Journal of Ethnopharmacology 127 (2010) 165–170

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Effects of Tribulus terrestris on endocrine sensitive organs in male and female

Wistar rats
Anderson J. Martino-Andrade a,∗ , Rosana N. Morais a , Katherinne M. Spercoski a , Stefani C. Rossi b ,
Marina F. Vechi b , Munisa Golin b , Natália F. Lombardi b , Cláudio S. Greca c , Paulo R. Dalsenter b
a
Departamento de Fisiologia, Universidade Federal do Paraná, Centro Politécnico, 81531-980 Curitiba, Brazil
b
Departamento de Farmacologia, Universidade Federal do Paraná, Centro Politécnico, 81531-980 Curitiba, Brazil
c
Departamento de Biologia Celular, Universidade Federal do Paraná, Centro Politécnico, 81531-980 Curitiba, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Aim of the study: Investigate the possible effects of Tribulus terrestris (TT) on endocrine sensitive organs in
Received 23 June 2009 intact and castrated male rats as well as in a post-menopausal rat model using ovariectomized females.
Received in revised form Materials and methods: Three different dose levels of TT (11, 42 and 110 mg/kg/day) were administered
15 September 2009 to castrated males for 7 days and to intact males and castrated females for 28 days. In addition to TT
Accepted 16 September 2009 treatment, all experiments also included a group of rats treated with dehydroepiandrosterone (DHEA).
Available online 23 September 2009
In experiments using castrated males and females we also used testosterone and 17␣-ethynylestradiol,
respectively, as positive controls for androgenicity and estrogenicity.
Keywords:
Results: Neither DHEA nor TT was able to stimulate androgen sensitive tissues like the prostate and
Tribulus terrestris
seminal vesicle in both intact and castrated male rats. In addition, administration of TT to intact male
Testosterone
Estrogenicity
rats for 28 days did not change serum testosterone levels as well as did not produce any quantitative
Androgenicity change in the fecal excretion of androgenic metabolites. However, a slight increase in the number of
homogenization-resistant spermatids was observed in rats treated with 11 mg/kg/day of TT extract. In
ovariectomized females, TT did not produce any stimulatory effects in uterine and vaginal epithelia.
Conclusions: Tribulus terrestris was not able to stimulate endocrine sensitive tissues such as the prostate,
seminal vesicle, uterus and vagina in Wistar rats, indicating lack of androgenic and estrogenic activity
in vivo. We also showed a positive effect of TT administration on rat sperm production, associated with
unchanged levels of circulating androgens.
© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction cating stimulatory effects of TT on sperm quantity and quality

Tribulus terrestris L. (Zygophyllaceae) is a perennial creeping
herb with a widespread distribution in Mediterranean, subtropi-
cal and desert climates worldwide. In traditional folk medicine, it
has been used since ancient times as an aphrodisiac as well as to
treat urinary infections, inflammation, oedema and other ailments
(Adaikan et al., 2001).
Although experimental and clinical studies have partially con-
firmed some effects of Tribulus terrestris (TT) on libido and sperm
production, there is still much debate regarding possible mech-
anisms of action as well as therapeutic applications. Results
published by Gauthaman et al. (2002, 2003) indicate that TT can
improve some aspects of male sexual behaviour and enhance
spermatogenesis in rats. In addition, there are clinical data indi-
∗ Corresponding author. Tel.: +55 41 33611719; fax: +55 41 33611714.
E-mail addresses: anderson.andrade@ufpr.br, martino.andrade@gmail.com
(A.J. Martino-Andrade).
and improved sexual response in men (Arsyad, 1996). Reports
of increased androgen levels have also been reported following
TT administration to nonhuman primates, rats and rabbits (El-
Tantawy et al., 2007; Gauthaman and Ganesan, 2008), but most of
these effects were short-lived and showed no clear dose–response
relationships. In addition, there is no consensus on the exact
mechanisms underlying TT effects on sexual performance and sper-
matogenesis. It is believed that the steroidal saponins present in TT
extracts, particularly protodioscin, can increase endogenous andro-
gen production by increasing luteinizing hormone (LH) release
from the pituitary gland. Alternatively, it has been proposed that
TT active components might be enzymatically converted into weak
androgens like dehydroepiandrosterone (DHEA), which could in
turn be converted into more potent androgens like testosterone in
the gonads and peripheral tissues (Adaikan et al., 2001). However,
changes in endogenous hormone levels following TT administra-
tion are still controversial (Neychev and Mitev, 2005).
The presence of steroidal saponins also raises the question
whether TT extracts might have intrinsic hormonal activity that

