Alzheimer's disease (AD) is a chronic neurodegenerative disease, which accounts for the majority of dementia cases (1). Common symptoms of AD include short-term memory loss, disorientation, loss of motivation, mood swings and behavioral issues (1,2). Patients with AD often withdraw from family and society as their condition declines (3,4). The cause of AD is not fully understood (1), and researchers have hypothesized that ~70% of the risk is due to genetic factors (4), followed by other factors, including head injuries, depression or hypertension (4–7). Patients with AD rely on caregivers for assistance, and in certain cases they may pose a burden to them (6). In developed countries, AD is considered to be one of the most financially challenging diseases (8–11). The underlying mechanisms of AD are not fully known and the majority of available drug therapies are based on the cholinergic hypothesis (12). The cholinergic hypothesis proposes that AD results from reduced synthesis of the neurotransmitter acetylcholine (ACh) (12). Houghton et al (13) indicated that inhibiting the enzyme acetylcholinesterase (AChE), which breaks down ACh, is a promising strategy for treating patients with AD. There are alternative hypotheses, such as the amyloid (14) and tau (15) hypotheses; however, AChE is a favorable enzyme target for numerous researchers. Furthermore, AChE has garnered more attention regarding its screening potential, and the identification of AChE novel inhibitors from various natural products, including plants and herbal extracts.

The enzyme AChE is a hydrolase of the carboxylesterase family, which hydrolyzes the neurotransmitter ACh (16,17). It is primarily present in the neuromuscular junctions and cholinergic brain synapses, assisting in the termination of synaptic transmissions. It belongs to the carboxylesterase family of enzymes (18,19). During neurotransmission, ACh is released from the nerve into the synaptic cleft where it binds to ACh receptors on the post-synaptic membrane, relaying the signal from the nerve (20). AChE is located on the post-synaptic membrane and terminates the signal transmission by hydrolyzing Ach (21). The liberated choline is then taken up by the pre-synaptic nerve and ACh is synthesized by combining with acetyl-CoA through the action of choline acetyltransferase (22).

The present study performed an inhibitory assay to determine the inhibitory activity of certain plant extracts against human AChE. In addition, computational research employing virtual screening of the compounds present in the plant extracts and AChE retrieved from the Protein Data Bank (PDB ID: 4PQE) was conducted, using the molecular docking simulation protocol. Subsequently, a protein-ligand interaction analysis was performed with 20 ns molecular dynamics (MD) simulation.

The AChE inhibitory activities of the plant extracts are presented in Table I. Total alkaloidal extracts from Nelumbo nucifera, Uncaria rhynchophylla and Portulaca oleracea exhibited 76.5, 78.4 and 74.2% inhibition against AChE, respectively (Table I). The compounds present in the herbal plants were retrieved from the NCBI PubChem database, and are presented in Table II. The molecular docking scores of the bioactive compounds present in the herbal plants against AChE are shown in Table III. The scores and results were based on the Rerank and MolDock scores, and interaction energy (24). Rerank Score, MolDock Score and Interaction Energy are measure based on the energy parameters E inter (steric, van der Waals, hydrogen bonding and electrostatic) between the ligand and the protein, and E intra (torsion, SP2, hydrogen bonding, van der Waals and electrostatic) (24). These energy terms are generated by the MolDock SE algorithm and based on the most stable E inter and E intra; the compounds were ranked accordingly. The results of the ligand-protein interaction analyses of the top docked compounds against AChE (PDB ID: 4PQE) are presented in Table IV. Images depicting the molecular interactions for the top docking hits are presented in Figs. 1–6, and the MD simulation investigating stability of the RMSD backbones of PDB ID: 4PQE and protein-ligand complexes is demonstrated in Fig. 7.

In the present study, AChE inhibitory assay of the plant extracts demonstrated that the total alkaloidal extracts from Uncaria rhynchophylla (78.4%), Nelumbo nucifera (76.5%) and Portulaca oleracea (74.2%) exhibited AChE inhibition (Table I). In addition, the alkaloidal extract from Areca catechu demonstrated the poorest AChE inhibition (19.3%; Table I).

Molecular docking analysis of the bioactive compounds present in the herbal plants confirmed that certain compounds in Portulaca oleracea, Zingiber officinale and Camellia sinensis possessed strong molecular interactions at the potential ligand binding site of AChE. The major interactions, including bonded and non-bonded interactions, were formed between the docked compounds and the binding cavity of the enzyme. The strength of the ligand-protein interaction was measured by the Rerank score. The Rerank score is a linear combination of E-inter (steric, Van der Waals, hydrogen bonding, electrostatic) between the ligand and the protein, and E-intra (torsion, sp2-sp2, hydrogen bonding, Van der Waals, electrostatic) of the ligand weighted by pre-defined coefficients (24). The Rerank score of the docked compounds along with their MolDock score and interaction energy are shown in Table III. In the present molecular docking simulation analysis, CID9064 (Uncaria rhynchophylla) CID681 (Portulaca oleracea), and CID439260 (Portulaca oleracea) and CID6047 (Portulaca oleracea) docked at the binding cavity having a Rerank score of −67.43, −64.76 and −60.40 kJmol⁻¹, respectively. Uncaria rhynchophylla and Portulaca oleracea exhibited 78.4 and 74.2% AChE inhibition, respectively. CID 31211 (Zingiber officinale) demonstrated a Rerank score of −59.39 and exhibited 60.5% AChE inhibition. Finally, CID65084 (Camellia sinensis) had a Rerank score of −53.40 and exhibited 40.2% AChE inhibition.

To understand the in-depth molecular interaction of these compounds with AChE, and its binding mechanism, a ligand-protein interaction analysis for the top 6 docking hits was conducted using a ligand energy inspector. The ligand-protein interaction, including the residues present, their interaction distances and energy, and the interacting atoms of the protein and ligand are presented in Table IV. The molecular docking simulation indicated that the top docking poses were demonstrated to be docked into the binding cavity displaying bonded and non-bonded interactions. Images depicting ligand-protein binding are shown in Fig. 1 (CID9064), Fig. 2 (CID681), Fig. 3 (CID6047) and Fig. 4 (CID31211), Fig. 5 (CID65084) and Fig. 6 (CID439260). The backbone RMSD values of the protein and protein-ligand complexes during 20 ns of MD simulation are presented in Fig. 7, and indicated that the RMSD values for AChE-ligand complexes were more stable suggesting a conformational flexibility and stability in dynamic behavior. Numerous herbal compounds have been demonstrated to have anti-cancer, anti-microbial and anti-inflammatory potential. However, this is not in case of AChE inhibition. In fact, there are few reports on the investigation of herbal compounds as inhibitors of AChE inhibition (26).

In conclusion, Uncaria rhynchophylla, Nelumbo nucifera and Portulaca oleracea possessed strong AChE inhibition. In addition, the molecular docking simulation analyses demonstrated that the active compounds present in Uncaria rhynchophylla (CID9061) and Portulaca oleracea (CID681 and CID439260) had strong molecular interactions, as evidenced by the molecular docking scores and ligand-protein interaction energy analyses. Furthermore, the MD simulation confirmed the stability of the protein-ligand docked complexes. The results of the present study suggested that CID9064 (catechin), CID681 (dopamine) and CID439260 (norepinephrine) may be key bioactive ingredients that may be prescribed to patients with AD.