Archives of Oral Biology 109 (2020) 104577

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Archives of Oral Biology

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Cytotoxicity and antimicrobial effects of citronella oil (Cymbopogon nardus) T

and commercial mouthwashes on S. aureus and C. albicans biofilms in
prosthetic materials
Bruno Guandalini Cunhaa, Cristiane Duqueb, Karina Sampaio Caiaffab, Loiane Massunarib,
Isabela Araguê Catanozea, Daniela Micheline dos Santosa, Sandra Helena Penha de Oliveirac,
⁎
Aimée Maria Guiottia,
a
Department Dental Materials and Prosthodontics, São Paulo State University (UNESP), School of Dentistry, Araçatuba, Brazil
b
Department of Pediatric and Public Health, São Paulo State University (UNESP), School of Dentistry, Araçatuba, Brazil
c
Department of Basic Sciences, Immunopharmacology Laboratory, São Paulo State University (UNESP), School of Dentistry, Araçatuba, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Although the effectiveness of some mouthwashes has been proven, phytotherapy is still a field to be explored as
Cymbopogon an alternative to commercial products. Objective: To evaluate, in vitro, the cytotoxicity and efficacy of two
Biofilms solutions based on citronella oil (CN), on S. aureus and C. albicans biofilms (in formation–adhesion phase and
Acrylic resins 24 h-biofilm formation) on acrylic resin and nickel-chromium alloy samples (one trademark of each material),
Candida albicans
compared to two alcohol-free commercial mouthwashes. Material and Methods: Two solutions containing CN at
Staphylococcus aureus
concentrations of 5x and 10x the minimum bactericidal/fungicidal concentration (MBC/MFC) were prepared by
microdilution. After contamination of the samples surfaces with these microorganisms, the mouthwashes (CN -
5x and 10x; CHX - 0,12% alcohol-free chlorhexidine and LT - alcohol-free essential oils) were evaluated.
Mouthwash simulation was performed for 1 min at two moments, the first simulation after 4 h of microbial
adhesion and 24 h-biofilm formation, and the second simulation, 6 h after the first simulation. For biofilm
quantification, the number of cultured cells was evaluated by CFUs. The cytotoxicity assay was performed on
HaCat epithelial cells and quantified by the MTT method. Results: Tested solutions completely inhibited the
growth of both microorganisms in the adhesion phase. All solutions showed inhibitory activity against 24 h-
biofilm formation. However, CN led to greater microbial reduction, regardless of the surface of the sample. All
solutions demonstrated a toxic effect. However, after serial dilution, CN presented the lowest cytotoxic effect.
Conclusion: Citronella had a lower cytotoxic effect and a higher action compared to commercial solutions.

1. Introduction disease, among others, and dentures offer a reservoir for microorgan-
isms associated with these infections (Carmona, Diz Dios, & Scully,
The implant-supported fixed complete dental prosthesis has become 2002). An effective oral hygiene regimen is important to control den-
the first treatment option for rehabilitation of edentulous patients. On ture plaque biofilm and contributes to the control of associated oral and
the other hand, it is a fact that the surface of the materials that make up systemic diseases. The microbiology of denture plaque has received
this type of prosthesis constitute niches for microorganisms, which, little attention in comparison with dental plaque, yet it differs in lo-
consequently, may lead to the colonization and formation of biofilms, cation and composition. Denture plaque and poor denture hygiene is
these being the etiological agents of diseases such as candidiasis, mu- associated with stomatitis (Candida infection), may also serve as a re-
cositis, peri-implantitis, and the consequent loss of implants (Lang, servoir of potentially infectious pathogens, and may contribute to oral
Wilson, & Corbet, 2000). Oral bacteria have been implicated in sys- malodour and to caries and periodontitis in people who have remaining
temic complication too, like bacterial endocarditis, aspiration pneu- natural teeth (Coulthwaite & Verran, 2007). In the human oral cavity,
monia, gastrointestinal infection and chronic obstructive pulmonary there are more than 750 different species of microorganisms living

⁎
Corresponding author at: Department of Dental Materials and Prosthodontics, São Paulo State University (UNESP), School of Dentistry, Araçatuba, José Bonifácio,
1153, Araçatuba, São Paulo, 16015-050, Brazil.
E-mail address: aimee@foa.unesp.br (A.M. Guiotti).

https://doi.org/10.1016/j.archoralbio.2019.104577
Received 31 May 2019; Received in revised form 20 September 2019; Accepted 25 September 2019
0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
B. Guandalini Cunha, et al. Archives of Oral Biology 109 (2020) 104577

together within dental plaque (Lang et al., 2000). treatment (Bunte, Hensel, & Beikler, 2019; Li et al., 2019; Moon, Kim, &
Candida albicans and Staphylococcus aureus are versatile and dan- Cha, 2011). Essential oils contain a complex mixture of odorous and
gerous pathogens under certain patient systemic conditions, with both volatile compounds from secondary plant metabolism, and are widely
polymer and metal adhesion (Salerno et al., 2011; Veyries, Faurisson, used in cosmetics as fragrance components, in the food industry as
Joly-Guillou, & Rouveix, 2000). Oral mycoses, mostly affecting mu- flavoring additives and in a variety of household products as scenting
cosae, are mainly caused by the opportunistic pathogen Candida albi- agents (Moon et al., 2011). The essential oils of many different plants
cans. They become relevant in denture wearers elderly people, in dia- have been previously tested in both in vitro and in vivo studies, as
betic patients, and in immunocompromised individuals. Differently, promising agents in the treatment of oral diseases and other infections
bacteria are responsible for other pathologies, such as dental caries, (Cha et al., 2005, 2007; Haffajee et al., 2009).
gingivitis and periodontitis, which affect even immune-competent in- The plant known as citronella (Cymbopogon nardus) is used to obtain
dividuals (Ardizzoni et al., 2018). The oral cavity should be considered an oil which contains the chemical components, citronellal, geraniol
a source of S. aureus in terms of cross-infection and dissemination to and citronellol, considered antiseptic (Nakahara, Alzoreky, Yoshihashi,
other body sites (McCormack et al., 2015; Petti & Polimeni, 2011). Nguyen, & Trakoontivakorn, 2003; Pereira et al., 2015; Singh, Fatima,
Reported isolation rates for Staphylococcus aureus vary with the popu- & Hameed, 2016). Studies have demonstrated its effectiveness in both
lation studied, with an incidence of 48% among the denture-wearing isolated and synergistic use, with good antimicrobial efficacy (Guiotti,
population (Tawara, Honma, & Naito, 1996). In addition, a number of Goiato et al., 2016, 2016b; Nakahara et al., 2003). In this way, ci-
distinct oral infections (eg, angular cheilitis, parotitis, staphylococcal tronella oil can be considered a phytotherapeutic potential for anti-
mucositis) are caused by this microorganism. More recently, it has also bacterial and antifungal action, opening new perspectives for the con-
been suggested that S. aureus may have a role in dental implant failure trol of oral biofilm. To date, the literature does not contain results of
(Kronström, Svenson, Hellman, & Persson, 2001; Rokadiya & Malden, citronella oil used as a mouthwash. Citronella essential oil was shown
2008). Numerous epidemiologic studies shown that 14%–20% of the to be a viable active phytocomplex for the formulation of a new
cases of bacterial endocarditis are associated with a possible oral origin mouthwash.
(Manford, Matharu, & Farrington, 1992; Sandre & Shafran, 1996; Therefore, the aim of this study was to evaluate, in vitro, the cy-
Sekido, Takano, Takayama, & Hayakama, 1999). The presence of Sta- totoxicity and efficacy of two citronella oil (CN) based solutions on
phylococcus species has considerably increased in the majority of bac- monospecies biofilms (in formation and formed) of S. aureus and C.
terial endocarditis over five decades (Slipczuk et al., 2013). S. aureus is albicans on samples of a thermally activated acrylic resin (TAAR) and a
especially virulent because, in addition to having a platelet-aggregating nickel-chromium alloy (NCA), compared to two alcohol-free commer-
action, it can adhere to specific receptors on the endothelium of healthy cial mouthwashes.
valves (Yeaman & Bayer, 2000). It is a skin and nasal commensal and a
nosocomial pathogen. It has also been identified in the oral cavities of 2. Material and methods
older patients with periodontitis (Jacobson, Patel, Asher, Woolliscroft,
& Schaberg, 1997; Owen, 1994; Younessi, Walker, Ellis, & Dwyer, 2.1. Materials
1998) and in relation to systemic disorders such as rheumatoid arthritis
associated with xerostomia. For this reason, S. aureus has been sug- The following products were evaluated in this study: two commer-
gested as being responsible for some bacterial endocarditis of oral cial mouthwashes - 0,12% alcohol-free chlorhexidine– CHX, Periogard,
origin (Jacobson et al., 1997). Colgate Palmolive Company, São Paulo, SP, Brazil; and alcohol-free
The oral hygiene required for patients with this type of prosthesis essential oils– LT, Listerine Zero Johnson & Johnson, São Paulo, SP,
should be performed through mechanical cleaning techniques and oral Brazil) and one phytotherapeutic product - citronella oil (Aphoticário,
mouthwashes, such as 0.12% chlorhexidine solution, minimizing the Araçatuba, SP, Brazil).
proliferation of microorganisms (da Costa, Amaral, Barbirato, Leão, &
Fogacci, 2017). However, the use of chlorhexidine must be indicated 2.2. Analysis of constituents of C. nardus essential oil
with caution, since it presents adverse effects when used in the long
term (James et al., 2017; Wyganowska-Swiatkowska et al., 2016). The The qualitative analysis of the chemical composition of the essential
presence of the alcohol on the formulation of gluconate chlorhexidine oil was performed by gas chromatography–mass spectroscopy (GC–MS),
mouthwashes does not seem to interfere with their antimicrobial po- in a Shimadzu/GC2010; GCMS-QP2010 Plus instrument (Shimadzu,
tential and with their taste perception (Cantarelli et al., 2017), but for Kyoto, Japan). The sample was diluted 50 times with hexane and 1 μL
another widely used commercial mouthwash without alcohol which is of the sample was injected into a column. A fused silica capillary
composed of essential oils (eucalyptol, menthol, thymol, methyl sali- column (Restek Rxi®-5 ms 5% diphenyl/95% dimethyl polysiloxane,
cylate - Listerine Zero Johnson & Johnson), the alcohol-free version still with 30 m lenght x 0,25 mm thickness and 0.25 μm diameter) was used.
deserves attention due to the lack of scientific evidence about their The mass transfer line temperature and the source were kept at 280 ◦C,
potential to inhibit microorganisms associated with oral diseases and 230◦C, respectively. Helium was the carrier gas and a split ratio of
(Vlachojannis, Al-Ahmad, Hellwig, & Chrubasik, 2016). In an update on 1/50 was used. The compounds of the test solution were identified by
the efficacy and safety for Listerine® products, the evidence of effec- analysis of the fragments patterns displayed in the mass spectra, and
tiveness, based on the bulk of three confirmatory studies and numerous their identities were confirmed by comparing their mass spectra with
exploratory studies carried out so far, is strong for Listerine with al- those present in the database (internal library, Wiley 8 e FFNSC 1.3)
cohol, but only moderate for Listerine® Zero. Three of the four 6-month (Kandimalla et al., 2016).
studies were of sound confirmatory design. Two of these investigated
Listerine® and only one investigated Listerine Zero® (Vlachojannis 2.3. Microbial strains
et al., 2016).
Thus, considering the necessity to search for new mouthwashes to The antibiofilm activity of two commercial mouthwashes (0,12%
aid in biofilm control, aiming at effectiveness with the minimum of alcohol-free chlorhexidine– CHX, Periogard, Colgate Palmolive
undesirable effects, phytotherapy becomes an alternative to be studied Company, São Paulo, SP, Brazil; and alcohol-free essential oils– LT,
(Li, Jiang, Hao, Zhang, & Huang, 2019; Rodrigues, Rodrigues, & Listerine Zero Johnson & Johnson, São Paulo, SP, Brazil) and citronella
Henriques, 2019). Essential oils and plant-derived polyphenols with oil (citronellal concentration = 70%; relative density = 0,862 g/ml;
antimicrobial and immunomodulatory characteristics appear to provide Aphoticário, Araçatuba, SP, Brazil) based mouthwashes was analyzed
a variety of oral health benefits and therapeutic effect in cancer in standard microbial species, provided by the Oswaldo Cruz

