Structural identification and distribution of proanthocyanidins in 13 different hops

Ten newly isolated hop proanthocyanidin oligomers and flavan-3-ol monomers from 13 different hops have been identified as gallocatechin, gallocatechin-(4alpha-->8)-catechin, gallocatechin-(4alpha-->6)-catechin, catechin-(4alpha-->8)-gallocatechin, catechin-(4alpha-->6)-gallocatechin, afzelechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-catechin-(4alpha-->8)-catechin, epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin, catechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, and gallocatechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, together with seven previously isolated oligomers, namely, catechin, epicatechin, epicatechin-(4beta-->8)-catechin, epicatechin-(4beta-->8)-epicatechin, catechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-epicatechin, and epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin. These compounds were subjected to acid-catalyzed degradation in the presence of phloroglucinol or by partial or complete acid-catalyzed degradation and reaction with benzyl mercaptan followed by desulfurization. The resultant adducts when compared to authentic samples by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry served to identify the precursors. The composition of proanthocyanidins from 13 different hops was similar, but the concentration of individual compounds showed some differences, which indicated that hop proanthocyanidin profiles are affected by geographic origin and are variable depending on the cultivars.     

Rapid reversed phase ultra-performance liquid chromatography analysis of the major cocoa polyphenols and inter-relationships of their concentrations in chocolate

Chocolate and other cocoa-containing products are a rich source of polyphenols. This paper describes an ultra-performance liquid chromatography (UPLC) method that can separate and quantify in 3 min six of the major chocolate polyphenols: catechin; epicatechin; B2 (epicatechin-4beta-8-epicatechin); B5 (epicatechin-4beta-6-epicatechin); C1 (epicatechin-4beta-8-epicatechin-4beta-8-epicatechin); and tetramer D (epicatechin-4beta-8-epicatechin-4beta-8-epicatechin-4beta-8-epicatechin). A survey of 68 chocolate samples indicated that there was a strongly predictive relationship between epicatechin and the other individual polyphenols, especially procyanidin B2 (R 2 = 0.989), even though the chocolates came from varied sources and manufacturers. The relationship was less strong with catechin, and so further work to explore the reasons for this difference was performed. Chiral analysis on a subset of 23 chocolates showed that (-)-epicatechin had a predictive relationship with (+)-catechin in line with the other polyphenols, but not with (-)-catechin (the predominant form). This indicates that (-)-catechin is the most affected by manufacturing conditions, possibly formed through epimerization from (-)-epicatechin during processing. The results show that epicatechin concentrations can be used to predict the content of other polyphenols, especially B2 and C1, and total polyphenols content. Finally, the (-)-catechin content is not predictable from the epicatechin content, and it is concluded that this is the main form of polyphenol that varies according to manufacturing conditions and cocoa origin.     

Monomeric, oligomeric, and polymeric flavan-3-ol composition of wines and grapes from Vitis vinifera L. Cv. Graciano, Tempranillo, and Cabernet Sauvignon

The monomeric, oligomeric, and polymeric flavan-3-ol composition of wines, grape seeds, and skins from Vitis vinifera L. cv. Graciano, Tempranillo, and Cabernet Sauvignon has been studied using (1) fractionation by polyamide column chromatography followed by HPLC/ESI-MS analysis, (2) fractionation on C(18) Sep-Pak cartridges followed by reaction with vanillin and acid-catalyzed degradation in the presence of toluene-alpha-thiol (thiolysis). The content of monomers ((+)-catechin and (-)-epicatechin), procyanidin dimers (B3, B1, B4, and B2), trimers (T2 and C1), and dimer gallates (B2-3-O-gallate, B2-3'-O-gallate, and B1-3-O-gallate) ranged from 76.93 to 133.18 mg/L in wines, from 2.30 to 8.21 mg/g in grape seeds, and from 0.14 to 0.38 mg/g in grape skins. In wines, the polymeric fraction represented 77-84% of total flavan-3-ols and showed a mean degree of polymerization (mDP) value of 6.3-13.0. In grapes, the polymeric fraction represented 75-81% of total flavan-3-ols in seeds and 94-98% in skins and showed mDP values of 6.4-7.3 in seeds and 33.8-85.7 in skins. All the monomeric flavan-3-ols and oligomeric procyanidins found in wines were also present in seeds, although differences in their relative abundances were seen. The skin polymeric proanthocyanidins participated in the equilibration of the wine polymeric proanthocyanidin fraction, especially contributing to the polymer subunit composition and mDP.     

