Condensed Tannin Fractions Research Articles

Characterization of phenolic constituents in hazelnut kernels.

Hazelnuts contain biologically active phenolic compounds and are widely used for their nutritional value. In this study, the phenolic compounds contained in hazelnuts were isolated from the kernels of Corylus avellana L. and investigated. Spectral analyses revealed 2 new acetophenone glycosides, characterized as 2',4',6'-trihydroxyacetophenone-4'-O-(2-O-β-d-apiosyl)-β-d-glucoside and 2',4',6'-trihydroxyacetophenone-4'-O-(2-O-β-d-apiosyl-6-O-α-l-arabinosyl)-β-d-glucoside, and 4 known compounds. Four high-molecular-mass condensed tannin fractions were detected in the water-soluble fraction of the extract, characterized as B-type procyanidin consisting of extension and terminal units. Gel permeation chromatography analyses revealed that the average molecular mass, based on the polystyrene standard, was approximately 15000-113000. These high-molecular-mass condensed tannin fractions were chemically characterized and exhibited different molecular weights. The fractions of high-molecular-mass condensed tannins were obtained from hazelnuts and tested for 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. The EC50 values indicated significant activity for all the fractions.

Microbial colonisation of tannin-rich tropical plants: Interplay between degradability, methane production and tannin disappearance in the rumen

Condensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonisation. This study explored the rumen degradation of tropical tannin-rich plants and the relationship between the disappearance of free and bound condensed tannin fractions and microbial communities colonising plant particles using in situ and in vitro experiments. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of three dairy cows to determine their degradability for up to 96 h. Tannin disappearance was determined at 24 h of incubation, and adherent microbial communities were examined at 3 and 12 h of incubation using a metataxonomic approach. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32–61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Free condensed tannins from all plants completely disappeared after 24-h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannin disappearance averaged ∼ 82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with the degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. whereas, archaea diversity was reduced in high-tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here, we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important.

Heather (Calluna vulgaris) supplementation does not reduce trapping ability of Duddingtonia flagrans in faeces of Haemonchus contortus infected lambs

Infection with gastro-intestinal nematodes (GIN) seriously impairs productivity and health of grazing animals. Due to the considerable rise in anthelmintic resistance and the increasing popularity of organic farming, alternative control strategies will replace or complement traditional anthelmintics. The efficacy of two potential alternatives (i) feeding the tanniferous forage heather (Calluna vulgaris) and (ii) the nematophagous fungus Duddingtonia flagrans (isolate FiBL-DF-P14), was tested in a feeding experiment with lambs artificially infected with Haemonchus contortus. Animals received hay supplemented with heather or with a late cut hay (ecohay) as a control feed ad libitum for three weeks. Two doses (1 × 105 and 5 × 104 chlsp/kg LW) of D. flagrans chlamydospores (chlsp) were administered to animals of each roughage treatment and H. contortus larval recovery from faecal cultures was compared with an untreated control (6 animals per D. flagrans-heather combination). Protein, crude fiber and energy contents of ecohay and heather were similar but heather contained approximately twice more fat, four times more lignin and ten times more of all condensed tannin fractions. Heather contained 17.3 mg Proanthocyanidin per g dry matter (DM) while contents of ecohay were 1.7 mg/g DM. Daily average feed intake across both treatments was 1.5 kg DM/animal/day, of which heather/ecohay intake accounted for 0.17/0.19 kg. Overall, there was no significant effect of heather on faecal egg counts (FEC). There was a tendency for a significant interaction between feed supplement and time and a significantly (p = 0.030) lower FEC of nominally 1799 EPG in the heather treatment at the end of the heather feeding period compared with the ecohay treatment. Lambs in this study consumed less heather than grazing sheep in other studies, even though condensed tannin contents were comparably low. Heather supplementation did not affect larval recovery in faecal cultures and trapping ability of D. flagrans. As compared with the untreated control, both doses of D. flagrans reduced larval recovery by 96.2 % and 95.5 %, respectively (p < 0.001), with no significant difference between the doses. The isolate FiBL-DF-P14 was at least as effective as isolates tested in other studies and achieved over 95 % reduction at a low dosage of 5 × 104 chlsp/kg LW. In conclusion, our results confirm the potential of and indicate no negative interactions between both alternative GIN control methods.

