INTRODUCTION

Bacterial skin infections, particularly those caused by Escherichia coli and Staphylococcus aureus, represent a significant global health challenge, especially in healthcare settings. These pathogens are a leading cause of healthcare-associated infections (HAIs), imposing a considerable socioeconomic burden due to prolonged treatment durations and escalating healthcare costs [1, 2].

Conventional antibiotic treatments against these bacteria face considerable challenges. Sophisticated bacterial resistance mechanisms, such as efflux pump systems that actively expel antimicrobial compounds, combined with the protective nature of biofilms, can increase bacterial tolerance by 10- to 1,000-fold [3, 4]. This has spurred the exploration of alternative antimicrobial strategies. Mentha piperita essential oil (EO) and its principal component, menthol, have demonstrated broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as anti-inflammatory properties, making them promising candidates for managing skin infections [5-7]. Menthol, a naturally occurring chemical found in numerous plant species, has garnered considerable interest due to its unique pharmacological properties. Menthol, widely recognized for its aromatic and cooling properties, demonstrates a diverse range of biological actions that highlight its medicinal potential [8-10]. To rationally guide their application, computational approaches such as molecular docking can provide valuable insights into the potential mechanisms of action by predicting the binding interactions and affinity of menthol with key bacterial proteins [11, 12].

Furthermore, EOs and their constituents are inherently lipophilic and volatile, which limits their direct topical application and efficacy. To enhance their usability, these compounds require formulation into hydrophilic delivery systems. Hydrogels, three-dimensional polymeric networks that can absorb large amounts of water while maintaining structural integrity, are among the most suitable platforms for topical applications due to their high biocompatibility, ease of application, and capacity for controlled release [13, 14].

A promising approach in hydrogel formulation involves polymer combination. In this strategy, the EO or active component is first dispersed within a bioactive polymer, such as chitosan. A secondary polymer is then used to gel the system, leveraging the synergistic properties of both materials [15, 16]. Chitosan, a natural polysaccharide derived from chitin, is widely recognized for its biocompatibility, biodegradability, and intrinsic antimicrobial properties [17]. Hydroxypropyl methylcellulose (HPMC), a semi-synthetic polymer, is another excellent candidate, known for its superior gel-forming capacity, biocompatibility, and stability in pharmaceutical formulations [18, 19]. Furthermore, with advancements in nanotechnology, loading bioactive compounds into nanostructures prior to hydrogel incorporation has emerged as a powerful strategy to enhance stability, bioavailability, and provide sustained release, leading to the development of efficient nanoparticle-based hydrogels [20, 21].

In this study, we developed and characterized advanced hydrogels containing M. piperita EO and menthol. Chitosan/HPMC hydrogels and nanoparticle-based chitosan/HPMC hydrogels containing either the EO or menthol (Chi/HPMC-EO, Chi/HPMC-Menthol, ChiNP/HPMC-EO, and ChiNP/HPMC-Menthol) were prepared, and their antibacterial efficacy was evaluated against E. coli and S. aureus. Following the promising results from these antibacterial assays, we performed molecular docking simulations to gain deeper insight into the molecular basis of the observed activity. Specifically, the interactions of menthol with a selection of vital protein targets from S. aureus and E. coli were investigated..

MATERIALS AND METHODS

Materials

Chitosan (medium molecular weight, 75–85% deacetylation), menthol, tween 20, and hydroxypropyl methylcellulose (HPMC) were obtained from Merck Chemicals (Germany). Bark-extracted M. piperita EO was supplied by Tabib Daru Company (Iran). Bacterial strains, namely S. aureus (ATCC 25923) and E. coli (ATCC 25922), were provided by the Paesture Institute (Iran).

M. piperita EO Analysis Using GC-MS

The chemical composition of M. piperita EO was characterized using gas chromatography-mass spectrometry (GC-MS). Analysis was performed on an Agilent 6890 gas chromatograph coupled with an Agilent 5973N mass selective detector. Volatile constituents were separated on an HP-5ms capillary column with helium as the carrier gas. The oven temperature was initially set at 50°C (held for 3 minutes) and then increased linearly at a rate of 10°C/min until it reached 300°C. Mass spectra were recorded in electron impact ionization mode over a mass range of 35–450 m/z, and compound identification was achieved by matching the obtained spectra with reference libraries (Wiley and Adams).

