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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 19  |  Issue : 3  |  Page : 217-223

A comparative evaluation of herbal and nonherbal mouthrinses on salivary pH levels and salivary Streptococcus mutans count in 6–12-year-old children: A randomized controlled trial


1 Department of Public Health Dentistry, Government Dental College and Research Institute, Bengaluru, Karnataka, India
2 Department of Microbiology, Bowring and Lady Curzon Hospital, BLCMC and RI, Bengaluru, Karnataka, India

Date of Submission05-Mar-2021
Date of Decision05-Apr-2021
Date of Acceptance18-Jul-2021
Date of Web Publication15-Oct-2021

Correspondence Address:
Avisek Mukherjee
Department of Public Health Dentistry, Government Dental College and Research Institute, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_31_21

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  Abstract 


Background: Dental caries is a disease of complex etiology, in which microorganisms play an important role. Salivary pH and Streptococcus mutans count influence the initiation and progression of dental caries. Objectives: This study aimed to assess the efficacy of the combined herbal and nonherbal rinse on salivary pH and salivary S. mutans count and comparing it with herbal and nonherbal mouthrinse in 6–12-year-old high caries-risk children. Methodology: Ninety high caries-risk children of age 6–12 years were selected and allocated randomly into three groups (herbal, nonherbal, combination mouthrinses = 30 each). Children were instructed to rinse their mouth twice daily with their assigned mouthrinses for 7 days. Salivary samples were collected at baseline, 1 h post rinsing, and at the end of 7th day of mouthrinsing for determining the salivary pH and S. mutans count. ANOVA and post hoc tests were applied. Statistical significance was considered at P < 0.05. Results: There was a statistically significant difference in salivary pH between baseline, 1 h, and 7 days. There was a statistical significant difference in mean S. mutans count among herbal, nonherbal, and combination mouthrinse at baseline and 1 h. There was a statistically significant intragroup difference for mean salivary pH and S. mutans at baseline and follow-ups in all three groups. Conclusion: Combination mouthrinse was more effective in increasing salivary pH level and reducing S. mutans count followed by herbal and nonherbal mouthrinse at 1 h. Nonherbal mouthrinse was more effective in increasing salivary pH level as compared to combination and herbal mouthrinse after 7 days. There was no statistically significant reduction in S. mutans count among groups at 7 days.

Keywords: Herbal, mouthrinse, nonherbal, salivary pH, salivary Streptococcus mutans


How to cite this article:
Mukherjee A, Yashoda R, Puranik M, Yadav K. A comparative evaluation of herbal and nonherbal mouthrinses on salivary pH levels and salivary Streptococcus mutans count in 6–12-year-old children: A randomized controlled trial. J Indian Assoc Public Health Dent 2021;19:217-23

How to cite this URL:
Mukherjee A, Yashoda R, Puranik M, Yadav K. A comparative evaluation of herbal and nonherbal mouthrinses on salivary pH levels and salivary Streptococcus mutans count in 6–12-year-old children: A randomized controlled trial. J Indian Assoc Public Health Dent [serial online] 2021 [cited 2021 Dec 7];19:217-23. Available from: https://www.jiaphd.org/text.asp?2021/19/3/217/328279




  Introduction Top


Dental caries is a major public health problem.[1] Endogenous oral bacterial species such as Streptococcus mutans and their metabolites have a complex role in the initiation and progression of dental caries.[2]

Various components of saliva play significant roles in modulating the properties of saliva. However, among these, pH of saliva maintains the integrity of the oral cavity.[3] When the pH increases, the remineralization of tooth surface occurs because of the degree of supersaturation, whereas acidic pH can cause demineralization and increase the incidence of dental caries.[3]

S. mutans has been known to be an etiological agent in the development of dental caries.[4],[5] Thus, evaluating the risk factors such as pH and S. mutans count in saliva of individuals at high caries risk could be of great use in the prevention of dental caries.

