*3.4. Ethanolic Extracts*

Five hundred milligrams of frozen onion samples were weighed and extracted at room temperature under continuous stirring for 1.5 h with ethanol (15 mL), as described in Muscolo et al. [17]. The samples were centrifuged at 2365× *g* for 15 min, and the supernatants were filtered dried and resuspended in 3 mL of ethanol.

#### *3.5. Determination of Total Phenolic Compounds and Total Flavonoids*

The Folin–Ciocalteu assay was used for evaluating the total phenol content as reported in Muscolo et al. [17]. The absorbance of the samples was recorded at 760 nm. A calibration curve was constructed with gallic acid, and the results were expressed as the gallic acid equivalent (GAE) in mg·g−<sup>1</sup> DW.

The total flavonoid was detected according to the spectrophotometric method, as reported in Muscolo et al. [17]. One milliliter of extract was mixed with 1 mL of 20 g L−<sup>1</sup> AlCl3 methanolic solution. After incubation at room temperature for 15 min, the absorbance was measured at 430 nm. The flavonoid content was calculated from a calibration curve of rutin and expressed as mg rutin g−<sup>1</sup> DW.

#### *3.6. Determination of Antioxidant Activities*

The antioxidant activity against the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical was determined according to [49]. The DPPH concentration in the cuvette was chosen to give absorbance values of ≈1.0. The reaction mixtures were composed of 10 μL of each extract, 700 μL of DPPH, and ethanol up to a final volume of 1 mL. A blank without ethanol extract was prepared for each sample. The change in absorbance of the violet solution was recorded at 517 nm after 30 min of incubation at 37 ◦C. The inhibition I (%) of radical scavenging activity was calculated as I (%) = [(A0 − AS)/A0] × 100, where A0 is the absorbance of the control, and AS is the absorbance of the sample after 30 min of incubation. The results were expressed as Trolox equivalents (TE).

The ABTS (2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay was performed according to [50]. Solutions of 7 mmol L−<sup>1</sup> ABTS+ (final concentration) and 2.45 mmol L−<sup>1</sup> ammonium persulfate (final concentration) in phosphate-buffered saline (PBS) were mixed and kept in the dark at room temperature for 12–16 h. Before use, the absorbance of the ABTS+ solution was fixed at 0.70 ± 0.02 at 734 nm. Aliquots of ethanol extract (25, 50 and 100 μL) were added to 0.5 mL of ABTS<sup>+</sup> solution and brought to a final volume of 600 μL with PBS. After 6 min of incubation in the dark at room temperature, the absorbance of the samples was recorded at 734 nm using a UV-Visible spectrophotometer. The inhibition I (%) of radical scavenging activity was calculated as I (%) = [(A0 − AS)/A0] × 100, where A0 is the absorbance of the control, and AS is the absorbance of the sample after 4 min of incubation. The results were expressed as <sup>μ</sup>mol·L−<sup>1</sup> TE using a Trolox (1–50 <sup>μ</sup>mol·L−1) calibration curve.

The oxygen radical absorbance capacity (ORAC) assay was performed according to [50]. A 20-μL aliquot of extract was added to 120 μL of fresh fluorescein solution (117 nmol L−1). After a preincubation time of 15 min at 37 ◦C, 60 μL of freshly prepared AAPH solution (40 mmol L−1) was added. Fluorescence was recorded every 30 s for 90 min (*λ*ex 485 nm, *λ*em 520 nm). A blank using 20 μL of methanol instead of the sample was also analyzed. ORAC values were expressed as μmol TE mg−<sup>1</sup> FW using a Trolox (10–100 μmol L<sup>−</sup>1) calibration curve.
