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INDO-GLOBAL RESEARCH LIBRARY

Indo-Global Research Journal of Pharmaceutical Sciences


Jan-Mar 2013 | Vol. 3 | Issue 1
Available online www.igrlinfo.com
Research Article ISSN: 2249-4189

CHRONIC EFFECT OF PHENOBARBITONE ADMINISTRATION ON UTEROTROPHIC ACTIVITY


OF ALBINO RATS
Sharangouda J. Patil1*, Sneha R. Banagar2, Ravikumar J. Banagar2 and Sarawati B. Patil1
1Reproductive Biology Laboratory, Department of Zoology, Gulbarga University Gulbarga-585 106, Karnataka, India
2Department of Zoology, L. V. D. College, Raichur -584 103, Karnataka, India

E-Mail: shajapatil@gmail.com
Abstract: The effect of chronic administration of phenobarbitone on uterotrophic potency, estrous cycle, and their gravimetrical, histological and
biochemical changes has been studied. Three groups of healthy adult female albino rats having six rats in each group were taken. The rats of groups
II and III were administered Phenobarbitone at the dose level 5.00mg and 7.5mg/100g body weight respectively intraperitoneally/daily for 30 days.
However, the rats of group I (Control) were given saline alone. Completions of the experimental periods, the rats were sacrificed and the histological
study of uterus was performed. The estrous cycles of these rats were irregular with prolonged diestrus and reduced oestrus, estrus and metaestrus
phases also support the decreased estrogen synthesis. Responsible for cornification of vaginal smear in phenobarbitone treated rats and
histometrical changes of uterine parameters like diameter, thickness of myometrium and endometrium and surface epithelial cell height were
decreased significantly. Biochemical changes of endocrine glands are parallel to the gravimetrical changes, the protein and glycogen contents are
decreased significantly and increased cholesterol content significantly due to the phenobarbitone administration.
Keywords: Phenobarbitone, Uterotrophic activity, Endocrine, Estrous cycle, Rat
INTRODUCTION
Reproduction is a complex and continuous process that starts before
birth and continues through puberty and a series of endocrine and
behavioral events that include estrous cycles, ovulation, breeding,
conception, gestation, parturition and lactation. Phenobarbitone is a
commonly used antiepileptic belonging to barbiturate group. The
exact mechanism of action of phenobarbitone is not known, but
enhancement of inhibitory processes and diminution of excitatory
transmission are known to contribute for the antiepileptic activity
[1]. The most common adverse effect following administration of
phenobarbitone includes sedation, depression, nystagmus, ataxia,
paradoxical
excitement,
restlessness,
and
confusion.
Hypersensitivity reactions occur in a small proportion of patients.
Phenobarbitone is also known to cause skin reactions in 1 to 3% of
patients, the common ones being maculopapular, morbilliform, or
scarlatiniform rashes [2]. Prolonged treatment of barbiturates to
immature or pregnant rats decreases the uterotrophic potency to
synthetic estrogens or cause foetal resorption or abortion
respectively [3-4]. Besides, these sedatives may interfere in the
conversion of pregnenolone to progesterone, thus altering the
steroidal environment essential for ovulation [5]. Secretion of
pituitary gonadotrophins are regulated by brain and neurons
situated in the anterior part of the hypothalamus which synthesize
the gonadotrophin releasing hormones (GnRH) [6]. Therefore, it is
aimed to study the chronic effects of phenobarbitone on female
reproductive activities like uterotrophic potency and estrous cycle
in adult female rats. The gravimetric, histological, micrometric and
biochemical methods are employed to analysis the uterotrophic
potency.
Phenobarbitone:
Phenobarbitone is a barbiturate, marketed between 1934-1945
under the brand name Luminal by Friedr Bayer et al., the first
barbiturate drug, barbital, was synthesized in 1902 by German
chemists Emil Fischer and Joseph Von Mering at Bayer. By 1904
several related drugs, including phenobarbitone, had been
synthesized by Fischer. Phenobarbitone was brought to market in
1912 by the drug company Bayer using the brand Luminal. It

