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BY 4.0 license Open Access Published by De Gruyter Open Access February 20, 2023

Essential oils of Origanum compactum Benth: Chemical characterization, in vitro, in silico, antioxidant, and antibacterial activities

  • Youness El Abdali EMAIL logo , Adil M. Mahraz , Ghada Beniaich , Ibrahim Mssillou , Mohamed Chebaibi , Yousef A. Bin Jardan , Amal Lahkimi , Hiba-Allah Nafidi , Mourad A. M. Aboul-Soud , Mohammed Bourhia EMAIL logo and Abdelhak Bouia
From the journal Open Chemistry

Abstract

This study was performed to investigate the phytochemical profile, and the, in vitro, and, in silico, antioxidant and antibacterial properties of the essential oil (EO) extracted from Origanum compactum. EO phytochemical screening was examined by gas chromatography coupled to mass spectrometry. The antioxidant potential, in vitro, was assessed using reducing power(FRAP), free 2,2 diphenylpicrylhydrazyl (DPPH) radical scavenging and total antioxidant capacity tests. Antibacterial properties against two Gram (−) and two Gram (+) bacteria were assessed using the minimal inhibitory concentration (MIC) and the disc diffusion methods. By use of molecular docking, antioxidant and antibacterial activities of oregano EO were also tested. Thymol (75.53%) was the major compound among the nine compounds identified in the EO of Origanum compactum, followed by carvacrol (18.26%). Oregano EO showed an important antioxidant capacity, as tested by FRAP and DPPH assays, with EC50 and IC50 values of 13.300 ± 0.200 and 0.690 ± 0.062 mg/mL, respectively. The same EO has a total antioxidant capacity of 173.900 ± 7.231 mg AAE/g EO. The antibacterial results showed significant activity of Origanum compactum EO against all tested bacteria, especially against S. aureus (MIC = 0.25 mg/mL) and B. subtilis (MIC = 0.06 mg/mL). In silico, carvacrol was the most active molecule against nicotinamide adenine dinucleotide phosphate oxidase (2CDU) and S. aureus nucleoside diphosphate kinase (3Q8U) with a glide score of −6.082, and −6.039 kcal/mol, respectively. Regarding the inhibition of E. coli beta-ketoacyl-[acyl carrier protein] synthase (1FJ4), piperitenone was the most active molecule with a glide score of −7.112 kcal/mol. In light of the results obtained, the EO of Origanum compactum Moroccan species can be used as promising natural food conservatives and an agent to fight antibiotic-resistant nosocomial microbes.

1 Introduction

Nowadays, antibiotic resistance has become an extremely determining problem in public health. It causes a crisis in many hospitals around the world and constitutes a great incidence of nosocomial diseases [1]. Consequently, the research of anti-infective agents is an unavoidable need [2]. In the conservation of food systems as well, global interest has increased recently due to the higher economic costs of spoilage and poisoning caused by oxidation and microbial pathogens [3]. On the other hand, the damaging consequences of oxidative stress on the human body have become a real problem. Synthetic antioxidants, like butylated hydroxytoluene (BHT), have been commonly employed in the food industry as antioxidants and can lead to liver damage and cancer [4], hence the interest in using naturally occurring antioxidants and antibacterials.

In the light of scientific advances, the medicinal qualities of aromatic plants have attracted much attention due to their pharmacological activities, low toxicity and economic efficiency [5,6]. In these natural resources, different bioactive molecules, such as flavonoids, phenolic acids, tannins, carotenoids, sterols and terpenoids, have been identified and studied [7]. Understanding the mechanism of action of these bioactive molecules in biological and pharmacological activities is, nowadays, necessary for more effective use and valorization. In this perspective, in silico ligand-protein docking, based on computer-assisted drug design (CADD) approaches, can be used to identify key sites and predict the predominant binding mode(s) of a ligand with a protein of known three-dimensional structure and consequently propose structural hypotheses of how the ligands inhibit the target [8,9].

