Phytochemical Shift from Condensed Tannins to Flavonoids in Transgenic Betula pendula Decreases Consumption and Growth but Improves Growth Efficiency of Epirrita autumnata Larvae.

Despite active research, antiherbivore activity of specific plant phenolics remains largely unresolved. We constructed silver birch (Betula pendula) lines with modified phenolic metabolism to study the effects of foliar flavonoids and condensed tannins on consumption and growth of larvae of a generalist herbivore, the autumnal moth (Epirrita autumnata). We conducted a feeding experiment using birch lines in which expression of dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) or anthocyanidin reductase (ANR) had been decreased by RNA interference. Modification-specific effects on plant phenolics, nutrients and phenotype, and on larval consumption and growth were analyzed using uni- and multivariate methods. Inhibiting DFR expression increased the concentration of flavonoids at the expense of condensed tannins, and silencing DFR and ANR decreased leaf and plant size. E. autumnata larvae consumed on average 82% less of DFRi plants than of unmodified controls, suggesting that flavonoids or glandular trichomes deter larval feeding. However, larval growth efficiency was highest on low-tannin DFRi plants, indicating that condensed tannins (or their monomers) are physiologically more harmful than non-tannin flavonoids for E. autumnata larvae. Our results show that genetic manipulation of the flavonoid pathway in plants can effectively be used to produce altered phenolic profiles required for elucidating the roles of low-molecular weight phenolics and condensed tannins in plant-herbivore relationships, and suggest that phenolic secondary metabolites participate in regulation of plant growth.

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.

Microbial polyphenol metabolism is part of the thawing permafrost carbon cycle.

With rising global temperatures, permafrost carbon stores are vulnerable to microbial degradation. The enzyme latch theory states that polyphenols should accumulate in saturated peatlands due to diminished phenol oxidase activity, inhibiting resident microbes and promoting carbon stabilization. Pairing microbiome and geochemical measurements along a permafrost thaw-induced saturation gradient in Stordalen Mire, a model Arctic peatland, we confirmed a negative relationship between phenol oxidase expression and saturation but failed to support other trends predicted by the enzyme latch. To inventory alternative polyphenol removal strategies, we built CAMPER, a gene annotation tool leveraging polyphenol enzyme knowledge gleaned across microbial ecosystems. Applying CAMPER to genome-resolved metatranscriptomes, we identified genes for diverse polyphenol-active enzymes expressed by various microbial lineages under a range of redox conditions. This shifts the paradigm that polyphenols stabilize carbon in saturated soils and highlights the need to consider both oxic and anoxic polyphenol metabolisms to understand carbon cycling in changing ecosystems.

Stability of Polyphenols Epigallocatechin Gallate and Pentagalloyl Glucose in a Simulated Digestive System.

Polyphenols found in foods and beverages are under intense scrutiny for their potential beneficial effects on human health. We examined the stability of two bioactive polyphenols, epigallocatechin-O-gallate (EGCg) and 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG), in a model digestive system at low oxygen tension with and without added digestive components and foods. Both compounds were stable at pH values of 5-6 and below, indicating gastric stability. Both compounds decomposed at pH 7.0. PGG was stabilized in a model system containing pepsin, pancreatin, bile and lipase, and/or baby food, but was not stabilized by dry cereal. EGCg was not stabilized by the addition of any biomolecule. The effects of polyphenols on human health should be evaluated in the context of their stability in the digestive tract with and without added digestive components and/or food.

Capillary electrophoresis methods for the determination of covalent polyphenol-protein complexes.

The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine.

Qualitative variation in proanthocyanidin composition of Populus species and hybrids: genetics is the key.