0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2009.09.031
166 A.J. Martino-Andrade et al. / Journal of Ethnopharmacology 127 (2010) 165–170
could directly stimulate male and/or female endocrine sensitive tis-
sues such as the prostate, seminal vesicle, uterus and vagina (Nian
et al., 2006). This is considered an important issue for both effi-
cacy and safety assessment of TT. Gauthaman et al. (2002) reported
that administration of TT to castrated rats was able to improve
sexual behaviour and increase prostate weight and intracavernous
pressure in relation to castrated controls. Currently, TT has been
also indicated as an alternative treatment to hormone replacement
therapy in aging men and women. In the present study, we inves-
tigated whether TT extract could affect endocrine sensitive organs
in intact and castrated male rats as well as in a post-menopausal
rat model using ovariectomized females. Moreover, we also inves-
tigated the effects of TT on sperm production and monitored fecal
androgen levels in intact treated males. In addition to TT treatment,
all experiments also included a group of rats treated with DHEA, an
endogenous hormone claimed to directly and/or indirectly increase
androgen effects and promote general well being in aging men and
women (Brown et al., 2000, 2001).
2. Materials and methods
2.1. Tribulus terrestris extract
The dry ethanolic extract of TT (Androsten® ) was supplied
by Herbarium Laboratório Botânico (Colombo, Brazil; lot number
047918) and contained 16.43% of protodioscin. A dried voucher
specimen is also available.
2.2. Animals
Wistar rats were obtained from the stock of the Universidade
Federal do Paraná and kept under a 12 h light/dark cycle and
controlled temperature (22 ± 2 ◦ C). Standard pellet food (Nuvital® ,
Curitiba/PR) and tap water were available ad libitum. All experimen-
tal protocols were approved by the Committee on Animal Research
and Ethics of the Universidade Federal do Paraná (Consent Num-
ber 331) which is in accordance with national and international
Guidelines of animal welfare.
For the test of androgenicity using castrated rats (Hershberger
Assay), 7-week-old male rats were castrated under anesthesia
induced by intraperitoneal injections of ketamin (90 mg/kg) and
xilazin (1.5 mg/kg). Treatment was not commenced until 7 days
after castration to allow for complete recovery.
For evaluation of females, bilateral ovariectomy was conducted
in 13-week-old rats that were anesthetized as described above for
males. Likewise, a recovery period of 7 days was observed before
administration of test compounds.
2.3. Chemicals and dose selection
The Tribulus terrestris dry extract was dissolved in distilled water
(vehicle) and administered by gavage in a volume of 5 mL/kg body
weight. In all experimental protocols, one group served as vehicle
control (distilled water) and three other groups received different
doses of TT extract: 11, 42 and 110 mg/kg/day. The lowest dose
level (11 mg/kg/day) corresponds to approximately the usual dose
indicated for humans (750 mg/day and assuming a body weight of
70 kg), while the highest dose (110 mg/kg/day) is a 10-fold extrap-
olation. The intermediate dose of 42 mg/kg/day corresponds to the
extrapolated total human dose (750 mg/day) to rats following an
allometric calculation (Nevill, 1994).
In addition, in all experimental protocols, one group of animals
was orally treated with a commercially available DHEA supple-
ment (CAS no. 53-43-0; Natrol Inc., Chatsworth, CA). The dose
of DHEA used was 5 mg/kg/day, which is a dose 10 times higher
than the usual supplementation dose in humans (Rhoden et al.,
2003). In experiments using castrated males and females we also
used testosterone propionate and 17-␣-ethynylestradiol, respec-
tively, as positive controls for androgenicity and estrogenicity (see
Sections 2.4 and 2.6 below). DHEA, testosterone propionate and
17-␣-ethynylestradiol were dissolved in canola oil. All drugs were
administered by oral route (gavage) in a volume of 5 mL/kg body
