Effect of Green Tea Mouthwash on Oral Malodor

This study aimed to determine the effect of green tea mouthwash on oral malodor, plaque, and gingival inflammation. Gingivitis subjects who had over 80 parts per billion of volatile sulfur compounds (VSC) in the morning breath were randomly assigned into green tea or placebo mouthwash group. At baseline, VSC, Plaque Index (PI) and Papillary Bleeding Index (PBI) were recorded. Participants were rinsed with the assigned mouthwash, and VSC level was remeasured at 30 minutes and 3 hours postrinsing. For the following 4 weeks, participants were asked to rinse with the assigned mouthwash twice daily. VSC, PI and PBI were remeasured at day 28. It was found that, at 30 minutes and 3 hours postrinsing, VSC was reduced by 36.76% and 33.18% in the green tea group and 19.83% and 9.17% in the placebo group, respectively. At day 28, VSC was reduced by 38.61% in the green tea group and 10.86% in the placebo group. VSC level in the green tea group was significantly different when compared to the placebo. PI and PBI were significantly reduced in both groups. However, no significant difference was found between groups. In conclusion, green tea mouthwash could significantly reduce VSC level in gingivitis subjects after rinsing for 4 weeks.


Introduction
Oral malodor is de�ned as an unpleasant breath odor whose causes originate from the mouth. is condition is caused mainly by the emanation of volatile sulfur compounds (VSC) especially hydrogen sul�de, methyl mercaptan, and dimethyl sul�de through the mouth air. ese foul-smelling compounds are produced through the degradation of sulfurcontaining amino acids by anaerobic Gram-negative bacteria. Other odoriferous compounds, for example, indole and cadaverine are also reported to cause oral malodor [1].
Oral malodor was found to correlate with periodontal disease. Quirynen et al. [2] found that 11% of 2,000 patients who suffered from bad breath had periodontal health problem. Takeuchi et al. [3] reported a positive association between periodontal parameters and the severity of oral malodor. e increase in oral malodor in subjects with periodontal disease is attributed to a higher number of Gram-negative, periodontal bacteria within the oral cavity. ese bacteria including Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Tannerella forsythia, and Eubacterium species and spirochetes are known to produce signi�cant amount of VSC [4]. In addition, desquamated cells or blood elements from the in�amed gingival tissue can provide sources for microbial putrefaction. In contrast, oral malodor was found to decrease aer subsidence of gingival in�ammation [3].
Mechanical cleaning methods including tooth brushing and tongue scraping have been found to reduce oral malodor [5]. However, such methods are not adequately practiced by large number of individuals. erefore, the use of mouthwash is provided as an alternative approach [6,7]. e mechanism of mouthwash to reduce odor intensity occurs through an antimicrobial activity against oral bacteria or the conversion of VSC into nonodorous compounds [1].

ISRN Preventive Medicine
Catechins showed an in vitro bactericidal activity against odor-producing, periodontal bacteria, P. gingivalis, and Prevotella spp., with an MIC of 1.0 mg/mL [8]. Green tea also inhibited adherence of P. gingivalis to oral epithelial cells at a concentration below 0.25 mg/mL [9]. Catechins and its derivatives could reduce periodontal breakdown by inhibiting collagenase [10] and cysteine proteinase activity of P. gingivalis [11]. EGCG has an inhibitory effect on protein tyrosine phosphatases (PTPase) activity in P. intermedia [12]. Immediately aer administered, green tea powder could reduce VSC concentration in the mouth air [13]. Green tea could also inhibit VSC production in a saliva-putrefaction system [13]. In this study, green tea mouthwash was formulated. We hypothesized that green tea mouthwash could reduce oral malodor aer one-time usage and could reduce oral malodor as well as gingival in�ammation aer 4-week usage in gingivitis subjects. us, the objectives of this study were to determine the effect of green tea mouthwash on the level of VSC aer one-time usage and on the level of VSC, plaque and gingival in�ammation aer 4-week usage in gingivitis subjects.

Method
is study was a double-blinded and placebo-controlled clinical trial. e study design was followed to the American Dental Association guideline for the assessment of efficacy of oral malodor products [15]. e study protocol was documentary proved by Mahidol University Institutional Review Board (COA. no. MU-IRB 2008/177.1211) and registered in ClinicalTrials.gov (NCT00932347). e study was performed at the Postgraduate Clinic, Faculty of Dentistry, Mahidol University, Bangkok, ailand.
Green tea mouthwash and placebo mouthwash were prepared from the Department of Pharmacology, Faculty of Dentistry, Mahidol University. Green tea mouthwash was a hydroalcoholic brownie solution. It contained green tea extract, propylene glycol, parabens, saccharin, and mint �avor. e green tea extract had more than 80% of total catechins. e placebo mouthwash was a hydroalcoholic brownie solution. It contained the same ingredients but without green tea extracts.

