Skip to content
BY 4.0 license Open Access Published by De Gruyter Open Access June 5, 2023

Study on antioxidant and antimicrobial potential of chemically profiled essential oils extracted from Juniperus phoenicea (L.) by use of in vitro and in silico approaches

  • Tarik Chelouati , Soufyane Lafraxo , Mohammed Bouslamti , Azeddin El Barnossi , Mohamed Chebaibi , Mohamed Akhazzane , Ahmad Mohammad Salamatullah , Hiba-Allah Nafidi , Mohammed Bourhia EMAIL logo , Badiaa Lyoussi and Ahmed Samir Benjelloun
From the journal Open Chemistry


Juniperus phoenicea (L.) is a medicinal plant that has been used in phytotherapy as a treatment of certain pathological infections. In this context, the present work aimed to valorize the essential oil of J. phoenicea seeds (EOGP) by studying its chemical composition, and antioxidant and antimicrobial activities. The EOGP was extracted by use of hydrodistillation and characterized by gas chromatography (GC–MS). The antioxidant power was evaluated by three methods (TAC, DPPH, and FRAP). The antimicrobial power was evaluated against Staphylococcus aureus (ATCC6633), Escherichia coli (K12), Bacillus subtilis (DSM6333), Proteus mirabilis (ATCC29906), Candida albicans (ATCC10231), Aspergillus niger (MTCC282), Aspergillus flavus (MTCC9606), and Fusarium oxysporum (MTCC9913). The GC/MS results revealed a total identification of 99.98% with a dominance of carvacrol (39.81%) followed by p-cymen-3-ol (34.44%) and o-cymene (13.60%). Findings showed that EOGP exhibited important antioxidant power as IC50 was determined to be 26 µg/mL for 2,2-diphenyl-1-picrylhydrazyl, while EC50 was 216.34 µg/mL for ferric reducing antioxidant power and total antioxidant capacity was 720 mg AAE/g. The antimicrobial power on solid medium revealed that the inhibition diameters ranged from 11.30 ± 0.58 to 20 mm for the bacterial strains and from 9.33 ± 0.57 to 54.43 ± 0.29 mm for fungi. Notably, minimum inhibitory concentrations ranged from 18 to 19 µg/mL for bacterial strains and from 5.04 to 10.09 µg/mL for fungal strains. Overall, our results demonstrated the importance of EOGP as a source of natural antioxidant and antibacterial medicines against clinically relevant pathogenic strains.

1 Introduction

The floristic diversity of the Mediterranean region is estimated at 25,000 species or 30,000 species and subspecies, which is equivalent to around 10% of the world’s higher plants [1]. The Iberian Peninsula and Morocco are located at one of two major poles of plant diversity, while Turkey and Greece are located at the other [2]. Morocco has a Mediterranean temperature and diversified flora due to its geographic location between the two seas. The plains and the lowlands are vast, and the upper mountain ranges (over 4,000 m altitudes) have a complicated and highly segmented structure. The Mediterranean region has all the identified bioclimates and bioclimatic variations, with an average annual precipitation range of 30 to over 2,000 mm [36].

Modern medicine has paid close attention to the potential of phytochemicals as a safer and more natural alternative [7,8]. Antimicrobial and antioxidant properties of essential oils have been known for a long time, and their derivatives have been utilized as natural antimicrobial and antioxidant agents in numerous fields, including pharmacology, plant pathology, pharmaceutical, medical and clinical microbiology, conservation of food, etc. An extensive study on essential oils and other extracts that exhibit antimicrobial and/or antioxidant activity has led to the screening of a vast array of plant species, each of which has been shown to have its own structurally distinct biologically active chemicals [9]. However, the activity of their primary components in the studied essential oils has received comparatively little study. The new natural agents’ primary benefit is that they do not display antibiotic resistance, a problem often seen with prolonged antibiotic treatment. Essential oils’ antibacterial and antioxidant properties may be traced back to a variety of tiny terpenoids and phenolic chemicals (such as thymol, carvacrol, and eugenol), which show potent antibacterial and antioxidant properties even when isolated [10].

The coniferous Juniperus are an essential plant of the dry and semi-arid ecosystems in the northern hemisphere [11]. It is one of the second-most diversified conifer genus. It is made up of roughly 67 species of conifers and 28 variations [11]. The family Cupressaceae includes the evergreen Juniperus phoenicea tree (red juniper), which is indigenous to North Africa. Leaves of the J. phoenicea species are used as a decoction to treat rheumatism and diarrhea [12]. Although little is known about J phoenicea’s antibacterial effect, its chemical composition has also not been sufficiently examined. Some bacteriostatic and bactericidal effectiveness has been demonstrated for J. phoenicea [13]. To the best of our knowledge no reports on the chemical composition, antioxidant and antimicrobial activities of J. phoenicea seeds collected in Morocco have been found; therefore, the present work aimed to investigate these biological activities.

2 Materials and methods

2.1 Extraction of essential oil of J. phoenicea seeds (EOGP)

J. phoenicea was manually collected in March 2022, in a mountainous population (33.75750029; −4.32896628) before being identified and deposited in the herbarium under a voucher number (LSNA/TC/JP-22). Next, 100 g of seeds was ground before the extraction of essential oils. Notably, the seeds were subjected to extraction with 750 mL of water using Clevenger apparatus set to 100°C for 120 min, the EOGP was stored and kept at 4°C until further use [14].

2.2 Chemical characterization of EOGP

The phytochemical compositions of EOGP were identified using gas chromatography–mass spectrometry (GC–MS, Thermo Fischer; GC/ULTRAS/N-20062969; Polaris QS/N-210729), equipped with a HP5MS non-polar fused silica capillary column (60 m × 320 µm, film thickness 0.25 µm) as detailed in previous works [15,16].

