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RESEARCH REPORT |
1 Department of Oral Health Promotion, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; and
2 Fundamental Research Laboratory, Asahi Breweries, Ltd., Ibaraki, Japan
* corresponding author, shinada.ohp{at}tmd.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: dental plaque • hop bract polyphenols • mouthrinse
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In recent years, polyphenols, commonly found in plants, have been reported to have many positive applications in humans. These applications include anti-oxidation, anti-allergy, fat reduction, and cancer prevention (Akazome, 2004; Crespy and Williamson, 2004). Additionally, some papers have reported that polyphenols had a preventive effect on dental caries. In vitro studies showed that polyphenols derived from green tea (Sakanaka et al., 1990) or oolong tea (Nakahara et al., 1993) inhibited the activity of glucosyltransferases and, therefore, the growth of mutans streptococci. One report investigated the inhibitory effects of hop bract polyphenols (HBP), extracted from the bract part of hops (Humulus lupulus L.), on cariogenic streptococci in vitro. This high-molecular-weight polyphenol (separated by an ultrafiltration method) potently inhibited the cellular adherence of Streptococcus mutans and Streptococcus sobrinus to a glass surface. Furthermore, this occurred at much lower concentrations than with the polyphenols extracted from green tea or oolong tea leaves (Tagashira et al., 1997); however, the effect of HBP in humans has not yet been reported. Thus, we performed this clinical study to test the hypothesis that HBP, delivered in a mouthrinse, suppresses dental plaque regrowth in humans.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Volunteers
Twenty-nine male volunteers, aged 20–38 yrs (mean age, 26.0 ± 5.9 yrs), in good general health, were selected for this study. They had no periodontitis and no removable oral appliances. None of the participants had taken any antibiotics during the month preceding this study. The participants were given verbal and written information about the study and signed consent forms to participate. The sample size was estimated where the expected near difference and variance were both 1 at a significance level of 5% and a power of 80%. The results showed that a sample size of around 30 individuals would be required.
Mouthrinse Formulation
HBP was prepared as previously described (Tagashira et al., 1997). The glucosyltransferase-inhibiting constituents of HBP are considered to be pro-anthocyanidines, which are made from between 8 and 22 catechins (Kurumatani et al., 2005). The percentage of proanthocyanidins in the HBP was estimated (by ultrafiltration) to be 35%. At a concentration of 0.01%, HBP has been reported to show more than 80% inhibition of the glucosyltransferase of cariogenic bacteria, but no antibiotic activity (Tagashira et al., 1997). A primary irritation test in rabbits showed that HBP had no stimulative nature (data not shown). With HBP considered as a mouthrinse component, a higher concentration was recommended, because the amount of HBP in the oral region would fall gradually after mouthrinsing. In contrast, an extremely high concentration (such as 1.0%) of HBP was not suitable for a mouthrinse, because of its bitterness. Thus, the concentration of HBP in the mouthrinse was determined as 0.1%. Two mouthrinses were prepared for the study: (1) the HBP mouthrinse, 10 g of HBP dissolved in 10 L of distilled water; and (2) a placebo, 10 L of distilled water colored with food dye to resemble the HBP mouthrinse. Both mouthrinses were membrane-filtered (pore size, 0.45 µm) and put into 20-mL plastic bottles. The bottles were sealed with a screw-cap and pasteurized in water at 60°C for 10 min.
Experimental Design
The clinical study was of a parallel-group, randomized, double-blind, and crossover design (Fig.
). The 29 male volunteers were randomly divided into two groups, A and B. Groups A and B included 14 and 15 volunteers, respectively. This division was fixed throughout the test period (from the beginning of the 1st test phase to the end of the 2nd test phase). Allocation was generated by Ms. Y. Akazome (Asahi Breweries, Ltd.), using a random numbers table, and was sealed until the end of the clinical experiment. At the beginning of the 1st test phase, following completion of a questionnaire about lifestyle, a baseline oral examination (decayed teeth, missing teeth, filled teeth, depth of pockets, and gingival index), and plaque assessment by the Patient Hygiene Performance method (Podshadley and Haley, 1968), participants received thorough prophylaxis to remove all plaque from their teeth. For the following 72 hrs, the participants were directed to refrain from all oral hygiene except for rinsing with the distributed mouthrinse 5 times a day (after waking, breakfast, lunch, and dinner, and before sleeping). The participants rinsed with 10 mL of mouthrinse for 1 min. Group A used the HBP mouthrinse, and group B used the placebo in the 1st test phase. On the 4th day, a questionnaire about behavior during the 1st test phase, an oral examination (depth of pockets and gingival index), and an assessment of plaque were performed. After a two-week washout period, the 2nd test phase was performed similarly to the 1st test phase. There were only three differences between the 1st and 2nd test phases: (1) The questionnaire at the beginning of the 2nd test phase asked about behavior during the washout period, (2) the baseline oral examination checked only the depth of pockets and gingival index, and (3) group A used the placebo and group B used the HBP mouthrinse in the 2nd test phase.
