In Vitro and In Vivo Wide-Spectrum Dual Antimycetomal Activity of Eight Essential Oils Coupled with Chemical Composition and Metabolomic Profiling

: Mycetoma, a neglected infection of subcutaneous tissues, poses a significant health burden, especially in tropical regions. It is caused by fungal (eumycetoma) and bacterial (actinomycetoma) pathogens, with current treatments often providing unsatisfactory outcomes. This study aims to discover novel broad-spectrum antimicrobial agents to circumvent the lengthy and costly diagnostic procedures. Eight essential oils (EOs) from the roots and aerial parts of Geigeria alata , Lavandula angustifolia , Melaleuca alternifolia , Myristica fragrans , Pimpinella anisum , Syzigum aromaticum , and Thymus vulgaris were prepared using steam distillation. The in vitro antimycetomal activity against Madurella mycetomatis and Actinomadura madurae strains was assessed using resazurin assays. The chemical compositions of the EOs were analyzed using gas chromatography and mass spectrometry (GC–MS). Promising EOs underwent further in vivo toxicity and efficacy testing in Galleria mellonella larvae models. EOs of G. alata roots, M. fragrans , P. anisum , S. aromaticum , and T. vulgaris showed wide-spectrum dual in vitro antimycetomal activity against all tested strains, with minimum inhibitory concentrations (MICs) ranging from 0.004 to 0.125% v / v . G. alata aerial parts and L. angustifolia EOs demonstrated activity predominantly against A. madurae , while M. alternifolia EO did not inhibit any tested strains. M. fragrans and P. anisum EOs significantly enhanced the survival of M. mycetomatis - infected larvae without inducing toxicity in uninfected larvae. Notably, P. anisum EO tended to enhance the survival of A. madurae -infected larvae, ranking it as the most promising EO among those tested. The investigated EOs, particularly P. anisum , exhibited promising broad-spectrum antimycetomal activity against fungal and bacterial pathogens responsible for mycetoma. These findings highlight the potential of essential oils as a basis for developing novel antimycetomal agents, offering hope for improved treatment strategies for this neglected disease.


Introduction
Mycetoma is a poverty-associated neglected tropical disease that is mainly prevalent in the "Mycetoma Belt" [1].The disease is characterized by a sizeable subcutaneous mass that contains grains.Mycetoma can be caused by either fungi (eumycetoma) or bacteria (actinomycetoma).The filamentous fungal pathogen Madurella mycetomatis is considered the most common causative agent of eumycetoma, while Actinomadura madurae is regarded as the most common causative agent of actinomycetoma [1].The causative agents are often identified using histology, culture, or molecular diagnosis [2].Unfortunately, most of these techniques can only be done in specialized centers and are currently unavailable in remote healthcare facilities in mycetoma endemic areas, where identification of the causative micro-organism is mandatory for appropriate treatment [3].
Eumycetoma is more challenging to treat.The standard therapy consists of medical treatment with synthetic antifungal azoles (e.g., itraconazole) and surgery [5].Despite prolonged treatment, only 25-30% cure rates were reported for eumycetoma [6].
Several studies are ongoing for both actinomycetoma and eumycetomato identify better treatment options.For actinomycetoma, the focus was mainly on repurposing antibacterial agents active against other bacterial pathogens [7,8].A drug repurposing attempt based on niclosamide and its ethanolamine salt demonstrated in vitro antimicrobial activity against both eumycetoma and actinomycetoma strains at MICs ranging between 0.39 and 1.6 µg/mL [9].For eumycetoma, the focus in the past decades has been on repurposing currently available antifungals and creating novel synthetic compounds using the Mycetoma Open-Source Drug discovery platform MycetOS [10].However, an effort has yet to be made to determine which drugs have wide-spectrum antimicrobial activity against both actinomycetoma and eumycetoma.This dual activity is highly needed since treatment often starts before the final identification of the causative micro-organism.A drug that is active against both forms of mycetoma would circumvent the need for diagnosis and is highly needed in mycetoma-endemic areas with low resources.
Essential oils (EOs) are promising antimicrobial agents due to the inherent chemical diversity in their wide range of mono-and sesquiterpenes.Hence, EOs represent a prominent reservoir of bioactivity against bacterial and fungal pathogens [11].Several EOs were demonstrated to attenuate fungal growth [11,12] and prevent biofilm formation through specific mechanisms [13][14][15].Based on their lipophilicity, EOs can easily penetrate fungal cells, accumulate inside, form a lipid bilayer of cytoplasmic membranes, attach themselves, and destabilize the cellular membranes [16][17][18][19].Subsequently, this process leads to the breakdown of the integrity of the phospholipid bilayer and an increase in its permeability.The enhanced lipophilicity of EOs enables EO penetration.Subsequently, it disrupts the normal cellular function, resulting in a leakage of the vital intracellular content such as ions, proteins, and nucleic acids; a disruption in the proton motive force (PMF); a reduction in the membrane potential; and a depletion of adenosine triphosphate (ATP) [20].Whereas, in bacterial infections, EOs primarily destabilize the cellular architecture, leading to the breakdown of membrane integrity, disrupting many cellular activities, including energy production and membrane transport, as well as leakage of cellular components and loss of ions [20].Therefore, EOs might be candidates for treatment of both actinomycetoma and eumycetoma.
EOs such as tea tree (Melaleuca alternifolia); plant extracts of Boswellia papyrifera, Acacia nubia, and Nigella sativa; and some synthetic derivatives of cinnamon oil exhibited inhibition of growth of M. mycetomatis [21][22][23].The coniferous pine tree EO's bicyclic monoterpene, α-pinene, has already demonstrated an appreciable in vitro activity toward three Sudanese clinical strains of A. madurae [24].However, no studies have focused on identifying EOs that would be active against both actinomycetoma and eumycetoma.
In this study, we followed a systematic approach to identify the therapeutic potential of eight chemically complex EOs against strains of both types of mycetoma.We determined the in vitro activity of eight EOs of seven taxonomically diverse aromatic medicinal plants belonging to five botanical families, including Apiaceae, Asteraceae, Lamiaceae, Myristicaceae, and Myrtaceae, against both M. mycetomatis and A. madurae strains.The EOs with the most in vitro activity were further subjected to in vivo assays based on the Galleria mellonella model to evaluate their toxicity and efficacy.Chemical profiles of all EOs were characterized by GC-MS, and chemometrics was applied to the data obtained from GC-MS.

