Antiepileptic Activity Of Aerial Parts Of Viscum Articulatum Biology Essay

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Based on the ethnopharmacological information of the plant, the methanolic extract of aerial parts of Viscum articulatum was evaluated for its anti epileptic activity in rats and mice. Anti epileptic activity was studied by maximum electric shock induced seizures in rats and pentylenetetrazole induced convulsions in rats. Preliminary phytochemical investigations revealed the presence of proteins, carbohydrates, flavonoids, glycosides, phenolic compounds, tannins and triterpenes. At 100mg/kg and 200mg/kg the extract exhibited significant anti epileptic activity (p<0.001) which may be attributed to the presence of various phytochemicals detected during the studies.

Key words: Viscum articulatum, Anti epileptic activity, maximum electric shock (MES), pentylenetetrazole (PTZ).

INTRODUCTION

Epilepsy is a common chronic neurological disorder characterized by recurrent unprovoked seizures.[1] These seizures are transient signs and/or symptoms of abnormal, excessive or synchronous neuronal activity in the brain.[2] About 50 million people worldwide have epilepsy, with almost 90% of these people being in developing countries.[3] Epilepsy is more likely to occur in young children, or people over the age of 65 years, however it can occur at any time. Epilepsy is usually controlled, but not cured, with medication, although surgery may be considered in difficult cases. However, over 30% of people with epilepsy do not have seizure control even with the best available medications.[4] The most common causes for the disorder are head injury, brain infections, encephalitis, meningitis and dementia. Environmental and socio-economic conditions may cause epilepsy. [5]

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The aerial parts of Viscum articulatum is used traditionally in bacillary dysentery, bronchitis, cachexia, epistaxis, fever, lactation deficiency, leucorrhea, lumbar muscle strain, psoriasis, rheumatoid arthritis, syphilis, tuberculosis, urinary tract infection and uterine bleeding. It have been reported that stem paste and decoction of Viscum articulatum is applied on cuts, wounds, bone fracture. It is also used in ulcers, blood diseases, epilepsy and sprain.

MATERIALS AND METHODS:

The aerial parts of Viscum articulatum used for the present studies were collected from Tirupati, Chittoor district of Andhra Pradesh. The plant was identified, confirmed and authenticated by Dr. K. Madhava Chetty, Assistant Professor, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh.

Albino rats (Wistar strain) of either sex weighing between 150-200 g and Albino mice of either sex weighting 20-30 g were procured from the Sri Raghavendra enterprises, Vijaya nagar post, Bangalore, Karnataka. The animals were acclimatized for seven days under laboratory conditions. The animals were fed with commercially available rat pelleted diet (Sai Durga feeds& foods, Bangalore). Water was allowed ad libitum under strict hygienic conditions. The study protocols were duly approved by the Institutional Animal Ethics Committee (IAEC, Approval No: DSCP/ M.Pharm col/ IAEC/ 21/ 09-10) of Dayananda Sagar College of pharmacy, Bangalore. Studies were performed in accordance with the CPCSEA guidelines.

Preparation of Methanolic Extract:

The powdered drug was dried and packed well in Soxhlet apparatus and extracted with 1500 ml of methanol for seven days. The extract was concentrated and dried using Rotary flash evaporator and stored in a refrigerator.

Phytochemical screening

A preliminary phytochemical screening of methanolic extract of Viscum articulatum (MEVA) was carried out [6] to detect the presence of important phytoconstituents.

Determination of acute toxicity (LD50):[7]

The procedure was divided into two phases, Phase I (observation made on day one), and Phase II (observed the animals since next 14 days). Two set of healthy female rats (each set of 3 rats) were used for the experiment. First set animals were divided and fasted for 18 hours deprived from food, water withdrawn before 4 hours of the dosing, body weights were noted before and after dosing with methanolic extract of Viscum articulatum (2000mg/kg) orally. Individually animals were observed for 4 hours to see any clinical symptoms, any change in behaviour or mortality. 6 hours post dosing again body weights recorded. Form the next day onwards, each day 1 hour the behavioural change, clinical symptoms or mortality was observed in the same animals for next 14 days and animal body weights were recorded on 8th and 14th day. The same procedure was repeated with another set of animals to nullify the errors. [8]

Selection and preparation of dose for pharmacological screening:

The MEVA was suspended in 0.3% CMC solution to prepare two dose levels, 100 and 200mg/kg body weight of the animals.

Anti epileptic activity:

Maximum Electric Shock induced seizures model: [9]

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Healthy albino rats of either sex weighing 180-220 g were divided into 4 groups of 6 animals each. Group A received distilled water (1ml/kg, p.o.) orally. Group B received Diazepam (4mg/kg, i.p.). Group C and Group D received 100mg/kg and 200 mg/kg of methanolic extract of Viscum articulatum respectively for seven days. A shock of 150 mA for 0.2 sec was given for rats to induce convulsions by using an electroconvulsiometer, 60 min. after the adiministration of standard drug and the extracts. The rats were then observed for 30 min. for various phases of epilepsy like flexion, extensor, convulsions, recovery/death. Reduction or abolition of extensor phase was taken as a parameter for the evaluation of anti-epileptic activity.

