Integrated LC-MS/MS profiling, network pharmacology, molecular docking, and enzyme inhibition assays reveal the antidiabetic potential of Mitragyna speciosa.

Mitragyna speciosa has gained attention as a medicinal plant with potential metabolic benefits, yet its antidiabetic mechanism remains unclear. This study characterized the metabolite profile of Mitragyna speciosa and explored its potential antidiabetic mechanisms using LC-MS/MS, network pharmacology, molecular docking, and in vitro enzyme inhibition assays. Metabolites were identified by LC-MS/MS, followed by target prediction and collection of diabetes-related targets to obtain overlapping genes. Protein-protein interaction (PPI) analysis and maximal clique centrality (MCC) were used to determine hub proteins. Molecular docking evaluated binding affinity (ΔG) and inhibition constant (Ki) against prioritized targets, and docking accuracy was confirmed by re-docking RMSD. The predicted antidiabetic activity was subsequently validated through in vitro α-glucosidase and α-amylase inhibitory assays. LC-MS/MS revealed mitrafoline (31.54%) and mitragynine (30.59%) as dominant constituents, followed by corynoxine (10.43%), methyl indoloquinolizine derivatives (8.41%), and isopaynantheine (6.27%). Minor metabolites included mitraphylline (2.58%) and corynantheidine (1.75%). Network pharmacology identified 28 shared targets between Mitragyna speciosa and diabetes mellitus, forming an interconnected PPI network. Hub analysis prioritized EGFR (915), PIK3CA (808), JAK2 (673), SRC (606), and ERBB2 (602). Docking showed that corynantheidine had the strongest affinity for EGFR (ΔG -9.01 kcal/mol; Ki 250.21 nM), while mitraphylline performed best against JAK2 (ΔG -8.77 kcal/mol; Ki 374.56 nM); native ligands remained superior for PIK3CA, SRC, and ERBB2. In vitro assays revealed moderate α-glucosidase inhibitory activity of the extract (IC₅₀ ≈ 48.49 ppm) and substantially weaker α-amylase inhibition (IC₅₀ ≈ 17,907.37 ppm), indicating a selective enzyme inhibition profile. These findings suggest that Mitragyna speciosa exerts antidiabetic effects through multitarget modulation of kinase-centered signaling pathways and selective inhibition of carbohydrate-digesting enzymes.