Chemicals
All solvents and chemicals used were of analytical grade.All EOs, standard drugs, and pure compounds were dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany).

Extraction of Essential Oils
The aerial parts and roots of Geigeria alata (DC) Oliv.& Hiern.(Asteraceae), seeds of Myristica fragrans L. (Myristicaceae), Pimpinella anisum L. fruits (Apiaceae), flower buds of Syzigum aromaticum L. (Myrtaceae), and leaves of Thymus vulgaris L. (Lamiaceae), were bought from local markets and subjected to hydrodistillation for 3-5 h using the Clevengertype apparatus to obtain the EOs.The oil phase was separated, dried over anhydrous sodium sulfate, and kept in a labelled dark glass bottle at 4 • C for further analysis.The essential oils of leaves of Lavandula angustifolia Mill.(Lamiaceae) and Melaleuca alternifolia Cheel.(Myrtaceae) were obtained commercially from Oxford Lab Chem, Maharashtra, India and Novasel, Skarup, Denmark, respectively.

Strains Used in This Study
The three Madurella mycetomatis strains SO1, AL1, and t606931 used in this study originated from Algeria, Somalia, and Switzerland, respectively.They were identified at the species level by sequencing the internally transcribed spacer (ITS) region.The corresponding ITS sequences, MW541888, MW493233, and MW541889, were deposited in GenBank.The strains were maintained in the mycetoma collection of the Erasmus Medical Centre, Rotterdam, The Netherlands, subcultured on Sabouraud dextrose (SDA) plates, and propagated at 37 • C for 2-3 weeks.
Two reference Actinomadura madurae strains (DSM43236 and DSM44005) were purchased from the German DSM depository of strains, which are human or soil-originated type strains that have been kept since the 1940s.The clinical strain (SAK-E03) was obtained from the University of Science and Technology (UST) depository of strains between 2018 and 2019.

In Vitro Antifungal Assay of Essential Oils
The fungal strains of M. mycetomatis (SO1, AL1, and t606931) were inoculated in RPMI 1640 medium for 1 week.Then, they were sonicated for 10 s and centrifuged at 2600× g for 5 min.The mycelia were washed and resuspended in fresh RPMI 1640 medium to obtain a fungal suspension of 70% transmission (range 68% to 72%) at 660 nm (Novaspec II; Pharmacia Biotech, Apeldoorn, The Netherlands).Fungal strains were subjected to EOs at final concentrations ranging between 0.004 and 0.25% v/v.Itraconazole (Janssen Pharmaceutical Products, Beerse, Belgium) was used as the positive control at concentrations ranging from 0.03 to 1 µg/mL.In total, 100 µL of a 70% transmission fungal suspension and 1 µL of the EO were added to each well.Then, 1 µL of resazurin viability dye was added to obtain a final concentration of 0.15 mg/mL.After being added, the readily soluble resazurin formed a deep blue solution.The plates were incubated at 37 • C for 7 days.During incubation, resazurin is transformed into pink fluorescent resorufin in viable metabolically active fungal cells.The quantity of resorufin produced is proportional to the number of viable cells and can be assessed spectrophotometrically at 600 nm or visually without the use of specialized equipment.Here we determined both the visual MICs by assessing the color change of resazurin dye from purple to pink color and by transferring the supernatant to a flatbottom 96-well plate and reading the absorbance at 600 nm (Epoch 2, BioTek, Shoreline, WA, USA).The percentage metabolic activity was determined using the following formula: Percentage metabolic activity = ABS 600nm NC − ABS 600nm TEST ABS 600nm NC − ABS 600nm GC × 100 In this formula, ABS stands for absorbance, NC stands for negative control consisting of growth media and viability dye, and GC stands for growth control consisting of fungal inoculum in growth media and resazurin dye without EOs.All assays were performed in three independent replicates [25].The minimal fungicidal concentration (MFC) was determined by culturing the hyphal fragments of all wells on SDA at 37 • C; wells with no visible growth was observed after 14 days of incubation were considered as the MFC.

