Comparative Study among Watermelon Crud Extract, Citrulline and
Lycopene on Some Reproductive Indices in Male Mice
Awatif Ibrahim Mohammed
Mohanad A. Al-Bayati; BVM, MSc PhD. Pharmacology and Toxicology
aumnmumu@yahoo.com
Department of Pharmacology, College of the Veterinary Medicine, Baghdad University, Iraq.
Abstract
Objectives of this project were to study the effect of 70% crude alcoholic extract of
watermelon Citrullus lanatus pulp on the reproductive system of male mice as
compared with two main component citrulline and lycopene. The mice were divided
into four groups, the first group were treated with watermelon 500mg/kg b.w., the
second group was treated with citrulline 30mg /kg b.w., third group was treated with
lycopene 5mg/kg b.w. and treatment continueous for 38 days while the fourth group
used as a control group non treated. The study showed improvement of most
parameters related to increase the reproductive efficiency in alcoholic extract of
watermelon and citrulline treated group higher than as compared to lycopne
andcontrol group.
Key word: watermelon, citrulline, lycopene, sperm parameter .
Introduction :
Nourishment has been linked with animal
reproductive success throughout history (1).
Dietary habits and essential nutrients to
promote successful reproductive out- comes
have been identified for the maternal periconceptional and peri-natal period, but
healthy dietary habits and essential nutrients
for paternal reproductive fitness are less clear
(2). Watermelon is a fruit of great economic
importance, with worldwide production
Watermelon is enjoyed by many people
across the world as a fresh fruit, partly owing
to it being low in calories and highly
nutritious
and
thirst-quenching
(3).
Watermelon is very rich in phytonutrients
such as lycopene a forerunner of β-carotene
and a carotenoid of excessive notice because
of its antioxidant capacity in scavenging
reactive oxygen species, which cause
oxidative tissue destruction and loss of proper
cell function (4). Epidemiological revisions
I
have established that high consumption of
fruits and vegetables that high
consumption of fruits and vegetables
containing lycopene is associated with
reduced incidence of coronary heart
disease (5) and some types of prostate and
kidney cancer furthermore, provoke
sexual and reproductive system. small
intestine of rats (6) enables arginine to be
a non-essential amino acid by converting
most (83%) of citrulline to arginine in the
kidney (7). Citrulline-L-arginine is an
essential amino acid that has a wide role
in reproductive, pulmonary, renal,
gastrointestinal, hepatic and immune
systems and expedites the healing of
wounds (8). Citrulline is used in the nitric
oxide system in humans and animals and
has
potential
antioxidant
and
vasodilatatory roles denoted to both of
lycopene and citrulline are promote
molecular
events at cellular levels (9) that’s
encouragements of cellular functions through
marvel production of nitric oxide and
Carotenoids. Now a days, peoples and
animals condition are concerned about their
health and physical shape and demand more
natural ingredient foods of fresh quality such
as ready-to-eat, minimally processed or fresh
cut fruits and vegetables .
Materials and methods:
Watermelon extraction preparation:
The watermelon Citrullus lanatus was
purchased from the local market Baghdad and
certified in the Iraqi National Herbarium in
Abou–graib,16/9/2013. The fresh watermelon
washed with tabe water to remove
contaminante on rind (10) and decortications
with deseeding manually. The pulp
segmented and blendering by electrical
blender for 3minutes then sieving with sieve
at mesh 250 µm which were forming slurry
shape and saved in clean container (11) after
a dried at 40C̊ mostly for 48±4hr. and the
final yeild brown dry cake, grinding and
Sieving then stored at refrigerator 4C̊ until
extraction. Preparation of crude alcoholic
extract (70%) and dry of watermelon
Citrullus lanatus pulp:
The powdered sample was weighed at 15g
and the extraction is employe by used of
soxhlet apparatus which contain two part
electrical heater and magnetic stirrer with a
round glass flask which filled with 300ml of
solvent with 50C̊ ,50 rotation in min. The dry
powdered sample put into a thimble part then
extraction was clarified with magnatic
sterring with vacume pump machine. The
filtrate was concentrated and remove the
alcohol from it by using rotary evaporator
with 50̊c and 200 rotation per minute for
4hrour (12&13). The concentrated extract
placed
in glass Petri dish with small
magnetic bar on magnetic stirrer machine
with 40C̊ and 40 rotation per few minute. All
the dried extract collected and kept in frozen
at -20C̊. The body weight of males were
calculated as follow according to (14):
Body weight (gm) = b.w. after treatment –
b.w.before. After 38 day of the treatment
period finished, the animals were
anesthetized and the testis excised and
cleaned from the fat tissue. Testes weight
and compared to body weight as following :
Testicular weight. to body weight. ratio =
Weight of testis (gm )X 100/Weight of
animal (gm). Then The volume of testis was
calculated by volumetric redual method
according to: Volume of testis = V2 volume
of testis after dip -V1 volume of testis
before dip .
