Effect Of Vitis Vinifera Leave Hydro-Alcoholic Extract On Reproductive Parameters In Adult Normal Male Rats

Abstract

Grape leaves are used in a traditional food and for treatment of diarrhea, bleeding; and also reported that administered grape was good to treatment of constipation, gout and etc. So, the aim of this study was to investigate the decrease effect of grape leave hydro-alcoholic extract (GLHE) on reproductive parameters in normal male rats. Its leaves were dried and powdered, and mixed with ethanol, after prepared extract, 250 and 500 mg/kg/B.W of this extract was administered into two experimental groups, control received 1ml NaCl 0.9% by gavage for 42 days. 24h after last dosage, all rats anesthetized, then, their couda epididymis and testis dissected on and kept its in NaCl 0.9% separately, for measured sperm motility, total sperm and daily sperm production, then counted them by microscope. The results showed a significant decrease in epididymal, testicular sperm counts and daily sperm production, likewise, percent of sperm-progressive and non-progressive motility vs. control. So, GLHE was significantly decreased effect on final body and ventral prostate weights vs. control, also weight of cauda epididymis in test group I was significantly decrease vs. control. So, it seems that administration of this extract affects on some of fertility parameters and decreases spermatogenesis in the rats.

Full text

Effects Of Vitis Vinifera Leave Hydro-Alcoholic Extract On
Reproductive Parameters In Adult Normal Male Rats.
Afzalzadeh Mohammad Reza, Amirzargar Ashraf, Ahangarpour Akram, KazemiVarnamkhasti
Mohammad, Ganjali Hadi, GharibMombeni Ehsan, Papahn Ahmad Ali.
J. Phys. Pharm. Adv. 2013, 3(6): 159-167.
DOI: 10.5455/jppa.20130626011300.
Online version is available on: www.grjournals.com
Journal of Physiology and pharmacology advances

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J. Phys. Pharm. Adv., 2013, 3(6): 159-167.
Effect Of Vitis Vinifera Leave Hydro-Alcoholic
Extract On Reproductive Parameters In Adult
Normal Male Rats.
*1Afzalzadeh Mohammad Reza, 2Amirzargar Ashraf, 2*Ahangarpour Akram, 1KazemiVarnamkhasti
Mohammad, Ganjali Hadi, 1GharibMombeni Ehsan, 3Papahn Ahmad Ali.
1Department of Physiology, Faculty of Veterinary Medicine, ShahidChamran University, Ahvaz, Iran; 2Department of
Physiology and Diabetes Research Center, Faculty of Medical Sciences, Ahvaz Jundishapur University of Medical Sciences, Ahvaz,
Iran; 3Department of Physiology, Faculty of Veterinary Medicine, ShahidChamran University of Ahvaz, Iran.
Abstract
Grape leaves are used in a traditional food and for treatment of diarrhea, bleeding; and also reported that
administered grape was good to treatment of constipation, gout and etc. So, the aim of this study was to
investigate the decrease effect of grape leave hydro-alcoholic extract (GLHE) on reproductive parameters in
normal male rats. Its leaves were dried and powdered, and mixed with ethanol, after prepared extract, 250 and
500 mg/kg/B.W of this extract was administered into two experimental groups, control received 1ml NaCl 0.9%
by gavage for 42 days. 24h after last dosage, all rats anesthetized, then, their couda epididymis and testis
dissected on and kept its in NaCl 0.9% separately, for measured sperm motility, total sperm and daily sperm
production, then counted them by microscope. The results showed a significant decrease in epididymal,
testicular sperm counts and daily sperm production, likewise, percent of sperm-progressive and non-progressive
motility vs. control. So, GLHE was significantly decreased effect on final body and ventral prostate weights vs.
control, also weight of cauda epididymis in test group I was significantly decrease vs. control. So, it seems that
administration of this extract affects on some of fertility parameters and decreases spermatogenesis in the rats.
Key words: Vitis vinifera, leave, Sperm, Rat.
*Corresponding Author: Department of Physiology and Diabetes Research Center, Faculty of Medical Sciences, Ahvaz Jundishapur University of Medical Sciences,
Ahvaz, Iran.
Received on: 28 Apr 2013.
Revised on: 21 Jun 2013.
Accepted on: 26 Jun 2013.
Online Published on: Jun 2013.
Original Article
ISSN: 2251-7693

