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

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 of Trolox [32].

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‐ pheric oxygen.

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 and after equilibration at 25o C stabilized with ethanol or an acetate buffer (pH 5.6). The experiment is conducted at 30o C and the absorbance of the reaction mixture is read out after 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 times lower, it could be successfully used as an alternative for the ABTS assay [33].

### *3.1.6. Folin-Ciocalteu assay*

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

104 Column Chromatography

the results achieved by different researchers.

ascorbic acid [26], FeSO4 [23, 25], Trolox [27,18].

*3.1.4. CUPric Reducing Antioxidant Capacity (CUPRAC) assay*

cally after incubation at 37o

*3.1.3. Ferric Ion Reducing Antioxidant Power (FRAP) 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

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 Fe3+ ions to Fe2+. At low pH, Fe(III)-TPTZ (ferric-tripyridltria‐ zine) complex is reduced to the ferrous (Fe2+) 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 (1-pyridyl)-5 triazine) solution (10 mM in 40mM HCl), 25 ml acetate buffer, pH 3.6, and 2.5 ml FeCl3•H2O (20 mM). The colour of Fe(II)(TPTZ)2 which appears in the solution is measured colorimetri‐

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

The CUPRAC assay, developed by Turkish researchers from Istanbul University [28], has undergone many modifications by which it has been adapted to wider applications [29, 30]. The mechanism of monitoring the antioxidant activity of the sample has remained unchanged. The assay is based on a coloured reaction during which copper ions in the CUPRAC reagent, Cu(II)-neocuproine (2,9-dimethyl-1,10-phenanthroline (Nc)), are reduced by antioxidants contained in the analysed sample. Chelates Cu(I)-Nc formed during the reaction have the maximum light absorption at the wavelength of 450 nm. The reaction runs at pH 7, which – as

C. The measurement results are compared to those of a blank

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 compounds on the assay results. One such method is to remove the solvent from the sample and to dissolve phenolic compounds in alcohol, which eliminates the compounds insoluble in that environment or ones which become denatured.

The reaction of antioxidants in a sample with the radicals generated by AAPH and fluorescein

progresses, antioxidants in the analysed sample react with the radicals. With an excess of radicals, the ability of antioxidants to reduce them becomes exhausted and radicals react with fluorescein, oxidising it to a non-fluorescing form. Observation of the fluorescence of the reaction mixture is conducted at the excitation wavelength of 485 nm and emission wavelength of 525 nm. Measurements are conducted every 60-90 seconds until the resulting curve reaches

**Figure 3.** ORAC antioxidant activity determination of *Echium vulgare* defatted seeds methanolic extract expressed as

Surface area under the curve (AUC) was calculated by Ou and co-workers [43] from the

where *fo* denotes fluorescence read out at the beginning of the assay and *fi* denoted the value

The area under curve for the sample (AUCsample), reduced by the area under curve plotted for the blank sample (AUCblanc) is referred to as "net AUC". Moreover, the net AUC is calculated for a series of dilutions of Trolox and a calibration curve is plotted, showing the relationship between net AUC and the concentration of Trolox. The results of the assay which refer the net

Determination of the antioxidant activity in the system comprising β-carotene and linoleic acid is based on competitive oxidation of β-carotene during heat-induced auto-oxidation of linoleic

AUC of the sample to the calibration curve are expressed as Trolox equivalent.

*AUC* =1 + *f* <sup>1</sup> / *f <sup>o</sup>* + *f* <sup>2</sup> / *f <sup>o</sup>* + *f* <sup>3</sup> / *f <sup>o</sup>* + *f* <sup>4</sup> / *f <sup>o</sup>* + … + *f* <sup>34</sup> / *f <sup>o</sup>* + *f* <sup>35</sup> / *f <sup>o</sup>*

C. As the reaction

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

Chromatography in Bioactivity Analysis of Compounds

is conducted in a phosphate buffer at pH 7.4 and at the temperature of 37o

a plateau.

net area under the curve (net AUC)

of fluorescence read after the time i.

*3.2.3. β-carotene bleaching test*

following formula:

Performing the assay is reduced to putting an alcoholic solution of the analysed sample, Folin-Ciocalteu reagent and solution of sodium carbonate into a reaction tube, which brings the pH of the reaction environment to approx. 10. According to various literature reports, the reaction runs in the darkness for 10 to 120 minutes. After that time, the blue colour of the solution is observed colorimetrically at 725 nm – 760 nm [34, 35, 36, 37, 38]. The results are expressed based on calibration curves prepared for catechol and gallic acid.
