RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

ANNEXURE-II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

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NAME OF THE CANDIDATE AND ADDRESS (IN BLOCK LETTERS)

Dr. RAMA SHARMA DEPARTMENT OF PREVENTIVE AND COMMUNITY DENTISTRY, COLLEGE OF DENTAL SCIENCES, DAVANGERE-577004 KARNATAKA. COLLEGE OF DENTAL SCIENCES, DAVANGERE-577004 KARNATAKA. MASTER OF DENTAL SURGERY (MDS) IN PREVENTIVE AND COMMUNITY DENTISTRY. 22/04/2009

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NAME OF THE INSTITUTION

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COURSE OF STUDY AND SUBJECT

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DATE OF ADMISSION TO COURSE TITLE OF THE TOPIC:

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COMPARATIVE EVALUATION OF CARIOGENIC POTENTIAL OF CHOCOLATES AVAILABLE IN THE INDIAN MARKET: AN IN VITRO AND IN VIVO STUDY.

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BRIEF RESUME OF INTENDED WORK

6.1 Need for the study: The science of dentistry has existed for long, ever since there has been theorizing about the cause of dental caries. During the past several years, there has been an increasing level of researches directed towards determining the cariogenic potential of specific foods. The term cariogenic potential is used deliberately because the ability of any particular foodstuff to contribute to the initiation of caries is determined by many factors, of which sugar content and the frequency of ingestion are of prime importance.1 Also cariogenic effects are, in part, related to the retention time of carbohydrates in the mouth as a result of their acidogenicity, the role of saliva in oral carbohydrate clearance is of primary interest.2 Chocolate, being a source of sugars, has been implicated as a cause for dental caries, but the relative cariogenicity of any food substance is not just because of the presence of sugars but is also dependent on their composition, texture, solubility, retentiveness and ability to stimulate salivary flow. Chocolates also usually contain cocoa in varying concentrations and it has been suggested that increasing concentrations of cocoa in chocolates prove to be less cariogenic.1 Cacao bean husk has been shown to possess two types of cariostatic substances, one showing anti-glucosyltransferase (GTF) activity and other antibacterial activity, and to inhibit experimental dental caries in rats infected with Mutans streptococci.3 Cocoa levels in chocolate seem to be related to the percentages of the other constituents, such as carbohydrates4 and only in some cases

have these proportions been studied for any relationship to plaque acidogenicity. Also, there are only few studies where the different types of chocolate have been studied whereby mainly milk and plain (dark) chocolates have been compared with a number of other foods, mainly snacks, or were used as products with known cariogenic potential in order to compare other foods or to assess methodologies.4 As there are a wide range of chocolates available in the market today of varying composition, texture, flavor and sweetness. It is thus natural that the cariogenic

potential of these chocolates would also vary.1 Also different cocoa proportions in chocolate confectionery might be a factor influencing the cariogenic response of different types and their acidogenic potential was considered worthy of study.4 Though there are several studies in western countries that assess the effect of chocolates available and its possible effects on caries by influencing the pH of saliva, there is paucity of similar studies on the chocolates available in the Indian market.1

6.2 Review of literature: A study was conducted on 30 dental volunteers to observe the bacterial cell growth in vitro and pH changes of saliva in vivo using some of the chocolates from the Indian market. It was concluded that filled chocolates are more cariogenic compared to unfilled chocolates, diet chocolate was found to have a least drop in pH and found to be least cariogenic, and chocolate with fruits and nuts were found to have a maximum and longer pH drop, making it most cariogenic.1

A study was conducted to examine the clearance from saliva of the carbohydrates glucose, fructose, sucrose, maltose and sorbitol after a mouthrinse and the clearance of relevant sugars after eating carbohydrate containing foods (bread, chocolate bar and banana). Of the sugar rinses studied, sucrose was removed from saliva most rapidly whilst appreciable levels of sorbitol remained even after 1 hour. Clearance of residual carbohydrates from bananas and chocolate bars seemed marginally faster than in the case of bread .2 A study was conducted to isolate the cariostatic substances from the cacao bean husk. Two types of cariostatic substances, one showing anti-glucosyltransferase (GTF) activity and the other showing antibacterial activity against Streptococcus mutans were determined.3 A study was conducted on 14 volunteers to assess the acidogenic response of plaque to European chocolates with varying cocoa contents, those containing hazelnuts and diet chocolates. The study showed that diet chocolate was found to have no acidogenic effect on dental plaque. Also, the plain European chocolate and milk chocolate with hazelnut had a lower acidogenic potential compared with sucrose.4 A study was conducted to evaluate a number of reference foods and snacks with subjects whose plaque pH response to fermentable carbohydrates was well documented. Short term telemetry (30 min) revealed that most of the substrates yielded both pH minima and total responses similar to that obtained with a 10% sucrose rinse. Cheddar cheese and skim milk were much less acidogenic than were the other foods.5

6.3 Objectives of the study: 1) To observe Streptococcus mutans, Lactobacilli and Candida albicans growth potential of various chocolates in vitro. 2) To evaluate pH changes in saliva after exposure to various chocolates in vivo. 3) To evaluate the clearance rate of various chocolates from the saliva in vivo.

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MATERIALS AND METHODS 7.1 Source of data Thirty subjects (15 males and 15 females) aged 18-25 years will be included. Students fulfilling the inclusion criteria will be selected randomly from College of Dental Sciences, Davangere, Karnataka.