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J. Phys. Pharm. Adv., 2013, 3(6): 159-167.
Introduction
Vitis vinifera (grape) is a perpetual woody vine
native to Anatolia, which was established in Europe
and other continents. In Iran, grape leaves are used
in a traditional food (grapevine leaf dolma) and for
treatment of diarrhea and bleeding (Zargari, 1993).
Since ancient times, the different parts of this plant
have been used because of many biological
activities in folk medicine; they also reported that
administered grape to treat constipation, gastritis,
enteritis, gout, and hemorrhagic diarrhea. Thus,
applied grape could injurious for bladder and
kidney, despite, it could decrease sperm
overflowing (Mirheydar, 1985). The active
principles such as flavanols, flavanol oligomers and
proanthrocyanidins were found in V. vinifera leaves
and it has been reported as antioxidant (Monagas et
al., 2006). Most of the medicinal properties of this
plant may be attributed to phenolic compounds
which mainly include flavonoids and these
compounds are widely distributed in grapes. Also,
the grape leaves are rich in tannins, flavonoids, and
procyanidins (Felicio et al., 2001; Qin Xia et al.,
2010). Grape leaves have been used to stop
bleeding, inflammation, and pain, such as the kind
brought on by hemorrhoids in the traditional
medicine (Pari and Suresh, 2008). Grape leaves
with antioxidant activity have been reported to treat
chronic venous inadequacy in human and
nephrotoxicosis induced by citrinin (Gharib Naseri
et al., 2006). It has also been exhibit that the grape
leaf hydro-alcoholic extract (GLHE) causes
spasmolytic effect on rat uterus precontracted by
oxytocin (Gharib Naseri and Ehsani, 2004), and the
same extract induces vasorelaxant effect on rat-
secluded aorta. The recent effect was contingent
upon morality of endothelium and NO and cGMP
productions (Gharib Naseri et al., 2005). Although,
this study was aimed to evaluate the effects of V.
vinifera leaf extract on productivity parameters in
male rats.
Material And Methods
Preparation of extract
The plant material was collected from center of
fruits and vegetables in Ahwaz-Iran as Vitis vinifera
(Linn). The plant was identified and the voucher
specimens are deposited in the Herbarium of Shahid
Chamran University Faculty of Agriculture. Plant
materials were dried under shade and powdered
roughly by grinder (Moulinex, France) before
extraction. The powder was mixed with 70%
ethanol for 72h at room temperature, and the flask
was covered by an aluminum cap. During these
hours the flask was shaken well. After that, the flask
contents were poured gradually on a Büchner funnel
and filter paper (Whatman No.1); then, the liquid
has drawn through the perforated plate by vacuum
suction and was poured into the new dish. At the
end, the extract placed into water bath (60ºC) to
evaporated the solvent of the filtrate, and GLHE
was obtained as a very concentrated dark green
extract. Before starting this experiment the effective
concentrations (250 and 500 mg/kg B.W.) of GLHE
were prepared, then there were stored at 4ºC until
be used (Şendoğdu et al., 2006).
Experimental animals
In this study, eighteen adult and healthy male
albino rats of Wistar strain (250-300 g) from Ahvaz
Jundishapur University of Medical Sciences
(AJUMS) animal facility were used in this study.
Procedures involving animals and their care were
conducted in conformity with Committee of Ethics
in Research of the AJUMS. The animals were
housed in standard conditions in polypropylene
cages (430×270×150 mm3) at 20±5 ºC and 12-h
light: 12-h dark cycle. They were allowed free
access to feed with pelleted standard rat feed, and
water was provided ad libitum. The cage floors
were covered with wood chipsand cleaned three
times each week. During the experiment, all cages
were washed all cages three times with water and
detergent. The rats were allowed to acclimatize in
the laboratory for a period of one week before the
beginning of the study.
Treatment protocol
18 colonies bred, healthy adult male rats were
randomly divided into three groups (six animals
each) and classified in a control group and two
experimental groups as fallow:
Control group: (no intervention, other than dry
food and water) only received1ml normal saline
orally with feeding needle (gavage) (Sarkaki et al.,

