Chemical composition, antioxidant and antibacterial activities of the essential oils of Juniperus phoenicea, Juniperus thurifera and Juniperus oxycedrus

: This study aimed to compare the chemical composition, the antioxidant activity and the antibacterial activity of essential oils (EOs) of Juniperus phoenicea, Juniperus thurifera and Juniperus oxycedrus , obtained from Ait Bouguemez region (Province of Azilal, Morocco). The analysis by GC/MS of essential oils led to identify 37, 54 and 38 components for J. phoenicea, J. thurifera, and J. oxycedrus , respectively . Monoterpenic fraction was found predominant in essential oils of the three samples. The DPPH free radical scavenging activity showed that essential oil of J. thurifera has the strongest antioxidant activity with an IC50 value of 12.07 μg/mL. The antibacterial activity showed that S. aureus was more sensitive than P. aeruginosa and E. coli for the three EOs tested.


Introduction
Juniperus species (genus Juniperus, family of Cupressaceae) belong to the coniferous plants is an important component of arid and semi-arid ecosystems throughout the northern hemisphere 1,2 . This genus is the second most diverse genus of the conifers, it is consisting of approximately 67 species and 28 varieties 1 . In Morocco, the genus Juniperus is present in several places among which the valley of Ait Bouguemez (Province of Azilal, Morocco) where we find the three species J. phoenicea, J. thurifera and J. oxycedrus. The valley of Ait Bouguemez is located in the central High Atlas limestone (Morocco). It extends at an altitude ranging from 1800 to 2200 m over nearly 30 km 3 .
Juniperus is one of the most used genus by the traditional healers in central High Atlas of Morocco [4][5][6][7] . Juniperus tar, leaves and fruits are employed to heal wounds, abdominal pain and stomachic disorders, metabolic diseases, and against fungal infections [4][5][6] . The treatment of infections and health disorders with herbal medicines is usually involves active natural products mostly of low molecular weight of great structural diversity. These substances traditionally referred to as secondary metabolites, often are differentially distributed among limited taxonomic groups within the plant kingdom 8,9 . The function or importance of secondary metabolites in plants is ecological. It is for interactions between the plants and their environment 10,11 . Secondary metabolites include three major classes: terpenoids, alkaloids and phenolics. They contain numerous natural products, some of which have good pharmacological properties especially essential oils (EOs) 12,13 .
Many studies around the world have been performed on the chemical composition of the EOs and extracts of Juniperus species. Juniperus Oils and extracts contain various chemotypical compounds: from 2,6dimethyloctane to sesquiterpene skeletons, and flavonoids and biflavonoids, but the main classes identified in almost all Juniperus are mono and sesquiterpenoids and their derivatives 14-18 . Many constituents present in Juniperus EOs are responsible for their biological properties 8 . According to Majewska 19 20 reported that EOs of Juniperus species have a strong antioxidant activity and it depends on the nature and concentration of EOs components and also the geographical origin of plant.
In this regard, the purpose of this research is both to explore the chemical composition of EOs of the Juniperus species growing in valley of Ait Bouguemez in comparison to the EOs from other Mediterranean countries and to evaluate the antioxidant and antibacterial activities of the EOs tested.

Plant material
To begin with, we selected samples of J. phoenicea, J. thurifera and J. oxycedrus from the wilderness of Ait Bouguemez valley (Province of Azilal, Morocco). This region is chosen because of the presence of the 3 species at the same ecological conditions. Table 1 shows the ecological traits of the samples' collection setting. Authentication and identification of the plants was done at Laboratory of Ecology and Environment Laboratory, Faculty of Sciences Ben M'Sik (University Hassan II of Casablanca, Morocco). After that, samples were air dried, at room temperature (25°C) and put them away from sunlight for 21 days until their weight was stable. Finally, they were packed in bags made of paper and kept in a dark, cool and dry place after which their analysis started.

