ANTICONVULSANT AND BEHAVIOURAL ACTIONS OF MYRISTICA FRAGRANS SEEDS

Indian Journal of Pharmacology 2002; 34: 332-338 RESEARCH PAPER ANTICONVULSANT AND BEHAVIOURAL ACTION OF M. FRAGRANS ANTICONVULSANT AND BEHAVIOURAL ACTIONS OF MYRISTICA FRAGRANS SEEDS G.S. SONAVANE, R.C. PALEKAR, V.S. KASTURE, S.B. KASTURE* Natural Products Laboratory, Division of Pharmacology, N.D.M.V.P. Samaj's College of Pharmacy, Nashik - 422 002. Manuscript Received: 30.5.2001 Revised: 12.3.2002 Accepted: 24.3.2002 ABSTRACT Objective: To investigate anticonvulsant, cataleptic and sedative properties of n-hexane fraction of acetone insoluble part of petroleum ether extract of Myristica fragrans (MF) seeds. Methods: The anticonvulsant activity of MF (10, 30, 100 mg/kg i.p.) was studied against seizures induced by maximum electroshock (MES), pentylenetetrazol (PTZ), picrotoxin, and lithium sulphate-pilocarpine nitrate (Li-Pilo). The effect on gross behaviour, motor coordination, haloperidol-induced catalepsy (using Bar test) and pentobarbitone-induced sleep was studied. Results: MF inhibited seizures induced by MES, PTZ, and Li-Pilo. However, picrotoxin-induced seizures were not inhibited. The haloperidol-induced catalepsy was potentiated but motor coordination and pentobarbitone-induced sleep were not affected significantly. Conclusion: MF has complex actions on the central nervous system. Although it exhibited anticonvulsant activity against MES, PTZ and lithium-pilocarpine, it failed to inhibit picrotoxin-induced seizures. MF reduced central dopaminergic activity but was without any effect on pentobarbitone-induced sleep. KEY WORDS Antiepileptic central nervous system effects MES INTRODUCTION Seizure is a characteristic feature in epilepsy and is associated with disordered and rhythmic high frequency discharge of impulses by a group of neurons in the brain and status epilepticus is characterized by repeated episodes of epilepsy without the patient having recovered from the previous attack. Although several drugs are available to treat epilepsy, the treatment of epilepsy is still far from adequate 1. Many attempts have been made in the past to obtain anticonvulsant from plant origin and these efforts will continue till a satisfactory treatment becomes available. During the neuropharmacological screening of the petroleum ether extract of seeds of Myristica fragrans (family: Myristicaceae, commonly known as nutmeg) the n-hexane fraction (MF) of acetone insoluble part of petroleum ether extract exhibited depressant and anxiogenic effect 2. We therefore assessed the spectrum of anticonvulsant activity of MF against seizures induced by MES, pentylenetetrazol (PTZ), picrotoxin and status epilepticus induced by lithium sulphate plus pilocarpine nitrate (Li-Pilo). Since many antiepileptic agents induce drowsiness, motor incoordination and ataxia 1, we also studied the effect of MF on gross behaviour, neuroleptic-induced catalepsy, motor coordination and pentobarbitoneinduced sleep. MATERIALS AND METHODS Animals: Male albino mice weighing 22-25 g and male albino rats weighing 125-150 g were obtained from National Institute of Toxicology, Pune. Animals were housed into groups of 6-8 per cage at a Correspondence: Sanjay B. Kasture e-mail: kasture_sb@hotmail.com

333 G.S. SONAVANE et al. temperature of 25 O ±1 O C and relative humidity of 45-55%. A 12:12 dark:light cycle was followed during the experiments and the experiments were carried out during 12.00-14.00 h. Animals had free access to food and water, however, food but not water was withdrawn 8 h before and during the experiments. The Institutional Animal Ethical Committee approved the protocol of this study. Preparation of extract and isolation of active fraction: The Myristica fragrans (nutmeg) seeds were purchased from commercial source. The seeds (1.0 kg) were crushed to a coarse powder and extracted with petroleum ether (60-80 O C) using Soxhlet's extractor. The extract was concentrated under reduced pressure and then dried in air (yield 470 g). The extract was then partitioned into acetone soluble (70.5 g) and acetone insoluble part (399 g). The acetone insoluble part was charged into the chromatography column of neutral alumina and the column was