*4.4. Antioxidant Capacity Assays*

The free radicals scavenging capacity of the extracts was determined [50]. For it, an ethanolic solution of 130 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH•, Sigma Aldrich, St. Luis, MO, USA) was mixed with the extracts (2–1000 μg/mL). Ascorbic acid (Sigma) 50 μg/mL was employed as standard. The reaction mixtures were incubated in the dark at room temperature for 30 min and the absorbance was measured at 515 nm. The inhibition percent of DPPH• radical was calculated by:

$$\text{Inhibition (\%)} = \text{(D.O. control - D.O. sample)} \text{(D.O. control)} \times 100\tag{1}$$

The concentration required to scavenge 50% of DPPH (IC50) was determined.

The reducing capacity of the plant extracts was measured according to the method of Benzie and Strain (1996) [51]. Briefly, acetate buffer (300 mM, pH = 3.6), TPTZ (2, 4, 6-tripyridyl-s-triazine; Sigma) 10 mM in 40 mM HCl and FeCl3 × 6H2O (20 mM) were mixed in the ratio of 10:1:1 to obtain FRAP reagent. The extracts (20 μL) were mixed with 900 μL of FRAP reagent. The mixtures were incubated at room temperature for 4 min and absorbances were measured at 593 nm. Ascorbic acid (100 μM) was used as standard.

To evaluate the inhibition of brain phospholipid peroxidation, rat brains were excised after decapitation, weighed and washed with 0.9% NaCl ice cold solution. Tissue homogenates were prepared in a ratio of 1 g of wet tissue to 9 mL of phosphate buffer (50 mM, pH 7.4), by using a tissue homogenizer (Qiagen). The homogenates were centrifuged at 800× *g* in a Sigma centrifuge at −4 ◦C during 15 min and the supernatants were kept at −70 ◦C until analysis. Thiobarbituric reactive substances (TBARS) measurement assay was carried out as previously described [52]. Brain homogenates (25 mL) were incubated with different extracts (5–125 μg/mL). Incubations were stopped by the addition of 350 mL of cold acetic acid 20% pH 3.5. Malondialdehyde (MDA) levels were determined by the addition of 600 mL of TBA 0.5% in acetic acid 20% pH 3.5. The mixtures were incubated at 90 ◦C for 1 h. Then, 50 mL of sodium dodecyl sulfate (SDS) were added, and samples were centrifuged at 500× *g* during 15 min at room temperature. The absorbance was measured at 532 nm. All the values are means of three determinations. Trolox-C (0.5–75 μg/mL), was used as standard. The extract concentration which is needed to achieve the 50% of inhibition of lipid peroxidation (IC50) was calculated.
