Natural Oral Care in Dental Therapy Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Publishers at Scrivener Martin Scrivener (martin@scrivenerpublishing.com) Phillip Carmical (pcarmical@scrivenerpublishing.com) Natural Oral Care in Dental Therapy Edited by Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah and Nagendra Singh Chauhan This edition first published 2020 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2020 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. 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Library of Congress Cataloging-in-Publication Data Names: Chauhan, Durgesh Nandini, editor. | Singh, Prabhu Raj, editor. | Shah, Kamal, editor. | Chauhan, Nagendra Singh, editor. Title: Natural oral care in dental therapy / edited by Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah and Nagendra Singh Chauhan. Description: Hoboken, New Jersey : Wiley-Scrivener, [2020] | Includes bibliographical references and index. Identifiers: LCCN 2019050429 (print) | LCCN 2019050430 (ebook) | ISBN 9781119614227 (cloth) | ISBN 9781119618935 (adobe pdf) | ISBN 9781119618904 (epub) Subjects: MESH: Mouth Diseases--drug therapy | Plants, Medicinal | Mouth Diseases--prevention & control | Dental Care | Medicine, Traditional Classification: LCC RK305 (print) | LCC RK305 (ebook) | NLM WU 166 | DDC 617.6/306--dc23 LC record available at https://lccn.loc.gov/2019050429 LC ebook record available at https://lccn.loc.gov/2019050430 ISBN 978-1-119-61422-7 Cover image: Pixabay.Com Cover design by Russell Richardson Set in size of 11pt and Minion Pro by Manila Typesetting Company, Makati, Philippines Printed in the USA 10 9 8 7 6 5 4 3 2 1 Contents Preface Foreword xix xxiii Part I: Natural Oral Care 1 1 3 2 3 Natural Oral Care in Dental Therapy: Current and Future Prospects Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah and Nagendra Singh Chauhan 1.1 Introduction 1.2 Safety of Natural Oral Care 1.3 Advantage of Natural Oral Care 1.4 Limitations of Natural Oral Care 1.5 Future Prospects of Natural Oral Care References 3 15 15 16 16 17 Herbal Products for Oral Hygiene: An Overview of Their Biological Activities Ummuhan Sebnem Harput 2.1 Introduction 2.2 Oral Hygiene and Current Treatments 2.3 Plants Traditionally Used in Oral Hygiene 2.4 Clinically Studied Plant Product for Oral Hygiene 2.5 In Vitro Studied Herbal Product for Oral Hygiene 2.6 Discussion 2.7 Conclusion References 31 Go Green—Periodontal Care in the Natural Way Siddhartha Varma and Sameer Anil Zope 3.1 Introduction 3.2 Plaque Control 3.3 Dant Dhavani (Brushing) 3.4 Jivha Lekhana (Tongue Scrapping) 3.5 Gandusha (Gargling) or Oil Pulling 3.6 Oxidative Stress in Periodontitis 3.7 Green Tea 45 31 33 33 35 37 40 41 41 45 46 46 47 48 48 48 v vi Contents 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 3.17 3.18 3.19 3.20 3.21 3.22 3.23 3.24 3.25 3.26 3.27 3.28 3.29 4 3.7.1 Components 3.7.2 Beneficial Effects of Various Tea Components 3.7.2.1 Antioxidative Effect 3.7.3 Role in Managing Periodontitis Turmeric (Curcumin longa, Haldi) 3.8.1 Applications of Turmeric in Dentistry Amala (Emblica officinalis, Amalaki, Phyllanthus emblica, Indian Gooseberry, Dhatriphala) Anar/Dalima (Punica granatum) Launga/Clove (Syzygium aromaticum) Gotu Kola (Centella asiatica) Amra/Mango (Magnifera indica) Neem (Azadirachta indica) Tulsi (Ocimum sanctum) Nilgiri (Eucalyptus globulus) Tila/Sesame (Sesamum indicum) Triphala Tea Tree Oil (Melaleuca Oil) Rumi Mastagi/Mastic Gum (Pistacia lentiscus) Wheat Grass Goldenseal (Hydrastis canadensis) Licorice Root Myrrh (Commiphora glileadenis) Psidium guajava Ginkbo Biloba Honey Other Herbs Which Can Be Potentially Used for Treating Periodontitis Conclusion References Role of Herbal and Natural Products in the Management of Potentially Malignant Oral Disorders P. Kalyana Chakravarthy, Komal Smriti and Sravan Kumar Yeturu 4.1 Introduction 4.2 Oral Submucous Fibrosis (OSMF) 4.2.1 Background 4.2.2 Beta-Carotene 4.2.3 Lycopene 4.2.4 Aloe Vera 4.2.5 Colchicine 4.2.6 Tea Pigments 4.2.7 Spirulina 4.2.8 Chinese Herbal Medicines 4.2.9 Turmeric and Derivatives, Nigella sativa, Ocimum 4.2.10 Polyherbal Formulations 4.2.11 Ayurvedic Formulations 4.2.12 Conclusion 48 49 49 49 49 49 50 50 50 51 51 51 51 52 52 52 52 53 53 53 53 54 54 54 54 55 55 56 61 61 62 62 63 64 65 66 66 66 67 68 68 69 69 Contents 4.3 Oral Leukoplakia (OL) 4.3.1 Background 4.3.2 Green Tea and Extracts 4.3.3 Beta-Carotene (βC) 4.3.4 Lycopene 4.3.5 Curcumin 4.3.6 Miscellaneous 4.3.6.1 Alpha-Tocopherol 4.3.6.2 Chinese Herbs 4.3.6.3 Bowman–Birk Inhibitor Concentrate (BBIC) 4.3.7 Conclusion 4.4 Oral Lichen Planus (OLP) 4.4.1 Conclusion References Part II: Studies of Plants Used in Dental Disease 5 6 Studies on the Anticariogenic Potential of Medicinal Plant Seed and Fruit Extracts Disha M. Patel, Jenabhai B. Chauhan and Kalpesh B. Ishnava 5.1 Introduction 5.2 Materials and Methods 5.2.1 Plant Materials 5.2.2 Preparation of Plant Seed and Fruit Extracts 5.2.3 Cariogenic Bacterial Strains 5.2.4 Preparation of Inoculums 5.2.5 Anticariogenic Activity Screening of Plant Extracts 5.2.5.1 Agar Well Diffusion Assay 5.2.5.2 Determination of Minimum Inhibitory Concentration (MIC) 5.2.6 Preliminary Phytochemical Analysis 5.2.7 Analytical Thin Layer Chromatography 5.2.8 TLC—Bioautography 5.3 Result and Discussion 5.3.1 MIC Value of Effective Plant Extracts 5.3.2 Phytochemical Screening and Bioautography 5.4 Conclusion Acknowledgments References Cytotoxic and Anti-Inflammatory Effect of Turmeric and Aloe Vera in a Gingivitis Model Karen Esperanza Almanza-Aranda, Miguel Aranda-Fonseca, Gabriela Velazquez-Plascencia and Rene Garcia-Contreras 6.1 Introduction 6.2 Gingivitis and Periodontitis 6.3 Aloe Vera 6.3.1 Aloe Vera for Gingivitis and Periodontitis 6.3.2 Aloe Vera: Other Oral Applications vii 70 70 70 71 72 72 73 73 73 73 73 74 75 75 81 83 83 85 85 85 85 86 87 87 87 87 87 88 88 91 92 94 95 95 97 97 98 99 100 100 viii Contents 6.4 Turmeric 6.4.1 Turmeric for Gingivitis and Periodontitis 6.4.2 Turmeric: Other Oral Applications 6.5 Methodology 6.5.1 Materials and Methods 6.5.1.1 Authorization 6.5.1.2 Cell Culture 6.5.1.3 Cell Subculture 6.5.1.4 Cytotoxicity Test 6.5.1.5 Anti-Inflammatory Activity in a Gingivitis Model 6.5.1.6 Statistical Analysis 6.5.2 Results 6.5.2.1 Cytotoxicity 6.5.2.2 Anti-Inflammatory Activity in a Gingivitis Model 6.5.3 Discussion 6.5.3.1 Cytotoxicity 6.5.3.2 Anti-Inflammatory Activity 6.6 Perspectives for the Future 6.7 Conclusions References 7 Effects of Bauhinia forficata Link in Reducing Streptococcus mutans Biofilm on Teeth Julio Cesar C. Ferreira-Filho, Mariana Leonel Martins, Andressa Temperini de Oliveira Marre, Juliana Soares de Sá Almeida, Leandro de Araújo Lobo, Adriano Gomes Cruz, Marlon Máximo de Andrade, Thiago Isidro Vieira, Maria Teresa Villela Romanos, Lucianne Cople Maia, Ana Maria Gondim Valença and Andréa Fonseca-Gonçalves 7.1 Introduction 7.2 Materials and Methods 7.2.1 Recognition, Production, and Chemical Characterization of Ethanolic Tincture From B. forficata L. Leaves 7.2.2 Microbial Strains and Preparation of Inoculum 7.2.3 Minimum Inhibitory Concentration and Minimum Bactericidal Concentration (MBC) 7.2.4 Kill-Kinetic Assay 7.2.5 Cytotoxic Potential 7.2.6 Tooth Selection and Preparation for Microbiologic Assay Using an S. mutans Biofilm 7.2.7 Statistical Analysis 7.3 Results and Discussion 7.4 Final Considerations Acknowledgments References 100 101 101 102 102 102 102 102 103 103 104 104 104 105 105 105 106 107 107 107 111 112 112 112 113 113 113 114 114 115 115 118 118 119 Contents 8 9 Antimicrobial Effect of a Cardamom Ethanolic Extract on Oral Biofilm: An Ex Vivo Study Marina Fernandes Binimeliz, Mariana Leonel Martins, Julio Cesar Campos Ferreira Filho, Lucio Mendes Cabral, Adriano Gomes da Cruz, Lucianne Cople Maia and Andréa Fonseca-Gonçalves 8.1 Introduction 8.2 Materials and Methods 8.2.1 Cardamom Extract Production 8.2.2 Physical Analyses 8.2.3 Bacterial Strains and Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration 8.2.4 Salivary Collection for Biofilm Formation (Ex Vivo Experiment) 8.2.5 Biofilm Formation and Treatment 8.2.6 Statistical Analyses 8.3 Results and Discussion 8.4 Final Considerations Acknowledgment References Effect of Punica granatum Peel Extract on Growth of Candida albicans in Oral Mucosa of Diabetic Male Rats Maryam Eidi and Fatemeh Noorbakhsh 9.1 Introduction 9.2 Materials and Methods 9.2.1 Hydro-Methanolic Extract 9.2.2 Candida albicans Inoculation 9.2.3 Animal 9.2.4 Statistical Analysis 9.3 Results and Discussion 9.4 Conclusion Acknowledgment References Part III: Applications of Natural Products in Oral Care 10 Effect of Oil Pulling on Oral Health Sameer Anil Zope and Siddhartha Varma 10.1 Introduction 10.2 What Is Oil Pulling (Snaihik Gandoosh)? 10.3 How Does Oil Pulling Work? 10.4 Composition and Various Activities of Most Commonly Used Oils for Oil Pulling 10.4.1 Sesame Oil 10.4.1.1 Antioxidant Activity 10.4.1.2 Antimicrobial Activity ix 121 121 122 122 123 123 124 124 127 127 129 129 129 133 133 134 134 134 134 135 135 136 136 137 139 141 141 142 143 143 143 143 144 x Contents 10.4.2 Coconut Oil 10.4.2.1 Antibacterial, Antifungal, and Antiviral Activity 10.4.2.2 Antinociceptive, Anti-Inflammatory, Antioxidant, and Anti-Ulcer Activity 10.5 Procedure of Oil Pulling 10.6 Effects of Oil Pulling on Oral Health 10.6.1 Dental Caries 10.6.2 Plaque-Induced Gingivitis 10.6.3 Halitosis 10.6.4 Oral Thrush 10.6.5 Xerostomia and Burning Mouth Syndrome 10.7 Drawbacks of Oil Pulling References 145 145 145 146 146 146 147 148 149 149 150 150 11 Role of Proteolytic Enzymes in Dental Care P. Kalyana Chakravarthy and Sravan Kumar Yeturu 11.1 Introduction 11.2 Role of Proteolytic Enzymes in Oral Surgery 11.2.1 Post-Extraction Management 11.2.2 Post-Surgical Facial Ecchymosis and or Edema 11.2.3 Enhanced the Action of Antibiotics 11.2.4 Effect of Bromelain on Blood Coagulation and Fibrinolysis 11.3 Role of Proteolytic Enzymes in Cancer and Oral Mucositis 11.3.1 Cancer 11.3.2 Management in Oral Mucositis 11.4 Osteoarthritis 11.5 Anti-Microbial Action 11.6 Treatment of Dental Carious Lesions 11.6.1 Laboratory Studies 11.6.2 Clinical Studies 11.7 Improvement in Bonding of Orthodontics Brackets 11.8 Role on Biofilm Control (Plaque, Gingivitis, and Oral Malodor) 11.9 Extrinsic Stain Removal on the Teeth 11.10 Role in Replantation of the Avulsed Tooth 11.11 Effect of Bromelain on Immunogenicity 11.12 Other Possible Applications and Scope for Future Research References 153 12 The Effect of Probiotic on Oral Health Patricia Nadelman, Marcela Baraúna Magno, Mariana Farias da Cruz, Adriano Gomes da Cruz, Matheus Melo Pithon, Andréa Fonseca-Gonçalves and Lucianne Cople Maia 12.1 Introduction 12.2 Overview of Oral Communities and Probiotic-Based Therapy to Oral Dysbiosis 12.3 Probiotics Mechanisms of Action 12.4 Dental Caries 171 153 154 154 155 156 156 156 156 157 157 158 159 159 160 161 163 164 165 165 165 165 171 172 175 176 Contents xi 12.4.1 Definition and Etiopathology 12.4.2 Probiotics and Dental Caries 12.4.3 Probiotic-Contained Dairy Products and Dental Caries 12.4.4 Probiotic Powder and Dental Caries 12.4.5 Probiotic Tablets and Lozenges and Dental Caries 12.4.6 Probiotic Mouthwashes and Dental Caries Periodontal Disease 12.5.1 Definition and Etiopathology 12.5.2 Probiotics and Periodontal Diseases Oral Candidiasis 12.6.1 Definition and Etiopathology 12.6.2 Probiotics and Oral Candidiasis Halitosis 12.7.1 Definition and Etiopathology 12.7.2 Probiotics and Halitosis Conclusion Acknowledgments References 176 179 179 180 180 181 181 181 182 183 183 184 185 185 185 186 186 186 13 Charcoal in Dentistry Abhilasha Thakur, Aditya Ganeshpurkar and Anupam Jaiswal 13.1 Introduction 13.2 Charcoal Production Methods 13.2.1 The Traditional Method 13.2.2 The Modern Methods 13.3 Uses of Charcoal 13.3.1 Medicinal Uses 13.3.2 Non-Medicinal Uses 13.4 Charcoal Containing Oral and Dental Care Products 13.5 Benefits of Using Charcoal Containing Oral and Dental Care Products 13.5.1 Removes Stains and Whitens Teeth 13.5.2 Removes Acidic Plaque 13.5.3 Gives Fresh Breath and Improves Halitosis 13.5.4 Remineralize Teeth 13.5.5 Helps Overall Dental Health 13.5.6 Protects From Infection 13.5.7 Cost Effective for Regular Basis Use 13.6 Precautions to be Taken While Using Charcoal Containing Oral and Dental Care Products 13.7 Conclusion References 197 12.5 12.6 12.7 12.8 197 199 199 199 200 200 201 201 204 204 204 204 205 205 205 205 206 207 207 xii Contents 14 Propolis Benefits in Oral Health Mariana Leonel Martins, Karla Lorene de França Leite, Yuri Wanderley Cavalcanti, Lucianne Cople Maia and Andréa Fonseca-Gonçalves 14.1 Introduction 14.2 Types of Propolis 14.2.1 Brown Propolis 14.2.2 Green Propolis 14.2.3 Red Propolis 14.3 Biological Properties of Propolis 14.3.1 Oral Antibacterial Activity 14.3.2 Oral Antifungal Activity 14.4 Other Biological Properties of Propolis 14.4.1 Anti-Inflammatory Activity 14.4.2 Antioxidant Activity 14.4.3 Anticancer Activity 14.5 Benefits for Oral Health and Applications in Dentistry 14.6 Final Considerations Acknowledgment References 211 15 Grape Seed Extract in Dental Therapy Anusuya V, Ashok Kumar Jena and Jitendra Sharan 15.1 Introduction 15.2 Part I: Basics About Grape Seed Extracts 15.2.1 Components of Grape Seed Extracts 15.2.2 Chemical Structure 15.2.3 Types of GSEs 15.2.4 Methods of Separation 15.2.5 Factors Influencing the Quality and Quantity of Polyphenols in the GSEs 15.2.6 Physical Properties of Polyphenols 15.2.7 Biochemical Properties (Biological and Pharmacological) 15.3 Part II: Biological Applications in Dentistry 15.3.1 GSEs in Dental Caries 15.3.2 Anti-Erosive Agent (Prevention of Enamel erosion) 15.3.3 Antiplaque Effect 15.3.4 Antibacterial Agent 15.3.5 Biomodifier 15.3.6 GSEs as a Remineralizing Agent—Existing Dilemma 15.4 GSEs in Restorative Dentistry 15.4.1 GSE as Cross-Linking Agent 15.4.2 GSE in Bonding 15.5 GSEs in Endodontic Treatment 15.5.1 Endodontic Irrigants 15.5.2 Post Endodontic Restorations 15.6 GSEs in Periodontics 229 211 213 213 214 214 215 216 219 220 220 221 221 221 222 223 223 229 230 230 231 232 232 234 235 236 240 240 242 243 244 245 247 248 248 249 250 250 251 251 Contents xiii 15.6.1 Anti-Inflammatory Action in Periodontitis 15.6.2 Anti-Oxidative Action in Periodontitis 15.6.3 Antibacterial Action Against Periodontal Pathogens 15.6.4 Antimicrobial Activity in Peri-Implantitis 15.7 GSEs in Oral Cancer 15.8 Conclusion References 16 Ocimum Sanctum L: Promising Agent for Oral Health Care Management Trinette Fernandes, Anisha D’souza and Sujata P. Sawarkar 16.1 Introduction 16.2 History of Ocimum sanctum 16.3 Chemical Constituents of Ocimum sanctum 16.4 Therapeutic Significance of Ocimum in Dental Health and Preventive Care Management 16.5 Novel Drug Delivery Formulations and Its Application in Dentistry 16.5.1 Nanofibers 16.5.2 β-Cyclodextrin Complexes 16.5.3 Nanoparticles of Biocompatible Ocimum sanctum-Coated Silver Nanoparticles 16.6 Conclusion References 17 Coconut Palm (Cocos nucifera L.): A Natural Gift to Humans for Dental Ministrations Navneet Kishore and Akhilesh Kumar Verma 17.1 Introduction 17.2 Traditional Usage and Ethnopharmacological Relevance 17.3 Pharmacological Properties of Coconut 17.4 Role of Coconut Tree in Dental Ministrations 17.5 Exemplary Potential of Coconut Water in Dentistry 17.6 Other Significance of Coconut 17.6.1 Economic Value of Coconut Leaves 17.6.2 Use of Coconut Heart 17.6.3 Significance of Spathe and Inflorescence 17.6.4 Potential of Coconut Fruits 17.6.5 Usage of Coconut Milk 17.6.6 Importance of Coconut Shell 17.6.7 Commercial Usage of Husk Fibers 17.6.8 Economic Importance of Coconut Stems 17.6.9 Convention of Coconut Roots 17.7 Active Constituent Identified from Coconut 17.8 Future Prospective 17.9 Conclusions Acknowledgments References 252 252 253 253 254 254 255 259 259 260 260 262 264 264 264 264 265 266 271 271 272 273 274 275 276 276 277 277 277 277 277 278 278 278 278 279 280 280 281 xiv Contents 18 Salvadora persica L. (Miswak): An Effective Folklore Toothbrush Sujata P. Sawarkar, Anisha D’souza and Trinette Fernandes 18.1 Introduction 18.2 History 18.3 Chemical Constituents 18.4 Extraction, Isolation, Identification of Chemical Constituents 18.5 Pharmacology—Therapeutic Activity of Salvadora persica L. 18.5.1 Theories for Miswak Activities 18.5.2 Antibacterial and Antifungal 18.5.3 Anti-Viral Effect 18.5.4 Anti-Cariogenic Effect 18.5.5 Antiplaque Effect 18.5.6 Antiperiodontitis Effect 18.5.7 Whitening Effect 18.6 Conclusion References 285 19 Triphala and Oral Health Kamal Shigli, Sushma S Nayak, Mrinal Shete, Vasanti Lagali Jirge and Veerendra Nanjwade 19.1 Introduction 19.2 Taxonomical Classification 19.3 Chief Phytoconstituents 19.4 Role of Triphala in Dentistry 19.4.1 Anti-Caries Activity 19.4.2 Triphala as a Root Canal Irrigant 19.4.3 Anti-Microbial and Anti-Oxidant Effect of Triphala 19.4.4 Role of Triphala in Periodontal Diseases 19.4.5 Triphala as a Mouth Rinse 19.4.6 Anti-Candida Activity of Triphala 19.4.7 Anti-Collagenase Activity of Triphala 19.5 Pharmacological Activities of Triphala and Future Research 19.5.1 Anticancer and Antioxidant Activity of Triphala 19.5.2 Wound Healing Properties 19.5.3 Antibacterial Activity of Triphala 19.5.4 Anti-Diabetic Effect 19.5.5 Anti-Inflammatory, Analgesic, and Antipyretic Effect 19.5.6 Immunomodulatory Effect 19.6 Public Health Importance 19.7 Formulation Using Triphala 19.6 Conclusion References 297 285 286 286 287 287 287 288 290 290 290 290 291 292 292 297 298 298 300 300 300 306 306 306 306 306 307 307 307 307 307 307 308 308 308 308 309 Contents xv 20 Azadirachta indica (Neem): An Ancient Indian Boon to the Contemporary World of Dentistry Sri Chandana Tanguturi, Sumanth Gunupati and Sreenivas Nagarakanti 20.1 Introduction 20.2 Vital Bioactive Compounds of Neem 20.2.1 Nimbidin 20.2.2 Azadirachtin 20.2.3 Nimbolide 20.2.4 Gedunin 20.2.5 Mahmoodin 20.2.6 Tannins 20.2.7 Diterpenoids 20.3 How to Distinguish Azadirachta Indica (Neem) from its Common Adulterant Melia Azedarach 20.4 Therapeutic Applications of Neem 20.4.1 Neem as an Anti-Inflammatory, Analgesic Agent 20.4.2 Antioxidant Activity 20.4.3 Anticancerous Activity 20.4.4 Antimicrobial Activity 20.4.4.1 Antibacterial Activity 20.4.4.2 Antiviral Activity 20.4.4.3 Antifungal Activity 20.4.4.4 Antimalarial Activity 20.4.5 Wound Healing Effect 20.5 Applications of Neem in Dentistry 20.5.1 Neem in Treatment of Periodontal Diseases 20.5.2 Role of Neem in Endodontics 20.5.3 Potent Role of Neem in Preventive Dentistry 20.5.3.1 Application in Dental Erosion Therapy 20.5.3.2 Anti-Microbial Activity 20.5.3.3 Anticaries Activity of Neem 20.5.3.4 Anti-Candidiasis Property 20.5.3.5 Anti-Cancer Property 20.6 Literature Supporting the Use of Neem in Dentistry 20.7 Toxicity and Safety 20.8 Contamination and Adulteration 20.9 Drug Interactions 20.10 Neem’s Prospects in Dentistry 20.11 Action Points and Recommendations for Health Care Professionals 20.12 Conclusion References 21 Ginger in Oral Care Aditya Ganeshpurkar, Abhilasha Thakur and Anupam Jaiswal 21.1 Introduction 21.2 Description 313 313 314 314 315 315 315 315 315 315 316 316 317 317 317 318 318 318 318 318 318 318 319 319 320 320 320 320 321 321 321 322 322 322 323 323 323 324 329 329 330 xvi Contents 21.3 21.4 21.5 21.6 Macroscopic Characteristics Pharmacognostic Standards Nutrient Composition Pharmacological and Medicinal Effects 21.6.1 Oral Analgesic Effect 21.6.2 Antimicrobial Effect 21.6.3 Anti-Carries Activity 21.6.4 Anti-Decay Effect 21.6.5 Healing Effect in Root Canal Therapy 21.6.6 Anti-Xerostomia Effect 21.6.7 Anti-Pyorrhea Effect 21.6.8 Anti-Thrush Effect 21.6.9 Anti-Herpes Effect 21.6.10 Tooth Polishing 21.6.11 Mouth Deodorizing Effect 21.6.12 Anticancer Effect 21.6.13 Protection Against Aphthous Stomatitis 21.6.14 Effect on Dentin Hardness 21.7 Pharmacokinetics 21.8 Toxicological Studies 21.9 Conclusion References 22 Effectiveness of Allium sativum on Bacterial Oral Infection Vesna Karic, Anupam Jaiswal, Heidi Abrahamse, Abhilasha Thakur and Aditya Ganeshpurkar 22.1 Introduction 22.1.1 History and Origin of Garlic 22.1.2 Medicinal Values of Garlic 22.2 Types of Allium sativum 22.2.1 Allium sativum Ophisocorodon/Hard-Necked Garlic 22.2.2 Allium sativum Sativum/Soft-Necked Garlic 22.3 Chemical Constituents 22.3.1 Allicin 22.3.2 Ajoenes 22.3.3 Alliin 22.4 Dental Infections and Epidemiology 22.5 Dental Infection and Antibiotic Resistance 22.6 The Antibacterial Application of Garlic in Dentistry 22.6.1 The Use of Garlic to Treat Oral Infections 22.6.1.1 Periodontitis 22.6.1.2 Pediatric Endodontitis 22.6.1.3 Dental Caries 22.6.1.4 Denture Stomatitis 22.6.1.5 Protection Against Fibrosis 22.6.1.6 Garlic Chewing Gum 22.6.1.7 Garlic Used as a Breath-Freshening Agent 330 330 331 331 331 332 333 333 334 334 335 335 336 336 336 338 338 338 339 339 339 340 345 345 347 348 349 349 349 351 351 351 351 352 352 354 354 354 356 357 358 359 359 359 Contents 22.7 Additional Use of Garlic in Dentistry—Oral Cancer 22.7.1 High Blood Pressure 22.7.2 Skin Disorders 22.7.3 Anti-Allergic 22.7.4 Anti-Obesity 22.8 Garlic Mechanism of Action 22.9 Conclusions and Recommendations Acknowledgments References Part IV: Ethnobotany and Ethanopharmacology xvii 360 361 362 362 362 362 362 364 364 371 23 Curative Plants Worn in the Healing of Mouth Evils P. Shivakumar Singh, Pindi Pavan Kumar and D. Srinivasulu 23.1 Introduction 23.2 Materials and Methods 23.3 Results and Discussion 23.4 Conclusion Acknowledgment References 373 24 Ethnopharmacological Applications of Chewing Sticks on Oral Health Care E. A. Akaji and U. Otakhoigbogie 24.1 Introduction 24.1.1 Background 24.1.2 Historical Perspectives 24.1.3 Sources and Types of Chewing Sticks 24.2 Applications of Chewing Sticks in Oral Health Care 24.2.1 Chewing Sticks for Oral Hygiene 24.2.2 Ethnopharmacological Applications of Chewing Sticks in Oral Health 24.2.2.1 Dental Caries (Tooth Decay) 24.2.2.2 Periodontal Disease 24.2.2.3 Oral Candidiasis 24.2.2.4 Oral Ulcers and Halitosis 24.2.2.5 Other Oral Conditions 24.3 Conclusions References 383 25 Ethnomedicine and Ethnopharmacology for Dental Diseases in Indochina Viroj Wiwanitkit 25.1 Introduction 25.2 Ethnomedicine and Ethnopharmacology in Indochina 25.3 Locally Available Naturally Derived Dental Products in Indochina 25.4 Ethnopharmacology for Dental Diseases in Indochina 25.5 Ethnomedicine for Dental Diseases in Indochina 393 373 374 375 381 381 381 383 383 384 384 384 384 387 387 389 389 390 390 390 391 393 394 396 397 402 xviii Contents 25.6 Future Trend of Ethnomedicine and Ethnopharmacology for Dental Diseases in Indochina 25.7 Conclusion References 403 404 404 26 Traditional Medicinal Plants Used in Anti-Halitosis P. Shivakumar Singh, Pindi Pavan Kumar and D. Srinivasulu 26.1 Introduction 26.2 Materials and Methods 26.3 Results and Discussion 26.4 Conclusion Acknowledgment References 407 Index 415 407 408 409 412 413 413 Preface For hundreds of years now the existence and utility of natural products have bolstered the idea that natural products are still the choice for therapy. Their structural diversity and exclusive pharmacological actions differentiate them from drugs of synthetic origin. Not only are they used in traditional ways but also in modern medicine for treating lifethreatening diseases. The discovery and design of new drugs from natural products always remain a challenging task. Still, they remain a choice due to their safety profile and negligible side effects. This book focuses on perspectives of natural medicine in various dental diseases. Oral diseases continue to be a major health problem worldwide. Oral health is integral to general well-being and relates to the quality of life that extends beyond the functions of the craniofacial complex. Standard Western medicine has had only limited success in the prevention of periodontal disease and treatment of a variety of oral diseases. The dentist needs to be more informed regarding the use, safety, and effectiveness of the various traditional medicines and over-the-counter products. For example, herbal extracts have been used in dentistry for reducing inflammation, as antimicrobial plaque agents, for preventing the release of histamine and as antiseptics, antioxidants, antimicrobials, antifungals, antibacterials, antivirals, and analgesics. They also aid in healing and are effective in controlling microbial plaque in gingivitis and periodontitis, thereby improving immunity. The 26 chapters of this book cover the chemistry, clinical and preclinical panorama of natural products used in oral care. In Chapter 1, Chauhan et al. provide an exhaustive list of natural oral care products used in oral diseases and classify them according to use and chemistry. Chapter 2, contributed by Harput, discusses the crucial role that oral hygiene plays in the prevention of oral diseases, including periodontitis, tooth decay, and oral candidiasis. In Chapter 3, Varma and Zope highlight the herbs and their extracts used as an adjuvant in periodontal disease treatment. Chapter 4, written by Chakravarthy et al., focuses on various modalities currently available and discusses the efficacy and safety of such herbal products and natural extracts in the management of potentially malignant oral disorders. In Chapter 5, Patel et al. discuss experimental studies that show the promising broad spectrum anticariogenic activity of ethyl acetate and methanolic extract of Quercus infectoria that may prove useful for the clinical evaluation and development of a formulation suitable for the treatment of dental caries. Chapter 6, by Almanza-Aranda et al., reports that turmeric and Aloe vera in culture with HGF show promising potential and have clinical use for patients with gingivitis and periodontitis. In a study reported in Chapter 7, Ferreira-Filho et al. evaluate the effects of a tincture made from Bauhinia forficata Link leaves (TBF) on Streptococcus mutans biofilm formed on teeth. In Chapter 8, Binimeliz et al. demonstrate the effect of an Elettaria cardamomum ethanolic extract (ECE) against oral biofilm bacteria through an in vitro study. Chapter 9, contributed by Eidi and Noorbakhsh, xix xx Preface reports on the effect of Punica granatum peel extract on the growth of Candida albicans in the oral mucosa of diabetic male rats. In Chapter 10, Zope and Varma give an overview of the evidence-based use of oil pulling therapy in the maintenance of oral health. The focus of Chapter 11 by Chakravarthy and Yeturu is plant-based proteolytic enzymes, including papain, bromelain, actinidin, and ficin; as well as the application of these enzymes in professional as well as personal oral health care. The aim of Chapter 12 by Nadelman et al. is to present an updated viewpoint of the effect of probiotics on oral health by describing the relationship between the administration/consumption of these bacteria and the main oral dysbiosis, the oral microbiota parameters, and the immune salivary components. Chapter 13, written by Thakur, highlights the role of charcoal in dentistry and the development of charcoal-based dentifrices. In Chapter 14, Martins et al. present an updated review of the benefits of propolis in oral health, and the subject of Chapter 15 by Anusuya et al. is the role of grape seed extract in dentistry. In Chapter 16, Fernandes et al. discuss the use of Ocimum sanctum L for oral care and halitosis, which, because of its anticariogenic properties, is also used in periodontal diseases such as periodontitis and gingivitis. This chapter not only discusses its conventional formulation but also O. sanctum L formulations loaded in a promising novel drug delivery system. Chapter 17, authored by Kishore and Verma, presents the latest information regarding traditional, ethnopharmacological, and bioactive phytochemicals and the significant use of coconut plant in oral cavity therapies. In Chapter 18, Sawarkar et al. discuss the use of miswak for maintaining oral health and its various commercial products. Shigli et al. write about triphala in Chapter 19 and demonstrate its traditional use and newer pharmacologic activities for use as an adjuvant mainstream drug in dentistry as well as in general medicine. The role that Azadirachta indica (neem) plays in dentistry is revealed by Tanguturi et al. in Chapter 20. In Chapter 21, Ganeshpurkar reveals the lethal effect ginger has on the growth of tooth-decaying bacteria since it is a good dental analgesic and promotes dentine remineralization. Moreover, the antiplaque and mouthdeodorizing effects of ginger are also well documented. The aim of Chapter 22 by Karic et al. is to raise awareness on the use of garlic formulations to reduce the risk of oral disease and dental caries. In Chapter 23, Singh and Rao discuss the traditional uses of plants in the treatment of mouth evils at Kosgimandal of Naryanapet District of Telangana State, India. Chapter 24 by Akaji and Otakhoigbogie reveals the healing power of plant materials, such as a chewing stick, primarily used for oral hygiene. In Chapter 25, Wiwanitkit discusses ethnomedicine and ethnopharmacology for dental diseases in Indochina, a tropical region in Asia, and Chapter 26 by Singh and Reddy enlists traditional medicinal plants with antihalitosis efficacy. This important new volume will be valuable to dentists, oral hygienists, pharmacognosy experts, and natural product formulation scientists alike, either as a textbook or a reference. It is a must-have addition to any dental or herbal industry library. The aim of this book is to be a reference for all those interested in the development of natural oral care as an alternative treatment. Preface xxi Last, but not the least, we would like to express our sincere gratitude to all the authors who have taken time out from their busy schedules to be part of this project and have written wonderful chapters that add both to the depth and value of this book. We welcome suggestions and criticisms from our readers. We also acknowledge our mentor Prof. V.K. Dixit Sir for his valuable guidance. Special thanks to our families for their support and encouragement. We express our gratitude to the publishing and production team, especially Martin Scrivener, for their kind, proficient, and encouraging guidance. Durgesh Nandini Chauhan Prabhu Raj Singh Kamal Shah Nagendra Singh Chauhan December 2019 Foreword Teeth are unique in the fact that, on one hand, they live for thousands of years and are often encountered in archeologic excavations, and on the other hand, we see rampant caries destroying almost all teeth in children. Dental and oral health remains a definitive part of one’s overall health and wellbeing and helps one present oneself with confidence. The care of the oral cavity has been described as early as the Sushruta Samhita and in Hippocrates’ works. With evolution, there has been a constant change in the environmental factors, the food, oral hygiene measures, as well as products used in dental care. As we look back in time, one can appreciate that the traditional methods of oral care and their incorporation in current measures may be the way forward to sustainable dental and oral health in the future. This book entitled Natural Oral Care in Dental Therapy by Chauhan et al. has come a long way in establishing this connection. Prevention is better than cure is a dictum that has repeatedly established itself. Modern dental science encourages oral hygiene measures based on mechanical and chemical cleansing, with continuous research on the chemical agents. Time and again, those agents and natural remedies described in history are proving to be fruitful. There are lots of research activities that have been started by academic institutions and research centers along with their industrial partners, for the development of natural product formulation in dental therapy. Thus, this book is a timely reference work for the scientific community. Natural Oral Care in Dental Therapy is an applications-oriented book in the field of dental science. The book is a perfect resource for dentists, oral hygienists, herbal experts, phytochemists, research professionals, and technology investors. It is edited by an experienced and interdisciplinary group comprising a well-respected dentist, clinician, natural product, and pharmacology expert. I hope this book will inspire many current and future generations of academic and industrial researchers to expand the use of natural products in dentistry. Dr. Santhosh Rao BDS, MDS, FIBOMS Oral & Maxillofacial Surgeon Associate Professor Department of Dentistry All India Institute of Medical Sciences, Raipur, India August 2019 xxiii Part I NATURAL ORAL CARE 1 Natural Oral Care in Dental Therapy: Current and Future Prospects Durgesh Nandini Chauhan1*, Prabhu Raj Singh2, Kamal Shah3 and Nagendra Singh Chauhan4 1 Columbia Institute of Pharmacy, Raipur, Chhattisgarh, India MKD Muti Speciality Dental Clinic, Annupur, Madhy Pradesh, India 3 Institute of Pharmaceutical Research, GLA University, Chaumuha, Mathura, (U.P.) India 4 Drugs Testing Laboratory Avam Anusandhan Kendra, Raipur Chhattisgarh, India 2 Abstract Nowadays, natural medicines like honey, clove, miswak, and propolis are a part of dental treatment due to their reduced toxicity, wide availability, and cost effectiveness. This chapter gives an insight to the reader about the potential use of natural products in current dentistry. They are in many forms and include chewing sticks, oils, herbal extracts, minerals, animal products (e.g., honey), herbs, herbal materials, herbal preparations, and finished herbal products that contain parts of plants or other plant materials as active ingredients. Natural medicines hold huge benefits as adjunctive therapeutic uses in dentistry. Use of these techniques with suitable dosage would benefit the general population by preventing various dental problems. Keywords: Antiplaque, dentistry, antimicrobial, natural, oral health care, herbal, phytochemical, medicinal plants 1.1 Introduction Oral health is a key indicator of overall health, well-being, and quality of life. The World Health Organization (WHO) defines oral health as “a state of being free from chronic mouth and facial pain, oral and throat cancer, oral infection and sores, periodontal (gum) disease, tooth decay, tooth loss, and other diseases and disorders that limit an individual’s capacity in biting, chewing, smiling, speaking, and psychosocial well-being” [1]. There are seven major oral diseases and conditions reported for most of the oral disease burden. 1. Dental caries (tooth decay) 2. Periodontal (gum) diseases 3. Oral cancers *Corresponding author: pharmanandini@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (3–30) © 2020 Scrivener Publishing LLC 3 4 Natural Oral Care in Dental Therapy 4. 5. 6. 7. Oral manifestations of HIV Oro-dental trauma Cleft lip and palate Noma Oral diseases affect at least 3.58 billion people worldwide, with caries of the permanent teeth being the most common of all conditions assessed. Worldwide, it is estimated that 486 million children suffer from caries of primary teeth, and 2.4 billion people suffer from caries of permanent teeth as per study on the Global Burden of Disease Study 2016 [2]. Natural oral care rises to a growing trend in the increased use of “over-the-counter” dental product. Nowadays, there is a huge range of natural oral care products and technology available that are self used without consulting an expert. The potential use of natural product in current dentistry is obtained from plants, animals, marine animals, and minerals sources (Figure 1.1). Minerals like alum, sodium bicarbonate, and sodium chloride are commonly used in dentistry. The use of an alum mouthrinse daily inhibited caries development in children with decay-prone teeth [3]. The saturated saline rinse and alum rinse showed statistically significant reductions in salivary S. mutans counts in children [4]. Intrinsic tooth stain was reduced using sodium chloride with vinegar [5]. Sodium bicarbonate acts as an antiadhering agent for bacteria [6]. Cheese and cow milk stimulates salivary secretion and increases plaque calcium concentration and protection from caries [7, 8]. Natural products have been used in dentistry as analgesic and local anesthetic, as antimicrobial plaque agents, as antiseptics, whitening agent, antibacterials, to prevent adhesion of bacteria, antianxiety, and anti-halitosis. Commonly used natural products are Azadirachta indica, Syzygium Aromaticum, Acacia catechu, Aloe vera, miswak, and propolis. Plants are also used in root canal irrigation, pulpal and dentin repair, solvents, sealer cements, natural antioxidants, and storage medium (Table 1.1). Morinda citrifolia, propolis, Triphala, and aloe vera, besides their oral care activity also act as additives in Animal Eg. Propolis, Honey Plants Probiotics Eg. Clove, Miswak, Neem etc Minerals Eg. sodium chloride, alum Figure 1.1 Sources of natural care used in dental. Eg. Lactobacillus Natural Care used in Dental Marine Eg. alginate, carrageenan, fucoidan Natural Oral Care in Dental Therapy 5 Table 1.1 Classification of natural oral care on the basis of their uses. Part used or type of formulation References Pterocarpus marsupium Roxburgh Stem wood [12] Syzygium aromaticum Flower buds [13] Piper betel (Piperaceae) Leaves [14] Spilathes acmella (Asteraceae) Aerial part [15] Vitex negundo Fresh leaves [16] Anacyclus pyrethrum Root [17] Lavender (Lavandula angustifolia Miller) Volatile oil [18, 19] Citrus aurantium and Citrus sinensis Volatile oil [20, 21] Acacia arabica Chewing stick, gum [22, 23] Amaranthus hybridus L. Spinach leaf [24] Papain Papain, [25] Pineapple Bromelain, extract [25, 26] Salvadora persica Roots [25, 27] Azadirachta indica Mouthwash [28, 29] Aloe vera Mouthwash [30] Ocimum sanctum Mouthwash [31] Irimedadi oil Polyherbal Ayurvedic formulation [32] Apis Honey [33] Camellia sinensis (green tea) Catechin [34–36] Mangifera indica Leaf [37] Garcinia mangostana L Pericarp [38] Triphala Polyherbal Ayurvedic formulation [39] Propolis Mouthwash [40] Uses Source Analgesic and local anesthetic Antianxiety Whitening agent, Antiplaque (Continued) 6 Natural Oral Care in Dental Therapy Table 1.1 Classification of natural oral care on the basis of their uses. (Continued) Uses Antiadhesion activity Anti-halitosis Source Part used or type of formulation References Probiotic Mouthwash [41] Calendula officinalis Tincture [42] Dill Seed oil [43] Turmeric Mouthwash [44] Vaccinium macrocarpon Cranberry juice [45] Polygonum cuspidatum Methanol extract from root [46] Andrographis paniculata, Cassia alata, Chinese black tea (Camellia sinensis) and Harrisonia perforata Ethanolic extract [47] Helichrysum italicum Flowering tops [48] Malus domestica (Apple) Fruit [49] Schinus terebinthifolius and Croton urucurana Hydroalcholic extract [50] Mushroom (Lentinus edodes) Extract [51] Bauhinia variegata L. var. variegata Lectin [52] Camellia sinensis (Green tea) Epigallocatechin-3gallate [53] Cinnamomum verum Bark essential oil [54] Chenopodium quinoa Willd Alkali-transformed saponin from quinoa husks [55] Bixa orellana Seeds [56] Rice bran, sesame Oil [57] Sasa senanensis Rehder Leaves [58] Melaleuca alternifolia Essential oil [59] Brassica juncea L. (mustard) Seed (allyl isothiocyanate) [60] (Continued) Natural Oral Care in Dental Therapy 7 Table 1.1 Classification of natural oral care on the basis of their uses. (Continued) Uses Root canal irrigation Part used or type of formulation References Juniperus cedrus, Hiba cedar wood (Thujopsis dolabrata), and Western red cedar (Thuja plicata) Hinokitiol [61] Magnolia officinalis Bark [62] Curcuma zedoaria Root [63] Glycyrrhiza uralensis Licoricidin and licorisoflavan A isolated from Root [64] Panax ginseng C.A. Meyer Root [65] Eucalyptus Extract [66] Morinda citrifolia and Triphala Juice [67] Chamomile (Matricaria recutita L.) Hydroalcholic extract [68] Apple Vinegar [69] Oregano Extract solution [70] Chitosan 0.2% solution [71] Morinda citrifolia Fruit juice [72] Morinda citrifolia, Aloe Vera, and Propolis Juice and solution [73] German chamomile (Marticaria recutita L.) and tea tree (Melaleuca alternifolia L.) Extract and Oil [74] Aroeira-da-praia (Schinus terebintifolius Raddi) and the quixabeira (Syderoxylum obtusifolium Roem & Schult) Hydroalcoholic extracts [75] Berberine Plant alkaloid [76] Azadirachta indica, Morinda citrifolia Solution [77] Salvia officinalis Extract [78] Source (Continued) 8 Natural Oral Care in Dental Therapy Table 1.1 Classification of natural oral care on the basis of their uses. (Continued) Uses Pulpal and dentin repair Storage medium Source Part used or type of formulation References Salvadora persica Ethanolic extract [79] Turmeric, Morinda citrifolia Extract [80] Allium sativum Extract [81] Satureja Khuzistanica Jamzad Essential oil [82] Fragaria vesca (wild strawberry) Extract [83] Ferula gummosa Essential oil [84] Peganum harmala Seed extract [85] Azadirachta indica Ethanolic leaf extract [86] Allium sativum Oil [87] Green propolis Extract [88] Scutellaria baicalensis Flavonoid baicalein [89] Panax ginseng Ginsenoside rg1 [90] gardenia fruit extract Genipin [91] Quercetin Flavanoids [92] Nigella sativa Oil [93] Tobaco Nicotine [94] Polyphenols found in various plants Epicatechin [95] Green tea Extract [96] Coconut Water [97] Thai propolis Extract [98] Propolis Extract [99] Probiotic Milk [100] Probiotic Yogurt (Bifidibacterium animalis DN 173010) [101] Goat Milk [102] Morus rubra Fruit juice [103] Salvia officinalis extract [104] (Continued) Natural Oral Care in Dental Therapy 9 Table 1.1 Classification of natural oral care on the basis of their uses. (Continued) Uses Sealer cements Natural antioxidants on the shear bond strength of composite Resin Solvents Source Part used or type of formulation References Coconut and soy Water and milk [105] Capparis spinosa Buds [106] Punica granatum Juice [107] Green tea Extract [108] Castor Oil [109] Neem (Azadirachta indica) and turmeric (Curcuma longa) Neem leaves and turmeric rhizomes [110] Aloe vera Leaf extract [111] Juniperus cedrus, Hiba cedar wood (Thujopsis dolabrata) and Western red cedar (Thuja plicata) Hinokitiol-modified calcium silicate (CS) cement [112] Curcuma longa Curcumin-loaded mesoporous calcium silicate [113] Green tea and white tea Extract [114] Aloe Vera, Pomegranate Peel, Grape Seed, Green Tea Extract [115] Amla (Indian gooseberry) Extract [116] Grape seed Extract [117] Rosemary Extracts [118] Mangosteen Peel extract [119] Eucalyptus, orange, clove oil Oil [120] Grapefruit, lemon Oils [121] dental treatment. In the last few decades, various phytochemicals are reported in dentistry showing antimicrobial, analgesic, local anesthetic, anti-halitosis, and teeth whitening activity (Table 1.2). The chemical constituents obtained from natural resources play a key role in dentistry. Phytochemicals like alkaloids, flavones, flavonoids, flavonols, terpenoids, terpenes, phenols, phenolic acids, saponins, glycosides, quinone derivatives, organosulfur compounds, alcohols, aldehydes, ketones, lectins, enzymes, and amino acids are widely used in oral care (Table 1.3). 10 Natural Oral Care in Dental Therapy Table 1.2 List of phytochemical reported in dental care. S. No Sources Active phytoconstituents Activity References 1. Aceriphyllum rossii Aceriphyllic acid a and 3-oxoolean-12-en-27oic acid Anticariogenic activity [122] 2. Albizia myriophylla Lupinifolin Anticariogenic activity [123] 3. Allium sativum Allicin, diallyl sulfide Antimicrobial activity [124, 125] 4. Bursera morelensis Ramirez Α-pinene, γ-terpinene Antifungal [126] 5. Cymbopogon nardus Citronellal Antifungal [127] 6. Dryopteris crassirhizoma Linoleic acid Antibiofilm activity [128] 7. Diospyros lycioides Juglone Antibacterial [129] 8. Erythrina variegata Erycristagallin Antibacterial property [130] 9. Eucalyptus globules Macrocarpals a, b, and c Antibacterial [131] 10. Garcinia kola Heckel (Clusiaceae) Biflavonoid gb1 Antibacterial [132] 11. Gnetum gnemon L Resveratrol Osteoclast activity [133] 12. Mentha piperita Menthol Antimicrobial [134] 13. Origanum onites Carvacrol and thymol Antimicrobial [135] 14. Scrophularia striata Gallic acid, quercetin, and apigenin Antimicrobial [136] 15. Rumex acetosa L Procyanidin-b2-di-gallate Antimicrobial [137] 16. Flavonoids present in many fruits and vegetables Quercetin and kaemferol Antimicrobial [138] 17. Nidus vespae (honeycomb) Quercetin and kaempferol Antimicrobial [139] (Continued) Natural Oral Care in Dental Therapy 11 Table 1.2 List of phytochemical reported in dental care. (Continued) S. No Sources Active phytoconstituents Activity References 18. Dodonaea viscosa var. angustifolia 5,6,8-trihydroxy-7methoxy-2-(4methoxyphenyl)-4hchromen-4-one Anti-S. mutans, antibiofilm, and antiacidogenic activity [140] 19. Curcuma longa Curcumin Antibacterial [141, 142] 20. Tea Epigallocatechin-3-gallate Antibiofilm [143, 144] 21. Galla rhois Methyl gallate (mg) and gallic acid (ga) Antimicrobial [145] 22. Grapes Ethyl gallate Anticaries agent [146] 23. Garcinia mangostana L α-Mangostin Antimicrobial [147] 24. Magnolia officinalis Magnolol and honokiol Antimicrobial [148, 149] 25. Myristica fragrans Macelignan Anticariogenic activity, antibiofilm [150, 151] 26. Mikania glomerata Ent-kaurenoic acid Antimicrobial [152, 153] 27. Morus alba Kuwanon g Antimicrobial activity [154] 28. Clove, nutmeg, cinnamon Eugenol Local anesthetic, analgesia [155–157] 29. Trachyspermum ammi 4as, 5r, 8as) 5, 8a-di-1-propyloctahydronaphthalen1-(2h)-one Antibiofilm activity [158] 30. Theobroma cacao (cacao bean husk) Epicatechins Antibacterial activity [159] 31. Ginkgo biloba Ginkgoneolic acid Antimicrobial activity [160] 32. Psidium guajava Linn. Quercetin-3-o-alphal-arabinopyranoside (guaijaverin) Antiplaque agent [161] (Continued) 12 Natural Oral Care in Dental Therapy Table 1.2 List of phytochemical reported in dental care. (Continued) S. No Sources Active phytoconstituents Activity References 33. Rabdosia trichocarpa Trichoranin Antibacterial activity [162] 34. Rhus coriaria L. Methyl gallate Antibiofilm activity [163] 35. Rosmarinus officinalis Carnosic acid and carnosol Antimicrobial activity [164] 36. Glycyrrhiza glabra Glycyrrhetinic acid, disodium succinoyl glycyrrhetinate Antimicrobial activity [165, 166] 37. Glycyrrhiza uralensis Icoricidin and licorisoflavan A Antibacterial activity [167] 38. Aralia cachemirica L. (Araliaceae) 4-epi-pimaric acid Antimicrobial activity [168] 39. Piper betle Hydroxychavicol Antimicrobial activity [169] 40. Piper cubeba (−)-Cubebin Antimicrobial activity [170] 41. Polyalthia longifolia var. pendula (Linn.) 16-Oxo-cleroda-3, 13(14) e-diene-15 oic acid, and kolavenic acid Antimicrobial activity [171] 42. Propolis Neovestitol-vestitol, apigenin, and tt-farnesol Antimicrobial activity [172, 173] 43. Fruits of Rheedia brasiliensis 7-Epiclusianone Antimicrobial activity [174] 44. Iostephane heterophylla Ent-trachyloban-19-oic acid Antibiofilm activity [175] 45. Symplocos racemosa Symploquinones a-c Antibiofilm activity [176] 46. Swartzia polyphylla Dihydrobiochanin a, ferreirin and darbergioidin, and dihydrocajanin Antibacterial activity [177] 47. Polygonum cuspidatum Emodin Antibacterial activity [178] (Continued) Natural Oral Care in Dental Therapy 13 Table 1.2 List of phytochemical reported in dental care. (Continued) S. No Sources Active phytoconstituents Activity References 48. Croton nepetaefolius Casbane diterpene Antimicrobial activity [179] 49. Viguiera arenaria Ent-pimara-8(14),15dien-19-oic acid Antimicrobial activity [180] 50. Mikania glomerata Sprengel Ent-kaurenoic acid Antibacterial activity [181] 51. Vaccinium macrocarpon Cranberry a-type proanthocyanidins and flavonols Antibacterial activity [182] 52. Curcuma xanthorrhizha (Javanese turmeric) Xanthorrrhizol Antibacterial activity [183] 53. Ipecacuanha, cinchona Emetine, quinine Antibacterial activity [184] 54. Corn husks or sugarcane straw Xylitol Antibacterial activity [185] 55. Birch bark Betulin Antiinflammatory activity [186, 187] 56. Berberis vulgaris Berberine Slow periodontal degradation [188] 57. Salvadora persica Benzyl isothiocyanate Anti-Gramnegative bacteria [189] 58. Kaempferia pandurata Roxb. Panduratin A, isopanduratin A Antimicrobial [190, 191] 59. Syzygium aromaticum (L.) Merr. & L. M. Perry Eugenol Antibiofilm activity [192] 60. Copaifera reticulata (−)-Copalic acid Antimicrobial activity [193] 61. Melaleuca alternifolia (tea tree ) Alpha-bisabolol Antibiofilm activity [194] 62. Eucalyptus 1,8-cineole Antibiofilm activity [195] Q3 14 Natural Oral Care in Dental Therapy Table 1.3 Chemical classification of natural oral care. Category Examples Alkaloids Atropine, morphine, sanguinarine, chelerythrine, colchicines, nicotine, pilocarpine, cotinine, theobromine, sanguinarine, scopolamine, cocaine, vincristine, berberine chloride, oxyacanthine sulfate, harmine, vasicine, Berberine, Salvadorine, Piperine, Emetine, quinine Flavones, flavonoids, and flavonols Quercetrin, naringenin, proanthocyanidin, myricetin, apigenin, luteolin, fisetin, catechin, epicatechin, pelargonidin, myricetin, kaempferol, hesperidin, catechins, epicatechin, gunistein, daidezin, gallocatechin, cyanidin, Icoricidin, licorisoflavan A, Kenferaido, galangin, isorhamnetin, rhamnetin, 3,7-dihydroxy-5-methoxy flavanone, 2,5-dihydroxy-7-methoxy flavanones Bruno, 3-methyl quercetin, 8-methyl kaempferol, Dihydrobiochanin a, ferreirin and darbergioidin, dihydrocajanin, pinobanksin, pinobanksin 3-acetate, Lupinifolin, Biflavonoid gb1, Procyanidin-b2-di-gallate, pinobanksin 7-methyl ether, pinocembrin, sakuranetin, isosakuranetin, Pinosutorobin, trihydroxymethoxy flavanone, tetrahydroxy flavanones, tetrahydroxy flavone, chrysin, acacetin, baicalein, tectonics chrysin, kaempferol, Erumanin, 3,5,7-trihydroxy-4 -methoxy flavanols, 5,6,7-trihydroxy-3,4 -dimethoxy flavone, 5,6,8-trihydroxy-7-methoxy-2-(4-methoxyphenyl)-4hchromen-4-one, 4as, 5r, 8as) 5,8a-di-1-propyl-octahydronaphthalen-1(2h)-one, Cranberry a-type proanthocyanidins, flavonols Terpenoid Aceriphyllic acid, Nimbidin, 1,8-cineole, ursolic acid, oleanolic acid, terpineol, nerolidol, bisabolol, melliferone, moronic acid, β-caryophyllene, santatol, abietic acid, lanosterol, cupressic acid, agathalic acid, communic acid, methyl isocupressate, tremetone, viscidone, Erycristagallin, Macrocarpals, ledol, guajol, humulene, bulnesol, Glycyrrhetinic acid, viscidone, betuletol, anwuweizonic acid, thymol, Xanthorrrhizol, betulonic acid, α-copaene, ent-kaurenoic acid, β-selinene, α-elemene, calamenene, β-amyrine, amyrine, α-muurolene, γ-muurolene, dehydroabietic acid, β-eudesmol, syringaldehyde, imbricatoloic acid, tt-famesol, Oridonin, Ursolic acid, Citronellal, 16-oxo-cleroda-3, 13(14) e-diene-15 oic acid and kolavenic acid, Enttrachyloban-19-oic acid, Ent-pimara-8(14),15-dien-19-oic acid, Entkaurenoic acid Terpenes Casbane diterpene, Betulin, alpha-pinene, menthol, Copalic acid, Alpha-bisabolol Phenols and phenolic acids Eugenol, Epigallocatechin-3 gallate, gallic acid, Α-mangostin, citric acid, Curcumin, Magnolol, Carvacrol, (6)-gingerol, Hydroxychavicol, (6)-shogaol, Resveratrol, honokiol, Methyl gallate, Ethyl gallate, Macelignan, Ginkgoneolic acid, 4-epi-pimaric acid, (−)-Cubebin Saponins Ginsenoside Rg1, ginsenoside Rh2, Glycyrrhizin, Buddlejasaponin IV, Diosgeninlactoside (Continued) Natural Oral Care in Dental Therapy 15 Table 1.3 Chemical classification of natural oral care. (Continued) Category Examples Glycoside Fomitoside-K, Divaricoside, rubusoside, C-xylopyranoside, stevioside, rutin, Quercetin-3-o-alpha-l-arabinopyranoside Quinone derivatives aloe emodin, Juglone, Symploquinones a-c, Emodin Organosulfur compound Allicin, diallyl sulfide, allyl isothiocyanate, Benzyl Isothiocyanate, phenethyl isothiocyanate, allyl isothiocyanate Alcohol Xylitol Aldehyde Cinnamaldehyde Ketones Panduratin A, isopanduratin A, 7-epiclusianone, Panduratin A, isopanduratin A Lectins Labramin, aloctin A, lectins from Canavaliaensi formis (ConA), Canavalia brasiliensis (ConBr), Canavalia maritima (ConM), Canavalia gladiata (CGL), and Canavalia boliviana (ConBol), Cratylia floribunda (CFL), Vatairea macrocarpa (VML), Bauhinia bauhinioides (BBL), Bryothamnion seaforthii (BSL), and Hypnea musciformis (HML) Enzymes Papain and bromelain Amino acids Arginine, Caseine 1.2 Safety of Natural Oral Care Herbal medicines or natural products are generally considered safe. Clinical studies usually assess the efficacy of products containing natural products. There are a few studies done on the safety and possible side effects of such products. Oral administration of clove oil developed urinary abnormalities, central nervous system depression, and a large anion-gap acidosis in an infant [9]. Neem oil in adults causes metabolic acidosis, vomiting, seizures, and toxic encephalopathy [10]. Repeated sesame oil pulling for several months caused lipoid pneumonia [11]. More safety study is needed when used in combination with allopathic drugs. 1.3 Advantage of Natural Oral Care • Development of antimicrobial resistant strains is increasing using synthetic chemicals. So natural antibacterial substances are as useful as alternative antimicrobials in oral care. • Current synthetic chemicals such as chlorhexidine, povidone iodine, triclosan fluoride, cetylpyridinium, zinc citrate, and sodium lauryl sulfate used in dentifrices and mouthrinses as antibacterial can be quite damaging to the 16 Natural Oral Care in Dental Therapy gums, teeth, and mouth. So their long-term use is limited. So natural oral care formulation is free from such side effect. • Use of traditional tooth cleaning methods includes efficacy, safety low cost, popularity, and availability. • Natural oral care is a safe option to kids, pregnant women, blood pressure patients, diabetics, and people with dry mouth. 1.4 Limitations of Natural Oral Care • • • • • • Low worldwide availability Standardization Some plants are useful when used in fresh form Complete toxic study data are not available Data of interaction with allopathy drugs are not available Very slow in curing the disease 1.5 Future Prospects of Natural Oral Care India is juvenile in handling dental problems. It is still a common problem worldwide. Dental caries and periodontal diseases can be treated with chemicals and allopatic drugs. The common side effects associated with them are nausea, vomiting, diarrhea, or teeth staining. The patient’s compatibility with the chemical uses is also not found to be appropriate. The dental problems are well taken using drugs of herbal origin. The utmost requirement in industries is to develop a safe and effective formulation. Prolonged use of synthetic chemical agents produces side effects, and for financial considerations, there is a need for natural agents that are effective, safe, and economical. The herbal drugs may be used externally as ointment, pastes, plasters, and poultices or internally as syrups, suspensions, or pills. Ancient techniques like oil pulling therapy, chewing stick, and aqueous herbal extract are an affordable option for its oral health benefits in rural communities. As verified by the examples, there is extensive proof that plant products have the potential to be used as preventative or treatment therapies for oral diseases. The effective advantages with herbal products are cheap, easy availability, greater shelf life, minimal toxicity, and lack of microbial resistance, which is the major advantage with herbal drugs. The drugs used in oral care for suppressing inflammation, may have antibacterial, antifungal, and analgesic activities. They can be recommended in gingivitis, mucositis, or any infection of fungal or bacterial origin. The active chemical constituents, which are found to be effective in dental caries, are flavanoids, phenolic acids, resins, triterpenes, carotenoids, or tannins. These active constituents are obtained from drugs, for example, amla, lemon, clove, neem, tulsi, etc. The ethnopharmacology and reporting of clinical data, systems with chemical and pharmacological characterization of extracts from promising sources will lead to new product development for dentistry. Molecules such as eugenol, quercitin, and kaempeferol were isolated from plants and have dental therapeutic potential. After development with phytochemistry and formulation technologies, and in combination with other active ingredients, a novel formulation should be made. The use of Natural Oral Care in Dental Therapy 17 strandardized herbal formulation helps in pharmacological activity and prevents from adulteration. It can be concluded that these herbal drugs must be included in everyday life so that the dental problem can be resolved. These drugs may be used in isolation or combination. These will surely suppress the growth of oral pathogens, minimize the development of dental plaque, and also help in curing the symptoms of oral diseases. The minimal toxicity and minimal cost of these herbal drugs should be promoted for further investigation. This may come with a noble drug leading to a better understanding of traditional Asian medicines and their uses for oral health. However, a health well-wisher should take this herculean task to confirm that individuals must promote herbal medicines to protect public health. References 1. 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Souza, A.B., de Souza, M.G., Moreira, M.A., Moreira, M.R., Furtado, N.A., Martins, C.H., Bastos, J.K., dos Santos, R.A., Heleno, V.C., Ambrosio, S.R., Veneziani, R.C., Antimicrobial evaluation of diterpenes from Copaifera langsdorffii oleoresin against periodontal anaerobic bacteria. Molecules, 16, 11, 9611–9, 2011. 194. Forrer, M., Kulik, E.M., Filippi, A., Waltimo, T., The antimicrobial activity of alpha-bisabolol and tea tree oil against Solobacterium moorei, a Gram-positive bacterium associated with halitosis. Arch. Oral Biol., 58, 1, 10–6, 2013. 195. Hendry, E.R., Worthington, T., Conway, B.R., Lambert, P.A., Antimicrobial efficacy of eucalyptus oil and 1,8-cineole alone and in combination with chlorhexidine digluconate against microorganisms grown in planktonic and biofilm cultures. J. Antimicrob. Chemother., 64, 6, 1219–25, 2009. 2 Herbal Products for Oral Hygiene: An Overview of Their Biological Activities Ummuhan Sebnem Harput * Independent Researcher, Ankara, Turkey Abstract Oral hygiene plays a crucial role in the prevention of oral diseases, including periodontitis, tooth decay, and oral candidiasis. In addition, many scientific studies show a relationship between oral health and cardiovascular diseases, diabetes, and even death. In order to prevent caries formation and periodontal diseases, effective oral hygiene should be performed and plaques should be removed, plaque formation should be prevented, and strains should be removed. Patients and dentists are faced with different oral hygiene products containing active and inactive ingredients. Although chemical-based products are good at oral hygiene, there are many adverse effects of dental hygiene products, which are sold in the market. Effective usage of toothbrush, floss, or rinse is found very important for making oral hygiene even without using any chemicals. In addition, although popular herbal products have helped to control dental plaque and gingivitis, their safety and efficacy is not researched in detail. Herbal products may offer significant advantages over the chemical ones with less side effects and high antimicrobial potential. In addition, people are aware of the effects of herbal products for oral care, and their interest in these products has increased recently. Traditional herbal-based treatments provide reduced adverse reactions of chemical counterparts such as resistance to antibiotics, corrosion, or staining of teeth. Herbal products such as clove and clove oil, coconut oil, pomegranate, green tea, Salvadora persica (meswak), Aloe vera, Acacia arabica, Melaleuca alternifolia (tea tree), Azadirahta indica (neem), and licorice are used to promote oral hygiene, and their inhibitory effect on biofilm formation is shown in different studies. According to bioactivity studies on these species, antibacterial, anti-inflammatory, anticariogenic, and astringent properties were observed for their components and/or extracts. If such herbal products can be formulated effectively, this may lead to an improvement in the general dental health of the population. Summarized here are such natural products, which may be used effectively in the commercial formulations and/or personal product. Keywords: Herbal medicine, oral hygiene, oral hygiene products, dental plaque 2.1 Introduction There is an increasing public awareness on personal oral hygiene. Apart from toothbrushes and toothpastes, toothpowder, mouthrinses, and similar products are also widely used in Email: sharput@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (31–44) © 2020 Scrivener Publishing LLC 31 32 Natural Oral Care in Dental Therapy the population. People brush and rinse their teeth to feel fresh, to avoid bad breath, dental caries, oral diseases, and to have a nice smile. Effective oral care is important for all individuals, especially for kids, pregnant women, and those who have an immune system deficiency, or undergoing chemotherapeutic or radiation therapy. In addition, many scientific studies show a relationship between oral health and cardiovascular diseases, diabetes, and even death. In order to prevent caries formation and periodontal diseases, effective oral hygiene should be performed and plaques should be removed, plaque formation should be prevented, and strains should be removed [1, 2]. The World Health Organization (WHO) is also promoting oral hygiene advice to be included in the school curriculum. If oral hygiene habit is neglected for the school ages, it will cause serious health problems in the future. Oral infections, periodontal disease, and dental caries are occurring very common all around the world. Effective oral hygiene prevents oral infections and diseases. Antibiotics, fluoride, and chlorhexidine derivatives are used successfully as oral hygiene products against oral diseases including infections and caries; however, long-term overuse of these chemical-based oral hygiene products caused some oral or systemic adverse reactions [3]. Dental treatments are expensive and usually not covered by health insurance services. Abrasives, preservatives, surfactants, sweeteners, or colorants found in the composition of dental products can cause not only corrosion on teeth but also other health problems. Their effectiveness is still questionable. Hence, there is an increasing demand for the use of herbal products and treatments as alternatives to chemical treatments for maintaining oral health [4]. Numerous different products or their mixture can be used for oral hygiene throughout the centuries. These old formulations had the potential oral or systemic side effects because of their toxic constituents such as sulfuric acid, mercuric perchloride, carbolic acid, and formaldehyde. With the recognition of these toxic effects, researches for new products have increased rapidly, and more reliable products have emerged over the years [5]. In recent years, herbal products in oral care, which are widely used in traditional medicine, have attracted the attention of researchers looking for a safer and more effective oral hygiene product. Traditionally, herbal products such as clove and clove oil, coconut oil, pomegranate, green tea, Salvadora persica (meswak), Aloe vera, and Acacia arabica are used to promote oral hygiene. Antibacterial, anti-inflammatory, anticariogenic, and astringent compounds are mostly isolated from these species [6, 7]. For example, as a result of the evaluation of traditional usage and scientific evidences of the extract of Salvadora persica, it has been included in many oral rinse and toothpaste products with the evidence of safety and effectiveness data [8]. Since herbal products for oral hygiene can be purchased OTC in many countries, they began to attract an increasing number of consumers who are looking for an alternative product. However, it is difficult to find a reliable, safe, and effective herbal oral hygiene product in the market. More research is needed to determine their effectiveness and safety [9]. There has been no comprehensive review summarizing the pharmacological properties, phytochemical characteristics, and appropriate usages of herbal oral hygiene products for researchers and for the producers who want to develop new commercial products. In this study, we will discuss prominent plant extracts and phytochemicals, which can be used as a safe and effective oral hygiene product. Clinical studies that have investigated the quality, efficacy, and safety of such herbal products and their phytochemical composition and pharmacological properties will also be described. Herbal Products for Oral Hygiene 33 2.2 Oral Hygiene and Current Treatments The mouth is inhabited by millions of bacteria. While we cannot sterilize the mouth, we should minimize bacterial panacea for preventing tooth decay. The most important thing here is inhibition to form organized bacterial colonies, which are referred to as “dental plaque”. Dental plaque is composed of over 500 bacterial species [10]. Generally, inadequate oral hygiene habits cause oral health problems such as caries and periodontal disease. Similarly, periodontal diseases are chronic infections caused by bacteria existing at the dental plaque. Bacterial invasion and colonies are the causes of inflammation and bleeding gums in the oral cavity. If it is not treated successfully, it leads to tissue destruction, tooth mobility, and even tooth loss. In the final stage, periodontal bacteria can enter the blood stream and may cause systemic reactions such as increased risk of heart disease, stroke, and diabetes. Good oral hygiene is very important in preventing and controlling of the above risks and dental decay [11]. Generally, oral hygiene is provided by daily tooth brushing with toothpaste. The possible forms of action of these agents can be discussed under two headings: antiplaque and antigingivitis. The antiplaque agents can be further subdivided as antiadhesives and antimicrobials. Chemotherapeutic agents used for plaque removal include anticalculus agents such as pyrophosphate, antibacterials such as Triclosan and chlorhexidine, anticaries agents such as fluoride, whitening agents such as alumina, and desensitizing agents such as strontium chloride [12, 13]. Some of the commercially available toothpastes contain plant products. The addition of peppermint, cloves, aniseed, and recently tea tree oil (Melaleuca alternifolia), aloe vera gel, olive leaf, and neem leaf extracts enhance the antimicrobial activity of the toothpastes [3]. For example, the extract of Salvadora persica incorporated into herbal toothpastes against dental plaque bacteria is well documented [14]. Xylitol and stevia extract, which are natural sweeteners, are used in some toothpaste formulations instead of fluoride to control plaque, as well as to strengthen the teeth and gums. Recently, commercially available oral preparations can be available, which contain several plant ingredients including green tea, aloe vera, garlic juice, thymol, eucalyptol, licorice, and rooibos [3, 15, 16]. Mouthrinses are also used together with mechanical or chemical oral hygiene products in order to prevent different kinds of oral diseases, treat high caries risk and infection, reduce inflammation, relieve pain, and reduce halitosis. It is one of the alternative methods to mechanical plaque control. Chlorhexidine is the main ingredient, which has much of the desirable characteristics of antiplaque mouthrinse; however, it is also responsible for major side effects, especially the extrinsic staining of hard and soft tissues. Similarly, the long-term use of ethanol-containing mouthrinses should be discouraged in patients with a high risk of caries [17, 18]. Hence, safety of these products has not yet been proven, and there is a need for long-term clinical trials to find an effective and safer alternative to chlorhexidine with all the good properties and without its side effects. 2.3 Plants Traditionally Used in Oral Hygiene The use of herbal products for the protection of oral health and treatment of oral diseases comes from the very old days of the world. A well-known example, Salvadora persica L. (miswak or siwak), is the most common traditional source of the material for oral hygiene. 34 Natural Oral Care in Dental Therapy Since Islam has emphasized the usage of miswak for oral hygiene, the use of Miswak is widespread in Islamic countries for basic oral hygiene. The antibacterial effect of Salvadora persica extracts against bacteria in dental plaque is well documented throughout the world [8, 19–21]. Benzyl isothiocyanate and its derivatives were identified as the major antibacterial component, and their amount reflects the efficacy of the miswak [21]. It is proposed by the WHO, and it is encouraged to do further research for its biological activities on oral health [8, 14]. Green tea, the leaves of Camellia sinensis is a popular drink that has a beneficial physiological effect. It is widely consumed all over the world and proven to have antioxidant and antibacterial effects against bacterial colony of dental plaque. It has been found that green tea polyphenols can eliminate halitosis by modifying the odorant sulfur components. Oxidative stress and inflammation in the oral cavity, due to heavy cigarette smoking and alcohol consumption, also decreased in the presence of green tea polyphenols [6, 14, 22]. There are many different plant species used for periodontal disease and oral hygiene in India. The dried powders of Acacia arabica (bark), Terminalia chebula (fruits), Terminalia bellerica (fruits), and Emblica officinalis have been used in their traditional tooth formulas for more than a century [23]. Rasingam et al. reported on the traditional herbal tooth sticks used by the inhabitants of Andaman and Nicobar Island in India [24]. Among these species, neem (Azadirachta indica), which is widely used as chewing stick throughout the world, is a native of India and is cultivated in all parts of the subcontinent because of its medicinal properties. It was reported that every part of the neem tree, such as branches, twigs, bark, and oil, was used for oral health in India, for centuries [6, 24, 25]. It is one of the traditional dental care practices in India to brush with neem and mango branches and chew the neem leaves and seeds after the meals. The stems of Azadirachta indica have antiinflammatory and antimicrobial activities due to the presence of substances such as nimbin and nimbidine as phytochemicals. In case of mango twigs (Mangifera indica), mangiferin, which is the natural C-glucoside xanthone, has been reported in various parts of the plant such as leaves, fruits, stem, bark, and roots. Antioxidant, radioprotective, immunomodulatory, antitumor, antiallergic, anti-inflammatory, antidiabetic, and antimicrobial properties of mangiferin are reported in many studies. It has also therapeutic potential in both the prevention and treatment of periodontitis [26, 27]. Different Artemisia (wormwood) species are used traditionally because of its medicinal properties. For example, Artemisia absynthium, Artemisia herba alba, and Artemisia siberia extracts can be used in cosmetic and toiletry products such as toothpastes, which not only cleans the teeth but also prevents decay, treats bad breath, and protects the oral hygiene [28, 29]. Another important method of traditional oral hygiene is oil pulling. which has been applied for a long time as a traditional folk medicine in India. Recently, people around the world are increasingly interested in this method, which is offering oral health benefits and oral hygiene. Based on current research, it was found that, when administered correctly and regularly, oil pulling improved oral hygiene. However, oil pulling does not replace dental treatments and is currently not recommended by the American Dental Association. According to the latest data, it can be safely used with brushing teeth and flossing as well as help maintain good oral hygiene and health [30, 31]. Herbal Products for Oral Hygiene 35 2.4 Clinically-Studied Plant Products for Oral Hygiene Because of the side effects of routinely used chemical-based dental hygiene products such as toothpastes, dentifrices, and mouthwash, researchers are trying to find herbal products that do not contain any chemical ingredients. Consumers often prefer herbal products rather than the products that contain chemical ingredients. Recently, clinical studies on efficacy and safety of plant extracts and herbal-derived dental products were increased to support their usage in oral care [32]. Some of the important clinical studies are summarized below. For example, Srinivasa et al. evaluated the plaque and the gingival status of children brushing with commercially available herbal dentifrice (containing sodium monofluorophosphate and chamomile, eucalyptus, myrrh, sage as active ingredients) in comparison with non-herbal dentifrice (sodium monofluorophosphate and triclosan as active ingredients) for a period of 21 days [33]. Although they found no significant difference between the two groups, the herbal group showed a significant reduction in gingival bleeding and inflammation compared to the non-herbal group. It could be because of the anti-inflammatory and astringent properties of the herbal constituents in the herbal dentifrice. Therefore, the tested herbal dentifrice can be considered as an alternative product to protect gingival health compared with the commercial one [33]. In addition to their effect on dental health, taste, smell, and the other flavor characteristics of the herbal dental products are very important for the consumer or patients to use it tastefully. The antimicrobial effect of Melaleuca alternifolia (tea tree) containing dental gel was studied in 34 orthodontic patients compared with the commercial toothpaste Colgate Total. While Melaleuca gel was found to be more effective in decreasing the dental biofilm and the number of bacterial colonies, it was not as good as Colgate Total regarding the taste, smell, and the other flavor characteristics. They found that Melaleuca gel is efficient in the control of bacterial growth but needs good formulation for improvement in taste and first sensation [34]. Plaque and gingivitis reduction were assessed with the toothpaste containing Azadirahta indica (neem) and the commercial toothpaste comparatively in randomized, double blind clinical trial. In this study, regular use of neem-containing toothpaste provided a significant reduction in plaque formation and improved the gingival health of the participants. It could be due to the antibacterial and anti-inflammatory effects of neem. This result indicated that regular brushing with neem toothpaste is important for the maintenance of good oral hygiene and improvement of oral health [35]. Similarly, the effect of neem and mango mouthwashes on oral health was assessed by Sharma et al. [26]. Three groups of children were tested for 21 days for their gingival and plaque conditions with neem, mango, and chlorhexidine mouthwash. According to the above study, the tested three mouthwashes have antiplaque and antigingivitis effect. They found that while clorhexidine and neem have equivalent efficacy in plaque reduction, chlorhexidine has superior antigingivitis properties. This study provided enough data to show that neem- and mango-based mouthwashes have a beneficial effect on oral health [26]. In another study, herbal dentifrice containing Macleya cordata and Prunella vulgaris was investigated for the effectiveness of controlling gingivitis in a double-blind, placebo-controlled clinical study. The extract was found to be effective in the control of gingivitis, while it did not show any antiplaque effect. It was 36 Natural Oral Care in Dental Therapy concluded that the tested herbal dentifrice has a significant anti-inflammatory effect and can be used in the conventional dentifrice formulations [36]. Recently, considerable researches have been conducted on commercial mouthrinses and toothpastes containing herbal extracts. Commercial herbal toothpaste Paradontax, which contains sodium bicarbonate, sodium fluoride, and herbal ingredients including chamomile, echinacea, sage, myrrh, rhatany, and peppermint oil was evaluated in a double-blind clinical trial for the reduction of plaque and gingivitis. It was found that Paradontax did not show significant clinical advantage over the conventional toothpaste with fluoride. This result indicated that commercial herbal toothpastes should be evaluated properly for their efficacy and advantage comparing the conventional toothpastes [37]. Similarly, the efficacy of Paradontax was also evaluated on the reduction of plaque and gingivitis in comparison to the dentifrice with triclosan and fluoride for 28 days. While a significant reduction was observed in the plaque formation and gingivitis in both groups, no significant difference was found between the two groups. The authors concluded that both toothpastes were effective in reducing dental plaque and gingivitis rate in subjects with gingivitis [38]. The anti-inflammatory effect of Paradontax was assessed comparatively with Colgate herbal toothpaste, which contains calcium carbonate, chamomile, sage, myrrh, eucalyptus, and sodium monoflurophosphate in plaque and gingival inflammation. In this study, both formulations reduced plaque formation levels and gingival inflammation. However, Paradontax did not show any additional benefits over Colgate herbal toothpaste [39]. In another study, ayurvedic dental cream was evaluated in comparison to fluoride dental cream for efficacy and safety in a randomized double-blind study. Herbal dental cream consists of different ayurvedic plant species such as powders of Ajamoda satva, Vaikranta bhasma, and Azadirachta indica; and extracts of Zanthoxylum alatum, Punica granatum, Acacia arabica, Vitex negundo, Embelia ribes, and Triphala. While significant protection was observed in both groups, their efficacy is not significantly different from each other. According to this result, the ayurvedic dental cream is as safe and effective as that of fluoride dental cream, but it is not superior to the prevention and management of dental plaque formation [40]. Another ayurvedic toothpaste containing herbal ingredients such as the extracts of Acacia chundra, Adhatoda vasica, Mimusops elengi, Piper nigrum, Pongamia pinnata, Quercus infectoria, Syzygium aromaticum, Terminalia chebula, and Zingiber officinale was tested for its efficacy on improving gingival health, oral hygiene, and salivary microbial flora. They found statistically significant reductions on gingival bleeding, salivary anaerobic bacterial counts, and better oral hygiene, while the placebo group did not show any significant improvement in oral health conditions [7]. The above studies show that herbs, which are used traditionally in oral care, are important sources of dental hygiene products, and further researches are needed to clarify the mechanism of action of each herb included in these products. Mouthwashes or rinses are also important oral hygiene products and are recently very popular in western societies. The efficacy of the mouthwash containing an extract of Salvadora persica, which is the most important herbal product for oral hygiene was investigated in a double-blind, cross-over trial for a 3-week period. Although PersicaTM mouthwash increased gingival health and lowered cariogenic bacterial transport rate, the placebo also significantly improved gingival health [41]. Similarly, the antiplaque effects of S. persica and 0.2% chlorhexidine rinse were compared by Gazi et al., and they Herbal Products for Oral Hygiene 37 found that chlorhexidine rinse was better than S. persica extract in inhibiting plaque formation [42]. On the other hand, Rahmani et al. also assessed the efficacy of mounthrinses containing S. persica and chlorhexidine on plaque formation; they found a comparable plaque inhibition by both rinses [43, 44]. As a result of the above studies, S. persica-containing herbal mouthrinses have provided good oral hygiene and plaque inhibitory potential, but they are not as efficacious as chlorhexidine in preventing bacterial plaque formation. Herbal oral rinse containing the extracts of echinacea, Hydrastis canadensis, calendula, aloe, Sanguinaria canadensis, grapefruit seed, cinnamon and spearmint oil, and peppermint oil was also tested in comparison with 0.12% chlorhexidine oral rinse. The effects of herbal rinse on restoring gingival health status were not statistically greater than those of the placebo. Chlorhexidine is more efficient than herbal rinse in reducing the clinical indicators of gingivitis when compared to the placebo. Therefore, individuals who are looking for a natural, sugar-free, and non-alcohol mouth rinse should be advised that there is still a need for researches to support the effectiveness of herbal oral rinses [45]. Licorice is one of the important traditional herbs used for different physiological conditions and as a food ingredient throughout the world. The effect of different licorice preparations on dental problems was evaluated by different authors. Recent researches suggest that licorice extracts and its phytochemicals have beneficial effects in oral hygiene and oral diseases. These effects have been attributed to the antiadherence, antimicrobial, and anti-inflammatory properties of its constituents. However, glycyrrhizin, which is one of the important components of licorice, is converted to glycyrrhetic acid in the human intestine and can induce severe hypertension and hypokalemia in the body. Therefore, licorice extract without glycyrrhizin should be preferred for use in order to prevent the side effects of licorice [46, 47]. In vitro studies have demonstrated the potential of licorice and its bioactive constituents to treat oral disease. On the other hand, clinical trials have generally inconsistent results. Therefore, oral hygiene products containing licorice root extracts and constituents need to be further investigated to verify the beneficial effects observed in in vitro experiments [46]. Licorice was also used as an herbal lollipop and was examined to investigate its effect on Streptococcus mutans bacteria, which cause dental caries. It was found that there was a reduction in the growth of S. mutans in the oral cavity with consumption of more lollipops during the experiment period [48]. Clinical studies have also shown that administration of licorice lollipop leads to reduction in dental biofilm and the number of bacterial colonies in the oral cavity among the tested individuals [49, 50]. Extracts of licorice, particularly when formulated as candy, lollipop, or similar others indicated significant inhibition of the formation of dental caries of human subjects. These studies show that different licorice products can be used in oral hygiene products to prevent gingival diseases and improve oral health [51]. 2.5 In Vitro-Studied Herbal Products for Oral Hygiene In vitro studied plant products for oral hygiene are summarized below (Table 2.1). Tested herbal product Method Result Conclusion Phytochemicals Ref Leaf-bud extracts of Populusnigra, P. x berolinensis, P. lasiocarpa Anti-inflammatory activity in gingival fibroblasts P. X berolinensis is effective Useful for oral hygiene and in the treatment of gingivitis, periodontitis Flavanons: pinocembrin, pinostrobin [52] Artemisia sieberi essential oil Antimicrobial activity against Gram +/− bacteria, yeast and fungi Active It can be used in toothpastes because of antibacterial effect against S. mutans Dried extract of Acacia arabica (bark), Terminalia chebula (fruits), Terminalia bellerica (fruits), Emblica officinalis (fruits) Antimicrobial, biofilm disruption and anticaries effect against cariogenic bacteria Inhibitory effects of cariogenic microorganisms and S. mutants biofilm formation Useful resource of anticariogenic dental products Combination of Salvadora persica and green tea extracts Effect on dental biofilm formation Synergistic antibacterial and antiadherence effects Useful active agent for oral care products. , thujone, camphor [28] [23] Green tea: catechins and flavonoids; S. persica: trimethylamine, salvadorine, thiocyanate, tannins, nitrate, sulfate [14] (Continued) 38 Natural Oral Care in Dental Therapy Table 2.1 In vitro studied plant products for oral hygiene. Table 2.1 In vitro studied plant products for oral hygiene. (Continued) Method Result Conclusion Phytochemicals Ref Pistacia vera oleoresin Antimicrobial properties Strong anti-virulence effect against S. mutans and reducing the ability to form biofilm P. vera oleoresin can be used in oral hygiene products Oleoresin, -pinen, triterpenes [53] 95% ethanol extract of Piper betle with 3 different commercial toothpastes Antimicrobial Effect against Escherichia coli, Staphylococcus aureus, S. mutans and Streptococcus salivarius, and Candida albicans Toothpaste— P. betle extract combinations is more effective than those exhibited by the toothpastes alone It can be used as effective toothpaste ingredients Phenolics [54] Deglycyrrhizinated licorice extract Antimicrobial activity against Streptococcus mutans biofilm formation of 16 mg/mL of extract is useful in the development of oral hygiene products Phenolics [47] Herbal Products for Oral Hygiene 39 Tested herbal product 40 Natural Oral Care in Dental Therapy 2.6 Discussion Oral diseases are one of the major health problems worldwide. Efficient oral hygiene is important not only to prevent tooth decay and periodontal diseases but also to improve various autoimmune conditions. Poor oral hygiene and oral diseases are associated with chronic and systemic diseases. Therefore, the use of various herbal products has been increasing in recent years in order to provide oral hygiene and prevention of oral diseases [54]. Today, the daily oral hygiene routine is based on toothbrushing and/or mouth rinsing with mouthwashes. The traditional roles of dentifrices, toothpastes, powders, gel, or mouthrinses are helping in the cleaning of teeth, oral cavity, and producing fresh breath. Eid and Talic [55] compared the effectiveness of tooth brushing with toothpaste to the effectiveness of tooth brushing with water. While a decrease of 67% in plaque formation was observed by brushing with toothpaste, 59% reduction in tooth brushing with water was reported. Similarly, the effectiveness of rinsing before brushing on plaque removal was examined by Binney et al. [56]. Rinsing then brushing removed more plaque than any other combination of mouth rinse and dentifrice. According to the above results, the function of different oral hygiene products in plaque formation and the removal of plaque is still questionable. Chemical-based toothpaste or other products should be considered to provide oral hygiene. Among dental hygiene products, dentifrices generally contain abrasive chemicals and/ or surfactants. There are many conflicting results about the plaque removal effect of these combinations. In addition, research has shown that abrasion of dentifrice does not lead to an increase in plaque removal. This result is also supported by the American Dental Association Division of Science. Similarly, there is not enough evidence of the role of detergents (surfactants) in the plaque removal activity of dentifrices. Fluoride content is also very important in oral hygiene products. The contribution of fluoride to caries prevention is well known. However, fluoride did not show a standard and sustained effect on the control of gingivitis. For this reason, it has been suggested that herbal products with antimicrobial, antiplaque, and anti-inflammatory properties be added to eliminate some of the insufficient properties of chemical-based dentifrices and mechanical plaque removal using toothbrush. Since the most commonly used oral hygiene device is the toothbrush, it would be possible to consider dentifrice as the best delivery method for herbal products that will provide oral hygiene. However, herbal products that can provide both oral hygiene and therapeutic properties in some oral diseases have not yet been successfully formulated to dentifrice with a few exceptions. Problems of their formulation with herbal products are finding compatible constituents to keep the effectiveness of the product in addition to improving the taste, smell, and the other flavor characteristics in the dentifrice formulation [57, 58]. Recently, it is well known that there is an enormous interest to use different traditional herbal products for oral hygiene and dental health. As we summarized above, plants and plant products have great potential in preventing oral diseases and maintaining dental health. Therefore, the use of herbal products for oral hygiene and especially the production of dentifrice containing herbal products should be promoted. Consumers, dentists, and health professionals that are more informed about the use, safety, and effectiveness of the various traditional medicines and over-the-counter products will increase the use of herbal Herbal Products for Oral Hygiene 41 products effectively [59]. Our study provides short information on herbal products that can be used for oral hygiene. The identification of the active ingredients of those plants and their mechanisms of action may provide some useful leads for the development of more effective and safer compounds or extracts in daily used oral hygiene products [60]. 2.7 Conclusion Nowadays, most of the population prefers oral hygiene preparations that contain plant extracts due to safety problems and increased interest of individuals in natural-based products. When it comes to dental hygiene products, plants have been found to be safer and effective in the growth of dental plaque and tooth decay. Therefore, increasing the use of herbal products in mouth rinses and toothpastes will be useful in the control of dental caries. However, the knowledge and understanding of herbal products and traditional uses is still an ongoing process with respect to their effectiveness and safety. Further long-term studies should be carried out to find suitable herbal formulations and provide their compliance with consumers and patients. References 1. van der Weijden, F. and Slot, D.E., Oral hygiene in the prevention of periodontal diseases: The evidence. Periodontol. 2000, 55, 104–23, 2011. 2. Müller, F., Shimazaki, Y., Kahabuka, F., Schimmel, M., Oral health for an ageing population: The importance of a natural dentition in older adults. Int. Dent. J., 2, 7–13, 2017. 3. Bodiba, D., Szuman, K.M., Lall, N., The Role of Medicinal Plants in Oral Care, Medicinal Plants for Holistic Health and Well-Being, Lall, N. (Ed.), Elsevier, pp. 183–222, 2018. 4. Farrimond, B. and Oral Hygiene & Nursing Care, CNS for Head & Neck Oncology, 2008. 5. Abbate, J. and Guynup, S., Oral health goes modern. J. Sci. Am., 19, 4–5, 2016. 6. Rajagopalan, A., Herbal Products in Oral Hygiene Maintenance—A Review. IOSR J. Pharm., 5, 48–51, 2015. 7. 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Van Der Veijden, F. and Slot, D.E., Oral hygiene in the prevention of periodontal diseases: The evidence. Periodontol. 2000, 55, 104–123, 2011. 58. Ahmed, J., Shenoy, N., Binnal, A., Mallya, L.P., Shenoy, A., Herbal oral care: An old concept or a new model? Int. J. Res. Med. Sci., 2, 818–821, 2014. 60. Srinivasan, K. and Chitra, S., Holistic Dentistry: Natural approaches to Oral Health. Sch. Bull., 1, 267–270, 2015. 3 Go Green—Periodontal Care in the Natural Way Siddhartha Varma* and Sameer Anil Zope Department of Periodontology, School of Dental Sciences, Krishna Institute of Medical Sciences Deemed to be University, Karad, India Abstract Ayurvedic medicine, a method of Hindu customary medicine indigenous to India, is gradually engrossing much interest in developed countries such as the United States, Europe, and Japan. Ayurveda basically encompasses the treatment of various ailments by the use of plant-based medicines. The naturally present phytochemicals in these medicines offer an efficient substitute to antibiotics and therefore signify a promising alternative approach in the prevention and remedial strategies for oral infections. Periodontal diseases, one of the most prevalent oral diseases among adults worldwide, are considered to be the commonest cause of tooth loss. A range of antimicrobials and chemotherapeutic agents like triclosan, cetylpyridinium chloride, and chlorhexidine have been used in the treatment of periodontal diseases. Dentists face an uphill task in treating periodontitis because of its complexity in disease process and multifactorial etiology. Hence, herbal therapy has been sought to achieve antimicrobial, anti-inflammatory, and other beneficial effects to tackle periodontal diseases. This gradually led to an increasing popularity of complementary and alternative medicine over conventional allopathic approaches. Publicity of such ancient practices through an appropriate platform would boost the confidence among the general population in maintaining good oral health. Keywords: Ayurveda, gingivitis, herbal medicine, periodontitis 3.1 Introduction Dental caries and periodontal diseases are known to be the two most significant oral health problems worldwide, even though other conditions like oral cancers are also of major concern [1]. Approximately 90% of the people across the globe show prevalence of periodontal diseases, which are considered to be one of the most common reasons for tooth loss amongs the adult population [2]. The prevalence of periodontal disease in India has been investigated by national research scholars since 1940. Akhilesh H et al. in his systematic review stated that some parts of Assam, Uttar Pradesh, and West Bengal reported more than 85% prevalence of periodontal disease with 97.51% as the highest prevalence [3, 4]. Periodontal diseases are known to be one of the most universal infectious diseases of mankind, which result in destruction of the supporting structures of the tooth. Periodontal *Corresponding author: siddhartha_varma@yahoo.co.in Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (45–60) © 2020 Scrivener Publishing LLC 45 46 Natural Oral Care in Dental Therapy disease is multi-factorial in nature with periodontal microbes playing a main role in the disease process. The presence of various types of microbes in the dental plaque eventually leads toward the activation and recruitment of neutrophils. These neutrophils additionally upregulate pro-inflammatory cytokines and release some reactive oxygen species (ROS) and neutrophilic enzymes. The chronic exposure of the connective tissue to such insults leads to the destruction of the periodontal ligament and the alveolar bone further resulting in tooth loss [5]. Management of the periodontal disease includes both surgical and non surgical means. The key to halt periodontal disease progression is by effective plaque control either mechanically by the use of toothbrush/floss or chemically by the use of mouthrinses [6]. Nevertheless, lack of required skill and motivation among the majority of the population renders mechanical plaque control ineffective; consequently highlighting the adjunctive use of chemicals for plaque control [7]. A range of antimicrobial and chemotherapeutic agents like cetylpyridinium chloride and chlorhexidine have proven to be effective in the treatment of periodontal diseases. Dentists face an uphill task in treating periodontitis because of its complexity in disease process and multifactorial etiology. The steady increase in incidence of periodontal disease coupled with augmented antibiotic resistance by periodontal pathogens warrants a comprehensive alternative approach which is safe, efficient, and cost effective. An array of ayurvedic and herbal preparations are being used for oral hygiene procedures as well as for management of different oral diseases, which include, chewing sticks, herbal brushes (Babool, miswalk, neem, and mango), aloe vera, turmeric, ginger, amla, and garlic, etc. [8]. Properties like broad biological action, low cost, and superior safety margin of the herbal products make them more preferable over conventional drugs. Active ingredients of plants and their materials used in herbal preparations or products are perceived to have therapeutic benefits, which can achieve antimicrobial, antiseptic, antioxidant, antiinflammatory, and anti-collagenase effects [9]. A gaining popularity of complementary and alternative medicine above traditional allopathic medicine is visible in the recent years, which could be attributed to the products being natural and safe. Considering the importance of herbal medicines, the present section highlights the potential role of Ayurveda in the treatment of various periodontal conditions. 3.2 Plaque Control It is a well-established fact that the most basic aspect in preventing periodontal diseases is an effective plaque control technique, which includes both mechanical means like brushing and chemical means like mouthwashes. Ayurveda advocates various therapeutic measures on a daily basis for managing oral health, which is comprised of Gandoosha (gargling) or oil pulling, Jivha Lekhana (Tongue scrapping), and Dant Dhavani (Brushing). 3.3 Dant Dhavani (Brushing) Avurveda suggests the use of chewing sticks daily in the morning and after every meal to thwart oral diseases. The use of chewing sticks was widely practised since ancient times in India, Middle East, and Africa. Datun is considered to be an excellent substitute for Go Green—Periodontal Care in the Natural Way 47 toothbrush for dental plaque removal and prevention of oro-dental diseases. Its major advantages are: it is economical, possesses numerous therapeutic properties, and its ease of availability in most of the rural regions of India. It is a tool that necessitates no proficiency or particular assets for its fabrication and marketing [10]. The fresh stems of neem (Azadirachta indica) and babool are famously used for chewing sticks. Liquorice (Glycyrrhiza glabra), milkweed plant (Calotropis procera), Arjuna tree (Termmalia arjuna), black catechu (Acacia catechu Linn.), and fever nut (Caesalipinia bouduc) are the other possible options for chewing sticks [11]. The twigs from a healthy tree ought to be soft and with no leaves or knots. They must exhibit properties like “kashaya” (astringent), “katu” (acrid), or “tikta” (bitter) in taste. As per ayurvedic practices, the herbal brushes need to be approximately 9 inches in length and width equalling a little finger. The technique of usage involves crushing one end of the stick followed by chewing and eating it gradually [12]. Chewing the stems allegedly assist salivary secretions thereby probably achieving better plaque control and a clean oral cavity. Neem extract contains the alkaloids margosine, resins, gum, chloride, fluoride, silica, sulfur, tannins, oils, saponins, flavonoids, sterols, and calcium thereby exhibiting significant and higher antiplaque efficacy compared to ayurvedic tooth powder and commercial toothpastes. Contemporary research has revealed the medicinal and anti-cariogenic properties of chewing sticks as initially depicted in Avurveda texts (Circa 200 BC) [13]. Mango leaves are also widely used for cleaning teeth in rural areas of southern India. Mango leaves contain mangiferin, a compound that has a considerable antibacterial property against few strains of Streptococci, Staphylococci, Pneumococci, and Lactobacillus acidophilus. Mangifera indica consists of tannins resins and bitter gum. Extract of mango chewing sticks at higher concentration showed more antimicrobial activity [14]. The use of miswak, a chewing stick obtained from the tree of Salvadora persica, is predominant in muslim countries and predates the inception of Islam. Al-Otaibi et al. concluded that using miswak was more effective than an ordinary toothbrush provided that proper instructions are followed regarding its usage [15]. An in vivo study by Almas and Zeid (2004) assessed the efficacy of miswak on S. mutans, and lactobacillus concluded the instant antimicrobial effect of miswak [16]. 3.4 Jivha Lekhana (Tongue Scrapping) Tongue scraping is one of the most commonly ignored ayurvedic recommendation. Tongue scraping helps in removing the tongue coating, which can lead to halitosis due to the presence of microorganisms, and which can also have an impact on the systemic health. As described in Ayurveda, inappropriate eating, digestive problems, or an evidence of discrepancy in the gastrointestinal system results in the buildup of residual toxins in the body known as Ama. An easy practice of tongue scraping helps in eradicating Ama from our physiology. As per Charaka Samhita, the tongue scrapers must be prepared either from silver, tin, gold, copper, and brass and should be blunt and curved to prevent any trauma to the tongue. Scraping the tongue results in the stimulation of the reflex points and thereby improving the taste sensation and increased secretion of digestive enzymes. Scientific data also confirms that regular use of a tongue scraper considerably eliminates anaerobic bacteria and decreases halitosis [17]. 48 Natural Oral Care in Dental Therapy 3.5 Gandusha (Gargling) or Oil Pulling Oil pulling as described in Ayurveda involves swishing the oral cavity with oil to maintain oral and systemic health. Gandusha as mentioned in Charaka Samhita claims to alleviate a range of systemic diseases like migrane, diabetes, and asthma, etc. Conventionally since many years, halitosis and gingival bleeding were widely treated by oil pulling. Oil prepared from sunflower and sesame is commonly used for oil pulling. Oil pulling therapy is extremely valuable in managing plaque-induced gingivitis when assessed in terms of clinical findings and microbial counts [18]. 3.6 Oxidative Stress in Periodontitis Oxidative stress is a significant source of cell damage, which is linked with the initiation and development of several chronic diseases including periodontitis [19]. The altered balance between levels of prooxidants and antioxidants in favor of the former results in probable tissue destruction. Molecules like superoxide anion, singlet oxygen, hydrogen peroxide, hydroxyl radical, hypochlorous acid, and nitric oxide produced by oxidation–reduction reactions of molecular oxygen by a variety of enzymes are termed as reactive oxygen species (ROS) [20]. In spite of the ability of all the body cells in generating ROS, polymorphonuclear neutrophils are primarily significant in relation to periodontitis. Evidence suggests that osteoclasts primarily involved in bone resorption process are regulated by ROS [21]. The NADPH oxidase system is majorly involved in cases of aggressive periodontitis and also plays a role in other periodontal pathologies [22]. All the cells of the human body are capable of storing antioxidants intrinsically, which can combat the oxidative stress by scavenging the free radicals as and when produced. A variety of antioxidant molecules consist of vitamins C, E, carotenoids, coenzyme Q, and enzymes like glutathione reductase, superoxide dismutase, glutathione transferase, and peroxiredoxin. Shirzaiy M et al. concluded that the patients who received periodontal therapy had a significantly low level of total antioxidant capacity compared to healthy controls [23]. Such observations have promoted the use of exogenous supplements in the form of herbal antioxidants for the management of periodontal diseases, which have been the focal point of research in current times. 3.7 Green Tea 3.7.1 Components The leaves of Camellia sinensis are processed with minimal oxidation to produce green tea. The health-promoting benefits of green tea are mainly attributed to the concentrated levels of polyphenols, which exhibit antioxidant properties [24]. These polyphenols can be primarily categorized into six types of catechins: catechin, gallocatechin, epicatechin, epigallocatechin, epicatechin gallate (ECg), and epigallocatechin gallate (EGCg). Among all the catechins, EGCg has been extensively researched owing to its extremely potent antioxidant property. In addition to the catechins, green tea includes compounds like carotenoids, Go Green—Periodontal Care in the Natural Way 49 tocopherols, ascorbic acid, minerals such as zinc, selenium, chromium, and certain phytochemicals [25]. 3.7.2 Beneficial Effects of Various Tea Components 3.7.2.1 Antioxidative Effect Green tea helps in inhibiting prooxidant enzymes and induces antioxidant enzymes, which lead to scavenging of ROS, nitrogen species. 3.7.3 Role in Managing Periodontitis Green tea catechins have proven to exhibit predominant effect on periodontopathogens. Literature suggests that catechins inactivate P. gingivalis-induced collagenase. Catechins in green tea successfully inhibit the growth of P. gingivalis, Prevotella intermedia, and Prevotella nigrescens and also thwarts adhesion of P. gingivalis onto buccal epithelial cells of humans as demonstrated in a few in vitro studies [26–28]. Bone resorption observed in periodontitis is due to interaction amid the osteoblasts and osteoclasts. EGCg was found to curb LPS-mediated gene expression of RANKL, cyclooxygenase-1 and the cytokine PGE2 in osteoblasts of mice. Catechins are remarkably effective in managing periodontal disease by suppressing the inflammatory response-mediated bone resorption [29]. Green tea when used in the form of a dentifrice or a local drug delivery agent, improved the oral health status of chronic periodontitis patients [30, 31]. Similarly, green tea when used as a mouthwash for a period of 1 week had a similar antiplaque efficiency to that of chlorhexidine gluconate, which is considered to be a gold standard [32]. These encouraging results demonstrate that additional research is essential to explore the advantages of green tea in the managing of periodontal diseases. 3.8 Turmeric (Curcumin longa, Haldi) Turmeric is well known as sarvoshadhi in sanskrit, meaning medicine for all diseases. Turmeric has been traditionally used in India for the past 2500 years and is known for its actions like antibacterial, anti-inflammatory antiseptic, pain killer, and hepatoprotector. Recent research in humans illustrates that the precancerous changes in oral submucous fibrosis patients can be reversed with the use of turmeric extract and turmeric oil [33]. 3.8.1 Applications of Turmeric in Dentistry Turmeric encloses roughly 3–4% curcumin by dry weight [34]. 1. Rinsing the oral cavity with turmeric water (5 g of turmeric powder, two cloves, and two dried leaves of guava in 200 g of water) provides an instant pain relief. 2. A powder made from burnt turmeric pieces and Bishop’s weed seed when used intra-orally provides strength to the teeth and gums. 50 Natural Oral Care in Dental Therapy 3. A paste prepared with 1 tsp of turmeric, ½ tsp of mustard oil, and ½ tsp of salt used two times daily by rubbing on the gums provides relief from gingivitis and periodontitis. 4. Roasted and ground turmeric when massaged on aching teeth reduces pain and swelling. 5. Dental plaque detection system: It consists of curcumin and turmeric extract and yellow pigment of beni-koji, along with a light-emanating apparatus, which emits a 250- to 500-nm wavelength light [35]. 3.9 Amala (Emblica officinalis, Amalaki, Phyllanthus emblica, Indian Gooseberry, Dhatriphala) Ayurveda considers amla, a well-known rasayana herb to maintain balance between all the three doshas (vat, pit, and kuff). Amla extract can be taken orally per day (0.5 g) for long-term benefits for the teeth and gums. Vitamin C present in amla acts as a cofactor in the conversion of proline into hydroxyproline, which is one of the essential constituents of connective tissue [36]. Chyawanprash and triphala are generally the two frequently utilized ayurvedic preparations containing amla. Its antioxidant and astringent properties are demonstrated to be valuable in the management of toothache, apthous stomatitis, mouth ulcers, and gingival inflammation [37]. 3.10 Anar/Dalima (Punica granatum) The pomegranate fruit including its peel, seeds, oil, powdered extracts, and juice have medicinal properties and exhibit no side effects. Pomegranate flavonoids have anti-inflammatory and antibacterial properties, and pomegranate polyphenols have antioxidant and antiviral properties [38]. The antibacterial action of Punica granatum is attributed to the presence of ellagitannin and punicalagin [39]. Studies have showed that pomegranate extract, as a mouthrinse, showed the capacity to remove dental plaque, increased antioxidant activity, decreased activity of aspartate aminotransferase and proteins [39, 40], and inhibited major periodontal pathogens like Aggregatibacter actionomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia [41–43]. Pomegranate extract gel when used as an adjunctive treatment option along with scaling and root planing significantly reduces the inflammatory status of gingiva [44]. 3.11 Launga/Clove (Syzygium aromaticum) Clove oil can be used to treat oral ulcers and sore gums due to its gentle anesthetic property. It also battles oral infections and inflammation because of its strong antibacterial activity and even decreases halitosis [45]. Eugenol is considered as an active component (including β-caryophyllene) and also contains a variety of flavonoids; hence, it is extensively used as temporary fillings, in conjunction with root canal therapy, intra-oral abscesses, and painful gums [46, 47]. Go Green—Periodontal Care in the Natural Way 51 3.12 Gotu Kola (Centella asiatica) Its property in promoting growth of connective tissue aids in wound healing and thus is effective in the treatment of oral ulcers. According to Sastravaha et al. treatment of chronic periodontitis with Centella asiatica and P. granatum in the form of biodegradable chips showed a significant improvement in decreasing plaque, periodontal pocket depth, and loss of attachment at the end of 3 months in comparison to a placebo [48]. Gotu kola can also be used in enhancing recuperation after laser surgical procedures used for treating severe periodontal disease. The daily suggested dosage is 30 mg of triterpenoids two times, and it may vary depending on the content of triterpenic acid [49]. 3.13 Amra/Mango (Magnifera indica) Mango leaves exhibit anti-bacterial properties due to the presence of ascorbic and phenolic acids. It was found to be effective against anaerobic microorganisms such as Prevotella intermedia and Porphyromonas gingivalis and therefore can be used as an adjunct in oral hygiene maintenance [50]. 3.14 Neem (Azadirachta indica) The anti-bacterial, anti-inflammatory, analgesic, antioxidant, antifungal, antiviral, and immunostimulant properties of neem have been well documented in literature [51]. It compasses mechanical and chemotherapeutic antiplaque agents. Anirban Chatterjee et al. evaluated the antiplaque efficacy of neem as oral rinse on gingivitis when used an adjunctive therapy. They concluded that the presence of gallotannins in neem could effectively decrease the bacterial count in dental plaque [52]. Antibacterial plant extracts have an advantage over antibiotics as they produce no allergy nor exhibit any resistance. An herbal mouthrinse formulation prepared with active fractions from Azadirachta indica, Citrullus colocynthis, and Cucumis sativus extract with a carrier or additive was effective in preventing gingivitis and periodontal diseases [53]. 3.15 Tulsi (Ocimum sanctum) Tulsi is found on a large scale in India, Malaysia, West Africa, Australia, and some of the Arab countries. Majority of the therapeutic actions of tulsi are present in its leaves, while other parts of the plant like flowers, stem, root seeds, etc., are also known to have some medicinal potential. The extract of tulsi leaves includes 0.7% volatile oil, which comprises 71% eugenol and 20% methyl eugenol. The anti-inflammatory activity of tulsi against prostaglandin E2 (PGE2), leukotriene, and arachidonic acid-induced edema has been found due to the presence of fixed oil and linolenic acid [54]. Tulsi leaves when chewed raw help in maintaining oral hygiene. Carracrol, Tetpene, and Sesquiterpene b-caryophyllene are the antibacterial agents present in this plant. The powder of sun-dried tulsi leaves or a mixture with mustard oil can be used as a dentrifice. Tulsi was also proven to be effective in 52 Natural Oral Care in Dental Therapy counteracting halitosis. The anti-inflammatory property of tulsi makes it an appropriate medication for treating gingivitis and periodontitis [55]. 3.16 Nilgiri (Eucalyptus globulus) Eucalyptus extract is known to have anti cariogenic and anti-plaque property. Chewing gum containing Nilgiri demonstrated a considerable positive consequence on dental plaque accumulation, gingival index, bleeding on probing, and periodontal probing depth [56]. Essential oils have been gathering recognition since the past decade especially for the prevention and management of several infections. The antibacterial action of Eucalyptus oil in the management of colds, influenza, other respiratory infections, rhinitis, and sinusitis has been well recognized for ages [57]. Ragul et al. stated that mouthwash containing eucalyptus oil was similarly effective as chlorhexidine mouthwash (0.12%) in plaque control, and therefore, it could be considered as a good and economical oral hygiene aid [58]. 3.17 Tila/Sesame (Sesamum indicum) It is rich in vitamin E and flavonoid phenolic antioxidants. Ashokan et al. conducted a study on oil pulling therapy using sesame oil in managing gingivitis. They concluded that sesame oil significantly decreased the number of aerobic microorganisms, plaque index, and modified gingival scores in adolescents suffering from plaque-induced gingivitis [59]. 3.18 Triphala The dry fruits of Amalaki (Emblica officinalis), Haritaki (Terminalia chebula), and Bahera (Terminalia belerica) together constitute Triphala. At a concentration of 1500 µg/ml, it potently inhibits PMN-type collagenase, especially MMP-9. It has shown good antimicrobial, antioxidant and anti-collagenase activities and is also used for strengthening the gums [60]. The antioxidants in Triphala decrease the oxidative burden and prevent free radical-induced cell damage. Among the three medicinal plants in triphala, Bahera is the strongest antioxidant followed by Amalaki and Haritaki. A randomized clinical study has demonstrated that Triphala mouthwash is as efficient as chlorhexidine (0.2%) in antiplaque and anti-inflammatory actions [61]. 3.19 Tea Tree Oil (Melaleuca Oil) Tea tree oil (TTO) with antibiotic properties helps in curing the infection of periodontal disease. It has been used to treat severe chronic gingivitis, bleeding gums, and halitosis, as they deeply penetrate into the skin. Elgendy et al. in their randomized clinical trial, used tea tree oil gel for local application in chronic periodontitis patients and proved it to be beneficial in augmenting the results of SRP. Furthermore, they emphasized on Go Green—Periodontal Care in the Natural Way 53 monitoring pentraxin (PTX3) levels in GCF as a marker of tissue healing following periodontal therapy [62]. 