J. Biomedical Science and Engineering, 2010, 3, 268-273
doi:10.4236/jbise.2010.33036 Published Online March 2010 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online March 2010 in SciRes. http://www.scirp.org/journal/jbise
Sedative effects of peanut (Arachis hypogaea L.) leaf aqueous
extracts on brain ATP, AMP, Adenosine and Glutamate/GABA
of rats
Xiao-Yan Zu, Zhen-Ya Zhang*, Ji-Qiang Liu, Hong-Hai Hu, Guo-Qing Xing, Ying Zhang, Di Guan
Graduate School of Life and Environmental Sciences, Tsukuba University, Tsukuba, Japan; *Corresponding author.
Received 26 December 2009; revised 10 January 2010; accepted 17 January 2010.
ABSTRACT
Peanut (Arachis hypogaea L.) leaf aqueous extracts
(PLAE) has been reputed to be a type of sleep-aid in
China. To investigate the sedative effects and effect
pathways of PLAE, rats (n = 31) were employed in
two experiments and intragastrically administrated
of (1) distilled water, PLAE (500 mg/kg body weight
(BW)) and peanut stem aqueous extracts (PSAE, 500
mg/kg BW); (2) 0, 100 or 500 mg/kg BW of PLAE,
respectively for at least 14 days. Six relevant neuro-
transmitters were measured finally. Experiment-1 (n
= 16) results showed that the brain Lactate were sig-
nificantly elevated (p < 0.05) in rat cerebrums after
PLAE administrations, compared with Control and
PSAE groups. In respect of brain energy system, sig-
nificant degradations of the brain adenosine triphos-
phate (ATP) (p < 0.05) were observed in the brain-
stems and even the whole brains of rats though
PLAE treatments. Moreover, we found that the brain
Adenosine monophosphate (AMP) were clearly de-
creased (p < 0.05) in rat cerebrum and brainstem
regions, while the brain Adenosine revealed an in-
creasing propensity (p = 0.076) in the cerebrums of
freely behaving rats. After experiment-2 (n = 15),
the γ-aminobutyric acid (GABA) concentrations
were statistically (p < 0.05) enhanced and the ratios of
Glutamate/GABA were simultaneously reduced (p <
0.05) in rat brainstems, no matter which one dose
(100 or 500 mg/kg BW) of PLAE were used. Results
indicated that PLAE could influence the target neu-
rotransmitters that related to rat circadian rhythms
in the specific brain regions, possessing the potenti-
alities as a sedative or sleep-aid for hypnic therapy
purposes.
Keywords: Arachis Hypogaea L. Leaf Aqueous Extracts;
Sedative Effects; Rats; Neurotransmitters
1. INTRODUCTION
Given the high prevalence of insomnia and hypnotics
addiction worldwide, herb sedatives have attracted in-
creasing research interests in terms of substituting the
drug-addictive hypnotics. However, to our best knowl-
edge, most of herb-sedative studies remain inconclusive
in support of their effectiveness [1]. Therefore, a novel
herb medicine that is demonstratively available to alleviate
insomnia or sleep disorders is highly desired nowadays.
Peanut (Arachis hypogaea L.) leaf aqueous extracts
(PLAE) have received a long reputation in china as an
abirritative remedy to ease various sleep disorders [2],
and clinically validated by modernistic medical ap-
proaches [3,4]. However, many those researches only
focus on the clinical effects, and relevant studies on their
deep effect mechanisms are still lacking. Our studies
therefore carried out two rat experiments which lied in
clarifying PLAE effects and pathways in the sleep regu-
lation as well as evaluating their efficacies on spontane-
ous circadian rhythms of freely behaving rats.
