Novel eugenol/limonene nanoplatform as a new remedy against bacterial lung infections
Bassma H. Elwakil, Marwa M. Shaaban, Basant A. Bakr, Moaaz T. Hamed, Keshav Raj Paudel, Adel M. Atia, Mohamed Zakaria
TL;DR
A new nanoemulsified platform combining eugenol and limonene from essential oils shows antimicrobial potential against lung infections, though safety concerns remain.
Contribution
A novel nanoemulsified nanoplatform combining eugenol and limonene from essential oils is proposed for treating bacterial lung infections.
Findings
The nanoemulsion inhibits DHFR with an IC50 of 8075 ± 0.96 μg/mL.
It shows broad antibacterial activity with inhibition zones of 24.0–29.0 mm and MICs of 8.0–32.0 μg/mL.
In vivo-like tests show reduced bacterial load but also airway changes indicating safety concerns.
Abstract
A combinational approach to antimicrobial design emerges from a nanoemulsified nanoplatform from the union of two Egyptian essential oils—orange peel and clove—whose distinct chemistries were revealed by GC–MS. Orange EO is dominated by D-limonene (63.25%), with supporting alkyl-benzenes (22.92%) and β-myrcene (1.41%), while clove EO centers on Eugenol (36.20%), Caryophyllene (19.30%), Eugenol acetate (18.71%), along with alkyl-benzenes (11.86%) and humulene (2.41%). The chemical consonance between Eugenol and Limonene hints at a cooperative assembly: their lipophilic profiles enable the formation of nanoemulsified nanoparticles from their emulsion blend, potentially amplifying bioavailability and multi-target action. Biologically, Eugenol/Limonene demonstrates tangible antimicrobial activity. In a direct DHFR inhibition assay, the nanoformulation inhibits DHFR with an IC50 of 8075 ±…
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TopicsEssential Oils and Antimicrobial Activity · Antimicrobial agents and applications · Inhalation and Respiratory Drug Delivery
Introduction
Bacterial lung infections are a major public health concern in humans. Pneumonia, for example, is responsible for more than 1.3 million child deaths each year^1^. The epithelial lining of the upper respiratory tract and the mucosal surface may be invaded by pneumonic pathogens, which can also escape the immune response^1,2^. In vulnerable hosts, such as those experiencing a severe environment, overcrowding, or being exposed to indoor air pollution, these bacteria may enter the lower respiratory tract and cause illnesses^2^. Chronic lung infections caused by Pseudomonas aeruginosa are common in people with a variety of lung illnesses, including cystic fibrosis (CF), bronchiectasis, and chronic obstructive pulmonary disease (COPD)^3,4^. Directing aerosolized antibiotics to the lungs is an intriguing strategy, especially given the possibility of an increased therapeutic/systemic side effect index^5^. The spread of multidrug-resistant (MDR) bacteria is a global health emergency that has a devastating effect on the health of those who encounter it^6,7^. This includes not only those who have the infection but also those who are admitted to the hospital, receive a transplant, or are being treated for cancer^7,8^. Antibiotic resistance was identified as a global health problem in a 2017 study by the World Health Organization’s Global Antimicrobial Surveillance System^9^. In addition to the shortage of novel antibiotic therapies entering the pipeline, the overuse and improper use of antibiotics worsen this public health hazard^10^. Hence, more attention was directed to natural products to avoid the possible side effects of the commonly used drugs.
Rationale study
Eugenol is a hydroxyphenyl propene; it is one of the major constituents of clove oil. Eugenol has become a popular subject of research due to its antimicrobial and anti-inflammatory properties^11^. The antimicrobial activity of eugenol can be attributed to its free hydroxyl group, which can bind to essential bacterial enzymes, preventing their action^12^. In addition, eugenol possesses comparable structural features to trimethoprim (Fig. 1), the potent dihydrofolate reductase (DHFR) inhibitor^13^. Thus, a docking study was carried out to predict the binding affinity and the binding mode of eugenol toward DHFR, the main enzyme converting dihydrofolate to tetrahydrofolate. Tetrahydrofolate is required for the action of folate-dependent enzymes and is thus essential for DNA synthesis and methylation^14^. The binding score of eugenols against platelet-activating factor receptor (PAFR) was also evaluated. PAFR is an essential receptor involved in inflammation and allergic responses, causing a dramatic inflammation of the airway^15^.Fig. 1. Representative structure of DHFR inhibitor trimethoprim, eugenol, and D-limonene.
Moreover, captopril (Fig. 2) was found to be a potent MBLs inhibitor due to the presence of thiol and carboxylate groups, which can coordinate with the zinc ions of the enzyme active site^16^. Eugenol-free hydroxyl was expected to interact with MBLs through chelation of zinc ions or hydrogen bond formation. Thus, our attention was focused on studying the binding features and affinity of eugenol toward the key MBLs IMP-1 and NDM-1.Fig. 2. Representative structure of MBLs inhibitor L-captopril.
D-limonene is one of the most common terpenes in nature with a pleasant citrus fragrance. It is a major constituent in several citrus oils (orange, lemon, grapefruit, and tangerine). The antimicrobial activity of limonene can be ascribed to changing the membrane fatty acid composition and damaging cell morphology^17^. Eugenol, the dominant component of clove essential oil (EO), exists as a minor component in EO extracted from orange peel, as specified from GC–MS analysis. In contrast, D-limonene coexists as traces in clove EO along with the high eugenol content. Therefore, such findings inspired us to combine both clove and orange active components of their essential oils, so that their antimicrobial effects against resistant Pseudomonas aeruginosa would be potentiated in pneumonia infections.
