The Syntheses of Catechin-glucosides by Transglycosylation with Leuconostoc mesenteroides Sucrose Phosphorylase

Sucrose phosphorylase from Leuconostoc mesenteroides was found to catalyze transglycosylation from sucrose to catechins. All catechins were efficient glycosyl acceptors and their transfer ratios were more than 40%. The acceptor specificity of the enzyme decreased in the following order: (?)-epicatechin gallate= (+)-catechin> (?)-epicatechin > (?)-epigallocatechin gallate> (?)-epigallocatechin. About 150 mg of the purified transfer product was obtained from 100 mg of (+)-catechin. Its structure was identified as (+)-catechin 3′-O-α-D-glucopyranoside (C-G) on the bases of the secondary ion mass spectrometry analysis, the component analyses of its enzymatic hydrolyzates, and the nulcear magnetic resonance analysis. The browning resistance of C-G to light irradiation was greatly increased compared to that of (+)-catechin. The solubility of C-G in water was 50-fold higher than that of (+)-catechin. The antioxidative activity of C-G in the aqueous system with riboflavin was almost equal to that of (+)-catechin. In addition, C-G strongly inhibited tyrosinase, in contrast with (+)-catechin, which is the substrate of tyrosinase. The inhibitory pattern of C-G was competitive using L-β-3,4-dihydroxyphenylalanine as a substrate.     

Biotransformation of (−)-epicatechin, (+)-epicatechin, (−)-catechin,and (+)-catechin by intestinal bacteria involved in isoflavone metabolism

Isoflavone-metabolizing bacteria, Adlercreutzia equolifaciens, Asaccharobacter celatus, Slackia equolifaciens, and Slackia isoflavoniconvertens catalyzed C-ring cleavage of (–)-epicatechin and (+)-catechin, (+)-epicatechin, and (–)-catechin in varying degrees. The cleaving abilities of (–)-epicatechin and (+)-catechin were enhanced by hydrogen, except (+)-catechin cleavage by S. equolifaciens, which was not accelerated. (?)-Catechin cleavage by Ad. equolifaciens was remarkably accelerated by hydrogen.     

Oxidation of phenolic compounds by the bifunctional catalase–phenol oxidase (CATPO) from Scytalidium thermophilum

The thermophilic fungus Scytalidium thermophilum produces a novel bifunctional catalase with an additional phenol oxidase activity (CATPO); however, its phenol oxidation spectrum is not known. Here, 14 phenolic compounds were selected as substrates, among which (+)-catechin, catechol, caffeic acid, and chlorogenic acid yielded distinct oxidation products examined by reversed-phase HPLC chromatography method. Characterization of the products by LC-ESI/MS and UV–vis spectroscopy suggests the formation of dimers of dehydrocatechin type B (hydrophilic) and type A (hydrophobic), as well as oligomers, namely, a trimer and tetramer from (+)-catechin, the formation of a dimer and oligomer of catechol, a dimer from caffeic acid with a caffeicin-like structure, as well as trimeric and tetrameric derivatives, and a single major product from chlorogenic acid suggested to be a dimer. Based on the results, CATPO oxidizes phenolic compounds ranging from simple phenols to polyphenols but all having an ortho-diphenolic structure in common. The enzyme also appears to have stereoselectivity due to the oxidation of (+)-catechin, but not that of epicatechin. It is suggested that CATPO may contribute to the antioxidant mechanism of the fungus and may be of value for future food and biotechnology applications where such a bifunctional activity would be desirable.     

On the Mechanism of Metabolism of (−)-Epicatechin

(?)-Epicatechin was administered orally to rabbits and vanillic acid, 3-hydroxybenzoic acid, protocatechuic acid, and three kinds of neutral substances were found to be excreted in the urine. The three kinds of neutral substances were identified as 1-δ-(3-methoxy-4-hydroxyphenyl)-, 1-δ-(3-hydroxyphenyl)-, and 1-δ-(3,4-dihydroxyphenyl)-γ-valerolactones, which are optical isomers of the three kinds of neutral substances excreted after administration of (+)-catechin. From the presence of these intermediate metabolites, it was verified that (?)-epicatechin is metabolized by the same mechanism as (+)-catechin described earlier.     

Antioxidant components of <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis> (Bull.: Fr.) Murr. fruit bodies

Antioxidant activity of fruit bodies of Laetiporus sulphureus (Bull.: Fr.) Murr. (Polyporales) obtained by the natural plantation growing method in Pribaikal’e (Irkutsk region) has been studied. It was determined that the ethyl acetate fraction of L. sulphureus, which was chromatographically separated into seven compounds identified as quercetin, kaempferol, (+)-catechin, p-coumaric, gallic, caffeic, and chlorogenic acids was characterized with more expressed antioxidant activity. All compounds were extracted from this basidiomycete species for the first time. The quantitative amount of the substances isolated from L. sulphureus was determined by HPLC. It was found that antioxidant activity of preparations obtained from L. sulphureus is conditioned by phenolic compounds.     

