August 2006 Notes Biol. Pharm. Bull.

29(8) 1767—1770 (2006) 1767

Antidepressant Effect of Extracts from Ginkgo biloba Leaves in Behavioral

Models
Hiroyuki SAKAKIBARA,a Kaori ISHIDA,b Oliver GRUNDMANN,c Jun-ichiro NAKAJIMA,d Shujiro SEO,d
Veronika BUTTERWECK,c Yuko MINAMI,a Satomi SAITO,a Yoshichika KAWAI,a Yutaka NAKAYA,b and
Junji TERAO*, a
a
Departments of Food Science, Institute of Health Biosciences, The University of Tokushima Graduate School; b Nutrition
and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School; 3–18–15 Kuramoto-cho,
Tokushima 770–8503, Japan: c Department of Pharmaceutics, University of Florida; Gainesville, FL 32610, U.S.A.: and
d
Tokiwa Phytochemical Co., Ltd.; 158, Kinoko, Sakura, Chiba 285–0801, Japan.
Received March 20, 2006; accepted May 18, 2006

Extracts of Ginkgo biloba (EGB) are a complex product prepared from green leaves of the Ginkgo biloba
tree. In the present study, the antidepressant effect of EGB was examined using two behavioral models, the
forced swimming test (FST) in rats and tail suspension test (TST) in mice. EGB significantly reduced immobility
time in the FST at a dosage of 10 and 50 mg/kg body weight after repeated oral treatment for 14 d, although no
change of motor dysfunction was observed with the same dosage in the open field test. These results indicate that
EGB might possess an antidepressant activity. In addition, EGB markedly shortened immobility time in the TST
after acute inter-peritoneal treatment at a dosage of 50 and 100 mg/kg body weight. The present study clearly
demonstrated that EGB exerts an antidepressant effect in these two behavioral models.
Key words Ginkgo biloba extracts; antidepressant effect; forced swimming test; tail suspension test; flavonoid

In recent years, depression has become recognized as a MATERIALS AND METHODS

major public health problem. It is estimated that in the US
approximately 20% of the population has some depressive
symptoms, and around 2—5% are thought to suffer from se-
vere forms of depression.1) Understanding how to prevent
and treat depression is therefore an urgent subject. Although
the mechanism provoking depression has not been clearly
elucidated, the main trigger is known to be exposure to
chronic stress.2,3) Many types of antidepressant drugs such as
tricyclic antidepressants, and selective serotonin reuptake in-
hibitors (SSRI), as well as antidepressant herbal medicines
like St. John’s wort are used to treat depression. However,
most of the synthetic drugs are not without side effects.4)
Furthermore disturbance of the drug metabolizing enzyme
systems were revealed with St. John’s wort,5) and thus, the
search for a new antidepressant herb without side effects is
deemed important.
The Ginkgo biloba tree has been used as a traditional Chi-
nese herbal medicine for thousands of years.6) Several re-
search groups have shown that extracts from the green leaves
of the G. biloba tree (EGB) have diverse effects on improve-
ment of mood and cognitive performance, protection of
memory deficits and central nervous system (CNS) disor-
ders, and alleviation of the symptoms of mild to moderate
Alzheimer-type dementia.7—12) Until now, no antidepressant
effect of EGB has yet been revealed. EGB is thought to pos-
sess anti-stress properties,13) and is safe to use without dis-
tinct side effects.8,14) EGB might be a useful option for the
prevention and treatment for stress-induced disorders such as
depression. In the present study, we evaluated the antidepres-
sant effect of EGB using two behavioral models for screen-
ing antidepressants, the forced swimming test (FST) in rats
and tail suspension test (TST) in mice.
∗ To whom correspondence should be addressed. e-mail: terao@nutr.med.tokushima-u.ac.jp
Chemicals The EGB used in the present study was
Ginkgolon-24 from Tokiwa Phytochemical Co., Ltd. (Chiba,
Japan), which was 75% ethanol extracts from the leaves of
Japanese G. biloba tree. This product contains 25.5% of
flavonoid glycosides, including more than 8.2% quercetin
glycosides, more than 6.4% kaempferol glycosides, 1.6%
methylmyricetin glycosides and 1.3% isorhamnetin glyco-
sides, and 6.5% of terpenoids in the form of 2.98% bilob-
alide, 1.59% ginkgolide A, 1.16% ginkgolide B, and 0.75%
ginkgolide C. The antidepressant drug imipramine (hy-
drochloride form) was purchased from Sigma-Aldrich Co.
(St. Louis, MO, U.S.A.).
Animals Male CD rats (240—260 g; Charles River
Japan, Inc., Yokohama, Japan) and male C57BL/6J mice
(22—26 g; SLC, Inc., Hamamatsu, Japan) were used in the
FST and TST, respectively. All animals were housed in a
controlled room (temperature, 25
1 °C; humidity, 45—50%;
light–dark cycle, 12 h each) with free access to laboratory
chow (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap
water. Rodents were divided randomly into control and ex-
perimental groups. This study was performed according to
the guidelines for the care and use of laboratory animals of
The University of Tokushima Graduate School, Institute of
Health Biosciences.
Drug Treatment Imipramine and EGB were dissolved
homogeneously in deionized water by sonication. Both solu-
tions were prepared daily just before administration. Control
animals received the same volume of deionized water only.
FST Procedure The FST was performed according to
the method of Porsolt et al.15) with some modifications.16)
Briefly, a group of 35 rats was divided at random into five
groups and treated as follows: one group was orally adminis-
tered imipramine (15 mg/kg body weight), three groups were
orally administered EGB (5, 10, and 50 mg/kg body weight,
© 2006 Pharmaceutical Society of Japan
1768 Vol. 29, No. 8

