The plant material utilized in this study consists of the leaves of M. vulgare and they were harvested from the region of Fez Meknes (Moulay Idriss Zerhoun) in April 2020 during the flowering period of the plant. Subsequently, the plant samples were carefully placed in ventilated bags and subjected to a thorough cleaning to eliminate any potential contaminants or debris that could compromise the purity of the plant samples, after which its identity was authenticated at the Department of Botany of the Scientific Institute of Rabat. Furthermore, the plant samples were subjected to a drying process to remove excess moisture; this drying was carried out in a well-ventilated room and away from sunlight at a temperature range of 30°C–40°C for 10 and 15 days. Table 1 presents information on the region where the plant was harvested while Figure 1 depicts Morphological appearance of the harvested plant.

The antimicrobial activity of the plant extract was evaluated against nine bacterial strains and seven fungal strains known for their high resistance, invasiveness, and pathogenic properties. The bacterial strains are Staphylococcus epidermidis, Enterobacter cloacae, Streptococcus agalactiae (B), Klebsiella pneumoniae, wild Escherichia coli, ESBL Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Staphylococcus aureus BLACT, while the fungal strains included Candida albicans, Candida krusei, Candida tropicalis, Candida parapsilosis, Candida dubliniensis, Saccharomyces cerevisiae and Aspergillus niger. Noteworthy, these microorganisms were obtained from the Mohamed V-Meknes Provincial Hospital and were initially stored in −80°C 20% glycerol stock. Before the utilization of the microorganisms, they were subcultured and grown on Mueller Hinton agar and Sabouraud agar for the bacterial and fungal strains, respectively.

30 g of powdered M. vulgare leaves was introduced into a 1-liter Erlenmeyer flask containing 600 mL of purified water. The mixture obtained was subjected to heat at 70°C for 1 h, after which it was filtered. Subsequently, the filtrate was subjected to an evaporation process under reduced pressure after cooling at room temperature. The residue obtained after the cooling was oven-dried at 50°C and subsequently weighed to calculate the extraction yield (Chavan et al., 2001).

To determine the phytochemicals present in M. vulgare leaves, various solvents including water, chloroform, methanol, and petroleum ether were utilized. To determine the presence of alkaloids, Dragendorff and Mayer reagents were employed, while Hydrochloric acid and isoamyl alcohol were used to detect the presence of catechin tannins, and tiasny’s reagent, sodium acetate, and ferric chloride were used to characterize gallic tannins. Also, the presence of sterols and triterpenes was detected using acetic anhydride and strong sulfuric acid, Magnesium chips, isoamyl alcohol, and diluted hydrochloric alcohol were used to detect the presence of flavonoids, while chloroform, dilute ammonia, and hydrochloric acid was used for the detection of quinone substances (Tamert et al., 2017).

To determine the number of total polyphenols in the M. vulgare extract, the Folin-Ciocalteu reagent was employed. 500 µL of freshly prepared 0.1X Folin-Ciocalteu reagent and 2 mL of sodium carbonate solution (20% Na₂CO₃) were mixed with 100 µL of the extract. The resulting mixture was incubated for 30 min at room temperature and the absorbance was measured at a wavelength of 760 nm with referce to a standard (Ali-Rachedi et al., 2018).

The quantification of the flavonoid content of M. vulgare extract was conducted using the AlCl₃ method, as described by Barros et al. (Barros et al., 2011). To perform the quantification, 2 μL of the extract was mixed with 10 μL of 10% aluminum chloride solution and 2 mL of distilled water, after which 3 mL of absolute methanol was added to the mixture. Subsequently, the mixture was incubated for 2 hours in the dark, and the absorbance was measured at 433 nm.

50 µL of M. vulgare leaves extract was added to 1,500 µL of 4% vanillin/methanol workaround and subjected to vigorous mixing. Subsequently, 750 µL of concentrated HCl was added to the mixture, and the resulting mixture was allowed to react at room temperature for 20 min. Ultimately, the absorbance of the mixture formed after the reaction was measured at 550 nm concerning a blank (Lorrain et al., 2011).

