Free serum testosterone level in male rats treated with Tribulus alatus extract

Abstract

The present study was undertaken to investigate the effect of Tribulus alatus extracts on free serum testosterone in male rats.
Free serum testosterone level was measured in male rats treated with alcoholic extracts of the aerial part without fruits, fruits of Tribulus alatus and their fractions.
All tested extracts showed significant increase in the level of free serum testosterone when compared to that of corresponding control, p < 0.05. Statistical comparison of all groups revealed that the maximum level was found in groups treated with chloroformic and ethanolic fractions of fruits extract.
Tribulus alatus extract appears to possess aphrodisiac activity due to its androgen increasing property.

Full text

554
Tribulus Alatus Extracts and Testosterone Level
Investigative Urology
International Braz J Urol Vol. 33 (4): 554-559, July - August, 2007
Free Serum Testosterone Level in Male Rats Treated with
Tribulus Alatus Extracts
Walid H. El-Tantawy, Abeer Temraz, Omayma D. El-Gindi
Drug Bioavailability Center, National Organization For Drug Control and Research, Cairo, Egypt,
Pharmacognosy Department, Faculty of Pharmacy for Girls, Al-Azhar University, Cairo, Egypt
ABSTRACT
Objective: The present study was undertaken to investigate the effect of Tribulus alatus extracts on free serum testosterone
in male rats.
Materials and Methods: Free serum testosterone level was measured in male rats treated with alcoholic extracts of the
aerial part without fruits, fruits of Tribulus alatus and their fractions.
Results: All tested extracts showed significant increase in the level of free serum testosterone when compared to that of
corresponding control, p < 0.05. Statistical comparison of all groups revealed that the maximum level was found in groups
treated with chloroformic and ethanolic fractions of fruits extract.
Conclusion: Tribulus alatus extract appears to possess aphrodisiac activity due to its androgen increasing property.
Key words: Tribulus; testosterone; aphrodisiacs; rats
Int Braz J Urol. 2007; 33: 554-9
INTRODUCTION
In traditional medicine a variety of plants have
been used as sex stimulants (1). For centuries, Arabs
have made use of herbal drugs to improve sexual
performance and increase libido (2). In African
traditional medicine, especially in Cameroon, Zingiber
officinale and Pentadiplan-dra brazzeana are used as
aphrodisiac and male sexual stimulation (3). In Egypt,
the pollen grains of dates (Phoenix dactylifera) and
seeds of hermala (Peganum harmala) are used to
restore sexual potency (4).
The genus Tribulus of the Zygophyllaceae
comprises ca 20 species that grow as shrubs or herbs in
subtropical areas around the world (5).Among the
Tribulus species T. terrestris, T. cistoides and T. alatus
have been phytochemically investigated and isolation of
steroidal saponins from these plants was reported (6-8).
The present study was undertaken to
investigate the effect of Tribulus alatus extracts on
free serum testosterone in male rats.
MATERIALS AND METHODS
Plant Material and Preparation of Extracts
Samples of Tribulus alatus were collected
from Al Azhar University, Nasr-city, Cairo and were
kindly identified by Department of Botany, Faculty of
science, Cairo University. A voucher specimen
(number 3978/1) was deposited at Herbarium Horti
Botanici Pisani (Flora Aegyptiaca), Pisa, Italy.

