|Year : 2016 | Volume
| Issue : 4 | Page : 315-323
Comparative evaluation of antimicrobial efficacy of three different formulations of mouth rinses with multi-herbal mouth rinse
Isha Jain1, Pankaj Jain2
1 Department of Pedodontics and Preventive Dentistry, ITS Centre for Dental Studies and Research, Ghaziabad, Uttar Pradesh, India
2 Department of Oral and Maxillofacial Pathology, ITS Centre for Dental Studies and Research, Ghaziabad, Uttar Pradesh, India
|Date of Web Publication||29-Sep-2016|
Senior Lecturer, Department of Pedodontics and Preventive Dentistry, ITS-Centre of Dental Studies and Research, National Highway 58, Murad Nagar, Ghaziabad, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: In response to the propagation of various anti-Streptococcus preventive agents, the discovery of newer and more efficient agents which are more economical, efficacious, and safe are gaining popularity in today's era. Aims: The purpose was to compare the antimicrobial efficacy of multi-herbal mouth rinse with essential oil-based, fluoride containing, and 0.2% chlorhexidine digluconate mouth rinses, well-evidenced chemical formulations, against Streptococcus mutans. Settings and Design: It is triple-blinded randomized controlled trial. Methodology: One hundred and twenty adolescents aged between 15 and 17 years were randomized into four groups: (a) multi-herbal mouth rinse, 15 ml twice a day; (b) 0.2% chlorhexidine mouth rinse, 15 ml twice a day; (c) essential oil mouth rinse, 15 ml twice a day (d) 0.2% sodium fluoride mouth rinse, twice a day. Salivary and plaque samples were collected from subjects and oral streptococci colony forming units (CFUs)/mL was assessed using TYCSB agar. Statistical Analysis Used: Repeated measures of ANOVA were used to compare the various mouthrinses followed by post hoc Bonferroni test for comparing multi-herbal mouthrinse with other mouthrinses. Significance was set at P < 0.05. Results: At baseline, there was no statistically significant difference in the distribution of baseline data groups, but reduction of S. mutans colony count of multi-herbal mouth rinse in comparison with the other mouthrinses had statistically significant values except Fluoride mouth rinse till 1 week postrinsing. Conclusion: Chlorhexidine and multi-herbal mouth rinses showed statistically significant reduction in the S. mutans CFU count, in terms of efficacy and substantivity both.
Keywords: Colony forming units, herbal preparations, mouthrinses, Streptococcus mutans
|How to cite this article:|
Jain I, Jain P. Comparative evaluation of antimicrobial efficacy of three different formulations of mouth rinses with multi-herbal mouth rinse. J Indian Soc Pedod Prev Dent 2016;34:315-23
|How to cite this URL:|
Jain I, Jain P. Comparative evaluation of antimicrobial efficacy of three different formulations of mouth rinses with multi-herbal mouth rinse. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2019 Aug 23];34:315-23. Available from: http://www.jisppd.com/text.asp?2016/34/4/315/191409
| Introduction|| |
After decades of research, it has been established that plaque accumulation is a potential causative agent of dental caries as well as periodontal diseases.  Plaque has been consistently targeted with interventions with an exponential growth in the field of preventive dentistry.  Oral inhabitants such as Streptococcus mutans colonize in human dental plaque and play a key role in the initiation and progression of dental diseases such as dental caries.  Various novel antimicrobial and chemotherapeutic agents have dramatically assisted in improving the oral health of individuals.  Consequently, the use of mouthrinses has gained popularity in developing countries as it is less time consuming, halitosis-being the prime concern (especially in young adults and teenagers).  Mouthrinses have always been considered as an important anti-plaque chemical agent against dental caries. ,
Chlorhexidine mouth rinse (0.2%), among a myriad of oral hygiene products, has acquired an eponym of "gold standard against oral infections" owing to its dramatic therapeutic effect,  but is also accompanied by some disquieting characteristics such as taste alteration, supragingival calculus formation, and desquamation of oral mucosa have restricted its usage in pediatric age group. , Moreover, it also causes extrinsic staining by attaching to the polyphenolic and tannin group of beverages such as tea and coffee.  Hence, many studies are being conducted for identifying therapeutic agents from plant-based sources for the management of dental disease.
