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Original article
08 2023
:16;
104939
doi:
10.1016/j.arabjc.2023.104939

Compounds from myrtle flowers as antibacterial agents and SARS-CoV-2 inhibitors: In-vitro and molecular docking studies

, , , ,
a Pharmaceutical Sciences Research Center CRSP, Constantine 25000, Algeria
b Centre de Recherche Scientifique et Technique en Analyses Physico-Chimiques CRAPC, BP 384, Zone Industrielle, Bou-ismail, Tipaza RP 42004, Algeria

⁎Corresponding author. barhouchi.badra@crsp.dz (Badra Barhouchi)

Received: , Accepted: ,
© The Author(s) 2023
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

  • 54 phytochemicals were identified from myrtle flowers essential oil (EO) mainly α-pinene (48.94%) and 1,8-cineole (28.3%).

  • The tested myrtle EO was significantly efficient against Gram-negative bacteria with bactericidal effects against E. coli, K.oxytoca and S. marcescens.

  • The MD for antibacterial (anti-E.coli) and anti-SARS-CoV-2 investigation showed that the top-ranked inhibitors are: 1,8-cineole, s-cbz-cysteine, mayurone and methylxanthine against the targets: 1KZN, 6LU7, 6ZLG and 1R42, respectively.

  • The ADME(Tox) analysis of the top-ranked inhibitors revealed good druggability with no Lipinski’s rule violation.

Abstract

Plants and their related phytochemicals play a key role in the treatment of bacterial and viral infections, which inspire scientists to design and develop more efficient drugs starting from the phytochemical active scaffold. This work aims to characterize the chemical compounds of Myrtus communis essential oil (EO) from Algeria and to evaluate its in vitro antibacterial effect, as well as the in silico anti-SARS-CoV-2 activity. The chemical profile of hydrodistilled EO from myrtle flowers was determined using GC/MS analysis. The results showed qualitative and quantitative fluctuations and 54 compounds were identified including the main components: α-pinene (48.94%) and 1,8-cineole (28.3%) whereas other minor compounds were detected. The in vitro antibacterial activity of myrtle EO against Gram-negative bacteria was carried out by using the disc diffusion method. The best inhibition zone values ranged between 11 and 25 mm. The results revealed that Escherichia coli (25 mm), Klebsiella oxytoca (20 mm) and Serratia marcescens (20 mm) are the most susceptible strains to the EO which is endowed with a bactericidal effect. Furthermore, the antibacterial and anti-SARS-CoV-2 activities were investigated by the means of molecular docking (MD) study, in addition to ADME(Tox) analysis. The phytochemicals were docked against four targets: E. coli topoisomerase II DNA gyrase B (PDB: 1KZN), SARS-CoV-2 Main protease (PDB: 6LU7), Spike (PDB: 6ZLG) and angiotensin-converting enzyme II ACE2 (PDB: 1R42). The MD investigation revealed that 1,8-cineole could be the main phytochemical associated with the antibacterial activity of EO; s-cbz-cysteine, mayurone and methylxanthine were found the most promising phytochemicals against SARS-CoV-2; The ADME(Tox) analysis has shown their good druggability with no Lipinski’s rule violation.

Keywords

Myrtus communis
Antibacterial activity
E.coli
Molecular docking
SARS-CoV-2
1

1 Introduction

Plants are the richest host for novel drug discovery and development. They have played a key role in the treatment of multiple ailments among them bacterial and viral infections (Rahman and Sarker., 2020; Musarra-Pizzo et al., 2021). The investigation of natural compounds from plants for therapeutic purposes is one line of scientific research followed to develop effective antiviral and antibacterial treatments, particularly with the emergence of the new threat called antimicrobial resistance (Rahman and Sarker., 2020; De Oliveira et al., 2020). In recent decades, the antimicrobial effect of many plants and isolated natural compounds has been tested with promising results (Lewis., 2017). The antibacterial and antiviral properties of plant products present potential for manufacturing and developing drugs that can reduce the pathogenicity of the microorganisms by neutralizing and blocking their absorption into the cell, as well as by inhibiting their reproduction in the cell (Lewis., 2017; Maginnis., 2018).

Essential oils produced by plants are known to possess antimicrobial activity; they have been traditionally used for respiratory tract infections (Inouye et al., 2001; Maruzzella et al., 1960; Shubina et al., 1990). In the medicinal field, inhalation therapy of essential oils has been used to treat acute and chronic bronchitis (von Schindl., 1972) and acute sinusitis (Federspil et al., 1997).