2.3 Antioxidant activity of EOGP

2.3.1 2,2-diphenyl-1-picrylhydrazyl (DPPH) test

EOGP (100 µL) at doses ranging from 1 to 1,000 µg/mL was added to a solution of DPPH (1 mmol) [17]. Next, the solutions were well shaken and allowed to incubate for 1 h at room temperature. A spectrophotometer was used to measure the absorbance at 517 nm against a blank methanol solution. For comparison, quercetin and butylated hydroxytoluene (BHT) used for references were prepared in the same questions as EOGP. The inhibition (I%) was determined using the following formula:

I (%) = (AbT − AbS/Ab) × 100.

In this expression, AbS represents the absorbance of the test sample and AbT represents the absorbance of the blank.

2.3.2 Ferric reducing antioxidant power (PRAP) test

The method of Oyaizu [18] was used to calculate the iron reduction potential of EOGP. Importantly, 200 µL of EOGP at varying concentrations (0.001–1 mg/mL) was combined with 500 µL of phosphate buffer of pH = 6.6 (0.2 M) and 500 µL of K3[Fe(CN)6]. After incubating the mixture for 30 min at 50°C with 500 µL TCA (10%), the resulting solution was centrifuged at 3,000 rpm for 10 min. Subsequently, 500 µL of each concentration’s supernatant was mixed with 500 µL of water (H2O) and 100 mL of FeCl3 (1 mg/mL). Using a spectrophotometer, we determined the absorbance at 700 nm. The absorbance of quercetin and BHT used as references was measured under the same conditions as EOGP. The graph of absorbance versus EOGP concentration was used to determine the concentration of EOGP that results in a 50% reduction in absorbance (EC50; µg/mL).

2.3.3 Total antioxidant capacity (TAC) method

This method was conducted by preparing a reaction consisting of sulfuric acid (600.0 mmol/L), sodium phosphate (PBS: 28.0 mmol/L), and ammonium molybdate (4.0 mmol/L). Subsequently, 50 µL of EOGP was added to 2 mL of this prepared reaction before being incubated at 95°C for 90 min. Absorbance measurement at 695 nm was performed. The results were reported in mg AAE/g [19].

2.4 Antimicrobial activity

The antimicrobial activity of EOGP was performed against Escherichia coli (K12), Candida albicans (ATCC10231), Aspergillus niger (MTCC282), Aspergillus flavus (MTCC9606), Fusarium oxysporum (MTCC9913), Staphylococcus aureus (ATCC6633), Bacillus subtilis (DSM6333), and Proteus mirabilis (ATCC29906). Bacterial and fungal strains were isolated and provided by USMBA, Fez, Morocco.

2.4.1 Testing antimicrobial efficacy of EOGP

Disk diffusion technique was used to test the antibacterial activity of EOGP [20,21]. The strains tested were inoculated into Petri dishes (20 mL) containing Mueller-Hinton (MH) and malt extract (ME) media. From these freshly grown cultures, dilutions were made in NaCl solution (9 mg/mL) to achieve a turbidity of 0.5 McFarland equivalent of 106–108 CFU/mL. After inoculating Petri dishes with MH and EM media, 0.100 mL was placed in 5 mL of agar (0.5%). EOGP of 15 µL was used to impregnate 6 mm diameter paper (no. 4) discs. Using EM medium, the antifungal activity of EOGP was evaluated for the studied fungal strains such as A. niger, F. oxysporum, and A. flavus. Briefly, 15 µL of EOGP was impregnated into a 6 mm diameter Whatman No. 4 paper disk, and deposited on the surface of Petri dishes containing EM medium with the fungal strains. Negative controls and positive controls containing conventional antibiotics were used in the same manner as the tests to determine the efficacy of EOGP; oxacillin (15 mg/mL) for bacterial strains and fluconazole for fungal strains. Bacteria, C. albicans, and filamentous fungus were incubated at 37 and 30°C, respectively. After 24 h post-inoculation, inhibitory diameters and inhibition percentages were obtained for the bacterial strains, while after 48 h for C. albicans and after 7 days for A. flavus, F. oxysporum, and A. niger [22,23].

2.4.2 Determination of the minimum inhibitory concentration (MIC)

The microplate dilution method (96 wells) was used to determine the microbial growth inhibitory concentration (MIC) of EOGP [24,25]. The first column of the plate received 100 µL of EOGP, while the remaining wells each received 50 µL. After creating serial dilutions using a multichannel pipette, 30 µL of microbial suspension was added to the plate, followed by 50 µL of sterile MH for bacterial strains and 50 µL of sterile EM for fungal strains. Finally, microbial suspensions of each strain (106–108 CFU/mL) were added to each well after serial dilution in a multichannel pipette to a final volume of 30 µL. Notably, after 24 h at 37°C for bacteria, 48 h for C. albicans, and 7 days at 30°C for A. niger, A. flavus, and F. oxysporium, a colorimetric of triphenyltetrazolium chloride (2 mg/mL) is used to calculate the MICs [24,26].

The study used molecular docking methods to assess the antioxidant and antimicrobial activities of essential oils from J. phoenicea against NADPH oxidase, S. aureus nucleoside diphosphate kinase, and E. coli beta-ketoacyl-[acyl carrier protein] synthase, respectively. The LigPrep tool in Schrödinger Software’s Maestro 11.5 version was used to process the compounds retrieved from the PubChem database in SDF format. The compounds were analyzed in 32 stereoisomers after taking into account ionization states at pH 7.0 ± 2.0.

