RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
EVALUATION OF THE ANTI-EPILEPTIC ACTIVITY OF HYDROALCOHOLIC
EXTRACT OF ACHYRANTHES ASPERA Linn LEAVES.
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE
BY
LALA MINAXI MAHENDRA
Under The Guidance of
Dr. SHIRISH KUMAR AMBAVADE.M.PHARM, PhD
P.G. DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
2009 – 2010
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
ANNEXURE-II
BANGALORE, KARNATAKA.
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.0
NAME AND ADDRESS OF THE CANDIDATE
LALA MINAXI MAHENDRA
T.C.X-NORTH-118
OPPOSITE HOTEL SHIV
GANDHIDHAM
KUTCH-370201
GUJARAT
2.0
NAME OF THE INSTITUTION
SRINIVAS COLLEGE OF PHARMACY
VALACHIL,MANGALORE.-574143
3.0
COURSE OF STUDY AND SUBJECT
MASTER OF PHARMACY IN PHARMACOLOGY.
4.0
DATE OF ADMISSION TO COURSE
15th October 2009
5.0
TITLE OF THE TOPIC
EVALUATION OF THE ANTI-EPILEPTIC ACTIVITY OF HYDROALCOHOLIC
EXTRACT OF ACHYRANTHES ASPERA Linn LEAVES.
6.0
BRIEF RESUME OF THE INTENDED WORK :
6.1 NEED FOR THE STUDY:
Epilepsy (greek word epilepsia — "to seize") is a common chronic
neurological disorder characterized by recurrent unprovoked
seizures.[1] These seizures are transient signs and/or symptoms of
abnormal, excessive or synchronous neuronal activity in the
brain.[2] About 50 million people worldwide have epilepsy, with
almost 90% being in developing countries. Epilepsy mostly occurs in
young children, or over the age of 65 years, however it can occur
at any time. Seizure types are organized firstly according to
whether the source of the seizure within the brain is localized
(partial or focal onset seizures) or distributed (generalized
seizures).partial seizures are further divided into simple partial
and complex partial. Generalized seizures are divided according to
the effect on the body but all involve loss of consciousness. These
include absence (petit mal), myoclonic, clonic, tonic, tonic-clonic
(grand mal) and atonic seizures.[3]
The exact causes of epilepsy being unknown, sometimes another
problem, such as a head injury, brain tumor, brain infection, or
stroke, causes epilepsy. Person gets exhausted and confused,
develops headache, lose bowel and bladder control, and vomits.
Currently there are 20 medications approved by the Food and Drug
Administration for the use of treatment of epileptic seizures in
the US: carbamazepine, clorazepate , clonazepam, ethosuximide,
felbamate, fosphenytoin, gabapentin, lamotrigine, phenobarbital,
phenytoin etc.
But these drugs posesses dose dependent side effects like mood
changes, sleepiness, or unsteadiness in gait, idiosyncratic
reactions, rashes, liver toxicity (hepatitis), or aplastic
anemia.[4]
Surgery is an option for patients resistant to treatment with
anticonvulsant medications for controling of epileptic
seizures.[5]Other therapy includes ketogenic diet and vagus nerve
stimulation.[6]
Thus due to prevalent side effects and tolerance development of
the presently available treatment for epilepsy, it is a necessary
to develop a new drug without or less side effect. Plants are the
well proven source of new chemicals for the treatment of various
diseases and disorders. Majority of the drugs are obtained from the
plant source with various constituents possessing activity against
large number of diseases. Many traditional herbal medicines have
been used for CNS disorders. This reveals that evaluation of herbal
drugs is still needed. Achyranthes aspera (amaranthaceae) is the
traditionally used plant for the treatment of epilepsy; however,
there is paucity of scientific data for its anticonvulsant and
antiepileptic activity.
The objective of present study is to investigate the safety
profile and efficacy of Achyranthes aspera (amaranthaceae) for its
anticonvulsant activity.
