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Caracterização De Cogumelos Silvestres Da Espécie Amanita Ponderosa

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Caracterização De Cogumelos Silvestres Da Espécie Amanita Ponderosa Caracterização de cogumelos silvestres da espécie Amanita ponderosa Produção de metabolitos com atividade biológica Cátia Sofia Clemente Salvador Tese apresentada à Universidade de Évora para obtenção do Grau de Doutor em Bioquímica ORIENTADORAS: Prof. Doutora Ana Teresa Fialho Caeiro Caldeira Prof. Doutora Maria do Rosário Caeiro Martins Doutora Maria de Fátima Pereira Duarte ÉVORA, DEZEMBRO 2014 INSTITUTO DE INVESTIGAÇÃO E FORMAÇÃO AVANÇADA Esta Tese teve o apoio financeiro da Fundação para a Ciência e a Tecnologia (FCT) (Bolsa de Doutoramento, SFRH/BD/61184/2009), através do programa QREN-POPH-tipologia 4.1., coparticipado pelo Fundo Social Europeu (FSE) e Fundo Nacional MCTES. “ A alegria de ver e compreender é a mais bela dádiva da natureza” Albert Einstein Agradecimentos À minha orientadora Professora Ana Teresa Caldeira e coorientadora Professora Maria do Rosário Martins apresento um profundo agradecimento pelo suporte prestado nesta longa etapa. Agradeço imenso pela permanente orientação, por todo o auxílio, dedicação e incansável disponibilidade durante a realização deste trabalho. Agradeço também a amizade, apoio e incentivo que me permitiu concluir este projeto. À minha coorientadora Doutora Maria de Fátima Duarte, do centro de investigação CEBAL, agradeço pela forma como me acolheu na sua equipa de investigação, pela disponibilidade e interesse que sempre mostrou e principalmente pela orientação numa relevante parte do meu trabalho que foram os ensaios em cultura de células. Ao Professor José Maria dos Santos Arteiro agradeço por todos os conhecimentos transmitidos desde que iniciei o trabalho no Laboratório de Biotecnologia e pelas importantes palavras de incentivo e encorajamento para continuar a apostar na investigação científica para a minha formação profissional. Ao Professor Júlio da Cruz Morais agradeço pelo apoio, incentivo e auxilio prestado durante a fase inicial do meu doutoramento. Diriijo também os meus agradecimentos aos Professores do Departamento de Química que contribuíram de alguma forma para o desenvolvimento do meu trabalho. Ao Professor Henrique Vicente agradeço pela transmissão de conhecimentos e importante colaboração na valorização de parte da minha investigação. À Professora Dora Teixeira e ao Professor António Pereira agradeço pela ajuda na realização de alguns ensaios experimentais e análises químicas de compostos, Agradeço ainda ao Professor António Candeias pela cedência de verbas que possibilitaram a divulgação do meu trabalho em congressos científicos. A todos agradeço pela disponibilização de equipamentos e espaços laboratoriais fundamentais ao desenvolvimento do meu projeto de Doutoramento. Agradeço também às Professoras Célia Antunes e Ana V Rodrigues Costa e ao Professor Luís Martins por me terem acolhido e integrado nos projetos em curso durante a fase inicial do doutoramento, tornando possível a aprendizagem de novas metodologias indispensáveis à concretização de algumas etapas do meu trabalho de investigação. Gostaria de agradecer ao grupo de investigação do Centro de Engenharia Química e Biotecnológica do Instituto Superior de Engenharia de Lisboa. Ao Professor Amin Karmali, gostaria de agradecer pelo interesse que manifestou pelo meu trabalho, incentivando à aplicação de metodologias imunológicas, o que permitiu enriquecer e valorizar a investigação em curso. Agradeço ainda a colaboração da Doutora Sónia Martins e da Eng. Magda Semedo, pela cedência de anticorpos que produziram no seu centro de investigação, assim como a partilha de metodologias nesta área. À Dr.ª Maria de Fátima Candeias quero agradecer pela importante colaboração na fase de realização dos ensaios toxicológicos em animais, pela sua constante disponibilidade, interesse e transmissão de conhecimentos nestas aplicações. Agradeço à Eng. Mónica Morais de Brito pela colaboração na obtenção das fotografias de microscópio, e à Eng. Maria do Céu Serralheiro pelo acompanhamento laboratorial durante a realização das análises inorgânicas. Um especial obrigado ao meu grupo de Laboratório: Doutora Marina González, Mestres Mara Silva e Sílvia Arantes, e Drs. Tânia Rosado, Ana Branco, Patrícia Nunes, Ana Fialho e Ricardo Vieira pela amizade e companheirismo Agradeço às técnicas de laboratório D. Maria Helena Carvalho, D. Anabela Cabeça e D. Esperança Alves por tornarem mais fácil a gestão de logística laboratorial. Aos meus pais Maria Vicência e José Salvador pelo incansável apoio, compreensão, e constantes palavras de incentivo. Foi esse suporte emocional que me permitiu chegar até aqui. Agradeço também ao Carlos Travanca pelo suporte e incentivo, que me fez continuar quando tudo parecia mais difícil. Obrigada a todos! VI Resumo Resumo Amanita ponderosa é uma espécie de cogumelos silvestres, comestível, característica de alguns microclimas mediterrânicos, existente na Península Ibérica. Neste estudo, avaliaram-se as propriedades biológicas e toxicológicas destes cogumelos e culturas, com vista a valorizar o seu potencial biotecnológico. Desenvolveu-se também uma metodologia de monitorização, utilizando técnicas de microanálise e imunológicas para screening e análise da especificidade de produção dos compostos bioativos. Os corpos de frutificação apresentaram um relevante conteúdo mineral e um perfil molecular correlacionável com o local de colheita. Tanto os cogumelos como as culturas e seus extratos revelaram baixa toxicidade, in vitro e in vivo, apresentaram propriedades antioxidantes, capacidade hepatoprotetora e efeito antiproliferativo em células MDA-MB-231. Estes resultados sugerem que A. ponderosa e/ou os seus extratos podem constituir uma importante fonte de compostos bioativos, com potencial valor nutracêutico e medicinal, podendo ser utilizados como suplementos alimentares, coadjuvantes no tratamento de doenças hepáticas e/ou tumorais. Palavras-Chave: Amanita ponderosa; cogumelos silvestres comestíveis, composição inorgânica, M13-PCR, complexos proteína-polissacárido, propriedades antioxidantes, efeito hepatoprotetor, atividade antitumoral. VII Abstract Characterisation of Amanita ponderosa wild mushrooms: production of metabolites with biological activity Abstract Amanita ponderosa is a species of wild edible mushrooms that grows in some Mediterranean microclimates in the Iberian Peninsula. In this study, we evaluated the biological and toxicological properties of these mushrooms and cultures, in order to enhance their biotechnological potential. A monitoring methodology was also developed using microanalysis and immunological techniques, for screening and specificity evaluation of bioactive compounds production. The fruiting bodies presented a relevant mineral content and a characteristic molecular profile correlated with the geographical location. Either mushrooms or cultures and extracts have shown low toxicity in vitro and in vivo, and presented antioxidant properties, hepatoprotective effect and MDA-MB-231 antiproliferative activity. These results suggest that A. ponderosa and their extracts may constitute an important source of bioactive compounds with antioxidant benefits, nutraceutical potential and medicinal value, that can be used as dietetic supplements and as co-adjuvant of liver and cancer disease treatments. Keywords: Amanita ponderosa; wild edible mushrooms, inorganic content, M13-PCR, protein-polysaccharide complexes, antioxidant properties, hepatoprotective effect, antitumoral activity. IX Índice Índice geral Agradecimentos V Resumo VII Abstract IX Índice geral XI Índice de figuras XIX Índice de tabelas XXIX Abreviaturas XXXIII Unidades de medida XXXVII Publicações relacionadas XXXIX Objetivos e metodologia do trabalho 1 Capítulo 1 – Cogumelos silvestres e seus metabolitos: propriedades biológicas 5 1.1 Introdução 7 1.2 Cogumelos: Algumas considerações 8 1.3 Género Amanita 10 1.3.1 Espécies venenosas do género Amanita 11 1.3.2 Espécies comestíveis do género Amanita 13 1.3.3 Amanita ponderosa 15 1.4 Cogumelos silvestres comestíveis 19 1.4.1 Valorização económica 20 1.4.2 Valor nutricional 21 1.4.3 Propriedades medicinais 24 1.5 Compostos biologicamente ativos 27 1.6 Atividades biológicas 32 1.6.1 Propriedades antioxidantes 32 1.6.2 Efeito hepatoprotetor 36 1.6.3 Atividade antitumoral 41 XI Capítulo 2 – Caracterização inorgânica, isolamento e análise molecular de estirpes de A. ponderosa 49 2.1 Introdução 51 2.2 Metodologia 55 2.2.1 Amostras 55 2.2.2 Determinação da composição mineral 57 2.2.2.1 Tratamento das amostras de corpos de frutificação 57 2.2.2.2 Tratamento das amostras de solo 58 2.2.2.3 Determinação do conteúdo mineral das amostras de corpos de frutificação e solo 59 2.2.3 Isolamento, identificação e cultura de culturas puras 60 2.2.4 Análise molecular das estirpes isoladas 61 2.2.4.1 Extração do DNA – Método das microesferas adaptado 62 2.2.4.2 Quantificação do DNA 63 2.2.4.3 Amplificação do DNA por M13-PCR 63 2.2.4.4 Amplificação de sequenciação da região ITS 65 2.2.5 Análise estatística 65 2.3 Resultados e discussão 67 2.3.1 Análise da composição mineral presente nos corpos de frutificação 67 2.3.2 Caracterização de culturas de A. ponderosa e estirpes fúngicas que vivem em associação com esta espécie 82 2.3.3 Perfil genético de cogumelos A. ponderosa e de estirpes fúngicas que vivem em associação no seu habitat natural 87 Capítulo 3 – Produção e atividade biológica de compostos bioativos produzidos em culturas de A. ponderosa 97 3.1 Introdução 99 3.2 Metodologia 102 3.2.1 Amostras 102 3.2.2 Culturas líquidas 102 XII Índice 3.2.2.1 Obtenção da biomassa 103 3.2.3 Extração dos
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