Evaluation of Anticonvulsant and Antioxidant Activity of Flacourtia indica on Pentylenetetrazole (PTZ) Induced Convulsions in Mice

 

C. Ayyanna*, B. Narmada, G. Suresh, D. Munaf, B. Radha, K. Bhavani, M. Anitha

Department of Pharmacology, CES College of Pharmacy, Kurnool, Andhra Pradesh, India.

 

ABSTRACT:

Aim: To evaluation of anticonvulsant and antioxidant activity of Flacourtia indica on Pentylenetetrazole (PTZ) induced convulsions in mice Methods: Present study animals are divided into 4 groups of albino mice (n=6) were used. The First group was received as control, second group as standard (diazepam, 4mg/kg); third to fifth groups were treated with ethanol extract Flacourtia indica (100, 200 and 400mg/kg). After 30 min of administration ethanolic extract of Flacourtia indica, in mice I. P injection of PTZ (75mg/kg). Anticonvulsant activity was determined by measuring the Latency, Jerky movements, Straub’s tail, Clonic convulsions and Death/Recovery of animals in PTZ induced convulsions in mice. Results: In PTZ model, Flacourtia indica ethanolic extract at a dose of 400mg/kg prolonged latency and onset to seizure activity compared to 100 and 200 mg/kg. Conclusion: Flacourtia indica possess anticonvulsant effect as they delay the latency of seizures produced by PTZ.

 

 

 

INTRODUCTION:

Epilepsy is a neurological disorders characterized by recurrent spontaneous seizures that apparently result from complex processes involving several neurotransmitter systems such the glutamatergic, cholinergic, and gabaergic system¹. seizures are thought to occur via an alteration in the behavior of neuronal networks in the brain that induce the spontaneous expression of periods of synchronized burst firing interspersed by periods of normal electrical activity². Glutamate and ˠ-amino butyric acid (GABA) are quantitatively the most important excitatory and inhibitory neurotransmitters, respectively, in the mammalian brain³. Thus, receptors for these two neurotransmitters are regarded as important targets for antiepileptic drugs. Approximately 30% of patients with partial epilepsy and 25% of patients with generalized epilepsy are not well controlled on medications^4. These patients often receive multiple medical treatments to control their seizures.

 

 

Thus, there is an unmet need for new antiepileptic drugs. Herbal medicine could be a source for new therapeutics⁵.

Flacourtia indica (Burm.f.) Merr. Synonymousto Flacourtia ramontchi L’ Herit. (Family Flacourtiaceae) commonly known as ‘Baichi’ or ‘Katai’. It is an indigenous medicinal plant widely distributed in Bangladesh and India. Chufa tubers are daily ingredients of the diet of many people in North Africa and Spain⁶. Several therapeutically important natural compounds have been isolated (such as alkaloids, flavonoids, carbohydrates, tannins, saponins, and steroids) and they can serve as very potent and reliable drug candidate for treatment of various disorders.

 

This study was undertaken to evaluate the possible anticonvulsant activities of areal parts of Flacourtia indica extract using Pentylenetetrazole (PTZ) -induced seizure in mice.

 

EXPERIMENTATION:

Plant material:

Aerial parts of Flacourtia indica were collected during December 2018 from, Thirupathi hills, Andhra Pradesh, India. It was identified and authenticated by Prof. Dr. Madhavasetty, Botanist, University, Thirupathi, Andhra Pradesh, India. The voucher specimen was maintained in our laboratory for the future reference.

Preparation of extract:

Areal parts of Flacourtia indica was dried in shade, separated and made to dry powder. It was then passed through the 40 mesh sieve. Sufficient quantity of of powder was subjected to continuous hot extraction in Soxhlet apparatus using ethanol as solvent at a temperature range of 60-70˚C. The extract was evaporated under reduced pressure using rotary evaporator until all the solvent has been removed to give an extract sample. Yield thick green semisolid residue (EECE, 5.8% w/w). The extract were subjected to phytochemical preliminary screening and revealed the presence alkaloids, flavonoids, carbohydrates, glycosides, tannins, terpenoids, and phenols⁷.

