Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-30T14:26:59Z No. of bitstreams: 1 2010 - Fernanda Peixoto Giori.pdf: 2362278 bytes, checksum: a15d423533272c981b15d7f22b714be1 (MD5) / Made available in DSpace on 2016-08-30T14:26:59Z (GMT). No. of bitstreams: 1 2010 - Fernanda Peixoto Giori.pdf: 2362278 bytes, checksum: a15d423533272c981b15d7f22b714be1 (MD5) Previous issue date: 2010-02-23 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior-CAPES / Brazil was adverted by the World Health Organization (WHO) as a sub-clinical area of serious vitamin A deficiency. Thus, the Brazilian Agricultural Research Corporation (EMBRAPA) is selecting and improving varieties of sweet potatoes with higher levels of Beta-carotene, pro-vitamin A. The carotenoids provitamin A amount of in foods does not necessarily correspond the amount that is absorbed and metabolized by the body. For a better determination of these values and knowledge of the mechanisms of its transport and absorption, it is necessary to understand the factors that lead to the food matrix release, until the absorption and the influence on the promotion and maintenance in human health. In order to perform preliminary studies of its absorption, this study aims to determine the efficiency of micellization of Beta -carotene in orange sweet potato (Ipomoea batatas, Lam), by applying a in vitro digestion as a tool for determining the bioaccessibility, which is the first step for bioavailability determination. This approach aims to simulate the oral, gastric and intestinal stages of human. The digestion was performed with 10 g of fresh samples, homozeneided with 5% (w/w) of canola oil. The extraction was performed with acetone and petroleum ether and the micellar fraction with petroleum ether, NaCl 10% (w / v) and NaSO4 2% (w/v) and involves the use of enzymes as -amylase , pepsin, bile, pancreatin, lipase and mucin, and inorganic compounds such as KCl, KSCN, NaH2PO4, Na3PO4, NaOH, NaCl, CaCl2, HCl, NaHCO3. The physiological variations are reproduced by the heating bath shaker with orbital gyrus (37?C) and centrifugation (5000g, 45 min). Quantification and determination of the profile of carotenoids were performed by high performance liquid chromatography (HPLC) with YCM ? C30 Carotenoid S-3 4.6 x 250mm column. Quantification of total carotenoids was performed by UV-VIs. The whole procedure was performed under controlled temperature (25 ? C) and light. The Beta -carotene was present mainly with levels of 86%, 73% and 82% for access 1, 2 and 3 and after digestion, the profile of Beta -carotene has set levels of 96%, 89% and 100%, respectively. The efficiency of micellization was 23.8%, 28% and 25% for 1.2 and 3 hits, indicating Beta -carotene transfer of the food matrix to micelles, corresponding to bioaccessibility of the compound. This methodology proved to be faster and cheaper, since the in vivo studies are costly, complex and require more time. / O Brasil foi classificado pela Organiza??o Mundial da Sa?de (OMS) como ?rea de car?ncia sub-cl?nica grave de vitamina A. Assim, a Empresa Brasileira de Pesquisa Agropecu?ria (EMBRAPA) v?m selecionando e melhorando variedades de batata-doce com teores maiores de Beta-caroteno, composto pr?-vitamina A. A quantidade de caroten?ides pr?-vitamina A presentes nos alimentos n?o corresponde necessariamente ?quela quantidade absorvida e metabolizada pelo organismo. Para uma melhor determina??o destes valores e conhecimento dos mecanismos de transporte e absor??o deste composto, faz-se necess?rio, o entendimento dos fatores que levam ? sua libera??o da matriz do alimento, at? a extens?o de sua absor??o, bem como a influ?ncia na promo??o e manuten??o da sa?de humana. A fim de realizar estudos preliminares de sua absor??o, este trabalho visa determinar a efici?ncia de miceliza??o de Beta-caroteno de batata-doce de polpa alaranjada (Ipomoea batatas, Lam.), atrav?s da aplica??o de digest?o in vitro, como ferramenta de determina??o da bioacessibilidade, etapa preliminar para a determina??o da biodisponibilidade. Esta metodologia visa simular as etapas de digest?o oral, g?strica e intestinal humana. Foram pesados 10g de amostra in natura e adicionados 5% (p/p) de ?leo de canola. A extra??o do alimento foi realizada com acetona e ?ter de petr?leo e a da fra??o micelar, com ?ter de petr?leo, NaCl 10%(p/v) e NaSO4 2%(p/v) . Enzimas como: a-amilase, pepsina, bile, pancreatina, lipase e mucina, bem como compostos inorg?nicos, tais como KCl, KSCN, NaH2PO4, Na3PO4, NaOH, NaCl, CaCl2, HCl, NaHCO3. As varia??es fisiol?gicas foram reproduzidas pelo banho de aquecimento com giro orbital (37?C) e centrifuga??o (5000g;45 min). A quantifica??o e determina??o do perfil de caroten?ides foi realizada por cromatografia l?quida de alta efici?ncia (CLAE), com coluna YCM? Carotenoid C30 S-3 de 4,6 x 250mm. A quantifica??o de caroten?ides totais foi realizada por espectrofotometria UV-VIs. Todo o procedimento foi executado sob temperatura (25?C) e luz controlada. O Beta-caroteno estava presente majoritariamente, com teores de 86%, 73% e 82%, para as variedades 1, 2 e 3 e ap?s a digest?o, o perfil do Beta-caroteno passou a configurar teores de 96%, 89% e 100%, respectivamente. A efici?ncia de miceliza??o foi de 23,8%, 28% e 28,9% para as variedades 1,2 e 3, indicando a transfer?ncia do - caroteno da matriz do alimento para as micelas, correspondendo a bioacessibilidade deste composto. Esta metodologia demonstrou-se mais r?pida e mais barata, quando comparada aos estudos in vivo, que s?o mais onerosos, complexos e demandam mais tempo.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-09-05T20:11:17Z No. of bitstreams: 1 2016 - Fernanda Marques Peixoto.pdf: 14003225 bytes, checksum: 89c95a9ad22b1e74cdf2bda273665230 (MD5) / Made available in DSpace on 2017-09-05T20:11:17Z (GMT). No. of bitstreams: 1 2016 - Fernanda Marques Peixoto.pdf: 14003225 bytes, checksum: 89c95a9ad22b1e74cdf2bda273665230 (MD5) Previous issue date: 2016-12-08 / A lot of interest in the consumption of anthocyanins increased after the association of their intake and reduced risk of chronic diseases. Despite of in vitro evidences of anthocyanins benefits to health, there is still a gap in the knowledge of the mechanisms of absorption of anthocyanins by the human body. It is known that concentration of food anthocyanins doesn't reflect the amount of these compounds which are absorbed, metabolized, distributed and biologically active in humans. Some in vitro models have been developed to evaluate the steps of cell release and transport ( uptake) of these compounds from food. The objective of this study was to evaluate the in vitro absorption of food anthocyanins using the in vitro digestion followed by uptake and transport in Caco-2 human intestinal cell line and MKN-28 human gastric cell line. Initially, anthocyanins bioaccessibility of diverse fruits was evaluated in order to select the better sources for transport assays. The bioaccessibility assays were performed using an in vitro digestion model, which mimics the human oral, gastric and intestinal stages. Quantification and characterization of anthocyanins profile were performed by high-performance liquid chromatography (HPLC) with Thermo Scientific? C1s 2.4 (4.6 x 10mm) column. After selection of the most promising fruits, the bioaccessibility tests were followed by transport assays. To assess gastric absorption, the product from gastric digestion was applied on the MKN-28 cell monolayer, which was obtained after 7 days of culture of 2.5 x 10^5 MKN-28 cells seeded in RPMI culture media in transwell? plates. The permeate was collected after 30, 60, 120 andl80 minutes oftransport. For evaluation of intestinal absorption after digestion, the digesta