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...
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