Rajiv gandhi university of health sciences bangalore, karnataka

Name of the Candidate and Address

Name of the Institution

Course of Study and Subject

Date of Admission to Course

Title of the topic:

“Cardioprotective activity of Matricaria recutita Linn. in Sprague- Dawley rats”

Brief resume of the intended work

1. 
   Need for the study:
   

Cardiovascular disease (CVD) is the most relevant serious disorder in the developed nation. The American Heart Association as report that in 2002, 62 million American, 32 million male and 30 million female and preliminary mortality data from 2005 show that CVD accounted 861,826 of all 2447,910 deaths in 2005 in United States. This is 35.2 % percent of all deaths around the global had a cardiovascular disease including hypertension, so discharge from various survey report from hospital, cardiovascular disease have been rising statistically now exceed 6 million per year^1-2. Ischemic heart disease is caused primarily by coronary atherosclerosis, and result in an imbalance between oxygen supply and demand with resulting ischemia³. Cardiovascular disease claimed 949,619 lives, or 1 of every 2-5 deaths, in United States in 1998. Coronary artery disease was responsible for 459,841 deaths from CVD⁴. Heart failure is resulting any disorder that impairs the ability of the ventricle to fill with or eject blood, thus rendering the heart unable to pump blood at a rate sufficient to demands of the body⁵. Approximately 5 million American have heart failure, with an additional 550,000 cases diagnosed per year⁶. Complications of Congenital Heart Disease are myocardial hypertrophy and other changes of cardial remodeling brought about by the congenital defect⁷. Rupture of the intraventricular septum is an uncommon and often fatal complication of myocardial infarction⁸. Chamomile prepared as a tea used in most of country and chamomile containing active constituent apigenin and other flavonoids. Apigenin alone used in several cardiovascular diseases and which was proved by scientifically. But chamomile plant extract scientific validation as a cardioprotective was not carried out, especially in Ischemic-reperfusion injury models⁹. In view of this, the present study was designed to evaluation of cardioprotective activity of Matricaria recutita Linn. in Isoproterenol-induced myocardial infarction and ischemic-reperfusion injury in isolated heart models.

6.2 Review of literature:

Plant profile:

Title of plant : Matricaria recutita Linn.

Family : Asteraceae (Compositae)

Synonyms : Matricaria, German chamomile, Sweet false chamomile.s

Matricaria recutita Linn. (Chamomile) has been used medicinally for thousands of year, and is widely used in Europe. It is a popular treatment for numerous ailments, including sleep disorders, anxiety, digestion , intestinal conditions, skin infections, inflammation (including eczema), wound healing, infantile colic, teething pains and diaper rash. In the United States, Chamomile is best known as an ingredient in herbal tea preparations advertised for mild sedative effects¹⁰. German chamomile contains terpenoids, flavonoids, coumarins, other constituents such as tannin, polysaccharide and choline. Chamomile used externally for wounds, ulcers, ecezema, gout, neuralgia, rheumatic pain, hemorrhoids, leg ulcers¹¹. Anxiolytic effect of Matricaria recutita Linn. flowers and branch lets were studied in mice¹² .

Pharmacological actions:

Recently it has been showed to posses Antioxidant¹³, Diabetic complication¹⁴, Cardiac effects¹⁵ , Anti microbial activity¹⁶, Anti proliferative and apoptic effects¹⁷. Cardiovascular effects as increase atrial rate, relaxed thoracic aorta, hemodynamic effects ^18,19,20 and anti-ulcer activity²¹. Chamomile’s main active constituents are apigenin and bisabolol. Apigenin having cardioprotective activity⁹.

6.3 Objectives of the study:

The traditional practitioners well known Matricaria recutita Linn. will be extracted by using polar and non polar solvents and these extracts will be used for its cardioprotective properties using Isopro- terenol induced myocardial infarction and Ischemic-reperfusion injury in isolated rat heart models.

.

The objective of the study:-

1. 
   Cardioprotective potential of Matricaria recutita L. by measuring hemodynamic parameters like Blood pressure, Systolic blood pressure, Mean arterial pressure in ISO-induced myocardial necrosis in rats.
   
2. 
   Cardioprotective activity of Matricaria recutita L. by measuring Biochemical, Myocardial injury markers and Histopathological studies in ISO-induced myocardial necrosis in rats.
   
3. 
   Cardioprotective potential of Matricaria recutita L. by measuring hemodynamic parameters like Blood pressure, Systolic blood pressure, Mean arterial pressure in Ischemia-reperfusion induced myocardial damage in isolated rat heart preparation.
   
4. 
   Cardioprotective activity of Matricaria recutita L. by measuring Biochemical, Myocardial injury markers and Histopathological studies in Ischemia-reperfusion induced myocardial damage in isolated rat heart preparation.
   

Methods and materials:

7.1 Source of data:

The data will be collected from animal experiments and standard parameters for the study. The selection of doses 100, 200 and 300 mg/kg of body weight will be based on earlier literature²².