A rekindled interest in the medicinal properties of alternative natural products has gained popularity due to increasing problems of resistance to synthetic antimicrobials.  Despite the innumerate commercial mouth rinses inundating domestic and international markets, little or perhaps negligible is what is currently known about the microbial canopy and more importantly, their durability and measurable preemptive efficacy of individual ingredients of the plant-based formulations. Although many studies have documented the efficacy of mouth rinses on plaque deposition and its composition (biochemical or microbiological), the evaluation of a mouth rinse on bacterial load count in oral cavity has received little or no attention in human studies in this environment. 
Considering the paucity of literature on antimicrobial effect of virtually most of the commercially available mouth rinses, this study is a pioneer in determining the efficacy and substantivity of active ingredients of most of the commercially available mouth rinses on bacterial load, and it is meticulously designed to reflect upon not only the accuracy and validity of information misleading consumers, but also the futuristic endeavors in terms of addressing public health concerns and to verify the claims made by the manufacturers for adoption of these mouth rinses. Hence, the aim is to comparatively evaluate the four mouth rinses against S. mutans (bacterial count) as well as to provide information to dental professionals about the efficacy of their products in vivo and to use these mouth rinse as a base for the evaluation of antimicrobial herbal mouth rinse.
| Methodology|| |
The present study was a triple-blinded randomized controlled field trial conducted to evaluate the effectiveness of four mouthrinses on 15-17 years old school children in Ghaziabad. 
Clinical evaluation of the products on the selected subjects was carried out according to the guidelines of the Declaration of Helsinki for biomedical research involving human subjects. Institutional ethical committee permission was obtained prior to commencing the study and all the parents of the participants signed the informed patient consent form. The study includes assessing the caries status of the selected subjects followed by use of the specified products.
Children were free to withdraw from the study at any point during the study period.
A pilot study was conducted on ten people to determine the acceptability, palatability, and safety of the mouth rinses. The required sample size was estimated based on the difference in the caries status (decayed-missing-filled-teeth [DMFT] scores) between the study group and by using G * Power analysis, the mean and standard deviation of two groups (Group 1 = 4.70, 0.356 and Group 2 = 4.41, 0.350) (Effect size [d] = 0.8649354, α error = 0.05, Power [1-β err prob] = 0.95), so sample size for each group came out to be thirty for each group.
A volunteer who belonged to different field, at the first place, added equal and negligible volume of edible black color in all the mouth rinse and measured 2 L of each mouth rinse and labelled the mouth rinse as groups 1, 2, 3, and 4.
Two examiners were selected to ensure blind evaluation of the study participants. The first examiner (Examiner 1) selected the schools, obtained permission from them, did primary screening (examination for inclusion and exclusion criteria, which included clinical examination) and selection, distributed the questionnaire, collected data and consent, and administered the mouth rinse (without the knowledge of the type) to Examiner 2 and the children for 2 weeks (night time rinsing), on weekly basis.
Examiner 2 took the plaque and salivary samples with the help of cotton swab (for saliva) before rinsing, immediately after rinsing on the 1 st day, after 24 h, after 48 h, 1 week and 2 weeks. Examiner 2 was also blinded to the type of mouth rinse administered, at different time intervals during the 2 weeks.
Finally, the colony count was done by the microbiologist and mean values of S. mutans counts were noted down.
The statistician remained blinded regarding the subject allocation to the four groups. Both the examiners were trained before the initiation of the study under the guidance of a senior professional in order to limit the intra- and inter-examiner variability. They were reassessed for satisfactory agreement at various time intervals during the clinical examinations.
Recording assistants were trained in documenting the readings accurately. For obtaining the study sample, two-stage random sampling was done.
In the first stage, a list of all the schools of Ghaziabad was obtained. From these schools, one school was selected by lottery method.
