Evaluating the Chemical Composition and Antibacterial and Antioxidant Effects of the Essential Oil of Melissa officinalis L.

Salman Heidarian1*, Mehrdad Ataie Kachoie1,2*, Sadegh Mousavi-Fard1,3 and Fariborz Moattar2,4

1Department of Medicinal Plants, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

2Medicinal Plants Processing Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

3Department of Horticultural Science, Faculty of Agriculture, Shahrekord University, Shahrekord P.O. Box 115, Iran

4Department of Pharmacology, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

ABSTRACT

The present study aimed to assess the antimicrobial effects of Melissa officinalis L. essential oil against pathogenic bacteria. Numerical data collected from the experiment were statistically analyzed using SPSS/21.0 software (SPSS Inc., Chicago, IL). Thirty-four (99.95%) chemical components were identified in the M. officinalis essential oil, the dominant compounds being Geranial (27.92%), Neral (22.2%), (Z)-Caryophyllene (11.77%), (E)-Caryophyllene (3.18%), and Caryophyllene oxide (3.85%). Dose-dependent antimicrobial effects were observed, with the highest diameters of growth inhibition zones being recorded against 4 mg ml-1 M. officinalis essential oil for Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii. Furthermore, the lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were obtained for the M. officinalis essential oil against P. aeruginosa and E. coli bacteria. Molecular docking revealed that the highest effect of Caryophyllene, Caryophyllene oxide, Geranial and Neral compounds against D-transpeptidase MrdA of E. coli was observed for Caryophyllene oxide, with a high energy binding of -6.78 kJ mol-1. Taking into account the high diameter of the growth inhibition zone and low MIC and MBC levels of 4 mg ml-1 M. officinalis essential oil may serve as an economical source of antimicrobials.

Article Information

Received 09 March 2024

Revised 06 April 2024

Accepted 18 April 2024

Available online 12 September 2024

(early access)

Published 20 August 2025

Authors’ Contribution

SH, SM-F and FM: carried out computational and experimental jobs. MAK: conceived of the presented study. MAK: Wrote the manuscript and discussed the results and contributed to the final manuscript.

Key words

Medicinal plant, Melissa officinalis, Essential oil, Antimicrobial, Molecular docking

DOI: https://dx.doi.org/10.17582/journal.pjz/20240309051129

* Corresponding author: [email protected], [email protected]

0030-9923/2025/0005-2349 $ 9.00/00

Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).


INTRODUCTION

Using of herbs with medicinal properties has been in various medical contexts from a long time ago (Widoyo et al., 2023; Ebrahimi et al., 2022; Bagherzadeh- Lakani et al., 2024; Kiani and Akbary, 2023). This is mainly because of the more availability, affordable, and low side effect of these natural sources. So, it consider some of them as antibacterial, antiviral, anticancer and antioxidant agents (Basati et al., 2019; Ghamari et al., 2017). For example, the nutraceutical sector with ability of creating new phyto complexes from food and plant-derived substances have beneficial effects (Abbasi et al., 2014; Aidy et al., 2020). That’s why, we focus on a plant (the Lamiaceae family) with antibacterial and anticancer in this study.

One of the perennial subshrub of this family is Melissa officinalis L. (lemon balm) that is endemic to Europe and Central Asia, and is widely cultivated in Romania, Spain, Bulgaria, and Turkey (Jamal-Omidi et al., 2018). M. officinalis plant is a multi-year herbaceous plant with many branches, 30-80 cm high, and wide, egg-shaped leaves, 3-6 cm long, and oppositely dark green with an uneven surface and numerous ridges, flowers in The upper part of the plant and formed in the corner of the leaves, the buds are usually pale, which turn into white or purple flowers after opening. Hazelnut fruit has 4 parts and its length is 1-1.5 mm. Flowers appear in mid-summer. The seed is dark and shiny (black). The small root is cylindrical, hard and relatively branched. There are about 1500 seeds in one gram and the weight of 1000 seeds is 0.6-0.7 grams (Marongiu et al., 2004; Rădulescuet al., 2021).

