Simula??o de digest?o in vitro acoplada a modelos de transporte g?strico e intestinal para estimar a capta??o e absor??o de antocianinas em frutos

• Main Author: PEIXOTO, Fernanda Marques
• Other Authors: Godoy, Ronoel Luiz de Oliveira
• Format: Others
• Language: Portuguese
• Published: Universidade Federal Rural do Rio de Janeiro 2017
• Subjects: Bioaccessibility; ln vitro digestion; Bioacesibilidade.; Caco-2; MKN-28; Digest?o in vitro; Ci?ncia de Alimentos
• Online Access: https://tede.ufrrj.br/jspui/handle/jspui/2016

Submitted by Jorge Silva ([e-mail]) 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.