Chemical and reagents

All the media components and chemicals were purchased from Sigma Aldrich. Solvents were of analytical grade and were from Merck (Germany).

Essential oils

The Eos used in this study were received from a local producer in Algeria (Aromabioil) and stored at +4 °C in amber glass bottles until analysis. Eos were obtained by hydrodistillation from the aerial parts (leaves and stems) of wild Salvia officinalis (sage), and Juniperus phoenicea (Phoenician juniper), growing in northern Algeria.

GC-MS analysis

The GC-MS analysis was performed on a Hewlett Packard Agilent 6890 plus (Agilent Technologies, USA). The column used was an HP-5MS column (30 m × 0.25 mm i.d. × 0.25 μm film thickness). The injector temperature was maintained at 260 °C. The column oven temperature was initially held at 40 °C for 10 min and then increased to 280 °C at 5 °C/min. The debit of the gas vector (helium) was fixed to 0.5 mL/min. Essential oils were dissolved in hexane at a concentration of (10 %), and a volume of 2 μL of the diluted Eos was injected in split mode (1:80). The ionization energy was 70 eV. The retention indices of individual components were calculated using a series of n-alkanes (C8-C28). The components were identified by comparing their retention indices and mass spectra with those reported in the literature [⁸], and with data on the MS library NIST (National Institute of Standards and Technology). The relative percentage of each compound in the Eos were obtained as percentages of a peak area from the total chromatogram.

Antioxidant property

Determination of antioxidant combination index (CI)

The classical isobologram-combination index equation, based on the IC50 values was used to determine the presence of synergy or antagonism between the Eos [¹⁰].

where (D)1 and (D)2 are the doses (IC50 values) of two active Eos in combination; (Dx)1 and (Dx)2 are the doses (IC50 values) of two active Eos individually. The type of antioxidant interactions was interpreted as follows: CI < 1: synergistic; CI = 1: additive; CI > 1: antagonistic. In this work, extracts were paired at 1:1 ratio.

Antibacterial assay

The antibacterial activity of Juniperus phoenicea and Salvia officinalis Eos was evaluated against four bacterial isolates (Pseudomonas aeruginosa ATCC27853, Proteus vulgaris ATCC13315, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC25923) using agar well diffusion method. Plates containing Mueller-Hinton Agar (MHA) were inoculated with 100 μL of the standardized suspension (106 CFU/mL) of pathogens. A sterilized cork borer was used to bore wells in the solid culture medium. Different concentrations (10 and 30 mg) of the tested Eos were loaded on the wells. After 24 h of incubation at 37 °C, inhibition zone diameters were determined in millimeters. Gentamicin and ampicillin were used as positive controls.

Antifungal activity

Determination of minimum inhibitory concentration (MIC)

The broth macrodilution method previously described by Shukla et al. [¹³] has been used for the determination of the minimum inhibitory concentration (MIC). First, fungal suspensions were prepared by transferring spores from 10 days-old cultures in 10 mL sterile distilled water, and by adjusting the final concentrations to 106 spores/mL. Then, Two-fold serial dilutions of Eos were made with acetone and mixed with sterile Potato Dextrose Broth (PDB). The tubes with various concentrations of Eos (4 to 0.03 μL/mL) were inoculated with 10 μL of spore suspension of each test strain and incubated for 7 days at 25 °C. In control tubes, only the fungal suspension was added to the medium. The MIC was defined as the lowest concentration required to prevent visible growth.

Statistical analysis

The experiments were conducted in triplicate, and the results are expressed as the mean ± standard deviation of the experiments.

Chemical composition of essential oils

In this study, the chemical composition of the Eos was determined by GC-MS. The identified constituents and their amounts are listed in Table 1. Oxygenated monoterpene and monoterpene hydrocarbons were the most abundant compounds found in S. officinalis and J. phoenicea, respectively. The Eo of S. officinalis consisted predominantly of cis-chrysanthenyl acetate (64.82 %) and α-thujone (14.7 %). In J. phoenicea Eo, α-Pinene (64.44 %) was found in a high percentage, followed by δ-3-carene (7.02 %).

Our results are comparable to those reported in Algeria and Tunisia by other reserchers, who described α-pinene as a dominant component of J. phoenicea L. Eos [¹⁴,¹⁵]. In another study, 1,8-cineol, camphor, borneol, α-pinene, β-pinene, camphene, β-myrcene, and caryophyllene were described as the major components of the Eos of S. officinalis collected in Syria [¹⁶]. In a different investigation conducted in Tunisia, camphor, α-thujone, 1,8-cineole, viridiflorol, β-thujone, and β-caryophyllene were reported as the major components in the Eo of S. officinalis [¹⁷]. Monoterpenes are a widespread group of plants secondary metabolites that are commonly found in essential oils. The variation in Eos composition is usually associated with differences in its geographical location or any other abiotic factors [¹⁸].