During the initial phase of study (before selecting the sample), a 29-item self-designed combination of closed and open-ended questionnaire and assessment form was prepared to collect information regarding sociodemographics, oral hygiene practices, and food habits from all the children aged 15-17 years. All the children were also clinically examined for the inclusion and exclusion criteria, as mentioned later. This form was divided into five parts:
- First part (13 questions) pertained to sociodemographic data
- Second part (9 questions) dealt with self-reported oral hygiene practices of children
- Third part (4 questions) was concerned with food habits
- Fourth part (3 questions) was concerned with their use of mouth rinse, medication, and presence of any systemic, chronic diseases
- Fifth and last part of this form was designed to record DMFT.
Inclusion and exclusion criteria used were as follows.
- Free from systemic diseases
- DMFT scores between 3 and 6
- Should not have used mouth rinse for the last 1 month
- Parents should give informed consent.
- Suffering from diseases which might affect the salivary flow
- History of antibiotic therapy in the previous 1 month till the start of the study
- Retained deciduous teeth
- Currently using any mouth rinse or has used mouth rinse in the past 1 month
- Suffering from any physical disability
- History of fluoride treatment in the past 2 weeks.
One hundred and twenty subjects were randomly selected from the eligible population (who fulfilled the inclusion criteria) and randomized into four groups, namely herbal, essential oil, fluoride, and chlorhexidine mouth rinse groups, having thirty participants each.
Randomization was done using lottery method by a person not involved with the study proceedings. All the parameters (including food habits, oral hygiene habits, etc.) were assessed statistically.
No statistically significant difference was found among the four groups.
All the children were administered herbal, essential oil, fluoride, and chlorhexidine mouth rinse (0.2%) according to the group they were assigned to, twice daily for 2 weeks.
All 120 children were randomly divided in four groups of 30 children each as follows:
- Group 1: Herbal mouthrinse containing active ingredient as mixture of garlic powder, aqueous extract of amla (gooseberry), and organic solvent based extract of ginger (20 mg each)
- Group 2: Essential oil mouth rinse containing menthol and eucalyptol
- Group 3: Fluoride mouthrinse containing 0.2% sodium fluoride
- Group 4: Chlorhexidine mouth rinse (0.2%).
S. mutans counts in the salivary sample were estimated prior to the use of mouthrinses and repeated again after a period of 24 h, 48 h, 1 week and 2 weeks. Pre- and postrinse salivary samples are collected at each time interval.
Measured amount (15 ml) of mouth rinse was poured in plastic cups and given to children. All the children were asked to take mouth rinse into their mouths and start swishing the mouth rinse upon a prompt from the investigator who stood with a stop watch to record the time. They were also asked not to eat or drink anything for 30 min. Children were instructed to use 15 ml of mouth rinse as prescribed, under parents' supervision at night time after dinner. Every participant was provided with 105 ml of their respective mouth rinse for home use on a weekly basis.
Before start of the intervention, all the children were instructed to rinse with the mouth rinse given to them in the night, before going to bed, and not to eat or drink anything for at least half an hour after rinsing. Positive reinforcements were given from time to time. No oral prophylaxis was done prior to the commencement of intervention. Children were allowed to follow their individual oral hygiene procedures.
Children were blinded as to which mouth rinse they were receiving. Second examiner was blinded to the group allocation, meaning he did not know which child was assigned to which mouth rinse group. The statistician remained blinded regarding the subject allocation to the four groups.
Preparation of herbal mouth rinse solution
Extracts made according to our previous studies , were taken and mixed, i.e., dried garlic powder, aqueous gooseberry (amla), and organic solvent-based extract of ginger and were weighed using Digiweigh® electronic weighing machine and 20 mg of each powder was mixed with sterile deionized distilled water to obtain a solution of final volume of 100 ml and were stored in airtight sterile containers at 4°C. After 48 h, the mixture was filtered using a filter paper. Sweetening agent (30% sucralose, code E955) and preservative (0.05% sodium benzoate, code 211 and 0.01% sodium methyl paraben, code 218) were added to obtain the final mouth rinse.