Preparation and Characterization of Chitosan nanoparticle and Chitosan solution-based HPMC hydrogels

Because menthol is difficult to disperse uniformly in aqueous media, ultrasound was also used in the ionic gelation method [22]. For both active compounds, 0.25% w/v of either M. piperita EO or menthol was emulsified with 0.5% w/v of Tween 20 by stirring continuously at room temperature for 5 min (2000 rpm). Next, 4.5 mL of a 0.25% (w/v) chitosan solution—prepared by dissolving chitosan in 1% (v/v) acetic acid—was added dropwise and then was sonicated for 30 min using a bath sonicator (37 kHz) to ensure uniform dispersion of M. piperita EO or menthol into the polymeric matrix. For nanoparticle formation, sodium tripolyphosphate (TPP) solution (0.04% w/v) was added to reach 5 mL to initiate ionic crosslinking, and the mixture was stirred (2000 rpm) for an additional 40 minutes to complete nanoparticle formation. Subsequently, HPMC powder (1.5% w/v) was gradually incorporated into the prepared chitosan nanoparticles with continuous stirring (2000 rpm) overnight at room temperature to achieve a homogeneous mixture with optimal viscoelastic properties. The prepared samples were named ChiNP/HPMC-EO and ChiNP/HPMC-Menthol.

The hydrogel based on chitosan solutions was prepared similarly, with the same amount of distilled water used instead of the TPP solution; these were named Chi/HPMC-EO and Chi/HPMC-Menthol. Additionally, to investigate the antibacterial properties of the components in the prepared hydrogels, blank hydrogels, i.e., those without M. piperita EO or menthol, were prepared using the same method; they were named ChiNP/HPMC and Chi/HPMC.

The resultant chitosan nanoparticles were analyzed using Dynamic Light Scattering (DLS) to confirm nanoparticle sizes below 200 nm and a narrow particle size distribution (SPAN < 1). In addition, Attenuated Total Reflectance-Fourier-Transform InfraRed (ATR-FTIR) spectroscopy was performed on multiple samples—namely A: M. piperita EO, B: menthol, C: HPMC, D: Chi/HPMC, E: Chi/HPMC-EO, F: Chi/HPMC-Menthol, G: ChiNP/HPMC, H: ChiNP/HPMC-EO, and I: ChiNP/HPMC-Menthol—to verify successful encapsulation and to identify potential interactions between the active compounds and the polymeric components.

Antimicrobial Evaluation

The antimicrobial efficacy of the formulations was evaluated using the AATCC 100 standard method. Bacterial suspensions were adjusted to a 0.5 McFarland standard for both S. aureus and E. coli. Depending on the amount of EO or menthol in the prepared hydrogels (0.25%), adding 1, 0.5, and 0.25 grams of each Chi/HPMC-EO, Chi/HPMC-Menthol, ChiNP/HPMC-EO, and ChiNP/HPMC-Menthol to 2 mL suspension of each bacteria in a 5 cm plate, their final concentrations reached 1250, 625, and 310 μg/ml. Also, the same number of samples without EO/menthol (i.e., Chi/HPMC and ChiNP/HPMC) was added to the plates to investigate the effects of other substances in the hydrogels. After 24 hours of incubation at 37 °C, 10 μl of the suspension from each plate was cultured on an agar plate. Finally, the bacterial growth rate was calculated by comparing the colony-forming units (CFU/mL) in the control group (untreated) with those in the treated samples. All experiments were conducted in triplicate, and results are expressed as the mean ± standard deviation (SD).

In silico Study

This research employed menthol as the ligand for molecular docking studies to evaluate its potential antibacterial interactions. We obtained menthol’s three-dimensional structure from the PubChem compound database (https://pubchem.ncbi.nlm.nih.gov/) and used the Avogadro program (Version 1.2, https://avogadro.cc/) to minimize energy.

We selected protein targets from two types of bacteria, S. aureus and E. coli, as they are crucial for virulence, resistance, and survival. The chosen targets comprised ClfA, FemA, FmtA, SasG, DDL, and Sortase A from S. aureus, as well as DHPS, DNA gyrase, and Topoisomerase IV from E. coli. Crystal structures of available proteins were obtained from the Protein Data Bank (PDB, https://www.rcsb.org/). Binding site prediction for proteins was performed using the PrankWeb server (https://prankweb.cz/) and also based on the positions of natural ligands present in the protein structure.

