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Baghdad Science Journal Vol.9(4)2012
Effect of Aqueous and Ethanolic Extracts of Tribulus
terrestris, Phoenix dactylifera and Nasturtium officinale
Mixture on Some Reproductive Parameters in Male Mice
Mohaisen H. Adaay * Amal G. Mattar**
Received 5, May, 2011
Accepted 21, February, 2012
Abstract:The present investigation was conducted to evaluate the effect of the crude extracts
mixture of three plants (Tribulus terrestris, Phoenix dactylifera and Nasturtium
officinale) on semen quality,sex hormones and reproductive performance of mature
male mice. A group of 25 male mice given 150mg/kg/day of the powder of the plants
mixture with the food for four weeks and another three groups of 25 animals each
given intraperitoneal injection from each of the aqueous and ethanolic extracts with a
doses 75, 150, and 300mg/kg/day for two weeks. A remarkable increase in sperm
concentration and motility with a decreased abnormal morphology was obtained in
the experimental groups. A significant increase in hormones level were recognized in
most groups. The results of mating untreated females with treated males of the four
experimental groups revealed a decreased gestation period and an increased litter
size. The results showed a dose dependent pattern of activity and the effect of the
extracts were enhanced with increasing the dose level. The ethanolic extract being
the more effective extract in all parameters.
Keywords: Tribulus terrestris, Phoenix dactylifera, Nasturtium officinale,
Reproduction, mice.
Introduction:
In the past, medicinal plants were
considered as the only form of health
care readily available to the majority
of human population [1]. At present,
it is estimated that about 80% of the
world population relies on botanical
preparations as medicine to meet their
health needs [2]. In recent years, in
view of their beneficial effects, the
use of spices and herbs has been
gradually increasing in developing
countries[2].Tribulus terrestris L.
(TT) is a member of the plant family
Zygophillaceae. The fruit is regarded
as tonic, diuretic and aphrodisiac. It is
also used to treat urinary disorder,
impotency and heart disease. The
seeds are recommended in
hemorrhages, kidney stone and gout
[3]. The extract of TT contains
protodioscin (PTN), a steroidal
saponin that has been extensively
used for the treatment of various
ailments, such as urinary [4] and
cardiovascular[5] disorder.
Administration of TT to human and
animals improves libido and
spermatogenesis [6, 7]. TT has a
proerctile effect [8].
Phoenix dactylifera (Pd) is a member
of the plant family Arecaceae. Date
extracts have been shown to increase
sperm count in guinea pigs and to
enhance spermatogenesis and increase
the concentration of testosterone, FSH
and LH in rats. The pollen grains of
date palm have been used by
Egyptians to improve fertility. Date
*Ph.D Institute of Embryo Researches and Infertility Treatment, Univ. of Al-Nahrain,
Baghdad, Iraq. . E-mail : dr_mohsin2004@ yahoo.com Tel: 009647901665974.
**M.Sc Ministry of Health, Baghdad, Iraq.
Baghdad Science Journal Vol.9(4)2012
pits have been included on the animal
food to enhance growth [9].
Nasturtium officinale (No) is a
member of the plant family
Brassicaceae.The complete German
Commission E Monographs improved
the No for the catarrh of the
respiratory tract. In Germany, it is
also used to treat urinary tract
infections in children [10]. The
powdered leaves are used in India as
an expectorant to treat bronchitis and
a number of conditions affecting
human liver [11].The fresh herb is
used as a blood purifier [12]. The
present investigation was conducted
to evaluate the possible effect of the
crude extracts mixture of the three
plants on semen quality, sex
hormones and reproductive
performance of mature male mice.
Materials and Methods:
Animals:
Mature Swiss albino male mice, nine
weeks old and 25-30 gm in weight,
were used in this study. The animals
were kept under standard conditions of
temperature (25cº) and lighting period
(14hrs light/10hrs dark). Food and
water were supplied ad libitum.
