GC-MS Profiling, In Vitro Antioxidant, Antimicrobial, and In Silico NADPH Oxidase Inhibition Studies of Essential Oil of Juniperus thurifera Bark

Juniperus thurifera is a native species to the mountains of the western Mediterranean region. It is used in traditional medicine as a natural treatment against infections. The present study aimed to carry out the chemical analysis and evaluate the antioxidant, antimicrobial, as well as in silico inhibition studies of the essential oils from Juniperus thurifera bark (EOEJT). Chemical characterization of EOEJT was done by gas chromatography (GC-MS). We have performed three antioxidant assays (Reducing power (FRAP), 2, 2-diphenylpicrylhydrazyl (DPPH), and total antioxidant capacity (TAC)) of the EOEJT. We next evaluated the antimicrobial activity against in silico study, which was carried out to help evaluate the inhibitory effect of EOEJT against NADPH oxidase. Results of the GC/MS analysis revealed seven major compounds in EOEJT wherein muurolol (36%) and elemol (26%) were the major components. Moreover, EOEJT possessed interesting antioxidant potential with an IC50 respectively of 21.25 ± 1.02 μg/mL, 481.02 ± 5.25 μg/mL, and 271 μg EAA/mg in DPPH, FRAP, and total antioxidant capacity systems. Molecular docking of EOEJT in NADPH oxidase active site showed inhibitory activity of α-cadinol and muurolol with a glide score of −6.041 and −5.956 Kcal/mol, respectively. As regards the antibacterial and antifungal capacities, EOEJT was active against all tested bacteria and all fungi, notably, against Escherichia coli K12 with an inhibition diameter of 21 mm and a MIC value of 0.67 mg/mL, as well as against Proteus mirabilis ATCC 29906 with an inhibition diameter of 18.33 ± 1.15 mm and a MIC value of 1.34 mg/mL. A more pronounced effect was recorded for the fungal pathogens Fusarium oxysporum MTCC 9913 with inhibition of 37.44 ± 0.28% and MIC value of 6.45 mg/mL, as well as against Candida albicans ATCC 10231 with an inhibition diameter of 20.33 ± 1.15 mm and a MIC value of 0.67 ± 0.00 mg/mL. Altogether, these results highlight the importance of EOEJT as a source of natural antibacterial and antioxidant drugs to fight clinically important pathogenic strains.


Introduction
Morocco is the most biodiverse country in the western Mediterranean region [1]. Within it, the eastern half of the forest formations of the Middle Atlas is a veritable mosaic, with a wide range of forest species including cedar, holm oak, Phoenician juniper as well as Juniperus thurifera [2].
e Bouiblane site made up of two distinct regions (Bouiblane I and Bouiblane II) is part of the Sites of Biological and Ecological Interest (SBEI) of the Moroccan Eastern Middle Atlas. is locality stands out by its different agro-climatic conditions, its floristic diversity, and its significant but unquantified rate of endemism, as well as its wide range of plant forms [3]. Juniperus thurifera is a variably sized tree that belongs to the family Cupressaceae.
is species is native to mountainous areas of the Mediterranean basin's western region [4]. In Morocco, Juniperus thurifera grows more particularly in the High and Middle-Atlas Mountains [5,6]. e two arboreal plants that can grow with it are Quercus ilex and Cedrus atlantica. Such hardy species may tolerate a wide range of weather conditions and may reach up to 19 meters tall and live for up to 800 years. e thurifer covers an estimated area of almost 20 000 hectares. It is the most regressed forest species in Morocco, with a 90 percent regression from its potential range [7]. Different phytoconstituents have been isolated from this species, and include mainly polyphenols, essential oils, and sterols [8,9]. Such metabolites exhibited potential bioactivities such as anti-inflammatory, and antibacterial agents among other wide therapeutic applications [8][9][10].
Multidrug-resistant organisms have become a major concern and the situation is far from being mastered. erefore, the research for antibacterial from natural origins has become a vivid area of exploration. Essential oils are combinations of volatile secondary metabolites that occur naturally in plants, which mostly represent the various smells and scents of a variety of plants [10]. A myriad of published reports has provided evidence of their potent bioactivities as antioxidants, antibacterials, antifungals, and insecticides [8][9][10][11][12].
is study aimed at characterizing the bark essential oils of Juniperus thurifera by GC/MS. Next, we conducted the antioxidant potential of essential oils in vitro and explore in silico as NADPH oxidase inhibitors. Finally, the antibacterial and antifungal effects against drug-resistant microbial strains were also described. (Figure 1) was collected from a mountainous area in the Middle Atlas, Morocco before being identified by a botanist and given the voucher number (FJT/02D20). Next, the bark was isolated from the plant branches, and then cleaned and dried by use of an oven at 35°C before grinding into powder by use of an electric grinder [13].

