Trolox

The free-radical scavenging activity of the extract was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [22 ]. Four hundred mL of the extract was mixed with 3.6 mL DPPH solution (0.025  g DPPH, Sigma-Aldrich; in 100  mL methanol, Centralchem). The absorbance of the reaction mixture was determined at 515 nm using the Jenway 6405 UV/Vis spectrophotometer (Cole-Pharmer Ltd.). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; 10–100  mg/L; Sigma-Aldrich) was used as the standard, and the results are expressed in mg Trolox equivalents (TEAC)/g d.w. The measurement was performed in triplicate [16 , 22 ].
Molybdenum-reducing antioxidant power (MRAP) of the extract was determined by the method of Prieto et al. [23 (link)] with slight modifications. The mixture of the extract (1  mL), monopotassium phosphate (2.8  mL, 0.1 M; Centralchem), sulfuric acid (6  mL, 1 M; Centralchem), ammonium heptamolybdate (0.4  mL, 0.1 M; Centralchem), and distilled water (0.8  mL) was incubated at 90°C for 120 min and, then, rapidly cooled. The absorbance was determined at 700 nm with the Jenway 6405 UV/Vis spectrophotometer (Cole-Pharmer Ltd.). Trolox (10–1000 mg/L; Sigma-Aldrich) was used as the standard, and the results are expressed in mg Trolox equivalents (TEAC)/g d.w. The assessment was performed in triplicates [16 , 23 (link)].

A 0.1 nM P2Y₂ imager strand buffer solution (5-TTGTGGT-3’-Atto643, Eurofins) and a 0.2 nM αV imager strand buffer solution (5-GGAGGA-3’-Atto643, Eurofins) were made using 1x PCA (Sigma-Aldrich), 1x PCD (Sigma-Aldrich), 1x Trolox (Sigma-Aldrich), 1x PBS, and 500  mM NaCl (Merck) which facilitates the establishment of an oxygen scavenging and triplet state quencher system. Solutions were incubated for 1 hr in the dark before use. Stock solutions of PCA, PCD, and Trolox were prepared as follows: 40x PCA (protocatechuic acid) stock was made from 154 mg of PCA (Sigma-Aldrich) in 10 mL of Ultrapure Distilled water (Invitrogen) adjusted to pH 9.0 with NaOH (Avantor, Radnor Township, PA, USA). 100 x PCD (protocatechuate 3,4-dioxygenase) solution was made by adding 2.2 mg of PCD (Sigma-Aldrich) to 3.4 mL of 50% glycerol (Sigma-Aldrich) with 50 mM KCl (Sigma-Aldrich), 1 mM EDTA (Invitrogen), and 100 mM Tris buffer (Avantor). 100 x Trolox solution was made by dissolving 100 mg of Trolox in 0.43 mL methanol (Sigma-Aldrich), 0.345 mL 1 M NaOH, and 3.2 mL of Ultrapure Distilled water.

The radical scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma Aldrich, Schnelldorf, Germany) was used to measure the antioxidant activity of TTEO. DPPH was dissolved in methanol to concentration 0.025 g/L and was adjusted to absorbance 0.8 at wavelength 515 nm (by Glomax spectrophotometer, Promega Inc., Madison, WI, USA). A 5 μL volume of EO sample was added to 195 μL DPPH solution in a 96-well microplate and was incubated for 30 min in the dark with shaking at 1000 rpm. The percentage of DPPH inhibition was calculated according to the formula (A0 − AA)/A0 × 100, where A0 is the absorbance of DPPH with methanol, and AA is the absorbance of the sample. The standard reference Trolox (Sigma Aldrich, Schnelldorf, Germany) was used for calculation of total antioxidant capacity. Trolox was dissolved in methanol (Uvasol^® for spectroscopy, Merck, Darmstadt, Germany) to the concentration range 0–100 µg/mL. Total antioxidant activity was expressed according to the calibration curve as 1 μg Trolox to 1 mL EO sample (TEAC).