Anticonvulsant Activity of Leaf Extracts of Anacardium occidentale. Linn.

Syed Safiullah Ghori¹*, Md. Shamim Qureshi¹, Mohammed Safwan Ali Khan², Asma Khanam¹, and Jitendra Patel³

¹Anwarul-Uloom College of Pharmacy, New Mallepally, Hyderabad – 500001, Andhra Pradesh, India.

²Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia – Serdang 43400, Selangor Darul Ehsan, Malaysia.

³Bharat Institute of Technology, Ibrahimpatnam, RR. Dist., Hyderabad - 500001. Andhra Pradesh, India.

ABSTRACT:

The aim of the present study was to investigate the anticonvulsant effect of methanolic and aqueous extracts of the leaves of Anacardium occidentale Linn. on electrically and chemically induced seizures. The methanolic and aqueous extracts of the leaves of A. occidentale ( 250 and 500 mg/kg.b.w. p.o.) were studied for the anticonvulsant effect on Maximum electroshock (MES) and Pentylene tetrazole (PTZ) induced convulsions in mice. The latency of onset of tonic-clonic convulsions and the number of animals protected from tonic convulsions were noted. AOMEE and AOAQE (500 mg/kg.b.w.p.o) significantly reduced the onset and duration of seizures induced by maximal electroshock (MES). The same dose also protected animals from pentylene tetrazole-induced tonic seizures and significantly delayed the onset of tonic seizures.

KEYWORDS: Anacardium occidentale, Anticonvulsant Activity, MES and PTZ induced convulsions.

INTRODUCTION:

Epilepsy is a major neurological disorder and up to 5% of the world population develops epilepsy in their lifetime.¹It is a disorder of brain characterized by unpredictable and periodic occurrence of a transient alteration of behavior due to the disordered, synchronous and rhythmic firing of populations of brain neurons (McNamara, 2001).The current therapy of epilepsy with modern antiepileptic drugs is associated with dose related side effects and chronic toxicity, as well as teratogenic effects and approximately 30% of the patients continue to have seizures with current antiepileptic drug therapy^2-4. It has been observed that the presently available antiepileptic drugs are unable to control seizures effectively in as many as 25% of the patients. (Mattson, 1992).

Currently available anti-epileptic drugs (AEDs) do not provide cures nor prevent relapse and they are often associated with serious side effects, including teratogenicity, chronic toxicity and adverse effects on cognition and behaviour (Samren et al., 1997). As majority of antiepileptic drugs are consumed life long, concomitant administration of other drugs predisposes to the risk of drug interactions. Consequently, many people living in developing countries still rely on herbal medicine for management of epilepsy. The number of plant based crude drugs finding regular use in the management of epilepsy is put around 400 and the number is increasing.

Anacardium occidentale Linn. (Anacardiaceae), commonly known as cashew , a plant used in South Cameroon, Africa as well as in other countries like Brazil, Malaysia, Mexico, Turkey, Venezuela and India as a folk remedy for diabetes mellitus, malaria, stomatitis, toothache, diarrhoea, dysentery, leprosy, asthma, psoriasis, mouth ulcers, wounds, fever, intestinal colic, urinary disorders, as a gargle and mouthwash.^5,6. The present work was undertaken to evaluate the anti-convulsant activity of leaves of Anacardium occidentale Linn. by Maximum electroshock and Pentylene tetrazole-induced convulsions in mice.

MATERIAL AND METHODS:

Plant material:

The leaves of Anacardium occidentale Linn. were collected from field areas of Chevella region of Ranga Reddy district, A.P and taxonomically identified and authenticated by Prof. S. Seeta Ram Rao, Head of the Dept. of Botany, Osmania University, Hyderabad, A.P as Anacardium occidentale Linn. (Anacardiaceae) and a voucher specimen bearing No. (0170) was deposited at the herbarium of the university for future reference.

Preparation of the extracts:

The collected leaves were cleaned, shade dried for 10-12 days and powdered and sieved. 150 gm. of the leaf powder was subjected to successive soxhlet extraction using solvents methanol and water. The extracts were filtered and the filtrates were concentrated under reduced pressure using rotary evaporator to obtain a greenish–black coloured sticky residue.

Preliminary Phytochemical screening:^{7, 8}

The methanolic and aqueous extracts of Anacardium occidentale leaves were subjected to various phytochemical tests to screen for the presence of alkaloids, glycosides, tannins, flavonoids, terpenoids, sterols, saponins, mucilage and gum.

