Dietary A- and B-type procyanidins : characterization and biofunctional potential of an abundant and diverse group of phenolics

Procyanidins (PCs) are phenolic compounds that belong to the class of flavonoids and are oligomers of monomeric (epi)catechin units. These monomeric units can be linked to each other by a single C4-C8 or C4-C6 linkage, which is referred to as B-type. Besides these single linkages an additional ether bond can be present, C2-O-C7 or C2-O-C5, which is referred to as A-type. PCs are highly abundant in our diet. Well known PC food sources are cocoa, apple, grape seeds, wine and nuts. After the intake of PC-rich sources health beneficial effects have been detected, which are mainly related to the prevention of cardiovascular diseases such as lowering of blood pressure. The aims of this thesis were to study the bioavailability, bioconversion and bioactivity of purified PCs. Therefore, we first developed techniques for the efficient purification of both A- and B-type PCs from peanut skins and grape seeds, respectively. Furthermore, tools were set-up to analyze and characterize individual PCs in complex mixtures. We showed that A-type PC dimers were absorbed from the small intestine of rats and that they were better absorbed than B-type PC dimers. The PC dimers were not conjugated or methylated upon absorption in contrast to their monomeric units (epi)catechin. Furthermore, the presence of A-type PC tetramers enhanced the absorption of B-type PC dimers. The microbial conversion of B-type PC dimers was studied by exposing them to human microbiota. The main microbial metabolites were 2-(3,4-dihydroxyphenylacetic acid and 5-(3,4-dihydroxyphenyl)-γ-valerolactone. Based on these and other metabolites that were detected, a tentative microbial degradation route was proposed for B-type PC dimers, in which the interflavanic bond does not need to be cleaved upon degradation. Subsequently, the vasorelaxing potential of purified PCs and their microbial metabolites was analyzed by measuring their effect on the NO production of endothelial cells. Both A- and B-type PCs showed a tendency (insignificant) to increase NO production with increasing degree of polymerization and several of their human microbial metabolites that were tested were inactive. Besides enhancing NO production, several other mechanisms could be targets of PCs and were also discussed. This thesis increased our knowledge on the absorption, biotransformation and bioactivity of A- and B-type PCs. A possible interaction between oligomers with a high and low degree of polymerization, influencing absorption processes has been discussed, which suggests that until now the biofunctional potential of PAs has been underestimated. <br/

Procyanidin Dimers A1, A2, and B2 Are Absorbed without Conjugation or Methylation from the Small Intestine of Rats1–3

Intervention studies with procyanidin (PC)-rich extracts and products such as cocoa and wine suggest protective effects of PC against cardiovascular diseases. However, there is no consensus on the absorption and metabolism of PC dimers. Interestingly, nothing is known about the absorption of A-type PC. In this study, the absorption and metabolism of purified PC dimers A1 [epicatechin-(2-O-7, 4–8)-catechin], A2 [epicatechin-(2-O-7, 4–8)-epicatechin], and B2 [epicatechin-(4–8)-epicatechin], A-type trimers, a mixture of A1, B2, and a tetrameric A-type, and monomeric epicatechin were compared by in situ perfusion of the small intestine of rats for 0–30 min. The rats had their bile duct, portal vein, and small intestine cannulated. Unmodified and methylated metabolites were distinguished from their conjugates by differential ß-glucuronidase treatment. A1 and A2 dimers were absorbed from the small intestine of rats and they were better absorbed than dimer B2. Absorption of the A-type dimers was only 5–10% of that of monomeric epicatechin. Dimers were not conjugated or methylated in contrast to epicatechin, which was partly methylated and 100% conjugated. A-type trimers were not absorbed. Furthermore, the presence of tetrameric PC enhanced the absorption of B2 but not that of A1. Epicatechin, methylated epicatechin, and their conjugates were not found as metabolites of the PC tested. In conclusion, dimers A1, A2, and B2 are slightly absorbed but are not conjugated or methylated, thus conserving their biological activity after absorption. Because PC contents of foods are relatively high, dimers may contribute to systemic effects of PC

Procyanidin dimers Are Metabolized by Human Microbiota with 2-(3,4-Dihydroxyphenyl)acetic Acid and 5-(3,4-Dihydroxyphenyl)-y-valerolactone as the Major Metabolites

