www.ijpsonline.com catalepsy, which is comparable to the standard 13. Zhang S, Li H, Yang SJ. Tribulosin protects rat hearts from drug trihexyphenidyl. Our study indicates ischemia/perfusion injury. Acta Pharmacol 2010;31:671‑88. 14. Amin A, Lotfy M, Shafiullah M, Adeghate E. The protective effect of that Tribulus terrestris could be used as an Tribulus terrestris in diabetes. Ann NY Acad Sci 2006;84:391‑401. alternative/adjuvant drug in preventing and treating 15. Gauthaman K, Ganesan AP. The hormonal effects of Tribulus terrestris the extrapyramidal side effects of antipsychotic agents and its role in the management of male erectile dysfunction‑An evaluation using primates, rabbit and rat. Phytomedicine 2008;15:44‑54. in clinical practice. However, it requires further 16. Balanathan K, Omar MH, Zainul MR, Ong FB, Nurshaireen A, preclinical and clinical studies to prove it. Jamil MA. A clinical study on the effect of Tribulus terrestris (Tribestan) on the semen profile in males with low sperm count and low motility. Malay J Obstet Gynaecol 2001;7:69‑78. REFERENCES 17. Pemminati S, Nair V, Dorababu P, Gopalakrishna HN, Pai MR. Effect of aqueous fruit extract of Emblica officinalis on haloperidol induced 1. Casey DE. Tardive dyskinesia: Pathophysiology and animal models. catalepsy in albino mice. J Clin Diagn Res 2009;3:1657‑62. J Clin Psychiatry 2000;6(Suppl 4):5‑9. 18. Ahtee L, Buncombe G. Metoclopramide induces catalepsy and 2. Kulkarni SK, Naidu PS. Tardive dyskinesia: An update. Drugs Today increases striatal homovanillic acid content in mice. Acta Pharmacol 2001;37:97‑119. Toxicol (Copenh) 1974;35:429‑32. 3. Somani RS, Kasture VS, Kasture SB. Haloperidol inhibits (‑) bicucullin 19. Farde L, Nordstrom AL, Wiesel FA, Pauli S, Halldin C, Sedvall G. induced seizures and bicucullin potentiates haloperidol induced Positron emission tomographic analysis of central D1 and D2 dopamine catalepsy in mice. Indian J Pharmacol 1999;31:434‑6. receptor occupancy in patients treated with classical neuroleptics and 4. Klemm WR. Evidence for a cholinergic role in haloperidol‑induced clozapine: Relation to extrapyramidal side effects. Arch Gen Psychiatry catalepsy. Psychopharmacology (Berl) 1985;85:139‑42. 1992;49:538‑44. 5. Silva SR, Futuro HA, Pires JG. Effects of 5‑HT3 receptor antagonists on 20. Sanberg PR. Haloperidol induced catalepsy is mediated by postsynaptic neuroleptic‑induced catalepsy in mice. Neuropharmacology 1995;34:97‑9. dopamine receptors. Nature 1980;284:472‑3. 6. Pires JG, Costa PG, Saraiva FP, Bonikovski V, Futuro Neto HA. 21. Klemm WR. Evidence for a cholinergic role in haloperidol induced Gender‑related differences in the effects of nitric oxide donors catalepsy. Psychopharmacology (Berl) 1985;85:139‑42. on neuroleptic‑induced catalepsy in mice. Braz J Med Biol Res 22. Silva SR, Futuro‑Neto HA, Pires JG. Effect of 5‑HT3 2003;36:239‑45. receptor antagonists on neuroleptic‑induced catalepsy in mice. 7. Al‑Bayati FA, Al‑Mola HF. Antibacterial and antifungal activities Neuropharmacology 1995;34:97‑9. of Tribulus Terrestris L. growing in Iraq. J Zhejiang Univ Sci B 23. Pires JG, Costa PG, Saraiva FP, Bonikovski V, Futuro Neto HA. 2008;9:154‑9. Gender‑related differences in the effects of nitric oxide donors 8. Dikova N, Ognyanova V. Pharmacokinetic studies of Tribestan on neuroleptic‑induced catalepsy in mice. Braz J Med Biol Res Anniversary Scientific Session‑35. Sofia: Chemical Pharmaceutical 2003;36:239‑45. Research Institute; 1983. p. 1‑7. 24. Ossowska K. Neuronal basis of neuroleptic induced extrapyramidal side 9. Singh S, Gupta YK. Aphrodisiac activity of Tribulus terrestris Linn. in effects. Pol J Pharmacol 2002;54:299‑312. experimental models in rats. J Men’s Health 2011;8:575‑7. 25. Polydoro M, Schroder N, Lima MN, Caldana F, Laranja DC, 10. Yan W, Ohtani K, Kasai R, Yamasaki K. Steroidal saponins from fruits Bromberg E, et al. Haloperidol and clozapine‑induced oxidative stress of Tribulus terrestris. Phytochemistry 1996;42:1417‑22. in the rat brain. Pharmacol Biochem Behav 2004;78:751‑66. 11. Rai S, Chowta MN, Prabhu NM, Nishchal BS, Belagali Y, Nishith RS. Evaluation of Tribulus Terrestris in Depression Models of Albino Mice. Am J Pharm Tech Res 2013;3:538‑46. Accepted 10 October 2014 12. Aggarwal A, Tandon S, Singla SK, Tandon C. Diminution of oxalate Revised 06 October 2014 induced renal tubular epithelial cell injury and inhibition of calcium Received 12 October 2013 oxalate crystallization in vitro by Tribulus terrestris aqueous extract of plant. Int Braz J Urol 2010;36:480‑8. Indian J Pharm Sci 2014;76(6):564-567

