Antifungal Constituents of Piper crocatum and Their Activities as Ergosterol Biosynthesis Inhibitors Discovered via In Silico Study Using ADMET and Drug-Likeness Analysis.

Along with the increasing resistance of Candida spp. to some antibiotics, it is necessary to find new antifungal drugs, one of which is from the medicinal plant Red Betel (Piper crocatum). The purpose of this research is to isolate antifungal constituents from P. crocatum and evaluate their activities as ergosterol biosynthesis inhibitors via an in silico study of ADMET and drug-likeness analysis. Two new active compounds 1 and 2 and a known compound 3 were isolated, and their structures were determined using spectroscopic methods, while their bioactivities were evaluated via in vitro and in silico studies, respectively. Antifungal compound 3 was the most active compared to 1 and 2 with zone inhibition values of 14.5, 11.9, and 13.0 mm, respectively, at a concentration of 10% w/v, together with MIC/MFC at 0.31/1.2% w/v. Further in silico study demonstrated that compound 3 had a stronger ΔG than the positive control and compounds 1 and 2 with -11.14, -12.78, -12.00, and -6.89 Kcal/mol against ERG1, ERG2, ERG11, and ERG24, respectively, and also that 3 had the best Ki with 6.8 × 10-3, 4 × 10-4, 1.6 × 10-3, and 8.88 µM. On the other hand, an ADMET analysis of 1-3 met five parameters, while 1 had one violation of Ro5. Based on the research data, the promising antifungal constituents of P. crocatum allow P. crocatum to be proposed as a new antifungal candidate to treat and cure infections due to C. albicans.

Sterols Content of Fruiting Bodies of Medicinal Artist's Bracket Mushroom Ganoderma applanatum (Agaricomycetes) Collected in Armenia.

The qualitative analysis of hexane extracts obtained from different trama layers (WT, T1-T4) of dried fruiting bodies of medicinal bracket fungus Ganoderma applanatum collected in the Tavoush region of North-East Armenia was performed by GC-MS analysis. Three sterols [(7.22-ergostadienon, ergosterol and ergosta-14.22-diene-3-ol (3ß, 5α, 22E)] have been identified. The results have shown that the content and ratio of sterols differ in analyzed trama samples. The highest amount of sterols was detected in middle parts of T2 and T3 layers, while content of sterols gradually decreased to the upper cortical (T4) and lower hymenial (T1) layers. The chromatographic profiles of identified compounds indicate that different sterols dominated in each layer: 7.22-ergostadienon in T4, ergosterol in T3, T2, and T1. The average weight loss of analyzed trama samples during six days of drying was about 40 wt.% (37.0-43.49 wt.%) of the total weight of basidiome, which decreased up to 5 wt.% in the next two days. The complete extraction of sterols lasted six days. Its further prolongation leads to stationary phase without an increase in the amount of extracted sterols.

4D printing induced by microwave and ultrasound for mushroom mixtures: Efficient conversion of ergosterol into vitamin D2.

This study hoped to use microwave and ultrasound combined with 4D printing technology to promote the conversion of ergosterol into vitamin D2 in printing model with mushroom scraps. Under the UV irradiation, the conversion was different in the printed model with different irradiation areas and different physical field pretreatment. Compared with raw materials, vitamin D2 concentrations in the printed models was 4.6 time higher. Vitamin D2 in the product after physical field pretreatment was 2.2-3.8 times higher than that without pretreatment. From partial least square regression (PLS) analysis, irradiation area had the greatest impact while ultrasound treatment had the least. Pretreatment enhanced vitamin D2 content, possibly because pretreatment meant ergosterol was more susceptible to UV radiation, and expansion of the irradiated area increased the beneficial effect. This study established an artificial neural network model to predict ergosterol and vitamin D2 content.

Whole genome sequencing of a clinical drug resistant Candida albicans isolate reveals known and novel mutations in genes involved in resistance acquisition mechanisms.