In Vitro Antibacterial Assay of Essential Oils
Three cultures of A. madurae (SAK-A03, DSM 43236, and DSM 44005) were subjected to EOs at concentrations ranging between 0.004 and 0.25% v/v.Bacterial suspensions in cation-adjusted Muller Hinton II (CAMHII) were adjusted to the absorbance of 0.08-0.1 at 625 nm (Novaspec II; Pharmacia Biotech).Amikacin (Sigma-Aldrich, St. Louis, MO, USA, A3650) and cotrimoxazole (Sigma-Aldrich, S7507, T7883) were employed as the positive controls at a starting concentration of 0.5 µg/mL and 4/76 µg/mL, respectively [26].Here, 100 µL of adjusted bacterial suspension and 1 µL of the EO were added to each well.Then, 1 µL of resazurin was added to a final concentration of 0.15 mg/mL.The plates were incubated at 35 • C for 5 days.Visual MICs (purple to pink color of resazurin dye) and spectrometric endpoints at 600 nm (Epoch 2, BioTek, Shoreline, WA, USA) were determined.Here, we determined both the visual MICs by assessing the color change of resazurin from purple to pink color and by transferring the supernatant to a flatbottom 96-well plate and reading the absorbance at 600 nm (Epoch 2, BioTek, Shoreline, WA, USA).The percentage metabolic activity was determined using the following formula: Percentage metabolic activity = ABS 600nm NC − ABS 600nm TEST ABS 600nm NC − ABS 600nm GC × 100 In this formula, ABS stands for absorbance, NC stands for negative control consisting of growth media and resazurin dye, and GC stands for growth control consisting of bacterial inoculum in growth media and resazurin dye without EOs.All assays were performed in three independent replicates [27].The minimal bactericidal concentration (MBC) was determined by culturing 10 µL of all wells on MHA at 35 • C. The MBC was considered the lowest EO concentration with no visible growth after 7 days.

In Vivo Studies in Galleria mellonella Model Galleria mellonella Larvae
Healthy 300-500 mg 5th instar larvae were purchased from the Blue Lagoon Pets Company, Keucheniusstraat 11, 3144 EM Maassluis, The Netherlands.

In Vivo Toxicity Studies of Essential Oils in Galleria mellonella Model
To determine the toxicity profiles of the promising EOs, 45 G. mellonella larvae per oil concentration were injected with the EO via the left last proleg and monitored for 10 days.The tested concentrations of EOs were 0.025, 0.010, and 0.006 mg/kg for M. fragrans; 0.145, 0.036 and 0.009 mg/kg for P. anisum; 0.021, 0.085, and 1.36 mg/kg S. aromaticum; and 0.017, 0.069, and 1.11 mg/kg for T. vulgaris.All essential oils were dissolved in 5% DMSO in phosphate-buffered saline (PBS; Gibco, Miami, FL, USA).PBS was used as a negative control.Three biological replicates were performed [23,28].

