Discussion
The objective of this study was to evaluate the antimicrobial efficacy and ability to inhibit biofilm formation of 16 Mediterranean herb extracts against eight common oral bacterial pathogens and the fungusC. albicans . Previous research in the literature primarily focused on investigating the antimicrobial activity of essential oils derived from these extracts against non-oral bacteria and fungi, with only a couple of studies reporting on their antibiofilm properties against a single oral bacterial strain (Tsai et al., 2007; Rasooli et al., 2008). To the best of our knowledge, this is the first study to examine the antimicrobial and antibiofilm activities of ethyl acetate extracts from the aforementioned herb species against a variety of oral pathogens.
The ethyl acetate extract of A. taygetea demonstrated significant inhibition of obligate anaerobic bacteria. In comparison to the essential oil (EO) of A. taygetea , the ethyl acetate extract exhibited stronger inhibitory effects on Gram-positive facultative anaerobic bacteria than on Gram-negative bacteria (Magiatis et al., 2002).
To date, various studies (Chinou et al., 1994; Demetzos et al., 1997; Anastasaki et al., 1999; Bouamama et al., 2006; Sassi et al., 2008; Hutschenreuther et al., 2010) have confirmed the extended antimicrobial activity of Cistus spp. against diverse “non-oral” bacteria and fungi.
Gram-negative bacteria, being equipped with an outer cell membrane, pose a dense permeability barrier that restricts the entry of lipophilic molecules, rendering them more resistant to Cistus spp. extracts (Pagès and Amaral, 2009; Page, 2012; Li et al., 2015), compared to Gram-positive microorganisms (Sassi et al., 2008; Hutschenreuther et al., 2010). Gram-positive bacteria employ defense mechanisms such as extracellular protease production and chemical modifications of cell membranes or cell walls, which enhance their resistance to antimicrobial agents (Nawrocki et al., 2014; Munita et al., 2015). The use of Cistus tea for rinsing the oral cavity has been shown to reduce adherent bacteria on enamel surfaces in situ(Hannig et al., 2009). Fungi, even after the application of high-concentrated extracts, could not be effectively killed. In contrast, C. albicans , as a representative fungus, could not be effectively eliminated even with high-concentration Cistus spp. extracts (Chinou et al., 1994; Demetzos et al., 1997; Anastasaki et al., 1999; Bouamama et al., 2006).
S. euboea has shown only moderate antimicrobial activity compared to other Sideritis spp. On the other hand, S. syriaca has been studied for its antibacterial properties, both as a decoction and as an essential oil. Despite their different compositions due to polarity, both forms have exhibited activity. The polar decoction contains components such as hypoelatin, isoscutellarein diglucosides, and chlorogenic acid, which contribute to its inhibitory effect on both Gram-positive and Gram-negative bacteria (Goulas et al., 2014).
Several studies have investigated the antibacterial activity of L. stoechas essential oil against both Gram-positive and Gram-negative bacteria (Dadalioglu and Evrendilek, 2004; Kirmizibekmez et al., 2009; Benabdelkader et al., 2011). However, a study specifically focused on oral bacteria found that the essential oil had limited effectiveness, with an MIC of 4 µl / ml (Gursoy et al., 2009).
When comparing different forms of O. vulgare extracts, such as decoction, infusion, and methanol-water extract, with varying amounts of compounds including luteolin O-glucuronide, luteolin 7-O-glucoside, and rosmarinic acid, it was observed that the ethyl acetate extract exhibited enhanced antibacterial activity against Gram-negative bacteria compared to Gram-positive microorganisms (Martins et al., 2014). Leaves of O. vulgare species collected in Mexico contained higher levels of α-pinene and terpinen-4-ol than thymol and carvacrol. (Hernández-Hernández et al., 2014).
The tested oral bacteria showed sensitivity to the rosemary extract, which is consistent with the findings of a study by Takarada et al.(2004) using rosemary essential oil (EO). Rosemary leaves were found to contain higher levels of inhibitory compounds compared to stems. The main components carnosic acid and carnosol exhibited MIC values of 0.09 mg / ml and 0.08 mg / ml against S. mutans and S. sobrinus , respectively. They also demonstrated eradication of E. faecalisat concentrations of 0.07 mg / ml and 0.10 mg / ml, respectively (Bernardes et al., 2010b). Significantly, a polyherbal mouthwash containingR. officinalis extract, among other hydroalcoholic extracts, demonstrated high antibacterial efficacy comparable to 0.2% (w/v) chlorhexidine (CHX) in the treatment of gingivitis in a randomized double-blind placebo-controlled trial (Mahyari et al., 2016). Another clinical study on periodontitis showed that a mouthrinse containing Rosmarinus spp. essential oils, including rosemary, supported the eradication of subgingival biofilm primarily composed of obligate anaerobes (Azad et al., 2016). The potential mechanism behind this could be the inhibition of quorum sensing (QS) signals by rosemary compounds (Szabó et al., 2010).
In the present study, the S. sclarea ethyl acetate extract exhibited high antibacterial activity against obligate anaerobic oral pathogens, surpassing the activity of the EO (Bardajί et al., 2016). Notably, S. sclarea has been shown to inhibit the growth of methicillin-resistant Staphylococcus epidermidis when combined with oxacillin, potentially through the action of diterpenes that inhibit the expression of penicillin-binding proteins (PBPs) (Chovanová et al., 2013).
