*3.3. Inhibition of Advanced Glycation End-Products (AGEs)*

AGE formation is common at the later glycation stages in proteins, and most of the end products are fluorogenic in nature [53]. Caffeic acid and coumaric acid were studied for their inhibitory effects on AGE formation (Figure 4). HSA was incubated with MG, fluorescence was taken of native HSA, and MG + HSA was incubated. HSA incubated with MG showed high fluorescence compared to the native HSA, concluding the formation of AGEs. In the presence of varying concentrations (50–200 µM) of ligands, a dose-dependent decrease in the fluorescence emission was evident, with maximum inhibition observed for 200 µM for both the ligands (Figure 4A).

**Figure 4.** Inhibition of fluorescent advance glycation product (AGEs) by (**A**) caffeic acid and (**B**) p-coumaric acid.

Treatment of MG incubated HSA in the presence of 50, 100 and 200 µM ligands showed a reduction in fluorescent AGEs. Caffeic and coumaric acid both showed a significant decrease (79.2% and 43.6%) respectively at 200 µM concentration (Figure 4A,B). The antiglycative properties of caffeic acid and coumaric acid have been investigated in previous studies [53,54]. AGEs are the key players in the pathophysiology and progression of many diseases highlighting their clinical significance [30]. Strong inhibition of AGEs by caffeic acid compared to coumaric acid could be due to structural differences among them, where caffeic acid possesses 1 more OH group and might play an important role in AGE inhibition.

The results indicated that the binding of caffeic acid and coumaric acid inhibits the formation of AGEs. Thus, a conclusion was obtained that both the ligands attenuate the effect of MG by networking with HSA and reduce the fluorescence by AGEs. AGEs not only create a menace in diabetes but also contribute to other fatal diseases [29,53], signifying an urgent need to stop AGEs formation [54]. Antiglycation activities of caffeic and coumaric acid could be due to its antioxidant, ROS scavenging activity and protein-stabilizing potential. Earlier, caffeic and ferulic acid have been found to attenuate glycation and thus diabetic complications [55,56]. Additionally, the binding analysis of these phenolics with αamylase hypothesized its inhibitory potential and thereby reduced glucose concentrations in serum.