Materials and methods
Microorganisms
Multi-drug-resistant Pseudomonas aeruginosa isolates utilized in this study were obtained from the Strain Bank of the Microbiology Department, affiliated with the Main University Hospital, Alexandria, Egypt. The strains were identified and curated by the department’s repository, ensuring standardized provenance and antimicrobial-resistance characterization for experimental reproducibility. The susceptibility pattern of the tested pathogens was added as a supplementary file (Table S1).
Extraction of essential oils
The fresh citrus peels (Citrus sinensis) were sourced from a local agricultural producer, while clove (Syzygium aromaticum) buds were procured from a regional marketplace in Alexandria, Egypt. The plant materials were identified and authenticated by a qualified taxonomist. Voucher specimens of Citrus sinensis (No. 984) and Syzygium aromaticum (No. 572) were deposited at the Herbarium of Alexandria University (ALEX, Heneidy et al. Collection). Essential oils (EOs) were isolated via hydro-distillation. The apparatus employed a round-bottom flask to generate steam, which was conveyed into a secondary vessel containing the plant material and distilled water. The steam permeates the plant matrices, entraining volatile constituents and yielding a water–oil vapor effluent. This condensable vapor subsequently traversed a condenser, where rapid thermal equilibration produced a liquid water–oil condensate. To maximize oil recovery, the distillation was conducted in successive 2-h cycles, continuing until the extraction plateaued. The condensate was collected in a round-bottom receiver, and phase separation was affected by decantation to remove the aqueous fraction after each collection. The crude EO was dried over anhydrous Na_2_SO_4_ to remove residual moisture and subsequently stored at 4 °C before automotive analysis by gas chromatography–mass spectrometry (GC–MS) for compositional profiling of both essential oils.
GC–MS analysis of extracted essential oils
Shimadzu QP-2010 ULTRA GC/MS instrumentation was employed to characterize both essential oils. The chromatographic program commenced with an oven temperature of 60 °C held for 3 min, followed by a ramp to 260°C through five calibrated rate steps, and a final isothermal hold of 10 min. Electron impact ionization (EI) mode served as the ionization source. Separation was achieved on a TRB-WAX column (30 m × 0.25 mm ID; film thickness 0.25 μm) with a helium carrier gas at a constant flow of 1.9 mL/min. The system operated in split injection mode with an injection temperature of 200 °C. Component identities were inferred by matching acquired mass spectra against the NIST11s reference library and supplementary literature to establish definitive spectral correlations.
Molecular docking
A docking investigation was conducted employing the Molecular Operating Environment (MOE) software suite (MOE 2019) to prognosticate both the binding affinity (binding score) and the binding modality (binding mode) of eugenol with respect to the target enzymatic proteins: dihydrofolate reductase (DHFR), β-lactamase IMP-1, metallo-β-lactamase NDM-1, and Platelet-Activating Factor Receptor (PAFR).
Preparation of the bacterial enzymes
The X-ray crystallographic structures of the target proteins—dihydrofolate reductase (DHFR; PDB ID: 4OR7)^18^ complexed with a pyrimidine co-crystallized ligand, metallo-β-lactamase NDM-1 (PDB ID: 4EXS)^19^, and IMP-1 (PDB ID: 4C1F)^20^ in complex with an L-captopril co-crystallized ligand, as well as Platelet-Activating Factor Receptor (PAFR; PDB ID: 5zkq)^21^ bound to an ABT-491 co-crystallized ligand, were retrieved from the Protein Data Bank (PDB) repository^22^ and employed as structural templates. Additionally, the preparatory phase for these proteins was conducted using the default settings of the “Structure preparation” workflow/module to ensure consistent initial conditioning.
Generation and optimization of the database
Test compounds were retrieved from the MOE database and subjected to initial optimization, including hydrogen display, partial charge calculations, and the default energy-minimization protocol to yield a converged, low-energy ensemble; ligand placement was then performed with the triangular matcher algorithm, from which five non-redundant poses with the lowest binding energies were selected using the default scoring function. The optimized ligand set underwent docking under an induced-fit framework to accommodate receptor flexibility, with docking scores reported in kcal·mol⁻^1^ and computed via two complementary axes: alpha-hydrogen bonding and London dispersion (London dG). Docking results were filtered by S-scores with RMSD < 2 Å to ensure pose reliability, and an essential check confirmed that the protocol could reproduce the binding mode of a known reference inhibitor for the target enzyme. Finally, the conformers exhibiting the highest binding affinity and most favorable ligand–enzyme interactions were identified for in-depth analysis.
Eugenol/limonene nanoparticles synthesis and characterization
Novel Eugenol/Limonene nanoparticles preparation was performed by the nanoemulsion technique. First, 50 mg/mL Eugenol was mixed with 10 mg/mL Limonene (co-oil) to prepare the active oil mixture phase. Then, a surfactant of 2.0% w/v Tween 80 as primary surfactant, 0.5% w/v Span 80 as a co-surfactant, was used with a ratio of 1:1 of surfactant and active oil phase. PBS (pH 7) was used as an aqueous phase. The oil phase was added dropwise into the aqueous phase under vigorous stirring (15,000 rpm). High-energy input was applied to achieve nanoemulsion using a Probe sonication (20 kHz, pulse 5–10 s on, 5–10 s off, for a total energy of 100–500 J per mL) for 10 min in an ice bath to avoid evaporation. The nanoparticles formation was characterized through the dynamic light scattering technique using a Zetasizer (Malvern Zetasizer Nano ZS, Cambridge, UK) and transmission electron microscope (TEM)^23^.
