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1.  Validation of ATP bioluminescence as a tool to assess antimicrobial effects of mouthrinses in an in vitro subgingival-biofilm model 
Objectives. The aim of this investigation was to evaluate whether the adenosine triphosphate (ATP) bioluminescence method is an appropriate tool to assess the efficacy of antiseptic mouthrinses in terms of quantitative reductions of total viable microbial counts in mixed biofilm populations in vitro. Study Design. Three mouthrinses, containing respectively, chlorhexidine and cetylpyridinium chloride (CHX/CPC), essential oils (EO) and amine fluoride/stannous fluoride (AFSF), as well as Phosphate Buffered Saline (PBS) used as control, were tested in an in vitro static biofilm model by ATP bioluminescence and compared to culture method. Biofilms were grown on saliva-coated hydroxyapatite disks for 72 hours and then exposed for 1 minute to the mouthrinse or control by immersion. The antibacterial effect of the rinses was tested by analysis of variance. The reliability of the ATP bioluminescence method was assessed by calculating the Pearson correlation coefficients when compared to the viable cell counts obtained by culture. Results. Using ATP bioluminescence, the antimicrobial activity of the tested mouthrinses was demonstrated when compared to the PBS control. The ATP bioluminescence values were significantly correlated (0.769, p<0.001) to the viable cell counts. CHX/CPC and AFSF showed similar antimicrobial activity, although AFSF had a less homogeneous effect, being both more effective than the EO rinse. Conclusion. ATP bioluminescence viability testing may be considered a useful tool to assess the in vitro efficacy of antibacterial compounds. In the proposed model, CHX/CPC and AFSF containing mouthrinses demonstrated superior antimicrobial activity, as compared to EO rinses, in a multispecies biofilm model.
Key words:Biofilm, ATP bioluminescence,mouthrinse, essential oils, chlorhexidine, amine fluoride/stannous fluoride.
doi:10.4317/medoral.18376
PMCID: PMC3548652  PMID: 23229259
2.  Chlorhexidine Substantivity on Salivary Flora and Plaque-Like Biofilm: An In Situ Model 
PLoS ONE  2013;8(12):e83522.
Objective
To evaluate the in situ antibacterial activity of a mouthrinse with 0.2% Chlorhexidine (M-0.2% CHX) on undisturbed de novo plaque-like biofilm (PL-biofilm) and on salivary flora up to 7 hours after its application.
Methods
A special acrylic appliance was designed, with 3 inserted glass disks on each buccal side, allowing for PL-biofilm growth. Fifteen healthy volunteers wore the appliance for 48 hours and then performed an M-0.2% CHX; disks were removed at 30 seconds and 1, 3, 5 and 7 hours after the mouth-rinsing. Applying a washout period, saliva samples were collected from each volunteer at 30 seconds and 1, 3, 5 and 7 hours after performing an M-0.2% CHX. The PL-biofilm and saliva samples were analysed by confocal laser scanning and epifluorescence microscopes, respectively.
Results
At 30 seconds after M-0.2% CHX, the levels of viable bacteria detected in saliva were significantly lower than those observed in PL-biofilm. The difference in the percentage of live bacteria detected in saliva was significantly higher than that observed in PL-biofilm at 5 and 7 hours after M-0.2% CHX.
Conclusion
After a single mouthrinse of the 0.2% CHX formulation tested in the present study, the 2-day PL-biofilm presented a significantly higher resistance to this antiseptic in situ than that observed in salivary flora. However, this 0.2% CHX formulation showed a higher substantivity on PL-biofilm than on salivary flora at 5 and 7 hours after mouth-rinsing, which could be related to the slower growth rate of PL-biofilm and the possible reservoir function for antimicrobial agents associated with the undisturbed de novo PL-biofilm.
doi:10.1371/journal.pone.0083522
PMCID: PMC3873939  PMID: 24386220
3.  Impact of an Anticaries Mouthrinse on In Vitro Remineralization and Microbial Control 
Objective. The objective of this research was to evaluate the caries control potential of a new fluoride mouthrinse that also contained antimicrobial agents and a biofilm disrupting agent using different in vitro models. Methods. Four in vitro studies were conducted to assess the performance of this three pronged approach to caries control: (1) traditional enamel fluoride uptake, (2) surface microhardness study using pH cycling model and subsequent fluoride uptake, (3) a salivary biofilm flow-through study to determine the anti-microbial activity, and (4) a single species biofilm model measuring effect on biofilm matrix disruption. Results. The data showed that a LISTERINE rinse with fluoride, essential oils and xylitol was superior in promoting enamel fluoride uptake and in enhancing antimicrobial activity over traditional commercially available fluoridated products. An increase of the surface microhardness was observed when the LISTERINE rinse was used in combination with fluoridated toothpaste versus the fluoridated toothpaste alone. Finally, it was demonstrated that xylitol solutions disrupted and reduced the biovolume of biofilm matrix of mature Streptococcus mutans. Conclusion. These in vitro studies demonstrated that a fluoride mouthrinse with antimicrobial agent and biofilm matrix disrupting agent provided multifaceted and enhanced anti-caries efficacy by promoting remineralization, reducing acidogenic bacteria and disrupting biofilm matrix.
doi:10.1155/2014/982071
PMCID: PMC3933167  PMID: 24648842
4.  A comparative study of antiplaque and antigingivitis effects of herbal mouthrinse containing tea tree oil, clove, and basil with commercially available essential oil mouthrinse 
Background:
The relatively safe nature and cost-effectiveness of herbal extracts have led to a resurgent interest in their utility as therapeutic agents. Therefore, this prospective, double-blind, randomly controlled clinical trial was designed to compare the antiplaque and antigingivitis effects of newly formulated mouthrinse containing tea tree oil (TTO), clove, and basil with those of commercially available essential oil (EO) mouthrinse.
