Radical Intermediates in the Degradation of Hop Acids.

Radical formation in isohumulones was investigated under different types of stress, including temperature, transition metal ions, and hydrogen peroxide. Including dihydroisohumulones and tetrahydroisohumulones, as relevant analogues, allowed us to evaluate critical functionalities in radical formation. Using spin-trapping methodology with 5,5-dimethyl-1-pyrroline N-oxide and N-tert-butyl-α-phenylnitrone as relevant traps, followed by simulation of corresponding spin adducts, identification of incipient radicals was attempted. The isohexenoyl side chain in isohumulones, but not present in dihydro- and tetrahydroisohumulones, was most sensitive to radical formation. Kinetic profiles further demonstrated that radical formation in this moiety was accelerated in the presence of ferrous ions. Reactivity of parent six-membered-ring humulones in radical formation was different, as scavenging of free radical species was more important. Lupulones, despite similarity with humulones, showed a different behavior with an obvious radical decay pathway during ageing, mainly ascribed to radical formation on the ring structure. Quantification of final spin adducts allowed us to determine absolute importance of the different degradation pathways. Eventually, mechanisms are presented explaining why isohumulones are more prone to radical processes in (aut)oxidation and thermal decay than close relatives such as dihydroisohumulones.

Flavonoids protecting food and beverages against light.

Flavonoids, which are ubiquitously present in the plant kingdom, preserve food and beverages at the parts per million level with minor perturbation of sensory impressions. Additionally, they are safe and possibly contribute positive health effects. Flavonoids should be further exploited for the protection of food and beverages against light-induced quality deterioration through: (1) direct absorption of photons as inner filters protecting sensitive food components; (2) deactivation of (triplet-)excited states of sensitisers like chlorophyll and riboflavin; (3) quenching of singlet oxygen from type II photosensitisation; and (iv) scavenging of radicals formed as reaction intermediates in type I photosensitisation. For absorption of light, combinations of flavonoids, as found in natural co-pigmentation, facilitate dissipation of photon energy to heat thus averting photodegradation. For protection against singlet oxygen and triplet sensitisers, chemical quenching gradually decreases efficiency hence the pathway to physical quenching should be optimised through product formulation. The feasibility of these protection strategies is further supported by kinetic data that are becoming available, allowing for calculation of threshold levels of flavonoids to prevent beer and dairy products from going off. On the other hand, increasing understanding of the interplay between light and matrix physicochemistry, for example the effect of aprotic microenvironments on phototautomerisation of compounds like quercetin, opens up for engineering better light-to-heat converting channels in processed food to eventually prevent quality loss.

Dietary intake of lycopene by the Belgian adult population.

OBJECTIVE: Lycopene is a potent antioxidant, and it has been suggested that intake of tomatoes and tomato products containing lycopene is associated with a decreased risk of various chronic diseases. The aim of the present study was to evaluate the distribution of dietary lycopene intake in the Belgian population and to determine the most important contributors to lycopene intake. DESIGN: Cross-sectional study. SETTING: National food consumption data from the Belgian Food Consumption Survey (BFCS) 2004 were used for the intake assessment. Determination of the lycopene content in foods was performed with HPLC-UV. Individual food consumption data were multiplied by the actual mean concentrations of lycopene per food. SUBJECTS: Individuals (n 3083) aged 15 years and older participated in the study and provided two 24 h recalls. RESULTS: The mean lycopene intake among Belgian adults was 4·1 (sd 2·3) mg/d or 0·059 (sd 0·033) mg/kg body weight per d. Lycopene intake among men (4·6 (sd 2·6) mg/d) was higher than among women (3·6 (sd 2·1) mg/d), and was higher in the younger compared with the older age groups. Cis-lycopene intake represented about one-third of the total lycopene intake. Tomatoes and tomato products (43%) and sauces and ready-to-eat meals containing tomato sauces (41%) were the main contributors to lycopene intake in Belgium. CONCLUSIONS: The lycopene intake of the Belgian adult population was comparable to intakes reported in neighbouring countries and was below the acceptable daily intake.

Flavonoid deactivation of excited state flavins: reaction monitoring by mass spectrometry.

