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ORIGINAL ARTICLE
Year : 2021  |  Volume : 39  |  Issue : 3  |  Page : 291-298
 

Antibacterial efficacy of Carica papaya leaf extract, probiotics, kidodent, and placebo mouthwashes in reduction of salivary Streptococcus mutans: A double-blinded parallel designed randomized controlled trial


1 Department of Pedodontics and Preventive Dentistry, AME's Dental College and Hospital, Raichur, Karnataka, India
2 Department of Pharmaceutics, V. L. College of Pharmacy, Raichur, Karnataka, India

Date of Submission24-Dec-2020
Date of Decision05-Aug-2021
Date of Acceptance27-Sep-2021
Date of Web Publication22-Nov-2021

Correspondence Address:
Dr. Raghavendra Havale
Department of Pedodontics and Preventive Dentistry, AME's Dental College and Hospital, Raichur - 584 103, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jisppd.jisppd_536_20

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   Abstract 


Aim: To compare the antibacterial efficacy of Kidodent, Probiotics, and Carica papaya Leaf extract mouthwashes in reducing Streptococcus mutans count in 8–12 years' old school children. Methodology: Sixty children of age group of 8–12 years were nominated and grouped as Group A (Kidodent mouthwash), Group B (probiotics mouthwash) Group C (C. papaya leaf extract mouthwash), and Group D (distilled water placebo). Probiotics sachets (Prebact) of about 1 g were diluted in 10 ml of water and given as mouthwash. C. papaya leaf extract was obtained by Soxhlet extraction using ethanol as a solvent. Participants were asked to rinse with mouthwashes for 30 s once daily for up to 15 days. Saliva samples were collected and inoculated using Salivarius Mitis and Agar Agar Type I at 38°C for 24 h and incubated, later colony-forming units per milliliter were determined by serial dilution and calculated using colony counter manually. Statistical Analysis: Data were statistically analyzed using the one-way ANOVA and t-test using the SPSS V.20 software. Results: Probiotics and C. papaya leaf extract mouthwashes were equally effective as Kidodent in reducing S. mutans count in saliva. Conclusion: Probiotics and C. papaya leaf extract mouthwashes manifested potential efficacy in reduction of S. mutans.


Keywords:  Carica papaya leaf, distilled water, Kidodent, mouthwash, probiotics, Streptococcus mutans


How to cite this article:
Rao DG, Havale R, Sara SS, Bemalgi N, Fatima BO, Kumar AY. Antibacterial efficacy of Carica papaya leaf extract, probiotics, kidodent, and placebo mouthwashes in reduction of salivary Streptococcus mutans: A double-blinded parallel designed randomized controlled trial. J Indian Soc Pedod Prev Dent 2021;39:291-8

How to cite this URL:
Rao DG, Havale R, Sara SS, Bemalgi N, Fatima BO, Kumar AY. Antibacterial efficacy of Carica papaya leaf extract, probiotics, kidodent, and placebo mouthwashes in reduction of salivary Streptococcus mutans: A double-blinded parallel designed randomized controlled trial. J Indian Soc Pedod Prev Dent [serial online] 2021 [cited 2021 Nov 29];39:291-8. Available from: https://www.jisppd.com/text.asp?2021/39/3/291/330718





