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British Journal of Pharmaceutical Research
6(1): 35-43, 2015, Article no.BJPR.2015.047
ISSN: 2231-2919
SCIENCEDOMAIN international
www.sciencedomain.org
Ameliorative Potential of Methanolic Extract of
Citrullus lanatus (Watermelon) Seeds on the Sperm
Parameters, Testosterone Level and Testicular
Cytoarchitecture of Male Albino Rats Induced with
Lead-Acetate
Onyeso Godspower
1
, Nkpaa Kpobari Williams
2*
and Nwaka Elochukwu
1
1
Department of Physiology, Faculty of Basic Medical Sciences, College of Medical Sciences,
Madonna University, Elele, Rivers State, Nigeria.
2
Department of Biochemistry (Toxicology Unit), Faculty of Chemical Science, College of Natural and
Applied Science, University of Port Harcourt, P.M.B. 5323, Choba, Rivers State, Nigeria.
Authors’ contributions
This work was carried out in collaboration between all authors. Author OG designed the study, wrote
the protocol, carried out the statistical analysis and wrote the first draft of the manuscript. Author NKW
managed the literature searches and experimental analysis and author NE also managed the
experimental process. All authors wrote, read and approved the final manuscript.
Article Information
DOI:10.9734/BJPR/2015/15358
Editor(s):
(1)
Wenbin Zeng, School of Pharmaceutical Sciences, Central South University, Hunan, China.
Reviewers:
(1)
Armando Zarrelli, Department of Chemical Sciences, University Federico II, Complesso Universitario Monte S. Angelo, Italy.
(2)
Ahmed E. Abdel Moneim, Department of Zoology, Helwan University, Egypt.
Complete Peer review History:
http://www.sciencedomain.org/review-history.php?iid=982&id=14&aid=8104
Received 22
nd
November 2014
Accepted 31
st
December 2014
Published 10
th
February 2015
ABSTRACT
The study aimed to evaluate the ameliorative potential of methanolic extract of Citrullus lanatus (C.
lanatus; watermelon) seeds on lead-acetate induced testicular toxicity considering the sperm
parameters, testosterone level and testicular cytoarchitecture on adult male albino rats. The results
showed statistically significant (p<0.05) decrease in serum level of testosterone and a deleterious
effect on the sperm motility, count, morphology, viabilityand seminiferous tubular derangement on
lead-acetate treated rats when compared with the control group. The methanolic extract of C.
lanatus seed from the results showed a corrective effect as against the lead-acetate treated group
in relative to the control group. In conclusion it was discovered that methanolic extract of C. lanatus
Original Research Article
Godspower et al.; BJPR, 6(1): 35-43, 2015; Article no.BJPR.2015.047
36
seed has ameliorative potentials to correct the deleterious effect of lead-acetate on male
reproductive system.
Keywords: Citrullus lanatus seeds; lead-acetate; testicular toxicity; sperm parameters; testosterone
level; testicular cytoarchitecture.
1. INTRODUCTION
In recent years, more and more attention is being
paid to the regulation of spermatogenesis and to
the possibilities of influencing therapeutically this
somatic function in a positive or negative way.
However, human fecundity appears to be on the
decline [1], which cannot solely be attributable to
an increase in contraception. Rather, a body of
data suggests that poor semen quality and low
sperm count is markedly increasing and is likely
to be a contributing factor [1]. Although many
people still think of infertility as a problem that
affect only women, but in about 40% to 50% of
infertile couples, the man is the sole cause or a
contributing cause of the inability to conceive [2].
Exposure of heavy metals during pregnancy has
been associated with adverse effects on
development of gonads. These substances may
act as testicular toxicants and correspond to
different compounds, which are related to social
habits, life conditions, working hazards or use of
drugs and medicines [3,4]. Although, many
studies have reported the toxic and carcinogenic
effects of metals in human and animals, it is also
well known that these metals form a crucial part
in normal biological functioning of cells [3].
