INTRODUCTION:

Epilepsy effects about 20-40 millions people worldwide. Epilepsy is the second most common neurological disorder, after stroke epilepsy is a disorder of the central nervous system characterized by excessive electrical charge ¹.

There are many drugs available for the treatment of epilepsy in Allopathic as well as in Ayurvedic system but most of the drugs are costly and many side effects of these drugs. Thus a need arises far a new agents with greater efficacy, negligible or reduce side effect and devoid of unfavorable drug interactions unlike most AEDs in the market. This has given rise stimulation in the search for investigating natural resources showing anticonvulsant activity. In spite of tremendous advances in the field of medicine, there is no truly and satisfactory drug for the treatment of epilepsy².

Celastrus paniculatus Willd. (CP) belongs to family Celastraceae is a large, woody, climbing shrub, distributed almost all over India up to an altitude of 1800 m is known for its ability to improve memory³. It also found in middle and South Andamans.

Ayurveda, the ancient Indian traditional system of medicine has used this plant seed for prevention and treatment of various diseases⁴. The bark is abortifacient, depurative and a brain tonic. The leaves are emmenagogue and the leaf sap is a good antidote for opium poisoning. The seeds are acrid, bitter, thermogenic, emollient, stimulant, intellect promoting, digestive, laxative, emetic, expectorant, appetizer, aphrodisiac, cardiotonic, anti-inflammatory, diuretic, emmenagogue, diaphoretic, febrifuge and tonic, abdominal disorders, leprosy, pruritus, skin diseases, paralysis, cephalalgia, arthralgia, asthma, leucoderma, cardiac debility, inflammation, nephropathy, amenorrhoea, dysmenorrhoea. The seed oil is bitter, thermogenic and intellect promoting and is useful in abdominal disorders, beri-beri and sores⁵. Hence, in the present study, design the seeds of the Celastrus paniculatus Willd. has been selected for anticonvulsant activity in mice.

MATERIALS AND METHODS:

Plant material:

The fresh seeds of Celastrus paniculatus Willd. were collected during the month of September 2009 from the local market in Jhansi (U.P.). The plant material was identified and authenticated by Dr. H.B. Singh, Scientist F and Head, National Institute of Science Communication and Information of Resources (NISCAIR) New Delhi, with reference number NISCAIR/ RHMD/consult/-2009-10/1291/94.

Preparation of extract:

The seeds of Celastrus paniculatus Willd were shaded dried, and then were made into coarsely powdered form using dry grinder. The powdered seeds of the plant (900 gm) was packed in Soxhlet apparatus and continuously extracted with petroleum ether (40-60ºC). The ethanolic extract was prepared using ethanol by Soxhlet method at temperature of (40-60ºC).The extract were concentrated under vacuum and dried over anhydrous sodium sulphate. The petroleum ether extracts yielded oil that was reddish brown in colour. The ethanolic extract yielded semisolid, viscous, dark colored mass⁶.

Preliminary Phytochemical screening:

The extract was then subjected to preliminary phytochemical screening to dectect the presence of various phytoconstituent. Qualitative chemical tests of (petroleum ether and ethanolic) extracts shows the presence of plant secondary plant metabolites such as alkaloid, tannins, phenolic compound, fatty acids steroids and flavonoids were carried out by using standard procedure^{7 ,8}. (Table 1)

Table 1: Phytochemical constituents of the seeds of Celastrus paniculatus Willd

Phytochemicals	Petroleum ether	Ethanol
Alkaloid	-	+
Glycosides	-	-
Saponins	-	+
Tannins	-	-
Anthraquinones	-	-
Fatty acid and oil	₊	-
Phenolic compound	+	+
Protens and amino acids	-	-
Flavanoids	-	+
Terpene	+	-
Steroids	-	+

- denotes the absence of the respective class of the compound

+ denotes the presence of the respective class of compound

Experimental Animals:

Mice (20-35gm) of either sex were used. The animals were obtained from animal house of the Institute of Pharmacy, Bundelkhand University, Jhansi, India. The animals were housed in standard cages with free access of food (Standard laboratory rodent’s chow) and water. The animal house temperature was maintained at 23± 3.0 with a 12-h light/dark cycle (light on from 6.00 A.M. to 6.00 P.M.). All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics Committee (IAEC) of the Institute (approved by CPCSEA Regd No.716/02/a/ CPCSEA) and BU/Pharm/IAEC/ 009/003).

