1H-13C HSQC-NMR Analysis of Cranberry (Vaccinium Macrocarpon) Juice Defines the Chemical Composition of Juice Precipitate.

Shelf-stable cranberry juice precipitate has not been well characterized. Here, we describe using 1H-13C heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) spectroscopy for cranberry juice analysis, focusing on proanthocyanidins and the precipitate. HSQC-NMR cross-peaks from juices were categorized as aliphatic, olefinic, aromatic, carbohydrate backbone, or anomeric signals. An average cranberry juice precipitate had significantly more aromatic and significantly less carbohydrate backbone signals than an average supernatant. The precipitate was a collection of biomolecules held together by a mix of weak and strong intermolecular forces. Proanthocyanidin signals from precipitates of juices showed 22 ± 2 to 29.9 ± 0.7% A-type interflavan linkages and 34 ± 2 to 48 ± 3% of flavan-3-ol units with trans stereochemistry between the C2 and C3 positions. Based on this work, 1H-13C HSQC-NMR is useful to analyze cranberry juice and reveals the complex chemical nature of components in the soluble and insoluble phases.

Effects of selected condensed tannins on Cryptosporidium parvum growth and proliferation in HCT-8 cell cultures.

Infections with Cryptosporidium spp. constitute a substantial public health burden and are responsible for widespread production losses in cattle herds. Reducing disease and shedding of Cryptosporidium spp. oocysts is an important One Health goal. There are very few therapeutic options available to treat cryptosporidiosis. Interest in plant bioactive compounds to mitigate the spread of anthelmintic resistance in ruminants has led to investigation of these phytocompounds against other parasitic taxa. Condensed tannins (CTs) are plant secondary metabolites that have shown potential against nematodes in vitro and in vivo but their applicability to Cryptosporidium spp. is comparatively under-explored. Cryptosporidium parvum infected human ileocecal colorectal adenocarcinoma (HCT)-8 cell cultures were treated with escalating doses of highly purified and well-characterized CTs from five plant species, big trefoil (Lotus pedunculatus), black currant (Ribes nigrum), sainfoin (Onobrychis viciifolia), white clover (Trifolium repens) and grapeseed (Vitis vinifera) for 44 h. Quantitative-PCR (qPCR) analysis revealed that none of the CTs examined demonstrated inhibitory potential against the parasite. Substantial inhibition of C. parvum by paromomycin was observed in positive controls in all assays (76.94-90.72% inhibition), proving the validity of the assay. Despite the lack of inhibition, these results represent an important step towards identifying alternative treatment options against this parasite.

Dietary proanthocyanidins promote localized antioxidant responses in porcine pulmonary and gastrointestinal tissues during Ascaris suum-induced type 2 inflammation.

Proanthocyanidins (PAC) are dietary polyphenols with putative anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated if diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type-2 biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. Whilst PAC had only minor effects on pulmonary immune responses, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function and antioxidant responses in the gut of both naïve and A. suum-infected animals. A. suum infection and dietary PAC induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC consumption substantially increased the abundance of Limosilactobacillus reuteri. In vitro experiments with porcine macrophages and intestinal epithelial cells supported a role for both PAC polymers and PAC-derived microbial metabolites in regulating oxidative stress responses in host tissues. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, while having a limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract.

Composition and Protein Precipitation Capacity of Condensed Tannins in Purple Prairie Clover (Dalea purpurea Vent.).

This study aimed to determine the concentration and composition of condensed tannins (CT) in different tissues of purple prairie clover (PPC; Dalea purpurea Vent.) at different maturities and to determine their protein-precipitating capacity. The compositions of CT were elucidated after thiolysis with benzyl mercaptan followed by high-performance liquid-chromatography (HPLC) and 1H-13C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The results indicated that PPC flowering heads contained the highest CT concentration. Purple prairie clover CT consisted mainly of epicatechin (EC) and epigallocatechin (EGC) subunits. CT in the leaves were composed of more EC and less EGC than CT in stems and flowering heads at both the early flowering (EF) and late flowering (LF) head stages. The mean degree of polymerization was the highest for CT in stems and increased with maturity. CT isolated from PPC leaves at the early flowering head stage exhibited the greatest biological activity in terms of protein precipitation. Overall, the CT in PPC were predominantly procyanidins and the concentration and composition varied among the plant tissues and with maturity.

Condensed Tannins in White Clover (Trifolium repens) Foliar Tissues Expressing the Transcription Factor TaMYB14-1 Bind to Forage Protein and Reduce Ammonia and Methane Emissions in vitro.

