2.7. Antioxidant Properties of Bee-Collected Artichoke Pollen

For examination of antioxidant properties lipid, extractable phenolic and alkaline hydrolyzable phenolic fractions were used. Determination of ABTS radical cation scavenging activity, total antioxidant capacity and ferrous-ion-chelating capacity were analyzed according to previously described methods [38]. For ABTS radical cation assay, 100 mL of lipid, extractable and alkaline hydrolyzable phenolic extracts of bee-collected artichoke pollen was mixed with 1 mL of ABTS^∙+ working solutions whose absorbance was in the range of 0.7–0.8. After 7 min of vigorous stirring, absorbance was measured at 734 nm. Obtained results were used to calculate the percentage of quenched radicals according to the following equation:

where Ac is the absorbance of ABTS radical cation working solution, As is the absorbance of the sample mixed with ABTS radical cation working solution.

For the determination of TAC 0.3 mL of samples was mixed and incubated (90 °C, 90 min) with 3 mL of phosphomolybdenum reagent. After that, the samples were cooled, and the absorbance was measured at 695 nm. The obtained results of pollen fractions were expressed as mg of ascorbic acid equivalents per g of dry weight sample (mg AAE/g DW).

For FCC, 200 mL of pre-prepared pollen fractions was mixed with milliQ water and 2 mM iron(II) sulfate. After 30 min, 5 mL of ferrozine was added and the absorbance of the mixture was measured after 10 min at 562 nm. Results for Fe²⁺ chelating capacity (%) were calculated according to the following equation:

where Ac is the absorbance of the blank; As is the absorbance of samples.

In addition to ABTS radical cation scavenging activity of pollen extracts DPPH radical assay was also applied. For that purpose, 105 μL of original extracts was mixed with 840 μL of DPPH^∙ working solution (0.062 g/100 mL) prepared in methanol according to literature data [40]. Samples were left in the dark for 30 min to react with DPPH radical at room temperature. After that, the resulting discoloration of DPPH radical working solution was determined by reading the absorbance at 517 nm. The obtained results were expressed as % of inhibition of DPPH radical and calculated according to the following equation:

where Ac is the absorbance of DPPH radical working solution, As is the absorbance of sample mixed with DPPH radical working solution.

In order to determine the ability of extracts to participate in redox processes, FRP was determined according to the previously described method [41] with some modifications. Briefly, 0.5 mL of original extracts was mixed with 0.5 mL of phosphate buffer solution (pH = 6.6) and 0.5 mL of 1% solution of K₃[Fe(CN)₆]. After heating for 20 min (t = 50 °C) 0.5 mL of 10% trichloroacetic acid solution was added to samples. After centrifugation 0.5 mL of clear supernatant was mixed with the same volume of distilled water and 0.1 mL of 0.1% ferric-chloride solution. The obtained green color of solution was read at 700 nm. The results were expressed as mg of AAE/g of DW.