Can mouth washes containing chlorhexidine 0 . 12 % be used as synonym of a water solution of chlorhexidine 0 . 12 % ?

Chlorhexidine digluconate (CHX) is a gold standard drug in dentistry and is widely used as a reference in both in vitro and in vivo experiments. Due to ease of access, mouth washes containing CHX 0.12% are used as a substitute for aqueous CHX 0.12% solution in laboratory experiments. Additionally, it is well known that for product flavor purposes, volatile compounds are added to mouth washes formulations. Volatiles added to CHX 0.12% may improve wash’s antibacterial ability. Volatiles add potency to the mouth wash formulation. Compared with an aqueous CHX 0.12% solution, it is proposed that CHX solutions and Periogard® would have antimicrobial activity. Antimicrobial activity was assessed in the present study via disk diffusion assays against Streptococcus mutans, Streptococcus sanguinis and Escherichia coli. Periogard® showed a significantly higher antibacterial activity in relation to CHX 0.12% (p<0.05) and a similar activity in relation to CHX 1% (p>0.05). Periogard® volatiles were analyzed by gas-chromatography/mass spectrometry (GCMS) and the presence of antibacterial menthol, menthone, isomenthol, menthyl acetate, trans-anethol and eugenol was verified. Finally, the use of Periogard® as a synonym of CHX 0.12% must be avoided, because its antibacterial activity is closely related to CHX 1%.


INTRODUCTION
Chlorhexidine digluconate (CHX) is considered a gold standard drug in dentistry (Van Strydonc et al., 2008;Barros et al., 1998) due to its extraordinary performance against several oral micro-organisms, despite some important side effects (Sivathasan et al., 2011).For this reason, CHX is frequently used in almost all in house antimicrobial in vitro and in vivo assays related to oral infectious diseases (Barros et al., 1998).CHX is also part of the composition of several mouth washes, such as Periogard ® , and acts as the active agent.CHX has also been suggested to improve the performance and efficacy of mouth washes compared to washes that do not contain it.
Periogard ® is a mouth wash that is used worldwide, and according to the manufacturer, it is composed by CHX 0.12% and some inactive components, such as water, glycerin, ethanol, polysorbate 20, an aromatic composition with a predominant peppermint flavor, sodium saccharynate and FD&C Blue #1.
Attention must be paid when using Periogard ® as a standard drug in place of pure chlorhexidine solutions diluted in water, for laboratory or clinical experiments.The present work aims to compare the antimicrobial activity of 0.12%, 1% and 2% CHX, anfothericin B and nystatin to that of Periogard ® against three micro-organisms that are commonly found in the mouth.The present work also claims that the effectiveness of Periogard ® might be related to a synergy between chlorhexidine and the terpenes/ phenylpropanes that are present its formula.

Drugs
CHX 2% was acquired (Fórmula & Ação, São Paulo, Brazil) and diluted with sterile distilled water to obtain both 0.12% and 1% dilutions.Solutions were kept in refrigeration until use.
Periogard ® was acquired from a local drugstore, and was used in its original formula, without being diluted in water.The formula without ethanol was tested in the present work.

Bacteria preparation
A frozen vial (Coastar) containing Streptococcus mutans ATCC ® 25175 TM and Streptococcus sanguinis ATCC ® 10556 TM (both from Microbiologics) suspended in broth medium containing 30% dimethylsulfoxide (Tedia Brazil, Rio de Janeiro, Brazil) was thawed and 300 µL of the suspension was surface-seeded in Brain Heart Infusion agar Petri dishes to obtain a mother-plaque.Petri dishes (J.Prolab, São José dos Pinhais, Brazil) were kept in an incubator (Fanem, Diadema, Brazil) at 36 °C for 48 h.Then, mother-plaques presenting bacterial growth were kept in a refrigerator for up to one month.Fresh colonies were obtained weekly from the mother-plaque, and a sufficient amount of bacteria was collected from Petri dishes to prepare a suspension for further testing.
Escherichia coli ATCC ® 25922 TM Culti Loop ® (Oxoid) was used to obtain mother-plaques by simply seeding a Müeller-Hinton agar Petri dish surface with lyophilized bacteria.The dish was kept in an incubator at 36 °C for 24 h.Fresh colonies were obtained weekly from the mother-plaque, and a sufficient amount of bacteria was collected from Petri dishes with fresh colonies to prepare a suspension for further testing.

Medium
Brain Heart Infusion agar (Oxoid) and Müeller-Hinton agar (Oxoid), used to test both Streptococci and Gram-negative bacteria, respectively.All media were prepared according to the manufacturer´s instruction (Oxoid) and were sterilized (Fanem) before use.