Epididymus Preparation:
epididymis were gently removed and freed
from fat tissue and immediately put in watch
glass with 0.1ml of ringer solution on warm
hot plate at 37 C˚ then incise by micro
scissor to 200 part (15)used by the
Spectrophotometric method to evaluate Lag
time, Velocity, fraction of rapidly moving
sperm as described by (16 and 17). Also
Add 0.1ml of fresh semen solutions on slide
to evaluated sperm counting after using of
hemocytometer with using according to
(18): Sperm concentration= Number of
sperms 1000 x4000/80. Also examining
sperm abnormalities by putting a drop of
semen with drop of eosin and negrosin stain
on edge of slide examining with bright field
optics at ×100 according to (19) associated
with Acrosomal abnormal- lities according
to (20) : Acrosomal abnormalities % =
number of counted abnormal acrosome (%)
/ 200 x 100. Evaluation of live and dead
sperms were identified according to (21):
Live sperm%= Number of live sperms
X100/ Total sperms number.
Animal groups:Total number of adult
healthy male mice (Swiss albino) 50
purchased from Laboratory Animal Collage
of Medicine University/ Baghdad, weighted
22-34g, and aged 8 weeks, they were housed
at a control system temperature 27◦C ± 2◦C,
light schedule 12:12, with ventilated vaccum.
Mice were incubated in plastic cages
(13x10x10) cm, and cleaned daily, with
substitute wood saw forth time weekly. Mice
left for adaptation period for 4 weeks.
Experimental design :
Group 1:Animals were served as control
group given orally distilled water for 38 days.
Group 2:Animals were received orally daily
watermelon alcoholic extract at dose 500mg
/kg B.W for 38 days (11 ).
Group 3:Animal were received orally daily
Citrulline at dose 30 mg/ kg B.W for 38 days
(7). Group 4:Animal were received orally
daily Lycopene at dose 5 mg/kg B.W. for 38
days (22).
Results &descusions :
This study showed the watermelon treated
group display significant p<0.05 increase in
body weight and testicular morphometric
parameter than both citrulline and lycopene
treated groupas in figure (1) ,table (1). In
table 2 there were significant p<0.05 increase
in sperm count in watermelon treated group
as compared to control group furthermore
significant p<0.05 higher than citrulline and
lycopene treated group. In table 3 there was
significant p<0.05 increase in sperm count
and viability & decrease in sperm
abnormalities in watermelon treated group as
compared to control group furthermore
significant p<0.05 higher than citrulline and
lycopene treated group. In table 4 there was
significant p<0.05 increase in turdidimetric
analysis of sperm motility in watermelon
treated group as compared to control group
furthermore significant p<0.05 higher than
citrulline and lycopene treated group . That
result finding in table (1) may be due to
ability of watermelon to ameliorate and
accelerate the metabolic process by improved
food intake (23) and feed behaviors in
animals under normal and stress conditions
(24) and had the ability to lowered the
subcutaneous adipose tissues and increase the
brown fat and muscle mass(25,26). In
citrulline-L-arginine considered as good
appetizer(27)play important role in stimulate
the synthesis and releasing of growth
hormone (28), essential for body building
by reducing triglyceride in subcutaneous
adipose tissue and increasing muscle mass
(29). That was clinically showed as
improvement in body weight (30)
&enhanced testicular parameter (31). While
Lycopene had little effect on body weight
may be due to the slight modulating in lean
body mass with reduction in fat tissue due
to decrease the production of cytokines,
chimiokins and adipokines proteins
secreted by adipose tissue (32). Farther
more lycopene considered one of causal
factor reduce insulin levels which led to
inhibition of Leptin was adipose tissue
products; led to reduction in hunger with
enhanced feelings of satiety (33). On other
hand, Sureda et al., (2010)(34) reported that
watermelon-citrulline-L-arginine-NO had
the ability to enhanced Insulin levels led to
significant increased of growth hormone
secretion which considered as the major
growth metabolic regulatory
hormone
interact with the receptor on the surface of
cell membrane led to activation of several
signaling pathway and increase the body
weight or lean body mass(35) . That
increase in Growth Hormone was acts
synergistically with testosterone synthesis
led to magnitude the change in synthesis led
to magnitude the change in body mass as
well as improvement the testicular
parameter that may (36) as in table (2).
