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DISEASES
Year : 2015  |  Volume : 5  |  Issue : 2  |  Page : 56-62

Screening of Pistacia integerrima extracts for their anticonvulsant activity in acute zebrafish and rodent models of epilepsy


Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Mumbai, Maharashtra, India

Date of Submission16-Oct-2014
Date of Acceptance10-Jan-2015
Date of Web Publication23-Mar-2015

Correspondence Address:
Sadhana Sathaye
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga (E), Mumbai - 400 019, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.153793

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   Abstract 

Background: Epilepsy is one of the most common, serious neurological conditions, affecting more than 50 million people worldwide. This disorder is characterized by the occurrence of spontaneous and recurrent seizures due to the abnormal, excessive, and synchronous electrical firing of neuronal networks Aim: The objective of the present study was to evaluate Pistacia integerrima (PI) gall extracts in acute models of epilepsy for their anticonvulsant activity. Materials and Methods: Extraction of galls of PI using a Soxhlet apparatus was carried out to yield petroleum ether and methanolic extracts. Both the extracts were screened in pentylenetetrazole (PTZ)-induced seizures in the zebrafish model. The active extract was further evaluated in the PTZ-induced seizures in mice. Both extracts were further screened in the maximal electroshock (MES) model in rats. Results: The petroleum ether extract of Pistacia integerrima (PEPI) exhibited dose-dependent delay at 50mg/kg, 100mg/kg, and 200 mg/kg in the onset of different seizure parameters in PTZ-induced seizures in the zebrafish model that was further confirmed in mice at 50 mg/kg and 100 mg/kg. In addition, the extract exhibited delay in the duration of hind limb extension (HLE) as well as protected from it at 50 mg/kg, 100mg/kg, and 150 mg/kg in the MES model in rats. The metabolic extract of Pistacia integerrima (MEPI) was unable to protect from seizures in both preliminary screening (PTZ and MES) models. Conclusion: The results showed that PEPI demonstrated potential anticonvulsant activity in zebrafish and rodent models of epilepsy. This may be attributed to the presence of essential oil and its phytoconstituents, indicating its role in suppressing generalized absence and tonic-clonic seizures.

Keywords: Epilepsy, maximal electroshock (MES), pentylenetetrazole (PTZ), Pistacia integerrima (PI), zebrafish


How to cite this article:
Jain PD, Tambe RM, Sancheti JS, Nahire MS, Bhardwaj AK, Sathaye S. Screening of Pistacia integerrima extracts for their anticonvulsant activity in acute zebrafish and rodent models of epilepsy. Int J Nutr Pharmacol Neurol Dis 2015;5:56-62

How to cite this URL:
Jain PD, Tambe RM, Sancheti JS, Nahire MS, Bhardwaj AK, Sathaye S. Screening of Pistacia integerrima extracts for their anticonvulsant activity in acute zebrafish and rodent models of epilepsy. Int J Nutr Pharmacol Neurol Dis [serial online] 2015 [cited 2019 Oct 13];5:56-62. Available from: http://www.ijnpnd.com/text.asp?2015/5/2/56/153793


   Introduction Top


Epilepsy is a one of the common neurological disorders, manifesting in recurrent spontaneous unprovoked epileptic seizures that occur due to abnormal firing or synchronous neuronal activity in the brain. [1] At the neuronal level, seizure activity usually occurs when glutamatergic excitatory neurotransmitters override the γ-aminobutyric acid (GABA)-mediated inhibition, which causes an imbalance between the two. [2] The currently available antiepileptic drugs (AEDs) provide a symptomatic cure and they are often associated with serious side effects and adverse effects on cognition and behavior. Consequently, many people still rely on herbal medicine for management of epilepsy in the developing countries. Herbal drugs have clinical effectiveness, relatively low costs, and a minimal side effect profile due to which they are used for various applications in traditional medicine. [3],[4] Zebra fish (Danio rerio) are small freshwater fish, which are emerging as an important model organism in genetics and developmental neurobiology. Although zebrafish are in widespread use in biomedicine, their usefulness in studying neurological disorders remains unexplored. [5]

