Chemical Composition, In Vitro Antifungal and Antimicrobial Activity, and Mechanism of Action of Essential Oils from Litsea cubeba, Elsholtzia blanda and Trachyspermum ammi Against Dermatophytes and Candida albicans

Essential oil chemistry and antifungal activity against dermatophytes

Abstract

Background: The escalating global burden of antimicrobial resistance, and the recent emergence in India of terbinafine-resistant Trichophyton indotineae, has renewed interest in plant-derived essential oils (EOs) as alternative antifungal agents that act through multiple, non-selective molecular targets. Objective: This study characterised the chemical composition and evaluated the antifungal and antimicrobial activity of EOs from Litsea cubeba (LC-EO), Elsholtzia blanda (EB-EO) and Trachyspermum ammi (TA-EO), and identified their principal bioactive constituent(s) and mode of antifungal action. Methods: EOs were obtained by Clevenger hydrodistillation (five batches per species) and characterised physicochemically and by GC-MS/GC-FID. Antifungal activity was evaluated against Trichophyton rubrum, Microsporum canis, Epidermophyton floccosum, Trichophyton violaceum and Candida albicans (reference strains and clinical isolates) by agar well diffusion and CLSI broth microdilution (MIC/MFC); antibacterial activity was evaluated against Staphylococcus aureus (including an MRSA isolate), Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The most active oil was subjected to bioassay-guided liquid-liquid fractionation, and the isolated active fraction was investigated mechanistically by ergosterol-binding and propidium iodide uptake assays. Checkerboard assays assessed pairwise synergy. Results: GC-MS identified citral (geranial + neral, 72.65%) as dominant in LC-EO, camphor and 1,8-cineole (43.13% combined) in EB-EO, and thymol (48.45%) in TA-EO. A consistent antifungal potency hierarchy, TA-EO > LC-EO > EB-EO, was observed across all fungal organisms (MIC: 62.5–125, 125–500 and 250–1000 µg/mL respectively), with MFC/MIC ratios ≤2 confirming fungicidal activity for all three oils. TA-EO at 50 µL/mL produced inhibition zones (27.6–35.4 mm) exceeding fluconazole (18.8–24.6 mm), and retained potent activity against an MRSA clinical isolate (MIC 62.5 µg/mL, identical to the susceptible strain). Bioassay-guided fractionation identified the thymol-rich ethyl acetate fraction of TA-EO (thymol 97.2%) as the principal antifungally active fraction (MIC 31.25 µg/mL), and pure thymol reproduced this potency (MIC 8–16 µg/mL). Thymol caused dose-dependent depletion of membrane ergosterol (52.8% reduction at MIC, p<0.001) and increased propidium iodide uptake (94.2% at 2×MIC), confirming membrane disruption as the principal mechanism of action. TA-EO showed synergy with LC-EO, EB-EO and fluconazole (FICI ≤ 0.50). Conclusion: Thymol-rich T. ammi EO possesses potent, fungicidal, membrane-targeted antifungal and antimicrobial activity that exceeds conventional azole comparators in vitro and synergises with both plant EOs and fluconazole, providing a strong rationale for its advancement into topical antifungal formulation development.