Presence of free gallic acid and gallate moieties reduces auto-oxidative browning of epicatechin (EC) and epicatechin gallate (ECg).

Auto-oxidation of flavan-3-ols leads to browning and consequently loss of product quality during storage of ready-to-drink (RTD) green tea. The mechanisms and products of auto-oxidation of galloylated catechins, the major flavan-3-ols in green tea, are still largely unknown. Therefore, we investigated auto-oxidation of epicatechin gallate (ECg) in aqueous model systems. Oxidation products tentatively identified based on MS included δ- or γ-type dehydrodicatechins (DhC2s) as the main contributors to browning. Additionally, various colourless products were detected, including epicatechin (EC) and gallic acid (GA) from degalloylation, ether-linked ε-type DhC2s, and 6 new coupling products of ECg and GA possessing a lactone interflavanic linkage. Supported by density function theory (DFT) calculations, we provide a mechanistic explanation on how presence of gallate moieties (D-ring) and GA affect the reaction pathway. Overall, presence of gallate moieties and GA resulted in a different product profile and less intense auto-oxidative browning of ECg compared to EC.

Separation of flavonoid isomers by cyclic ion mobility mass spectrometry.

Analytical techniques, such as liquid chromatography coupled to mass spectrometry (LC-MS) or nuclear magnetic resonance (NMR), are widely used for characterization of complex mixtures of (isomeric) proteins, carbohydrates, lipids, and phytochemicals in food. Food can contain isomers that are challenging to separate, but can possess different reactivity and bioactivity. Catechins are the main phenolic compounds in tea; they can be present as various stereoisomers, which differ in their chemical properties. Currently, there is a lack of fast and direct methods to monitor interconversion and individual reactivity of these epimers (e.g. epicatechin (EC) and catechin (C)). In this study, cyclic ion mobility mass spectrometry (cIMS-MS) was explored as a potential tool for the separation of catechin epimers. Formation of sodium and lithium adducts enhanced IMS separation of catechin epimers, compared to deprotonation and protonation. Baseline separation of the sodium adducts of catechin epimers was achieved. Moreover, we developed a fast method for the identification and semi-quantification of cIMS-MS separated catechin epimers. With this method, it is possible to semi-quantify the ratio between EC and C (1:5 to 5:1, within 50-1200 ng mL-1) in food samples, such as tea. Finally, the newly developed approach for cIMS-MS separation of flavonoids was demonstrated to be successful in separation of two sets of positional isomers (i.e. morin, tricetin, and quercetin; and kaempferol, fisetin, luteolin, and scutellarein). To conclude, we showed that both epimers and positional isomers of flavonoids can be separated using cIMS-MS, and established the potential of this method for challenging flavonoid separations.

Reactivity of Fe(III)-containing pyrophosphate salts with phenolics: complexation, oxidation, and surface interaction.

Mixed pyrophosphate salts with the general formula Ca2(1-x)Fe4x(P2O7)(1+2x) potentially possess less iron-phenolic reactivity compared to ferric pyrophosphate (FePP), due to decreased soluble Fe in the food-relevant pH range 3-7. We investigated reactivity (i.e., complexation, oxidation, and surface interaction) of FePP and mixed salts (with x = 0.14, 0.15, 0.18, and 0.35) in presence of structurally diverse phenolics. At pH 5-7, increased soluble iron from all salts was observed in presence of water-soluble phenolics. XPS confirmed that water-soluble phenolics solubilize iron after coordination at the salt surface, resulting in increased discoloration. However, color changes for mixed salts with x ≤ 0.18 remained acceptable for slightly water-soluble and insoluble phenolics. Furthermore, phenolic oxidation in presence of mixed salts was significantly reduced compared to FePP at pH 6. In conclusion, these mixed Ca-Fe(III) pyrophosphate salts with x ≤ 0.18 can potentially be used in designing iron-fortified foods containing slightly water-soluble and/or insoluble phenolics.

