Life Sciences 71 (2002) 1385 – 1396
www.elsevier.com/locate/lifescie

Aphrodisiac properties of Tribulus Terrestris extract (Protodioscin)in normal and castrated rats
K. Gauthaman, P.G. Adaikan *, R.N.V. Prasad
Department of Obstetrics and Gynaecology, National University Hospital, National University of Singapore,
Singapore 119074, Singapore
Received 15 November 2001; accepted 1 May 2002

Abstract
Tribulus terrestris (TT) has long been used in the traditional Chinese and Indian systems of medicine for thetreatment of various ailments and is popularly claimed to improve sexual functions in man. Sexual behaviour andintracavernous pressure (ICP) were studied in both normal and castrated rats to further understand the role of TT
containing protodioscin (PTN) as an aphrodisiac. Adult Sprague-Dawley rats were divided into five groups of 8each that included distilled water treated (normal and castrated), testosterone treated (normal and castrated, 10 mg/
kg body weight, subcutaneously, bi-weekly) and TT treated (castrated, 5 mg/kg body weight, orally once daily).
Decreases in body weight, prostate weight and ICP were observed among the castrated groups of rats compared tothe intact group. There was an overall reduction in the sexual behaviour parameters in the castrated groups of rats
as reflected by decrease in mount and intromission frequencies (MF and IF) and increase in mount, intromission,
ejaculation latencies (ML, IL, EL) as well as post-ejaculatory interval (PEI). Compared to the castrated control,
treatment of castrated rats (with either testosterone or TT extract) showed increase in prostate weight and ICP thatwere statistically significant. There was also a mild to moderate improvement of the sexual behaviour parameters
as evidenced by increase in MF and IF; decrease in ML, IL and PEI. These results were statistically significant. It
is concluded that TT extract appears to possess aphrodisiac activity probably due to androgen increasing propertyof TT (observed in our earlier study on primates). D 2002 Elsevier Science Inc. All rights reserved.
Keywords: Tribulus terrestris; Protodioscin; Castration; Sexual behaviour; Intracavernous pressure; Androgens; Aphrodisiac*

Corresponding author. Tel.: +65-6772-4128; fax: +65-6779-4753.
E-mail address: obgadaik@nus.edu.sg (P.G. Adaikan).
0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 8 5 8 - 1

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Introduction
The role of androgens in sexuality is unequivocal. Embryonic differentiation of the fetus into a maleand its subsequent growth along this line is essentially due to the presence of physiological amounts ofandrogens (especially testosterone and its metabolite dihydrotestosterone) in the body [1]. Apart fromthese two major androgens, dehydroepiandrosterone secreted from the adrenals also contributesignificantly to the overall androgenic status and therefore influence the sexual characteristics [2].
Reduction in the levels of these steroids (hypogonadism) at the early developmental phase leads tostructural abnormalities of the penis [3,4]. Hypogonadism in adulthood may result in loss of libido andsexual activity and androgen replacement has been found to be effective in restoration of these
conditions [5 – 8]. Androgens contribute to penile erection by acting in concert with the other
determinants of penile erectile physiology. Androgen receptors mediate the effects of androgens andtheir presence in various tissues has been demonstrated [9–11]. Using the rat model it has been
demonstrated previously that sexual behaviour and erection are androgen dependent (acting both
centrally and peripherally) and that treatment with testosterone of the castrated rats helped in restorationof both sexual behaviour and penile erectile capacity [3,6,12]. It has also been demonstrated by various
studies that androgens regulate corporal nitric oxide synthase (NOS) activity [13–15].
The aphrodisiac properties of Tribulus terrestris extract (TT) that contains protodioscin (PTN), a
steroidal saponin that forms 45% (dry weight) of the extract was explored in castrated rats. The plant
TT or its products have been extensively used both in the Chinese and Indian traditional medicine forthe treatment of various ailments such as urinary, cardiovascular, and gastrointestinal disorders [16–18]. Administration of TT to humans and animals improves libido and spermatogenesis [19]. PTN isalso found to increase the levels of testosterone, leutinizing hormone [20], dehydroepiandrosterone[21], dihydrotestosterone and dehydroepiandrosterone sulphate [22]. The corpus cavernosal tissues
obtained from New Zealand White rabbits following treatment with TT were tested in vitro withvarious pharmacological agents and electrical field stimulation and was found to have a proerectile
effect [23]. The present study was carried out to further understand the androgen releasing property of
the TT extract (PTN) and its relation to sexual behaviour and intracavernous pressure (ICP) using
castrated rats.

