# In-Depth Study ofThymus vulgaris Essential Oil: Towards Understanding the Antibacterial Target Mechanism and Toxicological and Pharmacological Aspects

Authors: Sarra Akermi; Slim Smaoui; Mariam Fourati; Khaoula Elhadef; Moufida Chaari; Ahlem Chakchouk Mtibaa; Lotfi Mellouli
Journal: BioMed Research International
 (2022)

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## Abstract

Questions have been raised apropos the emerging problem of microbial resistance, which may pose a great hazard to the human health. Among biosafe compounds are essential oils which captured consumer draw due to their multifunctional properties compared to chemical medication drugs. Here, we examined the chemical profile and the mechanism(s) of action of theThymus vulgaris essential oil (TVEO) against a Gram-negative bacterium Salmonella enterica Typhimurium ATTCC 10028 (S. enterica Typhimurium ATTCC 10028) and two Gram-positive bacteria Staphyloccocus aureus ATCC 6538 (S. aureus ATCC 6538) and Listeria monocytogenes ATCC 19117 (L. monocytogenes ATCC 19117). Findings showed that TVEO was principally composed of thymol, o-cymene, and γ-terpinene with 47.44, 16.55, and 7.80%, respectively. Molecular docking simulations stipulated that thymol and β-sesquiphellandrene (a minor compound at 1.37%) could target multiple bacterial pathways including topoisomerase II and DNA and RNA polymerases of the three tested bacteria. This result pointed plausible impairments of the pathogenic bacteria cell replication and transcription processes. Through computational approach, the VEGA quantitative structure–activity relationship (QSAR) model, we revealed that among twenty-six TVEO compounds, sixteen had no toxic effects and could be safe for human consumption as compared to the Food and Drug Administration (FDA) approved drugs (ciprofloxacin and rifamycin SV). Assessed by the SwissADME server, the pharmacokinetic profile of all identified TVEO compounds define their absorption, distribution, metabolism, and excretion (ADME) properties and were assessed. In order to predict their biological activity spectrum based on their chemical structure, all TVEO compounds were subjected to PASS (Prediction of Activity Spectra for Substances) online tool. Results indicated that the tested compounds could have multiple biological activities and various enzymatic targets. Findings of our study support that identified compounds of TVEO can be a safe and effective alternative to synthetic drugs and can easily combats hazardous multidrug-resistant bacteria.


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## Body

## 1. Introduction

In recent years, antimicrobial-resistant (AMR) bacteria have been admitted as a public health risk that could cause an increase in the global burden of infectious disease [1–4]. For instance, each year, more than 670,000 infections and 700,000 deaths worldwide were provoked by AMR [5, 6]. Mutually controlled by host immune condition, microflora organization, and antimicrobial interventions, AMR evolution occurs with the multidrug resistance [7, 8]. While AMR could not be pragmatically eradicated, antimicrobials will endure to miss their potency, and, in the close future, more people may die from infections [9–11]. Therefore, it is crucial to explore original effective and broad-spectrum antibacterial agents to control bacteria, which can be antibiotic resistant, highly virulent, and high costs for the medication. Among the leading strategies for the exploration and detection of new targets in pathogens are the masterfully using bioinformatics tools. These tools can provide useful information for better comprehension of the interactions between targets and biomolecules and therefore help to anticipate new treatment targets for pathogenic microorganisms [12, 13].Extracted from natural plants, essential oils (EOs) can be exploited as a practical alternative. More specifically, thyme (Thymus vulgaris L.), appertained to the family of Lamiaceae and the genus of Thymus, was consumed for centuries because of its medicinal properties and was generally recognized as safe (GRAS) by the FDA [14–17]. In addition, essential oil acquired from Thymus vulgaris L. had an extensive range of biological activities [17]. Thyme EO contains high levels of phenolic compounds, like thymol, carvacrol, p-cymene, and γ-terpinene [17, 18]. Interestingly, numerous studies reported that thymol, the major antibacterial component occurring in TEO, can destroy the bacterial cell membrane [19, 20]. Liu et al. revealed that thymol powerfully inhibit Pseudomans aeruginosa and directly change the cell structure [15]. P. aeruginosa cell membrane integrity is destroyed as evidenced by an increase in permeability of the inner/outer membranes. In other studies, Wang et al. and Lade et al. noted that thymol disrupts the Staphylococcus aureus membrane integrity to achieve the inner structure of the bacterial cell and joints to the minor groove of bacterial DNA, ensuring in a destabilization of the DNA secondary structure [21, 22].In extension of our research to disclose the potential of natural therapeutic agents [13], this current investigation is aimed at elucidating the molecular docking interactions of all components of Thymus EO with bacterial DNA and RNA polymerases, as well as the topoisomerase II of S. aureus, S. enterica Typhimurium, and L. monocytogenes were profoundly reviewed in silico. As a part of our endeavor to increase the potential and exploration of these activities, a pharmacokinetics and computational toxicological studies were well discussed.


## 2. Materials and Methods

### 2.1. Plant Material, Essential Oil Extraction, and Gas Chromatography–Mass Spectrometry (GC–MS) Analysis

#### 2.1.1. Plant Material Collection and EO Extraction

Thymus vulgaris L. plant was collected from the region of Sfax, Tunisia (N: 34.4426°, E: 10.4537°) which is characterized by a semiarid climate. Aerial parts were harvested during the flowering stage in April 2022 and were air-dried in obscurity at room temperature. The EO of dried samples of T. vulgaris (TVEO) aerial parts was hydrodistilled for 3 h using a Clevenger apparatus.


#### 2.1.2. EO Analysis Using Gas Chromatography–Mass Spectrometry (GC–MS)

The analysis of TVEO was accomplished using a GC/MS HP model 6980 inert MSD, equipped with an Agilent Technologies capillary HP-5MS column (60m×0.25mm, 0.25 mm film thickness) coupled to a mass selective detector (MSD5973, ionization voltage 70 eV, all Agilent, Santa Clara, CA, USA). The carrier gas was helium and has been maintained at 1.2 mL/min flow rate. The oven temperature program was as follows: 1 min at 100°C ramped from 100 to 280°C at 5°C/min and 25 min at 280°C. The chromatograph was equipped with a split/split less injector used in the split less mode. Identification of TVEO components was achieved by matching their mass spectra with Wiley Registry of Mass Spectral Data 7th edition (Agilent Technologies) and National Institute of Standards and Technology 05 MS (NIST) library data.


### 2.2. Antibacterial Activity

#### 2.2.1. Microorganisms and Growth Conditions

In order to evaluate TVEO antibacterial activity, two Gram-positive bacteria:S. aureus ATCC 6538 and L. monocytogenes ATCC 19117, and two Gram-negative bacteria: S. enterica Typhimurium ATCC 14028 and E. coli ATCC 8739, were selected. Bacterial cultures were deposited in 3 mL of Luria-Bertani (LB) agar medium composed of (g/L): peptone, 10; yeast extract, 5; NaCl, 5; and agar, 20 at pH 7.2; then, the bacterial strains were incubated at 37°C according to the method described by [23].


#### 2.2.2. Agar Diffusion Method

Antimicrobial activity of TVEO was evaluated by agar-well diffusion assay according to a method proposed by Güven et al. [24]. Fifteen milliliters of the molten agar (45°C) were flowed into sterile Petri dishes (Ø 90 mm). Bacterial cell suspensions were prepared, and 100 μL was evenly deposited onto the surface of plates containing LB agar medium. Plates were aseptically dried, and then, 5 mm wells were punched into the agar with a sterile Pasteur pipette. TVEO was dissolved in DMSO, water (1/9; v/v) to a final concentration of 1 mg/mL and then filtered through 0.22 μm pore size black polycarbonate filters. 100 μL of this filtered solution was placed into each well, and the plates were incubated at 37°C. Gentamicin (10 μg/wells) was used as a positive control.


#### 2.2.3. Minimal Inhibitory Concentration (MIC)

MIC is defined as the lowest concentration that could inhibit the visible growth of the tested microorganism. In this context, MIC of TVEO was tested against four pathogenic bacteria which are as follows: two Gram-positive bacteria:S. aureus ATCC 6538 and L. monocytogenes ATCC 19117, and two Gram-negative bacteria: S. enterica Typhimurium ATCC 14028 and E. coli ATCC 8739, using the microdilution method with serial dilution described by Chandrasekaran and Venkatesalu [25]. Then, bacterial suspension was added with a final inoculum concentration of 106 CF/mL. The contents of the tubes were mixed by pipetting and were incubated for 24 h at 37°C. For the antibacterial activity determination (inhibition zones and CMIs), each experiment was carried out simultaneously in triplicate under same conditions. The obtained diameters of inhibition zones were measured in mm and the MIC values were reported in mg/mL.


### 2.3. Interaction Study between TVEO Compounds and Selected Bacterial Targets by Molecular Docking

Receptor targets Fasta sequences ofS. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115 were obtained from UniProt database [26] and NCBI database [27]. Protein models were projected to SWISS-MODEL server [28] for molecular homology modeling approach [29]. Validation of the obtained models was performed by checking Ramachandran plots and the QMEAN values [30], using PROCHECK analysis tool integrated in Profunc server ([31]. SMILES (simplified molecular input line entry systems) strings of TVEO compounds were obtained from the PubChem database [32] and controls (rifamycin SV and ciprofloxacin). SMILES structures were downloaded from the DrugBank database [33], and all were converted into 3D structure using CORINA demo webserver [34] and then saved in pdb file format. Molecular docking was performed using the AutoDock Vina software (version 1.2.0) to calculate free energy of binding (kcal/mol) scores according to the methodology proposed by [35]. The docking position results were visualized using Discovery Studio version 16.1.0 (Dassault Systemes BIOVIA, 2016).


### 2.4. Toxicity Prediction of Compounds by VEGA HUB Software Using QSAR Method

All TVEO compounds and the two controls (rifamycin SV and ciprofloxacin) were subjected to 8 toxicity measurements in a view to assess carcinogenicity, mutagenicity, developmental/reproductive toxicity, endocrine disrupting ability, and genotoxicity. All tests were performed by VEGA software version 1.1.5 using the QSAR (quantitative structure–activity relationship) approach [36].


### 2.5. ADME Analysis

Pharmacokinetics, drug-likeness, and medicinal chemistry properties of TVEO compounds were predicted using SwissADME which is an open access software for ADME parameters evaluation and profiling [37].


### 2.6.In Silico Prediction of Possible Bioactivities

The PASS software was used to predict bioactivity of molecules based on the structural similarity to the large data base of known active substances in order to find new TVEO molecule targets [38, 39].


### 2.7. Statistical Analysis

All tests were assayed in triplicate and expressed as themean±standarddeviation of the measurements. The statistical program SPSS version 21.00 for Windows (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Variance was analyzed by one-way ANOVA and Student’s t-test was applied to compare each parameter at p<0.05.


## 2.1. Plant Material, Essential Oil Extraction, and Gas Chromatography–Mass Spectrometry (GC–MS) Analysis

### 2.1.1. Plant Material Collection and EO Extraction

Thymus vulgaris L. plant was collected from the region of Sfax, Tunisia (N: 34.4426°, E: 10.4537°) which is characterized by a semiarid climate. Aerial parts were harvested during the flowering stage in April 2022 and were air-dried in obscurity at room temperature. The EO of dried samples of T. vulgaris (TVEO) aerial parts was hydrodistilled for 3 h using a Clevenger apparatus.


