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* Corresponding author, Address: Anatomical Sciences Research Center, Kashan University of Medical
Sciences, Kashan, Iran
Tel: +98 361 550480 Email: nikzad_h@yahoo.com
61
The effect of aqueous extract of Salep Tubers on the structure of testis and sexual
hormones in male mice
Faraji Z1, Nikzad H*2, Parivar K3, Nikzad M4
Received: 03/27/2012 Revised: 01/13/2013 Accepted: 01/22/2013
1. Dehkhoda High School, Kashan, Iran
2. Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran
3. Dept. of Biology, Science & Research Branch of Islamic Azad University, Tehran, Iran
4. School of Medicine, Kashan University of Medical Sciences, Kashan, Iran
Journal of Jahrom University of Medical Sciences, Vol. 11, No. 1, Spring 2013
Abstract:
Introduction:
Salep tubers have been used in traditional medicine as a drug for improving sexual function and
vigor. We did not find sufficient scientific evidence to support it. The present study was designed
to evaluate the effect of Salep tuber extract on the structure of testis and sexual hormones in adult
male mice.
Materials and Methods:
In this experimental study, we used 18 adult male mice (mean age of 4 weeks) and randomly
divided them into three groups: the control group did not receive anything, the placebo group
received only 200 µl deionized water, and the experimental group received 40 mg salep
extract/200µl deionized water intraperitoneally for 7 days. Two weeks after the last injection, we
took blood sample for biochemical analysis of LH, FSH and Testosterone and removed the testis
of animals for histopathological study.
Results:
The results showed a significant increase in LH (ng/ml) in the experimental group (0.6 ± 0.09)
compared to the placebo (0.39 ± 0.006) and control (0.38 ± 0.007) groups (p<0.05). Also, the
level of testosterone hormone (ng/ml) showed a significant difference in the experimental group
(0.38 ± 0.52) compared to placebo (0.37 ± 0.03) and control (0.36 ± 0.02) groups (P<0.05). The
number of germinal and leydig cells revealed a significant difference in the experimental group
compared to the placebo and control groups (P<0.01). Amongst the other parameters, no
significant differences were observed in all groups.
Conclusion:
Injection of salep tuber extract could significantly be effective on spermatogenesis and
concentration of LH and testosterone hormones.
Keywords: Plant Extracts, Testis, BALB C Mice
Introduction
Infertility is one of the problems of
mankind. According to the World Health
Organization (WHO) report, 10 to 15% of
young couples are faced with infertility
problem. Male infertility factors contribute
to approximately 40% of all infertility
cases (1-2). There are many different
causes in the incidence of male infertility
including: genetic disorders, obstruction of
the genital tract, varicocele, reduced sperm
production, reduced semen quality
parameters, erectile dysfunction, and
men’s sexual impotence (3). Studies have
J Jahrom Univ Med Sci 2013; 11(1): 61-6
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Journal of Jahrom University of Medical Sciences, Vol. 11, No. 1, Spring 2013
62
shown that semen parameters in 25 to 40%
of young people are below the WHO
standard indicators (4). Treatment is
performed through surgery, chemical and
herbal medication, and laboratory assisted
reproductive techniques. In Iran and other
countries, prescribing herbal drugs by
herbal medicine practitioners is
commonplace for treatment of many
diseases. One of these plants that is highly
used in India, Nepal, China, Europe and
other parts of the world is the “Salep”
(wild orchid) (5-6).
Salep plant or marshland finger tuber
(Dactylorhiza lancibracteata (C.koch)
Renz) with the old name of “Orchis
maculata L.”, which belongs to the Orchid
family, has many species and grows
almost all over the world. Its tuber is
usually harvested in early summer and
maintains its medicinal properties for two
years (7-9). This plant contains such
chemicals as: glucomannan, nitrogenous
substances, starch, protein, sugar, hydroxy
benzaldehyde, ferulic acid, Quercetin,
Daucosterol, Cirsilineol, and steroid (5-10,
12). In traditional medicine, this plant is
used for dressing and treating glottal
inflammation and in treatment of bowel
disorders, tuberculosis, diarrhea,
Parkinson’s, cancer, fever, and especially
as a regenerative in sexual activity and
erectile dysfunction, and it is also
prescribed for increased stamina and as an
energizer (13-14, 5-6). This plant is also
used in ice-cream, soft drinks, and
confectionary industries (15-16).
Studies of Thakur et al. on rats showed
that consumption of salep plant root
extract increases attraction to the opposite
sex, frequency of erection and ejaculation,
the animal’s body weight, reproductive
organs weight, fructose and testosterone
(17-19). In a review study by Shamloul, it
was reported that despite increased
tendency to use medicinal plants to
improve sexual activity and erectile
dysfunction, but, there are no scientific
evidence to support such effects (20).
Given all the above, this study was
conducted to examine effects of Salep
tubers aqueous extract on the testis tissue
and sexual hormones in adult male mice.