weight, except for testosterone propionate, which was adminis-
tered subcutaneously to castrated rats in a volume of 1 mL/kg body
weight.
2.4. Test of androgenicity using castrated males – Hershberger
Assay
Castrated male rats (12–14 animals/group) were treated with
distilled water (vehicle control), DHEA (5 mg/kg/day) or three dose
levels of TT extract (11, 42 and 110 mg/kg/day) by oral route for 7
days (Owens et al., 2006). In addition, a group of castrated rats was
treated subcutaneously with 0.25 mg/kg/day of testosterone pro-
pionate (CAS no. 57-85-2; Sigma–Aldrich, Steinheim, Germany) to
serve as a positive control for androgenicity (Andrade et al., 2002a).
One day after the final administration, all rats were weighed and
killed by cervical dislocation. After careful trimming to remove fat
and other contiguous tissues in a uniform manner, ventral prostate
and seminal vesicle (with coagulating glands) were weighed. Sem-
inal vesicle was weighed without seminal fluid. Ventral prostate
and seminal vesicle weights were expressed as relative weights (%
of body weight).
2.5. Evaluation of TT effects in intact male rats treated during 28
days
To evaluate the effects of TT on sperm production, reproductive
organ weights and androgen levels, 13-week old rats were treated
daily by oral gavage with distilled water (vehicle control), DHEA
or three dose levels of TT extract (11, 42 and 110 mg/kg/day) for
28 days. One day after the final administration, males were decap-
itated and trunk blood was collected and allowed to clot at room
temperature. After centrifugation, serum samples were transferred
to clean tubes and kept at −20 ◦ C until analysis of testosterone
levels by enzyme immunoassay (Section 2.7). In addition, fecal pel-
lets were collected from individual rats on days 1, 7, 14, 21 and
28 of treatment for evaluation of fecal androgenic metabolites.
Body weight was recorded daily and at the end of treatment testes,
epididymides, ventral prostate, seminal vesicle, liver, kidneys and
adrenal glands were removed and weighed. Organ weights were
expressed as both absolute and relative weights (% of body weight).
Weight of paired organs is presented as the mean of left and right
sides, except for testes, for which only the right testis was weighed.
Immediately after sacrifice, the left testes were immersed-fixed
in Bouin’s solution for 24 h, and subsequently dehydrated and
embedded in paraffin. Testicular sections of 5 ␮m were stained
with hematoxilin and eosin and analyzed microscopically. Twenty
round seminiferous tubules per testis were randomly selected for
measurement of the minor diameter and seminiferous epithelium
height using the 3.0 version of UTHSCSA ImageTool software (UTH-
SCSA, San Antonio, TX). Images were acquired on a Zweiss Axiophot
microscope (Oberkochen, Germany) coupled with an imaging sys-
tem (ASI, Vista, CA) at 200× magnification. The right testes were
used for determination of the number of homogenization-resistant
spermatids in a Neubauer cell counting chamber as previously
described (Amann, 1982; Andrade et al., 2002b).
2.6. Evaluation of TT effects in ovariectomized female rats treated
during 28 days
Ovariectomized females (n = 5–12/group) were treated daily
by oral gavage with distilled water (vehicle control), DHEA
 A.J. Martino-Andrade et al. / Journal of Ethnopharmacology 127 (2010) 165–170
Fig. 1. Ventral prostate (A) and seminal vesicle (B) relative weights of castrated male rats treated with TT, DHEA or testosterone propionate for 7 days. Number of animals
used is in parentheses. *Significantly different from control group (p < 0.05; ANOVA – Dunnett).
(5 mg/kg/day) or three dose levels of TT extract (11, 42 and
110 mg/kg/day) for 28 days. An additional group was treated orally
with 0.1 mg/kg/day of 17-␣-ethynylestradiol (CAS no. 57-67-3;
Sigma–Aldrich, Steinheim, Germany) to serve as a positive control
for estrogenicity. Body weight was monitored throughout the treat-
ment period. One day after the final administration, females were
