Neuro-pharmacological and analgesic effects of Arnica montana extract

Abstract

Anxiety and disorders associated with anxiety are increasing rapidly throughout the world. In the present research Arnica montana (Asteraceae) was studied on neuro-pharmacological and analgesic effect in mice. Decreased locomotor and exploratory activities (open field, head dip cage, stationary rod, cage cross, forced swimming and light and dark) were observed in mice at the dose of 100 mg/kg. These results were found significant at P<0.05 when compared with Diazepam 2mg/kg. A. montana crude extract also exhibited potent analgesic effect at the doses of 50 and 100 mg/kg (P<0.05). Aspirin 300 mg/kg was used as a reference drug. Highest % of inhibition (57.6%) was observed in the third phase of acetic acid induced writhing test at the dose of 100 mg/kg of A. montana. Whereas, significant analgesic response was also observed at the low dose (50mg/kg). The percentage inhibition by formalin induced licking biting was found significant in the 2nd (76.1%) and 3rd (19.8%) phase at 50 mg/kg. These results were suggested that the extract of A. montana possess significant analgesic and anxiolytic effect.

Full text

Research Article
NEURO-PHARMACOLOGICAL AND ANALGESIC EFFECTS OF ARNICA MONTANA EXTRACT
MANSOOR AHMAD*1, FARAH SAEED2, MEHJABEEN3, NOOR JAHAN4
1Research Institute of Pharmaceutical Sciences, University of Karachi, 2Department of Pharmacognosy, Dow College of Pharmacy, Dow
University of Health Sciences, 3Department of Pharmacology, Faculty of Pharmacy, Federal Urdu University of Arts Science and
Technology, 4Department of Pharmacology, Dow College of Pharmacy, Dow University of Health Sciences.
Email: herbalist53@yahoo.com, mehjbn1@gmail.com
Received: 05 Aug 2013, Revised and Accepted: 07 Sep 2013
ABSTRACT
Anxiety and disorders associated with anxiety are increasing rapidly throughout the world . In the present research Arnica montana (Asteraceae)
was studied on neuro-pharmacological and analgesic effect in mice. Decreased locomotor and exploratory activities (open field, head dip cage,
stationary rod, cage cross, forced swimming and light and dark) were observed in mice at the dose of 100 mg/kg. These results were found
significant at P<0.05 when compared with Diazepam 2mg/kg. A. montana crude extract also exhibited potent analgesic effect at the doses of 50 and
100 mg/kg (P<0.05). Aspirin 300 mg/kg was used as a reference drug. Highest % of inhibition (57.6%) was observed in the third phase of acetic
acid induced writhing test at the dose of 100 mg/kg of A. montana. Whereas, significant analgesic response was also observed at the low dose
(50mg/kg). The percentage inhibition by formalin induced licking biting was found significant in the 2nd (76.1%) and 3rd (19.8%) phase at 50
mg/kg. These results were suggested that the extract of A. montana possess significant analgesic and anxiolytic effect.
Keywords: Arnica montana, Anxiolytic, Neuro-pharmacological activities, Diazepam, Analgesic.
INTRODUCTION
Anxiety and its related disorders have become a global problem and
affected most of the population world-wide [1]. Benzodiazepines are
commonly used for the treatment of anxiety [2-3]. They have
pronounced anxiolytic effect along with numerous unpleasant side-
effects. The low safety profile of benzodiazepines initiated the
researchers to explore natural world and discover new compounds
with lesser adverse effects [4-5].
Arnica montana (Wolf's Bane, Leopard's bane), belongs to family
Asteraceae. It contains volatile oil, carotenoids, flavonoids, tannins,
resins and triterpenic alcohol. A. montana, has antiseptic, anti-
inflammatory, anti-bacterial, decongestive and anti-fungal
properties. It also stimulates the forming the granular tissues and
thus accelerating the healing process [6-9].
