?rea de concentra??o: Farmacologia de produtos naturais e plantas medicinais. / Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-10-23T16:44:04Z No. of bitstreams: 2 valeria_gomes_almeida.pdf: 1888901 bytes, checksum: 721c755514ecbe7426926c79618f632a (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-10-23T16:45:38Z (GMT) No. of bitstreams: 2 valeria_gomes_almeida.pdf: 1888901 bytes, checksum: 721c755514ecbe7426926c79618f632a (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Made available in DSpace on 2015-10-23T16:45:38Z (GMT). No. of bitstreams: 2 valeria_gomes_almeida.pdf: 1888901 bytes, checksum: 721c755514ecbe7426926c79618f632a (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Previous issue date: 2012 / A Pseudobrickellia brasiliensis (Asteraceae), conhecida como arnica-do-campo, ? uma planta nativa da flora brasileira cuja prepara??o alco?lica tem sido utilizada como anti-inflamat?rio pela medicina popular. No entanto, os mecanismos biol?gicos envolvidos nessa atividade, ainda n?o foram devidamente elucidados. Nesse contexto, o presente estudo avaliou o potencial anti-inflamat?rio de P. brasiliensis atrav?s da an?lise da produ??o de citocinas anti e pr?-inflamat?rias por leuc?citos mononucleares do sangue perif?rico. Foram utilizados extratos de P. brasiliensis ap?s extra??o com os solventes Acetato de Etila (ACE) e Etanol (ETA), ambos dilu?dos em Dimetilsulf?xido (DMSO), bem como o extrato aquoso dilu?do em PBS (AQU). A citotoxicidade dos extratos, ap?s 24h ou 5 dias de cultura celular, foi avaliada por meio da t?cnica de exclus?o com Azul de Tripan e, tais resultados foram confirmados por meio de ensaios de apoptose e necrose, ap?s marca??o com Anexina V-FITC e Iodeto de Prop?deo. No ensaio de inibi??o da produ??o de citocinas, C?lulas Mononucleares do Sangue Perif?rico humano (PBMC) foram incubadas em meio de cultura, contendo Miristato-Acetato de Forbol (PMA) e Ionomicina como agentes estimuladores, na aus?ncia ou presen?a do extrato AQU nas concentra??es finais de 0,025 mg/mL, 0,05 mg/mL ou 0,1 mg/mL. Tamb?m, foi avaliado o efeito do extrato AQU sobre a resposta proliferativa de linf?citos ao est?mulo mitog?nico Fitohemaglutinina (PHA) como teste complementar na an?lise de ativa??o celular. De acordo com os dados obtidos, os extratos ACE e ETA apresentaram um alto grau de citotoxicidade, sendo, portanto exclu?dos das an?lises posteriores, que avaliaram o perfil de produ??o de citocinas e prolifera??o. O extrato AQU, al?m de n?o induzir apoptose e necrose, n?o demonstrou a??o t?xica, mesmo ap?s 5 dias de cultura. Nas culturas tratadas com AQU na concentra??o final de 0,1 mg/mL foi observada redu??o significativa da express?o de IFN-? e TNF-? em resposta ? estimula??o com PMA e ionomicina. O efeito inibit?rio do extrato AQU, tamb?m, pode ser observado por meio da diminui??o da resposta proliferativa de linf?citos ao PHA. Estes achados sugerem que o efeito anti-inflamat?rio atribu?do ? P.brasiliensis na medicina popular pode, pelo menos em parte, ser devido ? inibi??o da produ??o de citocinas pr?-inflamat?rias por linf?citos, bem como da resposta proliferativa. Ensaios futuros, utilizando subst?ncias isoladas e identificadas a partir do extrato aquoso, s?o necess?rios para fortalecer os dados aqui apresentados e para demonstrar quais os mecanismos moleculares envolvidos nas a??es inibit?rias apresentadas no presente trabalho. / Disserta??o (Mestrado) ? Programa Multic?ntrico de P?s-gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, [2012]. / ABSTRACT Pseudobrickellia brasiliensis (Asteraceae), also known as ?Arnica-do-Campo? is a native plant from Brazilian flora and its alcoholic preparation has been used as anti-inflammatory in folk medicine. However, the