Download proforma for registration of subjects for dissertation

“EFFECT OF ISORHAMNETIN ON GLOBAL ISCHEMIC
REPERFUSION (IRI) INDUCED MYOCARDIAL DAMAGE
AND ISOPROTERENOL INDUCED MYOCARDIAL DAMAGE”
Synopsis for registration of M.Pharm Dissertation
Submitted to
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE
KARNATAKA
In partial fulfillment
of the requirement for the Degree of
Master of pharmacy In Pharmacology
Under the Guidance of
Dr. RAJU KONERI
DEAN
BY
GOPALA K.P
Department of Pharmacology,
Karnataka College of Pharmacy, Bangalore-64
2010-11
0
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
Name of the candidate and address
GOPALA K.P
s/o Puttaswamy, Kudukundi,Gandasi(Ho),
Arasikere Tq, Hassan Dist-573164
Karnataka, India
Karnataka College of Pharmacy,
2.
Thirumenahalli ,Hegdenagar main road,
Name of the Institution
Yelahanka hobli,
Bangalore-560064
Karnataka, India
3.
4.
5.
Course of study and subject
M.Pharm-Pharmacology
Date of the admission
07-07-2010
Title of the topic
“Effect of isorhamnetin on global ichemic reperfusion induced myocardial
damage and Isoproterenol induced myocardial damage”
1
6.0
Brief resume of the intended work
6.1 - Need for the study:
Myocardial ischemia occurs when there is a reduction of blood flow as a result of
narrowing of the coronary arteries during atherosclerosis, blockage of perfusion as a
result of thrombosis, or development of coronary spasm. It is well known that myocardial
ischemia results in the loss of contractile function and produces myocardial damage as
a consequence of cell death.1 Oxygen-free radical formation and intracellular Ca2+
overload are strongly implicated as important pathophysiological mechanisms mediating
myocardial ischemia-reperfusion injury.2
Reperfusion of ischemic myocardium is accompanied by the development of oxidative
Stress and the generation of free radicals which plays
a major role in the
etiopathogenesis of reperfusion injury.3 Myocardial infarction (MI) is the rapid development
of myocardial necrosis that occurs when a coronary artery is severely blocked so that there
is a significant imbalance between the oxygen supply and the demand of the myocardium,
causing damage or death of a portion of the myocardium.13
Animals develop 'infarct-like' lesions when injected with isoproterenol (ISO), a potent
synthetic catecholamine. These lesions are morphologically similar to those of 'coagulative
myocytolysis' (COAM) or myofibrillar degeneration, one of the findings in acute myocardial
infarction in humans . Diabetic patients are more vulnerable to myocardial damage resulting in
heart failure than nondiabetic
patients . It is suggested that heart failure subsequent to
myocardial infarction may be associated with an antioxidant deficit, as well as increased
myocardial oxidative stress . Higher serum concentrations of enzymes aspartate transaminase
(AST) and creatinine kinase (CK) act as markers and are associated with higher incidence of
stroke after acute myocardial infarction . Elevated serum uric acid lactate dehydrogenase
(LDH), creatinine kinase-MB fraction (CK-MB) may also act as marker enzymes of tissue
ischemia . An increase in the concentration of total and LDL cholesterol and a decrease in HDL
cholesterol are associated with raised risk of myocardial infarction.4
2
Antiinflmmatory and antioedematous activities , Anti HIV activity, Immunostimulant
activity, Antiviral activity, Antibacterial and antifungal activities, Anticancer and lymphocyte
activation dual activities, Hepato protective activity, Antioxidant activity.5
Hence
the present
study
we
are
elucidating
the
cardioprotective
activities
of
isorhamnetin on Ischemic reperfusion induced myocardial damage and Isoproterenol induced
myocardial damage.
6.2 REVIEW OF LITERATURE:
Flavonoids belong to a group of natural substances with variable phenolic
structures and are found in fruit, vegetables, grains, bark, roots, stems, flowers, tea, and wine.
