*3.7. Antioxidant Activity*

The total antioxidant activity was determined spectrophotometrically by the ABTS free radical decolorization assay developed by Re et al. [52], with some modifications. In brief, a solution of the radical cation ABTS+ was prepared by mixing a solution of ABST (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (7 mM) and a solution of potassium persulfate (2.45 mM) in H2O. The mixture was kept in the dark overnight before use. Then, the ABTS+ solution was diluted with EtOH to an absorbance of 0.70 ± 0.02 at 734 nm. Trolox was used as standard. The samples were prepared by adding 100 μL of extracts to 2 mL of ABTS+ solution and the control by adding 100 μL of PBS solution to 2 mL of ABTS+ solution. The measure at the absorbance at 734 nm was registered after 6 min of reaction. The percentage of inhibition of the absorbance was calculated by the following equation:

$$\% \text{inhibition} = ((\text{A}\_0 - \text{A}\_1)/\text{A}\_0) \times 100 \tag{1}$$

where A0 is the absorbance of the control and A1 is the absorbance of the tested samples. Results were expressed as nmol of Trolox equivalents (TE) per gram of sample.

### *3.8. Total Lipid Extraction*

Total lipids were extracted using the modified Bligh and Dyer method [53]. Lyophilized powder (100 mg) was mixed with a total of 10 mL solvent added in the sequence of chloroform, methanol, water, to achieve a final chloroform/methanol/water ratio of 1:2:0.8 (by volume). Samples were shaken for 15 s after addition of each solvent, and incubated overnight at 4 ◦C. After centrifugation at 6500× *g* for 10 min, the supernatant was transferred into a separating funnel, and phase separation of the biomass-solvent mixtures was achieved by adding chloroform and water to obtain a final chloroform/methanol/water ratio of 2:2:1.8 (by volume). After settling, the bottom phase was collected and evaporated under vacuum.

### Fatty Acid Profiles Determination

Fatty acid methyl esters (FAMEs) were obtained using boron trifluoride (BF3) according to [54] with some modifications. Total lipid extract was saponified at 90 ◦C for 20 min with 0.5 M KOH in methanol (3 mL). Forty-nine micrograms of the internal standard (methyl tricosanoate) were added before saponification. The fatty acids were methylated by adding 14% BF3 in MeOH (2 mL) and heating at 90 ◦C for 10 min. After cooling, the mixture was extracted with hexane (1 mL × 2). After separation, the hexane layer was collected, taken to dryness under vacuum, dissolved in 1.0 mL of CH2Cl2 and analyzed by gas chromatography-mass spectrometry (GC-MS).
