Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link

Detalhes bibliográficos
Ano de defesa: 2021
Autor(a) principal: FIGUEREDO, Aline Santana lattes
Orientador(a): SILVA, Mayara Cristina Pinto da lattes
Banca de defesa: SILVA, Mayara Cristina Pinto da lattes, ANDRADE, Marcelo Souza de lattes, FREITAS, Dayanne da Silva lattes, BATISTA, Marisa Cristina Aranha lattes
Tipo de documento: Dissertação
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Universidade Federal do Maranhão
Programa de Pós-Graduação: PROGRAMA DE PÓS-GRADUAÇÃO EM SAÚDE DO ADULTO
Departamento: DEPARTAMENTO DE MEDICINA II/CCBS
País: Brasil
Palavras-chave em Português:
Palavras-chave em Inglês:
Área do conhecimento CNPq:
Link de acesso: https://tedebc.ufma.br/jspui/handle/tede/tede/3760
Resumo: Introduction: Leishmaniasis is part of the group of neglected diseases, caused by protozoa of the genus Leishmania, being endemic in 98 countries. The therapy used for this disease is associated with high toxicity, difficult administration, high costs and parasitic resistance. Therefore, the search for new drug alternatives, more effective and less toxic is necessary and protease inhibitors are crucial molecules, since Leishmania proteases are involved in several physiological and pathological mechanisms. Aim: To identify the chemical constituents of the Bauhinia forficata Link extract, as well as the cytotoxic effect of the extract and the aspartic protease inhibitor fraction on Leishmania (L.) amazonensis. Materials and methods: firstly, the preparation of the extract was carried out, followed by the dosage of proteins, through the Bradford method, and then these proteins were identified through the electrophoresis method. Another method performed was the study of the chemical constituents present in the extract, in which the Matos phytochemical screening test was used, followed by thin layer chromatography, and the flavonoid contents were determined by high performance liquid chromatography. After chemical characterization, in vitro cytotoxicity tests were performed using sheep erythrocytes and immortalized cells of the RAW lineage, and in vivo toxicity tests in Tenebrio molitor larvae. As for the leishmanicidal activity in Leishmania amazonensis promastigotes, the assay was performed using the MTT method (3-(4,5-dimethyithiazol2-yl) -2,5- diphenyltetrazolium bromide), and the antiamastigote activity was performed with RAW cells. Results and discussion: Derivatives of quercetin and kaempferol were the main metabolites found in BF-CA. After isolation of the inhibitor of aspartic profease (BFI), 130.6 ± 29.94 μg of protein was obtained, with a yield of approximately 4.41 ± 0.77 %, that is, 4.41% of the proteins of BF-CA. The extract and BFI did not show cytotoxicity to sheep erythrocytes. In RAW cells, the extract and the inhibitor tested (BF-CA and BFI) showed a cytotoxic concentration for 50% of the cells (CC50) of 166.5 μg/mL and 371.3 μg/mL, respectively, and for pentamidine with CC50 of 15.24 μg/ml. By analyzing the inhibitory concentration in 50% (IC50) of the extracts on the promastigote forms of Leishmania amazonensis, we obtained an IC50 of 54.9 μg/mL for BF-CA and 8.33 μg/mL for BFI, which exhibited toxicity at almost all concentrations tested. When evaluating the inhibition of proteases, BFI inhibited 44.66% of the activity of commercial pepsin protease and inhibited 40% of the serine protease secreted by L. amazonensis, LSP III. Conclusion: Therefore, BF-CA has a composition rich in flavonoids, with proven leishmanicidal action in promastigote and amastigote forms, evidenced by the reduction of infection. As for BFI, it inhibited the activity of the serine protease secreted by L. amazonensis (LSPIII), making it a potential therapeutic target, as it is capable of interfering in the pathophysiology of leishmaniasis
