Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-17T15:38:43Z No. of bitstreams: 2 bethania_alves_avelar_freitas.pdf: 67938565 bytes, checksum: 4f06416afedd4040fb61d7bad494c673 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-17T15:40:36Z (GMT) No. of bitstreams: 2 bethania_alves_avelar_freitas.pdf: 67938565 bytes, checksum: 4f06416afedd4040fb61d7bad494c673 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-17T15:41:09Z (GMT) No. of bitstreams: 2 bethania_alves_avelar_freitas.pdf: 67938565 bytes, checksum: 4f06416afedd4040fb61d7bad494c673 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Made available in DSpace on 2014-12-17T15:41:09Z (GMT). No. of bitstreams: 2 bethania_alves_avelar_freitas.pdf: 67938565 bytes, checksum: 4f06416afedd4040fb61d7bad494c673 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Previous issue date: 2013 / Ageratum fastigiatum ? uma planta utilizada na medicina popular como anti-inflamat?rio e analg?sico, no entanto, poucos estudos foram realizados a fim de detalhar os mecanismos envolvidos nessa atividade. O objetivo deste estudo foi avaliar a atividade anti-inflamat?ria in vitro do ?leo essencial e do extrato dicloromet?nico de A. fastigiatum. Pela t?cnica de exclus?o do azul de tripam por citometria de fluxo foram determinadas concentra??es n?o t?xicas das prepara??es de A. fastigiatum. As concentra??es n?o t?xicas do ?leo essencial foram 5x10-3 e 1x10-2 ?L/mL. Essas concentra??es foram utilizadas para a pesquisa do potencial anti-inflamat?rio do ?leo essencial, medido por meio da an?lise do perfil de citocinas pro (TNF- ? e IFN- ?) e anti-inflamat?rias (IL-10), em culturas de leuc?citos humanos estimulados e n?o estimulados com PMA (acetato de forbol miristato) . Os dados demonstraram que ambas as concentra??es inibiram o percentual de linf?citos-TNF+ nas culturas estimuladas com PMA. A an?lise cromatogr?fica em fase gasosa acoplada a espectrometria de massas (CG/EM) revelou como principais constituintes no ?leo essencial as subst?ncias ?-pineno (7,51%), limoneno (5,9%), ?xido de cariofileno (13,59%), 1,2 ep?xido humuleno (8,41%) e 1,6-humulanodien-3-ol (17,71%). O extrato dicloromet?nico de A. fastigiatum, na concentra??o 20 ?g/mL, n?o apresentou toxicidade aos leuc?citos do sangue perif?rico humano e reduziu o percentual de linf?citos-TNF-?+ e linf?citos-IFN-?+ nas culturas estimuladas com o PMA. Este extrato foi fracionado em coluna de Sephadex LH-20 (150 g). Na an?lise de citocinas, a fra??o 10 (AFDM 10), na concentra??o 10 ?g/mL, demonstrou efeito anti-inflamat?rio in vitro reduzindo a frequ?ncia de linf?citos-TNF-?+ e a prolifera??o de linf?citos estimulados com PHA (fitohemaglutinina). Sugere-se que parte da atividade anti-inflamat?ria de A. fastigiatum se d? pela inibi??o que os constituintes da planta promovem sobre a ativa??o de leuc?citos. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013. / ABSTRACT Ageratum fastigiatum is a plant used in folk medicine as topical anti-inflammatory and analgesic agent. However, few studies have been conducted with this plant in order to detail the molecular mechanisms involved in its biological activities. Therefore, main purpose of the present study was to investigate the anti-inflammatory activity of the essential oil and leaf rinse dichloromethane of A. fastigiatum extract. The cytotoxicity of the compounds was assessed by means of trypan blue exclusion. The essential oil at 5x10-3 e 1x10-2 ?L/ did not present cytotoxic activity. These two concentrations were used for investigating the anti-inflammatory potential of the essential oil, by analyzing the pro-inflammatory (TNF-? and IFN-?) and anti-inflammatory (IL-10) cytokine profiles on human leukocyte cultures stimulated and non-stimulated with PMA (phorbol myristate acetate). The data obtained by flow cytometry demonstrated that both concentrations decreased the percentage of lymphocytes ?TNF-? + on the cell cultures stimulated with PMA. In the non-stimulated cell cultures there were no changes in the percentage of IL-10+ cells. The gas phase chromatography coupled with mass spectrometry analysis (GC/MS) revealed that the main compounds present in oil samples were: ?