In Vivo Efficacy of Essential Oils in M. mycetomatis Galleria mellonella Model
M. fragrans and P. anisum EOs, which demonstrated no toxic effects on G. mellonella larvae, were selected for further efficacy testing in the Galleria model.
In short, mycelia from M. mycetomatis strain (SO1) were obtained from 2-weekold cultures grown on Sabouraud agar plates, scraped from the plate and inoculated in RPMI 1640 culture medium supplemented with L-glutamine (0.3 g/L; Capricorn-Scientific, Ebsdorfergrund, Germany), 20 mM mopholinepropanesulfonic acid (MOPS; Sigma, St. Louis, MO, USA), and chloramphenicol (100 mg/L; Oxoid, Basingstroke, UK).After 2 weeks of incubation at 37 • C, hyphae were collected by vacuum filtration through a 0.22 µm filter (Nalgene, Abcoude, The Netherlands) and washed with phosphate-buffered saline (PBS; Gibco, Miami, FL, USA).The wet biomass was scraped from the filter, weighed, resuspended in PBS, and sonicated for 2 min at 28 microns.Followed by centrifugation at 3400 rpm for 5 min, the biomass was adjusted with PBS to a concentration of 1 g/10 mL.A total of 40 µL of this inoculum was injected into the last left proleg of the larvae with an insulin 29 G U-100 needle (BD Diagnostics, Sparsk, MD, USA), resulting in a final inoculum of 4 mg SO1/larvae.A total of 15 larvae per group were used, and three biological replicates were performed.Each larvae group was treated with the EOs for three consecutive days at 4, 28, and 52 h post-infection.The same doses were used as for toxicity testing of M. fragrans and P. anisum EOs.Additionally, 5.71 mg/kg itraconazole, and solvent only control groups (5% DMSO in PBS) were included.Larvae were then incubated at 37 • C, and survival was monitored and recorded for 10 days [23,28].

In Vivo Efficacy of Essential Oils in A. madurae Galleria mellonella Model
Actinomycetoma infection was induced in Galleria larvae by injecting 40 µL of 10 5 CFU [29] of an A. madurae (DSM 44005) suspension into the last left proleg, as previously described [28].A total of 15 larvae per group were used, and three biological replicates were performed.Each larvae group was treated on three consecutive days at 4, 28, and 52 h post-infection.The same doses as used for toxicity testing of M. fragrans and P. anisum EOs.Additionally, 15 mg/kg amikacin, 7/35 mg/kg cotrimoxazole, and solvent only control groups (5% DMSO in PBS) were included.Larvae were then incubated at 37 • C, and survival was monitored and recorded for 10 days.

Statistical Analysis
The Log-rank test was performed with GraphPad Prism 8 (version 8.2.0, GraphPad Inc., Boston, MA, USA) to compare the survival lines and determine if there was a statistical difference between the different treatment groups.A p-value less than 0.05 was deemed significant [23].

Measurement of Free Radical Scavenging Activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging activity was estimated by measuring the decrease in the absorbance of the methanolic solution of DPPH• (257621, Sigma).In a 96-well plate, extracts (10 mg/mL) were allowed to react with 2,2 di (4-tertoctylphenyl)-1-picryl-hydrazyl stable free radical (DPPH) for half an hour at 37 • C. The concentration of DPPH was kept at (80 µg/mL).The test EOs and DPPH were dissolved in methanol.After incubation, a decrease in absorbance was measured at 517 nm using a Thermo Scientific Multiskcan spectrophotometer.The percentage radical scavenging activity (% RSA) by samples was determined compared to a methanol-treated control (negative control).At the same time, ascorbic acid (0.5 mM) and propyl gallate (100 µM) were used as positive controls [30].All determinations were performed in triplicate.Scavenging ability (%) was calculated by using the following formula: % Radical scavenging activity = 100 − (Rate of change in the absorbance of test) × 100 (Rate of change in the absorbance of control)

GC-MS Analysis of the Oils
The essential oils were analyzed with GC-MS using a Shimadzu GC-MS-QP2010 Ultra operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software, version 4.1.The GC column was a ZB-5 fused silica capillary column with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm.The carrier gas was helium with a column head pressure of 552 kPa and a 1.37 mL/min flow rate.The injector temperature was 250 • C, and the ion source temperature was 200 • C. The GC oven temperature was programmed for 50 • C initial temperature; the temperature increased at 2 • C/min to 260 • C. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared, and 0.1 µL was injected with a splitting mode (30:1).The identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes and by comparison of their mass spectral fragmentation patterns with those reported in the literature and stored in our in-house MS library [31].

Chemometric Analysis
Multivariate analysis was applied using principal component analysis (PCA) and hierarchical cluster analysis (HCA) to explore the similarities and differences between various essential oils based on GC-MS data analysis and antifungal activity.Principal component analysis (PCA) was first performed to explore the chemical and biological variability and to investigate the overall similarities and differences between oil samples [32].Hierarchical cluster analysis (HCA) was then employed to show the clustering of different oil samples.All multivariate statistical analysis was done using MetaboAnalyst 5.0 [33,34].