Flavanone as Acetylcholinesterase Inhibitors: Computational and Experimental Evidence

C. REMYA, K. V. DILEEP, I. TINTU, E. J. VARIYAR AND C. SADASIVAN* Department of Biotechnology and Microbiology and Inter‑University Centre for Bioscience, Kannur University, Thalassery Campus, Palayad‑670 661, India

Remya, et al.: Inhibitory Effect of Glycosides on AChE

Acetylcholinesterase hydrolyzes the neurotransmitter called acetylcholine and is crucially involved in the regulation of neurotransmission. One of the observable facts in the neurodegenerative disorders like Alzheimer’s disease is the decrease in the level of acetylcholine. Available drugs that are used for the treatment of Alzheimer’s disease are

*Address for correspondence E‑mail: [email protected]

November - December 2014 Indian Journal of Pharmaceutical Sciences 567 www.ijpsonline.com primarily acetylcholinesterase inhibitors with multiple activities. They maintain the level of acetylcholine in the brain by inhibiting the acetylcholinesterase function. Hence acetylcholinesterase inhibitors can be used as lead compounds for the development of drugs against AD. In the present study, the binding potential of four flavanone glycosides such as , , and sakuranin against acetylcholinesterase was analysed by using the method of molecular modeling and docking. The activity of the top scored compound, naringin was further

investigated by enzyme inhibition studies and its inhibitory concentration (IC50) towards acetylcholinesterase was also determined.

Key words: Acetylcholinesterase, Alzheimer’s disease, flavanone glycosides, naringin, induced fit docking

