Synergistic effects of tea polyphenol epigallocatechin 3-O-gallate and azole drugs against oral Candida isolates.

BACKGROUND: The antifungal drug resistance has become an emerging problem in the management of candida infections worldwide. The objective of this study was to examine the efficacy of epigallocatechin 3-O-gallate (EGCG) alone and in combination with fluconazole/ketoconazole drugs against oral Candida isolates. METHODS: Minimum inhibitory concentration (MIC) and minimum fungicidal concentrations (MFC) of EGCG against 60 oral Candida isolates and 4 ATCC strains were determined. Synergism of EGCG with azole drugs was evaluated by checkerboard micro-dilution method and calculated fractional inhibitory concentration index (FICI). Candida cells' ultrastructure was studied by electron microscopy. RESULTS: MIC and MFC values of EGCG were in the range of 3.91-15.63 and 15.63-31.25µg/mL, respectively. Minimum biofilm inhibitory concentration (MBIC) range of EGCG (62.5-125µg/mL), was less than the ketoconazole (64-256µg/mL) and fluconazole (128-512µg/mL). The combination of EGCG with fluconazole/ketoconazole exhibited synergistic effects (ΣFICI≤0.50). EGCG with azole drugs showed high sensitivity against the tested isolates in growth curve assays. Against the biofilm, the susceptibility of fluconazole/ketoconazole significantly increased (3 to 5 fold), after combination with EGCG (MBIC/4) (P≤0.001). Electron microscopy of EGCG treated cells showed deformation of cell structure, ruptured cell wall and release of intracellular content. In molecular docking experiments, a strong interaction was observed between EGCG and fungal cell membrane molecule ergosterol. CONCLUSION: We conclude that EGCG synergistically enhanced the antifungal potential of azole drugs. The synergistic potential of EGCG might be helpful in preventing the development of drug resistance, in lowering the drug dosage, and thus minimizing adverse effects.

Anticandidal activity of ethanolic root extract of Juglans regia (L.): Effect on growth, cell morphology, and key virulence factors.

The emergence of drug-resistant strains has encouraged several studies on natural products with antifungal activity and low toxicity. In this study, the antifungal effect of methanolic root extract of Juglans regia (JRE) was investigated against 9 strains of Candida (one reference and 8 clinical strains) through MIC90 and spot assays. To gain insight into the mechanism of antifungal action, we carried out confocal scanning laser microscopy (CLSM), transmission electron microscopy (TEM), and then examined the effect of JRE on hydrolytic enzyme secretion. Additionally, JRE was subjected to various phytochemical tests, chemically characterized by gas chromatography-mass spectrometry analysis (GC-MS) and its toxicity was tested against H9c2 rat cardiac myoblasts. The phytochemical tests showed the presence of phenols, alkaloids, steroids, saponins, and tannins in JRE. In the GC-MS analysis, a total of 40 compounds were identified. JRE was found to be effective in liquid media with MICs ranging from 300 to 700µg/mL. Spot assay results revealed that Candida cells show increased sensitivity to JRE. CSLM experiments showed that cells exposed to JRE (MIC) exhibited cell membrane disruption. TEM micrograph of treated cells showed extensive breakage in the cell wall and cell membrane. Average inhibition of proteinase and phospholipase secretion (of five C. albicans strains) at MIC/2 values of JRE was 45.17%, and 34.29%, respectively. Cellular toxicity of JRE against H9c2 rat cardiac myoblasts was less than 10% at the highest MIC value. These findings encourage further development of JRE.

Curcumin as a promising anticandidal of clinical interest.

Curcumin, an important Asian spice, is part of many Indian food preparations. This work evaluates the antifungal activity of curcumin against 14 strains of Candida (10 clinical and 4 standard). Curcumin displayed antifungal properties against all tested Candida strains, with minimum inhibitory concentrations (MICs) varying from 250 to 2000 µg·mL⁻¹. The in vitro effect of curcumin on growth, sterol content, proteinase secretion, and H+ extrusion by plasma membrane ATPase was investigated for 2 standard strains Candida albicans ATCC 10261 and Candida glabrata ATCC 90030 and compared with the effect of fluconazole. At MIC, curcumin inhibited H+ extrusion in 2 species of Candida by 42% and 32% in the absence of glucose and by 28% and 18% in the presence of glucose. Respective inhibition of H+ extrusion caused by the MIC of fluconazole was 85% and 89% in the absence of glucose and 61% and 66% in its presence. Ergosterol content decreased by 70% and 53% for the 2 strains following exposure to curcumin at MIC; comparative values for fluconazole at MIC were 93% and 98%. Curcumin and fluconazole decreased proteinase secretion by 49% and 53%, respectively, in C. albicans and by 39% and 46%, respectively, in C. glabrata. In conclusion, curcumin is found to be active against all tested clinical and standard strains but is less effective than fluconazole. Antifungal activity of curcumin might be originating from alteration of membrane-associated properties of ATPase activity, ergosterol biosynthesis, and proteinase secretion.