Anti-Streptococcus mutans property of Uvaria chamae, and its anticariogenecity Madiba Mukonazwothe Degree of Master of Science in Medicine by research only Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of the Master of Science in Medicine Johannesburg, 2022 https://www.google.com/url?sa=i&url=http://www.bdssouthafrica.com/academic-boycott/wits-right-to-protest/wits-university-workers-issue-ultimatum-to-new-vice-chancellor-professor-adam-habib/&psig=AOvVaw13Bhh4AEyUsRbXOYOzXWhd&ust=1607759665298000&source=images&cd=vfe&ved=0CAIQjRxqFwoTCKj_r9G5xe0CFQAAAAAdAAAAABAR i DECLARATION I, Madiba Mukonazwothe declare that this dissertation is my own. It is being submitted for the Degree of Master of Science in Medicine at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at any other University and has been complemented by referenced works duly acknowledged. I further declare that this dissertation has the approval by The Human Research Ethics Committee, University of The Witwatersrand. Ethical waiver (W-CBP-200529-03) 12/04/2022 Signature of candidate Date m.madiba ii DEDICATION I dedicate this dissertation to my family. Thank you for investing in my future. Your words of encouragement will forever be remembered. iii PUBLICATIONS AND PRESENTATIONS Poster presentation Anti-Streptococcus mutans property of Uvaria chamae, and its anticariogenicity. The International Association for Dental Research (IADR) South African Division, 51 st Scientific meeting, Cape town (online), 20-21 October 2021 Oral presentation Anti-Streptococcus mutans property of Uvaria chamae, and its anticariogenicity. The International Association for Dental Research (IADR), South African Division, 51 st Scientific meeting, Cape town (online), 20-21 October 2021 iv ACKNOWLEDGMENT This dissertation is a result of the combined efforts of many people and institutions who made invaluable contributions to make sure it was a success. I wish to express my sincere gratitude to my supervisors Prof. M Patel and Dr. ZM Gulube. Thank you for your commitment in guiding me through the journey of completing this dissertation. I will forever be grateful for your constant supervision, constructive criticism, expert guidance, and enthusiastic encouragement to pursue new ideas and never-ending inspiration. I would also like to express my gratitude to Dr. MP Ngoepe for assistance with the statistical analysis of the results and Dr. MD Marimani for assistance with the RT-qPCR experiment. To Sibongile Nciki, thank you for all the assistance and support provided during my study. I would also like to acknowledge my colleagues in the Department of Oral Biological Sciences and Clinical Microbiology and Infectious Diseases for the fruitful atmosphere you accorded me with. I would also like to appreciate my sponsor from the Council of Scientific and Industrial Research (CSIR) for financial support. v ABSTRACT Introduction Dental caries is the most prevalent oral infection. Streptococcus mutans is a major cause of dental caries in humans. These bacteria form biofilms and produce acids and extracellular polysaccharides which contribute to the development of dental caries. Many oral hygiene products containing antimicrobial chemicals have been used to prevent dental caries. In recent years, medicinal plants have been researched for their beneficial properties in the prevention of dental caries. Uvaria chamae have been used to treat various infections. It has proven antiparasitic, antiplasmodial, antidiabetic, antimicrobial, and antioxidant properties. Although the anti-S. mutans activity of this plant has been reported, its effect on the virulence properties has not been studied. Therefore, this study aimed to investigate the antimicrobial activity of U. chamae roots extracts on S. mutans virulence factors. Methods and materials Stock cultures of S. mutans were obtained from the Oral microbiology laboratory, the University of the Witwatersrand, and the plant extracts were provided by Dr. Ogunyemi Olajide Oderinlo from Nigeria. The plant extracts were prepared using methanol, dichloromethane, hexane, ethanol, and methanol: water. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined using the microdilution technique. Based on the MIC, the solvent with the best results was selected and three different concentrations were evaluated for their effect on biofilm formation, acid, and extracellular polysaccharides production in S. mutans. The effect of the plant extract on the expression of virulence genes (gtfB, gtfC, spaP, IDH, atpD, vicR, brpA, and gbpB) was also investigated using RT-qPCR. The results were analyzed using the one-way ANOVA and Wilcoxon Rank Sum Test. vi Results The mean MIC of U. chamae roots extracts against S. mutans ranged between 0.02 and 1.25 mg/ml and the MBC ranged between 0.04 and 1.25 mg/ml. The dichloromethane plant extract showed the best antibacterial activity against all the five cariogenic S. mutans strains with an average MIC and MBC of 0.02 and 0.04 mg/ml respectively and was used in the subsequent experiments such as the biofilm, acid, EPS, and RT-qPCR assay. At 6 hours, exposure to 0.005, 0.01, and 0.02 mg/ml of the plant extract reduced biofilm formation by 39.70, 59.17, and 76.82 % respectively. At 24 hours, the percentage reduction of the biofilm counts significantly improved up to 91 %. Not much difference in the test results was observed between 24 and 30 hours. The plant extract also significantly inhibited acid production (p < 0.01). The roots extract did not inhibit the production of soluble and insoluble extracellular polysaccharides. Furthermore, a significant decline in the transcription of virulence genes (gbpB, vicR, brpA, spaP, gtfB, gtfC, atpD, and IDH) was observed in the presence of the plant extract. Conclusion The dichloromethane extracts showed the best antibacterial activity. At subinhibitory concentrations, this plant extract significantly inhibited biofilm formation, acid production, and virulent gene expression by S. mutans. Therefore, this suggests that U. chamae has the potential to control and prevent dental caries. vii TABLE OF CONTENT DECLARATION............................................................................................................................ i DEDICATION............................................................................................................................... ii PUBLICATIONS AND PRESENTATIONS ............................................................................. iii ACKNOWLEDGMENT ............................................................................................................. iv ABSTRACT ................................................................................................................................... v TABLE OF CONTENT .............................................................................................................. vii LIST OF TABLES ...................................................................................................................... xii LIST OF FIGURES ................................................................................................................... xiv LIST OF ABBREVIATIONS AND ACRONYMS ................................................................. xvi Chapter 1: Introduction and literature review .......................................................................... 1 Introduction ............................................................................................................................................... 1 1. Literature review ................................................................................................................................... 2 1.1. Dental caries .................................................................................................................................. 2 1.2. Classification of dental caries ....................................................................................................... 4 1.3. Microbiology of dental caries ....................................................................................................... 7 1.3.1. Streptococcus mutans ............................................................................................................ 7 1.4. Virulence factors of Streptococcus mutans ................................................................................... 9 1.4.1. Biofilm production .............................................................................................................. 10 1.4.2. Acid production................................................................................................................... 11 1.4.3. Extracellular polysaccharides (EPS) production ................................................................. 13 1.5. Lactobacillus spp. ....................................................................................................................... 15 viii 1.6. The role of diet in the occurrence of dental caries ...................................................................... 15 1.7. Role of saliva in the caries process ............................................................................................. 16 1.8. Prevention of dental caries .......................................................................................................... 17 1.9. Uvaria chamae ............................................................................................................................ 18 1.9.1. Taxonomy of Uvaria chamae ............................................................................................. 18 1.9.2. Description of Uvaria chamae ............................................................................................ 18 1.9.3. Origin and distribution of Uvaria chamae .......................................................................... 19 1.9.4. Traditional uses of Uvaria chamae ..................................................................................... 19 1.9.5. Major chemical constituents of Uvaria chamae ................................................................. 20 1.10. The role of medicinal plants in oral diseases .......................................................................... 24 1.11. Anticariogenic effects of plant extracts against Streptococcus mutans .................................. 25 1.11.1. Artemisia princeps .............................................................................................................. 25 1.11.2. Ethyl gallate ........................................................................................................................ 26 1.11.3. Rhodiola rosea .................................................................................................................... 26 1.11.4. Chamaecyparis obtusa ........................................................................................................ 27 1.11.5. Prangos acaulis Bornm. ...................................................................................................... 27 1.12. Aim ......................................................................................................................................... 27 1.13. Objectives ............................................................................................................................... 28 Chapter 2: The antibacterial effect of Uvaria chamae dichloromethane extract against Streptococcus mutans .................................................................................................................. 29 2.1. Introduction ................................................................................................................................. 29 2.2. Methods and materials ................................................................................................................ 