Isolation, structure determination, synthesis, and sensory activity of N-phenylpropenoyl-L-amino acids from cocoa (Theobroma cacao)

Application of chromatographic separation and taste dilution analyses recently revealed besides procyanidins a series of N-phenylpropenoyl amino acids as the key contributors to the astringent taste of nonfermented cocoa beans as well as roasted cocoa nibs. Because these amides have as yet not been reported as key taste compounds, this paper presents the isolation, structure determination, and sensory activity of these amino acid amides. Besides the previously reported (-)-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, seven additional amides, namely, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4'-hydroxy-(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, were identified for the first time in cocoa products by means of LC-MS/MS, 1D/2D-NMR, UV-vis, CD spectroscopy, and polarimetry, as well as independent enantiopure synthesis. Using the recently developed half-tongue test, human recognition thresholds for the astringent and mouth-drying oral sensation were determined to be between 26 and 220 micromol/L (water) depending on the amino acid moiety. In addition, exposure to light rapidly converted these [E]-configured N-phenylpropenoyl amino acids into the corresponding [Z]-isomers, thus indicating that analysis of these compounds in food and plant materials needs to be performed very carefully in the absence of light to prevent artifact formation.     

Quantitative analysis of N-phenylpropenoyl-L-amino acids in roasted coffee and cocoa powder by means of a stable isotope dilution assay

Since recent reports on the role of N-phenylpropenoyl-L-amino acids as powerful antioxidants and key contributors to the astringent taste of cocoa nibs, there is an increasing interest in the concentrations of these phytochemicals in plant-derived foods. A versatile analytical method for the accurate quantitative analysis of N-phenylpropenoyl-L-amino acids in plant-derived foods by means of HPLC-MS/MS and synthetic stable isotope labeled N-phenylpropenoyl-L-amino acids as internal standards was developed. By means of the developed stable isotope dilution assay (SIDA), showing recovery rates of 95-102%, 14 N-phenylpropenoyl-L-amino acids were quantified for the first time in cocoa and coffee samples. On the basis of the results of LC-MS/MS experiments as well as cochromatography with the synthetic reference compounds N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tryptophan, N-[4'-hydroxy-(E)-cinnamoyl]-L-tryptophan, and N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tyrosine, respectively, were detected for the first time in cocoa powder, and (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tyrosine, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tryptophan, N-[4'-hydroxy-(E)-cinnamoyl]-L-tryptophan, and N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tryptophan, respectively, were detected for the first time in coffee beverages.     

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.     

Content of polyphenolic compounds in the Nigerian stimulants Cola nitida ssp. alba, Cola nitida ssp. rubra A. Chev, and Cola acuminata Schott & Endl and their antioxidant capacity

Varieties of kola nuts (Cola nitida alba, Cola nitida rubra A. Chev, and Cola acuminata Schott & Endl), a group of popular Nigerian and West African stimulants, were analyzed for their content of secondary plant metabolites. The three varieties of the kola nuts contained appreciable levels of (+)-catechin (27-37 g/kg), caffeine (18-24 g/kg), (-)-epicatechin (20-21 g/kg), procyanidin B 1 [epicatechin-(4beta-->8)-catechin] (15-19 g/kg), and procyanidin B2 [epicatechin-(4beta-->8)-epicatechin] (7-10 g/kg). Antioxidant capacity of the extracts and purified metabolites was assessed by two HPLC-based and two colorimetric in vitro assays. Extracts of all varieties exhibited antioxidant capacity with IC 50 values in the range 1.70-2.83 and 2.74-4.08 mg/mL in the hypoxanthine/xanthine oxidase and 2-deoxyguanosine HPLC-based assays, respectively. Utilization of HPLC-based assays designed to reflect in situ generation of free radicals (e.g., HO(*)), as opposed to general assays (DPPH, FRAP) in common use which do not, indicate that, of the major secondary plant metabolites present in kola nut extracts, caffeine is potentially the more effective cancer chemopreventive metabolite in terms of its antioxidant capacity.     

