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

Abstract

In this study, we aim to evaluate the anti-diabetic potential of Thunbergia mysorensis 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 IC₅₀: 27.86 ± 1.0, AM IC₅₀: 12.00 ± 0.0, AR IC₅₀: 4.50 ± 0.09 μg/mL) and showed effective radical ion scavenging activity during the antioxidant assay (DPPH EC₅₀: 30.10 ± 0.75, ABTS EC₅₀: 27.25 ± 1.00, Superoxide EC₅₀: 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 T. mysorensis leaves. Both DMBA (IC₅₀ AG: 27.00 ± 1.05, IC₅₀ AM: 12.15 ± 0.10, IC₅₀ AR: 4.86 ± 0.30 μg/mL) and DMCA (IC₅₀ AG: 27.25 ± 0.98, IC₅₀ AM: 12.50 ± 0.20, IC₅₀ 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 EC₅₀: 30.50 ± 0.99, ABTS EC₅₀: 27.86 ± 0.16, Superoxide EC₅₀: 36.10 ± 0.24 μg/mL), and DMCA (DPPH EC₅₀: 31.00 ± 1.00, ABTS EC₅₀: 28.00 ± 0.25, Superoxide EC₅₀: 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 in silico 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 in vitro and in silico. 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.

Communicated by Ramaswamy H. Sarma

Keywords:

• α-Amylase inhibitors
• Thunbergia mysorensis
• polyol pathway
• bioassay-guided fractionation
• phenolic acids
• DMBA
• DMCA
• enzyme inhibition
• enzyme kinetics
• molecular docking simulation
• molecular dynamics simulation

Disclosure Statement

All the authors declare that there are no relevant financial or non-financial competing interests to report.

Additional information

Funding

We acknowledge (i) Vision Group of Science and Technology; Department of IT, BT and S&T; GOK, (GRD-325, 2014-15 (2015-16)) India for the support (ii) JSS Academy of Technical Education, Bengaluru and JSS Academy of Higher Education and Research (JSS AHER), Mysuru for the support and encouragement.