30 2.2.1. Plant materials ..................................................................................................................... 30 2.2.2. Bacterial cultures................................................................................................................. 30 ix 2.2.3. Antimicrobial activity ......................................................................................................... 31 2.3. Results ......................................................................................................................................... 32 2.4. Discussion ................................................................................................................................... 34 2.5. Conclusion .................................................................................................................................. 35 Chapter 3: The effect of Uvaria chamae dichloromethane extract on the biofilm formation by Streptococcus mutans ............................................................................................................. 36 3.1. Introduction ................................................................................................................................. 36 3.2. Methods and materials ................................................................................................................ 37 3.2.1. Bacterial cultures................................................................................................................. 37 3.2.2. Plant materials ..................................................................................................................... 38 3.2.3. Biofilm assay ...................................................................................................................... 38 3.2.4. Statistical analysis ............................................................................................................... 39 3.3. Results ......................................................................................................................................... 39 3.4. Discussion ................................................................................................................................... 44 3.5. Conclusion .................................................................................................................................. 45 Chapter 4: The effect of Uvaria chamae dichloromethane extract on Streptococcus mutans acid production............................................................................................................................ 46 4.1. Introduction ................................................................................................................................. 46 4.2. Method and materials .................................................................................................................. 48 4.2.1. Plant extractions .................................................................................................................. 48 4.2.2. Bacterial cultures................................................................................................................. 48 4.2.3. Acid production assay ......................................................................................................... 48 4.2.4. Statistical analysis ............................................................................................................... 49 4.3. Results ......................................................................................................................................... 49 x 4.4. Discussion ................................................................................................................................... 57 4.5. Conclusion .................................................................................................................................. 58 Chapter 5: The effect of Uvaria chamae dichloromethane extract on soluble and insoluble extracellular polysaccharides production by Streptococcus mutans ...................................... 60 5.1. Introduction ................................................................................................................................. 60 5.2. Method and materials .................................................................................................................. 62 5.2.1. Bacterial cultures................................................................................................................. 62 5.2.2. Plant materials and extraction ............................................................................................. 62 5.2.3. Soluble extracellular polysaccharide production assay ....................................................... 63 5.2.4. Insoluble Extracellular Polysaccharide production assay ................................................... 63 5.2.5. Statistical analysis ............................................................................................................... 64 5.3. Results ......................................................................................................................................... 64 5.4. Discussion ................................................................................................................................... 67 5.5. Conclusion .................................................................................................................................. 68 Chapter 6: The effect of Uvaria chamae extract on the expression of virulence genes in Streptococcus mutans .................................................................................................................. 69 6.1. Introduction ................................................................................................................................. 69 6.2. Methods and materials ................................................................................................................ 71 6.2.1. Plant materials and preparation ........................................................................................... 71 6.2.2. Bacterial cultures and growth conditions ............................................................................ 71 6.2.3. Total RNA extraction from microbial cultures ................................................................... 71 6.2.4. Generation of complementary DNA by reverse transcription ............................................ 72 6.2.5. Examining the expression level of virulence genes by RT-qPCR ...................................... 72 6.3. Data analysis ............................................................................................................................... 73 xi 6.4. Results ......................................................................................................................................... 74 6.4.1. Variable virulence gene expression in treated and untreated S. mutans strains .................. 74 6.5. Discussion ................................................................................................................................... 78 6.6. Conclusion .................................................................................................................................. 79 Chapter 7: Overall discussion, conclusion, limitations of the study, and future research ... 80 7.1. Overall discussion ....................................................................................................................... 80 7.2. Clinical implications ................................................................................................................... 81 7.3. Conclusion .................................................................................................................................. 82 7.4. Limitations to the study............................................................................................................... 82 7.5. Future research ............................................................................................................................ 83 References .................................................................................................................................... 84 Appendices ................................................................................................................................... 99 Appendix I: Media composition and preparation .................................................................................. 99 Appendix II: Primer preparation .......................................................................................................... 101 Appendix III: Statistical analysis ........................................................................................................ 104 Appendix IV: Ethical clearance ........................................................................................................... 116 Appendix VI: Turn it in report ............................................................................................................. 117 xii LIST OF TABLES Table 2.1 MIC and MBC of U. chamae roots extract against S. mutans. 33 Table 2.2 Summary results of mean MBC and MIC of U. chamae roots extract against S. mutans 34 Table 3.1 The effect of 0.005, 0.01, and 0.02 mg/ml of U. chamae roots extract on S. mutans biofilm formation 41 Table 3.2 Statistical analysis of the data obtained in the biofilm formation assay 42 Table 4.1 The effect of 0.005, 0.01 and 0.02 mg/ml of U. chamae extract on S. mutans acid production 51 Table 4. 2 The effect of 0.005 mg/ml of U. chamae on S. mutans counts in acid assay 52 Table 4.3 The effect of 0.01 mg/ml of U. chamae on S. mutans counts in acid assay 53 Table 4.4 The effect of 0.02 mg/ml of U. chamae extract on S. mutans counts in acid assay 54 Table 4.5 Statistical analysis of the data obtained in the acid production assay 55 Table 5.1 The effect of 0.005, 0.01, and 0.02 mg/ml of U. chamae on the production of soluble and insoluble extracellular polysaccharides by planktonic cells of S. mutans. 65 Table 5.2 Statistical analysis of the data obtained in the extracellular polysaccharides production assay 66 Table 6.1 Primer sequences used to amplify the virulence genes and the 16 S ribosomal RNA reference genes in S. mutans RT-qPCR 73 xiii Table 6.2 Fold change in gene expression in SM1 strain 75 Table 6.3 Fold change in gene expression in SM12 strain 75 Table 6.4 Statistical analysis of SM1 and SM12 strains obtained in RT-qPCR 76 xiv LIST OF FIGURES Figure 1.1 a) Carious lesions on different sites of teeth and b) patient with white spot lesions at the Bucco-cervical surfaces (Deveci et al., 2018) 4 Figure 1.2 Classification of dental caries according to the American Dental Association Classification System (Ng and Fida, 2016). 6 Figure 1.3 Microscopic depiction of S. mutans (Ranganathan and Akhila, 2019b). 9 Figure 1.4 Schematic illustration of cariogenic biofilm formation in the presence of fermentable sugar (Kalesinskas et al., 2014). 11 Figure 1.5 Schematic illustration of sucrose metabolism by S. mutans (Lemos et al., 2019). 13 Figure 1.6 The percentage composition of polysaccharide matrix (Krzyściak et al., 2014) 14 Figure 1.7 Stephan’s curve illustrating the changes in plaque pH over time following a sucrose rinse (Bilbilova, 2020). 16 Figure 1.8 U. chamae tree and fruits (Teanpaisan et al., 2014) 19 Figure 1.9 Basic structure of alkaloids (Achilonu and Umesiobi, 2015) 21 Figure 1.10 Basic structure of triterpenoid (Xia et al., 2014). 