From,
Dr. Mohandas Rai,
Professor and Head (Department of Pharmacology),
A.J. Institute of Medical Sciences,
Mangalore- 575004
To,
The Member Secretary,
Ethics committee A.J.I.M.S,
A.J. Institute of Medical Sciences,
Mangalore- 575004
Respected sir,
Subject: Submission of protocol of Dissertation for Ethics Committee Clearance.
Herewith, I am forwarding the protocol of the dissertation work of Dr. Smitha Sivan, Postgraduate Resident in the department of pharmacology, titled:“Evaluation of Anti-ulcer Activity of Vanillin Using Stress and Aspirin Induced Ulcer Models in Wistar Albino Rats” for ethics committee clearance of A.J. Institute of Medical Sciences.
Kindly accept the same and oblige.
Thanking You
Signature of the Head of the Department
(Dr. Mohandas Rai)
Signature of the Postgraduate
(Dr. Smitha Sivan
Place: Mangalore
Date:
From,
Dr. Manohar V.R.
Professor (Department of Pharmacology),
A.J. Institute of Medical Sciences,
Mangalore- 575004
To,
The Member Secretary,
Ethics committee A.J.I.M.S,
A.J. Institute of Medical Sciences,
Mangalore- 575004
Respected sir,
Subject: Submission of protocol of Dissertation for Ethics Committee Clearance.
Herewith, I am forwarding the protocol of the dissertation work of Dr. Smitha Sivan, Postgraduate Resident in the department of pharmacology, titled: “Evaluation of Anti-ulcer Activity of Vanillin Using Stress and Aspirin Induced Ulcer Models in Wistar Albino Rats” for ethics committee clearance of A.J. Institute of Medical Sciences.
Kindly accept the same and oblige.
Thanking You
Signature of the Guide
(Dr. Manohar V.R.)
Signature of the Postgraduate
(Dr. Smitha Sivan)
Place: Mangalore
Date:
From,
Dr. Smitha Sivan
Post Graduate- Department of Pharmacology,
A.J. Institute of Medical Sciences,
Mangalore- 575004
To,
The Member Secretary,
Ethics Committee A.J.I.M.S,
A.J. Institute of Medical Sciences,
Mangalore- 575004
Respected sir,
Subject: Submission of protocol of Dissertation for Ethics Committee Clearance.
Herewith, I am submitting protocol of my dissertation work: “Evaluation of Anti-ulcer Activity of Vanillin Using Stress And Aspirin Induced Ulcer Models in Wistar Albino Rats” for Ethics Committee clearance of A.J. Institute of Medical Sciences, Mangalore.
Enclosed here with the copies of:-
Protocol of dissertation
Curriculum vitae of guide
Curriculum vitae of candidate
Kindly accept the same and oblige.
Thanking you,
Yours faithfully,
(Dr. Smitha Sivan)
Place: Mangalore
Date:
From,
Dr. Smitha Sivan
Post Graduate - Department of Pharmacology,
A.J. Institute of Medical Sciences,
Mangalore- 575004
To,
The Registrar,
Rajiv Gandhi University of Health Sciences,
Bangalore
(Through proper channel)
Sub: Submission of synopsis of Dissertation
Respected Sir,
Herewith, I am submitting synopsis of my dissertation work: “Evaluation of Anti-ulcer Activity of Vanillin Using Stress And Aspirin Induced Ulcer Models in Wistar Albino Rats” for the registration in Rajiv Gandhi University of Health Sciences, Bangalore.
Kindly accept the same and oblige.
Thanking you,
Yours faithfully,
(Dr. Smitha Sivan)
Place: Mangalore
Date:
CURRICULUM VITAE OF THE PRINCIPAL INVESTIGATOR
Name : Dr. Smitha Sivan
Address : Abhiman Palace, Flat no. 901,
Mannagudda
Mangalore- 575003
E- Mail :
Telephone : 9632126549
Date of birth : 20-05-1989
Marital Status : Married
Nationality : Indian
Academic Qualifications:
Name of the Institution/ University / Year of PassingM.B.B.S / A. J Institute of Medical Sciences
Mangalore/ RGUHS / 2012
Internship / A. J Institute of Medical Sciences
Mangalore/ RGUHS / 2013
PG /Tutor / A. J Institute of Medical Sciences
Mangalore/ RGUHS
CURRICULUM VITAE OF THE GUIDE
Name : Dr. Manohar V.R.
