**1. Introduction**

Thuriferous juniper (*Juniperus thurifera* L.) is a monoecious, dioecious, conifer tree or shrub with scaly leaves of the cypress family (Cupressaceae) [1]. It plays an important role in the western Mediterranean basin and is regarded as a keystone species of lowtemperature-adapted open woodlands, with steppe-like undercover [2]. In Morocco, the area of this species, which constitutes the upper limit of the forest in the Atlas Mountains, has been considerably restricted and, the vast majority of the stands have been degraded by over-exploitation and over-grazing, aggravated by an almost total absence of regeneration. Its current surface area, in the Atlas Mountains of Morocco, is estimated to be 20,000 ha [3,4]. Thuriferous juniper, with its extraordinary resistance, remarkable ability to withstand very severe climatic conditions, and indifference to the physical nature of soils, longevity of as much as 500 years, is unquestionably the predominant tree of the high Moroccan mountains [5]. Development of these natural plant resources is mainly based on extraction of essential oils (EOs), which are high value-added products, employed in the pharmaceutical, cosmetics and food industries [6–8]. The genus *Juniperus* contains essential medicinal plants with a long history of usage in traditional medicine. Its leaves are used to treat diabetes, diarrhoea, and rheumatism as a decoction [9]. Leaves and berries of *Juniperus* are utilized as an oral hypoglycemic medication [10], while leaves are used to treat bronchitis and as a diuretic [9]. Studies of the biological and biotechnological activities of the phytochemical compounds of plants is of interest and the antimicrobial activities of EOs have been reported [6,11–13]. These activities are attributed to oxygenated mono-terpenes [6]. Use of natural molecules to inhibit oxidation of fat, its consequences on health and its economic repercussions have been the subject of several studies [6,14]. Results of several studies of the antioxidant activities of EOs from a variety of aromatic plants have demonstrated that these properties are mainly ascribed to the presence of compounds containing hydroxyl group(s) [15–17]. Recently, the EOs and aqueous extracts of plants have attracted interest because of their richness in natural biologically active constituents including antioxidant, antimicrobial and insecticidal properties.

Under the existing restricted and inadequate arsenal of new therapies, the list of microorganisms that are becoming resistant to all commonly used antibiotics is growing, prompting the discovery of alternative classes of medications to prevent significant public health concerns, unconventional therapeutic interventions derived from natural resource exploitation have been intensively investigated [18,19]. Objectives of this study were to describe the chemical composition of the EOs of leaves of *J. thurifera* collected from the Jbel lakraa Massif in the Eastern, Middle Atlas of Morocco and to compare the results to those of previous studies and investigate the antioxidant, antibacterial and antifungal activities of the EOs, so that an evaluation of the economic value of the EOs of *J. thurifera* and their potential as replacements for antibiotics available on the market to combat microbial resistance could be conducted.

### **2. Materials and Methods**

### *2.1. Extraction of EOs from J. thurifera*

*J. thurifera* was harvested from the mountains (lat: 33.68093368; long: 4.30823143) during October 2021, which was autumn. Specimens were identified by a botanist in the department of biology, Faculty of Sciences-FSDM-USMBA-Fez, and the plant is deposited in the Herbarium under number (FJT/02D20). Leaves were cleaned and subsequently dried at 35 ◦C for 72 h in a ventilated oven. Dried leaves were crushed with an electric blender, then EOs extracted by hydro-distillation on a Clevenger-type extractor [15]. Briefly, 200 g of the ground leaf material was mixed with 750 mL distilled water (dH2O) and extracted for about 120 min. Samples were partitioned into hexane (10%). At least three replicates were performed in this study.

### *2.2. GC/MS/MS Analysis of EOs*

Constituents of EOs were identified and quantified by use of gas chromatography (TQ8040 NX; Shimadzu, Tokyo, Japan) attached to a triple quadrupole, tandem mass spectrometer (GC-MS). Chromatography was conducted on an apolar, capillary column RTxi-5 Sil MS column (30 m × 0.25 mm ID × 0.25 μm). Helium was used as carrier gas and the injection volume was 1 μL. Temperatures of the source and the interface were 200 ◦C and 280 ◦C, respectively. The chromatographic system was programmed with splitless injection (split opening at 4 min), injection temperature of 250 ◦C and pressure of 37.1 kPa. Temperature was programmed with an initial temperature of 50 ◦C for 2 min, ramp 1 was 5 ◦C/min to 160 ◦C for 2 min and ramp 2 was 5 ◦C/min to 280 ◦C for 2 min. Identification of phytochemicals in EOs was conducted by comparing the obtained retention indices with those of chemical compounds in the literature database [20].