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B. Guandalini Cunha, et al. Archives of Oral Biology 109 (2020) 104577

Foundation – FIOCRUZ (Rio de Janeiro, RJ, Brazil): Staphylococcus CCAT. Sterile saline solution (Samtec, Ribeirão Preto, SP, Brazil) was
aureus (ATCC 14458) and Candida albicans (ATCC 26790). used for the negative control.
For the biofilm tests, 200 samples of TAAR (VipiCril Plus; VIPI
2.4. Growth condition Dental Products, Pirassununga, SP, Brazil) were constructed using a
3 mm thick cast metal matrix, with dimensions of 10 mm in diameter,
In order to obtain the microbial cultures, these species were re- according to the manufacturer's specifications, and 200 samples of NCA
activated and cultured in Mannitol Salt Agar (MAS; Difco Laboratories, (Ligga N; VIPI Dental Products) were constructed using “the lost wax
Kansas City, MO, USA) for S. aureus and Sabouraud Dextrose Agar technique”, method in which the molten metal is poured into a mold
(DAS; Difco Laboratories) for C. albicans, incubated aerobically at 37 °C that has been created by means of a wax model (Anusavice, Shen, &
for 24 h (Binder GmbH; Tuttlingen, Germany). Next, they were trans- Rawls, 2013). For this, wax patterns (Rainbow branco opaca RW-02;
ferred to Brain Heart Infusion broth (BHI; Difco Laboratories) supple- Ceras Rainbow Ltda, Porto Ferreira, SP, Brasil) were made using a
mented with 1% glucose (BHI +; Sigma-Aldrich, St. Louis, Missouri, custom-made acrylic matrix with holes 6 mm in diameter and 3 mm
EUA) for S. aureus and Sabouraud Dextrose Broth (SDB; Difco thick. These circular wax patterns were incorporated in phosphate
Laboratories) for C. albicans, incubated at 37 °C for 18–24 h before the coating (VIPI Dental Products) inside a silicone ring. The samples of
day of the experiment, aerobically and under agitation (orbital agitator TAAR and NCA were then subjected to polishing with metallographic
- TE-420, Tecnal; Piracicaba, SP, Brazil), with luminosity and tem- sandpapers (Buehler, Lake Bluff, IL, EUA) and the edges regularized
perature control (Kern & Blevins, 1997). For the standardization of with discs and rubbers (Andreotti et al., 2014). Samples were stan-
microbiological assays, from overnight cultures, new cultures were dardized to 2.5–2.6 mm thickness for TAAR and 2.8–2.9 mm for NCA,
standardized until reaching an optical density (OD) of 0.5 for S. aureus with the aid of a digital caliper (Mitutoyo Sul Americana Ltda, São
(approximately 1–5 × 108 CFU/mL) and 0.3 for C. albicans (approxi- Paulo, SP, Brazil). Finally, the samples were submitted to an ultrasonic
mately1–5 × 106 CFU/mL), measured in a spectrophotometer (Eon bath (Ultracleaner 1400; UNIQUE, Indaiatuba, SP, Brasil) for 20 min.
Microplate, BioTek; Winooski, Vermont, EUA), considering the absor- The samples were then positioned and glued in the center of the
bance of 550 nm. All the subsequents assays were performed in tripli- well bottom of 24-well plates with the polished surface facing upward
cate in two different days (Microbiological and Cytotoxicity assays). (Guiotti, Goiato et al., 2016, 2016b). These plates were packaged and
sterilized with ethylene oxide (Oximed, São José do Rio Preto, SP,
2.5. Determination of minimum inhibitory concentration (MIC) Brazil). The distribution of samples for each material (n = 200) in their
respective groups is shown in Fig. 1.
The minimum inhibitory concentration (MIC) and minimum bac-
tericidal/fungicidal concentration (MBC/MFC) were obtained by the 2.6. Biofilm assays
microdilution method using sterile 96-well microplates (Kasvi; Curitiba,
PR, Brasil) based on the National Committee for Clinical Laboratory According to Fig. 1 and Table 1, each sample (TAAR or NCA) re-
(NCCL) criteria for bacteria and fungi, and modifications proposed by ceived only one mouthwash, totalizing 200 samples of each material,
Mor et al (CLSI - Clinical & Laboratory Standard Institute, 2008; CLSI & which were divided into 5 different groups (n = 10) of solutions, for
Clinical & Laboratory Standard Institute, 2012; Mor, Hani, & Nicolas, each microorganism separately.
1994).
The CN oil was then filtered on a 0.22 μm (Kasvi) filter to ensure 2.7. Biofilm in formation (adhesion phase)
sterility, and serial dilution was performed in a water-soluble vehicle
(sterile deionized water) and a liposoluble (caprylic capric acid trigly- For the biofilm assay, 1 mL of artificial saliva was added to the wells
ceride [CCAT]) vehicle, in order to test which vehicle promoted greater of a 24-well plate to form the salivary film on the TAAR and NCA
antimicrobial effectiveness of the oil (Traul, Driedger, Ingle, & Nakhasi, samples, in order to facilitate microbial adhesion. After 2 h, the saliva
2000). was removed and 1 mL of the culture medium (SDB or 1% glucose BHI,
The final diluted concentrations of pipetted microbial suspensions according to the microorganism) was added and 2 μL per well of the
were 1–5 × 105 CFU/mL for S. aureus and 1–5 × 103 CFU/mL for C. culture of previously standardized C. albicans or S. aureus, dilution of
albicans (Caiaffa et al., 2017). Serially diluted 2% chlorhexidine di- 10-1000x of culture in OD (0.3 and 0.5 respectively). The plates were
gluconate was used as a positive control; and cultures without anti- then placed on an orbital shaker at 37 °C at 120 rpm to represent the
microbial agents as negative control. In this way, it was possible to fluid dynamics in the buccal medium (Monteiro et al., 2011).
obtain concentrations of citronella oil ranging from 13.76% to 0,006% After 4 h of growth, the group of biofilm in formation (adhesion
for citronellal; 12.5% to 0.006% for citronellol; and 10.9% to 0.005% phase) underwent the first mouthwash simulation, i.e., the culture was
for nerol. After inoculation and the incubation period, 0.02% resazurin carefully removed, and 1 mL of the selected mouthwash (CN 5x and
(R7017; Sigma-Aldrich) was applied in each well for 4 h to determine 10x, CHX, and LT) was added. The solutions were kept in contact with
cell viability (Caiaffa et al., 2017; Hahnel et al., 2012). After incuba- the samples for 1 min, without stirring. The solutions were then re-
tion, the last blue well (MIC) and at least two previous wells were se- moved and the biofilm carefully washed with sterile 0.9% saline.
rially diluted, plated, and incubated for 20–24 h under the same con- Subsequently, each well was filled with 1 mL of 1% glucose BHI or SDB,
ditions (Caiaffa et al., 2017). After counting in a stereoscopic and the samples were maintained for another 6 h in an orbital shaker
magnifying glass (CP602, Phoenix Luferco; Araraquara, SP, Brasil), the with luminosity and temperature controlled at 37 °C. After this period, a
number of colonies (CFU/mL) was determined. MBC/MFC was con- new mouthwash simulation was performed under the same conditions.
sidered when antimicrobial agents eliminated 99.9% of the microbial
strains tested. All experiments were performed in triplicate. 2.8. 24 h-biofilm formation
For the biofilm assay, two alcohol-free commercial mouthwashes
and two CN-based mouthwashes were selected (Table 1): 0,12% al- In the formed biofilm group, for both microorganisms, the plates
cohol-free chlorhexidine (CHX) and alcohol-free essential oils (LT). For were incubated initially for 24 h, at which time the first mouthwash
the phytotherapics two different concentrations were formulated, simulation was performed. Next, after another 6 h of incubation, the
multiplying 5x MBC/MFC and 10x MBC/MFC of sterile CN oil. Next, the second mouthwash simulation was performed.
calculated amount was diluted in sterile deionized water as this vehicle After the final simulation, samples were transferred to a sterile tube
was the most effective to promote the antimicrobial effect of CN oil, containing 1 mL of sterile 0.9% saline, and submitted to ultrasound for
requiring less concentrated dilutions to reach MBC/MFC compared to 10 min, followed by vortexing (AP 56, Phoenix Luferco; Araraquara, SP,