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spelling SILVA, Mayara Cristina Pinto dahttp://lattes.cnpq.br/9507590466760552LÓPES, Raquel Elisa da Silvahttp://lattes.cnpq.br/9701239960925787SILVA, Mayara Cristina Pinto dahttp://lattes.cnpq.br/9507590466760552ANDRADE, Marcelo Souza dehttp://lattes.cnpq.br/6267637354657076FREITAS, Dayanne da Silvahttp://lattes.cnpq.br/7697882500616368BATISTA, Marisa Cristina Aranhahttp://lattes.cnpq.br/7775556248972778http://lattes.cnpq.br/6129359948262855FIGUEREDO, Aline Santana2022-06-24T17:30:56Z2021-10-13FIGUEREDO, Aline Santana. Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link. 2021. 105 f. Dissertação (Programa de Pós-Graduação em Saúde do Adulto) - Universidade Federal do Maranhão, São Luís, 2021.https://tedebc.ufma.br/jspui/handle/tede/tede/3760Introduction: Leishmaniasis is part of the group of neglected diseases, caused by protozoa of the genus Leishmania, being endemic in 98 countries. The therapy used for this disease is associated with high toxicity, difficult administration, high costs and parasitic resistance. Therefore, the search for new drug alternatives, more effective and less toxic is necessary and protease inhibitors are crucial molecules, since Leishmania proteases are involved in several physiological and pathological mechanisms. Aim: To identify the chemical constituents of the Bauhinia forficata Link extract, as well as the cytotoxic effect of the extract and the aspartic protease inhibitor fraction on Leishmania (L.) amazonensis. Materials and methods: firstly, the preparation of the extract was carried out, followed by the dosage of proteins, through the Bradford method, and then these proteins were identified through the electrophoresis method. Another method performed was the study of the chemical constituents present in the extract, in which the Matos phytochemical screening test was used, followed by thin layer chromatography, and the flavonoid contents were determined by high performance liquid chromatography. After chemical characterization, in vitro cytotoxicity tests were performed using sheep erythrocytes and immortalized cells of the RAW lineage, and in vivo toxicity tests in Tenebrio molitor larvae. As for the leishmanicidal activity in Leishmania amazonensis promastigotes, the assay was performed using the MTT method (3-(4,5-dimethyithiazol2-yl) -2,5- diphenyltetrazolium bromide), and the antiamastigote activity was performed with RAW cells. Results and discussion: Derivatives of quercetin and kaempferol were the main metabolites found in BF-CA. After isolation of the inhibitor of aspartic profease (BFI), 130.6 ± 29.94 μg of protein was obtained, with a yield of approximately 4.41 ± 0.77 %, that is, 4.41% of the proteins of BF-CA. The extract and BFI did not show cytotoxicity to sheep erythrocytes. In RAW cells, the extract and the inhibitor tested (BF-CA and BFI) showed a cytotoxic concentration for 50% of the cells (CC50) of 166.5 μg/mL and 371.3 μg/mL, respectively, and for pentamidine with CC50 of 15.24 μg/ml. By analyzing the inhibitory concentration in 50% (IC50) of the extracts on the promastigote forms of Leishmania amazonensis, we obtained an IC50 of 54.9 μg/mL for BF-CA and 8.33 μg/mL for BFI, which exhibited toxicity at almost all concentrations tested. When evaluating the inhibition of proteases, BFI inhibited 44.66% of the activity of commercial pepsin protease and inhibited 40% of the serine protease secreted by L. amazonensis, LSP III. Conclusion: Therefore, BF-CA has a composition rich in flavonoids, with proven leishmanicidal action in promastigote and amastigote forms, evidenced by the reduction of infection. As for BFI, it inhibited the activity of the serine protease secreted by L. amazonensis (LSPIII), making