“EFFECT OF URSOLIC ACID AGAINST CYCLOPHOSPHAMIDE AND DOXORUBICIN INDUCED CARDIOTOXICITY”

M. Pharm Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore– 560 041

By

MR. BINU SEBASTIAN

Under the Guidance of

DR. JAGADISH V KAMATH

Department of Pharmacology,

Shree Devi College of Pharmacy

Air port Road, Kenjar Village

Malavoor Panchayath Mangalore – 574 142.

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. / Name of the Candidate
and Address / BINU SEBASTIAN
Kandathinkara
Seethamount P.O
Pulpally
Wayanad. Kerala
2. /

Name of the Institute

/ Shree Devi College Of Pharmacy
Airport Road, Kenjar Village,
Malavoor Panchayath,
Mangalore Tq D.K.-574 142
3. /

Course of Study and Subject

/ Master of Pharmacy in Pharmacology
4. /

Date of Admission to Course

/
November 2012
5. / Title of the Topic:
“EFFECT OF URSOLIC ACID AGAINST CYCLOPHOSPHAMIDE AND DOXORUBICIN INDUCED CARDIOTOXICITY”
6. / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study
The advanced life style, different diseased conditions such as diabetes, atherosclerosis, toxic effect of different antibiotics and cytotoxic drugs are leading cause of development of cardiovascular diseases. All over the world myocardial infarction (MI) and the resultant complication in cardiac function represent the leading cause of morbidity and mortality.1
However, the heart can be temporarily or permanently damaged by exposure to toxic effect of anti-cancer molecules like cyclophosphamide and doxorubicin.2
Doxorubicin ,which is an anthracycline antibiotics are among the most effective anticancer agent and is still widely used in the treatment of cancer. The predominant mechanism of doxorubicin cardiomyopathy involves oxidative damage, and leads to membrane disruption, widespread cellular dysfunction, and ultimately, cell death.3,4
Cyclophosphamide is an alkylating agent that promotes cross linking of DNA. Cyclophosphamide demonstrates a form of cardio toxicity that can result in myocyte death, unlike the cardiac manifestations associated with anthracyclines, the toxicity of cyclophosphamide is related more to the magnitude of the dose administered during a single cycle rather than the cumulative dose over time.5
More than 2000 plants have been listed in the Traditional (Herbal/ Alternative) systems of medicine and some of these are providing comprehensive relief to the people suffering from cardio-vascular diseases.6
Some of the herbs such as Allium sativum, Asparagus racemosus, Curcuma longa, Emblica officinalis, Ocimum sanctum, Phyllanthus amarus, Terminalia arjuna, Withania somnifera and Zingiber officinalis already have proven themselves as potential cardioprotective agent. Potent phytoconstituents like triterpenoids, polyflavonoids by exhibiting potent antioxidant effects causes beneficial role in myocardial abnormalities.7
Ursolic acid, a natural pentacyclic triterpenoid carboxylicacid, is a potential phyto constituent isolated majorly from tulsi, apples, prunes, cranberries, and blueberries. It is known that ursolic acid exerts a wide range of biological activities, such as anti-inflammatory, antioxidant, nephroprotective and hepatoprotective effects.8 Apart from that cardioprotective activity of Ursolic acid has been reported against isoproterenol induced cardiotoxicity.9
Till now there is no study reporting the effect of Ursolic acid on cyclophosphamide and doxorubicin induced toxicity in myocardial cells. Hence, the present study is designed to evaluate the effect of Ursolic acid against cyclophosphamide and doxorubicin induced cardio toxicity.
6.2 Review of literature
Ursolic Acid which is a pentacyclic triterpenoid that is very widely distributed in many herbs such as Ocimum sanctum(Tulsi), Boerhaavia Diffusa, Boswellia Serrata, Asparagus Racemosus10, Apples (high in the peels)11,Cranberry juices12,and Grape skins which is responsible for wide variety of medicinal properties.
Some of the anti-diabetic plants like Banaba Leaf,Gynostemma pentaphyllum, andCrataegus pinnatifida having ursolic acid for their said activity.13
Anti-inflammatory plants like Athranthus roseus and Mentha spicata having ursolic acid which is responsible for the action.13
Hepatoprotective plants like Plantago major and Clerodendrum serratum L are having ursolic acid as major constituents.14
Nephroprotective plants like Momordica dioica Roxb shows said action because of the presence of ursolic acid.15
1.2. Structure
Ursolic acid is a natural pentacyclic triterpenoid carboxylic acid.

