“EFFECT OF STEVIOSIDE ON VERAPAMIL AGAINST CYCLOPHOSPHAMIDE AND

DOXORUBICIN INDUCED CARDIOTOXICITY”

M. Pharm Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore– 560 041

By

Mr. SIVIN VARGHESE

Under the Guidance of

Mr. ABHILASH.D

Department of Pharmacology,

Shree Devi College of Pharmacy

Airport Road, Kenjar Village

Malavoor PanchayathMangalore – 574 142.

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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. Name of the Candidate

and Address

SIVIN VARGHESE

PULINTHARA (H)

Chittadi P.O, Peringala, Kannur , 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 JULY 2013

5. Title of the project:

EFFECT OF STEVIOSIDE ON VERAPAMIL AGAINST CYCLOPHOSPHAMIDE

AND DOXORUBICIN INDUCED CARDIOTOXICITY

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6.

BRIEF RESUME OF THE INTENDED WORK:

6.1 Need of study

Cardiovascular diseases (CVD) are the major cause for the death globally. The most

common reason for CVD are cholesterol deposition, inflammation, and thrombus

formation.1

Myocardial infarction (MI) is defined in pathology as myocardial cell death due to

prolonged ischemia which can be diagnosed by electrocardiographic (ECG) findings,

elevated values of biochemical markers (biomarkers) of myocardial necrosis and by

imaging.2

In cancer chemotherapy most often practiced agents are like cyclophosphamide,

doxorubicin, epirubicin, cisplatin. Though they are beneficial against cancer treatment the

serious side effects affecting the major organs such as heart, kidney and liver brings them

under prosecution. Earlier studies reported that cyclophosphamide (CP) and doxorubicin

(DOX) due to their side effects can cause serious cardiotoxicity.3

Cyclophosphamide (CP) is an alkylating agent and its tumor Cell-killing activity is mainly

due to its DNA alkylation. Phosphoramide mustard and acrolein are the two active

metabolites of CP. CP metabolites can react with carboxyl (-C[O]OH), mercapto (-SH),

amino (-NH2), phosphate (-PO3H2) and hydroxyl (-OH) groups and can form cross-links

with DNA and proteins CP exposure can disrupt the redox balance of tissues, the main

cause of cardio toxicity by cyclophosphamide is due to oxidative stress.3

Doxorubicin is a drug used in cancer chemotherapy. It is an anthracycline antibiotic

closely related to the natural product daunomycin, and like all anthracyclines, it works

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by intercalating DNA, with the most serious adverse effect being life-threatening heart

damage.4

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bert)Bertoni

Belongs to the family Asteraceae. Research with rodents indicates that stevioside a

constituent of Stevia reboundiana leaves can reduces the systemic blood pressure, as well

as produces diuresis and natriuresis.5

Studies proved that stevioside may potentiate the calcium antagonist action of verapamil

if the two substance are taken at the same time.6

Verapamil is a calcium channel blocker(CCB).CCB is chemicals that block the movement

of calcium (Ca2+) through channels. Though it is used in the treatment of hypertension, it

carries many side effect such as dizziness, slow heartbeat, constipation, nausea, headache,

tiredness, swelling of ankles/feet, shortness of breath, unexplained/sudden weight gain,

fainting, very slow heartbeat, severe stomach/abdominal pain, dark urine, yellowing

eyes/skin, persistent nausea/vomiting. 7

Till now there is no study reporting the combined cardio protective effect of stevioside

and Verapamil against anticancer drugs like cyclophosphamide and doxorubicin induced

cardio toxicity. So the present study has been designed to evaluate the combination of

Stevioside and Verapamil in different dose levels against DOX and CP induced

cardiotoxicity.

6.2 Review of literature

Stevioside is a natural sweetener obtained from the leaves of Stevia rebaudiana belongs to

family Asteraceae  well-known for its intense sweetness (250–300 times sweeter than

sucrose) and is used as a non-caloric sweetener.8

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Chemistry

Stevioside is an ent-kaurene type of diterpenoid glycoside, white to light yellow powder,

odourless or having a slight characteristic odour, Freely soluble in water.9

Structure of stevioside (C38H60O18)

COMPOUND NAME R1 R2 Stevioside β-Glc β-Glc-β-Glc(2→1)

Anti-hyperglycemic activity

Combination of glybenclamide and Stevia rebaudiana leaf extract (200 and 400 mg/kg)

orally administered for 10 days in rodents produced a delayed but significant decrease in the

blood glucose level, together with lesser loss in the body weight.10

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Antihypertensive activity

Stevioside tablets (50,100mg/kg i.p) significantly reduces the blood pressure.Stevioside

demonstrate its antihypertensive activity by inhibiting the extracellular calcium influx.11

Hepatoprotective activity

Stevia rebaudiana with initial dose of 200 mg/kg exhibit hepatoprotective activity on

thioacetamide induced hepatotoxicity.12

Vasodilation property

Stevioside reduces blood pressure by decreasing the vascular resistance via inhibition of

extracellular Ca2+ influx and by stimulating the release of a Vasodilator prostaglandin.

