Abstract Apigenin, a natural flavone, has been well characterized for its their anticarcinogenic property; however, its bioactivity against pathogenic fungi has not been investigated in detail. In this study, we examined the antifungal activity and mode of action of apigenin. Apigenin inhibited the growth of fungal pathogens, which induced superficial infection and reduced biofilm mass. Three-dimensional flow cytometric analysis demonstrated that apigenin induced morphological changes, especially cell shrinkage, in Candida albicans. We investigated the cause of cell shrinkage using the cyanine dye 3,3΄-dipropylthiacarbocyanine iodide. Results revealed that apigenin altered the cell membrane potential. Apigenin also induced membrane dysfunction, and increased cell permeability to 1,6-diphenyl-1,3,5-hexatriene and propidium iodide. We observed the influx and efflux of fluorescent molecules of varying molecular weights and radii across large unilamellar vesicles and live cells that had been treated with apigenin. Membrane disruption facilitates the release of small intracellular constituents such as ions and sugars, but not proteins. These findings suggested that apigenin exerted an antifungal activity by inducing membrane disturbances, which led to cell shrinkage and leakage of intracellular components. antifungal activity, apigenin, flavone, membrane active mechanism INTRODUCTION Historically, natural products derived from various organisms, such as microorganisms, plants and animals, have played important roles in healthcare and have been widely used for treating human diseases (Strobel and Daisy 2003; Cragg and Newman 2013). Secondary metabolites obtained from natural sources, referred to as natural products, have revolutionized the diagnosis, prevention and treatment of human diseases (Cragg, Newman and Snader 1997; Demain 2014). They have been employed as medicinal agents and continue to contribute toward drug discovery programs in the pharmaceutical industry and other research organizations (David, Wolfender and Dias 2015). Therefore, natural products have received considerable attention and act as attractive sources of potential drug molecules (David, Wolfender and Dias 2015). Secondary metabolites present in terrestrial and marine plants are called phytochemicals (Goossens et al. 2003; Mehdinezhad, Ghannadi and Yegdaneh 2016), and are commercially used as dietary supplements (Krochmal et al. 2004). These plant metabolites possess therapeutic potential for treating human diseases, and have been particularly used in Asia (Alves and Rosa 2007; Choi et al. 2014). They can be classified into several categories. Among these, carotenoids and polyphenols are well known. Carotenoids, which absorb light energy, are beneficial for preventing certain cancers and eye diseases (Krinsky and Johnson 2005; Rao and Rao 2007). Polyphenols, the simplest bioactive phytochemicals, are ubiquitous and help in treating degenerative diseases (Meyer et al. 2006). Flavonoids consist of a large group of polyphenolic compounds that exhibit several bioactivities such as antioxidant activity and regulation of reactive oxygen species (Manach et al. 2004). Apigenin, a dietary flavonoid found in parsley and flowers of the chamomile plant (Shukla and Gupta 2010; Kumar and Pandey 2013), has been reported to possess medicinal properties including antioxidant, anti-inflammatory and antitumor properties (Chuang et al. 2009; Lii et al. 2010; Cardenas et al. 2016). Apigenin-mediated antimicrobial activity has also been reported (Yordanov et al. 2008; Ozcelik, Kartal and Orhan 2011). However, the antifungal mechanism of action of apigenin needs to be elucidated (Yordanov et al. 2008; Cheah, Lim and Sandai 2014; Singh, Kumar and Joshi 2014). In this study, we purified apigenin from Aster yomena (also called Kalimeris yomena), a herb traditionally used as a food ingredient and in the treatment of inflammation, cold and asthma (Choi et al. 2014; Kim et al. 2014). Here, we attempted to investigate the antifungal activity and mode of action of apigenin extracted from A. yomena. MATERIALS AND METHODS Isolation of apigenin Aerial parts of A. yomena Makino (Asteraceae) were collected, air-dried (yield = 1.9 kg) and subjected to three washes (under reflux) with methanol (MeOH), resulting in the production of 120.1 g residue. The MeOH extract was resuspended in water and partitioned sequentially using equal volumes of dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (BuOH). Each fraction was subjected to vacuum evaporation, which yielded CH2Cl2 (23.6 g), EtOAc (15.2 g), n-BuOH (48.8 g) and