3.20 Rumi Mastagi/Mastic Gum (Pistacia lentiscus) It is recognized for its strong anti-inflammatory, antioxidant, and bactericidal effects. Koychev et al. studied the possible use of mastic gum for improvement of periodontal health. They concluded that mastic extract significantly inhibited potential periodontal pathogens, namely, P. gingivalis, S. oralis, A. actinomycetemcomitans, F. nucleatum, and P. intermedia and also exhibited a valuable effect on cell viability on comparison with H2О2. Therefore, it could be considered a viable antibacterial agent in the management of periodontal disease [63]. 3.21 Wheat Grass Wheatgrass is the young grass shoots of the Triticum aestivum plant and exhibits antioxidant, antibacterial, and anti-inflammatory properties. Wheatgrass juice or wheatgrass supplement is known to prevent gingivitis and periodontal disease thereby eliminating bacteria in the oral cavity [64]. 3.22 Goldenseal (Hydrastis canadensis) Bererine in goldenseal is an effective antibacterial, antiviral, and antifungal agent. Goldenseal acts as an astringent and helps in treating gingivitis. Using a rinse made from one teaspoon dissolved in water or applying the goldenseal powder directly to the gums is believed to be effective for many gum problems, including canker sores. Bhandari et al. in their in vitro study, established that goldenseals extract exhibits promising antibacterial activity against selected periodontal pathogens [65]. 3.23 Licorice Root The American Dental Association considers that the antibacterial and antiviral properties of licorice restrains plaque buildup and therefore helps in the management of periodontal disease, oral ulcers, and canker sores. Licorice root powder, paste, and decoction for mouthwash are one of the best for managing inflammation in periodontal disease [66]. Licorice root can cause severe side effects and some unfavorable drug interactions and therefore should be used with a degree of caution and in consultation with a health care specialist. Alaa Omran Ali et al. in their recent study, concluded that a mouthwash containing liquorice extract reduced the amount of plaque and gingival inflammation, and it can be used for a prolonged duration with no side effects as an adjunct to scaling and root planing in the management of periodontal disease [67]. 54 Natural Oral Care in Dental Therapy 3.24 Myrrh (Commiphora glileadenis) Myrrh has been used since prehistoric times for a variety of medicinal remedies like inflamed gums, canker sores, throat and nasal infections, and breathing issues. Hence, one of the excellent methods to lessen bacteria and preserve oral health is using a mixture of myrrh oil with warm water as a mouthwash. Hossam A Eid et al. stated that the myrrh plant extract showed potential antibacterial property. It also exhibited significant results in controlling the biofilm of S. mutans, which supposedly plays a major role in the etiopathogenesis of dental caries and periodontal diseases [68]. 3.25 Psidium guajava Guava is principally rich in Vitamin C (ascorbic acid) and therefore exhibits an excellent antioxidant property. Its antioxidant action is augmented by the presence of carotenoids, quercetin, and polyphenols [69]. The extract of guava leaves and essential oil from its stem act as scavengers and aid in the inhibition of hydroxyl radicals [70, 71]. A decoction prepared from the root bark or leaves can be recommended as a mouthrinse, which can effectively mange bleeding gums [72]. 3.26 Ginkbo Biloba G. biloba (EGb) leaf extract is used extensively as an herbal nutritional supplement in the US. It is composed of terpenoids, ginkgo flavone glycosides, and ginkgolic acid (less than 5 ppm). It acts as a scavenger of the free radicals, lowers oxidative stress, and exhibits antiinflammatory effect [73]. Sezer U et al. stated in ligature-induced periodontitis rat model, systemic administration of EGb (28–56 mg/kg/day) reduced osteoclastic counts, decreased inflammation, and induced osteoblastic activity [74]. 3.27 Honey Honey was recommended since primeval times in various cultures to manage infections and many medical ailments. The antibacterial factors of honey are mainly due to the hyper osmolarity effect (>80% sugar content), bee defensin-1, methylglyoxal, hydrogen peroxide, acidic pH, a range of phenolic and proteinaceous compounds, and flavonoids. However, the primary antimicrobial activity of honey is due to the presence of hydrogen peroxide [75]. It helps in the maintenance of healthy gums because of the rich presence of minerals, vitamins, and other vital nutrients. Duailibe et al. concluded that propolis mouthwash exhibited potent antimicrobial activity against Streptococcus mutans and can be prescribed as an alternative to conventional mouthwashes in the management of dental caries and periodontal diseases [76]. Based on clinical and microbiological parameters, Amita Coutinho et al. reported that sub-gingival irrigation with propolis extract (20%) as an adjunct to periodontal treatment Go Green—Periodontal Care in the Natural Way 55 was more effective than scaling and root planing alone [77]. Similarly Atwa AD et al. suggested that honey (topical application/chewing) is an effective substitute to conventional remedies in preventing gingivitis and dental caries following orthodontic treatment [78]. 3.28 Other Herbs Which Can Be Potentially Used for Treating Periodontitis Cymbopogon citratus (lemon grass), Allium sativum (garlic), Eucalyptus globulus (eucalyptus), Murraya koenigii (curry leaves), Shiitake mushrooms lentinan, etc., can also be used. Even though several studies have revealed the effectiveness of herbal medicines as a substitute to established therapeutic procedures, the clinical application of such agents is yet to be supported by ample evidence. 3.29 Conclusion As per Ayurvedic classics, a variety of herbs are recommended for plaque control and management of periodontal diseases. According to estimates of the World health Organization (WHO), around 75% people worldwide use herbs for their fundamental health care needs. WHO has recommended for the incorporation of the traditional systems of medicine like Ayurveda into the primary health care system, among communities who accept it (Table 3.1). All the ayurvedic medicines and local remedies discussed above are easily accessible in the rural areas where socioeconomic conditions of the people are not good. Ayurveda must be reinterpreted in the light of new emerging knowledge, and it must be Table 3.1 Classification of Herbal Medicines [79]. Category I: Indigenous herbal medicines Includes medications well recognized by the local inhabitants/society since historic times in perspective to their dosage, content, and treatment options. Category 2: Herbal medicines in systems Medicines appropriately recognized by individual countries on the basis of well-established concepts and theories. E.g.: Ayurveda, Siddha, and Unani Category 3: Modified herbal medicines Herbal medicines, which are customized with respect to their composition, administration mode, dosage, and shape and fulfilling the safety and efficacy requirements of respective national regulatory bodies along with those depicted in categories 1 and 2. Category 4: Imported products with an herbal medicine base Herbal medicines either in the form of final products or raw materials with essential efficacy and safety information at an authorized national center in the country of import. 56 Natural Oral Care in Dental Therapy incorporated in modern medicine along with other forms of traditional medicine. The key is to figure out what works best for the treatment of the patient. Additional studies on plaque-inhibiting effect of various herbal formulations are obligatory to establish their duration of action. 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J. Periodontol., 79, 8, 1378–1385, 2008. 57. Warnke, P.H., Sherry, E., Russo, P.A., Antibacterial essential oils in malodorous cancer patients: Clinical observations in 30 patients. Phytomedicine, 13, 463–467, 2006. 58. Ragul, P., Dhanraj, M., Jain, A. R., Efficacy of eucalyptus oil over chlorhexidine mouthwash in dental practice. Drug Invent. Today, 10, 5, 638–641, 2018. 59. Asokan, S., Oil pulling therapy. Indian J. Dent. Res., 19, 169, 2008. 60. Abraham, S., Kumar, M.S., Sehgal, P.K., Nitish, S., Jayakumar, N.D., Evaluation of the inhibitory effect of Triphala on PMN-type Matrix Metalloproteinase (MMP-9). J. Periodontol., 76, 497–502, 2005. 61. Naiktari, R.S., Gaonkar, P., Gurav, A.N., Khiste, S.V., A randomized clinical trial to evaluate and compare the efficacy of triphala mouthwash with 0.2% chlorhexidine in hospitalized patients with periodontal diseases. J. Periodontal. Implant. Sci., 44, 134–140, 2014. 62. 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Kalyana Chakravarthy1*, Komal Smriti2 and Sravan Kumar Yeturu3 1 Department of Public Health Dentistry, Manipal College of Dental Sciences, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India 2 Department of Oral Medicine and Radiology, Manipal College of Dental Sciences, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India 3 Public Health Dentistry, Amrita School of Dentistry, Amrita Vishwa Vidyapeetham, Cochin, Kerala, India Abstract Potentially malignant oral disorders are visibly abnormal areas of the oral mucosa, which are white keratotic, raised roughened areas, which have the probability of malignant transformation. These may be a source of significant anxiety for the patient and the clinician. Though not all premalignant lesions transform into oral carcinomas, certain high-risk lesions have been identified such as leukoplakia, lichen planus, and oral submucous fibrosis. Their management remains controversially polarized between surgical excision to prevent malignant change and conservative medical or surveillance techniques. Drugs obtained from plant extracts, like lycopene, have been recently used in the treatment of leukoplakia and oral submucous fibrosis (OSMF). Beta-carotene and the retinoids are the most commonly used antioxidant supplements for chemoprevention. Beta-carotene is a carotenoid found primarily in dark green, orange, or yellow vegetables. Turmeric and its active ingredient “curcumin” are being studied upon as chemopreventive agents in the management of OSMF. Tulsi and turmeric offers a safe and efficacious combination of natural products for symptomatic treatment of both burning sensation and mouth opening in patients of OSMF. Ayurvedic preparations like Erandabhrishta Haritak and Pippalyadi Choorna have been used in OSMF. This chapter focuses on various modalities currently available and discusses the efficacy and safety of such herbal products and natural extracts in the management of potentially malignant oral disorders. Keywords: Potential, malignant, oral, dental, fibrosis, submucous, lichen planus, leukoplakia 4.1 Introduction Potentially malignant oral disorders are visibly abnormal areas of oral mucosa, which are white keratotic, raised roughened areas, which have a probability of malignant *Corresponding author: drkalyan81@gmail.com; kalyan.cp@manipal.edu Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (61–80) © 2020 Scrivener Publishing LLC 61 62 Natural Oral Care in Dental Therapy Natural or herbal products Potentially malignant disorders OSMF OLP OL Aloe vera Green tea extracts Aloe vera Beta–carotene Beta carotene Lycopene Lycopene Lycopene Turmeric extracts Colchicine Turmeric extracts Raspberry leaf extract Tea pigments Alpha-tocopherol Purslane Spirulina Chinese Herbal Medicines Chinese herbs Bowman-Birk inhibitor concentrate Turmeric extracts Nigella sativa Ocimum (Tulsi) Polyherbal formulations Ayurwedic formulations Figure 4.1 List of natural products for pharmacological management of potentially malignant disorders of the oral cavity. transformation. These may be a source of significant anxiety for the patient and the clinician. Many such lesions like oral submucous fibrosis, leukoplakia, and lichen planus have high risk of transformation to malignancies. Their management has a wide range of options, which include surgical excision, intra-lesional, topical, or systemic medication. Drugs obtained from plant extracts (beta-carotene and the retinoids) have been recently used in the treatment of leukoplakia and oral submucous fibrosis (OSMF). Tulsi, turmeric, and extracts are used in the management of OSMF. Pure ayurvedic preparations like Erandabhrishta Haritak and Pippalyadi Choorna have also been tried in OSMF (Figures 4.1 and 4.2). This chapter focuses on various modalities currently available and discusses the efficacy of herbal and natural extracts in the management of potentially malignant oral disorders. 4.2 Oral Submucous Fibrosis (OSMF) 4.2.1 Background OSMF is defined as an “insidious chronic condition of multifactorial etiology affecting the oral mucosa characterized by dense collagen tissue deposition within submucosa, occasionally extending to the pharynx and esophagus”. It has distinct clinical characteristics viz., “blanching and stiffness of oral mucosa, trismus, BS, loss of mobility of tongue and loss of gustatory sensation” [1]. The main etiological agents for OSMF are areca nut with or without tobacco and tobacco chewing in all forms. There is no ideal treatment protocol for the management of OSMF. Cessation of the habit is a prerequisite for planning the treatment. Khanna and Andrade suggested that early stages of OSMF can be treated effectively with various medical intervention, and advanced stages often require surgical Role of Herbal and Natural Products 63 TOBACCO AT WORK LEUKOPLAKIA ORAL SUB MUCOUS FIBROSIS ORAL CANCER LUNG BRAIN DEXTER CLUB I AM PLANNING TO TAKE ORAL ROUTE HEART HOW?? MAN .. I CAN ....... KILL A HUMAN RIGHT NOW ... BUT THERE ARE “THE GUARDIANS”... FOODS TO FIGHT CANCER PLANT EXTRACTS TO IMPROVE IMMUNE SYSTEM TOMATOS BERRY CARROT TURMERIC TULSI PEPPER SPIRULINA ALOE VERA TEA PIGMENTS Figure 4.2 Illustration showing the role of risk factors and natural products in potentially malignant disorders of the oral cavity. management. Medical interventions are administered as a variety of modalities: orally for systemic absorption, intra-lesional, or topical as single or combination of agents [2]. Many herbal formulations have been tried for the treatment of OSMF with variable efficacy. 4.2.2 Beta-Carotene Beta-carotene was shown to have properties that help in cancer prevention like anti-oxidant, immunomodulation, inhibition of mutagenesis, and growth of cancer cells. 64 Natural Oral Care in Dental Therapy Aggarwal et al. [3] analyzed serum beta carotene in OSMF patients and compared with age- and gender-matched controls. A significantly lower mean serum beta carotene was seen in OSMF than the control group. Similarly, lower levels of serum beta carotene were evident in grade III compared with grades I and II OSMF patients. Jirge et al. [4] evaluated the clinical and serum immunoglobulins (IgG, IgA, and IgM) effects of levamisole and Antoxid (beta carotene, zinc sulfate monohydrate, selenium dioxide, manganese, copper). Participants were divided into three groups, viz., levamisole only (50 mg thrice daily for 3 alternate weeks), Antoxid (twice daily for 6 weeks), combination of levamisole and Antoxid. Significant improvement in the mouth opening (MO) and reduction in burning sensation (BS) was seen in all the groups. Serum IgG, IgA, and IgM were significantly reduced in levamisole and combination groups. The Antoxid group showed significant reduction in serum IgA and IgM. Thriveni et al. [5] evaluated the effect of natural beta-carotene and revox in the oral management of OSMF patients. A total of 50 patients were divided into group A [hyaluronidase + hydrocortisone (bilaterally alternative week) and natural carotene (12 weeks)] and Group B [hyaluronidase + hydrocortisone (bilaterally in alternative week) and revox capsule (12 weeks)]. Both the groups were advised ice cream stick mouth exercises. Significant improvement was seen in mouth opening; reduced BS was seen in all the patients with early response in group A than in B. 4.2.3 Lycopene Lycopene is a potent antioxidant with very effective free radical scavenging property. Alternate anti-carcinogenic effects is through the regulation of communication at gap junction [6], induction of apoptosis, and modulation of carcinogen-metabolizing enzyme [7]. Kumar et al. [8] evaluated the efficacy of oral lycopene in 58 OSMF patients who were randomized into three groups. Patients of groups A, B, and C received lycopene, lycopene + intra-lesional steroids, and placebo, respectively. Significant improvement in mouth opening and BS was seen only in groups A and B. BS was relieved within 2 weeks use of oral lycopene in groups A and B. Karemore et al. [9] evaluated the role of lycopene in OSMF patients. A total of 92 patients were divided into two groups (lycopene and placebo). Significant improvement in mouth opening and reduced BS was seen with lycopene. Patil et al. [10] evaluated lycopene and AV in 120 OSMF patients. They were divided equally into the lycopene group (8 mg in two divided doses) and AV group (5 mg topical gel thrice daily for 3 months). Significant improvements in MO and tongue protrusion (TP) were seen in the lycopene group. BS, pain, swallowing, and speech improved in both the groups. Selvam et al. [11] aimed to evaluate oral lycopene therapy + conventional intra-lesional steroid injections in the management of OSMF. Forty-five OSMF patients (grades III and IV) were randomly divided into three groups viz., oral lycopene (16 mg/day), oral antioxidant capsules, and control group. All the three groups received twice a week intra-lesional steroids + hyaluronidase. There was complete relief of BS and significant increase in MO within 3 weeks among all the patients. Significant improvement in MO was seen with oral lycopene and oral antioxidant capsules compared with the control group. Supplementation of oral lycopene or oral antioxidants to the control intervention (intra-lesional steroids + hyaluronidase) was shown to be beneficial. Role of Herbal and Natural Products 65 Piyush et al. [12] compared lycopene, curcumin, and placebo in a randomized controlled parallel trial in 90 OSMF patients. They were randomly divided into three groups viz., curcumin (300 mg twice daily), lycopene (8 mg twice daily), and placebo groups, respectively. The mean difference in the MO, BS, and cheek flexibility (CF) scores was significantly higher in curcumin and lycopene groups than placebo. However, there was no significant difference in the mean difference in the TP among the groups. Saran et al. [13] compared the efficacy of lycopene and curcumin (Curcuma longa and Piper nigrum) given orally in 60 OSMF patients divided into two groups. After 3 months, there was complete cessation of BS in both the groups. No significant difference was seen between the groups with respect to the increase in the MO. Lycopene showed significantly higher improvement than curcumin. 4.2.4 Aloe Vera Aloe vera (AV) is an “emollient resin with many multitude of benefits like analgesic, antiinflammatory, immune modulatory, antioxidant, anti-neoplastic, and wound-healing properties” [14]. Considering the above properties, it was evaluated as a natural herbal alternative for the management of OSF. Alam et al. [15] conducted a randomized double blind placebo controlled trial to evaluate AV gel as an adjuvant in the treatment of OSMF. Sixty subjects were divided into medicinal (hyaluronidase + dexamethasone injections) and surgical groups. Each category was sub-divided into with and without AV supplementation. AV supplementation had a significant improvement in most symptoms than without AV in both medical and surgical groups. Anuradha et al. [16] evaluated AV in the treatment of OSMF in 74 patients randomly divided into two groups (systemic juice + topical AV for 3 months versus intra-lesional hydrocortisone + hyaluronidase injections for 6 weeks with antioxidants for 3 months). Both the groups showed significant reduction in BS and increased MO, CF, and TP at the end of 3 months. Patil et al. [17] compared spirulina and AV in the management of OSMF. All the subjects (n = 42) were randomly divided into spirulina (500 mg in two divided doses for 3 months) and AV groups (5 mg topical gel thrice daily for 3 months). The spirulina group showed significant improvement in MO and ulcers/erosions/vesicles. There was no difference in the improvement in BS and pain associated with the lesion between the groups. Patil et al. [18] compared the oxitard (two capsules twice daily) and AV (5 mg topical gel thrice daily for 3 months) in 120 OSMF subjects. Significant improvements in MO, TP, pain associated with the lesion, difficulty in swallowing, and speech were seen in the oxitard group. No difference in the BS was seen between the groups. Singh et al. [19] compared AV versus antioxidant supplementation with physiotherapy in the management of OSMF. A total of 40 subjects were divided into AV with physiotherapy and antioxidant with physiotherapy. Significant improvement in MO, TP, CF, and reduction in burning scores was seen with AV gel with physiotherapy in comparison with antioxidants with physiotherapy. Sudarshan et al. [20] compared the AV and antioxidants in the treatment for OSMF. Twenty OSMF subjects were divided into two groups (5 mg of topical AV gel, thrice daily for 3 months versus antioxidant capsules twice daily for 3 months). There was significant reduction in the BS and improvements in MO and CF in the AV group than in the antioxidants. 66 Natural Oral Care in Dental Therapy Al-Maweri et al. [14] conducted a systematic review on the effectiveness of AV in the treatment of OSMF. Six randomized controlled trials were identified out of which five studies used only topical gel, while one study used both topical and systemic AV routes. Metaanalysis showed significant differences in favor of AV in reducing pain/BS at the end of the first and second months, but no such effect was seen at the end of the third month. No significant difference was seen with respect to objective clinical outcomes between the groups. AV reduced pain/BS. However, marked heterogeneity was seen among the included studies. 4.2.5 Colchicine Colchicine is well known for its antifibrotic property. It inhibits collagen synthesis and disrupts the microtubule formation and depolymerizes microtubules preventing the extrusion of collagen fibers from the fibroblasts. Krishnamoorthy and Khan [21] studied the effects of colchicine in 50 OSMF who were divided randomly into two groups viz., colchicine group (0.5 mg twice daily and intralesional hyaluronidase once a week) and Hyaluronidase + Hydrocortisone group (once a week intra-lesional, alternatively). Inter-group comparisons with respect to MO and histological parameters indicated that colchicine, with hyaluronidase responded well than hyaluronidase + hydrocortisone. In both the groups, BS was relieved in all the patients. Daga et al. [22] compared the effectiveness of oral colchicine with intra-lesional injection of hyaluronidase or injection triamcinolone acetonide in 30 grade II OSMF patients randomly divided into two groups (colchicine 0.5 mg twice daily + weekly intra-lesional hyaluronidase versus colchicine 0.5 mg twice daily + weekly intra-lesional triamcinolone for 12 weeks). Improvement in MO and reduction in BS was significant in oral colchicine with hyaluronidase. However, improvement in blanching of mucosa was seen in both the groups. 4.2.6 Tea Pigments Tea pigments have polyphenols that are derived from tea leaves. Li et al. [23] conducted a study in 39 OSMF patients to evaluate tea pigments. The control group was administered with vitamins A, D, E, and B complex, and the experimental group was administered tea pigments along with the vitamins in the control group. Tea pigments have antioxidant property and improve microcirculation in these patients. The results of the experimental group were significantly better than that of the control group. The results indicated that tea pigment may possibly become a better therapy for the OSF patients with abnormal hematological parameters. 4.2.7 Spirulina Spirulina is a microalgae, which is rich in proteins, carotenoids and micronutrients that are known to have antioxidant properties [17]. Shetty et al. [24] evaluated spirulina as an adjunct in the management of OSMF among 40 patients divided into spirulina group (500 mg twice daily + biweekly intra-lesional Betamethasone) and placebo group (placebo capsules twice daily + biweekly intra-lesional Betamethasone). Significant improvement in MO and BS was Role of Herbal and Natural Products 67 seen in both the groups. Inter-group comparison showed that MO and BS were significantly in favor of the spirulina group at the end of 3 months. Patil et al. [17] compared the spirulina and AV in the management of OSMF. Subjects were divided into the spirulina group (500 mg spirulina in two divided doses for 3 months) and AV group (5 mg AV topical gel, thrice daily for 3 months). The spirulina group showed significant improvement in MO and ulcers/erosions/vesicles, while no improvement was seen with BS and pain associated with the lesion between the two groups. Mulk et al. [25] studied the efficacy of spirulina and pentoxyfilline in the treatment of OSMF in 40 subjects divided into Pentoxyfilline and Spirulina groups. Both the groups showed significant results in the improvement of MO and TP and reduction in BS. Intergroup comparisons showed no significant difference in the improvement of MO and TP between the two groups. A significant higher reduction in BS was seen in spirulina. Zwiri et al. [26] compared pentoxifylline and spirulina in the management of OSMF in 112 subjects. Subjects were divided into pentoxifylline (400 mg pentoxifylline twice daily) and spirulina (500 mg spirulina in two divided doses) groups. The patients in both the groups showed improvement in all the parameters measured. Significant improvement in MO, reduction in pain associated with the lesion, and BS were seen in the pentoxifylline group, while the spirulina group showed significant improvement in ulcers/erosions/ vesicles. 4.2.8 Chinese Herbal Medicines Salvianolic acid B [27] is the most bioactive component of Radix salvia miltiorrhizae, which is a popular traditional Chinese medicine. Previous in vitro study have shown the antifibrotic activity of salvianolic acids A and B [28]. Panax notoginseng saponin is another Chinese herb, which also showed excellent anti-fibrotic activity in vitro [29]. Wu et al. [30] evaluated the therapeutic effect of Salvia miltiorrhiza and prednisolone in 120 medium and advanced stage OSMF patients. Subjects were divided into Salvia miltiorrhiza + prednisolone and prednisolone groups. Significant decrease in the lesion area and increase in MO were seen in both medium and advanced stages in the Salvia miltiorrhiza + prednisolone group. However, such improvements were seen only in the medium stages of OSMF when treated with prednisolone only. No difference was seen in the advanced stages of OSMF. Combined Salvia miltiorrhiza + prednisolone therapy showed better efficacy in advanced stages of OSMF than prednisolone only, while medium stages of OSMF did not have any difference between the groups. Jiang et al. [31] conducted a randomized clinical trial for the effectiveness of intra-lesional triamcinolone acetonide, intra-lesional salvianolic acid B, and combined intra-lesional triamcinolone acetonide + salvianolic acid B in the treatment of OSMF for 20 weeks in 42 subjects. Combined therapy (triamcinolone acetonide + salvianolic acid B) was more effective than intra-lesional triamcinolone acetonide or salvianolic acid B alone in the improvement of MO and reduction of BS. Jiang et al. [32] evaluated the efficacy of allicin in a randomized clinical trial among 48 stage II OSMF subjects. Subjects were randomized into intra-lesional Triamcinolone acetonide or allicin groups. Allicin group significantly improved MO, BS, and oral health-related quality of life than Triamcinolone acetonide at the end of 40 weeks. 68 Natural Oral Care in Dental Therapy 4.2.9 Turmeric and Derivatives, Nigella sativa, Ocimum The active ingredient in turmeric is curcumin, which is a chemopreventive agent. It can act both as a scavenger or catalyzes the formation of hydroxyl radicals. Hastak et al. [33] studied the effect of turmeric, turmeric oil, and turmeric oleoresin in OSMF patients for 3 months. All the three derivatives decreased the number of micronucleated cells both in exfoliated cells of oral mucosal and circulating lymphocytes with Turmeric oleoresin as the more effective. Das et al. [34] evaluated the efficacy of curcumin capsules and turmeric oil in 48 OSMF patients. Subjects were divided into curcumin, turmeric oil, and control (multinal tablets) groups. A significant reduction in BS, pain, and improved MO was seen with curcumin and turmeric oil groups at the end of 3 months. The curcumin group showed faster improvements, whereas the turmeric group showed long-term effects on the symptoms. Hazarey et al. [35] evaluated curcumin in the treatment of OSMF. Thirty OSMF patients were divided into curcumin lozenges and control (clobetasol propionate 0.05%) groups. Curcumin group showed improved MO and decreased BS compared to the control group. The effect was sustained in the curcumin group, while relapse was seen in the control group at the end of 6 months. Pipalia et al. [36] compared turmeric with black pepper and Nigella sativa in 40 OSMF patients. Subjects were randomized into turmeric + black pepper and Nigella sativa groups with a follow-up of 3 months. Both the groups showed improvement in MO, reduction in BS, and improvement in serum superoxide dismutase levels. Piyush et al. [12] compared lycopene and curcumin with placebo in a randomized controlled parallel clinical trial on 90 OSMF patients. The mean difference in the MO, BS, and CF scores was significantly higher in the curcumin and lycopene groups than the placebo. However, there was no significant difference in the mean difference in the TP among the groups. Saran et al. [13] compared the efficacy of lycopene and curcumin (Curcuma longa and Piper nigrum) given orally in 60 OSMF patients divided into two groups. After 3 months, there was complete cessation of BS in both the groups. No significant difference was seen between the groups with respect to the increase in the MO. Lycopene showed significantly higher improvement than curcumin. Srivastava et al. [37] evaluated the clinical efficacy of tulsi and turmeric mixed for the treatment of OSMF in 41 patients. Significant improvement was seen in BS and MO when compared with baseline scores. Yadav et al. [38] evaluated the efficacy of curcumin in OSMF patients in a randomized open label, interventional trial in 40 OSMF patients randomized into intra-lesional Dexamethasone + Hyaluronidase group and Curcumin tablets group. BS improved in both the groups with complete resolution in the curcumin group. The MO improved in both the groups, but significant improvement was seen at the end of 1 month in the intra-lesional group. TP showed greater recovery on the first month in the intra-lesional group when compared with the curcumin group. 4.2.10 Polyherbal Formulations The formulation of the oxitard capsules have been tried in the treatment of OSMF. It contains the extracts of various plants viz., Mangifera indica, Withania somnifera, Daucus Role of Herbal and Natural Products 69 carota, Glycyrrhiza glabra, Vitis vinifera, Emblica officinalis, and Yashada bhasma; and oils of Triticum sativum. Patil et al. [39] evaluated Oxitard capsules in the management of OSMF in 120 subjects divided equally into the Oxitard and placebo groups. Significant improvements in MO and TP, reduction in BS, pain associated with the lesion, difficulty in swallowing and speech were seen in the Oxitard group. Patil et al. [18] evaluated the efficacy of Oxitard and AV in the management of OSMF in 120 subjects. Subjects were divided equally into Oxitard (two capsules twice daily) and AV groups (5 mg topical gel thrice daily). Significant improvements in MO, TP, pain associated with the lesion, difficulty in swallowing, and speech were in favor of the Oxitard group. However, there was no difference in the improvement of BS between the groups. 4.2.11 Ayurvedic Formulations PRAK–20 is an herbo-mineral combination of 19 herbs having diverse actions like antifibrotic, anti-inflammatory, and immunomodulatory properties. It contains extracts of “Zingiber officinale, Piper nigrum, Piper longum, Terminalia chebulia, Termnallia bellirica, Emblica officinalis, Plumbago zeylanica, Cyperus rotundus, Picrorrhiza kurroa, Cedrus deodara, Embellia ribes, Saussuria lappa, Curcuma longa, Berberis aristata, Baliospermum montanum, Holarrhena antidysentrica, Piper longum, Ipomoea turpethum, Boerhavia diffusa” along with ferric oxide. Rajeshwari and Jadhav [40] conducted a clinical study in 40 subjects with OSMF who were divided into the garlic group (two pearls; thrice daily) and PRAK-20 group (500 mg, three times a day). Both the groups showed a significant reduction in BS and improvement in MO and TP. The garlic group had better reduction in BS, while PRAK-20 showed in all other parameters. Dash and Sharma [41] conducted an open-label non-randomized trial with ayurvedic approach in 30 OSMF patients. They were treated with MukhaPralepa (external application) with turmeric and AV followed by kaval (gargling) with Dashamulataila for 1 month and followed for 1 month. A significant relief in all signs and symptoms with improvement in MO was seen. Patel et al. [42] evaluated the effect of ayurvedic treatment protocol in an open-label non-randomized trial with black box design in OSMF patients. “Koshthashuddhi (mild purgation) and ShodhanaNasya (errhine therapy); Pratisarana (external application) with Madhupippalyadi Yoga, Kavala (gargling) with Ksheerabala Taila and internally Rasayana Yoga” were given for 2 months and followed for 1 month. Significant relief in almost all signs and symptoms and improvement in MO were seen. 4.2.12 Conclusion OSMF has been treated with a variety of therapeutic modalities with natural and allopathic combinations of topical, systemic, or intra-lesional injections, habit cessation, physiotherapy exercises, and surgical modalities. There is a high heterogeneity among the studies and the participants as well. OSMF has a spectrum of clinical outcomes due to which there is heterogeneity in the assessment of subjective and objective criteria. This makes comparison of treatment modalities and evaluating their clinical effectiveness very difficult. Very 70 Natural Oral Care in Dental Therapy few studies exist that have used only natural products for the treatment of OSMF. Most studies emphasized habit cessation before the initiation of treatment. Also, the participants in most of the studies have diverse symptoms with different clinical staging. This becomes a daunting task for the researcher or clinician or health care provider to understand the effectiveness of the interventions used. Many studies used natural products in conjunction with standard of care. Currently, there is no consensus on the standard of care in the management of OSMF. Many clinicians rely on periodic intra-lesional steroid injections with or without hyaluronidase. There is no consensus in the type of steroid, dose, and regimen leading to numerous possible combinations. Within the limitations of this review, it is not possible to conclude which natural product would be a suitable alternative in the treatment of OSMF. Natural products do have a role in the management of OSMF. They can alleviate most of the symptoms of OSMF without any adverse reactions that are common with the use of intra-lesional steroid injections. However, all natural products do not alleviate all the symptoms of OSMF. Clinicians and health care providers should carefully understand the spectrum of symptoms in the patients and then decide the line of treatment. A single protocol may not be feasible for the management of OSMF with natural products. Further, high-quality studies standardized studies with patient reported outcomes are recommended. 4.3 Oral Leukoplakia (OL) 4.3.1 Background The term Leukoplakia should be used to recognize white plaques of questionable risk having excluded (other) known diseases or disorders that carry no increased risk for cancer. [43]. It is mostly asymptomatic and has a tendency to change into squamous cell carcinoma. Treatment of OL should emphasize on the elimination of risk factors (tobacco, betel quid, alcohol), superimposed candida infection over the lesion, etc. [44]. Up to 60% of the lesions regress or totally disappear after cessation of the habit [45]. In view of the evidence linking alcohol, tobacco, betel nut, and diet to the development of potentially malignant and malignant oral epithelial lesions, such deleterious habits should be discouraged [45]. A recent Cochrane review [46] have included only four trials, which had interventions with herbal extracts. However, we have reviewed most of the existing literature related to natural and herbal products to get an overall glimpse of the current status of the same. 4.3.2 Green Tea and Extracts Epigallocatechingallate (EGCG) is a polyphenol, which has antioxidant and chemopreventive properties. Li et al. [47] conducted a randomized double blind trial in 59 patients with OL. They were randomly divided into mixed tea group (3 g/day oral capsules, in four divided doses, plus 10% mixed tea ointment in glycerine topically) or placebo group (placebo plus topical glycerine). The size of oral lesions decreased in 37.9% and increased in 3.4% in the mixed tea group. The size decreased in 10.0% and increased in 6.7% in the placebo group. Tsao et al. [48] conducted a phase II trial with green tea capsules on 41 patients with oral premalignant lesions. They were randomized to receive green tea extract at Role of Herbal and Natural Products 71 concentrations of 1.0, 0.75, and 0.5 g/m2 or placebo, three times daily for 12 weeks. The clinical response rate was higher in all the patients who received green tea extracts (50%) versus placebo (18.2%). Patients receiving higher dosage (1 and 0.75 g/m2) had higher response than lower dose group and placebo. Yoon et al. [49] used “swish-and-spit” green tea extract mouthwash as an oral cancer chemopreventive agent. They found a detectable level of epigallocatechin-3-gallate in the saliva after 1 week of treatment. After 1 week of treatment, the immunohistochemical expression of markers like phosphoactivated epidermal growth factor receptor, cyclooxygenase-2, and ki-67 have been found in lower levels than baseline. 4.3.3 Beta-Carotene (βC) Stich et al. [50, 51] conducted a trial to evaluate vitamin A/βC supplementation in 33 betel quid chewers. They received capsules of retinol (1,00,000 IU/week) and βC (3,00,000 IU/ week) for 3 months. The frequency of micronucleated buccal mucosa cells was decreased, and no difference was seen in patients not receiving vitamin pills. Stich et al. [52] administered vitamin A (60 mg/week to 6 months) or βC (2.2 mmol/ week) in Indian fishermen who chewed betel quid + tobacco before and during the course of the study. The vitamin A group had complete remission of OL in more than half (57%) and reduction of micronucleated cells in almost all the participants. The βC group had remission in only 14.8% and reduction of micronucleated cells in almost all the participants. New OL formation was completely suppressed by vitamin A with only 50% suppression by βC in the study period. After cessation of the treatment, OL reappeared, and there was an increase in the frequency of micronuclei, and nuclear textures reverted baseline features. Stich et al. [53] conducted a trial in Indian fishermen who chewed betel quid + tobacco and had OL. Participants were divided into βC (180 mg/week, 6 months), βC + vitamin A (180 mg/week + 1,00,000 IU/week, 6 months), and placebo groups. At 3 months, no significant difference in the remission rates of OL was seen among the groups. Remission rates of OL in βC and βC + vitamin A groups differed significantly than the placebo. βC + vitamin A followed by βC had strong inhibition of new lesions than the placebo. βC and βC + vitamin A were successful in the remission and inhibition of new OL in continuous betel quid + tobacco chewers. Garewal et al. [54] conducted a phase 2 trial of βC in 24 patients with OL. Two patients had complete and 15 had partial remission with no significant toxicity after administration of βC for 3 months. Toma et al. [55] conducted a phase 2 trial of βC in 23 patients of OL. Patients have not modified any of their tobacco or alcohol habits. Eighteen patients completed follow-ups out of which six, two, and three patients showed complete, partial, and minimal response to treatment. No signs of toxicity were detected, but nine cases had side effects like orange pigmentation of palms and face, elevated cholesterol, renal colic, cephalgia, burning sensation, hypersensitivity of oral mucosa, and hypersalivation were observed. Sankaranarayanan et al. [56] conducted a randomized controlled trial to evaluate vitamin A or βC in the management of OL. Subjects were divided into oral vitamin A (retinyl acetate 300,000 IU/week for 1 year) or βC (360 mg/week for 1 year) or placebo groups. There was a significant difference in the complete regression rates in vitamin A, βC, and 72 Natural Oral Care in Dental Therapy placebo (52%, 33%, and 10%, respectively). Relapse after cessation of the supplementation was seen in two-thirds of vitamin A and half of βC groups. Liede et al. [57] studied the effect of supplements of alpha-tocopherol or βC on the prevalence of oral mucosal lesions in smokers. A total of 409 subjects were divided into alpha-tocopherol (50 mg/day) or βC (20 mg/day), both alpha-tocopherol + βC or placebo groups. No significant differences in the prevalence of oral mucosal lesions. Garewal et al. [58] conducted a randomized double blind trial duration to evaluate the response and the need for maintenance therapy in subjects responded to βC for the management of OL. Initially, subjects were asked to use βC for 6 months to see the response rates. Responders were randomized βC or placebo groups for 1 year. At 6 months, 52% of the subjects had a clinical response. Relapse was seen only in 2 of 11 in the βC and 2 of 12 in the placebo groups. Barth et al. [59] studied the effect of βC, and vitamins E and C on 24 patients with OL and 24 patients after radical resection of a primary oral cancer. With the intervention, the redifferentiation of the oral mucosa was intense when there was cessation of alcohol and tobacco than with the persistence of the alcohol and tobacco abuse. However, long-term prevention does not seem to be effective. Nagao T et al. [60] conducted a randomized, double-blind controlled trial to evaluate the use of low-dose βC combined + vitamin C in the management and prevention of malignant transformation of OL. A total of 46 were randomly divided into βC + vitamin C or placebo groups with supplementation for 12 months. There was no significant difference in the response rate in the study (17.4%) and placebo (4.3%) groups. Within the 5-year follow-up, two in the study and three in the placebo progressed to malignancy. 4.3.4 Lycopene Lycopene appears to be a very promising antioxidant as a treatment modality in OL and can protect cells against damage. It has an antioxidant property, modify intercellular exchange junctions, and has a protective role in dysplasia [61]. Singh et al. [62] studied the efficacy of different concentrations of lycopene in the treatment of OL with a placebo in 58 OL patients who were randomly divided into lycopene (8 mg/day), lycopene (4 mg/day), and placebo. The mean response of 80%, 66.25%, and 12.5% was seen in lycopene 8 mg, lycopene 4 mg, and placebo, respectively, which was highly significant in favor of lycopene. Patel et al. [63] conducted a randomized placebo controlled trial to evaluate antioxidants (lycopene + vitamin E + selenium) in the management of 41 OL subjects. Subjects receiving antioxidants showed significant clinical (lesion size) and histological improvements compared with the placebo. Complete and partial improvement was seen in 5 and 14 subjects in group A, while only 3 showed partial improvement. 4.3.5 Curcumin Cheng et al. [64] conducted a phase-I study in patients with any of the five different premalignant conditions among 25 participants. Histologic improvement was seen in two out of seven patients of OL. They reported no toxicity and adverse effects. Kuriakose et al. [65] conducted a randomized double-blind placebo-controlled (phase IIB) trial among 223 OL Role of Herbal and Natural Products 73 subjects randomized into curcumin and placebo groups. A significantly higher proportion of clinical response was observed in the curcumin group (67.5%) compared with the placebo group (55.3%) with a durable response in both groups and no relapse at 6 months follow-up. Among partial responders, continued therapy has not yielded any benefit. 4.3.6 Miscellaneous 4.3.6.1 Alpha-Tocopherol Benner et al. [66] also studied the micronuclei frequency with α-tocopherol supplementation in OL patients. Supplementation showed a significant reduction in the mean micronuclei frequencies in lesions in the normal mucosa. Benner et al. [67] conducted a phase II trial of α-tocopherol in the management of 43 OL. A total of 46% had clinical and 21% had histologic response, respectively. Kaugars et al. [68] studied antioxidant supplementation (βC + ascorbic acid + α-tocopherol) for 9 months in 79 patients with OL. Clinical improvement was seen in 55.7% and is more evident in subjects with reduced habits. 4.3.6.2 Chinese Herbs Zeng Sheng Ping [69] (ZSP) is a mixture of six medicinal herbs. Sun et al. [69] studied the chemopreventive action of ZSP mixture among animal models and human patients. With favorable results, they compared ZSP with placebo in a randomized controlled trial among subjects with OL. The lesion reduced in size significantly in the ZSP group (67.8%) compared with the placebo (17%). 4.3.6.3 Bowman–Birk Inhibitor Concentrate (BBIC) It is a protease inhibitor which showed a chemopreventive activity in vitro in animal and clinical trials (phase IIa and IIb). Armstrong et al. [70] conducted a clinical trial (phase IIa) with BBIC in 32 subjects with OL for 1 month. A total of 31% showed clinical response (two complete and eight partial). The lesion area decreased significantly after 1 month when compared to the baseline with an overall decrease of 24.2% in the lesion at all doses. This was followed by a phase IIb placebo-controlled trial by Armstrong et al. [71] with 100 subjects randomized into BBIC or placebo. Only 89 subjects completed the 6 months follow-up. Both the groups showed significant decrease in the mean lesion area with no significant difference. 4.3.7 Conclusion OL is asymptomatic unlike other potentially malignant disorders. The aim of the diagnosing and treatment is to prevent its transformation into malignancy. Many approaches have been tried in the management of OL that includes topical and systemic medications, surgery, cessation of habits, and only surveillance. There is no stringent consensus on the standard of care, and it is highly dependent on the decision of the clinician or the health care provider. All these established treatment protocols have their own advantages and disadvantages, 74 Natural Oral Care in Dental Therapy which also has a role in the decision process of the treatment. Natural products have been tried in the treatment of OL with variable efficacy rates and minimal or no adverse effects. They have been supplemented topically or systemically with variable, follow-up time periods with variable level of emphasis on habit cessation, which is a strong predictor for lesion regression. Since the condition is asymptomatic, not many clinical outcomes are recorded during the trials. Moreover, many studies were still in the early phases of the clinical trials without a comparative group. Hence, it is difficult to conclude the efficacy of these natural products. High-quality studies are recommended to have conclusive evidence in the efficacy of these products over long-term follow-up. 4.4 Oral Lichen Planus (OLP) OLP is a chronic T-cell-mediated autoimmune disease with [72] substantial fluctuation in disease activity within and between individuals [73]. Various treatment modalities with different regimens have been tried in the management of OLP but have not showed complete resolution. Choonhakaran et al. [74] compared AV and placebo in randomized, double-blind, controlled trial for the management of 54 OLP. They were randomized into AV gel or placebo. At the end of 8 weeks, a significantly higher proportion of good response was observed in the AV group (81%) compared with the placebo (4%). Complete remission was seen in two patients of the AV group. There was complete disappearance of burning pain in 33% treated with AV gel and in one in the placebo group, which was significant. Salazar-Sanchez et al. [75] conducted a randomized controlled trial to compare topical AV and placebo among 64 OLP patients. No significant differences in mean pain scores were recorded between both groups at the end of 6 and 12 weeks. Complete pain remission was seen in 31.2% and 61% at 6 and 12 weeks in the AV group, while only 17.2% and 41.6% were seen in placebo group. There was significant difference in the total score of oral health-related quality of life and psychological disability domain in favor of the AV group. Mansourian et al. [76] compared AV mouthwash with triamcinolone among 46 OLP patients randomly divided into AV or triamcinolone groups. Both the groups significantly reduced VAS, Thongprasom scores, and size of the lesions after treatment and after discontinuation of the treatment (2 months). In both the groups, three-fourths of the patients showed various degrees of healing at the end of follow-up. Reddy et al. [77] conducted a randomized clinical trial to evaluate the effectiveness of the AV gel over triamcinolone acetonide in 40 OLP patients. Subjects had erosive and atrophic lichen planus. Over 8 weeks of treatment, it was seen that the AV group showed more effectiveness than triamcinolone with respect to clinical signs and symptoms. Sawaarn et al. [78] conducted a randomized placebo-controlled trial with systemic lycopene in the management of OLP patients. They were randomly divided into lycopene (8 mg/day) and placebo. Reduction in BS was higher in the lycopene group (84%) than in the placebo group (67%). Among the lycopene group, all patients showed more than 50% benefit, while such benefit is seen only in 10 patients of the placebo group. Singh et al. [79] used turmeric extracts in the form of ointment to treat OLP. At the end of 3 months, almost all the patients had complete remission with respect to various symptoms like BS, intolerance to spices, redness, ulceration, and striae. Nine of the 10 patients Role of Herbal and Natural Products 75 showed healing at the end of 3 months. With respect to the Thongprasom sign score, 9 of the 10 patients reported a score of 0 at the end of 3 months. Thomas et al. [80] compared 1% curcumin gel with 0.1% triamcinolone acetonide in the management of OLP. A total of 75 subjects were divided into triamcinolone, curcumin gel (thrice daily), and curcumin gel (six times daily). All groups showed significant reduction in the BS, erythema, and ulceration with maximum reduction in triamcinolone. Curcumin gel when applied six times daily was beneficial than when applied three times daily. Vickers and Woodcock [81] used raspberry leaf extract to treat 10 OLP patients over 6 months. There was significant reduction in pain intensity and clinical features of reticulation, erosion, and ulceration at the end of 6 months. No adverse clinical or systemic effects were observed in the subjects. Agha-Hosseini et al. [82] conducted a randomized placebo-controlled trial to evaluate purslane in the treatment of in 37 OLP patients. All subjects were divided into purslane or placebo for 3 months. Partial to complete clinical improvement was seen in 83% of the participants in purslane, while only 17% had partial improvement in the placebo group. Similarly, with respect to the VAS scores, partial to complete response was seen in all the purslane group patients, while only 71% showed only partial response in placebo group. 4.4.1 Conclusion OLP has been treated with mainly topical steroids as the first line of treatment. There was a limited number of studies on the treatment of OLP with herbal or natural extracts. OLP has many clinical outcomes due to which there is heterogeneity in the assessment of subjective and objective criteria. Hence, evaluating their clinical effectiveness is difficult for the researcher or clinician or health care provider. A recent Cochrane review [73] included 28 studies but have included two trials that were done with AV. The review concluded with an insufficient evidence for any of the treatments that have been used. Within the limitations of this review, it is not possible to conclude the effectiveness of these natural products as a suitable alternative. Natural products might alleviate symptoms of OLP without any adverse reactions. Further, studies that are high quality with standardized outcomes and emphasis on patient-reported outcomes are needed with long-term follow-ups and adverse effects. References 1. Gupta, P.C., Hebert, J.R., Bhonsle, R.B. et al., Dietary factors in oral leukoplakia and submucous fibrosis in a population-based case control study in Gujarat, India. Oral Dis., 4, 200–6, 1998. 2. Borle, R.M. and Borle, S.R., Management of oral submucous fibrosis: A conservative approach. J. Oral Maxillofac. Surg., 49, 788–91, 1991. 3. 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Stich, H.F., Rosin, M.P., Vallejera, M.O., Reduction with vitamin A and beta-carotene administration of proportion of micronucleated buccal mucosal cells in Asian betal nut and tobacco chewers. Lancet, London, England, 1, 1204–6, 1984. 52. Stich, H.F., Mathew, B., Sankaranarayanan, R., Nair, M.K., Remission of precancerous lesions in the oral cavity of tobacco chewers and maintenance of the protective effect of β-carotene or vitamin A. Am. J. Clin. Nutr., 53, 298S–304S, 1991. 53. Stich, H.F., Rosin, M.P., Hornby, A.P. et al., Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with beta-carotene and with beta-carotene plus vitamin A. Int. J. Cancer, 42, 195–9, 1988. 54. Garewal, H.S., Meyskens, F.L., Killen, D. et al., Response of oral leukoplakia to beta-carotene. J. Clin. Oncol., 8, 1715–1720, 1990. 55. Toma, S., Benso, S., Albanese, E. et al., Treatment of Oral Leukoplakia with Beta-Carotene. Oncology, 49, 77–81, 1992. 56. Sankaranarayanan, R., Mathew, B., Varghese, C. et al., Chemoprevention of oral leukoplakia with vitamin A and beta carotene: an assessment. Oral Oncol., 33, 231–6, 1997. 57. Liede, K., Hietanen, J., Saxen, L. et al., Long-term supplementation with alpha-tocopherol and beta-carotene and prevalence of oral mucosal lesions in smokers. Oral Dis., 4, 78–83, 1998. 58. Garewal, H.S., Katz, R.V., Meyskens, F. et al., Beta-carotene produces sustained remissions in patients with oral leukoplakia: results of a multicenter prospective trial. Arch. Otolaryngol. Head Neck Surg., 125, 1305–10, 1999. 59. Barth, T.J., Zöller, J., Kübler, A., Born, I.A., Osswald, H., Redifferentiation of oral dysplastic mucosa by the application of the antioxidants beta-carotene, alpha-tocopherol and vitamin C. Int. J. Vitam. Nutr. Res., 67, 368–76, 1997. 60. Nagao, T., Warnakulasuriya, S., Nakamura, T. et al., Treatment of oral leukoplakia with a lowdose of beta-carotene and vitamin C supplements: A randomized controlled trial. Int. J. Cancer, 136, 1708–1717, 2015. 61. Arruda, J.A.A., Álvares, P.R., A., S., R., M., A Review of the Surgical and Nonsurgical Treatment of Oral Leukoplakia. J. Dent. Oral Disord., 2, 1009, 2016. 62. Singh, M., Krishanappa, R., Bagewadi, A., Keluskar, V., Efficacy of oral lycopene in the treatment of oral leukoplakia. Oral Oncol., 40, 591–596, 2004. 63. Patel, J., Dhokar, A., Panda, A. et al., Randomized controlled trial to evaluate the efficacy of oral lycopene in combination with vitamin E and selenium in the treatment of oral leukoplakia. J. Indian Acad. Oral Med. Radiol., 26, 369, 2014. 64. Cheng, A.L., Hsu, C.H., Lin, J.K. et al., Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res., 21, 2895–900, 2001. Role of Herbal and Natural Products 79 65. Kuriakose, M.A., Ramdas, K., Dey, B. et al., A Randomized Double-Blind Placebo-Controlled Phase IIB Trial of Curcumin in Oral Leukoplakia. Cancer Prev. Res., 9, 683–691, 2016. 66. Benner, S.E., Wargovich, M.J., Lippman, S.M. et al., Reduction in oral mucosa micronuclei frequency following alpha-tocopherol treatment of oral leukoplakia. Cancer Epidemiol. Biomarkers Prev., 3, 73–6, 1994. 67. Benner, S.E., Winn, R.J., Lippman, S.M. et al., Regression of oral leukoplakia with alphatocopherol: A community clinical oncology program chemoprevention study. J. Natl. Cancer Inst., 85, 44–7, 1993. 68. Kaugars, G.E., Silverman, S., Lovas, J.G. et al., A clinical trial of antioxidant supplements in the treatment of oral leukoplakia. Oral Surg. Oral Med. Oral Pathol., 78, 462–8, 1994. 69. Sun, Z., Guan, X., Li, N., Liu, X., Chen, X., Chemoprevention of oral cancer in animal models, and effect on leukoplakias in human patients with ZengShengPing, a mixture of medicinal herbs. Oral Oncol., 46, 105–110, 2010. 70. Armstrong, W.B., Kennedy, A.R., Wan, X.S. et al., Clinical modulation of oral leukoplakia and protease activity by Bowman–Birk inhibitor concentrate in a phase IIa chemoprevention trial. Clin. Cancer Res., 6, 4684–91, 2000. 71. Armstrong, W.B., Taylor, T.H., Kennedy, A.R. et al., Bowman Birk Inhibitor Concentrate and Oral Leukoplakia: A Randomized Phase IIb Trial. Cancer Prev. Res., 6, 410–418, 2013. 72. Scully, C., Eisen, D., Carrozzo, M., Management of Oral Lichen Planus. Am. J. Clin. Dermatol., 1, 287–306, 2000. 73. Thongprasom, K., Carrozzo, M., Furness, S., Lodi, G., Interventions for treating oral lichen planus. Cochrane Database Syst. Rev., 6, CD001168, 2011. 74. Choonhakarn, C., Busaracome, P., Sripanidkulchai, B., Sarakarn, P., The efficacy of aloe vera gel in the treatment of oral lichen planus: A randomized controlled trial. Br. J. Dermatol., 158, 573–577, 2007. 75. Salazar-Sánchez, N., López-Jornet, P., Camacho-Alonso, F., Sánchez-Siles, M., Efficacy of topical Aloe vera in patients with oral lichen planus: A randomized double-blind study. J. Oral Pathol. Med., 39, 735–740, 2010. 76. Mansourian, A., Saheb-Jamee, M., Momen-Beitollahi, J. et al., Comparison of Aloe Vera Mouthwash With Triamcinolone Acetonide 01% on Oral Lichen Planus: A Randomized Double-Blinded Clinical Trial. Am. J. Med. Sci., 342, 447–451, 2011. 77. Reddy, R.L., Reddy, R.S., Ramesh, T. et al., Randomized trial of aloe vera gel vs triamcinolone acetonide ointment in the treatment of oral lichen planus. Quintessence Int., 43, 793–800, 2012. 78. Saawarn, N., Saawarn, S., Chaitanya, N. et al., Lycopene in the management of oral lichen planus: A placebo-controlled study. Indian J. Dent. Res., 22, 639, 2011. 79. Singh, V., Tiwari, S., Das, S. et al., Turmeric – A new treatment option for lichen planus: A pilot study. Natl. J. Maxillofac. Surg., 4, 198, 2013. 80. Thomas, A., Varma, B., Kurup, S. et al., Evaluation of Efficacy of 1% Curcuminoids as Local Application in Management of Oral Lichen Planus – Interventional Study. J. Clin. Diagn. Res., 11, ZC89–ZC93, 2017. 81. Vickers, E.R. and Woodcock, K.L., Raspberry Leaf Herbal Extract Significantly Reduces Pain and Inflammation in Oral Lichen Planus Patients – A Case Series Analysis. Open J. Dent. Oral Med., 3, 73–81, 2015. 82. Agha-Hosseini, F., Borhan-Mojabi, K., Monsef-Esfahani, H.-R. et al., Efficacy of purslane in the treatment of oral lichen planus. Phyther. Res., 24, 240–244, 2009. Part II STUDIES OF PLANTS USED IN DENTAL DISEASE 5 Studies on the Anticariogenic Potential of Medicinal Plant Seed and Fruit Extracts Disha M. Patel, Jenabhai B. Chauhan* and Kalpesh B. Ishnava† Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar, Gujarat, India Abstract Oral diseases are common health problems worldwide. There is a global need for the alternative, safe, and effective treatment for oral diseases. Compounds from medicinal plants have been proven to be an effective alternative. To screen the anticariogenic activity, organic solvents (hexane, ethyl acetate, methanol) and aqueous extracts of 19 medicinal plant seeds and fruits were assayed against five different cariogenic bacteria by agar well diffusion assay and MIC. ethyl acetate (E), methanol (M) and aqueous extract of Quercus infecteria exhibited significant growth inhibition in all the selected cariogenic bacteria, which is comparable with the standard antibiotic drug erythromycine. The MIC values of 78, 625, and 156 µg/mL were obtained against L. casei when Q. infectoria (E), P. granatum (E), and Q. infectoria (M) extracts were used. Ethyl acetate and methanolic extract of Q. infectoria contains tannins, cardiac glycosides, steroids, terpenoids, phenolic compounds, and alkaloids. Bioactive compounds from all the effective crude extracts were separated using TLC and localized bioautographically. Ethyl acetate and methanolic extract of Q. infectoria were found to be useful for clinical evaluation and development of potential alternatives for the treatment against cariogenic bacteria responsible for dental caries. Keywords: Dental disease, medicinal plants, anticariogenic activity, TLC-Bioautography 5.1 Introduction Oral diseases are common and a growing problem across the globe. There are many bacteria present in the oral cavity, and a few of them are responsible for oral diseases especially dental caries. In Asia and worldwide, dental caries are most prevalent in adults and children. It is the major pathological cause of tooth loss in children [1]. Dental caries affect all the age groups, but the percentage varies. In India, one of two persons/individuals experience dental caries. The human oral cavity contains both Gram-positive and Gram-negative bacilli as well as spirochetes. They are distributed on various sites in the human mouth [2]. Gram-positive *Corresponding author: jbc109@yahoo.co.in † Corresponding author: ishnavakb203@yahoo.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (83–96) © 2020 Scrivener Publishing LLC 83 84 Natural Oral Care in Dental Therapy bacteria include cocci (facultative and anaerobic) and rods (facultative and anaerobic). Gram-negative bacteria include both streptococci (S. mutans, S. sanguis, S. mitis, S. salivarius, and S. mitis) and staphylococci. The proportions of each group of bacteria vary at different sites such as plaque, tongue, saliva, and gingival region. The bacterial composition also varies with age; for example, at the age of 6–9 months in humans, the oral cavity contains a large numbers of Streptococcus salivarius (98% of the total bacteria). Once teeth appear, the S. mutans and S. sanguis colonize the teeth surface (non-epithelial) and form a dental plaque or biofilm [3]. The oral flora of humans may harm their host since some of these bacteria are parasites or opportunistic pathogens. These bacteria also enter the bone, lungs, brain, and breasts through wounds created by dental manipulation or treatments [4]. Though plants are common in traditional health practice, there is a growing concern and use of plant extracts and plant-derived compounds for the treatment of different oral/dental conditions, namely, toothache, dental caries, ulcerative gingivitis, gingivitis, mouth ulcers, oral thrush, swollen tonsils, black tongue, and tonsillitis [5]. Common plants in the field include Citrus aurantifolia, Xylopia aethiopica, Aframomum melegueta, and Piper guineense. For ordinary oral hygiene in the morning, roots or stems of certain plants are used to make brush-like ends to clean the teeth [6]. The individual chewing sticks has been proven to have variable activity against different oral microbial flora. The presence of benzoic acid derivatives of Zanthoxylun zanthoxyloides is attributed to antimicrobial action [7]. The phenolic acids and alkaloids (canthin, berberine, and chelerythrine) are active at pH 5 and pH 7.5, respectively. Roots of certain medicinal plants contain anticariogenic compounds that are active at both alkaline pH and acidic pH. Tella (1976) reported that the root of Vernonia amygdalina is effective against gingivitis and toothache [8]. Many authors reported the activity of plant seeds against cariogenic and other bacteria like Azadirachta indica [9], Quercus infectoria [10], and Areca catechu [11]. In order to preserve the teeth, coloring or blackening of the teeth is done using plants, sometimes up to 1 year. This, in turn, helps in controlling dental caries and keeping the teeth strong and healthy. Examples of blackening plant species include Duroia hirsute, Neea parviflora (Family—Nyctaginaceae), Manettia divaricata, and Manetta glandulosa. It was reported that the cacao bean husk extract significantly reduces the growth rate with 69–72% reduction and inhibited synthesis of insoluble glucan in S. mutans. It also inhibits sucrose-dependent adhesion in S. mutans and S. sobrinus to a glass surface with 85% inhibition at >5 mg ml−1 concentration under in vitro conditions [12]. In vivo experiments in healthy rats infected with the above bacteria showed that the cacao bean husk extract exerted significant cariostatic activity. Ethanolic extract of flower of Helichrysum italicum (Compositae) exerted antimicrobial activity against S. mutans, S. sanguis, and S. sobrinus [13]. This extract, in the presence of dextran, reduced cell surface hydrophobicity, adherence to glass, and cellular aggregation of S. mutans. There are many studies on the activity of plant extracts and products against a wide variety of microbial pathogens of the oral cavity. Some studies also focused on the ability of plant-derived products to inhibit the formation of dental biofilms by interfering and reducing the adhesion of microbial pathogens to the tooth surface, a primary event in the formation of dental plaque and the progression to tooth decay and periodontal diseases. It has been demonstrated that crude plant extracts and purified phytochemicals act through cidal activity (inhibit any or all stages) or interfere with adherence/aggregation/biofilm formation and inhibit the production of glycolytic acid in cariogenic bacteria. Compared with Anticariogenic Potential of Medicinal Plants 85 seed extracts, there were more number of reports on antibacterial activity of aerial parts of the leaves, stem, flowers, roots, and rhizome seed extracts. Screening for various ethano-botanical plants were undertaken in Gujarat, India, against common cariogenic bacteria responsible for oral disease/dental caries, and specific plant part extracts were characterized for their anticariogenic potential [14–20]. Characterization of bioactive compounds responsible for anticariogenic activity was also performed from various plant part extracts [21, 22]. Some studies also focused on the anticariogenic activity of seed protein and oils [23, 24]. There is a report on herbal formulation against anticariogenic activity [25]. The present study was undertaken to screen the anticariogenic activity of selected medicinal plants seed extracts and fruit extract (only Querus infectoria) against a group of cariogenic bacteria. 5.2 Materials and Methods 5.2.1 Plant Materials Most of the seed materials were procured from the local market of Vidyanagar and Vadodara, Gujarat. Seeds of three plants (Andrographis peniculata, Cassia tora, and Solanum surattense) were a gift by Dr. Ram Kumar, Department of Medicinal and Aromatic Plants, Anand Agriculture University (AAU), Anand. Some of the plant seeds were collected from a botanical garden located in our institutional campus, New Vallabh Vidyanagar. The identity of plant specimens was confirmed through plant taxonomist Dr. Ishnava of our institute. Details of the plant materials are given in Table 5.1. 5.2.2 Preparation of Plant Seed and Fruit Extracts The seeds and fruits of all the selected plants were thoroughly washed with distilled water. It was surface sterilized using sodium hypochlorite (10%), rinsed with sterile distilled water, and air dried under laminar air flow. The powder of the plant materials was made using a household electric grinder, and 50 g of this materials was mixed in 125 mL of solvent (hexane). The content was kept on a shaker (130–140 rpm) for 24 h at room temperature. The extract was then filtered using Whatman filter paper. The resulting filtrate was transferred to glass petri-plates and dried aseptically. The methanol, ethyl acetate, and aqueous extract of all the plants were prepared as described above. The resultant dried material from petridishes was stored in an eppendorf tube and was used for the anticariogenic activity screening. 5.2.3 Cariogenic Bacterial Strains Bacteria causing dental caries/tooth decay used in this study were purchased from Microbial Type Culture Collection (MTCC), Chandigarh. Freeze-dried pure culture first revived as per MTCC specified growth conditions and preserved as glycerol stocks. Five cariogenic bacteria includes Staphylococus aureus (MTCC-96), Lactobacillus acidophilus (MTCC-*447), Streptococcus mutans (MTCC-890), Lactobacillus casei (MTCC-1423), and Actinomyces viscosus (MTCC-7345). 86 Natural Oral Care in Dental Therapy Table 5.1 Seeds and fruits used for anticariogenic activity. Sr. no. Botanical name Common name Family Collection site 1 Piper nigrum L. Meri Piperaceae Vadodara 2 Areca catechu L. Sopari Arecaceae Vadodara 3 Trigonella foenum-graceum L. Methi Papilionaceae Vadodara 4 Andrographis paniculata (Burm.f.) Wall. Kariyatu Acanthaceae AAU, Anand 5 Coriandrum sativum L. Dhanadal Apiaceae Vidyanagar 6 Derris indica (Lam.) Bennet. Karanj Papilionaceae IICP Garden 7 Juglans regia L. Akhrot Juglandanceae Vidyanagar 8 Prunus amygdalus Batsch. Badam Rosaceae Vidyanagar 9 Punica granatum L. Dadam Punicaeae Vidyanagar 10 Mimusops elengi L. Borsali Sapotaceae IICP Garden 11 Madhuca indica J. F. Gmel. Mahudo Sapotaceae IICP Garden 12 Sesamum indicum L. Tal Pedaliaceae Vidyanagar 13 Cassia tora L. Kuvandio Caesalpiniaceae AAU, Anand 14 Sapindus laurifolius Vahl. Aritha Sapindaceae IICP Garden 15 Azadirachta indica A.juss. Limdo Meliaceae IICP Garden 16 Solanum surattense Burr.f. Bhoyringni Solanaceae AAU, Anand 17 Cassia fistula L. Garmalo Caesalpiniacee IICP Garden 18 Quercus infectoria Oliv. Mayafal Fagaceae Vadodara 19 Elettaria cardamomum (L.) Maton Elaichi Zingiberaceae Vadodara 5.2.4 Preparation of Inoculums In order to make fresh bacterial cultures, loopful of bacterial cell suspension streaked in to specific liquid growth media (Hi-media) followed by incubation at optimal temperature. This step was simultaneously done for each bacteria. Cell growth (1 × 108 bacterial cell count per mL) was quantified by comparing it with 0.5 McFarland turbidity standard, and it is maintained throughout the experiment [26]. Anticariogenic Potential of Medicinal Plants 87 5.2.5 Anticariogenic Activity Screening of Plant Extracts 5.2.5.1 Agar Well Diffusion Assay Agar well diffusion assay used to access antibacterial activity of seed and fruit extracts. The stock of each plant extract was prepared by suspending 100 mg of solvent-extracted dried materials in 1 ml of dimethyl sulfoxide (DMSO). Petriplates were thoroughly washed using detergent, dried and sterilized using autoclave at 15 lbs pressure for 15 min and used for the reparation of agar plates. Approximately 25 mL of sterilized selective medium was poured in to each petridish and solidified at room temperature and incubated overnight at 37°C for sterility checking. Agar plates were first divided into four equal parts and labeled with H (Hexane), E (Ethyl acetate), M (Methanol), and D (Distilled water) and specific bacteria. The center of the plate was labeled with the serial number of the plant. One hundred microliters (100 µL) of fresh bacterial culture having 108 Cells/mL was spread on agar plates using a glass spreader. Using a sterile cork borer, a well of 10-mm diameter was made in the above petri plates, and it was then filled with 100 µL of the respective plant extracts. Agar plates were kept for half an hour at 4°C in a refrigerator for pre-diffusion and then incubated at 37°C/specified temperature for 24 h or more as per the strain of the bacteria. The actual zone of inhibition was measured and recorded. Commercially available standard antibiotics Cefadroxil, Tetracycline, and Erythromycine (100 µg/mL) was used as a positive control, whereas 100% DMSO was used as the negative control. The experiment was set up in three replicates and repeated twice. 5.2.5.2 Determination of Minimum Inhibitory Concentration (MIC) Minimum inhibitory concentration was evaluated by twofold serial broth dilution method and using 2,3,5-triphenyl tetrazolium chloride as described by Ishnava et al., 2003 and Barad et al., 2014 [19, 22, 26]. Plant extracts with greater than 10-mm zone of growth inhibition were subjected to determination of MIC. Bacteria-specific broth medium was used for preparing the inoculums and dilutions. The bacterial cell density (1 × 108 Cells /mL) was maintained uniformly throughout the experimentation. 5.2.6 Preliminary Phytochemical Analysis Presence of phytochemicals in the potential plant extracts was determined according to Parekh and Chanda, 2008 [28]. 5.2.7 Analytical Thin Layer Chromatography It is performed to find a suitable solvent system for the development of chromatogram. The details of the solvent system are finalized for different plants using ready-made TLC plates, silica gel 60, 0.25 mm, and F254 plate (Merck). The solvent systems for Areca catechu [methanol:benzene (4:1)], Punica granatum [ethyl acetate:methanol:distilled water (1:5:1)], Cassia tora [ethyl acetate:methanol:distilled water (6:1:0.5)], Quercus infectoria [ethyl acetate:methanol:distilled water (a) 2:10:0.5 (b) 5:3:0.5], and Elettaria cardamomum [Methanol: chloroform (4:1)] were tested. 88 Natural Oral Care in Dental Therapy 5.2.8 TLC—Bioautography Among the 19 plant seeds and fruit extracts tested for anticariogenic activity, only 1 plant extract showed the highest inhibition zones against Actinomyces viscosus and Staphylococcus aureus, 3 plant extracts showed inhibitory activity against Streptococcus mutans, and 2 plant extracts each for Lactobacillus casei and Lactobacillus acidophilus were subjected to bioautography. TLC—Bioautography was performed as per Ishnava et al., 2013 and Rajput and Ishnava, 2017 [20, 22]. 5.3 Result and Discussion The distilled water, ethyl acetate, hexane, and methanolic extracts were assayed against five selected cariogenic bacteria by agar well diffusion assay. None of the selected cariogenic bacterial growth was inhibited by dimethyl sulfoxide. The antimicrobial activity of the different solvent extracts of the plant seeds and fruits showed a variable pattern with the highest efficacy in the ethyl acetate extract followed by methanol, aqueous, and hexane extracts (Table 5.2). One of the important observations in the present study is that ethyl acetate, methanol, and aqueous extract of Quercus infecteria exhibited significant growth inhibition in all the selected cariogenic bacteria except Actinomyces viscosus, which is comparable with the standard antibiotic drug erythromycin (Table 5.2). The result obtain from the hexanolic extract of plant seeds showed that only 5 plants (26%) out of the total plants (19) are active against Staphylococcus aureus (3–5 mm zone), Actinomyces viscosus (8–9 mm zone), and Streptococcus mutans (5–8 mm zone). No growth inhibition was found in Lactobacillus acidophilus and Lactobacillus casei (Table 5.2). The plants showing growth-inhibitory activity include Piper nigrum, Areca catechu, Trigonella foenum-graceu, Derris indica, and Elettaria cardamomum. The zone of inhibition (9 mm) was found in A. viscosus when A. catechu and E. cardamomum extracts were used (Table 5.2). Comparatively the highest number of plants (47%) showed growth inhibition when seeds of the selected plants were extracted with ethyl acetate (Table 5.2). The plant materials showing zone of inhibition includes, P. nigrum, A. catechu, T. foenum-graceu, Punica granatum, Madhuca indica, Cassia tora, Cassia fistula, E. cardamomum, and Quercus infectoria. Growth of all the cariogenic bacteria was inhibited with a maximum zone of inhibition of 13, 20, 13, 18, and 18 mm in AV, SM, LA, LC, and SA, respectively (Table 5.2). These zones of inhibition are highly significant and comparable with one of the standard antibiotic erythromycin. One of the interesting finding is that the ethyl acetate extract of Q. infectoria exhibited the highest zone of inhibition in SM, LA, LC, and SA compared to the hexane, methanol, and aqueous extracts (Table 5.2 and Figure 5.1). Only E. cardamomum was found effective against A. viscosus among the 19 plant extracts evaluated in the present study. The effective plant extracts were subjected to MIC determination. Methanolic seed extracts of 8 plants (P. nigrum, A. Catechu, Derris indica, Juglans regia, P. granatum, M. indica, C. tora, and Quercus infectoria) from a total of 19 plants exhibited variable growth inhibition in SM, LA, LC, and SA with growth inhibiting ranges of 5–19 mm, 3–13 mm, 2–17 mm, and 2–17 mm, respectively (Table 5.2). None of the plant Table 5.2 Anticariogenic activity of solvent extracts of seeds and fruit. Bacteria Zone of inhibition (in mm) Hexane extract Ethyl Acetate extract Methanol extract Aqueous extract Plant name A V S M L A L C S A A V S M L A L C S A A V S M L A L C S A A V S M L A L C S A 1 Piper nigrum – – – – 05 – 05 – – – – – – – 02 – – – – 02 2 Areca catechu 09 08 – – 03 – – – 01 05 – 10 03 08 08 – 10 03 08 08 3 Trigonella foenum-graceum 08 – – – 05 – – – – 01 – – – – – – – – – – 4 Andrographis paniculata – – – – – – – – – – – – – – – – – – – – 5 Coriandrum sativum – – – – – – – – – – – – – – – – – – – – 6 Derris indica – 05 – – 05 – – – – – – 05 – 02 – – 05 – 02 – 7 Juglans regia – – – – – – – – – – – – – 08 – – – – 08 – 8 Prunus amygdalus – – – – – – – – – – – – – – – – – – – – 9 Mimusops elengi – – – – – – – – – – – – – – – – – – – – 10 Punica granatum – – – – – – – – 11 – – – – 09 – – – – 09 – 11 Madhuca indica – – – – – – – – 03 – – – – 03 – – – – 03 – 12 Sesamum indicum – – – – – – – – – – – – – – – – – – – – 13 Cassia tora – – – – – – 10 – 02 05 – – 03 03 – – – 03 03 – (Continued) Anticariogenic Potential of Medicinal Plants 89 Sr. No. Bacteria Zone of inhibition (in mm) Hexane extract Ethyl Acetate extract Methanol extract Aqueous extract Sr. No. Plant name A V S M L A L C S A A V S M L A L C S A A V S M L A L C S A A V S M L A L C S A 14 Sapindus laurifolius – – – – – – – – – – – – – – – – – – – – 15 Azadirachta indica – – – – – – – – – – – – – – – – – – – – 16 Solanum surattense – – – – – – – – – – – – – – – – – – – – 17 Cassia fistula – – – – – – 08 10 02 09 – – – – – – – – – – 18 Quercus infectoria – – – – – – 20 13 18 18 – 19 13 17 17 – 19 13 17 17 19 Elettaria cardamomum 09 – – – – 13 – – 07 – – – – – – – – – – – 20 Tetracycline – 28 28 41 26 – 28 28 41 26 – 28 28 41 26 – 28 28 41 26 21 Cefadroxil – 12 36 41 31 – 12 36 41 31 – 12 36 41 31 – 12 36 41 31 22 Erythromycine – 15 23 19 19 – 15 23 19 19 – 15 23 19 19 – 15 23 19 19 23 DMSO – 00 00 00 00 – 00 00 00 00 – 00 00 00 00 – 00 00 00 00 Actinomyces viscosus (AV), Streptococcus mutans (SM), Lactobacillus acidophilus (LA), Lactobacillus casei (LC), Staphylococcus aureus (SA) 90 Natural Oral Care in Dental Therapy Table 5.2 Anticariogenic activity of solvent extracts of seeds and fruit. (Continued) Anticariogenic Potential of Medicinal Plants 91 Staphylococcus aureus Streptococcus mutans Lactobacillus acidophilus Lactobacillus casei Figure 5.1 Anticariogenic activity of Quercus infectoria distilled water (D), ethyl acetate (E), hexane (H), and methanol (M) extracts. extracts inhibited the growth of AV (Table 5.2). Unlike the hexanolic extract, the methanolic extract of Q. infectoria remains highly active against all the cariogenic bacteria except AV, which was once again comparable with the standard antibiotic drug erythromycin. The growth inhibitory zone of the methanolic extract of Q. infectoria against key bacteria (S. mutans) responsible for dental caries was 19 mm, which is higher than that of the two standard drugs, cefadroxil (12 mm) and erythromycin (15 mm) (Table 5.2 and Figure 5.1). The effective extract showing a zone of inhibition of 10 mm or more was subjected to MIC determination. Aqueous or distilled water extract of plant seeds also showed variable growth inhibitory pattern in selected carcinogenic bacteria (Table 5.2). A total of eight plants showed activity where Q. infectoria is active against SM, LA, LC, and SA (Table 5.2). Both the extracts of A. catechu are active against SM, SA, and LC, whereas P. granatum is active against AV, LA, and LC (Table 5.2). The minimum zone of inhibition was 1 mm in LC by C. tora. The maximum inhibition zone of 13 mm was exerted by Q. infectoria aqueous extract both in SM and LC (Table 5.2 and Figure 5.1). Only one plant (P. granatum) exhibited activity against AV, whereas seven plants exhibited activity against LC (Table 5.2). Recently, due to the development of multiple drug resistance in the human pathogenic organisms by modern pharmaceuticals, there is an increase in the reports related to antimicrobial properties of plants from all parts of the world [29]. For the development of new antimicrobial drugs from the plant, it is necessary that the compound should inhibit the growth of pathogen or kill them, and it should not be toxic to the host cells. Traditionally, various parts of plants supply affordable medicine to the Indian population and are traditionally utilized for the treatment of various human diseases. Plant-derived compounds were successfully used to prevent oral and plaque-related diseases including dental caries [30]. Our result showed that crude ethyl acetate extract of Quercus infectoria, Cassia tora, Cassia fistula, Punica granatum, and methanol extracts of Q. infectoria and Areca catechu showed very good anticariogenic activity against L. casei, S. mutans, S. aureus, L. acidophilus, and A. viscosus. Determination of MIC and phytochemical screening of these extract provides valuable information for drug discovery. 5.3.1 MIC Value of Effective Plant Extracts All the selected plant extracts showing maximum zone of inhibition against targeted organisms were subjected to minimum inhibitory concentration (MIC) values and summarized in Table 5.3. 92 Natural Oral Care in Dental Therapy Table 5.3 MIC (expressed in µg/ml) of effective seed extracts against cariogenic bacteria. Bacteria MIC (µg/ml) Sr. no. Plant name (Extracts) AV SM LA LC SA 2 Areca catechu (M) – 312 – – – 10 Punica granatum (E) – – – 625 – 13 Cassia tora (E) – 312 – – – 17 Cassia fistula (E) – – 625 – – 18 Quercus infectoria (E) – 312 625 78 156 18 Quercus infectoria (M) – 312 625 156 625 19 Elettaria cardamomum (E) 625 – – – – Methanol (M), Ethyl acetate (E). Determination of the MIC values of ethyl acetate and methanolic extract of seven plants generated the data where the maximum MIC value was found to be 625 µg/mL and the minimum value as 78 µg/mL (Table 5.3). The MIC value of the methanolic extract of E. cardamomum against A. viscosus was 625 µg/mL (Table 5.3). The minimum inhibitory concentration value of 312 µg/mL was achieved against S. mutans, when ethyl acetate extract of Cassia tora, Q. infectoria and methanolic extract of Areca catechu, Q. infectoria were used (Table 5.3). Ethyl acetate extract of C. fistula and ethyl acetate as well as methanolic extract of Q. infectoria exhibited an MIC value of 625 µg/mL against L. acidophilus (Table 5.3). The MIC values of 78, 625, and 156 µg/mL were obtained against L. casei when Q. infectoria (E), P. granatum (E), and Q. infectoria (M) were used (Table 5.3). The minimum inhibitory concentration values of 156 µg/mL and 625 µg/mL were found when ethyl acetate and methanolic extracts of Q. infectoria were assessed against S. aureus (Table 5.3). 5.3.2 Phytochemical Screening and Bioautography The presence of phytochemical substances like alkaloids, cardiac glycosides, phenolic compounds, tannins, terpenoids, and steroids were tested qualitatively as per Ahmed & Beg, 2001 [29]. The ethyl acetate and methanolic extracts of Q. infectoria contain all these bioactive compounds, whereas in P. granatum, only terpenoids are absent (Table 5.4). Casia fistula was found positive for alkaloids, steroids, phenolic compounds, and cardiac glycosides. Only terpenoids and phenolic compounds were found positive in E. cardamomum (Table 5.4). For the presence of bioactive substances in the ethyl acetate and methanolic extracts of effective plants, various TLC solvent system were used and standardized using TLC plates, silica gel 60, 0.25 mm, and F254 plate (Merck). The standardized solvent systems are Areca catechu—methanol:benzene (4:1), Punica granatum—ethyl acetate:methanol:distilled water (1:5:1), Cassia tora—ethyl acetate:methanol:distilled water (6:1:0.5), Quercus Anticariogenic Potential of Medicinal Plants 93 Table 5.4 Phytochemical constituents of crude solvent extracts of effective plants. Sr. no. Plant name (Extracts) 1 2 3 4 5 6 2 Areca catechu (M) + – – + + – 10 Punica granatum (E) + + + – + + 13 Cassia tora (E) – – + – + + 17 Cassia fistula (E) – + + + + 18 Quercus infectoria (E) + + + + + + 18 Quercus infectoria (M) + + + + + + 19 Elettaria cardamomum (E) – – – + + – Absent = (−); Present = (+); 1 (Tannins), 2 (Cardiac Glycosides), 3 (Steroids), 4 (Terpenoids), 5 (Phenolic Compounds), and 6 (Alkaloids). infectoria—ethyl acetate:methanol:distilled water (a) 2:10:0.5 (b) 5:3:0.5 and for Elettaria cardamomum—methanol:chloroform (4:1). Bioactive substances are successfully separated from crude solvent extracts using TLC. For the determination of single or multiple substances in the various plant extracts, they were exposed under iodine vapor and UV rays (254 nm). The yellow brown spots and blue fluorescence was obtained when TLC chromatogram was exposed to iodine vapor and UV light, respectively. When TLC-bioautography was performed to identify the bioactive substances associated with anticariogenic activity, the chromatograms used were against AV, LA, LC, SA, and SM. TLC bioautography, iodine, and UV reaction of TLC fractionated ethyl extract of P. granatum and C. tora were used against L. Casei and S. mutans. Bioautography, separation of active principle, and UV fluorescence of ethyl acetate extract of E. cardamomum were used against L. acidophilus. The bioautographical location of active substances present in the methanolic extract of Q. infectoria, which is active against S. mutans is shown. Figure 5.2 shows the bioautographical location of the active substances present in ethyl acetate extracts of Q. infectoria, which is active against Streptococcus mutans (SM) and Figure 5.2 Bioautography of ethyl acetate extract of Quercus infectoria against Streptococcus mutans (SM) and Lactobacillus acidophilus (LA) and TLC of ethyl acetate extract. 94 Natural Oral Care in Dental Therapy Lactobacillus acidophilus (LA). The result of the present study revealed that ethyl acetate was found as a highly effective solvent against all the four cariogenic bacteria compared to methanol, hexane, and distilled water. This is due to its extraction capacity. Prabhat et al., 2010 studied four different solvent (acetone, methanol, petroleum ether, and aqueous) extracts of six medicinal plants, namely, Terminalia chebula, Mimusops elengi, Achyranthes aspera, Acacia catechu, A. arabica, and Glycyrrhiza glabra [31]. Individual extracts were tested for their antimicrobial activity against Staphylococcus aureus, S. salivarius, S. sanguis, S. mutans, Lactobacillus acidophilus, and Candida albicans using the well diffusion method. All the plant extracts showed significant activity against these pathogens. Among them all, the methanolic extract of T. chebula showed the highest (27 mm) inhibition zone against S. aureus. A minimum zone (9 mm) of inhibition was recorded when the petroleum ether extract of M. elengi and A. aspera was evaluated against S. aureus, S. mutans, and Candida albicans. Nazia Masood, 2008, evaluated the antibacterial activity of aqueous infusions and decoctions of cumin (Cuminum cyminum L, Umbelliferae), kalonji (Nigella sativa L., Ranunculaceae), and poppy seed (Papaver somniferum L., Papaveraceae) against 188 Gram-positive and Gram-Negative bacterial isolates found in the oral cavity of healthy individuals belonging to 11 different genera [32]. Variable growth inhibition was observed with the highest percent (73%) from the aqueous decoction of cumin, followed by 51% in the aqueous decoctions of kalonji, and the very least in the poppy seed (14.4%) in tested organisms. We found a promising activity of Q. infectoria in SM, LA, LC, and SA with maximum efficacy in L. casei (MIC 78 µg/mL). This is five times lesser than what were reported by Muskhazli et al. (2008) in C. cellulans (500 µg mL−1), which act as bacteriostatic agents rather than as bactericidal for C. cellulans [33]. In the present study, we could not find the activity of P. granatum against S. aureus. This is contradictory to the report of Jang et al. (2009) where they found presence of alkaloids, tannins, and sterols, which was effective against mithicillin-resistant S. aureus. This could be due to the concentration of the extract; a higher antibacterial activity would be obtained with a concentrated extract. In this study, the ethyl acetate extract of E. cardamomum was only active against A. viscosus. This may be due to the presence of volatile oil because it was reported in the literature. There is comparatively very meager information on anticariogenic activity of seed extracts, as majority of reports focus on the antibacterial activity of aerial parts of the leaves, stem, flowers, roots, and rhizomes extracts. Crude extract of Querecus infectoria was found to be highly effective against four cariogenic bacteria except A. viscosus. Therefore, the present study may encourage researchers to search the anticariogenic principle found in the plant seeds. Further chromatographic and spectroscopic analyses of these plant extracts are necessary for the determination of structures of bioactive compounds responsible for anticariogenic activity. 5.4 Conclusion In recent years, plant extracts have shown great potential to purify antimicrobial substances that are effective against a variety of pathogens, which in turn are used to treat diseases. Our results on the promising and broad-spectrum anticariogenic activity of ethyl acetate Anticariogenic Potential of Medicinal Plants 95 and methanolic extracts of Q. infectoria may prove to be useful for clinical evaluation and development of a suitable formulation for the treatment of dental caries. Acknowledgments The authors are thankful to the Director, ARIBAS, and Charutar Vidya Mandal, Vallabh Vidyanagar for providing the necessary support and research laboratory facilities. References 1. World health organization (WHO), Worldwide day (2001): Oral health. 2, 2001, www.who.int/ water_sanitation_health/en/oralhealth.htm 2. Hamada, S. and Slade, H., Biology, immunology and carcinogenic of streptococcus mutans. Microbiol. Rev., 44, 545–638, 1980. 3. Takahashi, N., Microbial ecosystem in the oral cavity: Metabolic diversity is an ecological riche and its relationship with oral diseases. Int. Congr. Ser., 1284, 103–112, 2005. 4. Meyer, D.H., Oral pathogen from dental plaque to cardiac diseases. Curr. Opin. Microbiol., 1, 1, 88–95, 1998. 5. Elujoba, A.A., Medicinal properties of plants with oral health implications. Proceedings of the 2nd Dr. David Barmes’ Memorial Public Health Symposium, 25th March 2003, organized by the Regional centre for oral health research & training for Africa. Jos in collaboration with WHO Regional Office, Brazzaville, 2003. 6. El-Said, F. et al., Nature cures in Nigeria. Part II: The antimicrobial properties of the buffer extracts of chewing sticks. Lloydia, 34, 1, 172, 1971. 7. Odebiyi, O.O. and Sofowora, A., Antimicrobial alkaloids from Nigeria chewing sticks. Planta Med., 36, 3, 204, 1979. 8. Tella, A., Analgesic and antimicrobial properties of Vernonia amygdalina. Br. J. Clin. Pharmacol., 7, 295–297, 1976. 9. Prashant, G.M., Chandu, G.N., Murulikrishna, K.S., Shafiulla, M.D., The effect of mango and neem extract on four organisms causing dental caries: Streptococcus mutants, Streptococcus mitis, and Streptococcus sanguis: An in vitro study. Indian J. Dent. Res., 18, 4, 148–151, 2007. 10. Muskhazil, M., Nurhafiza, Y., Azwady Nor, A.A., Dalilah Nor, E., Comparative study on the in vitro antibacterial efficacy of aqueous and methanolic extracts of Quercus infectoria gall’s against Cellulosi microbium cellulans. J. Boil. Science, 8, 634–638, 2008. 11. Joseph, I. and Ranjisingh, A.J.A., Antimicrobial activity of selected medicinal plants, Craetva magna (Linn.), Pongamia glabra (Linn.) and Areca catechu (Linn.). Ethnobot. Leaflets, 12, 995– 1002, 2008. 12. Ooshima, T., Osaka, Y., Sasaki, H., Osawa, K., Yasuda, H., Matsumura, M. et al., Caries inhibitory activity of cacao bean husk extract in in-vitro and animal experiments. Arch. Oral Biol., 45, 639–645, 2000. 13. Nostro, A., Cannatelli, M.A., Crisafi, G., Musolino, A.D., Procopio, F., Alonzo, V., Modifications of hydrophobicity, in vitro adherence and cellular aggregation of Streptococcus mutans by Helichrysum italicum extract. Lett. Appl. Microbiol., 38, 423–427, 2004. 14. Ishnava, K., Chauhan, J., Kachhiya, J., Patel, N., Screening and evaluation of ethano-botanical plants for their efficacy against cariogenic bacteria. J. Med. Aromat. Plant Sci., 34, 1–2, 57–62, 2012. 96 Natural Oral Care in Dental Therapy 15. Soni, H., Ishnava, K., Patel, K., Anticariogenic activity and hemolytic study of some medicinal plants leaf extract against six oral pathogen in in vitro condition. Int. J. Appl. Sci. Biotechnol., 2, 3, 253–259, 2014. 16. Bhattacharyaa, I., Ishnava, K., Chauhana, J., In vitro Anticariogenic activity of some Indian medicinal plants towards human oral pathogen. Asian J. Tradit. Med., 11, 4, 88–98, 2016. 17. Ishnava, K., Role of herbal medicine in dental health. J. Environ. Chem. Toxicol., 2, 1, 41–42, 2018. 18. Trivedi, P., Chauhan, J., Ishnava, K., In vitro assessment of inhibition potential of ethanomedicinal plants against cariogenic bacteria. Acta Sci. Microbiol., 1, 6, 43–49, 2018. 19. Barad, M., Ishnava, K., Chauhan, J., Anticariogenic activity and phytochemical studies of crude extracts from some Indian plant leaves. J. Intercult. Ethanoparmacol., 3, 2, 85–90, 2014. 20. Rajput, D. and Ishnava, K., Anticariogenic activity of some Indian medicinal plants. Eur. J. Dent. Med., 9, 1, 1–8, 2017. 21. Ishnava, K., Chauhan, J., Garg, A., Thakkar, A., Antibacterial and phytochemical studies on Calotropis gigantea (L.) R. Br. latex against selected cariogenic bacteria. Saudi J. Biol. Sci., 19, 87–91, 2012. 22. Ishnava, K., Chauhan, J., Barad, M., Anticariogenic and phytochemical evaluation of Eucalyptus globules Labill. Saudi J. Biol. Sci., 20, 69–74, 2013. 23. Ishnava, K., Shah, P., Anticariogenic and hemolytic activity of selected seed protein extracts in vitro condition. J. Dent. Tehran University of Medical Sciences, 11, 5, 576–586, 2014. 24. Bhoot, N. and Ishnava, K., Antimicrobial activity of medicinally important essential oils against selected dental microorganisms. Int. J. Curr. Microbiol. App. Sci., 6, 6, 1562–1575, 2017. 25. Shouche, S. and Ishanva, K., Screening of herbal formulation for anticariogenic activity. J. Med. Plants Stud., 6, 1, 243–249, 2018. 26. Perilla, M.J., Manual for the laboratory identification and antimicrobial susceptibility testing of bacterial pathogens of public health importance in this developing world, pp. 209–214, WHO, Atlanta, Georgia, USA, 2003. 27. Yogesh, M. and Mohan, J.S.S., Screening of plants for their potential antibacterial activity against Staphylococcus and Salmonella Spp. Nat. Prod. Rad., 6, 4, 301–305, 2007. 28. Parekh, J. and Chanda, S.V., In vitro antimicrobial and phytochemical analysis of some Indian medicinal plants. Turk. J. Biotechnol., 31, 53–58, 2008. 29. Ahmad, I. and Beg, A.Z., Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens. J. Ethnopharmacol., 74, 113–23, 2001. 30. Koo, H., Hayacibara, M.F., Schobel, B.D., Cury, J.A., Rosalen, P.L., Park, Y.K., Vacca-Smith, A.M., Bowen, W.H., Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol. J. Antimicrob. Chemother., 52, 782–789, 2003. 31. Ajaybhan, P., Chauhan, N., Chauhan, A., Evaluation of antimicrobial activity of six medicinal plants against dental pathogens. Rep. Opinion, 2, 6, 37–42, 2010. 32. Chaudhry, N.M.A. and Tariq, P., In vitro antibacterial activities of Kalonji, Cumin and Poppy seed. Pak. J. Bot., 40, 1, 461–467, 2008. 33. Choi, J.-G., Kang, O.-H., Lee, Y.-S., Chae, H.-S., Oh, Y.-C., Brice, O.-O., Kim, M.-S., Sohn, D.-H., Kim, H.-S., Park, H., Shin, D.-W., Rho, J.-R., Kwon, D.-Y., In vitro and in vivo antibacterial activity of Punica granatum peel ethanol extract against Salmonella. Evid.-Based Complementary Altern. Med., 2011, 1–8, 2009. 6 Cytotoxic and Anti-Inflammatory Effect of Turmeric and Aloe Vera in a Gingivitis Model Karen Esperanza Almanza-Aranda†, Miguel Aranda-Fonseca, Gabriela Velazquez-Plascencia and Rene Garcia-Contreras* * † Interdisciplinary Research Laboratory, Nanostructures and Biomaterials Area, National School of Higher Studies (ENES) Leon Unit, National Autonomous University of Mexico (UNAM), León, Guanajuato, México Abstract The indiscriminate use of synthetic medicines has generated resistance to antimicrobials, and in response to this, special attention has been placed on medicinal plants. Objective: To know the cytotoxic and anti-inflammatory effects of turmeric and Aloe vera in human gingivitis-model. Materials and methods: Human gingival fibroblast (HGF) was subcultured in DMEMsupplemented medium. Turmeric and Aloe vera were inoculated (0–100 mg/mL and 0–25%). Viable cell number was determined by the MTT method and the mean cytotoxic concentration (CC50). Interleukin-1β (IL-1β) was used to induce a proinflammatory state. The anti-inflammatory effect was evaluated by the expression of prostaglandin E2 by an ELISA assay. Data were interpreted with ISO-10993-5 and analyzed with the normality tests of Shapiro–Wilk and Student t tests with a significance of 0.05. The data were made in triplicate from three independent experiments. Results: The cell viability of turmeric and Aloe vera showed a moderate cytotoxicity (p < 0.05) with CC50 values of 44.98 ± 1.4 mg/mL and 18.42 ± 2.1%, respectively. The expression of PGE2 significantly reduced the contact with turmeric and Aloe vera. Conclusions: Turmeric and Aloe vera have a moderate cytotoxic effect with an anti-inflammatory effect in HGF gingivitis model. The use of these medicinal plants has a promising potential clinical use for patients with gingivitis and periodontitis. Keywords: Medicinal plants, cytotoxicity, anti-inflammatory, Aloe vera, turmeric, gingivitis 6.1 Introduction A medicinal plant is a natural resource, whose part or extracts are used as a medicinal drug for the treatment of some condition. Its use dates back to prehistoric times and was one of the most widespread forms of medicine [1]. *Corresponding author: dentist.garcia@gmail.com † Corresponding author: karen_esp13@hotmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (97–110) © 2020 Scrivener Publishing LLC 97 98 Natural Oral Care in Dental Therapy Gingivitis is the most prevalent inflammatory periodontal disease. Some reports have estimated that it affects more than 80% of the world’s population [2]. The implicated pathogens in this disease can induce the formation of biofilm around the teeth that initiates the disease process; the activated host immune inflammatory response leads to pathogenesis; first, there is gingivitis and then periodontitis. Some of the implicated inflammatory cytokines in this progressive and destructive disease are especially, interleukins (IL) and tumor necrosis factor-α (TNF-α) that activate the prostanoid mechanisms by the expression of prostaglandins (PG) and cyclooxygenase. It has been demonstrated that prostaglandin E2 (PGE2) levels in the gingival crevicular fluid are significantly higher in patients exhibiting gingivitis and periodontitis than those in gingivally and periodontally healthy subjects [3]. In order to solve this disease, the addition of a chemical agent as an adjunct has been suggested to improve the conventional methods of plaque control. It has now been found that some plants have a preventive and curative effect toward the most common oral conditions. This has been studied clinically in several groups of people with gingivitis using plants such as turmeric and aloe vera with different application methods [4]. The use of certain herbs for therapeutic purposes in stomatology has persisted today, despite the advances recorded in the field of pharmacological or allopathic therapy. There are references on the use of medicinal plants for oral diseases since pre-Hispanic times among the Maya. The use of plants to alleviate common ailments at the domestic level is a knowledge that refuses to disappear; hence, there is a record, either as an alternative or as a complement to pharmacological therapy in stomatology, of more than 30 plant species [5]. The present chapter summarizes the uses of Aloe vera and turmeric to treat dental inflammatory diseases highlighting their potential application in dental practice and the evaluation of cytotoxic and anti-inflammatory effect in a gingivitis model to show evidence to support these properties in order of the viable cell number and expression of PGE2. 6.2 Gingivitis and Periodontitis Gingivitis is an inflammatory process limited to the mucosal epithelial tissue surrounding the cervical portion of the teeth and the alveolar processes. Gingivitis has been classified by clinical appearance (e.g., ulcerative, hemorrhagic, necrotizing, purulent), etiology (e.g., drug-induced, hormonal, nutritional, infectious, plaque-induced), and duration (acute, chronic). The most common type of gingivitis is a chronic form induced by plaque [6–8]. Periodontitis is one of the most ubiquitous diseases and is characterized by the destruction of connective tissue and dental bone support following an inflammatory host response secondary to infection by periodontal bacteria. Severe periodontitis, which may result in tooth loss, is found in 5–20% of most adult populations worldwide [9]. Gingivitis and periodontal diseases comprise a number of infectious and inflammatory conditions brought about by the interaction between supragingival and subgingival biofilms and the host inflammatory response. The presence of biofilms of the microorganisms that compose them, and of bacterial metabolites produced following colonization of the subgingival area, elicits an inflammatory response. Activation of the host immune system, primarily for protective purposes, ultimately results in tissue destruction by triggering the synthesis and release of cytokines, proinflammatory mediators, and matrix metalloproteinases. The progression and severity of the periodontal destruction caused by periodontitis Turmeric and A V for Gingivitis 99 depend on the balance between the virulence of the local biofilm and the host immune response. A variety of systemic factors and conditions may interfere with and modulate the relationship between the microbial challenge and the host response. Smoking and diabetes have classically been recognized as risk factors for periodontitis; smokers and diabetics are at higher odds of developing periodontitis compared with individuals unaffected by either condition. Furthermore, longitudinal studies have shown that smokers are at a significantly higher risk of periodontal attachment loss than are never-smokers [10, 11]. 6.3 Aloe Vera Aloe vera (Figure 6.1a and b) is a native plant from arid regions that has been used as a medicinal plant for therapeutic reasons since ancient times. Its uses target different pathologies depending on the biological issues like the variety of species of the plant, and even of cultural issues (traditions and empirical knowledge). There are more than 360 species of aloe, and there is scientific support for the medicinal properties of only some of them with aloe vera being the most studied and currently marketed due to the aloe that contains polyphenol-like and anthracene-like substances that possess beneficial health properties for different diseases and therapeutic therapies. The beneficial effects on the different diseases are associated with their antioxidant and anti-inflammatory response. These beneficial effects have been observed in several studies both in vivo and in vitro. Although there is a lot of scientific evidence that support these properties and beneficial effects for aloe, there are still many aspects to be studied to fully understand the mechanisms of the beneficial action of Aloe vera [12]. Aloe vera contains 75 potentially active constituents: vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids, and amino acids. Polysaccharides are considered to be the (a) (b) (c) (d) Figure 6.1 Aloe vera plant (a–b) and turmeric from which the extract was taken (c–d). Source: Direct. 100 Natural Oral Care in Dental Therapy active ingredients of Aloe’s anti-inflammation and immune-modulation effects. Inflammation is a tissue reaction by the body to injury and typically follows burns or other skin insults. It is classically characterized by swelling, pain, redness, and heat, as well as loss of function [13]. The anti-inflammatory mechanisms of Aloe vera act by inhibiting the synthesis of prostaglandins and reduce the migration and infiltration of leukocytes, the release of histamine, and the synthesis and secretion of leukotrienes. 6.3.1 Aloe Vera for Gingivitis and Periodontitis Aloe vera can be very useful to help improve the periodontal health provided that the state of the denture has been previously checked. It is rather a way to prevent, regenerate, and eliminate bacteria that affect the gums. The dental uses of Aloe vera are numerous. There is increased interest among researchers to analyze the use of Aloe vera in dentistry, and various studies have proven the effectiveness of this plant [13]. Some of the effective properties are related to its good antiseptic and anti-inflammatory properties. They are used in the treatment of gingivitis and periodontitis. They readily reduce the gingival inflammation and pain associated with it. Clinical studies have shown that mouthrinses and dentifrices containing Aloe vera have shown a remarkable reduction in gingivitis and plaque accumulation after its use [14, 15]. Studies by Geetha Bhatt et al. have proven the use of Aloe vera gel as a subgingival administrator in the treatment of periodontal pockets that reduce the periodontal pathogens and enhance significantly the anti-inflammatory effect. The wound healing and antiinflammatory property of this gel is proven by the reduction in the incidence of alveolar osteitis in patients who received Aloe vera gel [16]. 6.3.2 Aloe Vera: Other Oral Applications Aloe vera gel incorporated in denitrificans reportedly inhibited the growth of Candida albicans, Streptococcus viscosus, Streptococcus mutans, and Streptococcus sanguis [17]. Also, the Aloe vera tooth gel does not contain abrasives, which are present in the normal dentifrices conferring a good alternative for sensitive teeth. Thus, acemannan, a complex mannose carbohydrate, which is derived from the Aloe vera plant, has an inherent viscosity, which makes it ideal to be used as a denture adhesive due to both its adhesive strength and minimal cytotoxicity properties. On the other hand, its anti-viral properties help in the treatment of herpes simplex and herpes zoster infections. The sore areas of the oral mucosa which are covered by dentures can be treated with Aloe gels as they are also a good antifungal agent. They also reduce the pain associated with ulcers in the commissures of the mouth. Finally, Aloe vera has demonstrated an antioxidant effect of some of the constituents of the aloe vera gel; three aloesin derivatives from Aloe, namely, i) isorabaichromone, ii) fer-uoylaloesin, and iii) p-coumaroylaloesin showed potent free radical and superoxide anion activities [18]. 6.4 Turmeric Turmeric (Figure 6.1c and d) is a product of Curcuma longa, a rhizomatous herbaceous perennial plant belonging to the ginger family Zingiberaceae, which is native to tropical South Asia. As many as 133 species of curcuma have been identified worldwide. Most of Turmeric and A V for Gingivitis 101 them have common local names and are used for various medicinal formulations. India produces nearly all of the world’s turmeric crop and consumes 80% of it. Turmeric has been put to use as a foodstuff, cosmetic, and medicine [1, 4]. Turmeric is used as an herbal medicine for rheumatoid arthritis, chronic anterior uveitis, conjunctivitis, skin cancer, smallpox, chicken pox, wound healing, urinary tract infections, and liver ailments. It is also used for digestive disorders; to reduce flatus, jaundice, menstrual difficulties, and colic; for abdominal pain and distension, and for dyspeptic conditions including loss of appetite, postprandial feelings of fullness, and liver and gallbladder complaints. It has anti-inflammatory, choleretic, antimicrobial, and carminative actions. The main clinical targets of turmeric are the digestive organs: in the intestine, for the treatment of diseases such as familial adenomatous polyposis; in the bowels, for the treatment of inflammatory bowel disease; and in the colon, for the treatment of colon cancer. For arthritis, dosages of 8–60 g of fresh turmeric root three times daily have been recommended. For dyspepsia, 1.3–3.0 g of turmeric root is recommended [19]. Since turmeric has antimicrobial, antioxidant, astringent, and other useful properties, it has a potential use in the field of dentistry and many of their specialties. 6.4.1 Turmeric for Gingivitis and Periodontitis Although chlorhexidine has been regarded as a “gold” standard in dentistry for the prevention of plaque and gingivitis, it has been found that turmeric modulates the cellular action of several growth factors, cytokines, and transcription factors that could be involved in the inflammatory process of gingivitis and periodontitis [20–23]. Bhandari and Shankwalker used turmeric in the form of mouthwash and found it to be an effective anti-inflammatory agent [24]. Also, its usefulness in the dental clinic has been described as preventive mouthwash of radiation injuries, disinfectant mouthwash, oral antimicrobial and, above all, a wide approach in relation to cervicofacial cancer. The antiplaque effect of turmeric gel was comparable to chlorhexidine gel. The anti-inflammatory effect on gingivitis and periodontitis of turmeric gel was similar to those reported in previous studies [25, 26]. The possible mechanism of action of turmeric as an anti-inflammatory agent could be due to the inhibitory action of inflammatory mediators of arachidonic acid metabolism. It selectively inhibits the synthesis of PGE2 and thromboxane while not affecting the synthesis of prostacyclin. Turmeric, by virtue of its anti-inflammatory property, reduces inflammatory mediators and causes shrinkage by reducing inflammatory edema and vascular engorgement of the connective tissue. The mechanisms are related to the incorporation in collagen, which acts as a supportive matrix for slow release, increases wound reduction, and enhances cellular proliferation [27, 28]. 6.4.2 Turmeric: Other Oral Applications Turmeric also has analgesic effects acting at the level of the central and peripheral nervous system; its possible mechanisms of action are the inhibition of certain transcription factors involved in inflammation and the alteration of pain signaling pathways through ion channels. It has been found that tinted pit and fissure sealant is useful for applying to tooth surfaces for the prevention or reduction of dental caries. This sealant can be produced from 102 Natural Oral Care in Dental Therapy a composition comprising a polymerizable resin system containing an acrylic monomer and at least one colorant selected from the group consisting of Annatto extract, turmeric extract, and β-Apo-8 -Carotenal. Accordingly, dental plaques are generally stained with dental-plaque staining agents, which contain dyes, to reveal their locations in order to uncover the attached dental plaques. The dental-plaque detection system includes a dentalplaque staining agent, which contains at least one selected from the yellow pigment of benikoji, turmeric extracts, and curcumin; and a light-emitting apparatus, which outputs light having a wavelength within the range of 250 to 500 nm to an object in the oral cavity where the dental-plaque staining agent is attached. The yellow pigments of beni-koji and turmeric are known as staining agents and are also used for other purposes [29]. 6.5 Methodology 6.5.1 Materials and Methods 6.5.1.1 Authorization To obtain human gingival fibroblasts (HGF), gingival tissue was collected without pathological damage from healthy patients, who underwent third molar surgical odontectomy at the National School of Higher Studies (ENES) Leon Unit of the National Autonomous University of Mexico (UNAM). To this end, this protocol was submitted to the Bioethics and Biosafety Committee of the ENES León Unit of the UNAM, where the patients gave their authorization so that their extracted tissues could be used in this investigation. Samples were stored immediately after extraction in Falcon tubes with 10 ml of phosphate buffer saline (PBS, pH 7.4) and 1% antibiotic (Gibco , Grand Island, NY, EU). 6.5.1.2 Cell Culture Once in the laboratory, the gingival tissue was washed twice with PBS. The HGF was obtained inside the laminar flow bench (Lumistell , Celaya Gto, Mexico). Explants, approximately 1 × 1 mm, were made with a No. 20 scalpel blade on a sterile glass slide and were inoculated in 10-cm-well sterile culture dishes (Thermo Fisher Scientific, Rochester, NY, USA), then 10 mL of DMEM culture medium supplemented with 20% sterile fetal bovine serum (FBS, Gibco ), 1% of antibiotic (10,000 IU/mL penicillin G and 10,000 mg/mL streptomycin, Gibco ), and 1% Glutamax (Gibco ) were subsequently allowed to incubate at 37°C with 5% CO2 and 95% humidity, for 3 weeks until a cellular monolayer of 80% of its cellular proliferation was obtained in an incubator (Binder, Tuttlingen, Germany). The culture medium was changed every third day after week 1. 