### 2.1.2. EO Analysis Using Gas Chromatography–Mass Spectrometry (GC–MS)

The analysis of TVEO was accomplished using a GC/MS HP model 6980 inert MSD, equipped with an Agilent Technologies capillary HP-5MS column (60m×0.25mm, 0.25 mm film thickness) coupled to a mass selective detector (MSD5973, ionization voltage 70 eV, all Agilent, Santa Clara, CA, USA). The carrier gas was helium and has been maintained at 1.2 mL/min flow rate. The oven temperature program was as follows: 1 min at 100°C ramped from 100 to 280°C at 5°C/min and 25 min at 280°C. The chromatograph was equipped with a split/split less injector used in the split less mode. Identification of TVEO components was achieved by matching their mass spectra with Wiley Registry of Mass Spectral Data 7th edition (Agilent Technologies) and National Institute of Standards and Technology 05 MS (NIST) library data.


## 2.1.1. Plant Material Collection and EO Extraction

Thymus vulgaris L. plant was collected from the region of Sfax, Tunisia (N: 34.4426°, E: 10.4537°) which is characterized by a semiarid climate. Aerial parts were harvested during the flowering stage in April 2022 and were air-dried in obscurity at room temperature. The EO of dried samples of T. vulgaris (TVEO) aerial parts was hydrodistilled for 3 h using a Clevenger apparatus.


## 2.1.2. EO Analysis Using Gas Chromatography–Mass Spectrometry (GC–MS)

The analysis of TVEO was accomplished using a GC/MS HP model 6980 inert MSD, equipped with an Agilent Technologies capillary HP-5MS column (60m×0.25mm, 0.25 mm film thickness) coupled to a mass selective detector (MSD5973, ionization voltage 70 eV, all Agilent, Santa Clara, CA, USA). The carrier gas was helium and has been maintained at 1.2 mL/min flow rate. The oven temperature program was as follows: 1 min at 100°C ramped from 100 to 280°C at 5°C/min and 25 min at 280°C. The chromatograph was equipped with a split/split less injector used in the split less mode. Identification of TVEO components was achieved by matching their mass spectra with Wiley Registry of Mass Spectral Data 7th edition (Agilent Technologies) and National Institute of Standards and Technology 05 MS (NIST) library data.


## 2.2. Antibacterial Activity

### 2.2.1. Microorganisms and Growth Conditions

In order to evaluate TVEO antibacterial activity, two Gram-positive bacteria:S. aureus ATCC 6538 and L. monocytogenes ATCC 19117, and two Gram-negative bacteria: S. enterica Typhimurium ATCC 14028 and E. coli ATCC 8739, were selected. Bacterial cultures were deposited in 3 mL of Luria-Bertani (LB) agar medium composed of (g/L): peptone, 10; yeast extract, 5; NaCl, 5; and agar, 20 at pH 7.2; then, the bacterial strains were incubated at 37°C according to the method described by [23].


### 2.2.2. Agar Diffusion Method

Antimicrobial activity of TVEO was evaluated by agar-well diffusion assay according to a method proposed by Güven et al. [24]. Fifteen milliliters of the molten agar (45°C) were flowed into sterile Petri dishes (Ø 90 mm). Bacterial cell suspensions were prepared, and 100 μL was evenly deposited onto the surface of plates containing LB agar medium. Plates were aseptically dried, and then, 5 mm wells were punched into the agar with a sterile Pasteur pipette. TVEO was dissolved in DMSO, water (1/9; v/v) to a final concentration of 1 mg/mL and then filtered through 0.22 μm pore size black polycarbonate filters. 100 μL of this filtered solution was placed into each well, and the plates were incubated at 37°C. Gentamicin (10 μg/wells) was used as a positive control.


### 2.2.3. Minimal Inhibitory Concentration (MIC)

MIC is defined as the lowest concentration that could inhibit the visible growth of the tested microorganism. In this context, MIC of TVEO was tested against four pathogenic bacteria which are as follows: two Gram-positive bacteria:S. aureus ATCC 6538 and L. monocytogenes ATCC 19117, and two Gram-negative bacteria: S. enterica Typhimurium ATCC 14028 and E. coli ATCC 8739, using the microdilution method with serial dilution described by Chandrasekaran and Venkatesalu [25]. Then, bacterial suspension was added with a final inoculum concentration of 106 CF/mL. The contents of the tubes were mixed by pipetting and were incubated for 24 h at 37°C. For the antibacterial activity determination (inhibition zones and CMIs), each experiment was carried out simultaneously in triplicate under same conditions. The obtained diameters of inhibition zones were measured in mm and the MIC values were reported in mg/mL.


## 2.2.1. Microorganisms and Growth Conditions

In order to evaluate TVEO antibacterial activity, two Gram-positive bacteria:S. aureus ATCC 6538 and L. monocytogenes ATCC 19117, and two Gram-negative bacteria: S. enterica Typhimurium ATCC 14028 and E. coli ATCC 8739, were selected. Bacterial cultures were deposited in 3 mL of Luria-Bertani (LB) agar medium composed of (g/L): peptone, 10; yeast extract, 5; NaCl, 5; and agar, 20 at pH 7.2; then, the bacterial strains were incubated at 37°C according to the method described by [23].


## 2.2.2. Agar Diffusion Method

Antimicrobial activity of TVEO was evaluated by agar-well diffusion assay according to a method proposed by Güven et al. [24]. Fifteen milliliters of the molten agar (45°C) were flowed into sterile Petri dishes (Ø 90 mm). Bacterial cell suspensions were prepared, and 100 μL was evenly deposited onto the surface of plates containing LB agar medium. Plates were aseptically dried, and then, 5 mm wells were punched into the agar with a sterile Pasteur pipette. TVEO was dissolved in DMSO, water (1/9; v/v) to a final concentration of 1 mg/mL and then filtered through 0.22 μm pore size black polycarbonate filters. 100 μL of this filtered solution was placed into each well, and the plates were incubated at 37°C. Gentamicin (10 μg/wells) was used as a positive control.


## 2.2.3. Minimal Inhibitory Concentration (MIC)

MIC is defined as the lowest concentration that could inhibit the visible growth of the tested microorganism. In this context, MIC of TVEO was tested against four pathogenic bacteria which are as follows: two Gram-positive bacteria:S. aureus ATCC 6538 and L. monocytogenes ATCC 19117, and two Gram-negative bacteria: S. enterica Typhimurium ATCC 14028 and E. coli ATCC 8739, using the microdilution method with serial dilution described by Chandrasekaran and Venkatesalu [25]. Then, bacterial suspension was added with a final inoculum concentration of 106 CF/mL. The contents of the tubes were mixed by pipetting and were incubated for 24 h at 37°C. For the antibacterial activity determination (inhibition zones and CMIs), each experiment was carried out simultaneously in triplicate under same conditions. The obtained diameters of inhibition zones were measured in mm and the MIC values were reported in mg/mL.


## 2.3. Interaction Study between TVEO Compounds and Selected Bacterial Targets by Molecular Docking

Receptor targets Fasta sequences ofS. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115 were obtained from UniProt database [26] and NCBI database [27]. Protein models were projected to SWISS-MODEL server [28] for molecular homology modeling approach [29]. Validation of the obtained models was performed by checking Ramachandran plots and the QMEAN values [30], using PROCHECK analysis tool integrated in Profunc server ([31]. SMILES (simplified molecular input line entry systems) strings of TVEO compounds were obtained from the PubChem database [32] and controls (rifamycin SV and ciprofloxacin). SMILES structures were downloaded from the DrugBank database [33], and all were converted into 3D structure using CORINA demo webserver [34] and then saved in pdb file format. Molecular docking was performed using the AutoDock Vina software (version 1.2.0) to calculate free energy of binding (kcal/mol) scores according to the methodology proposed by [35]. The docking position results were visualized using Discovery Studio version 16.1.0 (Dassault Systemes BIOVIA, 2016).


## 2.4. Toxicity Prediction of Compounds by VEGA HUB Software Using QSAR Method

All TVEO compounds and the two controls (rifamycin SV and ciprofloxacin) were subjected to 8 toxicity measurements in a view to assess carcinogenicity, mutagenicity, developmental/reproductive toxicity, endocrine disrupting ability, and genotoxicity. All tests were performed by VEGA software version 1.1.5 using the QSAR (quantitative structure–activity relationship) approach [36].


## 2.5. ADME Analysis

Pharmacokinetics, drug-likeness, and medicinal chemistry properties of TVEO compounds were predicted using SwissADME which is an open access software for ADME parameters evaluation and profiling [37].


## 2.6.In Silico Prediction of Possible Bioactivities

The PASS software was used to predict bioactivity of molecules based on the structural similarity to the large data base of known active substances in order to find new TVEO molecule targets [38, 39].


## 2.7. Statistical Analysis

All tests were assayed in triplicate and expressed as themean±standarddeviation of the measurements. The statistical program SPSS version 21.00 for Windows (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Variance was analyzed by one-way ANOVA and Student’s t-test was applied to compare each parameter at p<0.05.


## 3. Results and Discussion

### 3.1. Chemical Composition Analysis of TVEO

GC-MS analysis of TVEO revealed the existence of 26 different components (Table1). The main components were thymol, the most abundant compound (47.44%), followed by o-cymene (16.55%), γ-terpinene (7.80%), and linalool (4.41%). Moreover, it detected the presence of 4 compounds which their percentages (EO %) were more than 2%. These later are the α-zingibirene (3.40%), β-myrcene (2.36%), caryophyllene (2.09%), and borneol (2.02%).Table 1
Chemical composition of TVEO.
CompoundMolar mass (g/mol)Molecular formulaRetention time (min)EO (%)α-Pinene136.23C10H165.991.74α-Thujene136.23C10H166.151.61Camphene136.23C10H166.661.60β-Pinene136.23C10H167.390.29β-Myrcene136.23C10H167.852.36α-Fellandrene136.23C10H168.170.343-Carene136.23C10H168.310.16D-Limonene136.23C10H168.521.61O-Cymene134.22C10H148.8816.55Cymol134.22C10H149.460.09γ-Terpinene136.23C10H169.797.80Terpinolene136.23C10H1610.560.10Linalool154.25C10H18O11.034.41Camphor152.23C10H16O12.141.15Borneol152.23C10H18O12.842.024-Terpinenol152.23C10H18O13.230.92α-Terpineol152.23C10H18O13.900.10Thymol methyl ether164.24C11H16O14.990.43Thymol150.22C10H14O16.4847.44Caryophyllene204.35C15H2419.592.09α-Humulene204.35C15H2420.430.03α-Amorphene204.35C15H2420.990.10α-Curcumene202.33C15H2221.170.71α-Zingibirene204.35C15H2421.493.40α-Bisabolene204.35C15H2421.791.12β-Sesquiphellandrene204.35C15H2422.171.34Monoterpenes hydrocarbons34.25%Oxygenated monoterpenes56.47%Sesquiterpens8.79%Total99.51%Thymol, known also by the chemical name 2-isopropyl-5-methylphenol, is a natural phenol monoterpene [40] importantly detected in Lamiacaeae family [41] including many plant species such as Thymus vulgaris L. [42], Ocimum gratissimum L. [43], Origanum L. [44], and Trachyspermum ammi L. [45] and other species of the genus Satureja L. [46] and Monarda L. [47]. This volatile monoterpenoid is largely used by nutraceutical, pharmaceutical, and cosmeceutical industries due to its multiple potential therapeutic properties [48–58]. In addition, thymol was globally recognized-as-safe food additive according to US department of Food and Drug Administration (FDA) [59].O-cymene, known as 1-isopropyl-2-methylbenzene, is an acyclic monoterpene which belongs to p-cymene isomers and has an orthosubstituted alkyle group [60]. Previous studies demonstrated that o-cimene has several therapeutic effects [61–66]. Several studies indicated the existence of a remarkable synergetic effect between ocimene and other terpenes such as α-pinene and myrcene which were noticed to produce more beneficial effects when combined [67, 68].γ-Terpinene, renowned also as p-mentha-1,4-diene, is a naturally occurring monoterpene hydrocarbon [69] that has been isolated from many botanical sources including Origanum vulgare, Citrus limon L, Melaleuca alternifolia, and Eucalyptus obliqua [70, 71]. This monoterpene is largely employed in food, cosmetics, and pharmaceutical industries [72]. Previous research showed that γ-terpinene has potential biological activities such as antioxidant [73], anti-inflammatory [74], and antimicrobial activities [75, 76].On the other hand, linalool, known as 3,7-dimethyl-1,6-octadien-3-ol [77], is a naturally occurring acyclic monoterpenoid and tertiary alcohol which is commonly found as a major active component in the essential oil of several aromatic plant species [78] principally in Lamiaceae and Lauraceae botanical families. It has been reported that this monoterpenol is broadly used in food industry as an aromatic and preservative agent, in cosmetic as a fragrance and antiseptic constituent [79–83].It should be noted that TVEO composition may depend on many biotic and abiotic factors including seasonal variations of temperature and humidity [84], phenological stages and different vegetation cycles [85], geographic location [86], environmental stress [87], and extraction technique [88]. Aljabeili et al. reported that the TVEO collected from KSA showed a significant composition variation and the major compounds were thymol (41.04%), 1,8-cineole (14.26%), γ-terpinene (12.06%), and p-cymene (10.50%) [89]. Another study revealed that Turkish TVEO has different components amounts such as thymol (49%), β-cymene (19.99%), carvacrol (7.63%), and trans-caryophyllene (6.79%) [90]. In addition, Moghaddam et al. reported that Iranian TVEO contains thymol (36.81%), ρ-cymene (30.90%), and carvacrol (3.16%) [91].