In fact, M. officinalis L. plant has three subspecies with commercial value such as Melissa officinalis, Inodona, and Altissima (Marongiu et al., 2004; Rădulescuet al., 2021). Among them, officinalis has been extensively cultivated for its characteristic lemon-scented essential oil due to its digestive, antispasmodic, antibacterial, antiparasitic, antioxidant, and antiviral activity (Mimica-Dukic et al., 2004; Ambreen et al., 2022; Lin et al., 2012). Leaves of M. officinalis contain 0.05–0.15% essential oils in fresh material and 0.1–0.45% essential oils in dried material, respectively (Ebadollahi et al., 2016) that it has mild abdominal disorders, biliary dyskinesia (Shakeri et al. 2016) and inhibitor the growth of pathogens (Noshad et al., 2018) properties (Shakeri et al. 2016). Besides, Melissa essential oils commonly used in the food and pharmaceutical industries (Miraj et al., 2017), and high-priority pathogens (Tacconelli, 2017).

Many research groups use from computational study for drug design due to high cost and time cunsuming of experimental methods (Darvishi, 2016; Negahdari et al., 2019). One of structure based drug design (SBDD) method is molecular docking. In this experimental study, chemical compounds, antibacterial and antioxidant activities of M. officinalis essential oil and their interaction with target proteins were investigated.

MATERIALS AND METHODS

Plant material and essential oils preparation

Aerial parts of M. officinalis plants were collected from Shahrekord city, Iran in 2020, and identifed by the Department of Medicinal and Aromatic Plants, Islamic Azad University, Shahrekord Branch. A voucher specimen (Herbarium No. 1966) was deposited in the Agricultural Research and Training Center and Natural Resources of Chaharmahal and Bakhtiari Province. The plant material was air-dried at an ambient temperature of 20±2°C. The essential oils was obtained through water distillation (UK Pharmacopoeia) using Cloninger apparatus. The 50 g of the dried aerial parts of M. officinalis plant were weighed and the essential oils were extracted for 3 h. The obtained essential oils was dehydrated using sodium sulfate and was stored at 4°C in dark glass vessels until further analysis.

Gas chromatography-mass spectrometry (GC-MS) analysis

The obtained essential oils, after being concentrated using a rotary evaporator device, stored at -18°C for 48 h. The liquid phase of the essential oils. isolated using filter paper and blended with an equal amount of hexane. Then, mixture was placed on a shaker at 186 rpm for 1 h before being left to settle for 15 min, producing two separate phases. The phase was containing the M. officinalis essential oils used for injection into a GC-MS device (Hewlett Packard, HP-6890, USA). The device was equipped with an HP-1MS (methyl silicon-cross link) column (60 m length, 0.2 mm diameter, and 0.25 µm film thickness). The carrier gas, helium of 99.99% purity, was kept at a constant flow rate of 1 ml min-1. In the GC analysis, the column temperature was adjusted to 35°C for 5 min and then increased to 160°C at a rate of 7°C per min (residence time 10 min) before further increasing to 230°C at a rate of 15°C per min (residence time 10 min). The injection ratio was 1:20 (split). The injection chamber and transmission line temperatures were set to 250°C. The MS device (Hewlett Packard, HP-5970, USA) operated at an ionization energy of 70 eV, with a full sweep status of 20–550 m z-1. The ionization chamber and mass decomposer temperatures were set to 230°C and 150°C, respectively. Alkanes identified relative to commercial standards, while other compounds were tentatively identified by comparing their MS spectra with those of commercially available libraries (Wiley Registry of Mass Spectral Data, sixth ed.; NIST/EPA/NIH Mass Spectral Library 1.5a). Molecular weights were determined either by identifying the (M)+ peaks or their (M−15)+ fragments. Acid (RCO2H2)+ fragments were used to identify homologous isomers of wax esters eluting in one peak, whose intensity was used to calculate the composition of the respective isomers. In some cases, the corresponding alcohol fragments (R’− 1)+ were also detected in the mass spectrum (Etame et al., 2018). The relative amounts of the identified compounds were calculated by integrating the peak area and expressed as a percentage of the total area of all recognized peaks in the TIC chromatogram. The values presented in this article are the means of three independent wax analyses.

Evaluation of antibacterial activity

Four pathogenic bacterial strains, namely Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 9144), Acinetobacter baumannii (ATCC 19606), and Pseudomonas aeruginosa (ATCC 25922) were sourced from the Microbiology Research Center of the Pasteur Institute of Iran, in Tehran, Iran. The pure cultures were cultured separately in tryptic soy broth (Merck, Germany) and incubated at 37°C for 24 h for regeneration.

