Antimicrobial Activity of Essential Oils against Streptococcus mutans and their Antiproliferative Effects

This study aimed to evaluate the activity of essential oils (EOs) against Streptococcus mutans biofilm by chemically characterizing their fractions responsible for biological and antiproliferative activity. Twenty EO were obtained by hydrodistillation and submitted to the antimicrobial assay (minimum inhibitory (MIC) and bactericidal (MBC) concentrations) against S. mutans UA159. Thin-layer chromatography and gas chromatography/mass spectrometry were used for phytochemical analyses. EOs were selected according to predetermined criteria and fractionated using dry column; the resulting fractions were assessed by MIC and MBC, selected as active fractions, and evaluated against S. mutans biofilm. Biofilms formed were examined using scanning electron microscopy. Selected EOs and their selected active fractions were evaluated for their antiproliferative activity against keratinocytes and seven human tumor cell lines. MIC and MBC values obtained for EO and their active fractions showed strong antimicrobial activity. Chemical analyses mainly showed the presence of terpenes. The selected active fractions inhibited S. mutans biofilm formation (P < 0.05) did not affect glycolytic pH drop and were inactive against keratinocytes, normal cell line. In conclusion, EO showed activity at low concentrations, and their selected active fractions were also effective against biofilm formed by S. mutans and human tumor cell lines.


Introduction
Despite the implementation of measures to control and treat dental caries with fluoride, they remain the most prevalent dental disease in many countries [1]. Caries are a multifactorial infectious disease caused by accumulation of biofilm on tooth surface [2]. Manifestations of the disease occur when there is an imbalance between the biofilm and the host due to changes in biofilm matrix pH caused by diet, microorganisms, or salivary flow and their components [3,4].
Streptococcus mutans is considered the most cariogenic of all oral streptococci [5]. S. mutans is able to colonize the tooth surface and to produce large amounts of extra and intra-cellular polysaccharides. This microorganism is also highly acidogenic and aciduric, and it metabolizes several 2 Evidence-Based Complementary and Alternative Medicine salivary glycoproteins, thus being responsible for the initial stage of oral biofilm formation and caries lesions [6].
Several products have been used to control dental caries, such as fluoride, chlorhexidine, and their associations [7]. However, natural products have contributed significantly to the discovery of chemical structures to create new medicaments to be used as innovative therapeutic agents against this prevalent disease [8,9].
Essential oils (EOs) are important for their detected antimicrobial activity [10][11][12] including that against S. mutans [13]. They are complex, volatile, natural compounds formed by aromatic plants as secondary metabolites [14]. They are known for their bactericidal, virucidal, fungicidal, sedative, anti-inflammatory, analgesic, spasmolytic, and locally anesthetic properties [14]. The presence of complex chemical structures constituted of several groups, such as terpenes and terpenoids, aromatic and aliphatic constituents, all characterized by low molecular weight, may explain their successful bacteriostatic and bactericidal action [14].
Additionally, it was attested that the antimicrobial activity of a natural product, such as EO, is important to evaluate its effects on human normal cell lines and also against human tumor cell lines in order to evidence potential toxicity on human healthy and tumor cell lines [15]. For this reason, it is important that extensive studies involving EO as well as other sources of natural medicines are carried out.
The aim of this study was to evaluate the activity of EO and fractions against planktonic cells of S. mutans and also the selected active fractions of EO were chemically characterized and evaluated against mutans biofilm and antiproliferative activity on human cells.

Medicinal Plants.
We studied 20 medicinal and aromatic plants (Table 1) The plants were collected from November 2009 to January 2011, during the morning, after the dew point has been reached. The vouchers of each species were deposited in the herbarium of the Institute of Biology, at UNICAMP-UEC, and also registered in the herbarium of CPQBA, receiving identification numbers (CPMA number).

Essential Oil Extraction.
EOs were obtained from 100 g of aerial fresh plant parts by hydrodistillation using a Clevenger-type system, for 3 hours. The aqueous phase was extracted with 50 mL of dichloromethane. Then, the organic layer was separated, dried over anhydrous sodium sulphate (Na 2 SO 4 ), and filtered; the solvent was removed by vacuum evaporation at room temperature, resulting in EO. Oil samples were stored at −25 • C in sealed glass vials [11].