Structural characterization and antioxidant activity of condensed tannins fractionated from sorghum grain

The consumption of sorghum grain could prevent some chronic diseases, mainly due to the abundant levels of polyphenol. In this study, condensed tannins from sorghum grain were separated into six fractions (F1, F2, F3, F4, P1 and P2). The mean degree of polymerization (mDP) and structural composition of each fraction isolated by thiolysis were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC), ultra-performance liquid chromatography with quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The total polyphenol content of these fractions was determined, and their antioxidant capacity was measured with chemical-based and cellular-based assays. The results showed that the mDP could significantly influence the total phenolic content (p < 0.05), and the F1 fraction had the highest values. In addition, the lower mDP fractions exhibited the higher antioxidant activities than other fractions, measured by both the chemical and cellular-based assays. Well-controlled animal or human clinical studies will be required to more accurately evaluate the potential health effects of condensed tannins fractions.

Effect of the method of preservation on the chemical composition and in vitro fermentation characteristics in two legumes rich in condensed tannins

The objective of the present study was to evaluate the effects of preservation (hay and silage) on the chemical composition and the in vitro fermentation characteristics in comparison with fresh forage in two legumes rich in condensed tannins (CT). Sainfoin (Onobrychis viciifolia) was collected at the late bloom stage and sulla (Hedysarum coronarium) at the early bloom stage. In each forage, a part was immediately freeze-dried, a part was dried at ambient temperature to obtain hay, and another part was ensiled in vacuum-packages for 82 days. An in vitro assay to study the fermentation was carried out with an Ankom system during 72 h. In both forages, the silages had different contents of polyphenols and condensed tannins fractions than fresh forage and hay (P<0.05). Sainfoin hay only had greater content of fibre-bound CT fraction (FBCT) than fresh sainfoin, whereas sulla hay had greater protein-bound CT and FBCT contents than fresh sulla (P<0.05). Sainfoin silage had lower gas and methane production than hay and fresh forage (P < 0.001), whereas fresh sulla had greater gas and methane production, followed by hay and silage, which produced the least (P<0.05). In both legumes, hay had lower in vitro organic matter digestibility (IVOMD) than silage and fresh forage (P<0.001). The method of preservation affected the total production of volatile fatty acids (VFA) only in sulla (P<0.05). Most of the proportions of individual VFA were affected by the method of preservation in both legumes (P<0.001). In conclusion, polyphenols content, total content and fractions of CT were more affected in the silages than in the hays when compared to fresh forages. However, according to gas and methane production and IVOMD, silage may have greater feed nutritive value than hay although further studies on animal performance are warranted before recommendation.

Evidence for the role and fate of water-insoluble condensed tannins in the short-term reduction of carbon loss during litter decay

Warmer temperatures associated with climate change have the potential to accelerate litter decay and subsequently release large amounts of carbon stored in soils. Condensed tannins are widespread secondary metabolites, which accumulate to high concentrations in many woody plants and play key roles in forest soil nutrient cycles. Future elevated atmospheric CO2 concentrations are predicted to reduce nitrogen content and increase tannin concentrations in plant tissues, thus reducing litter quality for microbial communities and slowing decomposition rates. How the distinct condensed tannin fractions (water-soluble, acetone:MeOH-soluble and solvent-insoluble) impact soil processes, has not been investigated. We tested the impact of condensed tannin and nitrogen concentrations on decay rates of poplar and Douglas-fir litter at sites spanning temperature and moisture gradients in coastal rainshadow forests in British Columbia, Canada. The three condensed tannin fractions were quantified using recent improvements on the butanol-HCl assay. Decay was assessed based on carbon remaining, while changes in litter chemistry were primarily observed using two methods for proximate chemical analyses. After 0.6 and 1 year of decay, more carbon remained in poplar litter with high, compared to low, condensed tannin concentrations. By contrast, more carbon remained in Douglas fir litter than poplar litter during this period, despite lower condensed tannin concentrations. Rapid early decay was especially attributed to loss of soluble compounds, including water-soluble condensed tannins. Water-insoluble condensed tannin fractions, which were transformed to acid-unhydrolyzable residues over time, were associated with reduced carbon loss in high condensed tannin litter.

