C. albicans SC5314 and other Candida standard strains were obtained from the China General Microbiological Culture Collection Center (CGMCC, Beijing) (Yang et al., 2024b). The C. albicans clinical isolates were provided by the Clinical Laboratory of the Second Hospital of Jilin University (Yang et al., 2024a). These strains were kept on yeast extract–peptone–dextrose (YPD) agar at 4 °C in a refrigerator, and before each assay, one colony was transferred into YPD liquid medium for overnight culture at 28 °C with a rotation of 140 rpm. AmB and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) were purchased from Sangon Biotech (Shanghai, China). Dioscin (SD8350) was purchased from Solarbio (Beijing, China). Propidium iodide (PI) was from Sigma Aldrich (Shanghai, China). Bis(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3)) was purchased from MCE Company (Shanghai, China).

Checkerboard method and CLSI-M27-A3 guidelines were followed to determine the combinational effects of dioscin and AmB, as described previously (Yang et al., 2019a). Briefly, Candida cells from overnight cultures were collected through centrifugation (3,000×g, 5 min) and diluted to 2 × 10³ cells/mL in Roswell Park Memorial Institute (RPMI) 1640 medium. A total of 100 μL of such cell suspension was added to each well of a 96-well plate, followed by the addition of dioscin (0.125–8 μg/mL) and AmB (0.156–10 μg/mL) using a two-times serial dilution. After 24-h incubation at 37 °C, the minimal inhibitory inhibition (MIC) of each agent was inspected by the naked eye. The interaction of the combination was classified as follows: synergistic when the fractional inhibitory concentration index (FICI) was ≤ 0.5; additive when FICI was > 0.5and ≤ 1; indifferent when FICI was > 1 and ≤ 4; and antagonistic when FICI was > 4 (Barchiesi et al., 1998).

To determine whether the combination of dioscin and AmB also produces a synergistic effect on biofilm formation, a checkerboard assay was performed within the framework of the biofilm formation assay. Freshly prepared C. albicans SC5314 cell suspension in RPMI 1640 medium (10⁶ cells/mL) was added to 96-well plates, followed by the addition of dioscin (0.125–8 μg/mL) and AmB (0.156–10 μg/mL) in a twofold serial dilution. Wells containing only medium were set as the blank control, while wells containing fungal suspension plus the same volume of dimethyl sulfoxide (DMSO) were set as the negative control. After 24 h of growth in the presence of different drug combinations, the biofilms were washed, and 100 μL XTT solution was added to each well. After a 2-h incubation in the dark, the supernatants (70 μL) were transferred to a new plate. The optical density (OD) of each well at 490 nm (OD₄₉₀) was detected with a multifunctional plate reader (VarioSkan, Thermo, Vantaa, Finland). The viability of fungal biofilms in each well was calculated as follows: viability = (OD_{490 treatment} − OD_{490 blank})/(OD_{490 control} – OD_{490 blank}) (Yang et al., 2018b).

After the synergistic effects on biofilm formation were confirmed, specific concentrations of dioscin (1 μg/mL) and AmB (0.625 μg/mL) were selected for further assays. This combination was then tested on both biofilm formation and biofilm development. For tests on biofilm development, biofilms formed in the absence of any drugs were washed with phosphate-buffered saline (PBS) and exposed to fresh RPMI 1640 medium containing no drug, dioscin (1 μg/mL), AmB (0.625 μg/mL), and dioscin (1 μg/mL) plus AmB (0.625 μg/mL). After 24 h, the biofilms were washed and subjected to the XTT assay, as described earlier. These assays were performed in triplicate and repeated three times.

To show the direct effects of this combination on biofilm formation, C. albicans biofilms formed in the presence of control (DMSO), dioscin (1 μg/mL), AmB (0.625 μg/mL), and dioscin (1 μg/mL) plus AmB (0.625 μg/mL) were stained with calcofluor white (CFW; final concentration: 20 μg/mL) and washed with PBS, followed by confocal laser scanning microscope (CLSM; Olympus FV3000 Tokyo, Japan) analysis. The recordings of biofilms were performed in a 3D scanning mode under a × 40 objective, with a step size of 2 µm. The reconstruction was performed with Imaris (version 7.2.3).

