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Phytopharmacology 2010, 1(1) 7-11

Acetylcholinesterase and Butyrylcholinesterase inhibitory activities of Berberis vulgaris


D. Kol1,*, L. Wimmerov2, R. Kdek2
Institute of Experimental Medicine, Department of Pharmacology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. 2Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackho 1/3, CZ-612 42 Brno, Czech Republic. *Corresponding Author: Email: trpkolar@chef.net
Received: 14 October, 2010, Revised: 16 November, 2010, Accepted: 17 November 2010
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Abstract
Berberis vulgaris is a famous medicinal plant used in folk medicine for a variety of pathological condition. Aim of the study was to determine cholinesterase inhibitory effects of crude extracts and fractions of Berberis vulgaris. Considerable inhibition of acetylcholinesterase was observed in majority cases. The hexane fraction was the most active fraction with the IC50 being 680.028 g/ml. In case of butyrylcholinesterase, crude extract of the leaves found to be the most active sample with (64%) inhibition and its IC50 value was 590.058 g/ml. In case of fractions, aqueous fraction of crude bark extract showed most potent inhibitory profile (IC50: 590.058 g/ml). Results indicated promising potential of B. vulgaris as source of new compounds for management of Alzheimers Disease..
neurotransmission; presynaptic; nicotinic; muscarinic; receptors; postsynaptic; cell function; AChE; EC

Keywords: Acetylcholinesterase; Butyrylcholinesterase; Berberis vulgaris


3.1.1.7; Ach; BuChE; EC 3.1.1.8; innervation; pseudocholinesterase; peptides; Alzheimers disease; neurons; nucleus basalis; basal forebrain; beta amyloid; synaptic vesicles; axon; synaptic cleft; subcortical; vascular; dementia; Parkinson's disease; Berberidaceae; antimicrobial; antiemetic; antipyretic; antipruritic; antioxidant; anti-inflammatory; hypotensive; antiarrhythmic; sedative; antinociceptive; anticholinergic; cholagogue; cholecystitis;cholelithiasis;jaundice;dysentery;leishmaniasis;malaria; gall stone; 5,5Dithiobis(2-nitrobenzoic acid); DTNB; Galanthamine;

Acetylcholine;

neurotransmitter;

cholinergic;

Introduction Acetylcholine (ACh) is the neurotransmitter involved in cholinergic neurotransmission, released by presynaptic cholinergic terminals and activating nicotinic and/or muscarinic receptors to modify postsynaptic cell function. Normally, acetylcholinesterase (AChE, EC 3.1.1.7) rapidly and efficiently degrades ACh, thereby terminating its signaling action (Shen et al., 2009; Zelket al., 2009). The majority of Cholinesterase in the human brain is AChE. However, it is now known that a related enzyme butyrylcholinesterase (BuChE, EC 3.1.1.8) has more widespread distribution than previously thought. In the normal brain, BuChE activity has been located in all regions that receive cholinergic innervation (Mesulam

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Kol et al.

et al., 2002). AChE is highly selective for ACh hydrolysis, while BuChE is able to metabolize different molecules including various neuroactive peptides (Lane et al., 2006). Alzheimers disease (AD) is characterized by a loss of cholinergic neurons and their cortical projections from the nucleus basalis and associated areas in the basal forebrain. Cholinergic synaptic function appears to be particularly susceptible to beta-amyloid (A) peptide toxicity, and loss of synaptic vesicles on axon terminals may precede cholinergic neuronal loss (Small et al., 2001). Reversible inhibition of acetylcholinesterase leads to an increase in neurotransmitter concentration within the synaptic cleft, which positively affects AD patients and related conditions such as Lewy Body dementia, subcortical vascular dementia and Parkinson's disease (Porcel J and Montalban X, 2006). All cholinesterase inhibitors have demonstrated short-term symptomatic effects which provide actively treated patients with a higher baseline from which they subsequently decline. This effect results in transiently delaying patient decline. However, ideally, agents should also slow decline demonstrated by a widening gap over time between the courses of treated and untreated patients. It is the latter effect only that provides evidence of disease modification. Therefore, there is need to search new molecules with larger perspective for which natural sources could be ideal. Berberis vulgaris is a member of family Berberidaceae. It is famous as a folk medicinal plant and used extensively in traditional medicine. Medicinal properties for all parts of the plant have been reported, including tonic, antimicrobial, antiemetic, antipyretic, antipruritic, antioxidant, anti-inflammatory, hypotensive, antiarrhythmic, sedative, antinociceptive, anticholinergic and cholagogue actions, and it has been used in some cases like cholecystitis, cholelithiasis, jaundice, dysentery, leishmaniasis, malaria and gall stones (Khosrokhavar et al., 2010). In order to identify its therapeutic potential in AD, we have made an effort to study cholinesterase inhibitory profile of Berberis vulgaris Materials and Methods Extraction: The shade dried plant material was chopped into small pieces and finally pulverized into fine powder. The powdered plant material (9Kg) was soaked in methanol with occasional shaking, at room temperature. After 15 days, the methanol soluble materials were filtered off. The filtrate was concentrated under vacuum at low temperature (40C) using rotary evaporator. A crude extract (967 g) was obtained. Fractionation The crude methanol extract (482g) was suspended in distilled water (500 mL) and sequentially partitioned with n-hexane (3 x 500 mL), chloroform (3 x 500 mL), ethyl acetate (3 x 500 mL) and n-butanol (3 x 500 mL) to yield the n- hexane (44 g), chloroform (102 g), ethyl acetate (87 g), n-butanol (35 g) and aqueous (214 g) fractions, respectively. The enzyme inhibition activity assays were performed by using different concentrations of the crude extract and various fractions as per the requirement of the individual assay method.