Fractionation of Essential Oils.
In order to select the EO that should be fractionated, we predetermined some criteria: best antimicrobial activity (MIC < 250 µg/mL), extract yield (>0.5%, except for Coriandrum sativum EO), commercial availability, presence of the EO in aerial parts of plants, and easy cultivation. The resulting fractions were also submitted to the antimicrobial assay.
Fractionation was performed using dry column chromatography (cellulose 2 cm × 20 cm) with Si gel 60 (Merck, Darmstadt, Germany) as the stationary phase and dichloromethane as the mobile phase, previously chosen by thin-layer chromatography (TLC), visualized under UV 254 nm, followed by anisaldehyde solution application and drying at 105 • C for 5 min. After elution, columns were cut into different parts for each EO, according to polarity and extraction, using dichloromethane. The fractions so obtained were analyzed using TLC and gas chromatography coupled to mass spectrometry (GC-MS) and then bioguided using the antimicrobial assays [16]. All chemical wastes generated during this study were treated according to the Environmental Ethics Committee of UNICAMP (324/2009).

Analyses of the Selected Active Fractions using GC-MS.
The chemical composition of each selected active fraction was evaluated using a Hewlett-Packard 6890 gas chromatograph equipped with an HP-5975 mass selective detector and HP-5 capillary column (30 m × 0.25 mm × 0.25 µm). GC-MS was performed using split injection with the injector set at 220 • C, the column set at 60 • C with a heating ramp of 3 • C/min and a final temperature of 240 • C, and the MS detector set at 250 • C. Helium was used as a carrier gas at 1 mL/min. The GC-MS electron ionization system was set at 70 eV. The quantitative analyses were performed using a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector under the same conditions previously described. A sample of each EO or its selected active fraction was solubilized in ethyl acetate (15 mg/mL) for the analysis. Retention indices (RIs) were determined using injection of hydrocarbon standards and EO samples under the same conditions described above. The oil components were identified by comparison with data described in the literature and the profiles in the NIST 05 mass spectral library [11,17].

Microorganisms.
For the development of this study, Streptococcus mutans UA159 was used.
2.6. Antimicrobial Assay. We tested 20 EOs using the antimicrobial assay and selected them according to pre-determined criteria (item 2.3) before being fractionated and continuing the bioguided study.
MIC test was carried out using tissue culture microplates (96 wells) containing 100 µL/well BHI (Brain Heart Infusion, Difco, Franklin Lakes, NJ, USA) medium [18]. The stock solutions of EO and fractions from selected EO (item 2.3) were diluted with propylene glycol (4 mg/mL), transferred to the first well, and serial dilutions were performed to obtain concentrations ranging from 7.81 to 1000 µg/mL. We  The EOs were considered bactericidal when the MBC : MIC ratio was between 1 : 1 to 2 : 1, and bacteriostatic if this ratio was higher than 2 : 1.
Evidence-Based Complementary and Alternative Medicine 5 used 0.12% chlorhexidine (Sigma-Aldrich, St. Louis, MO, USA) as positive control and propylene glycol 6.25% as negative control. The bacterial inoculum (1 × 10 6 UFC/mL) was added to all wells, and the plates were incubated at 37 • C and 5% CO 2 for 24 hours. MIC was defined as the lowest concentration of EO or fraction from selected EO that inhibited microorganism visible growth indicated by resazurin 0.01% (Sigma-Aldrich, St. Louis, MO, USA) [19].
To determine MBC, an aliquot of each incubated well with concentrations higher than MIC was subcultured on BHI medium supplemented with 5% defibrinated sheep blood using a Whitley Automatic Spiral Plater (Don Whitley Scientific Limited, Shipley, West Yorkshire, UK). MBC was defined as the lowest concentration of EO or fraction that allowed no visible growth on the test medium.
To determine the nature of antibacterial effect of EO and fractions, the MBC : MIC ratio for bacteria was used [20]. When MBC : MIC ratio for S. mutans was between 1 : 1 and 2 : 1, the EO or fraction from selected EO was considered bactericidal against this microorganism [20], and when the ratio was higher than 2 : 1, it was considered bacteriostatic.

Action of Selected Active Fractions from Selected EO against S. mutans Biofilm.
We tested 20 EOs, and those that fulfilled the pre-determined criteria (item 2.3) were selected to be chemically fractionated. The resulting fractions were also tested using the antimicrobial assay and selected according to MIC and MBC results and yields. The selected active fractions were then assessed regarding their action against S. mutans biofilm.

