Inter. J. of Phytotherapy / Vol 3 / Issue 1 / 2013 / 18-23.

e - ISSN - 2249-7722
Print ISSN - 2249-7730

International Journal of Phytotherapy

www.phytotherapyjournal.com

EVALUATION OF ANTIDEPRESSANT ACTIVITY OF TRAMADOL

AND TRAMADOL PLUS IMIPRAMINE USING RESERPINE
INDUCED HYPOTHERMIA MODEL ON EXPERIMENTAL
ANIMALS
Mohd Riyaz1, Vinit Raj1, Ashish Kumar2, Satyajeet Singh2
1,2

Roorkee College of Pharmacy (RCP Universe) 09 milestone Roorkee-Dehradun Highway, Vill. Kishanpur, P.B.NO.104,
Roorkee, Distt. Haridwar Uttarakhand - 247667, India.

ABSTRACT
The present study was carried out to evaluated antidepressant activity of Tramadol and Tramadol plus
Imipramine in experimental animals by using Reserpine Induced Hypothermia Test. Standard drug used in tests was
Imipramine (10 mg/kg p.o.). It is a tricyclic antidepressant and it is a monoamine reuptake inhibitor. It inhibits the
reuptake of noradrenalin and serotonin (5-HT) by monoaminergic nerve terminals, and thus it facilitates transmission.
So, the possible mechanism of Tramadol and combination of Tramadol and Imipramine to decrease the immobility
time may be due to the inhibition of monoamine reuptake. These models suggest that Tramadol and combination of
Tramadol and Imipramine increases availability of biogenic amines Hence, Tramadol may produces antidepressant
due to inhibition of reuptake of noradrenalin and 5-HT (serotonin).
Key words: Serotonin, Dopamine transport, Biogenic amines, Reserpine Induced Hypothermia Test, Imipramine, and
Tramadol.
INTRODUCTION
The cause of depression was linked to
decreased brain levels of the neurotransmitters NE, 5HT, and DA, although the actual cause remains unknown
[1, 2]. Although the reuptake blockade of monoamines
(e.g., NE and 5-HT) occurs immediately upon
administration of an antidepressant, the clinical
antidepressant effects generally are not observed until
after 4 weeks of dosing It is apparent that no single
neurotransmitter theory of depressions adequate.[3, 5].
The 5-HT or NE link hypothesis maintains that both the
serotonergic and noradrenergic systems need to be
functional for an antidepressant effect to be exerted [6].
The 5-HTor NE link hypothesis is also consistent with
the rationale of the postsynaptic alteration theory of
depression, which emphasizes the importance of alpha

adrenergic receptor downregulation for achieving an

antidepressant effect [7]. Again, it has been proposed
that both NE and 5-HT are necessary for homologous
desensitization of central a-adrenergic receptors by
antidepressants. Tramadol is a synthetic centrally acting
opioid analgesic used mainly for the treatment of
moderate to severe pain. It is a weak opioid receptor
agonist and also produces analgesia by inhibiting uptake
of norepinephrine and serotonin [8].
MATERIALS AND METRHODS
All the drugs used in present study were of
pharmaceutical grade. Tramadol was gifted by Prolaboratories Pvt, Ltd, Roorkee, India, used as a test drug.
All solvents were of analytical grade and procured from

Corresponding Author:- Mohd Riyaz Email: riyaztyagi1986@gmail.com,

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Inter. J. of Phytotherapy / Vol 3 / Issue 1 / 2013 / 18-23.

