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Content uploaded by Dr. Pravin Tirgar
Author content
All content in this area was uploaded by Dr. Pravin Tirgar
Content may be subject to copyright.
Study of Sedative, Anxiolytic, CNS – Depressant and Skeletal Muscle
Relaxant Effects of Methanolic Extract of Hibiscus Rosa-Sinensis on
Laboratory Animals
Ganatra TH*, Joshi UH, Patel MN, , Desai TR Tirgar PR
Department of Pharmacology, R. K. College of Pharmacy, Kasturbadham, Bhavanagar Road, Rajkot, India - 360020
Abstract:
Aim: Methanolic extract of Hibiscus rosasinensis (MEHR) shows sedative, anxiolytic, skeletal muscle relaxant and
CNS – depressant effect.
Methods: Hibiscus rosasinensis, found in south Asia, was extracted by methanol and the practical yield of extract was
found to be 7.2%w/w.
Models:
1. Anxiolytic action was studied by the open field behavior model in which it significantly increase in rearing and
crossing, elevated plus-maze model in which MEHR significantly increase the entry in both arm, dark & light field
model in which MEHR significantly increase the time spent in dark field, compared with control-CMC solution
[using diazepam (25 mg/kg, i.p.)].
2. Sedative effect was seen by Phenobarbital induce sleep model.
3. Skeletal muscle relaxant effect was studied using rota rod model, in which MEHR significantly increase the time of
fall, compared with control-CMC solution [using diazepam (25 mg/kg, i.p.)].
4. CNS - depressant effect was studied using Actophotometer model in which MEHR significantly decrease the
number of cut-off, compared with control-CMC solution [using diazepam (25 mg/kg, i.p.)].
Conclusion: The MEHR shows seadative, anxiolytic, skeletal muscle relaxant and CNS – depressant effects.
Keywords: Hibiscus, sedative, anxiolytic, skeletal muscle relaxant, rota-rod and CNS – depressant effects,
actophotometer, etc.
INTRODUCTION:
[1,2,5]
Biological Name: The dried ripe flowers,
leaves of Hibiscus rosa-sinenis belongs to
family Malvaceae.
[1]
Source: Widely spread all over the world,
majorly in tropical and subtropical areas.
[2]
Description: The leaves are alternate, simple
and ovate to lanceolate, often with a toothed or
lobed margin. The flowers are large,
conspicuous, trumpet-shaped, with five or
more petals, ranging from white to pink, red,
orange, purple or yellow, and from 4–18 cm
broad.
[2, 6]
Synonyms: Aloala [Hawai]; red hibiscus;
China rose [English]; da hong hua (big red
flower) [China]; Jasud [Gujarati], japapushpam
[India]; shoeflower [Jamaica].
[5]
Nomenclature: Hibiscus is taken from the
Greek "hibiscos," a name for mallow.
[6]
Pharmacological Actions: Abortifacient
[5]
;
analgesic
[5]
; antidiarrhoic
[5]
; antiestrogenic
[1]
;
antifungal
[5]
; anti-infectious; anti-inflammatory;
antipyretic; astringent
[5]
; CNS depressant
[1]
;
constipating
[1]
; contraceptive; demulcent
[5]
;
dentifrice
[6]
; diuretic
[6]
; expectorant
[5]
;
hemostat; hypoglycemic
[5]
; hypotensive;
hypothermic; insect attractant
[1]
; promotes hair
growth and color
[1,2,,5,6]
; purgative
[1]
;
refrigerant
[5]
; relaxes spasm
[1]
; soothes irritated
tissue.
[5]
Collection of materials and Method for
extraction:
The herb of Hibiscus was collected from local
region in Rajkot district of Gujarat &
morphological & microscopy of pant was
authentified by pharmarcognosy department of
R.K. College of pharmacy. The leaves were
separated and dried between 55 º to 60º C and
then pulvirized to very fine powder. The
powder was extracted using Soxhlet apparatus
Ganatra TH et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1146-1155
1146
using methanol as a solvent. The % yield was
found to be 7.2%W/W.