Pistacia integerrima (PI) Stew. (Anacardiaceae) is a moderate-sized deciduous tree widely distributed in the subalpine regions of the Himalayas ranging from the Indus to the Kumaon, and is also cultivated in the plains. [6] Extracts of PI galls have been dispensed by traditional practitioners of the subcontinent for chest diseases as well as for aches and pains in the body. The extracts from galls of PI are known to have expectorant, bronchodilator, antiemetic, appetizer, diuretic, anti-inflammatory, and analgesic properties. [7],[8] The ethanolic extract of the galls of PI has been reported for its anticonvulsant activity. [9] An attempt was further made to biofractionate and screen the petroleum ether extract of Pistacia integerrima (PEPI) and the methanolic extract of Pistacia integerrima (MEPI) of the galls of PI and to explore their anticonvulsant activity in zebrafish and rodent models of epilepsy.

The present study was constructed to evaluate the anticonvulsant potential of PI extracts using pentylenetetrazole (PTZ) and maximal electroshock (MES) models of epilepsy. Zebrafish were used to preliminarily screen the extracts in PTZ-induced seizures, and the active extract was further evaluated to confirm its anticonvulsant activity in the mice. Further the extracts were screened in MES model of epilepsy.


   Materials and methods Top


Plant material

Galls of the PI tree were procured from a local market in Mumbai. Professor Ganesh Iyer, Department of Botany, Ruia College, Mumbai, India authenticated the specimen. The voucher specimen (No. SSS/0909/15) was deposited at the Institute of Chemical Technology for future reference.

Drugs and chemicals

PTZ (Sigma Chemical Co., St. Louis, MO, U.S.A.), diazepam (Mumbai, Roche Pharmaceuticals, India), and phenytoin (Ahmedabad, Zydus Pharmaceuticals, India) were used in the present study. The drugs were dissolved in water and subsequently used for injection. Petroleum ether and methanol (S.D. Fine-Chem, Mumbai, India) were the solvents used for the extraction. Tween 80 (S.D. Fine-Chem, Mumbai, India) was used as surfactant to solubilize the extracts. All other chemicals and reagents used in the experiments were of analytical grade.

Preparation of the extracts

The galls of PI were dried in shade and stored at 30°C, crushed to coarse powder, and passed through a sieve (no.40). The dried powdered galls of PI (500g) were first defatted with petroleum ether (60-80°C) and later extracted with methanol (60-80°C) for 24 h using the Soxhlet extraction assembly. After completion of the extraction, the solvent was removed by distillation and concentrated in vacuum (40°C) to yield the PEPI and MEPI, respectively.

Experimental animals

Zebrafish

Adult 4-6-months-old male and female zebrafish (50:50) of the heterozygous wild type were obtained from local commercial distributors (Lakeland Aquarium, Mumbai, India). Fish were housed in 50-L aquariums (40-50 fish per aquarium) for at least 1 week prior to the experiments in order for them to acclimatize to the new environmental conditions. Zebrafish were maintained under standard laboratory conditions, using a light schedule of 12 h on and 12 h off at a temperature of 25-28°C. the fish were fed twice daily using a combination of dry food and brine shrimp. All animals used in this study were experimentally naive, healthy, and free of any signs of disease. All behavioral tests took place between 11 am and 4 pm to ensure consistency and reduce potential variation in behavior. [10]

Rodents

Swiss male albino mice (20-25 g) and adult male Wistar rats (150-200 g) were procured from Haffkine Institute, Mumbai, India and were acclimatized inside the animal house of the Institute of Chemical Technology (ICT), Matunga, Mumbai. Young healthy male mice and rats were housed six per cage and maintained at a temperature of 23 ± 2°C, at a humidity of 51 ± 10% and in a 12:12-h light/dark cycle with free access to rodent chow (Amrut Laboratory Animal Feeds, Pranava Agro Industries Ltd, Sangli, India) and tap water ad libitum. The experiments were carried out between 9 AM and 6 PM. The animal study was approved by the Institutional Animal Ethics Committee (IAEC) (ICT/IAEC/2012/P48), Mumbai.

