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RESEARCH ARTICLE Am. J. PharmTech Res. 2014; 4(1) ISSN: 2249-3387
Please cite this article in press as: Pai PG. et al., Evaluation of Anxiolytic Effect of Chronic
Administration of Mucuna Pruriens In Wistar Albino Rats. American Journal of PharmTech Research
2014.
Evaluation of Anxiolytic Effect of Chronic Administration of
Mucuna Pruriens In Wistar Albino Rats
Seema Rai1, Preethi G Pai*1, Rajeshwari S1, Ullal Sheetal D1, Nishith RS1, Yogesh Belagali1
1.Department of Pharmacology, Kasturba Medical College, Mangalore, Manipal University
ABSTRACT
Mucuna pruriens commonly known as cowhage plant has been claimed to possess various
beneficial effects like anti-parkinsonian, anti-tumor, neuroprotective, antioxidant, anti-diabetic
and antidepressant activities. Previous studies have reported that Mucuna pruriens contains L-
DOPA and 5-hydroxy tryptophan (5-HT) as a major constituent with higher concentration in
seeds. However, literature search revealed no scientific data on its anxiolytic activity. So the
present study was designed to evaluate the anxiolytic activity of Mucuna pruriens in a murine
model. Wistar albino rats were divided into five groups (n=6). Mucuna pruriens administered at
doses of 250,500,750mg/kg/day orally, was compared with the standard drug Diazepam
(1.0mg/kg/day, oral) fed for 14 days. Three pharmacologically validated models elevated plus
maze, bright and dark arena and open field test were used. The data presented was analyzed
using one way ANOVA followed by Dunnett’s post hoc test. A value of p<0.05 was considered
as statistically significant. Mucuna pruriensat all three doses used significantly reduced the time
spent in closed arm, increased the entries into open arm in elevated plus maze (p < 0.05). In
bright & dark arena model, there was an increase in number of entries, time spent in bright
chamber (p < 0.05) and in open field test, the time spent and squares crossed in central chambers
were increased (p < 0.05). To conclude, the present study demonstrates the anxiolytic activity of
chronic administration of Mucuna pruriens in Wistar Albino rats.
Keywords: Anxiolytic, Mucuna pruriens, diazepam, elevated plus maze, bright & dark arena
model, open field test.
*Corresponding Author Email: meddocpai2@yahoo.com
Received 30 October 2013, Accepted 04 January 2014
Journal home page: http://www.ajptr.com/
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INTRODUCTION
Anxiety is an unpleasant emotional experience of daily living characterized by a sense of
apprehension, uneasiness or impending distress; this feeling is usually associated with changes in
the autonomic nervous system and behaviour. It affects one-eighth of the total population
worldwide and has become a very important area of research interest in psychopharmacology
during this decade1,2. Anxiety is a cardinal symptom of many psychiatric disorders and an almost
inevitable component of many medical and surgical conditions3. It is a normal emotional
behaviour; when it is severe and/or chronic and disturbs the day-to-day activities, it becomes
pathological and can precipitate or aggravate cardiovascular and psychiatric disorders.Although
many drugs are available in modern medicine to treat anxiety disorders, they produce various
systemic side effects or exhibit tolerance upon chronic use4.
Mucuna pruriensLinn. (Fabaceae) is commonly known as cowhage plant or kapikacho or kevach
in Hindi. It is an annual, climbing shrub grown in tropical regions. In Ayurvedic system of
medicine, Mucuna pruriens is used in rheumatoid arthritis, diabetes, atherosclerosis, male
infertility and nervous disorders5. Various activities attributed to this indigenous shrub include
anti-parkinsonian6,anti-tumor7,neuroprotective8, antioxidant9, learning and memory enhancement
and anti-diabetic activity10.Mucunapruriens also showed significant antidepressant activity in
rodent models11.Previous studies have reported that Mucuna pruriens contains L-DOPA and 5-
hydroxy tryptophan (5-HTP) as a major constituent with higher concentration in seeds12. The
recognition of anxiolytic effects of non-benzodiazepine azapirones agents (buspirone, gepirone,
and ipsapirone), which act as 5HT1A partial agonists and their therapeutic role in clinical anxiety
and mood disorders has further focused attention on the 5-HT1A receptor. Although the
azapirones interact with other neurotransmitter systems, such as the dopaminergic and
noradrenergic, they display nanomolar affinity for 5HT1A receptor sites13.Buspirone appears to
only interact with the dopaminergic system with reasonable potency and exhibits properties of
both a dopamine agonist and a dopamine antagonist. This suggests that dopamine is implicated
in the etiology and expression of anxiety. Based on earlier studies it has been postulated that
dopaminergic agents may play an important role in the pharmacotherapy of anxiety14.