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).     

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.     

Polyphenolic constituents of Cynomorium songaricum Rupr. and antibacterial effect of polymeric proanthocyanidin on methicillin-resistant Staphylococcus aureus

Oligomeric and polymeric flavan-3-ols were obtained by chromatographic fractionation of extracts from Cynomorium songaricum Rupr. The structure of the polymeric constituent, cynomoriitannin, was characterized using spectral and chemical data. Results from acid-catalyzed degradation indicated that cynomoriitannin is a polymeric proanthocyanidin predominantly composed of epicatechin, together with low proportions of epicatechin-3-O-gallate and catechin as extension units. The terminal unit was chiefly composed of catechin, with an admixture of epicatechin. Size exclusion chromatographic analysis demonstrated a mean polymerization degree of 14. Two new phloroglucinol adducts (cynomoriitannin-phloroglucinol adducts A and B) obtained by acid-catalyzed degradation of cynomoriitannin in the presence of phloroglucinol were characterized using spectral analyses. Six oligomeric flavan-3-ols were also identified as follows: procyanidin B3, catechin-(6'-8)-catechin, catechin-(6'-6)-catechin, epicatechin-(4β-8)- epicatechin-(4β-8)-catechin, epicatechin-(4β-6)-epicatechin-(4β-8)-catechin, and arecatannin A1, respectively. These flavan-3-ols were isolated from C. songaricum. This is the first time that this procedure has been described. The antibacterial activity of the fractions and constituents was tested against methicillin-resistant Staphylococcus aureus (MRSA). The crude acetone-water (7:3) extract had moderate activity against MRSA. Cynomoriitannin was the most effective of the plant constituents against MRSA.     

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.     

Phenolic constituents in the fruits of Cinnamomum zeylanicum and their antioxidant activity

Defatted cinnamon fruit powder was successively extracted with benzene ethyl acetate, acetone, MeOH, and water. The concentrated water extract contained the maximum amount of phenolics and showed the highest antioxidant activities. Hence, it was fractionated by Diaion HP-20SS, Diaion HP-20, and Sephadex LH-20 column chromatographies. It gave five purified compounds, the purities of which were analyzed by HPLC. Compounds 1-5 were identified as 3,4-dihydroxybenzoic acid (protocatechuic acid), epicatechin-(2beta-->O-7,4beta-->8)-epicatechin-(4beta-->8)-epicatechin (cinnamtannin B-1), 4-[2,3-dihydro-3-(hydroxymethyl)-5-(3-hydroxypropyl)-7-(methoxy)benzofuranyl]-2-methoxyphenyl beta-d-glucopyranoside (urolignoside), quercetin-3-O-(6-O-alpha-l-rhamnopyranosyl)-beta-d-glucopyranoside (rutin), and quercetin-3-O-alpha-l-rhamnopyranoside by using extensive spectral studies. The antioxidant activities of purified compounds were screened for their antioxidative potential using beta-carotene-linoleate and 1,1-diphenyl-2-picrylhydrazyl model systems. All of the compounds showed antioxidant and radical scavenging activities. This is the first report of the isolation and identification of nonvolatile constituents and as well as antioxidant activities from cinnamon fruits.     

Enzymatic dehydrogenative polymerization of urushiols in fresh exudates from the lacquer tree, Rhus vernicifera DC