The Inhibition of α-Amylase by Tea Polyphenols

The inhibition of α-amylase from human saliva by polyphenolic components of tea and its specificity was investigated in vitro. Four kinds of green tea catechins, and their isomers and four kinds of their dimeric compounds (theaflavins) produced oxidatively during black tea production were isolated. They were (?)-epicatechin (EC), (?)-epigallocatechin (EGC), (?)-epicatechin gallate (ECg), (?)-epigallocatechin gallate (EGCg), (?)-catechin (C), (?)-gallocatechin (GC), (?)-catechin gallate (Cg), (?)-gallocatechin gallate (GCg), theaflavin (TF1), theaflavin monogallates (TF2A and TF2B), and theaflavin digallate (TF3). Among the samples tested, EC, EGC, and their isomers did not have significant effects on the activity of α-amylase. All the other samples were potent inhibitors and the inhibitory effects were in the order of TF3>TF2A>TF2B>TFl>Cg> GCg > ECg > EGCg. The inhibitory patterns were noncompetitive except for TF3.     

Sensitivity to vinyl phenol derivatives produced by phenolic acid decarboxylase activity in Escherichia coli and several food-borne Gram-negative species

Ferulic, p-coumaric, and caffeic acids are phenolic acids present in soil, food, and gut, which have antimicrobial effects. Some Gram (+) bacteria metabolize these phenolic acids into vinyl derivatives due to phenolic acid decarboxylase activity (PAD) involved in the phenolic acid stress response (PASR). In this study, the antimicrobial activity of phenolic acids and their vinyl derivatives was tested on a panel of desirable and undesirable food-borne bacteria, especially Gram (?) species of Salmonella, Enterobacter, Klebsiella, and Pseudomonas, most of them without PAD activity. Native and engineered Escherichia coli strains either expressing or not PAD activity were included. Gram (?) bacteria of the panel were not significantly inhibited by phenolic acids at 3 mM, but were dramatically inhibited by the corresponding vinyl derivatives. On the contrary, Gram (+) bacteria displaying the PASR face the toxicity of phenolic acids by PAD activity and are not inhibited by vinyl phenols. In E. coli, the genes aaeB and marA, encoding efflux pumps for antimicrobial compounds, are upregulated by the addition of p-coumaric acid, but not by its derivative 4-vinyl phenol (p-hydroxystyrene). These results suggest that phenolic acids and their vinyl phenol derivatives produced by PAD (+) species could have a significant impact on undesirable or pathogenic food-borne Gram (?) bacteria in complex microbial ecosystems.     

In vitro effects of some flavonoids and phenolic acids on human pyruvate kinase isoenzyme M2

Pyruvate kinase isoenzyme M2 (PKM2) is one of the most important control point enzyme in glycolysis pathway. Hence, its inhibitors and activators are currently considered as the potential anticancer agents. The effect of 28 polyphenolic compounds on the enzyme activity was investigated in vitro. Among these compounds, neoeriocitrin, (?)-catechin gallate, fisetin, (±)-taxifolin and (?)-epicatechin have the highest inhibition effect with IC₅₀ value within 0.65–1.33?µM range. Myricetin and quercetin 3-β-d-glucoside exhibited the highest activation effect with 0.51 and 1.34?µM AC₅₀ values, respectively. Twelve of the compounds showed inhibition effect within 7–38?µM range of IC₅₀ value. Sinapinic acid and p-coumaric acid showed an activation effect with 26.2 and 22.2?µM AC₅₀ values, respectively. The results propose that the polyphenolics may be the potential PKM2 inhibitors/activators, and they may be used as lead compounds for the synthesis of new inhibitors or activators of this enzyme.     

X-Ray and IR-Studies on the Dehydrated Fish Flesh

Many actinomycetes, newly isolated from soil, converted (?)-carvone to either (?)-trans-carveol or (?)-cis-carveol or to a mixture of both compounds, and known metabolic products, such as (+)-dihydrocarvone, (+)-isodihydrocarvone, (+)-neodihydrocarveol, (?)-dihydrocarveol, (+)-isodihydrocarveol and (+)-neoisodihydrocarveol.