The ethyl acetate extract of M. aquatica exhibited weak inhibitory effects on facultative anaerobic bacteria, which aligns with the findings of an essential oil (EO) study that showed limited activity against E. coli and S. aureus strains, as well as minimal effect on C. albicans(Mimica-Dukić et al., 2003). It seems that the antimicrobial activity of M. longifolia is not solely dependent on the higher amount of monoterpene hydrocarbons, but rather on a balanced combination of monoterpene hydrocarbons and oxygenated monoterpenes (Schillaci et al., 2013)
In a comparative study of M. longifolia ethyl acetate and aqueous extracts, the ethyl acetate extract demonstrated slightly stronger bactericidal effects against S. aureus(Saeidi et al., 2014), which is consistent with our findings. Interestingly, coccoid-shaped bacteria, such as S. aureus , tend to show less cell damage at MIC values compared to rod-shaped bacteria like E. coli(Hafedh et al., 2010). Although the ethyl acetate extract of M. longifolia inhibited the growth of S. mutans in the current study, it did not completely eradicate the bacteria, unlike a hydroalcoholic extract that achieved a MBC value of 0.1 mg / ml, as reported by Kermanshah et al.(2014). Overall, the extract of M. longifolia demonstrated stronger inhibitory effects compared to the M. aquatica extract, consistent with the findings of Mimica-Dukić et al.(2003).
The antimicrobial activity of the ethyl acetate extract of S. spinosa has not been previously investigated. However, the observed inhibitory effects of the extract on various bacterial species can potentially be attributed to terpenes such as thymol and carvacrol (Conforti et al., 2009).
The ethyl acetate extract of P. cretica exhibited inhibitory effects on the growth of S. oralis and obligate anaerobic bacteria. These results are consistent with a previous study that used an EO of P. cretica and reported relatively high MIC values forS. aureus and E. coli . The observed trend in our study may be attributed, among other factors, to the presence of α-pinene in the extract, which has been shown to have an impact on the growth of these bacterial strains, rather than caryophyllene (Aligiannis et al., 2004).
Investigating the impact of plant collection time on EO activity, it was observed that both S. parnassica and S. thymbra collected in full flower had the lowest MIC values against the foodborne pathogensSalmonella enterica and Listeria monocytogenes(Chorianopoulos et al., 2006). It is worth noting that the relative proportions of carvacrol and thymol, rather than their absolute quantities, seem to play a role in determining the activity, with an optimum ratio near 3:2 (carvacrol:thymol). The ethyl acetate extract of S. thymbraexhibited stronger inhibition against Gram-positive bacteria such asS. aureus and E. faecalis compared to the Gram-negative bacterium E. coli . This finding is not fully consistent with the EOs tested by Markovic et al.(2011) and Giweli et al . (2012), which showed slightly lower MIC values against S. aureus compared to E. coli . (Chorianopoulos et al., 2008).
The ethyl acetate extract of T. longicaulis exhibited significant inhibitory activity against Gram-positive bacteria, including S. aureus and S. mutans . However, this aromatic herb also showed activity against Gram-negative bacteria, as demonstrated with an essential oil in a study by De Martino et al . (2009), which examined herbs from two different regions. Interestingly, the EO with higher quantities of thymol and carvacrol, and nearly a 2:3 ratio, exhibited weaker inhibition against all strains. In general, encapsulating extracts may be a promising technique to enhance their effectiveness, as indicated by the comparison of the more active methanolic extract to the dichloromethane extract (Gortzi et al., 2006).
The available studies on the antibiofilm activity of the tested plant species are limited but provide valuable insights. (Kuźma et al., 2007; Tsai et al., 2007; Walencka et al., 2007; Quave et al., 2008; Rasooli et al., 2008; Schillaci et al., 2013). For example, the components salvipisone and aethiopinone from S. sclareaeffectively reduced biofilm quantities produced by S. aureus andS. epidermidis(Kuźma et al., 2007; Walencka et al., 2007). Rosemary extracts inhibited the glucosyltransferase (GTase) of S. sobrinus , resulting in lower biofilm formation on tooth surfaces (Tsai et al., 2007). Methicillin-resistant S. aureus biofilm was also reduced by a rosemary ethanolic extract (Quave et al., 2008). Even in ten-fold lower concentrations than CHX, rosemary EO as a toothpaste component had higher antibiofilm formation activity against S. mutans(Rasooli et al., 2008).
In summary, the outcomes of this investigation underscore the potent inhibitory capabilities of the Mediterranean herbs under scrutiny against the assessed obligate anaerobic microorganisms found in the oral environment. These findings suggest a promising avenue for developing these herbs into natural agents with antimicrobial and antibiofilm properties, particularly targeted against oral pathogens. The specific composition of compounds within the herbs exerts a pivotal influence on their antimicrobial efficacy, thereby necessitating careful consideration of factors such as collection timing, geographical origin, and extraction methodologies.
Remarkably, the ethyl acetate extracts derived from Rosmarinus officinalis and Origanum vulgare exhibited noteworthy antimicrobial effects against the entire spectrum of oral pathogens examined. Furthermore, the Lavandula stoechas extract demonstrated marked potential in countering biofilm formation byS. mutans . The strategic combination of these plant extracts could conceivably serve as a foundational element in alternative antibacterial formulations, thereby contributing to the mitigation of biofilm-associated oral afflictions like caries and periodontitis.