Inhibition of S. mutans Biofilm Growth.
In order to evaluate the antimicrobial activity of EO selected active fractions against the formation of S. mutans biofilm, the samples were placed, at different concentrations (7.81-1000 µg/mL), in the wells of sterile polystyrene U-bottom microtiter plates, previously treated with saliva (the use of human saliva in this study was approved by the Research Ethics Committee of the Piracicaba Dental School, State University of Campinas (UNICAMP) (Approval 087/2011)) [21]. S. mutans cells (1.0 × 10 7 cells/mL in BHI medium) were added to wells containing BHI medium with 2% sucrose and the samples were incubated at 37 • C for 18 hours. Biofilm growth was revealed and quantified using the crystal violet staining method and measuring absorbance at 575 nm [11,22].
After 18 hours of incubation, the spent medium was aspirated, nonadhered cells were removed, the wells were washed three times with sterile distilled water, and the plates were dried for 45 min before carrying out biofilm quantification [22].

Glycolytic pH-Drop
Assay. The effect of EO selected active fractions against S. mutans biofilm was measured using the standard glycolytic pH-drop assay [23]. Biofilm growth was carried out as previously described (item 2.7.1), in sterile polystyrene U-bottom microtiter plates without fractions. The biofilms so obtained were washed twice with 0.9% NaCl solution and salt solution (50 mM KCl + 1.0 mM MgCl 2 ), containing EO selected active fractions at different concentrations (1000, 500, and 250 µg/mL), and vehicle (25% propylene glycol, v/v) was added. The pH was adjusted to 7.2 with 0.1 M KOH solution, and glucose was added to a final concentration of 1%, and pH-drop was assessed using Orion pH glass electrode attached to Orion 290 A + pHmeter (Orion Scientific, Houston, TX, USA) for 90 min.

Scanning Electron Microscopy (SEM).
In order to evaluate S. mutans integrity using SEM, biofilms were first developed in Lab-Tek chambered coverglass (Nunc, Naperville, IL, USA), as described previously (item 2.7.1), were treated with vehicle (6.12% propylene glycol) or had their active fractions selected at concentrations able to inhibit more than 90% of S. mutans biofilm formation. Samples were fixed in 4% glutaraldehyde (v/v) in phosphate-buffered saline (PBS) at room temperature for 12-24 hours. After this procedure, the biofilms were dehydrated through a graded series of ethanol (50% to 100%), dried to a critical point, coated with gold, and observed using a scanning electron microscope JEOL JSM5600LV (JEOL Ltd., Tokyo, Japan) [11,24].

Statistical
Analysis. An exploratory data analysis was performed to determine the most appropriate statistical test. Inhibition of biofilm growth, and glycolytic pH-drop data were compared using the nonparametric Kruskal-Wallis test. P value < 0.05 was considered statistically significant. Triplicates from at least three separated experiments were conducted in each assay.

Essential Oils and Fraction
Yields. The EO yields, expressed in relation to dry weight of plant material (%, w/w), are shown in Table 1.
According to pre-determined criteria (item 2.3), four EOs were selected to be fractionated using dry column as follows: A. gratissima, B. dracunculifolia, C. sativum, and L. sidoides.
The yields of the fractions from selected EO were expressed as a function of the respective EO yield (%, w/w) and are shown in Table (Table 2). Based on yield and antimicrobial activity, Ag 4 , Bd 2 , Cs 4 , and Ls 3 fractions were selected for further evaluations. Figure 1 shows the development of S. mutans biofilm inhibiton after treatment with selected active fractions. Their growth was measured by optic density at 575 nm. The result showed that the selected active fractions tested at different concentrations were significantly different (P < 0.05) from the vehicle. Moreover, Cs 4 and Bd 2 fractions presented a better performance since they inhibited more than 90% of biofilm formation at lower concentrations (31.2 µg/mL). formation in the presence of excess glucose was not significant (P > 0.05) for all fractions tested (Ag 4 , Bd 2 , Cs 4 , and Ls 3 ).