Effects of condensed tannin fractions of different molecular weights on population and diversity of bovine rumen methanogenic archaea in vitro, as determined by high-throughput sequencing

It has been suggested that condensed tannin (CT) fractions of different molecular weights (MWs) affect the level of methane (CH4) production in ruminant by altering the ruminal methanogenic diversity. In this study, the effects of unfractionated CTs (F0) and five CT fractions (F1⿿F5) of different MWs from Leucaena leucocephala hybrid-Rendang (LLR) on the population and diversity of rumen methanogenic archaea in vitro were investigated using real-time PCR and high throughput sequencing. Real-time PCR analysis showed that higher-MW CT fractions (fractions F1 and F2) significantly (P<0.05) decreased more total methanogens and methanogens from the order Methanobacteriales than the control (without CTs), unfractionated CTs F0 and lower-MW CT fractions (fractions F3-F5). Population of methanogens from the order Methanomicrobiales was not significantly (P>0.05) different among all CT fractions and the control. Amplification of archaeal 16S RNA genes (targeting the V3 region) of seven samples (obtained from in vitro gas production test) using Illumina Miseq sequencer, recovered 642,244 sequences (mean of 91,763 sequences per sample) which consisted of a number of OTUs ranging from 91 to 155 and were assigned to 12 genera. Bioinformatic analysis illustrated that the natural rumen archaeal community of a cattle (without supplementation of CTs) was predominated by an unclassified genus, VadinCA11 gut group which is Thermoplasmata-associated (56.3% of the total sequence reads), followed by Methanobrevibacter (25.1%) and Methanomicrobium (15.3%), while the remaining genera were below 1% of relative abundance. Relative abundance of the unclassified Thermoplasmata-associated group (VadinCA11 gut group) increased significantly (P<0.05) with increasing MWs of the CT fractions, whereas the predominant methanogen genus Methanobrevibacter was significantly (P<0.05) decreased. Condensed tannin fraction F4, but not the other CT fractions, significantly (P<0.05) increased the second predominant methongen genus Methanomicrobium. Condensed tannin fractions of different MWs from LLR could alter the size of populations and diversity of rumen methanogenic archaea in vitro, which could contribute to the reduction of CH4 production, and the impact was more pronounced for CT fractions with higher-MWs.

Bioactive constituents from the leaves of Quercus phillyraeoides A. Gray for α-glucosidase inhibitor activity with concurrent antioxidant activity

Several α-glucosidase inhibitory constituents were isolated from the methanolic extract of the leaves of Quercus phillyraeoides A. Gray (Q. phillyraeoides) using a bioassay-guided fractionation technique. Further separation and purification of the methanol-soluble fraction led to the isolation of constituents with moderate and strong inhibitory activities against α-glucosidase: β-sitosterol-d-glucoside (1) and condensed tannin fractions (2, 3, 4, 5, and 6). Compound 1 and fractions 2–6 had inhibitory concentration (IC50) values against α-glucosidase from Saccharomyces cerevisiae of 118.8, 2.79, 2.78, 3.10, 2.60, and 3.14μg/mL, respectively, while quercetin as the standard had an IC50 value of 4.80μg/mL. Furthermore, the significant antioxidant activities were evaluated using several assays, such as the DPPH radical scavenging, hydrogen peroxide radical scavenging, reducing power, and β-carotene-linoleate bleaching assays, and the results suggested that the isolated constituents showed their possible application for treating the hyperglycemia-induced oxidative stress. The results of the present study showed the potential of Q. phillyraeoides as a rich source of natural antidiabetic medicine.