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2007). Experimental groupI: rats were administered
the minimum dose of 250 mg/kg B.W orally with
gavage. Experimental groupII: received a maximum
dose of 500 mg/kg B.W orally with gavage. Each
rat received a daily dose every day for 42
consecutive days (Gonzales et al., 2006).
Before administrating, calculated doses were
dissolved separately in 1ml distilled water. 24 hours
after last dose animals were sacrificed by deep
anesthesia with ketamine (%10)/ Xylazine (%2),
80-90 mg/kg B.W) [intraperitoneal (i.p.)] (Alfasan-
Holand) (Gehring et al., 2008). After autopsy the
cauda epididymis were collected and minced in 20
ml fresh physiological saline at laboratory
temperature (25ºC). Then, added, 10 ml
physiological saline to this suspension until
produced 10 times attenuated suspension. One drop
of evenly mixed suspension was transferred to the
improved Neuberger’s chamber, (haemocytometer)
(Deep 1/10 mm, LABART, Germany). According
to the practical methods for sperm count which was
described by WHO (2010), white blood cell counts
in the regions considered aperm motility was
determined by counting both progressive and no
progressive motile spermatozoa by the light
microscope at ×400 magnifications (Olympus,
Japan) and expressed as percent. Also, total
epididymal sperm count was performedat the same
method. Testis was placed in 50ml of fresh
physiological saline (37°C) and homogenized by
homogenizer perfectly. Then, put one drop of it on
the chamber of Neubauer, after 2min. spermatozoa
were counted by an optical microscope with a ×40
magnification. Cauda epididymal and testicular
sperm counts were expressed as (106/mm3) of
suspension. Then, according to appropriate
conversion factor and using the following formula:
Sperm numbers in testis= Sperm count in 400 small
squares×Dilution×10000.
The total number of sperm per testis was
calculated. Then, these values were divided by
testicular weight to obtain the number of sperm per
gram of testis and these values divided by 6.3 to
convert them to daily sperm production (DSP) and
expressed as (DSP/g.t) (Parandin et al., 2008).
Body And Organ Weights
The initial and final body weights of the animals
were recorded. Twenty-four hours after last drug
administration, rats in each group were
anaesthetized with ketamine and xylazine. Then, the
rats were killed and the testis, epididymis, accessory
sex organs (seminal vesicles and prostate glands),
were dissected out and freed from adherent tissues
and blood carefully, grossly examined and weighed.
The index weight (I.W.) of the organ was calculated
by I.W. = organ weight/body weight × 100 Ibrahim
(et al., 2007).
Statistical Analysis
In order to determine the differences between
experimental and control groups, the results were
analyzed by the one-way analysis variance
(ANOVA), followed by Tukey test with TUKEY,
LSD with SPSS v.17 programmed. Data are
expressed as mean standard error of mean (SEM)
and the results were considered significant at the
p≤0.05 level.
Results
Body and organ weight
The values of initial and final body weight, testis,
epididymis, accessory glands (seminal vesicles and
prostate glands), and weights has shown in table 2.
The initial body weights were no significantly vs.
control, but final body weights was shown
significantly decreased in experimental groups I and
II (p≤0.001) when compared with control animals
(Fig. 1). On the other hand, no effect was observed
in testis and seminal vesicle weights, but couda
epididymis (p≤0.017) and prostate (p≤0.039)
weights in experimental group I was significantly
decreased when compared with control group
animals, also prostate weight (p≤0.044) in
experimental group II was observed significantly
decrease vs. control animals (Fig. 2) (Table 1).