2.5 Molecular docking

The PDB format of NADPH oxidase (PDB:2CDU), E. coli beta-ketoacyl-[acyl carrier protein] synthase (PDB: 1FJ4), and S. aureus nucleoside diphosphate kinase (PDB: 3Q8U) three-dimensional crystal structures were obtained from the protein data bank. Schrödinger-Maestro v11.5’s Protein Preparation Wizard was used to enhance the structures by adding hydrogens to heavy atoms, converting selenomethionines to methionines, and deleting all waters. The minimization was performed using the OPLS3 force field, with a maximum heavy atom root mean square displacement of 0.30 Å. A grid box with a volumetric spacing of 20 × 20 × 20 was employed to link the ligand with the protein-derived grid box. The SP method was used to perform this coupling, and the resulting outcomes were evaluated using the SP GScore. Using Schrödinger-Maestro v11.5’s Glide, flexible ligand docking was conducted utilizing the SP method. Non-cis/trans amide bonds were subject to penalties. Ligand atoms were assigned a Van der Waals scaling factor of 0.80 and a partial charge cutoff of 0.15. The glide score was computed using energy-minimized poses and recorded as the final score. The best-docked pose with the lowest glide score value was registered for each ligand.

2.6 Statistical analysis

The results of the study were presented as mean and standard deviation based on three separate sets of tests. Multiple comparisons were handled using analysis of variance and Tukey’s HSD test (a post hoc analysis of variance test). The significance was considered when p-value is 0.05.

3 Results and discussion

3.1 Phytochemical analysis of EOGP

The hydrodistillation used to extract the EOGP allowed to extract 8.24% of the total mass of crude plant material. This yield was comparable to that found by Medini et al. [27], who reported that the extraction yield of essential oils from J. phoenicea seeds ranged from 2 to 7%. In comparison with the literature data, this yield was higher than those previously reported [9,28,29]. GC/MS analysis of EOGP revealed that J. phoenicea seeds were rich in volatile compounds with a dominance of carvacrol (39.81%) followed by p-cymen-3-ol (34.44%) and o-cymene (13.60%) (Figure 1 and Table 1). Classification of identified phytochemicals revealed that EOGP was rich in monoterpene compounds (C10) and sesquiterpenes (C15). These results were comparable to the chemical composition of essential oils from J. phoenicea reported in previous work [30,31]. Another study on the essential oil of J. phoenicea revealed its richness in monoterpenes (74%), which is in agreement with our findings. Notably, literature reported a difference in chemical composition between J. phoenicea growing in different areas [27]. The phytochemical composition and the extraction yield can vary from one plant to another in the same species, depending on the variation of climatic factors (altitude, soil, and exposure) and the period of harvesting [32]. The molecular structure analysis of terpenic compounds revealed that the majority of these molecules presented hydroxyl functional group (p-cymen-3-ol, carvacrol, and terpinen-4-ol). On the other hand, some molecules present peroxide radicals (O) like linalool and isothymol methyl ether. The presence of these types of molecules and their heterogeneities in natural compounds can give these compounds pharmacological efficacy in vivo and in vitro [33].

Figure 1 
                  Chromatogram (GC–MS) of EOGP.
Figure 1

Chromatogram (GC–MS) of EOGP.

Table 1

Phytochemicals of EOGP by gas chromatography (GC–MS)

Pk RT Compound C.C RI.C RI.L A (%) Structure
1 10.543 o-Cymene MO 1,022 1,026 13.60
2 11.567 ϒ-Terpinene MO 1,156 1,059 3.75
3 12.777 β-Linalool MO 1,072 1,072 1.86
4 14.974 Endo-borneol MO 1,160 1,160 0.97
5 15.224 Terpinen-4-ol MO 1,173 1,177 0.56
6 16.869 Isothymol methyl ether O 1,231 1,235 2.0
7 18.318 Carvacrol MO 1,292 1,299 39.81
8 18.559 p-Cymen-3-ol MO 1,420 1,426 34.44
9 21.869 Caryophyllene SQ 1,461 1,466 2.28
10 26.116 Caryophyllene oxide SQ 1,467 1,667 0.71
Chemical classes
Monoterpenes (MO) 94.99
Sesquiterpenes (ST) 2.99
Others (O) 2.0
Total identified (%) 99.98%
  1. Note: Peak (Pk), chemical class (C.C), retention time (RT), area (A), retention index literature (RI.L), and retention index calculate (RI.C).

3.2 Antioxidant activity of EOGP

The antioxidant activity of EOGP was in a dose–response relationship (Figures 24). Importantly, from these figures, it can be seen that the antiradical activity increases by increasing the doses of EOGP, i.e., a concentration of 12 µg/mL has a percentage inhibition of 36% and 50 µg/mL of EOGP has a percentage inhibition of 68%, while 91% was noted for a concentration of 1 mg/mL. However, these results are less important than those recorded for quercetin and BHT. The IC50 of EOGP was 26 µg/mL against 19.52 µg/mL (BHT) and 22.84 µg/mL (quercetin). Recent studies revealed that the IC50 of EOs extracted from Juniperus was 21.25, 23, and 5.36 µg/mL [9,34,35]. These results may support our findings of the antioxidant power of EOGP.

Figure 2 
                  Antioxidant power of EOGP and control by DPPH assay.
Figure 2

Antioxidant power of EOGP and control by DPPH assay.

Figure 3 
                  Antioxidant power of EOGP, BHT, and quercetin by FRAP test.
Figure 3

Antioxidant power of EOGP, BHT, and quercetin by FRAP test.

Figure 4 
                  Antioxidant power of EOGP, BHT, and quercetin by determination of ammonium phosphomolybdate (TAC).
Figure 4

Antioxidant power of EOGP, BHT, and quercetin by determination of ammonium phosphomolybdate (TAC).

The FRAP test showed that EOGP has good antioxidant capacity, such that the EC50 was 216.34 µg/mL for EOGP, 256.52 µg/mL for BHT, and 240.06 µg/mL for quercetin (Figure 3). These results are in accordance with previous works on Juniperus antioxidant power wherein it was stated that leaves and bark of this plant possessed antioxidant power with EC50 of 190 and 481 µg/mL, respectively [34,35].

The antioxidant capacity of EOGP by the phosphomolybdenum method revealed that there is a dose effect. Notably, total antioxidant activity was determined to be 185.36 µg AAE/mg versus 312 and 263 AAE/mg for BHT and quercetin, respectively (Figure 4). The TAC of essential oils extracted from Juniperus was 930 µg AAE/mg (leaves) and 271 µg AAE/mg (barks), recorded in literature [34,35].

