Active Flavonoids from Colubrina greggii var. greggii S. Watson against Clinical Isolates of Candida spp.

Candida albicans is the most commonly implicated agent in invasive human fungal infections. The disease could be presented as minimal symptomatic candidemia or can be fulminant sepsis. Candidemia is associated with a high rate of mortality and high healthcare and hospitalization costs. The surveillance programs have reported the distribution of other Candida species reflecting the trends and antifungal susceptibilities. Previous studies have demonstrated that C. glabrata more frequently presents fluconazole-resistant strains. Extracts from Mexican plants have been reported with activity against pulmonary mycosis, among them Colubrina greggii. In the present study, extracts from the aerial parts (leaves, flowers, and fruits) of this plant were evaluated against clinical isolates of several species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. krusei, and C. tropicalis) by the broth microdilution assay. Through bioassay-guided fractionation, three antifungal glycosylated flavonoids were isolated and characterized. The isolated compounds showed antifungal activity only against C. glabrata resistant to fluconazole, and were non-toxic toward brine shrimp lethality bioassay and in vitro Vero cell line assay. The ethyl acetate and butanol extracts, as well as the fractions containing the mixture of flavonoids, were more active against Candida spp.     

Chemical Composition,Antifungal and Anti-Biofilm Activities of Volatile Fractions of Convolvulus althaeoides L. Roots from Tunisia

The antifungal drugs currently available and mostly used for the treatment of candidiasis exhibit the phenomena of toxicity and increasing resistance. In this context, plant materials might represent promising sources of antifungal agents. The aim of this study is to evaluate for the first time the chemical content of the volatile fractions (VFs) along with the antifungal and anti-biofilm of Convolvulus althaeoides L. roots. The chemical composition was determined by gas chromatography coupled to a flame ionization detector and mass spectrometry. In total, 73 and 86 chemical compounds were detected in the n-hexane (VF1) and chloroform (VF2) fractions, respectively. Analysis revealed the presence of four main compounds: n-hexadecenoic acid (29.77%), 4-vinyl guaiacol (12.2%), bis(2-ethylhexyl)-adipate (9.69%) and eicosane (3.98%) in the VF extracted by hexane (VF1). n-hexadecenoic acid (34.04%), benzyl alcohol (7.86%) and linoleic acid (7.30%) were the main compounds found in the VF extracted with chloroform (VF2). The antifungal minimum inhibitory concentrations (MICs) of the obtained fractions against Candida albicans, Candida glabrata and Candida tropicalis were determined by the micro-dilution technique and values against Candida spp. ranged from 0.87 to 3.5 mg/mL. The biofilm inhibitory concentrations (IBF) and sustained inhibition (BSI) assays on C. albicans, C. glabrata and C. tropicalis were also investigated. The VFs inhibited biofilm formation up to 0.87 mg/mL for C. albicans, up to 1.75 mg/mL against C. glabrata and up to 0.87 mg/mL against C. tropicalis. The obtained results highlighted the synergistic mechanism of the detected molecules in the prevention of candidosic biofilm formation.     

Molecular Umbrella as a Nanocarrier for Antifungals

A molecular umbrella composed of two O-sulfated cholic acid residues was applied for the construction of conjugates with cispentacin, containing a “trimethyl lock” (TML) or o-dithiobenzylcarbamoyl moiety as a cleavable linker. Three out of five conjugates demonstrated antifungal in vitro activity against C. albicans and C. glabrata but not against C. krusei, with MIC₉₀ values in the 0.22–0.99 mM range and were not hemolytic. Antifungal activity of the most active conjugate 24c, containing the TML–pimelate linker, was comparable to that of intact cispentacin. A structural analogue of 24c, containing the Nap-NH₂ fluorescent probe, was accumulated in Candida cells, and TML-containing conjugates were cleaved in cell-free extract of C. albicans cells. These results suggest that a molecular umbrella can be successfully applied as a nanocarrier for the construction of cleavable antifungal conjugates.     