In vitro enzyme activity
All cells, including eukaryotic and prokaryotic ones, contain the enzyme dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate NADP oxidoreductase; EC 1.5.1.3), which is essential for proper folate metabolism. Cell death, a decrease in tetrahydrofolate levels inside the cell, and disruption of RNA and DNA synthesis all arise from DHFR inhibition. This is why DHFR has been such a vital therapeutic target for cancer treatments. Dihydrofolate Reductase Inhibitor Screening Kit for the purpose of testing potential DHFR inhibitors (Biovision, Waltham, MA, USA). The drop in absorbance at OD340 nm may be used as a proxy for DHFR activity, with the inhibition of this decline by putative inhibitors providing an indication of their effectiveness.
Antibacterial activity
Antibacterial efficacy was assessed via the disc-diffusion assay, whereby sterile filter paper discs were saturated with 25 μL^23^ of each essential oil and gently positioned onto the surface of inoculated Mueller–Hinton agar plates. Complementary antimicrobial evaluation proceeded through determination of the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) values, following standard protocols^24^, to delineate the inhibitory and bactericidal thresholds of the test oils. The minimum inhibitory concentration (MIC) denotes the lowest concentration of essential oil that prevents any visible microbial growth. The minimum bactericidal concentration (MBC) is defined as the lowest concentration at which 99.9% of the bacterial population is eradicated. The MIC index, calculated as the ratio MBC/MIC, serves as a proxy for elucidating the antibacterial mechanism of action. An MIC index of 1 or 2 indicates a bactericidal effect, whereas an index of 4 or higher characterizes the effect as bacteriostatic^25^.
Cytotoxicity study in lung (BEAS-2Bs) cell line
Cytotoxic potential was evaluated in the BEAS-2B human bronchial epithelial cell line (obtained from the American Type Culture Collection (ATCC)). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, and maintained in a humidified atmosphere containing 5% CO₂ at 37 °C. Test samples were solubilized in DMEM to produce stock solutions, and a two-fold serial dilution series was prepared to yield ten concentrations, as requested. Confluent BEAS-2B monolayers were seeded into 96-well microplates and allowed to stabilize for 24 h. The cells were exposed to the diluted test compounds in triplicate, under standard incubator conditions, for 72 h. Subsequently, 20 μL of a 5 mg/mL MTT solution was added to each well and incubated at 37 °C for 4 h to allow for formazan formation. The medium was removed, and 150 μL of fresh MTT solution was added, followed by a 15-min incubation. Absorbance was measured at 570 nm using a BMGLABTECH FLUOstar Omega microplate reader to derive cytotoxicity metrics, with references^26,27^.
In vivo study
Animals
Male Sprague–Dawley rats (6–8 weeks old, 250–350 g, institutional breeding facility) were employed in both standard (imaging studies) and pneumonic models and housed individually with ad libitum access to food and water in ventilated cages. Group sizes were determined using the hazard ratio approach. USP Grade A (medical) CO₂ euthanasia was conducted at the study’s termination (100% CO_2_ at a fill rate of 30–70% was added to the existing air in the chamber. This gas composition aimed to achieve a homogenous mixture to support the study’s objectives while prioritizing reduction of distress and adherence to established animal welfare guidelines)^28^. Disease progression was monitored every 12 h for 7 days by recording body temperature, body weight, and clinical signs, including pallor, nasal bleeding, unresponsiveness, lethargy, instability, and abnormal respiration. All procedures were performed in accordance with the Animal Care and Use Committee (ACUC) guidelines of the Institutional Review Board of the Faculty of Science, Alexandria University (approval number: AU/04/23/04/27/637), and the study adhered to ARRIVE guidelines for in vivo reporting^29^. The animal group was divided into:
- Negative control group: normal non-infected group
- Positive control group: infected non-treated
- Eugenol-treated group: 160 mg/kg body wt in 0.2 ml saline^30^ using endotracheal aerosolization ^26^
- Limonene-treated group: 160 mg/kg body wt in 0.2 ml saline using endotracheal aerosolization^26^
- Eugenol/Limonene nanoformulated treated group: 160 mg/kg body wt in 0.2 ml saline using endotracheal aerosolization^26^
Pneumonia model
Pneumonia was induced by intranasal inoculation of a bacterial suspension (3% w/v in mucin) prepared from a subculture grown for < 20 h to achieve a density of 10^7^ CFU/mL; inoculum density was confirmed via serial dilution. Anesthesia was induced with inhaled isoflurane (3–5%), followed by intranasal administration of 50 μL of the bacterial suspension to each rat^31^.
Bacterial load assessment
Bacterial burden was quantified by aseptically harvesting the lungs, perfusing to remove blood, homogenizing in sterile PBS, performing serial dilutions, and plating on HIA to determine CFU per tissue weight^31^.
Histopathological studies
Histopathological evaluation included: (i) light microscopy, with lungs fixed in 10% formalin for ≥ 48 h, paraffin-embedded, sectioned, and stained with hematoxylin and eosin^31^; and (ii) transmission electron microscopy, with lung tissue fixed in 2.5% glutaraldehyde, processed through acetone and embedding medium, sectioned to 50–60 nm, stained with uranyl acetate and lead citrate, and examined on a JEM-100CX transmission electron microscope^32^.
Ethical statement
All procedures were performed in accordance with the Animal Care and Use Committee (ACUC) guidelines of the Institutional Review Board of the Faculty of Science, Alexandria University (approval number: AU/04/23/04/27/637), and the study adhered to ARRIVE guidelines for in vivo reporting^29^.
Statistical analysis
The data obtained by the different afore-mentioned analysis methods were statistically analyzed by an analysis of variance (ANOVA) combined with a Tukey’s multiple comparisons test via the GraphPad Prism 7.0d software (GraphPad Software Inc., San Diego, CA, USA). Statistical differences were designated as significant if p-values were less than 0.05 (p ≤ 0.05), and highly significant if p-values were less than 0.01 (* p ≤ 0.01) or less than 0.001 (*** p ≤ 0.001).
