NOVEL TRYPTOPHYLLIN-LIKE ANTIOXIDANT PEPTIDES OBTAINED FROM THE SKIN SECRETIONS OF THE CENTRAL DWARF FROG, PHYSALAEMUS CENTRALIS
(Novos peptídeos antioxidantes do tipo triptofilina, obtidos das secreções cutâneas do sapo Physalaemus centralis)
Ariane NOGUEIRA1, José BRANGO-VANEGAS1, Andreanne G. VASCONCELOS1, Éder A. BARBOSA1,2, Daniel C. MOREIRA1, Jhones D. NASCIMENTO3,4, Guilherme D. BRAND2, Alexandra PLÁCIDO5, José Roberto S. A. LEITE1*
1Applied Immunology and Morphology Research Centre, NuPMIA, Morphology Area, Faculty of Medicine, University of Brasília, Campus Universitário Darcy Ribeiro, Asa Norte. CEP: 70910900. Brasília, DF, Brazil. 2Laboratory of Synthesis and Analysis of Biomolecules, LSAB, Institute of Chemistry, IQ, University of Brasília, Brasília, Brazil. 3Department of Cell Biology, Institute of Biological Sciences, University of Brasília, Brasília, Brazil. 4Biomedicine Course, Federal University of Delta do Parnaíba, UFDPar, Parnaíba, Brazil. 5LAQV/REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences of the University of Porto, Porto, Portugal. E-mail: jrsaleite@gmail.com
ABSTRACT
The skin of amphibians is a morphologically and biochemically complex organ that has been studied for expressing different types of bioactive molecules, including biogenic amines, bufadienolides, alkaloids, steroids, peptides and proteins. Tryptophyllins represent a family of peptides discovered for the first time in skin secretions of Phyllomedusa spp. In this work, we report two tryptophyllin-like peptides with antioxidant activity, called PcT-2 and PcT-3, in the cutaneous secretions of Physalaemus centralis. Adult specimens of P. centralis were captured in Formoso do Araguaia, Tocantins, Brazil. Skin secretions were collected by biostimulation, analyzed by RP-HPLC and subsequently by MALDI TOF/MS for de novo peptide sequencing. Primary sequences of P. centralis peptides were confirmed based on a cDNA library. Subsequently, the chemical synthesis in solid phase F-moc and purification of the identified peptides were carried out. Chromatographic and spectrophotometric analyzes were performed to confirm the chemical structure of the synthesized peptides. Chemical tests of antioxidant activity (ABTS, DPPH and NO) were carried out together with toxicity tests in Galleria mellonella larvae and red blood cells. The molecular mass of PcT-2 and PcT-3 was estimated to be 1,030.52 Da and 761.41 Da, respectively. PcT-2 and PcT-3 showed free radical scavenging activity against DPPH (0.001 and 0.000 Trolox equivalents/mg, respectively), ABTS (0.269 and 0.305 Trolox equivalents / mg, respectively) and NO (0.031 and 0.039 equivalents of Trolox/mg, respectively). In the toxicity tests using G. mellonella larvae, a 100% survival rate was observed at concentrations of 10, 20 and 40 mg/kg. In the hemolytic assay, cytotoxicity in erythrocytes was not observed at any concentrations tested. These results suggest that this class of peptides may be related to antioxidant activity, thus increasing the opportunities for new applications mainly related to prevention of oxidative damage in the area of skin health. A.N. thanks CAPES for the doctoral scholarship linked to the graduate program in Medical Sciences.
Keywords: Amphibians, bioactive peptides, antioxidant activity.