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Phytopharmacology 2010, 1(1) 7-11

Figure 1. (Left) Percent Inhibition activity of acetylcholinesterase by the crude extracts of different parts of Berberis vulgaris. Concentrations of samples: wood (100g/ml), Bark (100g/ml), Leaves (100g/ml) and Galanthamine as a standard (0.50.01M). Fig2. (Right) Inhibition activity of acetylcholinesterase by the fractions of Berberis vulgaris. Concentrations of samples: Hexane (100 g/ml), Chloroform (100g/ml), Ethyl acetate (100g/ml) and aqueous fraction (100g/ml).

In-vitro Cholinesterase Inhibition Assay Acetylcholinesterase and butyrylcholinesterase inhibiting activities were measured by slightly modifying the spectrophotometric method previously developed [23]. Electric-eel AChE (type VI-S, Sigma) and horseserum BChE (Sigma) were used as source of the cholinesterases and acetylthiocholine iodide and butyrylthiocholine chloride (Sigma), respecttively, were used as substrates in the reaction. 5, 5 Dithiobis (2-nitrobenzoic acid) (DTNB, Sigma) was used for the measurement of cholinesterase activity. 140 mL of sodium phosphate buffer 100mM, (pH 8.0), 10mL of DTNB, 20 mL of the test samples solutions and 20mL of acetylcholinesterase/ butyrylcholinesterase solution were mixed and incubated for 15min at 258 0C. The reactions were then initiated by the addition of l0 mL acetylthiocholine/butyrylthiocholine, respectively. The hydrolysis of acetylthiocholine and butyrylthiocholine was monitored at a wavelength of 412nm by the formation of the yellow

Figure 2. (Left) Percent Inhibition activity of butyrylcholinesterase by the crude extracts of different parts of Berberis vulgaris. Concentrations of samples: wood (100g/ml), Bark (100g/ml), Leaves (100g/ml) and Galanthamine as a standard (8.50.01M). (Right) Percent Inhibition activity of butyrylcholinesterase by the fractions of Berberis vulgaris. Concentrations of samples: Hexane (100g/ml), Chloroform (100 g/ml), Ethyl acetate (100 g/ml) and aqueous fraction (100g/ml).

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Kol et al. Table 1. In Vitro quantitative Inhibition of acetylcholinesterase and Butyrylcholinesterase by the crude extracts and various fractions of bark extract. IC50 values are the mean S.E.M. of three assays. Drug / Fractions Wood (Crude) Leaves (Crude) Bark (Crude) Hexane Chloroform Ethyl acetate Aqueous Galanthamine IC50 SEM (g/ml) Acetylcholinesterase Butyrylcholinesterase 87 0.013 980.037 94 0.041 890.054 82 0.036 910.019 68 0.028 820.069 890.019 94 0.017 640.031 970.062 590.058 0.50.01M 8.50.01M

5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine respectively. The samples and the control were dissolved in 50% ethanol. All the reactions were performed in triplicate. Percent (%) Inhibition was calculated according to MichaelisMenten model by using EZ-Fit. Software (EZ-Fit:Enzyme Kinetics, Perrella Scientific, Inc., USA). Results and Discussion Ethanopharmaceuticals have a great history and plants are highly rich sources of new drugs. Many synthetic drugs owe their origin to plant-based complementary medicine. Since AD, one of the most common cause of death worldwide, has become a threat to public health, new treatment strategies based on medicinal plants have been focused. Therefore, the crude extract and fractions of Berberis vulgaris was subjected to acetylcholinesterase and butyrylcholinesterase activities. Results of the crude extracts against acetylcholinesterase inhibition in comparison to Galanthamine (standard drug) are given in Figure 1. Crude extract of wood displayed (53%) inhibition and the IC50 was 870.13 g/ml. Crude extract of bark showed relatively more potent inhibition (59%) inhibition and the IC50 was 820.36 g/ml. however the leaves exhibited 58% inhibition with IC50 value as 940.41 g/ml. Results of acetylcholinesterase inhibition by the fractions of crude methanol bark extract are shown in Figure 1. Hexane fraction showed the highest inhibition (64%) with IC50 being 72 g/ml. It was followed by the aqueous fraction having 57% inhibition and IC50 value was 680.28 g/ml. Moreover chloroform and ethyl acetate fractions displayed (51%) and (42%) inhibition, respectively. Similarly the crude extract of the different parts and fraction of the Berberis vulgaris were tested against butyrylcholinesterase. As shown in Figure 2, the crude bark extract showed 57% inhibition of butyrylcholinesterase and its IC50 value was 910.19 g/ml. Crude leaves extract was relatively more potent inhibitory profile with 64% inhibition and its IC50 value was 890.54 g/ml. Methanolic extract of wood was the least potent and exhibited 51% inhibition and its IC50 value was 980.37 g/ml. Results of the different fraction of bark extract against butyrylcholinesterase are presented in Figure 2. In case of fractions, aqueous fraction offered the highest (61%) inhibition and its IC50 value was 590.58 g/ml.

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Phytopharmacology 2010, 1(1) 7-11

Moreover, the ethyl acetate fraction was also found active and showed 52% inhibition with an IC50 value being 640.31 g/ml. The hexane and chloroform fractions revealed 51% and 43% inhibition of butyrylcholinesterase, respectively. These considerable results indicated that Berberis vulgaris should be screened for potential new lead compounds for better management of AD and related pathological conditions.

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