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Sedative Effect of Intraperitoneal Diazepam in Mice
Marsha Vania, Shinta Dewi Permata Sari, Bambang Siswitono, Endin Nokik Stujanna

Faculty of Medicine, Universitas Muhammadiyah Prof. DR. HAMKA, Jakarta, Indonesia

Introduction

Induction of anesthesia is the administration 
of drugs approximately 1-2 hours before 
anesthesia to assist the process of anesthesia. 
Pre-medication can be given before general 
or local anesthesia by intravenous (IV), 
intramuscular (IM), or subcutaneous (SC). 
Pre-medication drugs are classified into 
sedatives (diazepam and phenobarbital) 
and non-sedative drugs such as atropine.1 
Generally, phenobarbital and diazepam can 
be delivered by IM injection. Meanwhile, the 
most frequently used injections for solutions 
or suspensions were SC, IP, and IV injections. 
IP injection is a simple technique because the 
large surface area of the peritoneal cavity and 
many blood vessels allow for rapid absorption.2

Phenobarbital is a derivative of the 
barbiturate group for hypnotic therapy and 
the treatment of epilepsy. Phenobarbital has a 
long-acting period, used as general anesthesia 
in rats with a small dose.3 Diazepam has the 
same use as phenobarbital and can be used 

as an anesthetic induction and antiepileptic 
therapy. Diazepam is widely used for 
anxiety disorder, short-term relief of anxiety 
symptoms, alcohol withdrawal, and insomnia.4 
Therefore, diazepam has largely replaced 
phenobarbital for anxiety and sleep disorders 
treatment due to fewer side effects. However, 
diazepam and phenobarbital have the same 
general side effect, sedation.4 Diazepam was 
also used frequently as a positive control 
in study behavior in mice. Meanwhile, 
phenobarbital is commonly used as a positive 
control in study anesthesia in rodents.3,5,6 The 
phenobarbital side effect was hepatotoxicity, 
which may affect the use of anesthesia that 
assess the effects on the liver.7,8 These may 
limit the use of phenobarbital as a positive 
control. The alternative positive control needs 
to be studied.

Marina’s study showed a diazepam sedative 
effect rather than an anxiolytic effect in mice.9 
This finding may use as basic information to 
develop diazepam use in experimental animal 
research. The study regarding the sedative 

Original ArticleInternational Journal of Integrated Health Sciences (IIJHS)ISSN Print: 2302-1381; ISSN Online: 2338-4506

Received: December 29 2022
Accepted: March 11, 2023
Published: March 30, 2023

Correspondence:
Shinta Dewi Permata Sari, 
Universitas Muhammadiyah 
Prof. DR. HAMKA, Jakarta, 
Indonesia
Email: shinta.dps@uhamka.ac.id

DOI: 10.15850/ijihs.v11n1.3139
IJIHS. 2023;11(1):32-36

Article History
 

Abstract

Objective: To determine the effectiveness of Diazepam in comparison 
with Phenobarbital.

Methods: Twenty-seven male Swiss Webster mice were used and 
randomly divided into three groups of negative control (NS), positive 
control (Phenobarbital), and diazepam group. Two tests were performed 
on these group: the traction Test and the Fireplace Test. Pupillary 
diameter was also observed.

Results: A significant difference based on the Kruskal - Wallis statistical 
test was observed between the positive control and the diazepam group 
(<0.05) in the traction test, which was also true for the fireplaced test 
(p<0.05). The pupillary diameter in the test animals in the positive control 
and diazepam group was not statistically significant (p>0.05).

Conclusion: Diazepam has a better sedative effect than Phenobarbital. 
The sedative effect produced by Diazepam is stronger, with faster onset 
and longer half-life than the Phenobarbital the positive control. However, 
different test methods and comparisons should be sought to support this 
conclusion.

