The heterocyclic ring fission and dehydroxylation of catechins and related compounds by Eubacterium sp. strain SDG-2, a human intestinal bacterium.

A human intestinal bacterium, Eubacterium (E.) sp. strain SDG-2, was tested for its ability to metabolize various (3R)- and (3S)-flavan-3-ols and their 3-O-gallates. This bacterium cleaved the C-ring of (3R)- and (3S)-flavan-3-ols to give 1,3-diphenylpropan-2-ol derivatives, but not their 3-O-gallates. Furthermore, E. sp. strain SDG-2 had the ability of p-dehydroxylation in the B-ring of (3R)-flavan-3-ols, such as (-)-catechin, (-)-epicatechin, (-)-gallocatechin and (-)-epigallocatechin, but not of (3S)-flavan-3-ols, such as (+)-catechin and (+)-epicatechin.     

Rotation planar extraction and rotation planar chromatography of oak (Quercus robur L.) bark

The versatile novel instrument for rotation planar extraction and rotation planar chromatography was exploited for the investigation of oak bark (Quercus robur L.). The same instrument enabled extraction of the bark, analytical proof of (+)-catechin directly in the crude extract and also its fractionation. Additionally, epimeric flavan-3-ols, (+)-catechin and (-)-epicatechin were separated by analytical ultra-micro rotation planar chromatography on cellulose plates with pure water as developing solvent. A comparison of the extraction of oak bark with 80% aqueous methanol by rotation planar extraction and medium pressure solid-liquid extraction was carried out and both techniques were shown to be suitable for the efficient extraction of oak bark. The raw extracts and fractions on thin-layer chromatography showed many compounds that possessed antioxidant activity after spraying with 1,1-diphenyl-2-picrylhydrazyl. Rotation planar fractionation of 840 mg of crude oak bark extract on silica gel gave 6.7 mg of pure (+)-catechin in one run.     

(-)-Catechin in cocoa and chocolate: occurrence and analysis of an atypical flavan-3-ol enantiomer

Cocoa contains high levels of different flavonoids. In the present study, the enantioseparation of catechin and epicatechin in cocoa and cocoa products by chiral capillary electrophoresis (CCE) was performed. A baseline separation of the catechin and epicatechin enantiomers was achieved by using 0.1 mol x L(-1) borate buffer (pH 8.5) with 12 mmol x L(-1) (2-hydroxypropyl)-gamma-cyclodextrin as chiral selector, a fused-silica capillary with 50 cm effective length (75 microm I.D.), +18 kV applied voltage, a temperature of 20 degrees C and direct UV detection at 280 nm. To avoid comigration or coelution of other similar substances, the flavan-3-ols were isolated and purified using polyamide-solid-phase-extraction and LC-MS analysis. As expected, we found (-)-epicatechin and (+)-catechin in unfermented, dried, unroasted cocoa beans. In contrast, roasted cocoa beans and cocoa products additionally contained the atypical flavan-3-ol (-)-catechin. This is generally formed during the manufacturing process by an epimerization which converts (-)-epicatechin to its epimer (-)-catechin. High temperatures during the cocoa bean roasting process and particularly the alkalization of the cocoa powder are the main factors inducing the epimerization reaction. In addition to the analysis of cocoa and cocoa products, peak ratios were calculated for a better differentiation of the cocoa products.     

Changes of flavan-3-ols with different degrees of polymerization in seeds of 'Shiraz', 'Cabernet Sauvignon' and 'Marselan' grapes after veraison

