Lavandula abrialis RAB114629 and L. stoechas RAB114630 (Figures 2A, B) were collected in May 2022 in locations listed with their geographical coordinates in Table 1. The botanical identity of these two species was finalized (Table 2) at the Department of Botany, Scientific Institute in Rabat. The samples of leaves and flowers were collected in burlap bags and stored away from light and humidity until needed to preserve the integrity of the molecules as much as possible.

The moisture content of the plant is measured by subjecting 5 g of each plant to a drying process in an oven at a temperature between 100°C and 104°C for a duration of 24 h, following the guidelines outlined in the AFNOR standard (NF - V03-402 1985) (AFNOR NF V03-402, 1985).

The following formula was used to determine the moisture content after each species underwent three repetitions (Equation 1): MC % = ( m i − m f / m i × 100 (1) With:

MC: moisture content of the dry plant in %.

m_i: Initial mass of the plant before drying in (g).

m_f: Final mass of the plant after drying in (g).

The pH of a specific product indicates its level of acidity or alkalinity. A volume of 10 mL of heated distilled water was introduced to 2 g of the experimental specimen. Following agitation, the liquid is subjected to filtration, then cooled, and the pH is determined using a pH meter.

Following the protocol, 10 g of the powder from each plant was mixed with 100 mL of distilled water and heated for 15 min while stirring. After the mixture had cooled, it was titrated with a solution of NaOH (N = 0.1) in the presence of phenolphthalein until a persistent pink color developed (AFNOR NF V03-402, 1985).

The acidity index is determined according to the following Equation (2): I a = V × 56.1 × N / TS (2)

With:

V: Volume of the KOH solution (0.1 mol/L) in ml.

N: Normality of the KOH solution (0.1 mol/L).

TS: mass of the test sample in g.

56.1: Constant FactorTa.

According to AFNOR NF V05-113 standard, 1972 (AFNOR NF V05-113,1972), the determination of the ash content is based on the destruction of any carbon particle of a 2 g sample of each plant, under a temperature of 550°C. The operation will only be completed when the color of the residue becomes grayish-white, which will turn into a white color after cooling.

Organic matter is calculated according to the following Equation (3): OM % = W 1 − W 2 / TS × 100 (3)

With:

OM%: Organic matter.

W₁: Weight of the capsule and the sample before calcination.

W₂: Weight of the capsule and the sample after calcination.

TS: test sample.

The ash content (or mineral matter content: MM) is determined as follows: MM = 100-OM%

To determine the content of seven heavy metals (As, Cd, Cr, Fe, Pb, Sb, and Ti) in the plant matter of studied plants, the standardized mineralization protocol (NF EN, 1999-1-1/A1, 1999) was used.

This process consists of mixing the crushed plant material (0.1 g) with 3 mL of aqua regia prepared from 1 mL of nitric acid HNO₃ (99%) and 2 mL of hydrochloric acid HCl (37%), The mixture refluxes at 200°C for 2 hours. Once liquid is chilled and separated from solid particles, it is collected and filtered through a 0.45 µm pore size filter. The mixture is diluted with distilled water to 15 mL. The concentrations of heavy metals are determined by the inductively coupled plasma atomic emission spectrophotometer ICP-AES (Ultima 2 Jobin Yvon) at the TSUSR laboratory (Technical Support Unit for Scientific Research) at CNRST in Rabat (Skujins, 1998).

The essential oils of the two lavenders were obtained using hydrodistillation using a Clevenger-type device (Clevenger, 1928). A mass of 100 g of plant material, of each dried and mixed lavender, is submerged in water (1 L) in a 2 L flask. This mixture was boiled for 3 h at a temperature of 90°C. Each plant material underwent three cycles. The oil was dehydrated using anhydrous sodium sulfate (Na2SO4) and then kept in an airtight brown glass container at a temperature of 4°C until it was ready to be used.

The EO Yield was calculated according to the following formula (Equation 4): Yield  % = V EO / M 0 × 100 (4)

With:

V (EO): Volume of EO recovered (g).

M₀: Mass of plant material (100 g).