6.5.1.3 Cell Subculture The cells were washed three times with phosphate buffer solution (PBS, 5 ml), 1 ml of 0.05% trypsin (Gibco ) EDTA-2Na was added and incubated for 5 min at 37°. The cell count was carried out with the cell count method where a Neubauer chamber (BOECO 1/10 mm, Germany) and automated cell counter (Bio-Rad 2000 TC10, Hercules, California, Turmeric and A V for Gingivitis 103 USA) were used by exclusion of blue from trypan (Trypan Blue Dye 0.40%, Bio-Rad, Hercules California: EU). The number of cells per milliliter of suspension allowed calculating the total number of cells. Cells were subcultured in the range of 5 × 105 cells/mL for each experiment. 6.5.1.4 Cytotoxicity Test The first experiment was performed where 96-well dishes were used in the HGF and left to incubate for 48 h at 37°C with 5% CO2 and 95% humidity. Turmeric (Figure 6.2d–f) and Aloe vera (Figure 6.2a–c) were inoculated at 0–100 mg/mL and 0–25%, respectively. They were allowed to incubate for 24 h at 37°C with 5% CO2 and 95% humidity. The viable cell number and the mean cytotoxic concentration (CC50) were calculated from the dose–response curve. Briefly, the culture medium was removed and replaced with the MTT reagent (Thiazolyl Blue Tetrazolium Bromide, Sigma Aldrich) at a concentration of 0.2 mg/ mL in DMEM for 7 h at 37°C with 5% CO2 and 95% humidity. The formazan crystals were dissolved with dimethyl sulfoxide (DMSO, J.T Baker). The plates were analyzed in microplate spectrophotometer (Thermo Scientific Multiskan GO, Rochester, NY, USA) at 570 nm. 6.5.1.5 Anti-Inflammatory Activity in a Gingivitis Model HGF cells were subcultured at the aforementioned density in 24-well dishes. Interleukin 1-beta (IL-1β, recombinant human; >97% Purity; Minneapolis, MN, USA) was reconstituted with Bovine Albumin (IMMUCOR GAMMA, Norcross , GA, USA). The IL-1β (3 ng/ mL) was inoculated in the plates to induce a pro-inflammatory state for 3 h and to function as a positive control. Turmeric and Aloe vera were inoculated at three concentrations, 0, 12.5, 25 mg/mL and 0%, 12.5%, 25%, and incubated for 24 h. The supernatant of the culture medium was stored in Eppendorf tubes, and PGE2 expression was performed with an (a) (b) (c) (d) (e) (f) Figure 6.2 Cell culture in contact to Aloe Vera and Turmeric: Aloe Vera: (a) Control group; (b) 0.8% concentration; (c) 2.5% concentration. Turmeric: (d) Control group; (e) 3.125 mg/mL concentration; (f) 100 mg/mL concentration. Source: Direct. 104 Natural Oral Care in Dental Therapy expression kit (R&D Systems, Minneapolis, USA) with ELISA according to the manufacturer’s instructions. 6.5.1.6 Statistical Analysis Each reported value represents the mean ± standard deviation (S.D.). The data were subject to Shapiro–Wilks normality test and paired t-test using Statistical Package for Social Science (Chicago, IL, USA). Differences were considered significant at p < 0.05 with an interval confidence of 95%. The experiments were carried out for triplicate from three different experiments resulting in n = 9 samples per group. The viable cell number from dose-response and CC50 values were interpreted based on ISO-10993-5: Tests for in vitro cytotoxicity of medical devices. 6.5.2 Results 6.5.2.1 Cytotoxicity Turmeric and Aloe vera exhibited essentially moderate cytotoxicity in a dose-dependent manner (p < 0.05) against HGFs cell over the concentration range from 0.098 to 100 mg/ 140 120 100 Viable Cell Number (%) Viable Cell Number (%) 120 80 60 40 20 (a) 100 80 60 40 20 (c) 25 3 .5 12 1 6. 6 3. 8 1. 4 0. 0. 1 2 0. 9 0. 04 0. 0 0. mg/mL % 20 25 18 16 IL-1β (+) 15 IL-1β (–) 10 5 (b) PGE2 (ng/mL) 20 PGE2 (ng/mL) 4 02 0 50 10 .5 25 12 5 25 6. 3 12 56 3. 1. 1 1 78 0. 5 39 0. 19 0. 0. 09 0 8 0 14 12 10 8 6 4 2 0 0 0 12.5 mg/mL 25 IL-1β (+) IL-1β (–) (d) 0 12.5 % 25 Figure 6.3 Viable cell number and anti-inflammatory effect of turmeric and aloe vera in culture with human gingival fibroblast (HGF) cells and gingivitis-model. Near-confluent HGF cells were incubated for 48 h with 0–100 mg/mL (a) or 0–50% (c), respectively. The viable cell number was then determined by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay (A). Synergistic stimulation of prostaglandin E2 (PGE2) production by turmeric (b) or aloe vera (d) interleukin-1β (IL-1β, 3 ng/mL). The concentration of PGE2 in the culture medium was determined by ELISA, according to the manufacturer’s instruction (B). Each value represents the mean ± S.D. of three independent experiments (each experiment was performed in triplicate). *p < 0.05, **p < 0.01 paired t-test. Source: Direct. Turmeric and A V for Gingivitis 105 mL and 0.024–25% with CC50 values of 44.98 ± 1.4 mg/mL (Figure 6.3a) and 18.42 ± 2.1% (Figure 6.3c), respectively. 6.5.2.2 Anti-Inflammatory Activity in a Gingivitis Model Regardless of the presence or absence of IL-1β (3 ng/ml), it significantly (p < 0.05) stimulated the production of PGE2 in HGF gingivitis model. Synergically, IL-1β and turmeric or aloe vera reduced significantly (p < 0.01) (Figure 6.3b and d) the PGE2 production and showed the anti-inflammatory effect of both samples. 6.5.3 Discussion Dentobacterial plaque is the main causative agent of periodontal disease. Oral hygiene and the elimination of this plaque are of the utmost importance in the prevention of periodontal disease. Adulthood, style and quality of life, and some psychomotor disability are the risk factors involved in proper oral and personal hygiene, which has stimulated the search for added chemotherapeutic agents to mouthwashes to improve plaque control dentobacteriana and prevent both gingival and periodontal diseases. Therefore, various studies have been suggested to reduce the bacterial load in the host. Herbal products are agents that have been widely used from our origin to reduce bacterial population. Different researches have studied these purposes, and their results showed very satisfactory conclusions. This allowed us to evaluate the efficacy of turmeric and aloe vera in oral health. 6.5.3.1 Cytotoxicity Some studies have reported the non-cytotoxic effect of Aloe vera in culture with Vero cells at concentrations from 0 to 5% compared with the untreated healthy cells. Based on the results, up to 5% of the gel extract was nontoxic for Vero cells. Here our results suggest that 18.4% reduce the 50% of the viable cell number in contact with HGF for 24 h with any side effect at 5% concentration of Aloe vera [30]. International toxicological studies establish an LD50 of 30 mg/kg of the aqueous extract of turmeric and warn that the continuous consumption of turmeric orally, in doses of 100 mg/kg (higher than those indicated in the indications and above the LD50 levels), has an ulcerogenic effect [31]. Similarly, a study conducted at the Center of Toxicology and Biomedicine of Santiago de Cuba, found no level of toxicity when evaluating the acute oral toxicity of Curcuma longa [32]. An in vivo study with rodents confirmed that turmeric modulates the formation of adducts (DNA and proteins) of aflatoxin B1, AFB, in addition to improving the activity of the enzymes GSHT and UGT1A1, which significantly reduced the activity of the enzyme CYP1A1. At the molecular level, turmeric decreased the urinary excretion of the AFB-Nguanine adduct, hepatic DNA, and serum albumin adduct. This is of great importance to counteract the toxicity of aflatoxin B1, through the consumption of turmeric in the diet of people exposed to this toxin [33]. Our results showed that turmeric possesses moderate cytotoxicity and is well tolerated by the human gingival fibroblast, comparable to the results mentioned above. 106 Natural Oral Care in Dental Therapy Another in vitro study evaluated the effect of Aloe vera associated with endodontic medication, with or without irradiation with laser photobiomodulation (FTL) in human pulp fibroblasts and concluded that Aloe vera allowed greater cell viability in human pulp fibroblasts in the presence of calcium hydroxide [34]. In our research, we can verify that at medium concentration, there is greater cell viability and proliferation, and at high concentration, there is a moderate level of cytotoxicity depending on the doses. Cell viability was decreased with both plants in high concentrations, probably because they modulate mechanisms related to cell proliferation, survival routes, caspase activation pathways, and tyrosine kinase activity. 6.5.3.2 Anti-Inflammatory Activity The anti-inflammatory property of turmeric has been studied and has shown a significant reduction in inflammation. Bhandari and Shankwalker used turmeric in the form of mouthwash and discovered that it was an effective anti-inflammatory agent. For the main ingredients of this formulation, each gram contains 10 mg of Curcuma longa extract together with erythrosine and titanium dioxide [35, 36]. Similarly, our results showed that 12.5 mg/mL reduced the expression of PGE2 with previous pro-inflammatory stimulation with IL-1β (3 ng/mL) and showed an effective anti-inflammatory effect in a dose-dependent manner. Mulikar S et al. and Waghmare PF et al. studied the efficacy of curcumin mouthwash as a complement to scaling and root planning in the treatment of chronic gingivitis and to compare chlorhexidine in terms of its anti-inflammatory and antimicrobial properties. They concluded that curcumin is comparable to chlorhexidine as an anti-inflammatory mouth rinse and is an effective adjunct to periodontal therapy [37, 38]. Bathini et al. and Gottumukkala et al. studied the effectiveness of subgingival irrigation of a 0.1% indigenous curcumin solution in patients with clinical and microbiological parameters: a randomized pilot clinical trial [39]. In the case of Aloe vera, a research group carried out a randomized, double-blind clinical study to evaluate the antiplaque and antigingivitis efficacy of the mouthwash [40]. In our study, the data analyzed showed that turmeric is a medicinal plant with a property that reduces inflammation. This can be attributed to its ability to inhibit both the biosynthesis of inflammatory prostaglandins of arachidonic acid and the function of neutrophils during inflammatory states. Aloe vera is a plant with anti-inflammatory activity: it inhibits the synthesis of prostaglandins and reduces the migration and infiltration of leukocytes, the release of histamine, and the synthesis and secretion of leukotrienes. Our results showed the potential anti-inflammatory effect of both turmeric and Aloe vera in a gingivitis model by reducing dose-dependently the expression of PGE2 in culture with HGF. On the other hand, future experiments should be focused on the evaluation of Aloe vera action against microorganisms such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Bacteroides fragilis by agar diffusion or microdilution as well as co-culture with HGF or human periodontal ligament fibroblast (HPLF) to a more realistic gingivitis or periodontitis model. The antibacterial effect as well as the beneficial effects of aloe vera in mouthwashes intended to reduce dentobacterial plaque and prevented gingivitis [41]. Turmeric and A V for Gingivitis 107 6.6 Perspectives for the Future The use of medicinal plants in dentistry are increasing lately because they have good properties, low economic cost and are quite accessible, and are a good alternative to reduce the demand for synthetic medications, which have important side effects such as cytotoxicity and teratogenicity. Interest is increasing in the use of Aloe vera and turmeric in dentistry, and this natural therapy has already proven its unlimited use in the field. More and more studies are carried out where they demonstrate the efficacy of their anti-inflammatory properties and how they can be applied for the benefit of the population, which is why these plants represent a promise for the future of dentistry. However, most of these studies are short term. More long-term studies are needed and with more population samples to determine how these plants act in oral health. 6.7 Conclusions The effect of turmeric and Aloe vera in culture with HGF showed moderate cytotoxicity with an anti-inflammatory effect in a human gingivitis model. The use of these medicinal plants has a promising potential clinical use for patients with gingivitis and periodontitis. References 1. Díaz, J.L., Use of Medicinal Plants of Mexico. IMEPLAM, 2, 1–329, 1976. 2. Slots, J., Periodontology: Past, present, perspectives. Periodontol., 62, 7–19, 2013. 3. Noguchi, K. and Ishikawa, I., The roles of cyclooxygenase-2 and prostaglandin E2 in periodontal disease. Periodontol., 43, 1, 85–101, 2007. 4. Jain, L., Jain, P., Bisht, D., Sharma, A., Srivastava, B., Gupta, N., Use of traditional Indian plants in the inhibition of caries-causing bacteria—Streptococcus mutans. Braz. Dent. J., 2, 5–110, 2015. 5. Waizel, J. and Martínez, I.M., Plants used in dentistry. 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Carvalho, N., Guedes, S., Albuquerque, R., Albuquerque, D., Araújo, A., Paranhos, L., Camargo, S., Gonzaga, M., Analysis of Aloe vera cytotoxicity and genotoxicity associated with endodontic Turmeric and A V for Gingivitis 109 35. 36. 37. 38. 39. 40. 41. medication and laser photobiomodulation. J. Photochem. Photobiol. B, Biol., 178, 348–354, 2018. Ammon, H.P., Safayhi, H., Mack, T., Sabieraj, J., Mechanism of anti-inflammatory action of curcumin and boswellic acids. J. Etnofarmacol., 38, 113–119, 1993. Bhandari, H. and Shankwalker, G.B., Clinical evaluation of the action of the combination of indigenous drugs on dental plaque, calculus and gingivitis. Dissertation presented to the University of Bombay, pp. 113–19, 1980. Muglikar, S., Patil, K.C., Shivswami, S., Hegde, R., The efficacy of curcumin in the treatment of chronic gingivitis: a pilot study. Salud Oral Prev. Dent., 11, 1, 81–86, 2013. Eigner, D. and Scholz, D., Asa-foetida splint and Curcuma longa in the treatment with traditional medicines and diet in Nepal. La revista de etnofarmacología, 67, 1–6, 1999. Gottumakkala, S.N., Koneru, S., Mannem, S., Efficacy of subgingival irrigation of a 1% indigenous curcumin solution in clinical and microbiological parameters in patients with chronic periodontitis. A pilot randomized clinical trial. Contemp. Clin Dent., 41, 186–191, 2013. Bathini, Ch., Avula, J., Anumala, N., Kalakonda, B., Krishnanjaneya, R., Tupili, M., A randomized, double-blind clinical study to evaluate antiplaque and antigingivitis. Indian Soc. Periodontol., 18, 624–28, 2013. López, O., Toledo, B., Veloz, M., López, I., Navas, A., Therapeutic application of Aloe vera in the chronic periodontal inflammatory disease. Rev. Méd. Electrón., 40, 3, 2018. 7 Effects of Bauhinia forficata Link in Reducing Streptococcus mutans Biofilm on Teeth Julio Cesar C. Ferreira-Filho1, Mariana Leonel Martins1, Andressa Temperini de Oliveira Marre2, Juliana Soares de Sá Almeida2, Leandro de Araújo Lobo2, Adriano Gomes Cruz3, Marlon Máximo de Andrade3, Thiago Isidro Vieira1, Maria Teresa Villela Romanos4, Lucianne Cople Maia1, Ana Maria Gondim Valença5 and Andréa Fonseca-Gonçalves1* 1 Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil 2 Department of Medical Microbiology, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil 3 Department of Foods, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Rio de Janeiro, Brazil 4 Department of Virology, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 5 Department of Clinic and Social Dentistry, School of Dentistry, Universidade Federal da Paraíba (UFPB), João Pessoa, PB, Brazil Abstract Bauhinia forficata Link has been described as having bioactive properties including antimicrobial activity. However, this natural product has not yet been tested against dental biofilm. This book chapter evaluated by means of a laboratory study the effects of a tincture made from Bauhinia forficata Link leaves (TBF) on Streptococcus mutans biofilm formed on teeth. Susceptibility tests were performed to observe the viability of S. mutans. Total solids (ºBrix), pH, mineral content, and cytotoxic potential of the TBF on oral fibroblasts were determined. S. mutans biofilm was cultivated on enamel tooth blocks (n = 48) in polystyrene plates. After biofilm formation (24 h, 37°C), the blocks were treated (50 µL/1 min for 3 days) with 0.12% chlorhexidine; 0.11% TBF, 0.388% ethanol, 0.233% TBF, and 0.816% ethanol. The treatments were compared by means of analysis of variance/Tukey’s test. The TBF was found to contain calcium, phosphorus, and magnesium among other elements and promoted an S. mutans optical reduction when compared with the control (p < 0.05). The TBF thus constitutes a promising adjuvant substance for the prevention of carious lesion due to its ability to reduce S. mutans biofilm on teeth. For this reason, this chapter aims to present a discussion of the oral antibiofilm effect of TBF. Keywords: Microbiology, dental plaque, hydrogen-ion concentration, tooth, phytotherapy *Corresponding author: andrea.goncalves@odonto.ufrj.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (111–120) © 2020 Scrivener Publishing LLC 111 112 Natural Oral Care in Dental Therapy 7.1 Introduction Numerous microorganisms in the oral cavity are able to trigger dental caries [1]. Streptococcus mutans, a Gram-positive anaerobic bacterium, contributes to biofilm formation and build-up, and it is the major microorganism involved in caries process [2]. Its relevance is to be attributed to its acidogenic and aciduric properties, which facilitates the survival, growth, and maintenance of its metabolism under acidic environments [3]. Wherefore, S. mutans biofilms have been largely studied in vitro due to difficulties in developing in vivo researches for controlled cariogenic conditions [3]. The use of medicinal plants as another treatment choice for oral diseases has received great scientific attention [4], particularly for the reason that they do not origin bacterial resistance, as may happen with antibiotics [4, 5]. Several studies have investigated the inhibitory properties of natural products versus the growth of oral microorganisms [6–9]; such plants may represent a positive strategy for caries control [8]. Bauhinia forficata Link (Family Leguminosae, Subfamily Caesalpinioideae) has been described as having outstanding bioactive properties including antioxidant activity [10], antimutagenic action [11], antilipidemic, hypocholesterolemic [12], and antimicrobial activity [11, 13]. This plant grows as a heliophilous medium-sized tree (5–9 m high), indifferent to soil moisture conditions, and possessing bilobed and elongated leaves [14]. The antimicrobial activity of B. forficata L. has already been reported against planktonic cells [11, 13]. The antimicrobial effects of its leaves, however, which are extensively used as an antidiabetic herbal medicine [15], have not yet been tested against dental biofilm. Thus, the objective of this book chapter was to present a discussion, by means of an in vitro study, of the anti-S. mutans biofilm effect of a tincture made from leaves of B. forficata L. (TBF). The hypothesis is that the extract of TBF reduces the S. mutans biofilm formed on teeth. 7.2 Materials and Methods 7.2.1 Recognition, Production, and Chemical Characterization of Ethanolic Tincture From B. forficata L. Leaves This plant was collected in the city of Ribeirão Preto, Brazil (21°07 S, 47°45 W, 523,874 m altitude). The leaves were identified, indexed, and stored in the Herbarium of the Universidade Federal do Rio de Janeiro in Rio de Janeiro, Brazil, with the registration number 40781. The TBF was made by the percolation method, using 20 g of the plant in 100 g of the tincture. Ethanol 70% was used as the solvent. The measurement of total soluble solids was performed by means of a digital refractometer (PAL-1; Atago Co., Ltd., Minato-ku, Japan) in which values were expressed as °Brix. The pH was evaluated with a pH meter (DM20 Digitalized; Digimed, Santo Amparo, SP, Brazil). Both investigations were done in triplicate, and the final results were showed as the average of the values. To quantify the mineral content, aliquots (3 mL) of the TBF at 20% were sampled and centrifuged (3,000 g, 3 min, 48°C). To the supernatant was added 250 mL of 65% nitric acid. The sample was then evaluated by atomic absorption spectroscopy (Aanalyst 300; PerkinElmer, Inc., Waltham, MA, USA) to quantify the content of sodium (Na), Oral Antibiofilm Effect of Bauhinia 113 potassium (K), magnesium (Mg), calcium (Ca), zinc (Zn), and phosphorus (P). All analyses were performed in quadruplicate. 7.2.2 Microbial Strains and Preparation of Inoculum Samples of S. mutans [American Type Culture Collection (ATCC) no. 25175] were used to form the inoculum. Colonies of the pure strain were selected; moved to phosphate-buffered saline; and was standardized at an absorbance of 0.15 at 520 nm (Libra S2 Colorimeter; Biochrom, Cambridge, UK), equivalent to approximately 108 colonies of the microorganisms [colony-forming units (CFU)/mL]. 7.2.3 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) were evaluated using the Clinical and Laboratory Standards Institute M100-S22 specifications [16] with a modification [7]. Microplates (96 wells) were inoculated (5 × 105 CFU/ mL of each strain) with 100 µL of brain heart infusion medium (BHI; Difco, Sparks, USA). The tested TBF concentrations ranged from 95.238 to 0.0272 mg/mL (9.5238–0.00272%) against S. mutans. Chlorhexidine digluconate at 0.12% (0.12% CHX) was the positive control, and ethanol (70%) was also tested (from 33.333% to 0.009516%), as it was the main tincture solvent. The MIC was considered as the lowest concentration of solution that did not exhibit turbidity in the culture medium, as confirmed by resazurin salt (Sigma-Aldrich, St. Louis, MO, USA). The MBC corresponds to the minimum concentration that did not show microorganism growth after subculturing 50 µL of concentrations higher than the MIC on the BHI agar. 7.2.4 Kill-Kinetic Assay The kill-kinetics were demonstrated by the growth curve of the tested microorganism (S. mutans ATCC no. 25175), based on the recommendations of the CLSI M100-S25 guide for broth microdilution, with anaerobe and aerobe antimicrobial susceptibility [17]. It was used to measure the effects of the TBF on bacterial growth. Initially, the BHI medium containing the 0.11% TBF (1.110 mg/mL) and 0.388% ethanol (3.88 mg/mL) was added, in triplicate, to the wells of a 96-well plate. For the inoculum, microorganisms were initially grown in that medium for 24 h, then inoculated into the fresh medium and allowed to grow to McFarland 0.5. The cultures were diluted (1:15) in the same medium, and an inoculum of 20 μL was inserted to each well, corresponding approximately 106 CFU/mL. Positive controls for microorganism growth were made in the medium without TBF, or with the excipient (ethanol) alone. Negative controls without the inoculum were used as a reference. The 96-well plates remained at 37°C under microaerophilic conditions for S. mutans. Cell growth was measured in arbitrary units, in relation to the optical density of the medium at 620 nm. Readings were taken every 60 min, using an Infinite F50 plate reader (Absorbance microplate reader F50 ; Tecan, Männedorf, Switzerland) over 48 h. Growth curves were plotted using the average readings of duplicates over time from a representative test. The experiment was performed in triplicate. 114 Natural Oral Care in Dental Therapy 7.2.5 Cytotoxic Potential The cytotoxic level of the TBF was investigated by the “dye uptake” technique [18]. Monolayers of confluent fibroblast cells L-929 (ATCC CCL-1) on polystyrene 96-well microplates were tested against alcohol and TBF solutions, using the following concentrations: 1.11 mg/mL (0.11%) and 2.33 mg/mL (0.233%). Eagle’s Minimum Essential Medium (Cultilab, Campinas, Brazil) was used as the diluent (cell maintenance medium), and the plates remained at 37°C during 15, 30, and 60 min in microaerophilic environment. The cell viability reading was performed with a spectrophotometer (model Elx800; BioTek Instruments, Winooski, VT, USA), using a wavelength of 492 nm. Viable cell percentages for each tested solution were found by means of optical density values. White cell control (wells with untreated L-929 cells), 0.12% CHX, and a positive control (1% Tween) were also analyzed. The cytotoxic potential was calculated based on the value of cell viability of the negative control (100%). 7.2.6 Tooth Selection and Preparation for Microbiologic Assay Using an S. mutans Biofilm Bovine incisors (n = 48) stored in 2% formaldehyde without caries, spots, cracks, or other defects in the enamel were selected. Cuts were made, using a water-cooled diamond saw (Bühler, Uzwill, Switzerland) to acquire blocks (4 × 4 × 2 mm). These enamel surfaces were polished, using 600- and 1,200-grit silicon carbide papers (Extec Corp., Enfield, CT, USA), followed by a 1-mm diamond abrasive slurry (Extec Corp., Enfield, CT, USA), and then were ultrasonically washed with distilled and deionized water (Merck Millipore, Darmstadt, Germany). Finally, the quality of the polished surfaces of the enamel was verified by means of an optical microscope with 50× magnification. Forty-eight bovine enamel blocks, with a surface microhardness mean of 312.73 ± 6.85 KHN, were randomly positioned in single wells in 24-well polystyrene plates (n = 2). These were then sterilized with ethylene oxide (Bioxxi Esterilização, Rio de Janeiro, Brazil). Next, added to each well was 1,492.5 µL of BHI broth with 2% sucrose (Oxoid Ltd., Basingstoke, UK) plus bacterial inoculum (7.5 µL of S. mutans), with a final concentration of 5 × 105 CFU/mL. Then, the system was incubated under microaerophilic conditions, for 24 h, so that biofilm formed on the enamel blocks (Figure 7.1). After biofilm growth, the blocks received the following treatment (G, n = 7): 0.12% chlorhexidine (G1), 0.11% TBF (G2), 0.388% ethanol (G3), 0.233% TBF (G4), and 0.889% ethanol (G5). Other two groups were separated out—a growth control (BHI medium with inoculum; G5) group and a block removed for S. mutans count after 24 h (G7) group, respectively—to register baseline values. Six other blocks, with BHI medium without inoculum, represented the sterilized control or blank control. Treatment was carried out after the culture medium removal from the wells with a pipette. The enamel blocks were treated with 50 μL of the test substance, which remained for 1 min on these fragments. After this period, the blocks/biofilms were washed twice with 1,000 μL of sterile deionized water. After washing, the culture medium was renewed (BHI supplemented with 2% sucrose, but without the inoculum). The plate/block/biofilm system was transferred to a microbiological oven, under 5% CO2, for 24 h, and the treatment was repeated at the same time in 3 days. Oral Antibiofilm Effect of Bauhinia 115 20kV X200 100 µm COPPE 20kV X200 100 µm COPPE Figure 7.1 Scanning electron microscopy pictures of two enamel samples: the left one is from G7- after 24-h biofilm formation (baseline control) and the right one is from the blank control (photomicrographs with magnification of 500×). At the end of the assay, the tooth blocks were removed from the plate and transferred to microtubes with 1 mL of saline solution. This block was vortexed (1 min) to separate the biofilm. From the suspension, 100 μL was seeded (dilutions of 10−1 to 10−7), in duplicate, spread over Petri dishes with BHI agar for S. mutans counts. The dishes were moved to a microbiological stove for 48 h under 5% CO2, and the results were shown in Log10 CFU/mL. 7.2.7 Statistical Analysis The Statistical Package for the Social Sciences version 21.0 software program (IBM Corp., Armonk, NY, USA) and Graph Pad PRISM 6.0 (Graph Pad Inc., San Diego, CA, USA) were used in the data analyses. For the microbiological assays, cytotoxic potential, and killkinetics, the normality was verified by Shapiro–Wilk test (CFU counts, % cell death, and optical density). One-way analysis of variance, followed by the Tukey post hoc test, was used to compare the means according to the treatment groups. For all analyses, a significance level of 5% was considered. 7.3 Results and Discussion The pH of the TBF was 5.8 ± 0.1, and the total solid content was 17 ± 0.0 °Brix. Considering the elemental content, high proportions of calcium (1,369.65 ± 22.54 mg/kg) and phosphorus (1,067.39 ± 9.51 mg/kg) were observed in the TBF. Also, the presence of magnesium (111.87 ± 3.53 mg/kg), a natural antimicrobial, was recorded (Table 7.1). The TBF showed bacteriostatic (MIC: 0.11%) and bactericidal (MBC: 0.11%) activity against S. mutans. Moreover, the tincture (0.233%) showed acceptable values of % cell death, even after 60 min of contact (Table 7.2). Actually, the TBF in all tested concentrations was significantly different from that in the positive control group (p < 0.00) over all analyzed periods. The 0.12% chlorhexidine promoted a considerable percentage of cell death (55.06–69.9%), with a reduction of the viable cells after 60 min (p < 0.05), and preserved fewer oral fibroblast cells, at all time-points, then the TBF in both concentrations (p < 0.01). 116 Natural Oral Care in Dental Therapy Table 7.1 Mineral contents of TBF at 20%. Minerals (mg/kg) TBF (mean ± SD) Sodium (Na) 663.43 ± 9.26 Potassium (K) 921.00 ± 16.19 Magnesium (Mg) 111.87 ± 3.53 Calcium (Ca) 1369.65 ± 22.54 Zinc (Zn) 2.73 ± 0.26 Phosphorus (P) 1067.39 ± 9.51 Note: SD—Standard deviation. Table 7.2 Potential cytotoxic in four time points (0, 15, 30, and 60 min) by percentage count of cell deaths after treatment with TBF, in different concentrations, and controls. % Cell death (± SD) Substances T0 T15 T30 T60 0.11% TBF 18.59 (2.26)a,A 12.99 (8.42)a,AB 9.39 (2.85)a,B 4.32 (3.13)a,C 0.388% Ethanol 8.36 (2.05)b,A 22.49 (6.99)a,B 15.58 (1.97)a,BC 10.96 (8.96)a,AC 0.233% TBF 26.93 (3.96)c,A 17.78 (5.91)a,AB 13.71 (4.34)a,B 14.47 (9.09)a,B 0.816% Ethanol 9.13 (3.34)b,A 20.08 (6.23)a,B 15.78 (7.91)a,ABC 7.55 (7.21)a.AC 0.12% CHX 69.90 (1.87)d,A 69.35 (2.87)b,A 69.43 (0.34)b,A 55.06 (5.73)b,B 1% Tween 81.45 (0.99)e,A 79.92 (0.85)b,A 83.81 (0.49)c,A 80.01 (1.05)c,A Note: Distinct lowercase letters show difference of % cell death at the same time, considering the columns. Distinct capital letters show difference of % cell death between times, considering the same rows. The alcohol in both concentrations promoted cell death less than 25% for all evaluated times (Table 7.2). In view of the relative increase in the number of microorganisms resistant to antiseptics and conventional antibiotics, there is a rising interest in the use of tests to investigate antimicrobials of natural origin [19]. Therefore, studies that address the use of natural plants are perfectly reasonable, mainly those where a biofilm/caries model is controlled by an in vitro environment, since they simulate a real condition [6, 20]. The present research investigated the antibacterial action of B. forficata L. against an S. mutans biofilm formed on dental surfaces. The TBF, at the highest concentration tested, expressed antibacterial activity against S. mutans biofilm, similar to chlorhexidine at 0.12% (p > 0.05). This is a promising result, since there are no reports, to our knowledge, of adverse effects concerning the topical use of TBF consumption. On the other hand, prolonged use of chlorhexidine may trigger a decrease in gustatory sensitivity, dental pigmentation, and bacterial resistance [21]. Separately, we highlight that the concentration of TBF tested in the assay was lower than Oral Antibiofilm Effect of Bauhinia 117 those previously reported as toxic [11, 13, 22]. This was confirmed by the high viability of oral fibroblasts after contact with TBF at all exposure times. Thus, TBF probably does not present any risk to human health. Laboratory studies suggest that the leaves of B. forficata L. are rich in polyphenols [10, 13, 23, 24]. Polyphenols have multiple mechanisms of antibacterial activity, including the suppression of the quorum sensing, or other bacterial global regulator systems. Another point that should be highlighted is the presence of Mg in the TBF; Mg has attracted interest as an antibacterial biomaterial [25], owing to its ability to alkalize the surrounding medium during degradation [26]. Therefore, it is plausible to associate the presence of polyphenols and Mg in the chemical constitution of the leaves of B. forficata L. as the possible source of the antimicrobial action found in this study against S. mutans biofilm. Although the polyphenol composition of B. forficata L. has already been disclosed, further studies using chromatographic analyses, in order to identify and quantify the phenolic content of the presented solution, are needed. Even with this limitation, however, the originality of the present work should be emphasized, since it is the pioneer study in the evaluation of the antibacterial effect of B. forficate L. on the dental biofilm. Considering the results of the growth curve, the TBF acted differently on S. mutans in comparison with the control (Figure 7.2); specifically, the TBF demonstrated an optical density reduction (0.32 nm) in the development of S. mutans after 48 h of treatment (p < 0.01). Considering the inhibitory properties of the TBF against S. mutans demonstrated in the growth curve over time, the authors observed lower bacterial levels, mainly after the first 18 h (p < 0.05), compared with those in the culture medium alone and ethanol. Thus, we suggest that the TBF presents considerable substantivity and a low degree of degradation over time. Further studies should be performed to confirm such a hypothesis, however. The biofilm assay on the dental enamel surface showed that the baseline group (G7) presented the largest counts of viable microorganisms (6.89 ± 0.19 Log10 CFU/mL; p < 0.01). The treatment with 0.233% TBF (G4: 4.51 ± 0.65 Log10 CFU/mL) was effective in reducing the biofilm compared with other groups (p < 0.01), except for G1 (4.62 ± 0.37 Log10 CFU/ mL; p = 0.996). G3 (5.76 ± 0.21 Log10 CFU/mL) and G5 (6.03 ± 0.2 Log10 CFU/mL) presented the same behavior (p = 0.856 and 0.98, respectively) as that of G6 (6.04 ± 0.32 Log10 CFU/mL), while G2 (0.11% TBF) presented better activity in reducing biofilm versus G3, G5, G7, and G8 (p < 0.01) (Table 7.3). Streptococcus mutans OD 600nm 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 5 10 15 20 25 30 Hours 35 40 45 50 Figure 7.2 Streptococcus mutans kill-kinetics of TBF and control for 48 h. The S. mutans optical density was evaluated at 60-min intervals after contact with ethanol (blue line) and TBF (green line). Bacterial growth was also verified (black line). 118 Natural Oral Care in Dental Therapy Table 7.3 S. mutans count (Log10 CFU/mL) of mature biofilm formed on enamel bovine blocks according to each studied group (G). Group Log10 Mean (± SD) G1 4.62 (0.37)a G2 5.23 (0.77)b G3 5.76 (0.21)bcd G4 4.51 (0.65)a G5 6.03 (0.20)de G6 6.04 (0.32)e G7 6.89 (0.19)f Note: Distinct letters show statistical difference in Log10 CFU counts between the groups. G1: 0.12% CHX, G2: TBF at the MBC concentration, G3: ethanol at the MBC concentration, G4: TBF at the MBC × 2 concentration, G5: ethanol at a MBC × 2 concentration, G6: growth control (BHI medium with inoculum), G7: S. mutans count after 24 h (baseline). The S. mutans is the principal bacteria of the mutans streptococci group that is involved with the initial development and severity of caries lesions [2, 27, 28]. Thus, in the present book chapter, S. mutans was chosen as the microbial parameter for the in vitro study. Moreover, the most concentrated TBF was chosen as a treatment option because isolated planktonic cells were found to be more vulnerable to treatment with antibacterial than a structured biofilm. Therefore, the authors also elected for a higher, nontoxic concentration of TBF for the biofilm assay. 7.4 Final Considerations The TBF has a pH value above that for enamel dissolution, contains Mg, has a low cytotoxic potential in oral fibroblasts, shows inhibitory activity against S. mutans, and appears to be an efficient adjuvant in the prevention of carious lesions due to it reducing microbial levels in formed dental biofilm. From the results of the present in vitro research, in order to investigate new techniques for approaching caries, ex vivo/in vivo studies with B. forficata L are required. Acknowledgments The authors thank the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for Oral Antibiofilm Effect of Bauhinia 119 financial support. The authors also thank PhD Adrielle Mangabeira for the Bauhinia forficata L. leaves and Cássia de Mattos de Lima for the elaboration of the schematic drawing presented in the methodology. This study constitutes the master’s degree thesis of the first author. References 1. Simón-Soro, A. and Mira, A., Solving the etiology of dental caries. Trends Microbiol., 23, 76–82, 2015. 2. Bowen, W.H. and Koo, H., Biology of Streptococcus mutans-derived glucosyltransferases: Role in extracellular matrix formation of cariogenic biofilms. Caries Res., 45, 69–86, 2011. 3. Queiroz, V.S., Ccahuana-Vásquez, R.A., Tedesco, A.F., Lyra, L., Cury, J.A., Schreiber, A.Z., Influence of the Culture Medium in Dose-Response Effect of the Chlorhexidine on Streptococcus mutans Biofilms. Scientifica (Cairo), 2816812, 7, 2016. 4. Kouidhi, B., Al Qurashi, Y.M., Chaieb, K., Drug resistance of bacterial dental biofilm and the potential use of natural compounds as alternative for prevention and treatment. Microb. 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Oral Biosci., 57, 18–26, 2016. 8 Antimicrobial Effect of a Cardamom Ethanolic Extract on Oral Biofilm: An Ex Vivo Study Marina Fernandes Binimeliz1, Mariana Leonel Martins1, Julio Cesar Campos Ferreira Filho1, Lucio Mendes Cabral2, Adriano Gomes da Cruz3, Lucianne Cople Maia1 and Andréa Fonseca-Gonçalves1* 1 Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 2 Department of Pharmaceutics, Faculty of Pharmacy, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil 3 Food Department, Federal Institute of Education, Science and Technology of Rio de Janeiro (IFRJ), Rio de Janeiro, RJ, Brazil Abstract Elettaria cardamomum, known as the small cardamom, belongs to the Zingiberaceae family and grows in rainforest. Its seeds contain a volatile oil that is used for culinary purposes but which also has pharmacological properties. However, knowledge of its antimicrobial potential against oral biofilm remains scarce. The present book chapter proposes to demonstrate the effect of an Elettaria cardamomum ethanolic extract (ECE) against oral biofilm bacteria through an in vitro study. The ECE’s composition (e.g., moisture, proteins, fat) was also evaluated. Antibacterial activity of the ECE for Streptococcus mutans and Lactobacillus casei was investigated as part of an ex vivo experiment. Salivary samples of children were collected, homogenized (saliva pool), and spread (20 µL) on cellulose membranes over brain heart infusion agar for biofilm formation (5% CO2, 37°C). After 48 h, the cellulose membranes were treated for 1 min with the following (n = 4), respectively: 0.12% chlorhexidine, ECE (7.34 mg/mL), ECE × 2 (15.45 mg/ mL), and deionized water. Microorganisms were quantified after treatment and analyzed by analysis of variance/Tukey’s test (α = 5%). The ECE presented moisture = 13.04 ± 0.48 g/100 g, proteins = 9.62 ± 0.54 g/100 g, and fat = 2.74 ± 0.40/100 g. The ECE thus presents antibacterial activity against the tested microorganisms and reduces the microbial viability levels of a biofilm formed from a pediatric saliva pool. As such, this chapter aims to present a review and discussion about the oral antibiofilm effect of cardamom. Keywords: Antibacterial agents, biofilms, laboratory research 8.1 Introduction Caries disease is a condition caused by acidophilic microorganisms that metabolize fermentable sugars. In this regard, dental caries is seen as a consequence of an imbalance in *Corresponding author: andrea.goncalves@odonto.ufrj.br; andreagantonio@yahoo.com.br Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (121–132) © 2020 Scrivener Publishing LLC 121 122 Natural Oral Care in Dental Therapy the resident microbiota due to frequent pH drops in the biofilm. Thus, situations of a diet rich in sugars or a reduction in the saliva amount, due to their ability to buffer the acids, are predisposing factors for the appearance of carious lesions [1, 2]. It is known that the removal of the dental biofilm, commonly performed by mechanical methods, is extremely important for the control of dental caries. However, in children and adolescents with orthodontic appliances, factors such as individual motivation and monitoring limit the effectiveness of brushing. These individuals also have difficulty in maintaining adequate control of the biofilm, particularly in interproximal sites, often requiring the use of chemical agents for the control of such [3]. Among the antimicrobials with action on several microorganisms, chlorhexidine is one of the most studied agents and is consecrated as the most effective and potent compound in buccal mouthwashes [4, 5]. This substance can still be found in dentifrices, gels, varnishes, or solutions [3, 6], being widely used as a positive control for tests of the efficacy of other antimicrobial agents [7, 8]. However, despite its proven efficacy, its use in dentifrices, for example, may be undue as a result of inactivation by anionic ingredients [3, 6] and by the presence of detergents such as sodium lauryl sulfate, which are incompatible with its composition and thus consequently reduce its antimicrobial potential. Another issue worth mentioning is the cytotoxicity produced and the amount of adverse effects caused by prolonged use of chlorhexidine. Among them, the following stand out: dental pigmentation, loss of gustatory sensitivity, desquamation of mucosa, and resistance of microorganisms with prolonged daily use [9]. In this perspective, new phytotherapeutic compounds have been investigated [10] and some studies claim that natural antimicrobial agents incorporated into dentifrices or mouthwashes may be as effective as chlorhexidine digluconate for immediate and delayed bacterial death in oral biofilms [10–12]. The Elettaria cardamomum, known as the small cardamom, belongs to the Zingiberaceae family and grows in rainforest with elevations ranging from 762 to 1524 m, with rain amounting to 381 cm annually. Commercially grown in India, Sri Lanka, Guatemala, and Tanzania, its fruits have thin walls and are smooth and oblong, containing 15 to 20 aromatic, reddish-brown seeds. These seeds contain a volatile oil that is used for culinary purposes but which also boasts pharmacological properties including in the management of gastrointestinal, cardiovascular, and neural disorders [13, 14]. In addition, there are studies that have reported anticarcinogenic, antiulcerogenic, and antimicrobial actions of Elettaria cardamomum against important colonizers of oral sites, such as Candida albicans and Streptococcus mutans [15]. However, knowledge of its antimicrobial potential against oral biofilm remains scarce. In the present book chapter, we present and discuss the results of an evaluation of the antimicrobial effect of the ethanolic extract of Elettaria cardamomum against isolated microorganisms and oral biofilm. 8.2 Materials and Methods 8.2.1 Cardamom Extract Production The seeds of Elettaria cardamomum, in the crude form, were crushed and sifted in a 0.6-mm sieve (Figures 8.1 and 8.2). The extract was then prepared at the Pharmaceutical Industrial Technology Laboratory at the Centro de Ciências da Saúde (CCS) of the Universidade Federal do Rio de Janeiro (UFRJ), as follows: 30 mL of 80% ethanol was added to 10 g of the Elettaria Antibiofilm Effect of Cardamom 123 Figure 8.1 Cardamom seeds. Personal file. Figure 8.2 Cardamom seeds. Personal file. cardamomum crushed plant. The mixture was heated at 60°C for 30 min with stirring and then filtered on Whatman paper no. 2 and centrifuged at 7,500 g at 5°C for 10 min. The final extract concentration was 33.3%, and the supernatant was directly used in the experiments. 8.2.2 Physical Analyses The physical analyses were also performed at the Pharmaceutical Industrial Technology Laboratory. The approximate compositions of total solids, proteins, and fat content of the extract were determined according to standard procedures [16]. The total solids were determined gravimetrically after 24 h of drying of the Elettaria cardamomum extract (ECE) in the oven (Micronal, São Paulo, Brazil). Protein levels were determined using the Kjeldahl method, based on total sample nitrogen, with subsequent correction using a conversion factor of 6.38. Fat was determined by the method of Gerber [17]. 8.2.3 Bacterial Strains and Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Strains of S. mutans (American Type Culture Collection no. 25175) and Lactobacillus casei (L. casei; American Type Culture Collection no. 393) were used to determine the 124 Natural Oral Care in Dental Therapy antibacterial activity of the extract. Initially, the degree of purity of the bacterial samples was checked. Then, isolated bacterial colonies were screened and transferred to 0.85% saline until reaching an optical density of 0.13 at a 625 nm wavelength (S2 Colorimeter; Biochrom, Cambridge, UK), corresponding approximately to an inoculum of 108 colony-forming units (CFU)/mL. In order to evaluate the antibacterial activity of the ECE against the mentioned microorganisms, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined, according to the protocol of the Clinical and Laboratory Standards Institute [18] and the modification proposed by da Cunha et al. [19]. In this perspective, 100 μL of brain heart infusion culture medium (BHI; Difco, Sparks, NV, USA) was inserted into each well of two 96-well microplates (Kasvi-São José dos Pinhais, Paraná, Brazil). Thereafter, each first test well received 100 μL of the ECE or 0.12% chlorhexidine digluconate (pharmacological control) and the microdilution process was performed, which consisted of transferring 100 μL of each first test well to the subsequent well. Then, 100 μL of the last well was discarded so that all had the same volume (100 μL). Finally, 5 μL of the inoculum was inserted into each well in order to obtain a final concentration of 5 × 105 CFU/mL. The tested concentrations of ECE ranged from 97.56 to 0.036 mg/mL, while that of chlorhexidine digluconate ranged from 58.536 to 0.022 mg/mL. The MIC was defined as the lowest concentration of the solution that did not allow visible microbial growth, which was confirmed by the application of a 0.01% suspension of resazurin (Sigma-Aldrich, St. Louis, MO, USA), while the MBC was determined as the lowest concentration that was able to cause the death of all microorganisms. This was verified by a subculture of 50 μL of MIC and above concentrations on BHI agar, which were incubated for 48 h (37°C, 5% CO2) [20]. 8.2.4 Salivary Collection for Biofilm Formation (Ex Vivo Experiment) The study was approved by the Ethics in Local Research Committee of the UFRJ (CAAE: 67020617.8.0000.5257, opinion 2.060.371), and for this stage, parents or guardians for the involved children signed a free consent term and approved the collection of the saliva of their children. Thus, children (n = 3) seen at the pediatric dentistry clinics of the Department of Pediatric Dentistry and Orthodontics of the UFRJ, with a mean age of 9.33 ± 0.57 years and who were in good health without the use of antibiotics or antibacterial mouthwash in the last 30 days prior to collection were selected. The mean number of decayed, missing, and filled teeth (DMFT) of these children (DMFT = 7.67 ± 3.05) and mean total salivary flow (0.64 ± 0.05 mL/min) were also recorded. The children were instructed not to consume food or drink except water within 1 h prior to saliva collection. The unstimulated saliva produced in the first 30 seconds was discarded and then collected for exactly 5 min in sterile test tubes. Saliva samples were immediately taken to the Multidisciplinary Laboratory of Dental Research (MLDR) at the Faculty of Dentistry of the UFRJ for the development of the biofilm formation stage. 8.2.5 Biofilm Formation and Treatment Microbiological assays for biofilm formation were conducted according to the methodology proposed by Antonio et al. [21]. Here, 1 mL of saliva from each volunteer was placed in a single tube, which was vortexed for one minute, resulting in a saliva pool. An aliquot Antibiofilm Effect of Cardamom 125 (20 μL) of the pool was placed on the surface of cellulose membranes (N = 20) with a 13 mm diameter (Millipore Corp., Billerica, MA, USA), located on Petri dishes with BHI medium. The system was incubated for 48 h (37°C, 5% CO2) to form a mature biofilm (Figure 8.3). Following biofilm formation, each membrane disk was carefully removed with sterile tweezers and placed for 1 min in tubes containing the test substances (2 mL) according to the following treatment groups (n = 4): 0.12% chlorhexidine, ECE (7.34 mg/mL), ECE × 2 (15.42 mg/mL), and deionized water (Figure 8.4). The biofilm formed on the membranes Figure 8.3 Biofilm formed over the surface of cellulose membranes placed on Petri dishes with BHI medium. Figure 8.4 Treatment of biofilm formed over a membrane disk for 1 min in a tube containing the test substance (2 mL) according to the following treatment groups: 0.12% chlorhexidine, ECE (7.34 mg/mL), ECE × 2 (15.42 mg/mL), and deionized water. 126 Natural Oral Care in Dental Therapy of the growth control group did not receive any type of treatment. Serial dilutions (100 to 10−8) were performed for viable cell counts, considering the number of total microorganisms. The experiment was performed in duplicate and the results expressed in Log10 CFU/ mL (Figure 8.5). (a) (b) (c) (d) Saliva pool Vortex 1min 48h at 37°C with 5% CO2 20 µL cellulose membrane disks 13 mm diameter N = 20 n=3 (e) (f) 2 mL 1 min F1) 0,12% Chlorhexidine F2) ECE (7.34 mg/mL) F3) ECE × 2 (15.42 mg/mL) F4) Deionized water F5) Growth control (without treatment) Treatment n=4 (g) Cardamom seeds (h) 50 µL 100 µL each 10−4 100 10−1 10−2 10−3 (i) (j) Log10 CFU/mL 10−5 10−6 10−7 10−8 48 at 37°C with 5% CO2 Figure 8.5 Schematic drawing of the experimental model with oral biofilm. (a) Collection of the saliva of three children; (b) Pediatric saliva pool formed from 1 mL of saliva from each volunteer; (c) An aliquot of the pool was placed on the surface of cellulose membranes disks; (d) The system was incubated in a microbiological oven; (e) Each membrane disk was placed for 1 min in tubes containing the test substances; (f) The biofilms formed were treated with: 0.12% chlorhexidine, ECE (7.34 mg/mL), ECE × 2 (15.42 mg/mL), and deionized water. The growth control group did not receive any type of treatment; (g) Serial dilutions were performed for viable cell count; (h) Subculture of 50 μL for evaluate the bacterial viability; (i) Incubation in microbiological oven; (j) The results were expressed in Log10 CFU/mL. Antibiofilm Effect of Cardamom 127 8.2.6 Statistical Analyses Data were analyzed descriptively and by statistical tests using the Statistical Package for the Social Sciences version 21.0 software program (IBM Corp., Armonk, NY, USA). The Shapiro–Wilk test was performed to evaluate the normality of the data and analysis of variance with a post hoc Tukey test (p < 0.05) were the tests used to analyze the differences of the means between the groups in the microbiological test of biofilm treatment. 8.3 Results and Discussion Although existing literature that has evaluated the antibiofilm activity of ECE with the methodology similar to the present study is scarce, it is known that the extract of the evaluated seed has as main chemical components the acetate of α-terpinol and 1,8-cineole [22, 23], among other highly bioactive compounds [23]. These compounds have medicinal properties, such as antimicrobial activity, that are used as phytotherapics. In this sense, E. cardamomum appears to present significant antibacterial activity and to be useful in the discovery of new antibiotics. Although there are few studies to date that have performed a chemical analysis of phenolic content in the composition of cardamom, ferulic, vanillic, caffeic, and p-coumaric acids [24] have been previously identified, which are associated with antibacterial activity against S. mutans and an influence on biofilm degradation [25]. The ECE showed a moisture value of 13.04 ± 0.48 g/100 g, a total protein equivalent to 9.62 ± 0.54 g/100 g, and total fat equal to 2.74 ± 0.40 g/100 g. This extract inhibited the growth of S. mutans at 7.34 mg/mL, which also corresponded to an MBC against S. mutans. For L. casei, the MIC was 15.42 mg/mL, but the MBC was not found in the concentration range investigated. Regarding inhibitory and bactericidal activity, in a study conducted in Turkey [26], the authors observed a large amount of eucalyptol (1,8-cineol), an oily component, in the investigated Cardamomum species. In that study, the plant species played an important antimicrobial role against Campylobacter spp. Although an investigation of this compound was not performed in the present study, it is suggested that a great part of the total fat content is also responsible for the antimicrobial activity demonstrated by the extract tested, since fats disturb biofilm adhesion [27]. However, further investigations to evaluate the eucalyptol content of the present extract should be conducted. In an earlier study, ECE showed inhibitory activity against S. mutans (5 mg/mL), Staphylococcus aureus (1.25 mg/mL), C. albicans (2.5 mg/mL), and Saccharomyces cerevisiae (5 mg/mL) [15]. Thus, there is agreement with the present study, since similar results of MIC were observed for S. mutans [15]. Another study [26] found MIC and MBC values to be between 0.012 and 0.05 μL/mL against Campylobacter jejuni and Campylobacter coli. However, the difference in comparison with the present study can be explained by the fact that different strains were tested. The antibacterial activity of several essential oils of the Zingiberaceae family was verified and, among them, the ECE had an MIC of 2,000 μg/mL, but no MBC value was found in the concentrations evaluated [25]. The divergence of the results can be justified by the different strains, microorganisms, and methodologies used; how the tested substances were prepared (essential oil, extract); and the different parts of the plant involved (seeds, leaves). 128 Natural Oral Care in Dental Therapy Considering the inhibitory action against the biofilm formed from the saliva pool, 0.12% chlorhexidine was the substance that presented the greatest reduction of viable total microorganisms from the biofilm (6.30 ± 0.22 Log10 CFU/mL; p = 0.000). The ECE × 2 finding (7.10 ± 0.36 Log10 CFU/mL) was better when compared with the untreated group (7.61 ± 0.18 Log10 CFU/mL; p = 0.006) and the group treated with deionized water (7.67 ± 0.16 Log10 CFU/mL). However, ECE, deionized water, and the growth control did not present statistically significant differences among them (p > 0.05) (Table 8.1). The 0.12% chlorhexidine was the agent with the greatest efficacy against biofilm microorganisms, ultimately reducing its count by 18.7% (p < 0.05), while ECE and ECE × 2 reduced the number of CFUs, respectively, by 6.4% and 8.3%, without any statistical difference between them. It is known that high levels of S. mutans are directly related to dental caries [28]. Therefore, a substance that interferes with growth or reduces the number of these species and also the total microorganisms of the biofilm are substances that aid in the prevention of carious lesions [20]. There are some studies in the literature that describe the anticariogenic potential of medicinal plants [29–32]; however, there is a need for new discoveries regarding the biological properties of these products. In the present study, it was observed that the ECE presented antimicrobial activity against strains of S. mutans and L. casei and was able to reduce the microbial viability in the biofilm, suggesting a potential anticariogenic activity of this product. In the biofilm assay formed through the saliva pool of children, a reduction in the counts of viable microorganisms after treatment with both extracts was observed in both the concentrated (ECE × 2) and diluted (ECE) groups, with no statistical difference between them. The 0.12% chlorhexidine had a higher antibiofilm effect after a single treatment versus the other groups, but the prolonged daily use of this substance may cause undesirable effects, such as a loss of taste, pigmentation of the dental elements, and desquamation of the mucosa mainly due to its toxicity [33]. In addition, it may cause resistance of several pathogenic oral microorganisms to antibiotics. Therefore, it is important to search for other alternatives such as natural products that can help to reduce these adverse effects in dentistry, since medicinal plants have been used for prophylactic and curative purposes since ancient times [34]. Table 8.1 Microbial levels in log10 CFU/mL for each group after the biofilm assay formed on cellulose membranes. Treatment group 0.12% chlorhexidine Bacterial count (SD)* 6.30 (0.22)a ECE (7.34 mg/mL) 7.25 (0.22)b,c ECE × 2 (15.42 mg/mL) 7.10 (0.36)c Deionized water 7.67 (0.16)b Growth control 7.61 (0.17)b Different lowercase letters indicate p < 0.05 in the column. Counts transformed into log10 CFU/mL. *SD: Standard deviation. Antibiofilm Effect of Cardamom 129 Some limitations of this study should be considered, including the reduced number of strains against which the MIC and MBC were evaluated or the number of samples per group and the surface on which the biofilm was formed (celluloid membranes), since in the literature studies bovine teeth have been more frequently used for biofilm assays [29, 31]. This is due to the great similarity between bovine and human enamel [35]. Therefore, future studies should be carried out to analyze the chemical composition and cytotoxic potential of ECE in different concentrations of the product and on tooth structure (enamel and/or dentin) in order to complement the present study and confirm the findings. 8.4 Final Considerations This chapter was carried out with the purpose of evaluating, by means of a study, the action of ECE on planktonic and oral biofilm microorganisms. No studies were found in the researched literature that tested ECE against a mixed oral biofilm formed on cellulose membranes; thus, this is an innovative proposal. In addition, the biofilm was formed from a saliva pool, representing a situation that is closer to what occurs in the oral cavity in comparison with tests performed with planktonic cells, such as the MIC and MBC. Ultimately, the ECE showed antimicrobial activity against the tested oral microorganisms and can reduce the microbial levels of a biofilm formed in vitro by a pediatric saliva pool. However, further studies are needed to confirm the antibiofilm potential of this product. Acknowledgment This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. The authors thank Cássia de Mattos de Lima for the elaboration of the schematic drawing presented in the methodology. References 1. Fejerskov, O. and Kidd, E., Dental caries: The disease and its clinical management. Oxford: Blackwell Munksgaard, 2nd ed., United Kingdom, 2008. 2. Marsh, P.D., Controlling the oral biofilm with antimicrobials. J. Dent., 38, 1, 11–5, 2010. 3. Davies, R.M., Toothpaste in the control of plaque/gingivitis and periodontitis. Periodontol. 2000, 48, 1, 23–30, 2008. 4. 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Mutlu-Ingok, A., Karbancioglu-Guler, F., Cardamom, Cumin, Dill Weed Essential Oils: Chemical Compositions, Antimicrobial Activities, and Mechanisms of Action against Campylobacter spp. Molecules, 22, 7, 1191, 2017. 27. Simões, M., Simões, L.C., Vieira, M.J., A review of current and emergent biofilm control strategies. J. Food Sci. Technol., 43, 573–583, 2010. 28. Banas, J.A. and Drake, D.R., Are the mutans streptococci still considered relevant to understanding the microbial etiology of dental caries? BMC Oral Health, 18, 1, 129, 2018. 29. Tanner, A.C., Anaerobic culture to detect periodontal and caries pathogens. J. Oral Biosci., 57, 18–26, 2015. 30. Leite, K.L.F., Martins, M.L., Medeiros, M.M.D., Iorio, N.L.P., Fonseca-Gonçalves, A., Cavalcanti, Y.W., Padilha, W.W.N., Antibacterial Activity of Melaleuca alternifolia (tea tree essential oil) on Bacteria of the Dental Biofilm. Pesqui. Bras. Odontopediatria Clin. Integr., 17, 1, e3857, 2017. 31. 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Oral Boil., 49, 11, 912–22, 2004. 9 Effect of Punica granatum Peel Extract on Growth of Candida albicans in Oral Mucosa of Diabetic Male Rats Maryam Eidi1* and Fatemeh Noorbakhsh2 1 Department of Biology, College of Biological Sciences, Varamin-Pishva Branch, Islamic Azad University, Varamin-Pishva, Iran 2 Department of Microbiology, College of Biological Sciences, Varamin-Pishva Branch, Islamic Azad University, Varamin-Pishva, Iran Abstract To survey the oral treatments of Punica granatum L. peel hydro-methanolic extract (0.025, 0.05, 0.1, and 0.2g/kg, body weight) and itraconazole (0.01 g/kg, body weight) on the growth of Candida albicans (ATCC 10231) in the oral mucosa of alloxan-induced diabetic male rats, the animal was made diabetic by alloxan (0.15 g/kg, intraperitoneally). After diabetes induction, they were contaminated by oral administration of Candida albicans. Fungus sampling, culturing in medium and colony count were done by swabs from mouths of the animal. The period of the experiment was 7 days. On the second day of plant treatment, the results showed that treatment of extract decreased colony count of C. albicans in diabetic rats (p < 0.001). The growth of C. albicans is suppressed on the 6th day. The effect of the plant is similar to itraconazole. So, the consumption of pomegranate extract inhibited the growth of Candida albicans in the month of diabetic rats. Keywords: Punica granatum, diabetes, Candida albicans, rat 9.1 Introduction Herbal medicine is known as the inexpensive and accessible source in the healthcare system [1]. For thousands of years, the ayurvedic medicine has reduced illness and promoted good health [2]. The flora of Iran includes abundant medicinal plants. Many medicinal plants have been consumed in folk medicinal systems and accounted in pharmacopoeia as materials for treating oral fungal pathogens [3]. The medicinal plants have alkaloids, tannins, essential oils, and flavonoids. There are many species of medicinal plants, which are being used, traditionally, to control fungal infections [4]. Candida albicans (C. albicans) is one of the chronic pathogens in humans that cause wounds in the mucosa of the upper digestive organs and vagina. The consumption of *Corresponding author: maryameidi@gmail.com; eidi@iauvaramin.ac.ir Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (133–138) © 2020 Scrivener Publishing LLC 133 134 Natural Oral Care in Dental Therapy antibiotics, steroids, immunosuppressive agents, and radiotherapy can cause Candida infections [5]. The conditions such as the immunodeficiency syndrome and iron and vitamin deficiency, which are common in diabetes mellitus (DM) disease, are related to the Candida infections. The lesion of oral epithelium due to Candida infection causes candidiasis, which is defined by the abnormal thickened oral mucosa and inflammation [6, 7]. Punica granatum L. (Lythraceae) is also called pomegranate and grown from Iran to Northern India. The pomegranate is a shrub or small tree [8] with spiny branches and 12–16 feet in height. The fruit is deep red, five inches wide, with thick peel, spherical, and with pointed calyx. The seeds of the fruit are parted by a white and thin pericarp and environed by small contents of tart and juice. The leaves of the pomegranate are shiny and 7–8 cm long [9]. The juice, peels, and seed oil of pomegranate contain cyaniding and delphinidin (anthocyanidins), chlorogenic acid, and caffeic acid (phenol compounds), gallic acid, ellagic acid, luteolin, quercetin, kaempferol, naringenin, 17-alpha-estradiol, estrone, estriol, testosterone, beta-sitosterol, coumestrol, gamma-tocopherol, punicic acid, campesterol, and stigmasterol. They are related to potentials of pomegranate for treating diseases [10, 11]. Since the prevalence of infective diseases in diabetic patients is more numerous, an investigative study was conducted on the antifungal activity of the hydro-methanolic extract of pomegranate peel in diabetic rats. 9.2 Materials and Methods 9.2.1 Hydro-Methanolic Extract Fresh Punica granatum L. peels were obtained from Central Province, Iran. A voucher specimen was validated in the herbarium of Islamic Azad University. The peels of the pomegranate were dried in the shade and ground by an Ultra-Torax apparatus. About 50 g of the powder was soaked in 480 mL of methanol (80%) and kept in the shadow for 72 h. The mixture was filtered and then condensed in low pressure by a rotary evaporator. The product was 153% (w/w) of the extract and was kept at –20°C until usage. The extract was diluted by saline, and concentrations of 0.025, 0.05, 0.1, and 0.2 g/kg body weight were made. The hydro-methanolic extract was treated orally to the alloxan-induced diabetic rats for 7 days. 9.2.2 Candida albicans Inoculation Candida albicans was purchased from the American Type culture collection (ATCC 10231) and kept at −70°C in TSA medium until usage. Twenty four hours before inoculation, C. albicans was cultured in the medium of sabouraud dextrose agar (SDA) and then incubated at 37°C. The grown colonies were suspended in saline (5 mL). The suspension was stirred by vortex for 10 s. The final inoculum concentration of the suspension was adjusted to 0.5–2.5 × 103 CFU/mL. 9.2.3 Animal Male Wistar rats (weighing 200–250 g) were bought from the Pasteur Institute and kept in an animal room at 22 ± 2°C, light/dark cycle at 7 AM to 19 PM and with humidity of Effect of Punica granatum Peel Extract 135 40–60%. Each animal was used once only. Experiments were performed according to the guidelines of Branch of Varamin-Pishva, Islamic Azad University, which is in accord with the institutional use and care of laboratory animal guidelines (NIH, Number of publication, 85-23; revised in 2010; 86/609/EEC European Communities Directive). Six groups were used in this study. The diabetic groups were made by alloxan (0.15 g/kg body weight, intraperitoneally). After 5 days, the rats in which the fasting blood glucose was higher than 180 mg/dL were selected for the research. The animals were contaminated by oral administration of Candida albicans. After the growth of fungus in the mouth of the animal, oral treatments of the extract (0.025, 0.05, 0.1, and 0.2 g/kg body weight) and itraconazole (0.01 g/kg body weight) were made for 7 days. The sampling, culturing in medium, and colony count of Candida albicans were done by swabs from mouths of all animals every day. The period of the experiment was 7 days. The control rats were treated with saline (as the vehicle). 9.2.4 Statistical Analysis Data were represented as mean ± SEM and analyzed by one-way analysis of variance followed by Tukey post hoc test. A value of p < 0.05 was determined significance. 9.3 Results and Discussion DM is a metabolic disease and is defined as a deficiency of insulin secretion, insulin action, or both of them [12, 13]. Many people in the world have DM in 2010, and subsequently, 300 million will have DM by 2025 [14, 15]. DM is related to the various inflammatory diseases and soft tissue lesions in the oral cavity; however, knowledge of these problems is absent worldwide [16]. The prevalence of Candida infections and oral candidiasis has been investigated for many years among diabetic people, frequently [17–19]. The present result showed that treatments of extract and itraconazole inhibited the growth of C. albicans in experimental diabetic groups compared to control diabetic rats on the second day of the treatment period, significantly. Extracts of pomegranate and itraconazole reduced the colony count of C. albicans, significantly (p < 0.001). The growth of C. albicans is suppressed on the 6th day. The significance of extract was similar to itraconazole (Table 9.1). In the agreement, Mansourian et al. reported a significant antifungal effect of Punica granatum extract against C. albicans by the well diffusion method (in vitro) [20]. Hayouni et al. showed the significant antifungal activity of pomegranate against C. albicans in guinea pigs. The plant extract also affected wound healing in guinea pigs [21]. Endo et al. isolated one of the significant components from pomegranate peel, which is called punicalagin. They showed its strong antifungal activity against C. albicans. It is reported that the treatment of punicalagin and fluconazole inhibited the growth of C. albicans (in vitro) and showed a synergistic effect [22]. Madugula et al. showed that the minimum inhibitory concentration of P. granatum peel is equal to clotrimazole in vitro, and both of them inhibited the growth of Candida species, significantly (p < 0.05) [23]. Tehranifar et al. showed that the pomegranate is a powerful antioxidant and antifungal substance. It is shown that the methanolic extract of pomegranate inhibited mycelial growth 136 Natural Oral Care in Dental Therapy Table 9.1 Effects of oral treatment of Punica granatum peel extract at concentrations of 0.025, 0.05, 0.1, and 0.2 g/kg and itraconazole (0.01 g/kg) on the colony count of C. albicans. Extract (g/kg) Day Control 5 0.025 5 0.05 5 0.1 0.2 5 Itraconazole 5 0 >10 >10 >10 >10 >10 >105 1 >105 2906 ± 1043 *** + 341 ± 11 *** 353 ± 30 *** 30 ± 3 *** 726 ± 165 *** 2 >105 1109 ± 334 *** ++ 132 ± 15 *** 87 ± 39 *** 8±1 *** 215 ± 93 *** 3 >105 204 ± 72 *** 101 ± 10 *** 14 ± 4 0 60 ± 37 *** 4 >105 166 ± 57 *** +++ 33 ± 10 *** 0 0 0 5 >105 8±3 *** 14 ± 4 *** +++ 0 0 0 6 >105 0 0 0 0 0 7 >105 0 0 0 0 0 ***p < 0.001 shows significance from control diabetic group, +p < 0.05, ++p < 0.01, +++p < 0.001 show significance from itraconazole group. and spore germination of Candida species, strongly. On the other hand, inhibitory effects of the seed and peel of pomegranate is higher than its leaf (2.8-fold). The antioxidant potential of the seed, peel, and leaf extracts were measured as 55%, 36%, and 16%, respectively. So, the great content of phenolic compounds in the extracts of the peel and seed could cause the potent antioxidant and antifungal and antioxidant activities of pomegranate [24]. 9.4 Conclusion The present study indicated that the hydro-methanolic extract of pomegranate peel has an antifungal effect against C. albicans and reduces the growth of C. albicans in diabetic rats. Acknowledgment The authors thank the Deputy Research of the Varamin-Pishva Branch, Islamic Azad University, for support of the project. Effect of Punica granatum Peel Extract 137 References 1. Yineger, H. and Yewhalaw, D., Traditional medicinal plant knowledge and use by local healers in Sekoru District, Jimma Zone, Southwestern Ethiopia. J. Ethnobiol. Ethnomed., 3, 24, 2007. 2. Samy, R.P., Pushparaj, P.N., Gopalakrishnakone, P.A., Compilation of bioactive compounds from Ayurveda. Bioinformation, 3, 100–110, 2008. 3. Devi, A., Singh, V., Bhatt, A.B., Comparative antibacterial study of different extract of pomegranate and its wild variety. I. J. P. S. R., 2, 2647–2650, 2011. 4. Bhardwaj, A. and Bhardwaj, S.V., Ethno-dentistry: Popular medicinal plants used for dental diseases in India. J. Intercult. Ethnopharmacol., 1, 62–65, 2012. 5. Sitheeque, M.A.M. and Samaranayake, L.P., Chronic hyperplastic candidosis/candidiasis (candidal leukoplakia). Crit. Rev. Oral Biol. Med., 14, 253–267, 2003. 6. Gainza-Cirauqui, M.L., Nieminen, M.T., Novak Frazer, L., Aguirre-Urizar, J.M., Moragues, M.D., Rautemaa, R., Production of carcinogenic acetaldehyde by Candida albicans from patients with potentially malignant oral mucosal disorders. J. Oral Pathol. Med., 42, 243–249, 2013. 7. Gall, F., Colella, G., Di Onofrio, V., Rossiello, R., Angelillo, I.F., Liguori, G., Candida spp. in oral cancer and oral precancerous lesions. New Microbiol., 36, 283–288, 2013. 8. Adler, L., Modin, C., Friskopp, J., Jansson, L., Relationship between smoking and periodontal probing pocket depth profile. Swed. Dent. J., 32, 157–163, 2008. 9. Qnais, E.Y., Elokda, A.S., Abu Ghalyun, Y.Y., Abdulla, F.A., Antidiarrheal activity of the aqueous extract of Punica granatum (Pomegranate) peels. Pharm. Biol., 45, 715–720, 2007. 10. Lansky, E.P. and Newman, R.A., Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J. Ethnopharmacol., 109, 177–206, 2007. 11. Kim, N.D., Mehta, R., Yu, W., Neeman, I., Livney, T., Amichay, A., Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer. Breast Cancer Res. Treat., 71, 203–217, 2002. 12. Beverley, B. and Eschwège, E., The diagnosis and classification of diabetes and impaired glucose tolerance, in: Textbook of Diabetes 1, Third edition; Pickup, J.C. and Williams, G. (Eds.), Blackwell Publishing, pp. 210–211, 2003. 13. Kumar, P.J. and Clark, M., Textbook of Clinical Medicine, pp. 1099–1121, Saunders, London, 2002. 14. King, H., Aubert, R., Herman, W., Global burden of diabetes, 1995–2025. Prevalence, numerical estimates and projections. Diabetes Care, 21, 1414–1431, 1998. 15. Zimmet, P., Globalization, coca-colonization and the chronic disease epidemic: Can the Doomsday scenario be averted? J. Med., 247, 301–310, 2000. 16. Al-Maskari, A.Y., Al-Maskari, M.Y., Al-Sudairy, S., Oral manifestations and complications of diabetes mellitus: A review. Sultan Qaboos Univ. Med. J., 11, 179–186, 2011. 17. Kumar, B.V., Padshetty, N.S., Bai, K.Y., Rao, M.S., Prevalence of Candida in the oral cavity of diabetic subjects. J. Assoc. Physicians India, 53, 599–602, 2005. 18. Shirmali, L., Astekar, M., Sowmya, G.V., Correlation of oral manifestations in controlled and uncontrolled diabetes mellitus. Int. J. Oral Maxillofac. Pathol., 2, 24–27, 2011. 19. Al-Attas, S.A. and Amro, S.O., Candidal colonization, strain diversity, and antifungal susceptibility among adult diabetic patients. Ann. Saudi Med., 30, 101–108, 2010. 20. Mansourian, A., Boojarpour, N., Ashnagar, S., Momen Beitollahi, J., Shamshiri, A.R., The comparative study of antifungal activity of Syzygium aromaticum, Punica granatum and nystatin on Candida albicans; An in vitro study. J. Med. Mycol., 24, e163–168, 2014. 21. Hayouni, E.A., Miled, K., Boubaker, S., Bellasfar, Z., Abedrabba, M., Iwaski, H., Oku, H., Matsui, T., Limam, F., Hamdi, M., Hydroalcoholic extract based-ointment from Punica granatum L. 138 Natural Oral Care in Dental Therapy peels with enhanced in vivo healing potential on dermal wounds. Phytomedicine, 15, 976–984, 2011. 22. Endo, E.H., Cortez, D.A.G., Ueda-Nakamura, T., Nakamura, C.V., Filho, B.P.D., Potent antifungal activity of extracts and pure compound isolated from pomegranate peels and synergism with fluconazole against Candida albicans. Res. Microbiol., 161, 534–540, 2010. 23. Madugula, P., Reddy, S., Koneru, J., Rao, A.S., Sruthi, R., Dalli, D.T., Rhetoric to reality—Efficacy of Punica granatum peel extract on oral candidiasis: An in vitro study. J. Clin. Diagn. Res., 11, ZC114–ZC117, 2017. 24. Tehranifar, A., Selahvarzi, Y., Kharrazi, M., Jahan Bakhsh, V., High potential of agro-industrial by products of pomegranate (Punica granatum L.) as the powerful antifungal and antioxidant substances. Ind. Crops Prod., 34, 1523–1527, 2011. Part III APPLICATIONS OF NATURAL PRODUCTS IN ORAL CARE 10 Effect of Oil Pulling on Oral Health Sameer Anil Zope* and Siddhartha Varma Department of Periodontology, School of Dental Sciences, Krishna Institute of Medical Sciences Deemed to be University, Karad, India Abstract Oil pulling has been used widely as a conventional Indian folk remedy for many years for strengthening teeth, gingiva, to prevent dental caries, halitosis, bleeding gums, dryness of mouth, and cracked lips. In the Ayurveda literature Charaka Samhita (Sutrasthana 5, 78–80), it is referred to as Gandoosha, Kavala, and Kavala Graha. It is claimed to treat about 30 systemic disorders ranging from diabetes to migraine and asthma. A Ukrainian medical practitioner, Dr. F. Karach, acquainted the remarkable notion of oil pulling in the 1990s. Recent studies on oil pulling therapy using sunflower and sesame oil were found to reduce dental caries and plaque-induced gingivitis. The most amazing part of oil pulling therapy is that it can be performed using any cold pressed oil easily at home such as coconut, sunflower, or sesame oil; hence, it becomes a very cost-effective modality. Since last decade, there are many studies available on the use of oil pulling for the maintenance of overall oral health. There is mounting evidence of oil pulling being as good as many other chemical-containing ointments, toothpastes, or mouthwashes in control of oral problems with no untoward side effects. This chapter overviews the evidence-based use of oil pulling therapy in the maintenance of oral health. Keywords: Ayurveda, dental caries, gingivitis, oral malodor, oil pulling, oral hygiene 10.1 Introduction The oral cavity is considered the reflection of systemic health. Oral health is an integral and fundamental part of the overall health and is considered as most important to everyone. The World Health Organization (WHO) defined oral health as a state of being free from chronic mouth and facial pain, oral and throat cancer, oral sores, birth defects such as cleft lip and palate, periodontal (gum) disease, tooth decay and tooth loss, and other diseases and disorders that affect the oral cavity [1]. This implies that oral health significantly impacts the general health and well being of an individual. In recent years, various studies have provided unequivocal evidence on the *Corresponding author: aoldentist@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (141–152) © 2020 Scrivener Publishing LLC 141 142 Natural Oral Care in Dental Therapy strong interrelationship between systemic and oral diseases. It is believed that this relationship is due to common risk factors shared between various diseases. Oral hygiene maintenance is very important for all individuals, and it includes mechanical tooth cleaning with the additional adjunctive use of a chemical/herbal agent for reducing plaque formation. Apart from mechanical plaque control methods, research has been focused on chemotherapeutic agents for preventing or reducing plaque-induced oral diseases. Widely used various chemical plaque control agents like chlorhexidine and cetylpyridinium chloride mouthwash have their own disadvantages like brown staining of teeth and oral appliances, increased formation of tartar, temporary alteration of taste, oral dryness, and burning sensation of the oral mucosa. Alcohol-containing mouthwash may result in dry mouth and worsening of halitosis [2]. Drug toxicity, adverse effects, and antibiotic resistance to modern drugs have encouraged scientists to research on natural products. A number of other alternatives have been put forward in order to overcome these problems; among those, the procedure of oil pulling is the one which has no side effects if used properly. It can be performed using readily available edible oils in the household. Recently, various forms of alternative or traditional medicinal treatments, such as Ayurveda, have started to gain popularity, due to their natural origin, cost effectiveness, negligible side effects, and improved patient compliance. Ayurveda is a form of traditional holistic medicinal system originating in the Indian subcontinent region. Its advent and practice in the region reportedly date back about 3000– 5000 years. Recently, it has gained popularity as a complementary medicine in other parts of the world [3]. 10.2 What is Oil Pulling (Snaihik Gandoosh)? Oil pulling has been an extensively used procedure as a conventional Indian folk remedy for many decades for strengthening of the gingiva, teeth, cracked lips, and dryness of the throat. In Ayurveda text Charaka Samhita (Sutrasthana 5, 78–80), it is mentioned as Kavala, Gandoosh/Kavala Graha, and it is claimed to treat about 30 systemic illnesses ranging from migraine headaches to asthma and diabetes. Dr. F. Karach popularized the oil pulling concept in the late 1990s in Russia. Oil pulling (Snaihik Gandoosh), as the name suggests, involves vigorous swishing of oil in the oral cavity to achieve local and systemic benefits, similar to the modern day use of mouthwashes and oral rinses. It has been used for centuries for the treatment and Figure 10.1 Procedure of oil pulling. Effect of Oil Pulling on Oral Health 143 prevention of various oral and systemic diseases, using edible oils derived from sesame, coconut, and sunflower [4]. Various studies on oil pulling therapy as an adjunct to conventional oral hygiene measures using sesame oil, sunflower, coconut, and olive oil have shown promising results in the control of oral diseases (Figure 10.1). Scientific evidence reports that oil pulling therapy may reduce plaque and gingival index scores by reducing the total oral bacterial count. 10.3 How Does Oil Pulling Work? While there are numerous theories, the exact mechanism of action is unclear. One theory speculates a mechanism involving alkali hydrolysis of fat, resulting in saponification or “soap making” process. The fat content of the oils used for oil pulling undergoes an alkali hydrolysis process and emulsifies the fat into bicarbonate ions, a normal constituent found in the saliva. The resultant soaps are effective cleaning agents, which blend in the oil, hence, increasing the total surface area of the oil, which in turn increases the cleansing action [5]. An in vitro study by Asokan et al. revealed that under the microscope, in the 5-min oil-pulled sample, the oil remained as relatively large globules. Very few numbers of Grampositive bacteria and foreign fibrous particles were seen. With the progressing time from 10 to 15 min, the oil droplets became smaller, revealing more colonies of Gram-positive bacteria and few squamous epithelial cells. The smaller fractionation of oil globules was suggestive of the ongoing emulsification process as a result of the agitation of the oil in the mouth. Hence, it was inferred that this vigorous swishing process may be responsible for the formation of a soapy layer. Another theory suggests that inhibition of plaque formation was due to the viscous nature and emulsification process of the oil, which interferes with microbial adhesion, coaggregation of bacteria, and removal of the superficial worn-out squamous cells [6]. Furthermore, the third theory hypothesizes that the inherent antioxidants found in the oil result in detoxification by prevention of lipid peroxidation and exerting an antimicrobial effect [7]. 10.4 Composition and Various Activities of Most Commonly Used Oils for Oil Pulling Composition and various activities of most commonly used oils for oil pulling are described in Tables 10.1 and 10.2. 10.4.1 10.4.1.1 Sesame Oil Antioxidant Activity Sesame (Sesamum indicum, Pedaliaceae) is a very old cultivated crop and thought to have originated in Africa. Antifungal activity has been demonstrated by Chlorosesamone 144 Natural Oral Care in Dental Therapy Table 10.1 Various edible oils used for oil pulling. Various edible oils used for oil pulling 1) Sesame oil 2) Coconut oil 3) Olive oil 4) Sunflower oil 5) Rice bran oil 6) Soya bean oil 7) Palm oil 8) Corn oil Table 10.2 Activities of oils used for oil pulling. General activities of oils used for oil pulling 1) Emollient 2) Antibacterial 3) Antifungal 4) Antiviral activity 5) Anti-nociceptive 6) Anti-inflammatory 7) Antioxidant 8) Anti-ulcer activity obtained from sesame roots, while Sesame lignans have antioxidant activities. Large amounts of unsaponifiable substances, sesamolin and sesamin, have been found in the sesame oil. Both sesamin and sesamolin increases both the peroxisomal and the hepatic mitochondrial fatty acid oxidation rate. The unsaponifiable fraction (sesaminol, sesamin, and sesamolin), a class of substances, which is not found in other oil fats, can probably protect the oral cavity from inflammation and infection by its antioxidant property [7]. Consumption of Sesame seed appears to enhance vitamin E activity and increases plasma gamma-tocopherol, which is believed to prevent heart disease and cancer [8]. 10.4.1.2 Antimicrobial Activity A study was conducted to assess the anti-microbial activity of sesame oil against selected gram-negative and gram-positive microorganism [Bacillus subtilis (NCIM 2480), Staphylococcus aureus (NCIM 2602), Escherichia coli (NCIM 2981), Salmonella typhi (NCIM 2493), Proteus vulgaris (NCIM 2813), Cornebacterium diphtheria, Streptomyces gresius]. The author reported that the sesame oil demonstrates excellent antimicrobial activity with a zone of inhibition range that equals with the standard Kenamycin [9]. A phytochemical screening of sesame oil using gas chromatography mass spectrometry (GC-MS) revealed the presence of essential oils primarily the carboxylic and phenolic acids groups, which are responsible for its antimicrobial activity [10]. Effect of Oil Pulling on Oral Health 10.4.2 145 Coconut Oil Coconut oil is one of the most commonly used oil in the soap industry as it has a high saponification value. It was also proposed that the salivary alkalis can react with the coconut oil leading to the saponification process and formation of a soap-like substance, which results in reduced adhesion of plaque [2]. 10.4.2.1 Antibacterial, Antifungal, and Antiviral Activity Oral microorganisms produce many long-chain fatty acids and medium-chain fatty acids as end products of their metabolism. These fatty acids can potentially contribute to biological and ecological interactions among the oral pathogens residing in the biofilm. The fatty acid-producing pathogens are present in both periodontitis and dental caries. Fatty acid production could contribute to a natural “nutrient reservoir” in oral plaque biofilms, resulting in mutual relationships with other oral bacteria. Free fatty acids (FFAs) present in coconut oil result in competitive inhibition of oral organisms by competing with fatty acids produced by pathogens [11]. Medium-chain monoglycerides, especially monolaurin found in coconut oil, are effective in destroying a wide variety of lipid-coated Gram-positive and Gram-negative microorganisms by disrupting their lipid membrane and inhibiting enzymes required in nutrient transfer and energy production, leading to the death of the bacteria. In a recent study, coconut oil has demonstrated a significant antifungal activity, which is comparable with ketoconazole against various strains of Candida spp [12]. Coconut oil is extremely effective against a variety of lipid-coated viruses such as influenza virus, visna virus, cytomegalovirus, Epstein–Barr virus, leukemia virus, hepatitis virus, and pneumonia virus. The virucidal activity of monolaurin is attributed to solubilization of the phospholipids and lipids in the envelope leading to the disintegration of the viral particles and interference with virus maturation [13]. 10.4.2.2 Antinociceptive, Anti-Inflammatory, Antioxidant, and Anti-Ulcer Activity Antinociceptive virgin coconut oil (VCO) most likely inhibits the prgressive phase of chronic inflammation. During the inflammatory process, release of lysosomal enzymes by phagocytic cells damage the surrounding cells. The anti-inflammatory activity of VCO may be attributed to its inhibitory effect on the activity of inflammatory cell and/or the lysosomal membrane stabilization. VCO inhibits the release and/or synthesis of these inflammatory mediators such as prostaglandins, bradykinin, and histamine responsible for pain and edema formation [14]. Antiulcer activity of VCO could be attributed to its FFAs content. Some of these FFAs possess antioxidant effects (i.e., palmitic acid and myristic acid) and anti-inflammatory activities (i.e., oleic acid, linoleic acid, and lauric acid). The antiulcer activity of the edible oils could be associated with their anti-inflammatory and antioxidant activities and the FFA composition. This activity may contribute to the reduction in gingival bleeding and healing time of ulcerative lesions of the oral cavity [15]. 146 Natural Oral Care in Dental Therapy 10.5 Procedure of Oil Pulling Ideally, the oil pulling procedure or snaihik gandoosh is usually carried out early in the morning and on an empty stomach by swishing a tablespoon full of oil vigorously around the mouth. In children older than 4 years of age, one teaspoon of oil is used. The ideal quantity is the “half-full mouth.” While swishing the oil all around the mouth, it is forced and “pulled” in between all the teeth. Oil is to be pulled inside the mouth cavity until there is uncontrollable salivation (the patient knows it as the quantity of the liquid inside the mouth cavity increases, which the patient feels by himself). At the end of this correctly done activity, the viscous oil usually becomes thinner and milky white. After that it is spit out and clean tap water or warm water is used to thoroughly wash the mouth. Then it is followed by teeth cleaning with the fingers or regular tooth brushing. If the jaw muscle fatigues, then the procedure can be carried just for 5 to 10 min. Importantly, during oil pulling, care should be taken to avoid swallowing or aspiration of oil as the pulled oil contains toxins and bacteria. Because of the risk of aspiration, it is contraindicated in children younger than 5 years. Ideally, oil pulling is practiced in a sitting position with chin up. To hasten the healing effects, it can be practiced three times daily on an empty stomach before every meal. In conditions like vomiting tendency, oral ulcers, asthma, fever, where tooth brushing is not easily possible, oil pulling can be beneficial in the maintenance of oral hygiene [16, 17]. 10.6 Effects of Oil Pulling on Oral Health The effects of oil pulling on oral health are found in Table 10.3. 10.6.1 Dental Caries The oral cavity is always covered with a biofilm. The chemical and mechanical removal of the oral biofilm is important in maintaining the ecological equilibrium of the oral cavity and preventing the initiation of the carious process. An estimated 700 different species of bacteria are found in the oral microbiome, inhabiting the oral biofilm. In dental caries or tooth decay, bacterial metabolic processes metabolized carbohydrates present in food stuck on the teeth to an acid, which in turn demineralizes the hard tooth structure. After the pH of plaque drops below the “critical value” (5.5 for hydroxyapatite, 4.5 for fluorapatite, and Table 10.3 Effect of oil pulling on oral health. Effect of oil pulling on oral health 1) Reduction in mucosal inflammation 2) Reduction in gingival bleeding 3) Reduction in halitosis 4) Reduction in caries activity 5) Early healing of ulcerative lesions 6) Soothing effect on oral mucosa 7) Reduction in burning mouth 8) Reduction in mucosal dryness Effect of Oil Pulling on Oral Health 147 6.7 for cementum), the demineralization process starts, causing disintegration of the calcium phosphate ions in the hydroxyapatite crystals. The demineralized form of enamel is known as dental caries [5]. Streptococcus mutans and Lactobacillus are two groups of lactic acid-producing pathogens that are mainly associated with dental caries [18]. Asokan S et al. compared the efficacy of sesame oil pulling and chlorhexidine mouthwash in controlling plaque-induced gingivitis. At the end of the study period, significant reduction was observed in the baseline and post follow-up values of the modified gingival, plaque index scores, and total bacterial colony count of aerobic dental plaque microorganisms in both the study and control groups. Intergroup comparison did not show statistical difference in any of the test parameter [6]. Anand et al. investigated the effects of sesame oil pulling on S. mutans and L. acidophilus colony counts in patients with dental caries. At the initial visit, participants were asked to gargle with a normal saline solution followed by the collection of salivary samples for analysis of the total colony counts. The colony counts were assessed at baseline and 40th day. Post follow-up data revealed an average reduction of 20% in the total colony count. The Snyder method was used to determine caries susceptibility. The antimicrobial activity of sesame oil against bacterial strains of L. acidophilus and S. mutans was assessed measuring the zone of inhibition. All the participants exhibited marked reduction in caries susceptibility and demonstrated moderate inhibitory effects against total bacteria counts, S. mutans and L. acidophilus [8]. Various other studies have reported that coconut oil gargling is as effective as using chlorhexidine mouthwash in decreasing S. mutans and Lactobacillus count. The synergistic effect of the saponification, emulsification, and the antimicrobial effects of medium-chain fatty acids present in coconut oil may contribute in the reduction of cariogenic microorganisms [19–21]. 10.6.2 Plaque-Induced Gingivitis Gingivitis is the mildest and most common form of the periodontal disease usually caused by poor oral hygiene. It is characterized by inflammation, bleeding, and swelling of the gums. The main etiology of gingivitis is an accumulation of plaque on the surface of the teeth and gums. In a global scenario, gingivitis begins in the early childhood aged 3–11 years, and the prevalence rises to 70–90% at puberty [2]. Kaliamoorthy et al. study to compare the effect of oil pulling utilizing coconut and sesame oil in 60 patients with plaque-induced gingivitis. All the patients were divided into three groups Each group was advised a different method of treatment like coconut oil for oil pulling, sesame oil for oil pulling, and routine toothbrushing alone. Clnical evaluation was done at baseline, 7th, 14th, and 21st day. At the end of the study, the authors concluded that coconut oil pulling is a useful and effective oral hygiene practice along with routine oral hygiene practice. Coconut oil is very effective compared to sesame oil in the reduction of severity of gingivitis [22]. Amith and colleagues assessed the efficacy, acceptability, and safety of oil pulling in controlling plaque-induced gingivitis. In a 45-day follow-up study, 10 male subjects were advised oil pulling with refined sunflower oil in addition to their routine oral hygiene practices throughout the study. All the clinical indices recorded at baseline, days 15, 30, and 45 revealed significant decrease in mean plaque scores from baseline to day 45. At the end of 148 Natural Oral Care in Dental Therapy the study, no adverse reactions to soft or hard tissues were reported. Even though the oil pulling procedure was time consuming and difficult to master, 80% of the survey participants expressed acceptability of the oil pulling regimen and were ready to use oil pulling therapy for the rest of their lives [23]. In a two-phase study, Busscher et al. investigated the antibacterial and clinical efficacy of a vegetable oil-based oral rinse. In vitro inhibition of bacterial growth was studied against microorganisms linked with dental caries and gingivitis. Bacterial strains of Streptococcus sanguis, Streptococcus mutans, Lactobacillus acidophilus, Veillonella alcalescens, and Actinomyces viscosus were isolated from humans and multiplied overnight in broth. Authors of the study reported that two strains commonly associated with dental caries (S. mutans and V. alcalescens) were strongly suppressed by the vegetable oil-based oral rinse. On the basis of these in vitro study findings, the authors conducted a short clinical trial to assess and compare the clinical efficacy of a vegetable oil-based oral rinse and six commercially available products: Prodent, Hibident, Meridol, Veadent, Merocet, and Listerine. All clinical parameters were assessed at baseline 14th and 20th day. Subjects were advised to continue routine brushing with non-fluoridated dentifrice along with the use of only their allocated rinse, twice daily, for 30 s. Researchers suggested that the almond oilbased mouth rinse had shown promising results in reducing gingival index scores, which are comparable to the tested commercially available products [24]. In a randomized controlled trial, Dani N et al. studied the antiplaque activity of sesame oil pulling and its effect on plaque-induced gingivitis. Forty subjects randomly divided in two groups were subjected to routine scaling and root planing. Thereafter, the first group was instructed to carry out oil pulling for 14 days; while the second group was given chlorhexidine mouthwash for 14 days. The authors reported that both the groups showed significant reduction in all parameters like gingival index scores, Plaque index scores, and total aerobic bacteria colony counts after 14 days. Sesame oil exhibited similar effectiveness as chlorhexidine in reducing plaque-induced gingivitis [25]. A clinico-microbiological study was carried out by Saravanan et al. to assess the effect of sesame oil pulling on dental plaque, colony-forming bacteria, and gingivitis. The results of the study stated that the oil pulling group demonstrated significant reduction in plaque index scores, bacterial counts, and gingival index scores compared to the group that used only routine oral hygiene methods [26]. In a recent study, the anti-inflammatory effect of coconut oil has been demonstrated in subjects with plaque-induced gingivitis. The authors reported a significant reduction in gingival inflammation, which was attributed to the aforementioned antimicrobial and anti-inflammatory activity, decreased accumulation of plaque, and the emollient effect of coconut oil with no untoward side effects [2]. 10.6.3 Halitosis Halitosis or bad breath is a common problem that can often cause social embarrassment. The malodor is produced from volatile sulfide compounds especially dimethyl sulfide, hydrogen sulfide, and methyl mercaptan, originating from the proteolytic degradation of the peptides present in food debris, saliva, plaque, and desquamated epithelial cells. Gramnegative proteolytic bacteria responsible for periodontitis and gingivitis are also known to produce sulfide compounds [5]. Effect of Oil Pulling on Oral Health 149 Asokan and colleagues conducted a randomized controlled study to evaluate the effecacy of oil pulling in controlling halitosis. Enrolled 20 study subjects were randomized equally into two groups. In addition to routine oral hygiene practice, group I was advised to perform sesame oil pulling for 10 to 15 min in the morning, while group II was instructed to use 0.2% chlorhexidine rinse for 1 min in the morning. Five clinical parameters (plaque index, modified gingival index, self-assessment of breath, organoleptic breath assessment, and the BANA test) were evaluated at baseline and day 14. At the end of the study, both groups showed a significant decrease in scores of all parameters. Hence, the authors concluded that oil pulling was as good as chlorhexidine in reducing microorganisms associated with malodor [27]. In a recent study, an especially formulated oil by Dentiste, Thailand (Dentiste’7M oil), demonstrated an in vitro inhibitory effect against the prominent VSC-producing bacterium, P. gingivalis. Consequently, swishing with this newly formulated oil may be beneficial in managing oral malodor [28]. 10.6.4 Oral Thrush Oral thrush or oral candidiasis is a non-contagious fungal infection caused by Candida species. It is commonly seen in individuals taking medications that may alter the oral microflora over extended periods. Denture wearers, patients undergoing prolonged antibiotic treatment or using inhaled corticosteroid for asthma, and patients undergoing chemotherapy or radiotherapy reportedly have a higher incidence of oral candidiasis. Evidence suggests that oil pulling therapy improves symptoms of oral thrush in two ways. First, it traps or pulls the toxins and other pathogens during oil swishing and therefore aids in the mechanical removal of the pathogens from the oral cavity. Second, the antifungal properties of the oils used, particularly coconut oil, kills the yeast in the oral cavity and therefore plays a role in eliminating the candida pathogens [5]. 10.6.5 Xerostomia and Burning Mouth Syndrome Xerostomia is defined as dry mouth resulting from reduced or absent saliva flow. Xerostomia is not a disorder, but it may be an indicator of some underlying medical illnesses, an adverse effect of several of medications, or an undesirable effect of a radiation to the head and neck. Decreased salivary gland function may or may not be associated with xerostomia. Burning mouth syndrome (BMS) is a condition that is commonly associated with burning sensation and dryness of mouth, altered taste, and mostly affects the women of menopause transition age. Despite the large number of available trials on the management of this condition, there seems to be no complete cure until date, which is mainly attributed to the uncertainty of this condition. In a double-blinded cross-over study, Xerolube (artificial saliva) and vegetable oil were compared as a treatment for xerostomia in patients with cancer of the head and neck. All enrolled subjects were randomly allotted to two groups: vegetable oil or Xerolube for a 2-week duration of treatment. After the washout period of 2 weeks, the products were switched among the groups for another 2 weeks. A 17-item Mouth Dryness Questionnaire (MDQ) was used to record the participants’ subjective experiences of dryness. Results revealed significant improvement in non-tobacco users with the utilization of oil and 150 Natural Oral Care in Dental Therapy emphasized a superior predilection for vegetable oil. The MDQ scores exhibited no significant differences between the two treatments. Hence, the authors concluded that vegetable oil can be used as an inexpensive and effective substitute treatment modality for patients with xerostomia induced by radiation [29]. Garg et al. assessed the efficacy of sesame oil pulling therapy in the treatment of patients suffering from primary BMS. All clinically diagnosed BMS patients were advised to perform oil pulling once daily for a period of 3 months. The Visual Analog Scale (VAS) was used to assess symptoms of stomatopyrosis and xerostomia at the end of every month. The results of the study revealed a significant improvement in the symptoms of both stomatopyrosis and xerostomia at the end of 3 months follow-up when compared with the baseline status [30]. 10.7 Drawbacks of Oil Pulling The drawbacks associated with the practice of oil pulling are [31]; a. Oil pulling cannot be used as monothearapy. b. It is a time-consuming procedure. c. Recently, few isolated case reports have highlighted that improper technique of oil pulling led to aspirational lipoid pneumonia in patients using this technique vigorously [32, 33]. Ayurvedic literature has reported possible negative side effects if improper technique is used, such as dryness of mouth, excessive thirst, dysgeusia, exhaustion, and muscular stiffness [4]. The first systematic review by Oghenekome Gbinigie at the Centre for Evidence-based Medicine, University of Oxford, UK, proposes that oil pulling may have advantageous effects on oral hygiene as observed from short-term investigations [34]. As stated that as oil pulling is a potentially low-cost therapeutic modality, this procedure might be of particular benefit. More meticulous long-term clinical trials are required to further evaluate local and systemic benefits of oil pulling therapy. References 1. WHO/Oral health. [Last accessed on 20/03/2019]. Available at http://www.who.int/topics/ oral_health/en/. 2. Chalke, S., Zope, S.A., Suragimath, G., Varma, S.A., Abbayya, K., Kale, V., Effect of coconut oil pulling on plaque-induced gingivitis: A prospective clinical study. Int. J. Green Pharm., 11, 04, S750–S755, 2017. 3. Nagilla, J., Kulkarni, S., Madupu, P.R., Doshi, D., Bandari, S.R., Srilatha, A., Comparative Evaluation of Antiplaque Efficacy of Coconut Oil Pulling and a Placebo, Among Dental College Students: A Randomized Controlled Trial. J. Clin. Diagn. Res., 11, 9, ZC08–ZC11, 2017. 4. Sooryavanshi, S. and Mardikar, B.R., Prevention and treatment of diseases of mouth by gandoosha and kavala. Anc. Sci. Life, 13, 3–4, 266–270, 1994. 5. Naseem, M., Khiyani, M.F., Nauman, H., Zafar, M.S., Shah, A.H., Khalil, H.S., Oil pulling and importance of traditional medicine in oral health maintenance. Int. J. Health Sci., 11, 4, 65, 2017. Effect of Oil Pulling on Oral Health 151 6. Asokan, S., Emmadi, P., Chamundeswari, R., Effect of oil pulling on plaque induced gingivitis: A randomized, controlled, triple-blind study. Indian J. Dent. Res., 20, 47–51.18, 2009. 7. Asokan, S., Rathinasamy, T.K., Inbamani, N., Menon, T., Kumar, S.S., Emmadi, P. et al., Mechanism of oil-pulling therapy—In vitro study. Indian J. Dent. Res., 22, 34–7, 2011. 8. Anand, T.D., Pothiraj, C., Gopinath, R.M., Kayalvizhi, B., Effect of oil-pulling on dental caries causing bacteria. Afr. J. Microbiol. Res., 2, 063–6, 2008. 9. Saleem, T.M., Anti-microbial activity of sesame oil. Int. J. Res. Phytochem. Pharmacol., 1, 1, 21–3, 2011. 10. Shittu, L.A.L., Bankole, M.A., Ahmed, T., Aile, K., Akinsanya, M.A., Bankole, M.N., Shittu, R.K., Ashiru, O.A., Differential antimicrobial activity of the various crude leaves extracts of Sesame radiatum against some common pathogenic micro-organisms. Sci. Res. Essay, 1, 108–11, 2006. 11. Huang, C.B., Alimova, Y., Myers, T.M., Ebersole, J.L., Short- and medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms. Arch. Oral Biol., 56, 650–4, 2011. 12. Shino, B., Peedikayil, F.C., Jaiprakash, S.R., Ahmed Bijapur, G., Kottayi, S., Jose, D. et al., Comparison of antimicrobial activity of chlorhexidine, coconut oil, probiotics, and ketoconazole on Candida albicans isolated in children with early childhood caries: An in vitro study. Scientifica (Cairo), 2016, 7061587, 2016. 13. Arora, R., Chawla, R., Marwah, R., Arora, P., Sharma, R.K., Kaushik, V. et al., Potential of complementary and alternative medicine in preventive management of novel H1N1 flu (Swine flu) pandemic: Thwarting potential disasters in the bud. Evid. Based Complement. Alternat. Med., 2011, 586506, 2011. 14. Intahphuak, S., Khonsung, P., Panthong, A., Anti-inflammatory, analgesic, and antipyretic activities of virgin coconut oil. Pharm. Biol., 48, 151–7, 2010. 15. Selverajah, M., Zakaria, Z.A., Long, K., Ahmad, Z., Yaacob, A., Somchit, M.N., Anti-ulcerogenic activity of virgin coconut oil contribute to the stomach health of humankind. Tang [Humanitas Medicine], 6, 11.1–7, 2016. 16. Sooryavanshi, S. and Mardikar, B.R., Prevention and treatment of diseases of mouth by gandoosha and kavala. Anc. Sci. Life, 13, 3–4, 266–270, 1994. 17. Shanbhag, V.K., Oil pulling for maintaining oral hygiene—A review. J. Tradit. Complement. Med., 7, 1, 106–109, 2016. 18. Lakshmi, T., Rajendran, R., Krishnan, V., Perspectives of oil pulling therapy in dental practice. Dent. Hypotheses, 4, 131–4, 2013. 19. Peedikayil, F.C., Remy, V., John, S., Chandru, T.P., Sreenivasan, P., Bijapur, G.A., Comparison of antibacterial efficacy of coconut oil and chlorhexidine on Streptococcus mutans: An in vivo study. J. Int. Soc. Prev. Community Dent., 6, 5, 447–452, 2016. 20. Singla, N., Acharya, S., Martena, S., Singla, R., Effect of oil gum massage therapy on common pathogenic oral microorganisms—A randomized controlled trial. J. Indian Soc. Periodontol., 18, 441–6, 2014. 21. Thaweboon, S., Nakaparksin, J., Thaweboon, B., Effect of oil-pulling on oral microorganisms in biofilm models. Asia J. Public Health, 2, 62–6, 2011. 22. Kaliamoorthy, S., Pazhani, A., Nagarajan, M., Meyyappan, A., Rayar, S., Mathivanan, S., Comparing the effect of coconut oil pulling practice with oil pulling using sesame oil in plaque-induced gingivitis: A prospective comparative interventional study. J. Nat. Sci. Biol. Med., 9, 165–8, 2018. 23. Amith, H.V., Ankola, A.V., Nagesh, L., Effect of oil pulling on plaque and gingivitis. J. Oral Community Dent., 1, 1, 12–8, 2007. 24. Busscher, H.J., Perdok, J.F., Mei, V.D., Bacterial growth inhibition and short-term clinical efficacy of a vegetable oil-based mouthrinse: Preliminary study. Clin. Prev. Dent., 14, 3, 5–8, 1992. 152 Natural Oral Care in Dental Therapy 25. Dani, N., Kale, T., Beldar, A., Raghavan, M., Thakkar, P., Oil pulling as an adjunct to scaling and root planing: A Clinico-Microbial study. Int. J. Pharm. Sci. Invent., 4, 38e44, 2015. 26. Saravanan, D., Ramkumar, S., Vineetha, K., Effect of oil pulling with sesame oil on plaqueinduced gingivitis: A microbiological study. J. Orofac. Res., 3, 175e180, 2013. 27. Asokan, S., Kumar, R.S., Emmadi, P., Raghuraman, R., Sivakumar, N., Effect of oil pulling on halitosis and microorganisms causing halitosis: A randomized controlled pilot trial. J. Indian Soc. Pedod. Prev. Dent., 29, 90–4, 2011. 28. Amornvit, P., Choonharuangdej, S., Pithayanukul, S., Phetdee, K., In vitro Efficacy of Newly Formulated Oil Pulling against Oral Malodor Related Microbiota. J. Adv. Med. Med. Res., 20, 1–5, 2017. 29. Walizer, E.M. and Ephraim, P.M., Double-blind cross-over controlled clinical trial of vegetable oil versus xerolube for xerostomia: An expanded study abstract. ORL Head Neck Nurs., 14, 1, 11–eoa, 1996. 30. Garg, A., Bhatnagar, A., Tayal, S., Singh, U.P., Merits of Oil Pulling Therapy in the Management of Xerostomia and Stomatopyrosis in Burning Mouth Syndrome. J. Clin. Diagn. Res., 11, ZC27– ZC29, 2017. 31. Selvam, P., Nandan, N., Raj, S., Oil Pulling—A Blessing in Disguise. J. Ayurveda Integr. Med. Sci., 4, 8–13, 2016. 32. Kim, J.Y., Jung, J.W., Choi, J.C., Shin, J.W., Park, I.W., Choi, B.W., Recurrent lipoid pneumonia associated with oil pulling. Int. J. Tuberc. Lung Dis., 18, 2, 251–2, 2014. 33. Kuroyama, M., Kagawa, H., Kitada, S., Maekura, R., Mori, M., Hirano, H., Exogenous lipoid pneumonia caused by repeated sesame oil pulling: A report of two cases. BMC Pulm. Med., 15, 1, 135, 2015. 34. Gbinigie, O., Onakpoya, I., Spencer, E., McCall MacBain, M., Heneghan, C., Effect of oil pulling in promoting oro dental hygiene: A systematic review of randomized clinical trials. Complement Ther. Med., 26, 47–54, 2016. 11 Role of Proteolytic Enzymes in Dental Care P. Kalyana Chakravarthy1* and Sravan Kumar Yeturu2 1 Department of Public Health Dentistry, Manipal College of Dental Sciences, Manipal, Manipal Academy of Higher Education, Manipal, India 2 Department of Public Health Dentistry, Amrita School of Dentistry, Amrita Vishwa Vidyapeetham, Kochi, India Abstract Plant-based proteolytic enzymes include papain, bromelain, actinidin, and ficin. These enzymes have been tried and tested for numerous applications in the professional as well as personal oral health care. The range of applications include basic oral hygiene maintenance via dentifrices, as antiinflammatory and adjuvant to antibiotics in postoperative management, possible role in management of cancer and oral mucositis, caries excavation, and increasing bond strength for bonding orthodontic brackets. Literature pertaining to these areas has been reviewed, and findings have been discussed with future scope for research. Keywords: Tooth whitening, plaque, gingivitis, halitosis, papain, bromelain, cancer, mucositis, caries removal 11.1 Introduction A strong desire among the consumers for natural products is steadily increasing, and manufacturers are constantly trying to meet these demands of the consumers. Currently, there is a wide range of herbal products that are targeted for dental care. One such category of products is plant-based proteolytic enzymes, which are mainly proteases. Common plant-based proteolytic enzymes that are of interest in the field of dentistry are papain and bromelain. Actinidin and ficin are some emerging newer products that are being researched for their possible applications in oral health. Many reviews exist in the literature that describe the potential applications of these products in only the medical field. However, many applications of these enzymes have been researched for use in the field of dentistry (Figure 11.1). This chapter reviews the literature pertaining to the applications and efficacy of these proteolytic enzymes for professional and personal oral health care. Papain is a proteolytic enzyme that comes from the latex of the leaves and fruits of the papaya. It has an anti-inflammatory, bacteriostatic, bactericidal characteristic and is effective against gram-positive and gram-negative organisms [1]. *Corresponding author: drkalyan81@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (153–170) © 2020 Scrivener Publishing LLC 153 154 Natural Oral Care in Dental Therapy Malodour Gingivitis Extrinsic Stains Increased bonding of Restorations Control of Biofilm Plaque Periodontics Children Differently Abled Less, Pain, Discomfort and Minimally Invasive Improved Action of Antibiotics Dry Socket Improved Healing Post Extraction Management Blood Coagulation and Fibrinolytics Oral Surgery Chemomechanical Caries Removal Plant based Proteolytic Enzymes Post Surgical Edema Post Surgical Ecchymosis Geriatric Patients Avulsed Teeth Anxious and Phobic Patients Orthodontics Removal of Smear Layer Less Microleakage ? Oral Medicine Increased Bonding of Brackets TMJ Osteoarthritis Antimicrobial Action Oral Submucous Fibrosis ? Oral Cancer, Oral Mucositis ? Figure 8.1 Overview of the potential applications of proteolytic enzymes in the field of dentistry and oral care. Bromelain is a complex natural mixture “thiol endopeptidases and non-protease components, including phosphatases, glycosidases, peroxidases, cellulases, glycoproteins, ribonuclease, carbohydrates, and others” [2]. It is derived from pineapple, which has numerous therapeutic applications. Actinidin or actinidain is an enzyme, which is commonly present in fruits like kiwi, pinapple, papaya, mango, and banana. Ficin or ficain is a latex derived from the trunk Ficus insipida. All these enzymes are cysteine proteases. 