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Effects of Cinnamon Water Extract As a Cariostatic Agent

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Effects of Cinnamon Water Extract As a Cariostatic Agent EFFECTS OF CINNAMON WATER EXTRACT AS A CARIOSTATIC AGENT ON NICOTINE-INDUCED STREPTOCOCCUS MUTANS BIOFILM by Abdulaziz Alshahrani Submitted to the Graduate Faculty of the School of Dentistry in partial fulfillment of the requirements for the degree of Master of Science in Dentistry, Indiana University School of Dentistry, March 2019. ii Thesis accepted by the faculty of the Department of Operative Dentistry, Indiana University, in partial fulfillment of the requirements for the degree of Master of Science in Dentistry. Frank Lippert Richard L. Gregory Chair of the Committee Norman Blaine Cook Program Director Date iii DEDICATION iv I dedicate this work to the Almighty Allah for his blessings, graces and virtues. In addition, I dedicate this work to my beloved parents, without whose prayers and support I would never have been who I am today, and to my lovely brothers and sisters for their love and encouragement during my studies. Lastly, I dedicate this work to my mentors and instructors throughout all my schooling. v ACKNOWLEDGMENTS vi My appreciation and thanks go to my research committee – Dr. Richard L. Gregory, Dr. Blaine Cook, and Dr. Frank Lippert – for their guidance and encouragement throughout the conduct of my research. Without their knowledge and thoughtful advice, this research project would never have been completed. I would like to extend thanks to Mr. George Eckert and Ms. Sruthi for their help in the biostatistics section. I am grateful to my amazing and loving parents for their endless support and encouragement. I would like to thank my wonderful brothers and sisters for their love and encouragement. Many thanks go to all my supportive friends and fellow residents. Finally, I extend my profound thanks to the government of the Kingdom of Saudi Arabia and to my university, the King Khalid University, for sponsoring me and for providing all I needed during this program. vii TABLE OF CONTENTS viii Introduction ....................................................................................................................1 Review of Literature ......................................................................................................7 Materials and Methods .................................................................................................19 Results ..........................................................................................................................24 Figures and Tables .......................................................................................................29 Discussion… ................................................................................................................51 Summary and Conclusion… ........................................................................................57 References ....................................................................................................................59 Abstract ........................................................................................................................70 Curriculum Vitae ix LIST OF ILLUSTRATIONS x FIGURE 1 S. mutans UA159 was grown in TSB at 37° C in 5% CO2 for 24 hours and stored with 10% glycerol at 80° C....................... 30 FIGURE 2 Cinnamon powder was mixed at 20 mg/ml with sterile deionized water, transferred into a test tube, placed in the autoclave and heated for 60 minutes at 121° C at 15 PSI of pressure.…................................................................................ 31 FIGURE 3 The sterility of the cinnamon solution was confirmed by streaking on a blood agar plate.……………………….…….... 32 FIGURE 4 The cinnamon solution was centrifuged (Beckman GS-6R Refrigerated Centrifuge) for 10 min.……………………......... 33 FIGURE 5 Cinnamon powder was mixed with sterile deionized water.… 34 FIGURE 6 Dilutions of 0, 0.25, 0.5, 1, 2, 4, 8, 16 and 32 mg/ml nicotine in TSBS with and without cinnamon water extract ……............ 35 FIGURE 7 Each nicotine concentration was aliquoted by pipetting 190 µl of TSBS containing the nicotine / cinnamon water extract into wells of a sterile 96-well flat bottom microtiter plate. Then, 10 µl of the fresh overnight TSB culture of S. mutans was added………………………………………………………… 36 FIGURE 8 Biofilm formation can be seen clearly in the control group (rows E, F, G and H/columns 1-7) and in the testing group (rows A, B, C and D/ columns 5-6) after 30 minutes following the application of formaldehyde…………………................... 37 FIGURE 9 Two hundred (200) µl of 0.5% crystal violet dye was added to each well and the biofilm cells stained for 30 min…………………………………………………………… 38 FIGURE 10 After crystal violet dye application, the wells were rinsed 3 times. The heavily stained wells are associated with more biofilm formation………………………………………….