Chemical Profiling and In Silico Study of Russelia equisetifomis Major Phytochemicals as Potential Dual-Site Acetylcholinesterase Inhibitors in Alzheimer’s Disease

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and memory impairment, associated with cholinergic dysfunction and β-amyloid aggregation. Acetylcholinesterase (AChE) plays a dual role in AD progression by hydrolysing acetylcholine (ACh) at the catalytic active site (CAS) and promoting β-amyloid aggregation at the peripheral anionic site (PAS), making dual-site inhibition a promising therapeutic strategy. This study aimed to evaluate the potential of major phytochemicals from the stem of Russelia equisetiformis as dual-site AChE inhibitors using an in silico approach. Chemical profiling of the ethanolic stem extract was performed using Liquid Chromatography-Mass Spectrophotometry (LC-MS), and compounds with relative concentrations above 2% were selected for further analysis.  Molecular docking was conducted using Autodock Vina implemented in PyRx against AChE (PDB ID: 4M0E), while ligand interactions were visualised using BIOVIA Discovery Studio Visualizer. Druglikeness was evaluated using SwissADME based on Lipinski’s Rule of Five, and Molecular Initiating Event (MIE) prediction was performed using ProTox 3.0. Docking validation produced an RMSD value of 1.672 Å, indicating acceptable reliability. Among the identified compounds, tilianin exhibited a binding affinity of -8.5 kcal/mol and interacted with key PAS residues (Tyr72, Trp286, and Tyr341) as well as residues extending toward the CAS, comparable to that of donepezil (-7.4 kcal/mol). Tilianin also fulfilled all Lipinski criteria and was predicted to be active toward AChE with a probability score of 0.65. These findings suggest that tilianin is a promising dual-site AChE inhibitor candidate that may simultaneously improve cholinergic neurotransmission and reduce β-amyloid (Aβ) aggregation. Nevertheless, further in vitro and in vivo validation studies are required to confirm its therapeutic potential against AD.