decapitated and the weights of liver, kidneys, adrenal glands and
uterus were recorded and reported as both absolute and relative
weights (% of body weight). Weight of paired organs is presented
as the mean of left and right sides.
A mid-portion of the right uterine horn as well as a mid-
fragment of the vagina was fixed in Bouin’s solution for 24 h.
After histological processing, uterine and vaginal sections of 5 ␮m
were stained with hematoxylin and eosin. Luminal epithelium cell
heights were measured in six uterine/vaginal sections per animal
using the 3.0 version of UTHSCSA ImageTool software (UTHSCSA,
San Antonio, TX). In each section, four different regions were ana-
lyzed, giving a total of 24 measurements per animal. Images were
acquired on a Zweiss Axiophot microscope (Oberkochen, Germany)
coupled with an imaging system (ASI, Vista, CA) at 200× magnifi-
cation.
2.7. Analysis of serum testosterone and fecal androgenic
metabolites
Fecal pellets were collected from intact male rats treated with
distilled water (control), TT extract (11, 42 and 110 mg/kg/day)
or DHEA (5 mg/kg/day) on days 1, 7, 14, 21 and 28 of treat-
ment. Fecal steroids were extracted according to the procedure
previously described by Touma and Palme (2005). Briefly, an
aliquot of approximately 0.5 g (±0.05 g) of the well-mixed wet
fecal sample was placed in a glass tube containing 5 mL of 80%
ethanol: 20% distilled water and vigorously shaken using Multi
Pulse vortexer (Glas-Col, Terre Haute, IN) for 30 min. Each sample
was centrifuged at 1000 × g for 15 min, and supernatant recov-
ered and diluted at 1:1 ratio with a phosphate buffered solution.
Fecal extracts were analyzed by enzyme immunoassay (Munro et
al., 1991) using a polyclonal anti-testosterone antibody (R 156/7
1:7500 dilution) obtained from Coralie Munro at the University
of California (Davis, CA, USA), cross reacting with testosterone
100%, 5␣-dihydrotestosterone 57.4%, androstenedione 0.27%, and
androsterone, dehydroepiandrosterone (DHEA), cholesterol, estra-
diol, progesterone and pregnenolone <0.05%. Serial dilutions of
pooled fecal extracts produced displacement curves parallel to
167
those of standards and the assay sensitivity was 2.3 pg/well. Serum
samples were analyzed as described above, but without previ-
ous extraction. The intra- and inter-assay coefficients of variation
were 2.8 and 9.74%, respectively. The general term “fecal andro-
genic metabolites” is used as metabolites of several androgens are
excreted in the feces and can be eventually recognized by the anti-
body of the enzyme immunoassay.
2.8. Statistical analysis
Data were analyzed using GraphPad Prism 5.0 (GraphPad Soft-
ware Inc, La Jolla, CA). Normality and homogeneity of variances
were evaluated prior to statistical analysis. Data were analyzed
by analysis of variance (ANOVA) followed by Dunnett’s multiple
comparison test. Serum testosterone levels were not normally dis-
tributed and, therefore, log-transformed values were used in the
ANOVA. For the analysis of fecal androgenic metabolites, two-way
ANOVA was performed with time and treatment as fixed factors.
Differences were considered to be statistically significant at a prob-
ability level of 5% (p < 0.05).
3. Results
3.1. Test of androgenicity using castrated males – Hershberger
Assay
An increase in both ventral prostate and seminal vesicle rela-
tive weights was seen in castrated rats treated with 0.25 mg/kg/day
testosterone propionate, when compared to castrated controls.
However, no significant effects were seen in rats treated with TT
or DHEA (Fig. 1).
3.2. Evaluation of TT effects in intact male rats
At the end of treatment, no changes were observed in serum
testosterone levels at any experimental group (Fig. 2). However,
fecal androgenic metabolites were increased in the group treated
with DHEA in all time points evaluated after beginning of treatment
(days 7, 14, 21 and 28) (Fig. 2).
When compared to control group, the number of
homogenization-resistant spermatids per testis was signifi-
