{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T05:17:00Z","timestamp":1788844620837,"version":"build-2803163510"},"reference-count":34,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T00:00:00Z","timestamp":1755216000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Pharmacol."],"abstract":"Background<\/jats:title>The increasing prevalence of azole-resistant Candida albicans<\/jats:italic> (RCa) poses a critical therapeutic challenge, necessitating innovative antifungal approaches. Natural deep eutectic solvents (NADES), derived from natural metabolites such as terpenes, provide a promising and sustainable platform for delivering bioactive compounds with intrinsic pharmacological properties.<\/jats:p><\/jats:sec>Purpose<\/jats:title>This study evaluated a eutectic system composed of menthol and thymol (MT NADES, 1:1\u00a0M ratio) for its antifungal efficacy against a multidrug-resistant clinical C. albicans<\/jats:italic> strain.<\/jats:p><\/jats:sec>Materials and methods<\/jats:title>The antifungal activity of MT NADES was evaluated against a clinical C. albicans<\/jats:italic> strain resistant to azole antifungals (RCa). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined using the broth microdilution method, following the CLSI M27-A4 guidelines. Synergistic effects with fluconazole were assessed through checkerboard microdilution and disc diffusion assays, with the fractional inhibitory concentration index (FICI) calculated to quantify interactions. Intracellular reactive oxygen species (ROS) levels were quantified using DCFH-DA staining and fluorescence spectrophotometry. Efflux pump inhibition was investigated via Nile red accumulation assay, analyzed by flow cytometry, using tacrolimus (100\u00a0\u00b5M) as a positive control. All experiments were performed in triplicate.<\/jats:p><\/jats:sec>Results and discussion<\/jats:title>MT NADES demonstrated potent fungicidal activity against resistant C<\/jats:italic>. albicans<\/jats:italic> with an MIC of 180\u00a0\u03bcg\/mL and MFC of 360\u00a0\u03bcg\/mL (MFC\/MIC = 2), outperforming its components (menthol: 1000\u00a0\u03bcg\/mL; thymol: 200\u00a0\u03bcg\/mL). Synergistic interaction with fluconazole (MIC: 32\u00a0\u03bcg\/mL) was confirmed by checkerboard and disc diffusion assays (FICI: 0.2839). Mechanistic studies revealed increased intracellular ROS, supporting oxidative stress as a key antifungal mechanism. Additionally, MT NADES at half its MIC enhanced Nile red retention 10-fold over the efflux pump inhibitor tacrolimus (100\u00a0\u00b5M), indicating strong inhibition of multidrug resistance (MDR)-related transporters. These findings highlight MT NADES as a promising chemosensitizing agent with superior efficacy over its individual components.<\/jats:p><\/jats:sec>Conclusion<\/jats:title>These findings underscore the therapeutic potential of menthol\u2013thymol NADES as a multifunctional, plant-derived antifungal strategy capable of overcoming multidrug resistance mechanisms and potentiating azole efficacy in C. albicans<\/jats:italic>.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fphar.2025.1643472","type":"journal-article","created":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T05:28:49Z","timestamp":1755235729000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":9,"title":["Menthol\u2013thymol NADES as a fungicidal and chemosensitizing agent against multidrug-resistant Candida albicans: ROS induction, efflux pump inhibition, and synergy with fluconazole"],"prefix":"10.3389","volume":"16","author":[{"given":"Melisa Fabiana","family":"Negro","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pamela Soledad","family":"Bustos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lautaro","family":"Bellezze","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda Gabriela","family":"Ortega","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Javier","family":"Echeverr\u00eda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda Fernanda","family":"Silva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariana Andrea","family":"Peralta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,8,15]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"80","DOI":"10.1016\/j.ejps.2012.09.016","article-title":"Reversal of efflux mediated antifungal resistance underlies synergistic activity of two monoterpenes with fluconazole","volume":"48","author":"Ahmad","year":"2013","journal-title":"Eur. 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