Effects and Mechanism of Action of a Tribulus terrestris Extract on Penile Erection

Abstract

Tribulus terrestris has been used as an aphrodisiac. However, little is known about the effects and mechanism of action of T. terrestris on penile erection. Therefore, the effect of a T. terrestris extract and the mechanism of action of the extract on relaxation of the corpus cavernosum (CC) were investigated. The erectogenic effects of an oral preparation of the extract were also assessed.
The relaxation effects and mechanism of action of the T. terrestris extract on rabbit CC were investigated in an organ bath. The intracavernous pressure (ICP) was calculated after oral administration of the extract for 1 month to evaluate whether the relaxation response of the CC shown in the organ bath occurred in vivo. Additionally, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured in the CC by immunoassay. Smooth muscle relaxation was expressed as the percentage decrease in precontraction induced by phenylephrine. The ICP was also assessed in rats after oral administration of the extract for 1 month, and changes in concentrations of cGMP and cAMP were monitored.
Concentration-dependent relaxation effects of the extract on the CC were detected in the organ bath study. Relaxation of the CC by the T. terrestris extract was inhibited in both an endothelium-removed group and an L-arginen methyl ester pretreatment group. The ICP measured after oral administration of the T. terrestris extract for 1 month was higher than that measured in the control group, and a significant increase in cAMP was observed in the T. terrestris extract group.
The T. terrestris extract induced concentration-dependent relaxation of the CC in an organ bath. The mechanism included a reaction involving the nitric oxide/nitric oxide synthase pathway and endothelium of the CC. Moreover, in an in vivo study, the T. terrestris extract showed a significant concentration-dependent increase in ICP. Accordingly, the T. terrestris extract may improve erectile function.

Full text

Korean Journal of Urology
Ⓒ The Korean Urological Association, 2013 183
Korean J Urol 2013;54:183-188
www.kjurology.org
http://dx.doi.org/10.4111/kju.2013.54.3.183
Sexual Dysfunction
Effects and Mechanism of Action of a Tribulus terrestris Extract on
Penile Erection
Jungmo Do, Seemin Choi, Jaehwi Choi, Jae Seog Hyun
Department of Urology, Gyeongsang National University Hospital, Jinju, Korea
Purpose: Tribulus terrestris has been used as an aphrodisiac. However, little is known
about the effects and mechanism of action of T. terrestris on penile erection. Therefore,
the effect of a T. terrestris extract and the mechanism of action of the extract on relaxa-
tion of the corpus cavernosum (CC) were investigated. The erectogenic effects of an oral
preparation of the extract were also assessed.
Materials and Methods: The relaxation effects and mechanism of action of the T. terrest-
ris extract on rabbit CC were investigated in an organ bath. The intracavernous pres-
sure (ICP) was calculated after oral administration of the extract for 1 month to evaluate
whether the relaxation response of the CC shown in the organ bath occurred in vivo.
Additionally, cyclic adenosine monophosphate (cAMP) and cyclic guanosine mono-
phosphate (cGMP) were measured in the CC by immunoassay. Smooth muscle relaxa-
tion was expressed as the percentage decrease in precontraction induced by
phenylephrine. The ICP was also assessed in rats after oral administration of the ex-
tract for 1 month, and changes in concentrations of cGMP and cAMP were monitored.
Results: Concentration-dependent relaxation effects of the extract on the CC were de-
tected in the organ bath study. Relaxation of the CC by the T. terrestris extract was
inhibited in both an endothelium-removed group and an L-arginen methyl ester pre-
treatment group. The ICP measured after oral administration of the T. terrestris extract
for 1 month was higher than that measured in the control group, and a significant in-
crease in cAMP was observed in the T. terrestris extract group.
Conclusions: The T. terrestris extract induced concentration-dependent relaxation of
the CC in an organ bath. The mechanism included a reaction involving the nitric ox-
ide/nitric oxide synthase pathway and endothelium of the CC. Moreover, in an in vivo
study, the T. terrestris extract showed a significant concentration-dependent increase
in ICP. Accordingly, the T. terrestris extract may improve erectile function.
