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Inhibitory effect of asiatic acid on acetylcholinesterase, excitatory post synapticpotential and locomotor activity.

ARTICLE INFO

Keywords: Centella asiatica Asiatic acid Acetylcholinesterase Excitatory postsynaptic potential Locomotor activity

ABSTRACT

The asiatic acid, a triterpenoids isolated from Centella asiatica was used to delineate its inhibitory effect on acetylcholinesterase (AChE) properties, excitatory post synaptic potential (EPSP) and locomotor activity. This study is consistent with asiatic acid having an effect on AChE, a selective GABAB receptor agonist and no sedative effect on locomotor.

Introduction

Acetylcholinesterase (ACNE) with EC 3.1.1.7 is a serine hydrolase that plays a vital role at cholinergic synapses (Cheng et al. 2008). The inhibition of AChE, which is the key enzyme in the breakdown of acetylcholine into acetyl and choline before the re-uptake process initiate. This process is considered as one of the treatment strategies by correcting the deficiency of the neurotransmitter acetylcholine in the synaptic cleft of the cerebral cortex against several neurological disorders such as Alzheimer's disease (AD), senile dementia, ataxia, and myasthenia gravis (Mukherjee et al. 2007; Savelev et al. 2004). In the recent years, some synthetic and natural compounds (tacrine, rivastigmine, donepezil, and galanthamine) have become available for clinical use (Orhan et al. 2007); however, they were accessible with a different bioavailability, where some compounds were found to possess moderate active structures, while some others with different inhibitory effects and possible side-effects (Jackson and Soliman 1996; Sienkiewicz et al. 2003).

On the other hand, [gamma]-aminobutyric acids (GABA) are the main inhibitory neurotransmitters in the brain. There is evidence that there are two different GABA receptors in the brain: [GABA.sub.A] and [GABA.sub.B] receptors (Bowery 1989; Matsumoto 1989). [GABA.sub.A] receptor is coupled with benzodiazepine receptor and [C1.sup.-] channels. On the other hand, [GABA.sub.B] receptor is coupled with G protein. The activation of GABA B receptors decreases the amplitude of [Ca.sup.2+] currents and increases the [K.sup.+] conductance. [GABA.sub.A] and [GABA.sub.B] receptors have different physiological actions (Nakagawa et al. 1995). [GABA.sub.A] receptor is mainly involved in anxiety and convulsion (Matsumoto 1989). In contrast, GABAB receptor is mainly related to depression and analgesia (Sawynok 1990). Regarding the abundance of the receptor subtypes, GABAB receptor represents 30% of the total GABA receptors (Stuchlik and Vales 2009). However, GABAB receptor represents a capable target for potential drugs that might enhance cognitive functions (Bowery 2006) since antagonists of [GABA.sub.A] receptor often induces epileptic paroxysms and convulsions.

Centella asiatica is an aromatically weak, slender and creeping perennial herbal plants used by diverse ancient cultures in Malaysia and other Asian countries. In the last decade, C. asiatica has been identified to possess the properties of cholinergic activity, anti oxidant activity or anti inflammatory activity (Sushma et al. 2008). The analytical studies have shown that C. asiatica contains triterpenoids, essential oils, amino acids and other compounds such as vellarin (Aziz et al. 2007). The terpenoids include asiaticoside, madecassoside, madeccasic acid and asiatic acid as shown in Fig. 1 (Phensri 2008).

Asiaticoside, one of the major terpenoids group from C. asiatica, has been patented by Hoechst Aktiengesellschaft as a cognitive enhancer useful in treating dementia (De Souza et al. 1992). However, asiaticoside is required at high doses and has to be given in long term to achieve its cognitive-enhancing activity as a putative anti-amnesic drug. Asiatic acid showed the good target as a cognitive enhancer and this can be demonstrated by increasing the inhibitory AChE activity via thin layer chromatographic (TLC) bioautographic technique, enhancing the EPSP without affecting the locomotor. For locomotor activity, asiaticoside did not show any effects on locomotor activity, suggesting these compounds do not have sedative effects on rodents (Wijeweera et al. 2006) since the increasing or decreasing locomotor may give wrong interpretation of the memory and learning test. Thus far, however, there is no report showing asiatic acid isolated from C. asiatica activity on the memory and learning. Therefore, the aims of the present study are to investigate the effects of asiatic acid on learning and memory correlating to AChE, EPSP and locomotor.