Materials and methods
Forty adult male Sprague-Dawley (SD) rats approximately 6 weeks old and weighing between
200–250 g were fed on standard rat pellets (Glen Forest Stock Feeders, Western Australia) and water
ad libitum. The animals were housed under standard laboratory conditions and maintained on areverse light- dark cycle (10 PM–10 AM) for a minimum of two weeks prior to the study to facilitate
adaptation. The rats were divided into five experimental groups of 8 each. Of these, two groups wereintact and three groups were castrated. The rats were treated either with distilled water, testosterone or
TT extract as follows. Group I: Intact + distilled water, Group II: Intact + testosterone, Group III:
Castrated + distilled water, Group IV: Castrated + testosterone and Group V: Castrated + TT extract.
Testosterone cypionate (Pharmacia and Upjohn) was administered in the dose of 10 mg/kg bodyweight, subcutaneously, bi-weekly for 8 weeks. TT (Sopharma, Bulgaria), was administered in thedose of 5 mg/kg body weight (the optimal dose that had significant effect in our previous studies),

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orally, once daily for 8 weeks. The weight of the animals was recorded at zero and eight weeks fromall the groups.
Ovariectomised SD rats were used for the copulatory studies. The female rats were brought to oestrus
by sequential administration of oestradiol benzoate (10 Ag/100 g body weight) and progesterone (500Ag/100 g body weight), through subcutaneous injections, 48 hours and 4 hours before the copulatory
studies respectively [24]. A baseline sexual behaviour study was carried out in rats from all groups torender them sexually experienced and were then repeated following the treatment schedule as mentioned
above.
Sexual behaviour studies were carried out in a separate room under dim red illumination according to
the standard procedure [25]. The male rat was placed in a rectangular plexiglass chamber, 10 minutes
before the introduction of a primed female, for it to get acclimatized to the chamber conditions. The
primed female was then introduced into the chamber and the following sexual behaviour parameters
were recorded:a) mount frequency (MF): the number of mounts without intromission from the time of introductionof the female until ejaculation,b) intromission frequency (IF): the number of intromissions from the time of introduction of thefemale until ejaculation,c) mount latency (ML): the time interval between the introduction of the female and the first mountby the male,d) intromission latency (IL): the interval from the time of introduction of the female to the first
intromission by the male (characterized by pelvic thrusting and springing dismount),e) ejaculation latency (EL): the time interval between the first intromission and ejaculation(characterized by longer, deeper pelvic thrusting and slow dismount followed by a period
of inactivity),f) post-ejaculatory interval (PEI): the time interval between ejaculation and the first intromission of
the following series.
The experiment was terminated when the male rat begins to mount the female followed byintromission after a brief period of inactivity (which normally results following ejaculation).
The male rat at the completion of behaviour studies were taken up for investigation on cavernous
nerve stimulation and measurement of ICP considered as a measure of penile erection [26]. The rats
were anaesthetized using pentobarbital sodium 45 mg/kg body weight, intraperitoneally with 5–10
mg/kg intravenous supplements, if necessary. The temperature was maintained between 36jC–37jC
using a heating source; an intravenous line established through the right external jugular vein, forsaline infusion and intravenous supplements of the anaesthetic agent if needed. Tracheal cannulation
was done to avoid respiratory embarrassment and to keep the animal stable throughout the procedure.
The left internal carotid artery was cannulated and blood pressure was recorded. Through a perineal
dissection, a 27 G needle filled with heparinised saline (250 units/ml) and fitted with PE10 tubing wasinserted into the right crus for recording ICP. Through abdominal dissection, cavernous nerve was
identified at its exit from the major pelvic ganglion on the medial aspect and traced towards the penis[27]. The cavernous nerve was gently teased from the prostatic capsule and hooked to a stainless steel
bipolar electrode for nerve stimulation. The two arms of the electrode were separated by a distance of
1 mm and each arm was 0.2 mm in diameter. The stimulation parameters were 2 volts and a frequency

