Search

Made available in DSpace on 2019-10-04T12:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-12-01 Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Leishmaniasis is one of the most important neglected tropical diseases (NTDs) that are especially common among low-income populations in developing regions of Africa, Asia, and the Americas. Many natural products, particularly alkaloids, have been reported to have inhibitory activity against arginase, the key enzyme in the pathology caused by Leishmania sp. In this way, piperidine alkaloids (-)-cassine (1), (-)-spectaline (2), (-)-3-O-acetylcassine (3), and (-)-3-O-acetylspectaline (4) were isolated from Senna spectabilis flowers. These compounds (1/2 and 3/4) initially present as homologous mixtures were separated by high performance liquid chromatography and evaluated against the promastigote phase of Leishmania amazonensis. In addition, molecular docking simulations were implemented in order to probe the binding modes of the ligands 1-4 to the amino acids in the active site of L. amazonensis arginase. Alkaloid 2 (IC50 15.81 mu g mL(-1)) was the most effective against L. amazonensis. Compounds 2 and 4, with larger side chain, were more effective against the parasite than compounds 1 and 3. The cell viability test on Vero cells revealed that compound 2 (CC50 66.67 mu g mL-1) was the most toxic. The acetyl group in the 3-O position of the parent structures reduced the leishmanicidal activity and the toxicity of the alkaloids. Further, molecular docking suggested that Asn143 is essential for arginase to interact with (-)-spectaline-derived compounds, which agreed with the IC50 measurements. Our findings revealed that S. spectabilis is an important source of piperidine alkaloids with leishmanicidal activity. Moreover, the natural compound 3 has been isolated for the first time. Experimental investigation combined with theoretical study advances knowledge about the enzyme binding site mode of interaction and contributes to the design of new bioactive drugs against Leishmania infection. Univ Fed Uberlandia, Inst Quim, Nucleo Pesquisa Prod Nat NuPPeN, BR-38400902 Uberlandia, MG, Brazil Univ Fed Uberlandia, Inst Ciencias Biomed, BR-38400902 Uberlandia, MG, Brazil Univ Fed Triangulo Mineiro, Inst Ciencias Exatas Nat & Educ, Dept Fis, Lab Biofis Teor, BR-38064200 Uberaba, MG, Brazil Univ Fed Alfenas, Inst Quim, Lab Pesquisa Quim Med PeQuiM, BR-37133840 Alfenas, MG, Brazil Univ Estadual Paulista, Inst Quim, Dept Quim Organ, Nucleo Bioensaios Biossintese & Ecofisiol Prod Na, POB 355, BR-14801970 Araraquara, SP, Brazil Univ Fed Triangulo Mineiro, Inst Ciencias Exatas Nat & Educ, Dept Quim, Nucleo Desenvolvimento Compostos Bioativos NDCBio, BR-38064200 Uberaba, MG, Brazil Univ Estadual Paulista, Inst Quim, Dept Quim Organ, Nucleo Bioensaios Biossintese & Ecofisiol Prod Na, POB 355, BR-14801970 Araraquara, SP, Brazil FAPEMIG: APQ-02481-14 CNPq: 449846/2014-8 FAPEMIG: REDE-113/10 FAPEMIG: CEX-RED-00010-14

O Brasil é detentor de uma das maiores biodiversidades do planeta e neste sentido não podemos abdicar desta posição privilegiada para o estudo químico dos produtos naturais visando à descoberta de novas moléculas bioativas. Ao longo do território nacional são encontrados seis biomas que apresentam uma grande diversidade de plantas. Neste trabalho, Senna spectabilis (Fabaceae) foi selecionada para o estudo químico por apresentar alcaloides piperidínicos do tipo 2,6-dialquil-piperidin-3-óis, raros na natureza e com grande potencial farmacológico. O estudo foi realizado a partir do extrato etanólico das flores e os compostos isolados foram submetidos aos ensaios para avaliação da atividade leishmanicida e citotoxicidade. Inicialmente, o extrato etanólico das flores de S. spectabilis foi submetido à extração líquido-líquido com n-hexano, clorofórmio e acetato de etila. As frações foram analisadas por cromatografia em camada delgada (CCD), onde foi constatado que a fração clorofórmio apresentou maior número de alcaloides com diferentes Rfs. Esta fração foi submetida a cromatografia em coluna (CC), de onde foi obtida a mistura dos homólogos (-)-cassina (1) e (-)-espectalina (6), que foram separados por cromatografia líquida de alta eficiência (CLAE) utilizando detector de light scattering. Estes compostos foram submetidos aos experimentos de ressonância magnética nuclear (RMN) e espectrometria de massas de alta resolução com ionização por eletrospray (IES-EM) para determinação das suas estruturas. Ao longo do fracionamento cromatográfico foram obtidas duas misturas de compostos homólogos (61a e 61b e 62a e 62b), mais polares que os alcaloides 1 e 6. Devido a pequena quantidade de massa, estes metabólitos foram identificados por EM-IES. Também foi obtida uma fração com uma mistura de alcaloides de polaridade menor que 1 e 6, que foi submetida a CCD preparativa, de onde foi possível obter uma mistura dos alcalóides homólogos (-)-3-O- acetilcassina (13) e (-)-3-O-acetilespectalina (14) que foram separados por CLAE-light scattering. Os compostos isolados foram submetidos ao ensaio para avaliação da atividade leishmanicida utilizando cepas do parasita Leishmania amazoniensis, sendo que 6 e 13 apresentaram valores de IC50 promissores (IC50 15,81 ± 0,47 pg mL-1 e 30,25 ± 1,74 pg mL-1, respectivamente), quando comparados com o padrão positivo anfotericina B (0,29 ± 0,01 pg mL-1). A partir do ensaio de citotoxicidade, foi observado que o alcaloide 6 apresenta certa toxicidade (CC50 66,67 ± 9,68 pg mL-1), porém, a partir do cálculo do índice de seletividade (IS), foi possível constatar que os compostos avaliados apresentam atividade leishmanicida em concentrações não citotóxicas. Brazil holds one of the greatest biodiversity on the planet and in this sense we cannot give up this privileged position for the chemical study of natural products for the discovery of new bioactive molecules. Along the national territory are six biomes that present a great diversity of plants. In this work, Senna spectabilis (Fabaceae) was selected for the chemical study because it presents 2,6-dialkyl-piperidin-3-ol type piperidine alkaloids, rare in nature and with great pharmacological potential. The study was carried out from the ethanolic extract of the flowers and the isolated compounds were submitted to the tests to evaluate the leishmanicidal activity and cytotoxicity. Initially, the ethanolic extract of S. spectabilis flowers was submitted to liquid-liquid extraction with n-hexane, chloroform and ethyl acetate. The fractions were analyzed by thin layer chromatography (TLC), where it was found that the chloroform fraction had a higher number of alkaloids with different Rfs. This fraction was subjected to column chromatography (CC), from which the mixture of (-)-cassine (1) and (-)- spectaline (6) homologues were obtained, which were separated by high performance liquid chromatography (HPLC) using light scattering detector. These compounds were submitted to nuclear magnetic resonance (NMR) experiments and high resolution mass spectrometry with electrospray ionization (ESI-MS) to determine their structures. During the chromatographic fractionation, two mixtures of homologous compounds (61a and 61b and 62a and 62b) were obtained, more polar than alkaloids 1 and 6. Due to the small amount of mass, these metabolites were identified by ESI-MS. Furthermore, a fraction with a mixture of alkaloids less polar than 1 and 6 was also obtained, which was subjected to preparative TLC, and was possible to obtain a mixture of the homologous alkaloids (-)-3-O-acetylcassine (13) and (-)- 3-O-acetylspectaline (14) which were separated by HPLC-light scattering. The isolated compounds were tested for leishmanicidal activity using strains of the parasite Leishmania amazoniensis, with 6 and 13 showing promising IC50 values ( IC50 15.81 ± 0.47 pg mL-1 e 30.25 ± 1.74 pg mL-1, respectively), when compared to the positive control amphotericin B (0.29 ± 0.01 pg mL-1). From the cytotoxicity assay, it was observed that alkaloid 6 presents some toxicity (CC50 66.67 ± 9.68 pg mL-1), but from the calculation of the selectivity index (SI), it was possible to verify that the evaluated compounds presented leishmanicidal activity at non-cytotoxic concentrations. Dissertação (Mestrado)