Antibacterial Activity of Essential Oils and Their Isolated Constituents against Cariogenic Bacteria: A Systematic Review

Dental caries remains the most prevalent and costly oral infectious disease worldwide. Several methods have been employed to prevent this biofilm-dependent disease, including the use of essential oils (EOs). In this systematic review, we discuss the antibacterial activity of EOs and their isolated constituents in view of a potential applicability in novel dental formulations. Seven databases were systematically searched for clinical trials, in situ, in vivo and in vitro studies addressing the topic published up to date. Most of the knowledge in the literature is based on in vitro studies assessing the effects of EOs on caries-related streptococci (mainly Streptococcus mutans) and lactobacilli, and on a limited number of clinical trials. The most promising species with antibacterial potential against cariogenic bacteria are: Achillea ligustica, Baccharis dracunculifolia, Croton cajucara, Cryptomeria japonica, Coriandrum sativum, Eugenia caryophyllata, Lippia sidoides, Ocimum americanum, and Rosmarinus officinalis. In some cases, the major phytochemical compounds determine the biological properties of EOs. Menthol and eugenol were considered outstanding compounds demonstrating an antibacterial potential. Only L. sidoides mouthwash (1%) has shown clinical antimicrobial effects against oral pathogens thus far. This review suggests avenues for further non-clinical and clinical studies with the most promising EOs and their isolated constituents bioprospected worldwide.


Results
According to a previously set strategy, literature searches resulted in 1405 articles, of which 25 met the inclusion criteria and were included in the final review after thorough analysis (Figure 1). A total of 22 in vitro studies and three clinical trials addressing the anti-caries properties of EOs and their isolated compounds were selected and will be further discussed herein.

Figure 1.
Flow diagram of the search strategy comprising the identification of potentially relevant material, and preliminary screening and final selection of the studies included in this review (based on PRISMA guidelines). * The leading reasons for exclusion of articles were: clinical trials-"score lower than 3 in Jadad's scale" (see Methods); in vitro studies-lack of critical information on chemical profiling, and methodological shortcomings.

In Vitro Studies
According to the in vitro studies analyzed, there was a predominance of tests with planktonic cultures (Tables 1-6) rather than mono-or multi-species biofilm cultures (Table 7). Of the 22 studies, 5 (22.72%) tested the effect of the EO on streptococci and lactobacilli biofilms. Studies included in qualiquantitative synthesis (total n = 24) In vitro studies (n = 21) Clinical trials: (n = 3)

Crude EOs and Biofilms of Streptococci and Lactobacilli
A total of eight species were tested against biofilm cultures of S. mutans, S. sobrinus and/or L. casei using different assays (Table 7). Interestingly, bioactive fractions of C. sativum and B. dracunculifolia inhibited 90% of S. mutans biofilm formation at concentrations as low as 31.2 μg/mL. Moreover, C. cajucara EO (100 μg/mL) and O. americanum EO (3%) inhibited S. mutans and L. lactis biofilms as effectively as chlorhexidine, used as positive control.
Overall, the majority of studies in this review tested the effectiveness of EO against S. mutans (35 out of 40 studies), followed in lower proportions by S. sobrinus, S. salivarius, S. sanguinis and Lactobacillus spp. As seen in Table 8, just a few studies carried out a comprehensive analysis of the effect of EO against a broad panel of caries-related species.

Randomized Clinical Trials
Three high quality randomized, double-blind clinical trials of herbal interventions with low risk of bias were included in this review ( Figure 2). The EOs from L. sidoides [35,36] and a multi-herbal formulation including Melaleuca alternifolia and Leptospermum scoparium oils (combined with Calendula officinalis and Camellia sinensis extracts) [37], were tested in humans for their effectiveness in reducing the amount of cariogenic biofilm, measured by means of plaque indexes. The experimental period of studies ranged from 1 week to 12 weeks, with different assessment checkpoints and dosing protocols. As seen in Table 9, only individuals treated with 1% L. sidoides EO mouthwash had a statistically significant reduction in their supragingival biofilm levels compared to chlorhexidine group (positive control) and to their baseline condition.
The EO from A. camphorata, B. sulphurea, L. alba, M. glomerata, O. gratissimum and S. guianenses were not chemically characterized by the studies included in this review. Therefore, 21.7% of the selected studies had no chemical control regarding the EO under test. Furthermore, only 60.8% of the studies proceeded with a botanical identification of the aromatic plants that served as source for the EO. Finally, only 56.52% of the studies showed any piece of information about georeferencing of the plant species and 47.82% reported the period of plant collection.