Agar disk diffusion assay

The antimicrobial activity of M. officinalis essential oils was evaluated using the disk diffusion method. After overnight incubation, the bacterial concentration reached 1×106 CFU ml-1. The bacteria were then cultured in Müller-Hinton agar medium. Blank discs of 6 mm diameter were placed on the medium, and 1000 μL of M. officinalis essential oils with concentrations of 0.5, 1, 2, and 4 mg ml-1 were applied to the discs. For comparison, ceftazidime (30 µg), imipenem (10 µg), gentamicin (10 µg), vancomycin (30 µg), penicillin (10 µg), ciprofloxacin (5 µg), tetracycline (30 µg), erythromycin (15 µg), ampicillin (10 µg), and azithromycin (15 µg) (Mast, UK) antibiotics were also tested. Each disc was placed at regular intervals on the plates containing the bacteria and the plates were then incubated for 24 h at 37 °C. The diameter of the growth inhibition zones around the discs was measured in millimeters (Khameneh et al., 2019).

Minimum inhibitory concentrations (MIC) and minimum bacterial concentrations (MBC)

Fresh cultures of bacteria were obtained in a Mueller–Hinton broth medium (Merck, Germany) to create a turbidity of 0.5 McFarland. This turbidity was then diluted to a ratio of 1 to 100, resulting in a concentration of 1×106 CFU ml-1. Subsequently, 8 µL of different dilutions of M. officinalis essential oil containing 2 µL of the bacterial suspension were added to polystyrene plates. Additionally, wells containing 4 μL of broth medium served as negative control, while wells containing culture medium and bacteria served as positive control. Wells composed of 2 μL of the medium and 1 μL of each dilution were also used as a control for turbidity assessment. The tests were performed in triplicates. The plates were then covered and incubated at 4 °C for 4 h. After incubation, the turbidity was read at 630 nm using an ELISA reader (Statfax 2100, USA). The lowest concentration of M. officinalis essential oils that reduced the turbidity by 90% compared to the control group was considered the MIC, while the lowest concentration of M. officinalis essential oils that caused complete turbidity removal was considered the MBC. For comparison, the MIC and MBC of antibiotics were also determined (Khalili et al., 2018). Ceftazidime, imipenem, gentamicin, vancomycin, penicillin, ciprofloxacin, tetracycline, erythromycin, ampicillin, and azithromycin (Mast, UK) antibiotics were prepared in powder form and then serial concentrations of 0.5, 1, 2, and 4 mg ml-1 were created using dilution in sterile water. A negative control experiment was conducted using only sterile water.

Molecular docking

Protein preparation

Three-dimensional structure of the D-transpeptidase enzyme from E. coli was obtained from the protein data bank (PDB) repository with the identification code 6G9P. The crystallographic water molecules were excluded from the protein structure. To ensure accuracy, the chemistry of the protein was modified by adding missing hydrogen atoms and correcting any crystallographic disorder and unfilled valence atoms utilizing the alternate conformations and valence monitor options. This particular structure was chosen due to its high structural similarity to that of Staphylococcus aureus (96%) and Acinetobacter baumannii (81.52%), as determined by the PDB (Fig. 2).

 

Ligand preparation

Three-dimensional structures of Caryophyllene, Caryophyllene oxide, Geranial, Geraniol, and Neral compounds of the M. officinalis essential oils were retrieved from scientific databases such as PubMed and OpenBabel. Subsequently, hydrogen bonds were added and energy optimization of the ligand was performed by using the MM+ force field. The optimized structures of the compounds are depicted in Figure 1. This force field is a widely used and well-validated force field in computationalstudies that applies classical mechanical potential to system. It provides a reliable approximation of the interaction energies of molecules in terms of electrostatic, van der Waals, and other non-bonded components. Therefore, the use of this force field is suitable for accurately describing three-dimensional structure of the M. officinalis essential oils compound in this study (Tsai et al., 2020).

 

Table I. Chemical compositions of M. officinalis essential oils by gas chromatography-mass spectrometry analysis.

No.