Results and discussions
Chemical analysis of essential oils
The GC–MS analysis of the pale-yellow oil extracted from Egyptian Valencia Orange Peel has revealed 54 chemical compounds (Table 1 and Fig. 3), representing 100% of the total essential oil (EO). The identified dominant chemical compounds were D-Limonene [(R)-1-methyl-4-(1-methylethenyl)-cyclohexene] with 63.25% of the relative area, different alkyl-benzenes (22.92%), and β-Myrcene (1.41%). Interestingly, Eugenol has also been found to be present in terms of trace amounts (0.11%) in orange extract, despite not being one of its main constituents. This finding can be linked to a metabolic mechanism that generates eugenol as a secondary metabolite. Regarding clove buds, GC–MS analysis of the extracted pale-yellow oil has shown 44 identified components representing 100% of the total oil (Table 2 and Fig. 4). The dominant components were Eugenol [2-methoxy-4-(2-propenyl)-phenol] with a relative area of 36.20%, Caryophyllene (19.30%), Eugenol acetate (18.71%), different alkyl-benzenes (11.86%), and Humulene (2.41%). D-limonene, the primary compound in orange essential oil, has also been confirmed in clove extract in minimal quantities of 0.18%. The chemical compatibility of Eugenol (as a hydrophilic phenolic compound) and Limonene (as a hydrophobic monoterpene hydrocarbon) facilitates the formation of an emulsion. In particular, the non-polar nature of Limonene allows it to form micelles or nanoparticles with Eugenol, which are stabilized by the polar Eugenol molecules, through exerting a hydrophilic shell around the hydrophobic Limonene core. Furthermore, the abundance of numerous alkyl-benzenes in both EOs, with their hydrophobic nature, may promote their encapsulation within the nanoparticles and contribute to the stability of these nanoparticles.Table 1. Chemical Composition of Egyptian Valencia Orange Peel Essential Oil.Peak NoCompoundRetention Time (min)% of Relative Area1Pentane1.0835.042Hexyl-8-chlorooctylsebacate1.1170.083Cyclohexane1.2792.034Acetone1.4171.4552-Butanone1.6311.7161,3,5,7-Cyclooctatetraene1.7500.437Benzene1.8000.178α-Pinene2.2640.299Toluene2.4080.70102-Hexanone2.8760.27112-Methyl-2-pentanol3.0630.59123-Methyl-3-pentanol3.2840.2213( +)-Sabinene3.4080.08143-Carene3.8180.1015β-Myrcene4.121****1.4116D-Limonene4.763****63.2517β-Phellandrene4.9060.4018Octanal6.8270.0819Decanal12.1330.1620β-Linalool13.5220.61211-Octanol13.7280.1022Caryophyllene14.3300.1023L-Terpinen-4-ol14.6850.1024L-α-Terpineol17.0380.5125Eremophila-1(10),11-diene17.2960.1326α-Citral17.8270.0627(1-Butylhexyl)-benzene18.0680.4728γ-Muurolene18.2950.0929(1-Propylheptyl)-benzene18.3920.4230(1-Ethyloctyl)-benzene18.9310.5331(1-Pentylhexyl)-benzene20.1980.5432(1-Methylnonyl)-benzene20.3020.7633(1-Butylheptyl)-benzene20.3591.1534(1-Propyloctyl)-benzene20.6881.0435(1-Ethylnonyl)-benzene21.2721.3136Hexadecane21.8170.0837(1-Pentylheptyl)-benzene22.3930.9338(1-Butyloctyl)-benzene22.5600.9639(1-Methyldecyl)-benzene22.6221.8840(1-Propylnonyl)-benzene22.9280.9041(1-Ethyldecyl)-benzene23.5271.0642Octadecane23.9590.0643(1-Pentyloctyl)-benzene24.4981.0444(1-Butylnonyl)-benzene24.7080.7145(1-Methylundecyl)-benzene24.8511.6346(1-Propyldecyl)-benzene25.0920.6947(1-Ethylundecyl)-benzene25.7030.8348(1-Methyldodecyl)-benzene26.9951.3049Eugenol27.194****0.1150DEHA (Di(2-ethylhexyl) adipate)38.7660.405112-Crown-443.3170.0652Estragole47.5710.30533,7-Dimethyl-1,7-octanediol48.4670.1254Tetradecyl-oxirane48.9740.56Total:****100.00Fig. 3GC–MS Chromatogram of the Egyptian Valencia orange essential oils along with a chart design of the components and 3D structure of D-Limonene compound.Table 2. Chemical Composition of Clove Essential Oil.Peak NoCompoundRetention Time (min)% of Relative Area12-Methylbutane1.0834.982Cyclohexane1.2811.133Toluene2.4650.4145-Methyl-3-hexanol3.0750.325D-Limonene4.744****0.186Copaene11.7420.557Caryophyllene14.394****19.308δ-Cadinene15.9470.109Humulene16.111****2.4110(1-Butylhexyl)-benzene18.0750.3311β-Cadinene18.3000.6912(1-Propylheptyl)-benzene18.4010.2813(1-Ethyloctyl)-benzene18.9400.3414L-Calamenene20.0000.1915(1-Pentylhexyl)-benzene20.2090.3516(1-Methylnonyl)-benzene20.3120.5017(1-Butylheptyl)-benzene20.3700.7818(1-Propyloctyl)-benzene20.7000.6919(1-Ethylnonyl)-benzene21.2840.8620(1-Pentylheptyl)-benzene22.4100.6021(1-Butyloctyl)-benzene22.5790.6222(1-Methyldecyl)-benzene22.6391.30234,8,8-Trimethyl-2-methylene-4-vinylbicyclo[5.2.0]nonane22.7860.1524(1-Propylnonyl)-benzene22.9490.6225Caryophyllene oxide23.1720.4326(1-Ethyldecyl)-benzene23.5500.7227(1-Pentyloctyl)-benzene24.5280.6828(1-Butylnonyl)-benzene24.7370.6929(1-Methylundecyl)-benzene24.8821.12301,2,3,4,4a,7-Hexahydro-1,6-dimethyl-4-(1-methylethyl)naphthalene24.9920.0931(1-Propyldecyl)-benzene25.1250.4432(1-Ethylundecyl)-benzene25.7460.5333Caryophyllene26.4240.09344-Methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinylcycloheptane26.7080.1135Eugenol27.241****36.2362-Isopropyl-5-methyl-9-methylene- bicyclo[4.4.0]dec-1-ene27.5080.6737Eugenol acetate28.995****18.7138Viridiflorol30.2930.12394-(2-Propenyl)-phenol30.5020.5040Diethyl phthalate30.7560.304110,12-Pentacosadiynoic acid31.0220.10422',3',4'-Trimethoxyacetophenone33.4740.1743n-Hexadecanoic acid40.0160.3844Oleic Acid43.5450.28Total:****100.00Fig. 4GC–MS Chromatogram of the Clove Essential Oils, along with a chart design of the components and 3D structure of Eugenol compound.
