C-Nitrosation, C-Nitration, and Coupling of Flavonoids with N-Acetyltryptophan Limit This Amine N-Nitrosation in a Simulated Cured and Cooked Meat.

Nitrite is a common additive in cured meat formulation that provides microbiological safety, lipid oxidation management, and typical organoleptic properties. However, it is associated with the formation of carcinogenic N-nitrosamines. In this context, the antinitrosating capacity of selected flavonoids and ascorbate was evaluated in a simulated cooked and cured meat under formulation and digestion conditions. N-Acetyltryptophan was used as a secondary amine target. (-)-Epicatechin, rutin, and quercetin were all able to limit the formation of N-acetyl-N-nitrosotryptophan (NO-AcTrp) at pH 2.5 and pH 5 although (-)-epicatechin was 2 to 3-fold more efficient. Kinetics for the newly identified compounds allowed us to unravel common mechanistic pathways, which are flavonoid oxidation by nitrite followed by C-nitration and an original covalent coupling between NO-AcTrp and flavonoids or their nitro and nitroso counterparts. C-nitrosation of the A-ring was evidenced only for (-)-epicatechin. These major findings suggest that flavonoids could help to manage N-nitrosamine formation during cured meat processing, storage, and digestion.

Advanced characterization of polyphenols from Myrciaria jaboticaba peel and lipid protection in in vitro gastrointestinal digestion.

Ultrasound-assisted and solvent extractions resulted in similar levels of hydrolyzable tannins (10.3-6.0 mg/g), anthocyanins (7.8-10.2 mg/g) and flavonols (0.24-0.32 mg/g) for dried Myrciaria jaboticaba peel (DJP). Ultrasound was efficient for the extraction of poorly soluble hydrolyzable tannins but affected the stability of anthocyanins and flavonols. UPLC-DAD-MSn allowed the identification of 44 hydrolyzable tannins as single and mixed hexosides bearing galloyl, HHDP and tergalloyl units. Twelve mixed HHDP-galloylgluconic acids and tergalloylated hexosides were newly discovered in this work. Acid hydrolysis of both ultrasonic extract and DJP yielded five major compounds, i.e. gallic acid, ellagic acid, gallic acid-C-hexoside, valoneic acid dilactone and sanguisorbic acid dilactone and pointed to higher contents in hydrolyzable tannins than by summing individual polyphenols after UPLC. Last, cyanidin-3-O-glucoside and hydrolyzable tannins from the ultrasonic extract inhibited lipid peroxidation of a Western type meal in in vitro digestion, suggesting a health benefit for these jabuticaba polyphenols.

Mechanisms Governing the Transfer of Pure and Plant Matrix Carotenoids Toward Emulsified Triglycerides.

SCOPE: The study aims to assess the role of factors assumed to be involved in the transfer of carotenoids from plant matrices to dietary emulsions in the upper digestive tract. METHODS AND RESULTS: Transfer is first measured as a function of time of pure ß-carotene (ßC), lutein (LUT), and lycopene (LYC) to triglyceride (TG) droplets dispersed in water. Then the transfer to TG droplets stabilized with either bovine serum albumin (BSA), phospholipids (PL), or both is measured. Finally, transfer of tomato and spinach puree carotenoids to these emulsions is measured. The maximal transfer efficiency of the pure carotenoids to uncoated emulsions is very efficient, ranging from 59% to 77%. However, it is dramatically impaired, ranging from 0.5% to 31% (p < 0.05), when emulsions are stabilized by the emulsifiers. Conversely, when LUT, and to a less extent ßC, but not LYC, is provided by the vegetable purees, its maximal transfer efficiency is significantly higher for the coated emulsions than for the uncoated one. CONCLUSIONS: Emulsifiers can dramatically impair the transfer of pure carotenoids to emulsion TG while they can facilitate the transfer of carotenoids from plant matrices. This suggests that specific interactions between plant matrix compounds and emulsifiers can enhance the transfer efficiency of carotenoids.

Increased diffusivity of lycopene in hot break vs. cold break purees may be due to bioconversion of associated phospholipids rather than differential destruction of fruit tissues or cell structures.

