Isolation, Acetylcholinesterase Inhibitory Activity, Molecular Docking, and ADME Analysis of compounds from (Afzel. ex G. Don) Benth

This study aimed to isolate, characterize, and evaluate the acetylcholinesterase (AChE) inhibitory potential of bioactive compounds from Crossopterix febrifuga, a plant of interest in Alzheimer's disease (AD) research. Crude extracts were obtained from the stem bark and trunk wood using ethanol/water (70:30) maceration. The trunk wood extract was subjected to silica gel column chromatography, leading to the isolation of three pure compounds: β-sitosterol (1), scopoletin (3), and D-mannitol (4) along with a mixture of stigmasterol (2) and β-sitosterol (1). Structural elucidation was performed using 1D and 2D 1H and 13C NMR spectroscopy, along with mass spectrometry (MS). The AChE inhibitory activity of both extracts and isolated compounds was assessed in vitro using a modified Ellman's colorimetric method. Additionally, molecular docking simulations were conducted to predict the binding affinities and interactions of the compounds with the AChE active site. In vitro assays revealed that the stem bark and trunk wood extracts exhibited moderate AChE inhibition with IC50 values of 424.3 µg/mL and 480.0 µg/mL, respectively. Among the isolated compounds, the mixture of stigmasterol and β-sitosterol showed the highest activity (IC₅₀ = 50.85 µg/mL), followed by β-sitosterol (57.25 µg/mL) and scopoletin (58.97 µg/mL). D-mannitol displayed very weak inhibition (IC₅₀ = 4955.64 µg/mL). Docking results supported these findings, with stigmasterol demonstrating the strongest binding affinity (–10.5 kcal/mol), followed by β-sitosterol (–10.5 kcal/mol), while D-Mannitol showed lower affinity (–5.0 kcal/mol). Despite its moderate inhibition, scopoletin exhibited favorable physicochemical properties, including small molecular size, high solubility, and predicted blood-brain barrier (BBB) permeability. These results suggest that C. febrifuga contains promising anti-AChE compounds, particularly phytosterols and coumarins, which may serve as lead candidates for the development of new therapies against Alzheimer's disease.