In Vitro Anti-Candida Activity and Action Mode of Benzoxazole Derivatives

A newly synthetized series of N-phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazole, were screened against Candida strains and the action mechanism was evaluated. 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanone (5d), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichloro-phenyl)ethanone (5i), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,4,6-trichlorophenyl)ethanone (5k) and 2-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]-1-phenylethanone (6a) showed anti-C. albicans SC5314 activity, where 5d displayed MIC_T = 16 µg/mL (%R = 100) and a weak anti-proliferative activity against the clinical strains: C. albicans resistant to azoles (Itr and Flu) and C. glabrata. Derivatives 5k and 6a displayed MIC_P = 16 µg/mL and %R = 64.2 ± 10.6, %R = 88.0 ± 9.7, respectively, against the C. albicans isolate. Derivative 5i was the most active against C. glabrata (%R = 53.0 ± 3.5 at 16 µg/mL). Benzoxazoles displayed no MIC against C. glabrata. Benzoxazoles showed a pleiotropic action mode: (1) the total sterols content was perturbed; (2) 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(3,4-dichlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichlorophenyl)ethanol (8h–i) at the lowest fungistatic conc. inhibited the efflux of the Rho123 tracker during the membrane transport process; (3) mitochondrial respiration was affected/inhibited by the benzoxazoles: 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-chlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanol 8c–d and 8i. Benzoxazoles showed comparable activity to commercially available azoles due to (1) the interaction with exogenous ergosterol, (2) endogenous ergosterol synthesis blocking as well as (3) membrane permeabilizing properties typical of AmB. Benzoxazoles display a broad spectrum of anti-Candida activity and action mode towards the membrane without cross-resistance with AmB; furthermore, they are safe to mammals.     

Synthesis,Biological Evaluation,and Structure–Activity Relationships of 4-Aminopiperidines as Novel Antifungal Agents Targeting Ergosterol Biosynthesis

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.     

In Vitro Activity of Essential Oils Distilled from Colombian Plants against Candida auris and Other Candida Species with Different Antifungal Susceptibility Profiles

Multi-drug resistant species such as Candida auris are a global health threat. This scenario has highlighted the need to search for antifungal alternatives. Essential oils (EOs), or some of their major compounds, could be a source of new antifungal molecules. The aim of this study was to evaluate the in vitro activity of EOs and some terpenes against C. auris and other Candida spp. The eleven EOs evaluated were obtained by hydro-distillation from different Colombian plants and the terpenes were purchased. EO chemical compositions were obtained by gas chromatography/mass spectrometry (GC/MS). Antifungal activity was evaluated following the CLSI standard M27, 4th Edition. Cytotoxicity was tested on the HaCaT cell line and fungal growth kinetics were tested by time–kill assays. Candida spp. showed different susceptibility to antifungals and the activity of EOs and terpenes was strain-dependent. The Lippia origanoides (thymol + p-cymene) chemotype EO, thymol, carvacrol, and limonene were the most active, mainly against drug-resistant strains. The most active EOs and terpenes were also slightly cytotoxic on the HaCaT cells. The findings of this study suggest that some EOs and commercial terpenes can be a source for the development of new anti-Candida products and aid the identification of new antifungal targets or action mechanisms.     

Lycorine Alkaloid and Crinum americanum L. (Amaryllidaceae) Extracts Display Antifungal Activity on Clinically Relevant Candida Species

Candida species are the main fungal agents causing infectious conditions in hospital patients. The development of new drugs with antifungal potential, increased efficacy, and reduced toxicity is essential to face the challenge of fungal resistance to standard treatments. The aim of this study is to evaluate the in vitro antifungal effects of two crude extracts of Crinum americanum L., a rich alkaloid fraction and lycorine alkaloid, on the Candida species. As such, we used a disk diffusion susceptibility test, determined the minimum inhibitory concentration (MIC), and characterized the components of the extracts using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI FT-ICR MS). The extracts were found to have antifungal activity against various Candida species. The chemical characterization of the extracts indicated the presence of alkaloids such as lycorine and crinine. The Amaryllidaceae family has a promising antifungal potential. Furthermore, it was found that the alkaloid lycorine directly contributes to the effects that were observed for the extracts and fraction of C. americanum.     

Synthesis and in vitro Antimicrobial Activity of Nalidixic Acid Hydrazones

A series of nalidixic acid‐based hydrazones have been synthesized and evaluated for their in vitro antimicrobial activity using the broth microdilution method against a panel of reference strains of microorganisms, including Gram‐positive bacteria, Gram‐negative bacteria, and fungi belonging to yeasts Candida spp. and molds Aspergillus spp., Penicillium spp., and Rhizopus spp. Nalidixic acid derivatives were obtained by condensation reaction of nalidixic acid hydrazide with substituted (hetero)aromatic aldehydes. All compounds have been characterized by ¹H NMR and ¹³C NMR spectra. The antimicrobial activity indicated that compound with indole substituent could be a promising lead for future development of active antifungal agents.     

