Promising antioxidant and antimicrobial e ﬀ ects of essential oils extracted from fruits of Juniperus thurifera : In vitro and in silico investigations

: The objective of this work was to characterize the phytochemical composition of essential oil from Juniperus thurifera ( L. ) fruits ( EOFT ) and study its antioxidant, anti - bacterial, and antifungal e ﬀ ects. EOFT was extracted by hydrodistillation and ﬁ ngerprinted by using GC – MS. The antioxidant e ﬀ ect of EOFT was evaluated using 2,2 - diphe - nylpicrylhydrazyl ( DPPH ) , ferric iron reduction assay ( FRAP ) , and total antioxidant capacity ( TAC ) assays. Importantly, the antimicrobial activity of EOFT was performed against Candida albicans, Aspergillus niger, Aspergillus ﬂ avus, Fusarium oxysporum, Staphylococcus aureus, Escherichia coli, Bacillus subtilis , and Proteus mirabilis . In addition, the inhibitory capacity of NADPH oxidase and human acetylcholines - terase was also investigated using molecular docking. The results of the chemical composition reveal that EOFT con - stituted 11 terpenic compounds with dominance of elemol ( 33.86% ) , terpinen - 4 - ol ( 27.80% ) , and cryptomeridiol ( 18.36% ) . The antioxidant power of EOFT recorded IC 50 values of 197.07 ± 0.09 μ g/mL ( DPPH ) and 216.34 ± 0.06 μ g/mL ( FRAP ) , while TAC of EOFT was determined to be 181.06 μ g AAE/mg. The antibacterial potency on solid medium revealed that EOFT induced inhibition zone diameters reaching 14 mm, and a minimum concentration up to 2.78 µg/mL against the stu - died bacterial strains. The EOFT also showed an important antifungal e ﬀ ect as the inhibition reached 42%, and the MIC was between 7.50 and 22.25 µg/mL. The in silico study showed that o - Cymene was the most active molecule against NAD ( P ) H oxidase followed by cadinol with a Glide score of − 5.344 and − 5.143 kcal/mol, respectively. Due to their promising results, the outcome of this work suggests that EOFT could be used as an interesting natural weapon to control microbial and freed radical - related diseases.


Introduction
Plants are used as medicines by a large number of people all over the globe.Traditional Moroccan medicine has been spread all over the country.Over 500 plant species have been identified via ethnopharmacological and ethnobotanical studies undertaken in various regions of Morocco.Herbal medicines are crucial in the development of contemporary medicine, particularly in the ideation of synthetic medications.In recent decades, researchers have made considerable progress in characterizing natural compounds isolated from plants that have biological effects.According to several studies, essential oils have shown strong antibacterial action against various clinically important microbes.These natural products are predicted to solve many issues in microbial resistance, which is overburdening the healthcare system worldwide [1,2].
Juniperus thurifera (L.) is one of the most outstanding species and dominates the Mediterranean basin [3].The Moroccan forest formations extend over an area of a surface of approximately 9 million hectares; Morocco has the most important stands of this species in North Africa with an estimated area of about 30000.00ha [4].At the upper limit of the Thuriferous stands, the vegetation landscapes of Morocco possess various plant groups of thorny xerophytes including the Erinacea anthyllis, Bupleurum spinosum, Arenaria pungens, Astragalus ibrahimianus, Cytisus balansae, and Alyssum spinosum [5,6].Indeed, Thinon and Alifriqui considered that the Thuriferous juniper is the last stage of degradation of tree groups based essentially on oaks, preceding the asylvatism [7].The rest of the Thuriferous forest is in continuous degradation, even extinction due to intensive grazing and excessive illegal and excessive illegal collection of firewood.The absence of natural regeneration of the species reinforces its situation of vulnerability [8].J. thurifera (L.), sometimes known as incense Juniper or Spanish Juniper, is a member of the Cupressaceae family [6].
The plant matures into a dioecious evergreen conifer with scale-like leaves and bluish-black berries.J. thurifera is a valuable resource indigenous to the western Mediterranean basin and has importance over its whole range in terms of ecology, economy, floristry, and medicine [4,9].J. thurifera is also popularly known as "Ar-ar fawah," a plant that grows in the Middle Atlas Mountains and is frequently used in traditional Moroccan medicine for its various therapeutic properties [10,11].
To the best of our knowledge, this is the first report on the phytochemical composition, antioxidant, and antibacterial activities of essential oils extracted from J. thurifera fruit collected in Morocco.Therefore, the main objective of the present work was to determine the phytochemical and antioxidant potential of essential oils from J. thurifera fruits.In addition, we evaluated their antimicrobial activity against clinically important microbes.