6.2 REVIEW OF LITERATURE:
6.2.1 ACHYRANTHES ASPERA.
Achyranthes aspera linn belongs to family amaranthaceae.
common name : apamarga, prickly chaff flower, safed aghedo,
latjira, uttaranee
Description: Chirchira is an erect herb, 0.3-1meter high with
stiff branches terete or absolutely quadrangular, striate,
pubescent, leaves few, usually thick, elliptic-obovate, petiolate,
acute and entire. Flowers are greenish white, numerous in small
dense auxiliary heads or spikes, bracts and bracteoles persisting
ending in a spine. Main root is long cylindrical thick; secondary
and tertiary roots present slightly ribbed, yellowish brown in
color; odor is slight, taste is slightly sweet and mucilaginous;
stem is yellow brownish, erect branched, cylindrical hairy about 60
cm high. Seeds are sub cylindrical, truncats at apex, rounded at
base, black and shining. The plant is distributed throughout India
up to an altitude of 3000ft.[7]
Chemical constituents: plant is reported to contain alkaloid,
flavonoid, saponin, steroids and terpenoids. It yield water soluble
base called betain.[8] Seed, stem, leaf and root are reported to
contain ecdysterone.[9] Long chain alcohol, viz. 17-penta
triacontanol, 27-cyclohexyl heptaeosan-7-ol, 16-hydroxyl 26-methyl
heptacosan-2one and 36, 47-dihydroxy hen-pentacontan-4one.[10]
Chloroform extract of stem led to isolation of pentatriacontan,
6-pentatriacontanone, hexatriacontane and triacontane.[11] Ethanol
extract contained alkaloid and saponin while flavonoid and tannins
were absent.[12]
Uses: Achyranthes aspera plant material is used in the Ayurvedic
formulations for the treatment of Epilepsy (Ayurvedic system of
medicine).[13] It is bitter, pungent, heating, laxative, stomachic,
carminative and useful for the treatment of vomiting, bronchitis,
heart disease, piles, itching abdominal pains, ascites, dyspepsia,
dysentery, blood diseases.[7] it is well known for its diuretic
effect.
Ethanolic extract of the drug in doses of 100-200mg/kg showed
anti-inflammatory and anti-arthritic activity.[14] Aqueous solution
of achyranthine and entire plant showed antibacterial activity
against S.aureus, Streptococuss heamolyticus and Bacillus
typhosus.[15] Extracts of whole plant showed anti-fertility
activity.[16] Plant extract of whole plant showed immunomodulatory
activity. Aqueous and methanolic extract of entire plant showed
anti-diabetic action.[17] Aqueous and alcoholic extract of roots
produces dieresis and hypotension.[18] Methanolic extract,
alkaloidal, and nonalkaloidal fractions of leaf possess
anticarcinogenic activity.[19]
6.3 OBJECTIVES OF THE STUDY:
1) Extraction of plant of Achyranthes aspera Linn.
2) Preliminary phytochemical investigations.
3) Assessment of acute toxicity of the plant extract.
4) To study the effect of Achyranthes aspera Linn. on chemical
induced convulsions.
Chemical induced convulsions
i) Pentylenetetrazole induced convulsions
ii) Picrotoxin induced convulsions
iii) Strychnine induced convulsions
5) To study the effect of Achyranthes aspera Linn on
electrically induced convulsions.
6) To study the effect of drug on the GABA level in mice.
7.0
MATERIALS AND METHODS:
7.1 SOURCE OF DATA:
Experiment will be performed as described in the standard
bibliography, literatures and text books. The reputed journals and
publications are obtained from college library.
7.2 COLLECTION OF MATERIAL:
7.2.1 Collection of plant material and extraction:
The plant of Achyranthes aspera Linn will be collected from the
local suppliers of mangalore. The powdered leaves (135 g) will be
extracted by cold maceration method using 70% ethanol solvent
system. The extract will be filtered through a cotton plug followed
by Whatman filter paper No.1 and then concentrated by using a
rotary evaporator at low temperature. The extract will be preserved
in airtight containers and kept at 4-5°C until further use.
All other chemicals like PTZ, picrotoxin, strychnine of pure
analytical grade and drugs like diazepam will be obtained from
hi-media suppliers.
7.2.2 ANIMALS
Wistar rats (175-200 g) and Swiss albino mice (17-25 g) of
either sex are procured from Indian Institute of Sciences. They are
maintained under standard conditions (temperature 22 ± 2oC,
relative humidity 50±5% and 12 h light/dark cycle).The animals were
housed in sanitized polypropylene cages containing sterile paddy
husk as bedding. They had free access to standard pellet diet and
water and libitum. The Institutional Animal Ethics Committee
approved the experimental protocol. All the animals received humane
care according to the criteria outlined in the “Guide for the Care
and Use of Laboratory Animals” prepared by the “National Academy of
Sciences” and published by the “National Institute of Health”.