Keywords: Penile erection; Smooth muscle; Tribulus
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial
License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Article History:
received
27 May, 2012
accepted
5 July, 2012
Corresponding Author:
Jae Seog Hyun
Department of Urology,
Gyeongsang National University,
79 Gangnam-ro, Jinju 660-702,
Korea
TEL: +82-55-750-8192
FAX: +82-55-750-8192
E-mail: hyunjs@gnu.ac.kr
INTRODUCTION
Recently developed phosphodiesterase type-5 (PDE-5) in-
hibitors have been widely used as first-line therapeutics to
treat erectile dysfunction (ED). Although large, multi-
center clinical trials have shown the efficacy and toler-
ability of these drugs in patients with ED of various etiol-
ogies with a broad range of severity, 30% to 35% of patients
fail to respond. The use of PDE-5 inhibitors may result in
side effects, including visual disturbances, headache, fa-
cial flushing, rhinitis, and indigestion. Other treatments
for ED include penile injection therapy or penile implants.
However, such methods are invasive and irreversible and
are not widely used [1]. Thus, there is a continuing need
for the development of new noninvasive and effective
therapies to treat patients with ED.
Despite the remarkable developments of modern medi-
cine, many people are still favorably disposed toward herb-

Korean J Urol 2013;54:183-188
184 Do et al
al medicines owing to the aggressive treatment protocols,
toxicity, and drug tolerance associated with modern
therapies. The widespread use of herbal medicines, how-
ever, requires scientific verification of their indications
and effects by use of modern medical analysis.
Tribulus terrestris is a perennial creeping herb that is
broadly distributed in Mediterranean, subtropical, and
desert climates worldwide. It has been used since ancient
times in traditional folk medicine as an aphrodisiac and to
treat urinary tract infections, inflammation, and other ail-
ments [2]. Animal studies have found that T. terrestris is
helpful as an aphrodisiac [3]. The treatment of castrated
rats with a Tribulus extract was previously shown to in-
crease the weight of the prostate and intracavernous pres-
sure (ICP). Improved sexual behavior was also detected, as
evidenced by an increase in mounting frequency. However,
much controversy surrounds the possible mechanisms of
action and the therapeutic applications of T. terrestris ex-
tracts [4]. Furthermore, little is known about the effects
and mechanism of action of T. terrestris on penile erection.
In this study, we investigated the effect of a T. terrestris
extract on relaxation of the corpus cavernosum (CC) in tis-
sue from rabbits and in rats. The mechanism of action of
the extract was also assessed. We further examined the
erectogenic effects of the extract after oral administration.
MATERIALS AND METHODS
1. T. terrestris extraction
T. terrestris was purchased at a local market in Korea. An
amount of 100 g of the T. terrestris fruit was ground and
extracted with 1 L of 90% ethanol at 80
o
C for 2 hours and
then filtered through Whatman No. 2 filter paper. The fil-
trates were evaporated in a rotary evaporator (Eyela-NE,
Rikakikai Co., Tokyo, Japan) under reduced pressure. The
yield of T. terrestris fruit extract was 25% (wt/wt). The aque-
ous extracts were filtered and lyophilized (Thermo Fisher
Scientific Inc., Rockford, IL, USA) to yield 55% (wt/wt) of
the crude extract. These extracts were preserved in a re-
frigerator and used in the studies described below.
2. Organ bath CC study
New Zealand white male rabbits weighing 2.5 to 3.0 kg
were used (n=8/group). All the animals were cared for in
accordance with the National Research Council pub-
lication Guide for the Care and Use of Laboratory Animals.
