2,2-Diphenylpicryl hydroxyl (DPPH) radical, butylated hydroxyl toluene (BHT), aluminum chloride (AlC_l3), quercetin, rutin, gallic acid, iron III chloride (FeC_l3), potassium ferricyanide (K₃Fe (CN)₆), sodium carbonate (Na₂CO₃), sodium nitrite (NaNO₂), and Folin–Ciocalteu reagent were purchased from Sigma-Aldrich (St. Louis, MO, United States). All the other chemicals and solvents used were of analytical grade.

The Ferula communis plant was harvested at its mature age in February 2022 from Taounate city in Morocco. Professor Abdelfattah Abdellaoui, a botanist from the Biology Department, Faculty of Science, University of Sidi Mohamed Ben Abdellah, Fez, Morocco, identified the plant material. A herbarium specimen with voucher number 2299/4-16-1/taw was deposited.

The leaves of Ferula communis were dried at room temperature (between 20°C and 25°C). For a full day, 18 g of powdered leaves were macerated in 150 mL of distilled water (we have used water as it is traditionally used in preparation methods in Morocco). The resultant macerate was filtered and concentrated using a rotating evaporator for dehydration below space at 40°C. The extracted materials were stored in sterilized Eppendorf sample tubes at 4°C in preparation for later use.

The total flavonoid content of the aqueous extract (AE) was ascertained by colorimetric analysis using aluminum chloride. A measure of 500 μL of aluminum chloride (20%) was mixed with 500 µL of the sample or quercetin, following a 1-h reaction under darkness at room temperature (14.0°C ± 2.00°C). Absorbance was read at 420 nm. The total flavonoid amount was presented as milligrams of quercetin equivalents (mg QE/g Dw) for each gram of dry weight of the extract. The calibration curve was constructed using quercetin as the standard (Kara et al., 2022). The phytochemical composition of a plant is under complex control and is affected by both external environmental factors and endogenous circadian rhythms. The environmental factors that directly affect phytochemical profiles and concentrations vary across time of day and time of year (Liebelt et al., 2019).

The Folin–Ciocalteu method (Slinkard and Singleton, 1977a) was used to determine the total polyphenol concentrations of the aqueous extracts. A known extract dilution of 0.5 mL and a sodium carbonate solution of 7% were combined with 2.5 mL of 10% Folin–Ciocalteu, following a 2-h reaction in darkness at ambient temperature (14.00°C ± 2.00°C). At 760 nm, absorbance was measured. Milligrams of gallic acid equivalents (mg GAE/g Dw) were used to express the extract’s total phenol concentration in grams of dry weight. Gallic acid served as the norm in the calibration curve’s building.

The chemical profile of the Ferula communis extract was established using UHPLC coupled with high-resolution mass spectrometry (LCMS-8060, Shimadzu Italy, Milan). Specifically, the source settings were configured as follows: a nebulizing gas flow rate of 2.9 L/min, a heating gas flow rate of 10 L/min, an interface temperature of 300°C, a linear ion trap (LIT) detector temperature of 250°C, a thermal block temperature of 400°C, and a drying gas flow rate of 10 L/min. LC–MS detection was set in the negative ionization mode. We developed an internal database that includes polyphenol derivatives through qualitative analysis. The separation of compounds and standards was carried out on a C18 column with dimensions of 3 × 100 mm and a particle size of 2.6 µm (Phenomenex, Torrance, CA, United States). The elution of the extract components was achieved under isocratic conditions using acetonitrile (A) and water containing 0.01% formic acid (B), with a total run time of 25 min. The mobile phase comprised acetonitrile (A) and water with 0.01% formic acid (B). The Ferula communis extract was added to a mixture of acetonitrile and water in a 1:1 ratio. The solution (20 µL) was then diluted with acetonitrile (980 µL) and injected into the instrument for analysis. A molecule was considered positive if its area under the curve was greater than that of the blank sample. In cases of very similar structures, the distinction was made using retention time, with the instrument configured to record the molecular mass in the third quadrupole (Kandsi et al., 2021).

