Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine:: CUPRAC method
Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine:: CUPRAC method
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DOI：
10.1021/jf048741x
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发表时间：
2004-12-29
影响因子：
6.1
通讯作者：
Karademir, SE
中科院分区：
文献类型：
--
作者：
Apak, R;Güçlu, K;Karademir, SE
抗氧化剂的化学多样性使得难以从蔬菜基质中分离和定量抗氧化剂。因此，需要建立一种可以直接从植物提取物中测量总抗氧化活性水平的方法。目前的文献明确指出，由于缺乏标准的定量方法，没有“总抗氧化剂”作为食品标签的营养指标。因此，这项工作报告的发展，一个简单的，广泛适用的抗氧化能力指数的膳食多酚和维生素C和E，利用铜（II）-新亚铜灵[Cu（II）-NC]试剂作为显色氧化剂。因为测量多酚的铜（II）（或二价铜）离子还原能力，所以该方法被我们的研究小组命名为“二价铜还原抗氧化能力”，缩写为CUPRAC方法。这种方法应优于铁还原抗氧化能力（FRAP）的方法，因为铜（II）的氧化还原化学-而不是铁离子-涉及更快的动力学。该方法包括将抗氧化剂溶液（直接或酸水解后）与氯化铜（II）溶液、新亚铜灵醇溶液和乙酸铵缓冲水溶液在pH 7下反应，并随后在30分钟后测量450 nm处的显色吸光度。由于化合物如抗坏血酸、没食子酸和槲皮素的显色快，但柚苷和柚皮素的显色慢，在50 ℃水浴上温育20分钟后[加入Cu（II）-Nc试剂后]测定后一种化合物，以促使氧化反应完成。在1.2 M盐酸中，含50%甲醇的乙醇中，黄酮苷水解为相应的糖苷配基，从而对Cu（II）-Nc发挥最大的还原力。某些化合物在酸水解后还需要温育以充分显示其还原能力。抗氧化剂的合成混合物的CUPRAC抗氧化能力的实验测量作为Trolox当量，并与理论上发现的那些进行比较，通过利用加和性的原理，假设混合物成分之间没有化学相互作用。由于抗坏血酸不耐高温孵育，因此最初应通过在加入Cu（II）-Nc试剂1 min结束时测量原始溶液和加入抗坏血酸氧化酶的混合溶液的吸光度（450 nm处）差异进行测定。因此，含有各种抗氧化剂的混合物的总CUPRAC抗氧化能力应该是在水解和孵育程序的合适组合之后最终测量的，加上由于抗坏血酸盐而最初测量的能力。测试的抗氧化剂多酚化合物表明，在CUPRAC方法中观察到表儿茶素没食子酸酯、表没食子儿茶素没食子酸酯、槲皮素、非瑟酮、表没食子儿茶素、儿茶素和咖啡酸的最高能力，这与理论预期一致，因为羟基的数量和位置以及整个分子的共轭程度是重要的。类黄酮的抗氧化效力几乎与-OH基团的总数成比例，并且受到B环中存在的o-二羟基部分的积极影响。β-胡萝卜素，它不与CUPRAC试剂在酒精水介质中反应，可以在二氯甲烷solvent.Linear校准曲线抗坏血酸和黄酮类化合物的合成溶液中含有的抗氧化剂的混合物，也在真实的矩阵，如葡萄和橙子汁，绿色茶，黑莓茶，显示出初始非零吸光度与CUPRAC试剂。在一个给定的复杂的矩阵中的纯化合物的线性校准曲线的平行性有效地表明，有溶液成分之间没有明显的化学相互作用，测试的抗氧化剂的抗氧化能力是加和的。与FRAP方法不同，CUPRAC试剂具有合理的选择性、稳定性、易于获得且对硫醇型氧化剂敏感。该反应在接近生理pH下进行，而不是FRAP的不切实际的酸性pH。
The chemical diversity of antioxidants makes it difficult to separate and quantify antioxidants from the vegetable matrix. Therefore, it is desirable to establish a method that can measure the total antioxidant activity level directly from vegetable extracts. The current literature clearly states that there is no "total antioxidant" as a nutritional index available for food labeling because of the lack of standard quantitation methods. Thus, this work reports the development of a simple, widely applicable antioxidant capacity index for dietary polyphenols and vitamins C and E, utilizing the copper(II)-neocuproine [Cu(II)-Nc] reagent as the chromogenic oxidizing agent. Because the copper(II) (or cupric) ion reducing ability of polyphenols is measured, the method is named by our research group "cupric reducing antioxidant capacity" abbreviated as the CUPRAC method. This method should be advantageous over the ferric reducing antioxidant power (FRAP) method because the redox chemistry of copper(II)-as opposed to that of ferric ion-involves faster kinetics. The method comprises mixing of the antioxidant solution (directly or after acid hydrolysis) with a copper(II) chloride solution, a neocuproine alcoholic solution, and an ammonium acetate aqueous buffer at pH 7 and subsequent measurement of the developed absorbance at 450 nm after 30 min. Because the color development is fast for compounds such as ascorbic acid, gallic acid, and quercetin but slow for naringin and naringenin, the latter compounds were assayed after incubation at 50degreesC on a water bath for 20 min [after Cu(II)-Nc reagent addition] so as to force the oxidation reaction to reach completion. The flavonoid glycosides were hydrolyzed to their corresponding aglycons by refluxing in 1.2 M HCl-containing 50% MeOH so as to exert maximal reducing power toward Cu(II)-Nc. Certain compounds also needed incubation after acid hydrolysis to fully exhibit their reducing capability. The CUPRAC antioxidant capacities of synthetic mixtures of antioxidants were experimentally measured as Trolox equivalents and compared to those theoretically found by making use of the principle of additivity of absorbances assuming no chemical interaction between the mixture constituents. Because ascorbic acid is not resistant to elevated temperature incubation, it should be assayed initially by measuring the absorbance (at 450 nm) difference of original and ascorbate oxidase-added mixture solutions at the end of 1 min of Cu(II)-Nc reagent addition. Thus, the total CUPRAC antioxidant capacity of a mixture containing various antioxidants should be that finally measured after a suitable combination of hydrolysis and incubation procedures, added to the initially measured capacity due to ascorbate. The antioxidant polyphenolic compounds tested demonstrate that the highest capacities in the CUPRAC method were observed for epicatechin gallate, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, and caffeic acid in this order, in accordance with theoretical expectations, because the number and position of the hydroxyl groups as well as the degree of conjugation of the whole molecule are important. The antioxidant potency of flavonoids is nearly proportional to the total number of -OH groups and is positively affected by the presence of an o-dihydroxy moiety in the B-ring. P-Carotene, which did not react with the CUPRAC reagent in alcoholic aqueous medium, could be assayed in dichloromethane solvent.Linear calibration curves for ascorbic acid and flavonoids were redrawn in synthetic solutions containing a mixture of antioxidants, and also in real matrices such as grape and orange juices, green tea, and blackberry tea, showing an initial nonzero absorbance with the CUPRAC reagent. The parallellism of the linear calibration curves of pure compounds in a given complex matrix effectively demonstrated that there were no interferent chemical interactions among the solution constituents and that the antioxidant capacities of the tested antioxidants were additive. The CUPRAC reagent is reasonably selective, stable, easily accessible, and sensitive toward thiol-type oxidants, unlike the FRAP method. The reaction is carried out at nearly physiological pH as opposed to the unrealistic acidic pH of FRAP.