It has been hypothesized that the hydroxyl function present in the phytochemical compositions of EOs accounts for their antioxidant effects. The antioxidant properties of EOs, terpenes, and phenolic components have been confirmed by our previous studies [3638]. The antioxidant efficacy of EOGP may be due to the wide variety of phytochemicals included in the extract, some of which may have a synergistic effect with one another. Terpinene, one of the chemicals thought to boost antioxidant capacity, has been found in abundance in J. thurifera in recent investigations [35,39].

Studies have shown the presence of hydroxy phenolic compounds in natural compounds (essential oils and plant extracts) considered as strong antioxidants and can scavenge free radicals [40]. Indeed, the richness of J. phoenicea essential oils (EOGP) in terpene compounds with different structures can explain the antioxidant power. Importantly, the presence of −OH radicals and −O radicals in some molecules identified in EOGP can explain the antioxidant power of EOGP, which was tested by various methods (TAC, DPPH, and FRAP).

3.3 Antimicrobial power of EOGP

Results showed that EOGP possessed potent antibacterial power against various strains used for testing. Notably, EOGP on solid medium revealed that the largest inhibition diameter was recorded against Proteus mirabilis with an inhibition zone of about 20.0 ± 0.0 mm followed by E. coli with an inhibition zone of about 18.33 ± 0.57 mm, while the smallest diameter was noted against S. aureus with an inhibition zone of about 11.30 ± 0.58 mm. These results showed that all the strains were sensitive to EOGP when compared to oxacillin used as positive control, which was almost ineffective except for E. coli with an inhibition zone of 18.0 ± 0.0. The inhibitory power of bacterial growth on a liquid medium by microdilution method showed that the most inhibitory concentration was recorded against E. coli and B. subtilis with a concentration of about 18.13 and 18.21 µg/mL, respectively (Figures 5 and 6).

Figure 5 
                  Antibacterial power of EOGP on solid medium by the disc method showing the inhibition zone in the presence of EOGP.
Figure 5

Antibacterial power of EOGP on solid medium by the disc method showing the inhibition zone in the presence of EOGP.

Figure 6 
                  Evaluation of the antibacterial power of EOGP on solid media by determining the inhibition diameter (in mm).
Figure 6

Evaluation of the antibacterial power of EOGP on solid media by determining the inhibition diameter (in mm).

It was reported that EO from J. phoenicea collected in Algeria was active against S. aureus and E. coli, resulting in inhibition zones up to 25 mm [31]. Essential oils of J. phoenicea bark collected in Morocco were also active against S. aureus, E. coli, and B. subtilis with inhibition zones of 24.0, 34.0, and 10.0 mm, respectively. Importantly, the minimum concentration of EO from J. phoenicea was 20 µg/mL for E. coli, 100 µg/mL for S. aureus, and 320 µg/mL for B. subtilis [30]. Additional literature showed that EO from Juniperus thurifera abolished E. coli and P. mirabilis with 21 and 18 mm in inhibitory zones, respectively. Moreover, EO from the genus Juniperus revealed a MIC of 670 and 1,340 µg/mL against E. coli and P. mirabilis [35]. Rahhal et al. [41] showed that the essential oil of species in the genus Juniperus was effective against S. aureus (31.12 mm), E. coli (13.23 mm), and P. aeruginosa (31.12 mm). Several studies, like the one by Mansouri et al. [45], showed that S. aureus is sensitive to the EO of Juniperus. Bahri et al. [42] showed that EO of Juniperus had antibacterial activity against S. aureus (27 mm; 450 µg/mL) and E. coli (18.8 mm).

The antifungal results of EOGP possessed important antifungal effects against fungi used for testing. Notably, the inhibition diameter against C. albicans and F. oxysporum was 9.33 ± 0.57 mm and 54.43 ± 0.29%, respectively; however, a resistance of A. niger to EOGP was noted (Table 2 and Figure 7). The minimum concentration of EOGP against fungi ranged from 5.04 ± 0.41 to 10.09 ± 0.84 µg/mL (Table 2). These results are in agreement with previous literature [35], wherein it was reported that essential oils from the genus Thurifera are active against C. albicans and A. niger with inhibition percentages of 10.75% and 37.44, respectively. Our findings are consistent with those of previous reports, including a study by El Jemli et al. [43], which demonstrated significant antifungal power against Aspergillus alternata, Fusarium solani, F. oxysporum, Vibrio dahliae, and Rhizopus solani, with inhibition percentages ranging from 24 to 92.1%.

Table 2

Antifungal power of EOGP against fungi

C.a A.n A.f F.o
EOGP Di (mm) 9.33 ± 0.57 0.0 ± 0.0 42.55 ± 0.85 54.43 ± 0.29
MIC (µg/mL) 5.04 ± 0.41 0.0 ± 0.0 10.09 ± 0.83 10.09 ± 0.84
Fluc Di (mm) 13.00 ± 1.00 20.67 ± 1.52 0.00 ± 0.00 30.67 ± 0.50
MIC (mg/mL) 7.50 ± 0.0 21.33 0.00 ± 0.0 7.50 ± 0.0

C.a: C. albicans; A.n: A. niger; A.f: A. flavus; F.o: F. oxysporum; Di: diameter of inhibition.