 

Experimental animals:

Albino mice, weighing 25-30g, were procured from the animal house of CES College of pharmacy, Chinnatekur, Kurnool (1305/Po/Re/S//09/CPCSEA). The animals were kept in polypropylene cages (6 in each cages) under standard laboratory condition (12 hr light and 12 hr dark day night cycle) and had free access to commercial pellet diet with water ad libitum. The temperature was maintained at 25 ± 1⁰C with relative humidity (50 ±15%). The study was approved by the institutional animal ethical committee (IAEC/CESCOP/AUG-2018-03). Ethical norms were strictly followed during all experiments.

 

Acute oral toxicity study:

The acute toxicity of ethanolic extract of Flacourtia indica was determined as per the OECD guideline no. 423 (Acute Toxic Class Method). Flacourtia indica linn ethanolic extract was observed to safe up to 2000mg/kg by oral route. After 24 hours animals were found to be well tolerated. There was no mortality and signs of toxicity. Hence 1/15^th(100mg/kg),1/10^th (200mg/kg) and 1/5^th (400mg/kg) of this dose were selected for biological study.⁸

 

PTZ INDUCED CONVULSIONS IN MICE⁸

1.       Group I - Served as a control.

2.       Group II - Received Diazepam (4mg/kg i.p) b.w. served as a standard

3.       Group III - Received EEFI (100mg/kg p.o) served as a test-I

4.       Group IV- Received EEFI (200mg/kg p.o) served as a test-II

5.       Group V – Received EEFI (400mg/kg p.o) served as a test-III

 

There are four distinct phases constituted the PTZ (75mg/kg i.p) seizure sequence i.e. Latency, Jerky movements, Straub’s tail, clonic convulsions and Death/Recovery.

 

The mice of 25-30g of either sex animals (n=6 in each group) were used for the study. The test groups (III, IV and V) received F.indica ethanol extract (100mg/kg), F.indica ethanol extract (200mg/kg) and F.indica ethanol extract (400mg/kg) orally for 14days. Respectively and test conducted for antiepileptic activity 1hr after the last dose of the extract. PTZ (75mg/kg) is used as the inducing agent. After the administration of PTZ, the animals were placed in an individual polypropelene cage for observation convulsions. Standard group animals were received Diazepam (4mg/kg) on 14^th day 1hr prior to PTZ administration.

 

STATISTICAL ANALYSIS:

Data were expressed as percentage (%) protection and mean ± SEM and were analyzed by one-way ANOVA followed by Dunnett's test for multiple comparisons using Graph pad prism version 5.03. Results were considered significant at p <0.05. (Prism graph pad 5.3 version).

 

RESULTS:

Table :1- Summary of Phytochemical Constituents

S. No	TEST	INFERENCE
1	Liebermann’s test	Steroids absent
2	Salwoski test	Steroids absent
3	Schinoda test	Flavonoids present
4	Ferric chloride test	Tannins present
5	Dragandroff’s test	Alkaloids absent
6	Brontanger’s test	Anthraquinone absent
7	Kedde’s test	Cardinolides absent
8	Legal’s test	Cardinolides absent

 

The percentage yield of ethanol extract of entire plant of Flacourtia indica linn was found to be 5.8% w/w respectively.

 

Acute oral toxicity:

The results obtained indicated that Flacourtia indica extract at oral doses up to 2000 mg/kg did not produce any symptom of acute toxicity and none of the rats died during 72 h of observation and up to 14 days.

 

ANTICONVULSANT ACTIVITY:

Table 2: EFFECT OF EEFI ON PTZ INDUCED CONVULSIONSINS IN MICE

Where n=6 the observation are Mean + SEM. *P<0.05, **P<0.01 and ***P<0.001 as compared to control All the data were analyzed by using one way ANOVA followed by Dunnett’s test. FIE – Ethanol extract of Flacourtia indica linn

 

PTZ INDUCED CONVULSIONS-ANTIOXIDANT STUDIES:

Table: 3- Effect of EEFI on Lipid Per oxidation, Glutathione and GABA levels in mice brain ^9,10and11

