ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10024-3127 |
Clinical Efficacy of Single Use of Three Different Mouthrinses on the Level of Streptococcus mutans in Saliva
1,2Department of Pediatric Dentistry and Orthodontics, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
3Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department of Microbiology and Immunology, Faculty of Pharmacy, Al Azhar University, Cairo, Egypt
4–6College of Dentistry, King Saud University, Riyadh, Saudi Arabia
Corresponding Author: Mannaa K Aldowsari, Department of Pediatric Dentistry and Orthodontics, College of Dentistry, King Saud University, Riyadh, Saudi Arabia, Phone: +96655338765, e-mail: maldowsari@ksu.edu.sa
How to cite this article: Aldowsari MK, Salama F, Al-Agamy MH, et al. Clinical Efficacy of Single Use of Three Different Mouthrinses on the Level of Streptococcus mutans in Saliva. J Contemp Dent Pract 2021;22(7):769–773.
Source of support: Nil
Conflict of interest: None
ABSTRACT
Aim and objective: The aim of this cross-sectional population-based clinical study was to assess the effect of single use of three different mouthrinses on the level of salivary Streptococcus mutans of 8 to 10-year-old Saudi children.
Materials and methods: Convenient samples of 52 Saudi children aged 8–10 years were randomly allocated into four groups of 13 each. Saliva samples were collected to assess the level of S. mutans at baseline before rinsing with the assigned mouthrinse or control. Three mouthrinses, Avalon Avohex, Listerine Miswak, and Optima Aloe Dent Mouthrinse, were randomly distributed to the children. Each participant was instructed to rinse for 2 minutes using 10 mL of the assigned mouthrinse. Saliva samples were collected after rinsing and colony forming unit (CFU) of S. mutans per mL of saliva was calculated. Statistical analysis was performed to compare S. mutans count at baselines and postintervention values of each experimental group and control using paired t-test and one-way ANOVA. All statistical analyses were set at a significance level of p < 0.05.
Results: All test groups showed a reduction in salivary S. mutans compared to that at baseline. Statistically significant reduction (p > 0.05) in bacterial count was seen in Avalon Avohex group.
Conclusion: A single-time rinse of chlorhexidine extract mouthrinse for 2 minutes effectively reduced the number of S. mutans of 8 to 10-year-old Saudi children.
Clinical significance: Rinsing with chlorhexidine extract mouthrinse should be considered as a potential method in prevention of dental caries in children.
Keywords: Antimicrobial agent, Caries, Chlorhexidine, Clinical study, Mouthrinse, Streptococcus mutans.
INTRODUCTION
The dental biofilm has a main role in the etiology of dental caries and periodontal diseases.1 Dental caries is one of the most predominant and preventable oral infectious diseases that affect the majority of the world’s population and might lead to pain, tooth destruction, or loss.2 It is a multifactorial disease that is characterized by demineralization of the dental hard tissues.3 Streptococcus mutans is a gram-positive, facultative, anaerobic round-shaped bacterium, which is considered a key contributor to the formation of cariogenic plaque.4 It is a highly acidogenic and acid-tolerant bacteria that use dietary sucrose to synthesize large amounts of extracellular polysaccharides and adheres firmly to glucan-coated surfaces.4 Besides that, S. mutans has the ability to consume extra- and intracellular polysaccharides as short-term storage mixtures, which offer an extra increase in the amount of acid production and extent of acidification.5 Genetic variability of S. mutans is still not well understood.6
A study assessed the genetic variability of S. mutans by extensive whole-genome sequencing revealed that the core genome size of S. mutans was determined to be around 1,370 genes by including 67 S. mutans genomes.6 Many antiplaque agents have been in practice as additional aids.7 It is thought that using mouthrinses might act as an effective and harmless way for the delivery of antimicrobial agents that inhibit microbial adhesion and colonization and disturb the bacterial growth.7 Chlorhexidine is the gold standard mouthrinse due to its antimicrobial properties.8,9 On the contrary, despite its side effects like teeth and soft tissue staining, taste alteration, and supragingival calculus formation, its continued use is supported.9–11 Concerning dental caries, a systematic review found that no randomized clinical trials assessed chlorhexidine mouthrinses for the purpose of the prevention of dental caries in children and adolescents.12
