*4.2. BSA-Glucose Assay*

The BSA-glucose assay was devised by Matsuura et al. [35] to screen for effective AGE inhibitors from natural product extracts. A total of 50 mL of 50 mM phosphate buffer (pH 7.4) and 200 mM glucose were mixed as a stock solution. Then, 500 μL of reaction mixture containing 400 μg of BSA and 200 mM glucose, with or without 10 μL of plant extracts, in 50 mM phosphate buffer at a pH of 7.4 in an Eppendorf tube was prepared. The reaction mixture was heated at 60 ◦C on a heat block for 48 h. A blank, unreacted solution sample without inhibitors was kept at 4 ◦C until measurement. Samples were cooled to room temperature. After cooling, 100 μL aliquots were transferred to new 1.5 mL plastic tubes, and 10 μL of TCA was added to each tube. The tubes were centrifuged at 15,000 rpm at 4 ◦C for 4 min. The supernatant was removed and AGE-BSA precipitate was dissolved in 800 μL of PBS. The fluorescence of the samples was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm, and inhibitory activity was calculated as a percentage using the following formula:

I% = [1 − (fluorescence of BSA + glucose + inhibitor − fluorescence of BSA +

inhibitor)/(fluorescence of BSA + glucose) − (fluorescence of BSA)] × 100%

#### *4.3. BSA-MGO Assay*

The middle stage of protein glycation was determined using the BSA-MGO assay described by Wu et al. [8], with a slight modification. BSA (50 mg/mL) was incubated with 100 mM MGO under sterile conditions in 0.1 M phosphate buffer (pH 7.4) at 37 ◦C for 9 days. Sample fractions and subfractions were added to the model system at final concentrations of 125 μg/mL and 250 μg/mL, respectively. The sample solutions were kept at 4 ◦C until measurement. After cooling, 100 μL of aliquots were transferred to 1.5 mL plastic tubes, and 10 μL of 100% TCA was added to each tube. Tubes were centrifuged at 15,000 rpm at 4 ◦C for 4 min. The supernatant was removed, and the precipitate of MGO-BSA was dissolved in 800 μL of PBS. The fluorescence of the samples was measured at the excitation and emission maxima of 330 and 410 nm, respectively, and compared with that of the unincubated blank that contained the protein, MGO, and inhibitors. The percent inhibition was calculated as follows:

I% = [1 − (fluorescence of BSA + MGO + inhibitor − fluorescence of BSA + inhibitor)/(fluorescence of BSA + MGO) <sup>−</sup> (fluorescence of BSA)] <sup>×</sup> 100% (1)
