Antiepileptic Effects of Sapindus mukorossi in rats

 

Pradeep Kumar Samal*, Shani Sharaf, N.R. Beck and J.S. Dangi

SLT Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh.

*Corresponding Author E-mail: samalpharmacology@rediffmail.com

 

ABSTRACT:

Plant for the present studies was selected on the bases of literature review. Plant was collected from local market Bilaspur, Chhattisgarh, The collected leaves were powdered in grinder and extracts were prepared in ethanol and chloroform solvents. These extracts were used for further studies. Phytochemical studies had been performed on the extracts and it was found to have like saponin, Sterols, Fatty acids and oils and carbohydrate. Acute oral toxicity study of ethanolic extract of Sapindus mukorossi (EESM) was conducted in rat by using OECD guideline no.425 and it was found to safe up to the dose label of 2000mg/kg of body weight. In Maximal electro-shock (MES) induced model, dose of EESM (200 and 400mg/kg) show highly significant anticonvulsant activity, but dose of chloroform extract of  Sapindus mukorossi (CESM) did not show any anticonvulsant activity as compared to control group. In Pentylenetetrazol (PTZ) model lower dose of EESM (200 and 400mg/kg) show good anticonvulsant activity as compared to control group. Locomotor study in actophotomotor extract of EESM exhibited the decreased motor activity in dose dependent manner, it is sign of sedation. ESM increase the threshold of MES induced convulsion in a dose dependent manner.

 

 

1. INTRODUCTION:

Epilepsy is the second most common neurological disorder which affects an estimated seven million people in India and 50 million worldwide i.e., approximately 1–2% of the world population. Epilepsy is a neurological disorder characterized by brief episodes of seizures and excessive EEG discharge which is occasional, sudden and excessive. Seizure is a hyperexcitation of neuron in the brain leading to altered behavior with or without violent motor activity. Despite the introduction of many new antiepileptic drugs (AEDs) but a significant percentage of patients with epilepsy continue to experience seizures. Hence, there continues to be an unmet clinical need for more effective and less toxic anti-epileptic drugs^(1-3). Sapindus mukorossi (Sapindaceae) is considered as one of the most valuable drug in traditional system of medicine from ancient time⁽⁴⁾.

 

2. Material and Methods:

2.1. Drugs and Chemicals:- Analytical grade Chemical were used in this study. Pentylene tetrazole (PTZ) HIMDIA CHEMICAL and Diazepam (Campose Inj. Ranbaxy Lab) were purchased from local market; while Phenytoin was obtain as a gift sample from Stallion Laboratories Pvt. Ltd. Ahmadabad (Gujarat).

 

2.2. Plant Materials:-

 The fruits of Sapindus mukorossi were procured from the local market (Amarnath shop Bilaspur, C.G., India). The Plant material was authenticated by Dr. H.B. Singh, Head, Raw Material Herbarium & Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. The voucher no. is NISCAIR/ RHMD/Consult/-2011-12/1813/113 dated 5 Sept 2011.

 

2.3. Animals:- Wistar albino rats (150-200g) purchased from IICB Kolkatta were maintained in the department animal house for experimental purpose.

 

Then all the animals were acclimatized for seven days under standard husbandry conditions, i.e.; room temperature of 25 ± 1°C; relative humidity 45-55% and a 12:12hr light/ dark cycle. The animals had free access to standard rat pellet (Vinod Agro Industries Ltd, Vadodara, India), with water supplied ad libitum under strict hygienic conditions.

 

 

Table-1 Effect of CESM and EESM on animals in locomotor activity

S.No.	Treatment dose (mg/ml)	Avg. Body wt (g)	Locomotor activity counts/5min	Mortality/Total rat	% Mortality
1.	Vehicle control	187.16+3.911	442+6.15	6/6	0%
2	Phenytoin (25mg/kg)	189+3.236	441.66+5.40	6/6	0%
3.	EESM (200mg/kg)	191.83+4.393	348+9.65**	6/6	0%
4.	EESM (400mg/kg)	192.16+3.701	247+13.58**	6/6	0%
5.	CESM (200mg/kg)	186.16+3.719	451.33+6.76	6/6	0%
6.	CESM (400mg/kg)	188.66+2.654	447.5+7.05	6/6	0%

Values are expressed as mean ± SEM, n=6

**P<0.01 Vs control using oneway ANOVA followed by dunnett’s test

 