A targeted prenylation analysis by a combination of IT-MS and HR-MS: Identification of prenyl number, configuration, and position in different subclasses of (iso)flavonoids.

Prenylated (iso)flavonoids are potent bioactive compounds found in the Fabaceae family. Analysis and quantification of this type of phytochemicals is challenging due to their large structural diversity. In this study, the fragmentation of prenylated (iso)flavonoids was investigated using electrospray ionization ion trap mass spectrometry (ESI-IT-MSn) with fragmentation by collision induced dissociation (CID) in combination and Orbitrap-MS (ESI-FT-MS2) with fragmentation by higher energy C-trap dissociation (HCD). With this combination of IT-MSn and high resolution MS (FT-MSn), it was possible to determine the fragmentation pathways and characteristic spectral features of different subclasses of prenylated (iso)flavonoid standards, as well as characteristic fragmentations and neutral losses of different prenyl configurations. Based on our findings, a decision guideline was developed to (i) identify (iso)flavonoid backbones, (ii) annotate prenyl number, (iii) configuration, and (iv) position of unknown prenylated (iso)flavonoids, in complex plant extracts. In this guideline, structural characteristics were identified based on: (i) UV absorbance of the compound, (ii) mass-to-charge (m/z) ratio of the parent compound; (iii) ratio of relative abundances between neutral losses 42 and 56 u in MSn; (iv) retro-Diels-Alder (RDA) fragments, neutral losses 54 and 68 u, and the ratio [M+H-C4H8]+/[M+H]+. Using this guideline, 196 prenylated (iso)flavonoids were annotated in a Glycyrrhiza glabra root extract. In total, 75 skeletons were single prenylated, 104 were double prenylated, and for merely 17 skeletons prenyl number could not unambiguously be annotated. Our prenylation guideline allows rapid screening for identification of prenylated (iso)flavonoids, including prenyl number, configuration, and position, in complex plant extracts. This guideline supports research on these bioactive compounds in the areas of plant metabolomics and natural products.

Insights in the Recalcitrance of Theasinensin A to Human Gut Microbial Degradation.

Due to low bioavailability of dietary phenolic compounds in small intestine, their metabolism by gut microbiota is gaining increasing attention. The microbial metabolism of theasinensin A (TSA), a bioactive catechin dimer found in black tea, has not been studied yet. Here, TSA was extracted and purified for in vitro fermentation by human fecal microbiota, and epigallocatechin gallate (EGCG) and procyanidin B2 (PCB2) were used for comparison. Despite the similarity in their flavan-3-ol skeletons, metabolic fate of TSA was distinctively different. After degalloylation, its core biphenyl-2,2',3,3',4,4'-hexaol structure remained intact during fermentation. Conversely, EGCG and PCB2 were promptly degraded into a series of hydroxylated phenylcarboxylic acids. Computational analyses comparing TSA and PCB2 revealed that TSA's stronger interflavanic bond and more compact stereo-configuration might underlie its lower fermentability. These insights in the recalcitrance of theasinensins to degradation by human gut microbiota are of key importance for a comprehensive understanding of its health benefits.

Microbial Metabolism of Theaflavin-3,3'-digallate and Its Gut Microbiota Composition Modulatory Effects.

Theaflavin-3,3'-digallate (TFDG), a bioactive black tea phenolic, is poorly absorbed in the small intestine, and it has been suggested that gut microbiota metabolism plays a crucial role in its bioactivities. However, information on its metabolic fate and impact on gut microbiota is limited. Here, TFDG was anaerobically fermented in vitro by human fecal microbiota, and epigallocatechin gallate (EGCG) was used for comparison. Despite the similar flavan-3-ol skeletons, TFDG was more slowly degraded and yielded a distinctively different metabolic profile. The formation of theanaphthoquinone as the main metabolites was unique to TFDG. Additionally, a number of hydroxylated phenylcarboxylic acids were formed with low concentrations, when comparing to EGCG metabolism. Microbiome profiling demonstrated several similarities in gut microbiota modulatory effects, including growth-promoting effects on Bacteroides, Faecalibacterium, Parabacteroides, and Bifidobacterium, and inhibitory effects on Prevotella and Fusobacterium. In conclusion, TFDG and EGCG underwent significantly different microbial metabolic fates, yet their gut microbiota modulatory effects were similar.