The literature on proanthocyanidins (tannins) in ecological systems is dominated by quantitative studies. Despite evidence that the qualitative characteristics (subunit type, polymer chain length) of these complex polyphenolics are important determinants of biological activity, little is known about genetic and environmental controls on the type of proanthocyanidins produced by plants. We tested the hypothesis that genetics, season, developmental stage, and environment determine proanthocyanidin qualitative characteristics by using four Populus "cross types" (narrowleaf [P. angustifolia], Fremont [P. fremontii], F1 hybrids, and backcrosses to narrowleaf). We used thiolysis and HPLC analysis to characterize the proanthocyanidins, and found that genetics strongly control composition. The narrowleaf plants accumulate mixed procyanidin/prodelphinidins with average composition epicatechin(11)-epigallocatechin(8)-catechin(2)-catechin((terminal)). Backcross genotypes produce mixed procyanidin/prodelphinidins similar to narrowleaf, while Fremont makes procyanidin dimers, and the F1 plants contain procyanidin heptamers. Less striking effects were noted for genotype × environment, while season and developmental zone had little effect on proanthocyanidin composition or chain length. We discuss the metabolic and ecological consequences of differences in condensed tannin qualitative traits.

Genetic modification of the flavonoid pathway alters growth and reveals flexible responses to enhanced UVB - Role of foliar condensed tannins.

Accumulation of certain phenolics is a well-known response of plants to enhanced UVB radiation (280-315 nm), but few experiments have compared the relative importance of different phenolic groups for UVB resilience. To study how an altered phenolic profile affects the responses and resilience of silver birch (Betula pendula) to enhanced UVB, we used RNA interference (RNAi) targeting dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), or anthocyanidin reductase (ANR) to change the accumulation of phenolics. The unmodified control line and RNAi-modified plants were grown for 51 days under ambient or +32% enhanced UVB dose in a greenhouse. RNAi greatly affected phenolic profile and plant growth. There were no interactive effects of RNAi and UVB on growth or photosynthesis, which indicates that the RNAi and unmodified control plants were equally resilient. UVB enhancement led to an accumulation of foliar flavonoids and condensed tannins, and an increase in the density of stem glands and glandular trichomes on upper leaf surfaces in both the control and RNAi-modified plants. Our results do not indicate a photoprotective role for condensed tannins. However, decreased growth of high-flavonoid low-tannin DFRi and ANRi plants implies that the balance of flavonoids and condensed tannins might be important for normal plant growth.

Decrypting bacterial polyphenol metabolism in an anoxic wetland soil.

Microorganisms play vital roles in modulating organic matter decomposition and nutrient cycling in soil ecosystems. The enzyme latch paradigm posits microbial degradation of polyphenols is hindered in anoxic peat leading to polyphenol accumulation, and consequently diminished microbial activity. This model assumes that polyphenols are microbially unavailable under anoxia, a supposition that has not been thoroughly investigated in any soil type. Here, we use anoxic soil reactors amended with and without a chemically defined polyphenol to test this hypothesis, employing metabolomics and genome-resolved metaproteomics to interrogate soil microbial polyphenol metabolism. Challenging the idea that polyphenols are not bioavailable under anoxia, we provide metabolite evidence that polyphenols are depolymerized, resulting in monomer accumulation, followed by the generation of small phenolic degradation products. Further, we show that soil microbiome function is maintained, and possibly enhanced, with polyphenol addition. In summary, this study provides chemical and enzymatic evidence that some soil microbiota can degrade polyphenols under anoxia and subvert the assumed polyphenol lock on soil microbial metabolism.

Acute physical activity effects on cardiac gene expression.

Regular bouts of physical activity may cause changes in gene expression that accumulate over time and ultimately affect phenotypes, such as body weight, blood lipid profile and tumour development. Furthermore, acute activity may affect gene expression and phenotypes differently depending on whether the individual is regularly inactive or active. One-month-old male Sprague-Dawley rats (n = 72) were equally divided into SED (standard laboratory cage, n = 24), PA (large activity box, n = 24) and EX groups (exercise wheel inside standard cage, n = 24). At 3 months of age, half the animals from each group were killed at rest and the other half following 30 min of physical activity. The RNA was extracted from cardiac tissue, and microarray analysis was performed on 27,000 genes. Select gene results were validated using quantitative PCR. No gene expression differences occurred when comparing all 3-month-old groups at rest. A relatively small percentage of genes (1.9%) were differentially expressed (P < 0.05) following acute swimming activity in all groups, but only 37 unique and identifiable genes reached or exceeded twofold differences in expression. The genes Atf3, Fos, Apold1 and Pxdn were expressed differently among SED, PA and EX following acute activity, with a clear separation of the magnitude in gene expression with SED > PA > EX. Differences in gene expression levels in young physically inactive and active animals following acute activity have different regulatory roles in gene networks that affect health-related phenotypes.