C. albicans SC5314 cells from an overnight culture were prepared in RPMI 1640 medium to get a density of 10⁶ cells/mL. Such suspensions were transferred into 96-well plates and exposed to control (0.5 μL DMSO), dioscin (1 μg/mL), AmB (0.625 μg/mL), and dioscin (1 μg/mL) plus AmB (0.625 μg/mL) at 37 °C for 1.5 h. Wells containing medium only were used as the blank control. PBS washing and XTT reduction assay, as described earlier, were then performed to detect the relative viability of the adherent cells remaining on the bottoms of the plates. The relative adhesion of each treatment was calculated as follows: (OD_{490 treatment} − OD_{490 blank})/(OD_{490 control} − OD_{490 blank}).

C. albicans cells in RPMI 1640 medium (10⁶ cells/mL), obtained through dilution from overnight YPD cultures in which the Candida cells were mainly in the yeast form, were exposed to control (DMSO), dioscin (1 μg/mL), AmB (0.625 μg/mL), and dioscin (1 μg/mL) plus AmB (0.625 μg/mL) at 37 °C for 4 h. Candida cells in the absence of hyphal growth-inhibiting agents grew into hyphae, whereas agents inhibiting hyphal growth caused reduced hyphae formation or shorter hyphae. Cell morphologies were observed under an inverted microscope (objective: × 40).

In this assay, freshly prepared C. albicans SC5314 suspensions in RPMI 1640 medium (at a density of 10⁶ cells/mL) were exposed to DMSO (control), dioscin (1 μg/mL), AmB (0.625 μg/mL), and dioscin (1 μg/mL) plus AmB (0.625 μg/mL). The suspensions were incubated at 28 °C, with rotation at 140 rpm. At 0, 2, 4, 6, 8, 12, and 24 h, 100 μL cultures were fetched from each treatment for 10 × serial dilution and spreading on Sabouraud dextrose (SD) agar plates. The number of colony-forming units (CFU) on each agar plate was counted manually after the plates were incubated at 37 °C for 48 h.

AmB was shown to induce reactive oxygen species (ROS) in C. albicans cells (Guirao-Abad et al., 2017), while dioscin can facilitate excessive ROS production in cancer cells (Zheng et al., 2022), and rimonabant can potentiate AmB by elevating cellular oxidative stress in C. albicans (Zhang et al., 2021). Based on these findings, we tested whether dioscin can enhance the ROS production caused by AmB. Fresh C. albicans suspensions in 96-well plates were treated with control (DMSO), dioscin (1 μg/mL), AmB (0.625 μg/mL), and dioscin (1 μg/mL) plus AmB (0.625 μg/mL) for 4 h at 37 °C. Cells were then stained with DCFH-DA (Solarbio, Beijing, China; final concentration: 10 μM) in the dark for 20 min. After washing with PBS three times to remove excessive dye, fungal cells were sent to CLSM (Olympus FV3000, Japan) for ROS detection, under a × 40 objective. The excitation wavelength of the laser used was 488 nm.

The freshly prepared C. albicans SC5314 suspensions (in RPMI 1640 medium, 10⁶ cells/mL) were transferred into wells of a 96-well plate and challenged with control (DMSO), dioscin (1 μg/mL), AmB (0.625 μg/mL), and dioscin (1 μg/mL) plus AmB (0.625 μg/mL), for 4 h. The fungal cells were then stained with PI (stock solution: 1 mg/mL in PBS; final working concentration: 10 μg/mL) for 10 min. After washing with PBS, the cells were sent for CLSM analysis under a × 40 objective, using the laser at 561 nm.

To detect whether the cell membrane potential was affected by dioscin and AmB, the voltage-sensitive fluorescent dye DiBAC₄(3) (stock solution: 10 mM in DMSO; final concentration: 1 μM) was used to stain C. albicans cells. This assay was performed in 96-well plates. Fungal cells (at a density of 10⁶ cells/mL in RPMI 1640 medium) were exposed to control (DMSO), dioscin (1 μg/mL), AmB (0.625 μg/mL), and dioscin (1 μg/mL) plus AmB (0.625 μg/mL) for 4h at 37 °C. The fungal cells were then stained with DiBAC₄(3) for 10 min. After washing with PBS to remove the residual dye DiBAC₄(3), samples were observed with CLSM under a × 40 objective using the laser at 488 nm.