cantly increased in rats treated with 11 mg/kg/day of TT extract,
but not at higher doses (Fig. 3). When these results were expressed
168 A.J. Martino-Andrade et al. / Journal of Ethnopharmacology 127 (2010) 165–170
Fig. 2. Androgenic hormone levels in adult male rats treated with TT or DHEA for
28 days. Serum testosterone levels (A) were measured at the end of the treatment
period while fecal androgenic metabolites (B) were analyzed at different time points
throughout treatment. Number of animals used is in parentheses. *Significantly
different from control group (p < 0.05; ANOVA – Dunnett).
in relation to testis weight (spermatid number/g testis), no sta-
tistically significant differences were detected for any treatment
group (Fig. 3). The mean diameter of seminiferous tubules and the
seminiferous epithelium height were not significantly different
from control for any treatment group (data not shown). Absolute
and relative organ weights (testis, epididymis, ventral prostate,
seminal vesicle, liver, kidney and adrenal gland) from TT groups
were not significantly different from those of control rats (Table 1).
Absolute liver and kidney weights of DHEA treated animals were
significant lower when compared to control group, but relative
Fig. 3. Number of homogenization-resistant spermatids in male rats treated with TT or DHEA for 28 days. Both absolute (A) and corrected for testis weight (B) numbers are
presented. Number of animals used is in parentheses. *Significantly different from control group (p < 0.05; ANOVA – Dunnett).
weights were not different. The absolute and relative weights of
all other organs of DHEA treated animals were not different from
controls. Body weight of rats treated with DHEA was lower than
that of control animals at the end of treatment, but DHEA rats were
already lighter at the beginning of treatment (Table 1).
3.3. Evaluation of TT effects in ovariectomized female rats
Treatment of ovariectomized rats with DHEA or three differ-
ent dose levels of TT extract during 28 days did not change either
absolute or relative uterus weight in relation to ovariectomized
controls (Table 2). In addition, no changes were observed in the
uterine and vaginal epithelium cell heights (Fig. 4). However, as
expected, administration of 0.1 mg/kg/day of ethynylestradiol was
able to increase absolute and relative uterus weight (Table 2) as
well as the uterine and vaginal epithelium cell heights (Fig. 4). Body
weight at the end of treatment was significantly reduced in animals
treated with ethynylestradiol. Moreover, relative liver and adrenal
weights were significantly increased while absolute kidney weight
was reduced in this group when compared to controls. Absolute
adrenal weights were significantly suppressed in animals treated
with DHEA.
4. Discussion
Tribulus terrestris (TT) has been popularly used as an aphrodisiac
and enhancer of sperm production and more recently as an alter-
native to hormone replacement therapy in aging men and women
(Gauthaman and Ganesan, 2008). In the present study, we demon-
strated that TT has no intrinsic hormonal activity, being unable to
stimulate endocrine sensitive organs in both male and female rats.
We also demonstrated that administration of TT to intact male rats
for 28 days did not change serum testosterone levels as well as
did not produce any quantitative change in the fecal excretion of
androgenic metabolites.
As previously mentioned, it is believed that active components
of TT extract might be enzymatically converted into weak andro-
gens like DHEA, which could in turn be converted into more potent
androgens in the gonads and peripheral tissues. In our results,
the tested doses of TT extract (11, 42 and 110 mg/kg/day) did
not produce any significant effect on serum testosterone levels in
intact male rats after 28 days of treatment. Moreover, treatment
with TT did not induce any quantitative change in the excre-
tion of fecal androgenic metabolites throughout treatment. The
 A.J. Martino-Andrade et al. / Journal of Ethnopharmacology 127 (2010) 165–170 169

