Fitoterapia 81 (2010) 546–551

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Fitoterapia

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Increased excitability and metabolism in pilocarpine induced epileptic rats:Effect of Bacopa monnieri

Jobin Mathew, Jes Paul, M.S. Nandhu, C.S. Paulose⁎Molecular Neurobiology and Cell Biology Unit, Centre for Neuroscience, Department of Biotechnology, Cochin University of Science and Technology, Cochin-682 022,Kerala, India

a r t i c l e i n f o

⁎ Corresponding author. Tel./fax: +91 484 2575588E-mail addresses: cspaulose@cusat.ac.in, paulosecs

(C.S. Paulose).

0367-326X/$ – see front matter © 2010 Elsevier B.V.doi:10.1016/j.fitote.2010.01.017

a b s t r a c t

Article history:Received 21 August 2009Accepted in revised form 12 January 2010Available online 1 February 2010

Wehave evaluated the acetylcholine esterase andmalate dehydrogenase activity in themuscle,epinephrine, norepinephrine, insulin and T3 content in the serum of epileptic rats.Acetylcholine esterase and malate dehydrogenase activity increased in the muscle anddecreased in the heart of the epileptic rats compared to control. Insulin and T3 content wereincreased significantly in the serum of the epileptic rats. Our results suggest that repetitiveseizures resulted in increased metabolism and excitability in epileptic rats. Bacopa monnieriand Bacoside-A treatment prevents the occurrence of seizures there by reducing theimpairment on peripheral nervous system.

© 2010 Elsevier B.V. All rights reserved.

Keywords:EpilepsyBacopa monnieriBacoside-APilocarpineCarbamazepine

1. Introduction

Epilepsy is the commonest serious neurological conditionaffecting 0.5–1% of the population. The most commonetiologic factors of epilepsy that can predispose a person toepilepsy are head traumas, neoplasms, degenerative diseases,infections, metabolic diseases, ischemia and hemorrhages [1].Certain brain areas, i.e. temporal and frontal lobes are moresusceptible to produce epileptic seizure activity than theother regions. However, there are also patients with unre-solved etiology of epilepsy [2]. Epilepsy is a seizure disorder.A seizure is an event that involves loss of consciousness andmotor (muscular) control. A person with a seizure disorderoften experiences repetitive muscle jerking called convul-sions. The condition is caused by a sudden change in electricalactivity in the brain. Systemic administration of pilocarpinehas been used as an animal model for temporal lobe epilepsyand has several features in commonwith the human complexpartial seizures. The most striking similarity was probablythat pilocarpine produced marked changes in morphology,

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membrane properties and synaptic responses of hippocampalrat neurons, comparable to those observed in humanepileptic hippocampal neurons [3]. The chemical compoundacetylcholine (ACh) is a neurotransmitter in both theperipheral nervous system (PNS) and central nervous system(CNS) in many organisms including humans. The brain sendssignals, in the form of action potentials, through the nervoussystem to the motor neuron that innervates the muscle fiber.These neurons release ACh which mediates muscle contrac-tion. Acetylcholine esterase (AChE) is an enzyme thatdegrades the neurotransmitter acetylcholine, producingcholine and acetate. 3, 5, 3′-triiodo-L-thyronine (T3) is amajor metabolic hormone fund to be alerting abnormally inepileptic patients. Insulin facilitate carbohydrate metabolism.Malate dehydrogenase (MDH) is the major enzyme in theenergy metabolism and considered this as a rate indicatingenzyme of energymetabolism [4]. Mitochondrial function is akey determinant of both excitability and viability of neurons[5]. The drugs of plant origin are gaining importance and arebeing investigated for remedies of a number of disorders.Since the introduction of adaptogen concept, several plantshave been investigated, which were used earlier as tonics dueto their adaptogenic and rejuvenating properties in tradi-tional medicine [6]. Bacopa monnieri has been reported to

547J. Mathew et al. / Fitoterapia 81 (2010) 546–551

possess anxiolytic, antidepressant and memory-enhancingactivity [7–9]. Our study indicated the increased metabolicrate and excitability in epileptic rats and the antiepilepticproperty of Bacopa monnieri.

2. Materials and methods

Biochemicals used in the present study were purchasedfrom Sigma Chemical Co., St. Louis, USA. All other reagentswere of analytical grade purchased locally.