water (48.2 g) extracts. The CH2Cl2 fraction (15 g) was subjected to silica gel column chromatography (CC) using a gradient solvent system of hexane:acetone (100:1→1:1), and 12 subfractions (D1–D12) were collected. Subfraction D11 (2.5 g) was subjected to YMC Sep-Pack (YMC, Kyoto, Japan) fractionation using 50%, 80% and 100% MeOH as elution solvents, resulting in three subfractions (D111–D113). Subfraction D113 (900.9 mg) was subjected to silica gel CC using a gradient solvent system of chloroform:MeOH (30:1→100% MeOH). Of the six subfractions that were collected (D1131–D1136), subfraction D1135 (113.2 mg) was purified by semipreparative high-performance liquid chromatography (75% MeOH), resulting in the isolation of compound 1 (apigenin, 3.9 mg) (Ersoz et al. 2002; Kim et al. 2014). Apigenin was dissolved in dimethyl sulfoxide (DMSO) to a reach a final concentration of 10 mg/mL. Apigenin (a yellow powder) was subjected to fast atom bombardment mass spectrometry (FAB-MS), proton nuclear magnetic resonance (1H NMR) and 13C NMR, yielding the following data: FAB-MS m/z: 271 [M+]; 1H-NMR (500 MHz, CD3OD) δ: 7.89 (2H, d, J = 8.8 Hz, H-2΄ and H-6΄), 6.92 (2H, d, J = 8.8 Hz, H-3΄ and H-5΄), 6.72 (1H, s, H-3), 6.45 (1H, d, J = 2.1 Hz, H-6), 6.16 (1H, d, J = 2.1 Hz, H-8); and 13C-NMR (125 MHz, CD3OD) δ: 181.7 (s, C-4), 165.2 (s, C-5), 163.8 (s, C-2), 161.5 (s, C-4΄), 161.4 (s, C-7), 157.5 (s, C-9), 128.5 (d, C-2΄,6΄), 121.2 (s, C-1΄), 116.1 (d, C-3΄, 5΄), 103.5 (s, C-10), 102.8 (d, C-3), 99.2 (d, C-6), 94.2 (d, C-8) (Fig. 1A). Figure 1. View largeDownload slide (A) Structure of apigenin. (B) Effects of apigenin and amphotericin B on biofilm biomass. Matured C. albicans biofilms were incubated with apigenin or amphotericin B at their respective MICs for 48 h. Biofilm biomass was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in microplates. Results are presented as the mean ± SD of OD570 values from three independent experiments. **P < 0.01; ***P < 0.001 (treated versus control; Student's t-test). Figure 1. View largeDownload slide (A) Structure of apigenin. (B) Effects of apigenin and amphotericin B on biofilm biomass. Matured C. albicans biofilms were incubated with apigenin or amphotericin B at their respective MICs for 48 h. Biofilm biomass was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in microplates. Results are presented as the mean ± SD of OD570 values from three independent experiments. **P < 0.01; ***P < 0.001 (treated versus control; Student's t-test). Fungal strains and antifungal susceptibility Candida albicans (ATCC 90028) and C. parapsilosis (ATCC 22019) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Malassezia furfur (KCTC 7744), Trichophyton rubrum (KCTC 6345) and Trichosporon beigelii (KCTC 7707) were obtained from the Korean Collection for Type Cultures (KCTC). All fungal strains were cultured in YPD broth (BD Diagnostics, Sparks, MD, USA) with aeration at 28°C, except M. furfur, which was cultured at 32°C in modified YM broth (BD Diagnostics) containing 1% olive oil. Growing fungal cells (1 × 103 cells/mL) were inoculated into YPD broth and dispensed into microtiter plates (0.1 mL/well). Minimum inhibitory concentration (MIC), defined as the concentration of drug inhibiting 90% of cell growth, apigenin and amphotericin B (Sigma-Aldrich, St. Louis, MO, USA), was determined by a 2-fold serial dilution via the Clinical and Laboratory Standards Institute method (Lee et al. 2015). Following incubation for 12–18 h, growth was measured by monitoring the absorption at 600 nm using a microtiter ELISA Reader (Molecular Devices Emax, Sunnyvale, CA, USA). MIC values were determined using three independent assays. Biofilm biomass assessment Candida albicans cell suspension (1 × 106 cells/mL in RPMI1640) was seeded into individual wells of a sterile, polystyrene, 96-well flat bottom plate (Falcon, Becton-Dickinson Labware, USA). After 48 h, apigenin or amphotericin B was added at their previously determined MICs to the respective wells. The matured C. albicans biofilms were allowed to incubate with either apigenin or amphotericin B for 24 h at 37°C. The wells were washed three times with phosphate-buffered saline (PBS) to remove free-floating fungi, and the biofilms formed by adherent organisms were stained with 0.1% (w/v) 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich) for 4 h. The plates were thoroughly rinsed with deionized water to remove excess MTT and air-dried. Next, DMSO was added to the plates and optical density (OD) of the stained adherent fungi was measured at 570 nm (OD570) using the BioTek ELx800 Absorbance