Acetylcholinesterase inhibitory activities of some flavonoids from the root bark of Pinus krempfii Lecomte: in vitro and in silico study

Abstract From the root bark of Pinus krempfii Lecomte, four flavonoids were isolated and evaluated for their inhibitory activities against AChE and BChE enzymes in vitro and in silico. Tectochrysin (1) was found to inhibit AChE with an IC50 value of 33.69 ± 2.80 μM. The docking study results also showed agreement with the in vitro test results. All four compounds also showed the best binding affinity for the AChE enzyme, characterised by binding energy (ΔG) values as low as −8.1 to −9.3 kcal/mol, in which, the compound tectochrysin had the best binding affinity for the AChE protein with a ΔG value of −9.329 kcal/mol. Tectochrysin (1) was also bound to the amino acid Phe295 of AChE with a bond length of 2.8 Å, similar to the control dihydrotanshinone-I. Galangin (2) also showed its in vitro inhibitory activity against BChE with an IC50 value of 82.21 ± 2.70 μM. In silico, it also had the best binding energy value of −9.072 kcal/mol with BChE and formed hydrogen bonds with the His438 (2.85 Å) residues of BChE like the positive control (tacrine). The steered molecular dynamics (SMD) simulation results of these two complexes revealed a mechanistic insight that the protein-ligand complexes showed stable trajectories throughout the 20 and 150 ns simulations. Moreover, the drug likeliness suggested that both flavonoids (1 and 2) were expected to be drug-like and have an LD50 toxicity level of 5. This study has contributed new results for drug discovery and the development of substances with neuroprotective effects, especially for the treatment of Alzheimer’s disease. Communicated by Ramaswamy H. Sarma


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia.It is characterised by severe memory loss and cognitive dysfunction (Alzheimer-Association-Alzheimer's Association Report, 2017; Essa et al., 2012).AD affects more than 35 million people worldwide, and this number is believed to reach 65.7 million by 2030.This degenerative brain illness is connected with ageing and is prevalent among those aged 65 and older (Chiroma et al., 2018).In the later stages of the disease, patients are bedridden, require 24-h care, and are ultimately fatal (Alzheimer-Association-Alzheimer's Association Report, 2019).
In Alzheimer's disease, neurons in various parts of the brain are eventually irreversibly damaged or destroyed, causing trouble with memory, language, thinking, and behavior, including trouble carrying out basic bodily functions such as walking and swallowing (Alzheimer-Association-Alzheimer's Association Report, 2017).The main features and symptoms of Alzheimer's pathology are the deposition of b-amyloid (Ab) plaques and neurofibrillary tangles (NFTs) composed of hyperphosphorylated TAU protein (pTAU) (Alzheimer-Association-Alzheimer's Association Report, 2017; Maramai et al., 2020), as well as an increase in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) levels (Miles & Ross, 2021).
AChE and BChE are two important substrate-specific cholinesterases that degrade the neurotransmitters acetylcholine (Ach) and butyrylcholine (BCh) in the nerve synapse (Taylor et al., 2009).Changes in AChE and BChE levels and their consequent imbalance ratios cause a neurotransmitter shortage in the brain (Mushtaq et al., 2014).Therefore, inhibiting the enzymatic activity of both AChE and BChE, or selective individual enzymes, is one of the potential therapeutic strategies to increase cholinergic levels in the brain.Accordingly, compounds with cholinesterase inhibitory activity can interact with the central cholinergic system, reduce the breakdown of acetylcholine at synaptic sites in the brain, and thus have potential therapeutic benefits in the treatment of AD in particular and other related neurological diseases in general.
There are currently available medications such as donepezil (AriceptV R ), galantamine (Razadyne V R ), and rivastigmine (Exelon V R ) commonly used to treat AD (Sharma, 2019).Products from natural resources such as medicinal plants have drawn especial attention for the drug discovery of AChE and BChE inhibitory drugs.Numerous natural compounds, such as huperzine A, a lycopodium alkaloid, isolated from the herb Huperzia serrata (Camps et al., 2000;Li et al., 2008), galantamine from the fruit of Galanthus nivalis (Olin & Schneider, 2002), cardanol, a phenolic lipid, obtained from Anacardium occidentale (de Paula et al., 2009), pycnogenol, a polyphenol-rich extract prepared from Pinus pinaster (Ustun et al., 2012), and especially flavonoid compounds with their free-radical-scavenging properties like galangin and those derived from the rhizomes of Alpiniae officinarum (Guo et al., 2010;Sharma, 2019) and the cones of Pinus sylvestris (Topal, 2019) have shown effective AChE inhibitory activities.
Pinus (Pinaceae) is the largest genus of conifers (Camps et al., 2000), containing more than 100 distinct species (Dziedzi� nski et al., 2021).Pinus krempfii Lecomte [synonym, Ducampopinus krempfii (Lecomte) A.Chev.], also known locally as "Thông hai l� a d _ et" or Thong Sr� e, is an endemic species of flat-needle pine with a restricted distribution at higher altitudes in the highlands of Tay Nguyen provinces of Vietnam (Phong et al., 2015).This plant has been designated as an endemic species in Vietnam and a rare "living fossil," or an ancient plant that has survived to the present day.Due to the fact that this species are on the list of protected plants, research into its chemical composition and biological activity is of greater scientific significance.
In previous studies, the isolation of six flavonoids, including pinostrobin, tectochrysin, pinobanksin, galangin, strobopinin, and cryptostrobin, from the root bark of this plant was described (Nhung et al., 2013).As part of our search for bioactive compounds isolated from Vietnamese medicinal plants, the aim of this study was to evaluate the anticholinesterase activities of selective flavonoids, namely tectochrysin (1), galangin (2), strobopinin (3), and cryptostrobin (4) isolated from this plant.The findings indicated that these flavonoids merited additional research for the development of neuroprotective drugs.

