Protective effect of combined pumpkin seed and ginger extracts on sperm characteristics, biochemical parameters and epididymal histology in adult male rats treated with cyclophosphamide

Abstract

Reproductive toxicity is one of the side effects of cyclophosphamide (CP) in cancer treatment. Pumpkin seeds and Zingiber officinale are natural sources of antioxidants. We investigated the possible protective effect of combined pumpkin seed and Zingiber officinale extracts on sperm characteristics, epididymal histology and biochemical parameters of CP-treated rats. Male adult Wistar rats were divided randomly into six groups. Group 1, as a control, received an isotonic saline solution injection intraperitoneally (IP). Group 2 were injected IP with a single dose of CP (100 mg/kg) once. Groups 3 and 4 received CP plus 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract (50:50). Groups 5 and 6 received only 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract. Six weeks after treatment, sperm characteristics, histopathological changes and biochemical parameters were assessed. In CP-treated rats, motile spermatozoa were decreased, and abnormal or dead spermatozoa increased significantly (P < 0.001) but administration of the mixed extract improved sperm parameters. Epididymal epithelium and fibromascular thickness were also improved in extract-treated rats compared to control or CP groups. Biochemical analysis showed that the administration of combined extracts could increase the total antioxidant capacity (TAC) level significantly in groups 3, 4, 5 and 6. Interestingly, the mixed extract could decrease most of the side effects of CP such as vacuolization and separation of epididymal tissue. Our findings indicated that the combined extracts might be used as a protective agent against CP-induced reproductive toxicity.

Full text

33
February 2014, Volume 11, Number 1
Combined Effect of Ginger and Pumpkin Seed Extracts
on Rat Testis and Serum Biochemical Parameters after
Cyclophosphamide Treatment
Forouzan Mohammadi 1, Hossein Nikzad 1, 2 *, Aliakbar Taherian 2, Mohsen Taghizadeh 3, Abolfazl Azami-Tameh 1, Homayon Naderian 1,
Mohammad Ali Atlasi 1
1. Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran.
2. Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran.
3. Research Center for Biochemistry and Nutrition in Metabolic Disorders. Kashan University of Medical Sciences, Kashan, Iran.
* Corresponding Author:
Hossein Nikzad, PhD
Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran.
Tel/Fax: +98 (31) 55621158
E-mail: nikzad_h@kaums.ac.ir
Introduction: Cyclophosphamide is a chemotherapy drug with several side effects on various
organs such as male reproductive system that can cause infertility. In this study, we assessed
combined effect of ginger and pumpkin extractson rat testis after CP injection.
Methods: Forty adult male rats were randomly divided into 4 groups: The control group
received intraperitoneal injection of isotonic saline solution. The cyclophosphamide (CP) group
received a single dose of cyclophosphamide (100 mg kg_1BW) intraperitoneally. Combined
extracts (ginger+ pumpkin) group received orally 300 mg combined extracts and combined
extracts(ginger+ pumpkin) +CP groups received orally 300 mg combined extracts for a period
of 6 weeks after CP injection.
Results: Our results showed that although ginger extract could not change testis weight,
testosterone, malondialdehyde (MDA) and ROS, antioxidant level in serum was increased
signicantly. Epithelium thickness and tube diameter were decreased in combined groups with
or without CP in comparison to control group. The combined extract could improve histological
changes in both combined extract and combined extract+ CP compared to CP group, which
could be attributed to the higher serum level of antioxidants.
Conclusion: The administration of combined extracts can increase the serum antioxidant level
and decrease the side effects of CP on testis.
A B S T R A C T
Article info:
Received: 14 Aug 2013
Accepted: 24 Dec 2013
Key Words:
Testis,
Ginger,
Pumpkin,
Cyclophosphamide,
Biochemical parameters.
Hossein Nikzad is an academic member (Professor) in Kashan University of Medical Sciences and teaches anatomy and
embryology courses for medical and paramedical students for 20 years. His interest research eld is clinical embryology,
IVF, reproducon and androloy. Unl now, he published more than 40 manuscript at the naonal and internaonal jour-
nals. Now, he is head of gametogenesis research center in Kashan University of Medical Sciences.