Watermelon-citrulline-L-arginine
had
diverse metabolites including ornithine,
polyamines, proline, glutamate, glutamite,
creatinine, agrnatine, dimethylarginine (37),
spermidine and spermine (38). These
metabolite considered as good regulator for
integrity and function of cell membrane of
sperm cell(39) led to super increase in grow
and count of sperm. That mean there was
positive correlations between increase in
citrulline-L-arginine concentration and sperm
concentration (40) as in (table 3). Also
watermelon had beneficial activation of the
AMP-activated protein kinase (41) led to
provoke the rate of glycolysis, resulting in
higher rates of ATP generation in
spermatozoa (42) and consequently induce
formation of stabilizing disulfide cross-links
(43) led to increase chromatin compaction
with DNA arrangement (44) which
considered important determinant factor for
sperm head morphology that mean there was
correlation between sperm DNA stability and
head morphology (45). That mean
watermelon and citrulline had the ability to
increase viability of sperm (46) and decrease
any disorder in sperm morphology. and that
result agreed with watermelon contain
lycopene which showed that increase in
sperm count due to increase the physiological
activity of testicular cell associated with
reduction in testicular cell associated with
reduction in the apoptosis (47). In table
(4)There was significant p<0.05 decrease in
watermelon and citrulline derived treated
group as compared to compared to control
group furthermore lesser than lycopene
treated group in lag time. Watermeloncitrulline-L-arginine pathway may be
increase sperm concentration associated with
increase in energy utilization that cause low
concentration of calcium medium which may
be effected the time lag required for the
initiated stimulation needed (48). That’s
mean there was negative relationship
between lag time and sperm concentration
(49). There was significant p<0.05 increase
Referancederived
:
in watermelon
citrulline treated
group as compared to control group
furthermore watermelon derived citrulline
more than lycopene treated group in fraction
of rapid movement, velocity and motility
index. In watermelon-citrulline-L-argine
there was increase in sperm concentration
and the motile will be the first groups to
swim upward into clear medium from a
concentrated cell suspension at the bottom
mainly dependent on time, increase
turbidity of the medium and increase in
absorbance. The strongly motile sperms
may be the best candidates for fertilization
(50) and increase velocity of sperm trough
enhance the rate of glycolysis and higher
rates of ATP generation (42) and [Ca+2 ]
increased the sperm motility and velocity
(51) as well as lycopene showed the same
increase increase in sperm motility and
velocity due to the antioxidant effect .
Conclusion:
The major aim of this thesis was to
determine the responsibility of nutrient on
increase and/or maximized the efficiency
of male reproductive function through via
loading dose of alcoholic extract of
watermelon comparable with two main
active ingredient citrulline and lycopene or
other constituted. In the last 38 days, this
study was developed and clarifies and
provide
a
good
indication
that
administration of watermelon fruit which
considered as good source of different
content e.g. vitamin, Carbohydrate, trace
salt beside the sufficient amount of Larginine-Nitric oxide which has agood role
in human health. So the endpoint of this
study was to reveal the major role of
watermelon and two main contain citrulline
and lycopene on testicular fitness and
fertilized semen.