Animals:
Male Swiss albino rat of weighing 220-
280 g were used for the study.
The animals were procured from Animal House
, Department of Pharmacology, R.K.College of
Pharmacy, Rajkot, India. The animals were
place at random and allocated to treatment
groups in polypropylene cages with paddy husk
as bedding. Animals were housed at a
temperature of 24±20˚C and relative humidity
of 30 – 70 %. A light and dark cycle was
followed. All animals were fed on standard
balance diet and provided with water ad
libitum.
Experiments were carried out between 09:00 an
d 14:00 h.
All the experimental procedures and protocols
used in the study were reviewed and
approved by the Institutional AnimalEthical
Committee(IAEC) and care of laboratory
animals was taken as per the guidelines of
Committee for the purpose of control and
supervision of experiments on
animals(CPCSEA), Govt. of India (Registration
No.1131/ac/07/CPCSEA.)
EXPERIMENTAL MODELS AND
STUDIES (METHODS):
The study includes the sedative, anxiolytic,
skeletal muscle relaxant effect and CNS-
depressant effect of Methanolic extract of
Hibiscus rosa-sinensis [MEHR].
A] For Sedative effect:
1. Phenobarbital induced sleep model:
The 18 animals (mice) divided in to 3 groups
containing 6 animals each.
[Normal: 0.5ml 1% CMC solution, i.p.;
standard: Phenobarbital: 5 mg/kg, i.p.; test:
MEHR: 200 mg/kg, p.o.]. Observe the onset of
time and duration of action.
[3,4,9,11]
. From the
given data in table: 1 we can say that drug
possesses sedative effect.[Chart 1]
B] For Anxiolytic effect:
1. Open-field behavior model:
Instrument: The apparatus consisted of a
wooden box (60 Χ 60 Χ 60 cm). The arena of
the open field was divided into 16 squares (15
Χ 15 cm): the four inner squares in the center
and 12 squares in the periphery along the walls.
The experimental room was a sound attenuated,
dark room.
Method: The 18 animals (mice) divided in to 3
groups containing 6 animals each. [Normal:
0.5ml 1% CMC solution, i.p.; standard:
diazepam: 25 mg/kg, i.p.; test: MEHR: 200
mg/kg, p.o.]. Allow the animal to freely move
in the model and note the number of crossing
and number of rearing. From the given data
table 2 the number of crossing increases in test
compare to normal.
[3,4]
[Chart 2]
2. Elevated Plus model:
Instrument: The EPMT apparatus consisted of
four arms elevated 30 cm above the floor, with
each arm positioned at 90° relative to the
adjacent arms. Two of the arms were enclosed
with high walls (30 Χ 7 Χ 20 cm), and the other
arms were connected via a central area (7 Χ 7
cm) to form a plus sign.
Method: The 18 animals (mice) divided in to 3
groups containing 6 animals each. [Normal:
0.5ml 1% CMC solution, i.p.; standard:
diazepam: 25 mg/kg, i.p.; test: MEHR: 200
mg/kg, p.o.]. Allow the animal to freely move
in the model and note the number of entry in
open arm and close arm. From the given data
table 3 the number entry in open arm increases
in test compare to normal.
[3,4]
[Chart 3]
3. Dark and light field model:
Instrument: It consists of open top wooden box.
Two distinct chambers, a black chamber (25 cm
long Χ 35 cm wide Χ 35 cm deep), painted
black and made dark by covering its top with
black plywood, and a bright chamber (25 cm
long Χ 35 cm wide Χ 35 cm deep), painted
white and brightly illuminated with 40-W white
light source, were placed 25 cm above the open
box. The two chambers were connected through
a small open doorway, (7.5 cm long Χ 5 cm
wide) situated on the floor level at the center of
the partition.
