Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal

Oral candidiasis is an important clinical manifestation in kidney transplant recipients. Candida spp. it has virulence factors that contribute to the infectious process, including the ability to adhere to epithelial cells and form biofilm on biotic and abiotic surfaces. Considering the limited arsen...

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Autor principal: Souza, Luanda Bárbara Ferreira Canário de
Outros Autores: Chaves, Guilherme Maranhão
Formato: doctoralThesis
Idioma:pt_BR
Publicado em: Universidade Federal do Rio Grande do Norte
Assuntos:
Candida spp.
Fatores de virulência
Eugenia uniflora
Mecanismo de ação
Sinergismo
Endereço do item:https://repositorio.ufrn.br/handle/123456789/45467
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id ri-123456789-45467
record_format dspace
institution Repositório Institucional
collection RI - UFRN
language pt_BR
topic Candida spp.
Fatores de virulência
Eugenia uniflora
Mecanismo de ação
Sinergismo
spellingShingle Candida spp.
Fatores de virulência
Eugenia uniflora
Mecanismo de ação
Sinergismo
Souza, Luanda Bárbara Ferreira Canário de
Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal
description Oral candidiasis is an important clinical manifestation in kidney transplant recipients. Candida spp. it has virulence factors that contribute to the infectious process, including the ability to adhere to epithelial cells and form biofilm on biotic and abiotic surfaces. Considering the limited arsenal of antifungals available on the market, such as adverse and resistant species surgeries, it becomes necessary to find new antifungals that reach alternative targets, or synergistically to antifungals and that have less toxicity. In this context, natural products have gained importance. The extract activated from the leaves of Eugenia uniflora [acetone: water (7: 3, v/v)] has demonstrated antifungal activity against Candida spp.. Therefore, this study aimed to evaluate the effect of E. uniflora extract on virulence factors in vitro and to characterize the antifungal action in Candida spp. from the oral cavity of kidney transplant recipients. The E. uniflora extract was characterized by high performance liquid chromatography. Strains of Candida spp. belonging to a microorganism bank were reactivated and used in the analysis. The determination of the minimum inhibitory concentration of the extract was carried out by the broth microdilution method. Toxicity tests against erythrocytes and human epithelial bucal cells (HEBC) were carried out by determining the hemolysis index and counting 150 HEBC in an optical microscope, respectively. For in vitro virulence assays, yeasts were grown in the presence and absence of 1000 µg/mL of the extract. Adherence was quantified using the number of Candida cells adhered to 150 CEBH determined by an optical microscope. The biofilm formation was evaluated using two methodologies: XTT (2,3-bis (2-methoxy-4-nitro5-sulfophenyl) -2H-tetrazolium-5-carboxanilide) and the violet crystal assay, being analyzed later by scanning electron microscopy. The cell surface hydrophobicity (CSH) was quantified with the microbial adhesion test to hydrocarbons. In silico analysis was performed to predict antifungal activity of the major compounds determined in the extract. MIC was determined when in the presence of exogenous ergosterol, as well as in an osmoprotected environment by sorbitol (0.8 M). The action of the extract on the cell wall was evaluated from the growth of the cells treated with E. uniflora extract in ASD plus calcofluor, Congo red and sodium dodecyl sulfate (SDS). The loss of membrane integrity was assessed using propidium iodide (PI) staining. The combined action of E. uniflora extract with Fluconazole, Micafungin and Gallic acid was determined by the checkerboard method and finally, the time-kill curve of Candida spp. treated with E. uniflora extract and in combination with others antifungals drugs. E. uniflora extract was able to inhibit the growth of Candida spp cells by 50% and it has two marjoritary compounds: myricitrin and gallic acid. It was shown to be noncytotoxic to erythrocytes and HEBC. Reduced adherence to HEBC and HSC for all Candida species was shown. There was also a statistically significant reduced ability to form biofilms using both methods of quantification. In silico analysis demonstrated that there are no molecular targets that correlate myricitrin to the antifungal action of E. uniflora extract, while gallic acid demonstrated a link to the CPY51 enzyme present in the fungal cell. The extract slightly increased MIC when the ergosterol binding assay was performed, discarding the direct binding of the extract with this molecule. In the assay in the presence of sorbitol, the MIC values for the extract were increased, suggesting that one of the possible mechanisms of action is to act on the fungal cell wall. The treatment of Candida cells with the extract conferred resistance to cell wall disturbers. There was a loss of fungal cell membrane integrity when cells treated with the extract were stained with PI. E. uniflora extract showed an additive synergistic action when combined with Fluconazole and Micafungin. The combination with gallic acid had an indeterminate effect (without interaction). The time-kill curve of the cells treated with the extract demonstrated a decrease in the number of CFU count in 48 hours and the combination of E. uniflora extract with fluconazole resulted in a significant drop in the CFU count. The E. uniflora extract may in some way be disturbing the cell membrane, having seen the loss of membrane integrity seen in the PI labeled assay. However, this process apparently does not occur through direct binding to ergosterol or only by inhibiting the enzyme CYP51 (14 α-demethylase), an action attributed to gallic acid in in silico analyzes. The extract interacts at the fungal cell wall level, having seen the increase in MIC in the sorbitol assay and the slight decrease in growth for some strains in the assay with cell wall disturbers. E. uniflora extract has shown promise as a great alternative therapy combined with fluconazole for the treatment of oral candidiasis. Further analysis should be carried out to confirm the evidence presented by the tests carried out regarding the mechanism of action of E. uniflora extract.
