Anti-trichophyton Rubrum Potential of (R)-(+)- Citronellal

Anti-trichophyton Rubrum Potential of (R)-(+)- Citronellal | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anti-trichophyton Rubrum Potential of (R)-(+)- Citronellal Francisco Patricio Andrade, Janiere Pereira Sousa, Gustavo Fernandes Queiroga Moraes, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4344964/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Trichophyton rubrum is a keratolytic and keratinophilic fungus responsible for causing dermatophytoses. These pathogens have been associated with resistance phenomena, which encourages the need to search for new substances with anti- Trichophyton rubrum activity. The present study aimed to investigate the antifungal activity of (R)-(+)- Citronellal (RCIT) against clinical isolates of T. rubrum . The antifungal potential of RCIT was evaluated from the Minimum Inhibitory Concentration (MIC), Minimum Fungicide Concentration (MFC), Association study, and assays with cholesterol, ergosterol and sorbitol. RCIT MIC values ranged from 4 to 512 µg/mL, while their MFC ranged from 4 to 512 µg/mL. When associating RCIT with the drug fluconazole, pharmacological indifference and antagonism were evidenced. It was shown that the mechanism of action is related to fungal ergosterol and showed interactions with exogenous cholesterol. The results obtained in this research demonstrate that RCIT has the potential to become a product for the treatment of dermatophytosis. filamentous fungi terpenes monoterpene antifungals INTRODUCTION Dermatophytoses are superficial infections, popularly known as tinea (from the Latin tinea ), caused by filamentous, hyaline, septate, keratinolytic and keratinophilic fungi. These conditions can be caused by fungi belonging to the genera: Epidermophyton , Microsporum and Trichophyton (Andrade Júnior et al. 2020 ; Jartarkar et al. 2022 ) with T. rubrum being the most prevalent species in tropical countries such as Brazil (Andrade Júnior et al. 2020 ; Lana et al. 2016 ). Pharmacological treatment can be performed using antifungals: griseofulvin, terbinafine and azoles. The therapeutic choice is usually associated with the type of microorganism and the anatomical site affected (Andrade Júnior et al. 2020 ). Furthermore, in dermatophytes of the genus Trichophyton , more specifically, due to discontinuity of pharmacological treatment, irrational use of antifungals, use of immunosuppressants and the presence of acquired immunodeficiency syndrome (AIDS), cases of resistance of these microorganisms have been observed, above all, against the azoles that represent the main antifungal agents used in the treatment of these pathogens (Lana et al. 2016 ; Appelt et al. 2021 ). Thus, the development of studies that seek new molecules with antifungal potential, against strains of the genus Trichophyton , are necessary, such as terpenes. Terpenes can be conceptualized as substances produced by plants that chemically appear as unsaturated hydrocarbons, formed by isoprene units (C 5 H 8 ), and may contain chemical functions such as acids, ketones, alcohols, aldehydes, ethers, phenols or terpene epoxides (Felipe and Bicas 2017 ; Salha et al. 2019 ). Among the terpenic compounds, it is possible to highlight citronellal. This natural product is a monoterpene found in the essential oil of Cymbopogon nardus (Felipe and Bicas 2017 ; Sharma et al. 2017). Numerous studies have shown the antifungal activity of citronellal and its respective optical isomers against fungi of the genera Aspergillus , Penicillium and Candida (Aguiar et al. 2014 ; Wu et al. 2016 ; Medeiros et al. 2017 ; Oliveira et al. 2017 ), however, studies involving ( R )-(+)-Citronellal against Trichophyton rubrum species were not found in the literature, which justifies the need for this research. MATERIALS AND METHODS Workplace The experiments were carried out at the Research Laboratory of Antibacterial and Antifungal Activity of Natural and Synthetic Bioactive Products, Department of Pharmaceutical Sciences (DCF), Health Sciences Center (CCS) of the Federal University of Paraíba (UFPB). Obtaining and preparing (R)-(+)- Citronellal and standard antifungals To carry out the in vitro studies, the terpene ( R )-(+)- Citronellal (RCIT) and the drugs fluconazole (FLU) and amphotericin B (AB), commercially purchased from Sigma Aldrich®, São Paulo, Brazil, were used. All substances were solubilized with 2% Tween 80® and dimethylsulfoxide (DMSO), in a percentage of up to 5%, completing the final volume of 5 mL, with sterilized distilled water to obtain an emulsion of the products at the concentration initial 1024 µg/mL (Cleeland and Squires 1991 ; Pereira et al. 2014 ). Culture mediums Sabouraud Dextrose Agar (ASD) media were used for the maintenance of fungal strains, and RPMI 1640 with L-glutamine and without bicarbonate, for antifungal activity assays, commercially purchased from Difco Laboratories Ltd (USA, France) and INLAB, São Paulo, Brazil, respectively. Both media were prepared according to the manufacturer's instructions. T. rubrum strains The T. rubrum strains were acquired from the isolation and identification of biological material from lesions on the skin, scalp and nails of patients with a hypothesis of diagnosis treated at the Clinical Pathology Laboratory - HEMATO, located in the city of João Pessoa, Paraíba, Brazil. T. rubrum species (LM-3, 4, 15, 28, 29, 32, 34, 63, 70, 104, 150, 234, 255, 314 and ATCC − 28188) were used. The microorganisms were kept stored in ASD medium, under refrigeration at 4ºC, and registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the number: ADF1D10. Inoculum To prepare the inoculum, the microorganisms were subcultured in sterile tubes containing ASD for 5 to 7 days at 35º C. After this incubation period, an aliquot of the medium containing the fungus was collected and then suspended in 0.9% sterile saline solution. The resulting suspension was stirred by vortexing and, soon after, the inoculum density was standardized according to the 0.5 McFarland scale to obtain 10 6 CFU/mL (Cleeland and Squires 1991 ). Antifungal activity assays Minimum Inhibitory Concentration (MIC) For the evaluation of the antifungal activity of ( R )-(+)- Citronellal and the antifungal controls, the method of microdilution in broth was used, made from sterile plates, containing 96 wells with a “U” bottom, suitable for microdilution. In each of the wells was added 100 µL of double concentrated RPMI broth. Subsequently, 100 µL of the investigated substance was dispensed into the wells of the first line of the microdilution plate. After this process, the monoterpene was serially diluted in a 1:2 ratio. Then, 10 µL of inoculum, suspension of microorganisms, was added to each of the wells. Finally, the viability of the microorganisms (culture medium with the fungal strains) and sterility (only the culture medium) were checked in the last two lines of the microplate. This test was performed in triplicate (Cleeland and Squires 1991 ; Ellof 1998 ; Klepser 1998). Then, the plates were sealed and incubated at room temperature (28ºC ± 2) for 5 to 7 days. After the delimited