Materials and Methods:
Forty patients were selected for a 21-day study period and randomly divided into two groups. The test group patients were given newly formulated herbal mouthrinse and the control group patients were given commercially available EO mouthrinse. The Plaque Index (PI), Gingival Index (GI), and Papillary Marginal Attachment (PMA) Index were recorded at baseline, 14 days, and 21 days. The microbial colony forming units (CFU) were assessed at baseline and 21 days.
Results:
Test group patients using herbal mouthrinse showed significant improvement in GI (0.16), PI (0.57), and PMA (0.02) scores. These improvements were comparable to those achieved with commercially available EO mouthrinse. However, the aerobic and anaerobic CFU of microbiota were reduced with the herbal mouthrinse (P = 0.0000).
Conclusion:
The newly formulated herbal mouthrinse and commercially available mouthrinse were beneficial clinically as antiplaque and antigingivitis agents. Newly formulated mouthrinses showed significant reduction in microbial CFU at 21 days. So, our findings support the regular use of herbal mouthrinse as an antiplaque, antigingivitis, and antimicrobial rinse for better efficacy.
doi:10.4103/0972-124X.134568
PMCID: PMC4095623  PMID: 25024544
Antigingivitis; antiplaque; basil; mouthrinse; tea tree oil
5.  Mouthrinses and dentifrices are effective antigingivitis and antiplaque agents 
Summary
Subjects
Systematic review of 6-month RCT that evaluated the antigingivitis and antiplaque properties of dentifrices or mouth rinses in adults 18 years and older. A total of 50 studies identified that met inclusion criteria. Subject characteristics not otherwise specified.
Exposure
Mouthrinse use or Dentifrice use
Main Outcome Measure
Gingivitis, as measured by the Gingival Index (GI) or Modified Gingival Index (MGI) and Plaque accumulation (as measured by Turesky modification of the Quigley-Hein Index)
Main Results
The meta-analytic results were expressed as the standardized mean effect (i.e., active agent minus control divided by the standard deviation). This was used as a measure of the relative strength of the active agent, and the summary results presented as the Standardized Difference (Std.Diff.).
As measured by the GI, mouthrinses containing 0.12 % chlorhexidine (Std.Diff. = 0.563), or essential oils (Std.Diff. = 0.306), had a significant antigingivitis effect. Dentifrices containing triclosan with 2% Gantrez copolymer (Std.Diff. = 0.858), or stannous fluoride (Std.Diff. = 0.441) also had a significant antigingivitis effect.
As measured by the MGI, essential oils (Std.Diff. = 0.762) had a significant antigingivitis effect.
Mouthrinses containing cetylpyridium chloride had significant antigingivitis effects in several individual studies, but no meta-analytic conclusion was reached due to “both statistical heterogeneity and a variety of formulations evaluated.”
Conclusions
Mouthrinses containing 0.12 % chlorhexidine or essential oils, and dentifrices containing triclosan with 2% Gantrez copolymer or stannous fluoride, , each have significant antigingivitis effects in adults after six months of use.
doi:10.1016/j.jebdp.2007.12.010
PMCID: PMC2323913  PMID: 18346694
6.  Chlorhexidine alcohol base mouthrinse versus Chlorhexidine formaldehyde base mouthrinse efficacy on plaque control: Double blind, randomized clinical trials 
Background: Chlorhexidine is well known for its antiplaque effect. However, the mouthrinse based chlorhexidine antiplaque efficiency may vary according to the formulation of the final product. The aim of the present study was to compare anti-plaque effectiveness of two commercial mouthrinses: 0.12 % Chlorhexidine alcohol base (CLX-A) versus a diluted 0.1% Chlorhexidine non-alcohol base with 0.1% of Formaldehyde (CLX-F). Material and Methods: the study was a seven day randomized, double-blind, placebo-controlled trial including 30 volunteers. At the start, all participants received a dental prophylaxis. Over 7 days experimental non-brushing period, during which subjects abstained from all forms of mechanical oral hygiene, one group test rinsed twice daily with 15ml of an alcohol base 0.12% Chlorhexidine mouthrinse. The second group test used 15ml of alcohol free 0.1% Chlorhexidine mouthrinse base 0.1% formaldehyde twice daily. The negative control group used a placebo. Plaque indexes were recorded in all volunteers prior to treatment at Day 0, 1 and 7. Results: After 7 days, the mean plaque index for the first group was 0.76±0.38 compared with a mean plaque index of 1.43±0.56 for the second group. The difference in plaque scores between the groups was statistically significant. Conclusion: the results of this study showed that rinsing with an alcohol base 0.12% Chlorhexidine mouthrinse is significantly different from rinsing with an alcohol free 0.1% Chlorhexidine mouthrinse on plaque inhibition.
Key words:Chlorhexidine, dental plaque, mouthrinse, alcohol, formaldehyde.
doi:10.4317/medoral.17863
PMCID: PMC3548633  PMID: 23229237
7.  Comparison of intraoral distribution of two commercially available chlorhexidine mouthrinses with and without alcohol at three different rinsing periods 
Objectives:
This study compared the intraoral distribution of 0.1% chlorhexidine with alcohol (CHX+Alc) and 0.2% chlorhexidine without alcohol (CHX-Alc) with shorter rinsing times (10s,20s,30s) following a 72h non brushing period.