Participants.
Sixty participants were recruited from gingivitis patients at the Faculty of Dentistry, Mahidol University. ose who had at least 20 teeth and had over 80 parts per billion (ppb) of VSC in the mouth air were invited to enroll in the study. Patients were excluded from the study if they had systemic complicating factors or oral mucosal lesions. ey were also excluded from the study if they were smokers, were denture wearers, or took an antibiotic 1 month prior to the study. Before entering the study, informed consents were obtained from all participants.

Sample Size Calculation.
Number of participants was calculated according to the level of VSC (primary outcome measure) aer using 0.2% chlorhexidine mouthwash or 0.03% triclosan mouthwash [16]. We calculated the sample size as follows: (1) A sample size of at least 25 participants in each group would provide 80% power of detection at a two-sided 5% signi�cance level. irty participants were enrolled in each group to compensate for the drop out participants.

Screening Examination.
Screening examination was done between 7.00-8.30 a.m. Patients were asked to refrain from tooth brushing, mouth rinsing, eating, or drinking for at least 2 hours prior to the measurement. ey were asked to rinse with water for at least 20 minutes before the measurement to protect dry mouth effect and stop talking for at least 5 minutes. VSC level was measured by portable sul�de monitor (R�-17, �alimeter, Interscan Corp, CA, USA) as described by the manufacturer. e measurements were performed 3 times, and the mean levels of VSC were recorded as peak ppb. Only gingivitis patients who had over 80 ppb of VSC were invited to enroll the study.

Experimental Procedure.
Aer screening examination, participants were strati�ed according to their VSC level. e participants in each strata were randomly allocated into the green tea mouthwash group (mouthwash containing C. sinensis extracts) or the placebo mouthwash group using sealed envelopes. Baseline parameters consisting of VSC level, Plaque Index; PI [17], and Papillary Bleeding Index; PBI [18] were recorded by one examiner (P. Phusudsawang) who was blind to the allocation until data collection was completely done. Participants were asked to refrain from tooth brushing, mouth rinsing, eating, or drinking for at least 2 hours prior to the measurement. ey were asked to rinse with water for at least 20 minutes before the measurement to protect dry mouth effect and stop talking for at least 5 minutes. VSC level was measured between 7.00-8.30 a.m. by the sul�de monitor as previously described. Subsequently, PI and PBI were scored on the Ramord teeth. Results were recorded as means of the buccal and lingual surfaces examined. e participants were thoroughly rinsed with 15 mL of the assigned mouthwash for 1 minute. To determine the effect of mouthwash on oral malodor aer one time usage, the level of VSC was remeasured at the following 30 minutes and 3 hours. Within this period, participants were refrained from tooth brushing, rinsing, eating, or drinking.
To determine the effect of mouthwash on oral malodor and plaque and gingival in�ammation aer 4-week usage, the participants were asked to rinse with the assigned mouthwash twice daily aer tooth brushing and were asked to refrain from drinking or water rinsing for at least 30 minutes aer mouthwash usage. At day 28, VSC, PI, and PBI were recorded again as previously described (Figure 1). During the course of the study, participants were asked to use the assigned toothbrush and toothpaste (M-Dent, Mahidol University) and continued their usual oral hygiene and dietary habits but refrain from rinsing with other mouthwash.

Evaluation of Participants' Compliance and Side Effects.
At day 14 and day 28, participants were asked to return the bottles of the assigned mouthwash. e remaining content of mouthwash was measured. At day 28, they were also asked to complete the questionnaire regarding xerostomia, burning sensation, and changes in taste perception and received oral examination to determine tooth staining and other signs of mucosal irritation. At the end of the experiment, oral hygiene instruction and oral prophylaxis were given to all participants.

Statistical Analysis.
For the comparison within group, paired -test was used to analyze the VSC level between baseline and 30 minutes, baseline and 3 hours, as well as baseline and day 28, while Wilcoxon sign rank test was used to analyze PI and PBI between baseline and Day 28. For the comparison between green tea mouthwash group and placebo mouthwash group, -test was used to compare VSC level, and Mann-Whitney test was used to compare PI and PBI. e signi�cance level was set at .

Study Population.
Sixty participants completed the study. ere was no dropout participant. e green tea mouthwash group comprised of 30 participants (27 women and 3 men) with an age range between 18-55 years (mean age of 27 2 ± 9 1 years). e placebo mouthwash group comprised of 30 participants (27 women and 3 men) with an age range between 19-42 years (mean age of 2 8 ± 7 6 years). During the experimental period, none of them received medical interventions which might in�uence the outcome of the study. At baseline, VSC, PI, and PBI were not signi�cantly different between groups.