Candida albicans is an opportunistic pathogen accounting for the majority of cases of Candida infections. Currently, C. albicans are developing resistance towards different classes of antifungal drugs and this has become a global health burden that does not spare Lebanon. This study aims at determining point mutations in genes known to be involved in resistance acquisition and correlating resistance to virulence and ergosterol content in the azole resistant C. albicans isolate CA77 from Lebanon. This pilot study is the first of its kind to be implemented in Lebanon. We carried out whole genome sequencing of the azole resistant C. albicans isolate CA77 and examined 18 genes involved in antifungal resistance. To correlate genotype to phenotype, we evaluated the virulence potential of this isolate by injecting it into BALB/c mice and we quantified membrane ergosterol. Whole genome sequencing revealed that eight out of 18 genes involved in antifungal resistance were mutated in previously reported and novel residues. These genotypic changes were associated with an increase in ergosterol content but no discrepancy in virulence potential was observed between our isolate and the susceptible C. albicans control strain SC5314. This suggests that antifungal resistance and virulence potential in this antifungal resistant isolate are not correlated and that resistance is a result of an increase in membrane ergosterol content and the occurrence of point mutations in genes involved in the ergosterol biosynthesis pathway.

Review on Research Progress and Prospects of Cicada Flower, Isaria cicadae (Ascomycetes).

Cicada flower, Isaria cicadae Miq., has been a traditional Chinese medicine for approximately 1600 years. Many works on its identification, bioactivities, and clinical use against some disorders have been published, but some inaccuracies and inconsistencies need to be further clarified. In combination with our > 20 years of research and application of cicada flower and examination of the literature and patents published in recent years, this article summarizes and reviews the life cycle and taxonomy, genome size and mating type, molecular systematic classification and cultivation, active ingredients, and pharmacological functions of I. cicadae.

Plant-derived isoquinoline alkaloids that target ergosterol biosynthesis discovered by using a novel antifungal screening tool.

The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-ß-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.

Near-infrared hyperspectral imaging for deoxynivalenol and ergosterol estimation in wheat samples.

The present study aimed to evaluate the use of hyperspectral imaging (HSI)-NIR spectroscopy to assess the presence of DON and ergosterol in wheat samples through prediction and classification models. To achieve these objectives, a first set of bulk samples was scanned by HSI-NIR and divided into two subsamples, one that was analysed for ergosterol and another that was analysed for DON by HPLC. This method was repeated for a second larger set to build prediction and classification models. All the spectra were pretreated and statistically processed by PLS and LDA. The prediction models presented a RMSEP of 1.17 mg/kg and 501 µg/kg for ergosterol and DON, respectively. Classification achieved an encouraging accuracy of 85.4% for an independent validation set of samples. The results confirm that HSI-NIR may be a suitable technique for ergosterol quantification and DON classification of samples according to the EU legal limit for DON.

Characterization of seven sterols in five different types of cattle feedstuffs.

This paper provides a method for the quantification of sterols in different types of calf feedstuffs based on soy, sunflower, hay, calf feed and a mixture of all of them. The free fraction and the total sterolic fraction, after saponification and acidic hydrolysis of the samples, are extracted by solvent and the sterols are identified/quantified by reversed phase HPLC coupled to tandem mass spectrometry by atmospheric pressure chemical ionization. After the recovery evaluation, the method is validated in terms of linearity (coefficient of determination R2), repeatability (coefficient of variation RSD), limit of detection and quantification. In most of the cases, the most representative phytosterol is ß-sitosterol, followed by campesterol or stigmasterol and by other minor sterols such as fucosterol, and Δ-5-avenasterol. In addition, also cholesterol and ergosterol, if present, are evaluated in all the samples. As far as we know, very little information is available on the investigated feeds, which are commonly used on farms. The results of this survey were compared to other studies, if present in literature, showing good agreement. The proposed method resulted to be simple, fast and suitable for application to other sterols, feedstuffs and derived foods. The knowledge of the sterolic content and composition is getting more and more important, both in terms of comprehension of the vegetal biochemistry and as basis for sterolomic studies.