Materials and Methods:
This study was designed as a single blind, randomized two group parallel experiment with a total of 30(15male,15 female) dental students. To disclose the plaque, erythrosine-containing disclosing agent was added to both the mouthrinses. Group I (0.1% CHX+Alc) & Group II (0.2% CHX-Alc) were asked to rinse with respective mouthrinse for cumulative periods of 10,20 and 30s, following which plaque was scored.
Results:
In Group I, comparison between 10&20, 10&30s was statistically significant but no significance was observed between 20&30s, whereas in Group II, comparison between all the time points were statistically significant. On comparison of plaque scores, plaque scores of both the groups at 10 & 30s sec, show no statistical significance but at 20 seconds was significant (P<0.001). The mean plaque scores of lingual surfaces were lesser in group II whereas in group I lingual surfaces recorded more plaque than the vestibular surfaces.
Conclusions:
Within the limitations of this study, it can be concluded that rinsing for 30 seconds with 0.2% CHX-Alc (Rexidin) is enough for intraoral spread of the mouthrinse whereas rinsing with 0.1% CHX+Alc(Eludril) for achieves the same in 20 seconds, for effective plaque inhibition, both of which will have a positive influence on patient compliance.
doi:10.4103/2231-0762.103450
PMCID: PMC3894089  PMID: 24478962
Chlorhexidine; alcohol; rinsing time; plaque
8.  Comparative study of 0.2% and 0.12% digluconate chlorhexidine mouth rinses on the level of dental staining and gingival indices 
Dental Research Journal  2012;9(3):305-308.
Background:
Chlorhexidine (CHX) as a gold standard chemical agent appears to be the most effective antimicrobial agent for reduction of both plaque and gingivitis. The aim of this study was to compare the efficacy of two concentrations of digluconate chlorhexidine (CHX) solutions (0.12% and 0.20%) on gingival indices and the level of dental staining during 14 days.
Materials and Methods:
in this double-blind controlled clinical trial study 60 patients with moderate to severe gingivitis aged 17–56 years were randomly selected and divided to three groups: Group I (placebo) Group II (0.12% CHX), and Group III (0.2% CHX). Patients rinsed their mouthwashes twice a day after brushing. Before the examination and after 14 days plaque index, gingival index, bleeding index, and stain index were evaluated. The data were analyzed by “Mann–Whitney” test and P value was 0.05.
Results:
the results showed that plaque index and gingival index significantly reduced in Groups II and III in comparison with the placebo group (P < 0.0001). However, the two concentrations did not differ significantly from each other (P = 0.552). Same results were observed in term of gingival bleeding index with this different that 0.2% CHX was significantly more efficient than 0.12% CHX (P < 0.0001). CHX mouthrinse, both concentrations, significantly increased the dental staining level (intensity and area) in comparison with the placebo group. Remarkable difference also was seen between 2 CHX concentrations so that the 0.2% CHX caused much more staining on the teeth than 0.12% CHX.
Conclusion:
based on the results of this study we can conclude that the lower concentrations of CHX should be prescribed, decreasing side effects, since higher concentrations do not seem to be more effective in controlling dental plaque and gingivitis.
PMCID: PMC3469897  PMID: 23087736
chlorhexidine; periodontal index; staining
9.  Effects of typified propolis on mutans streptococci and lactobacilli: a randomized clinical trial 
Brazilian dental science  2013;16(2):31-36.
Objective
The aim of this study was to determine in a randomized, double-blind, placebo-controlled clinical trial the effects of typified propolis and chlorhexidine rinses on salivary levels of mutans streptococci (MS) and lactobacilli (LACT).
Methods
One hundred patients were screened for salivary levels of MS >100,000 CFUs/mL of saliva. All patients presented with at least one cavitated decayed surface. Sixty patients met entry criteria. Subjects were adults 18–55 years old. After restoration of cavitated lesions patients were randomized to 3 experimental groups: 1) PROP-alcohol-free 2% typified propolis rinse (n = 20); 2) CHX- 0.12% chlorhexidine rinse; 3) PL-placebo mouthrinse. Patients rinsed unsupervised 15 mL of respective rinses twice a day for 1 min for 28 days. Patients were assessed for the salivary levels of MS (Dentocult SM) and LACT (Dentocult LB) at baseline, 7-day, 14-day, and at 28-day visits (experimental effects) and at 45-day visit (residual effects). General linear models were employed to analyze the data.
Results
PROP was superior to CHX at 14-day and 28-day visits in suppressing the salivary levels of MS (p < .05). PROP was superior to PL at all visits (p < .01). The residual effects of PROP in suppressing the salivary levels of MS could still be observed at the 45-day visit, where significant differences between PROP and CHX (p < .05), were demonstrated. PROP was significantly superior than CHX in suppressing the levels of salivary LACT at the 28-day visit (p < .05).
Conclusion
Typified propolis rinse was effective in suppressing cariogenic infections in caries-active patients when compared to existing and placebo therapies.
PMCID: PMC3909523  PMID: 24494174
Randomized clinical trial; Typified propolis; Chlorhexidine; Mutans Streptococci; Lactobacilli
10.  In vitro antibacterial effect of carbamide peroxide on oral biofilm 
Journal of Oral Microbiology  2013;5:10.3402/jom.v5i0.20392.