Extraction of EOEJT.
Using a Clevenger device, 200 g of the plant powder was immersed in 800 mL of water before being mixed and heated at boiling temperature for two hours. Next, the essential oils (EOEJT) were recovered and placed at 4°C until further use [13,14].

Chromatographic Analysis of EOEJT by GC/MS.
Analysis was done using a gas chromatograph (GCMS-TQ8040 NX Shimadzu brand) with an apolar capillary column (RTxi-5 Sil MS-30.00 m × 0.250 mm ID × 0.250 μm Film thickness). One microliter of EOEJT was used for chromatographic analysis after being diluted in hexane (10 : 100 dilution). e oven temperature program was initially set to 40°C for 2 minutes and followed by 260°C for 10 minutes, then increased by 5°C/min up to 280°C. Finally, the temperature was held at 280°C for 10 minutes. e carrier gas was nitrogen and the flow rate was 1 mL/min. e temperatures of the injector and detector were adjusted to 250 and 280°C. e ionization energy was set to 70 eV, the ion source temperature was set at 200°C, the interface line temperature was 280°C, and the scan mass range was m/z 40-650.00. Volatile compounds of essential oil were identified by the use of the Kovats index and NIST-MS Search Version 2.0 [13,15].

Antioxidant Activity of EOEJT.
e antioxidant capacity of EOEJTs was assayed by the use of three systems DPPH, TAC test, and FRAP, respectively. EOEJTs were prepared in a dilution series from 1 μg/mL to 1000 μg/mL in methanol; thus, positive controls of quercetin and BHT were used in this evaluation and were prepared under the same conditions as the EOEJTs.

DPPH Test.
Free radical powder was prepared in methanol [16,17]. Briefly, 0.004 mg in 100 mL. 100 μL of the sample (EOEJT, BHT, and quercetin) were diluted in methanol before being added to 750 μL DPPH, while a solution consisting of 750 μL DPPH and 100 μL methanol was used as a negative control. e reaction media were 2 Evidence-Based Complementary and Alternative Medicine incubated for 25 minutes, and the optical density (DO) was measured at λ � 517 nm. e antioxidant efficiency was determined according to the following formula: EA: antioxidant efficiency in percent (%); Ds: optical density of sample; Ct: optical density of negative control (White).

FRAP Test.
is test was assayed by preparing a reaction medium using phosphate buffer with 200 mM-pH � 6.6 and K 3 Fe(CN)6 (1%) [14,18]. Briefly, 200 µL of each solution was mixed with 50 μL EOEJT before being incubated at 50°C for 30 minutes. To prepare for analysis, the sample was remixed with 200 μL of trichloroacetic acid (10 mg/mL), 500 μL of distilled water (H 2 O), and 100 μL of FeCl 3 (1 mg/mL). At 700 nm, absorbance was measured against a negative control (50 μL of methanol without EOEJT). e EC-50 effective concentration was used to present the results.

TAC Test by Use of Phosphomolybdenum Method.
Briefly, EOEJT was combined with 1 mL of a reagent solution before being heated at 95°C for one hour and then cooled at room temperature for 20 minutes. e optical density was measured at λ � 695 nm against a negative control containing 25 μL of methanol without EOEJT, while ascorbic acid was used as a positive reference. Results of the TAC assay were expressed in micrograms equivalent to ascorbic acid per milligram of the sample (μg AAE/mg) [19].

