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Chemico-Biological Interactions 181 (2009) 383–389

Contents lists available at ScienceDirect

Chemico-Biological Interactions

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holinesterase inhibitory activities of some flavonoid derivatives and chosenanthone and their molecular docking studies

ahmud Tareq Hassan Khan a,∗, I. Orhan b, F.S. Senol b, M. Kartal c, B. Sener b, M. Dvorská d,. Smejkal d, T. Slapetová d

Department of Pharmacology, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, NorwayDepartment of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, TurkeyDepartment of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100 Ankara, TurkeyDepartment of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1-3, 61242 Brno, Czech Republic

r t i c l e i n f o

rticle history:eceived 7 May 2009eceived in revised form 28 June 2009ccepted 29 June 2009vailable online 9 July 2009

eywords:lzheimer’s diseasecetylcholinesteraseutyrylcholinesterase

a b s t r a c t

Flavonoids are one of the largest classes of plant secondary metabolites and are known to possess anumber of significant biological activities for human health. In this study, we examined in vitro acetyl-cholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of four flavonoid derivatives– quercetin, rutin, kaempferol 3-O-�-d-galactoside and macluraxanthone. The in vitro results showedthat quercetin and macluraxanthone displayed a concentration-dependant inhibition of AChE and BChE.Macluraxanthone showed to be the most potent and specific inhibitor of both the enzymes having theIC50 values of 8.47 and 29.8 �M, respectively. The enzyme kinetic studies revealed that quercetin inhibitedboth the enzymes in competitive manner, whereas the mode of inhibition of macluraxanthone was non-competitive against AChE and competitive against BChE. The inhibitory profiles of the compounds have

lavonoidsanthoneolecular docking

been compared with standard AChE inhibitor galanthamine. To get insight of the intermolecular interac-tions, the molecular docking studies of these two compounds were performed at the active site 3D spaceof both the enzymes, using ICM-DockTM module. Docking studies exhibited that macluraxanthone bindsmuch more tightly with both the enzymes than quercetin. The calculated docking and binding energiesalso supported the in vitro inhibitory profiles (IC50 values). Both the compounds showed several stronghydrogen bonds to several important amino acid residues of both the enzymes. A number of hydrophobic

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interactions could also ex

. Introduction

Flavonoids refer to a large class of plant secondary metabolitesaving a polyphenolic structure and when widely found in fruits,egetables, and certain beverages, they have miscellaneous favor-ble biochemical and antioxidant effects associated with variousiseases such as cancer, Alzheimer’s disease, atherosclerosis, etc.1–3].

Alzheimer’s disease (AD), the most frequent form of dementia,s a neurodegenerative disease characterized by deficiency in theeuromediator acetylcholine or �-amyloid plaque formation. AD

as become one of the major health problems among elderly peo-le, especially in the industrialized countries. The recent statistics

ndicate that AD affects approximately 13 million people worldwidend, with the aging of Western countries population, represents

∗ Corresponding author. Tel.: +47 97794171.E-mail addresses: mahmud.khan@uit.no, mthkhan2002@yahoo.com

M.T.H. Khan).

009-2797/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Ireland Ltoi:10.1016/j.cbi.2009.06.024

the potency of the compounds to inhibit AChE and BChE.n Copyright © 2009 Published by Elsevier Ireland Ltd. All rights reserved.

one of the main public health issues. The disease has been also pro-posed to be associated with oxidative stress [4,5]. The cholinergichypothesis, “a deficit in central cholinergic transmission caused bydegeneration of the basal forebrain nuclei”, was established as themost known speculation of AD symptoms and the interference ofacetylcholine insufficiency has been accepted the most applicabletreatment strategy for AD. The drugs approved and licensed for thedisease have been generally acetylcholinesterase (AChE) inhibitorssuch as rivastigmin, donepezil, and galanthamine. Although thesedrugs are part of the same therapeutic class, they differ in theirpharmacology and pharmacokinetics and possess some side effects.Besides, there is still no cure for severe type of AD. Therefore, it iscompulsory to discover new anti-Alzheimer drug candidates. In therecent years, a number of potential candidates have been tested forthe development of drugs against AD. Compounds have been stud-

ied using molecular docking and some other in silico studies havebeen reported targeting AChE and BChE [6–8].

In the present work, we have aimed on investigation of invitro AChE and butyrylcholinesterase (BChE) enzyme inhibitoryactivity of several flavonoids; quercetin, rutin, kaempferol

d. All rights reserved.

