Integrated bioactivity-guided isolation, network pharmacology, and experimental validation to reveal the key constituents and potential mechanisms of Tectona grandis against diabetes and lipid disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Tectona grandis L. f. (T. grandis) is traditionally used for the management of diabetes and lipid disorders in the Ayurvedic system of medicine.

AIM OF THE STUDY: To investigate the bioactive compounds and potential mechanisms responsible for the efficacy of T. grandis against diabetes and lipid disorders.

METHODS

The active ingredients were isolated through bioactivity-guided fractionation based on glucose uptake and pancreatic lipase (PL) inhibition assays. Subsequently, the mechanism for enhancing glucose uptake was elucidated by integrating network pharmacology with experimental validation (immunofluorescence, Western blot, inhibitor interference assay, and cellular thermal shift assays (CETSA)), and the in vivo glucose-lowering efficacy was evaluated by an oral glucose tolerance test (OGTT). In addition, the inhibition type and quenching mechanism against PL were characterized by enzyme kinetics and fluorescence quenching analysis. Furthermore, molecular docking and dynamics simulations were employed to investigate the molecular interactions and complex stability.

RESULTS

A rare diquinone (tectograndone, TG1) with glucose uptake-stimulating activity and PL inhibition was identified from T. grandis. Mechanistic studies revealed that TG1 (10-40 μM) dose-dependently upregulated the expression levels of p-AMPK (Thr172) and plasma membrane GLUT4 (p < 0.001; p < 0.01). Treatment with the AMPK inhibitor Compound C markedly attenuated the TG1-promoted glucose uptake in 3T3-L1 adipocytes. CETSA results demonstrated that TG1 (40 μM) enhanced the thermal stability of AMPK in 3T3-L1 adipocyte lysates, as evidenced by a distinct rightward shift of its thermal denaturation curve. The OGTT test results indicated that both low-dose (10 mg/kg, i.p.) and high-dose (40 mg/kg, i.p.) TG1 significantly improved glucose tolerance compared to the vehicle control group (p < 0.01, p < 0.001). The enzyme kinetics and fluorescence quenching assays indicated that TG1 acted as a mixed-type inhibitor and formed a ground-state complex with PL (static quenching), thereby inhibiting its catalytic activity. Furthermore, molecular docking analysis suggested that TG1 bound to AMPK primarily via hydrogen bonds and hydrophobic interactions (-6.6 kcal/mol), while its interaction with PL was dominated by π-π stacking and hydrophobic forces (-10.2 kcal/mol). Molecular dynamics simulations further validated the structural stability of both AMPK-TG1 and PL-TG1 complexes.

CONCLUSION

This study demonstrated that TG1 from T. grandis exerted dual mechanisms: Firstly, it directly binds to and activates AMPK, thereby mediating the translocation of GLUT4 to the plasma membrane and enhancing glucose uptake in 3T3-L1 adipocytes; secondly, it acts as a mixed-type inhibitor of PL, effectively suppressing its catalytic activity via a static quenching mechanism. These findings provide a scientific validation for the traditional use of T. grandis in Indian medicine and identify TG1 as a promising multi-target lead candidate for the treatment of diabetes and lipid disorders.