*3.1.2. Trolox Equivalent Antioxidant Capacity (TEAC) assay*

An assay employing the ABTS•+ cation-radical was proposed by Miller and co-workers [20]. It is based on a colour reaction, in which the stable cation-radical ABTS•+ is formed from 2,2' azinobis-(3-ethyl-benzothiazoline-6-sulfonic) acid (ABTS) with metmyoglobin and hydrogen peroxide. The reaction runs in phosphate-buffered saline, pH 7.4 (PBS). In a modification of the method proposed by Ozgen and co-workers [21], pH is equal to 4.5, which is to make it closer to that of the materials under analysis. A solution of the prepared radical turns bluegreen, with the absorption spectrum within the range from approx 490 to 900 nm. When the antioxidants contained in the solution quench the ABTS•+ cation-radical, the solution absorb‐ ance decreases, which is observed by colorimetry after 6 minutes of the reaction at the temperature of 30ºC and the wavelength of 734 nm. In the method modification proposed by Re and co-workers [22], the ABTS•+ radical is generated in the reaction of 22'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and potassium persulfate in dark at room temperature for 12-16 hours. The analysis results are expressed as an equivalent of the reference substance, e.g. vitamin C, gallic acid, and, most frequently, Trolox. Trolox, which is water-soluble vitamin E analogue, is used to plot the standard curve. Due to this, it is possible to express the strength of antioxidants under analysis in a unified scale TEAC and to compare the results achieved by different researchers.

the authors of the method have pointed out – is closer to the natural physiological environment, unlike in the FRAP assay (pH 3.6) and the Folin-Ciocalteu assay (pH 10) [29, 30]. Those same authors have pointed out the low cost of the method, its simplicity, response to thiol groups of antioxidants and, importantly, its flexibility, which enables using it – by changing a solvent – to examine both lipophilic and hydrophilic antioxidants. The reaction of Cu(II)-Nc with an antioxidant runs vigorously at 37ºC, but certain compounds, such as naringine, require

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http://dx.doi.org/10.5772/55620

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A method of analysing antioxidant activity with respect to the DMPD•+ cation-radical (N,Ndimethyl-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

Asghar and Khan [33] modified the method by adding K2S2O8 (potassium persulfate) in an acetate buffer at pH 5.6 as the initiator of DMPD•+. They abandoned ferric chloride, due to the presence of metal ions in the analysed material which could – as a result of the Fenton Reaction – induce formation of hydroxyl radicals, which affects the antioxidant activity which is being determined. They also noted that the DMPD•+ radical obtained in the reaction with K2S2O8 is more stable than that obtained with FeCl3 as iron ions are susceptible to oxidation by atmos‐

The improved DPMD•+ decolorization assay is suitable for water-soluble as well as lipidsoluble antioxidants [33]. A stock solution of DMPD cation radical is diluted to A517.5nm =0.7÷0.8

6 minutes. The measurement values obtained by the method with the cation radical DMPD are comparable with those obtained in the ABTS assay. As the cost of the DPMD is several

The Falin-Ciocalteu reagent (FCR) is a complex formed in a reaction between sodium tungstate and sodium molybdenate in hydrochloric acid and phosphoric acid, which turns yellow after lithium sulphate is added. The reagent reacts in an alkaline environment with reducing compounds. Such a reaction gives a blue chromophore which is observed by colorimetry. The Folin-Ciocalteu method is highly sensitive – both to phenolic and non-phenolic compounds, e.g. proteins, vitamin C, vitamin B1, folic acid, Cu(I). The method is applied most frequently to determine the total content of phenolic compounds [34, 35]. If that is the case, a sample for determination should be prepared in a proper manner to minimise the effect of non-phenolic

times lower, it could be successfully used as an alternative for the ABTS assay [33].

C stabilized with ethanol or an acetate buffer (pH 5.6). The

C and the absorbance of the reaction mixture is read out after

*3.1.5. DMPD (N,N-dimethyl-p-phenylenediamine) radical cation decolorization assay*

previous acidic hydrolysis.

of Trolox [32].

pheric oxygen.

and after equilibration at 25o

experiment is conducted at 30o

*3.1.6. Folin-Ciocalteu assay*