Design,synthesis, and in vitro antifungal evaluation of novel triazole derivatives bearing alkynyl side chains

In order to explore novel antifungal agents, twenty-seven triazole derivatives featuring an alkyne linker in the side chain were designed and synthesized by the Sonogashira reaction. Most of the target compounds exhibited good antifungal activity against eight human pathogenic fungi, especially excellent activity against Candida and Cryptococcus species, comparing with the reference drugs fluconazole, voriconazole and ravuconazole. Compounds A2 and A3 exhibited in vitro activity against all the tested fungi with MIC₈₀ values ranging from 0.0156 μg/mL to 0.5 μg/mL, which are superior to ravuconazole and fluconazole. SAR and molecular docking study give a clear conclusion that para-fluoro, para-chloro, and para-cyano substituted phenylalkynyl or pyridinylalkynyl side chains may promote triazole antifungal activity.     

Assessment of Dentifrices Against Candida Biofilm

The invasion of opportunistic pleiomorphic Candida albicans into oral cavity environment leads to development and progression of its resistance to both naturally occurring antifungal peptides in human saliva as well as commercially available antifungal therapies. As a result of this, the usage and popularity of natural medicine and dentifrices had increased significantly in the last decade. In the present investigation, we have assessed the action of locally available dentifrices against C. albicans biofilm. Disk diffusion test showed maximum zone of inhibition (20?mm) by herbal dentifrice (D-5) as compared to other dentifrices when incubated at 37?°C and 48?h. Assessment of dentifrice D-5 for its effectiveness against C. albicans was further shown in MIC₉₀ (3.12?mg?mL^?1) and SMIC₉₀ (6.2?mg?mL^?1) values for planktonic and sessile cells (biofilm forming), respectively. Our data depicted 80% reduction in the cell surface hydrophobicity when 6.2?mg?mL^?1 of herbal dentifrice D-5 was used against 48-h grown Candida biofilm at 37?°C. Visualization of herbal dentifrice D-5-treated C. albicans biofilm under SEM revealed drastic reduction in the dense network of yeast, hyphae, and pseudohyphae enclosed in its ECM as compared to its control biofilm. The data were further supported by CLSM analysis which depicted C. albicans architecture disruption by herbal dentifrices. From the above data, it is inferred that these studies would provide researchers and medical practitioners with better insight into the antifungal effect of natural herbal dentifrices.     

The Inhibition of Non-albicans Candida Species and Uncommon Yeast Pathogens by Selected Essential Oils and Their Major Compounds

The epidemiology of yeast infections and resistance to available antifungal drugs are rapidly increasing, and non-albicans Candida species and rare yeast species are increasingly emerging as major opportunistic pathogens. In order to identify new strategies to counter the threat of antimicrobial resistant microorganisms, essential oils (EOs) have become an important potential in the treatment of fungal infections. EOs and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities. We investigated the in vitro antifungal activity of nine commercial EOs such as Thymus vulgaris (thyme red), Origanum vulgare (oregano), Lavandula vera (lavender), Pinus sylvestris (pine), Foeniculum vulgare (fennel), Melissa officinalis (lemon balm), Salvia officinalis (sage), Eugenia caryophyllata (clove) and Pelargonium asperum (geranium), and some of their main components (α-pinene, carvacrol, citronellal, eugenol, γ-terpinene, linalool, linalylacetate, terpinen-4-ol, thymol) against non-albicans Candida strains and uncommon yeasts. The EOs were analyzed by GC-MS, and their antifungal properties were evaluated by minimum inhibitory concentration and minimum fungicidal concentration parameters, in accordance with CLSI guidelines, with some modifications for EOs. Pine exhibited strong antifungal activity against the selected non-albicans Candida isolates and uncommon yeasts. In addition, lemon balm EOs and α-pinene exhibited strong antifungal activity against the selected non-albicans Candida yeasts. Thymol inhibited the growth of all uncommon yeasts. These data showed a promising potential application of EOs as natural adjuvant for management of infections by emerging non-albicans Candida species and uncommon pathogenic yeasts.     

In Vitro Antifungal Activity and Mechanism of Ag3PW12O40 Composites against Candida Species

Fungal infections pose a serious threat to human health. Polyoxometalates (POMs) are metal–oxygen clusters with potential application in the control of microbial infections. Herein, the Ag₃PW₁₂O₄₀ composites have been synthesized and verified by Fourier transform infrared (FT-IR) spectrum, transmission electron microscopy (TEM), scanning electron microscope (SEM), elemental analysis, and X-ray diffraction (XRD). The antifungal activities of Ag₃PW₁₂O₄₀ were screened in 19 Candida species strains through the determination of minimum inhibitory concentration (MIC) by the microdilution checkerboard technique. The minimum inhibitory concentration (MIC₅₀) values of Ag₃PW₁₂O₄₀ are 2~32 μg/mL to the Candida species. The MIC₈₀ value of Ag₃PW₁₂O₄₀ to resistant clinical isolates C. albicans HL963 is 8 μg/mL, which is lower than the positive control, fluconazole (FLC). The mechanism against C. albicans HL963 results show that Ag₃PW₁₂O₄₀ can decrease the ergosterol content. The expressions of ERG1, ERG7, and ERG11, which impact on the synthesis of ergosterol, are all prominently upregulated by Ag₃PW₁₂O₄₀. It indicates that Ag₃PW₁₂O₄₀ is a candidate in the development of new antifungal agents.     

