First report of an anti-tumor, anti-fungal, anti-yeast and anti-bacterial hemolysin from Albizia lebbeck seeds

Phytomedicine 18 (2011) 601–608

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Phytomedicine journal homepage: www.elsevier.de/phymed

First report of an anti-tumor, anti-fungal, anti-yeast and anti-bacterial hemolysin from Albizia lebbeck seeds Sze Kwan Lam ∗ , Tzi Bun Ng ∗ School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

a r t i c l e Keywords: Albizia lebbeck Hemolysin Anti-tumor Anti-fungal Anti-yeast Anti-bacterial

i n f o

a b s t r a c t A monomeric 5.5-kDa protein with hemolytic activity toward rabbit erythrocytes was isolated from seeds of Albizia lebbeck by using a protocol that involved ion-exchange chromatography on Q-Sepharose and SPSepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on both Q-Sepharose and SP-Sepharose, but adsorbed on Phenyl-Sepharose. Its hemolytic activity was fully preserved in the pH range 0–14 and in the temperature range 0–100 ◦ C, and unaffected in the presence of a variety of metal ions and carbohydrates. The hemolysin reduced viability of murine splenocytes and inhibited proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells with an IC50 of 0.21, 0.97, and 1.37 ␮M, respectively. It impeded mycelial growth in the fungi Rhizoctonia solani with an IC50 of 39 ␮M but there was no effect on a variety of other filamentous fungi, including Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Mycosphaerella arachidicola. Lebbeckalysin inhibited growth of Escherichia coli with an IC50 of 0.52 ␮M. © 2010 Elsevier GmbH. All rights reserved.

Introduction Hemolysins are produced by bacteria in order to obtain nutrients from host cells. The concentration of free iron within the body of an animal is very low. However, red blood cells are rich in ironcontaining heme. Release of heme by lysing red blood cells with hemolysin allows the bacteria to take up the free iron (Sritharan 2006). Furthermore, many bacterial pathogens produce toxins that kill and lyse host red blood cells. Most of them are pore-forming proteins (Alouf 2001). So, most of the hemolysins were purified from bacteria (Mudenda Hang’ombe et al. 2006; Rózalska and Szewczyk 2008). Apart from pore-forming activity, hemolysins may have antifungal (Abe and Nakazawa 1994), anti-bacterial (Ngai and Ng 2006), and antiproliferative (Berne et al. 2009) activities. So, they are classified as defense proteins. Albizia lebbeck is a species of Albizia, native to tropical southern Asia, and widely cultivated and naturalized in other tropical and subtropical regions. It has various common names including Lebbeck, Lebbek Tree, Flea Tree, Frywood, Koko and Woman’s tongues Tree. Its uses comprise environmental management, forage, medicine and wood. Plantation of A. lebbeck can reduce heavy

∗ Corresponding authors. Tel.: +852 26098031; fax: +852 26035123. E-mail addresses: [email protected] (S.K. Lam), [email protected] (T.B. Ng). 0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.08.009

metal concentrations in the redeveloping soil of mine spoil (Singh et al. 2005). The bark of A. lebbeck demonstrates anti-inflammatory (Babu et al. 2009; Saha and Ahmed 2009) and analgesic (Saha and Ahmed 2009) activities. Saponins (Gupta et al. 2005) isolated from bark show antispermatogenic and antiandrogenic activities (Gupta et al. 2006). Different saponins have been identified from the leaves (el-Mousallamy 1998) and bark (Pal et al. 1995) of A. lebbeck. Furthermore, it is one of the ingredients in a formulation for allergic rhinitis (Amit et al. 2003). In view of the diversity of hemolysins and the meager information about the protein constituents of A. lebbeck seeds that is available, we undertook the present investigation to isolate a hemolysin from this source. The results indicate that the isolated hemolysin possesses some distinctive characteristics. Materials and methods Purification of hemolysin Fresh seeds of A. lebbeck (130 g) collected from an A. lebbeck tree were homogenized. Following centrifugation, the supernatant was applied on a 14 cm × 5 cm Q-Sepharose (GE Healthcare) column in 10 mM NH4 HCO3 (pH 9.4). Adsorbed proteins were eluted with 1 M NaCl added to the NH4 HCO3 buffer. The unadsorbed fraction was dialyzed, and then chromatographed on a 14 cm × 5 cm SPSepharose (GE Healthcare) column in 10 mM ammonium acetate