In the present work, the chemical composition of Ferula communis leaf was examined using in silico predictions of physicochemical and ADME-Tox pharmacokinetic features, followed by the molecular docking simulation for p-hydroxybenzoic acid (M8) as the major compound of the studied extract, which was complexed to NADPH oxidase protein to explore the chemical interactions mode that could provide a rationale for the antioxidant activity of Ferula communis leaf (Assaggaf et al., 2023; Benkhaira et al., 2023; Jeddi et al., 2023). Initially, pkCSM and SwissADME servers were properly used to predict 11 chemical compounds’ physicochemical properties and absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics (El fadili et al., 2023a, El fadili et al., 2023b; Abechi et al., 2024; Er-rahmani et al., 2024; Nouioura et al., 2024e). Second, AutoDock software was equally used to explore the inhibition mechanism with corresponding binding energies in kcal/mol (Nouioura et al., 2024a; Nouioura et al., 2024d; Nouioura et al., 2024c), in which the targeted protein coded in the protein data bank by 2CDU.pdb was prepared by adding the Gasteiger charges and removing all suspended ligands bound to the targeted protein (El fadili et al., 2022b, El fadili et al., 2022a, El fadili et al., 2023c, El fadili et al., 2024). Finally, Discovery Studio 2021 software was also employed to visualize the produced intermolecular interactions in two and three dimensions (Bouzammit et al., 2024c; Bouzammit et al., 2024b; Bouzammit et al., 2024a). The thermodynamic stability of the produced interactions was equally investigated by the molecular dynamics technique using the Desmond program, a package of Schrodinger software (Er-rajy et al., 2023), in which the output file of molecular docking was used as an input file of molecular dynamics (El fadili et al., 2023d).

The polyphenolic compounds and their antioxidant activity are crucial factors in assessing the samples’ antioxidant capability. Utilizing the Folin–Ciocalteu and aluminum chloride colorimetry techniques, respectively, to ascertain the total phenolic and total flavonoid contents, Table 1 presents the results of the investigations. The outcomes indicate that the extract of F. communis leaves has a high level of flavonoid content (0.32 ± 0.04 mg eq AG/mg) and total polyphenol (0.257 ± 0.003 mg eq AG/mg). When comparing our research to other studies, our results were in line with those of Aydin et al. (2021), who used an ethanol–water (50:50) extract.

In Nouioura et al. (2024a), F. communis fruits were macerated with various solvents, including methanol, ethanol, water, hexane, acetone, ethyl acetate, and chloroform, which were used individually. The ethanol extract exhibited the highest total polyphenol content (62.20 ± 0.11 mg GAE/g DW), followed by the methanol extracts (60.82 ± 0.32 mg GAE/g DW) and aqueous extracts (44.04 ± 0.22 mg GAE/g DW), respectively. The flavonoid content exhibited the highest extraction with the ethanol extract (17.09 mg QE/g DW), while the aqueous extract showed lower extraction yields with 9.31 ± 0.24 and 8.97 ± 0.47 mg GAE/g DW. In a previous study, F. communis roots (Ed-dahmani et al., 2024d) were tested with distilled water. The results show that the extract contains 0.194 ± 0.004 mg polyphenols, expressed as GA equivalent/mg, and 0.820 ± 0.031 mg flavonoids, expressed as GA equivalent/mg.

These high quantities of polyphenols and flavonoids make the plant a powerful antioxidant. Numerous investigations have verified the affirmative correlation between high levels of phenol and flavonoids, robust overall antioxidant potential, and the ability to mitigate free radical damage (Sarikurkcu et al., 2018; Tangjitjaroenkun, 2018).

The results we present are from an analysis conducted by LC–MS-MS, revealing the composition of the aqueous extract of Ferula communis. This analysis highlights the presence of 11 compounds, the most abundant of which is p-hydroxybenzoic/salicylic acid, representing 53.65%. The main molecular families within this extract are flavonoids, glucosides, triterpenoids, and phenolic acids. These conclusions are supported by the empirical data provided in Table 2. Additionally, Figure 1 represents the chromatogram, further underscoring the critical importance of the aqueous extract in preserving and enriching these compounds.

We had no information on the phenolic composition of the aqueous extract of F. communis leaves until now. This study represents the first qualitative and quantitative analysis of the leaf polyphenols of this plant, conducted using UHPLC. In total, 11 compounds have been identified in the aqueous extract of F. communis leaves. These include hesperetin, trimethoxyflavone, arbutin, ursolic acid, luteolin, kaempferol-3-O-pentoside, vanillic acid, p-hydroxybenzoic acid (or salicylic acid), catechin (or epicatechin), gallocatechin (or epigallocatechin), and kaempferol (Table 2). The fruits of the same plant exhibit 15 phenolic compounds, among which gallic acid, caffeic acid, catechin, 4-hydroxybenzoic acid, catechin hydrate, succinic acid, syringic acid, 3-hydroxybenzoic acid, naringin, cinnamic acid, ferulic acid, p-coumaric acid, sinapic acid, quercetin 3-O-β-D-glucoside, and rutin were identified and quantified using HPLC-DAD (Nouioura et al., 2024a). In addition, in Al-Yahya et al. (1998), various phenolic compounds such as resorcinol, ferulic acid, syringic acid, and coumarin were identified as the predominant components in the fruits of F. communis. Another study conducted by Rahali et al. (2019) using RP-HPLC revealed that methanolic extracts from the flowers, fruits, and stems of F. communis collected from northern Tunisia were primarily composed of resorcinol, ferulic acid, syringic acid, and coumarin.

