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.     

Stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant

The microbial transformation of five flavans (1-5) by endophytic fungi isolated from the tea plant Camellia sinensis was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. oxidized stereoselectively at C-4 position of (+)-catechin (1) and (-)-epicatechin (2) to give the correspondent 3,4-cis-dihydroxyflavan derivatives (6, 10), respectively. (-)-Epicatechin 3-O-gallate (3) and (-)-epigallocatechin 3-O-gallate (4) were also oxidized by the fungus into 3,4-dihydroxyflavan derivatives (10, 12) via (-)-epicatechin (2) and (-)-epigallocatechin (11), respectively. Meanwhile, (-)-gallocatechin 3-O-gallate (5), (-)-catechin (ent-1) and (+)-epicatechin (ent-2), which possess a 2S-phenyl substitution, resisted the biotransformation.     

Enzymatic sulfation of polyphenols related to tannins by arylsulfotransferase.

This report discusses a novel type of arylsulfotransferase (AST) which was derived from human intestinal bacterium sulfated polyphenolic compounds when p-nitrophenyl sulfate (PNS) was taken as a donor substrate. (+)-Catechin, (+/-)-catechin, (-)-epicatechin and (-)-epicatechin gallate were better substrates than tyramine. (-)-Epigallocatechin and (-)-epigallocatechin gallate were slightly worse substrates than tyramine. Although gallic acid was a bad substrate, alkyl gallate esters were better substrates than tyramine. The degree of acceptor specificity increased in proportion to the length of the alkyl group up to the carbon number of five. Pedunculagin, geraniin and corilagin were less effective than tyramine. Rosmarinic acid and penta-O-galloyl-beta-D-glucose were similarly well sulfated. Two products, 4'-monosulfate and 4',5-disulfate of (+)-catechin, were detected at a two-fold molar excess of PNS over (+)-catechin. When (+)-catechin-4'-monosulfate as an acceptor was enzymatically sulfated with PNS as a donor, only the 4',5-disulfate was produced. Thus, arylsulfotransferase was useful for the convenient preparation of sulfate esters of polyphenols at their specific hydroxyl groups.     

Anticomplement and antimicrobial activities of flavonoids from Entada phaseoloides

Seventeen flavonoids isolated from the extracts of the stem of Entada phaseoloides were investigated for their anticomplement (both classic and alternative pathways) and antimicrobial activities against Gram-positive bacteria (MRSA, MSSA, Standard Enterococcus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeuroginosa) and the yeast-like pathogenic fungus Candida albicans. The anticomplement studies revealed a dose-dependent activity among isolated quercetin, luteolin, apigenin, galangin, 5,2',5'-trihydroxy-3,7,4'-trimethoxyflavone-2'-O-beta-D-glucoside (+)-3,3',5',5,7-pentahydroflavanone, (+)-dihydrokaempferol, (-)-epicatechin, (+)-catechin, naringenin, and 5,7,3',5'-tetrahydroxyflavanone, and the antimicrobial results indicated that quercetin, 5,7,4'-trihydroxy-3'-methoxyflavonol and galangin produced the inhibitory activities against MRSA, MSSA, and Standard Enterococcus, while luteolin and rhamnocitrin displayed inhibition against only MRSA and MSSA. To the best of our knowledge, this is the first report that describes the anticomplement and antimicrobial activities of the stem of E. phaseoloides.     

Cytotoxicity of constituents from Mexican propolis against a panel of six different cancer cell lines

The cytotoxicity of 39 compounds, including eighteen flavonoids (flavanones, 1-10; flavones, 11-17; flavanol, 18), sixteen phenolic acid derivatives (aromatic acids, 19-24; aldehyde, 25; esters, 26-34) and five glycerides (35-39), isolated from Mexican propolis, were evaluated against a panel of six different cancer cell lines; murine colon 26-L5 carcinoma, murine B16-BL6 melanoma, murine Lewis lung carcinoma, human lung A549 adenocarcinoma, human cervix HeLa adenocarcinoma and human HT-1080 fibrosarcoma. A phenylpropanoid-substituted flavanol, (2R,3S)-8-[4-phenylprop-2-en-1-one]-4',7-dihydroxy-3',5-dimethoxyflavan-3-ol (18), showed the most potent cytotoxicity against A549 cells (IC50, 6.2 microM) and HT-1080 cells (IC50, 3.9 microM), stronger than those of the clinically used anticancer drug, 5-fluorouracil (IC50, 7.5 microM and 5.4 microM, respectively). Based on the observed results, the structure-activity relationships are discussed.     

