Department of Pedodontics and Preventive Dentistry, A. J. Institute of Dental Sciences, Mangalore, Karnataka, India

Transcription

1 Original Article Anti-microbial efficacy of green tea and chlorhexidine mouth rinses against Streptococcus mutans, Lactobacilli spp. and Candida albicans in children with severe early childhood caries: A randomized clinical study Ann Thomas, Sneha R Thakur, Sowmya B Shetty Department of Pedodontics and Preventive Dentistry, A. J. Institute of Dental Sciences, Mangalore, Karnataka, India ABSTRACT Introduction: Green tea is a beverage which is consumed worldwide and is reported to have anti-cariogenic effect. So, if it was as effective as chlorhexidine (CHX) mouth rinse against cariogenic microbes it could be considered a natural, economical alternative. The purpose of this study was to evaluate and compare the anti-microbial efficacy of 0.5% green tea and 0.2% CHX mouth rinses against Streptococcus Mutans, Lactobacilli spp. and Candida Albicans. Materials and Methods: 30 children aged 4-6 years with S-ECC (based ondefs score) were selected. Children were divided randomly into 2 equal groups and were asked to rinse with the prescribed mouth rinse once daily for 2 weeks after breakfast under supervision. A base-line and post rinsing non-stimulated whole salivary sample (2 ml) was collected and tested for the number of colony forming units. The data was statistically analyzed using SPSS v16.0 software with one-way ANOVA and Tukey sposthoc test. Results: A statistically significant fall in colony count was found with both the mouth rinses in Streptococcus Mutans (P < 0.001, P < 0.001) and lactobacilli (P < 0.001, P < 0.001) but not against Candida albicans (P = 0.264, P = 0.264). Against Streptococcus Mutans, green tea mouth rinse was found to be significantly better than CHX mouth rinse (P = 0.005). Against lactobacilli spp, CHX mouth rinse was significantly better than green tea mouth rinse (P < 0.001). Conclusion: Green tea mouth rinse can be considered safe, economical and used without much concern. However, further studies are recommended. KEYWORDS: Herbal mouth wash, severe early childhood caries, home oral hygiene, caries prevention Introduction The use of mouth rinses as an aide to other oral hygiene measures was first propagated by ancient Egyptians Address for correspondence: Dr. Sneha R Thakur, Department of Pedodontics and Preventive Dentistry, A. J. Institute of Dental Sciences, Kuntikana, Mangalore , Karnataka, India. snehathakur68@gmail.com Access this article online Quick response code Website: and Romans. As early as A. D, Pedanius Dioscorides, a Greek physician suggested a concoction of olive juice, pomegranate fillings, wine, and gum myrrh to treat halitosis, while Hippocrates is known to have advocated mouth rinsing with a mixture of alum, salt, and vinegar in the ancient times. [1] Mouth rinses are used generally for their analgesic, antiinflammatory, anti-microbial, and anti-cariogenic This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: reprints@medknow.com DOI: / PMID: How to cite this article: Thomas A, Thakur SR, Shetty SB. Anti-microbial efficacy of green tea and chlorhexidine mouth rinses against Streptococcus mutans, Lactobacilli spp. and Candida albicans in children with severe early childhood caries: A randomized clinical study. J Indian Soc Pedod Prev Dent 2016;34: Journal of Indian Society of Pedodontics and Preventive Dentistry Published by Wolters Kluwer - Medknow 65

2 activities. Currently, a wide range of mouth rinses such as chlorhexidine (CHX), sodium fluoride, and essential oils are available commercially. The American Dental Association (ADA) recommends that mouth rinses should be effective at modifying the micro-biota by selectively eliminating pathogens without negatively affecting the normal commensals of oral cavity. [2] Evidence illustrates that the long-term twice daily use of anti-plaque and anti-gingivitis mouth rinses, namely 0.12% CHX gluconate and essential oils with methyl salicylate which are approved by the Council on Dental Therapeutics of the ADA, do not have any negative effect on the oral microbial flora. [2] Studies have also demonstrated that the use of mouth rinses in children provided a significant reduction in the decayed extracted filled surface (defs) index. [2,3] The most common plaque-mediated disease in children is dental caries, which is also one of the most common childhood diseases. It is a preventable, localized infectious, multi-factorial disease resulting from the interaction among host, diet, and microflora on the tooth surface over a period of time, resulting in localized de-mineralization of hard tissues. The most implicated bacteria in its etiology