Degradation of procyanidins by Aspergillus fumigatus: identification of a novel aromatic ring cleavage product

Aspergillus fumigatus was able to grow on apple-purified procyanidins (PCs). PCs concentration decreased 30% over the first 60 h of liquid fermentation. The mean degree of polymerization (DPn) of apple-purified PCs increased from 8 to 15 during the fermentation. A fungal enzyme extract from the liquid fermentation was used to study procyanidin B2 [(-)-epicatechin-(4beta-8)-(-)-epicatechin] degradation. The major degradation product (PB2-X) had a retention time of 10.5 min and a molecular mass at m/z 609. High-performance liquid chromatography/multiple fragment mass spectrometry (HPLC/MS(n)) was used for the structural characterization of PB2-X as well as that of thiolysis-treated PB2-X. Twelve fragment ions at m/z 565, 547, 457, 439 (two fragment ions), 421, 413, 377, 395, 351, 287 and 277 were completely identified. It was therefore deduced that the terminal unit of procyanidin B2 dimer was modified by an oxygenase from A. fumigatus leaving the extension unit intact. In addition, FT-IR analysis confirmed a lactone formation in (-)-epicatechin moiety involved in oxidative degradation. Two reaction schemes were postulated for the interpretation of the results.     

Inhibitory effects of cocoa flavanols and procyanidin oligomers on free radical-induced erythrocyte hemolysis

Excessive peroxidation of biomembranes is thought to contribute to the initiation and progression of numerous degenerative diseases. The present study examined the inhibitory effects of a cocoa extract, individual cocoa flavanols (-)-epicatechin and (+)-catechin, and procyanidin oligomers (dimer to decamer) isolated from cocoa on rat erythrocyte hemolysis. In vitro, the flavanols and the procyanidin oligomers exhibited dose-dependent protection against 2,2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis between concentrations of 2.5 and 40 microM. Dimer, trimer, and tetramer showed the strongest inhibitory effects at 10 microM, 59.4%, 66.2%, 70.9%; 20 microM, 84.1%, 87.6%, 81.0%; and 40 microM, 90.2%, 88.9%, 78.6%, respectively. In a subsequent experiment, male Sprague-Dawley rats (approximately 200 g; n = 5-6) were given a 100-mg intragastric dose of a cocoa extract. Blood was collected over a 4-hr time period. Epicatechin and catechin, and the dimers (-)-epicatechin-(4beta>8)-epicatechin (Dimer B2) and (-)-epicatechin-(4beta>6)-epicatechin (Dimer B5) were detected in the plasma with concentrations of 6.4 microM, and 217.6, 248.2, and 55.4 nM, respectively. Plasma antioxidant capacity (as measured by the total antioxidant potential [TRAP] assay) was elevated (P < 0.05) between 30 and 240 min following the cocoa extract feeding. Erythrocytes obtained from the cocoa extract-fed animals showed an enhanced resistance to hemolysis (P < 0.05). This enhanced resistance was also observed when erythrocytes from animals fed the cocoa extract were mixed with plasma obtained from animals given water only. Conversely, plasma obtained from rats given the cocoa extract improved the resistance of erythrocytes obtained from rats given water only. These results show cocoa flavanols and procyanidins can provide membrane protective effects.     

The relationship between antiglycation activity and procyanidin and phenolic content in commercial grape seed products

Eight commercial grape seed products (GSPs) were assessed for their inhibition of the formation of advanced glycation end-products in vitro. All 8 commercial GSPs included in this study were potent inhibitors of advanced glycation end-product formation with IC(50) values ranging from 2.93 to 20.0?μg/mL. Total procyanidin content ranged from 60% to 73%. HPLC-DAD-ELSD results indicate that (+)-catechin, (-)-epicatechin, procyanidin B1, and procyanidin B2 were predominant and ubiquitously present in all the products under study, while gallic acid and procyanidin B4 were present in relatively minor amounts. The IC(50) values correlated with total phenolic content, and multiple regression analysis indicated that IC(50) is a linear function of the concentration of gallic acid and procyanidins B1, B2, and B4. Based on this study, GSPs have the potential to complement conventional diabetes medication toward disease management and prevention.     