Ursolic acid have been demonstrated earlier to possess cardioprotective activity.16 Several other pharmacological activities such as antitumor, hepatoprotective, anti-inflammatory (oral & topical), anti-ulcer, antimicrobial, anti-hyperlipidemic, and anti-viral activities, have also been attributed to ursolic acid.17
6.3 Objective of study:
The objective of the present investigation is to investigate the cardioprotective effect of ursolic acid on cyclophosphamide and doxorubicin induced toxicity using different experimental models in animals.
SPECIFIC OBJECTIVES
·  To explore the cardioprotective role of Ursolic Acid on Cyclophosphamide induced cardiotoxicity in rat.
·  To explore the cardioprotective role of Ursolic Acid on Doxirubicin induced cardiotoxicity in rat.
·  To study the protective effect of Ursolic Acid during ECG changes.
7. / MATERIALS AND METHODS:
7.1 Source of Data:
Data will be obtained from laboratory based studies by using albino rats of either sex weighing between 150-200 g maintained at room temperature having free access to food (standard pellet diet), tap water ad libitum. These studies will be carried out in intact animal as well as on isolated tissues that will be supported by biochemical data and histopathological studies.
7.2 Method of Collection of Data:
Chemicals and reagents will be procured from standard companies. Following models will be used for interactive studies:
·  Cyclophosphamide induced myocardial infarction
·  Doxirubicin induced myocardial infarction
·  ECG also will be studied in treated and controlled groups.
Suitable biochemical and histological investigation will be carried out in each animal model.
EXPERIMENTAL MODELS:
Cyclophosphamide (CYP)induced cardiotoxicity18 :-
Cardiotoxicitywill beinducedin Albino rats by administeringCYP (200 mg/kg, i.p.) single injection on first day of experimental period. Ursolic acid (40mg/kg p.o) will be administered immediately after administration ofCYP on first day and daily for 10 days. Symptoms and mortality in each group will be recorded and compared with those of the rats given CYP alone. Twenty four hour after the last administration biochemical analysis will be done in isolated serum and heart tissue homogenate. Apart from that isolated heart will be embedded for histological examination.
The Albino rats of either sex will be divided into four groups of six animals each as following:
o  Group-I - normal controlwithout pretreatment
o  Group-II- CYP (200 mg/kg, i.p.) on the first day of experimental period
o  Group-III- Ursolic acid (40mg/kg, p.o) for 10 days
o  Group-IV- Ursolic acid (40mg/kg, p.o) for 10 days + CYP (200 mg/kg, i.p.) on the first day of experimental period
The endogenous biological markers such as Lactate Dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), CKNAC, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total cholesterol(TC), triglyceride(TG), and antioxidant [Superoxide dismutase (SOD), reduced glutathione (GSH) and catalase] will be determined in serum as well as in heart tissue homogenate.
Doxorubicin induced Cardiac Stress19:-
Cardiotoxicity will be induced in Albino rats by administering Doxorubicin (DOX) with total cumulative dose of 15mg/kg i.p for 2 weeks in six divided dosage.
The Albino rats of either sex will be divided into FOUR groups of five animals each as following:
o  Group-I - normal controlwithout pretreatment
o  Group-II - Doxorubicin total cumulative dose of 15mg/kg i.p for 2 weeks in six divided dosage
o  Group-III - Ursolic acid (40mg/kg, p.o)
o  Group-IV - Ursolic acid (40mg/kg, p.o) + DOX treatment.
Groups II & IV will receive DOX at alternate days for a period of 2 weeks. The days that are selected for DOX administration are 8th, 10th, 14th, 16th, 18th & 21st day after 7 days pretreatment with Ursolic acid and the treatment will be conducted for 21 days.
On 22nd day biochemical analysis will be done in isolated serum and heart tissue homogenate. Then the rats will be sacrificed and autopsied. The hearts will be removed and weighed, and the frontal sections will be embedded for histological examination.
The endogenous biological markers such as Lactate Dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), CKNAC, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total cholesterol(TC), triglyceride(TG), and antioxidant [Superoxide dismutase (SOD), reduced glutathione (GSH) and catalase] will be determined in serum as well as in heart tissue homogenate.
ECG Studies20.
After anesthetizing the rat with a combination of ketamine hydrochloride (75mg/kg, i.p) and xylazine (8.0mg/kg, i.p), leads will be attached to the dermal layer of both the front paws and hind legs and recording will be made with the help of computerized ambulatory ECG system. Animal grouping and treatment will be same as CYP, DOX model.
7.3 Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:
YES
Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats and mice as experimental animals.
7.4 Has ethical clearance been obtained from your institute
Ethical Committee approval letter is enclosed.
8. / List of References:

1.  Vakili H, Sadeghi R, Tabkhi M, Safi M. Corrected thrombolysis in myocardial infarction frame count and ejection fraction in patients undergoing primary percutaneous coronary intervention for myocardial infarction. ARYA Atheroscler. 2013 Mar;9(2):134-139..

2.  Lao J, Madani J, Puértolas T, Alvarez M, Hernández A, Pazo-Cid R, Artal A, Antón Torres A. Liposomal Doxorubicin in the treatment of breast cancer patients: a review. J Drug Deliv. 2013;2013:456409.

3.  Hydock DS, Lien CY, Jensen BT, Parry TL, Schneider CM, Hayward R. Rehabilitative exercise in a rat model of doxorubicin cardiotoxicity. Exp Biol Med (Maywood). 2012 Dec;237(12):1483-92.