Reference :
1. Jill, E. Schneider (2004) Energy
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2. Tomoko, Fujiwara; Natsuyo, Sato;
g
body weight changes
8
6
4
2
0
A
B
C
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
control watermelon citrulline lycopene
Figure 1:The body weight changes parameter of male mice under loading doses of watermelon, citrulline and
lycopene for 38 days.
Table 2:The testicular morphometrical in male mice under loading doses watermelon, citrulline and lycopene for
38 days.
Control
Watermelon
500mg/Kg.Bw
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
Testicular weight g
Testicular
weight/body
weight ratio
Testicular volume cm3
0.020±0.001
0.077±0.020
0.058±0.216 A
1.895±0.060A
0.053±0.017AB
0.189±0.045B
0.030±0.008B
0.113±0.040C
1.75±0.04089
3.16±2.116A
Testicular dimensions cm
0.018±0.10
0.307±0.3213A
0.272±0.3310 B
0.033±0.2102C
Testicular density g/cm3
0.0114±0.120
0.0183±1.264A
0.0203±0.216B
0.0120±0.155C
2.60±1.000B
2.50±0.489B
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Table 3: The Spermatogram parameter of male mice under loading doses of watermelon, citrulline and lycopene
for 38 days.
Control
Sperm concentration 106/ml
11.99±0.70
Sperm viability%
85.77±1.36
Total spermabnormalities%
16.53±0.614
Primary%
7.05±0.58
Secondary%
9.37±0.872
Acrosome%
5.86±0.010
Watermelon
500mg/kgB.w
18.71±0.42
A
94.18±1.52
A
3.72±0.131
A
0.61±0.101
A
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A
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A
Citrulline
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5mg/Kg.Bw
17.31±0.22
BA
91.02±1.46
BA
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B
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B
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B
0.17±0.0031
B
16.66±0.97
B
90.10±0.53
CB
5.79±0.083
C
0.27±0.22
C
4.08±0.011
B
3.39±0.0047
C
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Table 4: The turbidimetric analysis of sperm motility parameter of male mice under loading doses of
watermelon, citrulline and lycopene for 38 days.
Control
Lag time sec.
Fraction of rapidly moving sperm
Velocity µm/sec
motility index %
Watermelon
500mg/kgB.w
1.88±0.0062
0.69±0.0095
A
0.0174±0.0010 0.1557±0.0037
A
9.07±0.176
15.09±1.08
A
Citrulline
30mg/Kg.Bw
0.85±0.013
B
0.1408±0.0026
B
14.81±1.19
B
Lycopene
5mg/Kg.Bw
0.92±0.017
C
0.0819±0.0058
C
11.57±0.210
C
1.20±0.048
2.44±0.039
B
2.00±0.761
C
2.97±0.067
A
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
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1.
2. Hiroyo, Awaji
and Rieko (2007)
Adverse Effect of
Dietary habits on
menstrual disorder in young women . The
Open Food Science Journal., 1: 24-30.
3. Reiner, Winkler (2009) Middlebury
Natural Foods Cooperative, Dedicated to
the Health and Wellbeing of the Whole
Community. MNFC Newsletter A Monthly
Publication of the Middlebury Natural
Foods Cooperative.
4. Dahl and Jeffrey, H.(2010) Antioxidant
Effects and Metabolism of Lycopene .
Ealth sciences center :1-133.
5. G., Riccioni; B., Mancini; E., Di Ilio; T.,
Bucciarelli and N., D’Orazio (2008)
Protective effect of lycopene in cardio
vascular disease European Review for
Medical and Pharmacological Sciences.,12:
183-190.
6. S.,Osowska; C., Moinard; C., Loï; N.,
Neveux and L., Cynober (2004) Citrulline
increases arginine pools and restores
nitrogen balance after massive intestinal
resection. December; 53(12): 1781–1786.
7. Collins, J.K.; Wu, G.; Perkins-Veazie,
P.; Spears, K.; Claypool, P.L.; Baker, R.A.