Northern Morocco, with its Mediterranean climate, is very rich in medicinal and aromatic plants, some of which have long been used in traditional medicine and diet. Among the medicinal plants of Morocco, the compact oregano (Origanum compactum Benth.), generally known as “Za’atar,” is an endemic plant of northern Morocco. Belonging to the family of Lamiaceae, this medicinal and aromatic plant is rich essentially in phenolic compounds and is commonly employed in traditional Moroccan medicine to cure certain diseases and pathologies, like diarrhea, respiratory, skin, and urinary infections [10]. The wide specter of applications for this compact flowered oregano depends on the region, the medication purposes, the parts used, and the mode of preparation [11]. Several works have shown the biological activities of Origanum compactum, like antibacterial [12,13], antifungal [14], antioxidant [13,15], insecticidal [11], cytotoxic [15,16], antimalarial [15], and antitumor activities [16,17]. However, reports on the medicinal properties of EOs extracted from the northern Moroccan Origanum compactum species and their mode of action in several biological activities are lacking. Therefore, the main objective of the current work was to evaluate, in vitro, and in silico, the antioxidant capacity and the antibacterial activity of Origanum compactum essential oil ([EO] from Taounate city) against several bacteria responsible for nosocomial infections and food spoilage, beside the identification of the chemical profile and the compounds involved, in order to a possible use in biomedicine and food preservation.

2 Materials and methods

2.1 Chemicals

Dimethylsulphoxide (DMSO), Butylated hydroxytoluene (BHT), Quercetin, ascorbic acid, sodium phosphate, ammonium molybdate, potassium ferricyanide (K3Fe(CN)6), iron III chloride (FeCl3), and DPPH were provided from Sigma Aldrich (Germany). Standard antibiotics and bacterial culture media were provided by Biokar Diagnostics (France).

2.2 Selection of plant material

The plant material was the total aerial parts including stems, leaves, and flowers of Origanum compactum Benth, growing spontaneously in the Taounate region (34°32′19.2″N; 4°38′03.4″W), Morocco. After identification at the biology department by botanists (DO12/04281), the samples collected were cleaned and then dried in the laboratory in a shaded, well-ventilated area at room temperature.

2.3 Extraction of EOs

The essential oils were hydrodistilled by a Clevenger-type apparatus for 3 h. A volume of 1 L of distilled water was mixed with 100 g of aerial parts of Origanum compactum. The EO obtained was collected with a micropipette and stored in an Eppendorf tube at 4°C and protected from light.

2.4 Chemical composition

The separation and identification of the different chemical compounds of Origanum compactum EO were conducted using a GC system (Agilent-Technologies 6890 N Network) equipped with a flame ionization detector. The capillary column used was an Agilent-Technologies HP-5MS, Little Falls, CA (30.00 m × 00.25 mm, film thickness of 00.25 µm). The column temperature was programmed between 35 and 250°C at 5 °C/min. The detector (ionization source) temperature was set to 280°C and the temperature of the injector was set to 250°C. Helium was used as a carrier gas with a flow rate of 1 mL/min; 1 µL of the EO diluted in hexane was injected in split mode (split ratio, 1:100). The compound quantification was calculated using a built-in data-handling program offered by the gas chromatograph company. The composition of EO was presented as a proportion of the overall peak area. The chemicals were identified by determining their retention indices (RI) referring to those of a homologous series of aliphatic hydrocarbons (n-alkanes) [18].

2.5 In vitro, antioxidant activity of EO

The antioxidant capacity of Origanum compactum EO was studied by three different tests, including the reducing power (FRAP), the DPPH inhibition, and the total antioxidant capacity (TAC).

2.5.1 DPPH free radical scavenging test

The scavenger capacity of DPPH (2,2-diphenyl-1-picryl hydrazine) radical was studied following the method of [19]. In the presence of the antioxidant, the DPPH free radical is reduced by accepting an electron or a hydrogen, the degree of discoloration of the DPPH purple solution reflects the scavenging potential of the extract [20].

The reaction medium for this assay consisted of 1 mL of EO (at different concentrations prepared in methanol) and 1 mL of the DPPH methanolic solution (0.01 M). The mixtures were incubated for 30 minutes in the darkness at ambient temperature, and then, the absorbance at 517 nm was measured against a negative control containing methanol instead of the EO. Butylated hydroxytoluene (BHT) was also employed as a positive control. The free radical scavenging capacity was calculated with the following formula:

DPPH inhibition ( % ) = [ ( Absorbance control Absorbance sample ) /Absorbance control ] × 100 .

The 50% inhibitory concentration of DPPH (IC50) was also calculated.

2.5.2 FRAP

The presence of the reducing compounds in samples leads to the reduction of Fe3+/ferricyanide complex to the ferrous form. The reducing power of the oregano EO was evaluated according to the method detailed by [19]. One milliliter of different EO concentrations (prepared in methanol) was added to 2.5 mL of a solution (1%) of potassium ferricyanide [K3Fe(CN)6] and 2.5 mL of phosphate buffer (0.2 M, pH 6.6). The mixture was incubated for 20 min in a water bath at 50°C. Therefore, 2.5 mL of trichloroacetic acid (10%) was added. After vigorous shaking, 2.5 mL of the resulting solution was added to 0.5 mL of 0.1% FeCl3 solution and 2.5 mL of distilled water. Finally, absorbance at 700 nm was measured relative to a blank. BHT and Quercetin were employed as standard antioxidants. The EC50 was the concentration of the sample that corresponds to an absorbance of 0.5 and was calculated from the equation of the absorbance versus sample concentration curve.

