Busca de inibidores de cisteíno proteases em espécies de Passiflora utilizando isolamento bioguiado

• Ano de defesa: 2022
• Autor(a) principal: Araújo, Izabel Pesqueira Ribeiro de
• Orientador(a): Silva, James Almada da
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Programa de Pós-Graduação: Pós-Graduação em Química
• Palavras-chave em Português: Química; Passiflora; Matéria médica vegetal; Fenóis; Flavonóides; Enzimas proteolíticas; Inibidores enzimáticos; Protease; Inibição enzimática
• Palavras-chave em Inglês: Passionfruit; Flavonoids; Phenols; Enzyme inhibition
• Área do conhecimento CNPq: CIENCIAS EXATAS E DA TERRA::QUIMICA
• Link de acesso: http://ri.ufs.br/jspui/handle/riufs/17138

The cysteine proteases, cathepsins B, C and K, are directly involved with depression and anxiety and therefore can be used as biological targets in the search for anxiolytic drugs. As Passiflora species are traditionally used for the treatment of anxiety, plants of this genus have potential to search for cysteine protease inhibitors, which can improve in the treatment of anxiety. Thus, the objective of this work was to isolate cysteine protease inhibitors, using papain as a model, through bioguided isolation of secondary metabolites with the species Passiflora cincinnata, P. edulis and P. foetida. Extracts of aerial parts of the species were obtained by maceration using ethanol (1:4, m/v; 72 h). The extract with the highest enzymatic inhibition was fractionated by liquid-liquid partition (PLL), using hexane, dichloromethane and ethyl acetate. The fraction with the highest enzymatic inhibition was subjected to classical liquid chromatography (CLC) and later the most active subfraction was analyzed and subjected to isolation by high performance liquid chromatography (HPLC). Extracts (125; 250 and 500 µg.mL-1), fractions and subfractions (500 µg.mL-1), and substances (250 to 15.62 µg.mL-1) were evaluated for papain inhibition, in wells constructed in a petri dish containing agar/milk, by a method developed in the laboratory. The total phenolic compounds and flavonoids of the extracts were quantified in a spectrophotometer using Folin-Ciocalteu and AlCl3 reagents, respectively. Among the extracts of the three species, it was observed that Passiflora foetida (PF) presented the highest enzyme inhibition (40.1% ± 2.16), and that the branches extract (PFG) of this species had the highest percentage of inhibition (47.2% ± 2.55). The PFG presented phenolic compounds and flavonoids content of 49.42 ± 0.92 mg EqAG/g, and 19.39 ± 1.98 mg EqQUE/g, respectively. In addition to the presence of flavonoids in this extract, anthraquinones, alkaloids, triterpenes and fatty acids was detected. As the dichloromethane fraction (PFGD), obtained from the PFG extract after PLL, showed the highest enzymatic inhibition (38.31% ± 0.09), it was submitted to CLC. The PFGD-E subfraction (45.83% ± 0.79) showed one of the best enzymatic inhibition results, and sufficient mass to follow the isolation process (HPLC), and so it was selected. After optimizing the chromatographic separation of this subfraction, six fractions were obtained (PFGD-E1 to PFGD-E6). The NMR analysis process showed that three were still in mixture (PFGD-E2 to PFGD-E4), and that the predominant class of this mixture are flavanones. The other three fractions (PFGD-E1, PFGDE5, PFGD-E6) were identified as flavonoids: 5,4'-dihydroxy-7-methoxy-flavanone (E1), 5-hydroxy-7,3',4'-trimethoxy-flavanone (E5) and 5-hydroxy-7,4'-dimethoxyflavone (E6). Substances E5 and E6 (250 µg.mL-1) showed enzyme inhibition of 13.66% ± 3.26 and 18.73% ± 4.27, respectively, a low inhibition when compared to the PFGD-E fraction (45.83% ± 0.79). Substances E1, E5 and E6 were subjected to molecular docking with cathepsins B, C and K, where a greater affinity towards cathepsin C was observed. In view of this, it is concluded that the species under study has the potential to inhibit cysteine proteases, however, these substances, alone, are not the main responsible for the activity observed in the extracts and fractions.