Flavan-3-ols consist of flavan-3-ol monomers and polymers with different degrees of polymerization (DP). In this study, flavan-3-ol extracts from grape seeds were well separated into three fractions including monomers, oligomers (2 < DP < 10) and polymers (DP > 10), by means of normal-phase HPLC-MS. The different patterns of these three fractions were analyzed in three Vitis vinifera cultivars ('Shiraz', 'Cabernet Sauvignon' and 'Marselan') seeds from veraison to harvest. The results showed: (1) polymers were the main form of flavan-3-ols in grape seeds and monomers accounted for only a small proportion; (2) the contents of flavan-3-ol monomers in the seeds of three grape cultivars all exhibited a gradually decreasing trend with a little fluctuation, whereas the patterns of the change of contents of oligomers and polymers were extremely different among grape cultivars; the contents of flavan-3-ol oligomers were enhanced in the seeds of 'Cabernet Sauvignon', but were reduced in the other two cultivars; (3) with regard to the proportion of flavan-3-ols with a certain DP to total flavan-3-ols, both flavan-3-ol monomers and flavan-3-ols with low DP fell in proportion, while the flavan-3-ols with high DP increased correspondingly. These findings indicate that flavan-3-ol polymerization in developing seeds is variety-dependent and may be genetically regulated.     

Extraction of flavan-3-ols from grape seed and skin into wine using simulated maceration

Simulated maceration assays were carried out in wine model systems using increasing ethanol percentages from 0 to 12.5%, in order to study the extraction of flavan-3-ol monomers and oligomers from grape skins and seeds. The amount of flavan-3-ols transferred to the solutions improves with the increase in the alcohol percentage, although flavanols were much more readily released from grape skins than from seeds. Maximum release of flavanols from the skins is reached after 24 h of maceration in 12.5% ethanol, whereas long maceration and higher ethanol percentages were required for extraction from the seeds, from which maximum flavanol extraction was observed after 2-3 weeks of maceration. Thus, the length of the maceration not only had an influence in the total levels of flavanols reached in the wines, but also in their qualitative composition. The contribution of the seeds to the flavanol composition of the model wines was found to range from about 40% at 24 h of maceration in 12.5% ethanol to as high as 90% after 3 weeks. Gallocatechin was the major flavanol contributed by the skins and catechin by the seeds of the grape variety used (Vitis vinifera cv. Viura). Flavanol extraction was also followed during winemaking of commercial red wines, reaching similar conclusions to that obtained from the model assays.     

High-performance liquid chromatography/electrospray ionization mass spectrometric characterization of new products formed by the reaction between flavanols and malvidin 3-glucoside in the presence of acetaldehyde

Monomer and polymer flavan-3-ols and anthocyanins are the two major groups of phenolic compounds in red wine and one of the most important reactions during red wine ageing is the condensation reaction, either direct or indirect, between these two classes of compounds. In this work, the reaction of (+)-catechin, (-)-epicatechin and dimer procyanidin B2 with malvidin 3-glucoside, in the presence of acetaldehyde, was carried out in wine model solutions at pH 1.7 and 3.2. Identification of the reaction products was carried out by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and MS(n) analysis. Various reaction products were detected. The structures of three condensation products - malvidin-3-glucoside-ethyl-(epi)catechin-1'-hydroxyethyl, malvidin-3-glucoside-ethyl-dimer-vinyl and malvidin-3-glucoside-ethyl-dimer-1'-hydroxyethyl - were identified in a model solution for the first time. Moreover, in the reaction solution containing dimer B2 at pH 3.2, monomer epicatechin, trimer and tetramer procyanidins were also detected indicating that, under these conditions, dimer procyanidins can either be hydrolyzed to monomers or polymerized to higher oligomer or polymers.     

Simultaneous determination of polyphenols and major purine alkaloids in Greek Sideritis species, herbal extracts, green tea, black tea, and coffee by high-performance liquid chromatography-diode array detection

Herein, a high-performance liquid chromatography-diode array detection method has been developed for the simultaneous determination of 15 phenolic antioxidants: flavan-3-ols, (-)-epigallocatechin, (+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin, a phenolic acid (gallic acid), a hydroxycinnamic acid (chlorogenic acid), flavones (apigenin), flavonols (kaempferol, quercetin, and myricetin), and purine alkaloids (caffeine theophylline, theobromine) in different herb extracts, tea, and coffee varieties. The developed method was validated and successfully applied in order to determine the polyphenolic content to estimate the antioxidant activity of the Sideritis species commonly known as Greek mountain tea. To the best of our knowledge, this is the first report on the quantitative determination of catechins and other polyphenols in Greek mountain tea. Acidic hydrolysis was necessary for the simultaneous determination of the aglycones of the target analytes. According to our results, chlorogenic acid, myricetin, apigenin, catechin, and epicatechin gallate are found in the Sideritis species.     