This study compared the effects of carbamide peroxide (CP) and chlorhexidine (CHX) on oral biofilm in vitro. Collagen-coated hydroxyapatite discs were inoculated with subgingival plaque. After 3 weeks, the emergent biofilms were subjected to 1-, 3-, and 10-min exposures of a 1% CHX gel, a 5% CP gel and rinse, and a 10% CP gel and rinse. Subsequently, the biofilms were stained using a two-colour fluorescent dye kit for confocal laser scanning microscopy, and the volume ratio of dead bacteria to all bacteria was analysed. Compared to a non-treated gel control, the active agents killed bacteria on all the discs, with higher concentration and longer exposure times killing more bacteria. The rinse form disrupted the biofilm quicker than the gel form. Overall, 10% CP showed more disruption of biofilm and a greater proportion of killed bacteria than 1% CHX (p<0.05).
doi:10.3402/jom.v5i0.20392
PMCID: PMC3682087  PMID: 23785567
antibacterial; biofilm; carbamide peroxide; confocal laser scanning microscopy
11.  Staining and calculus formation after 0.12% chlorhexidine rinses in plaque-free and plaque covered surfaces: a randomized trial 
Journal of Applied Oral Science  2010;18(5):515-521.
Objectives
Studies concerning side effects of chlorhexidine as related to the presence of plaque are scarce. The purpose of this study was to compare the side effects of 0.12% chlorhexidine gluconate (CHX) on previously plaque-free (control group) and plaque-covered surfaces (test group).
Methods
This study had a single-blind, randomized, split-mouth, 21 days-experimental gingivitis design, including 20 individuals who abandoned all mechanical plaque control methods during 25 days. After 4 days of plaque accumulation, the individuals had 2 randomized quadrants cleaned, remaining 2 quadrants with plaque-covered dental surfaces. On the fourth day, the individuals started with 0.12% CHX rinsing lasting for 21 days. Stain index intensity and extent as well as calculus formation were evaluated during the experimental period.
Results
Intergroup comparisons showed statistically higher (p<0.05) stain intensity and extent index as well as calculus formation over the study in test surfaces as compared to control surfaces. Thus, 26.19% of test surfaces presented calculus, whereas calculus was observed in 4.52% in control surfaces.
Conclusion
The presence of plaque increased 0.12% CHX side effects. These results strengthen the necessity of biofilm disruption prior to the start of CHX mouthrinses in order to reduce side effects.
doi:10.1590/S1678-77572010000500015
PMCID: PMC4246385  PMID: 21085810
Chlorhexidine; Adverse effects; Staining; Tooth discoloration; Dental calculus
12.  The effect of adjunctive chlorhexidine mouthrinse on GCF MMP-8 and TIMP-1 levels in gingivitis: a randomized placebo-controlled study 
BMC Oral Health  2014;14:55.
Background
The aim of the present study was to evaluate the effect of adjunctive chlorhexidine (CHX) mouthrinse on gingival crevicular fluid (GCF) MMP-8 and TIMP-1 levels in plaque-associated gingivitis.
Methods
A total of 50 gingivitis patients were included in the present study. In addition to daily plaque control, CHX group rinsed with CHX, while placebo group rinsed with placebo mouthrinse for 4 weeks. GCF samples were collected, and clinical parameters including plaque index, papillary bleeding index, calculus index and pocket depth were recorded at baseline and 4 weeks. GCF MMP-8 and TIMP-1 levels were determined by immunofluorometric assay (IFMA) and enzyme-linked immunosorbent assay (ELISA), respectively.
Results
In both groups, GCF MMP-8 levels of anterior and posterior sites at four weeks were not different from baseline (p > 0.05). There were no significant differences in GCF MMP-8 levels between the study groups at four weeks (p > 0.05). GCF TIMP-1 levels of anterior and posterior sites at four weeks were higher compared to baseline in both groups (p < 0.05). There was no significant difference in GCF TIMP level between the study groups at four weeks (p > 0.05).
Conclusions
CHX usage had no significant effects on the GCF MMP-8 and TIMP-1 levels in plaque-associate gingivitis. However, daily plaque control resulted in the increase of GCF TIMP-1 levels regardless of CHX usage.
doi:10.1186/1472-6831-14-55
PMCID: PMC4045876  PMID: 24886536
Chlorhexidine; Gingivitis; Gingival crevicular fluid; MMP-8; TIMP-1
13.  Antimicrobial Penetration and Efficacy in an In Vitro Oral Biofilm Model ▿ † 
The penetration and overall efficacy of six mouthrinse actives was evaluated by using an in vitro flow cell oral biofilm model. The technique involved preloading biofilm cells with a green fluorescent dye that leaked out as the cells were permeabilized by a treatment. The loss of green color, and of biomass, was observed by time-lapse microscopy during 60 min of treatment under continuous flow conditions. The six actives analyzed were ethanol, sodium lauryl sulfate, triclosan, chlorhexidine digluconate (CHX), cetylpyridinium chloride, and nisin. Each of these agents effected loss of green fluorescence throughout biofilm cell clusters, with faster action at the edge of a cell cluster and slower action in the cluster center. The time to reach half of the initial fluorescent intensity at the center of a cell cluster, which can be viewed as a combined penetration and biological action time, ranged from 0.6 to 19 min for the various agents. These times are much longer than the predicted penetration time based on diffusion alone, suggesting that anti-biofilm action was controlled more by the biological action time than by the penetration time of the active. None of the agents tested caused any removal of the biofilm. The extent of fluorescence loss after 1 h of exposure to an active ranged from 87 to 99.5%, with CHX being the most effective. The extent of fluorescence loss in vitro, but not penetration and action time, correlated well with the relative efficacy data from published clinical trials.
doi:10.1128/AAC.00206-11
PMCID: PMC3122404  PMID: 21537022
14.  Does human saliva decrease the antimicrobial activity of chlorhexidine against oral bacteria? 
BMC Research Notes  2014;7(1):711.
