SYNOPSIS

For approval of topic of the thesis to be submitted in the partial fulfillment of the
requirement for the degree of Master of Pharmacy in Pharmacology.

Evaluation of role of Withania Somnifera in premenstrual

syndrome associated depression in female rats

By

Vrushali G.Sawarkar
B. Pharm

Guide

N.R.Kotagale
M. Pharm., Ph.D.

SMT. KISHORITAI BHOYAR COLLEGE OF PHARMACY,

NEW KAMPTEE, NAGPUR, MAHARASHTRA, 441 002 (INDIA)

2015-2016
Rashtrasant Tukdoji Maharaj Nagpur University, Nagpur-14.

Introduction:
Beginning in adolescence, females experience affective disorder at higher
rates than males, partially due to sex specific disorder such as premenstrual
syndrome (Li et al., 2012). It is characterized by range of physical and affective
symptoms including anxiety, irritability, anhedonia, depression, social withdrawal
which is reduced in the luteal phase of menstrual cycle. The cause of PMS is not
clear, but the major factor involved is changes in hormones during the menstrual
cycle which seems to be important chemical change in the brain such as stress,
emotional problems, depression, do not seem to cause PMS but they may make it
worse((McGrath et.al.,1990, Stener et al.,2003).
The prevalence of PMS on the basis of epidemiologic surveys have shown
that PMS is consistently affecting 25% to 50% of women in the age group of 2040 yrs(Y. Li et al., 2012); having at least 1 child, a family history of depression and
a past medical history of either postpartum depression or a mood disorder.
Many research papers elucidated that there are strong evidences of
underlying the changes in hormones observed in the PMS (McGrath et
al.,1990,Steineret al.,2003). Infact the disregulation of hormones may be the
common pathway in the depression associated with the PMS (Barbaccia et al.,
2001; Girdleret al., 2001; Holsboer, 2001; Morrow et al., 1995;Steimer et al.,
1997; Young and Korszun, 1998; Young et al., 2000; Zahn et al., 1991; Zurita et
al.,2000 ). It has been examined as ovarian hormones have an effect on many
neurotransmitters in the brain, but interactions between oestrogen, progesterone
and serotonin, noradrenaline and dopamine are of most interest in premenstrual
syndrome. Women with premenstrual syndrome may have abnormal functioning
of the serotonergic system which seems to be related to lower serotonin levels
and altered serotonin transmission. Antidepressants appear to work by increasing
brain chemical (dopamine, serotonin, and others) levels that are affected by the
ovarian hormones. Declining oestrogen and progesterone during menopause
seems to lead to lower levels of serotonergic activity, which might contribute to

the depression and mood changes that are common during this phase( Eriksson
and Humble 1990).
The serotonin reuptake inhibitor group of antidepressants seems to be the
most

effective.

Fluoxetine (Prozac)

and paroxetine (Paxil)

are

examples

of

antidepressant medications from this group that have been found to be effective
in treating the mood changes associated with PMS. But the major complications
associated with SSRIs and TCAs is sexual dysfunction, insomnia, weight gain,
restlessness, migraine etc. these drugs having many side-effects which are been
marked during research and the efficacies of various classes of antidepressants
have not been well studied in rodent models of hormonally induced depression (Y.
Li et al., 2012).
As

the naturally occurring drug

folk medicine Withania Somnifera

commonly known as Ashwagandha has anti-inflammatory, antitumor,antistress,

antioxidant,

mind-boosting,immune-enhancing,

and

rejuvenating

properties(Singh et al., 1982;Ageel et al., 1987; Al-Yahya et al., 1990) also it has
an ability to restore sexual health and improve overall vitality while promoting a
calm state of mind which has been already patented as an antidepressant agent
as it