Topoisomerase-II-inhibitory principles from the stems of Spatholobus suberectus

Bioassay-guided fractionation of the stems of Spatholobus suberectus led to the isolation of procyanidin B4 (= (+)-catechin-(4-->8)-(-)-epicatechin) (1) and (+)-catechin-(4-->8)-(+)-catechin-(4-->8)-(-)-epicatechin (2). These compounds, isolated before from other sources, were found to be highly potent inhibitors of DNA-topoisomerase-II (Topo-II)-mediated KDNA decatenation, with IC50 values of 22.5+/-2.3 and 21.9+/-2.2 nM, resp.     

Systematic synthesis of four epicatechin series procyanidin trimers and their inhibitory activity on the Maillard reaction and antioxidant activity

A systematic synthesis of four natural epicatechin series procyanidin trimers [[4,8:4",8"]-2,3-cis-3,4-trans: 2",3"-cis-3",4"-trans: 2,3-trans-(-)-epi-catechin-(-)-epicatechin-(+)-catechin, [4,8:4",8"]-2,3-cis-3,4-trans: 2",3"-cis-3",4"-trans: 2,3-cis-tri-(-)-epicatechin: procyanidin C1, [4,8:4",8"]-2,3-cis-3,4-trans: 2",3"-trans-3",4"-trans: 2,3-trans-(-)-epicatechin-(+)-catechin-(+)-catechin: procyanidin C4, and [4,8:4",8"]-2,3-cis-3,4-trans: 2",3"-trans-3",4"-trans: 2,3-cis-(-)-epicatechin-(+)-catechin-(-)-epicatechin] is described. Condensation of (2R,3R,4S)-5,7,3'4'-tetra-O-benzyl-4-(2"-ethoxyethyloxy)flavan derived from (-)-epicatechin as an electrophile with the dimeric nucleophiles in the presence of TMSOTf followed by deprotection yielded trimers. Inhibitory activities on the Maillard reaction and antioxidant activity on lipid peroxide of the synthesized oligomers were also investigated.     

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.     

Qualitative analysis and HPLC isolation and identification of procyanidins from Vicia faba

The soluble proanthocyanidins of the coloured seed coats of Vicia faba L. were isolated and separated by solvent partition. The chemical characteristics of the proanthocyanidins were elucidated by total oxidation and partial degradation in the presence of phloroglucinol followed by HPLC analysis. The native extract of proanthocyanidins contained (+)-gallocatechin, (-)-epigallocatechin, (+)-catechin and (-)-epicatechin units. Oligomeric procyanidins were purified by chromatography on Sephadex LH-20 and the accessible compounds were isolated by RP-HPLC using a Licrospher Li 100 Column. The structures of the purified oligomeric procyanidins were elucidated using a procedure involving TLC, UV spectroscopy, ESI-MS and HPLC analysis of the products from the phloroglucinol reaction. The major condensed tannins of Vicia faba comprise six compounds identified as two A-type procyanidin dimers, the procyanidin dimers B1, B2 and B3, and a procyanidin trimer.     

Procyanidin B2 has anti- and pro-oxidant effects on metal-mediated DNA damage

Procyanidin B2 (epicatechin-(4beta-8)-epicatechin), which is present in grape seeds, apples, and cacao beans, has antioxidant properties. We investigated the mechanism of preventive action of procyanidin B2 against oxidative DNA damage in human cultured cells and isolated DNA. Procyanidin B2 inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in the human leukemia cell line HL-60 treated with an H2O2-generating system. In contrast, a high concentration of procyanidin B2 increased the formation of 8-oxodG in HL-60 cells. Experiments with calf thymus DNA also revealed that procyanidin B2 decreased 8-oxodG formation by Fe(II)/H2O2, whereas procyanidin B2 induced DNA damage in the presence of Cu(II), and H2O2 extensively enhanced it. An electron spin resonance spin trapping study utilizing 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) demonstrated that procyanidin B2 decreased the signal of M4PO-OH from H2O2 and Fe(II), whereas procyanidin B2 enhanced the signal from H2O2 and Cu(II). As an antioxidant mechanism, UV-visible spectroscopy showed that procyanidin B2 chelated Fe(II) at equivalent concentrations. As a pro-oxidant property, we examined DNA damage induced by procyanidin B2, using 32P-labeled DNA fragments obtained from genes relevant to human cancer. Our results raise the possibility that procyanidin B2 exerts both antioxidant and pro-oxidant properties by interacting with H2O2 and metal ions.     

Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4β-8)-epicatechin) – a major polyphenol in cocoa – against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H₂O₂). CPF (1 and 5 μg/ml) and procyanidin B2 (1 and 5 μM) reduced PC12 cell death caused by H₂O₂, as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H₂O₂-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H₂O₂ induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X_L and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H₂O₂ treatment diminished PARP cleavage and increased Bcl-X_L and Bcl-2 expression compared with those only treated with H₂O₂. Activation of caspase-3 by H₂O₂ was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H₂O₂-induced apoptosis involve inhibiting the downregulation of Bcl-X_L and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.     

Changes and importance of oligomeric procyanidins during maturation of grape seeds

Flavans and procyanidins from the seeds of different grape varieties were separated and identified using HPLC techniques. The compounds identified were (+)-catechin and (?)-epicatechin, dimeric procyanidins B1, B2, B3 and B4, trimeric procyanidin C2 and gallic acid. During maturation of the grape berries, the flavan-3-ol content fell in the seeds whereas procyanidin levels increased. This suggests an interrelationship between the compounds. There was also evidence of varietal differences in the amounts of phenolic compounds in grape seeds.     

Peculiarities of polyphenolic profile of fruits of Siberian crab apple and its hybrids with <Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">Domestica</Emphasis> Borkh

For the first time, we have studied the polyphenolic profile of fruits of Siberian crabapple and its hybrids (F1, F2, and F3) with domestic apple cultivated in East Siberia. It is shown that the chemical composition of polyphenolics of Siberian crabapple fruits is overwhelmingly characteristic of the genus Malus; on the other hand, it has clearly identifiable features: low content of flavan-3-ols and derivatives of cinnamic acid; high content of procyanidin B1, phloridzin, anthocyanes, and quercetin glycosides. Procyanidin B2 was not found in both peel and flesh of Siberian crabapple. In the case of hybridization with domestic apple, the fruits of resulting cultivars show a substantial change in the flavan-3-ol content ratio because of the synthesizing of procyanidin B2 in tissues and an increase in (?)-epicatechin content.     

(-)-Epicatechin inhibits nitration and dimerization of tyrosine in hydrophilic as well as hydrophobic environments.

The flavanol (-)-epicatechin is known to protect against peroxynitrite-induced nitration and oxidation reactions. This study investigated the protection afforded by (-)-epicatechin against both these reaction types on one target molecule, the aminoacid tyrosine, in a hydrophilic milieu as well as with a lipophilic tyrosine derivative, N-t-BOC l-tyrosine tert-butyl ester (BTBE), bound to liposomes. The flavanol efficiently attenuated both tyrosine nitration and tyrosine dimerization (which is based on an initial oxidation reaction) and was active in the hydrophilic and hydrophobic systems at similar IC(50) values, approximately 0.02-0.05 mol (-)-epicatechin/mol peroxynitrite. Related procyanidin oligomers of different chain-length (dimer to octamer) were also tested for their protective properties, and exhibited protection that, on a monomer basis, was in the same order of magnitude as those for (-)-epicatechin.     

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba

A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.     

