Evaluation of the Antimicrobial Activity of Some Components of the Essential Oils of Plants Used in the Traditional Medicine of the Tehuacán-Cuicatlán Valley, Puebla, México

In Tehuacán-Cuicatlán valley (Mexico), studies have been carried out on the essential oils of medicinal plants with antimicrobial activity and it was found that they present compounds in common such as: α-pinene, β-pinene, carvacrol, eugenol, limonene, myrcene, ocimene, cineole, methyl salicylate, farnesene, and thymol. The goal of this study was to assess the antimicrobial activity of essential oils’ compounds. The qualitative evaluation was carried out by the Kirby Baüer agar diffusion technique in Gram-positive bacteria (11 strains), Gram-negative bacteria (18 strains), and yeasts (8 strains). For the determination of the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), the agar dilution method was used. All the evaluated compounds presented antimicrobial activity. The compounds eugenol and carvacrol showed the largest inhibition zones. Regarding yeasts, the compounds ocimene, cineole, and farnesene did not show any activity. The compounds eugenol, carvacrol, and thymol presented the lowest MIC; bactericidal effect was observed at MIC level for S. aureus 75MR, E. coli 128 MR, and C albicans CUSI, for different compounds, eugenol, carvacrol, and thymol. Finally, this study shows that the essential oils of plants used by the population of Tehuacán-Cuicatlán valley share compounds and some of them have antibacterial and fungicidal activity.


Introduction
One of the major health concerns today in the world is the emergence of new microbial strains resistant to the chemical substances used for their control [1]. During the last years, new strains resistant to commonly used antibiotics have appeared; for example, Candida auris is today an emergent and extremely dangerous strain difficult to control [2,3], as well as some other strains of Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Streptococcus pneumoniae, and Vibrio cholerae, among others, causing serious health problems that are difficult to control and causing global concerns on the subject.
On the other hand, the essential oils of plants have been developed as a commercial and pharmacological alternative for the treatment of various diseases, and their importance is based on the selective effects towards resistant strains that are recently reported. For example, it has been observed that clove oil, for which main component is eugenol, has antibacterial activity affecting respiratory metabolism, structural changes of DNA, and cell membrane permeability [4,5].
Therefore, it is important to carry out studies that validate the use of essential oils and to know the components that confer antimicrobial activity.
In this way, this study aims to evaluate the antimicrobial activity of several compounds in the essential oils of plants used in the Mexican Traditional Medicine of the Valley of Tehuacán-Cuicatlán, Puebla, that include α-pinene, β-pinene, carvacrol, eugenol, limonene, myrcene, ocimene, cineole, methyl salicylate, farnesene, and thymol.

Qualitative Evaluation of Antibacterial Activity
The results obtained in the evaluations of the antibacterial activity are shown in Tables 1-3. As it can be seen, bacterial strains (Gram-positive and Gram-negative bacteria) and yeasts were sensitive to the tested compounds, and the ones that showed the highest inhibition zones for Gram-positive and Gram-negative bacteria were carvacrol and eugenol; α-pinene, β-pinene, and myrcene were the compounds that presented the lowest inhibition zones (Tables 1 and 2). Regarding yeasts, the ocimene, cineole, and farnesene compounds did not show activity on any yeast strain (Table 3).
Considering the size of the inhibition zone with respect to the bacterial group (Grampositive and Gram-negative) and yeast, an analysis of variance (ANOVA) was carried out. It can be observed that there are no significant differences (p < 0.05), i.e., the compounds inhibited the growth of both bacterial and yeast types.

Quantitative Evaluation
Once the antimicrobial activity of the different compounds had been verified, the minimum inhibitory concentrations (MIC) were determined. The results obtained are presented in Tables 4-6.
The compounds carvacrol and eugenol presented the smallest minimum inhibitory concentrations for the bacterial strains (Tables 4 and 5), i.e., low concentrations were required to drastically inhibit growth (0.03 to 0.75 mg /mL). The α-pinene and β-pinene compounds presented MICs above 4 mg /mL, these being the highest values for both bacterial types. The compounds carvacrol, eugenol, and thymol presented the lowest MICs in yeast (Table 6), and low concentrations of 0.062 to 0.25 mg/mL were required to drastically inhibit the growth of challenged populations. The other compounds that showed activity presented concentrations greater than or equal to 2 mg/mL.
As it can be seen in Tables 4-6, six of the tested compounds showed activity in multi-resistant strains (eugenol, limonene, carvacrol, methyl salicylate, farmesene, and thymol).

Time-Killing Curves
Once the MIC, MBC and MFC parameters were established, the effects of the compounds with the highest antimicrobial activity on a Gram-positive bacterium (S. aureus 75 MR), a Gram-negative bacterium (E. coli 128 MR) and a yeast (C albicans CUSI), for the compounds eugenol (Figure 1a-c), carvacrol (Figure 2a-c), and thymol (Figure 3a-c) were evaluated (the strains were chosen for being multi-resistant and the compounds for presenting the lowest MIC values).