biological mechanisms involved in the anti-inflammatory activity are not well elucidated. In this context, the present study evaluated the anti-inflammatory potential of P. brasiliensis by performing an anti- and pro-inflammatory cytokine analysis on human peripheral blood leucocytes as well as cell activation, measured by proliferation assay. We used P. brasiliensis extracts utilizing different solvents including Ethyl Acetate (ACE) and ethanol (ETA), both diluted in dimethylsulfoxide (DMSO), and aqueous extract (AQU) diluted in PBS. After 24h or 5 days, in vitro cytotoxicity promoted by those extracts was evaluated using the exclusion technique with Trypan Blue. Furthermore, apoptosis and necrosis was measured by Annexin V-FITC and propidium iodide staining, after 4 hours of culture. For cytokine production inhibitory assay, humans Peripheral Blood Mononuclear Cells (PBMC) were incubated in medium containing Phorbol Myristate Acetate (PMA) plus ionomycin as stimulating agents in the absence or presence of AQU extract at the final concentrations 0.025 mg/ml, 0.05 mg/ml or 0.1 mg/ml. We also assessed, as a complementary test for cellular activation analysis, the effect of AQU extract on lymphocytes proliferative response after mitogenic stimulation with Phytohemagglutinin (PHA). According to the obtained data, ACE and ETA showed a high cytotoxicity rate and, therefore, they were not used in cytokine and proliferative analysis. The AQU extracts, besides not inducing apoptosis and necrosis, showed no toxic effects even after 5 days of culture. In cultures treated with AQU extract at final concentration of 0.1 mg / ml was observed a significant reduction in IFN-? and TNF-? expression in response to PMA stimulation. The AQU extract inhibitory effect can also be observed by the decrease of lymphocytes proliferative rate in response to PHA stimulation. These findings suggest that P.brasiliensis anti-inflammatory effect can be, at least in part, due PBMC pro-inflammatory cytokines inhibition as well as the proliferative response. Further assays, using aqueous extract isolated and identified substances are needed to strengthen the presented data and to demonstrate the molecular mechanisms involved in inhibitory actions reported in this study.

?rea de concentra??o: Ci?ncias fisiol?gicas. / Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-01-04T17:58:44Z No. of bitstreams: 2 val?ria_gomes_lmeida.pdf: 4026192 bytes, checksum: b5681abc4a430487b1ac936cd81615c3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-01-04T17:59:53Z (GMT) No. of bitstreams: 2 val?ria_gomes_lmeida.pdf: 4026192 bytes, checksum: b5681abc4a430487b1ac936cd81615c3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-01-04T17:59:53Z (GMT). No. of bitstreams: 2 val?ria_gomes_lmeida.pdf: 4026192 bytes, checksum: b5681abc4a430487b1ac936cd81615c3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015 / A Pseudobrickellia brasiliensis (Spreng) R. M. King & H. Rob., (Asteraceae) ? nativa da flora brasileira, e sua prepara??o alco?lica tem sido utilizada de forma t?pica como anti-inflamat?rio na medicina popular. Tendo em vista a aus?ncia de estudos acerca da bioatividade e composi??o qu?mica dessa esp?cie, o objetivo do presente estudo foi avaliar o efeito de prepara??es de partes a?reas de P.brasiliensis sobre par?metros inflamat?rios, in vitro, e realizar a an?lise dos seus constituintes qu?micos. A triagem fitoqu?mica dos caules, flores e folhas da planta foi realizada por meio de rea??es cromog?nicas, fluorog?nicas e de precipita??o e sugeriu a presen?a de saponinas, terpenos, taninos e flavonoides nas partes a?reas da planta. Os extratos etan?licos de caules (PBETca), flores (PBETfl) e folhas (PBETfo) foram avaliados por cromatografia l?quida de alta efici?ncia acoplada a detector de arranjo de diodos (CLAE-DAD). Essa an?lise sugeriu a presen?a de flavonoides nos extratos, especialmente das classes das flavonas e flavon?is. Os ?leos essenciais de flores (PBOEfl) e de folhas (PBOEfo) foram analisados por Cromatografia Gasosa acoplada a Espectr?metro de Massas (CG-EM), sendo poss?vel a identifica??o de 22 compostos no PBOEfl e 27 no PBOEfo, dentre os quais, o ?