21 Figure 1.11 Basic structure of common tannins (Amorim et al., 2012) 22 Figure 1.12 Basic structure of flavonoid (Nishiumi et al., 2011) 23 xv Figure 3.1 Biofilm assay (a) Beaker containing glass slides after incubation at 37oC (b) Washing of biofilm c) Removal of the biofilm by scraping off the attached cells with a clean slide, (d) S. mutans colonies on blood agar. 39 Figure 3.2 The effect of U. chamae on S. mutans biofilm formation 42 Figure 3.3 Percentage reduction in S. mutans biofilm formation by U. chamae 43 Figure 4.1 Acid production assay a) Grow S. mutans at 37oC for 48 hours b) pH measurements c) S. mutans colonies on blood agar 49 Figure 4.2 The effect of U. chamae on S. mutans acid production in acid assay 55 Figure 4.3 The effect of U. chamae on the growth of S. mutans in acid production assay 56 Figure 5.1 Illustration of the efficient metabolism of sucrose by S. mutans 62 Figure 5.2 The effect of U. chamae on soluble and insoluble extracellular polysaccharides by S. mutans 66 Figure 5.3 Figure 6.1 Percentage reduction of S. mutans soluble and insoluble extracellular polysaccharides production by U. chamae Virulence genes in Streptococcus mutans 67 70 Figure 6.2 Target gene expression in treated and untreated S. mutans (SM1) strain 76 Figure 6.3 Target gene expression in treated and untreated S. mutans (SM12) strain 77 xvi LIST OF ABBREVIATIONS AND ACRONYMS µl ABS brpA CDC cDNA CFU CHX CO2 DCM DNA ECM eDNA EPS F-ATPase Fruct-6-P gbpA gbpD GlcN-6-P gtfB gtfC gtfD Hrs ICDAS INT IPS LDH LTA Microliters ATP-binding cassette Biofilm regulatory protein Centre for disease control and prevention Complementary DNA Colony Forming Unit Chlorohexidine Carbon dioxide Dichloromethane Deoxyribonucleic acid Extracellular matrix Extracellular DNA Extracellular polysaccharides ATP synthase Fructose-Phosphate Glucan binding protein A Glucan binding protein D Glucosamine-6-phosphate Glucosyltransferase B Glucosyltransferase B Glucosyltransferase C Glucosyltransferase D hours International Caries Detection and Assessment System Iodonitrotetrazolium chloride Intracellular polysaccharides Lactate dehydrogenase Lipoteichoic acid xvii MBC mg mg/ml MIC mins mL nm OD PBS QS RNA RT-qPCR S. mutans spp TB TSB U. chamae Minimum Bactericidal Concentration Milligrams Milligrams per milliliter Minimum Inhibitory Concentration Minutes Milliliter Nanometers Optical density Phosphate Buffered Saline Quorum sensing Ribonucleic acid Real-Time Polymerase Chain Reaction Streptococcus mutans Species Tryptone Broth Tryptone Soy Broth Uvaria chamae 1 Chapter 1: Introduction and literature review Introduction The human oral cavity harbour up to 700 different types of commensal bacteria, and only a few cause infections such as dental caries (Abid et al., 2015). Dental caries is the most common chronic infectious disease in the oral cavity and the most prevalent bacterial infection affecting all age groups (Yactayo-Alburquerque et al., 2021). In South Africa, 60% of 6 years old have had dental caries (Bhayat and Chikte, 2019). Factors such as the inadequate ratio of dentists per 1,000 population, cost of service, lack of oral health service providers due to geographic isolation, and shortage of healthcare providers play an important role in delayed diagnosis and compromised health status (Tiwari et al., 2021). Dental caries is caused by the oral bacteria Streptococcus mutans which ferments dietary carbohydrates and produces organic acids that demineralize the enamel of the tooth (Culp et al., 2021). This bacterium alters the ecology of dental plaque primarily due to its acidic metabolic products. In addition, S. mutans may express certain virulence factors to maintain their ecological niche in the oral cavity (Li et al., 2016). These bacteria form biofilms and produce extracellular polysaccharides which also contribute to the development of caries. Although dental caries is not a life-threatening infection, it has become a major financial burden to the public health system due to its costly and ongoing treatment, which consumes between 5 to 10 % of the health budget in developed countries (Ngabaza et al., 2018;Lee et al., 2019). The prevention of dental caries is based on reducing oral bacteria, maintaining an alkaline environment, and providing appropriate dietary advice (Jacob and Nivedhitha, 2018). Antibacterial agents are recommended for the prevention of dental caries. Chlorhexidine (CHX) is an antibacterial compound against most bacterial species found in the oral cavity. However, there is controversy on the use of chlorhexidine for caries prevention due to common side effects such as irritation of the mucosa, tooth staining, taste alteration, and formation of calculus on the tooth surfaces (Moghadam et al., 2020). Fluoride is widely used as a highly effective anticaries 2 agent (Villa et al., 2018) because it promotes caries lesion remineralization and inhibits demineralization of tooth surfaces subjected to organic acids (Almohefer et al., 2018). In contrast, natural products have been proven to be safe and they contain bioactive compounds with potential therapeutic applications in dentistry (Jacob and Nivedhitha, 2018). Uvaria chamae have been used throughout the world to treat infections. This plant synthesizes various secondary metabolites from its root, stem, leaf, and fruit which has beneficial medicinal properties (Abu et al., 2018). It has proven antiparasitic, antiplasmodial, antidiabetic, antimicrobial, and antioxidant activity. Although the anti-S. mutans activity of this plant has been reported, its effect on the virulence properties has not been studied. Therefore, this study aimed to investigate the antimicrobial effect of U. chamae roots extracts on acid production, biofilm formation, extracellular polysaccharides production, and virulence gene expressions by S. mutans. Antibacterial activity was studied using a microdilution technique. Biofilm assay was performed using a glass slide technique. In the acid assay, sequential pH measurements were performed. Extracellular polysaccharides assay was performed using the phenol-sulphuric acid technique. The expression of virulence genes was quantified using Real- Time Polymerase Chain Reaction (RT-qPCR). 1. Literature review 1.1. Dental caries Dental caries as shown in Figure 1.1a is a chronic bacterial disease that involves the destruction of tooth hard tissue structure. The term dental caries originates from the Latin word “caries”, which means decay (Rathee and Sapra, 2019). Untreated dental caries in permanent teeth affect 2.3 billion people, and it is the most prevalent non-fatal non-communicable disease worldwide (Chikte et al., 2020). Dental caries is caused by acid-producing bacteria e.g Streptococcus mutans, Lactobacillus acidophilus, Actinomyces viscosus, and Nocardia spp that ferment dietary carbohydrates in the dental plaque and produces organic acids such as lactic, acetic, formic, and propionic acids (Chu et al., 2016;Kabra et al., 2012). These acids have been reported to readily dissolve the enamel and 3 dentine of the teeth (Featherstone, 2008). Lifestyle behavioral factors such as poor oral hygiene, frequent consumption of refined carbohydrates, frequent use of oral medications that contain sugar, and inappropriate methods of feeding infants contribute to the risk of developing dental caries (Pitts et al., 2017a). Figure 1.1b presents the first clinical sign of dental caries called the “white spot”. This is the first sign that can be seen with human eyes and as the disease progress into the enamel, they can be detected by radiographs (Featherstone, 2008). During the disease process, the organic substances on the tooth surfaces are broken down and demineralization of the calcified tissues of the tooth occurs (El Sherbiny, 2014). If caries progresses it can lead to cavitation, the condition can cause considerable pain and discomfort. Dental caries can also spread to the dental pulp and cause infection and ultimately sepsis and tooth loss (Peres et al., 2019). The progression of dental caries is determined by the equilibrium status between protective factors which are components of the saliva e.g carbonate (Ca2+), phosphates, fluoride, protective proteins of the pellicle, saliva antibacterial components, and pathological factors (cariogenic bacteria, a dysfunction of the salivary glands, frequent consumption of carbohydrates). A preponderance of pathological factors results in the processes of demineralization and dental caries (Strużycka, 2014). Over time, this process leads to either caries lesions- or the repair and reversal of a lesion. Reversal of the lesions occurs through the process of remineralization, in which calcium, phosphate, and fluoride are incorporated in the areas damaged due to demineralization processes, resulting in a stronger, fluoridated mineral (Pitts et al., 2017a). 4 Figure 1.1 a) Carious lesions on different sites of teeth adapted from https://image.shutterstock.com/z/stock- photo-neglected-teeth-cleaned-for-restoration-98850518.jpg and b) patient with white spot lesions at the Bucco-cervical surfaces (Deveci et al., 2018). 1.2. Classification of dental caries The oldest dental caries lesions classification is the G.V Blacks classification. The G.Vs Black classification was first introduced in the year 1908 (Macri and Chitlall, 2017). This classification was based on the type of tooth and the tooth surface where caries was more likely to begin e.g occlusal fissures, proximal contacts, and cervical areas. Based on these observations, he developed a classification of caries lesions dependent on the position of a lesion and prescribed a cavity design regardless of the size and extent of the lesion (Nagarajan and Anjaneyulu, 2019). Other epidemiologic studies have used the modified versions of Klein and colleagues, decayed, missing, and filled (DMF) method to measure the prevalence and severity of caries. The Decayed-Missing- Filled (DMF) was proposed by the World Health Organisation (WHO) and it is the most common method used in oral health epidemiology for assessing and measuring dental caries (Campus et al., 2019). This method records both current and past caries experiences for an individual or group by numerically counting affected teeth per individual collected at either tooth (DMFT) or tooth surface level (DMFS) (Tellez and Lim, 2020). https://image.shutterstock.com/z/stock-photo-neglected-teeth-cleaned-for-restoration-98850518.jpg https://image.shutterstock.com/z/stock-photo-neglected-teeth-cleaned-for-restoration-98850518.jpg 5 Other indexes were designed to describe additional stages of the caries (Young et al., 2015). The International Caries Detection and Assessment System (ICDAS) was developed to create a caries detection method that might be used universally to measure caries at different stages (Pradeep, 2020). This method assesses both the presence of caries lesions and their severity from initial and reversible states to cavitated lesions, and classify them in increasing severity codes from 0 to 6 (Coelho, 2020). In 2008, the American Dental Association (ADA) convened a group of experts and stakeholders to begin the development of a Caries Classification System (CCS) that would be useful in clinical practice while incorporating up-to-date scientific evidence (Campus et al., 2019). The ADA CCS scores each surface of the dentition based on the tooth surface, presence or absence of a caries lesion, anatomic site of origin, the severity of the change, and estimation of lesion activity. The ADA CCS uses categories such as sound, initial, moderate, advanced to score a tooth surface’s clinical appearance. The initial, moderate, and advanced categories are each subdivided to account for variations in appearance- Figure 1.2.(Young et al., 2015). 6 Figure 1.2: Classification of dental caries according to the American Dental Association Classification System (Ng and Fida, 2016). 