Fresh exudates from the lacquer tree, Rhus vernicifera DC, were extracted with acetone and the solution was chromatographed to isolate monomer, dimer, trimer, and oligomer fractions of urushiols. Constituents of the monomeric and dimeric fractions were then identified by two-dimensional (2D) 1H-13C heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC) NMR spectroscopic techniques. The results showed that the monomeric fraction contained 3-[8'Z,11'E,13'Z-pentadecatrienyl]catechol (1), 3-[8'Z,11'Z,14'-pentadecatrienyl]catechol (2), and 3-pentadecanyl]catechol (3), which was verified by HPLC analysis. The dimeric fraction contained 8'-(3' ',4' '-dihydroxy-5' '-alkenyl)phenyl-3-[9'E,11'E,13'Z-pentadecatrienyl]catechol (4), 14'-(3' ',4' '-dihydroxy-5' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadecatrienyl]catechol (5), 2-hydroxyl-3- or -6-alkenylphenyl ethyl ether (6), 14'-(3' ',4' '-dihydroxy-2' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadeca-trienyl]catechol (7), 15'-(2' '-hydroxy-3' '- or -6' '-alkenyl)phenyloxy-3-[8'Z,11'Z,13'E)-pentadecatrienyl]catechol (8), 14'-(2' ',3' '-dihydroxy-4' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadecantrienyl]catechol (9), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-4,3'-biphenyl (10), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-4,4'-biphenyl (11), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-5,4'-biphenyl (12), and 1,2,1'-trihydroxy-6,6'-dialkenyldibenzofuran (13) as constituents. In addition, dimeric ethers and peroxides, such as compounds 14 and 15, were produced by autoxidation of monomeric urushiols in atmospheric air. The possible reaction mechanisms for the dehydrogenative polymerization of urushiols by Rhus laccase present in the fresh raw exudates under the atmospheric oxygen are discussed on the basis of structures identified. This is of primary importance because the use of the urushi exudates as coating materials does not involve organic solvents and is an environmentally friendly process.     

Molecular definition of the taste of roasted cocoa nibs (Theobroma cacao) by means of quantitative studies and sensory experiments

Sensory-guided decomposition of roasted cocoa nibs revealed that, besides theobromine and caffeine, a series of bitter-tasting 2,5-diketopiperazines and flavan-3-ols were the key inducers of the bitter taste as well as the astringent mouthfeel imparted upon consumption of roasted cocoa. In addition, a number of polyphenol glycopyranosides as well as a series of N-phenylpropenoyl-l-amino acids have been identified as key astringent compounds of roasted cocoa. In the present investigation, a total of 84 putative taste compounds were quantified in roasted cocoa beans and then rated for the taste contribution on the basis of dose-over-threshold (DoT) factors to bridge the gap between pure structural chemistry and human taste perception. To verify these quantitative results, an aqueous taste reconstitute was prepared by blending aqueous solutions of the individual taste compounds in their "natural" concentrations. Sensory analyses revealed that the taste profile of this artificial cocktail was very close to the taste profile of an aqueous suspension of roasted cocoa nibs. To further narrow down the number of key taste compounds, finally, taste omission experiments and human dose/response functions were performed, demonstrating that the bitter-tasting alkaloids theobromine and caffeine, seven bitter-tasting diketopiperazines, seven bitter- and astringent-tasting flavan-3-ols, six puckering astringent N-phenylpropenoyl-l-amino acids, four velvety astringent flavonol glycosides, gamma-aminobutyric acid, beta-aminoisobutyric acid, and six organic acids are the key organoleptics of the roasted cocoa nibs.     

Orosensory-directed identification of astringent mouthfeel and bitter-tasting compounds in red wine

Application of sequential solvent extraction, followed by HPLC combined with the taste dilution analysis, enabled the localization of the most intense velvety astringent, drying, and puckering astringent, as well as bitter-tasting, compounds in red wine, respectively. Isolation of the taste components involving gel adsorption chromatography, ultrafiltration, and synthesis revealed the identification of 26 sensory-active nonvolatiles, among which several hydroxybenzoic acids, hydroxycinnamic acids, flavon-3-ol glycosides, and dihydroflavon-3-ol rhamnosides as well as a structurally undefined polymeric fraction (>5 kDa) were identified as the key astringent components. In contradiction to literature suggestions, flavan-3-ols were found to be not of major importance for astringency and bitter taste, respectively. Surprisingly, a series of hydroxybenzoic acid ethyl esters and hydroxycinnamic acid ethyl esters were identified as bitter compounds in wine. Taste qualities and taste threshold concentrations of the individual wine components were determined by means of a three-alternative forced-choice test and the half-mouth test, respectively.     

