|Year : 2015 | Volume
| Issue : 2 | Page : 128-133
Effect of high-molecular-weight component of Cranberry on plaque and salivary Streptococcus mutans counts in children: An in vivo study
Akanksha Gupta, Kalpana Bansal, Mohita Marwaha
Department of Pedodontics and Preventive Dentistry, Sri Gobind Tricentenary Dental College and Research Institute, Budhera, Gurgaon, Haryana, India
|Date of Web Publication||15-Apr-2015|
Gurgaon Kalpana Bansal
Department of Pedodontics and Preventive Dentistry, Sri Gobind Tricentenary Dental College and Research Institute, Budhera, Gurgaon - 122 505, Haryana
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Previous investigations showed that a high-molecular-weight, nondialyzable material (NDM) from cranberries inhibits the adhesion of a number of bacterial species and prevents the coaggregation of many oral bacterial pairs. Aim: In the present study, the effect of mouthrinse containing high-molecular-weight component of cranberry was evaluated on colonization of Streptococcus mutans in children and compared it with a control mouthrinse without high-molecular-weight component on Streptococcus mutans counts. Materials and Methods: A high-molecular-weight NDM was isolated from cranberry juice concentrate after the dialysis of the cranberry concentrate; followed by lyophilization. A mouthwash was prepared especially for the study having NDM in the concentration of 3 mg/ml. Following 4 weeks of daily usage of cranberry-containing mouthwash by the children of an experimental group (n = 20), the Streptococcus mutans counts in plaque and saliva were compared with that in control group using placebo mouthwash (n = 20) with the help of Dentocult SM strips. Results: There was a highlysignificant reduction in Streptococcus mutans counts in saliva and plaque of children using mouthwash containing cranberry NDM (P < 0.05) compared to control. Conclusion: The data suggest that the high-molecular-weight cranberry extract in mouthwash has a significant potential in reducing the Streptococcus counts in the oral environment.
Keywords: Anti-adhesion, cranberry, dental caries, dentocult SM, nondialyzable material, Streptococcus mutans
|How to cite this article:|
Gupta A, Bansal K, Marwaha M. Effect of high-molecular-weight component of Cranberry on plaque and salivary Streptococcus mutans counts in children: An in vivo study. J Indian Soc Pedod Prev Dent 2015;33:128-33
|How to cite this URL:|
Gupta A, Bansal K, Marwaha M. Effect of high-molecular-weight component of Cranberry on plaque and salivary Streptococcus mutans counts in children: An in vivo study. J Indian Soc Pedod Prev Dent [serial online] 2015 [cited 2019 Jul 17];33:128-33. Available from: http://www.jisppd.com/text.asp?2015/33/2/128/155125
| Introduction|| |
There are over 750 species of bacteria that inhabit the humanoral cavity, but only a small fraction of those are attributed to causing plaque related diseases such as caries.  The development of dental caries involves acidogenic and aciduric gram-positive bacteria, primarily the mutans streptococci mainly Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus). 
The antibacterial agents used in the prevention and treatment of oral diseases, including cetylpyridinium chloride, chlorhexidine, amine fluorides, or products containing such agents, are reported to exhibit toxicity, cause staining of teeth, or in the case of ethanol (commonly found in mouthwashes) have been linked to oral cancer. ,, There has been a rising interest in naturally-derived biologically active compounds that may have potential therapeutic uses in medicine and dentistry such as aloe vera (Asphodelaceae Juss), green and black tea (Camellia sinensis), garlic (Allium sativum), castor oil (Ricinnus communis), propolis, orange oil, etc. 
