Anticandidal Activity of Capsaicin and Its Effect on Ergosterol Biosynthesis and Membrane Integrity of Candida albicans

2.2. Synergistic Activity of Capsaicin with Fluconazole

Various combinations of Capsaicin and Fluconazole were used to test the combined effect against the endodontic isolates. The combination of Capsaicin with Fluconazole showed a remarkable decrease in the MIC values compared to each drug alone (Table 2). The MIC of Fluconazole significantly decreased from 16–128 to 4–16 µg/mL, whereas the MIC of Capsaicin significantly decreased from 25 to 6.25 or 12.5 µg/mL (p < 0.01). The combinations of Capsaicin and Fluconazole exhibited synergism effects (FICI ≤ 0.50). The combination did not show an antagonistic effect against the tested isolates. The antiCandidal activities of Capsaicin and Fluconazole alone and in combination against the 30 oral isolates are given in Supplementary Table S1.

2.3. Biofilm Inhibition

The effect of Capsaicin (MIC) was tested on the pre-formed mature biofilm of C. albicans. In Supplementary Figure S1, the left panel represents the CLSM image of the mature biofilm of C. albicans, whereas the right panel represents the biofilm morphology after the treatment. The CLSM image clearly showed the eradication of biofilm of the treated sample. The metabolic activity of biofilm assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) metabolic reduction assay demonstrated an average 73.6% reduction in the biofilm of endodontic Candida isolates, while for ATCC strains, it was 71.2% at the MIC level (Figure 1). In summary, our data indicate that Capsaicin significantly inhibits the mature biofilm (p < 0.01).

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Figure 1

Effect of Capsaicin on the mature biofilm of C. albicans. The standard deviations (SD) of each sample are shown in the graph. The mean differences between the control and test molecules at the MIC level were statistically significant (p-level, 0.01). MIC/2—half of the MIC concentration; Endo—endodontic isolates.

2.4. Inhibitory Activity on Hyphae Formation

A hyphae-inducing medium was used to examine the hyphae inhibition by Capsaicin. The live cell observer microscope recorded live images of cells’ transitions from yeast form to filaments form. In Figure 2, the left panel shows the untreated C. albicans (control) cells, and the right panel shows the cells’ morphology after the treatment with Capsaicin (MIC). The untreated controls showed massive hyphae growth after three hours, but in the presence of Capsaicin (MIC), hyphae growth was not observed. Cells treated with Capsaicin showed minor changes in cell morphology. The results clearly showed that Capsaicin inhibits the growth of hyphae.

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Figure 2

Effect of Capsaicin (MIC) on the hyphae growth of C. albicans (Endo-902). The live cells photograph was recorded by the cell observer microscope (20×). The photograph of untreated cells (1a–1d) and treated cells (2a–2d) were recorded at three-hour time points, respectively.

2.5. Inhibition Activity of Ergosterol Synthesis

The ergosterol content of treated and untreated C. albicans cells’ membranes was estimated. In Figure 3, the flat curve represents the significant reduction in ergosterol content in Capsaicin (MIC)- and Ketoconazole (64 µg/mL)-treated cells (p < 0.05). However, untreated control cells represented the sharp peaks of ergosterol. The mean reduction in the ergosterol content of the cells treated with MIC/4, MIC/2, and MIC of Capsaicin and 64 µg/mL of Ketoconazole was 42.4%, 88.7%, 98.6%, and 99.1%, respectively. The dose-dependent decrease in ergosterol content indicated that the antifungal effect of Capsaicin was dependent on its concentrations, and its primary mode of action was through the inhibition of ergosterol biosynthesis.

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Figure 3

Spectrophotometric sterol profiles of C. albicans (Endo-904, Endo-906, and Endo-908). Sterols were extracted from the cells and spectral profiles between 230 and 300 nm were determined.

