In phytochemical research, the most critical step in the extraction of phenolic and other bioactive constituents from vegetables, fruits, and plants is solvent selection. In summary, different factors, such as solvent polarity, temperature, and time, influence the extraction efficiency of phenolic components, and their effects can be independent or coupled27. In this work, results of the preliminary screening showed that three solvents (ethyl acetate, chloroform, and hexane) showed the most minimal effect in the antiradical scavenging activity, the reducing power, and the antioxidant activity. These three solvent extracts showed also a lower amount of total phenolic compounds. According to our preliminary results, the antioxidant power of M. longifolia was greatly impacted by the polarity of the extraction solvent which was confirmed by Aazza et al.28. The total phenolic and flavonoid content of the different assessed extracts varied between 7.130 ± 0.786 to 23.524 ± 0.139 mg GAE/g DW, and from 3.757 ± 0.255 to 17.622 ± 0.359 mg QE/g DW respectively. It should be noted that the hydroethanolic extract exhibited greater TPC and TFC levels. Considering the preliminary screening performed based on solvent polarity, we decided to select the stronger extracts (hydroethanolic, acetonic, and water) to perform the phenolic composition and antibacterial activities, and their results will be represented and discussed in the remaining sections of this paper.
Choosing the best extraction solvent mixture depending on material properties is crucial to attaining high yields. As solvents for extraction, ethanol 70%, acetone, and water were used in Table 1. The findings indicate that the high extraction yield was recorded during extraction with 70% EtOH with a rate of 10.5 ± 0.074%, followed by water with a rate of 2.25 ± 0.016%, however, acetone solvent achieved the lowest yield with a rate of 1.9 ± 0.013%. According to researchers, the polarity of the solvent has a substantial effect on extract yield29. The extract yield in polar solvents (water and EtOH 70%) was reported to be greater than in nonpolar solvents (acetone). Brahmi and coworkers reported that hydro-alcoholic solvents had the maximum extraction yield. Whereas ethanol (at 75%) provided the best extraction rates for Algerian Mentha spicata (20.02%), acetone gave the lowest rate (2.6%)30.
HPLC–DAD examination of phenolic compounds
HPLC–DAD chromatograms of the aqueous, hydroethanolic, and acetonic extracts are shown in Table 2 and Figs. 1 (a, b, and c), respectively. The chemical profiles of the three extracts vary considerably, indicating the distinct efficacy of each extraction method. The aqueous extract contains mainly compounds with a retention time of around 19.732 min, as shown by the highest peak in Fig. 1. These are rather polar molecules, which is quite normal in an aqueous extract. This extract contains gallic acid, known for its antioxidant properties. Gallic acid has been associated with a variety of health advantages, including potential antioxidant effects4. The hydroethanolic extract reveals a diverse range of compounds, with significant peaks around 16.281 min (non-identified) and 17.975 min (kaempferol). A range of moderately polar to apolar molecules, demonstrating the efficiency of the mixed ethanol/water solvent. Kaempferol, present in hydroethanolic extract, is a flavonoid known for its antioxidant and anti-inflammatory potential. The acetone extract shows a distinct chemical profile, with significant peaks at around 16.258 min (non-identified) and 17.988 min (kaempferol). This is an extract containing molecules that are rather medium in polarity. The presence of chlorobenzoic acid and kaempferol in the acetone extract shows that the acetone extract may contain compounds with antimicrobial, and antioxidant properties31, similar to those observed in the hydroethanolic extract. Our data are in line with studies4,32, of other plant extracts. Our findings highlight the distinct chemical profiles of the three extracts, suggesting the effectiveness of each extraction method. These results confirm the value of Mentha longifolia extracts as potential sources of bioactive compounds with diversified therapeutic potential, mainly antioxidant and possibly anti-bacterial activity.