Time-Killing Curves
Once the MIC, MBC and MFC parameters were established, the effects of the compounds with the highest antimicrobial activity on a Gram-positive bacterium (S. aureus 75 MR), a Gram-negative bacterium (E. coli 128 MR) and a yeast (C albicans CUSI), for the compounds eugenol (Figure 1a  For the eugenol compound in the bacterial population of S. aureus 75MR, a bacteriostatic effect was observed at MIC level and a bactericidal effect in MBC at the first hour of exposure. For the populations of E. coli 128 MR and C. albicans, a bactericidal or fungicidal effect was observed on MIC level at 2 h and 4 h, respectively (Figure 1a-c).
Carvacrol completely inhibited bacterial growth at 4 h for the strain of E. coli 128 MR; for S. aureus 75MR, the growth inhibition occurred at 2 h, and for the yeast strain of C. albicans CUSI, at 4 h (Figure 2a-c).
The compound thymol completely inhibited bacterial growth at the first hour of exposure for E. coli 128 MR, while for S. aureus 75MR, it was at 4 h, and for the C. albicans strain CUSI, the inhibition occurred at 2 h (Figure 3a-c).
As shown in the figures, carvacrol and thymol compounds presented a bactericidal or fungicidal behavior on the bacterial and yeast growth curves at MIC level.
It can be of interest that these compounds are good antimicrobial agents, since at different concentrations, they interrupted the growth of a bacterial population of E. coli 128 MR (Gram-negative), S. aureus 75MR (Gram-positive), and C. albicans CUSI (yeast strain).
Regarding the sensitivity of yeast to the essential oil compound exposure, it was observed that they were more sensitive to carvacrol, presenting the lowest MIC values (0.03-0.06 mg/mL), followed by carvacrol (0.03-0.25 mg/mL), and finally eugenol (0.125-0.25 mg/mL).

Discussion
The results show that the use of plants and their essential oils used in traditional medicine in the Tehuacan-Cuicatlan valley Puebla can be validated, since several compounds have antimicrobial activity against Gram-positive, Gram-negative bacteria, and yeasts. Several essential oils compounds are lipophilic and can interact with bacterial cytoplasmic membranes, increasing membrane permeability [4,[22][23][24][25].
The compounds eugenol, carvacrol, and thymol showed the highest antimicrobial activity against Gran positive bacteria, Gran negative bacteria, and yeast. Statistical analysis revealed no significant differences between the compounds and the different groups of microorganisms (bacteria and yeast), indicating that the essential oil compounds can act in different ways. Some authors mention that the sensitivity of Gram-negative bacteria to the different essential oil compounds is due to their outer membrane of lipopolysaccharides, which restricts the diffusion to hydrophobic compounds [15,[26][27][28].
The structural differences between the compounds are also the cause of the different activities observed. Myrcene (7-methyl-3 -methylene-1,6-octadiene) and ocimene (3,7dimethyl-1,3,6-octatriene) differ in the position of a double bond (C-3), causing one to present activity on yeast (myrcene) and the other not (ocimeno); carvacrol and thymol differ in the position of a hydroxyl group (in thymol is in ortho position, in carvacrol it is in meta position), causing the deterioration of enzyme systems by binding to the active sites of the enzymes responsible for producing energy and synthesis of structural components, and the destruction or inactivity of genetic material [17,29,30]. Thymol and carvacrol are lipophilic and can enter between the fatty acid chains that make up the lipid bilayers of the membrane, altering the fluidity and permeability of cell membranes; in the case of yeast, compounds such as carvacrol can affect the regulation and the functioning of membrane enzymes that catalyze the synthesis of a series of important polysaccharide components of the cell wall, such as β-glucans, chitin, and mannan, which are involved in cell growth and morphogenesis [14,15].
In regards to time-killing curves, it was observed that the effect of eugenol, at MIC level on the populations of E. coli 128 MR and C. albicans CUSI, showed a bactericidal and fungicidal effect at 2 and 4 h, respectively. Eugenol showed a bactericidal effect against S. aureus 75MR after 1 h of contact. Carvacrol and thymol showed a bactericidal and fungicidal effect at MIC level. This behavior is characteristic of a multiple mechanism of action [31].
Although the antimicrobial properties of essential oils and their components have been reviewed in the past, the mechanism of action has not been studied in detail [32]. Components of essential oils such as thymol and carvacrol have previously been reported to have properties that cause membrane disruption in E. coli and S. typhimurium [12,33]. On the other hand, it was shown that eugenol can disintegrate in the membrane and increase its permeability, and thus causes the death of the organism [4,34].
One of the main contributions of this work was to verify the antimicrobial activity of some essential oils compounds present in plants used in traditional medicine, validating their use in the treatment of diseases of possible infectious origin.
The determination of the specific antimicrobial action of essential oil compounds is important to define possible synergistic activities against resistant microorganisms. In addition, the determination of the specific antimicrobial activity of essential oil compounds is important to define possible synergistic activities against resistant strains, as demonstrated with the thyme essential oil compounds [35][36][37].