                 [R.sub.1]  [R.sub.2]        Mol. formula  Mol. wt.

Aslaticoside     H          O-glu-giu-rharn    [C.sub.48]       958
                                               [H.sub.78]
                                               [O.sub.19]

Madecassoside    OH         O-gfu-glu-rham     [C.sub.48]       974
                                               [H.sub.78]
                                               [O.sub.20]

Madecassic acid  OH         OH                 [C.sub.30]       504
                                               [H.sub.48]
                                                [O.sub.6]

Asiatic acid     H          OH                 [C.sub.30]       488
                                               [H.sub.48]
                                                [O.sub.5]

Fig. 1. Structure of asiaticoside, madecassoside, rnadeccasic acid
and Asiatic add. Adapted from Phensri (2008).


Materials and methods

Sample preparation and animals and drugs preparation

Sample preparation

The preparation of samples was performed as previously described by Nasir et al. (2010). C. asiatica whole plants were purchased from a local producer in Kuala Terengganu, Terengganu Darul Iman, Malaysia. Then, the samples were subjected to botanical identity by herbal expertise from Universiti Malaysia Terengganu (UMT), before the compound was tested in Universiti Sains Malaysia (USM). A voucher specimen is deposited at Chemistry Laboratory, Faculty of Science and Technology at UMT as CA 204. The samples were collected and weighted. Thereafter, the whole plants were grounded with an electric blender to obtain powder form. The grounded samples then underwent extraction phase.

The elution process was preceded in increasing polarity (step gradient). The fractions were collected in 100 ml each (first CC process) and 5 ml each (for repeat CC process). The interest fraction was monitored by TLC. Fractions with the same TLC pattern/profile were combined. The fraction containing the same chemical compound was purified by repeat CC (70.0 cm x 1.0 cm). The compound was identified using Infrared spectroscopy (IR spectroscopy) and Nuclear Magnetic Resonance (NMR)-Varian Unity Inova-500. For Bioautographic TLC, the procedure was performed using the method previously described by Marston et al. (2002).

Electrophysiology (excitatory-postsynaptic potential)

The preparation of hippocampal slices were performed as previously described by Anja et al. (2007). Briefly, hippocampal slices were prepared from young adult (4-6 weeks) male Spraque-Dawley rats from Laboratory Animal Research. Unit, Universiti Sains Malaysia (LARUSM). All experiments were performed in accordance with the guidelines of the Ethical Committee on the Use and Care of Animals (Animal Ethics Committee, Universiti Sains Malaysia). Animals were anesthetized with isoflurane and decapitated.

The brains were rapidly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): NaCI 125, KCI 2.5, NaHC[O.sub.3] 25, Ca[C1.sub2] 2, Mg[Cl.sub.2] 1, D-glucose 25, Na[H.sub.2]P[O.sub.4] 1.25 (pH 7.4), and bubbled with a 95% [0.sub.2]/5% C[O.sub.2] mixture. Transversal slices of the hippocampus (350 pm thick) were prepared using a microtome (Microm, Germany). After incubation in a holding chamber with aCSF (22-25[degrees]C) for at least 60 min, the slices were placed in the recording chamber and superfused with aCSF at a flow rate of 1.5 ml/min and were incubated for 30 min to decrease the stress of the brain and stable the process enzymatically.