   Introduction Top


Dental caries is one of those infectious diseases worldwide that are a major threat to human health. It is a chronic infectious disease, which shows multifaceted etiology with slow rise leading to the destruction of dental hard tissues. Distinct microorganisms are associated with dental caries initiation and progressions and the primary ones are S. mutans responsible for dental caries initiation and Lactobacilli with that of the progression.[1] S. mutans are acidogenic capable of forming long-chain high-molecular mass polymers referred to as extracellular polysaccharides in the presence of sucrose, fructose, and glucose[2],[3],[4] which serve as an important factor in the cariogenecity of S. mutans.[5] At present, irreparable and indicative treatment modality of drill and fill is employed in dental caries management, while on the contrary caries prevention is refuse to be acknowledged. Hence, focus is to be redirected toward the dismissal of the disease by intercepting its etiology and hazardous elements and dealing with the current and future caries initiation. A fundamental change to a medical model from a surgical is in need to obtain an ultimate goal of maintaining a dentition that is caries free and also to promote quality of life in terms of true oral health. The concept put forward by G. V. Black, i.e., “Extension for prevention” to “Prevention of extension” or also referred to as “constriction with conviction” is being practiced by adapting to various new techniques and materials.[6],[7] In today's antibiotic dominating field, probiotics are emerging with their beneficial advantages of being a good bacteria. At the turn of the 20th century, Elie Metchnikoff, a Nobel Prize-winning Russian, was the one who stated that these Probiotics could provide a health benefit.[8] It was Lilly and Stillwell in 1965 who first used the terminology Probiotics.[9] In 2001, FAO/WHO defined Probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host.”[10] In 1984, L. acidophilus by Hull was the very first probiotic species to be introduced in research following  Bifidobacterium bifidum Scientific Name Search bh et al. in 1991.[4],[11]

Carica papaya is owned by the Caricaceae family. Fermented Papaya Preparation has been beneficial in the treatment of the following, i.e., to reduce the liability of progressing pathologies of the oral cavity such as caries process and gingival inflammation[12] due to its broad-spectrum pharmacological activities.

Kidodent mouthwash (INDOCO REMEDIES LTD) is composed of the following active salt ingredients Sodium monofluorophosphate topical 0.05% w/v, Triclosan topical 0.03% w/v, and Xylitol topical 5% w/v.

Since teeth comprise only one quarter of the mouth and tooth brushing alone could not be helpful to eliminate or prevent malodour, gingival inflammation, and dental caries, hence the use of mouthwashes is a must to help bring things into balance. They help in minimizing the amount of acid present in the mouth, wipes inaccessible areas surrounding the periodontium, and also remineralizes the teeth.


   Methodology Top


Study design

The present in vivo study is a double-blinded parallel design, randomized control trial with an allocation the ratio of 1:1:1:1 was calculated with a total of 60 participants, 15 in each group age 8–12 years, including both gender selected from the Morarji Desai Residential School, Raichur, Karnataka by random sampling and divided into four equal groups as [Flowchart 1]:



  • Group A: consists of 15 participants who will be given C. papaya mouthwash
  • Group B consists of 15 participants who will be given probiotics mouthwash
  • Group C consists of 15 participants who will be given distilled water (placebo)
  • Group D consists of 15 participants who will be given Kidodent mouthwash (control).


Before commencement of the research, ethical clearance had been obtained from the Ethical committee of Academy of Medical Education society Dental College and Hospital Raichur (AME/DC/218/19-20).

Inclusion criteria

Children with the age group of 8–12 years.

  1. Good general health
  2. Devoid of any infectious diseases
  3. Willing for the study and are co-operative.


Exclusion criteria

  1. Children who cannot expectorate completely
  2. Children with a history of taking antibiotics 1 month before and amidst the clinical trial
  3. Children receiving orthodontic treatment or with any sort of intraoral prosthesis
  4. Children any intraoral pathology
  5. Children who were not available for the follow-up phase
  6. Children who are using any oral hygiene aids.


Collection of Carica papaya leaves

The leaves of C. papaya were collected from the University of Agriculture Science Raichur, Karnataka.

Preparation of plant extract

Fresh C. papaya leaves were first rinsed with tap water followed by distilled water and then shade dried, the same were later ground to coarse powder and sieved to remove impurities.

Soxhlet extraction

40 g of dried papaya powder was filled in a strong filter paper, wrapped, and placed in the chamber of the Soxhlet apparatus. Ethanol (200 ml) in the flask which is an extracting solvent was heated until its vapors condensed in a condenser. Later the extractant dripped into the thimble which had the crude drug and it came in contact with the level of liquid in the chamber that raised to the top of the siphon tube, finally the liquid content of the chamber drops down into the flask. Until the drop of solvent from the siphon tube did not leave the residue on evaporation, the above procedure was continued. The resultant was placed in a water bath to completely evaporate the solvent. The obtained residue was stored in ambered color bottles and refrigerated for further use.

