Download Pseudarthria viscida Pseudarthria viscida

EVALUATION OF CARDIOPROTECTIVE ACTIVITY OF
ETHANOLIC EXTARACT OF PSEUDARTHRIA VISCIDA(L.) WIGHT
& ARNOTT ROOTS
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE
BY
KORIA HARDIK MUKESHKUMAR
Under The Guidance of
Mr. MOSES SAMUEL RAJAN, M.PHARM
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
2011 – 2013
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
2.0
NAME OF THE
INSTITUTION
3.0
COURSE OF STUDY AND
SUBJECT
4.0
DATE OF ADMISSION TO
KORIA HARDIK MUKESHKUMAR
‘KORIA NIVAS’, AMBICANAGAR,
RAMNAGAR, SABARMATI,
AHMEDABAD-380005
GUJARAT
SRINIVAS COLLEGE OF PHARMACY
VALACHIL,
MANGALORE.-574143
MASTER OF PHARMACY IN
PHARMACOLOGY
14/05/2011
COURSE
EVALUATION OF CARDIOPROTECTIVE
5.0
TITLE OF THE TOPIC
ACTIVITY OF ETHANOLIC EXTRACT OF
PSEUDARTHRIA VISCIDA (L.) WIGHT AND
ARNOTT ROOTS
6.0
BRIEF RESUME OF THE INTENDED WORK :
6.1 NEED FOR THE STUDY:
According to the findings of the National Vital Statistics Report (2008) and the
Morbidity and Mortality Weekly Report (2007) of the Centers for Disease Control
and Prevention (CDC), cardiovascular diseases including myocardial infarction (MI)
and the resultant complication in cardiac function represent the leading cause of
morbidity and mortality in developed countries1,2. Moreover, with advanced life style
in developing countries, such as India, particularly in metropolitan cities, MI is
making increasingly important contribution to mortality statistics of such countries3.
Although clinical care is improved, public awareness is raised and health innovations
are widely used, myocardial infarction remains the leading cause of death worldwide4.
It is the acute condition of myocardial necrosis that occurs as a result of imbalance
between coronary blood supply and myocardial demand. Experimental and clinical
studies have shown that there is increased generation of reactive oxygen species such
as superoxide anion (.O-2) and hydroxyl radicals (.OH-) in heart failure, which are
involved in the formation of lipid peroxides, damage of cell membrane, and
destruction of antioxidative defense system 5,6.
Cell injury occurring after ischemia-reperfusion (I/R) in the heart is attributed to
necrosis caused by calcium overload, acidosis, and oxidative stress. After I/R,
myocardial cell die by necrosis and/or apoptosis. Free radicals and antioxidants play
an important role in cellular damage that can cause atherosclerosis and myocardial
infarction. Both neutrophils and free radicals, such as superoxide anions, hydroxyl
radicals and hydrogen peroxide, can cause oxidative damage to cell lipids, proteins,
and nucleic acids. A consensus still exists that reactive oxygen species play an
important role in the pathogenesis of cardiac reperfusion injury.7
Because of high incidence of morbidity, various drugs and regimes have been
advocated for the control of CVS diseases. Many new drugs have been introduced
which may demonstrate better efficacy but possess side effects.8 Recently attention
has been focused towards herbal and mineral preparations which are traditionally used
as potential therapeutic agents in the prevention and management of cardiovascular
diseases such as Terminalia arjuna (arjun), Trigonella foenum-graecum (fenugreek),
Curcuma longa (turmeric), Garcinia indica (kokum) and Vitis vinifera (grapes).9
CRITERIA FOR SELECTION OF THE PLANT:
Roots of Pseudarthria viscida was proved for its anti diabetic, antioxidant, antidiarrhoeal activity and neuroprotective activity. Pseudarthria viscida consists of
phytochemical constituents like alkaloids, glycosides, flavonoids, tannins, phenolic
compounds and saponins. Alkaloids, glycosides, saponins and flavonoids were
reported to show cardioprotective activity.10Also in Ayurveda Pseudarthria viscida
was found in many formulations used for cardio protective action.11In present study,
cardioprotective effect of Pseudarthria viscida will be assessed by using different
experimental models in animals.