respectively), and the final group received vehicle (deionized Table 1. Body Weight Gain
water). Test solutions were administered once daily between
Body weight (g)
1—3 p.m. over a period of 14 d. Treatment
Rats were placed in an acrylic cylinder (450192 mm i.d.) Day 1 Day 14
filled with water at 25
1 °C to a depth of 17 cm for 15 min
(pre-test session) after 14 d treatment. Twenty-four hours Control 135
8 244
9
after the pre-test session, the animals were once again ex- Imipramine 133
17 240
17
EGB 5 mg/kg 131
15 253
11
posed to the same conditions for 5 min (test session). Be- EGB 10 mg/kg 132
15 242
17
tween the pre-test session and main session drug solutions EGB 50 mg/kg 138
6 258
13
were administered orally three times as follows: just after the
pre-test session, 5 h before the main test, and 1 h before the
main test. A rat was judged immobile if it remained floating
in the water, except for small movements to keep its head
above the water. The FST was performed between 1—3 p.m.
and recorded using a video camera. The tapes were evaluated
by observers not informed about the kind of treatment each
animal had received.
Measurement of Locomotor Activity Another set of
rats who received the same drugs using the same treatment
regimen as in the FST underwent an open field test according
to Carlini et al.17) Rats were placed in an open field apparatus
Fig. 1. Effect of EGB on Immobility Time in the FST Using Rats
composed of an arena 70 cm in diameter divided into 18 ap-
Test solutions were administered orally once daily over a period of 14 d. Values rep-
proximately equal areas. For open field observations, each rat resent the mean
S.E. (n7). ∗ p0.05 vs. control group.
was individually placed in the center of the arena 15 h after
the last treatment. Hand-operated counters were employed to Table 2. Locomotor Activity in the Open Field Test
score the following behavioral parameters: locomotion (num-
ber of line crossings), rearing frequencies (number of times Treatment Locomotion Rearing Defecation
seen standing on hind legs), and number of defecations
Control 83
6 14
4 1.6
0.6
within 5 min. Open field observations were made between Imipramine 70
14 18
3 2.4
0.5
8—10 a.m. EGB 5 mg/kg 84
15 14
5 2.0
0.4
TST Procedure The TST was performed according to EGB 10 mg/kg 84
2 16
3 1.8
0.5
Steru et al.18) with slight modifications. In brief, a group of EGB 50 mg/kg 92
8 14
3 1.2
0.7
50 mice was divided at random into five groups and treated The behavioral parameters were recorded for 5 min. Locomotion: number of line
as follows: one group was inter-peritoneally administered crossings. Rearing: number of times seen standing on hind legs. Values represent the
mean
S.E.
imipramine (30 mg/kg body weight), three groups were inter-
peritoneally administered EGB (10, 50, and 100 mg/kg body
weight, respectively), and the final group received vehicle
(deionized water). Test solutions were injected 30 min prior
to the TST.
Four stands, each with a clamp located 450 mm from the
floor, were placed at intervals of 300 mm. A mouse was hung
20 mm from the end of its tail on a stand, and recorded with a
video camera for 6 min. The TST was performed between
1—3 p.m. Immobility time was evaluated by observers not
informed about the kind of treatment each animal had re-
ceived. Fig. 2. Effect of EGB on Immobility Time in the TST Using Mice
Statistics Differences were ascertained with analysis of Test solutions were administered intraperitoneally 30 min prior to the test. Values
variance (ANOVA). Multiple comparisons among treatments represent the mean
S.E. (n10). ∗ p0.05 vs. control group.
were checked with the Fisher-PLSD. The results were con-
sidered significant if the probability of error was 5% or less.
the imipramine treatment group. No effect was observed
RESULTS after administration of 5 mg EGB/kg. Neither administration
of imipramine (15 mg/kg) nor EGB (5, 10, 50 mg/kg) re-
FST Using Rats EGB (5, 10, 50 mg/kg body weight) or sulted in any overt behavioral changes or motor dysfunction
the synthetic antidepressant, imipramine (15 mg/kg), were in the open field test (Table 2).