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of 20 Hz that produced consistent pressure recordings. The contact time was 45 seconds/stimulation.
At the end of the study the animal was sacrificed and the wet weight of prostate measured from all thegroups.
Statistical methods
The independent variables for subjects in the different experimental groups were compared andanalyzed by two factors ANOVA with repeated measures of one factor and comparisons were madeusing Bonferroni procedure. All the results were expressed as mean F SEM and the level of significancefor comparisons set at P<0.05.

Results
Body and prostate weight
Considerable decreases in both the body weight (by 11.80%, 6.09% and 9.63%) and prostate weight(by 41.29%, 8.04% and 26.70%) for the groups III, IV and V respectively were observed among thecastrated groups of rats compared to the intact group. These parameters were statistically significant.
Within the castrated group the increase in prostate weight (by 43.28% and 29.85% for group IV and V
respectively) were statistically significant compared to the castrated control (Table 1).
Sexual behaviour
The rats from both the castrated and intact groups were paired with females that were brought tooestrus prior to experimentation. The males from the intact group readily responded while the castrated
group showed a diminished sexual response to the females. Unlike the intact group, the castrated rats
were mostly withdrawn towards the common pre-copulatory behaviours such as anogenital sniffing,nosing and chasing.

Table 1
Weight recorded in different groups of rats (n=8)
Group (n=8 SD rats)
N+V
N+T
C+V
C+T
C + TT

Body weight (G) at 8 weeks

Prostate Weight (G) at 8 weeks

Mean F SEM

Mean F SEM

636.25 F 15.80
650.00 F 29.22
560.00 F 11.80y597.50 F 11.91
575.00 F 8.66y

1.02 F 0.03
1.24 F 0.06
0.67 F 0.01y
0.96 F 0.02*
0.87 F 0.01y,*

N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. The results are compared
between a) the intact control and rest of the groups, b) the castrated control and rest of the castrated groups. The values are
expressed as mean F SEM. y and * indicates significant differences (p < 0.05) from control for a and b respectively.

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Fig. 1. N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. Number of mounts
recorded in different groups of rats (n = 8). The results are compared between a) the intact control and rest of the groups, b) the
castrated control and rest of the castrated groups. The values are expressed as mean F SEM. y and * indicates significant
differences (p < 0.05) from control for a and b respectively.

Fig. 2. N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. Number of
intromissions recorded in different groups of rats (n = 8). The results are compared between a) the intact control and rest of thegroups, b) the castrated control and rest of the castrated groups. The values are expressed as mean F SEM. y and * indicatessignificant differences (p < 0.05) from control for a and b respectively.

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Castrated groups showed a decrease in mount and intromission frequencies (Figs. 1 and 2). Decreasein MF (by 49.57%, 34.19% and 43.59%) and IF (by 51.13%, 38.35% and 42.11%) for the groups III, IV
and V respectively were statistically significant. In addition within the castrated group, both testosterone
and TT treated groups showed mild to moderate increase in both MF (by 30.51% and 11.86%) and IF(by 26.15% and 18.46%) for the groups IV and V respectively that were statistically significant.
Mount and intromission latencies showed an increase in both these parameters among the castrated
groups of rats compared to the intact group that were statistically significant (Figs. 3 and 4). Increase in
ML (by 70.76%, 39.95% and 52.71%) and IL (by 88.74%, 38.05% and 49.77%) were observed for thegroups III, IV and V respectively. Within the castrated group, both the testosterone and TT treated groups
showed minimal decrease in both ML (by 18.04% and 10.57%) and IL (by 26.86% and 20.65%) for thegroups IV and V respectively that were statistically significant.
Ejaculation latency and the post-ejaculatory interval also showed considerable increase among thecastrated groups of rats compared to the intact group (Figs. 5 and 6). The increase in EL (by 40.57%,
11.86% and 17.50%) and PEI (by 65.13%, 33.66% and 59.84%) for the groups III, IV and V were
statistically significant. Among the castrated group, both the testosterone and TT treated groups showed
moderate decrease in both EL (by 20.42% and 16.41% for the groups IV and V respectively) and PEI (by19.06% for group IV) that were statistically significant.