2.1. Animals

Wistar rats of 250–300 g body weight purchased fromKerala Agriculture University, Mannuthy were used for allexperiments. They were housed in separate cages under 12 hlight and 12 h dark periods and were maintained on standardfood pellets and water ad libitum. All animal care andprocedures were taken in accordance with the Institutional,National Institute of Health and CPCSEA guidelines.

2.2. Epilepsy induction

Adult maleWistar rats, weighing 250 to 300 g, were housedfor 1 to 2 weeks before experiments were performed. Experi-mental animalswere injectedwith pilocarpine (350 mg/kg i.p.),preceded by 30 min with atropine (1 mg/kg i.p.) to reduceperipheral pilocarpine effects. 24 days after pilocarpine treat-ment, the rats were continuously videomonitored for 72 h. Thebehavior and seizures were captured with a CCD camera and aPinnacle PCTV capturing software card.

2.3. Extraction of Bacopa monnieri

Bacopa monnieri dried in shade and then powdered, andthe plant material was extracted with distilled water. Theaqueous extract was discarded and the residual plantmaterial was extracted thrice with 90% ethanol. The residueobtained after removing the solvent was dried in vacuum andmacerated with acetone to give a free-flowing powder. Theextract of Bacopa monnieri contained 45% bacoside. Theestimation method involves acid hydrolysis of bacosides,which yields quantitatively a transformed aglycone-ebelinlactone which contained a conjugated triene system and wasestimated by UV spectrophotometer at 278 nm (Pal & Sarin,1992).

Structure of Bacoside-A.

2.4. Treatment

Animals were divided in to Group 1: Control, Group 2:Epileptic, Group 3: Epileptic rats treated with Bacopamonnieri, Group 4: Epileptic rats treated with Bacoside-Aand Group 5: Epileptic rats treated with Carbamazepine.Extract of Bacopa monnieriwas given orally to the third groupof epileptic rats, 300 mg/kg body weight/day for 15 days.Bacoside-A was given orally to the 4th group of epileptic ratsin the dosage of 150 mg/kg body weight/day for 15 days.Carbamazepine was given orally to the 5th group of epilepticrats in the dosage of 150 mg/kg body weight/day for 15 days.The rats were sacrificed and the tissueswere stored in−80 °Cfor all experiments.

2.5. Acetylcholine esterase activity

Acetylcholine esterase activity in the heart and musclewere done using the spectrophotometric method of Ellmanet al. [10]. Homogenate (10%) of heart and muscle wereprepared in 30 Mm sodium phosphate buffer, pH 8.0,containing 1% Triton X 100 to release the membrane boundenzyme and it was centrifuged at 12,500 ×g for 30 min at4 °C. Acetylthiocholie iodide of different concentrations,0.25–0.5 mM for heart and 0.1–0.8 Mm for muscle wereused as substrate. The mercaptan formed as a result of thehydrolysis of the ester reacts with an oxidizing agent 5,5′-dithiobis(2-nitrobenzoate) read at 412 nm in shimadzu UVspectrophotometer.

2.6. Quantification of epinephrine and norepinephrine in theexperimental groups of rat

The monoamines were assayed according to the modifiedprocedure of Paulose et al. [11]. The heart of experimentalgroups of rats was homogenised in 0.4 N perchloric acid. Thehomogenate was then centrifuged at 5000 ×g for 10 min at4 °C in a Sigma 3K30 refrigerated centrifuge and the clearsupernatant was filtered through 0.22 µm HPLC grade filtersand used for HPLC analysis.

Epinephrine and norepinephrine contents were deter-mined in high performance liquid chromatography (HPLC)with electrochemical detector (ECD) (Waters, USA) fittedwith CLC-ODS reverse phase column of 5 µm particle size.The mobile phase consisted of 50 mM sodium phosphatedibasic, 0.03 M citric acid, 0.1 mM EDTA, 0.6 mM sodiumoctyl sulfonate, and 15% methanol. The pH was adjusted to3.25 with orthophosphoric acid, filtered through 0.22 µmfilter (Millipore) and degassed. AWatersmodel 515,Milford,USA, pump was used to deliver the solvent at a rate of 1 ml/min. The neurotransmitters and their metabolites wereidentified by amperometric detection using an electrochem-ical detector (Waters, model 2465) with a reductionpotential of +0.80 V. Twenty microlitre aliquots of theacidified supernatant were injected into the system forquantification. The peaks were identified by relative reten-tion times compared with external standards and quantita-tively estimated using an integrator (Empower software)interfaced with the detector. Data from different brainregions of the experimental and control rats were statisti-cally analyzed and tabulated.

