*3.5. Free–SH Groups Content*

Free sulfhydryl group alterations are an important parameter to estimate oxidative modification. A disulfide bond between the sulfhydryl groups is formed due to oxidation. The oxidative modifications in HSA resultant of glycation were correlated with that of free sulfhydryl content. Figure 5B shows the free thiol group estimated for native HSA, protein incubated with MG and different concentrations of caffeic acid and coumarin. Native HSA was used as a control and was found to have 48.23 nmol/mg protein, while MG treated protein had 6.68 nmol/mg protein. Free sulfhydryl contents relate directly to the oxidation of HSA [53]. With the increase in the concentration of both the ligands, an increase in the free thiol group of HSA was observed. The maximum value was obtained at 200 µM of caffeic acid and coumaric acid. Caffeic acid increased the content of thiol to 17.8, 26.2 and 44.3 nmol/mg for 50, 100 and 200 µM, respectively. Similarly, for the same concentration of coumaric acid, the values were 5.8, 7.2 and 9.7 nmol/mg, respectively (Tables 3 and 4).

**Table 4.** Effect of different concentrations of p-coumaric acid on alpha amylase.


Glycation reactions generate ROS that works against the oxidative defense mechanism of the protein group [57]. Thus, the above observations show that caffeic acid and coumarin have the potential to reduce free thiol group, resisting glycation. The results that are presented here are supported by previously published results [29,57,58].
