The samples were harvested in the spring of 2021 in the Zaër area. After harvesting, the plant material was cleaned and rinsed to remove all debris. Drying was conducted under shade in an airy location and subsequently stored in dark, well-closed boxes until use. The botanical identification of the samples was performed by a team from Ibn Tofail University, Laboratory of Plant, Animal, and Agro-Industrial Production, Faculty of Science, under the supervision of Prof. ZIDANE Lahcen.

0.1 Kg of plant material (Mentha pulegium L) and distilled water (600 cm³) were placed in a flask, and the steam distillation process was carried out using the Clevenger device for 3 h. Our collected essential oil (volatile part) was stored in the dark at 277.15 K for later use. Oil storage required well-capped bottles protected from air and sunlight in order to prevent any risk of oxidation and polymerization. We calculated the essential oil content in relation to the total dry mass of the plant (Rached et al., 2022, 2023, 2024). The yield of EO was obtained by applying the following formula:

Where M₁: mass of EO (Kg). M₀: dry plant mass (Kg).

Physico-chemical properties enabled us to control and manage the quality of essential oils. For this reason, we calculated the following physico-chemical indices: refractive index, ethanol miscibility, peroxide value, acid value, ester value, and saponification value according to a methodology described in international standards (NM ISO) and the protocols of Rached et al. (2022).

Our EO was analyzed at the Center Universitaire of Analysis, Expertise, Technology Transfer and Incubation CUE2TI, Université IBN Tofail Kenitra, and Technical Support Units for Scientific Research UATRS, CNRST Rabat, using a type 456-GC gas chromatograph coupled to an EVOQ TQ mass spectrometer. The energy applied was 70 eV, and the impact of the electrons caused fragmentation. The column is a capillary Rxi-5Sil MS column (30 m 0.25 mm ID 0.25 μm df). The column temperature was initially set at 313.15 K for 8 min, then increased by 275.15 K/min for the next 20 min. Helium was used as the carrier gas, with a flow rate of 1.50 mL/min. The injection volume was 1 μL and the injection temperature was maintained at 523.15 K. The instrument was managed by MS Data review software and connected to a computer system hosting a NIST MS Search 2.0 mass spectra collection.

The study protocol was approved by the Ethical Institutional Committee of the Faculty of Sciences at University Ibn Tofail, Kenitra, Morocco. All experimental procedures involving laboratory animals was carried out in compliance with the organization for economic cooperation and development (OECD) guidelines no. 42 (OCDE, 2002). An esophageal probed was used to administer the test substance in a single dosed. Three healthy, non-pregnant female swiss mice fasted for 3 to 4 h with watered were used at each stage of the experiment. The weight of the animals was measured after fasting, and the test substance was administered. In our case, a dosed of 300 mg/kg was administered, followed by a dosed of 2000 mg/kg, while distilled watered was administered to another group (the controlled group). After administration, the animals was again deprived of food for 1 to 2 h (OCDE, 2002; Oubihi et al., 2023).

Lethal dosed 50, often referred to as LD₅₀, was a measured of a compound’s acute toxicity or short-term hazardous potential. The quantity of active ingredient was measured in milligrams per kilogram of animal body weight; the lowered the figure, the greater the toxicity of the product (OCDE, 2002). Mortality was recorded for 14 days after administration of the extracts tested, and the LD₅₀ was determined.

The fungal strains studied, Fusarium pseudograminearum and Fusarium moniliforme, belong to the mycotheque of the plant, animal and agro-industrial production laboratory at the Faculty of Science, Kenitra. They were isolated from saffron and regularly transplanted onto PDA (potato dextrose agar) culture medium. Morphological characteristics was used for identification, which was then confirmed at CNRST in rabat.

The antifungal power of our essential oil was tested in our study using the direct contact method (Mzioud et al., 2023; Rached et al., 2023) on isolated mold species, namely Fusarium pseudograminearum and Fusarium moniliforme. The procedure involved pouring a volume V of essential oil or extract into 20 cm³ of PDA medium in test tubes. After shaking, glass Petri dishes with a diameter of nine cm was filled from the medium. For inoculation, a disk of mycelium measuring 0.6 cm in diameter was placed in the middle of the dish after preculture for 3 to 7 days at 301.15 K. As a control, a Petri dish containing 20 cm³ of PDA without EO was inoculated. For each concentration, three tests were performed. The antifungal index was determined after 7 days incubation at 28°C, taking into account the expansion of the control mycelium:

Where, Da and D_b represent the dimension of the test growth zone and is the dimension of the control growth zone.