7 1.3. Microbiology of dental caries Dental caries was believed to be caused by only a few Gram-positive bacterial species, such as Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus spp. These findings were based on cultivation studies and the determination of the cariogenecity of these bacteria. This is termed the specific plaque hypothesis. Recent studies have shown that different population groups and individuals are susceptible to dental caries even when they have a low level of S. mutans, and vice versa. This led to the formulation of the ecological plaque hypothesis which was proposed by Phil Marsh with an attempt to explore dental caries etiology (Al-Shahrani, 2019). According to the "Ecological Plaque Hypothesis", the disease is caused by an imbalance in the total microflora due to ecological stress, resulting in an enrichment of oral pathogens (Rosier et al., 2014). Recently, the ecological hypothesis was extended and highlighted the role of the metabolic activity of the oral microbiota rather than its composition as the principal modulator of the environment (Nyvad and Takahashi, 2020). According to the extended ecological plaque hypothesis, changes in the local environment, e.g. frequent carbohydrate availability, assumedly favour certain representatives of the oral biofilm and thus lead to a distinct shift in the microbiota composition towards a higher proportion of acidogenic and acid- tolerant species (Anderson et al., 2018). Previous studies suggested that the main pathogenic bacteria in dental caries are Streptococcus mutans and Lactobacillus spp (Ahirwar et al., 2019a). 1.3.1. Streptococcus mutans Streptococcus mutans is a Gram-positive bacterium with thick cell walls that are composed of a layer of peptidoglycan (murein) and teichoic acids. These thick walls are important in preventing osmotic lysis of the cell protoplast and they provide the rigidity needed to maintain the shape of the cell (Ramawat and Mérillon, 2015). S. mutans are commonly found in the human oral cavity and contribute significantly to tooth decay (David et al., 2011). These bacteria generally appear as pairs or chains: spherical under a light microscope (Hardie and Whiley, 1997). Their diagram is presented in Figure 1.3. On Mitis Salivarius Agar (MSA), they grow as highly convex colonies. Unlike other oral streptococci, majority of S. mutans strains can grow on a selective media, Mitis Salivarius agar containing 20% sucrose and 0.2% units/ml of bacitracin (Rajendran, 2009). 8 Streptococcus mutans was first isolated from human cavities and received the name S. mutans because Clarke believed that it was a mutant version of Streptococcus (Clarke, 1924). The mutans streptococci comprise a group of seven species, of which S. mutans and S. sobrinus are the predominant species isolated from human saliva and dental plaque (Banas, 2004a). This bacterium is first acquired by infants soon after their first tooth emerges and mothers are considered to be a major source since identical genotypic profiles of the isolated strains are shared between mother and child (Matsui and Cvitkovitch, 2010). In some cases, S. mutans can coexist with S. sobrinus, and their collaboration leads to various oral clinical conditions (Ranganathan and Akhila, 2019a). Strains of S. mutans are classified into four different serological groups, which are c, e, f, and k. This classification is based on the composition of the cell-surface rhamose-glucose polysaccharide, which is composed of a backbone of rhamnose and side chains of a1,2 (serotype c), b1,2-(serotype e), and a1,3-(serotype f) glucosidic residues (Nakano et al., 2008). Serotype c accounts for approximately 70 to 80% of S. mutans strains in the oral cavity, followed by e (20%) and less than 5% of serotype f (Nakano et al., 2010). Up to date, the genome of only two serotypes of S. mutans are known. The genome analysis provides further insight into how S. mutans have adapted to surviving the oral environment through resource acquisition, defense against host factors, and the use of gene products that maintain its niche against microbial competitors. The recent completion of three S. mutans genome sequences (UA159, NN2025, and LJ23) indicates a large degree of diversity and genome rearrangement within the species (Al-Shahrani, 2019). Of the four S. mutans serotypes, serotype c is the most predominant oral isolate, with over 70% of strains isolated from dental plaque (Al-Shahrani, 2019). The cariogenic potential of S. mutans includes the ability to metabolize numerous dietary carbohydrates into organic acids, and survive under low pH environmental stress conditions. This bacterium also synthesizes extracellular polysaccharides polymers which promote bacterial adherence and accumulation on tooth surfaces (Garcia et al., 2021). 9 Figure 1.3: Microscopic depiction of Streptococcus mutans (Ranganathan and Akhila, 2019b). 1.4. Virulence factors of Streptococcus mutans Streptococcus mutans possess various virulence factors that enable them to ferment numerous carbohydrates and produce organic acids. Unlike other oral plaque, S. mutans can accumulate in large numbers in the presence of sucrose (Alejandra and Daniel, 2020). These bacteria secrete glucosyltransferase on their cell wall, which allows the bacteria to produce polysaccharides from sucrose. The sticky, highly hydrated extracellular matrix created by dental plaque bacteria, largely comprised of glucans, which act as a glue that encourages plaque formation (Chen et al., 2016). Glucans mediate the attachment of bacteria to the tooth surface and other members of the oral biota and form biofilm (Argimón et al., 2013). Each of these virulence factors works coordinately to change dental plaque ecology. The ecological changes are characterized by increased proportions of S. mutans and other species that are similarly acidogenic and aciduric. The selection for a cariogenic flora increases the magnitude of the drop in pH following the fermentation of available carbohydrates and increases the probability of enamel demineralization (Banas, 2004b). The main virulence factors associated with cariogenicity include adhesion, biofilm formation, acidogenicity, acid tolerance, and extracellular polysaccharides production (Li et al., 2020). 10 1.4.1. Biofilm production Biofilm is an organized aggregate of microorganisms living within an extracellular polymeric matrix that they produce and irreversibly attached to the living surface which will not be cleared unless rinsed quickly (Jamal et al., 2018). The formation of cariogenic biofilm is regulated by genes that are responsible for microbial adhesion and biofilm formation. The genes gbpB, sacB (ftf), and vicR are involved in sucrose-dependent adhesion, and spaP is involved in sucrose- independent adhesion. In addition to the genes, S. mutans encode several surface-associated glucan-binding proteins, gbpA, gbpB, gbpC, and gbpD (Gabe et al., 2019). The major role of the gbpB includes promoting bacterial adhesion, cell aggregation, and biofilm maturation process (Fujita et al., 2011). The gene ftf catalyzes the formation of fructans from sucrose. Fructan polymers serve as the storage of extracellular nutrients, which may be utilized during periods of nutrient deprivation. The vicR gene encodes a vicR response regulator, which is an essential part of VicRKX TCS. It is known to regulate a set of genes (gtfB, gtfC, gtfD, and gbpB) that code for the synthesis of glucan matrix, which is crucial for adhesion to a smooth tooth surface (Senadheera et al., 2005). spaP gene participates in bacterial adherence to teeth via interaction with the salivary pellicle (Lemos et al., 2019). The formation of biofilm involves several stages as presented in Figure 1.4. In the beginning, salivary proteins are selectively adsorbed to the tooth enamel. This forms an initial layer of material called salivary pellicle, which acts as a substrate for the fixation of cariogenic bacteria such as S. mutans. The salivary pellicle is formed by salivary components namely proline-rich proteins, amylase, lysozyme, histatin, peroxidase, mucin, and bacterial components, e.g., ftf, Gtf and lipoteichoic acid (Krzyściak et al., 2014;Castro et al., 2006a). The second step is bacterial adhesion to the pellicle. Early colonizers such as oral streptococci and Actinomyces spp recognize the binding proteins i.e., α-amylase and proline-rich glycoproteins in the acquired pellicle and bind to the pellicle. The early attachments are primarily based on hydrogen bonds, hydrophobic interactions, calcium bridges, van der Waals forces, acid-base interactions, and electrostatic interactions. Chemical forces become predominant at a later stage (Huang et al., 2011). Specific interactions between the pathogen and components of the salivary coating are mediated by the streptococcal antigen I/II protein. This polypeptide plays an important role in the binding activities, such as binding with the salivary glycoproteins, host cell receptors, and soluble extracellular matrix glycoproteins (Xu et al., 2007). Secondary colonizer species such 11 as Prevetella intermedia, Prevetella loescheii, Capnocytophaga spp, and Fusobacterium nucleatum adhere to the microbes that are already present in the dental plaque. After the adherence of the oral pathogen into the tooth surfaces, the dental biofilm undergoes maturation and numerous microbial interactions such as synergistic and antagonistic microbial interactions occur (Pitts et al., 2017a;Aruni et al., 2015). Figure 1.4: Schematic illustration of cariogenic biofilm formation in the presence of fermentable sugar (Kalesinskas et al., 2014). 1.4.2. Acid production The production of organic acids such as lactic acid from dietary carbohydrates metabolism is another major virulence factor in S. mutans. Lactic acid is produced via the glycolysis pathway, a biochemical reaction that occurs in the cytoplasm of a cell (Gomes et al., 2018). This acid erodes the hydroxyapatite of dentin and enamel thereby leading to the initiation and progression of caries. When acid is released into the environment, it dissociates as anions and conjugates protons, thereby reducing the pH of the environment (Kianoush et al., 2014). These protons may also diffuse into the cytoplasm of a cell through bacterial membranes, thereby acidifying the cytoplasm (Quivey et al. 2001). As S. mutans carry out glycolysis at a pH lower than 4.0, the organism protects its acid-sensitive glycolytic enzymes by transporting the protons across the cell membrane through the membrane-associated F-ATPase (Matsui and Cvitkovitch, 2010). S. mutans is also capable of surviving at low pH and continue with metabolic activities. 12 This is termed aciduricity (Alejandra and Daniel, 2020). The genes atpD, aguD, brpA, and relA in S. mutans enable it to overcome an acidic environment (You, 2019). The gene atpD in S. mutans encodes the F1F0ATPase. F1F0-ATPase is a proton pump that discharges H+ from within the bacteria to the outside, to overcome acid stress and maintain acid tolerance, which may confer a selective advantage over other members of dental biofilm (Argimón and Caufield, 2011). The gene relA and brpA are involved in the formation of dental plaque, acid, and oxidative stress tolerance mechanisms (He et al., 2019). In addition, aguD encodes the agmatine deiminase system, which produces alkali by converting agmatine to carbon dioxide, ammonia, and putrescine, enabling it to overcome acid stress and maintain acid tolerance (Griswold et al. 2006). During sucrose metabolism (Figure 1.5), sucrose is broken down into fructans and glucans by the action of fructosyltransferase and glucosyltransferase enzymes respectively. The enzyme DexA breaks glucans down the α1, 6-linkage thereby yielding maltodextrans whereas fructans are degraded by the enzyme fructanase enzyme FruA to yield fructose which is used for energy production (Lemos et al., 2019). After being transported into the cell, oligosaccharides (e.g maltodextrans) are degraded into monosaccharides by the enzyme DexB glucosidase. The transport of oligosaccharides is primarily conducted by the activity of ATP binding cassette (ABC) transporters encoded in the genome of S. mutans. The ATP binding cassette (ABC) transporters include multiple sugar metabolism (MSM) and malXFGK transport systems. The predominant route for the uptake of mono- and disaccharides by S. mutans is the phosphoenolpyruvate: sugar phosphotransferase system (Moye et al., 2014). In the intracellular environment, carbohydrates are phosphorylated and processed to fructose-6-phosphate (Fru-6- P) and fermented by glycolysis with the production of organic acids, mainly lactic acid. Furthermore, glucosamine-6-phosphate (GlcN-6-P) is synthesized from Fru-6-P, which is a precursor for cell wall biosynthesis. When carbohydrates are abundant, cells can produce an intracellular polysaccharide (IPS), a glycogen-amylopectin polymer that can be stored as intracellular granules and is used as an energy source reserve during starvation (Lemos et al., 2019). 