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Table 1. Body and organ weights after 30 days of treatment with 70% ethanol extract of Vitis vinifera leave on
male rats.
Treatment groups
Body weight (gm)
Testisa
Couda
epididymisa
prostatea
Seminal
vesiclea
Initial
Final
Control
257.35±10.29
311.16±13.64
495.12±45.44
109.85±8.45
217.83±19.86
518.68±79.43
Experimental Group I
267.33±22.43
268.33±16.41*
506.33±43.62
92.53±11.89*
183.22±28.79*
615.94±128.17
Experimental GroupII
267.16±12.4
264.33±17.53*
507.63±46.49
110.08±26.47
174.71±38.76*
552.68±87.36
Data are expressed as means’s.
a mg/100gm of body weight.
*Indicates significant difference (p≤0.05) between control and experimental groups.
Sperm motility and count
The frequencies of sperm counts, sperm motility,
and daily sperm production (DSP) were
demonstrated in Table 2. There was a significant
decrease (p≤0.05) in the mean frequency percentage
of progressive and non-progressive sperms in cauda
epididymis of experimental groups I (progressive:
p≤0.021, non-progressive: p≤0.003) and II
(progressive: p≤0.009, non-progressive: p≤0.01)
when compared with control animals (Fig. 3). On
the other hand, there was a significant decrease
(p≤0.05) million/mm3 testicular sperm count
(treatment group I: p≤0.005, treatment group II:
p≤0.004) (Fig. 4), cauda epididymal sperm count
(treatment group I: p≤0.001, treatment group II:
p≤0) million/mm3 (Fig. 4) and daily sperm
production (treatment group I: p≤0.017, treatment
group II: p≤0.014) (DSP/g.t) were evident in
experimental groups I and II animals when
compared with control group animals (Fig. 5)
(Table 2).
Table 2. Sperm characteristics after 30 days of treatment with 70% ethanol extract of Vitis vinifer leave male
adult rats.
Treatment groups
Sperm motility (%) Sperm counts (Million/mm3)
Cauda epididymis
Testis Cauda epididymis DSPa(DSP/g.tb)
Progressive Non-progressive
Control
Experimental Group I
Experimental Group II
86.66±6.66
59.9±6.66*
28.8±9.3*
83.07±4.34
37.5±2.84*
49.16±2.65*
39.19±2.09
14.64±5.3*
13.86±5.33*
42.36±9.74
26.16±5.62*
23.3±2.94*
45.03±2.93
17.38±0.65*
16.46±0.68*
Data are expressed as means’s, n=6.
a Daily sperm production.
b gm.testis.
*Indicates significant difference (p≤0.05) between control and experimental groups.
Figure 1. The effect of hydro-alcoholic grape leaves extract (250 and 500 mg/kg B.W) on
decrease mean frequency weights of final body weight. The decrease effect of extract on these
experimental groups were not dose dependent (ANOVA, *(p≤0.001), n=6).

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Figure 3. Effect of hydro-alcoholic grape leave extract (250 and 500 mg/kg B.W) on decrease
mean frequency percentage of progressive and non-progressive sperms motility. The decrease
effect of extract on these experimental groups were not dose dependent (ANOVA, *(p≤0.021),
•(p≤0.003), ▪(p≤0.009) and (p≤0.01) n= 6).

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Discussion
Because of the problems caused by unwanted
pregnancies in the worldwide, some of the new
methods of male contraception have been discussed.
Recently, many research centers are focused to
developing methods of contraception from medical
herbs (Jensen, 2002). Herbal products as
contraceptives will be more affordable in populous
countries than contraceptive pills to prevent
pregnancy. However, studies on the effects of
herbal products on male reproductive and fertility
are few. So the present study took to consideration
the effects of GLHE on some of the most important
male reproductive organs in the rat. All the
parameters were assayed according to the standard
protocols.
In the studies published, the active principles of
grape leaves have been mentioned and some of
them are included as follow: flavanoid, tannin,
terpenes, coumarine, quercitrin, quercetin;
kaempferol and resveratrol (Şendoğdu, 2004).
Figure 4. Effect of hydro-alcoholic grape leave extract (250 and 500 mg/kg B.W) on decrease
sperm count in testis and cauda epididymis (Million/mm3). The decrease effect of extract on these
experimental groups are not dose dependent (ANOVA, * p≤0.005, * * p≤0.004, p≤0.001, 
p≤0 n= 6).
Figure 5. Effect of hydro-alcoholic grape seed extracts (250 and 500 mg/kg B.W) on decrease
of daily sperm production per gram of testis (DSP/g.t). The decrease effect of extract on these
experimental groups are not dose dependent (ANOVA, *p≤0.017, * *p≤0.014, n= 6).

