Antimicrobial activity of the essential oil of Tetradenia riparia (Hochst.) Codd. (Lamiaceae) against cariogenic bacteria

In Brazilian folk medicine, Tetradenia riparia (Hochst.) Codd. (Lamiaceae) is used to treat toothaches and dental abscesses and diseases induced by worms, bacteria, or fungi. This paper aims to investigate the chemical composition and the antibacterial effects of the essential oil obtained from Tetradenia riparia leaves (TR-EO) grown in Southeastern Brazil against a representative panel of oral pathogens. We evaluated the antibacterial activity of TR-EO in terms of the minimal inhibitory concentration (MIC). We identified aromadendrene oxide (14.0%), (E,E)-farnesol (13.6%), dronabinol (12.5%), and fenchone (6.2%) as the major constituents of TR-EO. TR-EO displayed MIC values between 31.2 and 500 μg/mL, with the lowest MIC value being obtained against Streptococcus mitis (31.2 μg/mL), S. mutans (62.5 μg/mL), S. sobrinus (31.2 μg/mL), and Lactobacillus casei (62.5 μg/mL). In time-kill experiments, TR-EO demonstrated bactericidal activity against S. mutans within the first 12 h, resulting in a curve profile similar to that of chlorhexidine. These results revealed that the essential oil of Tetradenia riparia displays promising activity against most of the selected cariogenic bacteria, including Streptococcus mutans.


Introduction
Dental caries is a major public health concern that affects many countries worldwide. This pathology and other periodontal diseases are associated with acidogenic and aciduric bacteria that adhere to the tooth surface as a structurally and functionally organized biofilm (dental plaque) (Marsh, 2003;Marsh, 2006). The most efficient procedure to prevent caries is to remove the biofilm by brushing and flossing; however, most people fail to maintain a sufficient level of control through mechanical removal only (Barnett, 2006). Therefore, the use of oral products containing antimicrobial ingredients as a complementary measure has become necessary and has great value in regard to diminishing tooth surface biofilm (Furiga et al., 2008;Sharma et al., 2004). Currently, chlorhexidine is considered to be the anticariogenic gold standard and has received the approval of the American Dental Association Council on Dental Therapeutics. Nevertheless, the regular use of oral care products containing this chemical often incurs several side effects (Greenberg et al., 2008;More et al., 2008). As a result, the search for new potential chemotherapeutic agents that can be incorporated into dental products has escalated in recent years (Palombo, 2011). phenylpropanoids, that can easily diffuse across cell membranes, a major advantage with regard to interactions with intracellular targets (Edris, 2007). Additionally, synergistic interactions between the components of EOs are possible and beneficial for their activities (Dorman and Deans, 2000).
Tetradenia riparia (Hochst.) Codd. (Lamiaceae), commonly known in Brazil as "false myrrh", is an herbaceous and aromatic shrub that originated in South Africa; it was introduced as an exotic ornamental plant in Brazil (Gazim et al., 2010;Phillipson and Steyn, 2008). In folk medicine, this species is used to treat toothaches and dental abscesses, malaria, and diseases induced by worms, bacteria, or fungi, among others (Scott et al., 2004;Vanpuyvelde et al., 1988;Vlietinck et al., 1995). The essential oil from T. riparia leaves displays repellent (Omolo et al., 2004), insecticidal (Dunkel et al., 1990), ascaricidal (Peter and Deogracious, 2006), antimalarial (Campbell et al., 1997), and antinociceptive actions (Gazim et al., 2010). Recently, the antimicrobial activity of this oil against Candida albicans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis, Klebsiella pneumonia, and Salmonella enterica was reported (Gazim et al., 2010). However, despite its use in folk medicine to treat toothaches and dental abscesses, the effects of this essential oil against oral pathogens have not yet been investigated. This paper reports the chemical composition and antimicrobial activity of the essential oil of T. riparia leaves (TR-EO) grown in Southeastern Brazil against a representative panel of cariogenic bacteria.