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B. Guandalini Cunha, et al. Archives of Oral Biology 109 (2020) 104577

Table 1
Mouthwashes tested microbiologically and cytotoxically.
GROUPS MOUTHWASHES MANUFACTURER

Control group Sterile 0,9% saline solution Samtec

Cymbopogom nardus based solution (5x MBC / Citronella oil Pharmacy Apothicário
MFC of each micro-organism – CN 5x
MBC/MFC)
Cymbopogom nardus based solution (10x MBC Citronella oil Pharmacy Apothicário
/ MFC of each micro-organism – CN 10x
MBC/MFC)
CHX Chlorhexidine gluconate 0.12% (digluconate formulated to a chlorhexidine- Periogard alcohol-free, Colgate Palmolive
free base at a concentration of 0.067%) and the following inactive Company
components: water, glycerine, propylene glycol, sorbitol, PEG-40
hydrogenated castor oil, cetylpyridinium chloride, citric acid, CI 42,090.
LT Water, thymol, menthol, methylsalicylate, sorbitol, eucalyptol, aroma (d- Listerine Zero, Johnson & Johnson of Brazil -
Limonene), sodium lauryl sulfate, Poloxamer 407, sucralose, benzoic acid, Industry and Commerce of Products for Health
sodium saccharin, sodium benzoate, propylene glycol, CI 42053. Ltda

at 570 nm in a spectrophotometer (Eon Microplate; BioTek, Winooski,

Vermont, EUA). The negative control (DMEM) was defined as 100% cell
viability (Kreling et al., 2016).

2.10. Statistical analysis

The microbial count data in CFU/mL were transformed into

log10+1 (CFU/mL) due to the amplitude of numerical data and sub-
mitted to the Kruskal-Wallis and Mann-Whitney tests. For the cell via-
bility assays, the data were submitted to two-way ANOVA tests, fol-
lowed by the Tukey test (α = .05). SPSS Software (v17.0; IBM Corp)
was used to perform these analyzes.

Fig. 1. Study design diagram. 3. Results

3.1. Chemical components of C. nardus essential oil (EO-CN)

Brasil) for 1 min (de Foggi et al., 2016). Subsequently, this solution was
serially diluted and inoculated in duplicate into MSA and SDA plates.
A total of 16 compounds were identified from GC–MS analysis of CN
The plates were incubated for 20–24 hours and the number of CFU/mL
essential oil and the main compounds were citronellal (27,53%), ci-
counted (Li, Liu, & Xu, 2012).
tronellol (25%) and nerol (21,89%). Chemical composition and per-
centages of compounds from EO-CN were shown in Table 2 and Fig. 5.
2.9. Citotoxicity assays
3.2. Microbiological analysis
For cytotoxicity analysis of the commercial and citronella oil based
mouthwashes, cell viability tests were performed using HaCaT epithe- Table 3 shows the MIC and MBC/MFC values (in %) for the CN oil
lial cells. HaCaT cells were grown in Dulbecco's modified Eagle's against S. aureus and C. albicans. Higher values of MIC and MBC/MFC
medium (DMEM; Gibco BRL, Carlsbad, CA, EUA) containing 10% fetal were obtained for S. aureus. Regardless of the surface tested, all
calf serum and 100 μg mL -1 penicillin G/streptomycin. The cells were
cultured at 37 °C in 5% CO2 until reaching confluence (Bedran, Mayer, Table 2
Spolidorio, & Grenier, 2014). - GC–MS analysis of EO-CN showing chemical compounds with percentages.
Epithelial cells were detached after trypsin-EDTA (Life Technologies
# Pico RT (min) Chemical compound RI Percentage (%)
Inc.; Waltham, MA, USA) treatment at 37 °C for 5 min. Then 3x the
DMEM volume was added and the cell culture was collected in tubes 1 11,80 Limonene 1029 3,50
and centrifuged at 500xg for 5 min. Subsequently, the cells were re- 2 14,34 Linalool 1100 0,69
suspended in fresh DMEM medium, counted in a Newbauer chamber 3 16,04 Isopulegol 1147 0,37
4 16,31 Citronellal 1154 27,53
and distributed in a 96-well microplate (200 μl/well, 1 × 105 cells/mL), 5 18,98 Citronellol 1230 25,00
incubated at 37 °C in 5% CO2. After 24 h, the culture medium was re- 6 19,41 Neral 1242 0,50
moved and the amount of 200 μL of each mouthwash solution (CN oil, 7 19,90 Nerol 1256 21,89
CHX and LT), serially diluted in DMEM (50 to 1.56%), applied in the 8 20,42 Geranial 1271 0,67
9 23,18 Citronellyl acetate 1353 5,75
wells. CN oil at 10x MBC/MFC (4.3% citronellal, 3.9% citronellol and
10 23,37 Eugenol 1359 0,50
3.4% nerol) was chosen based on microbiological results to run the 11 24,17 Geranyl acetate 1384 4,53
cytotoxicity assays. The mouthwashes remained in contact with the 12 24,58 Elemene < beta- > 1396 0,87
cells for 1 min at 37 °C in 5% CO2. After exposure to the mouthwashes, 13 27,44 Germacrene D 1488 3,14
the media from each well were aspirated and replaced with 180 μL of 14 27,99 Muurolene < alpha- > 1505 0,49
15 28,68 Cadinene < delta- > 1529 2,78
DMEM added to 20 μL of MTT solution (5 mg/mL sterile PBS; Sigma 16 29,44 Elemol < alpha- > 1554 1,79
Aldrich), and the cells were incubated at 37 °C in 5% CO2 for 4 h,
protected from light (Kreling, Aida, Massunari, Caiaffa, & Percinoto, RI: Retention indice calculated based on C7-C30 alkane series/ RT: Retention
2016). The solution was transferred to a 96-well plate to measure D.O. time in minutes.