The phytochemicals present in the M. vulgare extract were chromatographically characterized using high-performance liquid chromatography (HPLC) by employing an UltiMate 3000 system. Notably, the system contained a reverse-phase C18 column (250 mm × 4 mm, id 5 μm, Lichro CART, Lichrospher, Germany) with a sample changer, and the tests were conducted at a maintained temperature of 5°C. The temperature of the column was maintained at 40°C, while the mobile phase consisted of ultrasonically degassed solutions of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and possessed gradient composition as follows: starting with 2% B at 0 min, followed by a linear change to 30% B at 20 min, 95% B at 25 min, and returning to 2% B at 26 and 30 min. Furthermore, the flow rate was set at 1 ml/min, and the injection volume was 20 µL. Detection of the compounds was performed using a Maxis Impact HD in MS/MS mode with negative electrospray ionization, while broadband collision-induced dissociation was employed for fragmentation. Additionally, UV detection was carried out using an L-2455 diode array detector, scanning in the wavelength range of 190–600 nm. Specific acquisition wavelengths of 280, 320, and 360 nm were utilized for analysis. Various parameter values were set for optimal performance, including a capillary voltage of 3000 V, the drying gas temperature of 200°C, dry gas flow rate of 8 L/min, vaporizing gas pressure of 2 bar, and an offset plate of −500 V. Nitrogen was used as the nebulization and desolvation gas, and mass spectrometry data were collected within the range of 50–750 m/z. Data acquisition and evaluation were performed using the Sysremsoftware program, specifically designed for chromatography data analysis in the Thermo ScientificTM ChromeleonTM 7.2 platform.

The Minimum Inhibitory Concentration (MIC) of the M. vulgare extract was determined using the microdilution method in a 96-well microplates, in accordance with the protocol utilized in the study by Kotan et al. (Kotan et al., 2008). The MIC represents the lowest concentration of the plant extract which visibly inhibit the growth of the microorganism being evaluated. To determine the MIC, standard solutions of the extracts were prepared by dilution in 10% dimethyl sulfoxide (DMSO) to obtain concentrations of extracting ranging from 75 to 2.35 mg/ml. For the evaluation of the bacteria susceptibility, the dilutions were incorporated into Mueller-Hinton basal medium, while for fungal susceptibility testing, the Sabouraud bouillon medium was utilized. Each well of the microplate was filled with 100 µL of the respective dilution, and subsequently, 100 µL of the inoculum containing a microbial count of 10⁶ colony-forming units per milliliter (CFU/ml) was sequentially introduced into each well. The microplate was subjected to a 24-h incubation period at 37°C after which 10 µL of resazurin, a colorimetric indicator of bacterial growth, was introduced into each well. Subsequently, the microplates were further incubated for 2 hours at 37°C, after which color change from deep violet to pink, was monitored as it indicates the absence of microbial growth, signifying the MIC. Noteworthy, the experimental setup included growth and sterility control wells and the MIC determination process was performed twice for the M. vulgare extract. The determination of the MBC/MFC was performed by aseptically transferring 10 µL of the contents from wells without visible growth onto Mueller Hinton agar (MHA) and Sabouraud agar plates for bacteria and fungi respectively. Subsequently, the plates were incubated for 24 h at 37°C and 30°C for bacteria and fungi respectively, and the MBC and MFC were defined as the lowest concentration of the extract that resulted in a 99.9% reduction in CFU/ml compared to the control. Noteworthy, 250 mg of Terbinafine was utilized as the standard antifungal by dissolving in 10% DMSO. The evaluation of the antimicrobial efficacy of the varying concentrations of the extract was done by calculating the MBC/MIC or MFC/MIC ratio, where a ratio of less than 4 indicated a bactericidal/fungicidal effect, a ratio greater than 4 indicated a bacteriostatic/fungistatic effect, and a ratio equal to 4 suggested an indeterminate effect (Bissel et al., 2014).

The animal model employed in this study consisted of Wistar rats weighing between 195–260 g and Albino mice weighing between 20–30 g. The animals were housed in a dedicated animal facility located within a biological laboratory. The housing conditions provided a controlled environment with a photoperiod of 12 h of light followed by 12 h of darkness, and a temperature maintained at 22°C. The rodents were kept under standard breeding conditions, with unrestricted access to food and water.

The procedures used to perform this study agree with the international guidelines used for the use of laboratory animals. The ethical committee of the Faculty of Sciences of Meknes, Morocco, revised and approved this work under the ethical clearance 04/2019/LBEAS.