Flavin mononucleotide (FMN, as a B(2) vitamin model) was shown to induce dimerization of flavonoids (flavanone, apigenin, naringenin, eriodictyol, taxifolin, catechin, kaempferol, luteolin, quercetin, rutin, and seven smaller model phenols studied) as the major photoreaction, when aqueous solutions were exposed to visible light using a new, real-time electrospray ionization mass-spectrometric (ESI-MS) technique supported by LC-MS and MS(2) analysis. Electrophilic intermediates such as transient radical cations, o-quinones, and p-quinone methide were proposed to be involved in the coupling process. The C(3)-OH in flavon-3-ols gave rise to atypical compounds such as a depside or a dioxane-linked dimer. Flavonoid dimers, formed in vegetal extracts added to food during storage in light and for which structures are proprosed based on MS and MS(2), may affect colloidal stability, color, astringency, and antioxidative capacity.

Dietary intake of artificial sweeteners by the Belgian population.

This study investigated whether the Belgian population older than 15 years is at risk of exceeding ADI levels for acesulfame-K, saccharin, cyclamate, aspartame and sucralose through an assessment of usual dietary intake of artificial sweeteners and specific consumption of table-top sweeteners. A conservative Tier 2 approach, for which an extensive label survey was performed, showed that mean usual intake was significantly lower than the respective ADIs for all sweeteners. Even consumers with high intakes were not exposed to excessive levels, as relative intakes at the 95th percentile (p95) were 31% for acesulfame-K, 13% for aspartame, 30% for cyclamate, 17% for saccharin, and 16% for sucralose of the respective ADIs. Assessment of intake using a Tier 3 approach was preceded by optimisation and validation of an analytical method based on liquid chromatography with mass spectrometric detection. Concentrations of sweeteners in various food matrices and table-top sweeteners were determined and mean positive concentration values were included in the Tier 3 approach, leading to relative intakes at p95 of 17% for acesulfame-K, 5% for aspartame, 25% for cyclamate, 11% for saccharin, and 7% for sucralose of the corresponding ADIs. The contribution of table-top sweeteners to the total usual intake (<1% of ADI) was negligible. A comparison of observed intake for the total population with intake for diabetics (acesulfame-K: 3.55 versus 3.75; aspartame: 6.77 versus 6.53; cyclamate: 1.97 versus 2.06; saccharine: 1.14 versus 0.97; sucralose: 3.08 versus 3.03, expressed as mg kg(-1) bodyweight day(-1) at p95) showed that the latter group was not exposed to higher levels. It was concluded that the Belgian population is not at risk of exceeding the established ADIs for sweeteners.

Antioxidant properties of green tea extract protect reduced fat soft cheese against oxidation induced by light exposure.

The effect of two different antioxidants, EDTA and green tea extract (GTE), used individually or in combination, on the light-induced oxidation of reduced fat soft cheeses (0.2 and 6% fat) was investigated. In samples with 0.2% fat, lipid hydroperoxides as primary lipid oxidation products were not detected, but their interference was suggested from the formation of secondary lipid oxidation products such as hexanal and heptanal. The occurrence of these oxidation markers was inhibited by spiking with 50 ppm EDTA or 750 ppm GTE, or a combination of the two prior to irradiation. In contrast, addition of 50 ppm EDTA to samples with 6% fat was ineffective, but 750 ppm GTE (alone or in combination with EDTA) strongly reduced levels of hexanal and heptanal. Accumulation of primary lipid hydroperoxides was not affected by GTE, hence antioxidative activity was ascribed to scavenging of hexanal and heptanal precursors. These radical intermediates result from hydroperoxide disintegration, and subsequent scavenging by GTE, which acts as a radical sink, corroborates the intense signal observed by electron paramagnetic resonance (EPR) spectroscopy.

Light-induced oxidation of unsaturated lipids as sensitized by flavins.