Table 1Acetylcholine esterase activity in the muscle, heart and malate dehydrogenase activity in the muscle of control and experimental rats.

Acetylcholine esterase activity in the muscle Acetylcholine esterase activity in the heart Malate dehydrogenase activity in the muscle

Animal status Vmax

(µmoles/min/mg protein)Km (µM) Vmax

(µmoles/min/mg protein)Km (µM) Vmax

(µmoles/min/mg protein)Km (µM)

C 0.872±0.03 0.025±0.02 2.01±0.05 1.34±0.07 0.872±0.03 0.025±0.02E 1.29±0.05** 0.023±0.025 1.53±0.05** 1.23±0.065 1.29±0.05** 0.023±0.025E+B 1.01±0.09@@ 0.0225±0.015 1.85±0.04@@ 1.25±0.025 1.01±0.09@@ 0.0225±0.015E+D 0.98±0.04@@ 0.0225±0.02 1.98±0.07@@ 1.45±0.062 0.98±0.04@@ 0.0225±0.02E+C 0.96±0.06@@ 0.0265±0.018 1.76±0.02@@ 1.16±0.078 0.96±0.06@@ 0.0265±0.018

Values are mean±SEM of 4–6 separate experiments. Each group consists of 6–8 rats. E+B Epileptic rats treated with Bacopa monnieri, E+D Epileptic rats treatedwith Bacoside-A and E+C Epileptic rats treated with Carbamazepine. **pb0.01 when compared to control and @@pb0.01 when compared to epileptic group.

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2.7. Measurement of T3 and insulin

T3 and insulin content of the serum of the experimentalrats were measured according to the kit protocol of BRACradioimmunoassay kit. T3 and insulin concentration in thesample were determined from the standard cure plottedusing MultiCalc™ software (Wallac, Finland).

2.8. Malate dehydrogenase activity

Malate dehydrogenase activity was assayed according toMehelar et al., [12]. Crude sample was prepared by making a5% homogenate of the muscle in phosphate buffer, pH 7.4using polytron homogenizer. The homogenate was centri-fuged at 1000 ×g for 10 min. The supernatant was collectedand centrifuged at 10,000 ×g for 20 min. The reactionmixturecontain phosphate buffer, pH 7.4, NADH, oxaloactate andenzyme. The reaction mixture of 1 ml was assayed at 340 nmin the spectrophotometer by measuring the decrease inoptical density due to oxidation of NMDA measured at 15 sinterval for 1 min at room temperature. One unit of enzymeactivity was equal to the change in OD of 0.1 for 100 s at334 nm. Kinetic parameters such as Vmax and Km werecalculated from the data of MDH activity measured atsubstrate concentration 0.0125–0.2 Mm.

3. Results

3.1. Acetylcholine esterase activity in the muscle of Control,Epileptic, Epileptic+Bacopa monnieri, Epileptic+Bacoside-Aand Epileptic+Carbamazepine, treated rat groups

Acetylcholine esterase kinetic studies showed that Vmax

significantly increased (pb0.01) in the muscle of epileptic

Table 2Epinephrine, norepinephrine content in the heart, T3 and insulin content in the ser

Animalstatus

Epinephrine (nmoles/g wet wt) Norepinephrine

C 19.54±0.13 13.34±0.77E 18.37±0.51 12.23±0.65E+B 17.85±0.43 11.25±0.25E+D 20.98±0.70 14.45±0.62E+C 18.76±0.25 12.16±0.78

Values are mean±SEM of 4–6 separate experiments. Each group consists of 6–8 ratswith Bacoside-A and E+C Epileptic rats treated with Carbamazepine. ***pb0.001epileptic group.

rats when compared to control. Carbamazepine, extract ofBacopa monnieri and Bacoside-A treatment significantlyreversed the Vmax (pb0.01) to near control. Km showed nosignificant change in the experimental groups (Table 1).

3.2. Acetylcholine esterase activity in the heart of Control,Epileptic, Epileptic+Bacopa monnieri, Epileptic+Bacoside-Aand Epileptic+Carbamazepine, treated rat groups

Acetylcholine esterase kinetic studies showed that Vmax

significantly decreased (pb0.01) in the heart of epileptic groupwhen compared to control. Carbamazepine, extract of Bacopamonnieri and Bacoside-A treatment significantly reversed theVmax (pb0.01) tonear control. Km showednosignificant changein the experimental groups (Table 1).