With this background we decided to explore the anxiolytic activity of chronic administration of
Mucuna pruriens in Wistar albino rats, using three pharmacologically validated experimental
models namely elevated plus maze, bright and dark arena and the open field test 15
MATERIALS AND METHOD
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Animals
Healthy adult Wistar albino rats of either sex, weighing 150-200 g inbred in our central animal
house (KMC, Mangalore)were used for the study. The rodents were housed in clean
polypropylene cages, with dust free rice husk as a bedding material; three rats per cage under
controlled laboratory conditions ( Temperature: 25° ± 2°C, humidity (60% ± 10%) and 12 h
light/dark cycle as per CPCSEA guidelines). The experimental animals were fed with standard
chow containing fat 4.15%, protein 22.15%, carbohydrates 4% (supplied by Amruth laboratory
animal feed manufactured by Pranav Agro industries ltd., Sangli) and water ad libitum. The
rodents were allowed to acclimatize to these conditions for one week prior to the commencement
of the study. Experiments were performed during the light phase of the cycle (10:00-17:00).The
study was approved by the institutional animal ethics committee.
Drugs and dosage
Mucuna pruriens Choorna powder was obtained from SDP Remedies & Research Centre, Puttur,
D.K. District, Karnataka. Standard anxiolytic drug, Diazepam was obtained from Ranbaxy
Laboratories Limited, Gurgaon, Haryana. The doses of each drug were selected on the basis of
earlier findings for diazepam16 and Mucuna pruriens17. All the drugswere freshly prepared in
distilled water andadministered orally in a constant volume of 10 ml/kg. Drugs, dosage and
number of animals used per treatment are shown in (Table 1). Drugs/vehicle wereadministered
once daily for fourteen days and the last dose was given on the fourteenth day, 60 minutes prior
to the experiment.
Table1. Treatment groups
Groups (n=6)
Treatment
Dose
I
Control – Distilled water
10.0 ml/kg
II
Diazepam
1.0 mg/kg
III
Mucuna pruriens
250 mg/kg
IV
Mucuna pruriens
500 mg/kg
V
Mucuna pruriens
750 mg/kg
ANIMAL MODELS FOR TESTING ANXIOLYTIC ACTIVITY:
Elevated plus maze:
The wooden maze consists of two open arms (length 50 cm X breadth 10 cm) and two closed
arms of the same size (height 40 cm). The arms of the same type are opposite to each other, with
a central square of 10 cm. The maze is elevated to height of 50 cm above the floor.
Bright and dark arena
The apparatus consists of an open top wooden box. Two distinct chambers, a black chamber (20
X30 X 35 cm) painted black and illuminated with dimmed red light and a bright chamber (30 X
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30 X 35 cm) painted white and brightly illuminated with a 100 W white light source located 17
cm above the box. The two chambers are connected through a small open doorway (7.5 X 5cm)
situated on the floor level at the centre of the partition.
Open field test
The apparatus consists of a large rectangular box (100 X 80 cm) with 60 cm high walls. The
floor is made of wire mesh and divided into twenty-five squares (outer 16 and central 9). The
box is illuminated with a 100 W bulb placed 60 cm above the centre of the field.
Behavioural assessment
Each animal was tested initially in elevated plus maze and then, in light and dark arena paradigm
and then, in open field test in a single setting. On the 14th day of drug or vehicle administration,
each animal was placed in the centre square of the plus maze, facing one of the open arms and
the observations were made 60 min after administering the last dose. The number of entries into
and the time spent in open and closed arms and the number of rears in each arm in a five-minute
period was noted.
Following the elevated plus maze test, the animal was placed at the centre of the brightly lit
arena in the light and dark box. The number of entries into and the time spent in the bright arena,
the number of rears in the bright and dark arenas and the duration of immobility was noted
during the 5minutes of observation.
In the open field test, each animal were placed in one of the peripheral corner squares of the box
and the number of peripheral and central squares crossed, the time spent in central squares and
the number of rears were observed for a five-minute period. Following each trial, the apparatus
was cleaned with hydrogen peroxide to mask the odour left by the animal in the previous
experiment. Hand operated counters and stop watches were used to score the behaviour of
animals.