Table 1
Body weight and absolute and relative (%) organ weights of intact male rats treated during 28 days with distilled water (control), Tribulus terrestris (TT) or DHEA.

Parameter Treatment (mg/kg/day)

Control TT 11 TT 42 TT 110 DHEA 5

n 13 13 13 13 12
Body weight day 1 (g) 291 ± 10.1 292 ± 9.8 289 ± 6.9 292 ± 10.2 255 ± 5.1*
Body weight day 28 (g) 334 ± 10.1 334 ± 9.5 341 ± 7.1 316 ± 11.7 291 ± 6.7*
Liver (g) 11.5 ± 0.39 11.4 ± 0.37 11.1 ± 0.39 10.4 ± 0.46 9.7 ± 0.44*
Liver (g%) 3.43 ± 0.07 3.42 ± 0.06 3.26 ± 0.07 3.29 ± 0.07 3.33 ± 0.10
Kidneys (g) 1.13 ± 0.03 1.14 ± 0.04 1.11 ± 0.02 1.05 ± 0.04 0.96 ± 0.03*
Kidneys (g%) 0.34 ± 0.004 0.34 ± 0.07 0.33 ± 0.006 0.33 ± 0.006 0.33 ± 0.006
Adrenal gland (mg) 23.8 ± 1.01 24.8 ± 0.92 24.0 ± 0.84 24.1 ± 1.50 21.04 ± 0.93
Adrenal gland (mg%) 7.11 ± 0.25 7.47 ± 0.24 7.06 ± 0.23 7.56 ± 0.32 7.23 ± 0.30
Testis (g) 1.70 ± 0.03 1.81 ± 0.04 1.66 ± 0.03 1.71 ± 0.05 1.63 ± 0.05
Testis (g%) 0.51 ± 0.013 0.55 ± 0.017 0.49 ± 0.013 0.55 ± 0.020 0.56 ± 0.019
Epididymis (mg) 522 ± 12.5 549 ± 14.0 527 ± 11.0 525 ± 13.0 497 ± 17.6
Epididymis (mg%) 158 ± 5.66 165 ± 4.08 155 ± 2.61 168 ± 5.45 171 ± 6.23
Ventral prostate (mg) 282 ± 21.4 284 ± 19.6 316 ± 24.7 302 ± 17.9 285 ± 17.6
Ventral prostate (mg%) 84.4 ± 5.51 84.9 ± 5.07 93.1 ± 7.08 95.8 ± 4.72 97.8 ± 5.87
Seminal vesicle (mg) 460 ± 17.8 508 ± 17.7 465 ± 19.8 443 ± 20.3 437 ± 14.4
Seminal vesicle (mg%) 139 ± 5.38 153 ± 5.29 138 ± 7.17 141 ± 5.96 150 ± 4.96
*
Significantly different from control group (p < 0.05; ANOVA – Dunnett).

Fig. 4. Uterine and vaginal luminal epithelial cell heights in ovariectomized female rats treated with TT, DHEA and 17-␣-ethynylestradiol (EE) for 28 days. Number of animals
used is in parentheses. *Significantly different from control group (p < 0.05; ANOVA – Dunnett).

unchanged weights of prostate and seminal vesicle in both intact
and castrated rats also indicate a lack of intrinsic androgenic activ-
ity of this natural product as well as an absence of an indirect
mechanism of androgenic stimulation. Fecal androgenic metabo-
lites were increased in the group treated with 5 mg/kg/day of DHEA
in all time points evaluated after beginning of treatment, indicating
that one or more metabolites formed from DHEA were detected in
our immunoassay. However, the lack of effects on serum testos-
terone and the unchanged weights of prostate and seminal vesicle
in both intact and castrated rats indicate that, similarly to TT, DHEA
has neither direct nor indirect androgenic activity at the dose level
tested here. Based on the results of fecal androgenic metabolites,
it is also possible to infer that TT did not change the endogenous
levels of DHEA, as an increase in these fecal metabolites, similar
to that observed in DHEA treated rats, was not observed in TT
groups.

Table 2
Body weight and absolute and relative (%) organ weights of ovariectomized female rats treated during 28 days with distilled water (control), Tribulus terrestris (TT), DHEA or
17-␣-ethynylestradiol (EE).

Parameter Treatment (mg/kg/day)