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Table 3 differed statistically from each other (p = 0.05) only for the acrylic
- MIC and MBC / MFC values obtained for the main compounds of citronella oil resin group.
(CN). For C. albicans biofilm, the lowest microbial counts were observed
MAIN COMPOUNDS MIC (%) MBC/MFC (%) 10x MBC/MFC after exposure to the CN 10x MFC, but did not differ statistically from
CN 5x MFC, regardless of the surface (resin, p = 0.109; metal,
C. albicans Citronellal 0.43 0.43 4.3 p = 0.364; Fig. 3). In Table 3, the reduction of 34.78% and 32.46% in
Citronellol 0.39 0.39 3.9
number of C. albicans cells on the surface of the resin and metal sam-
Nerol 0.34 0.34 3.4
ples, respectively, can also be observed using the CN 5x MFC solution.
S. aureus Citronellal 0.86 0.86 8.6
For the CN 10x MFC solution, there was a reduction of 46.72% and
Citronellol 0.78 0.78 7.8
Nerol 0.68 0.68 6.8 30.02%, respectively for the resin and metal samples. The CHX and LT
presented lower antibiofilm activity compared to CN, regardless of
Note: MIC – Minimal inhibitory concentration, MBC –Minimum bactericidal concentration.
concentration, MFC – Minimal fungicidal concentration.

3.3. Cytotoxicity results

solutions tested in this study, CN 5x MBC/MFC, CN 10x MBC/MFC,
CHX, and LT prevented the formation of S. aureus and C. albicans bio-
All solutions diluted at 50 and 25% caused cellular toxicity (Fig. 4).
films, when applied at the beginning of their formation (4 h of adhe-
At 12.5% only the CN solution allowed cell growth by 40%. When the
sion). In the control group (Sterile 0,9% NaCl solution), the mean
solutions were diluted at 6.25%, the rate of cell viability in the presence
(standard deviation) of S. aureus growth on resin and metal surfaces (in
of CN and LT were higher when compared to CHX. From 3.12% dilu-
log) was 4.77 (0.86) and 5.22 (0.33), respectively; and for C. albicans
tion, all solutions allowed more than 70% cell viability. The higher the
was 3.83 (0.43) and 3.74 (0.67), respectively.
dilution, the greater the cell viability in the presence of all solutions.
Fig. 2 shows the results for 24 h-biofilm formation of S. aureus and
Considering the concentrations of active principles of each tested
Fig. 3 for 24 h-biofilm formation of C. albicans. All solutions differed
mouthwash solution, as shown in Table 5, CN had the lowest cytotoxic
from controls, on both materials, demonstrating the activity on S.
effect. This data can be observed, for example, at the 3.12% dilution,
aureus and C. albicans biofilms.
and the concentrations of CN components (0.13% for citronellal, 0.12%
Considering the phytotherapeutic solutions for S. aureus (Fig. 2), CN
for citronellol and 0.11% for nerol) was higher than CHX (0.00375%)
5x MBC and CN 10x MBC presented the best results, not statistically
and LT components (0.00187% for thymol, 0.0025% for eucalyptol,
different from each other, independent of the material tested (resin,
0.0187% for methyl salicylate, and 0.0012% for menthol).
p = 0.971; metal, p = 0.114). These results can also be observed in
percentage of microbial reduction (Table 4). Compared to the control
group, the CN 5x MBC solution reduced the number of S. aureus cells on 4. Discussion
the surface of the resin and metal samples by 40.09% and 22.88%,
respectively. For the CN10x MBC solution, there was a reduction of Regarding the biofilms in adhesion phase, even without mechanical
38.19% and 36.79%, respectively for resin and metal. It was also ob- friction or agitation of the solution, all the commercial and phy-
served that both CN concentrations had a superior effect to the CHX and totherapeutic mouthwashes tested (both concentrations of 5x MBC/
LT solutions, regardless of the surface (p = 0.00). The CHX and LT MFC and 10x MBC/MFC) were effective, eliminating 100% of the bio-
film at the beginning of their formation, i.e., 4 h after initial adhesion.

Fig. 2. Box-plots graph of the activity of solutions on S. aureus formed biofilm.

Note: CN – Citronella, MBC – Minimum bactericidal concentration, CHX, LT. Different letters show difference between groups of solutions and tested surfaces, resin
or metal, according to Mann−Whitney test (p≤0.05).

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B. Guandalini Cunha, et al. Archives of Oral Biology 109 (2020) 104577

Fig. 3. Box-plots graph of the activity of solutions on C. albicans formed biofilm.

Note: CN – Citronella, MBC – Minimum fungicidal concentration, CHX, LT· Different letters show difference between groups of solutions and tested surfaces, resin or
metal, according to Mann−Whitney test (p≤0.05).

Table 4 effect. According to some studies, the chlorhexidine does not present
– Medians of microbial reduction (in percentage %) of 24 h-biofilm formation in good effectiveness in biofilms with a degree of organization, and this
relation to the control for S. aureus and C. albicans, in both material’s surfaces. can be explained by the fact that these biofilms have higher maturation,
Mouthwashes Microbial Reduction in % and consequently a higher extracellular matrix, making it difficult to for
these types of mouthwash to penetrate and act (Shen, Stojicic, &
S. aureus C. albicans Haapasalo, 2011).
Although the literature considers chlorhexidine solutions as the gold
Resin Metal Resin Metal
CN 5x MBC/MFC 40.09 Aa 22.88 Ab 34.78 Aa 32.46 Aa
standard in research, some studies have reported inferior or similar
CN 10x MBC/MFC 38.19 Aa 36.79 Aa 46.72 Aa 30.02 Aa effectiveness to the conventional form of LT, such as antimicrobial and
CHX 21.14 Ba 15.07 Ba 6.07 Ba 11.88 Ba antibiofilm effectiveness and control of calculus and gingivitis (Van
LT 13.81 Ba 10.65 Ba 5.39 Ba 11.29 Ba Leeuwen, Slot, & Van der Weijden, 2011). A study evaluating MIC and
MBC/MFC of the 4 essential oils contained in the formulation of LT
Note: Different upper letters show difference between the groups of solutions
(eucalyptol, menthol, thymol, methyl salicylate) showed that to offer
for each tested surface, resin or metal, according to the Mann-Whitney tests
(p < 0.05). Different lower letters show difference between the materials greater antimicrobial effectiveness, these active principles should be in
surfaces for each solution, according to the Mann-Whitney tests (p < 0.05). higher concentrations than those contained in the product
(Vlachojannis, Chrubasik-Hausmann, Hellwig, & Al-Ahmad, 2015).
The effectiveness of 100% reduction in the biofilm in adhesion phase The Cymbopogon nardus plant contains some active principles, called
can be explained by the low number of cells adhered, the weak binding monoterpenes (citronellal, citronellol, nerol, geraniol and others),
of the microorganisms to the substrate, and the low protection due to constituents of citronella oil. These principles have clearly demon-
the insufficient amount of the glycol polysaccharide matrix to minimize strated antifungal efficacy (Nakahara et al., 2003). It is reported that
the antimicrobial activity of the mouthwashes, causing greater contact the mechanism of action of these compounds occurs by increasing the
with the cell, breaking the cell wall, or inhibiting the enzyme actions of membrane fluidity and permeability of the microorganisms, inducing
microorganisms, as reported for LT (Busscher & van der Mei, 1997; cellular disturbances or lysis (Di Pasqua et al., 2007). A study con-
Mandel, 1994). In the case of CHX, damage occurs in the absorption ducted with Trichophyton rubrum, a dermatophyte fungus, showed that
barrier of the membrane, causing precipitation and coagulation of all geraniol and citronellol induced damage and loss of cytoplasmic
cytoplasmic content (da Silva et al., 2011). membrane integrity of these fungi (Pereira et al., 2015). Also in this
For the 24 h-biofilm formation the mouthwash solutions sig- same study, it was verified that these constituents alter the biosynthesis
nificantly reduced the number of CFU/mL. These results corroborate of ergosterol, an important regulator of the membrane fluidity of fungi
clinical and microbiological studies, which showed good results in the (Pereira et al., 2015). In other studies, the efficacy of geraniol and ci-
use of mouthwashes for biofilm control, showing statistically significant tronellal in C. albicans and other Candida species was evidenced
reduction in microorganisms when LT and CHX solutions were used, through the same mechanisms already mentioned, justifying the high
although this microbial reduction was greatly reduced in the present effectiveness of CN based solutions in this study (Pereira et al., 2015;
study, mainly against C. albicans (Cortelli, Cortelli, Shang, McGuire, & Singh et al., 2016). The action of citronellal and citronellol has also
Charles, 2013; da Costa et al., 2017; Wyganowska-Swiatkowska et al., been reported in bacteria such as S. aureus, in which there was a change
2016). in cytoplasmic membranes and cell walls, as a consequence of perme-
The LT and CHX solutions had similar antibiofilm effects, except for ability related to changes in physicochemical properties (hydro-
S. aureus on the acrylic resin surface, where LT had a statistically lower phobicity and surface charge) (Lopez-Romero, González-Ríos, Borges, &

