An Experimental Evaluation of Anticonvulsant Activity of Ethanolic Extract of Seeds of Holarrhena antidysenterica In Mice.

Jiban Debnath¹, Santosh B. Dighe^2*, Nachiket S. Dighe³ and Mana Supriya⁴

¹Department of Pharmacology, Girijananda Chowdhury Institute of Pharmaceutical Science, Azara, Guwahati, Assam, (India)

²Department of Pharmacology, Pravara Rural College of Pharmacy, Pravaranagar, Maharashtra, (India)

³Department of Medicinal Chemistry, Pravara Rural College of Pharmacy, Pravaranagar, Maharashtra, (India)

⁴Department of Pharmacology, NSHM, College of Pharmaceutical Technology, Kolkata. (India)

ABSTRACT:

In present study the anticonvulsant activity of ethanolic extract of Holarrhena antidysenterica was carried out in swiss albino mice. The anticonvulsant activity of ethanolic extract of seeds of Holarrhena antidysenterica (250 and 500 mg/kg, p.o.) in mice was assessed by using maximum electroshock seizure (MES) test, pentylenetetrazol (PTZ), and bicuculine (BC) test. The ethanolic extract of H. antidysenterica significantly reduced the duration of seizures induced by maximal electroshock (MES). The ethanol extract in doses of 250 and 500 mg/kg conferred protection (17 and 50%, respectively) on the mice. The same doses also protected animals from tonic seizures induced by Pentylenetetrazol and significantly delayed the onset of tonic seizures produced by Pentylenetetrazol. The extract had no effect on bicuculine induced seizure. The ethanolic extract of H. antidysenterica (EEHA) reduced MES, PTZ induced convulsions.

KEYWORDS: Anticonvulsant, H. antidysenterica, MES, PTZ, BC.

1. INTRODUCTION:

The present study aims to study the anticonvulsant activity of ethanolic extract of Holarrhena antidysenterica in swiss albino mice. Epilepsy is a major neurological disorder and up to 5% of the world population develops epilepsy in their lifetime¹. The current therapy of epilepsy with modern antiepileptic drugs is associated with side effects, dose-related and chronic toxicity, as well as teratogenic effects, and approximately 30% of the patients continue to have seizures with current antiepileptic drugs therapy^2-3. Traditional system is believed to be an important source of new chemical substances with potential therapeutic effects. Several plants used for the treatment of epilepsy in different systems of traditional medicine have shown activity when tested in modern bioassays for the detection of anticonvulsant activity and many such plants are yet to be scientifically investigated. Concurrently, phytochemicals identified from traditional medicinal plants are presenting an exciting opportunity for the development of new types of therapeutics. This has accelerated the global effort to harness and harvest those medicinal plants that bear substantial amount of potential phytochemicals showing multiple beneficial effects in convulsion.

Holarrhena antidysenterica Wall (Family: Apocynaceae)is a plant is common in the forests of India, indigenous to the tropical Himalayas, Assam, Uttar Pradesh, down to Travancore^{4, 5}. The literature survey reveals that various parts of this plant are used in diarrhoea, blood dysentery, haematemesis, acute rheumatism, astringent, diabetes and convulsion^{6, 7}. The fresh bark and seeds of this plant were reportedly used for antibacterial, antidiarrhoeal activity^{8, 9}. However, scientific data on anticonvulsant activity of Holarrhena antidysenterica is not available. So, in the present study we have made attempt to explore the anti-convulsant effect of Holarrhena antidysenterica in mice with a view to providing a pharmacological justification (or otherwise) for the ethnomedical use of the plant’s seeds in the management of convulsions and epilepsy in some rural communities of India.

2. MATERIALS AND METHODS:

Plant material:

The seeds of Holarrhena antidysenterica were procured commercially from “Amruth Kashri”, Bangalore. The seeds were authenticated by Dr. Shiddamallayya N, Regional Research Institute (Ay.) Govt. Central Pharmacy Annexe, Ashoka Pilar, Bangalore-11. The Holarrhena antidysenterica seeds extract was prepared by soxhlet extraction with ethanol. The extract was concentrated, dried in vacuum and residue stored in refrigerator at 2-8^οC for used in subsequent experiments.

