Investigation of Antiepileptic Activity of Hibiscus vitifolius
leaves
Pradeep Kumar Samal*
SLT Institute of Pharmaceutical Sciences,
Guru Ghasidas Vishwavidyalaya,
Bilaspur, Chhattisgarh.
*Corresponding
Author E-mail: samalpharmacology@rediffmail.com
ABSTRACT:
The leaves of Hibiscus vitifolius (Malvaceae) for the present studies was
selected on the basis of literature review. The fresh leaves were collected
from Thakur Chhedilal
barrister Agriculture College and Research Centre, Bilaspur,
Chhattisgarh. The Plant material was authenticated by Dr. H. B. Singh. Ref No.
is NISCAIR/RHMD/Consult/-2011-12/1868/168 on 24 Oct 2011 New Delhi. Shade dried leaves were powdered in a hand grinder and
extracts were prepared in chloroform and ethanol solvents. Phytochemical
studies found that the presence of flavionoids, glycosids, Sterols, Fatty acids and carbohydrate. Acute
oral toxicity study of plant extracts was conducted in mice by using OECD
guideline no.425 and it was found to safe up to the dose label of 6000mg/kg of
body weight. In Maximal electro-shock (MES) induced model, dose of EEHV (200,
400 and 600 mg/kg) show significant anticonvulsant activity, but dose of
chloroform extract of Hibiscus
vitifolius (CEHV)
did not show any anticonvulsant activity as compared to control group. In Pentylenetetrazol (PTZ) model lower dose of EEHV (200, 400
and 600 mg/kg) show good anticonvulsant activity as compared to control group. Locomotor study in actophotomotor
extract of EEHV exhibited the decreased motor activity in dose dependent
manner, it is sign of sedation. EEHV increase the threshold of MES induced
convulsion in a dose dependent manner.
KEY WORDS: Hibiscus vitifolius, PTZ, Maximal electro-shock, Diazepam and Phenytoin
1. INTRODUCTION:
Epilepsy is one of the major
brain disorders worldwide. Epilepsy is a medical condition that produces
seizures affecting a variety of mental and physical functions. The condition is
characterized by repeated seizures or fits. Patients with epilepsy often
require lifelong pharmacotherapy. Current drug therapy of epilepsy is
complicated by side-effects, tolerance, teratogenic
effects, dependence and long term toxicity. Medicinal
plants used for the treatment of epilepsy in traditional medicine practice
possess promising anticonvulsant activities in animal models of anticonvulsant
screening and these can be an invaluable source for search for new
antiepileptic compounds. Hibiscus vitifolius (Malveceae)
is a common plant of Indian Territory and well-adapted species in the USA and
other country. Traditionally Hibiscus
vitifolius leaves are used for the
treatment of anti-inflammatory, hypoglycemic, and epilepsy1
The
aim of the present study is to investigate
the antiepileptic activity of Hibiscus vitifolius leaves.
2. MATERIAL AND
METHODS: -
2.1. Drugs and
Chemicals:-
Analytical grade chemicals were used in this
study. Pentylenetetrazole
(PTZ) (Himdia chemical) and Diazepam (Calmpose inj.) were purchased from local market, while Phenytoin was obtained as a gift sample from Stallion
laboratories Pvt. Ltd. Ahmadabad (Gujarat). All phytochemical
reagents and other chemicals were procured from the Institute.
2.2. Plant Materials :-
The fresh leaves of Hibiscus vitifolius
(Malvaceae
) were collect from Thakur
Chhedilal Barrister Agriculture College and Research
Centre, Bilaspur, Chhattisgarh in the month of
November 2011. The Plant was identified by Dr. H. B. Singh Chief Scientist Head
of the Raw Materials Herbarium and Museum NISCAIR, New Delhi (Ref. - NISCAIR/RMHD/Consult/2011-12/1868/168).
Leaves were shade
dried for 15 days, grind in a mechanical grinder and extracted with ethanol solvent.