time, the experiments were read for data recording. The MIC was considered the lowest concentration of the antifungal agent, responsible for preventing the visible growth of the microorganism, while the MIC 50 was defined the concentration responsible for inhibiting the visible growth of 50% of the microorganisms, taking into account the total number of tested strains. The substance was classified as having strong activity when the MIC was up to 600 µg/mL; moderate activity when MIC ranged from 600 to 1500 µg/mL; above 1500 µg/mL was called weakly active or inactive (Sartoratto et al. 2004 ). Minimum Fungicide Concentration (MFC) The MFC was determined from the MIC. After obtaining the MIC, 10 µL were removed from each of the wells in which there was no visible growth and, soon after, they were added to a new microplate containing 100 µL of double concentrated RPMI medium in each of its wells. The plates were then incubated at 28°C for 24 hours (Ncube et al. 2008 ). Finally, MFC was considered as the lowest concentration necessary to prevent fungus growth in the subculture. The mode of action was classified employing the MFC/MIC ratio, in which it was considered fungicidal when MFC/MIC < 4 and fungistatic when MFC/MIC ≥ 4 (Siddiqui et al. 2013 ). Association test using the Checkerboard method The association assay was performed using the checkerboard technique, using microdilution plates with 96 wells and a conical bottom (INLAB, São Paulo-SP, Brazil). Initially, based on the MIC values obtained, solutions of drug A (( R )-(+)- Citronelall) and drug B (fluconazole) were prepared at different concentrations (8XMIC, 4XMIC; 2XMIC; MIC; MIC/2; MIC/4; MIC/8). Then, 100 µL of RPMI 1640 with L-glutamine and without bicarbonate was added to all plate wells. Soon after, 50 µL of drug A was distributed horizontally in different concentrations, while for drug B, 50 µL was distributed in different concentrations vertically. Thus, all different concentrations were tested and combined between drugs (White et al. 1996 ; D’arrigo et al. 2010 ). Finally, 20 µl of the inoculum was added. The measurement of the antimicrobial potential was obtained through the association between drugs and was determined through Fractional Inhibitory Concentration (FIC): $${FIC}_{A }= \frac{MIC of drug A in association}{MIC of drug A individually}$$ $${FIC}_{B }= \frac{MIC of drug B in association}{MIC of drug B individually}$$ Subsequently, the Fractional Inhibitory Concentration Index was calculated from the following equation: $$FICI= {FIC}_{A}+ {FIC}_{B}$$ Thus, a synergistic association was considered when the FICI was ≤ 0.5; indifferent association when FICI > 0.5 or ≤ 4.0 and antagonistic association when FICI > 4.0 (White et al. 1996 ; Tobudic et al. 2010 ). Cholesterol and Ergosterol Assay To verify possible interactions with fungal ergosterol and exogenous cholesterol, the MIC for citronellal and Amphotericin B was determined, against strains of Trichophyton that were pre-selected, in the presence and absence of ergosterol and cholesterol, through the microdilution technique. For this, ergosterol was added and solubilized at a concentration of 400 µg/mL in RPMI medium, while cholesterol was solubilized at the same concentration. The microdilution plates were previously incubated at 28ºC ± 2ºC and the reading was performed after 7 days of incubation. Then, the MIC was compared in the presence and absence of cholesterol and ergosterol (Teixeira et al. 2018 ; Escalante et al. 2008 ). Sorbitol Assay The MIC of ( R )-(+)- Citronellal was determined for pre-selected strains of Trichophyton , in the presence and absence of sorbitol, using the microdilution technique, which was previously described. For this purpose, sorbitol (MW = 182.17) was added to the RPMI medium at 0.8 M. The microdilution plates were previously incubated at 28ºC ± 2ºC and the reading was performed after 7 days of incubation. Soon after, the MIC was compared in the presence and absence of sorbitol (Frost et al. 1995 ). RESULTS AND DISCUSSION In Table 1 it is possible to observe the Minimum Inhibitory Concentration of ( R )-(+)- Citronellal against strains of T. rubrum . Table 1 Mean MIC (µg/mL) of ( R )-(+)-Citronellal and fluconazole against T. rubrum strains. Microorganisms ( R )-(+)- Citronellal Fluconazole Trichophyton rubrum MIC (µg/mL) MIC (µg/mL) ATCC − 28188 32 64 LM-03 4 256 LM-04 4 256 LM-15 4 16 LM-28 256 8 LM-29 4 512 LM-32 256 512 LM-34 4 512 LM-63 4 16 LM-70 512 64 LM-104 256 16 LM-150 128 8 LM-234 256 8 LM-255 512 256 LM-314 128 256 Source: Research data, 2024. MIC, through microdilution, is an interesting technique, since it saves reagents, time and resources, in addition to allowing relatively fast and sensitive results with a large number of replicates (Lira et al. 2020 ). RCIT MIC values ranged from 4 to 512 µg/mL, while the MIC 50 was 128 µg/mL. Thus, RCIT showed strong activity against T. rubrum species (Sartoratto et al. 2004 ). In other studies involving RCIT, there was also evidence of strong activity of this monoterpene against C. albicans yeasts with MICs ranging from 16 to 32 µg/mL (Medeiros et al. 2017 ). Fluconazole was the drug used as a control due to its wide usability in clinical practice against fungi of the genus Trichophyton . This active principle is fungistatic and acts by inhibiting the enzyme sterol 14α-demethylase. Thus, FLU presented a MIC that ranged from 8 to 512 µg/mL, with 64 µg/mL as the necessary concentration to inhibit 50% of the investigated microorganisms. In this context, high MIC values may be associated with antifungal resistance (Pérez-Cantero et al. 2020 ). In Table 2 it is possible to observe the MFC of ( R )-(+)- Citronellal, against T. rubrum strains. Table 2 MFC means (µg/mL) of (R)-(+)-citronellal against T. rubrum strains. Microorganisms Monoterpene ( R )-(+)-Citronellal Trichophyton rubrum MFC (µg/mL) MFC/MIC (µg/mL) Activity ATCC − 28188 32 1 Fungicide LM-03 4 1 Fungicide LM-04 4 1 Fungicide LM-15 4 1 Fungicide LM-28 256 1 Fungicide LM-29 4 1 Fungicide LM-32 512 2 Fungicide LM-34 4 1 Fungicide LM-63 4 1 Fungicide LM-70 512 1 Fungicide LM-104 1024 4 Fungicide LM-150 512 4 Fungicide LM-234 256 1 Fungicide LM-255 512 1 Fungicide LM-314 128 1 Fungicide Source: Research data, 2024. The CFM allows understanding at which concentration a given substance is fungicidal, which can result in concentrations greater than the MIC (Andrade Júnior et al. 2023 ). Thus, it is interesting to note that the CFM for RCIT ranged from 4 to 1024 µg/mL. Similar results were evidenced in the literature, since RCIT against C. albicans strains presented CFM was 32 µg/mL (Medeiros et al. 2017 ). Furthermore, when comparing the MFC/MIC ratio, it is considered that ratios ≤ 4 demonstrate fungicidal characteristics, while values > 4 are associated with fungistatic potential (Siddiqui et al. 2013 ). In this respect, RCIT was fungicidal. In this context, it is evident that the results were interesting since substances that have a fungistatic character are more favorable to the development of resistance when compared to fungicidal substances. This occurs because, by inhibiting only