Procyanidins (PCs) are highly abundant phenolic compounds in the human diet and might be responsible for the health effects of chocolate and wine. Due to low absorption of intact PCs, microbial metabolism might play an important role. So far, only a few studies, with crude extracts rich in PCs but also containing a multitude of other phenolic compounds, have been performed to reveal human microbial PC metabolites. Therefore, the origin of the metabolites remains questionable. This study included in vitro fermentation of purified PC dimers with human microbiota. The main metabolites identified were 2-(3,4-dihydroxyphenyl)acetic acid and 5-(3,4-dihydroxyphenyl)-y-valerolactone. Other metabolites detected were 3-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 3-hydroxyphenylpropionic acid, phenylvaleric acids, monohydroxylated phenylvalerolactone, and 1-(3',4'-dihydroxyphenyl)-3-(2",4",6"-trihydroxyphenyl)propan-2-ol. Metabolites that could be quantified accounted for at least 12 mol % of the dimers, assuming 1 mol of dimers is converted into 2 mol of metabolite. A degradation pathway, partly different from that of monomeric flavan-3-ols, is proposed

Combined normal-phase and reversed-phase liquid chromatography/ESI-MS as a tool to determine the molecular diversity of A-type procyanidins in peanut skins

Peanut skins, a byproduct of the peanut butter industry, are a rich source of proanthocyanidins, which might be used in food supplements. Data on the molecular diversity of proanthocyanidins in peanut skins is limited and conflicting with respect to the ratio of double- (A-type) versus single-linked (B-type) flavan-3-ol units. NP- and RP-HPLC-MS were used as tools to analyze the molecular diversity of proanthocyanidins in a 20% (v/v) methanol extract of peanut skins. NP-HPLC was used to prepurify monomeric to pentameric fractions, which were further separated and characterized by RP-HPLC-MS. With this method, 83 different proanthocyanidin molecular species were characterized and quantified. Furthermore, it was possible to determine that A-type procyanidin oligomers were predominant and represented 95.0% (w/w) of the extract. In addition, the position of the A-linkages in 16 trimers and 27 tetramers could be determined, which in this case appeared to occur at all possible positions. The majority of trimers and tetramers with one or more A-linkage always had an A-linkage at the terminal uni

Characterization of Oligomeric Xylan Structures from Corn Fiber Resistant to Pretreatment and Simultaneous Saccharification and Fermentation

Corn fiber, a byproduct from the corn industry, would be a good source for bioethanol production if the hemicellulose, consisting of polymeric glucoronoarabinoxylans, can be degraded into fermentable sugars. Structural knowledge of the hemicellulose is needed to improve the enzymatic hydrolyses of corn fiber. Oligosaccharides that resisted a mild acid pretreatment and subsequent enzymatic hydrolysis, representing 50% of the starting material, were fractionated on reversed phase and size exclusion material and characterized. The oligosaccharides within each fraction were highly substituted by various compounds. Oligosaccharides containing uronic acid were accumulated in two polar fractions unless also a feruloyl group was present. Feruloylated oligosaccharides, containing mono- and/or diferulic acid, were accumulated within four more apolar fractions. All fractions contained high amounts of acetyl substituents. The data show that complex xylan oligomers are present in which ferulic acid, diferulates, acetic acid, galactose, arabinose, and uronic acids were combined within an oligomer. Hypothetical structures are discussed, demonstrating which enzyme activities are lacking to fully degrade corn glucuronoarabinoxylans

Enzyme resistant feruloylated xylooligomer analogues from thermochemically treated corn fiber contain large side chains, ethyl glycosides and novel sites of acetylation

In order to use corn fiber as a source for bioethanol production the enzymatic hydrolysis of the complex glucuronoarabinoxylans present has to be improved. Several oligosaccharides present in the supernatant of mild acid pretreated and enzymatically saccharified corn fiber that resist the current available enzymes were (semi)purified for structural analysis by NMR or ESI-MSn. The structural features of 21 recalcitrant oligosaccharides are presented. A common feature of almost all these oligosaccharides is that they contain (part of) an a-l-galactopyranosyl-(1¿2)-ß-d-xylopyranosyl-(1¿2)-5-O-trans-feruloyl-l-arabinofuranose side chain attached to the O-3 position of the ß-1–4 linked xylose backbone. Several of the identified oligosaccharides contained an ethyl group at the reducing end hypothesized to be formed during SSF. The ethyl glycosides found are far more complex than previously described structures. A new feature present in more than half of the oligosaccharides is an acetyl group attached to the O-2 position of the same xylose to which the oligomeric side chain was attached to the O-3 position. Finding enzymes attacking these large side chains and the dense substituted xylan backbone will boost the hydrolysis of corn fiber glucuronoxyla