Molecular docking simulations were performed using the Molegro Virtual Docker (MVD) software. We used MolDock and Re-rank scores to determine binding affinities and hydrogen bond energies, thereby calculating the strength of the interactions. We used BIOVIA Discovery Studio Visualizer to visualize and understand protein-ligand interactions, which include hydrogen bonds, hydrophobic contacts, and π-π interactions.

RESULT

The GC-MS analysis of the M. piperita EO identified 14 distinct components (Table 1). After menthol (21.249%), the most predominant compounds were germacrene D, trans-caryophyllene, and camphane, constituting most of the EOs profile.

Chitosan Nanoparticles Containing Menthol and M. piperita EO

DLS analysis revealed that chitosan nanoparticles containing menthol had an average size of 179 ± 8 nm with a SPAN value of 0.96. In comparison, chitosan nanoparticles containing M. piperita EO exhibited an average size of 195 ± 6 nm with a SPAN value of 0.97 (Figure 1).

ATR-FTIR spectroscopy was used to investigate the successful incorporation of M. piperita EO and menthol within the prepared hydrogels. The resulting spectra are illustrated in Figure 2. ATR-FTIR analysis of M. piperita EO reveals key absorption bands, including a broad band at 3441 cm-1, characteristic of hydrogen-bonded OH groups (alcohols and phenols like menthol), and bands at 2952, 2922, and 2868 cm-1 for C-H stretching vibrations. A distinct absorption at 1706 cm-1 and a shoulder at 1734 cm-1 provide evidence of the presence of carbonyl groups. Furthermore, the absorption bands at 1452 and 1369 cm-1 can be attributed to C-O-H, CH3, and CH2 bending vibrations. The bands observed at 1245, 1098, 1045, and 1024 cm-1 correspond to characteristic vibrations associated with C-O-C stretching and/or -CH2- deformation modes. Additionally, the bands identified at 988, 919, 881, and 844 cm-1 can be attributed to out-of-plane (oop) C-H bending vibrations, typically associated with alkenes and aromatic ring structures.

The menthol spectrum exhibits the typical O-H and C-H bond absorptions at 3243, 2954, 2924, and 2869 cm-1, respectively. Bending vibrations associated with C-O-H, CH2, and CH3 groups are identified within the 1451-1224 cm-1 range. Intense absorption bands in the 1174-995 cm-1 region are attributed to C-O stretching vibrations. The characteristic absorption bands of HPMC are observed at 3421 cm-1 (O-H stretching), 2896 cm-1 (C-H stretching), 1452-1312 cm-1 (CH2, CH3, and C-O-H bending), and 1051 cm-1 (C-O-C stretching).

The spectrum of Chi/HPMC provides significant insights into its chemical composition. A broad band at 3653- 3252 cm-1 indicates O-H stretching from HPMC, chitosan, and tween 20, as well as the NH2 stretching from chitosan. Peaks at 3023, 2925, and 2856 cm-1 correspond to the C-H stretching vibration. A medium band at 1711 cm-1 is characteristic of the carbonyl groups. Other peaks at 1546, 1459, 1378, 1281, and 1073 cm-1 correspond to the NH, CH2, CH3, and C-O-H bending and C-O-C stretching vibrations, respectively.

The spectrum of Chi/HPMC-EO shows absorption bands at 3456, 2922, 1709, 1546, 1453, 1381, 1278, 1064, and 946 cm-1, corresponding to O-H/NH2, C-H, and C=O stretching, NH, CH2, and CH3 bending, and C-O stretching vibrations, respectively. Pique intensity, position, and shape changes indicate interactions between the hydrogel matrix and M. piperita EO. A red shift in the O-H stretching peak suggests hydrogen bond formation, and increased intensities of the C-H and C-O bands confirm the EO’s incorporation into the hydrogel.

The Chi/HPMC-Menthol spectrum exhibits characteristic bands of both hydrogel and menthol, confirming successful menthol encapsulation. The characteristic peaks at 3487, 2979, 1715, 1556, 1469, 1054, and 863 cm-1 correspond to O-H/NH2, C-H, C=O stretching, NH, C-O-H and CH2/ CH3 bending, C-O stretching, and oop C-H bending vibrations, respectively. A red shift in the O-H/NH2 peak, increased absorption near 863 cm-1, and a carbonyl peak at 1715 cm-1 indicate the interaction between the hydrogel matrix and menthol.