Phenolic constituents from the rhizomes of Dryopteris crassirhizoma

A new phenolic glycoside, dryopteroside (1), was isolated from the rhizomes of Dryopteris crassirhizoma (Dryopteridaceae), together with five known compounds, 4beta-carboxymethyl-(-)-epicatechin (2), isobiflorin (3), biflorin (4), 1-beta-D-glucopyranosyloxy-3-methoxy-5-hydroxybenzene (5) and (+)-catechin-6-C-beta-D-glucopyranoside (6). The new compound was elucidated to be 1-butanoyl-3-C-beta-D-glucopyranosyl-5-methyl-phloroglucinyl-6-O-beta-D-glucopyranoside (1) by chemical and various spectroscopic analyses. The known compounds 2-6 were first reported from the genus Dryopteris.     

Biotransformation of pinoresinol diglucoside to mammalian lignans by human intestinal microflora,and isolation of Enterococcus faecalis strain PDG-1 responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol

By anaerobic incubation of pinoresinol diglucoside (1) from the bark of Eucommia ulmoides with a fecal suspension of humans, eleven metabolites were formed, and their structures were identified as (+)-pinoresinol (2), (+)-lariciresinol (3), 3'-demethyl-(+)-lariciresinol (4), (-)-secoisolariciresinol (5), (-)-3-(3", 4"-dihydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butane-1, 4-diol (6), 2-(3', 4'-dihydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butane-1, 4-diol (7), 3-(3"-hydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butane-1, 4-diol (8), 2-(3', 4'-dihydroxybenzyl)-3-(3"-hydroxybenzyl)butane-1, 4-diol (9), (-)-enterodiol (10), (-)-(2R, 3R)-3-(3", 4"-dihydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butyrolactone (11), (-)-(2R, 3R)-2-(3', 4'-dihydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butyrolactone (12), (-)-(2R, 3R)-3-(3"-hydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butyrolactone (13), 2-(3', 4'-dihydroxybenzyl)-3-(3"-hydroxybenzyl)butyrolactone (14), 2-(3'-hydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butyrolactone (15) and (-)-(2R, 3R)-enterolactone (16) by various spectroscopic means, including two dimensional (2D)-NMR, mass spectrometry and circular dichroism. A possible metabolic pathway was proposed on the basis of their structures and time course experiments monitored by thin-layer chromatography. Furthermore, a bacterial strain responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol was isolated from a human fecal suspension and identified as Enterococcus faecalis strain PDG-1.     

Chemical Constltuents from the Roots of Zizyphus jujuba Mill. var. spinosa (I)

The chemical constituents of the roots of Zizyphus jujuba Mill. var. spinosa were investigated with a combination of Sephadex LH-20 column, centrifugal partition chromatography and RP-18 low pressure liquid chromatography. We isolated seven flavonoids-quercetin ( 1 ), (+)-dihydroquercetin ( 2 ), (+)-dihydrokaempferol ( 7 ), quercetin-3-0-β-glucoside ( 8 ), 2-hydroxynaringenin ( 11 ), rutin ( 12 ), and quercetin 3-0-(2^G-β-D-xylopyranosylrutinoside) ( 13 ); four catechins-(-)-gallocatechin ( 3 ), (-)-epigallocatechin ( 4 ), (-)-catechin ( 5 ), and (-)-epicatechin ( 6 ); and two phenolic carboxylic acids-p-hydroxybenzoic acid ( 9 ) and protocatechuic acid ( 10 ) from the water soluble fraction of the ethanolic extract NMR date of 13 were assigned by 2D NMR techniques.     

