Fig 1.
Structure of lichochalcone-A.
Table 1.
Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of lichochalcone-A against Candida albicans.
Fig 2.
Time Kill of C. albicans (MYA 2876) inoculum (10³ CFU/ml) tested against lichochalcone-A (at 10x MIC, and 20x MIC), fluconazole 32 μM, (positive control), 1% ethanol (vehicle control), and medium with inoculum only (negative control), plot expressed as average values for log10 of the numbers of CFU/milliliter versus time (hrs.).
Fig 3.
Fungal viability of C. albicans biofilm expressed in CFU/ml/ grams of dry weight after treatment with lichochalcone-A.
The antifungal activity of lichochalcone-A (625 μM; 10x MIC) against C. albicans MYA 2876 biofilms was compared to the vehicle control group (1% ethanol) and positive control group (fluconazole 320 μM; 10x MIC) The standard deviations of each sample are shown in the graph, and all the mean differences between the control groups and test (lichochalcone-A at 625 μM) were statistically significant (*p<0.05).
Fig 4.
Co-culture fluorescence microscopy stained with calcofluor white stain (Blue: C. albicans) and Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (Green: live fibroblast cells; Red: dead fibroblast cells).
A. Vehicle control (ethanol 1%); B. Positive control (fluconazole 32.2 μM); and C. Lichochalcone-A at 62.5 μM. Lichochalcone-A displayed low candida (blue fluorescence) growth with even distribution of live fibroblast cells (green fluorescence). Scale bars are set in μm.
Fig 5.
C. albicans enzymes secretion expressed in U/grams of dry weight after treatment with lichochalcone-A at concentrations of 625 μM and 1500 μM: A. Proteinase enzyme activity showed 50% reduction in enzyme activity at 1500 μM concentration; B. Phospholipase enzyme activity showed up to 30% decrease in 1500 μM lichochalcone treated biofilms. Significant reduction in proteinases and phospholipases enzyme activities were observed in lichochalcone-A treated biofilms (at concentrations of 625 μM and 1500 μM; equivalent to 10x MIC) in comparison to vehicle control group. *p<0.05.
Fig 6.
Pro-inflammatory cytokines expression of IL-1α and IL-1β, and anti-inflammatory cytokine expression of IL-10 in C. albicans treated with lichochalcone-A (62.5 μM and 150 μM), fluconazole (32 μM) (positive control), and 1% ethanol (vehicle control).
Significant decrease in IL-1α and IL-1β (*p<0.05), and an increase in IL-10 (results were not statistically significant; p>0.05) in the treated groups compared to the vehicle group. All values are expressed as normalized average expression percentages ± S.e.m (normalized based on the vehicle control groups).
Fig 7.
Longitudinal In vivo imaging of OC after 1 day, 4 and 5 days post infection with C. albicans ACTgLUC23 (1x107/ml).
A. Mice (-C) treated with 1% ethanol as a vehicle control. B. Treated (+C) with nystatin, as positive control. C. Treated (LA) with lichochalcone-A. D. Total photon flux from oral cavities in the images (ROI) of each mouse was quantified with Living ImageR software package. Longitudinal monitoring of fungal load of the mice, grouped as vehicle control, positive control (treated with nystatin), and lichochalcone-A treated group after day 1, 4, and 5 post-infection. Baseline imaging of infection at day 1 post-infection and prior to topical treatments. Significant decrease in total photon flux was observed at days 4 and 5 in lichochalcone-A and nystatin treated groups (*p<0.05).
Fig 8.
Ex-vivo microbiology analysis of tongue samples of mice grouped as vehicle control (1% ethanol), positive control (nystatin treated) and lichochalcone-A treated group.
Fungal load of the tongues are expressed as colony formation unit (CFU)/ml/mg of tissue. Lichochalcone-A group showed more than 50% reduction in colony count compared to vehicle control group (*p<0.05).
Fig 9.
Histopathology of tongue sections at 4x magnification (top) and (400x magnification) bottom from A. Vehicle control, B. Positive control treated with nystatin, and C. Lichochalcone-A. Circles on top images shows a section of the epithelium layer that is enlarged below at 400x magnification. Tongue section (A, left panel) from vehicle control showed yeasts and hyphae (arrows) invasion of the dorsal papillary architecture. Presence of C. albicans is indicated by arrows pointing to pink staining (A, lower left). Tongue sections from panel B, positive control, and panel C, lichochalcone-A, did not show signs of fungal burden.