Lycopene bioaccessibility is enhanced by processing, as explained by the destructuration of plant tissues, making diffusion easier. However, in tomato, the relationship between grinding intensity and lycopene release from purees suffers from uncertainty. In particular, hot break puree exhibited twice as much diffusible lycopene as compared to cold break, while both were processed with the same grinding intensity. To explain the difference, we systematically studied the diffusivity of particles according to their size and integrity, and used microscopic and physical analyses to reveal structural differences. Neither particle size distribution, nor cell destruction, nor plastid transformation exhibited any correlation to the differences in diffusivity. However, Raman microspectroscopy combined with a chemometric analysis revealed significant changes in lycopene spectra and a putative linkage to phospholipid transformation. Phospholipid profiling of five pairs of contrasted purees revealed that, during the cold break, a transition from complex phospholipids to more simple phosphatidic acid molecules systematically occurred.

Carotenoids: Experimental Ionization Energies and Capacity at Inhibiting Lipid Peroxidation in a Chemical Model of Dietary Oxidative Stress.

Carotenoids are important natural pigments and micronutrients contributing to health prevention by several mechanisms, including their electron-donating (antioxidant) activity. In this work, a large series of carotenoids, including 11 carotenes and 14 xanthophylls, have been investigated by wavelength-resolved atmospheric pressure photoionization mass spectrometry (DISCO line of SOLEIL synchrotron), thus allowing the experimental determination of their ionization energy (IE) for the first time. On the other hand, the carotenoids have been also investigated for their ability to inhibit the heme iron-induced peroxidation of linoleic acid in mildly acidic micelles, a simple but relevant chemical model of oxidative stress in the gastric compartment. Thus, the carotenoids can be easily classified from IC50 concentrations deduced from the time dependence of the lipid hydroperoxide concentration. With a selection of two carotenes and three xanthophylls a quantitative analysis is also provided to extract stoichio-kinetic parameters. The influence of the carotenoid structure (number of conjugated carbon-carbon double bonds, presence of terminal six-membered rings, hydroxyl, keto, and/or epoxy groups) on the IE, IC50, and stoichio-kinetic parameters is discussed in details. The data show that the antioxidant activity of carotenes is well correlated to their electron-donating capacity, which itself largely depends on the length of the conjugated polyene chain. By contrast, the IE of xanthophylls is poorly correlated to the polyene chain length because of the strong, and sometimes unexpected, electronic effects of the O-atoms. Although IE remains an approximate predictor of the antioxidant activity of xanthophylls, other factors (interaction with the aqueous phase, competing radical-scavenging mechanisms, the residual activity of the antioxidant's oxidation products) probably play a significant role.

Opposite Effects of the Spinach Food Matrix on Lutein Bioaccessibility and Intestinal Uptake Lead to Unchanged Bioavailability Compared to Pure Lutein.

SCOPE: Food matrix is generally believed to alter carotenoid bioavailability, but its effect on xanthophylls is usually limited. This study thus aims to decipher the digestion-absorption process of lutein in the presence or not of a food matrix. METHODS: Lutein transfer to gastric-like lipid droplets or artificial mixed micelles was assessed when lutein was added to test meals either as a pure molecule ((all-E)-lutein) or in canned spinach ((Z) + (all-E)-lutein). The obtained mixed micelles were delivered to Caco-2 cells to evaluate lutein uptake. Finally postprandial plasma lutein responses were compared in minipigs after the two test meals. RESULTS: Lutein transfer to gastric-like lipid droplets and to mixed micelles was higher when lutein was added in spinach than when it was added as pure lutein (+614% and +147%, respectively, p < 0.05). Conversely, lutein uptake was less effective when micellar lutein was from a meal containing spinach than from a meal containing its pure form (-55%, p < 0.05). In minipigs, postprandial lutein response was delayed with spinach but not significantly different after the two test meals. CONCLUSION: Opposite effects at the micellarization and intestinal cell uptake steps explain the lack of effect of spinach matrix on lutein bioavailability.

Intestinal scavenger receptors are involved in vitamin K1 absorption.

Vitamin K1 (phylloquinone) intestinal absorption is thought to be mediated by a carrier protein that still remains to be identified. Apical transport of vitamin K1 was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium and in transfected HEK cells. Phylloquinone uptake was then measured ex vivo using mouse intestinal explants. Finally, vitamin K1 absorption was compared between wild-type mice and mice overexpressing scavenger receptor class B type I (SR-BI) in the intestine and mice deficient in cluster determinant 36 (CD36). Phylloquinone uptake by Caco-2 cells was saturable and was significantly impaired by co-incubation with α-tocopherol (and vice versa). Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 85% of vitamin K1 uptake. BLT1 also decreased phylloquinone apical efflux by ∼80%. Transfection of HEK cells with SR-BI and CD36 significantly enhanced vitamin K1 uptake, which was subsequently decreased by the addition of BLT1 or sulfo-N-succinimidyl oleate (CD36 inhibitor), respectively. Similar results were obtained in mouse intestinal explants. In vivo, the phylloquinone postprandial response was significantly higher, and the proximal intestine mucosa phylloquinone content 4 h after gavage was increased in mice overexpressing SR-BI compared with controls. Phylloquinone postprandial response was also significantly increased in CD36-deficient mice compared with wild-type mice, but their vitamin K1 intestinal content remained unchanged. Overall, the present data demonstrate for the first time that intestinal scavenger receptors participate in the absorption of dietary phylloquinone.