Method: The 18 animals (mice) divided in to 3
groups containing 6 animals each. [Normal:
0.5ml 1% CMC solution, i.p.; standard:
diazepam: 25 mg/kg, i.p.; test: MEHR: 200
mg/kg, p.o.]. Allow the animal to freely move
Ganatra TH et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1146-1155
1147
in the model and note the number of entry in
light field as well as count the time spent in
light field. From the given data table 4a & 4b
the number entry as well as time spent in light
field increases in test compare to normal.
[7]
[Chart 4 a & b]
C] For CNS-depressant effect:
1. Actophotometer:
The 18 animals (mice) divided in to 3 groups
containing 6 animals each. [Normal: 0.5ml 1%
CMC solution, i.p.; standard: diazepam: 25
mg/kg, i.p.; test: MEHR: 250 mg/kg, p.o.].
Allow the animal to freely move in the model
and note the number of cut off (crossing) of
lesser for 2 minutes. From the given data in
table 5 the number of cut off decreases in test
compare to normal.
[3,4,10]
[Chart 5]
D] For Skeletal muscle relaxant effect:
1. Rota rod model:
Instrument: Rota rod apparatus consisted of a
base platform and an iron rod of 3 cm diameter
and 30 cm length, with a non-slippery surface.
This rod was divided into two equal sections by
two disks, thus enabling two mice to walk on
the rod at the same time at the speed of 25 rpm.
Method: The 18 animals (mice) divided in to 3
groups containing 6 animals each. [Normal:
0.5ml 1% CMC solution, i.p.; standard:
diazepam: 25 mg/kg, i.p.; test: MEHR: 200
mg/kg, p.o.].
The animal is put on the rotating rod and the
time required to fall down from the rod is
measured. From the given data, in table 6 the
time required to fall down is lesser in test
compared to normal.
[3,4,11]
[Chart 6]
Statistical Analysis: Results were expressed as
mean ± SEM. Difference in means were
compared using one way analysis of variance
(ANOVA) followed by Tukey’s test. P<0.001
were considered statistically significant.
Result:
Activity and
Model
Parameters
Control Standard Test
Before
Reading
±SEM
After
Reading
±SEM
Before
Reading
±SEM
After
Reading
±SEM
Before
Reading
±SEM
After
Reading
±SEM
Sedative
(Phenobarbital
induce sleep)
Onset of action
(Minutes)
--- --- --- 26.5±0.84 --- 33.16±1.25
Duration of action
(Minutes)
--- --- --- 187.66±4.75 --- 159±3.41
Anxiolytic
(open field model)
Number of crossing
18.75±1.1 21.5±1.5 19±1.04 48.5±1.73 16±1.42 35.83±0.97
Anxiolytic
(Elevated plus
model)
Entries in open arm
8.16±0.49 8.5±0.27 7.5±0.52 18.33±2.12 8±0.44 14±0.89
Anxiolytic
(Light/Dark
model)
Number of Entry in
light area
10.5±0.64 11±0.70 9±0.40 20.5±0.6 10.66±0.4 18±0.40
Time spent in light
area (seconds)
25±0.70 24±0.70 24±0.70 112±0.9 22±0.70 72.5±0.93
CNS-depressant
(Actophotometer)
Number of cut-off
52.5±0.76 53±0.63 51.5±0.8 9.5±0.62 51±0.96 20±0.36
SKM-relaxant
(Rota-rod model)
Time of fall
(Seconds)
--- 246.6±3.3 --- 5.83±0.60 --- 17.5±0.80
Ganatra TH et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1146-1155
1148
Images of Models used in work:
Table 1: Sedative effect of MEHR on rat using Phenobarbital induced sleep model
For Sedative: phenobarbiatal induced sleep model
Sr. No. Group
Control (Seconds) Standard (Seconds) Test (Seconds)
Onset of
time
Duration
of action
Onset of
time
Duration of
action
Onset of
time
Duration of
action
1 Head --- --- 26 190 30 155
2 Tail --- --- 27 180 34 154