Identification of dimeric anthocyanins and new oligomeric pigments in red wine by means of HPLC-DAD-ESI/MSn

High-pressure liquid chromatography-diode array detector-electrospray ionisation/ion trap mass spectrometry (HPLC-DAD-ESI/MS(n)) analyses carried out in red wine fractions revealed the existence of dimeric anthocyanins (A-A(+)), previously detected in grape skin, and allowed the detection and identification, for the first time, of other derived oligomers. The structure of these compounds was characterised according to their MS(n)(n = 1-4) fragmentation patterns. The newly detected oligomers consisted of a flavanol, (epi)catechin or (epi)gallocatechin, linked through its C(4) position to the nucleophilic positions of the upper unit of a dimeric anthocyanin (F-A-A(+)). All the compounds contained malvidin as one of the anthocyanin subunits, whereas the other anthocyanin moiety could be either delphinidin, cyanidin, petunidin, peonidin or malvidin. With the fractionation method employed, the dimeric anthocyanins eluted in the same fractions as the monomeric anthocyanins. However, the new F-A-A(+) oligomers were found in the same fractions as F-A(+) dimers, which might indicate a structural similarity between both types of compounds. ESI/MS(n) analyses, coupled or not to HPLC, showed to be a useful and powerful tool for detecting and identifying these oligomers in wines, which usually elute from reversed-phase columns as humps and/or are overlapped by the peaks of other compounds. The detection of these oligomeric pigments in wine has provided more information about the complex pigments responsible for the elevation of the base line observed in the chromatograms of wines and has also revealed that oligomeric anthocyanins can take part in the reactions usually undergone by monomeric anthocyanins.     

Chiral separation of (+)/(−)-catechin from sulfated and glucuronidated metabolites in human plasma after cocoa consumption

Cocoa is well-known to be rich in flavan-3-ols. Previous analyses have established that alkaline treatment of cocoa beans results in epimerization of (−)-epicatechin to (−)-catechin and (+)-catechin to (+)-epicatechin. Now, the question is whether both epimers can be absorbed by the human organism. This paper describes sample preparation and an HPLC method for chiral determination of (+)/(−)-catechin from sulfated and glucuronidated metabolites in human plasma. The sample preparation includes enzymatic hydrolysis of the catechin metabolites, and solid-phase extraction (SPE). A PM-γ-cyclodextrin column is used with a coulometric electrode-array detection (CEAD) system. The recovery of catechin ranges from 89.9 to 96.8%. The limit of detection is 5.9 ng mL⁻¹ for (−)-catechin and 6.8 ng mL⁻¹ for (+)-catechin, and the limit of quantification is 12.8 ng mL⁻¹ for (−)-catechin and 16.9 ng mL⁻¹ for (+)-catechin. The relative standard deviation of the method ranges from 0.9 to 1.5%. This method was successfully applied to human plasma after consumption of a cocoa drink. In one human self-experiment, (+)-catechin and (−)-catechin were found in human plasma, but metabolism of the two enantiomers differed.     

Selective O-methylation of polyhydroxyflavan-3-ols via benzyl carbonates

The flavan-3-ols (+)-catechin and (−)-epicatechin were selectively transformed to their 3′,4′-di-O-methyl- and 5,7-di-Omethyl-ethers respectively via benzyl carbonates. Such a regioselectivity is facilitated by marked differences in the pKa values of the phenolic hydroxyl groups and by the ability of the o-dihydroxy funtionality of the pyrocatechol B-ring to form borate complexes under mild basic conditions.     

