Biomoléculas de Nectandra barbellata Coe-Teixeira (Lauraceae): caracterização molecular e avaliação do potencial antiparasitário
Data
2022-03-24
Autores
Londero, Vinicius Silva 
Orientadores
Lago, João Henrique Ghilardi
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Com base no potencial em estudos de bioprospecção é que este estudo propôs investigar, pela primeira vez, o potencial químico e farmacológico da espécie Nectandra barbellata (Lauraceae). Para tanto, foi realizado o estudo fitoquímico biomonitorado pela atividade antiprotozoária (Leishmania infantum e Trypanosoma cruzi) dos extratos dos galhos e folhas de N. barbellata, os quais foram obtidos a partir de método alternativo e sustentável, empregando o líquido iônico brometo de 1-butil-3-metilimidazólio (BMImBr). A análise por RMN de 1H e 13C dos extratos obtidos com BMImBr em meio aquoso, bem como de seu fracionamento, levou ao isolamento de quatro compostos (1 – 4) oriundos dos galhos e quatro compostos (5 – 8), das folhas, cujas estruturas foram determinadas por métodos como EM, RMN, IV e DC. Após a elucidação de três butanolídeos relacionados inéditos (1 – 3), o potencial antiparasitário in vitro foi avaliado e mostrou CE50 de 13,1, 4,0 e 2,1 μM, respectivamente, para formas tripomastigostas de T. cruzi. Dentre os butanolídeos, cabe destacar que o composto 3 apresentou uma potente seletividade (IS > 95), superior ao do controle positivo benznidazol. A atividade antileishmania também foi determinada e os compostos 2 e 3 mostraram CE50 de 67,4 e 27,0 μM, respectivamente, para promastigotas. O composto 4 foi caracterizado como sendo o sesquiterpeno ácido cóstico e seu potencial anti-T. cruzi foi avaliado sendo determinados valores de CE50 de 7,9 μM para amastigotas e CC50 > 200 μM frente a células NCTC (IS > 25). Ainda, o estudo fitoquímico biomonitorado do extrato das folhas de N. barbellata em BMImBr resultou no isolamento de dois sesquiterpenos do tipo eudesmano (5 e 6), um sesquiterpeno rearranjado do tipo ifionano (7) e um pseudodissesquiterpeno inédito (8). Os resultados da avaliação biológica revelam que os compostos 5 – 7 não apresentaram toxicidade frente a células NCTC (CC50 > 200 μM) e mostraram-se inativos para as formas extracelulares de L. infantum e T. cruzi. Por outro lado, o composto 8 apresentou-se ativo com um baixo efeito antileishmania para promastigotas (CE50 = 75,6 μM) e citotóxico para NCTC (CC50 = 93,9 μM), resultando em IS de 1,2. Em T. cruzi, o composto 8 demonstrou CE50 = 13,2 μM para as formas tripomastigotas e IS de 7,1. O estudo do mecanismo de ação frente ao T. cruzi foi investigado para os butanolídeos 1 – 3 e revelou que o ΔΨp de tripomastigotas foi alterado pelos compostos 1 e 2. Os butanolídeos 1 e 3 promoveram alcalinização dos acidocalcissomas, liberando reservas de Ca2+ intracitoplasmáticas, que pode ter contribuído para a despolarização do ΔΨm, resultando em um desequilíbrio dos níveis de ATP e EROs e levando à morte do parasita. Finalmente, a análise in silico dos compostos 1 – 3 pela ferramenta Swiss ADME demonstrou que nenhum deles se assemelha a PAINS. O estudo mecanístico do composto 4 frente às formas tripomastigotas de T. cruzi mostrou uma despolarização do ΔΨp e do ΔΨm. Estudos de microscopia eletrônica revelaram dano ultraestrutural e desorganização das organelas citoplasmáticas, presença de vesículas autofágicas e formação de grandes vacúolos. A avaliação dos parâmetros de ADME in silico demonstraram uma previsão promissora para quase todas as propriedades físico-químicas analisadas para o ácido cóstico. O ensaio de mecanismo de morte com o composto 8 mostrou um efeito tempo-dependente de alteração na permeabilidade da membrana plasmática do parasita com reduzida atividade hemolítica. Assim, estes resultados contribuem para o uso da biodiversidade de espécies da Mata Atlântica através de um procedimento alternativo e sustentável, que forneceu diversas substâncias bioativas inéditas (1 – 3 e 8), bem como agregou valor a compostos conhecidos (4 – 7) e ocorrentes em Lauraceae. Diante dos resultados obtidos até então, foi possível inferir que essa espécie produz metabólitos com atividade biológica e que podem, portanto, vir a se tornarem protótipos ou modelos químicos para futuros fármacos antiparasitários.