Interactions between flavan-3-ols and poly(L-proline) studied by isothermal titration calorimetry: effect of the tannin structure

Interactions of proline-rich proteins (PRPs) with flavan-3-ols was studied using poly(L-proline) as a model protein by means of isothermal titration calorimetry (ITC). Several parameters were varied: (i) the galloylation and B-ring trihydroxylation of the flavan-3-ols (catechin, epicatechin, epicatechin gallate, and epigallocatechin gallate) and (ii) the degree of polymerization (monomers were compared to a mixture of oligomers with average degree of polymerization of 3.85). Large differences were observed between the flavan-3-ol monomers: no enthalpy change was measured when catechin and epicatechin were titrated by poly(L-proline), whereas thermodynamic parameters were determined in the case of galloylated monomers and mixture of oligomers. Stoichiometry ranged from 1 oligomer bound for each 12 proline units to 1 galloylated monomer for each 8 or 10 proline units. Association constants were in the range of 10(4)-10(5) M(-1), indicating a relatively high affinity of galloylated flavanols toward poly(L-proline), and the coexistence of both enthalpy- and entropy-driven phenomena was suggested. Finally, the binding of grape seed tannins to proteins was shown to be a cooperative process.     

A-type proanthocyanidins from lychee seeds and their antioxidant and antiviral activities

Two new A-type trimeric proanthocyanidins with two doubly bonded interflavanoid linkages, litchitannin A1 [epicatechin-(2β→O→7,4β→6)-epicatechin-(2β→O→7,4β→8)-catechin] (1) and litchitannin A2 [epicatechin-(2β→O→7,4β→6)-epicatechin-(2β→O→7,4β→6)-epicatechin] (2), were isolated from lychee (Litchi chinensis Sonn. cv. Heiye) seeds together with aesculitannin A (3), epicatechin-(2β→O→7,4β→8)-epiafzelechin-(4α→8)-epicatechin (4), proanthocyanidin A1 (5), proanthocyanidin A2 (6), proanthocyanidin A6 (7), epicatechin-(7,8-bc)-4β-(4-hydroxyphenyl)-dihydro-2(3H)-pyranone (8), and epicatechin (9). Their structures were elucidated on the basis of spectroscopic and chemical evidence. It is the first time that compounds 1-4, 7, and 8 have been reported in this species. Compounds 1-9 showed more potent antioxidant activity than L-ascorbic acid with ferric reducing antioxidant power (FRAP) values of 3.71-24.18 mmol/g and IC50 values of 5.25-20.07 μM toward DPPH radicals. Moreover, litchitannin A2 (2) was found to exhibit in vitro antiviral activity against coxsackie virus B3 (CVB3) and compounds 3 and 6 displayed antiherpes simplex virus 1 (HSV-1) activity.     

Application of a molecular sensory science approach to alkalized cocoa (Theobroma cacao): structure determination and sensory activity of nonenzymatically C-glycosylated flavan-3-ols

Application of comparative taste dilution analyses on nonalkalized and alkalized cocoa powder revealed the detection of a velvety, smoothly astringent tasting fraction, which was predominantly present in the alkalized sample. LC-MS/MS analysis, 1D- and 2D-NMR, and CD spectroscopy as well as model alkalization reactions led to the unequivocal identification of the velvety, smoothly astringent molecules as a series of catechin- and epicatechin-C-glycopyranosides. Besides the previously reported (-)-epicatechin-8-C-beta-D-galactopyranoside, additional flavan-3-ol-C-glycosides, namely, (-)-epicatechin-8-C-beta-D-glucopyranoside, (-)-catechin-8-C-beta-D-glucopyranoside, (-)-catechin-6-C-beta-D-glucopyranoside, (-)-epicatechin-6-C-beta-D-glucopyranoside, (-)-catechin-8-C-beta-D-galactopyranoside, (-)-catechin-6-C-beta-D-galactopyranoside, (-)-catechin-6-C,8-C-beta-D-diglucopyranoside, (-)-epicatechin-6-C,8-C-beta-D-digalactopyranoside, (-)-catechin-6-C,8-C-beta-D-digalactopyranoside, and epicatechin-6-C,8-C-beta-D-diglucopyranoside, were identified for the first time in cocoa. Most surprisingly, these phenol glycoconjugates were demonstrated by model experiments to be formed via a novel nonenzymatic C-glycosylation of flavan-3-ols. Using the recently developed half-tongue test, human recognition thresholds for the astringent and mouth-drying oral sensation were determined to be between 1.1 and 99.5 micro mol/L (water) depending on the sugar and the intramolecular binding position as well as the aglycone.     