Plant material
Plant materials were collected in Lam Dong province, Viet Nam, in August 2012.The plant Pinus krempfii Lecomte (Figure 1) was identified by taxonomist Dr. Nguyen Tien Hiep, Vietnam National Museum of Nature, VAST.A voucher specimen (No.CPC-4711) was deposited at the same place.

Extraction and isolation of compounds
The extraction and isolation of natural flavonoids have been described previously (Nhung et al., 2013).Briefly, the powdered root barks of P. krempfii (1.1 kg) were extracted with 80% aqueous MeOH (4x, 3.0 À 4.5 L each) and the solvent was removed under reduced atmosphere.The crude methanol extract was suspended in water and subsequently fractionated with n-hexane, chloroform, and ethyl acetate.From the ethyl acetate fraction, six flavonoids were subsequently isolated through column chromatography, including pinostrobin, pinobanksin, tectochrysin, galangin, strobopinin, and cryptostrobin (Nhung et al., 2013).The AChE/BChE inhibitory activity of the last four flavonoids, tectochrysin (1), galangin (2), strobopinin (3) and cryptostrobin (4) was investigated.Their spectral data were presented in detail in supporting information.

In vitro assay to evaluate the AChE and BChE inhibitory activities
The in vitro photometric assay used to evaluate the inhibitory effect of the acetylcholinesterase enzyme was first developed by Ellman in 1961(Ellman et al., 1961).Briefly, a reaction mixture consisting of 700 mL sodium phosphate buffer (pH 8.0), 100 mL of test solution at different concentrations, and 100 mL of 0.5 IU/mL AChE enzyme solution was mixed well and incubated for 15 min at 25 � C. The tested extracts and the positive standard (tacrine) were then dissolved in 10% dimethyl sulfoxide (DMSO) and 50 mL of 2.5 mM DTNB and 50 mL 2.5 mM ACTI were added.The mixture was mixed well for 1 min.and continued to incubate for 10 min.at 25 � C.Then, the solution was measured for absorbance at 412 nm.All experiments were repeated three times.
The percentage inhibition of AChE enzyme activity (%I) was calculated by the formula: where

