The composition of the essential oil and aqueous distillate of Origanum vulgare L. growing in Saudi Arabia and evaluation of their antibacterial activity

2.1 Plant material

The aerial parts of O. vulgare L. grown in Al-Kharj, central province of Saudi Arabia, were collected before flowering stage in the month of March 2013. Authentication of the plant material was assured by a plant taxonomist (Dr. Jacob Thomas Pandalayil, Herbarium Division, King Saud University, Riyadh, Saudi Arabia). A token sample of the plant materials with voucher specimen number (OVHZK-303) is retained in our laboratory.

2.2 Isolation of essential oils by hydro-distillation

The freshly collected aerial parts of O. vulgare L. were sliced into tiny sections and air-dried in the shade at 20 °C. The resultant dried aerial parts of O. vulgare L. (326.4 g) were subjected to hydro-distillation in a Clevenger apparatus for 3 h to give a light-yellow oil. The yield of the oil was 1.7% (w/w) on a dry weight basis. The organic constituents recovered from the aqueous distillate were dried using anhydrous Na₂SO₄ as the dehydrating agent and stored at 4 °C until further use. The aqueous distillate (300 mL) obtained during hydro-distillation of the dried aerial parts of O. vulgare L. was exhaustively extracted (three times) with ethyl acetate (50 mL) in a separatory funnel. The combined ethyl acetate extracts (150 mL) were then dried using anhydrous Na₂SO₄, filtered, and the solvent was removed by rotary evaporation under reduced pressure to acquire the volatile oil.

2.3 Gas chromatography (GC) and gas chromatography−mass spectrometry (GC-MS) analysis of the essential oil and organic content in the aqueous distillate

The volatile oils were analyzed by using GC–MS and GC–FID techniques with two different stationary phase columns. For instance, the DB-Wax column was used for polar column analysis and the HP-5MS column was used for apolar column analysis. GC–MS analysis was executed on an Agilent single-quadrupole mass spectrometer fitted with an inert mass selective detector (MSD-5975C detector, Agilent Technologies, USA) attached directly to an Agilent 7890A gas chromatograph that was equipped with an auto-sampler (Agilent model 7693), a quickswap assembly, a split–splitless injector, and a HP-5MS column (5% phenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm i.d., film thickness: 0.25 μm, Agilent Technologies, USA). Additional analyses were performed on the same instrument using the polar DB-Wax column (polyethylene glycol, 30 m × 0.25 mm i.d., film thickness: 0.25 μm, Agilent Technologies, USA). The non-polar column was utilized at an injector temperature of 250 °C with the following oven temperature programming: initially, the oven temperature was kept isothermal for 4 min at 50 °C and was then raised to 220 °C at a rate of 4 °C·min⁻¹, followed by another isothermal hold for 2 min; in the second ramp, the temperature was raised to 280 °C at a rate of 20 °C·min⁻¹, and finally, the temperature was kept isothermal for 15 min. In contrast, the polar column was utilized at an injector temperature of 250 °C with the following oven temperature programming: initially, the oven temperature was maintained isothermal for 4 min at 40 °C, followed by a temperature increment of 4 °C·min⁻¹ to 220 °C, and then finally kept isothermal for 10 min.

Approximately 0.2 μL of the respective O. vulgare L. oils dissolved in acetone (5% O. vulgare L. oil solution in acetone) was injected by utilizing the split injection approach; the split flow ratio was 10:1. Helium was used as the carrier gas at a rate of 1 mL·min⁻¹. The mass spectra and gas chromatography-total ion chromatogram (GC–TIC) profiles were acquired with the help of ChemStation data analysis software (version E-02.00.493, Agilent). All mass spectra were obtained in the electron ionization (EI) mode by using an ionization energy of 70 eV and an m/z scan range of 45–600. The temperature of the electronic-impact ion source was maintained at 230 °C, whereas the MS quadrupole temperature was kept at 150 °C. The MSD transfer line temperature was held at 280 °C for both the polar and nonpolar analysis. GC analysis was performed on an Agilent GC-7890A dual-channel gas chromatograph (Agilent Technologies, USA) connected to a flame ionization detector (FID) using both polar and nonpolar columns by applying the aforementioned parameters. The temperature of the FID was kept at 300 °C for all analyses. The relative percentage of the volatile oil constituents was computed on the basis of the areas of the peaks in the GC–FID profile, acquired by using the non-polar column without applying a correction factor. The results obtained for the O. vulgare oils are recorded in Table 1 based on the order of elution of each component on the non-polar column.

2.4 Identification of oil constituents

The GC–FID chromatograms of the volatile oils obtained from the dried aerial parts of O. vulgare L. and its aqueous distillates and the identified peaks of the oil constituents separated on the non-polar (HP-5MS) column are displayed in Figs. 1 and 2, respectively. The volatile oil components were characterized by matching their mass spectra with the library entries in the mass spectra databases (NIST-08 MS Library, version 2.0 f, WILEY 9th edition, Flavor and Adams libraries) and by comparing their mass spectra and LRIs with the data reported using both the DB-Wax and HP-5MS columns (Adams, 2007; Babushok et al., 2011; NIST 2017), and by co-injection of genuine standards accessible in our laboratory.

