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Isocryptomerin, a novel membrane-active antifungal compound from Selaginella tamariscina
Biochemical and Biophysical Research Communications 379 (2009) 676–680
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Isocryptomerin, a novel membrane-active antifungal compound from Selaginella tamariscina Juneyoung Lee a,1, Yunjung Choi a,1, Eun-Rhan Woo b, Dong Gun Lee a,* a b
School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Puk-ku, Daegu 702-701, South Korea College of Pharmacy, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, South Korea
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Article history: Received 19 November 2008 Available online 25 December 2008
Keywords: Isocryptomerin Biflavonoid Antifungal activity Membrane-disruption mechanism(s)
a b s t r a c t Isocryptomerin is a biflavonoid isolated from Selaginella tamariscina used in traditional medicine. In this study, we investigated novel antifungal properties of isocryptomerin. The results indicated that isocryptomerin exerted antifungal activity in an energy-independent manner without remarkable hemolytic effects. To understand mode of action(s) of isocryptomerin, we conducted experiments on Candida albicans, a noted human pathogenic fungal strain. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], a translational membrane potential dye, regeneration test with fungal protoplasts, and fluorescence analysis with 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe for membrane studies by depolarization, indicated that isocryptomerin could depolarize fungal plasma membrane. In conclusion, the results suggested that the antifungal activities of isocryptomerin might be due to its membrane-disruption mechanism(s). Ó 2009 Published by Elsevier Inc.
The overgrowth of organisms resistant to antimicrobial agents demands the discovery of new therapeutic agents. So, many people have tried to find antimicrobial agents from natural products for drug discovery. Currently, natural products and their derivatives represent more than 50% of all the drugs in clinical use in the world [1]. The interest in nature as a source of potential chemotherapeutic agents continues and natural product compounds have served as the most significant source of new leads against microbial pathogens. From this point of view, traditional material medica (medicinal plants and other materials) are being used as a guide to lead scientists towards different sources and classes of compounds. Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years and continue to provide mankind with new remedies [1]. For example, several Selaginella species are used in traditional medicine in various countries to treat a variety of diseases such as cancer [2], cardiovascular problems [2], diabetes [3], hepatitis [4], skin diseases [5] and urinary tract infections [6]. The Selaginella species contain a large number of active compounds, the most important being biflavonoids. Biflavonoids are flavonoid dimers connected with a C–C or a C–O–C bond and are known to display a variety of biological activities, such as anti-inflammatory activity [7–10], inhibitory * Corresponding author. Fax: +82 53 955 5522. E-mail address: [email protected] (D.G. Lee). 1 These authors should be considered co-first authors 0006-291X/$ - see front matter Ó 2009 Published by Elsevier Inc. doi:10.1016/j.bbrc.2008.12.030
activity of mast cell histamine release [11,12], anti-tumor activity [13,14], phospholipase A2 inhibitory activity [15], and inhibition of matrix metalloproteinase-1 production in fibroblast cells [16]. Distribution of biflavonoids in the plant kingdom is limited to several species such as Ginkgo biloba, Selaginella species, and Garcinia kola [17]. Selaginella tamariscina, used in this research, belongs to Selaginellaceae which is a traditional medicinal plant for therapy agents in the Orient. Isocryptomerin is one kind of biflavonoids isolated from S. tamariscina. Previously, isocryptomerin was reported to suppress lymphocyte proliferation and the cytotoxicity against human tumor cell lines [18,19]. Although several biological activities of isocryptomerin are known, antimicrobial effects of isocryptomerin have not yet been studied. Therefore, in this study, we investigated novel antifungal activity and mode of action of isocryptomerin and suggested potential as therapeutic agents for fungal infections in humans. Materials and methods Plant material, extraction and isolation. Dried whole plants of S. tamariscina Spring, which belongs to the Selaginellaceae family, were obtained from herbal drug stores in Gwangju. The plants were authenticated by the Department of Pharmacognosy, Chosun University. Voucher specimens (853-16) were deposited in the Herbarium of the College of Pharmacy, Chosun University. The whole plant of S. tamariscina (0.6 kg) was extracted three times with MeOH at room temperature and evaporated to dryness under
J. Lee et al. / Biochemical and Biophysical Research Communications 379 (2009) 676–680