Saliva collection procedure
Baseline salivary samples were collected. The subjects were informed not to eat or drink anything (except water) 2 h prior to saliva collection. Saliva collection was done in the morning between 10 and 10:30 am to match the circadian rhythm. Care was also taken to avoid saliva collection before heavy physical exercise. Children were told to swallow the preexisting saliva in order to clear mouth of any residual unstimulated saliva. Subsequently, each subject was asked to collect saliva in the mouth for a minute and then it was collected using a sterile disposable syringe. The saliva was later transferred to vial containing thioglycollate transport medium.
Salivary sample was taken by rolling the sterile cotton swab. Sample was evenly distributed onto the surface of TYCSB agar (selective media for S. mutans) agar surface using sterile spreaders (Oxoid, UniPath Ltd., Baskingstoke, Hampshire, England) using sterile spreaders (Oxoid, UniPath Ltd., Baskingstoke, Hampshire, England). Plates were incubated at 37°C under anaerobic conditions (AnaeroPacks) for 72 h. The experimental procedure was conducted three times, using triplicate plates for each dilution. The colonies on plates were enumerated using a colony counter (Quebec Darkfield 3328, American Optical Corp., New York, USA) and the mean colony-forming units/mL were noted down. Culture plates which were negative for streptococcal growth were left on the bench for further 72 h and then examined again before being discarded as negative. Biochemical tests were performed, including raffinose fermentation, melibiose, and esculin hydrolysis. When all three are positive, they indicate presence of S. mutans.
Results were expressed as mean ± standard deviation. Data were entered and analyzed using SPSS version 17 (SPSS Inc., Chicago, IL, USA). Repeated measures of ANOVA was used to compare the various groups of mouthrinses at baseline (before rinsing) and at various postrinsing intervals followed by post hoc Bonferroni test for multiple comparisons of multi-herbal mouthrinse with other mouthrinses. Significance was set at P < 0.05. While Student's t-test was applied for comparison of baseline samples with other time intervals in all the groups.
For all the tests, a P ≤ 0.05 was considered statistically significant.
| Results|| |
Repeated measures of ANOVA revealed statistically significant reduction of S. mutans colony count using all four mouthrinses at different time intervals (P = 0.003) [Table 1], followed by Post hoc Bonferroni test revealed that the multi-herbal and fluoride mouthrinses exhibited no statistically significant differences at almost all the time intervals except at 1 week and 2 weeks (P < 0.05) [Table 2]. Student's t-test revealed that only Group 1 (multi-herbal) and Group 4 (chlorhexidine) showed statistically significant reduction (from the baseline till 2 weeks) at all the time intervals, whereas Group 2 (essential oil) worked till 48 h and Group 3 (fluoride) till 1 weeks at statistically significant values [Table 3],[Table 4],[Table 5] and [Table 6].
|Table 1: Descriptive statistics showing the intergroup comparison of mean bacterial colonies (CFU × 106/ml) among four different groups of mouthrinses at different time intervals|
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|Table 2: Comparison of multi - herbal mouthwash with other mouthwashes at different intervals of rinsing by post-hoc Bonferroni test|
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|Table 3: Descriptive statistics showing the intergroup comparison of difference between the before rinsing (baseline) mean bacterial colony values with mean values at different time intervals among group 1 (Multi-herbal)|
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|Table 4: Descriptive statistics showing the intergroup comparison of difference between the before rinsing (baseline) mean bacterial colony values with mean values at different time intervals among group 2 (Essential oil)|
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|Table 5: Descriptive statistics showing the intergroup comparison of difference between the before rinsing (baseline) mean bacterial colony values with mean values at different time intervals among group 3 (Fluoride mouthrinse)|
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|Table 6: Descriptive statistics showing the intergroup comparison of difference between the before rinsing (baseline) mean bacterial colony values with mean values at different time intervals among group 4 (Chlorhexidine mouthrinse)|
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| Discussion|| |
In the past several years, there has been a backlog of unprecedented interest in the root cause of dental caries and other plaque-induced disorders. Consequently, the market has been spirally flooded with oral hygiene products that have been unremittingly introduced to purportedly attempt and curb oral microflora at fault such as S. mutans. S. mutans bind to the acquired pellicle to form first stage of plaque formation. The removal of streptococci prevents plaque formation and disease extension. 