The activity of our essential oil on sporulation was evaluated with the cultures where the fungistatic effect was observed. The Malassez cell was used to count the spores recovered after dilution in distilled water, and the results was expressed as the number of spores/mL of suspension. The following formula was used to calculate the number of spores:

The Muller-Hinton agar diffusion method was used to measured antibacterial activity. The agar medium was placed on a petri dished with a microbiological solution having an optical density of 1 McFarland (Božović et al., 2015). The EO has been assessed was placed on 6 mm diameter Whatman absorbent paper disks. The disks were then placed on the surface of the seeded agar, and incubated for 24 h at 310.15 K. After incubation, the appearance of an area of inhibition surrounding the impregnated disks, in which no micro-organisms develop, demonstrates their sensitivity to EO. EO diffuses uniformly as soon as the impregnated disks were applied. The size of the inhibition area determines the sensitivity of a given pathogen. Penicillin (5 μg), oxacillin (5 μg) and amoxicillin (25 μg) were used individually as positive controls (Oubihi et al., 2020).

The docking technique was equipped with tools that facilitate understanding the interactions between a presumed rigid active site and a ligand molecule, making it a valuable approach in drug discovery (Er-rajy et al., 2023a, 2023b). Molecular docking investigations was conducted to evaluate the interactions of compounds, identified through GC–MS based on major peak areas, focusing on three activities: antifungal, antoxidant and antibacterial (Ahmed et al., 2018).

The two major ligands, Menthone and Pulegone, was downloaded from the PubChem database, and biomacromolecules (Code ID: 6PXX, 2BJI, 3K4Q and 2CDU) was acquired in protein data bank (PDB) format (Gill et al., 2005; Papp-Wallace et al., 2019). Ligands was prepared as PDB files to facilitate molecular research using various software tools, including Discovery Studio and ChemOffice (Baroroh et al., 2023). In addition, Discovery Studio was used to prepare the proteins obtained from the PDB base. The ligands’ structures was docked into the enzyme’s active site using AutoDockTools (Morris et al., 2008). The interactions between enzyme active-site residues and ligand molecules was analyzed using Discovery Studio Visualizer, which included examining 2D interactions (Er-rajy et al., 2023a).

To represent yield, a percentage is used, calculated on the basis of 100 g of dry plant material. By hydrodistillation, the essential oil yield for Mentha pulegium L., is 2.31%. This proportion is the same as that found by Bouhaddouda et al. (2.69%; Bouhaddouda et al., 2016). and higher than that reported by Derwich et al., (1.66%; Derwich et al., 2010) and Aljaiyash et al., between 1.10 and 1.78% (Ahmed et al., 2018). So if we compare the yields for the same species according to the condition of the fresh plant (1.43%) or dry, MP in the dry state is more reproducible (Rached et al., 2024). Several factors may explain this difference in performance, including variation in botanical origin, environmental factors and cultivation practices, maturity and the part of the plant used, external factors (temperature, humidity, drought, etc.), seasonal variation and photoperiod.

In the field of pharmacology, the capacity of a plant or even a natural principle to act is not enough to justify its therapeutic use. Indeed, any bioactive molecule runs the risk of having negative effects on human health, at least over the long term and in high doses. Beyond its performance, the active dose must be purified of any toxicity and made to appear harmless. It is essential to determine the benefit/risk ratio of any substance within the framework of a therapeutic indication (El Omari, 2021; Maniben et al., 2023). Toxicity tests are essential for estimating the level of damage caused by compounds to biological and non-biological materials. This type of testing is often carried out on potential products in order to develop new drugs and determine the therapeutic potency of a drug molecule. Acute toxicity tests define the effect of a single dose on certain species of laboratory animals (Falya et al., 2020; Oubihi et al., 2023).

In accordance with OECD Guideline 423, the acute toxicity of MP HE was determined. The results are summarized in Figures 2A,B. General behavioral control is one of the essential indicators to be checked, as are changes in body weight, which are among the early signs of toxicity. During the 14 days of EO administration, the behavior of our mice was monitored and mortality recorded. We emphasize that our essential oil administered at levels of 300 mg/kg and 2000 mg/kg showed no symptoms of toxicity or mortality, and all animals tested survived the 14 days of observation.