;
Clevidence,
B.A.)2007)Watermelon
consumption increases plasma arginine
concentrations in adults. Nutrition
(burbank, los Angeles country,
califurnia) . Mar.,23(3):261-6.
g
body weight changes
8
6
4
2
0
A
B
C
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
control watermelon citrulline lycopene
Figure 1:The body weight changes parameter of male mice under loading doses of watermelon, citrulline and
lycopene for 38 days.
Table 2:The testicular morphometrical in male mice under loading doses watermelon, citrulline and lycopene for
38 days.
Control
Watermelon
500mg/Kg.Bw
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
Testicular weight g
Testicular
weight/body
weight ratio
Testicular volume cm3
0.020±0.001
0.077±0.020
0.058±0.216 A
1.895±0.060A
0.053±0.017AB
0.189±0.045B
0.030±0.008B
0.113±0.040C
1.75±0.04089
3.16±2.116A
Testicular dimensions cm
0.018±0.10
0.307±0.3213A
0.272±0.3310 B
0.033±0.2102C
Testicular density g/cm3
0.0114±0.120
0.0183±1.264A
0.0203±0.216B
0.0120±0.155C
2.60±1.000B
2.50±0.489B
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Table 3: The Spermatogram parameter of male mice under loading doses of watermelon, citrulline and lycopene
for 38 days.
Control
Sperm concentration 106/ml
11.99±0.70
Sperm viability%
85.77±1.36
Total spermabnormalities%
16.53±0.614
Primary%
7.05±0.58
Secondary%
9.37±0.872
Acrosome%
5.86±0.010
Watermelon
500mg/kgB.w
18.71±0.42
A
94.18±1.52
A
3.72±0.131
A
0.61±0.101
A
2.50±0.045
A
0.23±0.0028
A
Citrulline
30mg/Kg.Bw
Lycopene
5mg/Kg.Bw
17.31±0.22
BA
91.02±1.46
BA
4.81±0.079
B
0.43±0.30
B
3.99±0.072
B
0.17±0.0031
B
16.66±0.97
B
90.10±0.53
CB
5.79±0.083
C
0.27±0.22
C
4.08±0.011
B
3.39±0.0047
C
Values are presented as mean ±SE
Letter p<0.05 significant different from other groups, n=10
Table 4: The turbidimetric analysis of sperm motility parameter of male mice under loading doses of
watermelon, citrulline and lycopene for 38 days.
Control
Lag time sec.
Fraction of rapidly moving sperm
Velocity µm/sec
motility index %
Values are presented as mean ±SE
Watermelon
500mg/kgB.w
1.88±0.0062
0.69±0.0095
A
0.0174±0.0010 0.1557±0.0037
A
9.07±0.176
15.09±1.08
A
Citrulline
30mg/Kg.Bw
0.85±0.013
B
0.1408±0.0026
B
14.81±1.19
B
Lycopene
5mg/Kg.Bw
0.92±0.017
C
0.0819±0.0058
C
11.57±0.210
C
1.20±0.048
2.44±0.039
B
2.00±0.761
C
2.97±0.067
A
Letter p<0.05 significant different from other groups, n=10
Conclusion
The major aim of this thesis was to determine the endorse of watermelon
nutrient capcitation and maximized functional property of male reproductive
performance during loading dose of alcoholic extract of watermelon in mice model, to
ultimately design a synergistic strategy for watermelon main active ingredient
citrulline, lycopene or other constituted vitamin, Carbohydrate and trace salt. In the
last 38 days, this study was developed and clarify supported boosting idea. The
watermelon administration and citrulline-L-arginine-Nitric oxide pathway on
spermatogenesis and spermatogram and motility indecies.
Characterize an upregulation of spermatogenic cells yield in watermelon mice
model as a synergestic yield of lycopene and citrulline, experiment joined and
determined the sexual hormone levels and their reflection on tissue morphometric and
stereological profile of peritubular and seminiferous tubule testes. Finally watermelon
and their active ingredient treated group play a DNA keeper and attendant in
epididymis extracted spermatozoa. That obviously marked reduction of oxidative
stress.
The endpoint increase fertility rate and refereed the approach compatibility the
watermelon derived citrulline and lycopene on male productivity of superior testicular
fitness and fertilized semen.