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84 M.T.H. Khan et al. / Chemico-Biolo

-O-�-d-galactoside, and of chosen xanthone – macluraxanthone.he kinetic studies have been performed on the compounds tostablish their modes of inhibition. Molecular docking calculationsere performed in order to explain their interactions with both the

nzymes.

. Materials and methods

.1. Flavonoid derivatives

Quercetin and rutin were obtained from Serva Chemical Com-any (Heidelberg, Germany). Kaempferol-3-O-�-d-galactoside wasbtained from the aerial parts of Calluna vulgaris L. (Ericaceae) asescribed in our previous publication [9]. Macluraxanthone was

solated from nonpolar fractions of methanolic extract from the rootark of Maclura pomifera (Rafin.) Schneider (Moraceae) using col-mn chromatography on silica gel (parts size 0.04–0.06 mm), in theixture of benzen:ethylacetate (8:2) and identified by analyzing

nd comparison of basic spectral characteristics, HRMS, 1H and 13CMR with those found in literature [10]. The molecular structuresf the compounds are shown in Fig. 1.

.2. Determination of AChE and BChE inhibitory activities

AChE and BChE inhibitory activities were measured by slightlyodified spectrophotometric method developed by Ellman et al.

11]. Torpedo californica (electric eel) AChE (Type-VI-S, EC 3.1.1.7,igma) and horse serum BChE (EC 3.1.1.8, Sigma) were used,hile acetylthiocholine iodide and butyrylthiocholine chloride

Sigma, St. Louis, MO, USA) were employed as reaction sub-trates. 5,5′-dithio-bis(2-nitrobenzoic)acid (DTNB, Sigma, St. Louis,O, USA) was used as chromogenic substrate for the measure-ent of the cholinesterase activity. All the other reagents and

onditions were the same as described in our earlier publica-

ions [12,13]. Briefly, in this method, 140 �l of 0.1 mM sodiumhosphate buffer (pH 8.0), 20 �l of DTNB, 20 �l of test solution,nd 20 �l of AChE/BChE solution were added by multichan-el automatic pipette (Gilson pipetman, France) into a 96-wellicroplate and incubated for 15 min at 25 ◦C. The reaction

Fig. 1. Chemical structures of the tested flavonoid derivatives; quercetin (1), m

Interactions 181 (2009) 383–389

was then initiated by the addition of 10 �l of acetylthiocholineiodide/butyrylthiocholine chloride. The hydrolysis of acetylthio-choline iodide/butyrylthiocholine chloride was monitored by theformation of the yellow 5-thio-2-nitrobenzoate anion as a resultof the reaction of DTNB with thiocholines, catalyzed by enzymes,measured at a wavelength of 412 nm using a 96-well microplatereader (VersaMax Molecular Devices, CA, USA). The measurementsand calculations were evaluated using Softmax PRO 4.3.2.LS soft-ware. Galanthamine, the anticholinesterase alkaloid-type of drugisolated from the bulbs of snowdrop (Galanthus sp.), was purchasedfrom Sigma (St. Louis, MO, USA) and used as reference. Percentageof inhibition of AChE/BChE was determined by using the formuladescribed below, where E and S are the activities of enzyme withoutand done in triplicate.% Inhibition = E−S

E×100

2.3. Kinetic studies of the compounds against AChE and BChE

The enzyme kinetic studies were performed for the compoundsshowing prominent inhibition of ChEs during initial screenings(quercetin and macluraxanthone). All methods were similar tothe initial studies (described above) except that multiple con-centrations (5, 10, 50, 100, 500, and 1000 �M) of the substrates(AChI and BChCl for AChE and BChE, respectively) were used.The kinetic parameters (Vmax, Km) were then calculated usingEnzyme KineticTM module of SigmaPlotTM version 10, integratedwith SigmaStatTM. Finally the Ki values were determined from IC50of the inhibitors values using the Cheng–Prusoff relation [14–16].

Ki = IC50

(1 + S/Km)(1)

In the similar fashion the enzyme kinetic studies of the standardAChE inhibitor galanthamine also been performed, in final concen-trations of 1.0, 0.1, and 0.01 mM, for comparison of the effects ofexperimental compounds (1 and 2).

2.4. Docking calculations with ICMTM (internal coordinatemechanics) dock

All the docking calculations of compounds quercetin andmacluraxanthone were performed using the ICMTM docking

acluraxanthone (2), kaempferol-3-O-�-d-galactoside (3), and rutin (4).

