Claire Dufour

A standardised static in vitro digestion method suitable for food-an international consensus

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

Antioxidant activity of olive phenols: mechanistic investigation and characterization of oxidation products by mass spectrometry

In this work, the antioxidant activity of olive phenols is first characterized by their stoichiometries n(tot)(number of radicals trapped per antioxidant molecule) and their rate constants for the first H-atom abstraction k(1) by the stable radical DPPH. It appears that oleuropein, hydroxytyrosol and caffeic acid have the largest k(1) values, whereas dihydrocaffeic acid, an intestinal metabolite of caffeic acid, is the best antioxidant in terms of n(tot). For phenols with a catechol moiety n(tot) is higher than two, implying an antioxidant effect of their primarily formed oxidation products. A HPLC-MS analysis of the main products formed in the AAPH-induced oxidation of olive phenols reveals the presence of dimers and trimers. With hydroxytyrosol and dihydrocaffeic acid, oligomerization can take place with the addition of water molecules.The antioxidant activity of olive phenols is then evaluated by their ability to inhibit the AAPH-induced peroxidation of linoleic acid in SDS micelles. It is shown that olive phenols and quercetin act as retardants rather than chain breakers like alpha-tocopherol. From a detailed mechanistic investigation, it appears that the inhibition of lipid peroxidation by olive phenols can be satisfactorily interpreted by assuming that they essentially reduce the AAPH-derived initiating radicals. Overall, olive phenols prove to be efficient scavengers of hydrophilic peroxyl radicals with a long lasting antioxidant effect owing to the residual activity of some of their oxidation products.

Vitamin D intestinal absorption is not a simple passive diffusion: Evidences for involvement of cholesterol transporters

SCOPE It is assumed that vitamin D is absorbed by passive diffusion. However, since cholecalciferol (vitamin D(3) ) and cholesterol display similar structures, we hypothesized that common absorption pathways may exist. METHODS AND RESULTS Cholecalciferol apical transport was first examined in human Caco-2 and transfected Human embryonic kidney (HEK) cells. Cholecalciferol uptake was then valuated ex vivo and in vivo, using either wild-type mice, mice overexpressing Scavenger Receptor class B type I (SR-BI) at the intestinal level or mice treated or not with ezetimibe. Cholecalciferol uptake was concentration-, temperature- and direction-dependent, and was significantly impaired by a co-incubation with cholesterol or tocopherol in Caco-2 cells. Moreover Block Lipid Transport-1 (SR-BI inhibitor) and ezetimibe glucuronide (Niemann-Pick C1 Like 1 inhibitor) significantly decreased cholecalciferol transport. Transfection of HEK cells with SR-BI, Cluster Determinant 36 and Niemann-Pick C1 Like 1 significantly enhanced vitamin D uptake, which was significantly decreased by the addition of Block Lipid Transport-1, sulfo-N-succinimidyl oleate (Cluster Determinant 36 inhibitor) or ezetimibe glucuronide, respectively. Similar results were obtained in mouse intestinal explants. In vivo, cholecalciferol uptake in proximal intestinal fragments was 60% higher in mice overexpressing SR-BI than in wild-type mice (p<0.05), while ezetimibe effect remained non-significant. CONCLUSION These data show for the first time that vitamin D intestinal absorption is not passive only but involves, at least partly, some cholesterol transporters.

One-electron oxidation of quercetin and quercetin derivatives in protic and non protic media

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) and quercetin derivatives (3-methylquercetin, rutin) are strong flavonoid antioxidants abundant in plants and in human diet. Their oxidation by DPPH, CAN or dioxygen (autoxidation) is studied in protic and non protic solvents. From kinetic investigations by UV–visible spectroscopy, oxidation rate constants are estimated. Fast disproportionation of flavonoid radicals is shown to give quinones which can be identified by their adducts with methanol (quercetin quinone) or sodium benzenesulfinate (rutin quinone). In strongly alkaline non aqueous conditions, the quercetin quinone can also be evidenced by strong charge transfer absorption bands in the range 700–800 nm.The consequences of these observations for the antioxidant properties of quercetin and quercetin derivatives are discussed.

Flavonol–serum albumin complexation. Two-electron oxidation of flavonols and their complexes with serum albumin

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) and quercetin derivatives (3-methylquercetin, isoquercitrin, rutin) are strong polyphenolic antioxidants abundant in plants and in the human diet. Recent investigations have shown that significant concentrations of albumin-bound quercetin conjugates are present in the plasma of humans fed a quercetin-rich diet.In this work, binding of quercetin and quercetin glycosides to bovine serum albumin (BSA) is quantitatively investigated by fluorescence spectroscopy. The strong fluorescence enhancement of quercetin upon binding points to the fact that a significant fraction of quercetin adopts a pyrylium-like structure in the complex. On the other hand, the observation of a very efficient quenching of tryptophan fluorescence by quercetin is consistent with a binding occurring in the IIA domain.Flavonoid-derived quinones may be formed upon quenching of reactive oxygen species by flavonoids (antioxidant activity). In this work, the quinones are conveniently formed upon periodate oxidation of the selected flavonoids in methanol and in aqueous buffers with and without BSA. A kinetic investigation by UV–visible spectroscopy shows that albumin-bound flavonoids are oxidized as quickly as free flavonoids. Interestingly, the quercetin quinone, which is merely detectable in the absence of BSA because of fast solvent addition, is efficiently stabilized in the complex by charge transfer interactions (pH 9). No evidence for quercetin–BSA conjugates could be found, thus showing that water addition (and subsequent degradation) remains the sole significant pathway of quinone transformation in the complex.

