Alkaloid Screening and HPTLC Fingerprinting of the Ethanolic Leaf Extract of Datura innoxia Mill. with In Silico Evaluation of Scopolamine at the Human M2 Muscarinic Receptor

Abstract

Objectives: To prepare the ethanolic leaf extract of Datura innoxia Mill., screen it for alkaloids, establish its high-performance thin-layer chromatography (HPTLC) fingerprint, and use molecular docking to examine how scopolamine binds the human M2 muscarinic receptor. Methods: Dried leaf powder was extracted with ethanol in a Soxhlet apparatus. Alkaloids were screened with Dragendorff's, Mayer's, Wagner's and Hager's reagents. HPTLC was performed on silica gel 60 F254 plates with toluene:ethyl acetate:formic acid (5:4:1, v/v/v) at five extract concentrations (25–150 mg/mL) in two independent runs, with detection at 254 and 366 nm and densitometric scanning. Scopolamine was docked into the M2 receptor orthosteric site (PDB ID: 3UON); the co-crystallised antagonist quinuclidinyl benzilate (QNB) served as a reference. Results: All four alkaloid reagents gave positive reactions. HPTLC produced a reproducible banding pattern in both runs, with band intensity rising with extract concentration Scopolamine docked with a best binding energy of −9.40 kcal/mol (predicted Ki 128.32 nM), compared with −11.86 kcal/mol (2.04 nM) for QNB. The ten poses formed one cluster (cluster RMSD ≤ 0.94 Å), stabilised by a salt bridge with Asp103, an aromatic cage formed by Tyr104, Trp400, Tyr403 and Tyr430, and a hydrogen bond with Asn404. Conclusion: The leaf extract contains alkaloids and yields a reproducible HPTLC fingerprint, and scopolamine is predicted to occupy the M2 orthosteric pocket in the canonical antagonist mode. The presence of scopolamine in the extract and any sedative effect need confirmation by co-chromatography or mass spectrometry and by in vivo testing.