These natural products were known for their beneficial effects on health long before flavonoids
were isolated as the effective compounds. Furthermore, epidemiologic studies suggest a
protective role of dietary flavonoids against coronary heart disease .The association between
flavonoid intake and the long-term effects on mortality was studied subsequently and it was
suggested that flavonoid intake is inversely correlated with mortality due to coronary heart
disease.6
Flavonoids are plant-specific secondary metabolites, compounds so named because they are not
apparently involved in the survival of the cell. Secondary metabolites are extraordinarily
diverse, with upwards of 105 structures reported to date, and they are also synthesized at a
considerable rate, Flavonoids represent one of the largest groups of secondary metabolites
Flavonoids are not synthesized in animal cells, thus their detection in animal tissues is
indicative of plant ingestion.1
The flavonoids in sea buckthorn are bioactive constituents,which are reported to suppress
platelet aggregation, relieve cardiac disease and have antioxidants, hepato-protective and
immunomodulatory properties.7
Flavonoids are capable of modulating the activity of many enzymes and possess a remarkable
spectrum of biochemical and pharmacological activities, some of which interfere with control
processes in carcinogenesis.These include antioxidant activities, the scavenging effect on
3
activated carcinogens and mutagens,the action on cell cycle progression and altered gene
expression.11
The flavonoids (ginkgo flavone glycosides, bioflavonoids) and terpenoids (ginkgolides and
bilobalide) in GbE have been found to possess antitumor, antiaging, hepatoprotective, and
cardioprotective properties. Ginkgo biloba in cardiovascular diseases are mediated through its
protective roles against free radical injury.8
Isorhamnetin prevent endothelial cell injuries. Isorhamnetin Prevent Endothelial Dysfunction,
Superoxide Production, and Overexpression of p47phox Induced by Angiotensin II in Rat
Aorta.12
The increase in Ca2þ in endothelial cells was inhibited by a mixture of superoxide dismutase
and catalase suggesting the involvement of O2. In addition, by scavenging O2 or by inhibiting
its synthesis, flavonoids protect NO from O2 -driven inactivation, increasing its half-life and its
biological activity. Due to its antioxidant properties, flavonoids can potentially avoid BH4
oxidation and eNOS uncoupling. Therefore, under conditions of high O2, flavonoids potentiate
NO- and endothelium-dependent relaxation, reverting oxidative stress-induced endothelial
dysfunction. However, the chemical relationships between flavonoids and NO is more complex
because flavonoids may also scavenge NO. This reaction involved the autooxidation of
flavonoids in aqueous buffers producing O2 which ultimately inactivates NO. Flavonoids may
also regulate NO activity at the level of eNOS mRNA and/or protein expression. Longterm
incubation of endothelial cells with red wine or the anthocyanins delphinidin, malvidin,
cyanidin and paeonidin increased eNOS expression while most isolated flavonols were without
effect flavonoids inhibit several ROS generating enzymes including xanthine oxidase and the
membrane NADPH oxidase complex in neutrophils. As mentioned above, by reducing cellular
O2 concentrations flavonoids protect NO and increase its bioactivity. flavonoids are not devoid
of prooxidantproperties.Quercetin can autooxidize in aqueous solutions to generate free
radicals and may also deplete intracellular thiols such as glutathione.14
4
6.3 OBJECTIVE OF THE STUDY:
1. To study the effect of Isorhamnetin on Global Ischemic Reperfusion (IRI) induced
myocardial damage.
2. To study the effect of Isorhamnetin on Isoproterenol induced myocardial damage.
7.0
MATERIALS AND METHODS:7.1 Global Ischemic Reperfusion Injury (IRI) induced myocardial damage
Rats will be randomly divided into three groups, each group having 6 rats.
Group 1: Control.
Group 2: Global Ischemic Reperfusion Injury (IRI) induced myocardial damaged animals.
