Abstract
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, (5β,12β)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12β,25,26-tetrahydroxy-7-oxo-5β-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.
Highlights
Brazil is the country with the richest biodiversity of anuran amphibians, with 1093 catalogued species, of which at least 42 are on the list of endangered species [1,2,3]
In view of the ability of Rhinella icterica parotoid secretion (RIPS) and Rhinella icterica icterica fraction fraction 23 (RI23) to cause muscle facilitation prior to blockade, a finding suggestive of ACh accumulation in the skeletal neuromuscular junction, we examined the ability of suggestive of ACh accumulation in the skeletal neuromuscular junction, we examined the ability of RIPS and its fractions to inhibit AChE activity in homogenates of chick biventer cervicis preparations
As a continuation of these investigations, in this study we investigated the activity of RIPS and some of its chromatographic fractions in avian central and peripheral nervous tissue and undertook chemical screening to identify the main compounds associated with the pharmacological activity of RIPS
Summary
Brazil is the country with the richest biodiversity of anuran amphibians, with 1093 catalogued species, of which at least 42 are on the list of endangered species [1,2,3]. Toads possess cutaneous glands that are broadly classified into two types: mucous glands, that produce mucus involved in cutaneous respiration, thermoregulation, and reproduction; and granular glands, that secrete noxious or toxic compounds used as a defense against predators [12,13,14]. These secretions consist mainly of alkaloids, steroids (bufadienolides and bufotoxins), biogenic amines (epinephrine, norepinephrine, serotonin, bufotenine, and dehydrobufotenine), proteins, and peptides [6,12,13,15], and act by direct contact with the mucous membranes of the mouth, eye, or throat of potential predators [9,16]. Many compounds that occur in toad secretions, e.g., bufalin, telocinobufagin, hellebrin, marinobufagin, and cinobufagin, can vary markedly among individuals, geographic regions, and species in response to environmental conditions, e.g., temperature, and dietary composition, and may involve morphological adaptations [14,35,36]
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