The fEPSPs were elicited by Schaffer collateral stimulation through a bipolar stimulation electrode and were recorded as extracellular field potentials with ACSF-filled glass recording electrodes (0.5-1.5 M[OMEGA]) placed in the stratum radiatum of the CA1 region as shown in Fig. 2. The synaptic response to a standard test stimulus (0.033 Hz) was monitored until a stable recording was obtained, and the input-output relationship was then determined. The stimulus strength (0.2-2.5 mA) producing a response of approximately 50% of the maximal response amplitude was determined and was laterused for all subsequent experiments. Only synaptic potentials with more than 0.2 mV and without superimposed population spikes were used for the experiments. After stable baseline recording of the responses for 20 min, GABA blocker bath was applied followed by asiatic acid to the hippocampal slices for 60 min and then washed out for a further 30 min. The evoked synaptic responses were recorded every 15 s during bath application of asiatic acid and for a further 30 min after washout.

The evoked synaptic responses were recorded and analyzed with a personal computer using custom-developed software (Cell-work version 5.0 and Igor Pro, version 2.30D). The fEPSPs were quantified by measurements of the amplitude of the synaptic responses. Each of the amplitudes of the fEPSPs obtained during asiatic acid application was normalized to the average amplitude of the 10 min baseline recordings of the fEPSPs acquired before asiatic acid application. The significance of the differences between the means was calculated for different points in time (t= 20,40,60, 80, 100, 120 and 140 min), using a t-test or Mann-Whitney rank sum test. Values were considered significantly different if p [less than or equal to] 0.05. In the text, values are shown as mean [+ or -]SEM.

Locomotor activity

The open-field test was used for estimation of locomotor activity of rats using the IR-Actimeter devices from Panlab, Spain as shown in Fig. 3. Briefly, the apparatus is consisted of a square of 100 cm x 100 cm black floor, which was divided by 8 lines into 25 equal squares, and surrounded by white walls of 47 cm high. Four plastic bars, 20 cm high each was located in four different line crossings in the central area of the floor. A single rat was placed inside the apparatus for 1 min of adaptation. Subsequently the length of crossings for 5 min. Asiatic acid, scopolamine and baclofen were administered 30 min before the test.

Drugs preparation and administration

Asiatic acid was dissolved in saline with some modifications. This is due to the dilution of asiatic acid with 100% saline had resulted in cloudy undiluted asiatic acid. Dimethyl sulfoxide (DMSO) is commonly used as a solvent for water-insoluble drugs. For that, DMSO was used with 0.05% DMSO in asiatic acid and saline. Qi et al. (2008) demonstrated that DMSO concentrations of 0.1% and 0.25% caused little or no toxicity to the cell. Baclofen and scopolamine were dissolved in saline and vortex in 10 min. For the locomotor activity, asiatic acid was injected with I.P. Baclofen and scopolamine were administered via I.P according to the method as previously described by Liang et al. (2006) and Pitsikas et al. (2003).

Statistical analysis

Results were expressed as means [+ or -]SEM of seven animals per group. The data was analyzed by Kruskall-Wallis and followed by Mann-Whitney and 1-way ANOVA using SPSS 11.0 software. In all the tests, the criterion for statistical significance was p < 0.05.

Results

Extraction, isolation and TLC bioautographic AChE inhibitory

The dried and finely ground leaves of C. asiatica (10 kg) were extracted at room temperature with MeOH for 3 days and were concentrated under vacuum to afford 445 g of crude extract. The asiatic acid (m. p 325-330 [degrees]C) compound was successfully isolated with a white powder and purity was confirmed by IR spectroscopy and NMR spectroscopy according to various chromatography tech-niques as described previously by Yu et al. (2006).

In TLC bioautographic AChE inhibitory, the test revealed that asiatic acid has inhibitory effect on AChE. In order to establish detection limits for the bioautographic assay, asiatic acid isolated from hexane partitioning were applied at 10 folds serial dilution onto the TLC plate, and the concentration that produced least intense white spot was noted. Result revealed that a 1 0-fold was not susceptible in detecting the minimal concentration needed for AChE inhibition. Within this limitation, a 2-fold serial dilution was designed. From these results, asiatic acid inhibited that enzyme down to 0.125 [micro]g (125 ng).

