Thymol, menthol and eucalyptol as agents for microbiological control in the oral cavity: A scoping review

Dental plaque is a complex environment that maintains a balance with certain microbial communities; however, this microhabitat can be disturbed by some endogenous species causing disease. An exploratory systematic review was carried out using the PubMed, Scopus, Lilacs, and Science Direct databases, identifying that the thymol, menthol, and eucalyptol compounds present varying antimicrobial activity, intraand interspecies discordance, and a strong antimicrobial intensity on Aggregatibacter actinomycetemcomitans, Candida albicans, Candida dubliniensis, Enterococcus faecalis, Escherichia coli, Lactobacillus plantarum, and Streptococcus mutans, indicating that these phytochemicals can be considered broad-spectrum antimicrobial substances, with an effect on microorganisms linked to oral diseases.

These effects have been evaluated and reported by different techniques, including dilutions and agar diffusion [11], which are chosen according to the nature or polarity of the substance of interest [11,12].
The dilution method includes the broth dilution and agar dilution techniques, which in turn can be prepared in micro-and macrodilutions, while the agar or Kirby Bauer diffusion method comprises the techniques of disk diffusion, well diffusion, and antimicrobial gradient (Etest), whose techniques report the results in inhibition diameters [13].
Due to the incidence and prevalence of oral diseases worldwide [2] and in the country [3], the development of oral hygiene products has been promoted, including toothpastes and mouth rinses with the addition of compounds derived from plants [3,5,10], such as thymol, menthol, and eucalyptol (figure 1), which according to the literature have anti-plaque effects [14][15][16][17][18][19]. However, while bibliographic support is available, it is necessary to clarify some aspects based on a broad literature review, which may be applicable to the experimental design of future research, in order to promote the adequate use of these substances in the formulation of products aimed at oral health care. For this reason, an exploratory systematic review was proposed in order to identify such scientific evidence, analyze it and further clarify the appropriate possible use of these compounds, identifying their origin, procurement techniques, concentrations, and the microorganisms on which they have been tested and effective.  [20].

Thymol (2-isopropyl-5-methylphenol)
Thymol is a monoterpene isomer with carvacrol, soluble in some organic solvents and alcohols, but with low solubility in water. It is colorless, with unpleasant taste and smell [6,[18][19][20]. Thymol is found in vegetable oils obtained from the species belonging to the Lamiaceae family and from other plants of the Ranunculaceae, Verbenaceae, Apiaceae and Scrophulariaceae families. This compound is well accepted among the scientific community because of its wide range of biological properties (antispasmodic, antimicrobial, anticarcinogenic, anti-inflammatory, antiseptic, antioxidant) [18,19,21]; in addition, due to its physicochemical properties, it has good absorption, with rapid metabolization and elimination [18,19,22]. It is not cytotoxic on human cells derived from a human colon adenocarcinoma Caco-2 at concentrations of ≤250 µM [23], but showed mild toxicity at concentrations of ≥100 µM on the human colorectal carcinoma cell line HT29 [24]; in animals, there was a lethal dose (LD50) above 250 mg/kg [25].
It is classified by the European Commission and the Food and Drug Administration (FDA) as "generally recognized as safe" (GRAS) [26,27]; however, it should be taken into account that prolonged exposure and intake of amounts higher than those recommended may be toxic [18,22,23,27]. Concerning antimicrobial properties, thymol has effect on cariogenic bacteria [15,17,28,29], periodontal pathogens [29], enterobacteria [17,28,30], and fungi of the Candida genus [21,31,32] (tables 1 y 2). The mechanisms of action used are exerted on the internal and external cytoplasmic membranes, which alter permeability, inducing their disintegration; in addition, it interferes with the synthesis of proteins of the outer membrane, enzymatic activity of the ATPase, and the citric metabolic pathways [6,12,33].

Menthol (2-isopropyl-5-methylcyclohexanol)
Menthol is a white, crystalline phenolic compound very soluble in alcohol, chloroform, ether, petroleum ether, and hexane, which can be isolated from essential oils extracted from some species of the Mentha genus and can also be synthetically manufactured [9,16,20]. It is classified by the European Commission and the FDA as GRAS [20,27]; however, the inhalation of ≥200 mg and intake of ≥ 8000 mg causes a burning sensation in the mucous membranes of the digestive tract, and other discomforts that in rare cases can become serious [20]. Menthol is used in the pharmaceutical and food industries for products such as mouth rinses, toothpastes, candies, and flavoring due to its organoleptic characteristics (refreshing smell and taste), because it has a short half-life that prevents its accumulation in organisms, being metabolized and quickly eliminated by the lungs [9,2 0]. It also has antimicrobial properties [9,16] reported with strong intensity on A.a [29], C. albicans [34], E.
coli [35], L. plantarum [15], and S. mutans [17] (table 3), and its effect is facilitated by its lipophilic characteristics, which allow it to migrate through the aqueous extracellular medium and interact with the phospholipid membranes, causing damage to these structures and even producing permeabilization and the subsequent leakage of intracellular material, thus destabilizing the microbial agent [33].

