Aim of the study: Epilepsy is a complex neurological disorder affecting 50 million of world's total population. Number of medicinal plants has been used to treat the convulsion. In ancient time morus alba was used in treatment of epilepsy and mental illness. The present study was designed to explore the effect of morusin, a flavonoid glycoside isolated from Morus alba as anticonvulsant activity along with biochemical mechanism.
Materials and methods: Morusin was isolated from Morus alba and acute toxicity study was determined. Anticonvulsant activity of Morusin (5 and 10 mg/kg, i.p.) was studied by using isoniazid (INH) and maximal electroshock (MES) induced convulsion models; diazepam (5 mg/kg) and phenytoin (20 mg/kg) were used as standards, respectively. Biochemical mechanism was investigated by estimating the GABA level in brain.
Results: The median lethal dose (LD50) of Morusin was found up to 20 mg/kg. . Treatment with morusin (5 and 10 mg/kg) delayed onset of convulsion and tonic hind limb extension along with duration of tonic-clonic convulsions as well as it signiï¬cantly reduced mortality in INH and MES induced convulsion. Rats treated with morusin (5 and 10 mg/kg) signiï¬cantly increased level of brain GABA at both doses.
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Conclusion: the ï¬ndings of present study provide pharmacological credibility to anticonvulsant activity of morusin. The protection against the convulsions and restoration of GABA level give a suggestion to its probable mechanism of action
Keywords: anticonvulsant activity, morusin, isoniazid, maximum electroshock, GABA
Incidence of epilepsy in developed countries is approximately 50 per 100,000 while that of developing countries is 100 per 100,000. The entire currently available antiepileptic drugs are synthetic molecules associated with side effects and approximately 30% of the patients continue to have seizures with this therapy . Thus, research for ï¬nding new drugs with less adverse effects and more efficacies, seems to be essential. As many of the herbal drugs have few adverse effects, assessment of herbal medications for their possible antiepileptic activity is worthwhile.
Mulberry, Morus alba L., as a non-toxic natural therapeutic agent, belongs with the family of Moraceae, and has been cultivated in many Asian countries such as China, India, Korea, Japan and Thailand where the leaves were used as food for silkworms , is a natural food additive having vitamins, carbohydrates, mineral, lipids, sugars, proteins, fibers, etc. in appropriate proportion . It is a potent antioxidant commonly used as a dietary supplement [4, 5]. Latest information shows that it can be used as a good pharmaceutical food [6, 7].
Number of active phytochemical constituents like alkaloids , flavonoids , glycosides [10, 11], terpenoids , steroids , volatile oils, tannins , etc has been reported from different parts of the plant. The plant has been reported to possess anti diabetic , hypolipidemic, antihypertensive , antimicrobial , antioxidant [18, 19], anti atherosclerotic , anticancer , neuroprotective , anxiolytic and antidepressant  etc. activities.
The anticonvulsant activity of Morusin, an isoprenylated flavone, isolated from Morus alba was evaluated against maximal electroshock (MES) induced and isoniazid (INH) induced convulsions in experimental animal models.
The animals were obtained from the animal house Siddhartha Institute of Pharmacy, Dehradun, India, maintained under standard conditions (12 h light/dark cycle; 25 ± 3 -C, 45-65% humidity), had free access to standard rat feed and water ad libitum and studies were performed as per CPCSEA, India.
The M. alba L. plant material was collected from local area of Dehradun, (U.K), India, in april 2011, were authenticated by Dr. Imran Kazmi, Department of Pharmacognosy, Siddhartha Institute of Pharmacy.
Extraction and isolation of Morusin
The air-dried stem bark of Morus alba (2 kg) was successively extracted with 70% ethanol. The extract was concentrated in vacuum to give a residue (451 g). 100 grams of the extract was fractionated over cellulose column chromatography eluted with 100% MeOH to give flavonoids. 25 grams of isolated fraction was subjected to silica gel column chromatography and eluted with a mixture of n-hexane and acetone, increasing the amount of acetone. Morusin was eluted on 3:2 fraction (2 g). Morusin was identified by the comparison with the authentic specimens .The structure of Morusin is depicted in Figure 1.
