Comprehensive in vitro and in silico analysis of Piliostigma thonningii’s bioactive compounds as multifunctional inhibitors of α-amylase, α-glucosidase, and oxidative stress for enhanced glycemic regulation

Type 2 diabetes mellitus is a growing global health crisis requiring effective management strategies, including the control of postprandial hyperglycemia. Inhibiting key carbohydrate-digesting enzymes, namely α-amylase and α-glucosidase, is a proven therapeutic approach. However, many current inhibitors have undesirable side effects. This study examines the anti-diabetic and antioxidant potential of bioactive compounds isolated from Piliostigma thonningii , a plant with a history of traditional use in treating diabetes, employing both in vitro and in silico approaches. A multifaceted approach was employed to elucidate the chemical profile and bioactive potential of the P. thonningii methanolic extract. This approach combined chromatographic separation, spectroscopic characterization, and in vitro antioxidant (DPPH, ABTS, HRSA, and FRAP assays) and anti-diabetic evaluations. Molecular docking and dynamics simulations were employed to predict the binding affinities and stabilities of isolated compounds with α-amylase and α-glucosidase. The methanolic extract exhibited notable antioxidant activity, comparable to that of the synthetic antioxidant BHT. The highest DPPH anti-radical activity was exhibited by kaempferol 3-O-α- L -rhamnopyranosyl-(1−2)-β- D -galactopyranoside ( 10 ) (IC 50 = 172.45 µg/mL, 69.48 % inhibition). In addition, the methanol (MeOH) extract demonstrated potent anti-diabetic activity in vitro , exhibiting the lowest IC 50 values for both α-amylase (184.45 µg/mL) and α-glucosidase (175.61 µg/mL) inhibition. Among the isolated compounds, genkwanin ( 5 ) displayed promising inhibitory activity against both enzymes (α-amylase IC 50 : 180.95 µg/mL, α-glucosidase IC₅₀: 174.95 µg/mL), while epicatechin ( 4 ) and shikimic acid ( 2 ) showed notable and moderate α-glucosidase inhibitory activities, respectively (epicatechin IC₅₀: 201.51 µg/mL, shikimic acid IC₅₀: 196.25 µg/mL). Vitexin ( 6 ) and genkwanin ( 5 ) were identified as the most potent α-amylase and α-glucosidase inhibitors in silico , with binding affinities of −8.6 kcal/mol and −8.5 kcal/mol, respectively. This study identifies compounds derived from P. thonningii , particularly vitexin ( 6 ) and genkwanin ( 5 ), as promising natural inhibitors of α-amylase and α-glucosidase. The findings indicate that the plant may serve as a valuable source of antioxidants and anti-diabetic compounds, thereby supporting its traditional medicinal applications and suggesting a pathway for the development of new, natural-based therapies for type 2 diabetes. • Piliostigma thonningii methanolic extract exhibits significant antioxidant properties. • Vitexin serve as an effective natural inhibitor of α-amylase and α-glucosidase. • Kaempferol 3-O-α- L -rhamnopyranosyl-(1−2)-β- D -galactopyranoside shows high DPPH anti-radical activity. • The identified compounds demontrate therapeutic promise for type 2 diabetes. • The study corroborates the plant's traditional uses.