Design,Synthesis and Antifungal Evaluation of Novel Pyrylium Salt In Vitro and In Vivo

Nowadays, discovering new skeleton antifungal drugs is the direct way to address clinical fungal infections. Pyrylium salt SM21 was screened from a library containing 50,240 small molecules. Several studies about the antifungal activity and mechanism of SM21 have been reported, but the structure–activity relationship of pyrylium salts was not clear. To explore the chemical space of antifungal pyrylium salt SM21, a series of pyrylium salt derivatives were designed and synthesized. Their antifungal activity and structure-activity relationships (SAR) were investigated. Compared with SM21, most of the synthesized compounds exhibited equivalent or improved antifungal activities against Candida albicans in vitro. The synthesized compounds, such as XY10, XY13, XY14, XY16 and XY17 exhibited comparable antifungal activities against C. albicans with MIC values ranging from 0.47 to 1.0 μM. Fortunately, a compound numbered XY12 showed stronger antifungal activities and lower cytotoxicity was obtained. The MIC of compound XY12 against C. albicans was 0.24 μM, and the cytotoxicity decreased 20-fold as compared to SM21. In addition, XY12 was effective against fluconazole-resistant C. albicans and other pathogenic Candida species. More importantly, XY12 could significantly increase the survival rate of mice with a systemic C. albicans infection, which suggested the good antifungal activities of XY12 in vitro and in vivo. Our results indicated that structural modification of pyrylium salts could lead to the discovery of new antifungal drugs.     

Heteroleptic transition metal complexes of Schiff‐base‐derived ligands exert their antifungal activity by disrupting membrane integrity

Development of new treatment strategies and chemotherapeutic agents is urgently needed to combat the growing multidrug resistant species of Candida. In this direction, a new series of Cu (II), Co (II), Ni (II) and Zn (II) heteroleptic complexes were synthesized, characterized and evaluated for antifungal activity. Based on spectral characterization and physical measurements, an octahedral geometry was assigned to [Co(L1)(L2)ClH₂O] ( C2 ), [Ni(L1)(L2)ClH₂O] ( C3 ), [Zn(L1)(L2)ClH₂O] ( C4 ) complexes, while a distorted octahedral geometry was assigned to [Cu(L1)(L2)ClH₂O] ( C1 ) complex. All the synthesized compounds were tested for antifungal activity against 11 Candida albicans isolates, including fluconazole (FLC)‐resistant isolates, by determining minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC), following CLSI guidelines. The mechanism of their antifungal activity was assessed by studying their effect on the plasma membrane using flow cytometry and quantifying the ergosterol contents. All the test compounds showed varying levels of antifungal activity. Both the ligands showed moderate antifungal activity with a median MIC value of 100 μg/mL with no fungicidal activity. Compound C3 was the most potent compound with median MIC and MFC values of 0.10 and 1.60 μg/mL, respectively. Flow cytometry analysis revealed that these compounds at MFC values disrupt the cell membrane, resulting in propidium iodide entering the cells. These compounds also reduced a considerable amount of ergosterol content after treating the cells with MIC and sub‐MIC values. This study indicates that these compounds have high antifungal activity against C. albicans, and have the potential to be developed as novel antifungal drugs.     

1,3-Oxazole derivatives of cytisine as potential inhibitors of glutathione reductase of Candida spp.: QSAR modeling,docking analysis and experimental study of new anti-Candida agents

Natural products as well as their derivatives play a significant role in the discovery of new biologically active compounds in the different areas of our life especially in the field of medicine. The synthesis of compounds produced from natural products including cytisine is one approach for the wider use of natural substances in the development of new drugs. QSAR modeling was used to predict and select of biologically active cytisine-containing 1,3-oxazoles. The eleven most promising compounds were identified, synthesized and tested. The activity of the synthesized compounds was evaluated using the disc diffusion method against C. albicans M 885 (ATCC 10,231) strain and clinical fluconazole-resistant Candida krusei strain. Molecular docking of the most active compounds as potential inhibitors of the Candida spp. glutathione reductase was performed using the AutoDock Vina. The built classification models demonstrated good stability, robustness and predictive power. The eleven cytisine-containing 1,3-oxazoles were synthesized and their activity against Candida spp. was evaluated. Compounds 10, 11 as potential inhibitors of the Candida spp. glutathione reductase demonstrated the high activity against C. albicans M 885 (ATCC 10,231) strain and clinical fluconazole-resistant Candida krusei strain. The studied compounds 10, 11 present the interesting scaffold for further investigation as potential inhibitors of the Candida spp. glutathione reductase with the promising antifungal properties. The developed models are publicly available online at http://ochem.eu/article/120720 and could be used by scientists for design of new more effective drugs.     