Diazepam (Calmpose Inj. Ranbaxy, India), Isoniazid (S.d.fine Chemicals) and Phenytoin (Zydus Neurosciences, India) were purchased. All other chemicals used were of analytical grade. Isoniazid was dissolved in normal saline and Morusin was suspended in 1% DMSO and used.
Acute toxicity study
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Acute toxicity study was performed in mice according to OECD guidelines. The isolated Morusin was administered intraperitoneally (i.p) at doses of 1.75, 5.5, 17.5, 55 mg/kg. They were then observed for signs of toxicity, continuously for 2 h, and for mortality up to 24 h, after injection .
INH induced convulsion model
Wistar albino rats (150-200 g) were divided into four groups (n = 6). Group I (vehicle control), received 1% DMSO, Group II (positive control), received Diazepam (5 mg/kg) Group III and IV received Morusin, 5.0 and 10.0 mg/kg. All animals were treated intraperitonially 30 min prior to administration of INH (300 mg/kg, p.o.).
Animals that did not convulse within 30 min were considered as protected and their results were expressed in terms of percentage. Each animal was observed for 60 min by placing in a separate cage for determination of percent protection. In unprotected animals, the latency to first convulsion and the durations of convulsions were recorded . Forty-five minute after vehicle or Morusin and 30 min after diazepam, rats were sacrificed. The animals were sacrificed as soon as onset of convulsions occurs or 65 min after INH treatment. Brain tissue was isolated and transferred to homogenization tube, containing 10 mL of 0.01 M hydrochloric acid. Homogenate was transferred in a bottle with 16 mL of ice cold absolute alcohol. The samples were subjected to centrifugation at 16,000 rpm for 10 min to obtain the precipitate. Precipitate was washed thrice with 10 mL of 90% alcohol. Washed liquids were combined with supernatant. The combination was transferred to petri plate for evaporation and dried at 70°C. 2 mL water and 4 mL of chloroform added to the dry mass and centrifuged at 2100 rpm. Upper phase containing GABA (4.0 mL) was separated and 20 µL of it was spotted on Whatman paper (No. 41). n-butanol (50 mL) acetic acid (12 mL) and water (60 mL) were selected as mobile phase. Ascending technique was adopted to develop the paper chromatogram. 0.5% ninhydrin solution in 95% ethanol was sprayed and it was dried for 1 h at 90°C. Blue color spot was developed, cut and heated with 2 mL ninhydrin solution on water bath for 5 min. Water (10.0 mL) was added to solution and kept for 1 h. Supernatant (4.0 mL) was decanted and absorbance was measured at 570 nm .
MES induced convulsion model
Maximal electroshock-induced convulsion model animals were divided as per the INH-induced seizure model. The positive control group animals were treated with phenytoin (20 mg/kg). After 30 min of treatments, the electroshock was induced in animals by passing the current of 150 mA for 0.2 second duration through auricular electrodes. The latency and incidence of tonic hind limb extension (THLE) and mortality rate was observed for 15 min.
The data were expressed as mean ± S.E.M. Statistical comparisons were performed by one-way ANOVA followed by Dunnett's-test using Graph Pad Prism version 5.0, USA. P < 0.05 was considered significant.
Intraperitoneal administrations of stepwise, escalated doses of isolated Morusin in mice gave an LD50 value as 20 mg/kg.
Orally administration of INH (300mg/kg) resulted in hind limb tonic-clonic convulsion along with lethality in rats. Pretreatment with morusin (5 and 10 mg/kg) signiï¬cantly and dose dependently (P < 0.01 and P < 0.001 respectively) delayed onset of convulsion, decrease duration of convulsion and reduce INH-induced mortality in rats as compared to control grouop rats. . It also signiï¬cantly reduced the total number of animals convulsed per group (P < 0.01 and P < 0.001 respectively) as compared to control group rats. When compared with control group rats diazepam (5mg/kg) treated rats showed signiï¬cant delayed (P < 0.001) onset of convulsion and it signiï¬cantly (P < 0.001) reduced the duration of convulsion. Mortality induced by INH was also signiï¬cantly attenuated (P < 0.001) by treatment with diazepam (5mg/kg) (Table 1).
