肌肽清除自由基及抗氧化性研究
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摘要
肌肽是本世纪初在脊椎动物骨骼肌和大脑组织中发现的一种二肽，组成为β-丙氨酰-L-组氨酸（β-Ala-L-His），在国外肌肽作为一种自由基清除剂和抗氧化剂已得到广泛研究，在国内这些方面的研究开展得比较少。本试验从肌肽清除自由基、肌肽抑制蛋白氧化修饰、肌肽抑制脂质氧化三个方面研究了肌肽在清除自由基及抗氧化方面的性质
系列Ⅰ研究了肌肽清除自由基的性质。利用稳定自由基DPPH研究了肌肽对DPPH的清除效果，并分析了介质pH、温度、冷藏时间对肌肽清除能力的影响，结果表明:与对照组相比，不同浓度的肌肽对DPPH自由基都有显著的清除效果（P＜0.01），随着肌肽浓度的增大，对DPPH自由基的清除效果也增强，并且不同浓度肌肽组间的作用效果也有极显著差异（P＜0.01），研究也发现肌肽的组成成分β-丙氨酸和L-组氨酸清除能力远低于肌肽；在不同pH值的情况下肌肽对DPPH的清除能力有所不同，100mmol/L 肌肽在pH6.3、pH7.4和pH8.4对DPPH的清除率分别为42.78%、42.55%，36.88%，其中以pH6.3的清除效果为最好；采用不同温度处理肌肽后发现，加热处理对肌肽清除自由基的活性无显著影响（P＞0.05），在60℃加热处理后对肌肽的清除能力有促进作用；在4℃冷藏放置不同时间后检测发现，肌肽溶液在4℃冷藏1-3周，对它清除自由基的性质没有显著影响（P＞0.05）；采用Fe3+-H2O2-脱氧核糖体系检测肌肽清除羟自由基的作用，发现肌肽能够通过清除·OH的生成，来减少脱氧核糖MDA的生成量，起到保护脱氧核糖的作用，肌肽1～100mmol/L的清除率为17.94%～81.30%；采用邻苯三酚自氧化体系检测肌肽对超氧阴离子的清除作用，通过对邻苯三酚吸光度随时间的变化曲线作图，结果表明肌肽
在低浓度（1mmol/L，10mmol/L，20mmol/L）能够有效的抑制超氧阴离子的生成，随着肌肽浓度的升高，抑制作用逐渐减弱。
系列Ⅱ研究了肌肽抑制蛋白氧化修饰的性质。采用铁氰化钾还原法检测肌肽还原力，结果表明：各浓度肌肽具有显著的还原力（P＜0.01），并且与肌肽浓度呈剂量依赖性，还原力的测定还发现L-组氨酸还原能力很低，100mmol/L时还原力只达到肌肽的14.14%，而丙氨酸的还原能力在100mmol/L时达到肌肽的64.01%；采用CuCl2-H2O2体系产生自由基，氧化修饰牛血清白蛋白，检测羰基含量，结果表明肌肽随浓度增加能显著抑制羰基的生成（P＜0.01），与对照组相比100mmol/L肌肽对羰基的抑制率可以达到82.74%；采用葡萄糖糖化修饰牛血清白蛋白体系，检测糖化产物5-羟甲基糠醛和果糖胺，发现随肌肽的浓度增加糖化产物的含量也随之显著增加（P＜0.01），并且实验表明肌肽能单独被葡萄糖糖化修饰。
系列Ⅲ研究了肌肽抑制脂质氧化的性质。采用红细胞、肝组织匀浆、线粒体体系，检测脂质氧化产物。结果表明：高浓度的肌肽能够显著的抑制体外自氧化，H2O2和紫外线照射所导致的红细胞膜脂质氧化（P＜0.05），肌肽100mmol/L时抑制率分别为34.07%、42.80%、83.67%；在体外自氧化肝组织匀浆体系中肌肽在100mmol/L时抑制率达到50%，而在CCl4诱导肝匀浆膜脂质氧化体系中肌肽作用不是十分明显，100mmol/L时抑制率为18%；超速离心制备线粒体，以Fenton反应产生羟自由基氧化线粒体膜脂质，结果表明肌肽对Fe2+催化产生的·OH具有显著的清除效果（P＜0.05），随肌肽浓度增加，抑制率呈线性上升，在1mmol/L时抑制率已达到13.71%，在高浓度100mmol/L时肌肽的抑制效果有所下降。
Carnosine is a dipeptide found in skeletal muscle and brain tissue of vertebrates that has been reported to possess radical scavenging and antioxidant function for a long time.Its structure（β-Alany-L-Histidine） was determined in the very beginning of the 20 century.In our contury,few people have studied on it.This thesis consisted of three parts:the radical scavenging ability of carnosine was studied in series Ⅰ;the inhibitory of BSA oxidation and glycationin in seriesⅡ;the inhibitory of lipid peroxidation in series Ⅲ.
