Potassium persulphate

The ABTS radical was generated by the oxidation of ABTS^●+ 7 mM with potassium persulphate 2.45 mM (Merck, Darmstadt, Germany) in water, and stored for 12 to 16 h under the light, at room temperature [45 (link)]. Posteriorly, buffered saline (PBS) at pH 7.0 was added to this solution to achieve an absorbance of 0.7 ± 0.02 at the wavelength of 734 nm. Then, 50 µL of the sample was mixed with 2 mL of ABTS^●+ and vortexed for 10 min. The reaction was incubated for 4 min being its absorbance measured at 734 nm. The correspondent IC₅₀ value was calculated. All the determinations were performed in triplicate.

Gallic acid (purity > 98%), quercetin, (purity > 97%), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (purity > 97%), 2,2′-azinobis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), aluminum chloride, iron (III) chloride hexahydrate, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA), adenosine 5′-triphosphate (ATP) disodium salt, acetylcholinesterase from Electrophorus electricus (electric eel) and butyrylcholinesterase from equine serum were from Sigma-Aldrich^® (Chile). The enzyme 5-lipoxygenase (human, recombinant) were from Cayman Chemical (Item No. 60402). Calcium chloride, sodium carbonate, sodium hydroxide, sodium nitrite, and potassium persulphate were obtained from Merck^® (Chile). Acetone, galanthamine, zileuton, sodium acetate trihydrate, and glacial acetic acid were from Merck^® (Chile). HPLC reagents, formic acid, methanol, ultrapure water, ethyl acetate, and n-hexane were form Merck^® (Chile). Deionized water was used for all the experiments.

The spices and herbs were tested for their ABTS radical scavenging activity according to the method of Re et al. [51 (link)], with some modifications. Two stock solutions of 7 mM ABTS (Sigma-Aldrich, Burlington, MA, USA) in double-distilled water and 2.45 mM potassium persulphate (Merck, Lethabong, South Africa) were mixed. The mixture was incubated in the dark for 12–16 h at room temperature and used as a working solution. The solution was adjusted with cold ethanol to obtain an absorbance of 0.700 (±0.02) at 732 nm using a microplate reader (Spectra Max M2, Molecular Devices Inc., Silicon Valley, CA, USA). A 100 µL volume of ABTS+ solution was added to the microtiter plate wells containing 100 µL of crude extracts or essential oils. After 30 min of incubation, the percentage of decolorization of ABTS+ at 734 nm was calculated for each concentration relative to the blank, according to Equation (2). Ascorbic acid (22.5 µg/mL) was used as a positive control.

where A = absorbance at 734 nm;

A_control = average absorbance of ABST⁺ − average absorbance of methanol;

A_methanol = average absorbance obtained in the wells containing methanol;

A_test = average absorbance obtained in the wells containing ABST⁺ and the test sample.

For determination of the serum TAS, ferric reducing antioxidant potential (FRAP) was performed as previously described [52 (link)], whereas the scavenging of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^●+) assay was employed as described by Gião et al. [53 (link)].
The ABTS^●+ stock solution was prepared by reacting equal amount of 7 mM ABTS diammonium salt (Sigma-Aldrich, St. Louis, MO, USA) and 2.45 mM potassium persulphate (Merck, Damstadt, Germany). The reaction was developed for 16 h in the dark. Aliquots of serum samples (10 µL), diluted when needed, were added to 1 mL of ABTS^●+ solution with an initial optical density (OD) of 0.70 ± 0.02 measured at 734 nm. After allowing the reaction to occur, the OD was recorded using an UV-Vis spectrophotometer (UVmini 1240, Shimadzu, Japan) and the results were calculated as inhibition percentage (IP) of ABTS^●+, according to the equation ABTS^●+ inhibition (%) = 100 − [(OD sample × DF)/OD ABTS] × 100, where OD sample indicates the sample absorbance following 6 min of reaction, DF is the dilution factor and OD ABTS refers to the initial absorbance of the diluted ABTS^●+ solution. All measurements were performed in triplicate.

Sodium carbonate (lot a0594092339), anhydrous sodium acetate (lot 882032), absolute ethanol (lot k4958171742), potassium persulphate (lot k40707991048), methanol (lot l823009611), acetic acid glacial (lot k47109963539), acetone (lot k43912814243), sodium hydroxide (lot b0895598319), concentrated hydrochloric acid (lot k41017317016), Folin–Ciocalteu phenol reagent (lot hc43368401), Mueller–Hinton broth (lot b1223098542), tetrahydrofuran (lot dg643), and 2,4,6-tri-(2-pyridyl)-1,3,5-triazine (lot l58155738107) were obtained from Merck (Darmstadt, Germany). Dimethyl sulfoxide (lot 190260) was obtained from PanReac AppliChem (Barcelona, Spain). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2, 2-diphenyl-1-picrylhydrazil (067k lot 1154), and diammonium salt (lot slbp9592v) were obtained from Sigma-Aldrich (St. Louis, MO, USA). (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (97% lot stbb6668) was obtained from Aldrich Chem (St. Louis, MO, USA). Ferric chloride. 6H₂O (lot 9n005099n) was obtained from Carlo Erba (Barcelona, Spain). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (lot p31b064) was obtained from Alfa Aesar (Haverhill, MA, USA). Annatto seeds were purchased from a farmers market of Medellín city (Colombia), which were sun-dried until reaching a moisture content of 10.58 ± 0.98%.

The ABTS assay was performed according to the methods used by Re et al. [35 (link)]. An aqueous solution of 7 mM ABTS^●+ and 2.45 mM potassium persulphate (Merck, Darmstadt, Germany) was prepared. After 16 h, in the dark at room temperature, this solution was diluted with phosphate-buffered saline (PBS) at pH 7 to achieve an absorbance of 0.7 ± 0.02 at 734 nm. The ABTS assay was performed by adding 50 µL of the extract to 2 mL of the ABTS ^●+ solution and vortexed for 10 s. After an incubation of 4 min, the absorbance of the reactional mixture was measured at the wavelength of 734 nm. The IC₅₀ value was calculated from the interpolation of the graph % of ABTS vs. concentration in µg/mL. Trolox was used as a positive control, and the results were also expressed as TE (Trolox equivalents). Three independent experiments in duplicate were performed for each sample.