Pentylenetetrazole induced convulsions model: [10]

The effect of the methanolic extract of Viscum articulatum on pentylenetetrazole induced convulsions was investigated in albino rats. Healthy albino rats of either sex weighing 180-220g were divided into 4 groups of 6 animals each. Group A received saline (1 ml/kg orally). Group B received Diazepam (4 mg /kg; i.p.). Group C received methanolic extract of Viscum articulatum (100 mg/kg orally). Group D received methanolic extract of Viscum articulatum (200mg/kg orally). All the extracts and standard drug were given for 7 days. Pentylenetetrazole (80 mg/kg, s.c) was injected 60 min after oral administration for all the groups. Latency to seizures and mortality were taken as parameters to assess the anti epileptic activity.

Statistical analysis:

The data are expressed as mean±SEM. Statistical differences between means were determined by one-way anova followed by Tukey Kramer's post hoc test. Values of P <0.05 were considered as significant.

RESULTS

The MEVA showed presence of proteins, carbohydrates, flavonoids, glycosides, phenolic compounds, tannins and triterpenes. The methanolic extract of Viscum articulatum was found to be nontoxic up to a dose of 2000 mg/kg.

On a shock of 150 mA for 0.2 sec, epilepsy was induced in the rats with an incidence of different phases like tonic flexion, hind limb extensor, tonic convulsions and recovery/death. Pretreatment with Diazepam, methanolic extract of Viscum articulatum reduced/abolished the hind limb extensor phase. (Table-I)

Administration of pentylenetetrazole at (80mg/kg, s.c) produced significant convulsions and death in rats. Pretreatment with Diazepam and methanolic extract of Viscum articulatum has increased the latency to convulsions. (Table-II)

DISCUSSIONS

MES induced seizures bear a semblance to grandmal epilepsy. Evidence indicates that imbalance between excitatory and inhibitory neurotransmission in the brain is a main cause contributing to seizure development in both, experimental and clinical conditions. Gamma-amino butyric acid (GABA) is the predominant inhibitory neurotransmitter in the CNS. Impairment of GABA function is widely recognized to provoke seizures, whereas facilitation has an anticonvulsant effect. GABA is synthesized from glutamate, exclusively in GABAergic neurons, by the action of the enzyme glutamic acid decarboxylase. Upon synaptic release, GABA acts on its three specific receptors, GABAA, GABAB, and the newly characterized GABAC. GABA is removed from the synaptic cleft into localized nerve terminals and glial cells, by specific membrane-bound transport molecules. After removal from the synapse, GABA is either recycled to the readily releasable neurotransmitter pool (GABAergic nerve terminals only) or metabolized (neurons and glial cells) to the inactive molecule succinic acid semialdehyde by the mitochondrial enzyme GABA-transaminase11. The convulsion in MES method is due to the disturbed activity of GABA in the brain.

PTZ a most frequently used substance as well as an acute experimental model in the preliminary screening to test potential anticonvulsant drugs. The mechanism by which PTZ is believed to exert its action is by acting as an antagonist at the GABAA receptor complex. Several biochemical hypotheses have been advanced involving the inhibitory GABAergic system and the system of the excitatory amino acid glutamate and aspartate. The mechanism by which PTZ is believed to exert its action is by acting as an antagonist at the GABAA receptor complex. Drugs protecting against tonic-clonic seizures induced by PTZ are considered to be useful to control myoclonic and absence seizures in humans. In this study methanolic extract of Viscum articulatum replicated the effect of this anti epileptic drug by delaying tonic convulsion and mortality. The benzodiazepine site in the GABAA receptor and T-type Ca2+ currents could be targets for further studies to know the mechanisms of action of methanolic extract of Viscum articulatum. It suggests that methanolic extract of Viscum articulatum is useful in suppressing generalized tonic-clonic seizures. Anticonvulsant activity of MEVA in inhibiting seizures may be by regulating GABA - mediated synaptic inhibition through action at distinct sites of the synapse. Summarizing the data obtained in this study, the results suggest a possible anticonvulsant effect of methanolic extract of Viscum articulatum in rodents. The precise mechanism of possible anti convulsant effect of methanolic extract of Viscum articulatum is not clear. Further research is in progress to isolate the compound responsible for this activity.

Acknowledgement:

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Authors are thankful to all the management members of Dayananda Sagar College of Pharmacy, for providing the necessary facilities to conduct this study

Table-I

EFFECT OF METHANOLIC EXTRACT OF VISCUM ARTICULATUM ON MAXIMUM ELECTRIC SHOCK INDUCED SEIZURES MODEL IN RATS:

Groups

Treatment

Dose

Duration of hind limb extension(sec)

Group A

Vehicle

1ml/kg, p.o

35.06 ± 1.54

Group B

Diazepam

4mg/kg, i.p.

9.35 ± 2.94

Group C

MEVA

100mg/kg, p.o

20.15 ± 0.55***

Group D

MEVA

200mg/kg, p.o.

15.32 ± 0.34***

Values are mean ± SEM (n=6) one way ANOVA followed by Tukey-kramer's test. Where, *** represents very significant at p<0.001.

Table II

EFFECT OF METHANOLIC EXTRACT OF VISCUM ARTICULATUM ON PENTYLENE TETRAZOLE INDUCED CONVULSIONS IN RATS:

Groups

Treatment

Dose

PTZ

Onset of spasm

(sec) ± SEM

Death

Group A

Vehicle

1ml/kg, p.o.

44.7 ± 2.52

2/6

Group B

Diazepam

4mg/kg, i.p.

72.4 ± 1.52

0/6

Group C

MEVA

100mg/kg, p.o.

77.7 ± 1.77***

0/6

Group D

MEVA

200mg/kg, p.o.

93.6 ± 0.42***

0/6

Values are mean ± SEM (n=6) one way ANOVA followed by Tukey-Kramer's test. Where, *** represents very significant at p<0.001.