Acute toxicity

PEPI and MEPI were evaluated at doses of 100 mg/kg, 500 mg/kg, and 800 mg/kg) in zebrafish intraperitoneally (i.p) and further at 175 mg/kg, 550 mg/kg, and 1000 mg/kg in mice (n = 6) at each dose. Mortality was observed after 24 h.

Assessment of anticonvulsant activity

Screening of PEPI and MEPI in PTZ-induced seizures in zebrafish model

Experimental groups

Group I: 0.2% Tween 80 in saline (vehicle control)

Group II: Negative control, PTZ 225 mg/kg [11]

Group III: PEPI, MEPI 50 mg/kg

Group IV: PEPI, MEPI 100 mg/kg

Group V: PEPI, MEPI 200 mg/kg

Group VI: Diazepam 10 mg/kg (positive control)

The doses and the duration of pretreatment and treatment were selected based on pilot studies on unpublished data from our lab.After anesthetizing the fish (n = 12)using ice, they were pretreated with the three concentrations of each extract(i.e., PEPI and MEPI) for 10 min. After pre-treatment, the proconvulsant agent i.e. PTZ (225 mg/kg) was injected i.p. The behavioral profile of zebrafish was observed for 10min after administrating PTZ for different phase scores (hyperactivity, onset of clonic seizure, onset of tonic seizure). The injection volume for each fish was calculated according to the fish weight.

Evaluation of PEPI in PTZ-induced seizure in mice

Experimental groups

Group I: 0.2% Tween 80 in saline (vehicle control)

Group II: Negative control, PTZ 100mg/kg,i.p.

Group III: PEPI 50 mg/kg

Group IV: PEPI 100 mg/kg

Group V: PEPI 150 mg/kg

Group VI: Diazepam 2 mg/kg,i.p. (positive control)

All the treatments were administered i.p. 30 min prior to the PTZ injection. Seizures were induced by administration of PTZ (100 mg/kg, i.p.) and the animals were observed during the first 30 min for parameters such as onset of myoclonic jerks, onset of clonic seizure, onset of hind limb extension (HLE), time taken for death, and percentage of animals protected from death. [12]

Evaluation of PEPI and MEPI in MES-induced seizures in rats

Experimental groups

Group I: 0.2% Tween 80 in saline (vehicle control)

Group II: PEPI, MEPI 50 mg/kg

Group IV: PEPI, MEPI 100 mg/kg

Group V: PEPI 150mg/kg, MEPI 200 mg/kg

Group VI: Phenytoin 25 mg/kg (positive control)

All the treatments were administered i.p., 30 min prior to the PTZ injection. After 30 min of pre-treatment, seizures were induced in rats by delivering electroshock of 150 mA for 0.2 s by means of electroconvulsiometer (Dolphin, Mumbai, India) through a pair of ear clip electrodes to all groups. Each animal was placed and observed in an individual transparent cage. Time in seconds for duration of HLE and percent protection from HLE were noted. [13]

Statistical analysis

Data of all the results were presented as mean ± SEM. The analyses of all the studies were done with the help of analysis of variance (ANOVA) followed by Dunnett's test. The difference in the results of PTZ and MES were considered statistically significant when *P < 0.05, **P < 0.01, and ***P < 0.001 compared to negative control.


   Results Top


Acute toxicity

PEPI and MEPI administration were found to be safe in zebrafish upto 800 mg/kg i.p with no mortality, while PEPI at a dose of 800 mg/kg i.p and MEPI at a dose of 1000 mg/kg i.p were safe in mice, with no mortality.

