Screening of Antiepileptic Activity of Leaves of Sapindus trifoliatus Linn.

 

Jennifer Fernandes*, Bhat K.Ishwar¹, Fernandes Ronald²

^*Professor, Dept. of Pharma. Chem., NGSM Institute of Pharmaceutical Sciences, Paneer-575018 India.

¹Professor & Head, Dept of Pharma. Chem., N.G.S.M. Institute of Pharmaceutical Sciences, Paneer-575018 India

²Professor, Dept of Pharma. Chem., N.G.S.M. Institute of Pharmaceutical Sciences, Paneer-575018 India.

*Corresponding Author E-mail: jennifer_pharma@yahoo.co.in

 

The aim of the present study was to investigate anticonvulsant effect of ethanolic extract of the leaves of Sapindus trifoliatus on electrically and chemically induced seizures. The ethanol extract of the leaves of Sapindus trifoliatus (100, 200 and 400 mg/kg) were studied for its anticonvulsant effect on maximal electroshock induced seizures and metrazol (pentylenetetrazole) induced seizures in rats. Ethanolic extract of Sapindus trifoliatus (100,200 and 400 mg/kg) significantly reduced the duration of seizures induced by maximal electroshock (MES) as well as protected animals from metrazole induced tonic seizures.

 

 

 

INTRODUCTION:

Epilepsy is a common chronic neurological disorder characterized by recurrent unprovoked seizures. [1] These seizures are transient signs and/or symptoms of abnormal, excessive or synchronous neuronal activity in the brain. [2] About 50million people worldwide have epilepsy, with almost 90% of these people being in developing countries.[3] Epilepsy is more likely to occur in young children, or people over the age of 65 years, however it can occur at any time. Epilepsy is usually controlled, but not cured, with medication, although surgery may be considered in difficult cases. However, over 30% of people with epilepsy do not have seizure control even with the best available medications. [4] The most common causes for the disorder are head injury, brain infections, encephalitis, meningitis and dementia. Environmental and socio-economic conditions may cause epilepsy.[5]

 

Sapindus trifoliatus (Sapindaceae) commonly known as Indian filbert and soap-nut tree. Fruits, leaves, roots and bark of the tree⁶are generally used for therapeutic purposes⁷.

The fruit of the plant is reported to have expectorant, emetic, abortificiant and spermicidal effects ⁸ and is also used in excessive salivation, epilepsy and chlorosis⁹.

 

So the objective of the present study was to evaluate antiepileptic activity of leaf extract of Sapindus trifoliatus against seizures induced by MES (maximal electroshock) and metetrazol.

 

MATERIALS AND METHODS:

Preparation of ethanolic extract:

The leaves of Sapindus trifoliatus were collected from the local areas near to Mangalore, Karnataka. The plant of Sapindus trifoliatus had been authenticated by botanist Mrs. Usha Nalini. Head of Botany Department, St. Agnes College, Mangalore. The leaves were cleaned, dried and broken down into pieces and powdered into a coarse powder by a mechanical grinder. The powder was then passed through sieve no.40 and extracted with ethanol in soxhlet extractor exhaustively for 20-24 hours. The extract was concentrated to dryness under reduced pressure and controlled temperature using flash evaporator.

 

Animals:

Male albino Wistar rats (200-250g) were selected and housed under standard conditions of temperature (25±1°C), relative humidity (30-60%) and 12 hours light/dark cycle. Animals were fed with standard pellet diet and water ad libitum. Animals had free access to food and water however water was withdrawn 8 hours before and during the experiment. The protocol of the experiment was approved by the Institutional ethical committee before the experiment.

 

Acute toxicity studies:

Acute toxicity study was carried out in female albino rats as per staircase method¹⁰ and OECD guidelines 425¹¹. All the experiments were performed within the guidelines of the Institutional ethical committee of Kshema. Derlakatte, Mangalore. (KSHEMA /IAEC / 032/2004)

 

Assessment of anticonvulsant activity:

Maximal Electroshock (MES) Induced Convulsions¹²:

The convulsive effect of MES^13,14 was considered to be analogues to grandmal type of convulsion in man. Five groups of 6 rats of body weight 200-250mg were selected for the study. Group III, IV, V received the test drug (100, 200, 400mg/kg) of leaf extract of S. trifoliatus respectively. Group II received the standard drug (Phenytoin 25mg/kg) and group I served as control. A supramaximal electrical stimulus of 150mA was given to the animals for 0.2 second through ear clip electrodes. Animals were observed and various phases of maximal electroshock seizures viz; tonic hind limb flexion, tonic hind limb extension and tonic clonic phases were noted. Abolition or decrease in the duration of extension phase was taken as an index of anticonvulsant activity. For statistical analysis of the data E/F (extensor / flexor) was used.

 

Metrozol Induced Convulsions:

Five groups of adult albino rats, each groups comprising of 6 animals were selected. For the group III, IV, V (test group) the test extract (100, 200, 400mg/kg body weight) was administered by oral route. Group II received standard drug Na valproiate, group I served as control. After 45 minutes, Metrazol was administered by ip route in a dose of 80mg/ kg of the body weight to all the groups and the animals were observed for onset of clonic convulsions up to 30 minutes after Metrazol administration.

 

Statistical Analysis:

The data are presented as mean ± SEM for metrzol and MES were analyzed by one way analysis of variance (ANOVA) followed by Dunnett's test. Differences were considered to be statistically significant when P<0.05

 

RESULTS:

The alcoholic extract has protected the animals from the seizures and reduced the duration of hind leg extension in the doses of 100, 200, 400 mg /kg body weight, where as the control did not shown any protection indicating the alcoholic extract is effective against seizures induced by MES (Table.1) The mechanism of inhibition of seizure may be due to enhancement of GABA activity or inhibition of sodium channels.

