“STUDY OF THE EFFECT OF MANGIFERIN AGAINST CYCLOPHOSPHAMIDE AND DOXORUBICIN INDUCED CARDIOTOXICITY”
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore – 560 041
By
MR. BHATT LAXIT KAUSHIKBHAI
Under the Guidance of
Mr. Licto Thomas
Asst. Professor
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 Candidateand Address / BHATT LAXIT KAUSHIKBHAI
S/o Kaushikbhai C Bhatt
44/B, Rudraksh Bunglows, Nr. Telghani
Kendra, Hariyala Plot,
Bhavnagar. – 364001
Gujarat.
2. /
Name of the Institute
/ Shree Devi College Of PharmacyAirport Road, Kenjar Village,
Malavoor Panchayath,
Mangalore Tq D.K.-574 142
3. /
Course of Study and Subject
/ Master of Pharmacy in Pharmacology4. /
Date of Admission to Course
/November 2012
5. / Title of the Topic:
“STUDY OF THE EFFECT OF MANGIFERIN AGAINST CYCLOPHOSPHAMIDE AND DOXORUBICIN INDUCED CARDIOTOXICITY”
6. / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study
Although there has been a decline in the incidence of ischemic heart disease in Western Europe, North America and Australia/New Zealand, it remains a major cause of morbidity and mortality worldwide due to rapidly increasing incidences in developing countries. Prevention is key to reducing the burden of this disease.1
Abnormal lipids, smoking, hypertension, diabetes, abdominal obesity, psychosocial factors, consumption of fruits, vegetables, and alcohol, and regular physical activity account for most of the risk ofmyocardial infarctionworldwide in both sexes and at all ages in all regions. This finding suggests that approaches to prevention can be based on similar principles worldwide and have the potential to prevent most premature cases ofmyocardial infarction.2
Selected cytotoxic chemicals can indeed elicit immunogenic cell death, a functionally peculiar type of apoptosis that stimulates tumor-specific cognate immune responses. Such immunogenic chemotherapeutics includecyclophosphamide and doxorubicin(which are approved by FDA for the treatment of various hematological and solid malignancies).3
Cyclophosphamide(CYC), primarily introduced into clinical practice as an anti-cancer substance, is a potent immunosuppressive drug. Today, it is used in a number of organ- or life -threatening autoimmune diseases such as systemic vasculitides or connective tissue diseases. While being effective, CYC has a small therapeutic index and is associated with significanttoxicity. CYC has been used in oncology in a variety of diseases and a lot of data has been derived from this area.4
Doxorubicinis a chemotherapeutic drug used to treat solid and hematopoietic tumors. Its use is limited by a major side effect ofcardiotoxicity. It was reported thatdoxorubicin-inducedcardiotoxicityis mediated through oxidative stress coupled with impaired NO bioavailability and NF-κB activation.5
Cardiotoxicity may occur in >20% of patients treated with doxorubicin. Cardiac events may include mild blood pressure changes, thrombosis, electrocardiographic changes, arrhythmias, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, cardiac failure (left ventricular failure) and congestive heart failure. These may occur during or shortly after treatment, within days or weeks after treatment, or may not be apparent until months, and sometimes years, after completion of chemotherapy.6
Herbaldrugsare increasingly being used in the treatment of cardiovascular disorders.7Long before the birth of orthodox Western medicine, medicinal herbs were applied to treat a wide range of disease categories. Due to emphasis on scientism and other complicated reasons, Western medicine now prevails over “traditional” forms of medicine including herbal medicine systems. Although herbal medicine systems are sometimes misinterpreted as being unscientific and anachronistic, their long-term existence proves they are able to compete with Western drugs at some level. 8 In recent trend researchers are isolating the phyto-constituents which are also called as marker compounds, which are equipotent of modern medicine with great deal of safety.9
Some of the herbs such as Terminalia arjuna10, Allium ursinum11, Aloe barbadensis miller12, Aegle marmelos13 and Azadirachta indica14 as sole or as a component of herbal formulation demonstrated remarkable protection in the pathological conditions of cardiovascular system due to the presence of some of active phyto-constituents.
Mangiferin, C19H18O11, a glucoxanthone (1, 3, 6, 7-tetrahydroxyxanthone-C2-β-D glucoside) has been reported to be present in various parts of Mangifera indica (Anacardiaceae) commonly known as Mango in most of the countries. Mangiferin is known for having potential effect such as anti-inflammatory, analgesic, antioxidant, immunomodulator and in the treatment of obesity. Apart from that cardioprotective effect of Mangiferin already has been proved against isoproterenol-induced myocardial infarction in rats.15
Till now there is no study reporting the cardioprotective effect of mangiferin against anticancer drugs like cyclophosphamide and doxorubicin induced cardiotoxicity. Hence, the present study is designed to demonstrate the effect of mangiferin against cyclophosphamide and doxorubicin induced cardiotoxicity.
