Accepted: 27 April 2016

DOI: 10.1111/and.12655

ORIGINAL ARTICLE

Effect of oral administration of Tribulus terrestris extract on

semen quality and body fat index of infertile men

R. M. Salgado | M. H. Marques-Silva | E. Gonçalves | A. C. Mathias | J. G. Aguiar |

P. Wolff

Genics – Reproductive Medicine and

Genomics, São Paulo, Brazil
Correspondence
Renato M. Salgado, Genics – Reproductive
Medicine and Genomics, São Paulo, Brazil.
Email: rmsalgado81@gmail.com
Summary
Male fertility can be evaluated through complete semen analysis. Plants belonging to
the Tribulus genus are known for their role in enhancing sex hormone levels and semen
quality. The aim of this study was to evaluate the effects of T. terrestris on semen qual-
ity and physiological parameters. Sixty-­five men with abnormal semen evaluation were
included in this study, in which they were prescribed with oral administration of
Androsten® (250 mg of Tribulus terrestris dried extract per capsule). Body fat percent-
age, lean muscle mass gain, fluctuation in steroid hormone levels and all semen param-
eters were analysed during the period of treatment. The results demonstrated that
decrease in the percentage of body fat and increase in lean mass were significant, as
well as increase in dihydrotestosterone levels. Complete semen analysis evaluated at
the end of treatment showed significant enhancement in sperm concentration, motil-
ity and liquefaction time. Protodioscin, the main phytochemical agent of the Tribulus
genus, acts on sertoli cells, germ cell proliferation and growth of seminiferous tubules.
This component is known to convert testosterone into dihydrotestosterone, which
plays important roles in male attributes. Our results indicate the therapeutic use of
Tribulus terrestris by men presenting altered semen parameters, and/or undergoing
infertility treatment.

KEYWORDS
dihydrotestosterone, male infertility, protodioscin, semen quality, Tribulus terrestris

1 | INTRODUCTION typically perform a physical examination and a series of tests including

Infertility in humans is defined by the failure to achieve a clinical preg-
nancy after 12 months or more of regular unprotected sexual inter-
course (Zegers-­Hochschild et al., 2009). Infertility affects society at
large and is intimately associated with emotional, psychological and
social issues for the patient. Male factor is involved in approximate-
ly 40% of all infertility cases registered worldwide. Many causes of
male infertility are still defined as idiopathic, and diagnosis tends to
be descriptive, often leading to ineffective medical approaches to
treatment or management (DeKretser & Baker, 1999; World Health
Organization, 2000). For diagnosis of male factor infertility, physicians
complete semen analysis, karyotyping, Y chromosome microdeletion,
sperm DNA fragmentation rate, endocrine profiling and, if necessary,
testicular biopsy.
Semen is a complex fluid composed of cellular elements and
plasma, primarily derived from the seminal vesicles and prostate
(Duncan & Thompason, 2007). Although several physiological and
environmental factors influence variability among semen samples,
semen analysis itself can serve as an overall predictor of male fer-
tility. Semen parameters are divided into macroscopic—qualities
of the semen as a whole (coagulation, colour, viscosity, pH and
volume)—and microscopic—qualities of the spermatozoa (sperm

Andrologia 2016; 1–6 wileyonlinelibrary.com/journal/and © 2016 Blackwell Verlag GmbH | 1