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B. Guandalini Cunha, et al. Archives of Oral Biology 109 (2020) 104577

Fig. 4. Bar chart of epithelial cell viability (HaCat) after

exposure to serial dilutions of solutions tested.
Note: A different upper case letters show statistical dif-
ference between the solutions, considering the same dilu-
tion (e.g. CN 50 x CHX 50), according to the ANOVA and
Tukey tests. a Different lowercase letters show a statistical
difference between the dilutions, considering the same
solution (e.g. CN 50 x CN 25), according to the ANOVA
and Tukey tests).

Fig. 5. Gas chromatography–mass spectroscopy (GC–MS) analysis.

Simões, 2015). This study showed the occurrence of 95 and 99% da- cellular viability for HaCat epidermal cells only from the 3.12% dilution
mage to the cytoplasmic membrane of S. aureus after exposure for 1 h to of CHX active principle (concentration of 0.00375% - 5 times more
citronellol and citronellal, respectively. diluted than the initial concentration of 0.12%). The cause of chlor-
An analysis of the cytotoxicity of the solutions that come into con- hexidine cytotoxicity in eukaryotic cells is still uncertain, but there are
tact with the oral mucosa, that complements the results of antimicrobial reports in the literature that this substance may induce the accumula-
effectiveness, is extremely important. Although HaCat cells were not tion of protein in the endoplasmic reticulum, causing cell stress and
specific for oral mucosa, it was reported in a study by Kreling et al. death by necrosis or apoptosis (Faria, Cardoso, Larson, Silva, & Rossi,
(2016) that these cells had a similar behavior to those of the OBA-9 line, 2009). CHX can also inhibit mitochondrial activity and protein and
which are specific cells of gingival epithelium (Kreling et al., 2016). DNA synthesis (Chang, Huang, Tai, & Chou, 2001). One study con-
In the present study, it was possible to obtain a level of acceptable firmed that chlorhexidine can induce apoptosis or necrosis from

Table 5
- Concentrations of constituents of solutions tested, according to each dilution performed for citotoxicity assays.
Solutions Main Compounds 100% 50% Dilution 25% Dilution 12.5% Dilution 6.25% Dilution 3.12% Dilution 1.56% Dilution

CN Citronellal 4.3% 2.15% 1.07% 0.54% 0.27% 0.13% 0.07%

Citronellol 3.9% 1.95% 0.98% 0.48% 0.24% 0.12% 0.06%
Nerol 3.4% 1.71% 0.85% 0.43% 0.21% 0.11% 0.05%
CHX Chlorhexidine gluconate 0.12% 0.06% 0.03% 0.015% 0.0075% 0.0037% 0.0002%
LT Thymol 0.6% 0.3% 0.15% 0.075% 0.0375% 0.0019% 0.0094%
Eucalyp. 0.092% 0.046% 0.023% 0.011% 0.005% 0.0025% 0.0012%
Methyl Sal. 0.06% 0.03% 0.015% 0.007% 0.037% 0.0187% 0.0093%
Menthol 0.042% 0.021% 0.010% 0.005% 0.0025% 0.0012% 0.0006%

Note: CN – Citronella oil, CHX, LT.

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B. Guandalini Cunha, et al. Archives of Oral Biology 109 (2020) 104577

mitochondrial disorder, increased intracellular Ca 2+, and oxidative for Research on Cancer. Lyon: IARC, 1999). Alcohol-containing
stress (Giannelli, Chellini, Margheri, Tonelli, & Tani, 2008). mouthwashes have shown to be genotoxic to normal and dysplastic oral
Thus, the results obtained corroborate some studies regarding the keratinocytes and induce widespread changes in gene expression.
cytotoxicity of CHX (even at low concentrations of chlorhexidine) that Dysplastic cells are more susceptible to the transcriptomic effects of
demonstrated their potent cytotoxic effects on human periodontal tis- mouthwash (Fox, Currie, Dalley, & Farah, 2018).
sues, such as gingival fibroblasts (Mariotti & Rumpf, 1999; Müller, Eick, An increased risk of oral cancer has been discussed for users of such
Moritz, Lussi, & Gruber, 2017; Wyganowska-Swiatkowska et al., 2016), mouthwashes; however, epidemiological evidence had remained in-
gingival epithelial cells (Babich, Wurzburger, Rubin, Sinensky, & Blau, conclusive (Werner & Seymour, 2009). While the bulk of the metabo-
1995; Müller et al., 2017), periodontal ligament cells (Chang et al., lism of alcohol is carried out in the liver, there is evidence that alcohol
2001), cultured alveolar bone cell (Cabral & Fernandes, 2007) and on metabolism could occur in the oral cavity and that various bacteria in
osteoblastic cells (Giannelli et al., 2008). It also reduces gingival fi- plaque can metabolize alcohol to acetaldehyde. This may support the
broblast adhesion to fibronectin (Balloni, Locci, Lumare, & Marinucci, only theory for why patients with poor oral hygiene are at an increased
2016; Cline & Layman, 1992) and prevents fibroblast attachment to risk of oral cancer. In addition to the possible risk of oral cancer, al-
root surfaces; thus, it can interfere with periodontal treatment and re- cohol-containing mouthwashes are also reported to have other adverse
generation (Giannelli et al., 2008). In addition to these in vitro studies, effects on oral structures and functions. These include burning mouth,
a systematic review with fifty-one randomized controlled trials (RCTs) drying of the oral mucosa, softening effects on composite filling mate-
involving a total of 5345 patients (James et al., 2017) showed the rials and mucosal pain (Settembrini, Penugonda, Scherer, Strassler, &
following adverse effects for the use of chlorhexidine: tooth staining, Hittelman, 1995; Weiner, Millstein, Hoang, & Marshall, 1997). The
supragingival calculus formation, changes in taste perception, parotid evidence suggests that the alcohol component of mouthwashes affords
gland swelling and oral mucosa symptoms, including soreness, irrita- little additional benefit to the other active ingredients in terms of
tion, mild desquamation and mucosal ulceration/erosions, and a gen- plaque and gingivitis control. In view of this outcome and the hy-
eral burning sensation or a burning tongue or both. Another clinical pothetical risk of oral cancer, it would seem prudent that members of
study comparatively evaluated DNA damage and cellular death in cells the dental team advise their patients accordingly (Werner & Seymour,
exposed to various commercially available mouthwashes. The study 2009). Thus, it was evident in the present study that regardless of the
suggested that Periogard® (0.12% chlorhexidine) can induce genetic presence or absence of alcohol in the composition, the use of these
damage. Since DNA damage is considered to be prime mechanism commercial solutions should be indicated with caution.
during chemical carcinogenesis, these data may be relevant in risk as- For the citronella test solution, the initial dilutions (50% and 25%)
sessment for protecting human health and preventing carcinogenesis were as cytotoxic as the first tested concentrations of CHX and LT. The
(Carlin et al., 2012). acceptable cell viability result for citronella was also obtained at the
On the other hand, only few in vitro studies were found evaluating dilution of 3.12%, i.e., at the concentration of 0.13% of the principal
the cytotoxicity of essential oil mouthwash (Balloni et al., 2016; Park, active compound (citronellal), where more than 70% of the cells re-
Lee, Yun, Kim, & Ko, 2014). A study evaluated the effects of commer- mained viable (Koba et al., 2009). It is worth pointing out that at this
cially available antiseptic mouthwashes on human gingival fibroblast same dilution (3.12%), the CHX and LT solutions presented much lower
and keratinocyte viability and metabolism. Three mouthwashes con- concentrations of their active principles than the CN, which shows that
taining essential oils, chlorhexidine and amine fluoride/stannous the active principles present in citronella are comparatively less toxic to
fluoride (AFSF) were tested. The results showed that chlorhexidine and epithelial cells. It was evident that all solutions are cytotoxic at the
essential oils mouthwashes inhibited cell proliferation better than AFSF concentrations where they are commercially available, although the
rinse. The gene expression of several matrix components and cell ad- presence of saliva in the clinical use of these mouthwashes may possibly
hesion receptors was downregulated in cells washed with chlorhexidine dilute and minimize this cytotoxic effect.
and essential oils compared with those washed with AFSF rinse (Balloni Thus, further studies should be carried out, aiming to isolate the
et al., 2016). Other study observed that the actual concentration of compounds responsible for the antimicrobial action of citronella, and
conventional LT has a cytotoxic effect on stem cells of the buccal adi- the combination of these active principles with other substances, in
pose, demonstrating a decrease in cell viability (Park et al., 2014). In order to maintain or improve the effectiveness observed in the present
this present study, the essential oil concentration for acceptable cell study and reduce cytotoxicity, thereby making possible the use of these
viability (IC 50) was 0.0375% for thymol, 0.005% for eucalyptol, compounds with the smallest possible adverse effects to the patient.
0.037% for methyl salicylate, and 0.0025% for menthol in contact with
HaCat cells, i.e., the solution had to be diluted four times from the
initial concentration in order to present satisfactory results. Based on 5. Conclusion
these studies, clinicians should be aware of the potentially adverse ef-
fects of mouthwashes and warn their patients against making improper In conclusion, both concentrations of the citronella-based
use of these products. mouthwash presented higher antibiofilm effects compared to com-
Thus, it was observed in the present study that, although the com- mercial alcohol-free solutions against S. aureus and C. albicans. At high
mercial mouthwashes studied (CHX and LT) did not contain alcohol in concentrations, all solutions tested demonstrated toxic effects on epi-
their composition, they were cytotoxic in their commercial concentra- thelial cells. However, after serial dilution, considering the concentra-
tion. It is suggested that the use of alcohol-free mouthwashes should be tions of the active principles of commercial mouthwashes, citronella
indicated for high risk populations (children, alcohol addicts, patients had the lowest cytotoxic effect.
with genetic deficiencies in ethanol metabolism), until the concerns
over its safety be clarified (Vlachojannis, Winsauer, & Chrubasik, Funding
2013). Increasing evidence suggested that acetaldehyde, the first and
genotoxic metabolite of ethanol, mediates the carcinogenicity of alco- This research did not receive any specific grant from funding
holic beverages. Ethanol is also contained in a number of ready-to-use agencies in the public, commercial, or not-for-profit sectors.
mouthwashes typically between 5 and 27% volume and these
mouthwashes with alcohol lead to salivary acetaldehyde concentrations
similar to those found after the consumption of alcoholic beverages Declaration of Competing Interest
(Lachenmeier et al., 2009). Animal studies have shown that acet-
aldehyde can be considered carcinogenic (WHO International Agency None.