The arnica flowers are used to treat the pale face skin complexion,
wounds, bruises and burns. The treatment of dislocations, bacterial
infections, skin cancer, bronchitis, tonsillitis, pharyngitis, flu, lung
cirhosis, cystitis, nephritis, kidney infections, coronary
insufficiencies, hypertension, angina, cerebral trauma, headaches,
paresis, semi-paresis, insomnia, heart palpitations, nightmares, night
terrors, moral depressions, neurosis, hysteria etc are the other uses
of A. montana [10-12].
MATERIAL AND METHOD
Arnica montana plant extract was purchased from Bioron suppliers.
The extracts obtained were stored in cool, dry place for further
studies. Male albino mice weighing 20-25g were maintained under
standard nutritional and environmental conditions throughout the
experiment. The animals had access to water and food ad libitum.
The animals were deprived of food 12 h before experimentation.
Diazepam (2 mg), aspirin (300 mg), formaldehyde and acetic acid
were purchased from the local Pharmacy and retail chemical stores.
All drugs and A. montana extract were dissolved in saline solution
which used as a vehicle to a desired concentration. Then, they were
filtered through gauze and given to the animals via the intra-gastric
feeding tube. All administered substances including the A. montana
suspension were freshly prepared.
Neuro-pharmacological activities were studied by open field,
traction, head dip, rearing test and swimming induced depression
test. All tests were performed in a calm and peaceful environment.
Animals were divided in to 5 groups (n= 6 in each group). Group I:
control group mice, Group II: Positive control treated group. In each
test, the positive control group was treated with the standard
reference drugs. In order to determine anxiolytic effect, the animals
were treated with diazepam (2 mg kg-1). Group III- V: The mice
treated with different doses of the test extract respectively
(500mg/kg, 300mg/kg, 100 mg/kg), via oral route [13].
Neuro-pharmacological activities
Open field test
Open-field test is a rodent model used for measuring anxiety and
exploration as well as locomotion in mice. Open field test area is
made of plastic walls and floor divided into nine square of equal
area. It is used to evaluate the exploratory activity of animal. The
observed parameters in this study are the number of square crossed
in 30 minutes [14].
Light dark test
Light and dark test is one of the apparatus designed to test anxiolytic
behavior in mice. The apparatus consists of a plastic box with two
compartments one of which is made of transparent plastic and the
other of black colour plastic. Each animal is placed at the center of
the transparent compartment and then the number of entries in
each space, as well as, time spent in each compartment is recorded
for 30 minutes [15-16].
Head-dip test
This study was conducted using wooded apparatus measuring
40×40 cm with 16 evenly spaced holes. Thirty minutes after
treatment, the mice were placed singly on a board with 16 evenly
spaced holes and the number of times the mice dipped their heads
into the holes was noted. The control and drug treated animals were
placed individually in the head dip box and the observations were
made for 30 minutes [17].
Cage crossing movement
The cage crossing test was performed in a specifically designed mice
cage having rectangular shape. Both control and treated mice were
placed separately in the cage and their movements were noted for
30 minutes [18].
Forced swimming test
In this test mice are forced to swim in a restricted space from which
there is no escape and become immobile. Individual mice were
forced to swim in an open cylindrical container containing 7 cm of
water at 22.0 ±1.0oC; the duration of immobility or struggling in a
period of 6 minutes was recorded. Immobility was evaluated as
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Ahmad et al.
Int J Pharm Pharm Sci, Vol 5, Issue 4, 590-593
591
when mice ceased to struggle and remained floating in the water,
making only necessary movements necessary to keep its head above
water. At the end of the session, the animal was removed from water
and dried gently [19-20].
Stationary rod test
The stationary rod test was used to assess learning ability and
locomotor activity. The test apparatus consisted of horizontal
stainless steel rods with the platform. At first the mice were trained
and then they were allowed to balance on stationary rod. During the
observation time mice maintained balance, travelled and fall from
the stationary rod was noted [21].