-pinene (7.51%), limonene (5.9%), caryophyllene oxide (13.59%), 1,2 humulene epoxide (8.41%) and 1,6-humulanodien-3-ol (17.71%). The dichloromethane extract of A. fastigiatum, obtained from the washing leaves, at the concentration 20 ?g/mL, did not presented cytotoxicic action on the human peripheral blood leukocytes. Following the same protocol performed to cytokines analysis used before, we observed that the dichloromethane A. fastigiatum extract also decreased the percentage of lymphocytes-TNF-? + and IFN-?+ on the cell cultures stimulated with PMA. This extract after fractionated on Sephadex LH-20 . The dichloromethane A. fastigiatum fraction (AFDM 10) at 10 ?g/mL reducing the frequency of lymphocytes-TNF-?+. Furthermore AFDM 10 decreasing the proliferation of lymphocytes stimulated with PHA (phytohemagglutinin). It is concluded that part of the anti-inflammatory activity of A. fastigiatum is give by inhibiting the plant constituents on promoting the activation of leukocytes.

?rea de concentra??o: Ci?ncias Fisiol?gicas. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-01-09T16:47:50Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lucas_abreu_costa.pdf: 2675995 bytes, checksum: b6686da3b0509ff9d2b21c38e1fdc5ce (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-01-10T13:46:07Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lucas_abreu_costa.pdf: 2675995 bytes, checksum: b6686da3b0509ff9d2b21c38e1fdc5ce (MD5) / Made available in DSpace on 2017-01-10T13:46:07Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lucas_abreu_costa.pdf: 2675995 bytes, checksum: b6686da3b0509ff9d2b21c38e1fdc5ce (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A planta Ageratum fastigiatum (Gardn.) R. M. King et H. Rob. ? comumente utilizada na medicina popular da regi?o do Vale do Jequitinhonha como anti-inflamat?rio e analg?sico. Em virtude do papel do sistema imune e de fatores inflamat?rios sol?veis na etiologia e no mecanismo fisiopatol?gico de diversas doen?as inflamat?rias, este trabalho teve como objetivo investigar par?metros relacionados ? atividade anti-inflamat?ria do extrato etan?lico de A. fastigiatum e de uma fra??o derivada desse extrato. A identifica??o dos constituintes qu?micos da fra??o do extrato etan?lico de A. fastigiatum foi realizada por meio da Cromatografia L?quida de Alta Efici?ncia com Detectores de Arranjo de Diodo acoplada ? espectrometria de massas (CLAE-DAD-MS) seguida pela Espectrometria de Massas em Tandem com ioniza??o por electrospray (ESI-MS/MS). A t?cnica de exclus?o com azul de tripan foi utilizada para determina??o da viabilidade das culturas de c?lulas mononucleares do sangue perif?rico (PBMC) incubadas por 8, 24 e 120 horas com o extrato etan?lico, a fra??o e o solvente Dimetilsulf?xido (DMSO). Na avalia??o do perfil proliferativo de linf?citos pela t?cnica de citometria de fluxo, PBMC estimuladas com o mit?geno Fitohemaglutinina (PHA) foram incubados por 120 horas na aus?ncia ou presen?a de diferentes concentra??es n?o t?xicas dos componentes citados. Para an?lise da produ??o das citocinas pr?-inflamat?rias, PBMC tratadas ou n?o com diferentes concentra??es n?o t?xicas do extrato etan?lico de A. fastigiatum, da fra??o e do solvente DMSO foram incubadas por 8 horas e estimulada nas ?ltimas 4 horas com Miristato Acetato de Forbol (PMA), utilizando a t?cnica de citometria de fluxo, linf?citos totais, c?lulas CD4+ e CD8+ foram avaliados quanto ? produ??o de IFN-?, TNF-? e IL-2. De acordo com nossos resultados, a concentra??o de DMSO, para todas as culturas celulares tratadas com o extrato etan?lico ou sua fra??o, foi definida como sendo 1% v/v para avalia??o da citotoxicidade e