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Ginkgo biloba extract enhances male

copulatory behavior and reduces serum
prolactin levels in rats

Article in Hormones and Behavior February 2008

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Hormones and Behavior 53 (2008) 225 231

www.elsevier.com/locate/yhbeh

Ginkgo biloba extract enhances male copulatory behavior and reduces

serum prolactin levels in rats
Kuei-Ying Yeh a , Hsiao-Fung Pu b , Krishna Kaphle c , Shih-Fan Lin a , Leang-Shin Wu c ,
Jen-Hsou Lin c , Yuan-Feen Tsai a,
a
Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
b
Department of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC
c
Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan, ROC
Received 11 September 2007; accepted 1 October 2007
Available online 10 October 2007

Abstract

The aim of this study was to investigate the effects of Ginkgo biloba extract (EGb 761) on male copulatory behavior in rats. EGb 761 (1 mg/ml)
induced significant production of testosterone (T) in rat Leydig cells in vitro. Its effects on sexual behavior were then tested in LongEvans male
rats after 7, 14, 21, or 28 days of oral gavage of vehicle (distilled water) or EGb 761 at doses of 10, 50, or 100 mg/kg. Administration of 50 mg/kg of
EGb 761 for 28 days and of 100 mg/kg for 14 or 21 days significantly increased intromission frequency compared to controls on the same day. An
increase in ejaculation frequency was seen after treatment with 50 mg/kg of EGb 761 for 14, 21, or 28 days when compared to either the control
group on the same day or the same group on day 0. A reduction in ejaculation latency was only seen after administration of 50 mg/kg of EGb 761
for 14 days compared to the vehicle-treated group. After treatment for 28 days, no significant difference was seen in mount latency, intromission
latency, serum T levels, reproductive organ weight, sperm number, or levels of the metabolite of dopamine, 3,4-dihydroxyphenylacetic acid in the
brain with any dose of EGb 761, but significantly reduced serum prolactin levels and increased dopamine levels in the medial preoptic area and
arcuate nucleus were seen at the dose of 50 mg/kg. These findings show that EGb 761 (especially at the dose of 50 mg/kg) enhances the copulatory
behavior of male rats and suggest that the dopaminergic system, which regulates prolactin secretion, may be involved in the facilitatory effect of
EGb 761.
2007 Elsevier Inc. All rights reserved.

Keywords: Ginkgo; Leydig cell; Sexual behavior; Testosterone; Prolactin; Male rat

Introduction Dopamine (DA) is widely believed to facilitate male sexual

function. L-DOPA treatment of Parkinsonian patients often results
Sexual behavior is mainly controlled by a well-organized in enhanced libido and sexual potency (Bowers et al., 1971), and
neural circuit that connects a variety of brain areas, but the acti- apomorphine, a DA agonist, has been used to correct erectile
vation of this circuit is highly dependent on gonadal hormones dysfunction (Lal et al., 1984; O'Sullivan and Hughes, 1998).
(Hull and Dominguez, 2007). It is well documented that Administration of DA agonists also facilitates male sexual behavior
testosterone (T) plays an essential role in the control of male in rodents (Bitran and Hull, 1987; Melis and Argiolas, 1995).
sexual behavior. Lack of T after castration abolishes sexual Prolactin (PRL) influences many different types of behavior
behavior in rodents, and the declined copulatory ability can be in various animal species. Peripheral administration of PRL has
restored by T replacement (Hull et al., 2002). been reported to markedly inhibit the mating behavior of male
rabbits (Hartmann et al., 1966). Subsequent studies showed that
Corresponding author. Department of Physiology, College of Medicine,
hyperprolactinemia impairs male sexual behavior in rats and
National Taiwan University, No.1 Jen-Ai Road, 1st Section, Taipei, Taiwan mice (Bailey and Herbert, 1982; Svare et al., 1979). Although
(100), ROC. Fax: +886 2 23225007. hyperprolactinemia is commonly associated with T deficiency
E-mail address: [email protected] (Y.-F. Tsai). in men and with ovarian dysfunction in women, it suppresses
0018-506X/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.yhbeh.2007.10.001
226 K.-Y. Yeh et al. / Hormones and Behavior 53 (2008) 225231