To exclude the interference of cellular morphologies that may be caused by the RPMI 1640 medium at 37 °C, C. albicans cells were cultured in YPD medium in scanning electron microscope (SEM) and transmission electron microscope (TEM) assays. For SEM analysis, C. albicans cells (10⁶ cells/mL) were treated with DMSO (control, same volume as in the treatment with dioscin plus AmB) or dioscin (4 μg/mL) plus AmB (2.5 μg/mL) at 37 °C for 4 h, followed by centrifugation and fixation in 2.5% glutaraldehyde at 4 °C overnight. Samples were then transferred to Huiceshi Company (Suzhou, China) for SEM scanning using a Quattro S system (Thermo Scientific, Eindhoven, Netherlands) under a × 10,000 magnification. Samples were scanned in a high-vacuum mode with a voltage of 15 kV; the spot size was 2.0, and the working distance (WD) was 9.1 mm.

For TEM analysis, C. albicans cells (10⁶ cells/mL) were treated with DMSO (control) or dioscin (4 μg/mL) plus AmB (2.5 μg/mL) at 37 °C for 16 h, and then the cells were centrifuged at 3,000×g for 5 min and fixed in 2.5% glutaraldehyde at 4 °C overnight. Samples were sent to Servicebio Inc. (Wuhan, China) for desiccation, embedding (SPI-Pon™ 812, SPI Supplies, West Chester, USA), and ultrathin section (Leica UC7, Leica Microsystems, Wetzlar, Germany) at 80 nm thickness. Sections were placed on copper grids and stained with 2% uranyl acetate for 8 min and then with lead citrate for 8 min, and photographed subsequently with a Hitachi TEM system (HT7800, Hitachi, Tokyo, Japan) under a ×10,000 magnification. The photos were obtained in a high-contrast mode with an accelerating voltage of 80 kV.

The results shown here were mean ± standard deviation from triplicates in three independent assays, and Student’s t-test (by GraphPad Prism 6.02) was used to analyze the statistical significance between drug-free controls and treatments. p < 0.05 was considered significant.

As a commonly used method to evaluate the effects of drug combinations, checkerboard assays were employed in the framework of CLSI-M27-A3-guided MIC tests. As shown in Table 1, the combination of dioscin and AmB could produce a synergistic effect in the standard strain C. albicans SC5314 and in the fluconazole-resistant strain C. albicans ATCC10231. Although this combination did not produce synergistic interactions in C. glabrata ATCC2001 and C. parapsilosis ATCC22019, their interactions were additive. While dioscin did not inhibit the planktonic growth of C. krusei ATCC6258 and C. tropicalis ATCC7349 (MIC higher than 2,048 μg/mL, as shown in Table 1), the combination of dioscin and AmB produced strong synergistic interactions in these two strains (the FICI in these two strains were both below 0.252). The interactions between dioscin and AmB were also tested in four clinical strains. The effects of this combination were synergistic (FICI = 0.5) in two isolates, while they were additive in another two isolates (FICI = 0.75). In general, the combination of dioscin and AmB can produce synergistic or additive effects in various Candida species and isolates.

To test whether dioscin and AmB also have synergistic effects on biofilm formation and development, we also performed a checkerboard assay within the framework of biofilm formation assays. The biofilms exposed to various concentration combinations of dioscin and AmB were subjected to an XTT assay to quantify the inhibition caused by those combinations. As shown in Figure 1A, the inhibition of AmB against C. albicans biofilm was gradually enhanced by increasing the concentration of dioscin. The combination of dioscin (1 μg/mL) and AmB (0.625 μg/mL) produced a synergistic effect. Thus, this combination was further tested on biofilm formation and development to quantify the inhibitory effects. As shown in Figure 1B, the biofilms exposed to dioscin (1 μg/mL) and AmB (0.625 μg/mL) showed only 16% and 49% inhibition on viability, respectively, while biofilms treated with dioscin (1 μg/mL) plus AmB (0.625 μg/mL) inhibited the viability by more than 97%. This confirmed the results from the checkerboard assay (Figure 1A). As for preformed biofilms, this combination also showed more suppression than the sum of each group (Figure 1C). Dioscin (1 μg/mL) and AmB (0.625 μg/mL) suppressed the development of biofilms by about 11% and 42%, respectively, while dioscin (1 μg/mL) plus AmB (0.625 μg/mL) showed approximately 60% inhibition, further confirming the synergy of this combination in suppressing biofilm development. The results of 3D structures from CLSM recordings also confirmed the synergy between dioscin and AmB in suppressing biofilm formation (Figure 1D). The biofilms formed in the presence of dioscin and AmB were much sparser and had fewer and shorter hyphae compared to others.