growth, it is not uncommon for mutations to occur and the subsequent emergence of resistance phenomena (Kumar et al. 2018 ). Table 3 shows the association between ( R )-(+)-citronellal and the drug fluconazole. Table 3 Fractional Inhibitory Concentration and Fractional Inhibitory Concentration Index of ( R )-(+)-citronellal associated with fluconazole against T. rubrum strains. T. rubrum *FIC A FIC B **IFIC Type of pharmacological association ( R )-(+)-citronellal Fluconazole ( R )-(+)-citronellal + Fluconazole ATCC − 28188 2.0 0.5 2.5 Indifference LM – 150 4.0 4.0 8.0 Antagonism * CIF - Fractional Inhibitory Concentration; ** ICIF - Fractional Inhibitory Concentration Index. Source: Research data, 2024. The association between drugs is one of the best options against fungal resistance, in addition to enhancing therapeutic effects and reducing adverse effects (Andrade Júnior et al. 2019 ). In this sense, it was observed that the association between fluconazole and ( R )-(+)-citronellal showed pharmacological indifference and antagonism. Differing from these results, in a study involving strains of C. albicans , a synergistic effect was shown when fluconazole was associated with the RCIT enantiomer (Medeiros et al. 2017 ). These differences between results may be associated with existing biological distinctions between different genera, species and fungal strains. Pharmacological indifference is related to the fact that one drug does not interact with the other's mechanism of action. In general, this finding is interesting, since it is possible that these drugs can be used in association, but in cases of pharmacological antagonism, one drug inhibits/disrupts the effectiveness of one another, which makes their use together impossible. Thus, the possible mechanism of action of RCIT against fungi was investigated through the ergosterol and sorbitol assays. Thus, below, it is possible to observe the MIC of RCIT in the presence and absence of ergosterol (Table 4 ). Table 4 Mean MIC (µg/mL) of ( R )-(+)-citronellal and amphotericin B in the presence and absence of ergosterol against T. rubrum strains. Trichophyton rubrum Monoterpene Control ( R )-(+)-Citronellal Amphotericin B Without ergosterol With ergosterol Without ergosterol With ergosterol ATCC − 28188 32 256 0.5 1024 LM – 150 128 1024 8 1024 Source: Research data, 2024. Ergosterol is the most abundant sterol present in fungal cells. This compound is responsible for the permeability and fluidity of the plasma membrane (Rodrigues, 2018 ). Currently, on the market, some molecules act directly or indirectly on fungal ergosterol. The azoles, such as fluconazole, are capable of inhibiting the synthesis of ergosterol by inhibiting the enzyme 14-α-sterol demethylase, while the polyenes, such as amphotericin B, are capable of chemically interacting with ergosterol causing the formation of membrane pores and the extravasation of intracellular content, which allows the death of microorganisms (Andrade Júnior et al. 2020 ; Appelt et al. 2021 ). In this context, it was observed that RCIT showed an increase in MIC in the presence of ergosterol, which indicates that this molecule interacts with this sterol, which is a very interesting finding, since the main drugs used in the treatment against fungi are capable of to act on this membrane lipid. However, more studies need to be carried out to understand, in more detail, the mechanism of action. Table 5 shows the MIC results of RCIT in the presence and absence of cholesterol. Table 5 Mean MIC (µg/mL) of ( R )-(+)-citronellal and amphotericin B in the presence and absence of cholesterol against T. rubrum strains. Trichophyton rubrum Monoterpene Control ( R )-(+)-citronellal Amphotericin B Without cholesterol With cholesterol Without cholesterol With cholesterol ATCC − 28188 32 64 0.5 512 LM – 150 128 512 8 512 Source: Research data, 2024. The fact that RCIT interacted with ergosterol fostered the hypothesis that this monoterpene is also capable of interacting with cholesterol, one of the main lipids present in the plasmatic membranes of eukaryotic cells (Nett and Andes 2016 ). Thus, based on the experiments carried out, it was observed that the MIC increased in the presence of cholesterol, indicating that RCIT, as well as the amphotericin B control, could bind to this lipid. This finding is worrying, because even though amphotericin B has a lower affinity for cholesterol than for fungal ergosterol, this drug is capable of causing several severe adverse effects, such as nephrotoxicity and hematotoxicity. In this sense, it is possible to predict that RCIT is possibly capable of causing damage to mammalian cells like amphotericin B, which is a terrible finding. However, pharmaceutical forms such as liposomes can considerably reduce toxicity, as they allow a reduction in the dose and adverse effects (Chávez-Fumagalli et al. 2015 ). Another structure widely studied by researchers, to elucidate possible mechanisms of action of antifungal agents, is the cell wall. Thus, in Table 6 it is possible to observe the MIC of RCIT in the presence and absence of sorbitol. Table 6 Mean MIC (µg/mL) of ( R )-(+)-citronellal in the presence and absence of sorbitol against T. rubrum strains. Trichophyton rubrum Monoterpene MIC (R)-(+)-citronellal Without sorbitol With sorbitol ATCC − 28188 32 32 LM – 150 128 128 Source: Research data, 2024. Currently, the search for drugs that present selective toxicity is essential. In this context, a molecule that acts on a structure that is not present in a human eukaryotic cell, such as the cell wall, is a sensational finding, since it will allow pharmacotherapy with fewer adverse effects due to its greater specificity. Nowadays, echinocandins are the class of drugs specifically used to act at the fungal cell wall level. Mechanistically, the active principles act as non-competitive inhibitors of the 1,3-β-glycan synthase enzyme, preventing the polymerization of glucose-uridine-diphosphate into 1,3-β-glycan, an important component of the wall, which generates osmotic instability and consequent fungal cell death (Nett and Andes 2016 ). Sorbitol is an osmotic protector. Therefore, it allows the stabilization of fungal protoplasts. If RCIT acts on cell wall components, an increase in MIC is observed in the presence of sorbitol (Frost et al. 1995 ). However, no variations were observed between the MIC values in the absence and presence of the osmotic protector, indicating that RCIT does not act on mechanisms related to the cell wall. CONCLUSIONS Thus, it is evident that ( R )-(+)-citronellal presents itself as a promising product against strains of T. rubrum , since it has demonstrated strong antifungal activity, fungicidal character and its mechanism of action seems to be associated with the cell membrane. However, the interaction of this molecule with exogenous cholesterol predicts possible toxicity to animal cells and encourages the need for further studies regarding the toxicity of this compound. Declarations Acknowledgements The authors acknowledge CAPES for granting the scholarship to conduct this research. Conflicts of interest The authors declare that there are no conflicts of interest. Author contribution F.P. Andrade Júnior, J. P. Sousa, H. D. S. Souza, G. F. Q. Moraes and J. M. Barbosa: responsible for carrying out the experiments and writing the manuscript. E. O. Lima: idealization and orientation of the project. References Aguiar RWS, Ootani MA, Ascencio SD, Ferreira TPS, Santos MM, Santos GR (2014). 