*3.2. Antioxidant Activity*

When antioxidant activities of EOs of *J. thurifera* were evaluated by three methods (Figure 3a), the percentage of inhibition of free radical (DPPH) was directly proportional to the concentrations of the EOs of *J. thurifera*. For a concentration of 13 μg/mL of EOs of *J. thurifera*, the percentage of DPPH inhibition was 85 ± 0.24% and for a concentration of 27 μg/mL the percentage of inhibition was approximately 91 ± 0.17% (Figure 3a). Antioxidant capacity was determined from the IC50, which is the concentration necessary to reduce 50% of the DPPH radical. The smaller the IC50 value, the greater the antioxidant activity of a compound [29]. Free radical activities of EOs of *J. thurifera*, BHT and quercetin revealed that IC50 of EOs of *J. thurifera* is of the order of 23.6 ± 0.71 μg/mL,

14.2 ± 0.14 μg/mL and 15.9 ± 0.56 μg/mL (Figure 3b), respectively. Evaluation of antioxidant capacity by use of the FRAP method revealed that the effective concentration (EC-50) is in the range of 0.19 ± 0.01 mg/mL (EOs of *J. thurifera*), 3.6 × 10−<sup>2</sup> ± 0.003 mg/mL (BHT) and 2.8 × 10−<sup>2</sup> ± 0.002 mg/mL (quercetin) (Figure 4a). Total antioxidant capacity (Figure 4b) of EOs of *J. thurifera* was 9.3 × 10<sup>2</sup> ± 38 mg EAA/g versus 5.3 × 10<sup>2</sup> ± 22 mg EAA/g (BHT) and 6.6 × 10<sup>2</sup> ± 46.67 mg EAA/g (quercetin).

**Figure 1.** Chromatograph of compounds identified by GC/MS in EOs of *J. thurifera*. Peaks represent absolute abundances, whereas numbers on the x-axis represent retention times in min.

**Figure 2.** Molecular structures of phytochemical compounds in EOs of *J. thurifera*.

**Figure 3.** Antioxidant activities of EOs of *J. thurifera* by the DPPH method ( **A**) and the concentration of IC-50 (**B**). Bars with the same letters do not differ significantly (*p* < 0.05).

**Figure 4.** Antioxidant activities of EOs of *J. thurifera* by the FRAP method ( **A**) and antioxidant capacity of EOs of *J. thurifera* (**B**). Bars with the same letters do not differ significantly (*p* < 0.05).

Assessment of oxidative stress (OS, oxidation in vivo) has become important since this type of oxidation can be involved in several health effects including rheumatoid arthritis, atherosclerosis, diabetes, aging and cancer [30–32]. Natural antioxidants present in plant extracts and EOs can provide protection against OS by two main mechanisms, namely scavenging reactive oxygen species (ROS) and blocking lipid peroxidation [33,34]. There is a correlation between the antioxidant power of EOs and its phytochemical composition. In this context, it has been previously documented that the antioxidant capacities of EOs are associated with their phytochemical composition via the hydroxyl function present in their constituents, and the richer an oil is in phenolic compounds and terpenes, the more effective its antioxidant capacity [35–40].

The phytochemical profile of EOs of *J. thurifera* (Table 1) revealed that EOs of *J. thurifera* are rich in terpenic compounds, such as thujene, γ-terpinene, cymene and linalool which are known for their antioxidant potentials [41]. Recent studies have shown that sabinene is an antioxidant compound [42]. Similarly, cymene possesses a potent anti-nociceptive behavior although it exhibited lesser antioxidant potential [43].

### *3.3. Antimicrobial Activity of J. thurifera EOs*

### 3.3.1. Antibacterial Activity of *J. thurifera* EOs

EOs extracted from leaves of *J. thurifera* exhibited antibacterial activity in comparison with the concentration used and which antibiotic was used, streptomycin sulphate or erythromycin especially against *P. aeruginosa* CIP A22 with an inhibition diameter of 27.67 ± 1.53 mm and a MIC of 0.0475 ± 0.00 μg/mL, against *S. aureus*, ATCC 6633 with an inhibition diameter of 20.33 ± 0.58 mm and a MIC of 0.095 ± 0.00 μg/mL, against *E. coli* K12 with an inhibition diameter of 15.67 ± 3.05 mm and a MIC of 0.095 ± 0.00 μg/mL and against *B. subtilis* DSM 6333 with an inhibition diameter of 14.33 ± 1.15 mm and a MIC of 0.095 ± 0.00 μg/mL (Table 2).

**Table 2.** Antibacterial activity of *J. thurifera* EOs in comparison with the antibiotics streptomycin and erythromycin.


Mean values (±SD, *n* = 3) followed by different letters in the same row are significantly different according to a mean analysis (Student *t*-test) and an analysis of variance (One-way ANOVA; Tukey's test, *p* ≤ 0.05). MIC: minimum inhibitory concentration; Rs: resistance.