Division of Pharmaceutical Sciences, Shri Guru Ram Rai

Institute of Technology and Science, Patel Nagar
Dehradun. Reserpine was purchase from Rajesh
Chemicals Mumbai, India and used for induction of
hypothermia. Imipramine was gifted from Unicure
Pharmaceuticals Pvt Ltd, Roorkee India.
Animals
Swiss albino mice weighing between 2025g.
All the animals were acclimatized at least under standard
husbandry conditions, i.e. room temperature of 24 1 C;
relative humidity 45 55% and a 12 : 12 h light/dark
cycle. The animals were free access to standard pellets of
rat with water supplied under strict hygienic conditions.
Each experimental group was separate set of animals and
care was taken to ensure that animals used for one
response was not employed anywhere. Animals were
habituated to laboratory conditions for 48 hours prior to
experimental protocol to minimize if any non- specific
stress [9, 13].
Experimental protocol
Experimental designs for Reserpine Induced
Hypothermia Test.
Animals divided in to nine groups and each group
containing six animals (n=6).
Group 1- (Negative control) Give Saline (1ml/100gm
body wt.)
Group 2-(Positive control) Give Reserpine (2mg/kg
body wt. s.c.)
Group 3-(Standard control) Give Reserpine (2 mg/kg
body wt. s.c.) and after 18 hr give Imipramine (10 mg/kg
body wt. p.o.)
Group 4- Give Reserpine (2 mg/kg body wt. s.c.) and
after 18 hr give Tramadol (10 mg/kg body wt.p.o.)
Group 5- Give Reserpine (2 mg/kg body wt. s.c.) and
after 18 hr give Tramadol (20 mg/kg body wt.p.o.)
Group 6- Give Reserpine (2 mg/kg body wt.s.c.) and
after 18 hr give Tramadol (40 mg/kg body wt. p.o.)
Group 7- Give Reserpine (2 mg/kg body wt.s.c.) and
after 18 hr give Imipramine (5 mg/kg body wt. p.o.) +
Tramadol (5mg/kg body wt.p.o.)
Group 8- Give Reserpine (2 mg/kg body wt.s.c.) and
after 18 hr give Imipramine (5 mg/kg body wt. p.o.) +
Tramadol (10mg/kg body wt.p.o.)
Group 9- Give Reserpine (2 mg/kg body wt.s.c.) and

after 18 hr give Imipramine (5 mg/kg body wt. p.o.) +

Tramadol (20mg/kg body wt. p.o.)
METHODS
Reserpine induced hypothermia
Depletion of biogenic amines (noradrenalin, 5hydroxytryptamine, and dopamine) in the brain induces
not only catalepsy and apoptosis but also hypothermia in
rodents. The decrease of body temperature induced by
Reserpine is antagonized by antidepressants, MAOinhibitors and central stimulants. The subcutaneous
administration of 2 mg/kg Reserpine leads to a decrease
of core temperature in mice to 2023 C after 18 h. The
fall in temperature can be antagonized by antidepressants.
Procedure
Groups of 6 Swiss albino mice (20-25 g body
weight) were used. On the day before testing, they were
dosed with 2 mg/kg Reserpine s.c. They were housed in a
climate controlled animal colony and have free access to
food and water. Eighteen hours after Reserpine
administration, the animals were placed into individual
cages. The initial rectal temperature was determined by
insertion of an electronic thermometer to a constant depth
of 2 cm. Following administration of the test compound
the rectal temperature were measured again at 60 min
intervals for 7 hrs [14].
Evaluation
Rectal temperature was recorded every hour. The
difference in temperature from vehicle controls were
calculated for each time and the maximal difference were
scored. The differences will be then statistically
compared.
Statistical analysis
The values were expressed as mean SEM. The
results were subjected to statistical analysis by using oneway ANOVA. Followed by Bonferroni Test, P<0.05 was
considered as significant.
RESULTS
In Reserpine Induced Hypothermia Model the
Reserpine treated groups showed decrease in body
temperature after 18 hours of administration. Imipramine
(10 mg/kg p.o.) used as standard drug significantly
increase body temperature of mice after 18 hours the
administration of Reserpine. Treatment of animals with
Tramadol (10 mg/kg, 20 mg/kg and 40 mg/kg p.o.) and
combination of Tramadol and Imipramine (5 mg/kg+5
mg/kg, 5 mg/kg+10 mg/kg and 5 mg/kg+20 mg/kg p.o.)
increase body temperature significantly (p<0.001) till 7
hrs. when compared with Positive control group after
treatment with Reserpine. Tramadol was found to be
comparable to Imipramine (10 mg/kg p.o.) for seven hrs.
treatment.

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Inter. J. of Phytotherapy / Vol 3 / Issue 1 / 2013 / 18-23.