reduced pressure to obtain 50.54 g of residue. The methanol extract was suspended in water and then partitioned by dichloromethane, ethyl acetate and n-butanol in turn. The EtOAc fraction (1.74 g) was subjected to column chromatography over a silica gel (Merck 43–60 and 63–200 lm, Germany; 200 g) eluting with a CHCl3–MeOH–H2O (12:1:0.1 ? 8:1:0.1 ? 5:1:0.1 ? 2:1:0.1 ? 1:1:0.1 ? MeOH only) gradient system. Fractions were combined based on their TLC pattern to yield subfractions designated as E1–E10. Subfraction E3 (30.49 mg) was purified by column chromatography over a Sephadex LH 20 eluting with a MeOH to give isocryptomerin (3.75 mg). The physical and chemical data, including MS, 1H NMR, 13C NMR and HSQC of isocryptomerin were identical with those reported previously (Fig. 1) [16,19]. Fungal strains and antifungal activity assay. Saccharomyces cerevisiae (KCTC 7296) and Trichosporon beigelii (KCTC 7707) were obtained from the Korean Collection for Type Cultures (KCTC), at the Korea Research Institute of Bioscience Biotechnology (KRIBB), Daejeon, Korea. Candida albicans (TIMM 1768) was obtained from the Center for Academic Societies, Osaka, Japan. Fungal cells grown at 28 °C in YPD (Dextrose 2%, Peptone 1%, Yeast extract 0.5%) media were seeded on 96-well microtiter plates at a density of 2 104 cells per well in 100 ll of YPD media. Serially diluted isocryptomerin was added to each fungal cells. The cell suspension was incubated at 28 °C for 48 h. After incubation of the cells, 5 ll of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution [5 mg/ml MTT in phosphate-buffered saline (PBS), pH 7.4] was added to each well [20]. The minimal compound concentration that prevented the growth of a given test organism was determined and was defined as the minimum inhibitory concentrations (MICs). Growth was assayed with a microtiter ELISA reader (Molecular Devices Emax, CA, USA) by monitoring absorption at 580 nm. MIC values were determined by three independent assays [21]. Kinetics of fungal killing. Exponential phased C. albicans cells (2 104 CFUs/ml) were incubated with 18.11 lM isocryptomerin or 10.82 lM amphotericin B used as a positive control. The cultures were obtained and spreaded on an YPD agar plate, and then the colony forming units (CFUs) were counted after incubation for 24 h at 28 °C [22]. The values were the average of triplicate measurements in three independent assays. Determination of intracellular trehalose. C. albicans cells containing 36.23 lM of isocryptomerin or 10.82 lM of amphotericin B were incubated for 2 h at 28 °C. Fungal cells were separated by centrifugation at 12,000 rpm for 10 min and dried. Three milligrams (dry weight) of dried fungal cells were destroyed by boiling in a 0.025 M potassium phosphate (pH 6.6). The crude neutral trehalose-containing fractions were extracted by removing the cell debris. The trehalose residue was then digested with 5 ll of 1 unit/mg trehalase (Sigma, T8778-1UN). After allowing the enzymatic reaction to proceed for 1 h at 37 °C, the reaction suspension
Fig. 1. Chemical structure of isocryptomerin derived from Selaginella tamariscina.
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was mixed with H2O and a 16% DNS reagent (1% 3,5-dinitrosalicylic acid, 2% NaOH, 20% sodium potassium tartrate) was added [23]. For the reaction between glucose and the DNS reagent, the mixture was boiled for 20 min and then cooled. Level of color formation was measured at a wavelength of 525 nm. The effect of sodium azide (NaN3) on antifungal activity. To examine effect of NaN3 on antifungal activity of isocryptomerin, the activity was investigated in presence or absence of 0.1 mM NaN3. Cell cultures were seeded on a 96-well microtiter plate at a density of 2 104 cells per well. Fungal cells were added 36.23 lM isocryptomerin with or without 0.1 mM NaN3, and an MTT assay was performed. The percentage of cell viability was determined by comparing the absorbance of compound-treated cell culture with that of compound-untreated cell culture. The values were the average of triplicate measurements in three independent assays. Flow cytometric analysis for plasma membrane potential. Exponential phased cells of C. albicans (1 108 cells) cultured in YPD media were harvested and resuspended with 500 ll of fresh YPD media containing 36.23 lM of isocryptomerin or 300 lM of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), which was used as a positive control [24]. After incubation for 3 h, the cells were washed with PBS three times. To detect depolarization of cell membrane, 1 ml of PBS containing 20 lg/ml of bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] (Molecular Probes Inc., Eugene, OR, USA), was added and the samples were incubated for 1 h at 4 °C in the dark [25]. A flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Cell wall regeneration of fungal protoplasts. For preparation of C. albicans protoplasts, cells (1 108) were digested with 10 mM phosphate buffer (pH 6.0) containing 1 M sorbitol, lysing enzyme (Sigma) (20 mg/ml), and cellulase (Sigma) (20 mg/ml) for 4 h at 28 °C by gentle agitation. The digests were filtered through a 3G3 glass filter, and protoplasts in the filtrate were gathered by centrifugation at 1500 rpm for 10 min. The protoplasts were resuspended in the washing