are Streptococcus mutans for its initiation and Lactobacillus acidophilus for its progression. Recently, a number of reports in the scientific literature show an association between Candida albicans, a fungi, which is an opportunistic pathogen and a normal commensal of the oral cavity and progression of dental caries. [4,5] Dental caries in children below 6 years is called early childhood caries (ECC), and its severe debilitating form is called severe-ecc (S-ECC). Of the commercially available mouth rinses, CHX mouth rinse is widely used and is considered to be the gold standard due to its broad spectrum antimicrobial activity. CHX de-stabilizes the bacterial cell wall and interferes with osmosis. In addition, the bacterial uptake of CHX is very rapid, which facilitates rupture of the cell wall and finally the cytoplasmic membrane causing cell death. [6-8] However, Lindhe et al., reported a number of local side-effects with CHX use such as discoloration of teeth, altered taste sensation, mucosal irritation, parotid swelling, and enhanced supra-gingival calculus formation due to precipitation of salivary proteins and organic salts, which limits its use as a therapeutic agent. [9] The ease of availability and affordability of commercial mouth rinses is a major hurdle faced by the low socioeconomic communities. Thus, to achieve better oral health for masses, there is always a constant quest to identify and promote newer, economical products that can be easily procured even by the people living in remote areas where advanced healthcare facilities are inadequate. Identification of an effective mouth rinse formulation containing a commonly used condiment would prove highly beneficial and currently a number of them are reported in the scientific literature. Worldwide, tea is consumed as a beverage for centuries and has shown many health benefits. It is also a condiment available in most homes. Some reports in the Japanese literature have quoted that those who continuously drink a large amount of green tea have less tooth decay [10] and drinking green tea makes the mouth clean. [11] Green tea is reported to be very rich in fluoride and catechin, a bioactive component, which exerts an anti-cariogenic effect by inhibiting the proliferation of the streptococcal agent, interfering with the process of bacterial adhesion to tooth enamel and also by inhibiting glucosyltransferase. [12-14] It also mediates protective salivary components such as secretory immunoglobulins, lysozymes, lactoferrin, oral peroxidases histatins, mucins, or others, thus exhibiting an indirect anti-cariogenic effect. [15] Pharmacokinetic studies have shown that after rinsing with tea, catechins can be found in the saliva for up to 60 min and that the enzymatic breakdown of starch on food particles trapped in the mouth is noticeably reduced. [14] Studies on the anti-cariogenic effect of green tea polyphenols have revealed that these compounds have a high inhibitory effect against S. mutans and the acids produced by it. [14] The cariogenic activity of catechins present in green tea was found to be related to its role in the depletion of thiol group which in turn exerted bactericidal effect. [14] To our knowledge, there is a paucity of reports on comparing the anti-microbial efficacy of green tea mouth rinse to that of CHX, the gold standard, and also no reports regarding its antifungal effect against C. albicans, which is recently being implicated in the etiology of caries. Thus, the main purpose of this study was to evaluate and compare the anti-microbial efficacy of green tea (0.5%) mouth rinse to that of CHX (0.2%) against S. mutans, Lactobacilli spp., and C. albicans. Materials and Methods A randomized, double-blind, active, controlled clinical trial was conducted at a local preschool between November 2013 and February The study protocol was reviewed and approved by the Ethical Committee of the Institution and the study was in compliance with The Code of Ethics of the World Medication (Declaration of Helsinki) for experiments involving human subjects. An informed consent was obtained from authorities of the preschool and the parents of the subjects before the onset of the study. To conduct the study, 30 children aged 4-6 years were randomly selected from the preschool. Physically fit children with S-ECC as per the definition given by the American Academy of Pediatric Dentistry [16] were included in the study. Children who were unable to expectorate completely or brush their teeth on their own, had positive history of taking antibiotics 3 months prior to and during the study period, undergoing orthodontic treatment or with an 66 Journal of Indian Society of Pedodontics and Preventive Dentistry Jan-Mar 2016 Vol 34 Issue 1