The spectrum of ChiNP/HPMC exhibits a broad band at 3670-3363 cm-1, corresponding to O-H stretching and intramolecular hydrogen bonding. Asymmetric and symmetric stretching vibrations of C-H are observed at 3023 and 2926 cm-1. The characteristic band at 1712 cm-1 is assigned to the carbonyl group. The band at 1548 cm-1 is likely associated with N-H bending of primary amine, while the bands at 1456, 1415, 1378, and 1280 cm-1 correspond to bending modes of CH2, CH3, and C-O-H, as well as P=O stretching vibrations of tripolyphosphate (TPP). A distinct strong band at 1068 cm-1 is also assigned to the stretching vibrations within various C-O bonds.

The ATR-FTIR spectra of ChiNP/HPMC and ChiNP/HPMC-EO show differences in peak position, shapes, and intensities, indicating interactions between the nanogel and M. piperita EO. Absorption bands at 3510, 2926, 1713, 1551, 1455, 1379, 1278, and 1072 cm-1 correspond to O-H/NH2, C-H, and C=O stretching, N-H, C-O-H, CH2, and CH3 bending, P=O, and C-O stretching vibrations. A shift in the O-H/NH2 peak suggests hydrogen bonding between the nanogel and the EO components. Enhanced C-H and C-O intensities, along with altered peak shapes, confirm the successful loading of EO into the nanogel matrix.

ATR-FTIR analysis of ChiNP/HPMC-Menthol reveals overlapping peaks from both nanogel and menthol, indicating successful encapsulation. The broad band at 3597-3373 cm-1 corresponds to O-H and N-H stretching with hydrogen bonding, while peaks at 2962 and 2921 cm-1 represent C-H vibrations. Key bands at 1709, 1546, 1455, 1395, 1274, 1067, 948, and 894 cm-1 are assigned to C=O and C=C stretching, N-H, C-H and O-H bending, and C-O/P=O stretching, as well as oop C-H bending vibrations. Distinct spectral changes, including a shift in the O-H/NH2 peak, altered C-H peak shape, increased C-O intensity, and the appearance of a peak at 894 cm-1, confirm the encapsulation of menthol within the nanogel.

Viscosity Analysis

Figure 3 (A–F) illustrates the viscosity profiles of the different formulations versus shear rates. In each panel, the square markers represent the experimental data, while the dashed lines indicate the Carreau–Yasuda model fitting. As observed, all formulations exhibit decreasing viscosity with increasing shear rate, indicative of a shear-thinning (non-Newtonian) behavior. This behavior is described in the Carreau–Yasuda model, which explains how changes in the structure and arrangement of polymer chains occur under various shear conditions.

Antibacterial Efficacy

The antibacterial effects of Chi/HPMC, Chi/HPMC-EO, Chi/HPMC-Menthol, ChiNP/HPMC, ChiNP/HPMC-EO, and ChiNP/HPMC-Menthol were evaluated against E. coli and S. aureus at concentrations of 310, 625, and 1250 µg/mL (Figures 4 and 5). For E. coli, the control group exhibited 100% bacterial viability across all concentrations, indicating no intrinsic resistance to the treatment. Similarly, Chi/HPMC and ChiNP/HPMC matrices showed minimal antibacterial effects, with viability exceeding 90%. In contrast, adding Chi/HPMC-EO and Chi/HPMC-Menthol significantly enhanced antibacterial activity, reducing E. coli viability to about 5% at 1250 µg/mL. The chitosan nanoparticle-based formulations (ChiNP/HPMC-EO and ChiNP/HPMC-Menthol) demonstrated even greater potency, with bacterial viability reduced to approximately 1%.

A comparable trend was observed against S. aureus, though the inhibition was generally more pronounced. The control group again demonstrated 100% viability at all concentrations, whereas the Chi/HPMC and ChiNP/HPMC exhibited minimal antibacterial effects. At 1250 µg/mL, Chi/HPMC-Menthol reduced S. aureus viability to around 5%, whereas Chi/HPMC-EO achieved complete bacterial eradication. Notably, the nanoparticle-based formulations (ChiNP/HPMC-Menthol and ChiNP/HPMC-EO) exhibited the most vigorous activity, reducing viability to approximately 5% at the lowest tested concentration (310 µg/mL) and nearly eliminating bacterial growth (0.5% viability) at higher concentrations. These findings indicate that S. aureus is more susceptible than E. coli, aligning with earlier reports that Gram-positive bacteria—owing to their more straightforward cell wall structure—are generally more vulnerable to EOs and natural antimicrobial agents.