-pineno, limoneno, ?-pineno, sabineno, ?xido de cariofileno e E-cariofileno est?o presentes em maior valor percentual. Foram confeccionadas culturas de 4h de sangue total para avaliar o efeito hemol?tico dos extratos, determinado pela densidade ?ptica do sobrenadante em 540 nm. Houve indu??o de hem?lise em culturas tratadas com PBETca ou PBETfo em concentra??es superiores a 100 ?g/mL. O extrato PBETfl induziu hem?lise em culturas na concentra??o final de 200 ?g/mL. A toxicidade dos produtos naturais ?s C?lulas Mononucleares do Sangue Perif?rico (PBMC) humano ap?s 24 h e 5 dias, foi avaliada por citometria de fluxo, utilizando Azul de Tripan ou Iodeto de Prop?deo. O efeito dos produtos naturais sobre a produ??o de TNF-?, IFN-? e IL-2 foi determinado por ELISA do sobrenadante de culturas de 24 h, ap?s est?mulo com Miristato Acetato de Forbol (PMA) + Ionomicina. Todos os extratos e ?leos essenciais reduziram a produ??o de TNF-?. A s?ntese de IFN-? foi inibida pelo extrato PBETfo e pelo ?leo essencial PBOEfl. Todos os extratos etan?licos e o PBOEfl, inibiram a produ??o de IL-2. O PBETfo foi fracionado por meio de parti??o em solventes com polaridade crescente e todas as fra??es foram eficientes em inibir a s?ntese de TNF-?, por PBMC, ap?s 24 h de cultura. Somente a fra??o hex?nica reduziu a s?ntese de IFN-? e IL-2. Foram confeccionadas culturas de sangue total estimuladas com lipopolissacar?deo (LPS) para investiga??o do efeito dos ?leos essenciais sobre a express?o de COX-2, por citometria de fluxo. Os ?leos essenciais n?o interferiram na express?o de COX-2. Nossos achados sugerem que o efeito anti-inflamat?rio atribu?do ? P.brasiliensis na medicina popular pode, pelo menos em parte, ser devido ? inibi??o da produ??o de citocinas pr?-inflamat?rias por linf?citos e mon?citos humanos. ? poss?vel que flavonoides das subclasses das flavonas e flavon?is possam contribuir para a atividade biol?gica dos extratos etan?licos de P. brasiliensis. Muitos monoterpenos e sesquiterpenos presentes nos ?leos essenciais de flores e folhas de P. brasiliensis j? tiveram a atividade anti-inflamat?ria descrita em outros trabalhos. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2015. / ABSTRACT The Pseudobrickellia brasiliensis (Spreng) R. M. King & H. Rob., (Asteraceae) is native from Brazilian flora, and its alcoholic preparation has been used topically as anti-inflammatory in folk medicine. Considering the lack of studies about the bioactivity and chemical composition of this specie, the aim of this study was to evaluate the effect of P. brasiliensis aerial parts preparations on in vitro inflammatory parameters, and perform analysis of its chemical constituents. The plant stems, flowers and leaves phytochemical screening was carried out by chromogenic, fluorogenic and precipitation reactions and suggested the presence of saponins, terpenes, tannins and flavonoids in the aerial parts of the plant. The ethanolic extracts of stems (PBETca), flowers (PBETfl) and leaves (PBETfo) were evaluated by high performance liquid chromatography coupled to diode array detector (HPLC-DAD). These analyses suggested the presence of flavonoids in the extracts, especially the flavones and flavonols subclasses. The flowers (PBOEfl) and leaves (PBOEfo) essential oils were analyzed by gas chromatography coupled to mass spectrometry (GC-MS), and it was possible to identify 22 and 27 compounds in PBOEfl and PBOEfo, respectively. Among the compounds the ?