author2 Chaves, Guilherme Maranhão
author_facet Chaves, Guilherme Maranhão
Souza, Luanda Bárbara Ferreira Canário de
format doctoralThesis
author Souza, Luanda Bárbara Ferreira Canário de
author_sort Souza, Luanda Bárbara Ferreira Canário de
title Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal
title_short Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal
title_full Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal
title_fullStr Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal
title_full_unstemmed Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal
title_sort efeito do extrato de eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de candida spp. oriundos da cavidade oral de receptores de transplante renal
publisher Universidade Federal do Rio Grande do Norte
publishDate 2021
url https://repositorio.ufrn.br/handle/123456789/45467
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spelling ri-123456789-454672022-05-02T15:15:11Z Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal Souza, Luanda Bárbara Ferreira Canário de Chaves, Guilherme Maranhão http://lattes.cnpq.br/0567484662111463 http://lattes.cnpq.br/1463249528959656 Padovan, Ana Carolina Barbosa http://lattes.cnpq.br/7106567615656883 Santos, Elizabeth Cristina Gomes dos http://lattes.cnpq.br/6990029952181366 Ururahy, Marcela Abbott Galvão http://lattes.cnpq.br/8016222352823817 Langassner, Silvana Maria Zucolotto http://lattes.cnpq.br/7390416147619446 Candida spp. Fatores de virulência Eugenia uniflora Mecanismo de ação Sinergismo Oral candidiasis is an important clinical manifestation in kidney transplant recipients. Candida spp. it has virulence factors that contribute to the infectious process, including the ability to adhere to epithelial cells and form biofilm on biotic and abiotic surfaces. Considering the limited arsenal of antifungals available on the market, such as adverse and resistant species surgeries, it becomes necessary to find new antifungals that reach alternative targets, or synergistically to antifungals and that have less toxicity. In this context, natural products have gained importance. The extract activated from the leaves of Eugenia uniflora [acetone: water (7: 3, v/v)] has demonstrated antifungal activity against Candida spp.. Therefore, this study aimed to evaluate the effect of E. uniflora extract on virulence factors in vitro and to characterize the antifungal action in Candida spp. from the oral cavity of kidney transplant recipients. The E. uniflora extract was characterized by high performance liquid chromatography. Strains of Candida spp. belonging to a microorganism bank were reactivated and used in the analysis. The determination of the minimum inhibitory concentration of the extract was carried out by the broth microdilution method. Toxicity tests against erythrocytes and human epithelial bucal cells (HEBC) were carried out by determining the hemolysis index and counting 150 HEBC in an optical microscope, respectively. For in vitro virulence assays, yeasts were grown in the presence and absence of 1000 µg/mL of the extract. Adherence was quantified using the number of Candida cells adhered to 150 CEBH determined by an optical microscope. The biofilm formation was evaluated using two methodologies: XTT (2,3-bis (2-methoxy-4-nitro5-sulfophenyl) -2H-tetrazolium-5-carboxanilide) and the violet crystal assay, being analyzed later by scanning electron microscopy. The cell surface hydrophobicity (CSH) was quantified with the microbial adhesion test to hydrocarbons. In silico analysis was performed to predict antifungal activity of the major compounds determined in the extract. MIC was determined when in the presence of exogenous ergosterol, as well as in an osmoprotected environment by sorbitol (0.8 M). The action of the extract on the cell wall was evaluated from the growth of the cells treated with E. uniflora extract in ASD plus calcofluor, Congo red and sodium dodecyl sulfate (SDS). The loss of membrane integrity was assessed using propidium iodide (PI) staining. The combined action of E. uniflora extract with Fluconazole, Micafungin and Gallic acid was determined by the checkerboard method and finally, the time-kill curve of Candida spp. treated with E. uniflora extract and in combination with others antifungals