Kinetic characterization of the inhibitory reaction of AChE and BChE enzymes
The kinetic characterizations of the AChE/BChE inhibitory reaction were obtained according to the method described previously (Sharma, 2019).The reaction mixture, consisting of 700 mL sodium phosphate buffer (pH 8.0); 100 mL of test solution at concentrations (0, 2.5, 5.0, and 10.0 mg/mL) and 100 mL of 0.5 IU/mL AChE (or BChE) enzyme solution, was mixed well and incubated for 15 min at 25 � C.Then, 50 mL of DTNB 2.5 mM and 50 mL with different concentrations of ACTI substrate (5, 2.5, and 1.25 mM) were added and incubated for 5 min.Measure the absorbance of the solution at 412 nm for 5 min.All experiments were repeated three times.Use the 1/[ACTI] and 1/V (1/reaction rate) graphs (Lineweaver-Burk plot) to determine the kinetics of enzyme inhibition.The inhibition constant K i was determined as the intersection of the [I] axis (inhibitor concentration) and 1/V (1/reaction rate) lines (Dixon plot).

Statistics
The research data were statistically processed by the student t-test method using SigmaPlot 10 software (Systat Software Inc, USA).The data are expressed as X ± SD.The difference is considered as significant when p < 0.05.

Ligand preparation
The 2D and 3D structures of ligands were prepared using Marvin Sketch v21.19 software (https://chemaxon.com/marvin).The optimal structures of ligands were performed with the MMFF94s force field using the steepest descent optimization algorithm during 10,000 minimization steps (Halgren, 1999).The pdbqt file formats of the ligands and target proteins were automatically generated using the free MGLTool v1.5.6 software (https://ccsb.scripps.edu/mgltools).

Steered-molecular dynamic simulation
From the results of molecular dockings, the selected compounds (1-2) with the AChE or BChE inhibitory potential were subjected to Steered Molecular Dynamics (SMD) simulations using the GROMACS v2021 package (Vuong et al., 2015).The process involves separating the ligand from the protein-ligand complex by applying a time-dependent external force to it (Parihar et al., 2022).The Caver v3.0.3 software was used to determine the pulling pathway of the ligands from the complex (Chovancova et al., 2012;Pet� rek et al., 2006).The complex was then rotated so that the pull-out path was oriented as the Z-axis using the PyMOL v2.3.0 software (DeLano, 2002).We used the Amber99SBILDN force field for protein and ionic parameterization in conjunction with the TIP3P water model (solvent density of 0.997 g/cm 3 (water solvent), Na þ /Cl -ions of 0.9%, pH of 7.4 and temperature of 298K) to simulate an explicit water molecule.The ligands were characterized using a general amber force field (GAFF) (Wang et al., 2004), where the restrained electrostatic potential (RESP) method was applied to determine atomic charge of the complexes through theoretical density calculation using the B3LYP function combination with the 6-31 G(d,p) basic set (Nhung et al., 2022).The AchE and BChE ligand complexes were set at the center of a triclinic box and the system charge was neutralized by the addition of counter ions.
The energy of the systems had been minimized through the steepest descent algorithm (Meza, 2010), and the maximum minimization step was 50,000 steps.Then, the following 500 ps position restriction simulations in both NVT and NPT combinations were performed.NPT simulations in 20 ns and 150 ns were set to relax the system.A standard SMD simulation with duration of 700 ps was conducted to bring the assembly to a steady state.The coordinates of the entire system were tracked every 10 ps.In total, 10 independent SMD trajectories were operated for each of the AchE and BChE ligand complexes.
In the SMD simulations, an external force F ¼ k(�t À z) was applied to the center of mass of the inhibitor along the Z direction (Figure 1, Supporting information), where k is the cantilever spring constant, m is the pulling rate, and z represents the displacement of the inhibitor's center of mass from its original position.Then the work of the external force is determined by the formula W ¼ v Ð t 0 FðtÞdt, where W is the work of the external force and F is an external force.The work of the pulling force is in principle proportional to the binding free energy of the ligand according to Jarzynski's equation e where k B is the Boltzmann constant, T is the absolute temperature of the system, W is the work of the external force, and DG is the Gibbs free energy difference between the bound and unbound states (Hummer & Szabo, 2001;Jarzynski, 1997).

