Antidepressant-Like Effect of Cordyceps sinensis in the Mouse Tail Suspension Tes

Antidepressant-Like Effect of Cordyceps sinensis in the Mouse Tail Suspension Tes

Cordyceps sinensis (CS) is a fungus parasitic to Hepialidae
larvae and has been known as a traditional medicine in
China. Many studies have shown that CS possesses anti-fatigue,
1) antitumor,2,3) antioxidant,4,5) immunomodulatory,6—8)
hypoglycemic,9,10) and vasorelaxant activities.11) Since a relationship
between depression and fatigue has been well established,
12—14) we determined to evaluate its antidepressive effect
using the tail suspension test (TST)15,16) that, in parallel
with the forced swimming test,17) has been proposed as a primary
screening test for antidepressant drugs.
The most prevalent theory for the pathogenesis of depression
is the “monoamine hypothesis” in which monoamines
(e.g., norepinephrine, dopamine, and serotonin) are thought
to play important roles in the development of symptoms.
Many antidepressant drugs work by modulating these neurotransmission
systems.18,19) Recently, behavioral tests have
been used to study mechanisms underlying some antidepressant-
like substances with inhibitors of neurotransmitters such
as alpha-1 adrenoreceptor antagonists, selective dopamine
D2 receptor antagonists, and serotonin synthesis inhibitors.
20—23) In addition to these screening behavioral tests of
antidepressants, counting the numbers of head twitches induced
by 5-hydroxytryptophan in mice is an effective method
to evaluate serotoninerigic effects of drugs in vivo.24) It has
been generally accepted that numbers of head twitches
represent the level of 5-hydroxytryptamine (5-HT) in the
synapses.
In the present study, we have evaluated the antidepressantlike
effects of hot water extract (HWCS) and supercritical
fluid extract (SCCS), obtained from CS, using the TST in
mice. We have also dissected the mechanism underlying the
antidepressant actions of SCCS by examining the effects of
pretreatment with an alpha-1 adrenoreceptor antagonist, a selective
dopamine D2 receptor antagonist, and an inhibitor of
serotonin synthesis. Furthermore, to determine whether the
antidepressive effect of SCCS is mediated by the serotoninergic
system, we have evaluated the effect of SCCS on 5-HTPinduced
head twitch response in mice.
MATERIALS AND METHODS
Animals Five-week-old male C57 BL/6 mice were purchased
from Japan SLC Inc. (Hamamatsu). They were
housed in clear plastic cages (152213 cm high) in groups
of 5 mice per cage with free access to food and water. The
temperature of the room in which the animals were housed,
treated, and observed was maintained at 231 °C with constant
humidity (557%). The room was illuminated from
8:00 a.m. to 8:00 p.m. The animals were acclimatized for at
least 5 d before behavioral experiments. All experiments
were performed according to guidelines for the care and use
of laboratory animals of the Keio University School of Medicine.
Drug Treatment The following drugs were used: prazosin
hydrochloride, ()-sulpiride, DL-p-chlorophenylalanine
(p-CPA), 5-hydroxy-L-tryptophan (5-HTP), fluoxetine hydrochloride,
and polyoxyethylene sorbitan monooleate
(Wako Pure Chemical Indusries, Ltd., Osaka); clorgyline hydrochloride
(MP Biomedicals, Inc., Solon, OH, U.S.A.); desipramine
hydrochloride (Sigma-Aldrich, St. Louis, MO,
U.S.A.); bupropion hydrochloride (Toronto Research Chemicals
Inc., North York, Canada). Prazosin hydrochloride, clorgyline
hydrochloride, desipramine hydrochloride, bupropion
hydrochloride and fluoxetine hydrochloride were dissolved in
saline (0.9% NaCl solution). ()-Sulpiride and p-CPA were
suspended in 1% polyoxyethylene sorbitan monooleatesaline.
All drugs were injected in a volume of 10 ml/kg. The
same amounts of vehicles were administered for control
1758 Vol. 30, No. 9
Antidepressant-Like Effect of Cordyceps sinensis in the Mouse Tail
Suspension Test
Koji NISHIZAWA,a Kosuke TORII,a Aya KAWASAKI,a Masanori KATADA,a Minoru ITO,a
Kenzo TERASHITA,b,c Sadakazu AISO,b,c and Masaaki MATSUOKA*,b
a Noevir-Keio Research Laboratory, Noevir Co., Ltd.; 35 Shinanomachi, Shinjuku-ku, Tokyo 160–8582, Japan:
b Department of Cell Biology and Neuroscience, KEIO University School of Medicine; and c Department of Anatomy, KEIO
University School of Medicine; 35 Shinanomachi, Shinjuku-ku, Tokyo 160–8582, Japan.
