## 2 Materials and Methods
### Methods
#### 2.2.2 Determination of Antioxidant/Pro-Oxidant Potential of Mountain Germander  
Extract on the Model Protein Macromolecule  
The derivatization method using 2,4-dinitrophenylhydrazine (DNPH) was employed to examine the influence of polyphenic extract on bovine serum protein oxidation [40]. The reaction mixture contained the following: 240 \(\mu\)L of BSA (1 mg mL\({}^{-1}\)), FeCl\({}_{3}\) solution (60 \(\mu\)L), ascorbic acid (60 \(\mu\)L), and 25 mM H\({}_{2}\)O\({}_{2}\) (24 \(\mu\)L), as well as the corresponding volumes of extract to obtain the previously defined extract concentrations, and demineralized water to reach a final volume of 600 \(\mu\)L, with an incubation time for 30 min at 37 \({}^{\circ}\)C. The negative control contained the same amount of water instead of the extract. After incubation, 2,4-dinitrophenylhydrazine (DNPH) was added (500 \(\mu\)L), and the mixture was incubated for 30 min at room temperature. In order to precipitate the carbonylated proteins, 500 \(\mu\)L of 10% trichloroacetic acid was added, with an incubation time of 15 min. After the centrifugation, the precipitate was washed with ethyl acetate (1 mL). Finally, the samples were resuspended in 500 \(\mu\)L of guanidine hydrochloride (6 M) and left overnight. The absorbance was measured at 370 nm, while the degree of protein oxidation was calculated in relation to the negative control (water), according to the Formula (1), which is as follows:  
\[\text{rate of protein oxidation }(\%)=\frac{\text{A}_{370}(\text{sample})}{\text{A}_{370 }(\text{control})}\times 100 \tag{1}\]  
2.3 Determination of Genotoxic/Genoprotective Effect of Mountain Germander Extract on Model DNA Macromolecule  
The genotoxic/genoprotective effect of MG extract on the model plasmid phi174 RF1 DNA was tested by electrophoresis in agarose gel, according to the modified protocol [41]. After preparing a 1% (\(w/w\)) agarose gel in a buffer solution of Tris base, acetic acid, and EDTA (TAE), and forming wells on the gel in an electrophoresis mold, the prepared reaction \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multicolumn{2}{c}{**TPC (mg GA g-1 *)**} & \multicolumn{6}{c}{**Total Hydroxycinnamic Acids (mg ECH g-1 *)**} \\ \hline \multicolumn{2}{c}{352.24 \(\pm\) 1.56} & \multicolumn{6}{c}{275.23 \(\pm\) 0.97} \\ \hline \multicolumn{2}{c}{phenylethanolg glycosides (mg g-1 **)**} & \\ \hline \hline \
\begin{tabular}{c} g-OH- \\ \end{tabular} &
\begin{tabular}{c} echinnacoside \\ \end{tabular} & jionoside A & teuplosioke & stabsysoside A & poliumoside & verbascoside & forysthoside B & isoverbascoside \\ \hline \(2.73\pm 0.03\) & \(75.37\pm 1.10\) & \(4.73\pm 0.10\) & \(22.19\pm 0.42\) & \(42.86\pm 0.83\) & \(29.27\pm 0.39\) & \(25.29\pm 0.46\) & \(9.03\pm 0.33\) & \(6.43\pm 0.24\) \\ \hline \multicolumn{2}{c}{flavonoids (mg g-1 **)**} & \\ \hline \multicolumn{2}{c}{vicnin-2} & \multicolumn{6}{c}{diosmin} & \multicolumn{6}{c}{accoint-7-O-nutinoside} \\ \hline \(1.86\pm 0.03\) & \multicolumn{6}{c}{\(1.76\pm 0.10\)} & \(1.19\pm 0.02\) \\ \hline \hline \end{tabular}  
* expressed in mg equivalents of gallic acid (GAE) or echinacoside (ECH) per g of lyophilized extract, ** expressed in mg per g of lyophilized extract.  
\end{table} 
Table 1: Bioactive characterization of Mountain Germander freeze-dried polyphenic extract. mixture (30 \(\mu\)L) consisted of: an extract at a certain concentration, 0.2% (_v/v_) of hydrogen peroxide, plasmid (0.1 mg mL\({}^{-1}\)) and TAE buffer. All samples are irradiated with UV radiation for 3 min. Simultaneously, a positive control consisting of plasmid and hydrogen peroxide is placed under a UV lamp and exposed to the radiation. In addition, three negative controls were prepared, as follows: (i) a sample containing only the plasmid, (ii) a plasmid and hydrogen peroxide without UV irradiation, and (iii) a plasmid irradiated with UV irradiation. After treatment, 1 \(\mu\)L of application buffer containing bromothymol blue and glycerol is added, allowing visual monitoring of the electrophoresis (1 h, 150 mA).