34
February 2014, Volume 11, Number 1
1. Introduction
n inability to conceive after 12 months of
sexual practice without using any contra-
ception is dened as infertility[1].World
Health Organization reported that 10–
15% of young couples are faced with in-
fertility and each gender shows 50% of the related caus-
ing factors[2]. It has been reported that environmental
factors such as pesticides, exogenous oestrogens, heavy
metals and chemotherapy are the reasons for declin-
ing male sperm count which may have a negative im-
pact on male fertility[3]. cyclophosphamide (N, N-bis
(2-chloroethyl) tetrahydro-2H-1, 3, 2-oxazaphospho-
rin-2-amine2-oxide), a cytotoxic alkylating agent is
a nitrogenous mustard belonging to the group of cy-
totoxic or cytostatic drugs[4]. Studies have shown
that generation of free radicals and reactive oxygen
species isassociated with CP treatment as well. It is
known that CP disrupts the redox balance of tissues
resulting in oxidative stress. It has been reported that
oxidative DNA damage is caused by hydroperoxide
derivatives of CP through generation of H2O2.Also,
acrolein, another component of CP, has been found to
interfere with the tissue antioxidant defense system
and produces highly reactive oxygen free radicals,
which are mutagenic to mammalian cells[5].Lipid per-
oxidation has been suggested to be closely related to
CP-induced testicular damage, and malondialdehyde
(MDA) is a good indicator of lipidperoxidation that
could induce sperm abnormality[4].CP treatment is
associated with oligozoospermia and azoospermia, as
well as biochemical and histological alterations in the
testis and epididymis of human and rats [6, 7].Further-
more, disturbance in gonadotropin secretion, testicular
damage, and decreased plasma testosterone levels are
found in patients enduring treatment with CP[8]. Me-
dicinal herbs have been popular among people from
ancient times, and in recent years, a new interest has
emerged to use medicines with natural and especially
herbal origin like pumpkin and ginger [9]. Medicinal
plants contain phytochemicals and numerous chemi-
cal compounds, which can be implemented in phar-
macology by isolating the active compounds to gen-
erate new medicines and provide alternative healing
methods[10]. In most cases, herbal medicine offers
less invasive and less costly physical and emotional
treatment compared with other procedures.
Ginger rhizome (ZingiberofcinaleR., family:
Zingiberaceae) is used worldwide as a spice. Both
antioxidative and androgenic activity of ginger have
been reported in animal models[11].It contains sev-
eral compounds including acids, resins, vitamin C
compounds, folic acid, inositol, choline, pantothenic
acid,gingerol, sesquiterpene, vitamin B3 and B6 vol-
atile oils and bio-trace elements like Ca, Mg, P and
K[12]. This plant has been considered a safe herbal
medicine with few side effects[9].Ginger has been pre-
viously shown to stimulate spermatogenesis[12]. Mor-
akinyo et al. (2008)suggested that ginger extract (500
and 1000 mg kg_1B.W doses)has a benecial effect
on male reproductive functions in rats, which is con-
rmed by other studies showing an increase in sperm
count, motility,testosterone, antioxidant enzymes, su-
peroxide dismutase (SOD), catalase, glutathione per-
oxidase (GSH) and a decrease in malonhydialdehyde
lipid peroxidation levels [13, 14].We recently showed
that ginger extract at doses of 300 and 600 mg/ kg BW
has a positive effect on recovery of spermatogenesis
in adult rats after cyclophosphamide (CP) treatment.
Also, we found that co-administration of this extract
with CP can counterbalance the negative effect of CP
on testis parameters demonstrated in our study[1].
Pumpkin (cucurbitapepo var. styriaca) family cucur-
bitacea is an important leaf and seedvegetable tropical
vine grown and highly reputed in traditional medicine
and largelyconsumed in many countries such as Iran
[15]. The seeds are a rich natural source of proteins,
phytosterols, polyunsaturated fatty acids, phytochemi-
cals, sterols, antioxidant vitamins such as carotenoids
and tocopherol and trace elements such aszinc and
selenium[4].It has been demonstrated that pumpkin
seeds and daily rich diet of zinc can decrease the un-
desirable side effect of lead contaminants and improve
the sexual health status[16].Pumpkin seeds improve
sexual stimulation and intromission and ejaculatory
latency[17]. Pumpkin causes a signicant reduction in
sperm count with primary and secondary abnormali-
ties by producing further zinc and protein. Therefore,
pumpkin is proposed for both the prevention and treat-
ment of infertility in male animals[18].The ndings of
our recent study indicated that pumpkin seed extract
could recover the side effects of CP, epididymis histol-
ogy and sperm parameters through preventing oxida-
tive stress[4].