2.5.3 TAC test

The TAC of oregano EO was assessed using the phosphomolybdenum assay [21]. A volume of 0.2 mL of the oregano EO was added to 3 mL of the reagent solution (4 mM ammonium molybdate, 28 mM sodium phosphate, and 0.6 M sulfuric acid). After incubation at 95°C for 90 min, the absorbance at 695 nm of the tubes was measured relative to the corresponding blank. The same protocol was performed for ascorbic acid at different concentrations to quantify the total antioxidant capacity, which was expressed as milligram equivalents of the ascorbic acid per gram of EO (mg EAA/g). All manipulations are repeated three times.

2.6 Antimicrobial activity of EO

2.6.1 Microbial strains

The evaluation of the antibacterial activity of oregano EO was performed against four strains: Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213), and Escherichia coli (ATCC 25922). All microbial strains were provided by the Microbiology Laboratory, Faculty of Medicine and Pharmacy of Fez (Morocco). These studied bacterial strains have been reported in many studies as multidrug-resistant [1,22]. The experiments were conducted following published procedures with some modifications [23,24].

2.6.2 Disk diffusion method

The objective of the disc diffusion method was to measure the inhibition zone diameter (mm) caused by the EO around the bacterial strain. For this purpose, disks of Whatman number 1 paper (0.6 cm diameter) were immersed with 10 µL of EO, streptomycin (0.5 mg/mL), and ampicillin (2.5 mg/mL). Then, the disks were placed on the surface of the agar culture medium Mueller Hinton (MH) inside the Petri dish inoculated with bacterial strains (1 × 108 to 2 × 108 CFU/mL). The antibacterial potential of EO was assessed after incubation (37°C) for 24 h.

2.6.3 Evaluation of the MIC

The MIC was evaluated using the microdilution method. The EO and the standards were diluted in DMSO. First, 50 µL of the MH culture medium was deposited in all wells of the microplate. Then, 100 µL of the EO solution and standard antibiotics were deposited in the first wells. Afterward, a microdilution was created by transferring 100 µL from the first well to the final one, excluding the positive growth control. Finally, except for the negative growth control, inoculation was performed by depositing 10 µL of the bacterial suspension prepared previously into all wells. After incubation (at 37°C) of the microplate for 24 h, the growth of bacteria was revealed by a white spot under the wells and also by adding 10 µL of Resazurin (5 mg/mL) for confirmation. MICs of EO, Ampicillin and Streptomycin, were identified as the lowest concentration of the sample that inhibited bacterial growth.

2.7 In silico molecular docking of antioxidant and antimicrobial activities of EO

In this molecular docking study, nicotinamide adenine dinucleotide phosphate oxidase (NADPH) was chosen to assess antioxidant activity, whereas Staphylococcus aureus nucleoside diphosphate kinase and Escherichia coli beta-ketoacyl-[acyl carrier protein] synthase were chosen to examine antibacterial activity.

All compounds identified in Origanum compactum EO were downloaded from the PubChem database in SDF format. Then, they were prepared by LigPrep tool in the Maestro 11.5 version of the Schrödinger Software program using the OPLS3 force field. A maximum of 32 stereoisomers were produced for each ligand after the ionization states at pH 7.0 ± 2.0 [25]. The three-dimensional crystal structures of NADPH oxidase, S. aureus nucleoside diphosphate kinase, and E. coli beta-ketoacyl-[acyl carrier protein] synthase were downloaded in PDB format from the protein data bank using the following PDB IDs: 2CDU, 3Q8U, and 1FJ4, respectively. All structures were prepared and refined using the Protein Preparation Wizard of Schrödinger-Maestro v11.5. The minimization of the structure was carried out using an OPLS3 force field [26,27].

The receptor grid was set at the following coordinates:

For NADPH oxidase, X = 19.853 Å, Y = −6.431 Å, and Z = −0.896 Å.

For S. aureus nucleoside diphosphate kinase, X = 10.035 Å, Y = 52.059 Å, and Z = −44.228 Å.

For E. coli beta-ketoacyl-[acyl carrier protein] synthase, X = 33.264 Å, Y = 28.978 Å, and Z = 30.303 Å.

The volumetric spacing performed was 20 × 20 × 20. SP flexible ligand docking was carried out in Glide of Schrödinger-Maestro v 11.5.