In-silico pharmacokinetics, drug-likeliness, and ADMET prediction
The SwissADME web server (http://www.swissadme.ch/index.php) was used to calculate drug-likeness parameters and ADME values of the lead molecules using Lipinski's rule of five (Lipinski et al., 2001).Compounds that did not violate the drug-like filter and had good blood-brain barrier penetration and water solubility were selected as the final candidate compounds.Parameters like human intestinal absorption (GI), blood-brain barrier permeability (BBB), and Pgp substrates were calculated using the Brain Or IntestinaL EstimateD permeation method (BOILED-egg model) (Daina et al., 2017).Different pharmacokinetic parameters of virtual screening candidates, such as enzyme (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4) inhibitors and logP, were also predicted.For each phytochemical, an input file with SMILES was obtained from PubChem (Kim et al., 2021).Total clearance (CL, log ml/min/kg) was predicted for the studied flavonoids using pkCSM (Pires et al., 2015).The predicted LD 50 (mg/kg) values and toxicity of the studied compounds were determined using the ProTox II web server (Banerjee et al., 2018).

AChE and BChE inhibitory activities
The in vitro inhibitory activity of the isolated flavonoids (1-4) from P. krempfii root bark was investigated based on the hydrolysis of the AChE/BChE on an artificial substrate, acetylthiocholine iodide (ACTI), to produce thiocholine, which reacted with the 5-5 0 -dithiobis-2-nitrobenzoic acid (DTNB) reagent to form a yellow 5-thio-2-nitrobenzoic acid (TNB) product.The amount of colored TNB compound produced was proportional to the AChE activity.For both AChE and BChE testing experiments, tacrine was used as a positive control (Table 1).At 100 mM, all flavonoids inhibited AChE by between 25.78 and 72.12% and BChE by between 14.45% and 56.20% (Figure 3A).Only tectochrysin (1) was found to inhibit AChE with an IC 50 value of 33.69 ± 2.80 lM, compared to tacrine (IC 50 126.70± 1.10 nM) served as a positive control (Figure 3A,B).Among the four compounds tested, galangin (2) exhibited the highest inhibitory activity against BChE with an IC 50 value of 82.21 ± 2.70 lM (tacrine) (IC 50 5.50 ± 1.70 nM) (Table 1, Figure 3A,C).Experimental kinetic parameters of the catalyzed reaction were also determined (Figure 4 and Table 2).Using the Dixon plot, the empirical inhibitor constant K i was determined as 16.90 lM for tectochrysin (1), and 10.5 lM for galangin (2).Both compounds showed their inhibition in the AChE and BChE, respectively, clinging to the competitive binding mode (Figure 4).These results indicate that galangin (2) is a potential selective inhibitor for BChE and tectochrysin (1) for AChE.