Over the past decade, cranberries and their molecular components have received increasing attention from researchers in human health for its therapeutic effect on urinary tract infections (UTIs) mainly by acting as a potential antiadherent agent.  Recently, attention is being focused on cranberry extracts as a potential therapeutic agent in various other areas of health, including infectious diseases, cancer, and cardiovascular disease. ,,
In terms of oral health; studies have shown that the polyphenols in cranberries have several properties that may be beneficial for treating and/or preventing dental caries  and periodontal disease.  Till date, not much research work has been carried out in evaluating the effect of cranberry NDM on salivary and plaque S. mutans count in vivo in pediatric population; hence the present study was planned to evaluate the effect of mouthrinse containing high-molecular-weight component of cranberry on colonization of S. mutans in children compared with that of a placebo mouthwash using Dentocult SM strips.
| Materials and Methods|| |
This school-based randomized comparative study was conducted in residential school near Sri Gobind Tricentenary Dental College and Hospital, Gurgaon, India. Parents/guardians of all participating children gave their written informed consent. The study was reviewed and approved by the University Ethical Board.
Selection of subjects
School children aged between 9 and 12 years were screened for caries status and 40 children were selected based on the following selection criteria:
- At least four decayed and/or missing due to caries or filled teeth (DMFT/dmft ≥4).
- Subjects adhering to a once-daily tooth brushing routine (using toothbrush and nonfluoridated toothpaste) and practicing no other oral hygiene measures, both professional and home based, other than the requisites of the research project.
- History of current or recent (at least for the past 1 month) antibiotic usage.
- Abscess, draining sinus, cellulitis, or other conditions requiring emergency dental treatment.
Subjects were randomly equally divided into two groups
- Experimental group (n = 20) - Subjects rinsed with a specially prepared mouthrinse with high-molecular-weight component of cranberry once daily in the morning at 9 am.
- Control group (n = 20) - Subjects rinsed with distilled water.
Preparation of mouthrinse containing high-molecular-weight component of cranberry
A high-molecular-weight nondialyzable material (NDM) was isolated from cranberry juice concentrate to avoid any effect of the low pH of cranberry juice on bacterial growth. Concentrated juice from the American cranberry, Vaccinium macrocarpon, was dialyzed against distilled water in dialysis bags with a molecular mass cut-off point of 12,000 at 4°C for 5 days [Figure 1]a. The NDM was lyophilized.  The retentate of the bagdesignated NDM was deep red in color and thick solid in consistency [Figure 1]b. The mouthrinse was freshly prepared by incorporating NDM at a final concentration of 3mg/ml into distilled water [Figure 1]c. The mouthrinse was tangy and palatable and had a neutral pH.
|Figure 1: (a) Dialysis procedure; (b) Non Dialyzed Material (NDM); (c) NDM mouthwash|
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Collection of plaque and salivary samples using Dentocult SM strips
Saliva and plaque samples were obtained from each individual, prior to the start of the experiment, to establish baseline S. mutans levels using Dentocult SM strips. Children were instructed to chew a piece of paraffin wax before saliva sample collection. For saliva collection the rough surface of the Dentocult SM saliva strip was pressed against the saliva on the tongue.
The plaque was collected with a sterile toothpick 1-2 hours after eating or brushing.  For the baseline status of S. mutans in both the control and study groups, plaque samples were collected from the following four sites:  a) Buccal surface of the maxillary right molar, b) labial surface of the maxillary incisor, c) lingual surface of the mandibular incisor, and d) lingual surface of the mandibular left molar. These samples were spread thoroughly but gently on the four sites of the rough surface of the plaque strip. The strips were then placed in the selective culture broth, with the smooth surfaces clipped and attached to the cap. The vials were labeled as per their lot numbers. Subsequent samples were obtained 30 days after the start of the experiment. Dentocult SM vials were incubated in an upright position at 37°C for 48 h with the cap opened one quarter of a turn to allow growth of microorganisms.
Mouth rinsing schedule
In this single-blinded study, the children were randomly divided into two groups of 20 children each.
All the subjects were made to rinse their mouth with 5 ml of the assigned mouthrinse for one minute daily at 9.00 am for duration of 30 days under the investigator's (AG) supervision in the school premises. The subjects were instructed not to eat/drink anything for 1 h after using the mouthrinse. A total of 100 ml of experimental mouthrinse was prepared for 1 day of mouth rinsing program. Same protocol for mouth rinsing was used for the control group as well.
Interpretation of results
Results obtained by culture in the incubator were read by the use of the model chart supplied by the manufacturer. The presence of S. mutans was confirmed by the detection of light-blue to dark-blue raised colonies on the inoculated surface of the strip classified as Class 0-3 as follows:
Class 0: <10,000 CFU/ml (CFU-colony forming unit).