sexual desire without causing obvious gonadal steroid defi- between 19:00 and 22:00 h under a dim red light. To allow acclimatization to the
ciency (Bancroft, 2005). test environment, each male rat was placed in a circular Plexiglas testing
chamber (45 cm diameter) 3 min before the introduction of a sexually receptive
EGb 761, a standardized extract of Ginkgo biloba leaves, has female, then the male was allowed to copulate for 30 min. The copulatory
been reported to have beneficial effects on memory, vigilance, behavior parameters recorded were mount frequency (MF, number of mounts
cognitive functions related to aging, and dementia (DeFeudis and without penile insertion during the 30-min test period), intromission frequency
Drieu, 2000). It has also been shown to correct antidepressant- (IF, number of mounts with penile insertion during the 30-min test period),
ejaculation frequency (EF, number of ejaculations during the 30-min test
induced sexual dysfunction (Cohen and Bartlik, 1998); however,
period), mount latency (ML, latency from the introduction of the female to the
these data were obtained by clinical interview and self-reporting first mount), intromission latency (IL, latency from the introduction of the
assessment by the patients. The effects of G. biloba on sexual female to the first intromission), and ejaculation latency (EL, latency from the
function have not yet been investigated in animal studies. The first intromission to ejaculation). If the animal was observed on a first
current study was performed using a rat model to determine the intromission prior to its first mount, or with intromission and ejaculation only
effects of EGb 761 on male copulatory behavior. Since T, PRL, during the 30-min testing period, we took the value of its IL as its ML for the
statistical analysis. Besides, the value was counted as one mount for its first
and DA play important roles in male sexual behavior, serum intromission. In addition, if a male rat failed to ejaculate within the 30-min
levels of T and PRL and brain levels of DA and its metabolite, 3,4- observational period, the EL for this animal was recorded as 30 min.
dihydroxyphenylacetic acid (DOPAC), were also measured.
Reproductive organ weight and sperm count
Materials and methods
After the last behavioral test, the males were decapitated using a guillotine
Subjects and the bilateral reproductive organs (seminal vesicles, epididymis, and testes)
were collected and weighed. Sperm head counting was carried out as described
previously (Blazak et al., 1993). After decapsulation, the testes from each rat
Male LongEvans rats (8 weeks old) were purchased from the Animal Center of
were homogenized in 20 ml of ice-cold 0.9% NaCl solution containing 0.01%
the National Science Council, Taipei, Taiwan. The animals were kept in groups of
Triton X-100 in a tissue mixer. The homogenate was allowed to settle for 1 min,
four in a cage (30 30 20 cm) in a temperature (22 1 C)- and humidity (55
then was gently mixed and placed in ice for 1 min. The number of sperm heads
10%)-controlled room on a 12-h light:dark cycle (lights off at 17:00 h). Food and
in five chambers of a hemocytometer was counted and the number of
water were available ad libitum. The experimental protocols were approved by the
spermatozoa produced per gram of testicular tissue calculated using the
Animal Care and Use Committee, College of Medicine, National Taiwan
equation: average number of sperm heads in five chambers hemocytometer
University, and all experimental procedures conformed to the National Institutes
factor (104) dilution (20) dilution volume (20) divided by testis weight (g).
of Health Guide for the Care and Use of Laboratory Animals. When the rats were
9 weeks old, an ovariectomized female LongEvans rat (912 weeks old) with an
estradiol capsule implant (see below) was placed in the males' home cage for 4 days Primary culture of rat Leydig cells
(about 90 h), then removed. The males were then screened for copulatory ability
using stimulus females of the same strain. Leydig cells were isolated from the left testes of another series of 90- to 100-
day-old male LongEvans rats. The rats were killed by CO2 inhalation and the
Stimulus females testes collected sterilely and placed in a 50-ml plastic tube (Falcon) containing
medium 199 (M199, Gibco). After careful decapsulation and tearing into smaller
fragments using forceps, the material was placed in a 10-cm dish containing
Female LongEvans rats aged 8 weeks were ovariectomized and immediately
Hanks balanced salt solution (HBSS, Gibco) and 1.2 IU/ml of type I collagenase
implanted with a 5-mm Silastic capsule (1.98 mm ID and 3.18 mm OD) filled with 17
(Sigma), 1% bovine serum albumin (BSA, Sigma), and 20 mM N-2-
-estradiol (Sigma) under 3.5% pentobarbital (1 ml/kg) anesthesia. Approximately
hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, Gibco) and the dish
1 week after surgery, the females were used as sexual partners for behavioral tests.
placed at 37 C in a 5% CO2 incubator for 30 min, then the dispersed tissues were
diluted with M199 and the suspension filtered through a nylon mesh (63 m) into
Copulation screening of test males a new 50-ml tube, which was centrifuged at 900g for 5 min at 4 C. The
precipitated interstitial cells were re-suspended in 5 ml of erythrocyte lysis buffer
The copulation screening test was performed when the male rats were 10 weeks [0.15 M NH4Cl, 0.1 mM Na2EDTA (both from Sigma), and 12 mM NaHCO3],
old during the dark phase of the cycle (2 h after lights off) and under a dim red light. then the sample was again centrifuged at 900g for 5 min at 4 C. The pellet was
Each male rat was placed in a circular Plexiglas testing chamber (45 cm diameter) and re-suspended in 5 ml of M199 and the suspension loaded on top of a step gradient
a stimulus female was introduced 3 min later, then the number and latency of mounts, (10%, 20%, 30%, 40%, and 50%) of Percoll (Pharmacia) and centrifuged at
intromissions, and ejaculations were recorded over a period of 15 min. Male rats were 170g for 10 min at 4 C. The Leydig cells, found in the 50% layer (Gale et al.,
tested three times at intervals of 56 days. Animals which had not ejaculated twice 1982), were characterized by a bright yellow halo on phase-contrast microscopy.
after three testing sessions were not used in the sexual behavior study. The cells were spun down, washed twice with M199, re-suspended in M199
containing 100 U/ml of penicillin/streptomycin and 1% BSA, plated at a density
Treatment of 106/ml on 24-well plates (Falcon), and incubated at 37 C in a 5% CO2
incubator. After 2 h, the culture medium was changed to phenol red free-M199
EGb 761 was purchased from Dr. Willmar Schwabe Pharmaceuticals and the effects of incubation for 24 h with different doses of EGb 761 on
(Karlsruhe, Germany). Male rats (approximately 12 weeks old) were randomly T production and viability tested, as described below.
divided into four groups, which were given 10, 50, or 100 mg/kg/day of EGb
761 or vehicle (distilled water) (n = 10 in each group) in a volume of 0.25 ml/ Enzyme immunoassay for testosterone in culture medium or serum
100 g of body weight via oral gavage for 28 days. The EGb 761 or vehicle was
given between 7:00 and 9:00 h each day. Leydig cells were incubated in the absence or presence of EGb 761 (50, 100,
250, 500, or 1000 g/ml) for 24 h, then the culture medium was removed and
Copulatory behavior testing stored at 80 C until assayed for T by enzyme immunoassay (EIA), and cell
viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
Copulatory behavior was measured after 7, 14, 21, and 28 days of oral diphenyl tetrazolium bromide, Sigma) reduction method. The viability of the
administration of EGb 761 or vehicle. All behavior tests were performed control and treated cells was greater than 90%.
 K.-Y. Yeh et al. / Hormones and Behavior 53 (2008) 225231 227

80 C. Serial 180-m-thick coronal sections were prepared using a cryostat at

14 C. The brain areas of the medial preoptic area (MPOA), arcuate nucleus
(ARC), and median eminence (ME) were microdissected according to Palkovits
(1973). Micropunched tissue samples were obtained bilaterally, homogenized at
room temperature in 0.1 N perchloric acid, and centrifuged at 7800g for 10 min
at 4 C, then the supernatant was assayed for DA and DOPAC by high pressure
liquid chromatography with electrochemical detection (Tsai et al., 1994).
Briefly, 20 l of supernatant was applied to a C18 reverse phase analytical
column filled with ODS-3 (3 m) (Bioanalytic System Inc., USA) using a
mobile phase of 8.65 mM heptanesulfonic acid (Sigma), 0.26 mM EDTA
(Sigma), 6.25% acetonitrile (v/v) (Merck), 0.35% triethylamine (v/v) (Merck),
and 0.4% orthophosphoric acid (v/v) (Merck), pH 2.73.1, and at a flow rate of
0.5 ml/min. The sensitivity of the LC-4C amperometric detector (Bioanalytic
System Inc., USA) was 50 nA full scale and the potential of the working
electrode was set at 0.75 V with respect to an Ag/AgCl reference electrode.
The pellets from the centrifugation were dissolved in 0.5 N NaOH and assayed
for protein by the method of Lowry et al. (1951) and the results were expressed as
ng/mg protein.
Fig. 1. Effect of EGb 761 on testosterone production by rat Leydig cells. Leydig
cells were incubated with different concentrations of EGb 761 for 24 h. The
columns and bars represent the mean SEM for seven independent experiments. Statistical analysis
P b 0.05 compared to vehicle-treated controls.
Statistic 6.0 (StatSoft Inc.) was used for statistical analyses. The data for sperm
counting, weight of the reproductive organs, serum T and PRL concentrations, and
The culture medium from the Leydig cells was assayed for T using an EIA as
DA and DOPAC levels were analyzed using one-way ANOVA. Copulatory
described previously (Yang et al., 2004). Briefly, 50 l of diluted test medium
behavioral results were evaluated by two-way mixed ANOVA with repeated
and 50 l of horseradish peroxidase-coupled testosterone were added to a 96-
measures followed by Fisher's post hoc test to establish the significance of
well microtiter plate (Costar 3590) coated with anti-T monoclonal antibody T-1.
After incubation at room temperature for 25 min and two washes with
phosphate-buffered saline (PBS), the color was developed using 200 l of
2.2 mM o-phenylenediamine in 0.003% H2O2 at room temperature for 30 min,
then the reaction was stopped by addition of 50 l of 8 N sulfuric acid and the
optical density measured at 490 nm and compared to a T standard curve. The
assay sensitivity was 15.6 pg/ml of T. The intra- and interassay coefficients of
variation were 510% and 1014%, respectively.
Serum samples were assayed for T in an identical fashion using a 50-l
sample of diluted serum instead of test medium.

Radioimmunoassay for serum prolactin

Male rats were sacrificed by decapitation approximately 14 h after the last

behavioral test, and trunk blood was collected into 15 ml test tubes, kept at room
temperature for 30 min, then centrifuged at 900g for 30 min at 2 C, and the serum
collected and stored at 80 C until assayed. Serum PRL concentrations were
determined by radioimmunoassay. Rat PRL (NIDDK-rPRL-I-6) was iodinated
using Na125I and chloramine-T and the labeled peptide separated from free iodine
on a Sephadex G-50 (superfine) column. Standard rat PRL (PRL-RP-3) and anti-
PRL antibodies (NIDDK-anti-rPRL-IC-5) were supplied by the NIDDK Hormone
Distribution Program. The assay sensitivity was 82 ng/ml of PRL and the intra- and
interassay coefficients of variation were 4.5% and 9%, respectively.

Measurement of dopamine and its metabolite

Approximately 14 h after the last behavioral test, the brain was rapidly
removed and immediately frozen in 20 C isopentane and then stored at

Table 1
Effect of EGb 761 on accessory sexual organ weights and sperm number
Group N Seminal Epididymis Testes Sperm
vesicle (g/kg BW) (g/kg BW) head count
(g/kg BW) (107/g testes)
Vehicle 10 1.94 0.16 3.09 0.14 8.03 0.22 9.29 0.54
EGb 761, 10 mg 10 2.04 0.13 2.98 0.12 8.01 0.41 10.34 0.94
EGb 761, 50 mg 10 2.06 0.13 3.08 0.10 8.29 0.22 10.31 1.05
Fig. 2. Intromission frequency and latency in the different groups. (A) intromission
EGb 761, 100 mg 10 2.10 0.13 3.12 0.09 8.02 0.29 10.37 0.78
frequency; (B) intromission latency. The data are presented as the mean SEM.
The data are presented as the mean SEM. No significant differences were P b 0.05, EGb 761-treated group compared to the vehicle-treated group on the
found. same day.
228 K.-Y. Yeh et al. / Hormones and Behavior 53 (2008) 225231

Fig. 4. Effect of 28 days of EGb 761 treatment on (A) serum T and (B) PRL
levels in male rats. The data are presented as the mean SEM. The numbers
above the bars indicate the number of animals used per group. P b 0.05
Fig. 3. Ejaculation frequency and latency in the different groups. (A) ejaculation compared to the vehicle-treated controls.
frequency; (B) ejaculation latency. The data are presented as the mean SEM
(a, EGb 761-treated group compared to the vehicle-treated group on the same
day; b, EGb 761-treated group compared to the same group on day 0; P b 0.05; the weight of the seminal vesicle [F(3,36) = 0.25, P = 0.86],
P b 0.01).
epididymis [F(3,36) = 0.31, P = 0.82], or testes [F(3,36) = 0.21,
P = 0.89] or on sperm numbers [F(3,36) = 0.38, P = 0.77] com-
differences between means. P values less than 0.05 were considered statistically pared to controls at the same time point.
significant. All quantitative data are given as the mean SEM.
Copulatory behavior
Results No significant differences in ML and MF were seen in any of
the EGb 761-treated groups at any time point when compared to
Testosterone production by primary Leydig cells
Table 2
Cultured rat Leydig cells were treated for 24 h with various Effect of treatment with 50 mg/kg EGb 761 for 28 days on brain DA and its
concentrations of EGb 761 (50, 100, 250, 500, or 1000 g/ml). metabolite levels
A significant increase in T production compared to controls Brain area Group DA DOPAC DOPAC/DA
was seen at the concentration of 1000 g/ml [F(5,36) = 2.86, (ng/mg protein) (ng/mg protein) ratio
P b 0.05] (Fig. 1). MPOA Vehicle 2.66 0.15 (9) 3.23 0.27 (7) 1.16 0.05
EGb 761 3.17 0.18 (8) 2.53 0.26 (8) 0.97 0.13
ARC Vehicle 2.22 0.26 (8) 3.41 0.32 (7) 1.14 0.13
In vivo studies
EGb 761 3.24 0.36 (7) 3.25 0.26 (7) 1.32 0.19
ME Vehicle 5.29 0.36 (9) 4.14 0.55 (8) 0.83 0.14
Reproductive organ weight and sperm numbers after 28 days EGb 761 6.13 0.61 (8) 4.62 0.50 (8) 0.83 0.09
The data for reproductive organ weights and sperm numbers The data are presented as the mean SEM. The number of animals per group is
after treatment for 28 days are shown in Table 1. No significant indicated in parentheses.
differences were seen. No dose of EGb 761 had any effect on P b 0.05 compared to vehicle treatment.
 K.-Y. Yeh et al. / Hormones and Behavior 53 (2008) 225231 229