The outer membrane structure of Gram-negative bacteria made them more resistant than Gram-positive bacteria to drugs. Therefore, unlike Gram-positive bacteria, Gram-negative bacteria have an outer membrane that is richer in lipo-polysaccharides and proteins, making it more hydrophilic and protecting the bacteria from the adhesion of hydrophobic terpenes [44,45]. However, low molecular weight phenolic chemicals can bind to the functional groups on these bacteria and hence attach to them. Studies on EOs showed that the hydroxyl group in carvacrol, which was the most abundant component in EOGP, possesses antibacterial properties [32,46]. When combined with charged groups in the membrane, carvacrol increases the permeability of the bacterial membrane [47] and modifies the cellular outer membrane [48]. Carvacrol can also inhibit other enzymes because of its ability to block ATPases, which results in the loss of proton motive force [49]. Due to their hydrophobic properties, some of the terpene chemicals found in essential oils can cross the outer membrane of Gram-negative bacteria, where they can enter into the phospholipid bilayer and alter the membrane’s structure and permeability. Numerous investigations have demonstrated the powerful antifungal action of the pinene compound. Notably, Shi et al. [50] revealed substantial antifungal power of pinene against five phytopathogens, including C. gloeosporioides, A. kikuchiana, F. proliferatum, and Phomopsis sp., while Nobrega et al. [51], demonstrated the potent antifungal effect of alpha pinene against Candida.

Recent studies support the use of EOs in classical antimicrobial applications against pathogenic illnesses. Literature showed that EOs are an important weapon for controlling bacterial resistance, even for broad-spectrum antibiotics [14,52]. Although in vitro studies have been used to evaluate the antibacterial potential of EOs via antagonistic/additive or synergistic actions against dangerous microorganisms, in vivo research is required to validate and investigate the therapeutic benefits of EOs. Antimicrobial or drug-resistance-reversing actions of EOs require more research to determine the specific EO components responsible for these effects, as well as an underlying mechanism of drug resistance. The claims and traditional usage of medicinal and aromatic plant EOs to treat human infections have recently been scientifically validated [16,52]. Several studies have demonstrated that essential oils possess potent antibacterial properties against a wide range of infectious diseases. A large body of research demonstrates that essential oils, both alone and in combination with medications, have antibacterial qualities. The transformation of these strong natural antibacterial agents into pharmaceuticals for therapeutic uses, however, requires more in vivo confirmations and further investigations of this kind, as well as preclinical and clinical trials. The screening can also increase the number of potent essential oils available for usage as lead molecules in medication discovery and development. Nonetheless, promising new research suggests these potent compounds will make drug development a reality [53,54].

Figure 7 
                  Antifungal power of EOGP on a solid medium by the disc method.
Figure 7

Antifungal power of EOGP on a solid medium by the disc method.

3.4 Molecular docking

In terms of antioxidant activity, carvacrol was the most active molecule against NADPH oxidase, followed by isothymol methyl ether with glide scores of −6.082 and −5.716 kcal/mol, respectively. Regarding antimicrobial activity, carvacrol exhibits the highest activity against S. aureus nucleoside diphosphate kinase, followed by terpinen-4-ol with glide scores of −6.039 and −6.008 kcal/mol, respectively. For E. coli beta-ketoacyl-[acyl carrier protein] synthase, p-Cymen-3-ol and carvacrol were the most active molecules with glide scores of −6.514 and −6.83 kcal/mol, respectively (Table 3). Figures 8 and 9 depict the number and types of bonds formed between ligands and active sites. Specifically, in the active site of NADPH oxidase, carvacrol established two hydrogen bonds with residues GLY 162 and CYS 242. Similarly, in the active site of S. aureus nucleoside diphosphate kinase, carvacrol formed two hydrogen bonds with residues GLY 110 and LYS 9. In the active site of E. coli beta-ketoacyl-[acyl carrier protein] synthase, p-cymen-3-ol established a single hydrogen bond with the THR 302 residue and a single Pi–Pi stacking bond with the residue HIE 298.

Table 3

Docking results with ligands in the active sites of 2CDU, 1FJ4, and 3Q8U

Variant 2CDU 1FJ4 3Q8U
Glide gscore Glide emodel Glide energy Glide gscore Glide emodel Glide energy Glide gscore Glide emodel Glide energy
Carvacrol −6.082 −32.356 −23.536 −6.514 −35.3 −25.646 −6.039 −26.981 −20.812
Isothymol methyl ether −5.716 −30.615 −22.382 −6.116 −33.932 −24.64 −5.003 −24.879 −19.477
o-Cymene −5.344 −23.239 −17.415 −6.055 −25.77 −19.363 −4.945 −21.146 −16.321
p-Cymen-3-ol −5.099 −29.022 −22.063 −6.83 −34.992 −24.811 −5.547 −27.257 −20.444
Terpinen-4-ol −4.944 −25.569 −19.655 −5.862 −29.94 −22.432 −6.008 −25.633 −18.404
Gamma-Terpinene −4.91 −24.19 −19.057 −5.905 −28.684 −21.486 −5.155 −21.683 −16.974
Caryophyllene −4.343 −11.897 −11.633 −5.064 −19.788 −11.924 −4.157 −20.099 −7.755
Caryophyllene oxide −4.144 −19.805 −18.17 −5.356 −27.944 −11.512 −4.194 −20.672 −17.414
Beta-Linalool −2.996 −22.954 −20.074 −4.187 −29.647 −24.469 −3.587 −25.871 −22.301
Endo-borneol −5.395 −23.663 −18.665 −3.432 −7.175 −7.097
Figure 8 
                  2D viewer of ligand interactions in the active sites. (a and b) Carvacrol interactions in the active sites of NADPH oxidase and S. aureus nucleoside diphosphate kinase. (c) p-Cymen-3-ol interactions in the active site of E. coli beta-ketoacyl-[acyl carrier protein] synthase.
Figure 8

2D viewer of ligand interactions in the active sites. (a and b) Carvacrol interactions in the active sites of NADPH oxidase and S. aureus nucleoside diphosphate kinase. (c) p-Cymen-3-ol interactions in the active site of E. coli beta-ketoacyl-[acyl carrier protein] synthase.