Eucalyptol (1, 8-cineole)
Eucalyptol is a liquid, colorless monoterpene soluble with alcohols. It is widely used in the pharmacological, cosmetics, and food industries due to its organoleptic properties (camphor smell and spicy refreshing flavor) [20] and is currently approved by the FDA for food preparation [27,36].
Eucalyptol is quickly absorbed in the gastrointestinal tract and does not show systemic absorption in the lungs; in addition, it did not produce ocular irritation in bovine corneas, its intake does not cause significant renal toxicity in humans, and an LD50 of 2480 mg/kg has been reported by oral administration in rats; in general, it is considered a safe chemical as long as it is taken in doses indicated as normal [20]. This compound shows therapeutic effects in the treatment of bronchial asthma, chronic obstructive pulmonary disease, and gastric inflammation; it improves blood circulation, causes vasodilation and bronchodilation, and has antitumorigenic, hepatoprotective, anti-inflammatory and antimicrobial effects [36].
The antimicrobial activity has been reported in Gram-positive and Gram-negative bacteria, and such effects occur because the compound alters the permeability and function of the cell membrane, inducing intracellular filtration and morphological alteration of the microbial cell [37]. The effect of eucalyptol is dependent on many variables, like dose, the microbial strain and the presence of other substances; thus, on S. mutans it shows MIC/MBC of 250/500 μg/mL, but for C. albicans, E. faecalis, and E. coli it requires MIC >2001 μg/mL (table 3). However, the mixture of eucalyptol, methyl salicylate and thymol on C. albicans decreases the MIC from 125 000 to 62 500 µg/mL, on S. mutans from 250 000 to 125 000 µg/mL and on E. faecalis from 500 000 to 125 000 µg/mL [16], while an additive effect occurs when eucalyptol is mixed with α-terpineol and linalool respectively, which decreases the MIC on E. coli from 7000 to 3000 μg/mL [37].

Methodology
An exploratory scoping review was carried out searching for studies evaluating the antimicrobial (antibacterial and antifungal) activity of the thymol, menthol, and eucalyptol compounds from January 01, 1997 to December 06, 2017 (20 years). A scoping review is a systematic process that helps explore or map the literature reported on a given subject, with results that validate the formulation of hypotheses to respond to research and to guide new studies that complement or strengthen the reported evidence [38,39].

Databases
A systematic search was carried out in order to find studies related to the antimicrobial activity of the thymol, menthol, and eucalyptol compounds in the PubMed, Scopus, Lilacs, and Science Direct databases.

Criteria of inclusion (by reviewing each record's title and abstract) and exclusion (by reviewing the full text of each article)
✓ Language: articles written in English, Spanish, and Portuguese were included.
✓ Topic: articles assessing the antimicrobial activity of the thymol, menthol, and eucalyptol compounds were included.
✓ Type of text: original articles were included. Reviews, books, chapters, and these were not included.
✓ Methodology and results: articles not fully describing the methodology and the respective results were excluded.
✓ Articles not reporting the antimicrobial activity of the thymol, menthol, and eucalyptol compounds individually were excluded.
✓ Articles evaluating the antimicrobial activity with methods other than dilutions (in broth and agar) and diffusion in agar or Kirby Bauer were excluded.
✓ Full text availability: articles with no full text available were excluded.

Extraction and analysis of data
The retrieved records were exported to a bibliographic manager. Duplicates were eliminated in the Zotero, EndNote and Colandr programs, in addition to a final manual revision by the operator. To guarantee the quality and reproducibility of the obtained data, two researchers, experts in the field, carried out independent reviews; discrepancies were resolved by means of a third-party's opinion.
After reading the abstracts, full texts were downloaded and saved in the Mendeley bibliographic manager in PDF. Then the following variables were entered in a Microsoft Office Excel 2013 worksheet: year, country, title, authors, compound evaluated, microorganism tested, strain or isolated used, technique used, result, and detail of the result. With this information, a qualitative synthesis was made, and a descriptive analysis was conducted including percentage frequency distribution and its 95% confidence interval (95% CI) in version 22.0 of the IBM SPSS statistical software.