Formulation of mouthwash

220 mg of obtained extract was uniformly mixed with 30 ml of distilled water in an ultrasonic vibrator. To this 15 ml of ethyl alcohol was added and was made up to 400 ml with distilled water. The obtained solution was filtered using filter paper and 20 ml of it was mixed with 80 ml of distilled water. The resultant mouthwash was dispensed into 100 ml bottles and labeled accordingly.

Probiotic mouthwash

Commercially available probiotic sachets named PREBACT 1 g (L. acidophilus; Lactobacillus rhamnosus; Bififobacterium longum; Saccharomyces boulardi.) was directly dispensed in 10 ml of water and given as a mouthwash.

Kidodent mouthwash

10 ml of the Kidodent mouthwash (INDOCO REMEDIES LTD) was given.

Mouth rinsing and sample collection

All the mouthwashes were given to the participants for 15 days and were asked to swish once daily for 30 s, half an hour before breakfast, and spit.

Saliva samples were collected before giving mouthwashes at baseline, 7th, 10th, and 15th day of the study period.

Microbiological analysis

Media preparation was done using selective media for S. mutans, Mitis Salivarius Agar by following the instructions given on the Mitis Salivarius Agar dispensing bottle, also Agar Agar Type I was used for solidification of media along with 1% of potassium tellurite solution for isolating streptococci microorganisms. About 90 g of the selective media along with 32 g of Agar Agar Type I was added to 1000 ml of distilled water and then autoclaved at 121°C (250°F) at 15 psi for 30 min. Later, it was cooled and about 1 ml of 1% potassium tellurite solution was added to it. (to specify S mutans). The resultant sterile media was poured onto the petri plates in the vicinity of the flame under the laminar airflow cabinet and kept for solidification of media. Serial dilution of saliva samples was done. A loop full of the resultant solution was streaked onto the petri plates cultured using Mitis Sailvarius Agar media by an inoculation loop of 4 mm diameter. The Mitis Salivarius Bacitracin agar culture plates were then incubated for 24 h. Postincubation colonies were examined which showed morphological characteristics of S. mutans.

Colony forming unit estimation

Within a given sample, the amount of viable bacterial cells forms the colony-forming unit. Colonies were counted using a magnifying glass under colony counter manually and were expressed as colony forming units per milliliter (CFU/ml) of saliva. Semi quantification of colony numbers was done by multiplying the actual colony count by 1 × 103. The readings obtained were noted down, tabulated and considered as the baseline data.

Statistical analysis

Data collected were tabulated using the Microsoft Excel. Data were analyzed using the Statistical Package for the Social Sciences software (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY, USA: IBM Corp). Descriptive statistics such as mean, standard deviation, and percentage were used. The Shapiro–Wilk test was used to assess the normality of distribution of all parameters. Data were found to be normally distributed. The comparison of means between the three groups was done using the one-way ANOVA with post hoc Tukey's HSD for data meeting the assumption of homogeneity of variances and post hoc Games Howell test for the data violating the assumption of similarity of variances. Any P < 0.05 was considered to be significant.


   Results Top


Demographic data

Out of 60 children selected in the study, 26 (43%) were female and 34 (57%) were male with a mean age of 9.33 ± 0.43.

Intragroup comparison

Carica papaya leaf extract mouthwash

On analyzing the efficacy of CPM in reduction of the Streptococcus mutans, the following mean values and SD observed are as follows 1.91 × 103 ± 0.20, 2.02 × 103 + 0.28

2.52 × 103 ± 0.36, 3.39 × 103 ± 0.532 which is highest from the 15th, 10th, and 7th day to baseline suggestive of a decline in reduction of S. mutans which showed to be significant statistically with (P = 0.00) [Table 1], [Figure 1] and [Graph 1].
Table 1: Intergroup comparison at different time intervals

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Figure 1: Soxhlet extraction and inoculation of salivary sample

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Probiotics mouthwash

Postanalysis of probiotic mouthwash in reducing S. mutans count showed decline highest at 15th day, followed by 10th, 7th, and baseline, the mean values and SD are as follows 1.77 × 103 ± 0.30, 1.78 × 103 ± 0.38, 2.13 × 103 ± 0.26, and 2.46 × 103 ± 0.27, respectively. The difference in the mean scores obtained showed to be statistically significant (P = 0.00) [Table 1], [Figure 1] and [Graph 1].