Table 1. Effect of Tramadol (p.o.) on Body Temperature in the Reserpine Induce Hypothermia Test using Swiss albino mice.
Basal temp.
Temperature after treatment (oC)
o
( C) after 18
S.
Treatment
Basal Temp.
hrs.
No.
1 hrs
2 hrs 3 hrs 4 hrs 5 hrs
6 hrs
7 hrs
of Reserpine
Treatment
37.4 37.52
37.46 37.44
37.56 37.36 37.76
Negative
20. 0.1
1
37.760.16
37.580.16
0.10### 0.09
0.04 0.09 0.07
Control
08
6
@@
@@@
@@@
@@@
@@@
@@@

Positive
Control

Standard
(IMP
10mg/kg)

TMD
(10mg/kg)

37.70.11

35.530.15

35.54
0.12
***
##

37.660.20

37.680.16

35.610.20

35.620.18

35.31
0.06*
**
##

36.23
0.12

36.66
0.09*

***

**@@@

35.89
0.08
***

35.4
10.
10***

36.37
0.18*
**@@@

###

35.560.13

TMD
(20mg/kg)

35.99
0.11
***

36.49
0.11*
**@@@

35.330.12

TMD
(40mg/kg)

36.06
0.12
***

36.79
0.12*
@@@

37.38
0.0
6

@@@

@@@

36.7

0.17*
*

36.7
3
0.11*
*
@@@

37.840.11

35.35
0.1
6***##

37.0
7
0.07

@@@

37.780.13

@@@

37.06
0.1
6
@@@

37.21

0.12

35.76
0.12*
**
###

36.12
0.19*
**

36.34
0.17*
**

###

###

37.68
006

37.67
0.14

@@@

@@@

37.71
0.15

37.48
0.18

37.83
0.16

37.97
0.15

@@@

@@@

@@@

37.61
0.09

37.80
0.08

38.08
0.07

@@@

@@@

@@@

38.07
0.12

37.99
0.16

37.99
0.14

@@@

@@@

@@@

@@@

37.4
1
0.09

37.86

0.09

@@@

@@@

IMP is Imipramine as a standard drug and TMD is Tramadol as a test drug. When compared with Negative Control group (p<
0.05 = *, p<0.01 = ** & p<0.001 = ***), When compared with Standard group (p<0.05 = #, p<0.01 = ## & p<0.001 = ###)
and When compared with Positive Control (p<0.05 = @, p<0.01 = @@ & p<0.001 =@@@). The results were analyzed for
statistical significance using one-way ANOVA followed by Bonferroni test. p<0.05 was considered a significant.
Table 2. Effect of combination of Tramadol and Imipramine (p.o.) on Body Temperature in the Reserpine
Hypothermia Test using Swiss albino mice using.
Basal temp. (oC)
Temperature after treatment (oC)
S.
Basal
after 18 hrs. of
Treatment
Temp.
No.
Reserpine
1 hrs
2 hrs 3 hrs 4 hrs
5 hrs
6 hrs
Treatment
37.42
37.46
37.44
37.52 37.56
37.36
37.760.1
0.0
Negative
37.580.16
0.10
0.09
0.16 0.04
0.09
1
6
8
Control
@@
@@@
@@@
@@@
@@@
@@@

###

Positive
Control

37.70.11

35.530.15

35.54
0.12

35.31
0.06*

***
##

**
##

~ 20 ~

35.41
0.1
0***
###

35.35
0.16
***
###

35.76
0.12*
**
###

36.12
0.19*
**
###

Induce

7 hrs
37.76
0.07
@@@

36.34
0.17*
**
###

Inter. J. of Phytotherapy / Vol 3 / Issue 1 / 2013 / 18-23.

Standard
(IMP
10mg/kg)

IMP+TMD
(5mg+5mg/k
g)

37.660.2
0

37.810.1
3

35.610.20

35.480.13

36.23
0.12

36.66
0.09*

***

**@@@

35.89
0.12
***

36.31
0.13*
@@@

37.07

0.07
@@@

36.70

0.15*
*
@@@

IMP+TMD
(5mg+10mg/
kg)

37.820.1
1

IMP+TMD
(5mg+20mg/
kg)