Plant materials:
Tribulus terrestris and Pd which were
collected from Baghdad (Al-
Khadimiya area) and identified by the
Iraqi National Herbarium Staff and No
seeds which were obtained from
herbalist, were mixed in a proportion
of TT (aerial parts, 40%), Pd (pollen
grain, 30%) and No (seeds, 30%) and
powdered by using an electrical
grinder. Preparation of the aqueous and
ethanolic crude extracts of the plants
materials was conducted according to
Mattar, 2005[13].
Treatmen:
1-Oral administration:
Two groups of 25 animals were used.
G1 considered as a control group and
fed the normal food only; G2 was the
experimental group which was given
the crude mixture of plants added with
the normal food in a dose of
150mg/kg/day for a period of four
weeks. Fifteen animals from each
group were killed at the end of the
fourth week for semen analysis (sperm
concentration, motility and
morphology). Blood serum was used to
evaluate hormone levels (testosterone,
FSH, LH) by the end.
of the fourth week by using a gamma
counter and a special hormonal
kits[14].
2-Intraperitoneal(ip) injection of the
aqueous and ethanolic extracts:
Four groups (per each extract) of 25
animals were treated for a period of
two weeks. G1 was the control group
given either water or olive oil injection
(0.5ml) daily, G2, G3 and G4 received
an ip injection in a dose of 75, 150 and
300mg/kg/day respectively from each
extract. Fifteen male mice from each
group of both treatment were killed at
the end of the 1st and 2nd week of
treatment for semen analysis (sperm
concentration, motility and
morphology), and evaluation of
hormones level "LH, FSH and
testosterone". Ten male mice from
each group of both treatments (oral and
ip) were mated, at the end of
treatments, with untreated mature
females at the ratio of one male /three
females to check the reproductive
performance. Timing of pregnancy was
performed by observing the presence
of vaginal plug which is regarded to be
day one of pregnancy.
Collection of blood sample:
Blood samples were collected under
light anesthesia by heart puncture
using a 22-19 mm gauge needle.
Serum for hormonal assay was
obtained by centrifugation for 10
minutes at 3000 rpm and kept at
(-20Cº) until use.
Microscopic examination of
epididymal sperms:
Baghdad Science Journal Vol.9(4)2012
Examination of epididymal sperms and
calculation of sperm concentration,
sperm motility and sperm morphology
was conducted according to Al-Dujaily
1996[15]
Statisticalanalysis:
Computerized statistical analysis was
performed using the SPSS (statistical
package of Social Sciences) version
10 under windows XP-2000(Inc,
Chicago, IL,USA) computer soft ware
and the use of excel program. Values
reported are means ±SE .Experimental
results were statistically analyzed
using the t-test, with P values less than
0.05 considered significant, less than
0.01 considered highly significant [16].
Results:
The effect of Oral administration of
plants mixture powder:
The effect of dry powder of plants
mixture on epididymal sperm
parameters is shown in table 1. A
significant (P<0.05) increase in sperm
concentration was found after the 2nd
and 4th week of treatment in
comparison with the controls.
A significant (P<0.05) increase in
sperm motility was shown after the 2nd
week of administration and a highly
significant (P<0.01) increase was
obtained after the 4th week of treatment
compared to controls. No significant
difference in abnormal sperm
morphology was found after the 2nd
week of treatment, whereas a highly
significant (P<0.01) decrease appeared
after the 4th week in comparison with
the controls.The effect of feeding
plants mixture powder on hormonal
levels is shown in table 2. A highly
significant (P<0.01) increase in LH
and FSH levels and a significant
(P<0.05) increase in testosterone level
were found after the 4th week of
treatment as compared with the control
group.Table 3 shows the effect of
feeding plants mixture on reproductive
performance. A highly significant
(P<0.01) increase in the number of
fetuses and a significant (P<0.05)
decrease in the gestation period was
found in the treated group in
comparison with the controls.