Nowadays, an interest in the naturally derived biologically active compounds has been increased.13,14 These compounds might have a helpful and therapeutic usage in medicine and dentistry fields.13,14 Herbal extracts and plant essential oils (EOs) have the potential to be used as therapeutic agents for chronic gingivitis and periodontitis conditions that have both bacterial and inflammatory components.15,16 These are useful, as their long-term daily usage has no side effects on the health of an individual.15,16 In addition, these are more cost-effective and easily available as over-the-counter products. Aloe vera has shown antibacterial properties against a variety of bacteria mainly against S. mutans, which accounts for its antiplaque action.16 Some of the components of Aloe vera like vitamin C, hyaluronic acid, and dermatan sulfate are involved in collagen synthesis and, hence, can relieve swelling and bleeding gum.17 A study evaluated the effect of Aloe vera mouthrinse on the dental plaque in the investigational period of four days reported that Aloe vera could prove an effective mouthrinse due to its capability in minimizing dental plaque.18 Traditional medicinal plants can show biological action that improves oral health. For example, Salvadora persica L., family: Salvadoraceae (Sp), root extract is well reported for its antibacterial effect properties against dental plaque.19,20
Miswak (S. persica) has been used as a natural way for tooth cleaning in many parts of the world for thousands of years.21 A number of scientific studies have confirmed that the miswak (S. persica) possesses not only antibacterial but also antifungal, antiviral, anticariogenic, and antiplaque properties. It was stated that those using chewing sticks have saliva with a significant lower level of primary plaque colonizers (including Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, and Streptococcus salivarius) as compared to those utilizing conventional toothbrushes.22 A randomized controlled crossover clinical trial evaluation of S. persica L. reported an antiplaque effect for a 24-hour period.23 A study evaluated the effectiveness of Miswak products on cariogenic bacteria in comparison with regular toothpaste reported that Miswak products, particularly mouthrinse, were more effective in minimizing the growth of cariogenic bacteria than ordinary toothpaste.24
Currently, a global trend has been seen for the usage of natural products due to their proven pharmacological effects on the oral environment as efficient caries-preventive agents. Therefore, clinical studies are the way to confirm the actual contributions of natural products to caries reduction in children. In summary, naturally derivative compounds appear to have potential for preventing and/or treating dental caries. However, more clinical studies in this area are therefore still warranted. Hence, exploring more caries-preventive approaches and agents is needed. Therefore, the aim of this cross-sectional population-based clinical study was to evaluate the effect of single use of three different mouthrinses, Avalon Avohex, Listerine Miswak, and Optima Aloe Dent Mouthrinses, on the level of salivary S. mutans of 8–10-year-old Saudi children. The null hypothesis tested was reported that there is no difference between the effect of the tested mouthrinses and the positive control mouthrinse against the level of salivary S. mutans.
MATERIALS AND METHODS
The study was approved by the Ethical Committee of Human Studies, College of Dentistry Research Center, King Saud University, and Medical City, King Saud University.
Sample Size Calculation
The sample size was calculated using OpenEpi software (version 3.01), through the formula: n = (Zα/2 + Zβ)2 × 2 × σ2/d2. The power of the sample size when the number of participants was equivalent or more than 16 was calculated. At the two-sided confidence interval = 95%, α = 0.05, SD in each group (σ) = 100 million bacteria and the minimum acceptable difference (d) = 100 million bacteria, the power of the study = 80% (β = 0.2), and the ratio of randomization groups = 1, the sample size was ≥16. To be familiar with the study protocol, a pilot study was conducted before the start of the actual study. Operators were trained and calibrations were undertaken throughout the pilot study. Intra-examiner reproducibility of the investigators who performed the clinical investigation was assessed during the pilot study by reassessing 10% of the participants.