Analgesic activity (by acetic acid)
The analgesic effect was assessed by acetic acid induced writhing
test in mice. The animals were administered orally with the test
samples and reference standard drug aspirin (300 mg/kg). 0.6%
acetic acid per kg was injected intra-peritoneal. The resultant
abdominal writhes consist of a contraction of body muscles and the
stretching of hind limb. These abdominal contractions were counted
over three periods of 10 min each after injection of acetic acid. The
anti-nociceptive activity was considered as the reduction of these
writhes in the treated animals. The % inhibition of the test samples
was calculated by using the following formula: (number of writhing
of control) − (no of writhing of test group)/ (number of writhing of
control) ×100 [22].
Analgesic activity (by Formalin)
In this test, 2.5% formalin solution (0.05 ml) was injected in the sub-
plantar region of right hind paw of the mice to induce pain. Aspirin
was administered (30 min before formalin injection) orally to serve as
a control. The A. montana extracts (50 and 100 mg/kg) were
administered orally 60 min before the reaction. The animals were
placed in transparent cage for recording observations and the time
spent in licking and biting responses of the injected paw was taken as
indicator of pain response. Responses were measured for 30 min after
formalin injection. The reduction in the number of licking and biting
responses is the inhibition of the pain score and the % inhibition can
be calculated by the following expression: (mean of control group) −
(mean of treated group)/ (mean of control group) ×100 [23].
Statistical analysis
The results were expressed as mean ± S.E.M. All statistical
comparisons were made by means of Student’s t-test and a P value
smaller than 0.05 was regarded as significant [24].
RESULTS
Locomotor and exploratory behavior of mice
The anxiolytic activity was assessed using open field, head dip and
stationary rod apparatus. The most significant CNS depression
effect was observed at the dose of 100 mg/kg of A. montana
extract. In open field activity the results were found to be
11.331.73 counts in 30 minutes. In head dip test, the mice dipped
head 242.65 times. Number of entries in light compartment is
5.51.51 times. The readings of cage cross is 24.672.41 times. In
forced swimming test (FST) the Mean forced mobility time was
1.780.12 seconds. Mean time of mobility on stationary rod was
12.50.83 seconds. Locomotor and exploratory activity was
reduced in comparison to control and standard drug Diazepam (2
mg/kg-1) (Table 1 and Graph 1).
Acetic Acid Test
Analgesic activity is widely assessed by the method of acetic acid
induced abdominal constrictions. 0.6% (10 ml/kg i.p) acetic acid
solution was administered in mice and the abdominal constrictions
(writhes) were observed after 05 minutes. The writhes were
counted for three phases, each of 10 minutes respectively. The
inhibition of acetic acid induced writhes by Aspirin was as follows;
1st; 66.7%, 2nd; 32.2%, 3rd phase; 35.5%. Where as A. montana, at the
dose of 100 mg/kg exhibited maximum inhibition (57.6 %) in 3rd
phase. (Table 2 and Graph 2).
Formalin Test
The results exhibited prominent analgesic effect in comparison with
aspirin. The analgesic effect of A. montana showed maximum
inhibition of the licking and biting response (76.1%) induced by
formalin at the dose of 50 mg/kg in 2nd phase. Aspirin in 1st, 2nd and
3rd phase showed was 21.7%, 84.3% and 22.8% inhibition
respectively (Table 3 and Graph 3).
Table 1: Neuro-pharmacological activities of Arnica montana
Treatment
Open field
Head Dip cage
Light and dark
Cage cross
FST (mobility time)
Stationary rod test
Control
209.833.97
109.53.67
17.51.99
68.333.68
1.7410.05
171.05
A .montana 500mg/kg
1257.94
65.334.19
15.831.55
26.334.46
0.660.16
22.161.27
A. montana 300mg/kg
187.672.98
67.832.90
212.28
97.832.76
2.380.03
18.161.03
A. montana
100mg/kg
11.331.73
242.65
5.51.51
24.672.41
1.780.12
12.50.83
Standard-Diazepam 2mg/kg
12.50.83
11.50.83
10.4
19.50.83
1.600.06
11.331.73
(fall)
Results were expressed along mean  standard error mean. Significance of data were evaluated at P  0.05 – student t test
Graph 1: Neuro-pharmacological activities of A. montana

Ahmad et al.