In Vitro Antifungal and Antibacterial Activity of Essential Oils
As shown in Table 1, seven of the eight selected essential oils inhibited the growth of the three M. mycetomatis isolates tested.Only the essential oil of M. alternifolia did not inhibit growth at 0.25% v/v in two out of three strains (AL1 and t606931).
Based on the lowest MICs obtained, the essential oils from Geigeria alata roots, Myristica fragrans, Pimpinella anisum, Syzigum aromaticum, and Thymus vulgaris seemed the most potent (MICs between 0.004-0.125%v/v).Among the three tested bacterial strains, G. alata roots, M. fragrans, P. anisum, S. aromaticum, and T. vulgaris EOs inhibited all strains as shown in Table 1 (MIC 0.004-0.031%v/v).L. angustifolia EO exhibited moderate antibacterial activity, while the EO from G. alata aerial parts showed moderate to weak antibacterial activity (MIC 0.063-0.125%v/v).In contrast, M. alternifolia exhibited weak activity against one strain (MIC 0.125% v/v), while no inhibition was observed toward the SAK-A03 and DSM 43236 strains.Taken together, M. fragrans, P. anisum, S. aromaticum, and T. vulgaris inhibited M. mycetomatis and A. madurae growth.These EOs were further tested for efficacy and toxicity.

Toxicity Studies in an Uninfected Galleria mellonella Model
As displayed in Figure 1A-D, 5.71 mg/kg itraconazole did not affect the G. mellonella larval survival rate without infection and was therefore considered nontoxic to the larvae.However, all concentrations of T. vulgaris EO and its major phenylpropanoid monoterpene, carvacrol (0.017, 0.069, and 1.11 mg/kg), decreased survival in uninfected larvae.The highest two concentrations of S. aromaticum EO (0.085 and 1.36 mg/kg) also appeared toxic to G. mellonella larvae (Figure 1C,D).No signs of toxicity were noted with the sub-MIC concentration of S. aromaticum EO (0.021 mg/kg) and its monoterpene alcohol, eugenol.Similarly, no signs of toxicity were noted with M. fragrans and P. anisum; all concentrations significantly differ from the PBS control (Figures 1A and 2B).However, all concentrations of T. vulgaris EO and its major phenylpropanoid monoterpene, carvacrol (0.017, 0.069, and 1.11 mg/kg), decreased survival in uninfected larvae.
The highest two concentrations of S. aromaticum EO (0.085 and 1.36 mg/kg) also appeared toxic to G. mellonella larvae (Figure 1C,D).No signs of toxicity were noted with the sub-MIC concentration of S. aromaticum EO (0.021 mg/kg) and its monoterpene alcohol, eugenol.Similarly, no signs of toxicity were noted with M. fragrans and P. anisum; all concentrations significantly differ from the PBS control (Figures 1A and 2B).

Efficacy of M. fragrans and P. anisum EOs in G. mellonella Larvae Infected with M. mycetomatis
As depicted in Figure 2A, when M. mycetomatis-infected larvae were treated with itraconazole, no enhanced survival was noted compared to the PBS-treated larvae (Log-Rank, p = 0.481).At a 0.025 mg/kg M. fragrans EO concentration, enhanced survival was noted compared to PBS-treated infected larvae (Log-rank, p = 0.004).This improved survival was no longer reported when M. mycetomatis-infected larvae were treated with 0.001 and 0.006 mg/kg M. fragrans EO (Log-rank, p = 0.363 and p = 0.226, respectively).

Efficacy of M. fragrans and P. anisum EOs in G. mellonella Larvae Infected with M. mycetomatis
As depicted in Figure 2A, when M. mycetomatis-infected larvae were treated with itraconazole, no enhanced survival was noted compared to the PBS-treated larvae (Log-Rank, p = 0.481).At a 0.025 mg/kg M. fragrans EO concentration, enhanced survival was noted compared to PBS-treated infected larvae (Log-rank, p = 0.004).This improved survival was no longer reported when M. mycetomatis-infected larvae were treated with 0.001 and 0.006 mg/kg M. fragrans EO (Log-rank, p = 0.363 and p = 0.226, respectively).

Antioxidant Activity of Essential Oils
To establish if there was a correlation between the antioxidant and antimicrobial activities of the essential oils, the DPPH radical scavenging assay was performed.As shown in Table 1, the highest % RSA was observed for P. anisum EO (% RSA ± SD 98.00 ± 0.003), indicating that it had the highest DPPH radical scavenging efficiency followed by G. alata root, T. vulgaris, M. fragrans, and S. aromaticum.EOs from L. angustifolia and G. alata aerial parts showed moderate DPPH radical scavenging activity.M. alternifolia essential oil had the weakest DPPH scavenging ability (% RSA ± SD, 20 ± 0.002).