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B. Guandalini Cunha, et al. Archives of Oral Biology 109 (2020) 104577

References C. E. (2016). Effect of surface roughness on the hydrophobicity of a denture-base

acrylic resin and Candida albicans colonization. Journal of Investigative and Clinical
Dentistry, 7, 141–148. https://doi.org/10.1111/jicd.12125.
Andreotti, A. M., Goiato, M. C., Moreno, A., Nobrega, A. S., Pesqueira, A. A., & dos Santos, Di Pasqua, R., Betts, G., Hoskins, N., Edwards, M., Ercolini, D., & Mauriello, G. (2007).
D. M. (2014). Influence of nanoparticles on color stability, microhardness, and Membrane toxicity of antimicrobial compounds from essential oils. Journal of
flexural strength of acrylic resins specific for ocular prosthesis. International Journal of Agricultural and Food Chemistry, 55, 4863–4870.
Nanomedicine, 9, 5779–5787. https://doi.org/10.2147/IJN.S71533. Faria, G., Cardoso, C. R., Larson, R. E., Silva, J. S., & Rossi, M. A. (2009). Chlorhexidine-
Anusavice, K. J., Shen, C., & Rawls, H. R. (2013). Phillips’ science of dental materials (12th induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum
ed.). St. Louis: Elsevier, Saunders. stress. Toxicology and Applied Pharmacology, 234, 256–265. https://doi.org/10.1016/
Ardizzoni, A., Pericolini, E., Paulone, S., Orsi, C. F., Castagnoli, A., Oliva, I., et al. (2018). j.taap.2008.10.012.
In vitro effects of commercial mouthwashes on several virulence traits of Candida Fox, S. A., Currie, S. S., Dalley, A. J., & Farah, C. S. (2018). Transcriptome changes in-
albicans, viridans streptococci and Enterococcus faecalis colonizing the oral cavity. duced in vitro by alcohol-containing mouthwashes in normal and dysplastic oral
PloS One, 15, e0207262. https://doi.org/10.1371/journal.pone.0207262. keratinocytes. Journal of Oral Pathology & Medicine : Official Publication of the
Babich, H., Wurzburger, B. J., Rubin, Y. L., Sinensky, M. C., & Blau, L. (1995). An in vitro International Association of Oral Pathologists and the American Academy of Oral
study on the cytotoxicity of chlorhexidine digluconate to human gingival cells. Cell Pathology, 47, 511–518. https://doi.org/10.1111/jop.12704.
Biology and Toxicology, 11, 79–88. Giannelli, M., Chellini, F., Margheri, M., Tonelli, P., & Tani, A. (2008). Effect of chlor-
Balloni, S., Locci, P., Lumare, A., & Marinucci, L. (2016). Cytotoxicity of three commercial hexidine digluconate on different cell types: A molecular and ultrastructural in-
mouthrinses on extracellular matrix metabolism and human gingival cell behaviour. vestigation. Toxicology in Vitro, 22, 308–317.
Toxicology in Vitro, 34, 88–96. https://doi.org/10.1016/j.tiv.2016.03.015. Guiotti, A. M., Cunha, B. G., Paulini, M. B., Goiato, M. C., Dos Santos, D. M., Duque, C.,
Bedran, T. B., Mayer, M. P., Spolidorio, D. P., & Grenier, D. (2014). Synergistic anti- et al. (2016). Antimicrobial activity of conventional and plant-extract disinfectant
inflammatory activity of the antimicrobial peptides human beta-defensin-3 (hBD-3) solutions on microbial biofilms on a maxillofacial polymer surface. The Journal of
and cathelicidin (LL-37) in a three-dimensional co-culture model of gingival epithe- Prosthetic Dentistry, 116, 136–143. https://doi.org/10.1016/j.prosdent.2015.12.014.
lial cells and fibroblasts. PloS One, 9, e106766. https://doi.org/10.1371/journal. Guiotti, A. M., Goiato, M. C., Dos Santos, D. M., Vechiato-Filho, A. J., Cunha, B. G.,
pone.0106766. Paulini, M. B., et al. (2016). Comparison of conventional and plant-extract disin-
Bunte, K., Hensel, A., & Beikler, T. (2019). Polyphenols in the prevention and treatment of fectant solutions on the hardness and color stability of a maxillofacial elastomer after
periodontal disease: A systematic review of in vivo, ex vivo and in vitro studies. artificial aging. The Journal of Prosthetic Dentistry, 115, 501–508. https://doi.org/10.
Fitoterapia, 132, 30–39. https://doi.org/10.1016/j.fitote.2018.11.012. 1016/j.prosdent.2015.09.009.
Busscher, H. J., & van der Mei, H. C. (1997). Physico-chemical interactions in initial Haffajee, A. D., Roberts, C., Murray, L., Veiga, N., Martin, L., Teles, R. P., et al. (2009).
microbial adhesion and relevance for biofilm formation. Advances in Dental Research, Effect of herbal, essential oil, and chlorhexidine mouthrinses on the composition of
11, 24–32. the subgingival microbiota and clinical periodontal parameters. The Journal of
Cabral, C. T., & Fernandes, M. H. (2007). In vitro comparison of chlorhexidine and po- Clinical Dentistry, 20, 211–217.
vidone-iodine on the long-term proliferation and functional activity of human al- Hahnel, S., Mühlbauer, G., Hoffmann, J., Ionescu, A., Bürgers, R., Rosentritt, M., et al.
veolar bone cells. Clinical Oral Investigations, 11, 155–164. https://doi.org/10.1007/ (2012). Streptococcus mutans and Streptococcus sobrinus biofilm formation and meta-
s00784-006-0094-8. bolic activity on dental materials. Acta Odontologica Scandinavica, 70, 114–121.
Caiaffa, K. S., Massunari, L., Danelon, M., Abuna, G. F., Bedran, T. B. L., Santos-Filho, N. https://doi.org/10.3109/00016357.2011.600703.
A., et al. (2017). KR-12-a5 is a non-cytotoxic agent with potent antimicrobial effects Jacobson, J. J., Patel, B., Asher, G., Woolliscroft, J. O., & Schaberg, D. (1997). Oral sta-
against oral pathogens. Biofouling, 12, 1–12. https://doi.org/10.1080/08927014. phylococcus in older subjects with rheumatoid arthritis. Journal of the American
2017.1370087. Geriatrics Society, 45, 590–593. https://doi.org/10.1111/j.1532-5415.1997.
Cantarelli, R., Negrini, T. C., Muniz, F. W., Oballe, H. J., Arthur, R. A., & Rösing, C. K. tb03092.x.
(2017). Antimicrobial potential and gustatory perception of chlorhexidine gluconate James, P., Worthington, H. V., Parnell, C., Harding, M., Lamont, T., Cheung, A., et al.
mouthwashes with or without alcohol after a single rinse - a randomized controlled (2017). Chlorhexidine mouthrinse as an adjunctive treatment for gingival health. The
crossover clinical trial. International Journal of Dental Hygiene, 15, 280–286. https:// Cochrane Database of Systematic Reviews, 3. https://doi.org/10.1002/14651858.
doi.org/10.1111/idh.12255. CD008676.pub2 CD008676.
