2.3.1. DPPH Test

Antioxidant activity was determined by use of the DPPH assay according to previously published method [21]. Briefly, 800 μL of a methanolic solution of DPPH (0.2 mM) was mixed with 200 μL of different dilutions of EOs of *J. thurifera* (0–1 mg/mL), and subsequently incubated in the dark at RT for 30 min. Absorbances of samples were recorded at 517 nm and compared to those of a control consisting of 800 μL of DPPH solution. Samples, positive controls, quercetin or BHT were prepared under the same operating conditions. Decay of absorbance was measured with a spectrophotometer and percent inhibition (I%) calculated (Equation (1)).

$$\text{I} \text{ (\%)}=\text{I} \text{(T0}-\text{Tx)}/\text{T0} \text{ } \text{\*}100\tag{1}$$

By performing kinetics of this activity, concentrations corresponding to 50% inhibition (IC50), expressed as μg/mL, were determined, where the least IC50 corresponds to the greatest efficiency of EOs.

## 2.3.2. TAC Test

Antioxidant activity was determined by placing 100 μL of EOs at various concentrations after adding 1000 μL of a reagen<sup>t</sup> composed of 0.6 M H2SO4, 28 mM Na2PO4 and 4 mM (NH4)2MoS4. Then, the tubes were tightly closed and incubated at 95 ◦C for 90 min. After cooling, absorbances were measured at 695 nm. The negative control consisted of 100 μL of methanol after the addition of 1000 μL of the above reagen<sup>t</sup> [22]. The samples and controls were incubated under the same conditions. The obtained results were represented in mg ascorbic acid equivalents per gram (mg EAA/g).

## 2.3.3. FRAP Test

Reducing power was recorded by placing 200 μL of sample at several concentrations, into 500 μL of 0.2 M phosphate buffer (pH = 6.6), followed by 500 μL of 1% K3Fe (CN)6 in dH2O. Mixtures were subsequently placed into a water bath and incubated at 50 ◦C for 20 min. Next, about 500 μL trichloroacetic acid (TCA, 10%) was added followed by centrifugation. A 500 μL aliquot of the supernatant was transferred to another tube followed by the addition of 500 μL of dH2O and 100 μL of freshly-prepared FeCl3 (1%) in dH2O. Similarly, a blank without sample was included by replacing EOs of *J. thurifera* with methanol. Absorbances of reaction media were recorded at 700 nm and compared to the methanol blank, which allowed calibration of the apparatus (UV-VIS spectrophotometer). Positive controls were a solution of the standard antioxidants BHT and quercetin [23].

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

### 2.4.1. Microbial Strains Tested

Antimicrobial activity of *J. thurifera* EOs against four fungal strains, *Candida albicans*, ATCC 10231; *Aspergillus niger*, MTCC 282; *Aspergillus flavus*, MTCC 9606 and *Fusarium*

*oxysporum*, MTCC 9913 and four strains of bacteria, *Staphylococcus aureus*, ATCC 6633; *Escherichia coli*, K12; *Bacillus subtilis*, DSM 6333 and *Pseudomonas aeruginosa*, CIP A22. The fungal and bacteria strains were provided by Sidi Mohammed Ben Abdellah University (Fez, Morocco) and Hassan II University Hospital (Fez, Morocco), respectively.

### 2.4.2. Assessment of Antimicrobial Activity

The antimicrobial activity of *J. thurifera* EOs was determined by use of the disc diffusion method [24]. Petri dishes containing Mueller–Hinton (MH) and Malt Extract (ME) culture media were inoculated with the four bacterial strains and *C. albicans*, respectively, by the double-layer method, from cultures freshly grown in MH and ME medium, decimal dilutions were made in sterile saline (0.9%) until turbidity of 0.5 McFarland (10<sup>8</sup> CFU/mL) was reached, 100 μL were added to tubes containing 5 mL of soft agar (0.5% agar), then the inoculated tubes were spread in Petri dishes containing MH and ME medium. For *A. niger*, *A. flavus*, and *F. oxysporum* the antifungal activity was determined by the direct confrontation method in the ME medium. Sterile 6 mm Whatman paper discs were positioned into the centre of the petri dish and then impregnated with 20 μL of *J. thurifera* EOs, and also with conventional antimicrobial drugs; streptomycin and erythromycin for bacterial strains and fluconazole for fungal strains according to the methodology of the European Committee for Antimicrobial Susceptibility Testing (EUCAST). Then, bacteria- and fungi-inoculated dishes were incubated at temperatures of 30 ◦C and 37 ◦C optimal for the bacterial and fungal strains and *C. albicans*, respectively. Inhibition diameters and percentages of inhibition were calculated 18–24 h post inoculation (hpi) for the bacterial strains and after 24–48 hpi for *C. albicans*, and 7 days post inoculation for *F. oxysporum*, *A. niger* and *A. flavus* [24,25].

### 2.4.3. Minimum Inhibitory Concentration (MIC) Determination

Minimum inhibitory concentrations (MIC) of *J. thurifera* EOs against the four bacterial and four fungal strains were determined by use of the microdilution as previously described [25]. Briefly, a sterile 96-well microplate was used and 50 μL of sterile MH or ME medium was added for bacterial and fungal strains, respectively. Serially diluted EOs of *J. thurifera* at a volume of 100 μL prepared in 10% (*v*/*v*) DMSO was pipetted into the first row. This was followed by the addition of 30 μL of microbial strains. Plates were incubated for 24 h, 48 h or 7 d for bacteria, *C. albicans* and fungi (*Fusarium oxysporum*, *A. niger*, *A. flavus)*, respectively; at 37 ◦C or 30 ◦C [25,26]. Each well received 20 μL of water 2,3,5-triphenyl tetrazolium chloride solution (0.2%) to visualize bacterial growth. MIC was defined as the least concentration that did not create a red colour [26].