Molecular docking results

Molecular docking results showed that menthol formed favorable interactions with all selected target proteins from S. aureus and E. coli. As shown in Table 2, menthol showed the highest binding affinity for the FmtA protein from S. aureus (with a MolDock score of −78.6 kcal/mol and a Rerank score of −70.1 kcal/mol). This was followed by DNA gyrase from E. coli (−71.1 kcal/mol; −61.6 kcal/mol) and ClfA from S. aureus (−70.1 kcal/mol; −55.0 kcal/mol). Additionally, the enzymes D-alanine–D-alanine ligase and Sortase A exhibited significant binding energies, suggesting the potential for inhibiting cell wall synthesis and protein binding to the membrane. The number of hydrogen bonds varied between 1 and 5, indicating the stability and specificity of the ligand-receptor complexes.

According to the observed protein-ligand complexes in Figures 6 and 7, menthol is located in the active site of the proteins in all the studied targets and establishes stable interactions. These interactions primarily involve hydrogen bonds with polar residues, as well as hydrophobic and π–π or π–alkyl contacts with nonpolar residues within the active site. In most targets, menthol is fixed deep in the active cavity. It is surrounded by several amino acid residues, which stabilize the complex through van der Waals forces and hydrophobic interactions. Examination of 3D models by docking reveals that this type of interaction pattern is similar in most of the studied proteins and plays a crucial role in maintaining the equilibrium and stability of the ligand in the binding environment.

DISCUSSION

Antimicrobial resistance (AMR) remains one of the most pressing challenges facing global healthcare systems. In this context, medicinal plants and their bioactive compounds have attracted growing attention as potential alternative antimicrobial agents [23, 24]. Essential oils (EOs), complex mixtures of volatile secondary metabolites derived from aromatic plants, exhibit diverse bioactivities that are often attributed to their major constituents [25, 26]. A central question in this field is whether the whole EO exerts greater efficacy than its isolated active components. The literature presents varied conclusions; for instance, while Tanacetum balsamita EO demonstrated greater larvicidal potency than its component carvone [27]. Similarly, the isolated compound α-pinene showed more substantial anticancer effects than its source, Rosmarinus officinalis EO [28]. In another study, Cymbopogon citratus EO and its major component, citral, exhibited IC50 values of 1760 and 380 µg/mL against MDA-MB-231 breast cancer cells [29]. Similarly, the antibacterial activity of Syzygium aromaticum EO and eugenol varies considerably depending on the specific microbial target [30, 31]. These comparative studies underscore the necessity of evaluating both whole EOs and their primary constituents.

In our study, both ChiNP/HPMC-Menthol and ChiNP/HPMC-EO exhibited potent antibacterial activity, with the latter showing marginally superior efficacy against S. aureus. This aligns with the existing body of evidence, suggesting that the relative effectiveness of an EO versus its principal components is highly dependent on the specific microorganism being targeted.

The antimicrobial mechanisms of EOs and plant-based compounds represent a dynamic area of research. These natural agents interact with microorganisms at the molecular level, offering novel strategies to counter antibiotic resistance [32, 33]. The activity of M. piperita EO and menthol involves multiple pathways that target bacterial viability. As a lipophilic monoterpene, menthol can integrate into and disrupt bacterial membrane integrity, increasing permeability and leading to the leakage of vital intracellular components, ultimately causing cell death [34, 35]. The whole EO, rich in monoterpenes, exerts a similar effect on membrane integrity. Gram-positive bacteria, such as S. aureus, are generally more susceptible to this action due to their simpler membrane architecture. In contrast, the complex outer membrane of Gram-negative bacteria, like E. coli, can confer greater resistance [36, 37]. Beyond membrane disruption, these compounds can interfere with biofilm formation [38, 39] and potentially disrupt intracellular targets, including enzymes and DNA replication processes [40, 41].