Chemical Profiles of the Essential Oils
To establish which compounds were present in the five most active and two moderately active EOs, we characterized the chemical profiles of the essential oils with GC-MS.As a comparison, we also profiled the least effective essential oil, Melaleuca alternifolia.In total 202 components were identified in the eight essential oils under study (Table S1).Most abundant compounds are oxygenated monoterpene hydrocarbons, in addition to sesquiterpenes, oxygenated mono-and sesquiterpenes, diterpenes, triterpenes, fatty acids, and others at varying concentrations.Focusing on the major components representing ≥0.5% of the total oil composition, 56 different major components were identified in the eight EOs (Table 2).Of the 56 major components, 24 were unique to one of the eight essential oils.The remaining 32 components appeared as major or minor components in one or more of the essential oils.The major component of the active P. anisum EO is trans-anethole (76.87%).In addition, estragole (1.23%), p-anisaldehyde (10.35%), γ-himachalane (1.07%), pseudoisoeugenyl 2-methylbutyrate (4.76%), and spathulenol (0.25%) were detected.M. fragrans EO comprised 39.5% of cyclic and acyclic terpenes, 23.31% oxygenated monoterpenes, and 5.28% sesquiterpenes.Sabinene is the major component (19.97%), followed by terpinen-4-ol (17.87%), α-pinene, and β-pinene (6.67%).In addition, β-caryophyllene and α-copaene comprise >1% quantities in the oil.
Exceptionally, EOs of Geigeria alata roots and aerial parts accumulated 34.69% and 39.51% oxygenated sesquiterpenes of the total EOs composition, respectively, with αand β-bisabolene constituting the highest percentage (Table 2).Monoterpene hydrocarbons were not detected in both oils, while negligible amounts of oxygenated monoterpenes, such as trans-anethole <1%, were reported in roots.Phytone, a monoterpene ketone, was detected in the EO from the aerial parts in a relatively appreciable amount (1.75%) compared to the roots.

Discrimination of Oil Samples by Chemometric Analysis
Chemometric analysis was applied to discriminate the oil samples based on their potential antimycetomal activity (MICs) against M. mycetomatis and A. madurae strains (Figure 4A).In the principal component analysis (PCA) score plot, the first two components described 95.2% of the data variance, where PC1 explained 73.8% and PC2 21.4%.As displayed in Figure 4A, M. alternifolia oil appeared isolated in a separate cluster from other EOs; this might indicate that M. alternifolia oil exhibited the least antimycetomal activity compared to all tested oils (Table 1).Meanwhile, oil samples from the L. angustifolia and G. alata aerial parts were grouped separately, showing moderate antimicrobial activity.Moreover, G. alata roots, M. fragrans, P. anisum, S. aromaticum, and T. vulgaris EOs appeared in one cluster due to their promising dual inhibition of both eumycetoma and actinomycetoma.The plot of VIP scores (Figure 4B) revealed the influence of individual components on the clustering patterns of the oils, potentially explaining the contribution of each component (VIP score > 1) to the overall antimycetomal activity of these oils.
Hierarchical cluster analysis (HCA) was further applied as an unsupervised pattern recognition technique to explore the closeness of the GC-MS obtained chemical components in the eight essential oils.The dendrogram (Figure 4C) revealed the segregation of various oils into clusters.It disclosed the similarity in chemical components between M. fragrans and L. angustifolia EOs as they are clustered in the same group.Moreover, the dendrogram showed the closeness of the four oil samples, M. alternifolia, P. anisum, S. aromaticum, and T. vulgaris, which was further confirmed by the positioning of the oils in the heat map.Additionally, EOs from G. alata roots and aerial parts showed a specific arrangement in the generated heat map (Figure 4D) that followed the same pattern obtained from HCA based on their common individual components.