Sensory-guided decomposition of roasted cocoa nibs (Theobroma cacao) and structure determination of taste-active polyphenols

Sequential application of solvent extraction, gel permeation chromatography, and RP-HPLC in combination with taste dilution analyses, followed by LC-MS and 1D/2D-NMR experiments and thiolytic degradation, revealed that, besides theobromine and caffeine, the flavan-3-ols epicatechin, catechin, procyanidin B-2, procyanidin B-5, procyanidin C-1, [epicatechin-(4beta-->8)](3)-epicatechin, and [epicatechin-(4beta-->8)](4)-epicatechin were among the key compounds contributing to the bitter taste as well as the astringent mouthfeel imparted upon consumption of roasted cocoa. In addition, a series of quercetin, naringenin, luteolin, and apigenin glycopyranosides as well as a family of not previously identified amino acid amides, namely, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine, (+)-N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-aspartic acid, and (+)-N-(E)-cinnamoyl-L-aspartic acid, have been identified as key astringent compounds of roasted cocoa. Furthermore, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-3-hydroxy-l-tyrosine (clovamide), (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine (deoxyclovamide), and (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, reported previously as antioxidants, have been found as contributors of cocoa's astringent taste. By means of the half-tongue test, the taste thresholds of flavan-3-ols and glycosides have been determined.     

Ethylidene-bridged Flavan-3-ols in red wine and correlation with wine age

Condensed tannins are responsible for astringency and bitterness and participate in the color stability of red wines. During wine making and aging, they undergo chemical changes including, for example, acetaldehyde-induced polymerization. Following this study, the ethylidene-bridged flavan-3-ols were monitored in different vintage wines made from grapes collected in the same vineyard in three wineries in Bordeaux, Pauillac, and Saint Julien. Flavan-3-ol ethylidene bridges were quantified by wine 2,2'-ethylidenediphloroglucinol (EDP) phloroglucinolysis. This method was based upon the analysis of EDP, a product formed after acid-catalyzed cleavage of wine flavan-3-ols in the presence of excess phloroglucinol. The flavan-3-ol ethylidene bridges were then compared to flavan-3-ol contents (phloroglucinolysis), phenolic contents, and color measurements. Low amounts of flavan-3-ol ethylidene bridges (0.8-2.5 mg L(-1)) were quantified in wines. Flavan-3-ol ethylidene bridges represent less than 4% of flavan-3-ol bonds, but the proportion of these linkages relative to native interflavan bonds increased with wine age. This proportion correlated with pigmented polymers.     

Analysis of ethylidene-bridged flavan-3-ols in wine

A method was developed to determine the amount of ethylidene-bridged flavan-3-ols in wine. The method was based upon the analysis of 2,2'-ethylidenediphloroglucinol (EDP), a product formed after acid-catalyzed cleavage of wine flavan-3-ols in the presence of excess phloroglucinol. In the developed analytical method, the wine was purified and concentrated using C18 solid-phase extraction before the phloroglucinolysis reaction was carried out. This procedure was used to quantify ethylidene-bridged flavan-3-ols in wine and the molar ratio between ethylidene-bridged linkages and native interflavan linkages. The method validation showed 9.2% repeatability. The recovery of the ethylidene-bridged flavanols in wine was 90% for concentrations up to 4.5 mg.L-1 of ethylidene-bridged linkages, and it decreases to 83% above and until the concentration reached 7.6 mg.L-1. Initial results showed that the concentration of ethylidene-bridged flavan-3-ols measured in wines was very low (less than 1.3 mg.L-1) and that they represented less than 1.3% of the total interflavonoid linkages on a molar ratio.     