Myrtus communis or common myrtle (family: Myrtaceae), native to the Mediterranean region is a wild aromatic plant that many works have demonstrated the biological activities of myrtle essential oils (EOs). According to the literature, myrtle exhibited several pharmacological activities including antioxidative, anticancer, anti-diabetic, antibacterial, antifungal, neuroprotective, hepatoprotective and antiviral activities (Alipour et al., 2014), especially against human papillomavirus (HPV), herpes simplex virus type 1 (HSV-1) and tobacco mosaic virus (TMV)) (Nikakhtar et al., 2018; Moradi et al., 2011). Different parts of the myrtle particularly its berries, leaves and flowers have long been used as a remedy for respiratory complaints like cough, gastrointestinal disorders, urinary diseases and skin ailments, as well as for microorganisms inactivation and wound healing (Alipour et al., 2014; Aleksic et al., 2014). The chemical composition of myrtle EO may change according to several factors; nevertheless, it was constant in practically all EOs, the presence of 1,8-cineole and α-pinene, as main components (Hennia et al., 2019). Although the contribution of the components on the biological activities was not determined, they were generally attributed to the presence of the main components (1,8-cineole, α-pinene, eugenol, methyleugenol, myrtenyl acetate, among other components) (Hennia et al., 2019). Myrtle parts have been broadly scrutinized by the scientific community where in Algeria; there is still a lack of scientific knowledge pertaining to the chemical composition and biological activities of myrtle flowers.

Traditional knowledge and experiential databases derived from clinical practice are instrumental in increasing the success rate of drug discovery by reducing the time consumed, money spent and toxicity occurrence which are the three main hurdles in drug development, when compared with the conventional approach adopting random screening and chemical synthesis. Current technology advancements have been able to land genetic, molecular, structural, and functional features in order to find effective targets against viruses (Wang et al., 2009). Computational analysis allows drug discovery from synthetic and natural compounds especially phytochemicals coming from medicinal plants, it makes predictions about the possible therapeutic effects of these compounds. Among the computational methods, molecular docking is most commonly used in the structure-based drug design for prediction of protein–ligand binding sites with binding affinity score (Lengauer et al., 1996).

In this study, myrtle flowers essential oil was experimentally investigated, firstly by identifying the existing phytochemicals, secondly by evaluating the essential oil antibacterial activity in vitro against 20 bacteria. Furthermore, the antibacterial and the eventual antiviral activity of the identified phytochemicals were rationalized by molecular docking against E. coli topoisomerase II DNA gyrase B, SARS-CoV-2 Main protease (Mpro), Spike and angiotensin-converting enzyme II ACE2 (PDB: 1R42). Absorption, Distribution, Metabolism, Excretion (ADME) and toxicity properties were also highlighted.

2

2 Methods and materials

2.1

2.1 Experimental details

2.1.1

2.1.1 Raw material

Fresh flowers of Myrtus communis L. were gathered during its flowering stage from northeastern Algeria, at the region of Annaba city (located at 36°53′59″ N, 7°46′00″ E). The voucher specimens were identified in the Biology Department, Badji Mokhtar University, Annaba. The plant material was dried in the shade at room temperature and conserved until the extraction process.

2.1.2

2.1.2 Essential oil isolation

According to the method recommended in the European Pharmacopoeia (2002), the volatile oils were extracted from 100 g of dried myrtle flowers by hydrodistillation for 2 h using a Clevenger-type apparatus. Anhydrous sodium sulfate was used to dry the aqueous phase and the obtained essential oil was filtered and kept cold at 4 °C in a hermetically sealed opaque bottle until the chemical analyzes were conducted. The EO yield was calculated using the dry weight of plant material based on the following equation: EO (% v/w) = volume of oil (mL)/weight of sample (g) × 100.

2.1.3

2.1.3 GC-MS analysis

The chemical composition of EO was analyzed by gas chromatography–mass spectrometry GC–MS (Agilent 6890 N; Agilent Technologies), equipped with a capillary HP-5MS column (60 m length, 0.25 mm diameter, 0.25 mm film thickness), and coupled with a mass selective detector. The electron impact ionization was 70 eV. The carrier gas was Helium with a flow rate of 1.2 mL/min and the injection (μL) was conducted manually in the split mode (1:50 split ratio). The operating conditions were as follows: the oven temperature was programmed for 1 min at 100 °C, increased from 100 to 280 °C at a rate of 5 °C/min, and then set at 280 °C for 25 min, the temperatures of the injector and detector were 250 and 310 °C, respectively. However, the mass-spectrometer conditions were the following: injection of 2 μL aliquot of the sample where the scan time and mass range were 1 s and 40–300 m/z, respectively. The components of myrtle essential oil were identified by a comparison of the fragmentation patterns in the mass spectra with those stored in the GC-MS databases and those from two libraries: the Wiley Registry of Mass Spectral Data 7th edition (Agilent Technologies) and the library of the national institute of standards and technology 05 MS (NIST). In addition, the percentages of the compounds were determined from their peak areas.

