Pharmacology & Pharmacy, 2013, 4, 535-541 Published Online October 2013 ( 535
Anticonvulsant, Anxiolytic and Neurotoxicity Profile of
Aqarqarha (Anacyclus pyrethrum) DC (Compositae) Root
Ethanolic Extract
Syed Mohd Abbas Zaidi1*, Shadab Ahmad Pathan2, Surender Singh3, Shakir Jamil4, Farhan Jalees
Ahmad2, Roop Krishen Khar2
1Department of Moalajat, HSZH Govt. Unani Medical College, Bhopal, India; 2Department of Pharmaceutics, Faculty of Pharmacy,
Jamia Hamdard, New Delhi, India; 3Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India;
4Department of Moalijat, Faculty of Medicine (Unani), Jamia Hamdard & CCRUM, New Delhi, India.
Email: *
Received July 27th, 2013; revised September 6th, 2013; accepted September 17th, 2013
Copyright © 2013 Syed Mohd Abbas Zaidi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ethnopharmacological relevance: Aqarqarha (Anacyclus pyrethrum) DC root has long been used as a traditional an-
tiepileptic remedy in Unani system of medicine over centuries. Aim of the Study: To rationalize the ethnomedical
claim and screen for anxiolytic and neurotoxicity profile of ethanolic extract of Aqarqarha (Anacyclus pyrethrum) root
(APE). Materials and Methods: The anticonvulsant and anxiolytic potential of APE (100 - 800 mg/kg) was evaluated
against Pentylenetetrazole (PTZ), Bicuculline (BCL), Increasing current electroshock (ICES) and Elevated plus
maze(EPM) models. Rotarod test was employed as neurotoxicity model including an additional higher dose (1600
mg/kg). Results: The APE showed significant anticonvulsant activity (p < 0.001) against PTZ (70 mg/kg, i.p.) in a
dose-dependent manner but against BCL (30 mg/kg, i.p.) at the dose 800 mg/kg only (p < 0.001). However, it did not
protect animals against ICES induced seizures (p > 0.05). The extract also showed anxiolytic behaviour in EPM (p <
0.001) and impaired motor coordination only at 1600 mg/kg in rotarod performance. HPTLC of the extract confirmed
the presence of eugenol in the extract. Conclusions: The results suggested significant anticonvulsant activity of APE
against PTZ and BCL but failure against ICES. Moreover, APE also exhibited anx iolytic poten tial without an y ev idence
of neurotoxicity at the effective dose level. We concluded that anticonvulsant effect of APE is probably mediated by
enhancing GA B Aer gi c ne u ro transmission.
Keywords: Aqarqarha; HPTLC; Epilepsy; Aqarqarha (Anacyclus pyrethru m)
1. Introduction
Epilepsy is one of the most common neurological dis-
orders with a prevalence of 5 to 8 cases per 1000 of po-
pulation in developed countries, and with even higher
rates in under-developed countries [1,2]. Currently avai-
lable antiepileptic drugs are synthetic compounds and
have dose-related and chronic toxicity, involving vir-
tually every major organ system, adverse effects on cog-
nition and behavior, reduced bone mineral density and te-
ratogenic effects [3-8]. The very high costs of the new
antiepileptic drugs have a major impact on the overall
cost of epilepsy therapy in both developed and under-
developed countries [9].
In the treasury of Unani system of medicine, there are
many herbal drugs which are reported to possess anti-
epileptic activity and are being used by the physicians
since the Stone Age. These drugs are relatively much
safer as evident by their long clinical use. The root of
Aqarqarha (Anacyclus pyrethrum) DC, popularly known
as “Aqarqarha” in the Unani system of medicine, is
reported to be used by the Unani physicians to manage,
control and treat epilepsy [10,11]. It is also used to
stimulate the salivary glands, cure chronic catarrh of the
head and nostrils, cure toothache, improve sexu a l debility
and clear the brain by exciting a free flow of nasal mucus,
tears and by stimulating blood flow to the tissues [12].
Recently it has been found to possess immunostimulating
[13] and aphrodisiac [14] action in various rodent
*Corresponding a uthor.
