Introduction
This study aimed to assess the anti-inflammatory and antimicrobial effects of mouthwashes with 0.12% chlorhexidine (CLX) and 0.5% Zingiber officinale essential oil (ZOEO).
Methods
The gas chromatography-mass spectrometry of ZOEO was developed, and the mouthwash was prepared. Thirty-one adult subjects of both sexes with fixed orthodontic appliances were selected. For 7 days, the mouthwashes with CLX, ZOEO, and flavored sterile water placebo were used randomly, with a 15-day interval between each solution. Saliva was collected before the first mouthwash, 1 minute and 15 minutes after it, and on the seventh day. The patients were subjected to clinical examinations of the bonded bracket index proposed by Ciancio, bleeding on probing, and sensory analysis (flavor). Generalized linear models were used to assess in vitro cell viability. The GENMOD procedure was used to assess the changes of bleeding on probing, and Friedman and Wilcoxon tests were used for data on colony-forming units per milliliter (CFU/mL), bonded bracket index, and flavor, at the 5% significance level.
Results
In the microbiologic analysis, the ZOEO mouthwash presented antimicrobial effectiveness for Streptococcus mutans as well as the CLX mouthwash, but it did not show the same substantivity. The ZOEO was efficient in controlling dental biofilm and reducing gingival bleeding. The sensory analyses showed that the flavor for ZOEO and CLX solutions presented low acceptability.
Conclusions
The ZOEO mouthwash has an anti-inflammatory property with an effect on reducing gingival bleeding. However, it requires adjustments to the formulation to improve flavor quality.
Highlights
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Anti-inflammatory and antimicrobial effects of mouthwash in an orthodontic appliance were assessed.
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0.12% chlorhexidine, 0.5% Zingiber officinale essential oil (ZOEO), and placebo were compared.
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ZOEO and chlorhexidine mouthwashes were efficient in controlling dental biofilm.
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ZOEO was efficient in reducing gingival bleeding.
The oral cavity is a rich ecosystem with countless microorganisms. However, some conditions include what is known as “ecological stress,” which refers to changes in the microbiologic balance, creating favorable conditions to the growth and appearance of cariogenic and/or periodontopathogenic bacteria. , The insertion of orthodontic appliances is the most important environmental change after tooth eruption that may result in qualitative and quantitative changes in the equilibrium state of oral microbiota, thus promoting an increase in microorganisms in both saliva and bacterial plaque.
During orthodontic treatment, professionals should prevent and control biofilm by introducing new habits to patients—added to the correction of malocclusion and esthetic improvement. In the oral cavity, the Streptococcus mutans species is usually considered the main cause of enamel demineralization. Candida albicans is another frequent microorganism in the oral cavity, and it may or may not be associated with pathologies. Both microorganisms are found in the presence of fixed orthodontic appliances. ,
Although the mechanical control of plaque is an effective strategy to prevent the progression of periodontal diseases, most people do not brush their teeth correctly and do not use dental floss. The daily use of mouthwash is usually considered a simple and effective strategy. In this context, patients with fixed orthodontic appliances who have difficulties in effectively controlling biofilm through mechanical methods require support for controlling inflammation and improving oral health using chemical control. , The chlorhexidine mouthwash can reduce around 90% of microorganisms. The main advantage is the extensive antimicrobial spectrum, with prolonged and continuous effect. Thus, it is considered the gold standard in research. , However, the prolonged use of chlorhexidine may cause side effects, , which limits and contraindicates its application in the long term. Thus, a potential supporting antimicrobial alternative with minimized side effects would be greatly valued to work on oral infections.
In this sense, phytotherapeutic products might be used as a complementary alternative for preventing and treating oral diseases. , Among the numerous existing plants used as supplementary therapy is ginger, which scientific name is Zingiber officinale . It has been used in the oral cavity through solutions and sprays because of its healing and anti-inflammatory actions —presenting activity against S mutans and C albicans as well as antioxidant and anticancer activities. It was verified that Z officinale oils and extracts inhibit Gram-positive and Gram-negative bacteria, but there are still few studies showing the effect of ginger on the microorganisms prevalent in the oral cavity.