orally administered to the rats once daily for 14 d. There were TST Using Mice As shown in Fig. 2, immobility time in
no differences in body weight gains after 14 d among all the TST using mice was markedly shortened after acute
treatment groups (Table 1). Figure 1 shows the activities of inter-peritoneal treatment with imipramine (30 mg/kg). Ad-
EGB in the FST using rats. EGB (10, 50 mg/kg) significantly ministration of EGB at dosages of 50 and 100 mg/kg signifi-
reduced the immobility time 14 d after daily treatment. The cantly reduced the immobility time, however, there was no
decrease in immobility for EGB was comparable to that of effect with the lower treatment dosage (10 mg/kg).
August 2006
DISCUSSION
The purpose of this study was to evaluate the antidepres-
sant effect of EGB using behavioral animal models. A major
problem in the screening for new antidepressants is the estab-
lishment of a valid animal model able to sufficiently and ac-
curately identify diverse antidepressant treatments.19) In this
study, we used two animal models, the forced swimming test
(FST) in rats developed by Porsolt et al.15) and tail suspen-
sion test (TST) in mice developed by Steru et al.18) The im-
mobility displayed by rodents when subjected to unavoidable
stress such as forced swimming is thought to reflect a state of
despair or lowered mood, which are thought to reflect depres-
sive disorders in humans. In addition, the immobility time
has been shown to be reduced by treatment with antidepres-
sant drugs.20) Moreover, a significant correlation was found
between the clinical efficacy of antidepressant drugs and
their potency in both models.18,20)
In the present study, EGB significantly reduced immobility
time in the FST after repeated administration of 10 and
50 mg/kg to rats for 14 d. The intensity of immobility reduc-
tion was stronger at a dosage of 10 mg/kg than 50 mg/kg.
Such U-shaped activity has been seen in St. Johns wort21) and
Apocynum extract.16) The efficacy of the decreasing immobil-
ity in the FST was also shown previously with psychostimu-
lants, which exert an indiscriminate motor stimulating activ-
ity.20) In order to exclude this false positive result, we em-
ployed an additional open field test to check the motor stimu-
lating activity of EGB. Both administration of EGB at a
dosage of 10 and 50 mg/kg for 14 d, active dosages in the
FST, resulted in no behavioral changes or motor dysfunction
in the open field test, indicating that the reduction in immo-
bility time after 14 d administration of EGB was attributed to
an inherent antidepressant effect. On the other hand, Porsolt
et al. reported that EGB administration at a dosage of 50 and
100 mg/kg was devoid of activity in the FST.22) They used
EGb761, which was the acetone extracts from G. biloba.23)
However we employed the ethanol extracts, Ginkgolon-24,
indicating that active compounds for antidepressant action
existed in both extracts might be not completely same. It
might be desirable experiments to compare the active com-
pounds in both extracts. In addition, in their study, the ad-
ministration period was only 5 d compared to 14 d in the
present study. Montgomery proposed that a treatment period
of 14 d is a valid interval for demonstrating antidepressant ef-
ficacy.24)
Our results also indicate that the activity of EGB in the
FST was more effective at a lower dosage (10 mg/kg). Thus,
the effective dosage of EGB in the FST seems to be around
10 mg/kg body weight, while treatment with more than 50
mg/kg will probably cause a decline in activity. The antide-
pressant effect of EGB was also revealed using the TST ac-
cording to the reduction in immobility of mice after acute
inter-peritoneal treatment with 50 and 100 mg EGB/kg, re-
spectively. Consequently, the present results clearly demon-
strate that EGB possesses antidepressant-like activity in the
behavioral models, FST and TST. The results in both FST
and TST indicated U-shaped activity, indicating to be neces-
sary to decide an effective dose in clinical use of EGB for
antidepressant. The Extract of Apocynum was reported to