Effects of asiatic acid on excitatory synaptic transmission in hippocampal slices

Effects of asiatic acid with [GABA.sub.B] blocker on fEPSP

In the CA1 region of hippocampus, extracellular potentials were elicited by stimulating the Schaffer collateral-commissural fibers and were recorded in the stratum radiatum. All recording slices were treated with phaclofen or bicuculine with asiatic acid various concentrations, while the other drug-untreated control was treated with phaclofen or bicuculine containing no asiatic acid. This procedure was adopted to determine the asiatic acid properties itself to give an inhibitory effect to EPSP according to early studies as shown in Fig. 4. Original recordings with and without application of 100 RM are shown in Fig. 4c. Perfusion of asiatic acid had no significant effect on fEPSP amplitudes, either in slices with concentration asiatic acid 30 [micro]M (Fig. 4a, n = 7) or from 100 p M (Fig. 4b, n=7). The fEPSP amplitudes are listed in Table 1. After t = 140, the one-way ANOVA revealed that asiatic acid with concentrations 1, 3, 10, 30 and 100 M decreases the EPSP amplitudes no differed significantly compared with drug-untreated control.

Table 1
Effects of asiatic acid compared to the drug untreated control.

fEPSP Point      r=20      r=40      t=60      r=80     r=100    t=tl20
in
time(min)

Drug              100       101       102       102       102       103
untreated    [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
control             3         3         2         2         2         2

30 [micro]M       100        99       100        99       101       102
asiatic      [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
acid with           3         3         2         3         3         1
phaclofen

100 [micro]       100       100       101       100       101       102
M asiatic    [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
acid with           3         2         3         3         4         1
phaclofen

1 [micro]M         99        99        99        97        98        98
asiatic      [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
acid with           3         3         4         3         3         2
bicucufine

3 [micro]M        100       100        96        89        89        88
asiatic      [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
acid with           3         4         3        3'        4'        1'
bicuculine

Vasiatic           99        96        89        87        86        S2
acid with    [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
bicuculine          2         3         3        3'        3'        3'

30 [micro]M        99        94        88        81        74        60
asiatic      [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
acid with           2         2        3'        4'        3'        3'
bicuculine

100 [micro]       100        83        71        61        46        29
M asiatic    [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
acid with           1        3'        2'        4'        3'        2'
bicuculine

fEPSP Point      t=140
in
time(min)

Drug               103
untreated     [+ or -]
control             12

30 [micro]M        103
asiatic       [+ or -]
acid with            3
phaclofen

100 [micro]M       102
asiatic       [+ or -]
acid with            3
phaclofen

1 [micro]M          98
asiatic       [+ or -]
acid with            2
bicucufine

3 [micro]M          88
asiatic       [+ or -]
acid with           2'
bicuculine

Vasiatic            75
acid with     [+ or -]
bicuculine          3'

30 [micro]M         51
asiatic       [+ or -]
acid with           4'
bicuculine

100 [micro]M        10
asiatic       [+ or -]
acid with           2'
bicuculine

Statistically significant (p < 0.01) compared with drug untreated
control, as determined by one-way ANOVA followed by Dunnett' test.