Based on the potential in bioprospecting studies, this study proposed to investigate, for the first time, the chemical and pharmacological potential of the species Nectandra barbellata (Lauraceae). Therefore, a phytochemical study was carried out, biomonitored by the antiprotozoal activity (Leishmania infantum and Trypanosoma cruzi) of the extracts from the N. barbellata twigs and leaves, which were obtained from an alternative and sustainable method, using the ionic liquid 1-butyl-3-methylimidazolium bromide (BMImBr). The 1H and 13C NMR analysis of the extracts obtained with BMImBr in aqueous medium, as well as their fractionation, led to the isolation of four compounds (1 – 4) from the twigs and four compounds (5 – 8) from the leaves, whose structures were determined by spectroscopic and spectrometric methods, such as MS, NMR, IR and CD. After the elucidation of three new related butanolides (1 – 3), the in vitro antiparasitic potential of these compounds was evaluated and showed EC50 of 13.1, 4.0 and 2.1 μM, respectively, for trypomastigote forms of T. cruzi. Among the butanolides, it is worth noting that compound 3 showed a potent selectivity (SI > 95), superior to the positive control benznidazole. The antileishmanial activity was also determined and compounds 2 and 3 showed EC50 of 67.4 and 27.0 μM, respectively, for promastigotes. Compound 4 was characterized as sesquiterpene costic acid and its anti-T. cruzi potential was evaluated and EC50 values of 7.9 μM were determined for amastigotes and CC50 > 200 μM against NCTC cells (SI > 25). Furthermore, the biomonitored phytochemical study of the extract of N. barbellata leaves in BMImBr resulted in the isolation of two eudesmane-type sesquiterpenes (5 and 6), an iphionane-type rearranged sesquiterpene (7) and a novel pseudo-disesquiterpene (8). The results of the biological evaluation reveal that compounds 5 – 7 did not show toxicity against mammalian cells (CC50 > 200 μM) and were shown to be inactive for the extracellular forms of L. infantum and T. cruzi. On the other hand, compound 8 was active with a low antileishmanial effect for promastigotes (EC50 = 75.6 μM) and cytotoxic effect for mammalian cells (CC50 = 93.9 μM), resulting in SI of 1.2. In T. cruzi, compound 8 showed an EC50 = 13.2 μM for the trypomastigote forms and SI of 7.1. The study of the mechanism of action against the T. cruzi parasite was investigated for butanolides 1 – 3 and revealed that the ΔΨp of trypomastigotes was altered by compounds 1 and 2. Butanolides 1 and 3 promoted alkalinization of acidocalcissomes, releasing intracytoplasmic Ca2+ reserves, which may have contributed to the depolarization of ΔΨm, resulting in an imbalance of ATP and ROS levels and leading to the death of the parasite. Finally, in silico analysis of compounds 1 – 3 by the Swiss ADME tool showed that none of them resemble PAINS. The mechanistic study of compound 4 against the trypomastigote forms of T. cruzi showed a depolarization of ΔΨp and ΔΨm. Electron microscopy studies revealed ultrastructural damage and disorganization of cytoplasmic organelles, presence of autophagic vesicles and formation of large vacuoles. Additionally, the evaluation of in silico ADME parameters was performed and the results showed a promising prediction for almost all physicochemical properties analyzed for costic acid. The mechanism of death assay with compound 8 showed a time-dependent effect of changing the permeability of the parasite's plasma membrane with reduced hemolytic activity. Thus, these results contribute to the use of the biodiversity of Atlantic Forest species through an alternative and sustainable procedure, which provided several new bioactive substances (1 – 3 and 8), as well as added value to known compounds (4 – 7) and occurring in Lauraceae. In view of the results obtained so far, it was possible to infer that this species produces metabolites with biological activity and that may, therefore, become prototypes or chemical models for future antiparasitic drugs.
