Segurança e biodisponibilidade de suplementos alimentares

Segurança e biodisponibilidade de suplementos alimentares Ivone Mariana da Costa Almeida Porto, 2014 Segurança e biodisponibilidade de suplementos alimentares Ivone Mariana da Costa Almeida Dissertação do 3º Ciclo de Estudos Conducente ao Grau de Doutoramento em Ciências Farmacêuticas - Especialidade de Nutrição e Química do Alimento Trabalho realizado sob a orientação da Professora Doutora Maria Beatriz Prior Pinto Oliveira Porto Fevereiro, 2014 iv © Autorizada a reprodução parcial desta dissertação (condicionada à autorização das editoras das revistas onde os artigos foram publicados) apenas para efeitos de investigação, mediante declaração escrita do interessado, que a tal se compromete. v O trabalho aqui apresentado foi apoiado pela Fundação para a Ciência e Tecnologia (FCT) através da bolsa n.º PEst-C/EQB/LA0006/2011 e de uma bolsa de doutoramento (SFRH/BD/66032/2009) financiadas pelo Programa Operacional Potencial Humano - Quadro de Referência Estratégico Nacional - Tipologia 4.1 - Formação Avançada (POPHQREN) subsidiado pelo Fundo Social Europeu (FSE) e fundos nacionais do Ministério da Ciência, Tecnologia e Ensino Superior (MCTES). vi Os estudos apresentados nesta tese foram realizados no Serviço de Bromatologia e Hidrologia, Departamento de Ciências Químicas, da Faculdade de Farmácia da Universidade do Porto, no Grupo de Reações e Análises Químicas (GRAQ) do Instituto Superior de Engenharia do Porto, e no Centro de Investigação de Montanha (CIMO), da Escola Superior Agrária do Instituto Politécnico de Bragança. vii AGRADECIMENTOS À Professora Doutora Maria Beatriz Oliveira, minha orientadora, por me ter proporcionado a possibilidade de realizar este trabalho, pela confiança, apoio e incentivo ao longo desta etapa, bem como a disponibilidade evidenciada. À Faculdade de Farmácia da Universidade do Porto por ter-me aceite como estudante de doutoramento e à Fundação para a Ciência e Tecnologia pela concessão da bolsa. À Professora Doutora Isabel C.F.R. Ferreira, pelo seu acolhimento no Centro de Investigação de Montanha (CIMO), bem como, pela sua disponibilidade e simpatia. Ao Doutor João Barreira pelo orientação e disponibilidade para partilhar os seus conhecimentos durante este período. À Professora Doutora Teresa Oliva-Teles, pela sua orientação, pelo apoio e pela disponibilidade demonstrada ao longo do meu trabalho no GRAQ, ISEP. À Professora Doutora Cristina Delerue-Matos, por me ter acolhido no GRAQ e pelas palavras de incentivo. Ao Professor Doutor Bruno Sarmento, pelos ensinamentos e apoio nos ensaios celulares. A todos os colaboradores, e especialmente, aos meus amigos do Laboratório de Bromatologia e Hidrologia da Faculdade de Farmácia da Universidade do Porto, pela boa disposição, apoio, amizade e pela ajuda, sempre presentes. Agradeço a todos os colaboradores do CIMO e do GRAQ, pelo bom acolhimento e por todo o apoio disponibilizado. A todos os que colaboraram no meu trabalho de doutoramento, especialmente ao João Barreira, ao Telmo Fernandes, à Rita Alves, e à Francisca Rodrigues. Aos meus amigos, que sem estarem diretamente ligados à ciência apoiaram-me e deramme alento e motivação nos problemas e ansiedades, ao longo destes quatro anos. Gostaria, principalmente, de agradecer a toda a minha família. Aos meus Pais, e aos meus irmãos, pelo apoio e incentivo constante para continuar a progredir na carreira académica, e investir num futuro melhor. viii RESUMO O consumo de suplementos alimentares está generalizado e em crescimento constante. Tem como função primordial a complementação, e não substituição, da dieta normal, garantindo a ingestão adequada de nutrientes. No entanto, estes produtos têm sido comercializados com outros objetivos, nomeadamente a prevenção da doença, a manutenção e melhoria da saúde, a melhoria do desempenho físico, ou a manutenção ou obtenção do peso corporal desejado. A regulamentação dos suplementos alimentares tem sido alvo de harmonização na União Europeia (UE) a fim de garantir a segurança dos consumidores e a livre circulação de produtos entre países membros. Os suplementos na UE são regulamentados por diretivas, as quais especificam os ingredientes passíveis de utilização na sua preparação. No entanto, estas listas positivas estão apenas definidas para vitaminas e minerais. Para além destes nutrientes, os suplementos alimentares podem ainda conter uma infinidade de outras substâncias com efeito nutricional ou fisiológico que não se encontram diretamente abrangidas pela diretiva, sendo regidas pela legislação nacional de cada Estado-Membro. Adicionalmente, os produtos alimentares não são sujeitos a um controlo rigoroso semelhante ao dos medicamentos, sendo da responsabilidade do operador económico garantir a conformidade com os requisitos da legislação vigente. A falta de conformidade com os valores rotulados tem sido reportada para vários produtos disponíveis no mercado. Torna-se, assim, necessário conhecer os produtos que se encontram no mercado e os seus ingredientes, garantir a segurança das dosagens recomendadas, e avaliar a real biodisponibilidade dos diversos compostos. O objetivo deste trabalho foi o de monitorizar os teores de compostos bioativos em determinados grupos de suplementos alimentares, procurando obter/melhorar a informação disponível. Esta informação poderá ser útil para o consumidor que assim pode ter uma noção dos riscos e benefícios do consumo de suplementos alimentares, mas também para os profissionais de saúde para que, com maior segurança, possam aconselhar a ingestão de suplementos alimentares. De entre os diferentes suplementos alimentares disponíveis no mercado, vários produtos (n=82) foram selecionados e analisados: suplementos à base de extratos vegetais, multivitaminas, minerais, ácidos gordos ómega-3 e fitoestrogénios (n=82). Foram avaliados vários compostos bioativos (compostos fenólicos totais, flavonoides ix totais, isoflavonas, ácido ascórbico, ácidos gordos ómega-3, vitamina E e selénio). Para tal, otimizaram-se e/ou aplicaram-se várias metodologias analíticas, nomeadamente técnicas espectrofotométricas, métodos cromatográficos acoplados a diferentes tipos de detetores (HPLC-DAD, HPLC-DAD-FL e GC-FID) e absorção atómica (HR-CS ETAAS). Foi ainda estudada a atividade anti-radicalar de alguns suplementos e a biodisponibilidade de isoflavonas em suplementos contendo fitoestrogénios utilizando a linha celular Caco-2. Os resultados mostraram que a rotulagem nem sempre descreve a composição do produto que contém. Como esperado, os resultados referentes à atividade antioxidante dos diferentes suplementos apresentaram uma grande variabilidade, o que reflete a sua diversificada composição e concentração. Em geral, os resultados obtidos confirmam o potencial antioxidante dos suplementos avaliados. Constatou-se que a associação de diferentes antioxidantes pode oferecer algumas vantagens adicionais. Relativamente aos suplementos alimentares à base de selénio, os resultados obtidos experimentalmente mostraram-se concordantes com os níveis médios reclamados nos rótulos dos produtos. Para a validação do método foi utilizado um material de referência certificado. Na análise dos suplementos alimentares enriquecidos com ácidos gordos ómega-3, verificaram-se diferenças significativas para os teores de óleo, EPA, DHA, ALA e vitamina E rotulados para a maioria das amostras estudadas. Finalmente, na análise de suplementos com fitoestrogénios na sua composição, verificaram-se diferenças significativas nas concentrações de isoflavonas determinadas e os teores rotulados. A biodisponibilidade das isoflavonas presentes nos extratos parece depender da concentração de isoflavonas presente e da matriz do produto. Palavras-chave: suplementos alimentares; controlo de qualidade; antioxidantes; selénio; ómega-3; isoflavonas. xvi ÍNDICE AGRADECIMENTOS ............................................................................................ vii RESUMO ..................................................................................................... viii ABSTRACT ........................................................................................................ x LISTA DE PUBLICAÇÕES .................................................................................... xii ÍNDICE ..................................................................................................... xvi ABREVIATURAS ............................................................................................... xviii 1. INTRODUÇÃO ..................................................................................................... 3 1.1 Suplementos Alimentares ............................................................................ 4 1.1.1 Enquadramento Legal e Definição de Suplemento Alimentar ............... 4 1.1.2 Uso de Suplementos Alimentares ......................................................... 5 1.1.2.1 Prevalência do uso de Suplementos Alimentares na população portuguesa ........ 6 1.2 Suplementos Alimentares em estudo na presente tese ............................... 7 1.2.1 Suplementos com ação Antioxidante .................................................... 7 1.2.1.1 Radicais livres ......................................................................................................... 7 1.2.1.2 Antioxidantes .......................................................................................................... 8 1.2.1.3 Suplementos com Selénio ...................................................................................... 9 1.2.2 Suplementos Alimentares com ácidos gordos ómega-3...................... 11 1.2.3 Suplementos Alimentares com Fitoestrogénios .................................. 13 1.3 OBJETIVOS E ORGANIZAÇÃO GERAL DA TESE ................................... 15 1.4 REFERÊNCIAS ......................................................................................... 19 2. RESULTADOS .................................................................................................. 29 2.1 – Suplementos Alimentares com atividade Antioxidante ............................ 31 Dietary antioxidant supplements: benefits of their combined use................... 33 Teas, dietary supplements and fruit juices: A comparative study regarding antioxidant activity and bioactive compounds .................................................. 49 Total selenium content of food supplements by microwave digestion and HRCS ETAAS. Label accuracy evaluation. .............................................................. 61 2.2 – Suplementos Alimentares com Ácidos Gordos Ómega-3 ........................ 75 Label compliance in omega-3 dietary supplements: oil, fatty acids and vitamin E contents analysis ............................................................................................. 77 2.3 – Suplementos Alimentares com Fitoestrogénios ............. 93 xvii Food supplements intended for menopause symptoms relief: isoflavones levels and bioavailability using Caco-2 cell model ............................................ 95 3. CONSIDERAÇÕES FINAIS ............................................................................. 115 3.1 Considerações Finais ............................................................................... 117 xviii ABREVIATURAS ADN ácido desoxirribonucleico AHA American Heart Association ALA ácido α-linolénico ASAE Autoridade de Segurança Alimentar e Económica DCV doença cardiovascular DAD deteção por díodos DAD/FLD detetores de díodos e fluorescência DHA ácido docosahexaenóico DPPH 1,1-diphenyl-2-picrylhydrazyl free radical CE Comunidade Europeia AESA/EFSA Autoridade Europeia para a Segurança Alimentar/ European Food Safety Authority EPA ácido eicosapentaenóico AG ácidos gordos FAME ésteres metílicos de ácidos gordos/ fatty acid methyl esters FD deteção de fluorescência FID deteção de ionização de chama/ flame ionization detection GC cromatografia gasosa HPLC cromatografia líquida de alta precisão/ high performance liquid chromatography HR-CS ETAAS espectrofotometria de absorção atómica de alta resolução de fonte contínua com atomização eletrotérmica LOD limite de deteção LOQ limite de quantificação xix MSPD dispersão da matriz em fase sólida/ matrix solid phase dispersion MUFA ácidos gordos monoinsaturados n-3 ómega-3 DDR dose diária recomendada ROS espécies reativas de oxigénio RNS espécies reativas de azoto PUFA ácidos gordos polinsaturados UE União Europeia UV / VIS ultravioleta-visível λ comprimento de onda 1. INTRODUÇÃO Capítulo 1 3 1. INTRODUÇÃO Um regime alimentar adequado, por definição, previne deficiências nutricionais, fornecendo nutrientes e energia suficientes para o crescimento e reprodução (1). A influência da dieta e do estado nutricional na etiologia de várias doenças crónicas está hoje bem estabelecida, não só a nível institucional, por organizações de saúde e entidades reguladoras, mas também por uma parcela crescente da população (2) que procura ter uma dieta mais saudável. Nas últimas décadas, as sociedades têm modificado os seus padrões de vida, e os consumidores estão cada vez mais interessados em assumir um papel ativo na sua saúde e bem-estar (3). A indústria alimentar e farmacêutica, atenta a estas tendências, vem desenvolvendo e lançando no mercado um número crescente de produtos com propriedades promotoras da saúde, incluindo alimentos funcionais, nutracêuticos e suplementos alimentares. A ingestão de suplementos alimentares visa complementar a dieta normal, garantindo a ingestão de nutrientes em quantidade e qualidade adequadas. No entanto, estes produtos têm sido utilizados com diferentes objetivos, nomeadamente a prevenção da doença, a manutenção e melhoria da saúde, a melhoria do desempenho físico, ou a obtenção e/ou a manutenção do peso corporal desejado. Cada vez mais pessoas recorrem a estes produtos em todo o mundo, tornando a avaliação da qualidade e segurança destes produtos uma questão premente para os governos e para a comunidade científica. A regulamentação dos suplementos alimentares tem sido alvo de harmonização na União Europeia (UE) a fim de garantir a segurança dos consumidores e a livre circulação de produtos entre países membros. Os suplementos na UE são regulamentados por diretivas e as suas respetivas alterações, as quais especificam os ingredientes passíveis de utilização na sua preparação. No entanto, estas listas positivas estão apenas definidas para vitaminas e minerais. Para além destes nutrientes, os suplementos alimentares podem ainda conter uma infinidade de outras substâncias com efeito nutricional ou fisiológico, incluindo aminoácidos, ácidos gordos essenciais, fibras, enzimas, várias plantas e extratos de plantas, probióticos e prebióticos, entre outros, que não se encontram regulamentados. Adicionalmente, estes produtos não são sujeitos a um controlo rigoroso, semelhante aos medicamentos, sendo da responsabilidade do operador económico garantir a conformidade com os requisitos da legislação vigente. Introdução 4 O número crescente e diversificado de suplementos alimentares disponíveis para consumo requer um controlo da qualidade destes produtos ao nível dos seus ingredientes e dosagens recomendadas, e da real biodisponibilidade dos diversos compostos presentes, por forma a demonstrar o seu efeito benéfico. 1.1 Suplementos Alimentares 1.1.1 Enquadramento Legal e Definição de Suplemento Alimentar Até 2002, os suplementos alimentares na UE estavam sujeitos às regulamentações nacionais, que variavam consideravelmente em toda a UE, criando obstáculos à sua livre comercialização entre os Estados-Membros. A 10 de junho de 2002, foi publicada a Diretiva nº 2002/46/CE do Parlamento Europeu e do Conselho, referente à regulamentação dos suplementos alimentares, e transposta em Portugal através do Decreto-Lei n º 136 /2003 de 28 de junho de 2003 (4, 5). Esta Diretiva tem como objetivo principal harmonizar a legislação relativa aos suplementos vitamínicos e minerais em toda a Europa, e garantir que os produtos disponibilizados no mercado são seguros, claramente rotulados, e não reivindicam propriedades medicinais, de forma a possibilitar uma escolha informada por parte dos consumidores. Os suplementos alimentares são definidos no Artigo 2.