Identification of the metabolic products and the metabolic pathways of (?)-carvone were described in a strain of Streptomyces, A-5–1 and a strain of Nocardia, 1-3-11. A summary shows the reaction mechanism pattern of (?)-carvone conversion by actinomycetes.     

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.     

Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41

A series of novel or known water-soluble derivatives of chiral gossypol were synthesized and screened in vitro for their anti-HIV-1 activity. (?)-gossypol derivative was more active against HIV-1 than the corresponding (+)-gossypol derivative, respectively. Among these derivatives, d-glucosamine derivative of (?)-gossypol, oligopeptide derivative of (?)-gossypol and taurine derivative of (?)-gossypol, such as compounds 1a, 3a and 14a, showed significant inhibitory activities against HIV-1 replication, HIV-1 mediated cell-cell fusion and HIV gp41 6-helix bundle formation as some amino acid derivatives of (?)-gossypol.     

Synthesis of Lipophilic Poly-Lauroyl-(+)-Catechins and Radical-Scavenging Activity

Lipophilic catechins were synthesized to improve absorption into living bodies and obtain new antioxidants effective in lipid bilayers. The hydroxyl (OH) groups of (+)-catechin was acylated randomly using lauroyl chloride. The mixture was separated by preparative HPLC, and 3-lauroyl-, 3′,4′-dilauroyl- and 3,3′,4′-trilauroyl-catechins (3-LC, 3′,4′-LC, and 3,3′,4′-LC) were obtained, their structures being determined by ¹H NMR. Their radical scavenging activity was measured in a ethanol solution using the 1,1-diphenyl-2-picrylhydrazyl radical, and was compared with that of (+)-catechin. The activity of 3-LC was almost same as that of (+)-catechin, but those of 3′,4′-LC and 3,3′,4′-LC were small, showing that the blocking of phenolic OH groups in the B ring lowered the activity. The scavenging activity on lipophilic radicals in a liposome system was also measured, and the activities were in the order of 3-LC > 3′,4′-LC = (+)-catechin. These results suggested that radical scavenging activity in the lipid membrane depended not only on the number and the relative positions of phenolic OH groups of catechins but also on affinity to the membrane.     

The Arabidopsis CrRLK1L protein kinases BUPS1 and BUPS2 are required for normal growth of pollen tubes in the pistil

In flowering plants, the interaction of pollen tubes with female tissues is important for the accomplishment of double fertilization. Little information is known about the mechanisms that underlie signalling between pollen tubes and female tissues. In this study, two Arabidopsis pollen tube‐expressed CrRLK1L protein kinases, Buddha's Paper Seal 1 (BUPS1) and BUPS2, were identified as being required for normal tip growth of pollen tubes in the pistil. They are expressed prolifically in pollen and pollen tubes and are localized on the plasma membrane of the pollen tube tip region. Mutations in BUPS1 drastically reduced seed set. Most of the bups1 mutant pollen tubes growing in the pistil exhibited a swollen pollen tube tip, leading to failure of fertilization. The bups2 pollen tubes had a slightly abnormal morphology but could still accomplish double fertilization. The bups1 bups2 double mutant exhibited a slightly enhanced phenotype compared to the single bups1 mutants. The BUPS1 proteins could form homomers and heteromers with BUPS2, whereas BUPS2 could only form heteromers with BUPS1. The BUPS proteins could interact with the Arabidopsis pollen‐expressed RopGEFs in the yeast two‐hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. The results indicated that the BUPSs may mediate normal polar growth of pollen tubes in the pistil.     

Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth

Reactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress responses and development. To better understand the function of NAD(P)H oxidases in plant development, we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip. Functional loss of RBOHH and RBOHJ in homozygous double mutants resulted in reduced fertility. Analyses of pollen tube growth revealed remarkable differences in growth dynamics between Col–0 and rbohh–1 rbohj–2 pollen tubes. Growth rate oscillations of rbohh–1 rbohj–2 pollen tubes showed strong fluctuations in amplitude and frequency, ultimately leading to pollen tube collapse. Prior to disintegration, rbohh–1 rbohj–2 pollen tubes exhibit high‐frequency growth oscillations, with significantly elevated growth rates, suggesting that an increase in the rate of cell‐wall exocytosis precedes pollen tube collapse. Time‐lapse imaging of exocytic dynamics revealed that NAD(P)H oxidases slow down pollen tube growth to coordinate the rate of cell expansion with the rate of exocytosis, thereby dampening the amplitude of intrinsic growth oscillations. Using the Ca²⁺ reporter Yellow Cameleon 3.6, we demonstrate that high‐amplitude growth rate oscillations in rbohh–1 rbohj–2 pollen tubes are correlated with growth‐dependent Ca²⁺ bursts. Electrophysiological experiments involving double mutant pollen tubes and pharmacological treatments also showed that ROS influence K⁺ homeostasis. Our results indicate that, by limiting pollen tube growth, ROS produced by NAD(P)H oxidases modulate the amplitude and frequency of pollen tube growth rate oscillations.     