Plant material
After being collected in November 2021 in the Eastern Middle Atlas of Morocco (33.68541448 north latitudes and −4.30734951 west longitude), J. thurifera was identified under reference FJT/02D20 by a botanist from Sidi Mohammed Ben Abdellah University.

Extraction of essential oils
EOFT was extracted using a Clevenger apparatus for 3 h.The essential obtained EOs were kept in the refrigerator (+4°C) in shaded and well-sealed tubes.
The yield of essential oil was (%) defined as the proportion of the quantity of oil in grams to the quantity of plant material in grams.
Quantity of oil in g Quantity of plant material in g 100.

Antioxidant activity of EOFT using DPPH radical
In this assay, the antioxidants reduce the purple-colored diphenyl picryl-hydrazyl to a yellow compound, the intensity of which is inversely proportional to the ability of the antioxidants present in the medium to donate protons.Briefly, 100 μL of the EOFT was added to 1,300 μL DPPH (0.004% prepared in methanol).In parallel, the negative control was made under identical circumstances.The reading of absorbance was done at 517 nm following 30 min of incubation at room temperature and darkness.The ascorbic acid solution was used as a positive control, which was measured under identical circumstances as the samples, and the test was conducted three times for each concentration.Using the equation below, the free radical scavenging activity is estimated [13]: Abs control Abs sample Abs control 100.
The concentration of the EO needed to neutralize 50% of the free radicals (IC 50 ) was determined graphically by linear regression.

Antioxidant activity of EOFT using ferric iron reduction assay (FRAP)
The FRAP assay is used to investigate the reduction potential of EOFT.Briefly, 1% K 3 [Fe(CN) 6 ] and 1 mL of phosphate buffer (0.2 M; pH = 6.6) are combined with 0.4 mL of the EOFT in various concentrations.About 1 mL of 10% trichloroacetic acid was added to the mixture before being incubated at 50°C for 30 min.Subsequently, the tubes were centrifuged at 3,000 rcf for 10 min.After combining 1 mL of the supernatant from each tube with 0.2 mL of a 0.1% FeCl 3 solution and letting them sit in the dark for 30 min, the absorbance at 700 nm was measured.The absorbance of the positive control (BHT) was measured under the same conditions as the EOFT [14].

Antioxidant activity of EOFT using total antioxidant capacity (TAC)
The phosphomolybdenum technique was used to calculate the TAC of EOFT.Briefly, 1,000 µL of a mixture of H 2 SO 4 , Na 2 PO 4 , and ammonium molybdate reagent was added to 100 µL of various concentrations of EOFT.These concentrations were in the range of 0.6 M, 28 mM, and 4 mM, respectively.For 90 min, the tubes were kept at a temperature of roughly 95°C.The absorbance was measured at 695 nm after cooling.The control contained 1,000 µL of reagent mixture and 100 µL of methanol [15].The settings for incubating samples and controls are the same.The data are displayed in terms of ascorbic acid equivalents per gram (µg EAA/g).

Antimicrobial activity 2.6.1 Microbial strains used for testing
The antimicrobial activity of EOFT was performed against four fungal strains which are Candida albicans ATCC10231, Aspergillus niger MTCC282, Aspergillus flavus MTCC9606, and Fusarium oxysporum MTCC9913, and four bacterial strains which are Staphylococcus aureus ATCC6633, Escherichia coli K12, Bacillus subtilis DSM6333, and Proteus mirabilis ATCC29906.

