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).

Over the last decade, a number of papers have reported the antimicrobial potential of essential oils (EOs) extracted from plants against oral pathogens (Aguiar et al., 2013; Alviano et al., 2005; Botelho et al., 2007; Filoche et al., 2005; Iscan et al., 2002; Maggi et al., 2009). EOs consist of mixtures of a variety of lipid-soluble and volatile compounds, such as monoterpenes, sesquiterpenes, and 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.

We obtained the essential oil extracted from T. riparia leaves (FV-EO) in 1.09 ± 0.15% yield (w/w). Table 1 depicts the chemical composition of TR-EO, as determined by GC-FID and GC-MS analyses. We identified a total of 37 compounds, with a predominance of oxygenated sesquiterpenes (42.7%). We verified that aromadendrene oxide (1, 14.0%), (E,E)-farnesol (2, 13.6%), dronabinol (3, 12.5%), and fenchone (4, 6.3%) were the major constituents in TR-EO (Figure 1).

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). Table 2 summarizes the obtained MIC values. TR-EO furnished MIC values ranging from 31.2 to 500 μg/mL against the main causative agents of dental caries. The lowest TR-EO MIC values were obtained against S. mitis (31.2 μg/mL), S. mutans (62.5 μg/mL), L. casei (62.5 μg/mL), and S. sobrinus (62.5 μg/mL).

Analysis of Figure 2 revealed that (1) at its MBC (62.5 μg/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.

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 α-terpineol (22.6%), fenchone (13.6%), β-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-9-epi-caryophyllene, cis-muurolol-5-en-4-α-ol, and α-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 μg/mL are interesting and 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 μg/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 μg/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 μg/mL and 20 μg/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.

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.