“STUDY OF THE COMBINATION EFFECT OF CURCUMIN AND PIPERINE AGAINST CYCLOPHOSPHAMIDE AND DOXORUBICIN INDUCED CARDIOTOXICITY”
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore – 560 041
By
Ms. SARANGA A.P
Under the Guidance of
Mr. ABHILASH .D
Asst. Professor
Department of Pharmacology,
Shree Devi College of Pharmacy
Air port Road, Kenjar Village
Malavoor Panchayath
Mangalor - 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 / SARANGA A.P
D/o of Manoj A.P
Kunimal house
c/o Meenangadi pharmacy
Meenangadi( po), Pin:673591
Wayanad (D),Kerala
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
/July 2013
5. / Title of the Topic:
“STUDY OF THE COMBINATION EFFECT OF CURCUMIN AND PIPERINE AGAINST CYCLOPHOSPHAMIDE AND DOXORUBICIN INDUCED CARDIOTOXICITY”
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study
The term Myocardial Infarction (MI) indicates the development of an area of myocardial necrosis caused by local ischemia. Abnormal lipids, smoking, hyper tension, diabetes, abdominal obesity, intake of alcohol, reduced consumption of fruits, vegetables and lack of regular exercise are the major risk factor of myocardial infarction. MI and related complication has become one of the world wide health problem affecting all economic group of the society. Moreover with advanced life style in developing countries such as India particularly in metropolitan cities MI making important contribution to mortality and morbidity statistics.1
The cytotoxic chemicals have ability to cause immunogenic cell death , impairing mitosis, effectively targeting fast dividing cells. This type of immunogenic chemotherapeutics includes cyclophosphamide and doxorubicin.2
Cyclophosphamide is a nitrogen mustard alkylating agent. It is used to treat cancer and autoimmune disorder. It induces cardiotoxicity that can result in myocardial cell death.3
Doxorubicin is an anthracyclin antibiotic used in cancer chemotherapy. Its most serious adverse effect is life threatening heart damage. It occurs when the cumulative dose is above 550 mg/ m2.
With the advancement of synthetic drugs the side effects associated with it demands medical attention and the need of alternative therapy.
From the ancient time to modern civilization herbs and herbal remedies play an important role to treat diseased conditions. Going a step further researcher across the globe witnessed that isolated phyto constituents which are also called as marker compounds are equipotent of modern medicine with great deal of safety.4
Curcumin is isolated from the plant source Curcuma longa belongs to the family Zingiberaceae. The earlier reported studies demonstrated that Curcumin is blessed with cardio protective, anti-ischemic, anti-inflammatory, hepato protective, wound healing, anti diabetic activities which provokes the need for further research on this potential phyto constituent. But from the beginning Curcumin is cursed with poor bioavailability which is a point of attention.5,6
Another potential phyto constituent, Piperin is isolated from fruits of Piper nigrum belongs to family Piperaceae. A study reported that curcumin when it was given alone in the dose 2mg/kg in rats, moderate serum concentrations were achieved over a period of 4 hr where as concomitant administration of piperine 20 mg/kg increased the serum concentration of curcumin for a short period of 1-2 hr post drug and bioavailability was increased by 154% in rats and increase in bioavailability was 2000% in human.7
Apart from that piperine is having anti-carcinogenic, anti-convulsant, anti-inflammatory, anti- malarial properties.8
Till now there is no study reporting the cardioprotective effect of curcumin and piperine against anticancer drugs like cyclophosphamide and doxorubicin induced cardiotoxicity. Hence the present study is designed to demonstrate the combination effect of curcumin and piperine in different doses against cyclophosphamide and doxorubicin induced cardiotoxicity.
6.2 Review of literature
Curcumin is the principle phyto constituent isolated from the rhizomes of Curcuma longa belongs to family Zingiberaceae. It is a small perennial herb bearing many rhizomes on its root system which are the source of its culinary spice known as turmeric. Curcumin the principle curcuminoids found in turmeric, is generally considered its most active constituents. Other two curcuminoids are desmethoxy curcumin and bis-des methoxy curcumin. Curcumin can exist in several tautomeric forms, including 1,3diketo form and two equivalent enol form The enol form is more stable in the solid phase and in solution.9
Antineoplastic.10
Curcumin at a dose of (110mg/kg) causes apoptosis of various cancer cell type including prostate, skin, colon, fore stomach, duodenum and ovary.
Anti diabetic.11
Curcumin at a dose of (10mg/kg) reported anti diabetic activity against streptozotocin induced type 1 diabetic.
Hepatoprotective.12
Curcumin at a dose of (100 and 200mg/kg) demonstrated hepatoprotective activity against CCl4 induced hepatotoxicity.
Cardioprotective.13
Curcumin with the therapeutic dose of (200mg/kg) reported cardioprotective potential against doxorubicin and isoprenaline induced cardiac damage.
Anti inflammatory.14
Curcumin at a dose of (50-200 mg/kg) reported anti inflammatory activity against carrageenan induced paw edema in mice.