Method for the evaluation of antimicrobial activity
The antibacterial investigation of the EOFT was evaluated using the disk diffusion method [16].The four bacterial strains and C. albicans were inoculated into Petri dishes containing malt extract (ME) and Mueller-Hinton (MH) culture media, respectively, using the double-layer technique.Decimal dilutions in sterile saline (0.9% NaCl) were produced from the fresh cultures grown on MH and ME media until a turbidity of 0.5 McFarland (10 6 -10 8 CFU/mL) was reached.Following that, 100 µL was transferred to tubes containing 5 mL of soft agar (0.5% agar), and the infected tubes were then emptied into Petri dishes with MH and EM medium.Whatman No. 4 paper discs of 6 mm were impregnated with 20 µL of EOFT diluted 1/5 in 10% DMSO.The antifungal activity of A. oxysporum, A. flavus, and A. niger was established using the confrontation technique in an EM medium between the tested EOFT and the fungi.In summary, 20 µL (1/5 dilution in 10% DMSO) of EOFT was applied to 6 mm diameter Whatman No. 4 paper discs, and an agar plate with the fungal strain was placed 1 cm away from the disc containing EOFT.Oxacillin for bacterial strains and fluconazole for fungal strains were used in the same way as the tests to evaluate the efficacy of EOFT positive controls and negative controls with conventional antibiotics.Petri plates containing bacteria and fungus were incubated at 37 and 30°C, respectively, for strains of bacteria, C. albicans, and filamentous fungi [17].
Inhibition diameters and inhibition percentages were calculated 24 h post-inoculation (hpi) for bacterial strains, 48 hpi for C. albicans, and 7 days post-inoculation for F. oxysporum, A. niger, and A. flavus [18].

Minimum inhibitory concentration (MIC)
The microdilution method, as described previously [18], was used to determine the MIC of EOFT against the four bacterial and four fungal strains.In short, sterile 96-well microplates were labeled, 100 µL of EOFT was pipetted into the first column of the plate, 50 µL of sterile MH for bacterial strains and 50 mL of sterile EM for fungal strains were added to the remaining wells, serial dilutions were made using a multichannel pipette, and finally 30 µL of microbial suspension of each strain (10 6 -10 8 CFU/mL) was added to all wells.After incubating 24 h for bacteria, 48 h for C. albicans, and 7 days for A. niger, A. flavus and F. oxysporum at 37 and 30°C, respectively, the MIC was determined using the colorimetric method (TTC 0.2% (w/v)) [19,20].

Molecular docking
Essential oil components of J. thurifera fruit were obtained in SDF format from the PubChem database.Following that, compounds were prepared for docking using the OPLS3 force field and the Schrödinger Software LigPrep tool.After considering various ionization states, 32 stereoisomers were created for each ligand at pH 7.0 ± 2.0.Using the RCSB Protein Data Bank, three-dimensional (3D) crystal structures of several enzymes, including human acetylcholinesterase (PDB ID: 4EY7), E. coli receptor (eta-ketoacyl-acyl carrier protein) (PDB ID: 1FJ4), S. aureus nucleoside diphosphatase (PDB ID: 3Q8U), NAD(P)H oxidase (PDB ID: 2CDU), and others, were determined.The protein preparation wizard in Schrödinger-Maestro v.11.5 was used to create the structures.All the water was eliminated once selenomethionines were changed into methionines.All the water was removed after selenomethionines were transformed into methionines, and heavy atoms containing hydrogen were then added.Charges and bond ordering were also assigned using the OPLS3 force field, and the root mean square displacement of heavy atoms was reduced to 0.30.The receptor grid was automatically created by clicking on any ligand atom.The volumetric space needed was 20 20 20.In the Glide package of Schrödinger-Maestro v.11.5 during SP flexible ligand docking, the non-cis/trans amide bond was penalized.The charge threshold was set at 0.150 and the van der Waals scaling factor was set at 0.80.The Glide score represented the final scoring, which was based on energy-efficient postures.For each ligand, the best-docked pose with the lowest Glide score value was noted.