Inclusion and Exclusion Criteria
The inclusion criteria were healthy Saudi children aged 8–10 years, currently not under using any medication, have no active periodontal diseases, not underneath active orthodontic treatment, and with Decayed, missing due to caries, and filled teeth in the primary teeth (dmft) and Decayed, missing due to caries, and filled teeth in the permanent teeth (DMFT) equal or fewer than four.
A convenient sample of 52 Saudi children aged 8–10 years who visited the clinics of the dental hospital is decided to participate in this clinical study, and then, a consent form was signed by their parents or legal guardian. Study protocol was emphasized about the importance of following the instructions for the participated children before the saliva collection visit that was given within approximately 1 week after the initial visit.
Randomization and Blindness
Participants were randomly assigned into four groups of 13 each. Group 1 was assigned to use Avalon Avohex mouthrinse (Avalon Pharma, Riyadh, Kingdom of Saudi Arabia), group 2 was assigned to use Listerine Miswak Mouthrinse (Johnson and Johnson, Kingdom of Saudi Arabia), group 3 was assigned to Optima Aloe Dent Mouthrinse (Optima Naturals, Kingdom of Saudi Arabia), and group 4 assigned to distilled water mouthrinse as a negative control group. The ingredients of each mouthrinse are shown in Table 1. Allocation concealment was used in the randomization method to avoid any participant from knowing in advance which group he or she was part of. Different participants were assigned to conduct the experiment and collect the saliva. To create unbiased study environment, the participants who did the microbiological investigation did not know which mouthrinse was used for each sample; saliva samples were given different codes.
| Mouthwash | Ingredients | Manufacturer |
|---|---|---|
| Avalon Avohex | Chlorhexidine gluconate 0.2% w/v. | Avalon Pharma, Riyadh, Kingdom of Saudi Arabia |
| Listerine Miswak | Aqua, sorbitol, propylene glycol, poloxamer 407, sodium lauryl sulfate, zinc chloride, benzoic acid, eucalyptol, aroma, sodium benzoate, methyl salicylate, thymol, sodium fluoride, menthol, sodium saccharin, caramel, sucralose, glycerin, Salvadora persica bark extract, sodium fluoride (220 ppm F) | Johnson and Johnson, Kingdom of Saudi Arabia |
| Optima Aloe Dent | Aqua, Aloe Barbadensis, sorbitol, polysorbate 20, Citrus paradisi (grapefruit seed extract), Mentha piperita, sodium lauroyl sarcosinate, aroma menthol, Melaleuca alternifolia (tea tree oil), Escin (horse chestnut), Centella asiatica (Indian pennywort), xylitol sodium hydroxymethylglycinate, sodium monofluorophosphate, citric acid | Optima Naturals, Kingdom of Saudi Arabia |
Methods for Assessment of Oral Hygiene and Caries
Demographic data and dental as well as medical history were collected from parents and subjects’ files. Clinical examination was carried out for dental caries and oral hygiene status. For caries assessment, incidence of decayed, missed, and filled surfaces was documented in the primary Decayed, missing due to caries, and filled surfaces in the primary teeth (dmfs) as well as in the permanent Decayed, missing due to caries, and filled surfaces in the permanent teeth (DMFS) dentition according to the World Health Organization.25
Oral hygiene was measured by using DI-S score, which describes the level of soft deposits and is one of the two components of the Simplified Oral Hygiene Index (OHI-S) introduced by Green and Vermillion.26–28
Sample Collection
The participants did not consume food or drink any liquids (including water and soft drink) for 60 minutes before the collection of saliva. Besides that, the participants should not brush their teeth for at least 8 hours before the saliva collection. The saliva was collected in an aseptic condition. Each participant was rinsed twice with 10mL of water for 30 seconds. After 2 minutes, each child was requested to spit normal (unstimulated) saliva (2mL) into a sterilized disposable 5-mL tube (before rinsing with the assigned mouthrinse) to use for baseline count of S. mutans.