Received June 5, 2007; accepted June 23, 2007; published online June 26, 2007
Cordyceps sinensis (CS) has been known as a component of traditional medicines that elicit various biological
effects such as anti-fatigue, immunomodulatory, and hypoglycemic actions. Since it has been well-established
that fatigue is closely related to depression, we used the tail suspension test (TST) in mice to examine the antidepressant-
like effects of hot water extract (HWCS) and supercritical fluid extract (SCCS) of CS. Immobility time
in the TST was reduced by administration of SCCS (2.5—10 ml/kg, p.o.) dose-dependently though it was not reduced
by treatment with HWCS (500—2000 mg/kg, p.o.). Neither HWCS nor SCCS altered locomotor activity in
the open field test, excluding the possibility that the effect of SCCS is due to activation of locomotion. Pretreatment
with prazosin (an adrenoreceptor antagonist) or sulpiride (a dopamine D2 receptor antagonist) reduced the
effect of SCCS on the immobility time. In contrast, pretreatment with p-chlorophenylalanine (p-CPA, a serotonin
synthesis inhibitor) did not alter the anti-immobility effect of SCCS. The last finding is consistent with an
additional observation that SCCS had no effect on head twitch response induced by 5-hydroxy-L-tryptophan in
mice. Taken altogether, these results suggest that SCCS may elicit an antidepressant-like effect by affecting the
adrenergic and dopaminergic systems, but not by affecting the serotonergic system.
Key words Cordyceps sinensis; depression; tail suspension test; noradrenaline; dopamine
Biol. Pharm. Bull. 30(9) 1758—1762 (2007)
∗ To whom correspondence should be addressed. e-mail: sakimatu@sc.itc.keio.ac.jp © 2007 Pharmaceutical Society of Japan
mice.
Preparation of HWCS A hot water extract of CS
(HWCS) was prepared by autoclaving 20 g of CS in 400 g of
water at approximately 120 °C for 20 min, followed by filtration
and freeze-drying (yield: approximately 30%). HWCS
was dissolved in distilled water and administered in a volume
of 10 ml/kg.
Preparation of SCCS A supercritical fluid extract
(SCCS, 4.82 kg) was obtained from 55.2 kg of CS by CO2
extraction conducted at a pressure of 25MPa, 40 °C using a
supercritical fluid extraction system (batch method, 300L,
UHDE GmbH, Germany).
Measurement of the Total Duration of Immobility in
the TST Immobility time during tail suspension was measured
according to the method described previously.15) The tail
suspension apparatus consisted of a gray polyethylene box
(353540 cm high) with a hook in the center of the ceiling.
Each mouse was individually suspended by the tail from
the hook with an adhesive tape. Total duration of immobility
during the 6-min test was calculated as immobility time. To
evaluate the effects of HWCS and SCCS, mice were treated
with each extract (HWCS: 500, 1000, 2000 mg/kg, p.o.,
SCCS: 2.5, 5, 10 ml/kg, p.o.) or with water for 5 consecutive
days. The last administration was conducted 1 h before the
test.
To dissect the mechanism underlying the antidepressantlike
effect of SCCS in the TST, we assessed the effects of
pretreatment with prazosin (an alpha 1 adrenoceptor antagonist,
1 mg/kg, i.p., administered 45 min before the test),
sulpiride (a selective dopamine D2 receptor antagonist,
20 mg/kg, i.p., administered 45 min before the test), or p-
CPA (a serotonin synthesis inhibitor, 300 mg/kg, i.p., administered
72, 48, and 24 h before the test), on the antidepressive
action of SCCS at the most effective dose (10 ml/kg, p.o. for
5 d). As a positive control, desipramine (30 mg/kg, i.p.),
bupropion (10 mg/kg, i.p.), or fluoxetine (20 mg/kg, i.p.) was
administered for 5 consecutive days. The last administration
was conducted 30 min before the test. Prazosin, sulpiride, or
p-CPA was administered according to the same schedule as
above.
Measurement of Locomotor Activity in the Open Field
Test (OFT) The open field apparatus was made of a gray
polyethylene box (907040 cm high). The floor of the apparatus
was divided into 63 squares of equal area (10
10 cm) by black lines. Each mouse was moved from its home
cage to the center square of the open field. Crossings of the
black lines were counted for 10 min. Mice were treated with
each extract (HWCS: 500, 1000, 2000 mg/kg, p.o., SCCS:
2.5, 5, 10 ml/kg, p.o.) or with water for 6 consecutive days.