### 3.2. Antibacterial Activity

As represented in Table2, TVEO displayed an interesting antibacterial activity against the four tested bacterial strains with inhibition zones ranging from of 21 to 23 mm against Gram-negative and Gram-positive bacteria, respectively. In addition, it is important to note that monoterpenes exhibit a broad-spectrum antibacterial activity against pathogenic bacteria [92, 93]. This fact could be explained by the presence of a lipophilic character which provides to monoterpenes the ability to adhere to bacterial cell membrane lipids and to deploy their antibacterial action [94]. In our study, the obtained MIC values mentioned in Table 2, indicated that TVEO was more potent (p<0.05) against Gram-positive bacteria (MICs=0.097 mg/mL) than Gram-negative bacteria (MICs=0.195 mg/mL). These findings were in agreement with previous studies which reported that Gram-positive bacteria are susceptible to be more sensitive to plant EOs than Gram-negative bacteria due to the existence of lipopolysaccharides which acts as a hydrophobic barrier [95, 96].Table 2
Antibacterial activity of TVEO and the control gentamicin. Zones of growth inhibition was expressed in mm, and minimum inhibitory concentrations (MICs) were expressed in mg/mL.
Bacterial strainsInhibition zone diameters (mm)MIC (mg/mL)TVEOGentamicinTVEOGentamicinS. aureus ATCC 653823±1.00b20±0.83a0.097±0.00b0.013±0.00bL. monocytogenes ATCCC 1911722±1.00b20±0.75a0.097±0.00b0.013±0.00bS. enterica Typhimurium ATCC 1402823±1.00a25±1.25b0.097±0.00b0.005 ± 0.00bE. coli ATCC 873921±0.83a25±1.25b0.195±0.00b0.005±0.00bA Studentt-test was used to determine the significant differences between inhibition zones and MIC.


### 3.3. Interactions between TVEO Molecules and Bacterial Topoisomerase II and DNA and RNA Polymerases

Computational modeling is a 3R-based approach and an attractive alternative to experiments in a view to understand bioactive compounds mechanism of action and their antibacterial inhibitory process [97]. Molecular docking was performed to predict different TVEO components that could bind specifically to select bacterial receptors active sites responsible for DNA replication and transcription processes. In this respect, we investigated TVEO antibacterial inhibitory effect on topoisomerase II (DNA gyrase) and DNA and RNA polymerases of three pathogenic bacteria S. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115. Molecular homology results indicated that selected templates can be used for molecular modeling (Table 3); their identities (%) are higher than 30% [98], and Ramachandran plot values of favored regions and allowed regions were over 90% [99].Table 3
Receptors models of the selected pathogenic bacteria used in molecular docking simulation with TVEO constituents.
Bacterial StrainBacterial TargetReceptorTemplateIdentity (%)Ramachandran PlotQMEANFavored regions (%)Additional allowed regions (%)S. aureus ATCC 700699DNA polymeraseP63979 (UniProt)4IQJ.1.L36.7787.910.1−3.26RNA polymeraseQ932F8 (UniProt)6WVK.1.C81.0985.512.7−2.36Topoisomerase IIP66936 (UniProt)6GAV.1.A54.4288.410.7−1.69S. enterica Typhimurium ATCC 700720DNA polymeraseP14567 (UniProt)5FKU.1.A96.7288.010.0−2.35RNA polymeraseP06173 (UniProt)4LLG.2.C98.6688.011.0−1.11Topoisomerase IIP0A213 (UniProt)4TMA.2.B95.4190.29.2−1.82L. monocytogenes ATCC 19115DNA polymeraseWP031669548 (NCBI)6VDE.1.A38.3081.616.4-3.91RNA polymeraseGAT39567 (NCBI)6WVK.1.C86.1087.111.4-2.24Topoisomerase IIGAT39106 (NCBI)2XCR.2.A69.5987.410.4-2.01Moreover, it is necessary to remind that DNA and RNA polymerases are crucial enzymes involved in the DNA replication, transcription, and translation as well as nucleic acid formation in bacterial cells [100]. On the other hand, topoisomerase II (DNA gyrase) is also implicated in DNA replication and transcription processes and has an imperative role characterized by its ability to catalyze the unwinding of supercoiled DNA strands [101]. These imperative enzymes constitute attractive and validated targets for antibacterial agents [102]. Molecular docking simulation results are elucidated by (Table 4). These later displayed that TVEO major compound thymol (47.44%) showed a good inhibitory effect on topoisomerase II, RNA polymerase, and DNA polymerase of the selected pathogenic bacteria.Table 4
Molecular docking results for complexes ofT. vulgaris EO compounds against DNA and DNA polymerases of S. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115 using Autodock Vina (kcal/mol).
Bacterial targetS. aureus ATCC 700699S. enterica Typhimurium ATCC 700720L.monocytogenes ATCC 19115CompoundDNA polymeraseRNA polymeraseTopoisomerase IIDNA polymeraseRNA polymeraseTopoisomerase IIDNA polymeraseRNA polymeraseTopoisomerase IIα-Pinene-5.1-6.1-5.3-5.4-5.4-5.2-5.9-6.0-5.4α-Thujene-4.9-5.4-5.5-5.3-5.2-5.3-5.5-5.3-5.6Camphene-5.2-6.0-5.6-5.2-5.2-5.0-6.1-5.9-5.3β-Pinene-5.2-5.7-5.2-5.5-5.7-5.3-5.9-6.0-5.2β-Myrcene-4.9-5.0-5.2-5.1-5.1-5.0-5.2-5.0-5.0α-Fellandrene-5.2-5.6-5.4-5.1-5.1-5.1-5.5-5.5-5.63-Carene-5.2-5.6-5.2-5.6-5.6-5.5-5.6-5.5-5.7D-Limonene-5.1-6.0-5.4-5.4-5.4-5.1-5.5-5.6-5.6O-Cymene-5.3-5.5-5.6-5.7-5.4-5.4-5.8-5.5-5.5Cymol-5.2-5.7-5.6-5.5-5.5-5.2-6.0-5.9-5.7γ-Terpinene-5.3-6.0-5.6-5.8-5.6-5.7-5.8-5.8-5.6Terpinolene-5.4-5.8-5.6-5.6-6.0-5.6-5.8-5.6-5.8Linalool-5.0-5.2-5.2-5.5-5.6-5.4-5.8-5.1-5.2Camphor-5.3-6.0-5.4-5.4-5.2-5.3-6.0-5.6-5.2Linderol (borneol)-5.4-5.7-5.5-5.3-5.3-5.2-5.8-5.7-5.34-Terpineol-5.4-6.2-5.8-5.8-5.8-5.5-5.9-6.0-5.8α-Terpineol-5.4-6.0-5.6-5.6-5.8-5.0-5.8-5.6-5.8Thymol methyl ether-5.5-5.8-5.6-5.3-5.4-4.9-5.6-5.5-5.7Thymol-6.3-6.2-6.6-6.4-6.6-6.2-6.8-6.7-6.6Caryophyllene-6.0-6.4-6.7-6.4-6.5-6.4-6.5-6.3-6.4α-Humulene-6.3-6.3-6.9-6.4-7.1-6.1-6.5-6.8-6.4α-Amorphene-6.1-6.2-6.3-6.5-6.0-6.1-6.6-6.6-6.2α-Curcumene-6.3-6.6-6.7-6.5-6.5-6.0-6.8-6.4-6.3α-Zingibirene-5.4-6.5-6.4-6.0-5.5-5.6-5.7-6.0-6.2α-Bisabolene-5.6-6.8-6.6-6.4-6.4-5.8-6.4-5.9-6.2β-Sesquiphellandrene-5.9-7.0-7.1-6.86.8-6.7-7.1-6.9-6.9Rifamycin SV-8.7-9.8—-8.7-8.6—-9.6-10.2—Ciprofloxacin——-7.4——-6.3——-6.5A previous study conducted by Liu et al. reported that thymol has a potential antibacterial activity againstP. aeruginosa [15]. This monoterpenoid can affect bacterial DNA normal function. It could block gene expression processes including replication, transcription, and expression by intercalation with bacterial DNA leading to bacterial death. The same study indicated that thymol could destroy bacterial membrane integrity by affecting its permeability and it could also hinder biofilm formation. Another research paper displayed that thymol could bind to bacterial DNA, modulate its structure, and prohibit its biological function [103]. Furthermore, dos Santos Barbosa et al. showed that the antibacterial inhibitory activity of Origanum vulgare EO was effective against Salmonella Enteritidis due to the presence of thymol which caused interference in protein regulation as well as DNA synthesis [104]. On the other hand, the minor compound β-sesquiphellandrene (1.34%) showed the lowest free energy of binding (Kcal/mol) and the highest inhibitory potential on topoisomerase II and DNA and RNA polymerases of the selected pathogenic bacteria (Table 4). A previous study reported that β-sesquiphellandrene was responsible for the high antibacterial and antioxidant activities of some pomelo varieties’ EOs [105]. Another study indicated that the presence of β-sesquiphellandrene presented in ginger (Zingiber officinale) essential oil could make this later as a potential antimicrobial agent by inhibiting mycobacterial acyl carrier protein reductase enzyme and Enoyl acyl carrier protein reductase activities [106].A recent study showed thatCupressus sempervirens EO had a great inhibitory effect on DNA gyrase and DNA and RNA polymerases of S. aureus and S. enterica owing to the presence of α-pinene, δ-3 carene, and borneol [13]. Moreover, a previous study conducted by [107] evaluated the inhibitory effect of Litsea cubeba EO on topoisomerase and DNA and RNA polymerases of E.coli. Another study revealed that the germacrene B, a minor compound in Siparuna guianensis EO, had an effective inhibitory activity against bacterial DNA and RNA polymerases of multiple pathogenic bacteria including E.coli, P. aeruginosa, S. aureus, and S. pyogenes [108]. Therefore, these findings confirmed that TVEO has a powerful inhibitory effect on pathogenic bacteria based on the inhibition of DNA replication and transcription processes. Consequently, we project in subsequent work to perform further in vitro assays based on the evaluation of protein-molecule binding assays.Interaction profiles details of thymol andβ-sesquiphellandrene with the active sites of the selected targets of S. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115 are summarized in Table 5.Table 5
Interactions details of thymol andβ-sesquiphellandrene with the selected bacterial targets.
BacteriaCompoundTargetsNumber of Residues InteractingResidues with H-bondS. aureus ATCC 700699ThymolDNA polymerase4LYS228RNA polymerase4—Topoisomerase II6GLU609, ASP610β-SesquiphellandreneDNA polymerase1—RNA polymerase7—Topoisomerase II8—S. enterica Typhimurium ATCC 700720ThymolDNA polymerase5—RNA polymerase1—Topoisomerase II6ASN588β-SesquiphellandreneDNA polymerase8—RNA polymerase5—Topoisomerase II6—L. monocytogenes ATCC 19115ThymolDNA polymerase8—RNA polymerase5—Topoisomerase II3VAL113β-SesquiphellandreneDNA polymerase6—RNA polymerase4—Topoisomerase II5—Interaction profile results of thymol andβ-sesquiphellandrene with topoisomerase II and DNA and RNA polymerases of S. aureus are presented by Figures 1 and 2. Thymol made a complex with DNA polymerase receptor via alkyl interaction and conventional hydrogen bond with LYS228 and van der Waals interactions with ASN84, LYS88, LEU87, GLU469, TYR91, LEU227, and ILE492 (Figure 1(a)). Additionally, RNA polymerase complexed with thymol had alkyl and Pi-alkyl interaction with VAL536 and LYS35; van der Waals interactions with GLU413, SER410, GLY412, SER36, and GLU538; and Pi-sigma interaction with TRP39 (Figure 1(b)). Likewise, it interacted with topoisomerase II via alkyl interaction with ALA614; van der Waals interactions with HIS46, ARG198, TRP49, ARG42, THR194, GLN197, THR617, and LEU608; and two conventional hydrogen bonds with ASP610 and GLU609 (Figure 1(c)).Figure 1
Thymol complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) ofS. aureus.
(a)(b)(c)Figure 2
β-Sesquiphellandrene complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) of S. aureus.
(a)(b)(c)β-Sesquiphellandrene complexed with DNA polymerase showed Pi-alkyl interaction with LEU641 and van der Waals interactions with ILE938, LYS901, PHE900, ASP936, GLU939, ILE899, SER903, LEU902, and GLN975 (Figure 2(a)). When complexed with RNA polymerase, it made Pi-alkyl interaction with VAL270, LYS35, and VAL536; van der Waals interactions with SER36, SER410, GLY412, ARG407, GLN416, GLN413, GLU538, and ILE32; and Pi-sigma interaction with TRP39 (Figure 2(b)). Concerning topoisomerase II, β-sesquiphellandrene displayed alkyl and Pi-alkyl interactions with VAL606, PHE618, ILE532, ALA614, LEU521, and LEU608 and van der Waals interactions with LYS607, ARG198, GLU609, GLU613, ASP610, and TYR525 (Figure 2(c)).On the other hand, interaction profiles between thymol andβ-sesquiphellandrene with topoisomerase II and DNA and RNA polymerases of S. enterica Typhimurium are outlined in Figures 3 and 4. DNA polymerase complexed with thymol showed alkyl and Pi-alkyl interactions with ARG18 and LYS17 and van der Waals interactions with ASP1188, THR657, SER656, PRO19, ASN622, ASN620, SER621, LEU623, and ALA 619 (Figure 3(a)). Thymol complex with RNA polymerase indicated the presence of alkyl interaction with MET768 and van der Waals interactions with ASP785, GLN767, ASN766, GLY786, PRO787, SER788, PRO691, THR692, ALA695, THR789, and LEU693 (Figure 3(b)). This monoterpene also made interactions with topoisomerase II via alkyl interactions with VAL727, ILE557, and ALA560; van der Waals interactions with LEU561, VAL584, LEU723, GLN591, and ASP553; and one conventional hydrogen bond with ASN588 (Figure 3(c)).Figure 3
Thymol complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) ofS. enterica Typhimurium.
(a)(b)(c)Figure 4
β-Sesquiphellandrene complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) of S. enterica Typhimurium.
(a)(b)(c)The complex ofβ-sesquiphellandrene and the DNA polymerase of S. enterica Typhimurium revealed the existence of alkyl and Pi-akyl interactions with LEU75, ALA94, LEU129, LEU32, PHE35, and ILE39 and van der Waals interactions with GLN41, GLN36, GLY76, and MET130 (Figure 4(a)). This sesquiterpene complexed with RNA polymerase showed alkyl and Pi-alkyl interactions with MET130, LEU32, LEU129, PHE35, ALA94, and LEU75 and van der Waals interactions with ILE39, GLN36, and ASP32 (Figure 4(b)). In addition, it made interactions with topoisomerase II via alkyl and Pi alkyl interactions with VAL467, PHE513, PHE777, ARG516, and LEU462 and van der Waals interactions with MET461, LEU509, and THR512 (Figure 4(c)).DNA polymerase receptor ofL. monocytogenes complexed with thymol displayed the existence of alkyl and Pi-alkyl interactions with MET389, LEU345, LEU394, ILE392, and PHE367 and van der Waals interactions with GLU359, THR357, LYS358, ILE343, THR360, and SER364 (Figure 5(a)). It also showed when complexed with RNA polymerase alkyl interactions with LYS283 and LYS284 and van der Waals interactions with VAL150, GLY149, TYR151, ASN410, ASP403, ASP571, and ASN289 (Figure 5(b)). Further, thymol made interactions with topoisomerase II via alkyl interaction with PHE97; van der Waals interactions with GLN95, SER98, GLN267, TYR266, THR220, VAL268, ASN269, GLY115, and SER112; and one conventional hydrogen bond with VAL113 (Figure 5(c)).Figure 5
Thymol complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) ofL. monocytogenes.
(a)(b)(c)Finally,β-sesquiphellandrene complex with DNA polymerase of L. monocytogenes indicated the existence of Alkyl interactions with MET386, ALA382, ILE399, and LEU488 and van der Waals interactions with SER397, PHE492, PRO489, GLU484, THR400, and THR491 (Figure 6(a)). It showed also with RNA polymerase alkyl interactions with LYS280, LYS284, and TYR151 and van der Waals interactions with VAL150, GLY149, ASP403, ASN410, ASP571, ASP404, ASP401, ASN289, and LYS283 (Figure 6(b)). In addition, β-sesquiphellandrene made a complex with topoisomerase II via alkyl and Pi-alkyl interactions with VAL113, ILE264, and PHE97 and van der Waals interactions with GLY115, SER112, GLN95, ARG92, GLY111, PHE88, SER98, ASN269, VAL268, THR220, PRO265, and TYR266 (Figure 6(c)).Figure 6
β-Sesquiphellandrene complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) of L. monocytogenes.
(a)(b)(c)


