Trolox®, antioxidant

The total antioxidant activity of teas and tea polyphenols in aqueous phase oxidation reactions has been deterrnined using an assay based on oxidation of 2,2 -azinobis-(3-ethylbenzothiazoline-sulfonate) (ABTS) by peroxyl radicals (114—117). Black and green tea extracts (2500 ppm) were found to be 8—12 times more effective antioxidants than a 1-mAf solution of the water-soluble form of vitamin E, Trolox. The most potent antioxidants of the tea flavonoids were found to be epicatechin gallate and epigallocatechin gallate. A 1-mAf solution of these flavanols were found respectively to be 4.9 and 4.8 times more potent than a 1-mAf solution of Trolox in scavenging an ABT radical cation. 

Gliszczyhska-Swigl, A. (2006). Antioxidant activity of water soluble vitamins in the TEAC (trolox equivalent antioxidant capacity) and the FRAP (ferric reducing antioxidant power) assays. Food Chemistry, Vol.96, No.l, (May 2006), pp. 131-136, ISSN 0308-8146. 

For aqueous solutions, ascorbate can be included in the hierarchy, while a-tocopherol has to be replaced by its water-soluble analogue trolox, which is often assumed to have the same standard reduction potential. The ordering of the antioxidants based on the two different determinations of E in water is rather similar, and it should be noted that ascorbate is the antioxidant which will regenerate the other antioxidants, with the ascorbate itself ending up being oxidised. In contrast to what was observed for DMF, the ordering in water predicts that quercetin could regenerate a-tocopherol from its oxidised form. 

Regarding antioxidants, the parameter total dietary antioxidant capacity (TDAC) can be taken to reflect antioxidant intake it is defined as the antioxidant capacity of all plant foods and beverages (alcoholic and nonalcoholic) consumed daily in a diet and may represent the amount of antioxidant units (Trolox equivalents) present daily in the human gut (Saura-Calixto and Goni 2006). 

TDAC in the Spanish diet has been estimated at 3,500 pimol Trolox equivalents by the ABTS method. The contribution of each specific food to the TDAC was dependent on both food intake and food antioxidant capacity. The largest contributors to the TDAC were beverages (about 68%) and fruits and vegetables (about 20%). 

Radical Cation 2,2-Azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS 1) TEAC Assay (Trolox Equivalent Antioxidant Capacity) or ABTS Assay... 

The Trolox equivalent antioxidant capacity (TEAC) assay was reported first by Miller and others (1993) and Rice-Evans and Miller (1994). They used the peroxidase activity of metmyoglobin to oxidize 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in the presence of hydrogen peroxide. The TEAC assay is based on the... 

Another assay that is very similar to the ABTS assay is the AGV-dimethyl-p-phenylenediamine (DMPD assay). In the presence of a suitable oxidant solution at an acidic pH, DMPD is converted to a stable and colored DMPD radical cation (DMPD +). Antioxidants capable of transferring a hydrogen atom to the radical cause the decol-orization of the solution, which is spectrophotometrically measured at 505 nm. The reaction is stable, and the endpoint is taken to be the measure of antioxidant efficiency. Antioxidant ability is expressed as Trolox equivalents using a calibration curve plotted with different amounts of Trolox (Fogliano and others 1999). This method is used to measure hydrophilic compounds. The presence of organic acids, especially citric acid, in some extracts may interfere with the DMPD assay, and so this assay should be used with caution in those extracts rich in organic acids (Gil and others 2000). 

A typical time course of PCL with luminol as the photosensitizer is shown in Figure 5, as blank. The presence of a water-soluble antioxidant leads to dose-dependent temporary inhibition of PCL. ACW (antioxidant capacity of water-soluble compounds) represents the effect of human blood plasma (2 p.L) on PCL all tested antioxidants, such as ascorbic acid, uric acid, Trolox, taurine, bilirubin, ceruloplasmin, etc., produced the same effects. 

