| Abstract|| |
Background: Antimicrobial agents serve as an effective adjunct with mechanical means in plaque control. Chlorhexidine has been the gold standard in the field of dentistry, but these days a growing number of dentists are embracing the philosophy that natural agents are better for children's oral health, and the focus is shifted toward biogenic agents for oral hygiene maintenance in children.
Aim: The aim was to evaluate antimicrobial and plaque inhibitory potential of herbal and probiotic rinses against Streptococcus viridans with commonly used antimicrobial agent like 0.2% chlorhexidine digluconate.
Materials and Methods: A randomized clinical trial was conducted on 60 subjects aged between 6 and 14 years and were divided into three groups comprising 20 subjects in each group. Three oral rinses were administered twice daily for a period of 1 week. Estimation of plaque scores and S. viridans counts was done before and after intervention, and the results were statistically analyzed.
Results: The change in mean plaque index in Groups A, B, and C was 0.28 ± 0.16, 1.37 ± 0.43, and 0.60 ± 0.35 respectively. Furthermore, change in mean log 10 colony forming unit (CFU)/ml of S. viridans in Groups A, B, and C was 0.13 ± 0.06, 0.53 ± 0.17, and 0.22 ± 0.06 CFU/ml, respectively.
Conclusion: Based on observations done during the course of study herbal rinse proved equally effective as 0.2% chlorhexidine digluconate in reducing S. viridans counts and plaque accumulation after 1 week of intervention, whereas probiotic rinse was least effective. However, long-term clinical trial with larger sample size needs to be undertaken, especially to evaluate beneficial effects of biogenic agents such as herbal and probiotic rinses.
Keywords: 0.2% Chlorhexidine rinse, dental plaque, herbal rinse, probiotic rinse, Streptococcus viridans
|How to cite this article:|
Mishra R, Tandon S, Rathore M, Banerjee M. Antimicrobial and plaque inhibitory potential of herbal and probiotic oral rinses in children: A randomized clinical trial. Indian J Dent Res 2014;25:485-92
Oral health is an integral component of general health. Daily oral home care helps in maintaining healthy oral environment. However, it is also a well-known fact by dental professionals that children do not consistently perform home care procedures at an acceptable level and plaque control by mechanical means like tooth brushing and flossing are sometimes not sufficient leading to plaque accumulation. In this process, environmental factors such as saliva, diet, and oral hygiene measures have a certain role.  Dental plaque has been proved to be a paramount factor in initiation and progression of gingival and oral diseases hence pediatric dentists often recommend chemical adjuncts in addition to tooth brushing and flossing for routine home care, which provides unique and beneficial approach in prevention of plaque formation and bacterial infection. ,
|How to cite this URL:|
Mishra R, Tandon S, Rathore M, Banerjee M. Antimicrobial and plaque inhibitory potential of herbal and probiotic oral rinses in children: A randomized clinical trial. Indian J Dent Res [serial online] 2014 [cited 2020 May 28];25:485-92. Available from: http://www.ijdr.in/text.asp?2014/25/4/485/142543
Arrays of antimicrobial agents are available. Chlorhexidine digluconate, a broad-spectrum antimicrobial agent with advantage of substantivity, is considered the gold standard in the field of dentistry.  However, few individuals experience bitter taste, light-brown stains on tooth, loss of taste sensitivity.  These side-effects limits long-term use of chlorhexidine as oral rinse and acceptability by patients. Therefore, search for new rinse continued and the scientific focus shifted toward biogenic agents.
Since times immemorial, herbal hygiene measures are practiced by different populations around the world. History of oral health care in India goes back to 5000 years BC in ancient literatures such as "Rig-Veda" and "Atharva-Veda", when oral care needs and diseases were noted, and use of plants and/or polyherbal formulations was highlighted for health care.  In almost all the traditional systems of medicine, medicinal plants play a major role and constitute their backbone. Chewing of neem sticks Azadiracta indica as a substitute of toothbrush is widely practiced in rural India.  Three myrobalans (Triphala) is extensively used in Ayurveda as anti-diabetic, antioxidant, anti-caries agent, and maintains gingival health. ,
Besides herbal agents, probiotics proved beneficial to host health by improving indigenous flora and play a role in our general health and wellbeing. "Live microorganisms when administered in adequate amounts confer a health benefit on the host."  It is well-documented in the literature that probiotics neutralizes intraoral acidic pH, release bacteriocins thus inhibit plaque accumulation. ,
With increasing antibiotic resistance, it has become important that we turn our attention to new ways to combat microbial infection. Henceforth, the objective of our study was to evaluate the antimicrobial and plaque inhibitory potential against Streptococcus viridans of herbal and probiotic oral rinses with commonly used antimicrobial agent like 0.2% chlorhexidine digluconate.
