Antioxidant and Neuroprotective Potential of Rhamnus alaternus Extract: Cholinesterase Modulation and Preliminary Safety Evaluation in Artemia salina

Reliable plant authentication is crucial for reproducible pharmacological investigations. In this study, ITS rDNA sequencing unequivocally confirmed the identity of Rhamnus alaternus (97.65% BLAST similarity) with robust phylogenetic support (99% bootstrap). Ultrasound-assisted extraction yielded a phenolic-rich leaf extract, characterized by high levels of total phenolics (215.36 ± 4.21 mg GAE/g), flavonoids (68.74 ± 2.85 mg QE/g), tannins (32.18 ± 1.47 mg CE/g), and saponins (45.92 ± 2.03 mg DE/g). LC–MS/MS analysis revealed a diverse phytochemical profile dominated by bioactive phenolic acids and flavonoids, including gallic acid, caffeic acid, quercetin, rutin, kaempferol, and isorhamnetin. Biological evaluation highlighted a dual bioactivity profile, with moderate general toxicity in the Artemia salina lethality assay (LC₅₀ = 285.7 µg mL-1) alongside marked cholinesterase inhibition against AChE (68.4%, IC₅₀ = 21.3 µg mL-1) and BChE (61.7%, IC₅₀ = 28.6 µg mL-1). The extract exhibited remarkable antioxidant capacity, with low IC₅₀ values in DPPH (7.80 µg mL-1) and ABTS (8.20 µg mL-1) assays, and competitive reducing power in FRAP and CUPRAC systems. Molecular docking suggested that rutin, apigenin-7-glucoside, and kaempferol-3-glucoside may interact favourably with AChE and BChE active-site regions, with rutin showing the most favourable predicted binding scores among the tested compounds. Collectively, these findings support the potential of R. alaternus leaf extract as a source of bioactive compounds with antioxidant and cholinesterase-modulating properties, warranting further studies on compound level activity, BBB permeability, in vivo efficacy, and safety.