Trolox, antioxidant activity

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. 

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

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]. 

According to the theory of free radical oxidation, antioxidizing activities of chlorogenic acids are stipulated by their participation in reaction with free radicals. Chlorogenic and caffeic acids have high stoichiometric numbers and reactivity with peroxyl radicals as compared with trolox, the water-soluble analogue of tocopherol [48]. Considering... 

Relative Antioxidant Activities of Various Compounds with Respect to That of Trolox... 

Antioxidant activity of estrogens was compared on the basis of their activity to inhibit formation of ABTS,+. Estriol, estrone, 17/3-estradiol, and 17a-ethinyl-estradiol showed more than twofold higher activity than diethylstilbestrol, 2-hydro-xyestradiol, and 4-hydroxyestradiol. Antioxidant activity of mestranol was negligible. On a molar basis, estrone was 2.43 times more effective than Trolox (R24). 

Antioxidant activities of caffeine (1,3,7-trimethylxanthine) and its main metabolites was compared. Caffeine, 1,7-dimethylxanthine, and 3,7-dimethylxanthine did not show any peroxyl radical-scavenging capacity at concentration up to 100 fxM. Flowever, the relative antioxidant activities (with respect to Trolox) of 1-methyl-xanthine and 1-methyluric acid were 0.82 and 0.58, respectively (L9). 

Some drugs were found to have antioxidant activity in standard assays of TAC. Aminoguanidine was found to have an antioxidant activity, although three orders of magnitude lower than Trolox on a molar basis (C36). Dimethyl sulfoxide (DMSO), used as a solvent for many compounds, also shows antioxidant activity and delays ABTS oxidation (Y5). 

Correlate the decrease observed in a dose-response curve with a standard antioxidant (e.g., trolox, ascorbic acid), expressing the antioxidant activity as equivalents of standard antioxidant, a well-established parameter in this respect being Trolox Equivalent Antioxidant Capacity (TEAC). ... 

Fig. 3 Dual-HPLC plots of two antioxidants trolox and resveratrol. Upper chromatograms show UV profiles registered at 250 nm and lower chromatograms ABTS scavenging (antioxidant activity) profiles registered at 600 nm (negative peak). In (A), trolox was detected with a retention time of 6.2 min. Inset Calibration curve of scavenging activity (peak areas at 600 nm) for different amounts of trolox. In (B), resveratrol was detected at 5.0 min.

Under these conditions, a study of the antioxidant potential of pure compounds could be carried out. Table 1 shows the values of antioxidant activity (expressed as TEAC) of different compounds of interest, determined by the on-hne method (HPLC-ARTS method) and compared with the values obtained by our conventional photometric end-point method. As can be observed, the two most important standard antioxidants, trolox and ascorbic acid, presented similar TEAC using either method. Thus, either can be used as reference to express antioxidant activity, except that trolox has the advantage because it can be used in both hydrophilic and lipophilic assays. The TEAC values of phenolic compounds were underestimated by approximately half when the HPLC-ARTS method was used as compared to the end-point method. This was due to the different reactivities of antioxidants with ARTS, and because, unfortunately, the time dependence of online scavenging activity determinations made it very difficult to obtain the total reaction for the slowest antioxidants, resulting in a partial estimation of this activity. Nevertheless, the HPLC-ARTS method provided important additional information in the form of correlation between the different peaks of a sample and their antioxidant activities. 

The antioxidant activity of a compound depends upon which free radical or oxidant is used in the assay (Halliwell and Gutteridge, 1995), and a different order of antioxidant activity is therefore to be expected when analyses are performed using different methods. This has been demonstrated by Tsuda et al. (1994) in their study of antioxidative activity of an anthocyanin (cyanidin-3-O-p-D-glucosidc) and an anthocyanidin (cyanidin) in four different lipophilic assay systems. Both compounds had antioxidative activity in all four systems, but the relative activity between them and their activity, compared with Trolox, varied with the method used. Fukumoto and Mazza (2000) reported that antioxidant activity of compounds with similar structures gave the same trends, although not always the same results, when measured by P-carotene bleaching, DPPH and HPLC detection of malonaldehyde formation in linoleic acid emulsion. 

Total antioxidant activities in 13 typical Italian wines were determined with an average of 12.3 pM Trolox equivalents for red wines and 1.6 pM for white wines (Simonetti et al., 1997). The values correlated well with total phenols (r = 0.9902) and flavanols (r = 0.9270) and clearly demonstrated that red wine polyphenols are significant in vitro antioxidants. The antioxidant capacity of 16 red wines from several countries was tested using electron spin resonance (ESR) spectroscopy (Bums et al., 2000). The antioxidant activity ranged from 4.13 x 1021 to 9.29 x 1021 number of Fremy s radicals reduced by 1 L of wine. The antioxidant capacity was associated with the phenolic content of the wines, determined either by Folin-Ciocalteau or by summation of the individual phenolics determined by HPLC or by spectroscopy. Total phenolics as measured by the Folin-Ciocalteau method ranged from 6.47 to 18.6 mM GAE per liter. The anthocyanin levels ranged from 101.5 to 325.7 pM. There were substantial differences in the proportion of polymeric pigments in the wines. 

NOTE A cup = 200 mL, prepared by steeping one tea bag in 200 mL of freshly boiled water for 5 min Antioxidant activity is expressed as pmole trolox equivalents (TE) per cup, using the fluorescein method Traditional = Fermented Values in columns mean SD of 10 samples done in triplicate. 

The grounded leaves (100 mg) were extracted using the same method that was used with the total phenols. ABTS method was used to evaluate the antioxidant activity and the absorbance was measured at 734 nm. The results were expressed as g of Trolox (a water soluble analog of vitamin E) on a dry weight basis (g Trolox /lOO g DW) (S). 

Different methods have been used for determination of antioxidant activity. Rice-Evans et al. (1997) have used a method based on the ability to quench the colored ABTS radicals to compare antioxidant activity of some carotenoids. The results were calculated as the Trolox equivalent of antioxidant capacity (TEAC). The activity of Trolox, the water soluble a-tocopherol analog, was given a value of 1. A higher value in this assay indicated a higher activity of the carotenoid (Table 9.3). 

Flavonoids as antioxidants have been reviewed several times 45s including an outline of many claims to their beneficial health effects . Due to their complex structures and different classes (eight thousand different compounds are known ), researchers often resorted to qualitative screening methods to evaluate their antioxidant potentials in mixed aqueous/lipid phases. For example, the so-called Trolox equivalent antioxidant capacity (TEAC), the concentration of Trolox with equivalent antioxidant activity of a 1 mM concentration of the substrate, is frequently used in heterogeneous systems. Unfortunately, this can be an unreliable measure of the activity of the substance, especially if initiation is also carried out in the aqueous phase. Nevertheless, there have been some efforts made to evaluate antioxidant activities of specific flavonoids using more quantitative methods in heterogeneous systems in order to mimic natural environments. A few examples are cited below to illustrate some approaches to determine flavonoid activities in micelles or lipid membranes. 

A sensitive and simple chemiluminescent (CL) method for measuring antioxidant activity was developed. The determination of TEAC (Trolox equivalent antioxidant capacity) is based on the inhibition of CL intensity of luminol by an antioxidant. Antimicrobial activity was tested as a minimal inhibitory concentration (MIC) by broth microdilution method on gram-positive Enterococcus faecalis. Staphylococcus aureus) and gram-negative Pseudomonas aeruginosa, Escherichia coli) bacterial strains. 

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