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Agents Chemother. 40: 1914-1918. doi: 10.1128/aac.40.8.1914. Wu Y, Yang QO, Tao N (2016). Plasma membrane damage contributes to antifungal activity of citronellal against Penicillium digitatum. J. Food Science Technol. 53: 3853-3858. doi: 10.1007/s13197-016-2358-x. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 30 Apr, 2024 Submission checks completed at journal 30 Apr, 2024 First submitted to journal 29 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4344964","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":297435261,"identity":"cab8627d-038e-447c-882f-a28223de57f2","order_by":0,"name":"Francisco Patricio Andrade","email":"data:image/png;base64,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","orcid":"","institution":"Federal University of Paraíba","correspondingAuthor":true,"prefix":"","firstName":"Francisco","middleName":"Patricio","lastName":"Andrade","suffix":""},{"id":297435262,"identity":"e0029462-d370-4945-9d22-7336eae9bcca","order_by":1,"name":"Janiere Pereira Sousa","email":"","orcid":"","institution":"Federal University of Paraíba","correspondingAuthor":false,"prefix":"","firstName":"Janiere","middleName":"Pereira","lastName":"Sousa","suffix":""},{"id":297435263,"identity":"714bd3ed-0cd7-4fba-9357-983faae95c47","order_by":2,"name":"Gustavo Fernandes Queiroga Moraes","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"Fernandes Queiroga","lastName":"Moraes","suffix":""},{"id":297435264,"identity":"681d683e-21eb-4efb-9159-ae341469dfcf","order_by":3,"name":"Edeltrudes Oliveira Lima","email":"","orcid":"","institution":"Federal University of Paraíba","correspondingAuthor":false,"prefix":"","firstName":"Edeltrudes","middleName":"Oliveira","lastName":"Lima","suffix":""}],"badges":[],"createdAt":"2024-04-29 20:25:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4344964/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4344964/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55924779,"identity":"025f2fd2-cb24-4e2d-aa57-515f4bdd41d7","added_by":"auto","created_at":"2024-05-06 11:06:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":613574,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4344964/v1/c7aa838b-feaa-4ac9-b430-427d361a7fc0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAnti-trichophyton Rubrum Potential of (R)-(+)- Citronellal\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDermatophytoses are superficial infections, popularly known as tinea (from the Latin \u003cem\u003etinea\u003c/em\u003e), caused by filamentous, hyaline, septate, keratinolytic and keratinophilic fungi. These conditions can be caused by fungi belonging to the genera: \u003cem\u003eEpidermophyton\u003c/em\u003e, \u003cem\u003eMicrosporum\u003c/em\u003e and \u003cem\u003eTrichophyton\u003c/em\u003e (Andrade J\u0026uacute;nior et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jartarkar et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) with \u003cem\u003eT. rubrum\u003c/em\u003e being the most prevalent species in tropical countries such as Brazil (Andrade J\u0026uacute;nior et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lana et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePharmacological treatment can be performed using antifungals: griseofulvin, terbinafine and azoles. The therapeutic choice is usually associated with the type of microorganism and the anatomical site affected (Andrade J\u0026uacute;nior et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, in dermatophytes of the genus \u003cem\u003eTrichophyton\u003c/em\u003e, more specifically, due to discontinuity of pharmacological treatment, irrational use of antifungals, use of immunosuppressants and the presence of acquired immunodeficiency syndrome (AIDS), cases of resistance of these microorganisms have been observed, above all, against the azoles that represent the main antifungal agents used in the treatment of these pathogens (Lana et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Appelt et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThus, the development of studies that seek new molecules with antifungal potential, against strains of the genus \u003cem\u003eTrichophyton\u003c/em\u003e, are necessary, such as terpenes. Terpenes can be conceptualized as substances produced by plants that chemically appear as unsaturated hydrocarbons, formed by isoprene units (C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003e), and may contain chemical functions such as acids, ketones, alcohols, aldehydes, ethers, phenols or terpene epoxides (Felipe and Bicas \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Salha et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the terpenic compounds, it is possible to highlight citronellal. This natural product is a monoterpene found in the essential oil of \u003cem\u003eCymbopogon nardus\u003c/em\u003e (Felipe and Bicas \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sharma et al. 2017).\u003c/p\u003e \u003cp\u003eNumerous studies have shown the antifungal activity of citronellal and its respective optical isomers against fungi of the genera \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e and \u003cem\u003eCandida\u003c/em\u003e (Aguiar et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Medeiros et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), however, studies involving (\u003cem\u003eR\u003c/em\u003e)-(+)-Citronellal against \u003cem\u003eTrichophyton rubrum\u003c/em\u003e species were not found in the literature, which justifies the need for this research.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eWorkplace\u003c/h2\u003e \u003cp\u003eThe experiments were carried out at the Research Laboratory of Antibacterial and Antifungal Activity of Natural and Synthetic Bioactive Products, Department of Pharmaceutical Sciences (DCF), Health Sciences Center (CCS) of the Federal University of Para\u0026iacute;ba (UFPB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eObtaining and preparing (R)-(+)- Citronellal and standard antifungals\u003c/h2\u003e \u003cp\u003eTo carry out the in vitro studies, the terpene (\u003cem\u003eR\u003c/em\u003e)-(+)- Citronellal (RCIT) and the drugs fluconazole (FLU) and amphotericin B (AB), commercially purchased from Sigma Aldrich\u0026reg;, S\u0026atilde;o Paulo, Brazil, were used.