| Materials and methods|| |
The present study was conducted at Department of Pedodontics and Preventive Dentistry in collaboration with Department of Microbiology, Babu Banarasi Das College of Dental Sciences (BBDCODS), Lucknow, India. Protocol approval was obtained from Institutional Ethics Committee, BBDCODS, Lucknow. Before the commencement of the study, the purpose of the study was explained to parents and written informed consent was obtained.
A randomized clinical trial was conducted with a sample size of 60 subjects of age between 6 and 14 years attending pedodontics out-patient unit for a period of 6 months. Trial subjects were randomly divided into three groups comprising 20 subjects in each group. Three oral agents were administered twice a day for 1 week with an aim to evaluate the efficacy of herbal and probiotic oral rinses against S. viridans and to determine their plaque inhibitory potential with a commercially available agent like 0.2% chlorhexidine oral rinse.
- Children with carious teeth
- Children having plaque index (PI) ≥0.9
- No history of any antimicrobial agent or drug used in 3 months.
- Patients on any other oral hygiene regimen other than routine tooth brushing
- Patients undergoing any specialized dental treatment, for example, orthodontic treatment, space maintainers or any other appliances.
The oral agents used for the present study were [Figure 1] probiotics mint tablets (Evora Plustm, Florida, USA, composed of Probiora3), 0.2% chlorhexidine digluconate oral rinse (Hexidine ® , IPCA Health Products Ltd., Andhra Pradesh, India), and herbal oral rinse (Herboral, M-Tech Innovations Ltd., Pune, India).
It was a double-blind study and the designated rinse was dispensed by investigator to each subject in a sealed bottle without knowing the identity of the rinse.
- Group A: Subjects were administered probiotic mint tablet by mixing the tablet with 5 ml of bottled or filtered water in a supplied measuring cup
- Group B: Subjects were administered 0.2% chlorhexidine digluconate oral rinse
- Group C: Subjects were administered herbal oral rinse.
Baseline scores of PI and 7 days after intervention scores were recorded for all the subjects based on the format of Lφe.  The designated oral rinses were administered to respective group. Parents were advised to administer 5 ml rinse twice daily half an hour after brushing in the morning and at night for a period of 1 week. Furthermore, subjects were instructed to retain rinse in the oral cavity for 1 min followed by expectoration of rinse under parent's supervision and not to consume any form of solid or liquid food stuff for another half an hour. The subjects were constantly monitored to check whether they are following the aforementioned regimen regularly. During the intervention period, the subjects were encouraged to maintain normal oral hygiene habits. Saliva sample through swab technique was collected at baseline and a day after 7 days of intervention for estimation of S. viridans colony forming unit (CFU)/ml [Figure 2]. Sterilized saliva sample vials were immediately transferred to Department of Microbiology, BBDCODS, Lucknow for further microbiological estimation.
About 4 ml of normal saline was added to each of sterile vials containing salivary samples and kept under refrigeration. About 0.1 ml sample of saliva in saline was transferred to the culture plate containing blood agar and incubated at 37°C for 24-48 h [Figure 3]. α-Hemolytic green bacterial colonies were isolated and were identified by Gram stain. Gram-positive cocci were further tested for catalase. Finally, Gram-positive catalase negative cocci were isolated and transferred to skim milk culture media and estimated for CFU count. 
|Figure 3: Preintervention and postintervention Streptococcus viridans culture plates|
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The statistical analysis was carried out using Statistical Package for Social Sciences (SPSS) Version 15.0 statistical analysis software (SPSS Inc., Chicago, IL, USA). The values were represented in number (%) and mean ± standard deviation (SD). Student's "t"- test (unpaired t-test) was performed to assess the significance of two means of each oral rinse and paired "t"- test to compare the change in a parameter at two different time intervals, that is, at baseline and after 1 week. Finally, analysis of variance (ANOVA) test was used to perform intragroup and intergroup variances amongst the study groups. "P" is level of significance, where
- P > 0.05: Not significant
- P < 0.05: Significant
- P < 0.01: Highly significant
- P < 0.001: Very highly significant.
| Results|| |
The mean age of subjects in Group A was minimum (8.65 ± 2.01 years) followed by Group B (8.90 ± 2.99 years) while in Group C it was maximum (9.70 ± 3.01 years). However, on statistical evaluation, no significant difference was seen among the groups (P = 0.410) [Table 1].