13 Figure 1.5: Schematic illustration of sucrose metabolism by S. mutans (Lemos et al., 2019). 1.4.3. Extracellular polysaccharides (EPS) production Extracellular polysaccharides are high molecular weight sugar-based polymers that are synthesized and secreted by different microbes. These sugar-based polymers are a critical virulence factor in the production of biofilm formed in the presence of sucrose (Bowen and Koo, 2011). The composition of the polysaccharide matrix is shown in Figure 1.6 (Krzyściak et al., 2014). The production of EPS promotes the cariogenic potential of dental biofilms and their resistance to oral hygiene measures (Decker et al., 2014). Various genes are involved in the formation of extracellular polysaccharides. The genes gtfs, gtfB, gtfC, and gtfD encode glycosyltransferase (GTF) B, GTF C, and GTF D respectively whereas ftf encode fructosyltransferase (Shemesh et al., 2007). The gene gtfB synthesizes water-insoluble polysaccharides containing α1,3-linked glucans, which contributes to the scaffolding of the EPS matrix and facilitates cell aggregation in stable biofilms. The gene gftC catalyzes the synthesis of a mixture of water-insoluble and alkali-soluble glucan from sucrose, with both α- 1,3 and α-1,6-linked glucans, which are required for plaque formation and structurally stable biofilms. GtfD forms a soluble polysaccharide that acts as a primer for GtfB (De et al., 2018). The gene ftf catalyzes the formation of fructans from sucrose. Fructan polymers serves as the 14 storage of extracellular nutrients, which may be utilized during periods of nutrient deprivation. In addition, EPS serves as a storage nutrient for the bacteria (Lemos et al., 2005). Streptococcus mutans decomposes sucrose in the oral cavity into glucose and fructose using bacterial invertase and then synthesizes glucan by polymerizing glucose using the enzyme glycosyltransferase whereas fructosyltransferase synthesizes polysaccharides such as fructan by polymerizing fructose. The Gtfs secreted by S. mutans binds avidly to the pellicle formed and bacterial surfaces. In the presence of sucrose, glucans are formed in situ within minutes (Leme et al., 2006b). The exopolymers contribute to the bulk and physical stability of the biofilm matrix. The glucan-mediated processes promote tight adherence and coherence of bacterial cells bound to each other and to the apatitic surface, which leads to the formation of microcolonies and thereby modulates the initial steps of cariogenic biofilm development (Kooi et al., 2010). As the biofilm develops, the EPS formed in situ enmeshes and surrounds the microorganisms while forming an insoluble matrix facilitating the assembly of spatially heterogeneous yet cohesive 3D multicellular structures. The spatial heterogeneities shaped by EPS synthesis form a complex 3D matrix architecture and create environmental and protective niches within biofilms that can directly modulate caries pathogenesis (Klein et al., 2015). Figure 1.6: The percentage composition of polysaccharide matrix (Krzyściak et al., 2014) 15 1.5. Lactobacillus spp. Lactobacillus spp. is considered the second most cariogenic bacteria of oral microflora and it plays a major role in the progression of caries. These bacteria are part of the normal flora of the oral cavity and they are present in a large number on the saliva, surfaces of mucous membranes, and tooth surfaces (Ahirwar et al., 2019b). The presence of lactobacilli in the oral cavity depends on the presence of ecological niches such as natural anfractuosities of the teeth, partly erupted third molars, or orthodontic devices (Badet and Thebaud, 2008). The main virulence property of Lactobacillus spp. includes adhesion, biofilm formation, and their ability to ferment glucose and produces lactic acid that demineralizes the enamel of the tooth. The dominant Lactobacillus species associated with the pathogenesis of dental caries are Lactobacillus gassier, Lactobacillus fermentum, and Lactobacillus casei. These bacteria use two types of metabolism methods including homo-fermentative and heterofermentative to produce lactic acid and acetic acid (Sachidananda and Mallya, 2020). Lactobacillus spp. carry out glycolysis at pH as low as 3. After colonizing the established dental plaque, the lactobacilli can further acidify the plaque and suppress the acid susceptible microorganism, further enriching acidogenic and aciduric bacteria (Salvetti et al., 2012). 1.6. The role of diet in the occurrence of dental caries Dental diet and nutrition play a crucial role in childhood caries (Tungare and Paranjpe, 2018). The consumption of fermentable carbohydrates such as sucrose, glucose, and starch have both local and systemic effects on dental caries. The local dietary effect is dependent on individual diet and it is influenced by factors such as overall dietary habits, biofilm composition, saliva, and fluoride (Hujoel and Lingström, 2017). Numerous studies conducted within different population groups have demonstrated the role of high intake of sugars and carbohydrates in the occurrence of dental caries (Palacios et al., 2016;Touger-Decker and Van Loveren, 2003;van Loveren, 2019). The World Health Organization (WHO) guideline recommends reducing the consumption of free sugars below 10% of the energy intake and below 5% of the total diet. Free sugars are defined as all monosaccharides and disaccharides added to foods by the manufacturer, cook, or consumer, and sugars naturally present in honey, syrups, fruit juices, and fruit juice concentrates (van Loveren, 2019). Figure 1.7 presents the changes in plaque pH following a sucrose rinse. This phenomenon creates a Stephan curve. After sucrose rinse, the plaque pH was reduced to less than 5.0. When the pH of the enamel is below the critical pH of 16 5.5, demineralization of the enamel occurs. The plaque pH stays below the critical pH for 15- 20 and then returns to a normal pH of 6.5 after 40 min. In the presence of saliva, the enamel is remineralized when the plaque pH recovers to a level above the critical pH (Bilbilova, 2020). Figure 1.7: Stephan’s curve illustrating the changes in plague pH over time following a sucrose rinse (Bilbilova, 2020). 1.7. Role of saliva in the caries process Saliva plays a significant role in maintaining oral health (Dodds et al., 2015). It contains water (99.5%), protein (0.3%), and inorganic and organic substances (0.2%). Organic constituents of the saliva include amylases, growth factors, peroxidases, lipases, lysozyme, kallikreins, mucins cystatins, lactoferrin, hormones, and, whereas inorganic constituents include salt, potassium, calcium, magnesium, chlorides, and carbonates (Marsh et al., 2016;Kubala et al., 2018). The functions of the saliva in the mouth include antibacterial action, buffering properties, cleansing effect, and maintenance of saliva supersaturated in calcium phosphate. Importantly, saliva has been recognized as having the ability to reduce the incidence of dental caries due to its antimicrobial compounds such as immunoglobulins, lactoperoxidases, and lactoferrin which inhibit the growth of bacteria (Kertiasih and Artawa, 2015). The presence of calcium, phosphate, and fluoride within the saliva enhances the resistance of the tooth surfaces to a cariogenic attack, therefore decreasing the chances of demineralization and promoting remineralization of previously demineralized enamel and root surfaces (Hicks et al., 17 2004;Stookey, 2008). Several salivary proteins attach to hydroxyapatite and help maintain saliva supersaturated state. This allows calcium and phosphate-containing mineral components in solution at saliva's resting pH (close to neutral) and prevents precipitation out of solution (Hicks et al., 2004). A dry mouth exposes the teeth to acidic challenges from food, drinks, and organic acids produced by acidogenic bacteria. When the pH in the oral cavity falls below the critical pH<5.5, demineralization occurs. In a dry mouth, natural remineralization and protection may not occur. These may be due to a lack or inadequate salivary calcium and phosphate ions (Su et al., 2011). 1.8. Prevention of dental caries Oral hygiene, healthy eating, increasing fluoride availability, and the placement of fissures and sealants are the four main principles to prevent dental caries (Morgan, 2008). Mechanical plaque control by brushing and flossing is the most widely used method in disrupting and eliminating oral biofilm on tooth surfaces (Hughes and Dean, 2015). However, mechanical plaque control alone may be insufficient in preventing the development or recurrence of caries (Figuero et al., 2019). With more than 50 years of clinical success, fluoride serves as the gold standard agent for preventing caries (Karlinsey and Pfarrer, 2012). Animals and in vitro systems have demonstrated that dairy products have hypoacidogenic, anti-acidogenic properties, and prevent demineralization, and enhance remineralization (Walsh, 2006). Consumption of sugar-free gums containing xylitol and sorbitol can reduce the acidogenic potential of dental plaque and neutralize lactase produced by dental plaque thereby promoting enamel remineralization. Xylitol reduces caries by keeping sucrose molecules from binding to the S. mutans, thereby blocking sucrose metabolism. Chewing gum can also prevent dental caries by stimulating salivary flow and also enhancing salivary function, especially for those people with low flow rates (Shen et al., 2001). Increasing availability of fluoride using methods such as water fluoridation, fluoride toothpaste, fluoride mouth rinse, dietary fluoride supplements, and professionally applied fluoride compounds such as gels and varnishes also reduces dental caries. Mechanisms of action of fluoride for caries control are based on inhibiting demineralization of the crystal structures inside the tooth and enhancing remineralization. Additionally, fluoride inhibits bacterial enzymes (Lee, 2013). Placement of fissure sealants is one of the methods which can be used to prevent and control dental caries. By restoring teeth with fissure sealants, occlusal pits and fissures become less morphologically 18 susceptible. This preventive measure is recommended in young patients with erupting teeth and adults with a high caries index. Sealants prevent food from collecting in molar pits and fissures and, therefore, prevent dental caries (Goršeta, 2015). Many medicinal plants have been studied for their anticariogenic activity. Punica granatum, Dodoneae viscosa var. angustifolia, Cedrus deodara, Terminalia chebula, Psidium guajava, Azadirachta indica and Pongamia pinnata are some of the plants that have shown a very good anticariogenic potential. The present study examined Uvaria chamae. 