**2.5.2 Pharmacological and toxicity evaluation**

#### **2.5.2.1 Open field activity**

Thirty minutes after the administration of vehicle or test compound a mouse was placed in the centre of a round open field of 30 cm diameter and 25 cm high and the open field activity were measured during 6 minutes recording how many times the animal stay in the centre of cage and the number of rising (Sukma et al., 2002; Tyler, 1982).

#### **2.5.2.2 Aggressive behavior**

A group of animal was isolated in individually cage and other group remained grouped during six week. The aggressive behaviors were evaluated through an intruder mouse into the isolated mice's home cage and were recorder the aggressive activity (biting attacks and wrestling) in isolated mice was measure as total fighting time during a 20 min period (Tyler, 1982).

#### **2.5.2.3 Thiopental-induced sleep**

Animals were divided into three groups: control (distilled water), diazepam (1 mg/kg) treated and SR-treated groups. Thiopental sodium (30 mg/kg) was injected intraperitoneally 30 min after administration of vehicle or test compound. An animal was placed on its back on a warmed (35 ºC) pad. The number of sleeping animals and the duration of loss of righting reflex were recorded. The duration from loss of righting reflex until a mouse regained its righting reflex was measured (Carlini et al., 1986).

#### **2.5.2.4 Drug-induced convulsion**

Mice were divided in groups of ten each. The animals were pre-treated with SR 30 min before the administration of PTZ (85 mg/kg, s.c.) or PTX (10 mg/kg, s.c.). The anticonvulsive effect was assessed by measuring the numbers of convulsing mice and deaths, and the latency of the appearance of the first episode of clonic seizure. The cut off time was set as 30 min. after the convulsant administration (Costa & Greengard, 1975; Fischer & Vander, 1998).

#### **2.5.2.5 Elevated plus-maze behavior**

The elevated plus-maze consisted of two closed arms (30\_/5\_/15 cm) and two open arms (30\_/5 cm) emanating from a common central platform (5\_/5 cm). The two pairs of identical arms were opposite each other. The entire apparatus was elevated to a height of 54 cm above floor level. Thirty minutes after test compound administration, the mouse was placed at the centre of the maze with its head facing an open arm and allowed to explore the maze for 5 min. Entry into an arm was defined as placement of all four paws into an arm and were recorded: number of entries into each type of arm, the percentage of time spent and the percentage of arm entries in open arms (Sukma et al., 2002).

#### **2.5.2.6 Amphetamine-induced behavioral stereotypy**

Amphetamine (1.5 mg/Kg) was injected subcutaneously 30 min after administration of vehicle or test compound in rats and the animals were collocated in individually cage to recorder the behavioral stereotypy each 5 min during 1 h. (Kuczenski et al., 1999).

#### **2.5.2.7 Amphetamine -induced sleep in mice**

Mice were divided in four groups of ten each. The animals were pre-treated with SR 30 min before the administration of amphetamine 5 mg/Kg (p sc). An animal was placed on its back on a warmed (35 ºC) pad and the number of sleeping animals and the duration of loss of righting reflex were recorded.

#### **2.5.2.8 Acute oral toxicity study**

A single dose of SR (2000 mg/kg) or distilled water was administered orally (10 mL/Kg) in equal number female (n=3) animals; and rats were returned to an *ad libitum* diet immediately after dosing. All animals were monitored continuously for 12 h after dosing for signs of toxicosis and daily for changes additional behavioural or clinical signs. The animal weights were recorded weekly. Rats were euthanized on day 14 by ether inhalation, and selected organs removed and examined macroscopically for toxicant-induced changes (OECD, 2001).

#### **2.5.2.9 Statistics**

522 Pharmacology

They were deprived of food but not water 6 h before the drug administration and each group consisted of ten animals. All experiments were carried out between 8:00 am and 11:00 am in accordance with the Institutional Animal Ethical Committee approved the study and

SR was administrated in three doses levels (100, 200 y 400 mg/Kg) in all experiment except the elevated plus-maze behaviour test (50, 100 y 150 mg/Kg). The volume of injection in mouse was 0.4 ml/20 g and in rat was 1 ml/100 g. The SR was dissolved in distilled water

Thirty minutes after the administration of vehicle or test compound a mouse was placed in the centre of a round open field of 30 cm diameter and 25 cm high and the open field activity were measured during 6 minutes recording how many times the animal stay in the

A group of animal was isolated in individually cage and other group remained grouped during six week. The aggressive behaviors were evaluated through an intruder mouse into the isolated mice's home cage and were recorder the aggressive activity (biting attacks and wrestling) in isolated mice was measure as total fighting time during a 20 min period (Tyler,

Animals were divided into three groups: control (distilled water), diazepam (1 mg/kg) treated and SR-treated groups. Thiopental sodium (30 mg/kg) was injected intraperitoneally 30 min after administration of vehicle or test compound. An animal was placed on its back on a warmed (35 ºC) pad. The number of sleeping animals and the duration of loss of righting reflex were recorded. The duration from loss of righting reflex

Mice were divided in groups of ten each. The animals were pre-treated with SR 30 min before the administration of PTZ (85 mg/kg, s.c.) or PTX (10 mg/kg, s.c.). The anticonvulsive effect was assessed by measuring the numbers of convulsing mice and deaths, and the latency of the appearance of the first episode of clonic seizure. The cut off time was set as 30 min. after the convulsant administration (Costa & Greengard, 1975;

The elevated plus-maze consisted of two closed arms (30\_/5\_/15 cm) and two open arms (30\_/5 cm) emanating from a common central platform (5\_/5 cm). The two pairs of identical arms were opposite each other. The entire apparatus was elevated to a height of 54 cm above floor level. Thirty minutes after test compound administration, the mouse was placed

until a mouse regained its righting reflex was measured (Carlini et al., 1986).

centre of cage and the number of rising (Sukma et al., 2002; Tyler, 1982).

animal care was in conformity with Canadian Council for Animal Care guidelines

and administered orally.

**2.5.2.1 Open field activity** 

**2.5.2.2 Aggressive behavior** 

**2.5.2.3 Thiopental-induced sleep** 

**2.5.2.4 Drug-induced convulsion** 

**2.5.2.5 Elevated plus-maze behavior** 

Fischer & Vander, 1998).

1982).

**2.5.2 Pharmacological and toxicity evaluation** 

Drug effects were assessed by single factor analysis of variance followed by the Student- /Newman-/Keuls post-hoc test. The level of significance was set at p<0.05.
