Cocoa procyanidins modulate transcriptional pathways linked to inflammation and metabolism in human dendritic cells.

Foods rich in polyphenols such as procyanidins (PC) have been proposed to have anti-inflammatory properties, and we have previously reported inhibition of lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in human dendritic cells (DCs) by PC derived from cocoa. To explore the mechanistic basis of this inhibition, here we conducted transcriptomic analysis on DCs cultured with either LPS or LPS combined with oligomeric cocoa PC. Procyanidins suppressed a number of genes encoding cytokines and chemokines such as CXCL1, but also genes involved in the cGMP pathway such as GUCY1A3 (encoding guanylate cyclase soluble subunit alpha-3). Upregulated genes were involved in diverse metabolic pathways, but notably two of the four most upregulated genes (NMB, encoding neuromedin B and ADCY3, encoding adenyl cyclase type 3) were involved in the cAMP signalling pathway. Gene-set enrichment analysis demonstrated that upregulated gene pathways were primarily involved in nutrient transport, carbohydrate metabolism and lysosome function, whereas down-regulated gene pathways involved cell cycle, signal transduction and gene transcription, as well as immune function. qPCR analysis verified differential expression of GUCY1A3, ADCY3, NMB as well as a number of other genes, and marked suppression of LPS-induced CXCL1 and IL-23 protein secretion was also observed. Thus, our results confirm a marked anti-inflammatory effect of PC in human DCs, which may be related mechanistically to second-messenger function and metabolic activity. Our results provide a foundation to further investigate metabolic pathways altered by PC during intestinal inflammation, and further encourage investigation of the health-promoting potential of PC-rich functional foods.

Facile Purification of Milligram to Gram Quantities of Condensed Tannins According to Mean Degree of Polymerization and Flavan-3-ol Subunit Composition.

Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts, and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP), and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a prepurification step, needed more time for large scale purifications, and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146-2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. 1H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation toward repurification or 1H-13C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples.

Identification of Structural Features of Condensed Tannins That Affect Protein Aggregation.

A diverse panel of condensed tannins was used to resolve the confounding effects of size and subunit composition seen previously in tannin-protein interactions. Turbidimetry revealed that size in terms of mean degree of polymerisation (mDP) or average molecular weight (amw) was the most important tannin parameter. The smallest tannin with the relatively largest effect on protein aggregation had an mDP of ~7. The average size was significantly correlated with aggregation of bovine serum albumin, BSA (mDP: r = -0.916; amw: r = -0.925; p<0.01; df = 27), and gelatin (mDP: r = -0.961; amw: r = -0.981; p<0.01; df = 12). The procyanidin/prodelphinidin and cis-/trans-flavan-3-ol ratios gave no significant correlations. Tryptophan fluorescence quenching indicated that procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface and in the hydrophobic binding pocket (r = 0.677; p<0.05; df = 9 and r = 0.887; p<0.01; df = 9, respectively). Circular dichroism revealed that higher proportions of prodelphinidins decreased the apparent α-helix content (r = -0.941; p<0.01; df = 5) and increased the apparent ß-sheet content (r = 0.916; p<0.05; df = 5) of BSA.

Co-operative suppression of inflammatory responses in human dendritic cells by plant proanthocyanidins and products from the parasitic nematode Trichuris suis.