Discussion
This study correlates the in vitro activity, in vivo toxicity, and efficacy of investigated EOs to the level of chemical profiling responsible for bioactivity, and the findings are reinforced with metabolomics analysis.
We demonstrated that the EOs of M. fragrans and P. anisum inhibited M. mycetomatis and A. madurae in vitro.Furthermore, at the highest concentrations, M. fragrans and P. anisum EOs could prolong the survival of M. mycetomatis-infected G. mellonella larvae.A trend toward enhanced survival was also noted in A. madurae-infected G. mellonella larvae when they were treated with the different concentrations of P. anisum EO, indicating that only P. anisum EO has a dual in vivo efficacy against eumycetoma and actinomycetoma causative agents in efficacy models of Galleria mellonella.
In vitro results are further supported by in vivo studies, which consolidated the correlations between in vitro results and in vivo toxicity and efficacy of the EOs under study.The significance of these findings is underscored by metabolomics analysis revealing the clustering patterns of the EOs based on their bioactivity associated with their chemical profiles.
The HCA of the component profiles of these oils demonstrated that the M. fragrans EO was closely related to L. angustifolia EOs, as they are clustered in the same group.Similarly, the P. anisum EO clustered together with M. alternifolia, S. aromaticum, and T. vulgaris.However, the in vivo efficacy of G. alata roots, S. aromaticum, and T. vulgaris could not be determined due to the observed toxicity in uninfected G. mellonella larvae.This toxicity could be explained in terms of the acute toxicity that usually takes place when grain insects such as Tribolium castaneum and Rhyzopertha dominica are exposed to phenylpropanoids, including thymol, carvacrol, eugenol, and trans-anethole [35].Principally, the EO of G. alata roots is rich in oxygenated sesquiterpenes (34.69%).It is worth noting that the essential oils of Ferula communis L. are toxic to some insects, including Simosyrphus aegyptius, Colletes latreille, and Apis mellifera [36].G. alata root EO shared some of the oxygenated sesquiterpenes detected in Ferula communis L. EO, which might be responsible for the toxicity of Galleria larvae.
Our findings demonstrated the dual activity of P. anisum EO against eumycetoma and actinomycetoma causative agents.In previous studies, this EO showed fungistatic activity against the filamentous fungi Aspergillus niger, Aspergillus oryzae, and Aspergillus ochraceous with MICs ranging from 0.5 to 1 mg/mL [37][38][39].It also had activity against the yeasts Candida albicans, Candida glabrata, and Candida parapsilosis with MIC values between 0.10 and 1.56% v/v [40].Furthermore, P. anisum EO also exhibited antibacterial activity.It effectively inhibited Staphylococcus aureus, Bacillus cereus, and Proteus vulgaris with MICs of 125, 62.5, and 62.5 µg/mL, respectively [31].Both nano and coarse emulsions of anise oil reduced E. coli O157:H7 and L. monocytogenes counts by 2.51 and 1.64 log cfu/mL, respectively, compared to bulk P. anisum oil (1.48 log cfu/mL and 0.47 log cfu/mL) under the same condition [41].
P. anisum EO has been reported to contain the highest percentage of monoterpene alcohols, trans-anethole, and estragole [37,42,43].The antimicrobial activity of this oil is most likely due to the major phenylpropanoid constituent of P. anisum EO, trans-anethole.Anethole is known to have a broad spectrum of antimicrobial activity that is relatively weaker than that of known antibiotics.It exhibited synergistic antifungal activity against the budding yeast Saccharomyces cerevisiae, and the human opportunistic pathogenic yeast, Candida albicans, when combined with polygonal, nagilactone E, and n-dodecanol with an MIC of 625 µM and a minimal fungicidal concentration (MFC) of 1250 µM against S. cerevisiae [44].Additionally, an antifungal activity of anethole at 625 µM against the pathogenic fungus A. fumigatus has been reported [44].Anethole induces growth inhibition and morphological changes in the filamentous fungi Mucor and Fusarium spp.via metabolic blockade of cell wall biosynthesis based on the uncompetitive inhibition of chitin synthase [45].Since G. mellonella integument also contains chitin, it was debatable if P. anisum EO would also inhibit the chitin biosynthesis in G. mellonella [46].
As depicted in Figure 2A, all tested concentrations of P. anisum EO showed no toxic effect on the uninfected Galleria larvae, and possibly chitin biosynthesis in G. mellonella was unaffected at the tested concentrations.It has been reported that P. anisum EO does not exert cytotoxicity on NIH/3T3 fibroblast cells at incubation times of 24, 48, and 72 h; it rather proved to induce fibroblast viability in a concentration dependent manner [47].Previous studies reported that trans-anethole protects against liver, lung, and gastrointestinal tract damage caused by various agents in animal models [48].It is worth noting that supplementation of trans-anethole in daily feed of broilers improved intestinal antioxidant status and immune function and enhanced liver lipid metabolism [49].
In vivo studies revealed that P. anisum EO at 0.145 mg/kg significantly enhanced the survival of Galleria larvae infected with M. mycetomatis.A trend toward enhanced survival was noted for Galleria larvae infected with A. madurae.In addition the direct effect on both M. mycetomatis and A. madurae, P. anisum EO is also known to influence the host immune system.In human, P. anisum EO significantly decreased the expression levels of IL-1 and IL-8 and increased the Muc5ac secretion in primary airway bronchial and tracheal epithelial cell lines (HBEpC and HTEpC) stimulated with lipopolysaccharide [50].In this study, we also showed that this EO had the highest antioxidant activity.
