Anticholinesterase activity of selected phenolic acids and flavonoids – interaction testing in model solutions
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Department of Biotechnology, Human Nutrition and Food Commodity Science, University of Life Sciences, Lublin, Poland
Dominik Szwajgier   

Department of Biotechnology, Human Nutrition and Food Commodity Science, University of Life Sciences, Lublin, Poland
Ann Agric Environ Med. 2015;22(4):690–694
Alzheimer’s disease is a progressively developing neurodegenerative disorder of the central nervous system. The only present treatment of this disease is the use of acetyl- and butyrylcholinesterase inhibitors. Previously, the neuroprotection of phenolic acids and flavonoids in the brain has been indicated.

Material and Methods:
This study measured anticholinesterase activities of 9 phenolic acids and 6 flavonoids, singly or in combination. The synergy/antagonism/zero interaction between compounds was evaluated taking into consideration the statistical significance. Ellman’s modified spectrophotometric method was used with the simultaneous measurement of the false-positive effect of compounds.

The anti-acetylcholinesterase activity of phenolic acids was as follows: homogentisic acid > 4-hydroxyphenylpyruvic acid > nordihydroguaiaretic acid > rosmarinic acid > caffeic acid > gallic acid = chlorogenic acid > homovanillic acid > sinapic acid. p-Hydroxyphenylpyruvic, caffeic, chlorogenic, gentisic, homogentisic, nordihydroguaiaretic and rosmarinic acids in pairs exhibited, in most cases, a lower inhibitory activity (at p>0.05), than the sum of the activities of single compounds. Also, phenolic acids in pairs with flavonoids (cyanidin, delphinidin, kaempferol, myricetin, phloridzin, pelargonidin or quercetin) presented, in most cases, a lower inhibitory activity than could be calculated for both compounds singly (at p>0.05). Only in the case of a few samples was the inhibitory activity of two compounds higher than the sum of inhibitions exerted by the same compounds tested singly (either at p>0.05 or p<0.05). The lack of synergy of pairs of inhibitors suggests one small binding site, making impossible to accommodate both inhibitors adjacent to one another.

AD, Alzheimer’s disease; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; 4-OH-PP, 4-hydroxyphenylpyruvic acid; CA, caffeic acid; CHA, chlorogenic acid; GA, gentisic acid; HGA, homogentisic acid; HVA, homovanillic acid; NDGA, nordihydroguaiaretic acid; RA, rosmarinic acid; SA, salicylic acid; KAE, kaempferol; PEL, pelargonidin; QUE, quercetin; PHL, phloridzin; DEL, delphinidin; CYA, cyanidin; MYR, myricetin; ATChI, acetylthiocholine iodide; BTCh, S-butyrylthiocholine chloride; DTNB, 5,5’-dithiobis-2-nitrobenzoic acid.