M.T.H. Khan et al. / Chemico-Biological Interactions 181 (2009) 383–389 385

F ChE. (a .

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ig. 2. The double reciprocal (Line-weaver Burk) plots of 1 and 2 against AChE and Bnd the panels C and D show the kinetics of 2 against AChE and BChE, respectively)

odule with the default setup [17]. The mmff charges weressigned.

.4.1. Preparations of the inhibitors and target moleculesTo the lowest energy conformers of compounds quercetin and

acluraxanthone were from the 3D energy minimization withinhe ICM space starting from mol files. The compounds were dockedoth into eAChE (PDB code 1c2b) and huBChE (PDB code 2pm8)18].

.4.2. Docking processAll the docking calculations were performed using the ‘inter-

ctive docking’ menu at the ICM environment. After docking thetack of docking poses were checked visually. Multiple stack con-ormations were selected based on their docking energies, rmsd

alues (compared between the docked model and X-ray conforma-ion) and similarities to closely related X-ray crystal structures fromDB. Then, the best conformations for each of the compounds werenally chosen, and their binding energies were calculated using

CM script (briefly described in the following Section 2.4.3). Bothhe docking energy (Edocking) and binding energies (Cal.�G) areresented in Section 3.

Here, the panels A and B show the kinetics of 1 against AChE and BChE, respectively,

2.4.3. Calculations of free energies of bindingFor each of the individual docked complexes the free energies of

binding (Cal.�G) between the protein and ligand were calculatedusing ICM script utilizing the Eqs. (2) and (3) [19].

�G = �GH + �GEL + �GS + C (2)

�G = �GH + �GCOUL + �GDESOLV + �GS + C (3)

Here �GH is the hydrophobic or cavity term, which accounts forthe variation of water/non-water interface area. �GEL is the elec-trostatic term composed of coulombic (�GCOUL) interactions anddesolvation (�GDESOLV) of partial charges transferred from an aque-ous medium to a protein core environment. �GS is the entropicterm which results from the decrease of the conformational free-dom of functional groups buried upon complexation; and finallythe C is a constant account for the change of entropy of the systemdue to the decrease of free molecules concentration (cratic factor),and due to the loss of rotational/translational degrees of freedom[19].

2.4.4. Interpretations of intermolecular interactionsTo study the intermolecular interactions between the targets

and the compounds, LigPlot [20] were used to plot the interactionsfrom 3D to 2D. Beside LigPlot, ICM (www.molsoft.com) and Discov-

386 M.T.H. Khan et al. / Chemico-Biological Interactions 181 (2009) 383–389

Fig. 3. Enzyme kinetic profile of Galanthamine, a standard Acetylcholinesterase inhibitor. (a) double reciprocal (Line-weaver Burk) plot, and (b) Michaelis–Menten plot.

Table 1Summary of the inhibiting profiles of 1 and 2 against AChE and BChE.

AChE BChE Specificity (B:A)

IC50 (�M) Ki (�M) Type IC50 (�M) Ki (�M) Type

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tight binding against both enzymes with higher docking and bind-ing energies. These docking results are also supported by the invitro binding data (IC50 values in Table 1). The calculated energyparameters are shown in Table 2.

Table 2Summary of the molecular docking studies of 1 and 2 against AChE and BChE.

AChE BChE

Denergya �Ga rmsdb Denergy

a �Ga rmsdb

353.86 3.79 Competitive8.47 3.43 Non-competitive

alanthamine 0.75 2.08 × 10−5 Competitive

ry Studio Visualizer (www.accelrys.com) also been used to analyzehe interactions in 3D space.

. Results

.1. Cholinesterase inhibitory profiling

Four different types of flavonoids – quercetin, rutin, kaempferol-O-�-d-galactoside, and the xanthone (macluraxanthone) werecreened for their in vitro AChE and BChE inhibitory activities atifferent concentrations using ELISA microplate reader by Ellmanethod. Among them, only the aglycones – quercetin and maclu-

axanthone were found to inhibit both enzymes with IC50 valuesf 353.86 and 8.47 �M against AChE, respectively, and 420.76 and9.8 �M against BChE, respectively. The other two flavonoids con-aining sugar moieties (rutin and kaempferol 3-O-�-d-galactoside)howed insignificant inhibition towards these enzymes below 50%t the higher concentration tested and therefore, their IC50 valuesere not feasible to calculate.