Curcumin
Piperine is an alkaloid isolated from the fruits of Piper nigrum, the source plant of both the black and white pepper grains. Piperine inhibit human CYP3A4 , p-glycoprotein, enzyme important for the metabolism and transport of xenobiotics and metabolite. In animal studies, Piperine also inhibited other enzymes involved in drug metabolism. Piperine enhances the bioavailability of structurally and therapeutically different drugs, either by increasing the absorption or by delaying the metabolism of the drug or by a combination of both the processes. Piperine increases the bioavailability of curcumin by 2000% in humans. Combination of curcumin with piperine is safe, non toxic and is more effective than curcumin.15
Antimutagenic.16
Piperine at dose of (100,400 and 800mg/kg body weight for 24 days) reported anti mutagenic effect on cyclophosphamide induced chromosome aberrations rat bone marrow cell.
Anti convulsant.17
Piperine at a dose of 25-86mg/kg exhibit Anti convulsant activity on seizures induced by excitatory aminoacid receptor agonist.
Anti depressant.18
Anti depressant and cognitive effects piperine - encapsulated liposomes(PL) were investigated in male wistar rats. Oral piperine (5mg/kg body weight/day) and intranasal PL (7.2 Ug/kg/day)were randomly assigned to daily administer for 14 days to rats.
Anti inflammatory and anti arthritic.19
Piperine at a dose of 20 and 100mg/kg/day for 8 days exhibit anti inflammatory and antiarthritic effects of piperine in human interleukin 1beta stimulated fibroblast like synoviocytes and in rat arthritis models.
Piperine
The plant is used in many Asian countries as a stimulant for the treatement of colic, rheumatism, headache, insomnia, liver desease dysentery, cholera. The three common uses of piperine in Indonesia include antipyretic, anti-epileptic and for the treatment for snake venom poisoning.20
6.3 Objective of study:
The objective of the present investigation is to investigate the cardioprotective effect of combination of curcumin and piperine on toxicity induced by anticancer drugs like cyclophosphamide and doxorubicin using different experimental models in animals.
SPECIFIC OBJECTIVES
· To explore the cardioprotective role of combination of curcumin and piperine on experimental models such as cyclophosphamide and doxorubicin induced cardiotoxicity.
· To study the action of curcumin and piperine on rat ECG.
7. / 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.
Ø The normal adult rats (200-250 g) of either sex will be procured from central animal house of Shree Devi collage of pharmacy.
Ø Cyclophosphamide and doxorubicin pure sample will be collected from manufactures.
Ø Curcumin and piperine will be procured from Yucca Enterprises, Mumbai
Ø Diagnostic kits will be purchased from Robonik (India) Pvt. Ltd.
Ø All other laboratory grade chemicals will be procured from Merck Limited, Mumbai.
7.2 Method of Collection of Data:
The study is planned on normal rats and work is designed in following steps.
· Treatment will be given by different dose combination of Curcumin and Piperine.
· The effect of different treatments will be evaluated against doxorubicin and cyclophosphamide induced cardiotoxicity.
· 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
1) Blood sampling will be done by retro orbital sinus and serum will be separated by centrifugation.
2) Tissue homogenate will be prepared by tissue homogenizer.
3) Biomarker levels will be checked in semiautomatic autoanalyzer.
4) Antioxidant levels will be checked by spectrophotometer
5) Statistical significance will be calculated by Anova followed by Tukey kramer multiple comparison.
6) The total duration will be approximately 10 months.
EXPERIMENTAL MODELS:
Cyclophosphamide (CYP)induced cardiotoxicity21:-
Cardiotoxicitywill beinducedin Albino rats by administeringCYP (200 mg/kg, i.p.) single injection on first day of experimental period. Curcumin, piperine and their combination will be administered in the respective groups 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- animals kept as normal control without pretreatment.
o Group-II- CYP only (200 mg/kg, i.p. on first day)
o Group-III- animals treated with Curcumin (Cur- 200 mg/kg, p.o) for 10 days + CYP toxicity.
o Group-IV-animals treated with Curcumin (100mg/kg, p.o) + piperine (20mg/kg, p.o) for 10 days + CYP toxicity.
o Group-V- animals treated with Curcumin (50mg/kg) + Piperine (20mg/kg) for 10 days + CYP toxicity.
o Group-VI- animals treated with Curcumin (25mg/kg) + Piperine (20mg/kg) for 10 days + CYP toxicity.
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 Stress13:-
Cardiotoxicity will be induced in Albino rats by administering Doxorubicin (DOX) with total cumulative dose of 15mg/kg i.p. in six devided dose of 2.5 mg/kg i.p alternatively for 2 weeks. Curcumin, piperine 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 four groups of six animals each as following
o Group-I- animals kept as normal control without pretreatment.
o Group-II- DOX only, (Cumulative dose of 15 mg/kg, i.p.)
o Group-III- animals treated with Curcumin (Cur- 200 mg/kg, p.o) for two weeks and subjected to DOX toxicity.
o Group-IV-animals treated with Curcumin (100mg/kg) + piperine (20mg/kg) for two weeks + DOX toxicity.
o Group-V- animals treated with Curcumin (50mg/kg) + Piperine (20mg/kg) for two weeks + DOX toxicity.
o Group-VI- animals treated with Curcumin (25mg/kg) + Piperine (20mg/kg) for two weeks + DOX toxicity.