The analysis of the lavender EO samples was carried out by a Thermo Electron type gas chromatograph (Trace GC Ultra) coupled to a Thermo Electron Trace MS system mass spectrometer (Thermo Electron: Trace GC Ultra; Polaris Q MS). Fragmentation is carried out by electronic impact with an intensity of 70 eV. The chromatograph is equipped with a DB-5 column (5% phenyl-methyl-siloxane) (30 m × 0.25 mm x 0.25 μm film thickness), and a flame ionization detector (FID) powered by a mixture of H₂/Air gas. The column temperature is programmed at a rate of 4°C/min from 50°C to 200°C for 5 min. The device has a split–splitless PVT (Programmed Vaporization Temperature) injector. Split injection is employed, with a leakage ratio of 1/70 and a flow rate of 1 mL/min for the vector gas nitrogen.

The identification of the constituents of essential oils was made based on the determination and comparison of the Kovats indices (KI) of the compounds with those of the standard products known and described in the databases of Kovats (1965) and Adams (2007), By conducting a comparison of the peak retention periods with the known legitimate standards present in the authors’ laboratory, as well as comparing the stated KI and MS data with the mass spectral database standards of WILEY and NIST 14, along with published literature.

The density of an essential oil is determined by using a pycnometer at a temperature of 20°C. The term refers to the ratio between the density of the oil and the density of pure water at the same temperature. The determination was made based on the following formula (Equation 5). d 20 = m 2 − m 0 / m 1 − m 0   (5)

With

m₀: Mass in grams of the empty pycnometer.

m₁: Mass in grams of the pycnometer filled with water.

m₂: Mass in grams of the pycnometer filled with oil.

The acid value is the number of mg of KOH necessary for the neutralization of the free acids contained in 1 g of EO. The free acids are neutralized with a titrated ethanolic solution of potassium hydroxide (KOH). In our case, we used a 0.1 N KOH solution. Phenolphthalein is used as a color indicator.

The acidity result is expressed in % oleic acid by the following formula (Equation 6): Acidity  % = V * N * M A / 10 * TS  and therefore , acid value = N * V − V 0   * M KOH / TS (6)

With:

V: Buret drop volume (in ml).

N: Normality of the KOH solution.

TS: Test sample in g.

M_A: Molecular weight of oleic acid = 282 g/mol.

The Iodine value (IV) is a crucial analytical feature of oil that serves as an indication of its unsaturation. It is calculated according to the ISO 3961 method, by determining the number of grams of iodine attached to the double bonds present in 100 g of fat.

A 0.1 g quantity of the oil is added to a 100 mL Erlenmeyer flask along with 4 mL of chloroform and 5 mL of iodine monochloride (Wijs reagent). The solution is agitated gently and then stored in a dark location for 1 h at a temperature of 20°C. Subsequently, 4 mL of a potassium iodide solution with a concentration of 10% and 30 mL of distilled water are introduced.

The resulting mixture is titrated with sodium thiosulfate (0.1 N) using a few drops of starch solution as a colored indicator. The titration is continued until the blue color disappears. A blank test without oil is conducted at the same time and under the same circumstances.

The results are expressed as follows (Equation 7): IV = gde  I 2 / 100  gd ’ oil = 126.9 * N * V 0 − V 1 / TS (7)

With:

IV: Iodine value.

V₀: Volume of sodium thiosulfate used for the blank titration.

V₁: Volume of the sodium thiosulfate solution.

N: Normality of the thiosulfate solution.

TS: Test sample.

Peroxide value (PV) is the most widely used chemical technique for evaluating the oxidative degradation of unsaturated fatty acids in essential oils.

According to regulation CEE/2568/9, the prescribed amounts for the combination are as follows: 0.5 g of oil in a container with a volume of 100 mL, 4 mL of dichloromethane, 6 mL of acetic acid, and 0.1 mL of an aqueous solution containing saturated potassium iodide (15 g of potassium iodide diluted in 10 mL of distilled water). Afterward, the bottle is tightly sealed and forcefully shaken for a period of 1 min. Afterwards, the balloon is left undisturbed in darkness for 5 min, and then 10 cc of distilled water is added.

The liberated iodine is quantitatively determined by aggressively swirling it with a 0.1 N thiosulfate solution in the presence of starch as a chromogenic indicator. A control experiment, devoid of any oil, is conducted simultaneously and under identical circumstances.

The peroxide index (PI) is expressed in milliequivalents of oxygen per kg of oil (Equation 8). PV  M eq   O 2 / Kg = V * 1000 * N   / ST (8)

With:

V: Volume poured with sodium thiosulfate (in ml).

TS: Oil sample to be analyzed in g.

N: Normality of sodium thiosulfate solution.

