Antioxidant assays

The free DPPH radical scavenging activity was detected according to the method described by Brand-Williams et al.⁶². Briefly, 10 µL of EOs was added to 190 µL of DPPH methanol solution, and the absorbance was measured at 517 nm after reacting in the dark for 20 min. The control was replaced with methanol and the same volume of essential oil. Each determination was performed in triplicate, and the results are expressed as micromole Trolox equivalents per millilitre of essential oil (μmol eq Trolox/mL EO).

The ABTS radical scavenging assay was performed according to the method of Re et al.⁶³ with some modifications. The ABTS radical cation was obtained by reacting 5 mL of 7 mM ABTS (2,2′-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) solution with 88 μL of 140 mM K2S2O8 in the dark for 12 h. The solution was diluted with ethanol until the absorbance at 734 nm was 0.70 ± 0.02, which was used as the ABTS solution. After mixing 10 μL of diluted EO with 990 μL of ABTS solution and incubating in the dark for 6 min, the residual absorbance of ABTS radicals was measured at 734 nm. In the control group, the essential oil was replaced with the same volume of deionised water. Each measurement was performed in triplicate, and the results are expressed as micromole Trolox equivalent per millilitre of essential oil (μmol eq Trolox/mL EO).

The FRAP assay was performed following the methods of Benzie and Strain⁶¹. A TPTZ (2,4,6 -tripyridine-s-triazine) solution was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ working solution and 20 mM FeCl₃·6H₂O solution at a ratio of 10:1:1, followed by heating to 37 °C. Briefly, 10 μL of EOs was added to 300 μL of TPTZ solution, the reaction was carried out at 37 °C for 10 min, and the absorbance was measured at 593 nm. In the control group, the essential oil was replaced with the same volume of deionized water. Each measurement was performed in triplicate, and the results are expressed as micromole Trolox equivalent per millilitre of essential oil (μmol eq Trolox/mL EO).

The TPC of the EOs was determined by the Folin-Ciocalteu method⁷⁶. Briefly, 2 mL Eppendorf tubes were filled with 10 µL of EOs, and then 200 µL of deionized water and 50 µL of Folin-Ciocalteu reagent were added and shaken thoroughly. After 6 min, 500 µl of 7% sodium carbonate and 400 µl of deionized water were added, the solution was mixed, the reaction was kept away from light for 90 min, and the absorbance was measured at 760 nm. The control was deionized water, and each treatment was repeated three times. The results are presented as the gallic acid content (mg GAE/mL EO).