As part of our study, we collected areal part of O. elongatum in the Targuist region (34°43′29″ N; 4°18′21″ W) (Fig. 1). The samples were collected aerial plant part at three key points in the OE life cycle: the vegetative period (April 2023), the flowering period (June 2023) and the post-flowering period in August 2023 (Fig. 2). From these samples, we were able to carry out chemical and biological analyses of EO in the early, middle and late stages of their development. The plant material was dried in the shade and subjected to hydrodistillation using a Clevenger apparatus. The obtained EO was stores at 4°C until use.

The climate of the region varies considerably during the months that are most important for the development of the plants. In April, during the vegetative stage, the average temperature is around 11°C, providing relatively mild conditions for plant growth. The plants start growing vigorously, with maximum temperatures reaching 16°C. However, the number of rainy days (a day on which measurable rainfall occurs, which is commonly defined as ≥1 mm of precipitation) in April was 6.6 days, with a total rainfall of 45.2 mm. This may affect the health and growth of the crop. In June, when the plants are flowering, the climate warms considerably, with an average temperature of 19°C and a maximum up to 24°C. This increase in temperature promotes flower blooming. In addition, the number of rainy days drops dramatically to an average of only 1.6 days with an average rainfall of 10.2 mm. In August, during the post-flowering period, temperatures remained warm with an average of 22°C, although the maximum and minimum values did not differ much from those of June (Fig. 3). On the other hand, the number of rainy days remained low, with an average of only 0.6 days and a minimum rainfall of 3.4 mm.

The GC-MS analyses were carried out on three samples of OEEO corresponding to three phenological stages (vegetative, flowering, and post-flowering). The gas chromatography-mass spectrometry was performed using a GC-MS-TQ8040 NX (Shimadzu) equipped with a triple quadrupole detector. The compounds were isolated on a non-polar RTxi-5 Sil MS capillary column (30 m × 0.25 mm ID × 0.25 µm). Helium was used as the carrier gas. The injection volume was set at 1 µL in splitless mode, with the split being opened after 4 min. The injection temperature was set at 250°C, with a pressure of 37.1 kPa. The temperature program started at 50°C/min (held for 2 min), followed by two ramps: 5°C/min to 160°C (held for 2 min), then 5°C/min to 280°C (held for 2 min), for a total analysis time of 50 min. The ion source and interface temperatures were 200°C and 280°C, respectively. The acquisition was performed in full scan mode, and the compounds were identified using the NIST library version 2019, and Hexane was used as a dilution solvent for the samples. To obtain the percentage of relative peak area for the EOs, firstly, we add up all the areas of all the peaks. Then we divided the area of each peak by the total area and multiplied the results by 100 to obtain the percentage of each EO component [17]. The GC-MS analyses were performed in triplicate to ensure methodological rigor and reproducibility.

All assays were conducted in triplicate, and the data are reported as mean ± standard deviation. Statistical analyses were carried out using GraphPad version 9. Mean differences were evaluated by one-way ANOVA followed by Tukey’s multiple comparison test, with significance accepted at p < 0.05.

The GC-MS analysis of the essential oils of O. elongatum from three different phenological stages, including vegetative, flowering, and post-flowering (Fig. 4 and Table 1), revealed a diverse range of chemical composition (22 compounds), with monoterpenes representing the dominant class of compounds. The extraction yields were found to be 4.9%, 5% and 2.1% for OEEO-VS, OEEO-FS, and OEEO-PFS, respectively. The predominant compounds identified in all three samples were p-cymene (26.83%_31.45%), γ-terpinene (8.46%_26.95%), thymol (13%_29.54%), and carvacrol (20.25%_37.26%), with notable variations according to the phenological stage of the samples. The predominant chemical group was oxygenated monoterpenes, representing 49.22%, 69.29% and 62.76% of the total composition, respectively, for OEEO-VS, OEEO-FS, and OEEO-PFS. This was followed by hydrocarbon monoterpenes, which constituted 41.92%, 25.16% and 34.63% of the total composition, respectively. The presence of sesquiterpenes was observed in minimal quantities in OEEO-VS (4.11%) and OEEO-FS (2.62%), whereas no such compounds were identified in OEEO-PFS (Table 2). However, OEEO-FS was characterized by particularly high levels of carvacrol (37.26%) and thymol (29.54%), while OEEO-PFS had the highest concentration of γ-terpinene (26.95%) and p-cymene (31.45%). These results highlight the significant influence of the phenological stage on the chemical composition of OEEO.