7.2 Materials :

Materials : Matricaria recutita linn.

Animals : Either sex (4 to 8 weeks), Sprague-Dawley rats.

Chemicals : All chemicals will be used analytical research grade.

Instruments : Spectrophotometer, Langendroff’s assembly, Refrigerator Centrifuge, Biopac (NIBP),

Tissue homogenizer, INCO-ECG, and enzyme kits.

Animals were subdivided into twelve containing Eight animals in each groups.

Group-I : Effect of vehicle on normal control group.

Group II : Effects of vehicle on ISO-induced myocardial damage.

Group-III : Effect of standard drug on ISO-induced myocardial damage.

Group-IV : Effect of 100 mg/kg of extract on ISO-induced myocardial damage.

Group-V : Effect of 200 mg/kg of extract on ISO-induced myocardial damage.

Group-VI : Effect of 300 mg/kg of extract on ISO-induced myocardial damage.

Group-VII : Effect of vehicle on normal control group.

Group-IX : Effect of standard drug on Ischemia-reperfusion induced injury in isolated heart.

Group-X : Effect of 100 mg/kg of extract on Ischemia-reperfusion induced injury in isolated heart.

Group-XI : Effect of 200 mg/kg of extract on Ischemia-reperfusion induced injury in isolated heart.

Group-XII : Effect of 300 mg/kg of extract on Ischemia-reperfusion induced injury in isolated heart.

7.3 Methods:

7.3.1 Isoproterenol (ISO) induced Myocardial necrosis:

Treatment protocol

The animals were divided into six groups. The first group, treated with normal Saline ( Normal control group), the second group received ISO and third, fourth, fifth and sixth groups received

Standard drug, 100, 200, 300 mg/kg of body weight dose of methanolic extract Matricaria recutita L. was given to each animal orally for 12 days and ISO administrated subcutaneously on 11^th and 12^th day. The animals were sacrificed 24 h after the second dose of ISO, under chloroform anesthesia.

Heart were removed and processed immediately for morphological and histopathological studies. The heart were homogenated ice cold buffer and used for biochemical estimation²³.

Determination of myocardial necrosis by direct staining:

Myocardiaum of rat was frozen immediately after removal. When the tissue was firm, the heart was sliced into 1 mm section and incubated at 37⁰ C for 20 min. in 1% TTC stain. The formazan precipitate resulting from the reaction of Lactate dehyrogenase in normal and ischemic region delineated the area at risk from the infracted tissue²⁴.

A Langendroff apparatus for the isolated perfused heart was set up as mentioned elsewhere²⁵. The heart was isolated from each animal 2 hrs after the last dose of the drug(s) under ketamine ( 70 mg/kg) and xylazine ( 10 mg/kg, i.p.) anesthesia. The isolated heart was perfused with Kreb-Henseleit (K-H) solu-

4. 
   Dose the study require any investigations or interventions to be conducted on patients or
   

4. 
   Has ethical clearance been obtained from your institution in case of 7.3?
   

Yes, study of cleared from institutional ethical committee and copy enclosed with the protocol.

1. 
   Eugene Braunwald. Disorders of cardiovascular system, Principle of Internal Medicine, edn 16^th, Volume-II, McGraw-Hiil, Medical Publishing division; 2005:1301.
   

American Heart Association Committee and stroke statistics sub committee. Heart Disease and

Stroke Statistics-2008:e26.
3. Joseph T., DiPiro., Robert L. Ischemic Heart Disease, Pharmacotherapy. A Pathophysiological

Approach, 6^th edn, Talbert;1999:261.

of chronic heart failure in the adult: Executive summary. A report of the American College

of Cardiology/American Heart Association Task Force on Practice Guideline (Committee to

Revise the 1995 Guideline for the Evaluation and Management of heart Failure): Developed in

Collaboration with International Society for Heart and Lung Transplantation; endorsed by the

Heart Failure Society of America. Circulation 2001;104:2996-3007.

12:564.
8. Szkutnik M., Kusa J., Bialkowskl J . The use of two Amplatzer “ Cribriform ” Septal Occluders

to close matiple postintaction ventricular septal defects. Text Heart Inst J 2008;35:362-4.

9. Ko F.N., Huang T.F., Teng C.M. Vasodilatory action mechanisms of apigenin isolated from

Apium graveolens in rat thoracic arota. Biochem Biophy Acta 1991;1115:69-74.

double-blind study. Z. Hautkr 1987; 62 (17):1262,1267-1271.

sionals. London, Pharmaceutical press 1996;9: 296.

and GABA in flower heads of Matricaria chamomilla. Phytother Res 1996;10:S177-179.

B.M., Burger M.E., Morel A.F., Morsch V.M., Rocha J.B. Antioxidant Effects of Different

Extracts from Melissa officinalis, Matricaria recutita and Cymbopogon citratus. Neurochem

Res 2008.

