总抗氧化能力测定FRAP法
抗氧化物活性测定方法总结
抗氧化物活性测定方法总结抗氧化物活性测定方法是通过对样品中的抗氧化物质含量和抗氧化活性进行定量分析，评估其对自由基的清除能力和抗氧化能力。
随着抗氧化研究的不断深入，测定方法也逐渐完善。
以下是对常见的抗氧化物活性测定方法的总结。
1. ORAC法（氧化应激反应活性测定法）：该方法通过测定样品清除自由基的能力来评估其抗氧化活性。
实验中，将样品与荧光试剂（如2,2'-azobis(2,4-dimethylvaleronitrile)）共同作用，观察其清除自由基的能力，并通过建立标准曲线计算样品的ORAC值。
2.DPPH法（1,1-二苯基-2-苦味基-苦味酸磷）：该方法是一种常用的快速测定抗氧化活性的方法。
实验中，将样品与DPPH稳定自由基共同作用，通过比色反应观察DPPH自由基被样品清除的程度，从而评估抗氧化活性。
3.ABTS法（2,2'-联氮双5-苯砜酸）：该方法通过ABTS离子自由基的生成和清除反应来测定样品的抗氧化活性。
实验中，ABTS与过氧化氢反应生成ABTS离子自由基，通过观察样品对其的清除能力来评估抗氧化活性。
4.FRAP法（亚铁离子还原能力）：该方法基于样品对人造抗坏血酸（Fe3+）的还原能力，通过测量还原后的Fe2+离子的生成量来评估抗氧化活性。
实验中，将样品与Fe3+离子反应生成Fe2+离子，通过比色反应来测定Fe2+的含量。
5. 碘标法（Iodine value）：该方法用于测定油脂、脂肪等样品的抗氧化活性。
实验中，将已知量的碘与样品中的不饱和化合物反应，在光反应下观察反应终点的颜色变化，并根据标准曲线计算样品的抗氧化活性。
6. 硝酸盐法（Nitrite method）：该方法用于测定样品中亚硝酸盐的含量，从而评估其抗氧化活性。
实验中，样品经过还原反应生成亚硝酸盐，然后与DANO（N-乙基-N-（2-苯基乙基）-对硝基苯胺）反应生成稳定的偶氮染料，通过比色测定反应终点的吸光度来计算样品中亚硝酸盐的含量。
抗氧化物活性测定方法总结
抗氧化物活性测定方法总结抗氧化物活性 (antioxidant activity)描述了化学物质在抑制或减少氧化反应中所起的作用。
抗氧化物是一类具有亲电子的分子，它们容易被氧化，从而中和自由基。
抗氧化物具有重要的生物学和医学意义，因为氧化损害是许多疾病和老化的主要原因。
因此，抗氧化物活性测定方法是目前研究的热点之一，现将抗氧化物活性测定方法进行总结:1. DPPH法：该方法是一种常用的体外抗氧化测定方法。
含有DPPH（1,1-二苯基-2-三硝基苯肼）的溶液表现为紫色，DPPH自由基上的氢原子被抗氧化物夺取后，DPPH自由基变成无色，从而可以通过紫外可见光谱测定其吸光度的降低来表示抗氧化物活性。
2. ABTS法：该法通过测定2,2’-联氮双(3-乙基苯并咪唑啉硫酸铵) (ABTS)自由基的消除能力来测定抗氧化活性。
该法也是一种体外抗氧化测定方法，溶液发生颜色变化，从而通过紫外可见光谱测定其吸光度的降低来表示抗氧化物活性。
3. ORAC法：ORAC（氧化还原能力值）法对不同化学物质的体内抗氧化活性进行定量测定，其原理是将抗氧化剂加入与有氧气气氛接触的荧光染料溶液中，由于受到氧自由基的攻击，染料随着时间流逝会逐渐减少。
为了确定不同化学物质的抗氧化活性，十分重要的是应该不断输入氧自由基。
4. FRAP法：铁还原能力 (FRAP) 方法测量样品对Fe3+的还原能力，其原理是将含有Fe3+的试液与抗氧化剂反应后，Fe3+被还原为Fe2+，测试Fe2+的含量即可评估抗氧化剂的抗氧化性能。
5. TBARS法：该方法是用于评估脂质过氧化物含量，从而推断抗氧化剂的能力。
该评估方法是通过测定细胞膜上的脂质过氧化产物（丙二醛）来分析抗氧化剂活性。
6. Total Phenolic Content (TPC)法：该方法最初是用来测定葡萄酒和咖啡中酚类化合物含量的。
后来发现大多数植物成分含有大量的酚类化合物，故也用于测定植物中的酚类含量。
总抗氧化能力（FRAP 法）试剂盒说明书
总抗氧化能力（FRAP 法）试剂盒说明书微量法100T/96S 注 意：正式测定之前选择2-3 个预期差异大的样本做预测定。
研究意义：测定对象中各种抗氧化物质和抗氧化酶等构成总抗氧化水平。
在生物学、医学和药学研究中常常检测血浆、血清、唾液、尿液等各种体液，细胞或组织等裂解液、植物或中草药抽提液及各种抗氧化物(antioxidant)溶液的总抗氧化能力。
测定原理：在酸性环境下，抗氧化物质还原Fe3+-三吡啶三吖嗪(Fe3+-TPTZ)产生蓝色的Fe2+-TPTZ 的能力反映了总抗氧化能力。
自备实验用品：恒温水浴锅、低温离心机、酶标仪、96 孔板和蒸馏水。
试剂组成和配制：提取液：液体120mL×1 瓶，使用前预冷。
试剂一：液体20 mL×1 瓶，避光保存。
试剂二：液体2 mL×1 瓶，4℃避光保存。
试剂三：液体2 mL×1 瓶，避光保存。
混合液(现配现用)：将试剂一、试剂二、试剂三按10:1:1 的比例混合，使用前37℃预温。
样品的制备：(1) 血清、血浆、唾液或尿液等液体样品血浆（制备时可以使用肝素或柠檬酸钠抗凝，不宜使用EDTA 抗凝）4℃，5000rpm 离心10min，取上清待测。
血清、唾液或尿液样品直接用于测定，也可以-80℃冻存（不宜超过30 d）后再测定。
(2) 组织样品按照组织质量（g）：提取液体积(mL)为1：5~10 的比例（建议称取约0.1g 组织，加入1mL 提取液）进行冰浴匀浆，然后10000g，4℃离心10min，取上清，置冰上待测。
(3) 细胞样品按照细胞数量（104个）：提取液体积（mL）为500~1000：1 的比例（建议500 万细胞加入1mL 提取液），冰浴超声波破碎（功率200W，超声3s，间隔10s，重复30 次）；10000g，4℃离心10min，取上清，置冰上待测。
操作步骤：1、酶标仪预热30min，调节波长至593nm。
DPPH和FRAP法测定41种中草药抗氧化活性
实验室研究与探索
RESEARCH AND EXPLORATION IN LABORATORY
Vol． 30 No． 6 Jun． 2011
DPPH 和 FRAP 法测定 41 种中草药抗氧化活性
陈玉霞， 刘建华， 林 峰， 杜向党
（ 河南农业大学 牧医工程学院，河南 郑州 450002）
0引言
人体产生的自由基及其诱导的氧化反应与多种疾 病密切相关，并可加速人体衰老，抗氧剂能有效清除自 由基，延缓人体衰老和预防疾病。目前，在医疗和食品 领域使用较多的是合成抗氧剂，而实验动物研究表明， 合成抗氧剂，如 BHT、BHA 对生物体有潜在的毒副作 用，加上人们日益追求环保，因此，近年来寻找天然、安
（ 3） 抗氧化活性的测定［1］。 ① 总抗氧化能力的测定（ FRAP 值测定） 。取各 供试品溶液，适当稀释后移液器取 100 μL，加入 FRAP 工作液 3 mL，混匀反应 20 min，于 593 nm 处读取吸光 度。以 FeSO4 为标准物质绘制标准曲线（ 见图 1） ，求 得回归方程为 Y = 0． 007X － 0． 019，相关系数为 R2 = 0． 994，样品的抗氧化能力以 FRAP 值表示： 1 FRAP单 位 = 1 mmol / L FeSO4 ，即 样 品 的 抗 氧 化 能 力 相 当 于 FeSO4 的 mmol / L 数。
中草药 白花蛇舌草
苍术 黑豆 茺蔚子 益母草 川芎 徐长卿 杜仲 黑芝麻 牛膝 龙葵 人参 黄芪 山药 葛根 生地 青黛 薏苡仁 韭子 大蒜
DPPH / % 15． 11 ± 3． 75 10． 77 ± 2． 29 9． 31 ± 0． 22 9． 13 ± 3． 67 9． 13 ± 1． 09 8． 47 ± 4． 31 7． 96 ± 3． 79 7． 85 ± 1． 55 7． 64 ± 1． 64 7． 55 ± 1． 75 7． 04 ± 3． 07 6． 89 ± 1． 65 6． 27 ± 3． 88 6． 56 ± 0． 05 6． 07 ± 3． 47 5． 71 ± 0． 60 5． 61 ± 2． 75 5． 20 ± 0． 10 4． 69 ± 0． 41 2． 75 ± 0． 98
总抗氧化能力测定(FRAP法)讲解学习
总抗氧化能力测定(F R A P法)小麦叶片总抗氧化能力测定（FRAP法）一、实验原理FRAP法测定总抗氧化能力的原理是酸性条件下抗氧化物可以还原Ferric-tri-pyridyl-tria-zine(Fe3+-TPTZ)产生蓝色的Fe2+-TPTZ，随后在593nm测定蓝色的Fe2+-TPTZ即可获得样品中的总抗氧化能力。
由于反应在酸性条件下进行，可以抑制内源性的一些干扰因素。
并且由于血浆等样品中的铁离子或亚铁离子的总浓度通常低于10μM，因此血浆等样品中的铁离子或亚铁离子不会显著干扰FRAP法的检测反应。
由于反应体系中的铁离子或亚铁离子是和TPTZ螯合的，样品本身含有的少量金属离子螯合剂通常也不会显著影响检测反应。
AntioxidantFe3+-TPTZ（橘黄色）——————> Fe2+-TPTZ (蓝色)二、实验步骤1.FRAP工作液配制：0.3 M pH 3.6 醋酸缓冲液：0.364g无水醋酸钠+3.2mL冰乙酸定容至200mL，用1M HCl调节pH至3.6；10mmol/L TPTZ溶液25mL：0.078g TPTZ用40mM 盐酸溶液定容至25mL；20mmol/L FeCl溶液50mL：2.78g用RO水定容至50mL；3上述溶液以10:1:1的比例混合（现配现用）。
2.取叶片0.1g，加入2.5mL蒸馏水研磨稍沉淀后取1.5mL 12000g离心10min（4o C），取上清液。
3.在反应管中加入100uL上清液，再加入 2.4mL工作液，37 o C条件下水浴10min，于593nm处测定吸光度，的标准液替代样品绘制标准曲4.标准曲线绘制：以0.1-1.6mmol/L的FeSO4线。
三、结果计算以1.0mmol/L的FeSO4为标准，样品抗氧化活性以达到同样吸光度值为一个FRAP值，计算结果。