2 |  Salgado et al.
concentration, motility, morphology and viability; Turek, 2012).
These parameters may be affected by many factors, including
certain medications, chemotherapy, pollution, toxic products and
smoking, as well as excessive alcohol consumption (du Plessis,
Agarwal, & Sabanegh, 2014).
There are a variety of plants thought to improve sexual perfor-
mance and male fertility. Among them are the members of the genus
Tribulus of the Zygophyllaceae family, which comprises about 20
species of shrubs or herbs that grow in subtropical areas around the
world (Hegnauer, 1973). Tribulus terrestris (TT), T. cistoides and T. ala-
tus have been extensively used as male sexual stimulants (El-­Tantawy,
Temraz, & El-­Gindi, 2007). Moreover, TT has been used as tonic, aph-
rodisiac, palliative, astringent, stomachic, antihypertensive, diuretic
and urinary disinfectant; the fruit of the herb is also used to treat sex-
ual dysfunction (review, Chhatre, Nesari, Somani, Kanchan, & Sathaye,
2014).
There are some data to support a positive effect of oral TT
administration on male sexual function (Tomova et al., 1978). Oral
administration of TT extract increased erectile function in rabbit
penile tissue (Adaikan, Gauthaman, Prasad, & Ng, 2000). In old-
er rats, oral TT extract increased sexual activity, increased serum
testosterone and increased epididymal sperm numbers, in a dose-­
dependent manner (Singh, Nair, & Gupta, 2012). Administration
of TT increased both testosterone and dihydrotestosterone (DHT)
concentrations in rats, rabbits and rhesus monkeys (Gauthaman &
Ganesan, 2008). Considering its various phytochemical components,
majorly present are furostanol glycosides, such as protodioscin and
protogracillin, of which protodioscin is the most abundant saponin
(Kostova & Dinchev, 2005). It is suggested that a major role of this
steroidal saponin is to induce the reduction in testosterone into the
potent dihydrotestosterone (DHT), which occurs by the action of
the enzyme 5-­alpha reductase (review, Chhatre et al., 2014). In men,
protodioscin appeared to increase serum dehydroepiandrostenedi-
one (DHEA) concentrations and increase erectile function, when
values were compared before and after administration (Adimoelja
& Adaikan, 1997).
However, other studies showed little effect of TT extract.
Frydrychová et al. (2011) fed boars with a preparation containing
extracts of three herbs, including TT, for 10 weeks. All boars had
normal semen parameters at the onset of treatment. The treated
group had low libido; libido returned to control levels after 10 weeks
of treatment. There was no significant effect of treatment on sperm
parameters.
Two other studies were conducted to analyse the effects of TT
extract in athletes; however, there was no effect of oral administra-
tion of TT extract on lean muscle mass gain or body strength enhance-
ment in men during physical training (Antonio, Uelmen, Rodruiguez, &
Earnest, 2000; Rogerson et al., 2007).
The aim of the present trial was to evaluate whether treatment
with daily oral administration of TT extract in men having male fac-
tor infertility significantly affects semen quality and the androgen-­
related physiological parameters of lean muscle mass and body fat
index.
2 | MATERIALS AND METHODS
2.1 | Study population and data collection
Sixty-­five male patients (n = 65) were enrolled in this clinical study for
a total duration of 12 consecutive weeks. The study was conducted
at Instituto de Pesquisa Aluisio Calil Mathias, a private assisted repro-
duction centre in Brazil. Patients were evaluated on days 0, 1, 7, 14,
28, 56 and 84 (total of seven appointments).
The inclusion criteria for patient selection were as follows:
18–50 years of age with no hormonal disorders or chronic diseas-
es, diagnosed with infertility (no pregnancies and/or diagnosed with
altered sperm parameters; according to World Health Organization,
2000). The exclusion criteria were as follows: patients who used ana-
bolic steroids, patients with hepatic abnormalities as determined by
elevated liver enzymes on blood sample evaluation and patients par-
ticipating in any other clinical study. All patients included in the trial
read and signed an informed consent form. For recording of data, sub-
jects were identified using 3-­digit sequential numbers. This study was
approved by Ethics Committee of the University of São Paulo.
All participating men underwent an initial evaluation (Day 0) then
started treatment with TT extract in the form of a commercially pre-
pared pill (Androsten® [Herbarium Laboratório Botânico Ltda., Brazil]).
Each pill contained 250 mg of extract, including 37.5 mg (15%) of ste-
roidal saponins (protodioscin), according to the manufacturer. Starting
on Day 1, the men were instructed to take one pill orally every 8 hr,
thus providing 112.5 mg of protodioscin per day.
2.2 | Parameters evaluated
Safety was evaluated on days 0 and 84 of the treatment. The follow-
ing exams were performed: complete blood count, fasting glycemia,
liver function tests (transaminases, gamma-­glutamyl transferase, alka-
line phosphatase and total bilirubin), urea, creatinine and uric acid. No
significant differences were found; however, when any of these tests
was altered at the onset of treatment, the patient was not included
in the study.
At each visit, participants were evaluated for weight, body fat
index and lean muscle mass, the latter two both by dual-­energy
X-­ray absorptiometry (DEXA). DEXA scanners use an X-­ray tube that
scans the entire body. DEXA measures bone density, fat mass and
lean mass separately, with a high degree of accuracy (St-­Onge et al.,
2004).
Blood samples from participants were collected in serum-­
separation tubes on days 0, 28 and 84. The samples were allowed
to clot at room temperature and then were centrifuged for 10 min;
after centrifugation, serum was isolated, frozen and stored at −20°C.
Blood serum concentrations of free testosterone, FSH, LH and pro-
lactin were evaluated by chemiluminescence immunoassays (ADVIA
Centaur XPT Immunoassay System – Siemens Healthcare Diagnostics
Inc., USA), according to the manufacturer’s instructions. Analytical
measurement ranges are 10–1,500 ng dl−1 (testosterone), 0.2–
200 mIU ml−1 (FSH), 0.2–150 mIU ml−1 (LH) and 0.2–250 ng ml−1
Salgado et al. | 3

