Effectiveness of three mouthwashes - Manuka honey, Raw honey, and Chlorhexidine on plaque and gingival scores of 12-15-year-old school children: A randomized controlled field trial.

OBJECTIVES: The aim of this study is to compare the effectiveness of three types of mouthwashes manuka honey (MH), raw honey (RH), and chlorhexidine (CHX) on plaque and gingival scores of 12-15-year-old government school children. STUDY
DESIGN: This study was a double-blind, randomized controlled field trial conducted in Belagavi city, India.
MATERIALS AND METHODS: One hundred and thirty-five government school children aged 12-15 years were randomly selected and allocated into three groups, RH, MH, and CHX mouthwash groups. Ten milliliters each of honey-based mouthwash formulation and CHX mouthwashes (0.2%) were administered according to the group allocation twice daily for 21 days. All the children were examined at baseline, 22nd day (after discontinuation of mouthwash) and 28th day (1 week after discontinuation of mouthwash) for Gingival (Loe and silness 1963) and Plaque Index (Silness and Loe, 1964).
RESULTS: Descriptive statistics was applied for distribution of study participants according to age and gender. One-way ANOVA followed by Tukey's post hoc test and repeated measures ANOVA test followed by Bonferroni's post hoc were applied for inter- and intragroup comparison, respectively. Statistically significant reductions (P < 0.001) in plaque and gingival scores were observed in all the three types of mouthwash groups at the end of the 22nd day and 28th day. MH and RH mouthwash demonstrated equal effectiveness, whereas CHX mouthwash showed the maximum reduction in clinical parameters.
CONCLUSION: Honey-based mouthwash showed a promising antimicrobial effect on dental caries and plaque and gingival scores.

INTRODUCTION

Dental plaque occurs naturally on all tooth surfaces and performs a protective action by preventing the colonization of exogenous microbial species.[1] However, low pH of plaque leads to shift in the microbial flora from commensal to pathogenic leading to the development of caries and periodontal disease.[2] The use of mechanical agents and chemical plaque control agents have played a suitable adjunct in tackling this problem. Chlorhexidine (CHX) has been the gold standard agent used for chemical plaque control, but displeasing taste, staining of teeth, and increased microbial resistance have made its use unpopular in recent times.[3]

Nature has been a source of medicinal treatments since millennia and plant-based systems continue to play an essential role. One such natural product is Honey. Studied have indicated the antibacterial effect of honey can be attributed to the presence of hydrogen peroxide, flavonoids, and bee defensin-1 among others.[4] These antibacterial properties are further increased on dilution.

Hence, the present study was planned to evaluate and compare the effectiveness of two types of mouthwash manuka honey (MH) and raw honey (RH) with CHX mouthwash on plaque scores and gingival scores among 12–15-year-old government school children in Belagavi city.

MATERIALS AND METHODS

The trial was conducted from January 2017 to April 2017 as a field trial in government schools of Belagavi city, Karnataka, in accordance with the Declaration of Helsinki, 1975 modified in 2000 after receiving ethical clearance from the institutional review board. The trial was registered at the WHO Clinical T0rial Registry India CTRI no: CTRI/2017/11/010565.

Methodology

Study design and study setting

The present study was a double-blind, randomized controlled field trial conducted to evaluate the effectiveness of three types of mouthwash (MH, RH, and CHX) on plaque and gingival scores of 12–15-year-old school children in Belagavi city. Permission was obtained to conduct the study from Deputy Director of Public Instruction, Belagavi, and the principal of the selected high school.

The purity of honey

Before the start of the study, both the honey were tested for purity at AYUSH certified center of KLE'S BM Kankanwadi Ayurveda Hospital and all the values were within the permissible level.

Determination of the mouthwash concentration

Streptococcus is the early colonizer of plaque and after its adherence to the tooth surface it is significant in providing attachment substrates for the subsequent colonizers and ultimately influencing the succeeding stages of biofilm formation. Therefore, both the mouthwashes were tested for their activity against it and final mouthwash formulations were made based on the findings of this result.[5]

Honey mouthwashes were prepared based on the in vitro study conducted by determining the minimum inhibitory concentrations and minimum bactericidal concentrations. In this study, 40% MH mouthwash and 20% RH mouthwash were found to be most effective in inhibiting Streptococcus mutans.

pH of honey mouthwash

The pH of mouthwash formulation was checked before conducting the study and both the mouthwashes were well above the critical pH of saliva (demineralizing action on teeth). pH of MH and RH mouthwash was 5.9 and 6.1, respectively.