The last administration was conducted 1 h before the test.
Measurement of Head Twitch Response (HTR) Induced
by 5-HTP Plus Clorgyline Clorgyline (1 mg/kg,
i.p.), a monoamine oxidase inhibitor, was given as pretreatment
1 h prior to injection of 5-HTP (150 mg/kg, i.p.). After
the administration of 5-HTP, each mouse was placed in a
clear plastic cage (152213 cm high). The frequencies of
head twitches were counted for 2 min at 10 min intervals
from 10 to 50 min after injection of 5-HTP as head twitch response
(HTR). SCCS (10 ml/kg, p.o.) was administered 1 h
before the injection of 5-HTP while fluoxetine (10 mg/kg,
i.p.) was administered as a positive control 30 min before the
5-HTP injection.
Statistics Data are shown as the meanS.E.M. The data
indicating the dose dependency of HWCS and SCCS in the
TST and the OFT were analyzed by the Dunnett’s test after
the Bartlett test and one-way analysis of variance (ANOVA).
The data indicating the effects of SCCS pretreated with the
inhibitors of neurotransmitters in the TST were evaluated
using the Newman–Keuls test after the Bartlett test and the
one-way ANOVA. The data for the effects of SCCS on the
HTR were analyzed by the Student t-test or the Aspin–Welch
t-test after dispersal analysis using the F-test. p values less
than 0.05 were considered to indicate statistical significance.
RESULTS
The Effects of HWCS and SCCS on Immobility Time
in the TST and on Normal Behaviour in the OFT in Mice
Administration of HWCS (500—2000 mg/kg, p.o. for 5 d)
showed no effects on immobility time in the TST (Fig. 1A).
In contrast, administration of SCCS (2.5—10 ml/kg, p.o. for
5 d) decreased the immobility time in a dose-dependent
manner (Fig. 1B). Meanwhile, neither HWCS (500—2000
mg/kg, p.o. for 6 d) nor SCCS (2.5—10 ml/kg, p.o. for 6 d)
affected the number of crossings in the OFT (Figs. 2A, B),
indicating that these extracts did not affect normal behavior
of mice. There were also no effects of HWCS and SCCS on
the frequencies of rearing, glooming, defecating and urinating
(data not shown).
The Effect of Pretreatment with Prazosin on the Antidepressive
Action of SCCS and Desipramine in Mice
Pretreatment with prazosin (1 mg/kg, i.p.), an alpha-1
adrenoceptor antagonist, significantly blocked the decrease in
immobility time elicited by SCCS in the TST (Fig. 3A). De-
September 2007 1759
Fig. 1. The Effects of HWCS (500—2000 mg/kg, p.o. for 5 d, Panel A
[ANOVA: F(3,64)0.104; p0.96]) and SCCS (2.5—10 ml/kg, p.o. for 5 d,
Panel B [ANOVA: F(3,64)3.49; p0.021]) on Immobility Time in the
Mouse TST
The last administration of HWCS or SCCS was conducted 1 h before the test. Values
are shown as meanS.E.M. (n17). ∗ p0.05 vs. vehicle-treated control group (C).
sipramine (30 mg/kg, i.p. for 5 d), a tricyclic antidepressant
known as a noradrenaline reuptake inhibitor, also showed an
anti-immobility effect. The anti-immobility effect of desipramine,
as well as SCCS, was blocked by pretreatment
with prazosin (Fig. 3B).
The Effect of Pretreatment with Sulpiride on the Antidepressive
Action of SCCS and Bupuropion in Mice
Pretreatment with sulpiride (20 mg/kg, i.p.), a selective
dopamine D2 antagonist, significantly blocked SCCS action
(Fig. 4A). Pretreatment with sulpiride also abolished the antiimmobility
effect of bupropion (10 mg/kg, i.p.), a dopamine
reuptake inhibitor (Fig. 4B).
The Effect of Pretreatment with p-CPA on the Antidepressive
Action of SCCS and Fluoxetine in Mice Pretreatment
with p-CPA (300 mg/kg, i.p. for 3 d) did not alter
the action of SCCS in the TST (Fig. 5A). In contrast, the antidepressive
effect of fluoxetine, a selective serotonin reuptake
inhibitor, was significantly blocked by the pretreatment with
p-CPA (Fig. 5B).