Comparative study on antioxidant activity of lycopene (Z)-isomers in different assays.

Several studies have implicated the potent antioxidant properties of lycopene. However, most of the studies used only the (all-E)-isomer. (Z)-Isomers of lycopene were found in substantial amounts in processed foods and in human tissues. In the present study, we investigated in vitro the antioxidant activity of (5Z)-, (9Z)-, and (13Z)-lycopene compared to the (all-E)-isomer. Additionally, prolycopene, the (7Z,9Z,7'Z,9'Z)-isomer found in tangerine tomatoes, was analyzed. No significant differences were found between the isomers in ferric reducing antioxidant power assay and in bleaching the radical cation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), both based on ET mechanisms. In contrast, scavenging activity against peroxyl radicals generated by thermal degradation of 2,2'-azobis(2-amidinopropane) (AAPH) was higher in the (Z)-isomers. (5Z)-Lycopene was most antioxidant in scavenging lipid peroxyl radicals, evaluated by analyzing the inhibition of MbFe(III) lipid peroxidation of linoleic acid in mildly acidic conditions (pH 5.8) in a micellar environment, modeling a possible antioxidant action in the gastric compartment.

Antioxidant activity of wine pigments derived from anthocyanins: hydrogen transfer reactions to the dpph radical and inhibition of the heme-induced peroxidation of linoleic acid.

The consumption of red wine can provide substantial concentrations of antioxidant polyphenols, in particular grape anthocyanins (e.g., malvidin-3-O-beta-d-glucoside (1)) and specific red wine pigments formed by reaction between anthocyanins and other wine components such as catechin (3), ethanol, and hydroxycinnamic acids. In this work, the antioxidant properties of red wine pigments (RWPs) are evaluated by the DPPH assay and by inhibition of the heme-induced peroxidation of linoleic acid in acidic conditions (a model of antioxidant action in the gastric compartment). RWPs having a 1 and 3 moieties linked via a CH(3)-CH bridge appear more potent than the pigment with a direct 1-3 linkage. Pyranoanthocyanins derived from 1 reduce more DPPH radicals than 1 irrespective of the substitution of their additional aromatic ring. Pyranoanthocyanins are also efficient inhibitors of the heme-induced lipid peroxidation, although the highly hydrophilic pigment derived from pyruvic acid appears less active.

Dietary antioxidants as inhibitors of the heme-induced peroxidation of linoleic acid: mechanism of action and synergism.

In this work, a quantitative kinetic model for investigating the heme-induced peroxidation of linoleic acid and its inhibition by two important dietary antioxidants, quercetin and alpha-tocopherol, is developed. The main conclusions of this work are: (1) The time dependence of the lipid hydroperoxide concentration is critically dependent on the rate constant for lipid hydroperoxide cleavage, initial fraction of lipid hydroperoxides among the pool of conjugated dienes, and rate of heme degradation. (2) The lipophilic antioxidant alpha-tocopherol acts as a chain-breaking antioxidant that quickly reduces 1-2 eq of lipid peroxyl radicals (inhibition of propagation), whereas the more hydrophilic antioxidant quercetin is only marginally chain-breaking but capable of reducing 4-5 eq of iron-oxo initiator (inhibition of initiation). (3) Based on comparisons between experimental peroxidation curves and simulated curves assuming additivity, it can be concluded that combinations of alpha-tocopherol and quercetin are generally synergistic. The kinetic analysis and HPLC titrations of the antioxidants both suggest that synergism mainly arises from a capacity of alpha-tocopherol to regenerate some quercetin oxidation products still endowed with a reducing activity.

Inhibition of the metmyoglobin-induced peroxidation of linoleic acid by dietary antioxidants: Action in the aqueous vs. lipid phase.

