Hydrogen peroxide masks the bitterness of chlorhexidine mouthwash without affecting its antibacterial activity.

BACKGROUND: Chlorhexidine (CHX) is an antiseptic mouthwash widely used as the gold standard for inhibiting plaque formation. However, the bitter taste of CHX limits patient compliance. We developed a 0.12% CHX and 1.5% hydrogen peroxide (H2O2) mouthwash that masked the bitter taste of CHX. This study evaluated the antibacterial activity and subject satisfaction of the developed mouthwash.
MATERIALS AND METHODS: Three mouthwashes were used as follows: (1) a commercial 0.12% CHX mouthwash, (2) a prepared 0.12% CHX mouthwash containing 1.5% H2O2, and (3) a prepared 0.12% CHX mouthwash. A disc diffusion assay was performed to determine the antibacterial activity of each mouthwash against Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. To assess subject satisfaction with each mouthwash, a satisfaction questionnaire was completed immediately after rinsing with each mouthwash.
RESULTS: The antibacterial activities of the three mouthwashes were similar. Moreover, the questionnaire results revealed that the level of satisfaction was significantly higher for the 0.12% CHX/1.5% H2O2 mouthwash compared with the other mouthwashes.
CONCLUSION: The 0.12% CHX/1.5% H2O2 mouthwash revealed a similar antibacterial activity as the CHX standard against periodontal disease pathogens. In addition, the subjects were more satisfied with the new formula compared with 0.12% CHX alone. These data suggest that the 0.12% CHX/1.5% H2O2 formulation is an alternative antibacterial mouthwash to avoid the unpleasant CHX side effects.

INTRODUCTION

Periodontitis is one of the most prevalent diseases in the oral cavity, affecting up to 30% of the US population and 40% of the Thailand population.[12] Periodontitis is initiated by the pathogenic bacteria in the dental plaque that deposits around cervical tooth surfaces. The main pathogenic bacteria associated with periodontal disease are Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.[3] P. gingivalis is a Gram-negative anaerobic bacterium that comprises a high proportion (79%) of the subgingival microbiota in a periodontal pocket especially in chronic periodontitis.[456] Although less A. actinomycetemcomitans, a Gram-negative facultative anaerobe, is present, it is highly associated with an aggressive form of periodontitis.[78] Thus, the effective plaque control is crucial in controlling periodontal disease progression.[9] Mechanical cleaning by toothbrushing and flossing is the best way to remove dental plaque deposits.[10] Adequate mechanical plaque control requires patient ability and motivation. However, most of the patients are not able to perform effective mechanical plaque removal.[11] Thus, the chemical plaque control using various chemotherapeutic agents has been employed as an adjunct to mechanical control in preventing dental plaque formation.[12] Currently, several types of mouthwashes are commercially available worldwide. Among these chemical agents, chlorhexidine (CHX) is approved by the American Dental Association Council due to its superior substantivity and prolonged antiplaque effect.[13141516] However, CHX has several undesirable side effects such as an unpleasant bitter taste and tooth staining.[1718] Therefore, the patient compliance is limited particularly when long-term CHX use is prescribed.[1718] Due to these unpleasant CHX effects, a CHX mouthwash with reduced side effects while maintaining its antibacterial efficacy is needed.

Hydrogen peroxide (H2O2), an oxygenating agent, mouthwash has been widely used in treating necrotizing ulcerative periodontal disease to remove supragingival plaque deposits.[19] H2O2 at a 1.5% concentration is effective in removing tooth discoloration, it is one of the major side effects of CHX through the release of oxygen and oxidation-reduction reactions.[1920] Moreover, many previous studies have demonstrated that using an oxidizing agent as an adjunct to CHX was more effective in decreasing plaque scores and in preventing tooth staining compared with CHX alone.[192021] Our pilot study was found that adding 1.5% H2O2 masked the bitter taste of CHX mouthwash. Therefore, mouthwash containing a mixture of CHX and H2O2 might be an alternative formulation with increased patient compliance when the use of CHX is recommended. However, there is no report evaluating the antibacterial effect of this mouthwash formulation. Thus, this study investigated that the in vitro antibacterial activity of the developed mouthwash containing 0.12% CHX and 1.5% H2O2, compared to 0.12% CHX. Moreover, user satisfaction with this mouthwash formula was subjectively evaluated.

MATERIALS AND METHODS

The study protocol was approved by the Ethics Committee, Faculty of Dentistry, Chulalongkorn University (HREC-DCU 2106-100).