It is quite evident that bacterial plaques have a crucial role in the etiology of dental caries and periodontal diseases. As mechanical methods of plaque control have some limitations, chemical methods were proposed. Therefore, the use of mouth rinses can be beneficial to reduce biofilms in conjunction with mechanical methods. The antimicrobial action of mouth rinse is by virtue of apoptosis, inhibition of bacterial growth, and/or cell metabolic inhibition; and depending on their concentration of the antimicrobial, their bactericidal and/or bacteriostatic properties vary. ,
The high prevalence of oral diseases may be attributed to the fact that majority of population neglect oral health, and oral health education and simple modalities of prevention are not available, especially underprivileged part of the society.
This silent outbreak of oral diseases disproportionately affects children irrespective of their socioeconomic status. The age group selected to carry out this study was 15-17 years, as the prevalence of dental caries is relatively more in this age group due to the changes in dietary habits and lifestyle. Since the intervention in the present study is a mouthrinse, it would be difficult for younger children to rinse and also the microbial flora in younger children varies during mixed dentition stage; hence, the present age group was selected, when it is likely for children to have all the permanent teeth present in the oral cavity.
Mean values for colony count of S. mutans on TYCSB agar were considered for the results as chairside cultural tests displayed considerable disparity between tests in recovering bacteria from pure cultures. The frequency of isolation of pure strains of bacteria other than MS was common in Dentocult SM strip mutans test. 
Plant extracts  and subsequent mouth rinse  were prepared according to the studies conducted earlier. All the extracts included in this study also have been generally recognized as safe for their intended use by U.S. Food and Drug Administration as per 21 CFR section and 21 CFR 184.1257.
Artificial sweetening agent was added to make the taste pleasant. No flavoring agent was added as most of the flavoring agents such as thymol and menthol are essential oils, which might create confusion in the study as already essential oil mouth rinse is studied as a separate group.
Sodium benzoate is the sodium salt of benzoic acid. It has long been used as a preservative in foods and other products, and its safety has been established. Sodium benzoate is used at a concentration of 0.03-0.1%.  Methyl paraben is a methyl ester of p-hydroxybenzoic acid. It is a stable, nonvolatile compound that has been used as an antimicrobial preservative in foods. ,
Unstimulated saliva was used to assess the parameters as unstimulated saliva represents the basal salivary flow rate. To avoid contamination, the saliva was collected in sterile syringes. Thioglycollate transport media was used as a transport media, so that the vitality of the organisms is maintained.
Each of the active agents has a different mechanism of action.
Plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen-substituted derivatives. Most are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be <10% of the total. In many cases, these substances serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Some, such as terpenoids, give plants their odors; others (quinones and tannins) are responsible for plant pigment. Many compounds are responsible for plant flavor (e.g., the terpenoid capsaicin from chili peppers), and some of the same herbs and spices used by humans to season food yield useful medicinal compounds. ,
The active ingredient in group 1 (herbal mouth rinse) used in the present study was a mixture of Garlic powder, aqueous extract of amla (gooseberry) and organic solvent based extract of Ginger. The active components of gooseberry are flavonoids which have anti glucosyl-transferase activity in addition to the binding of active components to the proteins associated with the cell surface of bacteria.  Likewise, the antibacterial activity of garlic is widely attributed to allicin, which interferes with RNA production and lipid synthesis that leads to improper formation of the phospholipid bilayer of the cell wall in both Gram-positive and Gram-negative bacteria.  Ginger possesses gingerols responsible for the cell membrane rupture, which leads to the direct inhibition of the bacteria. 
EO has a complex mechanism of antibacterial action; the antimicrobial actions of the EO are intimately attached to their major characteristic, hydrophobicity, which produces an increase in the bacterial membrane permeability and the consequent loss of their main cellular elements. 