Grazing ruminants contribute to global climate change through enteric methane and nitrous oxide emissions. However, animal consumption of the plant polyphenolics, proanthocyanidins, or condensed tannins (CTs) can decrease both methane emissions and urine nitrogen levels, leading to reduced nitrous oxide emissions, and concomitantly increase animal health and production. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover (Trifolium repens), but found in flowers and seed coats. Attempts at enhancing levels of CT expression in white clover leaves by mutagenesis and breeding have not been successful. However, the transformation of white clover with the TaMYB14-1 transcription factor from Trifolium arvense has resulted in the production of CTs in leaves up to 1.2% of dry matter (DM). In this study, two generations of breeding elevated foliar CTs to >2% of DM. The CTs consisted predominantly of prodelphinidins (PD, 75-93%) and procyanidins (PC, 17-25%) and had a mean degree of polymerization (mDP) of approximately 10 flavan-3-ol subunits. In vitro studies showed that foliar CTs were bound to bovine serum albumin and white clover proteins at pH 6.5 and were released at pH 2.-2.5. Using rumen in vitro assays, white clover leaves containing soluble CTs of 1.6-2.4% of DM significantly reduced methane production by 19% (p ≤0.01) and ammonia production by 60% (p ≤ 0.01) relative to non-transformed wild type (WT) controls after 6 h of incubation. These results provide valuable information for further studies using CT expressing white clover leaves for bloat prevention and reduced greenhouse gas emissions in vivo.

Proanthocyanidin Structural Details Revealed by Ultrahigh Resolution FT-ICR MALDI-Mass Spectrometry, 1H-13C HSQC NMR, and Thiolysis-HPLC-DAD.

Proanthocyanidins (condensed tannins) are important in food chemistry, agriculture, and health, driving demand for improvements in structure determination. We used ultrahigh resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) methods to determine the exact composition of individual species in heterogeneous mixtures of proanthocyanidin polymers from Sorghum bicolor grain and Neptunia lutea leaves. Fragmentation patterns obtained with FT-ICR ESI MS-MS (electrospray ionization) confirmed structural details from thiolysis-high-performance liquid chromatography (HPLC)-diode array detection (DAD) and 1H-13C heteronuclear single quantum coherence (HSQC) NMR. We found that A-type linkages were characteristic of shorter polymers in predominantly B-linked proanthocyanidin. We suggest that supramolecular complex formation between proanthocyanidins and matrix components such as 2,5-dihydroxybenzoic acid was responsible for anomalous 152 dalton peaks, incorrectly assigned as 3-O-galloylation, when using FT-ICR matrix-assisted laser desorption ionization (MALDI-MS). Our data illustrate the power of the ultrahigh resolution FT-ICR methods but include the caveat that MALDI-MS must be paired with complementary analytical tools to avoid artifacts.

Proanthocyanidin Block Arrays (PACBAR) for Comprehensive Capture and Delineation of Proanthocyanidin Structures.

Proanthocyanidins (PACs) are near-ubiquitous and chemically complex metabolites, prototypical of higher plants. Their roles in food/feed/nutrition and ethnomedicine are widely recognized but poorly understood. With the analysis of evidence that underlies this challenge, this perspective identifies shortcomings in capturing and delineating PAC structures as key factors. While several groups have forwarded new representations, a consensus method that captures PAC structures concisely and offers high integrity for electronic storage is required to reduce confusion in this expansive field. The PAC block arrays (PACBAR) system fills this gap by providing precise and human- and machine-readable structural descriptors that capture PAC metabolomic structural diversity. PACBAR enables communication of PAC structures for the development of precise structure-activity relationships and will assist in advancing PAC research to the next level.

Comparison of Protein Precipitation Ability of Structurally Diverse Procyanidin-Rich Condensed Tannins in Two Buffer Systems.

The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by four procyanidin-rich condensed tannin (CT) samples in both 2-[N-morpholino]ethanesulfonic acid (MES) and a modified Goering-Van Soest (GVS) buffer is described. Purified CT samples examined included Vitis vinifera seed (mean degree of polymerization [mDP] 4.1, 16.5% galloylated), Tilia sp. flowers (B-type linkages, mDP 5.9), Vaccinium macrocarpon berries (mDP 8.7, 31.7% A-type linkages). and Trifolium pratense flowers (B-type linkages, mDP 12.3) and were characterized by 2D NMR (>90% purity). In general, CTs precipitated ALF > LYS ≥ BSA. PP in GVS buffer was 1 to 2.25 times greater than that in MES buffer (25 °C). The GVS buffer system better reflects the results/conclusions from the literature on the impacts mDP, galloylation, and A-type linkages have on PP. Determinations of PP using the MES buffer at 37 °C indicated that some of these differences may be attributed to the temperature at which GVS buffer determinations are conducted. In vitro PP studies using the GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.

