Erythrocytes were collected from healthy blood donors upon informed consent for the use of residual blood for research purposes, according to the Italian regulations and, in particular, the regulations of “Fondazione G. Monasterio CNR-Regione Toscana”. Blood samples were collected in ethylene-diamine-tetra acetic acid (EDTA)-treated tubes and centrifuged 10 min at 2300× g at 4 °C. Following plasma and buffy coat removal, erythrocytes were washed twice with PBS pH 7.4.

The antioxidant activity of ethanolic bee pollen extracts (100 μg/mL) was detected ex vivo on human erythrocytes under mild oxidation conditions as described by Frassinetti et al. [16]. Quercetin (8μM) was used as a standard, and the fluorescence was read at λex = 485 nm and λem = 535 nm using a VictorTM X3 Multilabel Plate Reader (Waltham, MA, USA). Each value was expressed according to the Wolfe and Liu [17] formula:

where ∫SA is the integrated area of the sample curve and ∫CA is the integrated area of the control curve.

The antihemolytic properties of increasing concentrations (20, 50, 100 and 200 μg/mL) of ethanolic bee pollen extracts were evaluated on oxidized human erythrocytes as described by Frassinetti et al. [16]. The erythrocytes hemolysis was induced by thermal decomposition of AAPH in peroxyl radicals and was spectrophotometrically recorded at 540 nm. Values were expressed as a percentage of hemolysis with respect to control corresponding to AAPH-treated erythrocytes.

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