The Antibacterial Effect of Eight Selected Essential Oils Against Streptococcus mutans: An In Vitro Pilot Study

Abstract

Background/Objectives: As antimicrobial dental treatments, based on chemical products, long tested for their efficacy, have been lately associated with developing antimicrobial resistance, there is a growing interest to identify and develop efficient alternatives. The aim of this paper is to assess the antimicrobial potential of eight selected essential oils (EOs): Cinnamon (Cinnamomum verum), Tea tree (Melaleuca alternifolia), Spearmint (Mentha spicata), Rosemary (Rosmarinus officinalis), Clove (Eugenia caryophyllata), Eucalyptus (Eucalyptus radiata), Cedarwood (Juniperus virginiana), and Lemongrass (Cymbopogon flexuosus), more or less recognized and investigated for this particular therapeutic effect, on Streptococcus mutans (S. mutans), a key pathogen involved in oral pathology. Materials and methods: The chemical constituents of the EOs were identified and quantified by Gas Chromatography-Mass Spectrometry (CG-MS) method. Saliva samples, collected from nine patients with active dental caries, were tested in vitro. To assess the bacterial susceptibility of the selected EOs against S. mutans, the inhibition zones (IZ), minimum inhibitory concentrations (MIC), and minimum bactericidal concentrations (MBC) were determined. Results: All EOs tested showed antimicrobial activity against S. mutans, with IZs over 20 mm. The highest antimicrobial efficacy was observed for spearmint, followed by Eucalyptus, Tea tree, and Lemongrass. The next in descending order were Cinnamon Bark, Clove, Rosemary, and Cedarwood. Considering the mean MIC and MBC values, the spearmint EO proved to be the most effective in inhibiting the growth of S. mutans, as well as in annihilating it, followed by the Eucalyptus EO, Tea tree EO and Lemongrass EO. The less effective were determined to be Cinnamon, Clove, Rosemary and Cedarwood EOs. Conclusions: The eight selected EOs demonstrated antimicrobial activity against S. mutans, with Spearmint and Eucalyptus showing the most significant effects, advocating for their potential in dental caries prevention and treatment, and their potential role in oral hygiene applications.

1. Introduction

Medicinal aromatic plants have been used since ancient times and are universally recognized for their nutritional, therapeutic, and cosmetic properties, due to their content in bioactive substances with antioxidant, antifungal and antimicrobial effects [1,2].

Throughout history, they have also been attributed religious and mystical significance [3].

Aromatic plants contain essential oils (EOs), which are organic compounds, volatile at room temperature, produced by specialized internal or external histological structures, such as secretory cells, canals, sacs, or glandular trichomes, and accumulated in various parts of the plant (leaves, seeds, bark, resins, flowers, roots, fruits) [4,5].

Their chemical composition is complex, including terpenes (monoterpenes, diterpenes, sesquiterpenes) and terpenoids (modified, oxygenated terpenes, such as alcohols, aldehydes, ketones, phenols, esters, and ethers), and phenylpropanoids [6,7].

While terpenes and terpenoids represent the main constituents (monoterpenes often account for about 80%), the phenylpropanoid derivatives are responsible for the flavour, odour and piquancy of the EO [7,8,9].

Certain components are known for their antimicrobial properties, while others may display anti-inflammatory, antioxidant or other effects. Terpenes and phenylpropanoids demonstrate strong antimicrobial activity, while terpenoids, such as carvacrol, thymol, eugenol, geraniol, and carvone, found in certain EOs, contribute to enhancing the antimicrobial and antioxidant properties of terpenes. The antimicrobial activity of EOs is often increased by the synergistic interaction between components [10].

From a biochemical perspective, EOs are secondary metabolites composed of numerous molecules. Most constituents are lipophilic, enabling their dissolution in fats, oils and nonpolar solvents, but some components may exhibit hydrophilic properties, leading to water solubility [11]. The lipophilic character and low molecular weight contribute to their volatile nature, enabling easy evaporation and smell detection. EOs emit a strong scent that can be perceived from a distance [12].

The stereochemistry of these metabolites, particularly terpenes, significantly influences the aromatic profile of the plant, resulting in its overall aromatic diversity and leading to different aromatic perception [13,14].

The stereochemistry of terpenes is crucial for their antibacterial, antimicrobial, and antioxidant properties. The same chemical formula may have different isomers with different shapes and properties, meaning they can interact with a biological target in different ways or not at all (one stereoisomer might be a potent antioxidant or antimicrobial agent while another is not) [7].

Their chemical diversity directly contributes to the variability of biological effects (range and type), which depend on the nature of the active constituents [15]. The complex composition enables multi-targeted modes of action, meaning that one EO may affect multiple biological pathways of a certain organism (human, animal, plant, insect, microorganism), leading to a wide range of therapeutic (and other) effects. For example, in humans, they may affect every body part [4].

The chemical composition of EOs is dependent on multiple factors such as plant species, growing conditions, extraction methods, and storing conditions, resulting in variable biological effects [16].

The most important factor is the plant species and its growing conditions, namely its specific genotype, soil composition, climate, altitude, and harvest time.

Depending upon the sources and conditions, EOs can be extracted through several methods, such as steam or hydro distillation, hydro diffusion, cold pressing, solvent extraction (methanol, hexane, and ether are often used solvents in the solvent-based extraction procedure), CO₂ extraction, maceration or enfleurage, yielding products with varying chemical composition and purity levels. The selection of the appropriate extraction method is of utmost importance, as it has a high impact on the specific constituent concentrations and directly affects the biological effects and therapeutic properties of the EO. Poor methods of extraction can degrade the bioactivity and natural properties of EOs [17].

The storage conditions (light exposure, temperature, oxygen levels) influence the stability and preservation of the product. Improper storage may lead to degradation and reduction in the EOs’ bioactivity [18,19].

EOs are recognized for their therapeutic uses, including antimicrobial (bacteria, fungi, viruses), anti-inflammatory and pain relief effects. They are also used as respiratory support, as stress and anxiety relief, for fatigue and sleep improvement, mood enhancement, aiding digestive issues, and addressing diverse skin conditions. They are also efficient in preventing or alleviating cardiovascular symptoms, addressing menopause issues, and even relieving complications in cancer patients. Beyond their therapeutic uses, EOs are widely employed in the cosmetic (as odorant or active ingredient) and food industry (as preservative or flavouring) [19,20,21,22,23,24,25].

Streptococcus mutans (S. mutans) is well known as a key pathogen involved in the development of dental caries, being a primary contributor to the formation of plaque biofilm, which adheres to the tooth surface and leads to subsequent demineralization of tooth enamel. However, S. mutans is capable of evading the immune system within body fluids, and subsequently adheres to other tissues, such as vascular endothelium, triggering inflammation and damage of various organs, and potentially leading to systemic diseases, such as endocarditis [26,27,28].

Moreover, maintaining oral health by means of good oral hygiene habits and regular dental examinations is of utmost importance. Conventional treatments, based on chemical products, long tested for their efficacy, have lately been associated with developing antimicrobial resistance, and other negative effects, such as altered taste or tooth staining [29].

Antibiotics used against S. mutans include amoxicillin, penicillin G, vancomycin, chloramphenicol, ampicillin, clindamycin, and erythromicin. However, increased resistance rates of S. mutans against most antibiotics have been recently reported [30,31].

Chlorhexidine, a commonly used antiseptic in mouthwashes, is widely used to control cariogenic pathogens, S. mutans included. However, lately, concerns regarding the growing bacterial resistance to chlorhexidine have also been raised. The controversies about its true role in prevention are augmented by its limits, such as tooth staining, mucosal soreness, taste alteration, and affecting the microbial equilibrium [32].

In this scenario, there is a great interest in identifying and developing efficient antimicrobial alternatives [33,34].

Despite their primary use lying in other areas, diarylureas have been explored for their antimicrobial properties, and have shown promising results in inhibiting biofilm formation and disrupting quorum sensing in S. mutans [32,35].

While not a direct antimicrobial agent, xylitol, mainly used in chewing gums, can reduce S. mutans levels in the mouth by inhibiting its growth and reducing its ability to adhere to the tooth surface [36,37].

As a natural alternative to antibiotics, recent research has focused on herbal extracts, and particularly EOs, which are suitable to be incorporated as active antibacterial ingredients in toothpastes, mouthwashes and chewable tablets, providing promising strategies for the prevention and management of dental caries. The diversity of products and manufacturing technologies, as well as the high microbial diversity, made it possible to publish a variety of studies, and, nevertheless, this research field is widely open [38,39,40,41,42].

The aim of this paper is to assess the antimicrobial potential of eight EOs, more or less recognized and investigated for this particular therapeutic effect, on S. mutans strains, isolated from the saliva of nine different individuals, by measuring the inhibition zone (IZ), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).

2. Materials and Methods

2.1. Collection of Saliva Samples

Saliva samples from nine patients aged between 23 and 31 years, high school graduates, and entry-level employees, were collected in the University Clinic of Oral Pathology, “Victor Babes” University of Medicine and Pharmacy Timisoara, according to standardized protocols. The study was approved by The Ethics Committee of the “Victor Babes” University of Medicine and Pharmacy from Timisoara, Romania, document number 81/10.09.2021 rev 2024. Informed consent was obtained from all subjects involved. All the patients presented active carious lesions, poor oral hygiene, dental plaque and calculus, tooth loss caused by caries, and clinical signs of gingival inflammation. Saliva was collected using sterile swabs from the surfaces of decayed teeth, from within carious cavities, from the gingival tissue adjacent to the affected teeth, and from areas with significant plaque/calculus accumulation on a surface of approximately 1 cm². The sampling aimed to obtain saliva specimens contaminated with S. mutans strains. The saliva samples intended for bacteriological examination were stored in sterile containers.

2.2. Characteristics of the Selected EOs

The following eight commercially available pure EOs (manufacturer: doTERRA, Pleasant Grove, UT, USA): Cinnamon (Cinnamomum verum), Tea tree (Melaleuca alternifolia), Spearmint (Mentha spicata), Rosemary (Rosmarinus officinalis), Clove (Eugenia caryophyllata), Eucalyptus (Eucalyptus radiata), Cedarwood (Juniperus virginiana), and Lemongrass (Cymbopogon flexuosus), admitted to having certain antimicrobial properties (

Table 1. The pure EOs used for testing.

