2.3.7. Determination of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

Total phenolic content (TPC) determination: 0.1 mL of bee pollen extract was mixed with 1.9 mL water, 0.1 mL of Folin Ciocalateau reagent, the mixture was homogenizated for 2 min and after it 0.8 mL of 5% sodium carbonate was added. The mixture was kept at 40 ◦C for 20 min and cooled down in an ice bath for stopping the reaction. The total phenolic content was determined at 750 nm, based on a gallic acid calibration curve expressing the results as gallic acid equivalent (mg GAE/g) dry mater [35]. All the measurements were carried out in triplicate.

Total flavonoid content (TFC) determination: 0.2 mL of bee pollen extract was mixed with 2 mL of methanol and 0.1 mL of 5% AlCl3 (prepared in methanol). The solution was left for 30 min at room temperature and its absorbance was measured at 425 nm. The concentration was determined based on a quercetin calibration curve expressing the results as mg quercetin equivalent/g (mg QE/g) dry mater [35]. All the measurement were carried out in triplicate.
