Polymeric grape-seed procyanidins, but not monomeric catechins and oligomeric procyanidins, impair degranulation and membrane ruffling in RBL-2H3 cells

Grape-seed proanthocyanidins (GSPs) are catechin polymers that are predicted to form helices in their global minimum-energy conformation and to have a mean degree of polymerization of seven (mDP = 7). The highly polymerized GSP-H fraction (mDP = 10) was found to impair degranulation in RBL-2H3 cells after stimulation with an antigen (Ag) and treatment with the Ca-ATPase inhibitor thapsigargin (Tg). In addition, GSP-H affected actin cytoskeleton and inhibited membrane ruffling in these cells, resulting in the suppression of exocytosis. By contrast, monomeric epicatechin, the dimeric procyanidins PA-1, PA-2, and PB-2, and the oligomerized GSP-L (mDP = 3) had no effect on membrane ruffling and degranulation. These findings indicate that the molecular size and length of GSP-H are needed for the inhibition of membrane ruffling and degranulation in RBL-2H3 mast-cell lines.     

Relationships between Degree of Polymerization and Antioxidant Activities: A Study on Proanthocyanidins from the Leaves of a Medicinal Mangrove Plant Ceriops tagal

Tannins from the leaves of a medicinal mangrove plant, Ceriops tagal, were purified and fractionated on Sephadex LH-20 columns. ¹³C nuclear magnetic resonance (¹³C-NMR), reversed/normal high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDT-TOF MS) analysis showed that the tannins were predominantly B-type procyanidins with minor A-type linkages, galloyl and glucosyl substitutions, and a degree of polymerization (DP) up to 33. Thirteen subfractions of the procyanidins were successfully obtained by a modified fractionation method, and their antioxidant activities were investigated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity and ferric reducing antioxidant power (FRAP) method. All these subfractions exhibited potent antioxidant activities, and eleven of them showed significantly different mean DP (mDP) ranging from 1.43±0.04 to 31.77±1.15. Regression analysis demonstrated that antioxidant activities were positively correlative with mDP when around mDP <10, while dropped and then remained at a level similar to mDP = 5 with around 95 µg ml⁻¹ for DPPH scavenging activity and 4 mmol AAE g⁻¹ for FRAP value.     

Latency of Botrytis cinerea Pers.: Fr. and Biochemical Studies During Growth and Ripening of Two Grape Berry Cultivars, Respectively Susceptible and Resistant to Grey Mould

Latency and development of Botrytis cinerea were assessed under field conditions and after artificial inoculation of two grape varieties, Gamay (susceptible) and Gamaret (resistant). When the percentage of latent Botrytis was the same for both varieties, severity of visible grey mould remained very low in Gamaret berries, while Gamay clusters were destroyed by the disease to a high percentage. Some biochemical parameters were measured in berries, such as constitutive and induced anti‐fungal compounds, polymeric proanthocyanidins and lipid peroxidation products as markers of senescence. Differences were observed in polymeric proanthocyanidins (PPRA) of Gamaret compared with those of Gamay. Concentration and mean degree of polymerization (mDP) of PPRA were always higher in the berries of the resistant variety. The inhibitory effect of Gamaret PPRA on enzyme activity remained until harvest whereas Gamay PPRA lost their inhibitory activity at the beginning of véraison. Based on these results, resistance to B. cinerea seems to be linked to the maintainance of the fungus in its latent form in berries, mainly due to the ability of Gamaret PPRA to inhibit macerating fungal enzyme activities.     