Direct versus Sequential Analysis of Procyanidin- and Prodelphinidin-Based Condensed Tannins by the HCl-Butanol-Acetone-Iron Assay.

In this study, we optimized the HCl-butanol-acetone-iron (HBAI) assay for the analysis of B-linked procyanidin (PC) and prodelphinidin (PD) condensed tannins (CTs) by direct analysis of whole tissue and sequential analysis of acetone-water extracts and insoluble residues prepared from forage, woody plant, food, and food byproduct samples. Yields of anthocyanidins (cyanidin and delphinidin) were optimized by heating ≤0.25 mg mL-1 CT standards, 1 mg mL-1 tissue, or 1-2 mg mL-1 acetone-water fractioned tissue for 3 h at 70 °C in medium containing 5% concentrated HCl, 6.7% total water, 50% acetone, 42% n-butanol, and 0.15% ammonium iron(III) sulfate dodecahydrate. Accurate quantitation required CT standards of known purity sourced from the same tissue being analyzed. Both analysis methods provided comparable estimates of total CTs for most PD-rich samples, but only the sequential method gave good recovery and accurate estimates of CTs in most PC-rich samples.

Influence of elevated protein and tannin-rich peanut skin supplementation on growth performance, blood metabolites, carcass traits and immune-related gene expression of grazing meat goats.

The aim of the present study was to define whether elevated rumen-undegradable protein (RUP) and tannin-rich peanut skin (PS) supplementation would affect animal growth performance, average daily gain (ADG), blood metabolites, carcass traits associated with lipogenic and immune-related gene expressions in meat goats grazing winter wheat (WW). Thirty-six Kiko-crossbreed male goats at approximately 6 months of age were blocked by body weight (BW; 25.6 ± 1.1 kg) and randomly assigned to one of the four treatments with two replicates based on a 2 × 2 factorial design. Diets contained PS replacing alfalfa meal (ALM), without or with RUP supplementation. Both PS and ALM were incorporated into grain mix portion of the diet and pelletized, with remaining diets fed ad libitum of WW forage for a period of 51 days. Lipogenic genes examined included SCD, ACLY, YWHAZ, PPIA and FABP4, while immune-related genes examined included ACTB (as a control gene), H3F3A, PPIA, IRF3, STAT2, HERC3 and IFIT3 antibody genes. The meat goats on PS-pellet-supplemented group with or without RUP supplementation grew 38.5% faster ADG (p < .001) when compared to control-supplemented group. When goats received PS diet, empty body weight, hot carcass, cold carcass, shoulder, hind shank, rack, loin and fat thickness were greater (p < .05) than control diet. Animals on PS-pellet had higher ACLY, YWHAZ, PPIA and FABP4 gene expression (p < .05) when compared to ALM-pellet control, with RUP by PS-pellet interactions (p < .01). Goats receiving additional RUP supplementation had increased (p < .05) STAT2 gene expression, whereas goats receiving PS-pellet supplementation showed increased STAT2 (p < .05) and a tendency to increase IRF3 (p = .07) gene expressions. In conclusion, the addition of PS-pellet or RUP supplementation has the potential to improve ADG and altered selected lipogenic and immune-related gene expressions.

Relationships between Structures of Condensed Tannins from Texas Legumes and Methane Production During In Vitro Rumen Digestion.

Previous studies showed that a series of purified condensed tannins (CTs) from warm-season perennial legumes exhibited high variability in their modulation of methane production during in vitro rumen digestion. The molecular weight differences between these CTs did not provide correlation with either the in vitro CH4 production or the ability to precipitate bovine serum albumin. In an effort to delineate other structure-activity relationships from these methane abatement experiments, the structures of purified CTs from these legumes were assessed with a combination of methanolysis, quantitative thiolysis, ¹H-13C HSQC NMR spectroscopy and ultrahigh-resolution MALDI-TOF MS. The composition of these CTs is very diverse: procyanidin/prodelphinidin (PC/PD) ratios ranged from 98/2 to 2/98; cis/trans ratios ranged from 98/2 to 34/66; mean degrees of polymerization ranged from 6 to 39; and % galloylation ranged from 0 to 75%. No strong correlation was observed between methane production and the protein precipitation capabilities of the CT towards three different proteins (BSA, lysozyme, and alfalfa leaf protein) at ruminal pH. However, a strong non-linear correlation was observed for the inhibition of methane production versus the antioxidant activity in plant sample containing typical PC- and PD-type CTs. The modulation of methane production could not be correlated to the CT structure (PC/PD or cis/trans ratios and extent of galloylation). The most active plant in methane abatement was Acacia angustissima, which contained CT, presenting an unusual challenge as it was resistant to standard thiolytic degradation conditions and exhibited an atypical set of cross-peak signals in the 2D NMR. The MALDI analysis supported a 5-deoxy flavan-3-ol-based structure for the CT from this plant.