Chemical Characterization of Fractions Constituents.
The chemical composition of the selected EO and the selected active fractions is shown in Table 3.
The analyses of EO and fractions indicated the presence of volatile compounds, mainly mono-and sesquiterpenes.
We identified 28 compounds in the EO of A. gratissima, representing 92.73% of the EO, 25 compounds in the EO of B. dracunculifolia, representing 93.45% of the EO, 15 compounds in the EO of C. sativum, representing 91.93% of the EO, and four compounds in the EO of L. sidoides, representing 100% of the EO. We also identified 19 compounds in fraction Ag 4 , representing 94.6% of the fraction, 10 compounds in fraction Bd 2 , representing 83.06% of the fraction, nine compounds in fraction Cs 4 , representing 89.71% of the fraction, and five compounds in fraction Ls 3 , representing 99.7% of the fraction.

Scanning Electron Microscopy (SEM).
The effect of selected active fractions against S. mutans biofilm formation was evaluated by SEM. Figure 2 shows a reduction in  1 The fractions from selected EO were considered bactericidal when the MBC : MIC ratio was between 1 : 1 to 2 : 1, and bacteriostatic if this ratio was higher than 2 : 1. 2 The fractions in bold font were selected as active fractions and evaluated against S. mutans biofilm and for their antiproliferative action. The subscript numbers of the fractions represent the numbers of parts obtained using the dry column fractionation.
biofilm formation. Biofilms were first developed as described previously (Section 2.7.1), were treated with vehicle, or had their active fractions selected at concentrations able to inhibit more than 90% of S. mutans biofilm formation (Ag 4 at 62.5 µg/mL, Bd 2 and Cs 4 at 31.2 µg/mL, and Ls 3 at 125 µg/mL).

