Comparative Evaluation of Antimicrobial Efficacy of Green Tea Herbal Mouth-wash and 0.2% Chlorhexidine Gluconate Mouthwash Against Streptococcus mutans in Dental Plaque - A Randomized Controlled Trial

Introduction

Dental caries occurs due to a change in the biofilm.¹ Streptococcus mutans is an acidogenic gram-positive bacteria which converts sucrose to organic acids, dissolves the calcium phosphate in teeth, and eventually leads to decalcification and decay. Several chemical agents can alter the profile of oral microflora and can cause undesirable effects. ² Chlorhexidine (CHX) is a broad-spectrum bis-biguanide antiseptic. Chlorhexidine is used as a mouth rinse, varnish, and spray. Mouth rinses have been used following mechanical tooth cleaning/ brushing. Also, post-surgery, during intermaxillary fixation, during fixed orthodontic treatments, and for physical and intellectual disability conditions.

Chlorhexidine mouth rinse is available in concentrations of 0.1%, 0.12%, or 0.2% chlorhexidine gluconate. Its effects on the microbial biofilm are dose-dependent. Chlorhexidine mouth rinse has been considered a gold standard mouth rinse. However, its use has been associated with several local adverse effects such as brown staining of teeth due to the precipitation of anions from dietary chromogens, supragingival calculus formation, oral mucosal lesions, and altered taste perception.³

Green tea (Camellia sinensis), which contains adequate amounts of catechins and various other polyphenol compounds, has demonstrated antioxidant, anti-inflammatory, antiviral, antimutagenic, anti-caries, and anti-bacterial properties against periodontal pathogens.^4,5 Catechins and theaflavins, polyphenolic compounds derived from tea (Camellia sinensis), have been reported to have biological activities including the prevention of tooth decay and oral cancer in several in vitro studies. These polyphenol components inhibit pathogenic microbial growth. There is a scarcity of studies assessing the antimicrobial efficacy of a commercial green tea (0.5%) mouth rinse with chlorhexidine gluconate. Therefore, the aim of this study was to comparatively assess the antimicrobial efficacy of 0.2% chlorhexidine gluconate mouthwash and green tea (0.5%) mouth rinse against Streptococcus mutans in dental plaque samples of school children.

Materials and Methods

Healthy, cooperative children aged between 9-12 years, from the government residential school, having similar socioeconomic and oral hygiene conditions were screened. Thirty participants were selected based on the following criteria of inclusion and exclusion and were called to report to the Department of Pediatric and Preventive Dentistry. The Plaque index, Gingival index, and DMFT/dmft index were recorded.

Inclusion criteria

• Healthy children aged 9-12 years (Late mixed dentition).
•  Children who can maintain oral hygiene themselves.
• Children with plaque index between 1 and 2 (fair), gingival index between 0 to 2 (mild to moderate), DMFT / deft score from 0 to 3, and similar oral hygiene practices.⁶

Exclusion criteria

• Children who recently used any mouthwash, topical fluoride, or antibiotics during the previous month.
• Children having rampant caries involving dental pulp or systemic diseases.
• Especially abled children.
• Children with orthodontic appliances and prostheses.  
• Children with any known allergies and allergies to the mouthwash assigned.

The size of the sample was determined based on the calculations and previous studies by the statistician. To overcome dropouts (if any), the size of the sample was fixed at thirty participants. The study protocol was approved by the institutional ethical committee, following which the study was registered under Clinical Trials Registry India. A double-blinded clinical trial was conducted, children from the government residential school were screened according to recruitment criteria, and 30 participants were selected for assigning two different types of mouthwashes. The possible discomfort, risks, and benefits regarding the procedure were explained and informed consent was taken from the respective authorities of the institution/parents/ guardians, and assent for participation was taken from the children. The study comprised thirty participants divided into two groups, Group A and Group B as shown in the CONSORT flow diagram (Figure 1).

