Ciclotídeos de Noisettia orchidiflora e Anchietea pyrifolia: isolamento, caracterização e avaliação de atividades biológicas
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Data
2020-03-10
Autores
Bobey, Antonio Fernández
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Universidade Estadual Paulista (Unesp)
Resumo
Ciclotídeos são peptídeos cíclicos de fonte vegetal biossintetizados via ribossomal, apresentam massa molecular entre 2,8-3,7 kDa, contendo seis resíduos de cisteína conservados e envolvidos na formação de um arranjo único conhecido como nó de cistina cíclico (cyclic cystine knot, CCK). Esse arranjo é responsável por sua estabilidade térmica, química e enzimática. Diversas atividades biológicas foram relatadas para esta classe de peptídeos, incluindo propriedades inseticidas, anti-helmínticas e moluscicidas, sugerindo que sua função biológica está relacionada à defesa de plantas. Até o momento, essas substâncias foram identificadas em cinco famílias vegetais: Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae e Fabaceae. Neste trabalho foram realizados o isolamento, caracterização e avaliação das atividades biológicas dos ciclotídeos presentes em duas espécies vegetais da família Violaceae: Anchietea pyrifolia e Noisettia orchidiflora. Após o processo de extração dos constituintes químicos, foi realizada uma pré-purificação por meio de cromatografia em coluna com octadecilsilano, como fase estacionária, permitindo a obtenção das frações peptídicas de cada espécie. Estas frações enriquecidas foram analisadas por meio da técnica LC-ESI-IT-MS confirmando a presença de peptídeos na faixa de massa de 2,8-3,7 kDa e a análise dos resultados permitiu a anotação de 35 ciclotídeos na espécie N. orchidiflora e 48 na A. pyrifolia. Este resultado motivou o isolamento por Cromatografia Líquida de Alta Eficiência de cinco substâncias da espécie A. pyrifolia (1-5) e quatro da N. orchidiflora (6-9). O sequenciamento de novo foi realizado por digestão enzimática com endoproteinase Glu-C, tripsina e quimiotripsina, seguido por análise de MALDI-TOF/TOF-MS. Os espectros foram cuidadosamente examinados e as sequências foram propostas, com base na presença das séries de íons b e y (fragmentos N e C-terminais); desta forma foi possível determinar a sequência dos ciclotídeos cyO4 (1), cyO17 (2), apy A (3), apy B (4), cter 15 (5), nor E (6), nor F (7), nor G (8) e vhr1 (9). Experimentos in vivo de injeção abdominal sobre a abelha africanizada Apis mellifera indicaram atividade inseticida para as substâncias 1 e 3. O nível de toxicidade destes ciclotídeos foi calculado de acordo com a análise dose-resposta pelo método Probit e expresso como concentração letal media (CL50), sendo suas CL50 de 76 e 262 μM, respectivamente. Citotoxicidade dos ciclotídeos 1, 2, 3 e 4 também foi avaliada em eritrócitos humanos. Apy A (3) apresentou dose hemolítica média (DH50) de 22 μM, enquanto que 1, 2 e 4 apresentaram baixa atividade na máxima concentração testada, com DH50 > 156 μM.