14. Kato A. , Minoshima Y., Yamamoto J., Adachi I., Watson A.A., Nash R. J. Protective effects of

dietary Chamomile tea on diabetic complication. J Agric Food chem 2008;56 (17):8206-11.

Tea. J Clin Pharmacol 1973;13:475.

16. Noqueira J.C., Diniz Mde F., Lima E.O. Anti microbial activity of plants in Acute Otiti Externa.

Braz J Otorhinolarygol 2008 ;74(1):118-24.

17. Srivastava J.K., Gupta S. Antiprliferative and apoptic effects of chamomile extract in various

human cancer cells. J Agric Food Chem 2007;55 (23): 9470-8.

18. Lorenzo P.S., Rubio M.C., Medina J.H., Alder-Graschinsky E. Involvement of monoamine

Oxidase and noradrenaline uptake in the positive chronotropic effects of apigenin in rat atria.

Euro J Pharmacol 1996; 312:203-7.

19. Ko F.N., Huang T.F., Teng C.M. Vasodilatory action mechanisms of apigenin isolated from

Apium graveollens in rat thoracic arota. Biochem Biophy Acta 1991; 1115:69-74.

20. Gould L, Reddy C.V.R., Gomprecht R.F. Cardiac effects of chamomile tea. J Clin Pharmacol

New Drugs 1973; 13:475-479.

21. Tamasdon S., Cristea E., Mihele D. Action upon gastric secretion of Rbiniae flores, chamomillae

flores and strobuli lupuli extracts. Farmacia 1981; 29:71-75.

J Pharmacol Toxi 2005:1-7.

Protective pontetial of Ocimum sanctum in isopreterenol induced myocardial infarction in rats,

Mol Cellular Biochem 2001;225:75-83.

24. Lie J.T., Pairolero P.C., Holley K.E..Macroscopic enzyme mapping verification of large, homo-

genous, experimental myocardial infarcts of predictable size and location in dogs. J Thorac

Cardiovasc Sug 1957;69:599-605.

25. Inamder M.N., Venkarataraman B.V., Aleem M.A. A simple and improved perfusion apparatus

for isolated hearts. Ind J Pharmacol 1994;26:262-265.

26. Mouhieddine S., Tresallet N., Boucher F., Leiris J.D. Ultrastructural basis of the free-radical

scavering effects of Indapamide in experimental myocardial ischemia and reperfusion. Cardio-

vasc Pharmacol 1993;22:S47-52.

global myocardial ischemia reperfusion injury in female rats. Am J Physiol Heart Circle 2000;

279:H2766-H2775.

fluid. Am J Clin Path 1958;30: 234-236.

29. Ellman G.L. Tissue sulfhydry groups. Arch Biochem Biophys 1959:82:70-77.

30. McCord J.M., Fridovich I. Superoxide dismutase: An enzyme fuction for erythrocuprcin ( hemo-

cuprcin). J Biol Chem 1969;244:6049-6055.

assay of super oxide dismutase. Analy Chem 1976; 70: 616-20.

32. Eva M.L. Mechanism of pH dependent hydrogen peroxide cytotoxicity in-vitro. Arch Biochem

Biophys 1988;365:362-72.

33. Carbonell L.F., Salazai F.J., Garcia Estan J., Ubeda M., Quesada T. Normal homodynamic

parameters in conscious rats, Rev Exp Physiol 1985;41:437-442.

Principal and professor

H.O.D. , Department of Pharmacology

H.S.K.College of Pharmacy,

B.V.V.S. Campus, Bagalkot-587101.

The above mentioned information is correct and I recommended the same for approval.

H.O.D., Department of Pharmacology

H.S.K.College of Pharmacy,

B.V.V.S. Campus, Bagalkot-587101.

Name and address of the Establishment:

BAGALKOT, KARNATAKA.

breeder from whom acquired and the date of

acquisition:

MINISTRY OF ENVIROMENT AND FOREST, 3^rd SEAWARD ROAD, VALMIKINAGAR,THRIRUVANMIYUR,

CHENNAI-600041.

Place where the animals are presently kept:

H.S.K. COLLAGE OF PHARMACY

BAGALKOT, KARNATAKA.

Place where the experiment is to be performed:

H.S.K. COLLAGE OF PHARMACY

BAGALKOT, KARNATAKA.

15 MAY 2009

Summary of project briefly summarize in laymen’s term the background, the objective and the experiment approach.

1. 
   Background:
   

2. 
   Objectives
   

Enclosed

Rationale for selection

first 12 months.

List all invasive Non Surgical Animal Procedures and Potentially Stressful Noninvasive procedures to be used (Example IM injection, foot pad injection , venapunctures).

Procedure and Approximate Frequency:

Enclosed

Anesthetic and/or Analgesic and Dosage:

Test substance injected and/or applied: