Biosynthesis of (+)-catechin glycosides using recombinant amylosucrase from Deinococcus geothermalis DSM 11300

Amylosucrase (ASase, EC 2.4.1.4) is a glucosyltransferase that hydrolyzes sucrose into glucose and fructose and produces amylose-like glucan polymers from the released glucose. (+)-Catechin is a plant polyphenolic metabolite having skin-whitening and antioxidant activities. In this study, the ASase gene from Deinococcus geothermalis (dgas) was expressed in Escherichia coli, while the recombinant DGAS enzyme was purified using a glutathione S-transferase fusion system. The (+)-catechin glycoside derivatives were synthesized from (+)-catechin using DGAS transglycosylation activity. We confirmed the presence of two major transglycosylation products using TLC. The (+)-catechin transglycosylation products were isolated using silica gel open column chromatography and recycling-HPLC. Two (+)-catechin major transfer products were determined through ¹H and ¹³C NMR to be (+)-catechin-3′-O-α-d-glucopyranoside with a glucose molecule linked to (+)-catechin and (+)-catechin-3′-O-α-D-maltoside with a maltose linked to (+)-catechin. The presence of (+)-catechin maltooligosaccharides in the DGAS reaction was also confirmed via recycling-HPLC and enzymatic analysis. The effects of various reaction conditions (temperature, enzyme concentration, and molar ratio of acceptor and donor) on the yield and type of (+)-catechin glycosides were investigated.     

Enzymic and nonenzymic reduction of (+)-dihydroquercetin to its 3,4,-diol

A NADPH-dependent reductase activity, capable of converting (+)-dihydroquercetin (2,3-trans) to its 3,4-diol (a leucocyanidin), has been demonstrated in crude, soluble protein extracts derived from cell suspension cultures of Douglas fir (Pseudotsuga menziessi). Neither NADH nor ascorbate substituted as the H-donor. Quantitative analyses were based on the production of cyanidin, the formation of an adduct with vanillin, and on absorbance at 280 nanometers. Nonenzymic reduction of (+)-dihydroquercetin with NaBH₄ produced two presumably isomeric flavan-3,4,-diols. One of these was similar to the enzymically produced diol, based on products isolated by chromatography on paper, on thin-layer cellulose and on C₁₈ reversed-phase columns (high performance liquid chromatography), and on the conversion of the diol to the all-trans dimer of (+)-catechin upon the addition of (+)-catechin.     

Degradation of (+)-catechin by Acinetobacter calcoaceticus MTC 127

Acinetobacter calcoaceticus MTC 127 was able to grow on catechin and protocatechuic acid (PCA) as sole carbon source. Cells induced with catechin oxidized catechin and PCA at rates higher than cells of uninduced cultures. Two aromatic compounds, PCA and phloroglucinol carboxylic acid (PGCA) were isolated from culture filtrate of cells grown in catechin and characterized by infrared spectrometry and high performance thin-layer chromatography. Moreover, A. calcoaceticus MTC 127 produced high levels of PCA compared to PGCA in the degradation of catechin. Based upon these results, a pathway for the degradation of (+)-catechin in A. calcoaceticus MTC 127 is proposed. Enzymes extracted from catechin-induced culture showed catechin oxygenase (cox) and protocatechuate 3,4-dioxygenase (pcd) activities. Catechin oxygenase was purified by column chromatography and SDS-PAGE analysis showed a single band with an apparent molecular weight of 47 kDa.     

Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)-catechin to cyanidin and a novel procyanidin

Anthocyanidins were proposed to derive from (+)-naringenin via (2R,3R)-dihydroflavonol(s) and (2R,3S,4S)-leucocyanidin(s) which are eventually oxidized by anthocyanidin synthase (ANS). Recently, the role of ANS has been put into question, because the recombinant enzyme from Arabidopsis exhibited primarily flavonol synthase (FLS) activity with negligible ANS activity. This and other studies led to the proposal that ANS as well as FLS may select for dihydroflavonoid substrates carrying a "beta-face" C-3 hydroxyl group and initially form the 3-geminal diol by "alpha-face" hydroxylation. Assays with recombinant ANS from Gerbera hybrida fully supported the proposal and were extended to catechin and epicatechin isomers as potential substrates to delineate the enzyme specificity. Gerbera ANS converted (+)-catechin to two major and one minor product, whereas ent(-)-catechin (2S,3R-trans-catechin), (-)-epicatechin, ent(+)-epicatechin (2S,3S-cis-epicatechin) and (-)-gallocatechin were not accepted. The K(m) value for (+)-catechin was determined at 175 microM, and the products were identified by LC-MS(n) and NMR as the 4,4-dimer of oxidized (+)-catechin (93%), cyanidin (7%) and quercetin (trace). When these incubations were repeated in the presence of UDP-glucose:flavonoid 3-O-glucosyltransferase from Fragariaxananassa (FaGT1), the product ratio shifted to cyanidin 3-O-glucoside (60%), cyanidin (14%) and dimeric oxidized (+)-catechin (26%) at an overall equivalent rate of conversion. The data appear to identify (+)-catechin as another substrate of ANS in vivo and shed new light on the mechanism of its catalysis. Moreover, the enzymatic dimerization of catechin monomers is reported for the first time suggesting a role for ANS beyond the oxidation of leucocyanidins.     

7-O-Galloyl-(+)-catechin and 3-O-galloylprocyanidin B-3 from Sanguisorba officinalis

7-O-Galloyl-(+)-catechin and 3-O-galloylprocyanidin B-3, along with gambiriins A-1 and B-3 and four polygalloylglucoses, have been isolated fro     

(+)-catechin 3-rhamnoside from Erythroxylum novogranatense

A new flavanol glycoside has been isolated from stems of Erythroxylum novogranatense and its structure has been elucidated on the basis of MS and ¹H NMR spectroscopy and hydrolytic studies as (+)-catechin 3-O-α-L-rhamnopyranoside. On a similar basis of chemical and spectroscopic evidence, the presence of ombuin 3-O-rutinoside has been established. Furthermore, the occurrence of procyanidin biflavanoids has been demonstrated by the characterization of B₁ and B₃ as the first representatives of B-type proanthocyanidins in the genus Erythroxylum.     

Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydroquercetin and Flavan-3,4-cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir

Extracts of cell suspension cultures from Douglas fir (Pseudotsuga menziesii) needles catalyzed the production of (+)-catechin (2,3-trans flavan-3-o1) from the 2,3-trans-flavan,3,4-cis-diol (leucocyanidin) in a NADPH-dependent reductase reaction at pH 7.4. Catechin was also produced, along with the 3,4-cis-diol, in a double step reduction from (+)-dihydroquercetin. It was necessary to eliminate any thiol such as 2-mercaptoethanol or dithiothreitol from the extract or assay mixture because these thiols apparently formed thioethers with the 3,4-diols.     

Interaction of (+)-catechin with a lipid bilayer studied by the spin probe method

The interaction of (+)-catechin with a lipid bilayer was examined by the spin probe method. The spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was dissolved in an aqueous dipalmitoylphosphatidylcholine (DPPC) dispersion containing (+)-catechin. The temperature dependence of the TEMPO parameter was measured. The increase of this parameter due to pretransition was eliminated by the addition of (+)-catechin, suggesting that it was adsorbed to the lipid membrane surface in the gel state, which hindered the change of the membrane from a flat to wavy structure. In the temperature region of the main transition, the TEMPO parameter increased rapidly, then gradually with increasing temperature, which could be explained by the eutectic phase diagram. The rotational correlation time of a spin probe 16-doxylstearic acid and the order parameter of 5-doxylstearic acid in the aqueous dispersion system of egg yolk phosphatidylcholine revealed that the motion of the alkyl chain in the liquid crystal state was hindered in the center of the membrane as well as near the surface by the adsorption of (+)-catechin.     

Determination of (+)-catechin in plasma by high-performance liquid chromatography using fluorescence detection

A high-performance liquid chromatographic method, using fluorescence detection, was developed for the determination of (+)-catechin in rabbit plasma. The procedure involved the precipitation of plasma protein using acetonitrile, followed by solid-phase adsorption onto alumina. After washing with water and methanol, the residue was vortex-mixed with perchloric acid solution to release the adsorbed (+)-catechin. Separation was performed on a reversed-phase column using an eluent consisting of phosphoric acid solution with 12% acetonitrile. The excitation and emission wavelengths were set at 280 and 310 nm, respectively. The retention times for (+)-catechin and the internal standard (deoxyhigenamine) were 6.87 and 8.47 min respectively, without any interference. Validations of accuracy and precision were satisfactory in both within- and between-run assays. All coefficients of variance were less than 6% and mean relative errors were within ± 3.75%. The average recovery was 73.77%. The limit of detection and quantitation were 1 ng and 0.02 μg/ml, respectively. Application of this method was successfully assessed by intravenous administration of a 15 mg/kg dose of (+)-catechin in rabbits. This new method provides a simple, specific and sensitive determination for (+)-catechin in rabbit plasma and is suitable for pharmacokinetic studies.     

Zn-polyphenol chelation: complexes with quercetin, (+)-catechin, and derivatives: II Electrochemical and EPR studies

In this work, we have studied the formation of complexes between flavonols, (quercetin, rutin, quercitrin, kaempferol, luteolin, tamarixetin) and flavanols ((+)-catechin, (−)-epicatechin), flavanonol, (+)-taxifolin, and Zn acetate in two hydro-organic media at neutral pH in the absence of oxygen. The complexation was first studied by cyclic voltammetry. Then preparative electrolysis have been attempted followed by coulometry, UV-Vis optical absorption and circular dicroism spectroscopies for characterizing the oxidized compounds. Spectroelectrochemistries monitored either in the UV-Vis or in the EPR spectrometers at room temperature have been also used and we have identified o-semi-quinone intermediates in some cases. Different behaviour vis-à-vis the complexation by Zn²⁺ according to the molecular structures of these different families of polyphenols have been found. Some of them are more easily oxidizible.     

Crystal structure,conformational analysis,and molecular dynamics of tetra-O-methyl-(+) -catechin

The structure of tetra-O-methyl- (+) -catechin has been determined in the crystalline state. Two independent molecules, denoted structure A and structure B, exist in the unit cell. Crystals are triclinic, space group P1, a = 4.8125(2) Å, b = 12.9148(8) Å, c = 13.8862(11) Å, α = 86.962(6) °, β = 89.120(5)°, γ = 88.044(5)°, Z = 2, D_c = 1.336 g cm^?3, R = 0.033 for 6830 observations. The heterocyclic rings of the crystal structures are compared to previous results for 8-bromotetra-O-methyl-(+)-catechin, penta-O-acetyl-(+)-catechin, and (?) -epicatechin. One of the two molecules has a heterocyclic ring conformation similar to that observed previously for (?)-epicatechin, and the other has a heterocyclic ring conformation similar to one predicted earlier in a theoretical analysis of dimers of (+)-catechin and (?) -epicatechin. Both structure A and structure B in the crystal have heterocyclic ring conformations that place the dimethoxyphenyl substituent at C(2) in the equatorial position. However, this heterocyclic ring conformation does not explain the proton nmr coupling constant measured in solution. Molecular dynamics simulations show an equatorial ? axial interconversion of the heterocyclic ring, which can explain the nmr results. © 1993 John Wiley & Sons, Inc.     

Studies on flavonoid metabolism. Metabolism of (+)-catechin in the guinea pig

1. Administration of (+)-catechin to the guinea pig gives rise to a number of phenolic acids and lactones, which have been identified by chromatographic and spectrophotometric methods. The major phenolic acid metabolite is m-hydroxybenzoic acid and the major lactone metabolite is delta-(3-hydroxyphenyl)-gamma-valerolactone. 2. The phenolic acid and lactone metabolites are excreted in both free and conjugated forms, including their glucuronides and to a lesser degree their ethereal sulphates. 3. Administration of certain of the metabolites isolated has permitted certain sequential relationships of these intermediates to be established. 4. Degradation of (+)-catechin in the guinea pig is effected at least in part by the gut microflora and is suppressed by aureomycin plus phthaloyl-sulphathiazole.     

Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering

With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief hurdle for the generation of efficient production platforms. As such, we employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. Specifically, two different model objectives were used to identify possible genotypes that exhibited either improved overall NADPH production or an improved flux through an artificial reaction coupling NADPH yield to biomass. The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin. Each is derived from their common precursor dihydroquercetin using two recombinant NADPH-dependent enzymes: dihydroflavonol 4-reductase and leucoanthocyanidin reductase. The best engineered strain carrying Δpgi, Δppc and ΔpldA deletions accumulated up to 817 mg/L of leucocyanidin and 39 mg/L (+)-catechin in batch culture with 10 g/L glucose in modified M9 medium, a 4-fold and 2-fold increase, respectively, compared to the wild-type control.     

Stimulation of heparan sulphate synthesis in cultured rat hepatocytes by (+)-catechin.

The secretion of heparan sulphate by cultured rat hepatocytes was increased in the presence of (+)-catechin. The increase was due to a new species of heparan sulphate that lacked the carbohydrate-protein linkage between xylose and serine in normal heparan sulphate proteoglycan. The mean molecular weight of this heparan sulphate varied between 6300 and 9500, was not affected by treatment with alkali or Pronase and was 2-3-fold lower than that of chains released from heparan sulphate proteoglycan. After digestion with Pronase, only a minor fraction of chains contained serine, and after treatment with alkali and NaB3H4 reduction less than 5% of the chains exposed [3H]xylitol at the reducing terminals. These results suggested that (+)-catechin or metabolites of it acted as acceptors of heparan sulphate synthesis. In cultures treated wih cycloheximide, synthesis of heparan sulphate decreased to less than 5%. (+)-Catechin could restore the heparan sulphate synthesis to almost normal values. The (+)-catechin-induced heparan sulphate was secreted. Only a small fraction was incorporated into the plasma membrane or other cellular compartments. This may indicate that the protein core is essential for association of heparan sulphate with cellular compartments.     

Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (−)-epicatechin to (+)-taxifolin

This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (?)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H₂O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (?)-epicatechin, which are major structural units in plants.     

(+)-Catechin is more bioavailable than (-)-catechin: relevance to the bioavailability of catechin from cocoa

Catechin is a flavonoid present in fruits, wine and cocoa products. Most foods contain the (+)-enantiomer of catechin but chocolate mainly contains ( - )-catechin, in addition to its major flavanol, ( - )-epicatechin. Previous studies have shown poor bioavailability of catechin when consumed in chocolate. We compared the absorption of ( - ) and (+)-catechin after in situ perfusion of 10, 30 or 50 micromol/l of each catechin enantiomer in the jejunum and ileum in the rat. We also assayed 23 samples of chocolate for (+) and ( - )-catechin. Samples were analyzed using HPLC with a Cyclobond I-2000 RSP chiral column. At all concentrations studied, the intestinal absorption of ( - )-catechin was lower than the intestinal absorption of (+)-catechin (p < 0.01). Plasma concentrations of ( - )-catechin were significantly reduced compared to (+)-catechin (p < 0.05). The mean concentration of ( - )-catechin in chocolate was 218 +/- 126 mg/kg compared to 25 +/- 15 mg/kg (+)-catechin. Our findings provide an explanation for the poor bioavailability of catechin when consumed in chocolate or other cocoa containing products.     

Flavonoid glycosides from needles of Pinus massoniana

Seven flavonoids have been isolated from Pinus massoniana needles and identified as taxifolin and its 3′-O-β-D-glucopyranoside, (+)-catechin, naringenin-7-O-β-D-glucopyranoside and three new flavonoid glycosides, 6-C-methylaromadendrin 7-O-β-D-glucopyranoside, taxifolin 3′-O-β-D-(6″-O-phenylacetyl)-glucopyranoside and eriodictyol 3′-O-β-D-glucopyranoside.     

Phenolic constituents from the roots of Rosa laevigata (Rosaceae)

Chemical investigation of the root of Rosa laevigata led to the isolation of sixteen phenolic compounds, including seven flavonoids (1–7), five condensed tannins (8–12), two stilbenes (13 and 14) and two benzoic acid derivatives (15 and 16). Their structures were identified as (+)-catechin (1), (+)-gallocatechin (2), (2R, 3S, 4S)-cis- leucocyanidin (3), (2R, 3S, 4S)-cis-leucofisetinidin (4), (2S, 3R, 4R)-cis- leucofisetinidin (5), dehydrodicatechin A (6), phloridzin (7), procyanidin B3 (8), fisetinidol-(4α, 8)-catechin (9), guibourtinidol- (4α, 8)-catechin (10), ent- isetinidol -(4α, 6)-catechin (11), fisetinidol-(4β, 8)-catechin (12), (Z)-3-methoxy-5-hydroxy- stilbene (13), (Z)-piceid (14), gallic acid (15) and 4-hydroxybenzoic acid- 4-O-β-D-glucopyranoside (16). Among them, compounds 3–7, 9–14, and 16 were isolated from R. laevigata for the first time, and compounds 3–7, 9, 10, 12–14 and 16 were reported for the first time from the genus Rosa. The chemotaxonomic significance of these compounds was summarized.     

The anti-invasive flavonoid (+)-catechin binds to laminin and abrogates the effect of laminin on cell morphology and adhesion

To study the effect of the flavonoid (+)-catechin on cell-matrix interactions two cell types with a different morphology on and adhesion to laminin were used. MO4 virally transformed fetal mouse cells adhere and spread when cultured on top of laminin-coated coverslips or on human amnion basement membrane. M5076 mouse reticulum cell sarcoma cells poorly adhere to these substrates and remain round. Both cell types are invasive in confronting cultures with embryonic chick heart fragments. (+)-Catechin binds to laminin in a pH-dependent way. Pretreatment of laminin-coated coverslips or amnion basement membrane with 0.5 mM (+)-catechin abrogates the effect of laminin on cell morphology and adhesion. MO4 cells do not adhere to the pretreated substrates and remain round, while M5076 cells now adhere and spread. (+)-Catechin inhibits the invasion of MO4 cells but not of M5076 cells into embryonic chick heart in vitro. We speculate that the anti-invasive activity of the flavonoid to MO4 cells is the result of its interference with MO4 cell adhesion to laminin. Invasion of M5076 cells does not imply adhesion to and spreading on laminin.     

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

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