EFFECT OF WITHANIA SOMNIFERA ON HALOPERIDOL-INDUCED CATALEPSY 243

Copyright 2007 John Wiley & Sons, Ltd. Phytother. Res. 22, 243246 (2008)
DOI: 10.1002/ptr
Copyright 2007 John Wiley & Sons, Ltd.
PHYTOTHERAPY RESEARCH
Phytother. Res. 22, 243246 (2008)
Published online 20 September 2007 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/ptr.2299
Effect of Withania somnifera Root Extract on
Haloperidol-induced Catalepsy in Albino Mice
Vinod Nair
1
*, Albina Arjuman
2
, H. N. Gopalakrishna
1
and M. Nandini
2
1
Department of Pharmacology, Kasturba Medical College, Mangalore, India
2
Department of Biochemistry, Kasturba Medical College, Mangalore, India
The objective of the study was to evaluate the anticataleptic effect of Withania somnifera (WS) extract, on
haloperidol-induced catalepsy in albino mice. Catalepsy was induced with haloperidol (1 mg/ kg) i.p. in ve
groups of male albino mice (n = == == 6). Three groups received Withania somnifera extract (1.7, 4.25, 8.5 mg/kg)
respectively, one group received scopolamine (1 mg/kg) and one group received the vehicle (1% gum acacia)
orally, 30 min prior to haloperidol administration. Catalepsy was measured by using standard bar test at 30, 60,
90, 120 and 240 min. This constituted the acute study. For the chronic study, the drugs were administered for
6 more days. Catalepsy was again measured on day 7. Animals were then sacriced by cervical dislocation
and superoxide dismutase (SOD) activity was estimated in the brain. In this study, Withania somnifera
extract treated groups showed a dose dependent reduction in cataleptic scores, both in the acute and chronic
study. The SOD activity in brain was also found to be lowered in the WS (4.25 mg, 8.5 mg/kg) treated
groups. In conclusion, Withania somnifera was found to be more efcacious than scopolamine in reversing
haloperidol induced catalepsy. A clear correlation between the SOD levels and cataleptic scores was observed.
We believe that the antioxidant properties of this drug could have contributed to the anticataleptic effect.
Copyright 2007 John Wiley & Sons, Ltd.
Keywords: Withania somnifera; haloperidol; catalepsy; oxidative stress; superoxide dismutase.
Received 13 March 2007
Accepted 22 June 2007
* Correspondence to: V. Nair, Department of Pharmacology, AIIMS,
Ansari Nagar, New Delhi, India, 110029.
E-mail: vinodnair1979@hotmail.com
INTRODUCTION
Catalepsy is a condition characterized most often by
rigidity of extremities and by decreased sensitivity to
pain. It is understood as a behavioural state in animals
in which the animal cannot rectify externally imposed
postures (Sanberg, 1980). This model is depictive of the
extrapyramidal side effects induced in neurodegenerative
disorders such as Parkinsons disease by blockade of
the dopamine receptors localized postsynaptically in
the striatum. Typical antipsychotics such as haloperidol
have been shown to exhibit similar effects by blocking
the dopaminergic action in the nigro-striatal pathway
leading to high frequency of extrapyramidal-motor side
effects (Farde et al., 1992) such as dystonias, pseudo-
parkinsonism, etc.
Withania somnifera (WS) (Ashwagandha, Indian
Ginseng; Family: Solanaceae) by virtue of its therapeu-
tic benets has been a popular name in Ayurvedic
medicine for centuries. This wonder herb had created
skepticism by reason of its widespread chemical prop-
erties, and as a result led researchers into extensive
study of its different properties and mode of action. It
has been shown to be an anti-inammatory, antitumour,
antistress, antioxidant and rejuvenating agent, apart
from many other characteristics (Thorne Research, Inc.,
2004; Mishra et al., 2000; Bhattacharya et al., 2001). It
has also been used in the treatment of diseases that
resemble the modern concept of neurodegenerative
disorders (Naidu et al., 2003; Weiner and Weiner, 1994).