Antioxidant activity

Free radical scavenging activity of Eos was determined through DPPH assay. The DPPH scavenging index and the half-maximal inhibitory concentration (IC50) values are summarized in Table 2. As can be seen from our data, J. phoenicea and S. officinalis Eos had very low‐antioxidant activities, especially when compared with ascorbic acid (positive control), which exhibited an IC50 value of 15 μg/mL in the same conditions. It is known from the literature that the antioxidant activity is related to the chemical composition of the Eos. The major compounds obtained from the investigated samples were monoterpenes. Terpenes are important components of Eos from medicinal plants that may contribute to the antioxidant properties [¹⁹]. In this work, the low antioxidant activities found might be explained by the absence of phenolic compounds. In fact, molecules like thymol and carvacrol play a notable role in inactivating free radicals and are responsible for the antioxidant activity of many Eos (20). Besides the dominant components, many constituents may contribute to the antioxidant activity due to the synergy of components’ action. In our case, J. phoenicea and S. officinalis Eos have shown to interact with each other as antagonistic agents (Table 2). Most studies attributed additive and synergistic effects to phenolic and alcohol compounds [⁴]. The predominance of monoterpenes and differences in the chemical composition of the investigated Eos could explain the observed result.

Antibacterial activity

Essential oils are known to possess antimicrobial properties against a wide range of organisms. In our work, the Eos were tested in vitro against four strains. The obtained data demonstrated that, Gram-negative bacteria were more resistant to the Eos compared to the Gram-positive strain (S. aureus) (Table 3). We noticed that S. officinalis was inactive against Gram-negative bacteria even at a concentration of 30 mg. Escherichia coli and Pseudomonas aeruginosa showed the highest levels of resistance to the tested Eos. In contrast, J. phoenicea was the only Eo active against the Gram-negative organism Proteus vulgaris.

Our observations are in concordance with those of previous experiences which reported an antibacterial activity of S. officinalis and J. phoenicea against Gram-negative and Gram-positive bacteria [²¹,²²]. In our study, the Gram-positive strain was more sensitive than the Gram-negative bacteria which is probably due to a difference in the cell wall composition. In fact, the lipopolysaccharides of the outer membrane in the Gram- negative strains are responsible for protecting the bacteria against the external environment [²³,²⁴]. Previous reports stated that, the antimicrobial activity depends on the chemical composition of the Eos. The mechanism of action of these compounds against bacteria lies mainly in their capacity to induce toxic effects on the membrane structure and functions [²⁵].

Antifungal activity

The obtained results indicated that all Eos had an inhibitory effect on the spore germination. Complete inhibition of all fungal strains was observed with thiophanate methyl at a concentration of 0.1 mg/mL (Table 4). The tested Eos showed percentages of inhibition ranging between 3.4% and 82.6%. The Eo of S. officinalis was characterized by a strong inhibitory effect on the mycelial growth of A. fumigatus (82.6%). In addition, J. phoenicea was less effective against the investigated fungi with a maximum inhibition rate of 14.5% in Penicillium sp. This antifungal activity could be due to the presence of bioactive chemical constituents. It has been previously reported that Eos of S. officinalis and J. phoenicea can suppress several plant pathogenic fungi [²⁶,²⁷].

Minimum inhibitory concentration (MIC) was determined by the broth macrodilution method. The obtained results are shown in Table 5. Our data demonstrated that all Eos had an antifungal activity against the tested strains. The growth of A. flavus and A. parasiticus was uniformly inhibited by the tested oils. A. carbonarius showed a strong sensitivity to S. officinalis Eo. However, Penicillium sp. Was more sensitive to the Eos treatments than the Aspergillus strains. This weak antifungal activity obtained with J. phoenicea and S. officinalis Eos in our work can be attributed to the predominance of α-pinene, β-pinene, and cis-chrysanthenyl acetate. These compounds are known for their moderate antimicrobial activity compared to alcoholic and phenolic monoterpenes [²⁵]. Several studies have shown that Eos can affect the fungal cell permeability and functions as well, through direct interaction with the cytomembrane [²⁸, ²⁹]. However, molecules like α-pinene, and β-pinene are characterized by a hydrophobicity that enables them to penetrate the cell membrane and exert their toxic effect [³⁰].