*2.6. HPLC Analysis*

After extraction, each extract was centrifuged for 15 min at 3000 rpm and the resulting supernatant filtered using a syringe filter (0.45 μm, Millipore, Molsheim, France) prior to analysis. Separation and identification of the main extract constituents was done by HPLC with a Varian system (Varian, Les Ulis, France) composed of: Prostar 230 pump, Metachem Degasit, Prostar 410 autosampler, Prostar 335 Photodiode Array Detector (PAD) and driven by Galaxie version 1.9.3.2 software (Varian, Les Ulis, France). A Purospher RP-18 column (250 × 4.0 mm internal diameter; 5 μm) (Merck Chemicals, Molsheim, France) was used for the separation performed at a temperature set at 35 ◦C. The mobile phase was a mixture of: (i) A, which was acidified HPLC grade water with acetic acid (0.2% (*v*/*v*)), and (ii) B, which was HPLC grade methanol. During the separation run, the mobile phase composition varied according to a nonlinear gradient as follows: 8% B (0 min), 12% B (11 min), 30% B (17 min), 33% B (28 min), 100% B (30–35 min), and 8% B (36 min) at a flow rate of 1 mL/min. Between each injection, a 10-min re-equilibration time was applied. The detection of compounds was set at 295 and 325 nm (corresponding to the λmax of the main compounds). Quantification was done based on assessment of retention times of commercial standards (Merck, Saint-Quentin Fallavier, France).

## *2.7. Antioxidant Activities*

2.7.1. In Vitro Cell Free DPPH Free Radical Scavenging Assay

The in vitro cell free DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was used to evaluate the free radical scavenging activity of the samples as described by the microplate protocol of Shah et al. [32].

#### 2.7.2. In Vitro Cell Free ABTS Antioxidant In Vitro Cell Free Assay

ABTS (2,2-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)) in vitro cell free antioxidant activity of each extract was determined as described by Ullah et al. [33].

2.7.3. Cupric Ion Reducing Antioxidant Capacity (CUPRAC) In Vitro Cell Free Assay

CUPRAC assay was performed in a microplate as described by Drouet et al. [8].

2.7.4. Determination of Membrane Lipid Peroxidation Using Thiobarbituric Acid-Reactive Substances (TBARS) Assay

An *in cellulo* antioxidant assay, using yeas<sup>t</sup> cells, based on the measurement of membrane lipid peroxide, was carried out with the thiobarbituric acid (TBA; Merck, Saint-Quentin Fallavier, France) method as described by Garros et al. [34].