Acute Toxicity Study:

The acute oral toxicity was carried out as per the guideline set by (OECD) received from the committee for the purpose of control and Supervision of experiments on animals (CPCSEA).One tenth of the medium lethal dose (LD₅₀) was taken as an effective dose.

From the acute toxicity study, the LD₅₀cut-off dose for pet-ether extract and alcoholic extract were found to be 5000 mg/kg and 3000 mg/kg body weight respectively⁹.

Dose and Treatment:

All compounds were prepared freshly each time and administered Phenytoin (25 mg/kg b.wt.). Phenytoin was used (Sain Medicament Pvt. Ltd., Hyderabad, A.P., India). The petroleum ether extract in 1% tween 80 and the ethanolic extract in 1% (w/v) Gum acacia of Celastrus paniculatus Willd. was prepared for administered p. o. and Control group normal saline (0.9% NaCl solution), was administered in a volume of 10 ml/kg b. wt.^10,11.

MES Induced Convulsion

Corneal electrodes were used for bilateral delivery of electrical stimulus. Electro convulsive shock (50 mA for 0.2 Sec.) was delivered through corneal electrode to induce Hind Limb Tonic Extensor (HLTE) phase in mice. The electrical stimulus was applied using a stimulator Apparatus (Biocraft Scientific System Pvt. Ltd., Agra, India) for eight groups of six mice each.

· Group I received normal saline control (0.9% NaCl solution)

· Group II received standard, Phenytoin as positive control (25 mg/kg i.p)

· Group III received EECP 200 mg/kg body weight

· Group IV received EECP 400 mg/kg body weight

· Group V received EECP 600 mg/kg body weight

· Group VI received PECP 200 mg/kg body weight

· Group VII received PECP 400 mg/kg body weight

· Group VII received PECP 600 mg/kg body weight^12,13,14.

For recording various parameters of the animals like Animal motor responses to seizure the pilot study of various stages of convulsions like Tonic flexion, extension, Stupor and mortality due to convulsion observed.

PTZ Induced convulsion

Pentylene tetrazole (PTZ) induced convulsion. The anticonvulsant activity of a compound is generally assessed by its ability to prevent the convulsions, to delay the onset of seizures or death and also shorten the duration of convulsions, spread of seizure discharge through neural tissue within the brain and central nervous system or elevating the seizure threshold¹⁵. PTZ is a central nervous system stimulant. It produces jerky type of clonic convulsions in rats and mice. The convulsive effect of this drug is considered to be analogue to petit mal type of convulsion in man. Activity in this model represents action on seizure focus itself. Twenty four mice of either sex, weighing between 28-35 g were divided into four groups, containing six in each.

• Group I received normal saline 10 ml/kg served as control.

• Group II received diazepam 2 mg/kg body weight intraperitoneally.

• Group III received PECP 200 mg/kg body weight.

• Group IV received PECP 500 mg/kg body weight.

Seizures were induced in mice with PTZ at 80 mg/kg i.p. which is the convulsive dose in 97% of the animals. PTZ was dissolved in 0.9% saline and injected i.p. in mice. All the extracts and standard drug are administered 60 min before the administration of PTZ and the mice were observed for onset of clonic convulsions¹⁶.

Statistical Analysis:

The data are expressed as mean ± SEM (n=6) using Analysis of variance followed by one way ANOVA and multiple comparisons test. Convulsion parameters were analyzed. The value of difference of P<0.05 P < 0.001, were considered as statistically significant with respect to control.

RESULT:

Effect on MES Induced Convulsion:

Poly herbal extract of exhibited a dose dependent significant reduction in various phases of epileptic seizure on comparison with reference standard Phenytoin (25 mg/kg. i.p. There was also a significant reduction in time required for the righting reflex (recovery) in the extract treated groups (Table 2, Fig. 1 and 3, Fig. 2).

Table 2: Effect of Ethanolic extract Celastrus paniculatus Willd. by MES Induced seizure in mice.

S. NO.	Treatment	Duration of HLTE	Mortality (%)	Recovery (%)
1.	Vehicle	15.06 ±0.26	75	25
2.	EECP-200	12.02±0.11	28	72 **
3.	EECP-400	10.03±0.39	25	75 **
4.	EECP-600	9.38±0.43	10	80 **
5.	Phenytoin	3.61±0.8	0	100 ***

Values are mean ± SEM (n=6) mice were pretreated with Vehicle and Ethanolic extract Celastrus paniculatus Willd. Orally 60 minutes before the electroconvulsive shock.

