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.