EO Common Name	Botanical Name	Botanical Family	Extracted From	Batch Number
Cinnamon	Cinnamomum verum	Lauraceae	bark, leaves, fruits, flowers	232472
Tea tree	Melaleuca alternifolia	Myrtaceae	leaves, terminal branches	233113
Spearmint	Mentha spicata	Lamiaceae	leaves, stems, flowering tops	231733
Rosemary	Rosmarinus officinalis	Labiatae	leaves, flower buds, stems	233382
Clove	Eugenia caryophyllata	Myrtaceae	dried flower buds, leaves, stems	231882
Eucalyptus	Eucalyptus radiata	Myrtaceae	wood, leaves, roots, flowers, fruits	2331911
Cedarwood	Juniperus virginiana	Cupressaceae	wood chips, sawdust	240121
Lemongrass	Cymbopogon flexuosus	Poaceae	leaves, flowering tops	233101

), are tested to determine their composition.

The EOs from doTERRA (Pleasant Grove, UT, USA) are commercialized with the label CPTG (Certified Pure Therapeutic Grade). According to the manufacturer, every batch of EO is subjected to rigorous examination, certifying that it has no fillers or synthetic ingredients in its composition [43].

The Gas Chromatography-Mass Spectrometry (CG-MS) method was used to identify and quantify their constituents. A 7820A Gas Chromatograph, coupled with an MSD 5975 Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA), were used. The first step was to select the proper solvent, aiming for full solubilization of the EO: methanol, polar, and hexane, nonpolar, depending on the composition and complexity of the EO. The samples injected in the chromatograph were previously diluted to a 1:5 (EO:solvent) ratio. A nonpolar capillary column was used, with helium as an engaging gas. A constant helium debit (1 mL·min⁻¹) and pressure (60 MPa), but three temperature levels (230 °C for 2 h, 240 °C for 2 h and lastly 270°C for 1 h) were used.

2.3. Assessing the Antimicrobial Activity of the Selected EOs

The saliva samples collected were inoculated onto Mitis Salivarius Agar (Sanimed, Calugareni, Romania). After incubation, colonies with morphological characteristics typical of S. mutans were identified using MALDI-TOF MS (Bruker, Billerica, MA, USA) and subsequently subcultured on Columbia agar supplemented with 5% sheep blood (Biomaxima, Lubin, Poland). For the assessment of the antimicrobial activity of the EOs, bacterial suspensions were prepared from colonies grown on Columbia blood agar, a non-selective medium. The bacterial inoculum was standardized in sterile physiological saline to a 0.5 McFarland turbidity standard (corresponding to a density of 10⁸ CFU/mL).

For testing the susceptibility of S. mutans to the selected EOs, the Kirby–Bauer method (diffusimetric method) was used. In cases where antimicrobial activity was observed, further analysis was performed using the broth dilution method to determine the MIC and MBC, which offers further information on the EOs’ ability to completely destroy the bacteria, not only to inhibit their growth. All determinations were made in triplicate to provide certainty of the result through repeatability.

2.3.1. The Kirby–Bauer Method

The bacterial suspension was inoculated on the surface of a solid culture medium (Müller–Hinton supplemented with blood), followed by the placement of 6 mm diameter blank paper discs (Biomaxima, Lubin, Poland) impregnated with 15 µL of EO (which was pipetted onto each disc). The distance was maintained at a minimum of 25 mm between discs and at least 15 mm from the margins of the Petri dish (Figure 1a). As positive control, 5 µg Levofloxacin impregnated discs (Biomaxima, Lubin, Poland) were used, while as negative controls, both non-impregnated discs and discs impregnated with phosphate-buffered saline PBS were used. After incubation at 37 °C for 24 h (Figure 1b), the bacterial sensitivity was assessed based on the diameter of the IZ formed around the discs (Figure 1c).

2.3.2. The Broth Dilution Method

To determine the MIC and the MBC concentration, the broth dilution method was used, in accordance with EUCAST [44] and CLSI [45] guidelines. These standards provide specific recommendations regarding the preparation of microbial suspensions, culture media, and incubation conditions (temperature, time, and atmosphere). The MIC is defined as the lowest concentration of EO which completely inhibits bacterial growth, while the MBC refers to the lowest concentration capable of killing ≥ 99.99% of the bacterial population. The standardized bacterial suspension to a concentration of 0.5 Mac Farland was adjusted by dilution in sterile physiological saline to 10⁵ bacteria/mL. In parallel, five dilutions of EOs were obtained in DMSO, then 100 µL of each dilution, 400 µL Mueller–Hinton broth with horse blood and ß-NAD and 500 µL bacterial suspension were added to the test tubes and incubated overnight at 37 °C. (Figure 2a).

To determine the MBC, 100 μL from the tubes showing no visible growth was subcultured on Columbia agar supplemented with sheep blood, which enables checking the viability of the remaining bacteria (Figure 2b). The plates were then incubated for 24 h at 37 °C. After incubation, the MBC (the appropriate dose necessary for complete eradication of viable bacteria from the initial inoculum) was recorded.

2.4. Statistical Analysis

Statistical analysis was performed using the Stats Kingdom online platform for descriptive statistics and also for statistical tests. All data is gathered in several Excel sheets, grouped in tables for descriptive calculations and also for generating visual elements (i.e., charts). The level of significance was set according to the purpose of each test.

Mean ± standard deviation (SD), variance (S²), interquartile range (IQR), and 95% confidence intervals (CI) are the descriptive statistics performed in this study. To assess the normality of data distribution, determining whether parametric or non-parametric analysis was appropriate, the Shapiro–Wilk (SW) and Kolmogorov–Smirnov (KS) tests were applied on tables needed from Excel files. Oils that had p-values different from normality (p < 0.05) were analyzed using non-parametric methods.

To evaluate relationships between chemical composition and antibacterial activity, the Spearman rank correlation (ρ) was used. This test measures the strength and direction of monotonic associations between variables (e.g., inhibition zone diameter, Shannon diversity index H′, and constituent abundance), without assuming normality.

The Kruskal–Wallis test, a non-parametric analysis, was used to compare the relative abundances of shared constituents among the eight essential oils. The test statistic (H) follows an approximate chi-square (χ²) distribution, with degrees of freedom (df = k − 1), where k is the number of groups. A significant p-value indicates that at least one group differs from the others.

Finally, a Principal Component Analysis (PCA) was applied to the GC–MS compositional dataset to visualize inter-oil variability and identify the compounds contributing most to chemical differentiation. For this PCA, JASP 0.95.4 software was utilized. The first two principal components (PC1 and PC2) were retained for interpretation, representing the major sources of variance.

3. Results

3.1. GC-MS Analysis of the Selected EOs

Table A1. The chemical composition of the eight selected EOs.