Although it is now broadly accepted that ginger and
pumpkin seeds have a positive effect on fertility, this
study is the rst study that evaluates the effects of the
combined extracts of ginger and pumpkin seeds on
CP-treated rat testis. Therefore, the present study was
designed to investigate any possible protective effects
A

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February 2014, Volume 11, Number 1
of the combined extracts of ginger and pumpkin on
biochemical parameters and testicular histology of
CP-treated male rats.
2. Materials and Methods
Cyclophosphamide was purchased from Baxter On-
cology GmbH, Frankfurt, Germany. Pumpkin seeds
(Cucurbita Pepo var. Styriaca) were purchased from
local Iranian markets. Ginger was obtained from Nat-
ural Remedies Company in India that has been stan-
dardized as ‘total Gingerol 5%’.MDA, ROS, antioxi-
dant and testosterone kits were purchased from Glory
Science Co. Ltd, China.
Animal experiment :In this study, forty healthy adult
male Wistar rats (8–10 weeks old, 300–350 g) were
obtained from Kashan University of Medical Sciences.
Rats were in wire-wooden cages under controlled light
schedule (12 h light and 12 h darkness). The animals
were allowed to acclimatize for a period of 7 days be-
fore starting the experiment. During the treatment pe-
riod (Six weeks) they were fed with the supplied food
pellets and had free access to water. All experiments
were implemented in accordance with the guidelines
and were approved by the Local Committee on Animal
Research in Kashan University of Medical Sciences.
Study design and treatment: The rats were randomly
divided into four groups of ten. Control group received
a single intraperitoneally injection of isotonic saline
solution (1 ml). CP Group received a single dose of
cyclophosphamide (100 mg kg_1body weight)intra-
peritoneally[19].Group ginger +pumpkin 300received
300 mg/kg BW of ginger extract plus pumpkin seed
extract. Group CP+ginger +pumpkin 300 received CP
plus300 mg/kg BW ginger and pumpkin seed extract
orally for a period of 6 weeks after CP injection.
Food regimens: The synthetic diet was purchased
from Behparvar Company in Iran. Pumpkin seed with-
out oil (cold pressed oil) was obtained from Barij Es-
sence Company in Kashan, Iran. The pumpkin seed
without oil was mixed with 70% alcohol in a blender
and incubated for 72 hours at room temperature. Then
ethanol liquid was separated with a lter paper. The
extract was standardized to contain amino acid, total
avonoid and total phenolic compound concentra-
tion[20, 21]. One kg of the synthetic diet was mixed
with 512cc of pumpkin seed extract (2.6%) for 300
mg/kg body weight dosage. Also, ginger extract was
dissolved in 50% ethanol and with pumpkin seed ex-
tract, was added to powdered food pellets of rats. The
mixture was dried in oven under 50°C and stored at
4°C. The animals were fed based on a daily consump-
tion of 15-17 gr of dried diet/rat. Before starting the
experiment, we measured the food intake in control
and CP-treated rats. In case of CP-injected rats, the
food intake was decreased to about 8 grams in the rst
5 days after injection. So, the ginger+ pumpkin content
of the food was adjusted to give the right dose during
the experiment. Fresh diet was prepared weekly.
Sample collection: Since spermatogenic period in
rats is 48 days[22], in our study, rats were weighed and
killed under anaesthesi a 42 days after CP injection.
Blood samples were collected from the left ventricle,
and plasma was immediately separated for biochemi-
cal examinations. After weighing, testis was xed with
Bouin’s solution for histology.