Class 1: <10,000-100,000CFU/ml.
Class 2: 100,000-1,000,000 CFU/ml.
Class 3: >1,000,000 CFU/ml.
Inspection of the growth was conducted with the strip held sideways against light and with a magnifying glass. The S. mutans counts in the saliva at baseline and 30 days post mouth rinse for both the groups were compared. Inter examiner agreement was performed for the reproducibility of outcome assessment by the semiquantitative chairside Dentocult SM test.
Method of record keeping and measurements
The name, age, sex, dmft, and baseline S. mutans scores of the subjects of both the groups were recorded on a Microsoft Excel sheet. After 30 days of mouth rinsing, the post-mouth rinsing S. mutans scores were again recorded on the same excel sheet.
The collected data were subjected to statistical analysis. For intragroup comparison of baseline and post mouthrinse plaque and salivary scores, the paired sample t-test was applied; while for the intergroup comparison, unpaired t-test was applied. The level of significance for all tests was P ≤ 0.05.
| Results|| |
The sample characteristics of the study are presented in [Table 1]. The mean plaque and salivary S. mutans counts of both the groups at baseline are presented in [Table 2]. The mean plaque and salivary scores at baseline for both the groups were compared using unpaired t-test and the differences were found to be nonsignificant.
|Table 2: Comparison between mean plaque and salivary scores of experimental and control group at baseline|
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Comparison between mean plaque and salivary score at baseline and after 30 days of mouth rinsing (intragroup comparison)
The intragroup statistical comparison between mean plaque and salivary S. mutans score at baseline and after 30 days of mouth rinsing in the experimental and the control group was done using paired t-test. In the experimental group, the mean plaque and salivary score declined post 30 days of mouth rinsing with the mouthrinse containing cranberry NDM. The two-tailed P - value was found to be less than 0.0001, which by conventional criteria was found to be extremely statistically significant. In the control group, this difference was found to be statistically nonsignificant [Table 3].
|Table 3: Intragroupcomparison between mean plaque and salivary score at baseline and after 30 days of mouth rinsing in the experimental and the control group using paired t-test|
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Comparison between the experimental and control mouthrinse (intergroup comparison)
The mean plaque and salivary scores post 30 days of mouth rinsing with the experimental mouthrinse and the placebo mouthrinse were compared using the unpaired t-test and by conventional criteria was found to be highly statistically significant [Table 4] and [Figure 2] and [Figure 3].
|Figure 2: Comparison between the plaque scores of experimental and control mouthrinse|
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|Figure 3: Comparison between the salivary scores of experimental and control mouthrinse|
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|Table 4: Intergroup comparison between the mean plaque and salivary scores post 30 days of mouth rinsing with the experimental mouthrinse and the placebo mouthrinse|
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| Discussion|| |
Cranberry (Vaccinium macrocarpon) is widely consumed in various food and natural products which originate from cranberry plants, which are a group of evergreen dwarf shrubs or trailing vines usually found in the northern hemisphere.  Cranberries contain more than 80% water and 10% carbohydrates. The single-strength cranberry juice is very acidic (pH, 2.5) and unpalatable.  The red cranberry fruit is a rich source of various classes of potentially bioactive phenolic compounds. Four classes of phenols have been identified in cranberry: Phenolic acids, anthocyanins (A), flavonols, and flavan-3-ols which consist of monomers and polymer classes of proanthocyanidins (PAC). 
It is unlikely that the consumption of cranberry juice on its own can benefit oral health, given the insufficient contact time between the oral tissues (the teeth and gingiva) and the cranberry polyphenols. In addition, the sugar that is added to cranberry drinks, as well as the acidity of these beverages, may have the counterproductive effect of contributing to the demineralization of tooth enamel. Therefore, in this study NDM was isolated from the cranberry concentrate. These molecules were integrated into a mouthrinse which proved to be efficacious.