The Effects of SCCS or Fluoxetine on 5-HTP Plus
Clorgyline-Induced HTR in Mice The effect of SCCS or
fluoxetine on 5-HTP plus clorgyline-induced HTR in mice is
demonstrated in Fig. 6A. SCCS treatment did not affect the
HTR pattern. In contrast, fluoxetine treatment at a dose of
10 mg/kg, i.p. significantly increased the number of 5-HTPinduced
HTR at 10—12 min and decreased the number of
HTR at 30—32 min (Fig. 6B).
1760 Vol. 30, No. 9
Fig. 2. The Effects of HWCS (500—2000 mg/kg, p.o. for 6 d, Panel A
[ANOVA: F(3,16)0.613; p0.62]) and SCCS (2.5—10 ml/kg, p.o. for 6 d,
Panel B [ANOVA: F(3,16)0.905; p0.46]) on Locomotor Activity in the
Mouse OFT
The last administration of HWCS or SCCS was conducted 1 h before the test. Values
are shown as meanS.E.M. (n5).
Fig. 3. The Effects of SCCS (10 ml/kg, p.o. for 5 d, n14—16, Panel A
[ANOVA: F(3,56)4.40; p0.0076]) or Desipramine (30 mg/kg, i.p. for 5 d,
n11, Panel B [ANOVA: F(3,40)10.4; p0.000033]) Alone and in Combination
with the Alpha-1 Adrenoceptor Antagonist Prazosin in the Mouse
TST
The last administration of SCCS or desipramine was conducted 1 h or 30 min before
the test, respectively. Prazosin (1 mg/kg, i.p.) was administered 45 min before the test.
Values are shown as meanS.E.M. ∗∗ p0.01 vs. vehicle-treated control group (C),
# p0.05, ## p0.01 vs. groups treated with SCCS or desipramine alone.
Fig. 4. The Effects of SCCS (10 ml/kg, p.o. for 5 d, n14—16, Panel A
[ANOVA: F(3,56)7.44; p0.00028]) or Bupropion (10 mg/kg, i.p. for 5 d,
n11—12, Panel B [ANOVA: F(3,42)6.92; p0.00069]) Alone and in
Combination with the Selective Dopamine D2 Receptor Antagonist
Sulpiride in the Mouse TST
The last administration of SCCS or bupropion was conducted 1 h or 30 min before
the test, respectively. Sulpiride (20 mg/kg, i.p.) was administered 45 min before the test.
Values are shown as meanS.E.M. ∗∗ p0.01 vs. vehicle-treated control group (C),
# p0.05, ## p0.01 vs. groups treated with SCCS or bupropion alone.
DISCUSSION
In this study, based on the findings that administration of
SCCS, but not that of HWCS, shortened immobility times in
the mouse TST without affecting the locomotor activity in
the mouse OFT, we have concluded that SCCS has significant
antidepressant-like activity. This is the first study showing
that the CS extract exerts an antidepressant-like effect.
The main constituents of SCCS are fat-soluble components
such as palmitic acid, oleic acid, triglyceride, ergosterol,
and cholesterol while those of HWCS are carbohydrate
and protein. Bioactivities of these main ingredients are
hardly known. Furthermore, it has been demonstrated that
minor components of CS such as cordycepin,25) polysaccharides,
4,26) and cordyglucan27) are responsible for some biological
activities. Recently, Wang B. J. et al.28) reported that the
supercritical CO2 fluid extractive fraction of CS had a strong
scavenging ability and selectively inhibited the growth of
cancer cells by promoting apoptosis. However, the constituents
responsible for these activities, especially psychotropic
activities, have not yet been determined. Further
investigation is necessary to identify which constituents of
SCCS have antidepressant-like activity.
TST is widely used as an animal behavior test to screen
antidepressant drugs.15,16) This test is quite sensitive and relatively
specific to the antidepressing activities of tricyclics,
serotonin-specific reuptake inhibitors, and monoamine oxidase
inhibitors.
It has been generally accepted that antidepressants elicit
their effects by modulating several neurotransmission systems,
including the noradrenergic, dopaminergic, and serotoninergic
systems. Therefore, the systems responsible for
their antidepressant activities have been investigated by using
specific inhibitors for these neurotransmission.20,29—32) Poncelet
M. et al.29) reported that prazosin, an alpha-1 adrenoreceptor
antagonist, reduced the effect of desipramine, a norepinephrine
and serotonin reuptake inhibitor. Yamada J. et al.30)
showed that anti-immobility effect of bupropion, a dopamin
reuptake inhibitors, was inhibited by sulpiride, a selective
dopamine D2 receptor antagonist, in the forced swimming
test.