By understanding how the animal circadian rhythms
are influenced via brain nerve system and neurotrans-
mitters [5,6], in our researches, PLAE efficacy can be
identified though brain neurotransmitter variation and
thereby we expect that its effect mechanisms can be re-
vealed to some extent though this way. Thus, after intr a-
gastric (i.g.) drug administrations we separated rat brain
tissues and then measured six neurotransmitters of Lac-
tate, Adenosine triphosphate (ATP), Adenosine mono-
phosphate (AMP), Adenosine (Ad), γ-aminobutyric acid
(GABA), and Glutamate (Glu) using HPLC (High per-
formance liquid chromatography) or Auto Amino Acid
Analyzer, to evaluate the hypnotic effects of PLAE and
further elucidate their effect roles correlated to PLAE on
rat sleep modulation.
2. MATERIALS AND METHODS
2.1. Animals, Plant Extracts and Reagents
Male Sprague-Dawley rats (8 weeks of age, weight of
270 ± 30 g) were used as research animals. They were

X. Y. Zu et al. / J. Biomedical Science and Engineering 3 (2010) 268-273
269
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housed at ambient circumstance of 25oC with 12 h
light/dark cycles (light on 08:00, light off 20:00), and
approached food and water ad libitum. All rat experi-
ments were carried out in a humane manner after re-
ceiving approval from Institutional Animal Experiment
Committee of the Tsukuba University (Japan), 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. The PLAE and peanut stem aqueous
extracts (PSAE) were extracted respectively from 98 oC
water (3 h, twice), following a filtration to remove the
residues. Their brown powders were obtained though
rotary evaporation and freeze drying. The regents of
ATP, AMP and Ad were purchased from Sigma Chemi-
cal Co. (St. Louis, MO, USA). Other chemicals used
were purchased from Wako Pure Chemical Industries,
Ltd. (Japan).
2.2. Experimental Protocols
Experiment-1: Sixteen rats were habituated in an animal
lab for at least 7 days, then randomly divided into the
following groups: Control (n = 5, distilled water), PLAE
(n = 6) and PSAE (n = 5). As described before [7], all
drug administrations were conducted intragastrically
(i.g.) for 14 days in a dose of 500 mg/kg body weight
(BW) before 8:00 (prior to the beginning of the light
phase). Experiment-2: After habituated for 7 days, the
rats (n = 15) employed were equally divided into three
groups, fed with 0, 100 or 500 mg/kg BW of PLAE (i.g.)
respectively for at least 14 days. After trials, the rats
were anaesthetized by Urethane (intra-peritoneal (i.p.)
injection, 200 mg/ml, 0.5 ml/100g BW), and dissected
rapidly to separate their whole brains. To verify the neu-
rotransmitter variation in diverse brain regions, the whole
brain was separated into three parts of cerebrum region,
brainstem region (midline involving thalamus and hypo-
thalamus) and cerebellum region. Considering the brain
responses to nucleoside and nucleotide synthesis, we
froze those brain tissues immediately and stored them at
–80oC till the target neurotransmitters were analyzed.
2.3. Neurotransmitter Analysis
After experiment-1, samples were firstly thawed and
homogenized with 4 oC saturated trichloroacetic acid
(TCA) in the sample volume of 25% (v/v), followed a
centrifugation to remove the protein sediments. The pH
values in cooled supernatants were then neutralized to
5-6 by NaOH. After filtered (0.45 µm membrane), the
supernatants were analyzed by the HPLC (Japanese
Jasco International Co., Ltd). Brain Lactates were meas-
ured though RI detector of HPLC on the basis of the
methods described before (Hallstrom et al., 1989), and
0.1% H3PO4 were used as the mobile phase. Analyzing
brain neurotransmitters of ATP, AMP and Ad, were per-
formed according to the methods reported previously [8]
with minor modifications. Simply, the samples were
analyzed by the HPLC with Capcell-Pak C18 column
(4.6 mm I.D. × 150 mm, particle size of 5 µm) in a flow
rate of 1 ml/min at the detection wavelength of 254 nm.
During testing, the mobile phase was 0.01 mol/L phos-
phate buffer solution (PBS, pH 6.5) that mixed with
methanol (99.7%) in a ratio of 85/15 (v/v). Concentra-
tions of ATP and its metabolites were calculated by
comparing peak areas with appropriate standards.