Time-kill assays were also performed on C. albicans SC5314. As shown in Figure 2, dioscin + AmB showed potent Candida-killing activity after 4 h, as the live fungal cell counts (CFU/mL) dropped 3 log₁₀ at 6 h and lowered further as the exposure time increased. The fungicidal effects were more pronounced if the concentrations of dioscin and AmB were both doubled, which obviously lowered the viable C. albicans cells from the second hour posttreatment, reaching a 3 log₁₀ (CFU/mL) reduction at 4 h. In contrast, the counts of viable cells exposed to 1 μg/mL dioscin were similar to those of the control, while treatment with 0.625 μg/mL AmB only slightly lowered the viable C. albicans cell numbers. These killing curves also confirmed the synergistic effects of dioscin and AmB, as well as the long-lasting killing effects of this combination on C. albicans (Deng et al., 2025).

As shown in Figure 3, although both dioscin and AmB could reduce the adhesion of C. albicans cells to polystyrene surfaces of 96-well plate bottoms by about 30% and 55% relative to the drug-free controls, respectively, the combination of dioscin and AmB caused a reduction of about 95% in adhesion. The higher adhesion inhibition of the combination compared with the sum of the inhibition by dioscin and that by AmB further confirmed the synergy between dioscin and AmB.

The effects of dioscin and AmB on hyphal growth were evaluated in RPMI 1640 medium at 37 °C. As shown in Figure 4, 1 μg/mL dioscin alone or 0.625 μg/mL AmB alone could hardly inhibit the growth. However, the combination of both agents could suppress hyphal growth and kept most of the C. albicans cells in yeast form. This suggested that dioscin and AmB produced a synergistic effect on inhibiting hyphal growth.

Although AmB can cause ROS production in C. albicans, as shown in our results and other reports, we did not notice an obvious rise in intracellular ROS production in cells exposed to either dioscin or AmB. Interestingly, dioscin alone did not induce ROS overproduction in C. albicans, although many publications have shown that dioscin can facilitate ROS overproduction in cancer cells. In fact, the combination of dioscin and AmB caused less ROS than AmB (Figure 5), indicating that the synergistic effects of this combination were not due to elevated intracellular oxidative stress.

To detect the cell membrane damage caused by dioscin and AmB, PI staining was employed. As shown in Figure 6, 1 μg/mL dioscin caused more cells to stain with PI compared with the drug-free control, while 0.625 μg/mL AmB almost did not increase cell membrane permeability. In contrast, the combination of dioscin and AmB caused most cells to be stained with PI. This suggested that dioscin and AmB caused much more damage to the cell membrane than either one alone. In other words, dioscin greatly increased the cell membrane damage caused by AmB.

To further explore the impact of dioscin and AmB on C. albicans cell membrane, the voltage-sensitive dye DiBAC₄(3) was used. As shown in Figure 7, cells from the drug-free control were almost not stained by DiBAC₄(3), indicating no depolarized cells. Some of the cells treated with dioscin or AmB for 4 h showed green fluorescence, indicating depolarized cell membrane potential. The combination of dioscin and AmB caused depolarization of the cell membrane potential in most cells, also showing a synergistic effect.

We also performed SEM analysis to show the changes in cell ultrastructure. As shown in Figure 8, C. albicans cells without drug treatment showed round or ovoid shapes and had smooth surfaces (Figures 8A–C). Treatment with dioscin and AmB for 16 h caused severe cellular changes, and rough cell surfaces could be seen. Even the collapse of cells was observed in dioscin plus AmB-treated cells (Figures 8D–F), which may indicate the loss of intracellular materials. This may suggest damage to the cell membrane, although cell membrane changes could not be observed directly by SEM. Under TEM (Figure 9), treatment with dioscin and AmB caused the loss of intracellular materials.