The pH of sodium fluoride is neutral and is available in a concentration of 0.05% (220 ppm) for daily use and it is one of the most important and effective components of preventive dental programs in children.[6]

Green tea is reported to be very rich in catechin, was found to be related to its role in the depletion of the thiol group which, in turn, exerted bactericidal effect.[7] It also mediates protective salivary components such as secretory immunoglobulins, lysozymes, lactoferrin, oral peroxidases histatins, mucins, or others.[8]

Tulsi, scientifically known as Ocimum sanctum, is a time-tested premier medicinal herb.[9] Studies have tested the efficacy of Triphala mouthrinse in the inhibition of Streptococcus counts.[10]

Neem (Azadirachta indica) due to its antiplaque, anticaries, and antibacterial effects and anti-inflammatory and analgesic actions has been widely used in different parts of the world as an oral hygiene tool.[11] Neem sticks are easily available and cost-effective, and efficacy is equally as good as common dentifrice.[12]

Previous studies have considered herbal products Triphala,[13],[14],[15],[16] tulsi,[9],[17] and green tea[18],[19] to assess its effectiveness on salivary pH and S. mutans and it was as effective as the nonherbal mouthrinse. However, there is a lack of studies of literature about the antimicrobial activity of combined herbal and nonherbal mouthrinse, against caries-causing microorganisms such as S. mutans and salivary pH levels.

Hence, this clinical study was planned to assess the efficacy of the combined herbal and nonherbal rinse on salivary pH and salivary S. mutans count and comparing it with herbal and nonherbal mouthrinse in 6–12-year-old high caries-risk children.


  Methodology Top


A triple-blind parallel clustered randomized controlled trial was conducted at Government Residential Schools in Bangalore city for a comparative evaluation of herbal and nonherbal mouthrinses on salivary pH levels and salivary S. mutans count among 6–12-year-old children in Bangalore city from July to September 2019. A protocol of the intended study was submitted to Institutional Ethical Committee and Review Board and ethical clearance was obtained. Permission was obtained from the concerned authorities. The written informed consent was obtained from the participant's parents. This study was carried out following the ethical standards of the World Medical Association for human experimentation 2008 version of the Helsinki Declaration.[20]

Investigator 1 was trained and calibrated in the Department of Public Health Dentistry.

The sample size was established using G* power version 3.1.9.2.[21] Effect size was 0.95 based on the mean of S. mutans count from the previous study.[2] Mean of S. mutans count: A group = 2.293; B group = 0.288; C group = 0.260; α = 0.05; Power (1− ß) = 0.9128. The sample size obtained was 18, considering 20% of lost to follow-up final sample size was rounded off to 30. Hence, 90 children between 6 and 12 years of age and with high caries risk according to the Cariesrisk Assessment Tool for >6 years old according to AAPD guidelines[22] 2011 in Bangalore city were included in the study. Children with any systemic disease, physically and mentally challenged, history of taking antibiotics up to 1 week before the study period and any intraoral soft tissue pathology were excluded from the study. The study participants were allocated into three groups herbal mouthrinse (Triphala 0.6%, green tea, neem), nonherbal mouthrinse (sodium fluoride 0.05%), and Group C – combination mouthrinse (tulsi 4% with sodium fluoride 0.05%) using research randomizer.[23] Allocation concealment was done by the investigator number 2. Throughout the study, the investigators, microbiologists, and children were blinded. The study design is given in the flowchart [Figure 1].
Figure 1: Flowchart of study design

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Data were collected using structured pro forma. Saliva collection was performed at baseline, 1 h, and after 7 days. The children were informed not to eat or drink anything (except water) 2 h before saliva collection. Saliva collection was done in the morning between 11 and 11.30 AM. The collection of unstimulated saliva (1.5 ml) was done by spitting method using sterile screw cap containers. Salivary pH analysis was done by pH meter immediately. The collected saliva was later transferred to a vial containing thioglycolate transport medium and the sample was sent to the laboratory for microbial analysis for estimation of salivary S. mutans count.

Intervention

Intervention was performed by instructing the children to rinse 5 ml of their assigned mouthrinse twice per day (morning and evening) for 7 days. They were informed to report in case of any adverse effect due to mouthrinse to the instructor/caretaker/warden. Compliance for the mouthrinse was assessed regularly.