Discussion
Essential oils have stood out as a promising source of bioactive molecules with potential application in the management of dental caries [40,41]. The data presented in this review suggest potential EO and constituents to be further tested as bioactive ingredients of anti-caries formulations. Moreover, the results of the reported chemical assessments of EO-isolated compounds could lead them to be used as chemical markers in future screening. Surprisingly, 20% and 60% of the studies do not provide any chemical or botanical information, respectively, which inevitably results in a biased and inconclusive analysis with reproducibility and traceability issues. Also, despite an understanding of the biological and physicochemical processes associated with the aetiopathogenesis of dental caries [8], great part (88%) of the current evidence on the anti-caries potential of EO is based on in vitro studies rather than clinical trials (see Section 3.3 in this Discussion). Altogether, the benefits and issues related to EO research suggest wide avenues for scientists to work on more comprehensive and trustworthy bioprospection studies.
According to our searches, the majority of in vitro studies have evaluated the effect of EO or isolated compounds against S. mutans, as expected. Considered the most cariogenic of the oral streptococci, S. mutans colonizes the tooth surfaces and produces significant amounts of extra-and intra-cellular polysaccharides [42], being responsible for the initial stage of oral biofilm formation and carious lesions [43]. Nevertheless, other streptococci and lactobacilli species are also implicated on the onset [44] and progression [45] of caries, respectively, thus playing a role in the aetiopathogenesis of this biofilm-dependent disease. An EO of interest to be included in a formulation should be that able to affect bacterial virulence without suppressing the resident oral species, as a more specific therapeutic approach [8]. However, most studies provide just preliminary evidence of anti-caries activity without further assessing the effects of EO on putative virulence factors in cariogenic bacteria (e.g., glycosyltransferase and F-ATPase activity). In addition, the cariogenic biofilm is composed of a multi-species microbial community, in which the predominance of different microorganisms is changed as a function of host, diet and microorganism factors [46]. These aspects are not considered in most studies evaluating only planktonic cultures and, at most, monospecies biofilm cultures.
Next, we provide a brief summary of the plant species whose EO and their isolated compounds were found to have significant in vitro anti-caries potential. Attention is given to the ethnopharmacological knowledge, biological properties and chemical composition. Despite our attempts to make inter-study comparisons, there are underlying distinctions related to extraction methods, georeferencing, seasonality, which should be taken into account.

