Biochemical and Biological Profile of Parotoid Secretion of the Amazonian Rhinella marina (Anura: Bufonidae)

Abstract

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

Figure 1

(a) Chromatographic profile of the filtrate (below 10 kDa) on reverse phase C18 column. The separation of the peaks C18 Rm-04 and Rm-08 ((b) and (c)) is observed. Linear gradient of 99.9 + 0.1% AC/TFA, monitoring at 280 and 215 nm absorbance.

Figure 2

Mass spectrometry profile (ESI-IT-TOF) of fraction Rm-04 (<10 kDa). Profile compatible with molecular weight of the steroid desacetylcinobufagin mass band of 400 Da (a), with protonated precursor 401 and fragments 383, 365, 319, and 251 [M + H]+(b).

Figure 3

Chromatographic profile of crude Rhinella marina poison and electrophoretic profile of fractions from molecular exclusion chromatography. (a) Chromatographic profile of the crude poison of R. marina in a column (Superdex Peptide 10x30 cm) of molecular exclusion. The interest fraction Superdex Rm01 and its electrophoretic profile in reducing conditions. (b) MW: 200 kDa (Myosin); 120 kDa (β-Galactosidase); 91 kDa (Bovine Serum); 62 kDa (Glutamate); 46 kDa (Ovalbumin); 38 kDa (Carbonic anhydrase); 26 kDa (Myoglobin); 19 kDa (lysozyme); and 9 kDa (Aprotinin).

Figure 4

(a) Chromatographic profile of the fraction Rm 01 (Gel Filtration) on reverse phase C18 column. The separation of the peaks C18 Rm02 and Rm 05 is highlighted by the arrows. (b) Rechromatography of the fraction C18 Rm01/02 and C18 Rm01/05. (c) 99.99 + linear gradient 0.1% ACN/TFA, 280 nm absorbance monitoring for the detection of aromatic amino acid side chains (Phenylalanine, Tyrosine, and Tryptophan). The purification profile of the sample is highlighted.

Figure 5

(a) Cytotoxic effects of crude venom upon human cells, after 72 h treatment, by MTT assay. Results are expressed as mean percentage in treated cells compared to control (solvent) ± SD of three independent experiments. In detail the IC50 value for each cell line. (b) Induction of DNA strand breaks by crude venom as evaluated by the comet assay in alkaline conditions. Bars represent the mean ± SD of three independent experiments. MMS is used as positive control. ^∗Significant difference as compared to negative control treatment at ^∗∗∗P < 0.001/one-way ANOVA Tukey's multiple comparison test.

Figure 6

Effect of Rhinella marina poison on inflammatory reaction.

Figure 7

(a) Effect of Rhinella marina poison on leukocytes phagocytosis. (b) Effect of Rhinella marina poison on superoxide anion.