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Tribulus Alatus Extracts and Testosterone Level
The dried aerial parts and fruits (400g and
100g, respectively) of Tribulus alatus were finely
powdered and were macerated separately in 70%
methanol. The alcoholic extract was evaporated to
dryness under vacuum. The residues were combined,
weighted (90g and 20g, respectively) suspended in
distilled water and successively extracted with
chloroform, ethylacetate and n-butanol saturated with
water. Each extract was collected and evaporated to
dryness under vacuum to give chloroformic extract
(4g and 2.1g, respectively ) ethylacetate extract (3.2g
and 1.5g, respectively) and n-butanolic extract (8g
and 3.8g, respectively) then the water was evaporated
to dryness and the residue was macerated in absolute
ethanol several times. The alcoholic extracts were
combined and evaporated to dryness under vacuum
to give ethanolic extract (30g and 8g, respectively)
Animals
Healthy, adult male Wistar albino rats weighing
200-250g, aged 4-5 months were used in this study.
The animals had free access to a standard commercial
diet, water and were kept in rooms maintained at
25 ± 1°C. The animals were divided randomly into
different groups; each group consisted of six rats.
Control groups treated with distilled water (2 mL/kg
p.o.) only.
Toxicity Study
On the basis of the toxicity study, the LD50
value of the extract of the aerial part without fruits in
mice was 812 and was 868 mg/kg body weight for
the extract of fruits. On the other hand, LD50 value
for extract of different fractions ranged from 155-
200 mg/kg body weight. The experiment was carried
out on three steps.
First Step
Control group: treated with distilled water (2
mL/kg p.o.). Group 1: received 70% alcoholic extract
of aerial part without fruits (n = 6). Group 2: received
70% alcoholic extract of fruits (n = 6). Group 3: received
70% alcoholic extract of total herb (n = 6). Reference
group: treated with 0.45 mg mestrolone (n = 6).
In the first step, groups received extracts
(suspended in water using Tween 20 as a surfactant)
at a dose of 50 mg / kg body weight orally by orogastric
catheter once a day for 40 days. A reference group
was treated with 0.45 mg mestrolone once a day for
40 days orally by orogastric catheter. After 40 days,
blood samples were collected from the tail veins of
the rats at the same time of the day and serum was
separated.
Second Step
Control group: treated with distilled water (2
mL/kg p.o.). Group 4: received chloroformic fraction
of aerial part without fruits (n = 6). Group 5: received
ethylacetate fraction of aerial part without fruits (n =
6). Group 6: received butanolic fraction of aerial part
without fruits (n = 6). Group 7: received ethanolic
fraction of aerial part without fruits (n = 6). Reference
group: treated with 0.45 mg mestrolone (n = 6).
Third Step
Control group: treated with distilled water (2
mL/kg p.o.). Group 8: received chloroformic fraction
of fruits (n = 6). Group 9: received ethylacetate
fraction of fruits (n = 6). Group 10: received butanolic
fraction of fruits (n = 6). Group 11: received ethanolic
fraction of fruits (n = 6). Reference group: treated
with 0.45 mg mestrolone (n = 6).
On the other hand, in the second and third
steps the groups received dose 12.5 mg / kg body
weight orally by orogastric catheter once a day for
40 days. A reference group was treated with 0.45 mg
mestrolone once a day for 40 days orally by orogastric
catheter. After 40 days, blood samples were collected
from the tail veins of the rats at the same time of the
day and serum was separated.
Determination of Free Serum Testosterone
The level of free serum testosterone was
measured by Enzyme-linked immunosorbant assay
(ELISA) according to (9), KAPD29:040318/
2KAPD2924 IN VITRO DIAGNOSTIC
USEenBioSource Europe SA - Nivelles, Belgium.
Statistical Analysis
Data were presented as the mean ± SE (n =
6). Statistical analysis used Student’s t-test to compare
differences between groups and the control. One-way
analysis of variance (ANOVA) was applied for

556
Tribulus Alatus Extracts and Testosterone Level
comparison between different treatments. Differences
were considered statistically significant at P < 0.05.
RESULTS
Table-1 represents mean free serum
testosterone level (pg/ mL) among group of rats
treated with 70 % alcoholic extracts of Tribulus alatus.
Testosterone level was significantly increased among
all groups, when compared to that of their
corresponding control, P < 0.05. The highest level was
found in the group treated with the reference drug
followed by the group treated with fruits extract,
followed by the one treated with the aerial part without
fruits and the lowest level was found in the group
treated with total herb.
Table-2 illustrates mean free serum
testosterone level (pg/mL) among groups of rats
treated with different fractions of 70% alcoholic
extracts of the aerial part without fruits of T. alatus.
Testosterone level showed significant increase among
all groups, when compared to that of their
corresponding control, p < 0.05.
The level of testosterone in the group treated
with the reference drug showed a significant increase
when compared to that of all other groups. The level
of testosterone in-group (5) showed a significant
increase when compared to that of other groups
including that treated by total aerial parts extract, group
(1).
Table-3 illustrates mean free serum
testosterone level (pg/mL) among groups of rats
treated with different fractions of 70 % alcoholic
Table 1 – Mean free serum testosterone level (pg/ mL) among groups of rats treated with 70% alcoholic extracts of
Tribulus alatus.
Values expressed as mean ± standard error of 6 animals/group. P < 0.05 in relation to control group (Student’s t-test). Different letters
mean significance.
Tested Parameter
Mean± SE
p Value
Control Group
0.75± 0.024
Group 1
2.96± 0.088
a
< 0.05
Group 2
3.9± 0.14
b
< 0.05
Group 3
1.85± 0.076
c
< 0.05
Reference Group
6.5± 0.98
d
< 0.05
Table 2 – Mean free serum testosterone level (pg/mL) among groups of rats treated with different fractions of 70%
alcoholic extract of aerial part without fruits of Tribulus alatus.
Values expressed as mean ± standard error of 6 animals/group. P < 0.05 in relation to control group (Student’s t-test). Different letters
mean significance.
Tested Parameter
Mean± SE
p Value
Control Group
0.8 ± 0.06
Group 4
3.88 ± 0.76
a
< 0.05
Group 5
5.7± 1.02
b
< 0.05
Group 6
2.93± 0.41
a
< 0.05
Group 7
3.38± 0.55
a
< 0.05
Reference Group
8± 0.86
d
< 0.05
Table 3 – Mean free serum testosterone level (pg/mL) among groups of rats treated with different fractions of 70%
alcoholic extract of fruits of Tribulus alatus.
Values expressed as mean ± standard error of 6 animals/group. P < 0.05 in relation to control group (Student’s t-test). Different letters
mean significance.
Tested Parameter
Mean± SE
p Value
Control Group
0.72 ± 0.048
Group 8
21.3 ± 0.882
a
< 0.05
Group 9
8 ± 0.577
b
< 0.05
Group 10
8.81 ± 0.079
b
< 0.05
Group 11
18.75± 1.88
c
< 0.05
Reference Group
7± 1.0
b
< 0.05