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 Studies22 :-
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. 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):937-52.
2. 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.
3. Brummaier T, Pohanka E, Studnicka Benke A, Pieringer H. Using cyclophosphamide in inflammatory rheumatic disease. Eur J Intern Med. 2013 Mar 22;6205(13):51-54.
4. Hade SN. Exploration of new therapeutic of phytoconstituents in anti-inflammatory plants by PASS. J Chem Pharm Res. 2012 Apr;4(4):1925-37.
5. Panchatcharam M, Miriyala D, Gayathri VS, Suguna L. Curcumin improves wound healing by modulating collagen and decreasing reactive oxygene species. Molecular and cellular biochemistry. 2006 Oct 2;290(1-2):87-96.
6. Lakshmanan AP, Watanabe K, Thandavarayan RA, Sari FR, Meileri H, Soetikno V et al. Curcumin attenuates hyperglycaemia- mediated AMPK activation and oxidative stress in cerebrum of streptozotocin- induced diabetic rat. Informa health care. 2011 Jul;45(7):788-95.
7. Shoba G,Joy D,Joseph T,Majeed M,Rajendran R,Srinivas PS. Influence of piperine on the pharmacokinetics ofcurcuminin animals and human volunteers. Planta Med.1998 May;64(4):353-6.
8. Kumar S, Kamboj J, Suman, Sharma S. Overview for various Aspect of the health benefits of Piper longum linn.fruit. J Acupunct Meridian Stud. 2011 Jun;4(2):134-40.
9. Kolev TM, Velcheva EA, Stamboliyska BA, Spiteller M, DFT and experimental studies of the structure and vibrational spectra of curcumin. Int J Quantum chem. 2005;102(6):1069-79.
10. Sundram V,Chauhan SC,Ebeling M,Jaggi M. CurcuminAttenuates β-catenin signaling in Prostate Cancer Cells through Activation of Protein Kinase D1. PloS One.2012 Apr 16;7(4):367-69.
11. Abdel Azi MT, El-Asmar MF, El-Ibrashy IN, Rezq AM, Al-Malki AL, Waseef MA et al. Effect of novel water soluble curcumin derivative on experimental type-1 diabetes mellitus(short term study). Diabetol Metb Syndr. 2012 Jul 4;4(1):30.
12. Park EJ,Jeon CH,Ko G,Kim J,Sohn DH. Protective effect of curcumin in rat liver injury induced by Carbon tetrachloride. Journal of pharmacy and pharmacology. 2000 Apr;52(4):437-40.
13. Swamy AV, Gulliaya S, Thippeswamy A, Koti BC, Manjula DV. Cardioprotective effect of curcumin against doxorubicin-induced myocardial toxicity in albino rat. Indian J pharmacology. 2012 Jan;44(1)73-37.
14. Jurenka JS, MT (ASCP) Anti-inflammatory Properties of Curcumin, a Major Constituentof Curcuma longa: A Review of Preclinical and Clinical research. Alternative Medicine Review. 2009 nov 2;14(3):146-47.
15. Bhardwaj RSK,Glaeser H,Becquemont L,Klotz U,Gupta SK,Fromm MF. Piperine, a major constituent ofblack pepper, inhibits human P-glycoprotein and CYP3A4. J Pharmacol Exp Ther.2002 Aug;302(2):645-50.
16. Wongpa S, Himakoun L, Soontornchai S, Temcharoen P. Antimutagenic Effects of Piperine on Cyclophosphamide induced Chromosome Aberrations in Rat Bone Marrow Cells. Asian Pacific J Cancer Prev. 2004 Aug;8(4):623-27.
17. D'Hooge R,Pei YQ,Raes A,Lebrun P,van Bogaert PP,de Deyn PP . Anticonvulsant activity of piperine on seizures induced by excitatory amino acid receptor agonists. Arzneimittelforschung.1996 Jun;46(6):557-60.
18. Priprem A, Chonpathompikunlert P, Sutthiparinyanont S, Wattanathorn J. Antidepressant and cognitive activity of intranasal piperine encapsulated liposomes. Biomedical and Life Science. 2011 Apr;2(2):108-06.
19. Bang JS, Oh da H, Choi HM, Sur BJ, Lim SJ, Kim JY, et al. Antiinflammatory and antiarthritic effects of piperine in human interleukin 1beta-stimulated fibroblast-like synoviocytes and in rat arthritis models. Arthritis Res Ther. 2009 Mar 30;11(2):2023-29.
20. Szallasi A, Piperine: Researchers discover new flavor in an ancient spice. Trends Pharmacol. Sci. 2005;26(9):437-39.
21. Swamy AH, Patel UM, Koti BC, Gadad PC, Patel NL, Thippewamy AH. Cardioprotective effect of Saraca indica against cyclophosphamide induce cardiotoxicity in rats: A biochemical,electrocardiographic and Histopathological study. Indian J Pharmacol. 2013 Jan-Feb;45(1):49-48.
22. 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
11.1 Guide / Mr. Abhilash .D
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.
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