…. 39 FIGURE 11 Two hundred (200) µl of 2-isopropanol was placed into each well for 1 h to lyse the biofilm cells and extract the crystal violet…………………………………….……………………. 40 xi FIGURE 12 Microtiter plate wells were read using a spectrophotometer… 41 FIGURE 13 Effect of cinnamon water extract on total absorbance of S. mutans. Asterisks indicate significant differences (p < 0.05) compared to samples without cinnamon water extract……..... 42 FIGURE 14 Effect of cinnamon water extract on biofilm formation of S. mutans. Asterisks indicate significant differences (p < 0.05) compared to samples without cinnamon water extract.……… 43 FIGURE 15 Combined effect of cinnamon water extract and nicotine on S. mutans total absorbance……………………………….…..…. 44 FIGURE 16 Combined effect of cinnamon water extract and nicotine on S. mutans planktonic growth………………………...…………. 45 FIGURE 17 Combined effect of cinnamon water extract and nicotine on S. mutans biofilm growth……………………………….………. 46 TABLE I Basic statistics for S. mutans total absorbance, by cinnamon water extract and nicotine concentration…………...………… 47 TABLE II Basic statistics for S. mutans planktonic growth, by cinnamon water extract and nicotine concentration…………………….. 48 TABLE III Basic statistics for S. mutans biofilm growth, by cinnamon water extract and nicotine concentration…………………….. 49 TABLE IV A two-way ANOVA test comparing the effects of cinnamon water extract, nicotine (concentrations ranging from 0 mg/ml to 32 mg/ml), and their interaction on S. mutans biofilm, planktonic cells, and total absorbance……………………….. 50 1 INTRODUCTION 2 Dental caries is considered one of the most widespread chronic bacterial infections in the world.1 Streptococcus mutans and Lactobacilli are considered the main bacteria involved in dental caries. However, other bacteria are involved in the caries process such as Actinomycetes and Veillonella species. The etiology of dental caries is attributed to differences in eating habits, especially sugar consumption, oral hygiene practices, the virulence of oral bacteria, and alterations in the oral protective mechanisms. Extracellular polysaccharide (EPS), composed of glucans, are an extracellular layer produced by oral streptococci in the presence of sucrose that enhances the adhesion and aids in dental plaque biofilm formation. S. mutans glucans are produced as a result of the cooperative effects of glucosyltransferase (GTF) and glucan-binding proteins (GBP).2 Tooth demineralization is primarily due to lactic acid that is produced specifically by cariogenic bacteria present in dental plaque.3 According to a 2003 World Oral Health report, 60 percent to 90 percent of schoolchildren and the majority of adults have experienced dental caries. Moreover, dental caries is a major public health concern in other countries, such as in Asian countries and Latin America.4 In 2007 around 91percent of adults in the US older than 20 years had dental caries and 27 percent had untreated carious lesions.5 A study by Dye et al. showed that one in five children aged five years to 11 years and one in seven adolescents aged 11 years to 19 years have at least one untreated dental caries lesion.6 Previous data showed that about 38 percent of children aged two years through eight years in the US had some dental caries, and 14 percent had untreated dental caries.7 3 Dental caries is a dynamic process of demineralization and remineralization cycles that affects the quantity of minerals in the tooth structure. If demineralization prevails, loss of minerals from the enamel occurs and teeth may become cavitated. However, if remineralization prevails, the lesion can be arrested, and teeth can gain the minerals that were lost during the demineralization process.8,9 It was proven that non-cavitated lesions could be remineralized without the need for tooth drilling and filling.10 Many approaches have been used to arrest or reverse the demineralization process. Fluoride is considered the most commonly used anti-caries agent. Studies indicate that it is effective in decreasing the amount of caries incidence around the world.11,12 In a systematic review of 70 clinical trials that compared fluoride dentifrice with a placebo, it was concluded that fluoride is capable of decreasing the DMFS score by 24 percent in permanent teeth.13 Fluoride has two modes of action, systemic and topical.14-16 During tooth development, systemic fluoride is incorporated into the tooth structure.16,17 After tooth formation is completed, fluoride is absorbed into the crystalline structure of the tooth through topical agents.16 Moreover, casein phosphopeptide (CPP) amorphous calcium-phosphate (ACP) complexes have shown the ability to inhibit demineralization and to enhance the remineralization
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