11.2 Role of Proteolytic Enzymes in Oral Surgery 11.2.1 Post-Extraction Management Postoperative complications after dental extraction include pain, dry socket, swelling, and limited mouth opening. Clinical management includes immediate cold and later hot compression, administration of analgesics and antibiotics as per the need. Proteolytic enzymes act at various sites of receptors on the cell surface that have variable role on the inflammatory components. It was shown that there was improvement in postoperative pain, swelling, and mouth opening and overall quality of life [3]. The possible mechanism of action suggested was due to the downregulation of inflammatory markers (IL-25 and TNF-α) and increased expression of EGFR and β-FGF. This led to the proliferation of fibroblasts, endothelial and epithelial cells, epithelization, and neovascularization, which hastened wound healing. Inchingolo et al. [4] concluded that the efficacy of bromelain is similar to that of ketoprofen in the reduction of pain and edema. Majid and Al-Mashhadani [5] reported on the analgesic and anti-inflammatory effects of bromelain, which is comparable to preemptive diclofenac and showed a significant positive effect on the quality of life measures with only a limited reduction of trismus. Ghensi et al. [6] evaluated the effect of oral administration of bromelain in postoperative discomfort after third molar surgery. They concluded that there was a 22% reduction in edema at day 2 compared to the control group. Bromelain showed anti-inflammatory properties, but the use of dexamethasone was inevitable. It was also suggested that the Proteolytic Enzymes in Dental Care 155 best results were seen when bromelain and dexamethasone were given in conjunction. The use of bromelain alone had minimal effect on pain, while the combination of bromelain and dexamethasone showed significant lower consumption of analgesics than the control group. They also concluded that bromelain had little effect on postoperative trismus. De la Barrera-Núñez et al. [7] assessed pain relief after third molar extraction and showed that the bromelain and placebo groups were the same in terms of the pain score. Eslami et al. concluded that oral administration of bromelain showed no effect on pain, swelling, and trismus after third mandibular molar removal [8]. Singh T et al. [9] concluded that there was overall effectiveness in terms of reduced pain, swelling, and healing time and also reported that there was effective reduction of pain and swelling in 70% of their patients. Tassman et al. [10] reported that bromelain was better than the placebo for the reduction of the degree and duration of edema and pain for 81% of the patients. Ordesi et al. [11] found a significant reduction in pain and swelling after extraction of the third molars in patients who have taken bromelain when compared with the controls. Vigano et al. [12] conducted a prospective and controlled study to evaluate the antiinflammatory and analgesic effect of bromelain with or without paracetamol and codeine during pre- and postoperative oral surgery (implants, impacted molars, sinus lift, and enucleation of cysts). Variations in the findings could be due to the heterogeneity in the dosage of bromelain, which was used in these studies (150 mg to 1000 mg/day). Other possible mechanisms of actions reported in the literature were through alteration of pain mediators (bradykinin), inhibiting the synthesis of pro-inflammatory prostaglandins (PGE2), and resolution of edema due to fibrinolytic action [9]. 11.2.2 Post-Surgical Facial Ecchymosis and/or Edema Seltzer et al. [10] evaluated the effect of bromelain on the resolution of ecchymosis and edema after rhinoplasty. They found that one to two tablets four times a day had fewer days of ecchymosis and edema than placebo. Seltzer et al. evaluated the effect of bromelain on ecchymosis and edema after surgical and nonsurgical facial trauma. They found a significant difference between bromelain and placebo for inhibition of ecchymosis and edema. Shetty and Mohan [13] evaluated the effect of systematic enzyme therapy on the thickness of soft tissue in bimaxillary orthognathic surgery patients. They found a significant improvement in the treatment than in the placebo group measured by ultrasound. Woolf et al. [14] evaluated the effect of bromelain on artificially induced hematoma and found a significant improvement of edema compared with the placebo. However, Gylling et al. [15] evaluated edema after facial plastic surgery and found that there was no significant difference between bromelain and control groups. Bromelain can be of use in wound debridement and removal of necrotic tissues. Bromelain has been shown to have wound debridement properties by hydrolysis (in vitro and in vivo) without effecting the normal tissue [2, 16]. Conventional surgical debridement can be simplified by complementing topical bromelain [2], and it was seen that it alters the wound microenvironment thus promoting healing. A systematic review on the effect of bromelain for edema and ecchymosis concluded that bromelain may not be efficacious for larger procedures [10]. Bromelain administered in multiple doses may have interactions with food consumption that may affect the absorption and the efficacy of bromelain [10]. 156 Natural Oral Care in Dental Therapy 11.2.3 Enhanced Action of Antibiotics Combined bromelain and antibiotic therapy was effective than only antibiotics in the treatment of cutaneous Staphylococcus infection, cellulitis, and sinusitis [17, 18] and has reduced the side effects [16]. Shahid et al. [19] evaluated the efficacy and safety of combined oral dose of bromelain, trypsin, and rutin as an adjuvant therapy in the treatment of sepsis among children in a double blind randomized controlled trial. The duration for subsiding fever and need for hemodynamic support were significantly lower in children with adjuvant therapy than in the placebo group. The study concluded that adjuvant therapy (bromelain, trypsin, and rutin) with antibiotics and support care was effective in the treatment of sepsis among children. 11.2.4 Effect of Bromelain on Blood Coagulation and Fibrinolysis Bromelain showed an effect on serum fibrinolytic ability [47]. Animal studies showed that higher concentration prolonged prothrombin time, and activated partial thromboplastin time was seen [48]. Hence, dentists and oral health care providers should be cautious in prescribing bromelain in subjects with coagulative disorders or those on anticoagulants. It is recommended that bromelain be administered before food intake, and daily dosage should be 750–1,000 mg/day in divided doses. Nevertheless, plant-based proteolytic enzymes have multitude of applications in the field of oral surgery. Clinicians should consider the benefits and harms in recommending these enzymes routinely to the patients. Further, high-quality clinical trials are needed to have high quality evidence to prescribe these enzymes for various applications in the field of oral surgery. 11.3 Role of Proteolytic Enzymes in Cancer and Oral Mucositis 11.3.1 Cancer Bromelain can be an alternative effective anticancer agent due to its system involving multiple cellular and molecular targets [20]. However, the exact mechanism of action is not clearly established. Currently, only literature review exists on their role as an anticancer agent. Bhui et al. [21] conducted laboratory studies to understand anti-tumor-initiating potential of bromelain using a tumorigenesis model. It was seen that there was a delay in initiation of tumorigenesis and increased tumor-free survival, which was linked to the apoptosis and anti-inflammatory activities. There was a so-called “preventative effect” due to the reduction of tumor volume (65%). Dhandayuthapani et al. [22] reported on dosedependent cytotoxic effects of bromelain in cell lines (breast cancer). It increased caspasecleaved cytokeratin concentration, which is a marker for apoptosis. In an in vivo study [23], bromelain inhibited translocation of nuclear factor-κB via G2/M arrest to apoptosis in epidermoid carcinoma and melanoma cells of humans. It was also seen that there was selectively induced apoptosis by upregulation by the expression of p53 and initiation of the mitochondrial apoptotic pathway in tumor cells [24] along with a decreased activity of cell survival regulators promoting apoptosis leading to cell death of tumor cells [25]. An ongoing phase II randomized, two-armed clinical study is being performed to evaluate the Proteolytic Enzymes in Dental Care 157 efficacy of oral high dose versus low dose bromelain (Comosain) in human subjects diagnosed with advanced late stage cancers [26]. Currently, mechanisms were proposed for the applications of these proteolytic enzymes as treatment modality for cancer. However, clinical research on their role is scant. Future studies are required and should focus on the clinical effectiveness of these enzymes in the treatment of head and neck cancers as well. 11.3.2 Management in Oral Mucositis Mucositis, which is the result of cancer chemotherapy and radiotherapy, can have symptoms like pain, lack of taste function, reduction in food intake leading to weight loss, treatment delays, dose adjustments, etc. Proteolytic enzymes have been used in the management of oral mucositis that are a result of chemotherapy and radiotherapy for the treatment of cancers. Beneficial effect of these proteolytic enzymes could be due to the anti-inflammatory action, but the mechanism is not clearly understood. Dorr et al. conducted a randomized placebo-controlled trial to evaluate the effectiveness of Wobe– Mugos E (mixture of proteolytic enzymes papain, trypsin, and chymotrypsin) to reduce the oral mucositis after radiotherapy [27]. No beneficial effect of this drug was seen in the treatment of oral mucositis over placebo. Similarly, a prospective randomized, open study by Gujral et al. evaluated proteolytic enzymes (Wobe–Mugos E) in conventional radiation therapy for oral mucositis [28]. They concluded that adjuvant enzyme therapy can have significant protection for radiation-induced side effects. Lower grades of mucositis scores were also reported by Kaul et al. with the use of pre- and post-radiotherapy proteolytic enzymes [29]. However, Cochrane review on interventions on mucositis reported a weak evidence [30]. 11.4 Osteoarthritis Osteoarthritis is a chronic degenerative disease of the hard and soft tissues around the temporomandibular joint. It involves the deterioration of the articular cartilage and synovial tissues and structural changes in bone. It can cause pain and/or dysfunction in functional movements of the jaw, and symptoms can be varied from one patient to another. Management is usually done by pharmacological (analgesics, anti-inflammatory, steroid injections) and non-pharmacological methods (hot and cold therapy, strengthening exercises, TENS, Lasers). A combination of bromelain, trypsin, and rutin in osteoarthritis patients of the knee and shoulder has been studied previously [31]. Not much literature exists on the role of bromelain on osteoarthritis of TMJ, but the analgesic properties (influence on bradykinin) could be [32] useful to treat osteoarthritis of TMJ. Jayachandran and Khobre [33] have evaluated the combination of bromelain, trypsin, and rutin with or without diclofenac in the treatment of osteoarthritis of TMJ. Subjects with combination therapy of enzymes and diclofenac responded better than those subjects with only diclofenac or enzyme therapy. Very limited research has been conducted to evaluate the effectiveness of these enzymes in the treatment of osteoarthritis of the temporomandibular joint. Further high-quality clinical trials are required to evaluate the potential role of these enzymes in the management of temporomandibular joint osteoarthritis. 158 Natural Oral Care in Dental Therapy 11.5 Anti-Microbial Action Papain and bromelain have been tested for their efficacy against numerous oral microorganisms in varied laboratory conditions. Silva et al. evaluated the role of bromelain in the reduction of preformed Candida biofilm biomass and metabolic activity. A total of nine Candida strains were tested to quantify biomass biofilm formation, and metabolic activity of sessile cells within the biofilms was measured. There was marked reduction in biofilm biomass in the majority of strains for the three highest concentrations of bromelain (0.5, 0.25, 0.125 mg/mL) used, while the effect on biofilm cell metabolism was not so evident [34]. In an in vitro experiment on Candida albicans, Bromelain stimulated phagocytosis and respiratory burst killing [35]. Bharadwaj et al. [36], in an in vitro study, compared the effectiveness of Morinda citrifolia, papain, aloe vera, 2% chlorhexidine gel, and calcium hydroxide in the control of Enterococcus faecalis. Results showed that the chlorhexidine gel showed the maximum, and calcium hydroxide showed the least, effectiveness. Morinda consistently exhibited good inhibition followed by aloe vera gel and papain gel. Basting et al. evaluated [37] the antimicrobial effect of papain-based gel followed by self-etching adhesive systems with and without MDPB monomer on Streptococcus mutans and Lactobacillus casei. With respect to Streptococcus mutans, papain-based gel followed by the application of adhesive systems had lower colony-forming units than papain-based gel only. No significant differences were seen between the two types of adhesive systems. Results for Lactobacillus casei showed that papain-based gel followed by the application of adhesive systems had lower colony-forming units than papain-based gel only. However, the control group was not included in the statistical analysis. On the contrary, Mendez et al. [38], in an in vitro study, reported that papain did not exhibit growth inhibition, while it showed increased growth in the liquid medium. Mota et al. [39] conducted an in vitro antimicrobial evaluation of Eucalyptus globulus, xylitol, papain, and chlorohexidine on Pseudomonas aeruginosa, Samonella sp., Staphylococus aureus, Proteus vulgaris, Escherichia coli, and Candida albicans. Papain (10%) showed a lower antimicrobial effect than chlorhexidine in relation to Candida albicans, and further investigation is recommended. Papain at a concentration of 20% showed no inhibition with respect to the microorganisms under study. Praveen et al. [40] tested the minimum inhibitory concentration of bromelain on Streptococcus mutans, Enterococcus fecalis, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. They found that Streptococcus mutans showed sensitivity at a concentration of 2 mg/ml, Porphyromonas gingivalis at 4.15 mg/ml, while Aggregatibacter actinomycetemcomitans showed at 16.6 mg/ml and Enterococcus fecalis showed at 31.25 mg/ ml. Bromelain showed its effectiveness on potent periodontal pathogens. Kulakarni et al. [41] compared the efficacy of papain-based gel (Papacarie) and conventional excavation in reducing cariogenic flora using excavated dentin samples. They reported similar reductions in cariogenic flora with both the techniques. Motta et al. [42] conducted a randomized, controlled split-mouth clinical trial to check the efficacy of papain-based gel and conventional method in reducing the bacterial counts. In both the groups, there were significant reductions in total bacteria, Streptococcus and Streptococcus mutans, while no such significant difference was seen with respect to Lactobacillus CFUs. Proteolytic Enzymes in Dental Care 159 Numerous studies have shown the anti-microbial actions of these enzymes via in vitro studies. Future research should focus on the effectiveness on biofilm models to corroborate clinical effectiveness of these enzymes. 11.6 Treatment of Dental Carious Lesions Carious dentine consists of two different layers. The outer layer (infected dentine) is irreversibly damaged and not remineralizable, which needs to be excavated or removed. The inner layer (affected dentine) can be remineralizable and can be left behind during the carious excavation procedures [43]. Conventional carious removal involves [44] the use of rotary instruments, which is rapid with inherent risks like damage to the sound tooth structure, excess heat generation, and sensitivity. They may also have noise and vibration, which might increase anxiety in young children making uncooperative to restorative procedures. An alternative technique like atraumatic restorative treatment involves the use of hand instruments only. It was also noticed that caries can be excavated using chemicals that are known as chemomechanical caries removal. This technique gained popularity in the treatment of carious lesion in anxious children, special children, geriatric patients, and medically compromised patients. It minimizes patient stress and need for local anesthetic injections [45–47]. Many advantages for chemomechanical caries removal are reported like viz., selective removal of infected dentine and atraumatic with antibacterial activity [48]. The principle that an active ingredient acts on the predegraded collagen of the lesion, promoting its softening, without acting on healthy adjacent tissue and without causing pain, has made this technique an effective alternative for treatment of carious injuries [1]. It acts by breaking partially degraded collagen fibrils and softens the infected dentin, which then can be removed using blunt hand instruments. Some commercial preparations like Papacarie and Carie-care, which are based on papain gel, are now available. 11.6.1 Laboratory Studies Chittem et al. [49] in an in vitro study compared the microshear bond strength of the caries-affected dentinal surface after treatment with papain-based gel and conventional techniques. The time duration was significantly more with papain-based technique than the conventional technique, which can be mitigated by an experienced user [50]. They reported that papain-based chemomechanical caries removal techniques are reliable methods for caries removal with conventional adhesive systems. Similarly, Aly Khattab et al. [51] aimed to evaluate papain-based gel (Papacarié) on microshear bond strength and microleakage and of two restorative materials. Composite restoration exhibited less microleakage and better micro-shear bond strength than glass ionomer cement. Gianini et al. evaluated the microtensile bond strength of two different adhesive systems to demineralized dentin after the use of a papain-based chemomechanical method. It was seen that there was no interference in the adhesion due to the use of papain-based gel. Hafez et al. [52] compared the microleakage of composite restorations after papain-based chemomechanical caries removal and conventional technique. There was no significant 160 Natural Oral Care in Dental Therapy difference in the degree of microleakage and the diameters of the resin tags, while a significant thick hybrid layer was seen in papain-based chemomechanical caries removal compared with the conventional technique. Alhumaid et al. [53] compared the volume of removed tissue and dentin mineral density after excavation between Carisolv and papain-based gel. Results showed that there was higher dentin mineral density, more sound dentin, and less removed tissue in the papain group. Nair et al. [54] compared the microtensile bond strength and chemical composition of the dentin of teeth after the use of three commercially available chemomechanical caries removal agents (Carisolv, Papacarie, and Carie-care). No significant difference in the mean bond strength and calcium–phosphate ratio was observed among the groups studied. Somani et al. [55] evaluated the efficiency of different chemomechanical caries removal agents in smear layer removal in an in vitro study using scanning electron microscopy. Smear layer removal was significantly higher in the sodium hypochlorite-based gel (Carisolv), followed by papain-based gel (Carie-care) with the least being in the control group (saline). Viral et al. [56], in an in vitro study, compared the efficiency, marginal leakage, and shear bond strength of Carisolv and Papacarie in primary molars. Papacarie took significantly less time than Carisolv. Higher proportion of teeth treated with Carisolv did not show any marginal leakage than Papacarie. The mean shear bond strength of Carisolv was more when compared to Papacarie. Papacarie was efficient in caries removal but showed more marginal leakage than Carisolv. Bittencourt et al. [57] quantified the mineral content removed from primary teeth after excavation of caries using papain-based gel. The authors concluded that the amount of calcium removed with papain-based gel affected only the carious component of the teeth. Kotb et al. [58] evaluated the topographic features of dentin after caries removal with papain-based gel and conventional techniques. Papain-based gel showed irregular, porous, rough, and globular dentin appearance. Papain-based gel produced partial or complete removal of the smear layer and with open dentinal tubules in contrast to the conventional technique. 11.6.2 Clinical Studies Kitsahawong et al. [59] comprehensively compared the efficacy of Papacarie gel and conventional drilling on various factors like caries removal, time, morphological changes, and microhardness of surface dentin, and microleakage of subsequent restorations. There was no difference in the caries removal between groups. The Papacarie group showed significantly longer time for caries removal, more microleakage, and lower dentin hardness than the drilling group. Carrillo et al. [60] conducted a randomized controlled open label study to evaluate caries removal time and patient acceptance of the chemomechanical caries removal agent and papain gel among disabled children. Papain gel had a completed caries removal time of 8 min per tooth and is well accepted by the patients in all phases and visits of treatment. Khalek et al. [61] compared pain and discomfort scores (Sound, Eye, and Motor scale) using Papacarie (papain-based gel) and conventional atraumatic restorative treatment. They have reported that Papacarie required significantly longer duration of time than atraumatic restorative treatment. Sound, eye and motor component scores were significantly lower in the Papacarie than in the atraumatic restorative treatment group. In an adjusted model, it Proteolytic Enzymes in Dental Care 161 was seen that the method of caries removal had significant effect on sound eye and motor component scores when adjusted for gender, age in years, arch, tooth, lesion type, and time to remove caries in minutes. Kotb et al. [62] clinically evaluated Papacarie in the primary teeth along with the need for local anesthesia, time for removal of caries, and sound, eye, and motor component scores. They found that the Papacarie group did not required local anesthesia, while it was required by majority of the participants in the conventional group. No significant difference was seen with respect to time required to perform the procedure between Papacarie and the conventional treatment. However, when the time required for administering local anesthesia was included, conventional procedure required significantly longer duration than the Papacarie gel. Similarly, sound, eye, and motor component scores were significantly lower in the Papacarie than in the conventional group. Konde et al. [50] compared the conventional and chemomechanical caries removal (papain gel) in primary molars. It was seen that the mean time taken and pain scores in the conventional method were significantly higher than in the papain gel. However, no significant difference in the secondary caries rate after a follow-up of 10 months between the groups. Matsumoto et al. [63] conducted a randomized clinical split-mouth trial to evaluate the effectiveness of Papacarie Duo gel in the removal of carious lesions in primary teeth. No significant differences were seen in terms of time required, pain, or status of the restoration status after 30 days. Motta et al. [64] compared the effectiveness of papain-based gel for the chemomechanical removal of carious lesions on primary teeth with regard to time, clinical aspects, and radiographic findings in a randomized controlled clinical split-mouth trial. No significant differences were seen between the groups regarding the time required and the radiographic follow-up. Authors concluded that papain-based gel was effective in carious removal of deciduous teeth. Laboratory studies using papain-based gel showed acceptable results with respect to bond strength, microleakage, mineral content, and smear layer removal. Most clinical studies have evaluated the time taken to perform the procedure, restoration status, and need for local anesthetics. Comparable results have been reported by papain-based gel in the treatment of carious lesions in primary and permanent teeth among children and adults. Papain-based gel can be a suitable alternative caries removal agent that preserves dentinal tissue. However, this technique of application is limited to cavitated carious lesions, which are accessible to the hand instruments, and the cavities do not comply with the required retention form and largely rely on the chemical bond of the restorative materials [59]. The loosely attached debris and smear layers with high organic content might interfere with the bonding ability of the restorative materials leading to microleakage. Further clinical studies are required to evaluate the retention rates of the different restorations after the use of papain-based gels. A recent meta-analysis also concluded that subsequent caries rate should also be evaluated as an outcome in future studies [65]. 11.7 Improvement in Bonding of Orthodontics Brackets Enamel etching with phosphoric acid is the preliminary step before the application of a bonding agent to bond orthodontic brackets. However, it was seen that ideal etching pattern 162 Natural Oral Care in Dental Therapy may not be attained on the entire surface of the enamel. It is attributed to the outer organic layer resulting in inconsistent and unreliable enamel surface for bonding. To overcome this problem, deproteinization of enamel surface was suggested with sodium hypochlorite prior to the bonding. Pithon et al. [66] reported that 10% papain gel can be an effective enamel deproteinizing agent by increasing the shear bond strength of the orthodontic brackets irrespective of the etching agent. In another study, Pithon et al. [67] evaluated various concentrations of papain gel (2%, 4%, 6%, 8%, and 10%). They found that 8% and 10% papain gel showed higher shear bond strength than the control group. Pithon et al. [68], in an in vitro study using bovine enamel, evaluated shear bond strength with different concentrations of bromelain in association with 10% papain. Results showed that 3% and 6% bromelain gel in combination with papain significantly increased the shear bond strength with acid etching followed by primer application and attachment using Transbond XT and without etching in resin modified glass ionomer cements. Agarwal et al. [69] studied the influence of application of Papacarie and indigenously prepared 10% papain gel as a deproteinizing agent on the shear bond strength of the orthodontic brackets before and after acid etching with phosphoric acid. They reported that both papacarie and indigenously prepared 10% papain gel when used before acid etching showed significantly higher shear bond strength than only the acid etching group. Also, they reported that no significant difference was seen when Papacarie and indigenously prepared 10% papain gel were used after acid etching with only the acid etching group. A similar trend in the findings was seen with the scanning electron microscope imaging of the enamel surface. The study also concluded that Papacarie and 10% papain gel before acid etching increases the types I–II etching patterns. Hasija et al. [70] reported on the deproteinizing effect of papain and bromelain after acid etching. Deproteinizing agents like papain and bromelain gel were applied for 60 s and rinsed and compared with the control and sodium hypochlorite groups in primary teeth. The authors concluded that shear bond strength was not affected significantly. The bromelain group showed higher mean shear bond strength than the other groups; however, the difference was not statistically significant. Dayem and Tameesh [71] evaluated the deproteinizing effect of bromelain enzyme and compared it with laser (Nd:YAG) and 10% sodium hypochlorite using Scanning Electron Microscope and polarized microscope. Bromelain was applied for 60 s after acid etching and was rinsed with distilled water. Bromelain enzyme removed collagen network and significantly reduced the global leakage scores. Chauhan et al. [72] assess the deproteinizing effect of bromelain enzyme on shear bond strength before the application of the adhesive system. Teeth were etched with 37% phosphoric acid for 15 s, rinsed with water, blot dried, and deproteinized with bromelain enzyme. The bromelain enzyme-treated group showed the highest bond strength compared with the control group. Zakarea et al. [73] evaluated the removal of a smear layer by a combination of castor and papain in the root canals. They found that partial removal of both organic and inorganic parts of the smear layer was seen at the middle, apical, and coronal parts of the root. However, the apical was significantly less clean than the middle and coronal parts. To conclude, studies conducted on the deproteinizing effect of proteolytic enzymes were in vitro studies and may not represent the complex clinical failure mechanism involved in real-life situations. Hence, the results have to be interpreted with caution and have to be Proteolytic Enzymes in Dental Care 163 correlated with clinical performance. Further, clinical trials are recommended to evaluate the bonding failure with the use of these techniques. 11.8 Role on Biofilm Control (Plaque, Gingivitis, and Oral Malodor) Dental plaque is a biofilm on the tooth surface, which is one of the chief causal agents responsible for two of the most common diseases (dental caries and gingivitis). Yao et al. [74] studied the effect of various pH values, ionic strength, and temperature on papain hydrolysis of salivary film. The authors concluded that the key aspects in the hydrolysis of salivary film were the hydrophobic and electrostatic interactions and hydrogen bonding. Further, the actions can be enhanced by modifying the pH, ionic strength, and temperature. Tadikonda et al. evaluated the anti-plaque and anti-gingivitis effect of papain-, bromelain-, miswak-, and neem-containing dentifrice among orthodontic patients. It was reported that the mean plaque and gingivitis were lower in papain- and bromelain-containing dentifrice than in the control group after adjusting for baseline values. Mugita et al. [75] conducted a double-blind, crossover, placebo-controlled study to evaluate the role of actinidin (cysteine protease derived from the kiwi fruit) on tongue coating removal. There was a higher reduction in the tongue coating for subjects taking the test tablets (actinidin) compared those taking placebo in both young and elderly subjects. Significant difference in the tongue coating was observed only in the elderly, but not in the young adults, with respect to baseline. Mugita et al. evaluated the in vitro effects of proteases (papain, actinidin, trypsin) on oral bacterial biofilm detachment on the tongue (monospecies biofilm). Papain reduced Actinomyces biofilms completely at 10 mg/mL after an incubation of 60 s and at 1 mg/mL after an incubation of 30 min. Dose-dependent removal of biofilm was seen with actinidin similar to papain. Similarly, in a later investigation on multispecies biofilm, actinidin, papain, and trypsin fully disrupted multispecies biofilms at a high concentration at the end of 60 min. These enzymes even inhibited the formation of the multispecies biofilms. Nohno et al. [76] evaluated the long-term use of candy tablets containing protease, actinidin tongue coating, and volatile sulfur compounds (double-blind randomized crossover trial). Measurements of volatile sulfur compounds significantly decreased only in the test group at the end of 7 days. However, no such difference was seen with respect to tongue coating. Yoshimatsu et al. [77] investigated the effect actinidin on the reduction of human tongue coating (crossover studies and double blind experiments) and observed that protease tablets were effective in biochemical cleaning of the tongue. Later [78], they investigated the effects of the same on the concentrations of volatile sulfur compounds in human mouth air. There was significant reduction in the concentrations of hydrogen sulfide, methylmercaptan, and total volatile sulfur compounds with the use of protease tablets at 90 min after administration, while no change in volatile sulfur compounds was seen in the placebo group. Sugimoto et al. [79] evaluated the surface roughness and actinidin enzyme tablet on the reduction of tongue coating (number of bacteria). The number of bacteria on the tongue was significantly reduced in the enzyme compared to the non-enzyme group. However, no difference was seen when the roughness of the tablet was taken into consideration. 164 Natural Oral Care in Dental Therapy Clinical studies conducted so far have evaluated the effectiveness of plaque, gingivitis, halitosis, and tongue coating. They showed effectiveness of these proteolytic enzymes and can be suitable alternatives in maintaining oral health. Nevertheless, further clinical trials are needed in diverse populations to gather more evidence on the effectiveness and also to monitor the adverse effects related to the use of these products. 11.9 Extrinsic Stain Removal on the Teeth Stain removal of toothpaste is mainly related to the abrasive content of the toothpaste. The mechanism of action of whitening toothpastes is by disrupting or removing the protein portion of the pellicle/plaque layer on the tooth surface. Kalyana et al. conducted an in vitro study to evaluate the stain removal efficacy of papain- and bromelain-containing toothpaste [80]. The mean lightness value after the brushing regimen was significantly higher for papain and bromelain toothpaste than the control group. Lyon Jr et al. [81] also evaluated Citroxain (mixture of papaya, alumina, and sodium citrate)-containing toothpaste and found similar results. Similarly, Munchow et al. [82] evaluated bromelain- and papain-based gels and found that they are effective in bleaching stained dental enamel. However, efficacy was lower than that of the carbamide peroxide group. Yao et al. [83] evaluated the potential of cysteine proteases (papain, stem bromelain, and ficin) on protein pigment removal. They concluded that effective elimination of theoflavin bound to dephosphorylated bovine β-casein was by hydrolyzing target substrates and preventing theoflavin readsorption after hydrolysis. Vejai Vekaash et al. [84], in an in vitro study, evaluated different concentrations of hydrogen peroxide-containing pineapple extract as an additive. Specimens bleached with pineapple extracts along with different concentrations of hydrogen peroxide (10%, 20%, and 30%) showed statistically significant whitening when compared to the specimens that were bleached only with hydrogen peroxide (10%, 20%, and 30%). Also, a 20-min protocol showed significantly higher color change than a 10-min protocol at all concentrations of hydrogen peroxide with pineapple extracts. However, no significant difference was seen among 10%, 20%, and 30% hydrogen peroxide with pineapple groups with respect to the color change. Chakravarthy and Acharya [85] evaluated the clinical stain removal efficacy of papain and bromelain toothpaste. They showed that papain and bromelain dentifrice showed significant stain removal when compared to the control and attributed this to papain and bromelain. Patil et al. [86] conducted a randomized, triple blind, parallel group study to evaluate the effectiveness of abrasive and enzymatic (papain and bromelain) whitening toothpastes. They showed that both the toothpastes were effective in the removal of extrinsic stains, and papain- and bromelain-based toothpaste showed better results compared to abrasive toothpaste. The summary of in vitro and clinical studies suggests the effectiveness of these proteolytic enzyme-based dentifrices in stain removal. These products can be suitable alternatives without having secondary complications seen with the use of conventional teeth whitening products. More studies are needed to reinforce the evidence on this effectiveness and also on the effectiveness in preventing the stain formation. Proteolytic Enzymes in Dental Care 165 11.10 Role in Replantation of the Avulsed Tooth Santos [87] studied the repair process in teeth of rats by delayed replantation after root surface treatment with the papain and sodium fluoride. Teeth were immersed in 50% papain solution for 20 min followed by immersion in 2% acidulated phosphate fluoride for 20 min. The results showed that this protocol can be a viable alternate option, but research is lacking in this area. 11.11 Effect of Bromelain on Immunogenicity Bromelain can be a potential alternative and adjuvant in the treatment of many chronic conditions [32]. Many in vitro studies showed the ability of bromelain to modulate surface adhesion molecules on various cells (T cells, macrophages, natural killer cells) and also induce the secretion of many mediators of biologic response by peripheral blood mononuclear cells [32]. Mansfield et al. [88] evaluated 500 allergy clinic patients who were prick skin tested with papain and many common aeroallergens. Among the seasonal allergic disease patients (n = 475), only five had positive skin tests to both papain and local pollens. Among the individuals with negative skin test (n = 25) to pollens, none had skin reactivity to papain. It was seen that all the papain-sensitive individuals have papain-specific IgE, but none in the controls. A total of 1.05% of allergic patients had papain sensitivity and also had cross-reacting antibodies with chymopapain. Literature reports that bromelain has very low toxicity. However, IgE-mediated hypersensitivity reactions cannot be ignored. 11.12 Other Possible Applications and Scope for Future Research Considering the broad scope and effectiveness of these plant-based proteolytic enzymes, further scope for research and development can be sought in areas like denture plaque removal and hygiene maintenance, inhibition of calculus formation, development of oral rinses, maintenance of toothbrush hygiene, management of dry socket after extraction, osteoradionecrosis, and management of oral submucous fibrosis. Future research should also address the possible adverse events, cost effectiveness, patient reported outcomes, and oral health-related quality of life in view of the current benefits. References 1. Jain, N., Rajwar, Y.C., Batra, M., Singh, H.P., Agarwal P., Dentistry: Turning towards Herbal Alternatives: A Review. Sch. J. App. Med. 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Rev, H. Worthington (Ed.), p. CD000978, John Wiley & Sons, Ltd, Chichester, UK, 2006. 31. Brien, S., Lewith, G., Walker, A., Hicks, S.M., Middleton, D., Bromelain as a Treatment for Osteoarthritis: A Review of Clinical Studies. Evid. Based Complement. Alternat. Med., 1, 251– 257, 2004. 32. Pavan, R., Jain, S., Shraddha, and Kumar, A., Properties and therapeutic application of bromelain: A review. Biotechnol. Res. Int., 2012, 976203, 2012. 33. Jayachandran, S. and Khobre, P., Efficacy of Bromelain along with Trypsin, Rutoside Trihydrate Enzymes and Diclofenac Sodium Combination Therapy for the treatment of TMJ Osteoarthritis—A Randomised Clinical Trial. J. Clin. Diagn. Res., 11, ZC09–ZC11, 2017. 34. Silva, J., Gaspar, C., Martinez-De-Oliveira, J., Palmeira-De-Oliveira, R., Palmeira-De-Oliveira, A., Antifungal activity of bromelain in Candida pre-formed biofilms, n.d. 35. Brakebusch, M., Wintergerst, U., Petropoulou, T. et al., Bromelain is an accelerator of phagocytosis, respiratory burst and Killing of Candida albicans by human granulocytes and monocytes. Eur. J. Med. Res., 6, 193–200, 2001. 36. Bhardwaj, A., Ballal, S., Velmurugan, N., Comparative evaluation of the antimicrobial activity of natural extracts of Morinda citrifolia, papain and aloe vera (all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against Enterococcus faecalis: An in vitro study. J. Conserv. Dent., 15, 293–7, 2012. 37. Basting, R.T., Gonçalves, F.R., França, F.M.G., do Amaral, F.L.B., Flório, F.M., Antimicrobial Potential of Papain Chemomechanical Agent on Streptococcus Mutans and Lactobacillus Casei Followed by the Use of Self-Etching Adhesive Systems. J. Clin. Pediatr. Dent., 40, 62–68, 2016. 38. Segura, E.P., Méndez, L., Márquez, E. et al., Effect of Carya illinoinensis, Quercus rubra and Smilax glyciphylla extracts, pectin, and papain on the dental biofilm microorganisms [Efecto de extractos de Carya illinoinensis, Quercus rubra y Smilax glyciphylla, pectina y papaina sobre los microorganis]. J. Pharm. Pharmacogn. Res., 3, 118–129, 2015. 39. Mota Vde, S., Turrini, R.N.T., Poveda Vde, B., Antimicrobial activity of Eucalyptus globulus oil, xylitol and papain: A pilot study. Rev. Esc. Enferm. USP, 49, 0216–0220, 2015. 40. Praveen, N.C., Rajesh, A., Madan, M. et al., In vitro Evaluation of Antibacterial Efficacy of Pineapple Extract (Bromelain) on Periodontal Pathogens. J. Int. Oral Health, 6, 96–8, 2014. 41. Kulkarni, G., Rane, D.C., V., M., Comparison of the Efficacy of Chemomechanical Caries Removal (Papacarie—A Papain Gel) and Conventional Excavation in Reducing Cariogenic Flora: An. In Vivo Study. J. Int. Oral Health, 8, 564–568, 2016. 168 Natural Oral Care in Dental Therapy 42. Motta, L.J., Bussadori, S.K., Campanelli, A.P. et al., Efficacy of Papacarie( ) in reduction of residual bacteria in deciduous teeth: A randomized, controlled clinical trial. Clinics (Sao Paulo), 69, 319–22, 2014. 43. Fusayama, T., Two layers of carious dentin; diagnosis and treatment. Oper. Dent., 4, 63–70, 1979. 44. Murdoch-Kinch, C.A. and McLean, M.E., Minimally invasive dentistry. J. Am. Dent. Assoc., 134, 87–95, 2003. 45. Beeley, J.A., Yip, H.K., Stevenson, A.G., Chemochemical caries removal: A review of the techniques and latest developments. Br. Dent. J., 188, 427–30, 2000. 46. Kavvadia, K., Karagianni, V., Polychronopoulou, A., Papagiannouli, L., Primary teeth caries removal using the Carisolv chemomechanical method: A clinical trial. Pediatr. Dent., 26, 23–8, 2004. 47. Ericson, D., Zimmerman, M., Raber, H. et al., Clinical Evaluation of Efficacy and Safety of a New Method for Chemo–Mechanical Removal of Caries. Caries Res., 33, 171–177, 1999. 48. Ansari, Beeley, J.A., Fung, D.E., Chemomechanical caries removal in primary teeth in a group of anxious children. J. Oral Rehabil., 30, 773–9, 2003. 49. Chittem, J., Sajjan, G.S., Varma, K.M., Comparative evaluation of microshear bond strength of the caries-affected dentinal surface treated with conventional method and chemomechanical method (papain). J. Conserv. Dent., 18, 369–73, 2015. 50. Sapna, K. and Pallavi, O, S, R., Efficacy of Papacarie for caries removal: An in vivo study. World J. Dent., 2, 183–6, 2011. 51. Mohamed Aly Khattab, N. and Omar, O.M., Papain-based gel for chemo-mechanical caries removal: Influence on microleakage and microshear bond strength of esthetic restorative materials. J. Am. Sci., 8, 391–399, 2012. 52. Hafez, M.A., Elkateb, M., El Shabrawy, S., Mahmoud, A., El Meligy, O., Microleakage Evaluation of Composite Restorations Following Papain-Based Chemo-Mechanical Caries Removal in Primary Teeth. J. Clin. Pediatr. Dent., 41, 53–61, 2017. 53. AlHumaid, J., Al-Harbi, F., El Tantawi, M., Elembaby, A., X-ray microtomography assessment of Carisolv and Papacarie effect on dentin mineral density and amount of removed tissue. Acta Odontol. Scand., 76, 236–240, 2018. 54. Nair, S., Nadig, R.R., Pai, V.S., Gowda, Y., Effect of a Papain-based Chemomechanical Agent on Structure of Dentin and Bond Strength: An in vitro Study. Int. J. Clin. Pediatr. Dent., 11, 161–166, 2018. 55. Somani, R., Jaidka, S., Jawa, D., Mishra, S., Comparative evaluation of smear layer removal by various chemomechanical caries removal agents: An in vitro SEM study. J. Indian Soc. Pedod. Prev. Dent., 33, 204–7, 2015. 56. Viral, P.M., Nagarathna, C., Shakuntala, B.S., Chemomechanical caries removal in primary molars: Evaluation of marginal leakage and shear bond strength in bonded restorations—An in vitro study. J. Clin. Pediatr. Dent., 37, 269–74, 2013. 57. Bittencourt, S.T., Pereira, J.R., Rosa, A.W. et al., Mineral content removal after Papacarie application in primary teeth: A quantitative analysis. J. Clin. Pediatr. Dent., 34, 229–31, 2010. 58. Kotb, R., Elkateb, M., Ahmed, A., Kawana, K., El Meligy, O., Dentin Topographic Features following Chemomechanical Caries Removal in Primary Teeth. J. Clin. Pediatr. Dent., 40, 472– 479, 2016. 59. Kitsahawong, K., Seminario, A.L., Pungchanchaikul, P., Rattanacharoenthum, A., Pitiphat, W., Chemomechanical versus drilling methods for caries removal: An in vitro study. Braz. Oral Res., 29, 1–8, 2015. 60. Carrillo, C.M., Tanaka, M.H., Cesar, M.F., Camargo, M.A., Juliano, Y., Novo, N.F., Use of papain gel in disabled patients. J. Dent. Child Chic. III, 75, 222–8, 2008. Proteolytic Enzymes in Dental Care 169 61. Abdul Khalek, A., Elkateb, M., Abdel Aziz, W., El Tantawi, M., Effect of Papacarie and Alternative Restorative Treatment on Pain Reaction during Caries Removal among Children: A Randomized Controlled Clinical Trial. J. Clin. Pediatr. Dent., 41, 219–224, 2017. 62. Kotb, R.M.S., Abdella, A.A., El Kateb, M.A., Ahmed, A.M., Clinical evaluation of Papacarie in primary teeth. J. Clin. Pediatr. Dent., 34, 117–23, 2009. 63. Alfaya, T., Cardoso Guedes, C., Fernandes, K.P. et al., Assessment of chemomechanical removal of carious lesions using Papacarie Duo : Randomized longitudinal clinical trial. Indian J. Dent. Res., 24, 488–92, 2013. 64. Motta, L.J., Bussadori, S.K., Campanelli, A.P. et al., Randomized controlled clinical trial of longterm chemo-mechanical caries removal using Papacarie gel. J. Appl. Oral Sci., 22, 307–13, 2014. 65. Deng, Y., Feng, G., Hu, B., Kuang, Y., Song, J., Effects of Papacarie on children with dental caries in primary teeth: A systematic review and meta-analysis. Int. J. Paediatr. Dent., 28, 361–372, 2018. 66. Pithon, M.M., de Souza Ferraz, C., do Couto de Oliveira, G. et al., Effect of 10% papain gel on enamel deproteinization before bonding procedure. Angle Orthod., 82, 541–545, 2012. 67. Pithon, M.M., Ferraz, C.S., Oliveira, G.D.C., Dos Santos, A.M., Effect of different concentrations of papain gel on orthodontic bracket bonding. Prog. Orthod., 14, 22, 2013. 68. Pithon, M.M., Campos, M.S., Coqueiro, R., da S., Effect of bromelain and papain gel on enamel deproteinisation before orthodontic bracket bonding. Aust. Orthod. J., 32, 23–30, 2016. 69. Agarwal, R., Yeluri, R., Singh, C., Munshi, A., Enamel Deproteinization using Papacarie and 10% Papain Gel on Shear Bond Strength of Orthodontic Brackets Before and After Acid Etching. J. Clin. Pediatr. Dent., 39, 348–357, 2015. 70. Hasija, P., Sachdev, V., Mathur, S., Rath, R., Deproteinizing Agents as an Effective Enamel Bond Enhancer—An in Vitro Study. J. Clin. Pediatr. Dent., 41, 280–283, 2017. 71. Dayem, R.N. and Tameesh, M.A., A new concept in hybridization: Bromelain enzyme for deproteinizing dentin before application of adhesive system. Contemp. Clin. Dent., 4, 421–6, 2013. 72. Chauhan, K., Shivanna, V., Basavanna, R., Effect of bromelain enzyme for dentin deproteinization on bond strength of adhesive system. J. Conserv. Dent., 18, 360, 2015. 73. Zakarea, N.A.A., Mohamad, T.H., Taqa, A.A., Chumbley, S., Al-Juaid, S., Baho, H., A Newly Prepared Solution for the Removal of the Smear Layer. Int. J. Dent. Sci. Res., 2, 19–26, 2014. 74. Yao, J.-W., Xiao, Y., Lin, F., Effect of various pH values, ionic strength, and temperature on papain hydrolysis of salivary film. Eur. J. Oral Sci., 120, 140–146, 2012. 75. Mugita, N., Nambu, T., Takahashi, K., Wang, P.-L., Komasa, Y., Proteases, actinidin, papain and trypsin reduce oral biofilm on the tongue in elderly subjects and in vitro. Arch. Oral Biol., 82, 233–240, 2017. 76. Nohno, K., Yamaga, T., Kaneko, N., Miyazaki, H., Tablets containing a cysteine protease, actinidine, reduce oral malodor: A crossover study. J. Breath Res., 6, 017107, 2012. 77. Yoshimatsu, D., Sugimura, S., Ioka, T. et al., P20 Effect of protease tablet on reduction of tongue coating. Oral Dis., 11, 112–112, 2005. 78. Yoshimatsu, D., Sugimura, S., Ioka, T. et al., Reduction of Volatile Sulfur Compounds in Mouth Air by Protease Tablet. J Dent Health, 57, 22–27, 2007. 79. Sugimoto, J., Takahashi, K., Komasa, Y., Effect of a protease-containing tablet with rough surface on the number of bacteria on the tongue. J. Osaka Dent. Univ., 49, 165–170, 2015. 80. Kalyana, P., Shashidhar, A., Meghashyam, B., Sreevidya, K.R., Sweta, S., Stain removal efficacy of a novel dentifrice containing papain and Bromelain extracts—An in vitro study. Int. J. Dent. Hyg., 9, 229–33, 2011. 81. Lyon, T.C., Parker, W.A., Barnes, G.P., Evaluation of effects of application of a citroxaincontaining dentifrice. J. Esthet. Dent., 3, 51–3, 1991. 170 Natural Oral Care in Dental Therapy 82. Münchow, E.A., Hamann, H.J., Carvajal, M.T., Pinal, R., Bottino, M.C., Stain removal effect of novel papain- and bromelain-containing gels applied to enamel. Clin. Oral Investig., 20, 2315– 2320, 2016. 83. Yao, J.-W., Xiao, Y., Zuo, Q. et al., Effectiveness of cysteine proteases on protein/pigment film removal. Arch. Oral Biol., 58, 1618–1626, 2013. 84. Vejai Vekaash, C.J., Kumar Reddy, T.V., Venkatesh, K.V., Effect of vital bleaching with solutions containing different concentrations of hydrogen peroxide and pineapple extract as an additive on human enamel using reflectance spectrophotometer: An in vitro study. J. Conserv. Dent., 20, 337–340, 2017. 85. Chakravarthy, P.K. and Acharya, S., Efficacy of extrinsic stain removal by novel dentifrice containing papain and bromelain extracts. J. Young Pharm., 4, 245–249, 2012. 86. Patil, P., Ankola, A., Hebbal, M., Patil, A., Comparison of effectiveness of abrasive and enzymatic action of whitening toothpastes in removal of extrinsic stains—A clinical trial. Int. J. Dent. Hyg., 13, 25–29, 2015. 87. Santos, C.L.V. and dos [UNESP], Efeito da solução de papaína na remoção do ligamento periodontal desvitalizado: estudo histomorfométrico em dentes de ratos. Aleph, 81 f.: il. + 1 CD-ROM, 2010. 88. Mansfield, L.E., Ting, S., Haverly, R.W., Yoo, T.J., The incidence and clinical implications of hypersensitivity to papain in an allergic population, confirmed by blinded oral challenge. Ann. Allergy, 55, 541–3, 1985. 12 The Effect of Probiotics on Oral Health Patricia Nadelman1, Marcela Baraúna Magno1, Mariana Farias da Cruz1, Adriano Gomes da Cruz2, Matheus Melo Pithon3, Andréa Fonseca-Gonçalves1 and Lucianne Cople Maia1* 1 Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 2 Department of Food, Federal Institute of Education Science and Technology of Rio de Janeiro, Rio de Janeiro, Brazil 3 Department of Health, School of Dentistry, Universidade Estadual do Sudoeste da Bahia (UESB), Bahia, Brazil Abstract The oral microbiome is a natural community of microorganisms that reside in different areas. Behavioral aspects of the host can impact this microbiome, destabilize it, and develop disorders. This last situation is called dysbiosis. Most prevalent oral dysbiosis biofilm-related include caries, gingivitis, periodontal and peri-implant diseases, and candidiasis. Additionally, halitosis is another oral condition related to a bacterial instability manifested in the tongue or other mouth sites. Hence, probiotic bacteria have been used to reestablish the microbiotas’ equilibrium. Concerning the oral environment, the common probiotic strains used are from Lactobacillus and Bifidobacterium genera, which can be administrated in different presentation forms (lozenge, tablets, powders, gums) and vehicles, including beverages (milks, yogurts, juices) and foods (cheese, kefir, ice cream, and chocolates bars). The use of these products with probiotics demonstrated optimistic results as an auxiliary method on the prevention and treatment of oral dysbiosis due to their direct and indirect effects against the pathogenic oral microorganisms growth and immunomodulation. In this sense, the present chapter aims to present an updated viewpoint of the probiotic effects on oral health, describing the relationship between the administration/consumption of these bacteria and the main oral dysbiosis, the oral microbiota parameters, and the immune salivary components. Keywords: Probiotics, dental caries, gingivitis, periodontitis, candidiasis, halitosis, oral health, dentistry 12.1 Introduction Probiotics are defined as microorganisms safe for human consumption that when ingested in sufficient quantities, result in beneficial effects on individual health [1]. These advantages *Corresponding author: rorefa@terra.com.br; maia_lc@ufrj.br Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (171–196) © 2020 Scrivener Publishing LLC 171 172 Natural Oral Care in Dental Therapy come from their capacity of inactivate toxins and their receptors, to provide bioactive or regulatory metabolites, produce bacteriocins that inhibit pathogenic bacteria, modify the environment, and compete with other microorganisms for adhesion sites and/or nutrients, preventing colonization by other microorganisms [2–7]. Thus, probiotics can inhibit pathogens and influence the host’s adaptive immune response [8, 9]. In this sense, new approaches to prevent and control oral diseases through the probiotics inhibitory effect on the progression of pathogens have been developed [6]. This could be a supporting strategy since the oral cavity has a large microflora, including microorganisms that cause disorders such as dental caries, gingivitis, periodontitis, candidiasis, and halitosis [10]. Moreover, saliva has immunological components that can be modulated by probiotics [11, 12]. Considering that differences between the intrinsic quality of saliva and individual oral environment vary based on eating habits, probiotics have been used for prophylactic and therapeutic goals [13–20]. There are several vehicles for probiotic administration including capsules, lozenges, chewing gum, powder, drinks such as juice, tea, and kombucha, and foods such as cheese, ice cream, and kefir [21]. The probiotics most frequently used for the oral environment are from the genera Lactobacillus and Bifidobacterium [22]. Considering the controversies about the effect of probiotics on oral health, discussing the issue is extremely necessary for finding scientific evidence about the subject. In this regard, the present chapter aims to update and discuss the scientific evidence available in the literature about the probiotic effects on oral health, describing the relationship between the administration/consumption of these bacteria and the main oral dysbiosis in the oral cavity, and the oral microbiota parameters. 12.2 Overview of Oral Communities and Probiotic-Based Therapy to Oral Dysbiosis The human microbiome is described as a natural community of different types of microorganisms, resident of the different compartments of the human body, together with its gene set [23]. Microbial communities of the body’s endogenous system participate in several essential functions for human health. Thus, changes in the composition of the microbiome could significantly affect the normal functions of the body. So, the dynamic microbiome– host relationship should be considered jointly for health maintenance and disease prevention. The highest microbial diversity is observed in the gastrointestinal tract and in the oral cavity [23]. Bacteria, fungi, archaea, and viruses are the microbial species that form the oral microbiome. They coexist in structured arrangements in the sites of the mouth [24]. Even the pathogenic microorganisms are also found in healthy individuals, however, in lower levels [25]. The behavioral aspects of the host can increase the number of pathogens, influencing this microbiome, unbalancing it, and developing disorders [24]. This imbalance is called dysbiosis, which orally can be characterized as a manifestation of pathogens that can grow the biofilm-dependent disorders [26]. Probiotic and Oral Health 173 This oral biofilm, in its turn, is described as a varied microscopic community located on the oral surfaces, surrounded in a matrix of polymers originating from microbial (Figure 12.1) and host [27], and oral microorganisms always form dental biofilm on tooth [28]. If, on one hand, this microbial organization can be found even without the disease [29], on the other hand, its accumulation may be accompanied by a change in bacterial composition, and it is the main cause for the initiation and progression of most oral dysbiosis, such as dental caries, gingivitis, periodontitis, and candidiasis [29, 30]. Regarding oral dysbiosis, oral candidiasis is an infection caused by commensal and opportunistic fungi belonging to the genus Candida. The species most commonly associated with this condition is Candida albicans—a natural inhabitant of the oral cavity [31]. Similarly to oral candidiasis, gingivitis and periodontitis are also caused by a group of (a) (b) (c) Figure 12.1 Scanning Electron Microscopy images of oral biofilm. (a) Cariogenic biofilm formed by S. mutans, S. paranguinis, S. salivarius. (b and c) Cariogenic biofilm + probiotic strains. 174 Natural Oral Care in Dental Therapy bacteria normally present in the oral biofilm [32]. However, in the periodontal diseases, the major pathogens are Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia [33]. However, only the presence of the microorganisms is not enough to cause these dysbioses, since they require conditions that compromise the host’s immune response, resulting in an imbalance of the biofilm’s microorganisms [34, 35]. Another example of an oral dysbiosis is dental caries, which is defined as a biofilm– sugar–dependent disease [36]. Regarding this disorder, it is known that this microbial imbalance has a predominance of gram-positive acidogenic and aciduric bacteria [37]. Similar to other diseases, biofilm accumulation is not enough for the progression of carious lesions. In this case, sugars are the fundamental negative factors for their development [38]. After the intake of sugar, sucrose-dependent microorganisms ferment the available substrate, generate acids, and, subsequently, decrease the biofilm pH [36]. This acidity not only causes dental demineralization but also selects the more cariogenic bacteria in the biofilm [39]. This association between the microbial communities and the oral cavity has been a subject of growing interest in science in order to develop therapies that can reestablish the healthy microbiota in individuals with those disorders. Therefore, new methods for preventing and restoring the balance of microbiota from the use of other microorganisms have been developed. Among these strategies using living cells as preventive and therapeutic agents, we highlight the use of probiotic bacteria [24]. Their effects are related to the ability to prevent the colonization by other microorganisms through competition with pathogens, reducing their adhesion to the oral surfaces, antagonizing its effects, inactivating biofilm microbial toxins, or modulating the host’s immune response [40]. As a result of the successful use of probiotics in the control of gastrointestinal tract balance [41, 42], these microorganisms have been tested in experiments in the oral cavity. In order to perform a beneficial effect in the mouth, the probiotic bacteria must have the ability of adhesion to mucosal or oral epithelial cells, becoming part of the biofilm [43]. In this sense, the ingestion of probiotics could be one of the possible strategies for the prevention and therapy of biofilm-dependent diseases [40], since they could interact with oral microorganisms [44] that cause dental caries, gingivitis, periodontitis, and candidiasis [10]. Among the probiotic strains, species from the genera Lactobacillus and Bifidobacterium spp. are the most recommended for human use due to its capacity of adhesion and colonization in the surfaces of the oral cavity [45]. The species can be used in association with other probiotic microorganisms [46] or as the only present probiotic microorganism in the vehicle [47]. Because of these positive effects, these bacteria have been supplemented in several foods, drinks, lozenges, and tablets [48], which are considered vehicles for their administration [49]. There are a wide variety of delivery vehicles including dairy products (milk, yogurt, curd, cheese, ice cream, kefir), food supplement, juices, tea, kombucha, chewing gum, powder, mouth-rinse, tablets, capsules, and lozenges [50]. Although they have potential benefits, there are still doubts and controversies about the exact mechanism of action and effect of these probiotic bacteria on the oral microorganisms and their main dysbiosis [51]. Probiotic and Oral Health 175 12.3 Probiotics Mechanisms of Action The probiotic mechanism of action is still poorly understood, but several studies have demonstrated results of possible probiotic modes of action [52]. According to Collins & Gibson [53], an effective probiotic product should be free of pathogenicity and toxicity, must contain an adequate number of viable cells, remain viable during storage, be detected in the host, and provoke beneficial effect. However, the mechanisms of action of probiotics could be resumed in three forms, proposed by Oelschlaege [54] and Busanello [55]: (i) antimicrobial action of products resulted from microbial metabolism, such as toxins, metabolites, and enzymes; (ii) competitive exclusion by nutrients and colonization sites; and (iii) immunomodulation of the innate and adaptive host immune system (Table 12.1). (i) Antimicrobial action of products resulted from microbial metabolism Some probiotics have the ability to suppress the growth of pathogenic bacteria producing bacteriocins that have broad spectrum [52]. According to Petri [56], probiotic bacteria can release organic acid compounds such as lactic acid, propionic acid, and hydrogen peroxide, which have antibacterial effects against pathogenic microorganisms—it creates a hostile environment for the pathogens. The author also reports that besides the biological effect, probiotics have also a chemical effect, since the acid’s production reduces the environment pH, interfering in the metabolism of pathogenic bacteria. In the oral microbiota, the pH reduction and the changes in the proteic composition of the salivary film transform the ecosystem, making adhesion difficult and reducing the number of pathogens in the formation of dental biofilm [2]. Table 12.1 Possible mechanisms of action of probiotics. Mechanism of action/effect Description (i) Antimicrobial action of products resulted from microbial metabolism (direct effect against bacteria) • Release of organic and inorganic acid compounds with consequent reduction of the medium pH • Creation of a hostile environment for the pathogenic bacteria and action on its cellular metabolism • Inactivation of biofilm microbial toxins and their receptors (ii) Competitive exclusion by nutrients and fixation sites (indirect effect against bacteria) Competition with pathogens for nutrients and for adhesion through obliteration to the cell fixation sites of the host, forming a physical barrier and preventing the pathogens adhesion, besides the release of mucous secretion (iii) Immunomodulation of the innate and adaptive immune system of the host Products of cellular metabolism generate immunomodulation or immunoregulation through alteration of cytokine and chemokines profiles, and activation of immune system cells 176 Natural Oral Care in Dental Therapy (ii) Competitive exclusion by nutrients and fixation sites Pathogenic microorganisms challenge the symbiotic relationship between host and normal flora. However, for the pathogenic colonization, free receptor sites—polysaccharide molecules—for its, adhesion must exist in the host cell wall [57]. When probiotic products are added to the host diet, these beneficial microorganisms adhere to the fixation sites, creating a physical barrier and decreasing the nutrients’ suppression for the pathogenic bacteria, and then probiotic bacteria can stop the pathogen colonization [55]. Another probiotic mechanism to prevent pathogen adhesion to the host is the secretion of a mucosal coverage that obliterates the binding sites [57]. In the oral microbiota, probiotics can alter the composition of the oral biofilm by colonizing microbial biofilms, co-aggregating and competing with pathogenic bacteria, subsequently substituting or reducing their numbers [5]. (iii) Immunomodulation of the innate and adaptive immune system of the host Probiotics alter the host’s immune system through two mechanisms: immunomodulation and immunoregulation [58]. Interferences of the immune system may occur through the products of the probiotic metabolism [54], which are characterized by the alteration of the cytokine, chemokine, and immune system cells involved in this process [58]. Immunostimulatory probiotics are able to interfere against cancer cells and infection, inducing the production of IL-12 that activates NK cells and develops T1 cells. Probiotic bacteria have also an effect against allergy, balancing T1 and T2 cells. On the other hand, immunoregulatory probiotics were characterized by the production of IL-10 and Treg cells that can also result in a reduction of allergies, autoimmune diseases, and inflammatory responses [58]. According to Oelschlaeger [54], the adhesion of probiotics to host epithelial cells triggers a signaling cascade that leads to immunomodulation through its contact, where an anti-inflammatory effect occurs, which is often a consequence of the IL-10 expression increase. 12.4 Dental Caries 12.4.1 Definition and Etiopathology Caries is a disorder characterized by the demineralization of the tooth surface caused after the production of acid resulting from the microbial fermentation of sugars [59]. Its etiological factors are mainly associated with the diet, hygiene, and microbiota [60]. Historically, dental caries has been defined as a multifactorial disease, depending on at least three essential factors: (i) the host, represented by the individual and his/her components such as the oral cavity, teeth, salivary flow, oral hygiene habits, etc; (ii) the microbiota, represented by the oral microorganisms; and (iii) the substrate, represented Probiotic and Oral Health 177 by the individual’s diet, characterized by frequency of consumption of fermented carbohydrates—mainly sucrose, which is the most cariogenic [61]. Nowadays, dental caries is considered a dysbiosis and can be defined as a biofilm–sugardependent disease [28] that affects both temporary and permanent teeth. In summary, the resident oral microorganisms form biofilms on teeth, fermenting the available sugars, produce acid from this fermentation and, the acidic products create localized demineralization of dental hard tissues [28]. This cariogenic biofilm is enriched by a large variety of acidogenic/aciduric microorganisms, including S. mutans, S. sobrinus, and species of Lactobacillus, Actinomyces, Atopobium, Bifidobacterium, Propionibacterium, and Scardovia [24]. The species involved in the caries process have the ability to (i) rapidly make acid from dietary carbohydrates, being considered acidogenic microorganisms; (ii) they have the capacity to tolerate low pH, being also considered aciduric microorganisms; and (iii) to have the ability to produce extracellular and intracellular polysaccharides, which leads to the adherence to dental surfaces and serve as a nutrient reserve, respectively [62]. Additionally, these cariogenic bacteria can (iv) compete successfully versus other oral microorganisms through an increase in its defenses against oxidative stress and its resistance to acid metabolites [63]. However, biofilm accumulation alone is not enough for caries lesion progression. In this process, sugars are fundamental for disease progression [38]. When sugar is ingested, oral sucrose-dependent microorganisms ferment the available substrates and produce acids. From this process, the biofilm fluid’s pH decreases to around pH 5.0 or below [36]. This acidic pH product also does a selection of the most cariogenic bacteria in the biofilm [39]. These factors contribute to the beginning and progression of dental caries, since the acidic oral environment caused by bacteria fermentation disturbs the Ca2+, (PO4)3−, and F−. The decrease in the pH and the increase in the concentration of these ions are serious problems in the loss and gain of minerals in the oral environment. The acid from sucrose fermentation binds to the Ca2+ and (PO4)3− causing dental demineralization, and consequently, a caries lesion develops [64]. Dietary fermentable sugars are known as primary factors responsible for modifications in the tooth biofilm [65, 66]. Comparing to glucose and fructose, sucrose is highlighted as the most cariogenic because microorganisms can easily ferment it into acids, and it has the ability to change the biofilm environment, making it more cariogenic [67]. Carbohydrate consumption stimulates the proliferation of S. mutans, S. sobrinus, and Lactobacillus [68, 69]. These bacteria catabolize dietary sucrose and convert it into polysaccharides. Many of the polysaccharides are hydrolyzed and subsequently used for growth during periods when there are no readily fermentable dietary carbohydrates available [70]. Clinically, the development of caries lesions in a healthy patient (Figure 12.2a) initiates with biofilm accumulation (Figure 12.2b), which is not removed and develops caries signs that may be presented in two forms: (i) the early stage characterized by active white spot lesions (non-cavitated lesions with enamel mineral loss) (Figure 12.2c) [71]; and (ii) its progression; when initial lesions are not controlled, it turns into advanced stages characterized by cavitated lesions (lesions with mineral and structural loss reaching enamel, dentin, or cementum) (Figure 12.2d and e) [72]. 178 Natural Oral Care in Dental Therapy (a) (b) (c) (d) (e) Figure 12.2 Development of caries lesion without treatment. (a) Healthy oral cavity without biofilm accumulation. (b) Oral cavity with poor hygiene and biofilm accumulation. (c) Oral cavity with early caries lesions (white spot lesions). (d) Oral cavity with cavitated caries lesions. (e) Oral cavity with dental destruction by caries lesions. Therefore, caries prevention is not a simple removal of microorganisms or an improvement in dental resistance. Prevention of complex disorders such as dental caries is directly related to biofilm control, eliminating residual food and microorganisms [36]. A physiologic equilibrium of the oral environment controls the caries development through mechanical methods of biofilm control (brushing teeth and using dental floss) and dietary monitoring (smart consumption of the sugar, decreasing its ingestion’s frequency) [67]. Despite all these scientific conceptions of patient protection against dental caries, it is known that certain individuals need more supervising than others to avoid caries lesion progression. Therefore, dental new approaches to improve oral health, aiming at inflammatory modulating effects, and reducing the amount of biofilm or microorganisms, have been developed and researched [24], and are still welcome. Probiotic and Oral Health 179 12.4.2 Probiotics and Dental Caries To limit or prevent dental caries, probiotics should be capable to compete with pathogens for nutrients and for adhesion, being part of the biofilm and forming a physical barrier to avoid the adhesion of pathogenic microorganisms [5, 55, 57]. Consequently, it prevents the pathogens’ proliferation and institutes a healthy oral colonization [33]. Additionally, some probiotics can also change the cariogenicity of S. mutans when they are in coexistence [73]. Another probiotics effect on the prevention and control of dental caries could be the antibacterial action of products resulting from microbial metabolism through bacterial coaggregation—the aggregation relationship between microorganisms of the same species or between different species, favoring their adhesion on the dental surface. Some probiotics strains were tested on an in vitro study and showed the capacity of coaggregate with oral microorganisms. Among these probiotic bacteria, L. acidophilus mostly coaggregated compared to the others [74]. Considering the crucial function of S. mutans and Lactobacillus in the caries process, several experiments have been done trying to disturb its cariogenic properties [22]. Several randomized clinical trials have been performed testing the probiotic administration to reduce cariogenic bacteria counts in saliva or plaque [75]. First of all, in vitro studies have shown the capacity of probiotics in the suppression and decrease of S. mutans, Lactobacillus, and other cariogenic bacteria [76–78]. After that, clinical reports were performed aiming to improve the evidences [75]. This anti-cariogenic effect may be associated with the probiotics capacity to create a hostile environment for the pathogenic bacteria, which specifically prevents the adherence of S. mutans [37]. Therefore, the use of probiotics with the target of conserving or renovating the biological environment against the pathogen proliferation associated with the development of the major oral dysbiosis. Several studies have already evaluated the effect of probiotics in dental caries using diverse probiotic strains: L. rhamnosus GG [79–81], L. paracasei [82], L. reuteri [83], Bifidobacterium spp., among others. The microorganisms’ effect was mainly evaluated on S. mutans and Lactobacillus count in saliva and/or plaque, pH values, and treatment of root caries lesions [40]. Researches on probiotic strains are still being carried out to recognize the strains that can perform a positive role against S. mutans and other cariogenic bacteria. Despite the fact that several studies are already demonstrating the influence of probiotic against the growth of cariogenic microorganisms, the results cannot be extrapolated because there is no standardization of probiotic strains and administration vehicles [37]. 12.4.3 Probiotic-Contained Dairy Products and Dental Caries Regarding the ingestion of dairy products as vehicles, it should be safe for uninterrupted daily consumption and suitable for all ages [84, 85]. Dairy products are interesting since they are not only nutritious foods but because of their natural content of proteins, fats, vitamins, and minerals (casein, calcium, and phosphate) [86]. The use of probiotics in dairy products could be considered an auxiliary method to control oral diseases [87, 88], since these bacteria can neutralize the acid conditions and interfere in the pathogenic species [33]. Therefore, studies have been carried out testing the oral effect of probiotics contained in dairy products [89]. It has already been reported that cheese prevents enamel mineral loss 180 Natural Oral Care in Dental Therapy and also helps in its remineralization [90]. Either probiotic ice cream has demonstrated a positive effect in reducing significantly the levels of S. mutans compared to control products [85, 91]. Additionally, yogurt is considered as a potential carrier of probiotics and has shown that it can impact the potential and colonization of the oral biofilm [74] by demonstrating good effects on reducing the S. mutans count [92, 93], as well as kefir drink [94] and curd [95] containing probiotics, which also exhibited good results in reduction of salivary bacteria counts and thus can be exploited for the prevention of enamel demineralization as a longterm resource keeping in mind its cost-effectiveness [95]. Regarding plain milk containing probiotics, studies reveal promising results aiming at the prevention, control, and treatment of dental caries. Milk containing L. rhamnosus has reduced statistically significantly the caries risk reducing S. mutans counts [79, 96], either clinically reversed caries lesions [80], and has also prevented the development of new lesions [81, 97]. A randomized controlled trial, published on the literature [98] examined the effect of probiotic milk intake on concentrations of S. mutans or progression of caries. It concluded that the milk powder containing L. paracasei SD1 was able to decrease S. mutans numbers and delayed new caries development, suggesting that probiotics might be a way for caries prevention. Finally, a recent systematic review and meta-analysis were conducted aiming to answer if dairy products containing probiotics have favorable outcomes on the oral health and salivary parameters presented that probiotics could decrease S. mutans counts [89]. These result concords with other systematic reviews [50, 51, 75], which also showed a reduction in the number of this cariogenic bacteria after the use of probiotics. It suggests that dairy products with probiotics might be a supporting form for prevention and treatment of dental caries. 12.4.4 Probiotic Powder and Dental Caries Beyond the addition of probiotics to the above food and drinks, probiotics can also be found in powder presentation. The product should be diluted in water and then administered orally. The consumption of a dried powdered combination of L. rhamnosus and Bifidobacterium species and the ingestion of freeze-dried powdered preparation of Bacillus coagulans have demonstrated positive results on the reduction of S. mutans counts [99]. 12.4.5 Probiotic Tablets and Lozenges and Dental Caries Vehicles such as lozenges and tablets can also deliver probiotics. The lozenge is an easy and acceptable vehicle for probiotic administration, it can be used safely in children or adults and, mainly, as it is dissolved inside the oral cavity, there is a probiotic’s substantivity of probiotics in the mouth [100]. Regarding the use of tablets, Çaglar et al. [101] examined if a short-term consumption of probiotic tablets containing L. reuteri could influence the numbers of S. mutans and Lactobacillus. The results suggest that the intake of probiotic tablets might reduce the levels of S. mutans. On the other hand, Keller et al. [102] investigated if tablets containing Probiotic and Oral Health 181 L. reuteri could avoid the regrowth of S. mutans after disinfection with chlorhexidine, concluding that the ingestion of these tablets did not alter or delay the bacteria regrowth. Concerning the use of lozenges, Burton et al. [103] compared the influence on active caries indices after the use of lozenges containing S. salivarius M18. The participants were assessed for changes to their numbers of S. salivarius, S. mutans, Lactobacillus, and B-hemolytic streptococci. This study demonstrated that the administration of probiotic lozenges did not show a diminution in S. mutans counts. 12.4.6 Probiotic Mouthwashes and Dental Caries As the proposed mechanisms of action of the probiotics appear to occur locally in the oral cavity through a biofilm alteration, mouthwashes could be interesting vehicles for the probiotic administration their ability to remain in the oral cavity for hours [104]. Moreover, a single-blind randomized control study [105] test the effectiveness of the use of probiotic mouthwash compared to chlorhexidine and fluoride mouthwashes on plaque S. mutans level. The study concluded that all the mouthwashes were able to reduce plaque S. mutans counts. Thus, the authors suggested that probiotic mouthwash could be effective as an auxiliary method for oral hygiene regimen. Accordingly, the mouthwash supplemented with L. rhamnosus B-445 demonstrated antibacterial effectiveness against S. mutans through an in vitro study [106]. The authors also suggested that the probiotic mouthwash has a potential preventive and therapeutic value. 12.5 Periodontal Disease 12.5.1 Definition and Etiopathology Periodontal diseases involve a variety of chronic inflammatory circumstances of the gingiva, ligament, and bone supporting the teeth. In summary, gingivitis is the dysbiosis initiation—gingiva inflammation—and progress to the periodontitis—characterized by the gingiva, ligament, and bone loss. Periodontitis is the more severe form of the disease, being progressive, destructive, and creating the deep periodontal “pockets” [107]. Periodontal diseases are started and persistent by the oral microorganisms [32]. The development of periodontitis is a microbial alteration of the subgingival biofilm, as a consequence of the interaction of the local microorganisms and the inflammatory response [108–110]. Although the microbiome is important to the beginning and sustaining of gingivitis and periodontitis, genetic and environmental host factors influence the dysbiosis proportion [111]. There are several risk factors for the development of periodontal disease including cigarette smoking, diabetes mellitus, socioeconomic and demographic variables, psychosocial factors such as stress, genetic predispositions, hormonal characteristics such as pregnancy and low immunity [112]. Regarding the periodontal microbial biofilm, it can comprise hundreds species in an individual [113]. Subgingival communities in healthy individuals are mainly formed by Gram-positive strains (Rothia spp. and Actinomyces spp). In contrast, the gingivitis microbial sets are formed by Gram-negative species (Prevotella, Selenomonas, and Fusobacterium) 182 Natural Oral Care in Dental Therapy [114, 115] and periodontitis sites are characterized by a diverse community including the species Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Filifactor alocis, and Fretibacterium spp., etc. [24]. Among these microorganisms, A. actinomycetemcomitans and P. gingivalis have been considered as mainly responsible in the onset of periodontal disease [116–118]. On the other hand, A. actinomycetemcomitans and P. gingivalis can also be identified in healthy subjects [119, 120]. The dysbiosis progression is determined by the capacity of resistance of the host immune response [107]. Prevention of gingivitis and chronic periodontitis comprises avoiding biofilm formation through correct individual oral hygiene and eliminating the biofilm through professional removal in regular intervals [111]. The overall recommendation is brushing the teeth twice daily, brushing the tongue, using dental floss and fluoride toothpaste. 12.5.2 Probiotics and Periodontal Diseases The use of probiotics in cases of periodontal diseases have been evaluated as they could act in the suppression of periodontitis microorganisms by the production of antimicrobial substances or through mechanisms of competition or exclusion, and also contribute to the modulation of immune responses [121, 122]. Thus, introducing beneficial microorganisms via probiotics products might be beneficial for the prevention of gingivitis and periodontitis [123]. The probiotic effects against periodontal bacteria have been already studied. The influence of Lactobacillus spp. in the treatment of periodontitis has been reported as being beneficial for the improvement of clinical periodontal parameters and reduction of periodontitis-associated species [24]. Regarding the use of a single probiotic microorganism, L. reuteri was able to improve periodontal clinical parameters including plaque index, bleeding on probing, and pocket depth [124, 125]. L. reuteri has also demonstrated the capacity of significantly reducing pocket depth and gain in moderate when used as adjunct to periodontal treatment [126]. L. salivarius has also improved periodontal clinical parameters in individuals that consumed the probiotic [127]; and has significantly reduced the number of periodontopathic bacteria (A. actinomycetemcomitans, P. gingivalis, T. forsythia, T. denticola, and P. intermedia) in their subgingival plaque [128]. Moreover, the ingestion of L. casei Shirota significantly reduced gingival inflammation and significantly decreased plaque index in the test group [14]. Concerning the use of the association of different species of probiotic microorganisms, the combination of L. rhamnosus and L. curvatus has reduced periodontal disease parameters after a short-term consumption [129]. However, on the other hand, the general plaque index was less affected, although there was a tendency of decreased plaque levels in the probiotic group [129]. Regardless of the differences between probiotic strains, a systematic review and meta-analysis [89] showed a significantly greater plaque index after the consumption of dairy products containing probiotics. This result may be explained because the ingestion of probiotics-contained dairy products increases the amount of carbohydrates available [15] in the mouth and, consequently, could increase the plaque index. A further systematic review and meta-analysis [51] revealed that the use of probiotics did not significantly disturb the levels of periodontitis microorganisms Probiotic and Oral Health 183 (A. actinomycetemcomitans, P. gingivalis, and P. intermedia), but it improved bleeding on probing, gingival index, and helped in reduction of pocket probing depth. Nevertheless, heterogeneity was elevated and publication bias suspected for all outcomes statically [51]. Two other systematic reviews focused on the effect of probiotics in periodontal disease, have also demonstrated large heterogeneity between the randomized clinical trials included, due to the variability of the probiotic’s strain used, the probiotic’s concentration, the delivery vehicle, the clinical characteristics of the tested population, among others [122, 130]. Considering the above-mentioned limitations, the evidence report reductions in total anaerobic bacteria, increases in total aerobic bacteria, reductions in black-pigmented bacteria, reductions in A. actinomycetemcomitans numbers, reductions in P. gingivalis numbers, and reductions in T. forsythia [130]. 12.6 Oral Candidiasis 12.6.1 Definition and Etiopathology Oral candidiasis is an oral mucosa inflammation caused by the increase in the quantity of the fungus Candida spp. [34]. Clinically, the oral candidiasis can be presented as the following forms: acute pseudomembranous candidiasis, chronic erythematous candidiasis, acute erythematous candidiasis, and chronic hyperplastic candidiasis [31]. The infection is initiated by commensal and opportunistic species of the yeast Candida, usually Candida albicans, which is a normal inhabitant of the oral cavity [22]. Other species have also been associated with the infection including C. glabrata, C. tropicalis, C. parapsilosis, C. kefyr, C. dubliniensis, C. lusitaniae, C. krusei, and C. guilliermondii [24]. The biofilm formation of Candida spp. is a process of several stages being initiated by adherence to the surface; followed by colonization, proliferation, and invasion; and finally the detachment of biofilm cells to promote colonization and infection of other sites [131]. Similarly to the other dysbiosis, the presence of the microorganisms exclusively is not capable to develop the disorder. The onset of oral candidiasis occurs when there is a drop of host immunity. This reduction in the immune response can be determined by systemic conditions including the use of medication, for example, immunosuppressive and broad-spectrum antibiotics, age (children or elderly), chemotherapy, radiotherapy, organ transplantation, systemic diseases, such as diabetes and malignancies, and HIV infection [24, 88]. Moreover, there are some local factors that can also contribute to the Candida growth including the use of removable prosthesis, poor oral hygiene, certain foods, tobacco, and hyposalivation [132]. The most common form of candidiasis is the acute pseudomembranous candidiasis. Clinically, its symptoms include white, curd-like patches in the mouth or throat and the disorder is frequently characterized by local discomfort, including altered taste sensation and burning pain [88]. When the infection becomes more serious, it may cause morbidity and also leads to death [133]. The prevention of candidiasis is associated with the use of systemic and local antifungal agents. However, antifungal drugs can manifest some adverse effects, such as vomiting, diarrhea, nausea, and hepatic and renal toxicity [134]. Besides that, the resistance of the 184 Natural Oral Care in Dental Therapy yeast to the antifungal prophylaxis caused an increase in strain number, and it remains problematic [135, 136]. Oral candidiasis also returns or becomes recurrent in immunosuppressed patients, elderly individuals, or those wearing dentures. Thus, searching for alternative agents effective against Candida, with no side effects and no possibility of resistance are needed [137, 138]. 12.6.2 Probiotics and Oral Candidiasis Probiotics have been used as an auxiliary to prevent and treat oral candidiasis in pediatric and adult patients, through in vitro and in vivo studies [139]. Starting from in vitro studies, the activity of probiotic microorganisms against Candida is important to select after an effective strain for clinical studies. However, the results remain controversial. Probiotics have been reported as effective agents in the reduction of C. albicans growth and against its virulence potential [82, 140–142], while it has also been related with no inhibition in the yeast growth [143]. Following the outcomes of clinical studies, trials have reported positive results of probiotic consumption in the reduction of oral candidiasis risk. Several investigations have shown that the use of probiotics decreased the salivary yeast levels in test subjects [144–148], and also expressively increased the anti-Candida IgA amounts [146, 147]. Additionally, the use of a mix of probiotics (B. longum, L. bulgaricus, and S. thermophiles) enabled oral pain and decline in Candida spp. counts in patients diagnosed with oral candidiasis to recover [149]. Furthermore, the consumption of capsules containing another probiotic mixture (L. rhamnosus, L. acidophillus, and B. bifidum) reduced the detection rate of oral Candida spp. in asymptomatic patients using oral appliances [150]. The clinical effect of dairy products containing probiotics on oral candidiasis has also been tested. Probiotic cheese significantly reduced the risk of candidiasis compared to the control group [144, 145]. Similarly, another cheese containing different probiotic strains tested and also presented a positive result against Candida infection [151]. On the other hand, the consumption of cheese containing another combination of different probiotic microorganisms could not reduce the yeast number compared to a cheese without probiotics [133]. Regarding the consumption of milk-containing probiotics, commercial fermented milk containing L. casei and Bifidobacterium breve decreased the occurrence of oral candidiasis in healthy individuals. Furthermore, the immunological analysis demonstrated an increment of anti-Candida IgA levels in this population, suggesting that this beverage could control oral candidiasis [146, 147]. It is worth noting that both were clinical studies without a control group. Finally, at the top of the evidence, a systematic review and meta-analysis aims to assess the efficacy of probiotics in the prevention of oral candidiasis in elderly individuals. It was found that probiotics could be an efficient method for the reduction of oral yeast counts in this population [88]. In contrast, another systematic review and meta-analysis focused on outcomes of probiotics-contained dairy products on oral health, showing no significant results in yeast identification [89]. The difference between the results of these two meta-analyses may be associated with the variation of vehicles tested. The first one [88] included studies testing cheese, capsules, and lozenges as probiotic vehicles, and the second study [89] included only dairy products containing probiotics. Probiotic and Oral Health 185 12.7 Halitosis 12.7.1 Definition and Etiopathology Bad breath or halitosis, also known as oral malodor, is an unpleasant odor exhaled from the mouth, nasal cavity, or facial and pharyngeal sinuses. Halitosis is clinically classified into genuine halitosis, halitophobia, and pseudo-halitosis [152]. It is estimated as the third most common complaint of patients about oral problems, following dental caries and periodontal disease [153]. Its etiology can include reduction of salivary flux, consumption of certain foods, presence of metabolic disorders, and respiratory tract infections [154]. Accumulation of food residues at different parts of the oral cavity and its interaction with oral bacteria is considered the major cause of halitosis [155]. Multiple bacteria may be related to halitosis development; among them, Atopobium parvulum, Eubacterium sulci, and Solobacterium moorei are the dominant species located in the tongue dorsum of people with oral halitosis [156]. Additionally, Gram-negative anaerobic bacteria (F. nucleatum, P. gingivalis, P. intermedia, and T. denticola) are also associated with halitosis since they have proteolytic abilities [154]. While halitosis prevention concerns the patients, the treatment of physiological halitosis, oral pathological halitosis, and pseudo-halitosis is the responsibility of the dentists [157]. The successful management of halitosis appears to rely on the removal or reduction of the total number of bacteria and food residuals. Mechanical interventions such as brushing teeth, using dental floss, and cleaning the tongue are intended to decrease the quantity of these bacteria and debris [158]. In some cases, professional tooth cleaning and periodontal therapies for the removal of colonization sites of these bacteria are also necessary [159]. However, mechanical cleaning appears to have limited benefits. The halitosis bacteria return to the same regions shortly after the termination of therapy [160]. Chemical products such as mouth-rinses, mainly with chlorhexidine, are widely used in the treatment of halitosis because they can get into the more inaccessible surfaces of the oral cavity, are refreshing, and possess a pleasant odor [161]. However, chlorhexidine cannot be used for long periods because it causes tooth and tongue discoloration and some reduction in taste sensation [162]. 12.7.2 Probiotics and Halitosis Specific replacement of the pathogenic microbiota with favorable bacteria, such as probiotics, should be an interesting way to reduce the halitosis and prevent the growth of malodor microorganisms [163]. As the dorsum of the tongue and periodontal tissues are the origins of most malodor problems, probiotic species capable of colonizing these surfaces would be a helpful method to control the halitosis [164–167]. The probiotic treatment of halitosis has already achieved promising outcomes. There are several studies confirming the reduction of halitosis in individuals who have used lozenges, tablets, or gums containing probiotics [164–167]. On the other hand, the supporting data showing the efficacy of consumption of probiotics contained in dairy products in malodor treatment are still deficient and remain poorly explored [14]. Vestman et al. [168] isolated the Lactobacillus species detected in the saliva and oral cavity of breastfed and formula-fed infants and evaluated the in vitro probiotic proprieties. 186 Natural Oral Care in Dental Therapy L. gasseri was the most prevalent Lactobacillus species, and this species inhibited growth of microorganisms mainly associated with the principal oral dysbiosis: S. mutans (caries), F. nucleatum (periodontal disease) that produces volatile sulfur compounds, C. albicans (candidiasis), and other microorganisms [168]. An in vitro and placebo-controlled trial showed that the consumption of yogurt including L. rhamnosus L8020 reduced the S. mutans counts and four periodontal pathogens: P. gingivalis, P. intermedia, T. forsythia, and Fusobacterium spp. It suggests that yogurt with probiotics may diminish the risk of dental caries and periodontal disease and also periodontal pathogens related to malodor [169]. A prospective RCT showed that the administration of chewing gum containing probiotics had positive results on the reduction of oral malodor through analysis of organoleptic scores [102]. Another clinical trial examined the influence of a probiotic tablet containing L. salivarius and xylitol on oral malodor [170]. The study found that organoleptic scores and concentration of volatile sulfur compounds declined in the probiotic group. Additionally, the levels of ubiquitous bacteria and Fusobacterium nucleatum were significantly lower. Although studies support the use of probiotics to manage halitosis, the available evidence is insufficient for more recommendations, especially regarding the administration approaches. 12.8 Conclusion There is no doubt about the benefits of the probiotic’s use for general health, and its use for this purpose is largely recommended. Due to these positive effects, probiotics have been reported as promising agents also for oral health, acting against some of the major biofilm-dependent oral dysbiosis. However, there are still controversies over their mechanisms of action and effects for the prevention and treatment of oral disorders, so their indication on oral health improvement is still inconclusive. Therefore, new studies testing different probiotics products are suggested, to recognize and describe probiotic strains that may, definitely, help in the control of dental caries, gingivitis, periodontitis, oral candidiasis, and halitosis. 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Li, D., Li, Q., Liu, C., Lin, M., Li, X., Xiao, X., Zhu, Z., Gong, Q., Zhou, H., Efficacy and safety of probiotics in the treatment of Candida associated stomatitis. Mycoses, 57, 141–146, 2014. 150. Ishikawa, K.H., Mayer, M.P., Miyazima, T.Y., Matsubara, V.H., Silva, E.G., Paula, C.R., Campos, T.T., Nakamae, A.E., A multispecies probiotic reduces oral Candida colonization in denture wearers. J. Prosthodont., 24, 194–199, 2015. 151. Miyazima, T.Y., Ishikawa, K.H., Mayer, M.P.A., Saad, S.M.I., Nakamae, A.E.M., Cheese supplemented with probiotics reduces Candida levels in denture wearers – RCT. Oral Dis., 23, 919–925, 2017. 152. Murata, T., Yamaga, T., Lida, T., Miyazaki, H., Classification and examination of halitosis. Int. Dent. J., 52, 181–186, 2002. 153. Loesche, W.J. and Kazor, C., Microbiology and treatment of halitosis. Periodontol. 2000, 28, 256–279, 2002. 154. Scully, C. and Greenman, J., Halitosis (breath odor). Periodontol. 2000, 48, 66–75, 2008. 155. Scully, C., el-Maaytah, M., Porter, S.R., Greenman, J., Breath odor: Etiopathogenesis, assessment and management. Eur. J. Oral Sci., 105, 287–293, 1997. 156. Kazor, C.E., Mitchell, P.M., Lee, A.M., Stokes, L.N., Loesche, W.J., Dewhirst, F.E., Paster, B.J., Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J. Clin. Microbiol., 41, 558–563, 2003. 157. Yaegaki, K. and Coil, J.M., Examination, classification, and treatment of halitosis; clinical perspectives. J. Can. Dent. Assoc., 66, 257–261, 2000. 158. Kuo, Y.W., Yen, M., Fetzer, S., Lee, J.D., Toothbrushing versus toothbrushing plus tongue cleaning in reducing halitosis and tongue coating: A systematic review and meta-analysis. Nurs. Res., 62, 422–429, 2013. Probiotic and Oral Health 195 159. Deutscher, H., Derman, S., Barbe, A.G., Seemann, R., Noack, M.J., The effect of professional tooth cleaning or non-surgical periodontal therapy on oral halitosis in patients with periodontal diseases. A systematic review. Int. J. Dent., 16, 1, 36–47, 2018. 160. Van der Sleen, M.I., Slot, D.E., Van Trijffel, E., Winkel, E.G., Van der Weijden, G.A., Effectiveness of mechanical tongue cleaning on breath odour and tongue coating: A systematic review. Int. J. Dent. Hyg., 8, 258–268, 2010. 161. Mishra, V., Shettar, L., Bajaj, M., Math, A.S., Comparison of a commercially available herbal and 0.2% chlorhexidine mouthrinse for prevention of oral malodor: A clinical trial. J. Int. Soc. Prev. Community Dent., 6, 6–11, 2016. 162. James, P., Worthigton, H.V., Parnell, C., Harding, M., Lamont, T., Cheung, A., Whelton, H., Riley, P. Chlorhexidine mouthrinse as an adjunctive treatment for ginginval health. Cochrane Database Syst. Rev., 31, 3, CD008676, 2017. 163. Faveri, M., Feres, M., Shibli, J.A., Hayacibara, R.F., Hayacibara, M.M., de Figueiredo, L.C., Microbiota of the dorsum of the tongue after plaque accumulation: An experimental study in humans. J. Periodontol., 77, 1539–46, 2006. 164. Burton, J.P., Chilcott, C.N., Tagg, J.R., The rationale and potential for the reduction of oral malodour using Streptococcus salivarius probiotics. Oral Dis., 11, 29–31, 2005. 165. Burton, J.P., Chilcott, C.N., Moore, C.J., Speiser, G., Tagg, J.R., A preliminary study of the effect of probiotic Streptococcus salivarius K12 on oral malodour parameters. J. Appl. Microbiol., 100, 754–764, 2006. 166. Jamali, Z., Aminabadi, N.A., Samiei, M., Sighari Deljavan, A., Shokravi, M., Shirazi, S., Impact of Chlorhexidine Pretreatment Followed by Probiotic Streptococcus salivarius Strain K12 on Halitosis in Children: A Randomised Controlled Clinical Trial. Oral Health Prev. Dent., 14, 305–13, 2016. 167. Zupancic, K., Kriksic, V., Kovacevic, I., Kovacevic, D., Influence of Oral Probiotic Streptococcus salivarius K12 on Ear and Oral Cavity Health in Humans: Systematic Review. Probiotics Antimicrob. Proteins, 9, 102–110, 2017. 168. Vestman, N.R., Timby, N., Holgerson, P.L., Kressirer, C.A., Claesson, R., Domellöf, M., Öhman, C., Tanner, A.C., Hernell, O., Johansson, I., Characterization and in vitro properties of oral lactobacilli in breastfed infants. BMC Microbiol., 13, 193–205, 2013. 169. Nikawa, H., Tomiyama, Y., Hiramatsu, M., Yushita, K., Takamoto, Y., Ishi, H., Mimura, S., Hiyama, A., Sasahara, H., Kawahara, K., Makihira, S., Satoda, T., Takemoto, T., Murata, H., Mine, Y., Taji, T., Bovine milk fermented with Lactobacillus rhamnosus L8020 decreases the oral carriage of mutans streptococci and the burden of periodontal pathogens. J. Investig. Clin. Dent., 2, 187–196, 2011. 170. Suzuki, N., Yoneda, M., Tanabe, K., Fujimoto, A., Iha, K., Seno, K., Yamada, K., Iwamoto, T., Masuo, Y., Hirofuji, T., Lactobacillus salivarius WB21-containing tablets for the treatment of oral malodor: A double-blind, randomized, placebo-controlled crossover trial. Oral Surg. Oral Med. Oral Pathol. Oral Radiol., 117, 4, 462–70, 2014. 13 Charcoal in Dentistry Abhilasha Thakur*, Aditya Ganeshpurkar and Anupam Jaiswal Shri Ram Institute of Technology-Pharmacy, Jabalpur, Madhya Pradesh, India Abstract Charcoal also called as “Black magic” dates its production and uses since the long ancient period. It is a black carbon and ash residue hydrocarbon, which is very lightweight and produced by removing water and other volatile constituents from substances of animal and vegetation origin. It is usually produced by a method called slow pyrolysis in which wood or other substances are heated in the absence of oxygen. The property of the product is determined by material being charred and also the temperature at which it is charred. The different types of charcoal include common charcoal, activated charcoal, sugar charcoal, lump charcoal, pillow-shaped briquettes, sawdust briquette charcoal, Japanese charcoal, and extruded charcoal. Charcoal has been eagerly adopted by the health and beauty industry. Other uses embrace it as a cooking fuel, syngas production, automotive fuel, metallurgical fuel, industrial fuel, pyrotechnics, carbon source for chemical reactions, art, horticulture, purification, and filtration purpose, etc. Recently, an array of charcoal dentifrices has appeared in the market and is being marketed through instafamous celebrity endorsements. These preparations remove stains, acidic plaque, and gives fresh breath, aiding in good dental health. The microbiological studies with charcoal-infused tooth bristles also claim lesser oral bacterial contamination. Charcoal is without doubt a health ingredient, which is in current fashion and is a rebirth of ancient medicine techniques. However, due to lack of sufficient clinical and laboratory data, the safety and efficacy of charcoal and charcoal-based dentifrices cannot be claimed and hence requires well-designed studies to be conducted on a larger scale for establishing the conclusive evidence. Keywords: Charcoal, dentifrice, antibacterial, stain, whitens 13.1 Introduction Oral and dental health is a necessary part of overall health and well being of an individual. It includes disease-free teeth and supporting tissues. A healthy oral cavity can help maintain a healthy body while its poor maintenance leads to dental cavities, gum diseases and may be associated with heart attack, stroke, poorly controlled diabetes and preterm labor. Oral health touches every aspect of our lives and can be called “a window into the health of one’s body”. It can affect issues like a person’s self-esteem, speech, nutrition, comfort and overall *Corresponding author: abhilashathakur27@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (197–210) © 2020 Scrivener Publishing LLC 197 198 Natural Oral Care in Dental Therapy quality of life [1–2]. The World Health Organization declared the theme “Oral Health for a Healthy Life” on World Health Day in 1994 [3]. Various materials and methods are suggested and are in use for maintaining oral hygiene. Table 13.1 enlist major over-the-counter (OTC) dental products with their main uses [4], and Figures 13.1 and 13.2 depict the different types of materials and instruments used for oral hygiene [1]. Table 13.1 OTC dental care products with their main use. OTC dental care products Use 1. Mouth fresheners Treatment method for halitosis. 2. Abrasive stain removers Along with removing dental stains, claims to remove plaque, tartar and tobacco stains. 3. Pregnancy toothpaste Specially formulated for pregnant and lactating women. 4. Charcoal toothpaste and charcoal brushes Charcoal has the ability to kill microbes and absorb gases causing malodor. 5. Whitening agents/pastes Stain removal and whitening of teeth. Datun Ayurvedic toothpowder-red Ayurvedic toothpaste Non-medicated toothpaste Materials for oral hygiene Non-abrasive white toothpowder Desensitizing toothpaste Figure 13.1 Different types of materials used for oral hygiene. Oral hygiene instruments Finger Toothbrush Proxa brush Figure 13.2 Different oral hygiene instruments. Charcoal in Dentistry 199 Charcoals containing oral and dental care preparations are a recent innovation in the oral healthcare product market. The marketing campaign for these preparations is fashionable, trendy and so is enjoying considerable success. So it can be said without doubt that charcoal is a current fashion in health ingredient and is the rebirth of ancient medicine techniques. Charcoal largely consists of black carbon and ash residue hydrocarbon produced by removing water and other volatile constituents from substances of animal and vegetation origin. It is generally produced by a method called slow pyrolysis in which the wood or other substances are heated in the absence of oxygen [5]. 13.2 Charcoal Production Methods The production of charcoal is being done since very ancient period. These methods can be broadly divided into: 13.2.1 The Traditional Method The method utilizes a clamp and is basically a pile of wooden logs placed in a circle, which leans against a chimney consisting of four wooden stakes being held up by ropes. These logs are then totally covered with soil and straw so that no air enters. Utilizing some burning fuel, the chimney is lit, and the logs are allowed to burn very slowly to transform into charcoal in a period of about 5 days. As the burning is complete, to prevent air from entering, the chimney is plugged [6]. However due to emissions of unburnt methane, this method has a strong inherent disadvantage of being hazardous to human health and environment [7]. Also, partial combustion of wood imparts low efficiency to this method. 13.2.2 The Modern Methods The methods provide a high yield of about 35–40% as it utilized retorting technology, i.e., process heat is recovered, rather than kilning [7]. The properties of the obtained charcoal depend on the material being charred and also the temperature at which it is charred. It contains different amounts of hydrogen and oxygen, ash, and other impurities. These, together with the structure, define the properties of the charcoal produced. The source material, which is free from non-volatile compounds, produces high purity product. 200 Natural Oral Care in Dental Therapy Table 13.2 Various types of charcoal. Common charcoal Made from wood, coal, coconut shell, peat, or petroleum. Sugar charcoal Obtained from the carbonization of sugar. Later to remove any mineral matter, purified by boiling with acids. Then after to remove the last traces of hydrogen, it is burned for a long time in a current of chlorine [8]. Activated charcoal Produced from coal, coconut shells, bone char, sawdust, peat, petroleum coke, or olive pits. By processing it at very high temperatures, the charcoal is activated leading to change in its internal structure, making it more porous, reducing the size of its pores, and increasing its surface area [9, 10]. Lump charcoal It is a conventional charcoal made from hardwood material and as compared with briquettes produces less ash. Japanese charcoal During the charcoal making pyroligneous acid is removed from it and hence when burned, it produces nearly no smoke or smell. Pillow shaped briquettes These are generally made from sawdust and other wood by-products, by compressing charcoal with a binder and other additives. Starch is used as binder and may include brown coal and mineral carbon as a heat source while borax, sodium nitrate, and raw sawdust are used as ignition aid. Other additives include limestone as ash-whitening agent. Sawdust briquette charcoal Produced by compressing sawdust without binders or other additives. It is used primarily for barbecue as it produces high heat but no smoke and odor with little ash. It has long burning hours which may exceed 4 h. Extruded charcoal Produced by extruding raw ground wood or carbonized wood into logs without using a binder. The charcoal is held together by the heat and pressure of the extruding process. 13.3 Uses of Charcoal Charcoal is being used since ancient times for different purposes including medicinal and nonmedicinal areas. 13.3.1 Medicinal Uses Teeth whitening, oral health charcoal toothpaste and dentifrices, Charcoal tablets, capsules or powder for digestive effects [11], To measure the mucociliary transport [12], Treatment of poisoning or drug overdose, To relieve kidney dialysis treatment related itching [13], To reduce high cholesterol, Charcoal in Dentistry 201 Mold cleansing [14] Treatment of skin infection [15] 13.3.2 Non-Medicinal Uses Metallurgical fuel ex-production of Iron [16] Industrial fuel ex-blast furnaces Cooking fuel ex-charcoal briquettes Syngas production ex-automotive propulsion Pyrotechnics ex-black powder for fireworks Cosmetic ex-bamboo charcoal for its absorbing properties Carbon source ex-chemical reactions Purification ex-sucrose from cane sugar Filtration ex-charcoal filters in gas masks Art ex-drawing and painting Horticulture ex-biochar Animal husbandry ex-mixed with feed 13.4 Charcoal Containing Oral and Dental Care Products A charcoal containing oral and dental care product may include charcoal as an ingredient as activated form of carbon, charcoal, raw charcoal, black charcoal, organic charcoal, premium charcoal, premium-grade charcoal, white charcoal, bamboo charcoal, all-natural bamboo charcoal, coconut charcoal, coconut shell charcoal, organic coconut charcoal, medical-grade charcoal, negative ionic charges of charcoal, pure hardwood charcoal, virgin carbon hardwood-derived, or active pine tree charcoal. Other forms include charcoal power, bamboo charcoal, natural bamboo charcoal, bamboo charcoal all-purpose, white charcoal, coconut charcoal, American eastern hardwood charcoal, and ecologic charcoal [17]. These products can be searched in the market as toothpowder (Healthvit coconut shellactivated charcoal instant teeth whitening powder), toothpaste (Healthvit-activated charcoal toothpaste, Colgate Total charcoal anticavity toothpaste, Beverly Hills white black formula perfect toothpaste), tooth tabs (Lush Boom Tooth Tabs), capsules (FineVine organic coconut activated charcoal capsules), dental creams [18], tooth polish (Diamond Whites Black Edition Tooth Polish) etc. John et al. 2017 reviewed the safety and efficacy of charcoal and charcoal-based dentifrices [17]. In certain countries nowadays, charcoal has been imparted to the bristles of toothbrushes, making appearance of the bristles black in color. Charcoal has inherent property of being absorbent for toxins, poisons, and noxious gases. Thus, the charcoal-infused tooth bristles can be considered a new product to prevent bacterial contamination [19]. Manufacturers of these charcoal toothbrushes claim that by using these charcoal blended nylon bristles, halitosis, and plaque can be reduced. Along with these advantages it also kills bacteria that may develop in the bristles during storage [19, 20]. Teraoka et al. 2004 studied the far infrared spectral characteristics of bamboo charcoal powder. Based on the study and its effect on cancer cells for use in the dental field they proved bamboo charcoal 202 Natural Oral Care in Dental Therapy Table 13.3 Patents for inventions/formulations containing charcoal. Formulation/invention Claim Ref. Bamboo charcoal toothpaste Bamboo charcoal fiber layer provides fresh taste and also reduces resource wastage. [26] Active carbon toothpaste Prevents and treats periodentitis, gingivitis, strengthened oral health, tooth decolorizing, and whitening effect. [27] A kind of Blumea balsamifera bamboo charcoal toothpaste Relieve bacteriostatic itching and eliminate oral peculiar smell. [28] Bamboo charcoal toothpaste capable of refreshing mouth smell and whitening teeth Refreshing mouth smell, whitens teeth, and highly stable. [29] Toothpaste using charcoal and negative ions Absorbs heavy metals and bacteria. Surfactants present in dentifrice generate anions. [30] Toothpaste composition Toothpaste composition at a low production cost. Bamboo charcoal and bamboo vinegar has good deodorizing and bactericidal power and tranquilizing effect. [31] Chinese body containing carbon black fashion toothpaste Dental cleaning and healthy gingival homeostatic efficacy. [32] A toothpaste having a charcoal Effective in periodentitis, whitening, and deodorization effect. [33] Bamboo charcoal-lavender toothpaste Bamboo charcoal adsorbs dirt from oral cavity and reduces bacteria survival. Lavender fragrance improves smell in the oral cavity, resists bacteria, protects nerves, and relieves oral cavity pains. [34] Bamboo charcoal toothbrush Gingival massage rings arranged on the toothbrush head remove food debris, cleans teeth, and remove dental plaque. The flexible bulges arranged at the back side of the toothbrush head remove leftover blots on inner walls and tongue. [35] Therapeutic chew device for cleaning teeth and breath of dogs Absorbs odors and bacteria to clean and freshen the dog’s breath. [36] Tooth paste composition Combination of a detergent base and a mixture of micronized colloidal silica gel and activated charcoal. [37] Double capsule composition for containing charcoal and manufacturing method for the same Uses charcoal powder to make a double capsule which maintains a color in a bright tone even after mixing in toothpaste. Original color revealed only when during use, capsule is destroyed. [38] (Continued) Charcoal in Dentistry 203 Table 13.3 Patents for inventions/formulations containing charcoal. (Continued) Formulation/invention Claim Ref. Outer remove intrinsic stains toothpaste Removes extrinsic stains, plaque, food debris, and dirt from the teeth surface. [39] Toothpaste containing bamboo activated carbon Removes foreign smell and stains deposited on the teeth. [40] Toothpaste adopting spherical activated carbon and preparation method of toothpaste Good cleaning and stain adsorbing effect. [41] Toothbrush having brush made of material comprising charcoal powder Antibacterial, deodorizing, and cleansing effect. [42] Hardwood charcoal toothpaste Removes mouth odor, prevents tooth decay, and periodontal disease. [43] Charcoal powder-containing dentifrice composition for removing nicotine Removes and prevents nicotine accumulation from the teeth. [44] Toothpastes containing powdered charcoal- having enhanced whitening effect on teeth Compared to conventional toothpastes increased whitening action, especially in heavy smokers. [45] Multi-functional bamboo charcoal toothbrush Toothpaste–toothbrush integration. [46] Dentifrice paste containing charcoal as abrasive has additional purifying action Satisfactory abrasive, absorbing, and purifying effect particularly for stains and bacteria. [47] Bamboo charcoal composition for removing bad breath as well as preparation method of composition Due to strong adsorption power, removes the bad breath. [48] Aqueous charcoal-containing slurry for removal of plaque, calculus or stains from teeth Capable of removing plaque, calculus and stains from teeth. [49] Aaaaa Alleviate toxicity and removes odor. [50] Dentifrice included charcoal Sterilizes microorganism generated in the mouth, deodorization effect. [51] Manufacturing process of charcoal toothpaste Charcoal toothpaste prepared by the method eliminates acid associated toxicity, produces good energy to human body by far-infrared raylike effect and anti-inflammatory effect without side effects. [52] 204 Natural Oral Care in Dental Therapy powder to be a promising dental filler material for prevention of cancer [21]. Thamke et al. 2018 evaluated the bacterial contamination and antimicrobial efficacy of charcoal bristles compared to noncharcoal bristles in used toothbrushes. The study showed a statistically significant difference in bacterial counts between bristle types and lower CFUs in the charcoal bristles compared with noncharcoal bristles, after 1 week of use. The zone of inhibition that was found around charcoal tooth bristles supported the antimicrobial properties of the charcoal toothbrush [22]. Through different studies, it has been proven that activated carbon has capacity to remove bacteria like Pseudomonas aeruginosa and Escherichia coli from fresh and potable water systems [23, 24]. Microorganisms get attached to activated carbon particles via strong Lifshitz van der Waals forces [25]. Although there is electrostatic repulsion between negatively charged microorganisms and carbon surfaces, there remains a possibility of enhancing the microorganism-removing efficacy of activated carbon efficacy by positive charge modification of the carbon particles surfaces. 