Interactions between dendritic cells (DCs) and environmental, dietary and pathogen antigens play a key role in immune homeostasis and regulation of inflammation. Dietary polyphenols such as proanthocyanidins (PAC) may reduce inflammation, and we therefore hypothesized that PAC may suppress lipopolysaccharide (LPS) -induced responses in human DCs and subsequent T helper type 1 (Th1) -type responses in naive T cells. Moreover, we proposed that, because DCs are likely to be exposed to multiple stimuli, the activity of PAC may synergise with other bioactive molecules that have anti-inflammatory activity, e.g. soluble products from the helminth parasite Trichuris suis (TsSP). We show that PAC are endocytosed by monocyte-derived DCs and selectively induce CD86 expression. Subsequently, PAC suppress the LPS-induced secretion of interleukin-6 (IL-6) and IL-12p70, while enhancing secretion of IL-10. Incubation of DCs with PAC did not affect lymphocyte proliferation; however, subsequent interferon-γ production was markedly suppressed, while IL-4 production was unaffected. The activity of PAC was confined to oligomers (degree of polymerization ≥ 4). Co-pulsing DCs with TsSP and PAC synergistically reduced secretion of tumour necrosis factor-α, IL-6 and IL-12p70 while increasing IL-10 secretion. Moreover, both TsSP and PAC alone induced Th2-associated OX40L expression in DCs, and together synergized to up-regulate OX40L. These data suggest that PAC induce an anti-inflammatory phenotype in human DCs that selectively down-regulates Th1 response in naive T cells, and that they also act cooperatively with TsSP. Our results indicate a novel interaction between dietary compounds and parasite products to influence immune function, and may suggest that combinations of PAC and TsSP can have therapeutic potential for inflammatory disorders.

Kinetics of the incorporation of the main phenolic compounds into the lignan macromolecule during flaxseed development.

The main flax lignan, secoisolariciresinol diglucoside, is stored in a macromolecule containing other ester-bound phenolic compounds. In this study, NMR and HPLC-UV analyses were performed on flaxseeds harvested at different developmental stages to identify and quantify the main phenolic compounds produced during seed development. Extraction was carried out with or without alkaline hydrolysis to determine if these molecules accumulate in the lignan macromolecule and/or in a free form. Monolignol glucosides accumulate in a free form up to 9.85mg/g dry matter at the early developmental stages. Hydroxycinnamic acid glucosides and flavonoid accumulate (up to 3.18 and 4.07mg/g dry matter, respectively) in the later developmental stages and are ester-bound in the lignan macromolecule. Secosiolariciresinol diglucoside accumulates (up to 28.65mg/g dry matter) in the later developmental stages in both forms, mainly ester-bound in the lignan macromolecule and slightly in a free form.

Laser Microdissection and Spatiotemporal Pinoresinol-Lariciresinol Reductase Gene Expression Assign the Cell Layer-Specific Accumulation of Secoisolariciresinol Diglucoside in Flaxseed Coats.

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed (Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum (LuPLR1), which is a key gene involved in SDG biosynthesis, was fused to a ß-glucuronidase (GUS) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS. The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.

Structure-Activity Relationship of Condensed Tannins and Synergism with trans-Cinnamaldehyde against Caenorhabditis elegans.

Parasitic gastrointestinal nematodes (GIN) of livestock are increasingly developing resistance to synthetic nematocidal drugs. Moreover, the use of nematocides can induce ecotoxicity by affecting free-living nematodes. Condensed tannins (CT) are a structurally diverse group of bioactive plant compounds possessing anthelmintic activity against GIN. We investigated the relationship between the chemical structure of contrasting, purified CT and nematocidal effects using Caenorhabditis elegans. We also explored whether the nematocidal activity of CT could synergize with trans-cinnamaldehyde (CIN). A nonsignificant correlation was evident between the ability of CT fractions to inhibit C. elegans motility and the molar proportion of prodelphinidin subunits in purified CT samples. Synergistic inhibition of motility was achieved by combinations of CT and CIN. Galloylation of procyanidins was also a key factor for synergy. To increase the nematocidal effect of CT, plant sources containing CT with specific structural features could be selected and combined with compounds acting in synergy.

Condensed tannins in extracts from European medicinal plants and herbal products.

Medicinal plant materials are not usually analysed for condensed tannins (CT). Thirty commercially available European medicinal plants and herbal products were screened for CT and fourteen CT samples were analysed in detail. This is also the first comprehensive CT analysis of pine buds, walnut leaves, heather flowers and great water dock roots. Acetone/water extracts contained between 3.2 and 25.9 g CT/100g of extract, had CT with mean degrees of polymerisation of 2.9 to 13.3, procyanidin/prodelphinidin ratios of 1.6/98.4 to 100/0 and cis/trans flavan-3-ol ratios of 17.7/82.3 to 97.3/2.7. The majority of samples contained procyanidins, four contained A-type linkages (blackthorn flowers, heather flowers, bilberry leaves and cowberry leaves) and one sample also had galloylated procyanidins (great water dock roots).