After experiment-2, the brain samples homogenized
were mixed with sulfosalicylic acid (3%) for 15 min
prior to centrifugation. The pH value of supernatants
were then adjusted to 2-3 using LiOH (3 mol/L). Brain
GABA and Glu in samples were determined by an auto
amino acid analyzer (JLC-500/V2, Jeol Ltd., Tokyo,
Japan) in accordance with the manufacturer’s specifica-
tions. Briefly, it was an ionic exchange chromatography
with a stepwise elution of free amino acids, and detec-
tion with ninhydrine. The final results were calculated
on the basis of the corresponding standards, and ex-
pressed as concentrations of nmol/mg in brain tissues.
2.4. Statistical Analysis
The obtained data were analyzed using a two-tailed Stu-
dent’s t-test, and results were expressed as mean ± SD.
Statistic difference was considered to be significant at
p < 0.05(*).
3. RESULTS
3.1. Brain ATP
As results shown in Figure 1, the ATP of control (Con.)
group in the cerebrum, brainstem, and whole brain were
0.114 ± 0.020, 0.109 ± 0.015 and 0.134 ± 0.022 nmol/ mg,
respectively. Contrasted to Control (Con.) Groups, the
brain ATP declined in both of the whole brain and the
single brain region after PLAE administrations. In detail,
The ATP from PLAE groups were slightly reduced in
cerebrums, while significant degradations were observed
(p < 0.05) in the brainstems and whole brains on com-
parison with Con. groups. In the case of PSAE treat-
ments, the enhancements of ATP were occurred in all
brain regions, especially in the cerebrums.
3.2. Brain AMP
In experiment-1, we also obtained the AMP concentra-
tions of 0.072 ± 0.013, 0.078 ± 0.005, 0.082 ± 0.007
nmol/mg respectively in the cerebrum, brainstem, and
whole brain from Con. groups. As can been seen from
Figure 2, after PLAE treated, the AMP were decreased
in almost all brain regions (cerebellum results were not
showed in this figure), particularly reduced (p < 0.05) in
the cerebrum and brainstem regions. The AMP from
PSAE groups showed no statistic changes compared

270 X. Y. Zu et al. / J. Biomedical Science and Engineering 3 (2010) 268-273
Figure 1. ATP concentrations in the cerebrum,
brainstem and whole brain of freely behaving rats
at experiment-1. Open bars, Con. (Control, distilled
water); closed bars, PLAE (500 mg/kg BW); di-
agonal bars, PSAE (500 mg/kg BW). Data were
expressed as mean ± SD of 5-6 independent sam-
ples (*p ＜ 0.05, vs. Con.).
Figure 2. AMP concentrations in the cerebrum,
brainstem and whole brain of freely behaving rats
at experiment-1. Open bars, Con. (Control, distilled
water); closed bars, PLAE (500 mg/kg BW); di-
agonal bars, PSAE (500 mg/kg BW). Data were
expressed as mean ± SD of 5-6 independent sam-
ples (*p ＜ 0.05, vs. Con.).
with group Con. though the trails, although the declines
were observed as well.
3.3. Brain Ad and Lactate
Figure 3 showed the results of Ad and lactate in Cere-
brum, and revealed that the Lactate changes were con-
sistent with Ad in cerebrum after PLAE administrations
in Experiment-1. As can be seen, compared with group
Con. and PSAE, the Ad amelioration (p = 0.076) in
PLAE group was occurred in cerebrum where played the
main roles on modulating animal sleepiness. And the
Lactate was elevated (p < 0.05) in cerebrum simultane-
ously after PLAE treated. On the other hand, PSAE re-
sults presented only slight differences in contrast to the
Con. groups.
