Busca por orbitídeos em espécies do gênero Croton (Euphorbiaceae) oriundas da Mata Atlântica
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Data
2019-04-30
Autores
Queiroz, Suzana Aparecida da Silva
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Universidade Estadual Paulista (Unesp)
Resumo
O gênero Croton, um dos maiores da família Euphorbiaceae, é conhecido pela sua riqueza de metabólitos secundários de origem terpênica, como os clerodânicos. No entanto, estudos recentes apontam para a presença de uma classe de compostos que vem ganhando visibilidade e interesse dos cientistas e de indústrias farmacêuticas, os peptídeos cíclicos. Os peptídeos naturais, especialmente aqueles de natureza cíclica e de baixo peso molecular (orbitídeos), constituem uma classe de moléculas promissoras para a identificação de novos hits e leads de fármacos. Nessa perspectiva, esse trabalho trata do estudo de duas espécies da Mata Atlântica: Croton floribundus e Croton campanulatus na busca por peptídeos cíclicos, mais precisamente por orbitídeos. Assim sendo, inicialmente investigou-se em pequena escala folhas e caules de ambas as espécies, raiz da espécie C. floribundus e látex da espécie C. campanulatus e após essas análises preliminares selecionou-se a raiz da de C. floribundus e o látex de C. campanulatus para dar continuidade com os estudos. O processo de extração das substâncias de interesse da raiz foi feito por maceração hidroetanólica e do látex, por partição líquido-líquido com acetato de etila. Em seguida esses extratos foram submetidos à cromatografia em coluna, empregando octadecilsilano (C18) como fase estacionária para a obtenção das frações contendo peptídeos. O processo de purificação dessas frações foi feito por HPLC em modo analítico e preparativo que permitiu o isolamento de 2 substâncias da espécie C. campanulatus e 4 substâncias da espécie C. floribundus. Após serem caracterizadas por HR-ESI- (+)-Q-TOF-MS/MS e RMN 1D e 2D observou-se que as substâncias isoladas da espécie C. campanulatus correspondem a 2 novos orbtídeos que foram denominados [1-7NαC] crocaorb A1 com m/z 827,4538 [M + H] + de sequência ciclo(-Pro1 -Val2 -Leu3 -Ile4 -Met5 -Ser6 -Trp7 -), e [1-7NαC] crocaorb A2 com m/z 843,4441 [M + H] + de sequência ciclo(-Pro1 -Val2 -Leu3 -Ile4 -Met[O]5 -Ser6 -Trp7 -). Com relação à espécie C. floribundus não foi possível identificar e nem isolar peptídeos e as 4 substâncias então obtidas foram caracterizadas por LC-ESI- (+)-IT-MS e RMN 1D e 2D como sendo 3 novos diterpenos clerodânicos denominados cascarillina J com m/z 431,16 [M + Na]+ , croflorin A com m/z 349,15 [M + H]+ e croflorin B com m/z 331,46 [M + H]+ . Também foi isolado o diterpeno da classe dos halimanos conhecido como 3β-hidroxi-5,10- didehidrociliolida, reportado pela primeira vez como um produto natural. A fração 80% da raiz de C. floribundus e a croflorin A isolada dessa fração foram submetidas a avaliação da atividade antiinflamatória e ambas as amostras apresentaram potencial anti-inflamatório.