º da Diretiva como “géneros alimentícios que se destinam a complementar e ou suplementar o regime alimentar normal e que constituem fontes concentradas de determinadas substâncias nutrientes ou outras com efeito nutricional ou fisiológico, estremes ou combinadas, comercializadas em forma doseada, tais como cápsulas, pastilhas, comprimidos, pílulas e outras formas semelhantes, saquetas de pó, ampolas de líquido, frascos com conta-gotas e outras formas similares de líquidos ou pós que se destinam a ser tomados em unidades medidas de quantidade reduzida, onde “nutrientes” são as seguintes substâncias: i) vitaminas, ii) minerais”. Na sua composição, além de vitaminas e sais minerais, podem ainda conter outras substâncias com efeito nutricional ou fisiológico, nomeadamente: aminoácidos, ácidos gordos essenciais, fibras e várias plantas e extratos de ervas. Capítulo 1 5 A lista de vitaminas e sais minerais permitidos para utilização nos suplementos alimentares, assim como a forma química em que se podem encontrar, estão presentes no Anexo I e no Anexo II da Diretiva. Esta lista foi posteriormente alterada pela Diretiva 2006/37/CE da Comissão (transposta para Portugal pelo Decreto-Lei n.º 296/2007, de 22 de Agosto) (6), o Regulamento (CE) N.º 1170/2009, o Regulamento (UE) N.º 1161/2011 e o Regulamento (UE) N.º 119/2014 para incluir novas substâncias (7-9). A Diretiva contempla cinco aspetos principais relativos à regulamentação dos suplementos vitamínicos e minerais: especificar as vitaminas e minerais permitidos e as suas fontes químicas; fixar as quantidades máximas e mínimas de vitaminas e minerais permitidas em suplementos; definir quais as regras para a rotulagem; proibir os fabricantes/distribuidores de suplementos de fazer quaisquer alegações terapêuticas nos seus produtos; proibir os Estados-Membros de restringir o comércio de produtos quando estes se encontram conformes com a diretiva. Na UE, a Autoridade Europeia para a Segurança dos Alimentos (AESA) é o organismo responsável pela implementação e monitorização destas substâncias, enquanto em Portugal esta função cabe ao Gabinete de Planeamento e Políticas (GPP), sob a tutela do Ministério da Agricultura e do Mar (6, 10). Para colocar um suplemento alimentar no mercado, o operador económico tem primeiro de notificar o GPP, enviando o rótulo para aprovação. De acordo com os princípios consagrados no Regulamento (CE) N.º 178/2002 de 28 de Janeiro, é da responsabilidade do operador económico garantir a conformidade com os requisitos da legislação vigente (11). A Autoridade de Segurança Alimentar e Económica (ASAE) é a autoridade nacional competente para a fiscalização, avaliação e comunicação dos riscos na cadeia alimentar, que será sempre feita retrospetivamente (não se trata de uma condição prévia à sua comercialização). 1.1.2 Uso de Suplementos Alimentares O crescente interesse e uso de suplementos alimentares pela população em geral, tornou a indústria de suplementos num dos negócios mais lucrativos do mercado da saúde atualmente, tendo sido avaliado globalmente em cerca de 84 mil milhões de dólares em 2011 (12). Os diversos estudos realizados para avaliar os hábitos de consumo de suplementos alimentares em diferentes populações têm revelado algumas tendências, nomeadamente no que se refere a atitudes e estilo de vida, além de outros aspetos sociodemográficos Introdução 12 nomeadamente, preferências alimentares, preço, preocupações ambientais, razões éticas, e as dificuldades na preparação (74). Nestes casos, os suplementos alimentares podem oferecer uma alternativa eficaz para aumentar a ingestão de n-3 PUFA. Os estudos sugerem que os seres humanos evoluíram com uma dieta contendo uma proporção de ácidos gordos essenciais n-6 e n-3 de cerca de 1-2:1 (75). As rápidas mudanças na dieta ocorridas a partir do século XX resultaram num aumento acentuado do consumo de gorduras totais, saturadas e de ácidos gordos da série n-6 e, simultaneamente, numa redução da ingestão de ácidos gordos n-3. A ingestão de teores elevados de n-6 PUFA, característica da dieta ocidental, pode aumentar a síntese de eicosanoides pró-inflamatórios, dando origem a um estado fisiológico promotor de doenças cardiovasculares (DCV) e de cancro. Pelo contrário, um maior consumo de n-3 LCPUFA está associado a uma maior proteção contra a inflamação (75). Os papéis opostos de n-3 e n6 PUFAs na regulação do processo inflamatório sugerem a importância da relação n-6/n-3 no desenvolvimento e na gravidade das doenças de natureza inflamatória. Isto é de particular importância quando se considera a profusão de ácidos gordos n-6 na dieta atual e na falta de n-3 (75). No entanto, é necessária uma maior evidência experimental, a fim de se recomendar um rácio específico dos ácidos gordos n-6 e n-3 (71, 76). O mercado oferece uma enorme variedade de produtos contendo n-3 PUFA, obtidos de óleos de peixe, de krill, de algas e de linhaça, ou misturas de óleos. Os n-3 PUFA em suplementos alimentares podem apresentar-se em várias formas, incluindo cápsulas moles, óleos e emulsões, e estão disponíveis a partir de vários canais: supermercados, lojas de saúde, ervanárias, na internet, e farmácias. Os suplementos de n-3 PUFA são geralmente bem tolerados, tendo-se observado alguns efeitos secundários ligeiros. Os efeitos adversos mais comuns incluem a eructação e um gosto a peixe, desconforto gastrointestinal e náuseas, e intolerância às cápsulas (77, 78). De uma forma geral, os suplementos de óleo de peixe são considerados seguros e livres de teores detetáveis de mercúrio (79). Apresentam, em geral, níveis reduzidos de bifenilos policlorados (PCB) (80, 81). Assim, os consumidores preocupados com a possibilidade da presença de contaminantes ambientais no peixe, podem optar pela suplementação. Estes produtos alegam manter as articulações e músculos saudáveis, a saúde cardiovascular, o desenvolvimento e funcionamento do cérebro, entre outros efeitos benéficos. Capítulo 1 13 1.2.3 Suplementos Alimentares com Fitoestrogénios As isoflavonas são uma classe de fitoestrogénios, compostos derivados de plantas com atividade estrogénica. A sua analogia estrutural com 17-β-estradiol confere-lhes efeitos hormonais, incluindo a capacidade de se ligar a recetores de estrogénio e de modular processos dependentes de hormonas (82). As isoflavonas têm uma distribuição restrita na natureza, ocorrendo principalmente em plantas da família Fabaceae, incluindo a soja, lentilhas, grão de bico, alfafa, trevo vermelho, e kudzu (83). A soja e os produtos à base de soja são a única fonte conhecida de quantidades significativas de isoflavonas na dieta humana (83). Estudos epidemiológicos têm relacionado o consumo de dietas ricas em isoflavonas, com a menor incidência dos principais cancros hormono-dependentes (84, 85), de doenças cardiovasculares (86), osteoporose, e da sintomatologia associada com a menopausa (87). Atualmente estão disponíveis vários suplementos alimentares com isoflavonas na sua composição, visando particularmente as mulheres na menopausa. Estes produtos têm sido referenciados como uma alternativa terapêutica para a sintomatologia da pósmenopausa em algumas situações de contra-indicação para a terapêutica hormonal de substituição (THS), como eventuais riscos associados e efeitos adversos da THS (88, 89). Os suplementos alimentares utilizados na menopausa frequentemente contêm extratos de soja, trevo vermelho, e kudzu, estremes ou em formulações multi-ingredientes com minerais, vitaminas, outros extratos vegetais, ácidos gordos ómega-3, 6 e 9, entre outros. As três principais isoflavonas da soja (Glycine max (L.) Merril) são a daidzeína, a genisteína e a gliciteína, que ocorrem principalmente como glicosídeos, acetilglicosídeos, e malonilglicosídeos. Nos derivados de soja, incluindo os suplementos alimentares, os glicosídeos e as agliconas são os componentes principais (90). O trevo vermelho (Trifolium pratense L.) contém numerosas isoflavonas, sendo a biochanina A e a formononetina e seus derivados os principais isoflavonóides presentes (91, 92). Outras plantas usadas pelo seu efeito estrogénico incluem diversas espécies do género Pueraria spp., utilizadas tradicionalmente na medicina popular asiática para atenuar os sintomas da menopausa. Vários isoflavonóides foram identificados nos tubérculos destas plantas incluindo daidzina, puerarina, daidzeína, e genisteína (93). As isoflavonas, como a maioria dos polifenóis, encontram-se nas plantas, principalmente como glicosídeos e ésteres de glicosídeos (94). Após a ingestão estes são Introdução 14 metabolizados pela flora do trato gastrointestinal, libertando as suas agliconas, os constituintes verdadeiramente bioativos (95, 96). Apesar de alguns estudos terem indicado, em alguns casos, a existência de contraindicações ao uso de isoflavonas (97), estudos epidemiológicos não têm observado efeitos adversos associados ao seu consumo. No entanto, uma vez que a atividade estrogénica destas moléculas tem sido comparada ao fármaco tamoxifeno, deverá ter-se em conta que o seu uso também pode aumentar o risco de se desenvolver cancro do endométrio (98). Capítulo 1 15 1.3 OBJETIVOS E ORGANIZAÇÃO GERAL DA TESE Nas últimas três décadas, observou-se um aumento acentuado e generalizado no consumo de suplementos alimentares. A promoção do bem-estar e a prevenção de doenças crónicas, o fortalecimento do sistema imunitário e a melhoria do desempenho desportivo ou da imagem corporal, estão entre os motivos geralmente apontados para a suplementação. Em Portugal, o seu consumo é crescente, acompanhando as tendências verificadas em outros países. Estes produtos encontram-se disponíveis numa grande variedade de formulações e são vendidos através de múltiplos canais, muitas vezes sem o apoio de um profissional. Os suplementos alimentares são considerados pelos consumidores como "naturais" e "seguros", em contraste com os medicamentos convencionais. No entanto, são cada vez mais frequentes as situações em que são postas em causa a qualidade, a segurança e a eficácia destes produtos, surgindo na bibliografia a descrição de efeitos adversos e algumas não conformidades. A regulamentação dos suplementos alimentares tem sido alvo de harmonização na União Europeia (UE), especialmente para vitaminas e minerais. O fabricante é o único responsável pela segurança/qualidade dos seus produtos, o que pode facilitar a ocorrência de fraude, adulteração, contaminação e a não conformidade relativamente às quantidades rotuladas. Vários estudos têm realçado a escassez de informação nos rótulos sobre ingredientes, concentrações, doses ou efeitos das substâncias presentes, o que pode comprometer a sua eficácia terapêutica e a segurança dos consumidores. A análise dos componentes que constituem os suplementos alimentares é, assim, uma etapa essencial para assegurar a sua utilização segura pelos consumidores. Nesse sentido, este trabalho teve os seguintes objetivos gerais:  Monitorizar os compostos bioativos e fornecer informações sobre diferentes suplementos alimentares disponíveis no mercado Português.  Contribuir para o melhor conhecimento destes produtos, a fim de beneficiar da presença dos compostos químicos bioativos presentes e minimizar os potenciais efeitos adversos na saúde. Para atingir os objetivos gerais acima enunciados, desenvolveu-se um conjunto de tarefas laboratoriais procurando dar resposta aos seguintes objetivos específicos:  Avaliar suplementos alimentares antioxidantes nomeadamente, quantificar alguns compostos bioativos (compostos fenólicos totais, flavonoides totais, ácido ascórbico, selénio) e determinar a atividade anti-radicalar dos Introdução 16 extratos. Avaliar vantagens/desvantagens da toma associada de diferentes produtos, e a conformidade com a rotulagem.  Analisar o perfil de ácidos gordos de suplementos à base de óleo de peixe, óleo de krill e/ou óleos vegetais e verificar a conformidade em relação aos valores rotulados relativamente aos teores de óleo, dos ácidos gordos ómega-3, EPA, DHA, ALA e vitamina E.  Determinar o perfil de isoflavonas de suplementos à base de fitoestrogénios provenientes de diferentes fontes vegetais, nomeadamente, soja, trevovermelho e Thai-kudzu. Verificar a conformidade em relação aos valores rotulados. Determinar a biodisponibilidade das isoflavonas nos extratos de suplementos selecionados, usando a linha celular Caco-2. A presente tese é constituída por 3 capítulos, que tratam respetivamente a Introdução à temática dos suplementos alimentares, os resultados obtidos e as considerações finais. Por sua vez, o capítulo referente aos resultados é constituído por 3 subcapítulos (artigos científicos). A tese está escrita em português, seguindo a "Regra de Vancouver" para as referências. No Capítulo 1 apresenta-se uma panorâmica geral sobre os suplementos alimentares, relativamente ao enquadramento legal e ao mercado, focando em particular a realidade portuguesa. É feita uma reflexão sobre o uso de suplementos alimentares e a necessidade de controlo da qualidade desses produtos, focando especificamente os suplementos com efeito antioxidante, os suplementos à base de ácidos gordos ómega-3, e os suplementos à base de isoflavonas. É feita uma descrição dos compostos bioativos característicos de cada classe de suplementos analisados, e são apresentados alguns resultados descritos na literatura. Segue-se o Capítulo 2, a parte experimental da tese, que é constituída por 3 partes, correspondendo cada uma delas a um grupo de suplementos alimentares: suplementos alimentares com ação antioxidante (2.1), suplementos contendo ácidos gordos ómega 3 (2.2) e suplementos alimentares contendo fitoestrogénios (2.3). Optou-se por manter a formatação original, adaptada ao corpo da tese, com a qual os textos foram submetidos ou publicados, de acordo com as normas específicas de cada revista. A primeira parte refere-se à avaliação de suplementos antioxidantes. É constituída por 3 artigos relativos à determinação de compostos bioativos presentes neste tipo de suplementos e avaliada a atividade antioxidante dos seus extratos. Capítulo 1 17 A segunda parte é dedicada ao estudo de suplementos alimentares contendo ácidos gordos ómega-3. Deste trabalho resultou um artigo referente à determinação do perfil dos ácidos gordos das amostras, aos teores de compostos bioativos e à comparação dos resultados obtidos com a informação indicada no rótulo. A terceira parte descreve o trabalho realizado com suplementos alimentares à base de fitoestrogénios nomeadamente, a determinação dos compostos bioativos e a avaliação da biodisponibilidade intestinal das isoflavonas presentes nos extratos destes produtos. O último capítulo refere-se às “considerações finais”, onde é feita uma síntese dos principais resultados obtidos para cada classe de suplementos alimentares. Tece-se um conjunto de reflexões sobre o trabalho realizado, e apresentam-se algumas questões que poderão ser retomadas em futuras investigações realizadas neste domínio. Introdução 18 Capítulo 1 19 1.4 REFERÊNCIAS 1. Thomas B, Bishop J. Manual of Dietetic Practice. 4th ed: Wiley-Blackwell; 2007. 2. WHO/FAO. Diet, nutrition and the prevention of chronic diseases: report of a joint WHO/FAO Expert Consultation. Geneva, Switzerland: World Health Organization, 2003. 3. Conner M, Kirk SF, Cade JE, Barrett JH. Why do women use dietary supplements? The use of the theory of planned behaviour to explore beliefs about their use. Soc Sci Med. 2001;52(4):621-33. 4. DIRECTIVA 2002/46/CE DO PARLAMENTO EUROPEU E DO CONSELHO de 10 de Junho de 2002 relativa à aproximação das legislações dos Estados-Membros respeitantes aos suplementos alimentares, (2002). 5. Decreto-Lei n.o 136/2003 de 28 de Junho, (2003). 6. Decreto-Lei n.º 296/2007 de 22 de Agosto, (2007). 7. REGULAMENTO (CE) N.º 1170/2009 DA COMISSÃO de 30 de Novembro de 2009 que altera a Directiva 2002/46/CE do Parlamento Europeu e do Conselho e o Regulamento (CE) n. o 1925/2006 do Parlamento Europeu e do Conselho no que se refere às listas de vitaminas, minerais e respectivas formas em que podem ser adicionados aos alimentos, incluindo suplementos alimentares (2009). 8. REGULAMENTO (UE) N. o 1161/2011 DA COMISSÃO de 14 de Novembro de 2011 que altera a Directiva 2002/46/CE do Parlamento Europeu e do Conselho, o Regulamento (CE) n. o 1925/2006 do Parlamento Europeu e do Conselho e o Regulamento (CE) n. o 953/2009 da Comissão no que se refere às listas de substâncias minerais que podem ser adicionadas aos alimentos (2011). 9. REGULAMENTO (UE) N.o 119/2014 DA COMISSÃO de 7 de fevereiro de 2014 que altera a Diretiva 2002/46/CE do Parlamento Europeu e do Conselho e o Regulamento (CE) n. o 1925/2006 do Parlamento Europeu e do Conselho no que se refere à levedura enriquecida em crómio utilizada no fabrico de suplementos alimentares e ao lactato de crómio (III) tri-hidratado adicionado aos alimentos, (2014). 10. EFSA. Food supplements: European Food Safety Authority; 2013 [cited 2014 23/02/ 2014]. Available from: http://www.efsa.europa.eu/en/topics/topic/supplements.htm. 11. REGULAMENTO (CE) N.o 178/2002 DO PARLAMENTO EUROPEU E DO CONSELHO de 28 de Janeiro de 2002 que determina os princípios e normas gerais da Introdução 20 legislação alimentar, cria a Autoridade Europeia para a Segurança dos Alimentos e estabelece procedimentos em matéria de segurança dos géneros alimentícios, (2002). 12. Euromonitor International 2014 [26/02/2014]. Available from: http://www.euromonitor.com/. 13. Felício J. Estudo de Mercado – Consumo de Suplementos Alimentares em Portugal. Centro de Estudos de Gestão do Instituto Superior de Economia e Gestão, 2006. 14. Lee JS, Kim J. Factors affecting the use of dietary supplements by Korean adults: data from the Korean National Health and Nutrition Examination Survey III. J Am Diet Assoc. 2009;109(9):1599-605. 15. Marques-Vidal P, Pecoud A, Hayoz D, Paccaud F, Mooser V, Waeber G, et al. Prevalence and characteristics of vitamin or dietary supplement users in Lausanne, Switzerland: the CoLaus study. Eur J Clin Nutr. 2009;63(2):273-81. 16. Li K, Kaaks R, Linseisen J, Rohrmann S. Consistency of vitamin and/or mineral supplement use and demographic, lifestyle and health-status predictors: findings from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg cohort. The British journal of nutrition. 2010;104(7):1058-64. 17. Balluz LS, Okoro CA, Bowman BA, Serdula MK, Mokdad AH. Vitamin or supplement use among adults, behavioral risk factor surveillance system, 13 states, 2001. Public Health Rep. 2005;120(2):117-23. 18. Raji MA, Kuo YF, Snih SA, Sharaf BM, Loera JA. Ethnic differences in herb and vitamin/mineral use in the elderly. Ann Pharmacother. 2005;39(6):1019-23. 19. Brustad M, Braaten T, Lund E. Predictors for cod-liver oil supplement use--the Norwegian Women and Cancer Study. Eur J Clin Nutr. 2004;58(1):128-36. 20. Ronsen O, Sundgot-Borgen J, Maehlum S. Supplement use and nutritional habits in Norwegian elite athletes. Scandinavian journal of medicine & science in sports. 1999;9(1):28-35. 21. Nieper A. Nutritional supplement practices in UK junior national track and field athletes. British Journal of Sports Medicine. 2005;39(9):645-9. 22. Touvier M, Kesse E, Volatier JL, Clavel-Chapelon F, Boutron-Ruault MC. Dietary and cancer-related behaviors of vitamin/mineral dietary supplement users in a large cohort of French women. Eur J Nutr. 2006;45(4):205-14. Capítulo 1 21 23. Reinert A, Rohrmann S, Becker N, Linseisen J. Lifestyle and diet in people using dietary supplements: a German cohort study. Eur J Nutr. 2007;46(3):165-73. 24. Rock CL, Newman VA, Neuhouser ML, Major J, Barnett MJ. Antioxidant supplement use in cancer survivors and the general population. The Journal of nutrition. 2004;134(11):3194s-5s. 25. Skeie G, Hjartaker A, Lund E. Diet among breast cancer survivors and healthy women. The Norwegian Women and Cancer Study. Eur J Clin Nutr. 2006;60(9):1046-54. 26. Blendon RJ, DesRoches CM, Benson JM, Brodie M, Altman DE. Americans' views on the use and regulation of dietary supplements. Arch Intern Med. 2001;161(6):805-10. 27. Klipstein-Grobusch K, Kroke A, Voss S, Boeing H. [Influence of lifestyle on the use of supplements in the Brandenburg nutrition and cancer study]. Z Ernahrungswiss. 1998;37(1):38-46. 28. Knudsen VK, Rasmussen LB, Haraldsdottir J, Ovesen L, Bulow I, Knudsen N, et al. Use of dietary supplements in Denmark is associated with health and former smoking. Public health nutrition. 2002;5(3):463-8. 29. Ford ES. Vitamin Supplement Use and Diabetes Mellitus Incidence among Adults in the United States. American journal of epidemiology. 2001;153(9):892-7. 30. Holmquist C, Larsson S, Wolk A, de Faire U. Multivitamin supplements are inversely associated with risk of myocardial infarction in men and women--Stockholm Heart Epidemiology Program (SHEEP). The Journal of nutrition. 2003;133(8):2650-4. 31. Marktest. Target Group Index (TGI). Target Group Index (TGI). 2013. 32. Marques-Vidal P. Vitamin supplement usage and nutritional knowledge in a sample of Portuguese health science students. Nutrition Research. 2004;24(2):165-72. 33. Botelho G, Melo AR, Aguiar M. Consumption of Dietary Supplements Among Undergraduate Students: Perceived Knowledge and Usage. REVISTA NUTRÍCIAS. 2013:14-7. 34. Sousa M, Fernandes MJ, Moreira P, Teixeira VH. Nutritional supplements usage by Portuguese athletes. Int J Vitam Nutr Res. 2013;83(1):48-58. 35. Gomes R, Veríssimo MT. Consumo de suplementos alimentares em frequentadores de ginásios na cidade de Coimbra. Rev Medicina Desportiva informa. 2012:20-2. 2. RESULTADOS Capítulo 2 31 2.1 – Suplementos Alimentares com atividade Antioxidante A. Dietary antioxidant supplements: Benefits of their combined use. Food and Chemical Toxicology, 2011, 49, 3232–3237 B. Teas, dietary supplements and fruit juices: A comparative study regarding antioxidant activity and bioactive compounds. LWT - Food Science and Technology, 2012, 49, 324-328 C. Total selenium content of food supplements by microwave digestion and HR-CS ETAAS. Label accuracy evaluation. (submitted) Resultados 32 Capítulo 2 33 Dietary antioxidant supplements: benefits of their combined use Ivone M.C. Almeidaa, João C.M. Barreiraa,b, M. Beatriz P.P. Oliveiraa, Isabel C.F.R. Ferreirab,* aREQUIMTE/ Depto. de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Aníbal Cunha, 164, 4050-047 Porto, Portugal bCIMO-ESA, Instituto Politécnico de Bragança Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal. * [email protected]. Abstract Several dietary supplements claim medicinal benefits due to their composition in hydrophilic and lipophilic molecules, natural extracts or synthetic compounds with antioxidant properties. In the present work, the antioxidant activity of selected supplements taken in pills, capsules or infusions were studied either individually or combined. Linear discriminant analysis (LDA) was used to categorize the condensed formulations (pills and capsules), infusion bags and combined samples according with their antioxidant activity measured by radical scavenging activity, reducing power and lipid peroxidation inhibition using brain homogenates as models. AAF proved to have the highest antioxidant activity in all the assayed methods, either singly taken or included in mixtures. Furthermore, the mixtures containing this supplement revealed synergistic effects in 92% of the cases. The intake of antioxidant mixtures might provide some additional benefits. Keywords: Dietary Supplements; Antioxidant activity; Synergistic Effects; Linear Discriminant Analysis. Resultados 34 1. Introduction In living systems, Reactive Oxygen/Nitrogen Species (ROS/RNS) are produced primarily during normal aerobic metabolism (Halliwell and Gutteridge, 2007). At physiological levels, these intermediates participate in numerous metabolic processes including cell signaling, energy production, gene transcription and immune defense, among others (Seifried et al., 2007). However, decline of antioxidant defense mechanisms or exposure to environmental factors (smoke, pollution, ultraviolet radiation, high-fat diet, etc.) and pathological conditions (chronic infection, inflammation, etc.) can lead to increased ROS/RNS production, resulting in oxidative stress (Valko et al., 2007). Oxidative stress can damage key organic substrates such as DNA, lipids and proteins, compromising cells physiological function (Nordberg and Arnér, 2001). This condition has been associated to the ageing process in general, and to the initiation and progression of a variety of chronic conditions related to it, such as cardiovascular disease and cancer (Valko et al., 2007). Protection against ROS/RNS-induced damage is provided by complex antioxidant defense systems, comprising endogenous enzymatic and non-enzymatic antioxidants (e.g., superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) and exogenous antioxidants (e.g., vitamin C, vitamin E, carotenoids and polyphenols), the latter provided mainly by the diet (Young and Woodside, 2001). Indeed, numerous epidemiological and clinical studies have linked high intake of fruits, vegetables, whole grains, and beverages of plant origin, which are rich in antioxidants, with lower incidence and mortality rates of chronic diseases including diabetes, atherosclerosis, rheumatoid arthritis, neurodegenerative and coronary diseases and cancer (Cerhan et al., 2003, de Kok et al., 2010, Esposito et al., 2002, Ford and Mokdad, 2001, Hertog et al., 1993 and Kris-Etherton et al., 2002). These potential physiological benefits of dietary antioxidants have lead, in recent years, to a dramatic growth of the market of functional foods and dietary supplements claiming “antioxidant power”, and to the widespread consumption of these products. Antioxidant dietary supplements are sold as isolated substances or as mixtures, from natural or synthetic origin, and are presented in a variety of forms including tablets, pills, capsules, powders, drinks and supplement bars. Antioxidant formulations use a plethora of ingredients, including antioxidant vitamins (tocopherols, ascorbic acid), bioactive compounds of plant origin (polyphenols and carotenoids), plant and algae extracts, fruits and vegetables concentrates, enzymes, minerals (selenium, zinc, manganese), polysaccharides, organosulfur compounds, etc. Capítulo 2 35 The antioxidant activity of foodstuffs as well as the purified bioactive compounds to be used in supplement formulations, has been intensely researched (Barreira et al., 2008, Borges et al., 2010, Gorinstein et al., 2011, Müller et al., 2011, Stratil et al., 2007 and Tabart et al., 2009). However, data regarding antioxidant activity of formulations already on the market is scarce. These products are promoted has antioxidant boosters but labels often lack information regarding effective antioxidant capacity values. Therefore, the present study aimed to evaluate the antioxidant activity of different commercial antioxidant dietary supplements available in Portuguese market, by three in vitro assays: scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, reducing power, and inhibition of lipid peroxidation using TBARS in brain homogenates. Moreover, some of the samples were mixed and further assayed in search of synergistic effects. 2. Materials and methods 2.1. Standards and reagents 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). Standards trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and αtocopherol were purchased from Sigma (St. Louis, MO, USA). Methanol and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Water was treated in a Mili-Q water purification system (TGI Pure Water Systems, USA). 2.2. Samples and samples preparation Samples were dietary supplements commercially available and labeled with antioxidant potential. In order to confirm and compare their antioxidant activity, the samples were prepared using the formulation available: pill, capsule or bag (Table 1). Each formulation was weighted and dissolved in 200 mL of distilled water in order to obtain the concentration of the stock-solution. Pills and the inner part of the capsules were dissolved in distilled water, while bags were used to prepare infusions. Several dilutions of each sample were prepared to perform the antioxidant activity assays. Some of the samples were mixed and further assayed in search of synergistic effects. Four mixtures were prepared: AAF + Res + EMCO (stock-solution 4.56 mg/mL) and the corresponding binary combinations: AAF + Res (stock-solution 3.60 mg/mL), AAF + EMCO (stock-solution 5.11 mg/mL) and Res + EMCO (stock-solution 4.99 mg/mL). Resultados 36 Table 1 Composition of the dietary supplements and concentrations of the stock-solution of each sample. Sample Composition Formulation Stock solution (mg/mL) SACE Disodium selenium (0.056%), vitamins A (retinol acetate: 0.74%), C (Lascorbic acid: 22.5%) and E (α-tocopherol: 15%) Pill (397 mg) 1.98 S200 Selenium: 200 μg, brewer’s yeast Pill (614 mg) 3.07 VB Vitamins A, C (L-ascorbic acid) and E (D-α-tocopherol succinate), broccoli sprouts powder, red fruit (grape, blueberry, cranberry, cherry, strawberry and raspberry) combined extract, selenium (yeast). Pill (578 mg) 2.89 BAPN Vitamins A (retinol: 864 μg), B1 (thiamine: 1.8 mg), B2 (riboflavine: 2.8 mg), B3 (nicotinamide ), B5 (pantothenic acid: 7.5 mg), B6 (pyridoxine), B7 (biotin: 100 μg), B11 (folacin: 200 μg), B12 (cyanocobalamin: 4.5 μg), C (ascorbic acid: 200 mg), D (calcipherol: 2.5 μg) and E (D-α-tocopherol: 30 mg), magnesium (75 mg), zinc (7.5 mg), Selenium (L-selenomethionine: 62.5 μg ), chromium (yeast: 50 μg), manganese (2.5 mg), copper (1 mg) Pill (1058 mg) 5.29 LLSC Soy isoflavones, vitamin C, Lycopersicon esculentum extract, lactoproteins, soy lecitin, Lacto-licopene Pill (737 mg) 3.68 KAG Aged garlic extract, Sylibum marianum extract, green tea (powder), vitamins A (β-carotene), C (L-ascorbic acid) and E (α-tocopherol succinate), grape seed extract, pine bark extract, selenium (L-selenomethionine) Capsule (431 mg) 2.16 SZCEA Sodium selenite (0.02%), zinc sulphate (4.8%), vitamins A (β-carotene: 7.5%), C (calcium L-ascorbate: 12%) and E (D-α-tocopherol acetate: 12%) Capsule (374 mg) 1.87 AAF Vitamins A (β-carotene: 4.5 mg), C (calcium L-ascorbate: 500 mg) and E (Dα-tocopherol succinate: 134 mg and other tocopherols: 20 mg), L-cysteine chloridrate ; food based antioxidants: powdered extracts of green tea (7.5 mg of polyphenols), red wine (4.5 mg of polyphenols) and Pycnogenol (3 mg of procyanidins), zinc glycinate (10 mg), taurine (50 mg), L-glutathione (50 mg), manganese glycinate (4 mg),powdered active plant base (Spirulina, Ginkgo biloba, Sylibum marianum and Gotu kola extracts), selenomethionine (50 μg), copper lysinate (1 mg) and riboflavin-5phosphate (6 mg) Capsule (744 mg) 3.72 Pyc Pycnogenol (Pinus maritima bark extract): 30 mg Capsule (247 mg) 1.24 Res Resveratrol(Polygonum cuspidatum root extract): 200 mg Capsule (695 mg) 3.48 GC Coffee arabica seeds (whole cryogrinded powder): 1% caffeine Capsule (288mg) 1.44 AA Vitamin C (L-ascorbic acid) and E (α-tocopherol: 50%), green tea powder, rosemary leaf powder, grape extract, propolis alcoholic extract, Pinus albicaulis Capsule (220 mg) 1.10 GBGT Vitamin A (retinol acetate: 0.083%), C (L-ascorbic acid: 13.9%) and E (D-αtocopherol: 5.6%), Lycopersicum esculentum fruit: 6.9%, Gingko biloba leaves (6.9%), Camelia sinensis (green tea): 1.9%; β-carotene: 0.7%. Capsule (650 mg) 3.25 GM Mangosteen 10:1 (Garcinia mangostana)Capsule (848 mg) 4.24 VRFR Vitis vinifera (red vine leaves: 35%), Hibiscus sabdariffa (flowers: 25%), Pyrus malus (fruit: 16%), orange and red fruits natural flavors Bag (1500 mg) 7.50 EA Equisetum arvense (30%), Olea europaea (30%), Crataegus laevigata (20%), Mentha piperita (20%) Bag (1300 mg) 6.5 EMCO Equisetum arvense, Mentha spicata, Crataegus monogyna, Olea europaea Bag (1300 mg) 6.5 Capítulo 2 37 2.3. Antioxidant activity assays 2.3.1. General The antioxidant activity of the individual and mixed samples was evaluated by DPPH radical-scavenging activity, reducing power and inhibition of lipid peroxidation using TBARS in brain homogenates. The sample concentrations providing 50% of antioxidant activity or 0.5 of absorbance (EC50) were calculated from the graphs of antioxidant activity percentages (DPPH and TBARS assays) or absorbance at 690 nm (reducing power assay) against sample concentrations. The concentrations range was defined in order to allow percentages of antioxidant activity from ∼10% to ∼90% (sock-solution and successive dilutions). Trolox and α-tocopherol were used as standards. 2.3.2. DPPH radical-scavenging activity This methodology was performed using an ELX800 Microplate Reader (Bio-Tek Instruments, Inc). The reaction mixture on 96 wells plate consisted of a solution by well of the different samples concentrations (30 μL) and methanolic solution (270 μL) containing DPPH radicals (6 × 10−5 mol/L). The mixture was left to stand for 30 min in the dark. The reduction of the DPPH radical was determined by measuring the absorption at 515 nm (Guimarães et al., 2010). The radical scavenging activity (RSA) was calculated as a percentage of DPPH discoloration using the equation: %RSA = [(ADPPH − AS)/ADPPH] × 100, where AS is the absorbance of the solution containing the sample, and ADPPH is the absorbance of the DPPH solution. 2.3.3. Reducing power The different concentrations of the samples solutions (0.5 mL) were mixed with sodium phosphate buffer (200 mmol/L, pH 6.6, 0.5 mL) and potassium ferricyanide (1% w/v, 0.5 mL). The mixture was incubated at 50 °C for 20 min, and trichloroacetic acid (10% w/v, 0.5 mL) was added. The mixture (0.8 mL) was poured in the 48 wells plate, as also deionised water (0.8 mL) and ferric chloride (0.1% w/v, 0.16 mL), and the absorbance was measured at 690 nm in the Microplate Reader described above (Guimarães et al., 2010). Resultados 44 compounds included in the pills or capsules. For the assayed combinations, the synergistic interaction was the main observed effect. Regarding LDA, the assayed dietary antioxidant supplements proved to have distinctive features, derived from being condensed (pills or capsules) or bags (infusions) formulas. 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Review of 97 bioavailability studies. Am. J. of Clin. Nutr. 81, 230S–242S. Marathe, S. A., Rajalakshmia, V., Jamdara, S. N., Sharma, A. 2011. Comparative study on antioxidant activity of different varieties of commonly consumed legumes in India. Food Chem. Toxicol. 49, 2005-2012. Moon, J.K., Shibamoto, T. 2009. Antioxidant assays for plant and food components. J. Agric. Food Chem. 57, 1655-1666. Müller, L., Fröhlich, K., Böhm, V. 2011. Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (αTEAC), DPPH assay and peroxyl radical scavenging assay. Food Chem.129, 139148. Nordberg, J., Arnér, E.S.J. 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31, 1287-1312. Prior, R.L., Cao, G. 1999. Variability in dietary antioxidant related natural product supplements: the need for methods of standardization. J. Am. Nutrient Assoc.2, 4656. Prior, R.L., Cao, G. 2000. Analysis of botanicals and dietary supplements for antioxidant capacity: a review. J. AOAC Int.83, 950-956. Rencher, A.C. 1995. Methods of Multivariate Analysis, John Willey, New York. Seifried, H.E., Anderson, D.E., Fisher, E.I., Milner, J.A. 2007. A review of the interaction among dietary antioxidants and reactive oxygen species. J.Nutr. Biochem. 18, 567579. Stratil, P., Klejdus, B., Kuban, V. 2007. Determination of phenolic compounds and their antioxidant activity in fruits and cereals. Talanta. 71, 1741–1751. Tabart, J., Kevers, C., Pincemail, J., Defraigne, J.O., Dommes, J. 2009. Comparative antioxidant capacities of phenolic compounds measured by various tests. Food Chem. 113, 1226–1233. Valko M., Leibfritz D., Moncol J., Cronin M.T., Mazur M., Telser, J. 2007. Free radicals and antioxidants in normal physiological functions and human disease.Review. Int. J. Biochem. Cell Biol.39, 44-84. Capítulo 2 47 Wang, S., Meckling, K. A., Marcone, M.F., Kakuda, Y, Tsao, R. 2011. Synergistic, additive, and antagonistic effects of food mixtures on total antioxidant capacities. J. Agric. Food Chem.59, 960–968. Young, I.S., Woodside, J.V. 2001. Antioxidants in health and disease. J Clin Pathol. 54, 176-186 Resultados 48 Capítulo 2 49 Teas, dietary supplements and fruit juices: A comparative study regarding antioxidant activity and bioactive compounds A.S.G. Costa a, M.A. Nunes a, I.M.C. Almeida a, M.R. Carvalho a, M.F. Barroso a,b, R.C. Alves a,b,*, M.B.P.P. Oliveira a a REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia da Universidade do Porto, Portugal b REQUIMTE, Instituto Superior de Engenharia do Porto, Portugal. *[email protected] Abstract Nowadays, new emerging products claiming antioxidant properties are becoming more frequent. However, information about this topic in their labels is usually scarce. In this paper, we analyzed total phenolics, total flavonoids and ascorbic acid contents, as well as DPPH scavenging activity of several commercial samples, namely green tea and other herbal infusions, dietary supplements, and fruit juices, available in the Portuguese market. In general, beverages containing green tea and hibiscus showed higher phenolics contents (including flavonoids) and antioxidant activity than those without these ingredients. A borututu infusion presented the lowest concentrations of bioactive compounds and scavenging activity, due to the low recommended amount of plant to prepare the beverage. Some juices without antioxidant claims in the label presented similar values to those with it. Keywords: Antioxidant activity; Ascorbic acid; Phenolic compounds; Tea; Dietary supplements; Juices Resultados 50 1. Introduction Modern society is facing a global problem of chronic diseases, such as cardio and cerebrovascular ones, diabetes or cancer. Although human body has a natural defense system that protects itself against harmful effects of free radicals and reactive oxygen species, unhealthy eating habits and day-to-day stress may lead to a decrease in body defenses with deleterious health consequences (Lang & Heasman, 2005). Phytochemicals (biologically active compounds from plants) have been receiving an increased attention due to their important and recognized health benefits, providing a potential protection against several injuries when moderately consumed (Oleszek, 2002). Indeed, antioxidant properties of several plant compounds (polyphenols, vitamin C, vitamin E, among others) have been widely publicized, since they may protect cellular systems of human body from oxidative damage through a variety of complementary and synergic mechanisms, and thus reduce the risk of chronic diseases (Kaur & Kapoor, 2001; Liu, Shi, Ibarra, Kakuda, & Xue, 2008; Serafini & Testa, 2009; Wojcik, BurzynskaPedziwiatr, & Wozniak, 2010). Modern consumers expect from food industry products easy and ready to consume, related with health and well-being. Besides, they also expect that processed foodstuffs have the same or more nutrients than natural foods. Many recent studies have noted and highlighted the importance of antioxidant constituents of beverages, namely coffee (Alves et al., 2010), red wine (Martín, GonzálezBurgos, Carretero, & GómezSerranillos, 2011), green tea (Lambert & Elias, 2010), fruit juices (Borges, Mullen, & Crozier, 2010), and flavored waters (Barroso, Noronha, DelerueMatos, & Oliveira, 2011). More and more, it is possible to find in the market several new products claiming their antioxidant properties. Indeed, these are becoming an important and emerging parameter to assess the quality of the product. Moreover, expansion of the global market and competition between food industries can make it part of the nutritional labeling. Nevertheless, and according to recent literature (Frankel & Finley, 2008) it has been highlighted that most phenolic compounds are poorly bioavailable and heavily metabolized in intestinal and hepatic cells and by the colonic microflora (with conjugation or elimination of phenolic -OH groups critical to the antioxidant activity). Indeed, the upper gastrointestinal tract might be the sole site where the antioxidant activity of phenolic compounds (in what concerns to direct scavenging of radical oxygen species) could be relevant. In this perspective, the term “antioxidant” should be used with precaution by food Capítulo 2 51 manufacturers to correctly inform consumers. The knowledge of the antioxidant profiles of commercial food products is very important in order to compare them. In this work, we evaluated the antioxidant activity (against DPPH radical) of several commercial samples (fruit juices, green tea and other herbal infusions, as well as dietary supplements), all available in the Portuguese market. Within the compounds potentially involved, ascorbic acid, total phenolics, and total flavonoids were evaluated. 2. Material and methods 2.1. Reagents and equipments Ascorbic acid, DPPH• (1,1-diphenyl-2-picrylhydrazyl) free radical, (-)-epicatechin, Folin-Ciocalteu’s reagent, gallic acid, iodine, trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E), and starch, were all purchased from Sigma-Aldrich (Steinheim, Germany). Ethanol of reagent grade, sodium acetate, sodium carbonate decahydrate, sodium nitrite, aluminum chloride, sodium hydroxide were purchased from Merck (Darmstadt, Germany). Spectrophotometric measurements were performed in a UV-visible spectrophotometer (UV-1800) from Shimadzu (Japan). 2.2. Samples Samples (n=19) were randomly selected from local supermarkets and herbalist shops in the area of Oporto, Portugal. Essentially, products claiming antioxidant properties or containing antioxidant constituents were selected. A detailed description of samples is presented in Table 1. 2.3. Samples preparation Samples as bags, leaves, roots or soluble granulates were used to prepare beverages according to the manufacturer’s instructions, as described in Table 1. These and the remaining commercial beverages were all filtered, analyzed immediately for ascorbic acid content, and stored in the dark at -20ºC, for the remaining determinations. Diluted extracts of samples were prepared as necessary. Resultados 52 Table 1. Samples description and methods of beverages preparation. Code Samples Composition Presentation Method of beverage preparation Teas and dietary supplements 1 Green tea (Camellia sinensis) (100%) Bags (2 g) Infusion of a bag in 200 ml of hot water (75 ºC), during 5 min 2 Green tea (Camellia sinensis) (100%) Dry leaves Infusion of leaves (1.62 g) in 200 ml of hot water (75ºC), during 3 min 3* Green tea (Camellia sinensis) extract (2%), sugar, dextrose, citric acid, ascorbic acid (0.225%) Soluble granulated powder Solubilization of 16.6 g of powder in 200 ml of hot water (75ºC) 4* Green tea (Camellia sinensis) extract (100%) 200 ml syrup (liquid concentrated extract) n.a. 5* Red fruits juice powder (10%), green tea extract (6%), citric acid, fruit aromas, hibiscus extract (1.5%), grape seed (1%), ascorbic acid Soluble powder Solubilization of 5 g in 200 ml of hot water (75 ºC) 6 Green tea (Camellia sinensis) (70%), lemon herb (Cymbopogon Citratus) (20%), and algae (Fucus Vesiculosus) (10%) Bags (1.5 g) Infusion of a bag in 200 ml of boiling water (100 ºC), during 3 minutes 7 Green tea (83.3%), aromas (12%), hibiscus (2.4%), pineapple (2.3%) Bags (2.3 g) Infusion of a bag in 200 ml of hot water (75 ºC), during 5 minutes 8* Borututu roots (Cochlospermum angolensis) (100%) Dry roots Infusion of roots (0.27 g) in 200 ml of hot water (75 ºC), during 3 minutes 9 Rooibos red tea leaves (Aspalathus linearis) (100%) Bags (1.5 g) Infusion of a bag in 200 ml of boiling water (100 ºC), during 4 minutes Commercial fuits juices 10 Pomegranate juice (16%), grape juice (4%), green tea extract (0,1%), carrot concentrated juice, hibiscus concentrated juice, ascorbic acid, citric acid Tetra Pak package (1L) n.a. 11 Blackberry juice (18%), grape juice (12%), red tea extract (0,11%), carrot concentrated juice, hibiscus concentrated juice, citric acid Tetra Pak package (1L) n.a. 12 Juice and pulp of raspberry (28%), juice and pulp of apple (22%), ascorbic acid Tetra Pak package (330 ml) n.a. 13 Red grape, raspberry, apple, strawberry, blackberry and cranberry juices, citric acid, ascorbic acid Tetra Pak package (330 ml) n.a. 14 Grape, cherry and blackberry juices, strawberry pulp, raspberry concentrated juice (40%), citric acid, ascorbic acid Tetra Pak package (330 ml) n.a. 15 Pineapple juice (35%) and grape juice (20%), citric acid, ascorbic acid Tetra Pak package (330 ml) n.a. 16 Juice and pulp of orange (23%), carrot (14%), mango (12%), lemon (1%) Tetra Pak package (330 ml) n.a. 17 Orange juice (27%), passion fruit (9%), lemon (2%), ascorbic acid Tetra Pak package (330 ml) n.a. 18 Orange juice (30%) and mango pulp (15%), ascorbic acid, citric acid Tetra Pak package (330 ml) n.a. 19 Tomato juice and pulp, citric acid, ascorbic acid Glass bottle (200 ml) n.a. n.a., not applicable. a Dietary supplement. Capítulo 2 53 2.4. Ascorbic acid A redox titration, involving an iodometric method, was used to perform ascorbic acid quantification. Briefly, 25.0 ml of sample were subjected to an iodimetric titration with 1 g/100 ml iodine solution, using a 1 g/100 ml starch solution as indicator. The endpoint was observed by the reaction of iodine with starch suspension, which produces a blue-black product. The standard ascorbic acid was used to plot the standard curve (linearity range = 0.2−1.0 mg/ml, r > 0.999). Ascorbic acid content was expressed as milligrams per 100 ml of beverage (or 10 ml of syrup). 2.5. Total phenolics Total phenolics were spectrophotometrically determined according to the FolinCiocalteu procedure (Singleton & Rossi, 1965) with minor modifications (Alves et al., 2010). Briefly, 500 ml of a diluted sample was mixed with 2.5 ml of the Folin-Ciocalteu reagent (1:10) and 2 ml of a Na2CO3.10H2O solution (7.5 g/ 100 ml). The mixture was incubated at 45 ºC, during 15 min, and after 30 min at room temperature, absorbance readings at 765 nm were performed, against a reagent blank. A calibration curve for the standard gallic acid was used to obtain a correlation between sample absorbance and standard concentration (linearity range = 5−100 μg/ml, r > 0.999). Total phenolics concentration was expressed as milligrams of gallic acid (GA) per 100 ml of beverage (or 10 ml of syrup). The results obtained were afterward corrected in order to eliminate the ascorbic acid influence. 2.6. Total flavonoids Total flavonoids content was determined by a colorimetric assay based on the formation of flavonoid-aluminum compound according to Barroso et al. (2011). Briefly, 1 ml of a diluted extract was mixed with 4 ml of ultrapure water and 300 μl of 5 g/100ml NaNO2 solution. After 5 min, 300 μl of 10 g/100ml AlCl3 solution were spiked, and after 1 min, 2 ml of 1mol/L NaOH and 2.4 ml of ultrapure water were also added. The solution was well mixed, and absorbance was read at 510 nm. Epicatechin was used to plot the standard curve (linearity range = 0−66 μg/ml, r > 0.999). Total flavonoids concentration was expressed as milligrams of epicatechin (E) per 100 ml of beverage (or 10 ml of syrup). Resultados 60 Liu, D., Shi, J., Ibarra, A. C., Kakuda, Y., & Xue, S. J. (2008). The scavenging capacity and synergistic effects of lycopene, vitamin E, vitamin C, and b-carotene mixtures on the DPPH free radical. LWT-Food Science and Technology, 41, 1344-1349. Martín, S., González-Burgos, E., Carretero, M. E., & Gómez-Serranillos, M. P. (2011). Neuroprotective properties of Spanish red wine and its isolated polyphenols on astrocytes. Food Chemistry, 128, 40-48. Oleszek, W. (2002). Dietary phytochemicals and human health. Phytochemistry Reviews, 1, 163-166. Rababah, T. M., Ereifej, K. I., Esoh, R. B., Al-u’datt, M. H., Alrababah, M. A., & Yang, W. (2011). Antioxidant activities, total phenolics and HPLC analyses of the phenolic compounds of extracts from common Mediterranean plants. Natural Product Research: Formerly Natural Product Letters, 25, 596-605. Ramirez-Rodrigues, M., Plaza, M. L., Azeredo, A., Balaban, M. O., & Marshall, M. R. (2011). Physicochemical and phytochemical properties of cold and hot water extraction from Hibiscus sabdariffa. Journal of Food Science, 76, C428-C435. Sánchez-Moreno, C., Plaza, L., Ancos, B., & Cano, M. P. (2003). Quantitative bioactive compounds assessment and their relative contribution to the antioxidant capacity of commercial orange juices. Journal of the Science of Food and Agriculture, 83, 430439. Serafini, M., & Testa, M. F. (2009). Redox ingredients for oxidative stress prevention: the unexplored potentiality of coffee. Clinics in Dermatology, 27, 225-229. Singleton, V. L., & Rossi, J. A., Jr. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144-158. Tsai, P.-J., Tsai, T.-H., Yu, C.-H., & Ho, S.-C. (2007). Comparison of NO-scavenging and NO-suppressing activities of different herbal teas with those of green tea. Food Chemistry, 103, 181-187. Wojcik, M., Burzynska-Pedziwiatr, I., & Wozniak, L. A. (2010). A review of natural and synthetic antioxidants important for health and longevity. Current Medicinal Chemistry, 17, 3262-3288. Zulueta, A., Esteve, M. J., Frasquet, I., & Frígola, A. (2007). Vitamin C, vitamin A, phenolic compounds and total antioxidant capacity of new fruit juice and skim milk mixture beverages marketed in Spain. Food Chemistry, 103, 1365-1374. Capítulo 2 61 Total selenium content of food supplements by microwave digestion and HR-CS ETAAS. Label accuracy evaluation. I.M.C. Almeidaa*, M.T. Oliva-Telesb, C. Delerue-Matosb, M.B.P.P. Oliveiraa aREQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal bREQUIMTE, Departamento de Engenharia Química, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Porto, Portugal *[email protected] Abstract The determination of selenium in food supplements is of major interest, due to the low range between beneficial and toxic effects of this element. The total selenium contents of eight different commercially available food supplements were determined. Microwaveassisted acid digestion was used for sample solubilisation and selenium was determined by high-resolution continuum source atomic absorption spectrometric with electrothermal atomization (HR-CS ETAAS). The electrothermal behaviour of selenium in the presence of different chemical modifiers, and pyrolysis and atomization temperatures, were optimized. Palladium nitrate-magnesium nitrate was selected as the matrix modifier. The optimum pyrolysis and atomization temperatures were 1050 °C and 2000 °C, respectively. The LOD and LOQ for selenium were 0.10 and 0.34 µg g-1, respectively. The intra-day precision showed a 3.2 % RSD and inter-day precision RSD did not exceeded 6.7 %. The accuracy of the method was checked with a certified reference material and good agreement was found between measured and certified total selenium content (p > 0.05). The total selenium contents were determined and compared with the labelled values. Selenium levels food supplements ranged from 15.4 ± 0.9 to 205.3 ± 9.9 μg/ unit. These results were in good agreement with the average levels claimed on product labels, with an error lower than ±15%. Also, all supplements were in compliance with the recent recommendations made by the European Community regarding the acceptable difference Resultados 62 between labelled and measured values for minerals and vitamins in food supplements, fixed in -20% to +45% of the declared on label. Keywords: Food supplements; Selenium; High-resolution continuum source electrothermal atomic absorption spectrometry; Label accuracy. 1. Introduction In the European Union (EU), the Food Supplements Directive [1] defines 'food supplements' as concentrated sources of nutrients or other substances with a nutritional or/and physiological effect whose purpose is to supplement the normal diet. They are marketed in dose form (capsules, tablets, pills, powders, liquids, etc.), alone or in combination, and are designed to be taken in measured small unit amounts [1]. Food supplements are generally used to overcome nutritional deficiencies, prevent or reduce the risk of disease, and/or to promote general well-being. Generally, consumers assume these products as natural and safe, using them in addition to, or as a replacement for, or alternative to pharmaceuticals. However, food supplements, unlike pharmaceutical drugs, do not require approval for safety and efficacy prior to their marketing. Manufacturers and/or distributors need only to notify the competent authority before marketing their product, and are responsible to ensure its compliance with the requirements of applicable legislation both in terms of safety and of consumer information [1]. With the widespread use of food supplements, it is essential to ensure the safety of these products for human consumption. There have been reports of the presence of impurities and the adulteration of several food supplements, lack of batch-to-batch consistency, and misformulated products [2, 3, 4]. Selenium is an essential trace element required for the normal growth, development and metabolism of both man and animals [5]. Selenium constitutes an integral part of important selenoproteins, including glutathione peroxidase (GSHPx), an antioxidant enzyme that protects cell membranes from free radicals damage, iodothyronine deiodinases, involved in the thyroid hormone metabolism, and thioredoxin reductase that, in conjunction with the compound thioredoxin, participates in the regeneration of several antioxidants from their oxidized forms, regulating cell growth and viability [6, 7]. Currently, the Recommended Dietary Allowance (RDA) for selenium is 55 µg/ day for healthy adult men and women, the estimated requirement necessary for maximization Capítulo 2 63 of plasma GSHPx activity [8]. However, clinical evidence reports that intakes of selenium in excess of the RDA (200–300 µg/ day) may prevent certain cancers [9, 10] and cardiovascular disease [11], and improve immune response and male fertility [12, 13]. In the United States, the Food and Nutrition Board of the Institute of Medicine [8] as derived a Tolerable Upper Intake Level (UL) of 400 µg selenium/ day for adults while the European Community (EC) Scientific Committee on Food (SCF) has set a UL of 300 µg/ day for the European Union (EU) [14]. The selenium content of foods and fodders depends of their geographical origin and the respective soil selenium content and availability. Consequently, the selenium intake by humans varies considerably between countries and regions [15]. Some European countries, including Portugal, register selenium dietary levels below RDA guidelines [16, 17, 15]. Although overt selenium deficiencies are rare, suboptimal selenium status can lead to cancer and heart disease, and an impaired immune system [18]. Supplementation, therefore, is becoming a common practice among consumers of developed countries to compensate for dietary deficiencies and/or to prevent certain cancers and aging. However, in view of the narrow range between deficiency, essentiality and toxicity of selenium in human nutrition, and the documented cases of intoxication caused by selenium supplements, makes particularly important the control of these products. Selenium has been determined in food supplements using different analytical techniques including inductively coupled plasma mass spectrometry (ICP-MS) [19], cathodic stripping voltammetry (CSV) [20], hydride generation atomic fluorescence spectrometry (HG-AFS) [21, 22], and electrothermal atomic absorption spectrometry (ETAAS) [23,24]. Line sourceETAAS has been extensively and successfully employed for the elemental analysis of several matrices [25] due to its versatility, low limits of detection, and selectivity. Recently, High-Resolution Continuum Source Atomic Absorption Spectrometry (HR-CS AAS) has extended the capabilities of conventional AAS methods. Novel features include a high-intensity xenon short-arc lamp used as a continuum radiation source and a high resolution double echelle monochromator, which allows performing fast-sequential multi-element measurements, and a linear chargecoupled device (CCD) array detector. The CCD contains 588 pixels, 200 of which are used for the monitoring the analytical signal, but also allows the simultaneous visualization at high resolution of the spectral environment around the analytical line, and in the background correction. Spectral interferences can be detected promptly and corrected if necessary, reducing noise levels and improving the detection limits [26]. HR-CS AAS has been employed for elemental analyses of diverse matrices [27, 28, 29]. Recently, Resultados 64 Krawczyk [30] determined macro and trace elements in multivitamin dietary supplements by HR-CS AAS, with slurry sampling. The aim of this work was to optimise and validate a method using microwaveassisted acid digestion and HR-CS ETAAS to quantify total selenium contents in commercially available food supplements and to compare the results against the amounts referred on the supplement label. 2. Materials and Methods 2.1 Equipment and HR-CS ETAAS measurement conditions Selenium determination was performed using an Analytik Jena contrAA 700 (Analytik Jena, Jena, Germany) high-resolution continuum source atomic absorption spectrometer. The equipment has a transversely heated graphite furnace, a high-intensity xenon short-arc lamp (XBO 301, GLE, Berlin, Germany), a high-resolution double monochromator, and a charge-coupled device (CCD) array detector and an MPE 60 autosampler. Pyrolytically coated graphite tubes with integrated platform (Analytik Jena, Jena, Germany) were used exclusively. Argon of 99.95% purity (Linde Sogás, Portugal) was used as purge gas. The optimised electrothermal program used for selenium determination in samples and the reference material is shown in Table 2. Quantification was performed using 5 µL matrix modifier volume and 10 µL sample volume, sequentially pipetted by the auto sampler. The measurements were performed in a spectral interval of 0.2209 nm (200 pixels) around the primary selenium line 196.0267 nm (pixel 101). The integrated absorbance (Aint) was optimised and it was selected the values obtained for seven pixels (the central pixel ± 3), corresponding to the wavelength range of 7.7 pm. A dynamic background correction, based on those pixels that do not significantly differ from baseline noise, was used. Analytical blanks and standards were checked routinely to check instrument performance. Four replicate measurements of absorbance were carried out for all solutions. Samples digestion was performed with a MARS X 1500W Microwave Accelerated Reaction System (CEM Corp., Mathews, NC, USA) and 100 mL Teflon HP-500 Plus closed-system vessels (CEM Corporation, Matthews, NC). All glassware and plastic materials were washed with an appropriate detergent, immersed in 10% HNO3 for 24 h and rinsed with ultrapure water, prior to use. Capítulo 2 65 2.2 Reagents and solutions Ultrapure water from a Simplicity 185 system (resistivity 18.2 MΩ.cm; Millipore, Belford, USA) was used for the preparation of samples and standards. Chemicals were of analytical reagent grade unless otherwise stated. Suprapur® grade nitric acid (65%) and hydrogen peroxide (30%) were obtained from Merck (Darmstadt, Germany). Selenium working standards were prepared by dilution of a 1000 mg L–1 selenium stock solution (Panreac, Barcelona, Spain). The Pd and Mg modifier solutions were made by dilution with water from commercially available stock solutions, 10.0 ± 0.2 g L−1 Pd in 15% (v/v) HNO3 (Merck, Darmstadt, Germany) and 10.0 ± 0.2 g L−1 Mg in 15% (v/v) HNO3 (Panreac, Barcelona, Spain), respectively. A 1% (m/v) nickel nitrate solution, used as chemical modifier, was prepared by dissolving an appropriated amount of Ni(NO3)2 · 6H2O (Merck, Darmstadt, Germany) in water. Table 1. Description of the analysed food supplements. Sample Description Claims RDD A Selenium (L-selenomethionine) (yeast free). No claim 1-2 tablets B Selenium (L-selenomethionine). Aged garlic extract, Sylibum marianum extract, green tea (powder), vitamins A, C and E, grape seed extract, pine bark extract. Antioxidant and anti-aging 4 capsules C Selenomethionine. Vitamins A, C, and E, L-cysteine chloridrate, powdered extracts of green tea, red wine and pycnogenol, zinc glycinate, taurine, L-glutathione, manganese glycinate, powdered active plant base (Spirulina, Ginkgo biloba, Sylibum marianum and Gotu kola extracts), copper lysinate, riboflavin-5-phosphate. Antioxidant 2 tablets D Selenium (L-selenomethionine). Vitamins A, B1, B2, B3, B, B6, B7, B11, B12, C, D and E, magnesium, zinc, chromium, manganese, copper. Antioxidant 1 tablet E Selenium: brewer´s yeast. Helps support the immune system 1 tablet F Selenium (yeast).Vitamins A, C and E, broccoli sprouts powder, red fruit (grape, blueberry, cranberry, cherry, strawberry and raspberry). Antioxidant 1 tablet G Disodium selenium. Vitamins A, C and E. No claim 1 tablet H Sodium selenite. Zinc sulphate, vitamins A, C and E. Antioxidant 1 capsule 2.3 Sampling and sample preparation Eight different food supplements containing selenium, for adult consumption, were purchased from local retail and herbal stores. The samples were selected to encompass Resultados 66 different selenium species (organic and inorganic) and formulations (tablet or capsule dosage). The supplements were designated as A, B, C, D, E, F, G, and H, respectively. The specifications of the selected supplements, according to the manufacturer, are summarised in Table 1. Ten tablets or capsules were taken from each product, and then crushed and homogenised manually in a mortar, after carefully removing tablets film coats, if present, and the hard-gelatine in capsules. Powdered samples were stored in screw-capped vials and kept at 4°C until analysis. Table 2. Optimized furnace program used for the determination of selenium by HR-CS ETAAS. Step Temperature (oC) Ramp (oC/s) Hold time (s) Ar flow rate L/min-1 Drying 90 3 20 Max Drying 110 5 10 Max Pyrolysis 1050 300 10 Max Atomization 2000 1500 4 Stop Cleaning 2450 500 4 Max 2.4 Microwave-assisted digestion Approximately 0.2 g of each powdered sample was weighed into 100 mL microwave Teflon vessels and 9 mL of concentrated nitric acid and 1 mL of hydroxide peroxide were added to each vessel. The vessels were left open for 15 minutes before sealing to allow samples to predigest, and were then positioned inside the microwave digestion system for a three-step microwave temperature program. First, samples were digested at 50 °C, with 3 min ramp to temperature, for 3 minutes. Subsequently, samples were irradiated to a temperature of 90 °C, with 10 min. ramp and 10 min hold. Finally, samples were digested at 190°C for 20 minutes, using a ramp time of 10 minutes. Once the vessels were cooled, the digested samples were transferred to volumetric flasks and diluted to 15 mL with MilliQ water. One reagent blank was run with each batch of samples. All the experiments were performed in triplicate. A certified reference material SELM-1 (selenium-enriched yeast) used in this work to validate results, was obtained from the National Research Council of Canada (NRCC) (Ottawa, Québec, Canada) and was submitted to the same procedure applied to samples. Capítulo 2 67 2.5 Statistical analysis Data are reported as mean ± standard deviation. One-sample t-test was used to compare means. Statistical analysis was carried out using IBM SPSS Statistics for Windows, version 21.0 (IBM Corp., Armonk, New York). Significant differences were considered when p < 0.05. 3. Results and discussion 3.1 Selenium measurements by HR-CS ETAAS The electrothermal behaviour of selenium in the presence of the chemical modifiers, and pyrolysis and atomization temperatures, were optimized to maximize absorbance signals and to minimize backgrounds and matrices interferences. The determination of selenium by ETAAS requires the use of adequate chemical modifiers to avoid elemental volatilization during the different stages of the electrothermal process, and the consequent loss of analyte. Nickel (1%), palladium (0.1%), magnesium (0.1%), and palladium-magnesium (0.1% Pd+0.05% Mg) nitrates solutions, prepared in water from the respective modifier stock solutions, were tested as chemical modifiers for the determination of selenium by HR-CS ETAAS. A 50 µg L-1 selenium standard solution and digested samples were analyzed with the modifiers, using the default cookbook values. The best sensitivity was achieved with 5 μL of the palladium nitrate-magnesium nitrate (0.1% Pd+0.05% Mg) mixed modifier, and was further used for the optimization of the electrothermal program. The optimization of pyrolysis and atomization temperatures was carried out automatically, using a 50 µg L-1 selenium standard solution and four digested sample solutions, in order to assure equal responses from selenium originated from selenite, selenomethionine and selenized yeast, present in the supplements. The optimum pyrolysis and atomization temperatures were 1050 °C and 2000 °C, respectively, and were used for all analyses. The optimized electrothermal program used for selenium determination by HR-CS ETAAS is shown in Table 2. The spectral environment, only available with this recent instrument, allowed detecting two selenium peaks (196.027 nm and 196.026 nm). Since the sensitivity and the precision of the methods depends on the wavelength integrated absorbance, and selenium has two overlapping peaks, it was necessary to evaluate the integrated Resultados 68 absorbance of a standard solution for different number of pixels. The best precision was achieved for 7 pixels, i.e. from central pixel (196.027 nm) ± 3. 3.2 Method validation Under the optimized conditions, the linear range, the limits of detection (LOD) and quantification (LOQ), the precision and the accuracy for the determination of selenium by HR-CS ETAAS were assessed. Calibration curves were obtained by using five selenium standard solutions (0, 12.5, 25, 50, 75, and 150 µg L-1). Correlation coefficients better than 0.998 were obtained. The LOD and LOQ were calculated as 3 and 10 times the standard deviation estimated from the regression line divided by the slope of the calibration curve, respectively, and taking into account the dilutions done, and are expressed in µg g-1. The LOD and LOQ for selenium were 0.10 and 0.34 µg g-1, respectively. The intraand interday precisions were evaluated by analysing one sample (supplement E) six times, under the same experimental conditions and on the same day, and on three different days. The intra-day RSD was 3.2 % and the inter-day RSD did not exceeded 6.7 %. The accuracy of the method was verified by the analysis of the certified reference material SELM-1, Selenium Enriched Yeast, which has a total certified amount of selenium of 2059 ± 64 µg g−1. The material was analysed in triplicate by the proposed method and an average concentration of 1999 ± 41 µg g-1 of total selenium was obtained (98.6 ± 2.8 % recovery). No significant differences were found between the certified and the experimental values (p > 0.05), confirming the accuracy of the method for the determination of total selenium food supplements with added selenium. 3.3 Quantification of total selenium in Food Supplements The market offers a plethora of food supplements with added selenium. In this work, samples were selected to encompass supplements with different selenium chemical species and different formulations. Samples A, B, C, and D contained Lselenomethionine, selenized yeast was present in samples E and F, and samples G and H contained sodium selenite. The selected samples comprised single-component selenium supplements and multi-ingredient formulations that, besides selenium, could contain minerals, vitamins, amino acids, and other antioxidants, in capsules or tablets (Table 1). The concentrations of total selenium were determined by HR-CS ETAAS, after microwave-assisted digestion of samples. Results are summarized in Table 3. Selenium levels in samples ranged from 15.4 ± 0.9 to 205.3 ± 9.9 μg/ unit. When compared with the Capítulo 2 69 declared value, the total selenium contents obtained were in good agreement with the label, with an error lower than ±15%. Table 3. Total selenium contents in 8 food supplements. Experimental results as mean value ± standard deviation (n=3). Sample Declared Se content/unit (µg) Measured Se content/ unit (µg) % difference from labelled Se/unit level* A 100 87.9±8.0 -12 B 17.5 15.4±0.9 -10 C 25 21.6±1.7 -14 D 62.5 70.6±3.3 +13 E 200 205.3±9.9 +3 F 100 102.0±7.3 +2 G 100 100.6±6.3 +1 H 50 49.4±4.8 -1 *Percent difference of label claim calculated as (measured Se content/ unit - Declared Se content/unit)/ Declared Se content/unit*1000 In 2012, the European Community Commission Standing Committee on the Food Chain and Animal Health has published guidance which established a tolerance threshold for nutrient values declared on nutrition labelling, defining this "tolerance threshold" as the acceptable differences between declared nutrient values on the label and those determined during official controls. The mineral content declared on label in food supplements should be in the range of -20 % to +45 % of the value declared [31]. According to this guidance, the differences between claimed and determined total selenium in all supplements are acceptable. The results obtained in our study are comparable to the difference range (–19% to +23%) reported by Feifer et al. [32] after analysing five selenium supplement brands used for prostate disease. In a previous study, eight different commercial supplements were analysed by GFAAS and results were found to be in good agreement with label contents [23]. B’Hymer et al. [19] analyzed six different brands of yeast-based supplements for total selenium contents using microwave digestion and ICP-MS. Reported results ranged from 91 to 111% of the label claim. A few studies have identified discrepancies between stated and actual contents in selenium supplements. Valiente et al. [33] analysed 3 different selenium supplements brands for total selenium by ETAAS and found significant differences between tablets and between batches of the same brand, with differences over 300% in one brand. More recently, a work by Stibilj et al. [34] revealed that 2 of the 9 Resultados 76 Capítulo 2 77 Label compliance in omega-3 dietary supplements: oil, fatty acids and vitamin E contents analysis Ivone M. C. Almeida, Telmo J. R. Fernandes, M. Beatriz P. P. Oliveira* REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. * beatol[email protected] Abstract This study aimed to verify the conformity of the label information of 16 omega-3 food supplements, regarding their oil, fatty acids and vitamin E contents. Fish oil, krill or vegetable oils are present as components on the analysed products, in a single or blend form. The levels of oil, EPA, DHA, ALA and vitamin E were determined and compared with label information. Significant differences between the experimental data and the displayed information on the label were verified for the majority of the studied samples. These results stress the need to a more effective labelling control of this type of products, in order to better inform the consumers. Keywords: Omega-3 fatty acids; ALA; DHA; EPA; Vitamin E; Food supplements. Abbreviations: n-3 PUFA, omega-3 polyunsaturated fatty acids; ALA, α-linolenic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl esters. Resultados 78 1. Introduction The omega-3 fatty acids (n-3 FA) are a family of polyunsaturated fatty acids (PUFA) commonly found in marine and a few vegetable oils. The most important n-3 PUFA involved in human nutrition are α-linolenic acid (ALA, 18: 3 n-3), and the long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) eicosapentaenoic acid (EPA, 20: 5 n-3), and docosahexaenoic acid (DHA, 22: 6 n-3) (Almeida et al., 2014). Epidemiological and clinical studies have shown that regular consumption of oily fish and omega-3 fatty acids decreases the risk of cardiovascular disease (CVD), including fatal coronary heart disease (CHD) and sudden cardiac death (Mozaffarian & Wu, 2011). Based on this evidence, most international groups and organizations recommend to the general population a daily intake of at least 250 mg of LC n-3 PUFA (mostly EPA and DHA), preferably through consumption of oily fish (1–2 servings per week) (EFSA, 2010; FAO/WHO, 2008; Lichtenstein et al., 2006). Despite consumer awareness of the health benefits of n-3 PUFA, in most Western countries the estimated intake levels are lower than the currently recommended (Abreu et al., 2012; Lucas et al., 2009). Barrier factors for consuming fish and/or seafood have been identified and include, among others, taste preferences, price, environmental concerns, ethical reasons, and difficulties in preparation (Brunso et al., 2009; Trondsen et al., 2003). In those cases, supplementation may offer an effective way to increase n-3 PUFA intake and status. Several products containing n-3 PUFAs are available in the market, and typically consist of fish, krill, algae or flaxseed oils or oil blends. n-3 PUFA in food supplements are delivered in various forms including soft gel capsules, oils and emulsions, and are available from multiple channels: retail stores, health stores, herbalists, in the internet, and pharmacies. Unlike medicines, which must be proven both safe and effective through clinical trials, dietary supplement manufacturers are responsible for the safety of their products (EC, 2013). Thus, sometimes these products lack consistency in dose and quality, according to several reports (Kolanowski, 2010; Opperman et al., 2011; Tatarczyk et al., 2007). Foods containing lipids are susceptible to oxidation during processing and storage. n-3 PUFAs are naturally highly unstable and prone to oxidation, due to their unsaturated nature. The oxidation of oils give rise to volatile compounds that confer undesirable taste and smell, loss of nutrients, and the development of toxic lipid oxidation products that might be harmful for humans (Kanner, 2007). These compounds are associated with the rancidity which contributes to consumer rejection of the products. Common complains to fish oils are the presence of unpleasant fishy taste and smell, as well as digestive issues, Capítulo 2 79 namely fishy eructation. Vitamin E is considered to be a major lipophilic antioxidant (Bramley et al., 20009) and it is commonly added to n-3 PUFA supplements to prevent oxidation and extend shelf life. The main function of vitamin E is to act as a primary antioxidant that scavenges lipid peroxyl radicals and neutralizes reactive oxygen and nitrogen species (Nelis et al., 2000). In humans, vitamin E has been associated with the prevention or delay of chronic diseases caused by oxidative stress including cardiovascular disease, cancer, cataracts, and Parkinson’s disease (Bramley et al., 2000; Nelis et al., 2000). The human body is not able to synthesise vitamin E and must obtain it through diet or supplementation. The Recommended Dietary Allowances (RDA) for vitamin E (αtocopherol) for men or women older than 14 years is 15 milligrams (or 22.4 IU) (IMFNB, 2000). Currently, to our knowledge, there is no available data on the evaluation of omega-3 supplements (from different origins) commercially available on the Portuguese market, regarding the quality and safety of these products. Thus, the aim of this study was to analyse 16 omega-3 supplements, concerning their FA profile, in particular, the EPA, DHA and ALA contents and also to quantify vitamin E amounts. Moreover, a comparison regarding these constituents was carried out, relating the analysed values with the manufacturers label information, important for the quality control and safety of these products. 2. Materials and methods 2.1. Reagents and standards FAME 37 standard mixture was purchased from Supelco (FAME 37, Bellefonte, PA, USA). Tocopherols (α, β, γ, and δ) and tocotrienols (α, β, γ, and δ) were from Calbiochem (La Jolla, CA). The internal standard tocol was obtained from Matreya Inc. (Pennsylvania, USA). HPLC grade n-hexane was obtained from Merck (Darmstad, Germany) and 1,4dioxane from Fluka. Butylated hydroxytoluene (BHT) was used as antioxidant and was from Aldrich (Madrid, Spain). All other chemicals were from analytical grade. Resultados 80 2.2. Samples Sixteen oil supplements were obtained from health and retail stores in Porto, Portugal, and analysed for its fatty acids composition and vitamin E contents. Twelve samples are fish oils, were designated as FO and numbered from 1 to 12; one sample contained krill oil (KO), and the remaining three brands were blends of fish oil and vegetable oils (BL1, BL2, and BL3). All products were encapsulated in soft gel capsules. Detailed information about the analysed food supplements trade name, origin, ingredients, and the recommended daily dose (RDD) is presented in Table 1. For sample preparation, six capsules of each brand were weighed, opened and the oil squeezed into a clean vial. The empty capsule was then washed with hexane, wiped and weighed again. Aliquots from the pooled oil were transferred to screw-capped vials for further analysis. Table 1. Trade name, country of origin, oil source, and manufacturer recommended daily dose (RDD) of analysed dietary supplements. Sample Name Oil source Recommended daily dose (RDD) FO1 ERGY 3 (France) Sardines and anchovies oil 4 FO2 MorEPA (Belgium) Deep sea fish oil 1 FO3 Omega 3 áreaviva (Portugal) Polyunsaturated omega-3 fatty acids 4 FO4 Omega 3 Family (Belgium) Fish oils 4 FO5 Aquamarine (USA) Fish oil and cod liver oil 2 FO6 Omega 3 Mood (USA) Fish oil concentrate (anchovy, sardine, mackerel, herring, salmon, tuna) 2 FO7 Omega 3 KAL (USA) Fish oil from cold water fish 2 FO8 Omega 3 Concentration (Portugal) Fish oil 1 FO9 Fish Oil Concentrate ( USA) Concentrated fish oil 1 FO10 Salmon Oil (Portugal) Salmon oil 3 FO11 Omega 3Fish Oil (Portugal) Salmon oil 2 FO12 Omega-3 Double Strengh (USA) Fish oil concentrate 4 KO Physalis Krill Omega 3 (Belgium) Krill oil 2 BL1 All-in-Fit Omega 3+Omega 6+Omega 9 (Portugal) Fish oil; flaxseed oil; sunflower seed oil 3 BL2 Omega 3-6-9 Forma+ (Portugal) Evening primrose oil; salmon oil; flaxseed oil 2 BL3 Omega-3-6-9 (Portugal) Borage oil; flaxseed oil; salmon oil 1 2.3. FA profile Fatty acid methyl esters (FAME) were prepared in duplicate by transmethylation using boron trifluoride (Sigma Aldrich St. Louis. MO. USA) according to Shanta & Ackman (1990) with some modifications, and analysed in a Shimadzu GC-2010 gas Capítulo 2 81 chromatograph with a flame ionization detector (Shimadzu, Columbia). The chromatograph was equipped with a CPSil 88 fused silica capillary column (Varian, Middelburg, The Netherlands; 50 m x 0.25 mm internal diameter, 0.19 µm film thickness). He was the carrier gas (120 kPa) and the temperature program was: 120ºC (5 min), 120 - 220 ºC (3ºC/min) and 220 ºC (10 min). Injector and detector temperatures were 250 °C and 270 °C, respectively. The split ratio was 1:50 and the injected volume was 1.0 μl, performed in duplicate assays, each one injected in duplicate. FAME’s were identified by comparison with standard mixtures (FAME 37, Supelco, Bellefonte, PA, USA) and data were analysed using GC Solution software (version 2.30, Shimadzu GC solution, Shimadzu, Columbia). Each FA was expressed as relative percentage of the individual FAMEs represented in the chromatogram (Casal & Oliveira, 2007). 2.4. Vitamin E determination Aliquots of the pooled oil were accurately weighed into microcentrifuge tubes. Then, 20 μl of the internal standard solution (tocol, 1mg/ml) was added and the volume made up to 1 ml with n-hexane. After vortex mixed and centrifuged (5 min., 13 000 rpm), the supernatant was transferred to amber glass vials and directly injected into the HPLC system. The chromatographic analysis was carried out in an HPLC integrated system equipped with an AS-950 automated injector, a PU-980 pump, an MD-910 multiwavelength diode array detector (DAD) and an FP-920 fluorescence detector (Jasco, Japan), programmed for excitation at 290 nm and emission at 330 nm. Normal-phase chromatography was run in a SupelcosilTM LC-SI column (3 µm; 75 x 3.0 mm; Supelco, Bellefonte, PA, USA), operating at room temperature (21 °C), according to Alves et al. (2009). 2.5. Statistical analysis Using the statistical program IBM SPSS STATISTICS (21.0 package, IBM Corporation, New York, USA), all experimental data were assessed for normality and homogeneity of variances by Kolmogorov-Smirnov and Leven’s tests. The results were then analysed using an One-sample T-test to compare the claimed and measured values of oil, EPA, DHA, ALA, EPA+DHA and vitamin E. Significant differences were considered when p<0.05. Resultados 82 3. Results Table 2 shows the FA profile of the sixteen food supplements analysed. Table 3 allows the comparison between the label information provided by the manufacturers and the obtained results (oil, EPA, DHA, ALA and vitamin E contents, mg per one capsule). There were significant differences (p<0.05) between the label information supplied and the experimental data in all analysed nutrients (Table 3) 3.1. Fish oil content For each sample, the oil content of six individual capsules was calculated as the weight difference between the filled and the empty capsules. The oil content ranged from 472 to 1179 mg per capsule. According with the manufacturer information the majority of the preparations (11 supplements) contained less oil than the label states, but only four had significantly lower values (p<0.05). The other five supplements held more oil than labelled, but only in two samples the oil contents were significantly higher. 3.2. Fatty Acid Composition The FA analysis of the supplements allowed the identification of 29 individual FAMES, though only 20 were considered significant in terms of weight percent (Tables 2 and 3). As expected, PUFA were the predominant class in all samples, ranging from 31.7 to 85.8 % of total FA. The second most representative group was SFA, with contents varying between 5.0 % and 38.7 %. In general, myristic (14:0), palmitic (16:0) and stearic (18:0) were the most prevalent SFA. The majority of the samples presented palmitoleic (16:1 n-7) and oleic (18:1 n-9) acids as the main MUFA and their sum varied from 2.2 to 23.7 %. As shown in Table 2, EPA and DHA were the major n-3 PUFA in all samples, except in BL1 and BL2, where ALA was found at higher concentrations. It was verified that: EPA contents ranged from 53.1 to 568.8 mg/capsule; DHA contents ranged from 35.7 to 292 mg/capsule; and ALA contents ranged from 3.3 to 186.4 mg/capsule. FO6 and BL2 samples contained the higher and the lower total amounts of LC n-3 PUFA per capsule (752 and 89 mg/capsule, respectively). Capítulo 2 83 Table 2. Fatty acid profiles of selected omega-3 supplements. Values represent mean ± standard deviation. SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids. 1Includes 10:0, 12:0, 15:0, 17:0 2Includes 14:1, 16:1 n-9, 18:1 n-9 t; 24:1 3Includes 20:2 n-6 Sample Fatty acid (%) FO1 FO2 FO3 FO4 FO5 FO6 FO7 FO8 14:0 6.89±0.17 0.07±0.00 0.12±0.00 6.28±0.07 5.68±0.44 0.27±0.04 8.80±0.36 - 16:0 15.16±0.47 0.08±0.01 2.78±0.11 17.33±1.97 13.97±0.94 0.52±0.06 18.51±1.40 0.59±0.12 18:0 3.27±0.14 0.32±0.03 3.45±0.38 3.44±0.09 2.78±0.00 0.66±0.10 3.24±0.01 2.21±0.42 20:0 0.39±0.01 0.30±0.01 0.64±0.06 0.66±0.04 0.28±0.03 0.69±0.10 0.55±0.02 0.33±0.02 21:0 2.92±0.15 2.53±0.10 1.60±0.07 3.55±0.19 2.31±0.00 0.89±0.09 2.83±0.00 1.72±0.12 23:0 0.88±0.07 1.90±0.05 1.35±0.22 1.17±0.20 0.77±0.01 1.93±0.06 0.68±0.00 1.63±0.08 ∑ SFA 131.28±0.08 5.45±0.17 10.29±0.85 33.30±2.23 27.07±1.50 4.95±0.33 35.87±1.68 6.96±0.67 16:1 n-7 8.31±0.02 0.49±0.02 1.32±0.10 8.21±0.09 7.77±0.60 0.42±0.07 9.86±0.17 0.39±0.04 17:1 1.06±0.00 0.14±0.01 0.11±0.00 0.93±0.04 0.85±0.06 0.07±0.01 1.20±0.00 0.18±0.01 18:1 n-9 7.94±0.16 1.81±0.05 7.39±0.44 8.54±0.42 11.16±0.02 1.16±0.08 8.87±0.02 9.29±0.70 18:1 n-7 3.31±0.05 0.59±0.04 2.66±0.25 2.80±0.19 3.59±0.01 0.36±0.06 3.01±0.03 2.46±0.18 ∑ MUFA 219.50±0.16 2.66±0.09 9.93±0.43 19.81±0.37 22.06±0.65 2.15±0.18 22.25±0.15 9.90±0.61 18:2 n-6 4.24±0.15 1.48±0.03 2.00±0.06 3.14±0.71 2.97±0.22 0.48±0.07 3.73±0.03 1.50±0.12 18:3 n-6 0.94±0.04 0.20±0.02 0.88±0.05 0.93±0.02 1.53±0.18 0.60±0.01 0.14±0.01 0.87±0.04 18:3 n-3 (ALA) 0.76±0.01 0.69±0.02 1.73±0.16 1.47±0.05 4.99±0.19 2.41±0.28 0.65±0.01 2.17±0.08 20:3 n-6 0.60±0.03 0.40±0.02 2.31±0.21 0.96±0.00 4.94±0.33 2.24±0.19 0.18±0.06 0.74±0.10 20:4 n-6 1.26±0.13 3.48±0.09 2.03±0.07 1.20±0.11 1.00±0.11 2.97±0.26 1.26±0.10 3.25±0.03 20:5 n-3 (EPA) 18.69±0.17 60.48±0.16 34.43±2.37 19.08±0.23 13.99±0.40 48.15±2.13 15.89±0.12 33.79±0.13 22:4 n-6 0.75±0.03 1.84±0.02 1.77±0.05 0.74±0.02 0.58±0.03 1.85±0.10 0.70±0.02 1.29±0.08 22:5 n-6 0.36±0.02 0.51±0.00 0.65±0.16 - 0.35±0.02 0.71±0.07 0.41±0.01 2.02±0.68 22:5 n-3 2.21±0.08 3.32±0.12 5.26±0.51 2.13±0.29 1.67±0.12 4.17±0.24 1.42±0.11 2.98±0.21 22:6 n-3 (DHA) 12.10±0.77 13.04±0.01 19.90±2.02 12.39±0.45 11.93±0.36 18.79±0.63 12.12±0.23 23.78±1.88 ∑ PUFA 342.19±1.01 85.81±0.10 70.96±5.53 42.04±0.13 44.13±1.13 82.93±4.00 36.70±0.15 72.74±1.47 ∑ n-3 PUFA 33.76±1.01 77.53±0.01 61.32±5.06 35.07±0.91 32.58±1.07 73.51±3.28 30.08±0.45 62.71±2.04 ∑ n-6 PUFA 8.14±0.00 7.91±0.11 9.64±0.47 6.97±0.78 11.38±0.10 8.86±0.69 6.42±0.21 9.67±0.59 Resultados 84 Table 2. (continuation) SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids. 1Includes 10:0, 12:0, 15:0, 17:0 2Includes 14:1, 16:1 n-9, 18:1 n-9 t; 24:1 3Includes 20:2 n-6 Sample Fatty acid (%) FO9 FO10 FO11 FO12 KO BL1 BL2 BL3 14:0 8.39±0.63 7.75±0.36 3.32±0.01 0.31±0.00 10.35±0.34 3.28±0.05 2.22±0.32 - 16:0 16.89±0.12 17.27±0.43 14.33±0.28 2.04±0.00 19.93±0.07 10.26±0.03 9.11±0.32 14.31±0.41 18:0 3.27±0.03 3.20±0.02 3.26±0.06 3.83±0.03 1.23±0.04 3.43±0.07 2.86±0.13 3.03±0.08 20:0 0.49±0.11 0.42±0.02 0.53±0.02 0.41±0.01 0.17±0.00 0.34±0.01 3.28±0.02 0.51±0.00 21:0 2.70±0.08 3.18±0.01 2.51±0.04 2.56±0.00 3.47±0.04 1.17±0.03 0.80±0.02 2.47±0.00 23:0 0.77±0.05 0.73±0.07 1.20±0.22 1.68±0.00 0.36±0.00 0.34±0.01 0.27±0.00 0.70±0.06 ∑ SFA 133.62±0.81 33.92±0.77 26.17±0.66 11.29±0.06 38.69±0.39 19.31±0.16 19.08±0.62 25.57±0.60 16:1 n-7 9.70±0.59 8.64±0.27 6.18±0.05 0.75±0.00 5.66±0.16 4.37±0.03 2.78±0.20 6.62±0.26 17:1 1.22±005 1.24±0.00 0.94±0.01 0.10±0.00 0.67±0.01 0.51±0.06 0.34±0.02 1.04±0.04 18:1 n-9 8.32±0.24 8.60±0.09 9.49±0.17 7.61±0.02 10.32±0.22 15.98±0.16 11.10±0.22 8.93±0.15 18:1 n-7 3.24±0.01 2.92±0.06 3.02±0.05 2.81±0.01 5.66±0.05 1.57±0.14 1.52±0.05 2.94±0.06 ∑ MUFA 221.42±0.26 20.86±0.40 19.32±0.23 12.14±0.03 23.69±0.43 21.56±0.05 14.78±0.02 19.63±0.47 18:2 n-6 3.65±0.04 3.83±0.07 3.26±0.07 1.04±0.00 3.00±0.07 24.03±0.95 30.85±0.00 3.43±0.05 18:3 n-6 0.84±0.01 0.75±0.03 1.21±0.10 0.17±0.00 0.23±0.00 - - 1.11±0.03 18:3 n-3 (ALA) 1.03±0.05 0.49±0.00 1.63±0.02 1.01±0.02 0.60±0.01 19.28±0.54 20.53±0.29 1.46±0.03 20:3 n-6 0.51±0.03 0.16±0.01 0.11±0.03 0.20±0.00 0.20±0.00 0.26±0.02 0.25±0.05 1.80±0.09 20:4 n-6 1.41±0.12 1.05±0.04 2.61±0.59 2.31±0.01 0.35±0.01 0.42±0.09 0.42±0.06 1.55±0.15 20:5 n-3 (EPA) 18.51±0.91 18.18±0.12 18.09±0.06 35.29±0.09 18.15±0.41 6.76±0.04 6.24±0.12 18.30±0.33 22:4 n-6 0.68±0.02 0.77±0.00 0.79±0.04 - - 0.31±0.00 0.24±0.01 0.83±0.03 22:5 n-6 0.38±0.02 0.40±0.03 0.49±0.00 - - 0.14±0.04 0.14±0.02 0.51±0.00 22:5 n-3 1.83±0.07 1.94±0.08 2.30±0.03 4.44±0.01 0.43±0.01 0.69±0.06 0.80±0.09 2.50±0.01 22:6 n-3 (DHA) 10.75±0.27 12.12±0.26 13.42±0.25 24.77±0.02 8.74±0.14 4.55±0.18 4.19±0.28 13.07±0.09 ∑ PUFA 339.78±0.51 39.90±0.46 44.07±0.92 69.87±0.07 31.71±0.50 56.44±1.05 63.67±0.33 44.80±0.05 ∑ n-3 PUFA 32.12±0.61 32.73±0.46 35.44±0.30 65.80±0.07 27.92±0.56 31.29±0.25 31.77±0.19 35.33±0.40 ∑ n-6 PUFA 7.48±0.10 6.96±0.01 8.47±0.61 4.07±0.00 3.79±0.06 25.15±0.80 31.90±0.14 9.23±0.35 Capítulo 2 85 The experimental EPA levels differ significantly from the ones given on the labels, except for samples FO2 and FO3. The differences verified ranged from 18% lower in FO7 to 71% higher in KO sample. Fourteen samples contained either equal or significantly higher levels of DHA compared to the label information, with sample FO6 containing almost 3 times the stated amount (211 mg vs. 75 mg, respectively). Samples FO10 and FO11 did not declare the amount of n-3 PUFA, although sample FO11 stated EPA+DHA content. Brands BL1, BL2, and BL3 are blends of fish oil and vegetable oils, formulated to supply omegas 3, 6 and 9. Samples BL1 and BL2 presented significantly higher ALA levels than claimed (24 and 5%, respectively) while BL3 contained significantly less ALA than the labelled amount (14.5 vs. 200 mg/capsule). FA profile of this sample (BL3) is very similar to the fish oil profile, indicating that, contrary to what is claimed, flaxseed oil is absent or in very low amounts. FO2, FO6 and FO12 samples contained higher EPA and DHA levels than the other samples, which is in accordance to the presence of fish oil concentrate referred on the label. 3.3. Recommended Daily Doses Current dietary recommendations for EPA and DHA are based on cardiovascular risk considerations and range between 250 and 500 mg/day for healthy adults (EFSA, 2010; Kris-Etherton et al., 2009). For individuals with established cardiovascular disease (CVD), the American Heart Association (AHA) recommends the intake of 1 g/day of EPA and DHA (Smith et al., 2006). Although recommended doses may be achieved by consuming two servings of (preferably oily) fish per week, supplementation of the diet with oil supplements can be an alternative to ensure an optimal n-3 PUFA intake (Smith et al., 2006). Based on the recommended daily doses (RDD) from manufacturers, sample BL2 fail to meet minimum concentration of LC n-3 PUFA recommended for primary prevention of coronary disease (250 mg/day), even when using the highest recommended dosage (± 180 mg/ 2 capsules). The ingestion of the RDD of FO5, FO9, KO, BL1, and BL3 samples provides less than 500 mg/day of EPA+DHA, with levels ranging from 287 to 328 mg/day. For all the other samples, the RDD exceeded the 500mg/day. FO3, FO6, and FO12 samples provided more than 1 g/day of total LC n-3 PUFA, the recommended daily intake by AHA for individuals with established CVD, with sample FO12 delivering almost three times that dose (2832.0 mg EPA and DHA). Resultados 92 Capítulo 2 93 2.3 – Suplementos Alimentares com Fitoestrogénios E. Food supplements intended for menopause symptoms relief: isoflavones levels and bioavailability using Caco-2 cell model. submitted Resultados 94 Capítulo 2 95 Food supplements intended for menopause symptoms relief: isoflavones levels and bioavailability using Caco-2 cell model I.M.C. Almeidaa, F. Rodriguesa, B. Sarmentob,c, R. C. Alvesd, M.B.P.P. Oliveira,a* aREQUIMTE / Dep. Ciências Químicas, Faculdade de Farmácia, Universidade do Porto. bIINFACTS - Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Dep. Pharmaceutical Sciences, CESPU. cINEB – Instituto de Engenharia Biomédica, Portugal. dREQUIMTE, ISEP, Instituto Politécnico do Porto, Portugal.* [email protected] Abstract A simple and efficient matrix solid-phase dispersion (MSPD) method was optimized and validated for the extraction of native isoflavones from food supplements (n=15) intended for menopause. The compounds were analysed RP-HPLC-DAD. The proposed method was successfully applied to the analysis of puerarin, daidzin and genistin, daidzein, glycitein, genistein, formononetin, prunetin, and biochanin A. Recoveries ranged from 90.1% to 101.2% for all isoflavones, and relative standard deviations were below 6%. Isoflavones were quantified and the results compared to the labelled information. Label claims were inconsistent with the determined isoflavones content for the majority of the assayed samples. Only four food supplements comply with the recommendations made by the European Community on the tolerable thresholds of the content of claimed constituent (80–145%). Isoflavones bioavailability of three food supplements (standardized isoflavonoids from soy, soy extract, and red clover) was further investigated using human intestinal epithelial Caco-2 cell monolayers. The apparent permeability coefficients (Papp) of selected isoflavonoids across the Caco-2 cell monolayers were found to be affected by the isoflavone concentration and the product matrix. Keywords: Food supplements, menopause, isoflavones, HPLC-DAD, bioavailability, Caco-2 cells Resultados 96 Introduction Today consumers are interested in taking an active role in their health and self-care, by selecting specific foods and other items, including food supplements, that they believe it can improve their health and quality of life. Food supplements are generally used to overcome nutritional deficiencies, prevent or reduce the risk of disease, and/or to promote general well-being. The wide and steadily growing consumption range and popularity of food supplements, constitutes a challenge for consumer protection. Food supplements are not subjected to standardized quality control measures, unlike pharmaceuticals, and, therefore, the presence of impurities, adulteration, and dosage inconsistency are more frequent. Several studies have shown a great variability in marketed products regarding the concentration and source of isoflavones, stressing the need of standardization and quality control of these products, considering its therapeutic use (Boniglia, Carratù, Gargiulo, Giammarioli, Mosca & Sanzini, 2009). Additionally, the biological effectiveness of these bioactive compounds depends greatly on the intestinal bioavailability, variable between isoflavones. Isoflavones are phytoestrogens, plant-derived compounds that have weak estrogenic activity. Their structural analogy to the estradiol molecule confers them hormonal effects, including the ability of binding to estrogen receptors and modulating hormone-dependent processes (Tham, Gardner & Haskell, 1998). Isoflavones are primarily found in plants of the Fabaceae family, including soy, lentils, bean plant, chickpeas, alfalfa and red clover (Fletcher, 2003). A number of epidemiological studies have linked the consumption of isoflavone-rich foods with low incidence of the major hormone-dependent cancers (Adlercreutz, 1995; Severson Nomura, Grove & Stemmerman, 1989), cardiovascular diseases (Clarks, Anthony & Hughes, 1995), osteoporosis (Tham et al., 1998), and climacteric complaints (Adlercreutz, Hämäläinen, Gorbach & Goldin, 1992). Driven by these purported health benefits, a plethora of products containing isoflavones have come on to the market, specifically targeting women in menopause. These preparations generally contain extracts from soy, red clover and kudzu, as single ingredients or in multi-ingredient formulations mixed with minerals, vitamins, other plant extracts, omega-3, 6, and 9 fatty acids, etc. The three major isoflavones found in soybean (Glycine max (L.) Merril) are daidzein, glycitein and genistein, which occur mainly as glycoside, acetylglycoside, and malonylglycoside forms. In soy derivates, including food supplements, the glycoside and aglycone forms are the main components (Coward, Smith, Kirk & Barnes, 1998). In Capítulo 2 97 contrast to soybean, red clover (Trifolium pratense L.) contains numerous isoflavones, being biochanin A and formononetin, and their glycosides and malonyl derivatives, the major isoflavonoids components (Rijke, Zafra-Gómez, Ariese, Brinkman & Gooijer, 2001; Wu, Wang, & Simon, 2003). Pueraria mirifica Airy Shaw et Suvatabhandu (Fabaceae), commonly known as White Kwao Krua and Thai kudzu, is an indigenous herb to Thailand, and has traditionally been used as folk medicine for rejuvenation and to attenuate menopausal symptoms. The dried powder of the plant tubers has also been used to prepare food supplements. Several isoflavonoids have been identified in P. mirifica tubers, including daidzin, puerarin, daidzein, genistin, and genistein (Malaivijitnond, 2012). Isoflavones, like the majority of polyphenols, are usually found in plants mainly as glycosides and glycoside esters (Tsao, 2010). After ingestion they are metabolised by bacteria in the gastrointestinal tract, releasing their aglycones, the truly bioactive constituents (Day, et al., 1998; Setchell et al., 2002). It is, therefore, very important to know the bioavailability of such compounds in order to understandand their biological activity. In this perspective, the Caco-2 cell line, derived from human colorectal adenocarcinoma, has been widely used as an in vitro model of the human small intestine for predicting drug intestinal absorption and excretion in humans (Artursson, Palm & Luthman, 2012). Several Caco-2 cell line studies have examined the intestinal absorption of isoflavones as pure compounds (Steensma, Noteborn, van der Jagt, Polman, Mengelers & Kuiper, 1999; Murota, Shimizu, Miyamoto, Izumi, Obata, Kikuchi & Terao, 2002; Chen, Lin & Hu, 2005; Tian, Yang, Yang & Wang, 2009), but studies using isoflavones-containing food supplements extracts are still very scarce (Wang, Chen, Joseph & Hu, 2008). The aim of the present study was to optimize and validate a simple analytical procedure for the quantification of nine isoflavones (puerarin, daidzin and genistin, daidzein, glycitein, genistein, formononetin, prunetin, and biochanin A) in 15 commercial food supplements, by Matrix Solid-Phase Dispersion (MSPD) and reversed-phase (RP) HPLC-DAD. The obtained quantitative data were compared with the labelled contents. Moreover, a study was undertaken to measure permeability in the human colon adenocarcinoma Caco-2 cell line of selected isoflavones from selected food supplements. 2. Material and methods 2.1 Chemicals and reagents Puerarin (≥99%), daidzin (≥95%), genistin (≥95%), glycitein (≥97%), daidzein (≥98%), genistein (≥98%), biochanin A (≥97%), prunetin (≥98%), and formononetin Resultados 98 (≥99%) and the internal standard 2-methoxyflavone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Preparative C18 sorbent (125 Å, 55-105 µm) was from Waters (Milford, MA, USA). Water was purified with a “Seradest LFM 20” system (Seral, Ransbach-Baumbach, Germany). The eluents were filtered through 0.45 µm filters and degassed under reduced pressure. Disposable cellulose acetated 0.45 µm was from OlimPeak, Teknokroma (Barcelona, Spain). HPLC grade solvents, methanol and acetonitrile, and analytical grade formic acid were from Merck (Darmstadt, Germany). Caco-2 (ATCC HTB-37) human colon adenocarcinoma cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose and GlutaMAX™, fetal bovine serum (FBS), 0.05% trypsin–EDTA, penicillin–streptomycin (Lonza Biowhittaker, Verviers, Belgium) and non-essential amino acids (NEAA) were obtained from Gibco (Life Technologies, Paisley, UK). Phosphate buffer saline (PBS) was purchased from Sigma Chemical Co., USA. Tissue-treated inserts (high density PET membrane, 23.1 mm, 0.3 μm pore size, 4.2 cm2) were from Becton Dickinson Falcon™ (Bedford, MA, USA) and 6 wells plates were purchased from Orange Scientific (Braine-l'Alleud, Belgium). 2.2 Standards Purity-corrected individual isoflavones stock solutions were prepared in DMSO (1 g/L) and then serially diluted in water: methanol (10:90, v/v) in order to obtain the standard concentrations for the calibration curves. A working 2 mg/L solution of the internal standard was also prepared in DMSO. All solutions were stored in amber glass vials at 4 °C. 2.3 Samples and sample preparation 2.3.1 Sampling Fifteen different food supplements containing isoflavones were purchased from local retail, herbal stores and a pharmacy. Their selection was based on availability on the market, trying to include a broad range of isoflavone profiles and concentrations. Table 1 shows the composition of the food supplements and the recommended daily dose (RDD) as indicated by the manufacturer. The food supplements were in the form of tablets and capsules. Ten tablets or capsules were taken from each product. Tablets were ground to a fine powder using a glass mortar and pestle, after carefully removing the colour coatings, if present. Shells from hard-gelatine capsules were removed and the content Capítulo 2 99 mixed with mortar and pestle. Contents of soft gel capsules were squeezed into a test tube and mixed. Table 1. Sample code and specifications of the analysed food supplements as provided by the manufacturers. Sample Code Country of origin and composition Dosage form Capsule/ tablet weight (g) S1 EU. Natural isoflavonoids from non-transgenic soy Capsule 0.394 S2 France. Soy extract (Glycine max); excipients Capsule 0.334 S3 France. Tomato extract, milk proteins, soy extract, vitamin C Capsule 0.740 S4 United Kingdom. Evening primrose oil; soy isoflavones; fish oil; vitamin E, excipients Gel capsule 0.564 S5 UE. Coral Calcium, soy extract rich in phytoestrogens, Passiflora; excipients Tablet 1.237 S6 France. Yam extract (Dioscorea opposita Thunb), soy extract (Glycine max), fructo Oligo Saccharides, hop (Humulus lupulus), meadowsweet (Spiraea ulmaria) , grape vine (Vitis vinifera), vitex (Vitex agnus castus); vitamin E; selenized yeast Capsule 0.307 S7 Portugal. Discorea opposite, wild yam, Soy (Glycine max) (pure isoflavones), primrose oil, Dong Quai (Angelica sinenis); melissa (Melissa officinalis); sage (Salvia officinalis), siberian ginseng (Eleutherococcus senticosus); hop (Humulus lupulus); vitex (Vitex agnus-castus); vitamins E, B6 Gel capsule 0.470 S8 Spain. Red algae (Lithothamnium calcareum); fermented soy; soy isoflavones Capsule 0.565 S9 EU. Evening primrose oil (Oenothera biennis); soy isoflavones; vitamin E; excipients Gel capsule 0.447 S10 Belgium. Sage extract; soy extract; saffron (Crocus sativus); vitamin B6 Tablet 0.772 S11 EU. Soy isoflavones; sage; oat; marine magnesium; vitamin E; excipients Capsule 0.538 S12 Italy. Soy isoflavones (with 55-72% genistin/genistein); excipients Capsule 0.623 RC Australia. Standardized red clover (Trifolium pratense) extract; excipients Tablet 0.351 SR Spain. Soy rich in isoflavones of retarded action; yam extract; red clover extract; vitamins A, C, B1, B2, B12, E, excipients Capsule 0.634 TK USA. Vitamin B12; standardized Pueraria mirifica root extract (Thai Kudzu): miroestrol, isoflavonoids; pyridoxal-5 phosphate; biotin; folic acid; excipients Capsule 0.519 Resultados 100 2.3.2. Matrix solid-phase dispersion An aliquot of 0.5 g of the previously homogenized samples, 2 g of C18 and 0.5 mL of the internal standard (2 g/L,) were placed in a glass mortar and blended together using a glass pestle to obtain a complete disruption and dispersion of the sample on the solid support. After blending was complete (1min), the sample was packed into an empty column containing a polyethylene frit at the bottom. A second frit was placed on the top of the sample by careful compression with a syringe plunger. The packed column was attached to a vacuum manifold (Visiprep, Supelco) coupled with a vacuum pump and the flow adjusted to 1mL/min. The column was rinsed with 10 mL water (discharged fraction) and the elution step was carried out with 2×5mL of water: methanol (10:90, v/v) (collected fraction). The extracts collected in amber vials were filtered through disposable 0.45 µm cellulose membranes before HPLC analysis. Sample extraction was performed in triplicate. 2.4. HPLC equipment The chromatographic analysis was performed using an HPLC unit (Jasco, Japan), consisting of two Jasco PU-2080 Plus HPLC pumps, an AS-950 automated injector (20 µL loop), and a MD-2010 Plus multiwavelength diode-array detector (DAD). The separation of the isoflavones was carried out on a reversed-phase Luna C18 column (4.60 mm × 150 mm, 5 µm particle size) from Teknokroma (Barcelona, Spain), maintained at 40 °C. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient program was previously developed in our laboratory (Visnevschi-Necrasov et al., 2009), and was used with some modifications: 0 min 0% B, 15min 32% B, 18 min 45% B, 23 min 50% B, 25 min 70% B, 35 min 10% B, maintaining these conditions for 5 min and returning to the initial conditions within 3 min. The flow rate of the mobile phase was 1 mL/min and the injection volume was 20 μL. UV spectral characteristics and peaks purity were evaluated trough DAD. Peak purity measurements of all compounds were based on spectral comparison at three different peak heights. Analytes were monitored at 254 nm and quantification was performed on the basis of the internal standard method. Chromatographic data were processed with ChromNAV Software (Jasco, Japan). Capítulo 2 101 2.5 Caco-2 cell culture The human colorectal carcinoma cell line Caco-2 was obtained from ATCC. Cells were routinely cultured in 75-cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM) containing D-glucose (4.5 g/L) and GlutaMAX™ and supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin (10,000 U/mL), and 1% MEM Amino Acids. The cells were maintained in a humidified atmosphere of 5% CO2/95% air at 37°C, and were supplied with fresh medium every 2 days. Cells were subcultured at 80-90% confluence. For experiments, Caco-2 cells were seeded in cell culture inserts in 6-well plates at a density of 4×104 cells/cm2. The basolateral and apical compartments contained 2.5 and 1.5 mL of culture medium DMEM, respectively. Culture medium was replaced every other day. The integrity of the Caco-2 cell monolayer was checked by Transepithelial Electrical Resistance (TEER) measurements using an epithelial voltammeter (EVOM, World Precision Instrument, Sarasota, FL, USA) for the whole period. 2.6 Isoflavones permeation experiments Cells used for the current study were from passage 65. Experiments in triplicate were performed 21 days after seeding. The cell monolayers were pre-equilibrated with fresh PBS pH 7.4 previously warmed at 37 °C, for 30 min, and the incubation medium was then discarded. Afterwards, 1.5 mL of the test solutions, prepared by dilution of the MSPD extracts in PBS, were added to the apical side of the Caco-2 monolayers and 2.5 mL of PBS to the basolateral side, and allowed to permeate for 120 min at 37 °C under 5% CO2, 95% of relative humidity. Samples (0.5 mL) were withdrawn from the receptor side at 0, 15, 30, 60, and 120 minutes for the determination of the isoflavone transported across the monolayer. After each sampling, the basolateral side was replenished with the same PBS volume. Samples were preserved at -20 °C for subsequent HPLC analysis as described in section 2.4. The apparent permeability to the isoflavones, expressed in cm/sec, was calculated from the following equation: Papp = Q/ (A x C x t) where, Q is the total amount of permeated isoflavones during the 120 minutes of experiment (μg), A is the diffusion area (cm2), C is the donor compartment concentration at time zero (μg/mL), and t is the time of experiment (s). In addition to Papp, the percentage of permeation (%) of the isoflavones daidzein, genistein, formononetin, and biochanin A was calculated as the proportion of the Resultados 108 due to the complexity of the extract compared to the pure solution, as the extracted matrix also possess additional components responsible for the diffusion saturation, resulting in the delay of permeability kinetics for genistein in the Caco-2 cells. Moreover, the commercial products also contain mucilage on their composition, which may act as a physical barrier against the free diffusion of compounds through the epithelia layer, as also observed by Jia, Chen, Lin and Hu (2004). This may also justify the Papp differences between different extracts. 4. Conclusions The proposed method to quantify isoflavones in food supplements is based on a MSPD extraction in the presence of an internal standard, followed by the direct HPLC/DAD analysis. As far as we know, this is the first time that MSDP is applied to isoflavones quantification in food supplements. The results obtained show significant differences between labelled and determined contents for the majority of supplements. The quantification of isoflavones in food supplements is important for the control of their contents, since they are choose by their therapeutic properties. Moreover, the apparent permeability coefficients (Papp) of the isoflavonoids across the Caco-2 cell monolayers were found to be affected by the isoflavone concentration and the product matrix. Reliable labelling information, better standardization, improved manufacturing practices and regulation of the market is required to assure consumers of the quality of isoflavone supplements. This study stresses the need for careful selection of isoflavonecontaining food supplements by consumers, retailers and health care professionals. Acknowledgments: I. Almeida, F. Rodrigues and R. Alves are grateful to FCT for PhD and a postdoctoral grants (SFRH/BD/66032/2009, SFRH/BDE/51385/2011, SFRH/BPD/68883/2010, respectively) financed by POPH-QREN and subsidised by European Science Foundation and Ministério da Ciência, Tecnologia e Ensino Superior. This work has been supported by FCT through grant no. PEst-C/EQB/LA0006/2013 and NORTE-07-0124-FEDER000069Food Science. References Adlercreutz, H. (1995). 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Journal of Chromatography A, 983, 271–275. 3. CONSIDERAÇÕES FINAIS Capítulo 3 117 3.1 Considerações Finais O controlo dos ingredientes dos suplementos alimentares está longe de ser assegurado por todos os fabricantes. A falta de conformidade com os valores rotulados tem sido reportada para vários produtos disponíveis no mercado, pondo em causa a função para que estão destinados. Tendo em conta o seu consumo generalizado, tornase necessário conhecer os produtos que se encontram no mercado e os seus ingredientes, garantir a segurança das dosagens recomendadas e avaliar a real biodisponibilidade dos diversos compostos. A regulamentação dos suplementos alimentares tem sido alvo de harmonização na UE a fim de melhorar a qualidade destes produtos e garantir a segurança dos consumidores. Têm sido publicadas listas positivas para vitaminas e minerais, relativamente às formas químicas e limites permitidos. O número de outras substâncias com efeito nutricional ou fisiológico que os suplementos alimentares podem conter está estimado em mais de 400, atualmente, no mercado europeu. Estas substâncias são regidas pela legislação nacional de cada Estado-Membro. O número avassalador de substâncias permitidas torna a tarefa da sua regulamentação e controlo um enorme desafio. O presente trabalho teve por objetivo melhorar o conhecimento sobre diferentes grupos de suplementos alimentares disponíveis em Portugal. Foram avaliados suplementos antioxidantes, suplementos contendo ácidos gordos e suplementos à base de isoflavonas. Tendo em conta a diversidade de compostos bioativos avaliados foi necessário recorrer a diferentes metodologias analíticas, as mais adequadas à quantificação dos compostos químicos de interesse nas diferentes matrizes. Atualmente encontram-se no mercado diversos produtos que reivindicam propriedades antioxidantes. No entanto, a informação rotulada é geralmente escassa. Os suplementos antioxidantes presentes no mercado apresentam uma composição extremamente variada, incluindo diferentes moléculas hidrofílicas e lipofílicas, extratos naturais ou compostos sintéticos com propriedades antioxidantes. Foi determinada a atividade antioxidante dos suplementos, individualmente ou combinados, através de três ensaios in vitro: a atividade captadora de radicais livres 1,1-difenil-2-picril-hidrazilo (DPPH), o poder redutor, e a inibição da peroxidação lipídica na presença de substâncias reativas do ácido tiobarbitúrico (TBARS). Posteriormente, utilizou-se uma análise