The animal experiments were approved by the Institutio-
nal Animal Care and Use Committee of the research in-
stitute at the university. The rabbits were sacrificed with
an overdose of ketamine hydrochloride (50 mg/kg) injected
into the marginal vein of the ear, the penis was dissected
immediately, and sections of the CC (2 mm×2 mm×10 mm)
were prepared. The sections were then transferred to an
organ bath; one end was connected to a muscle fixation ring
and the other end was connected to an isometric tension
transducer (SG-10). The sections were connected to a force
displacement transducer (TSD 125C, Biopac Inc., Goleta,
CA, USA), which is an isometric tension transformer, and
the signals were recorded on a personal computer by using
a four-channel data acquisition and analysis system for
Windows (MP36R 4-Channel Systems, Biopac Inc.). The
signals were relayed to a physiography instrument (Power-
Lab, ADI Instruments, Sydney, Australia) and measured.
Chart 5 software (ADI Instruments) was used for real-time
monitoring of tension. Krebs-Henseleit (KH) solution was
used for the organ bath at 37
o
C and pH 7.4 and gassed con-
tinuously with 95% O
2
/5% CO
2
. With the initial tension of
each section maintained at approximately 2 g, the KH sol-
ution was changed approximately every 30 minutes (total
equilibration period, 2 hours) and allowed to reach a stable
condition. Once a stable condition was reached, phenyl-
ephrine (PE) was added and the contraction level was
observed. Each section was progressively stretched to the
optimal point on its length-tension curve as determined by
the active tension developed in response to PE. A stable con-
dition was restored by washing the preparation with KH
solution three times. These steps were repeated, and con-
tracture within 100%±10% of the previous contraction was
defined as the ideal resting optimal isometric tension.
While maintaining resting optimal isometric tension, the
tissues were contracted by PE (5×10
-6
M) pretreatment
within the bath. A T. terrestris extract in five-fold dilutions
(0.25 to 4 mg/mL) was added, and the relaxation level was
assessed by observing the change in the tension curve.
The relaxation reactions were investigated in endothe-
lium-intact or -denuded tissue groups to examine the mech-
anism by which the T. terrestris extract induced CC
relaxation. Endothelium-intact or -denuded tissues from
the CC were incubated for 30 minutes in KH solution con-
taining various drugs. Next, the tissues were contracted
with PE (5×10
-6
M), during which plateau state contraction
was attained. The relaxation reaction was then observed
following the addition of increasing concentrations of T. ter-
restris extract. The drug probes included N(G)-nitro-L-argi-
nine methyl ester (L-NAME; 10
-5
M, nitric oxide [NO] syn-
thesis inhibitor), propronolol (10
-5
M, β-receptor blocker),
indomethacin (3×10
-5
M, cyclooxygenase inhibitor), gliben-
clamide (10
-5
M, K
+
ATP
channel inhibitor), 4-aminopyridine
(10
-5
M, membrane potential-dependent K
+
channel in-
hibitor), and methylene blue (10
-5
M, guanyl cyclase in-
hibitor) [5-7]. Eight sections were used for each group. The
CC sections were pretreated with 3 mL of 0.3% 3-[(3-chol-
amide propyl)]-1-propane sulfonate (CHAPS) for 20 sec-
onds to remove all vascular endothelial cells. The sections
were rubbed lightly by using the thumb and index finger for
20 seconds and washed with KH solution, and the cells were
removed by gently rolling the sections on dry paper.
Removal of the vascular endothelial cells was confirmed by
the failure of the loss of tissue contraction induced by ace-
tylcholine (10
-5
M) in response to PE (5×10
-6
M).
3. Measurement of the ICP after long-term oral admin-
istration
The CC relaxation response shown in the organ bath was

Korean J Urol 2013;54:183-188
Effect of a Tribulus Extract on Penile Erection
185
FIG. 1. Relaxation effects of the Tribulus terrestris extract
(mg/mL) on phenylephrine (PE) contraction (5×10
-6
M) in rabbit
corpus cavernosum. The T. terrestris extract showed a concen-
tration-dependent relaxation response beginning at 0.25 mg/mL
(p＜0.05).
studied to determine whether it was expressed in vivo.
Forty Sprague-Dawley male rats weighing 280 to 320 g
were allocated to six groups according to the oral dosage re-
ceived: control, 2.5, 5, 10, 50, and 100 mg/kg (n=8 each).