Molecular docking results
Molecular docking with the AutoDock Vina software was used to find out how the active flavonoids attach to the cholinesterase enzymes.
First, to demonstrate the docking protocol's efficacy, the bound crystallized inhibitor was manually re-docked into the active site of the proteins, and the random mean-square deviation (RMSD) value of the formed protein-ligand complex was measured.Re-docking simulation results for dihydrotanshinone I-AChE (4M0E) and tacrine-BChE (4BDS) yielded RMSD values of 0.307897 < 2 (Å) and 0.438639 < 2 (Å), respectively (Figure 5).The re-dock RMSD values for both complex systems were less than 2 Å, indicating a good spatial match between the crystal structure and the protein.This means that the Autodock Vina docking program is highly reliable and can be used for AChE and BChE docking studies.
Then, flavonoid compounds isolated from P. krempfii were docked to two protein targets, AChE and BChE.According to Table 2, the binding affinities of the investigated compounds for the AChE target ranged from À 8.192 to À 9.332 kcal/mol.Galangin (2), with binding affinities of À 9.047 kcal/mol displayed three hydrogen bonds in the binding pocket of the AChE protein at the amino acid residues Phe295, Ser293 and Tyr337 with bond lengths of 3.21, 3.02, 3.05, and 2.88 Å, respectively.The hydrophobic interactions between galangin and the AChE protein involved the residues Tyr124, Trp 286, Val294, Phe 338, and Tyr341 (Table 2).Cryptostrobin (4) and strobopinin (3) also demonstrated potential AChE inhibitory activity with binding affinities of À 8.192, and À 8.635 kcal mol À 1 , respectively.Hydrogen bonds were observed between cryptostrobin (4) and the residues Tyr124 and Ser293 with bond distances of 3.08 Å and 3.19 Å, respectively (Table 2).Strobopinin (3) also formed a single hydrogen bond with Tyr124 (2.82 Å) of the AChE enzyme (Table 2).Although these molecules have the potential to be AChE inhibitors due to their hydrogen bonding and hydrophobic interactions, they lack interactions at key sites like the control dihydrotanshinone-I.Moreover, the length of the hydrogen bond makes the ligand-protein complex less stable than tectochrysin (1).
With a DG value of À 9.329 kcal/mol, tectochrysin (1) possessed the least binding affinity and was regarded as the most stable ligand for the AChE protein among the tested compounds.The detailed molecular interaction analysis revealed that AChE amino acid residues, including Tyr72, Tyr124, Trp286, Ser293, Val294, Tyr337, Phe338, and Tyr341, interacted with tectochrysin (1) and dihydrotanshinone-I (Figure 1, supporting information).The length of the hydrogen bond established by tectochrysin (1) with the AChE protein's Phe295 amino acid residue was 2.80 Å, compared to 2.65 Å for the control compound dihydrotanshinone-I.These docking outcomes, similar to those of the control dihydrotanshinone I, revealed that tectochrysin (1) was the most effective inhibitor of the AChE target protein among the four flavonoids tested in the laboratory.It also demonstrated that tectochrysin (1) had a high potential to inhibit the activity of the AChE target protein (Figure 6).