The high-molecular-weight NDM, an active ingredient of cranberry juice, has been shown to reverse the coaggregation of the majority of bacterial pairs.  Precoatings of the bacteria with NDM have shown to reduce their ability to form biofilm.  The influence of extracts of flavonols (FLAV), A, and PAC from cranberry on virulence factors involved in S. mutans development and acidogenicity has been reported.  PAC and FLAV, alone or in combination, inhibit the surface-adsorbed glucosyltransferases and F-ATPases activities and the acid production by S. mutans cells. 
This is a school-based, randomized, comparative investigation which aims at exploring the antibacterial effect of NDM extract from cranberry in a specially prepared mouthrinse. So far, only one study has been reported in literature to investigate in vivo effect of NDM mouthrinse on cariogenic microorganisms. 
In the present study, the experimental group showed significant reduction in S. mutans score in both plaque and saliva, comparing the baseline sample with post 30 days sample after using NDM containing mouthrinse; whereas in the control group, the reduction in the mean plaque and salivary S. mutans score was observed, but not found to be statistically significant. The little reduction in the S. mutans counts in control group may be attributed to the mechanical dislodging or "washing away" of loose supragingival plaque that formed within the 24-h period or to the inactivation of viable bacteria by the distilled water. 
The results of our study are in accordance with the study conducted by Weiss et al., (2004)  in which an experimental group (n = 29) after 6 weeks of daily usage of cranberry-containing mouthwash showed significant reduction in the salivary mutans streptococci count as well as the total bacterial count on comparing with the control group (n = 30) using placebo mouthwash. In support of these in vivo results, in vitro studies by the same authors  showed that the NDM fraction inhibited the adhesion of S. sobrinusto a hydroxyapatite surface pretreated with saliva.
Yamanaka et al., (2004)  treated S. mutans and S. sobrinus cells with cranberry juice and found that it reduced cell surface hydrophobicity of the cells, interfering with adhesion and the initial stages of biofilm formation. Steinberg et al., (2004)  used a high-molecular-weight, nondialyzable constituent (NDM) of cranberry juice to study its effects on S. mutans GTF activity and found that NDM significantly reduced the activities of GTF as well as fructosyltransferase, both in solution and immobilized on saliva-coated hydroxyapatite. Duarte et al., (2006)  examined the influence of extracts of FLAV, A, and PAC fromcranberryon virulence factors involved in S. mutans bio film development andacidogenicity. They concluded that PAC and FLAV, alone or in combination, inhibited the surface-adsorbed glucosyltransferases and F-ATPases activities, and the acid production by S. mutans cells.
There were no undesirable effects of the mouthwash observed during the course of the study. The mouthwash was palatable, did not cause any staining, and was well accepted by the children. The advantages of using NDM mouthwash are that it is an herbal agent so there are no chances of toxicity. Secondly, since it is not affecting the viability of oral bacteria, bacterial resistance will not develop. Thirdly, it does not cause any staining of the teeth. The main disadvantages are that it is not readily available in the market. Moreover, the procedure involved in extraction and preparation of NDM mouthwash is expensive and requires laboratory set up.
This study used a simple and reliable method of S. mutans evaluation, the Dentocult SM Strip mutans test.  Shi et al.,  showed that the Dentocult SM Strip mutans test is useful in the diagnosis of caries and for monitoring its progression based on S. mutans counts.
| Conclusion|| |
The study suggested that the high-molecular-weight cranberry extract is highly efficacious in reducing the S. mutans counts in the oral environment. The bacterial counts were done after 30 days of mouth rinsing with NDM mouthwash. So the effect of cranberry mouthrinse could be cumulative as it lowered the bacterial counts in experimental group significantly.
Since this is a preliminary study on a newly introduced NDM mouthrinse for children, further long-term studies should be conducted to evaluate the potential benefits of NDM (cranberry extract).
It is also recommended that the comparative efficacy studies need to be performed against the known effective mouthwashes such as chlorhexidine.
| Acknowledgement|| |
The authors acknowledge the help and support of Department of Biochemistry, All India Institute of Medical Sciences, New Delhi for providing the facilities of dialysis and lyophilization of cranberry concentrate to extract NDM.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]