7.3 EXPERIMENTAL METHODS :
7.3.1. Assessment of acute toxicity:
The acute toxicity study of the fractions and isolated
phytoconstituents will be performed as per the OECD guidelines 425
at a limit dose of 2000 mg/kg or 5000 mg/kg. The doses will be
administered by oral route in mouse as per scheduled in OECD
guidelines 425. Animals will observed individually at least once
during the first 30 minutes after dosing, periodically during the
first 24 hours (with special attention given during the first 4
hours), and daily thereafter, for total 14 days for sign of
toxicity and/or mortality if any. The LD50 will be calculated by
using OECD 425 software.[20]
7.3.2 PENTYLENETETRAZOLE (METRAZOL) INDUCED CONVULSIONS
Experimental Design :
The animals will be randomly divided into 5 groups of 6 each.
The different groups will be assigned as described below. Mice of
either sex weighing 18 and 22g will be used. The test compound of
low, medium, and high dose or the reference drug diazepam (5mg/kg)
will be injected i.p. to groups of 6 mice. Another group of serves
as control. 30 min after i.p. injection, 80 mg/kg MTZ (Metrazol)
will be injected i.p. Animal will be placed into an individual cage
and observed for 1 h. Seizures and tonic-clonic convulsions will be
recorded.[21]
Group I : Vehicle control (0.9% saline).
Group II : diazepam (5mg/kg) + MTZ (80mg/kg).
Group III : test drug (low dose) + MTZ (80mg/kg).
Group IV : test drug (medium dose) + MTZ (80mg/kg).
Group V : test drug (high dose) + MTZ (80mg/kg).
EVALUATION:
The time interval between MTZ-injection and occurrence of
seizures can be measured. The delay of onset is calculated in
comparison with the control group.
7.3.3 PICROTOXIN INDUCED CONVULSIONS.
Experimental Design :
The animals will be randomly divided into 5 groups of 6 each.
The different groups will be assigned as described below. Groups of
6 mice weighing 18 and 22 g will be treated i.p. with the test
compound (low, medium, high dose) or the standard (10 mg/kg
diazepam i.p.).Thirty min after i.p. treatment administration the
animals will be injected with 3.5 mg/kg s.c. picrotoxin and
observed for the following symptoms during the next 30 min: clonic
seizures, tonic seizures, death. Time of onset of seizures and time
to death will be recorded.[21]
Group I : Vehicle control (0.9% saline).
Group II : diazepam (10mg/kg) + picrotoxin (3.5mg/kg).
Group III : test drug (low dose) + picrotoxin (3.5mg/kg).
Group IV : test drug (medium dose) + picrotoxin (3.5mg/kg).
Group V : test drug (high dose) + picrotoxin (3.5mg/kg).
EVALUATION:
Time of onset of seizures and time to death will be recorded.
The increase in latency of the onset of convulsions will be
evaluated as anticonvulsant activity.
7.3.4 STRYCHNINE INDUCED CONVULSIONS
Experimental Design :
The animals will be randomly divided into 5 groups of 6 each.
The different groups will be assigned as described below. Groups of
6 mice of either sex weighing 18 and 22 g will be used. They will
be treated orally with the test compound (low, medium, high dose)
or the standard (e.g. diazepam 5 mg/kg). One hour later the mice
will be injected with (2 mg/kg, i.p.) strychnine nitrate. The time
until occurrence of tonic extensor convulsions and death will be
noted during a 1 h period.[21]
Group I : Vehicle control (0.9% saline).
Group II : diazepam (5mg/kg) + strychnine (2mg/kg).
Group III : test drug (low dose) + strychnine (2mg/kg).
Group IV : test drug (medium dose) + strychnine (2mg/kg).
Group V : test drug (high dose) + strychnine (2mg/kg).
EVALUATION:
The increase in latency for onset of convulsions and decrease in
death rate will be evaluated as anticonvulsant activity.
