Eucalypti Globuli Folium was collected in Cao Pu Zhen, Anning City, Kunming, Yunnan Province, China and authenticated by Dr. Gao Li of the Yunnan Institute of Materia Medica according to the Flora Reipublicae Popularis Sinicae . Herbarium voucher specimens were deposited at the museums of Yunnan Institute of Materia Medica, Yunnan Province, China and the Institute of Chinese Medicine, The Chinese University of Hong Kong with the voucher specimen numbers 20100074 and 2012-3358, respectively.
All of the chemicals used in the current study were purchased as the reagent grade from Sigma-Aldrich (St. Louis, MO, USA). An In Situ Cell Death Detection Kit was purchased from Roche Diagnostics NZ Ltd (Mt. Wellington, Auckland, New Zealand). SYTOX® Green and LIVE/DEAD® yeast viability kits were purchased from Life Technologies Corporation (Carlsbad, CA, USA).
mentagrophytes ATCC 9129 (ATCC, Manassas, VA, USA) was used throughout this study. This material was cultured at 25 °C on modified Sabouraud dextrose agar (MSDA) slants containing mycological peptone 10 g/L, glucose 40 g/L and agar 15 g/L at a pH in the range of 5.4–5.8.
Isolation of macrocarpal C
Fresh E. globulus leaves were extracted two times for 1 h with 95 % ethanol under reflux conditions. The resulting ethanolic extracts were combined, dried and partitioned with n-hexane. This procedure was repeated one more time, and the resulting n-hexane fractions were combined and subjected to chromatographic purification for the isolation of macrocarpal C, as previously described . The purity of the macrocarpal C obtained in this way was higher than 95 %, as determined by HPLC.
In vitro antifungal susceptibility test
Cultures of the dermatophyte
Pure cultures of the dermatophytes were produced by aerobically culturing the fungi at 35 °C for 1 week on modified Sabouraud dextrose agar (MSDA) slants. A 3-mL portion of saline was added to the slants and the subsequent probing of the colonies with a sterile cotton-tipped swab resulted in the formation of a suspension, which was filtered twice through sterile gauze to eliminate hyphal filaments. The final inoculum of conidia was acquired by the dilution of the mixture with RPMI-1640 (buffered with MOPS) medium to 1–3 × 103 CFU/mL.
Determination of the minimum inhibitory concentration (MIC)
The MIC was defined as the lowest concentration at which the growth of the microorganism was completely inhibited. The M38-A2 broth dilution method was used in the current study according to a slightly modified version of the procedure described by the Clinical Laboratory Standards Institute (CLSI) . Macrocarpal C was diluted with RPMI-1640 to achieve concentrations ranging from 500 to 0.06 mg/L in the U-shaped wells of a 96-well microtiter plate. Equal volumes of drug solution and microbial suspension were mixed to make up a final volume of 200 µL. Growth control wells containing RPMI-1640 and microorganisms on each microtiter plate were used for quality control. Sterility control wells containing drug and RPMI but no microorganism were also included in each plate. The plates were then wrapped with paraffin to prevent losses due to evaporation and incubated at 35 °C. The endpoints were visually read every 24 h with a reading mirror and compared with the growth control for up to 4 days. All of these experiments were independently performed in duplicates thrice.
In vitro mechanistic studies
Effects on the membrane permeability
This assay was carried out by examining the uptake of SYTOX® Green, which is a high-affinity nuclear stain capable of entering cells with compromised membranes . Fungal cells were incubated in the presence of macrocarpal C, terbinafine hydrochloride, nystatin (positive control) or phosphate buffer saline (negative control) at 37 °C for 24 h. At the end of the incubation, SYTOX® Green was added to the fungal cultures at 0.5 µM (final concentration). After 10 min, the fluorescence emitted by the fungal cells was measured with a fluorescence spectrometer (SpectraMax i3 Multi-Mode Detection Platform, Molecular Devices, LLC, Sunnyvale, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 540 nm.
Effect on the production of reactive oxygen species (ROS)
The cell-permeable and fluorogenic probe 5-(and-6)-carboxy-2′,7′-dihydrodichlorofluorescein diacetate (carboxy-H2DCFDA) was used to determine ROS production in the fungal cells . The fungal cells were cultured for 3 h in the presence or absence of macrocarpal C. The medium was then replaced with PBS. After incubation with 25 µM carboxy-H2DCFDA in PBS at 37 °C for 30 min, the cells were washed twice with fresh pre-warmed PBS. The fluorescence emitted by the fungal cells was quantified with a fluorescence spectrometer at an excitation wavelength of 488 nm and an emission wavelength of 540 nm.
Effects on DNA fragmentation
A TUNEL assay was conducted to detect the occurrence of any DNA fragmentation . Briefly, T.
mentagrophytes cells were incubated with macrocarpal C at 1 × MIC for 3 h, washed twice with PBS and fixed with a fixative solution of 4 % paraformaldehyde in PBS (pH 7.4) for 1 h at 20 °C. The cells were then rinsed twice with PBS and incubated on ice for 2 min with the permeabilization solution described above. The cells were subsequently rinsed with PBS and labeled using an In Situ Cell Death Detection Kit according to the manufacturer’s instructions. Briefly, a 50 µL sample of the TUNEL reaction mixture was added to the cells, and the resulting mixture was incubated at 37 °C for 60 min in the dark. The cells were then rinsed with PBS and examined using a fluorescence spectrometer.
All of the data in this study have been presented as the mean values ± standard deviations (SD) and analyzed using Student’s t test. The differences between control and test groups were considered statistically significant for P < 0.05. Multiple comparisons among groups were conducted using one-way analysis of variance (ANOVA) followed by post hoc Tukey test, with significance level α set at 0.05. Dose-dependent changes were determined by visually inspecting the graphs.