2.1. Ferric reduction antioxidative power (FRAP) assay

To identify the strongest and most potentially useful antioxidative nutraceutical, the ferric reducing/antioxidant power (FRAP) assay was performed [33] in the absence of chondrocytes. In the FRAP assay, aqueous ferric tripyridyltriazine (Fe³⁺-TPTZ) is added in excess to be reduced by antioxidants. The redox reaction is coupled with a color change from pale yellow to blue that is maximally absorbed at 593 nm. The higher the antioxidative power of a nutraceutical, the better its ability to reduce iron from its ferric to ferrous form, and the larger the measured absorbance. Here, the assay was conducted following the procedure described by Benzie and Strain [34]. Briefly, the FRAP reagent was prepared by mixing 10 mL of 0.3 M acetate buffer at a pH of 3.6, with 1 mL of 10 mM tripyridyl-S-triazine (TPTZ) in 40 mM hydrochloric acid (HCl), and 1 mL of 20 mM ferric chloride. An amount of 100 μL of each nutraceutical, i.e. gallic acid, catechin hydrate, ascorbic acid, curcumin, α-tocopherol, or carvacrol, sample was placed inside a well of a 96-well plate, and to that, 300 μL of FRAP reagent was added. Mixtures were allowed to react and equilibrate for 6 min at 37 °C. The absorbance was then read at 594 nm using a Cytation 5 multi-plate reader (BioTek Instruments, Inc). A standard curve was prepared using an aqueous solution of ferrous sulfate at 7 different dilutions, 50, 100, 250, 500, 170, and 1000 μM, and deionized water was used as a blank. FRAP values were reported as mM equivalents of ferrous (Fe²⁺) species and were calculated using the following equation [35].