Effect of Green Tea Mouthwash on Level of Volatile Sulfur Compounds (VSC).
At baseline, VSC level in the green tea group and the placebo group was 187 7 ± 9 3 ppb  Figure 2). e reduction of VSC at 30 minutes and 3 hours postrinsing was 36 76 ± 22 % and 33 18 ± 32 29% in the green tea group and 19 83 ± 2 2 % and 9 17 ± 27 81% in the placebo group, respectively. Compared to baseline, VSC level was signi�cantly different in the green tea group at 30 minutes ( ) and 3 hours ( ) postrinsing. It appeared that the green tea mouthwash could more effectively reduce VSC level when compared to the placebo mouthwash; however, no signi�cant difference was found between groups at any time points ( 17 at 30 minutes; 8 at 3 hours). At day 28, VSC level was 1 ± 66 6 ppb (95% CI: 81.6-129.3 ppb; 38 61 ± 36 18% reduction from baseline) in the green tea group and 162 4 ± 11 7 ppb (95% CI: 121-203.8 ppb; 1 86 ± 2 4 % reduction from baseline) in the placebo group (Figure 2). VSC level in the green tea group was signi�cantly different when compared to baseline ( ) and to the placebo group ( 23).

Evaluation of Participants' Compliance and Side Effects.
e remaining content of the mouthwash was not different at day 14 and day 28 in both groups (data not shown) and thus indirectly indicated good participants' compliance. In this study, tooth staining, oral tissue irritation and desquamation, and burning sensation as well as abnormality in the taste perception aer using green tea mouthwash or placebo mouthwash for 4 weeks were not found.

Discussion
e pharmacological activity of green tea has been well documented in the �eld of dentistry [8][9][10][11][12]. In this study, green tea mouthwash was formulated, and its effect on oral malodor was assessed at different periods aer applying. At 30 minutes and 3 hours postrinsing, green tea mouthwash could reduce VSC level by 36.76% and 33.18%, respectively. e level of VSC at 30 minutes and 3 hours postrinsing was signi�cantly different when compared to baseline. However, no signi�cant difference in the VSC level was found when compared to the placebo mouthwash. e reduction of VSC level aer using green tea or placebo mouthwash may be explained by the action of rinsing which is capable of removing intraoral bacteria, desquamated cells, or tissue debris. Although green tea catechins can transform VSC to nonodorous compounds through the reaction with sulydryl and amino groups of VSC [19], this ability might be masked by the washout effect.
To assess the effect of green tea mouthwash on VSC level and gingival in�ammation, the mouthwash was assigned to use twice daily for 4 weeks in gingivitis subjects. e result showed that VSC level was reduced by 38.61% aer using green tea mouthwash for 4 weeks. Green tea mouthwash could signi�cantly reduce VSC level when compared to baseline and to the placebo.e ability of green tea mouthwash in reducing oral malodor may result from an antimicrobial activity of green tea catechins, especially on P. gingivalis [8,9] as well as the ability of green tea catechins to transform VSC to non-odorous compounds [19].
Regarding the effects of green tea mouthwash on plaque level, the reduction of plaque was found in both groups without signi�cant difference between groups at the end of the experiment. is may be explained by the Hawthorne's effect. e reduction of VSC was not associated with the reduction of plaque in this study. Because plaque index used in this study could not detect qualitative difference in plaque, the effect of green tea mouthwash on certain periodontal bacteria needs to be further elucidated.
e interproximal area of the teeth is the area where plaque accumulation is commonly found. In this study, Papillary Bleeding Index (PBI) which re�ected the condition of the interproximal marginal periodontium was used. At day 28, a signi�cant reduction of PBI as compared to baseline was found in both groups. When compared between groups, signi�cant difference in PBI could not be detected. It has been shown that catechins and its derivatives could inhibit tissue breakdown through the proteinase activity of P. gingivalis [10,11]. In doing so, catechins and its derivatives could reduce source for microbial putrefaction. However, the effect of green tea mouthwash on the reduction of gingival in�ammation could not be found in the present study. e time and the number of subjects enrolled in the study might be insufficient to detect the intergroup difference in PBI. us, the long-term clinical research involving large number of subjects may also be considered in the future studies.
Oral malodor can be assessed by several methods. Organoleptic assessment (the direct sniffing of the exhaled air by human nose) most closely resembled daily situations in which malodor is detected. Due to its uncomfortability, the sul�de monitor was used in this study. Previous work by Rosenberg et al. [20] reported a signi�cant correlation between the measurements obtained from the sul�de monitor and the organoleptic assessment. It should be noted that oral malodor is also caused, to a lesser extent, by other odoriferous compounds. e limitation of using the sul�de monitor is its inability to detect compounds other than VSC. us, the organoleptic assessment may be used in the future studies.

Conclusion
Aer using for 4 weeks, green tea mouthwash could signi�cantly reduce VSC level in gingivitis subjects without causing remarkable side effects.

Con�ict of �nterests
e authors declared that they had no con�ict of interests.