Vasoconstrictor and hemodynamic effects of a methanolic extract from Rhinella marina toad poison.

Rhinella marina toad is abundant in Brazil. Its poison contains cardiac glycosides called bufadienolides, which are extensively investigated for their bioactivity. Our aim was to characterize the vasoactivity of Rhinella marina poison (RmP) on the aorta of male Wistar rats. For this, the RmP was first collected and processed to obtain an alcoholic extract. To determine cardiovascular effects of RmP, we performed in vivo tests by administering RmP intravenously in doses of 0.1-0.8 mg/kg. Vascular reactivity was also performed through concentration-response curves to RmP (10 ng/mL to 200 µg/mL) in aortic segments with and without endothelium. RmP induced a concentration-dependent contraction in rat aorta which was partly endothelium-mediated. Nitric oxide contributes with this response in view that incubation with L-NAME increased the contractile response. Additionally, treatment with indomethacin [cyclooxygenase, (COX) inhibitor], nifedipine (L-type voltage-gated calcium channels blocker), and BQ-123 (ETA receptors antagonist) decreased maximum response, and ketanserin (5-HT2 receptors antagonist) decreased pEC50, suggesting active participation of these pathways in the contractile response. On the other hand, apocynin (NADPH oxidase inhibitor) did not alter contractility. Incubation with prazosin (α1-adrenergic receptor antagonist) abolished the contractile response, suggesting that the RmP-induced contraction is dependent on the adrenergic pathway. In the Na+/K+ ATPase protocol, a higher Emax was observed in the RmP experimental group, suggesting that RmP potentiated Na+/K+ATPase hyperpolarizing response. When this extract was injected (i.v.) in vivo, increase in blood pressure and decrease in heart rate were observed. The results were immediate and transitory, and occurred in a dose-dependent manner. Overall, these data suggest that the poison extract of R. marina toad has an important vasoconstrictor action and subsequent vasopressor effects, and its use can be investigated to some cardiovascular disorders.

Chemical and functional analyses of Rhinella icterica (Spix, 1824) toad secretion screened on contractions of the heart and oviduct in Locusta migratoria.

Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 µg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001-100 µg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 µg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC50 values ranging from 0.15 (leukemia) to 7.35 (larynx) µM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC50: 10.88 µM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC50: 7.5 µM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer.

Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.

Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens.

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 RP-HPLC. The molecular structure and mass determination were done by (1)H and (13)C 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 microg/mL for E. coli and both 128 microg/mL for S. aureus. Besides the antimicrobial activity both bufadienolides promoted an increase of the contraction force in isolated frog ventricle strips.