Effects of ergosteroside combined risedronate on fracture healing and BMP-2, BMP-7 and VEGF expression in rats

Purpose To evaluate the effect of ergosterol combined with risedronate on fracture healing. Methods Sixty male Sprague Dawley fracture model rats were assigned into group A (n=20), group B (n=20), and group C (n=20) at random. All rats were fed by gavage until their sacrifice as it follows: group A with ergosteroside and risedronate, group B with risedronate, and group C with saline solution. At weeks 2 and 4, 10 rats of each group were sacrificed. Healing effect and bone tissue changes in the fractures site were assessed by using hematoxylin and eosin stain histology. Enzyme-linked immunosorbent assay was used to detect the expression of serum bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF). Reverse transcriptase polymerase chain reaction was applied to detect the expression of osteoprotegerin (OPG) mRNA, osteocalcin (OCN) mRNA and core-binding factor subunit-?1 (CBF-?1) mRNA. Results In terms of serum BMP-2, BMP-7, and VEGF expression at weeks 2 and 4 after gavage, group A < group B < group C (P<0.05). At week 4 after gavage, serum VEGF expression in the three groups harbored positive relationship with serum BMP-2 and BMP-7 expression (P<0.05). Regarding serum OPG, OCN and CBF-?1 mRNA expression at weeks 2 and 4 after gavage, group A

A sensitive, precise and rapid LC-MS/MS method for determination of ergosterol peroxide in Paecilomyces cicadae mycelium.

Ergosterol peroxide (EP) has considerable potential effect against the proliferation of tumor cells. Here, we established a new approach for EP content detection through liquid chromatography-tandem mass spectrometry. The specificity, limit of detection (LOD)/quantitative (LOQ), linearity and range, accuracy, repeatability, and intermediate precision were tested. The EP retention time was 7.18 min. The linear relationship between the mass concentration of nonylphenol and the chromatographic peak area was good within the EP concentration range of 0.1-2.0 µg/mL. The correlation coefficient was 0.994, the regression equation was Y = 27 409.8 × X - 1114.67, the average recovery rate was 82.77%, the relative standard deviation was 11.1%, the LOQ was 50 ng/mL, and the LOD was 20 ng/mL. The detection technique was convenient, accurate, reproducible, and rapid. Therefore, this method could be used for deep liquid fermentation, providing a basis for EP to serve as a quality standard for the fermentation of Paecilomyces cicadae.

Isolation and characterization of ergosterol from Monascus anka for anti-lipid peroxidation properties.

Unbalanced lipid peroxidation damages the human body, and is associated with the formation of tumors, infections, inflammations, autoimmune diseases, and cardiovascular and cerebrovascular diseases. However, food and drugs that contain anti-lipid peroxidation active substances, can help to protect against these negative health impacts. We observed lipid peroxidation inhibition in the metabolites of fermented Monascus anka, in media with Dendrobium nobile Lindl. The anti-lipid peroxidation ability of the extracts was strongest in ethyl acetate, so this was selected for further purifications. A crystal with strong antioxidant properties was obtained by column chromatography. Based on its spectroscopic analysis by Electron Bombardment Ion Source and Mass Spectrometry (EI-MS), 1H-Nuclear Magnetic Resonance (1H-NMR), and 13C-Nuclear Magnetic Resonance (13C-NMR), the isolated crystal was identified as ergosterol. The inhibition rates of the lipid peroxide due to the ergosterol were 57.42%, at 2µg/mL in vitro. Simultaneously, the survival rates of the damaged cells treated with 0.3mmol/L H2O2 were significantly improved with the ergosterol, up to 43.88% (200µg/mL) and 46.64% (400µg/mL), compared to 36.47% for the injured cells. The survival rate of the cells was 78.32% (400µg/mL), with ergosterol as a prevention. Cell injury can increase the level of intracellular ROS, but its levels in the damaged cells were reduced after the ergosterol treatments, and the reduction increased with the increasing concentrations. A 400µg/mL concentration resulted in the lowest fluorescence intensity; 33421.11 AU below the normal level. Ergosterol significantly reduced the ROS levels, to reduce the cell damage. Ergosterol from Monascus anka was thus found to have strong anti-lipid peroxidation and antioxidant capabilities, and the ability to protect and repair damaged cells. It may consequently serve as a potential natural antioxidant and will play an important role in human anti-lipid peroxide.