13.5 Benefits of Using Charcoal Containing Oral and Dental Care Products 13.5.1 Removes Stains and Whitens Teeth The activated charcoal in a toothpaste works by mechanism of combination of mild abrasion and absorption of extrinsic tooth surface stains. Charcoal present in these products does not stick to the teeth surface and hence could be easily washed away. Its absorbent properties allow it to only bind with the surface stains like those from coffee and tea. However, it may not be beneficial on yellowed teeth due to antibiotics or other internal problems [53, 54]. So these products are an effective means to remove extrinsic stains, i.e., discoloration of outside surface of the tooth from substances like coffee, wine, berries, and other staining foods [55]. These products may be found to be most effective when used to delay the recurrence of surface staining on intact teeth following professional cleaning and polishing [56]. Also brushing teeth with the activated charcoal regularly has been reported to enhance the teeth appearance making them lighter by up to three shades [57]. 13.5.2 Removes Acidic Plaque Charcoal has binding capacity to the acidic elements present in mouth and thus increases their rate of excretion from the body. Hence brushing with these types of toothpaste raises the pH of mouth and helps in reducing the buildup of acidic plaque [58]. 13.5.3 Gives Fresh Breath and Improves Halitosis Due to its absorbent qualities, charcoal helps in controlling halitosis. Charcoal in Dentistry 205 Removes stains and whitens teeth Removes acidic plaque Improves halitosis Activated Charcoal Remineralizes teeth Protects from infection Cost effective for regular basis use Figure 13.3 Benefits of using charcoal containing oral and dental care products. 13.5.4 Remineralize Teeth Activated charcoal has been used by many people as part of their teeth remineralizing protocol. However, there is a myth that charcoal demineralizes the teeth. However, as activated charcoal binds mostly to organic compounds and not minerals, it does not pull out the calcium from the teeth [59]. 13.5.5 Helps Overall Dental Health Using these products is quite messy and makes mouth look black, but as charcoal present in these products does not stick to the teeth surface, the product can be completely washed out by rinsing leaving teeth clean and smooth. 13.5.6 Protects From Infection There have been some suggestions that charcoal particles left in the mouth after brushing may have certain antimicrobial effects [56]. Charcoal has the property to pull toxins from the surface of teeth and the mouth. It binds with the toxins and come out of the oral cavity during rinsing of the mouth. As it has capacity to change the pH of the mouth, it does not allow disease causing germs and bacteria to thrive and reproduce within the mouth rendering it safe and clean. So these products protect teeth from infections causing bacteria and other microorganisms [59]. Also, there are millions of tiny pores present in activated charcoal, which trap toxins and harmful chemicals [55]. 13.5.7 Cost Effective for Regular Basis Use Apart from the above-mentioned benefits, one important advantage of these charcoal containing dental products is that it is a cost-effective solution for a regular basis use. 206 Natural Oral Care in Dental Therapy 13.6 Precautions to be Taken While Using Charcoal Containing Oral and Dental Care Products Clinicians, researchers, and patients have shown alarm in using charcoal and charcoalcontaining products for dental care due to the risk of thinning of the dental structure. Unlike materials such as salt, charcoal was not found to abrade teeth. However, these products are black in color, and brushing off the color tends to prolong brushing, or the use of excessive brushing force, which may lead to the abrasion of teeth [56]. If activated charcoal is used very frequently or in an incorrect manner, it can cause eroding of the enamel. Also, a regular use of abrasive material like charcoal may scrub out surface stains making teeth look whiter. However, this effect is for short term. Later, regular use of these product leads to teeth eventually looking yellower due to wearing of enamel causing permanent damage. A loss of enamel can lead to exposure of dentin, increased sensitivity, and increased susceptibility to dental decay [60–62]. Particles of charcoal included in charcoal powder and toothpaste may accumulate in crevices and other defects in teeth. These particles of charcoal may also build up in gaps between the teeth and dental restorations resulting in a grey or black line around restoration margins. This would create negative effects on dental attractiveness and may necessitate the replacement of the affected fillings, veneers, or crowns [56]. The powdered formulations generally require a longer brushing time to acquire the whitening effect and its use tend to be messy. Therefore, compared with pastes, the charcoal-containing dental powders are recommended for shorter period use, which is on an average once a day for 3 to 5 days rather than every day [63]. Many of these products contain agents like sodium laurel sulfate (SLS), which may cause gingival irritation in some individuals. So a good quality charcoal toothpaste products should be free of agents like sodium lauryl sulfate and artificial sweeteners like erythritol and fluoride [55, 63]. While using a charcoal dentifrice instead of scrubbing it hard on teeth surface, one should brush gently in a circular motion and then after mouth should be rinsed until it spits out totally clear of any discoloration from the toothpaste. These may also be tried smearing it on teeth, letting it sit for 5 to 10 min and then allowing it to do its adsorptive action. This procedure is particularly advisable with issues like childhood illnesses, medication reactions, etc., and related enamel erosion [55]. Charcoal dentifrices should not be used every day. As a rule of thumb, in order to avoid any negative side effects, 2–3 days a week is the optimum schedule [55]. Many dental specialists suggest the use of activated charcoal toothpaste with caution and not for extended periods. Other reasons for cautions involve recession of gum tissue due to the abrasive quality of charcoal toothpaste causing teeth sensitivity. Hence, dentists recommend using charcoal toothpaste manufactured by reputable brands, and proper note should be taken of any unusual symptoms that might appear during its use like increased sensitivity or bleeding gums [59, 64]. Charcoal in Dentistry 207 13.7 Conclusion Charcoal has been eagerly adopted by the health and beauty industry. The marketing campaigns for these toothpastes are fashionable and trendy, and so considerable success was enjoyed. It is undoubtedly the current fashionable health ingredient and is a rebirth of ancient medicine techniques. However, there is still a lack of sufficient scientific evidence, which can authenticate the cosmetic, health benefits including antibacterial, antifungal, or antiviral activity, reduced caries, tooth whitening, oral detoxification, or safety claims of marketed charcoal-based oral and dental care products. Also, controlled clinical trials and laboratory investigations of such products are needed to determine product efficacy and safety. To our knowledge, till date, no charcoal-based dental preparation has been approved by any dental organizations. References 1. 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Quinlivan, A., Li, L., Knappe, D., Predicting adsorption isotherms for aqueous organic micropollutants from activated carbon and pollutant properties. Water. Res., 39, 1663–1673, 2005. 25. Jucker, F.M., Heus, H.A., Yip, P.F., Moors, E.H., Pardi, A., A network of heterogeneous hydrogen bonds in GNRA tetraloops. J. Mol. Biol., 264, 5, 968–80, 1996. 26. B. Ligen, C. Huiwu, Z. Qisheng, Z. Jianbin, Bamboo charcoal toothpaste, China Patent CN203473468U, 2013. 27. Z. Yongkang and X. Ke, Active carbon tooth paste, China Patent CN1899243A, 2006. 28. L. Xian, A kind of Blumea balsamifera bamboo charcoal toothpaste, China Patent CN109125235A, 2017. 29. Z. Yanping and M. Jianlin, Bamboo charcoal toothpaste capable of refreshing mouth smell and whitening teeth, China Patent CN108721190A, 2018. 30. Choi, Toothpaste Using Charcoal and Negative Ions, South Korea Patent KR20040032716A, 2002. 31. G. Yamada, Toothpaste composition, Japan Patent JP2000143471A, 1998. 32. G. Huating, H. Qianle, Z. Zhenhai, L. Li, C. Yiyi, C. Manli, W. Wei, L. Xidi, Chinese body containing carbon black fashion toothpaste, China Patent CN105168058B, 2015. 33. K. Yeol, A toothpaste having a charcoal, South Korea Patent KR20000071954A, 2000. 34. W. Yu, Bamboo charcoal–lavender toothpaste, China Patent CN105267083A, 2014. 35. B. Ligen and C. Huiwu, Bamboo charcoal toothbrush, China patent CN201920026U, 2010. 36. D.M. Owens, Therapeutic chew device for cleaning teeth and breath of dogs, United States Patent US6050224A, 1999. 37. G. Rialdi, Tooth paste composition, United States Patent US4181712A, 1978. 38. S. Taesung, S. Hyun-gyu, P. Jung-ho, Double capsule composition for containing charcoal and manufacturing method for the same, South Korea Patent KR101914051B1, 2018. 39. T. Xianlan, M. Yanqiang, Wensheng, Outer remove intrinsic stains toothpaste, China Patent CN104586644B, 2015. 40. G. Aimin, Toothpaste containing bamboo activated carbon, China Patent CN106109361A, 2016. 41. W. Xinghe, D. 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Burhenne, M., Activated charcoal toothpaste: Benefits and precautions, plus a recipe, https:// askthedentist.com, 2019, as assessed on 17 June 2019. 56. Greenwall, L. and Wilson, H.F., Charcoal toothpastes: What we know so far. Clin. Pharm., 9, 8, 20203167, 2017. 57. Middleton, A., Tooth whitening versus stain removal. BDJ, 49, 17175, 2017. 58. Karkkainen, S. and Neuvonen, P.J., Pharmacokinetics of amitriptyline influenced by oral charcoal and urine pH. Int. J. Clin. Pharmacol. Ther. Toxicol., 24, 6, 326–332, 1986. 59. Panda, S., Mishra, S.R., Kar, P.K., A review on activated charcoal tooth paste. Int. J. Sci. Res., 7, 1, 253–254, 2018. 60. Magalhaes, C.A., Wiegand, A., Rioss, D., Honorio, H.M., Buzalaf, M.A.R., Insights into prevention measures for dental erosion. J. Appl. Oral. Sci., 17, 2, 75–86, 2009. 61. Brucculieri, J., Is Charcoal Toothpaste Safe? Dentists Explain The Risks, Style & Beauty, beauty. org., 2018. 62. Haywood, V.B. and Boyleston, E., http://www.vanhaywood.com/uploads/articlespa1ge/2017ActivatedCharcoal.pdf. 63. Potts, K., Marketing ingenuity or beneficial dentifrice? RDH magazine, 38, 7, 16408136, 2018. 64. Lewis, J., Charcoal Toothpaste: Benefits and Side Effects, https://www.hlbenefits.com/ charcoaltoothpaste-benefitsside-effects/, 2017. 14 Propolis Benefits in Oral Health Mariana Leonel Martins1, Karla Lorene de França Leite1, Yuri Wanderley Cavalcanti2, Lucianne Cople Maia1 and Andréa Fonseca-Gonçalves1* 1 Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 2 Department of Clinical and Social Dentistry, Universidade Federal da Paraíba, João Pessoa, PB, Brazil Abstract The use of phytotherapy in medicine has given prominence to some natural products such as propolis, which consists of a resin produced by bees that is gathered from the shoots and exudates of plants. This product can be found in different countries such as Russia, China, Brazil, Cuba, Chile, and Turkey. The specific physicochemical characteristics of propolis are determined by its chemical composition, especially its phenolic compounds, and its main botanical origin, being classified as either red (Dalbergia ecastophyllum), green (Baccharis dracunculifolia), or brown (without specific main botanical origin). The variety of propolis compounds, such as flavonoids, appears to confer different biological properties to this product resulting in activities from antibacterial, antifungal, and antioxidant to anti-inflammatory, and anticancer. However, its bioactive compounds vary according to the climatic conditions, such as temperature and season. Different types of propolis have been evaluated in order to be applied in dentistry, mainly due to its effectiveness on the cariogenic biofilm control and fungal infections and in the case of periodontal disease or root canal contamination. Laboratory studies and clinical trials with propolis in different presentation forms have demonstrated a positive effect of this product against oral pathogens. In this sense, the present chapter aims to offer an updated review of the benefits of propolis from the perspective in oral health. Keywords: Propolis, phytotherapy, antibacterial agents, anti-infective agents, oral health 14.1 Introduction Propolis consists of a resinous matrix arising from the extraction of botanical compounds by bees that are added to substances secreted by their glandular metabolism in order to reduce the size of the entrance of the hive and protect it from invaders (Figure 14.1). This product also serves as an antiseptic inside the alveoli, where the queen bee lays the eggs (Figure 14.2), and for wrapping up intruders slaughtered inside the hive to prevent them from rotting and contaminating the nest (Figure 14.3). Propolis contains waxes, resins, balsams, oils, and pollen and may present variable color, taste, odor, consistency, and chemical *Corresponding author: andrea.goncalves@odonto.ufrj.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (211–228) © 2020 Scrivener Publishing LLC 211 212 Natural Oral Care in Dental Therapy Figure 14.1 Worker of Apis mellifera in sunflower blossom. Fototeca Cristiano Menezes (FCM) available at speciesLink network (http://www.splink.org.br) in 2019, Feb 22. Figure 14.2 Larvae of Apis mellifera in the honeycomb with propolis. Fototeca Cristiano Menezes (FCM) available at speciesLink network (http://www.splink.org.br) in 2019, Feb 22. Figure 14.3 Entrance of Tetragonisca angustula with carcasses of ants mummified with propolis. Fototeca Cristiano Menezes (FCM) available at speciesLink network (http://www.splink.org.br) in 2019, Feb 22. Propolis Benefits in Oral Health 213 composition as well as biological activity depending mainly on the plant species of origin and the season in which the product was created [1–3]. This product can be found in different countries, such as Russia [4], China [5], Brazil [6–8], Cuba [9], Chile [10, 11], and Turkey [12]. In addition, it is known that various types of propolis can be found in the same country as a result of biodiversity, but the quality varies from one region to another due to environmental factors. Therefore, it is recommended that the determination of the type of propolis be established according to its main plant source [13]. Propolis has been prominent in the international trade of apicultural products due to the diverse biological properties attributed to its chemical components, which consequently increase its aggregated value and the interest in performing propolis-centered research studies [14]. Thus, an increase in propolis use has been observed principally due to the major interest in natural products. The applicability of propolis may constitute a viable alternative for the treatment of oral diseases, since many types of propolis have proven to present biological properties [15–18]. However, it is necessary to know the composition and mechanism of action of these products in order to potentiate the expected results and avoid adverse effects [8]. 14.2 Types of Propolis As mentioned before, there exist different types of propolis (i.e., yellow, dark yellow, light brown, dark brown, greenish-brown, reddish-brown, green, and red), which can be classified by botanical origin and, consequently, by the main chemical components [13]. The chemical compounds isolated from propolis can be organized into the following main groups: aromatic esters, sugars, alcohols, aldehydes, fatty acids, amino acids, steroids, flavonoids (i.e., flavones, flavonols, flavonones, flavanones), terpenoids, proteins, vitamins, and minerals [19–24]. In Brazil specifically, propolis can be classified into 13 different types according to its physicochemical properties and the geographic area in which it was found and presents a color variation from yellow to brown. However, of these, the main types of propolis are brown, green, and red [13, 25], and these types can be also found in other countries such as Mexico [26], Cuba [9], Taiwan [27], and Turkey [12]. The chemical analysis of propolis is difficult because it is a mixture of substances that presents high variability. This analysis has been more thoroughly performed involving the aqueous and ethanolic extracts (Figure 14.4), since they are the most used presentation forms in several types of therapeutic applications [5–8, 28]. 14.2.1 Brown Propolis Brown propolis does not present a specific main botanical origin. The European brown propolis comes from the resins of the Populus spp. (P. alba, P. nigra, P. tremula) [20], while the Cuban brown propolis has its major origin source attributed to the Clusia spp. [29]. 214 Natural Oral Care in Dental Therapy Figure 14.4 Ethanolic extracts of propolis from southeastern Brazil, southern Chile, and northeastern Brazil (from left to right). Personal file. In its composition are found artepillin C, kaempferide, p-coumaric acid, chlorogenic acid, pinocembrin, p-hydroxybenzoic acid, galangin, chrisin, and pinobanksin, among others [25, 30]. These compounds are associated with the preventive effect of brown propolis in healthy subjects, avoiding the development of cardiovascular disease, diabetes, and cancer [30] and presenting activity against oral bacteria [31]. 14.2.2 Green Propolis Green propolis originates from Brazil and has its major plant source present in the Baccharis spp., especially the Baccharis dracunculifolia (called “wild rosemary” in Brazil). This vegetable is very common to find in vacant lots and other open places because it needs a lot of light to survive. The most prevalent compounds of Brazilian green propolis are kaempferide, p-coumaric acid, kaempferol, artepillin C, ferulic acid, caffeic acid, and pinocembrin [7, 25]. The overvaluation of this propolis is mainly explained due to the antibacterial, antitumor, and anti-inflammatory activities of artepillin C and p-coumaric acid, which can be found in higher concentrations in this type of propolis as compared with any other type [25]. 14.2.3 Red Propolis Red propolis comes mainly from plants of the gender Dalbergia, which are found in different species such as Dalbergia odorifera and Dalbergia ecastophyllum (popularly known as “howler monkey’s tail” in Brazil). These plants can be found near beaches and riverbanks and mangroves as a result of their preference for saline environments [2, 22] (Figures 14.5 and 14.6). This product is composed of several classes of flavonoids, such as flavone (rutin, luteolin), isoflavone (formononetin, daidzein), and dihydroflavonol (pinobanksin, Propolis Benefits in Oral Health 215 Figure 14.5 Raw red propolis from Southeastern Brazil. Personal file. Figure 14.6 Raw red propolis from Northeastern Brazil. Personal file. pinobanksin-3-acetate), which are mainly compounds associated with the biological activities of this group of propolis [6, 8, 22]. It is known that antioxidant and antimicrobial substances exist in higher concentrations in the hexane fraction than in the crude extract of red propolis [32]. A phenolic acid and seven flavonoids present in crude propolis as well as the extract and hexane fraction were quantified in a laboratory study, which revealed that formononetin and pinocembrin were the main constituents [33] (Table 14.1). 14.3 Biological Properties of Propolis The significant use of the propolis extract has been associated with its antimicrobial, antiinflammatory, healing, anesthetic, and antioxidant actions, supporting its diverse applicability [6, 11, 34–36]. The preparation and presentation form of this product, among other factors, may result in biological properties with different potentials. Ethanol is considered 216 Natural Oral Care in Dental Therapy Table 14.1 Mean concentration (μg/mL) of compounds isolated from raw red propolis (powder), the ethanolic extract, and the hexane fraction. Compounds isolated from red propolis Mean concentration (μg/mL) Raw Ethanolic extract Hexane fraction Ferulic acid 1.17 0.54 0.25 Biochanin A 0.54 0.36 0.32 Daidzein 0.19 0.01 0.03 Formononetin 7.21 2.28 2.47 Luteolin 0.65 1.06 0.6 Pinocembrin 3.32 2.19 2.33 Rutin 0.36 0.20 0.19 Quercetin 1.13 0.38 0.39 Total 14.57 7.02 6.58 Source: Das Neves et al. (2016) [33]. the most used solvent to obtain propolis extracts, because of the interaction of the chemical characteristics with the propolis matrix [37], in addition solvents such as ethyl ether, water and chloroform can be considered good constituents for the production of extracts [22, 37]. 14.3.1 Oral Antibacterial Activity Given that synthetic products, such as antimicrobials, when used for a long period, cause microbial resistance, loss of gustatory sensitivity, and pigmentation of the teeth and mucosa, the search for equivalent biocompatible alternatives that control the imbalance of the oral microbiota homeostasis becomes relevant [38]. In this way, propolis appears as an alternative treatment, since it has demonstrated antimicrobial activity and, for this reason, the product has been an object of frequent study in the design of new products with potential clinical application [6–8, 39–42]. In addition, propolis inhibits glycosyltransferases and oral bacteria involved in the caries process such as Streptococcus mutans [7, 8] and Streptococcus sanguinis [43]. These effects may be observed at varying levels depending on seasonality and the site of product collection, which interfere with the concentration of biologically active compounds present in the propolis [6, 11]. The antibacterial activity of propolis can be attributed to phenolic compounds, which inhibit the adhesion of bacteria to the surface of the teeth, reducing the production of the enzyme glycosyltransferase, an enzyme that is responsible for the production of extracellular polysaccharides [44], amylase, and acids derived from food fermentation besides controlling the demineralization of the enamel [45, 46]. A Brazilian brown propolis (20% and 40%) was previously tested as an intracanal medication in association or not with calcium hydroxide paste and successfully reduced the Propolis Benefits in Oral Health 217 number of Enterococcus faecalis viable after 14 days of assay [31]. Another study verified that brown propolis extracts exhibited the lowest antimicrobial activity against Staphylococcus aureus (ATCC no. 25923) compared with green and red propolis extracts and showed a similar effect as that of the green extract against Enterococcus spp. (ATCC no. 2912), Klebsiella spp. (ATCC no. 1706/700603), and Escherichia coli (ATCC no. 25922) [47]. A Taiwanese green propolis extract exhibited significant antibacterial activity, principally against Gram-positive bacterial strains [27], similar to that observed with an ethanolic extract of Brazilian green propolis (3%) used to control oral hygiene after minor oral surgery [48]. Separately, another extract of Brazilian green propolis reduced the number of bacteria present in the S. mutans biofilm and inhibited the demineralization process [7] in a manner similar to the outcome observed with a red propolis extract [8]. The antimicrobial activity of red propolis was verified against several microorganisms related to the oral cavity, such as S. mutans [6–8], Streptococcus sobrinus [6, 14], Actinomyces naeslundii [6], Enterococcus spp. [31, 47], and Staphylococcus aureus [6, 47]. An ethanolic extract of red propolis presented activity against both Gram-positive (100%) and Gramnegative (62.5%) strains [49]. Other research revealed that purified vestitol and neovestitol, isoflavonoids isolated from a Brazilian red propolis, presented better antimicrobial activity than did the raw, chloroform, or hexane extracts [39, 40]. In a study, a solution containing alcohol-free propolis (2%) was evaluated for its efficacy in reducing the salivary levels of the mutans streptococci and lactobacilli and regarding patient acceptability of the product. After 28 days of treatment (2×/day), it was found that the propolis group presented, at different times, better results than either the chlorhexidine group or the placebo group for both microorganisms. In addition, propolis presented a greater residual effect after 17 days of discontinuation of its use and was the product with a greater percentage of patient satisfaction and acceptability [50]. Elsewhere, a propolis tincture, even at low concentrations, reduced more than 99% of microorganisms after 10 min of exposure, suggesting its use in controlling biofilmassociated infections [51], while in other study in vivo, a dentifrice with propolis led to a reduction in the microbial of S. mutans in the oral cavities of young patients [52]. A review showed that different presentation forms of propolis (e.g., extracts, isolated fractions, purified compounds) reduced strains of S. mutans, besides that glucosyltransferase activity and their adhesion capacity [3]. Other studies in vivo have demonstrated reductions in S. mutans counts in saliva [53, 54] as well as decreases insoluble polysaccharide formation and plaque index [55] with the use of propolis. Therefore, propolis can be considered as a promising cariostatic agent for routine clinical use [3]. Considering the propolis mechanism of action in relation to its antibacterial properties, pinocembrin, galangin, and caffeic acid phenylethyl ester are some phenolic compounds found in propolis that probably inhibit the bacterial RNA polymerase [56]. Other propolis components with antimicrobial properties such as flavonoids, benzoic acid, and cinnamic acid as well as caffeic acid interact specifically with the cell wall of bacteria, causing cell lysis and bacterial death [4]. This finding suggests that propolis causes bacterial death through functional and structural damage, but it is known that the antibacterial activity is greater against Gram-negative bacteria and limited against Gram-positive bacteria, probably the greatest resistance is due to the presence of the chemical composition of the wall being more complex and having a higher lipid content (Figure 14.7A, B). The mechanism of propolis to inhibit the growth of Gram-positive strains remains unclear to date [27]. 218 Natural Oral Care in Dental Therapy (a) (b) Antibacterial Action Mechanism Raw Propolis and Extracts Site of propolis action B1) RNA polymerase 3’ 5’ 5’ DNA RNA polymerase 3’ RNAtranscript 5’ B2) Bacterial Cell Wall Gram-positive bacteria Lipopolysaccharide Peptidoglycan Gram-negative bacteria Outer Membrane Cell wall Cell wall Site of propolis action Periplasmic gel Site of propolis action Plasma membrane Plasma membrane Protein Gram-negative cell wall Gram-positive cell wall (c) Antifungal Action Mechanism (d) Anticancer Action Mechanism Effect on the transition from yeast to hyphae D1) Suppress cancer cell proliferation via anti-inflammatory effects D2) Reduces the cancer stem cell population Hyphae Yeasts D3) Blocks specific oncogene-signalling pathways D4) Exerts antiangiogenic effects Pseudohyphae D5) Modulating the tumour microenvironment Figure 14.7 Action mechanism of propolis: A) raw propolis and extracts; B) antibacterial action mechanism by inhibition of the bacterial RNA polymerase—B1 (Adapted from Carbonaro, T.M., 2011) or via bacterial cell wall, causing cell lysis and bacterial death—B2 (Adapted from Person Education. Inc., University of Colorado, Denver, publishing in Benjamin Cummings); C) Antifungal action for inhibiting the initial stages of infection processes; D) Anticancer action mechanism (Adapted from Instituto Vencer o Câncer). Propolis Benefits in Oral Health 219 14.3.2 Oral Antifungal Activity Systemic candidiasis is a fungal infection considered a public health problem due to the growing proliferation of multidrug-resistant Candida species [57]. The mechanisms of resistance of these microorganisms may be related to alterations in drug targets involving changes in membrane sterol, membrane-localized drug efflux pump assays at the functional and transcriptional levels, and reduced or limited drug penetration through biofilms [58]. To overcome fungal resistance, it is necessary to control the proliferation of Candida species without the drug causing a fungicidal effect [59]—that is, the microorganism must be in symbiosis with the human microbiota instead of being eliminated [60]. In this sense, the antifungal action of propolis is related to reducing, controlling, or removing the fungal agent; despite being a natural product, it is expected to interfere with the production of virulence factors and resistance to drugs at low doses [35, 61]. Probably, this antifungal action may be related to the synergistic activity between the phenolic compounds and flavonoids present in propolis [62]. Thus, there is an urgent need for new therapeutic approaches for candidiasis as a complementary treatment to synthetic fungicides, including strategies that use natural products such as propolis. Investigations must be carried out not only to find new compounds with action against Candida but also to elucidate the mechanisms of action of natural products like propolis, since this microorganism is a dimorphic fungus, possessing a yeast form that is associated with asymptomatic colonization and a filamentous form involved in pathological processes and favors adaptation in different biological niches [58]. For this reason, the formation of hyphae contributes to the fungus penetrating the host tissues, leading to the establishment of infection [63]. Thus, the action of propolis is involved in the initial stages of infection processes, in which its effect affects the transition from yeasts to hyphae, considered a critical attribute in the virulence of C. albicans (Figure 14.7C) [64]. Due to the difficulty that exists in comparing the antifungal activity among the propolis varieties, there is evidence that supports the use of this natural product as a therapeutic for this problem. In studies that evaluated the action of the hydroalcoholic extract of propolis green (30%; Brazil) against Candida isolates, a greater effect against C. albicans and C. parapsilosis was observed when compared with C. tropicalis cells. In addition, propolis prevented the biofilm growth of Candida spp. and was able to eradicate the more mature biofilms, as well as to reduce the filamentation of C. tropicalis and C. albicans [64]. In one study, 50 ethanolic extracts of propolis (EEPs) were tested against 69 clinical strains isolated from C. albicans. Most of the EEPs showed satisfactory activity and eradication of the biofilm formed by C. glabrata and C. krusei on the surfaces of polyvinyl chloride and silicone catheters. The EEPs at the subinhibitory concentrations inhibited the morphological transformation of yeast into the mycelium of C. albicans in liquid medium and mycelial growth in solid medium, respectively. An additional effect was observed when the EEPs were combined with fluconazole and voriconazole against C. albicans [65]. In another study, the action of the hydroalcoholic extract of commercial propolis (20%; Brazil) against strains of C. albicans isolated from patients with human immunodeficiency virus and oral candidiasis was compared with the inhibitory action of the synthetic antifungals as nystatin, clotrimazole, econazole, and fluconazole. No difference was found between the results of propolis and nystatin, whereas better results were observed for propolis than for the other antifungals [66]. 220 Natural Oral Care in Dental Therapy Regarding propolis products in the dental area, it has been observed that their performance is related to the control of denture stomatitis, a chronic infection caused by the excessive proliferation of Candida species [67]. Due to the peculiarities of this strain and the ability of this biofilm to adhere to the tissues and surfaces of dental prostheses [68], this condition has a high prevalence among the elderly population and in individuals who do not adequately clean these surfaces [67]. With regard to hygiene, it is known that the combination of mechanical and chemical methods of denture cleaning is more effective than the isolated mechanical method [69]. In this sense, in the search of sanitation methods that cause less surface changes such as abrasion and porosity, propolis products have been used due to their antifungal activity and low influence on microbial resistance [35, 64]. In a randomized clinical trial, Brazilian green propolis (2%) was evaluated to control prosthetic stomatitis in the elderly, and its effect was compared to miconazole gel (2%). There were significant reductions in the colony-forming unit count of 70% in the miconazole group and 25% in the propolis group [70]. In another study where the same oral miconazole gel was evaluated, the efficacy of a gel (2.5%) and mouthwash (24%) composed of Brazilian green propolis in patients diagnosed with prosthetic stomatitis produced a significant reduction or complete remission of (p > 0.05) and significant reduction of Candida colonies for miconazole oral gel, propolis gel and propolis for mouthwash, but without difference between the treatment groups (p > 0.05) [71]. In another study, patients received Daktarin (miconazole gel, Janssen-Cilag, USA) and Brazilian propolis gel (3%), and were instructed to use the product four times a day for 1 week. The patients were again examined after treatment, and it was observed that the two products tested caused complete clinical remission of palatal edema and erythema [72]. Despite significant progress in the applicability of propolis, the area of dental products is still growing, being important to encourage innovation and development of new products with the addition of natural products such as propolis, based on knowledge of its composition and therapeutic properties. 14.4 Other Biological Properties of Propolis 14.4.1 Anti-Inflammatory Activity The association of propolis and chlorhexidine may improve the recognition of antigens by monocytes, mildly activate the transcription factor nuclear factors kappa B, and increase the bactericidal action of human monocytes against S. mutans [16]. In a clinical study, an Indian propolis extract was used as an adjuvant to scaling and root planning in the treatment of periodontitis and showed promising results when assessed according to clinical and microbiological parameters [73]. The flavonoids present in propolis are considered representative phenolic groups among products of natural origin and are suggested to be associated with the anti-inflammatory activity observed in propolis. Among these flavonoids, we can mention galangin, which probably acts by inhibiting the enzyme cyclooxygenase (COX), which is responsible for the formation of prostaglandins. The immune system is modulated by thymus activation and inhibition of prostaglandin production, stimulating cellular immunity and promoting Propolis Benefits in Oral Health 221 phagocytosis. Another flavonoid present in propolis is the caffeic acid phenethyl ester, which boasts of anti-inflammatory activity to prevent the release of arachidonic acid from the cell membrane, suppressing the activities of COX-1 and COX-2 enzymes [74]. Therefore, flavonoids act by modulating cells involved with inflammation, inhibiting the production of proinflammatory cytokines, modulating the activity of the enzymes of the arachidonic acid pathway, and modulating nitric oxide-forming enzymes [75]. 14.4.2 Antioxidant Activity In addition to its antimicrobial and anti-inflammatory activities, propolis contributes to tissue reorganization and is a promissory product to be added to topical formulations due to its antioxidant properties [19]. The high content of flavonoids and phenolic compounds are associated with antioxidant activity. Red propolis has the highest overall antioxidant potential when compared with the brown and green propolis types, and when the extract is obtained by the phenol extraction method, a higher antioxidant activity is observed [47]. However, the relationship of bioavailability of propolis and the continued use of its oral administration is not well defined in the literature. In a study that used a brown propolis extract for determining the bioavailability in mice, the investigators observed that the glucuronide metabolite of galangin was measurable at 5 min after management in the plasma of mice. This result suggests that the galangin, present in the composition of the brown propolis extract, is rapidly absorbed and metabolized, promoting adaptations in the first line defense antioxidant system [76]. 14.4.3 Anticancer Activity Oral cancer can be treated conventionally by means of surgery, radiotherapy, and chemotherapy. However, due the disadvantages associated with these treatments, the literature has detailed an increasing interest in natural products that can combat cancer and its side effects, prevent them from occurring, and increase the lifespan and quality of life of affected patients. Propolis is one of the natural agents that have been studied for these purposes, and its antitumor action is attributed to its flavonoids [19]. It is suggested that the flavonoids found in propolis present antitumor action through different mechanisms such as suppressing cancer cell proliferation via anti-inflammatory action, reducing the cancer stem cell population, blocking specific oncogene-signaling pathways, exhibited antiangiogenic effects, and modulating the tumor microenvironment (Figure 14.7D). Good bioavailability through oral administration and its safety profile make propolis an ideal adjunct agent for use in future immunomodulatory or anticancer treatments [77]. 14.5 Benefits for Oral Health and Applications in Dentistry Laboratory studies and clinical trials with propolis in different presentation forms including spray, extract, mouthwash, gel, toothpaste, chewing gum, and tablet (Figure 14.8) have demonstrated its positive effect against oral pathogens. However, the production of easy 222 Natural Oral Care in Dental Therapy Figure 14.8 Tablets of red propolis. Personal file. and fast tests is necessary to further evaluate the composition and quality of propolis before widespread adoption by the industry. In dentistry, propolis can be used in different ways; for example, it can be incorporated into endodontic materials such as intracanal medications [31] or used for the healing of oral surgical wounds, as irrigating solutions for the treatment of periodontal diseases [78, 79], and for treatment of prosthetic stomatitis [70]. It further can be incorporated into chewing gums [54], mouthwashes [80], or varnishes [81, 82] associated or not with fluoride for the prevention and treatment of early stages of caries or in adhesive systems, restorative materials such as glass ionomer cement, or in the treatment of dentin hypersensitivity. In addition to these multiple applications, the use of propolis in storage medium for avulsed teeth has been highlighted, because it can increase the survival time of the cells of the periodontal ligament [83–85]. In addition, due to the adhesion characteristics of resinous products, propolis has been employed in the pulp capping agent [86–89]. Another effect that deserves to be highlighted is its action on recurrent aphthous stomatitis by helping to stimulate the immune system [90, 91]. It is also observed that propolis has contributed to the formation aiding osseointegration [92, 93]. Therefore, there exists a wide range of possibilities for propolis application in dentistry and the use of products with propolis to promote oral healthcare [51]. Importantly, though, biocompatibility should be considered to ensure safety during its clinical usage. This characteristic can be evaluated to determine the quality of formulations derived from the natural product. 14.6 Final Considerations Even with the variability of the natural products and the different designs of studies found in the literature, it can be affirmed that propolis has the potential to be used as complementary therapy in caries prevention, dental biofilm control, treatment of periodontal diseases, control of fungal infections such as prosthetic stomatitis, surgical wound healing, pulpal therapies, and restorative treatments when incorporated into materials such as glass ionomer cement or adhesive systems. However, one should know that its use may causes allergic reactions in some patients, so it should be marked as contraindicated for patients with a propolis allergy or allergy to any components resulting from beekeeping or bees. Propolis Benefits in Oral Health 223 Despite the growing number of investigations about natural products, additional studies are still needed to assess the toxicity and mechanism of action of propolis, besides its interactions with other drugs and its cost-effectiveness in comparison with commonly used synthetic products and/or other natural products. In addition, it is necessary to control the quality of these products so that they can be marketed with greater security and better efficacy. For this, the prominent interest of the industry in natural products such as propolis can favor its large-scale use, both regarding products for professional and commercial use, thus promoting improvements in the oral health of different population groups. 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Moradi, S., Saghravanian, N., Moushekhian, S., Fatemi, S., Forghani, M., Immunohistochemical Evaluation of Fibronectin and Tenascin Following Direct Pulp Capping with Mineral Trioxide Aggregate, Platelet-Rich Plasma and Propolis in Dogs’ Teeth. Iran. Endod. J., 10, 3, 188–92, 2015. 88. Lima, R.V., Esmeraldo, M.R., de Carvalho, M.G., de Oliveira, P.T., de Carvalho, R.A., da Silva, F.L. Jr, de Brito Costa, E.M., Pulp repair after pulpotomy using different pulp capping agents: A comparative histologic analysis. Pediatr. Dent., 33, 1, 14–8, 2011. 89. Parolia, A., Kundabala, M., Rao, N.N., Acharya, S.R., Agrawal, P., Mohan, M., Thomas, M., A comparative histological analysis of human pulp following direct pulp capping with Propolis, mineral trioxide aggregate and Dycal. Aust. Dent. J., 55, 1, 59–64, 2010. 90. Sulaiman, G.M., Ad’hiah, A.H., Al-Sammarrae, K.W., Bagnati, R., Frapolli, R., Bello, E. et al., Assessing the anti-tumour properties of Iraqi propolis in vitro and in vivo. Food Chem. Toxicol., 50, 5, 1632–1641, 2012. 91. Atanasovska, S.A., Popovska, M., Muratovska, I., Mitic, K., Stefanovska, E., Radojkova, N.V., Therapeutic effect of proaftol in treatment of recurrent aphthous stomatitis. Pril (Makedon Akad Nauk Umet Odd Med Nauki), 35, 3, 195–202, 2014. 92. Sowmya, 56. Redefining the precision of utilising propolis in the field of osseointergration. J. Indian Prosthodont. Soc., 18, Suppl 2, S91, 2018. 93. Somsanith, N., Kim, Y.K., Jang, Y.S., Lee, Y.H., Yi, H.K., Jang, J.H., Kim, K.A., Bae, T.S., Lee, M.H., Enhancing of Osseointegration with Propolis-Loaded TiO2; Nanotubes in Rat Mandible for Dental Implants. Materials (Basel), 11, 1, pii: E61, 2018. 15 Grape Seed Extracts in Dental Therapy Anusuya V, Ashok Kumar Jena and Jitendra Sharan * Department of Dentistry, AIIMS, Bhubaneswar, Odisha, India Abstract Grapes are one of the majorly consumed fruits worldwide. They are a rich source of polyphenols, carbohydrates and fruit acids. The presence of polyphenols and flavonoids increases the medicinal value of the grape products. Grape seed extracts are used in the dental therapy for its antioxidant, anti-proliferative, anti-inflammatory, anti-apoptotic, cytoprotective effects and collagen-cross linking ability with proteins. Different levels of trials and studies are going on the applications of grape seed extracts in the field of medicine. In dentistry, the application is quite less and more focused on the restorative dentistry and cariology, and applications in the remaining fields have also been tried in various disease conditions. Though there is a need for enormous studies, clinical trials, and development, this chapter throws important insights into the various aspects of grape seed extracts. Keywords: Grape seed extracts, polyphenols, proanthocyanidines, cross-linking agent, antioxidants, remineralizing agent, dentistry, cariology, anti-cancer effects 15.1 Introduction Grapes are one of the oldest and majorly cultivated fruits around the world. The majority of grapes are grown in vineyards and used for the production of wine. Next to wine production, a major portion of the grapes is used as table grapes, i.e., whole grapes for fresh consumption. Also, grapes are used in various forms like raisins, grape seed extracts (GSEs), grape seed oils, extract of grape skins, etc. Medicinal applications of grapes are mainly due to the presence of flavonoids in it. Grapes are a rich source of polyphenols, present next to carbohydrates and fruit acids [1]. Polyphenols are of prime importance in its role as antioxidant, anti-proliferative, anti-inflammatory, anti-apoptotic, cytoprotective effects, and collagen-cross-linking ability with proteins. Grapes belong to the genus Vitis, which was first studied by Linnaeus in 1735. Different species of Vitis originated from different parts of the world, among which Vitis vinifera is one of the oldest variety used for winery and others, and is of European origin. Grape seeds are eliminated as waste products from the wine industry. In grapes, seeds constitute about *Corresponding author: vanusuya91@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (229–258) © 2020 Scrivener Publishing LLC 229 230 Natural Oral Care in Dental Therapy 10% of the weight of the fruit [2]. During growth, they are a rich source of hormones, so they contribute to the regulation of berry development and ripening. The seeds contain proteins, lipids, carbohydrates, and phenolic compounds in various proportions depending on the variety of the fruit. The phenolic compounds present in grape seeds are polyphenols, are the important therapeutic content, which makes the grape seed extract possible to use in medicine and dentistry. These phenolic compounds represent the third most abundant constituents in grapes [3]. They are mainly flavonoids including gallic acids, monomeric and polymeric forms of phenolic compounds. GSE is known for its potent antioxidant properties that help in the protection against aging, disease conditions, and its anti-inflammatory properties help in reducing inflammation [4]. The presence of proanthocyanidins (PAs) in the grape seed extracts a natural cross-linking agent and strengthens the collagen fibers of dentinal tubules [5]. Also, the biomimetic properties of proanthocyanidins help in the biomodification of the dentinal structure, which in turn help in improving the mechanical properties of tooth structure. Thus, the GSE is an evolving therapeutic agent for its various applications in dentistry. The present chapter elaborates on the basics of GSEs and its use in all the spectrum of dental therapy. The first half of the chapter explains about the basics of GSEs, its biochemistry, properties, the method of preparation, and molecular mechanism of biological actions, and the second half elaborates on the various uses of GSE in dentistry. 15.2 Part I: Basics About Grape Seed Extracts Grape seed extracts (GSEs) are the crude extracts of various phenolic compounds and its mixture or concentrated extract of an individual phenolic component through chemical processing of grape seeds to be used for different purposes. GSEs used in dentistry are preferably prepared from the seeds obtained from the Vitis vinifera species. Because grape seed varieties belonging to Vitis vinifera have the highest amount of flavonoids [6]. Flavon-3-ols are the most common flavonoids present in grape seeds and occur in various forms including monomers, oligomers, and polymers. The amount and type of phenolic compounds in the grape seeds are influenced by various factors like the location of production, cultivar, season, and degree of maturation. 15.2.1 Components of Grape Seed Extracts Phenolic compounds are the basic bioactive natural compounds found in the GSEs. Among the wide variety of phenolic compounds, two groups are found in the GSEs, i.e., flavonoids and phenolic acids. Flavan-3-ols, otherwise known as catechins, are the most predominantly found flavonoids in grape seeds. Phenolic acids include various proportions of gallic acid either in free form or esterified with flavonoids. Polymerization of these monomer compounds produces oligomer, dimer, and polymers [1]. Three types of flavan-3-ol found in grape seeds are monomer, dimers, and trimmers, also in galloylated forms. The monomers include catechin, gallocatechin, epicatechin, and epigallocatechin, etc. Catechins condense into oligomeric procyanidins, i.e., dimers found in the seeds and polymeric compounds otherwise called as condensed tannins [7]. More than 14 dimeric, 11 trimeric, and 1 tetrameric procyanidin were separated from the grape seeds, and highly polymerized procyanidins also present in it. Nearly 55% of the proanthocyanidins in the GSEs consisted of ≥5 monomer units [8]. Grape Seed Extracts 231 HO HO HO HO B HO HO O A O* R C HO HO HO (a) HO (b) Figure 15.1 Molecular structure of (a) catechin and (b) cyanidin. Condensed tannins, commonly known as proanthocyanidins, are the essential constituent of grape seed extracts. This polymeric form is the active content for its cross-linking property with the collagen fibers. The degree of polymerization affects the ability to crosslink. Condensed tannins are responsible for the organoleptic properties of wines. It adds to the astringency of wines and grape juice that is felt as puckering sensation in the mouth just after drinking. This is due to the precipitation of salivary proteins by the tannins [9]. 15.2.2 Chemical Structure The structure of PAs has been studied widely. The molecular structure of dimeric and trimeric PAs was studied using high-performance liquid chromatography (HPLC) analysis. It consist of catechin and epicatechin units. Catechin is built upon the diphenylpropane skeleton; the three-carbon bridge (C6–C3–C6) between the phenyl groups is usually cyclized with oxygen [10]. Figure 15.1a, b shows the chemical structure of catechins and cyanidin. The catechins are present in both trans- and cis-configuration. Catechins have three rings named A, B, C with five hydroxyl groups. Based on the R in the third position of the B-ring, it could be either catechin or gallocatechin. Cyanidin has identical arrangements of five hydroxyl groups with catechin, but there is presence of a central anthocyanidin C ring, which is unsaturated. This unsaturated C ring, allowing conjugation, doubles the antioxidant potential of cyanidin [11]. Catechins polymerize into dimers and trimers. Polymerization occurs at the fourth position of the central C ring. They are termed as the B-series and C-series, respectively. The B-series includes B1, B2, B3, B4, and B5, and the C-series includes C1 and C2 in the GSEs. Fuleki and Silva identified 11 monomers with the use of the reverse-phase HPLC from the red grape seeds [12]. The degree of polymerization (DP) may reach up to 16 units [8]. Gallic acid (GA) structure is present in Figure 15.2 GA is, 3,4,5-trihydroxybenzoic acid, one of the commonly found phenolic acids in the GSE. It occurs either in free forms or in the form of esters. Both increases the efficacy of GSE in protecting the body from various oxidative damages. The amount and site of galloylation play an important role in the cross-linking process [13]. Figure 15.3a to 15.3f shows the chemical structure of some commonly present procyanidins in GSEs. 232 Natural Oral Care in Dental Therapy 15.2.3 Types of GSEs GSEs can be categorized into the following types based on the different factors and applications in dentistry. Based on degree of polymerization: a. Monomers include • Catechins • Epicatechins b. Polymers include • Dimers—dimeric procyanidins • Trimers—trimeric procyanidins • Tetrameric procyanidins • Polymeric procyanidins—condensed tannins Based on the molecular weight: • PALM—Proanthocyanidin low molecular weight fraction • PAHM—Proanthocyanidin high molecular weight fraction Based on the Galloylation: a. Non-galloylated species include • CT—Catechin • EC—Epicatechin b. Galloylated species include • EGC—Epigallocatechin • ECG—Epicatechingallate • EGCG—Epigallocatechin gallate Based on the percentage of GSE mixtures GSE mixtures include both monomers and polymers. Based on the percentage of the proanthocyanidin in the solution, it could be 2%, 5%, 10% weight of the resin adhesives. Based on the percentage of GSE in the distal water diluent, it is used in 6.5%, 10%, 25%, 50% irrigant solution in the endodontic irrigants. 15.2.4 Methods of Separation GSEs are prepared most commonly from the seeds of species Vitis vinifera. The preparation of crude extract from grape seeds is explained below in the flow diagram. HO HO COOH HO Figure 15.2 Molecular structure of Gallic acid. Grape Seed Extracts 233 HO HO HO HO HO O O OH R OG HO HO HO (b) (a) OH OH OH OH O HO O HO OH OH OH OH HO HO OH OH O HO O HO H OH HO OH H OH (c) (d) OH OH OH OH O HO O HO OH OH OH HO OH OH HO O HO OH O HO H OH OH (e) H OH HO (f) Figure 15.3 Molecular structure of (a) epicatechin, (b) epicatechin-3-gallate, G=gallic acid, (c) procyanidin dimer B1, (d) procyanidin dimer B2, (e) procyanidin dimer B3 and (f) procyanidin dimer B4. 234 Natural Oral Care in Dental Therapy Grapes (Vitis vinifera) Manual separation of seeds Washing the seeds (preferably with distilled water) Drying process (If air drying then storage at -30˚C until used / if sun drying then storage at room temperature) Pulverization of stored seeds under liquid nitrogen (N2) Extraction of powder (approximately 50 g) 2 times successively in acetone / water with proportion 7:3 (under N2) Removal of the plant debris by centrifuge Evaporation of acetone under vacuum and freeze-drying of aqueous solution Blending of the obtained extracts together (approximately 2.1 g and 1.3 g each) Crude tannin extract (grape seed extract is ready) The crude extract is ready for preparation of various forms of GSEs and separation of polyphenols. Different polyphenols and procyanidins are fractioned from this crude extract. The following methods are used for the fractionation and separation of various constituents of GSEs. • • • • • High-performance liquid chromatography (HPLC) [12] Reverse HPLC [14] Gel permeation chromatography (GPC) [8] Liquid/liquid extraction Thiolysis monitored HPLC [15] • Ultrafiltration [16] In GPC, filtration helps to selectively separate the individual components based on the molecular size. The selective removal of targeted components from the complex mixture is called chemical subtraction. Chemical subtraction is done mainly by the method called countercurrent separation, which specifically separates only the targeted components. This method is further developed for selective enrichment or depletion of extracts and coined as DESIGNER (Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources). Phansalkar et al. modified the DESIGNER CCS technique using centrifugal partition chromatography (CPC) [17]. The CPC was a reliable tool for the development of procyanidin-based dental biomaterials in dental application. 15.2.5 Factors Influencing the Quality and Quantity of Polyphenols in the GSEs Polyphenols, being the third most abundant content in the grapes, are distributed in different amounts in the different parts of the grapes. Phenolic compounds are broadly distributed among the whole pulp, grape seeds, and the skin. The total extractable phenolics are present around 10% in pulp and about 60–70% in the seeds. Skin phenolics contribute around 28–35%. The total content of phenolics in seeds range from 5% to 8% by weight [18]. Production of polyphenols in the grapes depends on many factors. Quality and quantity of Grape Seed Extracts 235 phenolic compounds vary depending on the grape variety. Total phenolic contents of table grapes or seedless grapes are lower than the grapes used for winemaking. Likewise, the composition of phenolics in the red grapes is different from that of white grapes. The following are the important agroecological variables that influence the total content of phenolics in grapes such as cultivar, the climatic condition and site of production, and the degree of maturation. The cultivar The variety of grape seed from which the GSE is prepared affects the quantity of polyphenols in it. Kovac et al. analyzed about 19 cultivars of the winemaking grapes to compare their phenolic contents in the year 1987 [19]. All the grapes were harvested at the same time with the same stage of maturity. On analysis, the total content of phenolics varied from 414 mg/kg to 2,593 mg/kg of the grape. Pinot Gris and Pinot Noir, varieties belonging to the Vitis vinifera, had the highest content of the catechins and procyanidins among the 19 cultivars studied. When comparing the table grapes with winemaking grapes, table grapes contain a lower total phenolic content. The type of phenols also varies according to the cultivar. Fuleki and Silva studied the individual phenolic components and their variation in the following varieties, vinifera, labrusca, and hybrid cultivars [12]. Compositional variation of procyanidin dimers, trimers, monomers (catechins and epicatechins), and dimer gallates were studied. Procyanidin trimers were found to be highest among the Pinot Noir variety (red grapes). Monomers were found to be highest in the hybrid (Vincent) red grapes and equally in the Pinot Noir variety [12]. Climatic condition and site of production The quantity of individual phenolics in the grape seeds from Muscat of Hambourg was studied by Revilla et al. [6]. It was found that the content of the phenolic was variable during the period 1992–1993, in spite of the degree of maturity. Changes in the climatic conditions affect the production and maturation of phenolics in the seeds. Along with climatic changes, the site of production and soil quality affect the quantity of polyphenols in the seeds. Tempranilo grapes cultivated in different sites of Madrid in Spain had total content of catechins and procyanidins from 108 to 225 mg/kg of grape. 15.2.6 Physical Properties of Polyphenols The following physical properties of polyphenols are studied: molecular weight, solubility, melting point, and optical rotation. Molecular weight of the various polyphenols is given in Table 15.1. Molecular weights of the monomer are based on their molecular structure, whereas the molecular weight differences in the polymers are based on the degree of polymerization [20]. This difference in the molecular weight is the fundamental principle in the development of gel permeation chromatography and membrane fractioning of polyphenols. Also, the molecular size affects the cross-linking ability of the GSE constituents with the dentin collagen and its stabilization. The solubility of polyphenols is one of the important physical properties. Some phenolic compounds are water soluble and some are lipid soluble. Most of the monomers are lipid soluble, i.e., catechin is lipid soluble, whereas procyanidins are water soluble. Thus, the difference in the solubility helps in the easy separation of procyanidin from catechins. Therefore, it is easy to prepare the water-soluble procyanidins by separating it without any 236 Natural Oral Care in Dental Therapy Table 15.1 Various physical properties of the polyphenols. S.no Properties Values 1. Molecular weight (MW) Catechin Epicatechin Epicatechin-3-o-gallate Procyanidin dimer Procyanidin trimer Procyanidin tetramer 293 294 445 580 870 1160 Solubility Catechin Procyanidins Lipid-soluble Water-soluble Melting point Catechin Epicatechin Epicatechin-3-o-gallate 174°C 236°C 236°C Optical rotation Catechin Epicatechin Epicatechin-3-o-gallate 0° 58.3° 188° 2. 3. 4. use of organic solvents. It adds more value and increases the safety of the food. This is one aspect of solubility. Another importance in the solubility of polyphenols is the role of polyphenols in scavenging superoxide radicals and understanding its antioxidant potential [11]. Watersoluble procyanidins and lipid-soluble catechins, both follow different pathways of antioxidant mechanisms. In the aqueous phase, the antioxidant activity of procyanidins is mainly due to its reducing activity in the o-dihydroxy structure. Whereas, polyphenols in the lipophilic phase work by either of the following modes: (a) chelating the copper ion, (b) hydrogen donors, and (c) regenerating alpha-tocopherol. Catechin lacking a carbonyl group as well as a 2,3-double bond is relatively ineffective in the inhibition of oxidation compared to procyanidins. 15.2.7 Biochemical Properties (Biological and Pharmacological) of polyphenols The characteristic biochemical properties of polyphenols are based on the ability to interact with different molecules such as macromolecules like proteins and polysaccharides, metal ions, and radical scavenging activities. Phenolic nuclei in the molecule are the principal key structure for its physical and chemical properties. These properties play complete, partial, or at least part of the role in their physiological and pharmacological actions. Among the different biochemical interactions of polyphenols, the following three distinctive characteristics of polyphenols have been proposed as a mode of action for their biochemical interactions as a therapeutic agent. Grape Seed Extracts 237 a. Polyphenol–protein interactions b. Antioxidant and radical scavenging activity c. Metal ion complexation a. Polyphenol–Protein Interactions: Various biological activities of polyphenols as medicinal agents revolve around the interaction between the protein and polyphenols, and protein binding. The puckering sensation/ astringency felt just after the intake of grape juice or wine is due to the precipitation of salivary glycoproteins by tannins [21]. Antioxidant activities, anti-inflammatory, and cytoprotective effects are dependent on the biological efficiency and interaction through different proteins in the body. In turn, the biological efficiency of polyphenols mainly depends on the bioavailability, which is influenced by one of the important factors, i.e., protein–polyphenol interaction. The application of GSEs in conservative dentistry is based on the collagen-stabilizing property. The molecular basis of this property is the interaction between polyphenols and protein. Protein–polyphenol interaction is fundamental for its application as a biomimetic agent for desensitization, as a cross-linking agent in adhesives, in improving the mechanical properties of the dentin and root structure, reducing the demineralization, and as a collagen-stabilizing etchant. The propensity of polyphenols to bind with proteins also accounts for the ability to inhibit various enzymes such as glucosyltransferases of Streptococcus mutans [22]. Polyphenols interact with proteins in a reversible and irreversible manner. Generally, in solution, an initial reversible interaction occurs between protein and phenol that produces soluble protein/tannin complexes until it reaches equilibrium. These soluble complexes may keep increasing in size, which no longer could be soluble and may aggregate or starts precipitating. This may still be in the reversible form, which redissolves or further undergo different processes, i.e., oxidation reaction with metal ions leads to irreversible precipitation of complexes [23]. The precipitation ability of polyphenols differs with respect to the different varieties of proteins. It was found that the relative affinity of condensed tannins varied more than 1,000-fold. The highest affinities were found for proteins, polymers rich in proline content, i.e., proline-rich proteins (PRP), and polypeptides, and the lowest affinity was found for small globular proteins like lysozymes. It has been found that large proteins with high proline contents and with lack of secondary or tertiary structure are readily precipitated by tannins. The polyphenol–protein complexation is specifically a molecular recognition phenomenon. The principle of molecular recognition in the complex formation is mainly of “hand-in-glove”. We need two sites for the complex formation, a donor molecule, and an acceptor molecule. In the hand-in-glove type, matching between the receptor and ligand is a dynamic process and also a time-dependent one. Both the molecules are mobile and flexible; thus, they acquire different shapes as complexation proceeds [23]. This complex formation is dependent on molecular size, protein type, pH, water solubility, composition and conformational flexibility of polyphenols. Table 15.2 outlines the different facets of the polyphenol–protein interaction. Hydrophobic Effects: Solubility in water is an important factor in polyphenol-protein complexation. Presence of hydrophilic groups on molecular surfaces, i.e., hydroxyl groups on polyphenols, and carbonyl 238 Natural Oral Care in Dental Therapy Table 15.2 Facets of the polyphenol–proteins interaction. S. no Different facets 1. Polyphenol–protein complex formation is a time dependent, dynamic surface phenomenon. Structural flexibility of both the molecules is important in the interaction. The molecular recognition is mainly “hand-in-glove” type. Unless further reactions take place, the primary interaction between protein and polyphenol is reversible. 2. Affinity for polyphenols is a highly variable factor. Small, compact proteins have very poor affinity whereas proteins with more open structure such as proline-rich proteins, gelatin, salivary proteins have high affinity toward polyphenols and make much stronger interactions. 3. Phenolic nuclei and presence of aromatic ring provides multidentate ligands to protein structure. Thus the molecular size of the phenolic group is important and the galloylation increases the efficacy of pholyphenol–protein complexation, i.e., tri < tetra < penta. Molecular size and galloylation are directly proportional to the binding efficacy. 4. The energetics of the complexation is mainly associated with “hydrophobic effects” followed by reinforcement via “hydrogen bonds”. and amide groups on peptides are responsible for solubility. The degree of solubility is approximately proportional to the availability and proximity to the hydrophilic groups, where water molecules are anchored between the donor and acceptor. Reorganization of water molecules drives the complexation process to takes place. Hydrophobic effects in the polyphenol–protein complexation are due to the presence of complex species of groups or regions that are hydrophobic in nature. Hydrophobic forces drive these structures in the various molecules to aggregate together and form the complex. The hydrophobic forces are contributed by van der Waals forces and the complex is stabilized further by the entropic effect. It is believed that aromatic rings of polyphenols and carbon-hydrogen skeleton of proteins provide multiple hydrophobic sites to participate in such interactions. So, if the polyphenol is highly water soluble then the formation of the complex with protein is less likely to take place. Thus the polyphenol/protein affinity is in an inverse relationship with its water solubility. Addition of chemical molecules, other substrates, or inorganic acids that alters the polyphenol’s solubility could be used to change the range of polyphenol–protein complexation [24]. Hydrogen Bonding: Hydrogen bonding plays a second key role in the protein complexation. Direct functionality, and presence of multiple hydrogen bonding sites in polyphenols, enhance the overall strength of the hydrogen bond that stabilizes the complex. In this process, molecular recognition occurs through the deployment of hydrogen bonds in water due to the interaction between solutes and forces of solvation. The potential hydrogen bonding groups in the substrate are also usually the water-soluble groups. So, they readily participate in the aqueous medium. Thus, for the hydrogen bonding in protein-polyphenol complex to take place, first, the hydrogen bonds formed by both polyphenols and proteins with the water must be broken. The overall energetics of interactions depend on the stability of hydrogen bonds, which are broken and made. Grape Seed Extracts 239 Carbonyl groups in the tertiary amides are much more effective in accepting the hydrogen bonding than those in the primary and secondary amides. Along with it, the methylene substituents of tertiary amide nitrogen readily donate the electron into the peptide bond, making it to be an electron-rich site. Thus, the carbonyl group has a higher capability for accepting the hydrogen bond. Hydrogen bonding networks are deployed and formed between the proton donors and proton acceptors (phenolic hydroxyl groups of polyphenol–proton donors and carbonyl groups of prolyl peptides-proton acceptors) [25]. b. Antioxidant Activity: In recent years, accumulation of toxins and oxidative damage leads to disease conditions and their pathophysiology has taken much attention. Considerable interest of medicine into the natural antioxidants is increasing day by day. Natural antioxidants are found in fruits, vegetables, green tea leaves, and seed oils. The polyphenolic flavonoids are the potent antioxidants [11]. Though the majority of the flavonoids that are consumed come from tea, wine contributes a considerable amount. The French paradox is a good example of the importance of natural antioxidants in the diet and reduction in coronary heart disease (CHD) [26]. Excessive dietary fatty acids undergoing lipid peroxidation, oxidative accumulation from pathogenic bacteria, and inflammatory process lead to further damage and progression of diseases. In cellular prooxidant states, reactive oxygen species concentration in the intracellular compartment is increased. This may be due to excessive production or reduced ability to eliminate them [27]. The reactive oxygen species are the activated form of oxygen via reduction of oxygen that produces superoxide radical O−2 . The major source of electrons is being leaked from the various electron transport chains in the intracellular components. Thus, the superoxide radical is formed in almost all the aerobic cells. In the aqueous medium, there is spontaneous dismutation of the superoxide radical into hydrogen peroxide formation. The superoxide dismutase enzyme accelerates the reaction. Further, it converts into a highly reactive hydroxyl radical. The oxidative damage occurs mainly due to hydroxyl and hydroperoxyl radicals. However, the less active superoxide and hydrogen peroxide are important because they can diffuse into remote cellular locations and cause distant damages. Other important activated species are reactive nitrogen species (RNS). It includes nitric oxide (NO) radicals and nitrogen dioxide radicals. NO have high affinity toward O−2 radicals, and the reaction between them produces a highly reactive molecule ONOO−. The highly reactive molecule formed, initiates the process of lipid peroxidation, eventually give rise to lipid hydroperoxide [28]. Oxidative stress arises when there is disturbance in the prooxidant/oxidant balance. Normally, cells have its defensive antioxidant mechanisms to remove the reactive molecules either endogenously or by dietary supplements. Polyphenols are known to inhibit the lipid peroxidation and lipoxygenases in-vitro, and they have the ability to scavenge the hydroxyl, superoxide, peroxyl, and nitric oxide radicals. Epidemiological data indicate that the consumption of food items rich in polyphenols increases the plasma antioxidant potential [29]. It was found that red wine contains up to 4 g/L of phenolics, mainly flavan-3-ols, oligomeric procyanidins, and anthocyanidins, and the regular consumption of red wine could reduce the incidence of CHD. Antioxidant activity of proanthocyanidins is superior to vitamin C and vitamin E. One thousand times diluted concentrations of PA from wine extracts were better than vitamin C and E in inhibiting lipid oxidation [30]. The assay of antioxidant potential is measured as total antioxidant activity (TAA) or Trolox equivalent antioxidant activity (TEAC). It could be defined as the 240 Natural Oral Care in Dental Therapy concentration of Trolox solution with an equivalent antioxidant potential to a standard concentration of a compound under investigation [11]. c. Metal Ion–Complexation: Tannins produce a blue-black color when treated with iron salts. It has been noted that the property of natural polyphenols with catechol nuclei forming strong complexes with metal ions such as iron, manganese, and calcium, etc., is important. When concerning the with iron, it is well known for its role in redox reactions, effect on microorganisms, iron balance in the body, and in iron-deficiency diseases. The antimicrobial activity of polyphenols over siderophore producing Escherichia coli occurs through iron depletion [31]. Likewise, the chelation of polyphenols with calcium ions produces the precipitation and deposition of minerals over dentine that acts as a scaffold for the remineralization process. It is one of the important applications in the reparative and therapeutic approach of the dental caries. Biocompatibility and Cytotoxicity of Polyphenols: PAs and other components of GSE are much more biocompatible than other synthetic components. As a natural derivative, and as the source is dietary in origin, it is well tolerated at high doses. The pharmacological actions of PAs are better at high doses. Majority of GSE application in dental therapy is on the prevention and repair of dental caries in vital teeth. GSE must be compatible with the pulpal tissues. Dose-response and time-response of pulpal cells for GSE were found to be biocompatible [32]. The lowest concentration was 0.0065%, and the highest tested was 6.5%, and direct and indirect contact tests were assessed. At the lowest concentration, an increase in cellular metabolism and increase in the extracellular matrix synthesis were found in the direct contact test. The highest cell viability was observed at both the concentrations. In the indirect contact test, the dentinal barrier was used. At a concentration of 0.65%, the viability of cells were maintained with an increase in cellular metabolism. Both the tests revealed no time-dependent response when performed at 24 to 72 h duration. 15.3 Part II: Biological Applications in Dentistry Grapes seed extract is used as a therapeutic agent in medicine for its known benefits. The presence of polyphenols in the GSEs is one of the important biologically active constituents of GSE increases in its medicinal values. Table 15.3 outlines some of the pharmacological and physiological actions of polyphenols. The characteristics of polyphenol such as protein complexation, metal ion complexation, and cross-linking of collagen make it possible to apply in various dental therapies. GSE application in dentistry and dental therapy was explained under different headings based on its uses in the different branches of dentistry for the convenience of understanding. 15.3.1 GSEs in Dental Caries The study about the development of dental caries, its prevention, and therapeutic technique is called cariology. During the process of dental caries, chronic destruction of the tooth structure takes place by demineralization of inorganic and degradation of organic Grape Seed Extracts 241 Table 15.3 The pharmacological and physiological actions of polyphenols. S. no In food industry a. Palatability b. Astringency c. Nutritional value d. Helps in ageing in winery In Medicine a. Antioxidants b. Anti-inflammatory c. Anti-carcinogenic d. Cross-linking activity e. Antibiotic f. Cytoprotective effect g. Anti-mutagenic by directly inhibiting the mutagens In Dentistry a. Cross-linker b. Remineralizing potential c. Anti-erosive capacity d. Antiplaque activity e. Antibacterial, both bactericidal and bacteriostatic f. Dentin biomodification g. Anti-carcinogenic against oral cancer cells h. Endodontic irrigant i. Therapeutic drug In periodontitis parts of the tooth. Dentin occupies the major portion of the tooth and is composed of inorganic minerals, organic matrices, and water. The collagen matrix comprises about 30 vol %, in which 90% are type I collagen, and 10% are non-collagenous substances. In the caries process, demineralization, i.e., dissolving the mineral, occurs via acids that expose the collagen to destruction by bacterially derived enzymes and also by host-derived enzymes. After demineralization, if protected, the collagen matrix will act as a major scaffold for remineralization and plays an important role in remineralization of carious dentin. Fluoride is a well-established remineralizing agent, which prevents and inhibits caries lesions. Fluoride forms flouro-hydroxyapatite crystals, which are more resistant to acid 242 Natural Oral Care in Dental Therapy attack [33]. Thus, the ideal approach in prevention and reduction of caries progression especially root caries should be focused on the use of substances that alters the dentin matrix to promote remineralization, and prevent and resist organic matrix degradation. Recently, various products have been investigated for its antibacterial and remineralizing potential on teeth. Grape seed extracts are used as a natural cross-linking agent that strengthens the dentin organic matrix, and it is also an effective bacterial enzyme inhibitor. The application of GSE in cariology could be divided into those in the prevention of dental caries and those in the reparative therapy of dental caries, which is covered under the topic of GSEs in conservative dentistry. Prevention of Dental Caries Grape seed extracts are used in the prevention of caries by acting on the various stages of caries development. GSE prevents enamel erosions by reducing mineral loss and enhancing re-uptake, antiplaque effect by inhibiting the adhesion of biofilm, antibacterial activity against S. mutans, and degradation of collagen matrix with improvement in the quality of dentin through biomodification. GSEs are used in the prevention of dental caries by the following activities. • • • • • 15.3.2 Anti-erosive agent Antiplaque agent Antibacterial agent Biomodifier Reminearlizing agent Anti-Erosive Agent (Prevention of Enamel Erosion) Enamel is the hardest part of the tooth with 90% of the minerals in the form of hydroxyapatite crystal and the remaining are organic portions and water. These minerals readily dissolve in the acidic environment with a critical pH of <4.5. The erosive pattern of enamel demineralization is different from dentin demineralization. Erosion is the process of tooth structure loss by a chemical process without the involvement of bacteria. Destruction occurs mainly due to the exposure of dental structures to acids, either extrinsic or intrinsic. Extrinsic sources such as occupational exposures and acidic beverages generally cause erosion on the labial surface. Whereas, intrinsic sources of acid are from the reflux of gastric acid into the oral cavity due to conditions like anorexia nervosa, gastroesophageal reflux disease, and bulimia. Intrinsic sources generally cause erosions of the enamel on the palatal and lingual surfaces of the teeth. Prevention of enamel erosion is mainly by increasing the acid-resistant crystal formations in the enamel. Remineralizing agents such as fluorides, bioactive glass, and casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) are used to produce strong hydroxyapatite crystals. GSE has been studied to evaluate the ability to reduce enamel erosions. Nandakumar and Nasim comparatively evaluated in vitro the GSE and cranberry extracts in the prevention of enamel erosion [34]. The evaluation was done using optical emission spectrometry, and stannous fluoride was taken as the control. The erosion remineralization cycle used was the immersion of enamel slab for 10-s in 20 ml of HCL, then 60 s in 20 ml of artificial saliva, then 30-s exposure to 20 ml of respective remineralizing Grape Seed Extracts 243 solutions followed by 60-min immersion in artificial saliva. After three erosive challenges, the amount of mineral loss was assessed through optical emission spectrometry. Exposure to 0.65% w/v solution of GSE for 30-s, considered as the minimal concentration of GSE required to produce remineralization, produced a remarkable amount of remineralization. The possible mechanism for the remineralizing effect is through superficial mineral deposition over the enamel lesion by formation of insoluble complexes. Mirkarimi et al. found that after treating with GSE solution, there was scaffold formation on the surface of the enamel with cluster-like structures resembling initiation of the remineralization process [35]. Prevention of enamel erosion is more of a metal–ion complexation process than decreasing demineralization. Though the GSE solution was producing remineralization, compared with the gold standard stannous fluoride, the effect was inferior. There were no significant or comparable results of GSE solution with stannous fluoride in enamel remineralization. However, GSE is more effective in the prevention of enamel erosion. When the effects on artificial enamel caries of primary teeth were studied, GSE had positive effects in vitro on the remineralization of enamel caries lesions. Both in permanent and primary caries prevention, GSE could be an effective non-invasive modality to treat caries. 15.3.3 Antiplaque Effect Dental plaque is a biofilm formed on the tooth surface and other structures of the oral cavity. It is the major etiological factor for oral diseases such as dental caries, gingivitis, and periodontitis. It is tenaciously adherent, gelatinous microbial colonies with hundreds of diverse species within this ecological niche. Microbial composition varies periodically and depending on the location in the oral cavity. However, it is clearly found that in the progression of plaque, streptococci are the predominant species, which pioneer the plaque formation, followed by actinomyces, and later with the mature colonies of gram-negative bacteria. Thus, an effective antiplaque agent works at different levels of biofilm such as anti-adhesive, preventing the bacterial cohesions, reduction in the growth of microbial colonies, and bactericidal effect. The distinct step in plaque development is the formation of a condensed layer of salivary pellicle at the base of the dental plaque. This pellicle serves as the receptor site for binding of various bacteria. Among the streptococci groups, two groups are important in the formation of plaque. One is S. sanguis and the other is S. mutans [36]. Though S. sanguis is the pioneer in the attachment of plaque, S. mutans is the major etiological agent in caries and other oral diseases. Thus, kinetics of oral bacterial adhesion and the adherence characteristics of S. mutans were the subjects of interest. Inhibition of Adhesion: Microbial adherence is the essential step for plaque formation. Gibbons suggested two processes of bacterial adhesion to the tooth surfaces. The first one is adsorption of the cells to the pellicle, and the second one is the binding of cells to each other to further building up of plaque. S. mutans bacteria are meant for its production of insoluble glucans that helps in binding of cells. Glucosyltransferases, which are produced by S. mutans, polymerize the glucosyl moiety from sucrose and starch carbohydrates into glucans. The insoluble glucans, alpha-1,3 branched glucans produces the cohesion of bacteria. Thus, controlling the bacterial adhesion by interfering these processes would be a better mode of antiplaque activity. 244 Natural Oral Care in Dental Therapy Major components of grape seed extracts are condensed tannins, which are composed mainly by different polyphenols. Polyphenols are well known for their interactions with proteins. One of the key characteristics of polyphenols for its biological activity is the ability to interact with proteins and form protein–polyphenol complexes. This complexation produces different effects in different ways depending on the type of protein–polyphenol complex. Polyphenol interaction with salivary proteins precipitates them, which produces the astringency of wine. While polyphenol binds with plasma proteins, then the bioavailability of polyphenols is reduced. Likewise, polyphenols bind with peptide enzymes and deactivate them. This important biochemical property of polyphenols is used to selectively inhibit unwanted enzymes and break their biochemical cycle. Polyphenols from the GSE binds with glucosyltransferase (GTF) enzyme and inhibits its action. Thus, the synthesis of insoluble glucans is inhibited, and biofilm formation is disturbed. This is the expected basic mechanism of action in the use of GSE as an antiplaque agent. GSE effects on GTF activity has been studied in vitro separately or in combination with Fluorinol (3-pyridinemethanol hydrofluoride) [37]. GSE separately inhibited the GTF by about 43.9%. When combined with the Fluorinol, the inhibition was more with 65.7% for the initial overall reaction. After 24 h of observation, the inhibitory activity did not improve significantly. It suggested the contribution of GSE in the inhibition of GTF that majority was done by GSE in the initial reaction itself. Biofilm inhibition and antibacterial activity of GSE is explained in the following sessions, which also contribute mainly to the antiplaque effect. Thus, GSE acts in multiple ways to reduce the plaque activity. 15.3.4 Antibacterial Agent GSEs also have antibacterial activity along with antiplaque activity. It was found that minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were high for GSE. MIC is the concentration at which no bacterial growth was visible after 24-h incubation at 37°C. MBC is the concentration at which the CFU number was reduced by 99.9%. The MIC value of GSE against S. mutans was 2 mg ml−1 and the MBC was 4 mg ml−1. Antibacterial effect of GSE reduces bacterial growth and biofilm formation. This accounts for the combined mechanism of antiplaque agent along with inhibition of glucosyltransferase enzymes. Biofilm assay was done to measure the antibacterial efficacy of GSE. After 64 h of incubation, the biofilm formation from the dental plaque sample produced around 33.6 ± 2.1 µm. When exposed to 2 mg ml−1 of GSE, a significant decrease in the microcolonies was observed with biofilm thickness 17.9 ± 3.2 µm. Considering the concentration of GSE used, it was found that the most successful concentration for inhibiting the biofilm is 2 mg ml−1, above which its effectiveness decreases due to the poor dissolution in water [37]. The procyanidin fraction of the polyphenols was the most active antibacterial component than other fractions, with MIC value against the S. mutans of 1.0 mg ml−1. Table 15.4 outlines the MIC values of procyanidin against different species [38]. Milan Swadas et al. comparatively tested the antimicrobial activity of GSE at various concentrations against the chlorhexidine gluconate by measuring the number of colony-forming units. GSE with concentrations 500 mg/mL and 250 mg/mL had an antibacterial effect on the S. mutans colonies, but they were not superior or equal to 2% chlorhexidine gluconate [39]. Apart from S. mutans, another important bacteria studied was A. actinomycetemcomitans ATCC 43718. Grape Seed Extracts 245 Table 15.4 MIC values of procyanidin against different species. S. no Microorganism MIC values, mg ml−1 1. Candida albicans >64 2. Enterococcus faecalis 32 3. Escherichia coli 32 4. Pseudomonas aeruginosa 32 5. Aggregatibacter actinomycetemcomitans 4 (3.84) 6. Streptococcus mutans 1 7. Streptococcus oralis 0.25 8. Streptococcus salivarius 0.25 9. Streptococcus sanguis 0.25 10. Lactobacillus acidophillus 0.25 The antioxidant activity of GSE also provides an additional benefit while used as an antiplaque agent. Generally, antiplaque agents are used in the form of mouthwashes. The availability of GSE to scavenge the superoxide radicals produced by plaque bacterial colonies helps in the reduction of the damage and inflammatory reaction from the gingivae and periodontium. 15.3.5 Biomodifier The biomimetic approach increases the quality of dentin structure by improving the organic and inorganic portions of tooth material to resist the process of dental caries along with enhancing the mechanical properties of the bio-improved microstructure of tooth. We saw that the caries process of enamel and dentin differs totally because of the difference in the composition of both. Dentin is composed of a more organic matrix relatively, which makes it important to improve and strengthen the collagen matrix, which will resist degradation as well as act as a scaffold for remineralization. The root caries is the same as that of dentinal caries in its progression, and it is difficult to approach to restore. The early involvement of pulp due to its proximity leads to prevention is it to be the best approach for root caries management. Prevention is possibly the best when there is inherent resistant of dentin to the bacterial acidic and enzymatic damage. Therefore, improving the mechanical and biochemical properties of the organic and inorganic constituents of dentin is a much evolving field and is an area of interest in the biomimetic approach. Dentin biomodifiers are natural or synthetic agents, used in the biomimetic approach to enhance the mechanical properties, decrease the biodegradation rates, and promote the remineralization of dentin [40]. The bioactive constituent of GSE is the basic catechin components scavenging the free radicals, which are required for calcium absorption. The PAs increase collagen synthesis and accelerate the formation of insoluble collagen by conversion; thus, they decrease the enzymatic degradation of organic matrices. 246 Natural Oral Care in Dental Therapy Pavan et al. studied (in vitro) the root dentin biomodification of GSE (Proanthocyanidins) and the effects on the demineralization using microradio analysis [41]. This study showed the direct effects of PAs on the root dentin. They compared the use of GSE alone and along with fluoride, the standard remineralizing agent. The following facets are the role of GSE in the biomimetic approach: • GSE when applied alone, reduces the progression of root dentinal caries, but compared to fluoride (F), i.e., the standard remineralizing agent, a lower scale of reduction of caries was found. • When combined with F, the GSE + F combination was better than the GSE alone, but the expected initial fluoride deposition on the surface of lesion is not allowing the GSE to effectively work. • GSE improves and maintains the integrity of the dentin matrix, which is comparable to the inner carious layer, thus, promoting remineralization and decreasing demineralization. • Mechanical properties of collagen matrices are modified by GSE, thus, improving the properties and reducing the degradation of collagen. • Formation of hydrogen bonding is the possible primary mechanism of PA to interact with collagen and improve insoluble collagen conversion. • Collagen–collagen interaction force was increased in the demineralized dentin matrix after treatment with GSE having PAs of >94%. There was an eightfold increase in the interaction force in the GSE-treated group, with glutaraldehyde having only 1.7-fold increase [42]. • GSE may also reduce the digestibility of collagen by interfering with various bacterial enzymes and proteases. • GSE binds to collagen via calcium (Ca2+) present in the remineralizing solutions that enhances the remineralizing process. • GSE is a potential MMP inhibitor, which prevents the release of non-collagenase proteins associated to the collagen fibrils of dentin, leading to inhibition of MMP. • The inhibition of NCP cleavage by GSE prevents further matrix degradation by protecting the collagen fibers [43]. • Antibacterial effects of GSE cannot be completely omitted in the reduction of root caries, but it accounts very minimal or secondary with respect to biomimetic approach. • GSE increases the modulus of elasticity of the dentin after treatment with crude extract containing 80% total polyphenol content, and the rate of biodegradation is reduced from 77% to 10%. • The ultimate tensile strength (UTS) of dentinal tubules increased with the 6.5% GSE treatment. The orientation of tubules significantly altered the UTS. Tubules oriented parallel to tensile forces had less UTS than those oriented perpendicular [44]. • After GSE treatment, the UTS of dentinal tubules reached about 15–20 MPa depending on the dentinal tubule orientation. • GSE treatment significantly increased the denaturation temperature (Td) in turn, it signifies the degree of cross-linking and increased stability of the biomodified matrix. Grape Seed Extracts 247 Liu and Wang determined the efficacy of the proanthocyanidins in stabilizing the dentin collagen against enzymatic degradation [45]. Different concentrations of solutions in weight percentage were tested against collagenase digestion following 1 h and 24 h. It was found that application of ≥2 wt% of PA for 30 s provides the optimal protection for dentin collagen, and also, the collagen was not digested significantly regardless of the digestion time. Zhao et al. supported the same with a GSE concentration ≥2 mg/mL significantly inhibiting the enamel caries lesion depth and mineral loss. Fawzy further improved the mechanical properties by adding GSE into a nanoparticle delivery system [46]. GSE-loaded nanoparticles were synthesized by nanoprecipitation, and loading was done into the biodegradable polymer poly-[lactic-co-glycolic acid] (PLGA) nanoparticles. Loading was done at the PLGA/GSE (w/w) ratios of 100:75, 100:50, and 100:25. Among the three ratios, sustained gradual release of GSE over 28 days with high cumulative release was found in the 100:75, approximately 2.47 mg. However, the drug release kinetics was similar in all. When it comes to resistance to biodegradation, significant increase in the surface mechanical properties was found when treated with the PLGA/GSE ratio of 100:75 even after a long storage time (1 month and 3 months). Drug-loaded nanoparticle size should be kept ≤200 nm to facilitate delivery into the dentinal tubules. Although there are possible proposed mechanisms of delivery of nanoparticles into the dentinal tubules, the PLGA/GSEloaded nanoparticles’ mode of delivery into the dentinal tubules needs to be studied further. 15.3.6 GSEs as a Remineralizing Agent—Existing Dilemma Facets of the biomimetic approach partially explain the role of GSE as a remineralizing agent. Polyphenols produce metal–ion complexation with various metals, which is one of the important modes of action in its pharmacological activity. The working principle is the same in case of enamel and dentin remineralization, along with collagen interaction of dentinal organic matrices. However, when talking about the efficacy of GSE as a remineralizing agent, different studies have given different results. When compared with the existing remineralizing agents, some studies found GSE to be superior to other agents, and some studies found it to be equal or less effective to use for remineralization. Benjamin et al. found significant difference in the remineralization of GSE compared to fluoride (F) and calcium glycerophosphate (CaGP). They compared 6.5% (w/v) GSE against 0.05% CaGP + 0.17% F, 0.5% CaGP and deionized water using confocal laser scanning microscopy (CSLM). It was found that relative optical density (ROD) for the GSE group was 78.37 and 45.32, 41.52 and 44.08 for others, respectively [47]. Jawale et al. used the same concentrations of solutions, and posttreatment evaluation was done by polarized light microscopy (PLM). They found a significantly small lesion depth and a wider mineral precipitation in the GSE group compared with the other groups. The percentage of proanthocyanidin used was also mentioned, i.e., 98% [48]. Xie et al. evaluated the effects 6.5% GSE, 1,000 ppm Fluoride, and deionized water [49]. Posttreatment specimens were analyzed for microhardness test using Knoop hardness indentation (KHN), PLM image analysis, and CSLM analysis. KHN revealed no significant difference in the hardness of remineralized lesion with GSE and F. In the PLM, the lesion depth was smaller in the F treatment group, and the mineral precipitation band was wider in the GSE treatment group. CSLM analysis revealed that ROD was almost double in the GSE group at 20 µm, 50 µm, and 80 µm with statistically significant difference. The ROD of the GSE-treated sample at 50 µm was 73.23. Pavan et al. found that GSE is less efficient compared 248 Natural Oral Care in Dental Therapy with the gold standard fluoride in the biomimetic approach. Though the GSE reduced the progression of root caries lesion in vitro, the results were relatively less than fluoride [41]. 15.4 GSEs in Restorative Dentistry Two important uses of GSEs in the conservative dentistry are as a cross-linking agent and in bonding of restorations and adhesives. Treatment of dental caries is mainly on the removal of the infected enamel and dentin through cavity preparation followed by restoring it with a biocompatible material. 15.4.1 GSEs as a Cross-Linking Agent Successful dentin bonding depends on the formation and integrity of the hybrid layer. After etching, the application of an adhesive on the exposed dentinal tubules made the adhesive to penetrate and entangle with the dentinal collagen fibrils, thus forming a hybrid layer. In the formation of achieving perfect seal and complete penetration of the adhesive may not possible, which leads to the exposure of the dentinal collagen, which in turn degrades over time. Exposed, unprotected collagen fibrils have relatively less stability and mechanical strength. It reduces the bond strength and gradually leads to bond failure. One specific approach to increase the resistance to degradation and strengthen the exposed collagen fibrils is the use of cross-linking agents. Cross-linking agents produce amino acid linkage between and within the molecules, thus, modifying and strengthening the collagen fibrils. Natural and synthetic cross-linking agents are used for this purpose. Natural cross-linking agents are more preferable over synthetic for their biocompatibility and negligible side effects. Various natural cross-linking agents such as tannic acid, UVA-activated riboflavin, and polyphenol extracts from plants are used. GSEs are used as cross-linking agents for its high polyphenol content, improved overall mechanical properties of dentin, and no cytotoxic effects on normal cells [32]. PAs produce the cross-links with the collagen of caries-affected dentin and reduce digestibility. Compared to glutaraldehyde, GSE-treated dentinal collagens showed significantly more resistance to digestibility by proteases [5]. PAs cross-link with collagen and mask the recognition sites for enzymes and/or retain the cleaved peptide fragments. Concerning the bond strength, caries-affected dentin reaches near the bond strength of sound dentin after treatment with GSE. It was found that 5% GSE inhibited the CTX release permanently. CTX is the C terminal peptide released by cathepsin K degradation. Potential benefits of GSE are • GSE is a potential natural cross-linking agent with comparable results over glutaraldehyde. • Improvement in the mechanical properties depends upon the degree of cross-linking. • Inhibits the biodegradation of dentin collagen fibrils at the bonded interface • Maintaining the dentin–resin bond strengths • Biocompatibility even at high concentrations • Antibacterial activity as an added benefit Grape Seed Extracts 249 Current concerns of GSE includes • Effect on the resin polymerization • Depth of adhesive penetration may be affected • Dentin discoloration and potential staining properties needs to be evaluated on long term 15.4.2 GSE in Bonding Application of GSE in bonding is based on the cross-linking ability of PAs to improve the adhesive–dentin interface in bonding. The PA treatment on teeth could be in the form of application as a separate step after etching, as a pre-conditioner, in the priming agent, in the self-etch primer, in the adhesive, or as a PA-combined etchant. Application of the PAs as a separate step after etching improves the tensile strength, decrease the collagen biodegradation of dentin matrix, and increase the bond strength. However, there is a need of separate washing to remove the excessive PAs, and excessive stiffness of inter-tubular collagen may restrict the depth of adhesive penetration. Transient application of PA-based pre-conditioners increased the bond strength. The bond strength was increased in a timeand concentration-dependent manner [50]. Treatment with 15% PAs for 120 s was found to best increase the bond strength. However, this application time of 120 s reduces the clinical applicability. In spite of the long application time, the concentration used did not interfere with the curing behaviors of adhesives [50]. GSE has been incorporated in the etchant to produce a collagen-stabilizing etchant. The benefits of GSE in bonding would be better if it is in the adhesive itself. So adding the GSE in self-etch adhesives reduces the need of multiple steps in bonding. However, the scavenging activity of PAs in GSE limited the incorporation of GSE in the self-etch adhesives, as it is hampering the polymerization of adhesives to some extent. Thus, the addition of GSE in the etchant would reduce this effect. Combining the GSE with the etchant will help in reducing the rinsing step, as both will be rinsed off together. However, when the efficiency is considered, it was found that phosphoric acid concentration should be maintained less than 20% [51]. As the collagen-stabilizing mechanism of PAs is not dependent on covalent bonds, it was possible to combine the phosphoric acid and the GSE, but the application of this combination on the polymerization of adhesive needs to be investigated. Cross-Linked Dry Bonding Cavity preparations are followed by acid etching that produces microporosities in the dentinal surface, which facilitate the infiltration of resin. The prepared cavity exposes the mineralized healthy dentin, which has a modulus of elasticity (ME) of 20,000 MPa. The processes of acid etching solubilize the crystallites to a depth of 10 um, leading to a reduction in modulus of elasticity. After etchant application, water rinsing is done to remove the excess acid and solubilized minerals. Thus, the exposed mineralized dentin matrix has only 3–5 MPa of elastic modulus. In the wet condition, the collagen fibrils are very pliable, which makes rapid interpeptide hydrogen bonds on dehydration. This leads to the formation of a membrane-like structure, which is impermeable for the infiltration of adhesive resins around the fibrils. To create a high resin–dentin bond strength by avoiding drying-induced shrinkage, Kanca developed the wet-bonding technique [52]. This technique involves the 250 Natural Oral Care in Dental Therapy wet demineralized dentin to float in 70% water during the monomer infiltration phase. This technique again has the disadvantage of excessive residual water in bonds. Excessive residual water fuels the endogenous hydrolytic proteases that slowly degrade the dentin collagen matrices. The goal is to replace the 70% residual water with resin infiltration during bonding, which is impossible with hydrophobic resin molecules. Thus, the modified wet-bonding technique was developed to rectify all the problems. Replacing the residual water by addition of a cross-linking agent will help to stiffen the collagen as well as scavenge the residual water. We have seen the role of hydrogen bond in the interaction of polyphenols and collagen peptides. The water is a known hydrogen-bonding solvent. The presence of the water molecules do help in stabilizing the protein–polyphenol complex, at the same time hindering the bonding between the adjacent collagen peptides. The balance between breaking the hydrogen bonds of the water molecule and making the hydrogen bonds of the collagen peptides is the most important factor in GSE–collagen interaction. After etching, pretreatment with GSE allows the dentin to completely air dry without collapse of the dentinal fibrils. The stiffness is inversely proportional to shrinkage. Cross-linking the collagen with GSE allows the individual collagen fibrils to get separate from each other and also allows the penetration of the adhesive. Five percent GSE pretreatment for 1 min allows the optimum cross-linked dry-bonding technique [53]. 15.5 GSEs in Endodontic Treatment GSE is used in root canal treatment as an endondontic irrigating solution and in the postendodontic restorations. 15.5.1 Endodontic Irrigants Root canal treatment includes three main steps, in which the most important step is cleaning and shaping. The aim of root canal preparation is to eliminate bacteria from the contaminated root canals and to provide the adequate shape to receive the final obturation by cleaning and shaping. GSE shows antibacterial activity against many aerobic and anaerobic bacteria. Among the different bacterial species, elimination of Enterococus faecalis from the root canals during endodontic treatment is challenging. E. faecalis is a facultative anaerobic Gram-positive bacterium and is isolated from about 67% of cases of endodontic failure and about 18% of primary endodontic lesions. GSE has been investigated as an endodontic irrigant for its antibacterial potential and found that it poses good bactericidal efficacy against E. faecalis [54]. When used with mechanical cleaning instruments such as ProTaper Next and Reciproc R25, it reduced 96% of the bacteria with no significant difference in the reduction compared to NaOCl and Ca(ClO)2. PAs in the GSE damage the microbial cells by altering the selective permeability of the membrane, causing leakage of the essential intracellular substances. An endodontic irrigant should be able to eliminate the bacteria, should have bactericidal activity, able to penetrate the dentinal tubules, should be a good solvent, biocompatible, should not produce harmful side effects, and should not interfere with obturative materials. Grape Seed Extracts 251 Soligo et al. suggested GSEs as a good auxiliary irrigant solution in endodontic treatment. The antibacterial activity against E. fecalis is seemingly dose dependent of GSEs [54]. They found good bactericidal activity on E. fecalis using 50% GSE solution, whereas Ghonmode et al. used much lesser concentration and found no antibacterial effect. Cecchin et al. also suggested using GSE as a good alternative auxiliary irrigating solution in the endodontic treatment [55]. They found an improvement in the mechanical properties of the dentinal walls after using GSE solution. One of the important disadvantages of sodium hypochlorite (NaOCl) is weakening the dentin surfaces by oxidizing effect during preparation. Thus, the vertical fractures are the leading failures in post-endodontic treatments. GSE, in turn, strengthens the dentin walls. Though it improves the dentin qualities in the biomimetic approach, when compared to NaOCl as an endodontic irrigant, no significant reduction in the flexural strength, ultimate tensile strength, and fracture resistance was found in the GSE group. Although additional improvement was not seen when compared to the control group, GSE could be used to reinforce the dentinal structure, especially in cases where potentially less thickness of the root dentin is found and the cases allergic to NaOCl. GSE does not fulfill all the criteria to be a clinically effective endodontic irrigant. The ability to penetrate the deep dentinal tubules needs to be studied, the bactericidal activity against different species of the root canal system needs to be evaluated, and optimal dissolving potential should be improved. Concerning the side effects of GSE as an endodontic irrigant, tooth staining may be a potential side effect that may need to be investigated. 15.5.2 Post Endodontic Restorations One important disadvantage of GSE as an irrigant is its inability to dissolve the smear layer and dentin shaves that is mechanically produced during instrumentation. As the proanthocyanidin produces complexes with the dentin collagen matrix, rather than dissolving, it produces strong complexes, which is good for further obturation. Cecchin et al. found that pretreatment with GSE in the root canal improves the long-term bond strength of fiber posts [56]. As we saw, the use of NaOCl produces oxidation, and superoxide radical damages the collagen and weakens the tooth structure. The remaining redox potential may also interfere with the bonding of obturative materials. Thus, the use of GSE restores the redox potential, and improves the quality of dentin and bonding of resin-based obturative materials. One study proposes the sequence of NaOCl, followed by EDTA and GSE as a final irrigant that would help in reducing the side effects of sodium hypochloride [57]. 15.6 GSEs in Periodontics Another important effect of biofilm in the oral cavity is the manifestation of periodontal diseases. The predominance of secondary colonizers leads to the development of gingivitis, periodontitis, and other inflammatory diseases of periodontium as a response to the microbial stimulus. The potential natural agent, GSE, has been studied widely for its protective effects from the inflammation, preventing fibroblast damage, for its antioxidant effects, as an antiplaque agent, and as an antibacterial drug. 252 Natural Oral Care in Dental Therapy 15.6.1 Anti-Inflammatory Action in Periodontitis Pro-inflammatory cytokines are synthesized as a response to periodontal pathogenic organisms. These cytokines induce and maintain the inflammation in periodontitis. Simultaneously, anti-inflammatory cytokines are produced and try to control the process as a part of healing. When there is imbalance between the two responses, this leads to periodontal breakdown. Altered immune response and/or overproduction of cytokines may lead to such conditions that can be improved with therapeutic compounds, which alter the host response. GSE has been proposed as a promising immune-modulator due to the PAs [58]. Animal studies were conducted to evaluate the effects of GSE on periodontal inflammation. Govindaraj et al. first demonstrated the antioxidant effects of PAs in the experimentally induced periodontitis by E. coli endotoxins [59]. Ozden et al. showed the anti-inflammatory effects on rats by a ligature-induced periodontitis model [60]. They used the dosage of 200 mg/kg body weight GSE without any side effects and estimated the interleukin-10 (IL-10) levels at various time periods. IL-10 is an anti-inflammatory cytokine, which mediates the control of periodontal disease progression and modulates the inflammatory response. It was found that administration of GSE prior to inflammation produced best results with increased production of IL-10 and protective effects compared with the remaining groups. PA has been shown to inhibit the tumor necrosis factor-alpha and interleukin-1 beta, decreases Th1 and Th17A levels, and increases Th2-released cytokines at the inflammation site. It demonstrates the anti-inflammatory action of GSE via regulating the pro- and anti-inflammatory cytokines. Another proposed mechanism is through the matrix metalloproteinase (MMP)-8 and hypoxia inducible factor (HIF)-1alpha [61]. PA administration of 200 mg/kg/day reduced the ligature-induced periodontitis in rats with diabetes mellitus (induced). HIF-1 is found more in diabetes mellitus, and PAs significantly reduced them. The decrease in HIF could be attributed to the improvement in the hypoxic state by administration of GSE. Along with this, PAs decrease the alveolar bone loss via osteoblast induction. GSE supplementation significantly improves the periodontitis condition in diabetes by decreasing inflammation, MMP-8 expression, increasing the osteoblast count and bone formation. Effects of GSE on the various MMPs have been studied [58]. PAs significantly reduce the MMP-1, -7, -8, -9, and -13 productions by A. actinomycetemcomitans-stimulated macrophages. Reduction of MMP production is found to be at the concentrations of 12, 59, and 100 µg/ml. Along with down regulating the MMP, GSE partially reduces the activity of MMP also. This dual effect of GSE on the MMP proposes it as a host-modulating natural agent and its application in the periodontal diseases. 