Mouthwash groups and preparation

Three mouthwashes were used in this study as follows: (1) a commercial 0.12% CHX antiseptic mouthwash (C20®, Bangkok, Thailand) (0.12%CHXPos) as positive control, (2) a prepared 0.12% CHX mouthwash containing 1.5% H2O2 (0.12%CHX/1.5%H2O2), and (3) a prepared 0.12% CHX mouthwash (0.12%CHXCon).

An aqueous 0.24% CHX solution containing 0.03% sodium saccharin, 4% propylene glycol, 0.1% menthol, 24% sorbitol, and 4% lychee-like flavoring agent was prepared. The test mouthwash (0.12%CHX/1.5%H2O2) was prepared by adding 3% H2O2 to the 0.24% CHX aqueous solution in equal volume, resulting in a final concentration of 0.12% CHX and 1.5% H2O2. The control mouthwash (0.12%CHXCon) was prepared by mixing equal volumes of distilled water with the prepared 0.24% CHX aqueous solution, generating a final concentration of 0.12% CHX.

In vitro antibacterial activity

The antibacterial effect of each mouthwash was determined against the periodontopathic bacteria, P. gingivalis ATCC33277 and A. actinomycetemcomitans Y4. P. gingivalis was cultured in anaerobic conditions. A P. gingivalis-80°C stock was inoculated on Brucella agar plate and incubated at 37°C for 5–7 days. A colony was inoculated into brain heart infusion (BHI) broth for 3–4 days. The culture optical density was adjusted to 0.5 McFarland standard (≈2.4 × 108 CFU/ml). A volume of 100 μl of the adjusted culture was spread on Brucella agar plates twice. For A. actinomycetemcomitans, −80°C stock was inoculated on blood agar plate and incubated at 37°C for 2 days. A colony was inoculated into BHI broth at 37°C for 24 h. The culture optical density was adjusted to 0.5 McFarland standard (≈0.5 × 108 CFU/ml). A volume of 100 μl of the adjusted culture was spread on a blood agar plate. A volume of 20 μl of each mouthwash was applied on individual paper discs. Distilled water was used as a negative control. The discs were placed on the prepared agar plates and then incubated at 37°C for 3 days in anaerobic condition (P. gingivalis) and 2 days (A. actinomycetemcomitans). The diameter of the zone of inhibition was measured with a finely calibrated ruler to determine the antibacterial action of each mouthwash formula. Each experiment was performed in triplicate.

Satisfaction assessment

The estimated sample size was calculated from the data obtained from our pilot study. The following assumptions were used: alpha error = 5%, study power = 80%, mean difference in satisfaction level = 1.111, and standard deviation = 2.522. The required sample size was calculated as 41, which was increased to 60 to compensate for unreturned questionnaires. The questionnaires were distributed to Thai healthy adult volunteers to evaluate each mouthwash regarding satisfaction. Individuals with a fever, systemic disease requiring long-term drug use, oral ulcer, pregnant or lactating, or CHX or H2O2 allergy were excluded from the study. The participants provided informed consent. Information on each participant's age, sex, education, occupation, history of drug allergy, and history of mouthwash use was collected. Each mouthwash was dispensed to each participant in identical bottles, which were labeled as Group A, Group B, or Group C. Each group consisted of two bottles as follows:

Group A (0.12%CHXPos):

Bottle 1A contained 7.5 ml of commercial 0.12% CHX antiseptic mouthwash (C20®)

Bottle 2A contained 7.5 ml of commercial 0.12% CHX antiseptic mouthwash (C20®).

Group B (0.12%CHX/1.5%H2O2):

Bottle 1B contained 7.5 ml of prepared 0.24% CHX mouthwash

Bottle 2B contained 7.5 ml of prepared 3% H2O2.

Group C (0.12%CHXCon):

Bottle 1C contained 7.5 ml of prepared 0.24% CHX mouthwash

Bottle 2C contained 7.5 ml of distilled water.