Antibacterial activity of *J. thurifera* EOs might be due to their chemical composition, *J. thurifera* EOs are rich in terpene compounds, especially thujene, γ-terpinene, cymene, and linalool which are well known for their antibacterial activity [41]. They are also rich with sabinene and cymene which are compounds with antibacterial activity [44]. Results of the study reported here were different from results of a previous study [44], which indicated that extracts of *J. thurifera* L. leaves growing in eastern Algeria were active only against *S. aureus*, ATCC and methicillin-resistant *S. aureus* bacteria and the greatest activity with an inhibition diameter of 14 mm for a concentration of 1 g/mL. However, no inhibition was detected for extracts against *E. coli* ATCC or *P. aeruginosa* ATCC. However, the antibacterial activity of extracts of leaves of *Juniperus phoenicea* L was observed against both Gram-positive and Gram-negative bacteria [45]. *J. thurifera* EOs exhibited significant antibacterial activity against Gram-positive and Gram-negative bacteria, especially against *S. aureus*, *E. coli*, and *P. aeruginosa* with inhibition diameters of 31.12 ± 3.11, 13.23 ± 2.59, and 18.27 ± 2.29 mm, respectively [46]. Those results were similar to those observed in the study, the results of which are presented here for *E. coli*, but are the opposite of the results for *S. aureus* and *P. aeruginosa*, for which the greater antibacterial activity might have been due to the different physicochemical composition of the EOs observed in the study reported here, which are consistent with results of several other studies [47], which found *S. aureus* was sensitive to the EOs of *J. thurifera* from Algeria. Furthermore, two strains of *Pseudomonas* proved to be resistant, [28]. In that study, the EOs from twigs of *J. thurifera* collected in the Eastern range of the Middle Atlas Mountains of Morocco exhibited significant antibacterial activity against *E. coli*, *B. subtilus*, *M. luteus*, and *S. aureus.* Similarly, EOs of *J. thurifera* had significant antibacterial activity against *S. aureus*, ATCC 33862 with an inhibition diameter of 27 mm and MIC of 450 μL/mL, against *E. coli*, ATCC 25922 with an inhibition diameter of 25.6 mm and MIC of 530 μL/mL and against *P. mirabilis*, ATCC 7002 with an inhibition diameter of 18.8 mm and MIC of 930 μL/mL [48].

### 3.3.2. Antifungal Activity of *J. thurifera* EOs

When compared with the fungicide fluconazole in the in vitro evaluation of antifungal activity of *J. thurifera* EOs against *A. niger*, *A. flavus*, *F. oxysporum*, and *C. albicans* in the disc diffusion test, these EOs exhibited significant activity against *F. oxysporum*, MTCC 9913 with percent inhibition of 32.47 ± 2.25 and MIC values of 0.095 μg/mL as well as with an inhibition diameter of 21.33 ± 2.08 mm and a MIC value of 0.095 μg/mL against *C. albicans*; ATCC 10231 (Table 3), In addition, *J. thurifera* EOs exhibited antifungal activities against *F. oxysporum* and *C. albicans*. However, *J. thurifera* EOs did not exhibit antifungal activity against *A. niger* or *A. flavus*. The antifungal activity of *J. thurifera* EOs may be mainly due to their chemical composition, *J. thurifera* EOs are particularly rich in thujene, pinene, and limonene which are well known for their antimicrobial activity, especially antifungal activity [16,17].

**Table 3.** Antifungal activity of *J. thurifera* EOs in comparison with fluconazole.


Mean values (±SD, *n* = 3) followed by different letters in the same row are significantly different according to a mean analysis (Student *t*-test) and an analysis of variance (One-way ANOVA; Tukey's test, *p* ≤ 0.05). MIC: minimum inhibitory concentration; Rs: resistance.

Several studies have been devoted to the control of pathogenic and phytopathogenic fungi in general, and *A. niger*, *A. flavus*, *F. oxysporum* and *C. albicans* in particular, through the use of various bioactive substances, either natural or synthetic. The results of this study are opposite of those of another study [49], in which sesquiterpenes of *J. thurifera* EOs did not present any antifungal activity against *C. albicans* CECT;1394. Similarly, in another study [28] the EOs of *J. thurifera* twigs collected from the Eastern sector of the Middle Atlas Mountains of Morocco exhibited antifungal activities against *A. niger*, *Penicillium expansum*, and *Penicillium digitatum*. The results of the study presented here are consistent with those focused on substances of bacterial and fungal origin [24], which reported an isolate from *Bacillus* sp. Gn-A11-18 exhibiting antifungal activity of 31.33 ± 0.58 mm against *C. albicans*; ATCC 10231 and a percentage of inhibition of 29.66 ± 0.57% against *A. niger.* Similarly, results of another study [50] showed significant antifungal activity mainly against *Alternaria alternata*, *F. oxysporum*, *F. solani*, *Rhizoctonia solani* and *Verticillium dahlia* with percentage inhibitions ranging from 24 to 92.1%.