### 3.4.In Silico TVEO Compound Toxicity Evaluation by VEGA QSAR Model

Toxicity evaluation of different TVEO compounds and the two selected FDA-approved antibiotics, used as controls (rifamycin SV and ciprofloxacin), was performed by the help of QSAR (quantitative structure–activity relationship) approach and using VEGA HUB software. We chose to assess compounds toxicity based on 8 different toxicity measurements. Results are represented by (Table6) and revealed that FDA-approved drugs could be toxic in several assays. In this context, rifamycin SV and ciprofloxacin are found to be mutagenic in the mutagenicity test/model (Ames test) and predicted to engender developmental/reproductive toxicity. These two antibiotics were also predicted to be genotoxic according to the in vitro micronucleus activity model. However, many TVEO compounds such as α-pinene, α-thujene, camphene, β-pinene, 3-carene, D-limonene, γ-terpinene, terpinolene, linalool, borneol, 4-terpineol, α-terpineol, thymol, caryophyllene, α-bisabolene, and β-sesquiphellandrene showed nontoxic effects. Computational toxicity results confirmed that TVEO molecules could be used as a safe antimicrobial agents and economically low-cost alternative as compared to synthetic antibiotics. It is important to mention that research related to toxicological profiles of different essential oil compounds are poorly studied due to experiments complexity, expensive cost, and difficulty to detect toxicity variation because of the chemical function and factor variability [109]. However, previous studies indicated that toxicity is a dose-/concentration-dependent manner and thymol could have a certain limit of toxicity ranging from 36 mg/mL to 49 mg/mL with less risks of accumulation in body tissues and suggested to replace synthetic drugs, which has more side effect [110]. In addition, Schönknecht et al. confirmed the safety and the effectiveness of the drug containing the extracts of thyme with the addition of thymol (Bronchosol®) in cough treatment instead of using the synthetic drug ambroxol [111]. Concerning β-sesquiphellandrene, it was reported that this sesquiterpenoid could have a great anticancer activity and can be safe to use as compared to synthetic chemotherapeutic agents velcade, thalidomide, and capecitabine [112]. Nevertheless, more toxicological in vitro/in vivo data are needed to validate the safety of these phytochemicals. Thus, computational toxicity assessment could be the best alternative that would give robust data, avoid unnecessary waste of reagents, and minimize cruelty and sacrifices of lab animal testing.Table 6
Toxicity predictions of TVEO compounds using the VEGA QSAR model.
Toxicity measurementsMutagenicity (Ames Test) Model (CAESAR) 2.1.13Carcinogenicity model (CAESAR) 2.1.9Developmental/Reproductive Toxicity Library (PG) 1.1.0Estrogen receptor relative binding affinity model (IRFMN)Androgen receptor-mediated effect (IRFMN/COMPARA) 1.0.0Thyroid receptor alpha effect (NRMEA) 1.0.0Thyroid receptor beta effect (NRMEA) 1.0.0In vitro micronucleus activity (IRFMN/VERMEER) 1.0.0Compoundα-PineneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveα-ThujeneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveCampheneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveβ-PineneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveβ-MyrceneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveα-FellandreneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveActive3-CareneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveD-LimoneneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveO-CymeneNonmutagenicNoncarcinogenToxicantInactiveNonactiveInactiveInactiveNot predictedCymolNonmutagenicNoncarcinogenToxicantInactiveNonactiveInactiveInactiveNot predictedγ-TerpineneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveTerpinoleneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveLinaloolNonmutagenicNon carcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveCamphorNonmutagenicNoncarcinogenToxicantInactiveNonactiveInactiveInactiveActiveBorneolNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactive4-TerpineolNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveα-TerpineolNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveThymol methyl etherNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveThymolNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveCaryophylleneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveα-HumuleneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveα-AmorpheneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveα-CurcumeneNonmutagenicNoncarcinogenToxicantInactiveActiveInactiveInactiveActiveα-ZingibireneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveα-BisaboleneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveβ-SesquiphellandreneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveRifamycin SVMutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveCiprofloxacinMutagenicNoncarcinogenToxicantInactiveNonactiveInactiveInactiveActive