Reader (BioTek Instruments, Winooski, VT, USA). The OD values indicated the degree of cell adhesion and biofilm formation. The percentage of biofilm inhibition was calculated using the following equation: [1 − (OD570 of cells treated with compound/OD570 of untreated control)] × 100 (Pierce et al. 2008). Measurement of morphological changes in cells Candida albicans cells (2 × 105 cells/mL) suspended in PBS were treated with apigenin or amphotericin B (5 μg/mL) for 4 h at 28°C. After incubation, the cells were harvested by centrifugation and resuspended in PBS. Morphological changes were analyzed using the FACSVerse flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). For each sample, non-stained live cells were evaluated by excitation with a 488-nm light from an argon ion laser by determining their position on a forward scatter (FSC) versus side scatter (SSC) contour plot. For microscopic analysis, live cells were resuspended in PBS, the compounds were added and all mixtures were incubated for 4 h. After incubation, the cells were harvested by centrifugation and resuspended in PBS. Morphological changes were observed using a fluorescence microscope (Nikon Eclipse Ti-S; Tokyo, Japan), and the average diameter was measured using the ImageJ software. Analysis of the membrane potential A potential-sensitive probe, namely 3,3΄-dipropyl thiacarbo cyanine iodide [DiSC3(5); Sigma-Aldrich], was used to determine the membrane electrical potential of C. albicans. The cells (2 × 105 cells/mL), cultured in YPD broth and aerated overnight at 28°C, were centrifuged at 12 000 rpm for 5 min and subsequently washed with PBS. To compare the antifungal mode of action, changes in fluorescence induced by apigenin or amphotericin B (5 μg/mL) were monitored using a spectrofluorophotometer at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. All measurements were repeated three times under the same conditions (Park et al. 2008). Propidium iodide influx assay Candida albicans cells (2 × 105 cells/mL), cultured in YPD broth and aerated overnight at 28°C, were centrifuged at 8000 rpm for 5 min and resuspended in PBS with apigenin or amphotericin B (5 μg/mL). After incubation for 4 h at 28°C, the cells were centrifuged, resuspended in PBS and incubated with 9 μM propidium iodide (PI; Sigma-Aldrich) for 5 min at room temperature. Membrane permeability of cells was analyzed using the FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) (Lee, Woo and Lee 2016). Assessment of plasma membrane fluorescence intensity Changes in C. albicans membrane dynamics were monitored by measuring the fluorescence emitted from the plasma membrane of cells labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH; Molecular Probes, Eugene, OR, USA). Candida albicans cells (2 × 105 cells/mL), cultured in YPD broth and aerated overnight at 28°C, were incubated with apigenin or amphotericin B (5 μg/mL) for 4 h at 28°C and fixed using 0.37% formaldehyde. The cells were washed with cold PBS, and subjected to two freeze–thaw cycles using liquid nitrogen and warm PBS. The cell suspensions were incubated with 0.6 mM DPH for 45 min at 28°C and washed three times with PBS. The fluorescence intensity of DPH was measured using the RF-5301PC spectrofluorophotometer (Shimadzu, Japan) at 350/425 nm (excitation/emission) (Lee, Woo and Lee 2016). Preparation of liposomes Large unilamellar vesicles (LUVs) containing fluorescein isothiocyanate (FITC)-labeled dextran (FD) (Sigma-Aldrich) were prepared at a concentration of 3 mg/mL by adding phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and ergosterol in a 5:4:1:2 (w/w/w/w) ratio (Makovitzki, Avrahami and Shai 2006) in chloroform. The lipid mixtures (1 mL) were evaporated in a round flask for 10 min under vacuum. After evaporation, the flask was filled with argon gas and incubated overnight. The chamber was filled with a dye buffer solution (pH 7.4) containing 10 mM Tris, 150 mM NaCl and 0.1 mM ethylenediaminetetraacetic acid. The suspension was subjected to 13 freeze–thaw cycles in liquid nitrogen and subsequently extruded through polycarbonate filters (two stacked, 200-nm pore filters) with the LiposoFast extruder (Avestin, Ottawa, Canada). Untrapped FD was removed by gel filtration using a Sephadex G-50 column (Lee et al. 2015). Estimation of damage size formed in artificial liposomes To evaluate the extent of membrane damage induced by apigenin and amphotericin B, the following FD molecules were used: FD4 [molecular weight (MW), 3.9 kDa; Stokes–Einstein radius, 1.4 nm], FD10 (MW, 9.9 kDa; Stokes–Einstein radius, 2.3 nm) and FD20 (MW, 19.8 