2.2. Preparation of Pollen Extracts

A scheme of extraction procedure is summarized in Figure 1 and explained in detail in further text. For the preparation of lipid fraction, bee-collected pollen (1 g) was extracted with 10 mL of hexane/isopropanol (60:40) mixture as it was suggested in literature [30].

The sheme of extraction procedure.

Extraction was performed for 30 min in an ultrasonic bath (VAB SB 3 LD, maximum power 440 W, operating frequency 40 Hz). After centrifugation, the supernatant was separated and the extraction procedure was repeated with another 10 mL of solvent mixture with vigorous stirring. The resulting supernatant was combined with the previous one, the solvents were evaporated to dryness at room temperature under a nitrogen atmosphere and the residual lipid fraction was finally dissolved in 10 mL of the mixture. By doing so, a lipid extract of bee-collected artichoke pollen was obtained, and it was ready for further GC-FID analysis. The extraction of lipids was performed separately for GC/FID analysis and for the determination of antioxidant properties. Hexane/isopropanol was used for extraction in both procedures. However, the preparation of the lipid fraction to determine the antioxidant properties additionally included rotary evaporation to dryness and final reconstitution in 10 mL of ethanol [31]. Defatted solid residue, which remained after the lipid extraction, was used for further extraction of phenolic compounds. For that purpose, 10 mL of 80% methanol (MeOH) was added and the extraction of free (extractable) phenolic fraction was performed for 60 min with vigorous stirring in a light-protected cuvette. After centrifugation, the supernatant was then separated and the extraction procedure was repeated. The two supernatants were combined, the solvent was evaporated to dryness and the residual solid was redissolved in 10 mL of 80% MeOH. Thus, an extractable phenolic fraction was obtained. This extract was used for further testing of this fraction. The solid residue remained after extractable phenolic fraction separation was used for preparation of alkaline hydrolyzable phenolic extract. Meaning, alkaline digestion was performed [32] by using 40 mL of 4 M NaOH solution and the extraction was performed for 4 h. After the extraction was completed, the obtained extract was neutralized with the required volume of 12M HCl until the pH of the solution was 2. Thereafter, the released phenolics were extracted three times with 10 mL of ethyl acetate. All supernatants were combined, the solvent was evaporated to dryness and the residual fraction was finally dissolved in 10 mL of 80% MeOH. In this way, an extract of alkaline hydrolyzable phenolics was prepared, which was used for further analyses.