6.2 Review of literature
Mangiferin (C19H18O11), a glucoxanthone chemically known as 1, 3, 6, 7-tetrahydroxyxanthone-C2-β-D glucoside has been reported to be present in various parts of Mangifera indica which belongs to family Anacardiaceae and was encountered for the first time by Wiechowski.15
It is commonly known as “Mango Tree”, however various other vernacular names are observed, like Aam, Alfonso mango, Alipriya, Am, Amm, Amra, Amva, Asm, Bhramavapriya, Bo-amb, Kamaphala, Kamayudha, Kamavallabha, Kokilavasa, Kires, Kokilananda, Maamidi, Mam-maram, Mango fruit, Mave, Oegkoti-tong, Pitavallabha.16
The conclusive structure of mangiferin has been established as 2-C-β-D-gluco-pyranosyl- 1,3,6,7-tetrahydroxyxanthone. Mangiferin occurs widely among angiosperms and has also been identified in ferns.15
Mangiferin
The mangiferin aglycone is a phenolic compound that arises from two different aromatization pathways, the shikimate (carbons C4b, C5, C6, C7, C8, and C8a) and the ketate (carbons C1, C2, C3, C4, C4a, and C8b) pathways.
Its structure fulfils the four requisites which have been reported to favor a high bioavailability by oral administration:
· Molecular weight below 500 daltons;
· Less than 5 donor functions for hydrogen bonds;
· Less than 10 acceptor functions for hydrogen bonds;
· Favorable octanol/water partition coefficient.16
The pharmacological evaluation of the research studies conducted on mangiferin as well as plant extract of M. indica reveals the fact that mangiferin being major chemical and representative constituent of M. indica exhibits similar pharmacological activities to that exhibited by the plant extract of M. indica.15
Mangiferin has prominent pharmacological actions corroborated by numerous research studies. Potential anti-inflammatory, analgesic, antipyretic, antioxidant, immunomodulator, antitumor, antiviral, anthelmintic, antiallergic, antihistaminic, cardioprotective, anticholinergic, antiamoebic and antidiabetic effects have been found to be exerted by mangiferin.15
Though, considerable research has been and are being done on mangiferin and its plant source. Yet, the potential therapeutic effects of mangiferin have not been fully exploited till now. There is an ample scope for researchers to work further in this area.15
6.3 Objective of study:
The objective of the present investigation is to investigate the cardioprotective effect of mangiferin on toxicity induced by anticancer drugs like cyclophosphamide and doxorubicin using different experimental models in animals.
SPECIFIC OBJECTIVES
· To explore the cardioprotective role of mangiferin on experimental models such as cyclophosphamide and doxorubicin induced cardiotoxicity.
· To study the action of mangiferin on rat ECG.
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 cardiac damage.
· Doxorubicin induced cardiac stress.
· ECG will also be studied in treated and control groups.
Suitable biochemical and histological investigation will be carried out in each animal model.
EXPERIMENTAL MODELS:
Cyclophosphamide (CYP)induced cardiotoxicity17 :-
Cardiotoxicitywill beinducedin Albino rats by administeringCYP (200 mg/kg, i.p.) single injection on first day of experimental period. Mangiferin (10mg/100gm body weight i.p) 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- Mangiferin (10mg/100gm, i.p) for 10 days
o Group-IV- Mangiferin (10mg/100gm, i.p) 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), Creatinine kinase N-acetyl cysteine (CKNAC), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), Triglyceride (TG), Total Cholesterol (TC) 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 Stress18:-
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 dosages.
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 - Mangiferin (10mg/100gm body weight i.p)
o Group-IV - Mangiferin (10mg/100gm body weight i.p) + 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 Mangiferin and the treatment will be continued for 21 days.
On 22nd day biochemical analysis will be done in isolated serum and heart tissue homogenate. After that 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), Creatinine kinase N-acetyl cysteine (CKNAC), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), Triglyceride (TG), Total Cholesterol (TC) and antioxidants [Superoxide dismutase (SOD), reduced glutathione (GSH) and catalase] will be determined in serum as well as in heart tissue homogenate.
ECG Studies19
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. Pais PS. Early intervention and prevention ofmyocardial infarction. J Hypertens Suppl.2006 Apr;24(2):S25-30.
2. Yusuf S,Hawken S,Ounpuu S,Dans T,Avezum A,Lanas Fet al. Effect of potentially modifiable risk factors associated withmyocardial infarctionin 52 countries (the INTERHEART study): case-control study. Lancet.2004 Sep 11-17;364(9438):937-952.
3. Vacchelli E,Senovilla L,Eggermont A,Fridman WH,Galon J,Zitvogel L et al. Trial watch: Chemotherapy with immunogenic cell death inducers. Oncoimmunology.2013 Mar 1;2(3):e23510.