Group 3: Isorhamnetin treated myocardial damaged animals.
Aqueous homogenate of Isorhamnetin will be fed by oral gavage everyday at a fixed time
(10.00 a.m) for 30 days. Control rats will be fed normal saline daily for 30 days.Changes in
bodyweight, food and water intake patterns of rats in all groups will be noted throughout the
experimental period. After 48 hours of the last dose, rats will be heparinised [375 units/200
gms i.p], anaesthetize after 30 min. with sodium pentobarbitone +(60 mg/kg i.p) and subjected
to the following protocol.9
7.11 Production of ischemic-reperfusion injury in isolated rat heart
After 30 days treatment Rat hearts will rapidly excise and wash in ice-cold saline, and then
perfuse with the non-recirculating Langendorff's technique (Hufesco, Hungary), in constant
pressure mode with modified Kreb's Henseleits buffer containing [in mM]: glucose 11.1; NaCl
118.5; NaHCO3 25; KCl 2.8; KH2PO4 1.2; CaCl2 1.2; MgSO4 0.6, with a pH of 7.4. The
buffer solution equilibrated with 95% O2+ 5% CO2 will be delivered to the aortic cannula at
370C under 60 mm Hg pressure. Following 5 min-equilibration period, hearts will be subjected
5
to 9 min. of zero-flow [ischemia] and 12 min of re-flow [reperfusion] (IR injury).
At the end of each experiment, myocardial tissue was stored in liquid nitrogen
for biochemical estimations, 10% buffered formalin for light microscopic studies and
Karnovsky fixative for electron microscopic studies.9
7.12 Biochemical parameters
Thiobarbituric acid reactive substances (TBARS) will be measured as a marker of lipid
peroxidation and endogenous antioxidants, superoxide dismutase (SOD), catalase, reduced
glutathione (GSH) & glutathione peroxidase (GPx) will be estimated.9
7.13 Histopathology
a) Light microscopic study
Myocardial tissue will be fixed in 10% formalin, routinely processed and embedded in paraffin.
Paraffin sections (3 m) will cut on glass slides and stained with hematoxylin and eosin (H&E),
periodic acid Schiff (PAS) reagent and examined under a light microscope.9
b) Ultrastructural study (transmission electron microscopy)
Small pieces of myocardial tissue will be washed in cold 0.1M phosphate buffer after fixation
(Karnovsky fixative) and post-fixed for two hours in 1% osmium tetraoxide in the same buffer
at 40C. After several washes in 0.1 M phosphate buffer, the specimens will be dehydrated using
graded acetone solutions and embedded in CY 212 araldite. Ultrathin sectioning (80–100 nm)
of the tissue blocks will be carried out using an ultracut E microtome. The sections will be
stained in alcohol uranyl acetate and lead citrate and viewed under a transmission electron
microscope operated at 60 KV.9
7.14 Statistical Analysis
All values will be expressd as mean SE. One way ANOVA will be applied to test for
significance of biochemical data of the different groups. Significance is set at p < 0.05.9
6
7.2 Isoproterenol induced myocardial damage
7.21 Experimental animals ;Eighteen male Wistar rats weighing 120-150 g. The animals will be housed in polypropylene
cages maintained in controlled temperature (27 ± 2°C) and light cycle (12 h light and 12 h
dark). They will be fed with standard rat pellet diet and water ad libitum.4
7.22 Experimental Procedure;
Rats will be divided into three groups.