Based on the potential in bioprospecting studies, this study proposed to investigate, for the first time, the chemical and pharmacological potential of the species Nectandra barbellata (Lauraceae). Therefore, a phytochemical study was carried out, biomonitored by the antiprotozoal activity (Leishmania infantum and Trypanosoma cruzi) of the extracts from the N. barbellata twigs and leaves, which were obtained from an alternative and sustainable method, using the ionic liquid 1-butyl-3-methylimidazolium bromide (BMImBr). The 1H and 13C NMR analysis of the extracts obtained with BMImBr in aqueous medium, as well as their fractionation, led to the isolation of four compounds (1 – 4) from the twigs and four compounds (5 – 8) from the leaves, whose structures were determined by spectroscopic and spectrometric methods, such as MS, NMR, IR and CD. After the elucidation of three new related butanolides (1 – 3), the in vitro antiparasitic potential of these compounds was evaluated and showed EC50 of 13.1, 4.0 and 2.1 μM, respectively, for trypomastigote forms of T. cruzi. Among the butanolides, it is worth noting that compound 3 showed a potent selectivity (SI > 95), superior to the positive control benznidazole. The antileishmanial activity was also determined and compounds 2 and 3 showed EC50 of 67.4 and 27.0 μM, respectively, for promastigotes. Compound 4 was characterized as sesquiterpene costic acid and its anti-T. cruzi potential was evaluated and EC50 values of 7.9 μM were determined for amastigotes and CC50 > 200 μM against NCTC cells (SI > 25). Furthermore, the biomonitored phytochemical study of the extract of N. barbellata leaves in BMImBr resulted in the isolation of two eudesmane-type sesquiterpenes (5 and 6), an iphionane-type rearranged sesquiterpene (7) and a novel pseudo-disesquiterpene (8). The results of the biological evaluation reveal that compounds 5 – 7 did not show toxicity against mammalian cells (CC50 > 200 μM) and were shown to be inactive for the extracellular forms of L. infantum and T. cruzi. On the other hand, compound 8 was active with a low antileishmanial effect for promastigotes (EC50 = 75.6 μM) and cytotoxic effect for mammalian cells (CC50 = 93.9 μM), resulting in SI of 1.2. In T. cruzi, compound 8 showed an EC50 = 13.2 μM for the trypomastigote forms and SI of 7.1. The study of the mechanism of action against the T. cruzi parasite was investigated for butanolides 1 – 3 and revealed that the ΔΨp of trypomastigotes was altered by compounds 1 and 2. Butanolides 1 and 3 promoted alkalinization of acidocalcissomes, releasing intracytoplasmic Ca2+ reserves, which may have contributed to the depolarization of ΔΨm, resulting in an imbalance of ATP and ROS levels and leading to the death of the parasite. Finally, in silico analysis of compounds 1 – 3 by the Swiss ADME tool showed that none of them resemble PAINS. The mechanistic study of compound 4 against the trypomastigote forms of T. cruzi showed a depolarization of ΔΨp and ΔΨm. Electron microscopy studies revealed ultrastructural damage and disorganization of cytoplasmic organelles, presence of autophagic vesicles and formation of large vacuoles. Additionally, the evaluation of in silico ADME parameters was performed and the results showed a promising prediction for almost all physicochemical properties analyzed for costic acid. The mechanism of death assay with compound 8 showed a time-dependent effect of changing the permeability of the parasite's plasma membrane with reduced hemolytic activity. Thus, these results contribute to the use of the biodiversity of Atlantic Forest species through an alternative and sustainable procedure, which provided several new bioactive substances (1 – 3 and 8), as well as added value to known compounds (4 – 7) and occurring in Lauraceae. In view of the results obtained so far, it was possible to infer that this species produces metabolites with biological activity and that may, therefore, become prototypes or chemical models for future antiparasitic drugs.
Descrição
Citação
LONDERO, V. S. Biomoléculas de Nectandra barbellata Coe-Teixeira (Lauraceae): Caracterização molecular e avaliação do potencial antiparasitário. 2022. 241 f. Tese (Doutorado em Biologia Química). Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, 2022.