Tandem mass spectrometry of the B-type procyanidins in wine and B-type dehydrodicatechins in an autoxidation mixture of (+)-catechin and (-)-epicatechin

We describe the characterization of the B-type procyanidins in wine and the B-type dehydrodicatechins (dimeric flavan-3-ols) obtained for the autoxidation of (+)-catechin and (-)-epicatechin by tandem mass spectrometry (MS/MS) coupled to reversed-phase high-performance liquid chromatography (HPLC). The MS/MS analysis demonstrates that the interesting major fragments derive from the dissociations of the C-ring on the catechin or epicatechin unit, such as retro-Diels-Alder reactions. The two kinds of dimers give completely different fragmentations because of the striking effect of the C-C interflavan linkage (IFL). For the natural dimers in wine, a catechin or epicatechin unit linking to the C-4 position stabilizes the product ions by forming a large pi-pi hyperconjugated system, whereas a similar pi-pi system is formed within dehydrodicatechin B through the C-C IFL. Thus dissociation in MS/MS experiments was inhibited. Apparently, the fragmentations of the dimers differ from that of the monomer, which is very important in the study of the gas-phase ion behaviour of the polymeric flavan-3-ols. In addition, two specific fragment ions at m/z 451 for native dimers and at m/z 393 for autoxidation species in HPLC/MS/MS were found to be very useful for analysing mixtures of B-type procyanidins and B-type dehydrodicatechins in food and beverages.     

Analysis of several phenolic compounds with potential antioxidant properties in grape extracts and wines by high-performance liquid chromatography-photodiode array detection without sample preparation

A RP-HPLC method that allows the separation of several types of phenolic compounds present in grapes and wines by direct injection of samples, using a binary gradient with solvents free of salts and photodiode array detection is described. Results show that more than 15 different phenolic molecules with antioxidant properties (flavan-3-ols, anthocyanins, cinnamic acid derivatives, flavonol derivatives and trans-resveratrol) may be separated in a single run by direct injection of red wine. The method is also valuable for the analysis of these compounds in white wine and in skins, seeds and pulp extracts of red and white grapes.     

Supercritical fluid extraction and HPLC determination of relevant polyphenolic compounds in grape skin

The polyphenols determined are: (+)-catechin, (-)-epicatechin, rutin, quercetin and trans-resveratrol. Suitable conditions of supercritical fluid extraction were established using ethanol as a modifier of the polarity solvent (supercritical carbon dioxide). Final extraction conditions were: 20% v/v ethanol, 60degreesC, 250 bars and flow rate 2 mL/min. Static step time and dynamic step time were established using a selected grape skin sample. The extract was collected in water; the more polar polyphenols ((+)-catechin and (-)-epicatechin) remain in solution but rutin, quercetin and trans-resveratrol precipitate in this medium, thereby the solution of the extracted polyphenols was filtered. (+)-Catechin and (-)-epicatechin were determined in the liquid fraction, while the solid fraction, containing rutin, quercetin and trans-resveratrol, was solved with ethanol/H20 (40:60). HPLC determination was carried out at C18 stationary phase, with ethanol/water/acetic acid as mobile phases and UV-visible diode array detection. Due to the significant differences between the polarity of the polyphenols, two different mobile phases were used. An ethanol/water/acetic acid (5:93:2) mobile phase was used to determine (+)-catechin (280 nm) and (-)-epicatechin (280 nm). On the other hand, rutin (254 nm), quercetin (254 nm) and trans-resveratrol (306 nm) were resolved using ethanol/water/acetic acid (40:58:2) as mobile phase. Instrumental parameters were optimised and analytical parameters obtained. The analytical method was validated and applied to five different varieties of Vitis vinifera from the geographical area of Valencia.     