Method of mouthwash preparation

Purified water was deionized and distilled water was collected from distillation unit. 0.01g of sodium methylparaben and 0.01 g of sodium propylparaben were weighed separately and dissolved in 25 ml of purified water. Stirring was continued till a uniform mix was obtained. To this, 0.5 g sodium benzoate was added and stirred for 15 min. A weighed quantity of MH (40 g/35 ml), RH (20 g/35 ml), and glycerin (5 g) was added to the above contents and stirred for 10 min.

Sample size estimation

A pilot study was conducted on 6 participants to determine the acceptability and safety of the mouthwashes and no adverse events were reported. A sample of 3 participants were divided into 2 groups (MH mouthwash group and CHX mouthwash group) each. The plaque index scores (Silness and Loe, 1964) were measured at baseline and after 1 week of using the mouthwashes and the difference in the scores were calculated. Based on these results, the sample size would be 40 in each group under 5% α-error and 90% power of the test. A dropout rate of 10% was considered, and hence, the final sample size was estimated out to be 45 in each group with the total sample size being 135.

Examiner calibrations

Two examiners were selected to ensure blind evaluation of the study participants. Examiner 1 selected the schools, obtained permission from them, performed primary screening (examination for inclusion and exclusion criteria, which included baseline clinical examination), collected the baseline data, and administered the mouthwashes for 21 days. Examiner 2 recorded the plaque and gingival scores after 21 days and 28th day after discontinuing the mouthwash. Examiner 2 was blinded with respect to the type of mouthwashes administered. Examiner calibration was done before the study. The intraexaminer and interexaminer variabilities were calculated using Kappa statistic and was calculated to range from 0.8 to 0.9 for plaque index and 0.7 to 0.8 for the gingival index during all the assessments.

Selection and allocation of participants

Randomization

For obtaining the study sample, simple random sampling was done in two stages [Figure 1].

Figure 1

Flow diagram showing randomization of participants' enrollment, allocation, follow-up, and analysis, n – No of participants

In the first stage, a list of all the schools was obtained from the local authorities. Three schools were selected randomly by lottery method. All the children were clinically examined for the inclusion and exclusion criteria, as mentioned later. Written and informed consent was obtained from parents and assent was obtained from the children.

In the second stage of randomization 135 participants were randomly selected using the table of random numbers from the eligible population and further allocated equally into three groups, that is, MH, RH, and CHX mouthwash groups, having 45 participants each using block randomization sequence generated with the help of statistician. Children were blinded as to which mouthwash they were receiving by allocation concealment using identical amber color-coded bottles.

All the participants were selected from a single hermitage to remove any bias due to dietary and lifestyle patterns.

Inclusion criteria

Participants who were free from systemic diseases

Having moderate gingival scores (Silness and Loe, 1963) and fair Plaque scores (Loe and Silness, 1964)[67]

All the index teeth completely erupted

Parents of the participants who gave informed consent

Participants who gave assent.

Exclusion criteria

Any participant who was suffering from any disease which might affect the salivary flow

Having the history of antibiotic therapy in the previous 1 month till the start of the study

Presented with retained deciduous teeth

Suffering from any physical disability.

Standardization of the oral hygiene practices was done before the start of the study by teaching the participants to brush twice daily using fluoridated toothpaste and toothbrush and Modified Bass brushing technique to eliminate confounders in the results.

Intervention

Group 1 (n = 45): MH (40%) mouthwash

Group 2 (n = 45): RH (20%) mouthwash

Group 3 (n = 45): CHX (0.2%) mouthwashes.

All the participants were administered mouthwashes according to the group they were assigned to, twice daily for 21 days. A measured amount (10 ml) of mouthwashes were poured into disposable cups and given to children.

After swishing the mouthwash for 30 s, as recorded in the stopwatch by the principal investigator, participants were asked to spit in the nearby wash basin. They were also asked not to eat or drink anything for 30 min after rinsing and were instructed to use 10 ml of mouthwash as prescribed under parents' supervision at night time before going to bed after dinner.

Primary outcome variables

After 21 days of mouthwash administration, Plaque index (Silness and Loe, 1964) and Gingival index (Silness and Loe, 1963) were reassessed at 22nd day and 28th day.