Lead represents a significant ecological and
public health concern due to its toxicity and its
ability to accumulate in living organisms. Earlier
studies have demonstrated that lead can pass
through the blood testis barrier, accumulate in
the testis and/or epididymis and seriously affect
the spermatogonia, primary spermatocytes,
spermatids or spermatozoa (germinal cells
different levels of differentiation) [5,6]. Several
studies assessed the genotoxic effect of lead
acetate (LA) by means of chromosomal
aberrations and micronucleus test. Regarding the
induction of chromosomal aberrations, LA
induced significant increase of aberrant cells and
numerical distortion in bone marrow cells of
Wistar rats [7,8]. Additionally, Aboul-Ela [9]
detected a significant increase of structural
chromosomal aberrations in bone marrow cells
and primary spermatocytes of albino mice
treated with LA. In addition, LA proved to be a
potent micronuclei inducer in vivo and in vitro test
systems: LA induced micronuclei in kidney cells
of Sprague–Dawley albino rats [10]; in human
melanoma cell [11]; in Chinese hamster V79
cells [12,13]; in Wistar rats’ leukocytes,
reticulocytes and erythrocytes [14,15]; in rats’
erythrocytes [16-18]; in peripheral blood
erythrocytes tissues, gill and fin epithelial cells of
Carassius auratus [19] and in bone marrow cells
of Algerian mice [20]. More so, the results of the
studies indicated that occupational exposure to
Pb has adverse effects on sperm parameters
(decreased sperm counts, lower and a lesser
motility and altered sperm morphology), studies
showed that exposure to inorganic lead greatly
impaired male reproductive functional activities
by decreasing sperm count or distorting sperm
motility and morphology [21].
The fruits of C. lanatus (watermelon) help in
Boosting Antioxidant Levels because it is
exceptionally rich in carotenoids such as
lycopene, lutein and β carotene [22]. A regular
watermelon juice consumption result in
significant increases in blood plasma
concentrations of lycopene and β carotene [23].
Also, lycopene is known to have over 40
potential health benefits and β carotene with
equally plentiful health benefits, which make this
finding very plausible. Moreover, watermelon-
induced is also known to increase plasma
antioxidant levels and may explain why
epidemiological studies of the Chinese found
greater watermelon intake to be associated with
a lower risk of cancer [24]. A study found that 6
weeks of treatment with a watermelon extract
containing 6 grams of L-citrulline and L-arginine
daily on middle-aged obese subjects with
prehypertension or stage 1 hypertension
experienced reduced ankle blood pressure and
altered carotid wave reflection, an indication of
improved arterial function of the individuals [25].
If watermelon can cure or ameliorate this
process, it would certainly provide a
breakthrough to many of the drugs used in the
market for primary prevention, such as the
cholesterol-lowering statin drug class, whose
side effects are numerous [26].
It is very important to note that all parts of the
watermelon have something to offer. For
Godspower et al.; BJPR, 6(1): 35-43, 2015; Article no.BJPR.2015.047
37
example, the seeds are excellent source of
protein. The good nutritional and functional
properties of watermelon seed meal proteins
suggest their potential use in food formulations
and diets [27]. C. lanatus possesses numerous
bioactivities from natural source which is of better
advantage than conventional therapies. This
study aimed to evaluate the ameliorative
potential of methanolic extract of C. lanatus
(watermelon) seeds on Lead-acetate induced
testicular toxicity considering the sperm
parameters, testosterone level and testicular
cytoarchitecture on adult male albino rats.
2. MATERIALS AND METHODS
2.1 Experimental Animals
A total number of 20 male Albino rats weighing
between 155 to 328 grams were used for the
study. The animals were bought from the animal
house of the Department of Pharmacology and
Toxicology, located in Niger Delta University
Wilberforce Island. It was kept in the animal
house of Department of Human Physiology
Madonna University, Elele Campus for 3 weeks
to acclimatize. The animals were kept under
normal room condition of temperature 25±2°C,
humidity of 50±5% and 12 hour light and dark
cycles. The animals were randomized into
experimental and control groups and housed in
sanitized wooden cages containing saw-dusts as
bedding. They were also fed with standard rat
chow pellet as diet and clean water ad-libitum
was supplied.