The saliva samples were homogenized manually by stirring using a stirrer. Hundred microliters of saliva was diluted in 500 μL of normal saline (1:5 dilution). Using an inoculation loop (2 mm inner diameter), 5 μl of the 1:5 dilution sample was streaked on mitis salivarius bacitracin agar (MSB), a selective medium for S. mutans. The MSB agar plates were incubated for 48 h at 37°C in an atmosphere of 5% carbon dioxide. The colony count of each plate was recorded and the mean streptococcal count was determined after multiplying the colony count of each plate with its respective dilution factor.[2] All the microbiological procedures were performed by a trained and calibrated microbiologist.

The data were analyzed using SPSS version 16.0, IBM, Armonk, NY, USA. P < 0.05 was considered as significant (confidence interval of 95%). Descriptive analysis comparing mean (standard deviation) and percentage (proportion) was done for continuous and ordinal data, respectively. One-way ANOVA test and post hoc Tukey test were used for comparison between the three groups. Repeated-measures ANOVA was used for intragroup comparison.


  Results Top


The mean age of the study children was 9.8 ± 2.05 years. The majority 54.4% were males and 45.6% were females. With herbal mouthrinse, mean salivary pH was 7.45 ± 0.244 at baseline which increased to 7.55 ± 0.25 at 1 h post mouthrinse and 7.66 ± 0.26 at 7 days after mouthrinse. With nonherbal mouthrinse, the mean salivary pH was 7.38 ± 0.28 at baseline, which increased to 7.44 ± 0.21 at 1 h post mouthrinse and 7.7 ± 0.25 at 7 days after mouthrinse. With combination mouthrinse, the mean salivary pH was 7.62 ± 0.18 at baseline, which increased to 7.79 ± 0.19 at 1 h post mouthrinse and 7.9 ± 0.18 at 7 days after mouthrinse. On intragroup comparison, there was a statistically significant increase in salivary pH levels among baseline and 1 h post mouthrinse; baseline and 7 days after mouthrinse; and 1 h postmouthrinse and 7 days after mouthrinse in all the groups. On intergroup comparison, at baseline, 1 h, and 7 days, there was a statistically significant decrease in salivary pH levels among the study groups [P ≤ 0.001; [Table 1] and [Figure 2]].
Table 1: Mean salivary pH at baseline and follow-up among study groups

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Figure 2: Mean salivary pH at baseline and follow-up among study groups

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With herbal mouthrinse, mean S. mutans count was 4.73 ± 0.05 at baseline which was reduced to a count of 4.70 ± 0.05 at 1 h post mouthrinse and 4.68 ± 0.04 at 7 days after mouthrinse.

With nonherbal mouthrinse, mean S. mutans count was 4.75 ± 0.075 at baseline, which reduced to 4.71 ± 0.062 at 1 h post mouthrinse and 4.69 ± 0.05 at 7 days after mouthrinse. With a combination mouthrinse, mean S. mutans count was 4.78 ± 0.06 at baseline, which was reduced to a count of 4.74 ± 0.05 at 1 h post mouthrinse and 4.70 ± 0.03 at 7 days after mouthrinse.

On intragroup comparison

There was a statistically significant reduction in S. mutans count among baseline and 1 h post mouthrinse; baseline and 7 days after mouthrinse; 1 h post mouthrinse; and 7 days after mouthrinse.

On Intergroup comparison

There was a statistically significant difference among herbal and combination groups at 1 h post mouthrinse. There was no statistically significant difference in S. mutans count among study groups at 7 days after mouthrinse [Table 2] and [Figure 3].
Table 2: Mean salivary Streptococcus mutans count at baseline and follow-up among study groups

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Figure 3: Mean salivary Streptococcus mutans count at baseline and follow-up among study groups

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During the study period, none of the children in all the three groups reported any adverse events and the compliance was satisfactory.


  Discussion Top


Numerous authors have interrelated S. mutans count with caries status of children and have consistently found a higher count in children having active caries.[24] Preventive strategies usually involve decreasing the growth or activity of S. mutans. Since caries is an infectious disease, it can be thought of as being treated with antimicrobials. The use of chemotherapeutic agents was prescribed for 7 days as changes in the bacterial composition and population have been noticed after short-term use of chemotherapeutic agents.[24]

Thus, in this study, herbal mouthrinse, nonherbal, and combination of herbal and nonherbal mouthrinses were evaluated for their antimicrobial efficacy by estimating the salivary pH levels and salivary S. mutans counts prerinse and postrinse for 1 h and 7 days. This clinical study was conducted in three residential schools; to ensure that all the individuals had the same dietary and oral hygiene practices. Besides, daily rinsing could be monitored by the local guardian and the researchers.