[1]Benzie I F F, Strain J J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay[J]. Analytical Biochemistry, 1996, 239(1):70-6.[2]Katarzyna Szafrańska, Rafał Szewczyk, Krystyna Maria Janas. Involvem ent of melatonin applied to Vigna radiata, L. seeds in plant response to chilling stress[J]. Central European Journal of Biology, 2014, 9(11):1117-1126.。
抗氧活性的测定
园艺科学研究方法——园艺植物育种研究Ⅱ抗氧活性的测定 一、实验目的：1、掌握FRAP 法测抗氧化活力的方法；2、常见水果、蔬菜的抗氧化能力比较。
二、实验原理：Ferric-tripyridyltriazine(Fe3+-TPTZ)三、仪器、耗材：离心机、离心管、离心管，移液器、分光光度计、水浴锅、塑料比色杯、试剂瓶，吸水纸，枪头、量筒、大研钵； 苹果、青椒、葡萄、胡萝卜、番茄、芒果。
四、实验内容：1.不同果、蔬抗氧化物质的提取用自来水、蒸馏水反复冲洗干净后，分离果肉称取1～5 g ，在研钵内按1：9比例加入蒸馏水，研磨成匀浆液，10 000 r ／min 离心10 min ，取上清按下述FRAP 法测定抗氧化活性，每份样品重复测定5次。
2.FRAP 测定方法操作Fe2+-TPTZ 样品中总抗氧化能力抗氧化剂antioxidant 593nm 测定取适量样品上清(必要时稀释)，加入FRAP试剂，混匀后37℃反应10 min，593 nm 测定吸光度，以1.0 mM FeSO4为标准．样品抗氧化活性以达到同样吸光度所需的FeSO4的毫摩尔数表示。
（1）FRAP试剂准备100 ml醋酸缓冲液+ 10 ml TPTZ溶液+ 10 ml三氯化铁溶液+ 12 ml双蒸水，混匀，37℃保温。
（1）2 ml FRAP试剂+双蒸水1 ml，4 min后593 nm调零。
（2）2 ml FRAP试剂+100 ul 提取液+双蒸水900 ul，4 min后593nm 测量。
五、数据整理：三次重复的不同浓度Fe2+对应的吸光度Fe2+浓度（mM）吸光度(A) 平均值（A）第一次第二次第三次1.0 1.181 1.182 1.177 1.180 0.8 1.198 1.196 1.185 1.193 0.6 1.195 1.192 1.185 1.191 0.4 1.167 1.168 1.163 1.166 0.2 1.006 1.021 1.009 1.012 0.1 0.631 0.620 0.623 0.625 各种材料FRAP法抗氧化活性的测定重测量值一测量值二测量值三平均值（A) 抗氧化活性种类青椒 1.170 1.294 1.257 1.240 ＞1.00胡萝卜0.266 0.366 0.456 0.363 0.04苹果0.912 1.039 1.011 0.987 0.16葡萄0.739 0.703 0.729 0.724 0.12番茄 1.021 1.012 1.034 1.022 0.21芒果 1.071 1.069 1.067 1.069 0.23六、结果分析由表格得，胡萝卜的抗氧化活性最低，青椒的抗氧化活性最高。
抗氧化物活性测定方法总结
抗氧化物活性测定方法（倾向于考虑DPPH法和ORAC法）1.FRAP法:铁离子还原抗氧化能力测定法[1]FRAP(ferric ion reducing antioxidant power)方法，在低pH值的溶液中，Fe3+-TPTz(Fe3+-三吡啶三嗪)被抗氧化剂还原成有色的Fe2+-TPTZ。
反应的结果常以Fe2+当量或标准物质的抗氧化能力表示。
该法快速简便、易于操作、重复性好，但FRAP反应属于电子转移(SET)反应，因此FRAP方法不能够测定氢转移反应(HAT)起作用的物质。
而且该法实际测定的是待测生物活性物质将Fe3+还原为Fe2+的能力，因此没有抗氧化能力的生物学相关性。
2.TEAC法(trolox equivalent antioxidant capacity)ABTS(2,2'-amino-di(2-ethyl-benzothiazoline sulphonic acid-6)ammonium salt,2,2'氨基-二(3-乙基-苯并噻唑啉磺酸-6)铵盐)与过氧化物酶和氢过氧化物在一起形成ABTS+阳离子自由基。
在抗氧化剂存在时，这种自由基混合物的光吸收值下降，下降程度取决于抗氧化剂的抗氧化能力，测得的结果以TEAC表示，即被测抗氧化剂清除ABTS·+的能力(吸光度大小的变化)与标准抗氧化剂trolox(VE的水溶性类似物)清除ABTS·+的能力的比值。
TEAC法十分简单，适用于大量样品的分析检测。
但是，ABTS·+并非生理自由基，缺乏生理相关性，而且与FRAP方法相似，ABTS·+与不同抗氧化剂问的氧化反应时间不同，因此，只能定性不能定量评价样品的抗氧化能力。
3.DPPH法[2，3](2，2-diphenyt-l-picrylhydrazyl radical scavenging capacity)DPPH·(二苯代苦味肼基自由基)法是较常用的方法之一。
总抗氧化能力测定(FRAP法)
精品文档小麦叶片总抗氧化能力测定（FRAP法）一、实验原理FRAP法测定总抗氧化能力的原理是酸性条件下抗氧化物可以还原Ferric-tri-pyridyl-tria-zine（Fe 3+-TPTZ）产生蓝色的 Fe2+-TPTZ,随后在 593nm测定蓝色的Fe2+-TPTZ即可获得样品中的总抗氧化能力。
由于反应在酸性条件下进行，可以抑制内源性的一些干扰因素。
并且由于血浆等样品中的铁离子或亚铁离子的总浓度通常低于10側，因此血浆等样品中的铁离子或亚铁离子不会显著干扰FRAP法的检测反应。
由于反应体系中的铁离子或亚铁离子是和TPTZ螯合的，样品本身含有的少量金属离子螯合剂通常也不会显著影响检测反应。
AntioxidantFe3+-TPTZ （橘黄色）---------- >Fe2+-TPTZ （蓝色）二、实验步骤1. FRAPT作液配制：0.3 M pH 3.6醋酸缓冲液：0.364g无水醋酸钠+3.2mL冰乙酸定容至200mL 用1M HCl调节pH至3.6 ;10mmol/L TPTZ溶液25mL 0.078g TPTZ 用40mM盐酸溶液定容至25mL 20mmol/L FeCl3溶液 50mL 2.78g 用 RO水定容至 50mL上述溶液以 10:1:1 的比例混合（现配现用）。
2. 取叶片0.1g，加入2.5mL蒸馏水研磨稍沉淀后取 1.5mL 12000g离心10min（4°C）,取上清液。
3. 在反应管中加入100uL上清液，再加入2.4mL工作液，37°C条件下水浴10min, 于593nm处测定吸光度，4. 标准曲线绘制：以0.1-1.6mmol/L的FeSQ的标准液替代样品绘制标准曲线。
三、结果计算以1.0mmol/L的FeSQ为标准，样品抗氧化活性以达到同样吸光度值为一个FRAP fi，计算结果。
精品文档2.8.2. Ferric Reducing/Antioxidant Power (FRAP) assayBriefly, the FRAP reagent contained 2.5 mL 10 mM L_1TPTZ solution in 40 mM L1HCI plus 2.5 mL 20 mM L1 FeCI^ and 25 mL 0+3 M L1acetate buffer, pH 3*6 was prepared freshly and warmed at 37°C・After addition of root ethanol extracts (prepared as in section 2.7) and incubation at 37°Cfor 5 min, absorbance of the reaction mixture was measured at 593 nm. The final result was expressed as the concentration of antioxidants having a ferric reducing ability equivalent to that of 1 mM L_1 FeSO4based on the standard curve for FeSO4 x 7H.0 at a concentration range between 100 and 1000 pM L_1 [3 口[1] Benzie I F F, Strain J J. The Ferric Reducing Ability of Plasma (FRAP) as aMeasure of “ntioxidant Power”： The FRAP Assay[J]. Analytical Biochemistry, 1996, 239(1):70-6.[2] Katarz yna Szafra nska, Rafa? Szewczyk, Kryst yna Maria Jan as. In volveme nt ofmelat onin applied to Vig na radiata, L. seeds in pla nt resp onse to chilli ng stress[J].Central European Journal of Biology, 2014, 9(11):1117-1126.精品文档欢迎您的下载，资料仅供参考!致力为企业和个人提供合同协议，策划案计划书，学习资料等打造全网一站式需求。
FRAP法测定大枣枣皮红色素的总抗氧化能力_赵文恩
司 ;RE-52AA旋 转蒸发 器 , 上 海亚 荣生化 仪器 厂 ;501型超级恒温水浴 , 上海浦东荣丰科技仪器 有限公司 ;SHZ-D循环水式真空泵 , 巩义市予华 仪器有限责任公司 ;PHS3 -3C型精密酸度计 , 上 海大普仪器有限公司 .
