Antimicrobial potential of Casearia sylvestris against oral bacteria

Aim: The aim of this study was to obtain Casearia sylvestris leave extracts by different extractive methods, including the obtention of essential oil, in order to compare their antimicrobial activities to conventional mouthwash chlorhexidine against oral bacteria. Material and method: For this evaluation, extracts from the leaves were obtained by different methods of extraction (infusion, decoction, maceration and percolation) using different solvent systems: water 100%, ethanol 100%, methanol 100%, water: ethanol 3:7; water: ethanol 7:3; water: methanol 7:3 and water: methanol 3:7. The essential oil, which corresponds to a volatile fraction, was obtained by hydrodistillation using Clevenger modified apparatus. The microdilution broth method was used to determine the values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the following microorganisms: Streptococcus mutans ATCC 25175, S. mitis ATCC 49456, S. sanguinis ATCC 10556, S. salivarius ATCC 25975, Lactobacillus casei ATCC 11578 and Enterococcus faecalis ATCC4082. Chlorhexidine gluconate was used as a positive control. Result: All extracts evaluated in the used protocol displayed MIC values higher than 400 μg/mL and few showed bactericidal activity. The antimicrobial activity of essential oil was higher than the activity of the extracts, and the best minimum inhibitory concentration and minimum bactericidal concentration values were obtained against L. casei (MIC of 0.023 μg/mL and MBC of 0.046 μg/mL) and S. mutans (MIC of 25 μg/mL and MBC of 50 μg/mL), respectively. Conclusion: The essential oil of Casearia sylvestris has significant antimicrobial activity against oral microorganisms. Descriptors: Laboratory research; dental caries; microorganisms; phytotherapy. 215 Rev Odontol UNESP. 2016 July-Aug; 45(4): 214-218 Antimicrobial potential of Casearia sylvestris...


INTRODUCTION
Dental plaque has been considered the main etiologic agent in the initiation of dental caries and periodontal disease, the most common form of which is gingivitis 1 .Periodontal diseases are bacterial infections that affect the supporting structure of the teeth 2 .Among these, dental caries is a localized and transmissible infectious process that ends in the destruction of hard dental tissue 2 .Normally, periodontal diseases are related to anaerobes such as Treponema denticola and Porphyromonas gingivalis 3 .Dental decay is biofilm dependent; one of the fundamental imbalances that cause the onset of cariogenic biofilm is a rich and frequent diet of fermentable carbohydrates, mainly sucrose 4 .
Many bacteria are involved in dental caries, mainly Streptococcus mutans 2 , which is capable of fermenting carbohydrates resulting in acid production and leading to the demineralization of the tooth.The tooth surface provides the necessary conditions for the adherence and establishment of bacteria, such as Streptococcus sanguis, Streptococcus mitis, Streptococcus gordonii, Streptococcus mutans and Actinomyces species, which can be considered early colonizers of the enamel surface 5 .Once the bacteria are established on the surface, plaque forms if the surface isn't adequately cleaned.Besides this, E. faecalis is also found in the mouth and may cause other diseases 6 .
The use of mouthrinses containing anti-plaque or those known to have antimicrobial contents 2 should complement toothbrushing as a potential prophylactic method of reducing plaque-mediated disease 2 .One of the chemical agents widely used against oral bacteria is chlorhexidine.It has higher solubility in water and dissociates to release a cationic component.
Chlorhexidine is the only compound that has substantivity, i.e., its active residence time in the buccal cavity is approximately 12 hours, which can be explained by its chemical structure.The interactions of charges in the film and with the bacteria exert a bactericidal action immediately after the initial rinse, combined with a prolonged bacteriostatic activity 7 .However, when used for an extended period, chlorhexidine causes undesirable side effects, such as stains on teeth and restorations, changes in taste, epithelial desquamation and a burning sensation on the tongue 8 .
Casearia sylvestris is considered a promising source of compounds that have an antimicrobial activity 9 .There are already patents to use these Brazilian plant species in a composition of drugs that promotes the healing of herpetic lip lesions, which represents a major breakthrough for herbal medicine 10 .Besides this, some phytotherapics are available in the public health system for medical treatments according to the law of 2006 11 .Maytenus ilicifolia, Mikania glomerata, Cynara scolymus and more than 9 plant species are approved until now 12 .However, only a few studies have compared the antimicrobial activity of chlorhexidine with different extracts and essential oil from Casearia sylvestris.The aim of this study was to obtain Casearia sylvestris leaf extracts by different extractive methods, including the obtention of essential oil, in order to compare their antimicrobial activities with the conventional mouthwash chlorhexidine against Streptococcus mutans, S. mitis, S. sanguinis, S. salivarius, Lactobacillus casei and Enterococcus faecalis.