Background
Several studies have shown the antibacterial effectiveness of 0.2% chlorhexidine (CHX) in both in vitro and in vivo studies. In this way, CHX comes directly in contact with saliva. This in vitro study aimed at investigating the possible neutralizing effect of saliva on CHX.
Methods
Saliva samples (12 ml) were collected from twenty healthy volunteers. The aerobic and anaerobic bacterial counts in saliva were determined on Colombia blood agar (CBA) and yeast cysteine agar (HCB), respectively. Saliva from each subject was divided among 4 experimental groups (3 ml/group). Samples were centrifuged at 4000 g for 10 min. The centrifuged salivary bacteria were incubated with the following solutions: 0.2% CHX in saliva, CHX in saliva with 7% ethanol, CHX in 0.9% NaCl, CHX in 0.9% NaCl with 7% ethanol. After exposure for 1 min or 3 min to these CHX solutions, the CHX was neutralized and the bacteria were cultivated, after which the number of colony forming units (aerobic and anaerobic) was determined.
Results
CHX reduced the CFU in all groups significantly (p = 0.0001). Therefore, CHX had a similar effect on both aerobic and anaerobic microorganisms. Significantly more bacteria survived the effect of CHX when kept in salivary solution. This effect from saliva could be compensated by the addition of ethanol. In the absence of saliva there was no significant difference observed in the effectiveness of CHX with respect to ethanol. Prolonging the exposure time to 3 min enhanced the effectiveness of CHX.
Conclusions
The effect of saliva on the antimicrobial activity of CHX was weak albeit statistically significant. However, addition of 7% ethanol compensates this effect. The impact of saliva on the reduction of the antimicrobial efficacy of mouthrinses such as CHX needs to be taken into consideration with regard to improving their antibacterial properties.
doi:10.1186/1756-0500-7-711
PMCID: PMC4200226  PMID: 25300308
Chlorhexidine (CHX); Saliva; Antimicrobial efficacy; Bacterial count
15.  Comparison of the effectiveness of a commercially available herbal mouthrinse with chlorhexidine gluconate at the clinical and patient level 
Background:
The key to good oral health is hidden in nature. Natural herbs like neem, tulsi, pudina, clove oil, ajwain, triphala and many more has been used since ages either as a whole single herb or as a combination against various oral health problems like bleeding gums, halitosis, mouth ulcers and preventing tooth decay. The aim of the study was to compare the efficacy of a commercially available herbal mouthrinse (Herboral) with that of chlorhexidine gluconate which is considered to be a gold standard as an anti-plaque agent.
Materials and Methods:
A randomized, two-group, parallel study as a ‘de novo’ plaque accumulation model was carried out on 50 subjects (23 males and 27 females). At baseline, all participants received a professional prophylaxis and were randomly assigned to the test (Herbal mouthrinse) and control (Chlorhexidine Gluconate) group. On the following three days, all subjects rinsed with 10 ml of the allocated mouthrinse twice daily for 1 min. They were asked to refrain from use of any other oral hygiene measures during the study. At the end of the experimental period, plaque was assessed and a questionnaire was filled by all subjects.
Results:
Chlorhexidine (mean plaque score=1.65) inhibited plaque growth significantly more than the herbal mouthrinse (mean plaque score=1.43, P<0.001). The results of the questionnaire showed that Herboral was preferred by patients for its taste, its convenience of use and taste duration (aftertaste). However, Chlorhexidine was considered to be more effective in reducing plaque as compared to Herboral.
Conclusion:
Herbal mouthrinse was found to be a potent plaque inhibitor, though less effective than Chlorhexidine Gluconate. However, it can serve as a good alternative for the patients with special needs as in case of diabetics, xerostomics, and so on.
doi:10.4103/0972-124X.92567
PMCID: PMC3283931  PMID: 22368358
Chlorhexidine; herbal; mouthrinse; plaque control
16.  Comparative antiplaque and antigingivitis effectiveness of tea tree oil mouthwash and a cetylpyridinium chloride mouthwash: A randomized controlled crossover study 
Contemporary Clinical Dentistry  2014;5(4):466-470.
Objective:
The aim of this study was to compare the antiplaque and antigingivitis effects of a mouthwash containing tea tree oil (TTO) with a cetylpyridinium chloride (CPC) mouthwash.
Materials and Methods:
This was a randomized 4 × 4, controlled, cross-over, involving 20 healthy volunteers in a 5-day plaque re-growth model. Test mouthwashes were TTO (Tebodont®) and a mouthwash containing CPC 0.05% (Aquafresh®). A 0.12% chlorhexidine (CHX) mouthwash (Oro-Clense®) was used as positive and colored water (placebo [PLB]) as negative controls. Gingival bleeding index (GBI) and plaque index (PI) scores were recorded before and after each test period. Test periods were separated with 2 weeks washout period.