3.4. Brain GABA
Brain GABA of PLAE-0 group were 0.767±0.180 and
0.767 ± 0.079 nmol/mg respectively in the brainstem and
Whole brain. And the GABA results of cerebrums and
cerebellums were monotonous and eliminated from
Figure 4. In the results, we found that the GABA con-
centrations exceeded statistically (p < 0.05) than PLAE-0
group in brainstems at both PLAE-100 and PLAE-500
groups. The GABA from PLAE-500 group showed sig-
nificant improvement (p < 0.05) even in the whole brain
of the experimental rats.
3.5. Brain Glu
After experiment-2, we investigated the brain Glu contents
in different brain regions simultaneously. As Figure 5
Figure 3. Concentrations of Ad and Lactate in the cerebrum of
freely behaving rats at experiment-1. Open bars, Con. (Control,
distilled water); closed bars, PLAE (500 mg/kg BW); diago-
nal bars, PSAE (500 mg/kg BW). Data were expressed as mean
± SD of 5-6 independent samples (*p ＜ 0.05, vs. Con.).

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271
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Figure 4. GABA concentrations in the
brainstem and whole brain of freely be-
having rats at experiment-2. Open bars,
PLAE-0 (Control, without PLAE); gray
bars, PLAE-100 (100 mg/kg BW of PLAE);
closed bars, PLAE-500 (500 mg/kg BW of
PLAE). Data were expressed as mean ± SD
of five independent samples (*p ＜ 0.05,
vs. PLAE-0).
Figure 5. Glu concentrations in the brain-
stem and whole brain of freely behaving
rats at experiment-2. Open bars, PLAE-0
(Control, without PLAE); gray bars,
PLAE-100 (100 mg/kg BW of PLAE);
closed bars, PLAE-500 (500 mg/kg BW of
PLAE). Data were expressed as mean ± SD
of five independent samples (*p ＜ 0.05,
vs. PLAE-0).
shown, the Brain Glu in the brainstem and whole brain
were 1.330 ± 0.139 and 1.361 ± 0.141 nmol/mg respec-
tively, which elucidated that the rats in PLAE-0 group
possessed the higher contents of initial brain Glu than
the brain GABA. Moreover, on comparison with
PLAE-0 groups, the Glu concentrations were greatly
raised (p < 0.05) in the brainstem as well as the whole
brain, no matter which one dosage (100 or 500 mg/kg
BW) of PLAE were administered in experiment-2.
3.6. The ratio of Glu/GABA
Considering the results in Figure 4 and 5, the ratios of
Glu/GABA was important to evaluate the excitatory or
suppressive state of the rat brain, we thereby calculated
it and presented the results in Figure 6. Available vari-
ances have not been observed in the whole brains of the
three groups. Moreover, compared to the PLAE-0
groups, the ratio of Glu/GABA in PLAE-100 and PLAE-
500 groups were specifically decreased (p < 0.05) in
brainstems where showed important relations with the
two neurotransmitters.
4. DISCUSSION
4.1. Effects of PLAE and PSAE on Brain Energy
Metabolism
ATP is the direct energy source of life activities. Its decline
(Figure 1) in rat brain on our investigations re flected
Figure 6. Ratios of Glu/GABA in the brain-
stem and whole brain of freely behaving rats
at experiment-2. Open bars, PLAE-0 (Control,
without PLAE); gray bars, PLAE-100 (100
mg/kg BW of PLAE); closed bars, PLAE-500
(500 mg/kg BW of PLAE). Data were ex-
pressed as mean ± SD of five independent
samples (*p ＜ 0.05, vs. PLAE-0).