Copyright © 2013 SciRes. PP
Anticonvulsant, Anxiolytic and Neurotoxicity Profile of Aqarqarha (Anacyclus pyrethrum)
DC (Compositae) Root Ethanolic Extract
The herb contains an essential oil and a pungent alka-
loid, pellitorin or pyrethrin. It has alkamides, lignane (in-
cluding sesamine), inulin (fructosan) and tannins. Alka-
mides include deca-2, 4-dien acid-isobutylamide, ana-
cycline, and dehydroanacycline [15,16]. The presence of
a polyacetylenic compound is also reported in the root
[17]. It is also reported to contain eugenol [18,19].
In an effort to scientifically validate an d rationa lize the
medicinal value of this drug, the present study was un-
dertaken to examine the anticonvulsant, anxiolytic and
neurotoxicity profile of this herb’s root ethanolic extract
in various experimental paradigms.
2. Materials and Methods
2.1. Plant Material
Fresh roots of Aqarqarha (Anacyclus pyrethrum) DC (Com-
positae) were purcha sed fro m an author ized supp lier (Green
Earth Products, G.K.-1, New Delhi) which was harv ested
in October from the Katra region of Jammu & Kashmir.
Identification and taxonomical authentication was per-
formed by a renowned taxonomist of NISCAIR, New Delhi
and a voucher specimen no. “NISCAIR/RHMD/CON-
SULT/-2008-09/1099/130/Aqarqarha (Anacyclus pyre-
thrum)/DATE 07NOV2008” was deposited in the her-
2.2. Preparation of Aqarqarha (Anacyclus pyre-
thrum) Root Ethanolic Extract (APE)
One kilogram (1 kg) of crushed, air dried roots of Ana-
cyclus pyrethrum was milled into a fine powder in a
commercial blender. The powder was extracted twice by
cold maceration on each occasion with 2.5 L of 99.9%
ethanol for 48 h at room temperature, with occasional
shaking. The combined extracts were concentrated to
dryness under reduced pressure at 40˚C ± 1˚C in a rotary
evaporator, finally giving 35.56 g (3.556% w/w yield) of
a dark brown, powdery crude APE. Aliquot portions of
the crude APE residue were weighed and dissolved in
0.5% w/v Tween 80 for use on each day of our experi-
2.3. HPTLC Fingerprinting of the Prepared
TLC profile of the APE was developed prior to going for
the HPTLC. Toluene: Ethyl acetate: Formic acid (3:2:0.5)
was selected as the mobile phase in the UV-mode. The
prepared extract was dissolved in ethanol and applied to
the pre-coated Silica gel 60F254 TLC plates (E. Merck)
using a CAMAG Linomat IV Automatic Sample Spotter.
After development in mobile phase, th e plates were dried
in air and scanned at 254 nm using a CAMAG TLC
Scanner and winCATS software. Eugenol was also
applied in the same manner to the HPTLC plates to find
out the presence of eugenol in the extract.
2.4. Drugs
The drugs used were sodium valproate (Wockhardt Ltd.,
Mumbai, India), Pentylenetetrazole (PTZ) and Bicucul-
line (BCL) from Sigma Aldrich, USA. Drugs were
freshly prepared by dissolving in distilled water. All i.p.
injections were given in volumes of not more than 10
ml/kg of the body weight for mice.
2.5. Animals
Swiss male albino mice weighing 20 - 35 g were pro-
cured from the central animal house, Jamia Hamdard,
New Delhi. Animals were housed in group s of 6 per cage
and maintained at 20˚C - 30˚C and 50% - 55% humidity
in a natural light an d dark cycle, with free access to food
and water. The experiments were performed during the
light cycle in awake, freely moving animals that were
adjusted to laboratory conditions before proceeding with
the experiments. The animals were divided into four
groups of plant’s extract (100, 200, 400 & 800 mg/kg) ,
reference anticonvulsan t drug-treated “test”, and distilled
water-treated “cont rol” groups of 6 animals per group.