\u003c/p\u003e \u003cp\u003eAll substances were solubilized with 2% Tween 80\u0026reg; and dimethylsulfoxide (DMSO), in a percentage of up to 5%, completing the final volume of 5 mL, with sterilized distilled water to obtain an emulsion of the products at the concentration initial 1024 \u0026micro;g/mL (Cleeland and Squires \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCulture mediums\u003c/h2\u003e \u003cp\u003eSabouraud Dextrose Agar (ASD) media were used for the maintenance of fungal strains, and RPMI 1640 with L-glutamine and without bicarbonate, for antifungal activity assays, commercially purchased from Difco Laboratories Ltd (USA, France) and INLAB, S\u0026atilde;o Paulo, Brazil, respectively. Both media were prepared according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eT. rubrum\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eT. rubrum\u003c/em\u003e strains were acquired from the isolation and identification of biological material from lesions on the skin, scalp and nails of patients with a hypothesis of diagnosis treated at the Clinical Pathology Laboratory - HEMATO, located in the city of Jo\u0026atilde;o Pessoa, Para\u0026iacute;ba, Brazil. \u003cem\u003eT. rubrum\u003c/em\u003e species (LM-3, 4, 15, 28, 29, 32, 34, 63, 70, 104, 150, 234, 255, 314 and ATCC \u0026minus;\u0026thinsp;28188) were used.\u003c/p\u003e \u003cp\u003eThe microorganisms were kept stored in ASD medium, under refrigeration at 4\u0026ordm;C, and registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the number: ADF1D10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eInoculum\u003c/h2\u003e \u003cp\u003eTo prepare the inoculum, the microorganisms were subcultured in sterile tubes containing ASD for 5 to 7 days at 35\u0026ordm; C. After this incubation period, an aliquot of the medium containing the fungus was collected and then suspended in 0.9% sterile saline solution. The resulting suspension was stirred by vortexing and, soon after, the inoculum density was standardized according to the 0.5 McFarland scale to obtain 10\u003csup\u003e6\u003c/sup\u003e CFU/mL (Cleeland and Squires \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAntifungal activity assays\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eMinimum Inhibitory Concentration (MIC)\u003c/h2\u003e \u003cp\u003eFor the evaluation of the antifungal activity of (\u003cem\u003eR\u003c/em\u003e)-(+)- Citronellal and the antifungal controls, the method of microdilution in broth was used, made from sterile plates, containing 96 wells with a \u0026ldquo;U\u0026rdquo; bottom, suitable for microdilution.\u003c/p\u003e \u003cp\u003eIn each of the wells was added 100 \u0026micro;L of double concentrated RPMI broth. Subsequently, 100 \u0026micro;L of the investigated substance was dispensed into the wells of the first line of the microdilution plate. After this process, the monoterpene was serially diluted in a 1:2 ratio. Then, 10 \u0026micro;L of inoculum, suspension of microorganisms, was added to each of the wells. Finally, the viability of the microorganisms (culture medium with the fungal strains) and sterility (only the culture medium) were checked in the last two lines of the microplate. This test was performed in triplicate (Cleeland and Squires \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Ellof \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Klepser 1998).\u003c/p\u003e \u003cp\u003eThen, the plates were sealed and incubated at room temperature (28\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2) for 5 to 7 days. After the delimited time, the experiments were read for data recording. The MIC was considered the lowest concentration of the antifungal agent, responsible for preventing the visible growth of the microorganism, while the MIC\u003csub\u003e50\u003c/sub\u003e was defined the concentration responsible for inhibiting the visible growth of 50% of the microorganisms, taking into account the total number of tested strains.\u003c/p\u003e \u003cp\u003eThe substance was classified as having strong activity when the MIC was up to 600 \u0026micro;g/mL; moderate activity when MIC ranged from 600 to 1500 \u0026micro;g/mL; above 1500 \u0026micro;g/mL was called weakly active or inactive (Sartoratto et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMinimum Fungicide Concentration (MFC)\u003c/h2\u003e \u003cp\u003eThe MFC was determined from the MIC. After obtaining the MIC, 10 \u0026micro;L were removed from each of the wells in which there was no visible growth and, soon after, they were added to a new microplate containing 100 \u0026micro;L of double concentrated RPMI medium in each of its wells. The plates were then incubated at 28\u0026deg;C for 24 hours (Ncube et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Finally, MFC was considered as the lowest concentration necessary to prevent fungus growth in the subculture.\u003c/p\u003e \u003cp\u003eThe mode of action was classified employing the MFC/MIC ratio, in which it was considered fungicidal when MFC/MIC\u0026thinsp;\u0026lt;\u0026thinsp;4 and fungistatic when MFC/MIC\u0026thinsp;\u0026ge;\u0026thinsp;4 (Siddiqui et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAssociation test using the Checkerboard method\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe association assay was performed using the checkerboard technique, using microdilution plates with 96 wells and a conical bottom (INLAB, S\u0026atilde;o Paulo-SP, Brazil).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eInitially, based on the MIC values obtained, solutions of drug A ((\u003cem\u003eR\u003c/em\u003e)-(+)- Citronelall) and drug B (fluconazole) were prepared at different concentrations (8XMIC, 4XMIC; 2XMIC; MIC; MIC/2; MIC/4; MIC/8). Then, 100 \u0026micro;L of RPMI 1640 with L-glutamine and without bicarbonate was added to all plate wells. Soon after, 50 \u0026micro;L of drug A was distributed horizontally in different concentrations, while for drug B, 50 \u0026micro;L was distributed in different concentrations vertically. Thus, all different concentrations were tested and combined between drugs (White et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; D\u0026rsquo;arrigo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Finally, 20 \u0026micro;l of the inoculum was added.\u003c/p\u003e \u003cp\u003eThe measurement of the antimicrobial potential was obtained through the association between drugs and was determined through Fractional Inhibitory Concentration (FIC):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$${FIC}_{A }= \\frac{MIC of drug A in association}{MIC of drug A individually}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$${FIC}_{B }= \\frac{MIC of drug B in association}{MIC of drug B individually}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eSubsequently, the Fractional Inhibitory Concentration Index was calculated from the following equation:\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$FICI= {FIC}_{A}+ {FIC}_{B}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThus, a synergistic association was considered when the FICI was \u0026le;\u0026thinsp;0.5; indifferent association when FICI\u0026thinsp;\u0026gt;\u0026thinsp;0.5 or \u0026le;\u0026thinsp;4.0 and antagonistic association when FICI\u0026thinsp;\u0026gt;\u0026thinsp;4.0 (White et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Tobudic et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCholesterol and Ergosterol Assay\u003c/h2\u003e \u003cp\u003eTo verify possible interactions with fungal ergosterol and exogenous