Our study showed that Shikonin (SK) could provide an action against almost all Candida albicans isolates tested. More importantly, to some Fluconazole (FCZ)-resistant Candida albicans, the action of SK (MIC 80 value 4 µg/mL) was shown to be >16 times higher than that of FCZ (MIC 80 >64 µg/mL). To clarify the mechanism underlying this action, we performed a comparative study in untreated control C. albicans and C. albicans treated with SK. In this study, we found that SK treatment increased generation of endogenous reactive oxygen species (ROS) and decreased mitochondrial membrane potential. Furthermore, anti-oxidants N-acetylcysteine (NAC) and glutathione (GSH) could reduce the antifungal activity of SK significantly in C. albicans. Our analyses also identified 9 differentially expressed genes, which were related to glycolysis-related genes (CDC19 and HXK2), fermentation-related genes (ALD5 and ADH1), antioxidant defense-related genes (SOD2 and SOD5), thioredoxin reductase-related gene (TRR1), mitochondrial respiratory electron transport chain-related gene (MRF1) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductase-related gene (EBP1). These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the action of SK against C. albicans. Key words Candida albicans; shikonin; reactive oxygen species; mitochondrial membrane potentialCandida albicans, the major opportunistic fungal pathogen of humans, normally exist as commensal. However, in patients with an impaired immune system such as those suffering from acquired immunodeficiency syndrome or cancer, C. albicans can cause recurrent superficial infection and fatal deep-seated and disseminated candidiasis. 1-4)Treatment for C. albicans includes antifungal drugs, mainly the azoles. Unfortunately, with the increasing clinical use of the azoles, azoles-resistant isolates are occurring more frequently. 5,6) Thus the development of new, effective antifungal agents is strongly needed in medicine.Shikonin (SK) (Fig. 1) is set in the orient, originally in China. This molecule is the major constituent of the red pigment extracts from the roots of the plant Lithospermum erythrorhizon SIEB. et ZUCC (LE). SK is widely used as a material to prepare an ointment called "Shiun-ko" which is used to treat wounds, burn and hemorrhoids in Japan. 7,8) Our study found that SK exhibit significant antifungal activities almost all C. albicans isolates tested. More importantly, to some Fluconazole (FCZ)-resistant C. albicans, the action of SK minimum inhibitory concentration (MIC 80 value 4 µg/mL) was shown to be >16 times higher than that of FCZ (MIC 80 >64 µg/mL). And the antifungal properties may relate to SKmediated oxidative damage and breakdown of mitochondrial membrane potential. Antifungal Susceptibility Testing Assays were performed on 10 isolates of C. albicans according to methods of the CLSI (formerly NCCLS) (M27-A). MATERIALS AND METHODS Strains and Agents9-11) The initial concentration of the fungal sus...

Candida albicans is the most important human fungal pathogen, causing various diseases from superficial mucosal infections to life-threatening systemic disorders.1,2) Compared to bacterial infections, few drugs are available to treat fungal infections. The polyene macrolide antibiotic amphotericin B (AmB) is effective in the treatment of many systemic mycoses. But due to its poor permeability across the membrane, an increased amount of AmB must be administered to patients in clinical situations, thus often resulting in severe side effects. [3][4][5] To lessen the severity of the side effects, AmB is often combined with other antifungal drugs such as the azoles, but coincident with the increased use of antifungal azole derivatives, the incidences of drug resistance have been recently increasing.6-8) Thus investigation of reducing the AmB dose by combining it with other compounds is necessary.Programmed cell death responses have been described in a variety of fungi after exposure of the organisms to a range of conditions such as weak acid stress, oxidative stress, or ultraviolet irradiation. [9][10][11] It was demonstrated that C. albicans could be triggered to undergo an apoptotic cell death response when exposed to H 2 O 2 , intracellular acidification, or AmB. A single-cell death assay was used to show that a majority of AmB-treated cells were nonviable and impermeable to propidium iodide. Apoptotic changes were further confirmed by electron microscopy and by the production of intracellular reactive oxygen species (ROS).12,13) However, the mechanism by which C. albicans undergoes apoptosis remains unclear. In mammalian cells, acquisition of an apoptotic morphology depends on the activation of cysteine dependent aspartate specific proteases (caspases). Functional analyses of genes in Saccharomyces cerevisiae have revealed similarities between fungal apoptosis and apoptosis in mammalian cells. A homologue of mammalian caspases, YCA1 (Yeast Caspase 1), has been identified. Overexpression of YCA1 increases hydrogen peroxide-induced caspase-like activity and apoptosis in S. cerevisiae.14-17) Caspase superfamily consists of three families; YCA1 belongs to the metacaspase family of enzymes which are found in plants, fungi and protozoa. We recently found that CaMCA1, a homologue of S. cerevisiae metacaspase YCA1, was involved in oxidative stress-induced apoptosis in C. albicans. 18)Baicalein (BE) is a compound that was originally extracted from the Scutellaria baicalensis root, and it is a promising herbal medicine in Chinese Pharmacopoeia (Fig. 1). BE exhibits antioxidant, antibacterial, antiviral, and antifungal activities. [19][20][21][22][23] In this study, we described a synergic effect of BE with AmB against C. albicans and the potential mechanism. Our results showed that combination of BE and AmB enhanced C. albicans apootosis accompanied with an increase of reactive oxygen species (ROS). We also found that AmB could induce the caspase activity and mRNA expression of the gene encoding C. albicans caspase, CaMCA1...