Biochemical studies: The level of rat testosterone, to-
tal antioxidant capacity (T-AOC), MDA and reactive
Groups
Parameters Control CP Combined Extract 300 CP+300 Combined Extract
Tube diameter(µm) 286.20±13.74 270.80±19.81 253.18±35.6* 245.57±18.72*
Epithelium thickness (µm) 152.79±7.21 131.16±6.60† 114.60±6.50† 111.42±2.8†
Lumen diameter(µm) 133.41±7.93 139.65±18.21 139.99±31.02 134.14±18.20
*P < 0.05 compared with control group.
†P < 0.001 compared with control group.
Table 1. Administration effect of the combined extracts of ginger and pumpkin seeds on histological parameters in rat
seminiferous tubes.

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February 2014, Volume 11, Number 1
oxygen species were determined using an enzyme-
linked immunosorbent assay (ELISA) method. Blood
was collected from left ventricle of anaesthetized rats
in a tube and centrifuged Immediately at 3000 rpm for
10 min. The separated serum was deposited in -20 °C
freezers for the biochemical tests. The biochemical
tests were carried out according to the kit instruction.
Briey, a series of standard solutions were prepared.
First, the primary antibody was added to the sample
wells of a 96-well plate (not to control or standard
wells). 40 ul of samples or 50 ul of standard solutions
were added to the designated wells. After adding the
secondary antibody (labelled with Strep-tavidin-HRP)
to the standard and sample wells, the plate was incubat-
ed at 37 °C for 60 min. Plate was washed;chromogenic
solutions (A, B) were added and incubated at 37 °C in
the dark for 10 min. To the control wells, only chro-
mogenic and stop solutions were added. After adding
the stop solutions to the wells, the absorbance of wells
at450 nm wavelengths was read by an Elisa Reader
(ModelStatFax 2100). A standard curve was extrapo-
lated based on the readings of the standard wells, and
linear regression equation was calculated. The concen-
tration levels of samples were calculated based on OD
readings of the samples and the standard curve.
Histology and light microscopy: The left testis was
carefully dissected, trimmed of all fats and blotted dry
to remove any blood. The testis was divided into three
parts, and the middle section was xed in Bouin’s uid
for 48 h. The xed samples were dehydrated in graded
levels of ethanol, cleared in xylene and embedded in
parafn wax for sectioning. Five μm thick sections
were prepared and stained with Hematoxylin and Eo-
sin (H&E) and observed under a light microscope.
Morphometric study:An optical microscope (Zeiss/
German) with an objective lens ×40 was used for cell
counting. Spermatogonia, primary spermatocyte, sper-
matid, spermatozoa and Sertoli cells were counted in
10 seminiferous tubules in stages VII or VIII in each
animal. The diameter of the round or nearly round
seminiferous tubules was estimated as the average of
two perpendicular longer and shorter diameters. Epi-
thelium thickness was obtained by the same method.
The size of the lumen was calculated by subtracting
epithelium thickness from the diameter. A mean value
of each of these parameters was calculated for each
group and was compared with the other groups.
Statistical analyses: The data are presented as mean
± SEM (standard error of mean). Statistical analyses
were carried out using ANOVA test and a P value of P
<0.05 was considered as statistically signicant.
3. Results
Morphometry: Testicular weight did not change sig-
nicantly between the control and other groups. The
general effects of CP were some hair loss and a de-
creased appetite in the rst5 days after CP injection
only seen in CP group. CP treatment decreased the
number of germ cells as well as epithelium thickness of
seminiferous tubules (Tables 1 and 2). Other features
Groups
Parameters Control CP Combined Extract 300 CP+300 Combined Extract
Spermatogonia(n) 63.15±8.15 51.55±3.51† 64.17±4.8†† 61.50±5.04††
Spermatocytes(n) 74.20±11.16 57.24±2.15* 71.03±6.5†† 71.41±4.5††
Spermad(n) 230.0±38.27 196±4.5 211.9±15.7 201.74±13.9
Sperm(n) 180.07±5.5 161.11±4.6†† 190.2±9.5*†† 175±4.57††
Sertoli(n) 17.75±3.89 15.22±2.99 18.82±2.81 18.56±1.47
*P < 0.05 compared with control group.
†P < 0.001 compared with control group.
††P < 0.001 compared with CP group.
Table 2. Administration effect of the combined extracts of ginger and pumpkin seeds on cell count of germ and Sertoli cell
parameters in rat seminiferous tubes.