3 Back --- --- 24 189 29 148
4 Head back --- --- 30 200 35 167
5 Back tail --- --- 27 169 34 160
6 No mark --- --- 25 198 37 170
Average --- --- 26.5 187.66 33.16 159
SD --- --- 2.073 11.60 3.06 8.34
SEM --- --- 0.84 4.75 1.25 3.41
variance --- --- 6.25 66.91 8.66 63.33
ACTOPHOTOMETER
ROTAROD
OPEN FIELD MODE
L
ELEVATED PLUS MODE
L
LIGHT DARK MODE
L
Ganatra TH et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1146-1155
1149
Table 2: Anxiolytic effect of MEHR on rat using Open field model
For Anxiolytic action: Open field model (number of crossing in 5 minutes)
Sr. No. Group
Control Standard Test
Before After Before After Before After
1 Head 15 17 22 51 20 39
2 Tail 18 21 20 54 18 36
3 Back 22 26 16 46 12 33
4 Head back 18 20 18 45 16 36
5 Back tail 19 23 20 49 15 36
6 No mark 20 22 18 46 14 35
Average 18.667 21.5 19 48.5 15.833 35.833
SD 2.3381 3.0166 2.0976 3.5071 2.8577 1.9408
SEM 1.169 1.5083 1.0488 1.7536 1.4289 0.9704
variance 5.46 9.1 4.4 12.3 8.16 3.76
Table 3: Anxiolytic effect of MEHR on rat using Elevated plus model
For Anxiolytic action: Elevated Plus method (number of entry in open arm 5 minutes)
Sr. No.
Group
Control Standard Test
Before After Before After Before After
1 Head 9 8 7 15 7 14
2 Tail 8 9 6 14 8 13
3 Back 9 9 9 19 9 15
4 head back 7 8 8 19 9 15
5 back tail 9 9 7 17 8 14
6 No mark 7 8 8 26 7 13
Average 8.16 8.5 7.5 18.33 8 14
SD 0.98 0.54 1.04 4.274 0.89 0.89
SEM 0.49 0.27 0.52 2.137 0.44 0.44
variance 0.96 0.3 1.1 18.26 0.8 0.8
Table 4 a: Anxiolytic effect of MEHR on rat using Light Dark model
For Anxiolytic action: Light Dark model (number of entry in light field 5 minutes)
Sr. No. Group
Control Standard Test
Before After Before After Before After
1 Head 11 10 10 22 10 18
2 Tail 12 13 9 20 12 18
3 Back 10 11 9 21 11 19
4 No mark 9 10 8 19 10 17
5 back tail 11 12 8 21 10 16
6 No mark 10 10 10 20 11 20
Average 10.5 11 9 20.5 10.66 18
SD 1.048 1.26 0.89 1.04 0.81 1.41
SEM 0.52 0.63 0.44 0.52 0.40 0.70
variance 1.66 2 0.66 1.66 0.91 0.66
Ganatra TH et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1146-1155
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Table 4 b: Anxiolytic effect of MEHR on rat using Light Dark model
For Anxiolytic action: Light Dark model (Time spent in light field 5 minutes)
Sr. No. Group
Control Standard Test
Before After Before After Before After
1 Head 25 23 24 115 21 73
2 Tail 26 22 23 113 22 75
3 Back 24 25 22 112 23 74
4 No mark 23 24 25 110 22 71
5 back tail 27 26 26 110 20 72
6 No mark 25 24 24 112 24 70
Average 25 24 24 112 22 72.5
SD 1.41 1.41 1.41 1.89 1.41 1.87
SEM 0.70 0.70 0.70 0.94 0.70 0.93
variance 1.66 1.66 1.66 4.33 0.66 2.91
Table 5: CNS -depressant effect of MEHR on rat using Actophotometer model
For CNS-Depressant: Actophometer model (number of cut-off in 2 minutes)
Sr. No. Group
Control Standard Test
Before After Before After Before After
1 Head 50 51 49 9 51 19
2 Tail 52 52 52 10 55 20
3 Back 55 54 51 8 50 21
4 head back 51 52 50 8 50 19
5 back tail 53 54 52 10 52 21
6 No mark 54 55 55 12 48 20
Average 52.5 53 51.5 9.5 51 20
SD 1.87 1.54 2.07 1.51 2.36 0.89
SEM 0.76 0.63 0.84 0.62 0.96 0.36
variance 3.5 2.4 4.3 2.3 5.6 0.8
Table 6: Skeletal Muscle Relaxant effect of MEHR on rat using Rota-rod model
For Skelatal muscle relaxant: Rota rod model (time required to fall down)
Sr. No. Group Control (seconds) Standard (seconds) Test (seconds)