The susceptibility of the brain tissue to free radical
damage is evident due to the presence of predominant
quantities of lipids, which are rudimentary in the process.
This cascade process of free radical damage is supple-
mented by the fact that the brain utilizes 20% of the
total body oxygen supply, leading to neuronal loss as in
cerebral ischemia and also neurodegenerative disorders
such as Parkinsonism (Mishra et al., 2000; Kedar, 2003).
As the use of WS in Ayurveda has been associated
with diseases related to oxidative damage, the bene-
cial effects have been shown to be attributed consider-
ably to its antioxidant activity. As haloperidol-induced
catalepsy is also associated with an elevation in the
antioxidant enzyme superoxide dismutase (SOD) in the
brain (Sagara, 1998), the aim was to establish a central
analogy between the anticataleptic and antioxidant
actions of WS.
MATERIALS AND METHODS
Prior permission was obtained from the Institutional
Animal Ethical Committee before starting the study.
Test drug. Water extract of WS was supplied by Natural
Remedies Pvt. Ltd, Bangalore. It was a light yellowish-
brown powder that was subjected to phytochemical
analysis by using the gravimetric method. It was found
to contain 2.1% w/w withanolides.
Animals. Adult male Swiss albino mice (2530 g)
from our breeding stock were used for the study. They
were housed in clean and transparent polypropylene
cages with three animals in each cage and maintained
Copyright 2007 John Wiley & Sons, Ltd. Phytother. Res. 22, 243246 (2008)
DOI: 10.1002/ptr
244 V. NAIR ET AL.
at 27 C with 12 h: 12 h lightdark cycle for a period of
7 days prior to the study. They were fed standard rat
chow and water ad libitum. The animals were divided
into ve groups with six animals in each group. All
observations were made between 10:00 hr and 16:00 hr.
The test drug WS and scopolamine were suspended in
1% gum acacia solution (vehicle). All drug solutions
were freshly prepared and administered orally using a
feeding tube. The rst group received vehicle and served
as the control, the second group received 1 mg/kg body
weight of scopolamine and the third, fourth and fth
groups received WS in doses of 1.7, 4.25 and 8.5 mg/kg
body weight respectively. Thirty minutes after adminis-
tration of vehicle/drugs, haloperidol in a dose of 1 mg/
kg body weight, in the form of an injectable solution
constituted in normal saline was administered by the
i.p. route to induce catalepsy. This dose of haloperidol
was chosen from our pilot study to produce a moderate
degree of catalepsy, so that attenuation or potentiation
of the phenomenon could be detected.
Acute study. The degree of catalepsy was measured
at 30, 60, 90, 120 and 240 min after haloperidol admin-
istration by using a method similar to the standard bar
test (Ahtee and Buncombe, 1974). Briey, catalepsy
was measured as the time the animal maintained an
imposed position with both front limbs extended and
resting on a 4 cm high wooden bar. The end point of
catalepsy was considered to occur when both front paws
were removed from the bar or if the animal moved
its head in an exploratory manner. If the animal main-
tained the imposed posture for at least 20 s it was said
to be cataleptic and given one point. For every further
20 s that the animal continued to maintain the cata-
leptic posture one extra point was given. Based on the
data obtained in our pilot study, a cut-off cataleptic
score of 60 (corresponding to 20 min) was used during
the recording of observations. Between determinations,
the animals were returned to their individual home
cages.