2.8 Statistical analysis

GraphPad Prism 8 a Microsoft Software (California, USA) was used to calculate mean values and standard deviations. Data from all tests were compared statistically using one-way ANOVA by a Tukey test. At p < 0.05, the difference was considered significant.

3 Results

3.1 Chemical composition and yield of EO

The aerial parts (leaves, flowers, and stems) of Origanum compactum. Benth from Taounate gave a limpid yellow EO with a characteristic smell, a density of 0.920 g/mL, and yielded 3.93% (w/w). In general, the yield of EO depends on the plant species, geographical distribution, anatomical organ, collection period, and extraction method [28]. For instance, the EO’s yield of Moroccan O. compactum extracted from leaves by Elbabili et al., Bouchra et al., and Rezouki et al., was 2.10, 5.4, and 4.47%, respectively [14,15,29]. This difference in yield is attributed to the geographical origin of the collection of plants [30].

The chemical composition and the gas chromatographic profile of EO extracted from Origanum compactum. Benth are shown in Figure 1 and Table 1. Nine compounds representing a total of 99.98% of EO were identified using gas chromatography coupled to mass spectrometry (GC–MS). The EO’s major compound was thymol (75.53%) followed by carvacrol (18.26%) and caryophyllene (1.76%). Monoterpene chemicals constitute 94.42% of the oregano EO compared to sesquiterpenes (1.82%). Chemical structures of the major compounds in the EO of O. compactum. Benth are shown in Figure 2. The composition of oregano EO has been extensively studied. Carvacrol (0.30–46.80%), thymol (2.20–79.01%), and β-caryophyllene (0.00–24.00%) were also reported as major compounds of oregano EO in previous studies, with different percentages [29,31,32]. Other terpenes like terpinen-4-ol, γ-terpinene, linalool, p-cymene, and β-myrcene were also present in different specimens of oregano [33]. The ratio of these and other compounds in the oregano EO within the same species determines the chemotype. It is also interesting to note that the contents and concentrations of oregano EO compounds were affected by a wide range of parameters, including species, harvest season, soil conditions, pests, climatic, geographical location, and growing circumstances. The authors also reported that the EO concentration is impacted by the drying method, as well as the aroma quality of the dried specimen [33]. Oregano EO compounds like thymol and carvacrol constitute promising bioactive agents which are accepted by consumers and exploited for prospective multi-purpose usage [34].

Figure 1 
                  GC–MS chromatographic profile of O. compactum EO.
Figure 1

GC–MS chromatographic profile of O. compactum EO.

Table 1

Phytochemical composition of O. compactum EO

Peak RT (min) Compounds RI RT (literature) Molecular formula Percentage (%)
1 5.157 p-Cymene 1,025 1,024 C10H16 0.54
2 5.438 Thymol 1,287 1,290 C10H14O 75.53
3 6.471 Carvacrol 1,296 1,299 C10H14O 18.26
4 6.762 Piperitenone 1,351 1,343 C10H14O 0.09
5 7.093 Caryophyllene 1,418 1,419 C15H24 1.76
6 7.224 Caryophyllene oxide 1,579 1,583 C15H24O 0.06
7 7.845 Methyl linoleate 2,099 2,092 C19H34O2 1.14
8 10.243 Ethyl linolenate 2,161 2,169 C20H34O2 0.75
9 12.249 Ethyl α-linolenate 2,164 2,169 C20H34O2 0.04
Monoterpenes 94.42
Sesquiterpenes 1.82
Others 1.93
Total 98.17
Figure 2 
                  Chemical structures of the major compounds of the O. compactum EO.
Figure 2

Chemical structures of the major compounds of the O. compactum EO.

3.2 In vitro antioxidant activity of EO

The action of reactive species radicals on the components of living organisms causes oxidative stress, which can lead to potential harm [35]. Lipid oxidation also causes deterioration of oils and fats, resulting in changes in flavor, color, and nutritional value [30]. Several EOs have been examined for their ability to neutralize oxidative stress by scavenging reactive species [33]. In the present study, a multi-assay approach was conducted to evaluate, in vitro, the antioxidant potential of O. compactum EO, including DPPH inhibition, FRAP, and phosphomolybdenum TAC tests. The results obtained are shown in Figures 3 and 4 and Table 2. As represented in Figure 3, the oregano EO showed interesting and comparative DPPH radical scavenging activity compared to BHT in a dose-dependent manner. In the same sense, the half maximal inhibitory DPPH radical concentration (IC50) value of O. compactum EO was 0.690 ± 0.062 mg/mL (Table 2). ANOVA analysis revealed that the IC50 values of the studied EO and BHT (0.122 ± 0.021 mg/mL) were not significantly different (p < 0.05).

