|Year : 2013 | Volume
| Issue : 4 | Page : 521-522
|Comparative evaluation of antibacterial property and substantivity of chlorhexidine containing dentifrices with sodium lauryl sulfate and Tween as surfactants: An in vivo study
V Venu1, AR Prabhakar2, N Basappa2
1 Department of Pedodontics and Preventive Dentistry, Army College of Dental Science, Secunderabad, Andhra Pradesh, India
2 Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India
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|Date of Submission||18-Jul-2012|
|Date of Decision||18-Jan-2013|
|Date of Acceptance||15-Mar-2013|
|Date of Web Publication||19-Sep-2013|
| Abstract|| |
Aim: The aim of the study was to determine the antibacterial property and substantivity of chlorhexidine containing dentifrices with sodium lauryl sulfate (SLS) and Tween as surfactants.
Materials and Methods: It is a double-blind cross over the study, a total of 20 children within their mixed dentition period (7-13 year) having Streptococci mutans count more than 10 6 were selected for the main study. Three types of chlorhexidine containing dentifrices were used with a washout period of 1 week. Out of the three toothpastes, one was without surfactant and other two toothpastes contained SLS and Tween as surfactants respectively. 20 volunteers brushed for
1 min during the study day with their assigned toothpaste. Saliva samples were collected before brushing, immediately after brushing and 1, 3, 5, and 7 hand sent for microbial analysis. The culture carried out by inoculating saliva sample onto Mitis salivarius agar for selective isolation of S. mutans followed by counting of colony forming unit.
Results: Group I and III (Chlorhexidine and CHX + Tween) had shown statistically significant reduction in bacterial count until 7 h when compared to their baseline values ( P < 0.001). Group II toothpaste (CHX + SLS) had shown significant reduction in bacterial count until 3 h only. On inter group comparison, Group III had shown good amount of percentage reduction in bacterial count when compared to other groups.
Conclusion: CHX + Tween toothpaste had shown statistically significant reduction in antibacterial activity and substantivity than other groups. These findings show chlorhexidine containing toothpaste with non-ionic surfactant will be able to maintain the antibacterial property and substantivity of chlorhexidine.
Chlorhexidine, S. mutans, sodium lauryl sulfate, Tween
|How to cite this article:|
Venu V, Prabhakar A R, Basappa N. Comparative evaluation of antibacterial property and substantivity of chlorhexidine containing dentifrices with sodium lauryl sulfate and Tween as surfactants: An in vivo study. Indian J Dent Res 2013;24:521-2
Dental plaque is a microbial film, which is invariably present on the hard and soft-tissues of the oral cavity and it contains a complex blend of various microorganisms. It is considered as the precursor of dental caries, gingivitis or periodontitis. The prevention and control of dental caries and as well the periodontal disease are dependent on optimal plaque control. 
|How to cite this URL:|
Venu V, Prabhakar A R, Basappa N. Comparative evaluation of antibacterial property and substantivity of chlorhexidine containing dentifrices with sodium lauryl sulfate and Tween as surfactants: An in vivo study. Indian J Dent Res [serial online] 2013 [cited 2019 Aug 23];24:521-2. Available from: http://www.ijdr.in/text.asp?2013/24/4/521/118367
The mechanical removal of plaque on a regular basis is one of the principle methods advocated by the researchers and clinicians. Regular tooth brushing habit helps in the reduction of cariogenic bacteria like mutans streptococci. 
Toothpaste can be used as a medium to deliver antimicrobial agents. A constant search for improvement in existing toothpastes has resulted in the addition of a number of chemicals to the toothpastes mainly antimicrobials, which offer the potential of adjunctive benefit to the gingival health such as chlorhexidine.  It can be incorporated into the toothpaste for enhanced antimicrobial action. Chlorhexidine is a cationibis-biguanide, with anti-plaque action and wide antibacterial activity against cariogenic bacteria. The property of substantivity gives prolonged action of chlorhexidine against oral micro-organisms. 
Till now chlorhexidine containing toothpastes are not available commercially because of its possible reaction with sodium lauryl sulfate (SLS) surfactant present in toothpaste, but according to few authors chlorhexidine containing dentifrices will be prepared by using ingredients, which are not antagonistic to chlorhexidine like Tween.  Until now, in most of the studies conducted, CHX and surfactants were present in dentifrices separately or alongwith surfactants in slurry form, but there are very few studies conducted, in corporating chlorhexidine into dentifrice with surfactants as in normal toothpaste form.