Triplet-excited riboflavin ((3)RF*) was found by laser flash photolysis to be quenched by polyunsaturated fatty acid methyl esters in tert-butanol/water (7:3, v/v) in a second-order reaction with k approximately 3.0 x 10(5) L mol(-1) s(-1) at 25 degrees C for methyl linoleate and 3.1 x 10(6) L mol(-1) s(-1), with DeltaH(double dagger) = 22.6 kJ mol(-1) and DeltaS(double dagger) = -62.3 J K(-1) mol(-1), for methyl linolenate in acetonitrile/water (8:2, v/v). For methyl oleate, k was <10(4) L mol(-1) s(-1). For comparison, beta-casein was found to have a rate constant k approximately 4.9 x 10(8) L mol(-1) s(-1). Singlet-excited flavin was not quenched by the esters as evidenced by insensitivity of steady-state fluorescence to their presence. Density functional theory (DFT) calculations showed that electron transfer from unsaturated fatty acid esters to triplet-excited flavins is endergonic, while a formal hydrogen atom transfer is exergonic (DeltaG(o)(HAT) = -114.3, -151.2, and -151.2 kJ mol(-1) for oleate, linoleate, and linolenate, respectively, in acetonitrile). The reaction is driven by acidity of the lipid cation radical for which a pK(a) approximately -0.12 was estimated by DFT calculations. Absence of electrochemical activity in acetonitrile during cyclic voltammetry up to 2.0 V versus NHE confirmed that DeltaG(o)(ET) > 0 for electron transfer. Interaction of methyl esters with (3)RF* is considered as initiation of the radical chain, which is subsequently propagated by combination reactions with residual oxygen. In this respect, carbon-centered and alkoxyl radicals were detected using the spin trapping technique in combination with electron paramagnetic resonance spectroscopy. Moreover, quenching of (3)RF* yields, directly or indirectly, radical species which are capable of initiating oxidation in unsaturated fatty acid methyl esters. Still, deactivation of triplet-excited flavins by lipid derivatives was slower than by proteins (factor up to 10(4)), which react preferentially by electron transfer. Depending on the reaction environment in biological systems (including food), protein radicals are expected to interfere in the mechanism of light-induced lipid oxidation.

Characterization of peroxides formed by riboflavin and light exposure of milk. Detection of urate hydroperoxide as a novel oxidation product.

Characterization of peroxides by size exclusion chromatography (SEC) of milk following exposure to riboflavin and light showed that hydrogen peroxide was the most abundant peroxide formed since it could be removed by catalase. Formation of peroxides after separation by SEC showed that hydrogen peroxide formation was primarily increased in the presence of caseins and ascorbate, although whey proteins also were found to contribute. Caseins and beta-lactoglobulin also formed catalase-resistant peroxides, presumably protein hydroperoxides. A catalase-resistant and unstable peroxide was observed in fractions containing urate. Experiments performed with pure urate suggested that urate radicals reacted further with superoxide leading to a urate hydroperoxide. Electron paramagnetic resonance spectroscopy using spin-traps showed that the presence of oxygen was required for urate radical formation, which could be assigned as nitrogen-centered radicals. These results suggest a new route during light-induced oxidation sensitized by flavins, in effect making urate pro-oxidative.

Quenching of triplet-excited flavins by flavonoids. Structural assessment of antioxidative activity.

The mechanism of flavin-mediated photooxidation of flavonoids was investigated for aqueous solutions. Interaction of triplet-excited flavin mononucleotide with phenols, as determined by laser flash photolysis, occurred at nearly diffusion-controlled rates (k approximately 1.6 x 10(9) L mol(-1) s(-1) for phenol at pH 7, 293 K), but protection of the phenolic function by methylation inhibited reaction. Still, electron transfer was proposed as the dominating mechanism due to the lack of primary kinetic hydrogen/deuterium isotope effect and the low activation enthalpy (<20 kJ mol(-1)) for photooxidation. Activation entropy worked compensating in a series of phenolic derivatives, supporting a common oxidation mechanism. An ortho-hydroxymethoxy pattern was equally reactive (k approximately 2.3 x 10(9) L mol(-1) s(-1) for guaiacol at pH 7) as compounds with ortho-dihydroxy substitution (k approximately 2.4 x 10(9) L mol(-1) s(-1) for catechol at pH 7), which are generally referred to as good antioxidants. This refutes the common belief that stabilization of incipient phenoxyl radicals through intramolecular hydrogen bonding is the driving force behind the reducing activity of catechol-like compounds. Instead, such bonding improves ionization characteristics of the substrates, hence the differences in reactivity with (photo)oxidation of isolated phenols. Despite the similar reactivity, radicals from ortho-dihydroxy compounds are detected in high steady-state concentrations by electron paramagnetic resonance (EPR) spectroscopy, while those resulting from oxidation of ortho-hydroxymethoxy (or isolated phenolic) patterns were too reactive to be observed. The ability to deprotonate and form the corresponding radical anions at neutral pH was proposed as the decisive factor for stabilization and, consequently, for antioxidative action. Thus, substituting other ionizable functions for the ortho- or para-hydroxyl in phenolic compounds resulted in stable radical anion formation, as demonstrated for para-hydroxybenzoic acid, in contrast to its methyl ester.