GC/MS analysis
The EOs were analyzed on a Hewlett-Packard gas chromatograph Model 5890, coupled to a Hewlett-Packard model 5971, equipped with a DB5 MS column (30 m X 0.25 mm; 0.25 μm), programming from 50°C (5 min) to 300°C at 5°C/min, with a 5 min hold. Helium was used as the carrier gas (1.0 mL/min); injection in split mode (1:30); injector and detector temperatures, 250 and 280°C, respectively. The mass spectrometer worked in EI mode at 70 eV; electron multiplier, 2500 V; ion source temperature, 180°C; MS data were acquired in the scan mode in the m/z range 33-450. The identification of the components was based on comparison of their mass spectra with those of NIST mass spectral library (NIST 2011) as well as on comparison of their retention indices either with those of authentic compounds or with literature values 1 .

Antioxidant activity
The slightly modified method of Pothitirat et al. 22 was of great help to assess the radical scavenging abilities of EOs. 0.5 mL of various concentrations of EO and standards butylhydroxytoluene (BHT), vitamin C and α-tocopherol separately were added to 1 mL of 100 μM methanol solution of DPPH. The reaction mixture was incubated in the dark at room temperature for 20 minutes. The optical density was monitored at 517 nm against blank containing methanol. The decrease in optical density of DPPH on addition of test samples in relation to the control was used to calculate the antioxidant activity as percentage of inhibition (%IP) of DPPH radical:
The antioxidant activity of samples was expressed as IC50 in μg/mL required inhibiting the formation of DPPH radicals by 50%. A low IC50 value represents a high antioxidant activity. Experiments were carried out in triplicate.

Antibacterial activity 2.5.1. Bacterial strains
To evaluate the antibacterial properties of the samples, three strains of pathogenic bacteria were used in the study: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853).

Disc diffusion method
The antibacterial activities of the EOs were determined by using paper disk diffusion method to screen the efficacy of Eos 23 . The EOs were diluted with dimethylsulfoxyde (DMSO 10%) at the concentration 100µg/disc. A volume of 10 μL of the concentration was impregnated into the paper disk with 6 mm diameter, and then placed onto Mueller-Hinton agar plates (90 mm), which were previously inoculated on the surface agar with 100 μL of 1.5 ×10 8 CFU/mL (equivalent to 0.5 McFarland) suspension for each tested bacterium. Respectively we used disc impregnated with DMSO 10% and standard disc of Ampicillin as negative and positive controls. In order to trigger excess prediffusion we kept the plates at room temperature for 30 minutes before incubation at 37°C for 24 h. We measured diameters of inhibition zones in mm. The experiments were carried out in triplicate.

Statistical analysis
In each test, the experimental data represent the mean ± Standard Deviation (SD). The IC50 was obtained with a computer program GraphPad (Prism 8.0.1). Statistical analyses were performed with XLSTAT V.7.1. The significance of the difference among groups was analyzed using ANOVA followed by the Fisher (LSD test) and Dunnett tests. Differences were considered significant at the level p < 0.05.

Essential Oils composition
The EOs' chemical composition of the three species of Juniperus is presented in Table 2.  24 , Egypt (0.36%) 25 and Algeria (0.70% to 0.92%) 26 . The yield of EO of J. thurifera is 0.93%. This yield was different from those reported in the literature. Zeraib et al. 27 reports values from 0.40 to 0.53 % in different populations of Algerian J. thurifera. The rate provided by J. thurifera is still higher than that obtained by El Hajjouji et al. 28 in Tizi N'tichka (High Atlas Mountains of Morocco) for this species, which did not exceed 0.67%. Finally, J. oxycedrus has the lowest yield among the three species (0.1%). This result is lower than that found for the same species in Kosovo (0.4 to 1.8 %) 29 , Spain (0.2%) 14 and Tunisia (0.15-0.21 %) 30 . EOs yield showed variations from site to another and these variations could be accounted for by pedoclimatic variations of the growing site or by differences in the genetic arsenal of plants 1,27 .  27 . In Morocco, Achak 15 reported that the major constituents of EO of Oukaimden (Western high Atlas, Morocco) were sabinene (16.5%) and γ-terpinene (9.3%). Another study showed that the EO of J. thurifera collected in eastern middle Atlas Mountains of Morocco are rich in monoterpenes (97.09 %) and the major constituent was β-pinene (36.26%) 33 .
The study of the EO of J. oxycedrus showed the presence of 38 components accounting for approximately 94.91%. Monoterpenic hydrocarbon fraction was found predominant in EO composition (81.60%). However, the contents of oxygenated monoterpenes, sesquiterpenes hydrocarbon and oxygenated sesquiterpenes were 8.07%, 4.93% and 0.31%, respectively. α-pinene (67.33%) and δ-3-carene (7.21%) were the major components. In Spain, Llorens-Molina 14 reported that monoterpenic hydrocarbon fraction was also found predominant in EO composition of leaves both for siliceous and calcicolous locations (72.5-71.4 %). α-pinene was also the main component accounting for 42% to 53.2% 14 . Another study of J. oxycedrus in Lebanon showed that EO also contains as majority components α-pinene with a rate of 27.4%, in addition to myrcene (18.9%), α -phellandrene (7.1%) and limonene (6.7%) 34 . Monoterpenes, with a percentage ranging between 34.94% and 81.25%, were the main class of constituents in Italy 35 . The EOs from leaves collected in Tunisia showed sesquiterpenes as main class of constituents, ranging from 34.78% to 44.16% 35 . Identification of the volatile constituents of the EO of J. oxycedrus from the north centre region of Morocco showed high contents of α-pinene (31.25%) 36 . The results obtained for the three species are in agreement with those announced by Adams 1 and Angioni 37 from the analysis of Juniperus genus, in which pinenes are generally dominant. Because of the genetic diversity 38 , different climatic conditions or of a large geographical diversity 1 the oils compositions of the same plant showed variability in different populations.