Results:
All four mouthwashes significantly (P < 0.001) reduced the GBI scores when compared to the baseline GBI scores. There was no significant difference between PLB and active mouthwashes in the GBI scores. CHX and CPC mouthwashes were found more effective in reducing the PI scores than TTO and PLB mouthwashes. There was no significant difference in PI scores of CHX and CPC mouthwashes.
Conclusion:
0.05% CPC mouthwash can be an alternative to CHX mouthwash since it is alcohol free and found as efficient as CHX in dental plaque reduction with lesser side effects. More studies are needed to test antigingivitis effects of the mouthwashes used in this study, preferably without initial scaling and polishing.
doi:10.4103/0976-237X.142813
PMCID: PMC4229754  PMID: 25395761
Antigingivitis; antiplaque; cetylepyridinium chloride; mouthwash; tea tree oil
17.  Direct Visualization of Spatial and Temporal Patterns of Antimicrobial Action within Model Oral Biofilms▿  
A microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm was developed and applied to describe spatial and temporal patterns of mouthrinse activity on model oral biofilms. Three species biofilms of Streptococcus oralis, Streptococcus gordonii, and Actinomyces naeslundii were grown in glass capillary flow cells. Bacterial cells were stained with the fluorogenic esterase substrate Calcien AM (CAM). Loss of green fluorescence upon exposure to an antimicrobial formulation was subsequently imaged by time-lapse confocal laser scanning microscopy. When an antimicrobial mouthrinse containing chlorhexidine digluconate was administered, a gradual loss of green fluorescence was observed that began at the periphery of cell clusters where they adjoined the flowing bulk fluid and progressed inward over a time period of several minutes. Image analysis was performed to quantify a penetration velocity of 4 μm/min. An enzyme-based antimicrobial formulation led to a gradual, continually slowing loss of fluorescence in a pattern that was qualitatively different from the behavior observed with chlorhexidine. Ethanol at 11.6% had little effect on the biofilm. None of these treatments resulted in the removal of biomass from the biofilm. Most methods to measure or visualize antimicrobial action in biofilms are destructive. Spatial information is important because biofilms are known for their structural and physiological heterogeneity. The CAM staining technique has the potential to provide information about the rate of antimicrobial penetration, the presence of tolerant subpopulations, and the extent of biomass removal effected by a treatment.
doi:10.1128/AEM.02218-07
PMCID: PMC2268315  PMID: 18223108
18.  Antimicrobial Efficacy of Different Toothpastes and Mouthrinses: An In Vitro Study 
Dental Research Journal  2011;8(2):85-94.
Background:
Anti-microbial agents have been used as a chemotherapeutic agent to improve oral health. This in vitro study was carried out to determine antimicrobial efficacy of different toothpastes and mouthrinses against the oral pathogens.
Methods:
A total of five toothpastes and five mouthrinses were tested for their antimicrobial activity against three oral pathogens namely, Streptococcus mutans (MTCC 890), Escherichia coli (MTCC 579) and Candida albicans (MTCC 854) by well agar diffusion assay. Statistical Analysis was performed using a statistical package, SPSS windows version 15, by applying mean values using analysis of variance (ANOVA) with post-hoc least square differences (LSD) method(α = 0.05).
Results:
Toothpaste formulation A showed maximum zones of inhibition against the test organism, Escherichia coli (P<60;0.001) compared to all other toothpastes formulations. Against Streptococcus mutans and Candida albicans, the zones of inhibition were less in comparison to E.coli but were significantly different at higher dilutions (1:8, 1:16 P<60;0.05) for toothpaste formulation A.
Mouthrinses formulation H showed maximum efficacy against the test organism, Escherichia coli (P<60;0.001) compared to all other mouthrinse formulations. Against Streptococcus mutans, mouthrinses formulations F, G and J showed significant antimicrobial activity (P<60;0.05) compared to formulation H and I.
Conclusion:
In the present study, it has been demonstrated that triclosan containing toothpastes formulations are more effective in control of oral microflora compared to non-triclosan containing synthetic toothpastes. Among mouthrinses formulations, chlorhexidine was found to be more effective than or as effective as triclosan against the organisms tested.
PMCID: PMC3177399  PMID: 22013468
Antimicrobial activity; Antimicrobial agents; Chlorhexidine gluconate; Mouthrinse; Toothpaste; Triclosan
19.  Development and pyrosequencing analysis of an in-vitro oral biofilm model 
BMC Microbiology  2015;15:24.
Background
Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers.
Results
Pyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms.
Conclusions
Diverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0364-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12866-015-0364-1
PMCID: PMC4332733
16S rRNA; Bacteria; Saliva; Plaque; Microbiome; Microbiota
20.  Antibacterial and physical properties of calcium-phosphate and calcium-fluoride nanocomposites with chlorhexidine 
Dental Materials  2012;28(5):573-583.
Objectives
Previous studies have developed calcium phosphate and fluoride releasing composites. Other studies have incorporated chlorhexidine (CHX) particles into dental composites. However, CHX has not been incorporated in calcium phosphate and fluoride composites. The objectives of this study were to develop nanocomposites containing amorphous calcium phosphate (ACP) or calcium fluoride (CaF2) nanoparticles and CHX particles, and investigate S. mutans biofilm formation and lactic acid production for the first time.
Methods
Chlorhexidine was frozen via liquid nitrogen and ground to obtain a particle size of 0.62 µm. Four nanocomposites were fabricated with fillers of: Nano ACP; nano ACP+10% CHX; nano CaF2; nano CaF2+10% CHX. Three commercial materials were tested as controls: A resin-modified glass ionomer, and two composites. S. mutans live/dead assay, colony-forming unit (CFU) counts, biofilm metabolic activity, and lactic acid were measured.
