Screening of the anticonvulsant activity of some plants from Fabaceae family in experimental seizure models in mice

Animals

Adult male NMRI mice, weighing 20–28 g, from Pasteur Institute of Iran were used throughout this study. The animals were housed in standard cages with free access to food (standard laboratory rodent›s chow) and water. The animal house temperature was maintained at 23±1 °C with a 12-h light/12-h dark cycle (light on from 06:00 AM to 18:00 PM). The study was approved by the ethics committee of Pasteur Institute of Iran and conforms to the European Communities Council Directive of 24th of November, 1986 (86/609/EEC). All animal experiments were carried out in a way that minimizes the number of animals and their sufferings. Each animal was tested once. All the injections were intraperitoneal (i.p.) in volume of 0.1 ml/10 g of mice body weight.

Chemicals

Pentylenetetrazole (PTZ), phenytoin sodium and ethosuximide were purchased from Sigma-Aldrich (Pool, UK). Tween 80, Dimethyl sulfoxide (DMSO), ethanol, dichloromethane, antimony trichloride, Dragendorrf's reagent, potassium hydroxide, glacial acetic acid, vanillin, sulphuric acid, ferric chloride, hydrochloric acid and sodium hydroxide were all from Merck (Darmstadt, Germany).

Plant materials

Botanical data of the plants are demonstrated in table 1. The plants were authenticated by Soroush Sardari and the voucher specimens were deposited in the herbarium of Pasteur Institute of Iran, Tehran.

Extract preparation

Air-dried parts (100 g) of each plant were grounded and extracted at the room temperature for 48 hrs, by percolation method using 80% ethanol (900 ml). The extracts were then concentrated by a rotary evaporator apparatus at temperature not exceeding 50°C. The yields of the extracts are demonstrated in table 1. The extracts were stored at 4°C throughout experiments.

Fractionation

The crude extract of A. obtusifolius was suspended in 200 ml of distilled water and extracted with dichloromethane (3×150 ml). The aqueous (f1) and dichloromethane (f2) fractions were collected separately, dried by rotary evaporator at 40 °C and stored at 4°C throughout experiments (Fig. 1).

Figure 1

Flow diagram of fractionation of the crude extract obtained from the aerial parts of Astragalus obtusifolius.

PTZ-induced seizures

The minimal i.p. dose of PTZ at which 99% of the animals showed general clonus was determined by a dose-percent effect curve. General clonus was considered as the criteria of clonic seizure, which was characterized by clonus of four limbs with transient loss of righting reflex (8). The extracts, fractions and the solvent of the extract [Tween 80 (25%): DMSO (2:1, v/v) 10 ml/kg], as control, ethosuximide (150 mg/kg), as positive control and saline (10 ml/kg), as control were injected to groups of 10 mice. After 30 min, PTZ (60 mg/kg) was injected to animals. If no general clonus occurred during a 30-min period of observation, the animals were considered protected.

MES-induced seizure

Electro-convulsive shock, inducing Hind Limb Tonic Extension (HLTE) in 99% of the animals (8) was determined by a current intensity-percent effect curve. The electrical stimulus (50 mA; 50 Hz; 1sec duration) was applied through ear-clip electrodes using a stimulator apparatus (MGH-777, Development of Electronic Industry, Iran). Different groups of 10 mice were pretreated i.p. with the extracts, the solvent of the extracts [Tween 80 (25%): DMSO (2:1, v/v) 10 ml/kg, as control], phenytoin sodium (25 mg/kg), as positive control and saline (10 ml/kg), as control.

After 30 min the animals received transauricular electroshock. If no HLTE was observed within 10 sec after delivery of the electroshock, the animals were considered protected.

Acute toxicity

Groups of 10 mice each were treated i.p. with the solvent of the extracts, different doses of the extracts or the fractions. The mortality rate was recorded after 24 hrs.

Preliminary phytochemical screening

The crude extract of A. obtusifolius and its fractions, f1 and f2, were screened for the presence of triterpens/sterols, alkaloids, flavonoids, anthraquinones, anthrones, coumarines, valepotriates, essential oils and tannins by thin layer chromatography using silica gel G (Merck) plates of 0.25 mm thickness. The extract and the fractions were dissolved in Tween 80 (25%): DMSO (2:1v/v). Development was carried out with ethyl acetate: methanol: water (100: 13.5: 10 v/v/v) and ethyl acetate: toluene (93: 7). After development, the plates were sprayed with the following reagents for detection of the respective classes of compounds: antimony trichloride (triterpens/sterols), Dragendorrf's reagent (alkaloids), potassium hydroxide (anthraquinones, anthrones and coumarins), hydrochloric acid and glacial acetic acid mixture (valpotriates), Vanillin and sulfuric acid mixture (essential oil), ferric chloride (tannins). Reagents were prepared according to Stahl method (7). Detection was carried out visually in visible light and under UV light (λ=365 nm).

Data analysis

The dose of the extract required to produce an anticonvulsant effect (ED50) or death (LD50) in 50% of the animals was calculated (8) using a commercial computer program (GRAPHPAD INSTAT 3, version 2003). Data obtained from convulsive tests were expressed as the percentage of the animals showing convulsions and Fisher's exact test was used to analyze the data. P value less than 0.05 was the critical criterion for statistical significance.