Figure 9 
                  3D viewer of ligand interactions in the active sites. (a and b) Carvacrol interactions in the active sites of NADPH oxidase and S. aureus nucleoside diphosphate kinase. (c) p-Cymen-3-ol interactions in the active site of E. coli beta-ketoacyl-[acyl carrier protein] synthase.
Figure 9

3D viewer of ligand interactions in the active sites. (a and b) Carvacrol interactions in the active sites of NADPH oxidase and S. aureus nucleoside diphosphate kinase. (c) p-Cymen-3-ol interactions in the active site of E. coli beta-ketoacyl-[acyl carrier protein] synthase.

4 Conclusion

The present study revealed that J. phoenicea EOs have strong antioxidant and antibacterial properties against clinically drug-resistant microorganisms. Importantly, J. phoenicea EOs could be used as a substitute for conventional antibacterial antioxidant therapies. However, toxicities on non-human primates and humans will be required before any prospective application of the investigated EOs as natural medicines


This work was funded by the Researchers Supporting Project number (RSP-2023R437), King Saud University, Riyadh, Saudi Arabia, and the authors are grateful for the support.

  1. Funding information: This work was funded by the Researchers Supporting Project number (RSP-2023R437), King Saud University, Riyadh, Saudi Arabia.

  2. Author contributions: Conceptualization, T.K. and S.L.; methodology, A.E.M.; software, M.B. (Mohammed Bouslamti) and M.C.; validation, A.E.B. and N.S.; formal analysis, T.K.; investigation, M.A.; resources, M.B. (Mohammed Bourhia); data curation, A.M.S.; writing – original draft preparation, M.B. (Mohammed Bourhia) and H.N.; writing – review and editing, B.L.; visualization, A.S.B.; supervision, A.S.B.; project administration, A.S.B.; funding acquisition, H.N. All authors have read and agreed to the published version of the manuscript.

  3. Conflict of interest: Authors declare no conflict of interest.

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

  5. Data availability statement: Derived data supporting the findings of this study are available from the corresponding author on request.


[1] Médail F, Quézel P. Hot-spots analysis for conservation of plant biodiversity in the Mediterranean Basin. Ann Mo Bot Gard. 1997;84:112–27. 10.2307/2399957.Search in Google Scholar

[2] Myers N, Mittermeler RA, Mittermeler CG, Da Fonseca GAB, Kent J. Biodiversity hotspots for conservation priorities. Nature. 2000;403:853–8. 10.1038/35002501.Search in Google Scholar PubMed

[3] Daget P. Le bioclimat Mediterraneen: Analyse des formes climatiques par le systeme d’Emberger. Vegetatio. 1977;34:87–103. 10.1007/BF00054477/METRICS.Search in Google Scholar

[4] Donadieu P. Contribution à une synthèse bioclimatique et phytogéographique au Maroc. Trav Inst Agron Vét Hassan II Rabat. 1977;1:15.Search in Google Scholar

[5] Emberger L. Une classification Biogéographique des Climats. Univ. Fac. Sci. Montpellier. vol. 7; 1955. p. 3–43.Search in Google Scholar

[6] Emberger L. La Position Phytogeographique Du Maroc Danis L’ensembi, E Mediterraneen. Al Awamia. 1964;12:15.Search in Google Scholar

[7] Smid EJ, Gorris, L. Natural antimicrobials for food preservation handbook of food preservation. N Y Marcel Dekker. 1999;1999:285–308.Search in Google Scholar

[8] Vaidya AB AV. New drugs from medicinal plants, opportunities and approaches. J Ass Phys India. 1994;42:221–8.Search in Google Scholar

[9] Ennajar M, Bouajila J, Lebrihi A, Mathieu F, Abderraba M, Raies A, et al. Chemical composition and antimicrobial and antioxidant activities of essential oils and various extracts of Juniperus phoenicea L. (Cupressacees). J Food Sci. 2009;74:M364–71. 10.1111/J.1750-3841.2009.01277.X.Search in Google Scholar PubMed

[10] Davidson P, Branen. AL. Naturally occurring compounds. In: Conner DE, editor. New York: Marcel Dek; 1993. p. 441–68.Search in Google Scholar

[11] Adams RP. Junipers of the world: the genus Juniperus. Bloomington, United States of America (USA): Trafford Pub. Co; 2011.Search in Google Scholar

[12] Bellakhdar J. The traditional Moroccan Pharmacopee, ancient Arab medicine and popular knowledge. Paris: IBIS Press; 1998.Search in Google Scholar

[13] Ait-Ouazzou A, Lorán S, Arakrak A, Laglaoui A, Rota C, Herrera A, et al. Evaluation of the chemical composition and antimicrobial activity of Mentha pulegium, Juniperus phoenicea, and Cyperus longus essential oils from Morocco. Food Res Int. 2012;45:313–9. 10.1016/J.FOODRES.2011.09.004.Search in Google Scholar

[14] Abdelfattah EM, Aimad A, Bourhia M, Chebbac K, Salamatullah AM, Soufan W, et al. Insecticidal and antifungal activities of chemically-characterized essential oils from the leaves of Withania frutescens L. Life. 2022;12:88. 10.3390/LIFE12010088.Search in Google Scholar

[15] Jalal Z, El Atki Y, Lyoussi B, Abdellaoui A. Phytochemistry of the essential oil of Melissa officinalis L. growing wild in Morocco: preventive approach against nosocomial infections. Asian Pac J Tropical Biomedicine. 2015;5:458–61.10.1016/j.apjtb.2015.03.003Search in Google Scholar

[16] Moussaoui A, El Bourhia M, Jawhari FZ, Salamatullah AM, Ullah R, Bari A, et al. Chemical profiling, antioxidant, and antimicrobial activity against drug-resistant microbes of essential oil from Withania frutescens L. Appl Sci. 2021;11:5168. 10.3390/APP11115168.Search in Google Scholar

[17] Agour A, Mssillou I, Saghrouchni H, Bari A, Lyoussi B, Derwich E. Tropical journal of natural product research essential oils of Haplophyllum tuberculatum (forsskal) a. juss from Morocco. Trop J Nat Prod Res Available. 2020;4:1108–15. 10.26538/tjnpr/v4i12.13.Search in Google Scholar