Therefore, this study is undertaken to compare the antibacterial property and substantivity of chlorhexidine containing dentifrices with SLS and Tween as surfactants.
| Materials and Methods|| |
One school was picked up randomly. List of all the students of 7-13 years age group from the selected school was made from school records. Those children whom the exclusion criteria asset were deleted from the group of remaining children. The selected school children were examined and those who had a minimum of one caries was selected.
One milliliter unstimulated saliva of the 60 selected children were then collected in a sterile test tube and assessed for S. Mutans colony forming units (CFU). 25 children who had S. mutans count of more than 10 6 were selected.  These children's names were written on chits of paper and 20 children (11 male, 9 female) were selected by employing lottery method. An informed written consent was obtained from the guardian of the volunteers, prior to the commencement of the study. Ethical clearance to conduct the study was obtained from the Institutional Ethical Review Board.
- Children in mixed dentition period of 7-13 years.
- Subjects having caries experience.
- Subjects having S. mutans count equal to or more than 10 6 CFU/ml.
During the investigation days
- History of taking antibiotic therapy within 7 days prior to the start of the study.
- of hyper sensitivity to any in gradients in all the selected dentifrices.
- Unable to comply with study appointment schedule.
- Mentally ill subjects.
- History of using chlorhexidine containing products.
- Students who didn't have the habit of using the toothbrush for their oral hygiene practices.
- Students who were using toothpowder for their oral hygiene practices.
- Students who were wearing intra-oral appliances or prosthesis.
- Subjects with severe crowding, malocclusion, and developmental disorders related to oral apparatus.
Test products used in the present study
- Avoid food and liquid (except water) during the evaluation period, being allowed to have normal breakfast and lunch between 5 h and 7 h.
- On the day of the study, they were instructed not to use the regular toothpaste, which they had been using daily and avoid any oral hygiene measures during the evaluation period.
- Avoid the use of chewing gum or sweets during the evaluation period. 
After taking permission from the ethical committee, three experimental toothpastes were prepared in Pharmacy College. The basic composition of all the three toothpastes [Figure 1] were the same (presence of CHX). Out of three, one toothpaste was surfactant free  other two had surfactants SLS (2%) and Tween (2%) respectively [Figure 2]. The absence of surfactant in toothpaste was supported by a study carried out by Van Strydonk et al.  The basic composition of toothpaste was taken from the study carried out by Olympio et al. 
During the study days, unstimulated saliva sample of approximately 1ml volume was obtained in the morning (atapproximately 8.30 a.m.), after which each participating child was provided with multi-tufted, medium textured, toothbrushes and were instructed to brush with respective assigned toothpaste for 1 m under the examiner's supervision. The amount of toothpaste used was standardized by placing a length of toothpaste covering the toothbrush head. 
After brushing with the allocated toothpaste, saliva samples were collected immediately after brushing and 1, 3, 5, and 7 h respectively and sent for microbial analysis for S. mutans count.
The same procedure was followed for the other two toothpastes also with a washout period of 1 week. The method is demonstrated in the flow chart below:
Samples were dispersed by vortex mixer to disrupt bacterial aggregation for 60 s then serially diluted to 10 3 fold in phosphate buffered saline and 0.005 ml [Figure 3] was taken with the help of inoculating loop and streaked on Mitis salivarius agar supplemented with bacitracin for selective enumeration of S. mutans [Figure 4].  The mitis salivarius bacitracin agar plates were incubated for 48 h at 37°C. Colony counting was done using an electronic colony counter. S. mutans were expressed as the numbe of CFU/ml (×10 6 CFU/ml). In this study, children brushed for 1 m under the examiner's supervision.  Washout period of 1 week was given between each group  to avoid the carry-over effects and to eliminate antimicrobial substance from the enamel and gingiva was carried out between each phase. 
Results were expressed as mean ± SD and percentage changes. Paired t-test was performed to compare the changes in the number of CFU/ml before and after brushing at various time periods. One-way ANOVA was used for multiple group comparison followed by post hoc Tukey's test for pairwise comparisons.
For all the tests, a P value of 0.05 or less was considered for statistical significance.
| Results|| |
In intra group comparison of antibacterial property and substantivity of CHX [Figure 5] and CHX + Tween [Figure 6] at different time intervals immediately after brushing and 1, 3, 5, and 7 h with before brushing bacterial counts, these both groups showed statistically significant (P < 0.001) reduction in bacterial count.