In series Ⅰ,we studied the scavenging activity of carnosine on free radicals. DPPH radical was used as a model system and we tested the scavenging ability of carnosine、β- alany 、L-histidine and the effect of different pH、temperature and time on the ability.The results illustrated that:compared with control group,different concerntration of carnosine had significant activity of scaveging DPPH radical in a dose-dependent manner( P＜0.01);the scaveging ability of DPPH radical of β- alany and L-histidine was notably under the carnosine;the scaveging ability of carnosine was not affected by pH value of reaction media,at pH 6.3、7.4、8.4,the scaveging rate was 42.78%,42.55%,36.88% respectively and at pH 6.3,the scaveging rate was the greastest;there was no significant difference in scaveging ability of different temperature treated groups(P＞0.05).The group of 60℃ water bath treatment for 15min could promote the activity;there was also no significant difference in the scaveging ability of carnosine solution being stored at 4℃ for three weeks.(P＞0.05)
The effect of carnosine on deoxyribose oxidation induced by FeCl3/H2O2 was invested through measured the diminishing of MDA.The results showed that carnosine could
scavege the ·OH radical and protect the deoxyribose from damage. 1-100mmol/L carnosine showed 17.94%～81.30% scaveging ability.The activity of carnosine as a quencher for superoxide anion radical was evaluated in the system of auto-oxidation of pyrogallol.The results indicated that Carnosine quenchered the superoxide anion radical on a low concentration(1,10,20mmol/L) and the ability decreased with a high concentration.
In series Ⅱ,about the inhibitory of carnosine on BSA oxidation and glycation.First,the reducing power of carnosine,β- alany and L-histidine was invested. Carnosine exhibited the greast reducing power among compounds.Its reducing power increased significantly with an increasing amount of carnosine(P＜0.01). L-histidine has little reducing power. 100mmol/L L-histidine only has 14.14% reducing power and 100mmol/Lβ- alany has 64.01% reducing power compared with the same concerntration of carnosine.In the CuCl2-H2O2 catalyzed BSA oxidation system,carnosine significantly decreased the protein carbonyl formation with the increasing amount of carnosine(P＜0.01), The inhibition rate of 100mmol/L carnosine on BSA oxidation was 82.74% .In the glucose catalyzed BSA glycation system，the amount of 5-HMF and Fructosamine increased with the increasing amount of carnosine(P＜0.01) .we draw a conclution carnosine glycated by the glucose.
In Series Ⅲ,about the inhibitory effect of carnosine of lipid peroxidation. High concerntration carnosine significantly inhibited the red blood cell membrane lipid peroxidation induced by auto-oxidation,H2O2,UV((P＜0.05).The inhibition rate of 100mmol/L carnosine on the three model system was 34.07%、42.80%、83.67% respectively. In liver homogate lipid peroxidation system induced by water bath in vivo, The inhibition rate of 100mmol/L carnosine was 50%，but in liver homogate lipid peroxidation system induced by CCl4 the inhibition rate was just 18%. In the mitochondria membrane lipid peroxidation induced by Fenton action，carnosine significant inhibited the
TBARS formation in a dose-dependent manner(P＜0.05). The inhibition rate reach the summit of 54.01% in 50mmol/Lcarnosine,but was decreased in 100mmol/L.