Baseline plaque sample collection and sample collection after 15 days was done in the morning by a single operator trained in Pediatric dentistry. The sample collection was done in the morning between 9.00 a.m. to 11.00 a.m. Baseline sample collection was done using a sterilized explorer (Figure 2) from the outer surfaces of the first permanent molars.⁷ Participants were asked to swallow just before plaque collection to minimize salivary contamination and during the sample collection, care was taken to avoid contamination with blood or saliva. The outer surfaces of the teeth were wiped with a cotton swab to remove saliva and prevent contamination. The sample collection was standardized by using four occlusal-directed strokes.⁸ Children were told not to drink/eat anything (except water) one hour before plaque collection.

The participants received a prenumbered identically masked mouth rinse bottle containing 140 mL of mouthwash (Figure 3). The mouth rinsing technique was demonstrated to all the participants and to the school nurse as well. The participants were asked to rinse the mouth using 10 mL of the designated mouth rinse for one minute after conventional tooth brushing done twice daily, morning and at night (just before bed) for 15 days. Mouth rinsing with the assigned mouthwash was carried out in front of the trained school nurse. A 10 mL measuring cup was provided for the same.

The baseline plaque samples collected were placed in a radiation-sterilized cryo vial tube (2 mL) containing 1 mL saline, then transported within two hours to the microbiological laboratory to ensure maximum live bacteria. During the clinical trial, compliance was evaluated, and were asked to report immediately via phone call in case of any adverse effects. The school nurse was instructed to supervise daily proper use of the mouth rinse and ensured that for a minimum of half an hour after rinsing, the child does not eat or drink anything. A checklist was provided to stick colourful stickers on days children used mouthwash and put an X on days they did not. Participants’ compliance was checked by the investigator on the seventh day by direct supervision. The mouth rinse bottles containing 140 mL of mouthwash were resupplied on the seventh day to all the participants.

The microbiological colony counting procedure was done in a standard laboratory. The samples were cultured in an MSB-specific medium containing 0.2 units per milliliter of Bacitracin by streaking with a calibrated loop. Each sample was streaked after flaming the calibrated loop until red hot to prevent cross-contamination which was then followed by incubation. The number of S. mutans colonies grown in the Bacitracin culture medium was counted after 24 hours by morphology, size, and colour (Figure 4).

The participants’ compliance was evaluated by measuring the remaining volume of the mouthwash they brought back on the 15th day and the checklist. All children were reviewed on the 15th day to collect the plaque samples once again using the same procedure as mentioned above for evaluation of S. mutans colony count. Later, all the participants underwent oral prophylaxis, oral hygiene instructions, and dietary instructions. The baseline and the follow-up data thus obtained were statistically analyzed, and compared. The study was conducted according to the CONSORT guidelines.

Results

Data were analyzed with the statistical package for social sciences (SPSS) for Windows, version 22.0. released 2013. Armonk, NY: IBM Corp. Descriptive statistics were performed to assess the mean, standard deviation in the respective groups. The normality of the data was assessed using the Shapiro-Wilkinson test. Cohens Kappa statistics were done to assess intra-examiner variability which showed near-perfect agreement (0.87). Inferential statistics to determine the difference in two groups was done using the Independent t-test. Paired t-test was used to find out the difference between and within the two groups. The level of significance (p value) was set at p <0.05.

Table 1 and Table 2 show the baseline and post-rinse S. mutans counts in Group A and Group B plaque samples, respectively. Within group, paired t-test analysis reported a significant reduction in S. mutans counts from baseline to post-rinse in both CHX and green tea groups (p <0.05).

Between groups independent t-test analysis showed no significant difference (p >0.05) both at baseline and post-rinse in Streptococcus mutans counts (Table 3 and 4). Thus, both 0.2% CHX and green tea (0.5%) mouthwash showed a significant reduction in Streptococcus mutans counts in the plaque samples of both the groups. However, 0.2% chlorhexidine was marginally better.