However, M. fragrans EO also had considerable antioxidant activity, and the in vitro and in vivo antimicrobial activities of M. fragrans EO against both eumycetoma and actinomycetoma pathogens might be associated with this antioxidant activity.Antioxidant and anti-inflammatory activity of M. fragrans was previously reported in different in vitro and in vivo models of carrageenan-induced rat paw edema by M. fragrans EO compared to indomethacin (p < 0.007, <0.05).With regard to acetic acid-induced writhing, the oil reduced the syndrome in a dose-dependent manner.Notably, M. fragrans oil is effective in pain due to its anti-inflammatory activity, but it is not effective on nociceptors [51].Moreover, in vitro and in vivo experimental protocols revealed that phenylpropanoids (cinnamaldehyde, eugenol, and safrole) act through different mechanisms of action as immunomodulators and suppressive agents in the inflammatory response [52].Nutmeg essential oil showed good antioxidant activity (EC50 = 1.35 ± 0.003 mg/mL) [53].Mace essential oil revealed % inhibition in a free radical scavenging assay (21.95 µg/mL) and % inhibition (67.9%) in the linoleic acid system [54].
M. fragrans EO showed no toxic effects on the uninfected larvae.However, a slight decrease in the survival rate at the highest concentration (0.025 mg/kg) was observed on day 10 (Figure 2B).Sabinene demonstrated a safety profile of up to 0.64 µL/mL in Raw and HaCat cells.Sabinene was less toxic, affecting mouse macrophage at 1.25 µL/mL, macrophage cell viability at 1.25 µL/mL, and human keratinocytes at 0.32 µL/mL [51].
EOs significantly reduce proinflammatory cytokines, immunoglobulins, or regulatory pathways and induce anti-inflammatory markers in vitro and in vivo [55].Some essential oils may stimulate the proliferation of immune-competent cells, including polymorphonuclear leukocytes, macrophages, dendritic cells, natural killer cells, and B and T lymphocytes [56].The antioxidant potential of EOs participates greatly in overcoming cellular oxidative damage caused by reactive oxygen species (ROS), which plays an additional anti-inflammatory role [57].It is worth noting that a combined antifungal and anti-inflammatory treatment with diclofenac was effective in treating patients infected with Madurella mycetomatis [58].Similarly, a good response was observed with diclofenac in addition to antibiotics combination therapy for actinomycetoma [59].
Comparative metabolomics indicated that the components from different essential oils obtained by GC-MS are qualitatively and quantitatively different.The chemometric analysis of terpenes distribution in the EOs of G. alata roots, M. fragrans, S. aromaticum, and T. vulgaris revealed that the accumulation of sesquiterpenes like β-caryophyllene and β-caryophyllene oxide might contribute to the activity of these EOs toward fungal and bacterial mycetoma (Table 1, Figure 4B,D).β-Caryophyllene oxide is reported to occur in the oils of Hypericum hyssopifolium and Hypericum heterophyllum and inhibits 33-85% of the growth of the pathogenic fungi Fusarium spp.and Rhizoctonia solani [60].The antifungal activity of the volatile oil of Eryngium duriaei sub spp.juresianum against pathogenic dermatophytes was also reported to be associated with caryophyllene-14-al, 14-hydroxy-βcaryophyllene, and caryophyllene oxide [61].The β-caryophyllene identified as responsible for Vernonia chalybaea EO-mediated inhibition of the growth of the dermatophytic fungi, Trichophyton rubrum, had an MIC value of 1.25 mg/mL.Seemingly, the essential oil of V. chalybaea and β-caryophyllene synergistically enhanced the antifungal action of ketoconazole with a fractional inhibitory concentration (FICI) value of 0.2 [62].Furthermore, the tricyclic sesquiterpene, 5,10-cycloaromadendrane, spathulenol, which was identified from the EOs of M. fragrans, P. anisum, and T. vulguris might be associated with their antifungal potential as depicted in the VIP score plot (Figure 4B).Spathulenol exhibited antifungal activity against some dermatophyte strains, Alternaria alternata, and Colletotrichum gloeosporioides [63,64].Additionally, the abundance of spathulenol in the essential oil of A. valentinus demonstrated a strong fungicidal activity against Fusarium sp., Aspergillus sp., and Penicillium sp.[65].

Conclusions
The inefficacy of existing antifungals and the inadequacy of antibacterial agents in treating both types of mycetoma has necessitated the search for novel and efficient therapeutic agents to combat these pathogens.The present study demonstrated that M. mycetomatis and A. madurae were susceptible to the same EOs both in vitro and in vivo.The empirical early use of these bioactive EOs as broad-spectrum dual antimycetomal agents against eumycetoma and actinomycetoma pathogens could save time and money by skipping the time-consuming diagnosis of the causative pathogen.This approach could pave the way for the next generation of antimycetomal agents.Further studies on a broad spectrum of antimicrobial treatment options for mycetoma based on essential oils offer a promising avenue for discovering and developing novel antimicrobial agents for therapeutic use.

Table 2 .
Major components identified by GC-MS in the eight essential oils under study (≥0.5% peak area).