cholesterol, the MIC for citronellal and Amphotericin B was determined, against strains of \u003cem\u003eTrichophyton\u003c/em\u003e that were pre-selected, in the presence and absence of ergosterol and cholesterol, through the microdilution technique. For this, ergosterol was added and solubilized at a concentration of 400 \u0026micro;g/mL in RPMI medium, while cholesterol was solubilized at the same concentration. The microdilution plates were previously incubated at 28\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C and the reading was performed after 7 days of incubation. Then, the MIC was compared in the presence and absence of cholesterol and ergosterol (Teixeira et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Escalante et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSorbitol Assay\u003c/h2\u003e \u003cp\u003eThe MIC of (\u003cem\u003eR\u003c/em\u003e)-(+)- Citronellal was determined for pre-selected strains of \u003cem\u003eTrichophyton\u003c/em\u003e, in the presence and absence of sorbitol, using the microdilution technique, which was previously described. For this purpose, sorbitol (MW\u0026thinsp;=\u0026thinsp;182.17) was added to the RPMI medium at 0.8 M. The microdilution plates were previously incubated at 28\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C and the reading was performed after 7 days of incubation. Soon after, the MIC was compared in the presence and absence of sorbitol (Frost et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eIn Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e it is possible to observe the Minimum Inhibitory Concentration of (\u003cem\u003eR\u003c/em\u003e)-(+)- Citronellal against strains of \u003cem\u003eT. rubrum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean MIC (\u0026micro;g/mL) of (\u003cem\u003eR\u003c/em\u003e)-(+)-Citronellal and fluconazole against \u003cem\u003eT. rubrum\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicroorganisms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-(+)- Citronellal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluconazole\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrichophyton rubrum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMIC (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATCC \u0026minus;\u0026thinsp;28188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSource: Research data, 2024.\u003c/p\u003e \u003cp\u003eMIC, through microdilution, is an interesting technique, since it saves reagents, time and resources, in addition to allowing relatively fast and sensitive results with a large number of replicates (Lira et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRCIT MIC values ranged from 4 to 512 \u0026micro;g/mL, while the MIC\u003csub\u003e50\u003c/sub\u003e was 128 \u0026micro;g/mL. Thus, RCIT showed strong activity against \u003cem\u003eT. rubrum\u003c/em\u003e species (Sartoratto et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn other studies involving RCIT, there was also evidence of strong activity of this monoterpene against \u003cem\u003eC. albicans\u003c/em\u003e yeasts with MICs ranging from 16 to 32 \u0026micro;g/mL (Medeiros et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFluconazole was the drug used as a control due to its wide usability in clinical practice against fungi of the genus \u003cem\u003eTrichophyton\u003c/em\u003e. This active principle is fungistatic and acts by inhibiting the enzyme sterol 14α-demethylase.\u003c/p\u003e \u003cp\u003eThus, FLU presented a MIC that ranged from 8 to 512 \u0026micro;g/mL, with 64 \u0026micro;g/mL as the necessary concentration to inhibit 50% of the investigated microorganisms. In this context, high MIC values may be associated with antifungal resistance (P\u0026eacute;rez-Cantero et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e it is possible to observe the MFC of (\u003cem\u003eR\u003c/em\u003e)-(+)- Citronellal, against \u003cem\u003eT. rubrum\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMFC means (\u0026micro;g/mL) of (R)-(+)-citronellal against \u003cem\u003eT. rubrum\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicroorganisms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMonoterpene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-(+)-Citronellal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrichophyton rubrum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMFC (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMFC/MIC (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eActivity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATCC \u0026minus;\u0026thinsp;28188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM-314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSource: Research data, 2024.\u003c/p\u003e \u003cp\u003eThe CFM allows understanding at which concentration a given substance is fungicidal, which can result in concentrations greater than the MIC (Andrade J\u0026uacute;nior et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThus, it is interesting to note that the CFM for RCIT ranged from 4 to 1024 \u0026micro;g/mL. Similar results were evidenced in the literature, since RCIT against \u003cem\u003eC. albicans\u003c/em\u003e strains presented CFM was 32 \u0026micro;g/mL (Medeiros et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, when comparing the MFC/MIC ratio, it is considered that ratios\u0026thinsp;\u0026le;\u0026thinsp;4 demonstrate fungicidal characteristics, while values\u0026thinsp;\u0026gt;\u0026thinsp;4 are associated with fungistatic potential (Siddiqui et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this respect, RCIT was fungicidal.\u003c/p\u003e \u003cp\u003eIn this context, it is evident that the results were interesting since substances that have a fungistatic character are more favorable to the development of resistance when compared to fungicidal substances. This occurs because, by inhibiting only growth, it is not uncommon for mutations to occur and the subsequent emergence of resistance phenomena (Kumar et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the association between (\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal and the drug fluconazole.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFractional Inhibitory Concentration and Fractional Inhibitory Concentration Index of (\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal associated with fluconazole against \u003cem\u003eT. rubrum\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eT. rubrum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*FIC\u003csub\u003eA\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFIC\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e**IFIC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eType of pharmacological association\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluconazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal\u0026thinsp;+\u0026thinsp;Fluconazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATCC \u0026minus;\u0026thinsp;28188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIndifference\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM \u0026ndash; 150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAntagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* CIF - Fractional Inhibitory Concentration; ** ICIF - Fractional Inhibitory Concentration Index.