Tyrosinase inhibitory constituents from the bark of Peltophorum dasyrachis (yellow batai)

Tyrosinase inhibitory activity-guided fractionation of the bark of Peltophorum dasyrachis (yellow batai) led to the isolation of the six active compounds which were characterised as six flavonoids: apigenin (1), (+)-2,3-trans-dihydrokaempferol (2), (+)-2,3-trans-dihydrokaempferol-3-O-α-L-rhamnoside (3), (+)-4',7-dimethoxy-2,3-trans-dihydroquercetin (4), (+)-2,3-trans-dihydroquercetin (5) and (-)-2,3-trans-dihydroquercetin-3-O-α-L-rhamnoside (6). All compounds were isolated for the first time from the bark of P. dasyrachis. Moreover, all compounds were evaluated for tyrosinase activities towards L-DOPA as the substrate. We observed that compounds 2 and 5 showed potent inhibitory effects (IC?? values were 126?±?3.2 and 210?±?5.8?μM, respectively). In general, for flavonoids the 3',4'-dihydroxy group's substituent is a more potent inhibitor than the 4'-hydroxy group substituent, i.e. quercetin?>?kaempferol. Interestingly, our result in the oxidation of L-DOPA showed that the 4'-hydroxy group substituent (compound 2) is a more potent inhibitor than the 3',4'-dihydroxy group substituent (compound 5). This result showed a new relationship between tyrosinase inhibitory activities and flavonoids. The kinetic analyses by Lineweaver-Burk plots showed that both the compounds 2 and 5 behaved as competitive inhibitors of L-DOPA oxidation.     

Anthocyanins and flavan-3-ols from grapes and wines of Vitis vinifera cv. Cesanese d'Affile

The objective of the present study was to evaluate the amount of some potential health-promoting phenols in the grape of Vitis vinifera cv. Cesanese d'Affile and in wines made from these grapes. The analyses were performed using HPLC/DAD/MS. The accumulation of anthocyanins in the skin and flavan-3-ols in the seed was determined at different stages of ripening of the grape (i.e. green, veraison, middle stage of ripening, and complete ripening). Thirteen anthocyanins were identified in the skin at all stages of ripening, except the green stage. With regard to flavan-3-ols, (+)-catechin, (-)-epicatechin, and (-)-epicatechin gallate were detected in all of the seed samples. The highest (+)-catechin content was found in the seeds of the green grape (2 mg g(-1) DW), whereas in the seeds from the completely ripe grape the content was more than ten times lower. The highest catechin content in the seed was correlated with the lowest anthocyanin content in the skin. The wines produced in the years 2004 and 2005 showed, at wavelengths of 520 and 280 nm, almost identical quali-quantitative chromatographic profiles, with high concentrations of anthocyanin 3-O-glucosides, low concentrations of acylated anthocyanins, and trace amounts of (+)-catechin and (-)-epicatechin.     

Conformational analysis of the sixteen C(4)-C(6) and C(4)-C(8) linked dimers of (+)-catechin and (−)-epicatechin

A conformational analysis has been performed for sixteen dimers of (+)-catechin and/or (?)-epicatechin using molecular mechanics (MM2). Monomer units are linked by 4α-6, 4α-8, 4β-6, and 4β-8 bonds. THe four possible combinations of (+)-catechin and/or(?)-epicatechin are used for each bonding pattern. The objectives are characterization of (1) the two rotational isomers at the bond between the two monomer units and (2) the conformations of the heterocyclic rings. There is a twofold rotation about the bond between monomer units. Differ4ences in the energies at the two minima range from a few tenths of a kcal/mol to several kcal/mol, depending on the dimer Heterocyclic rings occupy a range of conformations that can be described as half chairs with varying degrees of distoration toward C(2) or C(3) sofas. The more frequent distortion is toward the C(2) sofa. Interconversion between most of the heterocyclic ring conformations can be obtained by coordinated motion of C(2) and C(3), over a range of about 40 pm, with respect to the mean plane of the fused aromatic ring system.     