2.1.4

2.1.4 Bacterial strains

In this study, two classified bacteria ATCC-American Type Culture Collection: Escherichia coli (EC) ATCC 25922 and Klebsiella pneumoniae (KP) ATCC 700603), and eighteen pathogenic microorganisms isolated from human urine samples were used as indicators including: Escherichia coli (EC), Klebsiella pneumoniae (KP), Klebsiella oxytoca (KO), Shigella sonnei (SS), Serratia marcescens (SM), Serratia fonticola (SF), Acinetobacter baumannii (AB), Citrobacter koseri (CK), Citrobacter freundii (CF), Enterobacter aerogenes (EA), Enterobacter cloacae (EL), Enterobacter intermedius (EI), Enterobacter sakazakii (ES), Proteus mirabilis (PM), Proteus vulgaris (PV), Morganella morganii (MM), Salmonella typhimurium (ST) and Salmonella sp. (S). Nutrient agar was used as the growth media.

2.1.5

2.1.5 Essential oil antibacterial assay

Solid diffusion method: An agar diffusion disc method was used to test the selected bacteria's susceptibility to the essential oil (Prabuseenivasan et al., 2006). The inoculum (DO=0.1/625 nm) was streaked into agar plates using a sterile swab after the Mueller Hinton Agar (MHA) had solidified, a sterile filter disc with a 5 mm diameter (Whatman paper N°3) was inserted on the surface of the MHA. Then, 10 μL of the essential oil (crude EO and diluted EO with equivalent concentrations: EO/DMSO: 50/50 v/v) was dropped onto each disc and left for 30 min at room temperature for antibacterial agent diffusion. The plates were incubated for 18 to 24 h at 37 °C. DMSO was employed as a negative control and Gentamicin (GEN) was used as positive control. The essential oil's effectiveness was determined by measuring the diameter of the zone of bacterial growth inhibition above the disc and recording the diameter in mm. An essential oil-inducing inhibition zone ≥ 3 mm around the disc was considered as antibacterial. All tests were performed in triplicate.

Macrodilution method: The Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration of the total essential oil at which the microorganism does not demonstrate visible growth (Wikler, 2006). Serial dilutions of myrtle essential oil in dimethylsulfoxide (DMSO) were prepared (10, 5, 2.5, 1.25 and 0.625 mg/mL). Each dilution was transferred in each tube containing the Mueller Hinton Broth (MHB) medium and the tested bacterial inoculum. The inoculated tubes were then incubated at 37 °C for 24 h. After incubation, the first tube without bacterial visible growth was determined as Minimum Inhibitory Concentration (MIC).

2.2

2.2 Computational details

2.2.1

2.2.1 Docking simulation and ADME(Tox)

The molecular docking investigation aims to predict the antibacterial (E.coli) and anti-SARS-CoV-2 activities of the main compounds coming from myrtle flowers. Each compound structure, reported in Table 1 was pre-optimized using the Steepest Descent algorithm with a convergence criterion of 10 e-6 at the MMFF94s force field theory level. Then, the best conformer was kept after a conformational search and after that an optimization was performed by the same level of theory as implemented in AVOGADRO software (Hanwell et al., 2012). The final optimization was done by MOPAC software at PM7 theory level (Stewart, 1990). To elucidate the mechanism by which the extracted molecules induce antibacterial activity, the inhibitory activities of identified compounds were examined against DNA gyrase. For that, the X-ray crystallographic structure of E. coli topoisomerase II DNA gyrase B along with co-crystallized ligand CBN (PDB: 1KZN) was utilized. The evaluation of the anti-SARS-CoV-2 activity was performed using the following proteins: SARS-CoV-2 Main protease (Mpro) (PDB: 6LU7) (Jin et al., 2020) also named chymotrypsin-like protease (3CLpro), Spike (PDB: 6ZLG) and angiotensin-converting enzyme II ACE2 (PDB: 1R42). The proteins were prepared by removing all solvent molecules and co-crystallized ligands and the molecular docking protocol was validated by redocking using the published crystal structures of protein–ligand complexes. The root-mean square deviations (RMSD) between the conformations of the ligands from the X-ray crystal structure and those from AutoDock Vina (Trott and Olson, 2010) were < 2 A˚, hence the AutoDock Vina docking protocol is adequate to reproduce and to predict experimental findings. The docking of each structure against (PDB ID: 1KZN, 6LU7, 6ZLG, 1R42), was performed by Autodock Vina as implemented in PyRx (Dallakyan and Olson, 2015) in the search spaces and sizes (1KZN: center ×  = 17.11, y = 28.14, z = 31.40; size ×  = 25.0, y = 25.0, z = 25.0); (6LU7: center ×  = -26.28, y = 12.60, z = 58.96; size ×  = 51.30, y = 66.93, z = 59.57); (6ZLG: center ×  = –32.36, y = 25.78, z = 21.01; size ×  = 25, y = 25, z = 25); (1R42: center ×  = 51.13, y = 74.24, z = 28.024; size ×  = 18.70, y = 16.58, z = 18.48). The visualization of the results were depicted by Discovery Studio Visualizer software (Biovia, 2017). Finally, the Adsorption, Digestion, Metabolism, Excretion and Toxicity (ADME-Tox) study of the shortlisted compounds was performed using SwissADME server (Daina et al., 2017) and Pro-Tox II (Banerjee et al., 2018).

