α-Amylase inhibitory potential of leaves extract and bioactive compounds by in vitro and computational approach.

In this study, we aim to evaluate the anti-diabetic potential of leaves methanolic extract (MeL) using inhibitory assays for α-glucosidase (AG), α-amylase (AM) (carbohydrate digestive enzymes) and aldose reductase (AR) (an enzyme involved in the polyol pathway responsible for glycation). In addition to antidiabetic studies, antioxidant studies were also performed due to the fact that reactive oxygen species (ROS) are produced by various pathways under diabetic conditions. Hyperglycemia induces ROS by activating the glycation reaction and the electron transport chain in mitochondria. The MeL effectively inhibited the enzymes (AG IC: 27.86 ± 1.0, AM IC: 12.00 ± 0.0, AR IC: 4.50 ± 0.09 μg/mL) and showed effective radical ion scavenging activity during the antioxidant assay (DPPH EC: 30.10 ± 0.75, ABTS EC: 27.25 ± 1.00, Superoxide EC: 35.00 ± 1.50 μg/mL). Using activity-guided repeated fractionation on a silica gel column chromatography, two compounds including 3,4-dimethoxy benzoic acid (DMBA) (101 mg) and 3,4-dimethoxy cinnamic acid (DMCA) (87 mg) with potent anti-diabetic activity were extracted from the MeL of leaves. Both DMBA (IC AG: 27.00 ± 1.05, IC AM: 12.15 ± 0.10, IC AR: 4.86 ± 0.30 μg/mL) and DMCA (IC AG: 27.25 ± 0.98, IC AM: 12.50 ± 0.20, IC AR: 5.00 ± 1.00 μg/mL) were subjected for enzyme inhibition. Since both compounds significantly inhibited AM, enzyme kinetics for AM inhibition was performed. The compounds also showed effective antioxidant potential (DPPH EC: 30.50 ± 0.99, ABTS EC: 27.86 ± 0.16, Superoxide EC: 36.10 ± 0.24 μg/mL), and DMCA (DPPH EC: 31.00 ± 1.00, ABTS EC: 28.00 ± 0.25, Superoxide EC: 36.25 ± 0.37 μg/mL). Further, to elucidate the role of DMBA and DMCA in enzyme inhibition and stability at the molecular level, both compounds were subjected for enzyme inhibitory studies using molecular docking simulation, molecular dynamics (MD) simulation, and binding free energy calculations. Compared to AR and AG, AM was the most significantly inhibited enzyme (DMBA: -6.6 and DMCA: -7.8 kcal/mol), and compounds combined with AM were subjected to MD simulation. Both compounds were stable in the binding pocket of AM till 100 ns and chiefly use Van der Waal's energy to bind. Compared to the controls, both DMBA and DMCA had a higher efficiency in the inhibition of target enzymes and . The presence of DMBA and DMCA is more likely to be associated with the potential of MeL in antihyperglycemic activity. This bio-computational study indicates DMBA and DMCA as potential lead inhibitors of AM and could be used as effective anti-diabetic drugs in the near future.