Table-2 Effect of CESM and EESM on animals in MES induced

Values are expressed as mean ± SEM, n=6

**P<0.01 Vs control using one way ANOVA followed by dunnett’s test

 

 

Each experimental group (n = 6) had separate set of animals and care was taken to ensure that animals used for one response were not employed elsewhere. Animals were habituated to laboratory conditions for 48 hours prior to experimental protocol to minimize if any of non-specific stress. The approval of the Institutional Animal Ethical Committee (IAEC) of SLT Institute of Pharmaceutical Sciences, Bilaspur (Chhattisgarh) (Ref. No. IAEC/ Pharmacy/2012/49 dated 31 May 2012) was taken prior to the experiments. All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Approval No. 994/a/GO/06/ CPCSEA).

 

2.4 Acute toxicity study (AOT):- Acute toxicity study was performed according to the procedure OECD guideline no. 425.⁽⁵⁾  AOT was performed on swiss albino mice and the animal were kept fasting for overnight providing water ad libitum, after which the ethanolic extract of Sapindus mukorossi (EESM) and Chloroform extact of  Sapindus mukorossi (CESM) extracts were administered orally 2000 mg/kg and observed the mortality of animals.

 

2.5. Experimental Design and pharmacological screening:-

2.5.1 Assessment of locomotor activity:

The sedative activity and its possible mechanism of extract of Sapindus mukorosi was investigated by determining the spontaneous locomotor activity of rats⁽⁶⁾. Locomotor activity was monitored by using  actophotometer.  Animal were individually put in actophotometer after 60 min of treatment with different dose level of Sapindus mukorossi extracts and vehical and total activity count was registered for 5 min. The locomotor activity was expressed in term of total photobeam interruption count/5 min⁽⁷⁾.

 

2.5.2 Antiepileptic activity⁽⁸⁾

MES induced convulsion:-

Maximal electroshock induced seizures: The seizure was induced by maximal electroshock in Swiss albino rats with the help of electroconvulsiometer by passing current of 90 mA for 0.2 second using ear clip electrodes. The animals were divided into six groups each containing 6 animals (n = 6). The test samples were given 1 hour prior to induction of convulsions.

Group I (Control): Received normal saline (1 ml/kg, po).

Group II (Standard): Received  phenytoin (25mg/kg body weight I.P)

Group III and IV: Received EESM (200 and 400 mg/kg, po).

Group V and VI: Received CESM (200 and 400 mg/kg, po).

The animals were observed for the extensor phase as well as its duration. The abolition of extensor (tonic phase) in groups treated with extracts and diazepam were considered as criteria for anticonvulsant activity when compared with the control group.

 

PTZ induced seizures:-

The animals were divided into six groups each containing 6 animals (n = 6). Treatment was same as that of MES. After 30 minutes of the dosing all the groups were injected with the convulsing agent pentylenetetrazole (60 mg/Kg) and animals were kept in individual plastic cages to observe convulsions for 1h.

 

2.6. Statistical Analysis:-

The values were expressed mean + SEM from 6 animal. The result were subjected to statistical analysis by using ANOVA followed by dunnett`s test to calculate the significance from control group. P<0.05 was considered significance.

 

 

 

 

Table-3 Effect of CESM and EESM on animals in PTZ induced convulsion.

Values are expressed as mean ± SEM, n=6

**P<0.01 Vs control using oneway ANOVA followed by dunnett’s test

 

3. RESULT:

3.1  Preliminary phytochemical Analysis :-

In this study studies extract of Sapindus mukorossi revealed the phytoconstituents like saponin, Sterols, Fatty acids and oils, carbohydrate.

 

3.2 Acute toxicity study:-

There was no mortality amongst the graded dose groups of animals and they did not show any toxicity or behavioural changes at a dose level of 4000 mg/kg. This finding suggests that EESM and CESM were safe in or non-toxic to rats up to 4000 mg/kg. Hence, the doses of 200 and 400 mg/kg were selected for the antiepileptic activity.

