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ORIGINAL ARTICLE
Year : 2017  |  Volume : 15  |  Issue : 4  |  Page : 306-311

Effect of tulsi extract and honey mouthrinses on salivary Streptococcus mutans count in comparison with 0.2% of chlorhexidine: A randomized controlled trial


Department of Public Health Dentistry, Narayana Dental College and Hospital, Nellore, Andhra Pradesh, India

Date of Web Publication13-Dec-2017

Correspondence Address:
Dr. Shaik Ijaz Ahmed
H. No. 24 1 1952, Dargamitta, Near Police Ground, Nellore, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_55_17

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  Abstract 

Introduction: Mouthrinses have been popularly used as a supplementary oral hygiene aid. A lot of commercially available mouthrinses possess few adverse effects, which has necessitated the search for alternative and herbal mouthrinses. Aim: This study aims to evaluate the effect of tulsi extract and honey mouthrinses on Streptococcus mutans count in comparison with 0.2% of chlorhexidine. Materials and Methods: A randomized controlled trial was conducted in various schools of Nellore District, Andhra Pradesh. A total of forty-five individuals aged 15 years who satisfied the inclusion criteria were randomly allocated to three group, i.e., 15 each for Group A (0.2% of chlorhexidine mouthrinse), Group B (honey mouthrinse), and Group C (tulsi extract mouthrinse) by lottery methods. Oral hygiene status was assessed and evaluation of S. mutans was done before and after giving the mouthrinse. The sample was streaked on mitis salivarious-bacitracin agar medium. Results: There was a reduction in S. mutans in all the three groups with 0.2% of chlorhexidine showing a maximum reduction. There was significant difference between Group A (0.2% chlorhexidine mouthrinse) and Group B (honey mouthrinse) and between Group A (0.2% chlorhexidine mouthrinse) and Group C (tulsi extract mouthrinse) (P < 0.001). There was no significant difference between Group B (honey mouthrinse) and Group C (tulsi extract mouthrinse) (P = 1.00) in S. mutans count. Conclusion: Herbal mouthrinses containing tulsi and honey, though as not as effective as chlorhexidine in its antimicrobial property, have its own value and it can be effectively used in areas where people cannot access to chlorhexidine.

Keywords: Chlorhexidine, honey, Streptococcus mutans, tulsi (Ocimum sanctum)


How to cite this article:
Ahmed SI, Sekhara Reddy V C, Sudhir KM, Kumar RK, Srinivasulu G. Effect of tulsi extract and honey mouthrinses on salivary Streptococcus mutans count in comparison with 0.2% of chlorhexidine: A randomized controlled trial. J Indian Assoc Public Health Dent 2017;15:306-11

How to cite this URL:
Ahmed SI, Sekhara Reddy V C, Sudhir KM, Kumar RK, Srinivasulu G. Effect of tulsi extract and honey mouthrinses on salivary Streptococcus mutans count in comparison with 0.2% of chlorhexidine: A randomized controlled trial. J Indian Assoc Public Health Dent [serial online] 2017 [cited 2020 Dec 5];15:306-11. Available from: https://www.jiaphd.org/text.asp?2017/15/4/306/220716


  Introduction Top


Dental caries is a major oral health problem in most industrialized countries, affecting 60%–90% of school children and the vast majority of adults. In the recent years, the prevalence of dental caries in most western countries has steadily declined; the most common reason for the decline of dental caries is attributed to the widespread use of fluorides. However, it has shown increasing trend in developing countries including India.[1]

Research in the field of caries prevention has been focusing on ways for reducing totally eradicating cariogenic flora from the oral cavity. Studies have shown that caries can be prevented by regular tooth brushing and flossing. However, most of the studies have shown that it is difficult to eliminate Streptococcus mutans from pits, fissures, and approximal surfaces by mechanical means alone. For effective caries control, these methods should be combined with the chemoprophylactic agents. These agents are generally delivered as mouth rinses, toothpastes, gels, and varnishes.[1]

