Tptz 2 4 6 tripyridyl s triazine solution

Antioxidant capacity was determined by FRAP (ferric reducing antioxidant power assay) method as described by Benzie & Strain (1996) (link). A total of 250 mL of acetate buffer (POCH, Gliwice) at pH 3.6 was mixed with 25 mL of TPTZ-2,4,6-tripyridyl-s-triazine solution (Sigma–Aldrich, St. Louis, MO, USA) and 25 ml of iron (III) chloride hexahydrate solution (Chempur, Piekary Śląskie, Poland). The reaction mixture was prepared immediately prior to assay. The solution was incubated at 37 °C, throughout the analysis. A total of 6 mL of reaction mixture was mixed with 200 µL of sample and 600 µL of H₂O. The absorbance of the mixture was measured at 593 nm (Shimadzu UV-1800, UV Spectrophotometer System, Kyoto, Japan) after 4 minutes. Acetate buffer was used as a control. The control sample absorbance value was subtracted from the test sample values. Based on the performed measurements, absorbance values’ dependence on sample concentrations was plotted. That was used to determine the absorbance value at the mean concentration of the applied dilutions, and anti-oxidative capacity was calculated at the same absorbance using the standard curve determined for Fe²⁺ ions.

Gallic acid, tannic acid, epicatechin, trolox, sodium acetate, Folin–Ciocalteu’s phenol reagent, DPPH• (2, 2-diphenyl-1-picrylhydrazyl), sodium nitrite, ferric chloride, aluminium chloride, TPTZ (2,4,6-tripyridyl-s-triazine) solution, and ferrous sulfate heptahydrate were all obtained from Sigma–Aldrich (St. Louis, MO, USA). Sodium carbonate anhydrous, sodium hydroxide and absolute ethanol were purchased from Merck (Darmstadt, Germany). Ultrapure water was treated in a Milli-Q water purification system (Millipore, Bedford, MA, USA) and used to prepare all aqueous solutions.