Antioxidant activity
The antioxidant activity of EOs of our samples is showed in Figure 1. The IC50 (half maximal Inhibitory Concentration) values of EOs and those of standards: BHT, vitamin C and α-tocopherol are represented in Table 3.   (22.14 μg/mL). The antioxidant activity of J. thurifera is significantly stronger than the three standards antioxidants: BHT, vitamin C and α-tocopherol. J. phoenicea has activity similar to vitamin C and higher than BHT and α-tocopherol. The antioxidant activity of J. oxycedrus is the lowest in comparison with both EOs and standards. Our results are in agreement with studies of Amorati 40 which showed that the antioxidant properties are in close relationship to the presence of oxygenated compounds (oxygenated monoterpenes and oxygenated sesquiterpenes) (23.28% for J. thurifera and 8.38% for J. oxycedrus). According to Amorati 40 , oxygenated monoterpenes have been found to have good antioxidant activity because they undergo antioxidation, characterized by very fast termination process thereby reducing overall rate of oxidation. However, it is difficult to assign this activity to the only oxygenated compounds because of the chemical complexity of EOs which can generate a synergy effect between the various compounds 41 .

Antibacterial activity
Results of antibacterial activity of EOs of our samples are shown in Table 4. Although, there were many differences in the chemical composition of the three species' EOs, their antibacterial activity was still the same. We also noticed that Gram(+) bacteria (S. aureus) are more sensitive to EOs of the three samples than Gram(-) bacteria (E. coli and P. aeruginosa). This is mainly due to the fact that Gram(-) bacteria possess an outer membrane that is more complex, rigid and rich in lipopolysaccharide (LPS), this outer membrane limits the diffusion of hydrophobic compounds. Yet, Gram(+) bacteria lacks such a membrane and possess instead peptidoglycan wall which is not thick enough to fight small antimicrobial molecules, thus making the cell membrane easily accessible 44,45 . Besides, Gram(+) bacteria can facilitate the penetration of hydrophobic compounds of EOs owing to the lipophilic ends of lipoteichoic acid which exists in cell membrane 46 . Furthermore, some constituents of our EOs such as α-pinene, α-terpineol, β-phellandrene, δ-3-carene, δ-2-carene and terpinen-4-ol should also be taken into account when dealing with the antibacterial activity of Juniperus species Eos 47,48 .

Conclusion
The EOs of J. phoenicea, J. thurifera and J. oxycedrus, obtained from Ait Bouguemez region (Province of Azilal, Morocco), showed a chemical composition rich in monoterpenes hydrocarbon and oxygenated monoterpenes. They showed an important antioxidant activity and a good antibacterial activity against S. aureus, which can justify the multiple uses of the three Juniperus in the traditional medicine of Ait Bouguemez region. Thanks to their antioxidant and antibacterial activities, EOsof J. thurifera, J. phoenicea and J. oxycedrus have to be valorized via several domains. However, numerous investigations should be carried out on their mode of action and their probable toxicological effects in order to optimize their potential uses.