Carlin, V., Matsumoto, M. A., Saraiva, P. P., Artioli, A., Oshima, C. T., & Ribeiro, D. A. Kandimalla, R., Kalita, S., Choudhury, B., Dash, S., Kalita, K., & Kotoky, J. (2016).
(2012). Cytogenetic damage induced by mouthrinses formulations in vivo and in Chemical composition and anti-candidiasis mediated wound healing property of
vitro. Clinical Oral Investigations, 16, 813–820. https://doi.org/10.1007/s00784-011- Cymbopogon nardus essential oil on chronic diabetic wounds. Frontiers in
0559-2. Pharmacology, 7, 198. https://doi.org/10.3389/fphar.2016.00198.
Carmona, I. T., Diz Dios, P., & Scully, C. (2002). An update on the controversies in Kern, M. E., & Blevins, K. S. (1997). Medical mycology: A self-instructional text (2 ed.). F.A.
bacterial endocarditis of oral origin. Oral Surgery, Oral Medicine, Oral Pathology, Oral Davis Company.
Radiology, and Endodontics, 93, 660–670. https://doi.org/10.1067/moe.2002. Koba, K., Sanda, K., Guyon, C., Raynaud, C., Chaumont, J. P., & Nicod, L. (2009). In vitro
122338. cytotoxic activity of Cymbopogon citratus L. And Cymbopogon nardus L. Essential oils
Cha, J. D., Jeong, M. R., Choi, H. J., Jeong, S. I., Moon, S. E., Yun, S. I., et al. (2005). from Togo. Bangladesh Journal of Pharmacology, 4, 29–34. https://doi.org/10.3329/
Chemical composition and antimicrobial activity of the essential oil of Artemisia la- bjp.v4i1.1040.
vandulaefolia. Planta Medica, 71, 575–577. https://doi.org/10.1055/s-2005-864164. Kreling, P. F., Aida, K. L., Massunari, L., Caiaffa, K. S., Percinoto, C., Bedran, T. B., et al.
Cha, J. D., Jeong, M. R., Jeong, S. I., Moon, S. E., Kil, B. S., Yun, S. I., et al. (2007). (2016). Cytotoxicity and the effect of cationic peptide fragments against cariogenic
Chemical composition and antimicrobial activity of the essential oil of Cryptomeria bacteria under planktonic and biofilm conditions. Biofouling, 32, 995–1006. https://
japonica. Phytotherapy Research: PTR, 21, 295–299. https://doi.org/10.1002/ptr. doi.org/10.1080/08927014.2016.1218850.
1864. Kronström, M., Svenson, B., Hellman, M., & Persson, G. R. (2001). Early implant failures
Chang, Y. C., Huang, F. M., Tai, K. W., & Chou, M. Y. (2001). The effect of sodium hy- in patients treated with Brånemark System titanium dental implants: A retrospective
pochlorite and chlorhexidine on cultured human periodontal ligament cells. Oral study. The International Journal of Oral & Maxillofacial Implants, 16, 201–207.
Surgery, Oral Medicine, Oral Pathology and Oral Radiology, 92, 446–450. Lachenmeier, D. W., Gumbel-Mako, S., Sohnius, E. M., Keck-Wilhelm, A., Kratz, E., &
Cline, N. V., & Layman, D. L. (1992). The effects of chlorhexidine on the attachment and Mildau, G. (2009). Salivary acetaldehyde increase due to alcohol-containing
growth of cultured human periodontal cells. The Journal of Periodontology, 63, mouthwash use: A risk factor for oral cancer. International Journal of Cancer, 125,
598–602. 730–735. https://doi.org/10.1002/ijc.24381.
CLSI - Clinical and Laboratory Standard Institute (2008). Method for broth dilution anti- Lang, N. P., Wilson, T. G., & Corbet, E. F. (2000). Biological complications with dental
fungal susceptibility testing of yeast; approved standard (3rd ed.). Wayne, PA: CLSI implants: Their prevention, diagnosis and treatment. Clinical Oral Implants Research,
document M27-A3. 11, 146–155. https://doi.org/10.1034/j.1600-0501.2000.011S1146.x.
CLSI – Clinical and Laboratory Standard Institute (2012). Methods for dilution antimicrobial Li, W., Liu, H., & Xu, Q. (2012). Extracellular dextran and DNA affect the formation of
susceptibility tests for bacteria that grow aerobically; approved standard (9th ed.). Wayne, Enterococcus faecalis biofilms and their susceptibility to 2% chlorhexidine. Journal of
PA: CLSI document M7-A9. Endodontics, 38, 894–898. https://doi.org/10.1016/j.joen.2012.04.007.
Cortelli, S. C., Cortelli, J. R., Shang, H., McGuire, J. A., & Charles, C. A. (2013). Long-term Li, Y., Jiang, X., Hao, J., Zhang, Y., & Huang, R. (2019). Tea polyphenols: Application in
management of plaque and gingivitis using an alcohol-free essential oil containing the control of oral microorganism infectious diseases. Archives of Oral Biology, 102,
mouthrinse: A 6-month randomized clinical trial. American Journal of Dentistry, 26, 74–82. https://doi.org/10.1016/j.archoralbio.2019.03.027.
149–155. Lopez-Romero, J. C., González-Ríos, H., Borges, A., & Simões, M. (2015). Antibacterial
Coulthwaite, L., & Verran, J. (2007). Potential pathogenic aspects of denture plaque. effects and mode of action of selected essential oils components against Escherichia
British Journal of Biomedical Science, 64, 180–189. https://doi.org/10.1080/ coli and Staphylococcus aureus. Evidence-based Complementary and Alternative Medicine,
09674845.2007.11732784. 2015, 795435. https://doi.org/10.1155/2015/795435.
da Costa, L. F., Amaral, C. D., Barbirato, D. D., Leão, A. T., & Fogacci, M. F. (2017). Mandel, I. D. (1994). Antimicrobial mouthrinses: Overview and update. The Journal of the
Chlorhexidine mouthwash as an adjunct to mechanical therapy in chronic period- American Dental Association, 125 2S-10S.
ontitis: A meta-analysis. The Journal of the American Dental Association, 148, 308–318. Manford, M., Matharu, J., & Farrington, K. (1992). Infective endocarditis in a district
https://doi.org/10.1016/j.adaj.2017. general hospital. Journal of the Royal Society of Medicine, 85, 262–266.
da Silva, P. M., Acosta, E. J., Pinto, L. R., Graeff, M., Spolidorio, D. M., Almeida, R. S., Mariotti, A. J., & Rumpf, D. A. (1999). Chlorhexidine-induced changes to human gingival
et al. (2011). Microscopical analysis of Candida albicans biofilms on heat-polymerised fibroblast collagen and non-collagen protein production. The Journal of
acrylic resin after chlorhexidine gluconate and sodium hypochlorite treatments. Periodontology, 70, 1443–1448. https://doi.org/10.1902/jop.1999.70.12.1443.
Mycoses, 54, e712–717. https://doi.org/10.1111/j.1439-0507.2010.02005.x. McCormack, M. G., Smith, A. J., Akram, A. N., Jackson, M., Robertson, D., & Edwards, G.
de Foggi, C. C., Machado, A. L., Zamperini, C. A., Fernandes, D., Wady, A. F., & Vergani, (2015). Staphylococcus aureus and the oral cavity: An overlooked source of carriage