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Fig. 1. Purification of Albizia lebbeck hemolysin. (A) Anion exchange chromatography of A. lebbeck seed extract on a Q-Sepharose column (14 cm × 5 cm). (B) Cation exchange chromatography of fraction Q1 on an SP-Sepharose column (14 cm × 5 cm). (C) Hydrophobic interaction chromatography of fraction SP1 on a Phenyl-Sepharose column (8 cm × 5 cm). (D). Gel filtration of fraction PS2 on a Superdex 75 10/300 GL column. S3 represents the purified A. lebbeck hemolysin, which is designated as lebbeckalysin. The vertical line and horizontal line in the right halves of panels A and B and in the left half of the panel C represent the stepwise NaCl concentration used to elute adsorbed proteins.

(pH 4.6). Adsorbed proteins were eluted with 1 M NaCl added to the NH4 OAc buffer. The unadsorbed fraction was dialyzed, and diluted with the same volume of 3 M ammonium sulfate containing 40 mM Tris–HCl buffer (pH 7.6) before chromatography on an 8 cm × 5 cm Phenyl-Sepharose (GE Healthcare) column. Adsorbed proteins, eluted with 20 mM Tris–HCl (pH 7.6), were collected, dialyzed and lyophilized before chromatography on a Superdex 75 10/300 GL column (GE Healthcare) in 150 mM ammonium bicarbonate (pH 7.2), previously calibrated with molecular mass markers. The last fraction represented purified hemolysin (5.5 kDa), which was designated as lebbeckalysin. Molecular mass determination using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and N-terminal amino acid sequencing Purified lebbeckalysin was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Laemmli and Favre, 1973). After electrophoresis, the gel was stained with Coomassie Blue R-250. Gel filtration on a fast protein liquid chromatography Superdex 75 10/300 GL column (GE Healthcare) using an AKTA Purifier (GE Healthcare) was conducted to determine the molecular mass of the lebbeckalysin. The column had been calibrated with molecular mass markers, including blue dextran 2000 (to determine void volume), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), myoglobulin (17.6 kDa), ribonuclease A (13.7 kDa), aprotinin (6.5 kDa) and vitamin B12 (1.3 kDa) (GE Healthcare). The N-terminal sequence of lebbeckalysin was determined as described by Lam and Ng (2009b).

centrifugation at 14,000 × g for 30 s. The amount of hemoglobin released from disrupted erythrocytes was determined spectrophotometrically. One hundred percent hemolysis was defined as OD540 of hemoglobin released from erythrocytes treated with 0.1% Triton X-100. One hemolysin unit (HU) was defined as the reciprocal of amount of hemolysin (in mg) eliciting 50% hemoglobin release (Andreeva et al. 2006). Effects of temperature and pH on hemolytic activity A solution of purified lebbeckalysin in capped 1.5-ml microcentrifuge tubes was incubated in PBS (pH 7.5) at various temperatures (4, 25, 40, 50, 60, 70, 80, 90 and 100 ◦ C) for 15 min. In another experiment, the incubation was conducted in buffers at various pH values (pH 0–14) at 25 ◦ C for 15 min. The tubes were then cooled down to room temperature or neutralized to pH 7, immediately before assay of hemolytic activity. Effects of different carbohydrates on hemolytic activity The carbohydrates tested included d-glucosamine, mannitol, d-xylose, sucrose, d-fucose, d-raffinose, ␣-lactose, d-fructose, l-arabinose, d-galacturonic acid, d-galactose, d-mannose, dglucuronic acid, d-glucose and d-sorbitol. A serial two-fold dilution of the carbohydrate solution in microtiter U-plates (50 ␮l) was mixed with a solution of lebbeckalysin (25 ␮l) with 16 HU at room temperature for 30 min. Then a 50 ␮l 2% suspension of rabbit erythrocytes was added. After further incubation for 30 min at room temperature, the concentrations of carbohydrate(s) that could inhibit the hemolysis were determined.