To test each mouthwash formula, the participants were instructed to mix bottle 1 and bottle 2 of each group together immediately before use because H2O2 is an unstable substance that easily degrades on light and heat exposure. The current study was a single-blind trial. The participants were blinded regarding each mouthwash. The participants were instructed to test one formula a day (starting with Group A, B, and then C) for 3 consecutive days at bedtime at least 30 min after toothbrushing. The participants were instructed to retain the mouthwash solution in their mouth for 1 min before expectoration. To evaluate the participants’ satisfaction, a Thai-language satisfaction questionnaire was administered. The participants used a visual analog scale (1–10) immediately after rinsing with 15 ml of each mouthwash to report their satisfaction on pleasant smell, degree of bitterness, fresh sensation, and overall satisfaction.[22]

Statistical analysis

Data were analyzed using Prism GraphPad (Version 7, GraphPad Software Inc., California, USA). To determine the antibacterial activity and the participant satisfaction level, the one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison tests was performed to determine the statistical significance. P < 0.05 was considered statistically significant.

RESULTS

Antibacterial activity of the different mouthwash formulas

A disc diffusion assay was performed to determine the antibacterial activity of each mouthwash against P. gingivalis ATCC33277 [Figure 1] and A. actinomycetemcomitans Y4 (data not shown). All mouthwash formations showed a similar-sized zone of inhibition against both P. gingivalis and A. actinomycetemcomitans [Table 1].

Figure 1

Representative image of Porphyromonas gingivalis ATCC33277 disc diffusion assay demonstrated the zone of inhibition. CHX – Chlorhexidine; Pos – Positive control; Con – Control; H2O2 – Hydrogen peroxide

Table 1

Mean of the zone of inhibition of each mouthwash formulation (analysis of variance)

Bacterial strainsMouthwashes	Porphyromonas gingivalis ATCC33277		Aggregatibacter actinomycetemcomitans Y4
	Mean*±SD (mm)	P-values	Mean*±SD (mm)	P-values
A: 0.12%CHXPos (C20®)	14.44±0.768	0.854	11.67±0.577	0.854
B: 0.12%CHX/1.5%H2O2	14.11±1.169		11.44±0.510
C: 0.12%CHXCon	14.00±1.000		11.67±0.577

*Included 6 mm of disc diameter. SD – Standard deviation; P-values – Probability values; CHX – Chlorhexidine; Pos – Positive control; Con – Control; H2O2 – Hydrogen peroxide

Participant satisfaction with the different mouthwash formulas

Fifty-five questionnaires were returned. The demographic characteristics of the participants are shown in Table 2. Each subject scored their level of satisfaction immediately after using each mouthwash formula. The participant satisfaction results for the different mouthwashes are shown in Figure 2 and Table 3. ANOVA demonstrated that the significantly highest levels of satisfaction on pleasant smell, fresh sensation, and overall satisfaction were recorded after rinsing with the 0.12%CHX/1.5%H2O2 mouthwash. Lower levels of satisfaction were found for the 0.12%CHXCon and 0.12%CHXPos mouthwashes. The degree of bitterness results indicated that the participants scored the 0.12%CHX/1.5%H2O2 mouthwash as significantly less bitter (2.57 ± 1.49) compared with the other mouthwashes. In contrast, the highest bitter taste score (7.09 ± 1.54) was recorded for the 0.12%CHXPos mouthwash. None of the participants reported any allergic reaction due to the mouthwashes.

Table 2

Subject demographic characteristics

Demographic characteristics
Variables	Subjects (n=55), n (%)
Age range (years)
21-30	50 (90.909)
31-40	2 (3.636)
41-50	1 (1.818)
51-60	2 (3.636)
Sex
Male	21 (38.182)
Female	34 (61.818)
Education
Bachelor degree	49 (89.091)
Postgraduate degree	1 (1.818)
Others	5 (9.091)
Occupation
Dental student	47 (85.455)
Dentist	2 (3.636)
Dental assistant	4 (7.273)
Others	2 (3.636)
History of drug allergy
Yes	2 (3.636)
No	53 (96.364)
History of mouthwash use
Yes	31 (58.182)
No	24 (41.818)

n – number

Figure 2

Participant satisfaction after rinsing with each mouthwash. Level of satisfaction with pleasant smell (a), degree of bitterness (b), fresh sensation, (c), and overall satisfaction (d) were scored using a visual analog scale immediately after rinsing with each mouthwash. The results are shown as the mean ± standard deviation. ***A statistically significant difference at P < 0.0001; **A statistically significant difference at P = 0.0082. CHX – Chlorhexidine; Pos – Positive control; Con – Control; H2O2 – Hydrogen peroxide; VAS – Visual analog scale

Table 3

Participant satisfaction after rinsing with each mouthwash (analysis of variance)