7.2 Method of collection of data (Including sample size, inclusion criteria, exclusion criteria, if any): Study design: A randomized clinical trial with crossover study design. Sample size and design: A total of thirty subjects (15 males and 15 females) aged 18-25 years will be selected randomly from the students of College of Dental Sciences, Davangere, Karnataka, fulfilling the inclusion criteria.

Inclusion criteria: Subjects with: 1. Age between 18-25 years. 2. Minimum of 20 natural teeth. 3. DMFT value ranging from 0-3 4. Patients co-operative and willing to participate in the study. Exclusion criteria: 1. Active carious lesion. 2. Any oral soft tissue pathology. 3. Subjects with any systemic illness. 4. Pregnancy. 5. Subjects on any antimicrobial therapy. Test products: A. Unfilled chocolates: 1. Diet chocolate 2. Dark chocolate 3. Milk chocolate 4. White chocolate B. Filled chocolates: 1. Chocolate with fruit and nut 2. Chocolate with caramel 3. Chocolate with coconut

4. Chocolate with rice crisps 5. Chocolate with wafers 6. Chocolate with cereals

Oral isolates growth: in vitro To observe the Streptococcus mutans, Lactobacilli and Candida albicans growth potential of the chocolates, 5mg of each chocolate will be taken in sterilized test tubes, to this 5ml of Brain Heart Infusion broth will be added. Seed culture of Streptococcus mutans, Lactobacilli and Candida albicans will be inoculated into the broth medium. Cell growth will be monitored turbidometrically by periodically measuring the optical density of the culture at 550nm, using a spectrophotometer at an interval from baseline to 30 min, 1hr to 12 hr respectively.1 Each microorganism will be subjected to each test chocolate and the procedure will be repeated seven times.

Salivary pH changes: in vivo Purpose of study will be explained to the study subjects and demographic details will be taken in a proforma along with consent from all subjects. Each of the study subjects will undergo oral prophylaxis 24 hours before the morning of sample collection. Saliva will be collected in glass vials after the subjects are given 15 grams of each chocolate. Each of these two groups of chocolates will be given on different days to the subjects. On each test day, the saliva will be taken at baseline and at 5, 10, 15, 20, and 30 min to evaluate the pH levels of saliva using a digital pH meter.1

Oral clearance rate of various chocolates: in vivo The subjects will be instructed not to eat or drink anything 2 hours 30 minutes before the test. Before intake (0min) and 1, 3, 5, 7, 9, 11, 15, 30 and 60 min after the intake, a circular paper disc punched from filter paper, absorbing approximately 20l of saliva, will be placed in the oral cavity. After 10 sec, when the disc has become thoroughly soaked with saliva, it will be taken out of the mouth and will be transferred to the test tube containing 1 ml of distilled water. The tube will be placed in boiling water for 10 min and then kept frozen (-20C) until analyzed. After thawing and centrifugation, the concentration of glucose will be analyzed enzymatically.2,6

Statistical analysis: One way Analysis of Variance (ANOVA) will be used for multiple group comparisons followed by post hoc Tukeys test for pair wise comparison.

Duration of the study: One year

7.3 Does the study require any investigation (or) intervention to be conducted on patients (or) other humans (or) animals? If so please describe briefly: Yes. All the study subjects will be provided with the test chocolates. The salivary pH and oral clearance rate of the various chocolates will be estimated using digital pH meter and enzymatic method over a period of 30 min and 60 min respectively.

7.4 Has ethical clearance been obtained from your institution? Yes.

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LIST OF REFERENCES :

1. Shetty R, Hedge AM, Sequeira AR. The cariogenicity of chocolates available in the Indian market: an in vitro and in vivo study. J Indian Dent Assoc 2009;3(6):185-188. 2. Luke GA, Gough H, Beeley JA, Geddes DAM. Human salivary sugar clearance after sugar rinses and intake of foodstuffs. Caries Res 1999;33:123129. 3. Osawa K, Miyazaki K, Shimura S, Okuda J, Matsumota M, Ooshima T. Identification of cariostatic substances in the cacao bean husk: their antiglucosyltransferace and antibacterial activities. J Dent Res 2001;80(11):20002004. 4. Verakaki E, Duggal MS. A comparison of different kinds of European chocolates on human plaque pH. Eur J Paediatr Dent 2003;4(4):203-210. 5. Jensen ME, Schachtele CF. The acidogenic potential of reference foods and snacks at interproximal sites in the human dentition. J Dent Res 1983;62(8):889892. 6. Gabre P, Norrman C, Birkhed D. Oral sugar clearance in individuals with oral motor dysfunctions. Caries res 2005;39:357-362.

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SIGNATURE OF THE CANDIDATE REMARKS OF THE GUIDE

(Dr. RAMA SHARMA)

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NAME AND DESIGNATION OF 11.1. GUIDE (IN BLOCK LETTERS)

Dr. PRASHANT G. M., M.D.S., READER, DEPARTMENT OF PREVENTIVE AND COMMUNITY DENTISTRY, COLLEGE OF DENTAL SCIENCES, DAVANGERE 577004.

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SIGNATURE

11.3. CO-GUIDE (IF ANY) 11.4. SIGNATURE Dr. CHANDU G.N., M.D.S., PROFESSOR AND HEAD OF THE DEPARTMENT, DEPARTMENT OF PREVENTIVE AND COMMUNITY DENTISTRY, COLLEGE OF DENTAL SCIENCES, DAVANGERE 577004.

11.5 HEAD OF THE DEPARTMENT

11.6. SIGNATURE

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12.1. REMARKS OF THE CHAIRMAN AND THE PRINCIPAL

12.2. SIGNATURE