15.6.2 Anti-Oxidative Action in Periodontitis Accumulation of pathogens and persistent release of their byproducts produce destruction of gingivae and periodontium either directly through oxidative damage or indirectly via inflammatory mediators. Reactive species are produced either by certain bacteria as a byproduct of their metabolism or by host as a part of immune response. These include reactive oxygen species (ROS) and reactive nitrogen species (RNS). This reactive species cause oxidative damage via DNA and protein damage, lipid peroxidation, and by initiating inflammation, eventually leads cell death and tissue destruction. PAs are known potent antioxidants. Grape Seed Extracts 253 Effects of PAs on the three periodontal pathogens were studied in vitro. Pretreatment of macrophages with 4 µg/ml of PAs reduced the ROS production when stimulated with lipid polysaccharides (LPS) that are produced by three bacteria. Intracellular ROS production was inhibited by 43%, 33%, and 48%, respectively, for LPS of A. actinomycetemcomitans, F. nucleatum, and S. typhosa. PAs showed a strong inhibitory effect on RNS production with 80%, 78%, and 62%, respectively [28]. Cytoprotective effect of GSE is related to its antioxidant potential. Pretreatment of human gingival fibroblasts with GSE for 1 min produced cytoprotective effects in unfavorable conditions [62]. Other than fibroblasts, human alveolar cells are also protected from cytotoxic effects. Katsuda et al. reported that the cytoprotective effects of GSE on gingival fibroblasts are likely to be independent of its antioxidant effects [63]. 15.6.3 Antibacterial Action Against Periodontal Pathogens Antibacterial activity of GSE against periodontal pathogens is found against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Lactobacillus rhamnosus, Actinomyces viscosus, and Streptococcus sorbinus. The MIC and MBC values of GSE against periodontal pathogens and anaerobes are relatively high compared to the MIC and MBC values against cariogenic bacteria, i.e., S. mutans. Different authors found different values of MIC and MBC. The inhibitory effects of GSE against A. actinomycetemcomitans are quite high with MIC = 3.84 mg/L and MBC = 7.68 mg/L [64]. The MIC value of F. nucleatum and A. viscosus is 2 mg/mL, and the MBC values are double that of MIC. The MIC and MBC values for P. gingivalis and L. rhamnosus are found to be higher and just double that of F. nucleatum and A. viscosus. Furiga et al. found high values of MBC for P. gingivalis and F. nucleatum, i.e., 8 mg/mL. Despite a high dose, GSEs are well tolerated without side effects that could be an added value to its clinical applications [37]. All the above studies are of mainly in vitro or animal studies with hypothetic proposals of GSE as a drug. The first randomized clinical control study on humans was conducted by Rayyan et al. [65]. They used subgingival application of 2% mucoahesive GSE gel in the deep periodontal pockets. In the clinical trial, GSE improved the condition of periodontitis significantly only in periodontal index and gingival index. The periodontal depth of the periodontal pockets did not improve. For the clinical applications, further trials and studies are needed. 15.6.4 Antimicrobial Activity in Peri-Implantitis Maxillofacial defects are rehabilitated with various esthetic prostheses. The seal around the maxillofacial implants is a crucial factor as it is dynamic, and there is a lack of physical barrier between the soft tissues and the skin. A lack of hygiene maintenance and compromised seal around the implants leads to altered skin microflora. Staphylococcus aureus is the most commonly isolated microorganism from the infected implant-abutment site [66]. GSE is effective against S. aureus. Two strains of S. aureus are found to be susceptible to GSE solutions, ATCC 6538 and a clinical strain. The MIC and MBC value of both strains are 0.625 mg/mL and 1.250 mg/mL, respectively. Also, GSE exhibits a dose-dependent inhibitory effect on S. aureus when combined with propylene glycol and polyethylene glycol [67]. 254 Natural Oral Care in Dental Therapy 15.7 GSEs in Oral Cancer We saw the cytoprotective effects of GSE and its application in the protective side until now. In the biochemical properties, it was found that proanthocyanidins exert dosedependent selective cytotoxic effects. These were well studied as anti-cancer activity in the colorectal cancer, anti-proliferative and apoptotic effects on the HCT-8 colon cancer cell lines, and in vitro human cancer cells [68]. The presence of gallic acids plays an important role in this. The degree of polymerization and galloylation affect the cyototoxic effects. On oral carcinoma, GSE shows an anti-proliferative activity through inhibition of cell growth. The Oral squamous cell carcinoma cells (KB cell line) are studied for the effects of GSE [69]. GSE, at the concentrations of 250 µg/mL and incubation at 24 h, exhibits a potent cytotoxic effect on KB cells. It is important to notice that any anti-cancer drug should not harm the normal cell line. The effects of GSE on the KB cell line along with the normal cell lines such as human umblical vein endothelial cells (HUVEC) were studied. The results are promising, with selective dose- and time-dependent cytotoxicity on the KB cell line without affecting HUVEC cells. The possible mechanism for this is the induction of apoptosis by GSE, subsequent DNA fragmentation, and chromatin condensation. The DNA fragmentation analysis revealed that GSE cytotoxicity on KB cell lines produced DNA fragmentation at the late stage of apoptosis. In vitro potential needs to be evaluated in further trials. Cell lines of human gingival cancer Ca9-22 and HGF-1 are also studied for the dose-dependent effects of GSE [70]. A dose of 150 µg/mL of GSE with 24 h incubation reduced the Ca9-22 cells to half. The viability of HGF-1 cells is maintained at both low and high concentrations up to 400 µg/mL. The anti-proliferative effects on oral cancer CAL 27 cell lines are shown at 30–80 µg/mL. Thus, different cell types require different concentrations of GSE to inhibit proliferation. Also, GSE has been reported to induce mRNA over expression of apoptosis-associated signaling. The other activities seen at the high concentration of GSE (around 400 µg/mL) are ROS production and induction of apoptosis through high oxidative stress, mitochondrial depolarization, and caspase activation in Ca9-22 cells. 15.8 Conclusion Grape seed extract (GSEs) is an emerging natural medicine in various therapeutic procedures in dentistry. It serves as a good alternative in the prevention and treatment of caries, and in periodontal diseases. The major role provided in the biomodification of dentine by GSE makes it a biomimetic agent. A paradigm shift is noticed in the use of natural cross-linkers from a synthetic one, after the development of GSE in the conservative dentistry. Although it is used as a potential antioxidant, anti-inflammatory, and anti-carcinogenic agent in medicine, for application in dental therapy, further studies are needed. Very few clinical studies, scarcity of human studies and clinical trials are clear evidence that there is a need for a huge research and subsequent development of GSE as a drug in the treatment of oral cancers, in the premalignant lesions, in the endodontic treatment, and in the periodontal therapies. Grape Seed Extracts 255 References 1. Spanos, A. and George, R.E.W., Phenolics of apple, pear, and white grape juices and their changes with processing and storage. A review. J. Agric. Food Chem., 40, 1478, 1992. 2. Kanellis, A.K. and Roubelakis-Angelakis, K.A., Grape, in: Biochemistry of fruit ripening, pp. 189–234, Springer, Dordrecht, 1993. 3. Singleton, V.L. 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Mirkarimi, M., Amin-Marashi, S.M., Bargrizan, M., Abtahi, A., Fooladi, I., Ali, A., The antimicrobial activity of grape seed extract against two important oral pathogens. Zahedan J. Res. Med. Sci., 15, 43, 2013. 65. Rayyan, M., Terkawi, T., Abdo, H., Abdel Azim, D., Khalaf, A., Alkhouli, Z. et al., Efficacy of grape seed extract gel in the treatment of chronic periodontitis: A randomized clinical study. J. Investig. Clin. Dent., 9, e12318, 2018. 66. Klein, M., Weisz, I., Camerer, C., Menneking, H., Kim, D.M., Therapy of percutaneous infection around craniofacial implants. Int. J. Prosthodont., 22, 594, 2009. 67. Shrestha, B., Theerathavaj, M.L.S., Thaweboon, S., Thaweboon, B., In vitro antimicrobial effects of grape seed extract on peri-implantitis microflora in craniofacial implants. Asian Pac. J. Trop. Biomed., 2, 822, 2012. 68. Kaur, M., Singh, R.P., Gu, M., Agarwal, R., Agarwal, C., Grape seed extract inhibits in vitro and in vivo growth of human colorectal carcinoma cells. Clin. Cancer Res., 12, 6194, 2006. 69. Aghbali, A., Hosseini, S.V., Delazar, A., Gharavi, N.K., Shahneh, F.Z., Orangi, M. et al., Induction of apoptosis by grape seed extract (Vitis vinifera) in oral squamous cell carcinoma. Bosn. J. Basic Med. Sci., 13, 186, 2013. 70. Yen, C.Y., Hou, M.F., Yang, Z.W., Tang, J.Y., Li, K.Y., Huang, H.W. et al., Concentration effects of grape seed extracts in anti-oral cancer cells involving differential apoptosis, oxidative stress, and DNA damage. BMC Complement. Altern. Med., 15, 94, 2015. 16 Ocimum Sanctum L: Promising Agent for Oral Health Care Management Trinette Fernandes1, Anisha D’souza2 and Sujata P. Sawarkar1* 1 Department of Pharmaceutics, SVKM’s Dr. Bhanuben Nanavati College of Pharmacy, University of Mumbai, Maharashtra, India 2 Formulation Development Laboratory, Piramal Enterprises Limited, Light Hall, Chandivali, Powai, Mumbai, India Abstract Dental caries was considered as the most prevalent condition affecting 2.4 billion people worldwide (2010), and the 10th most prevalent condition affecting 621 million children on a global scale. Bacteria like Streptococcus mutans, Streptococcus sobrinus, Enterococcus faecalis, and Lactobacilli are responsible for formation of dental plaque. Natural products such as Ocimum sanctum L. extract have been used for treatment of various ailments since ancient times and have been mentioned in ancient texts such as Atharvaveda. The mention of Ocimum sanctum L. in literature dates back to 1200–1000 BC wherein it is called as the “elixir of life”. Ocimum sanctum L was reported to be used for oral care and halitosis as they have anticariogenic properties. The anti-inflammatory agents in Ocimum sanctum (Tulsi) also contribute to the reduction of inflammation of gums, which is characteristic of periodontal diseases such as periodontitis and gingivitis. In vitro tests on oral pathogenic bacteria shows that Ocimum sanctum L extract in the form of toothpastes, mouthwash, and gels have antibacterial activity against Streptococcus mutans and Enterococcus faecalis. Recently the formulations of Ocimum sanctum L loaded in novel drug delivery system have been developed for the treatment of various conditions such as acne, arthritis, etc. Mouthwashes, gels, and toothpastes containing Ocimum sanctum L-loaded particulate systems can potentially be researched in the future. Keywords: Ocimum sanctum L, tulsi, oral health, antibacterial, antibiofilm, antifungal, periodontal diseases 16.1 Introduction Oral Hygiene and dental public health have always been neglected issues although it is a well-known fact that oral cavity is the gateway to external environment and the first entry point of plethora of microorganisms to the human body. It is required to be well understood that dental health, if neglected, can lead to fatal ailments like cardiovascular diseases, *Corresponding author: sujata.sawarkar@bncp.ac.in; sujatasawarkar19@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (259–270) © 2020 Scrivener Publishing LLC 259 260 Natural Oral Care in Dental Therapy pneumonia, stroke, etc. Bacteria are major causative organisms for dental diseases. Dental caries and periodontal diseases are the most common chronic diseases. Dental caries, also known as cavities, often lead to dental plaque and bio-film formed by Streptococci and Lactobacilli species [1]. Formation of bio-film around the gingiva causes pathogenic conditions like gingivitis, inflammation of gingiva, softening of gums, and bad breath. Further erosion of enamel spreads to tooth pulp further leading to abscess and periodontitis. Several noninvasive or invasive surgical treatment modalities are adopted initially depending on the severity of the disease followed by local administration of antimicrobials and antibiotics like doxycycline, minocycline, chlorhexidine, cetylpyridinium chloride, or triclosan either as powders or gels, implants, or in the form of tablets and capsules for oral administration. Chemically synthesized drugs have their own share of drawbacks like poor bioavailability and therefore low therapeutic efficacy, systemic side effects, staining of teeth, and calculus formation as commonly associated with chlorhexidine and cetylpyridinium chloride. Considering the present scenario and the pros and cons of modern allopathic drugs of synthetic origin, researchers should revisit and employ traditional medicine for mitigation of dental disease [2]. 16.2 History of Ocimum sanctum Plants are the inevitable segment and quintessential source of drug or therapy in different traditional medicinal systems be it Ayurveda, Unani, or Traditional Chinese Medicine. Like Traditional Chinese Medicine, Ayurveda and Unani System also use different parts of plants, which are formulated as pills, powders, decoctions, extracts, juices, oils, and infusions. Many synthetic analogs have been prepared from prototype compounds isolated from plants. Ayurveda includes the uses of about 2,000 plant species, Unani literature mentions about 1,000 plant species, and Chinese Herbal Medicine mentions about 11,000 plant species [3]. Among the drugs of ancient times, Ocimum sanctum and other Ocimum species have been extensively researched in medical science. Ocimum sanctum L. or Ocimum tenuiflorum L, known as the Holy Basil in English or Tulsi in the various Indian languages is a well-known medicinal plant in the various traditional and folk systems of medicine in Southeast Asia. Ocimum sanctum L. is indigenous to tropical areas and is perennial in nature. The shrub grows up to 18 inches in height and blooms flowers in June to August (or about 150 days from planting the seed). The leaves are 5-cm long, green to purple in color, and ovate in shape. The border of the leaves is serrated and form reddish brown fruits. The Ocimum genus consists of at least 65 species, from which Ocimum sanctum, Ocimum bascilicum, Ocimum gratissimum, Ocimum kilimandschricum, Ocimum americanu, Ocimum camphora, and Ocimum micranthum are known for their therapeutic activity. The current chapter will discuss briefly about Ocimum sanctum Land and its chemical constituents. Various applications of Ocimum sanctum in dentistry and various marketed products will also be briefed upon in the further sections. 16.3 Chemical Constituents of Ocimum sanctum The plants coming under the genus Ocimum belong to the family Lamiaceae. There are about 150 aromatic annual and perennial herbs and shrubs that grow naturally or are cultivated in Ocimum Sanctum L. 261 tropical and subtropical regions of the world. Ocimum sanctum L. or Ocimum tenuiflorum, popularly known as Holy basil, is used as a whole plant or in individual parts of the plant (leaves, flowers, root, seeds, and stem) in various forms such as decoction/tea, powdered extract, or fresh leaves. The shrub grows up to 18 inches in height and blooms flowers in June to August (or about 150 days from planting the seed). The leaves are 5-cm long, green to purple in color and ovate in shape. The borders of the leaves are serrated and form reddish brown fruits. The other species of the Ocimum genus comprised of Ocimum bascilicum, Ocimum gratissimum, Ocimum kilimandschricum, Ocimum americanu, Ocimum camphora, and Ocimum micranthum, are known for their therapeutic activity [4, 5]. Based on the research studies conducted so far, Ocimum extracts have shown to have antibacterial, antifungal, anti-inflammatory, analgesic, antipyretic, antidiabetic, hepatoprotective, hypolipidemic, immunomodulatory, and cardioprotective activities [4–8]. Tulsi leaves primarily contain volatile oil, about 0.7%. The major constituents of volatile oil are 71% eugenol and 20% methyl eugenol. Apart from these, the other constituents present are carvacrol, linalool, limmatrol, sesquiterpine hydrocarbon, caryophyllene, cirsilineol, circimaritin, isothymusin, and apigenin. In addition, the leaves also contain orientin, vicenin, ursolic acid, apigenin, luteolin, apigenin-7-O-glucuronide, luteolin-7-O glucuronide, molludistin, and sesquiterpenes and monoterpenes like bornyl acetate, α-elemene, neral, myrtenal, α- and β-pinenes, camphene, campesterol, stigmasterol, and β-sitosterol. All these constituents exhibit a wide plethora of therapeutic effects [4, 9, 10]. The quantities of each of these constituents are highly variable as primarily it depends on harvesting, treatment, and storage conditions, geographic, and biodiversity. The essential oils present in Ocimum extracts have promising antibacterial activity. The major constituents that have been isolated from the different Ocimum oils include 1,8-cineol, linalool, pinene, eugenol, camphor, methyl chavicol, ocimene, terpinene, limonene, etc. Researchers have extensively studied the relationship between the composition of oil and biological activity. In India, there are two main subspecies of Ocimum sanctum obtained, viz. Ocimum sanctum L. green (Tulsi) and Ocimum sanctum purple (Krishna Tulsi). The essential oils obtained from both these subspecies differ in their composition. The green type of subspecies contains eugenol, limonene, and E-caryophyllene, whereas the purple contains eugenol and E-caryophyllene. These essential oil components show antibacterial activities by varied mechanistic pathways. Eugenol shows a mechanism of action by membrane disruption by inhibiting ATPase activity, possible efflux pump blocker, and reduction of several virulence factors at subinhibitory concentrations. Thymol causes membrane disruption with potential intracellular targets, along with disruption of citrate metabolic pathway. Carvacrol exhibits membrane disruption, inhibition of ATPase activity, membrane destabilization, leakage of cell ions, fluidization of membrane lipids, and reduction of proton motive force [11]. The table 16.1 below mentions the chemical constituents and their pharmacological activity: Literature reports have shown that several phyto constituents in essential oils obtained from Ocimum extracts have shown promising therapeutic activity. It is quintessential to extract, isolate, and identify them. The following section mentions the details of various extraction procedures reported. Extraction of Ocimum sanctum L can be carried out by Maceration, Soxhlet, Supercritical fluid extraction, Ultrasonic extraction, and Microwave-assisted extraction [12–14]. 262 Natural Oral Care in Dental Therapy Table 16.1 Pharmacological activity of phyto constituents of Ocimum. Sr no Chemical constituents Pharmacological activity 1.) Eugenol Anti-inflammatory, antitumor, antibacterial, antiviral 2.) Ursonic acid Anti-inflammatory 3.) Rosmarinic acid Antioxidant 4.) Limonene Antioxidant 5.) E. Caryophyllene Anti-inflammatory and antioxidant 6.) Isothymustin Anti-inflammatory Various analytical methods are used for isolation and identification of chemical constituents in different types of Ocimum sanctum L. LC-MS is the most extensively used method for isolation of flavonoids [15, 16]. In addition, GC-MS is also used for separation of constituents [17]. Eugenol is the major constituent detected by this method. HPTLC and NMR are also used for identification of the active constituents in Ocimum sanctum L. For characterization of total flavonoid and flavone content, spectrophotometric assay can be carried out. Total phenolic content can be quantified by modified Folin–Ciocalteu’s method. 16.4 Therapeutic Significance of Ocimum in Dental Health and Preventive Care Management Ocimum sanctum has been used as an active agent and is an age-old remedy for various ailments. In the field of dentistry, it has been mainly researched for its use as an antibacterial and anti-inflammatory agent for plagues, dental caries, and periodontitis. Ocimum sanctum has multifaceted activities mainly attributed to constituents such as Eugenol, Methyl eugenol, Ursolic acid, and rosmarinic acid. As mentioned in an earlier section, the therapeutic constituents show antibacterial activities by varied mechanistic pathways, viz. membrane disruption by inhibiting ATPase activity, membrane disruption, efflux pump blockage, reduction of several virulence factors. Ocimum sanctum extract has been tested for antibacterial activity against periodontal pathogens, such as Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. The extract showed superior activity when compared to other herbal agents and also was equivalent/superior to established gold standards i.e. Chlorhexidine, Triclosan, and CetylPyridinium Chloride. The superior activity was attributed to the presence of Eugenol in Ocimum extract [12, 18–27]. The activity of essential oils derived from Ocimum has been examined extensively for their capacity to control oral cariogenic bacteria and biofilm-forming microorganisms like Streptococci and Lactobacilli species [28–30, 49]. Ocimum can be used in various forms for its anticariogenic activity. The most common traditional method has been chewing whole leaves. Lolayekar, N.V. and Kadkhodayan, S.S conducted a study involving the evaluation of the eradication of Streptococcus mutans from the oral cavity. The study showed a statistically significant (P < 0.05) difference in the colony Ocimum Sanctum L. 263 count of Streptococcus mutans in saliva in the clinical volunteers aged between 9 and 12 years [31]. In another study, the researchers evaluated the thickness of tongue coating and salivary pH before and after chewing Ocimum sanctum (Tulsi) leaves. The study collected the saliva sample and checked the tongue coating on the first, second, and seventh day. The study showed an increase in saliva pH level after 30 min of chewing Tulsi leaves. The observations were correlated to decrease in acidic conditions required for induction of cariogenesis and tongue coating and halitosis [32]. The dental activity of Ocimum sanctum has been profoundly envisaged when administered in the form of mouthwash, gargles, and mouth irrigants. In recent times, several commercial oral health care products available in domestic and international markets contain essential oils derived from Ocimum species. Pereira SL et al. and Pimenta MS prepared a mouthrinse from the extract of Ocimum gratissimum and compared it with Chlorhexidine Digluconate solution in a clinical study conducted on human volunteers. The investigators evaluated the formulations with respect to biofilm and gingivitis control. The study showed a reduction in plaque and inflammation in the volunteers. In addition to this, it also showed a reduction in recurrence in the formation of new biofilm for up to 3 days after removal of the plaque [33, 34]. Gupta et al. conducted a randomized controlled clinical trial of Ocimum sanctum and Chlorhexidine mouthwash on dental plaque and gingival inflammation in 36 patients. The study results concluded that Ocimum mouthwash was equally effective as Chlorhexidine in mitigating plague and gingivitis. The interpretations were made on the basis of measurement of plaque index and reduction in gingival bleeding and inflammation. Ocimum mouth rinse was well accepted by participants as it did not give any burning sensation as commonly associated with the use of Chlorhexidine mouthwash [35]. The presence of residual bacteria after root canal treatment often causes periradicular lesion often leading to failure of therapy [36]. In order to eradicate these residual bacteria, dental irrigants are employed. Dental irrigants or mouth irrigants involve the use of fluids sprayed at high pressure in the mouth cavity to provide a scaling-like action and help with blood flow and promote healing of the alveolar tissue. Tulsi extract, as the main active ingredient or in combination with other herbal active constituents, has been used for disinfecting the dental cavity and subgingival region. Ocimum sanctum extract has also shown evidence for intracanal medication as dental irrigant. Gupta-Wadhwa J et al. conducted a clinical trial on 108 volunteers to check the reduction in Enterococcus faecalis posttreatment with Cinnamomum zeylanicum, Syzygium aromaticum, and Ocimum sanctum extract. The treatment showed significant reduction in bacterial levels in the dental cavity [37]. However, the effect of the extract failed to provide superior disinfection compared to strong chemical agents such as Sodium Hypochlorite. In another study, it was concluded that the increase in disinfectant activity of Sodium Hypochlorite along with herbal extracts can help to control the bacterial colonies [38, 39]. In addition to gingivitis and root canal disinfection, Tulsi was also researched for activity in the treatment of oral mucosal fibrosis. The treatment showed significant increase in mouth opening and reduction in burning in the volunteers. Reduction in symptoms confirmed the potential of Tulsi extract as adjuvant therapy in the management of oral submucosal fibrosis [40]. 264 Natural Oral Care in Dental Therapy Ocimum sanctum, being an antibacterial, is also reported for its use in periodontitis. Ocimum sanctum gel (2%) was studied in Wistar rat model after inducing periodontitis by a ligature model. Gel was made of Carbopol (2 g) and Hydroxypropyl methyl cellulose HPMC (2 g) with propylene glycol (5 ml) with a preservative. The gel inhibited 33.66% of edema with maximum activity observed at 24 h. The gingival index and probing pocket depth were significantly improved with no toxic effects [41]. 16.5 Novel Drug Delivery Formulations and Its Application in Dentistry Application of novel formulation strategy and nanotechnology for herbal drugs and their phyto constituents can help in promoting their therapeutic benefits. The various drug delivery systems studied for the delivery of Ocimum or explored for its activities is mentioned in the following sections. 16.5.1 Nanofibers Resorbable nanofibers containing Ocimum sanctum (1–20% w/w)-loaded polyvinyl vinyl acetate have also been studied for periodontitis. The nonwoven nanofibers were prepared by electrospinning to give high tunable porous and large surface area. Viscosity of solution diameter of needle (12 mm, 22 mm, 0.91 mm, 0.4 mm), solution flow rate (500 µl/h, 0.8 ml/h, 1 ml/h, 1.5 ml/h), and tip to collector distance (12 cm, 22 cm, 16–18.5 cm), the parameters of the nanofibers, can be controlled to obtain nanofibers of required parameters. Beadless and uniform fibers were formed with 10% of Ocimum concentration when the tip-to-collector distance was 12 cm, the diameter of the needle was 12 mm, with a solution flow rate of 500 µl/h, and with an applied voltage of 13 kV [42]. 16.5.2 β-Cyclodextrin Complexes To improve the stability of essential oil from Ocimum sanctum, against environmental conditions like oxygen and temperature, supramolecular complexation with β-cyclodextrin with essential oil was studied. The supramolecular structure protected the oil against degradation especially linalool and methyl chavical (estragole) as determined by GC-MS [43]. The supramolecular structure exhibited an anti-inflammatory effect in acute and chronic inflammation mice models by decreasing vascular permeability, leukocyte recruitment to peritoneal cavity, and granuloma formation. The activity can be explored for anti-inflammation in periodontitis [44]. 16.5.3 Biocompatible Ocimum sanctum-Coated Silver Nanoparticles Plant extracts have been popularly used for green synthesis of silver nanoparticles. They facilitate synthesis of the inorganic nanoparticles making them more biocompatible and eco-friendlier, besides being cost-effective. Plant extracts reduce the Ag+ ions to Ag(0) in the form of nanoparticles. Silver nitrate is reduced when kept at 80°C temperature within Ocimum Sanctum L. 265 45 min. Similarly the leaves of Ocimum sanctum assisted in the formation of silver nanoparticles ranging from size 5 nm to 40 nm depending upon the concentration of silver nitrate and leaf extracts. Eugenol, the phenolic compound is responsible for the capping activity and reduction process due to their redox property of neutralizing and absorption of free radicals. The capping agent prevents the agglomeration of nanoparticles due to the high affinity of the carbonyl group in the amino acid residues in Ocimum with silver. Besides, other antioxidants like Vitamin-C also play a major role. Air-dried leaves of Ocimum sanctum, usually done by keeping at 60°C for 48 h in a hot air oven followed by grinding and extraction with water, were used for reduction. The rate of reduction is faster with the extract of Ocimum sanctum compared to other plant leaf- as well as microbe (bacteria and fungi)-based synthesis. Formation of silver nanoparticles is confirmed by strong plasmon resonance at 440 nm. The silver nanoparticles give a characteristic yellowish-brown color [20, 45]. The silver nanoparticles thus synthesized have usually a cubic lattice structure of nano scales and a coat of biocompatible plant proteins [46]. However, the silver nanoparticles with desired characteristics can be obtained by changing the plant culture conditions. For instance, spherical-shaped crystalline silver nanoparticles with a size of 13.82 nm could be obtained from the extract of Ocimum basilicum, which was grown in vitro. The antibacterial effect against Gram-negative and -positive bacteria was retained [47]. Likewise, the presence of Cetyltrimethylammonium bromide revealed spherical nanoparticles ranging from size 18 to 35 nm but with triangular truncated nanoplates, which looked like a silver locket capped with O. sanctum biomolecules. The polydispersity of the nanoparticles was very high. A research group successfully evaluated both the green and purple varieties of Tulsi, i.e., Ocimum tenuiflorum L. green (known as Sri Tulsi in India) and Ocimum tenuiflorum L. purple (known as Krishna Tulsi in India) for bio reduction of silver nitrate to <100 nm-sized silver nanoparticles and found no significant difference. The presence of C–H vibration contributed by aromatic ring and stretch C–O vibrations from carbonyl group and flavonoids confirmed the same [50]. Extract of Ocimum basilicum leaves and Ocimum gratissimum Linn also have been successfully used in the generation of silver nanoparticles [48, 49]. Ocimum tenuiflorum leaf also yielded silver nanoparticles with good antioxidant potential and good catalytic reduction [51]. The synthesized nanoparticles were stable up to 4 months at room temperature and exhibited a lower minimum inhibition concentration compared to the nanoparticles biosynthesized using Garcinia mangostana against Staphylococcus aureus and E. coli [52]. Another research indicated that the antimicrobial activity of such silver nanoparticles was highly enhanced against Escherichia coli and Staphylococcus aureus when Ocimum was used for reduction. Fresh leaves of Tulsi were used for extraction. However, conversion to silver nanoparticles took almost 2 h [53]. 16.6 Conclusion The uses of Ocimum sanctum in dentistry has been documented in various ancient literatures. The mention of Ocimum sanctum L. in literature dates back to 1200–1000 BC wherein it was called as the “elixir of life”. The amount of active chemical constituents isolated from the crude extract depends on the type of extraction process implemented. Chemical 266 Natural Oral Care in Dental Therapy constituents such as Eugenol, Rosmarinic acid, Isothymusin, and Ursolic acid were proven to have antibacterial and anti-inflammatory activities. Tulsi has been reported to be used for oral care and halitosis as they have anticariogenic properties. The anti-inflammatory agents in Tulsi also contribute to the reduction in inflammation of gums, which is characteristic of periodontal diseases such as periodontitis and gingivitis. Ocimum sanctum L is also used as a wound healing agent. This property is proven beneficial in alveolar bone regeneration and postsurgical periodontium healing. The potential of Ocimum sanctum extract for use in various formulations such as gels and mouthwashes has been explored. The use of nanoparticles for the delivery of Ocimum sanctum helps in localized delivery of the extract. Metal nanoparticles such as silver, iron oxide, gold, and platinum are prepared using Ocimum sanctum extract. These nanoparticles are potent antibacterial and can be potentially incorporated in the formulation of mouthwash, toothpastes, gels, etc. References 1. Paster, B.J. et al., Bacterial Diversity in Human Subgingival Plaque. J. Bacteriol., 183, 12, 3770– 3783, 2001. 2. Fernandes, T., Bhavsar, C., Sawarkar, S., D’souza, A., Current and novel approaches for control of dental biofilm. Int. J. Pharm., 536, 199–210, 2018. 3. 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Suppakul, P., Miltz, J., Sonneveld, K., Bigger, S.W., Antimicrobial properties of basil and its possible application in food packaging. J. Agric. Food Chem., 21, 51, 11, 3197–207, 2003. 19. Prakash, P. and Gupta, N., Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: A short review. Indian J. Physiol. Pharmacol., 49, 2, 125–31, 2005. 20. Mallikarjun, S., Rao, A., Rajesh, G., Shenoy, R., Pai, M., Antimicrobial efficacy of Tulsi leaf (Ocimum sanctum) extract on periodontal pathogens: An in vitro study. J. Indian Soc. Periodontol., 20, 2, 145–50, 2016. 21. Jayanti, I., Jalaluddin, M., Avijeeta, A., Ramanna, P.K., Rai, P.M., Nair, R.A., In vitro Antimicrobial Activity of Ocimum sanctum (Tulsi) Extract on Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. J. Contemp. Dent. Pract., 19, 4, 415–419, 2018. 22. 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Dent. Res., Jul-Sep, 21, 3, 357–9, 2010. 27. Eswar, P., Devaraj, C.G., Agarwal, P., Anti-microbial Activity of Tulsi {Ocimum Sanctum (Linn.)} Extract on a Periodontal Pathogen in Human Dental Plaque: An In vitro Study. J. Clin. Diagn. Res., Mar, 10, 3, ZC53–6, 2016. 28. Freires, I.A., Denny, C., Benso, B., de Alencar, S.M., Rosalen, P.L., Antibacterial Activity of Essential Oils and Their Isolated Constituents against Cariogenic Bacteria: A Systematic Review. Molecules, 22, 20, 4, 7329–58, 2015. 29. Wiwattanarattanabut, K., Choonharuangdej, S., Srithavaj, T., In Vitro Anti-Cariogenic Plaque Effects of Essential Oils Extracted from Culinary Herbs. J. Clin. Diagn. Res., Sep, 11, 9, DC30– DC35, 2017. 30. Hugar, S., Patel P, M., Nagmoti, J., Uppin, C., Mistry, L., Dhariwal, N., An in vitro Comparative Evaluation of Efficacy of Disinfecting Ability of Garlic Oil, Neem Oil, Clove Oil, and Tulsi Oil with autoclaving on Endodontic K Files tested against Enterococcus faecalis. Int. J. Clin. Pediatr. Dent., 10, 3, 283–288, 2017. 31. Lolayekar, N.V. and Kadkhodayan, S.S., Estimation of salivary pH and viability of Streptococcus mutans on chewing of Tulsi leaves in children. J. Indian Soc. Pedod. Prev. Dent., 37, 1, 87–91, 2019. 268 Natural Oral Care in Dental Therapy 32. Ramesh, G., Nagarajappa, R., Madhusudan, A.S., Sandesh, N., Batra, M., Sharma, A., Patel, S.A., Estimation of salivary and tongue coating pH on chewing household herbal leaves: A randomized controlled trial. Anc. Sci. Life, 32, 2, 69–75, 2012. 33. Pereira, S.L., de Oliveira, J.W., Angelo, K.K., da Costa, A.M., Costa, F., Clinical effect of a mouth rinse containing Ocimum gratissimum on plaque and gingivitis control. J. Contemp. Dent. Pract., 1;12, 5, 350–5, 2011. 34. Pimenta, M.S., Lobo, N.S., Vieira, V.C., Costa, Â.M., Costa, F.N., Pereira, S.L., Effect of Ocimum gratissimum in Mouth rinses on De Novo Plaque Formation. A Randomized Clinical Trial. Braz. Dent. J., 27, 6, 646–651, 2016. 35. Gupta, D., Bhaskar, D.J., Gupta, R.K., Karim, B., Jain, A., Singh, R., Karim, W., A randomized controlled clinical trial of Ocimum sanctum and chlorhexidine mouthwash on dental plaque and gingival inflammation. J. Ayurveda Integr. Med., 5, 2, 109–16, 2014. 36. Bhardwaj, A., Srivastava, N., Rana, V., Adlakha, V.K., Asthana, A.K., How efficacious are Neem, Tulsi, Guduchi extracts and chlorhexidine as intracanal disinfectants? A comparative ex vivo study. Ayu, 38, 1–2, 70–75, 2017. 37. Gupta, A., Wadhwa, J., Duhan, J., Comparative evaluation of antimicrobial efficacy of three herbal irrigants in reducing intracanal E. faecalis populations: An in vitro study. J. Clin. Exp. Dent., 1, 8, 3, 230–235, 2016. 38. Pradhan, M.S., Gunwal, M., Shenoi, P., Sonarkar, S., Bhattacharya, S., Badole, G., Evaluation of pH and Chlorine Content of a Novel Herbal Sodium Hypochlorite for Root Canal Disinfection: An Experimental In vitro Study. Contemp. Clin. Dent., 9, Suppl 1, S74–S78, 2018. 39. Shenoy, A., Bolla, N., Sayish, Sarath, R.K., Ram, C.H., Sumlatha, Assessment of precipitate formation on interaction of irrigants used in different combinations: An in vitro study. Indian J. Dent. Res., 24, 4, 451–5, 2013. 40. Srivastava, A., Agarwal, R., Chaturvedi, T.P., Chandra, A., Singh, O.P., Clinical evaluation of the role of tulsi and turmeric in the management of oral submucous fibrosis: A pilot, prospective observational study. J. Ayurveda Integr. Med., 6, 1, 45–9, 2015. 41. Hosadurga, R.R. et al., Evaluation of the efficacy of 2% Ocimum sanctum gel in the treatment of experimental periodontitis. Int. J. Pharm. Investig., 5, 1, 35–42, 2015. 42. George, P.M. and Varghese, S.S., Electrospun Ocimum Sanctum Loaded Fibres With Potential Biomedical Applications – Periodontal Therapeutic Perspective. Biomed. Pharmacol. J., 11, 3, 1731–1736, 2018. 43. Hădărugă, D.I., Hădărugă, N.G., Costescu, C.I., David, I., Gruia, A.T., Thermal and oxidative stability of the Ocimum basilicum L. essential oil/β-cyclodextrin supramolecular system. Beilstein J. Org. Chem., 10, 1, 2809–2820, 2014. 44. Rodrigues, L.B. et al., Anti-inflammatory activity of the essential oil obtained from Ocimum basilicum complexed with β-cyclodextrin (β-CD) in mice. Food Chem. Toxicol., 109, 836–846, 2017. 45. Bindhani, B.K. and Panigrahi, A.K., Biosynthesis and Characterization of Silver Nanoparticles (Snps) by using Leaf Extracts of Ocimum Sanctum L (Tulsi) and Study of its Antibacterial Activities. J. Nanomed. Nanotechnol., s6, 6, 1–5, 2015. 46. Jacob, J.M. et al., Bactericidal coating of paper towels via sustainable biosynthesis of silver nanoparticles using Ocimum sanctum leaf extract. Mater. Res. Express, 6, 4, 045401, 2019. 47. Pirtarighat, S., Ghannadnia, M., Baghshahi, S., Biosynthesis of silver nanoparticles using Ocimum basilicum cultured under controlled conditions for bactericidal application. Mater. Sci. Eng: C, 98, 250–255, 2019. 48. Deepa, M.K., Eco friendly green synthsized silver nanoparticle with Ocimum Basilicum leaves aqueous extract. J. Innov. Pharm. Biol. Sci., 4, 3, 75–79, 2017. Ocimum Sanctum L. 269 49. El-Soud, N.H., Deabes, M., El-Kassem, L.A., Khalil, M., Chemical Composition and Antifungal Activity of Ocimum basilicum L. Essential Oil. Maced J. Med. Sci., 15, 3, 3, 374–9, 2015. 50. Anuradha, G., Syama Sundar, B., Ramana, M.V., Sreekanthkumar, J., Sujatha, T., Single step synthesis and characterization of Silver nanoparticles from Ocimum tenuiflorum L. Green and Purple. IOSR J. Appl. Chem., 7, 5, 123–127, 2014. 51. Dhaliwal, A.S. and Singh, J., Green synthesis of silver nanoparticles using Ocimum tenuiflorum leaf extract and their antioxidant and catalytic activity, Proceedings of Academics World 95th International Conference, 2018. 52. Gupta, N. et al., Comparative study of antibacterial activity of standard antibiotic with silver nanoparticles synthesized using Ocimum tenuiflorum and garcinia mangostana leaves. Chem. Biol. Lett., 2, 41–44, 2015. 53. Ramteke, C., Chakrabarti, T., Sarangi, B.K., Pandey, R.A., Synthesis of silver nanoparticles from the aqueous extract of leaves of Ocimum sanctum for enhanced antibacterial activity. J. Chem., 2013, 1–7, 2012. 17 Coconut Palm (Cocos nucifera L.): A Natural Gift to Humans for Dental Ministration Navneet Kishore and Akhilesh Kumar Verma* Department of Chemistry, University of Delhi, North Campus, New Delhi Abstract Coconut palm is scientifically known as Cocos nucifera L. (family: Arecaceae), which originated from southeast Asia and also across the Indian and Pacific islands. This plant has been used for a long time to treat a diverse range of human ailments in Ayurveda. According to Ayurveda, this plant is designated as a tree that provides all necessities to live. Approximately all parts of this plant are useful either medically or in other approach. Scientific reports disclose that it is one of the most valuable trees for dental care therapy. Coconut oil is one of the most important constituents to overcome various human ailments in traditional therapies. This oil has significant antibacterial, antifungal, and antiviral properties, which help to eradicate harmful microbes from oral cavity during oil pulling. Coconut water is an important component to preserve periodontal ligament cell viability and transport media to tear off a tooth. It is also defined as a nutritional power booster drink and has achieved international acclaim as natural sport drink. It is a clear liquid containing various nutrients with electrolytic property. It improves the skin tone and reduce the swelling through the advancement in immune function. It is also a better root canal irrigant due to its antimicrobial potential. Hence, this plant is accredited with several potential remedial actions in, for example, oral cavity disorders. The present chapter focused on up-to-date entries regarding traditional, ethnopharmacological, and bioactive phytochemicals and on significant use of coconut plant in oral cavity therapies. Keywords: Cocos nucifera L., medicinal value, coconut oil, coconut water, dental care 17.1 Introduction Oral cavity disorders are associated with the bacterial infections which originate serious health delinquencies in human beings around the world [1, 2]. The most prominent bacterial species accountable for periodontal diseases include Streptococcus mutans, Porphyromonas gingivalis, Prevotella intermedia, and other related bacterial species. All these species are responsible for various dental disorders that eventually lead to tooth decay, because they were isolated and identified from dental eruptions of human oral cavities [3, 4]. Suppressed concentrations of the aforesaid bacterial species in oral cavity could be the great achievement to overcome periodontal diseases. The inhibition of oral *Corresponding author: akhilesh682000@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (271–284) © 2020 Scrivener Publishing LLC 271 272 Natural Oral Care in Dental Therapy cavity ailments can be done by the eradication of these microbial species. There is approximate 90% of dental detonations encountered in teenagers and adult populaces which is preposterous. The plant-based drug development has constantly been an exceptional source for the invention of novel scaffolds [5, 6]. Hence, the medicinal plants provided a number of secondary metabolites for periodontal care to date, and many more are identified from the day-by-day research going on the respective field. Consequently, today, the universal requirements for the assessment of plant-derived compounds could be used in the treatment of dental diseases and the creation of alternative therapies from medicinal plants. Coconut palm (Cocos nucifera L.) belongs to the family Arecaceae, which originated from southeast Asia and also across the Indian and Pacific islands [7]. This plant has been used for a long time to treat a diverse range of human ailments in Ayurveda. According to Ayurveda, this plant is designated as a tree that provides all the necessities to live. Approximately all parts of this plant are useful either medically or in other approach. Its annual production is 61 million tons, which is encountered in approximately 90 countries worldwide. An estimated 73% of production is attributed to India, Indonesia, and the Philippines [8]. Several edible products made from coconut tree are orally taken as a tradition of many communities to maintain smooth natural life. Coconut milk is very operative to enhance in the growth of plant. Coconut water is a very efficient pure drink full of natural sugars and is 99% fat free with a lower level of carbohydrates. The electrolytic nature of coconut water due to the presence of ionic mineral, its constituent, is comparable with human plasma. This resemblance made it a global sport drink to cure oral dehydration [9]. Various parts of this tree, including leaves, fruits, spathe, flowers, flesh, husk fibers, coconut water, coconut oil, trunk, coconut milk, coconut shell, and roots, are very significant to make several medicinal and household products. 17.2 Traditional Usage and Ethnopharmacological Relevance Coconut is extensively used in traditional medicine for the treatment of a large assortment of human and animal ailments, which is also illustrated in Ayurveda. Coconut oil is applied on the backbone and stiff joints to relieve back pain and arthritis. It is also rubbed on the body as an ointment to preserve soft and smooth skin [10]. A mixture of coconut oil with turmeric powder is applied on newly born babies as well as mothers after delivery. Women desolated by complicated pregnancies are advised to take green coconut juice. Fruit juice helps in kidney nuisance and roots are applied on stomach pain, whereas a decoction with the root of Ruellia tuberose is a good remedy for bladder treatment. An extract from stem is proficient to overcome weakness after childbirth. Skin disorders like scabies, sores, and ulcers are treated with external application of apical bud in the form of poultice. Some parts of the plants are also used in the treatment of diarrhea and dysentery [11]. South Indian village people use husk fibers to brush their teeth. It is also used in folklore therapies to treat skin-related disorders such as skin burns, toothache, sunburns, sores, and ringworms. Massage using coconut oil makes skin soft and youthful and eradicates the cracks of heel and blackening portion of armpits. It eradicates dandruff and improves the scalp and hair. It is used to relieve stomach complications such as acidity and colitis. It is also quite effective in treating urinary glitches. It is helpful to treat the inflammation of mucosal membrane Coconut Palm (Cocos nucifera L.) 273 and measles. An intake of the combination of coconut milk and its flesh with honey by males and females strengthens their sexual appetite [12]. 17.3 Pharmacological Properties of Coconut Coconut tree has been recognized as a medication for a diverse range of sicknesses in various populaces due to its rich fiber contents, minerals, and vitamins with higher nutritional value. The presence of diverse mineral constituents in coconut water is used as an efficient storage medium and for tissue culture [13]. Diseases associated with oral cavity including gingivitis and tooth decay disturb approximately 60–90% of youngsters as well as a massive community of adults. Prophylactic effects of coconut water have also been observed from the ethylene glycol induced in mice [14]. Previous literature reports disclose that the antibacterial potential of coconut oil counters several strains of the genus Streptococcus including S. mutans. S. mutans is a species that causes tooth decay and extreme gingivitis. An antibacterial investigation on coconut alcoholic extract displayed good zone of inhibition against many bacterial species such as S. mutans, Prevotella intermedia, S. salivarius, S. mitis, Lactobacillus acidophilus, and Candida albicans (fungal) in the concentrationdependent method [11]. The pro-inflammatory properties of coconut are non-toxic in nature, and they are also helpful in the treatment of diabetic patients and prostatic hyperplasia [15]. When used in diet, Cocos nucifera is competent to overcome diabetes mellitus [16]. In a recent investigation, it has been proved that coconut extract exhibits significant antidiabetes property. Results showed that a combination of 250 mg/kg extract with 22.5 mg/ kg of metformin expressively lowered the level of PGL in the duration of 7th, 14th, 21st, and 28th days. It has also been confirmed that, it halts pancreatic diminishing after the treatment with coconut water [17]. Coconut fruits are helpful in the treatment of disorders in cardiac metabolism [18]. They are also very helpful in the restoration of islets of Langerhans b cells due to the presence of amino acids [19]. Other significant pharmacological properties include aphrodisiac, antiseptic, antioxidant, antihelminthic, antitumor, stomachic, and bactericidal [20]. The rich compositions of polyphenols regulate the metabolism of carbohydrate and lipid as well as insulin management. Coconut tree also helps to improve the metabolism of adipose tissue and signalling pathways [21]. Cocos fruits are enriched with lauric acid, which is responsible for the antimicrobial actions against some oral cavity contagions. The chemical components glycolipids and sucrose monolaurate existing in coconut are also liable for anti-dental carries. It is supposed to inhibit dental plaque in the tooth caused by S. mutans via reduced glycolysis and oxidation of sucrose. In recent reports, ethanolic leaf extract displayed protection against paralysis effect, inhibition of acetylcholinesterase as well as scavenger of free radicals [22], anthelmintic action [23], and antioxidant potential against reactive oxygen species [24]. There is a report on coconut water that showed that it significantly helps to protect cardiac problems [25]. Tender coconut water (TCW) is an efficient drink that has several health reimbursements without any adverse effect on the human body. The zone of inhibition is observed with values of 14.23 and 3.08 by the sterilized TCW and fresh TCW, respectively. The value of the zone of inhibition was clearly inferior to chlorhexidine, used as a standard drug [26]. An in vitro analysis of green coconut water was conducted against fungal species Candida 274 Natural Oral Care in Dental Therapy Anti-atherosclerotic action Anti-inflammatory action Anti-leishmanial activity Anti-cholecystitic action Anti-malarial activity Anti-diabetic activity Anti-neoplastic activity Cardio protective effect Anti-oxidant activity Hepatoprotective activity Anti-parasitic activity Hypolipidemic effect Anti-thrombotic action Gastrointestinal disorders Anthelmintic activity Anti-hypertensive action Immune stimulatory effect Antidote Action Endosperm Hormonal effect Disinfectant activity Antiprotozoal action Best electrolyte Protect teeth and gums. A better storage medium for avulsed tooth Virgin Coconut Oil Coconut Oil Fresh Coconut Water Anti-bacterial action Anticonvulsant action Figure 17.1 Represents the incredible medicinal value of the coconut tree. albicans over broth microdilution assay in an agar culture. The antifungal potential of this water was observed to be comparable to standard drug amphotericin B at a concentration of 1000 μg/mL and repressed the progress of fungal species by 14.0±03 and 15.0±03 for positive control [27]. 17.4 Role of a Coconut Tree in Dental Ministrations A systematic analysis of the endocarp of coconut proved that it is an affluent source of flavonoids and phenolic content. With the occurrence of these compounds, endocarp displays very good antimicrobial, antioxidant, and antihypertensive effects along with the inhibition of bacterial species existing in the oral cavity [28, 29]. The antimicrobial potential of alcoholic extract from husk fibers against oral pathogens, namely, Fusobacterium nucleatum, Lactobacillus casei, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus mutans, has been evaluated [30]. The result displayed significant potential against oral dentistry pathogens [31]. The antimicrobial susceptibility and cytotoxicity action have also been evaluated for husk ethanolic extract against three endodontic pathogens species, viz., Porphyromonas gingivalis, Prevotella intermedia, and Enterococcus faecalis [32]. The most efficient practice from the earliest periods to cure teeth is oil pulling. In this practice, coconut oil is used and helps to preserve healthy teeth. Extra virgin coconut oil of about 5–10 mL is taken in the mouth to rinse the oral cavity for 15–20 minutes [29]. This action removes all Coconut Palm (Cocos nucifera L.) 275 1. Coconut Leaves 2. Coconut Spathe & Flowers 3. Coconut Young Fruits 4. Coconut Flesh 5. Coconut Endosperm 6. Coconut Shells 7. Coconut Husk Fibres 8. Coconut Trunk 9. Coconut Roots Figure 17.2 Represents the various parts of coconut (Cocos nucifera L.) tree. the harmful bacterial species as well as other microorganisms and also reconciles the gums. A strong electrolyte inside the fruits contains chief essential minerals such as calcium, magnesium, and potassium, which are rich in amount. Coconut water also contains cytokinin hormone, which normalizes growth and aging; it encourages the use of coconut water in tissue culture. With the feasibility of periodontal ligament cells in coconut water, it is used in emergency cases for the transportation of avulsed tooth [33]. 17.5 Exemplary Potential of Coconut Water in Dentistry The consumption of coconut in everyday regime in several traditions not only affords good taste but also preserves healthy life devoid of various illnesses. The electrolytic liquid inside the fruits is a good rehydrating drink, a transference mediator for avulsed tooth, blood exchange, and a culture medium in cell culture laboratories [34]. Earlier lessons suggested coconut oil to be used in folk therapies for various dental complaints, and it achieved excellent recognition for folk solidification of teeth and exudates, deterrence of tooth falloff, bad smell from oral cavity, bloody gums, and splintered lips [35]. The usage of plant-based folklore remedies is documented in the Ayurveda; hence, these tactics need scientific authentication of herbal products in oral cavity disorders [36]. Hence, it exhibits excellent relief of pain and swelling and is used as a root canal irrigant due to its antimicrobial properties. It is used as storage medium for avulsed tooth. Moreover, coconut water has been recognized as a new enhanced choice to store avulsed teeth because of its significant viability and preservation of periodontal ligament cells [9]. 276 Natural Oral Care in Dental Therapy A number of herbal decoctions displayed extensive accomplishment in dentistry after treating different dental disorders. Plant-based natural compounds are a paramount alternative source to prevent several oral illnesses. However, the prolonged used of these herbal formulations is much safer and convenient compared to synthetic drugs. The assessments of available herbal remedies are still mysterious; an enormous use of these products is expected in the future. There are several aspects to acquire more data through further investigation with regards to these herbal products assured for dental disorders [37]. Several reports on coconut water in dentistry published in very recent years are discussed here. The viability of PDL fibroblasts was observed in skimmed milk followed by salt solution and coconut water [38]. A study conducted on the pH-adjusted coconut water showed significant viability of periodontal ligament cells, and it can preserve the viability of avulsed teeth for up to 24 hours [39]. Another lab report also supported the cell viability of periodontal ligament cells in coconut water [40]. In a recent report, it was revealed that the new formula developed from the powdered coconut water (ACP-404) showed significant PDL cell viability. This study was carried out on 60 teeth of a dog that were successfully replanted after the storage of newly formulated ACP-404 storage media [41]. In another research group, a setup of an in vitro experiment for 69 human premolars along with normal periodontium was divided into three groups of 23 each. They were preserved into three different storage media, viz., coconut milk, Hanks’ balanced salt solution (HBSS), and probiotic milk. Results showed noteworthy dissimilarity in the maintenance of cell viability between the three storage media used. Hence, this study determined that the coconut milk is not appropriate due to poor cell viability for interim media for transportation. However, HBSS and probiotic milk showed good preservation of PDL cell viability [42]. 17.6 Other Significance of Coconut A number of reimbursements have been attributed to coconut tree starting from the top of the leaves to the bottom of its roots in numerous civilizations of different countries. The wide range of products is accountable for their medicinal persistence, building material for houses, food stuff, decoration etc. [43]. The significance of various parts of coconut tree has been discussed in this segment. 17.6.1 Economic Value of Coconut Leaves In Maldives, leaves of coconut tree are used as a household material for making roof, and in the Philippines, they are used for cooking, to wrap rice, and for storing rice. The leaves of coconut tree are generally durable for 1 year. The leaves are substantial to make brooms and toys and are burnt to make lime in India; leaf midribs are used to make skewers and toothpicks. Stems of coconut tree are used to make boats, canoes, drums, furniture and, houses as timber. These leaves are very long and disbursed to generate inimitable look in landscape design. Coconut leaves are also a very favorite fodder for elephants. The leaves are also useful to make trays, hats, paper pulps, umbrellas, ropes, barbecue skewers, and cover for books. Coconut Palm (Cocos nucifera L.) 277 17.6.2 Use of Coconut Heart The coconut heart is generally known as “Palmis,” and it is very demandable for invitees and resident societies due to its delightful taste. These are mainly taken in salads as well as other cookeries. 17.6.3 Significance of Spathe and Inflorescence The covering of coconut inflorescence is termed as the spathe. The whole spathe and flowers are used to make containers for picnics [44]. The dead inflorescence is used to make brooms [45]. The young flowers with spathe are full of sap, which is used to make “Kalou” drink after the fermentation of extracted sap [46]. 17.6.4 Potential of Coconut Fruits Coconut flesh is a very significant natural laxative enriched with protein. It is used to make coco milk, coco flour, candies, copra, coco chips, and animal feeds. Also it is used to make several sweets and is a salad ingredient. Water inside the fruit is enriched with ascorbic acid, proteins, and vitamin B [47]. Also it is used as a main ingredient for salad and other sweet delicacies [48]. Coconut oil is a natural moisturizer, softener, and anti-wrinkle, avoids skin contagion, and retains skin elasticity with enhanced skin tone. With the presence of longchain fatty alcohol and glycerol in coconut oil, it is utilized in the formulation of candles, cosmetics, detergents, and soaps. 17.6.5 Usage of Coconut Milk Coconut milk contains rich ingredients of saturated fat. It is used in Brazil to cook several seafood dishes as well as baking of other dishes. The 1-week fermentation of coconut milk and sugar by yeast directed to the manufacture of home beverage [49]. The well-known cocktail Pina colada also has coconut milk as a component. It is also used all over Southeast Asian communities in making curries. 17.6.6 Importance of Coconut Shell The very hard outer cover of coconut fruits is called the coconut shell. These shells are very in demand in making significant art items by handicraft industries nowadays. The charcoal made from coconut shells is used in several localities and industries for various purposes. Apart from several usage of activated charcoal, medicinally it is used for the absorption of poisons and toxins. It is also used in making activated carbon. This activated carbon is safe and 100% natural, which is obtained by burning coconut shell in various ways. It produces only very little ash after burning and does not cause pollution. A whole lot of eco-friendly uses have made it preferable in many industries like manufacturing of air and water purifiers, odor eliminators, and even building golf courses. The coconut shell powder is used in the production of mosquito coils and is very good in timber industries due to its resistance to fungal and water outbreak. 278 Natural Oral Care in Dental Therapy 17.6.7 Commercial Usage of Husk Fibers The long life of husk fibers has attracted the attention of several industries to make several home and handicraft products. They are considered as they could normally survive for 20 years [50]. It is natural, eco-friendly, and safe. These fibers are basically used to make ropes due to their longevity and resistance to water [51]. They are used to make fishing net, to wash pots, as fuels, to make toys, in the production of mattresses, in irrigation arrangement due to its water holding capacity, in hydroponic industries, to make mats, as an absorbent, as automobile seats, to make concrete, to make brushes and brooms, in soil erosion, and as an insulated material [52] due to its upright temperature holding strength. 17.6.8 Economic Importance of Coconut Stems The main usage of coconut trunk is as a house building material. The wood is very durable and is used to make tables, chairs, walls, doors, and window frames with other minor usage. Coconut stems are used to make poles in telecommunication and other purposes. The trunk is also used to produce wood planks, which are used in the partition of buildings to make chambers. It is also valuable to make paper pulp. 17.6.9 Convention of Coconut Roots The roots of this tree have been used traditionally for brushing the teeth, as mouth wash, and for making dyes. The roots are also used to make beverage and various medicinal products. Roots are used to treat urinary tract contamination and gall bladder and kidney problems, to treat eczema, to omit blood clotting, and to stop painful hemorrhoidal bleeding in the human body. It is very effective in skin disorders, itching of toes, fever, diarrhea, and dysentery. 17.7 Active Constituent Identified from Coconut The flower extract of coconut in different solvents led to the identification of several classes of bioactive secondary metabolites, which include anthraquinones, alkaloids, amino acids, carbohydrates, flavonoids, phenols, saponins, and tannins [53, 54]. The oil obtained from the coconut shells is used for the treatment of skin disorders and abrasions. This oil was extracted into the solvent of different polarity fractions, which were analyzed for their phytochemicals, and show the way to the presence of alkaloids, amino acids, carbohydrates, carboxylic acid, flavonoids, phenols, proteins, quinones, oxalate, tannins, and terpenoids [55]. The oil obtained from the coconut mainly comprises triglycerides mixture of saturated fatty acids with short to medium chain and limited amount of unsaturated fatty acids. Lauric acid, one of the chief components of coconut oil, holds about 50% of total composition [56]. Apart from the lauric acid, its composition includes arachidonic acid, capric acid, caproic acid, caprylic acid, eicosinoic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid [57, 58]. This composition of coconut oil is accountable for the strong antibacterial, antifungal, antiprotozoal, and antiviral potential. Coconut Palm (Cocos nucifera L.) 279 CH2 CH2 H 3C CH3 H3CO CH3 H3CO H 3C H3C CH3 Lupeol Methyl Ether CH3 Skimmiwallin Isoskimmiwallin CH3 HO HO H CH3 H CH3 CH3 OH O O HO OH OH Ascarbic Acid a-tocopherol NH CH3 HO O OH O O HO OH HO CH3 O CH3 CH2 CH3 CH3 H3C H2N N H OH CH3 OH NH2 Lauric Acid L-Arginine Catechin O O HO CH3 HO CH3 Arachidonic acid Capric acid O O HO CH3 HO CH3 Linoleic acid Caprylic acid O HO CH3 CH3 CH3 COOH CH3 H3CO H 3C CH3 H 3C CH3 CH3 CH3 H 3C H 3C O CH3 HO CH3 Linolenic acid Caproic acid O CH3 O CH3 HO HO Oleic acid Myristic acid CH3 O HO CH3 O HO Stearic acid Palmitic acid 17.8 Future Prospective Coconut palm tree is distinguished for its therapeutic values as well as resourceful nourishments. Homemade and herbal formulations of coconut have extra plenteous devotion in the last decade, which has been continued since the ancient time. There are many bioactive 280 Natural Oral Care in Dental Therapy compounds that have been identified from coconut that exhibit significant pharmacological properties including extraordinary potential to preserve and sustain strong teeth. Coconut tree produces fresh coconut water, coconut milk, coconut oil, endosperms, husk fibers, leaves, roots, and trunks. All these parts of coconut have their own significant values in innumerable traditions. Coconut water is an important component to preserve periodontal ligament cell viability and transport media to tear off tooth. It is also defined as a nutritional power booster drink and has achieved international acclaim as a natural sport drink. It is a clear liquid containing various nutrients with electrolytic property. It improves the skin and reduces swelling, and one gets better immune function and root canal irrigant via its antimicrobial potential. Coconut oil has an amazing composition of saturated and unsaturated fatty acids, attributed to the anti-microbial properties. An alcoholic extract of coconut as well as coconut oil shows very good antibacterial potential against S. mutans including other oral bacterial species. A previous literature reports on traditional, ethnopharmacological, pharmacological, phytochemical, and valuable importance of coconut at different community levels; it is clear that there is hitherto scantiness of scientific investigation with respect to the mode of action inside the human body. However, a number of active metabolites have been identified and there may still be many more unknown compounds that could be accountable for biological properties. Hereafter, more explorations are needed for the credentials of mysterious metabolites and their respective bioactivity. Therefore, coconut tree could be of interest to many researcher; they should consider this tree for future research to discover the unseen facets of this enchanted tree. 17.9 Conclusions The current section of this book compiles the economic usage of coconut tree in oral cavity disorders. Apart from the significant potential to overcome on dental complications, we have also summarized the other advantage of the coconut tree. Its traditional usage, ethnopharmacological relevance, pharmacological properties and identified phytochemical profile of this plant have been compiled in this assignment. This tree is one of the most extensive species explored at the national and international level for its medical implication. More than 100 research articles have been published on C. nucifera tree. We have assembled all updates on coconut tree in this chapter with documentation of potential secondary metabolites and usage in dentistry. The designated evidences were composed of the several research articles published on this plant and from internet sources. A very recent report proved its antidiabetic potential in combination with metformin, a standard drug available in the market for diabetic patients. Hence, additional study is compulsory to fix the unidentified aspects regarding the coconut tree by organized examination and upscaling of the precise evidences. This chapter will provide a platform to the researches to convey the supplementary steps in further research. Acknowledgments The authors gratefully acknowledge the University of Delhi, India for help and support. University of Grant Commission (UGC), New Delhi, is also gratefully acknowledged for the postdoctoral fellowship award of Dr. D.S. Kothari and for financial support. Coconut Palm (Cocos nucifera L.) 281 References 1. 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Sawarkar1*, Anisha D’souza2 and Trinette Fernandes1 1 Department of Pharmaceutics, SVKM’s Dr. Bhanuben Nanavati College of Pharmacy, University of Mumbai, Maharashtra, India 2 Formulation Development Laboratory, Piramal Enterprises Limited, Light Hall, Chandivali, Powai, Mumbai, India Abstract Miswak (Misswak, meswak, miswaki, siwaki, sewak,) is one such ancient age-old extensively used chewing stick obtained from Salvadora persica L (Arak Tree) or toothbrush tree. The shrub is commonly cultivated in Asia, Middle East, and Africa. Miswak is obtained from Salvadora persica from the family Salvadoraceae. The plant has several phytoconstituents that have shown promising effects in maintaining oral hygiene. The chemical constituents of Salvadora persica or Arak (Miswak) include glycosides, flavonoids, resins, Benzyl isothiocyanate, volatile oils, terpenes, silica, and mineral salts. Miswak has several therapeutic activities, which include antibacterial, antifungal, anticariogenic, and anti-plaque effect and, hence, acts on the pathogens. The World Health Organization has recommended more use of miswak for promotion and maintenance of oral health. Traditionally, miswak is used as chewing sticks, but it is available in modern applications. The commercial products of miswak that are available are toothpastes, mouthwashes, irrigants, and chewing gums. It is essential to promote the usage of miswak to promote oral hygiene and health care management. Keywords: Miswak, oral hygiene, Salvadora Persica, volatile oils, Benzyl isothiocyanate 18.1 Introduction Oral cavity is a major entry portal for many diseases affecting health. Poor maintenance of craniofacial and oral health affects the psychosocial and functional quality of life. The easy strategy of maintaining oral hygiene is mechanical cleaning of tooth using dentifrice and toothbrush [1]. Natural and medicinal plants are complementary therapy due to their numerous beneficial effects [2]. Natural cleansers include chewing sticks from anti-microbialexhibiting shrubs, plants, and trees [3] instead of the currently used plastic-bristle brushes. Miswak (Misswak, meswak, miswaki, siwaki, sewak) is one such ancient age-old extensively used chewing stick obtained from Salvadora persica L (Arak Tree) or toothbrush tree. The *Corresponding author: sujata.sawarkar@bncp.ac.in; sujatasawarkar19@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (285–296) © 2020 Scrivener Publishing LLC 285 286 Natural Oral Care in Dental Therapy entire plant, i.e., the twigs, roots, and stems have been used as chewing sticks for oral hygiene. This shrub is commonly obtained in Asia, Middle East, and Africa [4]. Miswak brushes are obtained from the roots of the Arak tree and can be used on the teeth and gums. The plant exhibits anti-bacterial and anti-fungal properties against those bacteria and fungi responsible for the development of periodontitis and dental plaque [5]. These chewing sticks are chewed till they are frayed brush-like form and can be used as brush for cleaning. After multiple use, the chewing stick brush gets shredded and becomes ineffective. The chewing stick miswak exerts efficacy due to the fiber’s mechanical effect as well as the release of vitamin C, trimethyleamine, resins, saponins, salvadorine, flavodine, and sterol [6]. The present chapter discusses about Salvadora persica L (Miswak), known in ancient times, and the current scientific understanding of varied Miswak (Salvadora persica L) activities. 18.2 History The use of mechanical devices for cleaning teeth has been a folklore tradition. The exact inception is unknown. Toothpicks have been known to originate from the Babylonians and used along with other toiletry articles to remove or clean the fibrous residue retaining between the teeth after meals. While the Romans also used mastic tree-originated toothpicks (Pistacia lentiscus), the Arabs used miswak or siwak. The importance and use of meswak has also been incorporated as a religious practice in Islam. The use of miswak has been suggested before and after sleeping, before and after having meals, after entering house, during fasting, and before reading or reciting prayers or holy texts. Hence, it is more popular among the Muslims globally [7]. Miswak is obtained from Salvadora persica from the family Salvadoraceae. It is an evergreen shrub, which grows up to a maximum of 3 m. The fruits are edible berry and barely noticeable. It can tolerate extreme conditions, dry environments, as well as saline soils [8]. 18.3 Chemical Constituents The chemical constituents of Salvadora persica or Arak (Miswak) include glycosides such as Salvadosides and Salvadorasides, flavonoids [9]. Silica present in miswak has abrasive effect on stains. Resins present in miswak provide protective effect on tooth enamels. Other prominent phytoconstituents are Benzyl isothiocyanate, volatile oils, terpenes. Alali F. et al. performed GC MS analysis to identify the components of the oils. The constituents identified were 1,8-cineole (eucalyptol), α-caryophellene, β-pinene (6.3%), and 9-epi-(E) caryophellene [10]. Benzyl isothiocyanate has a potent activity against dental caries. M.S. Abdel-Kader et al. quantified benzyl isothiocyanate in root extracts of Salvadora persica by gas chromatography (GC) [11]. In addition to benzyl isothiocyanate, miswak also contains benzyl nitrile, carvacrol, aniline, benzaldehyde, naphthalene [12]. The root bark of miswak contains chlorine. In addition, it contains lauric acid, palmitic, and myristic acid and lignin derivatives of phenols and furans, syringins, liriodendrin, and sitosterol-O-glucopyranoside [12]. Chlorine acts as dentrifice to remove tartar. − Antimicrobial substances present in miswak are sulfate SO2− 4 and thiocyanate SCN . SCN− is acted upon by lactoperoxidase present in the saliva in the presence of hydrogen peroxide to Salvadora persica L. (Miswak) 287 form hypothiocyanate (OSCN−). Hypothiocyanate interacts with bacterial enzymes leading to cell death. β-Sitosterol and ascorbic acid show anticholesterolemic properties. Ascorbic acid (Vitamin C) showed an antiscorbutic activity. Trimethylamine and Salvadorine have shown potent antibacterial and gingiva-stimulating effects. The presence of trimethylamine, alkaloid resin, and sulfur compounds has shown potent antimycotic effect. Halib et al. performed morphological and elemental analysis of fibers of Salvadora persica. Energy-dispersive X-ray spectroscopy (EDX) and scanning electron microscopy was used to identify mineral crystals present in these fibers. The rhomboidal crystals were composed of O, C, S, Ca, Na, and K, whereas the irregular crystals consisted of C, O, Al, Si, S, K, Ca, and Fe [13]. Mohamed et al. investigated the presence of five α-amylases in miswak. The methodology adopted for purification was chromatography on DEAE Sepharose column and Sepharyl S-200 column and molecular weight determination by Gel filtration and SDS-PAGE. The five α-amylases identified were A1, A4a, A4b, A5a, and A5b. The researchers concluded that the presence of Ca2+ activated the enzymes, whereas Ni2+, Co2+, and Zn2+ activated or inhibited the enzymes [9]. 18.4 Extraction, Isolation, Identification of Chemical Constituents Benzyl isothiocyanate is obtained as an essential oil by steam distillation. Gas chromatographymass spectrometry confirmed the presence of benzyl isothiocyanate. It has a better cidal effect on gram-negative periodontal pathogens like A. actinomycetemcomitans, P. ginigivalis, Salmonella enterica, Haemophilus influenzae, etc. The roots of Miswak is cut and ground using stone mill and steam distilled at 80°C followed by extraction with hexane. Solid-phase microextraction of Miswak was also used for collecting the volatile Benzyl isothiocyanate using polydimethylsiloxane/divinylbenzene-coated SPME-fibers. The volatile oil was characterized by GC-MS and TLC [14]. Benzyl isothiocyanate composed 73.8% of volatile oil, while benzyl nitrile was 26.2%. The roots are devoid of any terpenes or hydrophobic chemicals [15]. 18.5 Pharmacology—Therapeutic Activity of Salvadora persica L. 18.5.1 Theories for Miswak Activities Mechanical cleaning of the chewing stick as toothbrush is one of the major reasons for cleaning teeth. Besides, the chemical constituents, Miswak exhibits antibacterial, antifungal, anticariogenic, and anti-plaque effect and, hence, acts on the pathogens disturbing the oral health. Another reason is the constant chewing of sticks, which increases salivation and also the chloride and calcium content with reduced phosphate and pH immediately after the chewing. This effect is not observed after 4 h of chewing. Calcium promotes the tooth enamel to mineralize, while chloride minimizes the formation of calculus [16]. Also, miswak extract elevated the plaque pH and also stimulated secretions from the parotid gland within 30 min of rinsing the oral cavity with the extract. Increasing the secretions increases the plaque pH and thus prevents caries [17]. Reports of antioxidant activity attributed to the various enzymes (catalase, peroxidase, polyphenol oxidase, etc.) and compounds like phenol, tocopherols [18], 288 Natural Oral Care in Dental Therapy beta-sitosterol, stigmasterol [19] in seed oil, and cake of Salvadora persica are synergistic for cleaning teeth [20]. A combination of miswak with other herbal extracts like kalonji and aloe vera provided synergestic effect as antimicrobial, antioxidant, and anti-proliferative [21]. 18.5.2 Antibacterial and Antifungal The ethanolic extracts have shown a higher antimicrobial activity. Among the different parts— root, twig, stem, it was found that the alcoholic root extract was more effective than the extract from the twig, and the least effective was the aqueous extract from the stem [22]. In vitro studies of miswak have shown antifungal and antibacterial effects on periodontal pathogens and cariogenic bacteria. They have been effective against both aerobic and anaerobic bacteria [23– 29]. Clinical studies additionally have shown that 10% of the aqueous extract of Miswak can be effectively used only if it is used as an irrigant clinically during endodontic therapy where the teeth have necrotic pulps [30–33]. However, the antimicrobial effect of crude miswak extract was low to moderate in comparison to tea tree oil and 0.2% aqueous chlorhexidine [22]. Zayed Al-Ayed et al. evaluated the antibacterial activity of Salvadora persica (Miswak) extracts against multidrug-resistant bacteria obtained from clinical isolates. Methanolic extracts of miswak showed a promising effect against methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia. In vitro studies of Miswak on 10 multidrug-resistant (MDR) bacterial clinical isolates excluding the oral pathogens have also been studied. These included a) methicillin-resistant Staphylococcus aureus, b) penicillin-resistant Streptococcus pyogenes, c) Klebsiella pneumoniae, d) Pseudomonas aeruginosa, e) Escherichia coli, f) Acinetobacter baumannii, g) Serratia marcescens, h) Stenotrophomonas maltophilia, i) methicillin-resistant Staphylococcus epidermidis, and j) Enterococcus faecalis. Activity was checked by agar diffusion as well as minimum inhibitory concentration method. The methanolic and water extracts were effective against all the MDR pathogens at concentrations of 400 mg/mL on all the strains, with methanolic extract being more effective than the aqueous extracts. The antibacterial effect was more prominent than the Gram-positive bacteria. The minimum inhibitory concentration was 0.39 mg/mL for Escherichia coli using the methanolic extract, while it was 1.56 mg/mL with the aqueous extract. Methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia, and Acinetobacter baumannii had high MIC of 12.5 mg/mL with the aqueous extract and 6.25 mg/mL with the methanolic extract. Vancomycin and Tobramycin were used as positive controls at concentrations of 30 µg/mL and 10 µg/mL, respectively [34]. The most probable reason could be that the methanolic extract has the capability of extracting flavonoids, tannins, alkaloids, sterols, and other reducing agents. However, aqueous extracts also have saponins, reducing components, and tannins [29]. Benzyl isothiocyanate is another volatile compound identified in the extracts, which have a quick and strong cidal effect against Gram-negative periodontal pathogens compared to the Gram-positive bacilli. The volatile ingredient could penetrate easily through the bacilli membrane and interfere with the redox systems failing to maintain the membrane potential [31]. Another such active ingredient of Salvadora persica is N-benzyl-2-phenylacetamide with moderate antimicrobial activity. At a concentration of 87 µL of N-benzyl-2-phenylacetamide, the activity was equivalent to 20 µL of gentamycin [35]. The results were, however, seen opposite to those studied with oral pathogens. With oral pathogens, the aqueous Miswak extract exhibited higher antimicrobial effect than the methanolic extracts. The oral Salvadora persica L. (Miswak) 289 pathogens studied were Candida albicans, Lactobacillus acidophilus, Pseudomonoas aeruginosa, Staphylococcus aureus, Streptococcus faecalis, Streptococcus mutans, and Streptococcus mutans. Streptococcus responded very quickly to aqueous extracts and exhibited an inhibitory activity against Streptococcus faecalis [36]. Simple addition of whole miswak pieces inside the agar or simply suspended above the agar plate exhibited an antibacterial effect against bacteria like Aggregatibacter actinomycetemcomitans, Haemophilus influenzae, Lactobacillus acidophilus, Poryphyromonas gingivalis, and Streptococcus mutans found during caries progression and periodontitis [37]. A comparison of different herbal extracts from Avicennia marina (Qurm), Aspho delustenui foils (Kufer), Fagonia indica (Shoka’a), Portulaca oleracea (Baq’lah), Lawsania inermis (henna), Salvadora persica (Souwak), and Ziziphus spina-Christi (Sidr) was studied with respect to the stability and toxicity, different dynamic growths of Candida albicans accompanied with secondary bacterial infections. Candida albicans have a complicated morphology switching between yeast and hyphae to form resistant biofilm harboring secondary bacilli. The dried ethanolic extracts of these plants were reconstituted with water before administration. The alcoholic extracts of S. persica, L. inermis, and P. oleracea significantly inhibited Candida albicans growth after 24 h and 72 h of incubation in liquid Luria–Bertani media, while it showed moderate antibacterial effect against secondary infection-related bacilli [38]. Salvadora persica extract showed superior activity against Lactobacillus acidophilus compared to Neem, which showed higher activity against Streptococcus mutans than the former [39]. An in vitro comparison of miswak extract, an Iranian toothpaste containing miswak extract, and another toothpaste from Switzerland also containing miswak extract was studied on Eikenella corrodens, Streptococcus sanguis, Porphyromonas gingivalis, and Streptococcus salivarius. All of them showed inhibition of dental plaque bacteria [40]. However, the presence of toothpaste containing miswak extract was more beneficial in Streptococcus mutans reduction compared to simple brushing. Lactobacilli were, however, not that affected [41]. The antibacterial effect of Miswak mouthwash (PersicaTM, Pursina Pharmaceutical Company, Tehran, Iran) though was effective against Streptococcus mutans; it was, however, not that potent as Chlorhexidine in orthodontic patients [42]. Moreover, Miswak mouthwash was found to be more effective in reducing the levels of Lactobacilli compared to ordinary toothwash within 2 weeks of use. The mouthwash was better than Miswak toothpaste also [43–45]. The effect was also pronounced than saline but not beyond 2 weeks [46]. Further addition of yarrow extracts and mint decreased the counts of Enterococcus faecalis and Candida albicans significantly in vivo [47]. In one study, it was observed that the antibacterial effect of fresh Miswak, 1-monthold miswak was unaffected [48]. The activity was found to be unaffected after 18 years of storage. The old Miswak extract demonstrated activity against Streptococcus faecalis [49]. Meanwhile, the antifungal effect of acetone extracts of (300 mg/mL) the dry stems of Salavadora persica had higher activity against Candida strains of Candida glabrata, Candida albicans, and Candida parapsilosis, while the methanolic and ethyl acetate extracts were effective on only one strain of the isolate [50]. Surprisingly, the dried Miswak was more effective as an antifungal rather than the fresh plant. The aqueous extract of the roots were antimycotic at concentrations greater than 15% for up to 48 h on Candida albicans [51]. Besides Candida albicans, a number of references cite about the antifungal activity of Salvadora persica against Enterococcus faecalis [52], Aspergillus strains [53, 54]. There have been mixed reports about efficacy of ethanolic extracts against Candida clinical isolates [54], while some researchers have reported strong to moderate activity [55–57]. 290 Natural Oral Care in Dental Therapy 18.5.3 Anti-Viral Effect BITC extracted from roots (133 µg/mL) have shown virucidal effect against Herpes simplex virus-1 [51]. 18.5.4 Anti-Cariogenic Effect Miswak has strong anti-decay effect with reduced plaque formation and development and thereby poor progression of caries in miswak adult users [58]. Besides, the rural population with a high tendency to use miswak exhibited lower caries prevalence compared to the urban population of Zanzibar [59]. The antidecay effect is attributed to its fluoride content [60, 61]. Besides, salivary secretion increases due to the chewing effect and hot taste of miswak, which subsequently increases the buffering capacity [62]. A clinical trial performed on second-year Iranian school children showed that miswak was able to prevent dental caries. The control group, using only toothbrush for cleaning, was 9.35 times more prone for the risk of dental caries [63]. Fortified fresh miswak chewing sticks with sodium fluoride have a remineralizing effect on the white spot lesions [64, 65]. Al-Dabbagh et al. compared the efficacy of Miswak toothpaste and mouthwash with other commercial dental products on cariogenic bacteria. The studies concluded that Miswak products especially mouthwash showed more efficacy against Lactobacilli and Streptococcus mutans than other products [66]. 18.5.5 Antiplaque Effect Miswak significantly reduced buccal and lingual gingivitis following brushing with Miswak five times a day compared to brushing with a conventional toothbrush. Brushing twice a day though showed significantly reduced buccal ginigivitis, but the effect was not much pronounced with lingual surfaces [67]. It is also effective against plaque [68] and dental caries [69] than tooth brushing. A research also reported that a toothpaste containing Salvadora persica was more effective than commercial toothpaste for plaque removal [62]. A single-blind, randomized clinical study showed that miswak was better in reducing Aggregatibacter actinomycetemcomitans in subgingival plaque [68]. A double blinded randomized trial of Salvadora persica extract chewing gum was conducted in volunteers having moderate gingivitis, reduced plaque index, bleeding index, and gingival index [70]. In one study, researchers performed a 24-h plaque re-growth, double-blinded randomized controlled crossover trial wherein they compared a Miswak and Green Tea combination mouthwash with Chlorhexidine mouthwash [71]. The plaque activity was measured by the modified Quigely Hein plaque index. The Miswak and Green tea combination mouthwash showed significant reduction in plaque accumulation compared to the placebo and Chlorhexidine mouthwash. 18.5.6 Antiperiodontitis Effect Clinical studies have shown that Miswak users showed fewer incidences of dental calculus and gingival bleeding. M. Hammad and A. K. Sallal studied the effect of mouth rinse with aqueous Salvadora twigs extract and Chlorhexidine digluconate on the adhesion of Streptococcus mutans bacterial cells to buccal epithelial cells [72]. Salvadora persica L. (Miswak) 291 18.5.7 Whitening Effect Salvadora persica (Miswak) extracts have shown potent whitening effect. Halib et al. evaluated the anti-staining activity of Miswak on extracted premolar teeth. The stains were developed using tea and coffee. The researchers formulated Miswak paste and compared it with commercial whitening toothpaste. The changes in teeth shade were assessed using VITAPAN Classical Shade. Miswak toothpaste showed a promising whitening effect. The whitening effect was attributed to the abrasive effect caused by silica and mineral crystals [73]. The therapeutic effects attributed by various constituents present in Salvadora persica (Miswak) are summarized in Table 18.1. Table 18.1 Phytoconstituents of Salvadora persica (Miswak) and their therapeutic activity on oral hygiene [74]. Sr. no. Phytoconstituent Therapeutic activity 1. Silica Abrasive−removes plaque and stain 2. Tannins (Tannic acid) Alleviates gingivitis Antifungal activity especially Candida albicans 3. Resins Forms protective layer on enamel surface, provides protective effect against dental caries 4. Alkaloids (Salvadorine) Antifungal, bactericidal, and gingival stimulatory effect 5. Essential volatile oils Antibacterial effect and stimulate saliva 6. Sulfur Bactericidal effect 7. Vitamin C Antiscorbutic, healing, and tissue repair 8. Sodium bicarbonate Mild abrasive, dentifrice 9. Calcium Prevention of demineralization, induction of remineralization of enamel 10. Fluoride Anticariogenic activity and tooth remineralization 11. Chloride Prevention of formation of tartar, calculus 12. N-benzyl-2phenylacetamide Inhibits human collagen-induced platelet aggregation and antibacterial activity against Escherichia coli. 13. Benzyl isothiocyanate Chemo-preventive agent, bactericidal activity, virucidal activity 14. Trimethylamine Antibacterial, antiphlogistic, and gum stimulating effects 292 Natural Oral Care in Dental Therapy 18.6 Conclusion Miswak has a good antibacterial and antifungal effect. The World Health Organization also promotes the use of miswak sticks for improving oral hygiene [75]. Extensive investigations and standardization of the extracts to study for its effect against various pathogens are required. Gingival recession (gingival recession index score) due to mechanical trauma is a main disadvantage of Miswak usage [76]. Accessibility of Miswak toothbrush to the entire tooth area limits the function of the stick during cleaning of the teeth. It is essential that miswak sticks should be designed in a manner to maximize the exposure of the chewing stick to the lingual surface and interdental spaces. For better oral hygiene, miswak should be promoted as an adjunct to traditional toothbrush not only due to its low cost and easy availability but because of its therapeutic effects also. Application of modern science and technology to develop contemporary dosages to avail optimum clinical benefits from this versatile, indigenous plant is the need of the hour. References 1. Halawany, H.S., A review on miswak (Salvadora persica) and its effect on various aspects of oral health. Saudi Dent. J., 24, 63–69, 2012. 2. Upadhyay, R.K., Ahmad, S., Tripathi, R., Rohtagi, L., Jain, S., Screening of antimicrobial potential of extracts and pure compounds isolated from Capparis decidua. J. Med. 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Shafiei-Bafti, L., Rad, M., Salehi-Soormaghi, M., Rezaei, M., An in vivo evaluation of antimicrobial effects of Salvadora persica herbal mouthwash on Candida albicans and Enterococcus faecalis. J. Oral Health Oral Epidemiol., 2, 64–69, 2013. 48. Almas, K., Al-Bagieh, N.H., Akpata, E.S., In vitro antimicrobial effects of extracts of freshly cut and 1-month-old miswak (chewing stick). Biomed. Lett., 56, 145–149, 1997. 49. Almas, K. and Stakiw, J.E., The effect of miswak extract from Salvadora persica stored for 18 years on microbes in vitro. Egyptian Dent. J., 46, 227–230, 2000. 50. Noumi, E., Snoussi, M., Hajlaoui, H., Valentin, E., Bakhrouf, A., Antifungal properties of Salvadora persica and Juglans regia L. extracts against oral Candida strains. Eur. J. Clin. Microbiol. Infect. Dis., 29, 81–88, 2010. 51. Al-Bagieh, N.H., Idowu, A., Salako, N.O., Effect of aqueous extract of miswak on the in vitro growth of Candida albicans. Microbios, 80, 107–113, 1994. 52. Al-Obaida, M.I., Al-Essa, M.A., Asiri, A.A., Al-Rahla, A.A., Effectiveness of a 20% miswak extract against a mixture of Candida albicans and Enterococcus faecalis. Saudi Med. J., 31, 640– 643, 2010. Salvadora persica L. (Miswak) 295 53. Saadabi, A.M.A., Antifungal activity of some Saudi plants used in traditional medicine. Asian J. Plant Sci., 5, 907–909, 2006. 54. Paliwal, S., Chauhan, R., Siddiqui, A.A., Paliwal, S., Sharma, J., Evaluation of antifungal activity of Salvadora persica Linn. Leaves. Nat. Prod. Rad., 6, 372–374, 2007. 55. Naeini, A., Naderi, N.J., Shokri, H., Analysis and in vitro anti-candidal antifungal activity of Cuminum cyminum and Salvadora persica herbs extracts against pathogenic Candida strains. J. Mycol. Med., 24, 13–18, 2014. 56. Balto, H., Al-Howiriny, T., Al-Somily, A., Siddiqui, Y.M., Al-Sowygh, Z., Halawany, H., Screening for the antimicrobial activity of Salvadora persica extracts against Enterococcus faecalis and Candida albicans. Int. J. 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Halib, N., Nuairy, N., Ramli, H., Ahmad, I., Othman, N.K., Salleh, S.M., Bakarudin, S.B., Preliminary Assessment of Salvadora persica Whitening Effects on Extracted Stained Teeth. J. Appl. Pharm. Sci., 7, 12, 121–125, 2017. 296 Natural Oral Care in Dental Therapy 74. Haque, M.M. and Alsareii, S.A., A review of the therapeutic effects of using miswak (Salvadora persica) on oral health. Saudi Med. J., 36, 5, 530–543, 2015. 75. World Health Organization, Prevention of oral diseases, WHO offset publication No. 103, p. 61, World Health Organization, Geneva, 1987, (http://www.who.int/iris/handle/10665/39046). 76. Eid, M., Selim, H., Al-Shammery, A., The relationship between chewing sticks (Miswak) and periodontal health, 3. Relationship to gingival recession. Quintessence Int., 22, 1, 61–64, 1991. 19 Triphala and Oral Health Kamal Shigli1*, Sushma S Nayak2, Mrinal Shete3, Vasanti Lagali Jirge4 and Veerendra Nanjwade5 1 Department of Prosthodontics, D.Y. Patil Dental School, Lohegaon, Pune, Maharashtra, India 2 Public Health Dentist, Bangalore, Karnataka, India 3 Department of Oral Pathology, D.Y. Patil Dental School, Lohegaon, Pune, Maharashtra, India 4 Department of Oral Medicine and Radiology, KLE VK Institute of Dental Sciences, Belgavi, Karnataka, India 5 Formulation Development, Unichem Laboratories Limited, Bangalore, India Abstract “Triphala” is a popular combination of three fruits, namely, Emblica officinalis (Amalaki), Terminalia chebula (Haritaki), and Terminalia bellerica (Bibhitaki) in equal proportions. The combination exerts several therapeutic effects on oral health, including anti-caries, antioxidant, anti-microbial, and anti-candida activities. It inhibits the growth of S. mutans. Tannic acid, which is one of the main components of triphala, is responsible for this effect. Triphala exhibits an antiplaque efficacy, which is almost equivalent to that of chlorhexidine. It has been suggested as an alternative to sodium hypochlorite (NaOCl) for irrigation of root canal. Triphala also exerts protective effect on gingival health. Thus, triphala could be an adjuvant to conventional drugs for overall improvement of oral health. Keywords: Triphala, oral health, dentistry, dental caries, periodontitis, candidiasis, oral cancer, root canal medicament 19.1 Introduction “Triphala’ is a popular herbal combination of three fruits namely Terminalia chebula, Terminalia bellerica, and Emblica officinalis or Phyllanthus emblica in equal proportion [2, 3]. These three myrobalans are also called as Harada, Bahera, and Amla, respectively, in vernacular language [2] (Figure 19.1). Triphala has about 47 tannins and 35 phytochemicals [2]. It could be used to treat various diseases of oral cavity with minimal or no side effects on oral tissues at a minimal cost compared to existing dentifrices. *Corresponding author: kamalshigli@yahoo.co.in Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (297–312) © 2020 Scrivener Publishing LLC 297 298 Natural Oral Care in Dental Therapy Triphala Terminalia chebula (Haritaki) Terminalia bellerica (Bibhitaki) Emblica officinalis or Phyllanthus emblica (Amalaki) Figure 19.1 Diagrammatic representation of components of Triphala. 19.2 Taxonomical Classification Terminalia chebula (Genus: Terminalia, Family Combretaceae) is commonly available in India, Sri Lanka, and Burma [1, 2, 4]. Terminalia bellerica (Genus: Terminalia L, Family Combretaceae) is found mainly in central Asia [1, 2]. Emblica officinalis (Genus: Phyllanthus, Family Euphorbiaceae) is a medium-to-large tree growing in India, Pakistan, China, Malaysia, and Thailand [2, 5]. 19.3 Chief Phytoconstituents Triphala mainly contains Tannins; Quinones; Flavones, flavonoids, and flavonols; Gallic acid and Vitamin C (Figure 19.2) [1]. Tannins Tannins are polymeric phenolic substances. They possess astringent property (ability to precipitate gelatin from solution) [1]. They exhibit anti-infective properties by deactivating microbial adhesins, enzymes, and cell envelope transport proteins [1, 2]. Quinones Quinones are aromatic rings (with two ketone substitutions) and are highly reactive. They are an initiator of stable free radicals [1, 2]. Anti-microbial effect of quinones results from inactivation of protein and loss of function caused by combining with the nucleophilic amino acids [1, 2]. Another mechanism is by making the substrates unavailable to the organisms [1, 2]. Triphala and Oral Health 299 Terminalia chebula (Haritaki) Tannin, chebulic acid, chebulagic acid, corrilagin, and gallic acid Anti-caries Antioxidant Efficient for gingivitis Antifungal Terminalia bellerica (Bibhitaki) Emblica officinalis or Phyllanthus emblica (Amalaki) Gallic acid, tannic acid, and glycosides Vitamin C, carotene, nicotinic acid, riboflavin, phenols, polyphenols, flavonoids, kaempferol and tannins Most active antioxidant Antifungal Cariostatic effect Antioxidant Gingivitis Antifungal Figure 19.2 Chief constituents of Triphala with their effect on the oral cavity. Flavones, flavonoids and flavonols Flavones are phenolic structures having one carbonyl group [1]. Adding a 3-hydroxyl group results in the formation of a flavonol. Flavonoids are produced against microbial infection and are effective against a range of organisms, as they bind with extracellular proteins [1, 2]. They also have the potential to bind with, disrupt bacterial cell walls and membranes thus exhibiting inhibitory effect on bacteria like Streptococcus mutans in in vitro conditions [1, 2]. Gallic acid Gallic acid is an antioxidant present in all constituents of Triphala [1, 2]. It possesses hepatoprotective, antioxidant activity, and suppresses growth of cancer cells [1]. Vitamin C Vitamin C is an antioxidant, and it hastens the healing process along with flavonoids [1, 2]. Emblica officinalis (EO) is rich in vitamin C and accounts for approximately 45–70% of the antioxidant activity exhibited by triphala [1, 2, 5]. Emblica officinalis (Amalaki) also contains carotene, niacin, vitamin B2, phenols, polyphenols, flavonoids, kaempferol, and tannins [1, 2, 5]. It has gallic acid derivatives like epigallocatechin gallate [2]. Terminalia chebula (Haritaki) has high tannin content, chebulic acid, chebulagic acid, corrilagin, and gallic acid [1, 4]. Terminalia bellerica (Bibhitaki) contains mainly ellagic and gallic acid. Other constituents are termilignan, anolignan B, β-sitosterol, arjungenin, belleric acid, bellericosidem, 300 Natural Oral Care in Dental Therapy flavonoids, saponins, amino acids, polyphenol, quinine, coumarin, glucosides, carbohydrates, alkaloids, ethylgallate, gallylglucose, chebulanic acid, and tannins [1, 2]. 19.4 Role of Triphala in Dentistry (Table 19.1) 19.4.1 Anti-Caries Activity T. chebula extract has demonstrated activity against dental caries, inhibiting the growth of S. mutans on the tooth surface, thus, reducing accumulation of acids and resulting demineralization and breakdown of tooth structure [1, 2, 6]. This activity could be due to tannins and flavonoid present in it [2]. Tannins exert cytotoxic action on the cell membranes of the microorganisms [2]. The astringent character of tannins inhibits electron transport system and causes complexation with enzymes or substrates [2]. Flavonoids, which are a group of phenols, are anticariogenic by the presence of antiglucosyl-transferases activity [2]. The therapeutic ingredients of E. officinalis bind to the surface of bacteria resulting in a decrease in the adherence of S. mutans to the surface of the tooth [2]. Also, the virulent genes that contribute to aggregation of plaque components are suppressed resulting in decreased caries of dental tissues [2]. Triphala extract prevents the formation of plaque on the tooth surface because of inhibition of sucrose-induced adherence and the glucan-induced aggregation, which mainly favors bacterial colonization on the tooth surface [1, 6]. The constituents of E. officinalis together exert a major cariostatic effect [2]. Individual constituents of E. officinalis may not be as potent as the whole composition [2]. The mixed herbal powder extracts (MHPE) containing Acacia arabica with Triphala retard the growth of cariogenic microorganisms at a very low concentration compared to chlorhexidine [7]. 19.4.2 Triphala as a Root Canal Irrigant Sodium hypochlorite (NaOCl) is a popular root canal irrigant. It removes debris, dissolves tissue remnants, disinfects the root canals, and removes E. faecalis biofilms. However, it has certain disadvantages like displeasing taste, high toxicity, and its inability to eliminate the smear layer. Compared to this, Triphala has shown strong antibacterial activity against 3-week and 6-week biofilms. The use of herbal agents for irrigation of root canals could be more beneficial as they seem to lack adverse effects [1]. Thomas et al. compared the antimicrobial efficacy of diode laser, triphala, and sodium hypochlorite against Enterococcus faecalis-contaminated primary root canals and concluded that Triphala could be used as an alternative disinfectant to Sodium hypochlorite (NaOCl) as it exhibited better antibacterial efficacy than Sodium hypochlorite [8]. Divia et al. concluded that Triphala exhibited antibacterial activity almost like that of NaOCl [9]. Another study found that the use of triphala juice as a root canal irrigant resulted in significant reduction in colony-forming units of E. faecalis and Candida albicans 1 day after irrigation compared to chlorhexidine, which inhibited the counts up to 3 days post irrigation in vitro [10]. Table 19.1 Depicting clinical studies and their outcome. Triphala or component used Duration of study Outcome measures Significant findings Usha et al. (2007) [15] T. chebula aqueous extract Immediate, 10 min, 30 min, 1 h Salivary pH, buffering capacity, & microbial activity Salivary pH, buffering capacity were higher than baseline up to 30 min after use of T. chebula mouthrinse. Streptococcus mutans and lactobacillus count reduced by 65% and 71%, respectively post rinsing. Nayak et al. (2010) [16] T. chebula aqueous extract Immediate Salivary pH & salivary S. mutans count Rinsing with T. chebula aqueous extract led to an increase in salivary pH, which remained high up to 60 min post rinsing. Salivary S. mutans count remained significantly low 5 min after rinsing. 60 min post rinsing sample also showed reduction in S. mutans however the difference in study and control group was not significant. Tandon et al. (2010) [17] Triphala mouthwash 3, 6, 9 months Caries status (DMFS scores) & Incipient caries (DSi index) The DMFS scores did not differ significantly at follow up when Triphala & chlorhexidine mouthwash groups were compared. There was no significant increase in incipient caries in Triphala group. Srinagesh et al. (2011) [18] Triphala Mouthwash 15 & 45 days Salivary mutans Streptococci A reduction of 83% and 67% in Mutans streptococci was observed in triphala group after 15 days and 45 days, respectively. Antimicrobial action of triphala was almost equivalent to chlorhexidine. (Continued) Triphala and Oral Health 301 Authors Authors Triphala or component used Duration of study Outcome measures Significant findings Bajaj et al. (2011) [19] Triphala mouthwash Baseline, 3, 6 & 9 months Plaque scores, gingival scores & microbiological analysis (Streptococcus & lactobacilli counts) Use of Triphala and chlorhexidine mouthwash led to a reduction in, plaque scores and Gingivitis scores, and also reduction in growth of Streptococcus mutans and Lactobacillus. In comparison to chlorhexidine, Triphala had more inhibitory effect on Lactobacillus, however the difference was not significant. Srinagesh et al. (2012) [20] Triphala mouthwash 48 h & 7 days Streptococci colony forming units A reduction of 17% and 44% was observed in oral Streptococcus colonies after 48 h and 7 days of triphala mouthwash use. Nayak et al. (2012) [4] T. chebula ethanol extract Immediate Salivary S. mutans counts Reduction of around 44% and 64% in S. mutans counts was observed 5 and 60 min after rinsing with ethanol extract of T. chebula. Narayan et al. (2012) [21] Triphala, Hi Ora, Chlorhexidine, & Colgate Plax 28-day washout period; 1 day Plaque formation Triphala and Hi Ora presented an anti-plaque efficacy similar to chlorhexidine and proved more effective than Colgate Plax mouthwash. Bhattacharjee et al. (2014) [22] Triphala aqueous extract Baseline & follow-up for 2 weeks Plaque & Gingival indices in children Triphala and chlorhexidine mouth rinses showed significant reduction of plaque and gingival scores at follow-up. Shetty et al. (2014) [23] Triphala Churna Effect of single use Total candida count Triphala Churna was as effective as denture cleansing tablet against Candida. (Continued) 302 Natural Oral Care in Dental Therapy Table 19.1 Depicting clinical studies and their outcome. (Continued) Table 19.1 Depicting clinical studies and their outcome. (Continued) Duration of study Outcome measures Significant findings Naiktari et al. (2014) [11] Triphala mouthwash 1st, 15th day Plaque index (PI) & Gingival index (GI) Triphala mouthwash reduced plaque accumulation and gingival inflammation, preventing periodontal disease. Its efficacy as an antiplaque agent was similar to Chlorhexidine. Chainani et al. (2014) [24] Triphala extract mouthrinse Three phases of 1-month duration each Plaque index & Gingival index The antiplaque and antigingivitis activity of triphala was almost equal to chlorhexidine. Gupta et al. (2014) [25] Terminalia chebula mouth rinse Baseline, 7 & 14 days Plaque; gingivitis indices; Salivary pH This study demonstrated that use of Terminalia chebula mouth rinse led to a decrease in plaque and gingivitis scores. Pradeep et al. (2016) [26] Triphala mouthwash Baseline, 7, 30 & 60 days Plaque index; gingival index; oral hygiene index-simplified; and microbiologic colony counts Use of Triphala mouthwash reduced gingival index scores and was comparable to that of chlorhexidine group. Mamgain et al. (2017) [27] Triphala and Ela decoction Baseline, 14th day, 21st day Plaque, Gingival Index & Halitosis percentage reduction Triphala and Ela decoction had similar efficacy as chlorhexidine. Sushma et al. (2017) [28] Triphala churna Effect of single use Total Candida counts Mean candida counts were lower in triphala group compared to chlorhexidine group. Significant decrease in candida counts was observed in triphala group. (Continued) Triphala and Oral Health 303 Triphala or component used Authors Authors Triphala or component used Duration of study Outcome measures Significant findings Vinod et al. (2017) [29] Herbal (combination of tulsi, neem, triphala, & turmeric) Immediate, 7th, 14th day Microbial colonies Chlorhexidine mouthwash was better at 7th day, while on prolonged usage for 14 days the herbal preparation was found to perform much better than Chlorhexidine in reducing the microbial colony count. Saxena et al. (2017) [30] T. chebula; T. bellerica; E. officinalis; & Triphala 5 min & 60 min Colony Forming Units (CFUs) of S. mutans T. chebula, T. bellerica, E officinalis, and Triphala mouthwash was effective in reducing S. mutans counts but among all Triphala mouthwash was more effective. Baratakke et al. (2017) [31] Alcoholic extract of Triphala 21 days Plaque and gingival status Triphala extract mouth rinse proved effective in reducing plaque accumulation and gingival inflammation with no side effects. Irfan et al. (2017) [32] Triphala mouthwash Baseline, 1 week, 1 month, and 45 days Gingival Index (GI) and Plaque Index (PI) Reduction in plaque scores was similar in Triphala and chlorhexidine groups. Slightly higher reduction in gingival scores was observed in triphala group. Shah et al. (2018) [33] Herbal mouthrinse (Oratreat) containing Terminalia chebula 2 weeks Colony Forming Unit count Higher reduction in Streptococcus mutans colony forming units was observed in triphala group when compared to chlorhexidine group. Naiktari et al. (2018) [34] Triphala mouthwash Sampling repeated after every 2 h for 12 h Colony forming units On use of Triphala mouthwash a reduction in the S. mutans counts was observed up to 4 h. (Continued) 304 Natural Oral Care in Dental Therapy Table 19.1 Depicting clinical studies and their outcome. (Continued) Table 19.1 Depicting clinical studies and their outcome. (Continued) Authors Triphala or component used Duration of study Outcome measures Significant findings Salivary Streptococcus mutans count & oral hygiene status Prevention of plaque accumulation was similar in garlic, Chlorhexidine gluconate, and triphala groups. Triphala showed decrease in plaque index scores. Slightly higher reduction in plaque index scores was observed in chlorhexidine group. Triphala, chlorhexidine gluconate & garlic extract mouthwash 1, 15, & 30 days Safiaghdam et al. (2018) [36] Review current clinical trials— effectiveness of herbal products in gingivitis Data collected from 2000 until January 2018 Triphala was effective in controlling gingivitis. Triphala and Oral Health 305 Padiyar et al. (2018) [35] 306 Natural Oral Care in Dental Therapy 19.4.3 Anti-Microbial and Anti-Oxidant Effect of Triphala In vitro studies have shown anti-microbial and anti-oxidant effects of Triphala by its inhibition of Streptococcus mutans at concentrations as low as 50 µg/ml [1]. Triphala is useful in the management of periodontal diseases because of its antimicrobial and antioxidant properties [2]. This could be attributed to the tannic acid content of Triphala. The tannic acid gets adsorbed on the bacterial cells, leading to protein denaturation and bacterial cell death [1]. Among the three constituents of Triphala, T. bellerica demonstrates the strongest antioxidant activity followed by E. officinalis and T. chebula [1]. 19.4.4 Role of Triphala in Periodontal Diseases Triphala possesses hemostatic, anti-inflammatory, analgesic, and wound healing properties. T. chebula is most effective in controlling gingivitis and gingival ulcers. On the other hand, Emblica officinalis contains a large amount of vitamin “C”, which helps in maintaining healthy gingiva and prevents gingivitis and bleeding [11]. Another study reported that triphala or metronidazole, provided partial relief to the patients with periodontal disease, and when combined and used, they demonstrated a synergistic effect. They recommended triphala and metronidazole as a combined treatment regimen for local (i.e., as mouth rinse) as well as systemic administration [12]. 19.4.5 Triphala as a Mouth Rinse The use of Triphala mouth rinse with scaling and root planing resulted in significant reduction in plaque, gingival, and oral hygiene indices at 1 week, 4 weeks, and 6 weeks, similar to the results obtained when chlorhexidine mouth rinse was used with scaling and root planing [1]. 19.4.6 Anti-Candida Activity of Triphala The ethanol extract of P. emblica inhibited the adhesion of C. albicans to human buccal epithelial cells and acrylic surfaces of dentures in in vitro studies [5]. The mechanism for impediment of adhesion may be due to tannins, lignans, flavonoids, alkaloids, and other phenolic compounds [5]. Compared to Aloe vera (50%), Triphala (12.5%) inhibited the growth of Candida albicans at a lower concentration [13]. T. chebula and T. bellerica are mainly responsible for the antifungal activity [13]. 19.4.7 Anti-Collagenase Activity of Triphala Triphala slows the polymorphonuclear neutrophils (PMNs)—type matrix metalloproteinase, which are involved in the extracellular matrix (ECM) degradation during periodontitis [1, 14]. Triphala and Oral Health 307 19.5 Pharmacological Activities of Triphala and Future Research All the three components of triphala have various beneficial effects on human health, when used alone and also when used in combination. The pharmacological activities of triphala and its constituents are as follows: 19.5.1 Anticancer and Antioxidant Activity of Triphala It exerts cytotoxic effect on cancer cells. This effect could be attributed to gallic acid present in triphala. Triphala extract exhibited antimutagenic activity on Ames histidine reversion assay. By increasing the antioxidant status of animals, the incidence of tumor formation is reduced. In addition, it also has a radioprotective effect. The polyphenols in triphala help in converting reactive oxygen free radicals to nonreactive products. It increases the nonspecific stress-tolerating capacity [37–41]. Studies have reported that Emblica officinalis fruit also has antioxidant and anti-aging properties [1–3, 5]. Terminalia bellerica is the most active antioxidant followed by Emblica officinalis and Terminalia chebula [2]. 19.5.2 Wound Healing Properties Triphala promotes wound healing by exerting antibacterial activity against wound pathogens. In addition, epigallocatechin gallate contributes to faster wound closure and improved tissue regeneration [41]. Terminalia chebula leaves extract, when applied on wounds in rats, caused wounds to heal faster mainly by improving rates of contraction and reduction in epithelialization period. 19.5.3 Antibacterial Activity of Triphala It exerts antibacterial activity against a wide range of bacteria including both gram-positive and gram-negative species. In addition to its activity on dental pathogens, it has antimicrobial activity on Klebsiella pneumonia, Shigella sonnei, Staphylococcus aureus, Vibrio cholerae, E. coli, Pseudomonas [41]. Fruits of Terminalia chebula, when used alone, also exhibited anti-bacterial, anti-fungal, and anti-viral properties [1]. 