2.3. Animals:
Wistar albino
rats (150-200g) purchased from IICB Kolkatta was
maintained in the department animal house for experimental purpose. All the
animals were acclimatized for seven days under standard animal husbandry
conditions, i.e.
room temperature of 25 ± 10 C,
relative humidity 45-55% and a 12:12hr light/ dark cycle. The animals were
feed with standard rat pellet (Pranav Agro Industries
Ltd, Vadodara, India), with water supplied ad libitum
under strict hygienic conditions. Each experimental group had separate set of
animals and care was taken to ensure that animals used for one response were
not employed elsewhere. Animals were habituated to laboratory conditions for 48
hours prior to experimental protocol to minimize if any of non-specific stress.
The approval of the Institutional Animal Ethical Committee (IAEC) of S.L.T.
Institute of Pharmaceutical Sciences, Bilaspur
(Chhattisgarh) was taken prior to the experiments. (Ref No.
IAEC / Pharamcy / 2012/41).
2.4 Acute
toxicity study (AOT):
Acute
toxicity study was performed according to the procedure OECD guideline no. 425.2
The acute oral toxicity of ethanolic
extract of Hibiscus vitifolius
leaves (EEHV) was
determined by using either sex albino
mice (Wistar strains) weighing between 35-50 gm. The animals were fasted 3 hrs
prior to the experiment. Animals were administered with single dose of extracts
dissolved in 2% w/v gum acacia and observed for its mortality during 48 hours
study period (short term) toxicity.
2.5. Experimental
Design and pharmacological screening:
2.5.1 Assessment
of locomotor activity:
The sedative
activity and its possible mechanism of EEHV
was investigated by determining the spontaneous locomotor activity of rats3. Locomotor
activity was monitored by using Actophotometer. All
groups of animals were put individual in the Actophotometer
after 60 min of treatment. The total activity count was registered for 5 min.
The locomotor activity was expressed in term of total
photo beam interruption count/5 min 4.
2.5.2
Antiepileptic activity 5
MES induced convulsion :-
Electrical shock
is an indicator, which shows the primary indication of grand mal epilepsy in
rats. Tonic limb extensions are evoked by electric stimuli which are suppressed
by antiepileptic but also by other centrally active drugs.6 The seizure was induced by maximal electroshock in swiss albino rats with the help of electroconvulsiometer
by passing current of 90 mA for 0.2 second using ear
clip electrodes. The animals were divided into six groups each containing 6
animals. The test samples were given 1 hour prior to induction of convulsions.
Group I (Control): Received vechile
(4% tween-80, 1 ml/kg body weight, p.o.),
Group II
(Standard): Received phenytoin (25mg/kg body weight i.p.),
Group III, IV and
V (Test): Received pretreated with single dose of EEHV 200 mg/kg, 400 mg/kg and 600 mg/kg body weight respectively.
Convulsion
was induced in rats by delivering trans-auricular electroshock of 90 mA for 0.2 sec by mean of convulsiometer
(INCO, Ambala, India). Duration of tonic limb
extension was noted in all groups. All the extract treated groups were compared
with control in order to determine the significant anticonvulsant activity7.
The rat was considered protected, if the drug prevented the hind leg
tonic extensor component of the convulsion.
PTZ induced
seizures: -
This is a widely used for preclinical evaluation
of anticonvulsant property of new drug. However, it has been shown that most anxiolytic agent is also able to prevent or antagonize PTZ
induced convulsio8. The animals were
divided into six groups each containing 6 animals. Treatment was same as that
of MES. After 1hr of the dosing all animals
were injected with the convulsing agent pentylenetetrazole
(PTZ )(60 mg/Kg) and animals were kept in individual plastic
cages to observe convulsions for 1h.
2.6
Statistical Analysis: - The values were expressed in
mean + SEM. The result were
subjected to statistical analysis by using one way ANOVA followed by dunnett’s test to calculate the significance from control
group. P<0.05 was considered significance.
3. RESULT:
3.1 Preliminary phytochemical Analysis :-
Preliminary phytochemical studies
of Hibiscus vitifolius leaves extract revealed the presence of phytoconstituents
like flavinoids, glycosids,
Sterols, Fatty acids, Tanin, oils and carbohydrate.