Antifungal Activity of Amphiphilic Perylene Bisimides

Perylene-based compounds, either naturally occurring or synthetic, have shown interesting biological activities. In this study, we report on the broad-spectrum antifungal properties of two lead amphiphilic perylene bisimides, compounds 4 and 5, which were synthesized from perylene-3,4,9,10-tetracarboxylic dianhydride by condensation with spermine and an ammonium salt formation. The antifungal activity was evaluated using a collection of fungal strains and clinical isolates from patients with onychomycosis or sporotrichosis. Both molecules displayed an interesting antifungal profile with MIC values in the range of 2–25 μM, being as active as several reference drugs, even more potent in some particular strains. The ammonium trifluoroacetate salt 5 showed the highest activity with a MIC value of 2.1 μM for all tested Candida spp., two Cryptococcus spp., two Fusarium spp., and one Neoscytalidium spp. strain. Therefore, these amphiphilic molecules with the perylene moiety and cationic ammonium side chains represent important structural features for the development of novel antifungals.     

Antifungal activity of Mongolian medicinal plant extracts

Abstract

The in vitro antifungal activity of extracts obtained from 14 medicinal plants of the mongolian flora were investigated by measuring their minimal inhibitory concentration (MIC) against fungi cause of cutaneous diseases such as Candida species, dermatophytes and Malassezia furfur. Among the species examined, Stellaria dichotoma L., Scutellaria scordifolia L. Aquilegia sibirica Fisch. Et Schrenk. and Hyoscyamus niger L. extracts demonstrated antifungal activity against all studied fungi. In particular, S. scordifolia L. methanol extract, obtained at room temperature, showed the best activity against Candida spp., Malassezia furfur and dermatophytes with GMMIC₅₀ values of 22?µg/mL, 64?µg/mL and 32?µg/mL, respectively. The flavones, luteolin and apigenin, identified in S. scordifolia extracts, and rutin identified in S. dichotoma and Hyoscyamus niger L. extracts, could be responsible of the observed antifungal activity.     

Synthesis and antibacterial properties of new phenothiazinyl- and phenyl-nitrones

The synthesis of new phenothiazinyl- and phenyl-nitrones under classical versus microwave heating conditions is described. Better yields were obtained under microwave irradiation in the condensation reactions of phenothiazyl-carbaldehyde with hydroxylamine derivatives. The structures of the new phenothiazinyl-nitrones were assigned on the basis of MS, FT–IR and NMR spectra. The new nitrones and some known phenyl-nitrones were screened for their antibacterial and antifungal activity against several Candida species, Gram negative bacteria, such as E. coli, Citrobacter spp, Morganella spp, Pseudomonas aeruginosa, Klebsiella pneumoniae (± ESBL), Proteus spp, Acinetobacter spp and the Gram positive bacterium Staphylococcus aureus, with moderate results.     

Mechanism of Antifungal Action of Monoterpene Isoespintanol against Clinical Isolates of Candida tropicalis

The growing increase in infections by Candida spp., non-albicans, coupled with expressed drug resistance and high mortality, especially in immunocompromised patients, have made candidemia a great challenge. The efficacy of compounds of plant origin with antifungal potential has recently been reported as an alternative to be used. Our objective was to evaluate the mechanism of the antifungal action of isoespintanol (ISO) against clinical isolates of Candida tropicalis. Microdilution assays revealed fungal growth inhibition, showing minimum inhibitory concentration (MIC) values between 326.6 and 500 µg/mL. The eradication of mature biofilms by ISO was between 20.3 and 25.8% after 1 h of exposure, being in all cases higher than the effect caused by amphotericin B (AFB), with values between 7.2 and 12.4%. Flow cytometry showed changes in the permeability of the plasma membrane, causing loss of intracellular material and osmotic balance; transmission electron microscopy (TEM) confirmed the damage to the integrity of the plasma membrane. Furthermore, ISO induced the production of intracellular reactive oxygen species (iROS). This indicates that the antifungal action of ISO is associated with damage to membrane integrity and the induction of iROS production, causing cell death.