An in silico evaluation of the compounds derived from the M. vulgare decoction was done against antimitotic, antidiabetic, and antimicrobial targets. Following the identification of the targets, their respective structures were retrieved from the RCSB protein data bank using the PDB IDs 1XO2, 4W93, and 1AJ6. The preparation of the protein before molecular docking was done using BIOVIA’s Discovery Studio Visualizer (Accelrys Software Inc, 2005) and Autodock tools (Morris et al., 2009). Notably, the preparatory steps included the removal of heteroatoms, cognate ligands, and water molecules, and subsequent optimization of the structure. Similarly, the structure of the ligands was drawn in ChemDraw Ultra (CambridgeSoft, 2009a), and was subjected to preparatory steps including energy minimization using Chem3D Pro (CambridgeSoft, 2009b). Subsequently, the ligands were converted to pdbqt files (Zentgraf et al., 2007) using OpenBabel (Morris et al., 2009; Ferreira et al., 2015), while the molecular docking was performed using AutoDock Vina (Yusuf et al., 2008; Trott and Olson, 2010; Eberhardt et al., 2021). Following the formation of the complexes after docking, the interactions between the ligands and the protein were visualized and analyzed using BIOVIA’s Discovery Studio Visualizer.

The data presented in this study are expressed as means ± standard error of measurement (SEM). All statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Noteworthy, significance levels were determined with p < 0.05, p < 0.01, and p < 0.001 considered as statistically significant.

The quality control assessments conducted on the retrieved leaves of M. vulgare included the determination of its moisture content (TH), pH, acidity, ash, and ICP-AVE. The results of this assessment are presented in Tables 2, 3. As evident in Table 2, the TH of M. vulgare leaves was found to be 22.56 ± 0.25%, indicating a high water content in the plant material. The pH of the M. vulgare extract was slightly acidic, with a value of approximately 5.945 ± 0.007, however, the pH value was in compliance with the quality standards of AFNOR (AFNOR, 1977; Kakoma et al., 2014). Determination of the titratable acidity revealed values of 0.1033 ± 0.9212, which was used to evaluate the characteristics, quality, absorption capacity, and solubility of many substances (Kroeze, 2020). Similarly, the percentage of ash in M. vulgare extract which provides information about the mineral content that remains after organic matter is volatilized at high temperatures was found to be 22.562 ± 0.25%. As presented in Table 3, analysis of the heavy metal content of the extract revealed Fe possessed the highest content with a value of 0.5498 mg/g, while Cu was the second-highest with a value of 0.0087 mg/g. Other heavy metals analyzed are presented in Table 3, interestingly, they were all found to be within the permitted range of FAO/WHO regulatory standards. Hence, rendering the extract suitable for direct consumption, as an ingredient in food processing, or for repackaging if necessary.

The qualitative screening of phytochemicals enables the identification of the classes of compounds present in an extract. To this end, qualitative reactions were employed to determine the phytochemicals present in the extract of M. vulgare, and the results are presented in Table 4. The results revealed the presence of primary metabolites including polysaccharides, reducing sugars (glucose and fructose), proteins, and lipids in varying concentrations. Similarly, the presence of secondary metabolites including flavonoids, tannins, mucilages, sterols and triterpenes, coumarins, and saponins were confirmed, however, alkaloids were found to be absent (Table 4). Interestingly, the results of this study are consistent with those of Akther et al. and Fayyad et al. who reported in their study that M. vulgare is rich in gallic tannins, catechin tannins, sterols, and triterpenes and flavonoids (Akther et al., 2013; Fayyad et al., 2014). Tannins and flavonoids are widely explored for their pharmacological properties including antioxidant, antiviral, antitumor, anti-inflammatory, anti-allergic and anti-cancer activities (Khanbabaee and van Ree, 2001; Crozier et al., 2009). Furthermore, numerous in vitro and in vivo studies show that polyphenols can modulate carbohydrate metabolism and have antidiabetic properties (Hanhineva et al., 2010; Martel et al., 2017).

