|Year : 2021 | Volume
| Issue : 3 | Page : 189-194
Efficacy of 4% tulsi leaf extract, fluoridated and placebo dentifrices against salivary Streptococcus mutans count among 14–15 years school children in Davangere City, Karnataka – A concurrent parallel trial
GV Usha1, Nagesh Lakshminarayan2, NG Bhuvaneshwari3
1 Department of Public Health Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India
2 Department of Public Health Dentistry, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India
3 Public Health Dentist, Oral Cancer Screening Vertical, Indian Cancer Society, Mumbai, Maharashtra, India
|Date of Submission||27-Sep-2020|
|Date of Decision||01-Apr-2021|
|Date of Acceptance||14-Sep-2021|
|Date of Web Publication||15-Oct-2021|
N G Bhuvaneshwari
Public Health Dentist, Oral Cancer Screening Vertical, Indian Cancer Society, Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Dental caries is a multifactorial disease, which is preventable by adopting healthy behaviors like toothbrushing. Tulsi extract mouthrinse has confirmed its antibacterial efficacy against Streptococcus mutans. Aim: The aim of this study was to assess and compare the antibacterial efficacy of 4% tulsi leaf extract, commercially available fluoridated (Colgate Total Care), and placebo dentifrices on salivary S. mutans counts among 14–15-year-old schoolchildren in Davangere city. Subjects and Methods: A triple-blinded randomized controlled trial consisted of 84 randomly selected participants aged 14–15 years. Baseline unstimulated 2-ml saliva was collected and the microorganisms were cultured using mitis salivarius selective media. The participants with 105 colony-forming units were considered eligible. Block randomization and concealed randomization method was adopted to allocate the participants into three groups containing 28 each. After 7 days, postassessment of salivary S. mutans count was done. Statistical Analysis: Wilcoxon signed-rank test for within-group comparison and Kruskal–Wallis analysis of variance for intergroup comparison were used. Missing data analysis was performed. Results: The study showed a significant reduction in salivary S. mutans counts from baseline to posttest (mean rank 37.95–28.45) in tulsi toothpaste group. Intergroup comparison showed a statistically significant difference (P = 0.035). Post hoc analysis showed a statistically significant difference between tulsi and placebo toothpaste group (P < 0.05). Conclusion: Four percent tulsi extract incorporated in the form of toothpastes have shown a maximum reduction in the salivary S. mutans count for a period of 7 days when compared to fluoridated toothpaste.
Keywords: Dental caries, dentifrice, randomization, Streptococcus mutans, tulsi
|How to cite this article:|
Usha G V, Lakshminarayan N, Bhuvaneshwari N G. Efficacy of 4% tulsi leaf extract, fluoridated and placebo dentifrices against salivary Streptococcus mutans count among 14–15 years school children in Davangere City, Karnataka – A concurrent parallel trial. J Indian Assoc Public Health Dent 2021;19:189-94
|How to cite this URL:|
Usha G V, Lakshminarayan N, Bhuvaneshwari N G. Efficacy of 4% tulsi leaf extract, fluoridated and placebo dentifrices against salivary Streptococcus mutans count among 14–15 years school children in Davangere City, Karnataka – A concurrent parallel trial. J Indian Assoc Public Health Dent [serial online] 2021 [cited 2021 Dec 7];19:189-94. Available from: https://www.jiaphd.org/text.asp?2021/19/3/189/328273
| Introduction|| |
Dental caries is the most common multifactorial infectious disease affecting humans. It is highly determined by one's “lifestyles” and Streptococcus mutans being one of the microbial causative agents. According to the National Oral Health Survey, the prevalence of dental caries among 15-year-old children was 61.4%. It is preventable by adopting healthy behaviors, such as regular toothbrushing, favorable dietary habits, and regular dental checkup. Toothbrushing is the main intervention universally performed in the home in order to mechanically remove and control the dental biofilm. It prevents caries and periodontal disease, but for many adults, toothbrushing alone is inadequate for this purpose. Because of its shortcomings, there arises the need for use of chemotherapeutic agents to improve plaque and oral hygiene. Chemotherapeutic agents in the form of dentifrice and mouthwash are used so as to efficiently maintain oral health.