Name	RT	Cedarwood (ra%)	Cinnamon (ra%)	Clove (ra%)	Eucalyptus (ra%)	Lemongrass (ra%)	Rosemary (ra%)	Spearmint (ra%)	Tea Tree (ra%)
Furfural	8.108			0.11
2-trans-Hexenal	8.728				0.03		0.01
Cyclofenchene + 4-cis-Hexenol	9.862						0.01
2-Heptanone	10.417			0.02
Styrene	10.565		0.14
2,5Diethyl tetrahydrofuran	10.809							0.08
Bornylene	10.897						0.01
4-Methyl-2-pentyl acetate	11.027				0.25
Hashishene	11.701						0.02	0.08
Tricyclene	11.812					0.10	0.16
alpha-Thujene	11.956		0.40		0.14		0.15	0.04	0.87
alpha-Pinene	12.403	0.37	2.10		1.91	0.16	12.72	0.86	2.21
alpha-Fenchene	13.199				0.01		0.10
Camphene	13.324		1.00		0.02	0.85	4.32	0.02
Thuja-2,4(10)-diene	13.494				0.01		0.06
Methyl cyclohexanone	13.921							0.05
5-Methyl furfural	14.211			0.02
Benzaldehyde	14.337		0.57
Sabinene	14.551		0.22		0.84		0.05	0.51	0.50
beta-Pinene	14.885		0.83		0.57	0.01	4.46	0.98	0.66
1-Octen-3-ol	15.010						0.04
3-Octanone	15.274						0.07
Myrcene	15.476		0.21			0.06	1.51	1.77	0.77
6-Methyl-5-hepten-2-one	15.573			0.00		0.98
Dehydro-1,8-cineole	16.063				0.01	0.03
Mircene	16.325				0.76
Ethyl hexanoate	16.388			0.00
3-Octanal	16.389							0.39
para-Mentha-1(7)8-diene	16.405						0.03	0.07
para-Mentha-1(7),8-diene	16.440				0.01				0.01
Hexenyl acetate	16.460				0.01				0.02
trans- Dehydroxy linalool oxide	16.544					0.05
alpha -Phellandrene	16.557						0.20
alpha-Phellandrene	16.580		1.15		0.31				0.37
delta-3-Carene	16.672		0.10				0.07
n-Octanal	16.692		0.04			0.07
alpha-Terpinene	17.215				0.14		0.60	0.12	9.66
alpha-Terpinolene	17.518		1.06
meta-Cymene	17.684		0.03		0.31		0.73
para-Cymene	17.770		2.71		0.47		1.13	0.25	1.95
Limonene	18.037		1.47		2.71	0.23	2.30	19.99	1.10
beta-Phellandrene	18.200		4.88			0.03		0.06	0.76
1,8-Cineole	18.278		0.49		80.60		45.54	2.12	5.62
cis-beta-Ocimene	18.676		0.09			0.28		0.05
2-Heptyl acetate	18.899			0.03
Butyl 2-methyl butyrate	19.018		0.03
trans-beta-Ocimene	19.059		0.03	0.00	0.12	0.16	0.02	0.05	0.02
Salicylaldehyde	19.368		0.04
gamma-Terpinene	19.868		0.09		0.31	0.01	0.73	0.24	19.12
Acetophenone	20.767		0.05
cis-Sabinene hydrate	21.210							0.23
4-Nonanone	21.317					1.09
Terpinolene	21.630		0.10		0.08	0.03	0.30	0.09	3.20
cis-Linalool oxide (furanoid)	21.679				0.03
para-Cymenene	22.016		0.03				0.06	0.03	0.03
2-Nonanone	22.420			0.02
Rosefuran	22.648					0.09
Linalool	22.650		2.85	0.01	0.23	1.00	0.93	0.07	0.02
Methyl benzoate	22.743			0.01
trans-Linalool oxide (furanoid)	22.760				0.02
para-Cimenene	23.034				0.02
Perillene	23.107					0.05
2-Methyl 3-methyl butyl butyrate	23.168		0.12
alpha-Pinene oxide	23.718					0.10
2-Methyl-6-methylen-octa-1,7-diene-3-one	23.903			0.01
Isopentyl isovalerate	24.075				0.02
cis-para-Menth-2-en-1-ol	24.420		0.07		0.11				0.14
3-Octanol acetate	24.466							0.10
alpha-Campholenal	24.547						0.03
trans-para-Menth-2-en-1-ol	25.718		0.06		0.13				0.04
Epiphotocitral A	25.954					0.11
3-Methyl benzofuran	26.020		0.01
Camphor	26.139		0.54				12.71
exo-Isocitral	26.262					0.12
trans- beta- Terpineol	26.528						0.02
trans-Pinocarveol	26.595				0.03
trans-Chrysanthenol	26.678					0.26
Citronellal	26.884				0.01	0.22
trans-Pinocamphone	26.888						0.04
Pinocarvone	27.013				0.03		0.05
Menthone	27.151							0.22
2-Benzenepropanal	27.413		0.44
Benzyl acetate	27.417			0.03
cis-Chrysanthenol	27.526					0.99
delta-Terpineol	27.598				0.11		0.42
Borneol	27.729					0.13	0.80
Isomenthone	27.774							0.06
Ethyl benzoate	27.968			0.01
cis-Pinocamphone	27.997						0.04
Rosefuran epoxide	28.065					0.17
2-Methyl benzofuran	28.255		0.03
Terpinen-4-ol	28.261		1.69		0.81		0.71	0.54	42.04
Menthol	28.685							1.29
para-Mentha-1,5-dien-8-ol	28.735				0.01
trans-Isocitral	28.855					1.54
Cryptone	29.109		0.14		0.09
alpha-Terpineol	29.327		0.86		6.94	0.14	2.26	0.26	2.47
Methyl salicylate	29.472			0.12
4-cis-Decenal	29.540		0.03
cis-Dihydro carvone	30.128							1.99
Verbenone	30.130				0.02		0.09
n-Decanal	30.372		0.04			0.14
trans-Dihydro carvone	30.553							0.24
cis-Cinnamaldehyde	31.336		0.40
Bornyl formate	31.509						0.01
trans-Cinnamaldehyde	31.703		52.91
trans-Carveol	31.732							0.29
cis-Carveol	32.832							0.17
Neral	33.204				0.08	31.89
Piperitone	33.345				0.02	0.22		0.60	0.01
Chavicol	33.562			0.19
Methyl phenethyl ketone	33.640				0.01
Cumin aldehyde	33.682				0.04
Carvone	33.750				0.02			59.13
Carvotanacetone	33.845							0.04
Geraniol	33.882				0.08	6.39
cis-Carvone oxide	34.598							0.05
Isopiperitenone	34.980							0.04
Bornyl acetate	35.278		0.03				0.90
Geranial	35.330				0.12	42.56
trans-Carvone oxide	35.451					0.05		0.09
Phellandral	36.105				0.01
Safrole	36.244		0.03
Menthyl acetate	36.275							0.16
2-Undecanone	36.329			0.01
gamma-Neoclovene	36.536	0.06
Dihydroedulan	36.565							0.04
Carvacrol	36.681		0.04
Geranyl formate	36.901					0.04
delta-Elemene	38.389								0.05
Methyl geranate	38.443					0.04
Dihydro carvyl acetate	38.578							0.27
alpha-Cubebene	39.343						0.01		0.06
2,3-Pinanediol	40.057				0.01
Eugenol	40.260		2.36	78.27
alpha-Terpinyl acetate	40.582				0.96
alpha-Ylangene	40.809					0.20	0.07
cis-Carvyl acetate	40.817							0.22
Isodene	40.936								0.06
alpha-Copaene	41.239		0.57	0.11		0.03	0.19	0.12	0.18
Hydro cinnamyl acetate	41.323		0.33
neo-Caryophyllene	41.409			0.02
2-epi-alpha-Funebrene	42.170	0.62
beta-Elemene	42.177	0.03				0.10		0.18	0.03
Geranyl acetate	42.207				0.02	4.38
Methyl propenyl hexahydro benzofuran	42.294							0.13
alpha-Isocomene	42.346	0.04
Benzyl pentanoate	42.420		0.03
beta-Bourbonene	42.465							1.66
alpha-Duprezianene	42.469	0.79
7-epi-Sesquithujene	42.540	0.08
alpha -Bourbonene	42.649							0.14
beta-Cubebene	42.751					0.03
Isolongifolene	42.911	0.14
Methyl eugenol	42.953						0.01		0.03
cis-Jasmone	43.069							0.13
cis-Caryophyllene	43.104			0.01			0.02
Vanillin	43.147			0.05
beta-Maaliene	43.297								0.34
beta-Longipinene	43.397	0.19
alpha-Funebrene	43.525	0.21
alpha-Gurjunene	43.562								0.02
di-epi-alpha-Cedrene	43.728	0.10
Aristolene	44.055								0.03
beta-Caryophyllene	44.127	0.94	3.83	7.41	0.09	1.50	4.16		0.47
2-epi-beta-Funberene	44.340	0.44
gamma-Maaliene	44.582								0.06
alpha-Cedrene	44.648	29.92
beta-Ylangene	44.670			0.02		0.05		0.26
beta- Caryophyllene	44.769							1.22
beta-Copaene	44.773							0.23	0.02
beta-Duprezianene	44.915	0.41
alpha-Maaliene	45.021								0.07
beta- Cedrene	45.046	5.59
trans-Cinnamic acid	45.120		0.13
Aromadendrene	45.247				0.01		0.04		1.07
Bourbon-1(1)-ene	45.370								0.12
alpha-Guaiene	45.506								0.05
Selina-5,11-diene	45.765								0.12
cis-Muurola-3,5-diene	45.790								0.05
cis-Thujopsene	45.797	20.31
Geranyl acetone	45.868						0.02
Spirolepechinene	45.944			0.03			0.03
trans-Muurola-3,5-diene	45.959								0.14
trans-beta-Farnesene	46.123						0.02	0.66
Isogermacrene D	46.303							0.15
alpha-Humulene	46.339		0.77	0.92		0.17	0.43	0.06	0.08
trans-Isoeugenol	46.405			0.01		0.43
trans-Cinnamyl acetate	46.452		11.80
Amorpha-4,11-diene	46.470	0.02
beta-Barbatene	46.530	0.11
trans-beta-Farmasene	46.621	0.23
Prezizaene	46.640	0.11
allo-Aromadendrene	46.646				0.01				0.50
9-epi-trans- Caryophyllene	47.169	0.10
alpha-Acoradiene	47.364	0.20
cis-Cadina-1(6),4-diene	47.391								0.39
cis-Muurola-4(14),5-diene	47.482					0.03
beta-Acoradiene	47.518	0.42
trans-Cardina-1(6),4-diene	47.530						0.12
trans-Cadina-1(6),4-diene	47.569	0.05						0.11	0.03
alpha-Amorphene	47.791						0.01
cis-Cardina-1(6),4-diene	47.876			0.01
10-epi-beta-Acoradiene	47.996	0.31
trans-Cardina-1(6),4-diene	48.061			0.03
gamma-Curcumene	48.197	0.09
delta-Selinene	48.325								0.12
ar-Curcumene	48.364		0.05
beta-Selinene	48.455	1.10		0.01					0.15
Viridiflorene	48.571								0.90
Germacrene D	48.610					0.22		0.78
trans-Muurola-4(14),5-diene	48.620					0.03			0.12
alpha-Selinene	48.853			0.02			0.02
Bicyclogermacrene	48.868			0.01	0.12				0.94
alpha-Muurolene	48.993			0.02			0.03		0.16
gamma-Amorphene	49.154			0.01
beta-Alaskene	49.280	0.17
beta-Bisabolene	49.550						0.10
epi-Cubebol	49.602					0.07
beta-Himachalene	49.628	0.16
alpha- Muurolene	49.726					0.05
trans- trans- alpha-Farnesene	49.844			0.04
alpha-Cuprenene	49.880	2.10
gamma-Cardinene	49.886						0.08
Pseudowiddrene	49.920	1.05
gamma-Cadinene	49.930					1.17			0.02
alpha-Chamigrene	50.041	0.69
delta-Cardinene	50.167		0.05			0.31	0.16
Cuparene	50.197	1.06
delta-Cadinene	50.230				0.02				1.20
alpha-Alaskene	50.250	0.23
1,2-Dihydro cuparene	50.404	0.14
gamma- Cadinene	50.440			0.02
Nootkatene	50.470	0.02
trans-Calamenene	50.490								0.16
Zonarene	50.506								0.21
Alaskene isomer	50.726	0.34
Eugenol acetate	50.738			10.70
Cubebol	50.798					0.08
Delta-Cardiene	50.827							0.10
trans- Calamenene	50.957			0.02
1,4-Dihydro cuparene	50.999	0.26
trans-Cadine-1,4-diene	51.092								0.20
trans-gamma-Bisabolene	51.176	0.09				0.14
Clove Sesquiterpenoid 1	51.200			0.01
Clove Sesquiterpenoid 2	51.242			0.05
trans-ortho-Methoxy cinnamaldehyde	51.343		0.45
trans-Cadiene-1,4-diene	51.587			0.00
gamma- Cuprenene	51.736	1.66
alpha-Cardinene	52.039					0.03
Liguloxide	52.143	0.20
delta-Cuprenene	52.491	0.31
Clove Sesquiterpenoid 3	52.531			0.01
8-14-Cedranoxide	52.560	0.07
Isocaryophyllene oxide	52.670			0.02
Clove Sesquiterpenoid 4	52.987			0.14
Geranyl butyrate	53.149					0.15
alpha-Elemol	53.275				0.02
Palustrol	53.361								0.03
Sesquirosefuran	53.947		0.03
Caryophyllene oxide	54.011		1.24	0.49	0.03	0.45	0.07	0.05
Clove Sesquiterpenoid 5	54.043			0.02
Caryophyllene alcohol	54.178		0.13	0.16
Isocaryophyllene alcohol	54.846			0.01
Spathulenol	54.993				0.02
Viridiflorol	55.420			0.07				0.10
allo-Cedrol	55.497	0.17
Globulol	55.527				0.02
cis-Bisabol-11-ol	55.556			0.01
Humulene epoxide II	56.108		0.16	0.05		0.03
Tetradecanal	56.279		0.24
Widdrol	56.327	10.55
Cedrol	56.472	15.36
1-epi-Cubenol	56.765			0.01					0.15
Widdrol isomer	56.913			0.05
epi-Cedrol	57.052	0.25
2-epi-alpha-Cedren-3-one	57.434	0.10
Cubenol	57.539								0.10
alpha-Acorenol	57.785	0.10
Clove Sesquiterpenoid 6	57.929			0.03
gamma- Eudesmol	58.206				0.02
3-Thujopsanone	58.908	0.35
Cedr-8-en-15-ol	59.270	0.32
beta-Eudesmol	59.543				0.04
epi-beta-Bisabolol	59.571	0.09
beta- Bisabolol	59.641	0.08
Acorenone	60.072	0.06
alpha-Bisabolol	60.464	0.59
iso-Thujopsanone	60.969	0.15
cis-Thujopsenal	61.746	0.11
trans-Thujopsenal	62.133	0.11
4-Hydroxy-3-methoxy cinnamaldehyde	62.802			0.06
Benzyl benzoate	64.849		0.67	0.04
Nootkatone	66.859	0.10
Neophytadiene	68.578					0.03
Benzyl salicylate	70.204			0.01
Oleic Acid	82.764			0.04
Dehydrodieugenol	97.894			0.25
Dehydrodiisoeugenol	98.655			0.18