Phenolic and flavonoid content of the different Mentha longifolia leaf extracts
Phenolic compounds are the most common type of phytochemical found in a wide range of plant-based products, including phenolic acids (hydroxycinnamic acids, and hydroxybenzoic), polyphenols (condensed tannins, and hydrolyzable), flavonoids (rutin, naringenin, quercetin, apigenin, kaempferol)33, because of its importance in the pharmaceutical, and food industries, these compounds have been extensively researched34. In this context, M. longifolia (Horsemint) is considered a potential source of bioactive compounds as well as micronutrients, which can be explored as a promising alternative for the formulation of nutraceutical products35. As previously confirmed by our results, the phenolic content strongly depends on the selected extraction solvent.
Figure 2 (a) demonstrates the data of the total phenolic content of the tested M. longifolia extracts, and found that aqueous extracts had the lowest value (17.90 0.49 mg GAE/g DW), whereas acetonic extracts had the greatest value (23.52 0.14 mg GAE/g DW). This study suggests that hydroethanolic was the most efficient extraction solvent for extracting polyphenols, followed by acetone and water, suggesting that polar solvents extract more phenolic content than apolar solvents. Our results are higher than those reported by Patonay et al.,36 and Motamed and coworkers37. In contrast, Ertaşet al. found that the phenolic content of methanol, acetone, and petroleum ether extracts ranged from 217.10 ± 1.82 to 225.65 ± 3.42 µg GAE/mg and were higher than our extracts38.
Regarding flavonoids content results are summarized in Fig. 2 (b) and suggested that the hydroethanolic extract showed the highest amount of flavonoids (17.622 ± 0.359 mg QE/g DW), followed by acetone extract (11.174 ± 0.330 mg QE/g DW), whereas aqueous extract showed the lowest amounts with (4.114 ± 0.497 mg QE/g DW), these findings were inferior to those found by Ertaş et al.38 and Hajlaoui et al.39.
Antioxidant activity of the different Mentha longifolia leaf extracts
Results of the antiradical property (DPPH), reducing power, (PR), and the total antioxidant capacity (TAC) of the selected Mentha longifolia leaf extracts were summarized in Fig. 3 and showed a significant variation between the different extracts. Our findings showed that hydroethanolic extract exhibited the highest total antioxidant capacity (74.40 ± 1.34 mg AAE/g) followed by acetone extract (52.40 ± 0.20 mg AAE/g), and aqueous extract which showed the lowest value (20.98 ± 0.08 mg AAE/g) (Fig. 3 (c)). In the same way, the aqueous extract presented the lowest radical scavenging inhibition with the highest IC50 = 306 ± 0.1 µg/mL for the DPPH test and the highest EC50 = 0.80 ± 0.03 mg/mL for the reducing power (RP) compared to standards. However, the hydroethanolic extract showed higher free radical scavenging activity (DPPH) and reducing power (RP) (IC50 = 39.00 ± 0.00 µg/mL and EC50 = 0.261 ± 0.00 mg/mL respectively) followed by the acetone extract (IC50 = 43.00 ± 0.00 µg/mL and EC50 = 0.324 ± 0.00 mg/mL respectively) showing no statistical significance with BHT and ascorbic acid used as standards (Fig. 3 (a, b)). These outcomes were stronger than those reported by Bahadoi et al.5 for the Iranian M. longifolia ethanolic and aqueous extracts in which they reported DPPH IC50 values ranged between 195.96 ± 0.94, and 162.08 ± 3.90 mg TEs/g extract, and RP EC50 values ranged between 239.87 ± 3.95, and 346.20 ± 0.17 mg TEs/g sample. The findings of this study showed that hydroethanolic solvent is the most effective for extraction and it had the highest levels of phenolic and flavonoid content, which were closely related to the strong antioxidant activities (DPPH, TAC, and RP) that were noticed. It was followed by acetone and water. Our findings are consistent with those previously published, which reported that high-polarity solvents are extensively used to extract antioxidant compounds such as hydroethanolic, water, acetone, and methanol. It is well known that ethanol, methanol, and water are polar solvents commonly used to extract polar molecules like phenolic and flavonoid components40.