Light-induced oxidation of tryptophan and histidine. Reactivity of aromatic N-heterocycles toward triplet-excited flavins.

Mechanisms of flavin-mediated photooxidation of electron-rich amino acids tryptophan and histidine were investigated for aqueous solutions. Indole, representing the tryptophan side chain in proteins, reacted at nearly diffusion controlled rates (k approximately 2.7 x 10(9) L mol(-1) s(-1) at 293 K) with the triplet-excited flavin state, but reactions of imidazole (and histidine) were significantly slower (k < 2.0 x 10(8) L mol(-1) s(-1)) as determined by laser flash photolysis. Oxidation rates of derivates were invariably susceptible to electronic factors affecting incipient radical cation stability, while no primary kinetic hydrogen/deuterium isotope effect was observed for imidazole. Thus reaction by electron transfer was proposed in contrast to a direct hydrogen abstraction. Unlike indole compounds, imidazole derivatives suffered from the presence of a basic imino nitrogen (=N-), which caused the rate constant of histidine free base (k approximately 1.8 x 10(8) L mol(-1) s(-1)) to drop considerably upon protonation. Complexation of the imino nitrogen with transition metals provoked changes in reactivity, as rate constants decreased after addition of Zn(2+) (k of 4-methylimidazole, as histidine model, decreased from 9.0 x 10(8) L mol(-1) s(-1) in the absence to 4.1 x 10(8) L mol(-1) s(-1) in the presence of ZnCl(2)). The pyrrole nitrogen (-NH-) was not directly involved in complexation reactions, but its electron density increased upon interaction with hydrogen bond-accepting anions and resulted in higher rate constants (k of 4-methylimidazole increased from 9.0 x 10(8) L mol(-1) s(-1) to 2.0 x 10(9) L mol(-1) s(-1) after addition of NaOAc). The high rate constants were in agreement with a large thermodynamical driving force, as calculated from oxidation peak potentials determined electrochemically. After oxidation, resulting radical cations were readily deprotonated and trapped by 2-methyl-2-nitrosopropane, as detected by electron paramagnetic resonance spectroscopy. Indole-derived spin adducts were attributed to selective trapping of C(3)-centered radicals, whereas spin adducts with imidazole-derivatives arose from both carbon and nitrogen-centered imidazolyl radicals.

Flavin-induced photodecomposition of sulfur-containing amino acids is decisive in the formation of beer lightstruck flavor.

Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light irradiation in the presence of flavins. The sulfur moiety seems pivotal for interaction, as was determined from kinetic analyses using laser flash photolysis spectroscopy. After photooxidation, the resulting radical intermediates were characterized by addition to a spin trap, followed by electron paramagnetic resonance spectroscopy and evaluation of the coupling constants. The presence of the proposed radical intermediates was strongly supported by the identification of the reaction products using mass spectrometry. Accordingly, feasible degradation pathways for various sulfur-containing amino acids and derivatives were proposed. It was finally proven that flavin-induced photoproduction of sulfhydryl radicals and recombination with a 3-methylbut-2-enyl radical, derived from the photodegradation of hop-derived isohumulones, are decisive in the formation of beer lightstruck flavor.

Regioselective synthesis and estrogenicity of (+/-)-8-alkyl-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarins.

Nine new (+/-)-8-alkyl-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarins have been synthesized from 2,4,6-trimethoxybenzaldehyde via a short, efficient, and regioselective pathway, together with the unsubstituted analogue (+/-)-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarin. The compounds were tested for estrogenic activity using a yeast-based estrogen screen. Weak estrogenicity was determined for seven members of the series.

Fate of flavins in sensitized photodegradation of isohumulones and reduced derivatives: studies on formation of radicals via EPR combined with detailed product analyses.

Photodegradation of isohumulones accounts for formation of the lightstruck flavor in beer. The reactions involved are mediated by riboflavin, a natural photosensitizer present in beer in ppb quantities. The results of an investigation of this sensitized degradation process are presented herein. Product analyses and electron paramagnetic resonance spectroscopy, in steady-state as well as in time-resolved mode, offer extensive insight into the photophysical and photochemical details of the degradation mechanism. In contrast to energy transfer and Norrish type I alpha-cleavage reactions that take place on direct irradiation of isohumulones, the sensitization pathway proceeds via one-electron redox chemistry involving the excited triplet state of riboflavin and derivatives. The flavin semiquinone radical thus formed could be readily detected, either by steady state or by time-resolved electron paramagnetic resonance spectroscopy. Superimposed signals in the spectra revealed the presence of radical fragments derived from isohumulones or tetrahydroisohumulones, which, on recombination with riboflavin semiquinone radicals, produced stable reaction products that were identified by HPLC-MS. However, no superimposed signals were observed on sensitized irradiation of dihydroisohumulones.