eluted with n-hexane. The n-hexane fraction (MF) was concentrated under reduced pressure and suspended in PEG 400 for administration. The volume of injection was kept at 0.1 ml per animal. Drugs and chemicals: Pentylenetetrazol, pentobarbitone (Sigma, USA), lithium sulphate (Glenmark Pharmaceuticals, India), Pilocarpine nitrate (FDC Limited, India), diazepam (Calmpose inj. Ranbaxy, India), were used in this study. The drugs were dissolved in water for injection and administered in a volume of 5 ml/kg. n-hexane, petroleum ether and acetone were obtained from Modern Scientific Co. Nashik, India. Acute toxicity: The MF was administered in doses of 10, 30, 100, 300, 1000, and 3000 mg/kg, i.p. to groups of mice, each consisting of 10 mice and percent mortality was observed after 24 h. Behavioural assessment: The central effects of MF were assessed using Irwin schedule as described by Turner 3. Briefly mice divided into groups of ten were administered vehicle or MF (10, 30, 100, 300 mg/kg) intraperitoneally. The procedure involved an initial phase of undisturbed observation and later a manipulative phase during which animals were subjected to the least provoking stimuli. In the initial phase, animals were observed for body position, locomotion, rearing, respiration, tremors, gait and in later phase, the effect on grip strength, passivity, pain response, righting reflex, lacrimation were observed. Observers were unaware of treatments. The effects were recorded using a scoring system suggested by Turner 3. Motor co-ordination: The effect of MF on motor co-ordination was studied using rota-rod as described by Dunham and Miya 4. In brief, mice divided into groups of five were trained to remain on the rod rotating at the speed of 25 rev/min. On the next day mice received MF (10, 30 and 100 mg/kg, i.p.) and the fall-off time of each animal from the rotating rod was tested after 30 min. Assessment of anticonvulsant activity Maximum electroshock-induced seizures: The MF was administered to groups of mice (n = 6) in varying doses (10, 30 and 100 mg/kg, i.p.) 30 min before application of electric shock (45 ma, 0.2 sec) using corneal electrodes 5. The duration of tonic extension of hindlimbs was noted. Pentylenetetrazol-induced seizures: Each group contained six mice. The MF was administered i.p. in varying doses (10, 30 and 100 mg/kg) 30 min before the subcutaneous injection of PTZ (80 mg/kg) and the animals were observed for onset of myoclonic spasm and clonic convulsions as described earlier 5. One group received vehicle while other group received diazepam (2.0 mg/kg, i.p.) as a reference standard. The animals were observed for onset of convulsion upto 30 min after PTZ. Picrotoxin-induced seizures: Albino mice, divided into two groups each containing five animals, were pretreated either with vehicle or MF (10 mg/kg, i.p.) 30 min before picrotoxin (3.0 mg/kg, i.p.) and the latency to clonic convulsions was noted as described earlier by Leewanich et al 6. Lithium-pilocarpine-induced status epilepticus: Albino rats were divided randomly into groups of six. Status epilepticus was induced by administration of pilocarpine (30 mg/kg, i.p.) 24 h after lithium sulphate (3 meq/kg, i.p.). The effect of MF (10, 30 and 100 mg/kg) was studied on the status epilepticus by administering MF 30 min before injection of pilocarpine nitrate. One group received only vehicle while other group received diazepam (1.0 mg/kg). The severity

ANTICONVULSANT AND BEHAVIOURAL ACTION OF M. FRAGRANS 334 of status epilepticus was observed every 15 min till 90 min and then every 30 min for next 90 min using the scoring system as described by Patel et al. 7 : no response-stage 0, fictive scratching-stage 1, tremors-stage 2, head nodding-3, forelimb clonus-stage 4, rearing and falling back-stage 5. Haloperidol-induced catalepsy: The groups of mice each consisting of 5 animals were treated with MF or vehicle 30 min before haloperidol (1.0 mg/kg, i.p.). The animals were observed for severity of catalepsy using 'Bar test' as described by Silva et al. 8 at 0, 30, 60 and 90 min after haloperidol. The phenomenon was measured as the time the mouse maintained an imposed position with both forepaws extended on a wooden bar (diameter, 0.9 cm) placed horizontally at a height of 3 cm. The test was performed twice and higher value was recorded. Pentobarbitone-induced sleep: Mice were pretreated intraperitoneally with either vehicle or MF (10,30,100 mg/kg) 30 min before pentobarbitone (40 mg/kg). The sleeping time was measured as the period that mice lost the righting reflex after receiving the hypnotic drug 3. Statistical analysis: The data were analyzed using one-way ANOVA followed by Dunnett's test. The nonpara-metric data was analyzed by Kruskal- Wallis ANOVA followed by Dunn's test. Differences were considered significant at the 5% level. RESULTS Acute toxicity and behavioral assessment: No mortality was observed in the animals treated with the n-hexane fraction of acetone insoluble part of petroleum ether extract of Myristica fragrans (MF). However, mice exhibited signs of depression of the central nervous system in a dose dependent manner. The behavioural assessment was carried out for 2 h after which the intensity of effect started waning. MF diminished alertness, grooming, reactivity, spontaneous motor activity, touch response, pain response and increased passivity and respiration. MF induced head twitches (Table 1). The same volume of PEG 400 was without any effect on the base line score of the parameters studied. Motor co-ordination: Animals treated with MF (10 mg/kg) remained on the rotating rod for 5 min whereas animals treated with higher doses (30 and 100 mg/kg) showed early fall from the rod but the effect could not reach significance (P>0.05). Assessment of anticonvulsant activity Maximum electroshock test: The duration of tonic hindlimbs extension in mice treated with vehicle was 15.2±1.3 sec. MF in doses of 10-100 mg/kg significantly reduced the duration of hindleg extension (P<0.05). MF was most effective in a dose of 10 mg/kg and the diminished response was noted with higher doses. The incidence of convulsions was 50% in the group that received MF in a dose of 10 mg/kg while 100% animals exhibited convulsions with 30 and 100 mg/kg dose. Mortality was not observed in any groups treated with MF. The observations are given in Table 2. PTZ-induced seizures: In animals treated with vehicle myoclonic jerks were observed 110.0±14.8 sec after PTZ and convulsions appeared 122.3±13.3 sec after PTZ. All animals died after seizures. MF in a dose of 10 mg/kg delayed myoclonic spasm unto 1020.0±350.5 sec and convulsions were completely inhibited. With higher doses (30 and 100 mg/kg) the anticonvulsant activity diminished and the onset of spasm was 516.0 ± 83.25 sec and 324.0 ± 44.8 sec respectively and the onset of convulsions was 804.0±92.0 sec and 1075.0±325.8 sec respectively. The incidence of convulsions was 100% in mice receiving 30 mg/kg and 50% in mice receiving 100 mg/ kg of MF. Diazepam (2 mg/kg) inhibited spasms as well as seizures completely (Table 2). Picrotoxin-induced seizures: In mice pretreated with vehicle, clonic convulsions were observed 11.9±1.9 min after picrotoxin. Whereas, mice pretreated with MF (10 mg/kg) exhibited convulsions after 13.6±1.6 min after picrotoxin (P>0.05, Student's ' t ' test). Lithium-pilocarpine - induced status epilepticus: In vehicle treated rats the intensity of status epilepticus increased gradually and peak effect was observed 120 min after pilocarpine. Pretreatment with MF in doses 10, 30, and 100 mg/kg significantly inhibited the progression and severity of status epilepticus. The dose of 30 mg/kg, i.p. was less effective than the other doses (10 and 100 mg/kg) in antagonizing the effect of lithium with pilocarpine. The stage of forelimb clonus (Stage 4) was not observed in rats treated with MF. The observations are given in Table 3.