The gastric digestion of food containing oxidizable lipids and iron catalysts for peroxide decomposition such as (met)myoglobin from muscle meat can be accompanied by an extensive formation of potentially toxic lipid hydroperoxides. An early protective action by dietary antioxidants in the gastro-intestinal tract is plausible, especially for poorly bioavailable antioxidants such as polyphenols. Hence, the ability of antioxidants to inhibit lipid peroxidation initiated by dietary iron in mildly acidic emulsions is a valuable and general model. In this work, the ability of some ubiquitous dietary antioxidants representative of the main antioxidant classes (alpha-tocopherol, the flavonol quercetin, beta-carotene) to inhibit the metmyoglobin-induced peroxidation of linoleic acid is investigated by UV-visible spectroscopy and HPLC in mildly acidic emulsions. The phenolic antioxidants quercetin and alpha-tocopherol come up as the most efficient peroxidation inhibitors. Inhibition by quercetin essentially proceeds in the aqueous phase via a fast reduction of an unidentified activated iron species (with a partially degraded heme) produced by reaction of metmyoglobin with the lipid hydroperoxides. This reaction is faster by, at least, a factor 40 than the reduction of ferrylmyoglobin (independently prepared by reacting metmyoglobin with hydrogen peroxide) by quercetin. By contrast, alpha-tocopherol mainly acts in the lipid phase by reducing the propagating lipid peroxyl radicals. The poorer inhibition afforded by beta-carotene may be related to both its slower reaction with the lipid peroxyl radicals and its competitive degradation by autoxidation and/or photo-oxidation.

Enrichment of tomato paste with 6% tomato peel increases lycopene and beta-carotene bioavailability in men.

A high intake of tomato products is associated with a lower incidence of upper aerodigestive tract and prostate cancers. This beneficial effect might be explained by a higher intake of carotenoids such as lycopene and/or beta-carotene. Because tomato peels, usually eliminated during tomato processing, are a valuable source of these carotenoids, we designed a study to examine whether a tomato paste enriched in tomato peels (ETP, 6% peel) increases the absorption of these carotenoids compared to a classically made tomato paste (CTP). Carotenoid bioaccessibility was evaluated using an in vitro digestion model by measuring the amount of carotenoids transferred from the pastes to micelles. Carotenoid absorption by human intestinal cells (Caco-2) was evaluated after the addition of carotenoid-rich micelles (obtained from the in vitro digestion of the 2 pastes). Carotenoid bioavailability in humans was assessed by measuring chylomicron carotenoid responses in a postprandial experiment in which 8 healthy men consumed 2 meals containing either the ETP or the CTP. ETP contained 47.6 mg lycopene (58% more than CTP) and 1.75 mg beta-carotene (99% more than CTP) per 100 g of paste. In micelles, 30% more lycopene and 81% more beta-carotene were recovered after ETP than after CTP in vitro digestion. The amount of carotenoids absorbed by Caco-2 cells was 75% greater (P < or = 0.05) for lycopene and 41% greater (P < or = 0.05) for beta-carotene after the addition of micelles from ETP than from CTP. After ETP intake the chylomicron beta-carotene response was 74% greater than after CTP intake, and the lycopene response tended to be greater (34.1%, P = 0.093). Peel enrichment of tomato paste with tomato peel is an interesting option for increasing lycopene and beta-carotene intakes.

Quantitative kinetic analysis of hydrogen transfer reactions from dietary polyphenols to the DPPH radical.

Diphenylpicrylhydrazyl (DPPH) is widely used for quickly assessing the ability of polyphenols to transfer labile H atoms to radicals, a likely mechanism of antioxidant protection. This popular test generally pays no attention to the kinetics of H atom transfer, which however could be even more important than the total H-atom-donating capacities (stoichiometry, EC50) typically evaluated. In the present work, a series of dietary polyphenols belonging to the most representative families (flavonols from onion, flavanol monomers and oligomers from barley, and caffeic acid and caffeoyl esters from artichoke and endive) are characterized not only by their total stoichiometries (n(tot)) but also by their rate constants of first H atom abstraction by DPPH (k(1)), deduced from the kinetic analysis of the decay of the DPPH visible band following addition of the antioxidant. The mildly reactive DPPH radical allows a good discrimation between polyphenols, as demonstrated by the relatively large ranges of k(1) (ca. 400-5000 M(-)(1) s(-)(1)) and n(tot) (ca. 1-5) values typically measured with antioxidants having a single polyphenolic nucleus. With antioxidants displaying more than one polyphenolic nucleus (procyanidin oligomers, dicaffeoyl esters), the kinetic analysis makes it possible to demonstrate significant differences in reactivity between the subunits (two distinct k(1) values whose ratio lies in the range 3-10) and nonadditive stoichiometries.