Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 μg/ml, and 100% inhibition at 1000 μg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05–0.01) larvae from passing through the LMI sieves. At 500 μg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200–500 μg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.     

Flavan-3-ols from the rhizomes of Drynaria fortunei

One monomer flavan-3-ol, 4α-carboxymethyl-(+)-catechin methyl ester, two monomer flavan-3-ol glycosides, (+)-afzelechin-3-O-β-allopyranoside, (+)-afzelechin-6-C-β-glucopyranoside, two dimer flavan-3-ols, (-)-epiafzelechin-(4β→8)-4β-carboxymethyl-(-)-epicatechin methyl ester, and -(-)-epiafzelechin-(4β→8)-4α-carboxymethyl-(-)epiafzelechin ethyl ester, and one trimer flavan-3-ol, (-)-epiafzelechin-(4β→8)-(-)-epiafzelechin-(4β→8)-4β-carboxymethyl-(-)-epiafzelechin methyl ester, together with thirteen known flavan-3-ols were isolated from the rhizomes of Drynaria fortunei (Kunze) J.Sm (Polypodiaceae). The structures were established by analysis of their HRESIMS, 1D, 2D NMR spectroscopic, and CD data. In order to obtain improved resolution, the high-resolution NMR spectra of the dimers and trimer were measured at -40 °C.     

New dibenzotropolone derivatives characterized from black tea using LC/MS/MS

Theaflavins and thearubigins are major pigments in black tea, and it is generally accepted that they are produced by oxidation of flavan-3-ols (catechins) during tea fermentation. In the course of studies on the oxidation mechanism of tea polyphenols, especially the formation of thearubigins, a method combining the enzymatic synthesis and LC/ESI-MS/MS analysis was developed to search for new higher molecular weight polymers from black tea. Three new dibenzotropolones, theadibenzotropolone A, B, and C, together with one new tribenzotropolone, theatribenzotropolone A, were formed by the reaction of theaflavins and tea catechins with horseradish peroxidase in the presence of H(2)O(2). The structures of these new benzotropolone derivatives were elucidated on the basis of MS and 2D NMR spectroscopic analyses. The existence of these compounds in black tea was characterized by LC/ESI-MS/MS. Theadibenzotropolone A and B were the first benzotropolone-type trimers of catechins found in the black tea extract. The observation that galloyl ester groups of theaflavins can be oxidized to form di- or tri-benzotropolone skeletons strongly implied that this type of oxidation is an important pathway to extend the molecular size of thearubigins.     

Characterisation of polyphenols by HPLC-PAD-ESI/MS and antioxidant activity in Equisetum telmateia

The antioxidant activity of an aqueous extract (infusion) and respective ethyl acetate fraction of Equisetum telmateia Ehrh. (Equisetaceae), a plant used in traditional medicine for its anti-inflammatory and diuretic properties, has been evaluated by DPPH, TEAC and TBARS assays. A high and significant antioxidant activity was detected in the ethyl acetate fraction. Analysis of the aqueous extract and the ethyl acetate fraction by HPLC-PAD-ESI/MS allowed the identification of the major phenolic compounds as flavan-3-ol, kaempferol and phenolic acid derivatives. Among the flavan-3-ols, A-type proanthocyanidins and afzelechin derivatives were detected as well as the more common B-type procyanidins, B2 and C1, whose identification was further confirmed by HPLC using detection involving chemical reaction with p-dimethylamino-cinnamaldehyde. The results suggest that the anti-inflammatory activity of E. telmateia could be due, at least in part, to the presence of compounds with antioxidant activity.     