Currently, there is an increasing search for orthodontic treatments to correct malocclusions while helping to control plaque at the same time. Therefore, finding an efficient phytotherapeutic agent for these patients becomes a major discovery. Thus, this study aimed to assess the anti-inflammatory and antimicrobial effects of mouthwashes with 0.12% chlorhexidine and 0.5% Z officinale essential oil (ZOEO) on patients with fixed orthodontic appliances.
Material and methods
The Research Ethics Committee of the University Center of Hermínio Ometto Foundation approved this double-blind clinical and prospective study (CAAE: 60778016.4.0000.5385; opinion no. 1.917.998). Initially, the essential oil was analyzed using gas chromatography-mass spectrometry (GC-MS) (Department of Organic Chemistry and Pharmaceutics of the Pluridisciplinary Center of Chemical, Biological, and Agricultural Research of University of Campinas) and later the mouthwashes were prepared (Laboratory of Microbiological Quality Control of the School of Pharmacy of University Center of Hermínio Ometto Foundation). Figure 1 presents the flowchart of the experimental design.
The ZOEO were acquired from the LASZLO company (Belo Horizonte, Minas Gerais, Brazil). The GC-MS analyses of the essential oil were performed according to the method modified and described by Markham et al and Proestos et al.
Ginger was used at a concentration of 0.5% according to the literature, and it presented solubility in the tensioactive mixture used, allowing the development of its solution, as observed in Table I . The manipulated formula was subjected to preliminary assessment on physical and chemical stability for the acceptance parameters and criteria previously defined.
| Components | Concentration (%) | Function |
|---|---|---|
| Sodium lauryl sulfate | 1 | Anionic tensioactive |
| Sorbitol | 20 | Sweetener and humectant |
| Polysorbate 80 | 1.2 | Tensioactive |
| Z officinale essential oil | 0.5 | Active ingredient |
| Saccharin | 0.5 | Sweetener |
| Distilled water qsp | 100 | Medium |
The in vitro cell viability of ZOEO was performed according to the method by Krishnan et al and analyzed using generalized linear models. This cell viability analysis showed that, in the experimental time of 24 hours, chlorhexidine was the substance with the lowest cell viability. The same cell viability of the essential oil, compared with chlorhexidine, was observed in the experimental time of 48 hours ( Fig 2 ).
The development and chemical and cytotoxic analyses of the ZOEO mouthwash allowed for the feasibility of the clinical study, as shown by the similarity in the selective toxicity of the solutions tested. This double-blind clinical study included 31 subjects of both sexes (17 women and 14 men) with an average age of 19.96 years (minimum age of 12 years and a maximum age of 35 years). These patients were under orthodontic treatment for at least 6 months. Each subject was their own control. The size of the sample (n = 31) was calculated using a pilot study with a power of 0.80, at the 0.05 significance level, and medium effect size. To satisfy the inclusion criteria, the subjects should use fixed, preadjusted, and conventional orthodontic appliances (Abzil, 3M do Brasil Ltda, São José do Rio Preto, São Paulo, Brazil) installed in both arches from the second premolar to the second premolar; and present good general health with low risk of periodontal disease assessed with the Periodontal Screening Record index. This index assessed regions from maxillary and mandibular right premolars to the left premolars, using a WHO 621 mm periodontal probe (Trinity Indústria e Comércio Ltda, São Paulo, Brazil) recommended by the World Health Organization. The exclusion criteria were the use of antibiotic therapy over the last 6 months, the use of anti-inflammatories over the last 3 months, smokers, and pregnant women.
Initially, all subjects of the study received an oral hygiene kit containing 1 toothbrush (Colgate Twister; Colgate-Palmolive Company, São Bernardo do Campo, São Paulo, Brazil) and Tandy toothpaste (Colgate-Palmolive Company) for having controlled concentrations of 1100 ppm sodium fluoride. The patients did not receive any type of additional instruction on oral hygiene. The mouthwashes analyzed were 0.12% chlorhexidine, 0.5% ZOEO, and placebo (flavored distilled water). The clinician and the participants involved in the study did not receive information on the products, which were visually similar and identified as mouthwash A, mouthwash B, and mouthwash C, respectively.