from the intestinal phase was applied on the Caco-2 cell monolayer, which was obtained after 21 days of culture of 2.5 x 105 Caco-2 cells seeded in DMEM culture media in TRANSWELL? plates. The permeate was collected after 30, 60 and 120 minutes of transport. All analyses were made by forming CLUE / photodiode array detector (Thermo? Scientific) at 520nm. Peel powder from jabuticaba, jambo and Jamel?o were the most promising sources. The bioaccessibility of anthocyanins after gastric digestion was 13% for jabuticaba, 45 % for jambo and 65 % for jamel?o. In addition, the intestinal bioaccessibility was 1 O % for jabuticaba, 15 % for jambo and 45 % for jamel?o. The transport assay with the MKN-28 gastric cell line, revealed 19.7%, 9.7 % and 14.1 % of transport efficiency, respectively, for jambo, jabuticaba and jamel?o digestion products. While Caco-2 intestinal cell model showed 0.8 %, 0.2 % and 0.3 % oftransport efficiency, respectively, for jambo, jabuticaba and jamel?o. These results suggest food anthocyanins are preferentially absorbed by the human gastric mucosa and to a lesser extent by the human intestinal epithelium. / O interesse pelo consumo das antocianinas aumentou ap?s o surgimento da rela??o entre o seu consumo e a redu??o do risco de doen?as cr?nicas. Apesar das evid?ncias in vitro quanto a esses beneficios ? sa?de, ainda h? uma lacuna que permanece sob investiga??o: o mecanismo de absor??o das antocianinas pelo organismo humano. Sabe-se que a quantidade desses compostos, nos alimentos, n?o reflete a quantidade absorvida, metabolizada, distribu?da e biologicamente ativa em humanos. Alguns modelos in vitro t?m sido desenvolvidos para avaliar as etapas de digest?o e transporte celular (absor??o) de compostos dos alimentos. Assim, o objetivo deste trabalho foi avaliar o transporte in vitro de antocianinas em alimentos utilizando modelos de digest?o in vitro seguido do transporte em c?lulas intestinais Caco-2 e c?lulas g?stricas MKN-28. Na 1? etapa, oito frutos foram analisados quanto aos valores de bioacessibilidade (BCSS) fornecidos pelas antocianinas presentes, para posterior sele??o para os ensaios de transporte. Os ensaios de BCSS foram realizados com um modelo de digest?o in vitro, para simula??o das fases oral, g?strica e intestinal humana. A quantifica??o e determina??o do perfil de antocianinas foram realizadas por Cromatografia l?quida de alta efici?ncia (CLAE), com coluna Thermo? Scientific C1s 2,4 (4,6 x 100mm). Na 2? etapa, realizou-se os ensaios de BCSS, anteriormente aos ensaios de transporte, nos frutos potencialmente mais promissores. Para a avalia??o do transporte g?strico, na sequ?ncia, o digerido g?strico foi aplicado sobre a monocamada de c?lulas MKN-28, com 2,5 x 10^5 c?lulas, em meio RPMI, em placa transwell? e, ap?s 7 dias de cultivo, o permeado foi coletado nos tempos 30, 60, 120, 180 minutos. Para o transporte intestinal, sequencial, o digerido intestinal foi aplicado sobre a monocamada celular Caco-2, com 2,5 x 105 c?lulas, em meio DMEM, em placas transwell? e, ap?s 21 dias de cultivo, o permeado foi coletado nos tempos 30, 60 e 120 minutos de transporte. Todas as an?lises foram realizadas por CLUE/detector de arranjo fotodiodo (Thermo? Scientific), a 520 nm. Os p?s da casca da jabuticaba, jambo e jamel?o foram as matrizes mais promissoras. A BCSS das antocianinas, ap?s a digest?o g?strica, foi de 13 % parajabuticaba, 45 % parajambo e 65 % parajamel?o, enquanto a BCSS intestinal foi de 10% para jabuticaba, 15 % para jambo e 45 % para jamel?o. Os ensaios de transporte (ET) com os modelos de c?lula MKN-28 resultaram em 19,7; 9,7 e 14,1 % de ET, respectivamente, para os p?s do jambo, jabuticaba, e jamel?o, enquanto que o modelo Caco-2, resultaram em 0,8, 0,2 e 0,3 % de ET, respectivamente. Estes resultados sugerem que as antocianinas s?o preferencialmente absorvidas pela mucosa g?strica.