Effect of condensed tannins on bacterial diversity and metabolic activity in the rat gastrointestinal tract

The effect of dietary condensed tannins (proanthocyanidins) on rat fecal bacterial populations was ascertained in order to determine whether the proportion on tannin-resistant bacteria increased and if there was a change in the predominant bacterial populations. After 3 weeks of tannin diets the proportion of tannin-resistant bacteria increased significantly (P < 0.05) from 0.3% +/- 5.5% to 25.3% +/- 8.3% with a 0.7% tannin diet and to 47.2% +/- 5.1% with a 2% tannin diet. The proportion of tannin-resistant bacteria returned to preexposure levels in the absence of dietary tannins. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations by denaturing gradient gel electrophoresis (DGGE). Posttreatment samples were generally still distinguishable from controls after 3.5 weeks. Sequence analysis of DGGE bands and characterization of tannin-resistant isolates indicated that tannins selected for Enterobacteriaceae and Bacteroides species. Dot blot quantification confirmed that these gram-negative bacterial groups predominated in the presence of dietary tannins and that there was a corresponding decrease in the gram-positive Clostridium leptum group and other groups. Metabolic fingerprint patterns revealed that functional activities of culturable fecal bacteria were affected by the presence of tannins. Condensed tannins of Acacia angustissima altered fecal bacterial populations in the rat gastrointestinal tract, resulting in a shift in the predominant bacteria towards tannin-resistant gram-negative Enterobacteriaceae and Bacteroides species.     

Polyphenol contents and antioxidant activity of soybean seed extracts

The antioxidant activity and contents of various polyphenol classes in the seeds of 20 soybean hybrids were evaluated. Total polyphenols, tannins and proanthocyanidins were determined after extraction of plant material with 70% aqueous acetone. In addition, flavonoid content was determined. Antioxidant activity of seed extracts was evaluated by DPPH free radical scavenging activity assay. A positive linear correlation between antioxidant activity and contents of total phenols, tannins and proanthocyanidins was established. The highest antioxidant activity was observed in the extracts of hybrids which have higher levels of all polyphenol classes examined. The most of the single-cross hybrids were poor in tannins which recommend them as good source for ensiled livestock feed. Results suggested that polyphenol content should be considered as an important feature of the soybean seed.     

Antioxidant capacity of flavanols and gallate esters: pulse radiolysis studies

Reactivities of several proanthocyanidins (monomers of condensed tannins) and gallate esters (representing hydrolyzable tannins) with hydroxyl radicals, azide radicals, and superoxide anions were investigated using pulse radiolysis combined with kinetic spectroscopy. We determined the scavenging rate constants and the decay kinetics of the aroxyl radicals both at the wavelength of the semiquinone absorption (275 nm) and the absorption band of the gallate ester ketyl radical (400-420 nm). For most compounds second-order decay kinetics were observed, which reflect disproportionation of the semiquinones. In the case of the oligomeric hydrolysable tannins, pentagalloyl glucose and tannic acid, the decay kinetics were more complex involving sequential first-order and second-order reactions, which could only be resolved by kinetic modeling. A correlation of the reaction rates with hydroxyl radicals (k*OH) with the number of adjacent aromatic hydroxyl groups (i.e., representing catechol and/or pyrogallol structures) was obtained for both condensed and hydrolyzable tannins. Similar correlation for the reactions with azide radicals and superoxide anions are less obvious, but exist as well. We consider proanthocyanidins superior radical scavenging agents as compared with the monomeric flavonols and flavones and propose that these substances rather than the flavonoids proper represent the antioxidative principle in red wine and green tea.     

Increasing the oxidative stress response allows Escherichia coli to overcome inhibitory effects of condensed tannins

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H(2)O(2). The oxidative stress response helps E. coli strains to overcome their inhibitory effect.     

Electron paramagnetic resonance studies of radical species of proanthocyanidins and gallate esters