The Croton genus, the largest of the Euphorbiaceae family, is known for its richness of secondary metabolites of terpenic origin, such as the clerodanic ones. However, recent studies point to the presence of a class of compounds that has gained visibility and interest from scientists and pharmaceutical industries, the cyclic peptides. Natural peptides, especially those of a cyclic and low molecular weight nature (orbitides), represent a class of promising molecules for identification of hits and leads in drug discovery. In this perspective, this research deals with the study of two species from the Atlantic Forest: Croton floribundus and Croton campanulatus in the search for cyclic peptides, more precisely for orbitides. Thus, leaves and stems of both species, root from C. floribundus and latex from C. campanulatus were investigated in a small scale, and after these preliminary analyzes, the C. froribundus’ root and the latex from C. campanulatus were selected to continue the studies. The extraction process of the root's compounds of interest was performed by hydroethanolic maceration, while the latex extraction consisted by liquid-liquid partition with ethyl acetate. Then, these extracts were submitted to a chromatographic column using octadecylsilane (C18) as a stationary phase to obtain fractions containing peptides. The fractions purification process was carried out by analytical and preparative HPLC, which allowed the isolation of 2 compounds from C. campanulatus and 4 compounds from C. floribundus. After being characterized by HR-ESI-(+)-Q-TOF-MS/MS and 1D and 2D NMR, it was observed that the compounds isolated from the C. campanulatus species correspond to 2 new orbitides that were named [1-7NαC] crocaorb A1 with m/z 827.4538 [M+H]+ and cyclo (-Pro1-Val2-Leu3-Ile4-Met5-Ser6-Trp7-) sequence, and [1-7 Nα C] crocaorb A2 with m/z 843.4441 [M+H]+ and cyclo (-Pro1-Val2- Leu3-Ile4-Met [O] 5-Ser6-Trp7-) sequence. With respect to the C. floribundus species, it was not possible to identify or isolate the expected peptides, and the 4 compounds obtained were characterized by LC-ESI-(+)-IT-MS and 1D and 2D NMR as 3 new clerodanic diterpenes called cascarillin J with m/z 431.16 [M+Na] + , crofllorin A with m/z 349.15 [M+H]+ and crofllorin B with m/z 331.46 [M+H]+ . A diterpene of the halimane class, known as 3β-hydroxy-5,10- didehydrociliolide, first reported as a natural product, was also isolated. The 80% fraction of the C. floribundus root and the croflorin A isolated from this fraction were evaluated for antiinflammatory activity and both samples presented interesting anti-inflammatory potential.
The Croton genus, the largest of the Euphorbiaceae family, is known for its richness of secondary metabolites of terpenic origin, such as the clerodanic ones. However, recent studies point to the presence of a class of compounds that has gained visibility and interest from scientists and pharmaceutical industries, the cyclic peptides. Natural peptides, especially those of a cyclic and low molecular weight nature (orbitides), represent a class of promising molecules for identification of hits and leads in drug discovery. In this perspective, this research deals with the study of two species from the Atlantic Forest: Croton floribundus and Croton campanulatus in the search for cyclic peptides, more precisely for orbitides. Thus, leaves and stems of both species, root from C. floribundus and latex from C. campanulatus were investigated in a small scale, and after these preliminary analyzes, the C. froribundus’ root and the latex from C. campanulatus were selected to continue the studies. The extraction process of the root's compounds of interest was performed by hydroethanolic maceration, while the latex extraction consisted by liquid-liquid partition with ethyl acetate. Then, these extracts were submitted to a chromatographic column using octadecylsilane (C18) as a stationary phase to obtain fractions containing peptides. The fractions purification process was carried out by analytical and preparative HPLC, which allowed the isolation of 2 compounds from C. campanulatus and 4 compounds from C. floribundus. After being characterized by HR-ESI-(+)-Q-TOF-MS/MS and 1D and 2D NMR, it was observed that the compounds isolated from the C. campanulatus species correspond to 2 new orbitides that were named [1-7NαC] crocaorb A1 with m/z 827.4538 [M+H]+ and cyclo (-Pro1-Val2-Leu3-Ile4-Met5-Ser6-Trp7-) sequence, and [1-7 Nα C] crocaorb A2 with m/z 843.4441 [M+H]+ and cyclo (-Pro1-Val2- Leu3-Ile4-Met [O] 5-Ser6-Trp7-) sequence. With respect to the C. floribundus species, it was not possible to identify or isolate the expected peptides, and the 4 compounds obtained were characterized by LC-ESI-(+)-IT-MS and 1D and 2D NMR as 3 new clerodanic diterpenes called cascarillin J with m/z 431.16 [M+Na] + , crofllorin A with m/z 349.15 [M+H]+ and crofllorin B with m/z 331.46 [M+H]+ . A diterpene of the halimane class, known as 3β-hydroxy-5,10- didehydrociliolide, first reported as a natural product, was also isolated. The 80% fraction of the C. floribundus root and the croflorin A isolated from this fraction were evaluated for antiinflammatory activity and both samples presented interesting anti-inflammatory potential.
Descrição
Palavras-chave
Euphorbiaceae, Croton (Genus), Agentes anti-inflamatórios, Metabólitos secundários, Peptídeos cíclicos, Cyclic peptides, Secondary metabolites, Terpenos, Terpenes