Fluoride dentifrices have been proven to be effective anticaries agents. There is substantial evidence that fluorides inhibit microbial enzyme systems and reduce the rate of growth of bacteria. India is considered as one of the highly fluoride endemic countries, with the level of fluoride in drinking water varying from 1 to 48 mg/l. Available data suggest that about 62 million people in India are reported to be affected by dental, skeletal, and nonskeletal fluorosis; out of these, six million are children below the age of 14 years. Therefore, the regular use of fluoridated dentifrices cannot be recommended for such population. The rise in the incidence of oral disease (particularly in developing countries), increased resistance by pathogenic bacteria to currently used chemotherapeutics, suggest a global need for phytotherapeutic agents with antimicrobial and anti inflammatory properties.
Among the plants known for medicinal value, Ocimum sanctum L. (tulsi) is one of the important species of genus Ocimum with known medicinal properties. In Indian materia medica, tulsi leaf extracts are described for the treatment of various oral conditions, hematological diseases, and parasitic infections because of its antimicrobial and anti-inflammatory properties. Tulsi was stated as a safe herbal intervention in the recent systematic review in 2017.
To the present date, there is no reported clinical trial evaluating the efficacy of a dentifrice containing tulsi against salivary S. mutans level. Therefore, the purpose of the present study is to assess the antibacterial efficacy of 4% tulsi extract dentifrice compared to a commercially available fluoridated dentifrice as positive control and a placebo dentifrice in reducing salivary S. mutans count among 14–15-year-old schoolchildren in Davangere city. The null hypothesis is there is no difference in the antibacterial efficacy of 4% tulsi extract, commercially available fluoridated, and placebo dentifrices against salivary S. mutans count among 14–15-year-old schoolchildren in Davangere city.
| Subjects and Methods|| |
The trial was designed, analyzed, and interpreted according to the extension of CONSORT for herbal intervention. The present study was a single-center, block-randomized, triple-blinded, placebo-controlled parallel-group study. The study was conducted for a period of 2 months from September to October 2017. The study protocol was approved by the ethical review board (Ref No. BDC/Exam/383/2017–18). Permission to conduct the study was obtained from the Deputy Director of Public Interest and school authorities. The trial was registered under Clinical Trials Registry India and trial no. CTRI/2017/12/010749. List of all the students of 14–15 years' age group from each of the three randomly selected schools was obtained from school records. Voluntary written informed consent and assent was obtained from parents and selected children. Dental caries was examined using the Nyvad's criteria. The participants were examined in the school premises under natural light using sterilized mouth mirror and community periodontal index probe. Only those children who had at least one cavitated active caries lesion (clinical selection criteria) and a salivary S. mutans count equal to or more than 105 colony-forming units (CFUs)/ml of saliva (microbiological criteria) were included in the study. Subjects with history of hypersensitivity to any products used in the study, suffering from any disease that may affect the salivary flow rate, having a history of antibiotic therapy in the previous 1 month till the start of the study, subjects undergoing orthodontic treatments, and those who were unable to comply with the study appointment schedules were excluded from the study.
Dried black tulsi leaves were subjected to cold maceration technique to prepare a 4% ethanolic extract [Figure 1] and [Figure 2]. The 4% concentration of tulsi extract was prepared because it showed a maximum inhibition zone of 22 mm against S. mutans. The three dentifrices, namely 4% tulsi extract, Colgate Total, and placebo dentifrices, constituted the three interventions. The standard ingredients for toothpaste preparation were included in the placebo dentifrice. All the three formulations were filled in identical tubes to ensure masking [Figure 3]. Each school was considered as one block or group. Computer-generated random numbers were used and concealed randomization in the form of precoding was followed. Random sequence generation and allocation of the dentifrices was done by a person not involved in the present study. All the participants and investigators were blinded with respect to the dentifrice groups. Along with the toothpaste, a soft bristle toothbrush was distributed and was instructed to refrain from any other oral hygiene aids for the next 7 days. Modified bass technique was demonstrated and they were instructed to brush for a period of 4–5 min twice daily using a pea-sized toothpaste. Baseline demographic details were obtained from patients by interview. All subjects were given clear instructions to refrain from eating food or beverage (water exempted) for 1 h before collection of saliva. Postsalivary assessment was done on the 8th day. Two milliliters of unstimulated saliva was collected using sterile cups in the morning between 9 and 11 am. The saliva samples were processed immediately and microorganisms were cultured using mitis salivarius-bacitracin selective agar and expressed in CFUs.