3 intraoral prosthesis, and had any intraoral pathology were medically compromised or for whom parental consent was not given were also not included in the study. The defs of the children was recorded using visible light, mouth mirror, and community periodontal index probe. The sum total of defs was considered and based on the caries experience, children were selected for the study. All the study participants were given a tube of nonfluoridated tooth paste and a tooth brush. The brushing and mouth rinsing technique was demonstrated to all and were advised to brush twice daily. The participants were randomly divided into two groups of 15 each by lottery method, i.e., they were asked to pick up chits with the name of the mouth rinse written on them. The children were given prenumbered identical mouth rinse bottles and advised to rinse the mouth for 1 min using 5 ml of the respective mouth rinse daily for 2 weeks in the school. Rinsing was carried out under the supervision of the investigator for six working days of the week and on Sundays under parental supervision. Preparation of mouth rinses Green tea mouth rinse was custom prepared by the pharmacist. To prepare green tea mouth rinse, dried green tea leaves (obtained by open air drying) were grounded to a desirable size using an electrical mill, and then extracted by percolation using distilled water as solvent. Green tea which is rich in phenolic compounds (6%) was diluted to obtain a concentration of 0.5% phenolic compound using double distilled water. Authorized additive, peppermint flavor (1 g/l), and sodium saccharine (1 g/l), a sweetening agent, were used to formulate the mouth rinse. [17] Commercially available CHX (0.2%) mouth rinse (CLOHEX, Dr. Reddy s) was used in this study. Saliva samples A volume of 2 ml saliva samples were collected prior to the commencement of mouth rinsing, i.e., at baseline and after 2 weeks rinsing, i.e., postrinsing. Un-stimulated whole saliva samples were collected by asking the children to drool into a sterile container for 3-5 min, sitting in an upright position in a well lit room with good ventilation. They were collected in the morning between and a.m. to prevent any bias in the concentration of saliva due to circadian rhythm. [18] Children were also informed not to eat or drink anything (except water) 1 h before saliva collection to minimize possible food debris and stimulation of saliva. Microbial evaluation The samples were transported to the microbiological laboratory in box with ice-packs. They were tested for the colony forming units (CFU) of S. mutans, Lactobacilli spp., and C. albicans using mitis-salivariusbacitracin agar, Rogosa agar, and HiCrome agar, respectively (HiMedia Laboratories, Mumbai). All the saliva samples were serially diluted using physiological saline to obtain 3 concentration. A volume of 0.1 ml saliva sample was spread on the selective agar plates using a sterile glass spreader. The plates were incubated for 48 h at 37 C in the incubation chamber (ROTEK) to obtain maximum growth of microbial colonies. The CFU were identified by morphology, size, and color, and counting was done with a handheld digital colony counter (HiMedia, Mumbai). The colony count was expressed as the number of CFUs per millimeter (CFU/ml) of saliva. Semi-quantification of the number of colonies was done by multiplying the actual colony count with to adjust for the dilution factor. The colony counting was done by the same observer under the same conditions and at the same time of the day to avoid the intraobserver variability in the results. Evaluation of acceptability After completion of the study, the study participants were given a self-administered, close-ended dichotomous questionnaire regarding the acceptability of mouth rinse prescribed to each of them. The questionnaire consisted of three questions with two mutually exclusive options (YES/NO) to answer. The questions were based on the acceptability of the mouth rinses in terms of flavor, smell, and willingness to continue using the mouth rinse. A paper and pencil method was used to administer the questionnaire. With the help of the caretaker, the participants response was elicited and the questionnaire was completed. Outcome measures and statistical methods The study had a single end point at 2 weeks. The data were statistically analyzed using one-way variance ANOVA and Tukey s post-hoc honestly significant difference (HSD) test in SPSS Software 16.0 (SPSS Inc., Chicago). The results were considered statistically significant at 0.05 probability level. One-way variance ANOVA test was used to compare the mean of differential colony counts in the two mouth rinse groups and to assess the