It would be desirable to establish and standardize methods that can measure the total antioxidant capacity level directly from vegetable extracts containing phenolics. Antioxidant capacity assays may be broadly classified as electron transfer (ET)− and hydrogen atom transfer (HAT)−based assays. The majority of HAT assays are kineticsbased, and involve a competitive reaction scheme in which antioxidant and substrate compete for peroxyl radicals thermally generated through the decomposition of azo compounds. ET−based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes colour when reduced. ET assays include the ABTS/TEAC, CUPRAC, DPPH, Folin-Ciocalteu and FRAP methods, each using different chromogenic redox reagents with different standard potentials. This review intends to offer a critical evaluation of existing antioxidant assays applied to phenolics, and reports the development by our research group of a simple and low-cost antioxidant capacity assay for dietary polyphenols, vitamins C and E, and human serum antioxidants, utilizing the copper(II)-neocuproine reagent as the chromogenic oxidizing agent, which we haved named the CUPRAC (cupric ion reducing antioxidant capacity) method. This method offers distinct advantages over other ET−based assays, namely the selection of working pH at Molecules 2007, 12 1497 physiological pH (as opposed to the Folin and FRAP methods, which work at alkaline and acidic pHs, respectively), applicability to both hydrophilic and lipophilic antioxidants (unlike Folin and DPPH), completion of the redox reactions for most common flavonoids (unlike FRAP), selective oxidation of antioxidant compounds without affecting sugars and citric acid commonly contained in foodstuffs and the capability to assay -SH bearing antioxidants (unlike FRAP). Other similar ET-based antioxidant assays that we have developed or modified for phenolics are the Fe(III)− and Ce(IV)−reducing capacity methods.

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The Folin-Ciocalteu (FC) method of performing a total phenolics assay, originally developed for protein determination, has recently evolved as a total antioxidant capacity assay but was found to be incapable of measuring lipophilic antioxidants due to the high affinity of the FC chromophore, that is, multivalent-charged phospho-tungsto-molybdate(V), toward water. Thus, the FC method was modified and standardized so as to enable simultaneous measurement of lipophilic and hydrophilic antioxidants in NaOH-added isobutanol-water medium. Optimal conditions were as follows: dilution ratio of aqueous FC reagent with iso-BuOH (1:2, v/v), final NaOH concentration of 3.5 × 10(-2) M, reaction time of 20 min, and maximum absorption wavelength of 665 nm. The modified procedure was successfully applied to the total antioxidant capacity assay of trolox, quercetin, ascorbic acid, gallic acid, catechin, caffeic acid, ferulic acid, rosmarinic acid, glutathione, and cysteine, as well as of lipophilic antioxidants such as α-tocopherol (vitamin E), butylated hydroxyanisole, butylated hydroxytoluene, tertiary butylhydroquinone, lauryl gallate, and β-carotene. The modified FC method reliably quantified ascorbic acid, whereas the conventional method could not. The modified method was reproducible and additive in terms of total antioxidant capacity values of constituents of complex mixtures such as olive oil extract and herbal tea infusion. The trolox equivalent antioxidant capacities of the tested antioxidant compounds correlated well with those found by the Cupric Reducing Antioxidant Capacity reference method.

Since antioxidants are health-beneficial compounds capable of removing reactive species, assay of total antioxidant capacity (TAC) by simple and low-cost methods is important. The magenta-coloured iron(II)-ferrozine (Fe(II)-FZ) complex showing an absorbance maximum at 562 nm has previously been utilized for iron-binding assays, but not for antioxidant determination. Ferrozine is a highly ferrousstabilizing ligand such that ferric ion in the presence of ferrozine easily oxidizes antioxidants and is itself reduced to Fe(II)-FZ, yielding a very high molar absorptivity and thus enhanced sensitivity for most antioxidants. The hierarchic order of antioxidant power for common antioxidants was in accordance with known structure-activity relationships. The Fe(III)-FZ assay was applied to synthetic antioxidant mixtures to yield additive absorbance values, which is a prerequisite for precise determination of antioxidant capacity of complex mixtures. The calibration curves (lines) of trolox and quercetin individually and in herbal infusions-by using the method of standard additions-were parallel, confirming that the herbal antioxidants and trolox did not chemically interact among each other so as to cause apparent deviations from Beer's law. The proposed method was applied to medicinal plant infusions for total antioxidant capacity assay as trolox-equivalents, and the results were compared to those found with CUPRAC (cupric reducing antioxidant capacity), FRAP (ferric reducing antioxidant power) and Folin total phenols assays, the highest correlation being achieved with CUPRAC. In short, a novel ferric reducing assay for food antioxidants was introduced, which was superior to FRAP in regard to its realistic pH, enhanced sensitivity, faster kinetics, and absence of free Fe(II)-which can cause Fenton-type oxidations-in the reaction products.

