4.4. Pentobarbital-Induced Sleep Test

The experimental procedure for sleep evaluation was based on the prolongation of pentobarbital-induced sleeping time [82]. The animals were randomly divided into five groups consisting of 8–12 mice each. All samples were dissolved in water prior to use. All experiments were performed between 13:00 and 17:00 h, and the mice were fasted for 12 h prior to the experiments. In the first experiment, to determine if MSE had a hypnotic effect, the following solutions were administered (p.o.) to mice using an oral Zonde needle 30 min prior to the intraperitoneal (i.p.) injection of pentobarbital: water as vehicle, valerian root extract (10 mg/kg) as a positive control, and MSE (110,100 mg/kg) for 7 days. In the second experiment, four groups of mice were administered (p.o.) with the following agents to determine the most effective fraction: water fraction (WF), butanol fraction (BuF), and ethyl acetate fraction (EAF) (10 mg/kg).

Sleep disturbance was induced by intraperitoneal injection of caffeine (10 mg/kg) 30 min before administering sodium pentobarbital [83]. Following pentobarbital injection (42 mg/kg, i.p.), mice were placed in individual cages and observed for measurements of sleep latency and sleep duration. The observers were blinded to the individual treatments. Mice were considered asleep if they stayed immobile and lost their righting reflex when positioned on their backs. Sleep latency was recorded from the time of pentobarbital injection to the time of sleep onset, and sleep duration was defined as the difference in time between the loss and recovery of righting reflex.