Enzyme resistant feruloylated xylooligomer analogues from thermochemically treated corn fiber contain large side chains, ethyl glycosides and novel sites of acetylation

In order to use corn fiber as a source for bioethanol production the enzymatic hydrolysis of the complex glucuronoarabinoxylans present has to be improved. Several oligosaccharides present in the supernatant of mild acid pretreated and enzymatically saccharified corn fiber that resist the current available enzymes were (semi)purified for structural analysis by NMR or ESI-MSn. The structural features of 21 recalcitrant oligosaccharides are presented. A common feature of almost all these oligosaccharides is that they contain (part of) an a-l-galactopyranosyl-(1¿2)-ß-d-xylopyranosyl-(1¿2)-5-O-trans-feruloyl-l-arabinofuranose side chain attached to the O-3 position of the ß-1–4 linked xylose backbone. Several of the identified oligosaccharides contained an ethyl group at the reducing end hypothesized to be formed during SSF. The ethyl glycosides found are far more complex than previously described structures. A new feature present in more than half of the oligosaccharides is an acetyl group attached to the O-2 position of the same xylose to which the oligomeric side chain was attached to the O-3 position. Finding enzymes attacking these large side chains and the dense substituted xylan backbone will boost the hydrolysis of corn fiber glucuronoxyla

The ferulic acid esterases of Chrysosporium lucknowense C1: Purification, characterization and their potential application in biorefinery

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6

Fast and Robust Method To Determine Phenoyl and Acetyl Esters of Polysaccharides by Quantitative 1H NMR

The acetyl (AcE), feruloyl (FE), and p-coumaroyl (pCE) ester contents of different cereal and grass polysaccharides were determined by a quantitative 1H NMR-based method. The repeatability and the robustness of the method were demonstrated by analyzing different plant polysaccharide preparations. Good sensitivity and selectivity for AcE, FE, and pCE were observed. Moreover, an optimized and easy sample preparation allowed for simultaneous quantification of AcE, FE, and pCE. The method is suitable for high-throughput analysis, and it is a good alternative for currently used analytical procedures. A comparison of the method presented to a conventional HPLC-based method showed that the results obtained are in good agreement, whereas the combination of the optimized sample preparation and analysis by the 1H NMR-based methodology results in significantly reduced analysis time

Some phenolic compounds increase the nitric oxide level in endothelial cells in vitro

The vasorelaxing properties of chocolate and wine might relate to the presence of phenolic compounds. One of the potential mechanisms involved is stimulation of endothelial nitric oxide (NO) production, as NO is a major regulator of vasodilatation. This study aimed to develop an in vitro assay using the hybrid human endothelial cell line EA.hy926 to rapidly screen phenolic compounds for their NO-stimulating potential. The assay was optimized, and a selection of 33 phenolics, namely, procyanidins, monomeric flavan-3-ols, flavonols, a flavone, a flavanone, a chalcone, a stilbene, and phenolic acids, was tested for their ability to enhance endothelial NO level. Resveratrol, a well-known enhancer of NO level, was included as a positive control. Of the 33 phenolics tested, only resveratrol (285% increase in NO level), quercetin (110% increase), epicatechingallate (ECg) (85% increase), and epigallocatechingallate (EGCg) (60% increase) were significant (P = 0.05) enhancers. Procyanidins showed a nonsignificant tendency to elevate NO level. Concentration-dependent correlations between enhanced NO level and endothelial nitric oxide synthase (eNOS) expression were demonstrated for the three polyphenols tested (resveratrol, ECg, and EGCg). Thus, an easy screening tool for change in cellular NO level was developed. Use of this assay showed that only a limited number of phenolic compounds might enhance NO level with an increased amount of eNOS enzyme as a possible contributing mechanism