The bisbiguanide chlorhexidine has been studied extensively for over 20 years and is currently the most potent chemotherapeutic agent against S. mutans. It has bactericidal activity against both Gram-positive and Gram-negative bacteria. Its effect against mutans streptococci is greater than against Streptococcus sanguis and lactobacilli. Studies have demonstrated that chlorhexidine has an antimicrobial activity against S. mutans.[2]

Tulsi is an aromatic shrub in the basil family Lamiaceae (Tribe ocimeae). Tulsi is one of the best examples of Ayurveda's holistic lifestyle approach to health. Tulsi tastes hot and bitter and is said to penetrate the deep tissue, dry tissue secretions. Its antimicrobial activity has been tested against plaque and salivary S. mutans, and it is found to be effective.[2]

Honey has been used as a source of nutrients as well as a medicine since ancient times. Recent publications indicating the effect of honey in the management of certain conditions have rekindled interest in honey as a natural therapeutic agent. The antibacterial properties of honey have been well documented. However, the specific antimicrobial mechanism of honey is still unclear. Among the possible mechanisms is the presence of inhibitory factors such as flavonoids and hydrogen peroxide, low pH, and high osmolarity. Honey may have a similar antibacterial effect on S. mutans, which is considered the main causative organism of dental caries.[3]

Mouth rinses have been popularly used as a supplementary oral hygiene aid. A lot of commercially available mouthrinses possess few adverse effects, which has necessitated the search for alternative and herbal mouth rinses. Only few in vivo studies have been documented on the antimicrobial properties of honey and tulsi extracts. Hence, this clinical study was conducted to confirm the antibacterial potency of honey and tulsi mouthrinses compared to 0.2% of chlorhexidine.

Aim

To evaluate the effect of tulsi extract and honey mouthrinses on S. mutans count in comparison with 0.2% of chlorhexidine.

Objectives

  • To assess the antimicrobial activity of 0.2% of chlorhexidine, honey mouthrinse, and tulsi extract mouth rinse against S. mutans
  • To assess the oral hygiene status (OHI-S) of high school children before and after taking the mouthrinses
  • To compare the antimicrobial activity of 0.2% of chlorhexidine, honey, and tulsi extract mouthrinses on S. mutans.



  Materials and Methods Top


A randomized controlled trial was conducted among children of various schools in Nellore District, Andhra Pradesh in September 2016 for a duration of 2 weeks. The ethical clearance for the study was obtained from the Institutional Ethical Committee.

Students of various schools of Nellore District who were willing to participate in the study, who gave a written consent, and who satisfied the inclusion criteria were included in the study. Individuals having systemic diseases, history of antibiotic therapy, and who were already using any mouth rinses were excluded from the study.

The sample size was calculated based on the difference obtained in the previous studies, with a standard deviation of 2.563, allowable mean error of <5% at 95% confidence interval using the following formula.[2]



N = Sample Size; At 95% confidence, Z value is 1.96; S = standard deviation = 2.563; D = least detectable difference = 0.1; n = 12.

The sample size was estimated to be 12. In the current study, the sample size was rounded up to 15 individuals in each group. As three groups were involved in the study, 15 individuals were allotted in each group, i.e., a total of 45 individuals participated in the study.[2]

Multistage sampling technique was followed. Nellore city was divided into four zones (North east, North West, South East, and South West). Three schools were selected randomly by employing lottery method. From the four zones, a total of forty-five individuals (proportionate number) who satisfied the inclusion criteria were randomly allocated to three groups, i.e., 15 each for GroupA (0.2% of chlorhexidine mouth rinse), Group B (honey mouth rinse), and Group C (Tulsi extract mouth rinse) by lottery method [Figure 1].
Figure 1: Randomization of study individuals

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Preparation of tulsi extract