Essential oil extraction, GC and GC-MS analysis
Fresh leaves (300 g) were submitted to hydrodistillation in a Clevenger-type apparatus for 3 h. To this end, 1,200 g of plant material was divided into three samples of 400 g each, and 500 mL of distilled water was added to each sample. After manual collection, traces of water remaining in the essential oil (EO) were removed using anhydrous sodium sulfate, which was followed by filtration. The EO was stored in an amber bottle and kept in the refrigerator at 4°C until further analysis. The EO yield was calculated from the weight of fresh leaves and expressed as the average of triplicate analysis.

GC-FID and GC-MS analyses
TR-EO was analyzed by gas chromatography (GC) on a Hewlett-Packard G1530A 6890 gas chromatograph fitted with FID and a data-handling processor. An HP-5 (Hewlett-Packard, Palo Alto, CA, USA) fused-silica capillary column (30 m x 0.25 mm i.d.; 0.33 mm film thickness) was employed. The operation conditions were as follows: column temperature programmed to rise from 60 to 240°C at 3°C/min and then held at 240°C for 5 min; carrier gas = H 2 , at 1.0 mL/min; injection mode; injection volume, 0.1 mL (split ratio of 1:10); and injector and detector temperatures = 240 and 280°C, respectively. Components relative concentrations were obtained by peak area normalization (%).The relative areas were the average of triplicate GC-FID analyses.
GC-MS analyses were carried out on a Shimadzu QP2010 Plus (Shimadzu Corporation, Kyoto, Japan) system equipped with an AOC-20i autosampler. The column was a Rtx-5MS (Restek Co., Bellefonte, PA, USA) fused silica capillary column (30 m x 0.25 mm i.d. x 0.25 mm film thickness). Electron ionization mode occurred at 70 eV. Helium (99.999%) was employed as the carrier gas at a constant flow of 1.0 mL/min. The injection volume was 0.1 mL (split ratio of 1:10). The temperatures of the injector and the ion-source temperature were set at 240 and 280°C, respectively. The oven temperature program was the same as the program used for GC. Mass spectra were taken with a scan interval of 0.5 s, in the mass range from 40 to 600 Da. TR-EO components identification was based on their retention indices on an Rtx-5MS capillary column under the same operating conditions as in the case of GC relative to a homologous series of n-alkanes (C 8 -C 24 ); structures were computer-matched with the Wiley 7, NIST 08, and FFNSC 1.2 spectra libraries, and their fragmentation patterns were compared with literature data (Adams, 2007).Standard compounds available in our laboratory were also co-eluted with TR-EO to confirm the identity of some essential oil components.