Polymerization degrees, molecular weights and protein-binding affinities of condensed tannin fractions from a Leucaena leucocephala hybrid.

Condensed tannins (CTs) form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP) and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs) from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1–F5) measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn) of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight—from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

Nutritional and fermentation quality of ensiled willow from an integrated feed and bioenergy agroforestry system in UK

Agroforestry, the integration of trees and agriculture, is valued as a multifunctional land use approach that balances the production of commodities (food, feed, fuel, fibre etc.) with non-commodity outputs such as environmental protection and cultural and landscape amenities. In this study, the possibilities for fodder production from a short rotation coppice of willow (Salix viminalis) used for wood chips to produce energy were investigated. The nutritional value and ensilability were assessed from first year regrowth of willow harvested on 29 June 2011 at Wakelyns Agroforestry, Suffolk, UK. The willow branches with a stem diameter less than 8 mm were manually harvested from 4 plots in two replicates. From 2 plots, another sample was prepared including leaves only. Both dried raw material and silage samples ensiled in evacuated polyethylene bags were analyzed. The crude protein concentration was relatively high in leaf + stem silage (182 g/kg dry matter (DM)) and even higher in leaf only silage (219 g/kg DM) and the fibre concentration was relatively low. However, the organic matter digestibility determined by in vitro pepsin-cellulase method was low (0.421 for leaf + stem silage and 0.511 for leaf only silage) and it cannot be considered as a suitable feed for lactating dairy cows. However, it might be suitable for other animal groups with lower energy requirements. The appearance and smell of the silage samples at opening of the vacuum plastic bags was rather pleasant with minor deteriorations (probably yeasts) visible. The extent of fermentation was low and pH high (5.79) for a rather low DM material (DM concentration 276 g/kg). The water soluble carbohydrates of the raw material (35 g/kg DM) and the residual water soluble carbohydrate concentration in silages was relatively low, which at least partly explains the restricted production of fermentation acids. The fermentation profile was heterofermentative (acetic acid dominated instead of lactic acid). The concentrations of total condensed tannins fractions were almost twice as high in the leaf only silage compared to leaf + stem silage. Although the feed values of willow were low, it may have a role in multifunctional systems, where it can provide additional values in grazing situations such as self-medication and microclimate benefits. For easy and efficient use in animal production, controlled browsing might be used; otherwise methods for harvesting and preservation need to be developed. There seems to be some scope for ensiling willow material. This work is part of an EU FP7 funded project “Sustainable organic and low input dairying” (SOLID, KBBE.2010.1.2-02). For more information on the project, see www.solidairy.eu

Condensed Tannins and Flavonoids from the Forage Legume Sulla (Hedysarum coronarium)

The condensed tannin concentrations and composition and the characterization of the phenolic constituents in the leaves of the forage legume sulla (Hedysarum coronarium), a biennial forage legume found in temperate agricultural regions, were studied. The colorimetric butanol-HCl assay was used for the quantitation of the seasonal condensed tannin concentrations in the leaves of sulla. Fractionation of extracts on Sephadex LH-20 using step elution with aqueous methanol, followed with aqueous acetone or gradient elution with water, aqueous methanol, and aqueous acetone, gave condensed tannin and flavonoid fractions. The chemical characteristics of the purified condensed tannin fractions were studied by acid-catalyzed degradation with benzyl mercaptan and electrospray ionization mass spectrometry (ESI-MS). Thiolysis revealed that epigallocatechin was the major extender unit (15-75%) while gallocatechin was the major terminal unit (50-66%), thus indicating the extractable sulla condensed tannin fraction as the prodelphinidin type. Condensed tannin oligomers to polymers obtained from Sephadex LH-20 gradient fractions ranged between 2.9 and 46 mDP. The homo- and heterogeneous oligomer ions in condensed tannin gradient fractions detected by ESI-MS ranged from 2 to 10 DP and are consistent with the values obtained by thiolysis (2.9-6.9 DP). Lower molecular weight phenolics, including flavonoids and phenolic acids, were characterized by liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI/MS) and ESI/MS/MS on a linear ion trap. The flavonoids extracted with aqueous acetone and methanol from sulla leaves and identified included kaempferol, rutin, quercetin-7-O-α-L-rhamnosyl-3-O-glucosylrhamnoside, quercetin-3-O-α-L-rhamnosyl-7-O-glucoside, kaempferol-3-O-β-D-glucoside-dirhamnoside, genistein-7-O-β-D-glucosyl-6″-O-malonate, formononetin-7-O-β-D-glucoside-6″-O-malonate, and afrormosin and the phenolic acid chlorogenic acid.