prevent acute stress-ind uced anxiety(Bha ttacha rya et aI., 1987) and

chronic stress-induced depression (Bhattacharya, 1998 ) in rats by significantly

increase in gonadotrophin (LS and FSH) levels. The above research suggest that
the Withania Somnifera may ultimately regulate the hormones that are essential
for the normal menstrual cycle .
In the view of this background it is hypothesized that Withania Somnifera
might modulate the normal functioning of hormones in framework of PMS to
regularise the stress response and their imbalance may be associated with
hormonal fluctuations in mammals. In addition the time course of antidepressant
action of drug in this model closely resembles to clinical time course (Willner et
al, 1991). Therefore the present study focuses on the induction of depression by
applying chronic treatment of progesterone and its modulation by exogenous
Withania Somnifera administration associated with the PMS in the female rats.

Objective :
To investigate the antidepressant action of Withania Somnifera using
Chronic Unpredictable Mild Stress model of depression.
To investigate the probable role of progesterone in the of depression
associated with the PMS in the female rats .

Material and methods

Subjects:
female sprauge-dawley rats weighing 160-180 g will be housed in polypropylene
cages in a temperature (252 0C) relative humidity (5070%) and maintained on
a 12:12-h light/dark cycle (lights on 07:0019:00 h). Food and water will be
provided

ad

libitum

except

during

specific

experimental

protocols.

The

experiments will be performed as per the protocol approved by Institutional

animal and ethical committee according to the guidelines of CPCSEA.

Drugs:
Withania Somnifera Extract
Progestrerone

Plan of Work:
Female sprauge-dawley rats weighing approximately 160-180 g were
subjected for daily IP injection of progesterone for 20 days and will be observed
for FST as a model of depression on same day.
After the chronic administration of progesterone, animals were withdrawn with progesterone
and tested for their depression level.

 Animals will be assessed for the administration of Withania somnifera extract (WSE), to
investigate its activity against depression via FST.

Methodology:
Female Sprague dawley rats weighing approx. 200g will be used. Animals will be
habituated to new housing environment (approximately 3-4 days).Vaginal smears
will be checked. Females at the stage of proestrous will be selected in which the
progesterone is expressed in greater amount. Females not showing complete
cycle will not be used. In the following morning, the progesterone will be replaced
with withania somnifera. These females will be divided into 4 groups.
1) 1st group of females will be provided with the continuous exposure of
progesterone for twenty days with the dose of 6mg/rat.
2)

2nd group of females will be exposed to progesterone and on the day 21 st

single administration of Withania somnifera with the dose of 60mg/kg will be

given.
3) 3rd group of females will be exposed to progesterone for 15 days and Withania
somnifera will be administered on the 16th to 19th days.
4) 4th group of females will be provided with progesterone alone for 15 days with
the same dose as above.
Further the antidepressant activity of Withania somnifera will be assessed
along with various biochemical parameters.

Behavioral Paradigms:
Forced Swim Test:
FST is specifically used for preclinical evaluation of antidepressant property of
drugs based upon the immobility time. Female rats will be individually forced to
swim into an 800ml plexiglass cylinder (40 cm height and 18 cm internal
diameter) containing 21cm of water (approximately 25C). Rats when placed first

time in cylinder were initially highly active, vigorously swim in circles, trying to
climb the wall or diving to bottom. After 2-3min this activity begins to subside
and gets interspersed with the phases of immobility or the floating of increasing
duration. After 5-6 min immobility reaches a plateau where the animal remains
immobile for approximately 80% of the time. After 15 min the animals are
removed and allowed to dry in a heated (32c) enclosure and returned to their
home cages. After 24hrs they are again placed in a cylinder and total duration of
immobility is measured during a 5 min test. An animal is considered to be
immobile whenever it remains floating passively in water in a slightly hunched
but upright position, its nose just above the surface. Test drug or standard are
administered 1hr prior to the testing.
Duration of immobility is recorded in controls and in animals of the various
treatment groups. Antidepressant significantly reduces the immobility and dose
responses can be evaluated. Rats have been found to be more suitable than mice
for detecting antidepressant activity.