Alzheimer’s disease (AD) is one of the progressive determined. It has already been reported that naringin neurodegenerative disorders characterized can alleviate cognitive impairment and oxidative stress by impairment in memory and cognition. AD is induced neuronal injury and it possesses memory associated with the loss of cholinergic neurons in enhancement property[11,12]. the brain regions related to memory and learning, and is also characterized by the presence of amyloid All the molecular docking studies were deposits and neurofibrillary tangles in the brain[1]. performed using Induced Fit Docking (IFD) Studies have proved that the memory loss and method (Schrödinger, LLC, New York, USA)[13]. The learning inabilities were linked with decreased level atomic coordinates of AChE (PDB ID: 1B41) were of acetylcholine (ACh), a neurotransmitter, in the downloaded from Protein Data Bank. The protein brain. This finding has led to research on methods to structure was prepared for docking studies using enhance the level of the cholinergic neurotransmitter the protein preparation wizard of Schrödinger suit. by the inhibition of acetylcholinesterase (AChE)[2]. The Initially, all the crystallographic water molecules were inhibition of AChE not only enhance the cholinergic deleted and hydrogen atoms were added. The bond transmission, but also reduce the aggregation of orders, partial charges and protonation states were amyloid beta (Aβ) peptide and the formation of the corrected properly. Finally the energy minimization neurotoxic fibrils in AD[3]. Recently, it has been shown was carried out using OPLS‑2005 force field. that AChE interacts with Aβ peptides and promotes the formation of amyloid fibril through a group of Similarly the structures of flavanone glycosides (fig. 1) amino acids located in the proximity of the peripheral were downloaded from PubChem database and the anionic binding site (PAS) of the enzyme[4]. It has geometries were optimized using LigPrep module. also been suggested that those molecules, which can OPLS‑2005 force field was again used for the energy interacts with PAS and catalytic sites may prevent the minimization process. The IFD protocol considers aggregation of Aβ mediated by AChE[5]. Aging is one of the main risk factors for AD and is reported that oxidative stress on the central nervous system increases upon aging. A number of studies has also been reported on the protective effects of various polyphenols against aging and related neurodegenerative diseases[6]. The main objective of the present study is to investigate whether selected flavone glycosides (naringin, hesperidin, poncirin and sakuranin) can inhibit the enzyme AChE. Molecular modeling and docking studies have been carried out to assess the relative binding affinity of the compounds towards AChE. The most promising compound has been tested using in vitro enzyme inhibition assay. The selected compounds have already been reported to possess anti oxidant and antiinflammatory properties[7‑10]. The top Fig. 1: Schematic representation of flavanone glycosides. scored compound in the docking studies was found The structures of flavanone and its different glycosides selected for to be naringin. The IC50 value of the compound was the study are shown.

568 Indian Journal of Pharmaceutical Sciences November - December 2014 www.ijpsonline.com flexibility to both compounds and protein. The at each concentration of naringin was calculated. It was following residues D74, Y86, G120, G122, Y124, plotted against the concentrations of naringin and the

S125, E202, S203, Y286, F295, E334, Y337, Y341 and IC50 value was calculated from the graph. H447 lining the binding site of human AChE (hAChE) were kept as flexible. The compounds were docked into AChE has a well‑defined active site. The residues the rigid protein using the softened potential and the S203, H447 and E334 of hAChE are essential for resulting top 20 poses of each ligand were taken. Then the catalysis and are being known as catalytic triad. the flexibility was applied to the protein residues, which The ligand‑binding site cleft in the cholinesterase is were within the 5Å of the docked ligands. The ligands extending from active site to PAS. The glide score and were redocked and glide XP (extra precision) was used binding energies obtained for all compounds are given for all docking calculations. After docking, the binding in Table 1. Naringin form several hydrogen bonds energy of all ligands was also calculated using Prime with the residues of AChE. The hydroxyl group of MM‑GBSA method. This method combines OPLS ‘B’ ring forms two hydrogen bonds with Gly121 and molecular mechanics energies (EMM), surface generalized Ser203, a catalytic residue located in the active site.

Born solvation model for polar solvation (GSGB) and Oxygen atom and keto group of ‘C’ ring is hydrogen a nonpolar solvation term (GNP). GNP term includes bonded with Tyr124 and Phe295, respectively. The the nonpolar solvent accessible surface area and van hydroxyl group attached to the ‘A’ ring also makes der Waals interactions. The binding free energy was a hydrogen bond with Phe295. The hydroxyl groups calculated using the following Eqn., ΔGbind=Gcomplex− of attached sugar moieties forms hydrogen bond with