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592
Table 2: Analgesic activity of A. montana (by acetic acid)
Treatment
Dose mg/kg
Phase 1
Phase 2
Phase 3
Mean No. of Writhes
% of
Inhibition
Mean No. of Writhes
% of
Inhibition
Mean No. of Writhes
% of
Inhibition
Control
0.5 ml saline
19.5±0.83
-
15.5±0.83
-
14.16±1.03
-
Aspirin (300mg)
6.5±0.83**
66.7
10.5±1.08
32.2
9.16±0.77
35.3
A. Montana (100 mg)
8.33±1.43*
57.4
10±1.41*
35.4
6±0.56**
57.6
A. Montana (50 mg)
10.67±0.96
45.1
8.83±0.86**
43
6.67±0.73*
52.8
Results were expressed along mean  standard error mean. Significance of data were evaluated at P  0.05 – student t test
Graph 2 Analgesic Activity of A. montana (by Acetic Acid)
Table 3: Analgesic activity of A. montana (by formalin)
Treatment
Dose mg/kg
Phase 1
Phase 2
Phase3
Mean No. of biting
& licking
% of Inhibition
Mean No. of
biting & licking
% of Inhibition
Mean No. of
biting & licking
% of Inhibition
Control
0.5 ml saline
19.16±1.03
-
10.67±0.54
-
5.83±1.03
-
Aspirin(300mg)
23.33±1.15
21.7
1.67±0.61
84.3
4.5±0.83**
22.8
A. montana (100 mg)
13.16±3.06**
45.5
1.16±0.52
73.1
6.16±1.86
5.6
A. montana(50 mg)
19±3.42
0.83
0.16±0.18**
76.1
4.67±1.40*
19.8
Results were expressed along mean  standard error mean. Significance of data were evaluated at P  0.05 – student t test
Graph 3: Analgesic Activity of A. montana (by Formalin)
DISCUSSION
Stress is the causative factor behind anxiety and depression. It is
estimated that by 2020, these disorders will become the second
number cause of disability [25]. In present study the neuro-
pharmacological screening like open field activity, stationary rod
activity test and head dip activity of A. montana were evaluated. The
extract decreased the exploratory and locomotor activities in mice.
Our observations indicated that A. montana treated mice spent more
time in light compartment and therefore, had anxiolytic effect. The
light and dark box test in mice is use to assess anxiolytic activity
[26]. The lesser number of entries in the light box reflects the
sedative and anxiolytic effect of the extract [26].
The development of immobility, during swimming test indicated
that the cessation of affective/motivational behavior. Plants rich in
tannins and flavonoid content have been found to be of therapeutic
significance in treating many CNS disorders [27]. A. montana also
contains tannins and flavonoids and maybe due to the presence of
these constituents it exhibited pronounced anxiolytic effect.
The present study revealed that A. montana possess significant
analgesic effect. The medicinal uses of A. montana are well
established and have been effectively used since ancient time for the
treatment of strains, sprains and bruises. A. montana contains
helenalin, a sesquiterpene lactone that is a major ingredient having
anti-inflammatory effect due to which it is mostly used for the

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treatment of bruises. It can be suggested that analgesic effect may be
due to the presence of volatile oil.
CONCLUSION
Our present study revealed that A. montana possesses significant
analgesic and anxiolytic effect, along with prominent decrease in
neuro-pharmacological activities due to its active chemical
constituents. However, further studies are required to confirm the
mechanism of action behind the effects observed in our study.
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