We previously showed that this compound has a remarkable antibacterial activity at low concentrations: 0.24 µg/mL against Staphylococcus aureus and 3.9 µg/mL against Escherichia coli . As such, we have strong reasons to consider that BrCl-flav has a good potential for the development of new antimicrobial agents. Therefore, further studies using four clinical Candida isolates were employed to investigate BrCl-flav antifungal properties and mechanism of action. A potent antifungal effect was evidenced for BrCl-flav against all Candida strains tested in vitro using the determination of minimum inhibitory concentration. The method allowed us to assess the lowest concentration of BrCl-flflav which inhibited the growth of the tested fungal strains—15.62 µg/mL.
When compared with the reference drug fluconazole, a considerable higher antifungal activity was recorded against fluconazole resistant C. albicans, C. krusei and C. glabrata isolates. Compared to most of the reported natural and synthetic flflavonoids, our compound displayed a stronger antifungal activity, being up to 28-fold more active against Candida spp.. We must emphasize that BrCl-flav exhibited an antifungal activity comparable to some chalcone, flavones and flavanones derivatives considered to be the most potent synthetic flavonoids against Candida reported so far. The fungistatic effect was dose-dependent, increasing concentrations of BrCl-flflav progressively inhibited the fungal growth of all tested Candida isolates.
The cells incubated with 7.81 µg/mL BrCl-flflav showed no significant growth inhibition compared with control. On the other hand, a signifificant growth delay represented by prolonged lag phases occurred when Candida spp. cells were incubated in the presence of BrCl-flav at 15.2 µg/mL, corresponding to MIC. We must point out that no turbidity was recorded by spectrophotometric measurements for all Candida cells exposed to 31.25 µg/mL within the time span of the experiments , denoting a strong fungicidal activity. A time-kill kinetics assay was employed to determine the fungistatic or fungicidal activity of BrCl-flav over time. The analysis confirmed also the important antifungal activity of BrCl-flav against all Candida spp. after exposure to concentrations equivalent to MIC and 2 × MIC . One exception occurred for C. albicans strain—a fungistatic effect was recorded by the growth experiments up to 24 h, while time kill studies revealed a fungicidal effect at 24 h.
We must emphasize that no viable cells were detected starting with 12 h after BrCl-flav exposure at 31.25 µg/mL , suggesting an important fungicidal potential. The fact that this activity has been recorded against fluconazole-resistant Candida strains makes BrCl-flav a more interesting compound for practical applications. Our conclusion is supported by a comparative literature survey which revealed that BrCl-flflav has higher fungicidal activity compared to many natural flavonoids.Also, the recorded activity was higher or comparablewith other synthetic flavonoids such as different chalcone, 1,3-thiazole and 2-hydrazinyl- 1,3-thiazole derivatives.We must emphasize that BrCl-flav acted as a more potent fungicidal compared with fluconazole, considered to be a gold standard of antifungal agents . Sorbitol binding affinity assay was used to investigate the interference of BrCl-flav with the Candida cell wall. Sorbitol is an osmotic protector which can support cell growth when the fungal wall is targeted by antifungal agents. In the absence of sorbitol, the fungal growth is inhibited due to the disruption of the cell wall. The effect is detected by an increase of the MIC value in the presence of sorbitol compared to the MIC value determined in medium without sorbitol.In our study, MIC values of BrCl-flav did not change in the presence of sorbitol, suggesting that the tested synthetic flavonoid does not target the cell wall. Therefore, other potential cellular targets were investigated. Penetration of PI into dead or injured C. albicans cells was evidenced using fluorescence microscopy. PI is a cell membrane-selective permeable dye that can only pass through damaged or permeabilized cell membranes, binding to DNA and exhibiting characteristic red fluorescence.
Our results revealed a gradual increase of the fluorescent cells number with the increasing concentration of the tested antifungal. After 24 h all cells exposed to BrCl-flav at a concentration equivalent to 5 × MIC were fluorescent, suggesting that the tested antifungal significantly damaged the fungal cell membrane integrity. Usually, damages to the cell membrane are related to cell lysis. SEM image analysis showed severe alteration of the cell morphology, with collapsed cells, wrinkled surfaces, along with cellular debris resulting from the disintegration of the BrCl-flav treated cells. These morphological changes are most likely caused by cell lysis.