Keywords: Diazepam, fireplace test, phenobarbital, traction test



International Journal of Integrated Health Sciences (IIJHS), Vol 11, Number 1, March 2023 33

effect of diazepam in experimental animals is 
still limited. Otherwise, diazepam is potentially 
used as an alternative positive control in 
study anesthesia and as a pre-medication 
anesthesia in the animal study. The route 
of administration is a critical factor for the 
availability of the drugs in plasma and could 
affect pharmacodynamics. In experimental 
animal research, IP injection used frequently 
because the simplicity of technique and 
minimal stress for the animal study.10 

The study regarding diazepam’s 
effectiveness in various injection routes is 
also still limited. At the same time, it can be 
informative to other researchers especially 
in experimental animal research. Further 
study needs to be conducted to assess how the 
effectiveness of diazepam sedative effect in 
experimental animals (mainly mice and rats) 
by various routes of administration. Therefore, 
this study aimed to determine the duration 
and effectiveness of diazepam sedative effect 
by IP injection in mice.

Methods

The experimental study was conducted 
in the Faculty of Medicine Universitas 
Muhammadiyah Prof. DR. HAMKA. This 
study used male Swiss-Webster mice (mus 
musculus) aged 3-6 months and weighed 
20-30 grams. The mice are cared for under 
standard conditions, by the temperature of 26 
– 28 °C, 12-hour light/dark cycle, and humidity. 
Animal acclimatization was carried out for ten 
days before the test, had free access to food and 
water in their cages. The animal experiments 
were carried out under the supervision of 
veterinarians to ensure their health during 
acclimatization until the tests of study. The 
animal experiments were eligible to undergo 
several study tests. The procedures of study 
were approved by Animal Ethics Committee, 
Universitas Muhammadiyah Prof. Dr. HAMKA 
(Protocol No.KEPKK/FK/024/01/2022). 

This animal research applies ethical 
principles 3R (replacement, reduction, and 
refinement). The ethical principles also refer 
to the 5F (Five freedom) consist of freedom 
from hunger and thirst (free from hunger 
and thirst), freedom from discomfort (free 
from discomfort), freedom from pain, injury, 
and disease (free from pain, injury, and 
illness), and freedom to express normal and 
natural behavior. The number of samples 
was calculated with the Federer formula, 
resulting in 27 mice for three groups (n=9) 
by random sampling. The groups were group 

I (normal saline as negative control), group 
II (phenobarbital as positive control), and 
group III (intervention with diazepam). All the 
interventions by IP injection. 

The dosage of 0.325 mg phenobarbital 
for 20 g mice is equivalent to 100 mg 
phenobarbital in humans and adjustable as 
the weight. Diazepam dosage use 0.0325 mg 
as for 20 g mice, equivalent with 10 mg of 
diazepam in humans. The animals performed 
several tests such as traction test, fireplace 
test, and examination of pupil diameter. The 
traction test was conducted first after the 
acclimatization and ensured all the animals 
were eligible for the study. The test was started 
when the mice was confirmed under sedation 
effect by touching the mice and showed less or 
no response. The onset of the drugs may vary 
each other. The traction test aimed to observe 
muscle relaxation activity by placing the mice 
with the anterior body position facing a wire 
stretched horizontally, then the tail is pulled 
up. The animal tests that fail to make a re-
establishment of at least one of its posterior 
limbs to reach the wire are considered under 
a sedative effect. The duration of mice to make 
re-establishment were recorded by stopwatch.

The fireplace tests were conducted three 
days after the fireplace tests were finished. 
The animal tests considered under sedation 
effect when showed no response or less 
response. The fireplace test was used to 
observe decreased activity and sensitivity 
toward the environment. The test was 
conducted by placing the mice individually in 
a cylindrical tube to assess the time mice get 
out quickly. The duration of mice attempting 
to escape the cylindrical tube was recorded 
by stopwatch. The pupillary reflex is used to 
determine the sedation effect by observing the 
pupil diameter. The normal pupil diameter in 
mice was 2 mm. This test is conducted during 
the fireplace test. 