Promising Essential Oils against Cariogenic Bacteria
Achillea ligustica (Asteraceae) is a small herbaceous plant rich in terpenes that grows in the Mediterranean region and has been used in folk medicine mainly for the treatment of gastrointestinal disorders [47]. The EO from different parts of this plant (inflorescences, leaves and flowers) is also found to have antimicrobial activity, particularly against S. mutans [19,22]. However, as it can be seen in this review, when the major compounds of A. ligustica EO are tested alone (e.g., γ-terpinene, β-pinene, 1,8-cineole, terpinen-4-ol), there is a decrease in their antimicrobial activity, which suggests a synergistic effect of the compounds present in the whole EO. Different EOs from the genus Achillea have been used in the cosmetic and liqueur industry as fragrances and flavoring agents, demonstrating commercial and economic relevance [22].
Baccharis dracunculifolia (Asteraceae) a native plant from Brazil, is widespread in the tropical areas of South America and is the botanical source of Southeastern (or green) propolis [48]. It has been widely used in folk medicine as febrifuge, anti-inflammatory, antiseptic and in the treatment of skin sores and gastrointestinal disorders [49]. The trans-nerolidol-and spathulenol-rich EO from B. dracunculifolia and its active fractions are bacteriostatic and have an in vitro anti-cariogenic activity by disrupting S. mutans biofilm at concentrations as low as 31.25 µg/mL [11].
Croton cajucara (Euphorbiaceae) is a common shrub growing in the Amazonian region commonly used in folk medicine as a tea for ailments such as diarrhea, diabetes and gastrointestinal disorders [50]. Alviano et al. [21] found that the EO of C. cajucara has significant antibacterial activity against S. mutans, S. sobrinus and L. casei in planktonic and monospecies biofilm cultures, unlike its isolated major compound linalool. This result disagrees with others reported in this review showing that linalool is considerably active against S. mutans [19,22,31]; however, it remains controversial.
Cryptomeria japonica (Cupressaceae) is an endemic and widely distributed coniferous plant in Japan, normally used for forestry, whose EO has been reported to have several pharmacological properties including larvicidal [51], antiulcer [52], antifungal [53] and antibacterial [20]. C. japonica EO is another example of how the complex mixture of chemical molecules plays a synergistic role in the antibacterial power of the EO over its isolated major compounds (sabinene, terpinen-4-ol, α-pinene and α-terpineol) [20]. In this review, we found significant inhibitory effects of the leaf EO against caries-related streptococci, warranting further investigation.
Coriandrum sativum (Apiaceae) popularly known as coriander, is an annual small plant whose leaves and seeds are widely used in folk medicine as anti-hypertensive, cholesterol-lowering and digestive stimulant [54], and also as food condiment. Moreover, other biological properties of C. sativum EO have also been reported: antifungal [55,56] antibacterial [11,56], antioxidant [57] and hepatoprotective [58]. The EO from C. sativum leaves contains mostly decanal, trans-2-decenal and 2-decen-1-ol [55], and has been shown to have in vitro anti-cariogenic potential against S. mutans biofilms and to be more active than its chemical fractions [11].
Eugenia caryophyllata (Myrtaceae) is widely cultivated in Indonesia, Sri Lanka, Madagascar, Tanzania and Brazil. E. caryophyllata EO (clove) has been described as having useful antiseptic, analgesic and anaesthetic effects. In community medicine, it serves as a topical pain-relieving and healing agent and in the industry as a fragrance and flavoring substance [59]. The main compounds of clove oil are phenylpropanoids such as eugenol and β-caryophyllene. According to our findings, eugenol was proven to be more active than the EO against S. mutans, i.e., showed lower MIC values. Nevertheless, the crude EO of E. caryophyllata, in general, showed strong antimicrobial activity against streptococci.
Lippia sidoides (Verbenaceae) is a typical shrub commonly found in the Northeastern Brazil, popularly used as topic skin and mucosal antiseptic [60]. L. sidoides EO also has anti-inflammatory, antioxidant and gastroprotective properties [61]. Its antimicrobial activity against cariogenic bacteria has been correlated with the presence of the phenolic monoterpenes thymol and carvacrol [62], and it may be considered of the most scientifically explored medicinal plants in Brazil, whose studies have reached the clinical phase. According to this review, L. sidoides EO showed both strong in vitro antibacterial activity and clinical efficacy as a mouthwash (see Section 3.3 in this Discussion), thus being considered a promising anti-plaque and anti-gingivitis phase II agent [37].
Ocimum americanum (Lamiaceae) popularly known as hoary basil, is an annual herbaceous plant native to Asia and Africa. O. americanum EO is reported to have anti-inflammatory, antinociceptive [63], antibacterial and insecticidal properties [64], and it is considered valuable for the cosmetic industry of soups and perfumes. The findings of this review showed that the leaf EO has strong antimicrobial activity against S. mutans and L. casei, either planktonic or biofilm cultures. The study by Thaweboon and Thaweboon [29] indicated that the 3% leaf EO is as effective as 0.2% chlorhexidine in reducing the bacterial counting of cariogenic biofilm cultures of S. mutans and L. lactis, thus highlighting its potential as an antiseptic agent for oral care. Other studies in vitro and in vivo are now encouraged to elucidate its effects on other aspects related to the aetiopathogenesis of tooth decay (e.g., glucosyltransferase activity, acid production, enamel demineralization, among others).
Rosmarinus officinalis (Lamiaceae) is a culinary evergreen shrub native to the Mediterranean region that has also been used for medicinal purposes to treat bacterial and fungal infections [65]. Unlike the other cases presented thus far, the major compounds of R. officinalis EO (camphor, verbenone, α-pinene, β-myrcene, 1,8-cineole and β-caryophyllene) showed better activity (lower MIC value) against cariogenic bacteria-particularly S. sobrinus and S. salivarius-than the crude EO.