3.3. Malate dehydrogenase activity in the muscle of Control,Epileptic, Epileptic+Bacopa monnieri, Epileptic+Bacoside-Aand Epileptic+Carbamazepine, treated rat groups

Vmax of malate dehydrogenase significantly increased(pb0.01) in the muscle of epileptic group when comparedto control. Carbamazepine extract of Bacopa monnieri andBacoside-A treatment significantly reversed Vmax (pb0.01) tonear control. Km showed no significant change in theexperimental groups (Table 1).

3.4. Epinephrine, norepinephrine and T3 content in the serum ofControl, Epileptic, Epileptic+Bacopa monnieri, Epileptic+Bacoside-A and Epileptic+Carbamazepine, treated rat groups

Epinephrine and norepinephrine content didn't sow signifi-cant change in the experimental group. T3 content in the serumwas significantly (pb0.001) increase in the epileptic groupwhen

um of control and experimental rats.

(nmoles/g wet wt) T3 (ng/ml) Insulin (µU/ml)

0.45±0.04 56.4±4.51.35±0.10*** 86.6±6.4***0.53±0.14@@@ 59.7±3.1@@@

0.59±0.07@@@ 54.2±5.8@@@

0.81±0.02@@ 68.9±4.2@@

. E+B Epileptic rats treated with Bacopa monnieri, E+D Epileptic rats treatedwhen compared to control, @@pb0.01 and @@@pb0.001when compared to

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compared to control. Carbamazepine extract of Bacopa monnieriand Bacoside-A treatment significantly reversed the T3 content(pb0.001) to near control (Table 2).

3.5. Insulin content in the serum of Control, Epileptic, Epileptic+Bacopa monnieri, Epileptic+Bacoside-A and Epileptic+Carbamazepine, treated rat groups

Insulin content in the serum was significantly (pb0.001)increase in the epileptic group when compared to control.Carbamazepine extract of Bacopa monnieri and Bacoside-Atreatment significantly reversed the insulin content (pb0.001)to near control (Table 2).

4. Discussion

In this study we have focused on the changes in energymetabolism and excitability of muscle during epilepsy.Epileptic seizures are episodes of disturbed brain functionthat cause changes in attention or behavior [13,14]. Seizuresare caused by abnormally excited electrical signals in thebrain. Generalized tonic–clonic seizure is a seizure involvingthe entire body, also called a grand mal seizure. Such seizuresusually involve muscle rigidity, violent muscle contractions,and loss of consciousness [15]. Epilepsy usually causemyoclonic twitches, usually caused by sudden musclecontractions; they also can result from brief lapses ofcontraction [16]. Acetylcholine induced seizure-like activityin chronically epileptic rats. Nicotinic acetylcholine receptorsare widespread ligand-gated ion channels that mediate fastcholinergic transmission at both the peripheral and centralnervous systems. In addition to their involvement inneuromuscular and autonomic ganglia synaptic transmission,they play an important role in cognitive and addictiveprocesses [17–19]. Furthermore, their dysfunction has beenlinked to a number of human diseases, including congenitalmyasthenia, schizophrenia, epilepsy and neurodegenerativedisorders such as Alzheimer's and Parkinson's diseases[18,20]. Nicotinic acetylcholine receptors are present inmany tissues in the body and are the best studied of theionotropic receptors. The neuronal receptors are found in thecentral nervous system and the peripheral nervous system.The neuromuscular receptors are found in the neuromuscularjunctions of somatic muscles; stimulation of these receptorscausesmuscular contraction [21]. Seizures are initiated by theexcessive neuronal activity in the brain which is transmittedthrough the peripheral nervous system and stimulate therelease of ACh in to the neuromuscular junction. Acetylcho-line released in to the neuromuscular junction from thepresynaptic neurons initiate the muscle contraction. On theother hand ACh inhibit heart beat and epinephrine andnorepinephrine initiate it [22]. AChE activity has been used asa marker for cholinergic activity [23]. The enzyme acetylcho-line esterase converts acetylcholine into the inactive meta-bolites choline and acetate. AChE is abundant in the synapticcleft, and its role in rapidly clearing free acetylcholine fromthe synapse is essential for proper muscle function. AChEplays a very important role in the ACh-cycle, including therelease of ACh [24]. The duration of ACh at the synaptic cleft iscritically dependent on AChE activity [25]. Our study showedincreased AChE activity in the muscle and decreased activity

in the heart of the epileptic rats. Increased cholinergic activityin the neuromuscular junction makes the muscle moresusceptible to seizures.