的碱水 提取效果 极浅黄色 橙黄色 深枣红色
0.2mol/L 的酸水
浅黄色
蒸馏水 黄色
采用 FRAP法测定不同溶剂提取的枣皮红色 素粗提物的抗氧化能力 , 结果如图 2所示 , 抗氧化 能力由高到低为 :0.8%NaOH粗提物 >50%乙醇粗 提物 >蒸馏水粗提物 >0.2 mol/LHCl粗提取 >乙 酸乙酯粗提物 .结果表明对于抗氧化活性物质的提 取 ,碱性水溶液效果最好 , 乙酸乙酯最差 , 并且这一 结果与枣皮红色素的提取效果是一致的 , 说明枣皮 红色素中含有抗氧化活性成分 , 并且枣皮红色素含 量与抗氧化活性存在一定的相关性 .
同样 , 按照上述方法 , 以 0.1 ～ 1.6 mmol/L的 FeSO4 的标准溶液代替样品绘制标准曲线 .
2 结果与讨论
2.1 FeSO4 标准曲线的绘制 图 1为 FeSO4 标准曲线 , 由图 1可知 , FeSO4
浓度在 0.1 ～ 1.6 mmol/L范围内与其在 593 nm 处的吸光度 成良好 线性 关系 .直 线方程 为 :Y= 0.003 55 +0.303 71X;Y为 593 nm处的吸光度 , X 为 FeSO4 浓度 , mmol/L;相关系数 R2 =0.999 6. 因此以 593 nm处的吸光值换算成 FeSO4 当量浓 度是可行的 , 如果需要 , 允许个别测定点的 FRAP 值不在线性范围内 [ 6] .即样品最终的总抗氧化能 力以硫酸亚铁的当量浓度表示 , 单位为 mmol/L.
DPPH、ABTS和FRAP微量法测定山奈酚的抗氧化能力
2. 1. 2 山奈酚对 DPPH·清除能力
称取适量山奈酚, 加入无水乙醇配制成浓度为 160 μmol / L
的溶液, 后用无 水 乙 醇 稀 释 成 浓 度 梯 度 为 160、 80、 40、 20、
10、 5、 2. 5 μmol / L 的溶液。 精密吸取梯度样品溶液 100 μL 和
to evaluate the scavenging ability of Kaempferol against DPPH free radical and ABTS free radical. The antioxidant activity
of Kaempferol was evaluated by measuring a micro - model of FRAP. The scavenges DPPH and ABTS free radicals of
∗
第 49 卷第 3 期
59
王荣, 等: DPPH、 ABTS 和 FRAP 微量法测定山奈酚的抗氧化能力
Hale Waihona Puke 2 方法与结果2. 1 DPPH·清除能力的测定
2. 1. 1 DPPH·标准曲线的绘制
避光称取 DPPH 粉末 82 mg, 无水乙醇定容于 50 mL 量瓶
中, 得 1. 64 mg / mL 的 DPPH 母液。 以无水乙醇为溶剂将上述母
Kaempferol increased with the increase of the concentration. IC50 of the scavenge DPPH and ABTS free radicals were
FRAP
分光光度计测：40mmol/L盐酸溶液：取浓盐酸(12mol/L)0.1ml加水至30ml，置于避光处，备用。
，用蒸馏水定容至50ml，置于避光处，备三氯化铁溶液：称取0.1625g的FeCl3用。
0.3mol/L醋酸钠缓冲溶液：称取醋酸钠5.1g，加冰醋酸20ml，用水稀释定容至250ml，置于避光处，备用。
10mmol/L TPTZ溶液：称取TPTZ样品31．233mg，用40mmol/L的盐酸定容至10ml，置于冰箱中冷藏备用。
(2)番茄乙醇提取物总抗氧化能力的测定FRAP法Fe3+吡啶三吖嗪可被样品中还原物质还原为二价铁形式，呈现出蓝色，并于593nm处具有最大光吸收，根据吸光度大小计算样品抗氧化活性的强弱。
取0.3ml样品，加2.7ml预热至37o C的FRAP工作液，摇匀后放置10min，于593nm 测其吸光度值(A值)。
样品分别作10倍、20倍、40倍稀释，做三个稀释度(用无水乙醇作为参比)，以无水乙醇代替样品加入FRAP工作液作为空白，每个稀释度做3次平行试验，求平均值。
根据反应后A值，在标准曲线上求得相应FeSO4的浓度(μmol/L)，定义为FRAP值。
FRAP值越大，抗氧化活性越强。
标准曲线的绘制：准确称取6．08mg硫酸亚铁溶于适量的水中，加入FeSO418mol/L的硫酸0.25ml，再加水稀释至50ml定容，并置入小铁钉。
取上述溶液标准溶液。
预先用小试5ml于5ml的水，定容至50ml，即为800μmol/L FeSO4(800μmol/L)标准溶液加入试管中，配制得400μmol/L 管装5ml水，取5mlFeSO4标准溶液，按此方法依次进行，配制200μmol/L、100μmol/L。
50μmol/L、25μmol/L标准溶液，绘制标准曲线(图1)，得回归方程为：Y=0．0023X+0．191，R2=0．9996。
[4]Benzie IFF，Strain J．The ferric reducing ability of plasma(FRAP)asa measure of“antioxidant power”：the FRAP assay．Analytical Biochemistry，1996，239(3)：70-76．参照Benzie etal.(1996)的方法，原理为Fe3+-三吡啶三吖嗪(TPTZ，sigma)可被样品中还原物质还原为二价铁形式，呈现出蓝色，并于593nm处具有最大光吸收，根据吸光度大小计算样品抗氧化活性的强弱。
抗氧化物活性测定方法总结
抗氧化物活性测定方法（倾向于考虑DPPH法和ORAC法）1.FRAP法:铁离子还原抗氧化能力测定法[1]FRAP(ferric ion reducing antioxidant power)方法，在低pH值的溶液中，Fe3+-TPTz(Fe3+-三吡啶三嗪)被抗氧化剂还原成有色的Fe2+-TPTZ。
反应的结果常以Fe2+当量或标准物质的抗氧化能力表示。
该法快速简便、易于操作、重复性好，但FRAP反应属于电子转移(SET)反应，因此FRAP方法不能够测定氢转移反应(HAT)起作用的物质。
而且该法实际测定的是待测生物活性物质将Fe3+还原为Fe2+的能力，因此没有抗氧化能力的生物学相关性。
2.TEAC法(trolox equivalent antioxidant capacity)ABTS(2,2'-amino-di(2-ethyl-benzothiazoline sulphonic acid-6)ammonium salt,2,2'氨基-二(3-乙基-苯并噻唑啉磺酸-6)铵盐)与过氧化物酶和氢过氧化物在一起形成ABTS+阳离子自由基。
在抗氧化剂存在时，这种自由基混合物的光吸收值下降，下降程度取决于抗氧化剂的抗氧化能力，测得的结果以TEAC表示，即被测抗氧化剂清除ABTS·+的能力(吸光度大小的变化)与标准抗氧化剂trolox(VE的水溶性类似物)清除ABTS·+的能力的比值。
TEAC法十分简单，适用于大量样品的分析检测。
但是，ABTS·+并非生理自由基，缺乏生理相关性，而且与FRAP方法相似，ABTS·+与不同抗氧化剂问的氧化反应时间不同，因此，只能定性不能定量评价样品的抗氧化能力。
3.DPPH法[2，3](2，2-diphenyt-l-picrylhydrazyl radical scavenging capacity)DPPH·(二苯代苦味肼基自由基)法是较常用的方法之一。
铁离子还原法(FRAP)测定中国被毛孢菌丝体提取物抗氧化活性
ｔｍｎＣ Ｃ ， Ｏ Ｈ 和 Ｈ １ ａ ｉ 、Ｈ Ｃ Ｏ Ｃ 均为 国产分 析纯试 剂 ． 精密 酸度测试 仪 Ｐ Ｓ一 Ｔ Ｈ ３ Ｃ为上海 天 达仪 器有 限公 司 产品 ， 紫外 一可见 分光光 度计 为 岛津 ｕ ２５ Ｖ一 ４０型．
１ ２ 样 品制 备 ．
冻干菌丝体研磨后过筛 （０目）７ ￣烘干 ３ｈ 准确称取 ２ｇ １０ｍ ５ ， Ｃ ０ ， 置 ０ Ｌ圆底烧瓶 ， 加入 ５ Ｌ９％ ０ｍ ５ 乙醇 ， 浸泡 ２ ， ℃加热 回流提取 １ｈ冷却室温后过滤 ， ｈ后 ７ ０ ， 取滤液 ， 滤渣继续用 ３ Ｌ ５ 乙醇 ， ０ｍ ％ ９ 再次 ７ ＇加热回流提取 １ ， ０ Ｅ 冷却室温后合并两次提取液定容至 １０ｍ ， ｈ ０ Ｌ 最后过滤取滤液置 ４Ｃ ￣ 冰箱保存． 乙 醇提取后的残渣置于通风橱内过夜 ， 让乙醇充分挥发 ， 然后用水替换 乙醇按 同样的方法提取 ， 提取温度 为 ９ｃ ， ５【 以获得水提物溶液 ， ４ ＝ 置 ℃冰箱保存． 各取少量提取溶液浓缩干燥用于计算其固形物浓度．
原力 的测定并探讨了该方法的适用性 ， 为比较研究天然冬虫夏草与人工培养菌丝体 的药理活性和物质 基础 、 制定质量分析和控制方法提供了实验依据．
１ 试剂仪器及样 品
１ １ 试剂 与 仪器 ．
６个 批 号 的 中国 被 毛孢 丝 体 由上 海 芝 草 生 物 技术 有 限公 司馈 赠 ． ， ，一 吡 啶 一１３ ５一三 吖 嗪 ２ ４ ６三 ，， （ ， ，－ｒ ２一 ｙｉｙ）ｓｔａｉｅＴ Ｔ ） 自 Ｓｇ ２ ４６Ｔｉ （ ｐｒ １－ ｒ ｚ ，Ｐ Ｚ 购 ｄ －ｉ ｎ ｉ ｍａ公 司 ． ｅ １ ６ ２ ＦＳ ４・ Ｈ Ｏ、 Ｈ Ｃ Ｏ ａ Ｖ． ＦＣ３・ Ｈ Ｏ、ｅＯ ７ ２ Ｃ ３ Ｏ Ｎ 、 ｉ
测定抗氧化的六种方法是
测定抗氧化的六种方法是
1.自由基清除能力测定法：通过测定样品对自由基的清除能力来评估其抗氧化能力。
常用的方法包括DPPH（2,2-二苯基-1-苦基肼）自由基清除法和ABTS（2,2'-联氨基二-(3-乙基苯并噻唑-6-磺酸)）自由基清除法。
2.氧化还原能力测定法：通过测定样品在氧化还原反应中的电子接受能力来评估其抗氧化能力。
常用的方法包括还原能力测定法和Ferric reducing antioxidant power（FRAP）法。
3.金属离子螯合能力测定法：通过测定样品对金属离子的螯合能力来评估其抗氧化能力。
常用的方法包括铁离子螯合能力测定法和铜离子螯合能力测定法。
4.脂质过氧化抑制能力测定法：通过测定样品对脂质过氧化的抑制能力来评估其抗氧化能力。
常用的方法包括脂质过氧化抑制能力测定法和TBARS（硫代巴比妥酸反应物）测定法。
5.蛋白质氧化抑制能力测定法：通过测定样品对蛋白质氧化的抑制能力来评估其抗氧化能力。
常用的方法包括蛋白质碳氧化酶活性测定法和蛋白质过氧化物酶活性测定法。
6.细胞抗氧化能力测定法：通过测定样品对细胞内氧化应激的保护作用来评估其抗氧化能力。
常用的方法包括细胞活力测定法和细胞内氧化应激指标测定法。
抗氧化性测定方法
抗氧化性测定方法
抗氧化性测定方法是一种用于测试物质对氧气自由基、过氧化物和其他氧化性物质的抵抗能力的方法。
以下是常见的抗氧化性测定方法：
1. DPPH自由基清除法：使用一种紫色自由基DPPH，通过测定反应前后DPPH 吸收峰的差异来评估样品的自由基清除能力。
2. ABTS自由基清除法：使用一种蓝色自由基ABTS，通过ABTS的光谱变化来衡量样品对自由基的清除能力。
3. ORAC法：利用ORAC反应器测量被测样品对氧化过程中产生的自由基的清除能力。
4. FRAP法：利用FRAP反应器测量被测样品对铁离子的还原能力。
5. TAC法：通过测量总抗氧化能力评价样品的抗氧化能力。
6. 超氧化物歧化酶(SOD)活力测定法：利用SOD对超氧化物的清除作用来评估物质的抗氧化能力。
以上方法中，DPPH和ABTS自由基清除法是较常用的评估抗氧化性的方法。
抗氧化性是很多食品和保健品评估其品质和功效的重要指标，因此，准确测定物质
的抗氧化性对于推广新产品、提高产品质量和开发功能性食品有着重要意义。
总抗氧化能力检测试剂盒 (FRAP 法 )
总抗氧化能力检测试剂盒(FRAP法)产品编号产品名称包装S0116 总抗氧化能力检测试剂盒(FRAP法) 100次产品简介：总抗氧化能力检测试剂盒(FRAP法)，即Total Antioxidant Capacity Assay Kit with FRAP method，简称T-AOC Assay Kit，是一种采用Ferric Reducing Ability of Plasma (FRAP)方法，可以对血浆、血清、唾液、尿液等各种体液，细胞或组织等裂解液、植物或中草药抽提液、或各种抗氧化物(antioxidant)溶液的总抗氧化能力进行检测的试剂盒。
活性氧(Reactive oxygen species, ROS)主要包括羟基自由基、超氧自由基和过氧化氢。
在细胞或组织的正常生理代谢过程中会产生活性氧，同时一些环境因子例如紫外照射、γ射线照射、吸烟、环境污染等也可以诱导活性氧的产生。
活性氧产生后，可以导致细胞内脂、蛋白和DNA等的氧化损伤，诱发氧化应激(Oxidative stress)，继而导致各种肿瘤、动脉粥样硬化、风湿性关节炎、糖尿病、肝损伤、以及中枢神经系统疾病等。
机体中存在多种抗氧化物，包括抗氧化大分子、抗氧化小分子和酶等，可以清除体内产生的各种活性氧，以阻止活性氧诱导的氧化应激(oxidative stress)的产生。
一个体系内的各种抗氧化大分子、抗氧化小分子和酶的总的水平即体现了该体系内的总抗氧化能力。
因此测定血浆、血清、尿液、唾液等各种体液，细胞或组织等裂解液中的总抗氧化能力具有非常重要的生物学意义。
植物或中草药抽提液、或各种抗氧化物溶液的总抗氧化能力的检测可以用于检测各种溶液的抗氧化能力的强弱，可以用于筛选强抗氧化能力的药物。
FRAP法测定总抗氧化能力的原理是酸性条件下抗氧化物可以还原Ferric-tripyridyltriazine (Fe3+-TPTZ)产生蓝色的Fe2+-TPTZ，随后在593nm测定蓝色的Fe2+-TPTZ即可获得样品中的总抗氧化能力。
总抗氧化能力测定(FRAP法)
小麦叶片总抗氧化能力测定（FRAP法）一、实验原理FRAP法测定总抗氧化能力的原理是酸性条件下抗氧化物可以还原Ferric-tri-pyridyl-tria-zine(Fe3+-TPTZ)产生蓝色的Fe2+-TPTZ，随后在593nm测定蓝色的Fe2+-TPTZ即可获得样品中的总抗氧化能力。
由于反应在酸性条件下进行，可以抑制内源性的一些干扰因素。
并且由于血浆等样品中的铁离子或亚铁离子的总浓度通常低于10μM，因此血浆等样品中的铁离子或亚铁离子不会显著干扰FRAP法的检测反应。
由于反应体系中的铁离子或亚铁离子是和TPTZ螯合的，样品本身含有的少量金属离子螯合剂通常也不会显著影响检测反应。
AntioxidantFe3+-TPTZ（橘黄色）——————> Fe2+-TPTZ (蓝色)二、实验步骤1.FRAP工作液配制：0.3 M pH 3.6 醋酸缓冲液：0.364g无水醋酸钠+3.2mL冰乙酸定容至200mL，用1M HCl调节pH至3.6；10mmol/L TPTZ溶液25mL：0.078g TPTZ用40mM 盐酸溶液定容至25mL；20mmol/L FeCl溶液50mL：2.78g用RO水定容至50mL；3上述溶液以10:1:1的比例混合（现配现用）。
2.取叶片0.1g，加入2.5mL蒸馏水研磨稍沉淀后取1.5mL 12000g离心10min（4o C），取上清液。
3.在反应管中加入100uL上清液，再加入2.4mL工作液，37o C条件下水浴10min，于593nm处测定吸光度，4.标准曲线绘制：以0.1-1.6mmol/L的FeSO的标准液替代样品绘制标准曲线。