Electromigration profiles of Cynomorium songaricum based on capillary electrophoresis with amperometric detection

A high-performance capillary electrophoresis with amperometric detection (CE-AD) method has been developed for the simultaneous determination of the pharmacologically active ingredients in Cynomorium songaricum in this work. Under the optimum conditions, phloridzin, epicatechin, catechin, naringenin, rutin, luteolin, quercetin, gallic acid, and protocatechuic acid can be well separated or nearly baseline separated (epicatechin and catechin peaks) within 31 min at the separation voltage of 14 kV in a 50 mmol L(-1) Borax running buffer (pH 9.0). Detection limits (S/N=3) ranged from 5.7 x 10(-8) to 8.5 x 10(-9) g mL(-1) for all nine analytes. This procedure was successfully used for the analysis and comparison of the content difference of C. songaricum samples collected from different places based on their electrophorograms or "electromigration profiles".     

Identification of adducts between an odoriferous volatile thiol and oxidized grape phenolic compounds: kinetic study of adduct formation under chemical and enzymatic oxidation conditions

HPLC-MS and (1)H, (13)C, and 2D NMR analyses were used to identify new addition products between 3-sulfanylhexan-1-ol (3SH) and o-quinones derived from (+)-catechin, (-)-epicatechin, and caftaric acid. The kinetics of formation of these adducts were monitored in a wine model solution and in a must-like medium by HPLC-UV-MS with the aim of understanding the chemical mechanism involved in reactions between volatile thiols and o-quinones. One o-quinone-caftaric acid/3SH adduct, three o-quinone-(+)-catechin/3SH adducts, and three o-quinone-(-)-epicatechin/3SH adducts were characterized. Caftaric acid was oxidized faster than (-)-epicatechin and (+)-catechin when these phenolic compounds were incubated in a one-component mixture with polyphenoloxidase (PPO) in the presence of 3SH. Consequently, o-quinone-caftaric acid formed adducts with 3SH more rapidly than o-quinone-(+)-catechin and o-quinone-(-)-epicatechin in the absence of other nucleophilic species. Furthermore, o-quinone-(-)-epicatechin reacted faster than o-quinone-(+)-catechin with 3SH. Sulfur dioxide decreased the yield of adduct formation to a significant extent. Under chemical oxidation conditions, the rates and yields of adduct formation were lower than those observed in the presence of PPO, and o-quinone-caftaric acid was slightly less reactive with 3SH, compared to oxidized flavan-3-ols. The identification of o-quinone-caftaric acid/3SH and o-quinone-(+)-catechin/3SH adducts in a must matrix suggests that the proposed reaction mechanism is responsible for 3SH loss in dry wines during their vinification and aging process.     

Structure-reactivity relationships of flavan-3-ols on product generation in aqueous glucose/glycine model systems

Ring structure-reactivity relationships of three flavan-3-ols [epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG)] and three simple phenolic compounds (1,3,5-trihydroxybenzene, 1,2,3-trihydroxybenzene, and methylgallate as the analogous individual A, B, and C benzene rings of EGCG) on product generation in an aqueous glucose-glycine reaction model system (125 degrees C and 30 min) were investigated. The addition of EC, ECG, or EGCG to a glucose-glycine model was reported to similarly significantly reduce the formation of pyrazine, methyl-substituted pyrazines, and cyclotene. All three flavan-3-ols were also reported to generate phenolic-C2, C3, C4, and C6 sugar fragment adducts and to statistically reduce the concentration of glyoxal, glycolaldehyde, methylglyoxal, hydroxyacetone, diacetyl, acetoin, and 3-deoxyglucosone during the reaction time course, except for the EGCG reaction where 3-deoxyglucosone was not statistically different from the control after 20 min. For the simple phenolic compounds, methylgallate followed by 1,2,3-trihydroxybenzene was the least reactive, while 1,3,5-trihydroxybenzene was reported as the most reactive phenolic structure for quenching or reducing the concentration of the alpha-hydroxy- and alpha-dicarbonyl sugar fragments during the reaction time course. These results imply that the main mechanism flavan-3-ols reduced product generation was phenolic-sugar fragment carbonyl trapping reactions primarily on the A ring (the meta-polyhydroxylated benzene ring) or not due to the alteration of the reaction reduction potential.     

