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Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens
Toxicon 45 (2005) 777–782 www.elsevier.com/locate/toxicon
Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens Geraldino A. Cunha Filhoa, Carlos Alberto Schwartza, Ineˆs S. Resckb, Maria Ma´rcia Murtab, Sebastia˜o S. Lemosb, Mariana S. Castroa, Cynthia Kyawc, Osmindo R. Pires Jr.a, Jose´ Roberto S. Leited, Carlos Bloch Jr.d, Elisabeth Ferroni Schwartza,* a
Laborato´rio de Toxinologia, Departamento de Cieˆncias Fisiolo´gicas, Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, Brası´lia, DF 70910-900, Brazil b Instituto de Quı´mica, Universidade de Brası´lia, Brası´lia, DF 70910-900, Brazil c Laborato´rio de Microbiologia, CEL, IB, Universidade de Brası´lia, Brası´lia, DF 70910-900, Brazil d Laborato´rio de Espectrometria de Massa, EMBRAPA-CENARGEN, P.O. Box 02372, Brası´lia, DF, Brazil Accepted 25 January 2005
Abstract The increase in the emergence of antibiotic-resistant microorganisms and difficult to treat infections caused by these pathogens stimulate research aiming the identification of novel antimicrobials. Skin secretion of amphibian contains a large number of biologically active compounds, including compounds that performance defense mechanisms against microorganisms. In the present work, two antimicrobial bufadienolides, telocinobufagin (402.1609 Da) and marinobufagin (400.1515 Da), were isolated from skin secretions of the Brazilian toad Bufo rubescens. The specimens were collected in Brasilia (Distrito Federal, Brazil), the skin secretions extracted by electric stimulation, and submitted to purification by RPHPLC. The molecular structure and mass determination were done by 1H and 13C NMR and mass spectrometry data, respectively. The antimicrobial activity was performed by liquid growth inhibition against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations of telocinobufagin and marinobufagin were, respectively, 64.0 and 16.0 mg/mL for E. coli and both 128 mg/mL for S. aureus. Besides the antimicrobial activity both bufadienolides promoted an increase of the contraction force in isolated frog ventricle strips. q 2005 Elsevier Ltd. All rights reserved. Keywords: Bufo rubescens; Bufadienolides; Skin secretion; Amphibian; Antimicrobial; Venom
1. Introduction The fast increase in the emergence of antibiotic-resistant microorganisms is a global problem that has threatened
* Corresponding author. Tel.: C55 61 307 2160; fax: C55 61 274 1251. E-mail address: [email protected] (E.F. Schwartz). 0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.01.017
the ability of physicians to treat serious infections. Studies have demonstrated that infections caused by antibioticresistant bacteria are more difficult to treat, resulting in excess morbidity and mortality as well as in higher health care costs. The growth of resistance to the antibiotics currently employed in clinical practice is a continual stimulus for further research aiming the identification of novel antimicrobial compounds (Boman, 1995; Hancock and Chapple, 1999; Zasloff, 2002).
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Skin secretion of amphibian contains a large number of biologically active compounds which are thought to play several roles, either in the regulation of the physiological functions of the skin or in defense mechanisms against predators or microorganisms (Clark, 1997). The synthesis of peptides with antimicrobial activity in granular glands located in the skin is a feature of several anuran (frog and toad) species, particularly those belonging to the families Bombinatoridae, Hylidae, Hyperoliidae, Myobatrachidae, Pipidae, and Ranidae (Conlon et al., 2004; Simmaco et al., 1998). A 39 amino acid peptide, buforin I, was isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans, a member of the Bufonidae family (Park et al., 1996). Two b-galactoside-binding lectins showing bacteriostatic activity against Gram negative and Gram positive bacteria were isolated from the skin of Bufo arenarum (Riera et al., 2003). The Chinese traditional drug Ch’an Su, called ‘Senso’ in Japanese, is a product of the skin secretions of local toads such as Bufo bufo gargarizans or Bufo melanostrictus (Nogawa et al., 2001; Bick et al., 2002). The principal biologically active components of Ch’an Su are bufadienolides, which have steroidal A/B cis and C/D cis ring junctures with a a-2-pyrone ring at C17-position, exhibiting a range of biological activities. The Ch’an Su has been traditionally used as a cardiotonic, diuretic, anodyne and hemostatic agent. Among the bufadienolides, resibufogenin is now used as a cardiotonic drug, and bufalin has recently been reported to have a strong surface anesthetic activity and cytotoxic effect and differentiation-apoptosis activity on murine leukemia HL-60 cells. Bufadienolides are typically polyhydroxy C24 steroids and their glycosides have been isolated from both plant and animal sources (Steyn and Heerden, 1998; Kren and Kopp, 1998). Over 250 bufadienolides have been identified so far, with almost 160 isolated from plants of six families (Crassulaceae, Hyacinthaceae, Iridaceae, Melianthaceae, Ranunculaceae, and Santalaceae). Bufadienolides and their conjugates may be found in free and conjugated forms in the tissues and body fluids of toads of the genus Bufo. The large variety of known genins is chiefly due to the number and position of substituents. Bufadienolides are important for their activity as cardiac glycosides, where they increase the contractile force of the heart by inhibiting NaC/KC-ATPase. Helleborin, isolated from Helleborus spp. was previously used medicinally in the treatment of heart rhythm irregularities (Meng et al., 2001). It has been suggested that endogenous bufadienolides and cardenolides are produced by the adrenal cortex in mammals, probably corresponding to a new class of steroid hormone involved in the regulation of hypertension (Schoner, 2002). Some bufadienolides possess antitumor activity, as described for bufalin, scillarein, bufotalin, gamabufotalin, cinobufotalin, cinobufagin, and others (Kamano et al., 2002; Nogawa et al., 2001). They also show toxic activity towards livestock, potent insect
antifeedant (Steyn and Heerden, 1998) and insecticidal properties (Supratman et al., 2001). Antimicrobial activity of bufadienolides is assigned to abyssinin and abyssinols, isolated from plant (Taniguchi and Kubo, 1993). In the present study we isolated and characterized two antimicrobial bufadienolides from the skin and parotoid gland secretions of Bufo rubescens.