Table 1 Chemical composition of essential oil extracted from myrtle flowers.
Peak Compound RT (min) Area (%) RI MM MF
1 Isobutyl isobutyrate 7.969 0.06 808 144.21 C8H16O2
2 α -Thujene 8.337 0.30 869 136,23 C10H16
3 α -Pinene 8.639 48.94 919 136,23 C10H16
4 β -Pinene 8.923 0.57 966 136,23 C10H16
5 1-Allyl tricyclo [4.1.0(2,7)] heptane 9.092 0.06 994 134.21 C10H14
6 Ocimene 10.252 0.15 1186 136,23 C10H16
7 α -Phellandrene 10.621 0.38 1247 136.24 C10H16
8 δ -3-Carene 10.784 0.48 1274 136,23 C10H16
9 α -Terpinene 11.020 1.76 1313 136,23 C10H16
10 1,8-Cineole 11.503 28.3 1393 154,24 C10H18O
11 γ -Terpinene 12.301 0.65 1525 136.23 C10H16
12 α -Terpinolene 13.183 0.75 1671 136.23 C10H16
13 Guajol 13.527 0.04 1728 124.13 C7H8O2
14 Linalol 13.654 2.37 1749 154,24 C10H18O
15 α-Campholenal 14.319 0.09 1859 152.23 C10H16O
16 2,5-Octadiene 14.754 0.05 1931 110.19 C8H14
17 2-Methyl-1,3-pentadiene 14.814 0.06 1941 82.14 C6H10
18 α -Phellandren-8-ol 15.678 0.06 2084 152.23 C10H16O
19 4-Terpineol 15.908 0.22 2122 154.24 C10H18O
20 α -Terpineol 16.379 2.02 2200 154,25 C10H18O
21 Cyclohexene 17.364 0.07 2363 82,143 C6H10
22 Carvon 17.799 0.43 2435 150,21 C10H14O
23 Linalyl acetate 18.016 0.40 2471 196.29 C12H20O2
24 Geraniol 18.131 0.60 2490 154.24 C10H18O
25 2-Undecanone 19.110 0.11 2652 170.29 C11H22O
26 S-cbz-cysteine 19.243 0.06 2674 178.22 C11H14O2
27 o-Acetyl-p-cresol 19.932 0.22 2788 150.17 C9H10O2
28 Exo-2-hydroxycineole acetate 20.367 0.11 2860 212.28 C12H20O3
29 2,4-Dimethyl-2,4-hexadiene 20.790 0.06 2930 110.19 C8H14
30 Eugenol 21.001 2.91 2965 164,20 C10H12O2
31 Geranyl acetate 21.503 2.65 3048 196.29 C12H20O2
32 2- Phenylbutyric acid 21.617 0.11 3067 164.20 C10H12O2
33 Thymoquinone 21.738 0.12 3087 164.20 C10H12O2
34 Methyleugenol 22.137 1.40 3153 178,22 C11H14O2
35 β -Caryophyllene 22.451 0.65 3205 204.36 C15H24
36 p-Ethoxyanisole 22.929 0.19 3284 152.19 C9H12O2
37 α-Humulene 23.321 0.17 3349 204.35 C15H24
38 Germacrene D 24.004 0.09 3462 204.35 C15H24
39 Aromadendrene 24.149 0.12 3486 204.35 C15H24
40 Ledene 24.367 0.12 3522 204.35 C15H24
41 Nerol 24.693 0.05 3576 154,24 C10H18O
42 Methylxanthine 24.916 0.26 3613 166.13 C6H6N4O2
43 Mayurone 25.478 0.14 3706 204.308 C14H20O
44 1-{2-[3-(2-Acetyloxiran-2-yl)-1,1-dimethyl propyl]cycloprop-2-enyl}ethanone 25.738 0.03 3749 236.30 C14H20O3
45 β -Selinene 25.841 0.62 3766 204.18 C15H24
46 Caryophyllene oxide 26.481 0.66 3872 220.35 C15H24O
47 2-Methyl-2 cyclopentenone 27.104 0.22 3975 96.13 C6H8O
48 4,6-Dimethyl-2-amino-1,3-diazine 27.230 0.15 3996 123.15 C6H9N3
49 1,4-Dihydrophenanthrene 27.412 0.18 4026 180.24 C14H12
50 Isolimonene 28.191 0.18 4155 136.23 C10H24
51 N-acetylpiperidone 29.490 0.30 4370 141.17 C7H11NO2
52 2-Nitro-1-decen-4-yne 29.744 0.07 4412 181.23 C10H15NO2
53 Aromadendrene oxide-(1) 30.705 0.15 4571 220.35 C15H24O
54 2-Butanoylthiazole 31.538 0.06 4709 155.21 C7H9NOS