Control TT 11 TT 42 TT 110 DHEA 5 EE 0.1

n 8 5 10 11 7 12
Body weight day 1 (g) 257 ± 9.7 273 ± 18.7 256 ± 4.7 239 ± 5.8 252 ± 2.8 248 ± 9.0
Body weight day 28 (g) 295 ± 9.3 307 ± 8.9 295 ± 6.7 281 ± 6.5 276 ± 6.6 240 ± 9.0*
Liver (g) 9.5 ± 0.30 10.7 ± 0.52 9.5 ± 0.41 9.7 ± 0.24 9.0 ± 0.47 9.76 ± 0.48
Liver (g%) 3.24 ± 0.10 3.48 ± 0.14 3.20 ± 0.08 3.45 ± 0.06 3.24 ± 0.10 4.07 ± 0.13*
Kidneys (mg) 868 ± 25.4 879 ± 29.5 852 ± 27.0 859 ± 26.9 800 ± 21.8 737 ± 30.4*
Kidneys (mg%) 295 ± 7.13 287 ± 8.84 289 ± 4.68 305 ± 5.58 290 ± 6.23 308 ± 6.87
Adrenal gland (mg) 33.4 ± 2.53 35.0 ± 2.65 33.2 ± 1.43 30.7 ± 2.26 24.0 ± 2.50* 34.8 ± 1.80
Adrenal gland (mg%) 11.20 ± 0.64 11.38 ± 0.70 11.25 ± 0.35 10.96 ± 0.81 8.67 ± 0.91 14.69 ± 0.90*
Uterus (g) 94 ± 4.28 81 ± 5.63 101 ± 5.73 85 ± 4.31 100 ± 3.22 372 ± 24.70*
Uterus (mg%) 32.0 ± 1.39 26.4 ± 1.78 34.4 ± 2.08 30.4 ± 1.53 36.4 ± 1.60 156 ± 9.82*
*
Significantly different from control group (p < 0.05; ANOVA – Dunnett).
170 A.J. Martino-Andrade et al. / Journal of Ethnopharmacology 127 (2010) 165–170
In relation to TT effects on sperm production, we demonstrated
that the lowest dose tested (TT 11 mg/kg/day) was able to increase
the number of homogenization-resistant spermatids without caus-
ing any alteration on the mean diameter of seminiferous tubules,
results that indicate a possible increase in the efficiency of sper-
matogenesis. The reason for an increase in spermatid number at
the lowest dose is unknown. However, in pharmacology it is rel-
atively common to observe nonmonotonic dose–responses, with
significant effects at low doses and no effects (or opposite effects)
at higher doses (Welshons et al., 2003; Andrade et al., 2006).
These responses are sometimes observed when testing crude plant
extracts, which contain multiple components that can interact in
many different ways (Belz, 2007).
Sex steroids also influence the growth, differentiation and func-
tion of female reproductive organs such as the uterus and vagina,
making them susceptible to endocrine disruption. In humans, these
tissues undergo severe atrophy following the decline of ovarian
function and the consequent fall in sex steroid hormone levels
during the postmenopausal period. In addition, typical symptoms
that result from the sudden decline in hormone levels, particularly
estrogens, are vasomotor changes, urogenital complications, mood
swings and sleep disturbances. Recently, the search for “natural”
alternative therapies to treat these symptoms has increased. How-
ever, one important aspect of the safety of natural products used as
alternatives to classical hormone replacement therapy is the deter-
mination of their intrinsic hormonal activity (e.g., estrogenicity), as
they could also overstimulate endocrine sensitive tissues such as
the endometrium and the mammary gland (Rice and Whitehead,
2006; Wuttke et al., 2007). In the present study, we observed, as
expected, a severe atrophy of both uterine and vaginal epithelia
in ovariectomized female rats. In addition, an effective reversion of
this atrophic state was seen after the oral administration of the syn-
thetic estrogen 17-␣-ethynylestradiol. However, administration of
TT to ovariectomized rats for 28 days was unable to promote prolif-
eration of both uterine and vaginal epithelia, indicating absence of
intrinsic estrogenic activity of this natural product. Likewise, DHEA
treatment did not change the cell height of both vaginal and uter-
ine epithelia of ovariectomized rats when compared to controls. In
addition, no treatment regimen was able to increase uterine weight,
except for 17-␣-ethynylestradiol.
Overall, our results demonstrate that Tribulus terrestris is not
able to stimulate endocrine sensitive tissues such as the prostate,
seminal vesicle, uterus and vagina in Wistar rats, indicating lack
of androgenic and estrogenic activity in vivo. We also showed a
positive effect of TT administration on rat sperm production, asso-
ciated with unchanged levels of circulating androgens. However,
it is important to note that conflicting results following TT admin-
istration in experimental animals may arise as a consequence of
differences in species or strains used, duration of treatment, and
content of active components in the extract. Additional clinical
and experimental studies using extended treatment periods and
a larger number of individuals are necessary in order to clearly
establish the effects of TT on sperm production and sex steroid
levels.
Acknowledgements
This work was financially supported by Herbarium Laboratório
Botânico (Colombo, Brazil) and by CNPq (Brazil).
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