Thymoquinone Exhibits Dose-Dependent, Multi-Enzyme Inhibitory Activity Against α-Amylase, α-Glucosidase, and Dipeptidyl Peptidase-4 in a High-Sucrose Diet-Induced Diabetic Drosophila Melanogaster Model

Abstract

Carbohydrate-hydrolyzing enzymes (α-amylase, α-glucosidase) and dipeptidyl peptidase-4 (DPP-4) are established pharmacological targets for postprandial and incretin-mediated glycemic control in type 2 diabetes mellitus (T2DM). Thymoquinone (TQ), is the principal bioactive constituent of the ethnomedicinal plant Nigella sativa. This study aimed to determine the dose-dependent inhibitory activity of thymoquinone against α-amylase, α-glucosidase, and DPP-4 in a high-sucrose diet (HSD)-induced diabetic Drosophila melanogaster model. Adult flies were assigned to seven dietary groups, including normal and diabetic (HSD) controls, two thymoquinone doses (2.0 and 4.0 mg/10 g diet), the relevant standard drug, and N. sativa oil. Enzyme inhibitory activity was determined in vitro from whole-body homogenates in biological triplicate for each group and expressed as percentage inhibition. HSD feeding alone produced minimal enzyme inhibition (5–15% across all three targets), while all active treatments produced highly significant increases in inhibitory activity relative to the diabetic control (p < 0.01). Thymoquinone's inhibitory effect was clearly dose-dependent. Critically, at the higher dose (4.0 mg/10 g diet), thymoquinone's inhibition of α-amylase (52.9%) and α-glucosidase (62.7%) was statistically indistinguishable from, and numerically exceeded, acarbose (47.1% and 58.5%, respectively; both p > 0.01), while its DPP-4 inhibition (72.0%) was statistically indistinguishable from sitagliptin (77.1%; p = 0.28). Thymoquinone exhibits dose-dependent multi-enzyme inhibitory activity in Drosophila melanogaster, achieving efficacy statistically equivalent to two distinct, mechanistically unrelated standard antidiabetic drugs across three separate enzymatic targets. These findings have shown that thymoquinone's antidiabetic mechanism extends to direct carbohydrate-and incretin-degrading enzyme inhibition, strengthening the case for its continued ethnopharmacological and translational development.