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The obtained results showed that GLHE is a
potent to diminish testicular and epididymal sperm
counts and causes to reduced body weight, cauda
epididymis, and accessory organs weights. The
treatment also reduced both progressive and non-
progressive sperm motility in cauda epididymis and
decreased daily sperm production after 42 days of
treatment. The weight reduction observed in the
treated groups may be due to effect of extract
polyphenols to diminished cholesterol (Frémont et
al., 2000). On the other hand, the decreased sex
organ weight may be due to reduced volume of
contents in them. Also atrophy of the seminiferous
tubules could also be one of the causes of testes
weight reduction by reducing their volume and
these discussed reasons have been accordance with
work done by other researchers (Ligha and Oyibo,
2012). Mali et al. (2002) reported that reduction size
of the seminiferous tubules is directly associated
with low testosterone production, also the lessened
weights of prostate further support the repressed
concentration of testosterone in the circulation. It is
known that the construction and operate of the
epididymis are dependent on androgens. The
weakened epididymal function also may caused by
diminished activity of the testis, which affects the
normal route of testicular fluid into the epididymis.
This is also approved by decreased cauda
epididymal weight. Likewise, it is renowned and
widely agreed thought that LH is responsible for
testosterone production (Modaresiet al., 2012). It is
plausible that the initial step in the mechanism of
the effects on testis induced by the GLHE was the
suppression of LH. At the testicular level, the
absence of stimulation by LH would secondarily
because leydig cell malfunction thereby may be
resulting in decline in testosterone secretion which
is responsible for reduced spermatogenesis and
then, reduction in sperm counts (Ali Ahmed et al.,
2012).
Several reports suggested that quercetin and
flavonoids compounds are found in this plant and
these compounds are also found in the
Apiumgraveolens and Achillea millefolium.
According to study of Takzaree et al.(2013),they
reported that the presence of quercetin and
flavonoids in this plant could be possible caused
inhibit replication of DNA and reducing
spermatocytes. A reduction in spermatocyte cells is
also responsible for diminished spermatid and
sperm cells (Ahmadi et al., 2007). The results of the
present study are revealed that two treatment doses
are diminished sperm counts in the treatment
groups. So, could be said that doses of the extract
are effective in reducing sperm counts.
On the other side,most of the herbal spermicidal
factors cause spermicidal effects by interrupting the
plasma membrane as they act on sperm surface
(Lohiya et al., 2000). This plant offshoot also cause
intense inhibition in sperm membrane-specific
enzymes like acrosin and hyaluronidase, the most
important enzymes in the act of fertilization
(Chakraborti et al., 2003). The above discussed
reasons have been accordance with work done by
Al-Sanabra et al. (2013) on the antifertility effect of
seed extract of celery in male rats.
At a research done by Gharib Naseri et al.(2006)
on GLHE,they concluded that the spasmolytic
effect of GLHE in the isolated rat colon through
blocking voltage relying on calcium channels
(VRCCs) and also activation of Ca2+-operated
potassium channels. Also, it has been said that
GLHE causes spasmolytic effect on rat uterus
(Gharib Naseri et al., 2004) and aorta. It is reported
that the vasorelaxant effect in rat aorta is mediated
by No and cGMP synthesis (Gharib Naseri et al.,
2005).Therefore, GLHE may be induces
spasmolytic effect in the smooth muscles in rat
testis by way of VDCCs and also activation of
Ca2+-operated potassium channels. So, the sperms
can’t be dispatched to epididymis from
seminiferous tubes and can’t be mature.
In summary, it has been demonstrated that the
hydro-alcoholic extract of grape leave possesses an
immobilizing factor that probably reduces motility
by causing sperm non-viability by interrupting the
membrane formation of the sperm cell. The active
component(s) in the extract responsible for
spermicidal activity will be delineated in future
researches. Furthermore, it is known that the
structure and operation of the epididymis are reliant
upon androgens. Thus, GLHE maybe influenced on
androgens (LH and FSH) that caused repression of
cauda epididymal sperm motility in all treatment
groups suggest an undersupply of testosterone to
epididymis and therefore, damaged epididymal

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function. The weakened epididymal operation may
also be caused by diminished activity of the testis,
which influences the normal passage of testicular
fluid into the epididymis (Mali et al. 2002). Also,
polyphenolic compounds such as flavonoids and
quercetin, which are found in GLHE could lead to
inhibition the replication of DNA and reducing
spermatocytes.
Conclusion
In conclusion, the oral administration of GLHE
to healthy male rat produced effects on
reproduction. The ethanolic extract of Vitis vinifera
L. leaves at 250 and 500 mg/kg dose has remarkable
effects on reproductive system in male rat, which
caused to diminished sperm motility and count and
also reduced body and reproductive organ weights.
These results are consistent with traditional usage of
the grape leaf for decreased sperm flow. Thus, it
seems that administration of this extract affectson
some of fertility parameters and decreases
spermatogenesis in the normal rats. However, the
specific mechanism(s) of these compound(s) on
human reproductive system requires to be clarified
in future studies.
Acknowledgment
This research is supported financially by
Shahid Chamran University of Ahvaz and is a part
of student research proposal approved in Research
Affairs of Ahvaz Shahid Chamran University. We
thank Ms. Fakhri Kiani Dehkordi and Diabetes
Research Center of AJUMS for her helps in extract
preparation.
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