RT (min): Retention time in minute, Area (%): Percentage of each compound, RI: Retention indices.

MM: Molecular Mass (g/moL). MF: Molecular formula.

3

3 Results and discussion

3.1

3.1 Chemical composition results

A Clevenger-type apparatus was used for the isolation of EOs of myrtle, with the following yield: 2.8 percent (v/w). Table 1 summarizes the identified components of myrtle flowers essential oil (n = 54), their percentages, retention times (Rt)and their associated retention indices (RI). Fifty-four components were identified in flowers, representing 89.65% of the total essential oil. The major constituents of the flower essential oil were α-pinene and 1,8-cineole with: 48,94% and 28.3%, respectively. Other representative compounds were detected as eugenol (2.91%), linalol (2,37%), geranyl acetate (2.65%) and α-terpineol (2,02%). Previous studies have reported the chemical composition of myrtle flowers essential oil (Aidi Wannes et al., 2010; Dhifi et al., 2020; Bouzabata et al., 2015; Jerkovic et al., 2002; Santana et al., 2014). However, the majority of compounds detected in this study coincide in earlier research, with some variations in ratios and the lack of some minority compounds. These differences could be attributable to a variety of circumstances, including where the species was grown, harvested, or how the oil was extracted. Similarly to our results, a Tunisian study conducted on myrtle flowers, confirmed that α-pinene and 1,8-cineole were reported as the main constituents (Dhifi et al., 2020; Djenane et al., 2011). Moreover, another Tunisian study showed that the essential oil isolated from myrtle flowers is rich in α-pinene and 1,8-cineole with the following yields: (22.50–15.15%) and (17.53–12.70%), respectively (Aidi Wannes et al., 2010). The chemical heterogeneity in myrtle essential oils appears to be dependent on their organ (leaves, berries, flowers), geographical origin, collection season and edaphoclimatic circumstances (Ben Hsouna et al., 2017; Pirbalouti et al., 2014; Brada et al., 2012).

3.2

3.2 Antibacterial activity results

The initial screening of in vitro antibacterial activity of M. communis EOs against twenty Gram-negative bacteria was carried out by using the disc diffusion method. Thus, in order to obtain more precise data about the antibacterial properties of the tested EOs, Minimum Inhibitory Concentrations (MICs) were determined using the macrodilution broth method. Table 2 showed that the myrtle essential oil had substantial antibacterial activity on all tested bacteria. The best inhibition zone diameter values (ID) were in the range of 11–25 mm. The MICs values of the myrtle essential oil ranged from 0.6 to 2.5 mg/mL. On the whole, Gram-negative bacteria were more sensitive to the crude essential oil than the diluted one with differentiated effects presented by eight sensible (ID≥10 mm), six intermediate (ID=10 mm) and six resistant (ID≤10 mm) strains. As illustrated in Fig. 1, the tested crude EO proved significantly efficient, with pronounced inhibitory action against eight Gram-negative strains with the following order of sensitivity:E.coli>E.coliATCC 25922 > K.oxytoca > S.marcescens > P.mirabilis > E.aerogenes > C.freundii > P.vulgaris. In addition, the myrtle oil was more potent than gentamicin against E.coli, M.morganii, P.mirabilis and P.vulgaris. However, it is important to consider that the differences in the sensitivity of the strains used in each study may justify the differences concerning the activity of the tested agent. The best zone of inhibition of 25 mm was recorded against E.coli, while the lowest one (08 mm) appeared against four microorganisms including: E.cloacae, M.morganii, S. sonnei and Salmonella sp. Consequently, our previous study showed that the leaves essential oil isolated from the same species of myrtle was characterized by a close chemical profile and was also colicidal with the best inhibition zone (35 mm) (Barhouchi et al., 2016). In agreement with our research, Algerian myrtle oil exhibited a remarkable effect against Escherichia coli with inhibition diameter greater than 20 mm, while it showed a low to moderate antibacterial capacity against other strains in comparison with the common antibacterial drug gentamicin (Boussak et al., 2022). These results justify the traditional use of the flowers of this plant species against many bacteria-related disorders including: cough, lung complaints, gastrointestinal disorders, wounds..etc. According to literature, Turkish reports proved the antibacterial effectviness of myrtle flowers EOs against two microorganisms: Parvimonas micra (Gram positive strain) and Aggregatibacter actinomycetemcomitans (Gram negative strain) (Gursoy et al., 2009). In Algeria, myrtle essential oils isolated from flowers were confirmed only as antifungal agents (Bouzabata et al., 2015).