In MES induced convulsion test, control group rats showed a distinctive seizure pattern. The tonic ï¬‚exion of the limbs occurred instantly after the shock, which then progressed into -THLE followed by either stupor and recovery or death. Rats treatment with morusin (5 and 10 mg/kg) signiï¬cantly reduced (P < 0.01 and P < 0.001) duration of MES induced THLE as compared to control group rats. When compared to control group rats, treatment with morusin (5 and 10 mg/kg) showed signiï¬cant protection (P < 0.01 and P < 0.001, respectively) against MES induced mortality. Morusin at a dose of 10 mg/kg showed complete protection against MES induced mortality as well as total number of rats convulsed per group. Treatment with phenytoin (20 mg/kg) showed signiï¬cant (P < 0.001) protection against MES induced mortality. It also significantly reduced (P < 0.001) duration of tonic extension of hind limbs in rats as compared to control group rats (Table 2).
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In INH induced convulsion, morusin dose dependently exerted a positive effect on GABA level, that is, the level of GABA was found to be significantly increased up to a dose of 10 mg/kg (Table 1).
Epilepsy is a general persistent brain disorder caused due to tumours, degenerative conditions or cerebrovascular diseases. The discrepancy between excitatory and inhibitory neurotransmission in the brain is a main distinguishing feature of experimental and clinical seizure . Nerve cell depolarization occurred due to the predominance of excitatory postsynaptic potentials (EPSP) over the inhibitory (IPSP) resulting in the generation of the seizures in the brain. Epilepsy is precipitated due to an group of factors together with elevated electrolytes (Na+ , K+, Ca2+) levels, excitatory amino acids (glutamic acid), and inhibitory amino acids (GABA), irregular interneuron links and abnormal afferent links from subcortical structures which transform various intertwining biochemical pathways giving augment to discharges of huge figures of neurons follow-on an epileptic seizure .
It is understood from the literature that GABAergic neurotransmission is closely associated with the induction of epilepsy in the animals [27, 28]. GABA is the major inhibitory neurotransmitter in the central nervous system and even slight deficiencies in GABAergic transmission may lead to hyperexcitability and pathological neuronal discharges leading to epilepsy. GABA is an endogenous agonist at GABAA receptor (ionotropic receptor) thereby opening the channels to Cl- ions in the neuronal membrane.
An enhancement of GABAergic inhibitory transmission is responsible for the antiepileptic effects of drugs that directly bind and activate GABAA receptors or influence GABA release, transport and metabolism. GABA mediated Cl- channel GABA-benzodiazepine receptor complex are closely associated with induction and onset of seizures . The benzodiazepines do not substitute for GABA but appear to enhance GABA's effects allosterically without directly activating GABAA receptors or opening the associated chloride channels.
In the current research the pharmacological screening models were elected on the source of mechanisms concerned in the anticonvulsant action of drugs. INH-induced convulsion in rats has been used to monitor a variety of drugs as it produces convulsion by interfering with GABA synthesis via down regulation of glutamic acid decarboxylase (GAD) activity, resulting in decreased level of GABA . Diazepam produces signiï¬cant protection against INH-induced convulsions as well as morusin also protect against INH-induced seizures and mortality indicating its action on the GABA synthesis mechanism. The drugs which produce effect on Na+ channels are widely used to screen against MES induced convulsions. These drugs are proven to be effective against partial and tonic-clonic seizures in humans . Phenytoin and morusin exhibited anticonvulsant proï¬le activity via inhibition of the Na2+ channels in partial and tonic-clonic seizures.
Nitric oxide (NO) is an endogenously formed intercellular signaling molecule  and depending upon the stimulants and NO associated chemicals it may act as an anticonvulsant or a proconvulsant [39,40]. Outcome of the current exploration reveals that by inhibiting the level of brain GABA, there is an increase glutamate release which may as a result increase NO synthesis in the hippocampus. Our results are in accordance with the previous ï¬ndings . In the present exploration, treatment with morusin restored the prominent level of nitric oxide.
It could be concluded from the current exploration that morusin modulates GABAergic conduction to show evidence of anticonvulsant result. Further studies are needed to unstitch its method of action.