TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat

The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV-visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.     

Proanthocyanidins Extracted from Rhododendron pulchrum Leaves as Source of Tyrosinase Inhibitors: Structure,Activity, and Mechanism

The objective of this study was to assess the structure, anti-tyrosinase activity, and mechanism of proanthocyanidins extracted from Rhododendron pulchrum leaves. Results obtained from mass spectra of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) revealed that proanthocyanidins were complex mixtures of procyanidins, prodelphinidins, propelargonidins, and their derivatives, among which procyanidins were the main components. The anti-tyrosinase analysis results indicated that the mixtures were reversible and mixed competitive inhibitors of tyrosinase. Interactions between proanthocyanidins with substrate (L-tyrosine and 3,4-dihydroxyphenylalanine) and with copper ions were the important molecular mechanisms for explaining their efficient inhibition. This research would provide scientific evidence for the use of R. pulchrum leaf proanthocyanidins as new novel tyrosinase inhibitors.     

Procyanidin B2 catabolism by human fecal microflora: Partial characterization of ‘dimeric’ intermediates

The catabolism by human fecal microflora of pure procyanidin B2 ((-)-epicatechin-4β → 8-(-)-epicatechin) has been investigated using a static in vitro culture model. For the first time, 24 catabolites were detected by LC-MS⁽ⁿ⁾ with M_r greater than 290 indicating that they could not have formed from just one of the epicatechin units in the procyanidin structure. Structures have been assigned on the basis of the fragmentation in the ion trap mass spectrometer and with regard to catabolic pathways known to occur in fecal microorganisms. Twenty of these ‘dimeric’ catabolites produced fragment ions characteristic of flavanols and/or proanthocyanidins. One catabolite was identified tentatively as either 6- or 8-hydroxy-procyanidin B2. Thirteen were characterized as having been microbially reduced in at least one of the epicatechin units. Five contained an apparently unmodified epicatechin unit but in at least one case this was shown to consist of the B-ring of the “upper” epicatechin unit and the A-ring of the “lower”. These ‘dimeric’ catabolites were detected up to 9 h after the start of the incubation and collectively accounted for ∼20% of the substrate.     

Reversed-phase HPLC-ESI/MS analysis of birch leaf proanthocyanidins after their acidic degradation in the presence of nucleophiles

Mountain birch leaves contain large amounts of structurally variable polymeric proanthocyanidins. Their isolation procedure was enhanced by the addition of liquid-liquid extractions prior to column chromatography over Sephadex LH-20. Isolated polymeric proanthocyanidins were depolymerised by acid-catalysis in the presence of benzyl mercaptan or phloroglucinol in order to study their composition. The resulting degradation products, flavan-3-ols and flavan-3-ol adducts, were analysed with reversed-phase high-performance liquid chromatography using UV photodiode array detection for quantification and electrospray ionisation mass spectrometry for identification. The results showed that polymeric proanthocyanidins contained (epi)gallocatechins and (epi)catechins as the extension units and, mainly, (+)-catechin as the terminal unit. The mean degree of polymerisation was found to be 26 based on thiolysis and 31 based on phloroglucinol degradation.     

Tissue-Specific and Development-Dependent Accumulation of Phenylpropanoids in Larch Mycorrhizas

The tissue-specific and development-dependent accumulation of secondary products in roots and mycorrhizas of larch (Larix decidua Mill.; Pinaceae) was studied using high-performance liquid chromatography and histochemical methods. The compounds identified were soluble catechin, epicatechin, quercetin 3-O-[alpha]-rhamnoside, cyanidin- and peonidin 3-O-[beta]-glucoside, 4-O-[beta]-hydroxybenzoyl-O-[beta]-glucose, 4-hydroxybenzoate 4-O-[beta]-glucoside, maltol 3-O-[beta]-glucoside, and the wall-bound 4-hydroxybenzaldehyde, vanillin, and ferulate. In addition, we partially identified a tetrahydroxystilbene monoglycoside, a quercetin glycoside, and eight oligomeric proanthocyanidins. Comparison between the compounds accumulating in the apical tissue of fine roots, long roots, and in vitro grown mycorrhizas (L. decidua-Suillus tridentinus) showed elevated levels of the major compounds catechin and epicatechin as well as the minor compound 4-hydroxybenzoate 4-O-[beta]-glucoside specifically in the root apex of young mycorrhizas. The amounts of wall-bound 4-hydroxybenzaldehyde and vanillin were increased in all of the mycorrhizal sections examined. During the early stages of mycorrhization the concentrations of these compounds increased rapidly, perhaps induced by the mycorrhizal fungus. In addition, studies of L. decidua-Boletinus cavipes mycorrhizas from a natural stand showed that the central part of the subapical cortex tissue and the endodermis both accumulate massive concentrations of catechin, epicatechin, and wall-bound ferulate compared with the outer part of the cortex, where the Hartig net is being formed.     