kDa; Stokes–Einstein radius, 3.3 nm). Liposomes containing 2 mg/mL FD were prepared in a dye-buffer solution (Lee et al. 2015). A suspension of liposomes treated with apigenin and amphotericin B (1 mL, final volume) was stirred for 10 min in the dark and centrifuged at 12 000 rpm for 10 min. The integrity of liposomes was monitored by measuring the fluorescence intensity at 494/520 nm (excitation/emission) using the RF-5301PC spectrofluorophotometer. To determine the maximum fluorescence intensity due to 100% FD leakage, 0.1% Triton X-100 (30 μL) was added to the vesicles. The percentage of FD leakage caused by the compounds was calculated as follows: dye leakage (%) = 100 × (F − F0)/(Ft − F0), where F represents the fluorescence intensity after addition of the compounds, and F0 and Ft represent the fluorescence intensities without the compounds and with Triton X-100, respectively (Lee et al. 2015). Measurement of flux of cytosolic components The effect of apigenin and amphotericin B activities on the flux of cell components across the membrane was estimated by measuring the efflux of potassium ions from C. albicans. Overnight-cultured C. albicans cells (2 × 105 cells/mL) were incubated with 5 μg/mL apigenin or amphotericin B for 4 h at 28°C, and centrifuged at 12 000 rpm for 5 min to remove cell debris. Potassium ion concentration in the supernatant was measured using the Orion Star A214 pH/ISE meter (Thermo Scientific, Singapore) and expressed as a percentage of the total number of free potassium ions released (Kanmani and Lim 2013). It was calculated as follows: potassium release (%) = 100 × ([K+] − [K+]0)/([K+]t − [K+]0), where [K+] represents the potassium release achieved after addition of the compounds, and [K+]0 and [K+]t represent the potassium release under conditions with medium and with sonicated cells, respectively. Cytosolic calcium levels were analyzed using the intracellular calcium indicator Fura-2 acetoxymethyl (AM) ester (Molecular Probes). Briefly, the apigenin- or amphotericin B-incubated cell suspension was washed twice in Krebs buffer (pH 7.4), and treated with 0.01% pluronic acid F-127 (Molecular Probes) and 1% bovine serum albumin. Cells were stained with 5 μM Fura-2AM at 37°C for 40 min, and washed twice in calcium-free Krebs buffer. The fluorescence intensity of Fura-2AM at 335/505 nm (excitation/emission) was detected using the RF-5301PC spectrofluorophotometer. The presence of reducing sugars in the supernatant of incubated cells was estimated as described by Masuko et al. (2005). Proteins in the supernatant were estimated using the Bradford assay. Statistical analysis Values are reported as the mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined using Student's t-test. Differences between the samples were considered to be significant at P-values < 0.05, <0.01 and <0.001. RESULTS Isolation of apigenin Repeated column chromatography of the extracts of aerial parts of A. yomena yielded a yellow amorphous powder (compound 1) from the soluble CH2Cl2 fraction. A [M + H]+ peak at 271 in the FAB-MS spectrum, along with the 13C NMR analysis, indicated the following molecular formula: C15H10O5. The 1H NMR spectrum of compound 1 exhibited aromatic AA’BB'-type protons at δ7.89 (2H, d, J = 8.8 Hz, H-2΄, and H-6΄) and 6.92 (2H, d, J = 8.8 Hz, H-3΄, and H-5΄), and ABX-type protons at δ6.45 (1H, d, J = 2.1 Hz, H-6) and 6.16 (1H, d, J = 2.1 Hz, H-8). In the 13C NMR spectrum, 15 carbon signals were observed, including one carbonyl carbon at dC 181.7(C-4) and three oxygenated quaternary carbons at dC 165.2(C-5), 161.5(C-4΄) and 161.4(C-7) (data not shown). These results confirmed the presence of a flavonoid skeleton in compound 1 (Fig. 1A). Based on these observations, which were in complete agreement with the literature (Ersoz et al. 2002), compound 1 was identified as apigenin. The purity of isolated apigenin was determined to be 99.8% using HPLC analysis (data not shown). Antifungal and antibiofilm effect of apigenin Apigenin is a secondary metabolite, specifically a flavone, isolated from A. yomena (Fig. 1A). The susceptibility of several pathogenic fungi to apigenin was assessed. Amphotericin B was used as the positive control. Amphotericin B, which is used to treat fungal infections (Ellis 2002), is widely known to form pores by binding sterol in the fungal cell membrane, and its immediate action induces cell death (Yang et al. 2013). As shown in Table 1, apigenin showed antifungal activity at a concentration of 5 μg/mL, while amphotericin B exhibited antifungal activity at 1.3–5 μg/mL. Among the tested strains, C. albicans