Drugs and chemicals:

Standard drugs like Phenytoin, Diazepam and other chemicals were purchased from S.D., .Fine chemicals Ltd. Mumbai. All the solvents used for the study were pure and of analytical grade.

Experimental Animals:

Albino mice weighing between 25-30 g. were used to study the effect of the standard drugs and test extracts (AOMEE-250 and 500 mg/kg.b.w and AOAQE-250 and 500 mg/kg.b.w) on MES and PTZ induced seizures . The animals were procured from the animal house, National Institute of Nutrition, Tarnaka, Hyderabad (A.P). and were housed in polypropylene cages with paddy husk as bedding at a temperature of 24 ± 2^oC and relative humidity of 30-70% under 12/12 light: dark cycle. All animals were allowed for free access to water and fed with standard commercial pelleted mice chaw (M/s. Hindustan Lever Ltd, Mumbai). All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics Committee (IAEC) and were in accordance with the guidelines of the IAEC.

Acute toxicity study:

Acute toxicity studies were conducted by using albino mice of either sex weighing 25-30 g. The animals were fasted overnight prior to the experiment. LD₅₀ determination was conducted using the method of Lorke (1983). Six groups of five animals each were treated with the (AOMEE) and (AOAQE) respectively at doses of 100, 500 and 1000, 2000 mg/kg b.w.p.o. and percent mortality was observed for 24 hours later. The LD₅₀ value was determined by calculating the geometric mean of the lowest dose that caused death and the highest dose for which the animal survived (0/1and 1/1).

Maximum electroshock-induced convulsion:

In the electrically-induced seizure experiment, the maximal electroshock (MES) method described previously by Swinyard was employed. Corneal electrodes were used for bilateral delivery of electrical stimulus (MES-50 mA; 50Hz; 0.2 Sec) convulsive shock including Hind Limb Tonic Extension (HLTE) in 99% of the animals, was previously determined. The electrical stimulus was applied using a stimulator apparatus (Biocraft Scientific Systems Pvt. Ltd., Agra, India) for eight groups of five mice each. In which one control (Group-I), treated with 0.9% w/v Normal saline solution (10 ml/Kg.b.w.p.o), one standard group(Group-II),treated with phenytoin as positive control (30 mg/Kg.b.w. i. p.) and four test groups (Group III,IV,and V, VI) , treated each with 250 and 500 mg/kg.b.w. p. o. of (AOMEE) and (AOAQE) respectively were used.The time of peak effect of phenytoin as 30 min after administration was previously established. The time for the extracts to reach its maximum effect was determined as 60 min after oral administration..The time for the onset of seizures, recovery time and % protection and there after % mortality was recorded. The orientation for the anticonvulsant effect was abolition of HLTE with in 10 sec. after delivery of the electroshock.⁹

Pentylene tetrazole-induced convulsions in mice :

The method of Swinyard et al., (1989) was employed. In each type of seizure model, the mice were divided into six groups with six animals. Group I served as solvent control, received 0.9% w/v of (10 mL/kg.b.w),treated as negative control Group II received standard drug diazepam (20 mg/kg.b.w), treated as positive control and Group III, IV and V, VI received test extracts (AOMEE and AOAQE each 250 and 500 mg/kg b.w.p.o) respectively. Thirty minutes later, mice in all the groups received 80 mg/kg.b.w. s.c dose of Pentylene tetrazole .The animals were observed for 1 hr. by placing in a separate cage to detect the onset of spasm and generalized tonic clonic seizures and further up to 2hrs. to detect mortality, if any animals devoid of seizures were considered as protected. The time of onset of seizures (tonic-clonic convulsions) and time taken for death were recorded (Yemitan and Salahdeen, 2005; Salahdeen and Yemiten,2006)

Table 1: Effect of Methanolic and Aqueous extracts of Anacardium occidentale leaves in Maximal Electro Shock (MES) – induced seizures in mice.

Group No.	Treatment	Dose (ml;mg/kg.b.w)	Mean onset of seizures (sec.)	Mean time of recovery (min.)	Quantal protection	Protection (%)	Mortality (%)
I	Normal saline	10	6.25 ± 1.00	5.85±1.5	0/6	0.0	0.00
II	Phenytoin	30	0.00	0.00	6/6	100.0	0.00
III	AOMEE	250	4.25±0.50	7.90±1.6	4/6	66.0	0.00
IV	AOMEE	500	9.50±0.45*	4.70±1.2*	5/6	83.0	0.00
V	AOAQE	250	3.52±0.50	8.30 ±3.2	2/6	33.0	0.00
VI	AOAQE	500	4.54±0.35*	2.50±2. 6*	3/6	50.0	0.00

Data presented as Mean±SEM: Values are *Significant at P < 0.01; n = 6, mice were pretreated with control and AOMEE and AOAQE before the induction of electroconvulsive shock. AOMEE: Anacardium occidentale Methanolic Extract, AOAQE: Anacardium occidentale Aqueous Extract.