4三、结果计算以1.0mmol/L的FeSO为标准，样品抗氧化活性以达到同样吸光度值为一个4FRAP值，计算结果。
[1]Benzie I F F, Strain J J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay[J]. Analytical Biochemistry, 1996, 239(1):70-6.[2]Katarzyna Szafrańska, Rafał Szewczyk, Krystyna Maria Janas. Involvement of melatonin applied to Vigna radiata, L. seeds in plant response to chilling stress[J]. Central European Journal of Biology, 2014, 9(11):1117-1126.。
FRAP法抗氧化
Journal of Chromatography A,1233 (2012) 8–15Contents lists available at SciVerse ScienceDirectJournal of ChromatographyAj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o maComparative evaluation of post-column free radical scavenging and ferricreducing antioxidant power assays for screening of antioxidants in strawberriesRaimondas Raudonis ∗,Lina Raudone 1,Valdas Jakstas 2,Valdimaras Janulis 3Department of Pharmacognosy,Lithuanian University of Health Sciences,Mickeviciaus Str.9,LT-44307,Lithuaniaa r t i c l ei n f oArticle history:Received 3November 2011Received in revised form 7February 2012Accepted 8February 2012Available online 15 February 2012Keywords:HPLC post-column ABTS FRAPAntioxidant activity Fragaria L.a b s t r a c tABTS and FRAP post-column techniques evaluate the antioxidant characteristics of HPLC separated compounds with specific reagents.ABTS characterize their ability to scavenge free radicals by electron-donating antioxidants,resulting in the absorbance decrease of the chromophoric radical.FRAP –is based on the reduction of Fe(III)–tripyridyltriazine complex to Fe(II)–tripyridyltriazine at low pH by electron-donating antioxidants,resulting in an absorbance increase.Both post-column assays were evaluated and compared according to the following validation parameters:specificity,precision,limit of detection (LoD),limit of quantitation (LoQ)and linearity.ABTS and FRAP post-column assays were specific,repeat-able and sensitive and thus can be used for the evaluation of antioxidant active compounds.Antioxidant active compounds were quantified according to TEAC for each assay and ABTS/FRAP ratio was derived.No previous records of antioxidative activity of leaves and fruits of strawberries (Fragaria viridis ,Fra-garia moschata )research have been found.The research results confirm the reliability of ABTS and FRAP post-column assays for screening of antioxidants in complex mixtures and the determination of radical scavenging and ferric reducing ability by their TEAC values.© 2012 Elsevier B.V. All rights reserved.1.IntroductionOxidative stress plays a pivotal role in pathogenesis of cardio-vascular,neurodegenerative diseases,cancer and aging [1].Antioxi-dants reduce oxidative stress by various mechanisms [2].Research of natural antioxidants has increased in area of functional foods,agriculture and disease prevention [3–5].Numerous assays with different mode of action have been established for the assessment of antioxidant activity [3,6,7].Spectrophotometric studies evaluat-ing the total antioxidant capacity are convenient and easy adapt-able.These assays depend on single electron transfer or hydrogen atom transfer [8].Most popular assays,based on single electron transfer,evaluate radical scavenging abilities (ABTS –2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid),DPPH –2,2-diphenyl-1-picrylhydrazyl)and potential of ferric or cupric reducing (FRAP –ferric reducing antioxidant power,CUPRAC –cupric reducing antioxidant capacity)capacity expressed as Trolox equivalents [3,9–12].Yet,they evaluate the additive and synergistic interre-lational impact of all compounds present in the sample as herbal∗Corresponding author.Tel.:+37067843678.E-mail addresses:raimondas.raudonis@ (R.Raudonis),raudone.lina@ (L.Raudone),valdas.jakstas@med.kmu.lt (V.Jakstas),farmakog@kmu.lt (V.Janulis).1Tel.:+37068241377.2Tel.:+37067200844.3Tel.:+37069802919.extracts contain a body of polyphenolic compounds with differ-ent structure and activity [13].To elucidate the activity of separate compounds in complex extracts is not possible.Structure–activity relationships well established in numerous studies confirmed different modes of action of separate polyphenolic compounds [14,15].It is purposeful to evaluate individual polyphenolic com-pounds in herbal extracts with antioxidant activity.Recently a body of researches has established on-line post-column methods for screening of antioxidants in complex mixtures [16–21].HPLC separation is coupled with rapid identification of antioxidative active compounds.The pivotal advantages of these post-column reaction methods are that the antioxidant activity of an individual compound can be measured and its contribution to the total activity of a complex mixture can be estimated,and also the activity of an individual compound can be compared to other constituents in the mixture and their structure–activity rela-tionships can be determined [21,22].These methods exclude the interactional effects of compounds as the detection occurs with separated analytes.Most of post-column assays use DPPH and ABTS radicals [16,21,23],while FRAP post-column assay,to the best of our knowledge,has not been installed previously.The latter assays can be performed in aqueous medium and low pH.As the assays are installed in the same conditions,it becomes possible to compare the radical scavenging and reducing abilities of antioxidant active compounds.Leaves and fruits of strawberries (Fragaria L.species)have long been used for medicinal and nutritional purposes [24].The herbal0021-9673/$–see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2012.02.019R.Raudonis et al./J.Chromatogr.A1233 (2012) 8–159material contains a complex mixture of various polyphenolic com-pounds(flavonoids,phenolic acids,tannins,anthocyanins).The potential health benefits have been associated with antioxidant effects[25].Therefore it is important to study polyphenolic-rich extracts and to identify the antioxidant active components.Thus,the aim of this study was to compare ABTS and FRAP post-column assays through the validation parameters and to apply the assays for the determination and evaluation of radical scavenging and ferric reducing abilities of antioxidants in strawberries.2.Materials and methods2.1.Chemicals and reagentsHPLC grade acetonitrile and HPLC grade methanol were pur-chased from Sigma–Aldrich(Buchs,Switzerland).Formic acid (98–100%),acetic acid(99.8%)and hydrochloric acid were obtained from Fluka Chemie(Buchs,Switzerland).Ethanol(96.3%)was provided by Stumbras(Kaunas,Lithuania).Ultrapure water was prepared using a Millipore water purification system(Bedford, MA).The following reagents were