2.6. Cell Membrane Permeability

A confocal laser scanning microscope (CLSM) was used to investigate individual C. albicans cells stained with propidium iodide. The CLSM result showed the membrane-impermeant propidium iodide uptake by the Capsaicin-treated Candida cells. Propidium iodide is a red color intercalating stain that is membrane-impermeant and is therefore excluded by healthy cells. In Figure 4, CLSM images in the left panel show the untreated C. albicans that did not take up the red dye, whereas the dye was taken up by the treated cells in the right panel. Our results confirm that propidium iodide penetrates Capsaicin-treated cells, suggesting that Capsaicin disrupted the structure of the cell membrane.

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Figure 4

Confocal scanning laser microscopy (CSLM) images of endodontic C. albicans cells (Endo) treated with Capsaicin (MIC) (right panel) and untreated control cells (left panel). To observe membrane damage, cells were stained with propidium iodide (red signals).

4.2. Sample Collection

Oral Candida species samples were collected from dental patients at the Kuwait University Dental Clinic (KUDC) using a standard oral rinse technique [29]. The collected samples were identified by using the CHROMagar medium (CHROMagar Candida, France) and VITEK-2 system assays (bioMerieux, Craponne, France), as listed in Supplementary Table S1. Endodontic C. albicans isolates (n = 8) were obtained from the Oral Microbiology Laboratory, Faculty of Dentistry, Kuwait University, Kuwait.

4.3. Minimum Inhibitory Concentration

The Clinical Laboratory Standards Institute (CLSI) for yeasts (M27-A3) method was used to estimate the minimum inhibitory concentration (MIC) of the test compound and the drug [30]. The MIC was determined in an RPMI-164 medium (Cat no. R6504, Sigma-Aldrich, St. Louis, MO, USA), and buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS). The overnight culture of Candida cells was diluted in media, and a 100 μL volume of this diluted inoculum was added to each well of the 96-well U-bottom tissue culture plate, resulting in a final inoculum of ~2.5 × 10³ cells/mL. The concentration range tested was 1.56 to 400 µg/mL for Capsaicin and 0.50–128 µg/mL for Fluconazole and Ketoconazole. The medium without the drugs was used as the control, and the blank control used contained only the medium. The plates were incubated at 37 °C for 48 h in the incubator. The plates were read visually, and the MIC was defined as the lowest concentration of the test agents that prevented visible growth with respect to the growth control. All the assays were performed in triplicate.

4.4. Synergistic Activity of Capsaicin and Fluconazole

Synergistic activity was measured by the checkerboard microtiter plate method [30,31]. Capsaicin and Fluconazole drugs were serially diluted in RPMI at different combinations. The final concentrations ranged from 1.56 to 100 µg/mL for Capsaicin and from 1 to 64µg/mL for Fluconazole. Candida cells were added to each well at a final concentration of ~2.5 × 10³ cells/mL and incubated for 48 h at 37 °C in the incubator. The growth of Candida cells was read visually. All assays were performed in triplicate.

The fractional inhibitory concentration index (FICI) was used to evaluate the interactions between Capsaicin and drugs. FICI = MIC (Capsaicin with Fluconazole)/MIC (Capsaicin alone) + MIC (Fluconazole with Capsaicin)/MIC (Fluconazole alone). FICI ≤ 0.5 indicated synergetic interaction between Capsaicin and Fluconazole, FICI > 0.5 to 4 indicated no interaction, and FICI > 4 indicated antagonistic interaction between both agents.

4.5. Biofilm Inhibition Assay

A standardized protocol was used in this study [4]. The mature biofilms of Candida were formed in the wells of polystyrene 96-well microtiter plates (Corning Inc., Corning, NY, USA). The MIC of Capsaicin was prepared separately in an RPMI 1640 medium and added to each well (200 µL/well) and incubated for 24 h at 37 °C. After the incubation period, the medium was aspirated and washed carefully with sterile PBS (pH 7). The density of biofilm was observed under confocal laser microscopy (Zeiss LSM 500) by using Cyto-9 fluorescence dye. The biofilm density was quantified by the MTT assay, and the percentage of biofilm inhibition was calculated [25]. All experiments were performed in triplicate.