The effect of ip injection of the
aqueous extract:
The effect of ip injection of the
aqueous extract on sperm parameters is
shown in table 4. No significant
difference in sperm concentration after
the 1st week of treatment in G2
compared to G1, whereas, G3 and G4
showed a highly significant (P<0.01)
increase in comparison with G1 after
the 1st week of treatment. A significant
(P<0.05) increase obtained in G2 and a
highly significant (P<0.01) increase
resulted in G3 and G4 after the 2nd week
of treatment compared to G1 with
respect to the same parameter.
After the 1st week of treatment, G2
showed a significant (P<0.05) increase
in the % motility and a highly
significant (P<0.01) increase in G3 and
G4 compared to G1. After the 2nd week,
there was a significant (P<0.05)
increase in G2, G3 and G4 in
comparison with the control group G1.
G2 showed a significantly (P<0.05)
decreased abnormal sperm
morphology, whereas G3 and G4
showed a highly significant (P<0.01)
decrease after the 1st week of treatment
compared to G1. After the 2nd week, a
highly significant (P<0.01) decrease
was observed in G3 and G4 compared to
G1.
The effect of ip injection on hormonal
levels is shown in table 5. G2 showed
no significant difference in LH, FSH
and testosterone levels after the 1st and
2nd weeks of treatment except a
significant (P<0.05) increase in
testosterone level after the 2nd week of
treatment compared with G1. G3
revealed a significant (P<0.05)
increase in LH level after the 1st and
2nd week and a significant (P<0.05)
increase in testosterone level after the
Baghdad Science Journal Vol.9(4)2012
2nd week in comparison with G1. G4
showed a highly significant (P<0.01)
increase in LH level and a significant
(P<0.05) increase in FSH and
testosterone levels after the 1st week of
treatment in comparison with G1,
whereas after the 2nd week, a
significant (P<0.05) increase in FSH
level and a highly significant (P<0.01)
increase was observed with respect to
LH and testosterone levels in
comparison with G1. Table (6) shows
the effect of ip injection of the aqueous
extract on fertility capacity. After the
2nd week of treatment, G2 and G3
showed a significant (P<0.05) increase
in number of fetuses and a significant
(P<0.05) decrease in gestation period
with a highly significant (P<0.01)
increase in No. of fetuses and a highly
significant (P<0.01) decrease in
gestation period in G4 in comparison
with G1.
Table 1: Effect of feeding crude mixture of TT, Pd and No on some semen
parameters in mice.
G2
G1
Group
Week
Abnormal
morphology
Motile%
Conc./ml
Abnormal
morphology
Motile%
Conc./ml
62.8±1.28
a
75.2±1.78
a
50.8±
1.95x106
66.4± 2.2
70.6±
0.89
40.6±
2.28x106
2nd
b
23.6±1.61
b
94.6±0.54
a
60.8±
3.10x106
62.8± 1.1
78.6±
1.19
44.8±
2.44x106
4th
Values = mean ±SE aP <0.05 compared to the control group. bP <0.01 compared to the control group.
Table 2: Effect of feeding crude mixture of TT, Pd and No on hormonal
level in mice.
G2
G1
Group
week
Testosteronepg/ml
FSH
mIU/ml
LH
mIU/ml
Testosterone pg/ml
FSH
mIU/ml
LH
mIU/ml
a
4.3±0.14
b
1.98±0.01
b
2.17±0.01
3.6±0.12
1.57±0.01
1.34±0.03
4th
Values = mean ±SE. aP <0.05 compared to the control group. bP <0.01 compared to the control group.
Baghdad Science Journal Vol.9(4)2012
Table 3: Effect of feeding crude mixture of TT, Pd and No on reproductive
performancein mice
Values = mean ±SE.
aP <0.05 compared to the control group.
bP <0.01 compared to the control group.
Table 4: Effect of ip injection of aqueous extract of TT, Pd and No on some
semen parameters in mice.
