Silver nanoparticles have been shown to be detrimental to fungal cells although the mechanism(s) of action have not been clearly established. In this study, we used Candida albicans cells to show that silver nanoparticles exert their antifungal effect through apoptosis. Many studies have shown that the accumulation of reactive oxygen species induces and regulates the induction of apoptosis. Furthermore, hydroxyl radicals are considered an important component of cell death. Therefore, we assumed that hydroxyl radicals were related to apoptosis and the effect of thiourea as a hydroxyl radical scavenger was investigated. We measured the production of reactive oxygen species and investigated whether silver nanoparticles induced the accumulation of hydroxyl radicals. A reduction in the mitochondrial membrane potential shown by flow cytometry analysis and the release of cytochrome c from mitochondria were also verified. In addition, the apoptotic effects of silver nanoparticles were detected by fluorescence microscopy using other confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, DNA and nuclear fragmentation, and the activation of metacaspases. Cells exposed to silver nanoparticles showed increased reactive oxygen species and hydroxyl radical production. All other phenomena of mitochondrial dysfunction and apoptotic features also appeared. The results indicate that silver nanoparticles possess antifungal effects with apoptotic features and we suggest that the hydroxyl radicals generated by silver nanoparticles have a significant role in mitochondrial dysfunctional apoptosis.

Silver nanoparticles (nano-Ags), which have well-known antimicrobial properties, are used extensively in various medical and general applications. In this study, the combination effects between nano-Ags and the conventional antibiotics ampicillin, chloramphenicol and kanamycin against various pathogenic bacteria were investigated. The MIC and fractional inhibitory concentration index (FICI) were determined to confirm antibacterial susceptibility and synergistic effects. The results showed that nano-Ags possessed antibacterial effects and synergistic activities. The antibiofilm activities of nano-Ags alone or in combination with antibiotics were also investigated. Formation of biofilm is associated with resistance to antimicrobial agents and chronic bacterial infections. The results indicated that nano-Ags also had antibiofilm activities. To understand these effects of nano-Ags, an ATPase inhibitor assay, permeability assay and hydroxyl radical assay were conducted. The antibacterial activity of nano-Ags was influenced by ATPassociated metabolism rather than by the permeability of the outer membrane. Additionally, nanoAgs generated hydroxyl radicals, a highly reactive oxygen species induced by bactericidal agents. It was concluded that nano-Ags have potential as a combination therapeutic agent for the treatment of infectious diseases by bacteria.

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Amentoflavone was isolated from an ethyl acetate extract of the whole plant of Selaginella tamariscina. It is a traditional herb for the therapy of chronic trachitis and exhibits some anti-tumor activity. Previously, we confirmed the antifungal effects of amentoflavone. The objective of this study was to investigate the antifungal mechanism(s) of amentoflavone, such as mitochondria-mediated apoptotic cell death. The cells that were treated with amentoflavone exhibited a series of cellular changes that were consistent with apoptosis: externalization of phosphatidylserine, DNA and nuclear fragmentation, accumulation of intracellular reactive oxygen species (ROS) and hydroxyl radicals, and activation of metacaspase. In addition, diagnostic markers of apoptosis, including the reduction of mitochondrial inner-membrane potential and the release of cytochrome c from mitochondria, were observed. These phenomena are important changes in mitochondria-mediated apoptosis. Furthermore, the effect of thiourea as hydroxyl radical scavenger on amentoflavone-induced apoptosis was evaluated. A hydroxyl radical is a more active ROS species. Mitochondrial dysfunction was inhibited, which was indicated by decreased levels of intracellular hydroxyl radicals. Taken together, our results present the first evidence that amentoflavone induces apoptosis in C. albicans cells and is associated with the mitochondrial dysfunction. Besides, amentoflavone-induced hydroxyl radicals may play a significant role in mitochondria-mediated apoptosis.