The study was performed with prior approval of the
Institutional Animal Ethics Committee (IAEC) of
CPCSEA (Committee for the Purpose of Control and
Supervision of Experiments on Animals) with approval
reg. no. JH/CPCSEA/31-01-2000/Project no.418/2007
2010. Utmost care was taken to ensure that animals were
treated in the most humane and ethically acceptable
2.6. Evaluation of Anticonvulsant Activity
2.6.1. PTZ and BCL Test
Seizures were induced chemically with PTZ as described
by Vohora et al. [20]. Standard convulsing agents, PTZ
(70 mg/kg, i.p.), and BCL (30 mg/kg, i.p.) were used to
induce convulsions in mice. Sodium valproate (300 mg/
kg, p.o.) was used as the reference anticonvulsant drug
for comparison. In the test group, the seizures were in-
duced after 60 min prior treatment with graded doses of
the APE (100 - 800 mg/kg, p.o.) and sodium valproate
(300 mg/kg, p.o.). The ability of the APE to prevent the
seizures or delay the latency or onset of the hind-limb
tonic extensions was considered as an indication of anti-
convulsant activity. The onset and duration of convul-
sions in the mice were noted and recorded, and percen-
tage protection was determined. Distilled water (10
ml/kg, p.o.) treated mice were used as “control” animals.
Copyright © 2013 SciRes. PP
Anticonvulsant, Anxiolytic and Neurotoxicity Profile of Aqarqarha (Anacyclus pyrethrum)
DC (Compositae) Root Ethanolic Extract 537
2.6.2. ICES
The ICES (Increasing Current Electroshock) test, as
proposed by Kitano et al. [21] and modified by Ali et al.
[22], was used to evaluate the an ticonvulsant effect of the
APE against generalized tonic clonic seizures. APE was
administered at various dose levels 60 min before
administering a current of 2 mA. If HLTE was not
observed with a current of 30 mA, electroshock was
terminated and this cut off current was used for the
2.7. Elevated Plus-Maze Test
The animals were trained before APE was administered
at various dose levels (100 - 800 mg/kg, p.o.) 60 min
before they were placed on maze. To begin a test session,
mice were placed on the open arm facing the center of
the maze. An entry into an arm was defined as the animal
placing all four paws over the line marking that area. The
number of entries and the time spent in the open and
closed arms were recorded during a 5min test period. The
percentage of open arm entries (100 ×open / total entries)
was calculated for each animal. Diazepam at a dose of
0.5 mg/kg, i.p. was used as the standard. Every precau-
tion was taken to ensure that no external stimuli, other
than the height of the plus-maze could invoke maze
2.8. Rotarod Test
The rotarod test according to Lima et al. [23] was used to
determine the effect of APE on motor coordination. The
trained animals were then evaluated for motor coordi-
nation at 30, 60, 90, and 120 minutes after oral ad-
ministration of 100, 400, 800, 1600 mg/kg of the APE.
The time each animal could walk continuously on the rod
was recorded.
2.9. Data Analysis
The results are presented as means ± SEM. Data were
analyzed using a one-way analysis of variance (ANOVA)
followed by Dunnett’s test (GraphPad) at the 95%
confidence level. P values < 0.05 were considered signi-
3. Results
3.1. HPTLC Fingerprinting of the APE
The HPTLC profile of the Aqarqarha (Anacyclus pyre-
thrum) ethanolic extract revealed the presence of six
spots at Rf 0.10, 0.15, 0.27, 0.34, 0.65 and 0.92. Rf 0.65
corresponds to the Rf value of the pure eugenol applied
and, therefore, confirmed the presence of eugenol in the
extract (Figure 1).
3.2. Effect of APE on PTZ-Induced Seizures
The study shows that APE markedly increases the
latency time for the onset of myoclonic jerks and clonic
seizures and offered significant percentage of protection
from 17% to 82% to mice against PTZ induced seizures
in a dose-dependent manner as compared to control value.
The latency time of stan dard was also increase to 98% as
compared to control value and was found satisfactory
significant as shown by Figures 2 and 3.
Figure 1. HPTLC fingerprinting of the Anacyclus pyre-
thrum ethanolic extract, [A] TLC Chromatogram of deve-
loped APE and pure eugenol; [B] HPTLC Chromatogram
of extract; [C] HPTLC Chromatogram of pure Eugenol
alone scanned at 254 nm.