The enzyme kinetic studies (shown in Fig. 2) pointed out thatuercetin and macluraxanthone are the competitive inhibitors ofChE. Macluraxanthone displayed a non-competitive inhibitiongainst AChE, while inhibition pattern of quercetin was found to beompetitive against it. The enzyme kinetic profile of galanthamine,standard acetylcholinesterase inhibitor, have been performed andresented. A double reciprocal (Line-weaver Burk) (in panel a) andichaelis–Menten (in panel b) plots are being shown in Fig. 3,hich showed that galanthamine is a strong competitive inhibitor

f AChE.The inhibition data and types of inhibitions of these two com-

ounds have been compared with the standard AChE inhibitoralanthamine and presented in Table 1.

.2. Specificity of the compounds

BChE is particularly expressed in serum, liver, heart, and in cen-ral nervous system (CNS). Unlike AChE, its physiologic function isot yet completely revealed [21]. It may not be an advantage for

420.76 3.43 Competitive 0.9129.8 2.12 Competitive 0.62

8.2

a cholinesterase inhibitor (ChEI) to be selective for AChE; on thecontrary, a good balance between AChE and BChE may result in ahigher efficacy. It is very prone that extracellularly diffused ACh maycome into contact with glial BChE and be effectively hydrolyzed,as demonstrated by the administration of the mixed AChE/BChEinhibitor rivastigmine in AD patients, because in these patientsBChE inhibition correlates significantly with cognitive improve-ment [22].

Our in vitro results demonstrated that both the flavonoid deriva-tives – quercetin and macluraxanthone, are the specific inhibitorsof AChE to different extents. The respective data are shown inTable 1.

3.3. Molecular docking studies

To get insight the intermolecular interactions, the moleculardocking studies were performed for quercetin and macluraxan-thone at the active site 3D space of both the AChE and BChE, usingICM-DockTM module. The docking calculations of quercetin at theactive sites of AChE and BChE revealed that the compounds bound toboth the enzyme with lower docking (Denergy) and binding energies(�G) when compared with macluraxanthone. The best conforma-tion of the second compound, macluraxanthone, exhibited quite

1 −64.14 −0.38 1.977 −67.35 −0.47 0.30682 −82.66 −15.49 0.277 −86.49 −11.61 0.819

a Energies in Kcal/mol.b The root mean square deviation of the starting and the final docked conforma-

tion.

M.T.H. Khan et al. / Chemico-Biological Interactions 181 (2009) 383–389 387

Fig. 4. Intermolecular interactions of 1 and 2 with AChE and BChE in 2D flattened space; where the panels A and B show the interactions of 1 with AChE and BChE, respectively,and the panels C and D show interaction of 2 with AChE and BChE, respectively. Brown stick models present the residues of the enzymes, thick purple stick models presentthe inhibitors (1 and 2), green dotted lines are hydrogen bonds and dashed half-moons present hydrophobic interactions with the corresponding amino acid residues of theenzymes.

388 M.T.H. Khan et al. / Chemico-Biological

Fasn

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ig. 5. Interactions of 2 with AChE at 3D space. Interactions with specific aminocid residues are shown in the box. The 3D ribbon represents the enzyme, yellowtick model is the lowest energy conforms of the inhibitor 2, and gray-sticks withumberings present the interacting amino acid residues of the enzyme.

.4. Putative intermolecular interactions

The main phenylchromen part of the quercetin showed severalydrogen bonds with the important amino acid residues of anionicubsite (AS) of AChE, as O3 (length = 2.58 Å) and O5 (2.26 Å) showedwo hydrogen bonds of the OH atom of Tyr133; and O4 (2.72 Å)howed hydrogen bond with the O atom of Trp86. The entireuercetin showed hydrophobic interactions with Ser125, His447,nd Glu202, which are some of the prominent residues of AS ofChE. The O atoms of the phenyl side chain of the same compoundlso showed hydrogen bonding to the important residues of periph-ral anionic subsite (PAS) of AChE, as O6 atom of the quercetinhowed hydrogen bonds with O (3.31 Å) and OE1 (2.88 Å) atoms ofhe Tyr72 as well as Gln71, respectively; and O7 (2.76 Å) with the

atom of Asp74. Lowest energy conformer of quercetin showedt least five hydrogen bonds at the active site of BChE. Atoms O3

3.0 Å) and O5 (2.2 Å) showed bonds with OH atom of Tyr128 and4 (3.16 Å) with the OG1 atom of Thr120; and O6 atom showedydrogen bonds with the OD1 (2.76 Å) and N (2.95 Å) atoms ofsn83 and Asp70, respectively. The 2D interactions of quercetin are

ig. 6. Interactions of 2 with BChE at 3D space. Interactions with specific amino acidesidues are shown in the box. The 3D ribbon represents enzyme, yellow stick models the lowest energy conform of the inhibitor 2, and gray-sticks with numberingsresent interacting amino acid residues of the enzyme.