 The statistical product and service solution 
(SPSS) program was used to analyze the results. 
The Kruskal–Wallis test was performed to 
measure the significance of differences among 
groups. A value of p<0.05 is considered a 
statistically significant difference.

Result

The animal tests weighed during the study 
period presents in Table 1. The weight of 
animal tests was not varied among groups 
because there was no significant elevated body 
weight during the study. The pupil diameter 
before tests were homogenous among groups 

Sedative Effect of Intraperitoneal Diazepam in Mice



34 International Journal of Integrated Health Sciences (IIJHS), Vol 11, Number 1, March 2023

with normal-sized diameter pupil for mice (2 
mm). There were no significant differences 
statistically in the body weight between 
groups. Table 1 also showed the onset of 
the drugs (phenobarbital and diazepam) 
in traction and fireplace tests were not far 
different. The onset of the drugs also may vary 
each other’s.

The animal test undergoes several 
interventions based on their group test. Several 
test parameters, such as traction test, fireplace 
test, and assessment of diameter pupil, 
collected the data results. Table 3 presents the 
results of those several parameters test. The 
tests were conducted to determine the effect 
of sedation in animal tests by the following 
results.

The negative control group took an average 
of two seconds in the Traction and Fireplace 
Test. Meanwhile, the positive control group 

took longer by an average of five seconds in the 
Traction test and 80 seconds in the Fireplace 
test. The Diazepam group showed a more 
prolonged sedation effect than the positive 
control by an average of seven seconds in 
the Traction Test and 500 seconds in the 
Fireplace Test (Table 2). The assessment of the 
diameter pupil in the negative control group, 
positive control group, and diazepam group, 
respectively, 2 mm, 1mm, and 1 mm (Table 2).

The statistical analysis presents data 
results that were not distributed normally 
(p<0.05) by the Shapiro - Wilk test. Table 3 
shows the traction test result’s significant 
difference between the positive control and 
the diazepam group (p<0.05). Moreover, the 
Fireplace test also performed a statistically 
significant difference between the positive 
control and the diazepam group (p<0.05) by 
the Kruskal – Wallis test. Pupillary diameter in 

Marsha Vania, Shinta Dewi Permata Sari, et al

Table 1 General Characteristics of Mice

Parameters
Groups

Negative Control 
(NS)

Positive Control 
(Phenobarbital) Diazepam

n 9 9 9
Body Weight (g) 32.9 33.2 32.8

Pupil Diameter (mm) 2 2 2
Onset:

Traction Test (min) 0 15 5
Fireplace Test (min) 0 10 3

Table 2 Parameter Tests Results of Sedation Effect

Parameter Test

Group

Negative Control
n=9

Positive Control
n=9

Diazepam
n=9

Traction Test 2 seconds ± 0.5 5 seconds ± 1.5 7 seconds ± 1.8
Fireplace Test 2 seconds ± 0.5 80 seconds ± 72.8 500 seconds ± 141.4
Pupil Diameter 2 mm 1 mm 1 mm

Table 3 Statistical Analysis Results in Several Parameter Tests

Groups
Traction Test Fireplace Test Pupillary Diameter

p p p

NS with Diazepam 0.000 0.000 0.000
Phenobarbital with Diazepam 0.015 0.001 1.000



International Journal of Integrated Health Sciences (IIJHS), Vol 11, Number 1, March 2023 35

the animal test was not statistically different 
between the positive control and diazepam 
groups (p>0.05). The negative control 
and diazepam groups showed a statistical 
difference in pupillary diameter (Table 3).

Discussion

A significance different among parameter 
tests between positive control and diazepam 
groups indicates a decrease in the activity 
of the animal’s test. These may show the 
occurrence of Central Nervous System (CNS) 
suppression.11 Mostly, the pharmacokinetics 
of sedative drugs are fat soluble, well 
absorbed, and distributed to the brain. High 
lipid solubility drugs can quickly enter the 
CNS. Sedative drugs are metabolized by liver 
enzymes and excreted by the kidney. However, 
there are different metabolic rates in each drug 
class. Diazepam belongs to benzodiazepine 
groups with anxiolytic action, hypnotic, 
muscle relaxation, and anti-convulsion, used 
in anesthesia and insomnia conditions.