Figure 2. Effect of APE and Sodium valproate on Latency
against PTZ-induced seizures in mice. *p < 0.05; **p <
0.01;***p < 0.001 as compared with control group.
Copyright © 2013 SciRes. PP
Anticonvulsant, Anxiolytic and Neurotoxicity Profile of Aqarqarha (Anacyclus pyrethrum)
DC (Compositae) Root Ethanolic Extract
Figure 3. Percentage anticonvulsant protective effect of
APE on PTZ-induced seizures in mice. *p < 0.05; **p < 0.01
and ***p < 0.001 as compared with control group.
3.3. Effect of APE on BCL-Induced Seizures
The study shows that 66% animals, treated with APE
(800 mg/kg) produced significant protection (p < 0.001)
against BCL induced seizures, while the animals treated
with lower d oses of AP E (100 - 4 00 mg/k g ) d id n o t show
any protection against BCL induced seizures. 100%
animals showed significant (p < 0 .001) protectio n again st
BCL induced seizures which were treated with standard
(Sodium valproate). The latency of tonic convulsions
was found 7.26%, 15.22 % 33.91% and 227.33% incr ease
in APE (100-800 mg/kg) treated animals and 530.79%
increase in sodium valproate treated animals as shown by
Figures 4 and 5.
3.4. Effect of APE on ICES-Induced Seizures
Aqarqarha (Anacyclus pyrethrum) root ethanolic extract
(APE, 100 - 800 mg/kg, p.o.) did not show any protective
effect against ICES-induced seizures.
3.5. Effect of APE on Elevated plus Maze
In the elevated plus maze, the observed behavior con-
firmed the anxiolytic activity of diazepam as reported.
The APE at the dose of 800 mg/kg increased the per-
centage of time spent and entries in the open arms (p <
0.001). However, APE (100 - 400 mg/kg) had no sig-
nificant effect on any of the measured parameters (Fig-
ures 6 and 7).
3.6. Effect of APE on Rotarod Test
APE did not produce the statistically significant distur-
Figure 4. Effects of APE and Sodium valproate on Latency
against BCL induced seizures in mice ***p < 0.001 as
compared wi t h control group.
Figure 5. Percentage anticonvulsant protective effect of
APE against BCL-induced seizures in mice. ***p < 0.001 as
compared wi t h control group.
bance in motor coordination or fall-off time up to the
maximum dose of 1600 mg/kg p.o. at 60 min post-
administration period. However, the dose of 1600 mg/kg
of APE at 90 and 120 min post administration exhibited a
significant impairment in motor coordination (Figure 8).
4. Discussion
There are a number of synthetic anticonvulsant drugs
currently available for use in the management, control
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Anticonvulsant, Anxiolytic and Neurotoxicity Profile of Aqarqarha (Anacyclus pyrethrum)
DC (Compositae) Root Ethanolic Extract 539
Figure 6. Number of open arm entries in Elevated plus
maze. ***p < 0.001 as compared with control group.
Figure 7. Percentage time spent in open arm in Elevated
plus maze ***p < 0.001 as compared with control group.
and/or treatment of individuals with epilepsy. Keeping in
view of their inaccessibility, higher cost and increased
toxicity, there is a dire need for the development of cheap,
effective and relatively safer anticonvulsant agents from
plants and other natural sources. Although Aqarqarha
(Anacyclus pyrethrum) root is widely used by the
practitioners of Unani system of medicine for epilepsy,
relatively little scientific information ex ists in biomedical
literature on the therapeutic efficacy of the plant.
The data obtained in this study also indicate that
relatively moderate and high doses of APE inhibited or
attenuated PTZ-induced seizures, while the reference
Figure 8. Effect of i.p. administration of APE on rotarod
test endurance time in seconds at different time intervals:
30, 60, 90, and 120 min post administration. *p < 0.05 and
**p < 0.01 as compared with control group.
anticonvulsant drug used, i.e. sodium valproate, com-
pletely abolished the seizures and protected all the ani-
mals against PTZ-induced seizures. PTZ has been re-
ported to produce seizures by inhibiting gamma amino-
butyric acid (GABA) neurotransmission. Favoring the
GABAergic hypothesis for sodium valproate is inhibition
of the onset of seizures induced by GABA antagonists
[24] which selectively enhances GABAergic inhib ition in
the cerebral cortex [25].