\u003c/p\u003e \u003cp\u003eSource: Research data, 2024.\u003c/p\u003e \u003cp\u003eThe association between drugs is one of the best options against fungal resistance, in addition to enhancing therapeutic effects and reducing adverse effects (Andrade J\u0026uacute;nior et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this sense, it was observed that the association between fluconazole and (\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal showed pharmacological indifference and antagonism. Differing from these results, in a study involving strains of \u003cem\u003eC. albicans\u003c/em\u003e, a synergistic effect was shown when fluconazole was associated with the RCIT enantiomer (Medeiros et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These differences between results may be associated with existing biological distinctions between different genera, species and fungal strains.\u003c/p\u003e \u003cp\u003ePharmacological indifference is related to the fact that one drug does not interact with the other's mechanism of action. In general, this finding is interesting, since it is possible that these drugs can be used in association, but in cases of pharmacological antagonism, one drug inhibits/disrupts the effectiveness of one another, which makes their use together impossible. Thus, the possible mechanism of action of RCIT against fungi was investigated through the ergosterol and sorbitol assays.\u003c/p\u003e \u003cp\u003eThus, below, it is possible to observe the MIC of RCIT in the presence and absence of ergosterol (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean MIC (\u0026micro;g/mL) of (\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal and amphotericin B in the presence and absence of ergosterol against T. rubrum strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrichophyton rubrum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMonoterpene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003e(\u003c/b\u003e\u003cb\u003eR\u003c/b\u003e\u003cb\u003e)-(+)-Citronellal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003eAmphotericin B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithout ergosterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWith\u003c/p\u003e \u003cp\u003eergosterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWithout ergosterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWith\u003c/p\u003e \u003cp\u003eergosterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATCC \u0026minus;\u0026thinsp;28188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM \u0026ndash; 150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSource: Research data, 2024.\u003c/p\u003e \u003cp\u003eErgosterol is the most abundant sterol present in fungal cells. This compound is responsible for the permeability and fluidity of the plasma membrane (Rodrigues, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrently, on the market, some molecules act directly or indirectly on fungal ergosterol. The azoles, such as fluconazole, are capable of inhibiting the synthesis of ergosterol by inhibiting the enzyme 14-α-sterol demethylase, while the polyenes, such as amphotericin B, are capable of chemically interacting with ergosterol causing the formation of membrane pores and the extravasation of intracellular content, which allows the death of microorganisms (Andrade J\u0026uacute;nior et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Appelt et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this context, it was observed that RCIT showed an increase in MIC in the presence of ergosterol, which indicates that this molecule interacts with this sterol, which is a very interesting finding, since the main drugs used in the treatment against fungi are capable of to act on this membrane lipid. However, more studies need to be carried out to understand, in more detail, the mechanism of action.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the MIC results of RCIT in the presence and absence of cholesterol.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean MIC (\u0026micro;g/mL) of (\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal and amphotericin B in the presence and absence of cholesterol against \u003cem\u003eT. rubrum\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrichophyton rubrum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMonoterpene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003e(\u003c/b\u003e\u003cb\u003eR\u003c/b\u003e\u003cb\u003e)-(+)-citronellal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003eAmphotericin B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithout cholesterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWith\u003c/p\u003e \u003cp\u003echolesterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWithout cholesterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWith\u003c/p\u003e \u003cp\u003echolesterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATCC \u0026minus;\u0026thinsp;28188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM \u0026ndash; 150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSource: Research data, 2024.\u003c/p\u003e \u003cp\u003eThe fact that RCIT interacted with ergosterol fostered the hypothesis that this monoterpene is also capable of interacting with cholesterol, one of the main lipids present in the plasmatic membranes of eukaryotic cells (Nett and Andes \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThus, based on the experiments carried out, it was observed that the MIC increased in the presence of cholesterol, indicating that RCIT, as well as the amphotericin B control, could bind to this lipid.\u003c/p\u003e \u003cp\u003eThis finding is worrying, because even though amphotericin B has a lower affinity for cholesterol than for fungal ergosterol, this drug is capable of causing several severe adverse effects, such as nephrotoxicity and hematotoxicity. In this sense, it is possible to predict that RCIT is possibly capable of causing damage to mammalian cells like amphotericin B, which is a terrible finding. However, pharmaceutical forms such as liposomes can considerably reduce toxicity, as they allow a reduction in the dose and adverse effects (Ch\u0026aacute;vez-Fumagalli et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother structure widely studied by researchers, to elucidate possible mechanisms of action of antifungal agents, is the cell wall. Thus, in Table \u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e it is possible to observe the MIC of RCIT in the presence and absence of sorbitol.