272 X. Y. Zu et al. / J. Biomedical Science and Engineering 3 (2010) 268-273
the requirement of energy reserves, and indicated the
potential of rat sleepiness. AMP can transfer into ATP or
degraded into Ad [9]. According to the deficiency of
ATP in the results, the reduction of AMP (Figure 2) in
group PLAE elucidated the tendency of its breaking
down to Ad. As reported previously, Ad is a critical
neurotransmitter in brain sleep system [10], which ac-
cumulates in the cholinergic basal forebrain of cerebrum,
has been proposed as one of the important homeostatic
sleep factors [11]. In our researches, as a result of the
deficiency of high-energy phosphates, the observed in-
creases of Ad concentrations in cerebrums reflected a
bio-energetic stress of animals. Energy depletion can
build up the sleep pressure [7] and it is an available
pathway and measure to explain the PLAE induced sleep
propensity. From experiment-1, the great decrease of the
ATP and AMP strongly indicated that the PLAE was
promising in driving homeostatic sleeps of freely be-
having rats. Besides, the insignificant accumulation of
Ad in the PLAE group suggested a mild effect of PLAE
on sleep ameliorating.
Lactate, an end product of metabolic system, can be
regarded as an additional metabolic marker of the sleep
pressure. Our results (Figure 3) illuminated that Lactate
changed together with Ad, in agreement with those re-
ported previously [12] and indicative of the PLAE con-
tributions in sedative or hypnic propensities.
In all PSAE investigations, the rat brain energy sys-
tem presented the great enhancement of ATP in cere-
brum compared with Control and PLAE groups. How-
ever, such variations were disadvantageous for estab-
lishing the sleep pressure and meliorating the sleep of
our experimental rats.
4.2. Effects of PLAE on Brain GABA, Glu and
the Ratio of Glu/GABA
GABA is a type of inhibitory neurotransmitter amino
acid in brain. To clarify the PLAE effects on brain
GABA, fifteen rats were employed in experiment-2 and
randomly divided into three groups, and treated with
PLAE in diverse dosages for 14 days. The GABA-
contained neurons in brainstem and thalamic nucleus
released GABA neurotransmitters [13,14], which can
response for inhibiting target neurons of the arousal sys-
tems in brainstem [15] and play an important role in
meliorating rats’ sleep [6]. Thus, the GABA increase in
our studies (Figure 4) not only suggested the specific
effect region (brainstem) of GABA-neurotransmitter,
and also testified the sedative efficacy of PLAE.
It is well demonstrated that Glu play an excitatory role
in animal brain, and then drives the animals to arousal.
Glu receptors, expressed in the basal forebrain [16,17],
stimulate gamma and theta electroencephalographic
(EEG) activities of arousal and then suppress rat sleepi-
ness [18,19]. In these researches, we found that PLAE
also aroused the neurons to release Glu neurotransmit-
ters (Figure 5) for exciting the rat brains.
In deed, GABA can be formed from Glu primarily via
the action of Glu decarboxylase [20]. Glu/GABA in
brainstem is a type of available parameters that can
characterize the state of sleep-wake system [21]. Since
GABA is inhibitory and Glu is excitatory, both of them
control many tranquilizing or exciting processes in rat
brain. Apparently, PLAE we used stimulated both the
brain Glu and the brain GABA. However, the
sleep-modulation effects of Glu sited specifically in
perifornical-lateral hypothalamic area [22] of the brain-
stem which is the main region that releases GABA to
inhibit the arousal of animal. Hence, the Glu/GABA
decrease in this area (Figure 6) indicated superior
GABA efficacy than Glu, and preferred to drive rats into
drowsiness. On the other hand, as a mild tranquilizer, it
was not surprising that PLAE significantly elevated
GABA-mediated neurotransmission, and reduced Glu/
GABA in target brain region. Thus, in profile, it was still
suggested the PLAE efficacy on spontaneous sleep im-
provement.
In summary, PLAE resulted in the great ATP con-
sumption, slight Ad accumulation, as well as the signifi-
cant decrease of Glu/GABA in corresponding brain areas.
It revealed a mild hypnotic effect of PLAE on sleep
ameliorations. In the case of sedatives, the PSAE was
considered to be ineffective on meliorating rats’ sleeps at
least view the matter from our investigations. On the
other hand, in these researches, more detailed works on
the effect components of such PLAE are desired to be
determined in the future. Moreover, apart from the
aqueous extracts of peanut leaves and stems, alcohol
extracts of them are needed as well to as the contrasts in
further investigations.
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