synopsis(myocardial ischemia)
TRANSCRIPT
Research proposal
Presented to: Dr Aalamgeer
Presented by :Moheer Fatima
Roll No: 07
Anti ischemic activity of some indeginous medicinal plants by using In-vivo and In-vitro models.
Contents: Introduction Aims & Objectives Plan of Work Methodology of Work (a) Plant Material (b)Animal Used (c)Experimental Procedures (d)Statistical Analysis Impact of Study References
Introduction: Cardiovascular diseases are an
important medical and public health issue through out the world[1,2]
CVS diseases the first killer for human being ,constitute the global economic burden .The updated data from WHO addresses that the mortality rank due to CVS diseases will increase from No.5 in 2000 to Top 1 till 2020 .And 30 % of overall mortality is due to CVS diseases [3].
Introduction: This is a bigger problem for developing
countries like Pakistan[4].
The rise in prevalence of CVS diseases in developing countries is mostly related to demographic changes ,urbanization ,life style modification and high risk factor levels [5].
In Pakistan, cardiac ailments contribute to about 25% deaths in the country [6].
Introduction: IHD is a condition in which the vascular supply to the heart is
impeded by atheroma, thrombosis or spasm of coronary artries.This may impair the supply of oxygenated blood to cardiac tissue sufficiently to cause Myocardial ischemia.
Pathogenesis:Myocardial ischemia occurs by 2 ways :
When oxygen demand exceeds myocardial oxygen supply.The resultant ischemic myocardium release adenosine,the main mediator of chest pain ,by stimulating the A1 receptors located on the cardiac nerve endings.
Myocardial Ischemia can also arise if oxygen demand is abnormally increased,as may occur in patients with thyrotoxicosis or severe ventricular hypertrophy due to hypertension.MI supply is dependent on the luminal cross sectional area of the coronary artery and coronary arteriolar tone .
Introduction: IHD is the leading cause of death and disability
worldword. It kills over 6.5 million people worldwide each year[7].
From 1990 to 2020 the rise in mortality due to ischemic heart disease (IHD) in developing countries (137% in men and 120% in women) is predicted to be much higher than that in the developed countries (48% in men and 29% in women ) [8].
Existing treatment for Myocardial Ischemia…….Nitrates, Beta blockers , Calcium channel blockers etc[9].
Introduction: But these synthetic drugs have ADRs like
Headache,constipation ,weakness,dry mouth etc and are expensive. Development of more influential anti-ischemic drugs with lesser ADRs is necessary. SO,the use of herbal medicine has been steadily increasing[10].
Natural products are easily biodegradable, possess least environmental hazards, represent minimum side effects and are available at affordable prices [11].
In different studies, Panax notoginseng, Tinospora cordifolia , Zingiber officinale exhibited to have anti-ischemic activity [12,13,14].
Aims & Objectives:The aim of this study will be:
To evaluate the anti-ischemic activity of plant extract.
To explore new novel medicinal plants which may be of commercial value.
To develop a herbal medicine and evaluate it scientifically.
Plan of work: Selection of
experimental animals
Selection of plants
Shade drying Preparation of crude extract
Fractionation of crude extract
Preliminary phytochemical
analysisAcute toxicity
test
Experimental procedures on
animals
Methodology of work:
(a) Instruments:Following intruments will be used during experimentation .
Spectrophotometer Electrocardiogram Techno positive pressure Respirator Teflon coated stainless steel electrode
Methodology of work:
(b) Standard drugs & Chemicals:The following drugs will be used in this investigation
Ramipril Sublingual Nitrates Sodium pentobarbital Solvents include saline solution,Ethanol ,Kreb solution 2,3,5 triphenyltetrazolium chloride (TTC) stain All chemicals will be purchased from Merck (Germany) ,Sigma Chemical Co (St. Louis, MO, USA).
Methodology of work:
(c) Animals:Following animals will be used for various experiments in-vivo and in-vitro .
Sprague-Dawley rats Dogs RabbitAll animals will be treated according to the standard procedures guided by national research council [15].
Methodology of work:
(d) Plant material:All plants for this study will be selected according to their Folkloric claim to cure the conditions of Ischemia.
Terminalia arjuna Carthamus tinctorius (Safflower)
Methodology of work: Preparation of crude extract: Dried flower petals will be extracted 3 times in 50%
ethanol. The extracts will be combined,filtered and evaporated in
evaporator to obtain condensed solution. Macroporous resin chromatography will be used to purify
the condensed extract solution . Finally the concentrated solution will be stored at cool
place in capped bottles. Extracts will be dissolved in normal saline before administration [16].
Macroporous Resin Chromatography:
Methodology of work: Fractionation of the crude extract: Activity directed fractionation of the crude extract will be carried out by using different organic solvents in order to separate or concentrate the activities in any one of the corresponding fraction [17].
