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. 2024 Dec 4;13(12):1174.
doi: 10.3390/antibiotics13121174.

Ellagic Acid Potentiates the Inhibitory Effects of Fluconazole Against Candida albicans

Amanda Graziela Gonçalves Mendes  1 , Carmem Duarte Lima Campos  1 , José Lima Pereira-Filho  1 , Aleania Polassa Almeida Pereira  1 , Gabriel Silva Abrantes Reis  1 , Árlon Wendel de Marinho Silva Araújo  1 , Pablo de Matos Monteiro  1 , Flávia Castello Branco Vidal  1 , Silvio Gomes Monteiro  1 , Isabella Fernandes da Silva Figueiredo  2   3 , Elizabeth Soares Fernandes  2   3 , Cristina de Andrade Monteiro  4 , Valério Monteiro-Neto  1
Affiliations

Ellagic Acid Potentiates the Inhibitory Effects of Fluconazole Against Candida albicans

Amanda Graziela Gonçalves Mendes et al. Antibiotics (Basel). .

Abstract

Background/Objectives: Antifungal resistance to azoles, coupled with the increasing prevalence of Candida albicans infections, represents a significant public health challenge and has driven the search for new natural compounds that can act as alternatives or adjuvants to the current antifungals. Ellagic acid (EA) has demonstrated antifungal activity; however, its effects are not fully understood. In this study, we investigated the in vitro anti-Candida activity of EA and its ability to potentiate the effects of fluconazole (FLZ) on C. albicans.Methods: The Minimum Inhibitory Concentration (MIC) of EA was determined by broth microdilution and its interaction with FLZ was assessed using a checkerboard assay. Additionally, we examined the effects of EA on yeast-to-hypha transition, inhibition of biofilm formation, time-kill kinetics, hemolytic activity, and cytotoxicity in HeLa ATCC® CCL-2™ cells. Results: EA exhibited MIC values ranging from 250 to 2000 µg/mL and showed synergistic and additive interactions with FLZ, resulting in a marked reduction in the MIC values of FLZ (up to 32-fold) and EA (up to 16-fold). In the time-kill assay, the most effective combinations were 4× EA MIC, 2× EA MIC, and FIC EA + FLZ, which showed fungicidal activity. Furthermore, EA did not show hemolytic activity and demonstrated low and dose-dependent cytotoxicity in HeLa cells, with no cytotoxic effects observed in combination with FLZ. EA and the synergistic combination of EA and FLZ interfered with both the yeast-to-hypha transition process in C. albicans cells and biofilm formation. In addition to its antifungal efficacy, EA demonstrated a favorable safety profile at the concentrations used. Conclusions: This study presents promising results regarding the potential use of EA in combination with FLZ for the treatment of C. albicans infections.

Keywords: anti-Candida activity; antibiofilm activity; ellagic acid; fluconazole.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Time-kill curve of C. albicans ATCC 90028 under the action of EA (A) and FLZ (B) and their combination (FIC EA + FLZ) (C) showing the medians. The Shapiro–Wilk test was used to assess the normality of the sample. Significant differences between groups were determined by Kruskal–Wallis analysis followed by Dunn’s test with p > 0.05 considered significant. Legend: FIC = fractional inhibitory combination, MIC = minimal inhibitory concentration, EA = ellagic acid, FLZ = fluconazole.
Figure 2
Figure 2
Time-kill curve of C. albicans CA 08 under the action of EA (A), FLZ (B) and in combination (FIC EA + FLZ) (C), at different concentrations and control. The Shapiro–Wilk test was applied to analyze normality. Significant differences between groups were determined by Kruskal-Walli’s analysis followed by Dunn’s test, presenting the medians test with (p > 0.05) considered significant. Legend: FIC = fractional inhibitory combinations, MIC = minimal inhibitory concentration, EA = ellagic acid, FLZ = fluconazole.
Figure 3
Figure 3
C. albicans hyphae formation. Clinical isolates: (A) CA 90028; (B) CA 013; (C) CA 010; (D) CA08. The Shapiro–Wilk normality test was applied. The results are presented as mean and standard deviation; significant differences between groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison where the value of p < 0.05 (*) was considered significant.
Figure 4
Figure 4
Cell viability of young C. albicans biofilms. Clinical isolates: (A) CA 90028; (B) CA 013; (C) CA 010; (D) CA08. The Shapiro–Wilk normality test was applied. Results are presented as mean and standard deviation; significant differences between groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison where the value of p < 0.05 (*) was significant.
Figure 5
Figure 5
Cell viability of mature C. albicans biofilms. Clinical isolates: (A) CA 90028; (B) CA 013; (C) CA 010; (D) CA08. The Shapiro–Wilk normality test was applied. Significant differences between groups were determined by Kruskal–Wallis analysis of variance followed by Dunn’s test, where the value of p < 0.05 (*) was significant.
Figure 6
Figure 6
Hemolytic activity. The Shapiro–Wilk normality test was applied. The test results are presented with mean and standard deviation; significant differences between specific groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison, where the value of (* p < 0.05) was significant.
Figure 7
Figure 7
Cytotoxicity assay in HeLa. Cell viability was evaluated after (A) 24 h, (B) 48 h, and (C) 72 h. The Shapiro–Wilk normality test was applied. Significant differences between groups were determined by Kruskal–Wallis analysis followed by Dunn’s multiple comparisons test, where the value of (* p < 0.05) was significant. EA = ellagic acid; FLZ = fluconazole.
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