Next, the groups were orally administered the same T. ter-
restris extract every day for 1 month before measurement
of the ICP.
The rats were anesthetized with ketamine (50 mg/kg) in-
jected intraperitoneally. The animals were placed in the
supine position on an operating table, a catheter (polyethy-
lene-50 tube) was inserted into the carotid artery, and blood
pressure (BP) was monitored. The prostate was exposed by
a midline incision of the abdomen. Additionally, the pelvic
ganglion, located on the posterolateral side of the prostate,
was assessed, and the pertinent pelvic nerves and cav-
ernosal nerves were assessed and dissected without injury.
The cavernosal nerves were stimulated by using an electric
stimulator and platinum electrodes. To assess an erection
in response to electrical stimulation of the nerves, the penis
was dissected up to the penile crura. The tunica albuginea
of the penis was assessed by dissecting the ischial corpus
cavernosal muscles surrounding the penile crura, and a
25-gauge needle was installed (polyethylene-50 tube pre-
treated with 250 U/mL heparin) in the CC [8].
BP and ICP were measured by using a BP manometer
transducer and recorder (PowerLab, ADI Instruments).
Chart 5 software (ADI Instruments) was used for real-time
BP monitoring. Electrical stimulation was performed at a
voltage of 5 V with a 60-second duration.
Pressure was measured before, during, and after stim-
ulation to examine the hemodynamic reaction in response
to autonomic nerve stimulation. Additionally, to examine
the reactions induced by nerve stimulation, the percentage
of maximum ICP/mean systemic arterial blood pressure
(max ICP/MAP×100) was measured by stimulating the CC
nerves, and differences among the groups were compared.
4. Measurement of cGMP and cAMP in rabbit CC
Contraction of the smooth muscle sections was equalized
for 1 hour in KH solution and then induced with PE (5×10
-6
M), after which the tissues were exposed to the T. terrestris
extract for 10 minutes. The tissues were frozen rapidly in
liquid nitrogen and homogenized in ice-cold 6% trichloro-
acetic acid. The homogenized tissues were centrifuged for
15 minutes, and cellular proteins in the supernatant were
separated by using conventional methods. The samples
were divided into a normal CC group and experimental
groups (0.25, 0.5, 1, 2, and 4 mg/mL T. terrestris extract),
and the concentrations of released cGMP (n=8/group) and
cAMP (n=8/group) were measured by immunoassay. The
immunoassay was based on the competitive binding tech-
nique, in which the cGMP and cAMP in a sample compete
with a fixed amount of horseradish peroxidase-labeled
cGMP and cAMP for sites on rabbit polyclonal antibodies.
During incubation, the polyclonal antibodies become
bound to a microplate coated with goat antirabbit anti-
bodies. Following a wash to remove excess conjugate and
unbound sample, a substrate solution was added to de-
termine the bound enzyme activity. Color development
was stopped, and the absorbance was read at 450 nm. The
color intensity was inversely proportional to the concen-
trations of cGMP and cAMP in the sample.
5. Drugs and solutions
PE, L-NAME, glibenclamide, 4-aminopyridine, indome-
thacin, methylene blue, propranolol, and CHAPS were
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
The composition of the KH solution (pH 7.4) was (mM/L)
as follows: NaCl, 118.1; NaHCO
3
, 25; KCl, 4.6; KH
2
PO
4
, 1.2;
CaCl
2
, 2.5; MgSO
4
, 1.21; and glucose, 11.0.
6. Data analysis
The statistical significance was analyzed by a t-test and
one-way analysis of variance followed by Scheffe’s F test
by using the SPSS ver. 14.0 (SPSS Inc., Chicago, IL, USA).
The results are expressed as the mean±standard devia-
tion. A p-value ＜0.05 indicated statistical significance.