-pinene, limonene, ?-pinene, sabinene, caryophyllene oxide and E-caryophyllene were present in high porcentage. A 4h culture of whole blood was prepared to evaluate the hemolytic effect of the extracts, determined by supernatant optical density at 540 nm. There were hemolysis induction in cultures treated with PBETca or PBETfo at concentrations exceeding 100 ?g/mL. The extract PBETfl induced hemolysis in cultures at a final concentration of 200 ?g/mL. The natural products toxicity to human Peripheral Blood Mononuclear Cells (PBMCs) after 24 h and 5 days were assessed by flow cytometry using trypan blue or propidium iodide. The effect of non-toxic concentrations of the natural products on TNF-?, IFN-? and IL-2 production was determined by ELISA of 24h supernatants culture after stimulation with phorbol myristate acetate (PMA) + Ionomycin. All extracts and essential oils reduced the TNF-? production. The IFN-? synthesis was inhibited by PETfo extract and PBOEfl essential oil.The PBETfo and PBOEfl inhibited the IFN-? synthesis. The PBOEfl and all ethanolic extracts inhibited IL-2 production. The PBETfo was fractionated by solvent partitioning of increasing polarity and all fractions were effective in inhibiting the TNF-? synthesis by PBMC after 24 h culture. Only the hexane fraction reduced the IFN-? and IL-2 synthesis. Whole blood cultures stimulated with lipopolysaccharide (LPS) were prepared to investigate the effect of essential oils on COX-2 expression by flow cytometry. Essential oils did not affect the expression of COX-2. Our findings suggest that the anti-inflammatory effect attributed to P.brasiliensis in folk medicine can, at least in part, be due to the inhibition of proinflammatory cytokines production by human lymphocytes and monocytes. It is possible that flavonoids of flavones and flavonols subclasses may contribute to the biological activity of ethanolic extracts of P. brasiliensis. Many monoterpenes and sesquiterpenes present in essential oils of P. brasiliensis flowers and leaves already had the anti-inflammatory activity described in other studies.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-19T13:03:29Z No. of bitstreams: 5 mercia.pdf: 7178804 bytes, checksum: 598f3cf54883c3dd3c56edfbb1797bca (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-20T10:47:16Z (GMT) No. of bitstreams: 5 mercia.pdf: 7178804 bytes, checksum: 598f3cf54883c3dd3c56edfbb1797bca (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) / Made available in DSpace on 2015-02-20T10:47:16Z (GMT). No. of bitstreams: 5 mercia.pdf: 7178804 bytes, checksum: 598f3cf54883c3dd3c56edfbb1797bca (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) Previous issue date: 2013-02-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes) / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / Pseudobrickellia brasiliensis (Spreng.) R.M. King & H. Rob (Asteraceae) ? uma esp?cie de Asteraceae conhecida popularmente como arnica-do-mato, arnica-do-campo ou simplesmente arnica. E utilizada na medicina popular contra machucados e dores no corpo, porem a literatura carece de informa??es cient?ficas, encontrando-se apenas um artigo sobre estudo fitoqu?mico. Com o objetivo de contribuir para o estudo de P. brasiliensis, esse trabalho relata o estudo fitoqu?mico e ensaios biol?gicos que investigaram a atividade antiinflamat?ria e antioxidante de extratos das folhas da planta. As folhas foram coletadas na cidade de Diamantina, no Campus JK da UFVJM. Uma parte das folhas ainda frescas foi utilizada para extra??o de ?leo essencial, e outra parte do material vegetal foi seco e submetido ? extra??o com solventes de diferentes polaridades ? hexano, acetato de etila, etanol e ?gua ? obtendo-se quatro extratos. Os constituintes do ?leo essencial foram identificados por CG-EM, encontrando-se 25 terpenos, sendo os majorit?rios os monoterpenos ?