Nanoparticle-based delivery systems present a promising strategy to enhance the stability and bioavailability of volatile compounds. Our Dynamic Light Scattering (DLS) analysis confirmed the formation of chitosan nanoparticles with a uniform and narrow size distribution, which is critical for controlled release and optimal bioavailability [42]. Encapsulating M. piperita EO and menthol within these nanoparticles serves to prevent volatilization, facilitate sustained release, and improve antimicrobial performance. The inherent physicochemical properties of chitosan further augment this effect by promoting interaction with bacterial membranes [43-45].

Rheological characterization revealed that our hydrogel formulations exhibit shear-thinning behavior, accurately described by the Carreau–Yasuda model [46]. This property is ideal for topical applications, as it ensures high viscosity at rest for stability while allowing easy spreadability under shear stress. The subsequent recovery of viscosity upon application prolongs the residence time of the active compounds at the site, thereby enhancing efficacy and enabling controlled release [47, 48].

A comparative evaluation with previous studies highlights the superior performance of our formulations. For example, while chitosan nanoparticles loaded with lemongrass EO required 1200 µg/mL to achieve 90% inhibition of P. aeruginosa and S. aureus [49]. Zingiber officinale EO-loaded chitosan nanoparticles showed only ~60% inhibition against E. coli and S. aureus [50]. Additionally, nanoemulsion-based gels containing camphor and thymol (1250 µg/mL) completely suppressed the growth of P. aeruginosa and S. aureus, while higher concentrations (1250–2500 µg/mL) were required to fully inhibit the growth of Listeria monocytogenes and E. coli [51]. Our formulations achieved total bacterial inhibition at significantly lower concentrations. This enhanced potency underscores the potential of our hydrogels as next-generation antimicrobial treatments.

To better understand the molecular basis of the observed antibacterial effects, molecular docking studies were performed for menthol against several essential protein targets of S. aureus and E. coli. The selected targets included S. aureus clumping factor A (ClfA), a surface-anchored adhesin that facilitates colonization and virulence [52]. D-alanine: D-alanine ligase (DDL), an enzyme crucial for bacterial cell wall synthesis [53]. Moreover, dihydropteroate synthase (DHPS), a key enzyme in folate biosynthesis, is inhibited by sulfonamide antibiotics that compete with p-aminobenzoic acid (pABA) [54].

Additional targets included FemA and FmtA—factors associated with methicillin resistance in S. aureus [55], as well as the biofilm-promoting surface protein SasG [56]. Moreover, sortase A is a transpeptidase responsible for anchoring surface proteins to the peptidoglycan layer [57]. Moreover, DNA gyrase and topoisomerase IV, both essential enzymes involved in bacterial DNA replication and supercoiling, were examined as critical antibacterial targets [58, 59].

Docking results revealed that menthol exhibited notable binding affinities with several of these proteins, stabilized through hydrogen bonding and hydrophobic interactions. These results suggest that menthol can effectively occupy active sites and potentially interfere with vital bacterial functions. This computational evidence corroborates previous experimental findings, which show menthol’s vigorous antibacterial activity against both Gram-positive and Gram-negative bacteria [60]. Furthermore, menthol has been reported to suppress the expression of key virulence factors such as hemolysins and toxins in S. aureus, indicating a possible dual mechanism of antimicrobial and anti-virulence action [61].

CONCLUSION

This study successfully demonstrates the development of chitosan nanoparticle-based hydrogels loaded with M. piperita EO and menthol as potent antimicrobial systems. These formulations exhibited strong antibacterial activity, achieving nearly complete inhibition of S. aureus and E. coli at low concentrations. The combination of experimental and in silico findings highlights menthol’s multifaceted antibacterial potential, involving both membrane disruption and enzyme inhibition mechanisms. Taken together, these results position menthol-loaded chitosan hydrogels as promising candidates for future topical antimicrobial therapies. Further investigations, particularly in vivo efficacy and toxicity studies, are necessary to validate their safety and clinical potential for managing resistant skin infections.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study has been ethically approved (IR.FUMS.REC.1403.037). Since this research did not involve a human study, an informed consent form was not used.

CONSENT FOR PUBLICATION

Not applicable.

ACKNOWLEDGMENTS

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

All data generated or analyzed during this study are included in this published article.

COMPETING INTERESTS

Not applicable.

FUNDING

Fasa University of Medical Sciences supported this study, grant No. 403024.