Discussion
The activity of natural products, especially EO, against microorganisms has been recently confirmed by several studies focusing on antimicrobial activity of EO against planktonic cells. However, bacteria growing in biofilms exhibit a specific phenotype and are often, but not always, more resistant to antimicrobial agents than their planktonic counterparts [10,11]. Thus, it is important to search for natural products that have antibiofilm properties and antimicrobial activity against oral pathogens [28].
This study aimed to evaluate the activity of EO and their fractions against planktonic cells of S. mutans, and the active fractions were evaluated against biofilm formed by S. mutans. Also, EO and their active fractions were chemically characterized and their activity against human normal and tumor cell lines proliferation were determined.
The antimicrobial assay revealed low MIC values for almost all 20 EOs and 15 fractions from the selected EO tested. EO and the selected active fractions presented strong activity against S. mutans, since natural products are considered strong inhibitors of microbial activity, when MIC values are lower than 500 µg/mL [29].  These results demonstrate that the EO studied and especially those selected (A. gratissima, B. dracunculifolia, C. sativum, and L. sidoides) have potential for bioprospection of new active biomolecules. The fractionation process adopted showed good results, since the fractions obtained were more active than the original EO (Table 2). This bioguided study is a model for bioprospecting new drugs [30], and it can be considered successful since we found active fractions presenting higher activity than their respective EO.
Most EO and fractions studied showed MBC : MIC ratio that enables them to be classified as bactericidal compounds. This could be explained by their hydrophobicity, an important characteristic that exists in EO and their fractions [31] and may allow them to partition the lipids of the bacterial cell membrane, turning them more permeable and leading to leakage of ions and other cell constituents [32,33]. On the other hand, B. dracunculifolia EO and its selected active fraction (Bd 2 ) present compounds that could be capable of infiltrating the cell and interact with cellular metabolic mechanisms [34], demonstrating their bacteriostatic effect. Nevertheless, despite presenting bactericidal or bacteriostatic effect, the selected EO proved to be active against both S. mutans planktonic cells and biofilm, demonstrating the effectiveness of the substances present in these EO, since it is difficult to disrupt S. mutans biofilm [35].
The selected active fractions were also tested against S. mutans biofilm, and they were able to disrupt its formation at all tested concentrations. This disruption was observed using SEM, which showed the change the selected active fractions caused in the structure of S. mutans biofilm.
At the concentrations tested, it was possible to observe huge failures in S. mutans biofilm surface treated with the active fractions when compared with the treatment with the vehicle, which presented a more homogeneous   Evidence-Based Complementary and Alternative Medicine biofilm surface. These changes were also observed in another study that tested the action of C. sativum and its bioactive fraction against Candida albicans [11]. Moreover, the simple conformational change in biofilm, caused by the action of the selected active fractions, could make it more susceptible and less virulent [4]. However, when the selected active fractions were tested in order to evaluate their ability to reduce S. mutans acid production, no significant results were observed (P > 0.05). Therefore, the selected active fractions could not act on this important virulence factor of S. mutans, different from the findings of another work with B. dracunculifolia extracts, which showed significant reduction in production of acid by this microorganism [36]. The difference between B. dracunculifolia EO and the active extracts from this plant may be attributed to the extraction method, which results in different compound mixtures with different mechanisms of action [37].
It is known that EOs are composed of numerous different chemical compounds, and their antimicrobial activity might be attributed to several different mechanisms, which could explain the variations in their mode of action [38].
The present data suggest the occurrence of a separation during the fractionation process of the selected EO in such a way that the selected active fractions presented higher amounts of bioactive compounds than their respective EO. The main biologically active compounds found in the selected active fractions were thymol, carvacrol, 2decen-1-ol, trans-nerolidol, spathulenol, ethyl ester benzenepropanoic, trans-pinocamphone, cis-pinocamphone, and guaiol. These compounds have been extensively described in the literature for their effect on microorganisms [39,40].
Both forms of trans-and cis-pinocamphone are major constituents of Ag 4 fraction and were also found in Hyssopus officinalis L. EO [41]. These compounds are responsible for the antibacterial, antifungal, and antioxidant activities of H. officinalis EO, demonstrating that they pass through the cell wall and the plasma membrane, disrupting their structure [41]. The bactericidal activity of Ag 4 fraction observed in the present study may be a consequence of this mode of action.
Trans-nerolidol and spathulenol, two compounds present in Bd 2 fraction, have been considered active against unknown Gram-positive and Gram-negative bacteria [13]. Although spathulenol shows activity against S. mutans, its mechanism of action still remains unknown [13].
Other studies showed that certain alcohols, such as 2decen-1-ol, have higher antimicrobial activity than aldehydes against Candida ssp. [11,16]. These alcohols were found in Cs 4 fraction and may be responsible for the action against S. mutans biofilm. Furthermore, considering the mode of action of C. sativum EO, it seems to result in bacterial cell permeabilization, leading to the impairment of other cell functions, such as membrane potential, respiratory activity, or efflux pump activity [42].
Thymol is an optic isomer of carvacrol, and both substances seem to make bacterial membrane more permeable [43]. In our study, both were found in Ls 3 fraction as its major components. Previous studies have shown that these compounds present antimicrobial activity against fungi and bacteria [44], including species of the genus Streptococcus [12].
After determining the antimicrobial activity of a natural product, it is important to verify if it also exhibits antiproliferative activity, mainly after its fractionation, a procedure that may concentrate toxic compounds in the fractions that present biological activity.
Based on TGI values, the selected EO and selected active fractions could be classified as inactive (TGI > 50 µg/mL), weakly active (15 µg/mL < TGI < 50 µg/mL), moderately active (6.25 µg/mL < TGI < 15 µg/mL), and strongly active (TGI < 6.25 µg/mL) [45]. The absence of activity was clearly observed in this study since all selected EO and selected active fractions were inactive against the human normal cell line tested.
All EOs tested were selective against the ovarian tumor cell line, showing potent activity. Ag 4 showed potent activity against the kidney tumor cell line, and Bd 2 and Cs 4 fractions showed only moderate activity against the ovarian tumor cell line. These results show the specificity of these EO and their fractions against some tumor cell lines, an important and desired characteristic for potential new chemotherapic drugs [15].
It is known that EO compounds, such as monoterpenes, have shown effects on mevalonate metabolism, linked to the maintenance of cell membrane, which could contribute to terpene tumor suppressive action [46]. Thereby, the presence of monoterpenes in the selected active fractions of our study may explain their antiproliferative actions against some tumor cell lines [47]; however, more studies are required to find the compounds of EO responsible for their anticancer activity, since little is known about essential oils and their antiproliferative activity.

Conclusion
The results of the present study indicate that all EO and fractions tested showed good antimicrobial activity, but only those showing activity at low concentrations were taken into consideration and fractionated for bioprospection of new agents against S. mutans. Among these fractions, the selected active fractions were able to disrupt S. mutans biofilm formation, did not inhibit normal cell line growth, and were more specific against human tumor cell lines. These features enable them to be tested in further studies and help the discovery of new bioactive molecules.