(PRL); insignificant cross-­reactivity has been reported for FSH, LH 3 | RESULTS

and prolactin, whereas low testosterone samples may present up
to 5% cross-­reactivity with DHT (as informed by the manufactur- 3.1 | Subjects
er). Blood serum concentrations of DHEA and DHT were measured
Initial semen analysis of patients enrolled in this trial revealed that the
by competitive ELISA, according to the manufacturer’s instructions
predominant alteration observed was oligozoospermia (32%), followed
(xyz kit—Diagnostic Biochem Canada Inc., Canada). Sensitivity of this
by azoospermia and oligoasthenozoospermia (14%). Of 65 men enrolled
assay is 6 pg ml−1 (DHT) and 0.1 ng ml−1 (DHEA), and the calibration
−1 in the study, 43 completed the study in its entirety with all physical exam-
range is 25–2,500 pg ml for DHT assay and 0.2–40 ng for DHEA
inations conducted, and only values from participants fully represented
assay. The DHT assay presents up to 8.7% cross-­reactivity with tes-
for that parameter were used for analysis of physiological parameters.
tosterone, whereas the DHEA assay presents 4.5% cross-­reactivity
with epiandrosterone.
On the same days as blood was collected, patients collected 3.2 | Physiological parameters
semen by masturbation. The following semen parameters were eval-
There were no significant differences in weight within patient over the
uated in fresh undiluted samples: ejaculate volume (ml), liquefac-
6 −1 seven examination points of the study (p > .1). There were also no sig-
tion time (min), sperm concentration (×10 ml ; evaluated visually
nificant differences in amount of fat tissue or total body mass (p > .1).
using a Makler chamber, under 200× magnification), total motility (%)
However, there was a significant increase in amount of lean tissue
and morphology (%). For morphological analyses, glass slides were
(p = .005) and a significant decrease in body fat percentage (p = .008)
smeared with 10 μl of fresh semen sample and stained with pan-
between Day 0 and Day 84 (Table 1).
optic staining technique; slides were observed at 1000× magnifica-
tion with immersion oil, and 200 sperm cells were counted. Samples
with sperm concentration ≤0.1 × 106 ml−1 were not considered for 3.3 | Hormone concentrations
statistical analysis, due to imprecision in using the Makler chamber;
morphology was not evaluated in samples with sperm concentration Blood serum concentrations of DHT increased significantly between
6
≤3 × 10 ml . −1 days 0 and 84 (p = .023); however, there were no significant differ-
ences in DHEA, free testosterone, FSH, LH and prolactin concentra-
tions between days 0 and 84 (Table 2).
2.3 | Statistical analysis
Statistical analyses were performed using SPSS Statistics for Windows, 3.4 | Semen parameters
version 17.0 (Chicago: SPSS Inc., USA). Minimum and maximum val-
ues, medians, and 25th and 75th percentiles were calculated. Normal Values for measured sperm parameters are presented in Table 3. There

distribution was determined using the Shapiro–Wilk test. In normally were significant increases in liquefaction time (p = .01), sperm concen-

distributed measures (DEXA evaluation), values on Day 0 and Day 84 tration (p = .007) and sperm motility (p < .001) between days 0 and 84.

