3.5.2. Aldose Reductase Inhibition Assay

A 96-well plate-based AR inhibition assay was used in this study, which was modified from a previous method [14]. In principle, the rate of the absorbance decrease of NADPH at 340 nm (OD340 nm) was used for evaluation of the AR inhibitory activity. In brief, 110 µL of 0.1 M PBS (pH 7.0), 20 μL of AR homogenate, 20 μL of NADPH (2.4 mM in 0.1 M PBS, pH 8.0), 10 μL of sample (extract 12.5–200 μg/mL; compounds 0.98–1000 μM), and 20 μL of ammonium sulfate solution (4 M in 0.1 M PBS, pH 7.0) were sequentially added into a 96-well plate. Then, as soon as 20 μL of the substrate (DL-glyceraldehyde dimer, 25 mM in 0.1 M PBS, pH 7.0) was added, the absorbance of the reaction solution at 340 nm was measured for 6 min using an Epoch microplate spectrophotometer (BioTek Instruments, Highland Park, IL, USA). Quercetin was used as a positive control. The samples were first prepared at high concentrations in DMSO or in a mixture of DMSO and water and then diluted using water shortly before the experiment. The concentration of DMSO was kept within 0.2% (v/v) in the reaction system. Particularly, in order to ensure the results obtained from different times of experiments are comparable, the concentration of AR stock homogenate was adjusted by a proper dilution using 0.1 M PBS (pH 6.2) to satisfy the value of |Slope_c| with about 0.038–0.042 as explained below. The AR inhibition ratio (%) for the samples was calculated using Equation (4):

where Slope_c, Slope_s, and Slope_b are the slopes obtained from the OD340 nm versus the reaction time (min) dotted lines of the control group (without sample), sample group (with enzyme and sample), and blank group (without enzyme or sample), respectively. |Slope| is the absolute value of Slope.