Each child was asked to rinse with 10 mL of the assigned mouthrinse or control for 2 minutes according to their assigned group. Saliva samples were collected after 15 minutes using the same procedures for the baseline. Each tube was then labeled with a unique identifier. The key of the unique identifier was known to the investigators who collected the samples but not to the laboratory personnel. Saliva samples were transported within 1 hour to the microbiology laboratory for preparation and analysis of salivary levels of S. mutans. At the end of the research, any leftover saliva samples were discarded. Ten tubes, which contain 4.5 mL sterile 0.9% (w/v) sodium chloride, were used for every sample. The tubes were labeled from 1–10. A 0.5 mL of saliva was added to the first tube to a variety of 10-fold dilution. The solution was mixed vigorously by vortex mixer (VWR International Global Exports, Arlington Heights, Illinois, USA). A 0.5 mL from the primary tube was moved into the second tube and mixed vigorously by vortex mixer (VWR International Global Exports, Arlington Heights, Illinois, USA). The process was repeated down to the tenth tube. A 100-μL volume from each dilution was plated into TYCBS agar plate and evenly distributed on the agar surface using sterile glass rod spreaders (Thomas Scientific, Swedesboro, New Jersey, USA). Each dilution was done in duplicate. The plates were incubated aerobically (Memmert 854; Memmert GmbH + Co.KG; Schwabach, Germany) for 48 hours at 37°C. After the incubation period, the colonies on the plates were calculated using a digital colony counter (Colony Counter, Gallenkamp, Co. Ltd., England), and the colony forming unit (CFU) of S. mutans per mL of saliva was calculated.
Statistical Analysis
Data were analyzed using SPSS version 20.0 statistical software (IBM, SPSS Inc., Chicago, Illinois). The mean and standard deviation of S. mutans count between baseline and postintervention (after rinsing) values of every experimental group and control were recorded and compared using paired t-test and one-way ANOVA. The level of significance was set at a p-value of <0.05.
RESULT
For intra-examiner reliability of the investigators who performed the clinical examination, kappa was 0.93 which indicates a very good agreement in doing the clinical examination. The mean and standard deviation of DMFS for Avalon Avohex, Optima Aloe Dent, and Listerine Miswak groups were 5.00, 4.92, and 4.30, respectively. None of the participant was plaque free.
The mean and standard deviation of the number of S. mutans at baseline and after rinsing for all mouthrinse groups are shown in Table 2. There was evidence of bacterial reduction of S. mutans rinsing with all of the mouthrinses. To assess whether the reduction in the bacterial count was significantly different within the same group, paired t-test was performed and the results showed that there was a statistically significant reduction in the mean number of S. mutans in the children who were assigned to Avalon Avohex in group 1 (p<0.004) (Table 2).
| Mean (SD) | |||
|---|---|---|---|
| Groups | Baseline | Postintervention | p value |
| Avalon Avohex | 1.65 × 108 (1.41 × 108) | 3.62 × 107 (4.44 × 107) | 0.004* |
| Optima Aloe Dent | 9.02 × 107 (1.63 × 108) | 1.50 × 107 (1.08 × 107) | 0.109 |
| Listerine Miswak | 1.03 × 108 (2.11 × 108) | 3.01 × 107 (3.79 × 107) | 0.215 |
| Distilled water | 8.10 × 107 (1.37 × 108) | 2.54 × 107 (3.30 × 107) | 0.089 |
A one-way ANOVA test was performed to evaluate whether the difference of the baseline and postintervention of the quantity of S. mutans was statistically significant among the four groups. The results showed that the mean difference in the reduction of the number of S. mutans was not statistically significant among the groups (p>0.05). Figure 1 shows the rank differences of the baseline and postintervention of the quantity of S. mutans in all the groups. Figure 2 shows the culture at baseline and after rinsing with the assigned mouthrinses.