7.3.5 ELECTROSHOCK INDUCED CONVULSIONS.
Experimental Design :
Electro-convulsive shock, induce Hind Limb Tonic Extension
(HLTE) in 99% of the animals. The electrical stimulus (50 mA; 50
Hz; 1-s duration) will be applied through ear-clip electrodes using
a stimulator apparatus. Animals will be divided in 5 groups of 6
each. Group of 6 mice will be pretreated with test drug (low,
medium, high dose), phenytoin (25 mg/kg, as positive control),
saline (10 ml/kg, as control) and after 30 min will receive the
transauricular electroshock. The criterion for the anticonvulsant
effect will be absence of HLTE within 10 s after delivery of the
electroshock.[22]
Group I : Vehicle control (0.9% saline).
Group II : phenytoin (25mg/kg) + electrical stimulus (50mA;
50Hz; 1secduration).
Group III : test drug (low dose) + electrical stimulus (50mA;
50Hz;1secduration).
Group IV : test drug (medium dose) + electrical stimulus (50mA;
50Hz;1secduration).
Group V : test drug (high dose) + electrical stimulus (50mA;
50Hz; 1secduration).
EVALUATION:
The animals will be observed closely for 2 min. Disappearance of
the hind leg extensor tonic convulsion is used as positive
criterion. Percent of inhibition of seizures relative to controls
will be calculated.
7.3.6 ESTIMATION OF GABA LEVEL IN MICE.
Five groups of 6 mice will be treated with diazepam (10mg/kg) or
vehicle or test drug (low, medium, high dose). After 45mins they
are sacrificed. Brain will be isolated immediately and transferred
to homogenization tube containing 5 ml of 0.01N hydrochloric acid
and homogenized. Brain homogenate will be transferred to bottle
containing 8 ml of ice cold absolute alcohol and will be kept for 1
hour at 0 0C. The content will be centrifuged for 10 min at
16000 rpm, supernatant collected in petridish. Precipitate will be
washed with 3-5 ml of 75% alcohol for three times and washes
combined with supernatant. Contents in petridish will be evaporated
to dryness at 70-90 0C on water bath under stream of air. To
the dry mass 1 ml water and 2 ml chloroform will be added and
centrifuged at 2000 rpm. Upper phase containing GABA will be
separated and 10 ul of it applied as spot on whatman paper (NO.
41).
Chromatographic conditions:
The mobile phase consist of n-butanol (50 ml) acetic acid (12
ml) and water (60 ml). The chamber will be saturated for half hour
with mobile phase. The paper chromatogram will be developed with
ascending technique. The paper dried in hot air and then spread
with 0.5% ninhydrin solution in 95% ethanol. The paper dried for 1
hr at 90 0C. Blue color spot developed on paper will be cut
and heated with 2 ml ninhydrin solution on water bath for 5 min.
water (5 ml) will be added to solution and will be kept for 1h.
Supernatant will be decanted and absorbance will be measured at 570
nm.
Standards and calculations:
Stock solution of standard GABA, 1 mg/ml will be prepared
in 0.01N HCl. Serial dilutions will be done to get
concentrations 1ng/10ul to 1000ng/10ul. To obtain a standard
concentration curve for GABA same procedure will be followed
replacing brain homogenate with standard GABA solutions.[23]
Group I : vehicle control (0.9% saline)
Group II : standard drug diazepam (10 mg/kg)
Group III : test drug (low dose)
Group IV : test drug (medium dose)
Group V : test drug (high dose)
7.4 STATISTICAL ANALYSIS:
The data will be expressed as Mean value + SEM and will be
analyzed by the one-way ANOVA followed by the Dennett’s test.
7.5 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS
TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO
PLEASE DESCRIBE BRIEFLY.
Yes. Study requires investigation on albino mice.
7.6 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR
INSTITUTION?
Yes. Ethical clearance has been obtained. (Copy enclosed)
8.00
LIST OF REFERENCES:
[1]. "Guidelines for epidemiologic studies on epilepsy.
Commission on Epidemiology and Prognosis, International League
against Epilepsy". Epilepsia 1993;34(4):592–6.
[2]. Fisher R, van Emde Boas W, Blume W, Elger C, Genton P, Lee
P, Engel J "Epileptic seizures and epilepsy: definitions proposed
by the International League Against Epilepsy (ILAE) and the
International Bureau for Epilepsy (IBE)". Epilepsia
2005;46(4):470–2.