Do the Main Components of the Sclerotia of Umbrella Polypore Mushroom, Polyporus umbellatus (Agaricomycetes), Correlate with Armillaria Associates?

Polyporus umbellatus is a traditional Chinese medicinal mushroom. The growth of P. umbellatus sclerotia requires the rhizomorphs of Armillaria spp. to supply nutrition. Whether the main components (MC) of sclerotia of P. umbellatus are related to the phylogeny of Armillaria associates or other environmental factors is largely unknown. In this study, we collected 17 sclerotia and soil samples from northeast to southwest China. In total, 17 Armillaria associates were isolated, and sclerotial MC contents and soil characteristics (total N, P, K, and organic matter) were determined. The analysis revealed that the MC content of P. umbellatus did not resemble a Brownian motion process in phylogeny of Armillaria associates, but were significantly influenced by the total N content of the soil. These results provide clear evidence that sclerotia of P. umbellatus associating with phylogenetic related Armillaria associates possess differing MC content. The mechanisms of nutrient exchange in P. umbellatus-Armillaria associations now require further elucidation.

Dimethyl disulfide exerts antifungal activity against Sclerotinia minor by damaging its membrane and induces systemic resistance in host plants.

Microbial volatile compounds (MVCs) significantly influence the growth of plants and phytopathogens. However, the practical application of MVCs at the field level is limited by the fact that the concentrations at which these compounds antagonize the pathogens are often toxic for the plants. In this study, we investigated the effect of dimethyl disulfide (DMDS), one of the MVCs produced by microorganisms, on the fitness of tomato plants and its fungicidal potential against a fungal phytopathogen, Sclerotinia minor. DMDS showed strong fungicidal and plant growth promoting activities with regard to the inhibition of mycelial growth, sclerotia formation, and germination, and reduction of disease symptoms in tomato plants infected with S. minor. DMDS exposure significantly upregulated the expression of genes related to growth and defense against the pathogen in tomato. Especially, the overexpression of PR1 and PR5 suggested the involvement of the salicylic acid pathway in the induction of systemic resistance. Several morphological and ultrastructural changes were observed in the cell membrane of S. minor and the expression of ergosterol biosynthesis gene was significantly downregulated, suggesting that DMDS damaged the membrane, thereby affecting the growth and pathogenicity of the fungus. In conclusion, the tripartite interaction studies among pathogenic fungus, DMDS, and tomato revealed that DMDS played roles in antagonizing pathogen as well as improving the growth and disease resistance of tomato. Our findings provide new insights into the potential of volatile DMDS as an effective tool against sclerotial rot disease.

Screening of bioactive compounds in truffles and evaluation of pressurized liquid extractions (PLE) to obtain fractions with biological activities.

Truffles, besides the appreciated aromatic compounds, contain other molecules with interesting bioactive properties. A screening of fungal sterols and ß-glucans within different truffle species and locations was carried out. These compounds were extracted with pressurized liquids (PLE) generating enriched fractions. Extraction efficiency was studied with a full-factorial experimental design (Response surface methodology, RSM), using water and ethanol as extraction solvents. Polysaccharides from truffle powder (TP) and the optimal PLE extract (EP) obtained were precipitated and analysed by NMR and GC-MS. THP-1 cell cultures were utilized to test immunomodulatory properties. With the optimal PLE conditions (16.7 MPa, 180 °C, 30 min) 64 and 22.5% yields were obtained respect, with water and ethanol, generating fractions containing respect, 9.1% ß-glucan and 4.5% ergosterol. NMR analyses detected (1 â†’ 3)-ß-glucan structures in truffle. The EP induced a reduction of 40% IL-1ß and 60% IL-6 pro-inflammatory cytokines secretion suggesting potential immunomodulatory activity.

Activities of Nerol, a natural plant active ingredient, against Candida albicans in vitro and in vivo.

