The Effects of Vitex agnus-castus L. Extract on Fertility of Male Rats

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Conference: International Conference on agriculture ,Food, biological and Sciences, August 22_24,2016, At Kuala Lumpur , Malaysia
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Abstract
Vitex agnus-custus L [Verbenaceae] in the category of flavonoids which is native phytoestrogen compounds in the middle east and southern Europe and in many countries it has medical use, , but it is not a herb which contains estrogenic compounds and does not impact directly on the ovaries and it seems by effecting on hypothalamic-pituitary axis apply its own effect. This herb cause decreases in releasing FSH and LH and prolactin from the pituitary and also it decrease LH, FSH and testosterone in male rat and due to reduction of testosterone it effects on spermatogenesis as well and reduces it. In this study for 48 days to experimental groups 1, 2 and 3 the extract of ethanol extract of vitex agnus-castus leaves at doses of 165, 265 and 365 mg/kg and divided into the 4 group as a sham group equivalent volume and duration of administration of the extract in experimental groups, the carrier and group 5 as a positive control of soybean at a dose of 120 mg/ kg were gavaged. After 49 days, the reproductive parameters such as sperm count, weight of testis, epididymis and levels of estradiol and testosterone, FSH and LH were measured and then histological studies were conducted. Administration of Vitex extract caused a significant reduction in the number of sperms. Serum factors indicate significant changes in the normal testicular function. Index Terms— Vitex ugnus-castus L, Fertility, Male Rats. Tahereh Naji (PhD) is with the
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Abstract Vitex agnus-custus L [Verbenaceae] in the category
of flavonoids which is native phytoestrogen compounds in the
middle east and southern Europe and in many countries it has
medical use, , but it is not a herb which contains estrogenic
compounds and does not impact directly on the ovaries and it
seems by effecting on hypothalamic-pituitary axis apply its own
effect. This herb cause decreases in releasing FSH and LH and
prolactin from the pituitary and also it decrease LH, FSH and
testosterone in male rat and due to reduction of testosterone it
effects on spermatogenesis as well and reduces it.
In this study for 48 days to experimental groups 1, 2 and 3 the
extract of ethanol extract of vitex agnus-castus leaves at doses of
165, 265 and 365 mg/kg and divided into the 4 group as a sham
group equivalent volume and duration of administration of the
extract in experimental groups, the carrier and group 5 as a
positive control of soybean at a dose of 120 mg/ kg were gavaged.
After 49 days, the reproductive parameters such as sperm count,
weight of testis, epididymis and levels of estradiol and
testosterone, FSH and LH were measured and then histological
studies were conducted.
Administration of Vitex extract caused a significant reduction in
the number of sperms. Serum factors indicate significant
changes in the normal testicular function.
Index Terms Vitex ugnus-castus L, Fertility, Male Rats.
Tahereh Naji (PhD) is with the Department of Molecular and
Cellular Sciences, Faculty of Advanced Sciences & Technology ,
Pharmaceutical Sciences Branch, Islamic Azad University,
Tehran-Iran (IAUPS). (e-mail: t.s.naji@yahoo.com).
Nima Rabie Nezhad Ganji (*corresponding author ) is with
Department of Molecular and Cellular Sciences, Faculty of
Advanced Sciences & Technology , Pharmaceutical Sciences
Branch, Islamic Azad University, Tehran-Iran(IAUPS).
(email: nimarabienezhadganji@gmail.com)
Fereshteh Ramezanloo is with Department of Medicine, Faculty
of Advanced Sciences & Technology, Pharmaceutical Sciences
Branch, Islamic Azad University, Tehran-Iran (IAUPS).
Jamil Kheirvari Khezerloo is with Department of Biochemistry,
Faculty of Advanced Sciences & Technology, Pharmaceutical
Sciences Branch, Islamic Azad University, Tehran-Iran (IAUPS).
(email:miladkheirvari@yahoo.com)
Mahya Daustany is with the Department of Molecular and
Cellular Sciences, Faculty of Advanced Sciences & Technology,
Pharmaceutical Sciences Branch, Islamic Azad University,
Tehran-Iran (IAUPS). (e-mail: monafras@yahoo.com).
I. INTRODUCTION
itex agnus-castus [Verbenaceae] is a deciduous tree
or a large shrub that is native to Europe but also
widely distributed in the Southern United States. The
fruits of V. agnus-castus [chaste berry, VAC] have a long
history [over 2000 years] of use as an herbal medicine.