used:2,2-azinobis(ethyl-2,3-dihydrobenzothiazoline-6-sulphonic acid)diammonium salt (ABTS),potassium persulfate from Fluka(Buchs,Switzerland); iron(III)chloride hexahydrate(FeCl3·6H2O),2,4,6-tripyridyl-s-triazine(TPTZ)and sodium acetate trihydrate from Sigma–Aldrich Chemie(Steinheim,Germany).The following standards were used:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox),chlorogenic acid,(−)-epicatechin,caffeic acid,(+)-catechin,rutin,ellagic acid,quercetin,isoquercitrin,quercitrin, hyperoside,epigallocatechin gallate were purchased from Fluka (Buchs,Switzerland),Roth(Karlsruhe,Germany),Sigma–Aldrich (Buchs,Switzerland).Individual stocks of standard solutions were prepared in ethanol.For HPLC post-column analysis the stock solutions were respectively diluted with ethanol to the required working concentrations.Ellagic acid was prepared and diluted with acetonitrile and purified water mixture(1:1).2.2.Sample materials and preparationFragaria viridis,Fragaria vesca and Fragaria moschata leaves and fruits,which were obtained from Vilnius University Botanical Gar-den,Lithuania,were the plant material used for this research. Strawberry leaves were collected in July and air-dried at room tem-perature(20–25◦C),in a ventilated chamber.Fully ripened fruits were frozen and stored in deep freezer at−30◦C.The air-dried parts of F.viridis,F.vesca,F.moschata leaves were milled.The fruits were homogenized using a rotating blade mixer.The milled strawberry leaves were extracted with70% ethanol(1:100,v/v)in ultrasonic bath(BioSonic UC100Mawai, USA)for30min.The homogenized fruits were extracted with100% methanol(1:10,v/v)in ultrasonic bath for10min.The extracts were centrifuged(13,000rpm,15min)and obtained supernatants were kept in the refrigerator at8◦C.All the samples werefiltered through 0.22m pore size membranefilters(Roth,Karlsruhe,Germany).2.3.HPLC post-column antioxidant detection system conditionsThe HPLC post-column equipment system for screening of indi-vidual antioxidants has been previously published by our group [12].The HPLC system applied consisted of Waters2695Alliance solvent manager(Waters,Milford,MA)equipped with a Waters 996photodiode array detector.Chromatographic separations were performed on an ACE C18analytical column(250mm×4.6mm, 5m)with guard column ACE C185-m(Aberdeen,Scotland).The chromatographic separation was performed using1%(v/v)formic acid solution in pure water(solvent A)and acetonitrile(solvent B).The solvent composition for the linear gradient elution was as follows:10–22%solvent B over30min;followed by22–80%sol-vent B from30to45min The mobile phaseflow rate in all analyses was set at1mL/min,and the injection volume of all samples was 10L.The confirmation of the chromatographic peaks identity was achieved by comparing the retention times and spectral character-istics( =200–600nm)of the eluting peaks with those of reference compounds.All samples were run in triplicate.HPLC post-column addition of ABTS and FRAP reagent was per-formed using a continuously working Waters Reagent Manager (Milford,MA)pump.Theflow rate of the individual reagents was set at0.5mL/min.The mobile phase with separated analytes and ABTS or FRAP reagentsflowed through a mixing tee to the reaction coil.The reaction coil was made of TFE(Teflon)tubing of the fol-lowing size:15m×0.3mm i.d.,1.58mm o.d.,∼1mL.The product chromatograms after ABTS and FRAP post-column reaction were registered at650and593nm respectively,using Waters2487dual absorbance(UV/Vis)detector(Milford,MA).Data received from experimental research was processed by Waters Empower soft-ware(Milford,MA).The stock ABTS solution was prepared by dissolving ABTS in aqueous potassium persulfate(0.7mM)to obtain the concentra-tion of2mM.The mixture was stored for16–17h in the dark at room temperatures before use[26].Acetate buffer was prepared of sodium acetate trihydrate,acetic acid and water up to the con-centration of300mM(pH3.6).The working solution of ABTS was prepared by diluting with acetate buffer up to the concentration of 0.11mM.The working FRAP solution comprised TPTZ(10mM dissolved in40mM HCl),FeCl3·6H2O(20mM in water)and acetate buffer (300mM,pH3.6)in the ratio of1:1:25.2.4.Antioxidant activity assessmentThe antioxidant activity of sample compounds was assessed by standard antioxidant Trolox.Calibration curves of Trolox(con-centrations5–400M)were made in ABTS(R2=0.999)and FRAP (R2=0.999)post-column assays.The radical scavenging and ferric reducing capacities of antioxidant active compounds in strawberry extracts were expressed as Trolox equivalent antioxidant capacity (TEAC).TEAC corresponds to Trolox quantity(mol),which at the equal conditions has the same antioxidant activity as the sample compound in1g of strawberries.TEAC was calculated according to the formula:TEAC=S comp.−b(M)×V sampl.(L)sampl.(g)(mol/g)S comp.is the peak area of antioxidant active compound in the post-column chromatogram;a is the slope,and b is the y-intercept from Trolox calibration curve regressive equation;V sampl.is the volume of herbal raw material extract;m sampl.is the weighed(precise) quantity of herbal raw material.TEAC rel value,that shows how many times the researched known antioxidant is more active than standard antioxidant Trolox. TEAC rel=a sample/a trolox,a is the slope of the sample compound and Trolox of the calibration curves[16].2.5.Statistical analysisStatistical analysis was performed using SPSS version11.0 (Chicago,USA)and Microsoft Excel.All determinations were done in triplicate,and results were calculated as mean±standard error (SE).Linear regression model was analyzed.For the suitability of each regression model determination coefficient R2and p-value obtained by checking hypothesis on non-linear regression were10R.Raudonis et al./J.Chromatogr.A 1233 (2012) 8–15used.The Mann–Whitney U test was performed for the hypothesis concerning equality of distributives.Level of significance ˛=0.05.3.Results and discussion3.1.Assessment of ABTS and FRAP post-column assay characteristicsABTS and FRAP post-column assays evaluate the qualitative characteristics of sample compounds with specific reagents (ABTS and FRAP).ABTS characterize the ability to scavenge free radi-cals by electron-donating antioxidants,resulting in the absorbance decrease of the chromophoric radical at 415,650,734and 815nm [26–28].FRAP is based on the reduction of Fe(III)–tripyridyltriazine (Fe(III)–TPTZ)complex to Fe(II)–tripyridyltriazine (Fe(II)–TPTZ)at low pH by electron-donating antioxidants,resulting in the absorbance increase at 593nm [28,29].While ABTS method mea-sures the active compound capacity against an oxidant,the FRAP assay directly