*3.5. Antioxidant Activity*

The antioxidant activity was estimated by two methods: ferric reducing power (FRAP) assay and the phosphomolybdate method (total antioxidant capacity, TAC).

#### 3.5.1. Ferric Reducing Power (FRAP) Assay

Ferric reducing power assay is a simple and inexpensive procedure that estimates the antioxidant level of a sample. It is based on the reducing potential of antioxidants in the extract that react with ferric ions (Fe3+) provided by potassium ferricyanide K3Fe(CN)6 and reduce them to ferrous ions (Fe2+). The method used is that described by Zovko Koncic [48].

Extract dilutions with concentrations ranging from 0 to 5 mg/mL were prepared. First, 0.5 mL of each solution was mixed with 2.5 mL of a phosphate buffer solution (0.2 M, pH 6.6) and 2.5 mL of a potassium ferricyanide solution K3Fe(CN)6 (1%). The mixtures were incubated in a water bath at 50 ◦C for 20 min. Afterwards, 2.5 mL of trichloroacetic acid (10%) was added to stop the reaction. The mixtures were then centrifuged at 3000 turns for 10 min. At the end, 2.5 mL of the supernatant of each concentration was mixed with

2.5 mL of distilled water and 0.5 mL of an aqueous solution of FeCl3 at 0.1%. The absorbance was measured at 700 nm. The increase in absorbance in the reaction medium indicates the increase in the reducing power of the sample. Ascorbic acid was used as a positive control, and its absorbance was measured under the same conditions as the sample.

The antioxidant capacity was expressed by the determination of the effective concentration (EC50), which corresponds to an absorbance equal to 0.5. This parameter was used to compare the reducing activity of the sample and the control.

#### 3.5.2. Phosphomolybdate Method (Total Antioxidant Capacity, TAC)

The total antioxidant capacity (TAC) of the ethanolic extract from *Origanum compactum* aerial parts was assessed using the phosphomolybdate method according to Prieto et al., 1999 [49]. This method is based on the use of the plant extract to reduce molybdenum (VI) into molybdenum (V) in an acidic medium. To tubes containing 10 μL of the plant extract with different concentrations, 1 mL of the phosphomolybdate reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) was added. After resting at room temperature for 20 min, the tubes were incubated for 90 min at 95 ◦C. At 695 nm, the absorbance was measured. The data are given as milligrams of ascorbic acid equivalent per gram of extract (mg EAA/g E).

The total antioxidant capacity (TAC) concentration of the analyzed extract was determined using the ascorbic acid calibration curve (y = 0.0411x + 0.0159. R2 = 0.9966) and the results are expressed in milligrams of ascorbic acid equivalent per gram of dry extract (mg EAA/1 g E).

#### *3.6. Antibacterial Activity*

#### 3.6.1. Bacterial Strains and Growth Conditions

The bacterial strains (*Escherichia coli*, *Salmonella typhimirium*, *Staphyloccocus aureus* and *Listeria monocytogenes*) used in this study were obtained from the Laboratory of Microbiology and Health, Faculty of Sciences at Moulay Ismail University of Morocco. Bacterial strains from the frozen stock (−80 ◦C) were spread on Mueller Hinton agar (Merck Life Science, Merck KGaA, Darmstadt, Germany) and incubated at 37 ◦C for 24 h. Then, bacterial suspensions were prepared in sterile distilled water and adjusted to the equivalent of 0.5 McFarland standard (108 cfu/mL).

### 3.6.2. Broth Microdilution Method

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of extract against four bacterial strains were determined by the broth microdilution method as described by Bouymajane et al. [21]. To sterile, flat-bottom 96-well microplates, 50 μL of Mueller Hinton broth and dimethyl sulfoxide (MHB-DMSO) was added. Then, 50 μL of dried extract mixed with DMSO (500 mg/mL) of *Origanum compactum* was added to the first microplate and mixed in order to determine cascade dilutions. Then, 50 μL of bacterial suspensions and 50 μL of MHB-DMSO were added to each well. The well containing the mixture of bacterial suspensions and MHB-DMSO served as a control, and the well containing the mixture of extract and MHB-DMSO was used as a blank. All microplates were incubated at 37 ◦C for 24 h. Afterward, 50 μL of TTC (2, 3, 5-triphenyl tetrazolium chloride) was added to each well of the microplates and re-incubated at 37 ◦C for 30 min. The MIC was determined as the lowest concentration of the extract that showed no visible bacterial growth. The MBC was determined as the lowest concentration of extract that did not produce any bacterial colony. The wells that showed no visible bacterial growth were streaked on Petri dishes containing MHA and incubated 37 ◦C for 30 min. The MBC/MIC ratio was used to determine the bacteriostatic and bactericidal effects of the extract. If MBC/MIC ≤ 4, the extract effect is bactericidal, and if MBC/MIC > 4, the extract effect is bacteriostatic. All the experiments were carried out in triplicate.