Effects of asiatic acid with [GABA.sub.A] blocker on fEPSP

Bath application of asiatic acid affected the amplitudes of the fEPSPs evoked in a concentration-dependent manner in slices from 1, 3, 10, 30 and 100 [micro]M asiatic acid. The onset of the blocking effect varied with the concentration of asiatic acid. The fEPSP amplitudes are listed in Table 1. Original recordings with and without application of 100 [micro]M are shown in Fig. Sc. Application of 1 [micro]M produced a slight decrease in the fEPSP amplitudes to about 97 [+ or -] 2 of the drug-untreated control amplitudes, which had no significance 140 min after application. Application of 3 M led to a sensible reduction of the fEPSP amplitudes to about 88 [+ or -] 2 of the drug-untreated control values, which was significant 80 min after application. Thus, the application of 10 [micro]M led to a moderate reduction of the fEPSP amplitudes to about 75 3 of the drug-untreated control values, which was significant 60 min after application. Application of 30 M (Fig. 5a, n=7) led to prominent reduction of the fEPSP amplitudes to about 51 [+ or -]4 of the drug-untreated control values, which was significant 60 min after application. Application of 100 M (Fig. 5b, n=7) led to a greater reduction of the fEPSP amplitudes to about 10 [+ or -] 2 of the drug-untreated control values, which was significant 40 min after application. The depressant effect was not reversible after a 30-min washout of the asiatic acid. After t=140, the one-way ANOVA revealed that asiatic acid with concentrations of 3, 10, 30 and 100 M decrease the EPSP amplitudes which differed significantly compared with drug-untreated control (F(4,30)=424.587, p<0.05).

Effects of locomotor activity

Locomotor activity was investigated to rule out the possibility that changes in cognitive paradigm are due to an alteration in locomotor activity. No significant differences in locomotor activity, assessed by comparing the distance in an open field test of asiatic acid group compared with saline group. The baclofen group showed significant lower distance compared with saline group and for scopolamine group, it showed significant increase in distance compared with saline group as shown in Fig. 6.

Discussions

The aim of the first study was to investigate the AChE properties of C. asiatica especially of active compound asiatic acid and its minimal concentration for inhibition of AChE. AChE essentially contains two special reactive sites; anionic site and the esteratic site. The anionic site attracts with quaternary nitrogen and esteratic site attracts to carbonyl carbon (nucleophilic) of AChE inhibitor compounds (Colletier et al. 2006). Regardless of their chemical structure of asiatic acid, it seems like there was no nitrogen group in asiatic acid structure.

From the structure, there were no formations of anionic site and inhibition of AChE by asiatic acid in vitro was due to an active competition with ACh and occurs at the esteratic site on the enzyme. Inhibition of AChE needs minimal one site of AChE gorge to inactivate the enzyme in that it degrades the activity of ACh. Previous studies showed that galanthamine inhibit AChE at the anionic site and neostigmine and physostigmine at both anionic and esteratic sites (Ghous and Townshend 1998). In this experiment, 0.125 [micro]g (125 ng), was sufficient to break down the AChE enzyme compared with well known AChE inhibitor physostigmine and galanthamine inhibited the enzyme down to 0.001 [micro]g (1 ng) and 0.01 [micro]g (10 ng) (Marston et al. 2002).

The aim of the second study was to delineate the effects of asiatic acid, a compound isolated from C. asiatica extract with blockers of [GABA.sub.A] and [GABA.sub.B], on GABAergic transmission in the hippocampus slices. The main finding of this experiment is asiatic acid blocks excitatory transmission at the hippocampal Schaffer collateral-CAI synapse in a concentration-dependent manner for [GABA.sub.A] antagonist. The blocking effects are considerably greater in slices with concentration of 100 [micro]M with GABAA antagonist, bicuculine. In contrast, asiatic acid, even at the high concentration of 100 [micro]M, exerted no effect with GABAB antagonist, phaclofen. With the [GABA.sub.B] antagonist in the bath solution, phaclofen inhibits the [GABA.sub.B] resulting in the inability of the asiatic acid to interact with the [GABA.sub.B] receptor to produce agonist response.

The impairment of excitatory synaptic transmission at the Schaffer collateral-CA1 synapse by asiatic acid indicates that this effect is caused by the direct action of this substance on GABAergic receptors. Asiatic acid interacts with specific [GABA.sub.B] receptors and lead to opening the [GABA.sub.B] receptor had caused the potassium ions of the neuronal cell to outflow from the cell. This lead to hyperpolarization of the neuronal cell and within time had caused the decrease in EPSP. The depressant effect was irreversible after a 30-min washout of the asiatic acid once the [GABA.sub.B] was activated, it will open the channel for hours to days and this is also parallel that [GABA.sub.B] receptors play a role for manifesting the metabotropic effect from this experiments.