Intra group comparison of antibacterial property and substantivity of CHX + SLS [Figure 7] at different time intervals with their base values showed statistically significant reduction until 3 h, but after that there is no significant difference observed.
|Figure 7: Intragroup comparison of Chlorhexidine + sodium lauryl sulphate paste|
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In inter group, comparison of CHX, CHX + SLS, and CHX + Tween [Figure 8], upto 3 h all the three groups had shown statistically significant bacterial reduction (P < 0.001) when compared to baseline values. After 3 h only CHX and CHX + Tween had shown statistically significant reduction in bacterial count until 7 h, but on inter-comparison of these two groups CHX + Tween had shown good amount of bacterial reduction [Table 1], [Table 2] and [Table 3].
|Table 1: Descriptive statistics showing the intergroup comparison of mean bacterial colonies (CFU × 106/ml) among three different groups at different time intervals|
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|Table 2: Descriptive statistics showing the intergroup comparison of difference between the before brushing mean bacterial colony values with mean values at different time intervals among the three groups|
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|Table 3: Descriptive statistics showing the intergroup comparison of percentage reduction in bacterial colonies (CFU × 106/ml) at different time intervals among the three groups|
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| Discussion|| |
CHX containing dentifrices are commercially not available in the market until now, due to the salt formation which occurs when it is added to SLS which is the most commonly used surfactant in dentifrices. Salt formation occurs due to cationic (CHX) and anionic (SLS) reaction and it leads to decreased efficiency of CHX paste.  The persistence or substantivity of chlorhexidine in the mouth is the mechanism by which plaque inhibition occurs.  The persistence of antimicrobial action of toothpastes on salivary bacterial counts following a single brushing appears to be a useful predictor of plaque inhibitory properties. 
However, there are only limited data evaluating the clinical efficacy because most of the studies until now conducted were using CHX and surfactants in liquid form only.
Generally in these studies the results were ascribed to the whole dentifrice, but here the basic composition of all the three pastes is same, except surfactant. Here, cross-over design  was used as it is considered as a useful predictor of antibacterial activity and substantivity. 
Statistical analysis of this experiment found CHX and Tween dentifrice had shown significant reduction in bacterial count and substantivity than other two groups.
The results of CHX paste showed that immediately after brushing there is a statistically significant reduction in bacterial counts, followed by a gradual increase in the count but still maintained till 7 h, same results were observed by Jenkin et al. 
The results of CHX + SLS, statistically significant reduction till 3 h, but later counts increased, similar findings observed by Barkvoll et al.  and Rathe et al. 
The results of CHX + Tween, showed statistically significant reduction in bacterial count and substantivity till 7 h, recently in a study conducted by Olympio et al. with the same composition toothpaste had shown significant reduction in different parameters such as gingivitis, bleeding and periodontal index in groups of orthopatients.  Here, the insoluble salt formation might be prevented by non-ionic Tween. ,
Hawthrone effect could be seen very often in studies which compared dentifrices with toothbrushing due to higher awareness of oral health care where subjects improve their oral hygiene as a result of study participation. ,,
There are some reasons to believe that CHX and dentifrices are incompatible, but the evidence for this does not allow the drawing of any definitive conclusion (Kolahi and Soolari, 2006).  Moreover, incompatibility is known to occur in aqueous solutions and all studies deal with SLS-dentifrice alongside CHX rinsing but not with both ingredients present together in single toothpaste. 
Manufacturing CHX regular toothpaste will decrease the side-effects of long-term use of mouthwashes such as staining, discoloration, impaired taste sensation, and supragingival calculus.  It would be ideal to incorporate chlorhexidine in a dentifrice formulation, thus combining mechanical cleaning, fluoride delivery, anti-plaque benefit, and resulting anti-gingivitis benefit with no added discomfort for patients. 
Within the limitations of this study, we can also infer that CHX containing dentifrices can be prepared with the ingredients which are not antagonistic to each other, so that antibacterial property and substantivity can be prolonged for a longer period but long-term studies with different surfactants and large study groups are required before going for commercial preparation of CHX containing regular dentifrice.
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Department of Pedodontics and Preventive Dentistry, Army College of Dental Science, Secunderabad, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3]
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