2.4. Aldose Reductase Activity Assay

Aldose Reductase Inhibitor Screening Kit (BioVision, CA, USA) was used to measure the aldose reductase activity toward (±)KU, (−)AR (lignan that reported as ARI), ZP (potential ARI), EP (positive control) and DMSO (solvent control). The procedure was conducted in accordance with the manufacturer’s instructions. Briefly, aldose reductase enzyme solution was added to a 96-well plate with sodium phosphate buffer (pH 6.2), 0.3 mM NADPH and tested compounds or DMSO. After 20 min incubation at 37 °C, substrate (glucose) was added and we immediately monitored a reduction in the absorbance reading at OD 340 nm in the kinetic mode for 100 min at 37 °C by the Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, MA, USA). All readings were subtracted by background control (enzyme free) and calculated using the following equation: