Evaluation of Humulus Lupulus L. Therapeutic Properties for the Treatment of Skin Diseases

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Evaluation of Humulus Lupulus L. Therapeutic Properties for the Treatment of Skin Diseases Evaluation of Humulus lupulus L. Therapeutic Properties for the Treatment of Skin Diseases Determination of Antimicrobial, Cytotoxic, Anti- inflammatory and Antioxidant Potential of H.lupulus Extracts Versão final após defesa Liliana Sofia Lima Guiomar Dissertação para obtenção do Grau de Mestre em Ciências Biomédicas (2º ciclo de estudos) Orientador (a): Profa. Doutora Ana Cristina Palmeira de Oliveira Co-orientador(a): Prof. Doutor José António Martinez Souto de Oliveira Co-orientador(a): Profa. Doutora Cátia Alexandra Vicente Vaz julho de 2020 Evaluation of Humulus lupulus L. Therapeutic Properties for the Treatment of Skin Diseases ii Evaluation of Humulus lupulus L. Therapeutic Properties for the Treatment of Skin Diseases Dedicatória Dedico este trabalho primeiramente a minha mãe, que muito lutou pela minha instrução académica, ao meu padrasto e aos meus avós maternos que sempre me apoiaram nesta longa trajetória, ao meu companheiro e a minha amada filha Luana que incondicionalmente transmitiram-me segurança e muita força para continuar lutando e de nunca desistir dos meus sonhos. A todos vocês meu profundo agradecimento. iii Evaluation of Humulus lupulus L. Therapeutic Properties for the Treatment of Skin Diseases iv Evaluation of Humulus lupulus L. Therapeutic Properties for the Treatment of Skin Diseases Agradecimentos É com enorme satisfação que expresso aqui os meus agradecimentos por todos aqueles que contribuíram para a realização desta dissertação. Em primeiro lugar quero agradecer à minha orientadora, Professora Doutora Ana Cristina Palmeira de Oliveira, pela oportunidade de integrar a equipa, pela paciência, disponibilidade, pela partilha de saber e pelas palavras de motivação e de incentivo que tanto contribuíram para o desenvolvimento deste trabalho. A minha Co-orientadora, Professora Doutora Cátia Alexandra Vicente Vaz, pelo conhecimento transmitido, pela disponibilidade, amizade, paciência, apoio, pela colaboração na resolução de dúvidas e problemas que foram surgindo ao longo do trabalho, pelas críticas construtivas e palavras de incentivo que fizeram com que fosse possível a realização deste trabalho. Ao meu Co-orientador, Professor Doutor José António Martinez Souto de Oliveira, pela boa disposição contagiante que nos inspira a todos, pela partilha de saber, pela disponibilidade, dedicação, paciência e sobretudo pelas palavras de incentivo e sugestões que tanto contribuíram para o meu aprimoramento ao longo deste percurso. Meus agradecimentos a Professora Doutora Joana Rita Gonçalves Rolo Mateus, pelo apoio, motivação, disponibilidade e colaboração no solucionar de dúvidas e problemas que foram surgindo ao longo deste trabalho. Agradeço a todos os meus colegas de laboratório de Microbiologia II, do Centro de Investigação em Ciências da Saúde (CICS-UBI), em especial à Ana Sofia Oliveira, à Paula Faria Gonçalves e ao Carlos Gaspar, pelos ensinamentos científicos, conselhos e sugestões transmitidos. Meus agradecimentos também se destinam à Professora Doutora Rita Palmeira de Oliveira e à Professora Doutora Sara Carina de Lima Correia, que mesmo não estando diretamente implicadas no desenvolvimento cientifico deste trabalho de dissertação muito me ajudaram pelos gestos de solidariedade, pela disponibilidade, apoio e pelos úteis conselhos que foram transmitindo-me ao longo desta trajetória. v Evaluation of Humulus lupulus L. Therapeutic Properties for the Treatment of Skin Diseases À Universidade da Beira Interior, e a todas as pessoas do Centro de Investigação em Ciências da Saúde (CICS-UBI), onde este projeto foi realizado, que de uma forma ou de outra contribuíram para a realização deste trabalho de dissertação. À minha querida mãe, Ana Lima, pelo apoio incondicional que sempre me transmitiu. Obrigada mãe, por sempre acreditar em mim e de nunca ter me deixado desistir nos momentos mais difíceis. Sem ti nada disto seria possível e as minhas palavras são insuficientes para expressar a minha profunda gratidão. À minha pequena e amada filha Luana cuja existência fez de mim uma pessoa melhor e faz nascer em mim uma vontade imensa de continuar lutando todos os dias. Agradeço com carinho aos meus avós maternos Hirondina Lima e Honorato da Luz, que mesmo estando longe sempre me apoiaram e cuidaram de mim, transmitindo-me boas energias para nunca eu desistir por mais obstáculos que eu possa encontrar no caminho. Ao meu padrasto José Guedes, por todo o apoio e pela contribuição na minha formação académica. A todos os meus familiares e amigos que me apoiaram nesta longa jornada, meu profundo e sincero agradecimento a todos vocês. Por último, este trabalho de dissertação enquadra-se no projeto INOVEP-Inovação com Extratos de Plantas (N°33815, Centro 2020), pelo que dirijo os meus agradecimentos pelo financiamento para o desenvolvimento desta dissertação. vi Evaluation of Humulus lupulus L. Therapeutic Properties for the Treatment of Skin Diseases Resumo A pele, é o maior órgão do corpo humano e é responsável por inúmeras funções vitais como barreira física protetora contra a invasão de microrganismos patogénicos, penetração de produtos químicos nocivos e de raios ultravioleta, mas também desempenha papéis importantes na homeostasia corporal e na elaboração de respostas imunes e sensitivas. A cooperação existente entre os vários elementos constitutivos da pele (queratinócitos, fibroblastos, células do sistema imunitário, fibras nervosas motoras e sensitivas, glândulas, vasos sanguíneos e linfáticos) e até mesmo com os microrganismos, da sua superfície é importante para manter o organismo saudável. Inúmeros distúrbios frequentes da pele como a dermatite atópica, a psoríase e a acne podem estar associadas a uma predisposição genética do indivíduo, mas também a um desequilíbrio da microflora da pele ou mesmo a uma ativação sem controlo de respostas do sistema imunitário inato e adaptativa devido muitas vezes a sobrexpressão de proteínas tais como a ciclooxigenase-2 (COX-2) responsáveis pela génese de processos inflamatórios e de stress oxidativo crónicos que levam à produção de moléculas como o óxido nítrico (NO) e espécies reativas de oxigénio (ROS) que quando em excesso se tornam tóxicos e deletérios para as células. O Humulus lupulus Linnaeus, pertencente a família dos Cannabaceae é popularmente utilizado na confeção da cerveja e na medicina tradicional pelas suas propriedades terapêuticas relaxantes, como, por exemplo, no tratamento de casos de insónia e ansiedade. A inflorescência do H.lupulus é a parte da planta mais utilizada por ser onde está localizada a glândula da lupulina, órgão constituído maioritariamente por resinas 15- 30% (resinas duras e macias), óleos essenciais, polifenóis, entre outros compostos minoritários responsáveis pelo carater medicinal da planta. Estes metabolitos secundários do H.lupulus são conhecidos por apresentarem um alto potencial anti-inflamatório, ansiolítico, antidepressivo, antioxidante e antimicrobiano. Na presente dissertação, foi estudada a capacidade de extratos aquosos da flor, do mix (mistura de caules, folhas e flores), do hidrolato da flor e do hidrolato do mix do H.lupulus na diminuição da multiplicação de estirpes bacterianas Gram-positivas (Staphylococcus aureus, Staphylococcus epidermidis e Cutibacterium acnes) e Gram-negativas (Escherichia coli e Pseudomonas aeruginosa) pelo método de microdiluição e na alteração da atividade metabólica de células de fibroblastos 3T3 e de macrófagos RAW provenientes da pele de ratinhos através do método colorimétrico, brometo de [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] (MTT). Foi também avaliado o impacto destas substâncias na produção de NO, através do método de Griess, produção de ROS vii Evaluation of Humulus lupulus L. Therapeutic Properties for the Treatment of Skin Diseases através de moléculas fluorescentes como o H2DCFDA, e na expressão da COX-2, agente interveniente nos processos de inflamação após ativação dos macrófagos pelo lipopolissacarideo (LPS), por Western blot. Os resultados obtidos demonstram que o hidrolato da flor do H.lupulus apresenta maiores capacidades antibacterianas, especialmente contra as bactérias Gram-positivas, forte capacidade antioxidante, porque diminui significativamente a produção de ROS, reduz a inflamação, diminuindo a produção de NO e a expressão da COX-2 pelos macrófagos ativados pelo LPS. Os restantes extratos apresentaram também efeito anti-inflamatório e antioxidante, mas menos evidente do que o hidrolato da flor. O hidrolato do mix e o extrato aquoso do mix demonstraram um baixo efeito antibacteriano para algumas das estirpes em estudo e não alteraram significativamente a atividade metabólica dos fibroblastos 3T3 e dos macrófagos RAW. Este estudo demonstrou que o H.lupulus, principalmente a inflorescência da planta (flores) possui elementos químicos com capacidades anti-inflamatórias, antioxidantes e antibacterianas, podendo ser bastante interessantes na conceção de produtos à base de lúpulo com o intuito terapêutico e de prevenção de doenças que possam surgir na pele respeitando ao mesmo tempo a sua constituição fisiológica. Contudo mais estudos são necessários de modo a se conhecer melhor o perfil dos diferentes compostos químicos do H.lupulus bem como de modo a se estabelecer concentrações não toxicas para as células e microrganismos que compõem a microbiota da pele. Palavras-chave Ciclooxigenase-2, Espécies reativas de oxigénio, Fibroblastos, Humulus lupulus L., Macrófagos, Metabolismo, Oxido nítrico. viii Evaluation of Humulus lupulus L. Therapeutic Properties for
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