Components

RI

Area (%)

1

1-Octen-3-ol

968

0.18

2

6-methyle-5-hepten-2-one

989

0.4

3

Linalool

1096

0.73

4

cis, cis- Photocitral A

1137

0.29

5

Exo-Isocitral

1142

0.15

6

cis-β-Terpineol

1147

0.42

7

Citronellal

1150

0.62

8

(Z)-Isocitral

1161

0.84

9

Neo-iso- Isopulegol

1172

0.37

10

(E)-Isocitral

1180

1.42

11

p-Cymen-8-ol

1185

0.49

12

Decanal

1206

0.99

13

Nerol

1228

5.76

14

Neral

1243

22.2

15

Geraniol

1255

6.81

16

Methyl citronellate

1258

0.48

17

Geranial

1274

27.92

18

Thymol

1291

1

19

Methyl geranate

1323

0.97

20

α-Copaene

1376

0.31

21

Geranyl acetate

1383

4.71

22

(Z)-Caryophyllene

1409

11.77

23

(E)-Caryophyllene

1421

3.18

24

α-Humulene

1455

0.9

25

Aromadendrene

1462

0.17

26

γ-Cadinene

1524

0.55

27

α-Calacorene

1554

0.19

28

Caryophyllene oxide

1586

3.85

29

Humulene epoxide II

1612

0.21

30

Caryophylla-4(12),8(13)-dien-5α-ol

1639

0.15

31

epi-α-Muurolol

1644

0.5

32

α-Cadinol

1657

0.93

33

Oplopanone

1673

0.25

34

Hexadecanoic acid

1958

0.24

Total

99.95

 

Docking procedure

In this study, Auto Dock (V. 4.2) was utilized to perform docking experiments (Pirbalouti et al., 2019) The parameters of the Lamarckian Genetic Algorithm (LGA) applied in the analysis included 30 independent runs, a population size of 150, a maximum number of 25 million energy evaluations, 27,000 generations, a mutation rate of 0.02, and a crossover rate of 0.8. Blind docking was employed and the grid size was adjusted to encompass the entire enzyme. The RMS cluster tolerance was set to 2.0. Autodock tools was utilized to compile, collect, and extract the generated conformations. The first and last conformation from a 100-ranked set of each complex were then analyzed using discover studio 2016. This experimental approach allowed us to explore and analyze the interactions between the compounds and the target enzyme in a comprehensive manner. Moreover, the parameters used for the docking experiments were optimized to allow for an efficient exploration of the conformational space of the enzyme-ligand complexes (Szliszka et al., 2009).

Statistical analysis

Numerical data collected from experiment were statistically analyzed using SPSS/21.0 software (SPSS Inc., Chicago, IL). The results were evaluated using a completely randomized design with Minitab 19 software. Qualitative data obtained from the tests were analyzed using the Chi-square test and Fisher’s exact two-tailed test. The significance level was determined as p-value less than 0.05.

RESULTS

In this study, antimicrobial effects of M. officinalis essential oils were assessed against pathogenic bacteria. First, chemical composition of M. officinalis essential oils was analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). Then, the antimicrobial effects were evaluated using disk diffusion, the MIC and MBC evaluation, and molecular docking. Table I presents the phytochemical compounds identified in the essential oils. Altogether, 34 (99.5%) chemical components were identified. Geranial (27.92%, C10H16O), Neral (22.2%, C10H16O), (Z)-Caryophyllene (11.77%, C15H24), (E)-Caryophyllene (3.18%, C15H24), and Caryophyllene oxide (3.85%, C15H24O) were the most abundant compounds in the essential oils. Citral, a mixture of Neral and Geranial, made up 50.12% of the essential oils compounds.

Diameters of growth inhibition zones of examined bacteria in response to the M. officinalis essential oils and antibiotic discs are given in Table II. Average diameter of tested bacteria’s growth inhibition zone increased with increasing concentrations of M. officinalis essential oils. The greatest diameter of the growth inhibition zone was observed against the 4 mg ml-1 concentration of M. officinalis essential oils for all of the examined bacteria. The largest diameters of the growth inhibition zones of P. aeruginosa, E. coli, S. aureus, and A. baumannii bacteria were found against 4 mg ml-1 of M. officinalis essential oils (14.21±0.74 mm), 4 mg ml-1 of M. officinalis essential oils (13.38±0.36 mm), imipenem (14.61±0.29 mm), and imipenem (10.01±0.31 mm), respectively. Statistically significant differences were observed in the diameter of the growth inhibition zone of the examined bacteria between some different concentrations of M. officinalis essential oils and antibiotic agents (P < 0.05).

 

Table II. The growth inhibition zone diameter of examined bacteria toward M. officinalis essential oils and antibiotic discs.