The assessment of the antibacterial efficacy of the essential oils (EOs) derived from two types of lavender was conducted on a total of 10 microorganisms (Table 3). All strains (bacterial and fungal) were isolated from the hospital environment: Mohamed V Provincial Hospital-Meknes. These specific microbes are pathogenic, renowned for their robust antibiotic resistance, as well as their ability to invade and produce toxins in humans. These strains were taken from a 20% glycerol stock at −80°C, rejuvenated on Mueller Hinton and Sabouraud broths, and subcultured before use (Ailli et al., 2023).

The minimum inhibitory concentration (MIC) is defined as the lowest concentration of essential oil capable of inhibiting the growth of a microorganism (Kuete,2 004). The determination of the MIC was carried out using the microdilution method (Balouiri et al., 2016; Ailli et al., 2023). The MIC value of each extract was determined using a sterile 96-well microtiter plate. A microtiter plate was prepared by transferring 100 µL of Mueller-Hinton broth for bacteria and Sabouraud for fungi to all wells. From a stock solution of the essential oil prepared in 10% DMSO, a dilution series was carried out to obtain concentrations of 5 to 0.93 10⁻² mg/mL of each EO. And then, 10 μL of inoculum 10⁶ CFU/mL (bacteria) or 10⁴ CFU/mL (fungi) was added to all wells. The microplates were incubated for 24–48 h at 37°C. After incubation, 10 µL of resazurin (5 mg/mL) is added to each well as an indicator of microbial growth. After a second incubation for 2 h at 37°C, microbial growth was revealed by the change in color from purple to pink. The MIC value is determined as the lowest concentration that prevents a color change of resazurin. To determine the minimum bactericidal concentration (MBC)/minimum fungicidal concentration (MFC), 10 µL was taken from each well without visible growth and inoculated in Mueller Hinton (MH) agar for bacteria or in Sabouraud for fungi. The boxes are incubated for 24 h at 37°C. The MBC and MFC were defined as the lowest concentrations of the analyzed samples that produced a 99.99% reduction in UFC/mL compared to the control (Bachiri et al., 2016). The MBC/MIC or MFC/MIC ratio is calculated to evaluate the antimicrobial potency. If this ratio is less than 4, the effect of the EO is bactericidal/fungicidal, if the ratio is greater than 4, the sample has a bacteriostatic/fungistatic effect.

The GC-MS analysis detected total 56 phytochemicals from L. stoechas and L. abrialis were used as a ligand for docking analysis. For virtual screening, all ligands are downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) in SDF format.

For in silico antibacterial and antioxidant analysis, the x-ray crystal structure of sarA protein of Staphylococcus aureus (PDB ID: 2fnp) and NADPH oxidase from Lavandula sanfranciscensis (PDB: 2CDU) were retrieved from the protein data bank (https://www.rcsb.org/). The Discovery Studio software was employed to clean the structure of the selected proteins obtained from the Protein Data Bank. Heteroatoms were eliminated during this process. Subsequently, utilizing the GROMOS96 43b1 force field and the SwissPDB Viewer software, the energy of the refined protein structures was subjected to minimization and optimization procedures.

The molecular docking analysis involving the interaction of phytochemicals derived from L. stoechas and L. abrialis with target proteins was performed using the PyRx software through the autodock wizard. The protein structure underwent conversion into a macromolecule, while the ligands were transformed into the PDBQT format. For the docked complexes, the center and grid box size were as on their binding pocket. The final docking calculations were executed using PyRx, and the selection of top molecules was based on lower binding energy. Subsequently, Discovery Studio software was employed to investigate the binding interactions and poses of the docked complexes.

The water content of L. stoechas and L. abrialis varied between 26.26% and 20.10% respectively. Several factors could influence the moisture content of plants such as the nature of the fibers, the age of the plants, the condition of the soil, and the shelf life of the plant after harvest (Bachiri et al., 2016).

The hydrogen potential provides information regarding the assimilation of nutrients by plants (Saidi et al., 2023). In the present work, the p H   o f   L . abrialis and L. stoechas ranged from 4.96 to 5.94, respectively. These values allowed these two lavenders to absorb nutrients such as iron, which was confirmed by the results of the ICP analysis.

The analyses performed on studied samples showed that the amounts of organic matter in L. stoechas and L. abrialis were approximately 91.83% and 91.04%, respectively. However, the amounts of mineral matter (or ash) were low, coming in at only 8.17% and 8.96%, respectively (Table 4). These findings showed that organic matter represented most of the weight of these plants.