Microbial Strains
The following strains of bacteria were used: , and V. cholerae (clinical strain pertaining to 01 group, Inaba serotype, El Tor biotype, and enterotoxin producer). These strains were maintained at 4 • C in Mueller Hinton agar (Bioxon), submitted to sensitivity tests (multidiscs Bigaux) and were subcultured twice prior the bioassays. The yeasts tested were: Candida albicans ATCC 10231, C. albicans ATCC 14065, C. albicans isolated from a clinical case (donated by the Clinical Analysis Laboratory of FES Iztacala), C. albicans, C. glabrata, and C. tropicalis (isolated from a clinical case and donated by Hospital Angeles Metropolitano). The stock culture was maintained on potato dextrose agar (PDA) and subcultured twice prior the bioassays.

Antibacterial Activity
The antibacterial activity was measured by the disc diffusion method [38,39]. The microorganisms were grown overnight at 37 • C in 10 mL of Mueller Hinton Broth (Bioxon). The cultures were adjusted with sterile saline solution to obtain turbidity comparable to that of McFarland no. 0.5 standard (10 8 CFU/mL). Petri dishes containing Mueller Hinton agar (Bioxon) were inoculated with these microbial suspensions. Discs of filter paper (Whatman no. 5) of 5-mm diameter were impregnated with 4 µL of each compound (α-pinene 3.4 mg, β-pinene 3.4 mg, carvacrol 3.9 mg, eugenol 4.3 mg, limonene 3.4 mg, myrcene 3.2 mg, ocimene 3.3 mg, cineole 3.7 mg, methyl salicylate 4.7 mg, farnesene 3.4 mg) and placed on the agar surface (for thymol, a solution was prepared at a concentration of 0.980 g/mL, discs were impregnated with 4 µL = 3.9 mg). Discs impregnated with chloramphenicol (25 µg) were used as positive controls. The plates were incubated overnight at 37 • C and the resulting inhibition zones were measured with a vernier. Each experiment was performed in triplicate. The estimation of the minimal inhibitory concentration (MIC) was carried out by the broth dilution method [40,41]. Ten dilutions of each compound (0.03-4.0 mg/mL) in broth were evaluated. The tubes were inoculated with 10 6 CFU/mL microorganism suspensions. MIC values were taken as the lowest essential oil concentration that prevents visible bacterial growth after 24 h of incubation at 37 • C and MBC as the lowest concentration that completely inhibited bacterial growth. Chloramphenicol was used as reference standard and compound-free plates as controls. Each experiment was repeated at least three times. The time-killing curve assay was performed using appropriate concentrations of the compounds (corresponding to 1 2 MIC, MIC, and MBC) in 10 mL of broth. One control tube was prepared without compound. Each tube was inoculated with 0.1 mL of a microbial suspension (10 6 CFU/mL) and incubated at 37 • C for 24 h. Of each tube, 50 µL aliquots were taken in ranges of 2 h. Aliquots were plated on Muller-Hinton agar and incubated for 24 h at 37 • C. Finally, the number of CFU was determined. Death kinetics was expressed in log10 reduction time kill plots [42].

Antifungal Activity
The evaluation of antifungal activity in yeast for each compound was evaluated following the same methodology as for the antibacterial test, but with potato dextrose agar and nystatin as a positive control [40].

Statistics
All experiments were performed in triplicate. The mean and standard deviation of the three experiments were determined. Statistical analysis of the differences between mean values obtained for experimental groups was done by an analysis of variance (ANOVA multifactorial model), where p-values of 0.05 or less were considered statistically significant [43].

Conclusions
There are no significant differences in the antimicrobial activity of the compounds on the different bacterial groups and yeasts. The compounds carvacrol, eugenol, and thymol were those that showed the highest antimicrobial activity. Carvacrol was the component with the highest antibacterial activity, followed by eugenol and thymol. Carvacrol was the component with the highest antifungal activity, followed by thymol and lastly eugenol. The compounds ocimene, cineole, and farnesene did not show activity on any yeast strain. A bactericidal effect was observed at concentrations equal to or greater than MIC for eugenol, thymol, and carvacrol. Finally, this study shows that the essential oils of plants used by the population of Tehuacán-Cuicatlán valley share compounds and some of them have antibacterial and fungicidal activity.

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.