Group 1:control (distilled water p.o.),
Group 2: ISO (60mg/kg, s.c.) at an interval of 24 h for two days
Group 3: Isorhamnetin for 45 days followed by ISO (60mg/kg, s.c.) at an interval of 24 h for
two days.4
7.23 Biochemical assessment
Marker Enzymes in serum
Twelve hours after the second injection of ISO, the animals will be sacrificed by cervical
decapitation, blood will be collected and the heart will be dissected out. The serum will be
separated immediately by cold centrifugation and used for determination of the myocardial
infarction marker enzymes LDH, CK-MB, AST, ALT, and ALP along with serum uric acid,
total cholesterol, triglycerides, LDL, and HDL. The enzymes, lipids and uric acid will be
estimated using commercial diagnostic kits.4
Oxidative Stress in Heart tissue
Heart will be immediately washed with ice-cold saline and a homogenate will be prepared in
0.1N Tris HCl buffer (pH 7.4). The homogenate will be centrifuged and supernatant will be
collected, which will be used for the assay of LPO, GSH, CAT, and SOD.4
7
Estimation of LPO
The extent of lipid peroxidation in tissues will be assessed by measuring the level of
malondialdehyde (MDA),1ml of trichloroacetic acid (TCA) 20% and 2 ml of thiobarbituric
acid (TBA) 0.67% will be added to 2 ml of homogenate supernatant. The absorbance of the
mixture will be recorded at 530 nm and the values will be expressed as ηM of MDA form /mg
of protein.4
Estimation of GSH activity
GSH in the cardiac tissue will be assayed by the method previously described by Ellman .0.02
ml of the homogenate supernatant will be added to 3 ml of Ellman reagent. The samples will be
mixed and kept at room temperature for at least 1 h. The changes in absorbance will be read at
412 ηm. The amount of glutathione will be expressed as μg of GSH/mg protein.4
Estimation of SOD activity
The level of SOD will be measured by the method of Kono. 1.3 ml of solution A (0.1 nM
EDTA containing 50 nM Na2CO3, pH 10.3), 0.5ml of solution B (90 M) nitro blue tetrazolium
dye (NBT) and 0.1 ml of solution C (20mM Hydroxylamine hydrochloride, pH 6.0) were
mixed and the rate of NBT reduction will be recorded for 1 min at 560 ηm. SOD activity will
be expressed as unit/mg protein change in optical density per min.4
Estimation of Catalase activity
Catalase activity will be estimated by determining the decomposition of H2O2 at 240 ηm in an
assay mixture containing phosphate buffer. The activity will be expressed in units as μM of
H2O2 consumed/min/mg of protein.4
7.24 Histopathology studies
A portion of the heart will be fixed in formalin (10%) and subjected to histopathology studies.
The section of the heart will be processed and embedded in paraffin wax. Sections of about 4-6
μm were made and stained with hematoxylin and eosin and photographed.4
8
7.25 Statistical Analysis:
The data will be analyzed using one-way ANOVA followed by Tukey Kramer multiplecomparison test and P values of 0.05 or less will be considered significant.4
7.3 - Does The Study Require Any Investigation To Be Conducted On Patients Or
Animals? If So, Please Describe Briefly.
Yes ; It is invivo study.
7.4- Has Ethical Clearance Been Obtained From Your Institution In Case Of 7.3?
Yes. Taken from Institutional Ethical Committee.
8.0
LIST OF REFERENCES:-
1. Naranjan SD, Todd AD. The paradoxes of reperfusion in the ischemic heart. Heart
Metab 2007;37:3.
2. Jiang DJ, Tan GS, Feng YE , YanHua DU, KangPing XU, YuanJian LI. Protective
effects of xanthones against myocardial ischemia-reperfusion injury in rats. Jiang DJ et
a l /Acta Pharmacol Sin 2003 Feb;24(2):175.
3. Karunakaran KG, Mohamed TSS, Peter TT, Vinoth VP, Karthikeyan KK, Niranjali SD
et al. Cardioprotective effect of the Hibiscus rosasinensis flowers in an oxidative stress
model of myocardial ischemic reperfusion injuryin rat. BMC Complementary and
Alternative Medicine 2006;6:32:2.
4. Raju K, Balaraman R, Hariprasad, Vinoth KM, Ali A. Cardioprotective Effect Of
Momordica Cymbalaria Fenzl In Rats With Isoproterenol-Induced Myocardial Injury.