Structural diversity and antioxidant activity of condensed tannins fractionated from mangosteen pericarp

The extract from mangosteen pericarp was separated into eight fractions, four by solvent extraction (hexane, FH; petroleum ether, FP; ethyl acetate, FE; water, FW) and the other four by fractionation on Sephadex LH-20 column (fractions F1, F2, F3, and F4). Phenolic compounds were identified as the major antioxidant components in each fraction. UV–VIS spectrum, reversed-phase HPLC–ESI-MS coupled with thiolysis, normal-phase HPLC–ESI-MS and MALDI-TOF-MS analyses complementally showed that the phenolic-rich fractions were condensed tannins with structural heterogeneity in monomer units, degree of polymerization and interflavan linkages. Mangosteen condensed tannins predominately contained procyanidins with a significant amount of propelargonidin but much lower signals of prodelphinidin. Eicosapentamers of condensed tannins were detected by MALDI-TOF-MS. Both B-type and A-type linkages were present. Condensed tannins fractions from mangosteen pericarp, especially fractions F3 and FE, can be explored as beneficial food antioxidants because of their high yields and potent antioxidant activities.

Chemical and pharmacological observations on some Hebe species

Abstract Examination of oven-dried leaves and twigs of four species of Hebe indigenous to New Zealand—H. stricta, H. odora, H. bollonsii and H. corriganii—has revealed D-mannitol and condensed tannins to be major constituents. Identification of some of the minor constituents, namely alkanes, fatty acids and aliphatic alcohols was achieved by means of gas-liquid chromatography. Pharmacological studies with the condensed tannin fractions of H. stricta and H. odora have indicated that the reputation of H. stricta as a constipatory agent is attributable to tannins; this agrees with earlier assumptions.

Tannin Oxidation: Intra- versus Intermolecular Reactions

Grape and apple condensed tannin fractions were autoxidized at high concentrations (5 g/L) in aqueous solutions and analyzed by thiolysis (depolymerization followed by HPLC analysis) and small angle X-ray scattering (SAXS). Structural parameters of native (unoxidized) tannin polymers were derived from SAXS according to the wormlike chain model: the length per monomer is 15 A, the length of the statistical segment 17 A, and the cross section of the macromolecule has a radius within the range 3-4.5 A. The rather short length of the statistical segment is an effect of the different location of interflavanol linkages, which cause a loss of orientational correlation between successive monomers. Oxidation created new bonds that were resistant to thiolysis, and, according to thiolysis, some of these new bonds were intramolecular. However, according to SAXS, oxidation at high tannin concentration caused the weight average degree of polymerization to increase, indicating that intermolecular reactions took place as well, creating larger macromolecules. In the case of the smaller grape seed tannins, these intermolecular reactions took place "end to end" leading to the formation of longer linear macromolecules, at least in the earlier stages of oxidation. In the case of the larger apple tannins, the SAXS patterns were characteristic of larger branched macromolecules. Accordingly, the intermolecular reactions were mainly "end to middle". This is in agreement with the higher probabilities of "end to middle" reactions arising from a higher ratio extension unit/terminal unit in the latter case.