Method for Assessing Antimicrobial Capacity.
e disk diffusion assay was used to determine whether or not EOEJT has antimicrobial capabilities [20]. In a nutshell, the double-layer approach was used in order to inoculate bacterial and fungal strains onto Petri plates that contained Mueller-Hinton (MH) agar and malt extract (ME), respectively. e inoculum was prepared from fresh cultures grown in MH and ME media with (0.9% NaCl). Next, Wattman paper discs measuring sex millimeters in diameter were immersed in 20 μL of EOEJT before being placed on Petri dishes surfaces that were previously inoculated with bacteria (10 6 to 10 8 CFU/mL) and fungi. Erythromycin and fluconazole served as positive controls, respectively, for bacteria and fungi. Subsequently, Petri dishes inoculated with bacteria and fungi were meticulously incubated at 37°C and 30°C for bacterial and fungi strains, respectively [21]. After 24 hours and 48 hours postinoculation, respectively, inhibition diameters and inhibition percentages were computed for bacterial strains and C. albicans. However, after 7 days postinoculation, these calculations were performed for A. flavus, F. oxysporum, and A. niger [13].

Minimum Inhibitory Concentration (MIC).
e MIC of EOEJT against microbial strains was determined using the microdilution method [22]. Briefly, the microplates were prepared in sterile conditions, each sterile 96-well microplate was labeled, and then a 100 μL of EOEJT with a 1 : 10 (V/V) dilution of DMSO (10 %) was pipetted into the first column of the plate to perform a series of dilution using sterile MH for bacteria and sterile ME for fungi, and lastly, 30 μL of microbial suspension of each strain (108 CFU/mL) was added to wells. After an incubation period ranging from 48 hours to 7 days for fungi and 24 hours for bacteria at 37°C [13,22], the MIC was ascertained by using the colorimetric method (TTC 0.2%), [22,23].

Ligand Preparations.
For ligand preparations, we used the PubChem database to download all the molecules identified by GC/MS in EOEJT in SDF format. Next, the OPLS3 force field was applied, and the LigPrep tool in Schrödinger's Maestro 11.5 software was used to prepare the structures. Based on the ionization states at pH 7.0 2.0, a total of 32 stereoisomers were generated for each ligand.

Protein Preparation.
For receptor preparation, the three-dimensional crystal structure of NADPH oxidase was downloaded from the PDB data bank in PDB format. Protein Preparation Wizard of Schrödinger-Maestro v11.5 was used to add hydrogen atoms to heavy atoms, transform selenomethionine into methionine, and remove water. Next, the OPLS3 force was used for minimization, and fixing the maximum heavy atoms RMSD (root mean square deviation) at 0.30Å [24].

Receptor Grid Generation.
e receptor grid was set at the following coordinates: X � 17.584, Y � 9.05, and Z � 51.541. When the volumetric spacing performed was

Receptor Grid
Generation. e receptor grid was set at the following coordinates: X � 17.584, Y � 9.05, and Z � 51.541. When the volumetric spacing performed was 20 × 20 × 20, the glide of Schrödinger-Maestro v11.5 was used for SP flexible ligand docking. e ligand was coupled to the grid box produced from protein using "Standard precision" (SP), while SP GScore was used to evaluate the results [24].

Glide Standard Precision (SP) Ligand Docking.
In ligand docking, the penalties were applied to noncis/trans amide bonds. e partial charge cutoff was set to 0.15 and the Van der Waals scaling factor and partial charge cutoff were set to 0.80 for each ligand atom, e energy-minimized poses presented by the glide score were used to calculate the final score. e best-docked pose with the lowest glide score value was recorded [25].

Statistical Analysis
Data were all provided as the mean value and standard deviation of tests performed in triplicate. GraphPad Prism was used to perform statistical analysis. Levine's test was used to verify homogeneity, while Shapiro-Wilks test was used to verify normality. Analysis of variance (ANOVA) flowed by Tukey's HSD test was used to arrange multiple comparisons. p values lower than 0.05 were regarded to indicate a statistically significant difference.