Exobasidium vexans infection of Camellia sinensis increased 2,3-cis isomerisation and gallate esterification of proanthocyanidins

Infection of leaves of tea (Camellia sinensis (Kuntze) L, cv TRI 2025) which was susceptible to blister blight (Exobasidium vexans Massee), resulted in a shift of the proanthocyanidin stereochemistry away from 2,3-trans (e.g. catechin and gallocatechin) and towards 2,3-cis (e.g. epicatechin and epigallocatechin). Infection also resulted in increased gallic acid esterification of the initiating subunits of proanthocyanidins. This was shown by both mass spectroscopy and phloroglucinolysis. Proanthocyanidins isolated from healthy tissue had a predominantly 2,3-trans stereochemistry which accounted for 53% and 61% of the total initiating and extension units of proanthocyanidin, respectively. Conversely in infected tissue, proanthocyanidin subunits with a 2,3-trans stereochemistry accounted for 26% and 40% of the total initiating and extension units, respectively. Infection had little impact on the hydroxylation state of the B-rings of proanthocyanidins. The products of acid hydrolysis under oxidative conditions had a slight excess of di-hydroxylated B-rings with cyanidin accounting for 58.3+/-0.05% and 60.4+/-0.2% of the total anthocyanidin recovered following hydrolysis of proanthocyanidin isolated from infected and healthy leaves, respectively. Similar results were obtained by phloroglucinolysis.     

Fractionation of grape tannins and analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Polymeric tannins, extracted from grape berries (Gamay variety), were fractionated according to their mean degree of polymerisation (mDP) on a styrene-divinylbenzene phase eluted with a gradient of methanol:chloroform. Increasing the percentage of methanol led to the solubilisation of higher molecular weight tannins. The mean mDP of each collected fraction was determined by acid-catalysed degradation in the presence of a nucleophilic reagent. The fractionation method produced a linear gradient of mDP varying between 1.84 and 19.34. The fractions were partially characterised by matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). The spectra showed a complex mixture of proanthocyanidins and galloylated proanthocyanidins up to 4000 amu.     

Identification of Proanthocyanidins from Litchi (Litchi chinensis Sonn.) Pulp by LC-ESI-Q-TOF-MS and Their Antioxidant Activity

Content of total proanthocyanidins as well as total phenolics, flavonoids, antioxidant activities were evaluated for litchi (Litchi chinensis Sonn.) pulp of 32 cultivars. One cultivar, Hemaoli, showed the highest total proanthocyanidins and total phenolics, and DPPH or ABTS radical scavenging activities. ESI-MS and NMR analysis of the Hemaoli pulp crude extracts (HPCE) showed that procyandins composed of (epi)catechin unites with degree of polymerization (DP) of 2–6 were dominant proanthocyanidins in HPCE. After the HPCE was fractionated by a Sephadex LH-20 column, 32 procyanidins were identified by LC-ESI-Q-TOF-MS in litchi pulp for the first time. Quantification of individual procyanidin in HPCE indicated that epicatechin, procyanidin B2, procyanidin C1 and A-type procyanidin trimer were the main procyanidins. The radical scavenging activities of different fractions of HPCE as well as six procyanidins standards were evaluated by both DPPH and ABTS assays. HPCE fractions showed similar antioxidant activities with those of Vc and six individual procyanidins, the IC₅₀ of which ranged from 1.88 ± 0.01 to 2.82 ± 0.10 μg/ml for DPPH assay, and from 1.52 ± 0.17 to 2.71 ± 0.15 μg/ml for ABTS assay. Such results indicate that litchi cultivars rich in proanthocyanidins are good resources of dietary antioxidants and have the potential to contribute to human health.     

Antioxidant activity of extract of Rhus oxyacantha root cortex

In the present study, the root extract of Rhus oxyacantha contained 25.33 mg of catechin equivalent per mg of fresh wt and was found rich in proanthocyanidins compared to vine shoot, grape pips and leaves. The chromatographic analysis of the extract suggested the presence of (+) catechin, (-) epicatechin -3-O-gallate as well as proanthocyanidinic oligomers and polymers. Root cortex inhibited the ascorbic acid oxidation by dioxygen. It also prevented DDT-induced thymocytes death in a dose-dependent manner. The results suggested antioxidant property of root extract of Rhus oxyacantha which could be ascribed to its free radical scavenging nature.     