### 3.5. TVEO Component ADME Analysis

In the present study, SwissADME server was used to determine some pharmacokinetics parameters included in absorption, distribution, metabolism, and excretion (ADME), drug-likeness, and medicinal chemistry characteristics for all TVEO compounds as represented in (Table7). Results revealed that all TVEO components possess slow passive gastrointestinal absorption (GI) except linalool, camphor, borneol, 4-terpineol, α-terpineol, thymol methyl ether, and thymol which were predicted to have high GI permeability. Additionally, only sesquiterpenes including caryophyllene, α-humulene, α-amorphene, α-curcumene, α-zingibirene, α-bisabolene, and β-sesquiphellandrene were found not to be blood-brain barrier (BBB) permanent due to their heavy molecular weight. However, rest of compounds could easily cross the blood-brain barrier for that reason it could be suggested as potent central nervous system antioxidants and effective drug candidates in the treatment of neurodegenerative diseases like Alzheimer’s and Parkinson’s [113]. On the other part, none of the tested compounds was predicted to be P-gp transporter substrate. Concerning Cytochrome p450 (CYP) isoenzymes which are involved in 50-90% of therapeutic molecules biotransformation processes in a view to reduce metabolites accumulation in blood/tissues and drug-drug interaction risks [114], thymol methyl ether and thymol were predicted to be CYP1A2 inhibitors. Many TVEO molecules were predicted to inhibit CYP2C9 and CYP2D6 inhibitors. However, none of the compounds showed an inhibitory effect towards the CYP3A4. Moreover, skin permeation coefficient (logKp) indicated that all compounds were impermeable through the skin barrier. Interestingly, TVEO drug-likeness score was acceptable with good bioavailability score (>10%) and the absence of violations related to known rules such as Lipinski’s rule of five that predicts drug permeability and absorption based on H-bond donors, H-bond acceptors, molecular weight (MW>500), and a calculated logp) [115], as well as Veber. Both rules are a mainstay of decision-making in drug design and development and in the present study; both were validated. Furthermore, medicinal chemistry parameters revealed that none of the selected molecules returns any pan-assay interference compounds (PAINS) alert. The synthetic accessibility values of TVEO compounds indicated that these later could be synthesized for pharmaceutical uses.Table 7
TVEO compounds pharmacokinetic properties prediction using SwissADME.
CompoundPharmacokineticsDrug-likeness and Medicinal chemistryGI absorptionBBB permeantP-gp substrateCYP1A2 inhibitorCYP2C9 inhibitorCYP2D6 inhibitorCYP3A4 inhibitorLogKp(cm/s)LipinskiVeberBioavailability scorePAINSSynthetic accessibilityα-PineneLowYesNoNoYesNoNo-3.95YesYes0.550 alert4.44α-ThujeneLowYesNoNoNoNoNo-5.11YesYes0.550 alert3.99CampheneLowYesNoNoYesNoNo-4.13YesYes0.550 alert3.50β-PineneLowYesNoNoYesNoNo-4.18YesYes0.550 alert3.73β-MyrceneLowYesNoNoNoNoNo-4.17YesYes0.550 alert2.85α-FellandreneLowYesNoNoNoNoNo-4.85YesYes0.550 alert4.153-CareneLowYesNoNoYesNoNo-4.02YesYes0.550 alert3.84D-LimoneneLowYesNoNoYesNoNo-3.89YesYes0.550 alert3.46O-CymeneLowYesNoNoNoYesNo-4.01YesYes0.550 alert1.00CymolLowYesNoNoNoYesNo-4.21YesYes0.550 alert1.00γ-TerpineneLowYesNoNoYesNoNo-3.94YesYes0.550 alert3.11TerpinoleneLowYesNoNoYesNoNo-3.96YesYes0.550 alert2.98LinaloolHighYesNoNoNoNoNo-5.13YesYes0.550 alert2.74CamphorHighYesNoNoNoNoNo-5.67YesYes0.550 alert3.22BorneolHighYesNoNoNoNoNo-5.31YesYes0.550 alert3.434-TerpineolHighYesNoNoNoNoNo-4.93YesYes0.550 alert3.28α-TerpineolHighYesNoNoNoNoNo-4.83YesYes0.550 alert3.24Thymol methyl etherHighYesNoYesNoYesNo-4.64YesYes0.550 alert1.09ThymolHighYesNoYesNoNoNo-4.87YesYes0.550 alert1.00CaryophylleneLowNoNoNoYesNoNo-4.44YesYes0.550 alert4.51α-HumuleneLowNoNoNoYesNoNo-4.32YesYes0.550 alert3.66α-AmorpheneLowNoNoNoYesNoNo-4.65YesYes0.550 alert4.35α-CurcumeneLowNoNoNoNoYesNo-3.71YesYes0.550 alert2.31α-ZingibireneLowNoNoNoYesNoNo-3.88YesYes0.550 alert4.81α-BisaboleneLowNoNoNoYesNoNo-3.03YesYes0.550 alert3.90β-SesquiphellandreneLowNoNoNoYesNoNo-3.71YesYes0.550 alert4.42


### 3.6. Prediction of Possible Activity Spectra of TVEO Components

All TVEO compounds were subjected to PASS (Prediction of Activity Spectra for Substances) online tool intending to predict their biological activity spectrum based on their chemical structure. Results indicated that the tested compounds could have multiple biological activities and various enzymatic targets (Table8). We have selected the top three activities which showed a Pa≥0.7. Pa and Pi values indicated the probability of the selected molecule to be active/ inactive towards the targeted receptor. Mojumdar et al. reported that the probability of experimental pharmacological action is high when Pa>0.7; however, the chance of finding the activity experimentally is less when Pa<0.5 [116]. Interestingly, TVEO major compounds such as thymol which were predicted, in earlier section by molecular docking, to have a potent antibacterial activity on pathogenic bacteria by inhibiting DNA replication and transcription processes, displayed the existence of other biological activities including the ability to inhibit bacterial membrane permeability (Pa=0.876) and it was anticipated to enhance AP0A1 expression (Pa=0.830) involved in the cellular synthesis of beneficial HDL [117]. In addition, O-cymene was predicted to be mitochondria ubiquinol-cytochrome-c reductase inhibitor (Pa=0.924) (antifungal activity), mucomembranous protector (Pa=0.842), and a fibrinolytic agent (Pa=0.778) which could stimulate the dissolution of blood clots. Concerning γ-terpinene, it was predicted to treat skin eczema (Pa=0.854) and phobic disorders (Pa=0.803) owing to its ability to cross the blood-brain barrier (BBB). Finally, PASS prediction revealed that the minor compound β-sesquiphellandrene has other possible biological activities including antineoplastic effect (Pa=0.827) and antipsoriatic (Pa=0.750) and could be also used as immunosuppressant agent during organ transplant (Pa=0.702) instead of using synthetic compounds such as cyclosporin A which was demonstrated to cause severe cholestatic liver disease [118]. These findings could provide more insights towards further in vivo and in vitro assays to validate the computational predictions.Table 8
PASS prediction of TVEO compounds activity spectrum.
CompoundPaPiPossible biological activitiesα-Pinene0.8210.004Cardiovascular analeptic0.7460.010Antidyskinetic0.7060.006Carminativeα-Thujene0.8660.008Antieczematic0.8070.006Anti-inflammatory0.7290.063Phobic disorder treatmentCamphene0.8820.006Antieczematic0.7820.040Phobic disorder treatment0.7380.015Alkylacetylglycerophosphatase inhibitorβ-Pinene0.9020.005Antieczematic0.7350.004Ovulation inhibitor0.7290.013Respiratory analepticβ-Myrcene0.9410.004Mucomembranous protector0.8920.004Antineoplastic (breast cancer)0.7560.002Antiviral (Rhinovirus)α-Fellandrene0.8690.012Ubiquinol-cytochrome c reductase inhibitor0.7530.009Fibrinolytic0.7270.005Adenomatous polyposis treatment3-Carene0.8150.005Antidyskinetic0.7180.034Antiseborrheic0.7130.004Transplant rejection treatmentD-Limonene0.9610.001Carminative0.7430.004Acetylcholine neuromuscular blocking agent0.7400.003ChemoprotectiveO-Cymene0.9240.004Ubiquinol-cytochrome c reductase inhibitor0.8420.010Mucomembranous protector0.7780.005FibrinolyticCymol0.8310.015Polyporopepsin inhibitor0.8220.005Omptin inhibitor0.7960.004Tpr proteinase (Porphyromonas gingivalis) inhibitorγ-Terpinene0.8540.009Antieczematic0.8030.033Phobic disorders treatment0.7560.023Sugar-phosphatase inhibitorTerpinolene0.9270.004Glutamate-5-semialdehyde dehydrogenase inhibitor0.8480.003Carminative0.7150.014Venombin AB inhibitorLinalool0.9130.003Cell adhesion molecule inhibitor0.8030.005Lipid metabolism regulator0.7250.004Gastrin inhibitorCamphor0.9220.004Respiratory analeptic0.8770.006Antiseborrheic0.7450.002PediculicideBorneol0.8720.003Vasoprotector0.8220.002Peptidoglycan glycosyltransferase inhibitor0.7810.004Alopecia treatment4-Terpineol0.8420.019Ubiquinol-cytochrome c reductase inhibitor0.7960.020Antiseborrheic0.7290.014Fibrinolyticα-Terpineol0.8250.014Antieczematic0.7630.023Alkenylglycerophosphocholine hydrolase inhibitor0.7500.048Aspulvinone dimethylallyltransferase inhibitorThymol methyl ether0.8910.005Mucomembranous protector0.7900.019Antineurotic0.7230.006Anesthetic generalThymol0.9130.003Antiseptic0.8760.004Membrane permeability inhibitor0.8300.003APOA1 expression enhancerCaryophyllene0.9150.005Antineoplastic (lung cancer)0.8470.005Apoptosis agonist0.7220.002NF-E2-related factor 2 stimulantα-Humulene0.8180.003MMP9 expression inhibitor0.7690.002Interleukin agonist0.7410.011Anti-inflammatoryα-Amorphene0.8500.003Carminative0.8210.009Antineoplastic0.7260.059Ubiquinol-cytochrome c reductase inhibitorα-Curcumene0.9420.004Mucomembranous protector0.7570.003Vitamin-K-epoxide reductase (warfarin-insensitive) inhibitor0.7230.003BRAF expression inhibitorα-Zingibirene0.8420.010Mucomembranous protector0.7850.008Fibrinolytic0.7110.002Antiviral (rhinovirus)α-Bisabolene0.9200.004Antieczematic0.8670.003Carminative0.7600.017Antineoplasticβ-Sesquiphellandrene0.8270.009Antineoplastic0.7500.004Antipsoriatic0.7020.016ImmunosuppressantPa∗ represents probability to be active; Pi∗ represents probability to be inactive.