was not only the most widespread fungal pathogen, but also the primary cause of candidiasis. Therefore, we selected C. albicans as a model organism for this study. To evaluate the antibiofilm activity of apigenin against C. albicans, matured biofilm was incubated for 24 h with apigenin and amphotericin B at 5 and 1.3 μg/mL, respectively. Next, the MTT assay was performed. The absorbance of MTT revealed that apigenin decreased metabolic activity by 31.8% (Fig. 1B). Amphotericin B also exhibited antibiofilm activity by decreasing metabolic activity by 71.9%. Altogether, these results indicated that apigenin shows a potent antifungal effect and reduces biofilm biomass, which is associated with pathogenicity under physiological conditions. Table 1. The antifungal effect of apigenin and amphotericin B. MIC (μg/mL) Fungal strains Apigenin Amphotericin B Candida albicans ATCC90028 5.0 1.3 Candida parapsilosis ATCC22019 5.0 1.3 Malassezia furfur KCTC7744 5.0 2.5 Trichophyton rubrum KCTC 6345 5.0 2.5 Trichosporon beigelii KCTC7707 5.0 1.3 MIC (μg/mL) Fungal strains Apigenin Amphotericin B Candida albicans ATCC90028 5.0 1.3 Candida parapsilosis ATCC22019 5.0 1.3 Malassezia furfur KCTC7744 5.0 2.5 Trichophyton rubrum KCTC 6345 5.0 2.5 Trichosporon beigelii KCTC7707 5.0 1.3 View Large Table 1. The antifungal effect of apigenin and amphotericin B. MIC (μg/mL) Fungal strains Apigenin Amphotericin B Candida albicans ATCC90028 5.0 1.3 Candida parapsilosis ATCC22019 5.0 1.3 Malassezia furfur KCTC7744 5.0 2.5 Trichophyton rubrum KCTC 6345 5.0 2.5 Trichosporon beigelii KCTC7707 5.0 1.3 MIC (μg/mL) Fungal strains Apigenin Amphotericin B Candida albicans ATCC90028 5.0 1.3 Candida parapsilosis ATCC22019 5.0 1.3 Malassezia furfur KCTC7744 5.0 2.5 Trichophyton rubrum KCTC 6345 5.0 2.5 Trichosporon beigelii KCTC7707 5.0 1.3 View Large Changes in cell morphology The effects of apigenin on C. albicans morphology were assessed using flow cytometry and microscopy. Morphological changes in the untreated and compound-treated cells were evaluated by flow cytometric analysis of their FSC (cell size, x-axis) and SSC (granularity, y-axis) values. FSC and SSC values lower than those of untreated cells were examined. It was observed that compared to untreated cells, values in the lower left quadrant increased in 13.2% of the cells treated with apigenin and 49.5% of cells treated with amphotericin B (Fig. 2A). Given the apparent cell shrinkage, apigenin-induced morphological changes in C. albicans were further assessed using microscopy. As shown in Fig. 2B and C, compared to untreated cells, treatment with apigenin or amphotericin B resulted in the reduction of cell size; however, the effect of amphotericin B on cell shrinkage was more pronounced than that of apigenin. Overall, these results indicated that apigenin decreased cell volume and granularity, leading to cell shrinkage in C. albicans. Figure 2. View largeDownload slide Morphological changes in C. albicans after apigenin and amphotericin B treatment. (A) Flow cytometric analysis of C. albicans cell size (FSC; forward scatter) and granularity (SSC; side scatter) after treatment with apigenin or amphotericin B. The indicated values refer to the percentage of fluorescent cells relative to the total number of cells. (B) Microscopic observation of C. albicans morphology after treatment with apigenin (b) or amphotericin B (c) [control (a)]. (C) The graph shows average diameter of a C. albicans cell. *P < 0.05; **P < 0.01; ***P < 0.001 (treated versus control; Student's t-test). Figure 2. View largeDownload slide Morphological changes in C. albicans after apigenin and amphotericin B treatment. (A) Flow cytometric analysis of C. albicans cell size (FSC; forward scatter) and granularity (SSC; side scatter) after treatment with apigenin or amphotericin B. The indicated values refer to the percentage of fluorescent cells relative to the total number of cells. (B) Microscopic observation of C. albicans morphology after treatment with apigenin (b) or amphotericin B (c) [control (a)]. (C) The graph shows average diameter of a C. albicans cell. *P < 0.05; **P < 0.01; ***P < 0.001 (treated versus control; Student's t-test). Cell depolarization and membrane permeabilization To investigate apigenin-mediated membrane disruption, DiSC3(5), a potentiometric fluorescent probe, was used. Accumulation of the probe in the cell membrane leads to self-quenching of its fluorescence, a process that is reversed when the probe is released from the membrane following alteration of the membrane potential. We added DiSC3(5) to a suspension of C. albicans