It would appear, therefore, that the complete protection
of the mice by the reference anticonvulsant drug used in
this study against PTZ-indu ced seizures is in consonance
with the above hypothesis. The findings of the present
study, therefore, tend to suggest that APE might have
inhibited and/or attenuated PTZ-induced seizures of the
mice by inhibiting high-frequency firing of neurons
and/or enhancing some ways interfering with GABA er-
gic neurotransmission.
BCL, a potent and selective GABAA-receptor antago-
nist, produces seizures by blocking the effect of GABA
at central GABAA-receptors, which have been associated
with epilepsy. Sodium valproate, which also acts by
enhancing GABA neurotransmission, antagonized BCL-
induced seizures in the present study. However, APE
only at dose 800 mg/kg, antagonized BCL-induced sei-
zures, which was less effective in this regard com- pared
with Sodium valproate. This observation also tends to
suggest that APE interferes with GABAergic neuro-
transmission. APE was also found to possess anxiolytic
potential when examined on the elevated plus-maze test
at the dose of 800 mg/kg. The magnitude of the anxio-
lytic effects of this dose was very close to that observed
with 0.5 mg/kg of diazepam. These anxiolytic properties
could be mediated by some components in the extract
interacting with the benzodiazepine/GABAA receptors as
Copyright © 2013 SciRes. PP
Anticonvulsant, Anxiolytic and Neurotoxicity Profile of Aqarqarha (Anacyclus pyrethrum)
DC (Compositae) Root Ethanolic Extract
agonists, with the 5-HT1A receptors agon ists, or with any
other mechanisms. Furthermore, APE did not show any
neurotoxic activities at the effective anticonvulsant dose
level (800 mg/kg) in the rotarod test. However, it
impaired motor coordination at 1600 mg/kg, 90 min after
the APE administration.
Our present state of knowledge of the chemical con-
stituents of the extract is limited. It is, therefore, im-
possible for us at this stage, to pinpoint and identify with
certainty, the anticonvulsant principle of the extract.
Nevertheless, eugenol (4-allyl-2-methoxy phenol, C 10H12O2,
an aromatic terpene compound, whose presence was
confirmed in the extract by HPTLC fingerprinting, is
likely to account for the observed anticonvulsant activity
of the APE. Szabadics and Erdelyi [26] have shown that
eugenol blocks the synaptic transmission in the snail,
Helix pomatia L., neurons both pre- and post-syn-
aptically. There are some studies indicating that eugenol
possesses an inhibitory effect on the events related to
NMDA receptor activation and also protects neuronal
cells from NMDA-induced excitotoxic and oxidative
injury [27]. Furthermore, it protects hippocampal neu-
rons from global ischemia through modulation of NMDA
receptors and also delays NMDA-induced convulsions
The experimental evidence obtained in the present
laboratory animal study indicates that APE significantly
delayed the onset of seizures induced by PTZ. Since
PTZ-induced seizures have been shown to be due to the
inhibition and/or attenuation of GABAergic neurotrans-
mission, it is not unreasonable to speculate that APE
probably produces its anticonvulsant activity by en-
hancing GABAergic neurotransmission.
In conclusion, the findings of the present laboratory
animal study lend a pharmacological support to the sug-
gested anecdotal, ethnomedical uses of Aqarqarha (Ana-
cyclus pyrethrum) roots in the treatment of epilepsy in
the Unani system of medicine. The results of the present
laboratory animal study provide evidence in favor of the
selective anticonvulsant activity of the herb, and show
that APE also possesses significant anxiolytic activity
without evidence of any neurotoxicity at the effective
dose level used. The effectiveness of the plant’s extract
in the experimental convulsion paradigm used probably
suggests that the herb could be used in complex partial
and absence types of epilepsy as supported by its failure
to protect the animals against ICES test. However,
extensive studies are needed to evaluate the precise
mechanism(s) of the plant as a medicinal remedy for
5. Acknowledgements
Authors are highly thankful to Dr Shadab Zafar (Medical
Officer, Municipal Corporation of Delhi) for his useful
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