37
February 2014, Volume 11, Number 1
of the CP-treated seminiferous tubules were exfolia-
tion of germ cells and vacuolated appearance of the
epithelium. The administration of combined extract to
CP-treated or normal rats could signicantly increase
germ cells count in seminiferous tubules (spermato-
gonia, spermatocytes, sperm) compared to CP group.
However, epithelium thickness and tube diameter were
decreased in combined groups with or without CP in
comparison to control group.
Biochemical Parameters
Antioxidant levels: Cyclophosphamide treatment
did not change the antioxidant level signicantly in
comparison with the control group. However, admin-
istration of the combined extracts 300 mg kg_1 BW_1
strongly increased antioxidant levels compared with
that of control or CP group (Table 3).
Testosterone, ROS and MDA: Testosterone, Ros and
MDA levels did not change signicantly in the differ-
ent groups (Table 3).
Histology: Cyclophosphamide treatment caused
a reduction in the size, epithelium thickness and the
number of different types of cells in the seminiferous
tubules. Degeneration, vacuolation and exfoliation of
germ cells into the lumen of seminiferous epithelium
were other features of the CP group samples. However,
administration of the combined extracts300 mg kg_1
BW_1to CP group rats and rats without CP caused an
improvement in the germ cells count of the seminifer-
ous tubules compared with the CP group but epithe-
lium thickness and tube diameter decreased in com-
parison to control group in these groups.
4. Discussion
Using chemotherapy drugs like CP for cancer treatment
is limited by their side effects. The side effects of che-
motherapy include reproductive toxicity that has been
documented in different studies[19].This study was
completed to investigate the effects ofthe combined
extracts of ginger plus pumpkin seeds on CP-injected
and normal rat testis. To our knowledge, this is the
rst study investigating the effects of this combined
extract against testicular damage caused by CP in rats.
Our results showed that in combined extract-treated
rats (300 mg kg_1BW_1), the number of germ cells
in seminiferous tubules was increased signicantly in
comparison to CP group alone. This result reconrms
our last report indicating that administration of ginger
or pumpkin increases the number of germ cells in sem-
iniferous tubes and has a positive effect on recovery of
spermatogenesis in adult rats after cyclophosphamide
(CP) treatment[1, 23].
Epithelium thickness and tube diameter decreased
in comparison to control group in these groups. Our
results support those of[24] Saalu reported decrease
in tubular diameter and epithelium thickness after ad-
ministration of uted pumpkin extract 400 mg/kg/day/
oral and our result in another study where we found
that administration of ginger extract 300 mg/kg/day/
oral decreased epithelium thickness in comparison to
control group [1].
In our study, the weight of the testis did not change
signicantly between the different groups. A decreased
testis weight has been reported in CP-treated rats [25].
In that study a dose of 15 mg kg_1 BW of CP was
Groups
Parameters Control CP Combined Extract 300 CP+300 Combined Extract
MDA(nmol/ml) 2.42±0.46 2.71±0.42 2.05±0.56 2.50±0.40
ROS 3.40±0.85 2.60±1.03 4.17±0.67 4.24±0.56
Anoxidant(µl) 5.51±3.52 5.20±2.43 11.97±3.6*†† 11.92±3.22*††
Testosterone(ng/dl) 7.38±2.09 8.39±1.43 9.45±1. 93 2.06 ±7.93
*P < 0.05 compared with control group.
††P < 0.001 compared with CP group.
Table 3. Administration effect of the combined extracts of ginger and pumpkin seeds on serum levels of testosterone, anti-
oxidant, Malondialdehyde (MDA) and ROS in rat seminiferous tubes.

38
February 2014, Volume 11, Number 1
given to rats by oral gavage once a week for 10 weeks
(in total 150 mg kg _1BW_1), while in our study, 100
mg kg_1 BW_1 of CP was injected in a single dose.