** = P < 0.001, (n=6) compare with control value.

Fig 1: Graphical representations of Ethanolic extract effect of Celastrus paniculatus Willd. on MES induce seizures in mice.

Table 3: Effect of Petroleum ether extract Celastrus paniculatus Willd. by MES

S. NO.	Treatment	Duration of HLTE	Mortality (%)	Recovery (%)
1.	Vehicle	14.84 ±0.26	75	25
2.	PECP-200	8.88 ±0.46	26	74**
3.	PECP-400	8.4 ±0.26	14	85**
4.	PECP-600	8.4 ±0.16	9	91***
5.	Phenytoin	3.61 ±0.8	0	100 ***

Induced seizure in mice.

Values are mean ± SEM (n=6) mice were pretreated with Vehicle and Petroleum ether extract of Celastrus paniculatus Willd. orally 60 minutes before the electroconvulsive shock. ** = P < 0.01, (n=6) *** = P < 0.001, (n=6) compare with control value.

Fig. 2: Graphical representations of Petroleum ether extract effect of Celastrus paniculatus Willd. on MES induces seizures in mice

PTZ (Pentylene tetrazole) Induced convulsion:

200 mg/kg, 500 mg/kg of PECP exhibited a significant anticonvulsant effect by increasing latency of seizures and decreased the duration of seizures. After 30 min of interval 80% and 90% animals survived as sown in (Table 4).

Table 4: Effect of Petroleum ether extract Celastrus paniculatus Willd. by Pentylene tetrazole (PTZ) induced convulsion

S. NO.	Treatment	Duration of HLTE	Mortality (%)	Recovery (%)
1.	Vehicle	14.84 ±0.26	75	25
2.	PECP-200	7.86 ±0.66	20	80**
3.	PECP-400	7.4 ±0.26	10	90***
5.	Diazepam	2.85 ±0.8	0	100 ***

Values are mean ± SEM (n=6) mice were pretreated with Vehicle and Petroleum ether extract Celastrus paniculatus Willd. Orally 60 minutes before the administration of pentylenetetrazole (i.p. 80 mg/kg). *** = P < 0.001, (n=6)

DISCUSSION:

Epilepsy is characterized by recurrent episodes of seizures. A seizure is due to abnormal discharge of some neurons in the brain. Antiepileptic drugs may have a stabilizing influence on neuronal membrane; prevent detonation of normal brain cells by the focal discharge, these drugs act only on those neurons which are firing repeatedly. Some drugs reduce low threshold Ca++ current and abolish absence seizures whereas some drugs increase GABA activity in the synapse causing neuronal inhibition hence antiseizure effect. Glutamic acid causes increased synaptic transmission. A variety of glutamic acid receptors is NMDA receptors and reduction of NMDA receptor activity reduces seizure development¹⁷. The ability of compound to prevent maximal electroshock seizures is believed to correlate with its ability to prevent spread of seizure discharge through neural tissue. Whereas the ability of compound to prevent threshold seizures (induced by pentylene tetrazole), has been correlated with the ability to raise the threshold for excitation of neural tissue¹⁸. Inhibition of the MES test predicts activity against generalized tonic-clonic and cortical focal seizures, lack of activity against MES induced seizures suggests that the drugs are ineffective in suppressing generalized tonic-clonic seizures.

In present investigation the extract was effective to protect the animals from seizures, both in MES test and PTZ test. Finally it is concluded that the MES (Maximal electroshock) induced convulsion test shown strong anticonvulsant effect by increasing onset of clonic convulsion time and by decreasing the time of extensor of test group reduced the significant level as compared to control group. These results indicate the strong protective effect of Celastrus paniculatus Willd. PTZ (Pentylene tetrazole) induced seizures test parameter like latency of seizures and onset of tonic convulsions, clonic convulsions and percent protection were observed in the test group (P < 0.001) showing strong antiepileptic effect. There are some evidences about fatty acids and flavonoids. Therefore it seems that antiseizure effect of Celastrus paniculatus Willd.may be due to part of linoleic acid, cinnamic acids and or flavonoids compound present in the extract¹⁹.

In conclusion, the seed extracts of petroleum ether and ethanolic Celastrus paniculatus Willd demonstrated potential anticonvulsant properties and less toxicity in the experimental animals at doses used, however further studies needed to be carried out on exposure in humans.

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Received on 14.07.2011 Modified on 29.07.2011

Asian J. Research Chem. 4(10): Oct., 2011; Page 1553-1556