The age group of 6–12 years was selected since the caries prevalence is considered to be high in this age group. This was similar to a Megalaa et al's study,[2] Whereas previous studies considered 4–6,[18] 7–13,[25] 8–14,[14] and 12–15[15] years age group.

Herbal products have been used since ancient times in folk medicine. Phytochemical constituents of herbal mouthwashes are responsible for its antimicrobial activity. A few in vitro[9] and clinical studies[17] have found tulsi to be effective against S. mutans. Black myrobalans/T. schedule one of the chemical constituents also exhibits antibacterial activity.[15]

Rinsing with green tea extract had valuable anticariogenic activities including inhibitory effect on cariogenic bacteria by inhibiting the adherence of bacterial cells to the tooth surfaces.[19],[26],[27] Green tea catechins maintain the salivary pH at a normal range, which is not a favorable condition for cariogenic bacteria to flourish.[28],[29] In an in vitro study, neem extracts have shown the maximum zone of inhibition against S. mutans. Even at a 5% concentration, this extract showed some antimicrobial activity with an inhibition zone of 3.1 mm.[12] Sheila et al. stated that neem has antiadhesive effects which minimize the plaque formation. The antiadhesive effect may be due to the presence of phytochemicals.[30] Studies have shown sodium fluoride mouthrinse to be effective in reducing S. mutans counts.[2],[31] Thus, sodium fluoride mouthrinse was selected as a nonherbal comparative in our study.

In the current study, tulsi was used as a herbal component in combination mouthrinse because tulsi is abundantly available, easily accessible, economically feasible, and culturally acceptable and may possess minimal side effects.

There was a significant difference in pH between baseline, 1 h, and 7 days among the groups. In the present study at 1 h post mouthrinse, an increase in salivary pH was more in the combination and herbal group followed by the nonherbal group, which was similar to the studies done by Megalaa et al.,[2] wherein increase in salivary pH was more in nonherbal group (sodium fluoride) followed by herbal group (tulsi group >black myrobalans), whereas a study done by Carounanidy et al.[32] showed no significant difference among the groups at 60 min. But at 90 min, there was a significant increase in salivary pH in the nonherbal (chlorhexidine) followed by the herbal (emblica > Terminalia chebula) group.

In the current study, after 7 days post mouthrinse, rise in salivary pH levels was more in a nonherbal group, followed by the combination group and the herbal group which was similar to the study done by Megalaa et al.[2] which showed an increase in salivary pH in nonherbal group (sodium fluoride) followed by herbal group (black myrobalans > tulsi group). With the use of herbal mouthrinse there was a steady increase in the salivary pH, which is similar with Nayak et al.'s study,[15] which observed an increase in salivary pH till 1 h. Carounanidy et al.[32] found T. chebula mouthrinse to be very effective in increasing the salivary pH up to 90 min and the peak increase was at 10 min post rinsing. Similar studies also found an increase in salivary pH at 10 min[16] and 5 h after rinsing.[33] This shows that both the green tea extract and Triphala extract mouthrinses could be tried as an effective mouthrinse.

Increase use of nonherbal mouthrinse for 7 days increased the salivary pH. These results are in line with three previous studies that used sodium fluoride.[31],[34],[35]

After using combination mouthrinse, there was an increase in the pH in an orderly manner, i.e. from baseline to 1 h post rinse and after 7th day post rinse in the present study. The results of this study cannot be compared with other studies because there is a lack of relevant literature, which assessed the salivary pH on using combination mouthrinse, whereas it was per the studies conducted with tulsi mouthrinse[2] and sodium fluoride mouthrinse[31],[34],[35] as both tulsi and sodium fluoride were components of the combination mouthrinse in the present study. Hence, it can be said that combination mouthrinse increased the salivary pH over some time. There was a significant difference in reduction in salivary S. mutans count between baseline and 1 h among the groups.

