The Arachis hypogaea L. plants were collected from the shores of lake Yezi (Wuhan, Hubei, China) in August, 2011. The plant was authenticated by the Agriculture and Forestry Research Center of Tsukuba University, Japan. AHSLE powder was obtained as previously described (6,7), comprising 1.92% protein and 65.31% carbohydrate. All the reagents used were of the highest available purity. Pentobarbital sodium was purchased from Rejuvenation Pharmaceutical Co., Ltd., (Fuzhou, China). Muscimol was purchased from Sigma (St. Louis, MO, USA). Flumazenil and other chemicals used were purchased from Wako Pure Chemical Industries, Ltd., (Osaka, Japan).

Male SD rats, aged 8 weeks and weighing 270±30 g, were provided by the Laboratory Animal Resource Center, University of Tsukuba, Japan. The animals were housed at ambient conditions of 25°C, with 12-h light/dark cycles (light on at 08:00, light off at 20:00) and were given ad libitum access to food and water. All the animal experiments were conducted humanely, following approval from the Institutional Animal Experiment Committee of Tsukuba University, Japan and in accordance with the regulations for Animal Experiments and Fundamental Guidelines under the jurisdiction of the Japanese Ministries of Education, Culture, Sports, Science and Technology.

The experiments were performed between 20:00 and 24:00. Rats (n=48) were employed and habituated in an animal lab for at least 7 days, then randomly divided into groups. In the 4 experiments, the number of rats in each group was 12, 9–12, 12 and 8, respectively. All the rats were reused after 2 weeks. The animals were fasted for 24 h prior to the experiment. AHSLE in physiological saline and flumazenil in 10% dimethyl sulfoxide were administered orally to the rats, whereas muscimol was administered intraperitoneally (i.p.). At 30 min following the administration of muscimol, flumazenil or AHSLE, pentobarbital sodium in physiological saline was administered i.p. to each rat to induce sleep. The animals that remained immobile for >1 min were considered to be asleep. The sleep latency was recorded from the pentobarbital injection to 1 min after the loss of the righting reflex and the sleep time was recorded from 1 min after the loss of the righting reflex to recovery (8).

The obtained data were analyzed using two-tailed Student’s t-test (version 2003, Microsoft excel) for comparisons and the results are expressed as means ± SD. P<0.05 was considered to indicate statistically significant differences.

The results (Table I) revealed that pentobarbital induced sleep at a dosage of 50 mg/kg. Muscimol was unsuccessful in inducing sleep. AHSLE alone, similar to muscimol, was unable to induce sleep directly, even at a high dose (500 mg/kg).

Following administration of muscimol or AHSLE, pentobarbital (50 mg/kg) was administered i.p. to the rats. AHSLE decreased sleep latency and prolonged sleep time in rats, particularly at a dose of 80 mg/kg, following sleep induction by pentobarbital. Muscimol (0.2 mg/kg) as a positive control also induced a decrease in sleep latency and a prolongation of the total sleep time (Fig. 1).

Prior to the administration of pentobarbital (50 mg/kg), low doses of muscimol (0.05 mg/kg) were used in the AHSLE (120 mg/kg)-treated groups (Fig. 2) to observe the direct association between the effects of muscimol and those of AHSLE. The results demonstrated that the pentobarbital-treated rats in the AHSLE+muscimol group exhibited significantly (P<0.01) increased sleep time and reduced sleep latency.

In pentobarbital (50 mg/kg)-treated rats, AHSLE decreased sleep latency and also significantly prolonged sleep time at doses of 80 mg/kg (Fig. 1A and B). These actions were significantly antagonized by flumazenil, a benzodiazepine receptor antagonist, at a dose of 3.5 mg/kg (Fig. 3A and B). This antagonistic effect of flumazenil was also observed in rats that were administered muscimol.