6.2 REVIEW OF LITERATURE:
6.2.1
Name of the plant : Pseudarthria viscida
Pseudarthria viscida (L.) Wight &Arnott
Family : Fabaceae
Sanskrit synonym : kalasi, prsniparni, salaparni, saliparni, sanaparni
Other name: Kannada (2) : antubelegida, antuparni
Malayalam (2) : muvila, muvvila
Tamil (8) : kotiottai, muvilai, muvilaippunnai, muvilaippunnaimaram,
neermali, nirmalli, pitani
Telugu (6) : muyak, muyakuponna, muyyakuponna, muyyakuponna,
nayakuponna, nayakuponna
Distribution: Globally the species is distributed in the Indo-Malaysian region and Sri
Lanka. In India it is recorded in the states of Gujarat, Orissa, Karnataka, Tamil Nadu
and Kerala
Plant Description: A semi erect, pubescent, perennial shrub grows up to 50 cm to 1
meter in height. Branches are covered with whitish hairs. Leaves are hairy,
compound; trifoliate, terminal leaflet rhomboid ovate, lateral ones are obliquely ovate
or oblong. Flowers are purplish or pink, small numerous, found on terminal and
axillary spikes. Fruits pods, flattened and covered with hairs, seeds 4-6.
Chemical constituents : alkaloids, glycosides, carbohydrates, proteins, steroids,
flavonoids, terpenoids, tannins & phenolic compounds, and fixed oils
Use :Plant is used in tridoshas, cough, asthma, fever, dysentery, cardiac ailments,
rheumatoid arthritis and aid in fast healing of fractured bone.
Useful part : Roots 11
6.3 OBJECTIVES OF THE STUDY:
1) Extraction of roots of Pseudarthria viscida
2) Preliminary phytochemical screening.
3) To study the cardioprotective activity of extracts using following animal
models
a) Isoproterenol (ISO) induced myocardial necrosis in rats
b) Ischemia-reperfusion induced (IRI) myocardial damage in isolated rat
heart.
4) Biochemical Estimation: Blood will be collected from the animals and
serum will be evaluated for
(a) LDH ( Lactate dehydrogenase )
(b) CK-MB ( Creatine phosphokinase-MB )
(c) Antioxidants ( Superoxide dismutase and Catalase )
5) Histopathological Examination: In all above models heart will be isolated
and examined for histopathological investigations.
6) In Isoproterenol (ISO) induced myocardial infarction model, ECG changes
will also be examined.
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 and through web search
7.2 COLLECTION OF MATERIAL:
7.2.1 COLLECTION OF PLANT MATERIAL:
The Pseudarthria viscida will be collected from local region of Mangalore district,
and authenticated by a Taxonomist.
7.2.2 DRUGS AND CHEMICALS:
Isoproterenol, Heparine, Ketamine hydrochloride, Pentobarbitone, Xylazine,
Propranolol, LDH kits, CK-MB kits will be procured from Hi-media suppliers. All
other chemicals are of pure analytical grade will be obtained from local suppliers.
7.2.3ANIMALS
Wistar rats (150-200 g)of either sex will be procured from Indian Institute of
Sciences. They will be maintained under standard conditions (temperature 22 ± 2oC,
relative humidity 50±5% and 12 h light/dark cycle).The animals will be housed in
sanitized polypropylene cages containing sterile paddy husk as bedding. They will
have free access to standard pellet diet and water ad libitum. The Institutional Animal
Ethics Committee approved the experimental protocol. All the animals received
human 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”. All the procedures will be performed in
accordance with Institutional Animal ethics committee constituted as per the direction
of the committee for the purpose of control and supervision of experiments on
animals (CPCSEA), under ministry of animal welfare division, Government of India,
New Delhi, India.
7.3 EXPERIMENTAL METHODS :
7.3.1.ISOPROTERENOL (ISO) INDUCED MYOCARDIAL NECROSIS IN
RATS12.
