Journal of Datta Meghe Institute of Medical Sciences University

: 2020  |  Volume : 15  |  Issue : 1  |  Page : 82--87

Effect of tulsi extract and probiotic mouthrinse on plaque and gingivitis among school children – A randomized controlled trial

Thippeswamy Honne Manjunathappa1, JJ Jojo2, Mruthunjaya Kenganora3, M Nanditha Kumar4,  
1 Department of Public Health Dentistry, JSS Dental College and Hospital, JSS Academy of Higher Education and Research, Mysore, Karnataka, India
2 Department of Prosthodontics, JSS Dental College and Hospital, Mysore, Karnataka, India
3 Department of Prosthodontics, JSS Pharmacy College, JSS Academy of Higher Education and Research, Mysore, Karnataka, India
4 Department of Prosthodontics, JSS Dental College and Hospital, JSS Academy of Higher Education and Research, Mysore, Karnataka, India

Correspondence Address:
Dr. M Nanditha Kumar
Department of Prosthodontics, JSS Dental College and Hospital, JSS Academy of Higher Education and Research, Mysore - 570 015, Karnataka


Background: Probiotics and tulsi extract mouthrinses are a breakthrough approach to preventing plaque and gingivitis through their different defence mechanism. Objective: Comparison of the effectiveness of chlorhexidine (CHX), tulsi extract and probiotic mouthrinses among 12–15-year-old school children of Mysore, India. Materials and Methods: Triple-blinded randomized controlled clinical trial-concurrent parallel design. The study was done in school which was selected by the lottery method. A total of 60 children who fulfilled the eligibility criteria were randomly selected for the study. After baseline examination using computer generated table divided into three groups – Group A: 0.2%CHX mouthrinse, Group B: tulsi extract mouthrinse, and Group C: probiotic mouthrinse. Results: There was a significant reduction in plaque and gingivitis among all three study products (P < 0.05). Compared to baseline data, plaque and gingivitis reduction was more pronounced in probiotic mouthrinse compared to chlorhexidine and tulsi extract mouthrinses. Conclusions: All the study products, i.e., CHX, tulsi extract and probiotic mouthrinses showed a significant reduction in plaque and gingivitis compared to baseline scores.

How to cite this article:
Manjunathappa TH, Jojo J J, Kenganora M, Kumar M N. Effect of tulsi extract and probiotic mouthrinse on plaque and gingivitis among school children – A randomized controlled trial.J Datta Meghe Inst Med Sci Univ 2020;15:82-87

How to cite this URL:
Manjunathappa TH, Jojo J J, Kenganora M, Kumar M N. Effect of tulsi extract and probiotic mouthrinse on plaque and gingivitis among school children – A randomized controlled trial. J Datta Meghe Inst Med Sci Univ [serial online] 2020 [cited 2020 Nov 27 ];15:82-87
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Plaque is known to be the initiating factor in the development of gingivitis.[1] Gingivitis is one of the most chronic infections and is caused by the accumulation of bacteria in the gingival crevices causing an inflammatory reaction. If the inflammation and degradation of collagen increase, gingivitis may develop into periodontitis.[2] Among the evaluated agents for chemical control, chlorhexidine (CHX) is the gold standard, as supported by a large body of evidence, produced since1970. Meta-analysis has indicated that oral care products containing CHX exert antiplaque effects. CHX is a cationic biguanide with a broad spectrum of antibacterial activity, encompassing Gram-positive and Gram-negative bacteria, yeasts, and some viruses.[3]

According to the World Health Organization, probiotic bacteria are defined as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Clinical trials have demonstrated a reduced prevalence of moderate-to-severe gingival inflammation as well as improved plaque index and probing depth in adults after regular use of probiotic chewing gums or tablets.[4]

Tulsi is one of the holiest and most sacred herbs grown widely in India. Natural herbs such as triphala, tulsipatra, jyestiamadh, neem, clove oil, pudina, ajwain, and many more used either as whole single herbs or in combination have been scientifically proven to be safe and effective medicines against various oral health problems such as bleeding gums, halitosis, mouth ulcers, and in preventing tooth decay. The major strength of these natural herbs is that their use has not been reported with any side effects till date.[1] It is a herb that is bestowed with enormous antimicrobial substance and is used to treat a variety of illnesses ranging from diabetes mellitus, arthritis, bronchitis, skin diseases, etc. Recent studies have also demonstrated significant anticancer properties of tulsi.[5] The impetus for the study was the nonavailability of literature on the effect of tulsi on plaque and gingivitis reduction. Null hypothesis of the present study was that no difference in reduction of plaque and gingivitis among 12–15 year old school children using CHX, probiotic, and tulsi extract mouthrinses. Hence, the aim of the present study was that comparison of the effectiveness of CHX, tulsi extract and probiotic mouthrinses against plaque and gingivitis among 12–15-year-old school children of Mysore city, India.