Assessment of anticonvulsant activity

Screening of PEPI and MEPI in PTZ-induced seizure in zebrafish model

Standardization, validation, and behavioural observation of PTZ-induced convulsions in the zebrafish model by i.p route were optimized. The zebrafish treated with 10mg/kg of diazepam, PEPI, and MEPI (50 mg/kg, 100 mg/kg, and 200 mg/kg) were compared with the PTZ control group (225 mg/kg, negative control).

In the diazepam (10 mg/kg)-treated group all the zebrafish exhibited 100% protection from seizures in the PTZ-induced convulsion model. PEPI treatment at all the doses exhibited dose-dependent delay in the onset of hyperactivity, clonus phase, and tonic phase. PEPI at doses of 50, 100, and 200 mg/kg showed delay in onset hyperactivity (P < 0.05, P < 0.05, and P < 0.001) [Figure 1]. PEPI at dose of 100 and 200 mg/kg showed delay in the onset of clonus (P < 0.001) and tonic phase (P < 0.01, P < 0.001) [Figure 2] and [Figure 3]. PEPI treatment at the dose of 200 mg/kg exhibited 83.33% protection from clonic and tonic phases in PTZ-induced seizures in zebrafish model [Table 1]. MEPI did not show any delay in either of the phases, thus it was not continued to treat PTZ-induced seizures in mice [Figure 4],[Figure 5] and [Figure 6], [Table 2].
Figure 1: Effect of PEPI on hyperactivity in PTZ-induced seizures in zebrafish

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Figure 2: Effect of PEPI on onset of clonic phase in PTZ-induced seizures in zebrafish

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Figure 3: Effect of PEPI on onset of tonic phase in PTZ-induced seizures in zebrafish

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Figure 4: Effect of MEPI on hyperactivity in PTZ-induced seizures in zebrafish

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Figure 5: Effect of MEPI on onset of clonic phase in PTZ-induced seizures in zebrafish

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Figure 6: Effect of MEPI on onset of tonic phase in PTZ-induced seizures in zebrafish

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Table 1: Effect of PEPI in PTZ-induced seizures on percent protection from clonic and tonic phases in zebrafi sh model

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Table 2: Effect of MEPI in PTZ-induced seizures on percent protection from clonic and tonic phases in zebrafi sh model

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PEPI and MEPI extracts were screened in the zebrafish model, while the PEPI extract exhibited anticonvulsant activity. To confirm its activity, it was evaluated in PTZ-induced seizures in mice.

Evaluation of PEPI in PTZ-induced seizures in mice

PEPI treatment at the dose of 150 mg/kg (P < 0.01) and diazepam (2 mg/kg) produced a significant (P < 0.001) increase in onset of cyclonic seizure and clonic seizure [Figure 7] and [Figure 8]. PEPI significantly delayed the onset of HLE and time taken for death at 100 and 150 mg/kg (P < 0.001) [Figure 9] and [Figure 10]. PEPI treatment showed 83.33% protection from death at the dose of 100 mg/kg, whereas diazepam showed 100% protection [Table 3].
Figure 7: Effect of PEPI on onset of myoclonic jerk in PTZ-induced seizures in mice

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Figure 8: Effect of PEPI on onset of clonic seizure in PTZ-induced seizures in mice

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Figure 9: Effect of PEPI on onset of HLE in PTZ-induced seizures in mice

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Figure 10: Effect of PEPI on time taken for death in PTZ-induced seizures in mice

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Table 3: Effect of PEPI in PTZ-induced seizure on percent protection from death in mice

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Evaluation of PEPI and MEPI in MES-induced seizures in rats