Chronic study. WS, scopolamine and haloperidol were
administered continuously for a period of 6 more days
in the manner described for the acute study. Catalepsy
was again measured on day 7 at 30, 60, 90, 120 and
240 min post-haloperidol administration. Soon after
the behavioural study, the animals were sacriced by
cervical dislocation and the SOD (EC 1.15.1.1;
superoxide:superoxide oxidoreductase) activity in the
whole brain was estimated (Beauchamp and Fridovich,
1971). Total protein (Peterson, 1979) in the brain was
estimated and the SOD activity was expressed in terms
of units/mg total protein. To give the normal range for
SOD activity in our testing conditions and to rule out
the involvement of the vehicle, three mice were admin-
istered 10 mL/kg vehicle for 7 days. On day 7 they were
sacriced by cervical dislocation 270 min after vehicle
administration and the SOD activity and total protein
in the brain was determined.
Statistical analysis. The data were analysed by one-way
ANOVA, followed by Dunnetts multiple comparison
test. p < 0.05 was considered signicant.
RESULTS
Acute study
In the acute phase of the study (Table 1), a dose de-
pendent decrease in the cataleptic score was observed
in the WS treated groups. When compared with the
control, a signicant reduction in cataleptic scores was
observed in the WS 4.25 and WS 8.5 mg/kg treated
groups from the start of the study, whereas in the WS
1.7 mg/kg treated groups there was an initial increase
in the cataleptic score. Signicant reduction in this group
was observed only after 120 min into the study. When
compared with scopolamine, only the WS 4.25 and WS
8.5 mg/kg treated groups showed a greater reduction in
cataleptic scores throughout the study.
Chronic study
In the chronic study also (Table 2), there was a dose
dependent decrease in the cataleptic scores in the WS
treated groups. A signicant reduction was observed in
WS 4.25 and 8.5 mg/kg treated groups from the start of
the study. A signicant reduction in the WS 1.7 mg/kg
and scopolamine treated groups was observed only
after 60 and 90 min, respectively. When compared with
scopolamine, only the WS 8.5 mg/kg treated group
showed a reduction in cataleptic score throughout the
observation period.
Table 1. Effect of WS on haloperidol-induced catalepsy: acute study
Time (min) after haloperidol administration
Group 30 60 90 120 240
Control 11.5 1.25 24.5 1.76 34 3.67 47.5 4.47 51.33 2.21
b
Scopolamine 1.0 12.66 0.95 17 1
b
21.5 1.14
b
26.83 0.98
b
17.83 1.37
b
WS 1.7 15.33 0.98 25.16 0.70 28.16 0.83 25.33 2.39
b
18.83 2.15
b
WS 4.25 5.66 0.61
b
9 0.81
b
14.5 0.99
b
16.33 2.39
b
15.33
b
WS 8.5 4.42 0.42
b
7.76 0.66
b
10.66 0.49
b
16.33 0.42
b
11.33 0.71
b
One-way ANOVA
F 27.577 59.804 27.489 24.942 109.54
p <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
df 4,25 4,25 4,25 4,25 4,25
All values are mean SEM (n = 6 in each group). Statistical analysis by one-way ANOVA followed by Dunnetts multiple comparison
test.
b
p < 0.01.
EFFECT OF WITHANIA SOMNIFERA ON HALOPERIDOL-INDUCED CATALEPSY 245
Copyright 2007 John Wiley & Sons, Ltd. Phytother. Res. 22, 243246 (2008)
DOI: 10.1002/ptr
Table 2. Effect of WS on haloperidol-induced catalepsy: chronic study
Time (min) after haloperidol administration
Group 30 60 90 120 240
Control 13.33 0.80 18.33 1.16 25.33 0.84 34.16 1.40 37.33 1.20
Scopolamine 1.0 13.66 0.49 15.33 0.47 18.33 0.40
b
16.66 0.49
b
14.5 0.50
b
WS 1.7 11.16 0.47 14.66 0.91
a
19.66 0.88
b
16.33 0.66
b
21.5 1.58
b
WS 4.25 9.83 0.47
b
13.16 0.87
b
17.83 0.83
b
14.16 0.47
b
16.16 0.47
b
WS 8.5 9.33 0.71
b
12.16 0.65
b
15.66 0.49
b
13.66 0.49
b
11.5 0.56
b
One way ANOVA
F 10.693 7.832 25.702 118.82 111.11
p <0.0001 0.0003 <0.0001 <0.0001 <0.0001
df 4,25 4,25 4,25 4,25 4,25
All values are mean SEM (n = 6 in each group). Statistical analysis by one-way ANOVA followed by Dunnetts multiple comparison
test.
a
p < 0.05,
b
p < 0.01.
