3.3. Antioxidant Activity by Spectrophotometric Methods

The colorimetric determination of antioxidant activity was carried out by standard SET mechanism assays employing ABTS and DPPH radicals as described previously [16,28] with minor modifications. Briefly, stock solutions of radicals were diluted in methanol before measurements until absorbance amounted to 1.00 ± 0.02 at λ = 734 nm in the case of the ABTS radical and 1.00 ± 0.02 at 515 nm for the DPPH radical. All reactions were carried out in 48-well plates at 37 °C. Stock solutions of anthocyanidins were prepared in analytical grade ethanol and then diluted with deionized water to a concentration of 10 mM. Then, stock solutions of antioxidants were diluted to concentrations falling within a linear range of the assay. The ABTS solution (1 mL) was mixed with solutions of anthocyanidins (30 μL). The absorbance of the mixtures was measured at 734 nm after 10 min of incubation in 37 °C. The DPPH solution (1 mL) was mixed with solutions of anthocyanidins (30 μL) and the absorbance was measured at 515 nm after 10 min. All absorbance measurements were performed with a Tecan Infinite M200 spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland). The results of antioxidant activity determinations for spectrophotometric tests were expressed as stoichiometry values (n₁₀), as described by Kusznierewicz et al. [15]. This parameter was determined as a regression coefficient, which was defined as the tangent of the line describing the relationship between concentrations of a radical scavenger and concentrations of the tested antioxidant present in the mixture after 10 min of reaction (n₁₀). The concentration of radicals scavenged by the tested antioxidants in reaction media was calculated with the use of the Beer–Lambert–Bouguer law (Beer’s law).

The ORAC (oxygen radical absorbance capacity) test is a fluorescent method using the HAT mechanism. In this method, a fluorescent probe is oxidized in the reaction with reactive oxygen species (ROS) to a form that does not emit fluorescence. Antioxidants present in the sample are able to inhibit the decrease in probe fluorescence. [20]. For the determination of antioxidant activity using the ORAC assay, first, 150 μL of fluorescein probe (0.4 nM in PBS) was added to inner wells of a 96-well black fluorescence plate. The outer wells were filled with 150 μL of deionized water and were not used in the measurement. Next, 25 μL of tested samples or standard solutions of trolox were added to each well and the plate was incubated for 30 min in 37 °C in a plate reader. After the incubation period, 25 μL of AAPH solution (153 mM) was added to each well in order to start the oxidation of the fluorescent probe. The fluorescence was measured every minute for 60 min at λ_ex = 485 nm and λ_em = 528 nm using the Tecan Infinite M200 spectrophotometer (Tecan Group Ltd., Switzerland). The plate reader generated the plot of fluorescence change in time for each sample. To determine the antioxidant capacity of the tested compounds, the area under the curve (AUC) for the obtained plots was calculated for tested samples and for standard solutions of trolox. To calculate the antioxidant activity of the tested samples, standard curves for trolox were plotted and antioxidant activity of the tested anthocyanidins was expressed as trolox equivalents (uM).