2. Materials and methods 2.1. Animals and venom extraction Adult specimens of B. rubescens captured in Brasilia, Distrito Federal, Brazil (IBAMA/RAN licenses 12/2001 and 054/02) were maintained in the laboratory and fed with Tenebrionidae larvae and mice. Parotoid secretion was obtained by gland compression, and skin secretion obtained by electrical stimulation (around 6 V). Both secretions were collected in milli-Q water, mixed, lyophilized, and then kept at K20 8C. 2.2. Bufadienolides purification The lyophilized secretion was denominated BrSS, and was submitted to a semi-purification as described by Zelnick et al. (1964). Briefly, the BrSS was resuspended in water and extracted three times with equal volume of CHCl3:CH3OH (9:1, v:v). The solvent of the combined CHCl3: CH3OH extract was removed in a rotary evaporator under vacuum. The resulting residue was dissolved in CH3OH:CH3CN (1:1, v:v), filtered and fractionated by RP-HPLC (Shimadzu Co.) using the Asahipac C4P-50 (250!10.0 mm) column from Shimadzu Corporation (Kyoto, Japan). The fractions were eluted in a 70 min linear gradient of H2O (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluroacetic acid (TFA) at a flow rate of 2.5 mL/min. The eluant was monitored at 297 nm and fractions were collected, lyophilized and stored at K20 8C. The antimicrobial fractions were submitted to a RP-HPLC using a Vydac 218TP510 column. The fractions were eluted in a 50 min linear gradient of H2O (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluroacetic acid (TFA). The experiment was monitored at 216 and 297 nm and samples were lyophilized and stored at K20 8C. 2.3. Antimicrobial activity The microorganisms used for the antimicrobial assays were purchased from American Type Culture Collection. They were Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25992. The microorganisms were cultured in stationary culture at 37 8C. Bacteria were grown in Mueller–Hinton liquid medium (NCCLS Approved Standard M100-S9).
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The bioassays by liquid growth inhibition were performed as described by Kim et al. (2000). The bufadienolides were dissolved and diluted eightfold in Mueller–Hinton broth. The highest concentration used for the assays was 128 mg/mL. The initial inoculum was approximately 1.5!105 colony forming units (CFU)/mL. The final volume was 200 mL:100 mL of the bufadienolides test in broth, 50 mL culture medium, and 50 mL of the inoculum in Mueller–Hinton. The minimal inhibitory concentration (MIC) was measured from optical density (OD595) 12 h after all microorganisms were grown in stationary culture at 37 8C. The lowest bufadienolide concentration in which no bacterial growth occurred was defined as the MIC. 2.4. Chemical characterization of the active compounds Aliquots of the antimicrobial bufadienolides were mass analyzed by a Q-Tof Ultima API (Waters, CO, UK) operating in data directed acquisition mode and with a Nano flow Z-spray source. NMR spectra were recorded at room temperature on a Varian Mercury Plus spectrometer (7.05 T) operating at 300 MHz for 1H and 75.46 MHz for 13C. Compounds were dissolved in CDCl3 containing TMS as internal reference and measured in 5 mm sample tubes. Chemical shifts were expressed in d (ppm) and coupling constants as J (Hz) with abbreviations d, doublet; dd, double dublet; t, triplet and br, broad. In the homonuclear and heteronuclear
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two-dimensional experiments (COSY and HMQC) we used the field gradient mode. 2.5. Cadiotonic activity The cardiotonic activity of the isolated bufadienolides was verified on the ventricle strip of frog’s heart as described previously (Schwartz et al., 1999). Frogs (Rana catesbeiana) were pithed and their abdominal cavity opened. The ventricle was dissected from isolated hearts in aerated Ringer at room temperature. The ventricle strips were electrically driven with square pulses of 1 ms duration, 0.2 Hz frequency and the lowest voltage that induced maximum contractions. The rate and strength of contraction were registered with the F-60 myograph (Narco Bio-Systems) and a recorder. Bufadienolides were added to the bath (10 ml) and the effects recorded for 15 min. The composition of Ringer solution was (in mM): 111.0 NaCl; 1.8 KCl; 1.1 CaCl2; 2.4 NaHCO3 and 6.76 glucose.