Puerins A and B, two new 8-C substituted flavan-3-ols from Pu-er tea

Pu-er tea is a special treated fermented tea produced from crude green tea, which is prepared from the leaves of Camellia sinensis var. assamica. It is a traditional beverage having been used in China, particularly the southern areas, for a long time. Chemical investigation led to the identification of two new 8-C substituted flavan-3-ols, puerins A (1) and B (2), and two known cinchonain-type phenols, epicatechin-[7,8-bc]-4alpha-(4-hydroxyphenyl)-dihydro-2(3H)-pyranone (3) and cinchonain Ib (4), together with other seven known phenolic compounds, 2,2',6,6'-tetrahydroxydiphenyl (5), (-)-epicatechin-8-C-beta-d-glucopyranoside (6), (-)-epicatechin (EC) (7), (-)-epigallocatechin (EGC) (8), (+/-)-gallocatechin (GC) (9), gallic acid (10), and myricetin (11), in addition to caffeine (12). Their structures were determined by spectroscopic methods. The compounds 1-5, which might be formed in the postfermentative process of Pu-er tea, were isolated from tea and theaceous plants for the first time.     

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.     

Isolation, structure determination, and sensory activity of mouth-drying and astringent nitrogen-containing phytochemicals isolated from red currants (Ribes rubrum)

Application of chromatographic separation and taste dilution analyses recently revealed, besides a series of flavon-3-ol glycosides and (E)/(Z)-aconitic acid, four nitrogen-containing phytochemicals as the key astringent and mouth-drying compounds in red currants (Ribes rubrum). The isolation and structure determination of the astringent indoles 3-carboxymethyl-indole-1-N-beta-D-glucopyranoside (1) and 3-methylcarboxymethyl-indole-1-N-beta-D-glucopyranoside (2), as well as the astringent, noncyanogenic nitriles 2-(4-hydroxybenzoyloxymethyl)-4-beta-D-glucopyranosyloxy-2(E)-butenenitrile (3) and 2-(4-hydroxy-3-methoxybenzoyloxymethyl)-4-beta-D-glucopyranosyloxy-2(E)-butenenitrile (4) by means of 1D/2D NMR, LC-MS/MS, and UV-vis spectroscopy are reported. The structures of compounds 1 and 2 were confirmed by synthesis. Using the recently developed half-tongue test, human recognition thresholds for the astringent and mouth-drying nitrogen compounds were determined to be between 0.0003 and 5.9 micromol/L (water). In particular, the extraordinarily low threshold of 0.0003 micromol/L evaluated for the indole 1 represents the lowest recognition threshold of any astringent phytochemical reported to date.     

Sensory-guided decomposition of red currant juice (Ribes rubrum) and structure determination of key astringent compounds

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, led to the discovery and structure determination of 25 key astringent compounds of red currant juice. Besides several flavonol glycosides, in particular, 3-carboxymethyl-indole-1-N-beta-D-glucopyranoside, 3-methylcarboxymethyl-indole-1-N-beta-D-glucopyranoside, and a family of previously not identified compounds, namely, 2-(4-hydroxybenzoyloxymethyl)-4-beta-D-glucopyranosyloxy-2(E)-butenenitrile, 2-(4-hydroxy-3-methoxybenzoyloxymethyl)-4-beta-D-glucopyranosyloxy-2(E)-butenenitrile, (E)-6-[3-hydroxy-4-(O-beta-D-glucopyranosyl)phenyl]-5-hexen-2-one named dehydrorubrumin, and (3E,5E)-6-[3-hydroxy-4-(O-beta-D-glucopyranosyl)phenyl]-3,5-hexadien-2-one named rubrumin, have been identified. Determination of the oral astringency thresholds by means of the half-tongue test revealed that the lowest thresholds of 0.3 and 1.0 nmol/L were found for the nitrogen-containing 3-carboxymethyl-indole-1-N-beta-D-glucopyranoside and 3-methylcarboxymethyl-indole-1-N-beta-D-glucopyranoside, which do not belong to the group of plant polyphenols.     