Preparation of an acid butanol standard from fresh apples.

We have developed a simple method for preparing and verifying suitable standards for the acid butanol assay from a readily available source. Phenolics were extracted from fresh apples with methanol, and sugars were removed from the crude extract by treatment with Amberlite resin before fractionating the proanthocyanidins into ethyl acetate. The ethyl acetate fraction was chromatographed on Toyopearl TSK HW-50F to yield about 50 mg of procyanidin dimer and 35 mg of trimer from 1 kg fresh apple fruit. The purity and identity of the standards was easily confirmed by using ESI-MS. In the acid butanol assay, the pure dimer, trimer and purified Sorghum procyanidin had similar color yields on a mass basis, and produced about three times more color than purified quebracho tannin. This new standard overcomes problems associated with overestimation of tannin due to use of the unreactive quebracho tannin standard. Use of the new standard will enable accurate comparisons of tannin levels between laboratories and will standardize comparisons between species, thus promoting our understanding of the role of condensed tannins in plants.

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.

Anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose.

1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose (PGG) is a polyphenolic compound highly enriched in a number of medicinal herbals. Several in vitro and a handful of in vivo studies have shown that PGG exhibits multiple biological activities which implicate a great potential for PGG in the therapy and prevention of several major diseases including cancer and diabetes. Chemically and functionally, PGG appears to be distinct from its constituent gallic acid or tea polyphenols. For anti-cancer activity, three published in vivo preclinical cancer model studies with PGG support promising efficacy to selectively inhibit malignancy without host toxicity. Potential mechanisms include anti-angiogenesis; anti-proliferative actions through inhibition of DNA replicative synthesis, S-phase arrest, and G(1) arrest; induction of apoptosis; anti-inflammation; and anti-oxidation. Putative molecular targets include p53, Stat3, Cox-2, VEGFR1, AP-1, SP-1, Nrf-2, and MMP-9. For anti-diabetic activity, PGG and analogues appear to improve glucose uptake. However, very little is known about the absorption, pharmacokinetics, and metabolism of PGG, or its toxicity profile. The lack of a large quantity of highly pure PGG has been a bottleneck limiting in vivo validation of cancer preventive and therapeutic efficacies in clinically relevant models.

Conformation and Aggregation of Human Serum Albumin in the Presence of Green Tea Polyphenol (EGCg) and/or Palmitic Acid.

Polyphenols such as epigallocatechin gallate (EGCg) may have roles in preventing some chronic diseases when they are ingested as components of plant-based foods and beverages. Human serum albumin (HSA) is a multi-domain protein that binds various ligands and aids in their transport, distribution, and metabolism in the circulatory system. In the present study, the HSA-EGCg interaction in the absence or presence of fatty acid has been investigated. Förster resonance energy transfer (FRET) was used to determine inter- and intra-domain distances in the protein with and without EGCg and palmitic acid (PA). By labeling Cys-34 with 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CPM), the distance between Trp-214 at domain IIA and CPM-Cys-34 at domain IA could be established. A small amount of PA decreased the distance, while a large amount increased the distance up to 5.4 Å. EGCg increased the inter-domain distance in HSA and HSA-PA up to 2.8 and 7.6 Å, respectively. We concluded that PA affects protein conformation more significantly compared to EGCg. Circular dichroism (CD) established that EGCg affects protein secondary structure more significantly than PA. PA had little effect on the α-helix content of HSA, while EGCg decreased the α-helix content in a dose-dependent fashion. Moreover, EGCg decreased α-helix content in HSA and HSA-PA to the same level. Dynamic light scattering (DLS) data revealed that both PA and EGCg increased HSA aggregation. EGCg increased HSA aggregation more significantly and promoted formation of aggregates that were more heterogenous. Any of these effects could impact the ability of serum albumin to transport and stabilize ligands including EGCg and other polyphenols.

Decreased polyphenol transport across cultured intestinal cells by a salivary proline-rich protein.