Address : Sri Ganesh kripa , 8-125/9(1), Kulashekhar Post , Mangalore-5.
E- Mail :
Telephone : 9448857263
Date of birth : 03-08-1970
Marital Status : Married
Nationality : Indian
Academic qualifications:
Name of the institution/ university / year of passingM.B.B.S / KMC, Mangalore/Mangalore University / 1993
Internship / KMC, Mangalore/Mangalore University / 1994
MD / KMC, Mangalore/MAHE / 2000
WORK EXPERIENCE
ASST. PROF / Yenepoya Medical College / 2000-2005ASSOC. PROF / Yenepoya Medical College / 2005-2013
PROFESSOR / AJ Institute of Medical Sciences, Mangalore. / Jan 2013 Till Date
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE II
SYNOPSIS FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1 / NAME OF THE CANDIDATE AND ADDRESS / DR. SMITHA SIVANPOSTGRADUATE STUDENT
DEPT OF PHARMACOLOGY
A.J.INSTITUTE OF MEDICAL SCIENCES
MANGALORE- 575004
2 / NAME OF THE INSTITUTION / A.J.INSTITUTE OF MEDICAL SCIENCES
MANGALORE- 575004
3 / COURSE OF STUDY AND SUBJECT / MD COURSE IN PHARMACOLOGY
4 / DATE OF ADMISSION TO COURSE / 11-07-2013
5 / TITLE OF THE TOPIC / Evaluation of Anti-ulcer Activity of Vanillin Using Stress and Aspirin Induced Ulcer Models in Wistar Albino Rats
6 / BRIEF RESUME OF INTENDED WORK:
6.1. NEED FOR THE STUDY:
Peptic ulcer, encompassing gastric and duodenal ulcers, is the most common gastrointestinal disorder.[1] The pathophysiology involves an imbalance between offensive (acid, pepsin and H. pylori) and defensive (mucin, bicarbonate, nitric oxide, prostaglandin and growth factors) mechanisms.[2]The main approaches to the treatment of peptic ulcer is reduction of gastric acid secretion and re-enforcing gastric mucosal protection. A number of drugs like proton pump inhibitors, H2-receptor antagonists, prostaglandin analogues are available for the treatment of the disease.[1,2] Although these drugs are effective for treatment, the magnitude in prevalence of this disease in the population has galvanized the ongoing search for better drugs with even more favourable profiles.
Vanilla planifolia is the scientific name of the plant commonly known as vanilla.[3] It belongs to the family orchidacea.[4]Vanilla planifolia yields the popular, commercial flavouring agent vanillin. Vanillin is the primary component of the extract of the vanilla bean. It is a phenolic aldehyde, an organic compound with the molecular formula C8H8O3. Its functional groups include aldehyde, ether and phenol.[5] .Phytochemistry of Vanilla planifolia has been studied and phenol was found to be the major constituent.Natural vanilla extract has been studied to have antioxidant properties.[3] Polyphenols display number of pharmacological properties in the GIT, acting as antisecretory, gastroprotective and anti-oxidant agents. The antioxidant properties of phenolic compounds have been widely studied but it has become clear that their mechanism of action has gone beyond modulation of oxidative stress. A study was conducted to evaluate the role of polyphenolic compounds in peptic ulcer and it was concluded that they possess gastroprotective and anti–ulcer properties.[1] Data evaluating the potency of vanillin as an ulcer protective is lacking. So this study will be conducted to evaluate the ulcer protective property of vanillin and to compare its potency with Omeprazole.