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.

The U.S. Dairy Forage Research Center (USDFRC) Condensed Tannin NMR Database.

This Perspective describes a solution-state NMR database for flavan-3-ol monomers and condensed tannin dimers through tetramers obtained from the literature to 2015, containing data searchable by structure, molecular formula, degrees of polymerization, and 1H and 13C chemical shifts of the condensed tannins. Citations for all literature references are provided and should serve as valuable resource for scientists working in the field of condensed tannin research. The database will be periodically updated as additional information becomes available, typically on a yearly basis and is available for use, free of charge, from the U.S. Dairy Forage Research Center (USDFRC) Website.

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.

Synthesis of 1-O-methylchlorogenic acid: reassignment of structure for MCGA3 isolated from bamboo (Phyllostachys edulis) leaves.

The first synthesis of 1-O-methylchlorogenic acid is described. The short and efficient synthesis of this compound provides laboratory-scale quantities of the material to investigate its biological properties. The synthesis involves C-1 alkylation of the known (-)-4,5-cyclohexylidenequinic acid lactone followed by methoxide opening to the hydroxyl ester. Acylation of the C-5 hydroxyl group followed by sequential removal of protecting groups afforded 1-O-methylchlorogenic acid. The NMR spectroscopic characteristics of this compound do not coincide with those reported for the original isolation from bamboo (Phyllostachys edulis) leaves of the compound designated MCGA3. Comparison of the published spectroscopic data reported for MCGA3, with both reported literature values and spectroscopic data obtained from an authentic sample, leads to the conclusion that the compound isolated from bamboo (Phyllostachys edulis) leaves is instead methyl chlorogenate.

Acetone enhances the direct analysis of procyanidin- and prodelphinidin-based condensed tannins in lotus species by the butanol-HCl-iron assay.

The butanol-HCl spectrophotometric assay is widely used for quantifying extractable and insoluble condensed tannins (CT, syn. proanthocyanidins) in foods, feeds, and foliage of herbaceous and woody plants, but the method underestimates total CT content when applied directly to plant material. To improve CT quantitation, we tested various cosolvents with butanol-HCl and found that acetone increased anthocyanidin yields from two forage Lotus species having contrasting procyanidin and prodelphinidin compositions. A butanol-HCl-iron assay run with 50% (v/v) acetone gave linear responses with Lotus CT standards and increased estimates of total CT in Lotus herbage and leaves by up to 3.2-fold over the conventional method run without acetone. The use of thiolysis to determine the purity of CT standards further improved quantitation. Gel-state (13)C and (1)H-(13)C HSQC NMR spectra of insoluble residues collected after butanol-HCl assays revealed that acetone increased anthocyanidin yields by facilitating complete solubilization of CT from tissue.

Efficacy of various naturally occurring caffeic acid derivatives in preventing post-harvest protein losses in forages.

BACKGROUND: In red clover, oxidation of endogenous o-diphenols by polyphenol oxidase (PPO) inhibits post-harvest proteolyis. This system is transferable to alfalfa by providing PPO (via a transgene) and o-diphenol PPO substrates (via exogenous application). To exploit the PPO system for protein protection, it would be advantageous to produce PPO substrates in alfalfa, which lacks them. We assessed the extent of PPO-mediated proteolytic inhibition by phenolic compounds, especially those whose biosynthesis could be engineered into alfalfa. RESULTS: Tested compounds included o-diphenols (caffeic acid, phaselic acid, chlorogenic acid, clovamide) and monophenols (p-coumaric acid, p-coumaroyl-malic acid). In the presence of PPO, 2 mmol o-diphenol g⁻¹ protein reduced 24 h proteolysis 68-87% (P < 0.001) and as little as 0.25 mmol g⁻¹ protein still decreased 24 h proteolysis 43-60% (P < 0.001). At high concentrations, clovamide inhibited 24 h proteolysis 50% (P < 0.001) in the absence of PPO, likely due to non-PPO oxidation. Monophenol p-coumaric acid did not inhibit 24 h proteolyis, although high levels of its malate ester did exhibit PPO- and oxygen-independent inhibition (37%, P < 0.001). CONCLUSIONS: For PPO-mediated proteolytic inhibition, pathways for both phaselic acid and chlorogenic acid may be good targets for engineering into alfalfa. Clovamide may be useful for inhibiting proteolysis without PPO.