The chemical composition of OEEO is comparable to that of other species within the Origanum genus, yet it possesses distinctive characteristics. In their studies of O. compactum, Bouyahya and colleagues observed that oxygenated monoterpenes (49.4–62.975%) and hydrocarbon monoterpenes (31.815–43.632%) constituted the main chemical groups, with carvacrol, thymol, p-cymene, and γ-terpinene identified as the major compounds. The proportions of carvacrol and thymol exhibited notable variation across the three phenological stages, at the vegetative stage, they constituted 27.71% and 15.32%, respectively. In contrast, at the flowering stage, carvacrol increased to 43.58%, while thymol decreased to 10.33%. At the post-flowering stage, this trend was reversed, with 38% thymol and 6.4% carvacrol being the predominant compounds [14,25]. A similar trend was observed in O. vulgare in a previous study, which reported that carvacrol was the predominant compound at all stages (61.08–83.37%), followed by p-cymene (3.02–9.87%) and γ-terpinene (4.13–6.34%) [22]. Morshedloo and colleagues additionally observed the preponderance of carvacrol (18.1–79.2%) in the EO vulgare subsp. gracile, with elevated concentrations in the flowers (79.2%), roots (70%), and early vegetative stage (67.34%) [26]. A further study revealed that thymol constituted the principal compound (46.90–62.26%) in O. vulgare EO at all stage of development, with γ-terpinene (1.11–11.75%) and p-cymene (3.11–5.32%) ranking as the next most abundant components [27]. However, Moradi and colleagues reported that the EO content was highest at the post-flowering stage (1.87%) and the lowest at the pre-flowering stage (1.01%) in O. vulgare, with carvacrol, p-cymene, and γ-terpinene as the main components [28].

It is of great importance to highlight that the chemical composition of EO is subject to variation due to a multitude of factors that influence the biosynthesis of secondary metabolites. The geographical origin of the plant, its exposure, the altitude of its habitat, climatic conditions, in particular temperature, the genotype of the plant, and edaphic factors linked to the soil are among the factors that contribute to the variation in chemical composition observed in EOs [29]. The aforementioned diversity of parameters provides an explanation for the observed variations in the chemical composition of our study in comparison with other oregano species analyzed in the scientific literature.

The results of the evolution of the antibacterial activity of OEEO as a function of its stage of development, using the disc diffusion method, show notable variations in antibacterial efficacy for the three phytopathogenic bacteria tested (Fig. 5a). The inhibition zones measured for E. amylovora were 47 ± 3.1 mm for OEEO-VS, 58 ± 2.01 mm for OEEO-FS, and 46 ± 1.13 mm for OEEO-PFS. When subjected to OEEO-VS, OEEO-FS, and OEEO-PFS, A. tumefaciens C58 exhibited inhibition zones of 45 ± 1.12, 47 ± 1.45, and 46 ± 1.32 mm, respectively. However, A. vitis S4 displayed inhibition zones of 34 ± 2.4, 52 ± 2.21, and 47 ± 3.32 mm for OEEO-VS, OEEO-FS, and OEEO-PFS, respectively. The findings showed that the OEEO-FS was the most effective, with the largest inhibition zones for the three phytopathogen tested, and the most sensitive to the essential oils tested was E. amylovora (Fig. 6). In the case of microatmospher assay, the inhibition zone caused by the volatile compounds present in OEEO are presented in Fig. 5b. With the OEEO-VS, E. amylovora and A. tumefaciens C58 displayed the most extensive zone of inhibition, measuring 30 ± 2.1 and 30 ± 3.2 mm, respectively. Additionally, E. amylovora reported a 26 ± 3.2 mm zone of inhibition for OEEO-FS. In summary, the biggest zone of inhibition for E. amylovora and A. tumefaciens C58 was 29 ± 1.07 and 29 ± 2.5 mm in the present of OEEO-PFS. On the other hand, the results of MIC demonstrate that OEEO exhibits antibacterial activity towards the three phytopathogen strains tested, including E. amylovora, A. tumefaciens C58, and A. vitis S4. However, the degree of sensitivity exhibited varies according to the phenological stage of OEEO (Table 3). Overall, OEEO-FS demonstrated the greatest efficacy, particularly against E. amylovora, exhibiting an MIC of 62.5 µg/mL, lower than that of the positive control. For A. vitis oils from vegetative and flowering stages exhibited equivalent activity (125 µg/mL), which was twice as potent as that observed in the post OEEO-PFS (250 µg/mL). A similar trend was observed for A. tumefaciens, with the OEEO-VS and OEEO-FS exhibiting more pronounced activity (140 µg/mL). In contrast, OEEO-PFS demonstrate reduced efficacy (300 µg/mL). In fact, a study has shown that O. elongatum EOs fractions obtained between 141 and 160 min of hydrodistillation exhibited the highest activity, with MICs ranging from 0.0312 to 0.125%(v/v) and MBCs ranging from 0.0312 to 0.25% (v/v) [30]. Furthermore, another study on the antibacterial efficacy of O. elongatum reported MICs ranging from 2 to 8 µL/mL against several bacterial strains, except for Staphylococcus aureus (MIC = 32 µL/mL) [5]