Figure 3 
                  DPPH free radical scavenging activity of O. compactum EO and BHT.
Figure 3

DPPH free radical scavenging activity of O. compactum EO and BHT.

Figure 4 
                  Total antioxidant capacity of O. compactum EO, BHT, and Quercetin. Bars with different letters are significantly different at p < 0.05.
Figure 4

Total antioxidant capacity of O. compactum EO, BHT, and Quercetin. Bars with different letters are significantly different at p < 0.05.

Table 2

Antioxidant activities of Oregano EO and standards (means ± SD)

DPPH (IC50 mg/mL) FRAP (EC50 mg/mL) TAC (mg AAE/g EO)
EO 0.690 ± 0.062a 13.300 ± 0.200a 173.900 ± 7.231a
BHT 0.122 ± 0.021a 0.362 ± 0.010b 48.530 ± 1.250b
Quercetin 0.032 ± 0.003c 29.470 ± 1.246b

In every column values with different letters are significantly different (p < 0.05).

The antioxidant potential of oregano EO was also evaluated using FRAP assay. Results demonstrated the potential of EO to reduce ferric iron (Fe3+) to ferrous iron (Fe2+) with an EC50 value of 13.300 ± 0.200 mg/mL (Table 2). However, the EO-reducing ability was significantly (p < 0.05) less effective than those of the standard antioxidants quercetin and BHT, which marked EC50 values of 0.032 ± 0.003 and 0.362 ± 0.010 mg/mL, respectively.

The total antioxidant capacity (TAC) of the studied EO was also assessed by the phosphomolybdenum assay. At an acidic pH, molybdenum Mo(vi) present as molybdate ions is reduced to molybdenum Mo(v) in the presence of the antioxidant, forming a green phosphate/Mo(v) complex [36]. Regarding the results obtained (Figure 4 and Table 2), the total antioxidant capacity (as ascorbic acid equivalents mg AAE/g EO) of oregano EO compared to reference antioxidants (BHT and quercetin) were 173.900 ± 7.231, 48.530 ± 1.250, and 29.470 ± 1.246 mg/g, respectively.

Numerous researchers have confirmed and demonstrated our data about the antioxidant property of O. compactum EO, which is due to the presence of some conjugated terpenes especially thymol and carvacrol [37]. In fact, Elbabili and collaborators have evaluated the antioxidant activity of Moroccan O. compatum EO (36.46% carvacrol, 29.74% thymol, and 24.31% p-cymene) using 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and DPPH free radical scavenging assays. Data showed that the EO exhibited a significant antioxidant activity with an IC50 = 2.00 ± 0.10 mg/L [15]. In another study, O. compactum EO showed comparable antioxidant activity (IC50 = 2.00 mg/L) to Vitamin C (IC50 = 1.90 mg/L) using ABTS assay [33]. Furthermore, the study of Quiroga et al. highlighted the significant DPPH radical scavenging activity (CI50 = 0.98 µg/mL and CI50 = 0.90 µg/mL) of EOs extracted from two oregano species in Argentina, O. vulgare ssp. Virens and ssp. vulgare, characterized by a higher content of thymol (29.70%) and (26.60%), respectively [38]. Likewise, the ferric reducing antioxidant power (FRAP) values of four oregano-type EOs with high contents of thymol (12.10–17.40%) and lower contents of carvacrol (0.10–3.50%) from southern and central regions of Argentina varied between 0.184 and 0.072 mM/mg [39]. On the other hand, studies have shown that O. glandulosum EOs from various locations in Tunisia exhibited low antioxidant potency when compared to ascorbic acid. The IC50 values of EOs ranged between 59.20 and 79.80 mg/L. Thus, the authors recommend that the EOs extracted from O. glandulosum as effective natural antioxidants could reduce lipid peroxidation [40]. EOs include combinations of a number of volatile and semi-volatile molecules with many functional groups and various polarities, resulting in a wide range of chemical characteristics. These characteristics vary according to the test used. The presence of phenolic molecules like thymol and carvacrol has been linked to the antioxidant effects of oregano EO in several investigations [37]. In fact, several studies comparing the antioxidant potencies of various compounds found that carvacrol, one of the main constituents of oregano EOs, has a stronger antioxidant potential than other compounds using the DPPH scavenging assay [41]. Similar research has reported that thymol and carvacrol exhibited a potent antioxidant activity than camphor, linalool, borneol, and 1.8 cineole throughout many tests [34]. In fact, the variety of molecules found in the investigated EO, as well as their various action modes and interactions, made difficult the attribution of the antioxidant activity to only one or a few bioactive components [42]. Furthermore, the EO of oregano also limited polyunsaturated fatty acid ester autoxidation obtained from the mouse brain, with the antioxidant components carvacrol and thymol [43]. In the same context, and through several tests, numerous studies have evaluated the antioxidant capacity using different food matrices by combining various films and EOs. The results were promising [44]. Practically, the antioxidant properties of EOs are expected to depend, at least in part, on the various mechanisms by which they act on oxidative stress. Some studied mechanisms are (i) free radical scavenging ability, (ii) modulation of antioxidant enzymes (such as superoxide dismutase), and (iii) control of pro-oxidation [33,45]. Besides the benefit of oregano EO in human health, and its ability to neutralize oxidative stress, the antioxidant properties of these essences offer the opportunity to use them as flavors and preservatives in food and nutraceutical products. It can also be utilized to substitute synthetic antioxidants into food products with high lipid content [46].