Quantitative analysis of the classes of phytochemicals present in the aqueous extract of M. vulgare was conducted and the results are presented in Table 5. Notably, the yield of the aqueous extract was found to be significant, with a value of 17.673 ± 0.48. Classes of phytochemicals present in the extract include a high content of polyphenols (488.432 ± 7.825 mg/EAG/g), flavonoids (25.5326 ± 1.317 mg/EQ/g), and tannins (23.966 ± 0.187 mg/EV/g). The results of the quantitative analysis were found to be consistent with that of the qualitative analysis, with the values also being much higher than that reported by Matkowski et al. and Wojdylo et al. whose study revealed that the methanolic extracts of M. vulgare had polyphenolic contents of 63.4 ± 1.7 mg/EAG/g and 3.86 ± 0.05 mg/EAG/g respectively (Wojdyło et al., 2007; Matkowski et al., 2008). Conversely, the flavonoid content of the extract was found to be lower than that reported in the study of Elberry et al. in which they reported a value of 15.53 ± 0.67 mg/EAG/g study (Elberry et al., 2015). Variations in the values could be due to many intrinsic and extrinsic factors like genetic and environmental factors, and this could influence the quality and quantity of chemical composition of phenolic compounds in plant material. Other factors related to harvesting, drying, and processing may also be responsible (Ouedraogo et al., 2021).

The identification of the phenolic composition of the aqueous of M. vulgare leaves was conducted using HPLC/UV-ESI-MS analysis. The resulting chromatographic profile, which reveals the presence of 19 chemical compounds presented in Table 6, is depicted in Supplementary Figure S1 (supplementary provided). Notably, the major phyto-compounds present in the extract were catechin (12.03%), maleic acid (11.18%), luteolin (10.55%), apigenin (10.55%), salicylic acid (8.01%), biotin (7.24%), caffeic acid (6.36%), and vanillic acid (5.03%), these eight compounds accounted for a significant proportion, totaling 70.95% of the total identified compounds (96.78%). Interestingly, a study by Benzidane et al., 2020 on the methanolic extract of M. vulgare leaves collected in Algeria revealed the presence of ferulic acid (Nadia Benzidane, Ridha Smahi, Boudjemaa Zabouche, Abdelhalim Makrouf, 2020), however, the compounds were found to be present in the current study with a very low percentage (0.77%). They also reported the presence of catechin in the hexane extract, which is in tandem with the compounds identified in our aqueous extract. A recent study in Saudi Arabia reported that the methanolic extract of the leaves of M. vulgare contains luteolin-7-O-D-glucoside as the third major compound, however, luteolin was found to be present in this and was not linked to any sugar moiety. Furthermore, a study by Rezgui et al. in Tunisia corroborated the presence of several phenolic molecules in M. vulgare leaves, including apigenin, ferulic acid, coumaric acid, caffeic acid, and luteolin (Rezgui et al., 2020), which are in line with the compounds identified in our study. These compounds have been extensively investigated for their pharmacological properties, such as anticancer, anti-inflammatory, antibacterial, antiviral, and antiseptic activities, as documented in previous scientific studies (Karryev et al., 1976; Pukalskas et al., 2012; Yousefi et al., 2013). Noteworthy, the observed variation in the reported compared to that of other studies could be attributed to intrinsic and/or extrinsic factors.

The assessment of the antioxidant activity of plant extract was done using three different methods namely DPPH, FRAP, and TAC tests, the results of which are presented in Table 7. The results revealed a positive correlation between the inhibitory effect of the free radical DPPH and the quantity of the M. vulgare extract. The antioxidant activities of the extract and ascorbic acid were quantified by the determination of their IC₅₀, which represents the concentration of the extract required to reduce 50% of the DPPH free radical. Noteworthy, a lower IC₅₀ value indicates a higher antioxidant effect. Interestingly, the results of this study revealed that the decoction of M. vulgare exhibited significant antioxidant power (IC50 = 1.1815 ± 0.621 mg/mL), although lower than that of the ascorbic acid (IC50 = 0.323 ± 0.411 mg/mL). The results of this study differ slightly from those reported in a study by Boudjelal et al. in which an IC50 value of 0.49 ± 0.517 mg/mL was reported for the aerial part of the methanolic extract of M. vulgare harvested in M'Sila, southern Algeria (Boudjelal et al., 2013). Evaluation of the reducing power via measuring its ability to transfer an electron or a hydrogen atom, thereby reducing Fe (III) to Fe (II) in the presence of the K₃Fe(CN)₆ complex, was done and the results are presented in Figure 3B. Notably, a remarkable increase in the reducing power of M. vulgare was noticed in concomitant with the increasing concentrations of the extract. The ascorbic acid and the decoction exhibited effective concentrations with respective values of EC50 = (0.15 ± 0.24) mg/mL and EC50 = (1.5 ± 0.203) mg/mL. An earlier study on M. vulgare extract reported that it possessed significant antioxidant activity, with an EC₅₀ of 0.51 ± 0.24 mg/mL (Ghedadba et al., 2014), this activity was attributed to the phenylpropanoid glycosides, which are reputed for their potent antioxidant activities. The TAC of M. vulgare extract was determined by the phosphomolybdate test which is based on the ability of the extract to reduce molybdenum Mo (VI) present as molybdate ions MoO₄ ²⁻, to molybdenum Mo(V) MoO₄ ²⁺, and the subsequent formation of a green complex [phosphate Mo(V)] at an acidic pH. The results of this study revealed that the decoction shows a TAC of 50.550 ± 3.746 mg EAA/gE, hence, indicative of the remarkable antioxidant capacity of the aqueous extract. However, the results of this study were not in tandem with that reported in a study by Amessis-Ouchemoukh et al., obtained from studying the TAC of the methanolic and acetone extracts of the aerial part of M. vulgare which showed high TAC with 101.82 ± 1.75 and 85.71 ± 1.35 (Amessis-Ouchemoukh et al., 2014). The antioxidant activities demonstrated by the aqueous extract of M. vulgare, as assessed through DPPH, FRAP, and TAC tests, hold potential applications in the food and pharmacological industries. These findings contribute to the understanding of the antioxidant properties of M. vulgare and its potential as a natural source of antioxidants.