Stevioside also induces diuresis, natriuresis and reduction of Na+ reabsorption resulting in

reduction of extracellular fluid volume.13

Cardioprotective Activity

In the isolated Langendorff perfused guineapig heart model, the

ischemia-reperfusion

Injury was partially alleviated by infusion pretreatment (50, 250 or 500

nmol) with

Isosteviol. Cardio protective effect of i.v. Pretreatment with

isosteviol(0.5, 1.0 and 2.0

Mg/kg) on rats with heart ischemia-reperfusion injury; hemodynamic

parameters were

Improved, and the myocardial infarct size, the activities of serum

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enzymes, and the

Incidences of ventricular tachycardia and ventricular fibrillation were

decreased compared

To the control group.14

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6.3 Objective of study:

The objective of the present investigation is to investigate the effect of stevioside on

cardioprotective effect of verapamil against cyclophosphamide and doxorubicin induced

cardio toxicity.

SPECIFIC OBJECTIVES

To explore the cardioprotective role of stevioside with verapamil on

Cyclophosphamide induced cardiotoxicity in rat.

To explore the cardioprotective role of stevioside with verapamil on Doxirubicin

induced cardiotoxicity in rat.

To study the cardioprotective effect of stevioside with verapamil during ECG

changes.

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Materials and methods

7.1 Source of Data:

Journals and publication of pharmacognosy and pharmacology.

Electronic data like CD-ROM and internet.

Helinet, Delnet.

Medicinal and aromatic plants Association.

National institute of science communication and information resources.

The normal adult rats (200-250 g) of either sex will be procured from central

animal house of Sreedevi College of pharmacy, Mangalore.

Cyclophosphamide and doxorubicin pure sample will be collected from

manufactures.

Standard drug verapamil will be procured from reputed manufacturer.

Stevioside will be procured fromYucca Enterprises, Mumbai

Diagnostic kits will be purchased from Robonik (India) Pvt. Ltd.

All other laboratory grade chemicals will be procured from Merck Limited,

Mumbai

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Method of Collection of Data

The study is planned on normal rats and work is designed in following steps.

o Treatment will be given by different dose combination of stevioside and

verapamil.

o The effect of different treatments will be evaluated against doxorubicin and

cyclophosphamide induced cardiotoxicity.

o Evaluation will be done by measuring serum biomarker level, antioxidant level in

heart tissue homogenate, electrocardiographic changes in normal adult Albino rats

of either sex.

Study Sampling

o Blood sampling will be done by retro orbital sinus and serum will be separated by

centrifugation.

o Tissue homogenate will be prepared by tissue homogenizer.

o Biomarker levels will be checked in semiautomatic autoanalyzer.

o Antioxidant levels will be checked by spectrophotometer

o Statistical significance will be calculated by ANOVA followed by Tukey Kramer

multiple comparison test.

o The total duration will be approximately 10 months.

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EXPERIMENTAL MODELS:

Cyclophosphamide (CYP)   induced cardiotoxicity 15 :-

Cardiotoxicity will be induced in Albino rats by administering CYP (200 mg/kg, i.p.)

single injection on first day of experimental period. Stevioside, verapamil and their

combination will be administered in the respective groups immediately after

administration of CYP 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 control without pretreatment

o Group-II- CYP (200 mg/kg, i.p.) on the first day of experimental period

o Group-III-Verapamil(5omg/kg, i.p)

o Group-IV- stevioside (100mg/kg, i.p) for 10 days

o Group-V- stevioside(50mg/kg,i.p)for 10 days

o Group-VI- Stevioside (200mg/kg, i.p) for 10 days + CYP (200 mg/kg, i.p.) on the

first day of experimental period.

o Group-VII-stevioside(100mg/kg,i.p)+verapamil(50mg/kg p.o)

The endogenous biological markers such as Lactate Dehydrogenase (LDH), creatine

kinase isoenzyme MB (CK-MB), CKNAC, alanine transaminase (ALT), aspartate

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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 Stress 16 :-

Cardiotoxicity will be induced in Albino rats by administering Doxorubicin (DOX) with

total cumulative dose of 15mg/kg i.p. in six divided dosages of 2.5mg/kg, i.p.

alternatively for 2 weeks.

Stevioside, verapamil and their combination will be administered in the respective groups

for 2 weeks.