Results
As a result of the exploratory systematic review, a total of 7065 records were retrieved, applying the      The compounds analyzed in this review show antimicrobial activity at variable intensities, as well as intra-and interspecies discrepancies (tables 2 and 3); it should be noted that these variations may depend on the phytochemicals' mechanism of action [6,10], the technical variations implemented by each research group, and the biological characteristics of the strains used (tables 2 and 3). On C. albicans, thymol at a concentration of 31 μg/mL can alter the morphogenesis of strain ATCC 90028 [49,50], while in exerting such an effect on the same specimen, menthol requires concentrations of 500 μg/mL [50].

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Similarly, menthol has an MBC of 2 μg/mL for E. coli ATCC 0157:H7 [30] and an MBC >2500 μg/mL for ATCC 10798 [29]; eucalyptol has an MBC of 500-1000 μg/mL for the strain ATCC 25175 of S. mutans [51,52], but an MBC of 10 000 μg/mL for menthol [29]. However, when the cells are clustered as biofilms, the effects of antimicrobial agents can vary, as in the case of thymol, which requires concentrations of 250-1000 μg/mL to inhibit cell adhesion and the formation of biofilms in strains ATCC 66396, 90028 and 3153 of C. albicans, while menthol needs concentrations higher than 2000 μg/mL [50]. In S. mutans ATCC 25175 with 100 μg/mL reduces the viability and biofilm formation [53], but for E. faecalis ATCC 4083 concentrations between 25 000 and 100 000 μg/mL are necessary [54,55].

Conclusions
Worldwide, natural products provide benefits thanks to their large chemical diversity; therefore, plants and their derivatives are gaining greater attention, encouraging and promoting scientific support for safe use [6,7,12]. The scientific articles retrieved and included in this scoping review using the described methodology show that the greatest interest exists in developed countries like the United States, Italy, Spain, and Brazil; however, other countries with smaller economies, like Serbia, India, and Iran, also invest on this area of research, as the aromatic, medicinal, and seasonal species are some of the most important natural resources in such territories, obtaining essential oils and various compounds that are terpenic in nature [100]. In Latin America, Brazil is one of the countries with the richest flora diversity and a population that generally accepts the pharmacopoeia; this country is also a leader in the publication of scientific studies on natural products and their derivatives, with tropical conditions similar to those of Colombia, meaning that both countries share the cultivation of certain species because of their geographic location [6,100].
The obtained results validate the implementation of studies with a biotechnological approach for a better use of the antimicrobial properties of the thymol, menthol, and eucalyptol compounds in the control of microbial agents that alter the homeostasis of the oral cavity; however, it is extremely important to take into account a few aspects that maximize the effectiveness and safety of the supplies, such as equivalence of the in vitro and in vivo antimicrobial activity, toxicity, mechanisms of action, and the specificity of the application pathway [12]; also, it should be kept in mind that while this study aimed to analyze each compound to determine their potential as active ingredients [15,34,86], it is important to conduct mixes with other substances and to evaluate the synergistic, neutral, antagonistic, and additive associations that may arise, taking into account the proportions and nature of the mixed substances [12,68], as phytochemicals can enhance their effect in the presence of other substances [64,77,90], but the effect can also be reduced [16,26] or even certain combinations may prove to be unnecessary [16].
These remarks should be considered and may even be essential for effective and safe products like mouth rinses and toothpastes mixed with active compounds that are terpenic in nature; for this reason, the scientific committees of several institutions have consolidated their agreements with research centers, in order to manufacture efficient, effective, safe products that can contribute to oral health by adequately controlling dental plaque without affecting patient's health.
This exploratory systematic review showed that the thymol, menthol, and eucalyptol compounds can be considered as broad-spectrum antimicrobial substances, with effect on microorganisms linked to various human diseases, including some that are closely related to oral diseases. This effect is dependent on a number of variables, such as species and used microbial strain, and highlights the need to standardize parameters related to the concentrations used in research, as the analyzed studies use diverse units for reporting the antimicrobial activity, complicating both the comparison and the analysis of results.
This scoping review showed that the thymol compound does not have significant synergies with the menthol and eucalyptol compounds in the evaluated concentrations; however, in other quantities and in the presence of other substances, with specific containers and carriers, this combination may be more relevant. S. sanguinis and S. sobrinus were the most resistant microorganisms, and eucalyptol was the least effective compound; however, it was important to identify that these compounds have a strong antimicrobial intensity on A.a, C. albicans, C. dubliniensis, E. faecalis, E. coli, L. plantarum, and S. mutans, showing that concentrations of 0.06-100 μg/mL are required for an antimicrobial effect, validating the possibility of formulating products with these compounds, as the FDA indicates that the lower the necessary amount of a substance to exert an effect, the lower the toxicity risks, and therefore the greater the safety for the exposed organisms.