Plant material
Dried leaves of Casearia sylvestris collected in August 2011 were acquired from Shop Herbs Natural Products Company (CNPJ 08.898.383/0001-79).The leaves were crushed and packed in a glass jar, were properly identified and were covered to avoid contact with air and moisture.

Extracts preparation
Extracts were prepared using the following extractive processes:

I. Infusion
The volume of 30 mL of distilled water was heated to boiling and immediately placed in a glass beaker, which contained one gram of the powdered leaves of Casearia sylvestris.This mixture was allowed to stand for 15 minutes and then filtered.The filtrate was transferred to lyophilization flasks and was then frozen and lyophilized.

II. Decoction
One gram of the powdered leaves of Casearia sylvestris was placed for heating in 30 mL of distilled water.After the start of boiling, we counted 15 minutes.The mixture was then filtered, and the filtrate was frozen and lyophilized in the same way as previously mentioned.

III. Maceration a) Static
One gram of the powdered leaves of Casearia sylvestris was placed with their respective solvent (water 100%, ethanol 100%, methanol 100%, water: ethanol 3:7; water: ethanol 7:3) into a beaker and allowed to stand for 24 hours.After this period, the suspension was filtered, the filtrate was frozen and a further 30 mL of the same solvent was added to the plant residue.This methodology was repeated three times consecutively over 3 days.

b) Exhaustive
The studied variables in this procedure were the same as in the previous method.However, the plant drug (one gram of powdered leaves of Casearia sylvestris) and the solvent were continuously stirred for 2 hours followed by filtration and solvent exchange three consecutive times, totaling an experiment that lasted 6 consecutive hours.After obtaining these statements, they were pooled, frozen and lyophilized.

IV. Percolation
The percolator was filled with one gram of powdered leaves of Casearia sylvestris and solvent (water 100%, ethanol 100%, methanol 100%, water: ethanol 3:7; water: ethanol 7:3; water: methanol 7:3 and water: methanol 3:7).This was left to rest (conceptually called maceration) for 6 hours and then the process of percolation was initiated at a rate of 30 drops/minute.Note: The ethanol and methanol were totally evaporated under vacuum conditions, and water was removed by lyophilization.

Essential oil obtention
Fraction of volatile compounds of plant leaves was obtained by hydrodistillation using Clevenger apparatus, following the parameters of the Brazilian Pharmacopoeia edition 13 .The oil obtained was packed in a properly sealed jar and was maintained in a freezer.

Bacteria
The following bacteria acquired from the American Type Culture Collection (ATCC) were used:

Antimicrobial activity assay
The minimum inhibitory concentration (MIC) values of the extracts and essential oil from Casearia sylvestris leaves were determined in triplicate using the broth microdilution method, according to CLSI (Clinical and Laboratory Standards Institute).The samples were dissolved in DMSO (dimethyl sulfoxide) at 1 mg/mL and were diluted in tryptone soya broth to achieve concentrations in the range 500-20 μg/mL.The final concentration of DMSO was 5% (v/v), and this solution was used as a negative control.
The inoculum was adjusted for each organism after 24h growing in blood agar to yield a cell concentration of 5 × 10 5 colony-forming units (CFU/mL) using the 0.5 MacFarland scale.One inoculated well was included to control the adequacy of the broth to support the growth of the organisms, and one non-inoculated well, free of antimicrobial agent, was also used to ensure the sterility of the medium.Chlorhexidine was used as a positive control.The microplates (96-well) were incubated at 37 °C for 24h.After that, 30 μL of resazurin (0.02%) in aqueous solution was added to indicate the viability of the microorganisms 14 .The MIC was determined as the lowest concentration of the extract capable of inhibiting microorganism growth.For the determination of either bacteriostatic or bactericidal activities, the entire volume of each well of all the incubated microplates was sub-cultured on blood agar at 37 °C for 24 h.The absence of viable cell growth indicated a bactericidal effect.The minimum bactericidal concentration was defined as the lowest concentration that enabled no growth on blood agar.

Analysis of essential oil by gas chromatography coupled to mass spectrometry
The essential oil sample obtained from the hydrodistillation process was submitted for analysis by gas chromatography coupled with mass spectrometry.Analyses were performed on Shimadzu model GC / MS QP-2010 equipped with a source of electrons by ionization (EI) and quadrupole-type analyzer.The chromatographic conditions used were as follows: ZB-5MS column (0.25mm × 0.25um 30mx)-Phenomenex, injector temperature of 230 °C and hydrogen in a mobile phase (gas flow rate: 1 mL.min -1 ).The column temperature was kept initially at 60 °C for 1 minute, was increased to 240 °C after 10 minutes and was then kept at this temperature for 60 minutes.
After elution, the flow divider was used to drive the samples into the ionization source with a ratio of 1:40.The source temperature was set at 250 °C and the energy of the electron beam was 70 eV.The analyzer was checked for separating ions of m / z 40 to 400.