19.5.4 Anti-Diabetic Effect Rat experiments have demonstrated a decrease in blood sugar level on administration of triphala extract [41]. 19.5.5 Anti-Inflammatory, Analgesic, and Antipyretic Effect Gallic acid present in triphala is a cyclooxygenase-2 (COX-2) inhibitor. In animal experiments, triphala, in combination with other herbs, was found to have analgesic and antipyretic effects [41]. The Emblica officinalis fruit has application as an antipyretic, analgesic, cytoprotective, anti-tussive, and gastroprotective agent [1–3, 5]. The fruit of Terminalia 308 Natural Oral Care in Dental Therapy chebula has shown anti-mutagenic/anti-carcinogenic, cytoprotective, and radioprotective activity [1]. Due to its anti-inflammatory, antioxidant, and antineoplastic activity, Triphala may play a role in the treatment of tobacco-induced oral mucosal lesions. 19.5.6 Immunomodulatory Effect The flavonoids, alkaloids, glycosides, saponins, and phenolic compounds present in triphala are responsible for its immunomodulatory effect [41]. The fruits of Emblica officinalis and Terminalia chebula have an immune-modulatory effect [1–3, 5]. The components of triphala exhibit the following activities individually: Terminalia chebula exhibits anti-anaphylactic and hypolipidemia/hypercholesterolemia activity. It also helps in improving gastro-intestinal motility. Terminalia bellerica possesses bronchodilator, cardioprotective, hepatoprotective, hypoglycemic, and hypotensive properties [1, 2]. Emblica officinalis has anti-pyretic, anti-tussive, and gastroprotective properties [1–3, 5]. 19.6 Public Health Importance Triphala has exhibited a range of protective activities on oral health. Few studies have reported it to be as effective as the conventional drugs. Future research should be directed toward assessing the effect of the combination of triphala with synthetic drugs on oral health. 19.7 Formulation Using Triphala Triphala tablet, Triphala churna (Dabur India Ltd, New Delhi, India), Triphala capsule (Himalaya Drug Company, Bangalore, India), Triphala mouth rinse, Salz Triphala toothpaste (Lion Corporation, Thailand), and Himalaya toothpaste with triphala are the formulations of Triphala. 19.8 Conclusion Triphala is an herb with a wide spectrum of beneficial effects on human health. It has been in use since time immemorial. The emerging scientific evidence supporting its traditional use and demonstration of newer pharmacologic activities could go a long way in triphala forming an adjuvant mainstream drug in dentistry as well as in general medicine. Triphala and Oral Health 309 References 1. Prakash, S. and Shelke, A.U., Role of Triphala in dentistry. J. Indian Soc. Periodontol., 18, 2, 132–5, 2014. 2. 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Phytochem., 5, 3, 23–27, 2016. 20 Azadirachta indica (Neem): An Ancient Indian Boon to the Contemporary World of Dentistry Sri Chandana Tanguturi*, Sumanth Gunupati and Sreenivas Nagarakanti Department of Periodontology, Narayana Dental College and Hospital, Nellore, Andhra Pradesh, India Abstract Known as the “Miracle tree” and “Arishta” (Sanskrit for Sickness reliever), Azadirachta indica (Neem), belonging to the Meliaceae family, from prehistoric Indian subcontinent, for its multitudinous medicinal properties. With globalization, it has gained even more recognition and acceptance worldwide, especially for having no side effects or complications. By virtue of its natural antibacterial and antimicrobial properties, it has diverse applications in dentistry, as most of the oral diseases are microbe induced. With active ingredients such as, Nimbidin, Azadirachtin, and Nimbinin that can eliminate oral aerobic and anaerobic bacteria, Neem is extensively used as a root canal irrigant, mouthwash for treatment of gingivitis, and toothpaste with good anti-cariogenic properties, among numerous other oral hygiene products. Though there are many scientific studies explaining Neem’s diverse applications in dentistry, literature that reviews them has been scarce. The aim of this chapter is to review Neem’s phytochemistry, mechanism of action, use of the tree’s various parts in dentistry, their chemical constituents, and biological effects. Keywords: Anti-inflammatory agent, Azadirachta, oral health, periodontal disease, root canal irrigant 20.1 Introduction Nowadays, the use of medicinal plants in the treatment of many diseases has gained significant importance, as people have become ever more cognizant of the side effects of synthetic drugs and, hence, are fascinated by the use of plant-based medications. The gaining popularity and demand for herbal medicines may lead to unmethodical and unscientific collection, misidentification, and adulteration without any standards for quality of the material, especially in developing countries. Ahmad et al. (2010) [1] noticed that in the markets of India and Pakistan, oftenly different products are being sold under the same trade name, which is dangerous. Hence, there is an immense need to describe the drugs in a systematic and scientific manner, and collect, process, extract, isolate, characterize, and identify its chemical compounds. Scientific methodologies should be developed to maintain standards *Corresponding author: drchandana87@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (313–328) © 2020 Scrivener Publishing LLC 313 314 Natural Oral Care in Dental Therapy and quality assurance of these herbal drugs. To help this cause, the authors have compiled and evaluated literature on Azadirachta indica (Neem), one of the most widely used therapeutic plants. Neem is native to the Indian subcontinent and grows well in tropical and subtropical regions. Owing to its innumerable medicinal benefits, Neem is appropriately called “Divine Tree” [2–5]. Mature trees grow up to 7–20m high and may live for more than 200 years [6]. Azadirachta indica is a Latin term that denotes “Azad” for “free”, “dirakht” for “tree”, and “I” for “Hind” meaning belonging to Indian origin. In Sanskrit language, “Neem” is called “arista” meaning “complete & imperishable” [7]. 20.2 Vital Bioactive Compounds of Neem Although numerous compounds (approximately 135) [8] have been extracted from different parts of Neem, only a few compounds’ biological activity was studied extensively, which are discussed in this chapter in detail, along with their medical and dental applications. The following are some of the biologically active compounds of neem with therapeutic role: 20.2.1 Nimbidin Nimbidin, the major bitter principle extracted from the oil of neem seed kernels, possess several therapeutic properties. Several other bioactive compounds, which are tetranortriterpenoids like nimbin, nimbinin, nimbidinin, nimbolide, and nimbidic acid, have been isolated from nimbidin [9, 10]. • Anti-inflammatory activity: In an animal study on rats, Nimbidin and Sodium Nimbidate showed significant anti-inflammatory activity [11, 12]. Nimbidin has the ability to suppress the macrophage and neutrophil functions and, thus, has anti-inflammatory action. Nimbidin has the ability to inhibit nitric oxide (NO) and prostaglandin E2 (PGE2) production in macrophages following in vitro exposure [13]. • Antipyretic activity: A study conducted by David S.N (1996) [14] confirms the antipyretic activity of Nimbidin. • Hypoglycemic effect: Oral administration of Nimbidin in fasting rabbits showed a significant difference [15]. • Antiulcer effect: Nimbidin prevents gastric lesions as well as duodenal ulcers [16, 17] and suppresses gastric acid output [18]. • Antibacterial and antifungal effect [19]: It is bactericidal as it has the ability to inhibit the growth of Mycobacterium tuberculosis, and it is a potent antifungal as it can inhibit the growth of Tinea rubrum. Role of Azadiraciita indica in Dentistry 315 20.2.2 Azadirachtin It belongs to the limonoid group and is a secondary metabolite present in neem seeds. Antiinflammatory activity: Azadirachtin possesses antiradical scavenging activity and reductive potential [20]. • Antimalarial activity: It also inhibits the development of malarial parasites and a very good antimalarial agent [21]. 20.2.3 Nimbolide Nimbolide is a limonoid tetranortriterpenoid with an α,β-unsaturated ketone system and a δ-lactone ring. • Anti-inflammatory activity: It possesses antiradical scavenging activity and reductive potential [20] • Anti-bacterial and anti-malarial effect: The growth of Plasmodium falciparum and Streptococcus aureus and coagulase [22, 23]. 20.2.4 Gedunin It is an active component extracted from neem seed oil. • Anti-neoplastic activity: Gedunin can inhibit the proliferation of cancer cells including those of the prostate, ovary, and colon [24, 25]. • Antimalarial and anti-fungal activity [26, 27] 20.2.5 Mahmoodin It is a deoxy-gedunin isolated from neem seed oil. • Antibacterial activity: It inhibits growth of various Gram-positive and Gram-negative organisms [28]. 20.2.6 Tannins • Anti-inflammatory activity During inflammation, Tannins have the ability to inhibit oxidative burst of PMN [22]. 20.2.7 Diterpenoids The three tricyclic diterpenoids are margolone, margolonone, and isomargolonone [29, 30]. 316 Natural Oral Care in Dental Therapy • Antibacterial activity: They are active against Staphylococcus, Klebsiella, and Serratia species. 20.3 How to Distinguish Azadirachta Indica (Neem) from its Common Adulterant Melia Azedarach Considering the high probability of accidental or deliberate misidentification of the neem tree, there should be some standards set for quality assurance. In a study conducted by Shazia sultana et al. (2011) [31], Azadirachta indica was distinguished from its adulterant Melia azedarach, based on morpho-palynological and foliar epidermal anatomy. Using pharmacognostic tests, the powdered drug can be distinguished based on solubility and fluorescence analysis. Solvents like HNO3 and C6H6 are recommended as a good test for authenticating the herbal drug Neem. 20.4 Therapeutic Applications of Neem Azadirachta indica (Neem) possesses plethora of therapeutic properties because of its rich source of its bioactive component, as shown in Figure 20.1. Various parts of the neem tree * Regulation of proinflammatory enzyme activities including cyclooxygenase (COS), and lipoxygenase (LOX) enzyme Neem Azadiracta Indica * Suppress the macrophage and neutrophil functions. Anti-Inflammatory Anti-Oxidant * Inhibits nitric oxide (NO) and prostaglandin E2 (PGE2) production Free radical scavenging activity * Activates apoptosis, suppression of NF-κB signaling, and cyclooxygenase pathway Anti-Cancerous Anti-Bacterial Disrupts the integrity of the bacterial cell membrane leading to disturbance in osmotic pressure that ultimately leads to cell death Anti-Viral Anti-Fungal * Activate the tumour suppressor genes and inactivate the activity of genes involved in the cancer development and progression such as VEGF, NF-κB, and PI3K/Akt • Interferes at the early events of virus replication * Inhibits cytoadhesion. • Increase superficial hydrophobicity on cells * Inhibits ergosterol biosynthesis. Wound-Healing Increased inflammatory response and neovascularization. Figure 20.1 Therapeutic activities of bioactive components of neem and their mechanism of action. Role of Azadiraciita indica in Dentistry 317 are used for the treatment of various ailments like infections, fever, respiratory disorders, leprosy, intestinal helminthiasis, constipation, skin, and oral diseases. 20.4.1 Neem as an Anti-Inflammatory, Analgesic Agent Most of the systemic diseases are inflammatory. Though there are many synthetic antiinflammatory drugs, many of them have serious side effects. Usage of herbal medicines is in practice for treating inflammatory conditions. Many natural anti-inflammatory drugs are available in the market, among them, Neem is considered to be the best, owing to its therapeutic benefits. Regulation of proinflammatory enzyme activities makes Neem a potent anti-inflammatory agent [32]. Nimbidin, isolated from seed oil, has the ability to suppress the macrophage and neutrophil functions. It also inhibits nitric oxide (NO) and prostaglandin E2 (PGE2) production in macrophages following in vitro exposure [13]. According to a study conducted by A. S. M. Mosaddek et al. (2008) [33], the neem leaf extract showed significant anti-inflammatory effect, but it is less efficacious than that of dexamethasone. A study conducted on albino rats showed that neem seed oil showed significant dose-dependent analgesic activity [34]. In a study conducted by K. Ilango et al. (2013) [35] showed significant antinociceptive and anti-inflammatory activities. 20.4.2 Antioxidant Activity Antioxidants deactivate free radicals often before they attack targets in biological cells and play a role in the activation of antioxidative enzyme control damage caused by free radicals/ reactive oxygen species [36]. Neem has free radical scavenging properties due to a rich source of antioxidants such as Azadirachtin and nimbolide [20]. According to a study conducted by A. K. Ghimeray et al. (2009) [37] on leaf and bark extracts of neem grown in the foothills of Nepal, they showed significant antioxidant properties. 20.4.3 Anticancerous Activity Cancer is one of the life-threatening diseases that have multifactorial etiology. The known fact is that drugs used for treating cancer cause after effects. Neem contains flavonoids and various other components that play a key role in the inhibition of cancer development and progression. A tumor suppressor gene, p53 plays an important role in the inhibition of abnormal cell proliferation and thus preventing the progression of cancer. A study conducted by Arumugam A et al. (2014) [38] showed that ethanolic fraction of neem leaf (EFNL) treatment effectively upregulated the proapoptotic genes and proteins including p53. In many tumors, pTEN in activation is noted. A study confirmed that the ethanolic fraction of neem leaf treatment significantly increased the expression of pTEN, which could inhibit mammary tumorigenesis through its inhibitory effect on Akt [38]. Nimbolide acts as a potent anticancer agent by inducing apoptosis and inhibiting cell proliferation [39]. 318 Natural Oral Care in Dental Therapy 20.4.4 Antimicrobial Activity Drug resistance is a side effect of antimicrobial drugs and is a serious issue globally. Usage of a natural antimicrobial agent like Neem is a viable solution. 20.4.4.1 Antibacterial Activity Oil from the leaves, seeds, and bark possesses a wide spectrum of antibacterial action against Gram-negative and Gram-positive microorganisms [40]. According to a study conducted by M. B. Yerima et al. (2012) [41], the bark extracts showed antibacterial activity at all the concentrations. 20.4.4.2 Antiviral Activity Neem leaf extracts have shown virucidal activity against B-4 of coxsackievirus [42]. Neem bark (NBE) extract significantly blocked the entry of HSV-1 into cells from 50- to 100-μg/mL concentrations [41]. 20.4.4.3 Antifungal Activity A study by Mondali NK et al. (2009) [43] confirmed that the growth of fungal species Aspergillus and Rhizopus was inhibited by both alcoholic and neem leaf water extracts. A study by Shrivastava DK (2014) [44] showed inhibition of Aspergillus flavus, Alternaria solani, and Cladosporium. According to study conducted by Raja Ratna Reddy Y et al. (2013) [45], the leaf extract exhibited strong antimicrobial activity against bacteria and fungi, namely, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis, Staphylococcus aureus, Aspergillus fumigates, and Candida albicans at all the concentrations tested. 20.4.4.4 Antimalarial Activity Udeinya JI et al. (2008) [46] concluded in a study that the number of parasites in the control crops was less than 50% when treated with crude acetone/water (50/50) leaf extract (IRAB). 20.4.5 Wound Healing Effect A study conducted by Barua CC et al. (2010) [47] showed the significance of the wound healing activity of neem leaf extracts in both excision and incision wound models. In addition, the tensile strength of healing tissue of both treatment groups of plants was found to be significantly higher in incision wound compared to the control group [48]. 20.5 Applications of Neem in Dentistry Nowadays, oral diseases are of major public health concern throughout the world. Based on scientific studies, there is a high correlation between poor oral hygiene status, dental plaque, and severity of dental problems. Therefore, the treatment of localized oral infections Role of Azadiraciita indica in Dentistry 319 has been prioritized in dental therapy. Owing to the side effects of synthetic drugs, usage of natural herbs is mushrooming. The dental benefits of Neem (Azadirachta indica) are described in detail below: 20.5.1 Neem in Treatment of Periodontal Diseases Periodontitis is an immunoinflammatory disease where there is overproduction of free radicals, in which antioxidants in our body will be unable to counteract, leading to periodontal tissue destruction. Eventually, antioxidant supplements must be used to help regulate overproduced free radicals in periodontal tissues. Hence, to treat such a complex disease, a drug with antimicrobial, anti-inflammatory, and antioxidant properties should be chosen. Though there are several commercially available drugs with such properties, they alter the oral microbiota and cause side effects like vomiting, diarrhea, bacterial resistance hypersensitivity, immunosuppressive and allergic reactions, and tooth staining. Hence, natural phytochemicals isolated from plants are considered as good alternatives in treating oral diseases and can be used as adjuvants for mechanical procedures. According to a recently published systematic review by Dhingra K et al. (2016) [49], the neem mouth rinse was as efficacious as an anti plaque and anti gingivitis agent when used as an adjunct to tooth brushing in gingivitis patients. In a study conducted by Abhishek KN et al. (2015) [50], the usage of neem-containing toothpaste showed a significant difference in plaque index and gingival index, and it was thereby suggested that it could be used as an adjunct for maintenance of oral hygiene. In a study performed by Sharma R et al. (2014) [51] on children, the authors concluded that when neem, mango, and chlorhexidine mouthwashes were compared, chlorhexidine and neem possess equivalent efficacy in reducing plaque, while chlorhexidine has superior antigingivitis properties. A double-blind, randomized, controlled trial confirmed that A. indica-based mouth rinse is equally efficacious with fewer side effects compared to chlorhexidine [52]. In conventional local drug delivery, herbal products can eliminate certain shortcomings in using synthetic drugs. In a clinico-microbiological study of 10% neem oil chip as a local drug delivery system, clinical parameters showed statistical improvement, and P. gingivalis strain load was significantly reduced in the periodontal pockets treated with the neem chip sites [53]. In a comparative study, the authors found that chlorhexidine gluconate gel and neem extract gel are clinically equally effective [54]. Neem extract has shown to reduce matrix metalloproteinases (MMP-2 & MMP-9) levels, though not more than that of doxycycline [55]. The above-mentioned scientific studies strongly put the onus on the role of Azadirachta indica-based herbal preparations (mouthrinse, chips, gel, and toothpaste) as effective antiplaque and anti-inflammatory agents. Being of herbal origin, A. indica can be used as a safe alternative to the gold standard chemical plaque-controlling agent, chlorhexidine gluconate. However, multicenter studies with larger sample sizes and long terms are to be conducted to establish its utility as an adjunct to periodontal therapies. 20.5.2 Role of Neem in Endodontics The endodontic treatment is said to be completed when there is complete elimination of microbes in the root canals, viz. maximum disinfection. The most commonly found 320 Natural Oral Care in Dental Therapy microorganisms in failed/infected root canals are Enterococcus faecalis and Candida albicans. Being a natural product with less side effects, it is preferable to use neem as an endodontic irrigant. While, more scientific studies need be conducted to emphasize its role in endodontics, we have reviewed the following studies showing the efficacy of Neem as a root canal disinfecting agent. • Chandrappa et al. (2015) [56] found that when neem extract, tulsi extract, and chlorhexidine are used as irrigants, all of them showed significant antibacterial efficiency against E. faecalis. • Babaji et al. (2016) [57] concluded that neem, M. citrifolia, and Aloe Vera are efficient root canal irrigating solutions. • Studies conducted by Sundaram et al. (2016) [58] and Prasad et al. (2016) [59] also proved the antibacterial efficacy of herbal irrigants with conventional irrigants. Dutta et al. (2014) [60] concluded that neem had good anti-microbial efficacy. 20.5.3 Potent Role of Neem in Preventive Dentistry Preventive dentistry saves a lot of time, money, and pain caused by dental problems. Using natural and efficient therapeutic drugs like neem extracts in preventive dentistry makes even more sense as we will also be avoiding any side effects. 20.5.3.1 Application in Dental Erosion Therapy Dental erosion is the wearing of dental hard tissues by means of acid etch, which is nonbacterial in origin that leads to its demineralization and dissolution. Sales Peres AC et al. (2013) [61] concluded that application of a Neem–fluoride gel combination is more efficient than the sole application of Neem gel in the prevention of dental erosion Therefore, these compounds might be considered to act synergistically or additively with respect to inhibition of demineralization. 20.5.3.2 Anti-Microbial Activity Based on the above-discussed literature [41–46, 49–60] we can excogitate that neem is an efficient and potent antimicrobial with negligible side effects. There is a plethora of its antibacterial applications in medicine and dentistry. It is successfully used as antiplaque and anti-gingivitis, which are the culprits for gingival and periodontal diseases. 20.5.3.3 Anticaries Activity of Neem The Neem extract has been found to be active against the caries-causing organisms with the capability of not only inhibiting their growth but also reversing incipient/initial caries. The following studies prove that neem extracts possess anticaries effect, thus, is useful in preventive dentistry. According to Vanka A et al. (2001) [62] the Azadirachta indica mouthwash has the ability to inhibit Streptococcus mutans and reverse incipient carious lesions. In an in vitro study conducted by Prashant GM et al. (2007) [63], the authors analyzed the effect Role of Azadiraciita indica in Dentistry 321 of mango and neem extract on four dental caries-causing microorganisms, Streptococcus mutans, Streptococcus salivarius, Streptococcus mitis, and Streptococcus sanguis. Neem extract produced a maximum zone of inhibition on S. mutans at a 50% concentration. 20.5.3.4 Anti-Candidiasis Property Candida species are opportunistic fungal infections that usually affect immune-compromised, immunosuppressed patients causing a wide range of infections like oral thrush, intestinal candidiasis, vaginal thrush, etc. Owing to the side effects caused by the standard local and systemic treatments, it is safe to opt for herbal remedies. Polaquini SR et al. (2007) [64] have shown that A. indica components have hydrophobic and anti-adhesive action against dental biofilm. 20.5.3.5 Anti-Cancer Property Flavonoids play a key role in the inhibition of cancer development and progression. The chemopreventive effects of neem in the oral mucosa are by modulating lipid peroxidation, antioxidants, and detoxification systems [65]. Harish Kumar, G et al. (2009) [66] found that nimbolide is a more potent antiproliferative and apoptosis-inducing agent. Ethanolic neem leaf extract (ENLE) exerted its anticancer properties by inhibiting cell proliferation and inducing differentiation and apoptosis [67]. The antioxidant properties of neem leaf fractions may be responsible for modulating key hallmark capabilities of cancer cells such as cell proliferation, angiogenesis, and apoptosis [68]. It is evident from the above discussion that studies are conducted only on animals till date on oral carcinomas, while preclinical and clinical studies with superior quality are warranted to establish the utility of neem in the treatment and prevention of oral carcinomas. 20.6 Literature Supporting the Use of Neem in Dentistry In a study conducted by Joy Sinha D et al. (2017) [69] neem is as effective as chlorhexidine or sodium hypochlorite in inhibiting the growth of E. faecalis. RC Patil et al. (2017) [70] confirmed the antibacterial activity of Neem against Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Enterobacter aerogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella typhi, Staphylococcus epidermidis, and Proteus vulgaris. When antimicrobial activity of Azadirachta indica (AI), Triphala, Curcuma longa, and Morinda citrifolia (MC) against E. faecalis were compared, Propolis and AI showed significant antimicrobial activity [71]. Neem extract is an effective antifungal against C. albicans and is comparable to 3% NaOCl [72]. When antimicrobial efficiency of Neem leaf extract, 2% chlorhexidine, 3% sodium hypochlorite were assessed, neem leaf extract showed comparable zones of inhibition with that of chlorhexidine and sodium hypochlorite, and thus, it can be used as an intracanal medication [73]. The authors concluded that neem mouthwash can be used as an alternative to chlorhexidine mouthwash [74]. When the antimicrobial effects of the aqueous extracts of neem, miswak, mango, and banyan chewing sticks were compared against Streptococcus mutans and Lactobacillus acidophilus, neem showed the most antimicrobial 322 Natural Oral Care in Dental Therapy activity against S. mutans [75]. When efficacy of Neem, Tulsi, and Guduchi extracts were compared as intracanal medicaments, Neem was found to be the most potent medicament followed by Tulsi and Guduchi [76]. A recent study conducted by Hamid SK et al. (2019) [76] suggested that adding neem powder to acrylic resin denture base materials reduces the adhesion of C. albicans and hence could be useful in preventing denture stomatitis. 20.7 Toxicity and Safety In the United States of America, 8% of hospital admissions are due to adverse or side effects of synthetic drugs [77]. The most important advantage of herbal medicine is the negligible side effects and is relatively less expensive than synthetic drugs. Though neem has multitudinous medicinal properties, its indiscriminate use, particularly when taken in overdoses, may cause undesirable side effects. Traditional ayurvedic practitioners recommend against the use of neem in people with obvious wasting or fatigue, infants, children, pregnant or nursing women, patients with impaired liver or kidney function. Excess doses of seed or seed supplements may be toxic [78]. Although numerous animal studies are conducted on the safety of various parts of neem, there are no quality-controlled studies on humans evaluating its safety. There is compelling necessity for human studies on neem extracts, so that it can be more generously utilized at granular and general levels in dental hygiene and therapy. 20.8 Contamination and Adulteration There is a good possibility that herbal products are contaminated or even deliberately adulterated. Such failures in quality control are rare, but when they occur, they can be serious effects. These types of safety hazards are seen more frequently in herbal supplements imported from India, China, or other countries where quality control procedures are less stringent than in the United States (Reports from University of Minnesota, 2007–2011). Safety of most herbal products is compromised due to lack of suitable quality controls, inadequate labeling, and the absence of appropriate patient information [79]. 20.9 Drug Interactions Owing to the assumed safety of herbal products, it is a common practice to often combine these products with synthetic drugs with resultant herb–drug interactions. Usually, most patients do not disclose this practice of co-administration to their health care providers, and thus, many of the health care providers being less informed of herbal–drug interactions may cause serious adverse effects. Most times, the potential risks of drug–herbal interaction, even when known, are often ignored or underestimated. One should be cautious while prescribing neem along with anti-diabetic drugs as neem has a hypoglycemic effect [15]. Taking neem along with diabetes medications might further lower the blood sugar levels more. Because of the immunostimulant property of neem [80], it is not advisable to use it in patients taking immunosuppressants. Role of Azadiraciita indica in Dentistry 323 20.10 Neem’s Prospects in Dentistry From literature reviewed so far, we can establish the current usage of neem in dentistry. Various Neem products that can be used or are being used for dental hygiene and therapy are: 1. Neem mouth rinse is a strong and effective antiplaque agent and can be used as an adjunct to tooth brushing in gingivitis patients with equivalent antiplaque efficacy to 0.2% Chlorhexidine mouthwash, but less effective antigingivitis property. 2. Neem chewing sticks as an adjuvant to oral brushing. 3. Neem gel is more effective than mouthwash. 4. Neem-based toothpastes are effective in reducing plaque and gingival index. 5. Neem chip (10%) can be used as a local drug delivery adjunctive to mechanical therapy in chronic periodontitis patients. 6. The antimicrobial property of Neem makes it a potent root canal irrigant. 7. Phytochemicals of A. indica have been shown to arrest tumor cell growth and proliferation, making it a drug of choice with minimal side effects for oral carcinomas. 20.11 Action Points and Recommendations for Health Care Professionals Healthcare Professionals, such as physicians, nurses, and pharmacists, often have little knowledge of properties of herbal medicines, their safety, mechanism of action, and interactions with synthetic drugs. Most patients use herbal products but do not reveal to the doctor about the herbal drugs as they assume that “Herbal” or “natural” means safe and benign in interaction with any other drug. Education of healthcare professionals is vital to prevent misuse of herbal medicines. It is mandatory to follow drug regulatory policies for herbal medicines to ensure safety, quality, and efficacy. It is well known that Neem was used since ages with minimal side effects. In the light of the above-discussed facts, Neem was shown with multitudinous medicinal applications making it a very useful drug for treating many ailments, especially oral. Hence, all the physicians, dentists, and pharmacists should focus and work intensively on developing, promoting, and including Neem extracts in their daily practice and, at the same time, considering its safety aspect and contraindications. During this process, they must take interest in procuring information about neem from physicians of Ayurveda that will make the process easy. 20.12 Conclusion Neem is considered a safe alternative and adjuvant to conventional and synthetic drugs that are being used in dentistry. Although many studies have shown promising results on the use of Neem, most of them are conducted on animals. Multicentre studies with 324 Natural Oral Care in Dental Therapy larger sample sizes and long terms are to be conducted to further establish and commercialize its role in dentistry. Regulatory policies are to be standardized in identification, extraction, isolation, and production of various neem formulations. When taken in overdoses or incorrect combination with other synthetic drugs, Neem products can cause some side effects. It is, therefore, advisable that these products are to be prescribed by qualified medical practitioners and dentists. Health care professionals would need to proactively motivate patients to help inculcate more natural products into their lifestyle and that Neem will do good to head their list. Publicizing Neem’s use cases on global healthcare platforms will help establish India’s contribution to the medical industry in general and Dentistry in particular. References 1. 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Ghimeray, A.K., Jin, C.W., Ghimire, B.K., Cho, D.H., Antioxidant activity and quantitative estimation of azadirachtin and nimbin in Azadirachta indica A. Juss grown in foothills of Nepal. Afr. J. Biotechnol., 8, 3084, 2009. 38. Arumugam, A., Agullo, P., Boopalan, T., Nandy, S., Lopez, R., Gutierrez, C., Narayan, M., Rajkumar, L., Neem leaf extract inhibits mammary carcinogenesis by altering cell proliferation, apoptosis, and angiogenesis. Cancer Biol. Ther., 15, 26, 2014. 39. Raja Singh, P., Arunkumar, R., Sivakamasundari, V., Sharmila, G., Elumalai, P., Suganthapriya, E., Brindha Mercy, A., Senthil Kumar, K., Arunakaran, J., Antiproliferative and apoptosis inducing effect of nimbolide by altering molecules involved in apoptosis and IGF signalling via PI3K/Akt in prostate cancer (PC-3) cell line. Cell Biochem. Funct., 32, 217, 2014. 40. Chopra, I.C., Gupta, K.C., Nair, B.N., Biological activities and medicinal properties of neem (Azadirachta indica). Indian J. Med. 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Raja Ratna Reddy, Y., Krishna Kumari, C., Lokanatha, O., Mamatha, S., Damodar Reddy, C., Antimicrobial activity of Azadirachta Indica (neem) leaf, bark and seed extracts. Int. J. Res. Phytochem. Pharmacol., 3, 1, 2013. 46. Udeinya, J.I., Shu, E.N., Quakyi, I., Ajayi, F.O., An antimalarial neem leaf extract has both schizonticidal and gametocytocidal activities. Am. J. Ther., 15, 108, 2008. 47. Barua, C.C., Talukdar, A., Barua, A.G., Chakraborty, A., Sarma, R.K., Bora, R.S., Evaluation of the wound healing activity of methanolic extract of Azadirachta Indica (Neem) and Tinospora cordifolia (Guduchi) in rats. Pharmacologyonline, 1, 70, 2010. 48. Ofusori, D.A., Falana, B.A., Ofusori, A.E., Abayomi, T.A., Ajayi, S.A., Ojo, G.B., Gastroprotective effect of aqueous extract of neem Azadirachta indica on induced gastric lesion in rats. Int. J. Biol. Med. Res., 1, 219, 2010. 49. Dhingra, K. and Vandana, K.L., Effectiveness of Azadirachta indica (Neem) mouthrinse in plaque and gingivitis control: A systematic review. Int. J. Dent. Hyg., 15, 4, 2016. 50. Abhishek, K.N., Supreetha, S., Sam, G., Khan, S.N., Chaithanya, K.H., Abdul, N., Effect of neem containing toothpaste on plaque and Gingivitis—A randomized double blind clinical trial. J. Contemp. Dent. Pract., 16, 880, 2015. 51. Sharma, R., Hebbal, M., Ankola, A.V., Murugaboopathy, V., Shetty, S.J., Effect of two herbal mouthwashes on gingival health of school children. J. Tradit. Complement. Med., 4, 272, 2014. 52. Chatterjee, A., Saluja, M., Singh, N., Kandwal, A., To evaluate the antigingivitis and antiplaque effect of an Azadirachta indica (neem) mouthrinse on plaque induced gingivitis: A doubleblind, randomized, controlled trial. J. Indian Soc. Periodontol., 15, 398, 2011. 53. Vennila, K., Elanchezhiyan, S., Ilavarasu, S., Efficacy of 10% whole Azadirachta indica (Neem) as adjunct to Scaling and root planing in chronic periodontitis. A clinical microbiological study. Indian J. Dent. Res., 27, 15, 2016. 54. Pai, M.R., Acharya, L.D., Udupa, N., The effect of two different dental gels and a mouthwash on plaque and gingival scores: A six-week clinical study. Int. Dent. J., 54, 219, 2004. Role of Azadiraciita indica in Dentistry 327 55. Kudalkar, M.D., Nayak, A., Bhat, K.S., Nayak, R.N., Effect of Azadirachta indica (Neem) and aloe vera as compared to subantimicrobial dose doxycycline on matrix metalloproteinases (MMP)-2 and MMP-9: An in-vitro study. Ayu, 35, 85, 2014. 56. Chandrappa, P.M., Dupper, A., Tripathi, P., Arroju, R., Sharma, P., Sulochana, K., Antimicrobial activity of herbal medicines (tulsi extract, neem extract) and chlorhexidine against Enterococcus faecalis in endodontics: An in vitro study. J. Int. Soc. Prev. Community Dent., 5, 89, 2015. 57. Babaji, P., Jagtap, K., Lau, H., Bansal, N., Thajuraj, S., Sondhi, P., Comparative evaluation of antimicrobial effect of herbal root canal irrigants (Morinda citrifolia, Azadirachta indica, aloe vera) with sodium hypochlorite: An in vitro study. J. Int. Soc. Prev. Community Dent., 6, 196, 2016. 58. Sundaram, D., Narayanan, R.K., Vadakkepurayil, K.A., Comparative evaluation on antimicrobial effect of honey, neem leaf extract and sodium hypochlorite as intracanal irrigant: An ex-vivo study. J. Clin. Diagn. Res., 10, 88, 2016. 59. Prasad, S.D., Goda, P.C., Reddy, K.S., Kumar, C.S., Hemadri, M., Ranga Reddy, D.S., Evaluation of antimicrobial efficacy of neem and aloe vera leaf extracts in comparison with 3% sodium hypochlorite and 2% chlorhexidine against E. faecalis and C. albicans. J NTR Univ. Health Sci., 5, 104, 2016. 60. Dutta, A. and Kundabala, M., Comparative anti-microbial efficacy of Azadirachta indica irrigant with standard endodontic irrigants: A preliminary study. J. Conserv. Dent., 17, 133, 2014. 61. Sales Peres, A.C., Marsicano, J.A., Garcia, R.P., Forim, M.R., Silva, M.F.G.F., Sales Peres, S.H.C., Effect of natural gel product on bovine dentin erosion in vitro. J. Appl. Oral Sci., 21, 597, 2016. 62. Vanka, A., Tandon, S., Rao, S.R., Udupa, N., The effect of indigenous neem mouthwash on S. mutans and Lactobacilli growth. Indian J. Dent. Res., 12, 133, 2001. 63. Prashant, G.M., Chandu, G.N., Murulikrishna, K.S., Shafiulla, M.D., The effect of mango and neem extract on four organisms causing dental caries: Streptococcus mutans, Streptococcus salivavius, Streptococcus mitis, and Streptococcus sanguis: An in vitro study. Indian J. Dent. Res., 18, 148, 2007. 64. Polaquini, S.R., Svidzinski, T.I., Kemmelmeier, C., Gasparetto, A., Effect of aqueous extract from neem (Azadirachta indica A. Juss) on hydrophobicity, biofilm formation and adhesion in composite resin by Candida albicans. Arch. Oral Biol., 51, 482, 2006. 65. Balasenthil, S., Arivazhagan, S., Ramachandran, C.R., Ramachandran, V., Nagini, S., Chemopreventive potential of neem (Azadirachta indica) on 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis. J. Ethnopharmacol., 67, 189, 1999. 66. Harish Kumar, G., Vidya Priyadarsini, R., Vinothini, G., Vidjaya Letchoumy, P., Nagini, S., The neem limonoids azadirachtin and nimbolide inhibit cell proliferation and induce apoptosis in an animal model of oral oncogenesis. Invest. New Drugs, 28, 392, 2010. 67. Subapriya, R., Kumaraguruparan, R., Nagini, S., Expression of PCNA, cytokeratin, Bcl-2 and p53 during chemoprevention of hamster buccal pouch carcinogenesis by ethanolic neem (Azadirachta indica) leaf extract. Clin. Biochem., 39, 1080, 2006. 68. Manikandan, P., Letchoumy, P.V., Gopalakrishnan, M., Nagini, S., Evaluation of Azadirachta indica leaf fractions for in vitro antioxidant potential and in vivo modulation of biomarkers of chemoprevention in the hamster buccal pouch carcinogenesis model. Food Chem. Toxicol., 46, 2332, 2008. 69. Joy Sinha, D., Nandha, K.D.S., Jaiswal, N., Vasudeva, A., Prabha Tyagi, S., Pratap Singh, U., Antibacterial Effect of Azadirachta indica (Neem) or Curcuma longa (Turmeric) against Enterococcus faecalis Compared with That of 5% Sodium Hypochlorite or 2% Chlorhexidine in vitro. Bull. Tokyo Dent. Coll., 58, 103, 2017. 328 Natural Oral Care in Dental Therapy 70. Patil, R.C., Kulkarni, C.P., Pandey, A., Antibacterial and phytochemical analysis of Tinospora cordifolia, Azarchita indica and Ocimum santum leaves extract against common human pathogens: An in vitro study. J. Pharmacogn. Phytochem., 6, 5, 702, 2017. 71. Saxena, D., Saha, S.G., Saha, M.K., Dubey, S., Khatri, M., An in vitro evaluation of antimicrobial activity of five herbal extracts and comparison of their activity with 2.5% sodium hypochlorite against Enterococcus faecalis. Indian J. Dent. Res., 26, 524, 2015. 72. Raghavendra, S.S. and Balsaraf, K.D., Antifungal efficacy of Azadirachta indica (neem)—An in vitro study. Braz. J. Oral Sci., 13, 3, 242, 2014. 73. Mustafa, M., Antibacterial Efficacy of Neem (Azadirachta indica) Extract against Enterococcus faecalis: An in vitro Study. J. Contemp. Dent. Pract., 17, 10, 791, 2016. 74. Jalaluddin, M., Rajasekaran, U.B., Paul, S., Dhanya, R.S., Sudeep, C.B., Adarsh, V.J., Comparative Evaluation of Neem Mouthwash on Plaque and Gingivitis: A Double-blind Crossover Study. J. Contemp. Dent. Pract., 18, 7, 567, 2017. 75. Elangovan, A., Muranga, J., Joseph, E., Comparative evaluation of the antimicrobial efficacy of four chewing sticks commonly used in South India: An in vitro study. Indian J. Dent. Res., 23, 840, 2012. 76. Hamid, S.K., Al-Dubayan, A.H., Al-Awami, H., Khan, S.Q., Gad, M.M., In vitro assessment of the antifungal effects of neem powder added to polymethylmethacrylate denture base material. J. Clin. Exp. Dent., 11, 2, e170, 2019. 77. Philomena, G., Concerns regarding the safety and toxicity of medicinal plants—An overview. J. Appl. Pharm. Sci., 1, 40, 2011. 78. Bandyopadhyay, U., Biswas, K., Sengupta, A., Moitra, P., Dutta, P., Sarka, D., Debnath, P., Ganguly, C.K., Banerjee, R.K., Clinical studies on the effect of Neem (Azadirachta indica) bark extract on gastric secretion and gastroduodenal ulcer. Life Sci., 75, 2867, 2004. 79. Raynor, D.K., Dickinson, R., Knapp, P., Long, A.F., Nicolson, D.J., Buyer beware? Does the information provided with herbal products available over the counter enable safe use? BMC Med., 9, 94, 2011. 80. Upadhyay, S.N., Dhawan, S., Garg, S., Talwar, G.P., Immunomodulatory Effects of Neem (Azadirachta indica) Oil. Int. J. Immunopharmacol., 14, 1187, 1992. 21 Ginger in Oral Care Aditya Ganeshpurkar*, Abhilasha Thakur and Anupam Jaiswal Shri Ram Institute of Technology-Pharmacy, Jabalpur, Madhya Pradesh, India Abstract Mankind has been using medicinal plants for the treatment of diseases from prehistoric era. The roots, leaves, stems, flowers, seeds, and tubers of the plants are utilized for isolation of various bioactive constituents. Ginger is an important medicinal herb, the medicinal benefits of which are mentioned in Ayurveda. Ginger is extensively used for its flavor. Various cuisines use ginger for its aroma. With the view of its biological and pharmacological effects, gingerols, and shogoals are among the few of its constituents responsible for its therapeutic effects. Ginger is scientifically validated for oral care. Ginger extracts are known to possess antimicrobial, antifungal, oral anticancer, anti-nausea, anti-carries, and antiplaque properties. Ginger is known to harden the teeth due to indirect remineralization property. Thus, overall, the health benefits of ginger in oral care make it useful for treatment of various oral disorders. Keywords: Ginger, antimicrobial, antifungal, oral anticancer, anti-nausea, anti-carries, antiplaque 21.1 Introduction The use of herbs for the treatment of disease dates back to prehistoric era. The use of complement and alternative medicine to cure diseases is followed by many parts of the globe. These therapies are more economical and are widely accepted. The medicinal plants have immunomodulatory [1–4], analgesic [5, 6], anabolic [7], antitumor [8, 9], and antimicrobial [8, 10] bioactives. The extensive phytochemical analysis has provided an in-depth knowledge about structural and functional properties of plant-derived bioactives. Ginger comprises the dried rhizome of Zingiber officinale Roscoe (Family Zingiberaceae) [11]. Ginger is one of the widely eaten dietary condiments worldwide. Ginger is spicy and aromatic. The aromatic flavor is predisposed due to the presence of gingerols in it. The use of ginger in the Asian subcontinent dates back 5000 years ago. Indian and Chinese food and medicine employ ginger as one of the key herbs for aroma and health. India is the second largest cultivator of ginger with an annual production of 385,000 tons after China. In India, Kerala, Orissa, Arunachal Pradesh, Karnataka, Sikkim, West Bengal, and Madhya Pradesh are the major ginger-producing states. *Corresponding author: adityaganeshpurkar@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (329–344) © 2020 Scrivener Publishing LLC 329 330 Natural Oral Care in Dental Therapy 21.2 Description It is a perennial herb with branched rhizome Table 21.1. The shoot grows up to 1.50 m in height. The leaves are lanceolate with an alternate arrangement. The stems of the flowers are shorter than the stems of the leaves. The flower has a superior tubular calyx. There is an orange yellow corolla differentiated above into three linear oblong, blunt lobes, with six staminodes in two rows. The fruit is a capsule with small arillate seeds [12, 13]. 21.3 Macroscopic Characteristics Table 21.1 Macroscopic characteristics of ginger. Character Inference Color Brown to pale yellow Odor Characteristic aromatic Taste Aromatic, Pungent Size 3–16 cm long, 3–4 cm wide, up to 2 cm thick Shape Horizontal, laterally flattened, irregularly branching pieces 21.4 Pharmacognostic Standards Table 21.2 Pharmacognostic standards of ginger. Standards Value Reference Foreign organic matter Not more than 2.0% [11] Total ash Not more than 6.0% [14] Acid-insoluble ash Not more than 2.0% [15] Water-soluble extractive Not less than 10% [14] Alcohol-soluble extractive Not less than 4.5% [14] Heavy metals Lead Cadmium 10 mg/kg 3 mg/kg [16] Ginger in Oral Care 331 Table 21.3 Proximate composition of ginger. Constituents [18] Percent Protein 5.8 Crude fiber 5.4 Fat 0.9 Ash 3.5 β-carotene 0.81 Ascorbic acid 3.8 Calcium 69.2 Iron 1.8 Copper 0.75 21.5 Nutrient Composition Ginger is used as an integral seasoning in various cuisines. Due to presence of essential oils, it has a characteristic aroma. It is used to impart flavor to tea, coffee, decoctions, confectionery, soft drinks, beverages, and many other dishes. Ginger is also used in pickles. Ginger can be desiccated to make ginger powder. The “so formed” ginger powder is stable for a long period of time compared to fresh ginger. The presence of a variety of constituents like 6-shogoals, 6-gingerol, bisabolene, and zingiberene imparts a pungent and aromatic taste to it [17]. Ginger can be regarded as a good source of mineral elements, essential amino acids, and nutrients, Table 21.2 and Table 21.3. The general nutrient composition of ginger is summarized in Table 21.3. 21.6 Pharmacological and Medicinal Effects 21.6.1 Oral Analgesic Effect A validated clinical study has been performed on ginger to evaluate its anti-inflammatory and analgesic property over postsurgical pain. In this study, 60 patients undergoing mandibular third molar removal participated in the study. In this study, patient volunteers were divided randomly into three groups. The first group received the capsule of ginger (rhizome) (500 mg). The second group received ibuprofen (400 mg), whereas the third group received placebo. In this study, the cardinal parameters like amount of mouth opening, pain, cheek swelling, and serum high-sensitivity C-reactive protein (hsCRP) were taken into consideration. On day 5, the maximal mouth opening offered due to ibuprofen treatment was 36.3 ± 11 where administration of ginger demonstrated a maximum mouth opening of 35.3 ± 10. There was a noteworthy decrement in cheek swelling due to administration of ginger capsule (32.6 ± 3.7) compared to that of the ibuprofen treatment (34.7 ± 4.4). Median pain 332 Natural Oral Care in Dental Therapy score after 12 h of ginger capsule administration was 24.6 ± 6.3 mm. There was no observable abnormal wound bleeding. There was insignificant difference in C-reactive protein levels. The outcomes of the study revealed the significant effectiveness of ginger in controlling postoperative dental pain [19]. 21.6.2 Antimicrobial Effect Streptococcus mutans is found in the oral cavity and forms a biofilm on the surface of the teeth. The formation of dental plaque prevents permeation of antimicrobial agents producing resistance to antibiotics [20]. In a study, the antimicrobial effect of ginger against Streptococcus mutans was evaluated. The minimum inhibitory concentration and minimum bactericidal concentration was found to be 256 μg/ml. The crude ginger extract revealed antibacterial activity with more than 5-log CFU/ml, whereas methanolic fraction showed a decrement of 4-log CFU/ ml, Figure 21.1. There was a noteworthy anti-adherence activity, which was due to a decrease in sucrose-dependent and sucrose-independent adherence. Crude ginger extract (128 µg/ ml) caused a decrement in sucrose-dependent and sucrose-independent adherence by 78% and 73%, respectively. Methanolic fraction revealed a decrease in sucrose-dependent and sucrose-independent adherence by 72% and 70%, respectively. While accessing glucosyltransferases inhibitory activity, it was seen that crude ginger extract caused a decrease in water soluble and insoluble glucans by 70%. Similar inhibition was observed in the case of methanolic extract. The hydrophobicity index of Streptococcus mutans was decreased by 75% and 71%, respectively. There was an inhibition in biofilm formation during active accumulated phase, initial plateau accumulated phase, and plateau accumulated phase of growth of bacteria. Initial pH drop was observed to be 7.25 to 6.83 in the case of crude extract, whereas methanolic fraction caused a drop from 7.25 to 5.41. F-ATPase activity (0.25 MIC) was decreased by 44% (methanolic fraction) and 53% (crude extract), respectively. Crude extract decreased protein surface antigen by 50%, whereas methanolic fraction reduced surface antigen by 70%. Distortion of bacterial biofilm was seen, which was evident from scanning electron microscopy and confocal scanning microscopy. Crude extract (85%) as well as methanolic fraction (68%) caused a decrement in expression of brpA gene. Thus, all growth and propagation of Streptococcus mutans was hampered by ginger extract [21]. Ginger extract was able to inhibit the growth of Streptococcus mutans (30.0.0 ± 1.5 mm) and Lactobacillus acidophilus (21.0 ± 0.7 mm) Figure 21.2, [22]. O HO OCH3 Ginger Figure 21.1 Antimicrobial effect of Ginger. Gingerol OH Ginger in Oral Care 333 Decrease in sucrose dependent and sucrose independent adherence Glucosyltransferases inhibitory activity Decreased hydrophobicity index of Streptococcus mutans Decreased biofilm formation Decreased protein surface antigen Decreased expression of brpA gene Figure 21.2 Mechanism of antimicrobial effect of Ginger extract. 21.6.3 Anti-Carries Activity Dental carries were developed in rats to study the anticariogenic effect of ginger extract. In this study, rats were supplemented with 5% sucrose diet and inoculated with Streptococcus mutans. Outcomes of the study revealed the beneficial effect of crude ginger extract in preventing carries in the animals. The persistence of smooth surface was about 80% in the ginger extract-treated group. Also, the incidence of formation was decreased [21]. Another independent study revealed a noteworthy inhibition (5 mm) over the microbes found over the tooth [23]. The combination of ginger, cloves, and mint extracts revealed a notable inhibition over caries forming bacteria, viz. Staphylococcus aureus (19 mm), Granulicatellaadiacens (11 mm), Pseudomonas oryzihabitans (20 mm), Enterobacter cloacae (19 mm), Stenotrophomonas maltophilia (19 mm), and Pseudomonas putida (21 mm) [24]. A study revealed the effectiveness of ginger as an auxillary chemical in association with calcium hydroxide and chlorhexidine [25]. One of the active constituents of ginger, zerumbone, showed a minimum inhibitory concentration of 250 μg/ml and a minimum bactericidal concentration of 500 μg/mL against Streptococcus mutans and proved to be a potential prophylactic anti-cariogenic agent [26]. 21.6.4 Anti-Decay Effect Tooth decay can be regarded as one of the most progressive diseases observed by infection in the mouth followed by bad breath and weakness of the tooth. This is predisposed due to accumulation of microbes on the surface of the enamel causing formation of a biofilm that 334 Natural Oral Care in Dental Therapy causes erosion as well as decay of teeth. If this condition is left untreated, it can result in injury to the pulp and loss of tooth [27]. Streptococcus sanguinis and Streptococcus mutans are two bacteria that are known to cause damage to the teeth and gums. These microbes biosynthesize the products, viz. levan and dextran, which are responsible for the formation of dental carries. In severe tooth decay, these bacteria find their way toward bold stream where they may cause endocarditis of the heart valves [28]. Ginger (drops) has been studied for its probable anti-decay effect. In a study, ginger (Arheumin drop, Iran) demonstrated a significant inhibition over the growth of Streptococcus sanguinis and Streptococcus mutans. The MIC and MBC values of the extract (drops) were 0.02 and 0.04 mg/ml for Streptococcus mutans and 0.3 and 0.6 mg/mL for Streptococcus sanguinis, respectively [29]. In another study, there was a decrement in bio-film formation during growth phase in Streptococcus mutans. There was inhibition and decreased adherence toward synthesis and tooth adherence of glucans. Similarly there was a significant decrement in sucrose-dependent adherence as well as insoluble glucan synthesis. The results of the study also revealed a downregulation of virulent gene [21]. Researchers suggested adding ginger (extract) into mouthwashes to wield the inhibition over the growth over cariogenic bacteria, thus promoting inhibition over tooth decay. Similarly in another study, ginger, in association with chlorhexidine and calcium hydroxide, demonstrated a notable inhibition over Enterococcus faecalis and Escherichia coli. The levels of endotoxins were also decreased significantly [23]. 21.6.5 Healing Effect in Root Canal Therapy Root canal treatment is utilized for the treatment of infected pulp underlying the tooth. It is followed by securing the tooth by a protective layer to prevent the tooth from future microbial attack. Inert fillings like eugenol-based cements and gutta-percha are utilized to fill the cavity. Sodium hypochlorite is a widely used irrigant in dental practice. It is an effective antimicrobial agent and also has the capability to dissolve tissues. The antimicrobial effect of sodium hypochlorite increases with an increase in its concentration. A study was designed to evaluate the antimicrobial and protective effect of sodium hypochlorite and ginger extract microorganisms and endotoxins in endodontic treatment of infected root canals. In this experiment, tooth was contaminated with bacteria and instrumentation for root canal treatment was performed. After a period of 28 days, the content of root canal was collected and analyzed for viable microorganisms. The results of studies indicated a synergy between sodium hypochlorite and ginger extract, due to which, there was complete elimination (100%) of microorganisms and reduction of endotoxins (88%) [30]. Another study aimed to determine the effect of propolis extract, ginger extract, calcium hydroxide, chlorhexidine gel, and their combinations against the growth of Candida albicans, Enterococcus faecalis, and Escherichia coli. The antimicrobial effect was possibly due to the presence of gingerol [31]. The outcomes of the present study revealed a synergistic decrease in microbial load and endotoxin [32]. 21.6.6 Anti-Xerostomia Effect Xerostomia is a condition observed by a decrease in salivary flow, which leads to difficulty in swallowing, chewing, tasting, and chewing. Reduced salivary flow is also responsible for teeth demineralization, decay of teeth, and sensitivity. The various reasons for xerostomia include Ginger in Oral Care 335 rheumatoid arthritis, aging, head and neck cancer, Parkinson’s disease, hyperthyroidism, anemia, and infectious disorders [33]. Ginger herbal spray has been evaluated for its probable effect on the management of dry mouth and associated disorders. In a clinical trial conducted on type II diabetic patients (n = 20), patients were given ginger extract (ethanolic; 70% ethanol for extraction) to administer. Schirmer test was used to measure saliva. The results of the study indicated a significant increase in the mean amount of saliva after ginger extract administration. The study seems to be useful for patients suffering from dry mouth [34]. 21.6.7 Anti-Pyorrhea Effect Pyorrhea is a periodontal disease observed by inflammation and infection of bones and ligaments that hold up the teeth. Gingivitis is also observed followed by plaque build-up in the gums. The decay in the tooth forms the holes and pockets. Pockets trap food particles, the decay of which causes deepening of cavities. This causes erosion of bone leading to tooth loss. Gingerol, an active component of ginger, is evaluated for inhibition over Porphyromonas gingivalis, Porphyromonas endodontalis, and Prevotella intermedia. The minimum inhibitory concentration for [10]-gingerol and [12]-gingerol was in the range of 6–30 µg/ml, and the minimum bactericidal concentration was 4–20 µg/ml, Figure 21.3. The antimicrobial effect of [10]-gingerol was more than that of [12]-gingerol. The outcomes of the present study concluded that the length and modification of the alkyl side chain of gingerol is a key factor in determining antimicrobial efficacy [31]. 21.6.8 Anti-Thrush Effect Thrush or oral candidiasis is candidiasis of the mouth. It is a condition in which Candida albicans accumulate at the linings of the mouth, observed by the production of white lesions Ginger Powder (in Tooth-paste/Tooth-powder) Analgesic effect Anti-gingival effect Anti-tooth sensitizing effect Figure 21.3 Analgesic effect of ginger. 336 Natural Oral Care in Dental Therapy on the inner check and tongue. It can also affect the gums, tonsils, and throat. In a laboratory investigation, it was observed that ginger extract exerted an antifungal effect on Candida albicans (Germ Tube formation Test). The extract at a concentration of 2 mg/ml (in the dilution) (1:5) revealed an antifungal effect [35]. In another independent study, Sunthi Churna (Ginger powder) extract (in 99% ethanol) was evaluated for its antifungal effect. The extract was prepared by maceration at cold temperature as well as maceration at room temperature. The lethality against Candida albicans was observed at log phase [36]. Supplementing ginger with chlorhexidine and calcium hydroxide reduced Candida albicans load [23]. 21.6.9 Anti-Herpes Effect Oral herpes is a viral infection, which is known to be caused by the Herpes Simplex virus. The symptoms of the disease include swollen lips, gums, palate, and cheeks followed by pain and inflammation. It is a contagious disease, which spread by saliva, mucus membrane, and skin. Essential oil from ginger (concentration 0.003%) was evaluated for its anti-herpes effect. The antiviral effect of ginger essential oil was due to the prevention of adsorption of the virus before entry to the host cell. The essential oil interferes with the structure of the virion envelop and possibly dissolved viral envelop. Microscopic investigation also revealed the disruption of the viral envelop due to pretreatment with essential oil. The lipophilic component of the essential oil interact with the lipid membrane, which influences envelop integrity [37]. 21.6.10 Tooth Polishing Brushing teeth has become a habit for man. Shining white teeth bestow confidence. Tooth polishing can be considered as an imperative division of dental procedures. Polishing of teeth includes the smoothening of the surface of the teeth with simultaneous removal of the deposition of plaque, biofilm, and acquired pellicle leading to the preservation of healthy tooth health [38]. The process of tooth polishing aids in the removal of various exogenous stains spawned due to food as well as environmental factors. Calcium carbonate, aluminum silicate, calcium phosphate, pumice, etc., are used for tooth polishing [39]. Use of ginger in cleaning and polishing tooth has been advocated by Pierre Fauchard (Father of Modern Dentistry). He suggested the use of a mixture of finely divided egg shell, ground coral with ginger to polish the teeth [40]. The toothpaste formulation of ginger extract has been patented. The toothpaste claims to provide better antibacterial and antiinflammatory effect when compared to conventional toothpaste formulation [41]. 21.6.11 Mouth Deodorizing Effect A mouthwash is a pharmaceutical preparation intended to be placed in the oral cavity and rustled by the action of perioral musculature in order to eradicate the oral pathogens. The history of mouthwash dates back to 460 BC. Hippocrates suggested the use of a mixture of alum, vinegar, and salt to clean the oral cavity. In the present time, systemic study on ginger mouthwash has been performed to evaluate its anti-gingival effect. A clinical study was Ginger in Oral Care 337 Table 21.4 The patented toothpaste formulations of ginger. Toothpaste formulation/invention Claim Reference Salted ginger toothpaste Against dental inflammation, bleeding, halitosis, [42] Ginger extract and ginger oil toothpaste Reduction of dentin hypersensitivity [43] Toothpaste with honey and ginger juice Anti-inflammatory, prevent carries [44] Ginger toothpaste Cleansing effect on teeth [45] Poly-herbal toothpaste containing ginger Against bad breath and gingival abscess [46] Attapulgite fresh ginger toothpaste Removal of odor, reduction of dental plaque, prevention of gingival bleeding [47] Poly-herbal toothpaste containing fresh ginger Reducing swelling and bleeding gums [48] Herbal toothpaste containing ginger Reduction in swelling and in sore gums [49] Polyherbal Chinese herbs toothpaste Prevention of tooth sensitivity and mitigation action [50] Folic acid toothpaste containing ginger Postpartum gingivitis, bleeding gums [51] Children’s toothpaste Oral care product for children with better acceptance [52] Antiphlogistic toothpaste Anti-inflammatory effect [53] Table 21.5 The patented toothpowder formulations of ginger. Toothpaste formulation/invention Claim Reference Polyherbal natural herbal tooth powder Yellowing/staining of teeth, treatment of pyorrhea, and Sensitivity of teeth [54] Dental powder with natural ingredients Retarding tooth decay, relief from tooth pain [55] Black attapulgite Chinese traditional medicine tooth powder containing ginger Prevention of bleeding gums [56] Antiphlogistic and antitussive medicine powder containing ginger Prevention of foul breath, preservation of dental health [57] 338 Natural Oral Care in Dental Therapy performed on 60 volunteers; 20 volunteers were divided into three groups and revived polyherbal mouthwash containing ginger, chlorhexidine mouthwash, and placebo mouthwash. Outcomes of the study revealed that the polyherbal mouthwash containing ginger significantly improved the symptoms of gingivitis as evident from decreased scores of modified gingival index, gingival bleeding, and plaque score [58]. 21.6.12 Anticancer Effect Ginger constituents have been extensively studied for anticancer effects. The protective effect of 6-Shogoal was evaluated over 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis (oral carcinoma). Administration of 6-shogoal (10, 20, and 40 mg/kg body weight) decreased over the expression of p53 and Bcl-2. There was restoration of antioxidant status in animals and decrement in lipid peroxidation. There was reversal of tumor incidences [59]. In another study of DMBA-induced oral carcinoma, there was inactivation of AP-1 and NF-κB followed by decrement in inflammation and cell proliferation [60]. In another study, [6]-paradol attenuated DMBA-induced hamster buccal pouch carcinogenesis. Administration of [6]-paradol (30 mg/kg) for 14 weeks resulted in a decrease in neoplastic changes as well as restoration of bcl-2, p53, TNF-α, and caspase-3 [61]. There was a decrease in lipid peroxidation and increase in reduced glutathione in DMBA-treated animals [62]. Similar effects were seen in the case of geraniol whereby peroxidation of lipids was decreased and restoration of phase I and II enzymes was seen [63]. Similar findings were seen in the case of geraniol, which proved to be anti-angiogenic, anti-cell proliferative, anti-inflammatory, and have apoptosisinducing properties [64]. 21.6.13 Protection Against Aphthous Stomatitis Aphthous stomatitis is a common painful condition, which affects the oral cavity. It is a condition observed by persistence of benign mouth ulcers. The small, round, and multiple ulcers are observed. It can develop due to trauma (dental procedures) [65], tobacco [66], and certain drugs (phenobarbitone, piroxicam, nicorandil, gold salts) [67]. In some studies, ginger has been evaluated for its analgesic and anti-arthritic effect [68, 69]. In a study, ginger-containing mucoadhesive system was evaluated for its efficacy in management of aphthous stomatitis. It was a double-blind placebo trail enrolling 15 patients. The placebo preparation contained tragacanth gum, and the ginger preparation consisted of an alcoholic extract of ginger. These preparations were circular discs of 1-cm diameter. Patient volunteers were asked to apply the preparation 20 min after every meal and before going to bed. The treatment continued for a period of 7 days. The parameters for evaluation included determination of ulcer diameter, pain, and inflammatory zone. The outcomes of study revealed a noteworthy decrease in pain. There was a significant decrease in pain on the fifth day. From the first day of treatment, there was a notable decrement in inflammatory zone [70]. 21.6.14 Effect on Dentin Hardness Dentin is a calcified tissue, yellow in color, and is found in teeth along with the enamel, pulp, and cementum. The enamel covers and protects the dentin [71]. The dentin is composed Ginger in Oral Care 339 of hydroxylapatite, minerals, and water. It is produced by odontoblast forming a layer over the pulp cavity. It forms the seat for the enamel [72]. The function of dentin is to protect the pulp of the tooth [73]. Ginger is studied for its modulatory effect on dentin rigidity. In a study, the effect of treatment with ginger (oil), sodium hypochlorite, and EDTA was evaluated on dentin hardness. The results revealed that ginger (oil) showed the highest reduction in dentin micro-hardness. The study suggested the incorporation of ginger oil as an alternative to chemical irrigants in endodontics [74]. 21.7 Pharmacokinetics Table 21.6 Pharmacokinetics of gingerol. Absorption Oral [75] Distribution Widely distributed [75] Metabolism 6-gingerdiol [76] Excretion Urine [77] 21.8 Toxicological Studies Ginger in moderate amounts (up to 2 g) is safe for human consumption. The phytoconstituents of ginger, viz. gingerol and shogaol, are well absorbed and bio-transformed after oral consumption [78]. 21.9 Conclusion Ginger has been extensively used in traditional medicines for treatment of various diseases. The use of ginger is documented to promote the process of food digestion, in flu and cold, and in preventing nausea. The various forms of ginger like ginger slices, grated ginger, ginger paste, ginger juice, and ginger oil are used. The contemporary scientific research has validated this claim. Ginger also possesses anti-colon cancer property. In view of oral health, ginger revealed a lethal effect on the growth of toothdecaying bacteria. It is a good dental analgesic and promotes dentine remineralization. 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Biomarkers, 17, 8, 1930–1936, 2008. 22 Effectiveness of Allium sativum on Bacterial Oral Infection Vesna Karic1,2, Anupam Jaiswal3, Heidi Abrahamse2*, Abhilasha Thakur3 and Aditya Ganeshpurkar3 1 Department of Prosthodontic and Oral Rehabilitation, and Laser Therapy in Dentistry Division, School of Oral Sciences, Health Sciences Faculty, WITS University, Johannesburg, South Africa 2 Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Doornfontein, Johannesburg, South Africa 3 Shri Ram Institute of Technology Pharmacy Jabalpur, Madhya Pradesh, India Abstract Garlic or Allium sativum is a species in the Allium genus. Its name is derived from an old English word that means spear and leek. Garlic is found all over the world. It has some important chemical compositions that show various activities. The health benefits of consuming garlic are very well known. The use of A. sativum as an antibacterial agent and its effects on oral flora are currently being studied in vitro and in vivo. The application of garlic in oral therapy has shown promising results against Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans and also on the proteases of Porphyromonas gingivalis that are found in periodontitis. Furthermore, in vivo studies have reported that mouthrinse containing garlic extract is efficient in the treatment of Streptococcus mutans bacteria by reducing their complete count in saliva. The ongoing interest in garlic as an oral antibacterial agent has grown since it appears that the resistance of bacteria to garlic is much less than conventional antibiotics. It has also been found that garlic has antifungal, anticancer, antiallergic, antiobesity, and antiviral properties. Garlic could conceivably become a treatment option for patients suffering from a variety of oral diseases. Keywords: Allium sativum, periodontitis, stomatitis, antibacterial, endodontitis, garlic, dental 22.1 Introduction Petersen et al. described diseases in the oral cavities as a worldwide public health problem, including dental caries and periodontal disease. Poor dental and oral health in patients has a wider effect on general health and on the general quality of life [1]. Furthermore, dental caries in most industrialized countries is considered a major health issue. It has been confirmed that it affects 60% to 90% of children of school-going age and also clearly adults in great numbers. *Corresponding author: habrahamse@uj.ac.za Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (345–370) © 2020 Scrivener Publishing LLC 345 346 Natural Oral Care in Dental Therapy Specific bacteria and constituents in the diet interact in the build-up of the biofilm plaque that forms on tooth surfaces, which results in the formation of dental caries [2]. The first suggestion that infection causes dental caries was communicated by Clark. He described the causation of dental caries: “by a hitherto undescribed streptococcus, Streptococus mutans.” Streptococcus mutans is a gram-positive cocci, facultative and anaerobic, which can use carbohydrates from food in the oral cavity to metabolize and grow. These microorganisms are the main cause of dental caries. The formation of a cariogenic biofilm starts and ends with S. mutans on the surface of the teeth because this bacterium uses sugar components, specifically sucrose, to form extracellular polysaccharides, which attach to glucan-coated surfaces [3]. S. mutans is also highly acidogenic [4]. Hence, brushing of the teeth is very important because it has a great impact on the removal of biofilm and reduces salivary levels of microorganisms, specifically S. mutans [5]. Dentists encourage a daily routine of brushing teeth at least two times per day. The mechanical effect of brushing is necessary to remove plaque with cariogenic bacteria, and brushing with fluoridated toothpaste is the first and most important line for the prevention of dental caries [6]. However, brushing of the teeth only will not be effective in completely removing bacteria in the mouth. Additional steps are necessary, such as reducing the sugar intake in the diet and rinsing the mouth with mouthwashes. The first recorded use of mouth rinsing is in ancient Chinese medicine in 2700 BC [7]. Currently, the most established mouth-rinsing liquid is chlorhexidine. This mouthwash has been in use for the last 20 years, with good results in the reduction of dental caries. However, using modern scientific concepts, it has become apparent that the formations of dental lesions are a result of complex interactions between inherited genetic factors and acquired environmental ones. Therefore, the fight against dental caries and the prevention of dental caries diseases is augmented using mouth-rinsing agents, which also play a key role in the prevention and treatment of periodontal disease. In addition, mouthwash is widely used for the treatment of pain, inflammation, and against the general population of pathogenic bacteria in the oral cavity [8]. Throughout recorded history, garlic (A. sativum) has shown efficacy in the field of dentistry due to its inhibitory effect on S. mutans in dental plaque [9]. The content of sulfur compounds found in garlic cloves such as allicin has been determined to be an antioxidant by the chemical analyses of garlic cloves. Furthermore, sulfur compounds have the ability to attach to the sulfhydryl (SH) groups in enzymes and proteins where it can modify their activities and inhibit the sulfhydryl enzymes [10, 11]. It also has the capacity to occupy cells through the cell membranes. The importance of finding better options for the treatment of dental infections is because of the complexity of the microorganisms involved in the formation of dental infections. An example is the Aggregatibacter actinomycetemcomitans, a gram-negative bacterium, which is a primary cause of a severe form of periodontal disease, localized aggressive periodontitis [12, 13]. This bacterium has been connected recently to systemic infections like urinary tract infection, osteomyelitis, and brain abscesses. The treatment of progressive periodontitis is very difficult due to constant strengthening of bacteria resistant to antibiotics [14]. A. actinomycetemcomitans is among other oral bacteria becoming resistant to antibiotics. To gain a better appreciation of the current global health situation regarding the antibiotic resistance of many bacteria, specifically in dentistry, the issues like dental infection epidemiology, dental infection, and antibiotic resistance and the antibacterial application of garlic in dentistry need special attention. The Indian subcontinent is the richest home to various plants, which have different medicinal properties. For the last few years, some of them were used as Ayurvedic drugs for Allium savitum on Bacterial Oral Infection 347 oral and dental care. The purpose of Ayurvedic medicine is to achieve physical, mental, and spiritual wellness by holistic approach [15]. The antimicrobial activity of garlic has been recognized for centuries. In 1500 BC, it was first identified in an Egyptian recipe known as Paprius Ebers. Garlic is used as a folk medicine for the treatment of many diseases [16]. It is also used for the preservation of food products [17]. The botanical name of garlic is Allium sativum belonging to the family Liliaceae (Table 22.1). Its various active constituents are S-allycystein, alliin, allinase, allymethyltrisulfide, allicin, and diallylsulfide. Alliin is an amino acid in the presence of alline– lyase catalyses, which is converted into allicin when the bulbs are crushed. Allicin, has a specific odor, flavor, and some important pharmacological properties, which is used to treat oral and dental problems because of the presence of sulfur-containing compounds. In the presence of air, allicin is converted into diallyldisulfide, which is responsible for the bactericidal effect, and is reduced by cysteine, which will disrupt the disulfide bond in microbial proteins. [18] Its bactericidal and bacteriostatic properties are mainly attributed to allicine and thiosulfonate present in garlic, but some sulfur-containing compound decreases the antibacterial activity [19, 20]. It has been reported that garlic has antibacterial, antifungal, antiviral, antihypertensive, blood glucose lowering, antithrombotic, antimutagenic, and antiplatelet properties [21]. Pure garlic extract has greater antimicrobial properties compared to tetracycline against Caecum bacteria. Garlic also has antifungal activity against Candida albicans, which is usually found in the oral cavity [22, 23]. 22.1.1 History and Origin of Garlic The name garlic is derived from the old English word that means a spear-shaped leek. A. sativum is a species from the genus Allium. Garlic is closely related to onion, shallot, leek, chive, and Chinese onion. There are several varieties of garlic, such as “wild garlic”, “crow garlic”, and the “field garlic” found in England. In addition, families of A. ursinum, A. vineale, and A. oleraceum demonstrate the types of garlic. There are about 120 different garlic types from Central Asia, which make their diversity more complicated. A type of wild garlic grows in North America as well [24]. Table 22.1 Scientific classification of garlic [41]. Kingdom Plantae Kingdom Angiosperm Phylum Monocots Order Asparagales Family Liliaceae Subfamily Allioideae Genus Allium Species A. sativum 348 Natural Oral Care in Dental Therapy It has been found that dietary content of 100 g of garlic has the highest percentage of water (59%), followed by carbohydrates (33%), protein (6%), dietary fiber (2%), and a minor content of fat (1%). It contains vitamins like B6 and C, as well as the dietary minerals manganese, iron, calcium, zinc, and phosphorous. Furthermore, garlic has certain B vitamins (B1, B2, B3, B5, and B9). The general health advantages of consuming garlic are therefore self-evident. However, disadvantages have emerged, too. For example, the longterm use of garlic may have side effects such as stomach discomfort or excessive excretion of water from the body through sweating. Furthermore, the overuse of garlic could cause increased bleeding and menstrual irregularities, as well as dizziness, and some people may have an allergic reaction to garlic [25]. It has been found that the anticoagulant properties of garlic in high doses may interact with certain anticoagulant medications, for example, warfarin. Garlic can also negatively interact with anti-hypertensive medications, calcium channel blockers, and the quinolone family of antibiotics (ciprofloxacin) [26]. Garlic has good medicinal properties. Since 2700 BC, garlic was used in China as one of the most useful ayurvedic medicines because of its healing and stimulating effect. Garlic is also used for the treatment of depression because of its stimulating effect. In Indian culture, garlic was used as a folk medicinal remedy for the treatment of skin disease, cough, weakness, rheumatism, hemorrhoids, lack of appetite, and many other conditions. India follows the various holistic concepts, and one of the very important holy books is Vedas. In this book, it is mentioned that garlic is a medicinal plant. Furthermore, the Egyptians were very familiar with so many aromatic, spices, poisons, and medicinal plants. Egyptians were very satisfied with their own medicinal plants (their flora) that were located around the Nile River. Among those, garlic was frequently used for the treatment of many different diseases [27, 28]. Garlic is an irreplaceable nutritional supplement of the Egyptians. They fed their vassal with garlic to make them powerful and able of do more efficient work. It was used to heal wounds, inflammation, and as a part of medicinal remedies in the ancient time. A carve of garlic bulbs have been dated from 3700 BC, while the first appearance of garlic has been found in another crypt from 3200 BC. In Ebers papyrus (around 1500 BC), lots of medicinal plants have been found, and among others, the much recognized garlic, having a good capability to heal injuries. Further on, the ancient Greeks’ garlic was first discovered from 1850 to 1400 BC. The earlier Greek army was used to be fed with garlic before battles. During the Olympic games, the Greek athletes ate garlic to increase the chance of getting a good score [29, 30]. In addition, the ancient Greeks used garlic as a medicinal remedy for the treatment of poor appetite, intestine parasites, anthelmintic, regulating the menstrual cycle, and other sickness. It has been used for snake bites and dog bites, too. Hence, the Greek garlic is known as snake grass [31]. 22.1.2 Medicinal Values of Garlic Garlic has a moderate and pungent odor until it has been peeled. After peeling and crushing, its smell intensifies because of the presence of sulfur glycosides. When it comes in contact with the skin, initially, the heat is felt on the skin followed by a high pain or irritation. If it is rubbed on the skin, the skin will get tanned. In 1892, it was reported that garlic contains various unsaturated aliphatic sulfur compounds. In 1944, the compound allicin was isolated from garlic by Cavallito and Bailey, and later on, it was confirmed that allicin has a powerful antibacterial properties (Figure 22.1) against some gram-negative and gram-positive bacteria. In 1947, the chemical formula of allicin was developed, and in the same year, isolation Allium savitum on Bacterial Oral Infection Alliinase 349 Anti bacterial effect Garlic Figure 22.1 Antibacterial activity of garlic. of alliin was done, which was crystalline in shape and having no smell. Alliin gave the antibacterial effect by an enzyme alliinase from fresh garlic. Research studies have shown that garlic has nutritional properties (Table 22.2) and can be used for the treatment of different diseases. Some important components that are responsible for nutritional and medicinal use of garlic (such as diallyl sulfide, ajoene, alliin, allicin, etc.) are cellulose, etheric oil, organic acids, minerals (Mg, Zn, Se, germanium), water, amino acids, vitamins (C, A, from B complex), lipids, complex of fructosans (carbohydrates), enzymes, steroid saponosides, etc. [32]. In vitro studies have confirmed that garlic contains allicin and other sulfur compounds that have effective antibiotic, antibacterial, and antimycotic properties [33, 34]. It was also found that garlic is beneficial for the treatment of respiratory tract diseases and pulmonary gangrene, and when treated with garlic tincture, the patient recovered within 17 days [35]. Research studies have shown that patients that used garlic were less prone to cold and more resistant against colds when compared to patients on placebos [36, 37]. Garlic contains allicin and other compounds that have hypolipidemic, antihypertensive activity, and hypocholesterolemic activity, and compound of Ajoenes that has an antithrombotic effect [38, 39]. 22.2 Types of Allium sativum Allium sativum (Figure 22.2) has two subtypes: Sativum, or soft-necked garlic and Ophioscorodon, or hard-necked garlic (Ophios for short). The hard-necked garlic is an original type of Allium sativum, and the soft necked is developed by cultivation. These are further classified into the following subtypes: 22.2.1 • • • • • 22.2.2 Allium sativum Ophisocorodon/Hard-Necked Garlic Purple Stripe Glazed Purple Stripe Rocambole Porcelain Marbled Purple Stripe Allium sativum Sativum/Soft-Necked Garlic • Turban • Artichoke 350 Natural Oral Care in Dental Therapy Table 22.2 Chemical composition of adult garlic [42]. Chemical composition Nutritional value per 100 g Carbohydrates 623 Kj Sugar 33.06 g Protein 0.5 g Fat 2.1 g Dietary fiber 1.00 g Beta-carotene 6.39 g Thiamine 5 µg Folate (Vit. B9) 1.235 mg Vitamin C 3 µg Riboflavin (Vit. B2) 2 mg Niacin (Vit. B3) 0.11 mg Pantothenic acid (Vit. B5) 0.7 mg Vitamin B6 0.596 mg Calcium 31.2 mg Iron 181 mg Magnesium 1.7 mg Phosphorus 25 mg Potassium 153 mg Sodium 401 mg Zinc 17 mg Selenium 14.2 µg Manganese 1.672 mg Figure 22.2 Buds/cloves of Allium sativum. Allium savitum on Bacterial Oral Infection 351 • Creole • Asiatic • Silver skin [40] 22.3 Chemical Constituents 22.3.1 Allicin Allicin is a sulfur-containing compound obtained from garlic. Allicin contains the alkane thiosulfinic acid ester functional group, R-S(O)-S-R (Figure 22.3). This is a very important compound responsible for its biological effect. This is formed by the enzymatic activity of alliinase on alliin. Allicin is a heat-sensitive compound, which is easily broken down, especially if heated. Conversely, its breakdown can be slowed down by refrigeration [43]. 22.3.2 Ajoenes Garlic has a medicinal compound (Figure 22.3) that contains allicin and some other compounds. They have, antihypertensive, hypolipidemic, and hypocholesterolemic activity [39], and the Ajoenes have an antithrombotic effect. 22.3.3 Alliin All natural drugs have nutritional properties as well as medicinal properties, which also contain various chemical compounds for the treatment of the different diseases. One of the important compounds of garlic is alliin (Figure 22.3). It is used to reduce bacterial infection, fungal infection, and for oral infection. Alliin gives the antibacterial effect by the presence of enzyme alliinase from fresh garlic [39]. O– S S+ Allicin O NH2 OH S O Alliin O S S S Ajoene Figure 22.3 Chemical structure of allicin, alliin and ajoene 352 Natural Oral Care in Dental Therapy 22.4 Dental Infections and Epidemiology Most of the head and neck space infections are of dental origin. The origin of these infections could be from the tooth and surrounding structures [44]. Dental infections, inter alia, comprise the following: tooth caries, oral mucosa infections, periapical abscesses, periodontal abscesses, and the infection of the head and neck area. To complicate matters, the general dental clinical health of the patient could be relevant, and a localized oral infection could be connected to a systemic infection. Vice versa, systemic infections could be the cause of dental infection. The dental infection could affect the tooth crown, tooth root structures, gingiva, and surrounding structures as seen in Figure 22.4. The complexity of odontogenic infection increases due to the generally accepted scientific fact that odontogenic infection is never caused by only one organism. Therefore, the polymicrobial nature of odontogenic infection complicates the treatment. For example, facultative anaerobes causing dental infection, specifically dentoalveolar abscesses, include Streptococci viridans, Streptococus anginosus, Peptostreptococci, Prevotella, and Fusobacterium species [45]. Dental abscesses are not a modern phenomenon, where microorganisms have been a part of the oral cavity in general. 22.5 Dental Infection and Antibiotic Resistance Penicillin was discovered in 1928 by Sir Alexander Fleming, the first step in the modern era of antibiotics. The first mass production of penicillin started in 1943, and after only 4 years, patients started developing antibiotic-resistant microorganisms. This process has been described as an evolutionary process [46]. The post World War II era has brought the world a variety of new medication, including penicillin, which has cured many infectious diseases and was perceived as a miracle by the people of that time. The infectious control measures Tooth Crown Gingiva Tooth Root Caries Lesion Periodontitis Figure 22.4 The radiographic view of dental caries, periodontis, root structures, and surrounding structures. Allium savitum on Bacterial Oral Infection 353 that used to be applied before antibiotics, such as isolation, quarantine, and the scrupulous application of aseptic technique, were largely discontinued, since antibiotics were curing all bacterial infections. However, the evolution of resistance to antibiotics had already started. When one antibiotic failed, another was prescribed, and so on. However, today, we are faced with a more brutal truth, that most bacteria are becoming resistant to antibiotics. Microorganisms have adapted to antibiotics, and they are evolving. In fact, bacteria are using antibiotics to boost their survival. Infectious disease treatment, previously effectively treated and caused by well-known microorganisms such as Staphylococci, Streptococci, Escherichia coli, and Mycobacterium tuberculosis, has lost the battle with the evolution of microorganisms, and they are now re-emerging as very dangerous diseases [47]. The infectious diseases of today are among the leading causes of patient deaths worldwide. In the European Union, about 25,000 patients die every year from an infection with the selected multidrug-resistant bacteria, and in the United States, every year, more than 63,000 patients die due to hospital-acquired bacterial infections. Some bacteria are resistant to most common antibiotics, and antibiotics are of no use. A further consequence is that normal bacterial flora is changing uncontrollably as well [48]. The genetic plasticity of bacteria has pushed pathogens into adaptation mutation. In Japan, during the 1950s, an epidemic of Shigellosis was induced by Shigella’s antibiotic resistance. Japanese scientists reported a few years later that resistance to “multiple antibiotics not only developed quickly and simultaneously, but also seemed to transfer from resistant to sensitive strains” [49]. The formation of the Antibiotic Resistance Genes Database (ARDB) has confirmed 20,000 possible resistant genes [50]. One microorganism is not the only resistant agent. All categories of microorganisms are resistant to antimicrobials today. The apocalypse of antibiotic resistance of microorganisms is causing a lot of issues with the medical and dental treatment of infection, and has changed fundamentally the antibiotic era’s way of treatment. There is a legitimate concern that we will return to the pre-antibiotic era. The root of the issue is the negligence of the medical and dental fraternity in the early years of antibiotic use, which has brought us to this no-exit situation. It is a well-known fact that dental surgeons in the past used to prescribe a wide range of antibiotics, and frequently. The overprescribing of antibiotics has promoted microbial resistance, hence, the permanent mutation and ongoing evolution of microorganisms. Human oral flora encompasses over 700 microorganisms. Some researchers have identified, with the aid of advanced technology, a rich microbial consortium and around 1000 diverse microbial species, which include bacteria, viruses, and fungi, in the dental biofilm. However, among microbes existing in the oral biofilm, many are natural inhabitants [51]. However, antibiotic overuse and the formation of resistant microorganisms to antibiotics in the treatment of dental infections have already caused severe complications. The appearance of penicillin in the market and clinical use has saved many lives throughout the planet, transformed medical science and its success in treating infectious disease. However, the abuse of antibiotics has taken the world on a different course, where antibiotic-resistant bacteria are increasing the possibility of antibiotic-associated adverse reactions. The surge of resistant bacteria has forced the medical and dental and scientific world to review the protocols of treatment for any infection with antibiotics. Even chronic oral and dental diseases can be very dangerous, especially among immunocompromised patients. Coronary heart disease, strokes, pneumonia, etc., are related to periodontal diseases and tooth loss 354 Natural Oral Care in Dental Therapy that then increases the importance of applying more strict protocols to the use of antibiotics for dental infections. For example, it has been found that endocarditis is linked to chronic oral infections, particularly periodontitis. Furthermore, several studies have shown that cardiovascular diseases such as coronary heart disease, stroke, peripheral vascular disease, cardiomyopathy, atherosclerosis, and myocardial infarction are linked to chronic infection and inflammation. It has been shown that severe chronic microbial dental infections, endocarditis, and meningitis are associated with cerebral infractions among male patients. Therefore, the need for antibacterial agents is obvious. The new protocols are limiting the treatment of infectious diseases with antibiotics but not offering any alternative solution. Even though the incorrect and inappropriate use of conventional antibiotics is contributing to the development of bacterial resistance that can endanger a patient’s health, we are left with only one solution, namely, antibiotics, for treatment of infections [52]. Increasing the need to develop natural therapies for the treatment of pathogenic microbial infection, one such application is the use of garlic against oral diseases. 22.6 The Antibacterial Application of Garlic in Dentistry The increase in bacterial resistance to antibiotics and the continuous struggle to control the spread of infection in the human population has brought the attention of dentistry more closely to herbal medication and more specifically to garlic. 22.6.1 The Use of Garlic to Treat Oral Infections The history of garlic and a closer look at its composition has seen A. sativum as a more favorable antibacterial medicament in dentistry, with specificity to periodontitis, dental caries, endodotitis, children’s endodontitis, candidiases, and even oral cancer. 22.6.1.1 Periodontitis Garlic is a known antimicrobial agent and its effects on oral flora have been studied in vitro and in vivo. A study that examined garlic as a mouthwash using a sample of 30 patients, who used a garlic solution mouthwash for 5 weeks, found inhibition of salivary S. mutans multi-drug-resistant bacteria, which continued for 2 weeks after the end of the mouthwash period. However, the downside of garlic mouthwash is the unpleasant taste and halitosis [53, 54]. Garlic has emerged as a possible option for the treatment of oral bacterial infections, since most oral diseases are due to bacterial infections. The primary content of garlic is sulfide, and this is the reason that garlic is so beneficial for the treatment of the oral bacterial infections. In dentistry, various mouthwashes like chlorhexidine have been used successfully for antibacterial oral treatment. There have been research studies that have tested garlic as an antibacterial mouthwash. In one study, they compared garlic extract and chlorhexidine mouthwash (0.12%), and it was found that they were both effective against salivary microbial populations. However, chlorhexidine mouthwash showed higher efficacy than garlic extract against salivary microbes [55]. The increase in ineffectiveness of antibiotics against microorganisms has brought wider scientific attention to herbal antibacterial treatments, and interest in garlic as an oral antibacterial has Allium savitum on Bacterial Oral Infection 355 grown since there have been no reports of resistance of microbes to garlic. Furthermore, garlic has antifungal and antiviral properties. The application of garlic in oral therapy has shown, in some studies, that garlic is effective against P. gingivalis and A. actinomycetemcomitans and on the proteases of P. gingivalis that are present in periodontitis. The garlic constituent allicin has a major disadvantage in the therapeutic usefulness of garlic extract in that it is unstable and breaks down at 23°C and within 16 h. This disadvantage of allicin could be improved by stabilizing the allicin molecule with the use of a water-based extract of allicin. However, when water-based extract of allicin is used, allicin can react with water to form diallyl sulfide, and that will result in allicin not having the same level of antibacterial effect as the allicin that is not water based. Therefore, patients that have periodontitis have bleeding gingiva, and this can compromise periodontal treatment with antibacterial garlic due to the water content in the blood diminishing allicin’s antimicrobial effects [56]. Another study used garlic extract to test its effects on oral bacteria and periodontal pathogens. The results have shown that garlic extract killed P. gingivalis immediately, and S. mutans was killed with a time delay. Furthermore, this study reported that the garlic extract also inhibited the trypsin-like and total protease activity of P. gingivalis by 92.7% and 94.88% [57]. Garlic has therapeutic value in the treatment of periodontitis and oral infections. Furthermore, another research study demonstrated that daily consumption of garlic will reduce gingival inflammation and gingival bleeding [58]. The search for potential herbal treatment of A. actinomycetemcomitans associated with oral and non-oral infections seems to stop with garlic extract because it significantly inhibits the growth of A. actinomycetemcomitans according to the research study that examined garlic extract against this microorganism [59]. In summary, out of the seven research studies that have been reported on the application of garlic in the treatment of periodontitis, only four studies have directed research to only bacteria that cause periodontitis, while the rest of the studies included oral bacteria, salivary bacteria, and S. mutans depicted in Table 22.3. The need for more research studies is obvious from this summary. Garlic’s potential as an antibacterial treatment for periodontitis should not be neglected. The study reported that garlic extract of (57.1% w/v), containing 220 µg/ml of allicin, inhibits the growth of gram-negative microorganisms, and the minimum bacteriostatic and bactericidal concentration for the gram-negative strains was found to be allicin mean MIC 4.1 µg/ml, MIC range 35.7–1.1 mg/ml, and mean MBC 7.9 µg/ml. These data clearly indicates that garlic extract has a capacity to inhibit the growth of bacteria and some pathogens that are present in the oral cavity, as well as proteases with therapeutic value for periodontitis [60]. Garlic shows antimicrobial activity. The disk diffusion method is used to determine antimicrobial activity; four different concentrations of garlic extract have been used for the evaluation (5%, 10%, 20%, and 100%) against Pseudomonas aeruginosa, Streptococcus mutans, Streptococcus salivarius, Lactobacillus spp, and Streptococcus sanguis. Papers soaked in 0.2% concentration chlorhexidine gluconate and saline were used as negative and positive controls, respectively. The result clearly indicated the zone of inhibition of the different concentrations of garlic extract had similar effect. It might be concluded that using proper concentrations of garlic extract in toothpastes or mouthwashes can be useful in the management of dental caries and periodontitis. The 5% of garlic extract is more useful as an antimicrobial for dental carries compared to the other concentrations of garlic extract [61]. 356 Natural Oral Care in Dental Therapy Table 22.3 Research articles with application of garlic for treatment of periodontitis. Disease/Microorganism Outcome [Ref] Streptococcus mutans Inhibition of organism in saliva [53] Streptococcus mutans Unpleasant taste, halitosis Garlic extract inhibited growth of Streptococcus mutans [54] Salivary microorganisms Both the garlic extract and CHX mouthwash (0.12%) were effective against salivary microbial [55] P. gingivalis, A. actinomycetemcomitans The antimicrobial activity of garlic extract against periodontal pathogens, P. gingivalis, A. actinomycetemcomitans. Its action against P. gingivalis includes inhibition of total protease activity. [56] P. ginigvalis & Streptococcus mutans Garlic inhibited growth of microorganisms. [57] Salivary bacteria Reduced gingival inflammation and bleeding [58] Aggregatibacter actinomycetemcomitans Growth inhibition [59] Various researchers are continuously working on garlic and their use in daily life. According to a patent study, the essential oil of garlic and onion oil are used for smokers for solubilizing and removing tobacco tars. Different concentrations of garlic and onion oil mixture are used for the treatment of teeth, gums, tongue, and other surfaces of the oral cavity as well as the dentures of a smoker [62]. 22.6.1.2 Pediatric Endodontitis A study researching the histopathological effects of garlic oil and formocresol on the pulp tissue of the permanent teeth in pulpotomies in children revealed that teeth treated with A. sativum oil showed inflammatory changes that had been resolved in the end of the study. On the other hand, pulp tissue treated with formocresol eventually produced pulp necrosis, and in some cases, pulp calcification was present [63]. This indicates that garlic offers less aggressive healing potential, leaving the remaining pulp tissue healthy and functioning. Garlic has been recognized as a natural remedy for many pathologic conditions because of its anti bacterial, antimicrobial, and antioxidant effects. In a recent study, garlic is found to be a novel therapeutic agent used to decrease inflammation of recurrent aphthous ulcer, which is a very common ulcer of oral non-keratinized mucosa. Allicin is the important component of garlic, which has an ability to decrease inflammation, pain, and heal ulcer to prevent the recurrence of recurrent aphthous ulcer [64]. The study investigated teething symptoms in infants based on the interviews with mothers of infants—107 mothers were interviewed and reported that various symptoms arise during teething. Furthermore, 91.6% of the mothers reported that teething symptoms arise Allium savitum on Bacterial Oral Infection 357 with different symptoms; 90.7% stated that during teething, diarrhea symptoms arise. One mother reported that she used paracetamol to relieve pain during teething, and 4.7% of the mothers gave their children a pacifier, while a few mothers used home remedies like garlic that they rubbed into the gum of the child. Dentists and health professionals should provide more information about teething problems of infants and their misconceptions [65]. Another comparative study on garlic extract with sodium hypochlorite was performed and evaluated for its possible smear layer removal. The study was carried out on 68 singlerooted mandibular premolars. They were divided into four groups, control (normal saline), NaOCl (5%) with ethylenediaminetetraacetic acid (17%), garlic extract with EDTA, and plane garlic extract. The outcomes of the study revealed that garlic extract as well as sodium chlorite demonstrated a noteworthy use for the removal of the smear layer of root canal [66]. 22.6.1.3 Dental Caries Garlic extract is an effective antibacterial agent against S. mutans when tested both in vitro and in vivo [67]. S. mutans is the main cause of dental caries, and it is of major interest to the oral health struggle against the global pandemic of this disease. Therefore, garlic could be an effective addition in the prolonged fight against resistant bacteria in the treatment of dental infectious diseases. The in vitro study that used ethanolic extract of black garlic on oral microorganism has confirmed good effects on microorganisms involved in oral diseases. Hence, black garlic is a promising candidate for use in oral therapy to control the biofilm associated with oral infections [68]. However, in another in vitro study, it was found that garlic did not have a significant effect on S. mutans bacteria compared to the effects seen using fig and mango [69]. The study that used plant extracts like garlic in crude form suggested that garlic has potential for the control of S. mutans and can be used as an alternative remedy for dental caries prevention in the form of mouthwash [70]. Lemon and garlic extract combination showed the greatest zone of inhibition against S. mutans compared to other combinations like ginger and lemon [71]. Additionally, the findings of the study that did research in children indicate that green tea and garlic with lime mouth rinse could be an economical alternative to fluoride mouthwash for prevention and therapeutics against S. mutans, Lactobacilli species, and Candida albicans [72]. In another study, clove oil was the most effective of all products against microorganisms causing caries followed by ginger– garlic paste and tea tree oil [73]. One research study has reported that garlic could be used as an alternative to chlorhexidine as a disinfectant for toothbrushes [74]. The study that used garlic extract that had a range of concentration between 40% and 70% assessed the inhibitory activity of garlic extracts on oral salivary microorganisms based on colony counts. The results have confirmed a reduction of oral salivary microorganisms in an applied time range of 30 and 60 s for 40% concentrations of garlic extracts in comparison to the control group. The reduction of the exposed colony-forming unit was at 78% and 83.5% and for the 70% concentrations, the reduction of oral salivary microorganisms was at 86.5% and 90.8% at 30 and 60 s. The conclusion of this study was that garlic extract is effective in the reduction of an oral salivary microbial population. Furthermore, this study has proven that new antibacterial treatment options with fewer side effects are available [75]. The decision of what dosage of A. sativum should be used for antibacterial therapy in dentistry is very important. For example, in one study, the broth dilution method revealed that planktonic growth of the cariogenic, gram-positive species S. mutans, S. sobrinus, and Actinomyces oris were 358 Natural Oral Care in Dental Therapy inhibited by an allicin concentration of 600 μg/mL or higher [76]. Therefore, more research studies should be exploring the dosages of A. sativum for use in antibacterial oral application. However, one research study that tested chlorhexidine and garlic against oral microbials have reported that chlorhexidine and garlic only had antimicrobial activity against S. mutans, but no positive results against other oral microorganisms [77]. However, there is a need for much more research studies in vitro and in vivo that has to be done to get closer to the proximity of regulated use of garlic for oral therapy. Garlic has a good capacity in inhibition of oral bacterial growth with a mixture of lime extract. This mixture inhibits the growth of seven bacterial species such as Norcadia asteroids, Veillonella alcaligens, Lactobacillus acidophilus, Pseudomonas aeruginosa, Actinomyces viscosus, Staphylococcus aureus, and Streptococcus mutans. This study used standard technique in vitro studies with carious teeth that were isolated from 240 extracted teeth. Compared with the standard drug of Gentamycin (18.0 ± 0.0)–(23.0 ± 0.5), the mean zone inhibition for mixture of both ranged from (15.0 ± 0.7)–(27.0 ± 1.0); (21.0 ± 1.0)–(27.0 ± 0.7) to (21.0 ± 0.7)–(30.0 ± 0.5), respectively, showing antibacterial activity. MICs for a mixture of both (garlic and lime) were 31.25–62.50 mg mL −1; 1:2 v/v and 15.63–31.25 mg mL −1, respectively, which coincided with the respective MBCs. The result of the investigation suggested that the mixture of both garlic and lime could be used as a mouthwash for mouth sores and sore throat, and it could also be used for the preparation of toothpaste to prevent dental caries [78, 79]. In another study, four different oils such as clove, garlic, neem, and tulsi were used to study the disinfectant effect against Enterococcus faecalis. The study used 50 endodontic K files and evaluated the antiseptic effect of different oils. The overview results of this study concluded that garlic oil has more effective antiseptic effects against Enterococcus faecalis compared to the other oil extracts [80]. 22.6.1.4 Denture Stomatitis Scientific research as early as 1955 has confirmed that C. albicans has became resistant to all available treatments. Additionally, the overuse of antibiotics by dentists has increased the chances of overgrowth of C. albicans [81, 82]. Denture stomatitis is a dental condition caused by C. albicans. Denture stomatitis is classified into three groups: - Petechiae dispersed throughout all or any part of palatal mucosa in contact with the denture as Newton type I. - Macular erythema without hyperplasia as Newton type II. - Diffuse or generalized erythema with papillary hyperplasia as Newton type III [83]. The study that followed-up 11 patients at 1 month and 3 months after delivery of a new prosthesis has confirmed the prevalence of denture stomatitis in 77.5% of patients [84]. A study that compared the antifungal effect of nystatin mouthwash with garlic aqueous extract in 40 patients has found that the erythematous lesions of denture stomatitis improved in patients and recommended that garlic could be regarded as a suitable replacement for nystatin in the treatment of denture stomatitis [85]. Another clinical study has used the essential oil of A. sativum and fluconazole for the treatment of each of 48 denture Allium savitum on Bacterial Oral Infection 359 isolates. Their results have confirmed that the essential oil of A. sativum was effective against all Candida types, but 4.2% of Candida species were resistant to fluconazole. This study has confirmed that the essential oil of A. sativum is an effective agent against all Candida species obtained from dentures. Additionally, an essential oil of A. sativum was effective to both biofilm and planktonic cells in vitro [86]. Patients that suffer from denture stomatitis will have severe signs and symptoms as follows: erythema, edema, and swelling of palatal mucosa. Patients will not be able to eat and that will then affect their digestive system. Therefore, the normal functioning of the patient will be interrupted as long as the treatment of denture stomatitis lasts. The use of garlic for the treatment of denture stomatitis could improve the outcomes of treatment. Garlic is used as a home remedy. Sulfur is a main component in garlic. Various scientists claimed that it is responsible for the treatment of oral and dental problem. Another study states that the oraganosulfur compound has a powerful antibacterial effect. The study demonstrated that garlic oraganosulfur compound, converted into nano-iron sulfide compound, has a capability of inhibiting topical bacterial infection on teeth and is also responsible for faster wound healing [87]. 22.6.1.5 Protection Against Fibrosis A study that examined the efficacy and safety of allicin treatment of stage II oral submucous fibrosis (OSF) reported a decrease in the burning sensation experienced by patients. The study was based on randomized clinical trials using allicin or Triamcinolone acetonide (TA) that was injected into the lesion weekly for 16 weeks. Forty-eight subjects were used, and at 40 weeks, it was found that the mouth opening was 5.16 ± 1.04 mm in the allicin group and 2.27 ± 0.84 mm in the TA group. Furthermore, the burning sensation improved by 4.33 ± 1.04 in the allicin group and by 2.79 ± 0.87 in the TA group. The OHIP14 score increased by 12.58 ± 9.82 in the allicin group and by 4.67 ± 2.94 in the TA group. This study showed a reduction in the burning sensation of the mouth as well as improved opening of the patients’ mouth after administration of Allicin intralesional injections [88]. 22.6.1.6 Garlic Chewing Gum Black garlic has rich antioxidant, vitamins, and nutrients compared to white garlic. It is more effective against heart disease. It has been patented for use in chewing gums. Raw materials are used in different weight ratios such as L-arabinose 8–15 1–7 3.1–2 part by tea polyphenols discretion 2–7 parts soya lecithin 10–20 sorbitol, 12–20 xylitol parts, citric acid, sodium erthorbate 1–4 1–3 40–50 parts gum base, which is used for many purposes like antibacterial, antimicrobial, lower cholesterol, reduce weight, stabilize blood pressure, etc. [89]. 22.6.1.7 Garlic Used as a Breath-Freshening Agent Scientists are frequently researching on most of the medicinal drugs, and one of the important components is garlic. Garlic has a medicinal as well as nutritional value. According to a recent study, garlic is used in breath freshening toothpaste. By weight, garlic extract is 0.005% to 0.05%, and the composition of toothpaste is calcium carbonate, glycerin, sodium carboxymethyl cellulose, sodium silicate, sodium lauryl sulfate, sorbitol, flavors, saccharin, garlic 360 Natural Oral Care in Dental Therapy oil, and deionized water. This invention says that garlic extract is not only used for respiratory and oral cavity drug stabilization but also as bactericidal antidrug toothpaste [90]. Halitosis is a quite unpleasant condition that makes the person uncomfortable. Halitosis is produced by bacteria, which are part of the oral cavity flora. Various natural food are available to overcome oral malodor. Halitosis is not only found in humans, but it is also present in animals. One of the patent studies says that the administration of raw garlic has a capacity to reduce and control oral malodor in pets such as dogs [91]. In a study, five toothbrush disinfectants containing tea tree oil, chlorhexidine gluconate, cetylpyridinium chloride, garlic extract and UV toothbrush-sanitizing device, and garlic were evaluated for its possible inhibitory effect against Streptococcus mutans. The study was carried out on dental students (120 were administered with the test formulations as mentioned above). The treatment continued for 2 weeks, and evaluation of antibacterial effect was determined. The outcomes of the study revealed that garlic extract was more effective over the growth of the bacteria compared to other antimicrobial agents [92]. In another study, three mouthwashes containing chlorhexidine gluconate, triphala, and garlic were evaluated for its possible inhibitory effect against Streptococcus mutans. The study was carried out on children (60), with ages from 9 to 12 years, who were administered with the test formulations as mentioned above. The treatment continued for 15 days and evaluation of inhibition of “plaque buildup” was determined. The outcomes of the study revealed that garlic extract as well as chlorhexidine demonstrated a noteworthy inhibition over the growth of the bacteria [93]. 22.7 Additional Use of Garlic in Dentistry—Oral Cancer The diet plays a significant role in cancer etiology and its prevention; this has been confirmed many times in the modern world. However, garlic has been used for medicinal purposes (Figure 22.5) over 5000 years. It is claimed that even the incidence of cancer can be reduced substantially by means of dietary modification including using garlic as part of the diet. History records the use of garlic in 1550 BC in Egypt as a treatment for tumors. When the Israelites fled Egypt, the Bible mentions garlic as a medicine. Even Hippocrates considered garlic to be a vital part of medicine [94]. The consumption of garlic extracts exerts a protective effect against various types of cancer, including prostate, colon, and oral cancers, this was established by epidemiological studies [95, 96]. The most prevalent type of cancer in the world is oral cancer. This is the sixth leading cause of cancer-related mortality in the United States of America with one death per hour [97]. The search for alternative treatments of oral cancers and use of garlic has been investigated in a few studies. The study that used S-allylcysteine to suppress tumor in mice has reported that oral cancer was significantly suppressed in growth and progression, in the mouse xenograft tumor model of oral cancer. In this study, human oral cancer CAL-27 cells were grown at 37°C in a 5% CO2 incubator. In this study, they used single-cell suspensions with a viability of 90% for the injections into the mouse. During the research period, the mouse was fed with Lab diet 5010 and the diet did not have S-allylcysteine. Additional to the findings of tumor inhibition in the mouse, S-allylcysteine also decreased the osteopontin plasma level in tumor-bearing mice [98]. Allium savitum on Bacterial Oral Infection 361 O OH H CARDIOPROTACTIVE (R) S (S) H2C CANCER NH2 O ANTI-ALLERGIC ANTI-OBESITY ANTI-OXIDANT ANTIFUNGAL Figure 22.5 Pharmacological potential of garlic. The very aggressive oral squamous cell carcinomas that originate from the mucosal surfaces of the oral cavity make out more than 90% of all head and neck cancers in the world [99]. The in vitro research study that applied S-allylcysteine to incubated oral squamous cancer cells at dosage levels of 0, 5, 10, and 20 mM for 24 h has confirmed that S-allylcysteine effectively inhibits the proliferation and malignant progression in human oral squamous cancer cells. Therefore, S-allylcysteine for human oral squamous cancer cells could be a future treatment option [100]. A. sativum could become one of the treatment options for oral cancer treatment. Crude garlic extract is used to reduce the risk of cancer. According to a research, mice were used for a study of cancer and were infected with cancer cells, and another group of mice was treated with garlic extract. The mice which were not given garlic died within 6 days; the other mice which were given garlic extract survived for 6 months [101]. 