Fig. 1: Ranks of the difference in the baseline and postintervention of the number of Streptococcus mutans in all the groups
Figs 2A to H: Culture showing results: (A) At baseline in distilled water group; (B) After rinsing with distilled water mouthrinse; (C) At baseline in Aloe Dent group; (D) After rinsing with Aloe Dent mouthrinse; (E) At baseline in Avalon Avohex group; (F) After rinsing with Avalon Avohex mouthrinse; (G) At baseline in Listerine Miswak group; (H) After rinsing with Listerine Miswak mouthrinse
DISCUSSION
The null hypothesis tested in the current study was accepted as there was no significant difference in the effect of the tested mouthrinses and the positive control group against the level of salivary S. mutans. The results of our study showed that there was a reduction in the number of S. mutans after using mouthrinses in all groups compared with the numbers at baseline. However, a statistically significant reduction was only seen in Avalon mouthrinse group. These findings were in agreement with the findings of another randomized clinical trial which showed that chlorhexidine extract mouthrinses were effective in the reduction of S. mutans.23 The active ingredients in Avalon mouthrinse are chlorhexidine gluconate. Chlorhexidine is the gold standard mouthrinse due to its antimicrobial properties.8,9 It was surprising that tested mouthrinse products did not show a statistically significant reduction of the number of S. mutans in comparison with the control group. This result is not expected but what is expected is to have more positive effect on the bacterial count. The findings are inconsistent with the findings of a study that showed Aloe vera mouthrinse can be an effective mouthrinse to reduce dental plaque.18 In addition, our findings are not in agreement with a study that demonstrated that Miswak products, especially mouthrinse, have a significant effect in reducing the growth of cariogenic bacteria.24 There are several possible explanations for these unexpected findings. One explanation could be the frequency of the exposure; the present study used a single use of mouthrinses. Several studies have reported significant effects when used more frequently over a longer duration.18,23 Another possible explanation could be related to the composition of the mouthrinses used in this study. In our study, we used alcohol-free mouthrinses. Several clinical studies demonstrated that alcohol-containing chlorhexidine mouthrinses showed a significant effect in preventing plaque regrowth and reducing bacterial activity when compared to alcohol-free chlorhexidine mouthrinses.29,30
The present study used an in vivo model to assess the ability of the tested mouthrinses to reduce the number of S. mutans in saliva. The model has several advantages. The main advantage of using this model is that it uses the natural oral environment that makes the findings more relevant. However, this model has some limitations. The main disadvantage is that it depends mainly on participants’ compliance. In addition, standardization is required to minimize the variations between the participants. In the current study, specific inclusion and exclusion criteria were applied to standardize the selection of the participants.
Several methods and techniques can be used for oral bacterial and sample collection. Saliva sample collection and processing are important factors that might have an effect on the observed values. Therefore, these factors should be taken into account when comparing the results of different studies. Microbiologic quantification has been shown to be the most accurate bacterial test. In the current study, bacterial culture from saliva at baseline and after rinsing with the assigned mouthrinses were obtained and used as microbiologic quantification. The technique showed the number of S. mutans within a reasonable time.
In the present study, mouth rinsing was performed for 2 minutes only. This protocol was used in a previous study and showed significant reduction of S. mutans after the use of tea mouthrinse.31 However, other study showed positive correlation between increased exposure time and lower level of S. mutans.32
There are some limitations to this study. The first limitation is that due to the current pandemic of coronavirus disease-2019 (COVID-19) and other unavoidable circumstances, we could not reach the calculated sample size. Future studies with a larger sample size would strengthen the research. In the present study, single use of assigned mouthrinse was used. It would be interesting to see the long-term effect of the assigned mouthrinses. We suggest further randomized studies with more extended period and repeated use rather than single use to evaluate the long-term effect of herbal extract mouthrinses on reducing bacterial biofilm.
CONCLUSION
Within the limitations of this study, it was concluded that in all groups, there was evidence of reduction of the number of S. mutans when compared to that at baseline. A statistically significant difference in terms of the number of S. mutans was only seen in the Avalon Avohex group. No statistically significant differences in the mean difference in bacterial reduction were found between the tested mouthrinses.