Preliminary Phytochemical analysis:
The fraction with highest activity will be selected & analyzed for the presence of different phytochemical groups such as, flavonoids, flavones, terpenoids, tannins, alkaloids by using phytochemical methods [18].
Methodolgy of work:
Acute toxicity test: Acute toxicity test will be performed on
rat as per OECD guidelines 2002 . [19].
The test will be done for observation of mortality, behavioral changes and toxic effects
Experimental procedures:
Animals models of Ischemia
In-vivo models
In-vitro models
In Vivo models of Ischemia:
Left coronary artery ligation method Open chest method Closed chest method Exercise induced Myocardial Ischemia Wire induced Myocardial Ischemia Sympathetic overactivity induced
myocardial ischemia Isoproterinol Induced myocardial ischemia Cocaine induced myocardial ischemia
Isoproterenol induced ischemia [20]:
Myocardial ischemia will be induced by subcutaneous injection of isoproterenol hydrochloride (ISO, 85 mg kg−1 bw), dissolved in physiological saline solution, for 2 consecutive days.
The rats will be randomly divided into 6 groups with 8 rats each. Group 1 will serve as control (received 5% DMSO sc, for 7 days). Group 2 rats will be administered isoproterenol (ISO, 85 mg kg−1
bw, sc, twice at an interval of 24 hours) on 8th and 9th days. Group 3 rats will be administered with plant extract(60 mg kg−1 bw,
sc) for 7 days. Groups 4, 5, and 6 rats will receive 20, 40, and 60 mg kg−1 b.w., of
plant extract subcutaneously, for 7 days and will receive ISO (85 mg kg−1 bw, sc, twice at an interval of 24 hours) on the 8th and 9th days, respectively.
Plasma cardiac Troponin T and I (cTnT and cTnI) will be quantitatively measured by means of a highly specific enzyme immunoassay using commercially available kits .
Sympathetic overactivity induced myocardial ischemia in rabbits[21].
In this experiment ,we will induce sympathetic hyperactivity by intracerebroventricular injection of excitatory amino acid L-Glutamate(10u mol)( in pentobarbital anaesthetized albino rabbits of either sex (2-3 kg),associated with inhibition of nitric oxide synthesis with L-NAME(40mg/kg, iv).
Glutamate will trigger ventricular arrythmia and persistent ST segment shifts in the ECG,it will indicate myocardial ischemia.
Protocol of study:
Divide in 4 Groups
Control group(1)=L-NAME(40mg/
kg) adm.then
after 1 hour L-
Glutamate(10u mol)
adm.ECG changes will be recorded
Pretreated group(2)=clonidine(10
ug/kg iv.)Rilmenidine(300ug/kg iv.)adm 10
mint before L-NAME.ECG changes will be observed
Post treated group(3)=Clonidine,Rilmenidine adm
.10 min. before L-
Glutamate.ECG chenges
will be noted.
4th group = adm only L-Glutamate
and will investigate effects of
sympathetic
hyperactivity in
absence of L-NAME.
Sympathetic overactivity induced myocardial ischemia in rabbits
The IV adm. Of clonidine(10ug/kg) and Rilmenidine(300ug/kg) will decrease the incidence of myocardial ischemia.
Coronary artery ligation method[22].Open chest
method:This procedure will be performed in larger animals like dog and pig etc ,in which major branch of coronary artery will be ligated.
By the use of anaesthesia ,chest will be opened for a long time,and by coronary artery ligation ischemia will be induced and we will map the ischemic areas by means of multi positioning of a surface electrode.Ischemic damage will be measured by ST segment elevation
Some metabolic changes in ischemic myocardium will be monitored by taking blood samples from the vein.
Coronary artery ligation method:
Closed chest method:
Myocardial ischemia in closed chest method will be brought about by 3 ways:
1. The production of thrombus will be done either by passing a current via an intracoronary catheter or simply by leaving a helical occlusion of the lumen by a balloon catheter.2. Copper wire will be inserted in the lumen of the coronary artery.3. Embolization will be done by radioopaque micro-spheres of different sizes or by intracoronary injection of mercury.
Balloon Catheter
In-Vitro models of Ischemia:
Cell culture model of seal induced Ischemia
Ischemia(blockage of circulation ;total or low flow).
Ischemia(global or regional)
Cell culture Model of seal induced Ischemia[23].
Neontal rat heart cells will be cultured in a sealed flask for 24 to 72 hours(cells will be exposed to hypoxia).PO2 and pH of medium will be decresed during the procedure
This will trigger severe cell injury including Morphological degeneration,ATP depletion,beating impairment.Apoptosis will also be occured in some cardiomyocytes after onset of ischemia.
This will be evident by using Hoechst 33258 .After 72 hr,DNA fragmentation will also be observed in some of the myocytes.
Impact of study: This study will :
be valuable for screening of various plant extracts having Anti-ischemic effect.
give scientific evidence about its tradtional use.