Cyclotides are ribosomal cyclic peptides found only in some plant taxa, they have a molecular mass among 2.8-3.7 kDa, with six conserved cysteine residues involved in the formation of a unique arrangement known as cyclic cystine knot (CCK). This arrangement is responsible for its thermal, chemical and enzymatic stability. Several biological activities have been reported for this class of peptides, including insecticidal, anthelmintic and molluscicidal properties, suggesting that their biological functions are related to plant defense. To date, these compounds have been identified in five taxonomic groups: Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae and Fabaceae. This research work deals on isolation, characterization and biological activities of cyclotides present in two plant species of the Violaceae: Anchietea pyrifolia and Noisettia orchidiflora. After initial extracting procedures, a pre-purification was performed by means of column chromatography with octadecylsilane, as a stationary phase, which allowed obtaining the peptide fractions of each species. These enriched fractions were analyzed using the LC-ESI-IT-MS technique to confirm the presence of peptides in the mass range of 2.8-3.7 kDa, the analysis of the results allowed the annotation of 35 cyclotides in species N. orchidiflora and 48 in A. pyrifolia. This result motivated the isolation by High Performance Liquid Chromatography of five compounds of the species A. pyrifolia (1-5) and four of N. orchidiflora (6-9). The de novo sequencing was performed by enzymatic digestion with endoproteinase Glu-C, trypsin and chymotrypsin, followed by MALDI-TOF/TOF-MS analysis. The spectra were carefully examined and the sequences were proposed, based on the presence of the series of ions b and y (fragments N and C-terminal); in this way it was possible to determine the sequence of the cyO4 (1), cyO17 (2), apy A (3), apy B (4), cter 15 (5), nor E (6), nor F (7), nor G (8) and vhr1 (9). In vivo experiments of abdominal injection on the Africanized bee Apis mellifera indicated insecticidal activity for compounds 1 and 3. The toxicity level of these cyclotides were calculated according to the dose-response analysis by the Probit method and expressed as average lethal concentration (LC50), with its LC50 of 76 and 262 μM, respectively. Cytotoxicity of cyclotides 1, 2, 3 and 4 was also evaluated in human erythrocytes. Apy A (3) had a mean hemolytic dose (HD50) of 22 μM, while 1, 2 and 4 had low activity at the maximum concentration tested, with HD50> 156 μM.
Cyclotides are ribosomal cyclic peptides found only in some plant taxa, they have a molecular mass among 2.8-3.7 kDa, with six conserved cysteine residues involved in the formation of a unique arrangement known as cyclic cystine knot (CCK). This arrangement is responsible for its thermal, chemical and enzymatic stability. Several biological activities have been reported for this class of peptides, including insecticidal, anthelmintic and molluscicidal properties, suggesting that their biological functions are related to plant defense. To date, these compounds have been identified in five taxonomic groups: Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae and Fabaceae. This research work deals on isolation, characterization and biological activities of cyclotides present in two plant species of the Violaceae: Anchietea pyrifolia and Noisettia orchidiflora. After initial extracting procedures, a pre-purification was performed by means of column chromatography with octadecylsilane, as a stationary phase, which allowed obtaining the peptide fractions of each species. These enriched fractions were analyzed using the LC-ESI-IT-MS technique to confirm the presence of peptides in the mass range of 2.8-3.7 kDa, the analysis of the results allowed the annotation of 35 cyclotides in species N. orchidiflora and 48 in A. pyrifolia. This result motivated the isolation by High Performance Liquid Chromatography of five compounds of the species A. pyrifolia (1-5) and four of N. orchidiflora (6-9). The de novo sequencing was performed by enzymatic digestion with endoproteinase Glu-C, trypsin and chymotrypsin, followed by MALDI-TOF/TOF-MS analysis. The spectra were carefully examined and the sequences were proposed, based on the presence of the series of ions b and y (fragments N and C-terminal); in this way it was possible to determine the sequence of the cyO4 (1), cyO17 (2), apy A (3), apy B (4), cter 15 (5), nor E (6), nor F (7), nor G (8) and vhr1 (9). In vivo experiments of abdominal injection on the Africanized bee Apis mellifera indicated insecticidal activity for compounds 1 and 3. The toxicity level of these cyclotides were calculated according to the dose-response analysis by the Probit method and expressed as average lethal concentration (LC50), with its LC50 of 76 and 262 μM, respectively. Cytotoxicity of cyclotides 1, 2, 3 and 4 was also evaluated in human erythrocytes. Apy A (3) had a mean hemolytic dose (HD50) of 22 μM, while 1, 2 and 4 had low activity at the maximum concentration tested, with HD50> 156 μM.
Descrição
Palavras-chave
Ciclotídeos, Apis mellifera, Violaceae, Atividade inseticida, Atividade hemolítica, Sequenciamento de novo, Noisettia orchidiflora, Anchietea pyrifolia