Antioxidant Activity

The extracts antioxidant capacity was evaluated by the in vitro assays of oxidative haemolysis inhibition (OxHLIA), thiobarbituric acid reactive substances formation inhibition (TBARS), and β-carotene bleaching inhibition (β-CBI), following methodologies previously described [28,29]. Extract concentrations ranging from 5 to 0.0159 mg/mL were used. Trolox was the positive control.

OxHLIA assay—An erythrocyte solution (2.8%, v/v; 200 μL) was mixed with 400 μL of either extract solution in PBS, PBS solution (control), or water (for complete haemolysis). After pre-incubation at 37 ◦C for 10 min with shaking, AAPH (200 μL, 160 mM in PBS, from Sigma-Aldrich) was added, and the optical density (690 nm) was measured every 10 min in a microplate reader (Bio-Tek Instruments, ELX800) until complete haemolysis [28]. The results were expressed as IC50 values (μg/mL) at a Δ*t* of 60 min, i.e., extract concentration required to keep 50% of the erythrocyte population intact for 60 min.

TBARS assay—A porcine brain cell solution (1:2, w/v; 0.1 mL) was incubated with the extract solutions (0.2 mL) plus FeSO4 (10 μM; 0.1 mL) and ascorbic acid (0.1 mM; 0.1 mL) at 37 ◦C for 1 h. Then, trichloroacetic (28% w/v, 0.5 mL) and thiobarbituric (TBA, 2%, w/v, 0.38 mL) acids were added, and the mixture was heated at 80 ◦C for 20 min. After centrifugation at 3000× *g* for 10 min, the malondialdehyde (MDA)-TBA complexes formed in the supernatant were monitored at 532 nm (Specord 200 spectrophotometer, Analytik Jena, Jena, Germany) [29]. The results were expressed as EC50 values (μg/mL), i.e., extract concentration providing 50% of antioxidant activity.

β-CBI assay—A β-carotene-linoleic acid emulsion (4.8 mL) was mixed with the extract solutions (0.2 mL) and the absorbance was measured at 470 nm as soon as mixed (AβT0) and after 2 h of incubation at 50 ◦C (AβT0). The β-CBI capacity was calculated as follows: (AβT2/AβT0) × 100 [29]. The results were expressed as EC50 values (μg/mL).
