*2.7. Protein Oxidation Measurement: Carbonyl and Free Thiol (SH) Content*

Carbonyl content was estimated to calculate the level of protein oxidation [42]. Briefly, aliquoted protein samples (100 µL) were mixed with 400 µL DNPH (10 mM). After thorough mixing, 500 µL of TCA (20% *w/v*) was added and centrifuged at 10,000× *g* for 10 min. The pellet was washed further with a 1 mL ethanol/ethyl acetate (1:1) mixture and resuspended in 1 mL of 6 M guanidine hydrochloride. The absorbance of the sample was recorded at 370 nm, and the concentration expressed as nanomoles of carbonyls per milligram of protein was determined using 22,000 M−<sup>1</sup> cm−<sup>1</sup> as molar absorptivity.

Ellman's reagent was used to calculate the free thiol content [44]. Native and glycated samples in the absence and presence of caffeic/coumaric acid (250 µL) were incubated with 750 µL of DTNB (0·5 mM) for 15 min, and the absorbance was measured at 412 nm. The concentration of free thiol groups was calculated using a standard curve of L-cysteine and expressed as nanomoles of L-cysteine per milligram of protein.