Select Plant Tannins Induce IL-2Rα Up-Regulation and Augment Cell Division in γδ T Cells

Gammadelta T cells are innate immune cells that participate in host responses against many pathogens and cancers. Recently, phosphoantigen-based drugs, capable of expanding gammadelta T cells in vivo, entered clinical trials with the goal of enhancing innate immune system functions. Potential shortcomings of these drugs include the induction of nonresponsiveness upon repeated use and the expansion of only the Vdelta2 subset of human gammadelta T cells. Vdelta1 T cells, the major tissue subset, are unaffected by phosphoantigen agonists. Using FACS-based assays, we screened primary bovine cells for novel gammadelta T cell agonists with activities not encompassed by the current treatments in an effort to realize the full therapeutic potential of gammadelta T cells. We identified gammadelta T cell agonists derived from the condensed tannin fractions of Uncaria tomentosa (Cat's Claw) and Malus domestica (apple). Based on superior potency, the apple extract was selected for detailed analyses on human cells. The apple extract was a potent agonist for both human Vdelta1 and Vdelta2 T cells and NK cells. Additionally, the extract greatly enhanced phosphoantigen-induced gammadelta T cell expansion. Our analyses suggest that a tannin-based drug may complement the phosphoantigen-based drugs, thereby enhancing the therapeutic potential of gammadelta T cells.

Application of flow nephelometry to the analysis of the influence of carbohydrates on protein–tannin interactions

AbstractThe flow nephelometric technique was recently introduced to study the interaction between tannins and proteins. This technique was employed here to study the ability of the polysaccharides xanthan, dextran and gum arabic to disrupt the interactions between condensed tannin fractions from grape seed and bovine serum albumin (BSA). Xanthan was shown to have a higher ability to inhibit the aggregate formation of tannins and BSA than the other polysaccharides. Xanthan's ability to inhibit protein‐tannin complex formation was higher for condensed tannin fractions of smaller size and lower complexity. Copyright © 2006 Society of Chemical Industry

Differential Activities of Plant Polyphenols on the Binding and Internalization of Cholera Toxin in Vero Cells

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.

Methane production from in vitro rumen incubations with Lotus pedunculatus and Medicago sativa, and effects of extractable condensed tannin fractions on methanogenesis

Inhibitory effects of condensed tannins (CT) on methane, hydrogen, total gas and volatile fatty acid (VFA) formation were explored for the forage legumes Lotus pedunculatus (also known as L. uliginosus; CT 0.10 of DM) and Medicago sativa (lucerne; CT < 0.001 of DM) in the presence and absence of polyethylene glycol (PEG; a tannin binding reagent) and 2-bromo-ethylsulfonic acid (BES; a methanogen inhibitor). Treatment × forage interactions were explored using a factorial design for in vitro batch incubations with rumen fluid and minced forages over a 12 h period. The accumulated methane volume (ml/0.5 g DM) at 12 h for M. sativa (12.5 ml) was higher than L. pedunculatus (8.8 ml; P < 0.05). PEG increased methane production for L. pedunculatus (17%, P < 0.001) but not for M. sativa. BES inhibited methane (>80%) and increased accumulated hydrogen for all treatments. For L. pedunculatus, addition of PEG in the presence of BES increased hydrogen accumulation (70%, P < 0.001) compared to L. pedunculatus and relative production (49%, P < 0.01), with no effect of PEG in combination with BES for M. sativa. The VFA (i.e., C2–C5) concentrations were determined at 12 h for the batch incubations of rumen fluid with forage. Addition of PEG increased total VFA production ( P < 0.01) for L. pedunculatus (16.9%) and M. sativa (1.8%). Addition of PEG increased C2–C5 concentrations for L. pedunculatus (5.9, 30, 22, 53%, respectively) with M. sativa having an increase only for C2 (13%). Branched chain VFA (bC4 and bC5) increased with PEG addition for L. pedunculatus (67 and 59%, respectively), but not for M. sativa. Inhibitory effects of extractable condensed tannin fractions from L. pedunculatus on the common rumen methanogens Methanobrevibacter ruminantium, strains YLM-1 and DSM1093, were evaluated. Oligomeric CT fractions were inactive against both strains in broth culture, as determined by methane production measurements. A polymeric fraction completely inhibited methane production. Inhibitory effects in broth culture for strain YLM1 were bacteriostatic, while strain DSM1093 did not recover growth, as indicated by methane production, even upon prolonged incubation. In a plate assay, the zone of inhibition with the polymeric fraction remained after a further week of incubation. This is the first demonstration of inhibition of growth of a rumen methanogen by a polymeric CT fraction. Results indicate that CT action on methanogenesis can be attributed to indirect effects via reduced hydrogen production (and presumably reduced forage digestibility) and via direct inhibitory effects on methanogens.