After 2 weeks
After 1 week
Week
Group
Abnormal
morphology
Motile%
Concen./ml
Abnormal
morphology
Motile%
Concen./ml
50.2±3.76
82.0±1.74
44.0± 3.29x106
63.2±1.95
76.6±1.30
44.6± 3.09x106
G1
48.2± 1.95
a 87.0± 1.74
a 58.8± 2.9x106
a
53.4± 1.30
a
84.2±1.38
52.8± 1.03x106
G2
b
37.6±1.51
a 94.0±2.24
b 69.0± 1.87x106
b
43.8±1.17
b
90.4±1.70
b
62.2± 1.27x106
G3
b
22.8±2.14
a 97.2±1.10
b
77.8± 1.1x106
b
31.0±3.48
b
92.8±1.50
b
71.0± 2.18x106
G4
Values = mean ±SE. aP <0.05 compared to the control group. bP <0.01 compared to the control group
Table 5: Effect of ip injection of aqueous extract of TT, Pd and No on hormonal
level in mice
Gestation period(day)
Average No. of
fetuses
Group
20.8±0.56
5.06±0.59
G1
a
19.8±0.67
b
9.0±0.92
G2
After 2 week
After 1 week
Week
Group
Testosteron
e
Pg/ml
FSH
mIU/ml
LH
mIU/ml
Testosteron
e
Pg/ml
FSH
mIU/ml
LH
mIU/ml
3.64±0.09
1.58±0.0
1
1.35±0.0
1
3.54± 0.09
1.58±0.0
1
1.34±0.0
2
G1
a
4.42±0.08
1.64±0.0
1
1.42±0.0
1
3.76±0.17
1.61±0.0
2
1.40±0.0
1
G2
a
4.84±0.05
1.93±0.0
2
a
2.38±0.0
8
4.26±0.11
1.86±0.0
2
a
2.25±0.0
3
G3
b
5.42±0.08
a
1.98±0.0
1
b
3.34±0.0
5
a
4.68±0.13
a
1.95±0.0
1
b
2.80±0.0
1
G4
Baghdad Science Journal Vol.9(4)2012
Values = mean±SE. aP <0.05 compared to the control group. bP <0.01 compared to the control group.
Table 6: Effect of ip injection of the
aqueous extract of TT, Pd and No on
reproductive performance in mice
Values = mean ±SE.aP <0.05 compared to the control group. bP <0.01 compared to the control group.
The effect of ip injection of ethanolic
extract:
The effect of ip injection of ethanolic
extract on epididymal sperm parameters
is shown in table (7). Treatment of the
animals with the dose levels used (G2
G3 and G4 ) showed a highly significant
(P<0.01) increase in sperm
concentration after the 1st and 2nd weeks
of treatment in comparison with the
control group G1. G2 showed a
significant <0.05) increase in the %
motility after the 1st and 2nd weeks of
treatment as compared to G1 ,whereas
G3 and G4 showed a highly significant
(P<0.01) increase after the 1st and 2nd
weeks of treatment compared to
G1.There was a significant (P<0.05)
decrease in the abnormal sperm
morphology in G2 after the 1st week of
treatment compared to G1, while there
was a highly significant (P<0.01)
decrease appeared in G3 and G4 after the
1st and 2nd weeks of treatment compared
to the control group.The effect of ip
injection of ethanolic extract on
hormonal levels is shown in table (8).
G2 showed no significant differences
with respect to all hormones after the 1st
and 2nd weeks in comparison with
control group. In G3, A highly
significant (P<0.01) increase in LH
level and a significant (P<0.05) increase
in FSH and testosterone levels were
found after the 1st week of treatment.