anti-microbial efficacy of green tea mouth rinse, which was the primary outcome of the study. Tukey s post-hoc HSD test was used for comparative analysis of two mouth rinse groups and to measure the secondary outcome, i.e., whether the newly formulated green tea mouth rinse was better than CHX mouth rinse. Results The mean age of the participants was 5 (standard deviation [SD]: 0.69) years and the mean defs was (SD: 8.58). Green tea showed a statistically significant fall in the colony counts of S. mutans and Lactobacilli spp. (P < 0.001, P < 0.001); whereas only a numerical fall in C. albicans colony count was found, which was not statistically significant (P = 0.264) [Table 1]. CHX showed a statistically significant fall in the colony counts of S. mutans and Lactobacilli spp. (P < 0.001, P < 0.001); whereas a numerical increase was observed Journal of Indian Society of Pedodontics and Preventive Dentistry Jan-Mar 2016 Vol 34 Issue 1 67

4 in the colony count of C. albicans, which was also not statistically significant (P = 0.264) [Table 1]. On comparison against S. mutans, green tea mouth rinse was found to be significantly better than CHX mouth rinse (P = 0.005). Against Lactobacilli spp., CHX mouth rinse was found to be significantly more effective than green tea mouth rinse (P < 0.001). Whereas against C. albicans, no statistically significant difference was found (P = 0.081) [Table 2]. Majority of the study participants in the green tea group had a positive response to flavor (n = 10; 66.7%); smell (n = 8; 55.3%); and willingness to rinse (n = 10; 66.7%). However, the response of the study participants in the CHX group was varying. Only 40% (n = 6) were positive to flavor, 66.7% (n = 10) were positive to smell, and 46.7% (n = 7) were positive to willingness to rinse. Discussion The purpose of this study was to evaluate and compare the anti-microbial efficacy of green tea mouth rinse to that of CHX mouth rinse on the level of salivary S. mutans, Lactobacilli spp., and C. albicans in children. The present study was carried out under real life conditions without altering the subjects routine oral hygiene practices except that they were advised to use a nonfluoridated dentifrice for cleaning their teeth. This was done to eliminate the bias resulting due to additional anti-microbial effect of fluoride from the dentifrice. To standardize rinsing, all the study participants were asked to rinse under monitoring at school. Thus, rinsing was carried out after breakfast and this could have also contributed toward reducing the microbial challenge. According to the results of this study, green tea was found to be a very effective anti-bacterial mouth rinse against S. mutans and Lactobacilli spp. with some antifungal activity against C. albicans. The antibacterial effect of green tea mouth rinse is in accordance to the previous reports where the authors stated that rinsing with green tea extract had valuable anti-cariogenic activities including inhibitory effect on cariogenic bacteria by inhibiting the adherence of bacterial cells to the tooth surfaces. [14,17,19,20] And also, green tea catechins maintain the salivary ph at a normal range, which is not a favorable condition for cariogenic bacteria to flourish. [21,22] The anti-fungal activity is in agreement to the earlier in vitro studies that have reported that green tea polyphenols and catechins inhibit the growth of C. albicans by 40% and 75%, respectively. [21,22] CHX mouth rinse is one of the best proven commercially available mouth rinses due to its broad spectrum anti-microbial activity and no systemic side effects [23] Scientific studies have reported that CHX mouth rinse was effective in reducing the colony counts of S. mutans in plaque biofilm; [18,23,24] Lactobacilli spp. colony counts, [23,25] and culturable microbes of tongue and saliva. [24] The present study also confirmed the anti-bacterial activity of CHX mouth rinse against S. mutans and Lactobacilli spp. However, no anticandida effect was found; in fact, a numerical increase was observed which was not statistically significant. This increase in C. albicans count could be assigned to various factors such as a direct trigger of C. albicans by CHX or due to depression of the bacterial counts aiding in the proliferation of this opportunistic pathogen. Another probable explanation is the basic differences in outer cell structure between bacteria and fungi, with the latter having a rigid outer chitin wall making it less amenable for lysis. [26] Bioavailability of CHX may also have been compromised in this study because rinsing was carried out only once daily and also, only 5 ml of mouth rinse was used, for reports have stated it to be about 12 h with 10 ml of 