1 Head 240 5 17
2 Tail 250 6 15
3 Back 260 8 21
4 head tail 250 5 17
5 back tail 240 7 18
6 No mark 240 4 17
Average 246.66 5.83 17.5
SD 8.16 1.47 1.97
SEM 3.34 0.60 0.80
variance 66.66 2.16 3.9
Ganatra TH et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1146-1155
1151
Chart 1: Sedative effect of MEHR on rat using Phenobarbital induced sleep model
* indicate significant difference from control (p<0.001)
Chart 2: Anxiolytic effect of MEHR on rat using Open field model
* indicate significant difference from control (p<0.001)
Chart 3: Anxiolytic effect of MEHR on rat using Elevated plus model
* indicate significant difference from control (p<0.001)
Ganatra TH et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1146-1155
1152
Chart 4 a: Anxiolytic effect of MEHR on rat using Light Dark Field model
* indicate significant difference from control (p<0.001)
Chart 4 b: Anxiolytic effect of MEHR on rat using Light Dark Field model
*
indicate significant difference from control (p<0.001)
Chart 5: CNS -depressant effect of MEHR on rat using Actophotometer model
* indicate significant difference from control (p<0.001)
Ganatra TH et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1146-1155
1153
Chart 6: Skeletal Muscle Relaxant effect of MEHR on rat using Rota-rod model
* indicate significant difference from control (p<0.001)
DISCUSSION:
The result of our study shows methanolic
extract of flower of Hibiscus rosa-sinensis can
markedly reduce fall of time (in Rota-rod
model), decrease in onset of time as well as
increase in duration of action (in phenobarbital
induced sleep model), decrease number of cut-
off (in Actophotometer) and increase in number
of crossing (in open-field behavior model),
increase time spent in light field as well as
increase number of entry in close and open arm
(in elevated plus model).
In conclusion, our data indicates that MEHR
can possess Sedative, anxiolytic, CNS -
depressant and skeletal muscle relaxant
activities.
The MEHR contains flavanoids (hibiscitin),
phenolic content as well as terpenoid
compounds like β – sitosterol, caemphesterol,
etc, which are probably responsible for the
actions.
[5]
CONCLUSION:
The methanolic extract of Hibiscus rosa-
sinensis can increase the duration of action so it
possesses sedative effect.It can also increase the
number of crossing in open field model,
increase the time spent in open arm, entry in
open arm (in elevated plus model) as well as
increase the time spent in light field (in Light
Dark field) thus we can conclude that it can also
possesses anxiolytic action.
The decrease in number of cut-off in
actophotometer model indicates that it can
possess CNS-depressant.It can also decrease the
time of fall from the rotating rod in rota-rod
model, thus it can possesses skeletal muscle
relaxant action.
ACKNOWLEDGEMENT
On the occasion of presenting this paper in
internet journal, I take this as a unique
opportunity to record my deep sense of
gratitude to my esteemed research guide Tirgar
PR, Professor and Head, Department of
Pharmacology, R. K. College of Pharmacy,
Rajkot and also to Dr. Desai TR, Principal, R.
K. College of Pharmacy, Rajkot.
I am also thankful to Pandiya DJ and Sheth DB
for his kind support for authentication and
identification of this plant.
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