Phytochemical Profiling, Green Synthesis of Metal Oxide Nanoparticles, and Multitarget Evaluation of Cotula cinerea and Origanum majorana L. as Potential Neuroprotective Agents

Abstract

Alzheimer’s disease (AD), the most common neurodegenerative disorder, is characterized by progressive cognitive decline, cholinergic deficits, oxidative stress, and neuroinflammation. Due to the limitations of current therapeutics, including Donepezil, Diclofenac, and α- tocopherol, the exploration of safer, multitarget alternatives is imperative. This study investigates the phytochemical composition, biological activity, and molecular mechanisms of Cotula cinerea and Origanum majorana L., two North African medicinal plants. Plant extracts were prepared using aqueous, methanolic, and butanolic solvents and characterized via UPLC–ESI–MS/MS, revealing the presence of bioactive flavonoids and phenolic acids, including quercetin, luteolin, and rosmarinic acid. In Vitro assays demonstrated potent antioxidant activity (up to 89.6% DPPH inhibition), anti-inflammatory activity (IC₅₀ of 42.1 μg/mL for C. cinerea), and significant anticholinesterase effects (IC₅₀ of 18.5 μg/mL for O. majorana extract). ZnO and AgO nanoparticles synthesized via green methods from aqueous extracts were confirmed using UV–Vis, FTIR, XRD, and SEM Molecular docking and Induced Fit Docking (IFD) revealed high binding affinities of quercetin (Glide score: −9.4 kcal/mol; IFD score: −11.2 kcal/mol) and rosmarinic acid (−9.0 and −10.8 kcal/mol, respectively) against AChE. These values were superior to those of Donepezil (−8.7 and −9.4 kcal/mol). Similar favorable interactions were observed for COX-1 and GR, surpassing Diclofenac and α-tocopherol. MD simulations over 100 ns confirmed complex stability, with low RMSD values (~2.1 Å for quercetin and ~1.6 Å for rosmarinic acid) and consistent hydrogen bonding. Free energy analyses (MM/GBSA) yielded ΔG values of −48.2 kcal/mol and −52.7 kcal/mol, respectively. Furthermore, DFT studies (FMO analysis) revealed HOMO–LUMO gaps of 4.74 eV (β- thujone) and 5.90 eV (caryophyllene oxide) in aqueous medium, supporting favorable chemical reactivity and electron transfer potential. An in Vivo AD model induced by AlCl₃ and D-gal was used to evaluate the plant extracts. Behavioral tests showed improved cognitive function, while hematological analysis indicated reduced systemic inflammation. Biochemical results revealed lower MDA levels and increased GSH, CAT, and SOD activity in the brain, liver, and kidneys. Histopathology confirmed reduced amyloid-beta (Aβ) deposition In summary, this multidisciplinary investigation provides compelling evidence for the neuroprotective potential of Cotula and Origanum . phytochemicals and their green-synthesized nanoparticles, offering promising scaffolds for further drug development targeting Alzheimer’s disease