MDH is an enzyme in the citric acid cycle that catalyzes theconversion of malate into oxaloacetate using NAD+ and viceversa. MDH is also involved in gluconeogenesis, the synthesisof glucose from smaller molecules. The activity of lactatedehydrogenase (LDH) and MDH was estimated as indicatorsof anaerobic metabolism [26]. As previously reported,mitochondrial MDH binds to purified complex I of theelectron transport system [27]. Mitochondrial function is akey determinant of both excitability and viability of neurons[28]. Brain mitochondrial membrane was somewhat morefluidized in epileptic animals possible consequences ofmitochondrial respiratory chain (MRC) dysfunction. Inconclusion, impairment of MRC function along with struc-tural alterations suggests novel pathophysiological mechan-isms important for chronic epileptic condition [28]. Alcoholicextract of Bacopamonnieriwas tested for its protective role onmorphine-induced brainmitochondrial enzyme status in rats.The level of the brain mitochondrial enzymes was signifi-cantly lower in the morphine-treated group when comparedwith control animals. These enzymes were maintained atnormal level when Bacopa extract was administered beforethe administration of morphine [29]. Our study showedincreased MDH activity in the muscle of the epileptic ratsindicative of the increased metabolic rate in the muscle of theepileptic rats.

Insulin facilitates the entrance of glucose in to the musclecells [30]. The only mechanism by which cells can take upglucose is by facilitated diffusion through a family of hexosetransporters. In muscle tissue the major transporter used forthe uptake of glucose is GLUT4 which made available in theplasma membrane through the action of insulin [31]. Insulinregulates the glucose uptake into these cells by recruitingmembrane vesicles containing the GLUT4 glucose transpor-ters from the interior of cells to the cell surface,where it allowsglucose to enter cells by facilitated diffusion [32]. Once in thecytoplasm, the glucose is phosphorylated and thereby trappedinside cells. The effect of insulin on GLUT4 distribution isreversible. Within an hour of insulin removal, GLUT4 isremoved from the membrane and restored intracellular invesicles ready to be re-recruited to the surface by insulin[33,34]. Increased insulin content in the serumof epileptic ratsfacilitates the rapid intake of glucose in to the muscle cells.

T3 is a major metabolic hormone which has effect on basalmetabolic rate (BMR) [35]. The thyroid gland secretes mostlyT4 and very little T3. Most of the T3 that drives cellmetabolism is produced by action of the enzyme named 5′-deiodinase, which converts T4 to T3, T3, therefore, is themetabolically active thyroid hormone. In adult life it exerts aprofound effect on basal metabolic rate, increasing respira-tion rate and simultaneously lowering metabolic efficiency[36]. It mainly acts through the coordinated and synergisticmodulation of both nuclear and mitochondrial genomeexpressions giving rise to a complex network of factors andcellular events [37]. Increased plasma T3 level is an indicatorof increased BMR [38]. Treatment of rats with T3 resulted in asignificant decrease in body weight [39], while the heartweight increased; indicate that T3- and T4-thyrotoxicosisresults in impaired energy metabolism in heart mitochondria

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[40]. T3 has the ability to uncouple oxidation of substratesfrom ATP production, results in reduced ATP production andan astounding production of heat [41,42]. Muscle glycogen isalso more rapidly depleted, and less efficiently stored duringhyperthyroidism, which may create muscle weakness.

Our results showed an increased MDH and AChE activityin the muscle, insulin and T3 content in the serum of epilepticrats indicated augmented energy metabolism in epileptic ratsand treatment using Bacopa monnieri reversing the changesto near control. Bacopa monnieri plant extract is a nerve tonicused extensively in the traditional Indian medicinal systemAyurveda. Bacopa monnieri has been used for centuries as amemory-enhancing, antioxidative, adaptogenic [43], analge-sic, antipyretic, sedative, and antiepileptic agent. Bacopamonnieri is currently recognized as being possibly active inthe treatment of mental illness and epilepsy [44]. Thus ourfindings suggest that repetitive seizures resulted in increasedmetabolism and excitability in epileptic rats. Bacopa monnieriand Bacoside-A treatment prevents the occurrence of seizuresthere by reducing the impairment on peripheral nervoussystem, indicating the potential medicinal value of Bacopamonnieri in epileptic treatment.

Acknowledgement

This work was supported by research grants from DBT,DST, ICMR, Govt. of India and KSCSTE, Govt. of Kerala to Dr. C. S.Paulose. Jobin Mathew thanks CSIR for SRF.

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