Materials and Methods
Collection and extraction method:
Samples of Salep plant or marshland
finger tuber were collected from suburbs
near Khomain city heights and after
preparation of herbarium specimens, they
were identified by the Kashan’s Barij
Essence Company’s botanist and
registered with code number 195-1. The
roots of this tuber plant were collected in
early summer and after washing and dust
removal, dried in the shade in laboratory.
Fully dried samples were turned into a
powder using an electric herb grinder. The
resulting powder was mixed with ethanol
96% at a rate of 5 times volume of plant
and thoroughly stirred in a Roto-Mix
mixer for 24 hours at ambient temperature
until a homogenous solution was obtained.
Next, the solution was filtered through and
dried in ambient conditions for 48 hours to
transform into a solid alcohol-free extract.
To prepare the aqueous extract, one gram
of the obtained extract was dissolved in
5cc double distilled water to achieve a
solution containing 20% extract (200 mgr
in 1 cc) and it was kept refrigerated.
Grouping of animals:
In this experimental study, 18 adult male
mice (one month old) of the Balb/c strain
with mean weight of 21-23 grams were
used. The mice were procured from the
Pasteur Institute, and accommodated in
Animal Care Center of Kashan University
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Journal of Jahrom University of Medical Sciences, Vol. 11, No. 1, Spring 2013 63
of Medical Sciences for a week to adapt to
the environment. During the study,
animals were kept under 12 hours light and
12 hours dark conditions at ambient
temperature (20-25 °C) and had free
access to food and water. Animals were
randomly divided into 3 equal groups of:
experimental, placebo, and control.
Experimental group received
intraperitoneal injections of 40 mg Salep
root extract in 200 µl double distilled
water daily for one week. The placebo
group received 200 µl double distilled
water intraperitoneally, and control group
did not receive any substance.
Blood collecting and hormonal tests:
Two weeks after the last injection and after
weighing the animals, blood samples were
obtained from their heart. After separating
the blood serum; FSH, LH, and
testosterone hormones were measured with
Immunoradiometric Assay using Gamma
counter LB951 Berthold model purchased
from Beckman Coulter Company-
Germany.
Microscopic examination of testis tissue:
Following the completion of test period,
mice were killed by cervical dislocation. In
sterile conditions, an incision was made in
the lower abdomen, right and left testes
were removed and placed in a
physiological serum.
After removing the surrounding fat, they
were weighed separately using Sartorius
scale (made in Germany, with accuracy of
0.0001 grams), and their volume was
measured by submerging them into
physiological serum in a 5 ml graduated
cylinder.
For the histological studies, testes were
fixed in Bouins solution for 24 hours and
preparation stages were performed with
standard procedure, and finally they were
embedded in paraffin. 5-micron serial
sections perpendicular to the longitudinal
axis of testes were prepared and stained
using Hematoxilin-Eosin method. Ten
slices were randomly selected from each
animal tissue sections, and in each slice, 5
round or nearly round seminiferous tubules
(50 in total) that were in stages VII and
VIII of seminiferous epithelium cycle,
were assessed. To measure seminiferous
tubule diameters, Zeiss microscope fitted
with an eye piece micrometer with
100×magnification and calibrated by a
stage micrometer was used. Measurements
were expressed in microns. Number of
seminiferous tubules’ cells including:
spermatogonia, spermatocyte I of
pachytene stage, spermatids, and mature
sperm were counted using an light
microscopy (with 400×magnifications) in
50 round seminiferous tubules in each
animal, and mean number of these cells in
each group was compared with other
groups. Leydig cells were counted using
light microscopy (400×magnification) in
10 visual fields (50 visual fields in total for
each animal), and average number of cells
in each group was compared with other
groups.
Statistical analysis:
Mean variables between the three groups
of experimental, placebo, and control were
performed using ANOVA and Tukey’s test
with confidence level of 95%. Differences
were considered significant at P<0.05.
Results
All the results obtained in this study are
presented in table 1. Comparison of
animal’s weight, testes weight, testes
volume, and seminiferous tubule diameters
between study groups revealed
insignificant differences.
No significant difference was observed in
mean FSH hormone between the 3 groups.
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64
Mean level of luteinizing hormone showed
a significant increase (P<0.05) in the
experimental group (0.6±0.09) compared
to placebo group (0.39±0.006) and control
group (0.38±0.007). Also, mean level of
testosterone hormone showed a significant
increase (P<0.05) in the experimental
group (1.25±0.52) compared to the
placebo group (0.37±0.03) and control
group (0.36±0.02).
