*2.10. UHPLC-MS Analysis*

The quantification of polygodial, citral and anethole, major compounds found in the essential oil of the tested herbs was performed by UHPLC-MS (ultra-high performance liquid chromatography/mass spectrometry) system. Extracts dissolved in methanol (HPLC Grade, Merck, Darmstadt, Germany) for analysis. The system consisted of Dionex UlitMate 3000 (Thermo-Fisher Scientific, Waltham, MA, USA) equipped with a quaternary solvent delivery system coupled to a Thermo-Fisher Q Exactive High Resolution Accurate Mass MS (Thermo-Fisher Scientific, Waltham, MA, USA). The instrument was fitted with an Acquity UHPLC BEH Shield 1.7 μm, 2.1 × 100 mm column (Waters, Milford, MA, USA). Chromatographic separation carried out with mobile phase A (Mili-Q water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid). The linear gradient program was as follows: 0–0.3 min, 5% B; 0.3–2 min, 5–25% B, 2–3.5 min 25–50%, 3.5–4 min 50–80%, 4–7.5 min 80%, 7.5–8 min 80–5% and 8–11 min 5%. The flow rate was 0.5 mL/min, the column temperature was 40 ◦C, the autosampler temperature was 15 ◦C and the injection volume was 1 μL. The UHPLC-MS run in ESI-positive ion mode. Parameters were as follows: capillary temperature 268 ◦C; auxiliary gas heater temperature 438 ◦C; spray voltage 3.5 kV; sheath gas flow rate 52; auxiliary gas flow rate 14; sweep gas flow rate 3; and S-lens RF level 50. Data was collected from 3 to 10 min, over a mass range of 50–300 *<sup>m</sup>*/*<sup>z</sup>*, with a maximum IT of 200 ms and a resolution setting of 70,000 at 200 *<sup>m</sup>*/*<sup>z</sup>*. Quantification of analytes performed by external calibration, using known standard solutions of polygodial (Sigma-Aldrich, St. Louis, MO, USA), citral (Sigma-Aldrich, St. Louis, MO, USA) and anethole (Sigma-Aldrich, St. Louis, MO, USA).

## *2.11. Statistical Analysis*

Results were expressed as mean ± standard deviation and all statistical analyses were performed using GraphPad Prism version 7.00 (GraphPad 2016, Version 7.03, GraphPad Software, Inc., San Diego, CA, USA) and figures were generated in Microsoft Excel (Office 2016, Microsoft, Redmond, WA, USA). Statistical significance of differences among treatment groups done by using one-way analysis of variance (ANOVA) followed by Tukey's test as a post hoc comparison and *p* < 0.05 is considered significant. Pearson's correlation coefficient was used to determine correlation between total phenolic content and antioxidant capacities.
