Cholinesterase Inhibitory Activity of Paeoniflorin: Molecular Dynamics Simulation, and In Vitro Mechanistic Investigation

Abstract

Alzheimer's disease (AD), a neurological disorder, is one of the major reasons for memory loss in the world. AD is characterized by a sequela of cognitive and functional decline caused by brain cell degeneration. Paeoniflorin is a monoterpenoid glycoside found in plants of the Paeoniaceae family, which are known for their medicinal properties including dementia. In this project, we report actions of paeoniflorin on the two related cholinesterases (ChE): acetylChE (AChE) and butyrylChE (BuChE). Paeoniflorin, in a dose-dependent (maximum inhibition at 1 mg/mL) manner, inhibited both AChE (0.06-1 mg/mL) and BuChE (0.007-1 mg/mL) enzymes with maximum inhibition of AChE enzyme at 90.3 ± 1.4%, while 99.4 ± 0.3% for BuChE enzyme. The EC₅₀ value for the inhibitory effect of the compound against AChE was 0.52 mg/mL (0.18-1.52), while against BuChE was 0.13 mg/mL (0.08-0.21). The observed ani-ChE action was like an effect also mediated by the known ChE blocker physostigmine. Molecular interactions between paeoniflorin and both ChE enzymes were additionally sought via molecular docking and molecular dynamics simulations for 100 ns, that showed paeoniflorin interacted with the active-site gorge of AChE and BuChE via hydrogen bonds and water bridging with the many amino acids of the AChE and BuChE enzymes. This study presents the ChE inhibitory potential of paeoniflorin against both AChE and BuChE enzymes. With this kind of inhibitory activity, the chemical can potentially increase ACh levels and may have use in the treatment of dementia of AD.

Keywords: Alzheimer's disease; Paeoniaceae; acetylcholinesterase; butyrylcholinesterase; docking; enzyme inhibition; memory.

Figure 1

2-Dimensional structure of paeoniflorin (analytical standard; obtained from Sigma-Aldrich, Inc.) and the 2-dimensional interaction pattern observed during 100 ns of MD production run with both enzymes: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).

Figure 2

Curves showing % inhibitory effect of paeoniflorin against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes tested via the in vitro enzyme assay for cholinesterase inhibition. Data presented are mean ± SEM, three observations. There is significant different between the two curves, two-way ANOVA followed by Bonferroni's test (p < 0.0001).

Figure 3

Molecular docking result of paeoniflorin binding into (a) acetylcholinesterase and (b) butyrylcholinesterase enzymes.

Figure 4

Deviation and fluctuation and lig-fit plots: (a) root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analysis of AChE-paeoniflorin complex, and (b) RMSD and RMSF analysis of BuChE-paeoniflorin complex projected in 100 ns MD run.

Figure 5

Histogram analysis of the interacted fraction pattern in (a) acetylcholinesterase (AChE) with paeoniflorin and (b) butyrylcholinesterase (BuChE) with paeoniflorin.

Figure 6

Molecular contact analyses of paeoniflorin against: (a, b) acetylcholinesterase (AChE), and (c, d) butyrylcholinesterase (BuChE), showing: (a, c) A timeline representation of the total no. of molecular interaction contacts in each trajectory frame and (b, d) the number of interactions with the active site residues in each frame of the simulated 100 trajectory frames.

Figure 7

Secondary structural element (SSE) analyses of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) complexed with paeoniflorin: (a) % secondary structure elements (SSE) in respect to residue index present in the complex, (b) % SSE in respect to time in nano-scale time frame, and (c) indicates influence of residual change with the variation of projected time frame.

Figure 8

Paeoniflorin property analyses against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE): (a) ligand root mean square deviation (RMSD), (b) radius of gyration (rGyr), (c) molecular surface area (MolSA), (d) solvent accessible surface area (SASA) and (e) polar surface area (PSA).