The antimitotic effect of M. vulgare extract was carried out by the Lepidium sativum method which was proposed in 1972 by GAGIU for a preliminary selection of molecules acting on plant growth without presuming their precise place of action. Several authors have contributed to the study of the validity of this test, and it is widely used as it is relatively simple and fast. Additionally, the seed of Lepidium sativum has only one rootlet, the appreciable growth of which allows easy measurement. This test is conducted by measuring the length of the root of a germinated Lepidium seed placed in a medium containing the molecules to be tested. The monitored percentage of growth inhibition is estimated by comparison with a control. Colchicine, with an already-known antimitotic effect, was used as a positive control. The antimitotic activity of the M. vulgare decoction was evaluated by determining the inhibition index. As evident in Table 8, an increase in the concentration of the extract was concomitant with an increase in the percentage inhibition, and the extract was found to possess better antimitotic activity compared to colchicine with 90.184 ± 0.164% and 95.37 ± 0.19% at 10 mg/mL and 1.5 mg/mL respectively. Conversely, the IC₅₀ values obtained for the decocted extract were higher for colchicine compared to the decoction with the values 1.8 ± 0.11 mg/mL and 3.688 ± 0.12 mg/mL respectively. The results of the mitotic index of the M. vulgare extract are presented in Table 9, with the extract exhibiting a higher value compared to the colchicine but lower compared to the negative control (distilled water). It is worth noting that a smaller mitotic index indicates a higher activity. Overall, the results of this study render the extract of M. vulgare fit for exploration as an antimitotic.

The docking scores of the eight major components of M. vulgare extract, which represents their binding affinities for the antimitotic protein (1XO2), antidiabetic protein (4W93), and antimicrobial protein (1AJ6, 2023) are presented in Supplementary Table S2 (Supplementary provided). It is worth noting that a lower docking score corresponds to higher binding affinities. The results showed that the binding energies of the ligands vary depending on the protein target. For instance, Leotulin has the lowest binding energy with the antimitotic protein (1XO2) with a value of −9.3 kcal/mol, indicating a strong binding affinity, whereas Maleic Acid has the lowest binding energy with the antimicrobial protein (1AJ6) with a value of −4.3 kcal/mol, indicating a relatively weaker binding affinity. Interestingly, Leotulin consistently exhibits the lowest binding energy across all three protein targets, suggesting that it has a high potential to be an effective drug candidate for these protein targets. Conversely, Maleic Acid consistently shows the weakest binding affinity among the ligands. Apigenin and Vanillic Acid have relatively consistent binding energies across the three protein targets, whereas the other ligands have more varied binding energies depending on the protein target. For example, Catechin has significantly lower binding energy with the antidiabetic protein (4W93) than with the other two protein targets. Biotin shows moderate binding energies across all three protein targets, with a slightly stronger binding affinity to the antimitotic protein (1XO2) than to the other two protein targets. Salicylic Acid has a similar binding energy to the antidiabetic protein (4W93, 2023) and antimicrobial protein (1AJ6), whereas it has a slightly weaker binding affinity to the antimitotic protein (1XO2). Leotulin consistently exhibits the strongest binding affinity across all three protein targets, while Maleic Acid shows the weakest binding affinity. The other ligands have varying binding energies depending on the protein target, with some showing consistent binding affinities across all three protein targets. Major interactions with good results are shown in Table 12.