In Group A, majority of subjects were females (55%), while in Group B and Group C 45% and 40% subjects were females. Despite proportional difference in gender, statistically no significant difference among groups could be seen (P = 0.626) [Table 2].
Preintervention S. viridans CFU/ml count was found to be ranging between 30,000 and 90,000 in study subjects. As there was a wide exponential within group variability, hence instead of comparing the absolute values, the log 10 values have been taken for comparison. The mean log 10 CFU/ml of S. viridans in Group A was 4.842 ± 0.095, which was maximum, followed by Group B with 4.841 ± 0.082 and then Group C 4.782 ± 0.143 [Table 3].
|Table 3: Preintervention and postintervention S. viridans CFU/ml count in three groups |
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The interquartile ranges in the box plot as depicted by the boxes show overlapping in the major part, more obviously in Group A and Group B with median values (as shown by dark horizontal line) falling at almost same plane. Though the interquartile range of Group C was widest, yet it was coinciding with both Group A and Group B [Figure 4].
|Figure 4: The interquartile ranges in box plot of preintervention mean colony forming unit/ml count of Streptococcus viridans in three groups|
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On comparing the data statistically by performing ANOVA, statistically no significant differences in mean preintervention CFU/ml count of S. viridans was seen in the three groups (F = 1.993; P = 0.146). As the differences among the groups were found to be not significant statistically on ANOVA, no further exploration was attempted.
Results of pairwise multiple comparison of three groups as regards preintervention CFU/ml count of S. viridans did not reveal a statistically significant intergroup difference (P > 0.05) [Table 4].
|Table 4: Pairwise multiple comparisons S. viridans mean CFU/ml count in three groups (values in log10) |
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Postintervention S. viridans CFU/ml count was found to be ranging between 10,000 and 50,000 in study subjects. The mean log 10 CFU/ml in Group A was 4.713 ± 0.104 which was maximum, followed by Group C with 4.562 ± 0.145 and then Group B with 4.314 ± 0.192 [Table 3].
The interquartile ranges in the box plot as depicted by the boxes show very less overlapping thereby implying significant differences among the groups. Some extreme values are also seen in Groups A and B graph [Figure 5].
|Figure 5: The interquartile ranges in box plot of postintervention mean colony forming unit/ml count of Streptococcus viridans in three groups|
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Analysis of variance revealed a statistically significant intergroup difference in postintervention mean CFU/ml count of S. viridans in three groups with Group A having maximum number, while Group B had a minimum number (P < 0.001) [Table 5].
|Table 5: Analysis of variance of postintervention mean CFU/ml count of S. viridans and mean PI in three groups |
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As the difference among the groups was found to be significant statistically on ANOVA, pairwise multiple comparisons were performed to see the intergroup differences more clearly. Pairwise multiple comparisons revealed that Group A had significantly higher mean CFU/ml of S. viridans when compared to Group B and Group C respectively, while Group B had significantly higher mean CFU/ml count when compared to Group C [Table 4].
The maximum change in mean CFU/ml counts of S. viridans was seen in Group B following intervention (0.53 ± 0.17) while the minimum was seen in Group A (0.13 ± 0.06). In all the three groups, a significant decrease in mean S. viridans CFU/ml count was seen (P < 0.001) [Table 6].
|Table 6: Comparison of change in S. viridans CFU/ml count and mean PI in three groups |
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In terms of change in S. viridans CFU/ml count, statistically significant results were obtained whereby, maximum reduction of S. viridans CFU/ml count was seen in Group B (chlorhexidine oral rinse) followed by Group C (herbal oral rinse) and least with Group A (probiotic oral rinse).
Preintervention PI was found to be ranging between 0.9 and 2.7 in study subjects. The mean PI in Group A was found to be 1.855 ± 0.322 which was the maximum while in Group B it was 1.680 ± 0.464 which was minimum. The mean value of the PI in Group C was 1.685 ± 0.509 [Table 7].
|Table 7: Preintervention and postintervention PI values in three groups |
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The interquartile ranges in the box plot as depicted by the boxes show complete overlapping of Group A by Group B and Group C. Group B and Group C too showed almost complete overlap with each other [Figure 6].
|Figure 6: The interquartile ranges in box plot of preintervention mean plaque index in three group|
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On comparing the data statistically by performing ANOVA, statistically no significant difference in mean PI was seen preintervention in the three groups (F = 1.030; P = 0.364). Pairwise multiple comparisons did not reveal a statistically significant intergroup difference (P < 0.001) [Table 8].