High-molecular-weight polyphenols from oolong tea and black tea: purification, some properties, and role in increasing mitochondrial membrane potential

High-molecular-weight polyphenols from oolong and black teas increased mitochondrial membrane potential, as measured by a method using ciliated protozoan Tetrahymena and rhodamine 123. These polyphenols, referred to as mitochondrial activation factors (MAFs), were purified from oolong and black teas by solvent extraction and Toyopearl column chromatography. The number-average molecular weights of the MAFs were 9,000 to 18,000, and the weight-average molecular weights were 15,000 to 25,000. The MAFs increased the mitochondrial membrane potential more than catechins did. Treatment of the MAFs with tannase indicated that they contained galloyl residues. When the MAFs were hydrolyzed with HCl-n-BuOH, cyanidin and delphinidin were detected. The partial structure of the MAFs was analyzed by pyrolysis-gas chromatography-mass spectrometry, and nine compounds were identified. These results suggest that MAFs are heterogeneous polymers of flavan-3-ols and flavan-3-ol gallates with intermonomeric linkages of B-ring to B-ring and C-ring to A-ring.     

Tyrosinase catalysed biphenyl construction from flavan-3-ol substrates

Mushroom tyrosinase catalysed oxidation of three flavan-3-ols, viz. catechin, fisetinidol and mesquitol, was conducted to construct biphenyl bonds. Exposure of the flavan-3-ols to tyrosinase and subsequent trapping of the o-quinone intermediates resulted in the formation of novel flavan-3-ol derivatives, the structures of which were elucidated by mono- and two-dimensional 1H-NMR experiments. Application of the methodology resulted in the improved synthesis of the natural flavan-3-ol dimer, mesquitol-[5-->8]-catechin, previously isolated from Prosopis glandulosa.     

Heterogeneity of the interflavanyl bond in proanthocyanidins from natural sources lacking C-4 (C-ring) deoxy flavonoid nucleophiles

The proanthocyanidin pool in the floral kingdom usually involves the presence of carbon-carbon bonds linking predominantly flavan-3-ol constituent moieties. Such an ensemble of flavan-3-ol units originates via electrophilic aromatic substitution of flavan-4-yl carbocations (or their equivalents) derived from flavan-4-ols and/or flavan-3,4-diols and the nucleophilic centers of the m-oxygenated A-rings of flavan-3-ol nucleophiles. In the absence of these potent flavan-3-ol nucleophiles with their aptitude for the formation of carbon-carbon bonds, alternative centers emerge as participants in interflavanyl bond formation. Such a phenomenon is demonstrated for the distribution of various profisetinidin-, prorobinetinidin-, proguibourtinidin-, promelacacinidin- and proteracacinidin-type pro- and leuco-anthocyanidins in several southern hemisphere heartwood species.     

Challenges and complexity of functionality evaluation of flavan-3-ol derivatives

Flavan-3-ol derivatives are common plant-derived bioactive compounds. In particular, (–)-epigallocatechin-3-O-gallate shows various moderate biological activities without severe toxicity, and its health-promoting effects have been widely studied because it is a main ingredient in green tea and is commercially available at low cost. Although various biologically active flavan-3-ol derivatives are present as minor constituents in plants as well as in green tea, their biological activities have yet to be revealed, mainly due to their relative unavailability. Here, I outline the major factors contributing to the complexity of functionality studies of flavan-3-ol derivatives, including proanthocyanidins and oligomeric flavan-3-ols. I emphasize the importance of conducting structure-activity relationship studies using synthesized flavan-3-ol derivatives that are difficult to obtain from plant extracts in pure form to overcome this challenge. Further discovery of these minor constituents showing strong biological activities is expected to produce useful information for the development of functional health foods.     

Antioxidative flavan-3-ol glycosides from stems of Rhizophora stylosa

Two flavan-3-ol glycosides together with seven flavan-3-ols were isolated from stems of the mangrove plant, Rhizophora stylosa. Their structures were established as glabraoside A (7) and glabraoside B (8) by spectroscopic and chemical methods. The antioxidative activity of these isolated compounds evaluated by the diphenylpicrylhydrazyl (DPPH) radical scavenging reaction was higher than that of L-ascorbic acid.     