The same trend was found in the 2nd
week, which showed a significant
(P<0.05) increase in LH and FSH levels
and a highly significant (P<0.01)
increase in testosterone level in
comparison with G1. G4 showed a
highly significant (P<0.01) increase in
LH level and a significant (P<0.05)
increase in FSH level after the two
periods in comparison with the control
group, as well as a significant
(P<0.05) increase was found after the
1st week and a highly significant
(P<0.01) increase after the 2nd week in
testosterone level as compared with the
control group. After two weeks of
treatment, G2 showed a significant
(P<0.05) increase in number of fetuses
and a significant (P<0.05) decrease in
gestation period, whereas, G3 and G4
exhibited a highly significant (P<0.01)
increase in the number of fetuses and a
significant (P<0.05) decrease in
gestation period in comparison with the
control group G1 (table 9).
Gestation
period
(day)
Average No. of
fetus
Group
21.8±1.01
5.4±0.34
G1
a 20.4±0.43
a 6.3±0.50
G2
a 20.4±0.33
a 6.4±0.44
G3
b20.2±0.22
b 7.7±0.40
G4
Baghdad Science Journal Vol.9(4)2012
Table 7: Effect of ip injection of ethanolic extract of TT, Pd and No on semen
parameters in mice.
After 2 weeks
After 1 week
Week
Group
Abnormal
morphology
Motile%
Concen./ml
Abnormal
morphology
Motile%
Concen./ml
50.2±4.76
82.0±1.74
44.0±
3.29x106
63.2±1.95
76.6±1.30
44.6±
3.09x106
G1
a
43.2± 1.25
a
88.0±
2.33
b
61.8±
1.8x106
a
51.4± 1.10
a
85.2±
1.18
b
58.8±
1.13x106
G2
b
33.6±1.37
b
94.5±2.04
b
72.1±
1.18x106
b
40.6±1.13
b
90.0±0.89
b
60.2±
1.05x106
G3
b
21.8±1.26
b
97.5±2.10
b
80.8±
1.51x106
b
28.6±1.06
b
94.8±1.57
b
77.0±
1.58x106
G4
Values = mean ±SE. aP <0.05 compared to the control group. bP <0.01 compared to the control group.
Table 8: Effect of ip injection of ethanolic extract of TT, Pd and No on hormonal
levels in mice.
After two weeks
After one week
Week
Group
TestosteronePg/ml
FSH
mIU/ml
LH
mIU/ml
Testosterone Pg/ml
FSH
mIU/ml
LH
mIU/ml
3.54±0.07
1.57±0.02
1.73±0.01
3.48±0.21
1.58±0.01
1.38±0.01
G1
4.06±0.11
1.66±0.02
1
1.46±0.01
3.76±0.05
1.63±0.01
1.42±0.02
G2
b
4.54±0.04
a
1.96±0.01
a 2.37±0.02
a
3.98±0.04
a
1.88±0.02
b
2.29±0.01
G3
b
5.58±0.13
a
2.16±0.05
b
3.50±0.04
a
4.22±0.07
a
1.98±0.01
b
2.92±0.03
G4
Values = mean ±SE. aP <0.05 compared to the control group. bP < 0.01 compared to the control group
Table 9: Effect of ip injection of the
ethanolic extract of TT, Pd and No
on reproductive performance in
mice.
Values = mean ±SE. aP <0.05 compared to the control group. bP <0.01 compared to the control
group.
Discussion: The three different methods of
treatment used in this study although
differ to a certain degree in their
effects on the different parameters, but
they showed that the plants extracts
have a good activity on reproduction in
male mice. The significant increase in
sperm concentration, motility and
improvement in sperm abnormal
morphology agrees with the report
traced in the literature that dealt with
Gestation
period
(day)
Average No.