Table 2 Minimum Inhibitory Concentrations MIC (mg/mL) and inhibition zone diameters ID (mm) of myrtle essential oil in comparison with gentamicin.
Bacterial strain Diluted EO (ID) Crude EO (ID) Gentamicin GEN (ID) Crude EO (MIC)
Escherichia coli EC (ATCC 25922) 15 (S) 23 (S) 30 (S) 2.5
Klebsiella pneumoniae KP (ATCC 700603) 09 (R) 10 (I) 19 (S) 2.5
Escherichia coli EC 10 (I) 25 (S) 21 (S) 1.25
Acinetobacter baumannii AB 05 (R) 10 (I) 11 (S) 1.25
Citrobacter freundii CF 05 (R) 11 (S) 22 (S) 2.5
Citrobacter koseri CK 05 (R) 10 (I) 20 (S) 2.5
Enterobacter aerogenes EA 10 (I) 18 (S) 21 (S) 0.6
Enterobacter cloacae EL 05 (R) 08 (R) 08 (R) 1.25
Enterobacter intermedius EI 09 (R) 10 (I) 19 (S) 1.25
Enterobacter sakazakii ES 09 (R) 10 (I) 22 (S) 1.25
Klebsiella pneumoniae KP 09 (R) 10 (I) 16 (I) 1.25
Klebsiella oxytoca KO 16 (S) 20 (S) 21 (S) 2.5
Morganella morganii MM 05 (R) 08 (R) 06 (R) 1.25
Proteus mirabilis PM 06 (R) 19 (S) 17 (I) 2.5
Proteus vulgaris PV 08 (R) 11 (S) 07 (R) 0.6
Salmonella sp. S 08 (R) 08 (R) 15 (R) 1.25
Salmonella typhimurium ST 05 (R) 09 (R) 13 (R) 1.25
Shigella sonnei SS 08 (R) 08 (R) 20 (S) 0.6
Serratia marcescens SM 08 (R) 20 (S) 26 (S) 1.25
Serratia fonticola SF 05 (R) 08 (R) 07 (R) 1.25
Negative control (DMSO) – – – –

Inhibition zone includes the diameter of the disk (5 mm). (05): No inhibition zone, ID: Inhibition zone diameter (mm),

(EO): Essential Oil, (MIC): Minimum Inhibitory Concentration (mg/mL), ATCC: American type culture collection,

(DMSO): Dimethylsulfoxide (negative control), (-): No inhibition zone of DMSO against all strains, GEN: Gentamicin (positive control), (R): Resistant, (I): Intermediate, (S): Sensible.

Inhibition zone diameters (mm) of myrtle essential oil in comparison with gentamicin
Fig. 1
Inhibition zone diameters (mm) of myrtle essential oil in comparison with gentamicin

The antibacterial activity of the essential oils investigated in the present study may be attributed to their major constituents namely: α-pinene and 1,8-cineole. As presented in previous studies, individual components of the EO such as α-pinene demonstrated a potent antibacterial activity (Stojkovic et al., 2008; Dhifi et al., 2020; Zanetti et al., 2010). It was also proven that 1,8-cineole exhibited also a strong antibacterial activity with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values in the range of 0.37 to 11.75 mg/mL and 0.73 to 11.75 mg/mL, respectively (Randrianarivelo et al., 2009). However, other minor constituents of myrtle can act synergistically with major components and play a key role in the observed antimicrobial activity (Dhifi et al., 2020). Hence, the essential oil of Eugenia jambolana, which has α-pinene as a primary constituent, presented synergistic effects when associated with gentamicin against E. coli, but not against P. aeruginosa (Pereira et al., 2017). Overall, the resistance of Gram-negative bacteria is attributed to the presence of an outer membrane with hydrophilic polysaccharide chains that operate as a barrier against hydrophobic essential oils (Mann et al., 2000). Thus, essential oils and their components have been demonstrated to act especially on bacterial membranes (Cox et al., 2000). In particular, monoterpenoids components would enhance the permeability of the cytoplasmic membrane by changing the order of proteins incorporated into the membrane, limiting cellular respiration, ion transportand nutritional absorption (Reichling et al., 2009). Further studies are required to identify the myrtle components responsible for its antimicrobial activity, to clarify how they work on their own or in combination with antibiotics and finally to ascertain the antiviral activity of these oils and/or their chemical constituents.

