AHSL were collected from the banks of Yezi Lake, Wuhan, Hubei, China in August 2011. The plant was authenticated by the Agriculture and Forestry Research Center of Tsukuba University, Japan. It was cleaned, air-dried and passed through a 40-mesh standard sieve for extraction. The AHSLE were obtained by a previously described method (4). The AHSL were extracted twice with 98°C water (3 h each time), followed by filtration to remove residue. Brown powders were obtained through rotary evaporation and freeze drying of the supernatant. ATP, ADP and Ad standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals used were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

All animals and experiments were supported by the Laboratory Animal Resource Center of the University of Tsukuba, Japan. Male Sprague-Dawley rats (8 weeks old, 270±30 g) were housed at 25°C with 12 h light/dark cycles (light on 08:00, light off 20:00), and food and water ad libitum. All rat experiments were performed in a humane manner after receiving approval from the Institutional Animal Experiment Committee of Tsukuba University, and in accordance with the regulations for animal experiments and fundamental guidelines under the jurisdiction of the Japanese Ministry of Education, Culture, Sports, Science and Technology.

In total, 34 rats were habituated in the animal lab for at least seven days, then randomly divided into two groups: the control group (water, n=16) and the AHSLE group [500 mg/kg body weight (BW), n=18]. Based on the method described previously (8) with certain modifications, the rats in the AHSLE group were randomly divided into three subgroups: SD-2.5 h (n=6), SD-5 h (n=6) and recovery (Rec)-3 h (n=6). All drug administration was conducted intragastrically (i.g.) for 14 days at a dose of 500 mg/kg BW before 8:00 a.m. (prior to the start of the light phase). On the 14th day, following drug administration, SD was achieved by the gentle handling method, as described previously (9). Rats were continuously inspected and SD was realized by tapping the cage lightly or touching the animal with a paintbrush. SD for 2.5 h or 5 h was started at 8:00 a.m. and performed under ‘lights-on’ conditions. The 3 h Rec started once the rats had been deprived of sleep for 5 h. The SD and Rec groups each had their own control group (n=5–6) of rats, which were treated under the same conditions.

Following the trials, the rats were anaesthetized with an intraperitoneal injection of urethane (200 mg/ml, 0.5 ml/100 g BW), and dissected rapidly to isolate the whole brains. To verify the differences in neurotransmitters at diverse brain regions, the entire brain was separated into three regions: the cerebrum, brainstem (midline involving thalamus and hypothalamus) and cerebellum. Considering the brain responses to nucleoside and nucleotide synthesis, the brain tissues were frozen immediately and stored at −80°C, prior to analyzing the target neurotransmitters.

ATP, ADP and Ad were analyzed by HPLC (JASCO International Co., Ltd, Tokyo, Japan). Rat brain samples were homogenized at 4°C with trichloroacetic acid (TCA) at a final concentration of 25% (v/v) and centrifuged to remove protein sediment. Supernatants were subsequently neutralized to pH 5–6 with NaOH, following filtration (0.45 μm membrane). Analyses of the brain neurotransmitters, ATP, ADP and Ad, were performed according to previously reported methods (10) with minor modifications. The samples were analyzed by HPLC with a Capcell-Pak C18 column [4.6 mm internal diameter (I.D.) × 150 mm, particle size 5 μm] at a flow rate of 1 ml/min and detected at 254 nm. The mobile phase consisted of a gradient mixture with solvent A (0.1 mol/l NH₄H₂PO₄ solution) and B [20% (v/v) methanol (99.7%) in solvent A]. During testing, the gradient was as follows: 0 min, 0% B; 6–18 min, 35% B; 26 min, 0% B. The flow-rate was 1 ml/min with an injection volume of 20 μl. The ATP, ADP and Ad standard were 100, 100 and 25 μmol/l, respectively, in concentration and diluted by five gradients, respectively. In the brain samples, ATP and its metabolites were calculated by comparing peak areas and the appropriate standard curves.