1.9. Uvaria chamae 1.9.1. Taxonomy of Uvaria chamae The name “Uvaria chamae” is derived from the Greek word chamai, which means "on the ground" (Bongers et al., 2005). This plant is a member of the Annonaceae family. The Annonaceae family consists of flowering plants such as trees, shrubs, and lians, and it contains 2106 species and over 130 genera. Several genera, most notably Annona, Anonidium, Asimina, Rollinia, and Uvaria, produce edible fruits (Tamokou et al., 2017). 1.9.2. Description of Uvaria chamae Uvaria chamae is an evergreen plant that grows to a height of 3.6-4.5 m. The leaves are stipulate, the leaf apex cuminate, and the leaf vestiture is glabrous (Monon et al., 2015). The fruit carpels are in finger-like clusters, the shape giving rise to many vernacular names translated as a bush banana. The fruit is yellow when ripe and has a sweet pulp which is widely eaten-Figure 1.8 (Abu et al., 2018). 19 Figure 1.8: Uvaria chamae tree and fruits (Teanpaisan et al., 2014) 1.9.3. Origin and distribution of Uvaria chamae Uvaria chamae is a Nigerian medicinal plant. It is commonly called Ayiloko by the Igala people of Kogi State, Kaskaifi by the Hausas, Oko Oja by the Yorubas in Nigeria as well as Akotompo by the Fula-fainte of Ghana (James et al., 2013). This plant is native to tropical West and Central Africa. It has been introduced to other parts of Africa and elsewhere in the tropics as a curiosity plant because of its finger-like, ornamental fruits (Lim, 2012a). 1.9.4. Traditional uses of Uvaria chamae In West Africa, U. chamae is mainly used to treat jaundice and intermittent fevers. The root bark is used for respiratory catarrh and dysentery (Kadiri et al., 2014). A root infusion of this plant is used to treat severe abdominal pain. The root decoction is also administered as a purgative for the treatment of hepatitis. The juice of the fresh leaves is applied to fresh wounds, sores, and into the eyes to treat conjunctivitis (Jalil et al., 2020). An alcoholic extract prepared from root bark, stem, or dried leaves is taken to treat inflammatory condition known as “Calabar swelling” (Iwu, 2014). The roots, bark, and leaves extracts are used to treat inflamed gums, gastroenteritis, malaria, fever, vomiting, wounds, and sore throat. The roots powder is consumed to treat hyperprolactinemia (Yakubu and Fayemo, 2021). 20 1.9.5. Major chemical constituents of Uvaria chamae The medicinal properties of a plant depend on its bioactive phytochemical constituents (Sheikh et al., 2013). The composition of essential bioactive compounds in medicinal plants depends on the plant species, the soil type, and as well as their association (Egamberdieva et al., 2017). Secondary metabolites isolated and characterized from the Annonaceae family include monoterpenes, diterpenes, triterpenes, lignans, flavonoids, asarone-derived phenylpropanoids, acetogenins, and primarily typical isoquinoline-derived alkaloids. Some of these secondary metabolites have been shown to have important biological activities, such as antimicrobial, anti-inflammatory, anticarcinogenic and urease-inhibiting properties (Costa et al., 2021). Phytochemical analysis of U. chamae reported the presence of bioactive compounds with potential antimicrobial activity against a variety of pathogens. The ethanolic roots extracts of U. chamae contain flavonoids, alkaloids, cardiac glycosides, terpenes, saponins, and tannins. 1.9.5.1. Alkaloids Alkaloids are low molecular weight compounds with a nitrogen atom in a heterocyclic ring. Alkaloids include neuroactive molecules like caffeine and nicotine, anti-tumoral vincristine, vinblastine as well as emetine, which is used to treat oral intoxication (Matsuura and Fett-Neto, 2015). Figure 1.9 shows the basic structures of common alkaloids. Alkaloids are classified as isoquinolines, quinolines, indoles, piperidine alkaloids, etc. This classification is based on the chemical core structures of these alkaloids. Various pharmacological properties such as anticancer, antiviral, anti-inflammatory, and antibacterial activities have been reported (Yan et al., 2021). The phytochemical analysis of U. chamae leaves extract resulted in the isolation of Uvaria of benzylisoquinoline alkaloids (+)-armepavine and racem O, O-dimethyl coclaurine. The aporphines nornantenine, nantenine, and corydine have been isolated for the first time for the species (Thomas et al., 2018). 21 Figure 1.9: Basic structure of alkaloids (Achilonu and Umesiobi, 2015) 1.9.5.2. Saponins Saponins are a group of naturally occurring plant glycosides. They are distinguished by their high foaming properties in aqueous solutions. Saponins have been isolated from over 100 plant families, and at least 150 different types of natural saponins have been shown to have significant anti-cancer properties. Saponins are classified into more than 11 distinct classes, including dammaranes, tirucallanes, lupanes, hopanes, oleananes, taraxasteranes, ursanes, cycloartanes, lanostanes, cucurbitanes, and steroids (Man et al., 2010). Figure 1.10 shows the basic structures of common saponins. The majority of plants used in traditional medicine worldwide contain saponins. Saponins are widely distributed in higher plants have a wide range of biological properties such as antimicrobial, anti-tumor, and anti-inflammatory properties (Sparg et al., 2004). Figure 1.10: Basic structure of triterpenoid (Xia et al., 2014). 22 1.9.5.3. Tannins Tannins are present in a wide range of fruits and vegetables and they have been isolated and characterized from several medicinal herbs such as cinnamon, thyme, black cohosh, and feverfew. Tannins are a common phenolic antioxidant that has been shown to have medicinal and therapeutic properties. Tannic acid is one such magical molecule with strong antioxidant properties. Tannic acid is one such magic molecule with potent antioxidant activity (Ghosh, 2015). Figure 1.11 shows a basic structure of common tannins. Their mechanism of action includes the inhibition of extracellular microbial enzymes, the deprivation of microbial growth substrates, or direct action on microbial metabolism via oxidative phosphorylation inhibition (Haslam, 1989). Figure 1.11: Basic structure of common tannins (Amorim et al., 2012) 1.9.5.4. Flavonoids Flavonoids are a group of natural substances that contains various phenolic structures and they are commonly found in fruits, vegetables, grains, bark, roots, stems, flowers, and wines. Flavonoids have long been recognized for their beneficial effects in nutraceutical, pharmaceutical, medicinal, and cosmetic applications (Panche et al., 2016). Figure 1.12 shows the basic structures of common flavonoids. The novel C- benzylated flavanones and C- benzylated dihydrochalcones have been obtained from several U. chamae (Thomas et al., 2018). The flavonoid pinocembrin, chamanetin, uvaretin, uvarinol, and pinostrobin isolated 23 from stem bark and root bark (Enin et al., 2021). The drug benzyl benzoate used as antifungal preparation has a mutagenic compound, chamuveritin, a benzyldihydrochalcone that was isolated from U. chamae (Olumese and Onoagbe, 2019). Chalcones isolated from Uvaria chamae have been found to have strong antimicrobial activity against Gram-positive cocci (Staphylococcus aureus and Streptococcus pyogenes) compared to Gram-negative bacteria (Salmonella typhimurium and Escherichia coli (Koudokpon et al., 2018). Figure 1.12: Basic structure of flavonoid (Nishiumi et al., 2011) 1.9.5.5. Terpenes Terpenes are the largest and the most diverse group of naturally occurring secondary metabolites. They are commonly found in plants but larger classes of terpenes such as sterols and squalene can be found in animals (Cox-Georgian et al., 2019). Terpenes are classified based on their distinct carbon skeleton. It consists of a basic five-carbon isoprene unit (2-methyl-1,3- butadiene). Terpenes are typically composed of two, three, four, or six isoprene units. These are known as monoterpenes, sesquiterpenes, diterpenes, and triterpenes respectively (Man et al., 2010). Sesquiterpenes were reported as the predominant constituents of U. chamae leaf. The major constituent of U. chamae leaf oil was D-cadinene while thymoquinoldimethyl ether 24 and benzyl benzoate were the major components of the root oil. Two benzyldihydrochalcones and the known benzyl benzoate were identified in the roots of U. chamae. A monobenzylated monoterpene, chamanen, and the dimethyl ether of thymoquinol were isolated from the root bark of U. chamae (Lim, 2012b). Essential oil constituents from the leaves and root also revealed the presence of terpenes of 1-Nitro-2-pheneylethane, Linalool, Germacrene D, (E)- Caryophyllene, (E)-β-Ocimene, (E)- erolidol, 1,8-Cineole, 1-epi-Cubebol, α-Humulene, α- Copaene and others in trace quantities (Enin et al., 2021). 1.10. The role of medicinal plants in oral diseases Medicinal plants include a variety of plants that are used in herbalism (Rasool Hassan, 2012). In developing countries, medicinal plants are used as a primary source of medicine (Palombo, 2011). These plants have been used throughout the world to treat various diseases since ancient times and they continue to play a vital role in the health care systems in many regions, especially Africa where modern drugs are not affordable (Agbor and Naidoo, 2019). Medicinal plants are inexpensive and available through traditional knowledge of medicinal plants that is passed from generation to generation (Nemudzivhadi and Masoko, 2015). The traditional healers typically diagnose and treats the psychological basis of an illness before prescribing medicines, particularly medicinal plants to treat the symptoms. The profound knowledge of herbal remedies in traditional cultures was developed through trial and error over many centuries, along with the most important cures was carefully passed on verbally from one generation to another (Mahomoodally, 2013). Other factors such as poverty, the inadequacy of health services, shortage of health care workers, and rampant shortage of drugs and equipment in existing health care facilities make traditional medicine an important component of health in Africa (Ashu Agbor and Naidoo, 2015). Various parts of these plants used in the preparation of traditional medicine include root, stem, seed, flower, fruit, and twig exudates (Kalaivani et al., 2012). A pilot study conducted in Limpopo, South Africa reported the use of more than 41 plant species belonging to 30 botanical families, mainly the Asteraceae and Solanaceae as remedies for different oral diseases. The results showed that the most common medicinal plants used to treat oral diseases in Limpopo province, Lepelle Nkumpi Municipality are Artemisia afra, Cannabis sativ, Carpobrotus edulis, Mentha longifolia, Nicotianatabacum, Punica granatum, Ricinus communis, Solanum panduriforme, Zanthoxylum capense, and Ziziphus mucron. In 25 some regions, medicinal plants are used traditionally as a toothbrush or chewing sticks. The plants such as Diospyros lycioides, the sticks of Salvadora persica (miswak), Salvadora persica leaves, Acacia mellifera, Jasminum Fluminense, Azadirachta indica as toothbrush or chewing sticks (Bodiba et al., 2018). Tapsoba and Deschamps. (2006) reported the use of 62 plant species belonging to 29 families. The study reported that these plant species are effective in the management of toothache, gingivitis, acute necrotizing gingivitis, loose teeth, dental abscesses, sores on the tongue and lips. All the plant parts are used as remedies and they are prepared in various ways. Vegetable materials are often boiled for drinking, mouth washing, gargling, or inhalation. The principal plant parts include fresh or dried roots, stems, leaves, and barks. Plants were used either alone or in association with other species. For instance, a decoction made with roots of Capparis tomentosa Lam., Cassia sieberiana DC., and Indigofera tinctoria L. is used for mouthwash against toothache (Tapsoba and Deschamps, 2006). 