The chronic low-dose administration of CP to male
rats could be the reason for decreased reproductive or-
gan weights[26]. In another study, two different doses
of CP (100 and 200 mg kg_1) were injected to male
rats. A decrease in the weight of testis was detectable
1 week after the injection for both doses, but after 5
weeks, the reduced weight of testis was only detect-
able in 200 mg kg_1injected rats[27]. In our study, the
samples were collected and studied 6 weeks after the
injection of 100 mg kg_1 of CP; therefore, we could
not see any difference in testis weight. Of course, the
reason that samples were studied after 6 weeks was
that spermatogenesis (development of mature sperma-
tozoa from diploid spermatogonial cells) in rats takes
48 days[22].
In CP treated rats the toxic effect was indicated by
signicant reduced spermatogonia,spermatocyte and
sperm count. Our results support those studies report-
ing irregular and diminished seminiferous tubules
containing only a few germ cells in the CP group[19].
Our results provided no evidence for those studies
[19]which reported that MDA and ROS levels were
increased signicantly in CP-treated rats. Since we ex-
amined the rats 42 days after chemotherapy, probably
the levels of these parameters have been recovered in
the rst or second weeks of the experiment. This re-
covery most probably has been done because of the
effective help of the mixed extract.In our study, tes-
tosterone, ROS and MDA levels neither changed sig-
nicantly in CP group nor in combined extract -treated
groups (with and without CP).
Our results showed that the combined extract with
dose (300 mg kg_1 BW_1) could increase antioxidant
to a higher level. This result conrms the antioxidant ef-
fects of Telfairia occidentalis (uted pumpkin) extracts
reported by Nwanna and Oboh[28] and zingiberreport-
ed by Morakinyo[12]. Antioxidant therapy improves
fertility parameters through a protective mechanism
against oxidative stress[11].Phenolic compounds such
as polyphenols avonoids as well as vitamins and zinc
in medicinal plants such as ginger and pumpkin are
attributed factors for antioxidant activity [4, 12]. In
studies by Tsai et al. (2006) and Gossell-Williams et
al.(2006), it was reported that Pumpkin seed oil is rich
in many powerful antioxidants and useful nutritional
supplements such as essential fatty acids and polyun-
saturated fatty acids including linoleic acid,oleic acid,
palmitic acid, omega 3, 6 and 9, carotenes, lutein,
gamma and P-tocopherols,phytosterols, chlorophyll,
selenium and zinc[29, 30].Also, the presence of oleic
acid, amonounsaturated fatty acid in pumpkin reduces
the susceptibility of the testis and epididymis to lipid
Peroxidation[31].Zinc in pumpkin seeds is an essen-
tial trace mineral that acts as an antioxidant by neu-
tralizing free radical generation. Also,Zinc could play
a direct antioxidant action by engrossing the iron or
copper binding sites of lipids, proteins, and DNA[14].
On the other hand,all major active ingredients of Z.
ofcinale such as Zingerone, Ginger¬diol, Zingibrene,
gingerols and shogaols, have antioxi¬dant proper-
ties[9]. Besides, other studies have shownthat ginger
oil has a protective effect on DNA damage against Hy-
drogen Peroxide (H2O2) and might decrease oxygen
radical and could be used as an antioxidant[32, 33].
In previous studies, we showed that the higher dose
of ginger extract (600 no 300mg kg_1 BW_1) alone
could increase testosterone level. Because only the
higher dose of ginger could induce the higher level of
testosterone. Ginger extract might act in a dose-depen-
dent manner[1].In our previous work, only the lower
dose (300 mg/kg) of pumpkin seed showed antioxidant
activity but not the higher dose (600 mg/kg), which
even increased serum free radical level. Based on these
results, the combined extract was prepared from the
lower dose of each plant. Since in Iranian family gath-
erings normally both pumpkin seeds and ginger a-
vored tea are served, we decided to study the effects
ofthe combined extract on testis after chemotherapy.
Conclusion
The combined extract of ginger and pumpkin (300
mg kg_1 BW_1) has an antioxidant activity and thus
can reduce the adverse effects of CP in testis. Also,
these results suggest further studies to evaluate the use
of the combined extracts of ginger and pumpkin as a
supplement drug to counterbalance the negative effect
of CP in human as well.
Acknowledgment
This research is nancially supported by the Deputy
for Research of Kashan University of Medical Sci-
ences (Kaums) and Kashan Anatomical Sciences Re-
search Center (Grant No 9110).

39
February 2014, Volume 11, Number 1
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