buffer (0.8 M NaCl, 10 mM CaCl2, and 50 mM Tris–HCl, pH 7.5) and centrifuged again. Isocryptomerin (36.23 lM) or amphotericin B (10.82 lM) was added to the protoplasts, then suspended in the washing buffer and incubated for 3 h at 28 °C. The protoplasts treated with compound were then transferred into YPD soft-agar solutions containing 1 M sorbitol and 2% agar. The regenerated colonies were counted following incubation of the plates at 28 °C for 3 days [26]. Measurement of plasma membrane fluorescence anisotropy. The anisotropy of fluorescence from exponential phased C. albicans cells labeled by 1,6-diphenyl-1,3,5-hexatriene (DPH) (Molecular Probes, Eugene, OR, USA) was used to monitor changes in membrane dynamics. The cells (1 108 cells in an YPD medium) containing 36.23 lM of isocryptomerin or 10.82 lM of amphotericin B used as a positive control were incubated at a physiological temperature of 28 °C on a rotary shaker at 140 rpm for 1 h. Control cells were incubated without compounds. The cells were fixed with formaldehyde (0.37%, v/v) for 30 min. And then they were washed with PBS and the pellets were frozen in liquid nitrogen. For DPH labeling, cells were thawed and resuspended in PBS. The suspension was incubated at 28 °C for 45 min in the presence of 0.6 mM DPH, followed by several washings in PBS. Steady-state fluorescence anisotropy was measured by using RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan) at 350 nm excitation and 425 nm emission wavelengths [27]. Hemolytic effects on human erythrocytes. The hemolytic activity of the compounds was evaluated by determining the release of hemoglobin from 4% suspension of fresh human erythrocytes at 414 nm with an ELISA plate Reader [28]. The percentage of hemolysis was calculated by employing the following equation: Percentage of
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hemolysis = [(Abs414 nm in the compound solution – Abs414 nm in PBS)/(Abs414 nm in 0.1% Triton X-100 – Abs414 nm in PBS)] 100. Results and discussion Antifungal activity of isocryptomerin The antifungal activities of isocryptomerin on human pathogenic fungi were investigated. In this study, amphotericin B, a fungicidal agent used to treat serious systemic infections, was used as a positive control for fungi [29]. Isocryptomerin, with an MIC value of 18.11 lM, exhibited antifungal activity against human pathogenic fungi, such as C. albicans and T. beigelii, exist in humans as commensals and are superficial contaminants that can variety of serious infections (Table 1). In addition, time-killing assay conducted with C. albicans demonstrated that the antifungal activity of isocryptomerin was due to not static but cidal action (Fig. 2A). Finally, the result of trehalose assay confirmed the antifungal activity of isocryptomerin. It could be explained by that the intracellular trehalose of isocryptomerin-treated cells was accumulated through the stress response caused by the antifungal activities of isocryptomerin (Fig. 2B). These results suggested that isocryptomerin exerted significant antifungal activities against human pathogenic fungi by fungicidal effects. Effect of energy metabolism on antifungal activity of isocryptomerin Cellular energy synthesis is important for the entry of essential molecules regarding cell growth and proliferation. The metabolic inhibitor for ATP depletion can inhibit the imported system from an extracellular environment into a cell and induce membrane depolarization by disrupting proton gradient [30]. Thus, to understand the relationship between cellular energy consumption and isocryptomerin transport pathway, an energy-dependence test was performed. C. albicans was incubated with isocryptomerin in the absence or presence of 0.1 mM NaN3 for the test. Sodium azide (NaN3) is a metabolic inhibitor that blocks intracellular ATP syn-
Table 1 Antifungal activity of isocryptomerin. Compound
Isocryptomerin Amphotericin B
MIC (lM) C. albicans
T. beigelii
S. cerevisiae
18.11 5.41
18.11 5.41–10.82
18.11 5.41
thesis and the ability of ATPase by inhibiting cytochrome oxidase, which further prevents membrane-active transport [31]. The result showed that the viability of the cells was not affected by the presence of 0.1 mM NaN3 (Fig. 3). However, it was affected by the presence of isocryptomerin, regardless of the presence of NaN3. The results demonstrated that the activity of isocryptomerin was independent to NaN3, suggesting that its effects were mediated by a cellular function, which does not need energy consumption. Membrane-disruption mechanism(s) of isocryptomerin To provide information about the mode of antifungal action of isocryptomerin, we selected C. albicans, which causes a variety of superficial and deep-seated mycoses such as candidiasis. This fungus is an important pathogen in opportunistic infections by fungi and is becoming of increasing importance in patients who are immunocompromised due to cancer chemotherapy, organ or bone marrow transplantation, or human immunodeficiency virus infection [32]. To assess whether isocryptomerin can affect the faculty of plasma membrane of C.albicans, the dissipation of plasma membrane potential was