With these neurotransmitter inhibitors, mechanisms underlying
the antidepressant-like effect of some herbal extracts
have been also investigated. Dhingra D. et al.21) reported that
the antidepressant-like effect of liquorice extract seems to
be mediated by an increase of brain norepinephrine and
dopamine, but not by an increase of serotonin. Rodrigues A.
L. et al.22) reported that the antidepressant-like effect of
Siphocampylus verticillatus extract seems to involve an interaction
with adrenergic, dopaminergic, glutamatergic, and
serotonergic systems.
In the present study, we evaluated the effects of pretreatment
with prazosin, sulpiride, and p-CPA on the antidepressant-
like actions of SCCS in the mouse TST and found
that these effects are mediated by both noradrenergic and
dopaminergic neurotransmissions, but not by serotoninergic
neurotransmission. We have confirmed the validity of these
September 2007 1761
Fig. 5. The Effect of SCCS (10 ml/kg, p.o. for 5 d, n21—23, Panel A
[ANOVA: F(3,84)5.05; p0.0029]) or Fluoxetine (20 mg/kg, i.p. for 5 d,
n10—11, Panel B [ANOVA: F(3,42)27.5; p0.00000000052]) Alone and
in Combination with the Serotonin Synthesis Inhibitor p-Chrolophenylalanine
(p-CPA) in the Mouse TST
The last administration of SCCS or fluoxetine was conducted 1 hr or 30 min before
the test, respectively. p-CPA (300 mg/kg, i.p.) was administered 72, 48, and 24 h before
the test. Values are shown as meanS.E.M. ∗ p0.05, ∗∗∗ p0.001 vs. vehicle-treated
control group (C), # p0.05 vs. the group treated with fluoxetine alone.
Fig. 6. The Effect of SCCS (10 ml/kg, p.o. for 5 d, n12, Panel A) and
Fluoxetine (10 mg/kg, i.p. for 5 d, n10, Panel B) on 5HTP and Clorgyline-
Induced Head Twitch Behavior in Mice
The last administration of SCCS or fluoxetine was conducted 1 h or 30 min before
the injection of 5-HTP. After the injection of 5-HTP, the number of HTR was counted
for 2 min at 10 min intervals from 10 to 50 min. Closed and open circles indicate treated
and non-treated mice, respectively. ∗ p0.05, ∗∗∗ p0.001 vs. vehicle-treated control
group (C).
inhibitors for these experiments by showing that they attenuated
the anti-immobility effects of desipramine, bupropion,
and fluoxetine.
Administration of large doses of 5-HTP, a precursor of 5-
HT, induces head twitches that occur spontaneously and irregularly,
probably via a central action of 5-HT. HTR, induced
by 5-HTP in mice, provides a simple method of determining
specific activities of potentiators and antagonists for
5-HT in the central nervous system.24) In this study, the administration
of SCCS did not affect 5-HTP-induced HTR in
mice. This finding is consistent with another finding that p-
CPA pretreatment did not attenuate the anti-immobility activity
of SCCS in the TST (Fig. 5). In contrast, fluoxetine, a
positive control compound, significantly increased the number
of 5-HTP-induced HTR at 10—12 min and decreased it
at 30—32 min. The potentiation of HTR by fluoxetine at
10—12 min may be due to the fluoxetine-mediated inhibition
of the 5-HT reuptake and resulting increase of the content of
5-HT in synapses. Subsequent rapid attenuations of HTR by
fluoxetine may be due to the exhaustion of 5-HT in synapses
or the negative-feedback inactivation of the neurotransmission
systems induced after high release of 5-HT into the
synaptic clefts.
One possible mechanism underlying antidepressant-like
activity of SCCS is that some constituents of SCCS might act
as adrenoceptor and dopamine D2 receptor agonists or noradrenaline/
dopamine reuptake inhibitors. This possibility
needs to be systematically addressed in the future investigation.
In summary, this study has demonstrated that SCCS has an
antidepressant-like activity and that its effect seems to originate
from SCCS-mediated alterations in the noradrenergic
and dopaminergic systems, but not in the serotonergic system.
Acknowledgements We thank Dr. Dovie Wylie for expert
technical assistance. We especially thank Ms. Takako
Hiraki, and Ms. Tomo Yoshida-Nishimoto for essential assistance.
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