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Fig. 1

GC–FID chromatogram of volatile oil from aerial parts of O. vulgare L. obtained using HP-5MS column. The characterized peaks are numbered according to their serial number in Table 1.

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Fig. 2

GC–FID chromatogram of essential oil from aqueous distillate of O. vulgare L. using HP-5MS column. The characterized peaks are numbered according to their serial number in Table 1.

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2.5 Isolation of major constituents from volatile oils

Column chromatography (CC) and thin layer chromatography (TLC) were used to purify carvacrol, a major component of the volatile oil from the aerial parts of O. vulgare L. A 1.0 g aliquot of the oil was subjected to CC using a silica gel column (60–120 mesh, 27 gm) and gradient elution was applied using hexane and chloroform mixtures in various ratios (100:0, 75:25, 65:35, 50:50, and 0:100), with increasing polarity, as the mobile phase. In total, forty-five fractions (15 mL each) were collected, of which fractions 29 to 33 corresponded to carvacrol based on the thin layer chromatography (TLC) profiles. These fractions were combined and the solvent was removed under reduced pressure using a rotary evaporator to produce pure carvacrol (1).

2.6 Carvacrol (1)

Dark-orange oil. ¹H NMR (400 MHz, CHCl₃-d): δ (ppm) = 1.38 (6H, m, H-8, H-9), 2.42 (3H, s, H-10), 2.99 (1H, m, H-7), 5.89 (1H, br s, OH), 6.83 (1H, s, H-6), 6.94 (1H, d, J = 7.3 Hz, H-4), 7.24 (1H, d, J = 7.3 Hz, H-3); ¹³C NMR (100 MHz, CHCl₃-d): δ 15.50 q (C-10), 24.02 q (C-8, C-9), 33.75 d (C-7),113.36 d (C-6), 119.05 d (C-4), 121.42 s (C-2), 131.04 d (C-3), 148.49 s (C-5), 153.48 s (C-1); EI-MS m/z: 150 ([M]⁺).

2.7 Chemicals

Analytical-grade 2-propanone (purchased from Sigma-Aldrich, Germany) was employed for dilution of the volatile oil samples. The constituents of the pure essential oil, such as β-pinene, γ-terpinene, α-pinene, and thymol, in addition to some volatile oil fractions mainly comprising compounds such as camphene, β-caryophyllene, p-cymene, 1-octen-3-ol, α-terpinene, terpinen-4-ol, cis-3-hexen-1-ol, 3-octanone, and caryophyllene oxide, were available and were used for co-injection/comparative analysis.

2.8 Retention indices

A mixture of an uninterrupted sequence of straight-chain hydrocarbons starting from C8 to C31 (C8–C20, 04070, Sigma-Aldrich, USA and C20–C31, S23747, AccuStandard, USA) was injected into both the DB-Wax (a polar column) and HP-5MS (a nonpolar column) columns using the conditions described above for the O. vulgare L. oil samples in order to calculate the linear retention indices (LRIs) of the O. vulgare L. oil components (Table 1). The LRIs of the oil components of O. vulgare L. were computed using van den Dool and Kratz’s equation.

2.9 Nuclear magnetic resonance (NMR) analysis

The ¹H and ¹³C NMR spectra of the O. vulgare L. oil samples and their purified compounds were obtained by using a JEOL ECP-400 spectrophotometer. The NMR samples were prepared in deuterated chloroform (CDCl₃) and tetramethylsilane (TMS) was used as an internal standard. The chemical shifts and coupling constants (J) are expressed in δ (ppm) and Hz, respectively. Thin layer chromatography (TLC) was carried out on pre-coated silica gel 60 F₂₅₄ (0.2 mm, Merck) plates and the compounds were detected under UV light.

2.10 Determination of antimicrobial activity

Inhibition of the growth of E. coli ATCC 25922, P. aeruginosa ATCC 75853, M. luteus ATCC 10240, and S. aureus ATCC 25,923 in the presence of the O. vulgare L. volatile oils and their purified compounds was evaluated by calculating the change in the optical density of cells grown with or without the test compounds. P. aeruginosa, E. coli, S. aureus, and M. luteus were grown in sterile nutrient broth, Luria broth, and Müeller-Hinton broth, respectively, at their optimal growth temperatures. A 10 µL aliquot of the cultures grown to the log phase was added to 90 µL of sterile broth. The test compounds were diluted and were added to the wells at final concentrations of 50, 100, 200, 300, and 500 μg mL⁻¹, where the test compounds were diluted in 5% DMSO. Finally, the plates were incubated for 12 h at the optimal growth temperatures (Haque et al., 2017). The absorbance was determined at 600 nm using a Multiskan microtiter plate reader (Multiskan Ex, Thermo Scientific, Finland).