Discussion

Chlorhexidine (CHX) is a very effective commercially available mouth rinse due to its broad spectrum antimicrobial activity and no systemic side effects.⁹ Scientific data reveals that chlorhexidine mouth rinse reduces the colony counts of S. mutans in plaque biofilm. ^10-12 Chlorhexidine mouth rinse is widely used and is accepted as the «gold standard» due to its broad spectrum antimicrobial activity. Chlorhexidine destabilizes the bacterial cell wall and also interferes with osmosis. In addition, rapid uptake causes rupture of bacterial cell wall resulting in cell death.^13-15

The bioavailability of chlorhexidine was not compromised in this study because rinsing was carried out twice daily using 10 mL of mouth rinse. Reports have stated it to be about 12 hours using 10 mL of 0.2% of chlorhexidine mouth rinse.^14,15 Rinse time of 30 seconds is effective and acceptable, while 60-seconds rinsing time is also advocated.³ However, Linde et al., reported a few local side effects with chlorhexidine 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.¹⁶ Hence, the search for newer herbal mouth rinses is ongoing to overcome the disadvantages of chlorhexidine.

Green tea plants are documented as Camellia sinensis by botanists. They are small, 3-4 feet high, and have bushy plants. Tea is an important dietary source of polyphenols, chiefly flavonoids which are phenol derivatives.¹⁷ Green tea has four major polyphenols namely (–) -epicatechin, (–)-epigallocatechin, (–)-epigallocatechin-3-gallate (EGCG), and (–)-epicatechin-3-gallate. The most common polyphenol in green tea is EGCG, which accounts for more than 50% of all the green tea components, followed by epigallocatechin and epicatechin-3-gallate. EGCG also shows the strongest antimicrobial activity of all catechins.

It has been reported that EGCG disrupts biofilm formation through different mechanisms such as, bacterial growth inhibition by preventing bacterial metabolism and by promoting bacterial aggregation, inhibition of glycosyltransferase (GTF) activity and other various cariogenic virulence factors of S. mutans at the transcriptional/enzymatic levels. EGCG regulates the oral ecosystem by the inhibition of acidification and acceleration of the bacterial clearance. Catechins damage the Gram-positive bacterial cell membranes and prevent their adhesion.¹⁷ EGCG also encourages the aggregation of plaque-forming Streptococci, which can help in accelerating salivary bacterial removal and reducing bacterial quantity.¹⁷

Kaur et al., reported that catechins within green tea (0.5%) help to maintain the neutral plaque pH, which inhibits the colonization of S. mutans. ^18,19 In the present clinical trial, both 0.2% CHX and green tea (0.5%) mouthwash showed a significant reduction in the Streptococcus mutans counts in plaque samples of both the groups. However, 0.2% chlorhexidine was marginally better. The participants’ acceptance was better for green tea (0.5%) mouth rinse, whereas CHX had a lower acceptance rate. These findings were also seen with many other studies.^13,14 Balappanavar et al.,¹⁹ also observed a significant reduction (p <0.001) in salivary S. mutans counts using green tea mouthwash. The results of the present study and many other studies strongly suggest that certain components of tea can exert significant anti-cariogenic effect by virtue of their inhibitory activity against S. mutans. ^19-21 Thus, the main outcome of the study was the significant reduction in the S. mutans counts in both 0.2% CHX and green tea (0.5%) mouthwash groups. The patient acceptance rate was better with green tea, and there was no taste disturbance, no tooth staining associated with green tea mouthwash.

Limitations

• Small sample size.
• he present study did not assess the substantivity of green tea when compared to chlorhexidine gluconate.
• It was a cross-sectional study that indicated the microbial count at a certain point in time.

Further recommendations

• More studies using a larger sample size and a longer duration can be performed.
• Studies comparing the substantivity of green tea to chlorhexidine gluconate can be performed.
• Since dental caries develop over a considerable period, longitudinal studies can be performed.

Conclusion

The following conclusions were derived from the present study:

• S. mutans count reduction was observed in both 0.2% CHX and green tea (0.5%) mouthwash groups. However, 0.2% Chlorhexidine mouthwash was marginally better.
• 0.5% green tea mouthwash is an herbal preparation and has the advantages of being non-staining, having no taste disturbance, good patient compliance, and being cost-effective.
• 0.5% green tea mouthwash can be recommended for child patients along with mechanical plaque control methods.

Financial support and sponsorship

Nil

Conflicts of interest

There are no conflicts of interest.