Statistical analysis
All findings were presented as the means of studies done in triplicate with standard deviation.The significance of the difference between the means was assessed using analysis of variance (two-way ANOVA).GraphPad Prism 9 was used to conduct Tukey's multiple range tests at p < 0.05.

Chemical composition of essential oils of J. thurifera
The essential oil obtained from the fruit of the J. thurifera, analyzed by GC-MS/MS, allowed us to identify 11 compounds, which represent 100% of the total mass of essential oil.The results are shown in Table 1.The major compounds identified are elemol (33.86%), terpinen-4-ol (27.80%), cryptomeridiol (18.36%), and selinenol (6.24%).It should be noted that the main components observed in this species are oxygenated sesquiterpenes (64.53%), followed by oxygenated monoterpenes (28.89%) and hydrocarbon monoterpenes (6.58%) (Figure 1).

Antioxidant activity
The findings showed that EOFT inhibited the DPPH radical in a dose-dependent manner (Figure 2a).Notably, the antioxidant potential of EOFT was compared to the activity of the reference antiradical compounds such as BHT and quercetin.The results obtained, expressed in terms of 50% radical inhibitory concentration (IC 50 ), showed that EOFT had a significant antioxidant capacity when compared with that of BHT and quercetin, which were 197.07 ± 0.09, 153.81 ± 0.001, and 189.47 ± 0.001 µg/mL, respectively.The results of the reducing power of EOFT, which was tested by using FRAP test are presented in Figure 2b.Findings showed that EOFT exhibited a dose-dependent reducing power.In this aspect, the EC 50 of the antioxidant capacity of EOFT, BHT, and quercetin are 216.34± 0.06, 256.52 ± 0.001, and 240.06 ± 0.001 μg/mL, respectively.EOFT also showed an important TAC, which was determined to be 181.06μg AAE/mg, while BHT and quercetin recorded 263 and 312 μg AAE/mg, respectively.

Antibacterial activity
The results demonstrated that EOFT had important antibacterial activity vs all bacteria used for testing (Table 2).Notably, large inhibition zones were recorded vs bacteria treated with EOFT.However, these microorganisms were found resistant to almost all oxacillin used as a commercial drug (p < 0.05).The MIC results of the EOFT antibacterial tests are summarized in Table 3.
In general, the most sensitive strains were found to be S. aureus and B. subtilis with MICs in the range of 2.78 ± 0.00 and 2.78 ± 0.00 µg/mL, respectively.The EOFT revealed a low activity vs E. coli K12 with a MIC of 5.57 ± 0.00 µg/mL.Furthermore, the sensitivity of P. mirabilis to EOFT was moderate when compared to the other strains with a MIC of 11.12 ± 0.00 µg/mL.Nevertheless, these bacteria were found to be resistant to oxacillin used as a drug reference, except in the case of E. coli K12 which was found to be sensitive to MIC in the range of 3.12 ± 0.00 µg/mL.

Antifungal activity
The results recorded for the antifungal activity of the EOFT are grouped in Table 4. Importantly, EOFT was active against C. albicans with an inhibition diameter of 16.33 ± 1.52 mm and F. oxysporum with an inhibition percentage of 42.00 ± 0.66%.However, EOFT was found to be inactive against A. niger and A. flavus.
The MIC results of the antifungal tests are summarized in Table 5.From this table, it can be seen that the most sensitive fungus to EOFT was C. albicans whose growth was strongly inhibited at 2.78 ± 0.00 µg/mL, while F. oxysporum was inhibited at 122.25 ± 0.00 µg/mL of Antioxidant and antimicrobial effects of essential oils from J. thurifera  5 EOFT.It is noted that both A. niger and A. flavus species were shown to be resistant to EOFT (Figure 3).

