays except O_2~-·. DSP1 exhibited strongest antioxidant activities among polysaccharide fractions in DPPH-scavenging activity, reducing power, anti-lipid peroxidation induced by Fe~(2+)-V_C, ABTS-scavenging activity and anti-erythrocyte hemolysis induced by H_2O_2. DSP had better antioxidant activities than others in·OH-scavenging activity and chelating metal ions. DSP3 showed better scavenging activities on H_2O_2 radical and NO radical than others. All the polysaccharide fractions showed excellent protection effect on DNA damage induced by·OH.
In conclusion, 4 polysaccharide fractions of D. candidum exhibited different antioxidant activities, which were possibly related with molecular weight and the construction of polysaccharides.
引文
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