Diazepam elevated GABA potential action 
by opening chloride channels, leading to 
hyperpolarization membrane, inducing CNS 
depression and sedative activity. The traction 
and fireplace tests were used as assessment 
parameters for the sedative effect in mice.12,13 
The traction test showed the length of time 
in mice to turn around and fall. The sedative 
effect becomes significant if the mice take 
longer to turn around. Moreover, the fireplace 
test showed the length of time mice jumped 
out of the tube. The sedative effect gets 
stronger if the mice take longer to jump 
out. Pupillary diameter changes may show 
decreased spontaneous activity in mice as a 
sedative effect.14The onset of the drugs varied 
among groups and could be affected by the 
route of administration. The administration 
by intraperitoneal injection was to avoid the 
potential degradation or modification of the 
drugs.2 Meanwhile, the IV injection was hard to 
use in rodents. The IP injection was minimally 
used in clinics but preferred in animal studies. 
Durk and colleagues’ studies showed that IP 
administration resulted in lower Tmax and 
higher Cmax than SC injection.15

The diazepam group’s traction and 
fireplace test results showed longer than 
the positive control (phenobarbital) group. 
These may cause the benzodiazepine group 
(diazepam) to become an active metabolite 
with a long half-life resulting in a more 
prolonged effect sedative than the positive 

control group. Meanwhile, barbiturates, 
especially phenobarbital, are partly excreted 
in the urine, and some undergo extensively 
metabolized.16 

Sadanandan’s study shows Diazepam has 
a long duration of sleep than Ganaxolone.17 
Diazepam is frequently used as a comparator 
drug in studies related to sedative effect 
and anesthesia (intravenously). In used to 
anesthesia, diazepam is mainly combined 
with other agents.16 In experimental animal 
models, benzodiazepines and older sedative-
hypnotic drugs can exert an anti-anxiety effect. 
However, not all sedative effects drugs have it.16 
The sedative-hypnotic drug group are dose-
dependent and induce sleep in high dose. The 
specific drug and administration frequency 
could affect the sleep stages in the sedative-
hypnotics effect.16 Phenobarbital has a long 
duration of action and long half-life (80-120 
hours), although the traction and fireplace test 
results showed a shorter duration of sedative 
effects in phenobarbital than diazepam. 
Phenobarbital has low lipid solubility, protein 
binding to albumin at approximately 55%, and 
a long onset delay.18 Meanwhile, diazepam has 
a 20-80 hours half-life, highly lipid soluble and 
highly protein bound, and easily crosses the 
blood-brain barrier. Diazepam is considered 
a better choice because of its rapid onset and 
long half-life compared to phenobarbital or in 
the benzodiazepine group.19 

Based on the traction and fireplace test 
results, this study concluded that diazepam 
has a better sedative effect than phenobarbital. 
The sedative effect produced by diazepam is 
more prolonged compared to phenobarbital 
as a positive control. These findings may be 
helpful to other researchers as an alternative 
agent to anesthesia the animal study or as 
a positive control in an anesthesia study. 
However, there were still several areas for 
improvement in this study, as we did not collect 
the blood sample to measure the drug plasma 
concentration or collect the organ to assess 
molecular parameters regarding the sedative 
effect. These could be used as complementary 
data to resulting comprehensive results. 
Despite that, this study followed the ethical 
principle of animal welfare, the sample 
size counted based on the Federer formula, 
the animal study health was under expert 
supervision, and used established methods. 
Therefore, suggestions for further research 
to be performed with different test methods, 
parameters, and comparisons.

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36 International Journal of Integrated Health Sciences (IIJHS), Vol 11, Number 1, March 2023

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References 

Marsha Vania, Shinta Dewi Permata Sari, et al