Results
Adding CHX fillers to ACP and CaF2 nanocomposites greatly increased their antimicrobial capability. ACP and CaF2 nanocomposites with CHX that were inoculated with S. mutans had a growth medium pH > 6.5 after 3 d, while the control commercial composites had a cariogenic pH of 4.2. Nanocomposites with CHX reduced the biofilm metabolic activity by 10–20 folds and reduced the acid production, compared to the controls. CFU on nanocomposites with CHX were three orders of magnitude less than that on commercial composite. Mechanical properties of nanocomposites with CHX matched a commercial composite without fluoride.
Significance
The novel calcium phosphate and fluoride nanocomposites could be rendered antibacterial with CHX to greatly reduce biofilm formation, acid production, CFU and metabolic activity. The antimicrobial and remineralizing nanocomposites with good mechanical properties may be promising for a wide range of tooth restorations with anti-caries capabilities.
doi:10.1016/j.dental.2012.01.006
PMCID: PMC3322264  PMID: 22317794
dental nanocomposite; calcium phosphate; calcium fluoride; chlorhexidine; stress-bearing; S. mutans biofilm; caries inhibition
21.  Improved Antibiotic-Impregnated Catheters with Extended-Spectrum Activity against Resistant Bacteria and Fungi 
Minocycline-rifampin-impregnated central venous catheters (M/R CVCs) have been shown to be efficacious in reducing catheter-related bloodstream infections (CRBSI) and inhibiting the biofilm adherence of resistant Gram-positive and Gram-negative pathogens, with the exception of Pseudomonas aeruginosa and Candida spp. To expand the spectrum of antimicrobial activity, a novel second-generation M/R catheter was developed by adding chlorhexidine (CHX-M/R). CVCs and peripherally inserted central catheters (PICCs) were impregnated with CHX-M/R and compared with first-generation M/R catheters, CHX-silver sulfadiazine-treated CVCs (CHX/SS-CVCs), chlorhexidine-treated PICCs, and uncoated catheters. A biofilm catheter colonization model was used to assess the efficacy of catheters against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), P. aeruginosa, Candida albicans, and Candida glabrata. CHX-M/R-impregnated CVCs were the only antimicrobial catheters that completely inhibited the biofilm colonization of all resistant bacterial and fungal organisms tested at all time intervals, and they were significantly superior to uncoated catheters (all P values were ≤0.003). Furthermore, CHX-M/R-coated CVCs had a significantly more effective and prolonged (up to 3 weeks) antimicrobial activity against MRSA and P. aeruginosa than M/R, CHX/SS, and uncoated CVCs (P < 0.0001). Similarly, CHX-M/R-coated PICCs were also superior to M/R-coated and CHX-coated PICCs in preventing biofilms of MRSA, VRE, P. aeruginosa, and Candida species (P value = 0.003 for all). Our study shows that novel CHX-M/R-coated catheters have unique properties in completely inhibiting biofilm colonization of MRSA, VRE, P. aeruginosa, and fungi in a manner superior to that of M/R- and chlorhexidine-treated catheters.
doi:10.1128/AAC.05836-11
PMCID: PMC3264266  PMID: 22123686
22.  Comparison of the Efficacy of Three Different Mouthrinse Solutions in Decreasing the Level of Streptococcus Mutans in Saliva 
European Journal of Dentistry  2009;3(1):57-61.
Objectives
The aim of this study was to evaluate the effectiveness of three different antiseptic mouthrinse solutions on the saliva samples obtained from the individuals, who had high caries activity rate.
Methods
The efficacy of three antiseptic mouthrinses were evaluated in a study with healthy volunteers. The three antiseptic solutions used in this study were 0.1% octenidine dihydrochloride (Octenisept, Schülke&Mayr, UK), 0.12% chlorhexidine digluconate (Kloroben, Drogsan, Turkey) and an antimicrobial enzymatic rinse (Biotene, Laclede, Inc, USA). A total of 27 adult volunteer subjects were participated in the study. The subjects were stratified into three balanced group. Then the mouth rinses were used by each group according to the manufacturer’s directions. The subjects were restricted for 60 minutes for food intake after using the prescribed mouthrinse. The saliva samples were collected from the volunteers at 1, 10 and 60 minutes after their usage in tubes. The tubes were kept in +4°C in a fridge till the evaluation. 10−3 and 10−5 dilutions were prepared for each solution and S. mutans were evaluated according to total number of colony forming unit (CFU) per ml. The dilutions were spreaded on the surface of Brucella agar plates for anaerobic incubation for 48 hours. The dilutions were 100, 10−3 and 10−5 of the solutions Kloroben, Biotene, Octenisept, and the time factor were 0, 1, 10 and 60 minutes. The statistical analyses were performed by Duncan and Bonferroni tests.
Results
Octenisept was found to be more effective over S. mutans than the other mouthrinse solutions (P<.05).
Conclusions
All mouthrinse solutions except Biotene were effective on oral microorganisms.
PMCID: PMC2647960  PMID: 19262732
Mouthrinse solutions; Saliva; S. mutans
23.  Grape seed extracts inhibit dentin matrix degradation by MMP-3 
Since Matrix metalloproteinases (MMPs) have been suggested to contribute to dentin caries progression, the hypothesis that MMP inhibition would affect the progression of dentin caries is clinically relevant. Grape seed extracts (GSE) have been previously reported to be natural inhibitors of MMPs.