were compared within patient using paired t test. When normal distri-
bution was rejected, nonparametric tests were used: Friedman test was
used to test the difference between several related samples (hormone 4 | DISCUSSION
levels on days 0, 28 and 84); Wilcoxon–Mann–Whitney test was used
to compare two independent samples (Semen parameters on days 0 The results of this study suggest that treatment with TT can increase
and 84). Values of p ≤ .05 were considered statistically significant. serum DHT concentrations and improve sperm count and motility, but

TABLE 1 Descriptive values of body composition measured by DEXA, in days 0 and 84

Variable Day N Average SD Minimum Maximum pa

Fat tissue (g) 0 43 20,125.53 9,926.86 1,574.00 44,662.00 .122

84 43 19,308.21 8,857.80 5,816.00 43,111.00
Total mass (g) 0 43 83,995.79 16,316.52 56,354.00 129,709.00 .435
84 43 84,159.14 16,275.82 56,043.00 131,980.00
Fat percentage (%) 0 43 22.88 7.28 10.00 36.00 .008
84 43 21.65 6.92 10.00 36.00
Lean tissue (g) 0 43 60,690.26 8,747.48 45,087.00 84,067.00 .005
84 43 61,909.37 9,485.95 44,773.00 85,926.00

g, grams.
Bold Values: Significant differences in the percentage of body fat and in lean muscle mass between days 0 and 84 of treatment.
a
Descriptive level of probability of the Student paired t test.
4 |  Salgado et al.

TABLE 2 Descriptive values of hormone levels, according to the day of evaluation

Variable Day n Average SD Minimum Maximum P25 Median P75 pa

DHEA (ng ml−1) 0 58 4.39 2.40 0.60 14.73 2.68 4.10 5.80
28 58 7.73 23.51 0.60 183.00 3.43 4.20 5.80 .426
84 58 12.43 59.03 1.10 454.00 3.38 4.30 5.58
DHT (pg ml−1) 0 58 469.49 319.26 26.00 1,905.00 359.50 455.00 586.23
28 58 561.38 297.91 0.60 1,891.00 395.25 481.00 712.00 .023
84 58 596.82 283.71 6.90 1,705.00 458.75 499.35 758.90
FSH (mUI ml−1) 0 60 8.27 5.02 1.17 22.62 4.83 6.74 10.40
28 60 8.34 5.14 1.33 25.18 4.72 6.74 11.28 .278
84 60 8.28 5.06 1.35 24.27 4.85 6.94 10.36
LH (mUI ml−1) 0 60 5.08 2.26 1.01 10.64 3.24 4.72 6.68
28 60 5.08 2.42 1.95 13.11 3.08 4.77 6.24 .971
84 60 5.35 3.32 1.66 24.73 3.51 4.59 6.50
Free testos terone 0 51 333.10 128.95 152.20 775.89 245.40 312.81 385.02
(ng dl−1) 28 51 318.94 103.82 107.09 543.93 242.92 303.59 379.65 .114
84 51 360.62 91.23 177.58 508.00 282.90 366.80 440.85
Prolactin (ng ml−1) 0 61 17.53 78.60 2.66 621.00 5.17 7.17 9.06
28 61 7.60 4.49 1.05 29.75 5.27 6.43 8.36 .916
84 61 7.67 3.08 2.63 17.23 5.44 7.18 9.35

Bold Values: Significant differences in DHT levels when comparing the beginning and the end of treatment.
a
Descriptive level of probability of the nonparametric Friedman test.