Fig. 3. N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. Mount latencies
recorded in different groups of rats (n = 8). The results are compared between a) the intact control and rest of the groups, b) the
castrated control and rest of the castrated groups. The values are expressed as mean F SEM. y and * indicates significant
differences (p < 0.05) from control for a and b respectively.

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Fig. 4. N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. Intromission latencies
recorded in different groups of rats (n = 8). The results are compared between a) the intact control and rest of the groups, b) the
castrated control and rest of the castrated groups. The values are expressed as mean F SEM. y and * indicates significant
differences (p < 0.05) from control for a and b respectively.

Fig. 5. N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. Ejaculatory latencies
recorded in different groups of rats (n = 8). The results are compared between a) the intact control and rest of the groups, b) the
castrated control and rest of the castrated groups. The values are expressed as mean F SEM. y and * indicates significant
differences (p < 0.05) from control for a and b respectively.

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Fig. 6. N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. Post-ejaculatoryinterval recorded in different groups of rats (n = 8). The results are compared between a) the intact control and rest of thegroups, b) the castrated control and rest of the castrated groups. The values are expressed as mean F SEM. y and * indicatessignificant differences (p < 0.05) from control for a and b respectively.

Fig. 7. N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. Blood pressurerecorded in the control and treated groups of rats (n = 8). The results are compared between the groups and the values are
expressed as mean F SEM.

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Fig. 8. N = Normal rat; V = Vehicle; C = Castrated rat; T = Testosterone; TT = Tribulus terrestris extract. Intracavernouspressure recorded in the control and treated groups of rats (n = 8). The results are compared between a) the intact control andrest of the groups, b) the castrated control and rest of the castrated groups. The values are expressed as mean F SEM. y and *indicates significant differences (p < 0.05) from control for a and b respectively.

Blood pressure and intracavernous pressure
There was a decrease in blood pressure among the castrated group of rats compared to the control by
11.64%, 7.8% and 6.51% for the groups III, IV and V respectively. The decrease observed were however
not statistically significant (Fig. 7). ICP was markedly decreased in the castrated group compared to theintact control. There was a decrease in ICP by 44.05%, 24.42% and 28.38% for the groups III, IV and V
respectively (Fig. 8) that were statistically significant. Treatment with testosterone and TT recorded anincrease in ICP (by 35.08% and 28.01%) for the groups IV and V that were statistically significant
compared to the castrated control (group III).

Discussion and conclusion
Animal studies from various centres indicate that sexuality decreases with low androgenic status andis restored following androgen replacement. In this current study we attempted to investigate the effectof TT using the castrated rat, a convenient model for studying the effect of androgens in relation to thesexual characteristics [3]. In this study the overall sexual behaviour parameters were markedly reduced
among the castrated groups of rats compared to the intact group. Castration leads to low androgenic
status affecting structural, biochemical, pharmacological or any of these components of erectile
physiology, which in turn cause reduction in erectile function as observed from studies on various
animal models [28,29]. Decrease in MF and increase in both ML and IL in castrated rats of this study