The polyphenols present in green tea or red wine comprise both regular flavon(ol)s and proanthocyanidins, i.e., derivatives of flavan-3-ols, whose distinct antioxidative potential is of great importance for explaining the beneficial effects of these nutrient beverages. Using EPR spectroscopy, we investigated radical structures obtained after oxidation of the parent compounds either by horseradish peroxidase/hydrogen peroxide or after autoxidation in moderately alkaline solutions. Both proanthocyanidins (monomers of condensed tannins, e.g., (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate, Pycnogenol) and gallate esters (hydrolyzable tannins, e.g., propylgallate, beta-glucogallin, pentagalloyl glucose and tannic acid) yielded predominantly semiquinone structures derived from the parent catechol or pyrogallol moieties. Evidence for a time-dependent oligomerization was obtained for (-)-epigallocatechin gallate, supporting our hypothesis that o-quinones formed from the initial semiquinone disproportionation are prone to nucleophilic addition reactions. Such phenolic coupling reactions would retain the numbers of reactive catechol/pyrogallol structures and thus the antioxidative potential. We therefore propose that proanthocyanidins are superior antioxidants as compared to flavon(ol)s proper, whose quinones are more likely to redox-cycle and act as prooxidants.     

Effect of Condensed Tannins on Bacterial Diversity and Metabolic Activity in the Rat Gastrointestinal Tract

The effect of dietary condensed tannins (proanthocyanidins) on rat fecal bacterial populations was ascertained in order to determine whether the proportion on tannin-resistant bacteria increased and if there was a change in the predominant bacterial populations. After 3 weeks of tannin diets the proportion of tannin-resistant bacteria increased significantly (P < 0.05) from 0.3% ± 5.5% to 25.3% ± 8.3% with a 0.7% tannin diet and to 47.2% ± 5.1% with a 2% tannin diet. The proportion of tannin-resistant bacteria returned to preexposure levels in the absence of dietary tannins. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations by denaturing gradient gel electrophoresis (DGGE). Posttreatment samples were generally still distinguishable from controls after 3.5 weeks. Sequence analysis of DGGE bands and characterization of tannin-resistant isolates indicated that tannins selected for Enterobacteriaceae and Bacteroides species. Dot blot quantification confirmed that these gram-negative bacterial groups predominated in the presence of dietary tannins and that there was a corresponding decrease in the gram-positive Clostridium leptum group and other groups. Metabolic fingerprint patterns revealed that functional activities of culturable fecal bacteria were affected by the presence of tannins. Condensed tannins of Acacia angustissima altered fecal bacterial populations in the rat gastrointestinal tract, resulting in a shift in the predominant bacteria towards tannin-resistant gram-negative Enterobacteriaceae and Bacteroides species.     

Qualitative analysis and HPLC isolation and identification of procyanidins from Vicia faba

The soluble proanthocyanidins of the coloured seed coats of Vicia faba L. were isolated and separated by solvent partition. The chemical characteristics of the proanthocyanidins were elucidated by total oxidation and partial degradation in the presence of phloroglucinol followed by HPLC analysis. The native extract of proanthocyanidins contained (+)-gallocatechin, (-)-epigallocatechin, (+)-catechin and (-)-epicatechin units. Oligomeric procyanidins were purified by chromatography on Sephadex LH-20 and the accessible compounds were isolated by RP-HPLC using a Licrospher Li 100 Column. The structures of the purified oligomeric procyanidins were elucidated using a procedure involving TLC, UV spectroscopy, ESI-MS and HPLC analysis of the products from the phloroglucinol reaction. The major condensed tannins of Vicia faba comprise six compounds identified as two A-type procyanidin dimers, the procyanidin dimers B1, B2 and B3, and a procyanidin trimer.     

Astringent tannins of Acer species

All 25 species of Acer examined contain condensed (proanthocyanidins) and hydrolysable (gallo- and/or ellagi-) tannins, but each of these varies ov     