[3]. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology.5th
ed.New Delhi:Elsevier; 2005. p. 550.
[4]. Baker GA, Jacoby A, Buck D, et al. "Quality in life in
people with epilepsy: a European study". Epilepsia
1997;38(3):353–362.
[5]. Birbeck GL, Hays RD, Cui X, Vickrey BG. "Seizure reduction
and quality of life improvements in people with epilepsy".
Epilepsia 2002;43(5):535–538. .
[6]. Theodore WH, Fisher RS. "Brain stimulation for epilepsy".
Lancet Neurol. 2004;3(2):111–118.
[7]. Kirtikar KR, Basu BP. Indian medicinal plants. 3rd ed.
Vol.3 Dehradhun:Bishen singh, Mahendra pal singh publications;
1991. P. 2065-2067.
[8]. Kapoor VK, Singh H. Isolation of betain from Achyranthes
aspera Linn. Indian J. Chem. 1966;4:461.
[9]. Banerjii A, Chintalwar GJ, Joshi, AK, Chandha MS. Isolation
of ecdysterone from Indian plants. Phytochem. 1971;10:2225-26.
[10]. Misra TN, Singh RS, Pandey HS, Prasad C, Singh BP. Two
long chain compounds from Achyranthes aspera. Phytochemistry
1993;33:221-23.
[11]. Ali M. Chemical investigation of Achyranthes aspera Linn.
Oriental J Chemistry 1993;9:84-85.
[12]. Kumar S, Singh JP, Kumar SS. Phyto chemical screening of
some plants of Manipur-I. J Econ Bot. Phytochemistry
1990;1:13-16
[13]. Gupta NNS. The Ayurvedic system of medicine. New Delhi:
Logas press 1984;2:411-416.
[14]. Gokhale AB, Damre AS, Kulkami KR, Saraf MN. Preliminary
evaluation of anti-inflammatory and anti-arthritic activity of S.
lappa, A. speciosa, & A. aspera. Phytomedicine
2002;9(5):433-37.
[15]. Basu NK, Neogi NC, Srivastava VP. Biological investigation
of Achyranthes aspera Linn and its constituent achyranthine. J proc
Inst Chem. 1957;29:161-65.
[16]. Pakrashi A, Bhattacharya N. Abortifacient principle of
Achyranthes aspera Linn. Indian J Exp Biol. 1977;15(10):856-87.
[17]. Akhtar MS, Iqbal J. Evaluation of the hypoglycemic effect
of Achyranthes aspera. J Ethnopharmacology 1991;31:49.
[18]. Neogi NC, Garg RD, Rathor RS. Preliminary
pharmacological studies on achyranthine. Indian J Pharmacology
1970;32:43-46.
[19]. Tahiliani P, Kar A. Achyranthes aspera elevates thyroid
levels and decreases hepatic lipid peroxidation in male rats.
Journal of Ethanopharmacology 2000; 71(3):527-32.
[20]. OCED 425 guidelines. OCED guidelines for testing animals
(2001); Decl/26:1-26.
[21]. Gerhard Vogel H (Ed.). “Drug Discovery and Evaluation”
pharmacological assays, 2nd ed. New York:Springer verlag
Heidelberg; 2002. P. 422-424.
[22]. Mohammad Sayyah, Leila Nadijafnia, Mohammad Kamalinejad.
Anticonvulsant activity and chemical composition of Artemisia
drancunculus L. essential oil. Journal of Ethnopharmacology
2004;94:283–287
[23]. MAynert EW, Klingman GI, Kaji HK. Tolerance to morphine.
II. Lack of effects on brain 5-hydroxytryptamine and
gamma-aminobutyric acid. J phar Exp Ther. 1962;135:296-299.
9.
SIGNATURE OF THE CANDIDATE
10.
REMARKS OF THE GUIDE
11.
NAME AND DESIGNATION
11.1 GUIDE
Dr. SHIRISH KUMAR AMBAVADE.
PROFESSOR,
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY)
11.4 SIGNATURE
11.5 HEAD OF THE
DEPARTMENT
Dr. SHIRISH KUMAR AMBAVADE.
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY,
MANGALORE – 574143
11.6 SIGNATURE
12
12.1 REMARKS OF THE CHAIRMAN AND THE PRINCIPAL
12.2 SIGNATURE