Several authors link phenolic chemicals, particularly the secondary metabolites of plants, to a range of biological characteristics. Because of this, disclosing such substances has evolved into an essential initial step toward ideal utilization. The antioxidant activity of Ferula communis extract leaves was evaluated using FRAP and DPPH techniques (Table 3).

The lowest IC₅₀ value (concentration capable of inhibiting 50% of DPPH) indicated the highest level of antioxidant activity. In light of this research’s results, the inhibitory power of the aqueous extract of Ferula communis leaves is greater, with an IC₅₀ value of the order of 0.263 ± 0.008 mg/mL. The activity level remains under the requirement for BHT (0.118 ± 0.0001 mg/mL) to be used as the reference.

Table 3 presents the FRAP assay results. The extracts studied show that the aqueous leaf extract of Ferula communis has a remarkable reducing power (EC₅₀ = 15.659 ± 0.087 mg/mL). However, its power is still less than the reference value of quercetin (EC₅₀ = 0.033 mg/mL).

DPPH^● is a radical widely used in model systems to investigate the neutralizing capabilities of various natural materials, such as anthocyanins, phenolic compounds, and pure plant extracts (Chang et al., 2007). Antioxidants can produce stable free radicals by neutralizing the free radical oxidation cycle, preventing further oxidation. Furthermore, antioxidants can scavenge DPPH radicals by donating hydrogen, resulting in the DPPH radical discoloring when extracts at increasing concentrations are added, creating decreased DPPH-H (Burits and Bucar, 2000).

Radical reactions are ubiquitous in all organisms and play a more or less direct role in gene modification, reproduction, and disease defense (Guillouty, 2016). Based on four different ways of action, antioxidant activity has been assessed. It is crucial to conduct a variety of assays to take the chemical makeup of the extract into account, as it acts through several methods. Variations in the way phenolic compounds react to the various antioxidant reaction mechanisms and the variety of resulting products. It was also noted that a good resulting product of these reactions could explain the differences in correlations found using applicable antioxidant tests (El Atki et al., 2019). Since most phenolic compounds are hydrophilic molecules, their low viscosity and smaller density allow for easier diffusion in polar to semi-polar solvents (Fernanda et al., 2016; Taroq et al., 2018).

According to published research, the FRAP approach is sensitive for determining the overall antioxidant power of fresh biological fluids, including medicinal plant compounds and plant homogenates (Szollosi and Szollosi Varga, 2002; Rattanachitthawat et al., 2010). The FRAP assay is used to examine the potential effects of medicinal plants by measuring their overall antioxidant power (Szollosi and Szollosi Varga, 2002).

When comparing our study with others on antioxidant activity using the DPPH method and the FRAP technique, Rahali et al. (2019) examined the antioxidant activity of the methanolic extract from the flowers, fruits, and stem of Ferula communis. The analysis revealed that the stem extract had the lowest scavenging activity (IC₅₀ = 168 mg/mL) by the DPPH method.

Ed-Dahmani et al. (2024c) assessed the antioxidant activity of the fruits of the Ferula communis plant by DPPH and FRAP. Extracts were prepared by maceration with methanol and distilled water. The results showed that the inhibitory power by DPPH of the methanol extracts of fruit (IC₅₀ = 0.076 ± 0.039 mg/mL) is greater compared to the aqueous extracts (IC₅₀ = 0.26 ± 0.006 mg/m). The FRAP test showed that the methanol extract of fruit (IC₅₀ = 202.71 ± 1.471 mg/mL) has greater antioxidant power than aqueous extracts (IC₅₀ = 208.04 ± 5.69 mg/mL). Ed-dahmani et al. (2024b) tested F. communis roots by the DPPH method. The extract exerted a considerable scavenging action on DPPH, with an IC₅₀ value of 0.820 ± 0.031 mg/mL.

The acute toxicity of the Ferula communis leaf aqueous extract was assessed by measuring its effects on the body weight, organ weight, and general behavior of mice.

Toxicity studies are performed on mice to estimate the toxicity of the plant and the right dose to determine the health risks associated with plants for use by humans. Since alterations in the blood system have a greater predictive value for human toxicity, hematological indices in animals are crucial for assessing the toxicity risk (Adinortey et al., 2012).

The current investigation examined the correlations between polyphenol content, flavonoid content, and antioxidant activity in the leaves and fruits of F. communis (Nouioura et al., 2024a; Ed-Dahmani et al., 2024b).

The results (Table 6) showed that the correlation test is considered an excellent tool for revealing relationships between different parameters studied, with a good correlation between flavonoids and polyphenols (0.981) and between FRAP and polyphenol (0.951). However, the correlation between FRAP and DPPH is weak (−0.491).