Bacterial strains and antimicrobial assays
The TR-EO minimum inhibitory concentration (MIC) values were calculated by using the broth microdilution method in 96-well microplates (CLSI, 2009). The following standard strains from the ATCC were used: Streptococcus salivarius (ATCC 25975), Streptococcus sobrinus (ATCC 33478), Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49456), Streptococcus sanguinis (ATCC 10556), and Lactobacillus casei (ATCC 11578). Individual 24-hour colonies from blood agar (Difco Labs, Detroit, Mich, USA) were suspended in 10.0 mL of tryptic soy broth (Difco). Standardization of each microorganism suspension was carried out using a spectrophotometer (Femto, São Paulo, Brazil) at a wavelength (l) of 625 nm, to match the transmittance of 81, equivalent to 0.5 on the McFarland scale (1.5 x 10 8 cfu/mL), followed by dilution to a final concentration of 5 x 10 5 cfu/mL. The samples were dissolved in DMSO (Merck, Darmstadt, Germany) at 4 mg/mL and were then diluted in tryptic soy broth (Difco), to yield concentrations between 3.9 and 4000 mg/mL. The final DMSO concentration was 5% (v/v), and this solution was used as a negative control. One inoculated well was included, to control broth adequacy for organism growth. One non-inoculated well free of antimicrobial agents was also included, to ensure medium sterility. Two-fold serial dilutions of chlorhexidine dihydrochloride (CHD) (Sigma-Aldrich, St. Louis) were made in tryptic soy broth (Difco), to obtain concentrations ranging from 59.0 to 0.115 g/mL. These dilutions were used as positive controls. The microplates (96 well) were sealed with parafilm and incubated at 37°C for 24 h. Before the addition of resazurin and the determination of the minimal bactericidal concentration (MBC), an aliquot of the inoculum was aseptically removed from each well presenting no apparent growth and then plated onto tryptic soy agar supplemented with 5% sheep blood. The plates were incubated as described above. After plating, 30 mL of 0.02% resazurinin aqueous solution (Sigma, St. Louis, MO, USA) was poured into each microplate reservoir, to indicate microorganism viability (Porto et al., 2009). The minimal inhibitory concentration (MIC) was determined as the lowest EO concentration capable of inhibiting microorganism growth. Three replicates were made for each microorganism.
The determination of MBC values (the lowest EO concentration in which 99.99% or more of the initial inoculum was killed) and the TR-EO time-kill-assays were conducted against S. mutans only because it is considered one of the primary causative agents of dental caries (Chung et al., 2006). Time-kill assays were performed in triplicate on the basis of the methodology established by D'Arrigo and co-workers (D'Arrigo et al., 2010). Tubes containing TR-EO at final concentrations of 62.5, 125, and 187.5 mg/mL (respectively one, two, and three times the TR-EO minimum bactericidal concentration for S. mutans) were inoculated with the tested microorganism, which resulted in an initial bacterial density of 5 x 10 5 cfu/mL, and then incubated at 37°C. Samples were removed, to determine viable strains at 0, 30 min, 6, 12, and 24 h after incubation, followed by dilution in sterile fresh medium when necessary. The diluted samples (50 mL) were spread onto tryptic soy agar plate supplemented with 5% sheep blood, incubated at 37°C, and counted after 48 h. Time-kill curves were constructed by plotting log 10 cfu/mL vs time. The assays were conducted in triplicate for each concentration and also for the positive (CHD, 0.92 mg/mL) and negative controls (suspension of S. mutans without added TR-EO).
We investigated the antibacterial activity of TR-EO against the main cariogenic bacteria in terms of their minimum inhibitory concentrations (MIC) values compared with chlorhexidine dihydrochloride (CHD, positive control). RI exp : Retention index determined relative to n-alkanes (C 8 -C 20 ) on the Rtx-5MS column. b) RI lit : Retention index from the literature (Adams, 2007). c) Calculated from the peak area relative to the total peak area. d) Compound identification: RL, comparison of the RI with those of the literature (Adams, 2007); RA: relative area (peak area relative to the total peak area in the GC-FID chromatogram), average of three replicates; MS, comparison of the mass spectra with those of the Wiley 7, NIST 08, and FFNSC 1.2 spectral libraries as well as with those of literature (Adams, 2007); Co: co-elution with standard compounds available in our laboratory; t: relative area lower than 0.1%. (31.2 mg/mL), S. mutans (62.5 mg/mL), L. casei (62.5 mg/mL), and S. sobrinus (62.5 mg/mL). Analysis of Figure 2 revealed that (1) at its MBC (62.5 mg/mL), TR-EO exhibited a bactericidal effect against S. mutans, the main cariogenic bacteria, within the first 12 h and that (2) its action became more pronounced after this period. We also constructed time-kill curves using two and three times the MBC value (data not shown). However, we did not verify any significant differences between the periods, indicating that no dose-dependent response effects existed for TR-EO in the assays conditions (p < 0.05). Moreover, the TR-EO and CHD time-kill curve profiles were very similar.