All the outcome measures were assessed at baseline and after 7 days of treatment at the follow-up visit. The primary outcome measures were reduction in the number of CFUs of salivary S. mutans count as assessed by microbiological analysis. The secondary outcome measures included the assessment of the patient compliance, acceptance, and any adverse effect through checklist and questionnaire. The sample was calculated using GPower software 18.104.22.168. We assumed a priori that 20% of subjects would be lost to follow-up by 7 days. The α, β and effect size were fixed as 0.05, 0.20 and 0.4 respectively and the calculated sample size was 84.
All the microbiological procedures were performed by an expert from oral pathology and microbiology department, who was blinded with respect to culture plates of different groups throughout the entire course of the study. Intra-examiner variability was assessed by kappa statistics (0.83). The subjects were followed up for a period of 7 days. On the 8th day, only 52 participants out of 84 turned up for the posttest assessment. Twenty children had their vacation and 12 children had their midterm examinations were the reasons for the dropout [Figure 4].
A checklist was provided to each subject to assess compliance. They were instructed to mark a tick (√) on both columns after using the assigned toothpaste twice daily; if they had missed or forgot to brush using the assigned toothpaste, they were asked to mark a cross (×). A provision was provided to write the reasons for not using the assigned toothpaste. Parents of the participants were instructed to supervise their children while brushing, so as to avoid excess use of toothpaste. None of the subjects reported any adverse effects. The participants were asked to return their used dentifrice tubes after 7 days to assess compliance indirectly. Data analysis was done using SPSS software version 20.0. (IBM Corp., Armonk, N.Y., USA). The statistical significance was fixed at P < 0.05. Nonparametric tests were used for analysis as the data were not normally distributed. Comparison of salivary S. mutans counts within groups and between groups was done by Wilcoxon signed-rank test (intragroup comparison) and Kruskal–Wallis analysis of variance (intergroup comparison), respectively, followed by Mann–Whitney U post hoc test. Post hoc analysis was performed to ascertain the significant difference between groups. Missing data were handled using the various commonly reported and naive statistical methods including (1) complete case analysis and (2) single-imputation-based procedures (best case, worst case, average, etc.). Sensitivity analysis using multiple imputation (MI) method was performed.
| Results|| |
A sample of 290 participants were examined and 84 participants satisfied the inclusion criteria. Out of which, 41 were male and 43 were female, and 59 participants belonged to 14 years' age group and 25 belonged to 15 years' age group. No significant difference was present between the three groups at baseline to ensure baseline comparability [Table 1].
|Table 1: Mean rank and comparison of baseline values between groups using Kruskal–Wallis ANOVA|
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Missing data analysis
Complete case analysis, average score, best score, worst score, and MI methods were employed to address the missing data. A complete case analysis by excluding all the dropouts revealed no significant difference between the groups. Average score analysis was done by assigning the average of the observed outcome in each of the groups, which showed a statistically significant difference between the groups. Similar results were observed in best and worst-case analysis by substituting the best and worst scores, respectively. Sensitivity analysis using MI also showed a statistically significant difference between the groups, and maximum reduction in microbial load was observed in tulsi extract group [Table 2]. MI analysis was sensitive enough for finding a significant difference between the groups even though missing data were present. Therefore, as compared to complete case analysis, the average, best, worst, and MI technique identified a significant inhibitory effect of tulsi extract dentifrice against salivary S. mutans counts. Furthermore, post hoc analysis showed a statistically significant (P < 0.05) reduction in tulsi group compared to placebo group when assessed through all missing data analysis [Table 2]. Intragroup comparison showed a significant reduction in S. mutans counts in tulsi group (mean rank 37.95–28.45) compared to placebo group (mean rank 32.91–42.05). The clinical effect size was calculated for the tulsi dentifrice, which showed a moderate effect of 0.34 for antimicrobial action [Table 2]. All the subjects in the present study used the dentifrice as per the instructions given by the investigator. Study subjects informed about the slight bitter taste of tulsi toothpaste and no adverse events were reported. Out of 52 participants, 39 participants brushed twice daily. However, the difference in frequency of brushing was not statistically significant.
| Discussion|| |
The present study is the first of its kind to evaluate the efficacy of 4% tulsi extract when incorporated in toothpaste form. The study showed a significant reduction in the salivary S. mutans count from baseline to posttest assessed after 7 days, i.e. 8th day. The therapeutic properties of tulsi are due to the presence of essential oils such as eugenol, methyl eugenol, and a-and b-caryophyllene., The antimicrobial activity can be attributed to constituents such as Carvacrol, terpene, and sesquiterpene b-caryophyllene present in this plant.