investigated by staining with DiBAC4(3). DiBAC4(3) has high voltage sensitivity and enters depolarized cells, where it binds to lipid-rich intracellular components [25]. CCCP was used as a positive control. It is an H+ ionophore which dissipates the H+ gradient and thus uncouples electron transport from ATP synthesis [24]. C. albicans cells were cultured in the presence or absence of isocryptomerin or CCCP and the amounts of accumulated DiBAC4(3) in cells were measured with flow cytometry. As shown in Fig. 4A, isocryptomerintreated cells caused the accumulation of DiBAC4(3) more than cells not treated with the compound. However, degrees of accumulation of DiBAC4(3) of isocryptomerin were less potent than those of CCCP. The result indicates that isocryptomerin affects C. albicans cells by membrane depolarization. To confirm whether isocryptomerin exerts an antifungal effect by damaging the plasma membrane of fungal cells, the cell wall regeneration of C. albicans protoplasts was investigated. After treatment of isocryptomerin and amphotericin B to the protoplast, frequency of regeneration (FR) values was measured. As shown in Table 2, FR values of isocryptomerin-treated protoplasts were lower than the control (no compound treated), and higher than amphotericin B. This indicated that the antifungal activity of isocryptomerin was due to the interaction between the compound and the plasma membrane, rather than cell wall. To elucidate the in vivo membrane fluidity by isocryptomerin in the plasma membrane of C. albicans, changes in membrane
Fig. 2. Antifungal activity of isocryptomerin for C. albicans. A: Time killing plots for C. albicans by isocryptomerin. B: Trehalose assay after adding isocryptomerin or amphotericin B. (A) control, (B) cells treated with amphotericin B, and (C) cells treated with isocryptomerin.
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dynamics were further investigated by using DPH as a membrane probe. If the antifungal activities exerted by isocryptomerin are at the level of the plasma membrane, DPH, which interacted with an acyl group of plasma membrane lipid bilayers, could not be inserted into the membrane [33]. As shown in Fig. 4B, plasma membrane DPH fluorescence anisotropy decreased in the presence of isocryptomerin, but was less than the decrease observed in the presence of amphotericin B. This result may confirm that isocryptomerin exerts its antimicrobial activities by perturbing fungal plasma membranes. Hemolytic activity of isocryptomerin From the standpoint of practical use, it is desirable that antimicrobial agents are devoid of cytotoxicity [34]. This compound is shown to be membrane-active mechanism, is able to discriminate
between microbial cells and mammalian red blood cell membranes. The hemolytic activity of membrane-active compounds against erythrocytes is often used as a measure for their cytotoxicity and to estimate their therapeutic index [35]. Therefore, the hemolytic activity of isocryptomerin was examined against human erythrocytes evaluated by the percentage of hemolysis in 4% suspension of hRBCs at various concentrations (from 2.26 to 36.23 lM) of isocryptomerin. Isocryptomerin exhibited no hemolytic activity except for at the highest concentration, however, amphotericin B exhibited high hemolytic activity at almost all concentrations (Table 3). This indicated that isocryptomerin, which showed almost no hemolytic activity, could be used in clinical use for human diseases. In conclusion, isocryptomerin exhibited novel antifungal effects on human pathogenic fungi with almost no hemolytic effects against human erythrocytes. Although exact mechanisms of isocryptomerin are not understood completely, this study suggests that this compound may act on the plasma membrane by depolarization or formation of pores. Therefore, it can be concluded that isocryptomerin has considerable antifungal effect, deserving further investigation for clinical applications.
Table 2 Effects of isocryptomerin on regeneration of C. albicans protoplasts. Compound
FRa
Controlb Isocryptomerin
80.9 36.3
Amphotericin B
2 10
1
a
Frequency of regeneration (FR) values were calculated by the formula: FR (%) = [(number of colonies on plate)/(number of protoplasts used)] 100. b Control indicates no compound treatment.
Table 3 Hemolytic activity of isocryptomerin against human erythrocytes.
Fig. 3. Effect of sodium azide (NaN3) on antifungal activity of isocryptomerin against C. albicans. (A) control, (B) cells treated with NaN3, (C) cells treated with isocryptomerin, and (D) cells treated with isocyptomerin and NaN3.
Compound
% Hemolysis (lM) 36.23
18.11
9.05
4.52
2.26
Isocryptomerin Amphotericin B
11.7 100
0 86.6
0 71.2
0 62.2
0 28.6
Fig. 4. Effects of isocryptomerin on membrane of C. albicans. A: FACScan analysis of DiBAC4(3) staining in C. albicans. Histograms showed the fluorescence intensity of stained DiBAC4(3) in C. albicans after treating compounds. (A) control, (B) cells treated with isocryptomerin, and (C) cells treated with CCCP. B: DPH fluorescence anisotropy after adding of compounds. (A) control, (B) cells treated with amphotericin B, and (C) cells treated with isocryptomerin.