9
B. Guandalini Cunha, et al. Archives of Oral Biology 109 (2020) 104577

and infection? American Journal of Infection Control, 43, 35–37. https://doi.org/10. Shen, Y., Stojicic, S., & Haapasalo, M. (2011). Antimicrobial efficacy of chlorhexidine
1016/j.ajic.2014.09.015. against bacteria in biofilms at different stages of development. Journal of Endodontics,
Monteiro, D. R., Gorup, L. F., Silva, S., Negri, M., de Camargo, E. R., Oliveira, R., et al. 37, 657–661. https://doi.org/10.1016/j.joen.2011.02.007.
(2011). Silver colloidal nanoparticles: Antifungal effect against adhered cells and Singh, S., Fatima, Z., & Hameed, S. (2016). Citronellal-induced disruption of membrane
biofilms of Candida albicans and Candida glabrata. Biofouling, 27, 711–719. https:// homeostasis in Candida albicans and attenuation of its virulence attributes. Revista Da
doi.org/10.1080/08927014.2011.599101. Sociedade Brasileira de Medicina Tropical, 49, 465–472. https://doi.org/10.1590/
Moon, S. E., Kim, H. Y., & Cha, J. D. (2011). Synergistic effect between clove oil and its 0037-8682-0190-2016.
major compounds and antibiotics against oral bacteria. Archives of Oral Biology, 56, Slipczuk, L., Codolosa, J. N., Davila, C. D., Romero-Corral, A., Yun, J., Pressman, G. S.,
907–916. https://doi.org/10.1016/j.archoralbio.2011.02.005. et al. (2013). Infective endocarditis epidemiology over five decades: A systematic
Mor, A., Hani, K., & Nicolas, P. J. (1994). The vertebrate peptide antibiotics dermaseptins review. PloS One, 9, e82665. https://doi.org/10.1371/journal.pone.0082665.
have overlapping structural features but target specific microorganisms. The Journal Tawara, Y., Honma, K., & Naito, Y. (1996). Methicillin-resistant Staphylococcus aureus and
of Biological Chemistry, 269, 31635–31641. Candida albicans on denture surfaces. The Bulletin of Tokyo Dental College, 37,
Müller, H. D., Eick, S., Moritz, A., Lussi, A., & Gruber, R. (2017). Cytotoxicity and anti- 119–128.
microbial activity of oral rinses in vitro. BioMed Research International, 2017, Traul, K. A., Driedger, A., Ingle, D. L., & Nakhasi, D. (2000). Review of the toxicologic
4019723. https://doi.org/10.1155/2017/4019723. properties of medium-chain triglycerides. Food and Chemical Toxicology, 38, 79–98.
Nakahara, K., Alzoreky, N. S., Yoshihashi, T., Nguyen, H. T. T., & Trakoontivakorn, G. Van Leeuwen, M. P., Slot, D. E., & Van der Weijden, G. A. (2011). Essential oils compared
(2003). Chemical composition and antifungal activity of essential oil from to chlorhexidine with respect to plaque and parameters of gingival inflammation: A
Cymbopogon nardus (Citronella grass). Japan Agricultural Research Quarterly, 37, systematic review. Journal of Perodontology, 82, 174–194. https://doi.org/10.1902/
249–252. https://doi.org/10.6090/jarq.37.249. jop.2010.100266.
Owen, M. K. (1994). Prevalence of oral methacillin-resistant Staphylococcus aureus in an Veyries, M. L., Faurisson, F., Joly-Guillou, M. L., & Rouveix, B. (2000). Control of sta-
institutionalized veterans population. Special Care in Dentistry: Official Publication of phylococcal adhesion to polymethylmethacrylate and enhancement of susceptibility
the American Association of Hospital Dentists, the Academy of Dentistry for the to antibiotics by poloxamer 407. Antimicrobial Agents and Chemotherapy, 44,
Handicapped, and the American Society for Geriatric Dentistry, 14, 75–79. https://doi. 1093–1096. https://doi.org/10.1128/AAC.44.4.1093-1096.2000.
org/10.1111/j.1754-4505.1994.tb01106.x. Vlachojannis, C., Winsauer, H., & Chrubasik, S. (2013). Effectiveness and safety of a
Park, J. B., Lee, G., Yun, B. G., Kim, C. H., & Ko, Y. (2014). Comparative effects of mouthwash containing essential oil ingredients. Phytotherapy Research, 27, 685–691.
chlorhexidine and essential oils containing mouth rinse on stem cells cultured on a https://doi.org/10.1002/ptr.4762.
titanium surface. Molecular Medicine Reports, 9, 1249–1253. https://doi.org/10. Vlachojannis, C., Chrubasik-Hausmann, S., Hellwig, E., & Al-Ahmad, A. (2015). A pre-
3892/mmr.2014.1971. liminary investigation on the antimicrobial activity of listerine®, its components, and
Pereira, F. O., Mendes, J. M., Lima, I. O., Mota, K. S., Oliveira, W. A., & Lima, E. O. (2015). of mixtures thereof. Phytotherapy Research, 29, 1590–1594. https://doi.org/10.1002/
Antifungal activity of geraniol and citronellol, two monoterpenes alcohols, against ptr.5399.
Trichophyton rubrum involves inhibition of ergosterol biosynthesis. Pharmaceutical Vlachojannis, C., Al-Ahmad, A., Hellwig, E., & Chrubasik, S. (2016). Listerine® products:
Biology, 53, 228–234. https://doi.org/10.3109/13880209.2014.913299. An update on the efficacy and safety. Phytotherapy Research, 30, 367–373. https://
Petti, S., & Polimeni, A. (2011). Risk of methicillin-resistant Staphylococcus aureus doi.org/10.1002/ptr.5555.
transmission in the dental healthcare setting: A narrative review. Infection Control and Weiner, R., Millstein, P., Hoang, E., & Marshall, D. (1997). The effect of alcoholic and
Hospital Epidemiology, 32, 1109–1115. https://doi.org/10.1086/662184. non-alcoholic mouthwashes on heat-treated composite resins. Operative Dentistry, 22,
Rodrigues, C. F., Rodrigues, M. E., & Henriques, M. C. R. (2019). Promising alternative 249–253.
therapeutics for oral candidiasis. Current Medicinal Chemistry, 26, 2515–2528. Werner, C. W., & Seymour, R. A. (2009). Are alcohol containing mouthwashes safe?
https://doi.org/10.2174/0929867325666180601102333. 207(10):E19, discussion 488-489https://doi.org/10.1038/sj.bdj.2009.1014.
Rokadiya, S., & Malden, N. J. (2008). An implant periapical lesion leading to acute os- WHO International Agency for Research on Cancer. Lyon: IARC (1999). Re-evaluation of
teomyelitis with isolation of Staphylococcus aureus. British Dental Journal, 205, some organic chemicals, hydrazine and hydrogen peroxide. IARC Monographs on the
489–491. https://doi.org/10.1038/sj.bdj.2008.935. Evaluation of Carcinogenic Risks to Humans, 71, 1–315.
Salerno, C., Pascale, M., Contaldo, M., Esposito, V., Busciolano, M., Milillo, L., et al. Wyganowska-Swiatkowska, M., Kotwicka, M., Urbaniak, P., Nowak, A., Skrzypczak-
(2011). Candida-associated denture stomatitis. Medicina Oral, Patologia Oral Y Cirugia Jankun, E., & Jankun, J. (2016). Clinical implications of the growth-suppressive ef-
Bucal, 16, e139–143. https://doi.org/10.4317/medoral.16.e139. fects of chlorhexidine at low and high concentrations on human gingival fibroblasts
Sandre, R. M., & Shafran, S. D. (1996). Infective endocarditis. Review of 135 cases over 9 and changes in morphology. International Journal of Molecular Medicine, 37,
years. Clinical Infectious Diseases: an Official Publication of the Infectious Diseases Society 1594–1600. https://doi.org/10.3892/ijmm.2016.2550.
of America, 22, 276–286. https://doi.org/10.1093/clinids/22.2.276. Yeaman, M. R., & Bayer, A. S. (2000). Staphylococcus aureus, platelets and the heart.
Sekido, M., Takano, T., Takayama, M., & Hayakama, H. (1999). Survey of infective en- Current Infectious Disease Reports, 2, 281–298.
docarditis in the last 10 years: Analysis of clinical, microbiological and therapeutic Younessi, O. J., Walker, D. M., Ellis, P., & Dwyer, D. E. (1998). Fatal Staphylococcus aureus
features. Journal of Cardiology, 33, 209–215. infective endocarditis: The dental implications. Oral Surgery, Oral Medicine, Oral
Settembrini, L., Penugonda, B., Scherer, W., Strassler, H., & Hittelman, E. (1995). Alcohol Pathology, Oral Radiology, and Endodontics, 85, 168–172. https://doi.org/10.1016/
containing mouthwashes: Effects on composite colour. Operative Dentistry, 20, 14–17. s1079-2104(98)90421-8.

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