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implies reduction in the desire component of sexuality. Physiologically low levels of androgen as seen in
hypogonadism is associated with decreased sexual desire and activity [30,31]. The observed decrease in
IF as well as the increase in EL among the castrated group of rats indicates reduction in performance.
Apart from desire that is essential for initiation of sex, penile tumescence and rigidity as well as theaccessory muscles that helps in providing additional penile rigidity and ejaculation are also dependent on
androgen for a normal sexual activity. Various neurotransmitters and their inter/intracellular signaling are
responsible for the relaxation of corpus cavernosal smooth muscle (CCSM). Androgens influence theseneurotransmitters and contribute to the regulation of penile erection. Restoration of apomorphineinduced erections in castrated rats following administration of exogenous testosterone indicates thecentral influence of androgens in penile erection. However, the exact mechanism of action is still to beelucidated [6]. The peripheral effects of androgens appear to be better understood. The decrease indensity of nonadrenergic noncholinergic fibres following castration and its reversal to near normal levels
as well as differential inhibition and restoration of NOS activity following testosterone supplementation
explains the association of androgens especially testosterone with nitric oxide (NO) mediated erectile
activity [13,15].
In the present study, upon treatment with either testosterone or TT the sexual behaviour in the
castrated groups of rats improved compared to the castrated control. The early loss of ejaculatory
capacity followed by cessation of intromission and mounting in castrated rats and the restoration of theseparameters with testosterone supplementation explain the role of testosterone in sexual function [32,33].
As regards to TT, its active ingredient PTN improves libido and spermatogenesis in humans and animals[19]; increase the levels of testosterone, leutinizing hormone [20] and dehydroepiandrosterone [21]. The
improvement in sexual behaviour as noted by increase in MF and IF, and decreases in ML, IL, EL and
PEI following TT administration to the castrated group of rats for eight weeks correlate well with that of
testosterone replacement although at the given dose it is not as effective as testosterone itself.
In addition the increase in body weight and prostate weight in both testosterone and TT treatedcastrated groups compared to the castrated control confirms the role of androgens and a possible
contribution by TT via similar mechanism. The role of androgens and its regulatory effect on prostate
have been studied earlier [34]. There were no significant changes as regards to blood pressure (BP)following treatment with TT.
The increase in ICP among the castrated group following testosterone and TT treatment implies theirrole on NO and erectile function. It has been studied earlier that stimulation of cavernous nerve leads toincrease in NO and cGMP signaling resulting in CCSM relaxation. The subsequent arteriolar dilatation
leading to increased arterial inflow and impaired venous return (due to engorgement of the cavernosum)builds up a pressure system within the corpora that results in penile tumescence and rigidity. Restorationof ICP has been observed in castrated rabbits and rats following testosterone replacement [15,35].
Inclusion of pharmacological agents such as prostaglandin E1, papaverine or sildenafil citrate (thatregulate the cAMP/cGMP levels needed for the initiation of erection) in this study could have provided
additional information to better understand the mechanism for the observed effects of TT. Blood pressure
remained more or less stable; mild decrease observed in the castrated group of rats implies that BP tosome extent is regulated by androgens. It has earlier been observed in dogs that the mean arterialpressure decreased following castration [36].
The continued administration of the extract for eight weeks in this study could have possibly
increased the androgenic status both centrally and peripherally. DHEA is a major circulating steroid inhuman plasma, mainly synthesized by the adrenal glands and to a lesser degree by the gonads; it is a

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common precursor for both androgens and oestrogens [37]. TT containing PTN, has been shown toincrease the DHEA level in man [21]. DHEA is considered as a neurosteroid; it acts centrally as a
gamma amino butyric acid antagonist to facilitate sexual function [38]. Possible increase in DHEA andits subsequent conversion to testosterone and its metabolites may account for the observed effects in thisstudy. It is far from clear how such increase in DHEA is brought about by this plant extract; themechanism for this effect needs to be clarified. Much detailed studies on the hormonal aspects as well as
the levels of intracellular nucleotides need to be studied to better understand the clinical usefulness of
TT. As such TT may still contribute as a conditioner for the treatment of mild to mid-level erectile
dysfunction. However, this study provides additional information as regards to androgens and its
regulation of penile erection and that the plant Tribulus terrestris is found to exert similar properties tothat of androgens. Results of this study provide evidence for the claimed role of this plant as an
aphrodisiac in the traditional medicine.

Acknowledgements
We would like to thank Sopharma Joint Stock Co., Bulgaria, for providing the Tribulus terrestris
extract used in this study.

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