1The bioaccessibility of phenolic compounds after in vitro gastro-intestinal digestion of dark 2 chocolate, dark chocolate enriched with Sakura green tea and dark chocolate enriched with turmeric 3 powder was studied. The phenolic profile, assessed by accurate mass spectrometry analysis, was 4 modified during in vitro gastro-intestinal digestion, with a considerable decrease of total and 5 individual phenolic compounds. Phenolic acids showed the highest bioaccessibility with 6 hydroxycinnamic acids displaying higher bioaccessibility (from 41.2% to 45.1%) respect to 7 hydroxybenzoic acids (from 28.1% to 43.5%). Isomerisation of caffeoyl-quinic acids and galloyl-8 quinic acids as well as dimerization of (epi)gallocatechin were also observed after in vitro gastro-9 intestinal digestion. Antioxidant activity increased after the gastric step and rose further at the end 10 of the digestion. Furthermore, in vitro digested phenolic-rich fractions showed anti-proliferative 11 activity against two models of human colon adenocarcinoma cell lines. Cell metabolism of digested 12 phenolic compounds resulted in the accumulation of coumaric and ferulic acids in the cell media. 13 14 Keywords: mass spectrometry, in vitro digestion, cell metabolism, functional foods, Caco-2, 15 SW480 16
Materials and methods 57
Materials 58Phenolic compound standards, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 59 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tri(2-pyridyl)-S-triazine 60 (TPTZ), Folin-Ciocalteau phenol reagent were purchased from Sigma (Milan, Italy). Methanol and 61 formic acid were obtained from Carlo Erba (Milan, Italy). All MS/MS reagents were from Bio-Rad 62 (Hercules, CA, U.S.A.). Chemicals and enzymes for the digestion procedure were purchased from 63Sigma-Aldrich (Milan, Italy). All the materials and chemicals for cell culture were from Euroclone 64 (Milan, Italy). MTS cell proliferation assay kit was purchased from Promega (Milan, Italy). Solid 65 phase extraction (SPE) columns (C18, 50 μm, 60 Å, 500 mg) were supplied by Waters (Milan, 66 Italy). Three different types of chocolate (dark 70% cocoa (DC), dark 70% cocoa and 8% turmeric 67 (TDC), dark 70% cocoa and 2% Sakura green tea (GTDC)) were bought from a local shop in 68Modena (Italy). The chocolates were all from the same manufacturer and had the same composition. 69The ingredients were cocoa mass, sugar, cocoa butter, soya lecithin and natural flavour vanilla. 70 GTDC and TDC were enriched with 2% Sakura green tea leaves and 8% turmeric powder, 71 respectively. Three chocolate bars for each sample were used in this study. 72 73
In vitro gastro-intestinal digestion of dark chocolates and preparation of the chemical extract 74For the in vitro digestion, the protocol previously developed within the COST Action INFOGEST 75 was followed (Minekus et al., 2014). The procedure consisted of three consecutive steps: oral, 76 gastric and intestinal phases. The three steps were carried out in absence of light. Simulated 77 salivary...