Photooxidative degradation of beer bittering principles: a key step on the route to lightstruck flavor formation in beer.

Isohumulones, dihydroisohumulones, tetrahydroisohumulones, and humulinones, important hop-derived bittering compounds in beer, were shown to give rise to reactive triacylmethyl radicals on interaction with triplet-excited riboflavin after spin trapping by 5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane, followed by electron paramagentic resonance spectroscopy combined with spectral simulation. Electron abstraction from the ionized beta-tricarbonyl chromophore, which is common to all five-membered ring hop derivatives, is the initial event on photoinduced degradation. Radicaloid decomposition of isohumulones leads to precursors for 3-methylbut-2-ene-1-thiol, the lightstruck constituent in beer. Interaction of reduced derivatives of isohumulones with triplet-excited riboflavin furnished radical precursors of volatile aldehydes, which may lead to the development of unpleasant stale or cardboard flavors.

Photooxidative degradation of beer bittering principles: product analysis with respect to lightstruck flavour formation.

Isohumulones, the main bittering agents in beer, are decomposed by light-induced reactions, thereby leading to radical precursors on the pathway to lightstruck flavour formation. Excited flavins, formed on visible-light irradiation, readily interact with isohumulones, as well as with reduced and oxidized derivatives thereof. From identification of both volatile and non-volatile reaction products thus formed, feasible degradation mechanisms are proposed.

Riboflavin-sensitized photooxidation of isohumulones and derivatives.

Isohumulones, the bitter principles in beer, are decomposed by light-induced reactions, thereby adversely affecting beer quality. Upon absorption of visible light, riboflavin is excited and interacts with isohumulones, as well as with oxidized and reduced derivatives thereof. Reaction kinetics were investigated by laser flash photolysis at 355 nm and at 440 nm, and analysis of kinetic data afforded detailed insights into the reaction mechanism.

Radicaloid-type oxidative decomposition of beer bittering agents revealed.

trans-Isohumulones, dihydroisohumulones, tetrahydroisohumulones, and humulinones, which are important hop-derived flavor components of beer, were found, by using electrolysis of organic solutions, to be stable against oxidation, like weak acids; however, they are readily oxidized in their anionic forms as present in beer. Oxygen- and carbon-centered radicals were formed by oxidation and identified by using spin trapping under aerobic and anaerobic conditions, followed by EPR (electron paramagnetic resonance) spectroscopy. Generated radicals were reactive, most likely degrading into products lacking the tricarbonyl chromophore; this is typical of five-membered-ring hop derivatives. Thus, flavor-active beer constituents may degrade oxidatively in the absence of oxygen, thereby leading to reaction products that escape UV detection.

Photolysis of hop-derived trans-iso-alpha-acids and trans-tetrahydroiso-alpha-acids: product identification in relation to the lightstruck flavour of beer.

The photolysis of hop-derived trans-iso-alpha-acids (2a-c; naturally occurring bitter compounds present in beer) and of trans-tetrahydroiso-alpha-acids (5a-c; semi-synthetic advanced hop products) was investigated at 300 nm in methanol. The complex photoreaction mixtures were separated by high-performance liquid chromatography (HPLC) using diode array detection and the major photoreaction products were identified by HPLC-mass spectroscopy. The main part of the mixture consisted of compounds, which originated from recombination of radicals derived from Norrish Type I photocleavage of the acyloin moiety in both trans-iso-alpha-acids and trans-tetrahydroiso-alpha-acids. The results confirm the intermediacy of radicals that were previously identified by time-resolved electron paramagnetic resonance and they bear relevance to the formation of the lightstruck flavour that is generated when beer is exposed to light. Additionally, new photoproducts were found that are formed by photochemical reactions hitherto undiscovered in hop chemistry, including photoenolization of trans-isohumulone (2a) leading to trans-alloisohumulone (13a) and a retro oxa-di-pi-methane rearrangement in trans-isohumulone (2a) and trans-tetrahydroiso-alpha-acids to humulone (1a) and tetrahydro-alpha-acids (23a-b), respectively.