Simultaneous distillation and extraction (SDE) was used to extract the essential oil from the AHSL. Petroleum ether and distilled water were used as low- and high-density solvents (contained 30 g AHSL/80 ml), respectively. The low- and high-density distillers for SDE were simultaneously heated by electric sets, so that the steam from the two distillers mixed with each other at the top of the glass instrument and subsequently divided into aqueous and petroleum ether layers. The petroleum ether layer with the essential oil from AHSL flowed to the low-density distiller and the aqueous layer flowed to the high-density distiller. The cyclical process was continued for 5–6 h, and three 30 g samples of AHSL were extracted by this method. The essential oil obtained was merged, following the removal of trace water by treatment with anhydrous Na₂SO₄. Following rotary evaporation, the essential oil in the petroleum ether was stored at −20°C.

The essential oil in the petroleum ether was analyzed using an Agilent 7890 A chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) coupled with a 5975 network mass selective detector and equipped with a HP-5 capillary fused silica column (30 m × 0.25 mm I.D. × 0.25 μm film thickness). The oven temperature was maintained at 80°C for 5 min, and then programmed to increase at a rate of 4°C/min to 280°C. Other operating conditions were as follows: carrier gas, He (99.999%) with a flow rate of 1 ml/min; injector temperature, 250°C; split ratio, 1:50; and injection volume, 1 μm. The mass spectra were recorded at 70 eV with a mass range from m/z 20–500.

The linalool standard was at concentrations of 6.25, 12.5, 25, 50, 100 and 200 μg/ml, with an internal standard of cyclohexanone with a final concentration of 100 μg/ml. The linalool standard was analyzed by the method mentioned previously with the modification that the oven temperature was maintained at 80°C for 5 min and was then programmed to increase at 4°C/min to 160°C. The concentration of linalool in the essential oil was calculated by comparing the peak area and the appropriate standard curve.

The data were analyzed using a two-tailed Student's t-test and the results are expressed as the means ± standard deviation. P<0.05, P<0.01 and P<0.001 are considered to indicate statistically significant differences.

It was demonstrated that in the AHSLE groups, brain ATP levels increased with SD time and decreased following 3 h Rec (Fig. 1). In the cerebrum and cerebellum, the ATP levels were reduced in the SD-2.5 h group, while they were increased in the SD-5 h group, compared with the respective controls. ATP levels were enhanced significantly (P<0.01) in the brainstems of the AHSLE-treated rats compared with the controls.

As the results (Fig. 2) demonstrated, the ADP levels decreased with increased SD time in the AHSLE and control groups. Following the administration of AHSLE, the ADP levels were enhanced during the SD phase compared with the respective control and the 3 h Rec did not increase the ADP values to normal levels.

According to the results (Fig. 3), the Ad values in the AHSLE groups increased with SD time in all brain regions in addition to the entire brain, and then reduced following 3 h Rec sleep. Following the administration of AHSLE, the Ad variation trend was similar to that of ATP; Ad accumulated in the brainstem and cerebellum during the SD phase. Contrasting results were observed in the control groups.

As can be seen in Table I, a total of 37 compounds were identified, and the major components were 3,7-dimethyl-1,6-octadien-3-ol (linalool, 16.17%), n-hexadecanoic acid (16.42%), 1-octen-3-ol (product of linalool decomposition, 8.48%,), 1,2,3-trimethylbenzene (6.56%), phytol (6.34%), 2-methoxy-4-vinylphenol (4.70%), 6,10,14-trimethyl-2-pentadecanone (3.96%), α,α,4-trimethyl-3 cyclohexene-1-methanol (3.80%), 1-ethyl-2-methylbenzene (3.74%), cis-linalool oxide (3.54%), 1,3,5-trimethylbenzene (2.94%) and benzeneacetaldehyde (2.81%; Table I). These 12 major components accounted for 79.46% of the yield, while other identified constituents represented <2%. The linalool standard curve obtained was Y=2926.4 × −16702, R²=0.9997. According to the peak area and standard curve, the linalool concentration in petroleum ether was ∼10.415 mg/ml, and yield was ∼578.611 mg/kg AHSL.