3.3 Antibacterial activity of EO

The antibacterial activity of O. compactum EO was evaluated, in vitro, qualitatively by the agar diffusion test (Table 3) and quantitatively by the microdilution method (MIC) to measure the MIC (Table 4). The bacterial strains (P. aeruginosa, S. aureus, B. subtilis, and E. coli) tested in the present study are among the most common bacteria to develop multidrug resistance, and they are classified as community- and hospital-acquired infections. [1,5,22]. They are also reported as responsible for most common diseases connected with food illness [44].

Table 3

Inhibition diameter (mm) of O. compactum EO against bacterial strains (means ± SEM)

Inhibition diameter (mm)
Gram (−) bacteria Gram (+) bacteria
E. coli (ATCC 25922) P. aeruginosa (ATCC 27853) B. subtilis (ATCC 6633) S. aureus (ATCC 29213)
Oregano EO 27.16 ± 1.01a 31.66 ± 0.88a TI 70.16 ± 2.34a
Ampicillin 25.33 ± 0.66a TI 33.00 ± 1.15b
Streptomycin 15.33 ± 0.33b 9.61 ± 1.20c

Inhibition zone includes diameter of the disk (6 mm); TI, total inhibition. In every column, values with different letters are significantly different at p < 0.05.

Table 4

Minimal inhibitory concentrations (mg/mL) of O. compactum EO against bacterial strains

Minimum concentration inhibition (mg/mL)
Gram (−) bacteria Gram (+) bacteria
E. coli (ATCC 25922) P. aeruginosa (ATCC 27853) B. subtilis (ATCC 6633) S. aureus (ATCC 29213)
Oregano EO 1.25 0.08 0.06 0.25
Streptomycin 0.50 3.13 3.13
Ampicillin 0.31 0.16

The results presented in Table 3 are expressed by measuring the growth inhibition zone diameter (mm). O. compactum EO exhibited a potent growth inhibitory effect on all tested strains marking different inhibition zone diameters. Gram-positive bacteria were the most sensitive to oregano EO, which completely inhibited the growth of Bacillus subtilis. The other inhibition zone diameters values ranged between 27.16 ± 1.01 and 70.16 ± 2.34 mm in the case of Gram (−) E. coli and Gram (+) S. aureus, respectively.

The results of MIC evaluating the bacteriostatic effect of O. compactum EO are listed in Table 4. The obtained data showed that the lowest inhibitory concentration of oregano OE was observed in the case of B. subtilis followed by P. aeruginosa with concentrations of 0.06 and 0.08 mg/mL, respectively. E. coli was the least sensitive marking 1.25 mg/mL MIC value. Furthermore, oregano OE was shown to be effective against both types of Gram (+) and Gram (−) bacteria, in comparison to streptomycin and ampicillin antibiotics, which were in some cases less effective.