Previous studies have been conducted to identify the phenolic and flavonoid profiles of M. longifolia and revealed a variety of natural antioxidant molecules such as phenolic acids (gallic acid, vanillic acid, rosmarinic acid, caffeic acid, syringic acid, ρ-coumaric acid, o-coumaric acid, ferulic acid, trans-cinnamic acid, and chlorogenic acid) and flavonoids (naringenin, rutin, quercetin, apigenin, kaempferol)36,41,42. Iranian authors5 stated that rosmarinic acid and cinnamic acid were principally identified in their M. longifolia areal part extracts. Furthermore, flavone glycosides such as apigenin and luteolin were the major flavonoids detected in the Moroccan M. longifolia areal part extracts43. The findings of the present work suggest that the strong antioxidant activity of M. longifolia extracts is mostly attributed to its phenolic profile5,44.
Evaluation of the antibacterial activity
The outcomes of the antibacterial effect of M. longifolia leaf extracts are represented in Tables 3 and 4. In this study, a total of 4 nosocomial pathogenic microbial strains (2 Gram-positive and 2 Gram-negative) were used to investigate the antibiotic potential of our samples. Results showed that all extracts revealed strong bacterial growth inhibition against all tested pathogenic bacteria. In particular, acetone extract showed the highest inhibitory effect against Bacillus cereus with a MIC = 1.17 ± 0.05 mg/mL, and MBC = 1.50 ± 0.05 mg/mL, and Staphylococcus aureus, (MIC = 2.34 ± 1.10 mg/mL, and MBC = 6.25 ± 0.43 mg/mL). Additionally, acetone extract exhibited higher activity against Escherichia coli with a MIC = 6.25 ± 0.00 mg/mL. It is crucial to record that acetone extract has exhibited a powerful antibacterial effect against Gram-positive bacteria, more than Gram-negative bacteria except Pseudomonas aeruginosa. Acetone plant extract tends to be more effective in terms of antibacterial activity; indeed, Felhi et al.45 reported that the acetone extract of fruit bark and seeds of E. elaterium signaled the highest activity against Staphylococcus aureus and Bacillus subtilis. However, Zhang et al.,46 have demonstrated in their research that the ethanolic extract of Mentha arvensis exhibited strong antibacterial activity against acinetobacter baumannii. Another study on methanolic extract of the aerial parts of M. longifolia ssp. had no antibacterial effect4. Preeti et al.47 evaluated the antibacterial efficacy of several Mentha piperita leaf solvent extracts against pathogenic bacteria notably Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, Proteus vulgaris, and results revealed that aqueous and ethyl acetate extracts exhibited the strongest antibacterial activity.
In our current study, we noticed that Gram-positive strains were more responsive to our extracts compared to Gram-negative strains. The reason for the lower sensitivity of Gram-negative bacteria could be attributed to the complexity of their double membrane, which includes a cell envelope made up of a lipoprotein and a lipopolysaccharide layer (LPS). Unlike Gram-positive bacteria’s single membrane, this structure works as a biological barrier to antibacterial drugs48. The demonstrated antibacterial activity of our Mentha longifolia extracts can be explained by its rich phytochemical profile, which includes caftaric acid, rosmarinic acid, cryptochlorogenic acid, ρ-coumaric acid, m-coumaric acid, chlorogenic acid, caffeic acid, gallic acid, luteolin, apigenin, quercetin, rutin, coumarins, and isocoumarins49,50. Indeed, previous studies have reported greater antimicrobial potency for caffeic, cryptochlorogenic, as well as chlorogenic acids49,51,52. Other phenolic compounds found in various species of the genus Mentha such as ellagic acid, ferulic acid, gallocatechin, epigallocatechin gallate, and catechins were reported to exhibit antibacterial or bacteriostatic effects against multiple bacterial strains including Escherichia coli, Staphylococcus aureus, Bacillus aureus, Bacillus pumilis, Bacillus subtilis, and Pseudomonas aeruginosa. This effect is associated with their capability to penetrate the bacterial wall and reach the bacterial cytoplasm52.
