Gallic acid 1 hydrate

Folin–Ciocalteu and gallic acid-1-hydrate (C₆H₂(OH)₃COOH·H₂O) were from PanReac (Cascais, Portugal). Quercetin dihydrate (C₁₅H₁₀O₇·2H₂O) was from Extrasynthese (Lyon, France). Anhydrous aluminum chloride, potassium acetate, sodium carbonate, and Tris(hydroxymethyl)amino methane were from Merck (Alges, Portugal). Phenazine methosulfate (PMS), nicotinamide-adenine dinucleotide hydride (NADH), and nitro-blue tetrazolium chloride (NBT) were from Sigma Aldrich (Lisbon, Portugal). All reagents were pro-analysis grade. All absorbance measurements were performed in a Perkin–Elmer (Lisbon, Portugal) Lambda 25. The reagents were weighed on an analytical balance (Sartorius, ±0.00001 g) (Lisbon, Portugal).

Folin–Ciocalteu and gallic acid-1-hydrate (C6H2(OH)3COOH.H2O) were from PanReac (Castellar del Vallès, Barcelona); tannic acid, TPTZ 2,4,6-tri (2-pyridyl)-s-triazine, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid), potassium persulfate (K2S2O8), NADH (nicotine-adenine dinucleotide reduced), NBT (nitro blue tetrazolium) ≥97%, and PMS (phenazine methosulfate) were from Sigma-Aldrich (Buenos Aires, Argentina); 1-butanol, NED (1- naphthyl ethylenediamine dihydrochloride), and sulfanilic acid were from Merck (Darmstadt, Germany); procyanidin A2 was from Extrasynthese (Genay, França); potassium iodate was from VWR Analar Normapur; and sodium nitroprusside was from Riedel-de Haën (Charlotte, NC, USA). All reagents were pro analysis grade. All absorbance measurements were performed in a Perkin-Elmer Lambda 25 (Watertown, MA, USA). The reagents were weighed on an analytical balance (Sartorius ± 0.0001 g).

Total polyphenols were determined using the procedure described by [26 (link)]. According to this, phenolic compounds were oxidized by the Folin–Ciocalteu reagent (Panreac, Barcelona, Spain), resulting in a blue color. The reaction was carried out in an alkaline medium; for this, sodium carbonate was added to the samples and the absorbance was measured at 725 nm by UV-vis spectrophotometry model Evolution 201 (Thermo Fisher Scientific, Walthman, MA, USA). Total content of polyphenols was obtained using a calibration line from a standard solution of gallic acid 1-hydrate (Panreac, Barcelona, Spain), and was expressed as mg-eq· gallic acid·L⁻¹.
Flavonoid compounds were determined by a spectrophotometric method described by [27 (link)]. For this, the following reagents were used: NaNO₂, AlCl₃·6H₂O and NaOH. As an external standard, a 1 g·L⁻¹ catechin stock, (+)-catechin hydrate (Sigma Aldrich, Darmstadt, Germany) in MilliQ water was used. The absorbance value was measured at 510 nm using UV-vis spectrophotometry model Evolution 201 (Thermo Fisher Scientific, Walthman, MA, USA). Total content of flavonoids was calculated by extrapolating the values obtained in the calibration line.