Assay of Hydroperoxides
The xylenol orange assay was used to evaluate the content of lipid hydroperoxides as described by Eymard and Genot (2003). The levels of hydroperoxides were expressed in mmol of cumenehydroperoxide equivalents per kg of bee pollen (mmolCuOOH per kg of bee pollen).
Thiobarbituric Acid (TBA) Test
The concentration of malonaldehyde (MDA) and other thiobarbituric acid reactive substance (TBARS) in bee pollen was analyzed by TBA test (Zacheo et al. 1998). After the sample (0.3 g) was homogenized with 1 mL of 2-trichloroacetic acid (0.3 M) at room temperature for 2 min, the homogenate was centrifuged for 10 min at 14,000 rpm and the intermediate phase was filtered through a 0.22-µm filter paper. The filtrate (500 µL) was then transferred to 500 µL of TBA (34 mM) in the screw capped tubes and the mixture was heated in a boiling water bath for 15 min. Afterward, the solution was cooled at room temperature and then mixed with 1 mL of 1-butanol for pink chromogen extraction. The absorbance at 532 nm was measured with a Shimadzu UV-2550 spectrophotometer. TBARS values were calculated using 1,1,3,3-tetraethoxypropane as standard. Results were expressed as mg MDA per kg of bee pollen.