The existing ferricyanide/Prussian blue assay of reducing capacity measurement was optimized so as to obtain a more reproducible, linear and additive response from antioxidants. The modification involved the simultaneous use of ferricyanide and iron(III) to regulate more favorable redox conditions for a greater variety of antioxidants. Prussian blue precipitation was hindered with the addition of sodium dodecyl sulfate, and the optimal pH was adjusted to 1.7 to maintain the redox activity of ferric ion while preventing its hydrolysis. Incubation of the reaction mixture at room temperature for 30 min enabled more complete oxidations than observed in the conventional ferricyanide method. The order of trolox equivalent antioxidant capacities was quercetin > rosmarinic acid > gallic acid > ferulic acid ≥ catechin > caffeic acid ≥ rutin ≥ ascorbic acid ≈ trolox. Synthetic antioxidant mixtures gave the theoretically expected total antioxidant capacities conforming to Beer's law. The assay was nonresponsive to simple sugars and citric acid (which are not true antioxidants) but responsive to biologically important thiols which are not oxidized by other Fe(III)-based assays. The assay was used in real sample solutions by using the method of standard additions to green tea, nettle, and sage, and validated against other similar antioxidant assays.

This study aims to investigate the total antioxidant capacity (TAC) as trolox equivalent (mmol g as a potential source of natural antioxidants to be incorporated in current diets to protect human health. CUPRAC method proved to be most effective among electron-transfer based TAC assays since it responded to a wide variety of hydrophilic and lipophilic antioxidants.-Keywords: ayvalik food plants, antioxidant capacity, CUPRAC (cupric reducing antioxidant capacity) assay *To whom correspondence should be addressed. Email: rapak@istanbul.edu.tr IntroductionThe quality of food has increased by using antioxidants which have prevented or delayed their oxidative deterioration during processing and storage (Cosio et al., 2006). Wild plants, vegetables and fruits have attracted much attention as sources of natural antioxidants. Many of the wild plants, vegetables and fruits contain antioxidants such as vitamins (β-carotene, vitamins C and E), and polyphenols (flavonoids, tannins, catechins) (Wong et al., 2006). Epidemiological studies have shown that a diet rich in vegetables and fruits can reduce the incidence of cardiovascular diseases and of certain cancers (Ames, 1983;Block et al., 1992).Ayvalik is located in the northwestern part of Anatolia (Turkey) where the use of wild edible plants is a practice that has developed over generations and is part of the local traditional knowledge system. The Maritime climate dominates in Ayvalik where the summers are hot and dry while the winters tepid and rainy (Temucin, 1993). The sub-humid climate around Ayvalik enables the widest distribution of plant species. The inhabitants in Ayvalik consume the aerial parts of various food plants listed in Table 1 with botanic, English, and local names. Esiyok et al. (2004) have reported a brief overview of important herbs in the Turkish flora, including fennel, mallow, chicory, nettle, wild radish, and wild mustard, as important food plants for promoting human health and preventing cancer.Total antioxidant capacity may better reflect the health beneficial quality of foods than individual measurements due to the possible cooperative action of antioxidants (Ghiselli et al., 2000). The aim of this study is to investigate the total antioxidant capacity as trolox equivalent (mmol g -1 ) of nine- et al., 2004; 2005; 2006; 2008), ABTS/persulfate (Re et al., 1999), FRAP (Benzie andStrain, 1996) and Folin (Singleton et al., 1999) assays. Additionally, three antioxidant assay methods with the exception of Folin were used to determine the antioxidant capacity of F. vulgare for the first time. Materials and MethodsMaterials Neocuproine and Folin-Ciocalteou reagent were purchased from Sigma Chemical Co. (Sigma-Aldrich GmbH, Steinheim, Germany). Trolox was obtained from Aldrich Chemicals Co. (Sigma-Aldrich GmbH, Steinheim, Germany). Ammonium acetate, copper (II) chloride, potassium persulfate, hydrochloric acid, sodium hydroxide, copper (II) sulfate, sodium carbonate, sodium potassium tartarate, glacial acetic acid, sodium acetate trihydrate, ferric c...

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