Effect of antioxidant oxidation potential in the oxygen radical absorption capacity (ORAC) assay (original) (raw)
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Oxygen-radical absorbance capacity assay for antioxidants
Free Radical Biology and Medicine, 1993
A relatively simple but sensitive and reliable method ofquantitating the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a few #l is described. In this assay system, ~-phycoerythrin (~-PE) is used as an indicator protein, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble vitamin E analogue) as a control standard. Results are expressed as ORAC units, where 10RAC unit equals the net protection produced by l ~M Trolox. The uniqueness of this assay is that total antioxidant capacity of a sample is estimated by taking the oxidation reaction to completion. At this point all of the nonprotein antioxidants (which include a-tocopherol, vitamin C, E-carotene, uric acid, and bilirubin) and most of the albumin in the sample are oxidized by the peroxyl radical. Results are quantified by measuring the protection produced by antioxidants. This solves many problems associated with kinetics or lag-time measurements. A linear correlation of ORAC value with concentration of serum, Trolox, vitamin C, uric acid, and bovine albumin is demonstrated. The coefficient of variation within a run is found to be about 2% and from run to run about 5%. Trolox, a-tocopherol, vitamin C,/3-carotene, uric acid, and bilirubin completely protect B-PE from oxidation, while bovine albumin protects/3-PE only partially. On a molar basis, the relative peroxyl radical absorbance capacity of Trolox, a-tocopherol acid succinate, uric acid, bilirubin, and vitamin C is l : 1 : 0.92 : 0.84 : 0.52. Bovine albumin per unit weight has a lower peroxyi absorbing capacity than these antioxidants. However, the serum protein fraction, containing some lipid-soluble antioxidants, represents the major contributor to the ORAC value found in whole serum. The minimum amount of vitamin C and uric acid which could still be detectable when added to a serum supernatant fraction is 1.5/~g and 0.59 #g, respectively, which account for about 1% of the total ORAC value of the serum supernatant fraction.
Processing and Impact on Antioxidants in Beverages, 2014
The potential activity of mango by-products to inhibit the peroxyl radical-mediated oxidation of tryptophan residues (Trp) of myofibrillar proteins (MP) was studied. Extracts of seeds, peels and pulps were studied in terms of their antioxidant activity, assessed by the oxygen radical absorbance capacity (ORAC) assay employing fluorescein and pyrogallol red as probes (ORAC-FL and ORAC-PGR, respectively). The effect of the extracts of mango by-products on the oxidation of Trp was evaluated by fluorescence spectroscopy. Data obtained employing the ORAC-FL assay showed a low discrimination of the antioxidant activity of the samples (Peel ≈ Seed > Pulp). However, employing the ORAC-PGR assay it was possible to differentiate the antioxidant activity of the samples (Seed > Peel >> Pulp). Extracts obtained from mango seed, peel, and pulp protected Trp of MP when the latter was exposed to AAPH (2,2′-azo-bis(2-amidinopropane) dihydrochloride)-derived free radicals. Such effect was in good agreement with the ORAC-PGR data, showing that this assay could be employed to predict the protection of Trp residues of MP.
Journal of Food Composition and Analysis, 2013
A novel ORAC (oxygen radical absorbance capacity) assay to assess antioxidant capacity of phenolic compounds in near-gastric conditions (pH 2.0) is presented. AAPH (2,2 0 -azo-bis(2-amidinopropane)dihydrochloride) was used as peroxyl radicals source, and fluorescein, pyranine and pyrogallol red were employed as target molecules. Only pyrogallol red (PGR) showed a behavior compatible with an ORAC assay under acidic conditions (ORAC-PGR a ). Excepting Trolox and ascorbic acid, phenolic compounds protected PGR, giving kinetic profiles without the presence of an induction time. ORAC-PGR a values, which reflect the reactivity of the antioxidants toward peroxyl radicals, ranged from 0.2 (caffeic acid) to 29.1 (myricetin) gallic acid equivalents. The ORAC-PGR a method showed analytical parameters in agreement with other ORAC-like assays and was applied to wines, teas, commercial juices and herb infusions, peach juice being the sample with the highest ORAC-PGR a value (7.1 mM gallic acid equivalents). In addition, ascorbic acid concentration in complex mixtures can be determined from kinetic profiles. ß
An improved method of oxygen radical absorbance capacity (ORAC) assay has been developed and validated using fluorescein (3′,6′-dihydroxyspiro[isobenzofuran-1[3H],9′[9H]-xanthen]-3-one) as the fluorescent probe. Our results demonstrate that fluorescein (FL) is superior to B-phycoerythrin. The oxidized FL products induced by peroxyl radical were identified by LC/MS, and the reaction mechanism was determined to follow a classic hydrogen atom transfer mechanism. In addition, methodological and mechanistic comparison of ORAC FL with other widely used methods was discussed. It is concluded that, unlike other popular methods, the improved ORAC FL assay provides a direct measure of hydrophilic chain-breaking antioxidant capacity against peroxyl radical.