2.2 Seeds Collection and Preparation
Ripe watermelon pods were obtained from the
local market in Elele (Eke-Onuma Market),
Rivers State, Nigeria (May, 2013). The seeds
extracted from the pods, only healthy looking
seeds were collected. The collected seeds were
oven-dried at 35°C, to a constant weight. The
dried seeds were reduced into fine powder using
a Laboratory grinding hand mill. The powder was
weighed and kept away from light before
extraction.
2.3 Seeds Extraction and Concentration
Extraction was by maceration over a 72 hours
period. 500 g of the powdered seeds material
was extracted with 1.5 liters of Methanol in 3
successive extractions (500 ml every 24 hours).
The jar was tightly closed and thoroughly shaken
intermittently. After 72 hours, the different
portions were combined and filtered using filter
paper. The filtrate was collected in a glass jar.
The extract was concentrated using a Rotary
Evaporator. The concentrated methanolic extract
of Citrullus lanatus seeds were then transferred
into bottles covered with aluminium foil and
stored in a refrigerator at 4°C before use. This is
to prevent it from losing its potency.
2.4 Experimental Procedure
On commencement of the experiment, the
animals were divided into 4 groups of 5 animals
each. The first group served as the control group
while the last 3 groups served as the
experimental groups.
2.4.1 Group 1
This group was the control group fed normal rat
chow and water.
2.4.2 Group 2
This group was fed normal rat chow and water
and received 2.25 mg/kg of Lead-acetate + 100
mg/kg (Low dose) of methanolic extract of C.
lanatus seeds.
2.4.3 Group 3
This group was fed normal rat chow and water
and received 2.25 mg/kg of Lead-acetate + 200
mg/kg (High dose) of methanolic extract of C.
lanatus seeds.
2.4.4 Group 4
This group was fed normal rat chow and water
ad-libitum and they received 2.25 mg/kg Lead-
acetate only.
2.5 Analytical Procedure
After 30 days of treatment, the animals were
fasted for 24 hours prior to sacrifice. The animals
were anaesthetized using chloroform and then
sacrificed. Thus, blood was obtained via cardiac
puncture and put in labeled EDTA bottles for
testosterone assay. The animals were then
dissected; the testes were removed along with
the caudal epididymis. The caudal epididymis
was separated from the testes and lacerated to
collect the semen with a microscope glass slide
for analysis of sperm characteristic and the
Godspower et al.; BJPR, 6(1): 35-43, 2015; Article no.BJPR.2015.047
38
testes for testicular cytoarchitecture
(histopathology).
2.5.1 Estimation of serum testosterone
Quantitative measurement of serum total
testosterone was carried out adopting ELISA
technique using kits specific for rats purchased
from Department of Pharmacology and
Toxicology, Niger Delta University Wilberforce
Island, Nigeria according to the protocol provided
with kit.
2.5.2 Histological examination
The testes tissues were collected and
immediately fixed in 1 ml PBS (pH 7.4) and
embedded in paraffin. Sections (5-µm) were
prepared and then stained with hematoxylin and
eosin (H & E) stain for photomicroscopic
observations.
2.6 Statistics
The result were expressed as mean ± standard
error of mean (S.E.M). The statistical evaluation
of data was performed by using a one-way
ANOVA (analysis of variance). The data were
considered significant at p˂0.05.
3. RESULTS
As shown in Table 3.1, the level of active sperm
cells in Lead-acetate + HDE of C. lanatus seed
(65.00±2.88%) was significantly higher (p<0.05)
than the Lead-acetate only (11.66±7.26%). The
level of dead cells increased significantly
(p<0.05) in Lead-acetate only rats
(81.67±10.14%) but showed no significant
difference (p<0.05) in the sluggish sperm cell
levelsthough it increased (18.33±1.66%) in the
Lead-acetate + LDE administered with C. lanatus
seed.