 

3.3 Assessment of locomotor activity:-

Locomotor activity of rats is measured in open field actophotometer. Behavioral changes were determined by different extract of Sapindus mukorossi. CESM (200 and 400mg/Kg) did not show any change while EESM (200mg/ml and 400 mg/Kg) show decreasing motor activity is significantly in dose-dependent manner. A drug-induced decrease in spontaneous motor activity is regarded as an indication of sedation. (Table- 1)

 

3.4  Antiepileptic activity :-

3.4.1  MES induced convulsion :

Different doses of ethanolic and chloroform extract (200 mg/kg, 400 mg/kg) were administered orally. As single dose of chloroform 200 mg/kg and 400mg /kg dose did not produce any significant effect on tonic extensior phase.  On way ANOVA showed significant effect  (p<0.01) on the duration of tonic hind limb extension at 200mg/kg and 400 mg/kg dose of the EESM respectively as compared to control group. Maximal protection was observed at 400 mg /kg dose. Mortality was not observed in any group. The phenytoin treated group (std gr.) showed highly significant (p<0.01) effect on the duration of tonic hind limb (Table- 2)

 

3.4.2  Pentylene tetrazole (PTZ) induced convulsions :-

In animals treated with vehicle, clonic convulsions appeared 69.33±2.96 sec after PTZ and 3 animals died after seizures. We examined the anticonvulsant effects of Sapindus mukorossi extract on the PTZ induced seizures in mice. EESM promoted a significant protection of PTZ-induced seizures in a dose-dependent manner (Table - 3). There was a significant decrease of the incidence of clonic convulsions and mortality in all doses of EESM tested (200and 400mg/kg) with maximum protection observed at 400 mg/kg (Table 3). Similarly, treatment with EESM significantly prolonged the latency for convulsions in a dose-dependent manner. But the highest dose prolonged the latency of the convulsions induced by PTZ. while CESM dose 200and 400 mg/kg did not show any significant effect against PTZ induce seizure. Standard dose of diazepam (2mg/kg) completely protected seizure. In the term of percentage protection EESM 200mg/kg exhibited 66.6% protection. diazepam and EESM 400mg/kg exhibited 100% protection from seizure. (Table - 3)

 

4.      Discussion:

Epilepsy is one of the most common brain diseases in human. About 1% of the population diagnosed with the disease. Several different types of human epilepsies have been characterized based on the classification of International League against Epilepsy (ILAE). According to this classification, epilepsy has been divided into partial epilepsy (simple and complex), generalized symptomatic epilepsy and unclassified epilepsy. An imbalance between the excitatory and inhibitory neurotransmitters is responsible for seizures. At neuronal level, seizures activity often occurs when glutamatergic excitatory neurotransmitters overrides gamma aminobutyric acid (GABA) mediated inhibition. In the assessment of antiepileptic study, several models have been developed. Many drugs that increase the brain contents of GABA have exhibited the antiepileptic against seizures induced by MES induced seizures and PTZ induced seizures⁽⁸⁾. Single dose treatment with EESM (200 mg/kg and 400 mg/kg, p.o.) spontaneous decrease of locomotor activity of rats in a dose dependent manner. Decrease the locomotors score, indicating the sedative effect of the extract. CESM (200 mg/kg and 400 mg/kg, p.o.) was not produce any significantly sedation.

 

The MES is probably the best validated method for assessment of antiepileptic drugs in generalized tonic- clonic seizures. At the highest tested dose (400 mg/ kg), EESM  was found to significantly decrease the duration of the hind limb tonic extensor phase whereas the lower dose (200 mg/ kg) shown less effect against seizures. The extracts of fruits of Sapindus mukorossi exhibited anticonvulsant activity by delaying the onset of PTZ induced seizures and protecting treated mice from mortality induced by seizures. Drugs protecting against tonic- clonic seizures induced by PTZ are considered as useful in controlling myoclonic and absence seizures in humans⁽⁸⁾. The phytochemical study of extracts revealed the presence of alkaloids, tannins, triterpene and steroids. The phytochemicals such as tannins, triterpene and steroids were reported as active substances for anticonvulsant activity ⁽⁸⁾. Hence, these phytochemicals might be contributing to the anticonvulsant activity of EESM. Further study is necessary to determine the mechanism of action and isolation of active principle(s) from ethanol extracts of fruits of Sapindus mukorossi  for antiepileptic activity.

 

5.      Acknowledgement:

The authors wish to thank Head, SLT. Institute of Pharm. Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur,  Chhattisgarh. for providing the necessary facilities to complete this work.

 

6.      Reference:

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Received on 01.10.2012       Modified on 15.10.2012

Accepted on 25.10.2012      © RJPT All right reserved