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Tribulus Alatus Extracts and Testosterone Level
extracts of the fruits of T. alatus. Testosterone level
showed significant increase among all groups, when
compared to that of their corresponding control, p <
0.05.
Testosterone level showed significant
increase in groups 8 and 11. As compared with that
of other fractions, total fruits extract and the group
treated with the reference drug.
COMMENTS
Some causes that are responsible for low
testosterone levels, include congenital problems such
as deficiencies of male hormones and rare
malformation syndromes, and acquired problems such
as aging, chronic illness, drugs, starvation, stress, head
trauma, infections, cancers, surgeries, alcoholism,
removal or trauma to the testicles, and infection or
twisting of the testicles in their sack.
The use of testosterone is widespread in the
treatment of many problems including infertility,
athletic enhancement, erectile dysfunction and libido
problems. Its application can have grave consequences
if not used properly. Androgen, or more specifically
testosterone, is widely utilized to treat erectile
dysfunction (10).
Various neurotransmitters and their inter/
intracellular signaling are responsible for the relaxation
of corpus cavernosal smooth muscle. Androgens
influence these neurotransmitters and contribute to
the regulation of penile erection. The classic theory
about testosterone treatment is that it stimulates the
sex drive and, by doing so, restores erectile functioning
(11).
Once a man is diagnosed as hypogonadic, or
having a low testosterone level, the next step is to
choose which form of treatment to utilize. As with all
medications, benefits should be evaluated against
potential risk. Age is one important factor in making
this decision.
In men less than fifty years old, the goal is
to restore libido and erections. Testosterone also
improves strength, physical stamina, and health status
(10). Many people are now relying on herbal
medicines for health care (12). Since other
treatments applied are becoming more expensive and
often carry serious side effects, there should be
scientific dissemination of information on the
therapeutic efficacy of these plants. Aphrodisiacs
are substances that enhance sex drive and/or sexual
pleasure or can arise sexual desire or libido (13).
They are also agents that can be used to modify
impaired sexual functions.
Studies have implicated the saponin
component of plants in enhancing aphrodisiac
properties due to their stimulatory effect of androgen
production (11).
A survey concerning the secondary
metabolites of genus Tribulus showed that steroidal
saponins are the typical constituents of the genus,
and in particular of T. terrestris (14). Saponins have
been implicated as possible bioactive agent
responsible for the aphrodisiac effect in Tribulus
terrestris extract (11). These saponins were found
to increase the levels of testosterone and luteinizing
hormone (15).
It was reported that Tribulus alatus contained
steroidal saponins (8), which might contribute to
increasing endogenous testosterone levels by raising
the level of luteinizing hormones (LH) as reported for
saponins isolated from T. terrestris (15).
In the present study, the significant increase
in the level of free serum testosterone is an indication
of the aphrodisiac potential of Tribulus alatus extract.
CONCLUSION
The alcoholic extracts of both parts of Tribulus
alatus produced a significant increase in the level of
free serum testosterone at dose 50 mg/Kg body
weight. Also different fractions of both parts of the
plant revealed significant increase in the level of free
serum testosterone at dose 12.5 mg/Kg body weight
when compared to their corresponding controls. It is
concluded that Tribulus alatus extract appears to
possess aphrodisiac activity due to its androgen
increasing property.
CONFLICT OF INTEREST
None declared.