0.2% of CHX mouth rinse. [7,8] Most scientific reports have shown fungicidal Table 1: Mean differential colony counts of Streptococcus mutans, Lactobacilli spp., and Candida albicans in both the mouth rinse groups Mouth rinse group n Microorganism Mean baseline (CFU/ml) Mean postrinse (CFU/ml) Mean difference (CFU/ml) Significance (P) CHX 15 S. mutans <0.001** Lactobacilli spp <0.001** C. albicans Green tea 15 S. mutans <0.001** Lactobacilli spp <0.001** C. albicans **Very highly significant (P < 0.001). S. mutans = Streptococcus mutans; C. albicans = Candida albicans; CFU/ml = Colony forming units per millimeter Table 2: Inter comparison of the mean difference of colony counts of both the mouth rinse groups for Streptococcus mutans, Lactobacilli spp., and Candida albicans Dependent variable Group (I) Group (J) Mean difference (I J) 10 3 (CFU/ml) SE P S. mutans CHX Green tea , * Lactobacilli spp. CHX Green tea <0.001** C. albicans CHX Green tea *Significant (P = 0.005); **Very highly significant (P < 0.001). SE = Standard error; CHX = Chlorhexidine; S. mutans = Streptococcus mutans; C. albicans = Candida albicans 68 Journal of Indian Society of Pedodontics and Preventive Dentistry Jan-Mar 2016 Vol 34 Issue 1

5 and fungi static action of CHX where it was used in combination with other chemical solutions such as cetylpyridinium chloride, fluconazole, and chitosan due to their synergistic action. [27-29] On comparison of the anti-microbial efficacy of green tea mouth rinse to that of 0.2% CHX, we found that it was significantly better against S. mutans, but less effective against Lactobacilli spp. and comparable against C. albicans. There is a paucity of reports comparing the anti-bacterial effect of green tea and CHX mouth rinses whereas the anti-plaque property of these mouth rinses has been reported to be similar. [19] While in another report, 0.5% green tea mouth rinse was reported to be better than 0.2% CHX mouth rinse. [30] Kaur et al., have suggested that green tea catechins helps to maintain the normal plaque ph, which in turn is an unfavorable condition for S. mutans to colonize and multiply. [19] In our study, the green tea mouth rinse was widely accepted by the study participants, whereas CHX mouth rinse had poor acceptance rate and this is a documented drawback of CHX. [6,8,31] Conclusion From the results of our study, it can be concluded that green tea mouth rinse could be very good costeffective mouth rinse. However, further studies would be beneficial to evaluate any potential adverse effects with long-term use of this mouth rinse. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. References 1. Kukreja BJ, Dodwad V. Herbal mouthwashes: A gift of nature. Int J Pharm Biol Sci 2012;3: Kocak MM, Ozcan S, Kocak S, Topuz O, Erten H. Comparison of the efficacy of three different mouthrinse solutions in decreasing the level of Streptococcus mutans in saliva. Eur J Dent 2009;3: Balagopal S, Arjunkumar R. Chlorhexidine: The gold standard antiplaque agent. J Pharm Sci Res 2013;12: Zafar S, Harnekar SY, Siddiqi A. 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6 25. Sreenivasan PK, Gittins E. The effects of a chlorhexidine mouthrinse on culturable microorganisms of the tongue and saliva. Microbiol Res 2004;159: Malhotra N, Rao SP, Acharya S, Vasudev B. Comparative in vitro evaluation of efficacy of mouthrinses against Streptococcus mutans, Lactobacilli and Candida albicans. Oral Health Prev Dent 2011;9: Fathilah AR, Himratul-Aznita WH, Fatheen AR, Suriani KR. The antifungal properties of chlorhexidine digluconate and cetylpyrinidinium chloride on oral Candida. J Dent 2012;40: Calamari SE, Bojanich MA, Barembaum SR, Berdicevski N, Azcurra AI. Antifungal and post-antifungal effects of chlorhexidine, fluconazole, chitosan and its combinations on Candida albicans. Med Oral Patol Oral Cir Bucal 2011;16: e Ellepola AN, Joseph BJ, Khan ZU. Effects of subtherapeutic concentrations of chlorhexidine gluconate on germ tube formation of oral Candida. Med Princ Pract 2012;21: Balappanavar AY, Sardana V, Singh M. Comparison of the effectiveness of 0.5% tea, 2% neem and 0.2% chlorhexidine mouthwashes on oral health: A randomized control trial. Indian J Dent Res 2013;24: Oyanagi T, Tagami J, Matin K. Potentials of mouthwashes in disinfecting cariogenic bacteria and biofilms leading to inhibition of caries. Open Dent J 2012;6: AUTHOR INSTITUTION MAP FOR THIS ISSUE Please note that not all the institutions may get mapped due to non-availability of requisite information in Google Map. For AIM of other issues, please check Archives/Back Issues page on the journal s website. 70 Journal of Indian Society of Pedodontics and Preventive Dentistry Jan-Mar 2016 Vol 34 Issue 1