The total sperm count was significantly (p<0.05)
higher in the Lead-acetate + HDE administered
rats (33.67±2.96X10
6
/ml) compared to the Lead-
acetate administered rats only (7.00±2.08
X10
6
/ml). The level of Sperm morphology (%) in
both Head, Mid-piece and Tail defect increased
significantly (p<0.05) in the Lead-acetate only
administered rats (compared 5.33 ± 2.40%, 2.33
±0.33% and 3.00±0.00% respectively) compared
to the Lead-acetate + HDE administered rats
(which had 0% Mid-piece defect, 0.66±0.33%
and 1.00±0.58% for Head and Tail defect
respectively).
Sperm viability (% viable) showed no significant
difference in either the Lead-acetate + HDE rats
Lead-acetate only rats or control rats. Though
the Sperm viability (% non-viable) of the Lead-
acetate only rats (10.67±2.33%) was significant
higher (p<0.05) as compared to the Lead-acetate
+ HDE albino male rats (1.67±.0.67%). The
Testosterone level of the Lead-acetate + HDE
rats (5.23±0.23ng/ml) was significantly higher
(p<0.05) compared to the Lead-acetate only rats
(0.73±0.12ng/ml).
Histological examination of rat testis in group 1
(control) as shown in Fig. 1 showed the
seminiferous tubules lined with stratified
epithelium composed of two major cells, which
are the sertoli cells and spermatogenic cells.
Seminiferous tubules depict normal architecture
with adequate cellularity; on the other hand,
histological examination in group 4 as shown in
Fig. 2 showed degeneration of the
spermatogenic cells, occlusion of the lumen and
hypertrophied seminiferous tubules. By contrast,
the lead acetate alone group depict shrunken
seminiferous tubules and reduced cellularity,
while group 2 shows increase in the
spermatogenic cells of the rats treated compared
with the control. This group showed a mild
thickening of the basement membrane as noted
in certain seminiferous tubules, but it appeared
generally healthy (Fig. 3). Finally, histological
examination of group 3 showed matured
spermatozoa in the seminiferous tubules of rats
treated. This group has a healthy testicular tissue
simila to the control, but with a slight increase in
the interstitial tissues. This shows that
methanolic extract of C. lanatus seed
administered as a treatment option may be
medicinally beneficial (Fig. 4).
4. DISCUSSION
Several studies show that alkaloids and terpenes
are widely spread in the genus citrullus [28].
These secondary metabolites are responsible for
the pharmacological activities such as antiulcer,
antimicrobial, antioxidant, analgesic, aphrodisiac
and many other ethno-medicinal uses [29].
Godspower et al.; BJPR, 6(1): 35-43, 2015; Article no.BJPR.2015.047
39
Table 3.1. Ameliorative potential of methanolic extracts of C. lanatus (watermelon) seeds on sperm parameters and testosterone level of lead-acetate induced male albino rats
Group
Sperm motility (%)
Sperm count (X10
6
/ml)
Sperm morphology (%)
Sperm viability (%)
Testosterone
(ng/ml)
AC
SC
DC
Sperm Count
HD
MPD
TD
VC
NVC
Testosterone
Control(1)
82.50±2.50 7.50±2.50 10.00±0.00 43. 50±1.50 0.50±0.50 0.00±0.00 0.50±0.50 99.00±1.00 1.00±1.00 2.75±1.45
Lead-acetate + LDE(2)
43.33±6.00 18.33±1.66* 38.33±4.41 20.00±1.55 2.33±1.20 1.00±0.58 1.00±0.58 95.67±0.33 4.33±0.33 1.77±0.52
Lead-acetate + HDE(3)
65.00±2.88* 15.00±5.00 20.0±2.88 33.67±2.96* 0.66±0.33 0.00±0.00 1.00±0.58 98.33±0.67 1.67±0.67 5.23±0.23*
Lead-acetate only(4)
11.66±7.26
6.66±3.33 81.66±10.14* 7.00±2.08 5.33±2.40* 2.33±0.33* 3.00±0.00* 89.33±2.33 10.67±2.33* 0.73±0.12
Data represented as Mean ± SEM; (*) p<0.05 significant difference; LDE=Low dose extract of C. lanatus seeds, HDE=High dose extract of C. lanatus seed; AC: Active cells; SC: Sluggish cells; Dead cells; HD: Head
defect; MPD: Mid-piece defect; TD: Tail defect; VC: Viable cells; NVC: Non-viable cells.
