3.1. Chemicals
Pentylenetetrazole was purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Diazepam was purchased as 10 mg/ml ampoule from Tolid-daru Co. Tehran, Iran.
3.2. Experimental Animals
The protocol for the study was based on animal ethical committee of Mazandaran University of Medical Sciences. Swiss male albino mice (20 - 25 g, Pasteur institute of Iran) were used for this study. The animals were housed in standard cages with free access to food and water. The temperature was maintained at 23 ± 1°C with a 12-hour light/12-hour dark cycle. Each animal was used once. Inclusion criteria for animals were their sex (male) and race (Swiss albino) and habitation in the lab environment one week before the exam. The exclusion criteria were being used previously for any behavioral exams and having underlying diseases.
3.3. Plant Material and Preparation of Freeze-Dried Extract
E. caucasicum inflorescence was collected from Sari, Iran, in June 2013. The sample was authenticated by Dr. Bahman Eslami, and the voucher specimen was deposited (No: 1725) in the herbarium of the Biology School of Gaemshahr Azad University. Plant materials were dried under dark conditions at the room temperature. The dry material was coarsely grounded (2 - 3 mm) and then extracted by methanol for 24 hours at the room temperature. The extracts were then separated from the sample residues by filtration. Extraction was repeated thrice. The resulting extracts were concentrated over a rotary vacuum at 35 - 40°C until crude solid extracts were obtained, which then were freeze-dried for complete solvent removal.
3.4. Anticonvulsant Activity
3.4.1. Convulsions Induced by Pentylenetetrazol
Seizure induced by PTZ was performed to evaluate the anticonvulsant property of the extract. Seventy-seven male mice were divided into eight groups, each of which comprising nine mice. The groups were treated with normal saline (10 ml kg-1), diazepam (4 mg kg-1), and methanolic extracts of E. caucasicum at doses of 125, 250, and 500 mg kg-1, and poly phenolic extracts at doses of 50,100, 200 mg kg-1 separately. Thirty minutes later, convulsions were induced by the intraperitoneally administration of 100 mg kg-1 of PTZ. Following the administration of PTZ, the mice were placed in separate transparent Plexiglas cages (25 × 15 × 10 cm) and were observed for the occurrence of seizures over a 30-minute period. The latency of the first convulsive episode, duration of tonic convulsions, and percentage of deaths were recorded (6).
3.5. Maximal Electroshock (MES) Induced Convulsions
Forty-eight male mice were divided into eight groups, each of which having six mice. The groups were treated with normal saline (10 mm kg-1), diazepam (4 mg kg-1), and methanolic extracts of E. caucasicum at doses of 125, 250, and 500 mg kg-1, and polyphenolic extracts at doses of 50, 100, 200 mg kg-1 separately. After 30 minutes, convulsions were induced in all the groups of animals, using electro-convulsometer. A 50 Hz alternating current of 60 mA for 0.2 seconds was delivered through the ear electrodes (7). The incidence and duration of tonic hind limb extension was recorded.
3.6. Statistical Analysis
Data as mean ± SD were analyzed by ANOVA. Duncan’s new multiple range test was used to determine the differences in means. All P values less than 0.05 were considered significant.
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