Tannins are polyphenols commonly found in plant-derived foods. When ingested they can have various harmful effects, but salivary proline-rich proteins (PRPs) may provide protection against dietary tannins. The aim of this study was to investigate whether basic PRPs, a major family of salivary proteins, can prevent intestinal absorption of tannin. To do so it was necessary first to characterize transport of pentagalloyl glucose (5GG), a hydrolysable tannin, across cultured epithelial cells. Using human intestinal epithelial cells (Caco-2 cells) it was found that a partial degradation of 5GG occurred during transepithelial transport resulting in the presence of 5GG as well as tetra- and trigalloyl glucose and glucose in the receiving compartment. The sodium-dependent glucose transporter SGLT1 played a role in apical (mucosal) to basolateral (serosal) transport and transport in the opposite direction was dependent on the multidrug resistance-associated protein MRP2. An increased uptake from the apical compartment was seen when the basolateral receiving solution was human serum rather than a balanced salt solution. Transport both in apical-basolateral and basolateral-apical directions was reduced when 1B4, a human basic PRP, was added to the 5GG-containing medium. This decrease closely paralleled the formation of insoluble 5GG-1B4 complexes. It appears that the formation of insoluble tannin-protein complexes diminishes the uptake of 5GG and its metabolites. There is little evidence of other biological activities of basic PRPs so in contrast to other salivary proteins they may exert a biological function in the intestines.

Exercise-induced changes in cardiac gene expression and its relation to spatial maze performance.

Cognitive performance is sensitive to both neural and non-neural changes induced by physical activity and inactivity. This study investigated whether access to physical activity outside a standard laboratory animal cage affected cognitive performance as measured by navigation of a spatial maze. It also examined gene expression in heart tissue for genes associated with cardiovascular function given recent reports of cognitive impairment associated with hyperlipidemia. Furthermore, we measured expression of neural-regulatory genes typically expressed in brain, but also found in cardiac tissue. Male Sprague-Dawley rats (n = 72) were separated into three groups having different access to physical activity: none outside a standard cage, twice-weekly physical activity, and every other day exercise on a running wheel. Compared with a sedentary group, spatial maze performance was enhanced in animals that had access to physical activity, either twice-weekly in a large box or every other day on a running wheel. Both the cardiovascular and neural-related genes expressed in the heart were distinguished by access to physical activity. Several genes that are associated with heart rate, cholesterol biosynthesis, blood pressure, and cell adhesion regulation, including GJA1, FDFT1, EDN1, and CD36, differed in animals based on access to physical activity. Neural-related genes expressed in cardiac tissue associated with neurite outgrowth, neuroplasticity, and neurogenesis including RTN4, HOMER2, ACTB, NCDN, KIF5B, and HMGB2, were expressed differently among the three groups. Significant shifts in ten cardiovascular and neural-related gene expressions in cardiac tissue were associated with physical activity and may have influenced learning and performance on a spatial maze.

Short-term isometric exercise reduces systolic blood pressure in hypertensive adults: possible role of reactive oxygen species.

OBJECTIVE: A short-term isometric exercise protocol was tested in ten hypertensive individuals to determine its efficacy as a high blood pressure-reducing intervention. DESIGN: The study was a prospective case study of 10 hypertensive individuals (8 men, 2 woman, mean age = 52 + 5 years) who underwent six weeks of isometric exercise training (three sessions/week). METHODS: Blood pressure, blood lipids and markers of oxidative stress were monitored before, during and following the isometric intervention. Electron spin resonance spectroscopy was used to directly measure radicals in the blood samples. RESULTS: After six weeks, systolic blood pressure decreased an average 13 mm Hg (p < 0.05) from a mean blood pressure of 146 to 133 mm Hg, a level that is below the usual 140 mm Hg hypertension threshold. Blood lipids were unchanged, but markers of oxidative stress were affected, with a dramatic decrease in exercise-induced oxygen centered radicals (-266%), (p < 0.05) and an increased resting whole blood glutathione:oxidized glutathione (+61%) in hypertensive adults following six weeks of isometric exercise. CONCLUSION: Six weeks of isometric exercise training was effective in lowering systolic but not diastolic blood pressure in pre-hypertensive and hypertensive individuals, and enhanced antioxidant protection is a likely underlying mechanism.