Yabe T, Hirahara H, Harada N, Ito N, Nagai T, Sanagi T, et al. Ferulic acid induces neural progenitor cell proliferation in vitro and in vivo. Neuroscience 2010; 165: 515–524.
Szwajgier D, Borowiec K. Phenolic acids from malt are efficient acetylcholinesterase and butyrylcholinesterase inhibitors. J Inst Brew. 2012a; 118: 40–48.
Karakida F, Ikeya Y, Tsunakawa M, Yamaguchi T, Ikarashi Y, Takeda S, et al. Cerebral protective and cognition-improving effects of sinapic acid in rodents. Biol Pharm Bull. 2007; 30: 514–519.
Hamaguchi T, Ono K, Murase A, Yamada M. Phenolic compounds prevent Alzheimer’s pathology through different effects on the amyloid-β aggregation pathway. Am J Pathol. 2009; 175: 2557–2565.
Ishge K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms. Free Rad Bio Med. 2001; 30: 433–446.
Szwajgier D. Anticholinesterase activities of selected polyphenols. Pol J Food Nutr Sci. 2014; 64, doi: 102478/v10222–012–0089-x.
Ono K, Yoshiike Y, Takashima A, Hasegawa K, Naiki H, Yamada M. Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer’s disease. J Neurochem. 2003; 87: 172–181.
Porat Y, Abramowitz A, Gazit E. Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism. Chem Biol Drug Des. 2006; 67: 27–37.
Isoda H, Talorete TPN, Kimura M, Maekawa T, Inamori Y, Nakajima N, et al. Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor. Cytotechnology 2002; 40: 117–123.
Young J, Wahle KWJ, Boyle SP. Cytoprotective effects of phenolic antioxidants and essential fatty acids in human blood monocyte and neuroblastoma cell lines: Surrogates for neurological damage in vivo. Prostag Leukotr Ess. 2008; 78: 45–59.
Choi Y-T, Jung C-H, Lee S-R, Bae J-H, Baek W-K, Suh M-H, et al. The green tea polyphenol (-)-epigallocatechingallate attenuates β-amyloid-induced neurotixicity in cultured hippocampal neurons. Life Sci. 2001; 70: 603–614.
Schroeter H, Williams RJ, Matin R, Iversen L, Rice-Evans CA. Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein. Free Rad Bio Med. 2000; 12: 1222–1233.
Whitehouse PJ, Price DL, Clark AW, Coyle JT, DeLong MR. Alzheimer’s disease: evidence for selective loss of cholinergic neurons in the nucleus basalis. Ann Neurol. 1981; 10: 115–126.
Rao AA, Sridhar GR, Das UN. Elevated butyrylcholinesterase and acetylcholinesterase may predict the development of type 2 diabetes mellitus and Alzheimer’s disease. Med Hyphoteses. 2007; 69: 1272–1276.
Ellman G L, Lourtney D K, Andres V, Gmelin G. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961; 7: 88–95.
Rhee IK, van Rijn RM, Verpoorte R. Qualitative determination of false-positive effects in the acetylcholinesterase assay using thin layer chromatography. Phytochem Analysis. 2003; 14: 127–131.
Szwajgier D, Borowiec K. Screening for cholinesterase inhibitors in selected fruits and vegetables. EJPAU 2012b; 15, #06, available online: (access: November 2015).
Szwajgier D. Anticholinesterase activity of phenolic acids and their derivatives. Z Naturforsch. 2013; 68c: 125–132.
Nachon F, Masson P, Nicolet Y, Lockridge O, Fontecilla-Camps JC. Comparison of structures of butyrylcholinesterase and acetylcholinesterase. In: Giacobini E. (Editor): Butyrylcholinesterase, its function and inhibitors, Martin Dunitz Ltd., London, 2003.p.39–54.
Johnson G, Moore SW. Why has butyrylcholinesterase been retained? Structural and functional diversification in a duplicated gene. Neurochem Int. 2012; 61: 783–797.
Wiesner J, Kriz Z, Kuca K, Jun D, Koca J. Acetylcholinesterases – the structural similarities and differences. J Enzyme Inhib Med Chem. 2007; 22: 417–424.
Ji H-F, Zhang H-Y. Theoretical evaluation of flavonoids as multipotent agents to combat Alzheimer‘s disease. J Mol Struct. (Theochem) 2006; 767: 3–9.
Khan MTH, Orhan I, Şenol FS, Kartal M, Şener B, Dvorská M, et al. Cholinesterase inhibitory activities of some flavonoid derivatives and chosen xantone and their molecular docking studies. Chem Biol Interact. 2009; 181: 383–389.
Akhtar MN, Lam KW, Abas F, Maulidiani, Ahmad S, Shah SAA, et al. New class of acetylcholinesterase inhibitors from the stem bark of Knema laurina and their structural insights. Bioorg & Med Chem Lett. 2011; 21: 4097–4103.
Srividhya R, Gayathri R, Kalaiselvi P. Impact of epigallo catechin-3-gallate on acetylcholine-acetylcholine esterase cycle in aged rat brain. Neurochem Int. 2012; 60: 517–522.
Anwar J, Spanevello M, Thomé G, Stefanello N, Schmatz R, Gutierres J, et al. Effects of caffeic acid on behavioral parameters and on the activity of acetylcholinesterase in different tissues from adult rats. Pharmacol Biochem Behav. 2012; 103: 386–394.
Rezg R, Mornagui B, El-Fazaa S, Gharbi N. Caffeic acid attenuates malathion induced metabolic disruption in rat liver, involvement of acetylcholinesterase activity. Toxicology 2008; 250: 27–31.