6.2. REVIEW OF LITERATURE:
Vanilla planifolia is a thick, tropical, leafy, evergreen vine climbing orchid.[3] Vanilla is native to the tropical region and its main producers are India, Madagascar, Mexico, Indonesia.[4]
The main constituent of Vanilla planifolia is vanillin. The extract of Vanilla planifolia with vanillin contains a number of related phenylpropanoid compounds.[3]
Vanillin inhibits mutation induced by hydrogen peroxide, N-methyl -N-nitrosoguanidine and mitomycin C.[6]
Vanillin has been shown to suppress in-vitro invasion and in-vivo metastasis of mouse breast cancer cells.Studies suggest that the antimetastatic activity of vanillin may be attributed to its aldehyde group. [7]
Vanillin extracts have been shown to possess antioxidant property.[8]
Vanillin improves and prevents trinitrobenzene sulphonic acid induced colitis in mice through inhibition of cytokines and various other mechanisms.[9]
Studies have shown that vanillin possesses cytolytic and cytostatic properties. It could induce apoptosis and inhibit cancerous cell growth.[10]
Experimental studies have demonstrated the antidepressant and anxiolytic effects of vanillin and the anxiolytic effect is attributed to its antioxidant property.[11,12]
Effects of vanillin on lipid profile has been studied. It was shown that vanillin at higher doses i.e.(200mg/kg and 400mg/kg) significantly lowered serum triglyceride and serum VLDL-C in high fat diet treated rats.[13]
But, there are no reports available regarding the anti-ulcer activities of vanillin. Since vanillin has been shown to have antioxidant property and also being a polyphenolic compound we expect it to have antiulcer activity. Hence, the present study is undertaken to evaluate the effect of vanillin against stress induced and aspirin induced ulcer models in rats and it will be compared with the standard drug, Omeprazole.
6.3. AIM & OBJECTIVES OF STUDY
1. To evaluate the anti-ulcer activity of vanillin in rats.
2. To compare the anti-ulcer activity of vanillin with Omeprazole in rats.
7 / MATERIALS AND METHODS:
7.1.SOURCE OF DATA:
Type of study : Animal study
Site of study : A J Institute of Medical Science, Mangalore
Animals used : Albino rats of Wistar strain
Weight : 150-250 grams
Material :Vanillin{4-Hydroxy-3-methoxybenzaldehyde}
Molecular formula: C8H8O3.
Standard Drug: Omeprazole will be powdered and dissolved in Normal Saline and administered orally.
Animals : 54 Albino wistar rats of either sex weighing 150-250grams will be bred and maintained under standard conditions in the central animal house in AJIMS. They will be kept at the animal house of the Pharmacology department of the institute for 7 days at 210 ± 10c with light/dark cycle of 12:12hours.
7.2. STUDY DESIGN:
Animals will be divided into 2 sets consisting of 5 groups with 6 animals in each group. Ulcer will be induced using two models i.e. Stress induced ( Restraint cage ) and Aspirin induced models. The drugs will be dissolved in normal saline and administered orally once daily for 10 days.
1.RESTRAINT CAGE MODEL (stress induced ulceration)
Group / No: of animals / Drug / Dosage & Route
I / 6 / Normal Saline / 2ml/kg
II / 6 / Normal Saline+Stress
III / 6 / Omeprazole / 20mg/kg Oral[14]
IV / 6 / Vanillin / 250mg/kg Oral
V / 6 / Vanilline / 500mg/kg Oral
Procedure : Wistar albino rats of either sex will be divided into five groups with six animals in each group as follows: Group I: Control (untreated) group. Group II: Stress control group Group III: Standard treatment group (Omeprazole 20 mg/kg) Group IV: Test treatment group ( Vanillin 250 mg/kg) Group V: Test treatment group ( Vanillin 500 mg/kg). The groups will be treated with the respective agents accordingly for ten days.On the tenth day thirty minutes after administering the respective agents, each animal will be subjected to stress by placing in separate restraint cages measuring approximately 11×6×6 cms for 24 hours. Next day the rats will be euthanised using Pentobarbitone 100mg/kg intra-peritoneal injection. Stomach will be removed and cut along the whole length of greater curvature, ulcers will be examined ,scored and ulcer index will be calculated.[15,16] Gastric juice will be used for the estimation of volume, pH and total acidity.