Currently, the fruit extract is used as a dietary supplement for
estrogen hormone imbalance which can produce menstrual
cycle disorders and premenstrual syndrome [PMS], such as
cyclical mastalgia, and corpus luteum insufficiency [1,2] as
well as for alleviating menopausal symptoms such as hot
flashes [3]. The classes of phytochemicals that have been
reported in VAC fruits include essential oils [4,5], flavonoids
[6,7], iridoids [8], and diterpenoids [910], as well as a
diterpene lactam, vitexlactam A [11]. Under investigation for
safety and efficacy at the UIC/NIH Center for Botanical
Dietary Supplements Research, preliminary screening of a
methanolic extract of VAC showed it contained ligands for
the estrogen receptor [ER] [3]. Since at that time, there had
been no literature reports of estrogenic constituents of VAC,
an ER binding assay was used to guide phytochemical. The
epididymis is one of the most important components of the
mammalian male reproductive system. It is only during transit
through the epididymal luminal microenvironment that
spermatozoa undergo maturation and acquire progressive
motility and the ability to fertilize oocytes [12]-[15]. The
mammalian epididymal duct can be subdivided into four
morphological regions: the initial segment, the caput, corpus
and cauda epididymidis, all of which are essential for sperm
maturation [16],[17].
II. MATERIAL AND METHOD
In this laboratory experimental study, Vitex agnus castus was
purchased from the Pharmacognosy herbarium of School of
Pharmacy, Tehran University of Medical Sciences and was
identified by experts of the sector of University of Medical
Sciences. The leaves were dried in room temperature
avoiding from direct sunlight and then ground into a fine
powder. Adult male Sprague-dawely rats, weighing 240-180
g of were used in this study. They were randomly housed in
polyethylene cages access to food and water in a room with
controlled temperature [18-24 °C] and a 12 h lightdark
cycle. Humidity of 45 to 50 animals were provided and they
were fed the pills provided from the company of Pars
livestock and poultry. In this study for 48 days to
experimental groups 1, 2 and 3 the extract of ethanol extract
of vitex agnus-castus leaves at doses of 165, 265 and 365
miligrams per kilogram and to the group 4 as a sham group
equivalent volume and duration of administration of the
extract in experimental groups, the carrier and group 5 as a
positive control of soybean at a dose of 120 mg/ kg were
gavaged. After 49 days, the reproductive parameters such as
sperm count, weight of testis, epididymis and levels of
estradiol and testosterone, FSH and LH were measured and
then histological studies were conducted. Statistical
significance was evaluated by one-way analysis of variance
(ANOVA) and following that using Turkey test. Differences
with P<0.05 were considered significant.
III. RESULTS
Figure I show the fertility of epididymal spermatozoa cells in
response to different doses of Vitex ugnus-castus L. Extract.
Our findings show that viability of Vitex extract caused a
significant reduction in the number of sperms. Serum factors
indicate significant changes in the normal testicular function.
The Effects of Vitex ugnus-castus L. Extract on Fertility of Male Rats
Naji T, Rabie Nezhad Ganji N*, Ramezanloo F, Kheirvari Khezerloo J, Daustany M
V
Fig I. fertility of epididymal spermatozoa cells in response to
different doses of Vitex ugnus-castus L. Extract
IV. DISCUSSION
Human and animal studies have determined Vitex to be safe
for most women of menstruating age, but they note that it
should not be used during pregnancy [18]. In mammals, the
epididymis is responsible for sperm transport, concentration,
storage, and maturation. Sperm maturation involves the
acquisition of forward motility and fertilizing ability.
Identification and characterization of differentially expressed
genes in epididymis have provided us additional insight into
the mechanisms of sperm maturation. Our previous study
demonstrated that several epididymal proteins, such as Bin1b
[19], HongrES1 [20], and Defb15 [21], are essential in sperm
motility, capacitation, and male fertility, respectively. In the
present study, we identified an epididymis-specific gene,
Spink13 and demonstrated its physiological function in
regulation of acrosome reaction. The biological role of
Spink13 in the sperm acrosome and how its down-regulation
causes the fertility reduction remains to be elucidated. One
possibility is that SPINK13 functions as a protease inhibitor
necessary for the regulation of critical proteases involved in
early signaling events during fertilization, which is consistent
with the hypothesis that the regulated serine protease activity
might be the key of sperm maturation [22]. Furthermore, the
fact that Spink13 is evolutionarily conserved from rodent to
human and has no expression in testis indicates that it is a
putative target for post-testicular male contraception. The
SPINK13 protein has the characteristic signature of serine
proteases inhibitors consisting of an N-terminal signal peptide
followed by a Kazal domain, but its serine protease inhibitory
activity is still unclear, rat spink13 showed potent inhibitory
effect on acrosin proteolytic activity using the gelatin
substrate film method. It is very likely that other target
proteases might exist in addition to acrosin. The active
spink13 recombinant protein after heterologous expression
with proper folding will be a key step in further studies.
However, the inhibitory target specificity by SPINK13 is
achieved primarily through temporal and spatial restrictions,
such as the site of expression, membrane anchorage, and the
microenvironment of epididymis, thus complicating the
identification of in vivo target proteases of SPINK13 using
conventional protease inhibitor assay. It is generally known
that gene expression in the epididymis is driven by androgens
signaling through the androgen receptor; many gene
expressions in the epididymis have been demonstrated to be
regulated by androgen [23.24]. This study shows that
androgen also regulates the Spink13 gene. Androgen and its
receptor form a complex that interacts with androgen
response elements in androgen-responsive genes and
regulates their expression. Considering the high homology of
rat Spink13 to its mouse homologue, we referred to the results
of ChIP-seq in mouse caput epididymis [25] to predict 12
potential androgen receptor-binding sites in intergenic and
intronic regions .Most of these androgen receptor-binding
sites harbored conserved androgen response element motifs
across rat and mouse based on whole genome alignment.