Tulsi leaves were obtained from courtyards and were dried in sunlight. The dried leaves were then powdered finely. Two hundred grams of finely powdered tulsi was then macerated with 100% ethanol. It was then subjected to filtration with Whatman filter paper to obtain a clear filtrate. The filtrate so obtained was reduced at a low temperature of <60°C to obtain a solid residue of tulsi extract. From 200 g of tulsi dissolved in 1 L of ethanol, 12 g of residue (extract) was obtained, and thus, the yield was 4% w/w.[2]

Preparation of honey mouthrinse

Ten grams of commercial honey was added to 100 ml of boiling distilled water and further boiled for 30 min. The solution obtained was reduced to 10 ml to obtain 100% (w/v) concentration. The solution of honey was cooled to the room temperature and stored in sterile individual containers before use.[4]

Saliva collection and microbial analysis

All the individuals were given instructions to follow same oral hygiene practices for 2 weeks. Clinical examinations were carried out for OHI-S. Simplified oral hygiene index was recorded before the collection of saliva and after using the mouth rinse. Individuals undergoing investigations were given clear instructions to refrain from eating for 1 h before collection of saliva. One ml of unstimulated saliva was collected by asking the children to let saliva collect in the floor of the mouth without swallowing it for at least 1 min and then to expectorate into the sterile container.

Saliva collection was done at baseline and after 2 weeks between 10 and 10.30 AM to match the circadian rhythm. The sterile container was then immediately transported to the microbiology laboratory, where it was processed immediately. Individuals were instructed to use 10 ml of mouth rinse twice daily for 2 weeks. After 2 weeks, the saliva was collected in the sterile container and then it was immediately transported to the microbiology laboratory, where it was also processed immediately.

The saliva sample was homogenized manually by stirring using a stirrer. Hundred microliters of saliva was diluted with 1 ml of normal saline (1:10 dilution). Using an inoculation loop (2 mm inner diameter), 5 of the 1:10 dilution sample was streaked on mitis salivarius-bacitracin (MSB) agar, a selective medium for S. mutans. The MSB agar plates were incubated for 48 h at 37°C.[2] The microbial count was done by the microbiologist and the values of S. mutans counts were noted down. The counting was performed using a digital colony counter and the counts of S. mutans were expressed as number of colony forming units/ml.

In an attempt to avoid bias, the study was blinded. Once the study was completed, the pharmacist of pharmaceutical college who did the blinding and supplied the mouthrinses in coded assignments did the decoding to reveal the contents of the mouthrinses as follows: Group A (0.2% of chlorhexidine), Group B (honey mouthrinse), Group C (tulsi extract mouthrinse).

Two examiners were selected to ensure blind evaluation of the study participants; the 1st examiner selected the schools, obtained permission for them, and did primary screening (examination for inclusion and exclusion criteria, which included clinical examination). Examiner 2 took the salivary samples before rinsing and immediately after rinsing on the 1st day and after 2 weeks. Examiner 2 was also blinded to the type of mouth rinse administered at different time intervals during the 2 weeks. The statistician remained blinded regarding the individual allocation to the three groups.

Both the examiners were trained before the initiation of the study under the guidance of a senior professional to limit the intra- and inter-examiner variability. They were reassessed for satisfactory agreement at various time intervals during the clinical examination. The Cronbach's alpha coefficient value for inter- and intra-examiner variability was 0.86 and 0.89. Since it is a randomized control trial, the study was conducted in adherence to the requirements for human trial under Declaration of Helsinki.

A 10 ml of the study solutions was dispensed in sterile disposable cups for each group and were given to the individuals as per their respective groups at baseline and after 2 weeks. Every participant was provided with their respective mouthrinse on home use on a weekly basis. Sodium benzoate is the sodium salt of benzoic acid. It has been used as a preservative in foods and other products, and its safety has been established. Artificial sweetening agent was added to make the taste pleasant in tulsi extract mouthrinse. Sodium benzoate is used at a concentration of 0.03%–0.1% in tulsi extract and honey mouthrises. Methylparaben is a methyl ester of p-hydroxybenzoic acid. It is a stable, nonvolatile compound that has been used as an antimicrobial preservative in foods. This methylparaben was also used in tulsi extract and honey mouthrinses.