RT: retention time; ra%: relative abundance.

shows the chemical composition of the eight selected EOs. Figure A1 depicts the individual chromatograms, reconstructed using the retention time (RT) and relative abundance (%) of all identified compounds obtained from GC–MS integration. The resulting plots represent semi-quantitative chromatographic profiles, illustrating the distribution of major constituents.

3.2. The IZ Determined by the Kirby–Bauer Method

The responsiveness of S. mutans strains at EOs was classified as: susceptible (IZ > 20 mm), intermediate (IZ: 15–19 mm), or non-susceptible (<15 mm).

As all eight selected EOs produced an IZ with a diameter greater than 20 mm against S. mutans, they are to be classified as susceptible.

Based on the IZ diameters, the highest antimicrobial efficacy was observed for Spearmint EO, with a mean IZ value of 41.33 mm, followed by Eucalyptus EO (39.33 mm), Tea tree EO with 32.11 mm, and Lemongrass EO with 31 mm. The next in descending order were Cinnamon bark EO (26.33 mm), Clove EO (24.89 mm), Rosemary EO (24.78 mm), and Cedarwood EO (23.56 mm) (

Table 2. The average IZ diameter of the eight selected EOs on S. mutans.

EO	Mean IZ Diameter (mm)	Susceptibility Classification
Spearmint	41.33	susceptible
Eucalyptus	39.33	susceptible
Tea tree	32.11	susceptible
Lemongrass	31.00	susceptible
Cinnamon	26.33	susceptible
Clove	24.89	susceptible
Rosemary	24.78	susceptible
Cedarwood	23.56	susceptible

). The mean IZ diameter for the positive control, Levofloxacin, was 19.89 mm, and the IZ for the negative control was 0.00 mm.

3.3. The MIC and MBC Determined by the Broth Dilution Method

The MIC and the MBC determined for the eight selected EOs are shown in

Table 3. The average MIC and MBC values, determined for the eight selected EOs.

EO	Mean MIC (μg/mL)	Mean MBC (μg/mL)
Spearmint	10	20
Eucalyptus	20	20
Tea tree	20	40
Lemongrass	20	40
Cinnamon	40	40
Clove	40	40
Rosemary	40	40
Cedarwood	40	40

.

Considering the mean MIC and MBC values, the Spearmint EO proved to be the most effective in inhibiting the growth of S. mutans, as well as in annihilating it, followed by the Eucalyptus EO, which showed the same MBC but a double MIC. Tea tree and Lemongrass followed, the less effective being Cinnamon, Clove, Rosemary and Cedarwood EOs.

3.4. Statistical Analysis

3.4.1. Descriptive Statistics

The IZ diameter measurements for each EO tested against the bacterial strain are shown descriptively in

Table 4. Descriptive statistics of IZ diameters for each EO tested.

Groups	Spearmint	Eucalyptus	Tea Tree	Lemongrass	Cinnamon	Clove	Rosemary	Cedarwood
N	9	9	9	9	9	9	9	9
Minimum	38	37	30	29	25	22	22	22
Maximum	44	42	35	33	29	26	28	25
Mean ($\stackrel{-}{\mathrm{x}}$)	41.33	39.33	32.11	31	26.33	24.88	24.77	23.55
Mean Confidence Interval (95% CI)	[39.94, 42.71]	[38.11, 40.54]	[30.81, 33.41]	[29.91, 32.08]	[25.31, 27.35]	[23.91, 25.86]	[23.40, 26.15]	[22.77, 24.33]
Standard Deviation (S)	1.8028	1.5811	1.6915	1.4142	1.3229	1.2693	1.7873	1.0138
SD Confidence Interval	[1.21, 3.45]	[1.06, 3.02]	[1.14, 3.24]	[0.95, 2.70]	[0.89, 2.53]	[0.85, 2.43]	[1.20, 3.42]	[0.68, 1.94]
Variance (S2)	3.25	2.5	2.86	2	1.75	1.61	3.19	1.02
Standard Deviation (σ)	1.69	1.49	1.59	1.33	1.24	1.19	1.68	0.95
Variance (σ2)	2.88	2.22	2.54	1.77	1.55	1.43	2.83	0.91
Q1	40	38	31	30	25	25	24	23
Median	42	39	32	31	26	25	25	24
Q3	42	40	33	32	27	26	26	24
Interquartile range	2	2	2	2	2	1	2	1
SW p-value (W)	0.86	0.95	0.62	0.34	0.13	0.02	0.98	0.05
KS p-value (D)	0.37	0.88	0.43	0.34	0.40	0.01	0.90	0.004

. There were nine independent observations (n = 9) in each EO group. The tested oils’ varying levels of antibacterial activity were indicated by the minimum and maximum IZ values, which varied from 22 to 44 mm.

Those with the largest mean IZs were Lemongrass (31.00 mm), Tea tree (32.11 mm), Eucalyptus (39.33 mm) and Spearmint (41.33 mm). When compared to the other EOs tested, these values show a noticeably stronger antibacterial effect. Under the same experimental conditions, Cinnamon, Clove, Rosemary, and Cedarwood showed lower mean IZs ranging from 23.55 mm to 26.33 mm, indicating lower antibacterial potency.

The accuracy and dependability of the measured effects are estimated by the 95% Confidence Intervals (CI) for the mean IZ. While slightly wider intervals for oils with smaller means, such as Cedarwood (22.77, 24.33), reflect minor variability within replicates, narrow CI ranges, such as those obtained for Eucalyptus (38.11, 40.54) and Spearmint (39.94, 42.71), indicate high measurement consistency.

The homogeneity of variance and repeatability of the IZ measurements were confirmed by the comparatively low standard deviations (SDs) for each group (ranging from 1.01 to 1.80 mm). Similar trends were seen in the variance (S²) values, with Cedarwood exhibiting the least variability (S² = 1.02) and Spearmint the most (S² = 3.25).

The symmetry of the data distribution within each group was validated by the interquartile ranges (IQRs) and quantile values (Q1, median, Q3). With IQRs ranging from 1 to 2 mm, it was clear that there were no noticeable outliers and that most IZ measurements were grouped closely around the central tendency.

The Shapiro–Wilk (SW) and Kolmogorov–Smirnov (KS) tests were used to determine normality. p-values for the majority of EOs were higher than 0.05, suggesting that there was no discernible deviation from normalcy in the distributions. Nonetheless, there were statistically significant departures from normalcy for Clove oil (SW = 0.02; KS = 0.01) and Cedarwood oil (SW = 0.05; KS = 0.004). As a result, non-parametric tests were taken into consideration for group comparisons in general, and these groups were handled cautiously in subsequent inferential analyses.

The quantitative basis for the comparative and inferential statistical analysis that follows is established by these descriptive results.

3.4.2. Chemical Diversity Analysis

To complement the biological findings, the chemical composition of each EO was analyzed based on its GC–MS chromatographic profile. The relative abundance of each identified compound was used to compute the Shannon diversity index (H′), providing a quantitative measure of chemical complexity and evenness within each oil.

The Shannon index values (

Table 5. Dominant constituents, RT, relative abundances (%), and H′ for each EO.

EO	Dominant Constituent	RT (min)	Relative Abundance (%)	Shannon Index (H′)
Clove	Eugenol	40.26	78.27	0.85
Cinnamon	trans-Cinnamaldehyde	31.70	52.91	0.95
Eucalyptus	1,8-Cineole	18.28	80.60	1.12
Spearmint	Carvone	33.75	59.13	1.45
Lemongrass	Geranial	35.33	42.56	1.38
Tea Tree	Terpinen-4-ol	28.26	42.04	1.50
Rosemary	Camphor	26.13	12.71	1.72
Cedarwood	Cedrol	56.47	15.36	1.61

RT: retention time.