335 G.S. SONAVANE et al. Table 1. Effect of n-hexane fraction of acetone insoluble part of petroleum ether extract of Myristica fragrans (MF) on gross behavior in mice. Behaviour Normal MF (mg/kg i.p.) score 10 30 100 300 H P Alertness 4 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 Visual placing 4 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 Passivity 0 0.0 ± 0.0 0.0 ± 0.0 2.0 ± 0.0* 2.0 ± 0.0* 39.0 0.0001 Stereotypy 0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Grooming 4 3.0 ± 0.0 2.7 ± 0.5 3.0 ± 0.0 1.8 ± 0.4* 21.3 0.0001 Restlessness 0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Reactivity 4 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0 2.5 ± 0.8 Spontaneous Activity 4 3.0 ± 0.0 3.0 ± 0.0 3.0 ± 0.0 2.5 ± 0.8 Touch response 4 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 Pain response 4 3.0 ± 0.0 3.0 ± 0.0 2.5 ± 0.2 1.7 ± 0.2* 23.3 0.0001 Tremors 0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Twitches 0 0.8 ± 0.4 1.4 ± 0.6 2.0 ± 0.0 3.0 ± 0.0* 23.7 0.0001 Body posture 4 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 Muscle tone 4 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 Reflexes 4 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 4.0 ± 0.0 Palpebral Opening 4 3.4 ± 0.4 3.0 ± 0.0 3.0 ± 0.0 2.2 ± 0.7* 8.49 0.04 n = 10, All values are mean±sem. The scores indicate the peak effect. The same volume of PEG 400 showed no effect on the base line scores of the behavior studied. The degree of freedom for H was 3. *P<0.05 compared to the base line score (Kruskal-Wallis test followed by Dunn's test). Table 2. Effect of n-hexane fraction of acetone insoluble part of petroleum ether extract of Myristica fragrans (MF) on seizures induced by maximum electroshock and pentylenetetrazol in mice. Treatment Duration of tonic Onset of myoclonic Onset of clonic (mg/kg, i.p.) hindlimb extension spasm in sec after convulsion in in sec (MES test) PTZ sec after PTZ Vehicle 15.2 ± 1.3 (100) 110.0 ± 14.8 122.3 ± 13.3 MF (10) 2.5 ± 1.2* (50) 1020.0 ± 350.5* -- MF(30) 9.0 ± 0.4* (100) 516.0 ± 83.25* 804.0 ± 92.0* MF(100) 8.0 ± 1.0* (100) 324.0 ± 44.8* 1075.0 ± 325.8* F 25.26 4.59 6.30 d.f. 3, 20 3, 20 2, 15 P 0.001 0.013 0.01 n = 6, Values are mean±sem. Figures in the parentheses indicate incidence of convulsions. The data were analyzed by one-way ANOVA followed by Dunnett's test. *P<0.05, compared to the vehicle treated group.

ANTICONVULSANT AND BEHAVIOURAL ACTION OF M. FRAGRANS 336 Table 3. Effect of n-hexane fraction of acetone insoluble part of petroleum ether extract of Myristica fragrans (MF) on lithiumpilocarpine-induced status epilepticus in rats. Time after MF in mg/kg i.p. pilocarpine Vehicle in min. 10 30 100 H P 15 1.5 ± 0.4 0.0 ± 0.0* 1.2 ± 0.3 1.0 ± 0.0 18.2 0.0001 30 2.7 ± 0.2 0.5 ± 0.3* 0.5 ± 0.3* 0.3 ± 0.2* 13.9 0.004 45 3.0 ± 0.4 1.0 ± 0.4* 1.2 ± 0.2* 0.5 ± 0.3* 15.4 0.002 60 3.8 ± 0.4 0.3 ± 0.2* 2.0 ± 0.7 0.3 ± 0.2* 17.5 0.0001 75 4.0 ± 0.4 0.7 ± 0.3* 1.7 ± 0.8* 1.0 ± 0.7* 13.6 0.004 90 4.3 ± 0.3 0.3 ± 0.2* 1.0 ± 0.4* 0.3 ± 0.2* 15.6 0.002 120 4.8 ± 0.2 0.0 ± 0.0* 1.2 ± 0.6* 1.0 ± 0.0 19.3 0.0001 150 2.0 ± 0.4 0.0 ± 0.0* 1.0 ± 0.4 0.7 ± 0.3* 12.5 0.007 180 1.0 ± 0.2 0.0 ± 0.0 1.0 ± 0.5 0.7 ± 0.3 7.79 0.06 (NS) n = 6, *P<0.05 compared to the vehicle treated group (Kruskal-Wallis test followed by Dunn's test). Values are mean+sem of scores representing severity of status epilepticus (see text). Table 4. Effect of n-hexane fraction of acetone insoluble part of petroleum ether extract of Myristica fragrans (MF) on haloperidolinduced catalepsy in mice. Treatment (mg/kg) Duration of catalepsy in sec (mean ± SEM) at 30 min 60 min 90 min 120 min Vehicle 78.0± 12.0 108.0 ± 18.0 113.4 ± 18.0 112.2 ± 15.0 MF (10) 180.0± 0.0* 180.0 ± 0.0* 180.0 ± 0.0* 134.1 ± 2.4 MF (30) 180.0± 0.0* 180.0 ± 0.0* 168.0 ± 6.0* 127.8 ± 3.0 MF (100) 180.0± 0.0* 180.0 ± 0.0* 180.0 ± 0.0* 150.0 ± 24 F 72.25 16 11.24 1.2 d.f. 3,16 3,16 3,16 3,16 P 0.0001 0.0001 0.0001 0.34 n = 5. *P<0.05 compared to vehicle treated group (one-way ANOVA followed by Dunnett's test). Haloperidol-induced catalepsy: Haloperidol gradually induced catalepsy in mice. The MF increased the severity of haloperidol-induced catalepsy in a dose dependent manner (P<0.05). The peak effect was observed at 90min and decreased after 120 min. MF did not induce catalepsy when used alone. The observations are given in Table 4. Pentobarbitone-induced sleep: In vehicle treated mice sleep was induced 313.0±16.6 sec after pentobarbitone and mice slept for 86.2±11.7 min. MF (10 mg/kg) significantly increased the onset of sleep to 459.6± 39.76 sec. Other doses of MF were without any significant effect on latency to sleep. MF alone was without any effect on the duration of sleep.