Candida albicans invasion is one of the most serious fungal infections in clinical history. In recent years, because of the widespread use of immunosuppressive drugs, chemotherapy drugs, glucocorticoids, and broad-spectrum antibiotics, serious drug resistance has been reported; therefore, a new type of antifungal drug needs to be developed. In this study, we found that Nerol (NEL) had strong antimicrobial activity and 0.77 µL/mL NEL was the minimum inhibitory concentration (MIC) effective against C. albicans. We determined the change of the growth curve of NEL for C. albicans, to identify the trend of NEL activity against C. albicans. Through the determination of the ergosterol content and glucose-induced extracellular fluid acidification of NEL on C. albicans, we found that NEL inhibits the growth of C. albicans by destroying cell membranes. This finding was also supported by the expression of SAP (secreted aspartyl proteinase) involved in cell membrane synthesis. Finally, demonstrations of phenotype investigation, colony-forming unit (CFU) counts, and PAS (periodic acid-Schiff) staining were conducted to prove that NEL had the ability to treated mouse oral C. albicans infection and vaginal C. albicans infection. This research may help us to investigate new antimicrobial agents for treating C. albicans infections. KEY POINTS: • NEL can inhibit the growth of C. albicans. • NEL destroys the cell membrane formation and permeability of C. albicans. • NEL can treat vulvovaginal candidiasis and oropharyngeal candidiasis in mice. • NEL could be used as a possible antifungal agent.

Systematic Review and Study of S Curves for Biomass Quantification in Solid-state Fermentation (SSF) and Digital Image Processing (DIP) Applied to Biomass Measurement in Food Processes.

BACKGROUND: There are several methods for the quantification of biomass in SSF, such as glucosamine measurement, ergosterol content, protein concentration, change in dry weight or evolution of CO2 production. However, all have drawbacks when obtaining accurate data on the progress of the SSF due to the dispersion in cell growth on the solid substrate, and the difficulty encountered in separating the biomass. Studying the disadvantages associated with the process of biomass quantification in SSF, the monitoring of the growth of biomass by a technique known as digital image processing (DIP), consists of obtaining information on the production of different compounds during fermentation, using colorimetric methods based on the pixels that are obtained from photographs. OBJECTIVE: The purpose of this study was to know about the state of the technology and the advantages of DIP. METHODS: The methodology employed four phases; the first describes the search equations for the SSF and the DIP. A search for patents related to SSF and DIP carried out in the Free Patents Online and Patent inspiration databases. Then there is the selection of the most relevant articles in each of the technologies. As a third step, modifications for obtaining the best adjustments were also carried out. Finally, the analysis of the results was done and the inflection years were determined by means of six mathematical models widely studied. RESULTS: For these models, the inflection years were 2018 and 2019 for both the SSF and the DIP. Additionally, the main methods for the measurement of biomass in SSF were found, and are also indicated in the review, as DIP measurement processes have already been carried out using the same technology. CONCLUSION: In addition, the DIP has shown satisfactory results and could be an interesting alternative for biomass measurement in SSF, due to its ease and versatility.

Detection of ergosterol using liquid chromatography/electrospray ionization mass spectrometry: Investigation of unusual in-source reactions.

RATIONALE: In the field of natural products, de-replication of complex mixtures has become a usual practice to annotate known compounds and avoid their re-isolation. For this purpose, many groups rely on liquid chromatography coupled to high-resolution mass spectrometry (HPLC/MS) to deduce molecular formulae of compounds allowing comparison with public or in-house databases. Electrospray ionization (ESI) is usually considered as the method of choice for investigating a large panel of compounds but, in some cases, it may lead to unusual results as described in this article for ergosterol. METHODS: Ergosterol and other fungal sterols in methanolic solution were analysed using various chromatographic gradients with HPLC/MS using both ion trap time-of-flight MS and Orbitrap MS instruments fitted with an ESI source. Further flow injection analyses were performed to investigate the influence of the solvent composition. MS/MS fragmentation data were acquired to annotate the various ions observed. RESULTS: Contrary to other fungal sterols, ergosterol was found to be highly sensitive to oxidation during ESI. Putative structures were proposed based on MS/MS studies and known oxidation mechanisms of ergosterol by reactive oxygen species that could be formed in the ESI process. The proportion of acetonitrile in the eluent was found to influence this in-source oxidation, with an increased proportion of oxidized sodium adducts with higher proportions of acetonitrile. CONCLUSIONS: While ergosterol is a major sterol found in fungi, this study investigates its ionization by electrospray for the first time. The results reported here will help further detection and annotation of this compound in fungal extracts after HPLC/ESI-MS analyses.