of fetus
Group
21.8±0.61
5.4±0.44
G1
a
21.26±0.39
a
6.26±0.40
G2
a
20.46±0.34
b
7.46±0.34
G3
a
20.22±0.39
b
8.53±0.44
G4
Baghdad Science Journal Vol.9(4)2012
this aspect on laboratory animals and
human [8, 9, 17, 18]. These changes
can be explained on the basis that the
plants mixture contains many materials
which act as a potent antioxidant like
vitamin C, E, A and B which may
protect sperm membrane against lipid
peroxidation thus lowering the
percentage of dead sperm and
maintaining normal sperm
morphology. The mixture also
contains many trace elements (Ca, Mg,
Mn, Na, K, Zn ions and others), these
minerals especially Ca++ is known to
inhibit the enzyme phosphate
diesterase , which prevents cAMP
degradation and consequently
increasing sperm motility and sperm
hyperactivity [19]. The presence of
zinc in the mixture leads to
improvement of the sperm count,
motility and morphology, because it
involved in hormone metabolism,
RNA and DNA organization, protein
synthesis, cell division biomembranes
and nuclear chromatin stabilization
[20]. The significant changes in serum
levels of the reproductive hormones
studied, FSH, LH and testosterone
played an important role in increasing
sperm concentration. Boukhliq and
Martin, 1997 [21] have shown that
aminoacids and fatty acids may
stimulate directly the secretion of Gn-
RH which in turn stimulates FSH and
LH secretion. FSH is known to be the
major hormone responsible for sperm
production and maturation [22]; it is
also an important factor in the final
stages of epididymal sperm maturation
[23]. FSH level showed a significant
increase at different groups of treated
animals.This hormone regulates the
Sertoli cell function including
increasing the production of androgen
binding protein and the latter works in
keeping a high concentration of
testosterone in the testes which is
necessary for normal spermatogenesis
and sperm maturation. FSH is also an
important factor in the initiation and
continuation of spermatogenesis [24].
The improvement in sperm motility
may have been brought about by
increasing intercellular cAMP, which
is known to be a very important factor
in stimulating sperm motility [25].
The PTN present in the mixture leads
to a direct increase in LH and
dehydroepiandrosteron (DHEA) levels
which leads to increase in testosterone
level , and thus the stimulation of
spermatogenesis with an increase in
concentration of spermatozoa [8,26].
TT may increase fertility by a direct
action on germinal and Sertoli cells by
improving spermatogenesis, it may
improve libido by increasing the level
of LH which activates the production
of testosterone from the Leydig cells
by increasing the androgen receptor
sensitivity or by stimulating the
enzyme 5-alpha-reductase which
increases the conversion of
testosterone into dihydrotestosterone
(DHT). In addition to its activity on
sexual performance.
The potassium ion present in the
mixture may have an important role in
increasing the LH and testosterone
levels by affecting the pulsatil release
of Gn-RH from the hypothalamus
which is an important factor in
regulating androgenic status and
fertility [27, 28]. The presence of
vitamin A is required for maximal
production of testosterone and vitamin
B helps to optimize macronutrient
metabolism, maximize muscle mass
and decrease the serum levels of
homocysteine, cholesterol and C-
reactive proteins, marker of heart
disease and inflammation in the body.
Decreasing inflammation helps to
decrease the cortisol level and thus
increase the anabolic effect of the
mixture [29]. The glycosides present in
No acts in the same way because it has
an anti-inflammatory effect [30]. All
these factors lead to improvement in
Baghdad Science Journal Vol.9(4)2012
the concentration and motility of sperm
and in some cases increase in the
volume of ejaculation [29] and this
may explain the significant increase in
the result of reproductive performance
in this study which is due to the
increase in sperm number, motility,
and the improvement in morphology
and erection function. The significant
decrease in gestation period is the most
common complication of multiple
pregnancies which leads to preterm
labor and premature rupture of the
membrane. This hypothesis originated
from the observation of the length of
gestation versus litter size which
demonstrated a direct correlation
between uterine stretching and the
initiation of parturition [31]. In
conclusion the results showed a dose
dependent pattern and the effect of the
extracts were enhanced with increasing
the dose. The ethanolic extract being
the more effective extract in all
parameters. The possible reason for the
variation in the effect between aqueous
and ethanolic extracts could be due to
the difference in the active principles
present in the two extracts.
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