Interactions 181 (2009) 383–389

shown in panels A and B of Fig. 4, where panel A represents AChEand B represents BChE. The atom numberings are also shown in theFig. 4.

The two oxygen atoms of macluraxanthone showed hydrogenbonds with the important amino acid residues of PAS site of AChE.There are bonds of O5 (2.49 Å) and O6 (2.5 Å) with the OH atom ofTyr124 and O atom of Tyr72. Other than this, the entire macluraxan-thone exhibited several hydrophobic interactions with the severalsubsites, like PAS, AS and acyl-binding pocket (ABP), of AChE. Proba-bly due to all these interactions the compound showed high potencyto inhibit AChE having IC50 value of 8.47 �M. The 2D and 3D interac-tions of macluraxanthone with AChE are shown in panel C of Fig. 4and in Fig. 5, respectively.

The O4 atom of the same compound showed hydrogen bondswith Thr120 of BChE and several hydrophobic interactions with theseveral important amino acid residues of the enzyme, like Asp70,Tyr332, Phe329, Trp82, Gly439, and some long distance interactionswith Ser198 and Glu197. The numbering of macluraxanthone atomsis shown in Fig. 4. The 2D interactions of macluraxanthone againstBChE are shown in panel D of Fig. 4 and in 3D shown in Fig. 6.

4. Discussion

Flavonoids are one of the largest compound classes widely foundin herbal sources. They have been reported to possess a variety ofdesired biological activities for human health. However, there hasbeen relatively lower number of reports on AChE/BChE inhibitoryactivity of flavonoids, which is the main strategy for the treatmentof AD. On the other hand, flavonoids as the polyphenolic substanceshave been known to have strong antioxidant activity, which is anadvantage for them in AD treatment [23]. In our recent study, wecarried out a preliminary screening on chemically diverse flavonoidderivatives for their AChE and BChE inhibitory effects at a concen-tration of 1 mg/ml [24] and amongst them, quercetin was found tobe the most active against AChE having 76.2% inhibition, whereasit showed 46.8% inhibition against BChE, which is in accordancewith our present data. Although Fan et al. [25] suggested that asugar moiety in the molecule is necessary for AChE inhibition, ourresults underlined that two flavonoids having glucose moieties,like rutin and kaempferol 3-O-�-d-galactoside, were shown to beineffective against both the enzymes. However, they also foundthat rutin was not inhibiting AChE. This report is consistent withour present data, and the result led these researchers to makeanother suggestion that the position of sugar is also quite impor-tant as well as the length and the interglycosidic linkages of sugarchain.

A number of xanthones have been found to inhibit monoamineoxidase (MAO) and AChE [26,27]. Particularly, Brühlmann et al.[27] found seven xanthone-type of compounds to be highly activeagainst AChE by a convenient microtiter plate assay, which is inagreement with our data obtained with macluraxanthone.

5. Conclusions

Previous reports indicated that quercetin, a flavonol deriva-tive widely found in foodstuff, has been approved to be effectivethrough different mechanisms including antioxidant, �-secretaseinhibition, and prevention of senile plaque formation [28–31]. Inaccordance with our results on AChE and BChE inhibitory activity ofquercetin, it has an enough solace to exert neuroprotective effect viavarious mechanisms. Our molecular docking studies exhibited that

macluraxanthone binds much more tightly with both the enzymesthan quercetin and exhibited a number of strong hydrogen bondswith several important amino acid residues of both the enzymesand a number of hydrophobic interactions against AChE and BChEwhich could explain the potency of the compound.

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In conclusion; the natural compounds tested herein, especiallyacluraxanthone, could be a good candidate as ‘lead’ molecule for

he further development of new drugs against AD.

onflict of interest

The authors declare that there are no conflicts of interest for thisork.

cknowledgements

One of the authors (F. Sezer Senol) would like to acknowledgehe scholarship for master program provided by the Turkish Scien-ific and Technical Research Council (TUBITAK). M.T.H. Khan wouldike to thank for the “2221-Fellowships for Visiting Scientists”nder the “Programme for Visiting Scientists provided by Scientificuman Resources Development of the Scientific and Technologicalesearch Council of Turkey (TUBITAK)”.

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