Atomic absorption spectrophotometry is used to determine the concentration of heavy metals (Cr, Sb, Pb, Cd, Fe, and Ti) in two studied lavender plants. According to the results shown in Table 5, several metals were undetectable and had very low and fluctuating quantities. Additionally, the analyzed samples had values that were below the FAO/WHO (2009) limit threshold.

Hydrodistillation was used to extract the essential oils from the aerial parts of L. stoechas and L. abrialis. The essential oils produced ranged in color from light yellow to clear yellow but were always extremely strongly scented.

The yield of essential oils varies greatly from one species to another, according to the results obtained in Table 6, we noted that Lavandula abrialisa gave a good yield of essential oil (2.9% ± 0.13%) and L. stoechas revealed almost 2.3% ± 0.11%.

These findings corroborate those of Bachiri et al. (2016) and N'Dédianhoua et al. (2014), who also used hydrodistillation to recover the aerial parts of lavender plants from Morocco’s Mid-Atlas region. However, research on L. stoechas from Turkey (Ahmet et al., 2002) and Algeria (Mohammedi and Fawzia, 2011). found that their respective EO yields were low at 1.33% and 1.2%. The essential oil content of L. abrialis was slightly higher than the findings of Zrira (2007). Various factors, such as the environment, genotype, geographical origin of the plant, cultural practices, and extraction method, may contribute to differences in essential oil production between species (Aberchane et al., 2001; Bourkhis et al., 2009).

The GC/MS analysis of the essential oil of L. abrialis revealed 56 chemical compounds, totaling 99.98% (Table 7), including 72.13% oxygenated monoterpenes, 18.59% oxygenated sesquiterpenes, 7.7% hydrocarbon monoterpenes, and 1.56% sesquiterpenes (Figure 5A).

The main volatile compounds of the essential oil extracted from the flowers and leaves of L. abrialis (Figure 3) were linalool (19.16%), linalool acetate (13.41%), camphor (11.24%), borneol (9.88%), and cineole <1.8->(9.28%). This EO also contained other compounds such as terpineol<α-> (5.99%), lavandulylacetate (2.88%), terpinen-4-ol (2.63%), geranylacetate (2.45%) and geraniol (2.12%).

Our results corroborate with studies carried out in Mediterranean areas with small differences in the percentages. N’Dédianhoua et al. (2014) showed that linalool (25.86%), linalool acetate (13.66%), camphor (16.06%), borneol (11.94%), and Cineole<1.8-> (16.04%) were the main components of L. abrialis EO collected in the Mid-Atlas of Morocco. Zrira (2007) mentioned that the EO of this species is dominated by linalool (33.7%), camphor (17.6%), cineole <1.8> (14.5%) and linalylacetate (13.5%).

The analysis of L. stoechas (Figure 4) EO also showed 56 compounds. The latter represents 100% of its total composition (Table 7). The main components of this oil were camphor (24.35%), cubenol (15.54%), fenchone (9.58%), Pinene<α-> (7.19%), camphene (6.07%) and borneol (4.45%). The compounds identified were classified into four chemical families, namely, oxygenated monoterpenes (49.93%), oxygenated sesquiterpenes (26.42%), monoterpenes (16.71%) and sesquiterpenes (6.94%) (Figure 5B).

Previous studies have noted a distinct chemical composition. According to Mohammedi and Fawzia (2011), the EO of L. stoechas collected in Telmsen, Algeria, is distinguished by the presence of 21 compounds, including camphor (18.1%), cineole (18.9%), and fenchone (27.6%). Our sample had different contents, according to the chemical analysis than those examined by Ahmet et al. (2002) for Turkish L. stoechas, where they discovered high concentrations of pulegone (40.37%), hexahydrothymol (menthol), and menthone (12.57%).

A comparison of the chemical composition of the EOs demonstrated that these two lavenders contained variable proportions of oxygenated monoterpenes and oxygenated sesquiterpenes. The EO of L. abrialis was rich in alcohol (47.1%) followed by esters (22.72%) and ketones (11.24%) (Figure 6A), while the EO of L. stoechas was dominated mainly by ketones (38.19%), alcohols (34.05%), and hydrocarbons (23.65%) (Figure 6B). In addition, other chemical families were found in the EOs of these plants but with low percentages, specifically ethers, aldehydes, and epoxides.

