2 20 azobis 2 amidinopropane dihydrochloride

The reagents used were 2,2-diphenyl-1-picrylhydrazil (DPPH^•), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,20-azobis(2-amidinopropane) dihydrochloride (AAPH), fluorescein sodium salt and Folin & Ciocalteu^’s phenol reagent, all purchased from Sigma-Aldrich. Ferrous sulfate heptahydrate and gallic acid were purchased from Vetec, and sodium carbonate from J.T Baker were also used. All solvents and chemicals were of analytical grade.

All reagents and chemicals used were of analytical or HPLC grade. The solvents used for extraction and the sodium hydroxide were obtained from J. T. Baker (Ecatepec, Estado de Mexico, Mexico), as were the solvents used for chromatographic mobile phases, and Millipore water was obtained from Merck (Merck, Darmstadt, Germany). Sodium carbonate, Folin-Ciocalteu reagent, gallic acid, quercetin, aluminum chloride and sodium nitrate were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,20-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were also from Sigma-Aldrich.

Antioxidant activity was determined using the oxygen radical absorbance capacity assay. Synthesized peptides were evaluated using a previously-described method [28 (link)]. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the standard (Sigma-Aldrich, St. Louis, MI, USA) with fluorescein (Sigma-Aldrich, Saint Louis, MI, USA). 2,20-Azobis (2-amidinopropane) dihydrochloride (Sigma-Aldrich, Saint Louis, MI, USA) was used to generate peroxyl radicals, and the fluorescence loss signal was monitored for 1 h using a microplate reader (Biotek Synergy HTX, Winooski, VT, USA). The absorbance of excitation and emission were set at 485 and 538 nm, respectively. The results were expressed as µmol of Trolox equivalents (TE) per gram of peptide.