It has been proposed [91] that nitric dioxide radical formation during the oxidation of nitrite by HRP or lactoperoxidase (LPO) can contribute to tyrosine nitration and be involved in cell and tissue injuries. This proposal was supported in the later work [92] where it has been shown that N02 formed in peroxide-catalyzed reactions is able to enter cells and induce tyrosyl nitration. Reszka et al. [93] demonstrated that N02 mediated the oxidation of biological electron donors and antioxidants (NADH, NADPH, cysteine, glutathione, ascorbate, and Trolox C) catalyzed by lactoperoxidase in the presence of nitrite. 

In addition to the well-known iron effects on peroxidative processes, there are also other mechanisms of iron-initiated free radical damage, one of them, the effect of iron ions on calcium metabolism. It has been shown that an increase in free cytosolic calcium may affect cellular redox balance. Stoyanovsky and Cederbaum [174] showed that in the presence of NADPH or ascorbic acid iron ions induced calcium release from liver microsomes. Calcium release occurred only under aerobic conditions and was inhibited by antioxidants Trolox C, glutathione, and ascorbate. It was suggested that the activation of calcium releasing channels by the redox cycling of iron ions may be an important factor in the stimulation of various hepatic disorders in humans with iron overload. 

The effects of antioxidants on protein oxidation were also studied in animal experiments. Barja et al. [73] demonstrated that feeding guinea pigs with vitamin C decreased endogenous protein oxidative damage in the liver. Administration of the mixture of antioxidants containing Trolox C, ascorbic palmitate, acetylcysteine, (3-carotene, ubiquinones 9 and 10, and (+)-catechin in addition to vitamin E and selenium to rats inhibited heme protein oxidation of kidney homogenates more efficiently than vitamin E + selenium [74]. 

In vitro Antioxidant capacity was measured by the method of Trolox equivalent antioxidative capacity (TEAC) [12]. The TEAC assay used 20 pL of the extract sample mixed with a 980 pL solution of ABTS + [2.2 -azobis-(3-ethylenebenzothia-zohne-6-srrlfonic) acid], the decolorization of ABTS ", which is a long-life radical cation and bine-green chromophore, measrrred at 734 nm. Results are expressed as millimoles of Trolox equivalent per kilograms of fresh weight. 

The in vitro antioxidant capacity was measured at 2, 4 and 6 min in duplicate. The results presented in Fig. 41.3 are obtained at 4 min (the best correlation coefficient for A Abs calculation, data not shown) and Trolox equivalents were calculated... 

Sample Total phenolics (mg CE /100 mg) Antioxidant activity (pM Trolox equivalents/100 mg)... 

Trolox Equivalent Antioxidant Capacity (TEAC) assay... 

Analysis of antioxidant activity by performing a FRAP assay was proposed by Benzie and Strain [23]. It involves colorimetric determination of the reaction mixture in which the oxidants contained in the sample reduce Fe ions to Fe. At low pH, Fe(in)-TPTZ (ferric-tripyridltria-zine) complex is reduced to the ferrous (Fe ) form and intense blue colour at 593 nm can be observed. The FRAP reagent is prepared by mixing 2.5 ml of TPTZ (2,4,6-tris (l-pyridyl)-5-triazine) solution (10 mM in 40mM HCl), 25 ml acetate buffer, pH 3.6, and 2.5 ml FeCl3 H20 (20 mM). The colour of Fe(II)(TPTZ)2 which appears in the solution is measured colorimetri-cally after incubation at 37°C. The measurement results are compared to those of a blank sample, which contains deionised water instead of the analysed sample. The duration of the assay differs from one study to another 4 min [23, 24], 10 min [25] to 15 min [26]. The analysis results are converted and expressed with reference to a standard substance, which can be ascorbic acid [26], FeS04 [23, 25], Trolox [27,18]. 

A method of analysing antioxidant activity with respect to the DMPD" cation-radical (N,N-dimethyl-p-phenylenediamine cation radical) has been proposed by Fogliano and co-workers [31]. The determination principle involves colorimetric observation of the disappearance of the cation-radical colour at the absorbed light wavelength of 505 nm after a reaction time of 10 min. Coloured cation-radical DMPD" in the assay is obtained by reaction of DMPD with iron chloride in an acetate buffer at pH 5.25. The decrease in absorbance of the reaction mixture caused by antioxidants is compared to the calibration curve, prepared with a series of dilutions of Trolox [32]. 

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