*3.3. Extraction of Polyphenols*

Extraction of polyphenols from aerial parts of *Origanum compactum* was performed by Soxhlet apparatus using an aqueous ethanolic mixture (70:30, *v*/*v*). First, 30 g of the plant powder was placed in a filter paper cartridge and introduced into the Soxhlet. The flask was filled with 350 mL of the hydroethanolic solvent and extracted several times until the plant was exhausted and the coloring disappeared. Afterwards, the solvent was evaporated and the extract was recovered with warm distilled water.

The yield of the crude extract was calculated in relation to m0, the mass of the dry matter at which the solid-liquid extraction was carried out.

$$\text{R\%} = \frac{\text{Mass of circle extracted}}{\text{Mass of dry matter power}} \times 100 = \frac{\text{m}\_0}{30} \times 100$$

3.3.1. Determination of Total Polyphenolic Contents in *Origanum compactum*

The total phenol content of the extract was determined by the Folin-Ciocalteu method [46]. In a 100 mL volumetric flask, 5 μL of the extract was mixed with 1.5 mL of Folin-Ciocalteu reagent (10%) and 1.5 mL of sodium carbonate (Na2CO3) at 7.5% (*m*/*v*). Then, the flask was filled with distilled water. The solution was left for 30 min at room temperature. The absorbance was measured at 760 nm

Gallic acid was used as a positive control. The polyphenol content of the studied extract is calculated from the regression equation of gallic acid calibration (y = 0.095x + 0.003). The results are expressed in milligrams of gallic acid equivalent per gram of dry matter (mg GAE/g dm). The polyphenol content is calculated using the following formula:

$$\mathbf{T} = \frac{\mathbf{C} \times \mathbf{V}}{\mathbf{m} \text{ (dry matter)}} \times \mathbf{D}$$

where C is the concentration measured by the regression equation of gallic acid calibration, V is the sample volume and D is the dilution factor.

#### 3.3.2. Determination of Flavonoids Contents in *Origanum compactum*

Flavonoid content was estimated by the aluminum trichloride (AlCl3) method [47]. First, 10 μL of each fraction was mixed with 0.1 ml of aluminum trichloride 10%, followed by 20 mL of distilled water and supplemented at 50 mL with absolute methanol. The solution was incubated in darkness at room temperature for two hours, and the absorbance was measured at 430 nm. Flavonoids were quantified using a calibration curve performed with the quercetin standard (y = 0.073x − 0.081). The flavonoid content is expressed in milligrams of quercetin equivalent per gram of dry matter (mg QE/g dm).