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Isocryptomerin, a novel membrane-active antifungal compound from Selaginella tamariscina Juneyoung Lee a,1, Yunjung Choi a,1, Eun-Rhan Woo b, Dong Gun Lee a,* a b
School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Puk-ku, Daegu 702-701, South Korea College of Pharmacy, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, South Korea
a r t i c l e
i n f o
Article history: Received 19 November 2008 Available online 25 December 2008
Keywords: Isocryptomerin Biflavonoid Antifungal activity Membrane-disruption mechanism(s)
a b s t r a c t Isocryptomerin is a biflavonoid isolated from Selaginella tamariscina used in traditional medicine. In this study, we investigated novel antifungal properties of isocryptomerin. The results indicated that isocryptomerin exerted antifungal activity in an energy-independent manner without remarkable hemolytic effects. To understand mode of action(s) of isocryptomerin, we conducted experiments on Candida albicans, a noted human pathogenic fungal strain. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], a translational membrane potential dye, regeneration test with fungal protoplasts, and fluorescence analysis with 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe for membrane studies by depolarization, indicated that isocryptomerin could depolarize fungal plasma membrane. In conclusion, the results suggested that the antifungal activities of isocryptomerin might be due to its membrane-disruption mechanism(s). Ó 2009 Published by Elsevier Inc.
The overgrowth of organisms resistant to antimicrobial agents demands the discovery of new therapeutic agents. So, many people have tried to find antimicrobial agents from natural products for drug discovery. Currently, natural products and their derivatives represent more than 50% of all the drugs in clinical use in the world [1]. The interest in nature as a source of potential chemotherapeutic agents continues and natural product compounds have served as the most significant source of new leads against microbial pathogens. From this point of view, traditional material medica (medicinal plants and other materials) are being used as a guide to lead scientists towards different sources and classes of compounds. Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years and continue to provide mankind with new remedies [1]. For example, several Selaginella species are used in traditional medicine in various countries to treat a variety of diseases such as cancer [2], cardiovascular problems [2], diabetes [3], hepatitis [4], skin diseases [5] and urinary tract infections [6]. The Selaginella species contain a large number of active compounds, the most important being biflavonoids. Biflavonoids are flavonoid dimers connected with a C–C or a C–O–C bond and are known to display a variety of biological activities, such as anti-inflammatory activity [7–10], inhibitory * Corresponding author. Fax: +82 53 955 5522. E-mail address: [email protected] (D.G. Lee). 1 These authors should be considered co-first authors 0006-291X/$ - see front matter Ó 2009 Published by Elsevier Inc. doi:10.1016/j.bbrc.2008.12.030
activity of mast cell histamine release [11,12], anti-tumor activity [13,14], phospholipase A2 inhibitory activity [15], and inhibition of matrix metalloproteinase-1 production in fibroblast cells [16]. Distribution of biflavonoids in the plant kingdom is limited to several species such as Ginkgo biloba, Selaginella species, and Garcinia kola [17]. Selaginella tamariscina, used in this research, belongs to Selaginellaceae which is a traditional medicinal plant for therapy agents in the Orient. Isocryptomerin is one kind of biflavonoids isolated from S. tamariscina. Previously, isocryptomerin was reported to suppress lymphocyte proliferation and the cytotoxicity against human tumor cell lines [18,19]. Although several biological activities of isocryptomerin are known, antimicrobial effects of isocryptomerin have not yet been studied. Therefore, in this study, we investigated novel antifungal activity and mode of action of isocryptomerin and suggested potential as therapeutic agents for fungal infections in humans. Materials and methods Plant material, extraction and isolation. Dried whole plants of S. tamariscina Spring, which belongs to the Selaginellaceae family, were obtained from herbal drug stores in Gwangju. The plants were authenticated by the Department of Pharmacognosy, Chosun University. Voucher specimens (853-16) were deposited in the Herbarium of the College of Pharmacy, Chosun University. The whole plant of S. tamariscina (0.6 kg) was extracted three times with MeOH at room temperature and evaporated to dryness under