 

Comparison of the control and test group by one way ANOVA, where P< 0.05 indicating the activity is significant. Phenytoin is the antiepileptic drug best known for its selective action in preventing maximal seizures.¹⁵

 

In case of Metrazol induced convulsions, the alcoholic extract in the doses of 100, 200 and 400mg/kg delayed the onset of clonic convulsion where as control treated animals, convulsions appeared in a short duration (Table.2). As the test group was compared with control group P<0.05 indicating the results were significant.

 

The ethanolic extract has protected the animals from the seizures and reduced the duration of hind leg extension at the doses of 100,200,400 mg/kg body weight, where as the control did not show any protection, indicating that the ethanolic extract is effective against seizures induced by MES. The mechanism of inhibition of GABA activity or inhibition of sodium channels. Comparison of control with standard and test groups by one way ANOVA, where P<0.05 indicating the activity is very highly significant. The activity of a compound to prevent maximal electroshock seizures is believed to correlate with its activity to prevent the spread of seizure discharge through neural tissue. Activity against maximal electroshock seizures is thought to indicate potential efficacy in the treatment of major motor (grandmal) seizures. Phenytoin is the antiepileptic drug best known for its selective action in preventing maximal seizures¹⁶.

 

In case of metrazole induced convulsions, the ethanolic extract of fruit/leaves at doses of 100,200,400 mg/kg body weight delayed the onset of clonic convulsions, whereas control group convulsions appeared in a short duration. As the test group is compared with control group P<0.05, indicating the results are very highly significant. The ability of a compound to prevent threshold seizures induced by subcutaneous metrazole injection has been correlated with the ability to raise the threshold excitation of neural tissue. Selective action of this is believed to indicate potential efficacy against absence (petit mal) seizures¹⁶. In both the test group animals as the dose increased, the protection from seizures was also increased.

 

 

 

Table 1: Maximal Electroshock (MES) Induced Convulsions

Groups	Duration in seconds(Mean±S.E.M)			E/F
	Flexor (F)	Extensor (E)	Tonic-clonic
Control	2.44±0.06	11.26±0.42	2.28±0.017	4.61±0.177
Phenytoin	2.06±0.015*	2.17±0.009*	2.59±0.029*	1.04±0.005*
Alcoholic extract of S.trifoliatus  100mg/kg	2.60±0.104*	8.94±0.129*	2.60±0.024*	3.43±0.06*
200mg/kg	2.57±0.006*	8.77±0.118*	2.52±0.005*	3.40±0.05*
400mg/kg	2.53±0.005*	4.14±0.111*	2.59±0.004*	1.63±0.042*

* The mean difference is significant at the 0.05 level, when compared to the control group

 

 

 

Table 2: Metrazole Induced Convulsions

Groups	Onset of clonic convulsions after metrazol administrations in seconds
Control	82.66±1.520
Sodium valproate	2108±46.70*
Alcoholic extract of S.trifoliatus 100mg/kg	709.5±2.55*
200mg/kg	964.1 ±6.50*
400mg/kg	1721.1±64.84*

*The mean difference is significant at the 0.05 level, when compared to the control group.

 

DISCUSSION:

MES induced seizures bear a semblance to grandmal epilepsy. Evidence indicates that imbalance between excitatory and inhibitory neurotransmission in the brain is a main cause contributing to seizure development in both, experimental and clinical conditions. Gamma-amino butyric acid (GABA) is the predominant inhibitory neurotransmitter in the CNS. Impairment of GABA function is widely recognized to provoke seizures, whereas facilitation has an anticonvulsant effect. GABA is synthesized from glutamate, exclusively in GABAergic neurons, by the action of the enzyme glutamic acid decarboxylase. Upon synaptic release, GABA acts on its three specific receptors, GABAA, GABAB, and the newly characterized GABAC. GABA is removed from the synaptic cleft into localized nerve terminals and glial cells, by specific membrane-bound transport molecules. After removal from the synapse, GABA is either recycled to the readily releasable neurotransmitter pool (GABAergic nerve terminals only) or metabolized (neurons and glial cells) to the inactive molecule succinic acid semialdehyde by the mitochondrial enzyme GABA-transaminase¹¹. The convulsion in MES method is due to the disturbed activity of GABA in the brain.

 

Metrazol/PTZ a most frequently used substance as well as an acute experimental model in the preliminary screening to test potential anticonvulsant drugs. The mechanism by which metrazol/PTZ is believed to exert its action is by acting as an antagonist at the GABAA receptor complex. Several biochemical hypotheses have been advanced involving the inhibitory GABAergic system and the system of the excitatory amino acid glutamate and aspartate. The mechanism by which metrazol /PTZ is believed to exert its action is by acting as an antagonist at the GABAA receptor complex.

 

Drugs protecting against tonic-clonic seizures induced by metrazol/PTZ are considered to be useful to control myoclonic and absence seizures in humans. In this study ethanolic extract of Sapindus trifoliatus replicated the effect of this anti epileptic drug by delaying tonic convulsion and mortality. The benzodiazepine site in the GABAA receptor and T-type Ca2+ currents could be targets for further studies to know the mechanisms of action of methanolic extract of Sapindus trifoliatus. It suggests that ethanolic extract of Sapindus trifoliatus is useful in suppressing generalized tonic-clonic seizures. Anticonvulsant activity of MEVA in inhibiting seizures may be by regulating GABA – mediated synaptic inhibition through action at distinct sites of the synapse. Summarizing the data obtained in this study, the results suggest a possible anticonvulsant effect of ethanolic extract of Sapindus trifoliatus in rodents.

 

ACKNOWLEDGEMENT:

Authors are thankful to Nitte Education Trust/ Nitte University for providing the necessary facilities in carrying out this study.

 

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