系列Ⅰ研究了肌肽清除自由基的性质。利用稳定自由基DPPH研究了肌肽对DPPH的清除效果，并分析了介质pH、温度、冷藏时间对肌肽清除能力的影响，结果表明:与对照组相比，不同浓度的肌肽对DPPH自由基都有显著的清除效果（P＜0.01），随着肌肽浓度的增大，对DPPH自由基的清除效果也增强，并且不同浓度肌肽组间的作用效果也有极显著差异（P＜0.01），研究也发现肌肽的组成成分β-丙氨酸和L-组氨酸清除能力远低于肌肽；在不同pH值的情况下肌肽对DPPH的清除能力有所不同，100mmol/L 肌肽在pH6.3、pH7.4和pH8.4对DPPH的清除率分别为42.78%、42.55%，36.88%，其中以pH6.3的清除效果为最好；采用不同温度处理肌肽后发现，加热处理对肌肽清除自由基的活性无显著影响（P＞0.05），在60℃加热处理后对肌肽的清除能力有促进作用；在4℃冷藏放置不同时间后检测发现，肌肽溶液在4℃冷藏1-3周，对它清除自由基的性质没有显著影响（P＞0.05）；采用Fe3+-H2O2-脱氧核糖体系检测肌肽清除羟自由基的作用，发现肌肽能够通过清除·OH的生成，来减少脱氧核糖MDA的生成量，起到保护脱氧核糖的作用，肌肽1～100mmol/L的清除率为17.94%～81.30%；采用邻苯三酚自氧化体系检测肌肽对超氧阴离子的清除作用，通过对邻苯三酚吸光度随时间的变化曲线作图，结果表明肌肽
在低浓度（1mmol/L，10mmol/L，20mmol/L）能够有效的抑制超氧阴离子的生成，随着肌肽浓度的升高，抑制作用逐渐减弱。
系列Ⅱ研究了肌肽抑制蛋白氧化修饰的性质。采用铁氰化钾还原法检测肌肽还原力，结果表明：各浓度肌肽具有显著的还原力（P＜0.01），并且与肌肽浓度呈剂量依赖性，还原力的测定还发现L-组氨酸还原能力很低，100mmol/L时还原力只达到肌肽的14.14%，而丙氨酸的还原能力在100mmol/L时达到肌肽的64.01%；采用CuCl2-H2O2体系产生自由基，氧化修饰牛血清白蛋白，检测羰基含量，结果表明肌肽随浓度增加能显著抑制羰基的生成（P＜0.01），与对照组相比100mmol/L肌肽对羰基的抑制率可以达到82.74%；采用葡萄糖糖化修饰牛血清白蛋白体系，检测糖化产物5-羟甲基糠醛和果糖胺，发现随肌肽的浓度增加糖化产物的含量也随之显著增加（P＜0.01），并且实验表明肌肽能单独被葡萄糖糖化修饰。
系列Ⅲ研究了肌肽抑制脂质氧化的性质。采用红细胞、肝组织匀浆、线粒体体系，检测脂质氧化产物。结果表明：高浓度的肌肽能够显著的抑制体外自氧化，H2O2和紫外线照射所导致的红细胞膜脂质氧化（P＜0.05），肌肽100mmol/L时抑制率分别为34.07%、42.80%、83.67%；在体外自氧化肝组织匀浆体系中肌肽在100mmol/L时抑制率达到50%，而在CCl4诱导肝匀浆膜脂质氧化体系中肌肽作用不是十分明显，100mmol/L时抑制率为18%；超速离心制备线粒体，以Fenton反应产生羟自由基氧化线粒体膜脂质，结果表明肌肽对Fe2+催化产生的·OH具有显著的清除效果（P＜0.05），随肌肽浓度增加，抑制率呈线性上升，在1mmol/L时抑制率已达到13.71%，在高浓度100mmol/L时肌肽的抑制效果有所下降。
Carnosine is a dipeptide found in skeletal muscle and brain tissue of vertebrates that has been reported to possess radical scavenging and antioxidant function for a long time.Its structure（β-Alany-L-Histidine） was determined in the very beginning of the 20 century.In our contury,few people have studied on it.This thesis consisted of three parts:the radical scavenging ability of carnosine was studied in series Ⅰ;the inhibitory of BSA oxidation and glycationin in seriesⅡ;the inhibitory of lipid peroxidation in series Ⅲ.