ACKNOWLEDGMENTS
The authors would like to thank the College of Pharmacy Research Center, Deanship of Scientific Research at King Saud University Saudi Arabia, and Wareef Charity, Saudi Arabia, for funding this research project. In addition, the authors would like to thank Dr. Fouad Salama for the idea of the research project.
ORCID
Mannaa K Aldowsari https://orcid.org/0000-0002-6265-729X
REFERENCES
1. Chandki R, Banthia P, Banthia R. Biofilms: a microbial home. J Indian Soc Periodontol 2011;15(2):111–114. DOI: 10.4103/0972-124X.84377.
2. Yadav K, Prakash S. Dental caries: a review. Asian J Biomed Pharm Sci 2016; 53(6): 1–7. DOI: 10.15272/ajbps.v6i53.773.
3. Selwitz R, Ismail A, Pitts N. Dental caries. Lancet 2007;369(9555): 51–59. DOI: 10.1016/S0140-6736(07)60031-2.
4. Hamada S, Slade H. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev 1980;44(2):331–384. DOI: 10.1128/mr.44.2.331-384.1980.
5. Busuioc M, Mackiewicz K, Buttaro B, et al. Role of intracellular polysaccharide in persistence of Streptococcus mutans. J Bacteriol 2009;191(23): 7315–7322. DOI: 10.1128/JB.00425-09.
6. Song L, Wang W, Conrads G, et al. Correction to genetic variability of mutans streptococci revealed by wide whole-genome sequencing. BMC Genom 2013;14:430. DOI: 10.1186/1471-2164-14-811.
7. Sharma U, Jain RL, Pathak A. A clinical assessment of effectiveness of mouthwashes in comparison to toothbrushing in children. J Indian Soc Pedod Prev Dent 2004;22(2):38–44.
8. Van der Weijden FA, Van der Sluijs E, Ciancio SG, et al. Can chemical mouthwash agents achieve plaque/gingivitis control? Dent Clin North Am 2015;59(4):799–829. DOI: 10.1016/j.cden.2015.06.002.
9. James P, Worthington HV, Parnell C, et al. Chlorhexidine mouthrinse as an adjunctive treatment for gingival health. Cochrane Database Syst Rev 2017;2017(3). DOI: 10.1002/14651858.CD008676.pub2.
10. Flötra L, Gjermo P, Rölla G, et al. Side effects of chlorhexidine mouth washes. Eur J Oral Sci 1971;79(2):119–125. DOI: 10.1111/j.1600-0722.1971.tb02001.x.
11. Ernst CP, Canbek K, Dillenburger A, et al. Clinical study on the effectiveness and side effects of hexetidine and chlorhexidine mouthrinses versus a negative control. Quintessence Int 2005;36(8):641–652.
12. Walsh T, Oliveira-Neto JM, Moore D. Chlorhexidine treatment for the prevention of dental caries in children and adolescents (Review). Cochrane Database Syst Rev 2015;(4):CD008457. DOI: 10.1002/14651858.CD008457.pub2.
13. Groppo F, Bergamaschi C, Cogo K, et al. Use of phytotherapy in dentistry. Phytother Res 2008;22(8):993–998. DOI: 10.1002/ptr.2471.
14. Newman DJ. Natural products as leads to potential drugs: an old process or the new hope for drug discovery? J Med Chem 2008;51(9):2589–2599. DOI: 10.1021/jm0704090.
15. Jain I, Jain P. Comparative evaluation of antimicrobial efficacy of three different formulations of mouth rinses with multi-herbal mouth rinse. J Indian Soc Pedod Prev Dent 2016;34(4):315–323. DOI: 10.4103/0970-4388.191409.
16. Fani M, Kohanteb J. Inhibitory activity of Aloe vera gel on some clinically isolated cariogenic and periodontopathic bacteria. J Oral Sci 2012;54(1):15–21. DOI: 10.2334/josnusd.54.15.