Steered molecular dynamic analysis
The binding properties of two best docked ligand-protein complexes (1-2) (AChE-tectochrysin and BChE-galangin), and the positive controls with AChE (AChE-dihydrotanshinone-I) and BChE (BChE-tacrine) were investigated using steered molecular dynamics (SMD) simulation.Using time-dependent external forces applied to a system, the SMD study implemented non-equilibrium statistical mechanics on a fully hydrated model using explicit triclinic boundary with harmonic restraints to quickly assess the ligand's binding pathways, from which the binding process may be predicted (Izrailev et al., 1999).Initially, SMD studies were performed to achieve a stable binding posture by loosening the solvation complex using NPT simulation in 20 ns.The all-atom RMSD (root-meansquare deviation) value was used to validate the stability of the complex.As shown in Figure 8, the mean RMSD values of AChE-tectochrysin(1), BChE-galangin (2), as well as positive control complexes, range from 0.37209 to 0.58958 Å (Table 4).Specifically, in the 20-ns simulation procedure, the complex AChE-(1) reached equilibrium and showed only a tiny amplitude of RMSD fluctuation (mean RMSD ¼ 0.37926 Å).This result was also similar to the control dihydrothanshinone-I with a RMSD value of 0.37209 Å (Figure 8 and Table 4).Meanwhile, the BChE complex with tacrine displayed considerable conformational changes, with RMSD values rising at intervals of 4 ns and 8 ns before progressively stabilizing at 9 ns after that.In contrast to BChE-tacrine, the BChE-(2) complex exhibited tiny and stable alterations that lasted until the end of the simulation with an RMSD value of 0.43336 Å (Table 4).All these RMSD values were less than 2 Å, which means that the backbone  deviation inside the studied ligand-protein complexes was low.
Secondly, the SMD study was applied to estimate the binding affinity of cholinesterase inhibitors after the system was fully relaxed after 20 ns NPT simulation.During this process, the inhibitors (tested flavonoids) were forced to separate from AChE and BChE by applying an external force on the ligand's center of mass in the direction of Z, which is  analogous to the predicted unbinding pathway (Figure 2, supporting information) (Nhung et al., 2022).Both the averaged rupture force (F max ) and the mean pulling work (W) obtained from 10 independent trajectories of SMD simulation processes were recorded every 0.02 ps (Figure 9 and Table 4).As shown in Table 4, the mean rupture force F max of AChE-(1), AchE-dihydrothanshinone-I, BChE-(2) and BChE-tacrine had values of 400.172 ± 34.796, 448,.995 ± 13.584, 432.367 ± 25.326, and 476.776 ± 34.980 pN, respectively, while the mean pulling work form was 46.030 ± 3.594, 49.192 ± 2.805, 54.747 ± 2.793, and 57.819 ± 5.205 kcal/mol, respectively (Table 4).DG SMD (kcal/mol) calculated by the SMD method according to the formula proposed previously (Thai et al., 2022) of the complexes AChE-tectochrysin and AChE-dihydrotanshinone-I were À 8.812 and À 8.982 kcal/mol, respectively.The DG value of the AChE-dihydrotanshinone-I similar to that obtained from experiment (À 8.54 kcal/mol) (Cheung et al., 2013) implied that the calculation by the SMD method was reliable.
Finally, to clarify whether the simulation time had an effect on the binding behavior of the studied complexes, an additional simulation at 150 ns was run, and the RMSD was determined.As shown in Table 4 the RMSD values of all complexes including AChE-tectochrysin, BChE-galangin, AChE-dihydrothanshinone-I and BChE-tacrine in 150 ns simulation were less than 2 Å and not significantly changed from those in 20 ns-simulation.The BChE-galangin complex, however, was not stable after 90 ns and only became unchanging after 120 ns, much like the positive control tacrine (Figure 8).
All these results indicated that (1) the structural change was not significant at either 20 ns or 150 ns, (2) the tested complexes reached their stableness and consistency inside the dynamic environment of a solvation model, and (3) the SMD simulation program was appropriate for investigation of these AChE and BChE targets.

ADMET calculation
For a more comprehensive overview of the tested flavonoids, drug-likeliness properties and ADME (absorption, distribution, metabolism, and excretion) calculations were performed.
The calculated results of Lipinski's rule of five for the drug-likeliness properties of the flavonoids and the positive control were displayed in Table 5.It revealed that all of the investigated compounds meet all five Lipinski criteria, including low molecular mass (MW) of less than 500 daltons, high lipophilicity represented by a LogP of less than 5, and a molar refractive index between 40 and 130. Thus, these compounds possess drug-like properties.Table 4.The mean rupture force F max (pN) and the mean pulling work W (kcal/mol) done by 2 flavonoids for 2 protein targets were obtained from 10 independent trajectories of SMD simulations (20 and 150 ns).DG SMD (kcal/mol) was calculated by SMD method and DG Exp (kcal/mol) was the binding free energy obtained from experiment. No.
Complexes  The BOILED-egg model (Figure 10 and Table 5) revealed that the three compounds (1, 3, and 4) had the highest probability of permeating to the brain (high blood-brain barrier (BBB) penetration ability) in humans.Only compound cryptostrobin (4) was an inhibitor of P-glycoprotein (PGPþ), which is a membrane protein that ejects foreign substances out of the cell and limits the delivery of therapeutic agents, indicating that it may be active in the central nervous system (CNS) (Nhung et al., 2022).Only galangin (2), which was readily absorbed through the human intestine (passive absorption through the stomach-intestine), was unlikely to cross the blood-brain barrier (Figure 10).
The ProTox-II software predicts oral toxicity based on 2D similarity and the recognition of toxic fragments.In Table 5, the predicted LD 50 (mg/kg) values for each studied flavonoid compound were displayed.Two compounds, tectochrysin (1) and galangin (2), with an LD 50 value of 3919 mg/kg were classified as having a toxicity level of 5 by oral administration.Two other flavonoids (3)(4) were marginally more toxic, with a predicted LD 50 of 2000 mg/kg, and were classified as having a toxicity of level 4.