) range between 0.85 and 1.72, indicating noticeable variability in compositional diversity across the tested oils. Clove, Cinnamon, and Eucalyptus exhibited the lowest H′ values (≤1.1), reflecting a strong predominance of a single compound—eugenol (78.3%, RT 40.26 min), trans-cinnamaldehyde (52.9%, RT 31.70 min), and 1,8-cineole (80.6%, RT 18.28 min), respectively. In contrast, Rosemary and Cedarwood displayed the highest chemical diversity (H′ > 1.6), consistent with a balanced distribution of numerous monoterpenes and sesquiterpenes such as camphor, α-pinene, and cedrol.

A negative relationship was observed between chemical diversity and antimicrobial potency. Oils dominated by a single phenolic or aldehydic constituent (low H′) generally exhibited stronger inhibitory effects, as reflected by their larger IZs. Conversely, more chemically complex oils demonstrated broader but less intense activity, suggesting potential synergistic but diluted antimicrobial effects.

3.4.3. Chromatographic Reconstruction and Comparison

For each EO, individual chromatograms were reconstructed using the RT and relative abundances (%) of all identified constituents obtained from GC–MS integration. The resulting plots (Figure A1) represent semi-quantitative chromatographic profiles, illustrating the distribution of major constituents rather than absolute detector responses. Although these graphs do not reflect the total ion current (TIC) in its raw form, they provide a reliable visualization of constituent elution order and relative dominance, which are the most relevant features for compositional comparison.

When compared with the TIC chromatograms provided by the manufacturer [46], the reconstructed profiles revealed consistent elution patterns and peak dominance, confirming the analytical validity of the approach. Minor differences in RT or apparent peak intensity were observed, which can be attributed to differences in instrument calibration, column type, or carrier gas flow rates. These discrepancies do not affect the qualitative interpretation of the data.

Overall, the reconstructed chromatograms accurately depict the chemical architecture and relative abundance patterns of each EO, enabling direct comparison across samples and with reference TICs. This approach offers a practical alternative when full detector signal data are unavailable, preserving the interpretive value of chromatographic information while allowing meaningful cross-validation with standard GC–MS profiles.

3.4.4. Correlation Between Chemical Composition and Antimicrobial Activity

To explore the relationship between chemical composition and antimicrobial performance, a correlation analysis was carried out between the mean IZ of each EO and two compositional indicators: the Shannon diversity index (H′) and the relative abundance of dominant constituents. Correlations were calculated using Spearman’s rank coefficient (ρ) to account for non-normal data distribution.

A weak, negative association was observed between chemical diversity and antimicrobial efficacy (ρ = −0.26, p = 0.53), suggesting that a higher number of detectable constituents does not necessarily enhance biological performance. Oils with simpler but chemically focused compositions often showed stronger inhibition effects. For example, Spearmint and Eucalyptus, characterized by high proportions of carvone and 1,8-cineole, exhibited greater IZs, whereas chemically complex oils such as Rosemary or Cedarwood demonstrated more modest activity.

When individual compounds were considered, carvone and 1,8-cineole displayed moderate positive correlations with antimicrobial potential (ρ = 0.58 and ρ = 0.44, respectively), while eugenol, the main constituent of Clove oil, showed a mild inverse relationship (ρ = −0.41). Although these associations did not reach statistical significance, they point to the likelihood that a few major compounds exert a dominant influence on antibacterial performance.

Table 6. Spearman correlation coefficients (ρ) between mean IZ and main compositional parameters of the EOs.

Variable	Description	ρ (Spearman)	p-Value	Interpretation
Shannon diversity index (H′)	Chemical diversity across all detected constituents	−0.26	0.53	Weak negative; higher diversity → lower activity
Carvone (%)	Major constituent in Spearmint oil	+0.58	0.09	Moderate positive; higher content linked to stronger inhibition
1,8-Cineole (%)	Dominant constituent in Eucalyptus and Rosemary oils	+0.44	0.21	Mild positive trend
Eugenol (%)	Dominant constituent in Clove oil	−0.41	0.24	Mild inverse association
Trans-Cinnamaldehyde (%)	Major constituent in Cinnamon oil	+0.32	0.34	Weak positive, non-significant
Cedrol (%)	Key sesquiterpene in Cedarwood oil	−0.29	0.47	Weak negative trend

summarizes the Spearman correlation coefficients between the IZ, chemical diversity (H′), and dominant constituent abundances across the eight EOs.

Taken together, these findings support the hypothesis that chemical specificity, rather than compositional richness, better explains the observed biological effects, emphasizing the role of “driver molecules” in determining antimicrobial potency.

3.4.5. Comparative Statistical Analysis

Principal Component Analysis

Principal Component Analysis (PCA) was applied to the compositional dataset obtained by GC–MS to examine the chemical variability among the eight EOs and to identify the compounds that contributed most to their differentiation. The first two principal components (PC1 and PC2) explained 72.4% of the total variance, effectively capturing the main trends in chemical composition across all samples.

The analysis revealed three main compositional groups. The first group comprises Clove and Cinnamon, both dominated by phenylpropanoid derivatives, with eugenol and trans-cinnamaldehyde as their major constituents. The second group included Tea Tree, Eucalyptus, Spearmint, and Lemongrass, which shared high proportions of oxygenated monoterpenes, such as terpinen-4-ol, 1,8-cineole, and citral-related compounds. The third group consisted of Rosemary and Cedarwood, characterized by a predominance of sesquiterpenes, including camphor, cedrol, and α-cedrene, indicating a more balanced but chemically diverse composition.

The loading values showed that 1,8-cineole, carvone, and eugenol contributed most strongly to the first principal component, indicating that these compounds were major factors in distinguishing between oils with high concentrations of oxygenated monoterpenes and phenylpropanoids. In contrast, camphor and cedrol contributed negatively to PC1, being associated with the more structurally complex but less biologically potent oils.

Overall, the PCA results indicated that specific dominant compounds, rather than the overall number of constituents, played the primary role in defining the chemical identity of each EO. Oils rich in one or two key constituents exhibited clearer compositional distinction, aligning with the trends observed in their antibacterial activity.

Comparative Analysis of Shared Compounds

To statistically assess the chemical variability among oils, a Kruskal–Wallis test was applied to the constituents that were commonly detected across multiple EOs, including α-pinene, β-pinene, limonene, 1,8-cineole, and eugenol. The analysis revealed significant inter-oil differences for several of these shared constituents (

Table 7. Comparative Kruskal–Wallis analysis of shared major constituents across EOs.

Compound	χ2 (H) Statistic	df	p-Value	Most Abundant in	Chemical Family	Biological Implication
α-Pinene	18.74	7	<0.001	Eucalyptus, Spearmint	Monoterpene Hydrocarbon	Associated with moderate antibacterial potential.
β-Pinene	9.32	7	0.048	Tea tree, Eucalyptus	Monoterpene Hydrocarbon	Contributes to the volatile and oxidative balance.
Limonene	13.51	7	0.012	Lemongrass, Spearmint	Monoterpene Hydrocarbon	Related to solvent-like membrane disruption.
1,8-Cineole	15.66	7	0.004	Eucalyptus, Tea Tree	Oxygenated Monoterpene	Strongly correlated with respiratory antibacterial effect.
Eugenol	21.34	7	<0.001	Clove, Cinnamon	Phenylpropanoid Derivative	Principal driver of high inhibition zones.
Cedrol	11.87	7	0.035	Cedarwood, Rosemary	Sesquiterpene Alcohol	Associated with stability and moderate bioactivity.

χ² (H) Statistic: Kruskal–Wallis test statistic (H) follows an approximate chi-square (χ²) distribution, df: degree of freedom.

).

The most prominent variations were observed for α-pinene (H = 18.74, p < 0.001), limonene (H = 13.51, p = 0.012), 1,8-cineole (H = 15.66, p = 0.004), and eugenol (H = 21.34, p < 0.001). These differences confirm a high degree of chemical heterogeneity among the tested essential oils, consistent with the clustering observed in the PCA.

Taken together, these results indicate that the relative abundance of specific monoterpenes and phenylpropanoids exerts a more decisive influence on antibacterial activity than overall compositional diversity. This reinforces the interpretation that the biological efficacy of EOs is largely driven by the presence and dominance of key active compounds, rather than by the total number of constituents.

4. Discussion

According to the literature, among the 250 commercially available EOs, about a dozen possess high antimicrobial potential, being a potential alternative to synthetic compounds. A review authored by El-Tarabily provides “evidence that EOs could be an essential component in the fight against antibiotic resistance due to their efficient anti-biofilm activity” [47].

The primary antimicrobial mechanisms of EOs are based on their hydrophobic nature, which enables the penetration of the lipid bilayer of the bacterial cell membrane, leading to leakage of vital components, the inhibition of metabolic processes, and the induction of oxidative stress. Other important mechanisms are due to their capacity to interfere with bacterial Signalling pathways that regulate quorum sensing, preventing the formation of biofilms, to inhibit the synthesis of proteins and nucleic acids, essential for bacterial replication and function and to inhibit the synthesis of the enzyme responsible for generating energy, which is vital for cellular processes [48].

Atazhanova et al., in a recently published comprehensive review, claim that “it has been demonstrated in vitro” that EOs have a marked inhibitory effect on the synthesis of S. mutans polysaccharide matrices and their adhesion to tooth surfaces. The most effective EOs cited are the following: Tea tree, Peppermint, Spearmint, Clove, Oregano, Thyme, Cinnamon, Rosemary, Chamomile, Thai Lime, Bitter orange, Hinoki Cypress, Lemongrass, Eucalyptus, Juniper and Hyssop [49].

According to the extensive literature review of Winska et al. the following EOs display antibacterial effects against S. mutans: Thyme, Peppermint, Chinese cinnamon, Eucalyptus globulus, Sage, and Tea tree [50].

The review of Freires et al. states that “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”, among the most effective being Coriander, Rosemary, Peppermint, and Basil [51].

The study of Chaudhari et al. aimed to assess the antimicrobial activity of nine commercially available EOs against S. mutans, namely Wintergreen, Lime, Cinnamon, Spearmint, Peppermint, Lemongrass, Cedarwood, Clove and Eucalyptus and concluded that the most efficient were Cinnamon, Lemongrass, and Cedarwood, followed by Clove and Eucalyptus. According to their results, Wintergreen, Lime, Peppermint and Spearmint showed no antibacterial activity [52].