Phytochemical Identification of EOEJTs by GC/MS.
e yield of EOEJTs was about 0.89%, and it is somewhat comparable to what was previously found in the leaves (0.96%) of Juniperus thurifera [13]. Seven compounds were identified with a dominance of eudesmane-type cryptomeridiol (37.02%) followed by a cadinane-type muurolol (36.31%) and elemane-type sesquiterpenoid which is elemol (26.93%). e representative GC-MS total ion chromatography (TIC) of EOEJT from the bark of Juniperus thurifera is shown in Figure 2 and Table 1.
Total antioxidant power was determined using the ammonium phosphomolybdate (TAC) method. Results showed that EOEJT had a good antioxidant capacity since it scored 271 μg AAE/mg, while PHT used as positive control scored 263 μg EAA/mg (Figure 3(e)). ese results were comparable to EOs from Dittrichia viscosa, which revealed 192 μg AAE/ mg as TAC [26]. In addition, the EOs of the leaves of Lavandula dentata scored 81.28 μg AAE/mg [27]. EOs from Withania frutescens also scored 91 μg AAE/mg [14].

Antibacterial Capacity.
EOEJT showed promising antibacterial results when compared to erythromycin, particularly vs. E. coli wherein we noted an inhibition zone diameter of 21 ± 0.00 mm and MIC of 0.67 ± 0.00 mg/mL, and against P. mirabilis with an inhibition diameter of 18.33 ± 1.15 mm and a MIC of 1.34 ± 0.00 mg/mL (Figure 4, Figure 5 and Table 2). e antibacterial activity of EOEJT may be due to their physicochemical compositions, most importantly the presence of bioactive molecules such as muurolol, elemol, and pinene.

Antifungal Capacity.
For antifungal susceptibility testing, in vitro evaluation of EOEJT against harmful fungal strains was conducted by the use of the diffusion assay. We found a potent antifungal potency with an inhibition percentage of 37.44 ± 0.28% and MIC value of 6.45 ± 0.00 mg/ mL against F. oxysporum compared to fluconazole ( Figure 6 and Table 3). Similarly, EOEJT exhibited significant activity against C. albicans with an inhibition zone diameter of 20.33 ± 1.15 mm and a MIC value of 0.67 ± 0.00 mg/mL (Table 3). Besides, EOEJT showed a fungistatic activity vs. A. niger and F. oxusporum and a fungicidal activity vs. C. albicans. e antimicrobial effect of EOEJT was significant in comparison with one bacterial (erythromycin) and one fungal (fluconazole) specific antimicrobials. e results showed that the EOEJT test was more effective against Gram-negative or Gram-positive bacterial strains. Moreover, the principal component analysis (Figure 7) indicated that all bacterial and fungal strains used for testing showed almost similar sensitivities to EOEJT except for A. flavus, which was resistant to both EOEJT and fluconazole.   Evidence-Based Complementary and Alternative Medicine       (Table 4).

Molecular
Regarding the nature of the bonds between EOEJT and the active site of NADPH oxidase, the molecules which present lower binding energy include α-cadinol, c-eudesmol, and muurolol, each one, establishing two hydrogen bonds with the ASP 282 and LYS 134 residues and elemol which formed a single hydrogen bond with residue ASP 282 (Figures 8 and 9).