RESULTS
1. CC relaxation response and the mechanism of action
The T. terrestris extract produced a concentration-depend-
ent relaxation response at 0.25 to 4 mg/mL (Fig. 1). Gliben-
clamide (10
-5
M, K
ATP
blocker), 4-aminopyridine (10
-5
M, K
+
channel blocker), methylene blue (10
-5
M, guanylate cy-
clase inhibitor), indomethacin (3×10
-5
M, prostaglandin
pathway cyclooxygenase inhibitor), and L-NAME (10
-5
M,
NO/NO synthase [NOS] inhibitor) were each administered
respectively before extract treatment to investigate the
mechanism of action of the relaxation response. The group
of endothelium-denuded tissues from the CC was also
investigated. Relaxation of the CC was inhibited in the en-
dothelium-removed and L-NAME pretreatment groups (p
＜0.05) (Fig. 2).
2. Changes in the ICP after oral administration of the T.
terrestris extract for 1 month
The T. terrestris extract was orally administered every day
at 2.5, 5, 10, 50, or 100 mg/kg for 1 month. Next, the

Korean J Urol 2013;54:183-188
186 Do et al
FIG. 3. Changes in intracavernous pressure (ICP) after oral
administration of the Tribulus terrestris extract for 1 month.
The graph shows an increase in ICP in response to carvernosal
nerve stimulation in all groups. The maximum ICP/mean syste-
mic arterial blood pressure (MAP) showed a significant concent-
ration-dependent increase beginning at 2.5 mg/kg compared
with that in the control group (p＜0.05, n=8/group).
*
p＜0.05.
FIG. 4. Changes in the cyclic adenosine monophosphate (cAMP) (A) and cyclic guanosine monophosphate (cGMP) (B) concentrations
in the corpus cavernosum (CC) of rats (n=8/group) after oral administration of the Tribulus terrestris extract for 1 month. The cAMP
concentration showed a statistically significant increase compared with that in the control group, but the difference was not
significant for cGMP.
*
p＜0.05.
FIG. 2. Effects of a Tribulus terrestris extract on the relaxation
response of the rat corpus cavernosum (CC). The relaxation
response of the CC was suppressed only by the nitric oxide
synthesis inhibitor L-arginine methyl ester (L-NAME) and
endothelium-denuded [E (-)] CC. Pretreatment with indometha-
cin (Indome), glibenclamide (GB), 4-aminopyridine (4-AP), or
methylene blue (MB) for 30 minutes did not suppress the
relaxation of the CC by the T. terrestris extract (p＜0.05,
n=8/group). PE, phenylephrine.
*,†
p＜0.05.
ICP/MAP (%) was measured during neural stimulation of
the CC. After 1 month of oral administration, a significant
concentration-dependent increase in ICP/MAP was ob-
served in accordance with the increasing dosage of the T.
terrestris extract (p＜0.05). The increase in ICP was ob-
served at a dose of 2.5 mg/kgㆍd T. terrestris extract, and
the maximum effect occurred at 100 mg/kgㆍd compared
with that in the control group (p＜0.05) (Fig. 3).
3. Changes in cAMP and cGMP in the CC
The cAMP concentration in the CC increased significantly
after administration of the T. terrestris extract. However,
the maximum concentration was observed at 0.5 mg; at 1,
2, and 4 mg, the values were lower than those at 0.5 mg.
The cAMP concentrations at 0.5 and 4 mg were lower than
those at 1 and 2 mg, revealing no concentration-dependent
pattern (Fig. 4A). The cGMP concentration in the CC after
administration of the T. terrestris extract was not statisti-
cally significant (Fig. 4B).
DISCUSSION
T. terrestris is a flowering plant in the family Zygophylla-
ceae. It is native to warm temperate and tropical regions
of the Old World in Southern Europe, Southern Asia,
Africa, and Australia. T. terrestris, also called “puncture
vine,” is used worldwide to improve sexual function in
humans. In Turkey, it is commonly used in folk medicine
to treat abnormal BP and cholesterol levels. In Europe, it

Korean J Urol 2013;54:183-188
Effect of a Tribulus Extract on Penile Erection
187
has been used in folk medicine throughout history, as far
back as the Greeks, for wide-ranging conditions such as
headache, nervous disorders, and sexual dysfunction. The
herb has also been touted for use as a liver, kidney, urinary,
and cardiovascular remedy in China and India.