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reduced pressure to obtain 50.54 g of residue. The methanol extract was suspended in water and then partitioned by dichloromethane, ethyl acetate and n-butanol in turn. The EtOAc fraction (1.74 g) was subjected to column chromatography over a silica gel (Merck 43–60 and 63–200 lm, Germany; 200 g) eluting with a CHCl3–MeOH–H2O (12:1:0.1 ? 8:1:0.1 ? 5:1:0.1 ? 2:1:0.1 ? 1:1:0.1 ? MeOH only) gradient system. Fractions were combined based on their TLC pattern to yield subfractions designated as E1–E10. Subfraction E3 (30.49 mg) was purified by column chromatography over a Sephadex LH 20 eluting with a MeOH to give isocryptomerin (3.75 mg). The physical and chemical data, including MS, 1H NMR, 13C NMR and HSQC of isocryptomerin were identical with those reported previously (Fig. 1) [16,19]. Fungal strains and antifungal activity assay. Saccharomyces cerevisiae (KCTC 7296) and Trichosporon beigelii (KCTC 7707) were obtained from the Korean Collection for Type Cultures (KCTC), at the Korea Research Institute of Bioscience Biotechnology (KRIBB), Daejeon, Korea. Candida albicans (TIMM 1768) was obtained from the Center for Academic Societies, Osaka, Japan. Fungal cells grown at 28 °C in YPD (Dextrose 2%, Peptone 1%, Yeast extract 0.5%) media were seeded on 96-well microtiter plates at a density of 2 104 cells per well in 100 ll of YPD media. Serially diluted isocryptomerin was added to each fungal cells. The cell suspension was incubated at 28 °C for 48 h. After incubation of the cells, 5 ll of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution [5 mg/ml MTT in phosphate-buffered saline (PBS), pH 7.4] was added to each well [20]. The minimal compound concentration that prevented the growth of a given test organism was determined and was defined as the minimum inhibitory concentrations (MICs). Growth was assayed with a microtiter ELISA reader (Molecular Devices Emax, CA, USA) by monitoring absorption at 580 nm. MIC values were determined by three independent assays [21]. Kinetics of fungal killing. Exponential phased C. albicans cells (2 104 CFUs/ml) were incubated with 18.11 lM isocryptomerin or 10.82 lM amphotericin B used as a positive control. The cultures were obtained and spreaded on an YPD agar plate, and then the colony forming units (CFUs) were counted after incubation for 24 h at 28 °C [22]. The values were the average of triplicate measurements in three independent assays. Determination of intracellular trehalose. C. albicans cells containing 36.23 lM of isocryptomerin or 10.82 lM of amphotericin B were incubated for 2 h at 28 °C. Fungal cells were separated by centrifugation at 12,000 rpm for 10 min and dried. Three milligrams (dry weight) of dried fungal cells were destroyed by boiling in a 0.025 M potassium phosphate (pH 6.6). The crude neutral trehalose-containing fractions were extracted by removing the cell debris. The trehalose residue was then digested with 5 ll of 1 unit/mg trehalase (Sigma, T8778-1UN). After allowing the enzymatic reaction to proceed for 1 h at 37 °C, the reaction suspension
Fig. 1. Chemical structure of isocryptomerin derived from Selaginella tamariscina.
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was mixed with H2O and a 16% DNS reagent (1% 3,5-dinitrosalicylic acid, 2% NaOH, 20% sodium potassium tartrate) was added [23]. For the reaction between glucose and the DNS reagent, the mixture was boiled for 20 min and then cooled. Level of color formation was measured at a wavelength of 525 nm. The effect of sodium azide (NaN3) on antifungal activity. To examine effect of NaN3 on antifungal activity of isocryptomerin, the activity was investigated in presence or absence of 0.1 mM NaN3. Cell cultures were seeded on a 96-well microtiter plate at a density of 2 104 cells per well. Fungal cells were added 36.23 lM isocryptomerin with or without 0.1 mM NaN3, and an MTT assay was performed. The percentage of cell viability was determined by comparing the absorbance of compound-treated cell culture with that of compound-untreated cell culture. The values were the average of triplicate measurements in three independent assays. Flow cytometric analysis for plasma membrane potential. Exponential phased cells of C. albicans (1 108 cells) cultured in YPD media were harvested and resuspended with 500 ll of fresh YPD media containing 36.23 lM of isocryptomerin or 300 lM of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), which was used as a positive control [24]. After incubation for 3 h, the cells were washed with PBS three times. To detect depolarization of cell membrane, 1 ml of PBS containing 20 lg/ml of bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] (Molecular Probes Inc., Eugene, OR, USA), was added and the samples were incubated for 1 h at 4 °C in the dark [25]. A flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Cell wall regeneration of fungal protoplasts. For preparation of C. albicans protoplasts, cells (1 108) were digested with 10 mM phosphate buffer (pH 6.0) containing 1 M sorbitol, lysing enzyme (Sigma) (20 mg/ml), and cellulase (Sigma) (20 mg/ml) for 4 h at 28 °C by gentle agitation. The digests were filtered through a 3G3 glass filter, and protoplasts in the filtrate were gathered by centrifugation at 1500 rpm for 10 min. The protoplasts were resuspended in the washing buffer (0.8 M NaCl, 10 mM CaCl2, and 50 mM Tris–HCl, pH 7.5) and centrifuged again. Isocryptomerin (36.23 lM) or amphotericin B (10.82 lM) was added to the