Assay of hemolytic activity

Effect of different ions on hemolytic activity

Rabbit erythrocytes were washed with phosphate-buffered saline (PBS, pH 7.5) and adjusted to a final concentration of 2% (v/v) in PBS. A sample solution (0.2 ml) was mixed with rabbit erythrocytes (0.2 ml) and incubated at 37 ◦ C for 30 min before

Ten microliters of purified lebbeckalysin (128 HU) were incubated with one of the following salts: AgNO3 , CaCl2 , CuCl2 , CuSO4 , FeCl3 , FeSO4 , MgCl2 , MgSO4 , MnSO4 , ZnSO4 , and Na tartrate all at 1 mM at room temperature for 15 min. The mixture was diluted

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with 150 ␮l deionized water. Hemolytic assay was then performed. The above salts, all 62.5 ␮M, were tested in hemolytic assay to confirm no hemolytic effect. Osmotic protection experiments Polyethylene glycol (PEG) 400 (30 mM), PEG 1500 (30 mM), PEG 4000 (30 mM), PEG 6000 (15 mM), PEG 10000 (15 mM), and PEG 20000 (15 mM) with molecular diameters 0.37, 139, 366, 5.86, 9.22, and 18.59 nm, respectively, were used as the osmotic protectants. Two hundred microliters of a 4% rabbit erythrocyte suspension containing an osmotic protectant were mixed with 200 ␮l of lebbeckalysin solution (8 HU). Protection from hemolysis was calculated as follows: protection (%) = (1 − hemolysis rate in the presence of osmotic protectant/hemolytic rate without osmotic protectant) × 100 (Berne et al. 2002). Assay of antiproliferative activity toward tumor cells The assay of the antiproliferative activity of lebbeckalysin was carried out by testing its inhibitory effect on the growth of human hepatoma HepG2 cells and human breast cancer MCF-7 cells as reported by Lam et al. (2009). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and 1% penicillin–streptomycin, in a humidified atmosphere of 5% CO2 at 37 ◦ C. The cells (10,000 cells/100 ␮l/well) were seeded in a 96-well culture plate and serial dilutions of a solution of the lebbeckalysin, or doxorubicin (as positive control) in 100 ␮l medium were added. Medium only was added as negative control. The cells were harvested after incubation for 24 h. Standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine the level of its inhibitory activity. All reported values are the means of triplicate samples. Annexin-V and propidium iodide (PI) staining MCF-7 cells (5 × 105 ) were seeded in a 6-well culture plate and treated with 0.1, 0.3 and 1 ␮M of lebbeckalysin for 24 h. Cells without lebbeckalysin treatment (0 ␮M) served as control. The cells were trypsinized, centrifuged (2000 × g, 4 min), rinsed with PBS, and then centrifuged again (2000 × g, 4 min). Cells were resuspended in 250 ␮l binding buffer (25 mM CaCl2 and 140 mM NaCl in 10 mM HEPES, pH 7.4). Staining solution [247 ␮l binding buffer with 0.5 ␮l PI (6 mg/ml) (Sigma) and 2.5 ␮l Annexin-V solution (BD Phamingen, CA, USA)] was added. The samples were incubated in darkness at room temperature for 20 min before analysis with a FACSort flow cytometer (Becton-Dickinson, Cowley, UK). The signal was discerned by FL-1 (530 nm) channel and data were analyzed with the program WinMDI (Version 2.8, Joseph Trotter, La Jolla, CA, USA) (Lam et al., 2009). Yeast survival assay The survival of Candida albicans after treatment with different concentrations of lebbeckalysin for 24 h at 37 ◦ C was monitored by counting viable yeast. This was done by counting the number of colony forming units (CFUs) after appropriate dilution on LB medium and calculating their number per milliliter. Nystatin (Sigma) was used as a positive control. Splenocyte survival assay C57BL/6 mice (20–25 g) were killed and the spleens were aseptically removed. Spleen cells were obtained by pressing the tissue through a sterilized 100-mesh stainless steel sieve. The cells were washed with PBS twice before resuspending to a density

