This study evaluated the antifungal activity and cytotoxicity profile of the ellagitannin punicalagin, a compound extracted from the A. in time-kill curves, but it was not able to achieve a statistically significant reduction of fungal growth suggesting a fungistatic effect of the compound. cytotoxicity studies using the two cell lines showed that punicalagin has low activity on these cells and no activity on sheep erythrocytes. Morphological changes were seen in the yeasts strains studied when treated with punicalagin. Therefore, punicalagin is a potential antifungal for important pathogenic yeasts and presents a low cytotoxicity profile associated with no hemolytic effects. CUDC-907 cost A. St.-Hil, Cytotoxicity, Antifungal activity, species Complex INTRODUCTION Punicalagin extracted from A. St.-Hil (Lythraceae) is a compound that has medicinal properties 1 . is a plant known in Brazil as dedaleiro ou pacari. In folk medicine, it is used as wound healing, antipyretic, antidiarrheal, and in the treatment of gastritis, ulcers and cancer 2 , 3 . The ellagitannin punicalagin has shown activity against species complex, dermatophytes and some species of have a protracted course of illness 10 , and it is well known that isolates are significantly less susceptible to azoles than the isolates of leaf against species complex, as well morphological change analyses induced in yeast strains by the presence of punicalagin. MATERIALS AND METHODS Leaves from A. St.-Hil (Lythraceae) were submitted to ultrasound extraction with acetone:water (70:30) and punicalagin was characterized by HPLC/UV and ESI-TOF MS, 1D and 2D NMR spectroscopic evaluations as described by Carneiro ATCC 28957, ATCC 24065, six clinical isolates of species complex obtained from patients with meningitis in a tertiary hospital of Goiania, Goias State, Brazil (three – L1, L9, and L20 and three – L3, L29, and L30). These isolates were collected in a previous study approved by the Bioethics Committee of Hospital de Doen?as Tropicais de Goias (protocol 027/07). Microdilution broth assay The activity of punicalagin was measured by means of the microdilution broth method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines M27-A3 (CLSI 2012 14 ) and M27- S4 (CLSI 2012 15 ) for yeasts. Serial twofold dilutions of the pure compound were prepared in 96-well microplates. The minimal inhibitory concentration CUDC-907 cost (MIC) was defined as the lowest concentration that resulted in the total inhibition growth analyzed by visual inspection, giving a numerical score in comparison with the growth present in the control (drug-free) sample. The minimal fungicidal concentration (MFC) was determined by an inoculum of 10 L from each well containing the MIC and up to 4 MIC seeded in petri plates containing Sabouraud dextrose agar (SDA), incubated for 72 h at 35 C. The MFC was defined as the lowest concentration of the compound that resulted in growth of less than two colonies representing the death of CUDC-907 cost 99% of the original inoculum. Fluconazole was used as the control. Time kill assay Cell growth and death rates of the yeast strains studied were analyzed according to the modified protocols of Klepser complex with a MIC range of 0.5 to 4.0 g/mL and MFC 256 g/mL for all isolates. At the concentration of 0.5 g/mL the compound inhibited 37% of the C. species Fes complex isolates. Fluconazole showed a MIC range of 0.5 to 8.0 and a MFC range of CUDC-907 cost 1 to 64 g/mL. Time kill curve The time-kill curve (Figures 1A and ?and1B)1B) showed reduction in the number of CFU/mL of the complex cells treated with punicalagin at concentrations corresponding to the MIC of 4 g/mL and 2 MIC (8 g/mL). The major reduction was observed with ATCC 28957 at a concentration corresponding to 2 MIC at 12 h. The difference was not statistically significant between treated and non-treated cells. Amphotericin B had maximum fungicidal activity after 12 h of incubation at concentrations equivalent to 1 g/mL for ATCC 24065 and ATCC 28957. Open in a separate window Figure 1 Growth curve of ATCC 24065 (A) and ATCC 28957 (B) without treatment and treated with punicalagin at the values corresponding to ? of the minimum.