Integrated In Vitro Evaluation of Orthosiphon stamineus Leaf Extract and Selenium Nanoparticles: Antidiabetic and Anticholinesterase Activities Supported by In Silico Analysis of Identified Phytochemicals

<title>Abstract</title> <p> Diabetes mellitus (DM) and diabetes-associated cognitive decline (DACD) are characterized by heightened oxidative stress and dysregulation of mammalian α-glucosidase (MAG) and cholinesterases [ChEs; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)]. This study evaluated the antioxidant, antidiabetic, and anticholinesterase potential of methanolic extract from <italic>Orthosiphon stamineus</italic> leaves (OSL-ME) and its selenium nanoparticles (OSL-SeNPs). OSL-SeNPs synthesized using OSL-ME underwent characterization by means of UV-Vis, FTIR, EDAX, XRD, and SEM analyses, revealing a crystallite size of 16.45 nm and physical size ranging from 30–60 nm. LC-ESI/MS/MS profiling identified 16 phytoconstituents. <italic>In vitro</italic> studies demonstrated that OSL-ME possessed considerable antioxidant activity and inhibited MAG and ChEs through a mixed-type inhibition mechanism, with IC ₅₀ values ranging from 121.50 to 173.41 µg/mL. In addition, both samples promoted concentration-dependent glucose uptake into yeast cells, supporting their antidiabetic potential. OSL-SeNPs showed no ChE inhibition but selective enhancement of MAG inhibitory activity towards sucrase, maltase, and glucoamylase, with IC ₅₀ values recorded at 74.58, 80.66, and 73.53 µg/mL, respectively, and demonstrated greater antioxidant potential than OSL-ME. Notably, both OSL-ME and OSL-SeNPs maintained &gt; 90% HEK293 cell viability, indicating low cytotoxicity. Molecular Dynamics, MM/GBSA, DCCM, and PCA analyses revealed stable binding of the LC-ESI/MS/MS identified compound Chrysin (CHR) and phylloflavan (PF), confirming target-dependent variations in binding stability within the active site of the MAG and ChEs. ADMET profiling indicated favourable pharmacokinetic properties. Collectively, these findings suggest that OSL-ME and OSL-SeNPs possess promising multifunctional potential that may contribute to the effective management of DM and DACD. <bold>Clinical Trial Number</bold> : Not applicable </p>