1.11. Anticariogenic effects of plant extracts against Streptococcus mutans There are nearly 500,000 species of plants that occur in all parts of the world, and only 1% has been investigated phytochemically (Agbor and Naidoo, 2019). In recent years, oral care products and medicinal plant extracts are gaining high interest because they are less toxic and have fewer side effects compared to synthetic drugs (Şener and Kiliç, 2019). With the increase in the development of resistant strains, there is a need for an alternative therapy since resistance microbes are difficult to treat and require a higher dose which is more expensive and toxic to the human body (Adedayo et al., 2020). These findings have led to the screening of natural products for possible pharmaceutical value, particularly for anti-inflammatory, cytotoxic, antimicrobial, and antioxidant properties. Several studies have investigated the activity of traditional plants against oral pathogens and the examination was mainly to validate the traditional use of the medicinal plants. 1.11.1. Artemisia princeps Artemisia princeps is originally native to eastern Asia (China, Japan, and Korea). It has been present for at least two decades in several localities in Belgium and the Netherland. This plant is frequently used as a medicine, culinary herb, essential oil, and for re-vegetation (Verloove 26 and Andeweg, 2020). Artemisia princeps extract shows antibacterial activity against S. mutans at a concentration of 0.4 mg/mL. The bacterial activity was observed at a concentration range of 0.05-0.4 mg/mL. This extract also reduces the expression of the gene gftB, gtfD, and relA at a concentration greater than 0.1 mg/mL, gtfC, and vicR at a concentration greater than 0.2 mg/mL, spaP, and brpA at the concentration greater of 0.005 mg/ml (Yang et al., 2019). 1.11.2. Ethyl gallate Ethyl gallate is a phenolic compound richly contained in Longan. Longan (also known as Dimocarpus longan) is a member of the soapberry family (Sapindaceae). It is commonly regarded as a "hot" fruit in traditional Chinese medicine because it induces inflammatory immunological responses. It is grown extensively in China and Southeast Asia, as well as in Australia, Florida, southern Europe, and southern Africa (Wang et al., 2018). Ethyl gallate significantly suppressed S. mutans biofilm build-up on polystyrene and glass surfaces by 68% and more than 91% and inhibit acidogenicity by 95% of S. mutans. The study also demonstrated that this extract produced a significant gene expression change in the genes gtfB and gbpB at the concentration of 3.53 mM (Gabe et al., 2019). 1.11.3. Rhodiola rosea Rhodiola rosea is a flowering biennial grown in high latitude and altitude regions of the world. It has been a part of traditional medicine systems in parts of Europe, Asia, and Russia for centuries. The traditional use of R. rosea as a treatment of cancer and Tuberculosis, and as a fertility booster has been documented previously in countries such as Mangolia and Siberia. In Norway, it has been used as food and hair wash (Ishaque et al., 2012). The extract significantly decreased the expression of the gft genes and slightly decreased the expression of comD and comE genes. Biofilm formation was strongly inhibited at a concentration of 0.25 μg/μL and 0.50 μg/μL on ex vivo bovine enamel (Zhang et al., 2020a). 27 1.11.4. Chamaecyparis obtusa Chamaecyparis obtusa is a tropical tree species found in Japan and the southern region of South Korea, and the essential oil is extracted from the leaves and twigs of the C. obtusa tree. The essential oil has several types of terpenes and has been commercially used in soaps, toothpaste, and cosmetics as a functional additive (Bae et al., 2012;Hong et al., 2004). Chamaecyparis obtusa oil extract significantly inhibited organic acid production from 5.30-7.40 produced by S. mutans at a concentration range of 0.025 mg/mL-0.2 mg/mL. The expression of virulence genes brpA, gbpA, gtfC, and gtfD were significantly decreased at a concentration of 0.025 mg/mL of C. obtuse (Kim et al., 2016). 1.11.5. Prangos acaulis Bornm. Prangos acaulis Bornm. is one of the important species of genus Prangos in Iran that is used in folk medicine as a sedative and anti-infective agent. The use of Prangos acaulis Bornm. as a traditional medicine for tooth whitener and pain relievers have been validated (Rustaiyan et al., 2006). The roots extracts showed a greater antibacterial activity compared to flowers, leaf, stem, and seed extracts. The MIC and MBC for the roots and seeds extract against S. mutans ranged between 500-1000 and 2000-3000 with the greatest antibacterial activity at 500-1000 and 2000-3000 µl/mL. The extract significantly reduced biofilm formation by 66.40% and 22 +/- 0.20% (Nosrati et al., 2018). Although the antibacterial activity of U. chamae against S. mutans is known, its effect on biofilm formation and the production of acid and extracellular polysaccharides has not been studied. 1.12. Aim This study aimed to evaluate the anti-S. mutans property of U. Chamae and its antivirulence activity. 28 1.13. Objectives a) To investigate the antibacterial effect of methanol, dichloromethane, ethanol, hexane, and methanol: water crude roots extracts against S. mutans using microdilution technique. b) To determine the effect of subinhibitory concentrations of the dichloromethane crude roots extracts on S. mutans biofilm formation using a glass slide technique. c) To determine the effect of subinhibitory concentrations of the dichloromethane crude roots extracts on S. mutans acid production using sequential pH measurements. d) To determine the effect of subinhibitory concentration of dichloromethane crude roots extract on S. mutans extracellular polysaccharides (EPS) production using phenol sulfuric acid assays. e) To analyze the effect of the dichloromethane crude roots extract on the expression of virulence genes using Real-Time Polymerase Chain Reaction (RT-qPCR). 29 Chapter 2: The antibacterial effect of Uvaria chamae dichloromethane extract against Streptococcus mutans 2.1. Introduction Streptococcus mutans, a Gram-positive, facultatively anaerobic bacterium is generally known as a major pathogen of dental caries and a possible causative agent of bacteremia and infective endocarditis (Nakano et al., 2008;Nakano and Ooshima, 2009). It is a part of the normal flora of the oral cavity and it resides primarily in the biofilm, also called plaque formed on the teeth surfaces (Metwalli et al., 2013;Lemos et al., 2013). In the prevention and control of dental caries, it is important to reduce the number of cariogenic bacteria such as S. mutans. There are several commercially available oral hygiene products such as toothpaste and mouth rinses containing fluoride, triclosan, and chlorhexidine with varied efficacy and a few have been shown to reduce dental plaque formation (Prasanth, 2011). The use of medicinal plants in the treatment and prevention of oral diseases including dental caries has been well documented in recent years. Herbal extracts have been used in dentistry for many years to reduce inflammation, inhibit the growth of oral pathogens, prevent the release of histamine, and as antiseptics, antioxidants, and analgesics (Megersa et al., 2019). These extracts consist of many bioactive compounds that are considered good alternatives in the management and treatment of various oral diseases (Semenya et al., 2019). In some countries, many synthetic and routine drugs are inaccessible. In addition, there is an increase in the loss of effectiveness and potency to multidrug resistance organisms to oral antibacterial agents. Therefore there is a need to source locally available drugs and alternative medicine to treat ailments (Udoh et al., 2019). Uvaria chamae is used in many African countries to treat bacterial infections. In addition to its antimicrobial activity, the plant has also been reported to have anti-inflammatory properties (Emordi et al., 2018;Abachi et al., 2016). Ogbulie et al. (2007) demonstrated that cold water extracts of U. chamae fresh leaves can moderately inhibit the growth of Staphylococcus aureus and Streptococcus pyogenes. The cold and hot ethanol extract profoundly inhibited Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Salmonella typhi (Ogbulie et al., 2007). Stem bark extracts were found to have the greatest antimicrobial activity 30 against Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella spp, and Proteus spp, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pneumonia aeruginosa. Leaf extracts showed the least antimicrobial activity against Escherichia coli, Klebsiella spp, and Proteus spp with MIC of 250 mg/mL (Oluremi et al., 2010). This chapter aimed to determine antimicrobial activity of U. chamae against S. mutans by investigating the minimum inhibitory concentration (MIC) and Minimal Bactericidal Concentrations (MBC). 2.2. Methods and materials 2.2.1. Plant materials Uvaria chamae roots extracts were provided by Dr. Ogunyemi Olajide Oderinlo from the Department of Chemistry, Faculty of Science, Federal University, Otuoke, Bayelsa, Nigeria. The plant was identified and authenticated by a qualified botanical expert in Nigeria. The voucher specimen number of the plant was FHI 107901. Plant extracts were prepared using methanol, methanol: water, hexane, ethanol, and dichloromethane. Briefly, the roots were harvested, washed, dried, milled, and stored at 4oC until required for use. The crude extracts were obtained by mixing 1g of the root powder separately with 10 ml of each solvent. The solution was shaken at 120 rpm at room temperature overnight and centrifuged at 10000 rpm for 10 minutes using a microcentrifuge. This procedure was done 3 times. Thereafter, the solvents were allowed to evaporate under a cold air stream and stored at 4o C until required. The crude extracts were weighed and reconstituted in 10% Dimethyl sulfoxide (DMSO) to obtain a final concentration of 50 mg/ml. 2.2.2. Bacterial cultures Five clinical strains (SM1, SM6, SM7, SM12, and SM13) of cariogenic S. mutans were obtained from the Oral Microbiology laboratory, University of Witwatersrand. An ethics 31 waiver (W-CBP-200529-03) for the use of stock cultures was granted by The Human Research Ethics Committee, University of The Witwatersrand. Cultures were grown on blood agar at 37oC for 48 hours. For each experiment, fresh cultures were used. To prepare inoculum which was used in this experiment, colonies were emulsified in phosphate-buffered saline and the suspension was adjusted to 0.5 McFarland standard containing approximately 105-106 cfu/ml. 2.2.3. Antimicrobial activity Antimicrobial activity of U. chamae roots extracts was performed using the microdilution technique as previously described by Gulube and Patel (2016) and Kuete et al (2012) with modifications. Dry U. chamae extracts were reconstituted in DMSO to obtain a concentration of 50 mg/ml. Two-fold dilutions of U. chamae roots extracts were prepared using tryptone broth to obtain concentrations of 5 mg/ml to 0.02 mg/ml. Thereafter, 50 µl of S. mutans inoculum prepared as a 0.5 McFarland standard containing approximately 105-106 cfu/ml cells was added to all the wells of the 96-microtiter plate containing 50 µl diluted plant extract. A well-containing 50 µl tryptone soy broth, inoculated with 50 µl S. mutans was used as a positive control, while different wells containing 50 µl of S. mutans inoculum and 50 µl of chlorohexidine was used as a negative control. The effect of 10 % DMSO on S. mutans growth was determined by adding 50 µl of the inoculum in a well containing 50 µl of DMSO. The 96- microtiter plates were incubated at 37oC for 48 hours under CO2 -enriched environment. After incubation, 5µl from each microtiter well was aliquoted on blood agar plates to determine the MBC. Thereafter, 0.2 % iodonitrotetrazolium chloride (INT) was added to each well. The microtiter plates were incubated for an additional 48 hours. The MIC was interpreted as the sample concentrations that prevent change in colour of the medium from colourless to pink and completely prevent microbial growth. The MBC was defined as the lowest concentration of the extract which did not produce any growth after 48 hours of incubation. The experiment was performed in triplicates for all 5 clinical strains of S. mutans. Based on the MIC concentrations, the solvent with the best results was selected and two subinhibitory concentrations were used for further studies, such as biofilm, acid assay, and extracellular polysaccharides study. 