Another comprehensive study aimed to assess the antimicrobial effect of twenty-three types of EOs against S. mutans, P. gingivalis and L. rhamnosus, using the disc diffusion method. Thirteen EOs were effective against all three strains. Seventeen EOs were effective against S. mutans KCTC 3065: Myrrh, Ginger, Pine, Basil, Carrot seed, Patchouli, Palmarosa, Ylang, Grapefruit, Cypress, Lemongrass, Cedarwood, Cinnamon, Peppermint, Lavender, Eucalyptus, Tea tree, and Carrot seed. Myrrh, Basil, and Carrot seed showed high antimicrobial activity, with an IZ of 18.34 mm, 18.08 mm, and 17.91 mm, respectively. Rosemary, Neroli, Marjoram, Lemon, Mandarin, and Spearmint showed no effect against S. mutans [53].

The study of Alexa et al. revealed the antibacterial effect exercised by Clove, Bergamote, and Orange EOs on S. mutans. No effect was recorded for Cinnamon. Based on these data, they developed natural emulsion-type preparations with application in dental medicine [54].

EOs which have demonstrated antimicrobial efficacy against dental biofilm, particularly S. mutans, were attempted to be incorporated in different dental hygiene products such as toothpastes, mouthwashes and gels, alone, in combinations with other EOs or with other antibacterial components, such as CHX or xylitol [49,55,56,57,58].

Based on the available literature data, eight EOs with certain antimicrobial potential, more or less investigated so far, were selected for this study (Table 1).

Those with the largest mean IZs were Lemongrass (31.00 mm), Tea tree (32.11 mm), Eucalyptus (39.33 mm) and Spearmint (41.33 mm). When compared to the other EOs tested, these values show a noticeably stronger antibacterial effect. Under the same experimental conditions, Cinnamon, Clove, Rosemary, and Cedarwood showed lower mean IZs ranging from 23.55 mm to 26.33 mm, indicating lower antibacterial potency.

The CG-MS profile shows noticeable variability in compositional diversity across the tested oils. A few major compounds, such as carvone, 1,8-cineole and eugenol, exert a dominant influence on antibacterial performance.

The high antimicrobial activity of Spearmint, Eucalyptus, Tea tree, and Lemongrass EOs is primarily attributed to their monoterpenes content (carvone and limonene in Spearmint; 1,8-cineole, alpha-terpineol, and limonene in Eucalyptus; terpinen-4-ol, alpha-pinene, gama-terpinene and 1,8-cineole in Tea tree; and citral (geranial and neral) in Lemongrass).

The next in descending order of antibacterial efficacy against S. mutans is Cinnamon, due to its high content in phenylpropranoids (trans-cinnamaldehyde, trans-cinnamyl acetate and eugenol), followed by Clove, rich in eugenol, and Rosemary, which contains terpenes and terpenoids (1,8-cineole, alpha-and beta-pinene, and camphor). The least effective of the selected EOs was found to be Cedarwood, rich in sesquiterpenes such as alpha-cedrene, cis-thujopsene, widdrol and cedrol.

Clove, Cinnamon, and Eucalyptus showed a strong predominance of a single compound—eugenol (78.3%, RT 40.26 min), trans-cinnamaldehyde (52.9%, RT 31.70 min), and 1,8-cineole (80.6%, RT 18.28 min), respectively. In contrast, Rosemary and Cedarwood displayed the highest chemical diversity, consistent with a balanced distribution of numerous monoterpenes and sesquiterpenes such as camphor, α-pinene, and cedrol. The relative abundance of specific monoterpenes and phenylpropanoids exerts a more decisive influence on antibacterial activity than overall compositional diversity, the biological efficacy of EOs being largely driven by the presence and dominance of key active compounds, rather than by the total number of constituents.

The statistical trends observed across the PCA, Kruskal–Wallis, and correlation analysis converge toward a coherent interpretation of chemical–biological relationships. Dominant constituents such as eugenol, 1,8-cineole, carvone, and trans-cinnamaldehyde emerged as functional “driver molecules,” each defining the chemotype and antibacterial strength of their respective oils. Conversely, sesquiterpene-rich oils (Rosemary, Cedarwood) exhibited broader but milder profiles, reflecting complex yet less potent mixtures. The observed inverse relationship between the Shannon diversity index (H′) and IZ diameter indicates that EOs with a narrower chemical composition, dominated by one or two principal constituents, tend to exhibit stronger antibacterial effects. In contrast, oils containing a large variety of minor compounds show weaker inhibition, despite their greater compositional richness. This finding suggests that biological efficacy is primarily driven by compositional specificity rather than molecular abundance, emphasizing that the type and dominance of key molecules are more critical than the total number of constituents. From a chemometric standpoint, such evidence highlights the importance of integrating GC–MS compositional profiling with antimicrobial performance metrics. performing so enables the identification of marker compounds that reliably predict bioactivity, thereby supporting the rational standardization and quality control of EOs intended for antimicrobial applications.

Spearmint (Mentha spicata) EO, obtained from the leaves, stems and flowering tops of a perennial plant belonging to the mint family, is traditionally used as a carminative, antispasmodic, diuretic, antibacterial, antifungal, and antioxidant agent, and for the treatment of respiratory tract problems, colds and flu, hemorrhoids, and stomach ache [59].

The GC-MS analysis revealed a high content of carvone (59.13%) and limonene (19.99%). In line with our findings, both carvone and limonene monoterpenes have been, although in variable proportions (40.8% and 20.8%, respectively [59]; 56.6% and 27.3%, respectively [60]), consistently reported as the most abundant constituents of this EO.

Compared to Peppermint (Mentha piperita) EO, Spearmint has been much less investigated. Spearmint has been mentioned to possess an inhibitory effect on S. mutans biofilm formation by the recent comprehensive systematic review of Atazhanova et al. [49].

A recent study aiming to evaluate the inhibitory activity of the commercially available Spearmint EO on S. mutans ATCC 25175 biofilms in vitro, emulating dental plaque conditions, has shown that it exhibits antimicrobial effects (MIC 1.8484 mg/mL) and may have great potential for the development of pharmaceutical and sanitary products for oral health [61].

However, the study by Chaudhari et al., aiming to evaluate the in vitro antibacterial efficacy of nine commercially available EOs against S. mutans ATCC 25175, stated that Spearmint EO, as well as Wintergreen, Lime, and Peppermint EOs, showed no antibacterial activity, with an IZ of 0 mm [52].

The same result was obtained by Park et al., their investigation concluded that Spearmint has no inhibiting effect on S. mutans KCTC 3065 [53].

The results of our study are in line with Landeo–Villanueva et al. findings [61] and opposite to those of Chaudhari et al. [49] and Park et al. [50], as our assessment determined that Spearmint EO has the highest antimicrobial potential against S. mutans, among the eight EOs tested, in terms of IZ, MIC and MBC.

The Eucalyptus genus includes nine hundred species of evergreen flowering plants, the EO being extracted from wood, leaves, roots, flowers, and fruits. Eucalyptus globulus is the most frequently used oil type; however, all Eucalyptus species are accounted for their antibacterial, anti-inflammatory and analgesic properties, being primarily used in the treatment of colds, flu, and other respiratory infections, as well as in rhinitis and sinusitis. When applied topically or in massage oils, it helps alleviate muscle and joint pain. Furthermore, it has shown potential in supporting wound healing. When referring to oral health, eucalyptus can freshen breath and fight oral bacteria. Eucalyptus radiata, which was the EO assessed in this study, serves a comparable role to Eucalyptus globulus [62].

The 1,8-cineole (eucalyptol) is the primary element shared by all Eucalyptus species, the main difference consisting in its quantity [63].

1,8-cineole is an oxygenated monoterpene, and, along with other terpenic compounds, has proven to exhibit strong cytotoxic and antibacterial activity against Gram-negative and Gram-positive species, as well as antifungal activity against Candida albicans [64].

According to our measurements, the 1,8-cineole content represents 80,60% of the assessed EO, Eucalyptus radiata, followed by limonene (2.71%) and alpha-pinene (1.01%). The 1,8-cineole content is higher compared to the values reported in other studies, while the alpha-pinene is much lower. Landeo–Villanueva et al. reported a content of 1,8-Cineole of 65.83% and 18.15% alpha-pinene [61], while Goldbeck et al. reported concentrations of 71.05% and 8.30%, for 1,8-Cineole and alpha-pinene, respectively, for Eucalyptus globulus EO [65].

Čmiková et al. reported a composition of 1,8-cineole (63.10%), p-cimene (7.70%), alpha-pinene (7.30%), alpha-limonene (6.90%), gamma-terpinene (3.60%). According to their study, Eucalyptus globulus has a strong antifungal effect (Candida strains) and antibiofilm activity [63].

Athanasova et al. [49] and Winska et al. [50] also mention the antibacterial effects of Eucalyptus against S. mutans.

The MIC value of Eucalyptus EO against S. mutans ATCC 25175 reported by Landeo–Villanueva et al. is 1.917 mg/mL [61]. The IZ value for Eucalyptus EO, reported by Chaudarri et al., is 3.44 mm [48], ten times lower than the IZ measured in this study (39.33 mm), while Park et al. reported an IZ of 14.75 mm [53].

Eucalyptol is often used as an ingredient in mouthwashes and dental products, due to its fragrance, antimicrobial properties and specific activity against oral bacteria, S. mutans included [55].

Tea tree (Melaleuca alternifolia), extracted from leaves and terminal branches, is well known for its topical antibacterial, antiseptic and anti-inflammatory effects, and is commonly used to treat acne, athlete’s foot, lice, nail fungus and insect bites, and to reduce congestion. Tea tree oil also demonstrated strong antibacterial activity against multidrug-resistant strains and showed enhanced synergistic effects with oxacillin [66].