(Gprotein+Gligand), where, G=EMM+GSGB+GNP Gln291, Asp74 and Thr75. Apart from these, a stacking interaction is found between the ‘A’ ring and Tyr341. The top scored compound, naringin was subjected to The hydroxyl and methoxy group of ‘B’ ring of enzyme inhibition assay using AMPLITETM AChE assay hesperidin form hydrogen bonds with Ser203, Gly121 kit in a 96 well microtiter plate based on Ellman’s and Gly122, respectively. The keto group located in method[14]. The electric eel AChE (EC 3.1.1.7) was the ‘C’ ring forms a hydrogen bond with Phe295. used for the study since it is structurally similar A π‑π stacking interaction was also observed between to the vertebrate’s nerve and muscle AChE. The ‘A’ ring and Tyr341. Hydroxyl groups of the sugar enzyme solution of about 1.37 µM concentration moieties form hydrogen bonds with Tyr72, Glu292 was prepared in double distilled water containing a and Ser293. A slightly different binding pattern was trace amount of BSA. Acetylthiocholine and Ellman’s observed for poncirin towards AChE. In this a classical reagent (5,5’‑dithiobis‑(2‑nitrobenzoic acid, DTNB) π‑π stacking interaction was observed between the ‘A’ were used as the substrate and chromogenic compound ring of poncirin and indole ring of Trp286. One of respectively. Acetylthiocholine reaction mixture the hydroxyl groups in the sugar moiety was found was prepared by adding 250 μl of each DTNB and to involve hydrogen bonding with the hydroxyl group acetylthiocholine stock solutions to 4.5 ml of assay of Tyr72. Similarly, the hydroxyl group of ‘B’ ring of buffer (pH 7.4). The activity of the enzyme was the sakuranin forms a hydrogen bond with His447, a assayed by mixing 25 µl of AChE, 25 μl of water catalytic residue. The keto group located in the ‘C’ ring and 50 μl of acetylthiocholine reaction mixture. The forms an interaction with Phe295. Apart from these, optical density was measured at 415 nm with 5 min the ‘A’ ring is involved in a stacking interaction with interval. The optical densities versus time graph were Tyr341. One of the hydroxyl groups of sugar moiety plotted. Naringin (Sigma Aldrich, Bangalore, India) mediates a hydrogen bond with Ser293. solutions with different concentrations such as 172, 344, 516, 688, and 860 µM were prepared in 0.01% TABLE 1: GLIDE SCORES AND BINDING ENERGIES OF of DMSO solution. The enzyme was incubated with 25 SELECTED FLAVANONE GLYCOSIDES µl of the naringin solution for 30 min and the assays Flavanone Glide score Binding free glycosides kcal/mol energy kcal/mol were repeated as given above in order to test whether Naringin −15.87 −54.81 the compound can inhibit enzyme activity. It was also Hesperidin −14.84 −53.18 confirmed that 0.01% DMSO has no inhibitory effect Poncirin −13.47 −48.54 on AChE. For each concentration of the ligand, reaction Sakuranin −11.84 −43.67 The affinity of the selected flavonones towards AChE in terms of Glide scores was carried out and the optical density was plotted and binding energies calculated using molecular mechanics, the generalized against time. From the graph, relative enzyme activity Born model and solvent accessibility method is shown

November - December 2014 Indian Journal of Pharmaceutical Sciences 569 www.ijpsonline.com

