## 2 Materials and Methods
### Methods
#### 2.2.2 Determination of Antioxidant/Pro-Oxidant Potential of Mountain Germander
At the end of the electrophoresis, the samples are stained with ethidium bromide solution (20 \(\mu\)g mL\({}^{-1}\)) and visualized with a transilluminator. The gel images showing the intensity of the supercoiled (lower band) and relaxed (upper band) forms of the plasmid were processed using the GelAnalyzer 19.0 software program. The results of the antioxidant potential of the MG extract were expressed as the ratio of the area of the supercoiled and relaxed forms of the band, along with the positive and negative controls. 2.4 Determination of Antioxidant/Pro-Oxidative Effect of Mountain Germander Extract on Human Cell Lines  
The DCFH-DA method (2\({}^{\prime}\),7\({}^{\prime}\)-dichlor fluorescein diacetate) was used to determine the reactive oxygen species (ROS) after treatment with the extract [42]. After treating the cells with the working concentrations of the extract (2 and 24 h), they are washed with PBS buffer, and 100 \(\mu\)L of DCFH-DA dye (0.05 mM in PBS) is added to the wells of the black microtiter plate. After treatment (15 min in a CO\({}_{2}\) incubator at 37 \({}^{\circ}\)C), the fluorescence intensity is proportional to the amount of ROS, and it is measured at 485 nm (emission) and 530 nm (excitation). The induction of free radicals is given in relation to the negative control water (100%) and calculated using Formula (2), which is as follows:  
\[\text{free radical induction }(\%)=\frac{\frac{\text{fluorescence }(\text{extract})}{\%\text{ of survival}}}{\frac{\text{fluorescence }(\text{control})}{100}} \tag{2}\]  
2.5 Determination of Cytotoxic/Proliferative Effect of Mountain Germander Extract on Human Cell Lines  
The Neutral Red method was used to analyze the cytotoxic or proliferative effect of the Mountain Germander extract [43]. Microtiter plates with 96 wells were inoculated with 100 \(\mu\)L of a specific cell suspension during 24 h cultivation. After removing the spent medium, cells were treated (100 \(\mu\)L) with previously dissolved Mountain Germander extract in the nutrient medium (0.025, 0.050, 0.150, and 0.500 mg mL\({}^{-1}\)). The control contained 100 \(\mu\)L of the appropriate nutrient medium instead of the extract. After the treatment (2 and 24 h), the spent nutrient medium with the extract was removed, and the Neutral Red dye working solution (100 \(\mu\)L) was added to each well. After incubation in a CO\({}_{2}\) incubator (37 \({}^{\circ}\)C, 3 h), the dye solution is removed from the wells, and the dye accumulated in the cells is extracted with a decolorizing solution, followed by a measurement of its absorbance at 540 nm. The intensity of the staining is proportional to the concentration of viable cells, and the percentage of surviving cells relative to the negative control (water) is calculated using the following Formula (3):  
\[\text{survival rate }(\%)=\frac{\text{A}_{540}\ (\text{sample})}{\text{A}_{540} (\text{control})}\times 100 \tag{3}\]  
2.6 Determination of Genotoxic/Genoprotective Effect of Mountain Germander Extract on Human Cell Lines  
Oxidative damage of the genetic material of continuous human cell lines in the presence of MG extract was tested using a comet assay with some modifications [44]. First, CAL 27, HepG2, AGS, and Caco-2 cells (initial concentration: 10\({}^{5}\) cells mL\({}^{-1}\), incubated at 37 \({}^{\circ}\)C under controlled atmosphere) were centrifuged from the spent culture medium,and then treated with MG extract in defined concentrations (3 mL) for 2 and 24 h. Simultaneously, the negative control contained only the same amount of corresponding fresh culture medium. After treatment, the medium containing the extract was removed from the Petri dishes, and the cells were washed with PBS buffer, trypsinized, and transferred to Eppendorf tubes. After centrifugation (5000 \(\times\)\(g\), 5 min), sediment with the cells is used for the rest of the experiment.