Molecular docking
Docking results (Table 3) showed that eugenol displayed high binding score with DHFR comparable with that exhibited by the pyrimidine co-crystallized ligand. It was also observed that eugenol hydroxyl group formed hydrogen bond interaction with the key amino acid Asp27. In addition, the phenyl group of eugenol formed л-stacking interaction with the proximal amino acid Phe31 (Fig. 5).Table 3. Calculated docking score (Kcal/mol) of the eugenol with DHFR enzyme.CompoundsDHFR binding scoreCo-crystallized ligand (pyrimidine derivative)−6.48Eugenol−5.39Fig. 5(A) 2D binding mode of eugenol, (B) 3D binding mode of eugenol within DHFR binding site.
MBLs inhibitory activity
NDM-1
As can be seen from Table 4, interestingly, eugenol was able to achieve better binding score with NDM-1 than the reference L-captopril. Moreover, eugenol displayed a fundamental hydrogen bond between the hydroxyl group and the amino acid Asp124 (Fig. 6).Table 4. Calculated docking score (Kcal/mol) of the eugenol with NDM-1 enzyme.CompoundsNDM-1 binding scoreCo-crystallized ligand (L-captopril)−6.95Eugenol−8.27Fig. 6(A) 2D binding mode of eugenol, (B) 3D binding mode of eugenol within NDM-1 binding site.
IMP-1
Regarding data in Table 5, eugenol exhibited a promising docking score toward IMP-1 compared with the reference L-captopril. It was also found that eugenol showed л-stacking interaction between the eugenol phenyl group and the essential amino acid His215 (Fig. 7).Table 5. Calculated docking score (Kcal/mol) of the eugenol with IMP-1 enzyme.CompoundsIMP-1 binding scoreCo-crystallized ligand (L-captopril)−8.07Eugenol−6.45Fig. 7(A) 2D binding mode of eugenol, (B) 3D binding mode of eugenol within IMP-1 binding site.
Anti-inflammatory activity
Results in Table 6 indicated that eugenol possessed moderate docking score toward PAFR according to ABT-491 co-crystallized ligand. Examining the eugenol-PAFR binding features revealed eugenol showed a hydrophobic pi-based interaction between the eugenol phenyl ring and the proximal amino acid His275 (Fig. 8).Table 6. Calculated docking score (Kcal/mol) of the eugenol with PAFR enzyme.CompoundsPAFR binding scoreCo-crystallized ligand (ABT-491)−8.77Eugenol−5.44Fig. 8(A) 2D binding mode of eugenol, (B) 3D binding mode of eugenol within PAFR binding site.
Nanoparticles characterization
Eugenol and limonene are natural, lipophilic bioactives with antimicrobial, anti-inflammatory, and anticancer potential, but both suffer from poor water solubility, volatility, and instability. Nanoplatforms (nanoemulsions, lipid nanoparticles, liposomes, polymeric NPs) are being widely explored to improve their stability, bioavailability, targeting, and controlled release in food, pharma, agrochemical, and cosmetic applications.
In the present formulated nanoparticles, the log-scale size distribution shows a single dominant peak centered around ~ 100 nm with a relatively narrow width (PDI 0.39). A monomodal, narrow distribution confirms uniform droplet size, essential for consistent rheology, appearance, and release profile. The peak around ~ 100 nm aligns with a typical nanoemulsion range (20–200 nm), supporting the intended nanoscale formulation. The Zeta potential distribution shows a sharp, narrow peak centered near a positive 48 mV value. A narrow zeta potential peak indicates a uniform surface charge, which is favorable for electrostatic stabilization of the nanoemulsion. The TEM image shows discrete, roughly spherical particles with a dense core and a lighter outline, dispersed in a darker background. Particle sizes appear in the 50 nm range. The bright core could indicate a relatively dense oil core with a surrounding surfactant shell (Fig. 9). The dispersion appears uniform, with limited agglomeration. The combination of TEM morphology, a well-defined zeta potential, and a narrow, mono-modal size distribution supports a stable Eugenol/Limonene nanoemulsion with nanoscale droplets.Fig. 9. Eugenol/limonene Nanoemulsion characteristics where (a) zeta size, (b) zeta potential and (c) transmission electron microscopic study of the synthesized nanoparticles.