Discussion
Campbell and co-workers have previously investigated the in vitro antimalarial activity and the chemical composition of the essential oil from Tetradenia riparia leaves collected in South Africa (Campbell et al., 1997), and they identified the monoterpenes a-terpineol (22.6%), fenchone (13.6%), b-fenchyl alcohol (10.7%), and perilla alcohol (6.0%) as major constituents. However, Omolo and co-workers identified fenchone (64.8%) and limonene (2.0%) as the main constituents of the repellent essential oil of T. riparia collected in Kenya (Omolo et al., 2004). More recently, Gazim and co-workers investigated the seasonal variation in the chemical composition and the antimicrobial activity of the essential oil from T. riparia leaves collected in Southern Brazil (Gazim et al., 2010) and verified that the most prevalent compounds in different seasons were the monoterpene fenchone; the sesquiterpenes 14-hydroxy-9epi-caryophyllene, cis-muurolol-5-en-4-a-ol, and a-cadinol; and the diterpene calyculone. In the present study, we also detected fenchone and aromadendrene oxide in the essential oil of T. riparia leaves (Gazim et al., 2010), but this is the first time that the presence of (E,E)-farnesol and dronabinol in this essential oil (Table 1) has been reported.
According to Rios and Recio (Rios and Recio, 2005) and Gibbons (Gibbons, 2004), EOs with MIC values higher than 1 mg/mL can be considered poorly active. However, EOs with MIC values below 100 mg/mL are interesting and Antimicrobial activity of T. riparia 523   very promising in the search for new antimicrobial agents. On the basis of these criteria and the data presented in Table  2, TR-EO MIC values ranged from 31.2 to 500 mg/mL against the main causative agents of dental caries. Among all of the tested bacteria, TR-EO gave one of the lowest MIC values against S. mutans (62.5 mg/mL). This is a noteworthy result because very few natural compounds are known to inhibit this microorganism,which is one of the primary causative agents of dental caries (Porto et al., 2009;Saleem et al., 2010).
The very promising MIC value of TR-EO against the main bacterial strain that causes caries disease (S. mutans) prompted us to investigate further aspects of the antimicrobial activity of this natural product, such as its minimal bactericidal concentration (MBC) and time-kill curve (Figure 2). Analysis of Figure 1 revealed that at its MBC, TR-EO exhibited its bactericidal effect within the first 12 h, and its action became more pronounced after this period. It is noteworthy that the time-kill curve profiles of TR-EO and CHD were very similar.
In the literature, two possible action mechanisms have been proposed to explain the biological activities of essential oils. Both mechanisms are associated with the hydrophobicity of monoterpenes and sesquiterpenes, which often are the main chemicals thereof. The hydrophobicity of terpenoids would allow these compounds to permeate the cell membranes easily, hence causing parasites or microorganisms death by affecting their metabolic pathways or organelles (Knobloch et al., 1989). These essential oils themselves could interact with the parasite membrane and cause drastic physiological changes, leading to reduced membrane permeability and culminating in cell death (Bakkali et al., 2008;Knobloch et al., 1989). However, considering the large number of chemical constituents and synergistic or antagonistic interactions between these constituents, the essential oils could also act on cellular targets other than cell membranes, such as lipids and proteins (Bakkali et al., 2008;Borges et al., 2012). In this context, the antimicrobial activity of TR-EO against the selected oral pathogens might be related to the sesquiterpene (E,E)-farnesol, one of the major constituents of the essential oil. This compound has been reported to be active in vitro against S. sobrinus and S. mutans at concentrations of 14 mg/mL and 20 mg/mL, respectively (Koo et al., 2002). The in vivo antimicrobial activity of (E,E)-farnesol in rodent teeth has also been evaluated by Koo and co-workers (Koo et al., 2002). The authors concluded that the topical application of this compound at a concentration of 1 mM caused a decrease in biomass accumulation and prevented S. mutans adherence, thus confirming the potential of (E,E)-farnesol in caries prevention. However, the mechanism by which TR-EO displayed antimicrobial activity and the compounds responsible for the essential oil activity are not clear if we consider only the data obtained in this study.

Conclusion
In summary, the essential oil of T. riparia (TR-EO) displays promising antimicrobial activity against some cariogenic bacteria, including Streptococcus mutans, which is one of the main causative agents of dental caries. The TR-EO chemical composition is slightly different from that reported in previous studies. Taken together, our results suggest that this essential oil might be promising for the development of new oral care products. Further studies aiming to identify the active chemical constituents of TR-EO are underway.