Distilled water mouthrinse

On comparison, DWM showed the following mean score for S. mutans with mean and SD from the 15th day to baseline as 4.00 × 103 ± 0.25, 4.75 × 103 ± 0.43, 6.81 × 103 ± 0.18, 6.77 × 103 ± 0.39. There is an increase in the mean score from the baseline to 7th day which could be attributed to dietary changes among the participants. Nevertheless, the difference among mean scores was found to be statistically significant (P = .00) [Table 1], [Figure 1] and [Graph 1].

Kidodent mouthwash

Reduction in S. mutans among the participants using KM was seen to be the highest on the 15th day followed by 10th, 7th, and baseline results in terms of mean values and SD are 1.50 × 103 ± 0.232, 1.71 × 103 ± 0.132, 1.80 × 103 ± 0.322, and 2.34 × 1 03 ± 0.438, respectively. The above mean score difference inferred the results to be statistically significant P = 0.00 [Table 1], [Figure 1] and [Graph 1].

Inter-group comparison

At baseline, the mean colony-forming units of S. mutans for CPM, PM, DWM, and KM are 3.39 × 103 ± 0.532, 2.46 × 103 ± 0.27, 6.77 × 103 ± 0.39, and 2.34 × 103 ± 0.438, respectively. The results on the 7th day showed to have the following mean scores of 2.52 × 103 ± 0.368, 2.13 × 103 ± 0.265, 6.81 × 103 ± 0.183, 1.80 × 103 ± 0.322 for CPM, PM, DWM, and KM, respectively followed by the 10th day which showed the following mean scores among CPM, PM, DWM, and KM as 2.02 × 103 ± 0.28, 1.78 × 103 ± 0.386, 4.75 × 103 ± 0.43, and 1.71 × 103 ± 0.132. At the end of the study period following mean scores with respect to CPM, PM, DWM and KM were 1.91 × 103 ± 0.20, 1.77 × 103 ± 0.37, 4.0 × 103 ± 0.25, and 1.5 × 103 ± 0.23, respectively. The difference between all four groups at baseline, 7th, 10th, and 15th day was found to be statistically significant with a P = 0.05 (P = 0.00) [Table 2], [Figure 2] and [Graph 2].
Table 2: Intragroup comparison at different time intervals

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Figure 2: Microscopic evaluation and colony count of Streptococcus mutans

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When analyzed using post hoc Bonferonni, CPM, PM, KM when compared with DWM at different time intervals showed the former three types of mouthwash were proven to be of more significance in reducing salivary S. mutans when compared to the latter and CPM and PM showed similar results to KM on comparison also KM showed a highly statistically significant reduction among all other from baseline to the 15th day (P = .0000) [Table 3].
Table 3: Post hoc Bonferroni

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   Discussion Top


This study has been performed to determine as well as compare the antibacterial efficacy of Probiotics and C. papaya leaves extract mouthwash with that of Kidodent and to evaluate whether the former would improve the suppressive effect on bacteria in association with caries in the saliva.

Dental caries, the most common chronic disease in the world is highly infectious one triggered due to bacterial colonization. The prevalence of dental caries remains high despite the numerous ways put forward in eradicating the same. Besides mechanical methods such as brushing and flossing, mouthrinses play an significant role in declining the oral bacterial counts including S. mutans.[13]

Herbal medicine is still the cornerstone approximating about 75%–80% of the whole population, mainly in developing countries such as India for primary health care attributable to its better cultural acceptability, not just that it is also compatible with the human body, and account for fewer or no adverse effects. Hence, efforts have been made for the advancement of an alternate mouthwash from natural products that could be safer, easily available, and the one which can substitute the standard pharmaceutical remedies. Literature speaks about the antimicrobial activity of plant phenolics and also their activity against various human pathogens. Researches on the same to distinguish and develop new healthy food ingredients, medical compounds, and pharmaceuticals is evident.[14]

Probiotics have numerous functions with their presence in the body. Some among them are as follows: (1) they act in the allergic process altering the structure of antigens and thereby reducing their immunogenicity. (2) They decrease tissue permeability and inhibit the release of pro-inflammatory agents from the immune response. (3) They stimulate the release of anti-inflammatory cytokines and bone remodeling factors linked to the synthesis of Vitamins D, C, and K, which participate in the metabolism of calcium, an important mineral in the formation of the bone matrix.