Statistical analysis:
Statistical analyses were carried out using the software package SPSS (Version 17.0). The data
was analysed by one-way ANOVA with drug treatment as the independent factor. Post hoc
comparisons were performed by applying Dunnet’s multiple comparison test. A value of P <
0.05 was considered to be statistically significant
RESULTS AND DISCUSSION:
In the present study, we have studied the effects of chronic administration of Mucuna pruriensin
three pharmacologically validated experimental models of anxiety –elevated plus maze, bright
and dark arena and open field test. All the studied models of anxiety are based on the theory that
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unfamiliar, brightly lit and an open environment induces stress which provokes anxiety, thereby
inhibiting normal behaviour in rats.
a. Elevated plus maze
In the elevated plus maze, the open arms are more fear provoking, unprotective and
anxiogenicthan the closed arms. The animal hence prefers to spend more time and shows normal
rearing behaviour in the closed arm. The ratio of entries, time spent and rearing behaviour in
open arms to closed arms reflects the safety of closed arms with relative fearfulness of open
arms18. The reduction in entry, time spent, rearing in open arms, ratio of open arm to total arm
entries and increased defecation are the indications of high level of fear or anxiety. Anxiolytic
drugs increase the proportion of entries, time spent and rearing in open arms. They also increase
the ratio of open arm to total arm entries.
The results as given in table 2 indicate that the diazepam (1.0 mg/kg) treated rats showed a
highly significant(p<0.001) increase in the number of open arm entries, percentage ratio of open
arm to total arm entries and time spent in the open arms. They showed a significant (p<0.05)
reduction in the time spent in the closed arms compared to control group. Mucuna pruriens
treated rats exhibited a highly significant (p<0.001) and significant (p<0.05) increase in time
spent in the open arms at doses, 500mg/kg, 750 mg/kg and 250mg/kg respectively.The increase
in the percentage ratio of open arm to total arm entries was significant (p<0.05) at 500mg/kg
dose level and highly significant (p<0.001)at 750 mg/kg dose used. Significant increase was also
noted in the number of open arm entries along with number of rears in open arms at 750mg/kg
dose. The time spent in the closed arms was significantly reduced (p<0.05).
Table 2: Effect of chronic administration of diazepam andMucuna pruriens on behaviour
of rats in elevated plus maze
Groups
(n=6)
Number of
open arm
entries
Percentage of
open /total
arm entries
Time spent in
open arms
(seconds)
Time spent in
closed arms
(seconds)
Number of
rears in
open arms
Control
4.83±0.30
51.92±3.84
76.33±4.30
222 ± 4.73
7.67±1.02
Diazepam
1.0 mg/kg
8.67±0.55**
76.48±2.42**
211.83±9.00**
88.17 ± 9.0*
11.8±1.30
Mucuna pruriens
(250mg/kg)
5.50±0.67
52.38±4.04
109.67±5.27*
273.83±15.1
9.33±1.33
Mucuna pruriens
(500mg/kg)
6.33±0.49
64.17±2.03*
156.33±5.78**
290.17±0.6
10.83±1.19
Mucuna pruriens
(750mg/kg)
7.00±0.63*
71.19±1.94**
207.33±8.52**
92.67±8.52*
12.33±1.33*
(All values are expressed as mean ± SEM; **P < 0.001 vs. control,*P < 0.05 vs. control)
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616
Mucuna pruriensin all the three doses studied, increased the number of entries, time spent and
rearing in the open arms compared to control. These behavioural changes were not only
comparable to those produced by diazepam but Mucuna pruriensat 750 mg/kg showed a
significant increase in time spent and rears in open arm than the diazepam treated group. This
suggests that Mucuna pruriens showed anxiolytic effect in animal models comparable to that of
diazepam.
b. Bright and dark arena
In the bright and dark arena, rodents tend to avoid entry into and reduce spontaneous exploratory
behaviour in the brightly illuminated area, a natural tendency when a rat is exposed to an
unfamiliar environment. Anxiolytics tend to show an increase in the number of entries, time
spent and rears in the bright arena.
The results given in table 3 indicate that the diazepam (1.0 mg/kg) treated rats showed a highly
significant (p<0.001) increase in the number of bright chamber entries, time spent and number of
rears in bright arena compared to control group. Mucuna pruriens treated rats at all the three
doses also significantly increased the time spent in the bright arena (250,500 and 750 mg/kg)
compared to control. They also showed significant(p<0.05) increase in the number of bright
chamber entries and rears in bright arena(750mg/kg).Moreover, the diazepam treated rats
showed significant increase in the duration of immobility compared tocontrol whereas, the
Mucuna pruriens treated groups in all the three doses reduced the immobility compared to the
diazepam treated groups. Moreover, at the dose of 750mg/kg Mucuna pruriens showed a
significant increase in the number of entries and time spent in the bright arena compared to
diazepam as well.