Groups	Drug treatment	Lipid peroxidation levels (nm)	Glutathione levels (nm)	GABA levels(nm)
I	Control	0.5200±0.01360	0.7249±0.06123	0.06758±0.001594
II	Diazepam(4mg/kg)	0.4025±0.004515***	1.407±0.06662***	0.04733±0.004349***
III	EEFI (100mg/kg)	0.3590±0.008470*	0.3557±0.07097*	0.04433±0.001473*
IV	EEFI (200mg/kg)	0.3182±0.007947**	1.060±0.009905**	0.03617±0.001493**
V	EEFI (400mg/kg)	0.3012±0.01058***	1.228±0.1467***	0.03133±0.001282***

Where n=6 the observation are Mean + SEM. *P<0.05, **P<0.01 and ***P<0.001 as compared to control All the data were analyzed by one-way ANOVA followed by Dunnett’s test. EEFI – Ethanol extract of Flacourtia indica linn.

 

 

DISCUSSION:

Results of phytochemical analysis revealed that ethanolic extract of Flacourtia indica linn showed considerable amount of flavonoids and phenolics which may be responsible for observed antioxidant and antiepileptic activities. Neuronal hyper excitability and unwarranted production of free radicals have been implicated in the pathogenesis of a substantial array of neurological disorders, including epilepsy¹². The high rate of oxidative metabolism, coupled with the low antioxidant defenses and the richness in polyunsaturated fatty acids, makes the brain highly vulnerable to free radical damage.

 

The antiepileptic activity of Flacourtia indica linn at three dose levels (100, 200 and 400mg/kg) was studied by PTZ induced seizure models. PTZ is a noncompetitive Gamma Amino Butyric Acid (GABA) receptor antagonist and works by inhibiting the activity of GABA receptor. PTZ is the most popular and widely used chemically induced seizure model and it represents an effective model for human generalized myoclonic and also absence seizure

 

Most of the anti-epileptic drugs act by enhancing GABA neurotransmission. In PTZ induced seizure model, PTZ produced Clonic convulsions and lethality in mice, while pre-treatment with EEFI at two different dose levels has resulted in to delayed onset of convulsions, prolonged latencies of tonic seizures and reduction in lethality.

 

It has been well reported in literature that flavonoids and phenolics enhance GABA neurotransmission and GABA is the main inhibitory neurotransmitter which is suppressed in epilepsy,¹³ thus the suppression of seizures by PTZ dose- dependently might be attributed to phenolics reach composition of the extract through enhancing GABA neurotransmission in brain or by blockade of glutamatergic neurotransmission mediated by the NMDA receptor. However effects of Flacourtia indica linn on glutamatergic neurotransmission were not studied in this study.

 

CONCLUSION:

In present study protective effect and anti-oxidant activity of ethanolic extract of Flacourtia indica linn against seizures induced by PTZ were evaluated. The observed antioxidant and antiepileptic activities are due to the presence of considerable amount of flavonoids and phenolics in the ethanolic extract of Flacourtia indica linn. Increased oxidative load is directly implicated as seizures can cause imbalance in oxidant, antioxidant system of brain which leads to oxidation of lipids, DNA and protein ultimately resulting into neurodegeneration. Ethanolic extract of 400mg/kg Flacourtia indica linn showed good antiepileptic activity in PTZ induced convulsions may be through the involvement of GABAergic and glutamatergic transmission and through glycine inhibitory property compared to 100mg/kgand200mg/kg. Thus results of our study showed promising anti-oxidant and antiepileptic effects of ethanolic extract of Flacourtia indica linn against both the toxicants and provided a scientific claim to the usefulness of this traditionalplant in neurological disorders like epilepsy. However, further studies are needed to develop the exact underlying mechanism of antiepileptic action of possible constituents of the plant after isolation of bioactive constituents.

 

CONFLICT OF INTEREST STATEMENT:

We declare that we have no conflict of interest.

 

ACKNOWLEDGEMENT:

This work was supported by Creative Educational Society’s College of Pharmacy, Kurnool, Andhra Pradesh.

 

ABBREVIATIONS:

PTZ-Pentylenetetrazole, EEFI- Ethanolic extract of Flacourtia indica linn., GABA- Gamma Amino Butyric Acid, OECD-Organization for Economic Co-operation and Development, NMDA- N-Methyl-D-aspartate

 

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Received on 09.08.2019         Modified on 10.10.2019

Accepted on 23.11.2019         © RJPT All right reserved