摘要
目的建立一种评价天然抗氧化剂对羟基自由基致DNA损伤保护作用的改进方法。方法将一定浓度的DNA(0.8mg/ml)、H2O2(0.2mmol/L)和抗坏血酸(1mmol/L)溶液混合,用50mmol/LpH7.4磷酸缓冲液(PBS)补足至终体积为1ml。样品组加入抗氧化剂,阳性组不加抗氧化剂。以预先与EDTA(1mmol/L)混合的FeSO4(1mmol/L)启动Fenton反应,37℃温育30min,温育结束后加入10%的三氯乙酸溶液1mL终止反应,再加入1.0%的硫代巴比妥酸溶液1ml,混匀,80℃水浴加热30min,冷却后用3ml正丁醇萃取,移取正丁醇萃取液在532nm处测定吸光度。结果在Fenton反应产生的羟基自由基攻击DNA的体系中加入羟基自由基清除剂,硫代巴比妥酸反应物的吸光度下降,表明自由基清除剂对羟基自由基致DNA氧化损伤有保护作用.结论该法可用于评价食品中的抗氧化剂对羟基自由基致DNA损伤的保护作用。
Objective To establish a modified DNA damage induced by hydroxyl radical. Method method to evaluate the protection of the antioxidant of Reaction mixture at a final volume of 1.0 ml contained 0.8mg/ml DNA, 0.2 mmol/L H2O2, 1.0 mmol/L ascorbic acid, in 50 mmol/L NaH2PO4-Na2HPO4 buffer （pH 7.4）. EDTA（1.0 mmol/L） and FeCl2 （1.0 mmol/L）were premixed and then dispensed into the reaction mixture to trigger the Fenton reaction, and the mixture was incubated at 37 ℃ for 30 min. The reaction was stopped by dispensing lml of 10% （w/v） trichloroacetic acid, and reaction mixture was mixed with lml of 1% （w/v） 2-thiobarbituric acid, then further heated at 80℃ for 30 min. The chromogen formed was extracted into n-1-butanol and absorbance was measured at 532 nm. Results Addition of hydroxyl radical scavenger can compete with DNA for the hydroxyl radicals and diminished TBARS formation at a defined time of reaction with dose-dependent effect. The result showed that hydroxyl radical scavenger exerted inhibition effects on hydroxyl radical-mediated DNA damage. Conclusion This method can be used to evaluate the protection of the antioxidant of DNA damage induced by hydroxyl radical.[ACTA NUTRIMENTA SINICA, 2008,30（1）： 74-77]
出处
《营养学报》
CAS
CSCD
北大核心
2008年第1期74-77,共4页
Acta Nutrimenta Sinica
基金
国家自然科学基金(No.20675062)
陕西省科技发展计划(No.2004K03-G8)
西北大学2004年度重点科研基地开放、基金(No.2004-29)
关键词
DNA损伤
羟基自由基
抗氧化作用
DNA damage
hydroxyl radical
Fenton reaction
antioxidant