Postintervention PI was found to be ranging between 0 and 2 in study subjects. The mean PI in Group A was found to be 1.58 ± 0.29 which was the maximum while in Group B it was 0.32 ± 0.25, which was minimum. The mean value of PI in Group C was 1.09 ± 0.35 [Table 7].
The interquartile ranges in the box plot as depicted by the boxes do not show an overlapping hence indicating existence of differences among the groups [Figure 7].
|Figure 7: The interquartile ranges in box plot of postintervention plaque index in three groups|
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On comparing the data statistically by performing ANOVA, a statistically significant difference in the mean PI was seen after intervention in the three groups (F = 92.102; P < 0.001). It was seen that Group A had the maximum mean PI value while Group B had minimum mean value [Table 5].
As the difference among the groups was found to be significant statistically on ANOVA, multiple comparisons were performed to see the intergroup differences more clearly. Pairwise multiple comparisons revealed that Group A had significantly higher mean PI as compared to Group B and Group C respectively while Group C had significantly higher mean PI as compared to Group B [Table 8].
As far as change in the mean PI was concerned, it was maximum in Group B (1.37 ± 0.43), followed by Group C (0.60 ± 0.35) and then Group A (0.28 ± 0.16). In all the three groups, the mean change was significant statistically (P < 0.001) [Table 6].
The order of efficacy of three groups was, maximum reduction was seen in Group B followed by Group C and least reduction was seen in Group A.
A < C< B
| Discussion|| |
Human oral cavity is usually sterile at birth soon it acquires a predominant streptococcal microbiota. The species most frequently related to "viridans streptococci" includes Streptococcus salivarius, Streptococcus sanguis, Streptococcus mitis, and Streptococcus mutans. 
Dental caries, a microbial disease is caused by α-hemolytic streptococci commonly referred to as S. viridans, a major component of dental plaque and solely responsible for issues concerning pediatric dental health. 
Failure to brush effectively and inappropriate interproximal flossing limits mechanical means of plaque control leading to gingivitis and dental caries. Subsequently, chemical means can serve an effective adjunct with mechanical means of plaque control.
Streptococcus viridans was selected for the present study as it is one the most commonly found Gram-positive species in the oral cavity and its prevalence ranges from 9% to 24% in healthy children. 
In our study, a significant reduction in S. viridans was observed in all the groups. Although, 0.2% chlorhexidine digluconate rinse showed better antimicrobial efficacy against S. viridans than herbal rinse, but no significant difference was noted between herbal and chlorhexidine group. Probiotic rinse was least effective.
The data obtained in our present study were in concurrence with Shapiro et al. they compared the efficacy of 12 different mouth rinses and inferred that herbal agents were substantially less effective than chlorhexidine rinses.  Later, Salehi and Momeni Danaie also observed significant reduction by chlorhexidine and herbal rinse.  Rosin et al. also proved that the reduction of bacterial counts with 0.12% chlorhexidine was significantly greater than with 0.04% polyhexamethylene biguanide. 
Considering the effects of herbal rinse on S. viridans, the present trial demonstrated a significant reduction in S. viridans counts after 1 week of intervention. The results of our present study were well in accordance with an earlier work conducted by Srikumar et al.  They revealed Triphala to be an effective antibacterial agent. Jagadish et al. evaluated the free radical scavenging property and antimicrobial activity of Triphala. 
In contrast to our present study, Tandon et al. observed no significant difference in antimicrobial efficacy of Triphala mouthwash in prevention of dental caries with chlorhexidine mouthwash. The difference in the two studies could be attributed to the difference in design, duration of the study or sample criteria selection. 
Triphala was a major ingredient in our herbal rinse. Apart from it, the other ingredients were A. indica, is effective in suppressing bacterial infections and is nontoxic, non-mutagenic, immuno-stimulant, with anti-oxidant properties;  Quercus infectoria has potential to generate herbal metabolites having anti carious activity; Tulsi (Ocimum sanctum) has several scientific evidences to prove its antimicrobial, antidiabetic, hepato-protective, anti-inflammatory, anti-carcinogenic, immunomodulatory and neuro-protective properties; Clove (Caryophyllus aromaticus), is widely used in dentistry due to its analgesic, local anesthetic, anti-inflammatory, and antibacterial property.
