All assays were made using 96-well microplates (Nunclon, Nunc, Roskilde, Denmark) and were measured in an ELISA Reader Infinite Pro 200F (Tecan Group Ltd., Männedorf, Switzerland).
The DPPH• (2,2-diphenyl-1-picryl-hydrazyl) radical scavenging activity was measured by a previously described method [57 (link)]. DPPH solution (180 μL of freshly prepared 0.07 mg/mL solution) was mixed with 20 μL of the examined extract in various concentrations in microplates. The absorbance at 517 nm was monitored after 30 min incubation at 28 °C and the results were expressed as an EC50 value. Ascorbic acid was used as the control. The antiradical potential was also analyzed using the previously described ABTS•+ (2,2′-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid) assay [57 (link)]. The absorbance was measured at 734 nm and the results were expressed as millimoles of Trolox equivalents per g of dry extract (TEAC).
The metal chelating activity was determined by the method described by Guo et al. [58 ] with some modifications. In this assay, 0.2 mM aqueous solution of ferric chloride and 0.5 mM aqueous solution of ferrozine were used. Twenty microliters of the 0.2 mM aqueous solution of ferric chloride (II) was mixed with 100 μL of extract at different concentrations. Next, 40 μL of 0.5 mM aqueous solution of ferrozine was added and microplates were shaken and incubated for 10 min in 24 °C. The absorbance was measured at 562 nm and the percentage of inhibition of ferrozine–Fe2+ complex formation was calculated using the following formula:
where Ac is the absorbance of the control (water instead of the extract), and As is the absorbance of the extract.
The results were presented as the concentration of the extract that causes metal chelating in 50% (EC50) calculated on the basis of the linear correlation between the inhibition of ferrozine–Fe2+ complex formation and the concentrations of the extract. EDTA was used as a positive control.
Antioxidant activity was also assayed using the β-carotene bleaching method described and modified by Deba and co-authors [51 (link)]. Twenty microliters of extract at different concentrations were mixed with freshly prepared β-carotene-linoleic acid emulsion, and incubated for 20 min at 40 °C. The absorbance was measured at 470 nm. BHT was used as a positive control.
The DPPH• (2,2-diphenyl-1-picryl-hydrazyl) radical scavenging activity was measured by a previously described method [57 (link)]. DPPH solution (180 μL of freshly prepared 0.07 mg/mL solution) was mixed with 20 μL of the examined extract in various concentrations in microplates. The absorbance at 517 nm was monitored after 30 min incubation at 28 °C and the results were expressed as an EC50 value. Ascorbic acid was used as the control. The antiradical potential was also analyzed using the previously described ABTS•+ (2,2′-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid) assay [57 (link)]. The absorbance was measured at 734 nm and the results were expressed as millimoles of Trolox equivalents per g of dry extract (TEAC).
The metal chelating activity was determined by the method described by Guo et al. [58 ] with some modifications. In this assay, 0.2 mM aqueous solution of ferric chloride and 0.5 mM aqueous solution of ferrozine were used. Twenty microliters of the 0.2 mM aqueous solution of ferric chloride (II) was mixed with 100 μL of extract at different concentrations. Next, 40 μL of 0.5 mM aqueous solution of ferrozine was added and microplates were shaken and incubated for 10 min in 24 °C. The absorbance was measured at 562 nm and the percentage of inhibition of ferrozine–Fe2+ complex formation was calculated using the following formula:
where Ac is the absorbance of the control (water instead of the extract), and As is the absorbance of the extract.
The results were presented as the concentration of the extract that causes metal chelating in 50% (EC50) calculated on the basis of the linear correlation between the inhibition of ferrozine–Fe2+ complex formation and the concentrations of the extract. EDTA was used as a positive control.
Antioxidant activity was also assayed using the β-carotene bleaching method described and modified by Deba and co-authors [51 (link)]. Twenty microliters of extract at different concentrations were mixed with freshly prepared β-carotene-linoleic acid emulsion, and incubated for 20 min at 40 °C. The absorbance was measured at 470 nm. BHT was used as a positive control.