measures the substance’s reducing capacity,which is an important parameter for a compound to be a good antioxi-dant [22,30].Since the antioxidant activity of a substance is usually correlated directly to its reducing capacity,the FRAP assay pro-vides a reliable method to study the antioxidant activity of various compounds [11].Antioxidant activity of sample compound is quantitatively assessed by TEAC value.For the objective and comprehensive comparison of ABTS and FRAP post-column assays at the equal experimental conditions,the assays need to be validated.Only the developed and validated method confirms that the analytical pro-cedure employed for a specific test is suitable for its intended use [31].ABTS and FRAP post-column assays were evaluated according to the following validation parameters:specificity,precision,limit of detection (LoD),limit of quantitation (LoQ)and linearity.Reaction kinetics between antioxidant active compound and ABTS or FRAP reagent is distinct,depending on the concentration of reagent,pH in the reactor’s medium,reaction time and tem-perature [12].FRAP method was modified from Benzie and Strain [32]and was adapted for post-column assay.Optimization of work-ing FRAP solution was performed by adjusting the concentration of TPTZ/FeCl 3·6H 2O in the reactor.Fixed concentration of Trolox (400M and 20M)was used for peak area assessment at different TPTZ/FeCl 3·6H 2O concentrations in the reactor (Fig.1).At the con-centration range of 123/246–278/556M no significant differences (p >0.05)in Trolox peak area were detected (4,268,514±1017and 275,210±930for 400M and 20M Trolox respectively).When the concentration of TPTZ/FeCl 3·6H 2O reached 104/208,peak area of Trolox significantly (p <0.05)decreased.TPTZ/FeCl 3·6H 2OFig.1.Dependence of Trolox (400M and 20M)peak area on TPTZ and FeCl 3·6H 2O reagent concentration in the reactor.Reaction coil –TFE (Teflon)15m ×0.3mm i.d.,1.58mm o.d.(volume ∼1mL).HPLC flow rate 1mL/min.FRAP solution flow rate 0.5mL/min.concentration of 123/246M in the reactor has been selected for the further research.Optimization of ABTS post-column assay (ABTS concentration in the reactor,reaction time,flow rate and reaction coil size)was performed in our previous study [12].The comparison of ABTS and FRAP post-column assays was performed at optimal concentrations of ABTS (35M ABTS radical cation)and FRAP (123M of TPTZ and 246M FeCl 3·6H 2O)in the reac-tor at low pH of 3.6.Reaction time depends on ABTS and FRAP reagents (0.5mL/min)and HPLC mobile phase (1mL/min)flow rates and volume of reaction coil.Reaction coil (TFE)of fixed size 15m ×0.3mm i.d.,1.58mm o.d.volume ∼1mL,was used for inves-tigations.Reaction between antioxidant and ABTS or FRAP reagents lasts for ∼40s in reactor at room temperature.Two phenolic acids (chlorogenic acid,caffeic acid),three flavanols ((+)-catechin,(−)-epicatechin,epigallocatechin gallate),five flavonols (quercetin,rutin,isoquercitrin,quercitrin,hyperoside),ellagic acid,and Trolox were chosen as reference compounds for the validation.ABTS and FRAP post-column assay specificity was assessed by identification test discriminating of active compounds [12].The reaction of chromophoric ABTS radical cation and particular active ingredient present in the sample results in the decrease of blue-green solution colour,conforming its radical scavenging identity [33].The reaction of Fe(III)–TPTZ complex with antioxidant com-pound results in the formation of a blue-colored ferrous chelate (Fe(II)–TPTZ)[34],confirming its ferric reducing identity.All the tested reference compounds had radical scavenging and ferric reducing abilities.The precision of the assays was evaluated by repeatability and intermediate precision by the repeated injection (Trolox 100M,chlorogenic acid 85M,caffeic acid 110M,(+)-catechin 100M,(−)-epicatechin 140M,epigallocatechin gallate 65M,quercetin 80M,rutin 40M,isoquercitrin 50M,quercitrin 50M,hyper-oside 50M and ellagic acid 100M).The intraday experiment was obtained by six replicates for a day,and the interday was determined by six injections for 3days for the post-column deriva-tization peak area.The mean value of relative standard deviations (RSD)of batch of samples within the same day (intraday)and at different days (interday)corresponds to repeatability and interme-diate precision,respectively.The experimental values obtained for the determination of ABTS and FRAP post-column assays of 12ref-erence compounds are presented in Table 1.The ABTS post-column assay repeatability RSD ranged from 1.18(ellagic acid)to 3.21%(chlorogenic acid),FRAP from 0.33(quercitrin)to 2.88%(caffeic acid).The ABTS post-column assay repeatability RSD values were greater than of FRAP method,but did not exceed 5%.RSD values of intermediate precision were <10%(maximum RSD was chlorogenic acid equals to 6.84%of ABTS and to 5.78%of FRAP method)suggest-ing that the post-column methods exhibited satisfactory results.The precision values are influenced by the instability of baseline,which is expressed as S/N (signal-to-noise)ratio.The instability of baseline increases with the decreasing S/N ratio and thus accuracy of compound determination decreases.Baseline stability is impor-tant in ABTS post-column assay,as ABTS is a colorful reagent [35].This problem is absent in FRAP post-column assay,as Fe(II)–TPTZ converts into colorful complex after reaction with active compound and therefore its baseline is stable.The same thing occurs with the limits of detection and quantitation that define the sensitivity of the assays,as the instability of the baseline makes the negative impact.The limit of detection was calculated as LoD =3.3 /a and the limit of quantitation as LoQ =10 /a ,where is the standard devia-tion of the response,a is the slope of the calibration curve [31].The lowest LoD and LoQ in ABTS post-column assay was of hyperoside 1.47M and 4.46M,respectively,while hyperoside in FRAP post-column assay accounted for 1.12M and 3.40M,respectively.The lowest LoD and LoQ in FRAP post-column assay was of quercetin 0.91M and 2.74M,respectively,while quercetin in ABTSR.Raudonis et al./J.Chromatogr.A1233 (2012) 8–1511 Table1Validation characteristics of ABTS and FRAP post-column assays.Reference compound LoD a(M)LoQ b(M)Intraday c RSD(%)Interday d RSD(%)Linear range(M)Calibration curve e R2(n)fABTS post-column assayTrolox 1.68 5.09 1.53 3.995–400y=6793.9x−7302.90.9998(7) Chlorogenic acid 2.29 6.94 3.21 6.845–285y=3394.4x−1735.10.9978(6) Caffeic acid 2.22 6.73 1.28 3.165–555y=6414.6x−6228.60.9994(6) Ellagic acid 3.289.94 1.18 4.565–200y=897.7x−2061.70.9994(6) Rutin 2.02 6.11 1.22 3.834–80y=3570.4x+1113.50.9994(6) Quercetin 