Nouioura et al. (2024a) assessed the antimicrobial properties of F. communis extracts in ethanol (EtOH), acetone (AcE), or water (AqE) against four bacterial strains, namely, Escherichia coli, Bacillus subtilis, Proteus mirabilis, and Staphylococcus aureus, as well as four fungal strains, namely, Fusarium oxysporum, Aspergillus niger, Aspergillus flavus, and Candida albicans.

The AcE extract displayed the highest inhibitory activity against P. mirabilis with an inhibition diameter of 19.00 ± 1.00 mm and an MIC value of 2.50 ± 0.00 mg/mL, followed by E. coli with 11.50 ± 1.50 mm of inhibition zone and an MIC value of 2.50 ± 0.00 mg/mL. The extract also exhibited inhibitions of 11.00 and 9.00 mm against S. aureus and B. subtilis, respectively.

The EtOH extract exhibited the greatest activity against E. coli, with an IC₅₀ inhibition diameter of 14.00 ± 1.00 mm and an MIC value of 0.312 ± 0.00 mg/mL, and the smallest inhibition zone of 9.00 ± 0.00 mm was observed for S. aureus. The antifungal activities of the F. communis extract against C. albicans, A. niger, A. flavus, and F. oxysporum were compared to that of the fungicide fluconazole. AcE exhibited significant activity against F. oxysporum, with a percent inhibition of 20.6% ± 1.4% and an MIC value of 5.0 ± 0.0 mg/mL.

In a previous study, the toxicity of the fruits of the Ferula communis plant was assessed using the hydroethanolic solvent. A single oral administration of hydroethanol at doses of 200, 300, and 400 mg/kg to mice showed no sign of toxicity (mobility, aggressiveness, vigilance, tremors, sleep, vomiting, or diarrhea), and no deaths occurred in the treated mice at the tested doses (Ed-Dahmani et al., 2024a). In another study, the toxicity of the F. communis roots was studied with distilled water. When given orally to mice at doses of 200, 300, and 400 mg/kg, F. communis roots did not induce any toxicity (mobility, aggressiveness, vigilance, tremors, sleep, vomiting, or diarrhea) or death in the treated animals at the tested doses (Ed-dahmani and Mssillou, 2024a).

The prediction of physicochemical properties reveals that all extracted molecules from the Ferula communis leaf meet all five rules of Lipinski (Aloui et al., 2024; Seddoqi et al., 2024), where the number of acceptors and donors of hydrogen bonds does not exceed ten and five, respectively. The molecular weights are less than 500 g/mol. The molar refractivity index falls within the range defined by (Kara et al. (2022) [130], and the lipophilicity in the octanol/water solvent is defined by a LogP value of less than 5, as presented in Table 7. Moreover, the prediction of the pharmacokinetic properties of ADMET confirms that extracted molecules are well-absorbed, with human intestinal absorption (HIA) ranging from 42% for arbutin (M3) to 100% for ursolic acid (M4); these molecules show good permeability to the central nervous system (CNS) and blood–brain barrier (BBB). The metabolism test also shows that M2 and M4 molecules are considered substrates of 3A4 cytochrome, and the chemical compounds labeled M2, M5, and M11 were predicted as potent agents to inhibit 1A2, 2C9, 2C19, and 3A4 cytochromes. The AMES toxicity test declares the safety of all extracted molecules except for M6, M9, and M10 molecules. However, all extracted molecules from the Ferula communis leaf are safe from any skin sensitization and hepatotoxicity effects except for M4, as presented in Table 8. The predictive model of Egan declares that M2, M7, and M8 are part of the yellow-boiled egg, so they are predicted to cross the BBB with the highest probability. M1, M3, M5, M9, M10, and M11 are part of the white Egan’s egg, so they were predicted to be passively absorbed by the gastrointestinal tract, as displayed in Figure 5.

The results of molecular docking simulation confirm that the major compound of Ferula communis leaf extract, p-hydroxybenzoic acid, was complexed with the NADPH oxidase protein (2CDU.pdb) with the lowest binding energy of −5.21 kcal/mol. This interaction produced four conventional hydrogen bonds with Ser115, Thr9, Gly12, and Lys134 amino acids residues in A chain and one amide–pi-stacked interaction with Thr113 amino acid residue; furthermore, one van der Waals bond was formed with Thr112 amino acid residue, and one Pi–alkyl bond was formed with Ala11 amino acid residue, as displayed in Figure 6.

The process of molecular docking is well-validated as the studied compound was docked to various active sites of the antioxidant protein, including Ser115, Thr9, Lys134, and Ala11 amino acid residues, which were the same active sites in the same chain that were obtained by the flavin adenine dinucleotide co-crystalized ligand, as presented in Figure 7.