References:1. Brauwald E, Zipes D, Libby P. Heart disease a textbook of cardiovascular medicine.
USA: W.B.Saunders; 6th ed. Vol.1, 2001. P. 1.
2. Lenfant C. Can we prevent cardiovascular diseases in low- and middle-income countries? Bull World Health Organ 2001; 79:980-2.
3. Murray CJL, Lopez AD, eds. The Global Burden of Disease: A Comprehensive Assessment of Mortality and Disability from Diseases, Injuries, and Risk Factors in 1990 and Projected to 2020. Boston, Mass: Harvard School of Public Health; 1996.
4. Sen K, Bonita R. Global health status: Two steps forward, one step back. Lancet 2000; 356:577-82.
5. Yusuf S, Reddy S, Ôunpuu S, Anand S. Global Burden of Cardiovascular Diseases: Part I: General Considerations, the Epidemiologic Transition, Risk Factors, and Impact of Urbanization. Circulation, 2001; 104:2746-53.
6. Ahn D, Cheng L, Moon C, Spurgeon H, Lakatta EG, Talan MI. Induction of myocardial infarcts of a predictable size and location by branch pattern probability-assisted coronary ligation in C57BL/6 mice. Am J Physiol Heart Circ Physiol. 2004;286:H1201-1207.
References:7. Walker R,Whittlesea C.Clinical Pharmacy and Therapeutics,5th edition.page no312.
8. Murray CJL, Lopez AD, eds. The Global Burden of Disease: A Comprehensive Assessment of Mortality and Disability from Diseases, Injuries, and Risk Factors in 1990 and Projected to 2020. Boston, Mass: Harvard School of Public Health; 1996.
9. Walker R,Whittlesea C.Clinical Pharmacy and Therapeutics,5th edition.page no312. 10. M. G. L. Hertog, E. J. M. Feskens, P. C. H. Hollam, M. B. Katan, and D. Kromhout, “Folate awareness among South American women”, THE LANCET., 19993, PP. 1007-1020.
11. Savitri S, Sundara RS, Sujan GPS, Ravi SBE, Dhananjaya BL. Evaluating the antimicrobial activity of methanolic extract of Rhus succedanea leaf cell. BioImpacts 2013;3:195-8.
12.. Chen FH. Panax notoginseng. In: Tang W, Disenbrand G, Eds.Chinese Drugs of Plant Origin. Chemistry, Pharmacology andUse in Traditional and Modern Medicine. Berlin, Heidelberg:Springer Verlag, 1992:745-51.
References:13. Ashish Kumar Sharma,a,* Kunal Kishore,a Divya Sharma,a B.P Srinivasan,b Shyam Sunder Agarwal,b Ashok Sharma,a Santosh Kumar Singh,a Samir Gaur,a and Vijay Singh Jatava Cardioprotective activity of alcoholic extract of Tinospora cordifolia J Biomed Res. 2011 Jul; 25(4): 280–286
14. Ansari MN, Bhandari U, Pillai KK. Ethanolic Zingiber officinale R. extractpretreatment alleviates isoproterenol-induced oxidative myocardial necrosis in rats.Indian J Exp Biol 2006;44: 892-7.
15. National research council, Guide for the care and use of laboratory animals. National academy press: Washington DC,1996. 16. Shu-Yan Hana,1, Hai-XiaLia,1, XuMaa, KeZhanga, Zhi-ZhongMab, Peng-FeiTu:Protective effects of purified Safflower extract on myocardial ischemia invivo and invitro, Phytomedicine16(2009)694–702. 32.
17. Williamson EM, Okpako DT, Evans FJ .Pharmacological methods in Phytotherapy research .John Wiley and sons:1998,pp.1533.
18. Edeoga HO, Okwu KE, Mbaebie BO. Phytochemical constituents of some Nigerian medicinal plants. African journal of Biotecnology 2005; 4:685-688.-23
References:19. Revised Document. October. 2000. Organization for Economic Co-operation and Development, Revised draft Guidelines 423. OECD guideline for the testing of Chemicals; pp. 1–26.
20 . S. SENTHIL, M. SRIDEVI, AND K. V. PUGALENDI . Cardioprotective Effect of Oleanolic Acid on Isoproterenol-Induced Myocardial Ischemiai Rats. Toxicologic Pathology, 35:418–423, 2007 21. M. Catelli1, J. Feldman2,P. Bousquet2 and E. Tibiriçá1Protective effects of centrally acting sympathomodulatory drugs on myocardial ischemia induced bysympathetic overactivity in rabbits(2003) 36: 85-95 .
22. Effect of inhibition of lipolysis on infarct size after experimental coronaryartery occlusion. Journal of Clinical Investigation, 52,1770-1778..
23 . 2001 Mar;53(3):379-86.Cellular characterization of an in-vitro cell culture model of seal-induced cardiac ischaemia.Takahashi K1, Ohyabu Y, Schaffer SW, Azuma J..