Senna alata leaves are a good source of propelargonidins.

Proanthocyanidins (PA) in Senna alata leaves were investigated by thiolysis with benzyl mercaptan, LC-MS and NMR and consisted of almost pure propelargonidins with <6% procyanidins, had B-type linkages and a mean degree of polymerisation of three. Epiafzelechin was the major flavan-3-ol subunit (>94%) and epicatechin a minor constituent (6.4%) in residual PA and mainly detected as an extension unit.

Sodium Hydroxide Enhances Extractability and Analysis of Proanthocyanidins in Ensiled Sainfoin (Onobrychis viciifolia).

Little information exists on the effects of ensiling on condensed tannins or proanthocyanidins. The acetone-butanol-HCl assay is suitable for measuring proanthocyanidin contents in a wide range of samples, silages included, but provides limited information on proanthocyanidin composition, which is of interest for deciphering the relationships between tannins and their bioactivities in terms of animal nutrition or health. Degradation with benzyl mercaptan (thiolysis) provides information on proanthocyanidin composition, but proanthocyanidins in several sainfoin silages have proved resistant to thiolysis. We now report that a pretreatment step with sodium hydroxide prior to thiolysis was needed to enable their analysis. This alkaline treatment increased their extractability from ensiled sainfoin and facilitated especially the release of larger proanthocyanidins. Ensiling reduced assayable proanthocyanidins by 29%, but the composition of the remaining proanthocyanidins in silage resembled that of the fresh plants.

Anthelmintic activity of trans-cinnamaldehyde and A- and B-type proanthocyanidins derived from cinnamon (Cinnamomum verum).

Cinnamon (Cinnamomum verum) has been shown to have anti-inflammatory and antimicrobial properties, but effects on parasitic worms of the intestine have not been investigated. Here, extracts of cinnamon bark were shown to have potent in vitro anthelmintic properties against the swine nematode Ascaris suum. Analysis of the extract revealed high concentrations of proanthocyanidins (PAC) and trans-cinnamaldehyde (CA). The PAC were subjected to thiolysis and HPLC-MS analysis which demonstrated that they were exclusively procyanidins, had a mean degree of polymerization of 5.2 and 21% of their inter-flavan-3-ol links were A-type linkages. Purification of the PAC revealed that whilst they had activity against A. suum, most of the potency of the extract derived from CA. Trichuris suis and Oesophagostomum dentatum larvae were similarly susceptible to CA. To test whether CA could reduce A. suum infection in pigs in vivo, CA was administered daily in the diet or as a targeted, encapsulated dose. However, infection was not significantly reduced. It is proposed that the rapid absorption or metabolism of CA in vivo may prevent it from being present in sufficient concentrations in situ to exert efficacy. Therefore, further work should focus on whether formulation of CA can enhance its activity against internal parasites.

Anthelmintic Activities against Haemonchus contortus or Trichostrongylus colubriformis from Small Ruminants Are Influenced by Structural Features of Condensed Tannins.

Plants containing condensed tannins (CTs) may hold promise as alternatives to synthetic anthelmintic (AH) drugs for controlling gastrointestinal nematodes (GINs). However, the structural features that contribute to the AH activities of CTs remain elusive. This study probed the relationships between CT structures and their AH activities. Eighteen plant resources were selected on the basis of their diverse CT structures. From each plant resource, two CT fractions were isolated and their in vitro AH activities were measured with the larval exsheathment inhibition assay, which was applied to Haemonchus contortus and Trichostrongylus colubriformis. Calculation of mean EC50 values indicated that H. contortus was more susceptible than T. colubriformis to the different fractions and that the F1 fractions were less efficient than the F2 ones, as indicated by the respective mean values for H. contortus, F1 = 136.9 ± 74.1 µg/mL and F2 = 108.1 ± 53.2 µg/mL, and for T. colubriformis, F1 = 233 ± 54.3 µg/mL and F2 = 166 ± 39.9 µg/mL. The results showed that the AH activity against H. contortus was associated with the monomeric subunits that give rise to prodelphinidins (P < 0.05) and with CT polymer size (P < 0.10). However, for T. colubriformis AH activity was correlated only with prodelphinidins (P < 0.05). These results suggest that CTs have different modes of action against different parasite species.