Protein binding and astringent taste of a polymeric procyanidin, 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose, castalagin, and grandinin.

The objective of the present investigation was to examine the oral astringency and protein-binding activity of four structurally well-defined tannins, namely, procyanidin [epicatechin16(4-->8)catechin], pentagalloyl glucose (1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose), castalagin, and grandinin, representing the three main structural categories of tannins, the proanthocyanidins, the gallotannins, and the ellagitannins. Astringency threshold and dos/response were determined by the half-tongue test using a trained human panel. Protein-binding stoichiometry and relative affinity were determined using radioiodinated bovine serum albumin in precipitation or competitive binding assays. Procyanidin and pentagalloyl glucose were perceived as highly astringent compounds and had relatively steep dose/response curves, but castalagin and grandinin had a lower mass threshold for detection. In vitro, procyanidin was the most effective protein-precipitating agent and grandinin the least. Increasing the temperature increased protein precipitation by the hydrolyzable tannins, especially grandinin. All four polyphenols had higher relative affinities for proline-rich proteins than for bovine serum albumin.

The Oxidative Activity of Ellagitannins Dictates Their Tendency To Form Highly Stabilized Complexes with Bovine Serum Albumin at Increased pH.

Many food and forage plants contain tannins, high molecular weight polyphenols that characteristically interact strongly with protein, forming complexes that affect taste, nutritional quality, and the health of the consumer. In the present study, the interaction between bovine serum albumin (BSA) and each of seven hydrolyzable tannins or epigallocatechin gallate was examined. The objective was to define the effect of tannin oxidation, measured as oxidative activity (browning) or as oxidizability (degradation monitored by HPLC), on the formation on highly stabilized tannin-protein complexes and to determine how the reaction depended on the pH conditions. Gel electrophoresis and MALDI-TOF-MS were used to assess the formation of tannin-protein complexes. The results showed that tannin oxidizability was directly correlated with the tendency of the tannins to form highly stabilized complexes with BSA at increased pH (7.6). However, at slightly lower pH (6.7), other tannin features, such as the size and flexibility of the tannin, appeared to dictate the formation of highly stabilized tannin-protein complexes.

Polyphenol-Aluminum Complex Formation: Implications for Aluminum Tolerance in Plants.

Natural polyphenols may play an important role in aluminum detoxification in some plants. We examined the interaction between Al(3+) and the purified high molecular weight polyphenols pentagalloyl glucose (940 Da) and oenothein B (1568 Da), and the related compound methyl gallate (184 Da) at pH 4 and 6. We used spectrophotometric titration and chemometric modeling to determine stability constants and stoichiometries for the aluminum-phenol (AlL) complexes. The structures and spectral features of aluminum-methyl gallate complexes were evaluated with quantum chemical calculations. The high molecular weight polyphenols formed Al3L2 complexes with conditional stability constants (ß) ∼ 1 × 10(23) at pH 6 and AlL complexes with ß âˆ¼ 1 × 10(5) at pH 4. Methyl gallate formed AlL complexes with ß = 1 × 10(6) at pH 6 but did not complex aluminum at pH 4. At intermediate metal-to-polyphenol ratios, high molecular weight polyphenols formed insoluble Al complexes but methyl gallate complexes were soluble. The high molecular weight polyphenols have high affinities and solubility features that are favorable for a role in aluminum detoxification in the environment.

Reaction pH and protein affect the oxidation products of beta-pentagalloyl glucose.

To better understand the biochemical consequences when polymeric polyphenols serve as biological antioxidants, we studied how reaction pH (pH 2.1-7.4) and protein affected the oxidation of pentagalloyl glucose (PGG) by NaIO4 in aqueous solution. PGG oxidation produced an o-semiquinone radical intermediate, which tended to form polymeric products at pH values below 5, and o-quinones at higher pH. The model protein bovine serum albumin promoted the formation of quinone even at low pH. Two other polyphenols, procyanidin (epicatechin16-(4-->8)-catechin) and epigallocatechin gallate, had similar pH-dependent oxidation patterns.