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Objective To evaluate the anti‐fungal activity of Syzygium aromaticum essential oil and its inhibition of a multispecies biofilm in patients with oral candidiasis. Background Inhibiting biofilm formation on the denture surface is an important practice for preventing denture stomatitis. Materials and Methods The anti‐fungal activity against Candida albicans and non‐albicans Candida species was evaluated through the microdilution method to define Minimal Inhibitory (MIC) and Fungicidal (MFC) Concentrations. Time‐kill assay assessed growth kinetics of C. albicans based on pre‐determined time points (0, 1, 2, 4, 6 and 24 hours). A multi‐species biofilm was formed using human saliva from patients with oral candidiasis and anti‐biofilm activity determined by Colony Forming Units per milliliter (CFU/mL) count, fluorescence microscopy with calcofluor white to observe yeast presence and structure, and metabolic activity by XTT (2,3‐Bis‐(2Methoxy‐4‐Nitro‐5‐Sulfophenyl)‐2H‐Tetrazolium‐5‐carboxanilide) reduction assay. Results The essential oil showed an anti‐fungal activity against all Candida species (MIC 500‐1000 µg/mL, MFC 1000‐2000 µg/mL), and the time‐kill assay showed that 2000 µg/mL (from 2 hours onward) and 1000 µg/mL (from 4 hours onward) concentrations had substantially lower yeast growth than the negative control. In the biofilm analysis, the essential oil had a lower CFU/mL count and a biofilm metabolic activity (91.4%) than seen with its negative control, and in both analyses, the essential oil was not significantly different from the positive control (chlorhexidine). Morphological analysis showed amorphous and fragmented cellular structures after treatment with the essential oil. Conclusion Syzygium aromaticum essential oil had anti‐fungal activities, reduced the Candida growth kinetics substantially and inhibited the multi‐species biofilm formation.

Oral candidiasis is one of the most common fungal infections in humans. Its incidence has increased widely, as well as the antifungal resistance, demanding for the search for novel antifungal therapeutic agents. Anadenanthera colubrina (Vell.) Brenan is a plant species that has been proven to possess pharmacological effects, including antifungal and anti-inflammatory activities. This study evaluated in vitro the effects of standardized A. colubrina extract on virulence factors of Candida albicans and its regulation on immune response through C. albicans-host interaction. Antifungal activity was evaluated by Broth Microdilution Method against reference Candida strains (C. albicans, C. glabrata, C. tropicalis; C. dubliniensis). Anti-biofilm effect was performed on C. albicans mature biofilm and quantified by CFU/mL/g of biofilm dry weight. Proleotlytic enzymatic activities of proteinase and phospholipase were assessed by Azocasein and Phosphatidylcholine assays, respectively. Cytotoxicity effect was determined by Cell Titer Blue Viability Assay on Human Gingival Fibroblasts. Co-cultured model was used to analyze C. albicans coexisting with HGF by Scanning Electron Microscopy and fluorescence microscopies; gene expression was assessed by RT-PCR of C. albicans enzymes (SAP-1, PLB-1) and of host inflammatory cytokines (IL-6, IL-8, IL-1β, IL-10). Cytokines secretion was analysed by Luminex. The extract presented antifungal effect with MIC<15.62 μg/ml against Candida strains. Biofilm and proteolytic activity were significant reduced at 312.4 μg/ml (20 × 15.62 μg/ml) extract concentration. Cell viability was maintained higher than 70% in concentrations up to 250 μg/ml (LD50 = 423.3 μg/ml). Co-culture microscopies demonstrated a substantial decreased in C. albicans growth and minimal toxicity against host cells. Gene expressions of SAP-1/PLB-1 were significantly down-regulated and host immune response was modulated by a significant decreased on IL-6 and IL-8 cytokines secretion. A. colubrina had antifungal activity on Candida strains, antibiofilm, and anti-proteolytic enzyme effects against C. albicans. Presented low cytotoxicity to the host cells and modulatory effects on the host immune response.