Fig. 2. SDS-polyacrylamide gel electrophoresis. Right lane: fraction S3 representing purified hemolysin, lebbeckalysin (5.5 kDa). Left lane: molecular mass markers from GE Healthcare. From top downward: carbonic anhydrase (37.7 kDa), soybean trypsin inhibitor (27.5 kDa), lysozyme (16.4 kDa), aprotinin (7.6 kDa), and insulin (4 kDa).

of 1 × 106 cells/ml in RPMI 1640 culture medium supplemented with 10% fetal bovine serum, 100 units penicillin/ml, and 100 ␮g streptomycin/ml. The cells (1 × 106 cells/100 ␮l/well) were seeded into a 96-well culture plate, and serial dilutions of a solution of lebbeckalysin in 100 ␮l medium were added. Medium only and Pleurotus eryngii hemolysin served as negative control and positive control, respectively. After incubation of the cells at 37 ◦ C for 24 h, the numbers of cells were counted. All reported values were means of triplicate samples (Wang et al. 2002). The reading of control was defined as 100% survival. Assay of anti-fungal activity The anti-fungal activity of lebbeckalysin was screened with an agar diffusion assay. Two hundred micrograms of lebbeckalysin were added to test its inhibitory effect on different fungi. The pathogenic fungi species used included Mycosphaerella arachidicola, Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Rhizoctonia solani. Nystatin (Sigma) was used as a positive control. The IC50 value for the anti-fungal activity of lebbeckalysin against R. solani was determined (Lam and Ng 2009b). Congo red and SYTOX green staining of R. solani hyphae Following incubation of R. solani with 80 ␮M lebbeckalysin for 4 h, Congo red or SYTOX green was added a final concentration of 50 and 5 ␮M, respectively. For Congo red staining, fluorescence was examined 30 min later by florescent microscopy using an excitation wavelength of 543 nm and an emission wavelength of 560–635 nm. For SYTOX green staining, an excitation wavelength of 504 nm and an emission wavelength of 523 nm were used (Moreno et al. 2006). Congo red and SYTOX green were used to detect chitin deposition at hyphal tips change in membrane permeability, respectively. Purple pole bean defensin was used as a positive control (Lin et al. 2009a,b).

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Table 1 Yields and hemolytic activity of lebbeckalysin at different stages of purification from 130 g fresh Albizia lebbeck seeds. Purification stages

Chromatographic fraction

Yield (mg)

Specific hemolytic activity (HU/mg)

Total hemolytic activity (HU)

Recovery of HU (%)

Extraction Ion-exchange chromatography on Q-Sepharose Ion-exchange chromatography on SP-Sepharose Hydrophobic interaction chromatography on Phenyl-Sepharose Gel filtration on Superdex 75

– Q1 SP1

1700 543 178

212 572 1620

360,400 310,600 288,400

87 80

PS2

62

4510

279,600

76

S3

16

13,650

218,400

61

HU: hemolytic unit.

Microscopic view of R. solani hyphal degradation after lebbeckalysin treatment The hyphae of R. solani were incubated with 80 ␮M lebbeckalysin at room temperature for 4 h. The hyphae were then observed under a light microscope. Assay of anti-bacterial activity Escherichia coli was collected in the exponential phase of growth and resuspended at a density of 1 × 108 cells/ml with PBS (pH 7.5). Different concentrations of lebbeckalysin in 200 ␮l of 0.2% (w/v) bovine serum albumin were then incubated in 10 ␮l with bacterial suspension with 190 ␮l of Luria–Bertani medium. The mixture was incubated with shaking at 37 ◦ C for 3 h. OD600 was measured. Rachycentron canadum ovary lectin was used as a positive control (Wong et al. 2006). Results and discussion Fresh seeds (130 g) collected locally from A. lebbeck trees were homogenized in 1 l of 10 mM NH4 HCO3 buffer (pH 8.8). The homogenate was centrifuged and the supernatant collected. The hemolysin was isolated using a protocol that entailed QSepharose (anion chromatography) (Fig. 1A), SP-Sepharose (cation chromatography) (Fig. 1B), Phenyl-Sepharose (hydrophobic interaction chromatography) (Fig. 1C) and Superdex 75 10/300 GL (gel filtration) (Fig. 1D). The active fractions were Q1, SP1, PS2 and S3, respectively. The purified hemolysin was designated as lebbeckalysin. Fractions S3 was chromatographed on Superdex peptide 10/300 GL and was eluted as a single homogeneous peak with the elution volume corresponding to a molecular mass of 5.5 kDa (not shown). It demonstrated a single 5.5-kDa band in SDS-PAGE (Fig. 2). The specific activity of lebbeckalysin was 13,650 hemolytic units/mg. A summary of the purification of lebbeckalysin is presented in Table 1. No sequence was obtained by sequencing on an automatic Edman degradation sequencer, suggesting the presence of a blocked N-terminus of hemolysin. Hemolysins have various molecular masses; some are as small as 2.1 kDa (Staphylococcus cohnii) (Rózalska and Szewczyk, 2008),

Fig. 3. Effects of lebbeckalysin on viability of breast cancer MCF-7 cells and hepatoma HepG2 cells. The viability of MCF-7, and HepG2 cells, after incubation for 24 h in the presence of various concentrations of lebbeckalysin, was determined as described in Materials and methods. Viability of cells in RPMI medium only was taken as 100%. Values are expressed as mean ± SD (N = 3). The IC50 values of lebbeckalysin against MCF-7 and HepG2 cells were 0.97 and 1.32 ␮M, respectively. Different alphabets next to the data points on the straight line indicate statistically significant difference (p < 0.05) from one another when the data were analyzed by analysis of variance followed by Duncan’s multiple range test.

while others are larger than 100 kDa, e.g. E. coli (107 kDa) (Aldick et al. 2007), and Vibrio parahemolyticus hemolysin (118 kDa) (Sakurai et al. 1973). The molecular mass of lebbeckalysin is 5.5 kDa, which is within the lower range. Most of the hemolysins were purified from bacteria. Some of them were isolated from animals, like fish (Zhong et al. 2006), sea anemone (Lanio et al. 2001; de Oliveira et al. 2006), jellyfish (Chung et al. 2001), toad (Gomes et al. 1994), and marine sponge (Mangel et al. 1992). There are only a few reports on hemolysin from mushrooms (Suzuki et al. 1990; Ngai and Ng 2006). The present account is the first report of hemolysin from seed. Lebbeckalysin was incubated at different temperatures in PBS and for 15 min. In another experiment, it was incubated in buffers at different pH values at 25 ◦ C for 15 min. Afterwards, it was assayed for hemolytic activity. It demonstrated that the hemolytic activity of the lebbeckalysin was stable throughout the pH range 0–14 and

Table 2 Comparison of biological potencies of lebbeckalysin, doxorubicin, nystatin and Rachycentron canadum ovary lectin. Results represent mean ± SD (N = 3). Lebbeckalysin (IC50 in ␮M) Antiproliferative activity against HepG2 cells Antiproliferative activity against MCF-7 cells Suppression of splenocyte growth Anti-fungal activity against R. solani Inhibitory activity against C. albicans Inhibitory activity against E. coli ND: not determined.

1.32 0.97 0.21 39 16.9 0.52

± ± ± ± ± ±

0.25 0.12 0.02 3 1.2 0.03

Doxorubicin (IC50 in ␮M)

Nystatin (IC50 in ␮M)

Pleurotus eryngii hemolysin (IC50 in ␮M)

Rachycentron canadum ovary lectin (IC50 in ␮M)

8.2 ± 2.1 0.99 ± 0.09 ND ND ND ND

ND ND ND 15.6 ± 1.2 1.3 ± 0.1 ND

ND ND 2.2 ± 0.4 ND ND ND

ND ND ND ND ND 10.5 ± 1.2

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Fig. 4. Annexin-V–FITC/PI staining of MCF-7 cells treated with different concentrations of lebbeckalysin (0.1, 0.3, and 1 ␮M) for 24 h. Cells collected were subjected to staining and then analyzed with a flow cytometer. The table summarizes the percentages of cells at each stage of apoptosis. The results shown here were from one of the three experiments conducted. Results in the other two experiments were similar.

in the temperature range 0–100 ◦ C after incubation for 15 min. The thermostability of most hemolysins has not been investigated. The thermostability of lebbeckalysin was the same as that of V. parahemolyticus hemolysin (Sakurai et al. 1973), which was stable at 100 ◦ C. The thermostability of P. eryngii hemolysin (eryngeolysin) is much lower, being stable only up to 40 ◦ C (Ngai and Ng, 2006). Eryngeolysin is stable over the pH range 4–12 (Ngai et al., 2006). Lebbeckalysin is stable over a wider pH range from 0 to 14. The stability of peptides is usually higher due to their confirmation stability. There are many examples of highly stable peptides, e.g. cabbage Brassica campestris anti-fungal peptide (Lin et al. 2009a,b) and Aspergillus clavatus anti-fungal peptide (Skouri-Gargouri and Gargouri 2008). The hemolytic activity of lebbeckalysin could not be inhibited by any sugars and ions tested. The hemolytic activity of eryngeolysin is inhibited by N-glycolylneuraminic acid, Na2 CO3 , Na3 PO4 , FeCl2 , and CuCl2 (Ngai et al., 2006). Ostreolysin is inhibited by HgCl2 (Berne et al. 2002). Vibrio fluvialis hemolysin is inhibited by the chlorides of Cd2+ , Cu2+ , Ni2+ and Zn2+ (Han et al. 2002). However, none of carbohydrates or ions could protect the red blood cells from lebbeckalysin. Hemolysis induced by lebbeckalysin could not be protected by any PEG tested. Hemolysis induced by eryngeolysin is osmotically protected by PEG 10000 with a mean hydrated diameter close to 9.3 nm (Ngai et al., 2006). Hemolysis induced by V. fluvialis hemolysis is osmotically protected by a mean hydrated diameter of 2.8–3.7 nm (Han et al. 2002). Leptospira interrogans hemolysinmediated hemolysis is osmotically protected by PEG 5000 (Lee et al. 2002). Hemolysis induced by lebbeckalysin could not be protected by any PEG tested, indicating that hemolysis induced by the hemolysin cannot be osmotically protected by a mean hydrated diameter from 0.32 to 18.59 nm.

Lebbeckalysin inhibited proliferation of MCF-7 and HepG2 tumor cells with an IC50 0.97 and 1.32 ␮M, respectively (Fig. 3). By comparison, the positive control doxorubicin exhibited an antiproliferative activity toward these tumor cells with an IC50 of 0.3 and 8.2 ␮M, respectively (Table 2). Asp-hemolysin (Kumagai et al. 2001), ostreolysin (Sepcic´ et al. 2003) and eryngeolysin (Ngai et al., 2006) exert an antiproliferative effect on different cell lines. The mechanism of anti-tumor activity was deduced by direct microscopic observations. Certain tumor cell lines exposed to ostreolysin colloid-osmotic alteration show swelling, blebbing and degranulation of cells (Sepcic´ et al. 2003).

Fig. 5. Effects of lebbeckalysin on survival of splenocytes. The viability of splenocytes after incubation for 24 h in the presence of various concentrations of lebbeckalysin was determined as described in Materials and methods. Viability of splenocytes in RPMI medium only was taken as 100%. Values are expressed as mean ± SD (N = 3). The IC50 value of lebbeckalysin against splenocytes is 0.21 ␮M. Different alphabets next to the data points indicate statistically significant difference (p < 0.05) from one another when the data were analyzed by analysis of variance followed by Duncan’s multiple range test.

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Fig. 6. Anti-fungal activity of lebbeckalysin toward R. solani. (A) Buffer control, (B) 7.5 ␮M lebbeckalysin. (C) 22 ␮M lebbeckalysin. (D) 66 ␮M lebbeckalysin. (E) 200 ␮M lebbeckalysin. (F) 600 ␮M lebbeckalysin. The IC50 value of lebbeckalysin against R. solani was 39 ␮M.

Incubation of 1 ␮M lebbeckalysin for 24 h led to an increase of 13.4% in the number of apoptotic cells and 20.5% in the number of necrotic cells (Fig. 4). The mechanism involved is probably colloidosmotic alternation (Sepcic´ et al. 2003). The results from annexin-V and propidium iodide staining of MCF-7 cells further have disclosed that the cells undergo necrosis. This result is greatly different from Pleurotus nebrodensis hemolysin (nebrodeolysin)-induced apoptosis in L929 and HeLa cells (Lv et al., 2008). Lebbeckalysin suppressed the growth of splenocytes with an IC50 of 0.21 ␮M (Fig. 5). Both eryngeolysin (Ngai et al., 2006) and lebbeckalysin suppress the growth of splenocytes. The mechanism involved is also probably colloid-osmotic alternation (Sepcic´ et al. 2003). Lebbeckalysin inhibited mycelial growth in R. solani with an IC50 of 39 ␮M (Fig. 6) but there was no effect on M. arachidicola, F. oxysporum, H. maydis, and V. mali when tested up to 200 ␮g in agar diffusion assay (not shown). There was neither Congo red staining in the hyphal tips of R. solani nor SYTOX green staining in the hyphae (not shown), while the result of positive control was similar with those previously reported. There are two major action mechanisms for anti-fungal proteins, including chitin deposition at hyphal tips and alteration of fungal membrane permeability. However, there was neither chitin deposition at hyphal tips of R. solani nor changes in membrane permeability as evidenced by lack of Congo red or SYTOX green staining in the hyphae. On the other hand, hyphal degradation in R. solani was observed after incubation with 80 ␮M lebbeckalysin for 4 h (Fig. 7). It may be due to the pore-forming property of lebbeckalysin, which causes the

degradation of fungal cell wall and ultimately hyphal disintegration. To date, none of the hemolysins has been reported to have anti-fungal activity. Lebbeckalysin exerted an anti-fungal action on R. solani, though there was no activity against four other fungal species tested. This finding is reminiscent of the observations that the shallot anti-fungal protein ascalin (Wang et al., 2002) and the anti-fungal amidase from Peltophorum pterocarpum (Lam and Ng 2009a) exert an anti-fungal action on only one out of the several fungi examined. Lebbeckalysin inhibited the growth of C. albicans with an IC50 value of 16.9 ␮M (Fig. 8). The number of Candida infections has been increasing over the period of several decades, especially for the elderly, critically ill patients, and immunosuppressed patients. As a consequence, some common superficial infections, such as oropharyngeal candidoses, in addition to serious deep candidoses have become important clinical problems (Ellepola and Samaranayake 2000). Furthermore, due to improper use of antibiotics and more invasive therapeutic medical procedures, C. albicans has developed resistance against commonly used anti-fungal agents (Richardson 2005). Information about growth inhibition of yeast by hemolysins is unavailable. The present report constitutes the first demonstration of growth inhibition of yeast by hemolysin. Lebbeckalysin can inhibit the growth of E. coli with an IC50 value of 0.52 ␮M (Fig. 9). Eryngeolysin does not exert anti-bacterial activity toward E. coli, but can suppress Bacillus megatarium and Bacillus subtilis (Ngai et al., 2006). To the best of our knowledge, no bacterial hemolysins have been reported with anti-bacterial activity. In fact, most of the hemolysins originate from bacteria. This is proba-

Fig. 7. Degradation of the hyphae of R. solani after treatment with lebbeckalysin for 4 h. (A) Buffer control. Picture was taken under the light microscope. (B) Treatment with 80 ␮M lebbeckalysin. Picture was taken under the light microscope. Degradation of hyphae was observed.

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Fig. 8. Inhibitory effect of lebbeckalysin toward Candida albicans. Values are expressed as mean ± SD (N = 3). The IC50 value of lebbeckalysin against C. albicans was 16.9 ␮M. Different alphabets next to the data points indicate statistically significant difference (p < 0.05) from one another when the data were analyzed by analysis of variance followed by Duncan’s multiple range test.

Fig. 9. Effects of lebbeckalysin on viability of Escherichia coli. The viability of E. coli after incubation for 3 h in the presence of various concentrations of lebbeckalysin was determined as described in Materials and methods. Viability of E. coli in Luria–Bertani medium only was taken as 100%. Values are expressed as mean ± SD (N = 3). The IC50 value of lebbeckalysin against E. coli was 0.52 ␮M. Different alphabets next to the data points indicate statistically significant difference (p < 0.05) from one another when the data were analyzed by analysis of variance followed by Duncan’s multiple range test.

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