1.55 4.69 2.68 5.304–330y=6471.9x+1917.30.9989(6) Quercitrin 1.83 5.56 2.11 4.73 5.5–110y=2106.8x+4838.50.9987(6) Isoquercitrin 2.377.18 2.41 4.97 5.5–110y=3625.4x+1267.10.9982(6) Hyperoside 1.47 4.46 1.63 3.07 2.75–110y=4842.7x+1178.40.9998(6) (+)-Catechin 1.71 4.93 1.35 4.114–410y=5503.1x+7823.60.9997(6) (−)-Epicatechin 2.287.19 1.42 4.395–550y=4484.2x+1193.70.9988(6) Epigallocatechin gallate 2.12 5.37 1.89 5.035–260y=10,190.0x−6715.20.9991(6) FRAP post-column assayTrolox 1.88 5.69 1.01 2.175–400y=10,984.1x+18,323.70.9997(7) Chlorogenic acid 2.17 6.58 2.59 5.785–340y=9165.2x−2426.80.9985(6) Caffeic acid 1.97 5.98 2.88 5.555–667y=13,244.8x−16,185.50.9978(6) Ellagic acid 1.20 3.63 2.65 4.99 2.5–400y=15,987.3x+96,401.20.9982(6) Rutin 1.21 3.660.47 2.102–160y=9894.6x−2812.10.9999(6) Quercetin0.91 2.74 1.60 3.672–330y=14,759.7x+65,949.30.9988(6) Quercitrin 1.01 3.070.33 2.41 2.75–220y=11,397.1x+2638.60.9999(6) Isoquercitrin 1.91 5.780.62 1.75 5.5–220y=10,879.4x+5093.30.9998(6) Hyperoside 1.12 3.400.94 3.12 2.75–220y=14,625.2x+16,564.50.9995(6) (+)-Catechin 1.08 3.260.90 2.582–410y=14,303.2x+16,836.90.9998(6) (−)-Epicatechin 1.82 5.510.88 1.935–550y=11,355.7x+41,959.40.9998(6) Epigallocatechin gallate 1.24 3.77 1.36 3.65 2.5–260y=16,987.1x−2621.70.9996(6)a Limit of detection.b Limit of quantitation.c Repeatability.d Intermediate precision.e In the calibration curve,x stands for the concentration of the antioxidant compound and y is the peak area.f Determination coefficient(data points in linear range).post-column assay accounted for1.55M and4.69M,respec-tively.Differences in sensitivity occur due to the affect of the baseline as explained hereinbefore.The LoD and LoQ values in Table1demonstrate that the ABTS and FRAP post-column assays proposed can be used for the evaluation of antioxidant active com-pounds as well as for their quantification according to TEAC.The linearity was tested by measuring the change of absorp-tion in both post-column assays of each antioxidant compound at known concentrations.Each measurement was repeated three times and the mean value was used for calculation of the regression line(Table1).The ABTS post-column assay lacked linearity for the higher concentrations of certain reference compounds.The linear-ity ranges can be expanded by increasing the ABTS concentration in the reactor.This ABTS assay usesfixed concentrations of ABTS radical cation(35M)and therefore greater concentrations of com-pounds reach the limits of detection.All12antioxidant compounds showed significant(p<0.0001)linear regression with a determina-tion coefficient higher than0.99for both post-column assays.The ABTS and FRAP post-column assays were fully linear over the con-centration range that were tested.The data obtained are presented in Table1.3.2.Analysis of reference compound antioxidant activityIn order to compare the efficacy of antioxidant activity eval-uation using ABTS and FRAP post-column assays,12samples of antioxidant compound were analyzed.The TEAC rel values of reference compounds from both of the post-column assays are presented in Table2.Various studies have determined that antioxidant activity of active compounds is pH dependent[36–39].Experiments by Lemanska et al.show that the radical scavenging ability increases with the increasing pH values[39].The ionization potential decreases with the increasing pH values,which reflects the higher electron-donating capacity with deprotonation[8].FRAP assay is established in pH of3.6.Therefore in order to compare the antiox-idant activity of the tested compounds ABTS assay was performed at same pH.Consequently,all the reference compounds in ABTS post-column assay possess lower radical scavenging activity than Trolox,except epigallocatechin gallate(TEAC rel1.50±0.07).Sci-entific studies propose thatflavanols andflavonols have greater activity compared to Trolox[16,22,37,39,11].Inconsistent results may occur due to different experimental pH values.In order to evaluate the radical scavenging and ferric reducing ability of antioxidant compound ABTS/FRAP TEAC rel ratio(A/F ratio) was estimated.If the ratio of sample antioxidant compound is less than1,the compound possesses greater ferric reducing abilities; if the ratio is greater than1,radical scavenging abilities are more expressed(Table2).Our results demonstrate that quercetin possesses greater rad-ical scavenging activity(TEAC rel0.95in ABTS post-column assay)Table2Comparison of relative Trolox equivalent antioxidant capacities(TEAC rel)of refer-ence compounds in ABTS and FRAP post-column assays.Reference compound TEAC rel in ABTS TEAC rel in FRAP A/F ratio aTrolox 1.00 1.00 1.00 Chlorogenic acid0.42±0.030.83±0.050.50Caffeic acid0.83±0.06 1.21±0.060.69Ellagic acid0.13±0.01 1.46±0.080.09Rutin0.53±0.040.90±0.050.58 Quercetin0.95±0.07 1.34±0.070.71 Quercitrin0.31±0.01 1.04±0.060.30 Isoquercitrin0.53±0.020.99±0.040.54 Hyperoside0.71±0.04 1.33±0.060.54(+)-Catechin0.81±0.05 1.30±0.050.62(−)-Epicatechin0.66±0.03 1.03±0.050.64 Epigallocatechin gallate 1.50±0.07 1.55±0.080.97a Ratio of TEACrelvalue in ABTS post-column assay and TEAC rel value in FRAP post-column assay.12R.Raudonis et al./J.Chromatogr.A 1233 (2012) 8–15bined chromatograms of F.viridis (1),F.vesca (2)and F.moschata (3)leaf extracts:chromatographic elution (a)and post-column reaction with ABTS (b)or FRAP (c)reagents.Numbers refer to identified antioxidant compounds:1–(+)-catechin,3–(−)-epicatechin,5,6,11–quercetin derivatives,7–ellagic acid,8–epigallocatechin gallate,9–hyperoside,10–isoquercitrin.and reduction power (TEAC rel 1.34in FRAP post-column assay)than its derivatives (rutin,quercitrin,isoquercitrin,hyperoside (Table 2))in both ABTS and FRAP post-column assays.This is in agreement with other scientific studies,showing that sugar moi-ety reduces the activity [22,40].Activity of quercetin derivatives analyzed with ABTS post-column assay had the following rank order:quercetin >hyperoside >isoquercitrin ≈rutin >quercitrin.In FRAP post-column the rank order is slightly different:quercetin >hyperoside >quercitrin >isoquercitrin >rutin.Iso-quercitrin and hyperoside have the same ABTS/FRAP TEAC rel ratio (0.54),but different TEAC rel values (p <0.05)in ABTS and FRAP post-column assays (0.53±0.02,0.71±0.04in ABTS and 0.99±0.04,1.33±0.06in FRAP,respectively).The ratio demon-strates that radical scavenging properties and ferric reducing abilities of both glycosides are proportional,while quercetin with galactosyde moiety (hyperoside)is more active in both assays.Antioxidant activity of quercetin (flavonol)has been confirmed in many studies [15,34,41,42]and its structure–activity relationship