protoplasts, then suspended in the washing buffer and incubated for 3 h at 28 °C. The protoplasts treated with compound were then transferred into YPD soft-agar solutions containing 1 M sorbitol and 2% agar. The regenerated colonies were counted following incubation of the plates at 28 °C for 3 days [26]. Measurement of plasma membrane fluorescence anisotropy. The anisotropy of fluorescence from exponential phased C. albicans cells labeled by 1,6-diphenyl-1,3,5-hexatriene (DPH) (Molecular Probes, Eugene, OR, USA) was used to monitor changes in membrane dynamics. The cells (1 108 cells in an YPD medium) containing 36.23 lM of isocryptomerin or 10.82 lM of amphotericin B used as a positive control were incubated at a physiological temperature of 28 °C on a rotary shaker at 140 rpm for 1 h. Control cells were incubated without compounds. The cells were fixed with formaldehyde (0.37%, v/v) for 30 min. And then they were washed with PBS and the pellets were frozen in liquid nitrogen. For DPH labeling, cells were thawed and resuspended in PBS. The suspension was incubated at 28 °C for 45 min in the presence of 0.6 mM DPH, followed by several washings in PBS. Steady-state fluorescence anisotropy was measured by using RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan) at 350 nm excitation and 425 nm emission wavelengths [27]. Hemolytic effects on human erythrocytes. The hemolytic activity of the compounds was evaluated by determining the release of hemoglobin from 4% suspension of fresh human erythrocytes at 414 nm with an ELISA plate Reader [28]. The percentage of hemolysis was calculated by employing the following equation: Percentage of
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hemolysis = [(Abs414 nm in the compound solution – Abs414 nm in PBS)/(Abs414 nm in 0.1% Triton X-100 – Abs414 nm in PBS)] 100. Results and discussion Antifungal activity of isocryptomerin The antifungal activities of isocryptomerin on human pathogenic fungi were investigated. In this study, amphotericin B, a fungicidal agent used to treat serious systemic infections, was used as a positive control for fungi [29]. Isocryptomerin, with an MIC value of 18.11 lM, exhibited antifungal activity against human pathogenic fungi, such as C. albicans and T. beigelii, exist in humans as commensals and are superficial contaminants that can variety of serious infections (Table 1). In addition, time-killing assay conducted with C. albicans demonstrated that the antifungal activity of isocryptomerin was due to not static but cidal action (Fig. 2A). Finally, the result of trehalose assay confirmed the antifungal activity of isocryptomerin. It could be explained by that the intracellular trehalose of isocryptomerin-treated cells was accumulated through the stress response caused by the antifungal activities of isocryptomerin (Fig. 2B). These results suggested that isocryptomerin exerted significant antifungal activities against human pathogenic fungi by fungicidal effects. Effect of energy metabolism on antifungal activity of isocryptomerin Cellular energy synthesis is important for the entry of essential molecules regarding cell growth and proliferation. The metabolic inhibitor for ATP depletion can inhibit the imported system from an extracellular environment into a cell and induce membrane depolarization by disrupting proton gradient [30]. Thus, to understand the relationship between cellular energy consumption and isocryptomerin transport pathway, an energy-dependence test was performed. C. albicans was incubated with isocryptomerin in the absence or presence of 0.1 mM NaN3 for the test. Sodium azide (NaN3) is a metabolic inhibitor that blocks intracellular ATP syn-
Table 1 Antifungal activity of isocryptomerin. Compound
Isocryptomerin Amphotericin B
MIC (lM) C. albicans
T. beigelii
S. cerevisiae
18.11 5.41
18.11 5.41–10.82
18.11 5.41
thesis and the ability of ATPase by inhibiting cytochrome oxidase, which further prevents membrane-active transport [31]. The result showed that the viability of the cells was not affected by the presence of 0.1 mM NaN3 (Fig. 3). However, it was affected by the presence of isocryptomerin, regardless of the presence of NaN3. The results demonstrated that the activity of isocryptomerin was independent to NaN3, suggesting that its effects were mediated by a cellular function, which does not need energy consumption. Membrane-disruption mechanism(s) of isocryptomerin To provide information about the mode of antifungal action of isocryptomerin, we selected C. albicans, which causes a variety of superficial and deep-seated mycoses such as candidiasis. This fungus is an important pathogen in opportunistic infections by fungi and is becoming of increasing importance in patients who are immunocompromised due to cancer chemotherapy, organ or bone marrow transplantation, or human immunodeficiency virus infection [32]. To assess whether isocryptomerin can affect the faculty of plasma membrane of C.albicans, the dissipation of plasma membrane potential was investigated by staining with DiBAC4(3). DiBAC4(3) has high voltage sensitivity and enters depolarized cells, where it binds to lipid-rich intracellular components [25]. CCCP was used as a positive control. It is an H+ ionophore which dissipates the H+ gradient and thus uncouples electron transport from ATP synthesis [24]. C. albicans cells were cultured in the presence or absence of isocryptomerin or CCCP and the amounts of accumulated DiBAC4(3) in cells were measured with flow cytometry. As shown in Fig. 4A, isocryptomerintreated cells caused the accumulation of DiBAC4(3) more than cells not treated with the compound. However, degrees of accumulation of DiBAC4(3) of isocryptomerin were less potent than those of CCCP. The result indicates that isocryptomerin affects C. albicans cells by membrane depolarization. To confirm whether isocryptomerin exerts an antifungal effect by damaging the plasma membrane of fungal cells, the cell wall regeneration of C. albicans protoplasts was investigated. After treatment of isocryptomerin and amphotericin B to the protoplast, frequency of regeneration (FR) values was measured. As shown in Table 2, FR values of isocryptomerin-treated protoplasts were lower than the control (no compound treated), and higher than amphotericin B. This indicated that the antifungal activity of isocryptomerin was due to the interaction between the compound and the plasma membrane, rather than cell wall. To elucidate the in vivo membrane fluidity by isocryptomerin in the plasma membrane of C. albicans, changes in membrane
Fig. 2. Antifungal activity of isocryptomerin for C. albicans. A: Time killing plots for C. albicans by isocryptomerin. B: Trehalose assay after adding isocryptomerin or amphotericin B. (A) control, (B) cells treated with amphotericin B, and (C) cells treated with isocryptomerin.
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dynamics were further investigated by using DPH as a membrane probe. If the antifungal activities exerted by isocryptomerin are at the level of the plasma membrane, DPH, which interacted with an acyl group of plasma membrane lipid bilayers, could not be inserted into the membrane [33]. As shown in Fig. 4B, plasma membrane DPH fluorescence anisotropy decreased in the presence of isocryptomerin, but was less than the decrease observed in the presence of amphotericin B. This result may confirm that isocryptomerin exerts its antimicrobial activities by perturbing fungal plasma membranes. Hemolytic activity of isocryptomerin From the standpoint of practical use, it is desirable that antimicrobial agents are devoid of cytotoxicity [34]. This compound is shown to be membrane-active mechanism, is able to discriminate
between microbial cells and mammalian red blood cell membranes. The hemolytic activity of membrane-active compounds against erythrocytes is often used as a measure for their cytotoxicity and to estimate their therapeutic index [35]. Therefore, the hemolytic activity of isocryptomerin was examined against human erythrocytes evaluated by the percentage of hemolysis in 4% suspension of hRBCs at various concentrations (from 2.26 to 36.23 lM) of isocryptomerin. Isocryptomerin exhibited no hemolytic activity except for at the highest concentration, however, amphotericin B exhibited high hemolytic activity at almost all concentrations (Table 3). This indicated that isocryptomerin, which showed almost no hemolytic activity, could be used in clinical use for human diseases. In conclusion, isocryptomerin exhibited novel antifungal effects on human pathogenic fungi with almost no hemolytic effects against human erythrocytes. Although exact mechanisms of isocryptomerin are not understood completely, this study suggests that this compound may act on the plasma membrane by depolarization or formation of pores. Therefore, it can be concluded that isocryptomerin has considerable antifungal effect, deserving further investigation for clinical applications.
Table 2 Effects of isocryptomerin on regeneration of C. albicans protoplasts. Compound
FRa
Controlb Isocryptomerin
80.9 36.3
Amphotericin B
2 10
1
a
Frequency of regeneration (FR) values were calculated by the formula: FR (%) = [(number of colonies on plate)/(number of protoplasts used)] 100. b Control indicates no compound treatment.
Table 3 Hemolytic activity of isocryptomerin against human erythrocytes.
Fig. 3. Effect of sodium azide (NaN3) on antifungal activity of isocryptomerin against C. albicans. (A) control, (B) cells treated with NaN3, (C) cells treated with isocryptomerin, and (D) cells treated with isocyptomerin and NaN3.
Compound
% Hemolysis (lM) 36.23
18.11
9.05
4.52
2.26
Isocryptomerin Amphotericin B
11.7 100
0 86.6
0 71.2
0 62.2
0 28.6
Fig. 4. Effects of isocryptomerin on membrane of C. albicans. A: FACScan analysis of DiBAC4(3) staining in C. albicans. Histograms showed the fluorescence intensity of stained DiBAC4(3) in C. albicans after treating compounds. (A) control, (B) cells treated with isocryptomerin, and (C) cells treated with CCCP. B: DPH fluorescence anisotropy after adding of compounds. (A) control, (B) cells treated with amphotericin B, and (C) cells treated with isocryptomerin.
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