High-yield anthocyanin biosynthesis in engineered Escherichia coli

Anthocyanins are red, purple, or blue plant water-soluble pigments. In the past two decades, anthocyanins have received extensive studies for their anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, and cardioprotective properties. In the present study, anthocyanin biosynthetic enzymes from different plant species were characterized and employed for pathway construction leading from inexpensive precursors such as flavanones and flavan-3-ols to anthocyanins in Escherichia coli. The recombinant E. coli cells successfully achieved milligram level production of two anthocyanins, pelargonidin 3-O-glucoside (0.98 mg/L) and cyanidin 3-O-gluside (2.07 mg/L) from their respective flavanone precursors naringenin and eriodictyol. Cyanidin 3-O-glucoside was produced at even higher yields (16.1 mg/L) from its flavan-3-ol, (+)-catechin precursor. Further studies demonstrated that availability of the glucosyl donor, UDP-glucose, was the key metabolic limitation, while product instability at normal pH was also identified as a barrier for production improvement. Therefore, various optimization strategies were employed for enhancing the homogenous synthesis of UDP-glucose in the host cells while at the same time stabilizing the final anthocyanin product. Such optimizations included culture medium pH adjustment, the creation of fusion proteins and the rational manipulation of E. coli metabolic network for improving the intracellular UDP-glucose metabolic pool. As a result, production of pelargonidin 3-O-glucoside at 78.9 mg/L and cyanidin 3-O-glucoside at 70.7 mg/L was achieved from their precursor flavan-3-ols without supplementation with extracellular UDP-glucose. These results demonstrate the efficient production of the core anthocyanins for the first time and open the possibility for their commercialization for pharmaceutical and nutraceutical applications.     

The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.     

Peroxidase-mediated oxidation of catechins

Theaflavins and thearubigins are major pigments in black tea, and they are produced by oxidation and polymerization of flavan-3-ols (catechins) during tea fermentation. In the course of studies on the mechanism of black tea polyphenol formation, we have systematically studied peroxidase-mediated oxidation of tea catechins. Individual catechins, appropriate pairs of catechins, and the combination of selected catechins and theaflavins have been used as substrates of peroxidase. Different dimers, trimers and a tetramer are formed. This work has led to the characterization of some of the new trimers in black tea by LC/ESI-MS/MS. The proposed mechanisms for the peroxidase-mediated reaction are discussed.     

Plant aging and excess light enhance flavan-3-ol content in Cistus clusii

Physiological studies on aging in perennials are mainly focused either on the primary metabolism or the hormonal regulation of the process. However, to our knowledge, the involvement of the secondary metabolism in this process has not yet been explored. Cistus clusii, a Mediterranean sclerophyllous evergreen bush, shows considerable amounts of flavan-3-ols in leaves. In the present study, we aimed at determining the impact of environmental conditions and plant aging in the flavan-3-ol content in C. clusii plants grown in field conditions, which included summer drought and recovery periods. Six-year-old plants suffered more from photo-oxidative stress, especially during excess light periods, and showed lower maximum photosynthetic rates than 1-year-old plants. C. clusii leaves accumulated (−)-epigallocatechin gallate in early summer, in a strong positive correlation with both the photon flux density and the photoperiod, but not with the plant water status. Moreover, C. clusii plants accumulated proanthocyanidins (polymeric flavan-3-ols) in leaves during summer. Older plants showed higher levels of proanthocyanidins and (−)-epicatechin, but only during late spring and summer. From the result of the present study, we conclude that excess light enhances flavan-3-ol content in C. clusii, a process enhanced as plants age due to increased excess light stress.     