4. Brummaier T,Pohanka E,Studnicka-Benke A,Pieringer H. Usingcyclophosphamidein inflammatory+y rheumatic diseases. Eur J Intern Med.2013 Mar 22;6205(13)51-54.
5. Abdel-Raheem IT,Taye A,Abouzied MM. Cardioprotective Effects of Nicorandil, a Mitochondrial Potassium Channel Opener againstDoxorubicin-induced Cardiotoxicityin Rats. Basic Clin Pharmacol Toxicol.2013 Apr 29.
6. Pai VB,Nahata MC. Cardiotoxicityof chemotherapeutic agents: incidence, treatment and prevention. Drug Saf.2000 Apr;22(4):263-302. [Epub ahead of print]
7. Khaliq F,Parveen A,Singh S,Gondal R,Hussain ME,Fahim M. Improvement in Myocardial Function by Terminalia arjuna in Streptozotocin-Induced Diabetic Rats: Possible Mechanisms. J Cardiovasc Pharmacol Ther.2013 May 14. [Epub ahead of print]
8. Cicero L. T. Chang,1Yenshou Lin,2Arlene P. Bartolome,3 ,4Yi-Ching Chen,4Shao-Chih Chiu,Wen-Chin Yang. Herbal Therapies for Type 2 Diabetes Mellitus: Chemistry, Biology, and Potential Application of Selected Plants and Compounds. Evid Based Complement Alternat Med.2013;2013:378657.
9. Hade SN. Exploration of new therapeutic of phytoconstituents in anti-inflammatory plants by PASS. J Chem Pharm Res 2012;4(4):1925-1937.
10. Singh G, Singh AT, Abraham A, Bhat B, Mukherjee A, Verma R et al. Protective effects ofTerminaliaarjunaagainst Doxorubicin-induced cardiotoxicity. J Ethno pharmacol. 2008 Apr 17;117(1):123-129.
11. Rietz B, Isensee H, Strobach H, Makdessi S, Jacob R. Cardioprotectiveactions of wild garlic (Alliumursinum) in ischemia and reperfusion. Mol Cell Biochem.1993 Feb 17;119(1-2):143-50.
12. Jain N,Vijayaraghavan R,Pant SC,Lomash V,Ali M. Aloe veragel alleviates cardiotoxicity in streptozocin-induced diabetes in rats. J Pharm Pharmacol.2010 Jan;62(1):115-23.
13. Krushna GS,Kareem MA,Reddy VD,Padmavathi P,Hussain SA,Devi Kodidhela L. Aegle marmelosfruit extract attenuates isoproterenol-induced oxidative stress in rats. J Clin Biochem Nutr.2012 May;50(3):199-204.
14. Peer PA,Trivedi PC,Nigade PB,Ghaisas MM,Deshpande AD. Cardioprotectiveeffect ofAzadirachta indicaA. Juss. on isoprenaline induced myocardial infarction in rats. Int J Cardiol.2008 May 7;126(1):123-126.
15. Singh SK, Sharma VK, Kumar Y, Sadish Kumar S, Sinha SK. Phytochemical and pharmacological investigations on mangiferin. HERBA POL 2009;55(1):126-138.
16. Wauthoz N, Balde A, Balde ES, Damme MV, Duez P. Ethnopharmacology of Mangifera indica L. Bark and Pharmacological Studies of its Main C Glucosylxanthone, Mangiferin. Intl. J. Biomed. Pharma. Sci 2007 August 15;1(2):112-119.
17. A. H. M. Viswanatha Swamy,U. M. Patel,B. C. Koti,P. C. Gadad,N. L. Patel,andA. H. M. Thippeswamy. Cardioprotective effect ofSaraca indicaagainst cyclophosphamide induced cardiotoxicity in rats: A biochemical, electrocardiographic and histopathological study. Indian J Pharmacol.2013 Jan-Feb;45(1): 44–48.
18. 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;44:178-183.
19. Singh PN, Athar MS. Simplified calculation of mean QRS vector (Mean electrical axis of heart) of electrocardiogram. Indian J Physiol Pharmacol 2003;47:212-216.
9. / SIGNATURE OF THE CANDIDATE:
10. / REMARKS OF THE GUIDE:
11. / Name and Designation
11.1 Guide / Mr. Licto Thomas
Assistant Professor
Department of Pharmacology.
Shree Devi College Of Pharmacy,
Mangalore.
11.2 Signature
11.3 Head of the Department / Dr. Jagadish V Kamath
Department of Pharmacology.
Shree Devi College Of Pharmacy,
Mangalore.
11.4 Signature
12. / 12.1 Remarks of the Principal.
12.2 Signature / Dr. Jagadish V. Kamath.
Principal,
Shree Devi College of Pharmacy,
Mangalore.
12