[18] Oyaizu M. Studies on products of browning reactions: antioxidative activities of product of browning reaction prepared from glucosamine. Jpn J Nutr. 1986;44:307–15.10.5264/eiyogakuzashi.44.307Search in Google Scholar

[19] Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 1999;269:337–41. 10.1006/ABIO.1999.4019.Search in Google Scholar

[20] Zouirech O, Alyousef AA, El Barnossi A, El Moussaoui A, Bourhia M, Salamatullah AM, et al. Phytochemical analysis and antioxidant, antibacterial, and antifungal effects of essential oil of black caraway (Nigella sativa L.) seeds against drug-resistant clinically pathogenic microorganisms. Biomed Res Int. 2022;2022:1–11. 10.1155/2022/5218950.Search in Google Scholar PubMed PubMed Central

[21] El Barnossi A, Moussaid F, Iraqi Housseini A. Antifungal activity of Bacillus sp. Gn-A11-18isolated from decomposing solid green household waste in water and soil against Candida albicans and Aspergillus Niger. In Proceedings of the E3S Web of Conferences; EDP Sciences. Vol. 150; 2020.10.1051/e3sconf/202015002003Search in Google Scholar

[22] Chebbac K, Ghneim HK, El Moussaoui A, Bourhia M, El Barnossi A, Ouaritini ZB, et al. Antioxidant and antimicrobial activities of chemically-characterized essential oil from Artemisia aragonensis Lam. against drug-resistant microbes. Molecules. 2022;27(3):1136. 10.3390/molecules27031136.Search in Google Scholar PubMed PubMed Central

[23] El Atki Y, Aouam I, El Kamari F, Taroq A, Lyoussi B, Oumokhtar B, et al. Phytochemistry, antioxidant and antibacterial activities of two Moroccan Teucrium polium L. subspecies: preventive approach against nosocomial infections. Arab J Chem. 2019;13(2):3866–74. 10.1016/j.arabjc.2019.04.001.Search in Google Scholar

[24] Sarker SD, Nahar L, Kumarasamy Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods. 2007;42:321–4. 10.1016/j.ymeth.2007.01.006.Search in Google Scholar PubMed PubMed Central

[25] El Moussaoui A, Jawhari FZ, Almehdi AM, Elmsellem H, Fikri Benbrahim K, Bousta D, et al. Antibacterial, antifungal and antioxidant activity of total polyphenols of Withania frutescens L. Bioorg Chem. 2019;93:103337. 10.1016/j.bioorg.2019.103337.Search in Google Scholar PubMed

[26] Chebbac K, Ghneim HK, Moussaoui A, El Bourhia M, El Barnossi A, Ouaritini ZB, et al. Antioxidant and antimicrobial activities of chemically-characterized essential oil from Artemisia aragonensis Lam. against drug-resistant microbes. Molecules. 2022;27(3):1136.10.3390/molecules27031136Search in Google Scholar

[27] Medini H, Elaissi A, Khouja ML, Piras A, Porcedda S, Falconieri D, et al. Chemical composition and antioxidant activity of the essential oil of Juniperus phoenicea L. berries. Nat Prod Res. 2011;25:1695–706. 10.1080/14786419.2010.535168.Search in Google Scholar PubMed

[28] Angioni A, Barra A, Russo MT, Coroneo V, Dessi S, Cabras P. Chemical composition of the essential oils of Juniperus from ripe and unripe berries and leaves and their antimicrobial activity. J Agric food Chem. 2003;51:373–4.10.1021/jf026203jSearch in Google Scholar PubMed

[29] El-Sawi SA, Motawae HM, Ali AM. Chemical composition, cytotoxic activity and antimicrobial activity of essential oils of leaves and berries of Juniperus phoenicea L. grown in Egypt. Afr J Tradit Complement Altern Med. 2007;4:417–26.10.4314/ajtcam.v4i4.31236Search in Google Scholar PubMed PubMed Central

[30] Derwich E, Benziane Z, Boukir A. Chemical composition of leaf essential oil of Juniperus phoenicea and evaluation of its antibacterial activity. Int J Agric Biol. 2010;12(2):199–204.Search in Google Scholar

[31] Ramdani M, Lograda T, Silini H, Zeraib A, Chalard P, Figueredo G, et al. Antibacterial activity of essential oils of Juniperus phoenicea from Eastern Algeria. J Appl Pharm Sci. 2013;3:22–28. 10.7324/JAPS.2013.31105.Search in Google Scholar

[32] Moussaoui A, El, Bourhia M, Jawhari FZ, Khalis H, Chedadi M, Agour A, et al. Responses of Withania frutescens (L.) pauquy (Solanaceae) growing in the Mediterranean area to changes in the environmental conditions: an approach of adaptation. Front Ecol Evol. 2021;470. 10.3389/FEVO.2021.666005.Search in Google Scholar

[33] Popovici C, Saykova I, Tylkowski B. Evaluation de l’activité antioxydant des composés phénoliques par la réactivité avec le radical libre DPPH-Food Safety for Healthy Living View project. Rev génie Ind. 2009;4:25–39.Search in Google Scholar

[34] Lafraxo S, Barnossi A, El, Moussaoui A, El Bourhia M, Salamatullah AM, Alzahrani A, et al. Essential oils from leaves of Juniperus thurifera l., exhibiting antioxidant, antifungal and antibacterial activities against antibiotic-resistant microbes. Hortic. 2022;8:321. 10.3390/HORTICULTURAE8040321.Search in Google Scholar

[35] Lafraxo S, El Moussaoui A, ABin Jardan, El Barnossi Y, Chebaibi A, Baammi MS, et al. GC–MS profiling, in vitro antioxidant, antimicrobial, and in silico NADPH oxidase inhibition studies of essential oil of Juniperus thurifera bark. Evid-based Complement Altern Med. 2022;2022:13. 10.1155/2022/6305672.Search in Google Scholar PubMed PubMed Central

