Six Spain Thymus essential oils composition analysis and their in vitro and in silico study against Streptococcus mutans

Abstract

Background: Streptococcus mutans is a well-known oral pathogen that plays a critical role in the development of dental caries. Many studies have been directed to discover the chemical compounds present in natural products to inhibit the growth and biofilm formation activity of S. mutans. Thymus essential oils exhibit good inhibition on the growth and pathogenesis of S. mutans. However, details about the active compounds in Thymus essential oil and the inhibition mechanism still remain unclear. The aim of this study was to investigate the antimicrobial activity of 6 Thymus species (Three samples of Thymus vulgaris, two samples of Thymus zygis, and one sample of Thymus satureioides essential oils) on S. mutans, to identify the potential active components, and to reveal the underlying mechanism.

Methods: The composition of Thymus essential oils was analyzed by gas chromatography-mass spectrometry. And its antibacterial effect was evaluated based on the bacterial growth, acid production, biofilm formation and genetic expression of virulence factors by S. mutans. Potential active components of the Thymus essential oil were identified using molecular docking and correlation analysis.

Results: GC-MS analysis showed that the major components in the 6 Spain Thymus essential oils were linalool, α-terpineol, p-cymene, thymol and carvacrol. MIC and MBC analysis showed that 3 Thymus essential oils showed very sensitive antimicrobial activity, and were chosen for further analysis. The 3 Thymus essential oil exhibited a significant inhibitory effect on acid production, adherence and biofilm formation of S. mutans and the expression of virulence genes, such as brpA, gbpB, gtfB, gtfC, gtfD, vicR, spaP and relA. Correlation analysis showed that phenolic components, such as carvacrol and thymol, were positively related to DIZ value, which suggests that they are the potential antimicrobial components. Molecular docking between the Thymus essential oil components and virulence proteins also found that carvacrol and thymol exhibited strong binding affinity with functional domains of virulence genes.

Conclusions: Thymus essential oil showed significant inhibition against the growth and pathogenesis of S. mutans depending on their composition and concentration. And phenolic compounds, such as carvacrol and thymol, are the major active components. Thymus essential oil could be used in oral healthcare products as a potential anti-caries ingredient.

Keywords: Gas chromatography-mass spectrometry; Molecular docking; Streptococcus mutans; Thymus essential oil; Virulence factor.

Fig. 1

Correlation of TEOs components class with DIZ Values. Network between TEOs chemical Classes and DIZ. The Spearman correlation coefficients between genes and metabolites were calculated. The gene-metabolite pair shown in the network was chosen based on an adjusted p-value of 1E-5. The circles represent metabolites, whereas the triangles represent genes with a different hue for each co-expression module. The metabolites were colored according to their major classes. The edges were colored based on whether the correlation coefficient between genes and metabolites was positive or negative

Fig. 2

Effects of TEOs on the pH and hydrophobic rate of S. mutans ATCC700610 cultures. A pH; B hydrophobic rate. Values are means ± STD. Statistical analysis was performed by one-way ANOVA with a Waller-Duncan test. “a”; “b”; “c”; and “d” indicate significant differences (p < 0.05)

Fig. 3

Effects of TEOs on biofilm formation inhibition of S. mutans ATCC700610. A crystal violet staining; B Safranine staining. Values are means ± STD. Statistical analysis was performed by one-way ANOVA with a Waller-Duncan test. “a”; “b”; “c”; and “d” indicate significant differences (p < 0.05)

Fig. 4

Effects of TEOs on genes expression of S. mutans ATCC700610. A: brpA; B: gbpB; C: gtfB; D gtfC; E gtfD; F vicR; G spaP; H relA. Values are means ± STD. Statistical analysis was performed by one-way ANOVA with a Waller-Duncan test. “a”; “b”; “c”; and “d” indicate significant differences (p < 0.05)

Fig. 5

Compounds present in all TEOs with good concentration. One Monoterpenoid, three Phenols and six Sesquiterpenoids were selected as ligands based on GC–MS and Pearson correlation

Fig. 6

Molecular docking affinities and interactions of carvacrol, thymol, p-cymene individually and as complex with 8 virulent proteins of S. mutans. A brpA protein with ligands; B gbpB protein with ligands; C gtfB protein with ligands; D gtfC protein with ligands; E gtfD protein with ligands; F vicR protein with lligands; G spaP protein with ligands; H relA proteins with ligands. The colored arrows were presented according to binding affinity from best to comparatively less good (red < brown < green < blue < yellow)