TABLE 3 Descriptive values of semen parameters, according to the day of evaluation

Variable Day n Average SD Minimum Maximum P25 Median P75 pa

Concentration 0 56 3.33 4.22 0.00 13.60 0.00 1.30 6.13 .007

(×106 ml−1) 84 56 6.40 10.36 0.00 48.00 0.00 1.55 7.53
Motility (%) 0 53 34.85 27.36 0.00 98.00 0.00 43.00 52.00 <.001
84 53 54.43 38.62 0.00 100.00 2.50 60.00 91.00
Morphology (%) 0 14 150 1.29 0.00 4.00 0.75 1.00 2.00 .863
84 14 1.43 1.09 0.00 4.00 1.00 1.00 2.00
Liquefaction time 0 57 13.95 3.87 10.00 30.00 10.00 15.00 15.00 .010
(min) 84 57 16.67 7.09 10.00 30.00 10.00 15.00 15.00
Ejaculate volume 0 58 2.87 1.60 0.10 9.50 1.78 2.75 3.50 .228
(ml) 84 58 3.30 2.51 0.50 18.60 2.15 2.80 3.63

Bold Values: Significant differences in sperm concentration and motility, as well as liquefaction time between days 0 and 84 of treatment.
a
Descriptive level of probability of the nonparametric Wilcoxon–Mann–Whitney test.

not sperm morphology, in men with reduced sperm quality in a clinical
ART program. These findings increase the support for investigation
of TT as a possible intervention in male factor infertility and serve as
a basis for future case-­controlled studies to evaluate the effect of TT
supplementation on both sperm parameters and pregnancy rates after
either ART or natural conception.
The effect of TT on DHT concentrations is in agreement with
a recent clinical trial, again comparing parameters before and after
treatment, with 30 male patients suffering from partial androgen
deficiency (Roaiah, El Khayat, GamalEl Din, & Abd El Salam, 2015).
In that trial, there was also a significant increase in free testoster-
one levels and a positive impact on erectile function after 3 months
of treatment with the same dose of TT extract as was used in our
study. Gauthaman and Ganesan (2008) showed an increase in serum
testosterone and DHT in primates. They stated that “The rise in the
level of DHT was proportionate to the increase observed with testos-
terone. Since DHT is the reduced form of testosterone, the observed
increase could probably be due to a primary increase in testosterone
level.” DHT has two or three times greater androgen receptor affinity
than testosterone and acts in the development of male secondary
Salgado et al.
sex characteristics, as well as prostate and sexual function (Amory
et al., 2008).
A recent review of the literature concluded that there is support
for a positive effect of TT on libido and erectile function, but that it
is inconclusive whether these effects are due to androgen-­enhancing
properties (Neychev & Mitev, 2016).
Arsyad (1996) reported a similar increase in sperm concentration
and motility after treatment of oligozoospermic men with an unspec-
ified amount of commercially prepared protodioscin. An increase in
sperm concentration, but not motility, was reported in a clinical trial
in oligozoospermic men (Moeloek, Adimoelja, Tanojo, & Pangkahila,
1994); pregnancies were achieved for 8/36 men in the TT treatment
group, vs. 0/9 in the control group. Treatment with TT extract may
improve sperm parameters in men with oligozoospermia by reversing
the negative effects of environmental toxins. Kumari and Singh (2015)
found that a dose of 200 mg kg−1 of TT fruit extract prevented testic-
ular damage induced by metronidazole treatment in mice. Similarly,
Rajendar et al. (2011) found that TT exerted a protective effect on
cadmium-­induced testicular damage in rats, which appeared to be
related to inhibition of peroxidation in testicular tissue. It has been
hypothesised that TT enhances sperm count by increasing testoster-
one levels via a prior increase in GnRH levels, which in turn stimu-
lates the production of LH and FSH (Antonio et al., 2000); however,
we found no indication of any changes in these gonadotropins in our
study.
Lean muscle mass gain and body fat were evaluated, as chang-
es in these parameters may be expected with increased androgen
levels. In 66% of the patients, DEXA evaluation demonstrated signif-
icant decreases in the percentage of body fat and increases in fat-­
free mass between days 0 and 84. Similar changes were not found
in a report on effects of a TT extract in trained athletes (Rogerson
et al., 2007).
In conclusion, the present clinical findings imply a possible thera-
peutic use of Tribulus terrestris extract in men presenting altered semen
parameters and/or infertility in a human ART program. Prospective
randomised controlled studies are needed to determine whether TT
is effective in improving sperm parameters and inducing gain in lean
muscle mass in this population of men.
ACKNOWLE DG E MEN TS
The authors wish to acknowledge the invaluable contribution of
Herbarium Laboratório Botânico, for supplying the TT extract
preparation.
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