Journal of Clinical and Diagnostic Research 2008 Feb;2(1):699-01.
5. Muley BP, Khadabadi SS, Banarase NB. Phytochemical Constituents and
Pharmacological Activities of Calendula officinalis Linn (Asteraceae): A Review.
Tropical Journal of Pharmaceutical Research 2009 oct;8(5):458-60.
9
6. Robert JN, Els VN, Danny ECVH, Petra GB, Klaske VN and Paul AMVL.
Flavonoids: a review of probable mechanisms of action and potential applications.
American Journal of Clinical Nutrition 2001 oct;74(4):418.
7. Yuangang Z, Chunying L, Yujie F, Zhao C. Simultaneous determination of catechin,
rutin, quercetin kaempferol and isorhamnetin in the extract of sea buckthorn
(Hippophae rhamnoides L.) leaves by RP-HPLC with DAD. Journal of Pharmaceutical
and Biomedical Analysis 2006;41:714.
8. Hsiu CO, Wen JL, I-Lee T, Tsan HC, Kun LT, Chih YL, Wayne HHS. Ginkgo biloba
extract attenuates oxLDL-induced oxidative functional damages in endothelial cells. J
Appl Physiol 2009 may;106:1674.
9. Sanjay KB, Amit KD, Subhash CM, Subir KM. Chronic garlic administration protects
rat heart against oxidative stress induced by ischemic reperfusion injury. BMC
Pharmacology 2002;2:16:6-7
10. Sabina P, Michela T, Raffaella F, Andreja V, Federica T, Enrico B
et.al .
Bioavailability of Flavonoids: A Review of Their Membrane Transport and the
Function of Bilitranslocase in Animal and Plant Organisms. Current Drug Metabolism
2009;10(4):369.
11. Gordana R, Herwig OG, Jutta LM. Effects of Structurally Related Flavonoids on hsp
Gene. Effects of Structurally Related Flavonoids on hsp Gene. Food Technol.
Biotechnol 2002;40(4):267-68.
12. Manuel S, Federica L, Rocio V, Inmaculada CV, Angel C, Rosario J, et al. Quercetin
and Isorhamnetin Prevent Endothelial Dysfunction, Superoxide Production, and
Overexpression of p47phox Induced by Angiotensin II hin Rat Aorta. The Journal of
Nutrition Biochemical, Molecular, and Genetic Mechanisms 2007;137:910.
13. Sevil K, Tamás R, Enik B, Kristóf H, Philipp N, Sivakkanan L, et al. Pharmacological
Activation of Soluble Guanylate Cyclase Protects the Heart Against Ischemic Injury.
Circulation 2009;120:679
14. Francisco PV, Juan D & Ramaroson A. Endothelial function and cardiovasculardisease.
Effects of quercetin and wine polyphenols. Free Radical Research 2006 oct;40(10):1057
-58.
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9.0
Signature of the candidate
(GOPALA K.P)
10
Remarks of the Guide: The topic selected for dissertation is satisfactory. Adequate
equipment and chemicals are available to carry out the project work.
11
Name and Designation of guide:
Dr.RAJU KONERI
Professor & Dean
Dept of Pharmacology,
Karnataka college of pharmacy,
Bangalore-64
Signature of the guide:
(Dr.RAJU KONERI)
12
Co-Guide:
Not applicable
13
Head of the Department:
Dr. RAJU KONERI
Professor & Dean.
Dept of Pharmacology,
Karnataka college of pharmacy.
Bangalore-64
11
Signature of HOD:
(Dr. RAJU KONERI)
14
Remarks of the Principal: All the required facilities will be provided to carry out
dissertation work under the supervision of guide.
Principal:
Dr. K. RAMESH,
Principal,
Karnataka College of Pharmacy,
Thirumenahalli,
Bangalore –64.
Signature of the principal:
(Dr. K. RAMESH)
12