2 4 6 tris pyridin 2 yl 1 3 5 triazine tptz

The FRAP (ferric reducing antioxidant power) assay measures the ability of antioxidants to reduce the ferric 2,4,6-tripyridyl-s-triazine complex [Fe (TPTZ)₂]³⁺ to the intensely blue ferrous complex [Fe (TPTZ)₂]²⁺ (λ = 593 nm) in acidic medium [58 (link),59 (link)]. 2,4,6-Tris(pyridin-2-yl)-1,3,5-triazine (TPTZ), FeCl₃·6H₂O, Trolox, quercetin and DMSO were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The FRAP reagent contained 2.5 mL of 10 mM TPTZ solution in 40 mM HCl, 2.5 mL of 20 mM FeCl₃·6H₂O water solution, and 25 mL of 0.3 M acetate buffer (pH 3.6). Aliquots of 10 µL of the test compound solution in DMSO (0.5 mM) were mixed with 240 µL of FRAP reagent, and the absorbance of the mixture was measured spectrophotometrically (FLUOStar OPTIMA microplate spectrophotometer (BMG Labtech, Ortenberg, Germany)) at 600 nm after 1 h incubation at 37 °C against a blank. Trolox was used as a reference compound. quercetin was used as a positive control. A calibration curve with linear formula y = 0.03x (R² = 0.998, p < 0.0001) was prepared for Trolox solutions in DMSO at a range of 2.5–100 µM, and the results are expressed as Trolox equivalents (TE), the values calculated as the ratio of the concentrations of Trolox and the test compound, resulting in the same effect.

Lipoid S75 (fat-free soybean phospholipids with 70% phosphatidylcholine; S75) was supplied by Lipoid GmbH (Ludwigshafen, Germany). Standards of phenolic compounds (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, peonidin-3-O-glucoside, petunidin-3-O-glucoside, gallic acid, ellagic acid, myricetin-3-O-galactoside, and myricetin-3-O-rhamnoside) were purchased from Extrasynthese (Genay Cedex, France). Ultrapure water (18 MΩ cm) was obtained with a Milli-Q Advantage A10 System apparatus (Millipore, Milan, Italy). Acetonitrile for ultra-phase liquid chromatography (UPLC, gradient grade) analyses, methanol, phosphoric acid, phosphate buffered saline (PBS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tris(pyridin-2-yl)-1,3,5-triazine (TPTZ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other reagents were purchased from Sigma-Aldrich/Merck (Milan, Italy), unless otherwise specified.

Phospholipid Lipoid S 75 (S75) was from Lipoid GmbH (Ludwigshafen, Germany). Malvidin-3-O-glucoside (Mv-3-O-glc) was provided by Extrasynthèse (Genay, France). HPLC-grade acetonitrile (MeCN) and methanol (MeOH) were purchased from Romil Chemical Ltd. (Heidelberg, Germany). Trifluoroacetic acid (TFA), Folin–Ciocalteu’s reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(pyridin-2-yl)-1,3,5-triazine (TPTZ) were from Merck/Sigma-Aldrich (Darmstadt, Germany).