37.780.1
2

35.340.11

35.92
0.03
***

35.350.13

36.15
0.04
*** @@

36.46
0.12*
**@@@

36.91
0.10*
**@@@

37
0.11
@@@

37.43

0.13
@@@

37.38
0.06

37.68
006@

37.67
0.14

@@@

@@

@@@

37.71
0.15

37.05

0.13

37.43
0.14

37.68
0.12

37.96
0.12

@@@

@@@

@@@

37.81
0.15

37.91
0.15

38.06
0.14

@@@

@@@

@@@

38.12
0.13*

38.21
0.14

*@@@

@@@

@@@

37.49

0.12
@@@

37.97
0.15

38.08
0.15
@@@

#
@@@

IMP is Imipramine as a standard drug and TMD is Tramadol as a test drug. When compared with Negative Control group (p<
0.05 = *, p<0.01 = ** & p<0.001 = ***), When compared with Standard group (p<0.05 = #, p<0.01 = ## & p<0.001 = ###)
and When compared with Positive Control (p<0.05 = @, p<0.01 = @@ & p<0.001 =@@@). The results were analyzed for
statistical significance using one-way ANOVA followed by Bonferroni test. p<0.05 was considered a significant.
Figure 1. Effect of Tramadol (p.o.) on Body Temperature in the Reserpine Induce Hypothermia Test using Swiss
albino mice.

38.5
38
37.5
Basal Temp.

Temperature (oC)

37

1 hrs

36.5

2 hrs

36

3 hrs

35.5

4 hrs
5 hrs

35

6 hrs

34.5

7 hrs

34
33.5

Negative
Control

Positive
Control

Standard(IMP
T MD(10mg/kg) T MD(20mg/kg) T MD(40mg/kg)
10mg/kg)

The effect of Tramadol (10, 20, 40 mg/kg p.o.) and Imipramine 10mg/kg p.o. on the five minute intervals in Reserpine
Induced Hypothermia test. The results were analyzed for statistical significance using one-way ANOVA followed by
Bonferroni test. p<0.05 was considered a significant effect as compared to control group 1st to 7th hrs. Treatment.

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Inter. J. of Phytotherapy / Vol 3 / Issue 1 / 2013 / 18-23.

Figure 2. Effect of combination of Tramadol and Imipramine (p.o.) on Body Temperature in the Reserpine Induce
Hypothermia Test using Swiss albino mice

39
38.5
38

Basal Temp.
1 hr
2 hr
3 hr
4 hr
5 hr
6 hr
7 hr

Temprature (oC)

37.5
37
36.5
36
35.5
35
34.5
34
33.5

Neg ative
Co ntro l

Po s itive Co ntro l

Stand ard
(IM P 10 mg /kg )

IM P+TM D
(5mg +5mg /kg )

IM P+TM D
(5mg +10 mg /kg )

IM P+TM D
(5mg +2 0 mg /kg )

The effect of combination of Imipramine and Tramadol (5+5, 5+10, 5+20 mg/kg p.o.) and Imipramine 10mg/kg p.o. in the
five minute intervals on Reserpine induced Hypothermia Test. The results were analyzed for statistical significance using
one-way ANOVA followed by Bonferroni test. p<0.05 was considered a significant effect as compared to control group 1 st to
7th hrs. Treatment.
DISCUSSION
In Reserpine Induced Hypothermia Model the
Reserpine causes depletion of biogenic amines in the
brain and which not only induces catalepsy and apoptosis
but also induces hypothermia in mice. Reserpine
decreases the body temperature. Antidepressant drugs
increases the body temperature and have slow onset of
action and action is long lasting. Tramadol and
Combination of Tramadol and Imipramine have
maintained the body temperature of mice significantly
(p<0.001) and this produces antidepressant effect as
compared to control animals. This model suggest that
Tramadol and combination of Tramadol and Imipramine
increases availability of biogenic amines Hence,
Tramadol may produces antidepressant due to inhibition
of reuptake of noradrenalin and 5-HT (serotonin).

CONCLUSION
In Present study of Evaluation of Antidepressant
Activity of Tramadol and Tramadol plus Imipramine
Using Reserpine Induced Hypothermia Model on
Experimental Animals shows that Tramadol is a potent
antidepressant. The study also revealed that combination
of Imipramine (5 mg/kg) with Tramadol (5 mg/kg, 10
mg/kg and 20 mg/kg) showed additive antidepressant
activity than the individual.
ACKNOWLEDGEMENTS
The authors would like to express their gratitude
to S. G. R. R. I. T. S. (Division of Pharmaceutical
Sciences) Dehradun for providing the animals, chemicals
and apparatus. One of the authors (Mohd .Riyaz) is also
thankful to Priya Pal for helping in research work.

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