Number of spermatogonia cells with
(P<0.01) and spermatocytes I in the stage
of pachytene, spermatids, and mature
sperm cells (P<0.001) in every
seminiferous tubule in the experimental
group showed significant differences
compared to the placebo and control
groups. However, number of Sertoli cells
per tubule was not significantly different
between the three groups. Leydig cells in
the experimental group (22±2.09) showed
a significant difference (P<0.01) compared
to placebo (15±1.46) and control
(16±1.01) groups. Comparison of study
variables between placebo and control
groups revealed insignificant differences.
Discussion
The results indicate a significant increase
induced by Salep plant root aqueous
extract injections in number of
spermatogonia, spermatocyte I, spermatid,
and mature sperm cells.
This is the first experimental study on
mice that shows effect of Salep plant on
the structure of testis tissue. Studies
conducted by Thakur et al. rather focused
on the sexual behavior effects of Salep in
mice, which caused an increase in
attraction to the opposite sex, frequency of
erection and ejaculation, testosterone
hormone, sperm count in epididymis, and
semen fructose (17, 19). On the other
hand, use of this plant in traditional
medicine is commonplace in various
societies and it is believed that this plant is
nourishing and energizing and effective in
improving male sexual libido (11, 15).
Table 1: Comparison of mean variables in the experimental, placebo, and control groups
Variables
Control
Placebo
Experimental
Test results
Animal weight (gr)
22±1.2
22±1.6
23±1.1
NS**
Testes weight (gr)
0.73±0.003
0.7±0.009
0.7±0.004
NS
Testes volume
(Cm3)
0.10±0.003
0.10±0.008
0.10±0.003
NS
Seminiferous duct
diameter (µm)
180.160±5.10
182.72±4.36
188.79±4.52
NS
Spermatogonia cells
(number/duct)
44±3.49
43±3.78
61±3.09
P<0.001
Spermatocyte I cells
(number/duct)
46±2.10
45±2.57
62±2.84
P<0.001
Spermatid cells
(number/duct)
126±4.90
125.7±2.96
180.5±7.45
P<0.001
Mature sperm cells
(number/duct)
53±5.3
60±8.33
134±7.04
P<0.001
Sertoli
(number/duct)
15±0.47
14±1.46
16±0.79
NS
Leydig cells
(number/microscope
field)
16±1.01
15±1.46
22±2.09
P<0.001
FSH (ng/ml)
0.39±0.23
0.41±0.03
37±0.03
NS
LH (ng/ml)
0.38±0.007
0.39±0.006
0.6±0.09
P<0.05
Testosterone (ng/ml)
0.36±0.02
0.37±0.03
1.25±0.52
P<0.05
* Findings are presented as mean±SD.
** NS stands for Not Significant
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Other results of this study include
increased sexual hormones of luteinizing
(LH) and testosterone in the experimental
group compared to the control group
(P<0.05). This increase has also been
reported in other studies (17).
An increase in the number of leydig cells
as a testosterone secreting cell in response
to luteinizing hormone could explain
changes created in the testis tissue and
increasing trend of spermatogenesis
through effects of chemicals in Salep plant
on the hypothalamic-pituitary-testis axis.
Increased testosterone could release
dopaminergic mediator chemicals in the
brain. It has been demonstrated that there
is a significant relationship between
dopamine release in the nucleus
accumbens and improving sexual activities
(21-23).
One of the factors proven to have a role in
increasing sperm count and sexual activity
is elimination of the oxidants in semen.
Quercetin and ferulic acid are chemicals
found in the Salep plant and are considered
to have an anti-inflammatory, anti-cancer,
and antioxidant role (10, 24-26). Young et
al. stated that consumption of natural
antioxidants protects sperm cells against
oxidative stress induced by lysed cells and
ultimately improve fertility (27).
Other compositions in the Salep plant
include glucomannan whose level in
various species varies from 7 to 61%.
Glucomannan is a polysaccharide that
provides the required energy for sperm
production in the seminiferous tubules.
This substance has a role in weight loss,
blood glucose control, and cholesterol
reduction (12, 28). Lack of animals’
weight gain could be due to relatively high
proportion of this substance in the Salep
root extract or short experiment duration.
In contrast to the results obtained in this
study, other studies have reported body
and sexual organs’ weight gain induced by
use of this plant (16). They believe that
anabolic activity of the body increases
with increasing testosterone, which leads
to increased body and sexual organs’
weight.
Conclusion
This study showed that Salep root aqueous
extract increased spermatogenesis and
improved libido by increasing testosterone
and LH. It is recommended that further
studies be conducted in order to find the
mechanism and the effect of this
substance, so that it can be used as an
effective medicine for improved sexual
activity and fertility.
Acknowledgements
This article is retrieved from student thesis
for the award of Master’s Degree from the
Tehran Islamic Azad University- science
and research division. We wish to express
our thanks to the personnel of the anatomy
and physiology departments and the
animal house of Kashan University of
Medical Sciences, Tehran Islamic Azad
University- science and research division’s
central laboratory, and Kashan city Barij
Essence Medicine Company that assisted
us in performing this study.
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