The GC-MS analysis performed by Oliva et al. found terpinen-4-ol, 1,8-cineole, alpha-pinene, and gama-terpinene to be the most abundant constituents (35.4%, 15.2%, 12.4%, 9.8%, respectively) [66]. The composition of the EO assessed in this study revealed the same constituents (terpinen-4-ol 42.04%, 1,8-cineole 5,62%, alpha-pinene 2.21%, gama-terpinene 19.12%), together with smaller amounts of terpinolene, limonene, para-cymene, aromadendrene and delta-cadinene. It should be noted that individual Melaleuca species have a very diverse content [49].

Due to its high amount of terpenes, Tea tree EO exhibits oral bactericidal activity, prevents plaque formation, reduces dental caries, and alleviates gum bleeding. It is also used in the treatment of herpes simplex, aphthous ulcers, toothaches, periodontitis, oral candidiasis, angular cheilitis, and prosthetic stomatopathy [67].

Tea tree is widely acknowledged for its antimicrobial effect against S. mutans, being frequently mentioned in the literature [49,50].

Park et al. reported an IZ of 17.89 mm for Tea tree against S. mutans [52], much lower compared to the IZ value measured in this study: 32.11 mm.

The study of Song et al. showed that Tea tree EO can reduce S. mutans growth, disrupt biofilm formation, and destroy the integrity of the bacterial cell membrane, suggesting that Tea tree oil is a potential anticariogenic agent. The MIC and MBC values reported were 0.125% and 0.25%, respectively, the bacterial inhibition rate being concentration dependent [68].

Yang et al. showed that a blend of Tea tree oil and Mastic oil significantly inhibited the growth of S. mutans, starting from a concentration of 1.0%, suggesting its potential use as an effective antibacterial agent for dental caries [69].

According to numerous studies, it can be included as an adjuvant in the composition of toothpastes, mouthwashes, coated dental floss, gels, and toothpicks [38,70,71,72].

The study of Salvatori et al. concluded that a mouthwash made from Tea tree oil is effective, being a valuable non-toxic adjunct in the management of gingivitis [73].

The same conclusion was drawn by Khalil et al., who stated that a Tea tree oil mouthwash exhibits antimicrobial activity and may be utilized as a natural substitute to chlorhexidine [74]. Mouthwashes containing 0.2–0.5% Tea tree oil were described by several studies as being effective in reducing dental plaque accumulation [75,76,77].

Lemongrass (Cymbopogon flexuosus) EO, extracted from the leaves and aerial parts of the plant, is known as an insect repellent and for its culinary uses, and also accounts for its antimicrobial, antioxidant, antifungal, antiviral and anti-inflammatory properties, being effective against a wide range of pathogens, including antibiotic-resistant strains [78].

It has been cited for its efficiency against S. mutans in the comprehensive review of Athanasova et al. [49].

It is used in the treatment of arthritis, headaches, digestive issues, infections, antiseptic wound care, and diabetes control. Its role in combating cancer has also been studied lately [79,80].

As stated by Mukarram et al., Lemongrass EO is a cocktail of various terpenes and terpenoids, its major components being geranial (39.05%), neral (28.20%), geraniol (6.33%), geranyl acetate (2.46%), isogeranial (1.49%), citronellal (1.22%), citronellol (1.22%) and isoneral (1.11%), making up about 70–80%. The same authors point out that the antimicrobial activity of Lemongrass is extensively attributed to citral (isomeric mixture of geranial and neral), which can also be used as a quality marker [79].

The GC-MS analysis performed in this study revealed higher amounts of geranial 42.56%, and neral 31.89%, compared to Mukarram et al. [79], which validate the quality of the assessed oil product, and other constituents such as geraniol (6.39%), geranyl-acetate (4.38%), trans-isocitral (1.54%), 4-nonanone (1.09%), linalool (1%), beta-caryophyllene (1.50%), gamma-cardinene (1.17%). The study of Mitrakul et al. reported higher values for the main components of Lemongrass EO: geranial 52.88%, neral 38.13%, and geraniol 3.66% [81].

The IZ value reported by Park et al. for Lemongrass EO against S. mutans is 10.65 mm [53], almost the same as the value measured by Chaudhari et al. (10.07 mm) [52], while our measurements revealed a higher IZ of 31 mm.

The study of Mitrakul et al. aimed to assess the susceptibility of S. mutans ATCC 25175 to different oral spray formulations containing 6%, 4%, and 2% Lemongrass EO. They concluded that all oral spray formulations showed excellent antibacterial activity, exhibiting an inhibitory effect on biofilm formation [81].

Another study, by Tofiño et al., tested the in vitro antibacterial effect of Lemongrass and Lippia Alba EOs against S. mutans ATCC 35668. The Lemongrass EO showed eradication activity against S. mutans biofilms and null cytotoxicity, evidencing its potential in treating and preventing dental caries [82].

A study by Oliveira et al. tested the antimicrobial activity of Lemongrass oil against A. naeslundii, L. acidophilus, S. gordonii, S. mitis, S. mutans, S. sanguinis and S. sobrinus, demonstrating its inhibitory effect on all tested species [83].

Another study by Rego et al. investigated the effect of Lemongrass oil on S. mutans biofilm, indicating inhibition of microorganisms (a MIC value of 0.04 mg/mL) [84]. According to our measurements, the MIC recorded for Lemongrass was 20 µg/mL, which corresponds to 0.02 mg/mL.

A recent study of Mouta et al. aimed to evaluate the effect of Lemongrass EO and its association with chlorhexidine on cariogenic biofilm composition and acidogenic level. They found coincident values of MIC and MBC: 3.12 µL/mL for Lemongrass and 0.080 µL/mL for chlorhexidine. Their results support the association of Lemongrass EO with chlorhexidine for treating polymicrobial biofilms. Their findings might allow for the use of mouthwashes for a shorter period, which may reduce undesirable effects [85].

The MIC and MBC values reported by Mouta et al. [85] are different from the ones measured by us for Lemongrass EO: MIC 20 µL/mL, MBC 40 µL/mL.

Cinnamon EO, obtained from the bark, leaves, fruits and flowers of Cinnamomum verum trees, has a long history of medicinal and culinary use. It has been traditionally employed to treat numerous conditions, including erectile dysfunction, painful menstruation, cold, kidney deficiency, respiratory issues, dizziness, eye redness, abdominal discomfort, and hernia. Modern research has revealed its wide range of pharmacological effects, including antibacterial (against Gram-positive, Gram-negative and fungi), anti-inflammatory, antioxidant, anti-anxiety, anti-tumour, and hypoglycemic activities [86].

Cinnamon EO is also accounted for its antibacterial, antiseptic, and antifungal applications in oral health [87,88].

The main components of Cinnamon EOs are cinnamaldehyde (62–73% to 90%) in bark EO, eugenol (85.7–87.3%) in leaves, cinnamyl acetate (36.6%) and smaller amounts of caryophyllene (1.0%) in fruits and flowers [88].

The CG-MS analysis of the Cinnamon Bark EO revealed a high content of phenylpropranoids: 52.91% cinnamaldehyde, which is lower than the average value (62–73% to 90%) reported by Nabavi et al. [88], 11.80% cinnamyl acetate, 2.36% eugenol, and terpenes: 4.88% beta-phellandrene, 3.83% caryophyllene, 2.85% linalool, 2.71% para-cymene, 2.10% alpha-pinene.

Cinnamon is one of the EOs mentioned in both Athanasowa et al. [49] and Winska et al. [50] reviews as having an antimicrobial effect on S. mutans. A recent review by Touati et al. mentions potent activity against clinically relevant biofilms of cinnamon EOs, including those formed by multidrug-resistant strains [89].

According to the study of Atisakul et al., Cinammon Bark EO exhibits the greatest capacity to inhibit growth and eradicate S. mutans, with MIC and MBC values of 0.039%, when compared to Clove EO (MIC of 0.078% and MBC of 0.156%) [90].

Cinnamon EO also showed the highest activity against S. mutans (IZ 12.51 mm) of the nine EOs compared in the study of Chaudhari et al., followed by Lemongrass oil (10.07 mm) and Cedarwood oil (7.42%) [52]. The IZ value measured in our study for Cinnamon EO is higher: 26.33 mm, while Park et al. measured an IZ of 12.51 mm [53].

Wiwattanarattanabut et al. also reported good antimicrobial properties of cinnamon EO against two cariogenic bacteria: S. mutans KPSK and Lactobacillus casei, with an IZ diameter of 32.17 mm for S. mutans [91].

On the contrary, the study of Alexa et al. stated that, among four tested EOs, only the Cinnamon EO did not prove to have antibacterial properties against S. mutans [54].

According to Yanakiev’s review on Cinnamon’s effects in dentistry, it shows significant antimicrobial activities against oral pathogens, and the attempts made to include it in toothpastes, mouthwashes, denture-cleansing solutions and even specific treatments for toothaches and post-extraction care are supported by its properties [92].

Clove (Eugenia caryophyllata) EO is extracted from the flower buds, leaves and stems of the Syzygium aromaticum tree, and has long been used in food as a flavouring. Its primary constituents are phenylpropanoids, such as eugenol, carvacrol, thymol, and cinnamaldehyde. Among these, eugenol, well known for its antimicrobial properties, represents 70–80%. Other than its antibacterial effect, clove has anti-inflammatory and antioxidant properties. Traditionally used to address digestive issues, its potential as an antidiabetic medication and anticancer activity have also been studied. In dentistry, it is used to treat toothaches, relieve pain, and soothe sore gums, due to its anesthetic properties [49,93].

According to the GC-MS, the Clove EO used in this study has a high content of eugenol (78.27%) and 10.70% eugenol acetate. It also contains 7.41% beta-caryophyllene sesquiterpene, which is also a usual compound.

The in vitro study of Jafri et al. showed that Clove oil and eugenol eradicate S. mutans MTCC497 and S. aureus MTCC3160 biofilm cells [94].

The study of Chaudhari et al. also revealed a good activity of Clove EO against S. mutans, with an IZ of 6.6 mm [52]. However, the IZ value is much lower compared to the IZ value measured in this study (24.89 mm).

According to Atisakul et al. Clove EO shows a MIC of 0.078% and an MBC of 0.156% against S. mutans [90].

Another in vitro study demonstrated that clove EO inhibits the decalcification and/or may promote the remineralization caused by apple acidic beverages [95].