A pilot study was conducted to determine the acceptability of the mouthrinses. With respect to adverse effects during the course of study, most individuals reported an alteration of taste sensation with 0.2% of chlorhexidine. There were no dropouts in the study. Mouthrinsing was supervised by the examiner in the morning by visiting the schools every alternate day. Parents were instructed to supervise the rinsing at night times.

Data were entered and analyzed using SPSS version 22 (SPSS Inc., Chicago, IL, USA). For comparison of S. mutans count at baseline and after 2 weeks, Chi-square test was used. For Comparison of OHI-S between each study groups, Kruskal–Wallis test and Mann–Whitney U-test were used.


  Results Top


[Table 1] shows that in Group A (0.2% chlorhexidine mouthrinse), Group B (honey mouthrinse), and Group C (tulsi extract mouthrinse), 53.3% of the individuals were males whereas 46.7% were females. No statistical significant difference in the gender distribution was observed among the study groups (P< 0.001).
Table 1: Distribution of the individuals according to gender in each study

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[Table 2] shows that there was no significant difference observed in the S. mutans count in the three study groups at baseline. After 2 weeks, distribution of the S. mutans count in between groups was significantly differing (P< 0.001). In Group A (0.2% chlorhexidine mouthrinse), all the individuals had a S. mutans count of <103, whereas in Group C (tulsi extract mouthrinse), 46.7% of the individuals and in Group B (honey mouthrinse), 26.7% of the individuals had S. mutans count of <103.
Table 2: Comparison of SM count between the study groups at baseline and after 2 weeks

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[Table 3] shows that at baseline, there was no significant difference in the OHI-S scores between the three study groups (P = 0.97). After 2 weeks, when intergroup comparison was made for OHI-S score using Kruskal–Wallis test, there was a significant difference in between three study groups (P = 0.02), with a highest mean reduction in Group A (0.2% chlorhexidine mouthrinse).
Table 3: Comparison of simplified oral hygiene index between each study groups

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When one to one comparison was made using Mann–Whitney U-test, after 2 weeks, there was significant difference between Group A (0.2% chlorhexidine mouthrinse) and Group B (honey mouthrinse), and no significant difference was observed between Group A (0.2% chlorhexidine mouthrinse) and Group C (tulsi extract mouthrinse) and between Group B (honey mouthrinse) and Group C (tulsi extract mouthrinse).


  Discussion Top


A randomized controlled double-blind study was designed to determine the effectiveness of tulsi, honey, and 0.2% of chlorhexidine as a mouthrinse among 15-year-old school children. The study was a randomized controlled trial conducted over a period of 2 weeks. The study was undertaken in three schools which were selected randomly from the Nellore City by lottery method.

The children's age was decided based on their mastery of their swallowing reflex that is convenience in easily rinsing the mouthwash without swallowing to avoid any side effects of mouthrinses.[5] The 15 years age group is considered as one of the index age groups. In this age, all permanent teeth will be erupted and they are exposed to oral environment for a sufficient period of time. Hence, children of 15 years of age were involved in the study procedure (WHO 1997).[5]

In the present study, we have made an attempt to assess the antimicrobial effect of 4% tulsi extract against S. mutans. Agarwal et al. found that 4% tulsi extract showed maximum antibacterial activity against S. mutans.[6] Subramanian et al. showed that among the various extracts of tulsi, ethanolic extract of tulsi (Ocimum sanctum) had higher antimicrobial activity when compared with other extract.[7] Eugenol (1-hydroxy-2 methoxy-4allylbenzene), the active constituent present in Tulsi (O. sanctum), has been found to be largely responsible for the therapeutic potentials of tulsi.[2]

Honey has antimicrobial activity against anaerobic, aerobic, Gram-negative and Gram-positive bacteria, molds and yeasts with unique properties because of its bacteriostatic and bactericidal effect. In addition, among the possible mechanisms of honey is the presence of inhibitory factors such as flavonoids, hydrogen peroxide, and low pH as well as high osmolality due to its sugar concentration. It was also demonstrated that hydrogen peroxide in the honey exerted bacteriostatic and DNA degrading activities to bacterial cells. However, the specific antimicrobial mechanism of honey is still unclear and need more research.[3]

Chlorhexidine gluconate 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.[8]

Alteration in taste sensation with the use of chlorhexidine is a common side effect of chlorhexidine and has been reported in many studies.[9],[10],[11],[12] The probable reasons for the alteration in taste sensation are that chlorhexidine binds to a specific sodium receptor molecule in the taste bud which is uniquely different from normal receptors.[13] Hence, it may lead to alteration in taste sensation.

There is limited literature available on mouthrinse containing tulsi and honey and there is no single study where all the three ingredients have been investigated as a mouthrinse. There was a reduction in S. mutans counts in all the three groups. However, 0.2% chlorhexidine has shown maximum reduction followed by tulsi extract and honey mouthrinses. Similar studies conducted by Jain et al., Briner et al., Jayaprakash et al.,[9],[11],[13] have shown the superiority of chlorhexidine when compared with other ingredients in reducing S. mutans count. Contradictory to the finding Jain et al.[14] that significantly reduced S. mutans in comparison with 0.2% of chlorhexidine.[2] The probable reason is due to the presence of Eugenol (1-hydroxy-2 methoxy-4allylbenzene), the active constituent present in Tulsi (O. sanctum), has been found to be largely responsible for the therapeutic potentials of tulsi.

There was a slight reduction in S. mutans count in honey mouthrinse group in the present study. These findings were in agreement with the findings of another study.[3] Similar studies conducted by Rupesh et al. and Atwa et al.[15],[16] found that the bacterial count significantly reduced in the honey group when compared with treatment group.[15],[16] The probable reason is due to the presence of inhibitory factors such as flavonoids, hydrogen peroxide, and low pH as well as high osmolality due to its sugar concentration.[3]

In the study, the chlorhexidine group showed a greater reduction of S. mutans count in saliva than tulsi extract mouthrinse. However, tulsi extract mouthrinse has some advantages over chlorhexidine as it caused no staining, no altered taste, or any other allergy reported by the individuals. Moreover, it can be considered as a good preventive home therapy in rural areas as it has added health benefits. Thus, herbal mouthrinse (tulsi extract mouthrinse) can be effectively used as an alternative chlorhexidine and can be prescribed for longer duration without any side effects for the management of oral problems.

Since the duration of study was short for the assessment of OHI-S, this study cannot comprehensively analyze the long-term benefits of mouthrinsing pertaining to OHI-S.

The duration of study was short for assessment of dental caries; hence, this study cannot comprehensively analyze the long-term benefits of mouthrinsing pertaining to dental caries. This study has not taken into consideration the financial implications on the individuals in case it has to be implemented at the community level on a daily basis. The study can be further carried out by more specific microbiological test to establish the more accurate efficacy of tulsi extract and honey as a mouthrinses. There is a need to study further on long-term effects of these products on oral health. The role of these mouthrinses needs to be assessed with emphasis on individuals with poor oral hygiene. As literature is totally devoid of any ill effects of tulsi on human beings, one can confidently recommend the use of tulsi as a mouthrinse, especially for rural population which has an easy access to tulsi and perhaps highly cost-effective.


  Conclusion Top


There was a reduction in S. mutans in all the three groups with 0.2% of chlorhexidine showing a maximum reduction. Herbal mouthrinses containing tulsi and honey, though as not as effective as chlorhexidine in its antimicrobial property, have its own value, and it can be effectively used in areas where people cannot access to chlorhexidine.

Acknowledgement

The author would like to thank his professors and colleagues for guiding him and for the support.

.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Bagramian RA, Garcia-Godoy F, Volpe AR. The global increase in dental caries. A pending public health crisis. Am J Dent 2009;22:3-8.  Back to cited text no. 1
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2.
Agarwal P, Nagesh L. Comparative evaluation of efficacy of 0.2% chlorhexidine, listerine and Tulsi extract mouth rinses on salivary Streptococcus mutans count of high school children – RCT. Contemp Clin Trials 2011;32:802-8.  Back to cited text no. 2
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Abdelmegid F, Al-Agamy M, Alwohaibi A, Ka'abi H, Salama F. Effect of honey and green tea solutions on Streptococcus mutans. J Clin Pediatr Dent 2015;39:435-41.  Back to cited text no. 3
    
4.
Ahmadi-Motamayel F, Hendi SS, Alikhani MY, Khamverdi Z. Antibacterial activity of honey on cariogenic bacteria. J Dent (Tehran) 2013;10:10-5.  Back to cited text no. 4
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WHO. Oral Health Surveys – Basic Methods. 4th ed. Delhi: A.I.T.B.S. Publishers and Distributors; 1997.  Back to cited text no. 5
    
6.
Agarwal P, Nagesh L, Murlikrishnan. Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study. Indian J Dent Res 2010;21:357-9.  Back to cited text no. 6
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Subramanian G, Tewari B, Gomathinayagam R. Studies of antimicrobial properties of different leaf extract of Tulsi (Ocimum tenuioflorum) against human pathogens. Am Int J Contemp Res 2014;4:149-57.  Back to cited text no. 7
    
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Löe H, Schiott CR. The effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. J Periodontal Res 1970;5:79-83.  Back to cited text no. 8
    
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Briner WW, Grossman E, Buckner R, Ebert ML. Effect of chlorhexidine gluconate mouthrinse on plaque bacteria. J Periodont Res 1986;21:44-52.  Back to cited text no. 9
    
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Axelsson P, Lindhe J. Efficacy of mouthrinses in inhibiting dental plaque and gingivitis in man. J Clin Periodontol 1987;14:205 12.  Back to cited text no. 10
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Jayaprakash R, Sharma A, Moses J. Comparative evaluation of the efficacy of different concentrations of chlorhexidine mouth rinses in reducing the mutans streptococci in saliva: An in vivo study. J Indian Soc Pedod Prev Dent 2010;28:162-6.  Back to cited text no. 11
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Yevenes I, Alvarez SR, Jara MN, Wolfenson PM, Smith L. Comparison of mouthrinses containing chlorhexidine and other active agents with chlorhexidine mouthrinse-gel: Effects on de novo plaque formation. Rev Odonto Cienc 2009;24:345-8.  Back to cited text no. 12
    
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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.  Back to cited text no. 13
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Jain I, Jain P, Bisht D, Sharma A, Srivastava B, Gupta N, et al. Use of traditional Indian plants in the inhibition of caries-causing bacteria – Streptococcus mutans. Braz Dent J 2015;26:110-5.  Back to cited text no. 14
    
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Rupesh S, Winnier JJ, Nayak UA, Rao AP, Reddy NV, Peter J, et al. Evaluation of the effects of manuka honey on salivary levels of mutans streptococci in children: A pilot study. J Indian Soc Pedod Prev Dent 2014;32:212-9.  Back to cited text no. 15
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Atwa AD, AbuShahba RY, Mostafa M, Hashem MI. Effect of honey in preventing gingivitis and dental caries in patients undergoing orthodontic treatment. Saudi Dent J 2014;26:108-14.  Back to cited text no. 16
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