Experimental animals:

Swiss mice of either sex, 8-10 weeks old, weighing about 25-30 g were used in experiments. Animals were housed in polypropylene cages maintained under standard condition (12 hours light / dark cycle; 25 ± 3⁰C, 45-65% humidity) and had free access to standard feed and water ad libitum. All the animals were acclimatized to laboratory condition for a week before commencement of experiment. All experimental protocols were reviewed and accepted by the Institutional Animal Ethical Committee (IAEC) prior to the initiation of the experiment.

Acute toxicity study:

The acute oral toxicity of ethanolic extract of H. antidysenterica (EEHA) were determined in 3 hours fasted female swiss albino mice by fixed dose method according to OECD guidelines No. 420¹⁰.

Assessment of Anticonvulsant activity:

1. Maximal Electrical Shock (MES) -induced seizures:

The animals were divided in four groups (n = 6). Group I served as vehicle control group. Groups II and III served as test groups treated with the ethanol extract (250 and 500 mg/kg, p.o.), respectively 60 min before application of electric shock (48 mA, 0.2 sec). Group IV served as reference standard, received phenytoin (25 mg/kg, i.p., 20 min), prior to the induction of convulsion. The number of animals protected from hind limb tonic extension seizure (HLTE) and the time spent in this position were determined for each dose group¹¹.

2. Pentylenetetrazol (PTZ) -induced seizures:

The animals were divided in four groups (n = 6). Group I served as vehicle control group. Groups II and III received ethanol extract in doses of (250 & 500 mg/kg, p.o.) 60 min before the subcutaneous injection of PTZ (80 mg/kg). Group IV received diazepam (2.0 mg/kg, i.p.) 20 min before the subcutaneous injection of PTZ (80 mg/kg) and act as a reference standard. The animals were observed for onset of convulsion upto 30 min after PTZ administration. Hind limb extension was taken as tonic convulsion. The onset of tonic convulsion and the number of animals convulsing or not convulsing within the observation period were noted. The ability of the plant extract to prevent or delay the onset of the hind limb extension exhibited by the animals was taken as an indication of anticonvulsant activity^11,
12.

3. Bicuculine (BC) -induced seizures:

The animals were divided in four groups (n = 6). Group I served as vehicle control group. Groups II and III received ethanol extract in doses of (250 & 500 mg/kg, p.o.) 60 min before injection of BC (40 mg/kg, i.p.). Group IV received diazepam (2.0 mg/kg, i.p.) 20 min before injection of BC (40 mg/kg, i.p.) and act as a reference standard. The animals were observed for onset of convulsion upto 30 min after BC administration. Hind limb extension was taken as tonic convulsion. The onset of tonic convulsion and the number of animals convulsing or not convulsing within the observation period were noted. The ability of the plant extract to prevent or delay the onset of the hind limb extension exhibited by the animals was taken as an indication of anticonvulsant activity^{11, 13}.

Statistical analysis:

All the values are expressed as mean ± SEM. Statistical differences between means were determined by one-way ANOVA followed by Dunnett’s post hoc test. p<0.05 was considered as significant.

3. RESULT:

Phytochemical screening:

The preliminary phytochemical screening of ethanol extract of revealed the presence of alkaloids, triterpeinods and phytosterol etc.

Acute toxicity study:

Acute oral toxicity studies of the ethanolic extract of H. antidysenterica did not exhibit any sign of toxicity up to 2000 mg/kg body weight. Since there was no mortality of the animals found at highest dose, hence 250 (1/8^th) and 500 (1/4^th)) mg/kg, p.o. doses of extract were selected for evaluation of Anticonvulsant activity.