Antioxidant properties of anthocyanins and tannins: a mechanistic investigation with catechin and the 3′,4′,7-trihydroxyflavylium ion

Plant polyphenols act as antioxidants mainly by trapping reactive oxygen species and by regenerating endogenous membrane-bound α-tocopherol (vitamin E). In both processes polyphenols are oxidized. Hence, knowledge of the oxidation mechanisms of polyphenols is important for an understanding of their antioxidant activity at the molecular level. This work focuses on anthocyanins (pigments) and flavanols (tannins), two important classes of polyphenols which are both relatively abundant in human diet. The oxidation of the 3′,4′,7-trihydroxyflavylium ion (1) and catechin (2), respectively taken as models for anthocyanins and tannins, has been investigated. From kinetic data and partial product analysis, the mechanisms for the reactions of 1 and 2 with sodium periodate and DPPH, a H atom-abstracting radical, are proposed. Both polyphenols are shown to form o-quinone intermediates upon H atom abstraction and subsequent radical disproportionation. In the case of 2, the quinone and a second molecule of antioxidant quickly couple to form dimers. By contrast, 1 is extensively degraded into coumarins by repeating sequences of oxidation–solvent addition, which consume several equivalents of oxidants. In aqueous solutions, 1 is typically a mixture of coloured and colourless forms. The latter (chalcones) are also shown to take part in the antioxidant activity. p

Chemical Modeling of Heme-Induced Lipid Oxidation in Gastric Conditions and Inhibition by Dietary Polyphenols

The gastric tract may be the first site exposed to diet-related oxidative stress. After food intake, dietary iron such as (met)myoglobin, the pigment of meat, oxygen, and polyunsaturated lipids come into close contact. The main goal of this work is the in vitro investigation of lipid oxidation taking place in the gastric compartment and its inhibition by dietary polyphenols. Oil-in-water emulsions stabilized either by bovine serum albumin (BSA) or egg yolk phospholipids (PL) were designed to model the gastric content. The metmyoglobin-initiated lipid oxidation led to the accumulation of lipid-derived conjugated dienes and volatile aldehydes. These reactions were faster in the BSA model than in the PL model, highlighting the influence of the interfacial composition. Quercetin, rutin, (+)-catechin, caffeic acid, and chlorogenic acid proved to be better inhibitors than alpha-tocopherol and ascorbic acid. Emulsions as models of the gastric environment are valuable tools to study the stability of macro- and micronutrients.

The impact of industrial processing on health-beneficial tomato microconstituents.

The effect of industrial processing was investigated on the stability of tomato carotenoids, phenolic compounds and ascorbic acid. A deep insight in the processed products allowed the quantification of caffeic acid hexosides, which are far more important contributors than the well-known chlorogenic acid, dicaffeoylquinic acids and quercetin oligosaccharides (new feruloyl, sinapoyl and syringoyl derivatives of quercetin apiosylrhamnosylglucoside). (E)-β-Carotene and (E)-lycopene were also quantified along with different mono- and di-(Z)-isomers of lycopene which were tentatively assigned. Processing of fresh tomato into paste had an overall positive effect on the contents in phenolic compounds, no effect on lycopene and a slight and high detrimental effect on β-carotene and ascorbic acid, respectively. The balance between the increase in tomato matrix extractability and microconstituent catabolism was further observed in two contrasted transformations of paste into sauce. Overall, the nutritional quality of tomato-processed products, except for ascorbic acid, is mainly preserved through manufacture.

Food Grade Lingonberry Extract: Polyphenolic Composition and In Vivo Protective Effect against Oxidative Stress

Fractionation of the polyphenols constituting a food grade lingonberry extract (Vaccinium vitis-idaea) highlighted a composition more complex than described until now in the berry. Procyanidins B1, B2, and A2 were identified by UPLC/ESI-MS(2) along with the presence of other flavanol oligomers. Processing induced the release of large amounts of aglycones for ferulic acid, p-coumaric acid, and quercetin. The described anthocyanic composition of lingonberry was completed with hexoside derivatives of peonidin, petunidin, malvidin, and delphinidin. Besides confirmation of in vitro antioxidant activity, in vivo study was performed on rats fed a diet inducing oxidative stress. Supplementation with lingonberry extract significantly decreased the total oxidant status and favorably affected antioxidant defense enzymes in red blood cells and liver. A drop in the serum reduced glutathione level was also prevented, and uric acid was maintained at low level, confirming the antioxidant activity of the extract (5% proanthocyanidins) from a dosage of 23 mg/kg of body weight.

Dietary Iron-Initiated Lipid Oxidation and Its Inhibition by Polyphenols in Gastric Conditions

The gastric tract may be the first site where food is exposed to postprandial oxidative stress and antioxidant activity by plant micronutrients. After food intake, dietary iron, dioxygen, and emulsified lipids come into close contact and lipid oxidation may take place. This study investigated lipid oxidation and its inhibition by dietary polyphenols in gastric-like conditions. Lipid oxidation induced by heme and nonheme iron was studied in acidic sunflower oil-in-water emulsions. The emulsifier type (bovine serum albumin, phospholipids), pH, and iron form were found to be factors governing the oxidation rates. Quercetin, rutin, and chlorogenic acid highly inhibited the metmyoglobin-initiated lipid oxidation in both emulsified systems at pH 5.8. Additionally, quercetin inhibited nonheme iron-initiated processes, while it was inefficient with hematin as an initiator. The presence of human gastric juice did not influence lipid oxidation, although it diminished the antioxidant activity of phenolics. Model emulsions may thus be valuable tools to study the gastric stability of polyunsaturated lipids.