Browning of Epicatechin (EC) and Epigallocatechin (EGC) by Auto-Oxidation.

Green tea catechins are well known for their health benefits. However, these compounds can easily be oxidized, resulting in brown color formation, even in the absence of active oxidative enzymes. Browning of catechin-rich beverages, such as green tea, during their shelf life is undesired. The mechanisms of auto-oxidation of catechins and the brown products formed are still largely unknown. Therefore, we studied auto-oxidative browning of epicatechin (EC) and epigallocatechin (EGC) in model systems. Products of EC and EGC auto-oxidation were analyzed by reversed-phase ultra-high-performance liquid chromatography with photodiode array detection coupled to mass spectrometry (RP-UHPLC-PDA-MS). In the EC model system, 11 δ-type dehydrodicatechins (DhC2s) and 18 δ-type dehydrotricatechins (DhC3s) that were related to browning could be tentatively identified by their MS2 signature fragments. In the EGC model system, auto-oxidation led to the formation of 13 dihydro-indene-carboxylic acid derivatives and 2 theaflagallins that were related to browning. Based on the products formed, we propose mechanisms for the auto-oxidative browning of EC and EGC. Furthermore, our results indicate that dimers and oligomers that possess a combination of an extended conjugated system, fused rings, and carbonyl groups are responsible for the brown color formation in the absence of oxidative enzymes.

Revealing the main factors and two-way interactions contributing to food discolouration caused by iron-catechol complexation.

Fortification of food with iron is considered to be an effective approach to counter the global health problem caused by iron deficiency. However, reactivity of iron with the catechol moiety of food phenolics leads to discolouration and impairs bioavailability. In this study, we investigated the interplay between intrinsic and extrinsic factors on food discolouration caused by iron-catechol complexation. To this end, a three-level fractional factorial design was implemented. Absorbance spectra were analysed using statistical methods, including PCA, HCA, and ANOVA. Furthermore, a direct link between absorbance spectra and stoichiometry of the iron-catechol complexes was confirmed by ESI-Q-TOF-MS. All statistical methods confirm that the main effects affecting discolouration were type of iron salt, pH, and temperature. Additionally, several two-way interactions, such as type of iron salt × pH, pH × temperature, and type of iron salt × concentration significantly affected iron-catechol complexation. Our findings provide insight into iron-phenolic complexation-mediated discolouration, and facilitate the design of iron-fortified foods.

Mass spectrometric characterisation of avenanthramides and enhancing their production by germination of oat (Avena sativa).

Avenanthramides are amides, with a phenylalkenoic acid (PA) and an anthranilic acid (AA) subunit, which are secondary metabolites of oat. Oat seeds were germinated, extracted, and the avenanthramides analysed by a combination of UHPLC with ion trap and high resolution ESI-MS. Typical fragmentation pathways with corresponding diagnostic fragments belonging to the PA and AA subunits were identified and summarised in a decision guideline. Based on these findings 28 unique avenanthramides were annotated in the oat seed(ling) extracts, including the new avenanthramide 6f (with a 4/5-methoxy AA subunit). Avenanthramide content increased by 25 times from seed to seedling. Avenanthramides 2p, 2c, and 2f, which are commonly described as the major avenanthramides, represented less than 20% of the total content in the seedlings. Future quantitative analyses should, therefore, include a wider range of avenanthramides to avoid underestimation of the total avenanthramide content.