Demonstration of the occurrence of flavanol-anthocyanin adducts in wine and in model solutions

Flavanol-anthocyanin (F-A⁺) adducts were detected in red wine. A mechanism involving acid-catalysed cleavage of flavanol oligomers followed by nucleophilic addition of the anthocyanin moiety (in its hemiketal form) to the resulting carbocation (F⁺) was postulated. To confirm this mechanism, reactions between malvidin 3-O-glucoside (Mv3glc) and procyanidin dimer epicatechin-(4-8)-epicatechin 3-O-gallate (B2-3′OG) were studied in a model solution system at pH 2. A new pigment with a UV-Vis spectrum similar to that of Mv3glc and a signal at m/z=781 in the positive ion mode was detected and was attributed to Ec-Mv3glc, in agreement with the proposed reaction pathway.A protocol adapted from the synthesis of procyanidin dimers, in which the terminal flavanol units was replaced with Mv3glc, was tested. Two new pigments were formed with a signal at m/z=781, in the positive ion mode. These ions were attributed to catechin-Mv3glc.Both adducts obtained by hemisynthesis showed exactly the same fragmentation pattern as that present in wine. In particular, the loss of a 126 amu fragment corresponding to the unsubstituted A-ring of the flavanol unit indicated that all of them were (epi)catechin-Mv3glc adducts.These results prove that reactions between the carbocations resulting from cleavage of tannin interflavanic bonds and anthocyanins occur in wine.     

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.     

Novel synthesis and oxidizing ability of tropylium ions annulated with two 2,4-dimethylfuro[2,3-d]pyrimidine-1(2H),3(4H)-diones

Convenient preparation of novel tropylium ions annulated with two 2,4-dimethylfuro[2,3-d]pyrimidine-1(2H),3(4H)-diones, 12a(+).BF(4)(-) and 12b(+)().BF(4)(-), consists of a reaction of 2-methoxytropone with dimethylbarbituric acid to give 7,9-dimethyl-3-[1',3'-dimethyl-2'(1'H),4'(3'H),6'(5'H)-trioxopyrimidin-5'-ylidene]cyclohepta[b]pyrimido[5,4-d]furan-8(7H),10(9H)-dione 8 and the following oxidative cyclization by using DDQ or photoirradiation under aerobic conditions. On the basis of the MO calculations, the selectivity of two types of oxidative cyclization reactions of 8 was rationalized. X-ray crystal analyses and MO calculations were carried out to clarify the structural characteristics of 12a(+). BF(4)(-) and 12b(+).BF(4)(-). The stability of cations 12a(+) and 12b(+) is expressed by the pK(R) + values which were determined spectrophotometrically as 8.8 and 8.6. The electrochemical reduction of 12a(+) and 12b(+) exhibited reduction potential at -0.63 and -0.62 (V vs Ag/AgNO(3)), respectively. Reactions of 12a(+)().BF(4)(-) and 12b(+)().BF(4)(-) with some nucleophiles, hydride and diethylamine, were carried out to clarify that the reactivity of 12a(+)().BF(4)(-) and 12b(+).BF(4)(-) was substantially dependent on the annulating position. The oxidizing ability of 12a(+).BF(4)(-) and 12b(+).BF(4)(-) toward alcohols and amines in the autorecycling process was demonstrated as well.     

(+)-Kunstlerone, a new antioxidant neolignan from the Leaves of Beilschmiedia kunstleri gamble

A new neolignan, 3,4-dimethoxy-3',4'-methylenedioxy-2,9-epoxy-6,7-cyclo-1,8-neolign-11-en-5(5H)-one, which has been named (+)-kunstlerone (1), together with six known alkaloids: (+)-norboldine (2), (+)-N-methylisococlaurine (3), (+)-cassythicine (4), (+)-laurotetanine (5), (+)-boldine (6) and (-)-pallidine (7), were isolated from the leaves of Beilschmiedia kunstleri. The structures were established through various spectroscopic methods notably 1D- and 2D-NMR, UV, IR and LCMS-IT-TOF. (+)- Kunstlerone (1) showed a strong antioxidant activity, with an SC(50) of 20.0 μg/mL.     