[36] Chun SS, Vattem DA, Lin YT, Shetty K. Phenolic antioxidants from clonal oregano (Origanum vulgare) with antimicrobial activity against Helicobacter pylori. Process Biochem. 2005;40:809–16. 10.1016/j.procbio.2004.02.018.Search in Google Scholar

[37] Fayed SA. Antioxidant and anticancer activities of citrus reticulate (Petitgrain mandarin) and Pelargonium graveolens (Geranium) essential oils. Res J Agric Biol Sci. 2009;5:740–7.Search in Google Scholar

[38] Zhuang SR, Chen SL, Tsai JH, Huang CC, Wu TC, Liu WS, et al. Effect of citronellol and the Chinese medical herb complex on cellular immunity of cancer patients receiving chemotherapy/radiotherapy. Phyther Res. 2009;23:785–90. 10.1002/ptr.Search in Google Scholar

[39] Momtaz S, Abdollahi M. An update on pharmacology of Satureja species; from antioxidant, antimicrobial, antidiabetes and anti-hyperlipidemic to reproductive stimulation. Int J Pharmacol. 2010;6:454–61. 10.3923/IJP.2010.346.353.Search in Google Scholar

[40] Bartosz G. Generation of reactive oxygen species in biological systems. Comm Toxicol. 2003;9:5–21. 10.1080/08865140302420.Search in Google Scholar

[41] Rahhal R, Hajjouji HEL, Gmouh S, Hsaine M, Fougrach H. Chemical composition, antioxidant and antibacterial activities of the essential oils of Juniperus phoenicea, Juniperus thurifera, Juniperus oxycedrus. Mediterr J Chem. 2019;9:190–8.10.13171/mjc93191002145hehSearch in Google Scholar

[42] Bahri F, Harrak R, Achak N, Romane A. Natural product research: formerly natural product letters chemical composition and antibacterial activities of the essential oils isolated from Juniperus thurifera L. var. Nat Prod Res. 2012;27:1789–94.10.1080/14786419.2012.755678Search in Google Scholar PubMed

[43] El Jemli M, Khattabi N, Lachqer K, Touati D, El Jemli Y, Marmouzi I, et al.Antifungal and insecticidal properties of Juniperus thurifera leaves. Nat Prod Commun. 2018;13:1047–9.10.1177/1934578X1801300831Search in Google Scholar

[44] El Abdali Y, Agour A, Allali A, Bourhia M, El Moussaoui A, Eloutassi N, et al. Lavandula dentata L.: phytochemical analysis, antioxidant, antifungal and insecticidal activities of its essential oil. Plants. 2022;11:311. 10.3390/PLANTS11030311.Search in Google Scholar PubMed PubMed Central

[45] Mansouri N, Satrani B, Ghanmi M, El Ghadraoui L, Aafi A, Farah A. Valorization of the essential oils of Moroccan Juniperus thurifera and Juniperus oxycedrus. Phytotherapie. 2010;8:166–70. 10.1007/s10298-010-0550-4.Search in Google Scholar

[46] Agati G, Azzarello E, Pollastri S, Tattini M. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 2012;196:67–76.10.1016/j.plantsci.2012.07.014Search in Google Scholar PubMed

[47] Liang D, Zhou Q, Gong W, Wang Y, Nie Z, He H, et al. Studies on the antioxidant and hepatoprotective activities of polysaccharides from Talinum triangulare. J Ethnopharmacol. 2011;136:316–21. 10.1016/J.JEP.2011.04.047.Search in Google Scholar PubMed

[48] Hybertson BM, Gao B, Bose SK, McCord JM. Oxidative stress in health and disease: the therapeutic potential of Nrf2 activation. Mol Asp Med. 2011;32:234–46. 10.1016/J.MAM.2011.10.006.Search in Google Scholar

[49] Rains JL, Jain SK. Oxidative stress, insulin signaling, and diabetes. Free Radic Biol Med. 2011;50:567–75. 10.1016/J.FREERADBIOMED.2010.12.006.Search in Google Scholar PubMed PubMed Central

[50] Shi Y, Si H, Wang P, Chen S, Shang S, Song Z, et al. Derivatization of natural compound β-pinene enhances its in vitro antifungal activity against plant pathogens. Molecules. 2019;24:3144. 10.3390/molecules24173144.Search in Google Scholar PubMed PubMed Central

[51] Nóbrega JR, Silva DDF, Patricio F, Júnior DA, Mariane P, Sousa S, et al. Antifungal action of α -pinene against Candida spp. isolated from patients with otomycosis and effects of its association with boric acid. Nat Prod Res. 2020;1–4. 10.1080/14786419.2020.1837803.Search in Google Scholar PubMed

[52] Chaves-López C, Usai D, Donadu MG, Serio A, González-Mina RT, Simeoni MC, et al. Potential of Borojoa patinoi cuatrecasas water extract to inhibit nosocomial antibiotic resistant bacteria and cancer cell proliferation in vitro. Food Funct. 2018;9:2725–34. 10.1039/c7fo01542a.Search in Google Scholar PubMed

[53] Ušjak L, Petrović S, Drobac M, Soković M, Stanojković T, Ćirić A, et al. Essential oils of three cow parsnips-composition and activity against nosocomial and foodborne pathogens and food contaminants. Food Funct. 2017;8:278–90. 10.1039/c6fo01698g.Search in Google Scholar PubMed

[54] Yu Z, Tang J, Khare T, Kumar V. The alarming antimicrobial resistance in ESKAPEE pathogens: can essential oils come to the rescue. Fitoterapia. 2020;140:104433. 10.1016/j.fitote.2019.104433.Search in Google Scholar PubMed

Received: 2023-03-08
Revised: 2023-04-14
Accepted: 2023-04-29
Published Online: 2023-06-05

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

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

Downloaded on 29.11.2023 from
Scroll to top button