2.Aspirin induced gastric ulcer :-
Group / No: of animals / Drug / Dose & Route
I / 6 / Normal Saline / 2ml/kg Oral
II / 6 / Normal Saline+Aspirin / 500mg/kg Oral[16]
III / 6 / Omeprazole / 20mg/kg Oral
IV / 6 / Vanillin / 250mg/kg Oral[17]
V / 6 / Vanillin / 500mg/kg Oral[17]
Procedure :
Wistar albino rats of either sex will be divided into five groups with six animals in each group as follows: Group I: Control (untreated) group. Group II: Aspirin control group Group III: Standard treatment group (Omeprazole 20 mg/kg) Group IV: Test treatment group ( Vanillin 250 mg/kg) Group V: Test treatment group ( Vanillin 500 mg/kg).The standard drug (Omeprazole 20 mg/kg) and the test drugs (Vanillin 250 and 500 mg/kg) will be administered orally to the respective groups for 10 days. On the tenth day, one hour after administration of test drugs, the animals will be treated with Aspirin (500 mg/kg). After 4 hours, they will be euthanised using Pentobarbitone 100mg/kg intra-peritoneal injection. The numbers of ulcer spots in the glandular portion of the stomach will be counted in both normal control and drug treated animals and the ulcer index will be calculated.[14,16]
Estimation of gastric volume, pH, and total acidity: The gastric content that is transferred into centrifuge tubes will be used for estimation of gastric volume, pH and total acidity. The tubes will be centrifuged at 1000 rpm for 10 minutes and the gastric volume will be directly read from the graduation on the tubes. The supernatant is then collected and pH will be determined by using a digital pH meter. Total acidity will be determined by titrating 1.0 ml of gastric juice against N/10 NaOH to pH 7 using phenolphthalein as the indicator and will be expressed in terms of clinical units, i.e., the amount (ml) of N/10 NaOH required to titrate 100 ml of gastric secretion.[18]
Determination of ulcer index: The tracing of the stomach boundary and the ulcerated area on the transparent film will be placed on top of a graph paper. The total surface area of the stomach and the lesions will be determined in mm2 from the graph paper. The ratio of total surface area and the total ulcerated area will be determined and scoring of the ulcer index will be done. Percentage protection will be calculated in the drug treated groups against control using the formula:
% protection = (1-ulcer index in test/ulcer index in control) x 100
Ulcer index= 10/x , where x= Total mucosal area/ total ulcerated area.[18]
Statistical Analysis:All data will be analysed using “one way ANOVA”
All data will be analysed using “one way ANOVA” followed by “Dunnett’s T Test”.
P values <0.05 will be considered as significant.
Effect of Vanillin on ulcer parameters in Restraint Cage Model in Rats
Treatment Groups/Doses / Mean Ulcer Score / Ulcer Index / % Inhibition
Stress Control/Group
Test Treatment Group (Vanillin 250mg/kg)
Test Treatment Group (Vanillin 500mg/kg)
Standard (20mg/kg) Group
Effect of Vanillin on Ulcer Parameters in Aspirin induced ulcer models rats
Treatment Groups/Doses / Mean Ulcer Score / Ulcer Index / % Inhibition
Toxicant Group
Test Treatment Group (Vanillin 250mg/kg)
Test Treatment Group (Vanillin 500mg/kg)
Standard (20mg/kg) Group
7.3 Does the study require any investigation or interventions to be conducted on patients or other human or animals? If so, please describe briefly.
Yes. In this study ulcer will be induced using stress and Aspirin. Drugs will be dissolved in normal saline and administered orally , once daily for ten days. Following which the animals will be euthanized humanely using Pentobarbitone 100mg intraperitoneal injection.
7.4 Has the ethical clearance obtained from your institution?
Obtained.
8 / LIST OF REFERENCES:
1)Sambul S, Ahmad MA, Mohd A. Role of phenolic compounds in peptic ulcer : An overview. J. Bioallied Sci. 2011 Jul;3(3):361-7.
2)Hoogerwerf WA, Pasricha P J. Agents used for control of gastric acidity and treatment of peptic ulcers and gastroesophageal reflux disease. In: Hardman JG, Limbird LE, Goodman Gilaman A, editors. Goodman and Gilman. The Pharmacological Basis of Therapeutics. 10th ed. New York: McGraw-Hill; 2001:1005-19.
3)Nayeem N, Menon S. Vanilla planifolia:A Review of a Plant Commonly Used as Flavouring agent:International Journal of Pharmaceutical Science.Rev.Res; 20(2), May-June 2013;42:225-8.