cells (at 50 s), and treated these cells with apigenin or amphotericin B after the probe had been quenched (at 200 s) (Fig. 3A). Treatment with apigenin or amphotericin B caused an increase in fluorescence, indicating an impairment in cell membrane maintenance, thereby affecting the electrical potential. To further investigate the impact of apigenin on the fungal plasma membrane, we monitored the influx of PI in apigenin-treated cells. Following exposure to apigenin or amphotericin B, we observed that 39.7% and 97.8% of cells, respectively, were PI+ compared to 21.2% of the control cells (Fig. 3B). Based on these results, we further examined the effects of apigenin on membrane permeability using DPH. The fluorescence intensity of DPH decreased in the apigenin- and amphotericin B-treated cells compared to that in the untreated cells (Fig. 3C). This decrease in DPH intensity revealed perturbations of the cell membrane following apigenin treatment. Figure 3. View largeDownload slide Effect of apigenin on cell membrane function. (A) Depolarization of membrane potential was detected using 3,3΄-dipropylthiacarbocyanine iodide [DiSC3(5)]. DiSC3(5) was added to cells at t = 50 s, and apigenin or amphotericin B was added at t = 200 s. (B) Flow cytometric analysis of C. albicans membrane permeabilization by PI staining. The indicated values refer to the percentage of fluorescent cells relative to the in relation to the respective total number of cells. Cells were treated with 5 μg/mL apigenin or amphotericin B for 4 h at 28°C, followed by incubation with 9 μM PI. (C) Fluorescence intensity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C. albicans cells treated with 5 μg/mL apigenin or amphotericin B. All results are presented as the mean ± SD from three independent experiments. ***P < 0.001 (treated versus control; Student's t-test). Figure 3. View largeDownload slide Effect of apigenin on cell membrane function. (A) Depolarization of membrane potential was detected using 3,3΄-dipropylthiacarbocyanine iodide [DiSC3(5)]. DiSC3(5) was added to cells at t = 50 s, and apigenin or amphotericin B was added at t = 200 s. (B) Flow cytometric analysis of C. albicans membrane permeabilization by PI staining. The indicated values refer to the percentage of fluorescent cells relative to the in relation to the respective total number of cells. Cells were treated with 5 μg/mL apigenin or amphotericin B for 4 h at 28°C, followed by incubation with 9 μM PI. (C) Fluorescence intensity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C. albicans cells treated with 5 μg/mL apigenin or amphotericin B. All results are presented as the mean ± SD from three independent experiments. ***P < 0.001 (treated versus control; Student's t-test). Assessment of the extent of apigenin-induced damage via FD influx and efflux We examined the mechanism of cell membrane disruption by apigenin using artificial membranes. LUVs [composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and ergosterol (5:4:1:2, w/w/w/w)] mimic the outer layer of plasma membrane of C. albicans. Therefore, the impact of potential membrane-altering compounds can be tested by measuring the leakage of internal contents of liposomes. The membrane disturbance induced by apigenin using various FD molecules of increasing weight and radii was examined. We monitored the efflux of FD4, FD10 and FD20 from liposomes (Fig. 4A), as well as their influx into live cells (Fig. S1, Supporting Information). Treatment with apigenin allowed FD4 and FD10 to translocate across membranes; however, FD20 translocation was observed to be very low (amphotericin B induced slightly more flux of FD20 than apigenin; Fig. 4). Based on these results, we estimated the maximum radius of apigenin-induced membrane damage to be 2.3 nm. Extensive damage would allow intracellular components to move freely into and out of the cells. These results indicated that apigenin induced membrane disturbance in fungal cells by generating damages of radius 2.3 nm. Figure 4. View largeDownload slide Analysis of the damage induced by antifungal agents. Percentage of FITC-labeled dextran (FD) translocated across the membrane of liposomes induced by 5 μg/mL of apigenin or amphotericin B. **P < 0.01; ***P < 0.001 (treated versus untreated liposome; Student's t-test). Figure 4. View largeDownload slide Analysis of the damage induced by antifungal agents. Percentage of FITC-labeled dextran (FD) translocated across the membrane of liposomes induced by 5 μg/mL of apigenin or amphotericin B. **P < 0.01; ***P < 0.001 (treated versus untreated liposome; Student's