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Tribulus Alatus Extracts and Testosterone Level
REFERENCES
1. Islam MW, Tariq M, Ageel AM, al-Said MS, al-Yhya
AM: Effect of Salvia haematodes on sexual behaviour
of male rats. J Ethnopharmacol. 1991; 33: 67-72.
2. Puri HS: Vegetable aphrodisiacs of India. Q J Crude
Drug Res. 1971; 11: 1742-1748.
3. Noumi E, Amvan ZPH, Lontsi D: Aphrodisiac plants
used in Cameroon. Fitotherapia 1998; 69: 125-34.
4. Amin ES, Awad O, El-Samad MA, Iskander MN:
Pharmacological studies on pollen grains of dates
(Phoenix dactylefera). Phytochemistry. 1969; 8: 295-8.
5. Hegnauer R: Chemotaxonomie der Pflanzen. Basel,
Birkhäuser-Verlag. 1973; 6: 707.
6. Mahato SB, Sahu NP, Ganguly AN, Miyahara K,
Kawasaki TJ: Steroidal glycosides of Tribulus
terrestris Linn. J Chem Soc Perkin Trans I. 1981; 1:
2405-10.
7. Achenbach H, Hubner H, Brandt W, Reiter M:
Cardioactive steroid saponins and other constituents
from the aerial parts of Tribulus cistoides.
Phytochemistry. 1994; 35: 1527-43.
8. Temraz A, El Gindi OD, Kadry HA, De Tommasi N,
Braca A: Steroidal saponins from the aerial parts of
Tribulus alatus Del. Phytochemistry. 2006; 67: 1011-8.
9. McCann D, Kirkish L: Evaluation of Free Testosterone
in serum. J Clin Immunoassay. 1985; 8: 234-6.
10. www.testosterones.com (access in August 2005).
11. Gauthaman K, Adaikan PG, Prasad RN: Aphrodisiac
properties of Tribulus Terrestris extract (Protodioscin)
in normal and castrated rats. Life Sci. 2002; 71: 1385-
96.
12. Tyler VE: A textbook of Herbs Of Choice: The
Therapeutic Use Of Phytomedicinals. Norwood,
Haworth Pr Inc. 1994; P 1.
13. Rosen RC, Ashton AK: Prosexual drugs: empirical
status of the “new aphrodisiacs”. Arch Sex Behav.
1993; 22: 521-43.
14. De Combarieu E, Fuzzati N, Lovati M, Mercalli E:
Furostanol saponins from Tribulus terrestris.
Fitoterapia. 2003; 74: 583-91.
15. Koumanov F, Bozadjieva E, Andreeva M, Platonva E,
Ankov V: Clinical trial of Tribestan. Experiment Med.
1982; 2-4.
EDITORIAL COMMENT
In this manuscript, the authors evaluated the
effect of Tribulus alatus extracts on free serum tes-
tosterone in male rats. The authors found that the
alcoholic extracts of both parts of Tribulus alatus pro-
duced a significant increase in the level of free se-
Accepted after revision:
December 23, 2006
Correspondence address:
Dr.Walid Hamdy Ali El-Tantawy
Drug Bioavailability Center
National Organization for Drug Control and Research
Dokki, P.O. 29, Cairo, Egypt
Fax: + 002023379445
E-mail: wldhmdy@yahoo.com
rum testosterone at dose 50 mg/kg body weight. Also
different fractions of both parts of the plant revealed
significant increase in the level of free serum test-
osterone at dose 12.5 mg/kg body weight. From these
studies, the authors concluded that the alcoholic ex-

559
Tribulus Alatus Extracts and Testosterone Level
tract of the Tribulus alatus might thus be used to modify
impaired sexual functions, especially those arising from
hypotestosteronemia. The paper is very original and
most part is carefully carried out. It presents some
interesting observations.
Dr. Q. T. Yang
Department of Urology
Second Affiliated Hospital
Shantou University Medical College
Shantou, 515041, China
E-mail: yang2001tao@126.com
EDITORIAL COMMENT
Male reproductive disorders and sexual dys-
function is a serious problem of recent society. In
this respect the current paper deal with topical ques-
tion of increasing incidences of male sexual dysfunc-
tion and potential risk of application of synthetic hor-
monal drugs. There is limited understanding in the
literature on aphrodisiac effect of plants, namely,
Tribulus on man and primates. The study is interest-
ing and informative and adds to our knowledge about
aphrodisiac properties of species belonging to genus
Tribulus. The authors develop a proper experimen-
tal model on rat and provide useful data on the ways
for stimulation of androgen production by natural
sources. The article contains new facts about isola-
tion of different organic fractions from fruits and
aerial part of Tribulus and their comparative effect
on testosterone production. The data are useful for
clinical practice and treatment of male sexual dys-
function. They will encourage further studies on find-
ing natural products for stimulation of testicular en-
docrine function that would be of interest for phar-
macology.
Dr. Nina Atanassova
Inst. of Exp. Morphology & Anthropology
Bulgarian Academy of Sciences
Sofia, Bulgaria
E-mail: ninaatanassova@yahoo.com