Flavanol binding of nuclei from tree species

Light microscopy was used to examine the nuclei of five tree species with respect to the presence of flavanols. Flavanols develop a blue colouration in the presence of a special p-dimethylaminocinnamaldehyde (DMACA) reagent that enables those nuclei loaded with flavanols to be recognized. Staining of the nuclei was most pronounced in both Tsuga canadensis and Taxus baccata, variable in Metasequoia glyptostroboides, faint in Coffea arabica and minimal in Prunus avium. HPLC analysis showed that the five species contained substantial amounts of different flavanols such as catechin, epicatechin and proanthocyanidins. Quantitatively, total flavanols were quite different among the species. The nuclei themselves, as studied in Tsuga seed wings, were found to contain mainly catechin, much lower amounts of epicatechin and traces of proanthocyanidins. Blue-coloured nuclei located centrally in small cells were often found to maximally occupy up to 90% of a cells radius, and the surrounding small rim of cytoplasm was visibly free of flavanols. A survey of 34 gymnosperm and angiosperm species indicated that the first group has much higher nuclear binding capacities for flavanols than the second group.Abbreviations DMACA p-Dimethylaminocinnamaldehyde Communicated by W. Barz     

Mass spectrometry studies of a novel digoxin-like substance (DLIS-2) isolated from human plasma ultrafiltrate

Two unique low molecular weight (531) compounds with both digoxin-like immunoreactivity and Na, K-ATPase inhibitory properties have been isolated from human plasma. One of these, digoxin-like substance 2, (DLIS-2), was studied by fast atom bombardment mass spectrometry and collisionally activated dissociation mass spectrometry/mass spectrometry. The fragment patterns were interpreted as being derived from a lysophosphatidyl serine containing a novel 19:4 fatty acid side chain. The molecular formula C25H42O9NP is consistent with these observations.     

Direct mass spectrometry approaches to characterize polyphenol composition of complex samples

Lower molecular weight polyphenols including proanthocyanidin oligomers can be analyzed after HPLC separation on either reversed-phase or normal phase columns. However, these techniques are time consuming and can have poor resolution as polymer chain length and structural diversity increase. The detection of higher molecular weight compounds, as well as the determination of molecular weight distributions, remain major challenges in polyphenol analysis.Approaches based on direct mass spectrometry (MS) analysis that are proposed to help overcome these problems are reviewed. Thus, direct flow injection electrospray ionization mass spectrometry analysis can be used to establish polyphenol fingerprints of complex extracts such as in wine. This technique enabled discrimination of samples on the basis of their phenolic (i.e. anthocyanin, phenolic acid and flavan-3-ol) compositions, but larger oligomers and polymers were poorly detectable. Detection of higher molecular weight proanthocyanidins was also restricted with matrix-assisted laser desorption ionization (MALDI) MS, suggesting that they are difficult to desorb as gas-phase ions. The mass distribution of polymeric fractions could, however, be determined by analyzing the mass distributions of bovine serum albumin/proanthocyanidin complexes using MALDI-TOF-MS.     

Grape Seed Polyphenols and Fatty Acids of Autochthonous Prokupac Vine Variety from Serbia

Grape pomace (grape skin, seeds, and stems) represents a valuable source of bioactive compounds, thus the interest in making use of these wine waste products is increasing. Prokupac is an autochthonous Serbian variety of black grape with a long tradition in production of red wine. Identification of the superficial polyphenolic compounds by LC/MS revealed presence of 19 compounds including hydroxybenzoic acid derivatives, ellagic acid derivatives, flavan‐3‐ol monomers, proanthocyanidins, and flavonols. Catechin and epicatechin together with proanthocyanidins were the most abundant compounds and the highest content of phenolic compounds was determined in Prokupac clones 43/1, 40/1, and 40/2. Grape seed oil consisted of dominantly UFA (87–95 %) where methyl linoleate was the most abundant in all samples (69–81 %). Palmitic (3–8 %) and stearic acid (2–4 %) methyl esters were dominant SFA. Prokupac clone 43/3 was the most abundant in UFA (95 %) with the lowest percentage of SFA (5 %), while clones 41/1 and 43/5 had the lowest percentage of UFA (87 %) and the highest content of SFA (13 %). Favorable chemical profile of Prokupac clone seeds implicates its potential to be used as a raw material in further processing and possibility to be included as one of the quality parameters in further selection of the most interesting Prokupac clone.     

