The UV–Vis spectra of the applied antioxidants and UV–Vis spectra of the DPPH and ABTS radicals, the concentration of which was monitored to determine antioxidant properties of the antioxidants are presented in Fig. 1. As results from the figure, none of the applied antioxidants absorbs light near 744 nm wavelength. Considering their absorption ability of 516 nm wavelength, the examined antioxidants can be divided into two groups. The first one consists of four antioxidants (BHA, BHT, Trolox and quercetin) which do not absorb 516 nm wavelength at all. Five other components—cyanidin, alizarin, thymol blue, E155 and E132—constitute the second group of antioxidants absorbing the mentioned wavelength. It is worth noting, however, that the extinction of 516 nm wavelength by these compounds varies (see the last column of Table 1). The greatest molar extinction of 516 nm is shown by cyanidin whereas the smallest by E132.
IC50 parameter is most frequently applied to express the antioxidant activity of compounds or mixtures by the DPPH and ABTS methods (Antolovich et al. 2002; Magalhăes et al. 2008; Dawidowicz et al. 2012). IC50 values for the examined antioxidants, estimated by monitoring absorbance changes of the measuring system at 516 nm (spectrophotometric measurements) and by monitoring DPPH concentration changes in the same system by chromatographic analysis, are collected in the second and third column of Table 1. As results from the data, the IC50 values estimated spectrophotometrically and chromatographically are almost the same only for BHA, BHT, Trolox and quercetin (i.e. for the antioxidants of the first group).
As to the components of the second group, IC50 values from spectrophotometric and chromatographic measurements could be calculated only for cyanidin and alizarin. It should be stressed, however, that spectrophotometrically estimated IC50 values for these two compounds are greater than those calculated from the chromatographic measurements. Thus, judging by the spectrophotometric data, the antioxidant properties of these compounds are worse than those established by monitoring the DPPH radical concentration by chromatography. Such discrepancies are explicable if one takes into account that some amount of the antioxidant absorbing 516 nm wavelength may still remain in the measuring system at the moment of measuring the DPPH radical concentration spectrophotometrically. In consequence, the remaining antioxidant precludes the estimation of the true concentration of the DPPH radicals in the measuring system. The greater difference between the spectrophotometrically and chromatographically estimated IC50 values for alizarin than for cyanidin, despite a significantly lower extinction of 516 nm wavelength by the former (see Table 1), indicate that there is another factor, beside the excitation value, responsible for the observed discrepancies.
It must be remembered that the IC50 values for given antioxidants are calculated from the relation illustrating the influence of antioxidant concentration on DPPH inhibition percent. Such dependencies determined chromatographically (solid lines) and spectrophotometrically (dotted lines), assuming that the absorbance at 516 nm in spectrophotometric measurements corresponds only with the DPPH radical concentration, are presented in Figs. 2 and 3. As results from Fig. 2, the run of the dependencies between inhibition percent and antioxidant concentration, determined spectrophotometrically and chromatographically, are the same for antioxidants of the first examined group, which do not exhibit any extinction at 516 nm. The same run of both dependencies confirms the reliability and utility of spectrophotometric monitoring of the DPPH radical concentration changes for the estimation of the antioxidant ability of these compounds.
For the antioxidants in the second group (see Fig. 3), the run of the dependencies between inhibition percent and antioxidant concentration determined in spectrophotometric and chromatographic measurements is different. The inhibition % values determined spectrophotometrically for these antioxidants are either significantly lower than those calculated from the chromatographic data (cyanidin, alizarin, E132) or even have negative values (E155, thymol blue). The observed discrepancies in the run of the curves obtained from the spectrophotometric and chromatographic measurements and in the shape of the curves obtained from the spectrophotometric measurements can be explained by analyzing the extinction of 516 nm wavelength by these antioxidants (see Table 1) and their residue in the measuring systems after the reactions with the DPPH radicals (see Table 2).
The smallest difference in the run of dependencies in Fig. 3 is observed for cyanidin despite its greatest molar extinction of 516 nm wavelength (see the last column of Table 1). Of all the examined colored antioxidants, this compound exhibits the greatest antioxidant activity and its remaining amount in the measuring system after the reaction with the DPPH radicals is relatively low: between 2.67 and 5.87% (see Table 2) at the concentration equal IC50 = 0.138 mg mL−1 (see HPLC data in Table 1). It was also observed that the run of the dependencies in Fig. 3 for alizarin and E132 showed even greater differences. Admittedly molar extinctions of 516 nm wavelength for these two antioxidants are the lowest in the second group (see the last column of Table 1); however, their residue in the measuring system after the reaction with the DPPH radicals is significantly greater. At concentration equal IC50 = 0.778 mg mL−1 for alizarin and IC50 = 4.746 mg mL−1 for E132 (see HPLC data in Table 1), their residues are between 14.07 and 21.50% and 67.07–69.52%, respectively (see Table 2).
Table 1 does not contain spectrophotometrically estimated IC50 values for three examined compounds of the second group: E132, thymol blue and E155. Although the run of the dependencies between inhibition percent and antioxidant concentration estimated spectrophotometrically for E132 has the classical shape, the curve is situated in the region of low inhibition % (below 30%). The extinction value of 516 nm for this weak antioxidant is the lowest in the second group (see Table 1), however, its residue in the measuring system is considerably larger. The concentration of the remaining E132 at the estimated IC50 = 4.746 mg mL−1 (see HPLC data in Table 1) is between 67.07 and 69.52% (see Table 2). The inflated absorbance of the reaction mixture, reflected by a low run of the dependencies between inhibition percent and antioxidant concentration, simulates small DPPH radical scavenging.
Quite different shape of the dependencies between spectrophotometrically estimated inhibition percent and antioxidant concentration is observed for thymol blue and E155 (see Fig. 3d, e). The negative values of the inhibition % for these antioxidants are due to greater absorption of 516 nm wavelength by the reaction mixtures than by the initial DPPH radical solution. This results from the high extinction of 516 nm by these antioxidants and from their significant concentration in the measuring system at the moment of spectrophotometric reading (see Tables 1, 2). In the case of E155, which exhibits higher extinction of 516 nm wavelength and more of it remains in the measuring system than of thymol blue, the number of spectrophotometrically estimated negative values of inhibition percent is greater.
IC50 values for the examined antioxidants, estimated by monitoring absorbance changes of the measuring system at 744 nm (spectrophotometric measurements) and by monitoring ABTS cation radical concentration changes in the same system by chromatographic analysis, are gathered in Table 3.
As results from the table, the IC50 values established for individual antioxidants using both monitoring ways of ABTS cation radical concentration changes are almost the same. It should be noticed, however, that in these experiments the applied monitored wavelength (744 nm) in spectrophotometric measurements is outside the spectra of examined antioxidants. Hence, the presence of antioxidant residues in the measuring system do not influence the estimation of true ABTS concentration.