Caffeic acid as a taxonomic marker in dicotyledons

Caffeic acid is plotted in the Dahlgren diagram and its distribution is compared to that of proanthocyanidins, the condensed tannins. The two chemicals derived from the same basic substance by a shift in the later steps in the pathway. Caffeic acid derivatives may be seen as more advanced constituents than proanthocyanidins.
Caffeic acid may function as a biosynthetical sink for surplus cinnamic acid in a system where the end-product, proanthocyanidins, has lost importance, and where lignin is not formed. Consequently, this may apply to all groups where a shift from woody to herbaceous habit has occurred, i.e. Asteridae, Araliiflorae and Ranuncu-liflorae. This is used as an argument in the presentation of a theory that evolution in dicotyledons was dichotomous. One main Magnoliiflorean-Caryophylliflorean group terminates in Ranunculiflorae and Caryophylliflorae with caffeic acid and ferulic acid, respectively. The other group, comprising most other dicotyledons, terminates in the Asteridae, also with caffeic acid.
There is much evidence pointing towards a similar protective effect of caffeic acid as has long been known for the condensed tannins. The advantages of caffeic acid as compared to condensed tannins is the shorter biosynthetic pathway, the less carbon bound in the molecule, and the possibility of re-entering the substance into the primary metabolism.     

Protein-proanthocyanidin interactions during extraction of scots pine needles

Successful protein extractions from Scots pine needles in different buffers (pH 5–9)with PVP-10 and Tween 80 suggest that both hydrogen bonds and hydrophobic interactions are responsible for the formation of insoluble protein-tannin complexes. Gel filtrations did not permit a separation of tannins from solubilized proteins. Contamination of the protein fractions was attributed to proanthocyanidins, both as such and when attached to the proteins. The ability of the soluble protein-proanthocyanidin complexes to adsorb on both Polyclar AT through hydrogen bonding and on Phenyl-Sepharose CL-4B through hydrophobic interactions suggests an amphiphilic capacity on the part of the proanthocyanidin. The factors causing persistent binding between proteins and proanthocyanidins through hydrophobic interactions are discussed. Although the vanillin test confirmed the presence of high molecular weight tannins, they occurred independently of the proteins.     

Condensed tannins: co-occurrence of procyanidins,prodelphinidins and profisetinidins in the heartwood of Acacia baileyana

The flavonoids and condensed tannins of the heartwood of Acacia baileyana var. purpurea are described. In conformity with other Acacia species, the hydroxylation pattern of the flavonoids is of the resorcinol type but, in sharp contrast, the tannins are heterogeneous consisting of a mixture of the resorcinol and phtoroglucinol series. Dimeric proanthocyanidins of the phloroglucinol type were absent and this exception to the general observation that they invariably co-occur with the polymers may be explained by the relative nucleophilicity of the aromatic A-rings.     

Proanthocyanidin biosynthesis--still more questions than answers?

Proanthocyanidins, also known as condensed tannins, are oligomers or polymers of flavan-3-ol units. In spite of important breakthroughs in our understanding of the biosynthesis of the major building blocks of proanthocyanidins, (+)-catechin and (-)-epicatechin, important questions still remain to be answered as to the exact nature of the molecular species that undergo polymerization, and the mechanisms of assembly. We review the structures of proanthocyanidins reported over the past 12 years in the context of biosynthesis, and summarize the outstanding questions concerning synthesis of proanthocyanidins from the chemical, biochemical and molecular genetic perspectives.     

Tannins and related compounds induce nitric oxide synthase and cytokines gene expressions in Leishmania major-infected macrophage-like RAW 264.7 cells

Some polyphenol-containing extracts (Pelargonium sidoides, Phyllanthus amarus) and representatives of simple phenols (shikimic acid 3- and 5-O-gallate), flavan-3-ols (epigallocatechin 3-gallate), proanthocyanidins (a hexamer) and hydrolysable tannins (corilagin, casuariin, geraniin) were studied for gene expressions (iNOS, IL-1, IL-10, IL-12, IL-18, TNF-alpha, IFN-alpha/gamma) by RT-PCR. All extracts and compounds were capable of enhancing the iNOS and cytokine mRNA levels in parasitised cells when compared with those in non-infected conditions.     