3.3

3.3 Molecular docking and ADME(Tox) results

The main goal of molecular docking research is to find an optimal conformation for a protein and a ligand with relative orientation between them which minimizes the total system's free energy. It is commonly accepted that the lower the predicted binding free energy, the more effective the binding and thus the more stable the protein–ligand complex is. The heat map presented in Table 3 demonstrates the range of the binding energies results obtained by the molecular docking. It gives an overview about the whole 54 extracted phytochemicals and their relative binding energies in colors, the values increase from green and yellow to red by column (target). In the last four rows of Table 3, the binding energies of Nelfinavir (Bolcato et al., 2020), Nirmatrelvir (Hammond et al., 2022), Gentamicin_C2 (Abdellattif et al., 2021) and Lopinavir (Bolcato et al., 2020) were added as positive controls for the following targets: 1R42, 6LU7, 1KZN and 6ZLG respectively. The three-dimensional representation of the co-crystal top phytochemicals having the lowest binding energies against E.coli (PDB: 1KZN); SARS-CoV-2 MPro (PDB: 6LU7); SARS-CoV-2 Spike (PDB: 6ZLG); ACE2 (PDB: 1R42) is shown in Table 4. The optimal binding energy of phytocompounds was compared with those reported by other researchers (Table 5) (Sisakht et al., 2021; Hakmi et al., 2020; Prasanth et al. 2021; Kandsi et al., 2022; Jianu et al., 2021). 1,8-cineole (-7.9 Kcal/Mol) has shown the lowest binding energy against E.coli (PDB: 1KZN) among all the 54 phytochemicals in addition to several phytochemicals i.e. trans-β-Terpinyl Butanoate, Carvyl acetate reported for Dysphania ambrosioides (L.) (Kandsi et al., 2022) and Mentha × smithiana respectively (Jianu et al., 2021), as shown in Table 5, it also has a lower energy compared to the positive control Gentamicin_C2 (-6.0 Kcal/Mol); 1,8-cineole/1KZN complex has only hydrophobic interactions with the residues ALA47 (4.68 Å), ALA47 (5.43 Å), VAL43 (5.45 Å), VAL167 (5.26 Å), ILE90 (5.22 Å), VAL120 (5.11 Å), ILE78 (5.17 Å) and ILE78(4.22 Å). S-cbz-cysteine has given the best binding energy (-6.0 Kcal/Mol) against SARS-CoV-2 MPro (PDB: 6LU7) between the compounds found in Myrtus communis L; the S-cbz-cysteine/6LU7 complex has the hydrogen bond interactions with the residues GLY143 (2.38 Å), SER144 (2.10 Å), LEU141 (2.71 Å) and hydrophobic interactions with the residues MET49 (4.64 Å), HIS41 (4.72 Å), MET165 (5.64 Å), HIS163 (1.65 Å), LEU141 (2.83 Å); In other research reports, some phytochemicals have given better binding energies such as Ginkgolide M (-11.2 Kcal/Mol) found in the nutshells of the Ginkgo biloba tree (Sisakht et al., 2021) in addition to the positive control Nirmatrelvir (-7.4 Kcal/Mol). Mayurone gave the best binding energy (-6.8 Kcal/Mol) against SARS-CoV-2 Spike (PDB: 6ZLG); Mayurone/6ZLG complex has the hydrogen bond interactions with the residues ARG355 (5.75 Å), SER514 (2.98 Å) and hydrophobic interactions with the residues PRO426 (5.44 Å) and PHE464 (5.01 Å); whereas some reported phytochemicals gave a better binding energy values such as Cinnamtannin-B1 (-10.2 Kcal/Mol) and Kaempferol (-8.7 Kcal/Mol) (Prasanth et al. 2021), in contrast, it has a better binding energy compared to its control Lopinavir (-5.1 Kcal/Mol). Methylxanthine gave the lowest binding energy (-5.0 Kcal/Mol) among the studied phytochemicals against ACE2 (PDB: 1R42); the Methylxanthine/1R42 complex has the hydrogen bond interactions with the residues ALA348 (2.56 Å), GLU37 (2.15 Å) and GLU402 (2.53 Å) in addition to the hydrophobic interactions with the residues ALA348 (4.59 Å), HIS401 (5.40 Å), ALA348 (1.86 Å), HIS401 (3.58 Å) and HIS378 (3.88 Å); some reported phytochemicals give a better binding energy values such as Bicuculline (-9.9 Kcal/Mol) (Xu et al., 2021) and Theaflavin (-8.6 Kcal/Mol) (Emon et al., 2021), in addition to the positive control Nelfinavir (-9.3 Kcal/Mol).  s-cbz-cysteine, mayurone and methylxanthine were found the most promising phytochemicals against SARS-CoV-2; The ADME(Tox) analysis has shown their good druggability with no Lipinski’s rule violation.