Monitoring the increase in body weight of mice given mint oil (Figure 2B), we noted a rapid and significant decrease in body weight, which stabilized from day 5 onwards. We know that the adverse effects of a drug or substance are accompanied by a change in body weight, which may explain the changes in body weight observed during the first 4 days of the experiment (Deyno et al., 2020). We assume that the majority pulegone in the essential oil (80.03%) is responsible for this effect. All these data imply that the lethal dose (LD₅₀) of our sample is greater than 2000 mg/kg.

According to numerous scientific studies, antioxidant drugs are the only approach to preventing and treating oxidative stress-related diseases such as Alzheimer’s and cancer. However, given their side-effects and high cost, most people choose to treat themselves with herbal remedies, which have fewer side-effects (Eddine et al., 2015; Haida et al., 2022; Rached et al., 2022).

Plants are known to generate a number of antioxidant molecules, such as phenolic acids, flavonoids and tannins, whose ability to prevent ROS-induced oxidative stress is very important (Eddine et al., 2013; Ahmed et al., 2018). Compared with synthetic antioxidants, these are used in preventive medicine with few or no negative side effects (Laouini and Ouahrani, 2017; Omari et al., 2019). Table 3 shows the IC₅₀ values representing the concentration of our EO to inhibit 50% of the DPPH / ABTS free radical. Analysis of the values obtained enabled us to conclude that ascorbic acid has a very powerful antiradical activity, with IC₅₀ = 0.086 mg/mL and linear regression (R₂ = 0.993). This value is similar to that obtained by Bentabet et al., (0.08 mg/mL; Bentabet et al., 2014).

In addition, our essential oil has a very interesting DPPH free radical scavenging capacity, with an IC₅₀ equal to 1.550 mg/mL. This can be explained by the nature and type of chemical constituents present in our oil, e.g., Menthone, Eugenol, Menthol, Pulegone and Limonene, etc., which are known for their antioxidant effects. In our case, we can assume that the IC₅₀ = 1.550 mg/mL of MP essential oil is due to pulegone (80.038%), the majority compound in this oil, which is known to be a good antioxidant, which may also justify the high antioxidant power of our EO, unlike that found by Bouhaddoda, where she noted the total absence of pulegone (Bouhaddouda, 2016). As a result, our findings on DPPH radical scavenging activity demonstrated that our tested plant could be used as a promising source of antioxidant molecules, while respecting the notion of dose dependency.

On the basis of the results illustrated in Table 3, we observe that the EO studied displays inhibition of ABTS^●+ radicals. The IC₅₀ value is 29.320 mg/mL. Nevertheless, it revealed a lower scavenging activity than that of the potent synthetic antioxidant ascorbic acid (IC₅₀ = 0.002 mg/mL). We share similar results to that found by Ait chaouche, who revealed that Lamiaceae, principally pennyroyal mint, had the most potent antioxidant activity (Ait Chaouche, 2018).

Compared with other Moroccan aromatic and medicinal plants, our EO is more effective than Myrtus communis (Myrtaceae) essential oil with IC₅₀ = 4748.33 μg/mL, Allium sativum (Liliaceae) essential oil with IC₅₀ = 3.630 mg/mL (Liliaceae) and almost equal to Artemisia herba alba (Lamiaceae) IC₅₀ = 1118.50 ± 3.97 μg/mL, Lippia citriodora (Verbenaceae) IC₅₀ = 1335.63 μg/mL and Marrubium vulgare L., (Lamiaceae) with a 1.136 mg/mL (Bouyahya et al., 2017; Rached et al., 2022; Mzioud et al., 2023).

The antifungal power of plant drugs has been the subject of numerous in vitro experiments. Many of these have focused on the potential application of EOs and plant extracts in the biological control of microscopic fungi (Rached et al., 2022; Mzioud et al., 2023). The results of our research show the inhibitory effect of our M. pulegium L. plant on the fungal strains selected, and can be applied to inhibit the growth of these fungi.

Interpretation of the mycelial growth results of molds exposed to the action of our volatile fraction of different concentrations tested reveals that our results are positive and that the degree of inhibition is a function of both the concentration of EO used and the type of mold tested. In terms of figures, inhibition values range from 17.977 to 100.00% (Figure 3). MP EO was fungicidal against both fungi tested at concentrations of 2.5 and 5.00 μL/mL in Fusarium moniliforme and Fusarium pseudograminearum, respectively. This suggests that Fusarium moniliforme is more sensitive than Fusarium pseudograminearum to MP EO.