Nitrogen concentration affects amphotericin B and fluconazole tolerance of pathogenic cryptococci.

Environmental stress often causes phenotypic changes among pathogenic cryptococci, such as altered antifungal susceptibility, changes in capsule and melanin formation, as well as altered levels of the membrane sterol and antifungal target, ergosterol. We therefore hypothesised that nitrogen limitation, a prevalent environmental stress in the natural habitat of these yeasts, might affect virulence and antifungal susceptibility. We tested the effect of different nitrogen concentrations on capsule, melanin and ergosterol biosynthesis, as well as amphotericin B (AmB) and fluconazole (FLU) susceptibility. This was achieved by culturing cryptococcal strains representing Cryptococcus neoformans and Cryptococcus gattii in media with high (0.53 g/l), control (0.42 g/l) and low (0.21 g/l) NH4Cl concentrations. India ink staining was used to determine capsule thickness microscopically, while melanin and ergosterol content were determined spectrophotometrically. We found that lower nitrogen concentrations enhanced both ergosterol and capsule biosynthesis, while a variable effect was observed on melanisation. Evaluation of drug tolerance using time-kill methodology, as well as tests for FLU heteroresistance, revealed that the low nitrogen cultures had the highest survival percentages in the presence of both AmB and FLU, and showed the highest frequency of FLU heteroresistance, suggesting that nitrogen concentration may indeed influence drug tolerance.

A multi-year survey of mycotoxins and ergosterol in Canadian oats.

Canadian oat harvest samples, deliveries to processors, and train shipments from primary elevators were collected from mid-2014 through mid-2017 and analyzed for 26 mycotoxins and the fungal biomarker ergosterol. Of the 26 mycotoxins, 7 were not detected in any sample. The most frequently measured mycotoxins were beauvericin (in over 95% of samples analyzed), followed by tentoxin, culmorin, alternariol, alternariol methyl ether, and deoxynivalenol. Median concentrations of the Fusarium-produced mycotoxins ranged from 68 to 1142 µg/kg for deoxynivalenol, 39 to 188 µg/kg for HT-2 and T-2 toxins, 66 to 232 µg/kg for nivalenol, and less than 35 µg/kg for beauvericin. Median concentrations of the sum of Alternaria-produced mycotoxins were all less than 250 µg/kg. Concentrations of analytes varied among years, as well as among growing areas, for the harvest samples. Ergosterol, Fusarium, and Alternaria mycotoxin concentrations appeared to increase from the west toward the eastern Prairies and the province of Quebec; the differences were not statistically significant though. Ochratoxin A in deliveries and train shipments showed annual cyclic increases in the late summer. The results of the survey demonstrate the general compliance of Canadian oats with existing maximum levels for mycotoxins and indicate that in late summer and in years with increased Fusarium infection, there can be a need for monitoring of ochratoxin A and deoxynivalenol, respectively, to mitigate risks of noncompliant grain.

Terpinen-4-ol inhibits the growth of Sporothrix schenckii complex and exhibits synergism with antifungal agents.

Aim: This study investigated the effect of terpinen-4-ol against Sporothrix schenckii complex and its interactions with antifungals. Materials & methods: The antifungal activity of terpinen-4-ol was evaluated by broth microdilution. The potential effect on cellular ergosterol concentration was evaluated by spectrophotometry. The antibiofilm activity was evaluated by violet crystal staining and XTT reduction assay. The potential pharmacological interactions with antifungals were evaluated by the checkerboard assay. Results: terpinen-4-ol (T-OH) showed minimal inhibitory concentrations ranging from 4 to 32 mg/l decreasing cellular ergosterol content and presented a SMIC ranging from 64 to 1024 mg/l for Sporothrix spp. The combinations of T-OH with itraconazole or terbinafine were synergistic. Conclusion: T-OH has antifungal activity against Sporothrix spp. and acts synergistically with standard antifungals.