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Number of Bacteria
To assess the number of bacteria, we collected a plaque sample with a sterile cotton swab from the buccal surface of the maxillary left first molar, placed it in a glycerine transport solution, and preserved it at –80°C. The samples were sonically dispersed for 30 sec, and appropriate dilutions were plated in duplicate on MSB agar (Gold et al., 1973) and brain-heart infusion agar (Difco Laboratories, Detroit, MI, USA). After 72 hrs of incubation at 37°C in an anaerobic chamber (BBLTM GasPakTM Anaerobic System, Becton, Dickinson and Company, Franklin Lakes, NJ, USA), the numbers of colony-forming units (CFU) on both types of plates were counted. The CFUs of mutans streptococci were identified by their characteristic colony morphology (Emilson, 1983). Representative and questionable colonies of mutans streptococci were isolated and tested by PCR analysis (BML, Inc., Shibuya, Tokyo, Japan).
Statistical Analysis
After completion of the study, statistical analysis was performed with the software program Statistical Package for the Social Sciences (SPSS 10.0J). Mean values and standard deviations of the clinical parameters were calculated. The HBP mouthrinse and placebo groups were compared with respect to their age and baseline index scores of variables by t test. The means of the plaque score, gingival index score, and the number of bacteria were analyzed by the paired t test, because of the crossover design, for the detection of significant differences between the HBP mouthrinse and the placebo.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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There were no significant differences between the two groups with respect to lifestyle habits or baseline oral healthcare markers such as smoking, floss use, DMFT, and depth of pocket levels (Table 1). From the questionnaire, the frequency of mouthrinsing during the test period when the HBP mouthrinse was used (12.1 ± 2.9 times) and that when the placebo was used (11.8 ± 2.9 times) also showed no significant differences. Mutans streptococci were observed in 27 participants (93.0%). Both group A and B contained only one participant who did not have mutans streptococci. In the baseline assessment of plaque, there were no significant differences in the number of bacteria between the two groups (Table 1). These results suggested that there were no unpredictable factors affecting plaque regrowth in this study.
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shows that the total mean plaque score after use of the HBP mouthrinse (2.5 ± 0.5) was significantly less than that after use of the placebo (3.3 ± 0.5) (p < 0.001). This inhibitory effect on accumulating plaque was significant on both the buccal and lingual surfaces of teeth (p < 0.001). When compared with the placebo, the total plaque reduction due to the HBP mouthrinse was 25.4%. The reducing ratios on the buccal and lingual surfaces were different (20.1% and 31.4%, respectively). The reducing effect by the HBP mouthrinse was seen on both sides of all teeth (APPENDIX Fig. 1). In comparison with the 1st and 2nd test phases, the HBP mouthrinse reduced the mean plaque score in each phase (APPENDIX Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we carried out a clinical test to evaluate the effect of a mouthrinse containing 0.1% HBP on dental plaque regrowth over 3 days. The results showed that the HBP mouthrinse was effective in reducing dental plaque regrowth, and lowered the number of mutans streptococci bacteria. There were no unexpected side-effects. This result shows the efficacy of HBP on dental hygiene in humans.
Plaque scores according to the Patient Hygiene Performance method were significantly lowered by the HBP mouthrinse in all oral regions. Apparently, the mouthrinse was more effective in reducing the plaque score in palatal and lingual regions than in buccal regions. The fact that the mouthrinse could more easily reach the palatal and lingual regions than the buccal region could explain this. The significant decrease in numbers of mutans streptococci bacteria seems to contradict previous reports that HBP had a potent inhibitory effect on glucosyltransferase, but little effect on the proliferation of mutans streptococci (Tagashira et al., 1997). The differences in experimental conditions may explain this. In Tagashira’s paper, the antibacterial effect of HBP on mutans streptococci was tested in Todd-Hewitt broth medium, but in the human mouth, mutans streptococci were in poorer nutritional conditions. The antibacterial effects of HBP on mutans streptococci may have been underestimated in the previous paper.
Although it is necessary to investigate further the efficacy and safety of HBP under normal oral-care conditions for a long experimental period, we concluded that HBP is a promising agent for the prevention of oral diseases and could be included in functional foods or oral hygiene items.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received October 14, 2005; Last revision December 25, 2006; Accepted May 7, 2007
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REFERENCES |
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