Thus, it is possible that androgen-androgen receptor acts
directly on Spink13 gene to regulate its gene expression. It is
noteworthy that the decline of Spink13 mRNA is not very
sharp in response to the rapid decrease of androgen level. We
speculate other regulation mechanisms are also involved,
such as mRNA stability. Three different nociceptive tests
[formalin test, acetic acid-induced writhing response, and tail
immersion test] were employed for evaluation of possible
peripheral and central effects of the Vitex agnus-castus
essential oil. Using these methods, it was revealed that
subcutaneous injection of EOVAC produced antinociceptive
effects in the rats. Subcutaneous injection of formalin 2.5%
into the ventral surface of right hind paw produced a biphasic
pattern of nociceptive responses in rats. Each phase of
formalin test has different mechanisms of nociception. The
first phase is produced by direct simulative effect of formalin
on myelenated and unmyelenated nociceptive afferent fibers,
mainly C fibers which corresponds to acute nociceptive
neurogenic pain. This phase of formalin pain is more sensitive
to opioidergic agent’s effect. The second phase of formalin
test is associated with release of several inflammatory
mediators and excitatory amino acids such as glutamate and
aspartate causing an inflammatory type of nociception and is
very sensitive to anti-inflammatory actions of non-steroid
anti-inflammatory drugs as the cyclooxygenase inhibitor.
Several studies suggested that the chemicals or drugs that act
as analgesic via activation of central mechanisms of analgesia
can inhibit both phases of formalin test whereas peripherally
acting drugs can inhibit only the late phase. Our results
showed that EOVAC significantly reduced licking and biting
behaviors in the both phases of formalin test and increased
latency time in the tail immersion test at various time points
post-treatment in rats. These findings suggested that the
analgesic activity of EOVAC is mediated by both peripheral
and central antinociceptive mechanisms in these models of
nociception. In the present study, intraperitoneal injection of
morphine (10 mg/kg) produced an inhibitory effect on both
phases of formalin pain and also increased latency time in the
tail immersion test. Moreover, pretreatment with naloxone (a
non-selective opioid receptors antagonist), at a dose that did
not produce any significant effect on the formalin pain or
thermal pain responses, completely prevented analgesia
induced by EOVAC on both phases of the formalin test as
well as tail immersion test. These results indicated that at least
part of the antinociceptive effect observed from EOVAC is
due to activation of endogenous opioidergic system.
Researchers reported that different fractions of Vitex
agnus-castus extract act as an agonist of µ and δ but not κ
opioid receptors. Opioidergic activity exhibited by VAC may
be one of the important mechanisms of action of VAC in
reduction of pain and treatment of PMS syndrome. In the
present study, it was revealed that pretreatment with atropine
(1 mg/kg) significantly prevented the analgesic effect of
EOVAC (50 mg/kg) in the formalin test as well as tail
immersion test. These results suggest an acetylcholine
muscarinic receptors involvement in the analgesic effect
induced by EOVAC. Cholinergic system has an important
role in the pain modulation. EOVAC contains some of
terpenes such as (-)-Linalool and α-phellandrene that produce
analgesia via activation of cholinergic system. Our results
showed that the EOVAC and piroxicam (non-selective
cyclooxygenase inhibitor) as a positive control significantly
reduced writhing response. This model of visceral
nociception is a typical model of inflammatory pain and also
accepted as a screening method for the assessment of
analgesic and/or anti-inflammatory properties of new
compounds and chemicals. Acetic acid promotes the release
of prostaglandins, serotonin, and histamine in the peritoneal
fluids. Therefore, the current results suggested that the
EOVAC significantly produced inhibitory effect in this
inflammatory model of pain, and this effect may be related to
its suppressive effect on the biosynthesis pathway of pro
inflammatory substances or reduction of endogenous
pro-inflammatory substances release. We can conclude that
the EOVAC produced analgesic effect in these models of
nociception and this effect seems to be mediated by activation
of endogenous opioidergic system and muscarinergic
receptors of cholinergic system. In addition, part of
antinociceptive activity of Vitex agnus castus essential oil
may be due to its anti-inflammatory effect.
V. CONCLUSION
Vitex extract can reduce the number of sperms in the
epididymis and damage to the testes, which the regularly
usage can lead to infertility.
ACKNOWLEDGMENT
We appreciate all who helped us to exert the present study.
This paper reports a part of results selected from research
work carried out by Fereshteh Ramezanloo supervised by Dr.
T . Naji and Dr. Gh. Amin at Department of Molecular and
Cellular Sciences, Faculty of Advanced Sciences &
Technology, Pharmaceutical Sciences Branch, Islamic Azad
University, Tehran-Iran (IAUPS).
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