Data of this study are in agreement with those in which treatment with O. compactum EO inhibited the growth of S. aureus and E. coli by low MIC values of 0.30 and 0.45 mg/mL, respectively [32]. Likewise, oregano oil exhibited an important antibacterial action against 11 multidrug-resistant clinical isolates including three P. aeruginosa (AF0001, IQ0042, and IQ0046), four A. baumannii (AF0004, AF0005, IQ0012, and IQ0013), and four methicillin-resistant S. aureus (MRSA strains of AF0003, IQ0211, IQ0103, and IQ0064) isolated from combat casualties and two luminescent strains of MRSA USA300 and PA01, with low MIC values ranging between 0.08 and 0.64 mg/mL [47]. The same study showed that oregano oil is also effective in eradicating biofilms composed of each of the pathogens studied at similar MIC values. Antibacterial activity of oregano EO has also been confirmed in other works against E. coli and S. aureus with low MIC values ranging between 1,600–1,800 ppm and 800–900 ppm, respectively [37]. In vivo, oil of oregano, applied topically (after 24 h), was also remarkably effective in reducing the bacterial load by 3log10 in 1 h, in third-degree burn wounds infected by 2 luminescent strains of MRSA USA300 and PA01 [47]. Different modes of action are implicated in the antimicrobial activity of oregano EO. These mechanisms seem to have a close relationship with many complex constituents in oregano EOs as well as with the pathogen studied [30]. In general, the biological properties of EOs are related to their major compounds [5]. In the case of oregano EO, the antimicrobial actions of phenolic molecules like thymol and carvacrol are expected to be attributed to functional and structural damages to the cytoplasmic membrane [5]. The metabolic pathway of citrate was also altered by thymol as well as many enzymes implicated in the synthesis of ATP. Inside the cell, it was also reported that the action of thymol affects important energy-generating mechanisms, thereby decreasing the ability of the bacterium to recover [48]. Carvacrol, like its closely related isomer thymol, possesses remarkable antibacterial potential. According to studies, carvacrol affects the outer membrane, while the cytoplasmic membrane is its site of action, generating passive ion transport across this membrane. Carvacrol has also been demonstrated to impact Gram-negative bacteria’s outer membrane [49]. As a result, attribution of antibacterial activity to one or more active molecules is complicated due to the diversity of chemicals found in the investigated EO, as well as their different antimicrobial efficacy. Oregano EO has already been tested as a food preservative. In this context, a study showed that incorporating oregano EO into an edible whey protein film was able to protect chicken breast fillets against food spoilage microorganisms like psychrotrophic bacteria and aerobic mesophilic, LAB, and Pseudomonas spp. [50]. In a similar work, experiments have shown that Kasar cheese inoculated with E. coli coated with whey protein isolate film containing oregano EO exhibited a 1.48 log reduction, while the same film reduced the count of S. aureus by 2.15 log in comparison with controls after 24 h [51]. Additionally, the food industry has recently started to show an increased interest in nanoencapsulation technology as a way to provide nutraceuticals and food preservatives based on plant EOs. These have the benefits of stability, protection against oxidation and pathogenic microorganisms, and minimal or no impact on the organoleptic qualities of applicable food products [52]. This was observed when specific EOs, such as thymol, were encapsulated in cholesterol and non-hydrogenated soybean PC lipoid S100 [53]. This type of study is of considerable importance because of the emerging antibiotic-resistant strains, the increasing population with low immunity, and the strong trend of using natural products for food preservation. This constitutes a promising perspective for the utilization of natural alternatives such as oregano EO to overcome the problem of antibiotic resistance as well as for preservation in the food industry.

3.4 In silico molecular docking of antioxidant and antimicrobial activities of EO

Currently, several research studies are based on molecular modeling, using CADD (computer-aided drug design) techniques to examine and predict the interactions of fixed and volatile compounds, such as EOs, with molecular targets involved in a variety of biological activities [8]. In silico, molecular docking enables the formulation of hypotheses on the mode of action of bioactive molecules by identifying the active sites in the three-dimensional structure and by calculating the affinity energy in the protein–ligand complexes [8,9]. In the present study, molecular docking was performed to examine the inhibitory effect of O. compactum EO compounds against enzymatic protein complexes involved in oxidative stress and the vitality of E. coli and S. aureus. Regarding antioxidant activity, NADPH oxidase constitutes a major enzyme source of oxygen free radicals in stimulated endothelial cells [54]. Consequently, its inhibition represents a significant factor in the defense against free radicals. Molecular docking of EO of O. compactum in the active site of NADPH oxidase (2CDU) showed a strong inhibitory effect of carvacrol, thymol, and p-cymene represented by glide scores of −6.082, −5.099, and −4.927 kcal/mol, respectively, and glide energy of −23.536, −22.063, and −19.846 kcal/mol, respectively (Table 5). Our results are in agreement with a study where carvacrol, thymol, and p-cymene showed remarkable antioxidant activity when tested using DPPH, ABTS, ORAC, and TBARS assays tests [34]. 2D and 3D viewers of O. compactum EO docked in the active site of NADPH oxidase (2CDU) showed that the hydroxyl group of carvacrol established two hydrogen bonds with residues GLY 161 and CYS 242 (Figures 5 and 6).