4)Azeez S, Parthasarathy VA, Chempakam B, Zachariah TJ. Chemistry of Spices, Vanilla: CAB International; 2008:287-89.
5)Kumar R, Sharma PK, Mishra PS. A Review on the Vanillin derivatives showing various Biological activities. International journal of Pharm Tech Research. 2012;4(1):266-79.
6)Gustafson DL, Franz HR, Uneno AM, Smith CJ, Doolittle DJ, Waldren CA.Vanillin (3-methoxy-4-hydroxybenzaldehyde) inhibits mutation induced by hydrogen peroxide,N-methyl-N-nitrosoguanidine and mitomycin C but not137Cs γ-radiation at theCD59locus in human–hamster hybrid ALcells. Mutagenesis. 2000May;15(3):207-13.
7)Lirdprapamongkol K, Sakurai H, Kawasaki N, Choo MK, Saitoh Y, Aozuka Y et al editors. Vanillin Supressess in-vitro invasion and in-vivo metastasis of mouse breast cancer cells. European J Pharm Sci. 2005 May;25(1):57-65.8)Shyamala BN, Naidu MM, Sulochanamma G, Srinivas P. Studies on the antioxidant activities of natural vanilla extract and its constituent compounds through in vitro models. J. Agric Food Chem. 2007 Sep 19;55(19):7238-43 Epub 2007 Aug 24.
9)Wu SL, Chen JC, Li CC, Lo HY, Ho TY, Hsiang CY. Vanillin Improves and Prevents Trinitrobenzene Sulfonic Acid- Induced Colitis In Mice. J Pharmacol Exp Ther. 2009Aug;330(2):370-76.
10)Ho K, Yazan LS, Ismail N, Ismail M. Apoptosis and cell cycle arrest of human colorectal cancer cell line HT-29 induced by vanillin. Cancer Epidemiol. 2009Aug;33(2):155-60.
11) Shoeb A, Chowta M, Pallempati G, Rai A, Singh A. Evaluation of Antidepressant activity of Vanillin in mice. Indian J of Pharmacol. 2013;Mar-Apr,45(2):141-44.
12)Bhagwat V, Chowta MN, Shoeb A, Makeri R, Venkatesh V, Rai A. Evaluation of anxiolytic activity of Vanillin in wistar albino rats.Indian Journal of Pharmacology. 2013;3(2):96-101.
13)Belagali Y, Ullal SD, Shoeb A, Bhagwath V, K R, Maskeri R. Effect of Vanillin on lipid profile in a model of hyperlipidemia, a preliminary study. Indian Journal of Experimental Biology. April 2013;51:288-91.
14)Panda V, Sonkamble M. Antiulcer activity of Ipomoea batata tubers(Sweet potato). Functional Foods in Health and Disease. 2012 April31;2(3):48-61.
15)Vogel GH, Vogel WH. Drug discovery and evaluation-pharmacological assays. Germany: Springer publication. 2002:868-69.
16)Parmar NS, Prakash S. Screening methods in pharmacology. Fifth ed. New Delhi: Narosa publishing house. 2011:264-66.
17)OECD SIDS UNEP Publications;1996[ Cited 2012 Dec 13]. Available from
18)Lahiri S, Palit G. An overview of the Current Methodologies Used for Evaluation of Gastric and Duodenal Anti-ulcer agents.Pharmacologia. 2012;3(8) :249-57.
9 / SIGNATURE OF CANDIDATE:
10 / REMARKS OF THE GUIDE
11 / NAME AND DESIGNATION OF
11.1. GUIDE / Dr Manohar V.R.
Professor (Department of Pharmacology)
A.J.Institute Of Medical Sciences
Kuntikana, Mangalore - 575004
11.2. SIGNATURE:
11.3. CO-GUIDE:
11.4. SIGNATURE:
11.3. HEAD OF THE DEPARTMENT: / Dr Mohandas Rai
Professor & HOD
Department of Pharmacology.
A.J.Institute Of Medical Sciences
Kuntikana, Mangalore - 575004
11.4. SIGNATURE:
12 / 12.1.REMARKS OF THE CHAIRMAN AND PRINCIPAL:
12.2.SIGNATURE OF THE PRINCIPAL:
1