Supplementary Table S3 (Supplementary provided) shows the interaction of the antimitotic protein target (1XO2, 2023) with the major components of M. vulgare extract ligands, representing the binding pocket residue amino acids with distances and types of bonding interactions. Each ligand is listed with the amino acid residues that it interacts with in the binding pocket of the antimitotic protein, along with the distance between the ligand and the amino acid residues in Å. The types of bonding interactions are also listed, which can be either hydrogen bonding, π-bonding, alkyl bonding, or hydrophobic interactions. Apigenin interacts with several amino acid residues, including GLU52, ARG46, GLN48, LEU56, TYR24, and PRO55. The distances of these interactions range from 1.98 to 4.97 Å. The type of bonding interactions involved include H-bonds, π-bonds, and alkyl bonds. Biotin interacts with VAL27, LYS43, ALA162, and PHE98 amino acid residues. The distance between the ligand and these residues ranges from 4.19 to 5.44 Å. The type of bonding interactions involved are mainly alkyl bonds and hydrophobic interactions. Caffeic acid interacts with GLU61, VAL101, PHE98, VAL27, LYS43, and ALA162 amino acid residues. The distances between the ligand and these residues range from 2.56 to 5.24 Å. The type of bonding interactions involved includes H-bonds, alkyl bonds, and hydrophobic interactions. Catechin interacts with GLN48, ARG46, GLY53, and PRO55 amino acid residues. The distance between the ligand and these residues ranges from 2.16 to 5.33 Å. The type of bonding interactions involved includes H-bonds, alkyl bonds, and hydrophobic interactions. Leotulin interacts with several amino acid residues, including GLU21, ASP163, ASP104, ILE19, VAL27, LEU152, and others. The distances of these interactions range from 2.06 to 4.96 Å. The type of bonding interactions involved includes H-bonds, π-bonds, and alkyl bonds. Maleic acid interacts with ASP163, GLU99, and H-O amino acid residues. The distances of these interactions range from 2.10 to 2.87 Å. The type of bonding interactions involved includes H-bonds. Salicylic acid interacts with several amino acid residues, including LYS43, ASP163, GLU61, PHE98, VAL27, and others. The distances of these interactions range from 2.34 to 5.44 Å. The type of bonding interactions involved include H-bonds, π-bonds, hydrophobic bonds. Vanillic Acid showed interactions with ASP163 and GLU61 both have hydrogen bonds, while ALA162, VAL27 (both occurrences), and LEU152 have hydrophobic properties. Meanwhile, VAL77 has a π-bond, and VAL27 and LEU152 have alkyl bonds. All type of interactions is also shown in Supplementary Table S4 (Supplementary provided) as 3D and 2D. Major interactions with good results are shown in Table 13.

Supplementary Table S5 (Supplementary provided) shows the results of an experiment investigating the interaction between antidiabetic protein and the major components of M. vulgare extract ligands. The first ligand presented in the table is apigenin. The antidiabetic protein interacts with GLN63, GLU233, TRP59, and TYR62 amino acid residues of apigenin, and the distances between the amino acid residues and the ligand range from 2.47 to 5.23 Å. The bonding interactions between the protein and the apigenin ligand are mainly hydrogen bonds, except for a hydrophobic interaction and two types of alkyl bonds. The second ligand is biotin, and the antidiabetic protein interacts with ARG252, ARG421, PRO332, SER289, ASP402, and GLY334 amino acid residues of biotin. The distance between the ligand and the interacting amino acid residues ranges from 1.85 to 3.76 Å. The bonding interactions between the protein and biotin are mainly hydrogen bonds, except for two types of π-bonds. The third ligand is caffeic acid, and the antidiabetic protein interacts with ASP197 and TYR62 amino acid residues of caffeic acid. The distance between the ligand and the interacting amino acid residues ranges from 2.51 to 4.35 Å. The bonding interactions between the protein and caffeic acid are a hydrogen bond and an alkyl bond. The fourth ligand is catechin, and the antidiabetic protein interacts with ASP197, GLU233, TRP59, and TYR62 amino acid residues of catechin. The distance between the ligand and the interacting amino acid residues ranges from 2.05 to 4.66 Å. The bonding interactions between the protein and catechin are mainly hydrogen bonds, except for three types of alkyl bonds.