3. Results 3.1. Bufadienolides purification The fractionation of the chloroform extract from B. rubescens skin secretion yielded the chromatographic profile shown in Fig. 1, indicating the presence of 12 main
Fig. 1. Chromatogram profile of the CHCl3:CH3OH extract filtered and fractionated by RP-HPLC (Shimadzu Co.) using the Asahipac C4P-50 (250!10.0 mm) column from Shimadzu Co. (Kyoto, Japan). The fractions were eluted in a 70 min linear gradient of H2O (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluroacetic acid (TFA) at a flow rate of 2.5 mL/min. The eluant was monitored at 297 nm.
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fractions. The prominent peaks designated 8 and 9 contained components that strongly inhibited the growth of S. aureus and E. coli (data not shown). The rechromatography of the fractions containing antimicrobial activity (fractions 8 and 9) led to the isolation of two pure bufadienolides named 8 0 and 9 0 , respectively (data not shown). 3.2. Spectrometric characterization The fragment ion spectrogram of bufadienolide 8 0 showed mass component at m/z 403.2610 Da (Fig. 2(A)), corresponding to the protonated [M(C24H34O5)CH]C of the molecular species. The spectrogram also exhibited specific fragment ions m/z 385.2498, 367.2357, 349.2249 and 307.1730 Da due to elimination of one, two and three water molecules and a-pyrone group (C5 H3O2 ), respectively. There was a interesting gas-phase reaction of protonated bufadienolide 8 0 , the mass spectrogram showed peaks at m/z 301.0783, 403.1609, 425.1171, 487.1324, 703.2789, 827.2839, 1025.9425, 1229.5254 Da, and ions were assigned as follows: 425.1171 Da (MCNaC); 827.2839 and 1229.5254 Da are addition of 403 Da in the aductor ion 425.1171 Da. This was confirmed by fragment pattern of 1229.5254 and 827.2839 Da (data not shown). The 1H NMR spectra of 8 0 agreed to the chemical structure of telocinobufagin with H-21, H-22 and H-23 signals [d 7.24 (dd, JZ2.6 and 1.0 Hz), d 7.82 (dd, JZ9.7 and 2.6 Hz) and d 6.27 (dd, JZ9.7 and 1.0 Hz)] characteristic of the a-pyrone ring of bufadienolides (Fig. 2(B)) (Shimada et al., 1977).
In the COSY spectrum, the correlations of these hydrogens were observed. HMQC experiment was carried out to determine the chemical shifts of C-21 (d 148.3), C-22 (d 145.8) and C-23 (d 114.7). Connectivity of H-3 (d 4.19, brt) of CH carbinylic group to C-3 (d 68.0) was also shown. The fragment ion spectrogram of bufadienolide 9 0 showed peak at m/z 401.2331 Da, corresponding to the protonated [M(C24H32O5)CH]C. The spectrogram also exhibited specific fragment ions m/z 383.2208, 365.2094, 347.1986 Da and 305.1916 due to elimination of one, two and three water molecules and a-pyrone group, respectively (Fig. 3(A)), similar to that obtained by the ion-spray MS/MS of marinobufagin reported by Bagrov et al. (1998). The same gas-phase reaction of protonated bufadienolide 9 0 was observed, the mass spectrogram showed peaks at m/z 301.0854, 401.1515, 423.1038, 823.2703, 1020.9385, 1223.5205 Da, and ions were assigned as follows: 423.1038 Da (MCNaC); 823.2703 and 1223.5205 Da are addition of 401 Da in the aductor ion 423.1038 Da. The 1H NMR spectrum of the compound 9 0 furnished the H-21, H-22 and H-23 signals [d 7.24 (dd, JZ1.0 and 2.6 Hz), d 7.78 (dd, JZ2.6 and 9.8 Hz) and d 6.26 (dd, JZ 1.0 and 9.7 Hz)] as characteristic of the a-pyrone ring of bufadienolides. The H-15 (d 3.49, d, JZ0.6 Hz) of epoxy group in the 1H NMR spectrum and 13C NMR data together with APT spectrum of the compound 9 0 were referred to marinobufagin (Fig. 3(B)) (Verpoorte et al., 1980). 3.3. Antimicrobial activity The bufadienolides telocinobufagin and marinobufagin exhibited antibacterial activity, as they inhibited the growth
Fig. 2. (A) Fragmentation ion profile in TOF MS/MS system of bufadienolide 8 0 , showing mass at 403.2610 Da [MCH]C similar to telocinobufagin (402.5238 Da). (B) Structure of telocinobufagin.
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Fig. 3. (A) Fragmentation ion profile in TOF MS/MS system of bufadienolide 9 0 , showing mass at 401.2331 Da [MCH]C similar to marinobufagin (400.5079 Da). (B) Structure of marinobufagin.
of the bacterial strains tested (S. aureus and E. coli) with significant differences in growth inhibition of them. The MIC obtained for both bufadienolides were compared with the ones obtained by the testing of conventional antibiotics (amoxicillim, imipenem e trimethoprim) (Table 1). 3.4. Cardiotonic activity The addition of bufadienolides telocinobufagin and marinobufagin (both 9.9 mg/mL) to the saline bathing the isolated ventricle strip, produced a positive inotropic effect, increasing the contraction force in 30.2G4.2 and 42.98G 3.30%, respectively.
4. Discussion Bufadienolides are the main biologically active components of Ch’an Su, a Chinese traditional drug. Various studies of the Ch’an Su and related bufadienolides have been
conducted previously in order to better characterize them as possible inhibitors of cancer cell growth (Kamano et al., 1998, 2002; Lee and Yoon, 1995) and inducers of apoptosis (Jing et al., 1994), to study their antiviral effects (Nakanishi et al., 1999), and to evaluate the possibility of an endogenous mammalian bufadienolide being involved in NaC,KC -ATPase activity related to the pathogenesis of arterial hypertension (Doris and Bagrov, 1998; Bagrov et al., 1998). In the present report, we described for the first time the antimicrobial activity of two bufadienolides isolated from skin secretions of the toad B. rubescens, telocinobufagin and marinobufagin. The mass fragmentation and NMR data provide the structure elucidation of both bufadienolides (Shimada et al., 1977; Verpoorte et al., 1980; Bagrov et al., 1998; Akizawa et al., 1994; Nogawa et al., 2001). Besides the antimicrobial activity, telocinobufagin and marinobufagin also promoted an increase of the contraction force in isolated frog ventricle strips. The presence of antimicrobial bufadienolides in the skin of B. rubescens led us to consider their possible biological
Table 1 Antimicrobial activity of telocinobufagin and marinobufagin isolated from skin secretion of Bufo rubescens Microorganisms
MICa (mg/mL) Telocinobufagin
Marinobufagin
Amoxillim
Imipenem
Trimethoprim
S. aureus ATCC 25213 E. coli ATCC 25922
128 64
128 16
N/Db !22
N/Db 14
!160 40
a MIC, minimal concentration of the compound required for total inhibition of bacteria growth in liquid medium. Experiments performed in triplicates. b N/D, no detectable activity.
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involvement in the defense mechanisms of Bufo species. Since, both bufadienolides also presented cardiac activity, additional studies comparing relative effective doses should be done. Besides, the utilization of these compounds as topic drugs in the treatment of superficial infections should not be discharged. Acknowledgements We thank Rodrigo A.V. Morales and Jorge A.T. Melo for helping with mass spectrometry analysis, Daniel N. Sifuentes for helping with ventricle strip assay, and Graciela Martins (Laborato´rio Sabin, Brası´lia, Brasil) for antimicrobial assay. These studies were supported by Brazilian government through the funding agencies CAPES and FINEP (process CT-INFRA 970/2001).
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Pettit, G.R., 2002. QSAR evaluation of the Ch’an Su and related bufadienolides against the colchicine-resistant primary liver carcinoma cell line PLC/PRF/5. J. Med. Chem. 45, 5440–5447. Kim, H.-S., Choi, B.-S., Kwon, K.-C., Lee, S.-O., Kwak, H.J., Lee, C.H., 2000. Synthesis and antimicrobial activity of squalamine analogue. Bioorg. Med. Chem. 8, 2059–2065. Krenn, L., Kopp, B., 1998. Bufadienolides from animal and plant sources. Phytochemistry 48, 1–29. Lee, D.Y., Yoon, H.J., 1995. Growth-inhibiting effect of bufadienolides on cultured vascular endothelial cells. Korean J. Toxicol. 11, 175–180. Meng, Y., Whiting, P., Sik, V., Rees, H.H., Dinan, L., 2001. Ecdiesteroids and bufadienolides from Helleborus torquatus (Ranunculaceae). Phytochemistry 47, 402–407. Nakanishi, T., Nishino, H., Ichiishi, E., Mukainaka, T., Okuda, M., Tokuda, H., 1999. Inhibitory effects of bufadienolides on Epstein–Barr virus early antigen activation and on growth of mouse skin and mouse pulmonary tumors. Nat. Med. 53, 324–328. Nogawa, T., Kamano, Y., Yamashita, A., Pettit, G.R., 2001. Isolation and structure of five mew cancer cell growth inhibitory bufadienolides from the Chinese tradicional drug Ch’an su. J. Nat. Prod. 64, 1148–1152. Park, C.B., Kim, M.S., Kim, S.C., 1996. Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem. Biophys. Res. Commun. 218, 408–413. Riera, A.S., Daud, A., Gallo, A., Genta, S., Ybar, M.A., Sa´nchez, S., 2003. Antibacterial activity of lactose-binding lectins from Bufo arenarum skin. Biocell 27 (1), 37–46. Schoner, W., 2002. Endogenous cardiac glycosides, a new class of steroid hormones. Eur. J. Biochem. 269, 2440–2448. Schwartz, E.N., Schwartz, C.A., Sebben, A., Largura, S.W., Mendes, E.G., 1999. Indirect cardiotoxic activity of the caecilian Siphonops paulensis (Gymnophiona, Amphibia) skin secretion. Toxicon 37 (1), 47–54. Shimada, K., Fujii, Y., Yamashita, E., Niizaki, Y., Sato, Y., Nambara, T., 1977. Studies on cardiotonic steroids from the skin of Japanese toad. Chem. Pharm. Bull. 25, 714–730. Simmaco, M., Mignogna, G., Barra, D., 1998. Antimicrobial peptides from amphibian skin: what do they tell us?. Biopolymers 47 (6), 435–450. Steyn, P.S., Heerden, F.R., 1998. Bufadienolides of plant and animal origin. Nat. Prod. Rep. 15 (4), 397–413. Supratman, U., Fujita, T., Akiyama, K., Hayashi, H., 2001. Inseticidal compounds from Kalanchoe daigremontiana!tubiflora. Phytochemistry 58, 311–314. Taniguchi, M., Kubo, I., 1993. Ethnobotanical drug discovery based on medicine men’s trials in the African savanna: screening of east African plants for antimicrobial activity II. J. Nat. Prod. 56 (9), 1539–1546. Verpoorte, R., Kinh, P.-Q., Svendsen, A.B., 1980. Chemical constituents of vietnamese toad venom, collected from Bufo melanostictus Schneider. Part II. The bufadienolides. J. Nat. Prod. 43, 347–352. Zasloff, M., 2002. Antimicrobial peptides in health and disease. N. Engl. J. Med. 347, 1199–1200. Zelnick, R., Ziti, L.M., Guimara˜es, C.V., 1964. A chromatographic study of the bufadienolides isolated from the venom of the parotoid glands of Bufo paracnemis Lutz 1925. J. Chromatogr. 15, 9–14.
Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens Geraldino A. Cunha Filhoa, Carlos Alberto Schwartza, Ineˆs S. Resckb, Maria Ma´rcia Murtab, Sebastia˜o S. Lemosb, Mariana S. Castroa, Cynthia Kyawc, Osmindo R. Pires Jr.a, Jose´ Roberto S. Leited, Carlos Bloch Jr.d, Elisabeth Ferroni Schwartza,* a
Laborato´rio de Toxinologia, Departamento de Cieˆncias Fisiolo´gicas, Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, Brası´lia, DF 70910-900, Brazil b Instituto de Quı´mica, Universidade de Brası´lia, Brası´lia, DF 70910-900, Brazil c Laborato´rio de Microbiologia, CEL, IB, Universidade de Brası´lia, Brası´lia, DF 70910-900, Brazil d Laborato´rio de Espectrometria de Massa, EMBRAPA-CENARGEN, P.O. Box 02372, Brası´lia, DF, Brazil Accepted 25 January 2005
Abstract The increase in the emergence of antibiotic-resistant microorganisms and difficult to treat infections caused by these pathogens stimulate research aiming the identification of novel antimicrobials. Skin secretion of amphibian contains a large number of biologically active compounds, including compounds that performance defense mechanisms against microorganisms. In the present work, two antimicrobial bufadienolides, telocinobufagin (402.1609 Da) and marinobufagin (400.1515 Da), were isolated from skin secretions of the Brazilian toad Bufo rubescens. The specimens were collected in Brasilia (Distrito Federal, Brazil), the skin secretions extracted by electric stimulation, and submitted to purification by RPHPLC. The molecular structure and mass determination were done by 1H and 13C NMR and mass spectrometry data, respectively. The antimicrobial activity was performed by liquid growth inhibition against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations of telocinobufagin and marinobufagin were, respectively, 64.0 and 16.0 mg/mL for E. coli and both 128 mg/mL for S. aureus. Besides the antimicrobial activity both bufadienolides promoted an increase of the contraction force in isolated frog ventricle strips. q 2005 Elsevier Ltd. All rights reserved. Keywords: Bufo rubescens; Bufadienolides; Skin secretion; Amphibian; Antimicrobial; Venom
1. Introduction The fast increase in the emergence of antibiotic-resistant microorganisms is a global problem that has threatened
* Corresponding author. Tel.: C55 61 307 2160; fax: C55 61 274 1251. E-mail address: [email protected] (E.F. Schwartz). 0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.01.017
the ability of physicians to treat serious infections. Studies have demonstrated that infections caused by antibioticresistant bacteria are more difficult to treat, resulting in excess morbidity and mortality as well as in higher health care costs. The growth of resistance to the antibiotics currently employed in clinical practice is a continual stimulus for further research aiming the identification of novel antimicrobial compounds (Boman, 1995; Hancock and Chapple, 1999; Zasloff, 2002).
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Skin secretion of amphibian contains a large number of biologically active compounds which are thought to play several roles, either in the regulation of the physiological functions of the skin or in defense mechanisms against predators or microorganisms (Clark, 1997). The synthesis of peptides with antimicrobial activity in granular glands located in the skin is a feature of several anuran (frog and toad) species, particularly those belonging to the families Bombinatoridae, Hylidae, Hyperoliidae, Myobatrachidae, Pipidae, and Ranidae (Conlon et al., 2004; Simmaco et al., 1998). A 39 amino acid peptide, buforin I, was isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans, a member of the Bufonidae family (Park et al., 1996). Two b-galactoside-binding lectins showing bacteriostatic activity against Gram negative and Gram positive bacteria were isolated from the skin of Bufo arenarum (Riera et al., 2003). The Chinese traditional drug Ch’an Su, called ‘Senso’ in Japanese, is a product of the skin secretions of local toads such as Bufo bufo gargarizans or Bufo melanostrictus (Nogawa et al., 2001; Bick et al., 2002). The principal biologically active components of Ch’an Su are bufadienolides, which have steroidal A/B cis and C/D cis ring junctures with a a-2-pyrone ring at C17-position, exhibiting a range of biological activities. The Ch’an Su has been traditionally used as a cardiotonic, diuretic, anodyne and hemostatic agent. Among the bufadienolides, resibufogenin is now used as a cardiotonic drug, and bufalin has recently been reported to have a strong surface anesthetic activity and cytotoxic effect and differentiation-apoptosis activity on murine leukemia HL-60 cells. Bufadienolides are typically polyhydroxy C24 steroids and their glycosides have been isolated from both plant and animal sources (Steyn and Heerden, 1998; Kren and Kopp, 1998). Over 250 bufadienolides have been identified so far, with almost 160 isolated from plants of six families (Crassulaceae, Hyacinthaceae, Iridaceae, Melianthaceae, Ranunculaceae, and Santalaceae). Bufadienolides and their conjugates may be found in free and conjugated forms in the tissues and body fluids of toads of the genus Bufo. The large variety of known genins is chiefly due to the number and position of substituents. Bufadienolides are important for their activity as cardiac glycosides, where they increase the contractile force of the heart by inhibiting NaC/KC-ATPase. Helleborin, isolated from Helleborus spp. was previously used medicinally in the treatment of heart rhythm irregularities (Meng et al., 2001). It has been suggested that endogenous bufadienolides and cardenolides are produced by the adrenal cortex in mammals, probably corresponding to a new class of steroid hormone involved in the regulation of hypertension (Schoner, 2002). Some bufadienolides possess antitumor activity, as described for bufalin, scillarein, bufotalin, gamabufotalin, cinobufotalin, cinobufagin, and others (Kamano et al., 2002; Nogawa et al., 2001). They also show toxic activity towards livestock, potent insect
antifeedant (Steyn and Heerden, 1998) and insecticidal properties (Supratman et al., 2001). Antimicrobial activity of bufadienolides is assigned to abyssinin and abyssinols, isolated from plant (Taniguchi and Kubo, 1993). In the present study we isolated and characterized two antimicrobial bufadienolides from the skin and parotoid gland secretions of Bufo rubescens.
2. Materials and methods 2.1. Animals and venom extraction Adult specimens of B. rubescens captured in Brasilia, Distrito Federal, Brazil (IBAMA/RAN licenses 12/2001 and 054/02) were maintained in the laboratory and fed with Tenebrionidae larvae and mice. Parotoid secretion was obtained by gland compression, and skin secretion obtained by electrical stimulation (around 6 V). Both secretions were collected in milli-Q water, mixed, lyophilized, and then kept at K20 8C. 2.2. Bufadienolides purification The lyophilized secretion was denominated BrSS, and was submitted to a semi-purification as described by Zelnick et al. (1964). Briefly, the BrSS was resuspended in water and extracted three times with equal volume of CHCl3:CH3OH (9:1, v:v). The solvent of the combined CHCl3: CH3OH extract was removed in a rotary evaporator under vacuum. The resulting residue was dissolved in CH3OH:CH3CN (1:1, v:v), filtered and fractionated by RP-HPLC (Shimadzu Co.) using the Asahipac C4P-50 (250!10.0 mm) column from Shimadzu Corporation (Kyoto, Japan). The fractions were eluted in a 70 min linear gradient of H2O (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluroacetic acid (TFA) at a flow rate of 2.5 mL/min. The eluant was monitored at 297 nm and fractions were collected, lyophilized and stored at K20 8C. The antimicrobial fractions were submitted to a RP-HPLC using a Vydac 218TP510 column. The fractions were eluted in a 50 min linear gradient of H2O (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluroacetic acid (TFA). The experiment was monitored at 216 and 297 nm and samples were lyophilized and stored at K20 8C. 2.3. Antimicrobial activity The microorganisms used for the antimicrobial assays were purchased from American Type Culture Collection. They were Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25992. The microorganisms were cultured in stationary culture at 37 8C. Bacteria were grown in Mueller–Hinton liquid medium (NCCLS Approved Standard M100-S9).
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The bioassays by liquid growth inhibition were performed as described by Kim et al. (2000). The bufadienolides were dissolved and diluted eightfold in Mueller–Hinton broth. The highest concentration used for the assays was 128 mg/mL. The initial inoculum was approximately 1.5!105 colony forming units (CFU)/mL. The final volume was 200 mL:100 mL of the bufadienolides test in broth, 50 mL culture medium, and 50 mL of the inoculum in Mueller–Hinton. The minimal inhibitory concentration (MIC) was measured from optical density (OD595) 12 h after all microorganisms were grown in stationary culture at 37 8C. The lowest bufadienolide concentration in which no bacterial growth occurred was defined as the MIC. 2.4. Chemical characterization of the active compounds Aliquots of the antimicrobial bufadienolides were mass analyzed by a Q-Tof Ultima API (Waters, CO, UK) operating in data directed acquisition mode and with a Nano flow Z-spray source. NMR spectra were recorded at room temperature on a Varian Mercury Plus spectrometer (7.05 T) operating at 300 MHz for 1H and 75.46 MHz for 13C. Compounds were dissolved in CDCl3 containing TMS as internal reference and measured in 5 mm sample tubes. Chemical shifts were expressed in d (ppm) and coupling constants as J (Hz) with abbreviations d, doublet; dd, double dublet; t, triplet and br, broad. In the homonuclear and heteronuclear
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two-dimensional experiments (COSY and HMQC) we used the field gradient mode. 2.5. Cadiotonic activity The cardiotonic activity of the isolated bufadienolides was verified on the ventricle strip of frog’s heart as described previously (Schwartz et al., 1999). Frogs (Rana catesbeiana) were pithed and their abdominal cavity opened. The ventricle was dissected from isolated hearts in aerated Ringer at room temperature. The ventricle strips were electrically driven with square pulses of 1 ms duration, 0.2 Hz frequency and the lowest voltage that induced maximum contractions. The rate and strength of contraction were registered with the F-60 myograph (Narco Bio-Systems) and a recorder. Bufadienolides were added to the bath (10 ml) and the effects recorded for 15 min. The composition of Ringer solution was (in mM): 111.0 NaCl; 1.8 KCl; 1.1 CaCl2; 2.4 NaHCO3 and 6.76 glucose.
3. Results 3.1. Bufadienolides purification The fractionation of the chloroform extract from B. rubescens skin secretion yielded the chromatographic profile shown in Fig. 1, indicating the presence of 12 main
Fig. 1. Chromatogram profile of the CHCl3:CH3OH extract filtered and fractionated by RP-HPLC (Shimadzu Co.) using the Asahipac C4P-50 (250!10.0 mm) column from Shimadzu Co. (Kyoto, Japan). The fractions were eluted in a 70 min linear gradient of H2O (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluroacetic acid (TFA) at a flow rate of 2.5 mL/min. The eluant was monitored at 297 nm.
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fractions. The prominent peaks designated 8 and 9 contained components that strongly inhibited the growth of S. aureus and E. coli (data not shown). The rechromatography of the fractions containing antimicrobial activity (fractions 8 and 9) led to the isolation of two pure bufadienolides named 8 0 and 9 0 , respectively (data not shown). 3.2. Spectrometric characterization The fragment ion spectrogram of bufadienolide 8 0 showed mass component at m/z 403.2610 Da (Fig. 2(A)), corresponding to the protonated [M(C24H34O5)CH]C of the molecular species. The spectrogram also exhibited specific fragment ions m/z 385.2498, 367.2357, 349.2249 and 307.1730 Da due to elimination of one, two and three water molecules and a-pyrone group (C5 H3O2 ), respectively. There was a interesting gas-phase reaction of protonated bufadienolide 8 0 , the mass spectrogram showed peaks at m/z 301.0783, 403.1609, 425.1171, 487.1324, 703.2789, 827.2839, 1025.9425, 1229.5254 Da, and ions were assigned as follows: 425.1171 Da (MCNaC); 827.2839 and 1229.5254 Da are addition of 403 Da in the aductor ion 425.1171 Da. This was confirmed by fragment pattern of 1229.5254 and 827.2839 Da (data not shown). The 1H NMR spectra of 8 0 agreed to the chemical structure of telocinobufagin with H-21, H-22 and H-23 signals [d 7.24 (dd, JZ2.6 and 1.0 Hz), d 7.82 (dd, JZ9.7 and 2.6 Hz) and d 6.27 (dd, JZ9.7 and 1.0 Hz)] characteristic of the a-pyrone ring of bufadienolides (Fig. 2(B)) (Shimada et al., 1977).
In the COSY spectrum, the correlations of these hydrogens were observed. HMQC experiment was carried out to determine the chemical shifts of C-21 (d 148.3), C-22 (d 145.8) and C-23 (d 114.7). Connectivity of H-3 (d 4.19, brt) of CH carbinylic group to C-3 (d 68.0) was also shown. The fragment ion spectrogram of bufadienolide 9 0 showed peak at m/z 401.2331 Da, corresponding to the protonated [M(C24H32O5)CH]C. The spectrogram also exhibited specific fragment ions m/z 383.2208, 365.2094, 347.1986 Da and 305.1916 due to elimination of one, two and three water molecules and a-pyrone group, respectively (Fig. 3(A)), similar to that obtained by the ion-spray MS/MS of marinobufagin reported by Bagrov et al. (1998). The same gas-phase reaction of protonated bufadienolide 9 0 was observed, the mass spectrogram showed peaks at m/z 301.0854, 401.1515, 423.1038, 823.2703, 1020.9385, 1223.5205 Da, and ions were assigned as follows: 423.1038 Da (MCNaC); 823.2703 and 1223.5205 Da are addition of 401 Da in the aductor ion 423.1038 Da. The 1H NMR spectrum of the compound 9 0 furnished the H-21, H-22 and H-23 signals [d 7.24 (dd, JZ1.0 and 2.6 Hz), d 7.78 (dd, JZ2.6 and 9.8 Hz) and d 6.26 (dd, JZ 1.0 and 9.7 Hz)] as characteristic of the a-pyrone ring of bufadienolides. The H-15 (d 3.49, d, JZ0.6 Hz) of epoxy group in the 1H NMR spectrum and 13C NMR data together with APT spectrum of the compound 9 0 were referred to marinobufagin (Fig. 3(B)) (Verpoorte et al., 1980). 3.3. Antimicrobial activity The bufadienolides telocinobufagin and marinobufagin exhibited antibacterial activity, as they inhibited the growth
Fig. 2. (A) Fragmentation ion profile in TOF MS/MS system of bufadienolide 8 0 , showing mass at 403.2610 Da [MCH]C similar to telocinobufagin (402.5238 Da). (B) Structure of telocinobufagin.
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Fig. 3. (A) Fragmentation ion profile in TOF MS/MS system of bufadienolide 9 0 , showing mass at 401.2331 Da [MCH]C similar to marinobufagin (400.5079 Da). (B) Structure of marinobufagin.
of the bacterial strains tested (S. aureus and E. coli) with significant differences in growth inhibition of them. The MIC obtained for both bufadienolides were compared with the ones obtained by the testing of conventional antibiotics (amoxicillim, imipenem e trimethoprim) (Table 1). 3.4. Cardiotonic activity The addition of bufadienolides telocinobufagin and marinobufagin (both 9.9 mg/mL) to the saline bathing the isolated ventricle strip, produced a positive inotropic effect, increasing the contraction force in 30.2G4.2 and 42.98G 3.30%, respectively.
4. Discussion Bufadienolides are the main biologically active components of Ch’an Su, a Chinese traditional drug. Various studies of the Ch’an Su and related bufadienolides have been
conducted previously in order to better characterize them as possible inhibitors of cancer cell growth (Kamano et al., 1998, 2002; Lee and Yoon, 1995) and inducers of apoptosis (Jing et al., 1994), to study their antiviral effects (Nakanishi et al., 1999), and to evaluate the possibility of an endogenous mammalian bufadienolide being involved in NaC,KC -ATPase activity related to the pathogenesis of arterial hypertension (Doris and Bagrov, 1998; Bagrov et al., 1998). In the present report, we described for the first time the antimicrobial activity of two bufadienolides isolated from skin secretions of the toad B. rubescens, telocinobufagin and marinobufagin. The mass fragmentation and NMR data provide the structure elucidation of both bufadienolides (Shimada et al., 1977; Verpoorte et al., 1980; Bagrov et al., 1998; Akizawa et al., 1994; Nogawa et al., 2001). Besides the antimicrobial activity, telocinobufagin and marinobufagin also promoted an increase of the contraction force in isolated frog ventricle strips. The presence of antimicrobial bufadienolides in the skin of B. rubescens led us to consider their possible biological
Table 1 Antimicrobial activity of telocinobufagin and marinobufagin isolated from skin secretion of Bufo rubescens Microorganisms
MICa (mg/mL) Telocinobufagin
Marinobufagin
Amoxillim
Imipenem
Trimethoprim
S. aureus ATCC 25213 E. coli ATCC 25922
128 64
128 16
N/Db !22
N/Db 14
!160 40
a MIC, minimal concentration of the compound required for total inhibition of bacteria growth in liquid medium. Experiments performed in triplicates. b N/D, no detectable activity.
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involvement in the defense mechanisms of Bufo species. Since, both bufadienolides also presented cardiac activity, additional studies comparing relative effective doses should be done. Besides, the utilization of these compounds as topic drugs in the treatment of superficial infections should not be discharged. Acknowledgements We thank Rodrigo A.V. Morales and Jorge A.T. Melo for helping with mass spectrometry analysis, Daniel N. Sifuentes for helping with ventricle strip assay, and Graciela Martins (Laborato´rio Sabin, Brası´lia, Brasil) for antimicrobial assay. These studies were supported by Brazilian government through the funding agencies CAPES and FINEP (process CT-INFRA 970/2001).
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