In another study, a eugenol-loaded nanoemulsion was crafted using Tween 80 and Tween 20 through spontaneous emulsification. The team assessed its physicochemical traits, long-term stability, and antimicrobial power across a broad spectrum of microbes. Among the tested formulations, the tiniest droplets—measuring 95, 101, 81, and 77 nm—were singled out, all carrying a surface charge of about − 25.0 ± 1.0 mV. Relative to the bulk solution, the eugenol nanoemulsion exhibited a slower release profile, hinting at potential for sustained delivery. Spontaneous nanoemulsion formation is highlighted as a cost-efficient, time-saving, and material-saving approach that yields stable products—well-suited for high-volume industrial production ^33^. In another work, all nanoformulations exhibited a light-brown hue, attributable to the RSV loading, with eugenol-containing formulations appearing marginally darker owing to the intrinsic color of eugenol. Eugenol-loaded nanoparticles possessed a vesicle size of 13.97 nm with a polydispersity index (PDI) of 0.055 ± 0.007. In contrast, D-limonene–containing nanoparticles displayed a vesicle size of 15.73 ± 0.07 nm and a PDI of 0.117 ± 0.003. Variation in the vehicle constituents and the incorporation of terpenes did not produce a statistically significant alteration in either the globule size or the size distribution^34^. Yin et al.^35^ prepared eugenol/citral emulsion with ideal physicochemical traits and antimicrobial power for strawberry preservation. A bilayer formulation, built on the interactions between whey protein isolate and chitosan, combined citral and eugenol. Through optimization of multiple parameters, an antimicrobial emulsion with superior stability and properties was achieved. The final bilayer consisted of 1.0% v/v total oil (eugenol and citral), 0.7% v/v whey protein, and 0.3% v/v chitosan. Assessments of appearance, structure, physicochemical behavior, and antimicrobial activity revealed that, compared with a single-layer counterpart, the pale-yellow bilayer emulsion behaved as a shear-thinning fluid with strong stability. Additionally, the emulsion demonstrated promising strawberry preservation performance^35^. Another study explored encapsulating varying amounts of eugenol within a Pickering emulsion stabilized by self-assembled chitosan nanoparticles, produced via ultrasound-assisted emulsification, and examined how different eugenol levels influenced the emulsion’s physical properties, stability over 60 days, antioxidant capacity, and antimicrobial performance. Increasing eugenol content enlarged droplet sizes from about 20 to 142 nm. Moreover, emulsion stability improved progressively with higher eugenol loading during storage^36^.
Dihydrofolate reductase enzyme inhibition assay
To evaluate the therapeutic potential of Eugenol/Limonene, an in vitro assay was conducted targeting dihydrofolate reductase (DHFR). The results demonstrated that the Eugenol/Limonene formulation exerted a measurable inhibitory effect on DHFR, with an IC50 of 8075 ± 0.96 μg/mL. By comparison, the reference pharmaceutical MTX (methotrexate) exhibited substantially stronger DHFR inhibition, recording an IC50 of 0.81 ± 0.07 μg/mL (Fig. 10 and Table 7). This juxtaposition underscores a meaningful difference in potency between the natural product duo and the marketed DHFR inhibitor, while still evidencing a tangible interaction with the DHFR catalytic system. A structure–activity relationship (SAR) interpretation of the data indicates that substituent positioning critically modulates inhibitory efficacy. Specifically, substituents at the para position appear optimal for DHFR engagement, suggesting that spatial orientation relative to the core scaffold governs binding interactions within the enzyme’s active site. Moreover, electron-withdrawing groups tend to favour stronger inhibition, aligning with a pattern wherein withdrawal of electron density enhances key contacts or stabilizes conformations conducive to DHFR blockade. However, the SAR also reveals that certain electron-donating groups can contribute meaningful inhibitory interactions, albeit generally to a lesser extent than their withdrawing counterparts. This nuance implies that DHFR recognition by Eugenol/Limonene encompasses a balance of electronic effects and steric factors, allowing for a subset of donors to still participate effectively in enzyme inhibition under favourable conformational contexts^37^. Collectively, the SAR outcomes suggest a dominant role for electron-withdrawing substituents in amplifying DHFR-inhibitory activity, while acknowledging that select electron-donating substituents can furnish beneficial interactions that sustain inhibition in a subset of structural scenarios. The implications are clear: these findings identify a framework for optimizing Eugenol/Limonene-derived candidates through targeted modification that enhances DHFR affinity and inhibitory performance. Future work should emphasize systematic exploration of substituent effects, expanding the panel of electron-withdrawing and -donating groups, and integrating computational docking with empirical SAR measurements to delineate the precise binding determinants. Such efforts will facilitate the rational design of improved DHFR inhibitors derived from natural product scaffolds, potentially yielding compounds with favourable pharmacodynamic and pharmacokinetic properties that can be advanced for therapeutic evaluation.Fig. 10. Plot graph of Dihydrofolate Reductase Enzyme inhibition assay.Table 7DHFR Enzyme Inhibition (%) against different sample concentrations.Sample conc. (µg/ml)DHFR Enzyme Inhibition (%) ± SDEugenol/LimoneneControl50092.51 ± 0.2596.87 ± 0.4725089.43 ± 0.1194.35 ± 0.1912583.62 ± 0.8493.14 ± 0.3262.576.59 ± 0.7388.23 ± 0.4131.2568.07 ± 0.9184.51 ± 0.1315.657.42 ± 0.6480.27 ± 0.197.848.96 ± 0.3269.24 ± 0.323.940.54 ± 0.6864.08 ± 0.68231.78 ± 0.4657.13 ± 0.39124.95 ± 0.3752.98 ± 0.640.513.06 ± 0.2245.31 ± 0.230.257.89 ± 0.1341.78 ± 0.14