Statistical analysis

Data were entered in Microsoft Excel and analyzed using SPSS for Windows, Version 21; SPSS Inc., (Chicago, IL, USA). Descriptive statistics were used to calculate frequencies, percentages, and mean values. One-way ANOVA followed by Tukey's post hoc test and repeated measures ANOVA followed by Bonferroni's post hoc test was carried out to determine the difference between and within groups, respectively.

RESULTS

At the onset of the study, there were 135 participants. Eleven participants dropped out, 4 from MH group, 4 from a RH group, and 3 from CHX group, thus at the termination of the study, 124 participants were present (CHX = 42, RH = 41, and MH = 41). Leave of absence, failure in following the study protocol, and astringent taste of CHX mouthwash were some of the factors for the exclusion of the participants. Tables 1 and 2 show the distribution of study participants according to age and gender. There was no statistically significant difference in the three groups at the baseline according to the age and gender.

Table 1

Distribution of study participants according to the age in the mouthwash group

Table 2

Distribution of study participants according to the gender in the mouthwash group

Intragroup comparison by repeated measure ANOVA followed by Bonferroni's post hoc test [Tables 3 and 4] showed a statistically significant reduction (P < 0.001) in plaque index and gingival index at 22nd day and 28th day for all the three mouthwash groups (MH, RH, and CHX).

Table 3

Comparison of mean±standard deviation plaque scores at baseline with plaque scores at 22nd day and 28th day, from the start of mouthwash administration in different mouthwash groups (intragroup comparison)

Table 4

Comparison of mean±standard deviation gingival score at baseline with gingival scores at 22nd day and 28th day, from the start of mouthwash administration in different mouthwash groups (intragroup comparison)

The mean plaque score for MH mouthwash reduced from 1.525 ± 0.2 at baseline to 0.72 ± 0.12 at 22nd day. The mean gingival score for MH mouthwash reduced from 1.457 ± 0.18 at baseline to 0.976 ± 0.15 at 22nd day.

The mean plaque score for RH mouthwash reduced from 1.525 ± 0.2 at baseline to 0.723 ± 0.11 at 22nd day. The mean gingival score for RH mouthwash reduced from 1.465 ± 0.17 at baseline to 0.927 ± 0.26 at 22nd day.

The mean plaque score for CHX mouthwash reduced from 1.505 ± 0.23 at baseline to 0.495 ± 0.13 at 22nd day and to 0.51 ± 0.13 at 28th day. The mean gingival score for CHX mouthwash reduced from 1.452 ± 0.19 at baseline to 0.498 ± 0.5 at 22nd day.

Intergroup comparison by one-way ANOVA revealed [Tables 5 and 6] statistically significant difference (P < 0.001) in plaque index and gingival index at 22nd and 28th day (1 week after discontinuation of mouthwash) between the three mouthwash group. Tukey's post hoc analysis revealed maximum reduction in clinical parameters by CHX followed by equivalent reduction shown by MH and RH at 22nd day and 28th day.

Table 5

Distribution of mean±standard deviation plaque scores at baseline, 22nd day, and 28th day from the start of mouthwash administration and comparison among the mouthwash group (intergroup comparison)

Table 6

Distribution of mean±standard deviation gingival scores at baseline, 22nd day, and 28th day from the start of mouthwash administration and comparison among the mouthwash group (intergroup comparison)

DISCUSSION

Honey is a natural product locally available in almost all countries and regions. It has a high nutritive profile. It is composed of various vitamins, minerals, antioxidants, enzymes, etc., A recent study has identified the antimicrobial properties of honey in vitro H2O2 and methylglyoxal (MGO) are distinct mechanisms involved in the bactericidal activity of honey.[8]

MH being a medical-grade honey is not easily available in all countries and regions. Although it is expensive, it has often been used in different studies as the “gold standard” to test and evaluate the various biochemical properties of different kinds of honey from different botanical and geographical origins.[9]

A few in vitro and in vivo experiments have studied the role of honey on plaque and dental microorganisms and most have used honey without dilution in its pure form to check the anti-plaque activity, but the current study was the first study that planned out the use honey as a mouthwash.[101112]