Tannins from Betel Nuts

From the acetone extract of fresh-betel nuts, two procyanidin pentamers, two procyanidin tetramers and three procyanidin trimers, along with (+)-catechin, (-)-epicatechin, procyanidin A-l, procyanidin B-l, procyanidin B-2, and procyanidin B-7 were isolated. Based on ₁H NMR and ₁₃C NMR spectral data as well as the results of thiolysis degradation, the chemical structures of two series of oligomeric arecatannins A and B have been established. The arecatannins from betel nuts were found to be condensed-type tannins which possess (-)-epicatechin unit and (+)-catechin units. All arecatannins had C₄-C₈ linked (-)-epicatechin in the upper units whereas the (+)-catechin in the terminal unit had C₄-C₈ or C₄-C₆ linkage.     

Ceramide and Cerebroside from the stem bark of Ficus mucuso (Moraceae)

Two new sphingolipids mucusamide (1) and mucusoside (2) have been isolated from methanol soluble part of the stem bark of Ficus mucuso WELW., together with fifteen known secondary metabolites including cellobiosylsterol (3), β-sitosterol (4), stigmasterol (5), β-sitosterol 3-O-β-D-glucopyranoside (6), lupeol acetate (7), ursolic acid (8), procatechuic acid (9), 2-methyl-5,7-dihydroxychromone 8-C-β-D-glucoside (10), apigenin (11), (-)-epicatechin (12), (+)-catechin (13), N-benzoyl-L-phenylalanilol (14), α-acetylamino-phenylpropyl α-benzoylamino-phenylpropionate (15), asperphenamate (16) and bejaminamide (17). Structures of compounds 1 and 2 were elucidated by spectroscopic analysis and chemical methods.     

Versatile synthesis of epicatechin series procyanidin oligomers, and their antioxidant and DNA polymerase inhibitory activity

Proanthocyanidins, known as condensed tannins or oligomeric flavonoids, exist in many edible plants and show various interesting biological activities. We have developed a simple and versatile method of synthesizing procyanidin oligomers consisting of (−)-epicatechin and (+)-catechin. This method is applicable to the synthesis of various 3-O-substituted oligomers. We report here the stereoselective and length controlled synthesis of [4-8]-condensed (−)-epicatechin series procyanidin oligomers. We described the details of the synthesis of an two tetramers, (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-(−)-epicatechin and (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-(+)-catechin (arecatannin A1), (−)-epicatechin pentamer and two 3,3″,3?-tri-O-galloyl trimers, (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-3,3″,3?-tri-O-gallate and (−)-epicatechin-(−)-epicatechin-(+)-catechin-3,3″,3?-tri-O-gallate with the condensation method using TMSOTf as a catalyst. The ability of DPPH radical scavenging activity and DNA polymerase inhibitory activity of these oligomeric compounds were investigated.     

(-)-Kunstleramide, a new antioxidant and cytotoxic dienamide from the bark of Beilschmiedia kunstleri gamble

A new dienamide, (2E,4E)-7-(3',4'-dimethoxyphenyl)-N-ethyl-6-(R)-hydroxyhepta- 2,4-dienamide, named (-)-kunstleramide (1), were isolated from the bark of Beilschmiedia kunstleri Gamble together with one neolignan: (+)-kunstlerone (2) and seven known alkaloids: (+)-nornuciferine (3), (-)-isocaryachine (4), (+)-cassythicine (5), (+)-laurotetanine (6), (+)-boldine (7), noratherosperminine (8), (+)-N-demethylphyllocaryptine (9). Their structures were established from spectroscopic techniques, most notably 1D- and 2D-NMR, UV, IR, OR, circular dichroism (CD) spectra and LCMS-IT-TOF. (-)-Kunstleramide (1) exhibited very poor dose-dependent inhibition of DPPH activity, with an IC?? value of 179.5 ± 4.4 μg/mL, but showed a moderate cytotoxic effect on MTT assays of A375, A549, HT-29, PC-3 and WRL-68 with EC?? values of 64.65, 44.74, 55.94, 73.87 and 70.95 μg/mL, respectively.