t-test). Effect of apigenin on intracellular contents To investigate apigenin-induced changes on intracellular components, we measured the release of potassium, calcium, sugars and proteins. Potassium release was measured using a potassium-sensitive electrode and was observed to be higher in the apigenin-treated C. albicans cells than in the untreated cells (Fig. 5A). The cytoplasmic free calcium ion concentration, measured using the membrane-permeable ratiometric calcium indicator Fura-2AM, was significantly lower in the apigenin-treated cells compared to that in the untreated cells (Fig. 5B). Furthermore, extracellular sugar levels slightly increased after apigenin treatment (Fig. 5C), confirming the efflux of intracellular contents. Nevertheless, apigenin-treated cells did not release proteins. These results suggested that apigenin induced intracellular content leakage, resulting in alteration of osmolarity. Figure 5. View largeDownload slide Effects of apigenin and amphotericin B on intracellular content leakage. (A) Potassium and (B) calcium leakage from C. albicans after 4 h incubation with MIC of apigenin or amphotericin B. (C) Cytoplasmic leakage of soluble sugars and proteins from C. albicans. Results are presented as the mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (treated versus control; Student's t-test). Figure 5. View largeDownload slide Effects of apigenin and amphotericin B on intracellular content leakage. (A) Potassium and (B) calcium leakage from C. albicans after 4 h incubation with MIC of apigenin or amphotericin B. (C) Cytoplasmic leakage of soluble sugars and proteins from C. albicans. Results are presented as the mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (treated versus control; Student's t-test). DISCUSSION Apigenin, a flavone found in fruits and vegetables, stimulates apoptosis and counteracts carcinogenesis (Shukla and Gupta 2010; Kumar and Pandey 2013; Harrison et al. 2014; Nayaka et al. 2014; Smiljkovic et al. 2017). It has been studied as an anticancer agent, but its effects on human pathogenic fungi have not yet been thoroughly investigated. Herein, we isolated apigenin from A. yonema and studied its antifungal mechanism of action. We confirmed that apigenin inhibited the growth of several pathogenic fungi. Among all the fungal strains associated with superficial infections, such as candidiasis, trichosporonosis and dermatophytosis, C. albicans induces a life-threatening systemic disease in predisposed patients. (Brillowska-Dabrowska, Saunte and Arendrup 2007; Choi and Lee 2014; Sathishkumar et al. 2016). Candida albicans biofilms are compact and structured communities composed of fungal cells enclosed by self-produced, extracellular polymeric substances that protect cells from the host immune system and antifungal agents (Wang et al. 2015). Given that apigenin reduced the biofilm biomass of C. albicans, we further investigated its mode of action. Flow cytometric analysis and microscopic observation of apigenin-treated cells revealed treatment-induced cell shrinkage, where apigenin-exposed cells were observed to be smaller than the untreated cells. This morphological change is associated with apoptosis, inhibition of biomass production and damage to the cell membrane (Narasimhan et al. 2001; Park et al. 2008; Pradhan et al. 2014). Additionally, cell membrane morphology was altered, which was consistent with the previous study that reported major damage to the cytoplasmic membrane of C. albicans (Li et al. 2013). Cell volume is controlled by ion movement and ion channel regulation (Remillard and Yuan 2004; Kondratskyi et al. 2015). Cell shrinkage reduces the ability of cells to maintain a volume balance, and causes extensive leakage of intracellular contents and a loss in osmolarity (Li et al. 2013). Changes in the ratio of cations to anions, which makes a cell electrically neutral, can induce cell shrinkage via loss of cell volume (Bortner and Cidlowski 2004, 2007). The intact cell membrane can prevent cell collapse (Li et al. 2013). In animal cells, apigenin affects the membrane ion transport, activates cystic fibrosis transmembrane conductance, penetrates biological membranes and induces loss of mitochondrial transmembrane potential in HL-60 cells causing apoptosis (Pawlikowska-Pawle et al. 2007; Chen et al. 2014; Zhu et al. 2016). Thus, we hypothesized that the apigenin-induced morphological changes were induced by the disruption of cytoplasmic membrane. Normal membrane potential, lipid fluidity and membrane dynamics are essential for cell survival, as they affect membrane functions such as biochemical