Data Analysis:
To observe the alteration in progesterone ,FSH, LH, prolactine, estrogen due
to chronic administration of progestrerone and sudden withdrawn with the
antidepressant activity of Withania Somnifera and compaire the same with
control groups.

Possible outcomes:
Withania somnifera may provide novel therapeutic options for treatment
of depression associated with premenstrual syndrome and Withania somnifera
could also project in the treatment of PMS and associated complications.

References:

Objective :
1. To study the effect of agmatine on hippocampal memory process in insulin resistance rat.
2. To study the role of imidazoline I2 receptor in the agmatine mediated effect.

Plan of work :
1.

The rats will undergo surgery for implantation of guide cannula in order to facilitate the intrahippocampal drug administration .The 8 day post-surgery recovery period will be given, during

2.

which they will be handled to adapt experimental conditions.

The induction of insulin resistance (T2DM) will be carried out in rats by giving high fat diet

3.

followed by sub effective dose of streptozotocin and will be evaluated for same.
Animals will then be trained for spatial learning in morris water maze, which includes
familiarization on 1st day followed by training on 2nd ,3rd & 4th day, and on 5th day retrieval will
be checked. All the three phases i.e. pretraining, post-training and retrieval phase will be

4.

evaluated for insulin treatment .

Depending upon this effect ,the either phase will be selected for administration of insulin and

5.

agmatine for assessment of insulin sensitivity.

Further the role of imidazoline I2 receptor will be evaluated by using I2 antagonist along with
agmatine.

Material and method :

Animals :
Male sprauge-dawley rats weighing 160-180 g will be used. . They will be housed in a
temperature- (22+1 C) and humidity-controlled (50+5%) environment and will have free access to
food and water. The animals in which T2DM is to be induced will be given high fat diet(HFD) for 2
weeks followed by low dose of streptozotocin (35mg/kg), after one week of which the drug testing can
be carried out. The animals will be treated in accordance with the CPCSEA guidelines for the care and
use of laboratory animals and in agreement with the institutional animal ethical committee. The
number of animals used and their suffering will minimized in all experiments designed.

Drugs:

The drugs used will be Insulin, Agmatine sulphate and BU224(I2-antagonist).The artificial
cerebrospinal fluid (aCSF) will be prepared in the laboratory.
Drug solutions and administration:
The aCSF will be used as vehicle for all drugs.The composition of aCSF is (composition: 125
mM NaCl, 10 mM glucose, 1.25 mM Na2HPO4 , 2.5 mM CaCl2 , 1.5 mM MgSO4 , 26 mM NaHCO2
treated for 30 min with 5% CO : 95% O, pH 7.4) . Microinjections will given to maze-tested animals
over 2 min in a total volume of 0.5 l, into the left hippocampus, 10 min prior to testing.

Intra-hippocampal Cannula Implantation:

The animal will be anaesthetized with pentobarbital (50 mg/kg i.p.) and the skull will be shaved
with hair remover. Iodine solution will be applied as an antiseptic to exposed skin. The animal will be
placed in a stereotaxic frame with flat skull position fixed in stereotaxic apparatus for implantation of
guide cannula. A midline incision will be made and skin and underlined periostium will be retracted.
Animals received either a microinjection guide cannula. into the left hippocampus as described
previously (McNay et al., 2000) with coordinates relative to bregma +3.8 mm AP, +5.0 mm lateral,
-4.5 mm from dura, nosebar at 5.0 mm above interaural line. After surgery, the animal will be placed
in individual cage for not less than 8 days, during which the animal will be handled to adapt the future
experimental conditions. The animal will be treated prophylactically with oxytetracyclin (25 mg/kg
i.p.) and Neosporin to avoid infection.