## 3.1. Chemical Composition Analysis of TVEO

GC-MS analysis of TVEO revealed the existence of 26 different components (Table1). The main components were thymol, the most abundant compound (47.44%), followed by o-cymene (16.55%), γ-terpinene (7.80%), and linalool (4.41%). Moreover, it detected the presence of 4 compounds which their percentages (EO %) were more than 2%. These later are the α-zingibirene (3.40%), β-myrcene (2.36%), caryophyllene (2.09%), and borneol (2.02%).Table 1
Chemical composition of TVEO.
CompoundMolar mass (g/mol)Molecular formulaRetention time (min)EO (%)α-Pinene136.23C10H165.991.74α-Thujene136.23C10H166.151.61Camphene136.23C10H166.661.60β-Pinene136.23C10H167.390.29β-Myrcene136.23C10H167.852.36α-Fellandrene136.23C10H168.170.343-Carene136.23C10H168.310.16D-Limonene136.23C10H168.521.61O-Cymene134.22C10H148.8816.55Cymol134.22C10H149.460.09γ-Terpinene136.23C10H169.797.80Terpinolene136.23C10H1610.560.10Linalool154.25C10H18O11.034.41Camphor152.23C10H16O12.141.15Borneol152.23C10H18O12.842.024-Terpinenol152.23C10H18O13.230.92α-Terpineol152.23C10H18O13.900.10Thymol methyl ether164.24C11H16O14.990.43Thymol150.22C10H14O16.4847.44Caryophyllene204.35C15H2419.592.09α-Humulene204.35C15H2420.430.03α-Amorphene204.35C15H2420.990.10α-Curcumene202.33C15H2221.170.71α-Zingibirene204.35C15H2421.493.40α-Bisabolene204.35C15H2421.791.12β-Sesquiphellandrene204.35C15H2422.171.34Monoterpenes hydrocarbons34.25%Oxygenated monoterpenes56.47%Sesquiterpens8.79%Total99.51%Thymol, known also by the chemical name 2-isopropyl-5-methylphenol, is a natural phenol monoterpene [40] importantly detected in Lamiacaeae family [41] including many plant species such as Thymus vulgaris L. [42], Ocimum gratissimum L. [43], Origanum L. [44], and Trachyspermum ammi L. [45] and other species of the genus Satureja L. [46] and Monarda L. [47]. This volatile monoterpenoid is largely used by nutraceutical, pharmaceutical, and cosmeceutical industries due to its multiple potential therapeutic properties [48–58]. In addition, thymol was globally recognized-as-safe food additive according to US department of Food and Drug Administration (FDA) [59].O-cymene, known as 1-isopropyl-2-methylbenzene, is an acyclic monoterpene which belongs to p-cymene isomers and has an orthosubstituted alkyle group [60]. Previous studies demonstrated that o-cimene has several therapeutic effects [61–66]. Several studies indicated the existence of a remarkable synergetic effect between ocimene and other terpenes such as α-pinene and myrcene which were noticed to produce more beneficial effects when combined [67, 68].γ-Terpinene, renowned also as p-mentha-1,4-diene, is a naturally occurring monoterpene hydrocarbon [69] that has been isolated from many botanical sources including Origanum vulgare, Citrus limon L, Melaleuca alternifolia, and Eucalyptus obliqua [70, 71]. This monoterpene is largely employed in food, cosmetics, and pharmaceutical industries [72]. Previous research showed that γ-terpinene has potential biological activities such as antioxidant [73], anti-inflammatory [74], and antimicrobial activities [75, 76].On the other hand, linalool, known as 3,7-dimethyl-1,6-octadien-3-ol [77], is a naturally occurring acyclic monoterpenoid and tertiary alcohol which is commonly found as a major active component in the essential oil of several aromatic plant species [78] principally in Lamiaceae and Lauraceae botanical families. It has been reported that this monoterpenol is broadly used in food industry as an aromatic and preservative agent, in cosmetic as a fragrance and antiseptic constituent [79–83].It should be noted that TVEO composition may depend on many biotic and abiotic factors including seasonal variations of temperature and humidity [84], phenological stages and different vegetation cycles [85], geographic location [86], environmental stress [87], and extraction technique [88]. Aljabeili et al. reported that the TVEO collected from KSA showed a significant composition variation and the major compounds were thymol (41.04%), 1,8-cineole (14.26%), γ-terpinene (12.06%), and p-cymene (10.50%) [89]. Another study revealed that Turkish TVEO has different components amounts such as thymol (49%), β-cymene (19.99%), carvacrol (7.63%), and trans-caryophyllene (6.79%) [90]. In addition, Moghaddam et al. reported that Iranian TVEO contains thymol (36.81%), ρ-cymene (30.90%), and carvacrol (3.16%) [91].


## 3.2. Antibacterial Activity

As represented in Table2, TVEO displayed an interesting antibacterial activity against the four tested bacterial strains with inhibition zones ranging from of 21 to 23 mm against Gram-negative and Gram-positive bacteria, respectively. In addition, it is important to note that monoterpenes exhibit a broad-spectrum antibacterial activity against pathogenic bacteria [92, 93]. This fact could be explained by the presence of a lipophilic character which provides to monoterpenes the ability to adhere to bacterial cell membrane lipids and to deploy their antibacterial action [94]. In our study, the obtained MIC values mentioned in Table 2, indicated that TVEO was more potent (p<0.05) against Gram-positive bacteria (MICs=0.097 mg/mL) than Gram-negative bacteria (MICs=0.195 mg/mL). These findings were in agreement with previous studies which reported that Gram-positive bacteria are susceptible to be more sensitive to plant EOs than Gram-negative bacteria due to the existence of lipopolysaccharides which acts as a hydrophobic barrier [95, 96].Table 2
Antibacterial activity of TVEO and the control gentamicin. Zones of growth inhibition was expressed in mm, and minimum inhibitory concentrations (MICs) were expressed in mg/mL.
Bacterial strainsInhibition zone diameters (mm)MIC (mg/mL)TVEOGentamicinTVEOGentamicinS. aureus ATCC 653823±1.00b20±0.83a0.097±0.00b0.013±0.00bL. monocytogenes ATCCC 1911722±1.00b20±0.75a0.097±0.00b0.013±0.00bS. enterica Typhimurium ATCC 1402823±1.00a25±1.25b0.097±0.00b0.005 ± 0.00bE. coli ATCC 873921±0.83a25±1.25b0.195±0.00b0.005±0.00bA Studentt-test was used to determine the significant differences between inhibition zones and MIC.


## 3.3. Interactions between TVEO Molecules and Bacterial Topoisomerase II and DNA and RNA Polymerases

Computational modeling is a 3R-based approach and an attractive alternative to experiments in a view to understand bioactive compounds mechanism of action and their antibacterial inhibitory process [97]. Molecular docking was performed to predict different TVEO components that could bind specifically to select bacterial receptors active sites responsible for DNA replication and transcription processes. In this respect, we investigated TVEO antibacterial inhibitory effect on topoisomerase II (DNA gyrase) and DNA and RNA polymerases of three pathogenic bacteria S. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115. Molecular homology results indicated that selected templates can be used for molecular modeling (Table 3); their identities (%) are higher than 30% [98], and Ramachandran plot values of favored regions and allowed regions were over 90% [99].Table 3
Receptors models of the selected pathogenic bacteria used in molecular docking simulation with TVEO constituents.
Bacterial StrainBacterial TargetReceptorTemplateIdentity (%)Ramachandran PlotQMEANFavored regions (%)Additional allowed regions (%)S. aureus ATCC 700699DNA polymeraseP63979 (UniProt)4IQJ.1.L36.7787.910.1−3.26RNA polymeraseQ932F8 (UniProt)6WVK.1.C81.0985.512.7−2.36Topoisomerase IIP66936 (UniProt)6GAV.1.A54.4288.410.7−1.69S. enterica Typhimurium ATCC 700720DNA polymeraseP14567 (UniProt)5FKU.1.A96.7288.010.0−2.35RNA polymeraseP06173 (UniProt)4LLG.2.C98.6688.011.0−1.11Topoisomerase IIP0A213 (UniProt)4TMA.2.B95.4190.29.2−1.82L. monocytogenes ATCC 19115DNA polymeraseWP031669548 (NCBI)6VDE.1.A38.3081.616.4-3.91RNA polymeraseGAT39567 (NCBI)6WVK.1.C86.1087.111.4-2.24Topoisomerase IIGAT39106 (NCBI)2XCR.2.A69.5987.410.4-2.01Moreover, it is necessary to remind that DNA and RNA polymerases are crucial enzymes involved in the DNA replication, transcription, and translation as well as nucleic acid formation in bacterial cells [100]. On the other hand, topoisomerase II (DNA gyrase) is also implicated in DNA replication and transcription processes and has an imperative role characterized by its ability to catalyze the unwinding of supercoiled DNA strands [101]. These imperative enzymes constitute attractive and validated targets for antibacterial agents [102]. Molecular docking simulation results are elucidated by (Table 4). These later displayed that TVEO major compound thymol (47.44%) showed a good inhibitory effect on topoisomerase II, RNA polymerase, and DNA polymerase of the selected pathogenic bacteria.Table 4
Molecular docking results for complexes ofT. vulgaris EO compounds against DNA and DNA polymerases of S. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115 using Autodock Vina (kcal/mol).
Bacterial targetS. aureus ATCC 700699S. enterica Typhimurium ATCC 700720L.monocytogenes ATCC 19115CompoundDNA polymeraseRNA polymeraseTopoisomerase IIDNA polymeraseRNA polymeraseTopoisomerase IIDNA polymeraseRNA polymeraseTopoisomerase IIα-Pinene-5.1-6.1-5.3-5.4-5.4-5.2-5.9-6.0-5.4α-Thujene-4.9-5.4-5.5-5.3-5.2-5.3-5.5-5.3-5.6Camphene-5.2-6.0-5.6-5.2-5.2-5.0-6.1-5.9-5.3β-Pinene-5.2-5.7-5.2-5.5-5.7-5.3-5.9-6.0-5.2β-Myrcene-4.9-5.0-5.2-5.1-5.1-5.0-5.2-5.0-5.0α-Fellandrene-5.2-5.6-5.4-5.1-5.1-5.1-5.5-5.5-5.63-Carene-5.2-5.6-5.2-5.6-5.6-5.5-5.6-5.5-5.7D-Limonene-5.1-6.0-5.4-5.4-5.4-5.1-5.5-5.6-5.6O-Cymene-5.3-5.5-5.6-5.7-5.4-5.4-5.8-5.5-5.5Cymol-5.2-5.7-5.6-5.5-5.5-5.2-6.0-5.9-5.7γ-Terpinene-5.3-6.0-5.6-5.8-5.6-5.7-5.8-5.8-5.6Terpinolene-5.4-5.8-5.6-5.6-6.0-5.6-5.8-5.6-5.8Linalool-5.0-5.2-5.2-5.5-5.6-5.4-5.8-5.1-5.2Camphor-5.3-6.0-5.4-5.4-5.2-5.3-6.0-5.6-5.2Linderol (borneol)-5.4-5.7-5.5-5.3-5.3-5.2-5.8-5.7-5.34-Terpineol-5.4-6.2-5.8-5.8-5.8-5.5-5.9-6.0-5.8α-Terpineol-5.4-6.0-5.6-5.6-5.8-5.0-5.8-5.6-5.8Thymol methyl ether-5.5-5.8-5.6-5.3-5.4-4.9-5.6-5.5-5.7Thymol-6.3-6.2-6.6-6.4-6.6-6.2-6.8-6.7-6.6Caryophyllene-6.0-6.4-6.7-6.4-6.5-6.4-6.5-6.3-6.4α-Humulene-6.3-6.3-6.9-6.4-7.1-6.1-6.5-6.8-6.4α-Amorphene-6.1-6.2-6.3-6.5-6.0-6.1-6.6-6.6-6.2α-Curcumene-6.3-6.6-6.7-6.5-6.5-6.0-6.8-6.4-6.3α-Zingibirene-5.4-6.5-6.4-6.0-5.5-5.6-5.7-6.0-6.2α-Bisabolene-5.6-6.8-6.6-6.4-6.4-5.8-6.4-5.9-6.2β-Sesquiphellandrene-5.9-7.0-7.1-6.86.8-6.7-7.1-6.9-6.9Rifamycin SV-8.7-9.8—-8.7-8.6—-9.6-10.2—Ciprofloxacin——-7.4——-6.3——-6.5A previous study conducted by Liu et al. reported that thymol has a potential antibacterial activity againstP. aeruginosa [15]. This monoterpenoid can affect bacterial DNA normal function. It could block gene expression processes including replication, transcription, and expression by intercalation with bacterial DNA leading to bacterial death. The same study indicated that thymol could destroy bacterial membrane integrity by affecting its permeability and it could also hinder biofilm formation. Another research paper displayed that thymol could bind to bacterial DNA, modulate its structure, and prohibit its biological function [103]. Furthermore, dos Santos Barbosa et al. showed that the antibacterial inhibitory activity of Origanum vulgare EO was effective against Salmonella Enteritidis due to the presence of thymol which caused interference in protein regulation as well as DNA synthesis [104]. On the other hand, the minor compound β-sesquiphellandrene (1.34%) showed the lowest free energy of binding (Kcal/mol) and the highest inhibitory potential on topoisomerase II and DNA and RNA polymerases of the selected pathogenic bacteria (Table 4). A previous study reported that β-sesquiphellandrene was responsible for the high antibacterial and antioxidant activities of some pomelo varieties’ EOs [105]. Another study indicated that the presence of β-sesquiphellandrene presented in ginger (Zingiber officinale) essential oil could make this later as a potential antimicrobial agent by inhibiting mycobacterial acyl carrier protein reductase enzyme and Enoyl acyl carrier protein reductase activities [106].A recent study showed thatCupressus sempervirens EO had a great inhibitory effect on DNA gyrase and DNA and RNA polymerases of S. aureus and S. enterica owing to the presence of α-pinene, δ-3 carene, and borneol [13]. Moreover, a previous study conducted by [107] evaluated the inhibitory effect of Litsea cubeba EO on topoisomerase and DNA and RNA polymerases of E.coli. Another study revealed that the germacrene B, a minor compound in Siparuna guianensis EO, had an effective inhibitory activity against bacterial DNA and RNA polymerases of multiple pathogenic bacteria including E.coli, P. aeruginosa, S. aureus, and S. pyogenes [108]. Therefore, these findings confirmed that TVEO has a powerful inhibitory effect on pathogenic bacteria based on the inhibition of DNA replication and transcription processes. Consequently, we project in subsequent work to perform further in vitro assays based on the evaluation of protein-molecule binding assays.Interaction profiles details of thymol andβ-sesquiphellandrene with the active sites of the selected targets of S. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115 are summarized in Table 5.Table 5
Interactions details of thymol andβ-sesquiphellandrene with the selected bacterial targets.
BacteriaCompoundTargetsNumber of Residues InteractingResidues with H-bondS. aureus ATCC 700699ThymolDNA polymerase4LYS228RNA polymerase4—Topoisomerase II6GLU609, ASP610β-SesquiphellandreneDNA polymerase1—RNA polymerase7—Topoisomerase II8—S. enterica Typhimurium ATCC 700720ThymolDNA polymerase5—RNA polymerase1—Topoisomerase II6ASN588β-SesquiphellandreneDNA polymerase8—RNA polymerase5—Topoisomerase II6—L. monocytogenes ATCC 19115ThymolDNA polymerase8—RNA polymerase5—Topoisomerase II3VAL113β-SesquiphellandreneDNA polymerase6—RNA polymerase4—Topoisomerase II5—Interaction profile results of thymol andβ-sesquiphellandrene with topoisomerase II and DNA and RNA polymerases of S. aureus are presented by Figures 1 and 2. Thymol made a complex with DNA polymerase receptor via alkyl interaction and conventional hydrogen bond with LYS228 and van der Waals interactions with ASN84, LYS88, LEU87, GLU469, TYR91, LEU227, and ILE492 (Figure 1(a)). Additionally, RNA polymerase complexed with thymol had alkyl and Pi-alkyl interaction with VAL536 and LYS35; van der Waals interactions with GLU413, SER410, GLY412, SER36, and GLU538; and Pi-sigma interaction with TRP39 (Figure 1(b)). Likewise, it interacted with topoisomerase II via alkyl interaction with ALA614; van der Waals interactions with HIS46, ARG198, TRP49, ARG42, THR194, GLN197, THR617, and LEU608; and two conventional hydrogen bonds with ASP610 and GLU609 (Figure 1(c)).Figure 1
Thymol complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) ofS. aureus.
(a)(b)(c)Figure 2
β-Sesquiphellandrene complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) of S. aureus.
(a)(b)(c)β-Sesquiphellandrene complexed with DNA polymerase showed Pi-alkyl interaction with LEU641 and van der Waals interactions with ILE938, LYS901, PHE900, ASP936, GLU939, ILE899, SER903, LEU902, and GLN975 (Figure 2(a)). When complexed with RNA polymerase, it made Pi-alkyl interaction with VAL270, LYS35, and VAL536; van der Waals interactions with SER36, SER410, GLY412, ARG407, GLN416, GLN413, GLU538, and ILE32; and Pi-sigma interaction with TRP39 (Figure 2(b)). Concerning topoisomerase II, β-sesquiphellandrene displayed alkyl and Pi-alkyl interactions with VAL606, PHE618, ILE532, ALA614, LEU521, and LEU608 and van der Waals interactions with LYS607, ARG198, GLU609, GLU613, ASP610, and TYR525 (Figure 2(c)).On the other hand, interaction profiles between thymol andβ-sesquiphellandrene with topoisomerase II and DNA and RNA polymerases of S. enterica Typhimurium are outlined in Figures 3 and 4. DNA polymerase complexed with thymol showed alkyl and Pi-alkyl interactions with ARG18 and LYS17 and van der Waals interactions with ASP1188, THR657, SER656, PRO19, ASN622, ASN620, SER621, LEU623, and ALA 619 (Figure 3(a)). Thymol complex with RNA polymerase indicated the presence of alkyl interaction with MET768 and van der Waals interactions with ASP785, GLN767, ASN766, GLY786, PRO787, SER788, PRO691, THR692, ALA695, THR789, and LEU693 (Figure 3(b)). This monoterpene also made interactions with topoisomerase II via alkyl interactions with VAL727, ILE557, and ALA560; van der Waals interactions with LEU561, VAL584, LEU723, GLN591, and ASP553; and one conventional hydrogen bond with ASN588 (Figure 3(c)).Figure 3
Thymol complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) ofS. enterica Typhimurium.
(a)(b)(c)Figure 4
β-Sesquiphellandrene complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) of S. enterica Typhimurium.
(a)(b)(c)The complex ofβ-sesquiphellandrene and the DNA polymerase of S. enterica Typhimurium revealed the existence of alkyl and Pi-akyl interactions with LEU75, ALA94, LEU129, LEU32, PHE35, and ILE39 and van der Waals interactions with GLN41, GLN36, GLY76, and MET130 (Figure 4(a)). This sesquiterpene complexed with RNA polymerase showed alkyl and Pi-alkyl interactions with MET130, LEU32, LEU129, PHE35, ALA94, and LEU75 and van der Waals interactions with ILE39, GLN36, and ASP32 (Figure 4(b)). In addition, it made interactions with topoisomerase II via alkyl and Pi alkyl interactions with VAL467, PHE513, PHE777, ARG516, and LEU462 and van der Waals interactions with MET461, LEU509, and THR512 (Figure 4(c)).DNA polymerase receptor ofL. monocytogenes complexed with thymol displayed the existence of alkyl and Pi-alkyl interactions with MET389, LEU345, LEU394, ILE392, and PHE367 and van der Waals interactions with GLU359, THR357, LYS358, ILE343, THR360, and SER364 (Figure 5(a)). It also showed when complexed with RNA polymerase alkyl interactions with LYS283 and LYS284 and van der Waals interactions with VAL150, GLY149, TYR151, ASN410, ASP403, ASP571, and ASN289 (Figure 5(b)). Further, thymol made interactions with topoisomerase II via alkyl interaction with PHE97; van der Waals interactions with GLN95, SER98, GLN267, TYR266, THR220, VAL268, ASN269, GLY115, and SER112; and one conventional hydrogen bond with VAL113 (Figure 5(c)).Figure 5
Thymol complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) ofL. monocytogenes.
(a)(b)(c)Finally,β-sesquiphellandrene complex with DNA polymerase of L. monocytogenes indicated the existence of Alkyl interactions with MET386, ALA382, ILE399, and LEU488 and van der Waals interactions with SER397, PHE492, PRO489, GLU484, THR400, and THR491 (Figure 6(a)). It showed also with RNA polymerase alkyl interactions with LYS280, LYS284, and TYR151 and van der Waals interactions with VAL150, GLY149, ASP403, ASN410, ASP571, ASP404, ASP401, ASN289, and LYS283 (Figure 6(b)). In addition, β-sesquiphellandrene made a complex with topoisomerase II via alkyl and Pi-alkyl interactions with VAL113, ILE264, and PHE97 and van der Waals interactions with GLY115, SER112, GLN95, ARG92, GLY111, PHE88, SER98, ASN269, VAL268, THR220, PRO265, and TYR266 (Figure 6(c)).Figure 6
β-Sesquiphellandrene complexed with DNA polymerase (a), RNA polymerase (b), and topoisomerase II (c) of L. monocytogenes.
(a)(b)(c)