an?lise da produ??o de citocinas em linf?citos e de 0.5% v/v no ensaio de prolifera??o celular. Na an?lise fitoqu?mica da fra??o do extrato etan?lico foram identificados dois flavon?is nunca antes descrito na constitui??o qu?mica da planta, a 7-metil-quercetina e a Rhamnocitrina, tais compostos n?o alteraram os par?metros anti-inflamat?rios avaliados neste estudo. O extrato etan?lico, por sua vez, nas concentra??es de 6,25 e 12,5 ?g/mL reduziu significativamente a prolifera??o de linf?citos, ao mesmo tempo, nas concentra??es de 50 e 75 ?g/mL inibiu a produ??o das citocinas TNF-? e IL-2 em c?lulas CD8+, na maior concentra??o inibiu ainda a produ??o de IL-2 na popula??o de linf?citos totais. Sugere-se que o extrato etan?lico de A. fastigiatum possui componentes ativos agindo sinergicamente ou de forma isolada que contribuem para atividade anti-inflamat?ria atribu?da ? planta em seu uso na medicina popular. / Disserta??o (Mestrado) ? Programa Multic?ntrico de P?s-gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / Ageratum fastigiatum (Gardner.) R. M. King et H. Rob. is a plant commonly used in the folk medicine of the Jequitinhonha Valley as anti-inflammatory and analgesic. Because of the role of the immune system and soluble inflammatory factors in the etiology and pathophysiological mechanism of various inflammatory diseases, this study aimed to investigate parameters related to the anti-inflammatory activity of the ethanol extract of A. fastigiatum and a fraction derived from this extract. The identification of the chemical constituents of the fraction of ethanol extract of A. fastigiatum was performed by High Performance Liquid Chromatography with Efficiency Diode Arrangement detectors coupled to mass spectrometry (HPLC-DAD-MS) followed by Mass Spectrometry in Tandem with electrospray ionization (ESI-MS/MS). The exclusion technique with Trypan blue was used to determine the viability of peripheral blood mononuclear cell (PBMC) cultures, previously incubated for 8, 24 and 120 hours with the ethanol extract, the fraction and the solvent dimethylsulfoxide (DMSO). In the evaluation of the proliferative profile of lymphocytes using the flow cytometry technique, PBMC stimulated with the mitogen Phytohemagglutinin (PHA) were incubated for 120 hours in the absence or presence of different non-toxic concentrations of the mentioned components. In order to analyze the production of pro-inflammatory cytokines, PBMC treated or not with different non-toxic concentrations of the ethanolic extract of A. fastigiatum, of the fraction, and of the solvent DMSO was incubated for 8 hours and stimulated with phorbol myristate acetate (PMA) in the last 4 hours. Subsequently, total lymphocytes, CD4+ and CD8+ cells were assessed for production of IFN-?, TNF-? and IL-2, using flow cytometry. According to our results, the concentration of DMSO, for all cell cultures treated with ethanolic extract and its fraction was defined as 1% v/v for evaluation of cytotoxicity and analysis of cytokine production by lymphocytes, and 0.5% v/v to be used in the cell proliferation assay. In the phytochemical analysis of the fraction of ethanol extract, were identified two flavonols never before described in the chemical constitution of the plant, 7-methyl-quercetin and Rhamnocitrina. Such compounds did not modified the anti-inflammatory parameters evaluated in this study. The ethanol extract, on the other hand, in concentrations of 6.25 and 12.5 ?g/ml significantly reduced the proliferation of lymphocytes, while at concentrations of 50 and 75 ug/mL, it inhibited the production of TNF-? and IL-2 in CD8+, in addition, at the highest concentration it inhibited IL-2 production by total lymphocytes population. It is suggested that the ethanol extract of A.fastigiatum has active components acting synergistically or separately, resulting in the anti-inflammatory activity attributed to the plant by the folk medicine.