The effects of tannins-containing ground pine bark diet upon nutrient digestion, nitrogen balance, and mineral retention in meat goats.

BACKGROUND: Pine bark is a rich source of phytochemical compounds including tannins, phenolic acids, anthocyanins, and fatty acids. These phytochemicals have potential to significantly impact on animal health and animal production. The goal of this work is to measure the effects of tannins in ground pine bark as a partial feed replacement on feed intake, dietary apparent digestibility, nitrogen balance, and mineral retention in meat goats. RESULTS: Eighteen Kiko cross goats (initial BW = 31.8 ± 1.49 kg) were randomly assigned to three treatment groups (n = 6). Dietary treatments were tested: control (0 % pine bark powder (PB) and 30 % wheat straw (WS)); 15 % PB and 15 % WS, and 30 % PB and 0 % WS. Although dry matter (DM) intake and digestibility were not affected (P > 0.10) by feeding PB, neutral detergent fiber (linear; P = 0.01), acid detergent fiber (linear; P = 0.001) and lignin digestibility (linear; P = 0.01) decreased, and crude protein (CP) digestibility tended to decrease (P = 0.09) as PB increased in the diet, apparent retention of Ca (P = 0.09), P (P = 0.03), Mg (P = 0.01), Mn (P = 0.01), Zn (P = 0.01) and Fe (P = 0.09) also increased linearly. Nitrogen intake and fecal N excretion were not affected (P > 0.05) by addition of PB in the diet, but N balance in the body was quadratically increased (P < 0.01) in the 15 % PB diet compared to other diets. This may be due to more rumen escape protein and less excreted N in the urine with the 15 % PB diet. The study showed that a moderate level of tannin-containing pine bark supplementation could improve gastrointestinal nitrogen balance with the aim of improving animal performance. CONCLUSION: These results suggest that tannin-containing PB has negative impact on fiber, lignin, and protein digestibility, but positively impacted on N-balance.

Protein Precipitation Behavior of Condensed Tannins from Lotus pedunculatus and Trifolium repens with Different Mean Degrees of Polymerization.

The precipitation of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited high-purity profiles by 1D/2D NMR and purities >90% (determined by thiolysis). At pH 6.5, large CTs with a mean degree of polymerization (mDP) of ∼18 exhibited similar protein precipitation behaviors and were significantly more effective than medium CTs (mDP ∼9). Medium CTs exhibited similar capacities to precipitate ALF or BSA, but showed small but significant differences in their capacity to precipitate LYS. All CTs precipitated ALF more effectively than BSA or LYS. Aggregation of CT-protein complexes likely aided precipitation of ALF and BSA, but not LYS. This study, one of the first to use CTs of confirmed high purity, demonstrates that the mDP of CTs influences protein precipitation efficacy.

¹H-¹³C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans-flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis.

Studies with a diverse array of 22 purified condensed tannin (CT) samples from nine plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans-flavan-3-ol ratios can be appraised by (1)H-(13)C HSQC NMR spectroscopy. The method was developed from samples containing 44-∼100% CT, PC/PD ratios ranging from 0/100 to 99/1, and cis/trans ratios ranging from 58/42 to 95/5 as determined by thiolysis with benzyl mercaptan. Integration of cross-peak contours of H/C-6' signals from PC and of H/C-2',6' signals from PD yielded nuclei-adjusted estimates that were highly correlated with PC/PD ratios obtained by thiolysis (R(2) = 0.99). cis/trans-Flavan-3-ol ratios, obtained by integration of the respective H/C-4 cross-peak contours, were also related to determinations made by thiolysis (R(2) = 0.89). Overall, (1)H-(13)C HSQC NMR spectroscopy appears to be a viable alternative to thiolysis for estimating PC/PD and cis/trans ratios of CT if precautions are taken to avoid integration of cross-peak contours of contaminants.

Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum.