Associations between flavan-3-ol intake and CVD risk in the Norfolk cohort of the European Prospective Investigation into Cancer (EPIC-Norfolk)

Dietary intervention studies suggest that flavan-3-ol intake can improve vascular function and reduce the risk of cardiovascular diseases (CVD). However, results from prospective studies failed to show a consistent beneficial effect. Associations between flavan-3-ol intake and CVD risk in the Norfolk arm of the European Prospective Investigation into Cancer and Nutrition (EPIC-Norfolk) were investigated. Data were available from 24,885 (11,252 men; 13,633 women) participants, recruited between 1993 and 1997 into the EPIC-Norfolk study. Flavan-3-ol intake was assessed using 7-day food diaries and the FLAVIOLA Flavanol Food Composition database. Missing data for plasma cholesterol and vitamin C were imputed using multiple imputation. Associations between flavan-3-ol intake and blood pressure at baseline were determined using linear regression models. Associations with CVD risk were estimated using Cox regression analyses. Median intake of total flavan-3-ols was 1034 mg/d (range: 0–8531 mg/d) for men and 970 mg/d (0–6695 mg/d) for women, median intake of flavan-3-ol monomers was 233 mg/d (0–3248 mg/d) for men and 217 (0–2712 mg/d) for women. There were no consistent associations between flavan-3-ol monomer intake and baseline systolic and diastolic blood pressure (BP). After 286,147 person-years of follow-up, there were 8463 cardiovascular events and 1987 CVD related deaths; no consistent association between flavan-3-ol intake and CVD risk (HR 0.93, 95% CI: 0.87; 1.00; Q1 vs Q5) or mortality was observed (HR 0.93, 95% CI: 0.84; 1.04). Flavan-3-ol intake in EPIC-Norfolk is not sufficient to achieve a statistically significant reduction in CVD risk.     

Reversed-phase HPLC-ESI/MS analysis of birch leaf proanthocyanidins after their acidic degradation in the presence of nucleophiles

Mountain birch leaves contain large amounts of structurally variable polymeric proanthocyanidins. Their isolation procedure was enhanced by the addition of liquid-liquid extractions prior to column chromatography over Sephadex LH-20. Isolated polymeric proanthocyanidins were depolymerised by acid-catalysis in the presence of benzyl mercaptan or phloroglucinol in order to study their composition. The resulting degradation products, flavan-3-ols and flavan-3-ol adducts, were analysed with reversed-phase high-performance liquid chromatography using UV photodiode array detection for quantification and electrospray ionisation mass spectrometry for identification. The results showed that polymeric proanthocyanidins contained (epi)gallocatechins and (epi)catechins as the extension units and, mainly, (+)-catechin as the terminal unit. The mean degree of polymerisation was found to be 26 based on thiolysis and 31 based on phloroglucinol degradation.     

Enhanced oxidation of flavan-3-ols and proanthocyanidin accumulation in water-stressed tea plants

(-)-Epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two major tea flavan-3-ols, have received attention in food science and biomedicine because of their potent antioxidant properties. In plants, flavan-3-ols serve as proanthocyanidin (PA) building blocks, and although both monomeric flavan-3-ols and PAs show antioxidant activity in vitro, their antioxidant function in vivo remains unclear. In the present study, EC quinone (ECQ) and EGCG quinone (EGCGQ), the oxidation products of EC and EGCG, increased up to 100- and 30-fold, respectively, in tea plants exposed to 19 days of water deficit. Oxidation of EC and EGCG preceded PAs accumulation in leaves, which increased from 35 to 53 mg gDW(-1) after 26 days of water deficit. Aside from the role monomeric flavan-3-ols may play in PAs biosynthesis, formation of ECQ and EGCGQ strongly negatively correlated with the extent of lipid peroxidation in leaves, thus supporting a protective role for these compounds in drought-stressed plants. Besides demonstrating flavonoid accumulation in drought-stressed tea plants, we show for the first time that EC and EGCG are oxidized to their respective quinones in plants in vivo.     

A new method for selective localization of flavan-3-ols in plant tissues involving glycolmethacrylate embedding and microwave irradiation

Summary A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed.     

A new method for selective localization of flavan-3-ols in plant tissues involving glycolmethacrylate embedding and microwave irradiation.

A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed.