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57
Original Article
The Eect of Oral Feeding of
Tribulus terrestris L.
on Sex
Hormone and Gonadotropin Levels in Addicted Male Rats
Mohammad Hassan Ghosian Moghaddam, Ph.D.
1
, Mohsen Khalili, Ph.D.
2
,
Maryam Maleki, M.D.
3
*, Mohammad Esmail Ahmad Abadi, M.D.
3
1. Department of Biochemistry, Shahed University, Tehran, Iran
2. Neurophysiology Research Center, Shahed University, Tehran, Iran
3. Shahed University, Tehran, Iran
Abstract
Background: Opioids can exert adverse effects on the body. Morphine, an opioid drug,
reduces hormone levels and fertility, and causes sexual activity disorders. Tribulus ter-
restris (TT) is a traditional herbal medicine used to enhance sexual activities. This study
investigates the possible role of TT on sex hormones and gonadotropins with the intent to
show its usefulness in treating fertility disorders in opioid users.
Materials and Methods: In this experimental study, we randomly divided 48 rats into
four groups: i. control, ii. TT-treated, iii. addicted and iv. TT-treated addicted. Water-
soluble morphine was administrated orally for 21 days to induce addiction, after which
the treated groups 2 and 4 received plant-mixed pelleted food (6.25%) orally for four
weeks. At the end of the treatment period, the sex hormone and gonadotropin levels of all
rats’ sera were determined by radioimmunoassay and Elisa kits. The data obtained were
statistically analyzed using the one-way analysis of variance, followed by post-hoc Tukey
test. P<0.05 was considered signicant.
Results: The addicted group had a signicantly lower luteinizing hormone (LH) level
than the control group (p<0.027). LH levels increased signicantly in the TT-treated ad-
dicted group (p<0.031). The testosterone level in the treated addicted group was lower
than the treated control group. The addicted group had a signicantly low testosterone
level (p<0.001). The estrogen level was signicantly (p<0.002) lower in the addicted
group than in the control group. In addition, there was a signicant difference between
the treated addicted group and the treated control group (p<0.048). The treated control
group had a signicant increase in its progesterone level (p<0.002). Overall, except for
follicle-stimulating hormone (FSH), morphine reduced most of the gonadotropins and
sexual hormones. Whereas TT caused a considerable increase (p<0.05) in the hormones
in the treated addicted group, there was only a slight increase in the treated control group.
Conclusion: Oral consumption of TT could markedly antagonize the reduction of sex
hormones and gonadotropins (except for FSH) due to morphine addiction.
Keywords: Morphine, Tribulus terrestris, FSH, LH, Sex Hormones
Citation: Ghosian Moghaddam MH, Khalili M, Maleki M, Ahmad Abadi ME. The effect of oral feeding of tribulus ter-
restris L. on sex hormone and gonadotropin levels in addicted male rats. Int J Fertil Steril. 2013; 7(1): 57-62.
Received: 25 Dec 2011, Accepted: 13 Oct 2012
* Corresponding Address: P.O.Box: 141557435, Shahed
University, Tehran, Iran
Email: mlk.mry@gmail.com
Royan Institute
International Journal of Fertility and Sterility
Vol 7, No 1, Apr-Jun 2013, Pages: 57-62
Introduction
Recent years have witnessed a dramatic rise in
the use of opioid drugs despite the documentation
of their numerous adverse effects in the literature.
One of these side effects is the negative impact on
sex hormone levels, libido, potency, and menor-
rhea (1-3). A study has shown that 96% of men
and 69% of women who receive opioid analgesic
IJFS, Vol 7, No 1, Apr-Jun 2013
58
drugs for pain management have decreased libido
or impotency (4). Also, it has been found that spi-
nal opiate analgesics reduce libido and cause dif-
culty in achieving or maintaining an erection in
men (5).
One of the most commonly used opioid drugs
is morphine. In a prospective, uncontrolled,
non-randomized study, a group of men with an
average pain duration of 11 years used intrathe-
cal morphine for 12 weeks. Most of the patients
reported poor libido and erectile difficulty to-
ward the end of the 12-week period; in addition,
testosterone and follicle-stimulating hormone
(FSH) levels significantly reduced. The intrath-
ecal opioid caused a reduction in hormone lev-
els (6).
Tribulus terrestris (TT), commonly known as
caltrop or devil’s eyelashes plays an important role
in traditional medicine. Most parts of this plant
are used in herbal medicine, for which TT can en-
hance sex drive and treat urolithiasis, menorrha-
gia, impotency, rheumatism pains, and premature
ejaculation (7-9). Khordadmehr and his colleagues
have used TT in an herbal formula, NOFODA, to
investigate its effect on infertile males and con-
cluded that it improved both sperm motility and
count (10).
In studies conducted on animals, TT is thought
to have a luteinizing hormone (LH)-like activity,
which can induce corpus luteum formation in fe-
male rats (11). LH induces the corpus luteum to
secrete progesterone. This results in an increase
in progesterone levels as well as some estrogen
secretion. Studies have shown that protodioscin,
which is found in TT extract, treats mild to mod-
erate erectile dysfunction and increases libido
(12, 13).
In light of the increasing fertility disorders in
opioid users, the present study investigated the ef-
fect of TT on the sex hormones and gonadotropins
of addicted male rats.
Materials and Methods
Experimental animals
Adult male Wister rats that weighed 200 ± 25 g
each (Razi Institute, Iran) were randomly divided
into four groups, of 12 animals each: i. control,
ii. TT-treated, iii. addicted and iv. TT-treated ad-
Ghosian Moghaddam et al.
dicted. The rats were housed in groups of three in
cages at a temperature of 22-25˚C, on a 12 hour
light/12 hour dark schedule and sufcient amounts
of food and water.
Preparation of Tribulus terrestris (TT)
After obtaining TT and verifying its suitability
for our study via the Department of Botany at Sha-
hid Beheshti University, we ground and combined
the plant with pelleted food at a weight ratio of
6.25%.
Study protocol
Morphine addiction was induced according to
the method of Moini Zanjani et al. (14). Groups
3 and 4 received oral administrations of water-
soluble morphine for 21 days. During this 21-day
period, the treated groups (2 and 4) also received
oral administration of TT plant-mixed pelleted
food (6.25%).
The water soluble morphine solution was given
in doses of 0.1, 0.2, and 0.3 mg/ml according to
the method of Swanston-Flatt et al. (15); each of
these doses was administered for 48 hours for the
rats to drink and then 0.4 mg/ml was administered
for the remaining 15 days. The bitterness of mor-
phine was eliminated by adding 3% sucrose to the
solution. In this experiment, the average amount
of water consumed, and therefore morphine, by
rats was approximately 60-80 mg/ml/day. Addic-
tion was conrmed by injecting a morphine an-
tagonist (naloxone) and observing for withdrawal
symptoms.
All techniques and methods were approved by
the Ethics Committee of Shahed University of
Medical Sciences. The laboratory animals were af-
forded due care in accordance with the regulations
of the Committee for the Purpose of Control and
Supervision on Experiments on Animals (CPC-
SEA).
Blood sampling
After 21 days of treatment, at treatment termi-
nation, blood samples (3-5cc) were obtained from
all the rats’ hearts to measure hormone levels. The
sera were subsequently separated via centrifuge
(Sigma 4-10, USA) at 2000 rpm for 10 minutes
and stored at -70˚C in a freezer until hormone
analysis.
59
The Effect of
Tribulus terrestris
on Hormone Levels
Plasma analysis
The plasma concentrations of the sex hormones
and gonadotropins were veried by the radioim-
munoassay (RIA) method using kits manufactured
by Monobind Inc. (USA), as well as ELISA kits
(Labsystem, Finland).
Statistical analysis
Obtained data were expressed as mean ± SEM
and statistically analyzed using the one-way analy-
sis of variance (ANOVA), followed by the post-
hoc Tukey test. p<0.05 was considered statistically
signicant.
Results
FSH analysis
Our FSH analysis showed that the TT-treated
control group had the least amount of FSH (0.271
± 0.025 mIU/ml), while the morphine-addicted
group had the highest FSH level (0.348 ± 0.022
mIU/ml). Morphine solely did not suppress FSH.
The TT-treated addicted group had a higher FSH
level (0.302 ± 0.01 mIU/ml) than did the TT-treat-
ed control group; there was, however, no signi-
cant change in any of the groups. Figure 1 illus-
trates these results.
FSH (mlU/ml)
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Control Con+TT Morphine Mor+TT
Test groups
Fig 1: The effect of Tribulus terrestris (TT) on FSH levels
in control and addicted groups. Bars depict mean ± SEM
of LH.
LH analysis
In LH analysis, the addicted group had decreased
LH levels. The highest amount of LH belonged to
the treated control group (0.207 ± 0.098 mIU/ml).
Figure 2 shows a signicantly lower LH level in
the addicted group than the control group (0.0125
± 0.017 mIU/ml), which was due to morphine sup-
pression (p<0.027). The TT-treated addicted group
had a signicantly higher amount of LH (0.273 ±
0.066 mIU/ml) than did the control group p<0.031.
LH (mlU/ml)
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Control Con+TT Morphine Mor+TT
Test groups
*
$
Fig 2: The effect of Tribulus terrestris (TT) on LH levels
in control and addicted groups. Bars depict mean ± SEM
of LH.* and $; P<0.05 compared between the control and
treated control groups.
Testosterone analysis
In this analysis, there was a reduction in the
addicted group (0.122 ± 0.058 ng/ml). The high-
est amount of testosterone belonged to the con-
trol group (0.399 ± 0.04 ng/ml). Figure 3 shows
that the addicted group had the least amount of
testosterone, as a result of morphine suppres-
sion. The treated addicted group had a signifi-
cantly lower hormone level (p<0.024) than did
the treated control group (0.193 ± 0.057 ng/ml).
The addicted group had a significantly lower
testosterone level than did the control group
(p<0.001).
Testestrone (ng/ml)
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Control Con+TT Morphine Mor+TT
Test groups
*
*
Fig 3: The effect of Tribulus terrestris (TT) on testosterone
levels in control and addicted groups.
Bars depict mean ± SEM of testosterone. *; P<0.05 com-
pared between the control and treated control groups.
Estrogen analysis
Our estrogen analysis revealed a decrease in
the addicted group compared to the control group
(22.70 ± 3.21 pg/ml). Figure 4 illustrates these
IJFS, Vol 7, No 1, Apr-Jun 2013
60
results. Compared to the control group, estro-
gen decreased in the addicted group; there was a
signicant difference between these two groups
(p<0.002). Figure 4 also shows a signicant dif-
ference between the treated addicted group and the
treated control group (p<0.048).
Estrogen (pg/ml)
60
50
40
30
20
10
0
Control Con+TT Morphine Mor+TT
Test groups
*
$
Fig 4: The effect of Tribulus terrestris (TT) on estrogen levels in
control and addicted groups. Bars depict mean ± SEM of LH.
* and $; P<0.05 compared between the control and treated
control groups.
Progesterone analysis
There was an increase in the treated control
group and a decrease in the addicted group
according to progesterone analysis. Figure 5
shows that the treated control group had the
highest hormone level (79.28 ± 5.2 pg/ml),
which was due to TT. The addicted group had
the least amount, which was due to the effects
of morphine. The addicted group had a lower
progesterone level than did the control group;
the difference, however, was not significant. In
comparison with the treated addicted group, the
treated control group had a significant increase
in hormone levels (p<0.002).
Progesterone (pg/ml)
90
80
70
60
50
40
30
20
10
0
Control Con+TT Morphine Mor+TT
Test groups
*
Fig 5: The effect of Tribulus terrestris (TT) on progesterone
levels in the control and addicted groups. Bars depict mean
± SEM of progesterone.
*; P<0.05 compared between the control and treated control
groups.
Discussion
In this study, sex hormone and gonadotropin
levels were evaluated based on the effect of TT
on morphine-addicted rats. FSH, a stimulating
hormone of the ovarian follicles, had a higher
level in our morphine-addicted groups. In the
treated morphine group, the FSH level had the
most decrease, which demonstrated that TT
suppressed FSH release in the addicted rats. A
similar result was observed in the treated con-
trol group; however the difference was not sig-
nificant.
Tabakova and his colleagues, in a compari-
son of endocrinal functions before and after
TT therapy, reported that TT acted on the hy-
pothalamus and reduced FSH levels but did not
decrease ovarian hormones (estrogen and pro-
gesterone). They concluded that TT could be
used to treat menopausal symptoms such as hot
flashes and increase sex drive. These research-
ers speculated that the presence of saponin in
TT was responsible for FSH suppression and
the resultant alleviation of hot flashes, irritabil-
ity, and depression in menopausal women (16).
However, in our study, no significant effect of
TT on FSH levels was found.
We observed a signicant decrease in LH lev-
els in the morphine-addicted group. The treated
addicted group had a relatively higher LH than
did the addicted group, which denoted that TT in-
creased LH levels. Similarly, the treated control
group had a signicantly higher LH level than did
the control group.
According to Neychev et al. (17) and a study
by Antonio et al. it was shown that TT induced
LH release. The upswing in LH led to a signal
for testosterone to increase (18). With regard to
testosterone levels, there was a significant de-
crease in our addicted rats. In the treated ad-
dicted group, the level was significantly higher
than that of the addicted group. Although TT
affected the increase in testosterone, a num-
ber of studies have demonstrated different re-
sults. Studies demonstrated that TT caused an
increase in testosterone, increase in cavernous
body pressure, increase in systolic pressure, and
increase in penile erection (19-22). In addition,
the effect of TT on castrated rats compared to
Ghosian Moghaddam et al.
61
normal rats was an increase in prostate weight,
which augmented sexual activities and potency
(22). In contrast, Neychev and his colleague re-
ported that TT did not influence androgen lev-
els in young men (17). Our results support the
results of a majority of similar studies in the
literature.
Based on our study, estrogen decreased sig-
nificantly in the addicted group but increased
significantly in the treated addicted group. It
can, therefore, be concluded that TT signifi-
cantly increased the estrogen hormone level in
male addicted rats. Of note, there was a dearth
of specic studies on estrogen in the literature.
Progesterone decreased in our addicted group;
the amount, however, was insignificant. In con-
trast, progesterone significantly increased in
the treated control group. We concluded that TT
increased progesterone in non-addicted cases;
however this increase was not significant in ad-
dicted cases. Mazaro-Costa and colleagues have
reported that not only could TT treat sexual dis-
orders and dysfunction in menopausal women
but it could also enhance vasomotor actions.
There are, however, no reports on progesterone
specifically (19).
Conclusion
The present study shows that addiction decreas-
es sex hormones and gonadotropins. Treatment
with TT can increase the hormone levels of tes-
tosterone, progesterone, estrogen, and LH. Never-
theless, TT did not increase the FSH levels in our
male rats.
Acknowledgments
This paper is the result of a medical student
thesis and was financially supported by the
Research Council of Shahed University. The
authors declare that they have no conflicts of
interest.
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