The albino rats (150-200 g) of either sex will be randomly divided into 5 groups of 6
each and assigned as below:
o Group-I- normal control (Normal saline)
o Group-II - toxic control (Isoproterenol: 150mg/Kg)
o Group-III–Reference standard (Propranolol: 10mg/Kg)
o Group-IV- Pseudarthria viscida roots extract (low dose: 200 mg/Kg)
o Group-V- Pseudarthria viscida roots extract(high dose: 400 mg/Kg)
Propranolol will be administered to group III animals one week before administration
of Isoproterenol. All the animals in group IV & V will be treated once daily post
orally for 4 weeks. On 29th and 30th day after the treatment, myocardial infarction will
be induced in group II – group V animals by sub cutaneous injection of Isoproterenol.
EVALUATION:
Symptoms and mortality in each group will be recorded and compared with animals
given with Isoproterenol alone. Forty-eight hours after the first Isoproterenol
administration, the rats will be sacrificed and autopsied. The hearts will be removed
and weighed and the frontal sections will be embedded for histopathological
examination.
BIOCHEMICAL ESTIMATION:
The collected blood as well as heart homogenate will be analyzed for endogenous
biological markers such as Lactate dehydrogenase (LDH), Creatine phosphokinaseMB (CK-MB) and antioxidant enzymes like Superoxide dismutase and Catalase.
7.3.2: ISCHEMIA-REPERFUSION INDUCED (IRI) MYOCARDIAL
DYSFUNCTION13
The albino rats (150-200 g) of either sex will be randomly divided into 5 groups of 3
each and assigned as below:
o Group-I-Normal control (saline)
o Group-II – Ischemia induced control
o Group-III – Ischemia induced + Reference standard (Propranolol: 10mg/Kg)
o Group-IV - Ischemia induced + Pseudarthria viscid roots extract (low dose:
200mg/Kg).
o Group-V- Ischemia induced + Pseudarthria viscid roots extract (high dose:
400 mg/Kg).
Propranolol will be administered to group III animals one week before producing
ischemia. All the animals in group IV & V will be treated once daily post orally for 4
weeks. The heart will be excised from deeply anesthetized rats (35 mg/kg sodium
pentobarbitone, i.p) and perfused with Kreb-Henseleit solution gassed with carbogen
at 37o C and a constant flow rate of 5ml/min by one-way circulation using modified
Langendorff setup. Measurement of contractile force will be done with displacement
transducer and recorded on Student physiograph. After a short period of equilibrium
(15 minutes), records will be taken for a control period of 15 minutes, followed by 15
minutes ischemia and then a washout period with Kreb-Henseleit solution. Recovery
in terms of inotropic and chronotropic effect will be studied and the extent of
cardioprotection due to Pseudarthria viscida will be evaluated by measuring the
developed tension.
BIOCHEMICAL ESTIMATION:
The collected blood as well as heart homogenate will be analysed for endogenous
biological markers such as Lactate dehydrogenase (LDH), Creatine phosphokinase
(CK) and antioxidant enzymes like Superoxide dismutase and Catalase.
7.3.3 ECG STUDIES14.
The albino rats (150-200 g) of either sex will be randomly divided into 5 groups of 3
each and assigned as below:
o Group-I- normal control (saline)
o Group-II - toxic control (Isoproterenol: 150mg/Kg)
o Group-III – Reference standard ( Propranolol : 10mg/Kg)
o Group-IV – Pseudarthria viscida roots extract (low dose: 200 mg/Kg ).
o Group-V- Pseudarthria viscida roots extract ( high dose: 400 mg/Kg).
Propranolol will be administered to group III animals one week before administration
of Isoproterenol. All the animals in group IV & V will be treated once daily post
orally for 4 weeks. On 29th and 30th day after the treatment, myocardial infarction will
be induced in group II – group V animals by sub cutaneous injection of Isoproterenol.
After anesthetizing the rat with a combination of Ketamine hydrochloride (75mg/kg,
i.p) and Xylazine (8.0mg/kg, i.p), electrical leads will be attached to the dermal layer
of all the limbs and recording will be done with the help of computerized ambulatory
ECG system.
7.4 STATISTICAL ANALYSIS:
The data will be expressed as Mean value + SEM and will be analyzed by the oneway ANOVA followed by the Dennett’s test.