In the present study at 1 h post mouthrinse, reduction in S. mutans count was more in the combination group followed by nonherbal group and herbal group. The results are contrary to a study[2] which showed a higher reduction in S. mutans count in herbal groups (tulsi group > black myrobalans) than nonherbal groups (sodium fluoride group). Similarly, a study done by Carounanidy et al.[32] showed a significant difference in reduction in salivary S. mutans count in herbal groups (Emblica officinalis > T. chebula group) than nonherbal group (chlorhexidine group) at 90 min.

However, in the present study, there was no significant difference in reduction of S. mutans count observed on post 7th day on intergroup comparison. This finding is contrary to a study which showed a higher reduction in S. mutans count in herbal groups (black myrobalans group > tulsi) than nonherbal group (sodium fluoride group).[2]

Similarly studies done by Mehta et al.[14] and Thomas et al.[18] for 10 days and 14 days duration, respectively, showed a significant reduction in salivary S. mutans count in herbal mouthrinse (chamomile and green tea respectively) followed by nonherbal mouthrinse (chlorhexidine), whereas studies[13],[17] found no significant reduction of S. mutans count among the groups. The difference in effectiveness among the groups in the current study and earlier studies could be attributed to a positive additive effect of combination of herbal and nonherbal, while a negative additive effect when different herbal products are combined also the duration of assessment was different. After using herbal mouthrinse, a significant reduction in the S. mutans count was observed on the 1 h when compared to 7 days post rinse. Our results are in comparison with previous studies.[15],[36] One of the constituents of which consists of tannic acid known to be well adsorbed to the hydroxyapatite of the tooth or the salivary mucins. Alternatively, it can bound to the anionic groups on the surface of the bacterial cells, which could result in protein denaturation and ultimately bacterial cell death. Jagtap and Karkera[33] have made similar observations up to 3 h post rinsing although they used 10% aqueous extract of T. chebula, while Carounanidy et al.[32] used 10% aqueous extract of black myrobalans and found a 65% decrease in the S. mutans count. In this study, the antibacterial effect of green tea mouthrinse is per the previous reports where the authors stated that rinsing with green tea extract had valuable anticariogenic activities which acts by inhibiting the adherence of bacterial cells to the tooth surfaces.[19],[26],[27],[28] However, studies regarding the antimicrobial effect of neem mouthrinse have been proven in in vitro studies, which is mainly due to inhibition of cell membrane synthesis in bacteria.[12] In this study, there was a significant reduction in the S. mutans count on using sodium fluoride mouthrinse at 1 h and 7 days post mouthrinse. This was similar to the results observed from other studies on sodium fluoride.[25],[34]

The effects of fluoride on streptococcal cells are partly ascribed to the inhibition of enolase, one of the series of glycolytic enzymes.[37] For combination mouthrinse, there is a lack of literature available for comparison within the groups. However, it can be reasonably argued that tulsi and sodium fluoride extracts present in the combination mouthrinse have a beneficial effect in terms of reducing S. mutans count. The antibacterial effect of tulsi may be attributed to the presence of methyl ester of salicylic acid which accelerates granulation and regeneration of tissues.[2] Furthermore, ursolic acid and carvacrol might be responsible for its antimicrobial activity.[38] Some authors have found its fixed oil to exhibit good antibacterial activity due to its higher linolenic acid content.[39]

To the best of our knowledge, this is the first study to include combination mouthrinse as a comparison group this was thought to provide the dual advantage of benefits of herbal and nonherbal products in one rinse. The present study was carried out under real-life conditions without altering the subjects' routine oral hygiene aids and practices of the children. The limitation of the study was that the present study was undertaken for 7 days. It was conducted among the age group of 6–12 years residential school children. Hence, the results should be interpreted with caution. Long-term research should be conducted involving combined different herbal and nonherbal mouthrinse with wide population subgroups.


  Conclusion Top


  • There was a statistically significant increase in salivary pH level and decrease in salivary S. mutans count in all three mouthrinse groups between baseline, 1 h, and 7 days
  • Combination mouthrinse was more effective in increasing salivary pH level and reducing S. mutans compared to herbal and nonherbal mouthrinse at 1 h. Nonherbal mouthrinse was more effective in increasing salivary pH level as compared to combination and herbal mouthrinse after 7 days.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

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