T. terrestris has been used as an aphrodisiac and en-
hancer of sperm production as well as an alternative to hor-
mone replacement therapy in aging men and women [2].
However, much debate surrounds the possible mecha-
nisms of action and the therapeutic applications of T. ter-
restris extracts. Results published by Gauthaman et al.
[3,9] indicate that T. terrestris improves some aspects of
male sexual behavior and enhances spermatogenesis in
rats. Additionally, clinical data indicate the stimulatory ef-
fects of T. terrestris on sperm quantity and quality and im-
proved sexual response in men [10]. Increased androgen
levels have also been reported following T. terrestris ad-
ministration in nonhuman primates, rats, and rabbits
[2,11], but most of these effects were short-lived and
showed no clear dose-response relationship. In addition,
there is no consensus on the mechanism underlying the ef-
fects of T. terrestris on sexual performance and spermato-
genesis. Recently, Martino-Andrade et al. [4] demonstra-
ted that T. terrestris has no intrinsic hormonal activity, be-
cause it was unable to stimulate endocrine-sensitive or-
gans in either male or female rats. They also demonstrated
that the administration of T. terrestris to intact male rats
for 28 days did not change serum testosterone levels and
did not produce quantitative changes in the fecal excretion
of androgenic metabolites.
Because T. terrestris has been used to improve sexual
function in various regions of the world, we presumed that
it might have a direct relaxation effect on the CC. The re-
sults of our study showed that the T. terrestris extract
caused relaxation of the cavernous smooth muscle in a con-
centration-dependent manner, and that the mechanism
included a reaction involving the NO/NOS pathway in the
CC endothelium. The traditional understanding of the ac-
tion of NO in the penis is that NO is constitutively produced
and released from autonomic nerve terminals and endo-
thelial cells in corporal tissue. It diffuses locally into ad-
jacent smooth muscle cells and binds with intracellular
guanylate cyclase, which serves as a physiological “recep-
tor” [12]. This binding induces a conformational change in
guanylate cyclase, activating the enzyme, so that it cata-
lyzes the conversion of guanosine triphosphate to cGMP.
cGMP then operates through a cGMP-dependent protein
kinase to regulate the contractile state of the corporal
smooth muscle [13].
Although the mechanism of action of the T. terrestris ex-
tract occurred through NO/NOS signaling in the CC in this
study, a significant increase in cGMP within the tissues
was not confirmed. The cAMP concentration was sig-
nificantly increased in the T. terrestris extract-treated
group compared with that in the control group.
However, the cAMP concentration did not reveal a con-
centration-dependent relaxation effect in accordance with
the concentration of the T. terrestris extract. Because the
T. terrestris extract was composed of many chemical com-
pounds rather than a single chemical compound, other CC
relaxation signal transduction pathways not confirmed in
this study might be necessary for the effect. Therefore, ad-
ditional experimental verification is needed to identify how
this phenomenon occurs.
A significant concentration-dependent increase in ICP
was observed in accordance with the increasing dosage of
the T. terrestris extract, similar to our organ bath ex-
perimental results. The oral administration of a T. terrest-
ris extract in clinical practice is expected to improve erec-
tile function. Although further clinical research is needed,
the extract shows promise as an erectogenic agent.
CONCLUSIONS
The T. terrestris extract showed a concentration-depend-
ent relaxation effect on the CC in an organ bath. The mecha-
nism involved NO/NOS signaling in the CC endothelium.
Moreover, an in vivo study after 1 month of oral admin-
istration of the T. terrestris extract showed a significant
concentration-dependent increase in ICP compared with
that in the control group. Accordingly, the T. terrestris ex-
tract may improve erectile function.
CONFLICTS OF INTEREST
The authors have nothing to disclose.
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