3.7. In Vitro Anti-Glycation Assay

Advanced glycation end-products (AGEs) were produced in vitro using a method described previously [17]. In brief, Bovine Serum Albumin (BSA, 20 mg/mL) in phosphate buffered-saline solution (PBS, pH 7.4) containing 0.02% sodium azide was incubated with glucose (500 mM) at 37 °C for 0, 7, 14, 21, and 28 days in the absence (control) and presence of each of the extracts (100, 200, and 400 μg/mL) or AG (200 μg/mL). Each solution was kept in the dark in a capped vial, and incubation was allowed to proceed in triplicated vials. For time course experiments involving fluorescent AGE formation, we measured the fluorescence (370-nm excitation wavelength and 440-nm emission wavelength) using an F-2500 Spectrofluorometer (Hitachi, Tokyo, Japan). The percentage inhibition was calculated using the following formula:

The effective concentration for 50% inhibition (IC₅₀) was obtained by interpolation of the linear regression analysis. All determinations were performed in triplicate, and the results were averaged and compared using the Duncan’s multiple range test (p < 0.05).