4.6. Study of Hyphae Formation and Cells Morphology

The aliquot of overnight culture cells (~10⁶ cells/mL) inoculated in YPD medium was supplemented with 10% fetal bovine serum (FBS) [23]. The MIC of Capsaicin was added into the medium and kept at 37 °C under the Eye lens (20 x) of the cell observer microscope (Zeiss LSM). The morphological change of Candida cells was live-recorded every 15 min. Cells without treatment were used as a positive control.

4.7. Ergosterol Extraction and Estimation

The total intracellular sterols were extracted by a standard method [32], with arrangements as described in our previous work [33]. Candida colony from an overnight Sabouraud dextrose agar plate culture was used to inoculate 10 mL of Sabouraud dextrose broth for the control and various concentrations of Capsaicin. Ketoconazole was used as a positive control. The cultures were incubated for 16 h and harvested by centrifugation at 4000× g for 5 min. The net weight of the cell pellet was determined. In all, 3 mL of 25% ethanolic potassium hydroxide solution was added to each pellet and vortex-mixed for 3 min. Cell suspensions were transferred to sterile borosilicate glass screw-cap tubes and were incubated at 85 °C in a water bath for one hour and then allowed to cool at room temperature. Sterols were then extracted by the addition of a mixture of 1 mL of sterile Milli-Q water and 3 mL of n-heptane followed by vigorous vortex mixing for 3 min. The n-heptane layer was transferred to a clean borosilicate glass screw-cap tube and stored at −20 °C.

The concentration of sterol was scanned spectrophotometrically between 230 and 300 nm with a spectrophotometer (Eppendorf, USA). The ergosterol content was calculated as a percentage of the wet weight of the cell using formula

The values 290 and 518 are the E values (in percentages per centimeter) determined for crystalline ergosterol and 24 (28) DHE, respectively.

4.8. Confocal Scanning Laser Microscopy (CSLM)

The disruptive effect of Capsaicin on Candida cell membranes was assessed using fluorescence dye Propidium iodide (PI) [4]. Candida cells (~1 × 10⁶) were suspended in a YPD medium and incubated with Capsaicin at 37 °C with constant shaking (120 rpm) for 12 h. To confirm cell membrane permeabilization, 1 µg/mL of dye was added, and the cell suspensions were then incubated again at 37 °C for 30 min. The dye uptake was visualized by confocal microscopy (Zeiss LSM 500).

4.9. Effect of Capsaicin on the Biofilm of Dentine Substrate

Freshly extracted teeth were collected from the Kuwait Dental Clinic. The teeth pulp chamber was opened by using a cutting machine (Accutom-100, USA). The teeth slice was washed and sterilized by the treatment with 17% EDTA, followed by 2.5% sodium hypochlorite for 4 min, and then air-dried for 10 min. The teeth slice was placed into the freshly prepared C. albicans cells’ suspension (1 × 10⁶) in YEPD medium and incubated with shaking (120 rpm) at 37 °C for 24 h. After the incubation period, the teeth slice was transferred to the fresh medium that contained the test molecule (MIC) and re-incubated for 24 h. After the end of the treatment period, the dental slice was washed thrice carefully with PBS and processed for scanning electron microscopy (JEOL, Carryscope JCM 5700).

We sincerely thank Nisha Sina Philip at the Nanoscopy Science Centre (College of Sciences, Kuwait University) for the SEM and TEM analyses (GE01/07), and Mohammad Arshad at the Research Core Facility, Kuwait University, for the assistance in the CSLM analysis (SRUL 02/13). We acknowledge the Oral Microbiology Research Laboratory for allowing us to utilize various equipment and other resources (SRUL 01/14).