Magellan v7

For ORAC the method of Davalos et al. [82 (link)] was used. Extracts were diluted with 75 mM phosphate buffer (pH 7.4). The assay was carried out in black-walled 96-well plates (Greiner-Bio One) and each well contained a final volume of 200 μL. To each well 20 μL of extract and 120 μL of fluorescein (FL; 70 nM final concentration) were added and the plate was incubated at 37 °C for 15 min. The AAPH (60 μL; 12 mM final concentration) was added to each well and fluorescence intensity was estimated using an Infinite200 Pro plate reader (Tecan, Männedorf, Switzerland), every minute for a total of 80 min using an excitation wavelength of 485/9 nm and an emission wavelength of 535/20 nm. A standard curve was constructed using 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, Sigma-Aldrich, Oakville, ON, Canada, 1.5–10.5 μM). A blank (fluorescein + AAPH) using phosphate buffer instead of the antioxidant solution was carried out in each assay. Results were determined by using Magellan v 7.2 software (Tecan, Männedorf, Switzerland), on the basis of the difference in area under the curve between the control and the sample and expressed as μmoles of Trolox equivalents (TE) per g of lipidic extract. All the reaction mixtures were prepared in triplicate and at least three independent assays were performed for each sample.

The TEAC assay was performed adapting the method described by Re et al. [100 (link)] to a microplate reader. 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma-Aldrich, Oakville, ON, Canada) radical cations were prepared by mixing potassium persulfate 2.45 mM (final concentration) and an aqueous solution of ABTS 7 mM (final concentration) in the dark at room temperature for 12–16 h. The ABTS radical cation solution was diluted in PBS (pH 7.4) to an absorbance of 0.40 at 734 nm ± 0.02. A standard calibration curve of Trolox (0–16 μM) was constructed. A volume of 10 μL of Trolox or extracts diluted in PBS were added in the wells of a 96 well-plate (Costar, MERCK, Darmstadt, Germany) with 200 μL of diluted ABTS. Afterwards the absorbance reading at 734 nm was taken 6 min after initial mixing using an Infinite200 Pro plate reader (Tecan, Männedorf, Swizerland). Appropriate solvent blanks were run in each plate. The lipidic extract was assayed in at least three separate dilutions and in triplicate. The inhibition of absorbance at 734 nm of the lipidic extract was plotted as a function of concentration of Trolox and the TEAC value expressed as Trolox equivalent (in micromolar) per g of lipidic extract, using Magellan v 7.2 software (Tecan, Männedorf, Switzerland).