17. Karim B, Bhaskar DJ, Agali C, et al. Effect of Aloe vera mouthwash on periodontal health: triple blind randomized control trial. Oral Health Dent Manag 2014;13(1):14–9.
18. Gupta RK, Gupta D, Bhaskar DJ, et al. Preliminary antiplaque efficacy of aloe vera mouthwash on 4 day plaque re-growth model: randomized control trial. Ethiop J Health Sci 2014;24(2):139–144. DOI: 10.4314/ejhs.v24i2.6.
19. Almas K, Skaug N, Ahmad I. An in vitro antimicrobial comparison of miswak extract with commercially available non-alcohol mouthrinses. Int J Dent Hyg 2005;3(1):18–24. DOI: 10.1111/j.1601-5037.2004.00111.x.
20. Chelli-Chentouf N, Tir Touil Meddah A, Mullié C, et al. In vitro and in vivo antimicrobial activity of Algerian Hoggar Salvadora persica L. extracts against microbial strains from children’s oral cavity. J Ethnopharmacol 2012;144(1):57–66. DOI: 10.1016/j.jep.2012.08.025.
21. Haque MM, Alsareii SA. A review of the therapeutic effects of using miswak (Salvadora persica) on oral health. Saudi Med J 2015;36(5):530–543. DOI: 10.15537/smj.2015.5.10785.
22. Darout IA, Albandar JM, Skaug N, et al. Salivary microbiota levels in relation to periodontal status, experience of caries and miswak use in Sudanese adults. J Clin Periodontol 2002;29(5):411–420. DOI: 10.1034/j.1600-051x.2002.290505.x.
23. Abdulbaqi HR, Himratul-Aznita WH, Baharuddin NA. Evaluation of Salvadora persica L. and green tea anti-plaque effect: A randomized controlled crossover clinical Trial. BMC Complement Altern Med 2016;16(1):1–7. DOI: 10.1186/s12906-016-1487-0.
24. Al-Dabbagh SA, Qasim HJ, Al-Derzi NA. Efficacy of Miswak toothpaste and mouthwash on cariogenic bacteria. Saudi Med J 2016;37(9):1009–1014. DOI: 10.15537/smj.2016.9.15855.
25. World Health Organization Oral Health Survey. Basic Methods, 5th ed. Geneva, Swizerland: WHO, 2013.
26. Martínez MC, Tolcachir B, Lescano de Ferrer A, et al. Comparative study of preventive protocols in children at high cariogenic risk. Acta Odontol Latinoam 2012;25(2):218–222.
27. Wilkins EM. Clinical practice of the dental hygienist. 10th ed. Baltimore, MD: Lippincott Williams and Wilkins; 2009. p. 333–335.
28. Greene JC, Vermillion JR. The simplified oral hygiene index. J Am Dent Assoc 1964;68(1):7–13. DOI: 10.14219/jada.archive.1964.0034.
29. Arweiler NB, Boehnke N, Sculean A, et al. Differences in efficacy of two commercial 0.2% chlorhexidine mouthrinse solutions: a 4-day plaque re-growth study. J Clin Periodontol 2006;33(5):334–339. DOI: 10.1111/j.1600-051X.2006.00917.x.
30. Abu-Obaid E, Salama F, Abu-Obaid A, et al. Comparative evaluation of the antimicrobial effects of different mouthrinses against streptococcus mutans: an in vitro study. J Clin Pediatr Dent 2019;43(6):398–407. DOI: 10.17796/1053-4625-43.6.7.
31. Abdelmegid F, Al-Agamy M, Alwohaibi A, et al. Effect of honey and green tea solutions on Streptococcus mutans. J Clin Pediatr Dent 2015;39(5):435–441. DOI: 10.17796/1053-4628-39.5.435.
32. Ferrazzano GF, Roberto L, Amato I, et al. Antimicrobial properties of green tea extract against cariogenic microflora: an in vivo study. J Med Food 2011;14(9):907–911. DOI: 10.1089/jmf.2010.0196.
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