4.  Gharanei M, Hussain A, Janneh O, Maddock HL. Doxorubicin induced myocardial injury is exacerbated following ischaemic stress via opening of the mitochondrial permeability transition pore. Toxicol Appl Pharmacol. 2013 Apr 15;268(2):149-56.

5.  Viswanatha Swamy AH, Patel UM, Koti BC, Gadad PC, Patel NL, Thippeswamy AH. Cardioprotective effect of Saraca indica against cyclophosphamide induced cardiotoxicity in rats: a biochemical, electrocardiographic and histopathological study. Indian J Pharmacol. 2013 Jan-Feb;45(1):44-8.

6.  Tilak-Jain JA, Devasagayam TPA. Cardioprotective and other beneficial effects of some Indian medicinal plants. J. Clin.Biochem. Nutr 2006; 38: 9-18.
7.  Arya V, Gupta VK. Chemistry and pharmacology of plant cardioproectives: A review. IJPSR 2011; 2:1156-1166.

8.  Shanmugam MK, Dai X, Kumar AP, Tan BK, Sethi G, Bishayee A. Ursolic acid in cancerprevention and treatment: Molecular targets, pharmacokinetics and clinical studies. Biochem Pharmacol. 2013 Jun 1;85(11):1579-87.

9.  Radhiga T,Rajamanickam C,Senthil S,Pugalendi KV. Effect ofursolic acidon cardiac marker enzymes, lipid profile and macroscopic enzyme mapping assay in isoproterenol-induced myocardial ischemicrats; Food Chem Toxicol.2012 Nov;50(11):3971-7.

10.  Kowalski R. Studies of selected plant raw materials as alternative sources of triterpenes of oleanolic and ursolic acid types. J Agric Food Chem. 2007 Feb 7;55(3):656-62.

11.  Denis MC, Furtos A, Dudonné S, Montoudis A, Garofalo C, Desjardins Y, Delvin E, Levy E. Apple peel polyphenols and their beneficial actions on oxidative stress and inflammation. PLoS One. 2013;8(1):e53725.

12.  Kim E, Sy-Cordero A, Graf TN, Brantley SJ, Paine MF, Oberlies NH. Isolation and identification of intestinal CYP3A inhibitors from cranberry (Vaccinium macrocarpon) using human intestinal microsomes. Planta Med. 2011 Feb;77(3):265-70

13.  Mitchell SA, Ahmad MH. A review of medicinal plant research at the University of the West Indies, Jamaica, 1948-2001. West Indian Med J. 2006 Sep;55(4):243-69.

14.  Vidya SM, Krishna V, Manjunatha BK, Mankani KL, Ahmed M, Singh SD. Evaluation of hepatoprotective activity of Clerodendrum serratum L. Indian J Exp Biol. 2007 Jun;45(6):538-42

15.  Jain A, Singhai AK. Nephroprotective activity of Momordica dioica Roxb. in cisplatin-induced nephrotoxicity. Nat Prod Res. 2010 May;24(9):846-54

16.  Senthil S, Sridevi M, Pugalendi KV. Protective effect of Ursolicacid against myocardial ischemia induced by isoproterenol in rats.Toxicol Mechm Methd; 2007;17:57-65

17.  Tang X, Gao J, Chen J, Fang F, Wang Y, Dou H, Xu Q, Qian Z. Inhibition by [corrected] ursolic acid of [corrected] calcium-induced mitochondrial permeability transition and release of two proapoptotic proteins. Biochem Biophys Res Commun. 2005 Nov 11;337(1):320-4.

18.  CardioprotectiveeffectofSaracaindicaagainstcyclophosphamideinducedcardiotoxicityinrats:abiochemical,electrocardiographicandhistopathologicalstudy. Indian J Pharmacol.2013 Jan-Feb;45(1):44-8.1

19.  .Momin FN, Kalai BR, Shikalgar TS, Naikwade NS. Cardioprotective effect of methanolic extract of Ixora coccinea Linn. Leaves on doxorubicin-induced cardiac toxicity in rats. Indian J Pharmacol 2012;44:178-83.
20.  Singh PN, Athar MS. Simplified calculation of mean QRS vector (Mean electrical axis of heart) of electrocardiogram. Indian J Physiol Pharmacol 2003;47:212-6.
9. / SIGNATURE OF THE CANDIDATE:
10. / REMARKS OF THE GUIDE:
11. / Name and Designation
11.1 Guide / Dr. Jagadish V Kamath
Department of Pharmacology.
Shree Devi College Of Pharmacy,
Mangalore. 574 142
11.2 Signature
11.3 Head of the Department / Dr. Jagadish V. Kamath
Department of Pharmacology.
Shree Devi College Of Pharmacy,
Mangalore. 574 142
11.4 Signature
12. / 12.1 Remarks of the Principal.
12.2 Signature / Dr. Jagadish V. Kamath.
Principal,
Shree Devi College of Pharmacy,
Manglore.574 142

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