Dextranase and Urfique are the enzymes produced by S. salivarius are characteristic of their anti-streptococcal action. They help in declining the aggregation and acidification of the placa bacteriana, thereby inhibiting the activity of S. mutans and glucan liberation by the same, hence preventing the biofilm production process.[15]

The basic mechanism of action by means of which probiotics act, can be divided into three (1) intestinal microbiota normalization, (2) immune response modulation, and (3) metabolic effect. (Kour S et al. 2015). The primary concept of probiotics is to focus on the prevention aspect of the diseases and it can be effective in selectively inhibiting oral pathogens. Hence, to prevent and fight against oral diseases, the potential capacity of probiotic bacteria and its mechanism of action is the need for the hour.[16]

C. papaya leaves encounter a have higher levels of protein (7.0 g), phosphorus (142 mg), Vitamin B and E (136 mg), calcium (334 mg), and sodium (16 mg),[1] Alkaloid like carpain, pseudocarpain and dehydrocarpaine I and II, choline, carposide, Vitamin C and E have also shown to be beneficial. Papaya leaf extracts also compose phenolic compounds, such as protocatechuic acid, p-coumaric acid, 5,7-dimethoxycoumarin, caffeic acid, kaempferol, quercetin, and chlorogenic acid which are shown to possess antimicrobial activity.[12] Fewer parts of Asia make use of the steamed young papaya leaves. It aids in treating dengue fever, cancer cell growth inhibition, facilitates digestion, and has antimalarial, antibacterial activity, and antiplasmodial activity.[17]

“Kidodent” the gold standard mouth rinse comprises fluorides and contributes to reducing the plaque S. mutans. It reduces the caries process by holding back the sugar intake of oral microorganisms by enolase enzyme blockage which results in a decrease in the intracellular level of phosphoenolpyruvate. The metabolic activity of bacteria is further reduced due to intracellular acidification due to the dissociation of unionized hydrofluoric acid into H+ and F in the cells. Analysis of the intracellular glycolytic pathway revealed that xylitol inhibits the upper half while fluoride inhibits the lower which is suggestive that both of these components have synergistic inhibition on acids produced by Streptococci mutans and xylitol enhances the properties of fluoride in terms of inhibition.[18]


   Conclusion Top


The present study indicates that C. papaya is a rich source of phytoconstituents which possesses immense antioxidant potential. Extraction procedures if not standardized may lead to the degradation of the phytochemical constituents in the plant and thereby lack reproducibility. Furthermore, the mechanisms by which the probiotic organisms exert their effects are still at the nascent stage. Therefore, it calls for a more appropriate research work that could focus on mastering and evaluating the exact mechanisms to promote health with the use of probiotics. On the basis of observations and analysis made in the course of the study, a significant decline in the mean CFU/ml of S. mutans. Count among all the mouthwash groups was appreciated. Herbal oral rinse (C. papaya leaf extract) and probiotics proved equally effective antimicrobial agents as Kidodent in reducing S. mutans count. Furthermore, clinical trials of long term and large sample size need to be performed to evaluate their efficacy.

Acknowledgment

The authors would like to thank Mrs. Kamala Navaratna Prof. and Head Department of Microbiology, Laxmi Venkatesh Desai College, Raichur, Karnataka, India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Tewari BB, Subramanian G, Gomathinayagm R. Antimicrobial properties of Carica papaya (Papaya) different leaf extract against E. coli, S. aureus and C. albicans. Am J Pharmacol Pharmacother 2014;1:025-39.  Back to cited text no. 1
    
2.
Alabi OA, Haruna MT, Anokwuru CP, Jegede T, Abia H, Okegbe VU, et al. Comparative studies on antimicrobial properties of extracts of fresh and dried leaves of Carica papaya (L) on clinical bacterial and fungal isolates. Adv Appl Sci Res 2012;3:3107-14.  Back to cited text no. 2
    
3.
Anibijuwon II, Udeze AO. Antimicrobial activity of Carica papaya (pawpaw leaf) on some pathogenic organisms of clinical origin from South-Western Nigeria. Ethnobotanical Leaflets 2009:4.  Back to cited text no. 3
    
4.
Chaturvedi S, Jain U. Importance of probiotics in orthodontics. J Orofac Res 2015;5:99-103.  Back to cited text no. 4
    
5.
Kour S, Verma VK, Sachan A, Singh K, Arora A, Kaur G. Role of probiotics in orthodontics. Rama Univ J Dent Sci 2015;2:26-31.  Back to cited text no. 5
    
6.
Megalaa N, Kayalvizhi G, Silas AJ, Sajeev R, Saravana Kumar MS. Role of herbal leaf extracts in caries prevention. Int J Contemp Med Res 2014;1:71-8.  Back to cited text no. 6
    
7.
Mello VJ, Gomes MT, Lemos FO, Delfino JL, Andrade SP, Lopes MT, et al. The gastric ulcer protective and healing role of cysteine proteinases from Carica candamarcensis. Phytomedicine 2008;15:237-44.  Back to cited text no. 7
    
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Metchnikoff E. Optimistic Studies. New York: Putman's Sons; 1908. p. 161-83.  Back to cited text no. 8
    
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Lilly DM, Stillwell RH. Probiotics: Growth-promoting factors produced by microorganisms. Science 1965;147:747-8.  Back to cited text no. 9
    
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World Health Organization. World Health Organization Report of a Joint FAO/WHO Expert Consultation of Evaluations of Health and Nutritional Properties of Probiotics in Food Including Powder Milk and Live Lactic Acid Bacteria. Geneva: World Health Organization; 2001.  Back to cited text no. 10
    
11.
Hull RR, Roberts AV and Mayes JJ. Survival of Lactobacillus acidophilus in yogurt Aust J. Dairy Technol. 1984;39:164-6.  Back to cited text no. 11
    
12.
Yogiraj V, Goyal PK, Chauhan CS, Goyal A, Vyas B. Carica papaya Linn: An overview. Int J Herbal Med 2014;2:1-8.  Back to cited text no. 12
    
13.
Hassan SA. Comparison of the efficacy of mouth rinses Camellia sinensis extract, guava leaves extract and sodium fluoride solution. Future Dent J 2019;5.  Back to cited text no. 13
    
14.
Helmy MM, Bakr RO. In vitro comparison of the antimicrobial activity of five herbal extracts, and selected mouthwashes marketed in Egypt against cariogenic Streptococcus mutans. Egypt J Med Microb 2014;23:49-58. doi: 10.12816/0024259.  Back to cited text no. 14
    
15.
Carneiro Pereira AL, de Lima LG, da Silva Maia SE, Melo de Matos JM, Melo de Matos JM, Lucena de Vasconcelos JE, et al. Co-aggregation of probiotics in the dental biofilm and inhibition of bacterial growth in caries prevention. Int J Oral Health Med Res 2017;4:4-7.  Back to cited text no. 15
    
16.
Maryam S, Chaudhry K, Singh C. Probiotics in dentistry-beneficial or baleful: A review. J Med Dent Sci Res 2018;5:56-65.  Back to cited text no. 16
    
17.
Pal A, Mazumder A. Carica papaya A Magic Herbal Remedy, Int J Adv Res 2017;5:2626-35.  Back to cited text no. 17
    
18.
Subramaniam P, Nandan N. Effect of xylitol, sodium fluoride and triclosan containing mouth rinse on Streptococcus mutans. Contemp Clin Dent 2011;2:287-90.  Back to cited text no. 18
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