32 2.3. Results The results of the MIC/MBC of U. chamae roots extracts are presented in Tables 2.1 and the summarized results are presented in Table 2.2. A total of five extracts, extracted using different solvents of varying polarity (ethanol, dichloromethane hexane, methanol, and methanol: water) were investigated. All five crude extracts had a degree of antibacterial activity against S. mutans. The MIC values of U. chamae against S. mutans ranged from 0.02 to 1.25 mg/ml whereas the MBC ranged from 0.04 to 1.25 mg/ml. The dichloromethane extracts of the roots showed the best antibacterial activity against all the five S. mutans strains with a MIC of 0.02 mg/ml and MBC of 0.04 mg/ml (Table 2.2). Ethanol and methanol showed a moderate activity with MIC of 0.63 mg/ml. Hexane and methanol: water extract showed the minimum antimicrobial activity with a MIC of 1.25 mg/ml. The positive control (chlorhexidine) killed S. mutans, and the negative control (tryptone soy broth) supported the growth of S. mutans. Dimethyl sulfoxide (DSMO) did not affect the S. mutans bacterial growth. 33 Table 2.1: Minimum Inhibitory concentrations (MIIC) and Minimum Bactericidal Concentrations (MBC) of U. chamae roots extract against S. mutans. Clinical strains Repeats Uvaria chamae MIC and MBC (mg/ml) Ethanol Dichloromethane Hexane Methanol Methanol: water MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC SM1 1 0.63 2.5 0.02 0.04 1.25 1.25 0.63 0.63 1.25 1.25 2 0.63 2.5 0.02 0.04 1.25 1.25 0.63 0.63 1.25 1.25 3 0.63 2.5 0.02 0.04 1.25 1.25 0.63 0.63 1.25 1.25 SM6 1 0.63 2.5 0.02 0.04 1.25 125 0.63 1.25 1.25 1.25 2 0.63 2.5 0.02 0.04 1.25 1.25 0.63 1.25 1.25 1.25 3 0.63 2.5 0.02 0.04 1.25 1.25 0.63 1.25 1.25 1.25 SM7 1 0.63 2.5 0.02 0.02 1.25 1.25 0.63 0.63 1.25 1.25 2 0.63 2.5 0.02 0.02 1.25 1.25 0.63 0.63 1.25 1.25 3 0.63 1.25 0.02 0.02 1.25 1.25 0.63 0.63 1.25 1.25 SM12 1 0.63 2.5 0.02 0.04 1.25 1.25 0.63 0.63 1.25 1.25 2 0.63 2.5 0.02 0.04 1.25 1.25 0.63 0.63 1.25 1.25 3 0.63 2.5 0.02 0.04 1.25 1.25 0.63 0.63 1.25 1.25 SM13 1 0.63 2.5 0.02 0.04 1.25 1.25 0.63 0.63 1.25 2.5 2 0.63 2.5 0.02 0.04 1.25 1.25 0.63 0.63 1.25 2.5 3 0.63 1.25 0.02 0.04 1.25 1.25 0.63 0.63 1.25 2,5 Mean n=15 0.63 2.33 0.02 0.04 1.25 1.25 0.63 0.75 1.25 1.50 ±SD 0.00 0.44 0.00 0.01 0.00 0.00 0.26 0.26 0.00 0.52 34 Table 2.2: Summary results of the mean MIC and MBC of U. chamae roots extract against S. mutans. Extraction solvents Mean MIC (mg/ml) n=15 Mean MBC (mg/ml) n=15 Ethanol 0.63 (moderate) 2.33 Dichloromethane 0.02 (good) 0.04 Hexane 1.25 (weak) 1.25 Methanol 0.63 (moderate) 0.75 Methanol: water 1.25 (weak) 1.50 2.4. Discussion It is well established that medicinal plants have antimicrobial activity against oral bacteria (Ghamari et al., 2017). The efficacy of natural products is significantly affected by the solvent and method used for extraction (Miliauskas et al., 2004). In this study, the MIC and MBC of U. chamae roots extracts against S. mutans were determined. The results showed that methanol: water, hexane, methanol, dichloromethane, and ethanol of U. chamae roots extracts had antimicrobial activity against S. mutans (Table 2.2). Antimicrobial activity of plant extracts has been classified previously as good (MIC< 0.1 mg/mL), moderate (0.1 ≤ MIC ≤0.625 mg/mL) and weak (MIC >0.625 mg/mL) (Famuyide et al., 2019). Based on these criteria, ethanol and methanol showed moderate antimicrobial activity against S. mutans. Hexane and methanol: water extracts showed weak antimicrobial activity against S. mutans. Dichloromethane extract showed a good activity with an average MIC of 0.02 mg/ml. These results suggest that bioactive compounds that inhibit the growth of S. mutans were better extracted with dichloromethane compared to other solvent. Earlier studies investigating aqueous root bark extracts of U. chamae have shown poor antimicrobial activity against S. mutans. They reported the MIC of 400 mg/ml (Amadi et al., 2007). The present study demonstrated that the mean MIC of U. chamae root extract against S. mutans ranged between 0.02 to 1.25 mg/ml. The difference in results may be due to the season of plant harvesting, solvents used for extraction, location, growth conditions, and storage duration of the plant extracts (Seleshe and Kang, 2019). 35 Generally, Gram-positive bacteria are more susceptible to antimicrobial drugs than Gram- negative bacteria (Kapoor et al., 2017). This can be attributed to the presence of an outer membrane layer that is rigid and rich in lipopolysaccharide (LPS) in Gram-negative bacteria. This protective layer limits the diffusion of hydrophobic compounds through it. Gram-positive bacteria cell is surrounded by thick peptidoglycan walls which are not dense enough to resist small antimicrobial molecules, thereby facilitating access to the cell membrane. Additionally, the presence of the lipophilic ends of lipoteichoic acids present in the cell membrane of Gram- positive may ease the infiltration of the hydrophobic bioactive compounds present in this plant extract (Chouhan et al., 2017) The activity of this plant extract may be attributed to alkaloids, tannins, steroids, terpenes, and flavonoids known to be present in the family Annonaceae. Annonaceae plants have been shown to possess antibacterial activities (Tamfu et al., 2019). A previous study by Lindsey reported that when active compounds are found in one species, the majority of species within the same genus contain active compounds of a similar nature (Lindsey et al., 1998). Similarly, the antimicrobial effect of this plant may be affiliated with the bioactive compounds present within the family Annonaceae (Attiq et al., 2017). These bioactive substances have different functions and mechanisms of action. For instance, the antimicrobial activity of flavonoids is due to their ability to complex with extracellular and soluble protein and to complex with bacterial cell wall while tannins may inactivate microbial adhesions, enzymes and cell envelop proteins (Mogana et al., 2020). A recent study (2019) reported that 2,4,60-trihydroxy-30-methylchalcone flavonoids lead S. mutans to leak intracellular substances such as protein and ions (Górniak et al., 2019). 2.5. Conclusion Uvaria chamae dichloromethane extract had the best antimicrobial activity followed by ethanol and methanol. Methanol: water and hexane extract had the least activity with MIC of 1.25 mg/ml. At a low MIC of 0.02 mg/ml, dichloromethane extract completely inhibited the growth of S. mutans. This study demonstrated that U. chamae dichloromethane extracts have the potential to be used to prevent dental caries. Further studies can be undertaken to study the effect of U. chamae on S. mutans virulence factors as well as its specific mechanism of action. These results also encourage further investigations on the active chemical compounds responsible for the observed antibacterial effect in this study. 36 Chapter 3: The effect of Uvaria chamae dichloromethane extract on the biofilm formation by Streptococcus mutans 3.1. Introduction Dental caries is a biofilm-induced oral disease that results due to a disruption of the microbial ecological balance in the oral cavity. This disturbance results in a population shift leading to the over-representation of pathogenic species in the oral cavities. Additionally, normal biota may also become opportunistic pathogens by the acquisition of gtfs genes through horizontal gene transfer thereby contributing to the onset and establishment of dental caries (Chen et al., 2016;Adedayo et al., 2020;Hoshino et al., 2012). Dental plaque is an oral microbial biofilm that forms on exposed tooth surfaces and is characterized by species diversity (Yu et al., 2017). Dental plaque comprises surface-attached bacterial communities encased in extracellular polysaccharides, proteins, and DNA (Davies, 2003) and they form through sequential events which may result in a structurally and functionally organized species-rich microbial community. The species composition of a plaque at a site is characterized by a degree of stability among the component species once it has formed. This stability is a result of a balance imposed by biochemical interactions where complex host glycoproteins catabolize to develop food chains and cell to cell signalling which leads to coordinated gene expression within the microbial community (Yadav and Prakash, 2017). Streptococcus mutans has been discovered as the primary etiological bacteria that predominantly proliferates in the dental biofilm (Akhalwaya et al., 2018). The pathogen S. mutans is a major bacterium producing the extracellular polysaccharide matrix in dental biofilms. During this biological process, S. mutans secretes the enzyme glucosyltransferases (GTFs) to synthesize glucans and binds them to the interface of bacteria and teeth leading to bacterial adhesion and biofilm formation. Bacteria in the plaque biofilm respond to many factors, such as cellular recognition of specific or non-specific attachment sites on a surface and nutritional signal using quorum sensing mechanisms (Yu et al., 2017;Yoshida and Kuramitsu, 2002;Zhang et al., 2020b). Quorum sensing plays a critical role in the formation of biofilm with its surrounding extracellular matrix (Munir et al., 2020;Lu et al., 2019). There are many treatments plans available in treating oral biofilms. These treatments include mechanical and chemical strategies. Mechanical strategies include physical removal of the dental plaque 37 by brushing and scrubbing. Chemical strategies include the use of mouthwashes and other products that contain chlorhexidine, stannous fluoride, and conventional antimicrobials (Sandasi et al., 2011). To date, several studies have investigated the effect of plant extracts and their products against different oral pathogens. Other researchers focused on the ability of the medicinal products to inhibit the formation of dental biofilms by reducing the adhesion of microbial pathogens to the tooth surface, which is a primary event in the formation of dental plaque and the progression to tooth decay (Palombo, 2011;Gabe et al., 2019;Nosrati et al., 2018;Teanpaisan et al., 2014). Many medicinal plants including Dodonaea viscosa var. angustifolia (Ngabaza et al., 2018), Curcuma (Kim et al., 2008), Prangos acaulis Bornm (Nosrati et al., 2018), Rhodiola rosea (Zhang et al., 2020b), Mikania (Yatsuda et al., 2005) have been shown to inhibit biofilm formation by S. mutans. The ability of these plant extracts to suppress and destruct mature biofilms has the potential to reduce microbial colonization of tooth surfaces and epithelial mucosa. In this chapter, the antibiofilm effects of U. chamae on S. mutans were investigated. Antibacterial effects of U. chamae have already been studied by some authors, but no report on the antibiofilm activity is available. Therefore, this chapter was to examine the effect of U. chamae extract on S. mutans biofilm formation. 3.2. Methods and materials 3.2.1. Bacterial cultures Five clinical isolates of cariogenic S. mutans (SM1, SM6, SM7, SM12, and SM13) were revived by culturing on blood agar plates. These cultures were grown at 37oC in a CO2-enriched environment for 48 hours. The inoculum was prepared by suspending the freshly grown S. mutans cells in phosphate buffered saline. 38 3.2.2. Plant materials The dichloromethane extract was selected based on its antimicrobial activity against S. mu