it a potential therapeutic target, as it is capable of interfering in the pathophysiology of leishmaniasisIntrodução: As leishmanioses fazem parte do grupo de doenças negligenciadas, causadas por protozoários do gênero Leishmania, sendo endêmicas em 98 países. A terapêutica utilizada para esta enfermidade está associada a alta toxicidade, dificuldade de administração, altos custos e resistência parasitária. Logo, a busca por novas alternativas medicamentosas mais eficazes e menos tóxicas se faz necessária e os inibidores de proteases são moléculas cruciais, visto que as proteases de Leishmania estão envolvidas em diversos mecanismos fisiológicos e patológicos. Objetivo: Identificar os constituintes químicos do extrato de Bauhinia forficata Link, bem como o efeito citotóxico do extrato e da fração de inibidor de aspártico-proteases sobre Leishmania amazonensis (L.). Materiais e métodos: primeiramente foi realizado a preparação do extrato, seguida da dosagem de proteínas, através do método de Bradford, e em seguida essas proteínas foram identificadas através do método de eletroforese. Outro método realizado foi o estudo dos constituintes químicos presentes no extrato, em que foi utilizado o teste de triagem fitoquímica de Matos, seguido da cromatografia de camada delgada, e a identificação dos constituintes químicos foram determinados através da cromatografia líquida de alta eficiência. Após a caracterização química, foram realizados os testes de citotoxicidade in vitro utilizando eritrócitos de carneiro e células imortalizadas de linhagem RAW, e ensaios de toxicidade in vivo em larvas de Tenebrio molitor. Quanto a atividade leishmanicida em promastigota de Leishmania amazonensis, o ensaio foi através do método de MTT (3-(4,5-dimethyithiazol2-yl) -2,5-diphenyltetrazolium bromide), e a atividade antiamastigota foi realizada com ensaio de infecção de macrófagos RAW. Resultados e discussão: Derivados de quercetina e kaempferol foram os principais metabólitos encontrados no BF-CA. Após o isolamento do inibidor de aspártico protease (BFI) foi obtido 130,6 ± 29,94 μg de proteína, com um rendimento aproximadamente de 4,41 ± 0,77 %, ou seja, 4,41% das proteínas de BF-CA. O extrato e BFI não apresentaram citotoxicidades para eritrócitos de carneiro. Em células RAW, o extrato e o inibidor testados (BF-CA e BFI) apresentaram uma concentração citotóxica para 50% das células (CC50) de 166.5 μg/mL e 371,3 μg/mL, respectivamente, e a pentamidina com CC50 de 15,24 μg/mL. Ao analisar a concentração inibitória em 50% (IC50) dos extratos, sobre as formas promastigotas de Leishmania amazonensis, obtivemos IC50 de 54,9 μg/mL para o BF-CA e de 8,33 μg/mL para o BFI, que exibiu toxicidade em quase todas as concentrações testadas. Ao avaliarmos a inibição de proteases o BFI inibiu 44,66% da atividade da pepsina protease comercial e inibiu 40% da serino protease secretada por L. amazonensis, a LSP III. Conclusão: O BF-CA possui uma composição rica em flavonoides, com comprovada ação leishmanicida em formas promastigotas, também foi identificado atividades leishmanicida contra as formas amastigotas, evidenciadas pela diminuição da infecção. Quanto ao BFI, o mesmo inibiu a atividade da serino protease secretada por L. amazonensis (LSPIII), o tornando um potencial alvo terapêutico, uma vez que esta enzima está associada a virulência e/ou patogenicidade do parasito. Sendo assim, os inibidores de proteases despertam interesse, uma vez que são capazes de inibir proteases específicas, interferindo no curso das leishmanioses.Submitted by Jonathan Sousa de Almeida (jonathan.sousa@ufma.br) on 2022-06-24T17:30:56Z No. of bitstreams: 1 ALINESANTANAFIGUEREDO.pdf: 3005846 bytes, checksum: 0dd14d47c6066461247ce9c7a3bfc6fb (MD5)Made available in DSpace on 2022-06-24T17:30:56Z (GMT). No. of