Antibacterial activity
The dataset summarized in Table 8 demonstrates that the inhibition zone (IZ) diameters produced by Eugenol/Limonene ranged from 24.0 to 29.0 mm across the tested pathogenic strains, indicating a broad spectrum of antibacterial activity. Across the evaluated pathogens, Eugenol/Limonene displayed bactericidal effects, with mean minimum inhibitory concentrations (MIC) spanning from 8.0 to 32.0 μg/mL and minimum bactericidal concentrations (MBC) extending from 64.0 to 512.0 μg/mL. Figure 11 shows the potent effect of the prepared Nanoemulsion in deforming the bacterial cells. These findings align with prior work by Mumu and Hossain^38^, who reported that essential oil (EO) preparations achieved complete (100%) growth inhibition against Staphylococcus aureus, Proteus vulgaris, and Aeromonas hydrophila, underscoring the potent antimicrobial potential of the EO formulations against diverse bacterial species. Taken together, these observations highlight substantial antimicrobial potential for EO-based formulations, albeit with considerable variability in reported efficacy across studies. Such differences are likely attributable to intrinsic disparities in the essential oil compositions employed (which can be influenced by botanical source, harvest conditions, and extraction methods) as well as divergences in experimental protocols (inoculum size, growth media, incubation times, and assay conditions). The observed heterogeneity in antimicrobial outcomes emphasizes the need for standardized methodologies and comprehensive chemical characterization of essential oil constituents to accurately interpret and compare results. Moreover, the data imply that while Eugenol/Limonene possess meaningful inhibitory and bactericidal capabilities against a range of pathogens, the precise MIC and MBC values—and hence the practical therapeutic index—are contingent upon both the specific microbial target and the compositional nuances of the EO preparation used.Table 8. Antibacterial activity of the extracted oils and the synthesized nanoparticles.Tested pathogensEugenolLimoneneEugenol/LimoneneIZ (mm)MIC (µg/mL)MBC (µg/mL)IZ (mm)MIC (µg/mL)MBC (µg/mL)IZ (mm)MIC (µg/mL)MBC (µg/mL)P. aeruginosa 17.0256.0512.010.0256.0512.024.032.0512.0P. aeruginosa 28.0256.0512.011.0128.0512.026.016.0128.0P. aeruginosa 37.0256.0512.010.0256.0512.024.532.0512.0P. aeruginosa 48.0256.0512.011.0128.0512.026.016.0128.0P. aeruginosa 58.0256.0512.010.5256.0512.025.032.0128.0P. aeruginosa 610.0128.0512.013.064.0512.029.08.064.0Fig. 11TEM study of treated bacterial cells.
dos Santos et al.^39^ reported that Pepper Rosemary essential oil and thymol, when tested separately, delivered the strongest antimicrobial punch, yielding the lowest MIC values across all tested microbes. The most potent ranges were 0.03–1.25 mg/mL for P. expansum and 0.31–1.25 mg/mL for S. Typhimurium. Blending Mandarin and Mexican lime boosted antimicrobial performance even further. The R-limonene component exhibited a synergistic additive effect, but only against Staphylococcus aureus strains. Using thymol and Lippia reduced S. aureus and S. Typhimurium growth by 3 log10 within 24 h. Overall, Pepper Rosemary essential oil—whether used alone or with thymol—shows greater promise as an antifungal and bactericidal agent against the microorganisms tested^39^. Another study delves into how eugenol fights against Bacillus cereus, including its possible action against spores and the impact on germination rates. The MIC of eugenol across six B. cereus strains was 0.6 mg/mL when compared to a control, with treated bacteria showing a noticeably extended lag phase. When eugenol exceeded half of the MIC, viable B. cereus counts dropped by about 5.7 log CFU/mL to below detectable levels within 2 h. At a thrice-MIC level, B. cereus in skim milk fell to roughly 5.9 log CFU/mL, again becoming undetectable within 30 min^40^. In another study, a eugenol-loaded nanoemulsion showed notably lower MICs and MBCs in pure oil and demonstrated effective antimicrobial and antifungal activity against various organisms. Encapsulated eugenol in nanoemulsions proved more potent than plain eugenol or nanoemulsions without eugenol. Beyond their antimicrobial edge, these nanoemulsions hold promise for diverse therapeutic uses, including transdermal delivery and food preservation^33^. Yin et al.^35^ reported the eugenol/citral emulsions antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Aspergillus niger, with MIC values of 25.6, 12.8, and 40 μL/mL, respectively. Another study of eugenol emulsion showed enhanced antimicrobial efficacy with inhibition zones of 14.1 mm against Staphylococcus aureus and 17 mm against Escherichia coli^36^.
In vitro cytotoxicity
The cytotoxic potential of the extracted essential oils was evaluated in the BEAS-2B human bronchial epithelial cell line under in vitro conditions. Cell viability was enhanced for the Eugenol/Limonene formulation, with the nanoformulation yielding an IC50 of 6000 μg/mL (Fig. 12). Collectively, these results indicate a favorable safety profile and imply potential biomedical utility for Eugenol/Limonene–derived nanoparticles. However, particle design matters: eugenol-loaded clay nanocarriers altered population dynamics of aquatic invertebrates, with carrier-specific adverse effects even when acute toxicity was reduced relative to free eugenol^41^Fig. 12. Cytotoxic effect of the extracted oils.