Fluoride alters the physiochemical properties of teeth by making them more resistant to acid dissolution due to the formation of fluorapatite. It also increases the posteruptive maturation, enhances remineralization, and inhibits demineralization, inhibits various enzymes, such as enolases and, therefore, the transport of glucose is inhibited. 
Chlorhexidine gluconate, which is charged positively, shows high affinity for negative ions found in cell membranes of microorganisms. It indirectly affects the enzymatic function of dehydrogenase and adenosine triphosphatase present in the cell wall of bacteria resulting in the disruption of cell membrane. It is evident in this study that the chlorhexidine showed a definite reduction in the microbial activity and an increase in the pH and buffering capacity resulting in marked anticariogenic effect. However, there is a lack of convincing clinical data and long-term clinical evidence for caries prevention with chlorhexidine. Moreover, chlorhexidine have reports of causing discoloration effects and an unpleasant taste. ,,
Analytically, this study had pragmatically tested all the four mouth rinses therein has shown interesting antimicrobial variations in terms of efficacies.
It has been validated through a series of studies that chlorhexidine mouth rinse is considered to be the most superior among all mouth rinses against oral microorganisms. , Most studies comparing chlorhexidine and other mouth rinses have shown the superiority of chlorhexidine, besides a few studied products which were capable to emulate chlorhexidine in terms of antibacterial properties. 
The present study also confirmed that antibacterial action of chlorhexidine mouth rinse can be simulated with a multi-herbal mouth rinse in terms of substantivity, although the rate of action seemed slow initially analogous to fluoride mouth rinse. It is in accordance to a study which demonstrated that green tea mouth rinse could reduce oral microorganisms due to tannins, and there is no significant difference between chlorhexidine mouthwashes and green tea extracts so that both material have the same antimicrobial effects. 
Moreover, substantivity is an important aspect of any mouthrinse as the active ingredients of the mouthrinse have to exert effect for a considerable period after rinsing. Various studies have assessed the substantivity of chlorhexidine. In the present study, the herbal mouthrinses showed significant substantivity whereas rate of reduction of S. mutans colony count significantly decreased in case of fluoride or chlorhexidine mouthrinses after 1 week. The essential oil mouthrinse, on the contrary, according to the present study was the least effective mouthrinse against S. mutans.
Periodic use, according to immediate need and under appropriate guidance of a specialized dental professional is important to obtain maximum benefit while avoiding potential chemical toxicity and subsequent oral tissue damage. These results indicate that the reduction in S. mutans is similar to that caused by chlorhexidine. In addition, the advantage of herbal mouthrinse would be its safety and reduced cost.
In this study, the chlorhexidine group showed a greater statistically significant reduction of S. mutans count in saliva at different time periods than the other groups. But it cannot be ignored that use of herbal mouth rinse has the equivalent potential to behave as an important aid in maintenance of oral health. Thus, herbal mouthwash can be effectively used as an alternative to chlorhexidine and can be prescribed for longer duration without any side effects for management of oral problems.
Limitations of this study are as follows:
- Two week period is insufficient to evaluate substantivity of multi-herbal mouth rinse
- A cross-over design with wash-out period would have been more authenticating as it eliminates the bias of variable host response.
Therefore, future studies should be directed towards evaluating the antibacterial efficacy in long-term period, which can elucidate more characteristics of this multi-herbal formulated mouth rinse.
| Conclusion|| |
To recapitulate, currently there is no standardized definition of what constitutes "natural," leaving it wide open for interpretation and confusion. According to the U.S. National Organics Program, a natural substance is derived from a plant, mineral, or animal source without undergoing a synthetic process. Natural substances still can result from physical and biological processes, even when the resulting substance cannot be found in nature independent of these processes.
Hence, the need of the hour is to refine the definition of herbal or natural, in that context, as they can still prove to be reliable and equally efficacious, as the synthetic products are, just as in this case multi-herbal formulation of mouthrinse simulates chlorhexidine in the long-term use.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]