Essential oil and antibiotics/ concentrations

Diameter of the growth inhibition zone (mm)

P. aeruginosa

E. coli

S. aureus

A. baumannii

M. officinalis essential oils

4

14.21±0.74 a***

13.38±0.36 a

10.72±0.10 b

9.19±0.14 a

2

13.70±1.02 a

12.99±0.18 a

9.88±0.15 c

8.20±0.29 b

1

12.03±0.55 b

11.01±0.29 b

9.22±0.41 c

7.95±0.19 bc

0.5

11.82±0.33 b

10.17±0.24 b

8.27±0.33 d

7.30±0.22 c

Antibiotics

Cef**

11.51±0.64 b

12.17±0.35 a

13.42±0.93 a

9.13±0.29 a

Imp

12.15±0.50 b

12.93±0.48 a

14.61±0.29 a

10.01±0.31 a

G

10.41±0.60 b

10.53±0.28 b

11.14±0.22 b

7.20±0.35 c

V

11.90±0.75 b

12.86±0.63 a

13.97±0.49 a

9.92±0.71 a

P

10.65±0.39 b

11.51±0.40 b

12.08±0.27 b

8.23±0.45 b

Cip

10.96±0.27 b

11.95±0.57 ab

12.58±0.39 b

8.77±0.41 b

Tet

10.37±0.56 b

10.60±0.14 b

11.02±0.18 b

7.07±0.27 c

Er

10.89±0.44 b

11.72±0.48 ab

12.39±0.26 b

8.61±0.53 b

Am

10.77±0.42 b

11.25±0.56 b

11.83±0.36 b

8.05±0.20 b

Az

11.30±0.55 b

12.26±0.18 a

13.22±0.46 a

9.19±0.14 a

 

Dissimilar letters in each column show statistically significant differences about P <0.05. Cef, Ceftazidime; Imp, Imipenem; G, Gentamicin; V, Vancomycin; P, Penicillin; Cip, Ciprofloxacin; Tet, Tetracycline; Er, Erythromycin; Am, Ampicillin; Az, Azithromycin.

 

Table III illustrates the MIC and MBC values of M. officinalis essential oils and antibiotic agents on the tested bacteria. Results indicate that the M. officinalis essential oils yielded the lowest MIC and MBC values against P. aeruginosa and E. coli bacteria, which is significantly lower than the MIC and MBC values of ampicillin, tetracycline, and gentamicin antibiotic agents. This suggests that M. officinalis essential oils is more effective than antibiotic agents in inhibiting the growth of P. aeruginosa and E. coli bacteria. In contrast, ampicillin, tetracycline, and gentamicin antibiotic agents exhibited the highest MIC and MBC values, indicating that their efficacy in controlling the growth of these bacterial species is relatively low.

 

Table III. The MIC and MBC indexes of M. officinalis essential oils and antibiotic agents.

Essential oil/ antibiotics

P. aeruginosa

E. coli

S. aureus

A. baumannii

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

Essential oil

0.5

1

0.5

1

1

2

1

2

Cef*

1

2

1

2

0.5

1

2

4

Imp

0.5

1

0.5

1

0.5

1

0.5

1

G

2

4

2

4

2

4

ND

ND

V

1

2

0.5

2

0.5

1

2

4

P

2

4

1

2

1

2

4

ND

Cip

2

4

1

2

1

2

4

ND

Tet

2

4

2

4

2

4

ND

ND

Er

2

4

2

4

1

2

4

ND

Am

2

4

2

4

2

4

-

ND

Az

1

2

50

100

1

2

4

ND

 

Molecular docking analysis of the essential oil of M. officinalis revealed that out of the four compounds tested, Caryophyllene oxide had the strongest affinity for the D-transpeptidase enzyme of E. coli bacteria. This was evidenced by the presence of multiple hydrogen bonds and hydrophobic interactions between two molecules. The high affinity of Caryophyllene oxide for the D-transpeptidase enzyme suggests that it has the potential to inhibit or disrupt the enzyme’s activity, which could in turn inhibit the growth of E. coli bacteria.