As stated previously, several solvent combinations have been applied to effectively extract phenolic components from plant material. Water, ethanol, methanol, acetone, and their aqueous mixes are the most commonly used solvents53. The significant antibacterial impact found in our investigation can be attributed to acetone’s ability to extract antimicrobial compounds due to its power to dissolve hydrophilic and lipophilic components54. Furthermore, acetone is the most effective extractor of plant material since it can extract molecules with a wide range of polarities and has low toxicity in biological assays, making it a highly valuable extraction solvent55. Interestingly, acetone was shown to be the most effective solvent for extracting polyphenols, such as hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, ρ-coumaric acid, chlorogenic acid, luteolin, quercetin, vanillic acid, and catechin), and hydroxybenzoic acids (gallic acid, and protocatechuic acid)56. These molecules have been mentioned to have a powerful antibacterial effect through several mechanisms, notably membrane instability, membrane hyper-permeabilization, hyper-acidification, enzyme inhibition, and the generation of reactive quinones57. According to Campos et al.,58 these acids might also inhibit the synthesis of nucleic acids by Gram-negative and Gram-positive bacteria. Due to their propenoid side chain, hydroxycinnamic acids are slightly more polar than corresponding hydroxybenzoic acids, which may assist their transit through the cell membrane increasing their toxic effect into the bacterial cell59,60. In the same context, gallic and ferulic acids have been shown to cause bacterial cell death by altering bacterial hydrophobicity (interacting with the surface of Gram-negative and Gram-positive bacteria), potentially acidifying their cytoplasm by increasing K + release and inducing protein denaturation, which can change the cytoplasmic membrane permeability, induce intracellular material release, and cause membrane damage.60. Moreover caffeic acid, and ρ-coumaric acid impact cell membrane structure through inflexibility and alterations in the phospholipid chains’ stability61.
In silico assessment
Molecular docking against Phospholipase C Bacillus cereus
Three compounds identified from M. longifolia leaf extracts through HPLC were subjected to molecular docking studies. The findings indicated that all three compounds bind to the same region within the active site (Fig. 4). Among these compounds, Kaempferol displayed the highest binding energy at −9.67 kcal/mol, followed by Ferulic acid at −9.5 kcal/mol and Gallic acid at −8.75 kcal/mol (Table 5).
The interaction analysis between Phospholipase C (PDB ID: 2huc) and the top-performing compound kaempferol revealed the formation of five hydrogen bonds at specific amino acid residues: Cys8 (2.83 Å), Thr9 (2.00 Å), Ala11 (2.30 Å), Thr112 (2.95 Å), and Lys134 (3.00 Å) (Fig. 4a). In the case of 2cdu and Ferulic acid, there were four hydrogen bonds formed with Thr9 (2.43 Å), Ala11 (2.40 Å), Gly12 (2.17 Å), and Thr112 (2.27 Å) (Fig. 4b). Notably, both Kaempferol and Ferulic acid shared a hydrogen bond at the same position, suggesting a similar interaction pattern with the target protein.
Additionally, the third compound, Gallic acid, exhibited interactions with the target protein 2huc, forming three hydrogen bonds at Thr9 (2.27 Å), Thr112 (2.78 Å), and Asp282 (2.12 Å) (Fig. 4c).
Molecular docking against NADPH oxidase
In terms of binding affinity to the target enzyme NADPH oxidase, Ferulic acid exhibited the strongest interaction with a binding energy of −8.4 kcal/mol, followed by Kaempferol and Gallic acid with binding energies of −8.0 and −7.9 kcal/mol, respectively (Table 6). Ferulic acid formed interactions with NADPH oxidase (PDB ID: 2cdu) through four hydrogen bonds, which significantly contributed to its binding stability. These hydrogen bonds were observed with specific amino acid residues: Trp1 (2.05 Å), Asp122 (2.72 Å), His128 (2.83 Å), and Thr65 (3.63 Å) (Fig. 5a).
On the other hand, both Kaempferol and Gallic acid exhibited an equivalent number of hydrogen bonds when interacting with the target protein. Specifically, they formed hydrogen bonds with Trp1 (at 2.28 Å) and His228 (at 2.67 Å) for Kaempferol (Fig. 5b), and with Trp1 (at 2.46 Å) and Asp55 (at 1.85 Å) for Gallic acid (Fig. 5c), respectively.