Elucidation of (-)-epicatechin metabolites after ingestion of chocolate by healthy humans

After absorption in the gastrointestinal tract, (-)-epicatechin is extensively transformed into various conjugated metabolites. These metabolites, chemically different from the aglycone forms found in foods, are the compounds that reach the circulatory system and the target organs. Therefore, it is imperative to identify and quantify these circulating metabolites to investigate their roles in the biological effects associated with (-)-epicatechin intake. Using authentic synthetic standards of (-)-epicatechin sulfates, glucuronides, and O-methyl sulfates, a novel LC-MS/MS-MRM analytical methodology to quantify (-)-epicatechin metabolites in biological matrices was developed and validated. The optimized method was subsequently applied to the analysis of plasma and urine metabolites after consumption of dark chocolate, an (-)-epicatechin-rich food, by humans. (-)-Epicatechin-3'-β-d-glucuronide (C(max) 290±49nM), (-)-epicatechin 3'-sulfate (C(max) 233±60nM), and 3'-O-methyl epicatechin sulfates substituted in the 4', 5, and 7 positions were the most relevant (-)-epicatechin metabolites in plasma. When plasmatic metabolites were divided into their substituent groups, it was revealed that (-)-epicatechin glucuronides, sulfates, and O-methyl sulfates represented 33±4, 28±5, and 33±4% of total metabolites (AUC(0-24)(h)), respectively, after dark chocolate consumption. Similar metabolites were found in urine samples collected over 24h. The total urine excretion of (-)-epicatechin was 20±2% of the amount ingested. In conclusion, we describe the entire metabolite profile and its degree of elimination after administration of (-)-epicatechin-containing food. These results will help us understand more precisely the mechanisms and the main metabolites involved in the beneficial physiological effects of flavanols.     

Identification of the major antioxidative metabolites in biological fluids of the rat with ingested (+)-catechin and (-)-epicatechin.

(+)-Catechin and (-)-epicatechin are known to be biologically effective antioxidants present in the human diet, particularly in wine and tea. We studied the metabolism of these compounds to elucidate the truly active structures in biological fluids by their oral administration to rats. Without any treatment with beta-glucuronidase and sulfatase, a pair of metabolites were detected at much higher concentrations in the plasma, bile, and urine than the originally ingested compounds. Each major metabolite found in the plasma at the highest concentration was excreted in both the bile and urine, and was purified from urine. Their chemical structures were established to be (+)-catechin 5-O-beta-glucuronide and (-)-epicatechin 5-O-beta-glucuronide by MS and NMR analyses. These glucuronide conjugates exhibited high antioxidative activities as superoxide anion radical scavengers like their parent compounds. It is concluded that (+)-catechin 5-O-beta-glucuronide and (-)-epicatechin 5-O-beta-glucuronide are the biologically active in vivo structures of the ingested polyphenolic antioxidants.     

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.     

Tannins from Bendo Eucalyptus

The gallic acid, (+)-catechin and myricetin glucoside have been isolated and indentified from Bendo Eucalyptus, and two kinds of dimeric flavan-3-ol B1 and B3 were isolated as their peracetates and identified by chromatography on TLC and 1H NMR spectroscopic cha- racteristics. It is showed that condensed tannin is a procyanidin with (+)-catechin as terminal units and (–)-epicatechin as extension units by hydrolyzation anthocyanidin reaction, phloroglucinol degradation and polarimetry, UV and IR spectrometry, HPLC, 3C NMR. Linkage is C4–C8. Degree of polymerization is about 4–5.     

Polyphenols of cocoa: inhibition of mammalian 15-lipoxygenase

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.     

High-performance liquid chromatography with chemiluminescence detection of penbutolol and its hydroxylated metabolite in rat plasma

This paper describes a new method of high-performance liquid chromatography with chemiluminescence detection for the analysis of penbutolol (PB) and its main metabolite, 4-hydroxy penbutolol (4-OH PB) in rat plasma. 4-Dimethylaminosulfonyl-7-(N-chloroformylmethyl-N-methyl) amino-2,1,3-benzoxadiazole (DBD-COCl) was used as a fluorogenic labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) in acetonitrile was used as a post-column chemiluminogenic reagent. The derivatives of PB and 4-OH PB with DBD-COCl were separated by isocratic effluent with 0.01 M imidazole buffer (pH 7.0)–acetonitrile within 10 min. The detection limits of the proposed method for PB and 4-OH PB were 9.9 and 15 fmol on column, respectively. After intravenous administration of PB in rats, its plasma concentration profiles of PB and 4-OH PB were determined by the proposed method. PB was demonstrated to be rapidly metabolized to 4-OH PB at the same rate as cardiac output.