## 3.4.In Silico TVEO Compound Toxicity Evaluation by VEGA QSAR Model

Toxicity evaluation of different TVEO compounds and the two selected FDA-approved antibiotics, used as controls (rifamycin SV and ciprofloxacin), was performed by the help of QSAR (quantitative structure–activity relationship) approach and using VEGA HUB software. We chose to assess compounds toxicity based on 8 different toxicity measurements. Results are represented by (Table6) and revealed that FDA-approved drugs could be toxic in several assays. In this context, rifamycin SV and ciprofloxacin are found to be mutagenic in the mutagenicity test/model (Ames test) and predicted to engender developmental/reproductive toxicity. These two antibiotics were also predicted to be genotoxic according to the in vitro micronucleus activity model. However, many TVEO compounds such as α-pinene, α-thujene, camphene, β-pinene, 3-carene, D-limonene, γ-terpinene, terpinolene, linalool, borneol, 4-terpineol, α-terpineol, thymol, caryophyllene, α-bisabolene, and β-sesquiphellandrene showed nontoxic effects. Computational toxicity results confirmed that TVEO molecules could be used as a safe antimicrobial agents and economically low-cost alternative as compared to synthetic antibiotics. It is important to mention that research related to toxicological profiles of different essential oil compounds are poorly studied due to experiments complexity, expensive cost, and difficulty to detect toxicity variation because of the chemical function and factor variability [109]. However, previous studies indicated that toxicity is a dose-/concentration-dependent manner and thymol could have a certain limit of toxicity ranging from 36 mg/mL to 49 mg/mL with less risks of accumulation in body tissues and suggested to replace synthetic drugs, which has more side effect [110]. In addition, Schönknecht et al. confirmed the safety and the effectiveness of the drug containing the extracts of thyme with the addition of thymol (Bronchosol®) in cough treatment instead of using the synthetic drug ambroxol [111]. Concerning β-sesquiphellandrene, it was reported that this sesquiterpenoid could have a great anticancer activity and can be safe to use as compared to synthetic chemotherapeutic agents velcade, thalidomide, and capecitabine [112]. Nevertheless, more toxicological in vitro/in vivo data are needed to validate the safety of these phytochemicals. Thus, computational toxicity assessment could be the best alternative that would give robust data, avoid unnecessary waste of reagents, and minimize cruelty and sacrifices of lab animal testing.Table 6
Toxicity predictions of TVEO compounds using the VEGA QSAR model.
Toxicity measurementsMutagenicity (Ames Test) Model (CAESAR) 2.1.13Carcinogenicity model (CAESAR) 2.1.9Developmental/Reproductive Toxicity Library (PG) 1.1.0Estrogen receptor relative binding affinity model (IRFMN)Androgen receptor-mediated effect (IRFMN/COMPARA) 1.0.0Thyroid receptor alpha effect (NRMEA) 1.0.0Thyroid receptor beta effect (NRMEA) 1.0.0In vitro micronucleus activity (IRFMN/VERMEER) 1.0.0Compoundα-PineneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveα-ThujeneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveCampheneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveβ-PineneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveβ-MyrceneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveα-FellandreneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveActive3-CareneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveD-LimoneneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveO-CymeneNonmutagenicNoncarcinogenToxicantInactiveNonactiveInactiveInactiveNot predictedCymolNonmutagenicNoncarcinogenToxicantInactiveNonactiveInactiveInactiveNot predictedγ-TerpineneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveTerpinoleneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveLinaloolNonmutagenicNon carcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveCamphorNonmutagenicNoncarcinogenToxicantInactiveNonactiveInactiveInactiveActiveBorneolNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactive4-TerpineolNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveα-TerpineolNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveThymol methyl etherNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveThymolNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveCaryophylleneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveα-HumuleneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveα-AmorpheneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveα-CurcumeneNonmutagenicNoncarcinogenToxicantInactiveActiveInactiveInactiveActiveα-ZingibireneNonmutagenicCarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveα-BisaboleneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveβ-SesquiphellandreneNonmutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveInactiveRifamycin SVMutagenicNoncarcinogenNontoxicantInactiveNonactiveInactiveInactiveActiveCiprofloxacinMutagenicNoncarcinogenToxicantInactiveNonactiveInactiveInactiveActive