Discussion
In the present work, EOs extracted from Juniperus thurifera were evaluated for their chemical composition, antioxidant, and antimicrobial effects as no previous work dealing with these pharmacological activities of EOs extracted from Juniperus thurifera grows in Morocco to the best of our knowledge. Regarding EOEJT, Juniperus thurifera leaves were more qualitatively richer in terpenic compounds and identified 31 compounds with a dominance of α-thujene (25%), elemol (12%), and muurolol (12%) [13]. Previously, 24 compounds were reported to account for 99.46% of the mass of Juniperus thurifera EOs [28]. Such differences may be due to climatic factors variation (altitude latitude, substrate, etc.), harvest period, organs explored (leaves, stem, and bark), as well as the extraction method used [29]. e assessment of oxidative stress is becoming increasingly significant since this particular type of oxidation has been connected to a wide range of health issues, including rheumatoid arthritis, atherosclerosis, diabetes, cancer, and aging [30][31][32]. It has been reported that the hydroxyl function present in the phytochemical compositions of EOs is responsible for their antioxidant capacities.
Terpenes and phenolic components in EOs are strong antioxidant agents [33][34][35][36][37][38], which is in agreement with our study. e phytochemical diversity in EOTJ (Table 1), may be responsible for the antioxidant efficiency, whether major or minor compounds may work in synergistic ways [16]. Recent studies have shown the richness of Juniperus thurifera of terpinene, which is considered among the compounds that have been shown to increase the antioxidant power [39].
Concerning the antibacterial power, taking a closer look at the published literature supported that muurolol exerted significant antibacterial activity against pathogenic bacteria [40]. Elemol compound, on the other side, has also shown some high biological activity [41]. e active ingredient pinene exhibited significant antibacterial capacity against P. aeruginosa and other multidrug-resistant bacteria [42,43]. More particularly, it also had strong antibacterial activity against S. aureus and E. coli strains [44]. e current results were in agreement with the results reported by Rahhal et al. [45], who showed that Juniperus thurifera essential oils exhibited substantial antibacterial capacity against bacteria strains, particularly against S. aureus (31.12 ± 3.11 mm of inhibition), E. coli (13.23 ± 2.59 mm of inhibition), and P. aeruginosa (18.27 ± 2.29 mm of inhibition). Our results are also in agreement with several other studies such as the study by Zeraib et al. [46], which reported that S. aureus was highly sensitive to the EO of Algerian Juniperus thurifera, and also the study by Bahri et al. [47] demonstrated that the essential oil of Juniperus thurifera had potent antibacterial activity against S. aureus ATCC 33862 (inhibition zone diameter: 27 mm; minimum inhibitory concentration: 450 μg/mL), E. coli ATCC 25922 (inhibition zone diameter: 25.6 mm; minimum inhibitory concentration: 530 μL/mL), and P. mirabilis ATCC 7002 (inhibition zone diameter: 18.8 mm).
with different letters in the same column are significantly different (Tow-way ANOVA; Tukey's test, p ≤ 0.05).
Negative control F.oxysporum C.albicans Figure 6: e antifungal capacity for EOEJT on solid media (disc method). 8 Evidence-Based Complementary and Alternative Medicine e interesting antifungal capacity of EOEJT against A. niger, C. albicans, and F. oxysporum may be due to their active ingredients, especially the high content of bioactive molecules in EOEJT such as muurolol, elemol, and pinene. Many studies have reported that these molecules have a strong antifungal activity, most notably, the study by Chang et al. [48], which showed that muurolol had strong antifungal activity against harmful fungi, whereas elemol was also shown to have strong antifungal activity [41]. For pinene compound, many studies have shown that this compound has significant antifungal activity. Nóbrega et al. [49], showed a significant antifungal action of α-pinene against Candida spp, and Shi et al. [50], reported also significant antifungal activity of pinene against five plant pathogens including C. gloeosporioides, F. proliferatum, A. kikuchiana, and Phomopsis sp.
Many strategies had been devoted to the control of A. flavus, F. oxysporum, A. niger, and C. albicans using different types of substances, either natural or chemically synthesized. Our results are in agreement with previous   reports e.g., the study of Jemli et al. [51], which showed a substantial antifungal activity against A. alternata, F. solani, F. oxysporum, V. dahlia, and R. solani with a percentage of inhibition ranging from 24% to 92.1%.
Lafraxo et al. [13] showed that the essential oil of Juniperus thurifera leaves exhibited potent antifungal activity vs. F. oxysporum and C. albicans at a concentration of 0.095 g/ mL.

Conclusion
is study showed that Juniperus thurifera bark essential is rich in terpene compounds, which were extracted and characterized by GC-MS. Additionally, the Juniperus thurifera essential oil also had proven antimicrobial, antifungal, and antioxidant capacity. is opens up the possibility of encapsulating this essential oil through complex biotechnology applications with antibiotics to enhance their effects against pathogen resistance. However, before any potential application, further studies dealing with toxicity are warranted.

Data Availability
Data used to support the findings are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest. Evidence-Based Complementary and Alternative Medicine 11