Table 3: Effect of chronic administration of diazepam andMucuna pruriens on behaviour
of rats in Bright and dark arena model:
Groups
(n=6)
Number of
bright
chamber
entries
Time spent in
bright
chamber
(seconds)
Number of
rears in
bright
chambers
Number
of rears in
dark
chambers
Immobility
Control
1±0
7.50±1.05
0.33±0.21
5.17±0.47
6.17 ± 3.12
Diazepam
1.0 mg/kg
3.17 ± 0.31**
25.17 ± 2.01**
3.00 ± 0.77*
13.00 ±
1.31**
24.50 ±
2.89**
Mucuna pruriens
(250mg/kg)
1.50±0.34
16.67±1.49*
1.33±0.42
6.83±0.47
1.00±0.51
Mucuna pruriens
(500mg/kg)
2.00±0.25
21.50±0.76**
1.17±0.40
6.67±0.55
0.67±0.66
Mucuna pruriens
(750mg/kg)
2.83±0.30**
25.17±1.13**
1.33±0.49*
12.50±0.99
**
0.50±0.34
(All values are expressed as mean ± SEM; **P < 0.001 vs control,*P < 0.05 vs control)
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c. Open field test
In open field test, forced confrontational situations, induce anxiety which makes the rodents
prefer the periphery, a behaviour called thigmotaxis. An increase in central locomotion or in time
spent in the central part of the device without increasing the total locomotion is interpreted as
anxiolytic activity.
The results given in table 4 indicate that the diazepam (1.0 mg/kg) treated rats and all the doses
of Mucuna pruriens (250,500 and 750 mg/kg) showed significant increase in the number of
squares crossed, rears and time spent in the central squares. There was also a significant
reduction in the number of squares crossed and time spent in the peripheral squares compared to
the control group.
Table 4: Effect of chronic administration of diazepam andMucuna pruriens on behaviour
of rats in open field test:
Groups
(n=6)
Number of
Peripheral
squaresCrossed
Time spent in
Peripheral
squares
Number of
Central
squaresCrossed
Time spent in
Central
squares
Number of
Central
squaresRears
Control
56.33±4.78
288.50±3.46
2.17±0.65
11.50±3.46
0.67±0.33
Diazepam
1.0 mg/kg
15.17 ±1.16**
148.67±6.78**
17.00±1.69**
151.33± 6.78**
7.83±0.91**
Mucuna pruriens
(250mg/kg)
23.83±3.07*
201.33±9.16
12.00±1.46*
98.67±9.16**
7.00±1.43**
Mucuna pruriens
(500mg/kg)
19.83±1.64**
180.83±5.38**
12.33±0.80**
119.17±5.38**
8.50±1.25**
Mucuna pruriens
(750mg/kg)
16.33±1.35**
153.00±3.47**
17.00±1.46**
147.00±3.47**
7.00±0.57**
GABAergic and serotoninergic systems are considered among the principal regulatory systems
of anxiety. Standard anxiolytics like BZDs act through the GABA A- BZD receptor – Chloride
channel complex. Then on-benzodiazepine agents (buspirone) act as 5HT1A partial agonists and
have therapeutic role in clinical anxiety and mood disorders. Buspirone exhibits properties of
both a dopamine agonist and a dopamine antagonist suggesting that dopamine may also be
implicated in the etiology and expression of anxiety. Earlier studies have also postulated that
dopaminergic agents may also play an important role in the pharmacotherapy of anxiety13.
Oxidative stress (OS) represents a loss of balance in oxidation-reduction reactions. It is
characterized by the reduced ability of the antioxidant defence system to efficiently eliminate the
excess of the oxygen-derived species production, eliciting the toxicity of oxygen and its
detrimental effects. Recent studies have shown that patients with anxiety disorders have higher
activity levels of the enzymes like superoxide dismutase and glutathione peroxidase as well as
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618
higher lipid peroxidation activity19. Hence oxidative metabolism is also regarded as a plausible
pathway that can affect the regulation of anxiety.
CONCLUSION:
The results of the present study indicate that Mucuna pruriens on chronic administration, showed
significant anxiolytic activity in the animal models studied. The presence of L-DOPA and 5-
hydroxy tryptophan (5-HTP) as a major constituent in Mucuna pruriens may suggest the role of
dopaminergic and/or serotonergic pathways in its anxiolytic activities. However, further studies
are required to elucidate the targets of action and the exact mechanism of action of Mucuna
pruriensas an antianxiety drug.
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