Table 3 Heat map of recorded docking scores (binding free energy in kcal/mol) of the essential oil of myrtle flowers components.
Table 4 Three-dimensional representation of the co-crystal top phytochemicals with E.coli (PDB: 1KZN); SARS-CoV-2 MPro (PDB: 6LU7); SARS-CoV-2 Spike (PDB: 6ZLG); ACE2 (PDB: 1R42): (a) interaction residues and (b) hydrogen bonding pocket.
Target/Top compound (Score) 2D interaction residues Hydrogen bonding surface
1,8-cineole/1KZN (-7.9 kcal/mol)
S-cbz-cysteine/6LU7 (-6.0 kcal/mol)
Mayurone/6ZLG (-6.8 kcal/mol)
Methylxanthine/1R42 (-5.0 kcal/mol)
Table 5 Interactions of E.coli (PDB: 1KZN); SARS-CoV-2 MPro (PDB: 6LU7); SARS-CoV-2 Spike (PDB: 6ZLG); ACE2 (PDB: 1R42) amino acid residues with the studied compound.
Compound PubChem ID Target Vina Score (Kcal/Mol) Residues in interaction* Ref
1,8-Cineole 1KZN −7.9 Thr165, Ile78, Ile90, Val120, Val43, Ala47, Val167, Ala47, Ile78 This work
trans-β-Terpinyl Butanoate −6.4 Thr165, Val120, Met91, Gly77, Arg136, Glu50, Ala47, Asn46, Asp73, Pro79, Ile78, Arg76, Val43,, Val167 (Kandsi et al., 2022)
Carvyl acetate −6.8 – (Jianu et al., 2021)
s-cbz-cysteine 6LU7 −6.0 Gly143, Ser144, Leu141, Met49, His41, Met165, His163 This work
Ginkgolide M 46173836 −11.2 Gly143, His163,Cys145, Glu166, Phe140, Asn142 (Sisakht et al., 2021)
Mayurone 538435 6ZLG −6.8 Arg355, Ser514, Pro426, Phe464 This work
Cinnamtannin-B1 −10.2 Phe390, Asn394, Arg393, Phe40, Trp349, Thr347 (Prasanth et al. 2021)
Kaempferol −8.7 Asp350, Tyr385, Asp382, His345, Hia374, Glu375, His378, His401, His378 (Prasanth et al. 2021)
Methylxanthine 1R42 −5.0 Glu375, Glu402, Ala348, Glu375, Glu402, Ala348, His378 This work
Bicuculline −9.9 – (Xu et al., 2021)
Theaflavin −8.6 Thr371, Arg518, Glu406, Glu375, Leu370 (Emon et al., 2021)

*The residues associated with hydrogen bonds are in bold.

4

4 Conclusion

Myrtle flowers have long been used as a remedy for respiratory complaints, as well as microorganisms inactivation. The phytochemical investigation showed that the EOs contained similar major components as previously reported by other studies although their percentages varied. The main compounds obtained in the flowers essential oil of Myrtus communis were: α-pinene, 1,8-cineole, eugenol, linalol, geranyl acetate and α-terpineol. The myrtle essential oil displayed significant antibacterial activity and showed a pronounced effect against Gram negative bacteria in comparison to the commercial drug. The bacterium most sensitive to the effect of the essential oil was Escherichia coli. The molecular docking investigation showed that 1,8-cineole is the best inhibitor against E. coli topoisomerase II DNA gyrase B and could be the main phytochemical associated with the antibacterial activity of EO. Among the EO compounds, s-cbz-cysteine, mayurone and methylxanthine were found as the most promising against SARS-CoV-2 by the molecular docking analysis. This work opens the gate to additional preclinical and clinical research of edible EOs to alleviate certain respiratory tract illnesses.

CRediT authorship contribution statement

Badra Barhouchi: Writing – original draft, Conceptualization, Methodology, Resources, Data curation, Writing – review & editing. Rafik Menacer: Conceptualization, Methodology, Resources, Data curation, Software, Investigation, Formal analysis, Visualization, Validation, Writing – review & editing. Saad Bouchkioua: Software, Investigation, Formal analysis, Visualization. Amira Mansour: Project administration. Nadjah Belattar: Data curation.

Acknowledgements

This study was supported by the Algerian Ministry of Higher Education and Scientific Research (MESRS) and the General Directorate of Scientific Research and Technological Development (DGRSDT). Many thanks are also addressed to Pr. Abdelhamid Djekoun, director of the Pharmaceutical Sciences Research Center (CRSP), for his great availability.

Declaration of Competing Interest

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

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