Table 2: Effect of Methanolic and Aqueous extracts of Anacardium occidentale leaves in Pentylenetetrazole (PTZ) -induced convulsions in mice.

Group No.	Treatment	Dose (ml;mg/kg.b.w)	Mean onset of seizures (sec.)	Mean time of death (sec.)	Quantal protection	Protection (%)	Mortality (%)
I	2 % Acacia solution	10	385.5±0.50	8.87±5.65	0/6	0.0	100.0
II	Diazepam	20	0.00	0.00	6/6	100.0	0.0
III	AOMEE	250	235.50±0.52	6.54±5.50	4/6	66.0	40.0
IV	AOMEE	500	198.5±0.50*	4.63±3.7^*	5/6	83.0	20.0
V	AOAQE	250	338.50±0.50	5.87±4.29	2/6	33.0	77.0
VI	AOAQE	500	226.5±0.60*	3.79±4.5*	3/6	50.0	50.0

Data presented as Mean±SEM: Values are *Significant at P<0.01; n = 6, mice were pretreated with control and AOMEE and AOAQE before the administration of PTZ. AOMEE: Anacardium occidentale Methanolic Extract, AOAQE: Anacardium occidentale Aqueous Extract.

Statistical analysis:

Assessment of response of anticonvulsant activity were done using graph pad software Inc. 11452EI Camino Real, 215 San Digeo 92130 USA. (Dunnett-t-test followed by one way ANOVA) considering significant variation account column means. P value of less than 0.01 was the critical criterion for statistical significance.

RESULTS AND DISCUSSION:

The phytochemical screening of the methanolic and aqueous extracts of A. occidentale revealed the presence of alkaloids, carbohydrates, gums, flavonoids, saponin glycosides and tannins. The extract (250 and 500 mg/kg) protected the rats against MES convulsions with highest percentage of protection observed at dose of 500 mg/kg (Table 1). The extract also significantly reduced the mean recovery time of convulsed animals. Phenytoin (20 mg/kg), the standard anticonvulsant used produced 100% inhibition of hind limb tonic extension (HLTE) of maximal electroshock test (MEST).

The methanolic and aqueous extracts of Anacardium occidentale Linn. significantly (P < 0.01) delayed the onset of seizure induced by PTZ exhibited protection of the animals against threshold seizure (Table 2). Diazepam (20 mg/kg), the standard anticonvulsant used protected the animals against threshold seizures.

Phenytoin and diazepam have been shown to exert their antiepileptic effects by enhancing the GABA-mediated inhibition in the brain (Porter and Meldrum, 2001). It is possible that diazepam and test extracts antagonize pentylene tetrazole convulsions in this study by enhancing GABA neurotransmission. Since the test extracts (AOMEE) and (AOAQE) delayed the occurrence of pentylene tetrazole-induced convulsion, it is probable that it may be interfering with GABA aminergic mechanism(s) to exert its anticonvulsant effect.

CONCLUSION:

As the study shows that methanolic and aqueous extracts of the leaves of A. occidentale protected some of the animals against seizures induced electrically by maximal electroshock method, and chemically by Pentylenetetrazole method, and from the results of the present study which indicate that methanolic and aqueous extracts (500 mg/kg) of the leaves of A. occidentale leaves possesses significant anticonvulsant activity in mice and the percent protection was found to be 83% and 50% respectively compared to that of the standard drugs Phenytoin (30mg/kg) and Diazepam(20mg/kg) which showed 100% protection in animals against MES and PTZ induced seizures . Phenytoin and diazepam have been shown to exert their antiepileptic effects by enhancing the GABA-mediated inhibition in the brain (Porter and Meldrum, 2001). Hence from the above study it was concluded that, the leaf extracts (AOMEE and AOAQE) contain predominantly flavonoids which are known to exhibit anticonvulsant activity and the probable mode of anticonvulsant action may be due to GABA minergic mediation, glycine inhibitory mechanism and inhibition of the electrical kindling effect. Further research studies can be carried out after isolation and characterisation of the flavonoid fraction in order to know the exact mechanism of anticonvulsant effect.

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Received on 10.01.2011

Accepted on 17.01.2011

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Research J. Pharmacology and Pharmacodynamics. 3(2): March –April, 2011, 72-76