remained a commonly prescribed sedative and hypnotic until the


introduction of benzodiazepines in the 1950s. Phenobarbitone is
soporific, sedative and hypnotic properties were well known in
1912, but nobody knew it was also an effective anticonvulsant. It is
the most widely used anticonvulsant worldwide and the oldest still
commonly used. It also has sedative and hypnotic properties but, as
with other benzodiazepines for these indications.
Barbiturates are the derivatives of barbituric acid obtained by
replacing the two hydrogen of the carbon atom in position 5 of
aromatic ring by alkyl group. An increase in the potency and
decrease in the duration of action can be produced by increasing the
length of alkyl group. Barbiturates are classified according to their
duration of action, this being used on animal experiments.
H
O

HN

Systematic (IUPAC) name 5-ethyl-5-pheylpyrimidine-2,4,6 (1H,


3H, 5H)-trione)

3d-Structure of Phenobarbitone
Materials Methods
Rat is used as experimental animal model since 1856. Since its
domestication, the rat has probably contributed more substantially
to the advancement of the biological sciences than any other
laboratory species. Therefore, in the present study the rat in used as
experimental model.

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Chronic effect of phenobarbitone administration on uterotrophic activity of albino rats.


Sharangouda J. Patil1*, Sneha R. Banagar2, Ravikumar J. Banagar2 and Sarawati B. Patil1

Sl. No.
1
2
3

Table 1: Effect of Phenobarbitone on the Body Weight of Albino Rats


Treatment / 100gm
Initial Body
Final Body
Percent Change
Body Weight
Weight
Weight
150.00 3.66
160.00 3.32
Control
6.66
Phenobarbitone
(5.00mg)
160.00 3.65
168.60 3.01
5.73
Phenobarbitone
180.00 3.65
186.16 3.85
3.42
(7.5mg)
M SE = Mean Standard error.
*P<0.05, **P<0.01, ***P<0.001, when compared to saline treated rats.

Sl. No.

Treatment

Control
(0.2ml Saline)
Phenobarbitone
(5.00mg)
Phenobarbitone
(7.5mg)

2
3

Table 2: Effect Phenobarbitone on Estrous Cycle in Albino Rats


Length
Duration of phases (days) / cycle
No. of
of cycle
cycles/rat
Estrus
Metaestrus
Diestrus
(days)
5.40 0.21
5.41
6.81 0.19
6.34 0.10
10.21 0.68
0.30
3.66 0.21
7.21
3.98***
5.21 0.29
15.82***
0.34
0.48
0.62
2.50 0.22
7.06
3.00***
4.92 0.43
16.42***
0.32
0.71
0.38

Proestrus
4.41 0.31
4.02 0.25
3.98 0.42

M SE = Mean Standard error.


*P<0.05, **P<0.01, ***P<0.001, when compared to saline treated rats.

Table 3: Effect of Phenobarbitone on Uterine Gravimetric and Biochemical Parameters in Albino Rats
Sl. No.

Treatment

1
2

Control
(0.2ml Saline)
Phenobarbitone (5.00mg)

Phenobarbitone (7.5mg)

Uterus Wt.
mg/100g body
wt.
205.91 8.59

Cholesterol
g/mg

Protein g/mg

Glycogen
g/mg

4.18 0.09

8.18 0.2

1.98 0.05

167.04** 3.84

5.89** 0.07

6.89*** 0.05

1.60* 0.05

148.47***
4.50

6.96*** 0.06

5.89*** 0.07

1.56* 0.08

M SE = Mean Standard error.


*P<0.05, **P<0.01, ***P<0.001, when compared to saline treated rats.

Table 4: Effect of Phenobarbitone on Uterine Histometric Parameters in Albino Rats


Sl. No.

Treatment

Uterus diameter
(
m)

Thickness of
Myometrium (
m)

Thickness of
Endometrium (
m)

Height of epithelial
cell (
m)

Control
(0.2ml Saline)
Phenobarbitone
(5.00mg)
Phenobarbitone (7.5mg)

631.96 22.16

169.61 8.04

390.78 7.56

32.33 1.40

462.23*** 17.03

105*** 1.88

310.82*** 2.26

26.22* 1.55

305.12*** 11.62

88.06*** 0.97

285.06*** 0.14

20.65*** 1.02

2
3

M SE = Mean Standard error.