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean MIC (\u0026micro;g/mL) of (\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal in the presence and absence of sorbitol against \u003cem\u003eT. rubrum\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrichophyton rubrum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMonoterpene\u003c/p\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003e(R)-(+)-citronellal\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWithout sorbitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWith sorbitol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATCC \u0026minus;\u0026thinsp;28188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLM \u0026ndash; 150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSource: Research data, 2024.\u003c/p\u003e \u003cp\u003eCurrently, the search for drugs that present selective toxicity is essential. In this context, a molecule that acts on a structure that is not present in a human eukaryotic cell, such as the cell wall, is a sensational finding, since it will allow pharmacotherapy with fewer adverse effects due to its greater specificity.\u003c/p\u003e \u003cp\u003eNowadays, echinocandins are the class of drugs specifically used to act at the fungal cell wall level. Mechanistically, the active principles act as non-competitive inhibitors of the 1,3-β-glycan synthase enzyme, preventing the polymerization of glucose-uridine-diphosphate into 1,3-β-glycan, an important component of the wall, which generates osmotic instability and consequent fungal cell death (Nett and Andes \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSorbitol is an osmotic protector. Therefore, it allows the stabilization of fungal protoplasts. If RCIT acts on cell wall components, an increase in MIC is observed in the presence of sorbitol (Frost et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). However, no variations were observed between the MIC values in the absence and presence of the osmotic protector, indicating that RCIT does not act on mechanisms related to the cell wall.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThus, it is evident that (\u003cem\u003eR\u003c/em\u003e)-(+)-citronellal presents itself as a promising product against strains of \u003cem\u003eT. rubrum\u003c/em\u003e, since it has demonstrated strong antifungal activity, fungicidal character and its mechanism of action seems to be associated with the cell membrane. However, the interaction of this molecule with exogenous cholesterol predicts possible toxicity to animal cells and encourages the need for further studies regarding the toxicity of this compound.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge CAPES for granting the scholarship to conduct this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.P. Andrade J\u0026uacute;nior, J. P. Sousa, H. D. S. Souza, G. F. Q. Moraes and J. M. Barbosa: responsible for carrying out the experiments and writing the manuscript.\u003c/p\u003e\n\u003cp\u003eE. O. Lima: idealization and orientation of the project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAguiar RWS, Ootani MA, Ascencio SD, Ferreira TPS, Santos MM, Santos GR (2014). Fumigant Antifungal Activity of Corymbia citriodora and Cymbopogon nardus Essential Oils and Citronellal against Three Fungal Species. Sci. World J. 492138. doi: 10.1155/2014/492138.\u003c/li\u003e\n \u003cli\u003eAndrade J\u0026uacute;nior FP, Souza HDS, Cordeiro LV, Silva DF, Lima EO (2020). \u003cem\u003eMicrosporum\u003c/em\u003e spp \u003cem\u003eMicrosporum\u003c/em\u003e spp as a cause of dermatophytosis: a review. Res., Soc. Dev. 9: e133953194. doi: 10.33448/rsd-v9i5.3194.\u003c/li\u003e\n \u003cli\u003eAndrade J\u0026uacute;nior FP, Gouveia RG, Medeiros CIS, Teixeira BA, Farias BKS, Oliveira NR, Silva DF, Lima EO. (2023). Antifungal activity of citronellal against Trichophyton rubrum and its predictive mechanism of action by CYP51 inhibition through molecular docking. 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Synthesis: characterization and antimicrobial evaluation of novel halopyrazole derivatives. J. Saudi Chem. Soc. 17: 237-243. doi: 10.1016/j.jscs.2011.03.016.\u003c/li\u003e\n \u003cli\u003eTeixeira APC, N\u0026oacute;brega RO, Lima EO, Ara\u0026uacute;jo WO, Lima IO (2018). Antifungal activity study of the monoterpene thymol Against \u003cem\u003eCryptococcus neoformans\u003c/em\u003e. Nat. Prod. Res. 34: 1-4. doi: 10.1080/14786419.2018.1547296.\u003c/li\u003e\n \u003cli\u003eTobudic S, Kratzer C, Lassnigg A, Graninger W, Presterl E (2010). In vitro activity of antifungal combinations against \u003cem\u003eCandida albicans\u003c/em\u003e Biofilms. J. Antimicrob. Chemother. 65: 271-274. doi: 10.1093/jac/dkp429.\u003c/li\u003e\n \u003cli\u003eWhite RL, Burgess DS, Manduru M, Bosso JA (1996). Comparasion of hree different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob. Agents Chemother. 40: 1914-1918. doi: 10.1128/aac.40.8.1914.\u003c/li\u003e\n \u003cli\u003eWu Y, Yang QO, Tao N (2016). Plasma membrane damage contributes to antifungal activity of citronellal against Penicillium digitatum. J. Food Science Technol. 53: 3853-3858. doi: 10.1007/s13197-016-2358-x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"filamentous fungi, terpenes, monoterpene, antifungals","lastPublishedDoi":"10.21203/rs.3.rs-4344964/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4344964/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eTrichophyton rubrum\u003c/em\u003e is a keratolytic and keratinophilic fungus responsible for causing dermatophytoses. These pathogens have been associated with resistance phenomena, which encourages the need to search for new substances with anti-\u003cem\u003eTrichophyton rubrum\u003c/em\u003e activity. The present study aimed to investigate the antifungal activity of (R)-(+)- Citronellal (RCIT) against clinical isolates of \u003cem\u003eT. rubrum\u003c/em\u003e. The antifungal potential of RCIT was evaluated from the Minimum Inhibitory Concentration (MIC), Minimum Fungicide Concentration (MFC), Association study, and assays with cholesterol, ergosterol and sorbitol. RCIT MIC values ranged from 4 to 512 \u0026micro;g/mL, while their MFC ranged from 4 to 512 \u0026micro;g/mL. When associating RCIT with the drug fluconazole, pharmacological indifference and antagonism were evidenced. It was shown that the mechanism of action is related to fungal ergosterol and showed interactions with exogenous cholesterol. The results obtained in this research demonstrate that RCIT has the potential to become a product for the treatment of dermatophytosis.\u003c/p\u003e","manuscriptTitle":"Anti-trichophyton Rubrum Potential of (R)-(+)- Citronellal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-06 10:58:06","doi":"10.21203/rs.3.rs-4344964/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-05-01T02:12:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-30T08:31:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2024-04-29T20:23:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e3558a2a-6194-4141-ba2d-501469b1f624","owner":[],"postedDate":"May 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-06T10:58:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-06 10:58:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4344964","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4344964","identity":"rs-4344964","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}