In series Ⅰ,we studied the scavenging activity of carnosine on free radicals. DPPH radical was used as a model system and we tested the scavenging ability of carnosine、β- alany 、L-histidine and the effect of different pH、temperature and time on the ability.The results illustrated that:compared with control group,different concerntration of carnosine had significant activity of scaveging DPPH radical in a dose-dependent manner( P＜0.01);the scaveging ability of DPPH radical of β- alany and L-histidine was notably under the carnosine;the scaveging ability of carnosine was not affected by pH value of reaction media,at pH 6.3、7.4、8.4,the scaveging rate was 42.78%,42.55%,36.88% respectively and at pH 6.3,the scaveging rate was the greastest;there was no significant difference in scaveging ability of different temperature treated groups(P＞0.05).The group of 60℃ water bath treatment for 15min could promote the activity;there was also no significant difference in the scaveging ability of carnosine solution being stored at 4℃ for three weeks.(P＞0.05)
The effect of carnosine on deoxyribose oxidation induced by FeCl3/H2O2 was invested through measured the diminishing of MDA.The results showed that carnosine could
scavege the ·OH radical and protect the deoxyribose from damage. 1-100mmol/L carnosine showed 17.94%～81.30% scaveging ability.The activity of carnosine as a quencher for superoxide anion radical was evaluated in the system of auto-oxidation of pyrogallol.The results indicated that Carnosine quenchered the superoxide anion radical on a low concentration(1,10,20mmol/L) and the ability decreased with a high concentration.
In series Ⅱ,about the inhibitory of carnosine on BSA oxidation and glycation.First,the reducing power of carnosine,β- alany and L-histidine was invested. Carnosine exhibited the greast reducing power among compounds.Its reducing power increased significantly with an increasing amount of carnosine(P＜0.01). L-histidine has little reducing power. 100mmol/L L-histidine only has 14.14% reducing power and 100mmol/Lβ- alany has 64.01% reducing power compared with the same concerntration of carnosine.In the CuCl2-H2O2 catalyzed BSA oxidation system,carnosine significantly decreased the protein carbonyl formation with the increasing amount of carnosine(P＜0.01), The inhibition rate of 100mmol/L carnosine on BSA oxidation was 82.74% .In the glucose catalyzed BSA glycation system，the amount of 5-HMF and Fructosamine increased with the increasing amount of carnosine(P＜0.01) .we draw a conclution carnosine glycated by the glucose.
In Series Ⅲ,about the inhibitory effect of carnosine of lipid peroxidation. High concerntration carnosine significantly inhibited the red blood cell membrane lipid peroxidation induced by auto-oxidation,H2O2,UV((P＜0.05).The inhibition rate of 100mmol/L carnosine on the three model system was 34.07%、42.80%、83.67% respectively. In liver homogate lipid peroxidation system induced by water bath in vivo, The inhibition rate of 100mmol/L carnosine was 50%，but in liver homogate lipid peroxidation system induced by CCl4 the inhibition rate was just 18%. In the mitochondria membrane lipid peroxidation induced by Fenton action，carnosine significant inhibited the
TBARS formation in a dose-dependent manner(P＜0.05). The inhibition rate reach the summit of 54.01% in 50mmol/Lcarnosine,but was decreased in 100mmol/L.
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