Characterization and function of wall-bound exo-β-glucanases of Lilium longiflorum pollen tubes

Two exo-β-glucanases (LP-ExoI, 83 kDa and LP-ExoII, 71 kDa) were extracted and partially purified from the cell wall of Lilium longiflorum pollen tubes. Both LP-ExoI and LP-ExoII hydrolyzed laminarin (1,3-β-glucan). These enzymes also exhibited some activity toward 1,3:1,4-β-glucans of Hordeum vulgare and Cetraria islandica and the 1,6-β-glucan of Umbilicaria papullosa. The pH for optimum activity for both exo-β-glucanases was 5.5. Methylation analysis of the reaction products revealed that purified LP-ExoI decreased both 1,3- and 1,4-glucosyl linkages in hemicellulosic polysaccharides isolated from the cell wall of lily pollen tubes. D-gluconolactone and nojirimycin, inhibitors of glucosidase, inhibited activities of both exo-β-glucanases, as well as growth of the lily pollen tubes. These results disclosed that the wall-bound exo-β-glucanases play an important role in the regulation of lily pollen tube growth. Received: 3 January 2000 / Revision accepted: 8 March 2000     

Exposing Arabidopsis seedlings to borneol and bornyl acetate affects root growth: Specificity due to the chemical and optical structures of the compounds

Abstract

Root growth of Arabidopsis seedlings on the surface of agar plates was measured after the seedlings were exposed to volatile organic compounds. Similar to the roots of unexposed seedlings, the roots of seedlings exposed to volatile methanol (control) grew straight down. On the other hand, seedlings exposed to volatile bornyl acetate produced wavy roots. Interestingly, the wavy roots from seedlings exposed to (+)-bornyl acetate were significantly longer than those from seedlings exposed to (?)-bornyl acetate. Exposure to either (+)- or (?)-borneol resulted in thick root tips and reduced root growth. The roots from seedlings treated with (+)-borneol were significantly longer than those from seedlings exposed to (?)-borneol. The interactions between root length and the concentrations of (+)- or (?)-borneol were significantly different, showing that the Arabidopsis seedlings specifically responded to the molecular configuration of the borneol.     

Anti-inflammatory phenolics isolated from Juniperus rigida leaves and twigs in lipopolysaccharide-stimulated RAW264.7 macrophage cells

Inflammation is an essential host defense system particularly in response to infection and injury; however, excessive or undesirable inflammatory responses contribute to acute and chronic human diseases. A high-throughput screening effort searching for anti-inflammatory compounds from medicinal plants deduced that the methanolic extract of Juniperus rigida S. et L. (Cupressaceae) inhibited significantly nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Activity-guided fractionation and isolation yielded 13 phenolic compounds, including one new phenylpropanoid glycosides, 3,4-dimethoxycinnamyl 9-O-β-D-glucopyranoside (1). Among the isolated compounds, phenylpropanoid glycosides with p-hydroxy group (2, 4) and massoniaside A (7), (+)-catechin (10), amentoflavone (11) effectively inhibited LPS-induced NO production in RAW264.7 cells.     

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.     

Biotransformation of (+)-catechin into taxifolin by a two-step oxidation: primary stage of (+)-catechin metabolism by a novel (+)-catechin-degrading bacteria, Burkholderia sp. KTC-1, isolated from tropical peat

Peat contains various persistent compounds derived from plant materials. We isolated a novel (+)-catechin-degrading bacterium, Burkholderia sp. KTC-1 (KTC-1), as an example of a bacterium capable of degrading persistent aromatic compounds present in tropical peat. This bacterium was isolated by an enrichment technique and grew on (+)-catechin as the sole carbon source under acidic conditions. The reaction of a crude enzyme extract and a structural study of its products showed that (+)-catechin is biotransformed into taxifolin during the preliminary stages of its metabolism by KTC-1. HPLC analysis showed that this transformation occurs in two oxidation steps: 4-hydroxylation and dehydrogenation. Furthermore, both (+)-catechin 4-hydroxylanase and leucocyanidin 4-dehydrogenase were localized in the cytosol of KTC-1. This is the first report on biotransformation of (+)-catechin into taxifolin via leucocyanidin by an aerobic bacterium. We suggest that tropical peat could become a unique resource for microorganisms that degrade natural aromatic compounds.