Promising Compounds Isolated from Essential Oils against Cariogenic Bacteria
Generally, the major phytochemical compounds determine the biological properties of EOs [66]. In these cases, the study of isolated compounds is meaningful to concentrate the active principle, enable industrial scale production and allow improvements in the chemical structure using molecular engineering approaches. Here, we provide a summary on menthol and eugenol as the most outstanding compounds isolated from EOs that possess an anti-caries potential.
Menthol is a compound that has raised interest of the pharmaceutical and food industry in the last decades. It is a terpenoid that can be found in the EO of the Mentha spp. genus, such as peppermint, with a crystalline, clear or white-colored aspect (Figure 3). Although there are several isomers of menthol available, only (-)-menthol occurs in nature [34].
In vitro [34,67] and in situ [68] studies have demonstrated that menthol inhibits the growth of both Gram-positive and -negative bacteria and yeasts, and that its mechanism of action may be related to membrane disruption leading to cell leakage. A number of clinical trials [18] have also supported the use of this compound as an ingredient of mouthwash formulations; some of which are already commercially available worldwide. Although menthol has been used more as a flavoring agent than an active principle, it has been proven to have a considerable antimicrobial activity and is considered as GRAS (Generally Regarded as Safe) by the FDA (US Food and Drug Administration). Eugenol is an amphipathic phenolic compound (Figure 4) representing the major constituent of EO from clove (Eugenia caryophillis) and cinnamon (Cinnamomum zeylanicum) leaves [12]. Eugenol has been reported to have antiseptic, antimicrobial, anesthetic, analgesic, antioxidant, anti-inflammatory, and cardiovascular activities [69]. In dentistry, it is used as component of a cement containing zinc oxide for provisional sealing of cavities or as base for definitive fillings [70]. According to our review, eugenol has a promising antimicrobial activity against streptococci, particularly S. mutans, and should be considered as an anti-cariogenic agent to further clinical testing. It is an interesting source of new drugs as it is classified as GRAS by the FDA. This compound has been commercially marketed. In addition to these three compounds, some others indicated in this review arouse attention for their antibacterial power with MIC values lower than 500 µg/mL, as follows: 1,8, cineole, terpinen-4-ol, linalool, β-myrcene, β-caryophyllene and caryophyllene oxide. As such, the presence of these compounds in the EO of a plant could predict its antibacterial properties.

Rational Clinical Use of Essential Oils and Isolated Compounds
Despite the large number of in vitro studies on the antimicrobial activity of EOs, just a few reach the clinical phase and even fewer lead to a commercial product. Indeed, there is a small number of clinical trials reported in the literature aiming at the development of an EO-containing dental formulation.
The most effective way to use the majority of EOs is by external application, such as mouthwashes for dental care. Topical application is generally safe [66] because most compounds are considered as GRAS by the FDA and have been long used in food preparation in several cultures. In case of eventual oral administration of a mouthwash, for instance, most EO compounds (such as (−)-menthol, thymol, carvacrol and eugenol) would be excreted renally or exhaled via the lungs [71,72], and their fast metabolism and short half-life highlight a minimal risk of accumulation in the organism [73]. However, although EOs have the advantage of being usually devoid of long-term cytotoxicity and genotoxic risks [12], the high volatility and chemical instability of some of their compounds in the presence of heat, humidity, light, or oxygen, may negatively impact their clinical use [74].
At the present time, the most popular EO-based formulation used in dental care in Western society is composed of a fixed combination of four EO-derived active ingredients: thymol (0.064%), eucalyptol (0.092%), methyl salicylate (0.060%) and menthol (0.042%). It is considered effective against cariogenic bacteria and relatively safe, although its 21%-27% alcoholic formula used to keep the constituents in solution is still controversial [75]. In some cases, such as with A. ligustica [19,22], C. japonica [20] and C. sativum [56], the synergism of compounds in the EO is critical for its biological properties as opposite to its isolated constituents. Such chemical complexity may favor solubility in vehicles other than ethanol (e.g., propylene glycol), with less likelihood of adverse effects.
According to our analysis, the mouthwash of thymol-and carvacrol-rich L. sidoides EO (ethanol-free) rinsed twice a day is an effective agent to prevent/disrupt the accumulation of cariogenic biofilm [36]. Furthermore, in a previous systematic review [76] we also found that such experimental mouthwash was effective against biofilm-induced gingivitis in adults. Altogether, these findings highlight the therapeutic potential of L. sidoides EO for dental care, but it is important to note that further studies are needed to investigate its effects on other aspects related to tooth decay, such as bacterial acid production, biofilm formation, enamel de-and remineralization, inhibition of glycosyltransferase production/activity, among others. Furthermore, the 10% gel of thymol-and carvacrol-rich L. sidoides EO was not effective to reduce the amount of biofilm in adults compared to a placebo [37], suggesting that the pharmaceutical preparation plays a crucial role in this clinical outcome.
The synergistic association of EOs with other topical agents, e.g., fluoride, should also be considered for the management of dental caries, combining both antimicrobial and remineralization properties. A study by Zero et al. [77] showed that an EO mouthrinse with 100 parts per million fluoride should be effective in promoting enamel remineralization and fluoride uptake, thus providing anti-caries efficacy.
In dentistry, EOs could be useful as preoperative rinses, in periodontal procedures (e.g., sub-gingival irrigation), post-treatment applications, as a conventional mouthwash etc. Nevertheless, the majority of studies in the literature up to date fail to indicate robust and translational data to support the clinical use of novel EOs as ingredients of dental formulations, particularly against dental caries. With that said, this review suggests further research on the EOs and their constituents described earlier due to their favorable potential against streptococci and lactobacilli. In addition, it is important to determine the effects of EO on bacterial virulence factors related to dental caries, such as synthesis of extracellular polysaccharides and ability to survive in and produce acidic environments [8]. The scientific validation of the anti-caries activity of EOs and isolated compounds could provide not only patentable preparations and advances in preventive dentistry, but also commercial value.