The OD₆₀₀ at 0 h was subtracted from the OD₆₀₀ at a given time interval to calculate the change in the optical density. The values presented are the mean ± standard error of three independent experiments. Moreover, the unpaired t-test implemented in GraphPad software was used to determine the statistical significance (p-values). The IC₅₀ values were calculated from the average OD₆₀₀ values for each treatment.

3.1 Screening for antimicrobial activity

There are several reports on O. vulgare L. essential oil and its biological activity (Afsharypour et al. 1997; Al-Kalaldeh et al., 2010; Camiletti et al., 2016; Ebani et al., 2016; Faleiro et al., 2005; Souza et al., 2007; Tommasi et al., 2009). However, reports on the isolation and identification of the active constituents responsible for the biological activity are very rare. Therefore, to identify the main constituent of Saudi O. vulgare L. oil exhibiting antimicrobial activity, the bactericidal activity of the volatile oils derived from the aerial parts and hydrosol of O. vulgare L., along with the isolated major compound carvacrol (1), was screened.

The antimicrobial activity was evaluated by measuring the change in the OD₆₀₀ for selected Gram-positive (GM+) and Gram-negative (GM−) bacteria. The antimicrobial potential of the tested oils and pure compound against two GM– bacteria, i.e., P. aeruginosa and E. coli, is shown in Fig. 5. Carvacrol was the most effective compound, and completely inhibited the growth of E. coli at 200 µg·mL⁻¹. In comparison, compared to the control, significant inhibition (P < 0.005) of the bacterial growth was obtained only at concentrations of 200 µg·mL⁻¹ and 100 µg·mL⁻¹ for the volatile oil from the aerial parts and aqueous distillates of O. vulgare L., respectively (Fig. 5A). The IC₅₀ values of the test compounds against E. coli are given in Table 3.

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Fig. 5

Inhibition of E. coli (A) and P. aeruginosa (B) growth, as measured by the change in the OD₆₀₀ following treatment with different test compounds. *Presents significant values that are different from the control (p value < 0.005).

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The decrease in the OD₆₀₀ of P. aeruginosa following treatment with various test compounds is shown in Fig. 5(B). Carvacrol (1) was also the most effective for retarding the growth of P. aeruginosa, with an IC₅₀ value of 151 µg·mL⁻¹. Significant inhibition (P < 0.005) of the growth of P. aeruginosa was obtained with 200 µg·mL⁻¹ of all the test compounds (Fig. 5B). Therefore, the compounds evaluated herein could be organized in the following order based on their bactericidal activity against the GM– bacteria (E. coli and P. aeruginosa) on the basis of their IC₅₀ values: carvacrol (1)>SOVADH > SOVAD.

The antimicrobial activity of these compounds against two GM+ bacteria was also determined. The antimicrobial activity of the test compounds against M. luteus is shown in Fig. 6A. Carvacrol (1) was clearly the most effective inhibitor of the growth of M. luteus, with significant growth inhibition at 200 µg·mL⁻¹ (Fig. 6A). A similar trend was obtained when the antimicrobial activity of these constituents against S. aureus, another GM+ bacteria, was determined. In fact, S. aureus was more sensitive to all three test compounds than M. luteus. Carvacrol (1) most effectively inhibited the growth of S. aureus (Fig. 6B) also. Based on the results presented in Fig. 6B and the IC₅₀ values presented in Table 3, the test compounds can be organized in the following order based on their activity against S. aureus: carvacrol (1) > SOVADH > SOVAD.

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Fig. 6

Inhibition of M. luteus (A) and S. aureus (B) growth, as measured by the change in the OD₆₀₀ following treatment with different test compounds. *Presents values that are significantly different from control (p value < 0.005).

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The trend in the antimicrobial activity was very similar for all four bacteria. Moreover, it is interesting that the bactericidal activity of the test compounds against the four selected organisms increased with an increase in the content of carvacrol in both essential oils, and the highest activity was observed with the purified compound carvacrol (1). Hence, based on the present results, it is suggested that the antimicrobial activity of Saudi O. vulgare L. oils is mainly due to the presence of carvacrol. Furthermore, it is notable that the IC₅₀ values of carvacrol (1) were comparable to those of the known antibiotic drug ampicillin (Table 3), whereas, the IC₅₀ value of carvacrol (1) was found to be 4–10 times higher than the minimum inhibitory concentration (MIC) of kanamycin, as determined in this study (Table 3). The mechanism of the antimicrobial activity of carvacrol was evaluated in our previous study by checking the cell membrane integrity through propidium iodide staining and scanning electron microscope (SEM) analysis. It was demonstrated that carvacrol damages the cell membranes of Streptococcus mutans, resulting in the leakage of intracellular materials, deformation of cells, leading to cell death (Khan et al., 2017). The genes involved in the apoptosis-like activity and oxidative stress were also over-expressed in the presence of carvacrol, suggesting that carvacrol induces general and oxidative stress in S. mutans. Therefore, carvacrol may have affected the growth of the microorganisms evaluated herein by the same mechanism of action. Because Saudi O. vulgare L. oils exhibited significant antimicrobial activity against all of the tested bacteria, its possible use in the control of food-borne pathogens, and the pathogens of skin and oral cavity, is suggested.