RESULT
The yield of each extract was evaluated and the largest was obtained by percolation process using, as the solvent, a mixture of water/methanol (3:7).The smallest was obtained by percolation using water.
The amount of essential oil obtained in the process of hydrodistillation of the dried leaves, which was collected in August 2011, was 0.85 mL/100 g of dry leaves.
All extracts displayed MIC values higher than 400 μg/mL and few showed bactericidal activity.In terms of their activity against S. mutans, the three solvent types-ethanol:water (7:3) in exhaustive maceration, 100% ethanol in percolation and water:methanol (7:3) in percolation-displayed values of 400 μg/mL for MBC.In terms of its activity against S. mitis, ethanol 100% in percolation also showed MBC values of 400 μg/mL.All the other extracts had MBC levels greater than 400 μg/mL.
All the values of MIC and MBC for the essential oil are presented in Table 1.
Due to the good activity displayed by the essential oil, a sample was analyzed using gas chromatography coupled with mass spectrometry in order to identify the constituents.The mass spectra gave the molecular weight and the fragmentation profile of each component.Therefore, by comparing the data obtained from the Wiley library, it was possible to identify the major constituents: β-caryophyllene, germacrene D, bicyclogermacrene, δ-cadidene, caryophyllene oxide, α-spathulenol and α-elemol.Figure 1 shows the chromatogram and the chemical names of the identified major constituents.

DISCUSSION
Mouth rinses have been used in dentistry as an adjuvant in oral hygiene.Among these, the most used rinse is chlorhexidine, which can be considered a gold standard anticariogenic, although regular use of this rinse is often associated with many negative side effects.Therefore, in order to find another antiplaque agent that can be used on a daily basis without the side effects of chlorhexidine, our research group has decided to investigate the antimicrobial potential of a shrub described in the Brazilian pharmacopoeia.
Studies with C. sylvestris reported in the literature were carried out with methanol, aqueous, and ethanol extracts.It was shown to have anti-hiperlipidemic, antinoceptive, wound healing and anti-inflammatory activities [15][16][17] .In this work, different solvents (water, ethanol and methanol in different proportions) were used in different extraction procedures to obtain different types of extracts in order to further analyze the antimicrobial potential.It was also decided to evaluate the essential oil extracted from the leaves through the Clevenger apparatus, because the anti-inflammatory activity of this oil has been described in the literature 18,19 .The MIC values of the obtained extracts were not promising, showing values above 400 µg/mL.Values below 100µg/mL for extracts and fractions can be considered promising 20 .
The essential oil displayed minimum inhibitory concentration values and minimum bactericidal concentration values, which is promising in terms of the development of formulations that demonstrate antimicrobial activity.The essential oil did not show activity in the evaluated concentrations against E. faecalis, which is an enteric facultative gram-positive bacterium that can grow independently in the root canal without the assistance of other bacteria and is also resistant to other agents such as calcium hydroxide 21 and sodium hipochlorite 22 .
Although chlorhexidine displayed lower values of minimum inhibitory concentration for all assayed microorganisms than those of essential oil, the obtained results seem to be satisfactory as the methanol extract of Hydrastis canadensis L. showed a minimum inhibitory concentration of 250 µg/mL against Streptococcus mutans and has been introduced into the formulation of a number of mouth rinses and toothpastes on the U.S. market 23 .
Essential oils are volatile odoriferous oils constituted by a complex mixture of compounds and produced as the secondary metabolites of some plants.Generally, components at a larger percentage determine the biological properties of the oil.
The essential oil composition depends on the stage of a plant's development and the presence of micronutrients, soil quality, temperature and seasonality.This work was carried out with C. sylvestris collected in August 2011 and which provided essential oil with a good antimicrobial activity.
Our results were qualitatively similar to others in the literature.They identified about 25 constituents, the most predominant of which was the presence of germacrene D, bicyclogermacrene, δ-cadinene, spathulenol, caryophyllene oxide and β-caryophyllene at different levels 24 .
Most of the compounds identified belong to the group of sesquiterpenes, such as bicyclogermacrene, germacrenes D, caryophyllene oxide and β-caryophyllene, which are defined in the literature as possessing anti-inflammatory, anti-swelling, antitumor, antihistaminic, bactericide and repellant properties 16,25 .

CONCLUSION
It can be concluded that the essential oil of Casearia sylvestris presents significant antimicrobial activity against oral bacteria.The results achieved in this work highlighted the antimicrobial potential of Casearia sylvestris essential oil, which could be introduced into the formulation of a number of mouth rinses, toothpastes or other types of phytotherapy for use in dental clinics.

Table 1 .
Values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Casearia sylvestris essential oil in µg/mL