Oxidative Transformation of Dihydroflavonols and Flavan-3-ols by Anthocyanidin Synthase from Vitis vinifera

Twelve polyphenols from three distinct families (dihydroflavonols, flavan-3-ols, and flavanones) were studied as potential substrates of anthocyanidin synthase from Vitis vinifera (VvANS). Only flavan-3-ols of (2R,3S) configuration having either a catechol or gallol group on ring B are accepted as substrates. Only dihydroflavonols of (2R,3R) configuration are accepted as substrates, but a catechol or gallol group is not mandatory. Flavanones are not substrates of VvANS. HPLC and MS/MS analyses of the enzymatic products showed that the VvANS-catalyzed oxidative transformation of (+)-dihydroflavonols, such as dihydroquercetin, dihydrokaempferol and dihydromyricetin, leads only to the corresponding flavonols. Among the flavan-3-ols recognized as substrates, (+)-gallocatechin was only transformed into delphinidin by VvANS, whereas (+)-catechin was transformed into three products, including two major products that were an ascorbate–cyanidin adduct and a dimer of oxidized catechin, and a minor product that was cyanidin. Data from real-time MS monitoring of the enzymatic transformation of (+)-catechin suggest that its products are all derived from the initial C₃-hydroxylation intermediate, i.e., a 3,3-gem-diol, and their most likely formation mechanism is discussed.     

Phenolic concentrations and antioxidant properties of wines made from north american grapes grown in china

The characteristics of wine phenolics found in several North American and (for comparison) European grape cultivars grown in China were analyzed. This was done to find non-Vitis vinifera wines with prominent features in order to diversify the kinds of wines. The phenolic richness and antioxidant activity decreased in the order: red > rose > white wines. In the red wines, the American grape 'Cynthiana' had the highest total concentrations of phenols, anthocyanins, flavonols and phenolic acids, as well as antioxidant capacity, followed by the French hybrid 'Chambourcin', the lowest were detected in two European grape varieties, 'Merlot' and 'Cabernet Sauvignon', while the total flavon-3-ols levels were reversed among these red grape cultivars. The highest concentration of stilbenes out of all the wines analyzed was found in the 'Merlot' variety. There were significant differences among wine phenolic compositions between North American and European grape cultivars. The antioxidant activities were significantly related to the concentrations of total phenols (r2 = 0.996), anthocyanins (r2 = 0.984), flavonols (r2 = 0.850) and gallic acid (r2 = 0.797). The prominent features of wine aroma and nutrition could make the American grape wines attractive to consumers. It is therefore necessary to perform further research on cultural practices and wine making involving these grapes.     

毛细管区带电泳法测定葡萄籽中儿茶素类化合物

 采用毛细管区带电泳法测定了 10种中国产葡萄籽中的 4个主要儿茶素类化合物 :(+)儿茶素、(- )表儿茶素、(± )表没食子儿茶素、(± )表儿茶素没食子酸酯的含量。在 0 0 2mol/L硼砂和 0 0 0 5mol/L磷酸盐的混合缓冲体系 (pH 10 0 )的背景缓冲液中 ,4个化合物在 10min内取得了令人满意的分离。迁移时间的重现性(RSD)小于 2 % ,峰面积的重现性 (RSD)小于 5 %。在质量浓度为 0 0 0 5g/L～ 0 5 g/L时 ,线性相关系数大于0 995。检测限为 3mg/L～ 10mg/L。该方法简单、快速、准确 ,可作为葡萄籽分析和药用开发过程中分析儿茶素类化合物的有效方法推广使用。     

Evaluation of Anthocyanin Profile and Color in Sweet Cherry Wine: Effect of Sinapic Acid and Grape Tannins during Aging

Cherries are rich in bioactive phenolic compounds and are often fermented into cherry wines. The degradation of anthocyanins during storage will cause color deterioration. The study aimed to utilize sinapic acid and grape tannins in cherry wine to maintain a high fraction in the colored forms of anthocyanins, in order to maximize the color intensity, the latter being associated with good product quality. The effects on the anthocyanin profile and on color parameters of copigments, utilizing spectral measurement combined with UPLC-MS quantitative analysis, have been evaluated in sweet cherry wines. The copigmentation effect of sinapic acid and grape tannin was accompanied by the bathochromic shift and the hyperchromic effect, which lead to an increase in color intensity (lower L*, higher a* and b*). During the aging process, sinapic and grape tannin increased the content of pyranoanthocyanins in cherry wine, especially the addition of sinapic acid makes the cherry wine generate 10-syringyl-pyranocyanidin-3-rutinoside. These results demonstrate that sinapic acid is suitable for adding before alcohol fermentation, while grape tannins can be added before aging.     