bitstreams: 1 ALINESANTANAFIGUEREDO.pdf: 3005846 bytes, checksum: 0dd14d47c6066461247ce9c7a3bfc6fb (MD5) Previous issue date: 2021-10-13application/pdfporUniversidade Federal do MaranhãoPROGRAMA DE PÓS-GRADUAÇÃO EM SAÚDE DO ADULTOUFMABrasilDEPARTAMENTO DE MEDICINA II/CCBSanti-Leishmania.flavonoides;inibidores de proteases;serino protease.serino protease.anti-leishmania;flavonoids;protease inhibitors;Doenças Infecciosas e ParasitáriasCiências da SaúdeProspecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata LinkProspecting the leishmanicidal action of protease inhibitors extracted from Bauhinia forficata Linkinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UFMAinstname:Universidade Federal do Maranhão (UFMA)instacron:UFMAORIGINALALINESANTANAFIGUEREDO.pdfALINESANTANAFIGUEREDO.pdfapplication/pdf3005846http://tedebc.ufma.br:8080/bitstream/tede/3760/2/ALINESANTANAFIGUEREDO.pdf0dd14d47c6066461247ce9c7a3bfc6fbMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82255http://tedebc.ufma.br:8080/bitstream/tede/3760/1/license.txt97eeade1fce43278e63fe063657f8083MD51tede/37602023-05-22 14:39:47.128oai:tede2:tede/3760Biblioteca Digital de Teses e Dissertaçõeshttps://tedebc.ufma.br/jspui/PUBhttp://tedebc.ufma.br:8080/oai/requestrepositorio@ufma.br||repositorio@ufma.bropendoar:21312023-05-22T17:39:47Biblioteca Digital de Teses e Dissertações da UFMA - Universidade Federal do Maranhão (UFMA)false
dc.title.por.fl_str_mv Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link
dc.title.alternative.eng.fl_str_mv Prospecting the leishmanicidal action of protease inhibitors extracted from Bauhinia forficata Link
title Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link
spellingShingle Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link
FIGUEREDO, Aline Santana
anti-Leishmania.
flavonoides;
inibidores de proteases;
serino protease.
serino protease.
anti-leishmania;
flavonoids;
protease inhibitors;
Doenças Infecciosas e Parasitárias
Ciências da Saúde
title_short Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link
title_full Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link
title_fullStr Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link
title_full_unstemmed Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link
title_sort Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link
author FIGUEREDO, Aline Santana
author_facet FIGUEREDO, Aline Santana
author_role author
dc.contributor.advisor1.fl_str_mv SILVA, Mayara Cristina Pinto da
dc.contributor.advisor1Lattes.fl_str_mv http://lattes.cnpq.br/9507590466760552
dc.contributor.advisor-co1.fl_str_mv LÓPES, Raquel Elisa da Silva
dc.contributor.advisor-co1Lattes.fl_str_mv http://lattes.cnpq.br/9701239960925787
dc.contributor.referee1.fl_str_mv SILVA, Mayara Cristina Pinto da
dc.contributor.referee1Lattes.fl_str_mv http://lattes.cnpq.br/9507590466760552
dc.contributor.referee2.fl_str_mv ANDRADE, Marcelo Souza de
dc.contributor.referee2Lattes.fl_str_mv http://lattes.cnpq.br/6267637354657076
dc.contributor.referee3.fl_str_mv FREITAS, Dayanne da Silva
dc.contributor.referee3Lattes.fl_str_mv http://lattes.cnpq.br/7697882500616368
dc.contributor.referee4.fl_str_mv BATISTA, Marisa Cristina Aranha
dc.contributor.referee4Lattes.fl_str_mv http://lattes.cnpq.br/7775556248972778
dc.contributor.authorLattes.fl_str_mv http://lattes.cnpq.br/6129359948262855
dc.contributor.author.fl_str_mv FIGUEREDO, Aline Santana
contributor_str_mv SILVA, Mayara Cristina Pinto da
LÓPES, Raquel Elisa da Silva
SILVA, Mayara Cristina Pinto da
ANDRADE, Marcelo Souza de
FREITAS, Dayanne da Silva
BATISTA, Marisa Cristina Aranha
dc.subject.por.fl_str_mv anti-Leishmania.
flavonoides;
inibidores de proteases;
serino protease.
serino protease.
topic anti-Leishmania.
flavonoides;
inibidores de proteases;
serino protease.
serino protease.