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 SEVEN groups of five animals each as

following:

o Group-I - normal control without pretreatment

o Group-II- DOX (15mg/kg, i.p.) on the first day of experimental period

o Group-III-Verapamil(50mg/kg, p.o)

o Group-IV- stevioside (100mg/kg, i.p) for 10 days

o Group-V- stevioside(50mg/kg,i.p)for 10 days

o Group-VI- Stevioside (200mg/kg, i.p) for 10 days + DOX (15 mg/kg, i.p.) on the

first day of experimental period.

o Group-VII-stevioside(100mg/kg,i.p)+verapamil(5omg/kg p.o)

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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 Studies17.

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.

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8.

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.

List of References:

1. Fuster V, Moreno PR, Fayad Z. Atherothrombosis and high risk plaque:partI: evolving

concepts. J AmColl Cardiol. 2005;46:937-54.

2. Tehrani DM, Seto AH. Third universal definition of myocardial infarction: Update,

caveats, differential diagnosis. Cleve clin J Med. 2013 Dec;80(12):777-86.

3. Kim SH, Lee IC, Baek HS, Shin IS, Moon C, Bae CS, Kim JC, Kim HC et al.

Mechanism for the protective effect of diallyl disulphide against cyclophosphamide

acute urotoxicity in rats. Food Chem Toxicol 2013 Nov 27;64:110-18.

4. Oliveira MS, Carvalho JL, Campos AC, Gomes DA, Degoes AM, Melo MM.

Doxorubicin has invivo toxicological effects on ex vivo cultured mesenchymal stem

cells. Toxicolett.2013Nov28;224(3):380-86.

5. Koyamma E, Kitazawa K, Ohori Y, Izawa O, Kakegawa K, Fujino A, Ui M et al.

In vitro metabolism of the glycoside sweeteners, stevia mixture and enzymatically

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modified stevia in human intestinal microflora. Food Chem Toxicol. 2003

Mar;41(3):359-74.

6. Melis MS, Sainati AR. Effect of calcium and verapamil on renal function of rats

during treatment with stevioside. J Ethnopharmacol. 1991 jul;33(3):

257-62.

7. Available from http://en.wikipedia.org/wiki/verapamil(online)retrived on 21/12/2013

at 11.30 a.m.

8. Wolwer-Rieck U. The leaves of Stevia rebaudiana(Bertoni),their constituents and the

analyses there of a review. J Agric Food Chem. 2012 Feb1;60(4):886-95.

9. Stevioside and related compounds Therapeutic benefits beyond sweetness.

Thailandpharmacol Ther. 2009 Jan; 121(1):41-54.

10. Mishra H, Soni M, Silawat N, Mehta D, Mehtha BK, Jain DC. Antidiabetic activity

of medium-polar extract from the leaves of stevia rebaudiana Bert(Bertoni) on

alloxan-induced diabetic rats. J Pharm Bioallied sci 2011Apr;3(2) :242-48.

11. Hsu YH. Liu JC. Kim PF. Lee CN. ChenYJ. Hsieh MH. Antihypertensive effect of

stevioside in different rat strains of hypertensive rats.Zhonghua Yi Xue Za

zhi(Taipei).2002 Jan;65(1):1-6.

12. Kundal D, Kumar A. Hepatoprotective activity of stevia rebaudiana Bert leaves

against thioacetamide induced toxicity. Turk J Pharm sci. 2012;9(3):343-52.

13. Takasu N, Hashimoto H, Miyazaki Y, Ito T, Ogawa K, Satake T. Effects of

phospholipase inhibitors and calcium antagonists on the changes in myocardial

phospholipids induced by isoproterenol. Basic Res Cardiol. 1988 sep-oct;83(5):567-

75.

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14. Xu D, Li Y, Wang J. Davey K, Zhang S, Evans AM. The cardioprotective effect of

isosteviol on rats with heart ischemia-reperfusion injury. Life sci. 2007 Jan

2;80(4):269-74.

15. Vishwanathaswamy 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.

16. Swamy AV, Gulliaya S, Thippeswamy A, Koti BC, Manjula DV. Cardioprotective

effect of curcumin against doxorubicin-induced myocardial toxicity in albino rat.

Indian J Pharmacol. 2012 Jan;44(1):73-74.

17. Singh PN, Athar MS. Simplified calculation of mean QRS vector (mean electrical axis

of heart) of electrocardiogram. Indian J physiol pharmacol. 2003;47:212-16.

9. Signature of the candidate:

10. Remarks of the guide:

11. Name and Designation of:

11.1 GuideAbhilash.DAsst. professorDepartment of Pharmacology.Shree Devi College Of Pharmacy, Mangalore. 574 142

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11.2 Signature of the guide

11.3 Head of the department Dr. Jagadish.V.KamathDepartment of pharmacology,Shreedevi college of pharmacy ,Mangalore574142

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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