3.7. Antioxidant Activity Evaluation of Thymol and Carvacrol

Thymol and carvacrol in vitro antioxidant activity was tested via DPPH and ABTS assays, according to the protocols described by Luo et al. [50]. Briefly, DPPH solution (100 µM) was prepared in methanol and its absorbance was measured at 517 nm. Calibration curves (r2 = 0.999) were obtained with thymol or carvacrol standard solutions (2 to 25 ppm). An amount of 3 mL of standard or sample solution was mixed with 1 mL of DPPH solution and their absorbance was measured at 517 nm. The radical scavenging activity percentages (% RSAs) were calculated with the following equation:

in which A_S represents the sample’s absorbance, whereas Arad is the absorbance of the bare DPPH radical. Each measurement was performed in triplicate and expressed as mean ± standard deviation. Ascorbic acid (50 µM) scavenging activity on DPPH radicals was also assessed and compared to the extract’s activity. All the assays were performed using a UV–visible Spectrophotometer UV-1900i, (Shimadzu, Milan, Italy).

Regarding the ABTS test, the radical ABTS was dissolved in PBS (0.01 M, pH 7.4) at a concentration of 7 mM, the radical cation was obtained after 16 h of reaction in the dark with 2.45 mM ammonium persulfate (APS), and it was then diluted to absorbance 0.70 ± 0.02 at 734 nm before use. An amount of 0.2 mL of the sample dissolved in ethanol (in the range 1–300 μg/mL), mixed with 2.0 mL of ABTS+, and the absorbance was measured at 734 nm after 6 min. The antioxidant activity was calculated using the following equation:

where A0 is the ABTS+ absorbance, As is the absorbance of ABTS+ with the sample, and Ab is the absorbance of the sample without the radical cation.

Furthermore, the DPPH and ABTS tests were also performed on Zn-modified clays after the loading of thymol or carvacrol. Composite samples were extracted in methanol or ethanol for DPPH or ABTS assays, respectively. Different extractions times were evaluated from 0 to 24 h; however, we observed no changes in the extraction performances depending on the time since methanol and ethanol proved to be good solvents for solubilizing both analytes and a complete extraction was obtained immediately. However, to allow for the correct comparison between free thymol or free carvacrol activity and that of the same molecules loaded in Zn-modified clays, the tests were performed considering the loading percentages of thymol or carvacrol in the clays evaluated by GC-MS (Section 3.5).