## 2 Materials and Methods
### Preparation of Extract
#### 2.4.4 Evaluation of Antioxidant Activity  
The antioxidant activity of different extracts of the aerial parts of _Cornulaca monacantua_ Del. and _Anabasis articulata_ (Frossk.) Moq. prepared with the optimized model validated in the first part of the study, was evaluated using five different antioxidant tests: DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS\({}^{+}\) (2,2\({}^{\prime}\)-Azino-bis(3-ethylbenzothazoline-6-sulfonic acid) free radical scavenging capacity, and copper reducing antioxidant capacity (CUPRAC), phenanthroline, and reducing power tests, as shown in Figure 1. Different concentrations were prepared for each extract and standard by serial dilution. The standards used in all tests were BHA and BHT at concentrations ranging from 100 \(\mu\)g/mL to 1.5625 \(\mu\)g/mL. DPPH Free-Radicals Scavenging Capacity  
The DPPH free-radical scavenging capacity of the standards and the extracts of the different plants studied was evaluated according to the method indicated by [PERSON] et al. [71]. Briefly, 40 \(\mu\)L of each extract or standard concentration was added to 160 \(\mu\)L of an ethanolic solution of DPPH (0.1 mM) in a 96-well microplate. The obtained reaction mixture was then incubated in the dark at room temperature for 30 min. Depending on the sample's potentiality, the reaction mixture's color changed from purple to pale yellow. The reduction in absorbance was measured at \(\lambda_{\text{OD}}\) = 517 nm using a Multiskan SkyHigh 96-well microplate reader (Thermo Fisher Scientific, A51119600C, Illkirch-Graffenstaden, France). The inhibition percentage of DPPH free radicals was calculated according to the following formula (Equation (3)):  
\[\%\text{ DPPH inhibition}=(\text{Ac}-\text{As})/\text{Ac}\times 100 \tag{3}\]  
where Ac represents the absorbance of the control, and As represents the absorbance of the sample. The results of this analysis were indicated as IC\({}_{50}\). It corresponds to the concentration of antioxidants in the extract or standard that can inhibit 50% of DPPH free radicals. It is determined based on the curve showing the DPPH free radical inhibition percentage as a function of various concentrations of the analyzed sample. The ABTS\({}^{+}\) scavenging capacity of the different analyzed samples was evaluated according to the method proposed by [PERSON] et al. [71]. ABTS cations were initially generated by reacting an aqueous ABTS solution (2.45 mM) with an aqueous potassium persulfate solution (K\({}_{2}\)S\({}_{2}\)O\({}_{8}\), 7.00 mM) at room temperature for 16 h under dark conditions. The absorbance of the resulting ABTS\({}^{+}\) stock solution was then adjusted by diluting it with ethanol to 0.70 \(\pm\) 0.02 at 734 nm before usage. A volume of 40 \(\mu\)L of each concentration of extract or standard (BHA and BHT) was added to 160 \(\mu\)L of ABTS\({}^{+}\) solution in a 96-well microplate. The obtained mixture was incubated for 10 min at room temperature in the dark. A Multiskan SkyHigh 96-well microplate reader (Thermo Fisher Scientific, A51119600C, Illkirch-Graffenstaden, France) was used to measure the reduction in absorbance at \(\lambda_{\text{OD}}\) = 734 nm. The inhibition percentage of ABTS\({}^{+}\) free radical was estimated according to the following Equation (4):  
\[\%\text{ ABTS}^{+}\text{ inhibition}=(\text{Ac}^{\prime}-\text{As}^{\prime})/ \text{Ac}^{\prime}\times 100 \tag{4}\]  
where Ac\({}^{\prime}\) corresponds to the absorbance of the control, and As\({}^{\prime}\) corresponds to the absorbance of the sample. The results of this test were estimated as IC\({}_{50}\), which indicates the concentration of antioxidants included in the examined extract or standard that can inhibit 50% of ABTS\({}^{+}\) free radicals. This value was measured based on the curve of the inhibition percentage of ABTS\({}^{+}\) as a function of various concentrations of the analyzed sample. Antioxidant Reducing Capacity of Copper (CUPRAC)  
Copper-reducing activity was tested by the method suggested by [PERSON] et al. [72].