a b

Fig. 3: Relative activity of AChE plotted against different concentration of naringin. c d The relative activity of the enzyme in the presence of different Fig. 2: Interactions of flavanone glycosides with human AChE. concentrations of naringin was calculated by assuming the activity The docked pose of (a) naringin, (b) hespiridin, (c) poncirin and of enzyme in the absence of naringin was 100%. IC50 value was (d) sakuranin at the active site of hAChE. The ligands and the protein determined from the graph. residues are shown in thick and thin lines respectively. The hydrogen bonds are shown as dashed lines. 1998;57:719‑31. 3. Selkoe DJ. Deciphering the genesis and fate of amyloid betaprotein From the detailed structural analysis, it was found yields novel therapies for Alzheimer disease. J Clin Invest 2002;110:375‑81. that all flavanone glycosides bind in such a way, the 4. Rees T, Hammond PI, Soreq H, Younkin S, Brimijoin S. flavanone moiety is pierced in to the active site cleft Acetylcholinesterase promotes beta‑amyloid plaques in cerebral cortex. and moieties are occupied at PAS near to the Neurobiol Aging 2003;24:777‑87. surface. The interactions formed by these ligands in the 5. Castro A, Martinez A. Targeting beta‑amyloid pathogenesis through acetylcholinesterase inhibitors. Curr Pharm Des 2006;12:4377‑87. active site and PAS may prevent the substrate and Aβ 6. Kay CD. The future of flavonoid research. Br J Nutr 2010;104:S91‑5. binding to the AChE respectively. The binding mode 7. Hirata A, Murakami Y, Shoji M, Kadoma Y, Fujisawa S. Kinetics of all flavanone glycosides in the active site of AChE of radical‑scavenging activity of and hesperidin and their inhibitory activity on COX‑2 expression. Anticancer Res 2005;25:3367‑74. is shown in fig. 2. From the analysis, it was found 8. Cavia‑Saiz M, Busto MD, Pilar‑Izquierdo MC, Ortega N, Perez‑Mateos M, naringin exhibit a strong binding affinity towards AChE Mu˜niz P. Antioxidant properties, radical scavenging activity and than other compounds. Hence an in vitro enzyme biomolecule protection capacity of flavonoid and its glycoside naringin: A comparative study. J Sci Food Agric 2010;90:1238‑44. inhibition study was carried out for naringin against 9. Kim JB, Han AR, Park EY, Kim JY, Cho W, Lee J, et al. Inhibition of LPS‑induced iNOS, COX‑2 and cytokines expression by poncirin AChE and the IC50 was found to be 446 µM (fig. 3). This is in accordance with the findings reported by through the NF‑kappa B inactivation in RAW 264.7 macrophage cells. Biol Pharm Bull 2007;30:2345‑51. Maratha et al. in 2012 that naringin can decreases 10. Zhang X, Hung TM, Phuong PT, Ngoc TM, Min BS, Song KS, et al. the brain AChE activity[11]. The current study reveals Antiinflammatory activity of flavonoids from Populus davidiana. Arch that naringin may be used as a lead compound for the Pharm Res 2006;29:1102‑8. 11. Maratha SR, Mahadevan N. Memory Enhancing Activity of Naringin development of drugs against AD. in Unstressed and Stressed Mice: Possible Cholinergic and Nitriergic Modulation. Neurochem Res 2012;37:2206‑12. ACKNOWLEDGEMENTS 12. Kumar A, Dogra S, Prakash A. Protective effect of naringin, a citrus flavonoid, against colchicine‑induced cognitive dysfunction and oxidative damage in rats. J Med Food 2010;13:976‑84. The authors gratefully acknowledge the use of computational 13. Sherman W, Day T, Jacobson MP, Friesner RA, Farid R. Novel facilities provided by Bioinformatics Infrastructure Facility Procedure for Modeling Ligand/Receptor Induced Fit Effects. J Med (supported by DBT, Government of India) at Kannur Chem 2006;49:534‑53. 14. Ellman GL, Courtney KD, Andres V Jr, Featherstone RM. A new University. Authors CR and KVD thank the ICMR for and rapid colorimetric determination of acetylcholinesterase activity. providing Senior Research Fellowships. Biochem Pharmacol 1961;7:88‑95.

REFERENCES Accepted 12 October 2014 1. Selkoe DJ. Alzheimer’s disease: genes, proteins and therapy. Phys Rev Revised 07 October 2014 2001;81:741‑66. Received 06 November 2013 2. Ladner CJ, Lee JM. Pharmacological drug treatment of Alzheimer disease: The cholinergic hypothesis revisited. J Neuropathol Exp Neurol Indian J Pharm Sci 2014;76(6):567-570

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