Focused Question
The aim of the present review was to answer the specific question: "Based on the current literature, which essential oils and/or isolated compounds are promising anti-caries agents warranting further investigation for clinical use?"

Search Strategy and Selection of the Studies
This systematic review of scientific studies followed the guidelines of the Transparent Reporting of Systematic Reviews and Meta-Analyses (PRISMA statement) [78]. Seven databases were systematically searched for clinical trials and in situ, in vivo and vitro studies (Table 10).

Eligibility Criteria
A systematic selection of the articles was carried out by three independent examiners based on the following inclusion criteria: (1) Biological activity: anti-caries activity against oral microorganisms involved in the etiology and progression of dental caries; (2) Plant material and chemical assessment: essential oils and/or isolated compounds from aromatic plants (their chemical assessment was not a restricted inclusion criteria, instead, it served as a point for discussion); (3) Study design: In vitro, in situ and/or in vivo laboratorial studies (planktonic and biofilm assays); randomized controlled clinical trials (outcome of interest: reduction in the amount of cariogenic biofilm); (4) Methodological quality: For clinical trials, Jadad scale [79] equal to or greater than 3, meeting high quality standards (see Section 4.4 for details); accuracy of outcomes; internal and external validity; (5) Language: Articles written in English, Spanish or Portuguese; (6) Novelty: Novel essential oils-containing dental formulations were included, if not currently marketed. Examiners agreed that in cases of inconsistence the final verdict on which articles should be included in this review would be reached by consensus.

Data Pooling and Analysis
The data were allocated into worksheets to proceed with exploratory analysis according to the study design. For in vitro studies, in order to standardize the susceptibility patterns of microorganisms to essential oils or isolated compounds, we used their minimum inhibitory concentration (MIC) range as a parameter to determine the intensity of antibacterial activity, based on the literature [80] and on our research experience (Table 11). The retrieved data were expressed according to the bacterial species related to different types of tooth decay, in terms of selectivity to specific surfaces: Streptococcus mutans (sulcus and fissure, smooth surface caries-main etiological agent of dental caries) [81]; S. sanguinis, S. sobrinus, S. salivarius play a secondary role and may be recovered from sulcus, fissure and smooth surface caries [82]; Lactobacillus spp. (dentin and root surface caries) [45], either in planktonic or biofilm assays. Table 11. Established parameters based on Minimum Inhibitory Concentrations of essential oils or related chemical constituents.

MIC Range
Intensity of Antibacterial Activity Score <100 µg/mL very strong activity (++++) 101-500 µg/mL strong activity (+++) 501-1000 µg/mL moderate activity (++) 1001-2000 µg/mL weak activity (+) >2001 µg/mL no activity (−) For clinical trials, the data were analyzed based on the CONSORT guidelines for reporting randomized, controlled trials of herbal interventions [83]. Jadad Scale [79] has also been adopted in this review as it checks the validity of evidence on interventions and evaluates methodological quality (randomization, blinding and loss of follow-up). Based on these criteria, we assigned scores to the studies ranging from 0 to 5. Studies reaching a score <3 were considered of poor quality and thus excluded from this review. Several studies, including systematic reviews, have already embraced this validated evaluation tool [84][85][86][87]. Furthermore, we used the risk-of-bias table proposed by Cochrane [88] to check the presence of selection, performance, detection, attrition and reporting biases in the selected clinical trials.

Conclusions
This review attempted to shed light on the anti-caries activity of EOs and their isolated constituents. Certainly, EOs extracted from a variety of aromatic plants worldwide can be considered promising sources of bioactive molecules effective against caries-related microorganisms, particularly S. mutans; however, most of the knowledge in the literature is based on in vitro studies and on a limited number of clinical trials. Overall, the studies have assessed the effects of EO and isolated compounds on microbial growth rather than virulence factors (e.g., bacterial EPS synthesis), which play a key role in the aetiopathogenesis of dental caries. Attention is also drawn to the fact that a number of studies do not provide any chemical or botanical characterization data, raising concern about the reproducibility and accuracy of their findings. Scientific journals should be more stringent in the adoption of criteria for the publication of studies with natural products, particularly EOs. Due to the gap between the in vitro biological properties identified in EOs and their clinical use for the prevention of dental caries, future researches should focus on translational approaches to advance the development of effective anti-caries products containing EO, given that most of them are considered as GRAS by the FDA.