 Materials and Methods

The triple-blind concurrent parallel study design was done in classrooms. The selection of the study participants using simple random sampling technique.

Sample size and selection of study participants

The study was done in a school which was selected by lottery method. The list of schools was obtained from Deputy Director of Public Instructions, Mysore, and one school was selected by lottery method. A total of 60 children who fulfilled the eligibility criteria were randomly selected for the study. The study participants were continuing their regular oral hygiene practices during the study. Ethical approval was obtained from institutional ethical committee. Before the beginning of the study, consent was obtained from the parents of the study participants and also from the study participants explaining the pros and cons of the study. Official permission was obtained from the head of the institution to conduct the study in a separate classroom. The sample size of 20 was selected in each group based on 95% confidence interval and 90% power and assuming the mean difference of 1.5 and Standard deviation of 1.35 (nMaster sample size calculation software).

Inclusion criteria

School children of 12–15 years of age as per school recordsSchool children willing to participateSchool children with parental consentSchool children with no antibiotic therapy.

Exclusion criteria

Children undergoing orthodontic treatmentChildren suffering from any systemic illnessChildren using any other commercially available probiotic productsChildren using any other oral hygiene aids other than routine tooth brushing.

Experimental design, allocation concealment

The medical and dental details of all participants were recorded using a questionnaire. In this triple-blinded randomized controlled trial, participants were enrolled and assigned to a computer generated table by the examiner who assigned the coded mouthrinses according to treatment groups after baseline examination into:

Group A – 0.2% CHX mouthrinseGroup B – Tulsi (Ocimum sanctum) extract mouthrinseGroup C – Probiotic mouthrinse.

The participants and the examiner were blinded to product allocation. Knowledge of the randomization list obtained by the computer generated table was limited to the study coordinator.

Preparation of the mouthrinses

Preparation of chlorhexidine mouthrinse

CHXgluconate mouthwash (Proprietary name: Clohex, concentration 0.2%) was procured from the market and given to the pharmacy manufacturing center. It was then diluted, and the final concentration of CHXgluconate was 0.1% such that 10 ml was dispensed at one time. Both solutions were made of identical colors to eliminate bias.

Preparation of tulsi extract mouthrinse

Collection and authentication of the plant material: Fresh bundles of leaves and aerial parts of O. sanctum were obtained from the market and identified and authenticated by an experienced botanist. Leaves and aerial parts which had turned yellow and old or black were removed and washed thoroughly with distilled water.

Preparation of tulsi extract

The extract of tulsi was prepared by hot maceration method using water. Accurately weighed quantity of tulsi was cut into small pieces, put into a round-bottomed flask and boiled over a water bath using a water condenser for 2 h. After extraction, the extract was filtered and concentrated in a flash rotary evaporator to a thick syrupy liquid, transferred to a 6-inch petriplate and dried over a water bath. The dried extract was placed in a vacuum desiccator until use.

Preparation of mouthwash

Accurately weighed tulsi extract was taken in a clean and sterile mortar. Accurately weighed sterile tween 80 (polyoxyethylene sorbitan monooleate) was taken in a sterile 25 ml beaker. Tulsi extracts and triturated continuously with the gradual addition of tween 80 to get homogeneous mass of extract. Menthol was dissolved in alcohol and added and triturated in the mortar. Accurately weighed sorbitol was dissolved in sterile water and was added to the mortar in increments of 10 ml and triturated to get a homogeneous mixture. The homogeneous mixture was transferred into a sterile glass bottle and volume adjusted with sterile water to make it up to the required volume.[6],[7]

The formula of the mouthwash is given below:

Tulsi extract 1 gTween 80 3 gMenthol 0.2 gSorbitol 3 gWater To make upto 100 ml.