Table 5

Docking results of O. compactum in active site of NADPH (PDB: 2CDU), S. aureus nucleoside diphosphate kinase (PDB: 3Q8U), and E. coli beta-ketoacyl-[acyl carrier protein] synthase (PDB: 1FJ4)

EO Compounds Antioxidant activity Antibacterial activity
2CDU receptor 3Q8U receptor 1FJ4 receptor
G. score (kcal/mol) G. energy (kcal/mol) G. score (kcal/mol) G. energy (kcal/mol) G. score (kcal/mol) G. energy (kcal/mol)
Carvacrol −6.082 −23.536 −6.039 −20.812 −6.514 −25.646
Thymol −5.099 −22.063 −5.547 −20.444 −6.83 −24.811
p-Cymene −4.927 −19.846 −5.152 −16.404 −5.794 −21.92
Piperitenone −6.075 −20.979 −4.497 −18.218 −7.112 −22.296
Caryophyllene oxide −4.144 −18.17 −4.194 −17.414 −5.356 −11.512
caryophyllene −4.343 −11.633 −4.157 −7.755 −5.064 −11.924
Ethyl linolenate −3.403 −35.847 −3.223 −30.518 −4.259 −39.557
Methyl linoleate −2.177 −41.04 −0.273 −30.402 −0.614 −35.007
Figure 5 
                  2D Diagrams of ligand interactions with the active site. (a) and (b) Carvacrol interactions with the active sites of NADPH oxidase and 3Q8U; (c) Piperitenone interactions with the active sites of 1FJ4.
Figure 5

2D Diagrams of ligand interactions with the active site. (a) and (b) Carvacrol interactions with the active sites of NADPH oxidase and 3Q8U; (c) Piperitenone interactions with the active sites of 1FJ4.

Figure 6 
                  3D Diagrams of ligands interactions with the active site. (a) and (b) Carvacrol interactions with the active sites of NADPH oxidase and 3Q8U; (c) piperitenone interactions with the active sites of 1FJ4.
Figure 6

3D Diagrams of ligands interactions with the active site. (a) and (b) Carvacrol interactions with the active sites of NADPH oxidase and 3Q8U; (c) piperitenone interactions with the active sites of 1FJ4.

On the other hand, nucleoside diphosphate kinases represent ubiquitous enzymes that catalyze the transfer of the γ phosphate from nucleoside triphosphates (NTP) to nucleoside diphosphates (NDP), thereby maintaining the proper NTP levels in bacteria cells [55]. However, beta-ketoacyl-[acyl carrier protein] synthase enzyme catalyzes the biosynthesis of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival [56]. The critical role of these enzymes in bacterial virulence renders them a potential target for drugs and bioactive molecules. In silico evaluation of the antibacterial activity of O. compactum EO showed that carvacrol was the most active molecule against S. aureus nucleoside diphosphate kinase (3Q8U) with a glide score and glide energy of −6.039 and −20.812 kcal/mol, respectively. Furthermore, piperitenone was the most active molecule against E. coli beta-ketoacyl-[acyl carrier protein] synthase (1FJ4) with a glide score and glide energy of −7.112, and −22.296 kcal/mol, respectively (Table 5). Our results confirm the antibacterial proprieties of carvacrol and piperitenone as previously reported [5,57]. 2D and 3D Viewers showed that carvacrol established two hydrogen bonds with residues GLY 110 and LYS9 in the active site of 3Q8U, when the piperitenone established two hydrogen bonds with residues GLY 180 and VAL 214 in the active site of 1FJ4 (Figures 5 and 6).

4 Conclusion

At last, Origanum compactum EOs have been demonstrated to exert significant antioxidant and antimicrobial potentials against a spectrum of multidrug-resistant microbes. Based on in silico studies, oregano EOs may be also a natural NADPH oxidase, S. aureus nucleoside diphosphate kinase, and E. coli beta-ketoacyl-[acyl carrier protein] synthase inhibitors. Consequently, they might be beneficial for therapeutic purposes to overcome the problem of antibiotic resistance. They could also prevent microbial deterioration and spoilage caused by the oxidation of food products. These biological properties could be specially attributed to the presence of oxygenated monoterpenes.

Acknowledgements

The authors would like to extend their sincere appreciation to the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia, for funding this work through project number (RSP2023R457).

  1. Funding information: This work was funded by the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia, through project number (RSP2023R457).

  2. Author contributions: All authors contributed to the writing of original draft, formal analysis, revising of the manuscript, and validation.

  3. Conflict of interest: The authors declare that there is no conflict of interest.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: All data generated or analyzed during this study are included in this published article.

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Received: 2022-11-03
Revised: 2023-01-01
Accepted: 2023-01-25
Published Online: 2023-02-20

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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