This is because antibacterial properties of honey are increased when diluted. This action of honey can be explained through the paradox that honey contains an enzyme glucose oxidase that produces hydrogen peroxide when diluted which is a potent antibacterial substance and used widely and popularly among dentists.[13] The probability of demineralizing action of honey-based mouthwash formulation due to the presence of intrinsic sugar such as glucose and fructose was reduced by the dilution, and this was further supported by a study done among the orthodontic patients which showed that pH of plaque sample did not drop below the critical pH up to 30 min after the use of undiluted honey.[14] Another study has shown that the pH of the saliva rapidly resumes to the normal level of 6.8 after swallowing of the honey. The honey mouthwash formulation can be safely used twice daily, even during the night when the salivary flow rate is less as it has shown to have negligible effect on enamel microhardness due to its components such as Ca, phosphate, and other ions, as well as the buffering capacity.[15]

Plaque scores

All the mouthwashes (MH, RH, and CHX mouthwash group) showed significant reductions in the plaque scores at 22nd day and 28th day as compared to the baseline.

The result of MH mouthwash in a reduction of mean plaque scores as compared from baseline was in accordance to the study done by English HK et al. and Nayak et al., after chewing lather made of honey and direct application into gingival sulcus, respectively.[12]

Dental plaque is a host-associated matrix-enclosed biofilm with different bacterial populations which adhere to the tooth or other surfaces. Inhibition of bacteria leads to impairment of plaque formation and can explain the antiplaque effect of the mouthwashes.[15]

The antiplaque effect of MH and RH can be attributed to the bactericidal activity of hydrogen peroxide which causes oxidative damages to the membrane, proteins, enzymes, and DNA.[16]

The reduction in plaque levels by honey-based formulations could also be attributed to the other antibacterial components of honey such as low pH, high osmolarity, flavonoids, and bee defensin-1.[17] Unique manuka factor known as MGO present specifically in MH is toxic toward pathogens at low concentrations, interrupts cell divisions, and specifically causes the degradation of bacterial DNA.[18]

Gingival scores

Reduction in a mean gingival index of all the three types of mouthwash (MH, RH, and CHX mouthwash group) was observed from baseline to 22nd day and 28th day. Results of MH mouthwash were in accordance to the study done by Atwa et al., where the anti-gingivitis effect of Honey on orthodontic patients was noticed after chewing lather made of honey due to which the number of Porphyromonas gingivalis in the plaque were significantly reduced by the honey group compared to the other treatment groups. In vitro studies were done by Eick et al. and Schmidlin et al. where MH inhibited the growth of the planktonic P. gingivalis also showed results in accordance with the present study.[131920] The simplest form of gingivitis is associated with the accumulation of supragingival plaque along the gingival margins of the teeth. Hence, the previously described antiplaque/antibiofilm potential of honey due to various antimicrobial components of the honey could have led to the decrease in the gingival bleeding scores.[21]

The maximum reduction in the mean gingival scores and plaque scores was demonstrated by CHX and the equal reduction was shown by MH and RH mouthwashes. Although no true comparison could be made to the existing literature since no study has compared all the three types of mouthwash, the lower effectiveness of the honey-based mouthwashes could be attributed to the heterogeneous nature of honey and interference in their action by the other local factors of the mouth.[22] Furthermore, significant reductions in plaque and gingival scores by RH and MH mouthwashes cannot be ignored.

Comparable reductions in plaque and gingival scores by RH honey and MH is a key finding in this study since MH is very expensive and not locally available, RH can serve as a good indigenous cost-effective alternative due to its equivalent reduction in plaque and gingival scores.

These mouthwash formulations showed no adverse effects and no staining of teeth with the usage which is a common drawback of CHX.[23]

Honey-based mouthwash formulation can also serve as a good alternative for pediatric patients who might experience taste alteration upon usage.[24]

Further, honey mouthwashes did not contain alcohol, artificial color, and artificial sweetener (such as saccharine). These ingredients in mouthwash formulation are easily fermentable by microorganisms, thereby releasing byproducts that cause halitosis.[25] Apart from improving dental health, the role of honey can be further tested on other type of mouth ailments such as aphthous ulcers, dry socket, and radiation mucositis.

CONCLUSION

Among the three types of mouthwash tested, 0.2% CHX was most effective in reduction of plaque and gingival scores. About 40% MH and 20% RH mouthwash also effectively decreased the amount of plaque and gingivitis from 0 day to 22nd day. Within the limits and scope of the study, it can be safely concluded that both RH and MH may be used as an adjunct to mechanical therapy for achieving a significant reduction in inflammatory periodontal changes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.