SOD activity
A reduction in SOD activity was observed in scopo-
lamine (Table 3), WS 4.25 and 8.5 mg/kg treated groups,
but a signicant reduction was observed only in the WS
8.5 mg/kg treated group.
DISCUSSION
Haloperidol is a well known neuroleptic, primarily acting
as a D
2
receptor antagonist in the mesolimbic-mesocortical
pathway. Due to its non-selective action, it also produces
blockade of post-synaptic D
2
receptors in the nigro-
striatal pathway leading to the development of extrapy-
ramidal side effects in humans (Farde et al., 1992) and
catalepsy in animals (Sanberg, 1980). Haloperidol-
induced catalepsy is also associated with an increase in
oxidative stress in the brain (Sagara, 1998).
Studies have reported the benecial effects of WS in
haloperidol-induced tardive dyskinesia (Bhattacharya
et al., 2002). In our study also a dose dependent decrease
in catalepsy was observed in the WS treated groups in
both acute and chronic studies. When compared with
the standard drug scopolamine, WS in doses of 4.25
and 8.5 mg/kg was found to be more effective in revers-
ing haloperidol-induced catalepsy. Similarly a dose
dependent decrease in SOD activity was observed
in the WS treated groups. Even though the reduction
was signicant only in the highest dose treated group,
the SOD activity in the WS 4.25 mg/kg treated group
was closer to the normal brain SOD values, compared
with the control which shows a decrease in oxidative
stress. This reduction in brain SOD activity could be
attributed to the antioxidant phytochemical constitu-
ents of WS (Mishra et al., 2000; Bhattacharya et al.,
2001). Paradoxically, WS 1.7 mg/kg showed a decrease
in the cataleptic score but an increase in brain SOD
activity compared with the control. The reason for
this increase cannot be explained by the present study.
A plausible explanation could be the involvement of
direct and indirect antioxidants in WS (Bhattacharya
et al., 2001; Naidu et al., 2003). A direct antioxidant is
one which possesses the ability to quench free radicals
directly, whereas an indirect antioxidant is one which
induces antioxidant enzymes. The net result of both
the processes is a decrease in the oxidative stress.
To ascertain the actual mechanisms involved, further
studies are required.
In conclusion, WS in the highest dose tested was
found to be more efcacious than the standard drug
scopolamine in reversing haloperidol-induced catalepsy.
A clear correlation between the SOD levels and
cataleptic scores can be observed. Based on our ndings
an analogy can be drawn between the antioxidant
and anticataleptic effects of WS, with a possible appli-
cation of the same as an adjuvant in the treatment in
drug-induced Parkinsonism.
Acknowledgement
Natural Remedies Pvt. Ltd, Bangalore provided the test drug Withania
somnifera.
Table 3. Effect of WS on brain SOD levels
SOD % reduction % increase
Group (U/mg TP) from control from control
Normal 5.28 0.28
Control 8.81 0.28
Scopolamine 8.49 0.66 3.63
WS 1.7 9.88 0.72 12.14
WS 4.25 6.99 0.28 20.66
WS 8.5 6.18 0.49
a
29.85
One way ANOVA
F 8.086
p 0.0035
df 4,10
All values are mean SEM (n = 3 in each group). Statistical analysis by one-way
ANOVA followed by Dunnetts multiple comparison test.
a
p < 0.05.
Copyright 2007 John Wiley & Sons, Ltd. Phytother. Res. 22, 243246 (2008)
DOI: 10.1002/ptr
246 V. NAIR ET AL.
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