It may induce deficit of place learning with the same effect on baclofen as previously reported by Nakagawa et al. (1995). The [GABA.sub.B] receptor agonist baclofen presumably increases presynaptic inhibition in the hippocampus as reported by Thompson (1994) possibly via an increased [K.sup.+] conductance and/or a decrease in voltage-dependent [Ca.sup.2+] conductance as shown in hippocampal neurons (Dutar and Nicoll, 1988). Because asiatic acid has also been shown to increase potassium conductance which is similar to baclofen, that could act via the similar process to decrease facilitation of perforant path synaptic transmission.

The aim of third study was to investigate the effects of asiatic acid on the locomotor activity. The experiment demonstrated that asiatic acid can enhance behavioral performance in the rats and this increase in performance was not due to an increase or decrease in locomotor activity. The results of the locomotor activity indicate that asiatic acid administrations did not alter the locomotor activity as compared with saline group (p < 0.05). On the other hand, scopolamine administrations induce hyperactivity and baclofen administrations reduce hyperactivity of locomotor in rats and this is also in partial agreement with the results of Stuchlik and Vales (2009), who demonstrated the effects of baclofen close-dependently disrupts learning and locomotor. These results suggested the existence of tonic [GABA.sub.B] receptor-mediated inhibitory control on locomotion (Colombo et al. 2001). Consistently, it has been reported by Schuler et al. (2001) that deletion of the gene encoding the [GABA.sub.B] receptor subunit had resulted in spontaneous hyper-locomotion in mice.

We conclude that asiatic administration has effective AChE inhibitory properties, although asiatic acid can give an inhibitory effect via GABAergic through [GABA.sub.B], this effect is less responsive compared with [GABA.sub.A]. For the locomotor assessment, asiatic acid had no effect on sedative properties which can be translated as giving good effect in performance in memory and learning study in future. The cholinergic and GABAergic pathways are intimately connected in the hippocampus and basal forebrain complex and may combine to exert their effects on cognition.

Acknowledgements

The authors are grateful to the Department of Neuroscience, School of Medical Sciences, Universiti Sains Malaysia and Department of Chemistry, Universiti Malaysia Terengganu staffs for work facilities and environment. The authors thank Mr. Hans Reiner Polder for advice in analyzing the EPSP. The authors also thank Dr. Muzaimi Mustapha and Ms. 11 ma for technical support. This grant is supported by Universiti Sains Malaysia, 304/PPSP/61314.

Abbreviations: ACh, acetylcholin; AChE, acetylcholinesterase; EPSP, excitatory postsynaptic potential; GABA, gamma amino butyric acid.

* Corresponding author. Tel.: +60 129888186; fax: +60 97672359.

E-mail address: brainsciences@gmail.com (J. Abdullah).

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M.N. Nasira (a), (b), J. Abdullah (a) *, M. Habsah (c), R.I. Ghani (a), G. Rammes (d), (e)

(a.) Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, Malaysia

(b.) Faculty of Medicine and Health Sciences, Universiti Sultan Zainal Abidin, Malaysia

(c.) Department of Chemistry, Faculty Science and Technology, Universiti Malaysia Terengganu, Malaysia

(d.) Max PIanck Institute of Psychiatry, Clinical Neuropharmacology, Kraepelinstr. 2-10, 80804 Munich, Germany

(e.) Department of Anaesthesiology, Technical University, Klinikum Rechts der Isar, lsmaningerstrasse 22, 80804 Munich, Germany

0944-71131$ -see front matter [c]2011 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2011.10.004
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Author:Nasira, M.N.; Abdullah, J.; Habsahc, M.; Ghani, R.I.; Rammes, G.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9MALA
Date:Mar 1, 2012
Words:4706
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