Before each mouthwash, 1 mL of unstimulated saliva was collected from each subject. They spat inside a previously sterilized cryotube (Kasvi MP e Distribuidora de produtos para laboratório Ltda, Curitiba, Paraná, Brazil), previously sterilized, containing 1 mL of 20% glycerol solution and sterile distilled water. Next, the participants were instructed to use 10 mL of the mouthwash for 60 seconds. After 1 minute and 15 minutes of using the mouthwash, an additional 1 mL of unstimulated saliva was collected. The solutions were selected randomly, and the patients used the same mouthwash in the same period. All participants repeated the mouthwash with each solution analyzed daily for 1 week. On the seventh day, the subjects would return for a new saliva collection. After a 15-day interval, the patients initiated the use of another mouthwash. , , The collected saliva was frozen for posterior microbiologic analyses.
The periodontal clinical assessment was also performed on the first and seventh days of each mouthwash use. One single calibrated examiner disclosed the biofilm using the method proposed by Ciancio that was modified by Elias et al and used by Santamaria Jr et al. Scores were used to determine the clinical biofilm disclosure (0 = no plaque; 1 = discontinuous points of the gingival plaque or plaque only in the bracket; 2 = continuous plate line less than 1 mm wide or plate only around the bracket without gingival extension; 3 = continuous plaque line near the gingiva, covering more than 1 mm; 4 = plaque covering one-third or more of the tooth surface; 5 = plaque covering more than two-thirds of tooth surface). For each patient, the average will be calculated by summing the index of each tooth divided by the number of teeth used. The gingival bleeding index was also part of the periodontal assessment of all subjects, including the maxillary and mandibular right premolars up to the left premolars, and the assessment was performed with a WHO 621 mm periodontal probe. The presence or absence of bleeding was verified and noted.
For the qualitative and sensory assessments of the mouthwashes, each subject in the study received a questionnaire on the flavor of the solutions tested, according to the method proposed by Nogueira et al in which the scores were written down for every flavor sensation of each solution on the first and seventh days. After obtaining scores, these values were categorized and distributed on a new scale.
The microbiologic analysis was performed after thawing the samples. All the materials used, which contained the samples, were specified before performing the procedures. The manipulation of samples for dilutions and seeding was performed inside the laminar flow chamber in a sterile environment free of potential contamination. For each saliva collection, serial dilutions were performed in sterile saline solution. Then, triplicate seedings of independent experiments were performed in disposable Petri dishes of 90 × 15 mm (Cral; Cralplast, Cotia, São Paulo, Brazil) containing 2 selective mediums: Brain Heart Infusion Agar for growing S mutans and Sabouraud Dextrose Agar for growing C albicans . All dishes were incubated in a 37°C stove in microaerophilia for S mutans and aerobiosis for C albicans for 48 hours, and the total number of colony-forming units per milliliter (CFU/mL) was expressed in decimal logarithm (log 10 CFU/mL).
Statistical analysis
Initially, a descriptive analysis of the GC-MS analysis of the ZOEO was performed. Considering the data of the study presented asymmetrical distribution, the comparisons between mouthwashes and times required the adjustment of generalized linear models according to a repeated measures design using the GENMOD procedure, for the variable of bleeding index. The data on CFUs, biofilm index proposed by Ciancio, and flavor did not allow adjusting to a model because of the distribution of data, whereas Friedman and Wilcoxon nonparametric tests were used. One-way analysis of variance and Tukey posttest were used to assess in vitro cell viability. The analyses were performed using SAS (version 9.3; SAS Institute Inc, Cary, NC) and Bioestat (version 5.0; Mamirauá Maintainable Development Institute, Belém, Pará, Brazil) software. A 5% significance level was used for all tests.
Results
The major chemical compounds presented by the GC-MS analysis of the ZOEO were α-zingiberene, β-sesquiphellandrene, ar-curcumene, and β-bisabolene ( Table II ). Among the compounds listed, α-zingiberene is the most expressive, comprising approximately 37.44% of the sample studied.