L.stoechas and L. abrialis both produced EOs with nearly identical densities. It fluctuated between 0.925 ± 0.001 and 0.929 ± 0.002 g/mL, respectively. These two EOs can be characterized as light oils because of their lower density (0.9982 g/mL) than water. The density of these oils complied with the AFNOR standard 2005 which sets a density of 0.906–0.990 as quality references (AFNOR, 2005; De Cliff and Harerimana, 2013).

The acid value indicates the content of free fatty acids in studied EOs. A low acidity value characterizes the purity and stability of an oil at room temperature [14]. The EOs of L. stoechas and L. abrialis had acid values between 10.09 ± 0.01 and 4.49 ± 0.003 respectively. These values indicate that L. stoechas oils were more susceptible to undergo alterations than L. abrialis EO.

The iodine value indicates the overall degree of unsaturation of the oils. The more unsaturated an oil is, the higher its iodine value (Wolff, 1968). The values of the iodine values of the EOs of the studied species (0.647 ± 0.01 for L. abrialis and 0.546 ± 0.01 for L. stoechas) were lower than those provided by the Codex Alimentarius standard (87–111 g of iodine/100 g) (Codex Alimentarius, 2017), and thus the conservation of these EOs could be done without running a significant risk of auto-oxidation.

The peroxide value is studied to assess the oxidation state of essential oils. The peroxide value of L. stoechas EO (18 ± 0.2) was higher than that of L. abrialis essential oil (14 ± 0.1). Comparing these values with those of the commercial standards CODEX STAN 210-1999, the two oils were categorized as good since they match the standards.

The in vitro analysis revealed that the extracts showed strong antibacterial activity against S. aureus, therefore we selected SarA protein of S. aureus for molecular docking analysis. The sarA locus regulates over 100 genes on the S. aureus chromosome, playing a role in the intrinsic multidrug resistance mechanism by regulating drug accumulation (O'Leary et al., 2004). This protein is considered a master regulator of biofilm formation in S. aureus, and targeting this protein has been explored by several researchers as a potential strategy for drug development (Arya et al., 2015; Hosen et al., 2023). Among the 56 phytochemicals from two extracts the top five best ligands were selected based on their lowest binding energy, interaction with target protein, pose and RMSD value shown in Table 11.

Whereas, the compounds CID: 15559789 and CID: 10398656 exhibited maximum binding energy with −9.8 kcal/mol and 9.3 kcal/mol respectively (Table 1). The complex between 2fnp + CID: 15559789 was stabilized with highest four hydrogen bonds at A:Phe110, A:Leu113, B:Tyr142, and B:His159 (Figure 10A) while complex 2fnp + CID: 10398656 exhibited three hydrogen bond at A:Phe110, A:Leu113, and B:His159 (Figure 10B). The binding of these compounds at the active site of SarA protein could inhibit the pathogenesis of S. aureus (EF1).

This study also involved molecular docking study of NADPH oxidase (PDB: 2CDU) from L. sanfranciscensis to screen phytochemicals with the goal of identifying the most specific and potent inhibitors. NADPH oxidase is recognized for generating reactive oxygen species (ROS) at normal physiological levels. However, under certain disease conditions, it can become excessively activated, leading to the production of an excess of ROS. Several compounds have demonstrated the capacity to inhibit the overactivity of the enzyme (Kumar Deb et al., 2022; Kandsi et al., 2022). Interestingly, there limited published study has been reported on the inhibitory activity of L. stoechas and L. abrialis phytochemicals on NADPH oxidase. In the molecular docking study, from 56 selected phytochemical compounds of L. stoechas and L. abrialis, top 5 compounds were chosen based on the lowest binding energy (Table 12).

In this case, against the target 2cdu, the highest binding energy was observed in the complex 2cdu + CID: 522296 with −10.8 kcal/mol followed by complex 2cdu + CID: 9855795 with binding energy −10.1 kcal/mol. The docking results revealed that to inhibit the 2cdu, the compound CID: 522296 showed five hydrogen bond at A:Pro432, B:His10, B:Phe14, B: Lys17, and B:Tyr62 (Figure 11A). Similarly, the complex 2cdu + CID: 9855795 stabilized with target protein with three hydrogen bond at A:Phe14, A:Lys17, B:Phe433, and B:Ile438 (Figure 11B).