Godspower et al.; BJPR, 6(1): 35-43, 2015; Article no.BJPR.2015.047
40
Fig. 1. Photomicrograph of albino rat testis in group 1
Fig. 2. Photomicrograph of albino rat testis in group 4
Fig. 3. Photomicrograph of albino rat testis in group 2
Godspower et al.; BJPR, 6(1): 35-43, 2015; Article no.BJPR.2015.047
41
Fig. 4. Photomicrograph of albino rat testis in group 3
This study indicates that even moderate
exposure to Lead-acetate can significantly
reduce semen parameters, reduce testosterone
level and cause toxicity to the testis. The
changes observed in the above agree with the
previous reports, which demonstrated that lead
acetate suppressed testosterone, follicle-
stimulating hormone (FSH) and luteinizing
hormone (LH) levels along with testicular
spermatogenesis, showing that lead acts at all
levels of reproduction [30]. The extract
administered resulted to improvement of sperm
characteristics; this may be as a result of the
presence of flavonoids.The presence of steroid in
the phytochemical screening of the crude extract
could be the resultant improved testosterone
level in group 3. The mechanisms by which
methanolic extract of C. lanatus seeds protect
against experimentally induced testicular toxicity
may be as a result of the rich source of vitamin
C, thiamine and including riboflavin which
contains a high level of polyphenolic compounds
present in the plant. High concentration of
vitamin C in C. lanatus seeds provides highly
effective anti-oxidants, reversing the negative
effect caused by the lead-acetate following the
administration of the extracts to the experimental
animal as seen in the group III of the different
parameters when compared to the group 4. This
effect may be influenced by the presence of
flavonoids in the extract which contains
antioxidants. Saponin that was found to be
present in the extract functions majorly at
stimulating an increase in the body’s natural
endogenous testosterone levels which helps to
maintain testosterone levels. C. lanatus seeds
are a rich source of flavonoids and phenol, with
ability to scavenge free radical and inhibit
hydrolytic and oxidative enzymes and anti-
inflammatory action [31]. However, findings from
these results are supported by the fact that
methanolic extracts of C. lanatus seeds contains
a high amount of anti-oxidant activity [32],
bringing about the promising result obtained in
this study.
5. CONCLUSION
Methanolic extracts of C. lanatus seeds has
shown to be a beneficial treatment option against
lead-acetate induced oxidative stress and toxicity
in testicular tissue. It was also deduced from the
study that among the treatment options,
administration of methanolic extracts of C.
lanatus seeds at a high dose on exposure to lead
result to the most beneficial result such as
increased sperm motility, well defined cellularity
of the testis, increased sperm viability, decreased
sperm morphological alterations, increased
sperm count, increased testosterone level. There
is need for further investigations because
methanolic extracts of C. lanatus seeds can be a
potential complimentary agent in treating lead
induced testicular toxicity.
CONSENT
It is not applicable.
ETHICAL APPROVAL
All authors hereby declare that principle of
laboratory animal care was dully followed, as well
as specific national laws where applicable in this
study.
Godspower et al.; BJPR, 6(1): 35-43, 2015; Article no.BJPR.2015.047
42
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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