Flavonoid content of U.S. fruits, vegetables, and nuts

Analytical data are reported for 20 flavonoids (as aglycones) determined for more than 60 fresh fruits, vegetables, and nuts collected from four regions across the United States at two times of the year. Sample collection was designed and implemented by the Nutrient Data Laboratory (USDA). Analyses of eight flavan-3-ols (catechin, catechin gallate, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, gallocatechin, and gallocatechin gallate), six anthocyanins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin), two flavanones (hesperetin and naringenin), two flavones (apigenin and luteolin), and two flavonols (myricetin and quercetin) were performed by the Food Composition Laboratory (USDA) using a hydrolysis method for the anthocyanidins, flavones, and flavonols and a direct extraction method for the flavan-3-ols and flavanones. Experimental results compare favorably (few statistically significant differences) to literature values in the flavonoid and proanthocyanidin database previously compiled by the Nutrient Data Laboratory. The results of this study showed a seasonal variation only for blueberries. This study also showed that the variation in the flavonoid content of foods, as purchased by the U.S. consumer, is very large. The relative standard deviation, averaged for each flavonoid in each food, was 168%.     

Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance

Procyanidin fractions from apple were separated according to the degree of polymerization using normal phase chromatography. Evaluation of physiological functionalities of procyanidins requires individual structural determination. However, it is difficult to elucidate the structure of procyanidins, in particular those with (+)-epicatechin (1) or (-)-catechin (2) units, and determine whether the interflavanoid bonds are 4beta-->8 or 4beta-->6 without cleavage and acetylation. Structural determination used LC-MS and low-temperature NMR. Nine procyanidins were separated by preparative HPLC consisting of three well-known procyanidins [procyanidin B1 (3), procyanidin B2 (4), and procyanidin C1 (5)] and six new procyanidins [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin (6); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-catechin (7); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin (8); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-catechin (9); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin (10); and epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin (11)]. Compounds 6-11 were detected for the first time as apple constituents.     

Identification and in vitro biological activities of hop proanthocyanidins: inhibition of nNOS activity and scavenging of reactive nitrogen species

Oligomeric proanthocyanidins constitute a group of water-soluble polyphenolic tannins that are present in the female inflorescences (up to 5% dry wt) of the hop plant (Humulus lupulus). Humans are exposed to hop proanthocyanidins through consumption of beer. Proanthocyanidins from hops were characterized for their chemical structure and their in vitro biological activities. Chemically, they consist mainly of oligomeric catechins ranging from dimers to octamers, with minor amounts of catechin oligomers containing one or two gallocatechin units. The chemical structures of four procyanidin dimers (B1, B2, B3, and B4) and one trimer, epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin (TR), were elucidated using mass spectrometry, NMR spectroscopy, and chemical degradation. When tested as a mixture, the hop oligomeric proanthocyanidins (PC) were found to be potent inhibitors of neuronal nitric oxide synthase (nNOS) activity. Among the oligomers tested, procyanidin B2 was most inhibitory against nNOS activity. Procyanidin B3, catechin, and epicatechin were noninhibitory against nNOS activity. PC and the individual oligomers were all strong inhibitors of 3-morpholinosydnonimine (SIN-1)-induced oxidation of LDL, with procyanidin B3 showing the highest antioxidant activity at 0.1 microg/mL. The catechin trimer (TR) exhibited antioxidant activity more than 1 order of magnitude greater than that of alpha-tocopherol or ascorbic acid on a molar basis.     

New approach for the synthesis and isolation of dimeric procyanidins

A semisynthetic approach for the strategic formation of various procyanidins has been developed. Procyanidin-rich grape seed extracts were reacted with flavan-3-ols under acid catalysis. The reaction enables the formation of dimeric procyanidins and the elimination of higher oligomeric and polymeric procyanidins through degradation. An easy and fast method for the isolation of large amounts of procyanidins after semisynthetic formation by high-speed countercurrent chromatography is presented. Dimeric procyanidins (B1, B2, B3, B4, B5, and B7) were obtained and isolated. Furthermore, galloylated dimeric procyanidins [(-)-epicatechin-3- O-gallate-4beta-->8-(+)-catechin, (-)-epicatechin-3- O-gallate-4beta-->8-(-)-epicatechin, (-)-epicatechin-3- O-gallate-4beta-->6-(-)-epicatechin, and (-)-epicatechin-4beta-->8-(-)-epicatechin-3- O-gallate], as well as trimeric procyanidins [C1, (-)-epicatechin-4beta-->6-(-)-epicatechin-4beta-->8-(-)-epicatechin, and (-)-epicatechin-4beta-->6-(-)-epicatechin-4beta-->6-(+)-catechin] were obtained and isolated as side products. This approach also afforded gambiriins A1 and A2, which were all isolated and unambiguously identified, and the novel 3-(2,4,6-trihydroxyphenyl)-1-(3,4-dihydroxyphenyl)-propan-2-ol-1beta-->8-(-)-epicatechin (gambiriin A4).     