Preparation of probiotic mouthrinse

The probiotic mouthrinse was prepared using a commercially available probiotic product, Sporlac (Uni Sankyo Ltd., India) containing 1 g powder of 150 million spores of bacillus coagulans. Powder from each sachet was dissolved in 20 ml of water in a measuring cup and used as a mouthrinse.[8]

The pharmacy college department prepared the mouthrinses in indistinguishable packets and sent them to the study coordinator, who marked the code number of each participant on the packets, according to the therapy assigned and gave them to the examiner. The random allocation sequence was generated by the clinical investigator. To maintain full blinding of the results, the randomization table was held by the study coordinator remotely from all the assessment and was not broken until the data were collected and analyzed. The randomization was concealed using sequentially numbered, identical appearing containers to participant assigned treatment. The mouthrinses were decoded after the data were analyzed.

Blinding and Investigators calibration

The triple-blinding technique was used. The investigator, study participants and the person who did the statistical analysis was unaware about the allocation details of the study participants.

Participated in a calibration exercise that was performed by taking measurements in duplicate of randomly chosen teeth in participants, who were not included in the study. Calibration was accepted and kappa value was found to be 0.92.

Clinical examination

Before the beginning of the trial, baseline data of Plaque index (Turesky modification of Quigley and Hein Plaque index[9] and Gingival index[10] were recorded by clinical examination. Gingivitis of the buccal and lingual marginal gingiva and interdental papillae of all scorable teeth was scored using the Loe-Silness Gingival Index in which the gingivae are scored on a four-point scale from 0 (the absence of inflammation) to 3 (severe inflammation). Supragingival plaque was scored on the buccal and lingual surfaces of all scorable teeth using the Turesky modification of the Quigley–Hein Plaque Index (Turesky et al. 1970). Following disclosing with an erythrosine solution, plaque was scored on a six-point scale from 0 (no plaque) to 5 (plaque covers two-thirds or more of the tooth surface).

Distribution of the mouthrinses

Each participant was given one of the test products with a given code according to the assigned group. Ten milliliter of mouthrinse was dispensed for each individual using a measuring cup and participants were instructed to swish the mouthrinse for 60 s and then expectorate. The procedure was performed once a daily morning, after breakfast and was supervised by the examiner by visiting the school every day in the morning. The participants assigned to each treatment group were given the mouthrinses in a packet and instructed to repeat the procedure before retiring to bed at night for the next 21 days. During the intervention period, no influence on personal oral hygiene procedures was exerted and the participants were encouraged to maintain routine oral hygiene and also instructed to maintain strict compliance. A day after the 21 days of intervention, gingival and plaque indices were recorded using the same indices by the same examiner. After the trial ended, group allocation details and results were revealed to the study participants.

Statistical analysis

Mean, standard deviation was done for descriptive statistics. A paired t-test was used to compare the mean difference of plaque and gingival indices scores between the baseline and after the intervention within each group. Kruskal–Wallis test was used to compare the mean difference scores in both plaque and gingiva. The normal distribution of the data was assessed using Kolmogorov–Smirnov test and Shapiro–Wilk test. The significance level was fixed at 5%. The data were analyzed using SPSS version 17 (Chicago, Illinois, USA).


Comparison of baseline data of plaque and gingiva

[Table 1] and [Table 2] show comparison of baseline data of plaque and gingival index. In both plaque and gingiva, the baseline data of all the mouthrinses were almost similar with minor differences. This was found statistically not significant [Figure 1].{Table 1}{Table 2}{Figure 1}

Effect on plaque

There was a significant reduction in plaque among all three study products (P < 0.05). The mean difference for probiotic mouthrinses was more compared to tulsi and CHX mouthrinses (mean difference scores 0.71, 0.66, 0.60). Compared to baseline data, the degree of plaque reduction was more pronounced in probiotic mouthrinses compared to CHX and tulsi extract mouthrinses. No significant differences were found after comparing mean difference scores of all the three study products [Table 3] and [Table 4].{Table 3}{Table 4}

Effect on gingiva

[Table 5] and [Table 6] show that all the study products were effective in reduction of inflammation of the gingiva. Comparing pre and post values all the products have shown significant reduction (P < 0.05), but probiotic mouthrinse showed greater mean difference followed by CHX and tulsi mouthrinses (0.53, 0.36, 0.28). No significant differences were found after comparing mean difference scores of all the three study products.{Table 5}{Table 6}


This study was undertaken to investigate the reduction in plaque and gingivitis in the oral cavity after short-term exposure to CHX, probiotic, and tulsi extract mouthrinses. It is well known that the effect of professional cleaning of teeth and gingiva is effective in the short-term treatment of gingivitis. In our experience, this effect usually lasts 1–3 weeks before plaque and gingivitis start to re-appear.