Molecular docking
For the evaluation of the antioxidant effect of EOFT using the molecular docking approach, we chose the inhibitory power of NADPH oxidase and human acetylcholinesterase.In the inhibition of NADPH, all the molecules studied presented an activity of inhibition ranging from −3.419 to −5.344 kcal/mol (Table 6).o-Cymene and cadinol were the most active molecules with a Glide score of −5.344 and −5.143 kcal/mol, respectively.Furthermore, arctiol was the most active molecules against human acetylcholinesterase followed by terpinen-4-ol with a Glide order of −6.75 and −6.377 kcal/mol, respectively (Table 6).Twodimensional (2D) and 3D viewers of the interaction between o-Cymene and the active sites of NADPH oxidase showed the formation of a PI-PI stacking type bond with the residue LYS 213, while the interaction between arctiol and the active site of human acetylcholinesterase presented the formation of hydrogen bonds with the residue TYR 124 (Figure 4).In microbial activity, alpha-terpineol was the most active molecule against E. coli receptor (eta-ketoacylacyl carrier protein) synthase followed by thujene with a Glide score of −6.313 and −6.156 kcal/mol, respectively (Table 6).Moreover, terpinen-4-ol and cadinol were the most active molecules with a Glide score of −6.008 and −5.714 kcal/mol against S. aureus nucleoside diphosphatase.The interaction between alpha-terpineol and the active site of E. coli receptor (eta-ketoacyl-acyl carrier protein) synthase showed the formation of a hydrogen bond with the residue THR 300, while the interaction between cadinol and the active site of S. aureus nucleoside diphosphatase showed the formation of a hydrogen bond with the residue VAL 109 (Figure 4).

Discussion
The synthesis of essential oils in J. thurifera takes place in various secretory tissues present in all the organs of the plant: leaves and also in bark and fruit.However, essential oils are highly variable both in terms of yield and composition [21].In the present work, the fruits of J. thurifera provided an essential oil yield of about 0.34% compared to 0.89% for the bark in a previous study [10], while in a study on the leaves, J. thurifera leaves provided an essential oil yield of about 0.96% [11].The chemical composition characterized by GC-MS/MS showed the richness of EOFT in elemol (33.86%), terpinen-4-ol (27.80%), and cryptomeridiol (18.36%) as the major components.For comparison, the chemical composition of EO obtained from the leaves of J. thurifera was found to be rich in α-thujene (25%), elemol (12%), and muurolol (12%) [11].The chemical composition of the EOs of the three studied parts of J. thurifera (bark, leaves, and fruit) was qualitatively and quantitatively different in terms of compounds.On the other hand, the essential oil of the bark was found to possess seven identical compounds, a dominant of eudesmane type cryptomeridol (37.02%), followed by a cadinan type muurol (36.31%) and a eudesmane type sesquiterpenoid, elemol [10].
Importantly, several factors can influence the yield and composition of essentials including intrinsic and extrinsic factors, the composition of secretory structures, and extraction and storage conditions [21][22][23][24].Generally, few plants give EOs of similar composition for their different organs (flowers, leaves).This difference in composition can be partly explained by the existence of different secretory structures in the same plant [23].Indeed, plants with internal secretory structures are characterized by a stable yield of essential oil [25].In addition to annual and monthly variations, there may also be daily fluctuations that appear to be related to pollinator activity [26].
The results of the antioxidant effect showed that EOFT had a very interesting antioxidant capacity.This free radical-reducing capacity of these oils may be due to their chemical profiles, which were found to be rich in phenols that act as reducing agents, hydrogen, and S. aureu  Alphaterpineol singular oxygen donors [27].However, the antioxidant activity of different parts tested separately (bark and leaves) showed a higher antioxidant capacity than that recorded for the essential oil obtained from the fruit [10,11].Inhibitory concentration values (IC 50 ) of EOFT was 197.07 ± 0.09, which is comparable to that of EO obtained from the bark (IC 50 = 21.25 ± 1.02 μg/mL) and leaves (IC 50 = 23.6 ± 0.71 µg/mL) [4,9].The FRAP (ferric reducing capacity of antioxidants) method revealed that EOFT had important antioxidant efficiency (EC 50 = 216.34± 0.06), which is comparable to that recorded for bark (190 ± 1.0 µg/mL).The TAC results showed that EOFT had an important TAC, which is determined to be 181.06μg AAE/mg, higher than that of the essential oils of the bark, 271 μg AAE/mg [10,11].It can be suggested that there is a correlation between these antioxidant activities and the molecules with antioxidant activity in the studied oils.We studied the antimicrobial power of EOFT using the disc diffusion method and MIC assays.The results we found showed a remarkable antimicrobial effect of this oil.Notably, a potent antibacterial effect was recorded against S. aureus, E. coli, B. subtilis, and P. mirabilis with MIC in the range of 2.78 ± 0.00, 5.57 ± 0.00, and 2.78 ± 0.00 and 11.125 ± 0.00 µg/mL, respectively.S. aureus and B. subtilis were the most sensitive strains to the action of the essential oil with a MIC of 2.78 ± 0.00 µg/mL.Importantly, the MICs recorded for EO of J. thurifera bark against S. aureus, E. coli, B. subtilis, and P. mirabilis were in the order of 1.34 ± 0.00, 0.67 ± 0.00, 2.69 ± 0.00, and 1.34 ± 0. 00 mg/mL, which are comparable to MIC recorded for leaves 0.095 ± 0.00, 0.095 ± 0.00, 0.095 ± 0.00, and 0.0475 ± 0.00 µg/mL, respectively.
As for the antifungal activity, the fungal strains C. albicans and F. oxysporum were sensitive to the action of the essential oil.Notably, a remarkable activity was detected, reflected by MIC ranging from 2.78 to 22.25 µg/mL, respectively.However, A. niger and A. flavus were resistant to EOFT.EOs from J. thurifera bark was found to be active against C. albicans, A. niger, and F. oxysporum strains and resulted in MICs of 0.67, 10.75, and 6.45 mg/mL, respectively; A. flavus was resistant, which is in agreement with our findings [10,11].Furthermore, the essential oils extracted from the leaves of this plant showed antifungal activity against C. albicans and F. oxysporum with MICs of 0.095 ± 0.00 and 0.095 ± 0.00 µg/mL, respectively, while A. niger and A. flavus were resistant.
This remarkable antimicrobial activity of the EOFT against some bacterial strains can be attributed largely to the presence of elemol and terpinen-4-ol as major compounds in this essential oil.The latter are widely studied for their interesting biological activities.The bactericidal potential of elemol and terpinen-4-ol has been reported [28,29].Other studies showed the inhibitory potential of elemol and terpinen-4-ol on the growth of some fungal strains [30,31].The antimicrobial mode of action of essential oils is non-specific due to the complexity of their phytocompounds [32].The hydrophobic character of essential oils enhances their action.As a result, several cellular targets are envisaged, the membrane being their preferred target.Generally, it is reported that the presence of phenols, aldehydes, and alcohols in essential oils is at the origin of this activity [33,34].