anti-leishmania;
flavonoids;
protease inhibitors;
Doenças Infecciosas e Parasitárias
Ciências da Saúde
dc.subject.eng.fl_str_mv anti-leishmania;
flavonoids;
protease inhibitors;
dc.subject.cnpq.fl_str_mv Doenças Infecciosas e Parasitárias
Ciências da Saúde
description Introduction: Leishmaniasis is part of the group of neglected diseases, caused by protozoa of the genus Leishmania, being endemic in 98 countries. The therapy used for this disease is associated with high toxicity, difficult administration, high costs and parasitic resistance. Therefore, the search for new drug alternatives, more effective and less toxic is necessary and protease inhibitors are crucial molecules, since Leishmania proteases are involved in several physiological and pathological mechanisms. Aim: To identify the chemical constituents of the Bauhinia forficata Link extract, as well as the cytotoxic effect of the extract and the aspartic protease inhibitor fraction on Leishmania (L.) amazonensis. Materials and methods: firstly, the preparation of the extract was carried out, followed by the dosage of proteins, through the Bradford method, and then these proteins were identified through the electrophoresis method. Another method performed was the study of the chemical constituents present in the extract, in which the Matos phytochemical screening test was used, followed by thin layer chromatography, and the flavonoid contents were determined by high performance liquid chromatography. After chemical characterization, in vitro cytotoxicity tests were performed using sheep erythrocytes and immortalized cells of the RAW lineage, and in vivo toxicity tests in Tenebrio molitor larvae. As for the leishmanicidal activity in Leishmania amazonensis promastigotes, the assay was performed using the MTT method (3-(4,5-dimethyithiazol2-yl) -2,5- diphenyltetrazolium bromide), and the antiamastigote activity was performed with RAW cells. Results and discussion: Derivatives of quercetin and kaempferol were the main metabolites found in BF-CA. After isolation of the inhibitor of aspartic profease (BFI), 130.6 ± 29.94 μg of protein was obtained, with a yield of approximately 4.41 ± 0.77 %, that is, 4.41% of the proteins of BF-CA. The extract and BFI did not show cytotoxicity to sheep erythrocytes. In RAW cells, the extract and the inhibitor tested (BF-CA and BFI) showed a cytotoxic concentration for 50% of the cells (CC50) of 166.5 μg/mL and 371.3 μg/mL, respectively, and for pentamidine with CC50 of 15.24 μg/ml. By analyzing the inhibitory concentration in 50% (IC50) of the extracts on the promastigote forms of Leishmania amazonensis, we obtained an IC50 of 54.9 μg/mL for BF-CA and 8.33 μg/mL for BFI, which exhibited toxicity at almost all concentrations tested. When evaluating the inhibition of proteases, BFI inhibited 44.66% of the activity of commercial pepsin protease and inhibited 40% of the serine protease secreted by L. amazonensis, LSP III. Conclusion: Therefore, BF-CA has a composition rich in flavonoids, with proven leishmanicidal action in promastigote and amastigote forms, evidenced by the reduction of infection. As for BFI, it inhibited the activity of the serine protease secreted by L. amazonensis (LSPIII), making it a potential therapeutic target, as it is capable of interfering in the pathophysiology of leishmaniasis
publishDate 2021
dc.date.issued.fl_str_mv 2021-10-13
dc.date.accessioned.fl_str_mv 2022-06-24T17:30:56Z
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dc.identifier.citation.fl_str_mv FIGUEREDO, Aline Santana. Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link. 2021. 105 f. Dissertação (Programa de Pós-Graduação em Saúde do Adulto) - Universidade Federal do Maranhão, São Luís, 2021.
dc.identifier.uri.fl_str_mv https://tedebc.ufma.br/jspui/handle/tede/tede/3760
identifier_str_mv FIGUEREDO, Aline Santana. Prospecção da ação leishmanicida de inibidores de proteases extraídas de Bauhinia forficata Link. 2021. 105 f. Dissertação (Programa de Pós-Graduação em Saúde do Adulto) - Universidade Federal do Maranhão, São Luís, 2021.
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