## 3.5. TVEO Component ADME Analysis

In the present study, SwissADME server was used to determine some pharmacokinetics parameters included in absorption, distribution, metabolism, and excretion (ADME), drug-likeness, and medicinal chemistry characteristics for all TVEO compounds as represented in (Table7). Results revealed that all TVEO components possess slow passive gastrointestinal absorption (GI) except linalool, camphor, borneol, 4-terpineol, α-terpineol, thymol methyl ether, and thymol which were predicted to have high GI permeability. Additionally, only sesquiterpenes including caryophyllene, α-humulene, α-amorphene, α-curcumene, α-zingibirene, α-bisabolene, and β-sesquiphellandrene were found not to be blood-brain barrier (BBB) permanent due to their heavy molecular weight. However, rest of compounds could easily cross the blood-brain barrier for that reason it could be suggested as potent central nervous system antioxidants and effective drug candidates in the treatment of neurodegenerative diseases like Alzheimer’s and Parkinson’s [113]. On the other part, none of the tested compounds was predicted to be P-gp transporter substrate. Concerning Cytochrome p450 (CYP) isoenzymes which are involved in 50-90% of therapeutic molecules biotransformation processes in a view to reduce metabolites accumulation in blood/tissues and drug-drug interaction risks [114], thymol methyl ether and thymol were predicted to be CYP1A2 inhibitors. Many TVEO molecules were predicted to inhibit CYP2C9 and CYP2D6 inhibitors. However, none of the compounds showed an inhibitory effect towards the CYP3A4. Moreover, skin permeation coefficient (logKp) indicated that all compounds were impermeable through the skin barrier. Interestingly, TVEO drug-likeness score was acceptable with good bioavailability score (>10%) and the absence of violations related to known rules such as Lipinski’s rule of five that predicts drug permeability and absorption based on H-bond donors, H-bond acceptors, molecular weight (MW>500), and a calculated logp) [115], as well as Veber. Both rules are a mainstay of decision-making in drug design and development and in the present study; both were validated. Furthermore, medicinal chemistry parameters revealed that none of the selected molecules returns any pan-assay interference compounds (PAINS) alert. The synthetic accessibility values of TVEO compounds indicated that these later could be synthesized for pharmaceutical uses.Table 7
TVEO compounds pharmacokinetic properties prediction using SwissADME.
CompoundPharmacokineticsDrug-likeness and Medicinal chemistryGI absorptionBBB permeantP-gp substrateCYP1A2 inhibitorCYP2C9 inhibitorCYP2D6 inhibitorCYP3A4 inhibitorLogKp(cm/s)LipinskiVeberBioavailability scorePAINSSynthetic accessibilityα-PineneLowYesNoNoYesNoNo-3.95YesYes0.550 alert4.44α-ThujeneLowYesNoNoNoNoNo-5.11YesYes0.550 alert3.99CampheneLowYesNoNoYesNoNo-4.13YesYes0.550 alert3.50β-PineneLowYesNoNoYesNoNo-4.18YesYes0.550 alert3.73β-MyrceneLowYesNoNoNoNoNo-4.17YesYes0.550 alert2.85α-FellandreneLowYesNoNoNoNoNo-4.85YesYes0.550 alert4.153-CareneLowYesNoNoYesNoNo-4.02YesYes0.550 alert3.84D-LimoneneLowYesNoNoYesNoNo-3.89YesYes0.550 alert3.46O-CymeneLowYesNoNoNoYesNo-4.01YesYes0.550 alert1.00CymolLowYesNoNoNoYesNo-4.21YesYes0.550 alert1.00γ-TerpineneLowYesNoNoYesNoNo-3.94YesYes0.550 alert3.11TerpinoleneLowYesNoNoYesNoNo-3.96YesYes0.550 alert2.98LinaloolHighYesNoNoNoNoNo-5.13YesYes0.550 alert2.74CamphorHighYesNoNoNoNoNo-5.67YesYes0.550 alert3.22BorneolHighYesNoNoNoNoNo-5.31YesYes0.550 alert3.434-TerpineolHighYesNoNoNoNoNo-4.93YesYes0.550 alert3.28α-TerpineolHighYesNoNoNoNoNo-4.83YesYes0.550 alert3.24Thymol methyl etherHighYesNoYesNoYesNo-4.64YesYes0.550 alert1.09ThymolHighYesNoYesNoNoNo-4.87YesYes0.550 alert1.00CaryophylleneLowNoNoNoYesNoNo-4.44YesYes0.550 alert4.51α-HumuleneLowNoNoNoYesNoNo-4.32YesYes0.550 alert3.66α-AmorpheneLowNoNoNoYesNoNo-4.65YesYes0.550 alert4.35α-CurcumeneLowNoNoNoNoYesNo-3.71YesYes0.550 alert2.31α-ZingibireneLowNoNoNoYesNoNo-3.88YesYes0.550 alert4.81α-BisaboleneLowNoNoNoYesNoNo-3.03YesYes0.550 alert3.90β-SesquiphellandreneLowNoNoNoYesNoNo-3.71YesYes0.550 alert4.42


## 3.6. Prediction of Possible Activity Spectra of TVEO Components

All TVEO compounds were subjected to PASS (Prediction of Activity Spectra for Substances) online tool intending to predict their biological activity spectrum based on their chemical structure. Results indicated that the tested compounds could have multiple biological activities and various enzymatic targets (Table8). We have selected the top three activities which showed a Pa≥0.7. Pa and Pi values indicated the probability of the selected molecule to be active/ inactive towards the targeted receptor. Mojumdar et al. reported that the probability of experimental pharmacological action is high when Pa>0.7; however, the chance of finding the activity experimentally is less when Pa<0.5 [116]. Interestingly, TVEO major compounds such as thymol which were predicted, in earlier section by molecular docking, to have a potent antibacterial activity on pathogenic bacteria by inhibiting DNA replication and transcription processes, displayed the existence of other biological activities including the ability to inhibit bacterial membrane permeability (Pa=0.876) and it was anticipated to enhance AP0A1 expression (Pa=0.830) involved in the cellular synthesis of beneficial HDL [117]. In addition, O-cymene was predicted to be mitochondria ubiquinol-cytochrome-c reductase inhibitor (Pa=0.924) (antifungal activity), mucomembranous protector (Pa=0.842), and a fibrinolytic agent (Pa=0.778) which could stimulate the dissolution of blood clots. Concerning γ-terpinene, it was predicted to treat skin eczema (Pa=0.854) and phobic disorders (Pa=0.803) owing to its ability to cross the blood-brain barrier (BBB). Finally, PASS prediction revealed that the minor compound β-sesquiphellandrene has other possible biological activities including antineoplastic effect (Pa=0.827) and antipsoriatic (Pa=0.750) and could be also used as immunosuppressant agent during organ transplant (Pa=0.702) instead of using synthetic compounds such as cyclosporin A which was demonstrated to cause severe cholestatic liver disease [118]. These findings could provide more insights towards further in vivo and in vitro assays to validate the computational predictions.Table 8
PASS prediction of TVEO compounds activity spectrum.
CompoundPaPiPossible biological activitiesα-Pinene0.8210.004Cardiovascular analeptic0.7460.010Antidyskinetic0.7060.006Carminativeα-Thujene0.8660.008Antieczematic0.8070.006Anti-inflammatory0.7290.063Phobic disorder treatmentCamphene0.8820.006Antieczematic0.7820.040Phobic disorder treatment0.7380.015Alkylacetylglycerophosphatase inhibitorβ-Pinene0.9020.005Antieczematic0.7350.004Ovulation inhibitor0.7290.013Respiratory analepticβ-Myrcene0.9410.004Mucomembranous protector0.8920.004Antineoplastic (breast cancer)0.7560.002Antiviral (Rhinovirus)α-Fellandrene0.8690.012Ubiquinol-cytochrome c reductase inhibitor0.7530.009Fibrinolytic0.7270.005Adenomatous polyposis treatment3-Carene0.8150.005Antidyskinetic0.7180.034Antiseborrheic0.7130.004Transplant rejection treatmentD-Limonene0.9610.001Carminative0.7430.004Acetylcholine neuromuscular blocking agent0.7400.003ChemoprotectiveO-Cymene0.9240.004Ubiquinol-cytochrome c reductase inhibitor0.8420.010Mucomembranous protector0.7780.005FibrinolyticCymol0.8310.015Polyporopepsin inhibitor0.8220.005Omptin inhibitor0.7960.004Tpr proteinase (Porphyromonas gingivalis) inhibitorγ-Terpinene0.8540.009Antieczematic0.8030.033Phobic disorders treatment0.7560.023Sugar-phosphatase inhibitorTerpinolene0.9270.004Glutamate-5-semialdehyde dehydrogenase inhibitor0.8480.003Carminative0.7150.014Venombin AB inhibitorLinalool0.9130.003Cell adhesion molecule inhibitor0.8030.005Lipid metabolism regulator0.7250.004Gastrin inhibitorCamphor0.9220.004Respiratory analeptic0.8770.006Antiseborrheic0.7450.002PediculicideBorneol0.8720.003Vasoprotector0.8220.002Peptidoglycan glycosyltransferase inhibitor0.7810.004Alopecia treatment4-Terpineol0.8420.019Ubiquinol-cytochrome c reductase inhibitor0.7960.020Antiseborrheic0.7290.014Fibrinolyticα-Terpineol0.8250.014Antieczematic0.7630.023Alkenylglycerophosphocholine hydrolase inhibitor0.7500.048Aspulvinone dimethylallyltransferase inhibitorThymol methyl ether0.8910.005Mucomembranous protector0.7900.019Antineurotic0.7230.006Anesthetic generalThymol0.9130.003Antiseptic0.8760.004Membrane permeability inhibitor0.8300.003APOA1 expression enhancerCaryophyllene0.9150.005Antineoplastic (lung cancer)0.8470.005Apoptosis agonist0.7220.002NF-E2-related factor 2 stimulantα-Humulene0.8180.003MMP9 expression inhibitor0.7690.002Interleukin agonist0.7410.011Anti-inflammatoryα-Amorphene0.8500.003Carminative0.8210.009Antineoplastic0.7260.059Ubiquinol-cytochrome c reductase inhibitorα-Curcumene0.9420.004Mucomembranous protector0.7570.003Vitamin-K-epoxide reductase (warfarin-insensitive) inhibitor0.7230.003BRAF expression inhibitorα-Zingibirene0.8420.010Mucomembranous protector0.7850.008Fibrinolytic0.7110.002Antiviral (rhinovirus)α-Bisabolene0.9200.004Antieczematic0.8670.003Carminative0.7600.017Antineoplasticβ-Sesquiphellandrene0.8270.009Antineoplastic0.7500.004Antipsoriatic0.7020.016ImmunosuppressantPa∗ represents probability to be active; Pi∗ represents probability to be inactive.


## 4. Conclusion

The dramatical increase of antibiotic resistance urged scientists to diverge towards the use of aromatic medicinal plants essential oils to tackle the spread of superbugs. In that regard, the present study is aimed at investigating the TVEO chemical composition and antibacterial mechanism of action againstS. aureus ATCC 6538, S. enterica Typhimurium ATCC 14028, and L. monocytogenes ATCC 19117. In addition, chemocomputational toxicological profile and pharmacological proprieties were developed. Interestingly, molecular docking simulations revealed that TVEO compounds such as thymol and β-sesquiphellandrene had an effective antibacterial activity against the tested bacteria by inhibiting topoisomerase II and DNA and RNA polymerase functions leading to vigorous impairment of bacterial DNA replication and transcription processes. Additionally, through VEGA QSAR, we demonstrated that TVEO could be a safe resource for potential antibacterial agents. Moreover, ADME analysis showed that both compounds fulfill the Lipinski’s rule of five and could be used as potential candidate to overcome antibiotic resistance. Likewise, the in silico PASS prediction studies disclosed the presence of other useful bioactivities and possible enzymatic targets of TVEO which would be applied in the future to reduce the impact of several lethal diseases.



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*Source: 3368883-2022-07-21.xml*