have antidepressant-like activity in the FST as dosage of
1769
30 mg/kg body weight/day for 14 d.16) The pharmacopoeia of
the People’s Republic of China recommends that a clinical
dose for beneficial effects of Apocynam is 6—12 g of the
dried leaves,25) which dose corresponds about 600 mg of
Apocynum extract. These suggest that effective clinical dose
of EGB for antidepressant action might be calculated as
around 200 mg per day, similarly to the recommended dose
of EGB for cognitive impairment and dementia.14) Adminis-
tration of EGB should be repeated, because Cryan et al. con-
cluded that the effects of antidepressants were augmented
following chronic treatment.26) The length of treatment is
recommended 4- and 6-week period for the demonstration of
acute efficacy in the guidelines of the European Union.27)
However, the onset of an antidepressant’s clinical effect can
be demonstrated early in the treatment period.24) In addition,
the significant differences seen at 2 weeks treatment do not
appear to be lost at 4 weeks. Hence, administration of EGB
for antidepressant action is repeatedly and probably at least
for 2 weeks.
EGB is a complex product prepared from green leaves of
the Ginkgo biloba tree. Major ingredients are polyphenols,
especially flavonoids including quercetin glycosides and
kaempferol glycosides.23) Recently, several studies have sug-
gested the antidepressant effect of quercetin glycosides such
as hyperoside, isoquercitrin and rutin using the positive re-
sults of FST.16,21,28) Flavonoid glycosides are mostly hy-
drolyzed into their aglycons by mucosal and bacterial en-
zymes in the intestines, and then converted to conjugated
metabolites during the absorption process.29,30) This perhaps
indicates that the active form for the antidepressant effect of
quercetin glycosides is the conjugated form, not the glyco-
side form.31) The EGB used in the present study, Ginkgolon-
24, contains more than 8.2% of quercetin glycosides, includ-
ing rutin and isoquercitrin. Quercetin glycosides are, hence,
thought to be one of the major ingredients contributing to the
antidepressant effect of EGB. Additionally, the present EGB
contained more than 6.4% kaempferol glycosides, 1.6%
methylmyricetin glycosides, and 1.3% isorhamnetin glyco-
sides. These flavonoid glycosides seem to appear as conju-
gated forms in the blood stream as with quercetin glycosides.
Transportation of these metabolites into the brain tissues via
the blood brain barrier and their effect on the CNS system
have been recently argued.32,33) Moreover, quercetin metabo-
lites were previously found in the brain tissues of rodents
after oral administration.34) Therefore, one of the antidepres-
sant mechanisms of EGB is thought to involve flavonoid gly-
cosides, which reach the brain tissues through the metaboliz-
ing process, protecting brain function from CNS disturbance,
and consequently, exerting an antidepressant effect.
Other candidates of EGB that exert an antidepressant ef-
fect are the 6.5% of terpenoids, known as bilobalide, and
ginkgolides A, B and C,23) which are known to have efficacy
in the CNS.35,36) Some of the antidepressant activity of EGB
might therefore be derived from these compounds. The pres-
ent study warrants further investigation into identification of
the active compounds in herbal medicines, in particular
EGB, with antidepressant effects. Porsolt indicated that cen-
tral levels of monoamine, such as dopamine and norepineph-
rine, were important factors for reduction of immobility in
the FST.20) Additionally, recent research reported that swim-
ming stress markedly enhanced a phosphorylation of mito-
1770
gen-activated protein kinases (MAPKs) in the brain.37) It is
likely that changes of monoamine levels and/or activation of
MAPKs in the brain are involved in the antidepressant mech-
anism of EGB.
Acknowledgments This study was performed as part of
the program of the 21st Century Center of Excellence on
“Human nutritional science on stress control” at the Univer-
sity of Tokushima.
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