In the MES-induced seizure model, a decrease in the duration of HLE was observed at all evaluated doses of PEPI (50 mg/kg, 100mg/kg, and 150 mg/kg). Phenytoin completely protected the animals from HLE when compared with vehicle control. PEPI reduced the duration of HLE significantly at the doses of 50 mg/kg and 100 mg/kg (P < 0.01) and at 150 mg/kg (P < 0.001) in a dose-dependent manner [Figure 11]. PEPI at 150 mg/kg showed 66.66% protection with respect to vehicle control [Table 4] MEPI was not effective in reducing the duration of HLE at any of the given doses (50-200 mg/kg) in the MES-induced seizure model [Figure 12], [Table 5]. Hence, it had no protective effect in MES-induced seizures.
Figure 11: Effect of PEPI on duration of HLE in rats

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Figure 12: Effect of MEPI on duration of HLE in rats

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Table 4: Effect of PEPI on percent protection from HLE in MES model in rats

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Table 5: Effect of MEPI on percent protection from HLE in MES model in rats

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   Discussion Top


The present study was undertaken to investigate the anticonvulsant potential of PEPI and MEPI. To our knowledge, this study presents the first report on the screening of herbal extracts administered i.p. for the treatment of PTZ-induced seizure in zebrafish.

PTZ and MES are the preliminary screening models used to evaluate the anticonvulsant potential. They have predictive relevance regarding the clinical spectrum of the experimental compounds. PTZ and MES tests are assumed to identify drugs effective against human generalized absence and tonic-clonic seizures. [14],[15] PTZ is a γ-aminobutyric acid type A (GABA A ) receptor antagonist acting at the picrotoxin site of the GABA A receptor [16] and it reduces chloride conductance, [17],[18],[19] which further leads to glutamatergic excitation. PTZ is a popular chemoconvulsant used for the evaluation of antiepileptic drugs [20] and for eliciting seizure activity in animal experiments. [21],[22],[23] Drugs that modulate GABA A receptor-mediated inhibitory neurotransmission, such as diazepam and phenobarbital, can prevent this type of seizure. Drugs that reduce T-type Ca++ currents, such asethosuximide, can prevent seizures induced by PTZ. [24] The observations made during the present study showed that the PEPI extract exhibited potential anticonvulsant activity in PTZ-induced seizures in zebrafish as well as in mice. This activity may be attributed to its ability to modulate the GABA A receptor, and it is probable that it may be interfering with GAB Aergic mechanisms by enhancing the activation of GABA A receptors, thus facilitating the GABA-mediated opening of chloride channels.

MES-induced seizures can be prevented by drugs that inhibit voltage-dependent Na+ channels, such as phenytoin. PEPI also showed good protection from HLE in the MES model, which may be due to its ability to block sodium channels. MEPI was unable to protect against the seizures and was ineffective in the PTZ and MES models, indicating its inability to suppress generalized absence and tonic-clonic seizures. The activity of the PEPI extract may be attributed to the presence of essential oil, which mainly contains α-pinene, β-pinene, and 4-carvomenthol.

Scores from docking studies carried out in our lab (unpublished data) showed that α-pinene and 4-carvomentholhadgood binding interaction with the GABA A receptor and the sodium channel, respectively. Previous studies showed that of the mentioned phytoconstituents, 4-carvomenthol has already been reported to be an anticonvulsant, [25] while the derivatives of α-pinene and β-pinene have been reported to have a neuroprotective effect. [26]


   Conclusion Top


In the present study, the PEPI extract demonstrated potential anticonvulsant activity in zebrafish and rodent models. This study suggests that the anticonvulsant activity of PEPI extract could be due to modulation of the GABA A receptor or because of its ability to block sodium channels. The essential oil components such as α-pinene, β-pinene, and 4-carvomentholthat are present in PEPI may be responsible for its anticonvulsant property. Further studies are required to explore the essential oil and its phytoconstituents present in PEPI to see if it can be safely used for the therapeutic treatment of epilepsy, which are ongoing in our laboratory.

 
   References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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