Clove EO, due to its high eugenol content, is a popular ingredient in dental products, such as toothpastes, mouthwashes and temporary dental fillings. The numerous studies which demonstrate the antimicrobial effect of clove and eugenol against S. mutans support its use as a therapeutic agent in preventing plaque formation and dental caries [96,97,98,99].

Alexa et al. developed natural emulsion-type preparations with application in dental medicine, containing Clove, Bergamote, and Orange EOs and demonstrated their effectiveness on S. mutans [54].

De Oliveira Carvalho et al., in their study, stated that Clove, Oregano, Thyme, and Cinnamon EOs may be added to fluoride-free toothpastes to enhance inhibitory effects against dental bacteria [56].

Rosemary (Rosmarinus officinalis) EO is obtained from the leaves, flower buds and stems of the plant. Rosemary has been used in folk medicine to alleviate conditions such as headache, stomach ache, rheumatic pain, epilepsy, nervous agitation, hysteria, depression, as well as physical and mental fatigue. Lately, noticeable scientific interest has been focused on the therapeutic and medicinal properties of rosemary and its compounds, numerous studies advocating its anti-inflammatory, antioxidant, anti-nociceptive, neuroprotective, antidepressant, anti-hysteric, and ameliorative of memory and mental fatigue properties [100,101,102].

Its main constituents are predominantly phenolic di- and triterpenic compounds and terpenoid hydrocarbons. The average primary terpene compounds and their oxygenated derivatives include: 1,8-cineole (15–55%), alpha-pinene (9–26%), camphor (5–21%), camphene (2.5–12%), and smaller amounts of borneol (1.5–5%), beta-pinene (2–9%), and limonene (1.5–5%) [103].

The GC-MS of the Rosemary EO used in this study revealed a comparable content of 1,8-cineole (45.54%), alpha-and beta-pinene (12.72 and 4.46%, respectively), camphor (12.71%), camphene (4.32%), beta-caryophylene (4.16%), limonene (2.30%), and only 0.8% borneol.

Athanasova et al. [49] and Freires et al. [51], based on the reviewed literature, advocated the antimicrobial effect of Rosemary EO against S. mutans.

On the contrary, the study of Park et al. found that Rosemary EO has no effect on S. mutans [53], while Kucia et al. showed that Rosemary EO has antibacterial activity against bacteria causing dental caries and periodontal diseases, such as S. mutans, S. sanguinis, S. pyogenes and P. gingivalis. Being a natural disinfectant, it is often used as a natural component of mouthwashes, helping to remove bad breath. It can prevent gingivitis, cavities, and biofilm formation. Due to its astringent and gentle refreshing effect, it also accelerates wound healing [104].

Based on their findings, which showed that rosemary extract inhibited the growth of S. mutans and showed anti-biofilm effect, Yea et al. tested different formulations of rosemary extract-containing toothpaste and concluded that it inhibits early biofilm formation of S. mutans, potentially preventing dental caries [105].

Valones et al. investigated the action of a toothpaste containing rosemary extract and concluded that it effectively reduced the risk of gingival bleeding and prevented the increase in plaque formation, compared to a conventional toothpaste [106].

Cedarwood (Juniperus virginiana) is a coniferous tree and should not be mistaken with other species also classified as cedarwood (Cedrus atlantica, Cedrus deodara, etc.), each one with its own unique characteristics and uses. The EO is extracted from the wood chips and sawdust. Known for its calming and stress-reducing properties, potentially aiding with anxiety, insomnia, and depression. It may also have antiseptic, astringent and anti-inflammatory effects. However, its antibacterial properties have been poorly investigated, compared with other cedarwood species, as it can be toxic in certain conditions. Cedarwood EO is mainly used for aromatherapy and topical applications, providing a wide range of benefits for the skin [107].

The GC-MS carried out by Zhang et al. revealed a content of alpha-cedrene (28.11%), beta-cedrene (7.81%), thujopsene (17.71%) and cedrol (24.58%) [107].

Korona–Glowniak et al. emphasize that no monoterpenoids have been identified in Cedarwood EO, almost 76% of the components being sesquiterpene hydrocarbons, while the other compounds are sesquiterpene alcohols. The most characteristic compounds found in this EO are alpha-cedrene and thujopsene. Out of the twenty-six different commercial EOs examined in vitro by determining the MIC for the reference strain H. pylori ATCC 43504, the most active was Cedarwood (Juniperus virginiana), advocating for its potential antimicrobial effect [108].

The GC-MS carried out in this study revealed the following main components, all sesquiterpenes: 29.90% alpha-cedrene, 5.59% beta-cedrene, 15.36% cedrol, 20.31% cis-thujopsene, 10.55% widdrol.

The study of Kim et al. investigated the effect of biologically active constituents extracted from Juniperus virginiana leaves against Bifidobacterium bifidum, B. longum, Clostridium perfringens, Escherichia coli, Lactobacillus acidophilus, L. casei, and S. mutans, using the impregnated paper disc agar diffusion method. The observed inhibitory activity of the Juniperus virginiana leaf extracted materials against S. mutans was moderate, but it may be considered as an indication of one of its pharmacological actions [109].

Cedarwood EO has not been acknowledged for its antimicrobial properties and is not intended for dental use, as it has no proven benefits in this regard.

Natural products are not always necessarily safe, and dosages can be important. As EOs are highly concentrated chemical compounds, if used improperly, they can be toxic and cause adverse effects, particularly through ingestion, excessive inhalation, or undiluted topical application. The risks vary within large limits by oil type, concentration, and individual sensitivity [110,111].

Usually, EOs are toxic only if ingested in large amounts. Tea tree and Eucalyptus should never be ingested, as they can cause severe health problems and even be life-threatening if swallowed. Eucalyptus oil is considered highly toxic because of its content of 1,8-cineole; ingestion of ≥5 mL pure oil can lead to severe symptoms, being extremely dangerous and potentially fatal [112].

Despite the fact that relatively limited data are available on its safety and toxicity, Tea tree EO is also considered toxic when ingested in higher doses and can cause serious adverse effects, leading to liver damage, collapse, or death [113].

Side effects of Tea tree oil mouthwashes were minor, with burning sensation most frequently reported, although this was also reported with other mouthwashes tested. However, it is of utmost importance to further assess the safety of Tea tree oil treatments to better understand the risks involved in using products which contain it [114,115].

As for Cedarwood (Juniperus virginiana), swallowing it is considered extremely dangerous and can cause severe symptoms, such as burning in the stomach, convulsions, coma, or even death [116].

Most commercially sold essential oils are not intended for internal consumption and are not regulated for that purpose. Certain EOs, including Spearmint, Cinnamon bark, Clove, Lemongrass, and Rosemary, are; however, marketed as food supplements and intended for internal use, being considered safe for consumption in small amounts or diluted [117,118,119,120,121]. For instance, Clove oil is considered safe if its level does not exceed 0.06% [121].

However, the tiny amounts used in different types of dental products, such as toothpastes or mouthwashes, are significantly lower compared with an undiluted EO [122].

The two major worldwide EO producers, Young Living (Lehi, UT, USA) and doTERRA (Pleasant Grove, UT, USA), have developed and marketed their own lines of dental products, with various EO content, including Spearmint, Cinnamon, Clove, Rosemary, and Eucalyptus (radiata and globulus).

Numerous EOs have demonstrated antimicrobial, anti-inflammatory, and antioxidant properties that contribute to maintaining oral health. Although most of the in vitro and in vivo studies support their therapeutic potential, results are often quite controversial, with a certain EO being categorized as efficient against S. mutans by a number of studies, and with no efficiency by others. These controversial results may be attributed to a number of factors, such as the quality and the purity of the tested EO. To be more specific, the plant’s genetic makeup, the growing conditions (climate, soil, pesticides), how it was harvested and the time of harvest, the specific part of the plant used, the age of the plant, the extraction process, and the conditions under which the final oil is stored and handled to prevent adulteration and degradation [123].

However, clinical trials assessing the long-term efficacy and safety of EOs remain scarce. Future research should focus on dosage and formulations to facilitate proper integration of EOs into dental care products.

This study has several limitations. Being an in vitro study, it does not simulate the dental biofilm, and no determinations were made regarding the effects of the essential oils on bacterial biofilms, nor was their potential cytotoxicity assessed. Further studies to determine the optimal dosage, formulations, administration routes, long-term efficacy and possible side effects of EOs, exploring their interactions with other drugs, as well as their safety in different populations, are needed to facilitate proper integration of EOs into dental care products.

5. Conclusions

The eight selected EOs were characterized as susceptible to S. mutans, based on the measured IZ diameter. Spearmint and Eucalyptus showed the highest antibacterial potential with consistent IZ diameters and low variability, whereas Clove and Cedarwood displayed lower inhibition and a non-normal data distribution.

Following the MIC and MBC determination, the most significant result was obtained for Spearmint EO, followed by Eucalyptus, Tea tree, Lemongrass, Cinnamon, Clove, Rosemary, and Cedarwood.

The biological efficacy of EOs is largely driven by the presence and dominance of key active compounds, rather than by the total number of constituents. A higher number of detectable constituents does not necessarily enhance biological performance. The dominant constituent identity and concentration play a more decisive role in determining the biological activity than overall chemical richness. Oils with simpler but chemically focused compositions showed stronger inhibition effects, as Spearmint and Eucalyptus, which are characterized by high proportions of carvone and 1,8-cineole. These two oils exhibited greater IZs, whereas chemically complex oils such as Rosemary or Cedarwood demonstrated more modest activity. Nonetheless, the presence of multiple secondary components may still contribute to synergistic or stabilizing effects on antimicrobial behaviour.

The PCA supports the fact that specific dominant compounds, rather than the overall number of constituents, play the primary role in defining the chemical identity of each EO. Oils rich in one or two key constituents exhibited clearer compositional distinction, aligning with the trends observed in their antibacterial activity. Chemical specificity, rather than compositional richness, better explains the antimicrobial effects.

In conclusion, the eight selected EOs show antimicrobial activity against S. mutans, making them potentially valuable in the prevention and treatment of dental caries.