Localization of Proanthocyanidins Using in Situ-Hydrolysis with Sulfuric Acid

A valuable method for specific localization of proanthocyanidins in plant tissues is given. The specificity is based on the acid hydrolysis of condensed tannins in situ via hot sulfuric acid, yielding bright red anthocyanidins. This treatment did not cause noticeable damage of tissue embedded in glycol methacrylate prior to sectioning. Further proof of specificity was achieved by control staining with the flavan-3-01-selective p-dimethylaminocinnamaldehyde (DMACA) reagent. In addition, comparison of adjacent sections stained with the DMACA reagent may give coarse information about the degree of polymerization of the proanthocyanidins.     

Increasing the Oxidative Stress Response Allows Escherichia coli To Overcome Inhibitory Effects of Condensed Tannins

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H₂O₂. The oxidative stress response helps E. coli strains to overcome their inhibitory effect.     

Putting the insect into the birch–insect interaction

Leaf maturation in mountain birch (Betula pubescens ssp. czerepanovii) is characterized by rapid shifts in the types of dominant phenolics: from carbon-economic flavonoids aglycons in flushing leaves, via hydrolysable tannins and flavonoid glycosides, to carbon-rich proanthocyanidins (condensed tannins) in mature foliage. This shift accords with the suggested trade-offs between carbon allocation to plant defense and growth, but may also relate to the simultaneous decline in nutritive leaf traits, such as water, proteins and sugars, which potentially limit insect growth. To elucidate how birch leaf quality translates into insect growth, I introduce a simple model that takes into account defensive compounds but also acknowledges insect demand for nutritive compounds. The effects of defensive compounds on insect growth depend strongly on background variation in nutritive leaf traits: compensatory feeding on low nutritive diets increases the intake of defensive compounds, and the availability of growth-limiting nutritive compounds may modify the effects of defenses. The ratio of consumption to larval growth (both in dry mass) increases very rapidly with leaf maturation: from 2.9 to 9.8 over 2 weeks in June-July, and to 15 by August. High concentrations in mature birch leaves of "quantitative" defenses, such as proanthocyanidins (15-20% of dry mass), presumably prevent further consumption. If the same compounds had also protected half-grown leaves (which supported the same larval growth with only one third of the dry matter consumption of older leaves), the same intake of proanthocyanidins would have demanded improbably high concentrations (close to 50%) in young leaves. The model thus suggests an adaptive explanation for the high levels of "quantitative" defenses, such as proanthocyanidins, in low-nutritive but not in high-nutritive leaves because of the behavioral responses of insect feeding to leaf nutritive levels.     

Wound-induced accumulations of condensed tannins in turtlegrass, Thalassia testudinum

Turtlegrass, Thalassia testudinum, produces high concentrations of proanthocyanidins (condensed tannins) which we hypothesized are induced by grazing, as a component of a general wound response. To test this we quantified condensed tannins in a variety of turtlegrass tissues following simulated fish grazing, grazing by the urchin Lytechinus variegatus, and treatment with the natural plant wound hormone jasmonic acid. We observed that simulated fish grazing triggered rapid induction of condensed tannins by an average of 10 mg tannin g⁻¹ dry mass (DM) after 5 days. Condensed tannin accumulations were correlated with a reduction of blade extension rates. Further, we observed that constitutive tannin levels in developing first-rank leaves were strongly correlated with the width of second-rank leaves on each shoot, with an increase of 7.7 mg tannin g⁻¹ tissue dry mass per millimeter blade width on average. We propose that wider source leaves provide additional resources for phenolic biosynthesis. There was no induction of tannins in leaves, meristematic or sheath tissues in response to grazing by the urchin L. variegatus, the presence of potential waterborne cues from nearby grazed plants, or to treatment with 5 mM jasmonic acid. However, urchin grazing did induce tannin production in root/rhizome tissues, where they accumulated to levels 3–4 times higher than in blades (up to 350 mg tannin g⁻¹ tissue dry mass). These results confirm the potential for rapid wound-induced condensed tannin accumulations in T. testudinum. The link between blade widths and the tannin content of new leaves indicates that leaf morphology may be a useful bioindicator for predicting herbivore and disease-resistance in the field.