controls on the same day or the same group on day 0 (data not DA ratio in these brain areas showed no significant differences
shown). between the control and EGb 761-treated groups. In addition,
As shown in Fig. 2A, ANOVA detected a significant dose EGb 761-treated rats showed no significant change in DA and
effect in IF [F(3,36) = 3.54, P b 0.05]. Post hoc comparison DOPAC levels in the ME compared to the control group.
revealed that animals treated with 50 mg/kg of EGb 761 for
28 days showed an increase in IF when compared to controls at Correlation analysis of EF and serum PRL levels on day 28
the same time point (P b 0.05). Treatment with 100 mg/kg on When the EF data for the vehicle-treated and all three EGb
day 14 and day 21 also resulted in more intromissions than in 761-treated groups combined were plotted against the serum PRL
the controls on the same day (P b 0.05). No significant main levels, a significant correlation was found (r = 0.45, P b 0.01)
effect of treatment [F(3,36) = 0.53, P = 0.67] or time [F(4,144) = (Fig. 5).
0.65, P = 0.63] was seen in IL (Fig. 2B).
Oral administration of EGb 761 induced an increase in EF. Discussion
ANOVA showed a significant dose effect [F(3,36) = 5.76, P b 0.01]
and time effect [F(4,144)= 2.54, P b 0.05] (Fig. 3A). Post hoc The present study demonstrated that, at the dose of 50 mg/kg
comparison showed that rats treated with 50 mg/kg of EGb 761 for body weight, EGb 761 significantly enhanced male copulatory
14, 21, or 28 days showed a significantly increased EF when behavior in rats, as revealed by an increase in both the IF and EF
compared to controls at the same time point (P b 0.05). Treatment and a decrease in the EL. The results of this study support the
with 50 mg/kg of EGb 761 for 14, 21, or 28 days also resulted in a clinical report that Ginkgo corrects male sexual dysfunction
greater EF than in the same group on day 0 (P b 0.01). In EL, (Cohen and Bartlik, 1998). Sexual behavior is composed of two
ANOVA detected a significant effect of treatment [F(3,36) = 5.78, major components, sexual motivation, and copulatory perfor-
P b 0.01] and post hoc comparison revealed that rats treated with mance. Sexual motivation can be measured in animals using the
50 mg/kg of EGb 761 for 14 days showed a significant decrease in behavioral parameters of ML, IL, and post-ejaculatory interval,
EL compared to vehicle-treated controls on the same day (P b 0.05) while copulatory performance is assessed by the IF, EF, and MF
(Fig. 3B). (Everitt, 1990). Interestingly, our results showed that Ginkgo
enhanced male copulatory performance, but not sexual motivation.
Serum hormone concentrations on day 28 In the present study, a high concentration of EGb 761
No significant difference in the serum T concentration was (1000 g/ml) stimulated primary rat Leydig cells to produce
seen in any of the EGb 761-treated groups compared to controls T directly in vitro (Fig. 1), and long-term treatment with 50 mg/
[F(3,35) = 1.58, P = 0.21] (Fig. 4A). In contrast, a significant kg of EGb 761 significantly enhanced male sexual behavior in
reduction in serum PRL levels was seen in the group treated rats. However, no difference in serum T levels was found
with 50 mg/kg of EGb 761 [F(3,33) = 3.06, P b 0.05] (Fig. 4B). between EGb 761-treated and control rats (Fig. 4A). A similar
finding that daily oral administration of G. biloba for 14 days
Concentrations of DA and its metabolite on day 28 does not affect serum androgen levels in men has been reported
As shown in Table 2, a significant increase in DA levels in (Markowitz et al., 2005). Our results that no changes in serum
the MPOA [F(1,15) = 4.92, P b 0.05] and ARC [F(1,13) = 5.52, T levels were seen after long-term administration of EGb 761
P b 0.05] was seen in rats treated with 50 mg/kg of EGb 761 may be explained by the androgen negative feedback
compared to the control group. DOPAC levels and the DOPAC/ mechanism. Although Ginkgo induced significant T production
in Leydig cells in vitro, excessive T levels attenuate GnRH
stimulation of LH and inhibit T synthesis and secretion in
Leydig cells in vivo. Serum androgen levels are important in
regulating sperm formation and in supporting the development
of androgen-dependent organs (Grumbach and Conte, 1992).
Since no significant increase in serum T levels after long-term
EGb 761 treatment of male rats was seen in the present study, it
is reasonable that EGb 761 failed to affect the reproductive
organ weight or increase sperm number in male rats (Table 1).
Our findings confirm a previous report that G. biloba treatment
does not affect male reproductive organ weight (except the
prostate gland) or sperm production in mice (Al-Yahya et al.,
2006).
EGb 761 contains 24% phytoestrogens, which are composed
of quercetin, kaempferol, and isorhamnetin glycosides (Gertz
and Kiefer, 2004). Quercetin significantly increases cAMP-
induced mRNA levels for steroidogenic acute regulatory (StAR)
protein in MA-10 mouse tumor Leydig cells (Chen et al., 2007).
Fig. 5. Linear regression of EF and serum PRL levels in male rats treated for Our result showing that EGb 761 increased T production in vitro
28 days with various doses of EGb 761 or distilled water. r = 0.45, P b 0.01. suggests that it may increase intracellular cAMP levels and StAR
230 K.-Y. Yeh et al. / Hormones and Behavior 53 (2008) 225231

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