The active control fluoridated toothpaste also showed a significant reduction in the salivary S. mutans count from baseline to posttest. The use of fluorides in caries prevention program has been the cornerstone and is well documented in the literature. Fluoride can accumulate in the interior of bacteria and cause metabolic changes inhibiting glycolysis, which is responsible for the acidogenic potential of S. mutans, thereby inhibiting the adhesion of this microorganism to hydroxyapatite crystals. This feature of fluoride toothpaste can be attributed to the antimicrobial efficiency. The effectiveness of fluoride toothpastes as an antimicrobial agent is concentration dependent. The results of complete case analysis performed by excluding the dropouts for the fluoridated toothpaste were similar to a study done by Petersson et al. The study showed no significant difference in the level of S. mutans between the subjects using fluoridated and nonfluoridated toothpaste. However, the concentration of fluoride used was 0.03% sodium fluoride, as in the present study, the concentration used is 0.2% sodium fluoride. The study by Sudha Patil also showed a significant reduction in the bacterial count from baseline to posttest by using herbal and fluoridated toothpaste when assessed after 14 days.
The present study has the advantage of a placebo group, which is the most rigorous test of evaluating treatment efficacy for a medical therapy. A significant reduction in bacterial count was seen in the placebo group. Children aged 14–15 years were selected, as this group represents a full complement of permanent dentition (except for third molars). Fifteen years is considered as the global indicator age and represents the end of mixed dentition period. At this age, the permanent dentition has been exposed to the oral cavity for 3–9 years, so the assessment of caries was found to be more meaningful. A study done by Honkala et al. revealed the strong effect of duration of toothbrushing on plaque removal. Therefore, the type of toothbrush, dentifrice, frequency, and method of brushing are controlled in the present study. Results of within-group comparison for the mean rank reduction in the salivary S. mutans colony counts from baseline to posttest showed a highly significant difference among all the three groups.
Attempts such as reminder phone calls and inflation of sample size to 20% were made to avoid missing data. Even then 38% dropouts were seen, which is a major limitation of the study. Inadequate handling of these missing data in the analysis can cause substantial bias in the treatment effect estimates. Randomized controlled trials are the cornerstone of evidence-based dentistry. This potential may be compromised when individuals in the trials have missing outcome data. Therefore, various statistical methods were followed to manage the missing data. The results obtained through complete case analysis are not biased, but it estimates the treatment effect among the subpopulation of participants who reported. Therefore, it can lead to loss of precision of reported estimates. The worst-case value approach very rarely leads to unbiased results for the treatment effects and is often too extreme.
MI statistical method has shown to generate less biased estimates with more statistical efficiency when compared with alternative methods of handling incomplete data (e.g., complete case analysis, single imputation, and missing indicator regression). The precision of parameter estimates and accuracy of standard errors make MI one of the best options for handling missing data. Sensitivity analysis using MI method was used to provide some assurance that neither the lost information nor the methods used to handle missing data had an important effect on the overall study conclusions. Results of MI method showed a statistically significant difference between tulsi and fluoridated toothpaste and also between tulsi and placebo toothpaste. The strengths of the study include random selection of the participants, block randomization method, concealed random allocation, and blinding of the investigator, participant, and statistician. These methods reduced selection bias, allocation bias, and confounder bias. Furthermore, the oral hygiene technique was standardized for all the participants. The compliance was assessed directly through the checklist and indirectly by assessing the dentifrice tubes of each participant. Dropouts, a major limitation of this study, occurred unequally among the three groups with dropout rate of 38%. Reasons for the significant dropout included midterm examinations and vacation because of which the study subjects could not adhere to the appointment schedule. The present study is single centered, thus our study findings can be generalized to those schoolchildren in Davangere city. However, future studies are recommended to evaluate the long-term effect of tulsi extract dentifrice and its effect among people of different age groups for wider generalizability.
| Conclusion|| |
Tulsi dentifrice was found to be more efficacious than fluoridated dentifrice in our study. As tulsi is culturally acceptable, affordable, and easily available along the courtyard, it can be used as regular home care preventive aid to combat caries among high caries risk adolescents.
We duly acknowledge Bapuji Pharmacy College for the preparation of tulsi extract dentifrice and placebo dentifrice and principals, teachers, parents, and students of the three schools for their cooperation. I extent my gratitude to Dr. Mahesh (public health dentist) for statistical assistance, Dr. Chandrabhaga (postgraduate – public health dentistry) for concealed randomization, and Dr. Mukund (postgraduate – oral pathology and microbiology) for microbiological assistance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]