Integrated biophysical, thermodynamic and cellular evaluation of prunetin as an α-glucosidase inhibitor: Insights from ITC, fluorescence quenching and molecular dynamics.

Inhibition of carbohydrate-hydrolysing enzymes, such as α-glucosidase, is an established method for controlling postprandial hyperglycemia in Type 2 diabetes (T2D). In the current work, a detailed mechanistic investigation of the interrelationship between α-glucosidase and prunetin was performed using an integrated approach that combined biochemical, biophysical, and computational techniques. Prunetin was found to possess potent inhibitory activity towards α-glucosidase, with an IC50 value of 40.53 ± 2.67 μg/mL. Fluorescence quenching studies demonstrated a static quenching model, which indicates the formation of a stable complex between the ligand and the enzyme. Most importantly, isothermal titration calorimetry studies demonstrated that the interaction between prunetin and α-glucosidase is primarily driven by hydrogen bonding and van der Waals interactions indicating a favourable binding process. Furthermore, molecular modelling studies demonstrated that prunetin is a stable ligand that is accommodated within the active site of the enzyme through the formation of hydrogen bonds and hydrophobic interactions. Prunetin was found to increase glucose uptake in HepG2 and L6 cells without inducing any cytotoxicity, thus emphasising its functional role. Overall, this work aimed to explore the dual role of prunetin as both an α-glucosidase inhibitor and modulator of glucose uptake. This study also provides a detailed mechanistic understanding of the interrelationship between prunetin and α-glucosidase.