Objective: To evaluate the capacity of a GSE mouthrinse to prevent the degradation of demineralized dentin matrix by MMP-3 (stromelysin-1).
Materials and Methods: Standardized blocks of dentin obtained from sound permanent teeth extracted for orthodontic reasons were demineralized with Ethylenediaminetetraacetic acid (EDTA) and pretreated either with (A) GSE (0.2% w/v), (B) amine fluoride (AmF) (20% w/v), (C) a mouthrinse which contains both, (D) placebo, (E) sodium fluoride (0.15 mg.ml−1), (F) PBS, (G) Chlorhexidine digluconate (CHX), or (H) zinc chloride (ZnCl2). The dentin blocks were then incubated with activated recombinant MMP-3. The supernatants were analyzed by Western Blot for several dentin matrix proteins known to be MMP-3 substrate. In parallel, scanning electron microscopy (SEM) was performed on resin replica of the dentin blocks.
Results: Western blot analysis of the supernatants revealed that MMP-3 released from the dentin matrix small proteoglycans (decorin and biglycan) and dentin sialoprotein (DSP) in the AmF, sodium fluoride, PBS and placebo pretreated groups, but not in the GSE and mouthrinse pretreated groups. SEM examination of resin replica showed that the mouthrinse and its active components not only had an anti-MMP action but also modified the dentin surface accessibility.
Conclusion: This study shows that GSE either alone or combined with AmF as in the evaluated mouthrinse limits dentin matrix degradation. This association may be promising to prevent the progression of caries within dentin. However, the procedure should be adapted to clinically relevant durations.
doi:10.3389/fphys.2014.00425
PMCID: PMC4215787  PMID: 25400590
grape seed extracts; dentin; matrix metalloproteinases; caries; mouthrinse
24.  Investigating the biological properties of carbohydrate derived fulvic acid (CHD-FA) as a potential novel therapy for the management of oral biofilm infections 
BMC Oral Health  2013;13:47.
Background
A number of oral diseases, including periodontitis, derive from microbial biofilms and are associated with increased antimicrobial resistance. Despite the widespread use of mouthwashes being used as adjunctive measures to control these biofilms, their prolonged use is not recommended due to various side effects. Therefore, alternative broad-spectrum antimicrobials that minimise these effects are highly sought after. Carbohydrate derived fulvic acid (CHD-FA) is an organic acid which has previously demonstrated to be microbiocidal against Candida albicans biofilms, therefore, the aims of this study were to evaluate the antibacterial activity of CHD-FA against orally derived biofilms and to investigate adjunctive biological effects.
Methods
Minimum inhibitory concentrations were evaluated for CHD-FA and chlorhexidine (CHX) against a range of oral bacteria using standardised microdilution testing for planktonic and sessile. Scanning electron microscopy was also employed to visualise changes in oral biofilms after antimicrobial treatment. Cytotoxicity of these compounds was assessed against oral epithelial cells, and the effect of CHD-FA on host inflammatory markers was assessed by measuring mRNA and protein expression.
Results
CHD-FA was highly active against all of the oral bacteria tested, including Porphyromonas gingivalis, with a sessile minimum inhibitory concentration of 0.5%. This concentration was shown to kill multi-species biofilms by approximately 90%, levels comparable to that of chlorhexidine (CHX). In a mammalian cell culture model, pretreatment of epithelial cells with buffered CHD-FA was shown to significantly down-regulate key inflammatory mediators, including interleukin-8 (IL-8), after stimulation with a multi-species biofilm.
Conclusions
Overall, CHD-FA was shown to possess broad-spectrum antibacterial activity, with a supplementary function of being able to down-regulate inflammation. These properties offer an attractive spectrum of function from a naturally derived compound, which could be used as an alternative topical treatment strategy for oral biofilm diseases. Further studies in vitro and in vivo are required to determine the precise mechanism by which CHD-FA modulates the host immune response.
doi:10.1186/1472-6831-13-47
PMCID: PMC3849008  PMID: 24063298
Fulvic acid; Chlorhexidine; Biofilm; Antibacterial; Periodontitis; Inflammation
25.  Effect of Mouthrinse Containing Propolis on Oral Microorganisms and Human Gingival Fibroblasts 
European journal of dentistry  2007;1(4):195-201.
Objectives
The aim of this study was to compare the effects of four different mouthrinse containing propolis solutions and mouthrinse containing 0.2% chlorhexidine (CHX) on oral microorganisms and human gingival fibroblasts.
Methods
Four different solutions of propolis were prepared and propylene glycol and alcohol were used as solvents for each propolis sample. Mouthrinse containing propolis was prepared at four different concentrations as 10%, 5%, 2.5% and 1%. Besides, CHX was used as control group. The antibacterial effects of five solutions on oral microorganisms were tested and their cytotoxic effects on human gingival fibroblasts were evaluated by agar diffusion test.
Results
At this concentrations effectiveness of mouthrinse containing propolis samples on oral microorganisms were not found as effective as CHX. On the contrary, samples found less cytotoxic on human gingival fibroblasts than CHX.
Conclusions
Standardized preparations of propolis can be used as a mouthrinse at appropriate concentrations. To obtain a standardized chemical composition, advanced researches are needed.
PMCID: PMC2609911  PMID: 19212467
Mouthrinse; Propolis; Chlorhexidine; Antibacterial activity; Cell culture

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