3.2 Acute toxicity study :-
Different
doses of ethanolic and chloroform extracts were screened for their acute oral
toxicity. No mortality was recorded till 6000 mg/kg with ethanolic extract, hence the extracts were found to be safe up to the
dose levels of 6000 mg/kg. Therefore 200, 400 and 600 mg/kg dose were selected
for the evaluation of antiepileptic activity.
3.3 Assessment of locomotor activity
:-
Locomotor activity of rats was measured in open field Actophotometer. Behavioral changes were determined by
different dose of EEHV. EEHV at a dose 200mg/kg did not show decrease motor
activity significantly whenever it was compare with normal control (P>0.05,
ns). While at dose of 400 mg/kg and 600 mg/kg body weight show
significantly decrease the motor activity in dose-dependent manner (P<0.05,
P<0.01). (Table No 1 and Fig No.1) A drug-induced decrease in spontaneous
motor activity is regarded as an indication of sedation.
Fig.1: Effect
of EEHV on animals in locomotors activity
3.4 Antiepileptic activity:-
3.4.1 MES induced
convulsion : -
On way ANOVA
showed significant effect
(P<0.05, P<0.01) on the duration of tonic hind limb
extension at 400 mg/ml and 600 mg/ml of EEHV respectively as compared to control group. Maximal protection was observed at
600 mg/ml dose. Mortality was not observed in any group. The phenytoin treated group (G II) showed highly significant
(p<0.01) effect on reduction of the
duration of tonic hind limb extension as compared to vehicle control and no
mortality was observed. In our investigation different components of maximal
electroshock seizure were simultaneously elicited in the following sequence:
tonic hind leg flexion, tonic hind leg extension follow by clonus
convulsion, stupor phase and recovery (Refer Table
2 and figure
No.2).
3.4.2 Pentylene tetrazole
(PTZ) induced convulsions: - EEHV show significant
anticonvulsant activity against PTZ induced convulsion in rats. It delayed the
onset of myoclonic jerks (fig. 3a) and decreased the
duration of tonic
convulsions (fig. 3b). In animals treated with vehicle, clonic convulsions appeared 121.66 ± 13.02 sec after PTZ
and 4 animals died after seizures. EEHV
significantly delayed the onset of myoclonic jerks at
doses of 200 mg/kg, 400mg/kg and 600 mg/kg (p<0.05, p<0.01 and p<0.01,
respectively) with maximum protection observed at 600 mg/kg (Table 3).
Reduction in the duration of tonic convulsions by the extract was profound at
the doses 400 mg/kg and 600 mg/kg (p<0.01, 0.01). Again, it was delay the
onset of PTZ-induced tonic convulsions and reduced the frequency of
convulsions. EEHV produce anticonvulsant effects similar to
that of Diazepam
against PTZ-induced
seizures. Diazepam
(2mg/kg) completely protected seizure. Also diazepam caused significant
reduction of the frequency and duration
of tonic convulsions.
Fig. 2:
Effect of EEHV on animals in MES induced
Fig. 3 Effect of EEHV on
animals in PTZ induced
Fig. 3.b:
Effect of EEHV on animals in PTZ induced
4.
DISCUSSION: -
Epilepsy is
one of the most common brain diseases in human. About 1% of world population
was affected from epilepsy. Several different types of human epilepsies have
been characterized based on the classification of International League against
Epilepsy (ILAE). An imbalance between the excitatory and inhibitory
neurotransmitters is responsible for seizures.The ability
of an agent
to prevent or
delay the onset
of tonic and
tonic-clonic convulsion induced
by PTZ in animals
is an indication
of anticonvulsant activity9. Ethanolic extract
screened for anti-epileptic activity against MES induced convulsions. In the
present study the single dose (400 mg/kg, and 600 mg/kg body weight)
administration of ethanolic extract of Hibiscus vitifolius show significant effect, but not in
the dose of 200 mg/kg body weight. Whereas, EEHV (600 mg/kg, p.o.)
has been highly significant in THE phase in MES model.
Single dose administration of EEHV (400 mg/kg and 600 mg/kg, p.o. respectively) show spontaneous decrease of locomotors
activity in rats in dose dependent manner. Decrease the locomotors
score, indicating the sedative effect of the extract. EEHV (200 mg/kg p.o.) was not producing any significantly effect.
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