The fifth ligand is leotulin, and the antidiabetic protein interacts with GLN63, H-O, TRP59, and TYR62 amino acid residues of botulin. The distance between the ligand and the interacting amino acid residues ranges from 1.98 to 5.34 Å. The bonding interactions between the protein and leotulin are mainly hydrogen bonds, except for four types of alkyl bonds. The sixth ligand is maleic acid, and the antidiabetic protein interacts with ARG346, ASP317, and GLN302 amino acid residues of maleic acid. The distance between the ligand and the interacting amino acid residues ranges from 1.95 to 2.12 Å. The bonding interactions between the protein and maleic acid are all hydrogen bonds. The seventh and final ligand presented in the table is salicylic acid, and the antidiabetic protein interacts with ARG195, ASP197, GLU233, and TYR62 amino acid residues of salicylic acid. The distance between the ligand and the interacting amino acid residues ranges from 2.10 to 4.98. Vanillic Acid interacts with the ASP197 residue of the protein target through a hydrogen bond, with 2.32 Å. It also forms a second hydrogen bond with another ASP197 residue with 2.48 Å. Additionally; the ligand interacts with the GLU233 residue of the protein through a pi-bond, with 3.73 Å. Vanillic Acid binds to the TYR62 residue of the protein through an alkyl-bond, with 4.50 Å. Finally, the ligand interacts with the ALA198 and LEU162 residues of the protein through pi-bonds and alkyl-bonds, respectively, with distances of 3.75 and 4.98 Å. These interactions may play a role in the potential antidiabetic properties of the M. vulgare extract, as the ligand is able to interact with specific binding pocket residues of the protein target as shown in Supplementary Table 6S (Supplementary provided) as 3D and 2D representations. Major interactions with good results are shown in Table 14.

Supplementary Table S7 (Supplementary provided) lists the interaction of various ligands with the major components of M. vulgare extract and the antimicrobial protein target. Apigenin interacts with the antimicrobial protein target through hydrogen bonding and pi-bonding interactions with multiple amino acid residues, including GLY113, ILE186, HIS38, ARG190, LYS189, and ARG190. The distances between the ligand and the amino acid residues range from 1.8454 to 5.34399 Å. Biotin interacts with the antimicrobial protein target through hydrogen bonding and hydrophobic interactions with amino acid residues GLY77, THR165, ASP73, ALA100, and ILE78. The distances between the ligand and the amino acid residues range from 2.32752 to 5.36772 Å. Caffeic Acid interacts with the antimicrobial protein target through hydrogen bonding and pi-bonding interactions with amino acid residues THR165, ASP73, GLY77, GLU50, and ILE78. The distances between the ligand and the amino acid residues range from 2.04553 to 3.81553 Å. Catechin interacts with the antimicrobial protein target through hydrogen bonding and hydrophobic interactions with amino acid residues ARG76, GLU50, THR165, and ALA47. The distances between the ligand and the amino acid residues range from 2.61913 to 5.25295 Å.

Leotulin interacts with the antimicrobial protein target through hydrogen bonding, pi-bonding interactions, and hydrophobic interactions with amino acid residues ARG76, GLU50, THR165, ASN46, ILE78, and ALA47. The distances between the ligand and the amino acid residues range from 2.56108 to 5.05026 Å. Maleic Acid interacts with the antimicrobial protein target through hydrogen bonding and hydrophobic interactions with amino acid residues HIS99 and ALA100. The distances between the ligand and the amino acid residues range from 2.33271 to 2.67941 Å. Salicylic Acid interacts with the antimicrobial protein target through hydrogen bonding and hydrophobic interactions with amino acid residues ASP73, VAL120, and VAL167. The distances between the ligand and the amino acid residues range from 2.39911 to 5.39453 Å.Vanillic Acid interacts with the antimicrobial protein target through hydrogen bonding, pi-bonding interactions, and hydrophobic interactions with amino acid residues THR165, ASP73, VAL71, ASP73, THR165, ALA47, VAL43, VAL71, VAL167, and ILE78. The distances between the ligand and the amino acid residues range from 1.97698 to 5.36435 Å. All type of interactions is also shown in Supplementary Table S8 (Supplementary provided) as 3D and 2D.