Conclusion
Results reported here may constitute a scientific justification for the use of these plants in traditional pharmacopeia in the treatment of infectious diseases and once again confirm the relevance of traditional remedies.However, toxicity on non-target organisms is required before any potential applications.The essential oils studied can potentially be used as a natural antimicrobial and antioxidant agent against infectious diseases in humans and for the preservation of food products.The development of such natural agents will also help to solve environmental problems caused by synthetic products and drugs such as pollution and resistance of certain microorganisms.

Figure 3 :
Figure 3: Pictures showing the antimicrobial activity of EOFT.

Figure 4 :
Figure 4: Interactions between ligands and active sites are shown in a 2D view: (a) interactions between o-Cymene and the 2CDU active site, (b) interactions between arctiol and the 4EY7 active site, (c) interactions between the active site of 1FJ4 and alpha-terpineol, and (d) interactions between the active site of 3Q8U and terpinen-4-ol.

Table 4 :
Antifungal activity of EOFTValues with different letters in the same column, the mean values (SD, n = 3) are noticeably different (two-way ANOVA; Tukey's test, p < 0.05).

Table 3 :
MIC of antibacterial activity of EOFT in µg/mL

Table 5 :
MIC of antifungal activity of EOFT (µg/mL)Values with different letters in the same column, the mean values (SD, n = 3) are noticeably different (two-way ANOVA; Tukey's test, p < 0.05).