Synthesis, analytical features, and biological relevance of 5-(3',4'-dihydroxyphenyl)-γ-valerolactone, a microbial metabolite derived from the catabolism of dietary flavan-3-ols

The physiological significance of 5-(3',4'-dihydroxyphenyl)-γ-valerolactone, an important metabolite derived from the catabolism of flavan-3-ols by gut microbiota, has been often overlooked due to the lack of the commercial standard. In the present work, this metabolite has been chemically synthesized, and its analytical parameters and antioxidant capacity have been determined in comparison to other chemical analogues [isomer 3-(3',4'-dihydroxyphenyl)-δ-valerolactone and γ-valerolactone] and other structurally related compounds [(+)-catechin, (-)-epicatechin, and 3-(3,4-dihydroxyphenyl)-propionic acid]. The synthesized compound was also used to perform a targeted analysis in samples collected during the in vitro fermentation of a grape seed flavan-3-ol extract with human fecal microbiota from three healthy volunteers. The time-course formation of 5-(3',4'-dihydroxyphenyl)-γ-valerolactone revealed large interindividual differences among volunteers, with concentrations ranging from 3.31 to 77.54 μM at 10 h of fermentation. These results are further discussed in view of the scarce reports quantifying 5-(3',4'-dihydroxyphenyl)-γ-valerolactone in in vitro fermentation studies, and pharmacokinetic and intervention studies.     

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.     

Structural analysis of a novel antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines.

Six compounds were isolated from fresh rhizomes of fingerroot (Boesenbergia pandurata Schult.) as strong antimutagens toward 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in Salmonella typhimurium TA98. These compounds were 2',4',6'-trihydroxychalcone (pinocembrin chalcone; 1), 2',4'-dihydroxy-6'-methoxychalcone (cardamonin; 2), 5,7-dihydroxyflavanone (pinocembrin; 3), 5-hydroxy-7-methoxyflavanone (pinostrobin; 4), (2,4,6-trihydroxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (5), and (2,6-dihydroxy-4-methoxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (panduratin A; 6). Compound 5 was a novel compound (tentatively termed 4-hydroxypanduratin A), and 1 was not previously reported in this plant, whereas 2-4 and 6 were known compounds. The antimutagenic IC(50) values of compounds 1-6 were 5.2 +/- 0.4, 5.9 +/- 0.7, 6.9 +/- 0.8, 5.3 +/- 1.0, 12.7 +/- 0.7, and 12.1 +/- 0.8 microM in the preincubation mixture, respectively. They also similarly inhibited the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All of them strongly inhibited the N-hydroxylation of Trp-P-2. Thus, the antimutagenic effect of compounds 1-6 was mainly due to the inhibition of the first step of enzymatic activation of heterocyclic amines.     

Regioselective dimerization of ferulic acid in a micellar solution.

Dehydrodimers of hydroxycinnamates play an important role in the cross-linking of plant cell walls. An aqueous solution of quaternary ammonium salts with a long aliphatic chain is known to spontaneously organize itself into micelles with the ionic part at the outer sphere. It is shown that regioisomeric ferulic acid dehydrodimers can be obtained in one step from trans-ferulic acid after attachment to these micelles and using the biomimetic peroxidase-H2O2 system. The surfactant hexadecyltrimethylammonium hydroxide yielded trans-4-(4-hydroxy-3-methoxybenzylidene)-2-(4-hydroxy-3-methoxyphenyl)-5-oxotetrahydrofuran-3-carboxylic acid (25%), (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (21%), and trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (14%), whereas the surfactant tetradecyltrimethylammonium bromide gave 4-cis, 8-cis-bis(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione (18%) as the main product. The use of micelles appears to be not only a new way to synthesize regioisomeric ferulic acid dehydrodimers but may also help to understand the regiospecificity of dimeric hydroxycinnamate formation in vivo.