Anticonvulsant activity:

Maximal electroshock produced hind limb tonic extension seizures (HLTE) in all the animals used. The vehicle-treated mice showed tonic hind limb extension for duration of 15.53 ± 0.40 sec.

Table 1: Effect of ethanol extract of the seeds of H. antidysenterica (EEHA) on maximal electroshock (MES) - induced seizures in mice.

Treatment	Dose	No of animal convulses/no of animal used	% protection	Duration of HLTE (sec) Mean ± SEM
Control	---	6/6	00	15.53 ± 0.40
Low Dose of EEHA	250 mg/kg, p.o.	5/6	17	11.50** ± 0.56
High Dose of EEHA	500 mg/kg, p.o.	3/6	50	8.50** ± 0.21
Standard (phenytoin)	25 mg/kg, .i.p.	o/6	100	---

*Results are expressed as Mean ± SEM; (n=6). Significance at P<0.05*, P<0.01** as compared to control.

Table 2: Effect of ethanol extract of the seeds of H. antidysenterica (EEHA) on Pentylenetetrazol (PTZ) - induced seizures in mice.

Treatment	Dose	No of animal convulses/no of animal used	% protection	Latency of tonic convulsion (min) Mean ± SEM
Control	---	6/6	00	5.83 ± 0.40
Low Dose of EEHA	250 mg/kg, p.o.	4/6	33	11.50** ± 0.47
High Dose of EEHA	500 mg/kg, p.o.	2/6	66	14.23** ± 0.21
Standard (diazepam)	2.0 mg/kg, .i.p.	o/6	100	---

*Results are expressed as Mean ± SEM; (n=6). Significance at P<0.05*, P<0.01** as compared to control.

EEHA at doses of 250 and 500 mg/kg, respectively, protected 17% and 50% of mice and significantly reduced the duration of the seizures. However, phenytoin completely inhibited the MES-induced tonic seizures in all the animals used. (Table 1). Pentylenetetrazol produced tonic seizures in all the animals used. EEHA, in doses of 250 and 500 mg/kg, respectively, protected 33% and 66% of mice against seizures, and significantly delayed the latency of the seizures. The standard antiepileptic drugs, diazepam completely protected the animals from seizures. (Table 2). Bicuculline induced tonic seizures in all the mice used. However, all the doses of EEHA (250 and 500 mg/kg) did not alter the incidence of seizures significantly. The standard antiepileptic drugs diazepam completely protected the animals from seizures.

4. DISCUSSION:

The results of the present study indicate that ethanol extract of H. antidysenterica (EEHA) possesses anticonvulsant activity in mice. GABA is the major inhibitory neurotransmitter in the brain while glutamic acid is an excitatory neurotransmitter in the brain. The inhibition of GABA neurotransmitter and the enhancement of the action of glutamic acid have been shown to be the underlying factors in epilepsy. The present study shows that ethanol extract of the seeds of H. antidysenterica protected some of the animals against seizures induced by maximal electroshock. Antiepileptic drugs that inhibit voltage-dependent Na⁺ channels, such as phenytoin can prevent MES-induced tonic extension^{14, 15}. Pentylenetetrazole induced seizures in all the mice used. Pentylenetetrazole may elicit seizures by inhibiting gabaergic mechanisms. In this study, diazepam shown to antagonize the effect of Pentylenetetrazole while the extract was also shown to delay the latency of Pentylenetetrazole –induced seizures, suggesting that the extract may be affecting gabaergic mechanisms, probably by opening the chloride channels associated with GABA receptors.

Thus, it concludes that H. antidysenterica possesses anticonvulsant property against the MES and PTZ induced seizures. Further research is needed to isolate the specific compound responsible for the anticonvulsant activity.

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13. Amabeoku GJ, Chikuni O. Cimetidine–inducedseizures in mice. Biochem Pharmacol, 1993; 46(12): 2171-75.

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Received on 23.11.2010

Accepted on 25.12.2010

Research J. Pharmacology and Pharmacodynamics. 3(1): Jan. –Feb. 2011, 33-33