*P<0.05, **P<0.01, ***P<0.001, when compared to saline treated rats.

Animals
Healthy, sexually matured, regularly cycling, colony bred virgin
female rats of Wistar strain (Rattus norvegicus) aged three months
and weighing 150 -200 gm were purchased from National Institute
of Nutrition, Hyderabad. The rats were housed in polypropylene
cages measuring 12x108, under well ventilated animal house
conditions (temperature: 28-31C, photoperiod: 12 hours natural
light and 12 hours natural dark, humidity: 50-55%). The rats were
fed with balanced diet as per CFTRI formula and tap water ad
libitum. They were maintained as per the principles of laboratory
animals care (NIH Publication No. 85-88, 1985).
The animals were divided into three groups, each consisting of six
rats in each group and treated as follows.
Group-I: Control, received 0.2 ml saline/ 100gm body weight.
Group-II: Received 5.0 mg phenobarbitone / 100gm body weight
Group- III: Received 7.5 mg phenobarbitone / 100gm body weight.
The body weight of each rat was recorded every day before the
treatment. The treatment for each rat was started at estrous phase.
Vaginal smears from each rats were monitored daily, only rats with
normal estrous cycles [7-8] were selected for the experiment, to study

the effect of phenobarbitone on the estrous cycle, the above selected


animals were divided into three groups containing 6 animals in each
groups. The behavior of animals after receiving drug treatment was
observed and compared with that of control.
All the experimental rats were sacrificed by cervical dislocation. The
uterus was dissected out immediately and separated free from the
adherent tissue and weighed to the nearest mg on an electronic
balance. Organ from one side of each animal were fixed in Bouins
fluid, embedded in paraffin wax, sectioned as 5 m, stained with
haematoxyleneosin for histological studies. The micrometric
measurements like diameter of uterus, thickness of endometrium
and myometrium and epithelial cell height were carried out as per
the method of Deb et al. [9]. Organ from the other side were used for
biochemical estimation like protein [10], glycogen [11] and cholesterol
[12].
Statistical Analysis
All the values were statistically analysed by Students-t test using
SPSS software [13]. Data are expressed as the Mean + S.E. Statistical
significance was set at p<0.05, p<0.01 and p<0.001.

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Chronic effect of phenobarbitone administration on uterotrophic activity of albino rats.


Sharangouda J. Patil1*, Sneha R. Banagar2, Ravikumar J. Banagar2 and Sarawati B. Patil1

Figure: 1. Part of cross section of the uterus of control treated rat


showing normal growth of uterine component (100).

Figure: 2. Part of cross section of uterus of phenobarbitone treated rat


showing decreased activity (x200).

RESULTS
Genaral Observations
Behavior
The rats which received the chronic treatment of phenobarbitone
were as active as the control rats. The phenobarbitone treatment
caused sedation for 3 to 6 hours. This sedation caused is dose
dependent. After period of sedation the animals show normal
activities. However, the total intake of feed/day is not much altered
due to drug administration.

Histometric Measurements (Table 4; Figure 1& 2):


The histometric measurements of the uterus are parallel to that of
protein content of uterus. The observations of phenobarbitone
treatment has reduced the diameter of uterus and thickness of its
myometerium which is highly significant (P<0.001). The thickness of
endometrium and its epithelial cell height are reduced significantly
(P<0.01) and highly significantly (P<0.001) with respect to
administration of low and high doses in comparison with that of
controls.

Mortality
No mortality is observed in either control group or in the
experimental group that received phenobarbitone.

DISCUSSION
The principle of CNS influencing drugs is the anticonvulsants,
antiparkinsonism drugs, opioid and non-opioid analgesics, appetite
suppressants, antiemetics, analgesics, antipyretics, certain
stimulants, neutolectics, transquilizers sedatives and hypnotics.
These drugs are known to interfere with the functions of CNS
including hypothalamus there by modify the activity of hypophysis
and gonads. The secretion and release of pituitary follicle
stimulating hormone (FSH), luteinizing hormone (LH) and prolactin
(PRL) are directly dependent on hypothalamic releasing hormonesGnRH. Therefore, the drugs which modify the functions of CNS may
also affect the pituitary functions.