337 G.S. SONAVANE et al. DISCUSSION The observations emanated in the present study indicated that MF possesses anticonvulsant activity against PTZ, MES and lithium-pilocarpine induced seizures and lower doses were more effective in inhibiting seizures. The MF was without any significant effect on picrotoxin-induced convulsions and motor coordination but potentiated haloperidolinduced catalepsy significantly. MF indicated signs of both CNS depression as well as stimulation. It diminished alertness, grooming, reactivity and spontaneous motor activity, touch response, pain response and increased passivity and respiration and it induced head twitches also. Motor co-ordination was not impaired to a significant extent. In various animal models of seizures used in this study i.e. MES, PTZ and status epilepticus, the anticonvulsant activity of MF decreased with increasing doses. In status epilepticus, the animals receiving MF in a dose of 10 mg/kg reduced the severity of seizures at much earlier time. These observations support the biphasic effect of MF on the central nervous system. MF was without any effect on the duration of pentobarbitone-induced sleep. Though the MES test predicts activity against generalized tonic-clonic and cortical focal seizures and the PTZ test against absence seizures, the underlying neuronal abnormality is poorly understood. Diminution of brain GABA level has been reported after subconvulsive dose of PTZ 9. Picrotoxin, the antagonist of GABA at the postsynaptic receptors, induced seizures in all the animals and its effect was not antagonized even at the dose of 100 mg/kg suggesting that GABA may not be involved in the anticonvulsant activity of MF. This observation is also supported by the finding that pentobarbitone -induced sleep is not increased by MF. Although lithium does not have general proconvulsant action in rats, its pretreatment provokes limbic seizures following administration of sub-convulsant doses of pilocarpine and other cholinergic agonist 10. The combined treatment with lithium and pilocarpine results in an accumulation of inositol monophosphate and reduction in cortical inositol that are about ten times greater than the effects obtained with either drugs alone 10,11. Neuroleptic-induced catalepsy remains a useful method to study central dopamine function. The neuroleptic-induced catalepsy produced by blockade of striatal D 1 and D 2 receptors 12,13 can be modified by cholinergic, histaminergic, and serotonergic manipulation 8,4,15. All these neurotransmitters also modulate epilepsy 1. D 1 receptors in the substantia nigra mediate proconvulsant action of dopamine 16. It has been shown that PTZ kindling enhances the basal activity and the sensitivity to PTZ of dopaminergic neurons in rat brain and nigrostriatal dopaminergic neurons contribute to the central alterations associated with experimental epilepsy 17. The blockade of D 1 and D 2 receptors by haloperidol inhibited (-) bicuculline-induced seizures 18. Thus the blockade of DA receptor may have some protective effect in epilepsy. Dopamine receptor antagonists like chlorpromazine are known to prolong pentobarbitone-induced sleep 3. Lack of potentiation of pentobarbitoneinduced sleep by MF suggests that MF does not directly involve dopaminergic mechanism in the potentiating haloperidol-induced catalepsy. A reciprocal relationship between 5-HT and DA has been reported earlier by Kuczenski and Segal 19. Further study is required to understand the mode of anticonvulsant action of MF. Thus, in conclusion, the n-hexane fraction of acetone insoluble part of petroleum ether extract of Myristica fragrans possesses anticonvulsant property against the animal models of Grand mal, Petit mal and status epilepticus. Decreased dopaminergic transmission may be partly responsible for its anticonvulsant effect. ACKNOWLEDGEMENT This work was supported in part by the All India Council for Technical Education, New Delhi, India. REFERENCES 1. McNamara JO. Drugs effective in the treatment of the epilepsies. In: Goodman and Gillman's The Pharmacological Basis of Therapeutics. 9 th edn. Hardman JG, Limbird JE, Molinoff PB, Ruddon RW, Gillman AG, eds. New York: McGraw Hill; 1996. p. 461-86. 2. Sonavane GS, Sarveiya VP, Kasture VS, Kasture SB. Anxiogenic activity of Myristica fragrans seeds. Pharmacol Biochem Behav 2002;71:247-52. 3. Turner RA. Screening procedures in Pharmacology. New York: Academic Press; 1972. p. 22-41.

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