Antibacterial activity
Disk diffusion assay was performed according to CLSI (formely NCCLS) standards (8 th edition, in Portuguese), with the following adaptations.A 0.5 MacFarland saline suspension was prepared from a fresh colony of each micro-organism.The assay was performed in sterile Brain Heart Infusion agar for both Streptococci, in Müeller-Hinton agar for E. coli, and then prepared in Petri dishes (12 cm diameter).Sterile swabs (Deltalab, Beijing, China) were used to seed micro-organisms on the medium surface.Six paper disks (Cefar Diagnostico, Sao Paulo, Brazil) measuring 6 mm in diameter were distributed over the inoculated medium surface.Then, 10 µL of drugs were added to each disk, in triplicate.Disks were incubated at 36 °C for 48 h (for Streptococci) and 24 h (for E. coli).Following this procedure, the diameter of growth inhibition zones was measured both horizontally and vertically using a caliper rule (Digital Caliper, Beijing, China).

Separation of the non polar content of Periogard ®
A non-polar fraction was partitioned from non alcoholic Periogard ® .Then, 100 mL of the mouth wash was transferred to a 250 mL funnel, and 20 mL of hexane (HEX, Synth, Diadema, Brazil) was added to the system.The system was stirred and put to rest up to the separation of both phases.The HEX residue was transferred to a 100 mL beaker.The operation was repeated two more times, and the HEX residues were combined in the beaker and left to evaporate.After that, the partition procedure was repeated with dichloromethane (DCM, Synth, Diadema, Brazil) with the remaining Periogard ® .The DCM residue was evaporated, and no emulsion was formed.Residue samples were closed with a cap and kept in the freezer until use.Crystals were formed during the freezing period and were then isolated by filtration, diluted in the proper solvent and sent for analysis in a gas chromatography-mass spectrometer.

Gas chromatography-mass spectrometry analysis of non polar residues obtained from Periogard ®
The HEX, DCM and MeOH residues were analyzed by a gas chromatographer (Shimatzu series 17A, Kyoto, Japan) coupled to a mass spectrometer (Shimatzu series QP5050A, Kyoto, Japan) (GC-MS).Non-polar residues were diluted in hexane or dichloromethane and injected into a gas chromatographer regulated to the following conditions: 1 µL of the sample was injected into a BPX5 column containing non-polar 5% phenylpolisylphenylene (30 m x 0.25 mm and a film of 0.25 µm); oven temperature of 60 °C; carrier gas of ultra-pure Helium at a flow of 2.5 mL/min; injector temperature of 280 °C; Mode Split; gradient starting at 60 °C (remaining for 2 min) increasing up to 320 °C (in 28 min) at a 10 °C/min rate and remaining at 320 °C for 6 min, totaling 34 min of development.The results relating to retention time 1 (7.82 min), retention time 3 (8.03min), retention time 4 (8.21 min), retention time 5 (9.79 min), retention time 6 (9.91 min) and retention time 7 (10.88min) were in accordance with NIST ® library.

Statistical analysis
Two-way ANOVA and Bonferroni's post test were used to analyze the efficacy of the treatments against different micro-organisms.Statistically significance among means was considered if p<0.05.
Periogard ® is one of the most effective mouth washes recommended in dentistry, particularly due to the presence of CHX at concentration of 0.12%.Periogard ® is commonly used as a standard drug (Barrella et al., 2012;Komiyama et al., 2010;Swaty et al., 2010;Botelho et al., 2009, Magalhães et al., 2009;Valera et al., 2009;Nascimento et al., 2008;Freitas et al., 2003) and as a synonym of 0.12% chlorhexidine digluconate.According to the manufacturer, Periogard ® is a gluconate chlorhexidine solution that is prepared at a concentration of 0.12%, but its free alkali, or the molecule without the gluconate ion, which is the active compound in the formulae, is found at concentrations of 0.067% in Periogard ® (Colgate-Palmolive, 2013).Thus, some authors use the concentration of 0.067% in their analysis (Felo et al., 1997).In the present manuscript, Periogard ® containing 0.12% of chlorhexidine was used.
Although the antimicrobial activity of CHX and mouth washes has been described many times, studies reporting not only the antibacterial potency comparison between both CHX and Periogard ® , but also the chemical composition analysis of Periogard ® , are scarce.Because the misuse of CHX as synonym of Periogard ® is frequent, the present work reports the results obtained from the comparison of the antibacterial analysis of CHX and Periogard ® .The present work also describes the main volatiles extracted from the mouth wash to avoid a misleading analysis of the results obtained from both in vivo and in vitro experiments.

CONCLUSIONS
According to the results obtained in the present work, all CHX solutions -except CHX 0.12% -and Periogard ® showed antimicrobial activity, but Periogard ® was as active as CHX 1%.This was perhaps due to the presence of the antibacterial active terpenes/phenylpropanoids menthol, menthone, isomenthol, menthyl acetate, transanethol and eugenol.