The aforementioned studies irrefutably demonstrate that the EOs of assorted medicinal plants exhibit pronounced antibacterial activity, with discernible variations contingent on the phenological phase. The analysis of Origanum vulgare subsp. glandulosum revealed zone of inhibition ranging from 9 to 36 mm and the MICs of 125 to 600 µg/mL. against Bacillus subtilis [22]. However, the efficacy of the vegetative and flowering stages of Origanum compactum against Staphylococcus aureus was also confirmed, with remarkable inhibition diameters of 47 mm and best bactericidal action (MIC = MBC = 0.0312%(v/v)) [23]. In a separate study, the researchers investigated the antibacterial activity of Thymus daenensis and Thymus fedtschenkoi EOs against diverse range of pathogenic bacteria. They observed that the plant exhibited notable antibacterial efficacy at various phenological stages, with MICs values ranging from 1 to 16 mg/mL and 2 to 32 mg/mL, respectively [31]. Nevertheless, a comprehensive examination of Salvia officinalis at all three developmental stages revealed a consistent and pronounced antibacterial efficacy. The most sensitive bacteria were Listeria monocytogenes, followed by Bacillus subtilis, Staphylococcus aureus, proteus mirabilis, Escherichia coli, and salmonella typhimurium, with slight superiority at the flowering stage [32]. This trend towards optimum efficacy at the flowering stage was also observed in Pelargonium graveolens against different bacterial strains, with zones of inhibition ranging from 11 to 17.30 and MICs of 0.25 to 2% (v/v) [33]. Notably, Thymus maroccanus yield particularly impressive results, demonstrating maximum activity against both Gram positive (21–48 mm and Gram negative (9.7–23.7 mm) bacteria, and the MICs (0.13 to 0.95 mg/mL at different stages of development [34].

The antioxidant potential of OEEO was evaluated using DPPH and FRAP tests, which yielded distinct activity profiling according to phenological stage (Table 4). This suggests an intriguing variation in antioxidant power. The DPPH test results demonstrated that the OEEO-PFS exhibited the most potent antioxidant effect, with an IC₅₀ value of 132.01 ± 2.06 µg/mL. This was followed by OEEO-VS (IC_{50 =} 147.01 ± 0.78 µg/mL), whereas OEEO-FS displayed the lowest activity with an IC₅₀ of 168.25 ± 1.14 µg/mL. However, although these results are promising, they are still less effective than the reference antioxidant BHT (IC_{50 =} 63.21 ± 0.03 µg/mL) and ascorbic acid (IC_{50 =} 41.32 ± 0.08 µg/mL). While the results of the FRAP test demonstrated a notable variation in the antioxidant activity of OEEO across the different phenological stages of the plant. The most pronounced antioxidant effect was observed in OEEO-FS, with an EC₅₀ of 164.22 ± 1.04 µg/mL, followed by OEEO-PFS (184.06 ± 0.95 µg/mL). In contrast, the OEEO-VS showed the lowest activity, with an EC₅₀ of 215.73 ± 1.48 µg/mL. While these values indicate an intriguing antioxidant potential, they remain inferior to that of BHT (89.44 ± 0.13 µg/mL), which serves as reference antioxidant.

A comparison of our results with those of previous studies revealed both similarities and differences in the antioxidant ability of our OEEO, depending on the phenological stage. However, for Origanum vulgare subsp. gracile, Morshedloo, and colleagues observed maximum antioxidant activity in the flowering stage (EC₅₀ = 0.86 mL/mL), while the vegetative and fruiting stages showed lower activity [26]. Conversely, another study demonstrated a progressive increase in antioxidant activity for Origanum compactum, with antioxidant capacity rising from EC₅₀ = 94.18 to 53.28 µg/mL and IC₅₀ values falling from 320 to 172.87 µg/mL, between the vegetative flowering and post flowering stages of growth [23]. In contrast, Origanum majorana exhibited higher antioxidant activity at the late vegetative stage (IC50 = 40–50 µg/mL) compared to the early vegetative and flowering period [35]. However, a study demonstrated that the antioxidant activity of Origanum vulgare ssp. Hirtum reaches its peak during the full flowering stage (7.711 ± 0.253 µmol/g), subsequently declining significantly at the post flowering phase [36]. Nevertheless, an investigation on Origanum vulgare L fined that the potential antioxidant of his EOs at various stages of growth range between EC₅₀ = 7.91 and 8.59 mg/mL [37]. Additionally, the anti-radical activity expressed by IC50 of Origanum vulgare subsp glandulosum (Desf.) Ietswaart was varied from 59.2 mg/L to 226.19 mg/L [38]. In another work on the effect of the development stage of Rosmarinus officinalis L. var. typicus Batt, find a high of ability of EOs to trap the free radicals of DPPH (IC₅₀ = 17.3 µg/mL for vegetative EO and IC₅₀ = 8.59 mg/mL for fruiting stage) [39]. The observed variations in antioxidant activity at different phenological growth stages may be ascribed to alterations in the chemical structure of the EOs occurring during the plant’s development. This hypothesis is supported by the findings of supplementary studies, including [37]. Geographical origin, exposure to sunlight, altitude, soil climate, and plant genetics can also modify the chemical content and therefore the antioxidant capacity of the plant [25,40,41].