Ascaris suum is one of the most prevalent nematode parasites in pigs and causes significant economic losses, and also serves as a good model for A. lumbricoides, the large roundworm of humans that is ubiquitous in developing countries and causes malnutrition, stunted growth and compromises immunity to other pathogens. New treatment options for Ascaris infections are urgently needed, to reduce reliance on the limited number of synthetic anthelmintic drugs. In areas where Ascaris infections are common, ethno-pharmacological practices such as treatment with natural plant extracts are still widely employed. However, scientific validation of these practices and identification of the active compounds are lacking, although observed effects are often ascribed to plant secondary metabolites such as tannins. Here, we extracted, purified and characterised a wide range of condensed tannins from diverse plant sources and investigated anthelmintic effects against A. suum in vitro. We show that condensed tannins can have potent, direct anthelmintic effects against A. suum, as evidenced by reduced migratory ability of newly hatched third-stage larvae and reduced motility and survival of fourth-stage larvae recovered from pigs. Transmission electron microscopy showed that CT caused significant damage to the cuticle and digestive tissues of the larvae. Furthermore, we provide evidence that the strength of the anthelmintic effect is related to the polymer size of the tannin molecule. Moreover, the identity of the monomeric structural units of tannin polymers may also have an influence as gallocatechin and epigallocatechin monomers exerted significant anthelmintic activity whereas catechin and epicatechin monomers did not. Therefore, our results clearly document direct anthelmintic effects of condensed tannins against Ascaris and encourage further in vivo investigation to determine optimal strategies for the use of these plant compounds for the prevention and/or treatment of ascariosis.

Microwave-assisted extraction of herbacetin diglucoside from flax (Linum usitatissimum L.) seed cakes and its quantification using an RP-HPLC-UV system.

Flax (Linum usitatissimum L.) seeds are widely used for oil extraction and the cold-pressed flaxseed (or linseed) cakes obtained during this process constitute a valuable by-product. The flavonol herbacetin diglucoside (HDG) has been previously reported as a constituent of the flaxseed lignan macromolecule linked through ester bonds to the linker molecule hydroxymethylglutaric acid. In this context, the development and validation of a new approach using microwave-assisted extraction (MAE) of HDG from flaxseed cakes followed by quantification with a reverse-phase HPLC system with UV detection was purposed. The experimental parameters affecting the HDG extraction yield, such as microwave power, extraction time and sodium hydroxide concentration, from the lignan macromolecule were optimized. A maximum HDG concentration of 5.76 mg/g DW in flaxseed cakes was measured following an irradiation time of 6 min, for a microwave power of 150 W using a direct extraction in 0.1 M NaOH in 70% (v/v) aqueous methanol. The optimized method was proven to be rapid and reliable in terms of precision, repeatability, stability and accuracy for the extraction of HDG. Comparison with a conventional extraction method demonstrated that MAE is more effective and less time-consuming.

Concentration kinetics of secoisolariciresinol diglucoside and its biosynthetic precursor coniferin in developing flaxseed.

INTRODUCTION: In the plant kingdom, flaxseed (Linum usitatissimum L.) is the richest source of secoisolariciresinol diglucoside (SDG), which is of great interest because of its potential health benefits for human beings. The information about the kinetics of SDG formation during flaxseed development is rare and incomplete. OBJECTIVE: In this study, a reversed-phase high-performance liquid chromatography-diode array detection (HPLC-DAD) method was developed to quantify SDG and coniferin, a key biosynthetic precursor of SDG in flaxseed. METHODOLOGY: Seeds from different developmental stages, which were scaled by days after flowering (DAF), were harvested. After alkaline hydrolysis, the validated HPLC method was applied to determine SDG and coniferin concentrations of flaxseed from different developing stages. RESULTS: Coniferin was found in the entire capsule as soon as flowering started and became undetectable 20 DAF. SDG was detected 6 DAF, and the concentration increased until maturity. On the other hand, the SDG amount in a single flaxseed approached the maximum around 25 DAF, before desiccation started. Concentration increase between 25 DAF and 35 DAF can be attributed to corresponding seed weight decrease. CONCLUSION: The biosynthesis of coniferin is not synchronous with that of SDG. Hence, the concentrations of SDG and coniferin change during flaxseed development.