Analyses of Polyphenols in Cacao Liquor,Cocoa, and Chocolate by Normal-Phase and Reversed-Phase HPLC

The antioxidant polyphenols in cacao liquor, a major ingredient of chocolate and cocoa, have been characterized as flavan-3-ols and proanthocyanidin oligomers. In this study, various cacao products were analyzed by normal-phase HPLC, and the profiles and quantities of the polyphenols present, grouped by molecular size (monomers~oligomers), were compared. Individual cacao polyphenols, flavan-3-ols (catechin and epicatechin), and dimeric (procyanidin B2), trimeric (procyanidin C1), and tetrameric (cinnamtannin A2) proanthocyanidins, and galactopyranosyl-ent-(-)-epicatechin (2α→7, 4α→8)-(-)-epicatechin (Gal-EC-EC), were analyzed by reversed-phase HPLC and/or HPLC/MS. The profile of monomers (catechins) and proanthocyanidin in dark chocolate was similar to that of cacao liquor, while the ratio of flavan-3-ols to the total amount of monomeric and oligomeric polyphenols in the case of pure cocoa powder was higher than that in the case of cacao liquor or chocolate.     

Analyses of polyphenols in cacao liquor, cocoa, and chocolate by normal-phase and reversed-phase HPLC

The antioxidant polyphenols in cacao liquor, a major ingredient of chocolate and cocoa, have been characterized as flavan-3-ols and proanthocyanidin oligomers. In this study, various cacao products were analyzed by normal-phase HPLC, and the profiles and quantities of the polyphenols present, grouped by molecular size (monomers to approximately oligomers), were compared. Individual cacao polyphenols, flavan-3-ols (catechin and epicatechin), and dimeric (procyanidin B2), trimeric (procyanidin C1), and tetrameric (cinnamtannin A2) proanthocyanidins, and galactopyranosyl-ent-(-)-epicatechin (2alpha-->7, 4alpha-->8)-(-)-epicatechin (Gal-EC-EC), were analyzed by reversed-phase HPLC and/or HPLC/MS. The profile of monomers (catechins) and proanthocyanidin in dark chocolate was similar to that of cacao liquor, while the ratio of flavan-3-ols to the total amount of monomeric and oligomeric polyphenols in the case of pure cocoa powder was higher than that in the case of cacao liquor or chocolate.     

Determination of procyanidins and their metabolites in plasma samples by improved liquid chromatography–tandem mass spectrometry

An off-line solid-phase extraction (SPE) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining procyanidins, catechin, epicatechin, dimer, and trimer in plasma samples. In the validation procedure of the analytical method, linearity, precision, accuracy, detection limits (LODs), quantification limits (LOQs), and the matrix effect were studied. Recoveries of the procyanidins were higher than 84%, except for the trimer, which was 65%. The LODs and LOQs were lower than 0.003 and 0.01 μM, respectively, for all the procyanidins studied, except for the trimers, which were 0.8 and 0.98 μM, respectively. This methodology was then applied for the analysis of rat plasma obtained 2 h after ingestion of grape seed phenolic extract. Monomers (catechin and epicatechin), dimer and trimer in their native form were detected and quantified in plasma samples, and their concentration ranged from 0.85 to 8.55 μM. Moreover, several metabolites, such as catechin and epicatechin glucuronide, catechin and epicatechin methyl glucuronide, and catechin and epicatechin methyl-sulphate were identified. These conjugated forms were quantified, in reference to the respective unconjugated form, showing concentrations between 0.06 and 23.90 μM.     

Dimeric anthocyanins from strawberry (Fragaria ananassa) consisting of pelargonidin 3-glucoside covalently linked to four flavan-3-ols

Flavanol-anthocyanin complexes were isolated by successive use of Amberlite XAD-7 chromatography, Sephadex LH-20 gel filtration and preparative HPLC from acidified, methanolic extract of strawberries (Fragaria ananassa Dutch.). These purple minor pigments were characterized by UV-Vis spectroscopy, 1D and 2D NMR techniques, and electrospray mass spectrometry to be catechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (1), epicatechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (2), afzelechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (3) and epiafzelechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (4). The stereochemistry at the 3- and 4-positions of the flavan-3-ol units was based on assumption of R-configuration at C-2. Each of the four pigments occurred in the NMR solvent as a pair of rotamers. Proved by cross-peaks in the 1H-1H NOESY NMR spectra of 1, 2 and 4, the two conformations within each rotameric pair were in equilibrium with each other. Even though 1 and 2 are based on a different aglycone, their structures may be similar to tentatively identified pigments, which have been assumed to contribute to the colour of red wines.     

The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.