Electrostatic energy and van der Waals energies, as well as the lowest binding energy from docking between Caryophyllene, Caryophyllene oxide, Geranial, and Neral and D-transpeptidase of E. coli are shown in Table IV. Geranial had the lowest binding energy against D-transpeptidase E. coli at -4.79 kJ mol-1. Caryophyllene oxide had the highest binding energy against A. baumannii at -6.78. Therefore, Caryophyllene oxide from M. officinalis essential oil demonstrated the strongest antimicrobial effects against E. coli. Additionally, the electrostatic energy was lower than van der Waals in this connection. In this table, results of the binding of the evaluated compounds with the receptor are shown. A hydrogen bond was observed between geranial and the amino acid phenylalanine 353 (Phe: 353), as well as between neral and the amino acid lysine 162 (Lys:162) with the D-transpeptidase.

DISCUSSION

Plant-derived antimicrobial agents are well-known for their ability to inhibit the growth of microorganisms, including bacteria, fungi and parasites. Inhibition of protein synthesis, interference with cell wall synthesis, inhibition of metabolic pathways, interference with nucleic acid synthesis and disruption of cell cytoplasmic membrane are the main mechanisms of their antimicrobial effects (Abdellatif et al., 2014). Furthermore, numerous studies have reported that geranial, neral, citronellal, and caryophyllene oxide were the predominant chemical

 

Table IV. Electrostatic energy and van der Waals energies and the lowest binding energy from docking Caryophyllene, Caryophyllene oxide, Geranial and Neral (D) of M. officinalis with D-transpeptidase of E. coli.

Molecule

Binding energy

Ki

(μM)

E vdw + H-bond + Desolvation energy

Electrostatic

energy

Hydrogen bonds

Hydrophobic bonds

Caryophyllene

-6.63

13.82

-6.63

+0.00

-

Ser(A):180, Lys(A):181, Asn(A):183, Gly(A):300

Caryophyllene oxide

-6.78

10.72

-6.74

-0.04

-

Pro(A):66, Ser(A):67, Asn(A):82, Lys(A):162, Arg(A):164, Asp(A):204, Arg(A):163, Ala(A):201

Geranial

-4.79

308.38

-5.99

+0.00

Phe(A):353

Leu(A):352, Asp(A):354, Gln(A):359, Thr(A):390

Neral

-4.89

261.99

-6.08

+0.00

Lys(A):162

Ala(A):65, Pro(A):66, Thr(A):161, Ala(A):201, Thr(A):202, Asp(A):204

Geraniol

-4.73

340.00

-6.04

-0.19

-

Asp(A):204, Arg(A):164, Asn(A):82, Ser(A):67, Thr(A):202

 

components in M. officinalis essential oil (Jalal et al., 2015; Hamad et al., 2021; Abers et al., 2021) with most frequently identified chemical components. Variations in the chemical composition of essential oil can be attributed to environmental and genetic factors, post-harvest processing, geographical area, climate, season of sampling, plant part, plant phenological stage, and method of chemical components identification (Klūga et al., 2017). In addition, the geographical area, climate, collection season, part of the plant (including aerial parts, stems, and roots), phenological stage of the plant at the time of collection, and method of component identification, variations in the chemical composition profile of medicinal and aromatic plants have been observed. This can be attributed to the aforementioned factors, as the environment and conditions under which the plants are grown can cause differences in the chemical composition of the plants. Furthermore, the method of component identification can affect the chemical composition profile, as different techniques may yield different results.

Our study demonstrated the potent antimicrobial properties of M. officinalis essential oil against two common clinical bacterial strains, namely P. aeruginosa and E. coli. By administering 4 mg ml-1 of M. officinalis essential oil, the highest antimicrobial effect was observed. The growth inhibition zone of P. aeruginosa treated with M. officinalis essential oil was larger than all antibiotic agents examined (P <0.05). Similarly, the growth inhibition zone of E. coli treated with 4 mg/mL M. officinalis essential oil was significantly larger than that of the gentamicin, penicillin, tetracycline, and ampicillin antibiotic agents (P <0.05).

The antimicrobial effects of M. officinalis essential oil can be attributed to the presence of chemical components such as citronellal, caryophyllene oxide, geranial, and neral, the antimicrobial effects of which have been reported in previous surveys (Ehsani et al., 2017; Jafarzadeh et al., 2020). Contrary to other findings indicating higher susceptibility of Gram-positive bacteria (e.g., S. aureus) to natural essential oils than gram-negative bacteria (e.g., E. coli and P. aeruginosa) (Yu et al., 2022; Arzhang et al., 2015), our results showed that M. officinalis essential oil had higher antimicrobial effects against P. aeruginosa and E. coli. Similar findings were obtained (Arzhang et al., 2015; Jafari-Sales et al., 2020).