7.5DOES 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 Wistar albino rats.
7.6HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR
INSTITUTION?
Yes. Ethical clearance has been obtained. (Copy enclosed)
8.0
LIST OF REFERENCES:
1. Kung HC, Hoyert DL, Xu J, Murphy SL. Deaths: final data for 2005.Natl
Vital Stat Rep 2008;56:1-120.
2. Burrows NR, Parekh S, Li Y, Geiss LS. Prevalence of self reported
cardiovascular disease among persons aged _35 years with diabetes –
United States, 1997–2005. MMWR. 2007;56:1129–1131. Available at:
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5643a2.htm Accessed
01 December 2010 3:30 pm.
3. Levy RI, Feinleib M. Heart disease. In: Text book of Cardiovascular
Medicine. W.B. Saunders Company, Philadelphia 1984;2:1205-34.
4. Aronow WS. Epidemiology, pathophysiology, prognosis and treatment of
systolic and diastolic heart failure. Cardiol 2006;14:108-24.
5. Biase D, Pignatelli P, Lenti L, Tocci G, Piccioni F. Enhanced TNF alpha
and oxidative stress in patients with heart failure: effect of TNF alpha on
platelet O-2 production. Thromb Haemost 2003;90:317-25.
6. Rajadurai M, Prince SM. Preventive effect of naringin on Isoproterenolinduced cardiotoxicity in Wistar rats: an in vivo and in vitro study.
Toxicology 2007;232:216-25.
7. AkcalıY, OzturkF,Ceyran H, Narin F, Narin N, Akgun H, Ceyran AB. The
Effect of High Dose Melatonin on Cardiac Ischemia reperfusion Injury.
Yonsei Med J 2008;49(5).
8. Sakat SS, Wankhede SS, Juvekar AR, Mali VR, Bodhankar SL.
Antihypertensive effect of aqueous extract of Elaeocarpus ganitrus Roxb.
Seeds in renal artery occluded hypertensive rats. International Journal of
Pharm Tech Research, 2009 sep;1:779-82
9. Jai Ashish Tilak-Jain and Thomas Paul Asir Devasagayam: Cardioprotective
and Other Beneficial Effects of Some Indian Medicinal Plants . J. Clin.
Biochem. Nutr., 38, 9-18, 2006
10. Arya and Gupta, IJPSR, 2011; Vol. 2(5): 1156-1167
11. Kirtikar & Basu, ”Indian medicinal plants”, second edition, volume 1,
2004, page no.748
12. Asdaq SMB, Inamdar MN, Asad M, Nanjundan PK. Interaction of
propranolol with garlic in Isoproterenol induced myocardial infarction in rat. J
PharmacolToxicol 2008;3 (6):414-24.
13. Asdaq SMB, Inamdar MN. Pharmacodynamic interaction of garlic
withcaptopril in Ischemia-reperfusion induced myocardial damage in rats.
Phamacology online 2008;2:875-88.
14. Singh PN, Athar MS. Simplified calculation of mean QRS vector(mean
electrical axis of heart) of electrocardiogram. Indian J Physiological
Pharmacological 2003;47:212-6.
9.
SIGNATURE OF THE
CANDIDATE
10.
REMARKS OF THE GUIDE
11.
NAME AND DESIGNATION
11.1 GUIDE
Recommended and Forwarded
MR. MOSES SAMUEL RAJAN
Associate Professor
Department of Pharmacology
Srinivas College of Pharmacy
Mangalore- 574 143
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY)
Not Applicable
11.4 SIGNATURE
11.5 HEAD OF THE
DEPARTMENT
MR. MOSES SAMUEL RAJAN
Asso. Professor
Department of Pharmacology
Srinivas College of Pharmacy
Mangalore- 574 143
11.6 SIGNATURE
12
12.1 REMARKS OF THE
PRINCIPAL
12.2 NAME & SIGNATURE OF
PRINCIPAL
DR. A. R. SHABARAYA
PRINCIPAL,
SRINIVAS COLLAGE OF PHARMACY
MANGALORE- 574 143