In the present study, a significant reduction of plaque and gingivitis took place in all the study participants. Prevention of periodontal disease is based on supragingival biofilm control, with mechanical plaque control as first-line option. CHX gluconate is today a thoroughly studied and the most effective antiplaque and antigingivitis agent. In oral use as a mouthrinse, CHX has been reported to have a number of local side effects. Staining was the most common observation. Other less frequent side effects were increased calculus formation, change of taste sensation, and hypersensitivity rarely mucosal lesions.[11]

In the present study, tulsi extract mouthrinse also showed a significant reduction of plaque and gingivitis. Tulsi was recognized thousands of years ago by ancient rishis to be one of the India's greatest healing herbs. They saw this herb is good for health and healing. Haffajee et al.,[12] compared the effectiveness of a herbal mouthrinse with 0.12% CHX gluconate and an essential oil mouthrinse. It was found that the herbal mouthrinse was inhibiting oral bacteria, predominantly Actinomyces sp, Eubacterium nodatum, Prevotella intermedia, Prevotella. melaninogenica, Prevotella nigrescens, and Tannerella forsythia. Malhotra et al.[1] proved that a herbal mouthrinse is less effective than chlorhexidine gluconate. However, in the present study, the inhibitory effect of probiotic rinse on the plaque is more than CHX. This may be due to the difference in age groups and combination of different herbal ingredients in the mouthrinse.

In the present study, the effectiveness of the probiotic mouthrinse is more than CHX and tulsi extract mouthrinse on both plaque and gingivitis. This was similar to a study by Harini and Anegundi,[11] the difference being that tulsi extract mouthrinse was not used in that study. Antibacterial mouthrinses act by nonspecifically reducing the levels of both friendly and harmful oral bacteria. In contrast, probiotic mouthrinse has been developed utilizing natural beneficial bacteria to promote a healthy balance of microorganisms in the mouth.

The present study had a parallel-group study design, where three different groups of participants received three different mouthrinses under similar conditions. In the crossover study designs, each participant receives each of the treatments in a randomized order. The drawback of the within-subjects or crossover study designs is contamination or carryover. It is entirely possible that some effect of the treatment used in one period might persist and alter the response observed in later treatment periods. Hence, a parallel design was chosen in this study.

The participants selected in this study were 12–15 years of age, which was important for the present study as this is the period when permanent teeth are erupting and new surfaces would be colonized by pathogenic bacteria. Any measure (s) directed toward prevention against early colonization might be beneficial in the long term for the prevention of dental disease. Another reason for selecting this age group was the intellectual ability of the child. In accordance with Jean Piaget, at the age of 7 years, a child largely corresponds to an increase in cognitive development whereby the child develops a sense of semi-logical reasoning to infer physical cause-effect relationships. Thus, in this age group, a positive compliance could be expected from a child.[8]

Further, more research is necessary to gain greater insight into the level of plaque inhibition achieved with this herbal mouthrinse. Studies of longer duration, in which the product in question is compared to other controls (positive or negative) or placebo products and where safety and microbiological parameters will be assessed are necessary to establish the effectiveness of this product and its place among the other agents used for chemical support of daily mechanical plaque removal.


All the study products i.e., CHX, tulsi extract, and probiotic mouthrinses showed a significant reduction in plaque and gingivitis compared to baseline scoresProbiotic mouthrinse was more effective in reducing plaque compared to tulsi extract and CHX mouthrinsesProbiotic mouthrinse is more effective in reducing gingivitis, followed by CHX and tulsi mouthrinses.

Financial support and sponsorship

This study has been funded by the Indian Council of Medical Research, Student Termship Project (STS).

Conflicts of interest

There are no conflicts of interest.


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