Triagem fitoqu?mica e atividade antiproliferativa do extrato diclorometano-etan?lico de ra?zes de Eriosema crinitum (Kunth) G. Don (Leguminosae)

?rea de concentra??o: Farmacologia de produtos naturais e plantas medicinais.A Eriosema crinitum (Kunth) G. Don ? utilizada no Vale do Jequitinhonha como planta anti-inflamat?ria na forma de decoc??o de suas ra?zes. Tendo em vista a complexidade do processo inflamat?rio, o qual envolve a ativa??o de c?lulas do sistema imune e produ??o de diversos mediadores, o objetivo desse estudo foi realizar uma triagem fitoqu?mica e avaliar a poss?vel atividade anti-inflamat?ria, in vitro, do extrato diclorometano-etan?lico de ra?zes de E. crinitum, atrav?s da avalia??o do efeito do mesmo sobre a prolifera??o de linf?citos, a produ??o de citocinas e da ciclooxigenase 2 (COX-2). A triagem fitoqu?mica do extrato foi realizada por meio de rea??es cromog?nicas, fluorog?nicas e de precipita??o, seguidas da an?lise em cromatografia l?quida de alta efici?ncia acoplada a detector de arranjo de diodos (CLAE-DAD). A citotoxicidade do extrato sobre hem?cias, ap?s 4 horas de cultura, e sobre leuc?citos do sangue perif?rico humano, ap?s 24 horas ou 5 dias de cultura, foi avaliada por meio do teste de atividade hemol?tica e pelo m?todo de exclus?o com azul de Tripan, respectivamente. Na avalia??o do efeito do extrato sobre a prolifera??o de linf?citos, por citometria de fluxo, c?lulas mononucleares do sangue perif?rico humano (PBMC) foram incubadas, por 5 dias, em meio de cultura contendo o mit?geno Fitohemaglutinina (PHA), na presen?a ou aus?ncia do extrato nas concentra??es de 25 ?g/mL, 12,5 ?g/mL e 6,25 ?g/mL. Essas mesmas culturas foram realizadas no tempo de 36 horas para an?lise da citocina IL-2, por ELISA. Para an?lise da produ??o das citocinas TNF-? e IFN-?, culturas de sangue total foram estimuladas com Miristato Acetato de Forbol (PMA) e tratadas com o extrato nas concentra??es de 50 ?g/mL, 25 ?g/mL e 12,5 ?g/mL. Essas concentra??es foram utilizadas em culturas de sangue total estimuladas com lipopolissacar?deo (LPS) para investiga??o do efeito do extrato sobre a express?o de COX-2. A produ??o de citocinas e de COX-2 foi analisada por citometria de fluxo. Os resultados evidenciaram a presen?a de terpenos e das subclasses dos flavonoides: flavonas e flavon?is no extrato. O extrato, nas concentra??es iguais ou inferiores a 50 ?g/mL, n?o foi t?xico para culturas celulares de 24 horas e, para culturas de 5 dias, as concentra??es n?o t?xicas foram iguais ou inferiores a 25 ?g/mL. Quanto ? a??o anti-inflamat?ria, o extrato n?o inibiu a produ??o das citocinas TNF-? e IFN-? e tamb?m n?o foi capaz de reduzir a express?o de COX-2. No entanto, o extrato inibiu a prolifera??o de linf?citos e suas subpopula??es T CD4 e TCD8, tendo uma efic?cia em rela??o ? Dexametasona de 71,65%, 53,14% e 167,94% para linf?citos totais, T CD4 e T CD8, respectivamente. O extrato tamb?m reduziu os n?veis de IL-2 nas culturas estimuladas com PHA. Esses achados sugerem que o extrato apresenta um efeito inibidor, principalmente sobre a prolifera??o de c?lulas T CD8, associado ? inibi??o na produ??o de IL-2. Diante dos resultados apresentados, os princ?pios ativos presentes na planta parecem exercer suas fun??es anti-inflamat?rias regulando a prolifera??o de linf?citos T, principalmente os linf?citos T CD8. Essa fun??o poderia ser explorada em desordens de natureza inflamat?ria-linfoproliferativa, bem como preven??o da rejei??o de transplantes de ?rg?os. Para tal, ensaios adicionais s?o necess?rios para investigar e identificar os constituintes ativos e os mecanismos moleculares envolvidos na a??o inibit?ria apresentada pelo extrato.Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014.ABSTRACT The Eriosema crinitum (Kunth) G. Don is used in Jequitinhonha Valley as anti-inflammatory plant in the form of decoction of its roots. Given the complexity of the inflammatory process, which involves the activation of immune cells and production of several mediators, the aim of this study was to perform a phytochemical screening and evaluate the potential anti-inflammatory activity, in vitro, the dichloromethane-ethanol extract of the roots of E. crinitum by assessing the effect of the same on lymphocyte proliferation, production of cytokines and cyclooxygenase-2 (COX-2). The phytochemical screening of the extract was performed using chromogenic reactions, fluorogenic and precipitation followed by analysis in high efficiency liquid chromatography coupled with a diode array detector (HPLC-DAD). The cytotoxicity of the extract after 24 hours or 5 days of culture on erythrocytes and leukocytes in human peripheral blood was evaluated by the test of hemolytic activity and the method of exclusion with Trypan blue, respectively. In evaluating of the effect of the extract on the proliferation of lymphocytes by flow cytometry, mononuclear cells from human peripheral blood (PBMC) have been incubated for 5 days in a culture medium containing the mitogen Phytohemagglutinin (PHA) in the presence or absence of extract at concentrations of 25 ?g/mL, 12.5 ?g/mL and 6.25 ?g/mL. These cultures too were performed in the time of 36 hours for analysis of IL -2 by ELISA. To analyze the production of cytokines TNF-? and IFN-?, whole blood cultures were stimulated with Phorbol Myristate acetate (PMA) and treated with the extract at concentrations of 50 ?g/mL, 25 ?g/mL and 12.5 ?g/mL. These concentrations were used in whole blood cultures stimulated with lipopolysaccharide (LPS) to investigate the effect of the extract on the expression of COX-2. Cytokine production and COX-2 was analyzed by flow cytometry. The results showed the presence of terpenes and subclasses of flavonoids: flavones and flavonols in extract. The extract, in concentration equal or greater than 50 ?g/mL was not toxic to cell cultures after 24 hours and in culture after 5 days, non- toxic concentrations were equal to or greater than 25 ?g/mL. In relation to the anti-inflammatory action, the extract did not inhibit the production of TNF-? and IFN-? and also was not able to reduce the expression of COX-2. Moreover, the extract inhibited the proliferation of lymphocytes and their subpopulations CD4 and CD8, and its efficacy compared to dexamethasone was 71.65%, 167.94% and 53.14% for total lymphocytes, CD4 and CD8, respectively. The extract also reduced the levels of IL-2 in cultures stimulated with PHA. These findings suggest that the extract has an inhibitory effect, especially on the proliferation of CD8 T cells associated with the inhibition of IL-2. Considering the presented results, the active molecules present in the plant seem to play their anti-inflammatory functions by regulating the T lymphocytes proliferation,?mainly T CD8 lymphocytes. This function could be exploited in inflammatory lymphoproliferative disorders, as well as in the prevention of transplant rejection. Further trials are needed to investigate the active constituents and the molecular mechanisms involved in the inhibitory action displayed by the extract