reactions, electrogenic transport of nutrients, protein secretion and permeability (Arora et al. 2000; Sharma, Bansal and Gupta 2002; Yuk and Marshall 2004; Steinmann et al. 2015). Assessment of alterations in the membrane potential (using DiSC3(5)), cell permeability (using PI) and membrane integrity (using DPH) after treatment with apigenin showed that the cell membrane of apigenin-treated cells was no longer able to perform these functions, and depolarization of membrane potential, increased permeability and disrupted lipid dynamics were observed. The enhanced membrane permeability could induce a disturbance of cytoplasmic membrane ion gradients and metabolic processes (Zhu et al. 2016). These observations indicated that the cytoplasmic membranes of C. albicans were damaged by apigenin. To assess the extent of damage caused by apigenin, we analyzed the leakage and influx of fluorescent-labeled molecules of varying weights and radii in both artificial and live cell membranes. Apigenin-induced membrane disruption allowed the flow of molecules within a radial limit of up to 2.3 nm. Amphotericin B was reported to produce membrane pores with radii ranging between 0.8 and 18 nm (Yang et al. 2013). The concentration of amphotericin B used in this study induced pores with radii between 2.3 and 3.3 nm. The damage caused by apigenin allowed the flux of FD4 and FD10 (radii 1.4 and 2.3 nm, respectively). A reduced flux of the larger FD20 (radius 3.3 nm) through artificial membranes was observed, along with an inability to cross the cell membranes of living cells. To determine whether apigenin interfered with ion migration, the potassium and calcium ion efflux was assessed (Ferreira Mdo et al. 2014). Potassium ion gradient is critical for cell growth and survival, since it regulates cytoplasmic pH and cell structure (Roosild et al. 2010). Loss of cytoplasmic potassium ions leads to cell death (Bolintineanu et al. 2010; Pena, Sanchez and Calahorra 2013). Therefore, leakage of potassium ions can be used to determine membrane lytic events, as the internal ionic environment in cells is typically potassium rich (Orlov, Nguyen and Lehrer 2002). Calcium ions, which are stored in organelles (endoplasmic reticulum, mitochondria and vacuoles), play an important role in regulating cellular processes. A gradient of cations, such as calcium, sodium and potassium, helps in maintaining normal membrane potential (Xiong et al. 2013). Consequently, the disruption of membrane potential may induce the release of intracellular ions. Our results demonstrated a higher efflux of potassium and calcium ions after treatment of C. albicans with apigenin. Several antifungal agents cause the leakage of cellular constituents, including sugars and proteins (Masin et al. 2013; Jeong et al. 2015; Jiang, Feng and Yang 2015). Our results showed that apigenin caused a decrease in cell membrane integrity, which, apart from resulting in the efflux of ions such as potassium and calcium, also led to the efflux of sugars. Nevertheless, no proteins were detected in the extracellular medium. This finding was in agreement with the results for the translocation of FD molecules in liposomes and live cells, which implied that apigenin-induced damage only permitted the passage of small compounds. The release of sugars, but not of molecules with radius larger than 2.3 nm (e.g. proteins), is consistent with an apigenin-induced antifungal action. An imbalance in membrane homeostasis caused by apigenin results in the leakage of intracellular ions and sugars, disturbing the osmotic balance and resulting in cell shrinkage. In conclusion, apigenin isolated from A. yomena exerted an antifungal activity by causing cell membrane perturbations, resulting in cell shrinkage and disruption of the ability of membranes to maintain the osmotic balance. Additionally, inhibition of C. albicans biofilm by apigenin may result in further alteration of the membrane. This study provides insights into the antifungal mechanisms of apigenin and suggests the use of this molecule as a therapeutic antifungal agent. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. FUNDING This work was supported by a grant from the Next-Generation BioGreen 21 Program (Project No. PJ01325603), Rural Development Administration, Republic of Korea. Conflict of interest. None declared. REFERENCES Alves RR, Rosa IM. Biodiversity, traditional medicine and public health: where do they meet? J Ethnobiol Ethnomed 2007; 3: 14. Google Scholar CrossRef Search ADS PubMed Arora A, Byrem TM, Nair MGet al. 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