Collected M. officinalis essential oil from Algeria was found to have remarkable antimicrobial effects against gram-negative bacteria such as B. subtilis, P. aeruginosa, E. coli, K. pneumonia, and S. enterica, as determined by the disk diffusion technique. This suggests that M. officinalis essential oil may be a promising alternative treatment for infections caused by gram-negative bacteria. Furthermore, essential oils have been found to have fewer side effects than traditional antibiotics (Islam et al., 2019) further reinforcing the potential of M. officinalis essential oil as a viable treatment option for gram-negative bacterial infections. Our results showed that the compound had a high negative binding energy, indicating its strong antibacterial activity against E. coli. This finding was further confirmed by disk diffusion and MIC and MBC analyses; these results are in good agreement with other report (Dorman and Deans, 2000). Antibiotic, anti-microbial, antioxidant properties of many medicinal plants due to the presence of phenolic compounds, flavonoids, flavonoids, tannins, anthocyanins and plant antioxidants (Shahsavari et al., 2022; Razmjoue et al., 2023; Sulieman et al., 2023).

CONCLUSION

This study demonstrated that the essential oil of M. officinalis possesses potent antimicrobial properties against multi-drug resistant bacteria, particularly P. aeruginosa and E. coli. The MIC and MBC of M. officinalis essential oil were found to be 4 mg ml-1 and 8 mg ml-1, respectively. This is significantly lower than the MIC and MBC of the majority of antibiotics tested, indicating that M. officinalis essential oil may be a more effective antimicrobial than the majority of antibiotics. Furthermore, the low MIC and MBC levels of M. officinalis essential oil suggest that it has specific antimicrobial properties even at low concentrations, making it a potential economical source of antimicrobials. This study has demonstrated the potential of M. officinalis essential oil as an alternative to conventional antibiotics, an attractive prospect given the growing prevalence of multi-drug resistant bacteria and the need for new antimicrobial solutions. Molecular docking studies have demonstrated that Caryophyllene oxide, a compound found in M.officinalis essential oil, has greater antimicrobial effects against E. coli bacteria when compared to other compounds. This was further corroborated by the results of disk diffusion, the MIC and MBC tests. As a result, M. officinalis essential oil is recommended for use as an oral antimicrobial agent in the food and medical industries due to its high efficacy against E. coli bacteria. Furthermore, its natural composition ensures that it is free from harmful chemicals, which could potentially cause adverse effects. Therefore, Caryophyllene oxide, found in M. officinalis essential oil, has the potential to be used as a safe and effective antimicrobial agent in the food and medical industries. Besides, the results of this research suggest that caryophyllene oxide is capable of binding to its receptor and inhibiting the enzyme, potentially making it a viable herbal compound for use with beta-lactamase antibiotics to reduce antibiotic resistance. However, further research is needed to confirm these findings.

Declarations

Acknowledgment

The authors would like to express their gratitude to the R & D team of the Goldaru-co Pharmaceutical Laboratory in Iran and the Agricultural Research and Training Center, as well as the Natural Resources of Chaharmahal Va Bakhtiari province in Iran, for providing substantial support. Without their help, this study would not have been possible. The team’s assistance enabled the authors to conduct a thorough investigation into the topic. This was a crucial factor in the successful completion of the research and its subsequent impact.

Funding

This study was supported by the authors, providing evidence of their commitment to furthering the research. Such an investment of time and effort is indicative of the authors’ dedication to advancing knowledge in the field and demonstrates their confidence in the results of the study.

Ethics approval and consent to participate

The Ethical Council of the Islamic Azad University, Shahrekord Branch, approved the study of Research of the Medicinal Plants Processing Center. This research project was verified and the licenses related to the sampling process were approved by Dr. Mehrdad Ataie Kachoie and Prof. Fariborz Moattar (Approval Ref Number 2020-552). This approval demonstrates that the research study was conducted in accordance with the ethical principles of research and is compliant with international standards. Furthermore, this approval serves as a testament to the fact that the research team was granted permission to carry out the sampling process in a safe and responsible manner.

Data availability

All of manuscript datas are presented in this manuscript.

Statement of conflict of interest

The authors have declared no conflict of interest.

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