22.7.1 High Blood Pressure High blood pressure is also known as hypertension. The patients often do not know that they have high blood pressure, and they will die. This is the reason why this condition is called a silent killer. The high blood pressure is a chronic condition with bad effect on the vascular system. Various home remedies that have medicinal properties are available in the market. Among all these remedies, garlic is one of the most effective medicinal remedies for high blood pressure [102]. 362 Natural Oral Care in Dental Therapy 22.7.2 Skin Disorders These days, acne is the most common problem in adult as well as in the elder person. Garlic has properties to reduce pimple spots, and it is helpful in clearing the skin from acnes. Acne is a chronic inflammatory disease of the pilosebaceous follicles involving altered follicular keratinization, androgen-induced sebaceous hyperplasia, hormonal imbalance, hypersensitivity of immune system, and bacteria (Propionibacterium acnes) colonization [103, 104]. Immunomodulator—Garlic has medicinal remedies. It is used as an immunity-boosting agent. Various scientists researched on garlic, and they concluded that garlic has an immunomodulatory effect [105]. 22.7.3 Anti-Allergic Garlic has been used in India since ancient time and all over the world. Garlic is used in many diseases because of the presence of many chemical compounds. One of the important properties of garlic its use as an anti-allergic for the treatment of allergy as well as an anti-oxidant—a substance that has a capability to reduce oxidation and prevent cell damage by free radicals [106]. 22.7.4 Anti-Obesity Obesity is an excess fat accumulated in the human body, and it could have various side effects on health. Obesity is the leading cause of various diseases such as heart attack, high blood pressure, asthma, etc. Garlic is used for reduction of body fat due to its good medicinal properties. Garlic can reduce cardiovascular risk—cardiovascular disease is a condition in which the risk of heart failure is due to blockage of blood vessels, narrowing of passage, and excess pressure on the heart. Garlic has been found to be a good medicinal remedy for the reduction of the risk of cardiovascular disease. A clinical study reported that garlic powder has a capacity to reduce multifunctional cardiovascular risk [107, 108]. 22.8 Garlic Mechanism of Action Garlic is an herb that contains various chemical compounds. Allicin is a compound that has a capability to inhibit bacterial growth (Figure 22.6). In the presence of allinelyase enzyme, allicin converted into diallyldisulfide inhibited the synthesis of DNA and RNA and stopped the growth of bacteria [109]. 22.9 Conclusions and Recommendations In conclusion, garlic has emerged as a possible option for the treatment of oral bacteria. Considering that most of the oral diseases are due to bacterial infections, this could be of great benefit to the patients. The crisis of antibiotic resistance has already forced dental Allium savitum on Bacterial Oral Infection 363 ALLICIN ALLINE-LYASE DIALLYLDISULPHIDE INHIBIT DNA & RNA SYNTHESIS INHIBIT GROWTH OF BACTERIA Figure 22.6 Mechanism of action in antibacterial effect of garlic. research to explore garlic as an option of treatment. A. sativum has been proven by many research studies as an available option for antibacterial treatment of oral diseases. The postulation that garlic is capable of killing oral flora in its complexity is one huge step forward for the treatment of oral diseases, bacterial, fungal, and oral cancer. However, the line between advantages and disadvantages of the use of garlic in dentistry needs to be explored. Additionally, formulation of appropriate dosage and delivery systems for antibacterial garlic application must be confirmed. The implication of garlic being used with any patient’s chronic medications has to be explored. A. sativum interaction with dental treatment medications and local anesthetic has to be investigated. Garlic, as an anticoagulant, and its effect on patient health need to be confirmed. The protocol for preventative and therapeutic application of garlic in dentistry needs to be developed and confirmed (Figure 22.7). Nausea Diarrhea Sweating Burning of the mouth Vomiting Figure 22.7 Side effects of garlic [110]. 364 Natural Oral Care in Dental Therapy Acknowledgments The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work is based on the research supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (Grant No. 98337). The authors sincerely thank Miss A. Crous and Dr. R. Chandran for all the editing support and the University of Johannesburg, Laser Research Centre, for the infrastructure. References 1. 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Nat. Commun. 13, 9, 1, 3713, 2018. 88. Jiang, X., Zhang, Y., Li, F., Zhu, Y., Chen, Y., Yang, S., Sun, G., Allicin as a possible adjunctive therapeutic drug for stage II oral submucous fibrosis a preliminary clinical trial in Chinese cohort. Elsevier December. Int. J. Oral Maxillofac Surg., 44, 12, 1540–1546, 2015. 89. K. Zhang and Z. Zhang, Black garlic chewing gum, CN Patent 102763759A,CN102763759A, 2012. 90. X. Li, Garlic antitoxic bactericidal tooth paste, CN Patent 1555777A, 2003. 91. J. Lewandowski, Method to reduce bad breath in a pet by administering raw garlic, US Patent 5976549A, 1999. 92. Chandrdas, D., Jayakumar, H.L., Chandra, M., Katodia, L., Sreedevi, A., Evaluation of antimicrobial efficacy of garlic, tea tree oil, cetylpyridinium chloride, chlorhexidine, and ultraviolet sanitizing device in the decontamination of toothbrush. Indian J. Dent., 5, 4, 183–189, 2014. 93. Thomas, A., Thakur, S., Mhambrey, S., Comparison of the antimicrobial efficacy of chlorhexidine, sodium fluoride, fluoride with essential oils, alum, green tea, and garlic with lime mouth rinses on cariogenic microbes. J. Int. Soc. Prev. Community Dent., 5, 4, 302–8, 2015. 94. Petrovska, B.B. and Svetlana Cekovska, S., Extracts from the history and medical properties of garlic. Pharmacogn. Rev., 4, 7, 106–110, 2010. 95. Fleischauer, A.T. and Arab, L., Garlic and cancer: A critical review of the epidemiologic literature. J. Nutr., 131, 1032S–1040S, 2001. 96. Hsing, A.W., Chokkalingam, A.P., Gao, Y.T. et al., Allium vegetables and risk of prostate cancer: A population-based study. J. Natl. Cancer Inst., 94, 1648–1651, 2002. 97. Kademani, D., Oral cancer. Mayo Clin. Proc., 82, 7, 878–87, 2007. 98. Vigneswaran, N. and Williams, M.D., Epidemiological Trends in Head and Neck Cancer and Aids in Diagnosis. Oral Maxillofac. Surg. Clin. North Am., 26, 2, 123–141, 2014. 99. Pai, M.H., Kuo, Y.H., Chiang, E.P.I., Tang, F.Y., S-Allylcysteine inhibits tumour progression and the epithelial–mesenchymal transition in a mouse xenograft model of oral cancer. Br. J. Nutr., 108, 28–38, 2012. 100. Tanga, F.Y., Chianga, E.P.I., Chungb, J.G., Hong-Zin Leec, H.Z., Chia-Yun Hsua, C.Y., S-Allylcysteine modulates the expression of E-cadherin and inhibits the malignant progression of human oral cancer. J. Nutr. Biochem., 20, 1013–1020, 2009. 101. Dorant, E., van den Brandt, P.A., Goldbohm, R.A., A prospective cohort study on the relationship between onion and leek consumption, garlic supplement use and the risk of colorectal carcinoma in The Netherlands. Carcinogenesis, 17, 3, 477–484, 1996. 102. Silagy, C.A. and Neil, H.A., A meta-analysis of the effect of garlic on blood pressure. J. Hypertens., 12, 463–468, 1994. Allium savitum on Bacterial Oral Infection 369 103. Williams, H.C., Dellavalle, R.P., Garner, S., Acne vulgaris. Lancet, 379, 9813, 361–72, 2012. 104. Coenye, T., Peeters, E., Nelis, H.J., Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Res. Microbiol., 158, 4, 386–92, 2007. 105. Chandrashekar, P.M., Prashanth, K.V., Venkatesh, Y.P., Isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract. Phytochemistry, 72, 2–3, 255–64, 2011. 106. Ryu, J.H. and Kang, D., Physicochemical Properties, Biological Activity, Health Benefits, and General Limitations of Aged Black Garlic, A Review. Molecules, 1, 22, 6, 2017. 107. Sobenin, I.A., Pryanishnikov, V.V., Kunnova, L.M., Rabinovich, Y.A., Martirosyan, D.M., Orekhov, A.N., The effects of time-released garlic powder tablets on multifunctional cardiovascular risk in patients with coronary artery disease. Lipids Health Dis., 19, 9, 119, 2010. 108. 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Shivakumar Singh1*, Pindi Pavan Kumar2 and Dasaiah Srinivasulu2 1 Department of Botany, Palamuru University, Mahabubnagar, Telangana State, India Department of Microbiology, Palamuru University, Mahabubnagar, Telangana State, India 2 Abstract Mouth evils, an unpleasant breath odor can become a serious problem affecting an individuals’ social communication and self-confidence. Furthermore, it is a discomforting issue for the people around the person affected because they consider it embarrassing to inform the person of the problem. This chapter reports on an ethno botanical survey that was conducted in the landscape of Kosgi Mandal of Naryanapet District, Telangana State, India, where these studies were nonexistent. The information is documented with a respective scientific questioner. At the time of field documentation, the location, season, and plant parts were collected, and the mode of preparation and usage of the medicine were taken via interview with the practitioners. In thi s study, a total of 32 traditional medicinal plants, which have been used in the treatment of mouth evils were documented. The results showed that the 32 plants species belong to 31 genus, and 22 families were occupied. The plant families are represented in their increasing order, i.e., Fabaceae, Euphorbiaceae, Apiaceae, Lythraceae, Lamiaceae, Myrtaceae, and Verbenaceae, and the 15 families correspond to the single species. The results give further ethno pharmacological studies for the discovery of potential new drugs. Keywords: Rural, curative plants, mouth evils, narayanapet, telangana 23.1 Introduction The skill of traditional medicinal treatment is very deep rooted in Indian culture, and the plants are used not only for remedial diseases but also during various ceremonies. Right through man’s history, people have relied on traditional medicine and folkloric plants, in particular, to promote and maintain good health. This dependency of man on plants made him obtain knowledge regarding the medicinal properties of plants by trial and error methods. The World Health Organization (WHO) has predicted that most of the world population is still reliant on traditional medicine for their primary health needs [1]. People living in developing countries rely quite effectively on traditional medicine for primary health care [2, 3]. The art of herbal treatment has very deep roots in Indian culture, and plants are used not only for curing diseases but also during several ceremonies. Nowadays, the world’s *Corresponding author: shivakumarsinghp@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (373–382) © 2020 Scrivener Publishing LLC 373 374 Natural Oral Care in Dental Therapy population follows the ethno medicinal system because of its effectiveness, security, and lesser side effects. Due to the rising national and international demand, medicinal plants are facing permanent exploitation from their natural habitats [4]. Medicinal plants are essential natural resources, which constitute one of the potential sources of new products and bioactive compounds for drug development [5]. Traditional medicinal uses contribute significantly to such drug development. It is estimated that about 60% of the world’s population and 80% of the population of developing countries rely on traditional medicine for their primary health care needs [6]. Nowadays, there is an increasing desire to unravel the role of ethno-botanical studies in trapping the centuries-old traditional folk knowledge as well as in penetrating new plant resources of food, drugs, etc. [7]. India is a warehouse of medicinal plants. At present, about 65% of Indians are dependent on the traditional system of medication [8]. Mouth evils are an extremely universal stipulation that influenced the common people in their everyday life. has gone a long way, i.e., mouth rinses, oral cleanliness, oral vigor, oral illness, mouth evils, dental caries, and periodontal illness [9]. 23.2 Materials and Methods The study area selected was a forest location, rural-indigenous communities, and lack of documentation. There were no previous reports on the medicinal plants used against mouth evils. Overall, there were 12 months in every session. Frequent visits Adilabad Kumuram Bheem Nirmal Mancherial Jagtial Nizamabad Kamareddy Medak Peddapalli Rajanna Sircilla Siddipet Bhadradri Kothagudem Jangaon Sangareddy Medahar Hydraband Yadadri Bhuwanagiri Nagarkurnool Figure 23.1 Study area, Telangana, India. t Nalgonda Jogulamba Gadwal Khammam e ap ry Rangareddy Mahabubnagar Mahabubabad Su Vikarabad Wanaparthy Jayashankar Bhupalpally Karimnagar Curative Plants Worn in the Healing of Mouth Evils 375 were made for the documentation of rural medicinal plants with the help of local practitioners in every season. These types of approach would elucidate the collections, preservations, and identifications. At the time of field documentation, the location, season, and plant parts were collected, and the mode of preparation and usage of the medicine were taken via interview with the practitioners using local flora [10–14]. The serenaded medication with plant samples was deposited at predictable herbarium centers. The voucher numbers allocated for the herbarium samples were deposited. The current chapter approach reveals the Telangana state, Narayanapet Dist., Kosgi Mandal (Figure 23.1). The Mandal area occupies 242.0 sq. km, has a population of 48,299, and a density of 200 (per sq. km). The plant diversity is very rich dealing with a variety of sickness together with mouth evils. Consequently, the current research outcome has given attention to the certification of rural medicinal plants in the healing of mouth evils. 23.3 Results and Discussion The present results reveal that a total of 32 rural therapeutic plants have been documented from the selected study area of Kosgi Mandal, Narayanapet Dist., Telangana State, India. Through the regular interactions of medicinal practitioners and healers in the rural areas, successful collection, preservation, and authentications were made with the help of local flora. The 32 therapeutics species belong to 31 genera covered, whereas 22 families have been occupied with their systematic positions in the botanical studies. Detailed information regarding rural medicinal plants used in treating mouth evils are given in Table 23.1. The name of the family, plant scientific name, plant local name, and part used are carefully documented and authenticated in the presence of a rural medicinal practitioner. After the collection of the specimen plants from the field, the plants were used in the herbarium preparation. The herbarium specimens have been documented, and they were carefully preserved in the Department of Botany, Palamuru University, Mahabubnagar Telangana State. The fraction allocation of a variety habitat was trees 13 sps., shrubs 10 sps., herbs 08 sps., and climbers 1 sps (Figure 23.2). The incidence allotment foundation of curative plants publicized 32 sps. comes under Cultivated, Wild 15 sps. Wild, 09 Cultivated 08 sps (Figure 23.3). The proportion of entity cluster was intended. The past testimonies of Lawsonia Maji Jose et al., Jatropha [15, 16], Hebber et al., [17] Chopra RN et al., [18] Cinnamon [19], and Sonowal [20] supported the in attendance donations. Data were compared with the available previous literature and found that many of the usages listed were not recorded earlier [21]. In the previous scenario, there were no reports on mouth ulcers from the current study area. This is the first and exclusive report. Frequency in distribution sources, percentage in the distribution of growth forms, distribution of therapeutic plants into taxonomic groups, and frequency in the distribution of plant parts of therapeutic plants against mouth ulcers were analyzed. In the present report, Lawsonia inermis L. leaves were used with some ingredients in the preparation of mouth ulcer-treating drugs, but in the previous, Maji Jose et al. used only leaves. In some of the previous reports that were also documented, Jatropha curcas L. latex was used in the treatment of mouth ulcers [22]. The Rajasthan rural Family Plant name Voucher number Parts used Local name Mode of use and suggested medical practitioner Annonaceae Annona squamosa L. HPU-19 F Seetha phalamu Leaf and young flower chew daily morning empty stomach for 4–5 days Papaveraceae Argemone mexicana L. HPU-61 L Pasupu alamu Young root juice is applied on top of sores Miliaceae Azadirachta indica HPU-14 B Vepa Olden bark beneath red bark juice is pertain on mouth evils thrice for 4 days Rutaceae Bergera koenigii L. HPU-51 L Karivepaku One spoon of unsullied bark juice with 1 spoon of honey is occupied two times a day for 3 days Fabaceae Butea monosperma (Lam.) Taub. HPU-50 L Moduga Petiole decoction mouth pulling twice a day Fabaceae Cajanus cajan (L.)Mill. HPU-515 L Kandulu 5 leaves twofold a day for 6 days Ceasalpiniaceae Cassia alata L. HPU-33 L Avisha Decoction of young leaves is worn mouth pulling for 5 days Arecaceae Coccus nucifera L. HPU-12 F Kobbara Dried up coconut squash thrice a day respite as of evils ache Apiaceaet Coriandrum sativum L. L. HPU-22 A Kottimira Gnaw ariel-stem fractions once a day until it is cured Apiaceae Cuminum cyminum L. HPU-74 S Jera 4 spoons of jera drenched in 200 ml of water with crystalloid sugar full of night and intake with drain stomach for 3 days Moraceae Ficus bengalensis L. HPU-80 L Merri Young leaves chomp bestows respite from mouth evils (Continued) 376 Natural Oral Care in Dental Therapy Table 23.1 The medicinal plants used in treating mouth evils. Table 23.1 The medicinal plants used in treating mouth evils. (Continued) Parts used Local name Mode of use and suggested medical practitioner Gymnosporia montana (Roth) Benth HPU-34 L Dantha Leaves chewed once a day for 3 days Euphorbiaceae Jatropha curcas L. HPU-27 Lt Adavi-amudamu Stem latex applied on top of mouth evils Asteraceae Launaea procumbens (Roxb). HPU-48 L Atavika gabi The boiled leaves juice like 10 ml is taken with empty appetite for 3 days Lythraceae Lawsonia inermis L. HPU-94 B Gorintaaku Young leaves of Lawsonia and equivalent sum of black jera were complete hooked on soft glue add crystal sugar combine fit, prepared tiny pills after that acquire every day two times morning with unfilled stomach & night sooner than departing to bed Anacardiaceae Mangifera indica L. HPU-52 S Mamedi Ripen fruit kernel paste is useful to the mouth evils every day 2 times Lamiaceae Mentha viridis L. HPU-10 L Pudina One spoon of dried leaves fine particles with humid water is used in unfilled stomach for 3 days Cucurbitaceae Momordica charantia L. HPU-84 F Kakara 2 spoons of dehydrated ripened produce soft tissue with half spoon of lemon sap assorted in roughly as regards 200 ml of water is in use two times a day for a 5 days Lamiaceae Ocimum sanctum L. HPU-532 L Thulasi Seeds, curcuma has taken equivalent quantity and finished soft paste put in one teaspoon of unsullied honey Plant name Celastraceae 377 (Continued) Curative Plants Worn in the Healing of Mouth Evils Voucher number Family Family Plant name Voucher number Parts used Local name Mode of use and suggested medical practitioner Euphorbiaceae Phyllanthus emblica L. HPU-92 F Nila usiri 1 spoon of young fruit juice & 1 spoon of sugar through 100 ml of water for two times a day are taken for 4 days. Euphorbiaceae Phyllanthus reticulates Poir. HPU--34 B Polichera Olden stalk bark cover masticate heal the mouth evils Fabaceae Pithacalobium dalsi L. HPU-77 F Sima Consume 3 juvenile fruits twice a day, it heals mouth evils without delay Myrtaceae Psidium guajava L. HPU-78 L Jaama Boiled leaf juice is whoosh each morning for a week Lythraceae Punica granatum L. HPU-79 S Danimma 2 teaspoons of young bark powder, smidgen of curcuma dust add to tea cup of cow milk & taken earlier than departure to bed, for 3 days Solanaceae Solanum nigrum L. HPU-83 L Keshobusa The fresh leaf paste, applied on mouth evils two times a day for 2 days Myrtaceae Syzygium aromaticum (L.) HPU-88 F Levangalu Young flower bud juice by honey is applied on evils once a day for 4 days (Continued) 378 Natural Oral Care in Dental Therapy Table 23.1 The medicinal plants used in treating mouth evils. (Continued) Table 23.1 The medicinal plants used in treating mouth evils. (Continued) Plant name Voucher number Parts used Local name Mode of use and suggested medical practitioner Verbenaceae Tectona grandis L. f. HPU-84 L Teeku Leaf base chew up cures mouth thrice a day, until it cures evils Combretaceae Terminalia chebula L. HPU-86 F Karakkye Olden fruit glue is assorted amid honey and applied on evils; it heals in 3 days Fabaceae Vachellia nilotica (L.) P.J.H. Hurter & Mabb. HPU-89 L Krishna thuma Chew up the fresh gum, alleviates mouth evils Verbenaceae Vitex negundo L. HPU-101 B Vaela Chew up 2 juvenile vegetation every day one time for 3 days Zingiberaceae Zingiber officinale Rosce. HPU- 100 R Allamu 1 teaspoon of clean rhizome sap through half tea spoon of honey intakes two times a day Rhamnaceae Zizyphus jujuba Lam. HPU- 60 L Niradu Drag of historic foliage boiled water for 3–4 days treats mouth evils Curative Plants Worn in the Healing of Mouth Evils Family 379 380 Natural Oral Care in Dental Therapy 50 40 30 20 10 0 Herbs erbs Shrubs Trees Climbers Figure 23.2 Fraction allocation of augmentation variety of remedial foliage adjacent to mouth sore. Figure 23.3 Fraction allocation of augmentation habitual of remedial foliage adjacent to mouth sore. people are using Jatropha species in routine oral treatments [23]. Hebber et al. [24] reported that 35 plants belonging to 26 families were used to treat different oral ailments from the Western Ghat region of Dharwad District of Karnataka. Chopra RN et al. [25] reported on the traditional therapeutic plants curing dental caries. Cinnamon has been reported to scientifically reduce the growth of oral microbes [26]. Sonowal Kachari [27] decrypted two therapeutic plants for mouth cleaning from the tribe of Dibrugarh District in Assam [28]. The ascending order-representing families are Fabaceae, Euphorbiaceae, Apiaceae, Lythraceae, Lamiaceae, Myrtaceae, and Verbenaceae. At the same time, 15 families represented single species. At the time of field documentation, the location, season, plant parts collected, the mode of preparation, and usage of the medicine were gathered via interview with the practitioners. The results give tremendous fundamentals for further authentication studies. Curative Plants Worn in the Healing of Mouth Evils 381 23.4 Conclusion Rural medicinal plants are the source for future research. The present chapter reveals rural therapeutic information. Rural medicinal plants, which were used against mouth evils, can be used in the rural area of the selected area. The documented information will be very much useful to upcoming researchers. Acknowledgment The authors are grateful to the rural healer citizens of Kosgi Mandal, Narayanapet Dist., Telangana State, for sharing their insights on counteractive foliages. References 1. Bannerma, R.H., Traditional medicine in modern health care. World Health Forum, 3, 1, 8–13, 1982. 2. Sullivan, K. and Shealy, C.N., Complete Natural Home Remedies, Element Books Limited, Shafts burry, UK, 1997. 3. Singh, J.S., The Biodiversity crisis, A multifaceted review. Curr. Sci., 82, 6, 638, 1997. 4. Badoni, A. and Badoni, K., Ethnomedicinal Heritage, in: Garhwal Himalaya: Nature, Culture and Society, O.P. Kandari and O.P. Gusain (Eds.), p. 242, Transmedia, Srinagar, 1997. 5. Gangwar, K.K., Deepali, Gangwar, R.S., Ethnomedicinal plant diversity in Kumaun Himalaya of Uttarakhand, India. Nat. Sci., 8, 66–78, 2010. 6. Shrestha, P.M. and Dhillion, S.S., Medicinal plant diversity and use in the highlands of Dolakha district, Nepal. J. Ethnopharmacol., 86, 81–96, 2003. 7. Jain, S.K., A Manual of Ethnobotany, Scientific Publication, Jodhpur, India, 1987. 8. Bhatt, D.C., Mitaliya, K.D., Patel, N.K., Ant, H.M., Herbal remedies for renal calculi. Adv. Plant Sci., 15, 1, 1–3, 2002. 9. Sheiham, A., Oral health, general health and quality of life. Bull. World Health Org., 83, 9, 641–720, 2005. 10. Shivakumar, Singh, P., Vidyasagar, G.M., Ethno medicinal plants used in the treatment of skin diseases in Hyderabad Karnataka region, Karnataka India, Asian Pacific Journal of Tropical Biomedicine,11, 882–886, 2012. 11. Pullaiah, T., Flora of Andhra Pradesh, Flora of Telangana., 1, 55–93, 2010. 12. Hooker, J.D., Flora of south India, Flora of British India., 3, 1–7, 1978. 13. Seetharam, Y.N., Kotresh, K., Uplaonkar, S.B., Flora of Gulbarga district, Gulbarga University, Gulbarga, 2010. 14. Rout, S.D., Panda, Mishra, N., Ethno-remidial plants Used to Cure Different Diseases by Tribals of Mayurbhanj District of North Orissa. Ethno-Med., 3, 1, 27–32, 2009. 15. Jose, M., Bhagya, B., Shantaram, M., Ethnomedicinal Herbs Used in Oral Health and Hygiene in Coastal Dakshina Kannada. J. Oral Health Community Dent., 5, 3, 119–123, 2011. 16. Bhasin, V., Oral Health Behaviour Among Bhils of Rajasthan. J. Soc. Sci., 8, 1, 1–5, 2004. 17. Hebbar, S.S., Harsha, V.H., Shripathi, V., Ethnomedicine of Dharwad district in Karnataka India. Plants used in oral health care. J. Ethnopharmacol., 94, 261–66, 2004. 18. Chopra, R.N., Chopra, I.C., Handa, K.L., Chopra’s indigenous drugs of India, 2nd ed., UN Dhur and Sons, Calcutta, 1958. 382 Natural Oral Care in Dental Therapy 19. Saeki Ito, Y. and Okuda, K., Antimicrobial action of natural substances on oral bacteria. Bull. Tokyo Dent. Coll., 30, 129–35, 1989. 20. Ripunjoy, S., Indigenous Knowledge on the Utilization of Remidial plants by the Sonowal Kachari Tribe of Dibrugarh District in Assam, North-East India. Int. Res. J. Biol. Sci., 2, 4, 44–50, 2013. 21. Rout, S.D., Panda, Mishra, N., Ethno-medicinal Plants Used to Cure Different Diseases by Tribals of Mayurbhanj District of North Orissa. EthnoMed., 3, 27–32, 2009. 22. Jose, M., Bhagya, B., Shantaram, M., Ethnomedicinal Herbs Used in Oral Health and Hygiene in Coastal Dakshina Kannada. J. Oral Health Community Dent., 5, 119–123, 2011. 23. Bhasin, V., Oral Health Behaviour among Bhils of Rajasthan. J. Soc. Sci., 8, 1–5, 2004. 24. Hebbar, S.S., Harsha, V.H., Shripathi, V., Hegde, G.R., Ethnomedicine of Dharwad district in Karnataka, India—Plants used in oral health care. J. Ethnopharmacol., 94, 261–266, 2004. 25. Chopra, R.N., Chopra, I.C., Handa, K.L., Chopra’s indigenous drugs of India, 2nd edition, UN Dhur and Sons, Calcutta, 1958. 26. Saeki, Y., Ito, Y., Shibata, M., Sato, Y., Okuda, K., Antimicrobial action of natural substances on oral bacteria. Bull. Tokyo Dent. Coll., 30, 129–135, 1989. 27. Ripunjoy, S., Indigenous Knowledge on the Utilization of Medicinal Plants by the Sonowal Kachari Tribe of Dibrugarh District in Assam, North-East India. Int. Res. J. Biol. Sci., 2, 44–50, 2013. 28. Pradeep Kumar, R., Ethno medicinal plants used for oral health care in India. Int. J. Herb. Med., 2, 81–87, 2014. 24 Ethnopharmacological Applications of Chewing Sticks on Oral Health Care E. A. Akaji 1* and U. Otakhoigbogie2 1 Department of Preventive Dentistry, Faculty of Dentistry, College of Medicine, University of Nigeria, Ituku-Ozalla, Enugu, Nigeria 2 Department of Oral Pathology and Oral Medicine, Faculty of Dentistry, College of Medicine, University of Nigeria, Ituku-Ozalla, Enugu, Nigeria Abstract The role of natural devices such as chewing sticks with a pedigree of good oral hygiene and antimicrobial activities in oral health therapy is apt. Chewing sticks are used worldwide for tooth-cleaning but more prominently in African and Asian communities, and their use dates back to prehistoric times. They are derived from a variety of plants containing bioactive agents with fewer side effects than the conventional antimicrobial agents. Constituents of these sticks such as tannins, anthraquinones, fluorides, salvadorine, resins, silica, trimethylamine, and essential oils reduce plaque formation and bacterial growth in the oral cavity; they are therefore invaluable in circumventing and controlling oral infections. Trimethylamine and salvadorine contents of Salvadora persica inhibit the activities of Steptococcus mutans reducing the development of tooth decay, while extracts from Azadirachta indica and Acacia nilotica demonstrate antimicrobial activity against Streptococcus mutans. SP was also effective against anaerobes implicated in periodontal diseases, and their tannin content reduced clinically visible gingivitis. The sap from Jatropha curcas was very useful in treating oral ulcers, herpes infection aside from candidal infection and toothache. Applications of chewing sticks and/or their products in managing oral infections could compare with established hospital oral care protocol used in modern health care. Keywords: Antimicrobial activity, chewing sticks, salvadora persica, caries, periodontal diseases, oral health 24.1 Introduction 24.1.1 Background The spate of oral infections and their attendant consequences cannot be overemphasized, so there is need for global action to address them using effective and affordable products. Typically, infections are caused by microorganisms so managed by antimicrobial *Corresponding author: ezi.akaji@unn.edu.ng Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (383–392) © 2020 Scrivener Publishing LLC 383 384 Natural Oral Care in Dental Therapy agents, but the indiscreet use of these antimicrobials calls for a revisit of the role of natural products in health care, the basis being that formulations of contemporary drugs used in today’s world are naturally derived [1, 2]. Hence, an understanding of natural devices such as chewing sticks, which in time past fostered good oral hygiene, and are still relevant in the present dispensation, is apt. The support received from the World Health Organization on alternate practices for oral hygiene with special reference to chewing sticks, together with the Consensus Report on Oral Hygiene in 2000 constituted a framework for further researches [3, 4]. Aside from these, chewing stick as an oral hygiene tool properly situates one of the fundamentals of primary health care, that is, “appropriate technology” serving as substitute for modern manual toothbrush in accomplishing the goal of prevention of oral diseases via tooth cleaning especially in resource-poor countries where availability and affordability of toothbrushes pose a challenge [5]. 24.1.2 Historical Perspectives The use of these sticks from plants dates back to prehistoric times by the early Arabs, Babylonian, Greek, and Roman societies. History records that a certain type of chewing stick for oral hygiene was promoted centuries ago by the Prophet Muhammad and upheld by some Muslims till date [6]. Also, toothpicks made from sticks were used to clean the mouth in ancient times, and many communities in developing countries still prefer natural methods of tooth cleaning over the conventional toothbrushes for their oral hygiene [7]. Chewing sticks, though used, in almost all continents of the world, are more common in the Middle East, African, and Asian communities (Table 24.1). 24.1.3 Sources and Types of Chewing Sticks Chewing sticks can be obtained from different types of plants. They include Salvadora, Acacia, Azadirachta, Terminalia, Ficus, Zanthoxylum, Butea, Pterocarpus, Pongamia, and Aegle. The common ones are Salvadora persica (SP), Acacia nicolitica (AN) and Azadirachta indica (AI) [8, 9]. The fresh young stem branches of SP are used to prepare miswak (an Arabic word for tooth cleaning stick), also known as meswak, miswaki, siwak, siwaki depending on the dialect [9, 10]. Miswak is used in most continents of the world, and in places where SP is absent as in West Africa, miswak is obtained from other sources such as citrus trees [3]. Being derived from plants, these sticks contain bioactive agents with fewer side effects than the conventional antimicrobial agents [1]. Table 24.1 summarizes the plant sources of common chewing sticks and their local names where they are found or applied. 24.2 Applications of Chewing Sticks in Oral Health Care 24.2.1 Chewing Sticks for Oral Hygiene The cleaning stick is washed and chewed at one end until it is frayed. The frayed end is then used like a “brush” to clean the teeth, gums, and tongue. The inclination of the bristles to the long axis of the teeth is an advantage for cleaning the surfaces especially the facial surfaces using a two- or five-finger grip [3]. One or two teeth are cleaned at Table 24.1 Some common chewing sticks and their local names. Common name Local name/language Part(s) used/remarks Reference(s) Salvadora persica (Salvadoraceae) Tooth brush tree Tooth brush tree of Orient Persian tooth brush tree’ Miswak/Arabic Datun/India & Pakistan Miswaki/Tanzania Root, stem, twigs or bark Wu et al., 2001 [3] Sagar, 2015 [9] Haque and Alsareii, 2015 [11] Azadirachta indica (Meliaceae) Neem, Nimtree, Magosa tree and Indian Lilac Datun/India* Dogonyaro/Hausa Stem/branches Sagar, 2015 [9] Acacia nilotica (Fabacaea) Indian gum Arabic tree Babul, Babool, Kikar/ Indian Twigs Sagar, 2015 [9] Idi odan; Pako pupa/ Yoruba Roots Flora of West Tropical Africa extending to East Africa Ndukwe et al., 2005 [1] Rotimi and Mosadomi, 1987 [12] Terminalia glaucescens/ (Combretaceae) Fagara zanthoxyloides (Rutacaea) Synonym: Zanthoxylum Zanthoxyloides Senegal prickly-ash Candle wood Orin Ata/Yoruba Roots Widely distributed in African countries Rotimi and Mosadomi, 1987 [12] Distemonanthus benthamianus (Caesalpiniaceae) African or yellow satinwood Orin Ayan/Yoruba Root and stem Widely but sparsely distributed throughout the high forests of West Africa Rotimi and Mosadomi, 1987 [12]; Aderinokun et al., 1999 [13] Massularia accuminata (Rubiacaea) Chewing stick tree Maiden breast tree Pako Ijebu/Yoruba Atu uhie/Igbo Stem Rotimi and Mosadomi, 1987 [12]; Aderinokun et al., 1999 [13] 385 (Continued) Applications of Chewing Stick in Oral Health Plant/source Plant/source Common name Anogeissus leiocarpus (Combretacaea) Local name/language Part(s) used/remarks Reference(s) Pako dudu/Yoruba Root Rotimi and Mosadomi, 1987 [12] Vernonia amygdalina (Asteracaea) Bitter leaf Ewuro/Yoruba Onugbu/Igbo Root and stem Rotimi and Mosadomi, 1987 [12] Garcinia kola (GK) (Clusiacaea) Bitter kola Orogbo/Yoruba Stems and twigs Ndukwe et al., 2005 [1] Gboyin Gboyin/Yoruba Stem and fruit Ndukwe et al., 2005 [1] Botuje, Lapalapa/Yoruba Sap Agbelusi et al., 2007 [14] Cnesti ferugenea (Connaraecaea) Jatropha curcas (Euphorbiaceae) American Purging tree/Big purge tree *Datun of Neem, also referred to as “village pharmacy” in India; Yoruba, Igbo, and Hausa are languages spoken in Nigeria. 386 Natural Oral Care in Dental Therapy Table 24.1 Some common chewing sticks and their local names. (Continued) Applications of Chewing Stick in Oral Health 387 Figure 24.1 Neem sticks and leaves. a time with the chewed bristles moving in perpendicular or parallel direction on the tooth surfaces. Cleaning motion should be directed away from the gum margins but should stimulate the gums and clean the tongue gently but conscientiously. It is advised that cleaning be done at least twice a day, morning and night [1]. An advantage of the use of a chewing stick is the length of time it is used, an average time of 5 to 10 min [1, 15]. Proper use of chewing sticks promote oral cleanliness and removal of plaque, a soft tenacious material, which develops naturally on the tooth surface, primarily implicated in dental and periodontal infections. In addition, the chewing stick from the toothbrush tree (miswak) is uniquely suited for brushing teeth because of the synergistic effect of silica and natural antiseptics found in the sap. Silica has an abrasive potential so help remove stains, cleaning the teeth the same way baking soda or manufactured toothpaste does [9]. Extracts especially those from SP (toothbrush tree) and AI (neem tree) have been used to prepare oral hygiene boosters like toothpaste and toothpowders and various herbal mouthwashes [6, 9]. 24.2.2 24.2.2.1 Ethnopharmacological Applications of Chewing Sticks in Oral Health Dental Caries (Tooth Decay) Caries occurs from demineralization and destruction of the tooth structure through the actions of oral microorganisms, mainly Streptococcus mutans and lactobacilli species. These bacteria act on the fermentable carbohydrates in the mouth to cause a drop in plaque pH 388 Natural Oral Care in Dental Therapy to <5.5, and dissolution of the tooth structures ensues [16]. Constituents of chewing sticks such as fluoride, salvadorine, tannins, anthraquinones, silica, trimethylamine, and essential oils reduce plaque formation and bacterial growth in the oral cavity [8, 9, 11]. Specifically, the trimethylamine and salvadorine contents of SP inhibit the activities of Steptococcus mutans thereby reducing the development of tooth decay. In a compendium culled from different studies, miswak was found very effective against the initiation and propagation of dental caries by various researchers [17]. In addition, an in vitro study using extracts from neem and babool showed that both extracts had antimicrobial activity against Streptococcus mutans, but the activity was significantly higher in aqueous extract of neem [18]. Also, aqueous extracts from Rhus vulgaris and Lantana trifolia, which are common chewing sticks used in Uganda, showed antibacterial activity against S. mutans comparable to benzyl penicillin effects [19]. In addition to silica, chewing sticks such as Garcina kola (GK), Fagara zanthoxyloides (FZ), and SP are rich in fluoride. The anticariogenic actions of fluoride are demonstrated via specific mechanisms: it reduces the solubility of enamel in acid by converting hydroxyapatite into less soluble fluoro-hydroxyapatite/fluorapatite; exerts an influence Table 24.2 Key constituents of chewing sticks and their functions. Constituents Functions Fluoride Gets incorporated into tooth structure and aids caries prevention Tannin Acts largely on the oral mucous membrane; inhibits the action of glucosyltransferase, an enzyme which catalyzes a process in dental plaque formation hence reducing plaque and inflammation of the gums Resins Serves a physical function by forming a layer over the enamel which protects it from microbial action Alkaloids Perform bactericidal action in the mouth Essential (volatile) oils Exhibits anticariogenic action by stimulating salivary flow so cleansing and maintaining optimum pH in the mouth Sulfur compounds Kill bacteria Vitamin C This is an antioxidant, thus, slows down oxidative processes in the body and helps in healing and tissue repair Sodium bicarbonate Has mild abrasive and germicidal properties; could be used as dentifrice Chloride In high concentration, chloride inhibits calculus formation and helps to remove extrinsic stains on the teeth Calcium Supports the process of re-mineralization of the tooth enamel Silica Performs an abrasive function by removing stains and deposits from tooth surface Applications of Chewing Stick in Oral Health 389 directly on dental plaque by reducing the ability of plaque organisms to produce acid, and promotes the remineralization or repair of tooth enamel in areas that have been demineralized by acids. A study on fluoride content of chewing sticks noted that Zanthoxylum zanthoxyloides (synonym of FZ) had a mean concentration of 1845 ppm fluoride, which is slightly higher than the average 1500 ppm fluoride content found in standard dentifrices [20]. The functions of key constituents of chewing sticks [8, 17] that help maintain good oral health are in Table 24.2. 24.2.2.2 Periodontal Disease Periodontal disease is an oral inflammatory disease caused by specific periodontal pathogens inhabiting periodontal pockets and is characterized by destruction of the attachment apparatus of the teeth [21]. It is a chronic reaction of gingival tissues in response to microorganisms associated with dental plaque accumulation; hence, to control periodontal disease, plaque, which is composed mainly of bacteria, needs to be removed. Chewing sticks have antibacterial agents, anti-inflammatory, abrasive qualities, and significantly plaque-inhibiting properties. Miswak, for instance, contains tannins, which reduced clinically visible gingivitis and antibacterial agents against a large spectrum of microorganisms including anaerobes implicated in periodontal diseases [9, 11]. A much older study observed that four species of anaerobic bacteria implicated in periodontitis were susceptible to extracts from nine regular tooth-cleaning sticks. The bactericidal activities against these anaerobes were attributed to the presence of tannins found at the bark of these chewing sticks. The astringent property of tannic acid on the oral mucous membrane helps in its antiplaque and anti-inflammatory activities [12]. Another study demonstrated that the aqueous extracts from most of the 10 cleaning sticks tested, had anti-microbial actions against various microorganisms that cause diseases; specifically, extracts from sticks derived from Vernonia amygdalina (bitter leaf), Massularia accuminata, and FZ showed significant activities against periopathogenic bacteria [22]. Hence, proper use of these chewing sticks could aid in the control of periodontal infections. 24.2.2.3 Oral Candidiasis This is a fungal infection appearing as soft friable and creamy colored plaques on the oral mucosa. Candida albicans is the most commonly implicated organism; however, other candida species like krusei, glabrata, tropicalis, and parapsilosis may be associated [23]. A study was done to determine the microbiological activity of Fagara zanthoxyloides, Anogeissus leiocarpus, and Distemonanthus benthamianus on clinical isolates of candida species; all species investigated were susceptible to extracts from the three tooth-cleaning sticks showing varying but significant inhibition zones [24]. Salvadora persica can also be used to check candidiasis because of its tannin and alkaloid (salvadorine) components, which are active against fungi [11]. Many African and Indian plants used for chewing stick exhibit antifungal (possibly from their sulfur content) and astringent properties, so they can offer great benefits in oral candidal infection 390 Natural Oral Care in Dental Therapy [8, 17]. The viscid sap from Jatropha curcas (JC) was found very useful in treating oral ulcers aside from candidal infection and toothache [14]. It can be postulated that oral lesions such as candidiasis commonly seen in immuno-compromised individuals would be alleviated with chewing stick extracts. 24.2.2.4 Oral Ulcers and Halitosis Ulceration is defined as a break in the continuity of an epithelial lining. It usually presents with pain and discomfort posing some difficulty in eating and drinking. The basic principles of treatment of ulcer are reduction of pain and inflammation, prevention of secondary infections, and reduction in duration and repetition especially in cases like aphthous ulcer [23]. The anti-inflammatory, analgesic, and astringent properties, together with their vitamin C content especially in SP and AI and JC, can soothe the discomfort from oral ulcers and promote healing [8, 9, 14]. An unpleasant odor from the mouth is called halitosis, also called oral malodor or bad breath. The origin can be from the mouth (intra-oral) or from outside (extra-oral). A wide range of bacteria are involved in intra-oral halitosis. Hence, one method to reasonably control bad breath is by the use of antiseptic mouth washes/rinses [25]. Natural antiseptics in Salvadora persica kill the bacteria that cause halitosis and mouth ulcers, just like storebought mouthwash. Astringent and antiseptic properties of neem oil and other essential oils prevent bad breath by destroying implicated microorganisms. Also, neem leaves can be used in mouthwash preparations; although bitter (especially to the new users), the individual adapts to the taste with time [9]. 24.2.2.5 Other Oral Conditions Chewing sticks help mitigate dentine hypersensitivity [26]. The desensitizing action could be due to the astringent properties of chewing stick extracts. Astringents contracts body tissues and canals including dentinal tubules by forming a covering layer over the tubules likened to the action of strontium chloride, thus, reducing the ability of the nerves to transmit pain as a result of the formed layer. Furthermore, there could also be respite from herpes infection, spontaneous bleeding from the mouth [14], sore throat [27, 28], and neoplastic influences [6, 11] through activities of alkaloids (salvadorine), flavonoids, and other antioxidant contents of chewing sticks. Therapeutic efficacy of Salvadora persica in an in vivo and in vitro study in mice illustrated some benefits in the treatment of recurrent oral herpes infections. Replication of Herpes simplex virus type 1 on the mice’s skin was inhibited by SP extracts; this result was attributed to the action of benzylisothiocyanate isolated from SP [29]. 24.3 Conclusions Inferences about the healing power of plant materials such as chewing stick serving primarily as an oral hygiene tool abound in literature; most have been investigated and substantiated scientifically. Chewing sticks materials or products were observed to offer enormous benefits in managing oral infections and other conditions. Their applications could compare Applications of Chewing Stick in Oral Health 391 with those of the established hospital oral care protocol of mouth rinses and drugs used in modern health care. References 1. Ndukwe, K.C., Okeke, I.N., Lamikanra, A., Adesina, S.K., Aboderin, O., Antimicrobial activity of aqueous extracts of selected chewing sticks. J. Contemp. Dent. Pract., 6, 86–94, 2005. 2. Douglas, K., Ethnobotanical Medicinal Plants Used as Chewing Sticks among the Kenyan Communities. BJPR, 13, 1, 1–8, 2016, Article no.BJPR.28833. 3. Wu, D., Darront, A., Skaug, N., Chewing sticks. Timeless natural toothbrushes for oral cleansing. J. Periodont. Res., 36, 275–84, 2001. 4. WHO, Consensus statement on oral hygiene. Int. Dent. J., 50, 139, 2000. 5. Malik, A.S., Shaukat, M.S., Qureshi, A.A., Abdun, R., Comparative Effectiveness of Chewing Stick and Toothbrush: A Randomized Clinical Trial. N. Am. J. Med. Sci., 6, 7, 333–7, Jul, 2014. 6. Niazi, F., Naseem, M., Khurshid, Z., Zafar, M.S., Almas, K., Role of Salvadora persica chewing stick (miswak): A natural toothbrush for holistic oral health. Eur. J. Dent., 10, 2, 301–8, Apr– Jun, 2016. 7. Akaji, E.A. and Uguru, N.P., Traditional Oral Health Practice in a Community in South-Eastern Nigeria. J. Coll. Med., 15, 1, 33–9, June 2010. 8. Hooda, A., Rathee, M., Singh, J., Chewing Sticks In The Era Of Toothbrush: A Review. Internet J. Fam. Pract., 9, 2, 2009. 9. Sagar, S., Role of natural toothbrushes in containing oral microbial flora—A review. Asian J. Pharm. Clin. Res., 8, 4, 29–33, 2015. 10. Sukkarwalla, A., Ali, S.M., Lundberg, P., Tarwin, F., Efficacy of Miswak on Oral Pathogens. Dent. Res. J. (Isfahan), 10, 3, 314–20, May–Jun 2013. 11. Haque, M.A. and Alsareii, S.A., A review of the therapeutic effects of using miswak (Salvadora Persica) on oral health. Saudi Med. J., 36, 5, 530–43, 2015. 12. Rotimi, V.O. and Mosadomi, H.A., The effect of crude extracts of nine African chewing sticks on oral anaerobes. J. Med. Microbiol., 23, 55–60, 1987. 13. Aderinokun, G.A., Lawoyin, J.O., Onyeaso, C.O., Effect of two common Nigerian chewing sticks on gingival health and oral hygiene. Odontostomatol. Trop., 22, 13–8, 1999. 14. Agbelusi, G.A., Odukoya, O.A., Otegbeye, A.F., In vitro Screening of Chewing Stick Extracts and Sap on Oral Pathogens: Immune Compromised Infections. Biotechnology, 6, 97–100, 2007. 15. Ezoddini-Ardakani, M., Shadkam, M.N., Fotouhi, H. et al., Study of the effects of natural toothbrush (Salvadora persica) in prevention of dental caries and plaque index. Health, 4, 09, 612–8, 2012. 16. Touger-Decker, R. and Loveren, C., Sugars and dental caries. Am. J. Clin. Nutr., 78, suppl, 881S– 92S, 2003. Retrieved from https://academic.oup.com/ajcn/article-abstract/78/4/881S/4690063, 12 December 2018. 17. Halawany, H.S., A review on miswak (Salvadora persica) and its effect on various aspects of oral health. Saudi Dent. J., 24, 2, 63–9, April 2012. 18. Sharma, A., Sankhla, B., Parkar, S.M., Hongal, S., Thanveer, K., Ajithkrishnan, C.G., Effect of Traditionally Used Neem and Babool Chewing Stick (Datun) on Streptococcus Mutans: An In-Vitro Study. J. Clin. Diagn. Res., 8, 7, ZC15–ZC17, 2014. 19. Odongo, C.O., Musisi, N.L., Waako, P., Obua, C., Chewing-Stick Practices Using Plants with Anti-Streptococcal Activity in a Ugandan Rural Community. Front. Pharmacol., 2, 13, 2011. 392 Natural Oral Care in Dental Therapy 20. Taiwo, J.O., Oladipupo, M.M., Denloye, O.O., Assessment of fluoride content of selected chewing stick used in Nigeria. Int. J. Public Health Dent., 3, 2, 1–8, 2012. 21. Popova, C., Dosseva-Panova, V., Panov, V., Microbiology of Periodontal Diseases. A Review. Biotechnol. Biotechnol. Equip., 27, 3, 3754–9, 2013. 22. Taiwo, O., Xu, H.X., Lee, S.F., Antibacterial activities of extracts from Nigerian chewing sticks. Phytother. Res., 13, 8, 675–9, 1999. 23. Chestnut, I.G. and Gibson, C., Churchill’s Pocketbook of Clinical Dentistry, 2nd edition, pp. 393– 5, Churchill Livingstone, Edinburgh, 2002. 24. Osho, A. and Adelani, O.A., The Antimicrobial Effect of Some Selected Nigerian Chewing Sticks on Clinical Isolates of Candida Species. J. Microbiol. Res., 2, 1, 1–5, 2012. 25. Akaji, E.A., Folaranmi, N., Ashiwaju, M.O., Halitosis: A Review of literature on Prevalence, Impact and Control. Oral Health Prev. Dent., 12, 4, 297–304, 2014. 26. Odukoya, O.A., Inya-Agha, S.I., Segun, F.I., Agbelusi, G.A., Sofidiya, M.O., Astringency as Antisensitivity Marker of Some Nigerian Chewing Sticks. J. Med. Sci., 7, 121–5, 2007. 27. Agbor, M.A. and Naidoo, S., Ethnomedicinal Plants Used by Traditional Healers to Treat Oral Health Problems in Cameroon. Evid. Based Complement. Alternat. Med., 10 pages, 2015, Article ID 649832, 2015, http://dx.doi.org/10.1155/2015/649832. 28. Idu, M., Umweni, A.A., Odaro, T., Ojelede, L., Ethnobotanical Plants Used for Oral Healthcare Among the Esan Tribe of Edo State, Nigeria. Ethnobot. Leaflets, 13, 548–63, 2009. 29. Taha, M.Y.M., Antiviral Effect of Ethanolic Extract of Salvadora Persica (Siwak) on Herpes Simplex Virus Infection. Al-Rafidain Dent. J., 8, 1, 50–5, 2008. 25 Ethnomedicine and Ethnopharmacology for Dental Diseases in Indochina Viroj Wiwanitkit1,2,3,4 * 1 Dr DY Patil University, Pune, India Faculty of Medicine, University of Nis, Serbia 3 Joseph Ayobabalola University, Nigeria 4 Hainan Medical University, Haikou, China 2 Abstract Ethnomedicine and ethnopharmacology are important alternative and complementary medicine systems for the management of health problems. Using ethnomedicine and ethnopharmacology for dental disease management is very interesting. In an area with a long local history, there are usually many interesting local ethnomedicine and ethnopharmacology practices. Here, the authors discuss the ethnomedicine and ethnopharmacology for dental diseases in Indochina, a tropical region in Asia. In this area, several interesting ethnomedicine and ethnopharmacology practices are observable and considered valuable local wisdoms that are merit for further systematic study. Keywords: Ethnomedicine, ethnopharmacology, dental, disease, Indochina 25.1 Introduction For a medical disorder, there are several factors that can affect the clinical presentation and clinical management. Of several factors, the effect of culture on disease manifestation and management is an interesting issue. The effect of culture on the dental disease presentation is widely mentioned and becomes an interesting issue for further investigation. The culturebounded health practice is usually observed in an area with rooted culture and belief. Indochina, a tropical region in Asia has plenty of local cultures, and some culture-bounded health practices are deeply rooted and widely practiced. Those health practices are aimed at not only medial but also dental problem management. Some practices are proven useful and accepted as a valuable naturopathy approach. Examples are the practiced based on locally derived natural medical products such as herbal plants and medicinal animals. Ethnomedicine and ethnopharmacology are important alternative and complementary medicine systems for the management of health as well as dental problems. Email: wviroj@yahoo.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (393–406) © 2020 Scrivener Publishing LLC 393 394 Natural Oral Care in Dental Therapy Using ethnomedicine and ethnopharmacology for dental health management is very interesting. In an area with a long local history, many interesting local ethnomedicine and ethnopharmacology practices are observable. Here, the authors discuss the ethnomedicine and ethnopharmacology for dental diseases in Indochina, a tropical region in Asia. In this area, several interesting ethnomedicine and ethnopharmacology practices considered valuable local wisdoms that merit further systematic studies. The specific interesting ethnomedicine and ethnopharmacology for dental diseases in Indochina are hereby summarized and presented. 25.2 Ethnomedicine and Ethnopharmacology in Indochina As already mentioned, ethnomedicine and ethnopharmacology are common health manipulation techniques seen worldwide. Both ethnomedicine and ethnopharmacology are considered culture-bounded practices. By definition, ethnomedicine is health manipulation based on locally available traditional medicine techniques practiced by local ethnic groups in each specific setting, and the practice is especially by indigenous peoples [1, 2]. For ethnopharmacology, it is a specific type of pharmacology based on locally available traditional drug regimens according to the local ethnomedicine [1, 2]. Hence, the background of both ethnomedicine and ethnopharmacology is specific to the setting. It might be said that both ethnomedicine and ethnopharmacology are place-specific phenomenon. There is no doubt that the pattern of ethnomedicine and ethnopharmacology might be different in different settings (Figure 25.1). Also, in each setting, the ethnomedicine and ethnopharmacology practice might vary. To understand the specific ethnomedicine and Local culture, belief and religious practice Local naturopathy and wisdom Ethnomedicine and ethnopharmacology Natural products resource such as herb Figure 25.1 Conceptual framework explaining ethnomedicine and ethnopharmacology (graphic drawn by Wiwanitkit, 2018). Ethnomedicine and Ethnopharmacology in Indochina 395 ethnopharmacology systems, the practitioner has to have a good knowledge background on medicine, pharmacology, and sociology. The effect of culture on ethnomedicine and ethnopharmacology in each specific setting is well clarified. As previously mentioned, the format of ethnomedicine and ethnopharmacology in each setting is considered a culture-bounded practice, hence, there must be consideration on the local culture when the practice uses or studies ethnomedicine and ethnopharmacology. Focusing on Indochina, it is a specific area in tropical Southeast Asia. There are many countries in the Indochina area including Thailand, Vietnam, Cambodia, Laos, and Myanmar. This area is one of the old settlements of local people in Asia for many centuries. There are many ancient legends, cultures, and architectures in Indochina. Several valuable local wisdoms are observable in Indochina. The local health wisdoms can also be seen. Good examples are several local traditional medicine systems (Table 25.1). Table 25.1 Some important local traditional medicine systems in Indochina. System Details Thai traditional medicine or Ayurvej Thai traditional medicine or Ayurvej is the specific traditional medicine system seen in Thailand. The system has been continuously practiced in Thailand for many centuries. The system has directly relationship to the Ayurveda in India. The important health manipulations in Ayurvej include Thai herbal regimen, Thai message, Thai herbal spa, and Thai acupressure. Khmer medicine Thai traditional medicine or Ayurvej is the specific traditional medicine system seen in Thailand. The system has been continuously practiced in Thailand for many centuries. The system has directly relationship to the Ayurveda in India. The important health manipulations in Ayurvej include Thai herbal regimen, Thai message, Thai herbal spa, and Thai acupressure. Vietnamese traditional medicine Vietnamese traditional medicine refers to the traditional medicine in Vietnam. The practice is directly adopted from Chinese traditional medicine. The most important health manipulation in Vietnamese traditional medicine is Vietnamese herbal regimen. Dongrek medicine Dongrek medicine is the specific traditional medicine seen at the border area between Thailand and Cambodia. This specific traditional medicine system is the hybrid between Khmer medicine and Thai traditional medicine [3]. 396 Natural Oral Care in Dental Therapy 25.3 Locally Available Naturally Derived Dental Products in Indochina A dental product is an important product. There are many kinds of dental products. In different areas of the world, there are also some specific interesting local dental products that are unique. The locally available naturally derived dental products in Indochina are very interesting. The first common product is the herbal formula toothpaste. In fact, toothpaste is a common dental product that everybody uses in tooth brushing process. Many locally produced toothpastes in Indochina are made from locally available materials including local herbal plants. Good examples are long-term available toothpastes in Thailand and Laos, ) and Dokbuakooor twin lotus (in Thai word namely, Wisesniyom (in Thai word ). Focusing on Wisesniyom, it is still produced in classical powder form toothpaste for direct rubbing at the teeth. Regarding Dokbuakoo, it is more advanced and developed into modern toothpaste cream form. Regarding Dokbuakoo, there is a scientific study on its efficacy. Vajrabhaya et al. performed a comparative study to observe changes in the permeability and morphology of dentine surfaces after brushing with this Thai herbal toothpaste [4]. Vajrabhaya et al. compared Dokbuakoo with a commercial desensitizing toothpaste and found no difference [4]. In addition to toothpaste, there are also locally available herbal mouthwash solutions. In fact, an interesting rooted traditional practice of the local people in Indochina is the use of salted water as mouthwash solution. The locally available mouthwash solutions usually contain salt and local herb as important ingredients. The way that a local naturally derived dental product is produced by the local company is interesting. Many local producers use only a simple technique such as grinding to crush the herbal plants into herbal powder and further use it to produce toothpaste powder. Some producers use the ground local herb powder to mix into the toothpaste crease directly to formulate a modern looked toothpaste dental product. The aim of adding herbal composition is also for attracting the local people who are attached to the concept of naturopathy, using natural product for health care, including dental care. An interesting point is the quality control of the standards of the locally available local herbal dental product. Many products are locally produced and registered as registered local products, but they usually lack medical scientific guarantee for effectiveness and safety. Only a few products passed the standard pharmaceutical study before manufacturing and marketing. At present, the promotion of local herbal product can be seen in some Indochina countries such as Vietnam and Thailand. In Thailand, a specific governmental department for alternative medicine, Thai Department of Alternative Medicine, is set, and there are some specific universities providing degree and performing researches on natural herbal products. Regarding herbal dental product, the well-known standardized certified product in Thailand is produced by a specific local hospital with, namely, Apaipubate brand. An example of an important product is Apaiphubate mouthwash, which is a modernized formulation with fluoride solution and finger root (Boesenbergia rotunda L.) extract. Similarly, there is also another important product, Apaiphubate toothpaste, which is a modernized herbal toothpaste with composition of finger root. Regarding finger use, a scientific study shows Ethnomedicine and Ethnopharmacology in Indochina 397 that this herb has antibactericidal activity, which might be useful for dental health promotion [5]. Comparing the price of those products to the generally available dental products, the price of the herbal dental product is slightly more expensive. Those locally herbal dental products are considered registered local products and already certified by the public health body. The standardization by the laboratory of the Thai Department of Alternative Medicine is available. 25.4 Ethnopharmacology for Dental Diseases in Indochina Ethnopharmacology is deeply rooted in Indochina. The use of ethnopharmacology for the management of dental diseases can be seen in several forms. The important forms will be hereby discussed. A. Herbal regimen The herbal regimen is the common alternative drug commonly used in Indochina. Many herbal products in Indochina are also ethnologically used for dental management. As previously mentioned in this chapter, the famous local herb claimed for the advantage on dental health is finger root. Finger root is used as an important composition in many locally manufactured dental products. The main effect of finger root is the antibactericidal activity, which is useful for the control of oral bacteria. The β-lactam-resistant staphylococci can be well managed by the finger root extract [5]. Another important herb is mangosteen (Garcinia mangostana L.), which is a tropical fruit seen in Indochina. There are some locally available herbal regimens made from mangosteen extracts. The effect of the extract on management of gum disease is mentioned. In a study by Rassameemasmaung et al., the effect of herbal mouthwash with important gradients of the pericarp extract of Garcinia mangostana L on plaque, papillary bleeding index, and halitosis is studied [6]. The effectiveness of the mangosteen extract for the management of gingivitis can be observed [6]. Rassameemasmaung et al. concluded that “Herbal mouthwash containing the pericarp extract of G. mangostana may be used as an adjunct in treating oral malodor [6].” Apart from the herbs that have anti-bacterial activity, some local herbs are also mentioned for its activity against fungus. The lemongrass (Cymbopogon citratus) is another important locally available herb in Indochina. This herb is proven for its fungicidal activity [7]. Amornvit et al. found that lemongrass essential oil could effectively kill Candida albicans [7]. The control of oral thrust or oral candidiasis is possible using lemongrass essential oil. Another interesting herb is Siamese rough bush (Streblus asper) or Koi [8]. This is a locally well-known medicinal plant in Indochina [8]. The lead extract of Siamese rough bush has antibacterial activity [9]. The high antioxidative activity of this herb is also reported [10]. Due to this property, Siamese rough brush has an advantage in the control of dental plaque [11]. Wongkham et al. studied this herb and proposed that the herb could be further developed into an anti-caries drug [10]. Indeed, the bark of this herb is also widely 398 Natural Oral Care in Dental Therapy used by local ethnic people in Indochina for tooth brushing. The tree is also known as tooth brushing tree. B. Animal product regimen Many animals products in Indochina are also ethnologically used for dental management. A good example is the shell of Tegillarca granosa, which is a tropical mollusk. This seashell has been used by the local people for the preparation of toothpaste. According to the local wisdom, the shell is burnt to get the ash for further use as toothpaste. Another important animal product is cuttlebone, which is a kind of marine skeleton. The cuttlebone is mentioned for its usefulness for the promotion of dental strength. It is also classified as an important marine drug composition. Local ethnic in the coastal areas of Thailand, Laos, and Cambodia has used cuttlebone as a composition in their locally produced toothpaste powder for a very long time. Scientifically, cuttlebone is proven useful in hard tissue repair and regeneration; hence, it might be useful for the case of dental erosion or caries [12]. C. Mineral product regimen Many local minerals in Indochina have been long term known to be used by local people for the management of oral health. Maine salt has been used for the preparation of mouthwash by the local people. In the remote landlocked area such as in the northern region of Thailand and Laos, the local ethnic people can use the available dirt salt for preparing a similar mouthwash. Regarding the effectiveness of saltwater solution, Aravinth et al. compared salt water rinse with chlorhexidine against oral microbes and found that “Salt water rinse can be used as adjunct to routine mechanical plaque control for prevention of oral diseases [13].” Hence, the use of salt for the preparation of salt mouthwash is an actual local wisdom of local ethnic people in Indochina for dental health management. Dental organ Animal product Herb Mineral product Figure 25.2 Ethnopharmacology in Indochina (Hand drawing and photograph by Wiwanitkit, 2018). Ethnomedicine and Ethnopharmacology in Indochina 399 Apart from the single herb, there are also many ethnological compound herbal regimens that are mentioned for their usefulness in the management of oral problems in Indochina. For example, “Manee” is a local herbal compound formula that is ethnologically used by the local people for the promotion of good dental health. Considering the formula, Manee consists of several local natural products including Tegillarca granosa shell, Siamese rough bush, Mimusopselengi, clove (Syzygium aromaticum), guava, mint, Pterocarpus santalinus, alum, marine salt, lemon, ebony (Diospyros rhodocalyx Kurz), cuttlebone, and Borneol camphor (Dryobalanops aromatica). The Manee compound is mentioned for its usefulness in the management of several oral problems including apthous ulcer, oral malodor, dental caries, and toothache. If we classify by oral disease, the important ethnopharmacology for each specific group of oral problem will be further detailed. A. Oral malodor As already mentioned, mangosteen is the tropical fruit that is proven for its usefulness in the control of unwanted oral odor [6]. In additional to mangosteen, the local people in Indochina also use guava (Psidium guajava L) extract for the control of mouth smell. Guava has high flavonoids and high vitamin C. Also, there is a report that guava has antibactericidal activity [14]. Shetty et al. recently studied the efficacy of guava extract as an antimicrobial agent on periodontal pathogens and found that the extract was effective against Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans [14]. The control of oral malodor is generally due to the antibactericidal activity of the ethnopharmacological regimen. Nevertheless, an important preventive method against oral malodor is the good dental care by tooth brushing and oral washing. The already mentioned locally available dental products, herbal toothpaste, and mouthwash, are also good ethnopharmacological natural products for the prevention of oral malodor problems. B. Gingivitis Gingivitis is the specific inflammation of the gums, and this is a common problem around the world. As previously mentioned, the mangosteen extract is an effective ethnopharmacological regimen for the management of gingivitis [6]. Another ethnopharmacological regimen against gingivitis is Wildbetal Leaf bush (Piper sarmentosum). This regimen has been widely used in Thailand, Cambodia, and Laos. Nevertheless, there is still no scientific proof on its usefulness in the management of gingivitis. Indeed, there are also other ethnopharmacological regimens that are mentioned for their usefulness in the management of periodontal disease. The important ones are listed in Table 25.2. C. Oral thrush Oral thrush is a common orodental problem. Candidiasis is an important infection causing oral thrush. As already mentioned, lemongrass essential oil is a famous local ethnopharmacological regimen for the management of oral thrush by the local ethnic people in Indochina [7]. 400 Natural Oral Care in Dental Therapy Table 25.2 Some important ethnopharmacological regimens that are mentioned for usefulness in management of periodontal disease. Regimens Details Galanga (Alpinia galanga) This is a single herbal regimen. For usage, local ethnical people will use fresh peeled galanga crushed with salt for direct chewing. Indian gooseberry (Phyllanthus emblica) This is a single herbal regimen. Indian gooseberry is a local tropical fruit in Indochina. For usage, local ethnical people will use the heat water extract of equal amount of Indian gooseberry for drinking in the early morning. White butterfly pea (Clitoria ternatea) This is a single herbal regimen. Butterfly pea is a local tropical flower in Indochina. The ethnic people in northern region of Thailand and Shan state of Myanmar regularly use the alcoholic (liquor) extract of white butterfly pea for management of gingivitis. D. Toothache Barleria cristata is an important herbal regimen that is widely used by the local people in Indochina for the management of toothache. As already mentioned, the traditional alternative medicine system gets a lot of effects from the Indian Ayurveda. A similar use of this Table 25.3 Some important ethnopharmacological regimens that are mentioned for usefulness in management of toothache. Regimens Details Galanga This is a single herbal regimen. For usage, similar way that local ethnical people use for management of gingivitis in is described. Tea Tea is the well-known plant that its leaf is used for preparation of tea beverage Tea drinking in common in Vietnam. The tea is also mentioned for its usefulness against toothache. It is traditionally as ethnopharmacological regimen for relief of toothache among East Asian ethnic [16]. Aloe vera This is a single herbal regimen. Aloe vera is a locally available herb that is well-known for its usefulness in burn wound healing. The aloe vera is also mentioned for its usefulness against toothache. Similar to the use in Brazila in South America [17], the aloe vera regimen is also an important ethnopharmacological regimen for management of toothache. Ethnomedicine and Ethnopharmacology in Indochina 401 herbal regimen as described for the management of toothache in the Indian Ayurveda can be seen in Indochina [15]. Indeed, there are also other ethnopharmacological regimens that are mentioned for their usefulness in the management of toothache. The important ones are listed in Table 25.3. E. Dental caries Some local ethnopharmacological regimens in Indochina are mentioned for their usefulness in the management of caries. Good examples are cuttlebone regimen [12] and Siamese rough bush [10]. Wiwattanarattanabut et al. recently studied several Thai culinary herbs for their anti-cariogenic plaque effects [17]. In that in vitro study, Wiwattanarattanabut et al. evaluated essential oils extracted from sweet basil (Ocimum basilicum), cinnamon bark (Cinnamomum zeylanicum), sweet fennel (Foeniculum vulgare), kaffir lime (Citrus hystrix), black pepper (Piper nigrum), peppermint (Mentha piperita), and spearmint (Mentha spicata) and found that cinnamon and sweet basil essential oils have acceptable in vitro anti-cariogenic bacteria and anti-plaque effects [18]. Joycharat et al. recently studied antibacterial substances from Albizia myriophylla wood, a common ethnopharmacological Thai herb, against cariogenic pathogens and found that the wood is effective in inhibiting the pathogen [19]. Based on the described evidence, there are several local herbs in Indochina that are reported for their activities and usefulness against dental caries. Conclusively, the promotion of tissue strength and the antibacterial activities of the local ethnopharmacological regimens are the main possible mechanisms against dental caries. Further in depth researches on the ethnopharmacological regimens that have potentials against dental rabies are recommended. F. Bleeding gum Bleeding gum is a common orodental problem. Bleeding gum is usually due to vitamin C deficiency. Several local ethnic people in Indochina know and usually use guava, tammarind, and citrus fruits that have high vitamin C content for the management of the problem. Intake of those natural products can help not only in the management of orodental problems but also prevents the problems. The high vitamin C in several locally available ethnopharmacological regimens, which are also tropical citrus fruits, is the main preventive factor against bleeding gums. Nevertheless, the simple use of saltwater solution for regular mouth washing by local people is also proven useful in the prevention of bleeding gums [13]. In some rare cases, bleeding gums might be the result of a system medical problem. For example, in Indochina, where the tropical mosquito-borne infectious disease is common, dengue might be the cause of bleeding gums. The patient with dengue might first present with bleeding gums [20]. The problem is directly due to the thrombocytopenia due to the illness. The use of ethnopharmacological regimen against bleeding gums due to dengue is very interesting. Papaya extract is reported for its usefulness in the management of bleeding gums and other bleeding disorders in cases with dengue [21]. For the pharmacological mechanism, the papaya leaf extract has antithrombocytopenic and immunomodulatory effects that can counteract the immunopathological process that results in thrombocytopenia and bleeding complications, including bleeding gums in dengue [22]. 402 Natural Oral Care in Dental Therapy 25.5 Ethnomedicine for Dental Diseases in Indochina As already mentioned, ethnomedicine practice is not uncommon in Indochina. There are many interesting ethnomedicine practices for dental management. The important ones will be further mentioned. A. Ethnodrug As already mentioned earlier in this chapter, there are many available ethnopharmacological regimens for the management of dental problems in Indochina. There are several available ethnodrugs. Many ethnodrugs are still ritual drugs or locally prepared herbal drugs that still need improvement in their pharmaceutical process. Focusing on standard produced ethnodrug by modern pharmaceutical process, there are some available regimens. In Thailand, a famous regimen named Fahtalayjones is registered and included in the national drug list as a modern herbal regimen that is allowed for use in the modern standard medical center. The regimen is made from the ethno drug regimen from of the herb named Andrographis paniculata. An important indication of Fahtalayjones is for the relief of toothache. B. Ethnodental procedure Ethnodental procedure is an interesting issue in traditional and alternative dentistry. In fact, several dentistry procedures have been performed for many centuries. The local methods for dental extraction by the local ethnic groups are interesting. The use of a local herb for the relief of pain during extraction is common. A good example is the use of aloe vera regimen. It is also reported that aloe vera also helps in promoting healing after dental extraction [23]. In additional to herbal regimen, some traditional alternative medicine practices are also applicable in helping relieve toothache. A good example is the use of acupuncture technique among the Chinese ethnics. Also, the construction of artificial tooth by a local classical technique is also observable in Indochina [24]. The practice is commonly seen among Chinese ethnic Indochina, and the classical tooth insertion service is usually available in China towns. Artificial tooth insertion by the classical technique is still observed at present, and some local people use this technique due to its inexpensive cost. In additional to dental extraction, dental fixation by ethnodental procedure is also observable in Indochina. In fact, due to the historical evidence appearing at the old Cambodian temple, it is evidenced that dental fixation technique might have already existed in the past millennium in Indochina [25]. The picture of orthodontic braces at the ancient temple reflects the long-lasting wisdom on dental procedure among the local people in Indochina [25]. C. Ethnodental cosmetics Ethnodental cosmetics is an interesting issue. The local culture-related style of dengue fashion becomes an important dental variant seen around the world. In Indochina, the fashion on betel nut chewing has ever been deeply rooted. At present, betel nut chewing is still common in Myanmar [26]. The black tooth is the previous dental fashion in Indochina. In the past, several local people in Indochina chewed betel nut aiming to have beautiful black Ethnomedicine and Ethnopharmacology in Indochina 403 Figure 25.3 Golden teeth procedure, an ethnocosmetics in Indochina (Photograph taken by Wiwanitkit, 2018). teeth [27]. In some remote areas of Indochina, some tribers have specific interesting ethnodental cosmetics belief and might perform interesting dental procedures such as cutting teeth aiming at a strange shape. Nevertheless, the specific more common ethnodental cosmetic practice seen in several cities in Indochina is the golden teeth procedure (Figure 25.3). This is a tooth manipulation by covering of the teeth with gold. This ethnodental cosmetic practice is developed from classical artificial tooth insertion procedure. Nevertheless, the additional ethnodental cosmetic procedure such as diamond implantation to the incisor is also observed among some rich ethnic people in Indochina. 25.6 Future Trend of Ethnomedicine and Ethnopharmacology for Dental Diseases in Indochina At present, there are still many ethnomedicine and ethnopharmacology systems for dental diseases in Indochina. Those practices are presently commonly used in several areas in this area. Those practiced are expected to still remain for a very long time in the future. With the merging new trend on the use of natural products and naturopathy, there will be many new researches and developments regarding the locally existing ethnomedicine and ethnopharmacology for dental diseases in Indochina. The development of research technologies and methodologies for better assessment of ethnomedicine and ethnopharmacology will be widely done. New modern products based on the existing ethnomedicine and ethnopharmacology for dental diseases in Indochina are expected to be successfully developed and continuously launched in the near future. With the trend in increased interest on ethnomedicine and ethnopharmacology for dental diseases in Indochina, there is a requirement for good control. The future trend of increased importance of quality control on pharmaceutical processes of ethnopharmacological products is expected. Quality accreditation and standardization are the coming general basic requirements. There will be the setting of a new health agency for specific responsibility regarding ethnomedicine and ethnopharmacology. Finally, the ethical control on the 404 Natural Oral Care in Dental Therapy practice will be another emerging important issue for dental practice relating to the use of ethnomedicine and ethnopharmacology in Indochina. 25.7 Conclusion Many ethnomedicine and ethnopharmacology systems for dental diseases in Indochina are very specific. Those practices can reflect the specific cultural and geographical background in Indochina. The proven advantages of many ethnomedicines and ethnopharmacology for dental diseases in Indochina can confirm the merit of those local health management wisdoms. There is merit in focusing on research and development regarding ethnomedicine and ethnopharmacology for dental diseases in Indochina aiming to find new modern developed formulations that will be effective in the management of the important dental problems. References 1. 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Rassameemasmaung, S., Sirikulsathean, A., Amornchat, C., Hirunrat, K., Rojanapanthu, P., Gritsanapan, W., Effects of herbal mouthwash containing the pericarp extract of Garcinia mangostana L on halitosis, plaque and papillary bleeding index. J. Int. Acad. Periodontol., 9, 19–25, 2007. 7. Amornvit, P., Choonharuangdej, S., Srithavaj, T., Lemongrass-Incorporated Tissue Conditioner Against Candida albicans Culture. J. Clin. Diagn. Res., 8, ZC50–2, 2014. 8. Wongkham, S. and Taweechaisupapong, S., Koi: Medicinal plant for oral hygiene. Thai J. Pharmacol., 27, 137–145, 2005. 9. Rao, D.S., Penmatsa, T., Kumar, A.K., Reddy, M.N., Gautam, N.S., Gautam, N.R., Antibacterial activity of aqueous extracts of Indian chewing sticks on dental plaque: An in vitro study. J. Pharm. Bioallied. Sci., 6, Suppl 1, S140–5, 2013. 10. Wongkham, S., Laupattarakasaem, P., Pienthaweechai, K., Areejitranusorn, P., Wongkham, C., Techanitiswad, T., Antimicrobial activity of Streblus asper leaf extract. Phytother. 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Joycharat, N., Thammavong, S., Limsuwan, S., Homlaead, S., Voravuthikunchai, S.P., Yingyongnarongkul, B.E., Dej-Adisai, S., Subhadhirasakul, S., Antibacterial substances from Albiziamyriophylla wood against cariogenic Streptococcus mutans. Arch. Pharm. Res., 36, 723– 30, 2013. 20. Joob, B. and Wiwanitkit, V., Oral manifestations of dengue viral infection. Braz. J. Otorhinolaryngol., 83, 605, 2017. 21. Wiwanitkit, V., Papaya and dengue. Anc. Sci. Life, 32, 263, 2013. 22. Anjum, V., Arora, P., Ansari, S.H., Najmi, A.K., Ahmad, S., Antithrombocytopenic and immunomodulatory potential of metabolically characterized aqueous extract of Carica papaya leaves. Pharm. Biol., 255, 2043–2056, 2017. 23. Boonyagul, S., Banlunara, W., Sangvanich, P., Thunyakitpisal, P., Effect of acemannan, an extracted polysaccharide from Aloe vera, on BMSCs proliferation, differentiation, extracellular matrix synthesis, mineralization, and bone formation in a tooth extraction model. Odontology, 102, 310–7, 2014. 24. Thanpaisan, K., Artificial teeth. Khon Kaen Med. J., 8, 47–9, 1987. 25. Wiwanitkit, V., Orthodontics braces: It might exist for a very long time—Evidence for ancient Khmer sculpture. Int. J. Prev. Clin. Dent. Res. Innov., 1, 1–2, 2018. 26. Reichart, P.A. and Way, T.H., Oral cancer and pre-cancer in Myanmar: A short review. J. Oral. Pathol. Med., 35, 193–6, 2006. 27. Pinchi, V., Barbieri, P., Pradella, F., Focardi, M., Bartolini, V., Norelli, G.A., Dental Ritual Mutilations and Forensic Odontologist Practice: A Review of the Literature. Acta Stomatol. Croat., 49, 3–13, 2015. 26 Traditional Medicinal Plants Used in Anti-Halitosis P. Shivakumar Singh1*, Pindi Pavan Kumar2 and Dasaiah Srinivasulu2 1 Department of Botany, Palamuru University, Mahabubnagar, Telangana State, India Department of Microbiology, Palamuru University, Mahabubnagar, Telangana State, India 2 Abstract Halitosis is an oral health condition characterized by unpleasant odors emanating constantly from the oral cavity. Almost 22–50% of the population experiences such a condition during lifespan, and about half of them suffer from personal discomfort and social embarrassment. The intrinsic causes of halitosis are both oral cavity and systemic disorder. In the current report, a total of 34 traditional medicinal plants used against halitosis from the Palmuru district of Telangana state, India, were documented. In this study, 34 natural plants species belonging to 29 genera and 19 families were found. The leading families like Anacardiaceae have four species followed by each of the three species from Annonaceae, Arecaceae, Moraceae, Cucurbitaceae, whereas two of each species were found in six families. The remaining nine families showed single species. Previous studies revealed that some of these plants have shown biological activities relating to anti-halitosis effect. The present study revealed the existence of indigenous knowledge of medicinal plants to treat halitosis. Keywords: Traditional medicinal plants, halitosis, oral cavity, Anacardiaceae 26.1 Introduction In the usual manner and in typical circumstances, a mouthful of air is odor-free, and the mouth ought to not have a scent. Normally, obviously, the mouth of a person has a meticulous stink called human smell, i.e., halitosis [1]. Halitosis is an unpleasant or offensive odor emanating from the oral cavity regardless of its origin. It is common in people of all ages with the prevalence of 22% to more than 50% [2–4]. Decomposition of proteins, mucin, and peptides by microbes of the tongue and dental plaque as well as anaerobic gram-negative bacteria can create unstable sulfur composites that are accountable for the awful odor [5]. Halitosis is a major pathologic constituent for periodontal and oral mucosa. There are 400 concerning microorganisms in the mouth [6]. The foul smell that is released from the mouth can be resulting from the oral hollow. *Corresponding author: shivakumarsinghp@gmail.com Durgesh Nandini Chauhan, Prabhu Raj Singh, Kamal Shah, and Nagendra Singh Chauhan (eds.) Natural Oral Care in Dental Therapy, (407–414) © 2020 Scrivener Publishing LLC 407 408 Natural Oral Care in Dental Therapy The reasons of oral malodor are abridged by ultimately the mouth, but the breath smell is not diminished by non-oral causes [7]. Mouth odor amended during the diverse days, level throughout dissimilar period of a day are diverge, and the quantity of mouth odor depends on the stream rate of saliva, food remains, accretion, and production of bacteria and conceivably metabolic alters [8, 9]. The World Health Organization (WHO) in 1978 has estimated that 80% of the populations of developing countries rely on traditional medicines, mostly plant drugs, for their primary health care needs. The use of traditional medicines and medicinal plants in most developing countries as therapeutic agents for the maintenance of good health has been widely observed [10]. In India, 65% of the population relies on ethnomedicine, which is the only source of their primary health care needs. India is one of the 12 mega-biodiversity countries of the world having rich vegetation with a wide variety of plants with medicinal value. Over 550 tribal communities are covered under 227 ethnic groups residing in about 5000 villages in India in different forests and vegetation types [11]. In many countries, scientific investigations of medicinal plants have been initiated because of their contribution to healthcare. It is also necessary to collect information about the knowledge of traditional medicines, preserved in tribal and rural communities of various parts of India before it is lost permanently. Recently, various ethno botanical studies have been reported to expose the knowledge from the various tribals of India. Documenting the indigenous knowledge through ethno botanical studies is important for the conservation of biological resources as well as their sustainable utilization. In such a way, the aim of the present study is to document the ethno medicinal plants and practices followed for oral health and diseases and to suggest that the traditional knowledge should be integrated with modern dental care practices to formulate their sustainable utilization [12]. 26.2 Materials and Methods Customary field trips were commenced in diverse localities of the study area throughout the period from May 2015 to June 2017. Localities were selected in such a way that they would correspond to the entire mandal counting rural areas and tribal pockets. The catalog of the traditional practitioners on the diverse areas was equipped. During the fieldwork, recurrent visits were completed by the herbal practitioners, and they were induced to reveal their traditional information about the curative plants. The in sequence about the foliage was evidenced by means of deliberations and meetings using standard questionnaire [13] with the informers, and the length of the pasture visits during compilation hours. The in sequence was teetered like local names of the plant, parts used, and methods of groundwork. The information concerning the components, added techniques of management or request, quantity and period of prescription, and connected medicated foodstuff were also recorded in a few events. The composed plant specimens were genuinely recognized with the help of flora such as Flora of Telangana Vol. I, II & III [14], Flora of British India, and Flora of Gulbarga District [15], and the voucher numbers were specified, and herbarium specimens were equipped. The people of the study area can talk four languages. The plant assortment is extremely affluent, and a good quality number of medicinal plants are used in the healing of a variety of illnesses including mouth discomforts. Consequently, the current study proved the rural knowledge on medicinal plants used against halitosis. Traditional Medicinal Plants Used in Anti-Halitosis 409 26.3 Results and Discussion The current report reveals the natural plants and their therapy for halitosis, revealing an immense sum of 34 natural plants used in the precise study area of Mahabubnagar district, Telangana state, India. A certification has been approved using a customary questioner. The study showed that 34 natural plants species belonging to 29 genera and 19 families were found valuable. The leading family was the Anacardiaceae with four species followed by each of the three species from Annonaceae, Arecaceae, Moraceae, and Cucurbitaceae, whereas two species were found in six families. The remaining nine families showed single species. The present report explained them thoroughly along with the habit, local name, botanical name, family, and therapeutic property. The medicinal plants used in anti halitosis were predicted (Table 26.1). The habitat, the part used graphically, is represented in figure forms (Figure 26.1, Figure 26.2, Figure 26.3, Figure 26.4). The backdrop of the therapeutic plants is shown in the figures. Oral rinses or mouthwashes are also effective products in preventing mouth bad odor. Mouthwashes, which have alcohol, make the mouth dry and worsen the problem. The plant remedies, such as black and green teas, due to having polyphenols, can reduce the sulfur compounds and decrease oral bacteria [16]. The plants presented in the present chapter are also rich in polyphenols. In most of the cases, polyphenols also performs antimicrobial Table 26.1 Traditional medicinal plants used in anti halitosis. Sl. no Botanical name Family Local name Habit Part used as anti halitosis 24 Semecarpus anacardium Anacardiaceae Nallajeedi Tree Root 25 Sicca acida Euphorbiaceae Rachausiri Tree Leaves 26 Solanum anguivi Solanaceae Mulla vankaya Herb Flower 27 Solanum nigrum Solanaceae Kamanchi Herb Fruit 28 Syzygium cuminii Myrtaceae Neradu Tree Seed powder 29 Tamarindus indica Caesalpiniaceae Chinta Tree Fruit, flower, leaves 30 Terminalia bellerica Combretaceae Tani Tree Bark, fruit 31 Terminalia chebula Combretaceae Karakaya Tree Bark, fruit 32 Vitis heyneana Vitaceae Adavi draksha Climber Leaves 33 Zizyphus mauritiana Rhamnaceae Pedda regu Tree Fruit, seed powder 34 Zizyphus jujuba Rhamnaceae Regu Tree Fruit, seed powder (Continued) 410 Natural Oral Care in Dental Therapy Table 26.1 Traditional medicinal plants used in anti halitosis. Sl. no Botanical name Family Local name Habit Part used as anti halitosis 1 Aegle marmelos Rutaceae Maredu Tree Fruit pulp & Seed 2 Alangium salvifolium Alangiaceae Uduga Tree Ripened fruit & Seed powder 3 Anacardium occidentale Anacardiaceae Jeedimamidi Tree Unripe fruit and root powder 4 Anana sqomosus Bromiliaceae Anasa Shrub Flower 5 Annona muricata Annonaceae Lakshmana phalum Tree Terminal buds 6 Annona reticulata Annonaceae Ramaphalum Tree Seed powder 7 Borassus flabellifer Arecaceae Tati Tree Seed juice 8 Buchanania lanzan Anacardiaceae Sarapappu Tree Leaf juice 9 Cucurbita maxima Cucurbitaceae Gummadi Climber Seeds 10 Coccinia grandis Cucurbitaceae Thonda Climber Unripe fruit 11 Ficus religiosa Moraceae Ravi Tree Terminal buds, bark and fruits 12 Ficus auriculata Moraceae Konda ravi Tree Roots 13 Ficus racemsoa Moraceae Medi Atti Tree Fruits, Bark juice 14 Limonia acidissima Rutaceae Velaga Tree Fruit pulp 15 Mangif era indica Anacardiaceae Mamidi Tree Flower 16 Manilkara hexandra Sapotaceae Palachettu Tree Bark 17 Mimordica dioica Cucurbitaceae Agakara Climber Climbed stem 18 Opuntia dillenii Cactaceae Nagajamudu Shrub Fruit 19 Pavonia odorata Malvaceae Tigabenda Herb Leaves juice 20 Phoenix loureiroi Arecaceae Chittietha Shrub Leaves juice 21 Phoenix sylvastris Arecaceae Etha Tree Flower 22 Phyllanthus emblica Euphorbiaceae Usiri Tree Fruit, bark, root 23 Pithecolobium dulce Mimosaceae Chimachinta Tree Flower Traditional Medicinal Plants Used in Anti-Halitosis 411 Figure 26.1 Study area, Mahabubnagar District, Telangana State, India. Percentage (%) Climbers 8% Herbs 8% Shrubs 8% Trees 76% Figure 26.2 Portion distribution of expansion forms of traditional medicinal plants used in anti halitosis and their habitat. Stem 5% Buds 6% Root 5% Fruit 29% Flower 15% Seed 15% Leaves 17% Bark 8% Percentage (%) Figure 26.3 Portion distribution of part of traditional medicinal plants used in anti halitosis. 412 Natural Oral Care in Dental Therapy Rhamnaceae Combretaceae Myrtaceae Mimosaceae Malvaceae Sapotaceae Cucurbitaceae Annonaceae Anacardiaceae Rutaceae 0 10 20 30 40 50 Figure 26.4 Segment allocation of expansion forms of traditional medicinal plants used in anti halitosis. activities and improved immune activities [17]. Other plants also contain similar types of agents [18], and this might be beneficial in resolving this health problem. These types of plants have a diversity of additional properties [19]. Therapeutic plants can be used for the treatment of infections [20, 21]. In the present report, the documentation work was done, whereas the previous report by Plerenpit Yasin et al. [22, 23] showed an antimicrobial activity, which proved the successive prohibiting of halitosis. In the future, studies presenting the outcome of the reported species should provide the scope to perform the extractions, isolations, characterizations, and antimicrobial activity. The present chapter has given evidences for the common plant usage in halitosis. It is an easy-to-use form and allows access to areas that are difficult to reach. It is practiced after brushing for the hygiene of the teeth, prevention of pathologies, gingivitis therapy, aesthetics, and well-being against halitosis. The ability of the herbal extract, in mouthwashes to reduce gingival inflammation and plaque formation and to be used as an irrigation agent to disinfect the root canal with less toxicity, has been well documented. 26.4 Conclusion Halitosis is a disagreeable odor emitted while some people are talking or inhaling, which can be a symbol of universal oral disorders. This can be controlled using the above-reported medicinal plants and their products. The present chapter demonstrates that plant molecules and extracts could be developed into products, which could be used by the common man to treat oral infections. Whereas the fluoride-based toothpaste and brushes results in Traditional Medicinal Plants Used in Anti-Halitosis 413 damage to the mouth tissue in adults, plant-based toothpaste gives positive results in curing halitosis. Furthermore, the current documented information on the medicinal plants of the Mahabubnagar district people can be used as baseline data for future studies of pharmacologically important medicinal plants and for phytochemical investigations. Acknowledgment The authors are thankful to the rural, folkloric, an ethnic people of the precise study area of Mahabubnagar district of Telangana state for their participation on innate therapeutics. References 1. Bear, D. and Cobb, P., Solitary psychosis. Br. J. Psych., 138, 64–66, 1981. 2. Lindhe, J., Clinical periodontology and implant dentistry, 5th Ed., vol. 24, pp. 512–516, WileyBlackwell, Munksgard, 2003. 3. Yaegaki, K. and Coil, J., Examination, classification and treatment of halitosis clinical perspectives. J. Can. Assoc., 66, 257–261, 2000. 4. Hoshmand, B., Yousefi, R., Khamvrdy, G., Irsha compare the efficacy of two disinfectants and antiplaque chlorhexidine 0/2% on the oral microbial flora. J. Islam. Dent. Dent., 18, 4, 20–60, 2006. 5. Fine, D.H., Furgang, D., Sinatra, K., In vivo antimicrobial effectiveness of an essential oilcontaining mouth rinse 12h after a single use and 14 days’ use. J. Clin. Periodontol., 32, 4, 335–40, 2005. 6. Lang, B. and Filippi, A., Halitosis—Part 1: Epidemiology and pathogenesis. Schweiz. Monatsschr. Zahnmed., 114, 10, 1037–50, 2004. 7. Heshmati, A. and Baharvand, M., Oral malodor (review). Undergraduate Thesis, Dental School, Azad University, Persian, 2000–2001. 8. Lang, N.P. and Lindhe, J., Clinical periodentology and ampler dentistry, 5th edition, pp. 183–202, Blackwell, Iowa (USA), 2008. 9. Bogdoasarian, R.S., Halitosis. Otolaryologic Med. Clin. North Am., 19, 1, 111–7, 1986. 10. Block, P. and Houston, G., Halitosis and fibroma. Ann. Dent., 46, 1, 20–2, b, 1985. 11. Bullinger, J., Disease of the Nose, throat, ear, head and neck, 13th ed., Saunders, Philadelphia, 1985. 12. Cranza, F.A., Glick man’s clinical periodontology, 6th edition, Saunders, Philadelphia, 1984. 13. Shivakumar, Singh, P., Vidyasagar, G.M., Ethno medicinal plants used in the treatment of skin diseases in Hyderabad Karnataka region, Karnataka India, Asian Pacific Journal of Tropical Biomedicine,11, 882–886, 2012. 14. Pullaiah, T., Flora of Telangana State, Astral Publishers, 3, 55–98, 2010. 15. Seetharam, Y.N., Flora of Gulbarga District, GUG Publishers, 2, 76–220, 2000. 16. Katz, H., How to stop and prevent bad breath, therabreath.com/how-to-stopprevent-badbreath, 1, 2–5, 2017. 17. Gupta, A., Shaikh, A.C., Chaphalkar, S.R., Aqueous extract of Calamus rotang as a novel immunoadjuvant enhances both humoral and cell mediated immune response. J. Herbmed Pharmacol., 6, 1, 43–48, 2017. 18. Rafieian-Kopaei, M., Baradaran, A., Rafieian, M., Oxidative stress and the paradoxical effects of antioxidants. J. Res. Med. Sci., 18, 7, 628, 2013. 414 Natural Oral Care in Dental Therapy 19. Heidarian, E. and Rafieian-Kopaei, M., Protective effect of artichoke (Cynara scolymus) leaf extract against lead toxicity in rat. Pharm. Biol., 51, 9, 1104–9, 2013. 20. Zarei, L., Naji-Haddadi, S., Pourjabali, M., Naghdi, N., Tasbih-Forosh, M., Shahsavari, S., Systematic Review of Anti-Rheumatic Medicinal Plants: An Overview of the Effectiveness of Articular Tissues and Joint Pain Associated with Rheumatoid Arthritis. J. Pharm. Sci. Res., 9, 5, 547–551, 2017. 21. Mohsenzadeh, A., Ahmadipour, S., Ahmadipour, S., Asadi-Samani, M., Iran’s medicinal plants effective on fever in children: A review. Der Pharm. Lett., 8, 1, 129–34, 2016. 22. Yasin, P., Wanachantararuk, P., Jidaphatinoi, Dumri, K., Antimicrobial activity of some Thai aromatic plants against oral pathogens inducing Halitosis. Asian J. Pharm. Clin. Res., 12, 1, 465–468, 2019. 23. Pourabbas, R., Delazar, A., Chitsaz, M.T., The effect of German chamomile mouthwash on dental plaque and gingival inflammation. Iran. J. Pharm. Res., 2, 105–109, 2005. Index 3,4,5-trihydroxybenzoic acid, 231 A. actinomycetemcomitans, 244, 252, 253 Acacia arabica, 32, 34, 38 Actinidin, 153, 154, 163 Actions of triphala, 300 Active constituent identified from coconut, 278 Agar well diffusion assay, 83, 87, 88 Alkaloids, 133 Allergy, 165 Allicin, 346–348, 351, 354–357, 359, 362 Alliin, 347, 348, 351 Alliinase, 348, 351 Aloe vera, 32, 33, 37, 62, 63, 65–67, 69, 74, 99, 100, 400 anti-inflammatory activity, 106 anti-inflammatory mechanisms, 100 citotoxicity, 105, 106 for Gingivitis and Periodontitis, 100 other oral applications, 100 Alpha Tocopherol, 62, 72, 73 Amalaki, 298, 299 Analgesic, 316 Analgesic and antipyretic action, 307 Animal, 398 Anti-cancer action, 307 Anti-candida effect, 301 Anti-collagenase action, 301 Anti-diabetic action, 307 Anti-gingivitis, 319, 320 Anti-inflammatory action, 307 Anti-microbial activity, 300 Anti-oxidant action, 300 Anti-plaque, 319, 320, 323 Antibacterial, 111–113, 115, 117, 121, 122, 127, 129, 241, 242, 244, 250, 251, 313–317, 320, 321 Antibiotic resistance, 346, 352, 353, 362 Antibiotics, 154, 156 Cefadroxil, 87, 88, 91 Erythromycine, 87, 88, 91 Tetracycline, 87, 88, 91 Anti-cancer, 156, 317, 321 Anti-candidiasis, 321 Anticaries, 321 Anticariogenic - activity-compound, 83–86, 88, 89, 91, 93, 94 Anti-erosive agent, 241, 242 Antifungal, 314, 318, 322 Anti-inflammatory, 45, 46, 49–54, 144, 145, 148, 236, 241, 252, 314–316, 318, 319 Antimalarial, 315, 318 Antimicrobial, 45–47, 52, 54, 111–113, 115, 117, 121, 122, 127–129, 143, 144, 147, 148, 313, 317–323 Antimicrobial-action -activity, 84, 85, 87, 88, 91, 94 Anti-neoplastic, 315 Antinociceptive, 316 Antioxidant, 48–54, 61, 64–66, 70, 72, 73, 143–145, 317, 319, 321 Antioxidant activity, 231, 236, 239, 244, 252, 253 Antiplaque, 3 Antiplaque effect, 243, 244 Anti-pyorrhea effect, 335 Antipyretic, 314 Antiradical, 315 Anti-thrush effect, 335 Anti-ulcer, 144, 145, 314 Antiviral, 317 Anti-xerostomia effect, 334 Artemisia, 34, 38 Astringency, 230, 236, 241, 244 Attributes of chewing sticks, anti-bacterial, 388, 389 anti-cariogenic, 388 anti-inflammatory, 389, 390 oral hygiene, 383, 384, 387, 390 415 416 Index Avulsed tooth, 165 Ayurveda, 141, 142 Ayurvedic, 61, 62, 69 Ayurvedic medicine, 133 Azadirachta indica, 33–35, 313, 314, 316, 319, 321 Azadirachtin, 313, 315, 317 Baccharis dracunculifolia, 211, 214 Bauhinia, Bauhinia forficata link, 111, 112, 119 Bees, 211, 222 Beta carotene, 61–64, 71 Betamethasone, 66 Bibhitaki, 298, 299 Bioactive compounds, 83, 85, 92 Bioautography, 83, 88, 93 Biocompatibility, 240, 248, 250 Biofilm, dental biofilm, 121, 122, 127–129, 211, 217, 219, 220, 222 Biological properties, antibacterial, 211, 214, 216–218 anticancer, 211, 218, 221 antifungal, 211, 218–220 anti-inflammatory, 211, 214, 220, 221 antimicrobial, 215–217, 221 antioxidant, 211, 215, 221 Biomimetic approach, 245–247, 251 Biomodifier, 242, 245 Black pepper, 68 Bonding, 249 Bowman–Birk inhibitor concentrate, 73 Burning mouth, 146, 149 Calendula, 37 Cancer, 154, 156, 157 Candida, 158 Candida albicans, 133 Candidiasis, 134 Cardamom, Elettaria cardamomum, 121, 122 Caries, 176–178, 216, 222, 401 Cariogenic bacteria - Staphylococus aureus Lactobacillus acidophilus - Streptococcus mutans - Lactobacillus casei -Actinomyces viscosus, 83–85, 88, 91, 92, 94 Cariology, 240, 241 Catechin, 230–232 Cellulose membranes, membrane disk, 121, 125, 126, 128, 129 Chamomile, 35, 36 Charcoal production methods, 199 Chemical classification of natural oral care, 14 alcohol, 15 aldehyde , 15 alkaloids, 14 amino acids, 15 enzymes, 15 flavones, 14 flavonoids, 14 flavonols, 14 glycoside, 15 ketones, 15 lectins, 15 organosulfur compound , 15 phenolic acids, 14 phenols, 14 quinone dervatives, 15 saponins, 14 terpenes, 14 terpenoid, 14 Chemical constituents, 286 Chemical constituents of Ocimum Sanctum, 260–262 Chewing sticks sources and types, acacia nicolitica, 383–385 azadiratcha indica, 383, 385 distemonanthus benthamianus, 385, 389 fagara zanthoxyloides, 385, 388, 389 garcinia kola, 386, 388 jatropha curcas, 386, 389 massularia accuminata, 385, 389 miswak, toothbrush tree, 384, 385, 387 – 389 neem, 385–390 salvadora persica, 383, 385, 387, 389 vernonia amygdalina, 386, 389 Children, 122, 124, 126, 128 Chinese herbs, 62, 73 Chlorhexidine, 300–306 Chlorhexidine, chlorhexidine digluconate, 111, 113–116, 121, 122, 124–126, 128, 217, 220 Chymotrypsin, 157 Cinnamon, 37 Classification of natural oral care, 5 analgesic, 5 antiadhesion activity, 6 antianxiety, 5 Index 417 anti-halitosis, 6 antiplaque, 5, 6 local anesthetic, 5 natural antioxidant, 9 pulpal and dentin repair, 8 root canal irrigation, 7 sealer cements, 9 solvents, 9 storage medium, 8, 9 whitening agent, 5 Clobetasol, 68 Clove, 32, 33 Coagulation, 156 Coconut oil, 32 Colchicine, 62, 66 Collagen, 159, 162 Condensed tannins, 230, 233, 237, 243 Confocal laser scanning microscopy (CSLM), 247 Constituents, astringent, 389, 390 extracts, 387, 389, 390 fluoride, 388, 389 resins, 383, 388 salvadorine, 383, 388–390 sap, 383, 387, 389, 390 silica, 383, 387, 388 sulfur compounds, 388, 389 tannins, 383, 388, 389 vitamin c, 388, 390 CPP-ACP, 242 Cross-linking agent, 229, 237, 241, 248, 250 Curative plants worn in the healing of mouth evils, 373–383 Curcumin, 49, 50, 61, 65, 68, 72, 73, 75 Cysteine proteases, 154, 164, 170 Cytoprotective, 241, 253, 254 Cytotoxic potential, 111, 114, 115, 118 Dalbergia, Dalbergia ecastophyllum, 211, 214 Dalbergia odorifera, 214 Dental abscesses, 352 Dental biofilm, 111, 112, 114–118 Dental caries, 32, 33, 37, 38, 83, 84, 91, 95, 121, 122, 128, 141, 145–148, 345, 346, 352, 354, 356–358 Dental caries, carious lesion, 111, 112 Dental plaque, 33–37, 243, 244 Dental plaque - biofilms, 84 Dentifrice, 163, 164 Dentistry, 3, 211, 221, 222 Desensitization, 236 Designer, 234 Diabetes mellitus, 134 Diterpenoids, 316 Divine tree, 314 Dry bonding, 249 Ecchymosis, 155, 166 Echinacea, 36, 37 Edema, 154, 155, 166 Emblica, 34, 38 Emblica officinalis, 297–299 Endodontic irrigants, 250, 253 Endodontics, 320 Endodontitis, 345, 354, 356 Enterococcus, 158 Enterococus faecalis, 245, 250 Epicatechin, 230–233, 235 Epigallocatechin, 70, 71 Essential oils, 133 Ethano-botanical, 85 Ethanol, 111–114, 116–118, 122, 213–217, 219 Ethnomedicine, 394 Ethnopharmacology, 394 Eucalyptus, 35 Ex vivo, 118, 121, 124 Exemplary potential of coconut water in dentistry, 275 Extract, 112, 122–124, 127, 215–217, 221 Extraction, 154, 155, 165, 166 Extraction, isolation, identification of chemical constituents, 287 Fatty acids, 145, 147 Fibrinolysis, 156 Ficin, 153, 154, 164 Flavonoids, 133, 211, 213–215, 217, 219–221, 229, 230, 239 Formocresol, 356, 359 Galanga, 400 Gallic acids, 230, 231 Gedunin, 315 Gel permeation chromatography, 234, 236 Gingival bleeding, 145, 146 Gingival region, 84 418 Index Gingivitis, 45, 48, 50–53, 55, 98, 141, 147, 148, 163, 164, 299, 301, 303, 304, 306, 319, 323, 399 Glucosyltransferases, 236, 237, 243 Gram-negative, 83, 84, 94 Gram-positive, 83, 94 Grape seed extracts, GSEs, chemical structure, 230 components, 230 methods of separation, 233 types, 231 Grapes, 229, 233–235 Green tea, 32, 34, 38, 48, 49, 62, 70, 71 Gum, 401 Halitosis, 34, 141, 142, 146–149, 164, 185, 186, 204, 354, 355, 360 Haritaki, 298, 299 Healing effect in root canal therapy, 334 Herbal formulation, 85 Herbal medicine, 133 High-performance liquid chromatography, HPLC, 230, 231, 234 History of Ocimum Sanctum, 260 Honey, 54–55 Human gingival fibroblasts, 102 anti-inflammatory activity in a Gingivitis model, 103 authorization, 102 cell culture, 102 cell subculture, 102 citotoxicity test, 103 statical Analysis, 103 HUVEC cells, 254 Hyaluronidase, 64–66, 68, 70 Hydrastis canadensis, 37 Hydrocortisone, 64–66 Hydrogen bonding, 238 Hydrophobic effects, 236 Hydrophobicity, 84 Hypersensitivity, 165 Hypoglycemic, 314, 323 Immunomodulatory action , 308 In vitro, 112, 116, 118, 129 Indochina, 394 Inflammation, 100 Interleukin, 252 Intra-lesional, 62–70 Itraconazole, 135 Lactobacillus, 158 Licorice, 37, 39 Limitations of natural oral care, 16 Local drug delivery, 319 Lozenges, 68 Lycopene, 61, 62, 64, 65, 72, 74 Macleya cordata, 35 Mahmoodin, 315 Malodor, 399 Mangifera indica, 34 Mango, 34, 46, 47, 51 Matrix metalloproteinase, 252 Medicinal plant, 97 Melaleuca alternifolia, 33, 35 Meswak, 32 Microhardness, 160 Microleakage, 159–161 Microorganisms, oral microorganisms, 112, 113, 116, 117 Actinomyces naeslundii, 217 C. glabrata, 219 C. krusei, 219 Campylobacter jejuni, Campylobacter coli, 127 Candida albicans, C. albicans, 122, 127, 219 Enterococcus spp., Enterococcus faecalis, 217 Klebsiella spp., 217 Lactobacillus casei, L. casei, 121, 123, 127, 128 planktonic cells, 112, 118, 129 Saccharomyces cerevisiae, 127 Staphylococcus aureus, 217 Streptococcus mutans, S. mutans, 111–118, 121–123, 127, 128, 216, 217, 220 Streptococcus sanguinis, 216 Streptococcus sobrinus, 217 Miswak, 47 Modulus of elasticity, 249 Molecular recognition, 236 Mouth deodorizing effect, 336 Mouth rinse, 301 Mucositis, 154, 156, 157 Myrrh, 35, 36 NaOCl, 250, 251 Neem, 33–35, 47, 51, 313–323 Nigella, 62, 68 Nimbidin, 313, 314, 316 Nimbolide, 314, 315, 317, 321 Index 419 Novel drug delivery formulations, nanofibers, 264, 265 nanoparticles of biocompatible Ocimum sanctum-coated silver nanoparticles, 264, 265 β-Cyclodextrin Ccomplexes, 264, 265 Ocimum, 62, 68 Oil, coconut oil, 144, 145, 147–149 corn oil, 144 oil pulling, 141–150 palm oil, 144 rice bran oil, 144 sesame Oil, 141, 143, 144, 147–150 soya bean oil, 144 sunflower oil, 141, 143, 144, 147 Oil pulling, 34 Oral antibiofilm, 111 Oral biofilm, actinomyces, 177 aggregatibacter actinomycetemcomitans, 174 candida albicans, 174 lactobacillus, 177 porphyromonas gingivalis, 174 streptococcus mutans, 177 streptococcus sobrinus, 177 tannerella forsythia, 174 treponema denticola, 174 Oral cancer, 3, 45, 71, 72, 154, 222, 254, 356, 360 Oral candidiasis, 183, 184 Oral cavity, 83–85, 94 Oral disease, 83, 85, 91 Oral fibroblasts, 111, 117, 118 Oral flora, 84 Oral health, 141, 146 Oral hygiene, 84, 141, 149 Oral malodor, 141, 149 Oral microbiome, 172–175 Oral thrush, 149 Organic solvents, 83 Orthognathic surgery, 155, 166 Osteoarthritis, 157 OTC dental care products, 198 Other significance of coconut, 276 commercial usage of husk fibers, 278 convention of coconut roots, 278 economic importance of coconut stem, 278 economic value of coconut leaves, 276 importance of coconut shell, 277 potential of coconut fruits, 277 significance of spathe and inflorescence, 277 use of coconut heart, 277 usage of coconut milk, 277 PAHM, 233 PALM, 233 Panaxnoto, 67 Pediatric dentistry, 121, 124, 126, 129 Pellicle, 164 Pentoxyfilline, 67 Peppermint oil, 36 Peri-implantitis, 253 Periodontal, 319, 320 Periodontal disease, 84, 181–183, 301 Periodontitis, 45, 46, 48–52, 54, 55, 98, 99, 319, 323, 345, 346, 353–356 Pharmacological properties of coconut, 273 Pharmacology—therapeutic activity of Salvadora persica L., 287–292 antibacterial and antifungal, 290 anti-cariogenic effect, 290 antiperiodontitis effect, 291 antiplaque effect, 290 anti-viral effect, 290 theories for Miswak activities, 287 whitening effect, 292 Phenolic, 211, 215–217, 219–221 Phytochemical, 10–15 Phytochemical constituents, 93 Phytotherapy, 211 Pictacia vera, 39 Pinot Gris, 234 Pinot Noir, 234, 235 Piper, 65, 68, 69 Piper betle, 39 Plant, 112, 116, 211, 213, 214 Plaque, 50–53, 55, 84, 91, 300, 301, 303–306, 319, 320 PLGA, 247 PMN, 315 Polyherbal, 62, 68 Polyphenols, 66, 70 biochemical properties, 236 cultivar, 234 factors, 233, 234 methods of separation, 233 physical properties, 235 420 Index Pomegranate, 32 Populus, 38 Prednisolone, 67 Premalignant, 61–63, 70, 73 Preventive dentistry, 320, 321 Proanthocyanidins, 229, 230, 245, 246, 250 Probiotics, 175 dairy products, 180 lactobacillus, 174 mechanisms of action, 179, 180 mouthwashes, 180 powder, 180, 181 tablets and lozenges, 181 Propolis, 211–223 Proteases, 153, 154, 163, 164 Protection against aphthous stomatitis, 338 Protein-polyphenol interactions, 236–238 Prunella vulgaris, 35 Punica granatum, 134 Punicalagin, 135 Quality of life, 154, 165, 166 Quercus infecteria, 83–87, 91–94 Radix salvia, 67 Redox reactions, 240 Replantation, 165 Role of coconut tree in dental ministrations, 274 Role of triphala in wound healing, 307 Root canal, 320, 323 Root canal irrigant, 300 S mutans, 299, 300 Safety of natural oral care, 15 Sage, 35, 36 Saliva, 84, 121, 122, 124, 126, 128 S-allycysteine, 361 Salvadora persica, 32, 33, 36, 38 Salvia miltiorrhiza, 67 Salvianolic acid, 67 Sanguinaria canadensis, 37 Saponification, 143, 145, 147 Sepsis, 156, 166 Sesame, 48, 52 Smear layer, 160, 162 Sodium hypochlorite, 85, 251 Sodium lauryl sulfate, 122 Spearmint oil, 37 Spirochetes, 83 Spirulina, 62, 65–67 Staphylococcus, 156 Stomatitis, 345, 358 Streptococcus, 158 Streptococcus mutans, 236, 237, 243, 245 Tannins, 133, 315 Tannins, quinones, 298 Tea, 400 Tea pigments, 62, 66 Tea tree, 33, 35 Teeth, 111, 112, 216, 222 Termibalia, 34, 38 Terminalia bellerica, 297–299 Terminalia chebula, 297–299 Therapeutic significance of ocimum in dental health and preventive care management, 262–264 Therapy, 400 Thin layer chromatography (TLC), 87, 88, 92, 93 Thrush, 399 TMJ/temporomandibular joint, 157 Tooth polishing, 336 Toothache, 84 Toothpaste, 164 Traditional medicinal plants used in antihalitosis, 407–415 Traditional usage and ethnopharmacological relevance, 272 Trend, 403 Triamcinolone, 66, 67, 74, 75 Triphala formulations, 308 Triphala phytoconstituents, 298, 299 Trismus, 154, 155, 166 Trolox equivalent antioxidant activity (TEAC), 239 Trypsin, 156, 157, 163 Tulsi, 51, 52, 61–63, 68 Turmeric, 46, 49, 50, 61–63, 68, 69, 74, 100, 101 for gingivitis and periodontitis, 101 other oral applications, 101 Ulcerative gingivitis, 84 Utimate tensile strength (UTS), 246 Index 421 Vitamin A, 71, 72 Vitamin C, 72 Vitamin E, 72 Vitis vinifera, 229, 230 Wound debridement, 155 Wound healing, 318 Wet bonding, 249, 250 Whatman filter paper, 85 Zingiberaceae, 121, 122, 127 Xerostomia, 145, 147