Analytical Biochemistry, 2006
Few methods for assessing total antioxidant capacity (TAC) include both the percentage of inhibition and the length of inhibition in the measurement. Available methods require above ambient constant temperature incubation, reaction preheating, and/or separate assays for testing hydrophilic and hydrophobic samples. We describe a high-throughput method, antioxidant inhibition of oxygen radicals (AIOR), that overcomes these difficulties. AIOR uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule, uroporphyrin I, which is delayed by the presence of antioxidants. The area under the curve is measured by a fluorescence spectrophotometer in a 96-well microplate format, and TAC results are expressed as millimole/liter Trolox equivalents. AIOR is performed at ambient temperature and is applicable to samples in either aqueous or common organic solvents. The reaction between uroporphyrin I and the peroxyl radicals generated from 2,2 0 -azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be of first-order kinetics with a mean rate constant (k) of 0.0254. Applications to measure antioxidant capacity are demonstrated on individual chemicals and biological samples. The method has good linearity, within-and between-assay precision, and recovery. Crown
Methods available for the measurement of antioxidant capacity are reviewed, presenting the general chemistry underlying the assays, the types of molecules detected, and the most important advantages and shortcomings of each method. This overview provides a basis and rationale for developing standardized antioxidant capacity methods for the food, nutraceutical, and dietary supplement industries. From evaluation of data presented at the First International Congress on Antioxidant Methods in 2004 and in the literature, as well as consideration of potential end uses of antioxidants, it is proposed that procedures and applications for three assays be considered for standardization: the oxygen radical absorbance capacity (ORAC) assay, the Folin-Ciocalteu method, and possibly the Trolox equivalent antioxidant capacity (TEAC) assay. ORAC represent a hydrogen atom transfer (HAT) reaction mechanism, which is most relevant to human biology. The Folin-Ciocalteu method is an electron transfer (ET) based assay and gives reducing capacity, which has normally been expressed as phenolic contents. The TEAC assay represents a second ET-based method. Other assays may need to be considered in the future as more is learned about some of the other radical sources and their importance to human biology. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.
Methods for the Determination of Antioxidant Capacity in Food and Raw Materials
A comprehensive description of the most frequently used methods to determine the antioxidant activity in food and raw materials is given. The methods are classified into two categories, depending on the type of the assessment carried out. Several methods for the assessment of antioxidant efficacy using free radical scavenging such as Oxygen Radical Absorbance Capacity Assay (ORAC), Total Radical Trapping Antioxidant Parameter assay (TEAC), Ferric reducing antioxidant power assay (FRAP) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay are described. An example of methods based on the assessment of antioxidant efficacy using significant biological substrates is also presented. Critical opinions concerning the proposed methods are presented.
Journal of Agricultural and Food Chemistry, 2008
A simple oxygraphic method, for which the theoretical and experimental bases have been recently revised, has been successfully applied to evaluate the peroxyl radical chain-breaking characteristics of some typical food antioxidants in micelle systems, among which is a system that reproduces conditions present in the upper part of the digestive tract, where the absorption and digestion of lipids occur. This method permits one to obtain from a single experimental run the peroxyl radical trapping capacity (PRTC, that is, the number of moles of peroxyl radicals trapped by a given amount of food), the peroxyl radical trapping efficiency (PRTE, that is, the reciprocal of the amount of food that reduces to half the steady-state concentration of peroxyl radicals), and the half-life of the antioxidant (t 1/2 ) when only a small fraction of peroxyl radicals reacts with the antioxidants present in foods. Examples of application of the method to various types of foodstuffs have been reported, assessing the general validity of the method in the simple and fast evaluation of the above-reported fundamental antioxidant characteristics of foods.
Journal of Agricultural and Food Chemistry, 2003
Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC FL ) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37°C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 ( 1.5 and 38.2 ( 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples.
The Chemistry behind Antioxidant Capacity Assays
Journal of Agricultural and Food Chemistry, 2005
This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays.