Bufotenine is able to block rabies virus infection in BHK-21 cells
© Vigerelli et al.; licensee BioMed Central Ltd. 2014
Received: 12 June 2014
Accepted: 6 October 2014
Published: 13 October 2014
Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.
Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity, Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.
In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.
This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.
KeywordsRabies Rhinella Bufotenine Alkaloids Toxins Mass spectrometry
According to the World Health Organization (WHO), rabies is a zoonotic neglected disease that occurs in more than 150 countries and territories, killing more than 55,000 people every year, mostly in Asia and Africa . It is caused by a virus that affects the central nervous system and, once symptoms have developed, is nearly always fatal [2, 3]. Different in vitro experiments showed evidences that some cells receptors, such as the muscular form of the nicotinic acetylcholine receptor (nAChR), are capable of participating on the entry of rabies virus into cells [4–7]. Other molecules – including the p75 neutrophin, the neuronal cell adhesion molecule (NCAM) and glicosides of the cell membrane – are also capable of participating in the entry of rabies virus into cells, maybe involved in different stages of the infection . Conformational studies on an internal tetrapeptide of rabies virus glycoprotein (Asn194-Ser195-Arg196-Gly197) considered this to be an essential part of the binding site of the virus to the acetylcholine receptor and have demonstrated that the side chains of asparagine and arginine can, apparently, mimic the acetylcholine spatial structure, being responsible for binding the virus to the acetylcholine receptor .
The skin secretion of some amphibians, on the other hand, is rich in alkaloids . Not only that, but also proteins, peptides, steroids and amines that act as chemical protection agents against predators and/or microorganisms, such as fungi and bacteria [8, 11]. Among those amphibians that are rich in alkaloids, there is the genus Bufo that was recently split into Bufo in the Old World and Rhinella in the New World . Their secretion contain a large number of alkaloids including bufotenine – a tryptamine alkaloid used as a defense mechanism due to its toxicity – that is also found in the Leguminosae family [13–16].
Taking into account that the rabies virus can bind to the nicotinic acetylcholine receptor, and that other alkaloids (nicotine, lobeline, cytisine, anabasine etc.) can also bind to these receptors, the aim of this study was to evaluate the effects of bufotenine as a possible interfering agent in the process of infection of the rabies virus in mammalian cells, using the simplified fluorescent inhibition microtest (SFIMT) and rapid fluorescent focus inhibition test (RFFIT) techniques (with adaptations). Both are serologic tests commonly used in rabies diagnosis that are considered viable tools for the evaluation of new natural compounds as potential antiviral agents.
All reagents were of analytical grade and were purchased from Sigma Aldrich (USA), unless otherwise stated.
Isolation of bufotenine from Rhinella jimi skin secretion
The collection and housing of Rhinella jimi specimens were performed under license number 15964-1 from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA). Skins secretions were collected through mechanical stimulation of the parotoid macroglands. A liquid-liquid partition (H2O:CH2Cl2) was performed and the aqueous partition was centrifuged at 4,794 g and the supernatant was analyzed by reversed phase high-performance liquid chromatography (RP-HPLC), using a binary HPLC system (20A Prominence, Shimadzu Co, Japan), coupled to a C18 column (ACE®, 250 mm × 7.75 mm). The mobile phase consisted of solvent A [H2O: trifluoroacetic acid (TFA); 1000:1] and solvent B [acetonitrile (ACN):H2O:TFA; 900:100:1] with a linear gradient of B over A (10% to 70%) in 35 minutes, at a constant flow rate of 1.7 mL.min-1 and monitored by UV absorbance at 214 nm. Fractions were manually collected and submitted to mass spectrometry (MS and MS2 ESI-IT-TOF) analyses. The fraction containing bufotenine was then purified using a C18 column (ACE®, 250 mm × 4.6 mm), with a linear gradient of B over A 13% to 15% in 15 minutes, at a constant flow rate of 1.1 mL.min-1, at 4°C, monitored by UV absorbance at 214 nm. Bufotenine peak was manually collected, dried and submitted to 1H-NMR spectroscopic analyses and/or biologic assays.
Isolation of bufotenine from Anadenanthera colubrine seeds
Since bufotenine is a long and well known alkaloid that can be also obtained from other biological sources, such as plants, we chose to purify this molecule from Anadenanthera colubrina seeds, which contain up to 2% of the seed dry weight in bufotenine. The seeds were obtained from the legitimate supplier Arbocenter Comércio de Sementes Ltda, Birigui, São Paulo (batch 0019), Brazil. The extraction of bufotenine was performed as described by Stromberg , with modifications. Twenty grams of seeds were powdered and subsequently deionized H2O and 5 g of sodium carbonate (Na2CO3) were added until a uniform mixture was obtained. This solution was then lyophilized and ressuspended in 50 mL of acetone (CH3COCH3). This extract was filtered and concentrated in a rotavapor system, following RP-HPLC analysis, in a C18 column (ACE®, 250 mm × 4.6 mm), with a linear gradient of B over A 0% to 100% in 20 minutes, at a constant flow rate of 1 mL/min and monitored by UV absorbance at 214 nm. Bufotenine peak was manually collected and dried for mass spectrometry and biologic assays.
Previously dried samples were dissolved into 50% ACN with 0.5% formic acid for MS and/or MS2 analyses in an ESI-IT-TOF mass spectrometer system (Shimadzu Co., Japan). Typically, 3 μL of samples were manually injected in a Rheodyne injector under a constant flow of 50 μL/min. The interface voltage used was 4.5 kV and detector voltage, 1.76 kV, with source temperature of 200°C. Data were acquired under positive mode and instrument control, data acquisition and processing were performed by the LCMS solution software (Shimadzu Co., Japan).
NMR screening was used to confirm the bufotenine structure from Rhinella jimi skin secretion. 1H NMR spectra were recorded on Bruker 500 MHz spectrometer (Bruker Co., Germany), with samples diluted in deuterated chloroform and achievement of 128 scans. The results were processed in TopSpin 1.3 software (Bruker Co., Germany).
Cells and viruses
Baby hamster kidney (BHK-21) cells (ATCC® CCL – 100) were cultured in Eagle’s minimum essential medium, supplemented with 10% fetal bovine serum (MEM-10), at 37°C under a humidified 5% CO2 atmosphere until the formation of the cell monolayer. Rabies virus strain Pasteur vaccine (PV), from Pasteur Institute, Brazil, was used to determine the antiviral activity of bufotenine. The titers were determined by plate assay in BHK-21 cells and expressed as 100% infecting dose in cell culture (IDCC100) and 50% focus-forming dose (FFD50) as described by Batista et al. .
The cytotoxicity evaluation of bufotenine was performed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method, according to Takeuchi et al.  and Mosmann , with modifications. Briefly, BHK-21 cells (5 × 104 cells/well) were deposited in 96-well tissue culture microtiter plates with 50 μL of different bufotenine concentrations diluted in MEM-10. For positive control of citotoxicity dimethyl sulfoxid (DMSO) 20% was used and the negative control was MEM-10 alone. After 24 hours at 37°C, under a humidified 5% CO2 atmosphere, the medium was removed and 50 μL of MTT (Sigma® 1 mg/mL) solution, prepared in MEM-10, was added to each well and the plates were incubated once more for four hours. After incubation, the MTT solution was removed and 100 μL of dimethyl sulfoxide (DMSO) were added to each well to solubilize the formazan crystals. After gently shaking the plates, the crystals were completely dissolved, and the absorbances were measured by using a spectrophotometer (Molecular Devices®, SpectraMax M2) at 540 nm. The CC50 was defined as the cytotoxic concentration of bufotenine that reduced the absorbance of treated cells to 50% when compared to the control.
Rabies virus does not cause cytopathic effects in cells cultured in vitro, being necessary some kind of additional test to monitor cell infection, such as tests with antibodies bound to fluorescent substances . For this reason, we choose these two tests adapted from commonly serological tests used for antirabies virus titration, being the fluorescence inhibition a qualitative test, for a better visualization of both the infection and its inhibition, and the fluorescence focus inhibition comprises a quantitative test, used for the statistical analyses.
Fluorescence inhibition test
This test was based on simplified fluorescent inhibition microtest (SFIMT), acording to Favoretto et al. , with modifications. Briefly, 50 μL of BHK-21 cells (5 × 104 cells/well) were deposited on 96-well tissue culture microtiter plates with 50 μL of each bufotenine dose (from 0.5 to 4 mg/mL) and 50 μL of PV virus previously diluted at 1:100 (IDCC100). As negative inhibition control, only 50 μL of MEM-10 was added to the cells and as positive inhibition control ketamine was used (Dopalen®, 23.4 μM), according to Lockhart et al. . After a 24-hour period of incubation at 37°C under a humidified 5% CO2 atmosphere, the medium was removed by suction from all wells and the microplates were cooled on ice. The cells were fixed by adding acetone 80% in water (kept at –20°C). After 15 minutes, the plates were emptied by inversion and dried at 37°C according to Smith et al.  and Chaves et al. . The staining was carried out by adding 40 μL of an optimal dilution of the antirabies fluorescent conjugate . After one hour of incubation, the microtiter plates were washed by immersion in PBS and then in distilled water. The microtiter plates reading was performed in inverted fluorescence microscope (Leica DMIL, 100× magnification) in a qualitative manner based on the fluorescence inhibition compared to negative and positive controls, where there are 100% and 0% of fluorescence, respectively.
Fluorescent focus inhibition test
This test was based on rapid fluorescent focus inhibition test (RFFIT) acording to Smith et al. , adapted to microtiter plates as described by Chaves et al. , with modifications. Briefly, 100 μL of BHK-21 cells (2.5 × 104 cells/well) were deposited on 96-well tissue culture microtiter plates containing different bufotenine doses (3.9, 1.95, 0.97 and 0.48 mg/mL), and 50 μL of PV virus previously diluted at thirty-fold the FFD50 value. As negative inhibition control, only 50 μL of MEM-10 was added to the cells and as positive inhibition control ketamine was used (Dopalen®, 23.4 μM). After a 20-hour period of incubation at 37°C under a humidified 5% CO2 atmosphere, the medium was removed by suction from all wells. The procedures of cells fixation and staining were the same described in “Fluorescence inhibition test” section. The microtiter plates reading was quantitatively performed in inverted fluorescence microscope (Leica DMIL, 200× magnification), where each field with fluorescence were counted, being 0 the minimum (positive control) and 18 the maximum (negative control) number of infected fields. The IC50 was defined as the inhibition concentration of bufotenine that reduced the number of fields with fluorescent foci to 50% when compared with the negative control.
Time course study
The time course effect of bufotenine was examined on PV virus with two minor modifications of the fluorescent focus inhibition test. First, to test a possible “protector effect”, bufotenine was added in different times (1, 3 and 6 hours), prior to the addition of the virus and, secondly, to test a possible “treatment effect”, bufotenine was added in different times (1, 3 and 6 hours), after incubation of cells and PV virus. All the other procedures were the same as described in “Fluorescent focus inhibition test” section.
The 50% cytotoxic (CC50) and inhibition (IC50) concentrations were calculated from concentration-effect curves after linear regression analysis. The results represent the mean ± standard error of the mean values of two different experiments (triplicates).
RP-HPLC and mass spectrometry analysis
Cytotoxicity effect on viability of BHK-21 cells
The 3.9 mg/mL dose of R. jimi bufotenine used on the virologic tests presented statistically significant cytotoxic effects on the viability of BHK-21 cells (66% of viable cells) when compared to the negative (cells + MEM-10) and positive controls (cells + DMSO 20%) (Additional file 5: Figure S5). Subsequent assays were performed with the seeds bufotenine, due to the high availability. In the MTT test (Additional file 6: Figure S6), it was possible to determine the CC50 value (7.6 mg/mL).
Fluorescence inhibition testBufotenine was able to inhibit the infection significantly, showing dose-response effect when tested at a concentration of 0.5 to 3 mg/mL (Figure 2).
Fluorescent focus inhibition test
Although bufotenine was isolated “only” in 1920, shamans and mystics have been employing toads’ skin secretions since ancient times as entheogens, which means “generating the divine within” . The chemical structure elucidation was published in 1934 by the group of the chemist Heinrich Otto Wieland and one can observe that the molecule is related to psilocin and dimethyltryptamine (DMT), which are known hallucinogens, as well to the neurotransmitter serotonin . Another early report on the molecule is the documentation of its toxicity, by the roman poet Decimus Junius Juvenalis (60-128 AD) in the first century.
In this work, we describe bufotenine as an inhibitor of rabies virus penetration in mammal cells. Initial bufotenine isolation and characterization was performed from the skin secretion of the toad R. jimi based on biological effect-driven assays, as previously done by the authors [13, 29–31]. After initial molecular identification and characterization, we have opted for obtaining this molecule from a more abundant source, so the seeds of Anadenanthera colubrina were chosen, according to early reports on the isolation of this alkaloid of Anadenanthera genus .
Bufotenine has been purified, described and synthesized many years ago [32–34]. However, the original isolation process includes several steps of extraction, filtering, partitioning and pH adjustments . In our work, on the other hand, we were able to demonstrate that the use of chromatographic techniques allows for greater efficiency in the purification of this molecule. Moreover, the association with mass spectrometry analyses allow further evaluation of the purity degree, as well as molecular identity confirmation, for the subsequent biological assays. From the toad skin secretion, pure bufotenine could be obtained after two chromatographic steps, and from the seeds, bufotenine was purified after acetone extraction employing only one chromatographic step, which increased the efficiency and the yield in the obtainment of the pure molecule.
There are recent described effects of hydroalcoholic extracts of different parts of A. colubrina, such as anti-inflammatory and peripheral antinociceptive activities in rodent models, antimicrobial (anti-Staphylococcus) and cutaneous wound healing in rats, among others [35–37]. However, these reports describe biological effects of extracts rather than pure molecules. Although the toxic and hallucinogens effects of bufotenine are widely discussed and studied, no reports about its antiviral activity are known to the best of our knowledge [38–40].Among the possible described mechanisms of rabies virus penetration known (or postulated), the bufotenine inhibition of rabies virus infection observed in this study appears to be related to the competition to the cells’ receptors, since it could only be observed when simultaneous administration of bufotenine and PV virus was performed (Figure 4). However, the elucidation of this mechanism depends on electrophysiological and/or patch clamp experiments to be performed in the future.
Numerous authors have described that there are high-affinity receptors at host cells specific for rabies virus, which include AChR – and/or associated molecules – that act as receptors. Although it was not possible to unequivocally demonstrate that the inhibitory effect of bufotenine was mediated by its binding to the acetylcholine receptor, the results of this work open important perspectives in the study of the mechanism of action of bufotenine, as well as the elucidation of the mechanisms of the virus pathogenicity. Altogether, our data may aid in the development of 2nd generation active molecules, since rabies is still a disease with no cure prognosis and it kills, every year, thousands of people around the world . Furthermore, bufotenine is cytotoxic, so medicinal chemistry and lead optimization will be required.
This work do not present or propose bufotenine as a drug for the treatment of rabies for several reasons, including the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule may lead to the choice or development of a tryptamine analogue presenting potential clinical use. These tryptophan derived alkaloids are very interesting building blocks in organic chemistry syntheses . Our next goal would be to evaluate such analogues as possible inhibitors of the viral infection, targeting the dissociation of toxic and psychotropic effects from the antiviral effect.
Ethics committee approval
The collection and housing of Rhinella jimi specimens were performed under license number 15964-1 from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA).
The authors would like to thank FAPESP, CNPq, CAPES and INCT-Tox for their funding of this research. Moreover, DCP is a CNPq fellow researcher.
- World Health Organization: Rabies fact sheet N° 99. [updated September 2014]. (Acessed: September 2014) http://www.who.int/mediacentre/factsheets/fs099/en/
- Consales CA, Bolzan VL: Rabies review: immunopathology, clinical aspects and treatment. J Venom Anim Toxins incl Trop Dis 2007, 13(1):5–38. http://dx.doi.org/10.1590/S1678–91992007000100002View ArticleGoogle Scholar
- Brandão PE: On the interference of clinical outcome on rabies transmission and perpetuation. J Venom Anim Toxins incl Trop Dis 2009, 15(2):190–203. http://dx.doi.org/10.1590/S1678–91992009000200003View ArticleGoogle Scholar
- Watson HD, Tignor GH, Smith AL: Entry of rabies virus into the peripheral nerves of mice. J Gen Virol 1981, 56(Pt 2):372–382.PubMedGoogle Scholar
- Lentz TL, Burrage TG, Smith AL, Crick J, Tignor GH: Is the acetylcholine receptor a rabies virus receptor? Science 1982, 215(4529):182–184. 10.1126/science.7053569View ArticlePubMedGoogle Scholar
- Burrage TG, Tignor GH, Smith AL: Rabies virus binding at neuromuscular junctions. Virus Res 1985, 2(3):273–389. 10.1016/0168-1702(85)90014-0View ArticlePubMedGoogle Scholar
- Lewis P, Fu Y, Lentz TL: Rabies virus entry at the neuromuscular junction in nerve-muscle cocultures. Muscle Nerve 2000, 23(5):720–730. 10.1002/(SICI)1097-4598(200005)23:5<720::AID-MUS9>3.0.CO;2-5View ArticlePubMedGoogle Scholar
- Lafon M: Rabies virus receptors. J Neurovirol 2005, 11(1):82–87. 10.1080/13550280590900427View ArticlePubMedGoogle Scholar
- Rustici M, Bracci L, Lozzi L, Neri P, Santucci A, Soldani P, Spreafico A, Niccolai N: A model of the rabies virus glycoprotein active site. Biopolymers 1993, 33(6):961–969. 10.1002/bip.360330612View ArticlePubMedGoogle Scholar
- Maciel NM, Schwartz CA, Rodrigues Pires Júnior O, Sebben A, Castro MS, Sousa MV, Fontes W, Ferroni Schwartz EN: Composition of indolealkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids with phylogenetic implications. Comp Biochem Physiol B Biochem Mol Biol 2003, 134(4):641–649. 10.1016/S1096-4959(03)00022-8View ArticlePubMedGoogle Scholar
- Daly JW, Highet RJ, Myers CW: Occurrence of skin alkaloids in non-dendrobatid frogs from Brazil (Bufonidae), Australia (Myobatrachidae) and Madagascar (Mantellinae). Toxicon 1984, 22(6):905–919. 10.1016/0041-0101(84)90182-XView ArticlePubMedGoogle Scholar
- Duellman WE, Trueb L: Biology of Amphibians. New York: McGraw-Hill; 1996.Google Scholar
- Sciani JM, Angeli CB, Antoniazzi MM, Jared C, Pimenta DC: Differences and similarities among parotoid macrogland secretions in South American toads: a preliminary biochemical delineation. Scient World J 2013, 2013(2013):1–9. doi:10.1155/2013/937407View ArticleGoogle Scholar
- Costa TO, Morales RA, Brito JP, Gordo M, Pinto AC, Blosh C Jr: Occurrence of bufotenin in the Osteocephalus genus (Anura: Hylidae). Toxicon 2005, 46(4):371–375. 10.1016/j.toxicon.2005.02.006View ArticlePubMedGoogle Scholar
- Smith TA: Tryptamine and related compounds in plants. Phytochemistry 1977, 16(2):171–175. 10.1016/S0031-9422(00)86778-3View ArticleGoogle Scholar
- Stromberg VL: The isolation of bufotenine from Piptadenia peregrina . J Am Chem Soc 1954, 76(6):1707.View ArticleGoogle Scholar
- Batista AM, Cruz PS, Almeida E, Costa AEB, Scheffer KC, Chaves LB, Silva ACR, Medeiros Caporale GM: Infection of BHK-21 cells cultivated in stationary monolayers by PV and CVS strains. Bol Epidemiol Paul (Online) 2009, 6(71):4–11.Google Scholar
- Takeuchi H, Baba M, Shigeta S: An application of tetrazolium (MTT) colorimetric assay for the screening of anti-herpes simplex virus compounds. J Virol Methods 1991, 33(1–2):61–71.View ArticlePubMedGoogle Scholar
- Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65(1–2):55–63.View ArticlePubMedGoogle Scholar
- Flores EF, Editor: Vírus da raiva e lyssavirus relacionados. In Virologia Veterinária. Rio Grande do Sul: UFSM; 2007:700–711.
- Favoretto SR, Carrieri ML, Tino MS, Zanetti CR, Pereira AO: Simplified fluorescent inhibition microtest for the titration of rabies neutralizing antibodies. Rev Inst Med Trop São Paulo 1993, 35(2):171–175. 10.1590/S0036-46651993000200009View ArticlePubMedGoogle Scholar
- Lockhart BP, Tordo N, Tsiang H: Inhibition of rabies virus transcription in rat cortical neurons with the dissociative anesthetic ketamine. Antimicrob Agents Chemother 1992, 36(8):1750–1755. 10.1128/AAC.36.8.1750PubMed CentralView ArticlePubMedGoogle Scholar
- Smith JS, Yager PA, Baer GM: A rapid reproducible test for determining rabies neutralizing antibody. Bull World Health Organ 1973, 48(5):535–541.PubMed CentralPubMedGoogle Scholar
- Chaves LB, Mazutti ALC, Medeiros Caporale GM, Scheffer KC, Silva ACR: Comparision of RFFIT performed in lab-tek® and in 96-well microtitre plates. In RITA XVII. Annales of the XVII International Conference on Rabies in the Americas: 15–20 octuber, 2006; Brasília, Brazil. Brasília: Ministério da Saúde; 2006:161.Google Scholar
- Medeiros Caporale GM, Rodrigues Da Silva A, De C, Peixoto ZM, Chaves LB, Carrieri ML, Vassao RC: First production of fluorescent anti-ribonucleoproteins conjugate for diagnostic of rabies in Brazil. J Clin Lab Anal 2009, 23(1):7–13. 10.1002/jcla.20275View ArticlePubMedGoogle Scholar
- McClean S, Robinson RC, Shaw C, Smyth WF: Characterisation and determination of indole alkaloids in frog-skin secretions by electrospray ionisation ion trap mass spectrometry. Rapid Commun Mass Spectrom 2002, 16(5):346–354. 10.1002/rcm.583View ArticlePubMedGoogle Scholar
- Handovsky H: Ein Alkaloid im Gifte von Bufo vulgaris . Arch Exp Pathol Pharmakol 1920, 86(1–2):138–158.View ArticleGoogle Scholar
- Blom JD: A Dictionary of Hallucinations. New York: Springer-Verlag; 2010:79–80.View ArticleGoogle Scholar
- Prates I, Antoniazzi MM, Sciani JM, Pimenta DC, Toledo LF, Haddad CF, Jared C: Skin glands, poison and mimicry in dendrobatid and leptodactylid amphibians. J Morphol 2012, 273(3):279–290. 10.1002/jmor.11021View ArticlePubMedGoogle Scholar
- Sousa JC, Berto RF, Gois EA, Fontenele-Cardi NC, Honório JE Jr, Konno K, Richardson M, Rocha MF, Camargo AA, Pimenta DC, Cardi BA, Carvalho KM: Leptoglycin: a new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae). Toxicon 2009, 54(1):23–32. 10.1016/j.toxicon.2009.03.011View ArticlePubMedGoogle Scholar
- Tempone AG, Pimenta DC, Lebrun I, Sartorelli P, Taniwaki NN, De Andrade HF, Jr AMM, Jared C: Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion. Toxicon 2008, 52(1):13–21. 10.1016/j.toxicon.2008.05.008View ArticlePubMedGoogle Scholar
- Evarts EV, Landau W, Freygang W Jr, Marshall WH: Some effects of lysergic acid diethylamide and bufotenine on electrical activity in the cat’s visual system. Am J Physiol 1955, 182: 594–598.PubMedGoogle Scholar
- Harley-Mason J, Jackson AH: A new synthesis of bufotenine. Chem Ind 1952, 39: 954.Google Scholar
- Wieland H, Konz W, Mittasch H: Die konstitution von Bufotenin und Bufotenidin. Über kröten-Giftstoffc. VII. Justus Liebigs Ann Chem 1934, 513(1):1–25. 10.1002/jlac.19345130102View ArticleGoogle Scholar
- Santos JS, Marinho RR, Ekundi-Valentim E, Rodrigues L, Yamamoto MH, Teixeira SA, Muscara MN, Costa SK, Thomazzi SM: Beneficial effects of Anadenanthera colubrina (Vell.) Brenan extract on the inflmmatory and nociceptive responses in rodent models. J Ethnopharmacol 2013, 148(1):218–222. 10.1016/j.jep.2013.04.012View ArticlePubMedGoogle Scholar
- Da Silva LC, Sandes JM, Paiva MM, De Araújo JM, De Figueiredo RC, Da Silva MV, Correia MT: Anti- Staphylococcus aureus action of three Caatinga fruits evaluated by electron microscopy. Nat Prod Res 2012, 27(16):1492–1496. doi:10.1080/14786419.2012.722090View ArticlePubMedGoogle Scholar
- Pessoa WS, Estevão LRM, Simões RS, Barros MEG, De Mendonça F S, Baratella-Evêncio L, Evêncio-Neto J: Effects of angico extract ( Anadenanthera colubrina var. cebil ) in cutaneous wound healing in rats. Acta Cir Bras 2012, 27(10):655–670. 10.1590/S0102-86502012001000001View ArticlePubMedGoogle Scholar
- Fabing HD, Hawkins Jr: Intravenous bufotenine injection in the human being. Science 1956, 123(3203):886–887. 10.1126/science.123.3203.886View ArticlePubMedGoogle Scholar
- Raymond-Hamet M: Sur les effects tenseurs et respiratoires de la bufoténine basique dissolute dans le diméthyl-acétonyl-carbinol. Seances Acad Sci 1944, 218: 54–56.Google Scholar
- Raymond-Hamet M: Sur les effets vasculaires de la bufoténine introduite dans la circulation générale. CR Hebd Seances Acad Sci 1942, 214: 506–508. Available at: http://gallica.bnf.fr/ark:/12148/bpt6k3166b/f506Google Scholar
- Stefani HA, Vasconcelos SNS, Souza FB, Manarin F, Zukerman-Sphector J: One-pot three-component synthesis of indole-3-glyoxyl derivatives and indole-3-glyoxyl triazoles. Tetrahedron Lett 2013, 54(43):5821–5825. 10.1016/j.tetlet.2013.08.064View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.