has been determined [34].The high activity of flavonols with catethol group can be explained by the bielectronic oxidation and formation of two highly stable quinonic structures [11].Flavanol compound,epigallocatechin gallate,has strong fer-ric reducing power (TEAC rel 1.55±0.08).This compound was determined as the most active in both assays.Other flavanols –(+)-catechin and (−)-epicatechin have lower TEAC rel (in both assays)than flavonol quercetin due to the lack of 2,3-double bond present in quercetin.Hydroxyl groups at C3,2,3-double bond and 4-oxo group are necessary for the antioxidant activity [14,15].These structural peculiarities are essential for both radical scavenging activity [14]and reducing activity [11].Ellagic acid was determined as a weak radical scavenger in our study (TEAC rel 0.13±0.01),but it possesses a strong ferric reducing ability –TEAC rel 1.46±0.08.Literature data on ellagic acid TEAC rel values in ABTS and FRAP systems are scarce.Most commonly the content of ellagic acid is being correlated with total antioxidant activity of complex extract test in ABTS,DPPH or FRAP systems [25,43–45].3.3.Analysis of antioxidant activity of Fragaria speciesEthanolic extracts of F.viridis , F.vesca , F.moschata leaves and fruits were analyzed in order to evaluate the efficacy of antioxidant determination using both ABTS and FRAP post-column assays.To the best of our knowledge the antioxidative activity of raw materials of F.viridis and F.moschata was ana-lyzed for the first time.In Fragaria leaf extracts six phenolic compounds –(+)-catechin,(−)-epicatechin,ellagic acid,epigal-locatechin gallate,hyperoside,isoquercitrin and three quercetin derivatives were identified (Fig.2).In Fragaria fruit extracts,besides previously mentioned compounds,two anthocyanins –cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside were determined (Fig.3).TEAC (mol/g)values of principal com-pounds and total of all quantitated compounds of F.viridis ,F.vesca and F.moschata leaf and fruit extracts were assessed and presented in Table 3.The results demonstrate that Fragaria leaf extracts possesses stronger antioxidant properties (range of total TEAC values 191.23–609.36mol/g and 178.63–642.20mol/g of ABTS and FRAP respectively)than Fragaria fruit extracts (8.24–25.11mol/g and 10.82–24.82mol/g of ABTS and FRAP respectively).Greater TEAC values represent the greater amounts of bioactive compounds.The main qualitative differences between leaves and fruits of Fragaria species are the anthocyanins.Pelargonidin and cyanidin glycosides are regarded as antiox-idant active compounds [46].In our study these compounds possessed lower activities due to low pH of the experimental medium.F.viridis (609.36mol/g and 642.20mol/g of ABTS and FRAP respectively)leaf extracts were the most active (p <0.05)among all the investigated species.F.vesca fruit extracts (18.05mol/g and 18.36mol/g of ABTS and FRAP respectively)showed signif-icantly (p <0.05)higher activity when compared to F.viridis and F.moschata .The calculated TEAC values of bioactive compounds in Fragaria leaf and fruit extracts confirmed that epigallocatechin gallate was the predominant radical scavenger and ferric reducer (Table 3).。
总抗氧化能力测定FRAP法
小麦叶片总抗氧化能力测定（ F R A P 法）一、实验原理FRAP 法测定总抗氧化能力的原理是酸性条件下抗氧化物可以还原Ferric-tri-pyridyl-tria-zine（Fe 3+-TPTZ）产生蓝色的Fe2+-TPTZ,随后在593nm测定蓝色的Fe2+-TPTZ即可获得样品中的总抗氧化能力。
由于反应在酸性条件下进行，可以抑制内源性的一些干扰因素。
并且由于血浆等样品中的铁离子或亚铁离子的总浓度通常低于10 yM,因此血浆等样品中的铁离子或亚铁离子不会显着干扰FRAP法的检测反应。
由于反应体系中的铁离子或亚铁离子是和TPTZ螯合的，样品本身含有的少量金属离子螯合剂通常也不会显着影响检测反应。
AntioxidantFe3+-TPTZ （橘黄色） ------------- > Fe2+-TPTZ （蓝色）二、实验步骤1.FRAP工作液配制：0.3 MpH3.6醋酸缓冲液：0.364g无水醋酸钠+3.2mL冰乙酸定容至200mL用1MHCI 调节pH至3.6 ;10mmol/L TPTZ溶液25mL 0.078g TPTZ 用40mM盐酸溶液定容至25mL20mmol/L FeCh溶液50mL 2.78g 用RO水定容至50mL上述溶液以10:1:1 的比例混合（现配现用）。
2.取叶片0.1g，加入2.5mL蒸馏水研磨稍沉淀后取1.5mL 12000g离心10min （4o C）,取上清液。
3.在反应管中加入100uL上清液，再加入2.4mL工作液，37°C条件下水浴10min,于593nm 处测定吸光度，4.标准曲线绘制：以0.1-1.6mmol/L的FeSO的标准液替代样品绘制标准曲线。
三、结果计算以1.0mmol/L的FeSQ为标准，样品抗氧化活性以达到同样吸光度值为一个FRAP fi,计算结果Z82 Ferric Reducmg/Antioxidant Power (FRAP) assay Briefly, the FRAP reagent contained 2.5 mL 10 mM L1 TPTZ solution in 40 mlVI L1 HGI plus 2,5 mL 20 mM L1 FeCI3 and 25 mL 0.3 M L1acetate buffer, pH 3.6 was prepared freshly and warmed at 37c C. After addition of root ethanol extracts (prepared as in section 2.7) and incub^tion at 37°C for5 absorbanee of the reactionmixture was measured at 593 nm. The final result was expressed as the concentration of antioxidants having a ferric reducing ability equivalent to that of 1 mM L_1 FeSO4 based on the standard curve for FeSO4 x 7H2O at a concentration range between 100 and 1000 uM L_1[31].[1]Be nzie IF F, Strain J J. The Ferric Reduci ng Ability of Plasma (FRAP) as a Measure of “ An tioxida ntPower” ： The FRAP Assay[J]. Analytical Biochemistry, 1996, 239(1):70 -6.[2]Katarz yna Szafra n ska, Rafa? Szewczyk, Krysty na Maria Jan as. In volveme nt of melato nin applied toVigna radiata, L. seeds in pla nt resp onse to chilli ng stress[J]. Cen tral Europea n Jour nal of Biology, 2014,9(11):1117-1126.。
farp抗氧化标准曲线y=
farp抗氧化标准曲线y=
FRAP抗氧化标准曲线的y值是通过测量一系列不同浓度的FeSO4的吸光度得到的。
这个过程包括，首先，吸取0.1、0.2、0.4、0.6、0.8和1.0mmol/L的FeSO4标准液，然后加入3ml FRAP工作液和0.3ml 超纯水，混匀后准确反应5min。
然后在593nm处测定其吸光度，并用超纯水调零。
通过这些步骤，就可以得到一个浓度-吸光值曲线，该曲线的数学模型通常为y=3.9242x-0.0006。
总抗氧化能力是以从标准曲线上获得的抗氧化剂Trolox的量来表示样本的总抗氧化能力。
计算公式如下： y = 2.4832x + 0.0134, R2 = 0.9996。
这里的x代表的是Trolox的浓度(μmol/mL)，y代表的是吸光值差值A。
这个公式可以用来计算样本的总抗氧化能力，根据需要，可以按样本质量或样本蛋白浓度来计算。
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
小麦叶片总抗氧化能力测定（F R A P法）
一、实验原理
FRAP法测定总抗氧化能力的原理是酸性条件下抗氧化物可以还原
Ferric-tri-pyridyl-tria-zine(Fe3+-TPTZ)产生蓝色的Fe2+-TPTZ，随后在593nm测定蓝色的Fe2+-TPTZ即可获得样品中的总抗氧化能力。
由于反应在酸性条件下进行，可以抑制内源性的一些干扰因素。
并且由于血浆等样品中的铁离子或亚铁离子的总浓度通常低于10μM，因此血浆等样品中的铁离子或亚铁离子不会显着干扰FRAP法的检测反应。
由于反应体系中的铁离子或亚铁离子是和TPTZ螯合的，样品本身含有的少量金属离子螯合剂通常也不会显着影响检测反应。
Antioxidant
Fe3+-TPTZ（橘黄色）——————> Fe2+-TPTZ (蓝色)
二、实验步骤
1.FRAP工作液配制：
0.3 M pH 3.6 醋酸缓冲液：0.364g无水醋酸钠+3.2mL冰乙酸定容至200mL，用1M HCl
调节pH至3.6；
10mmol/L TPTZ溶液25mL：0.078g TPTZ用40mM 盐酸溶液定容至25mL；
20mmol/L FeCl
溶液50mL：2.78g用RO水定容至50mL；
3
上述溶液以10:1:1的比例混合（现配现用）。
2.取叶片0.1g，加入2.5mL蒸馏水研磨稍沉淀后取1.5mL 12000g离心10min（4o C），取上
清液。
3.在反应管中加入100uL上清液，再加入2.4mL工作液，37o C条件下水浴10min，于593nm
处测定吸光度，
4.标准曲线绘制：以0.1-1.6mmol/L的FeSO
的标准液替代样品绘制标准曲线。
4
三、结果计算
以1.0mmol/L的FeSO
为标准，样品抗氧化活性以达到同样吸光度值为一个FRAP值，计
4
算结果。
[1]Benzie I F F, Strain J J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay[J]. Analytical Biochemistry, 1996, 239(1):70-6.
[2]Katarzyna Szafrańska, Rafa? Szewczyk, Krystyna Maria Janas. Involvement of melatonin applied to Vigna radiata, L. seeds in plant response to chilling stress[J]. Central European Journal of Biology, 2014,
9(11):1117-1126.。