In vivo study
Bacterial load assessment
At study initiation, Sprague–Dawley rats received an intradermal inoculum of 1.0 × 10^7^ CFU mL⁻^1^ of Pseudomonas aeruginosa to establish cutaneous infection; at predetermined time points, viable P. aeruginosa cells were enumerated as CFU g⁻^1^ to monitor bacterial burden and treatment efficacy, and the reduction in bacterial load within infected skin wounds following intervention was calculated for each group, with data in Fig. 13 showing a statistically significant decrease in bacterial counts across all treatment cohorts relative to their respective controls, indicating robust antimicrobial effects.Fig. 13. Bacterial load assessment of the experimental pneumonic model.
Histopathological study
The normal architecture of lung tissue was detected in lung slices using a light microscope, where thin inter-alveolar septa and patent alveoli were observed in the control group. Extensive degenerative alterations and deformation of lung architecture were found in the negative control group in a patchwork pattern, with most alveoli appearing collapsed, exacerbating emphysema. Furthermore, inter-alveolar septa were found to be significantly thickened in most locations of the lung tissue, with considerable cellular infiltration without tissue necrosis. Furthermore, the results of the group (3) treated with eugenol demonstrated significant airway congestion, which impeded gas passage to the lung due to bronchiolar desquamation. Furthermore, higher alveolar disintegration was seen in this group as compared to the negative control group. Lung damage was more severe in Group 4 (limonene-treated group) than in Group 5 (eugenol/limonene Nps treated group), where the alveolar histoarchitecture remained mainly unaltered, with only a few isolated alveoli filled with desquamated epithelial cells and widespread alveolar dilated capillary congestion. In comparison to the control group, the eugenol/limonene Nps-treated group had a typical alveolar lumen lined with typical pneumocyte architecture. Some dilated alveolar capillaries occur, indicating inflammatory lung tissue damage. The second interval demonstrated the most severe substantial degradation in the lung tissue of the positive control group, demonstrating severe emphysema as well as airway congestion, which led to respiratory failure shortly after. Not surprisingly, this interval was associated with considerable damaging lung deterioration in rats treated alone with NPs. Furthermore, the lung tissue in this group was edematous. Group 5 has the most significant findings with normal cyto-architecture compared to the control group, which does not show any symptoms of infection. Furthermore, inter-alveolar septa were found to be significantly thickened in most locations of the lung tissue, with extensive cellular infiltration. The preceding interval’s blood vessel congestion was still visible. The tissue showed thickness and some symptoms of edema, indicating the importance of nanoparticles in fighting bacterial infection (Fig. 14).Fig. 14. Photomicrograph illustrates pulmonary parenchyma at two-time intervals in different experimental groups stained with H&E; magnification X 200; in which control group (A); positive control group (B); Eugenol treated group (C); Limonene Treated group (D); Eugenol/Limonene group (E). Where: Labelle’s: collapsed alveoli (Red arrows); dilated congested blood vessels (green arrows); bronchiolar epithelium desquamation (blue arrow); tissue edema (yellow arrows); patent alveoli (Al); alveolar disintegration (asterisk); air way (A).
Transmission electron microscopy (TEM) study of lung alveoli
Because of their importance in breathing and surfactant production, the majority of these studies identified cells of type II pneumocytes. The alveolar walls were intact with a distinct border in the Control group, type II alveolar epithelial microvilli were well organized, and the quantity of lamellar bodies, many mitochondria, the air-blood barrier, and endothelial structure seemed normal, as found in the eugenol-treated group, indicating the involvement of eugenol as an antibacterial agent, boosting normal structures two days after infection. Conversely, the limonene group had a disordered nuclear membrane, showing the significance of the oil in trapping bacteria that had not been damaged. Despite the reality that the eugenol/limonene Nps-treated groups possessed vesicular nuclei with lamellar bodies in cells type II, they had substantial congestion in septal capillaries. The positive control group’s nuclear membrane was disordered, and the lamellar bodies lacked lamellation. The second interval indicates the Nps’ role in tissue repairing, which has been enhanced compared to the eugenol-treated group. Eugenol/limonene Nps treated group’s alveolar compartments were increased while their electron density was reduced. Similarly, the eugenol-treated group had a typical appearance with few microvilli, indicating that the Eugenol/limonene Nps group achieved similar effects over a longer period of time. To some extent, both the negative control group and the nano-treated group displayed symptoms of nuclear membrane congestion and disorganization (Fig. 15).Fig. 15. Transmission electron microscopy (TEM) micrographs of lung alveoli stained by uranyl acetate and lead citrate in which nucleus (N); alveolar lumen (L); cellular debris (D); congestion (C); luminal micro villi (blue arrows); lamellars bodies (green arrows).
Conclusion
A combinational antimicrobial strategy emerges from nanoemulsified nanoplatforms formed by merging two Egyptian essential oils—orange peel and clove—whose chemistries were mapped by GC–MS. Orange EO centers on D-limonene (63.25%), while clove EO is rich in Eugenol (36.20%) and Caryophyllene (19.30%). Their lipophilic harmony supports nanoemulsion-derived nanoparticles, potentially boosting bioavailability and multi-target action. The Eugenol/Limonene nanoemulsion exhibits broad antibacterial activity (IZ 24.0–29.0 mm; MIC 8.0–32.0 μg/mL; MBC 64.0–512.0 μg/mL) and DHFR inhibition (IC50 ≈ 8075 μg/mL), far surpassed by MTX. In vivo-like studies reveal reduced bacterial load but some bronchiolar/alveolar changes, underscoring both promise and pulmonary safety considerations.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1https://www.rcsb.org/, in.
- 2Hamid, K. H. A., Fauzi, M. A., Ajit, A., Arzmi, M. H., & Azman, N. A. M. (2025). Eugenol Pickering emulsion stabilized by chitosan self‐assembled nanoparticles: fabrication, emulsion stability, antioxidant and antimicrobial activity. Journal of Chemical Technology & Biotechnology.