-tujeno (17,21%) e ?-pineno (32,61%). Os extratos foram submetidos a t?cnicas cromatogr?ficas cl?ssicas e suas fra??es foram analisadas por CG-EM, IV, RMN de 1H e 13C, identificando-se o diterpeno acido cauren?ico; os triterpenos ?-amirina, acetato de ?-amirina, ?-amirina, acetato de ?-amirina, lupeol, acetato de lupeol, pseudotaraxasterol, taraxasterol; e possivelmente uma lactona sesquiterp?nica. Em ensaios de triagem fitoqu?mica realizados com os extratos, foram detectadas as classes de metab?litos secund?rios: cumarinas, flavon?ides, taninos condens?veis, antocianinas, antraquinonas, saponinas, compostos redutores e triterpenos/ester?ides. A esp?cie apresentou um potencial antiinflamat?rio, uma vez que os extratos aquoso e etan?lico modularam a produ??o da citocina IFN-?, envolvida diretamente na inicializa??o e amplifica??o da resposta inflamat?ria. Os extratos apresentaram baixo potencial antioxidante, nas concentra??es avaliadas e nos ensaios de atividade de retirada de radical, e de poder de redu??o do ?on met?lico Fe3+, apesar dos extratos aquoso e etan?lico possu?rem compostos fen?licos. Sendo assim, o presente trabalho foi uma contribui??o para o estudo fitoqu?mico e de atividades biol?gicas desta planta. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2012. / ABSTRACT Pseudobrickellia brasiliensis (Spreng.) R.M. King & H. Rob (Asteraceae) is a species of Asteraceae popularly known as arnica-do-mato, arnica-do-campo or arnica simply. It is used in folk medicine against wounds and body aches, but there lack of scientific literature and found only one article on phytochemical study. Aiming to contribute to the study of P. brasiliensis, this work reports the phytochemical and biological tests that investigated the anti-inflammatory and antioxidant activity of extracts of the leaves. The leaves were collected in the city of Diamantina, in the Campus JK of UFVJM. Part of the leaves still fresh, were used for extraction of essential oil, and another part of the plant material was dried and subjected to extraction with solvents of different polarities - hexane, ethyl acetate, ethanol and water - resulting in four extracts. The constituents of the essential oil were identified by GC-MS and was found 25 terpenes, of which the major was the monoterpenes ?-thujene (17.21%) and ?-pinene (32.61%). The extracts were subjected to classical chromatographic techniques and their fractions were analyzed by GC-MS, IR, 1H and 13C NMR, identifying the diterpene kaurenoic acid, triterpenes ?-amyrin acetate, ?-amyrin, ?-amyrin, ?-amyrin acetate, lupeol acetate, lupeol, pseudotaraxasterol, taraxasterol, and possibly a sesquiterpene lactone. In tests conducted with phytochemical extracts were detected classes of secondary metabolites, coumarins, flavonoids, condensed tannins, anthocyanins, anthraquinones, saponins, reducing compounds and triterpenes/steroids. The species had an anti-inflammatory potential, because the aqueous and ethanol extracts modulate the production of the cytokine IFN-?, directly involved in the startup and amplification of the inflammatory response. The extracts showed lower antioxidant potential, and concentrations evaluated in the trials of withdrawal of radical activity, and power reduction of Fe3+ metal ion, despite the aqueous and ethanol extracts possess phenolic compounds. Thus, the present work was a contribution to the study phytochemical and biological activities of this plant.