Changes in the Body Weight: (Table 1):


The body weight of the rats which received low or high doses of
phenobarbitone have shown non-significant reduction in the body
weight for treatment of 30 days.
Changes in the estrous cycle (Table 2):
The control rats show regular estrous cycle. Therefore, 5.30 0.21
cycles were observed with length of 5.41 0.30 days within 30 days.
Phenobarbitone treatment has decreased the number of cycles and
increased its length highly significantly (P<0.001). The duration of
proestrus and metaestrus though decreased it is not significant with
phenobarbitone. Contrarily the duration of estrus increases highly
significantly (P<0.001) and that of diestrus increases (P<0.01)
significantly.
Changes in Uterus:
The growth of the uterus and its biochemical contents like protein,
glycogen and cholesterol depends on the ovarian hormone
production.
Gravimetric Changes (Table 3):
The administration of low and high of doses of phenobarbitone, the
uterine weight has decreased significantly (P<0.01) and highly
significantly (P<0.001) respectively.
Biochemical Changes (Table 3):
Cholesterol
The cholesterol level is increased significantly (P<0.01) and highly
significantly (P<0.001) in respect to low and high doses of
phenobarbitone treatment.
Protein
The protein content is increased almost significantly (P<0.05) and
highly significantly (P<0.001) with respect to treatment of low and
high dose of phenobarbitone. In contrast, both the doses of
phenobarbitone have decreased the protein content of the uterus
highly significantly (P<0.001).
Glycogen
But both the doses of phenobarbitone have decreased the uterine
glycogen content which is almost significant (P<0.05).

Uterine growth depends upon the ovarian estrogen secretion.


Estrogen primarily acts upon the surface epithelium and the glands
within endometrium [14]. Progesterone acts on estrogen epithelium
from proliferative to secretory state [14]. In the present investigation,
reduction in the uterine diameter, reduced thickness of its
myometrium and endometrium, and reduced secretions from
endometrial glands indicates the inhibition of ovarian steroid
biosynthesis necessary for growth of the uterus and reproductive
cyclicity. When the estrogen levels are elevated due to the action of
FSH in a given period is known to trigger the LH release which
usually occurs in the late afternoon of proestrus [15-17]. Concomitant
with the release of LH from the pitutary, FSH and prolactin are
released [18-21]. This surge of gonadotrophins initiates the output of
progestins and is immediately followed by a sharp decrease in
estrogen secretion before midnight of proestrus [21-22]. In the
hamster, the pattern of estrogen and progesterone secretion is
similar to that of rat and mice [23-25].
As the uterine growth and reproductive cyclicity requires rhythmic
production of ovarian steroid hormones [26-28], uterine growth and
reproduction cyclicity is hampered in phenobarbitone treated rats.
Estrogens are known to increase the glycogen content in the uterus
of rats [29]. The decreased glycogen and protein content of the uterus
indicate retarded uterine growth and lowered availability of
estrogen to the major reproductive tract.
Oestrous cycle is regulated by the secretion and release of ovarian
estrogen and progesterone production which in turn controlled by
pituitary gonadotrophins [30]. The administration of phenobarbitone
prolongs the length of diestrus phase significantly resulting in
decrease in number of cycles. This may be the effect of reduced
steroidogenesis of the ovary as the estrogen synthesis which is

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Chronic effect of phenobarbitone administration on uterotrophic activity of albino rats.


Sharangouda J. Patil1*, Sneha R. Banagar2, Ravikumar J. Banagar2 and Sarawati B. Patil1

essential for cornification of vaginal epithelial cell during estrus


phase.
CONCLUSION
The aim of the present investigation conclude that significant
prolongation of diestrus phase in treated rats indicates the decrease
in the levels of hormone synthesize and secretion may be due to the
administration of phenobarbitone and suppress the uterine
activities and their histoarchitecture.
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