Discussion
Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases, brings about severe progressive memory loss and cognitive impairments in elderly people (Karmakar et al., 2019).
It has been recognized that the causes of the disease may be due to a decrease in the concentration of ACh through hydrolytic degradation of AChE and BChE and an excessive production of reactive oxygen species (ROS) in the brain cells (Karmakar et al., 2019).Thus, multifunctional AD drugs that combine the activities of AChE inhibitors as well as ROS  production inhibitors (antioxidants) are likely to be highly effective and beneficial in AD treatment.Flavonoids (synthetic or naturally occurring), especially polyhydroxy flavones, are such compounds that meet the above requirements for potential multifunctional anti-AD drugs.They are well known as plant-derived antioxidants and readily scavenge ROS that induce oxidative stress in living cells.Flavonoids were reported to possess in vitro anti-AChE and BChE activities and also prevent the aggregation of b-amyloid peptide (Luo et al., 2013).Therefore, in the search for a new scaffold for AD treatment, we screened a series of flavonoids, performed in vitro and in silico experiments to find out that the flavonoids isolated from Pinus krempfii showed its remarkable inhibitory activity against AChE and BChE and the potential to develop non-toxic, multifunctional anti-AD drugs.
In silico experiments also confirmed that tectochrysin (1) had the lowest binding affinity for the AChE protein with a DG value of À 9.329 kcal/mol and galangin (2) also had the lowest binding energy value of À 9.072 kcal/mol for BChE.The amino acid residue Phe 295 of the AChE protein formed a hydrogen bond with (1) with a binding distance of 2.80 Å.Additionally, the amino acid residue Arg296 also formed a new hydrogen bond with this molecule (3.30 Å).This may imply that hydrogen bond formation at residue Phe 295 plays a substantial role in Tectochrysin's capacity to inhibit AChE.In the case of galangin (2), after a thorough simulation with BChE, this still retains the hydrogen bond at the Tyr128 position with a distance of 2.95 Å.It can be seen that the Tyr128 site plays an important role in Galangin's ability to inhibit BChE.
Additionally, the revised binding pose produced by MD simulation was examined.At the molecular level, SMD is thus a valuable method to determine biomolecule's properties, especially its binding and unbinding characteristics.This method was previously evaluated against a group of ten AChE complexes that resulted in a significant correlation coefficient, R W ¼ À 0.92 ± 0.05 (Thai et al., 2022).This is also a convenient protocol for estimating the affinity of AChE inhibitors, therefore, in this study, we chose the SMD method to perform our experiment.All SMD results are shown in Table 4 and Figures 8-9.
In the time of the simulation procedure (20 ns), the complex AChE-(1) reached equilibrium and showed an average RMSD of 0.37926 Å, which was similar to that of the control dihydrothanshinone-I (RMSD value of 0.37209 Å).Meanwhile, the BChE-(2) complex showed stabilization after 9 ns, with an RMSD value of 0.43336 Å, even lower than that of the BChE-tacrine complex (RMSD value of 0.48542 Å) (Figure 8).At a simulation study at 150 ns, these complexes' RMSD values were also < 2 Å (ranging from 0.37529 to 0.58959 Å).
The mean rupture force F max and the mean pulling work W were obtained for 2 tested flavonoids (1-2) and varied from compounds range from 400.172 ± 34.796 to 432.367 ± 25.326 pN and from 46.030 ± 3.594 to 54.747 ± 2.793 kcal/mol, respectively (Table 4).The values of the external pulling forces of (1) had nearly no maxima but successively oscillated around zero after the unbinding contacts between the ligand and the protein were ended (Figure 9).However, these values of the two positive controls, dihydrotanshinone-I (with AChE) and tacrine (with BChE), were higher, ranging from 448.995 ± 13.584 to 476.776 6 34.980 pN and from 49.192 6 2.805 to 57.819 6 5.205 kcal/mol, respectively.This suggests that they might have had more robust binding to their respective proteins (Parihar et al., 2022).This calculation was consistent to the molecular docking results, as the binding free energy of dihydrothanshinone-I with AChE was À 8.982 kcal/mol (experimental À 8.540 kcal/mol), which was slightly smaller than that of tectochrysin (À 8.812 kcal/mol, calculated according to the formula proposed previously (Thai et al., 2022) (Table 4).Therefore, both molecular docking and SMD results suggested that both AChE-tectochrysin and BChE-galangin complexes remained stable throughout the molecular dynamics simulation.
This present work is our preliminary study to determine the neuroprotective activity, especially anticholinesterase activity in vitro and in silico, of several flavonoids from Pinus krempfii root barks.Further research on the potential impacts on other targets or pathways associated with Alzheimer's disease as well as in vivo experiments with natural and modified flavonoids is worthy of exploration.