Furthermore, Haffajee et al. investigated the antimicrobial effectiveness of herbal and essential oil mouth rinses in comparison with 0.12% chlorhexidine gluconate rinse against predominant oral bacteria and found that although less potent than chlorhexidine gluconate rinse, herbal rinse was more effective than essential oil rinse in inhibiting the growth of oral bacteria in vitro. 
World Health Organization defined probiotics as "live microorganisms that when administered in adequate amounts confer a health benefit on the host". 
Probiotics rinse in the present study showed minimum effect on S. viridans counts after one-week of intervention. These findings were in contrast to observations documented by Caglar et al., they reported statistically significant reduction of S. viridans after administration of Lactobacillus reuteri probiotic strains namely ATCC 55730 and ATCC PTA 5289, when compared to xylitol chewing gum during 3-week time span.  Jindal et al. also demonstrated that probiotics are effective in caries prevention by decreasing the number of mutans streptococci. 
Twetman et al. concluded that all probiotic strains derived from consumer products showed coaggregation abilities with the oral pathogens and the results were strain specific and time-dependent. They also demonstrated anti-caries properties of lactobacilli-derived probiotic bacteria by coaggregating with selected oral streptococci. 
Furthermore, Streptococcus uberis KJ2, Streptococcus oralis KJ3, and Streptococcus rattus JH145 strains (probiotic, ProBiora3) used in our present study are susceptible to commonly used antibiotics with no-observed-adverse-effect level.  Zahradnik et al. also demonstrated that these strains (probiotic, Probiora3) are able to substantially affect the levels of pathogens in saliva and in subgingival plaque. 
In our present study, a decrease in S. viridans counts was observed in Group C and Group A which correlated well with Mehanna et al. they demonstrated an inhibitory effect of Meswak plant and probiotic strain (Lactobacillus rahmonosus) on Streptococcus mutans and Porphyromonas gingivalis counts. 
As far as change in mean PI was concerned, the maximum reduction was seen in Group B (i.e. chlorhexidine rinse) followed by Group C (i.e. herbal rinse) and the least reduction was seen in Group A (i.e. probiotic oral rinse). Our results co-related well with the research conducted by Chitsazi et al. who elucidated effect of three rinses two herbal, Matrica, Persica; and chlorhexidine and found a statistically significant reduction in PI in all groups but it was prominent with chlorhexidine rinse.  In the same year, Van Strydonck et al. inferred that use of 0.2% chlorhexidine mouth rinse (in combination with brushing) remains the gold standard for plaque control. 
Rassameemasmaung et al. determined effects of herbal mouthwash containing pericarp extract of Carcinia mangostana L. on PI and observed a significant reduction in PI after 15 days of use.  In the current study, herbal rinse was used for a period of 7 days, and similar results were observed. Amrutesh et al. also observed a reduction in PI, gingival index and oral hygiene index by herbal dental cream in comparison to fluoride dental cream that was statistically not significant. 
Furthermore, Botelho et al. compared efficacy and safety of A. indica mouth rinse on gingival inflammation and microbial plaque, with 0.12% chlorhexidine. And concluded that A. indica-based mouth rinse is highly efficacious, and it may be used as an alternative therapy in the treatment of periodontal diseases. 
Our present study also demonstrated a reduction in PI to be concomitant with a reduction in S. viridans counts and was maximum reduction was in Group B (chlorhexidine rinse) and minimum in Group A (probiotic). The reason for lower efficacy of probiotics could be explained by theory of mutual exchange of microbes in plaque layer.  It is also possible that while the S. viridans count might have reduced, at the same time it might have been replaced by probiotic themselves. Harini and Anegundi  in a pilot study, done on 45 children aged 6-8 years observed no significant difference between probiotics and chlorhexidine rinse in reduction of PI within a time span of 14 days. 
Knowledge of biogenic agents has entered a new phase of research and progression in this field will offer novel and useful means for prevention and treatment of oral diseases.
| Conclusion|| |
On the basis of observations made during the course of study and their analysis, a significant reduction in mean CFU/ml count of all the three groups was observed for S. viridans and plaque accumulation. Herbal oral rinse proved equally effective antimicrobial agent as 0.2% chlorhexidine digluconate in reducing S. viridans counts and plaque accumulation whereas probiotic oral rinse prove to be less effective. However, long-term clinical trial with a larger sample size needs to be undertaken, especially to evaluate the beneficial effect of probiotics.
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Department of Pedodontics and Preventive Dentistry, Purvanchal Institute of Dental Sciences, Gorakhpur
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]