drugs. E. uniflora extract was able to inhibit the growth of Candida spp cells by 50% and it has two marjoritary compounds: myricitrin and gallic acid. It was shown to be noncytotoxic to erythrocytes and HEBC. Reduced adherence to HEBC and HSC for all Candida species was shown. There was also a statistically significant reduced ability to form biofilms using both methods of quantification. In silico analysis demonstrated that there are no molecular targets that correlate myricitrin to the antifungal action of E. uniflora extract, while gallic acid demonstrated a link to the CPY51 enzyme present in the fungal cell. The extract slightly increased MIC when the ergosterol binding assay was performed, discarding the direct binding of the extract with this molecule. In the assay in the presence of sorbitol, the MIC values for the extract were increased, suggesting that one of the possible mechanisms of action is to act on the fungal cell wall. The treatment of Candida cells with the extract conferred resistance to cell wall disturbers. There was a loss of fungal cell membrane integrity when cells treated with the extract were stained with PI. E. uniflora extract showed an additive synergistic action when combined with Fluconazole and Micafungin. The combination with gallic acid had an indeterminate effect (without interaction). The time-kill curve of the cells treated with the extract demonstrated a decrease in the number of CFU count in 48 hours and the combination of E. uniflora extract with fluconazole resulted in a significant drop in the CFU count. The E. uniflora extract may in some way be disturbing the cell membrane, having seen the loss of membrane integrity seen in the PI labeled assay. However, this process apparently does not occur through direct binding to ergosterol or only by inhibiting the enzyme CYP51 (14 α-demethylase), an action attributed to gallic acid in in silico analyzes. The extract interacts at the fungal cell wall level, having seen the increase in MIC in the sorbitol assay and the slight decrease in growth for some strains in the assay with cell wall disturbers. E. uniflora extract has shown promise as a great alternative therapy combined with fluconazole for the treatment of oral candidiasis. Further analysis should be carried out to confirm the evidence presented by the tests carried out regarding the mechanism of action of E. uniflora extract. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq A candidíase oral é uma importante manifestação clínica em receptores de transplante renal. Candida spp. possuem fatores de virulência que contribuem para o processo infeccioso, incluindo a capacidade de aderir a células epiteliais e de formar de biofilme em superfícies bióticas e abióticas. Devido ao limitado arsenal de antifúngicos disponíveis no mercado, as reações adversas e o surgimento de espécies resistentes, torna-se necessário encontrar novos antifúngicos que atuem em alvos alternativos, ou de forma sinérgica aos atuais antifúngicos e que possuam menor toxicidade. Nesse contexto, os produtos naturais têm ganhado importância. O extrato obtido a partir das folhas de Eugenia uniflora [acetona: água (7:3, v/v)] tem demonstrado atividade antifúngica contra Candida spp.. Portanto, este estudo objetivou avaliar o efeito do extrato de E. uniflora sobre fatores de virulência in vitro e caracterizar a ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal. O extrato de E. uniflora foi caracterizado por cromatografia líquida de alta eficiência e as cepas de Candida spp. pertencentes a um banco de microrganimos foram reativadas e utilizadas nas análises. A determinação da concentração inibitória mínima do extrato foi realizada pelo método de microdiluição em caldo, assim como ensaios de toxicidade frente a eritrócitos e células epiteliais bucais humanas (CEBH) foram realizados a partir da determinação do índice de hemólise e da contagem de 150 CEBH em microscópio óptico, respectivamente. Para os ensaios de virulência in vitro, as leveduras foram cultivadas na presença e ausência de 1000 µg/mL do extrato. A adesão foi quantificada usando o número de células de Candida spp. aderidas a 150 CEBH determinadas por microscópio óptico. A formação de biofilme foi avaliada usando duas metodologias: XTT (2,3-bis (2-metoxi-4-nitro-5-sulfofenil) -2H-tetrazólio-5-carboxanilida) e o ensaio do cristal violeta, sendo analisado posteriormente por microscopia eletrônica de varredura. A hidrofobicidade da superfície celular (HSC) foi quantificada com o teste de adesão microbiana a hidrocarbonetos. Realizaou-se análise in silico para predição de atividade antifúngica dos compostos majortitarios determinados no extrato. Determinou-se a CIM quando na presença de ergosterol exógeno, bem como em ambiente osmoprotegido por sorbitol (0,8 M). A ação do extrato na parede celular foi avaliada a partir do crescimento das células tratadas com extratdo de E. uniflora em Ágar Sabouraud Dextrose (ASD) acrescido de calcofluor, vermelho congo e dodecil sulfato de sódio (SDS). Avaliou-se a perda de integridade da membrana utilizandose a marcação com o iodeto de propídio (IP). A ação combinada do extrato de E. uniflora com o Fluconazol, a Micafungina e o Ácido gálico foi determinada pelo método checkerboard e por fim foi determinada a curva de morte de Candida spp. tratadas com o extrato de E. uniflora e em combinação em função do tempo. O extrato de E. uniflora foi capaz de inibir em 50% o crescimento das células de Candida spp. e possui dois compostos majoritários: miricitrina e ácido gálico. Além disso, mostrou-se não citotóxico aos eritrócitos e CEBH. Reduziu a adesão as CEBH e HSC para as espécies de Candida analisadas, e observou-se uma capacidade reduzida estatisticamente significativa para formar biofilmes usando os dois métodos de quantificação. A análise in silico demonstrou que não há alvos moleculares que correlacionem a miricitrina à ação antifúngica do extrato de E. uniflora, já o ácido gálico demonstrou ligação a enzima CPY51 presente na célula fúngica. O extrato aumentou discretamente as CIM quando realizado o ensaio de ligação ao ergosterol. Porém, no ensaio na presença do sorbitol, os valores de CIM para o extrato foram aumentados, sugerindo-se que um dos possíveis mecanismos de ação seja atuar na parede celular fúngica. O tratamento das células de Candida com o extrato conferiu resistência frente aos perturbadores de parede celular, e houve perda de integridade da membrana celular fúngica quando as células tratadas com o extrato foram coradas com IP. O extrato de E. uniflora apresentou ação sinérgica aditiva quando combinado com o Fluconazol e a Micafungina. Entretanto, a combinação com o ácido gálico apresentou um efeito indeterminado (sem interação). A curva de morte das células tratadas com o extrato demonstrou uma diminuição na contagem do número de UFC em 48 horas e a combinação do extrato de E. uniflora com o fluconazol resultou em uma queda significativa na contagem de UFC. O extrato de E. uniflora pode de alguma forma estar perturbando a membrana celular, tendo visto a perda de integridade da membrana verificada no ensaio com marcação com IP. Entretanto, aparentemente esse processo não se dá, exclusivamente, através da ligação direta ao ergosterol, ou somente pela inibição da enzima CYP51 (14 α-demetilase), ação atribuída ao ácido gálico nas análises in silico. O extrato interage em nível de parede celular fúngica, haja visto o aumento da CIM no ensaio com o sorbitol e a discreta diminuição de crescimento para algumas cepas no ensaio com os perturbadores de parede celular. O extrato de E. uniflora mostrou-se promissor como uma plausível alternativa de terapia combinada com o fluconazol para o tratamento de candidíase oral. Entretanto, mais análises devem ser realizadas para confirmação das evidências apresentadas pelos ensaios realizados quanto ao mecanismo de ação do extrato de E. uniflora. 2021-12-17T19:01:15Z 2021-12-17T19:01:15Z 2020-06-10 doctoralThesis SOUZA, Luanda Bárbara Ferreira Canário de. Efeito do extrato de Eugenia uniflora sobre fatores de virulência e caracterização da ação antifúngica em isolados de Candida spp. oriundos da cavidade oral de receptores de transplante renal. 2020. 113f. Tese (Doutorado em Ciências Farmacêuticas) - Centro de Ciências da Saúde, Universidade Federal do Rio Grande do Norte, Natal, 2020. https://repositorio.ufrn.br/handle/123456789/45467 pt_BR Acesso Aberto application/pdf Universidade Federal do Rio Grande do Norte Brasil UFRN PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS

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