4 amino 3 hydrazino 5 mercapto 1 2 4 triazole

We purchased 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), acetone, acetonitrile, citric acid, DPPH, ferrous-ion-o-dianisidine, Folin–Ciocalteu phenol reagent, formic acid, gallic acid, hydrochloric acid, methanol, β-nicotinamide adenine dinucleotide 2-phosphate reduced tetrasodium salt (NADPH), ultrapure phenolic standards, 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald), sodium acetate, superoxide dismutase determination kit, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Ascorbic acid, boric acid, ferric chloride, ferrous sulfate, hydrogen peroxide, potassium chloride, sodium carbonate, sodium hydroxide, and sulfuric acid were obtained from Vetec (Rio de Janeiro, RJ, Brazil). Cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, delphinidin, quercetin-3-O-glucoside, 3-O-caffeoyl-quinic acid, and myricetin were purchased from Extrasynthese (Genay, France). HA, PCA, VA, isovanillic acid (iVA), ellagic acid, FA, and kaempferolwere purchased from Sigma-Aldrich (St Louis, MO, USA). The kit to analyze urine creatinine was purchased from Labtest Diagnóstica S.A. (Lagoa Santa, MG, Brazil).

Catalase activity was determined according to the method described [17 (link)]. Standard concentrations of 5, 15, 30, 45, 60, and 75 μM were prepared in sample buffer (250 mM potassium phosphate buffer, pH 7.5 containing 0.1% bovine serum albumin, and 1 mM ethylenediamine-tetra acetic acid [EDTA]) using a formaldehyde standard (99.9%, VWR Prolabo®, France). A 0.5095 mg/mL bovine liver CAT (Sigma-Aldrich) was used as a positive control. aliquots of 0.1 mL of assay buffer (100 mM potassium phosphate buffer, pH 7.0) and 0.02 mL of each serum, standard, and positive control were mixed with 0.03 mL of methanol in designated reaction wells in a microplate to perform the assay. The reaction was initiated by adding 0.02 mL of 40 mM hydrogen peroxide and incubating it on an orbital shaker (VRN-360, Gemmy, Taiwan) at 1× g for 20 min at room temperature (25°C). A total of 0.03 mL each of 0.01 mM potassium hydroxide and 63 mM of 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Sigma-Aldrich) was added to each reaction well and incubated for 10 min. A solution of 0.01 mL of potassium periodate (20 mM) was added to all reaction wells and incubated on an orbital shaker at 1× g for 5 min. The microplate was transferred to a spectrophotometer (Multiskan Sky, Thermo Fisher Scientific, USA) for ABSR.