Application of Elicitors in Two Ripening Periods of Vitis vinifera L. cv Monastrell: Influence on Anthocyanin Concentration of Grapes and Wines

In recent years, it has been demonstrated that the application of elicitors such as methyl-jasmonate (MeJ) and benzothiadiazole (BTH) to wine grapes can increase their phenolic and aromatic compounds if they are treated at the beginning of ripening (veraison). However, the veraison period is short, and it is not always possible to apply the treatments in a few days. Therefore, it would be of great interest to optimize the moment of elicitor application or extend the treatment period. The aim of this paper was to analyze during two consecutive years (2016–2017) the foliar application of MeJ, BTH, and a combination of both, during two different ripening periods of Monastrell grapes (veraison and mid-ripening), and determine the more appropriate moment to increase the concentration of anthocyanins. To carry out this aim, analysis of anthocyanins by HPLC in grapes and wines was mainly performed. The most suitable period for the application of MeJ, BTH, and MeJ + BTH was at mid-ripening, since the grapes showed a greater accumulation of anthocyanins at harvest. However, the MeJ + BTH treatment applied during veraison also obtained similar results, which would allow extending the application period if necessary. However, the increase in the anthocyanin content of grapes was not reflected in all the wines, which may have been due to reinforcement of the skin cell wall as a result of the application of elicitors. Further analysis is needed to improve the maceration process of the Monastrell grapes and the extraction of the anthocyanins that were increased by the treatments applied in the vineyard.     

Biooxidation of (+)-catechin and (-)-epicatechin into 3,4-dihydroxyflavan derivatives by the endophytic fungus Diaporthe sp. isolated from a tea plant

The microbial transformation of (+)-catechin (1) and (-)-epicatechin (2) by endophytic fungi isolated from a tea plant was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. transformed them (1, 2) into the 3,4-cis-dihydroxyflavan derivatives, (+)-(2R,3S,4S)-3,4,5,7,3',4'-hexahydroxyflavan (3) and (-)-(2R,3R,4R)-3,4,5,7,3',4'-hexahydroxyflavan (7), respectively, whereas (-)-catechin (ent-1) and (+)-epicatechin (ent-2) with a 2S-phenyl group resisted the biooxidation.     

Method for the determination of catechin and epicatechin enantiomers in cocoa-based ingredients and products by high-performance liquid chromatography: single-laboratory validation

A single-laboratory validation study was performed for an HPLC method to identify and quantify the flavanol enantiomers (+)- and (-)-epicatechin and (+)- and (-)-catechin in cocoa-based ingredients and products. These compounds were eluted isocratically with an ammonium acetate-methanol mobile phase applied to a modified beta-cyclodextrin chiral stationary phase and detected using fluorescence. Spike recovery experiments using appropriate matrix blanks, along with cocoa extract, cocoa powder, and dark chocolate, were used to evaluate accuracy, repeatability, specificity, LOD, LOQ, and linearity of the method as performed by a single analyst on multiple days. In all samples analyzed, (-)-epicatechin was the predominant flavanol and represented 68-91% of the total monomeric flavanols detected. For the cocoa-based products, within-day (intraday) precision for (-)-epicatechin was between 1.46-3.22%, for (+)-catechin between 3.66-6.90%, and for (-)-catechin between 1.69-6.89%; (+)-epicatechin was not detected in these samples. Recoveries for the three sample types investigated ranged from 82.2 to 102.1% at the 50% spiking level, 83.7 to 102.0% at the 100% spiking level, and 80.4 to 101.1% at the 200% spiking level. Based on performance results, this method may be suitable for routine laboratory use in analysis of cocoa-based ingredients and products.