Apparatus:

Morris water maze

Morris water maze is the circular tank with 110 cm diameter, about 60 cm high filled with
water up to 30 cm maintained at 2510C. Water will be made opaque using water soluble, non-irritant
white color. The maze will be equipped with platform (9 cm diameter) 0.5 cm above or below water
level as per day of trial. The tank will be divided into four quadrants and marked accordingly, all
quadrants will be equipped with cues. The Morris water maze will be prepared and standardized in our
laboratory conditions.
Training:
It consisted of different phases such as:
1) Day 1: Familiarization
Animal will be kept on the platform for 60 seconds, which will be 0.5 cm above water
level. After 60 seconds it will be returned to its home cage. (During this period, sometimes
animal used to jump in water and explore through the maze so that it should be familiarized with
the water maze and cues inside it).
2) Day 2,3,4: Training
It consists of four sessions of three trials each with inter session delay of 15-20 minutes. All
conditions will be same as on day 1 except platform submerged 0.5 cm below water level. In each
trial animal will be introduced from starting point in the tank and allowed to locate and get on the
platform. The animal not finding platform within 60 seconds will be guided by stick to reach the
platform. Maximum latency period of 60 seconds will be recorded. Those animals not finding
platform within 60 seconds on day 3 and 4 will be excluded from the study. In each session,
starting point will be randomly changed. When animal will found platform or after 60 seconds, it
will be removed from the maze and kept under warm light to prevent hypothermia, which may
affect the next training trial. After this animal will be shifted back to its home cage.
3) Day 5: Probe test/ Retrieval
It will start exactly 24 hours after last trial on day 4. Here platform will be removed and animal
will be introduced from the starting point exactly opposite to the platform quadrant with its face
towards wall of the tank. Different treatments will be given starting from 30 minutes prior to
retrieval.

Mice will be evaluated during probe trail for the following parameters:
1) Time spent in quadrant in which platform will be placed.
2

2) Escape latency, i.e. time taken by animal to reach the position of platform.
3) Number of entries in platform quadrant.
4) Number of crossing over the position of platform.

Treatment groups:
A) Effect of insulin on different phases of memory
1)Pre-training administration
a) aCSF
b) insulin(100U,1mU) in normally fed animal
2)Post-training administration
a) aCSF
b) insulin(100U,1mU) in normally fed animal
3)Pre-testing administration
a) aCSF
b) insulin(100U, 1mU) in normally fed animal
B) Effect of agmatine on insulin sensitivity
Depending on the observed effect of insulin on different phases of memory, the agmatine
and insulin will be given in either of the three phases in HFD (high fat diet) animal.
1) Insulin (effective dose) in normally fed animal
2) Insulin (effective dose) in HFD animal
3) Agmatine (100,50 mol/kg) + insulin in HFD animal

C) Role of imidazoline I2 receptor in agmatine mediated effect

To check the effect mediated by agmatine is through imidazoline receptor or not,the I2imidazoline antagonist is given before agmatine.
1) BU224( 10mg/kg,i.p)+ Agmatine + insulin

Verification of cannulae placements:

After completion of experimental sessions, each animal will be sacrificed with an overdose of
pentobarbital sodium. Mice received intra-hippocampal injection of ink (1% methylene blue, 0.5
l/side). Brain will be removed and sections will be examined to determine the location of cannulae.
The cannula placement will be verified using the atlas of Paxinos and Franklin, (2004). Only data form
animals with correct cannulae placement will be considered.

Data analysis:
To observe the spatial learning, comparison between control and agmatine treated groups
will be done by unpaired t-test. For analysis of other data, two way analysis of variance (ANNOVA)
followed by by post hoc Bonferroni multiple comparison test will be used. A nonparametric test will
be used because of cut off time of 60 seconds. The data is expressed as a mean SEM (standard error
mean) and a value of P < 0.05 will be considered to be statistically significant.
Possible outcomes:
Agmatine may increase the insulin sensitivity in hippocampus and thereby facilitate memory
processing in T2DM.

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Signature of guide

B G. Taksande

Date:
Place:

Signature of candidate

Manish M. Aglawe