Conclusion
In this study, the in vitro and in silico AChE and BChE inhibitory activities of four flavonoids, including tectochrysin (1), galangin (2), strobopinin (3), and cryptostrobin (4), isolated from the root bark of Pinus krempfii Lecomte, were evaluated.Tectochrysin (1) was found to be the best AChE inhibitory compound, with an IC 50 value of 33.69 ± 2.80 lM and K i ¼16.90 lM.Galangin (2) also showed its highest inhibitory activity against BChE, with an IC 50 value of 82.21 ± 2.70 lM and K i ¼ 10.5 lM.In silico experiments also confirmed that tectochrysin (1) had the lowest binding affinity for the AChE protein with a DG value of À 9,329 kcal/mol and galangin (2) also had the lowest binding energy value of À 9.072 kcal/mol to for BChE.MD simulation studies (20 and 150 ns) also suggest that AChE-(1) and BChE-(2) complexes might not have binding affinity to their respective proteins as robust and stable as those complexes of the positive controls.The in silico ADME parameters of (1) and (2) were expected to be drug-like and showed good bioavailability based on their absorption, distribution, metabolism, and excretion parameters.In addition, the oral toxicity of these compounds was determined to be low and safe (LD 50 of 3919 mg/kg at a toxicity level of 5 by oral administration).These results indicate that galangin (2) is a potential selective inhibitor for BChE and tectochrysin (1) is for AChE.However, further studies are needed to determine their safety and efficacy in vivo.Both compounds are potential new agents for the development of drugs with neuroprotective effects, and especially for the treatment of Alzheimer's disease.

Figure 5 .
Figure 5. Re-dock results of: A. dihydrotanshinone I inhibitor with AChE receptor and of B. tacrine with BChE receptor.Dihydrotanshinone I and tacrine after redocking simulation (blue), initial (native) (green).

Table 1 .
In vitro AChE and BChE inhibitory activities with IC 50 values and kinetic study of isolated flavonoids (1-4) from Pinus krempfii with binding mode and K i (mM) value of compound (1).
a All compounds examined in a set of triplicated experiment.b Positive control.ND: Not determined.

Table 3 .
Results of docking between flavonoids and BChE protein (PDB ID 4BDS) and interactions with amino residues in the active sites.