Plant extracts, and especially essential oils, have long been recognized for their antifungal properties, and the first biological control strategy was introduced with Antoferine®, a vine extract rich in polyphenols. Today, chemists, doctors, biologists and the food industry are increasingly interested in essential oils as a means of treating certain infectious diseases, for which synthetic antibiotics are becoming less and less effective, or as a means of preserving foodstuffs against oxidation, replacing synthesized chemical substances. These studies are part of the “Could plant extracts be the answer to mycotoxins, ask the plants: “Plants for plants” concept (Uwineza et al., 2018; Chelaghema, 2021).

In order to determine whether the effect of our essential oil and extracts also influenced the sporulation of our fungi, or only mycelial growth, we adopted a sporulation study. The results presented in Table 4 show sporulation rates decreasing in proportion to the concentration of the essential oil tested in the medium. However, at 0.08 and 2.50 μL/mL, the sporulation reduction rate is 100% for Fusarium pseudograminearum and Fusarium moniliforme, respectively. The quantity of spores is often greater in Fusarium moniliforme, which is more resistant to the antifungal activity of our essential oil. Fusarium pseudograminearum is therefore more sensitive to our essential oil than Fusarium moniliforme. Our essential oil therefore showed satisfactory results and excellent effects on sporulation of the two strains tested, and can be used as an antifungal agent, since the results reveal significant inhibition of spore production and mycelial growth of the two Fusarium genera tested. Nevertheless, their actions on sporulation are rather favorable.

We have determined that an essential oil is antibacterial if its zone of inhibition is greater than or equal to 8 mm, in accordance with the literature (Ponce et al., 2003). Our study revealed that MP EO was highly effective against all isolates, with inhibition zones of up to 14 mm against Staphylococcus epidermidis; 11 mm against Klebsiella pneumoniae; 10 mm against Staphylococcus aureus; and 9 mm against Escherichia coli; Acinetobacter baumannii; Enterobacter cloacae (Table 5). In addition, this EO revealed a significant antibacterial power to the strains examined in comparison with the antibiotics chosen as controls, namely Penicillin (5 μg), Oxacillin (5 μg) and Amoxicillin (25 μg). In general, Gram- bacteria were more resistant. It is known that EOs have a greater effect on Gram+ bacteria than on Gram- bacteria.

The limited antibacterial activity of our EO against Gram negative bacteria observed in this experiment is, however, consistent with previous research. This observation has been attributed to the property of these bacteria of possessing a hydrophilic polysaccharide chain that forms a barrier against hydrophobic essential oils (Boukhebti et al., 2011; Mahendran et al., 2021). EOs enriched with phenolic compounds are known to possess a high level of antimicrobial activity. In fact, these compounds are considered to have the highest antimicrobial activity, followed by aldehydes, ketones and alcohols.

The experimental data collected in Table 6 show that compared with the control group, we observed a drop in the aggressiveness caused by the bacteria tested in the presence of our essential oil.

By inhibiting bacterial growth according to a dose–response relationship, we demonstrated that MP EO has antibacterial activity. These results enabled us to identify the antibacterial parameters MIC and MBC. Staphylococcus aureus and Staphylococcus epidermidis showed a MIC = 2 μL/mL, Escherichia coli and Acinetobacter baumannii recorded MICs of 10 μL/mL and 4 μL/mL respectively, while Klebsiella pneumoniae showed a MIC>10 μL/mL. The bacteriostatic effect of our HE therefore varies according to the strain tested and the concentration. However, the bactericidal effect (MBC) against Staphylococcus aureus, Staphylococcus epidermidis and Acinetobacter baumannii is recorded at a concentration of 4 μL/mL. However, for Escherichia coli and Enterobacter cloacae, a concentration of 10 μL/mL is needed to completely kill these microbial germs, which are more resistant to our oil. MP EO is therefore active against the strains tested.

Zekri et al. (2017) found that minimum inhibitory doses differed according to the species tested and the germ targeted in his study of numerous Moroccan mints. They ranged from 0.30 to 1.2 μL/mL for Mentha spicata, from 1.20 to 9.60 μL/mL for MP, and from 9.60 to 19.20 μL/mL for Mentha suaveolens. Bacterial concentrations ranging from 19.20 to 76.80 μL/mL for MP, from 19.20 to 76.80 μL/mL for Mentha spicata, and from 38.40 to 76.80 μL/mL for Mentha suaveolens were used to destroy the bacteria. Meanwhile, Medjekal et al., (Samir et al., 2017) have noted that this oil has an antibacterial effect at a concentration of 20% against E. coli and Staphylococcus. However, MP of Portuguese origin demonstrated an inhibitory action with a MIC of 1.2 mg/mL.