Characterization of a colorless anthocyanin-flavan-3-ol dimer containing both carbon-carbon and ether interflavanoid linkages by NMR and mass spectrometry

Direct addition of anthocyanins and flavan-3-ols was investigated in a model system by incubating malvidin 3-glucoside and (-)-epicatechin in ethanol. Analysis of reaction products by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS) before and after thiolysis showed the formation of colorless dimers detected at m/z 781 in the negative ion mode, with retention times and spectroscopic characteristics identical to those of compounds detected in wine, which contain one malvidin 3-glucoside unit and one flavanol unit. On the basis of their resistance to thiolysis, these compounds were postulated to be bicyclic dimers linked with both carbon-carbon and ether bonds as observed in the case of A type proanthocyanidins. The major dimer analyzed by NMR experiments was identified as malvidin 3-glucoside(C2-O-C7,C4-C8)epicatechin, confirming this hypothesis. A similar assay was performed with (+)-catechin instead of (-)-epicatechin, and the formation of bicyclic dimers was also observed.     

Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3'-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.     

Floral Trifolium proanthocyanidins: polyphenol formation and compositional diversity

Recent studies on the biosynthesis of proanthocyandins have identified key genes and enzymes in the formation of 2,3-cis-flavan-3-ols (epiafzelechin, epicatechin, and epigallocatechin). However, the enzymes that catalyze the polymerization of monomer units remain unknown. Studies of proanthocyanidin synthesis have involved the reference plant, Arabidopsis thaliana, forage legumes, tobacco, and grape. For this paper the floral proanthocyanidins of 10 Trifolium species were studied to identify candidates with contrasting proanthocyanidin chemistry, for the identification of factors involved in polymerization. Proanthocyandins were present in the floral portions (3.1-12.2 g/100 g of dry matter) of Trifolium spp. Thiolyic cleavage of proanthocyanidin fractions liberated flavan-3-ol extension units dominated by cis stereochemistry. The terminal units' stereochemistry of T. michelianum was exclusively trans (catechin), that of T. hirtum mixed trans (catechin and gallocatechin), and that of T. vesiculosum equal proportions of trans and cis (catechin and epicatechin). Compositional dispersion for oligomers was determined by MALDI-TOF MS, which showed a range of ions up to approximately 2200 Da. The three Trifolium spp. highlighted, all annuals, may warrant investigation for insights into proanthocyanidins biosynthesis.     

Antioxidant oligomeric proanthocyanidins from sea buckthorn (Hippophae rhamnoides) Pomace

After flavonol glycosides, monomeric flavan-3-ols, and dimeric and trimeric proanthocyanidins were fractionated from an extract of sea buckthorn (Hippophae rhamnoides) pomace by Sephadex LH-20 gel chromatography, oligomeric proanthocyanidins were eluted. The oligomeric fraction accounted for 84% of the total proanthocyanidins and 75% of the total antioxidant activity of the sea buckthorn pomace extract. To elucidate the structure of the oligomeric fraction, it was depolymerized by acid catalysis in the presence of phloroglucinol. The structure of the resulting flavan-3-ol monomers and flavan-3-ol-phloroglucinol adducts was determined by electrospray ionization mass spectrometry (ESI-MS) and (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy. Quantitative high-performance liquid chromatography investigations demonstrated (+)-gallocatechin as the predominating subunit in the oligomeric fraction. This observation was confirmed by ESI-MS, matrix-assisted laser desorption/ionization mass spectrometry, and (13)C NMR spectroscopy. The results showed that the majority of the flavan-3-ol subunits possessed a 2,3-trans configuration. The oligomers consisted mainly of prodelphinidin subunits whereas procyanidins were present in smaller amounts, indicating a very uncommon composition of the sea buckthorn proanthocyanidins. The mean degree of polymerization of the oligomeric proanthocyanidins was between 6 and 9.