MALDI-TOF mass spectrometry and PSD fragmentation as means for the analysis of condensed tannins in plant leaves and needles

MALDI-TOF mass spectrometry and 13C NMR spectroscopy were applied to unveil typical characteristics of condensed tannins of leaves and needles from willow ( Salix alba), spruce ( Picea abies) and beech ( Fagus sylvatica) of three tree species that are ubiquitous in German forests and landscapes. For further evaluation, lime ( Tilia cordata) was included. The 13C NMR spectroscopy confirmed the purity of the condensed tannin fractions and the efficiency of the procedure used for their extraction. While signals representative for procyanidin units are observable in all liquid-state 13C NMR spectra, resonance lines of prodelphinidin were only detected in those obtained from the condensed tannins of spruce needles and beech leaves. Typical signals in the chemical shift region between 70 and 90 ppm demonstrated the presence of stereoisomers (catechin/epicatechin; gallocatechin/ epigallocatechin). The MALDI-TOF mass spectra of the condensed tannins show signals of polymers of up to undecamers. Supporting the observations from the NMR spectroscopy, the mass spectra of the willow and lime leaf condensed tannins were identified as polymers with mainly procyanidin units, while the polymers of the spruce needle and beech leaves exhibit varying procyanidin/prodelphinidin ratios. Post source decay (PSD) fragmentation lead to a sequential loss of monomers and allowed a detailed characterization and sequencing of individual chains. In the case of the condensed tannins of lime this technique clearly excludes a pelargonidin terminal unit followed by a prodelphinidin unit, which would result in the same molecular masses as a polymer solely built up of procyanidin units.

Influence of balsam poplar tannin fractions on carbon and nitrogen dynamics in Alaskan taiga floodplain soils

The feedbacks between plant and soil processes play an important role in driving forest succession. One poorly understood feedback mechanism is the interaction between plant secondary chemicals and soil microbes. In the Alaskan taiga, changes in nutrient cycling caused by balsam poplar ( Populus balsamifera) secondary chemicals may affect the transition from alder ( Alnus tenuifolia) to balsam poplar on river floodplains. We examined the effects of four poplar condensed tannin fractions on N cycling in alder and poplar soils. Tannins were added to forest floor samples from both poplar and alder sites. Samples were incubated for 1 month in the laboratory with soil respiration rates measured over the course of the incubation. At the end of the incubation we measured both net and gross nitrogen mineralization and nitrification, microbial biomass C and N, and the activity of various exoenzymes. In all soils, tannin additions reduced N availability, however, the mechanisms differed depending on the molecular weight of the tannin and the native soil microbial community. Low molecular weight tannin fractions served as a labile C source in poplar Oi, poplar Oe, and alder Oe horizons but were toxic to microbes in alder Oi. High molecular weight tannin fractions appeared to act primarily by binding extracellular substrates and thus limiting C and N mineralization, with the strongest effects observed in the alder soils.