Bioaccessibility, bioactivity and cell metabolism of dark chocolate phenolic compounds after in vitro gastro-intestinal digestion

Abstract

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...

Antiproliferative Activity and Cell Metabolism of Hydroxycinnamic Acids in Human Colon Adenocarcinoma Cell Lines

“…Coumaroylquinic acids and hydroxycinnamoyl aspartates enriched fractions were extracted from in vitro digested sweet cherry (Celeste cultivar) and dark chocolate, respectively, following the protocol reported in Tagliazucchi et al with some modifications. 17 The two food matrices were digested as reported in Martini et al 14 Firstly, SPE column were preconditioned with 4 mL of methanol, followed by 5 mL of water adjusted to pH 7.4 with NaOH 1 mol/L. Then, 1 mL of the in vitro digested fractions were passed through the pre-conditioned C-18 column to adsorb neutral phenolic compounds.…”

“…[11][12] Recently, our research group thoroughly investigated the effect of in vitro gastro-intestinal digestion on the bioaccessibility of phenolic compounds from two different cherry cultivars (Celeste and Durone Nero I) and three different types of dark chocolates. [13][14] Phenolic acids and especially hydroxycinnamic acids showed the highest bioaccessibility in all the food matrices. The highest release after incubation of cherries with digestive fluids was found for coumaroylquinic acids, followed by feruloylquinic and caffeoylquinic acids; a similar trend was seen for the conjugate forms of hydroxycinnamic acids with aspartic acid after in vitro digestion of dark chocolate.…”

“…We also demonstrated that hydroxycinnamic acids tended to accumulate in cell media suggesting a possible role in the anti-proliferative activity of in vitro digested cherry and dark chocolate against colon adenocarcinoma cell lines. [13][14] The stability of phenolic compounds in cell culture media as well as their cellular metabolism are two aspects to be critically addressed when testing their anti-proliferative activity. Their instability in cell media may hinder their biological activity, severely changing the concentrations and/or the chemical structures of the tested compounds.…”

Physicochemical, Antioxidant, Microstructural Properties and Bioaccessibility of Dark Chocolate with Plant Extracts

“…On the other hand, in vitro digestion models are becoming useful tools for studying the digestive properties of chocolates and cocoa-based products and for understanding the mechanisms of lipid absorption and the bioaccessibility of amino acids, bioactive amines, polyphenols, and other antioxidants [ 6 , 12 , 13 ].…”

Solvent extraction and in vitro simulated gastrointestinal digestion of phenolic compounds from purple sweet potato

“…In vitro gastrointestinal digestion is a method of simulating the digestion of an organism in a laboratory, often used as an alternative to biological environment (Van et al, 2004). Gastrointestinal digestion could significantly increase the bioaccessibility of phenolics and the antioxidant activity (Martini et al, 2018). The release of polyphenols mainly occurs during the gastric phase; however, different plants showed different results for the intestinal phase.…”