Satisfaction assessment	0.12%CHXPos (C20®)	0.12%CHX/1.5%H2O2	0.12%CHXCon	P-values
Pleasant smell	5.33±1.57	8.03±1.15	7.32±1.43	<0.01
Degree of bitterness	7.09±1.54	2.57±1.49	4.05±1.21	<0.0001
Fresh sensation	4.59±1.72	7.74±0.98	6.31±1.38	<0.0001
Overall satisfaction	4.98±1.48	8.25±1.02	6.76±0.88	<0.0001

Data are shown as mean±SD. Statistical significance was considered at P<0.05. ANOVA indicated significant differences between the three mouthwash groups. P-values – Probability values; CHX – Chlorhexidine; Pos – Positive control; Con – Control; H2O2 – Hydrogen peroxide; SD – Standard deviation; ANOVA – Analysis of variance

DISCUSSION

CHX is a chemical agent recommended as an adjunctive treatment to maintain dental and periodontal health.[12] However, its unpleasant bitter taste and tooth discoloration are common side effects that limit patient compliance. Thus, we developed a new CHX mouthwash formula that reduced these side effects without affecting its antibacterial property. Boyd[23] found that rinsing with 1.5% H2O2 and 0.05% sodium fluoride (NaF) was more effective in preventing the development of gingivitis in orthodontic patients compared with rinsing with 0.05% NaF alone. Moreover, rinsing with H2O2 after CHX decreased plaque scores and prevented tooth staining.[192021] Our pilot study was found that mixing H2O2 with CHX reduced the bitter taste of CHX. Therefore, H2O2 was chosen to use in combination with CHX in our developed formula. To further improve the taste of CHX mouthwash, 0.03% sodium saccharin and 24% sorbitol were added as sweetening agents to reduce the extremely bitter taste of CHX. Saccharin and sorbitol are approved as low-cariogenic additives that do not affect the antibacterial activity of CHX.[2425] Cury et al.[26] demonstrated in vitro that a low concentration of saccharin (<0.5%) had no effect on the antimicrobial efficacy of CHX. Thus, these two sweetening agents are commonly used to prepare many CHX-containing dental products.[27]

In the current study, the antibacterial activity of the developed formula (0.12% CHX/1.5% H2O2) against two periodontal disease pathogens was evaluated. The results indicated that the antibacterial activity of CHX and CHX with H2O2 were similar. These findings indicate that H2O2 does not affect the antibacterial activity of the mouthwash. These results are in accordance those of Jhingta et al.,[20] which demonstrated that the adjunctive use of H2O2 to CHX reduced plaque formation. The plaque-inhibiting efficacy of CHX was not affected by subsequent rinsing with H2O2.[20] Moreover, H2O2 minimized stain intensity without any harmful effects on oral tissues.[20] Clinically, bacteria aggregate on the tooth surface in a community-based lifestyle of dental biofilm to promote their survival.[28] Thus, further in vivo studies are required to determine the antibacterial effectiveness of the developed mouthwash on dental biofilm. Furthermore, a prospective clinical trial should also be performed to evaluate the clinical safety and efficacy of the new mouthwash formula.

Bitter taste is one of the most unpleasant side effects of CHX.[1718] The developed mouthwash tasted significantly less bitter compared with the other mouthwash formulas. Moreover, compared with CHX mouthwash, higher satisfaction levels were reported after rinsing with the developed mouthwash for all subjective parameters evaluated. Therefore, the CHX/H2O2 mouthwash might be an alternative formula to increase patient compliance when the CHX use is recommended. Taste sensitivity differs between individuals, thus more pharmaceutical tests should be conducted. Currently, various automated instruments have been developed, such as an electronic tongue, to assess the bitterness of many pharmaceutical substances.[29] Therefore, in vitro taste assessment with the electronic tongue might be a good approach to characterize the taste and bitterness of our developed formula.

CONCLUSION

The developed mouthwash containing 0.12% CHX and 1.5% H2O2 demonstrated antibacterial activity comparable with that of the gold standard 0.12% CHX mouthwash. The satisfaction evaluation results indicated that this developed formula tasted less bitter and scored higher on the subjective parameters evaluated compared with 0.12% CHX. Therefore, the developed mouthwash might be an alternative antibacterial agent that minimizes the unpleasant CHX side effects and increases patient compliance. However, more in vivo and long-term clinical trials need to be performed to further evaluate the efficacy and clinical safety of this mouthwash formula.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.