Phytoestrogen consumption and association with breast, prostate and colorectal cancer in EPIC Norfolk

Phytoestrogens are polyphenolic secondary plant metabolites that have structural and functional similarities to 17β-oestradiol and have been associated with a protective effect against hormone-related cancers. Most foods in the UK only contain small amounts of phytoestrogens (median content 21 μg/100 g) and the highest content is found in soya and soya-containing foods. The highest phytoestrogen content in commonly consumed foods is found in breads (average content 450 μg/100 g), the main source of isoflavones in the UK diet. The phytoestrogen consumption in cases and controls was considerably lower than in Asian countries. No significant associations between phytoestrogen intake and breast cancer risk in a nested case-control study in EPIC Norfolk were found. Conversely, colorectal cancer risk was inversely associated with enterolignan intake in women but not in men. Prostate cancer risk was positively associated with enterolignan intake, however this association became non-significant when adjusting for dairy intake, suggesting that enterolignans can act as a surrogate marker for dairy or calcium intake.

Mass spectrometry imaging of glucosinolates in arabidopsis flowers and siliques

Glucosinolates are multi-functional plant secondary metabolites which play a vital role in plant defence and are, as dietary compounds, important to human health and livestock well-being. Knowledge of the tissue-specific regulation of their biosynthesis and accumulation is essential for plant breeding programs. Here, we report that in Arabidopsis thaliana, glucosinolates are accumulated differentially in specific cells of reproductive organs. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), distribution patterns of three selected compounds, 4-methylsulfinylbutyl (glucoraphanin), indol-3-ylmethyl (glucobrassicin), and 4-benzoyloxybutyl glucosinolates, were mapped in the tissues of whole flower buds, sepals and siliques. The results show that tissue localization patterns of aliphatic glucosinolate glucoraphanin and 4-benzoyloxybutyl glucosinolate were similar, but indole glucosinolate glucobrassicin had different localisation, indicating a possible difference in function. The high resolution images obtained by a complementary approach, cryo-SEM Energy Dispersive X-ray analysis (cryo-SEM-EDX), confirmed increased concentration of sulphur in areas with elevated amounts of glucosinolates, and allowed identifying the cell types implicated in accumulation of glucosinolates. High concentration of sulphur was found in S-cells adjacent to the phloem in pedicels and siliques, indicating the presence of glucosinolates. Moreover, both MALDI MSI and cryo-SEM-EDX analyses indicated accumulation of glucosinolates in cells on the outer surface of the sepals, suggesting that a layer of glucosinolate-accumulating epidermal cells protects the whole of the developing flower, in addition to the S-cells, which protect the phloem. This research demonstrates the high potential of MALDI MSI for understanding the cell-specific compartmentation of plant metabolites and its regulation.

Flavonoid intake in European adults (18 to 64 years)

Background Flavonoids are a group of phenolic secondary plant metabolites that are ubiquitous in plant-based diets. Data from anthropological, observational and intervention studies have shown that many flavonoids are bioactive. For this reason, there is an increasing interest in investigating the potential health effects of these compounds. The translation of these findings into the context of the health of the general public requires detailed information on habitual dietary intake. However, only limited data are currently available for European populations. Objective The objective of this study is to determine the habitual intake and main sources of anthocyanidins, flavanols, flavanones, flavones, flavonols, proanthocyanidins, theaflavins and thearubigins in the European Union. Design We use food consumption data from the European Food Safety Authority (EFSA) and the FLAVIOLA Food Composition Database to estimate intake of flavonoids. Results Mean (±SEM) intake of total flavonoids in Europe was 428±49 mg/d, of which 136±14 mg/d were monomeric compounds. Gallated flavan-3-ols (53±12 mg/d) were the main contributor. The lowest flavonoid intake was observed in Mediterranean countries (monomeric compounds: 95±11 mg/d). The distribution of intake was skewed in many countries, especially in Germany (monomeric flavonoids; mean intake: 181 mg/d; median intake: 3 mg/d). Conclusions The habitual intake of flavonoids in Europe is below the amounts found to have a significant health effect.

An evaluation of the costs of making specific secondary metabolites: does the yield penalty incurred by host plant resistance to insects due to competition for resources?

The presumption that the synthesis of 'defence' compounds in plants must incur some 'trade-off' or penalty in terms of annual crop yields has been used to explain observed inverse correlations between resistance to herbivores and rates of growth or photosynthesis. An analysis of the cost of making secondary compounds suggests that this accounts for only a small part of the overall carbon budget of annual crop plants. Even the highest reported amounts of secondary metabolites found in different crop species (flavonoids, allylisothiocyanates, hydroxamic acids, 2-tridecanone) represent a carbon demand that can be satisfied by less than an hour's photosynthesis. Similar considerations apply to secondary compounds containing nitrogen or sulphur, which are unlikely to represent a major investment compared to the cost of making proteins, the major demand for these elements. Decreases in growth and photosynthesis in response to stress are more likely the result of programmed down-regulation. Observed correlations between yield and low contents of unpalatable or toxic compounds may be the result of parallel selection during the refinement of crop species by humans.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL), for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins constituting the vast majority of species in any proteome, as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis. Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification / detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL capture is at least twice that of control, untreated sample.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL, for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins Constituting the vast majority of species in any proteome. as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis, Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic Compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification I detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL Capture is at least twice that of control, untreated sample. (c) 2008 Elsevier B.V. All rights reserved.

Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics

Quantitative analysis by mass spectrometry (MS) is a major challenge in proteomics as the correlation between analyte concentration and signal intensity is often poor due to varying ionisation efficiencies in the presence of molecular competitors. However, relative quantitation methods that utilise differential stable isotope labelling and mass spectrometric detection are available. Many drawbacks inherent to chemical labelling methods (ICAT, iTRAQ) can be overcome by metabolic labelling with amino acids containing stable isotopes (e.g. 13C and/or 15N) in methods such as Stable Isotope Labelling with Amino acids in Cell culture (SILAC). SILAC has also been used for labelling of proteins in plant cell cultures (1) but is not suitable for whole plant labelling. Plants are usually autotrophic (fixing carbon from atmospheric CO2) and, thus, labelling with carbon isotopes becomes impractical. In addition, SILAC is expensive. Recently, Arabidopsis cell cultures were labelled with 15N in a medium containing nitrate as sole nitrogen source. This was shown to be suitable for quantifying proteins and nitrogen-containing metabolites from this cell culture (2,3). Labelling whole plants, however, offers the advantage of studying quantitatively the response to stimulation or disease of a whole multicellular organism or multi-organism systems at the molecular level. Furthermore, plant metabolism enables the use of inexpensive labelling media without introducing additional stress to the organism. And finally, hydroponics is ideal to undertake metabolic labelling under extremely well-controlled conditions. We demonstrate the suitability of metabolic 15N hydroponic isotope labelling of entire plants (HILEP) for relative quantitative proteomic analysis by mass spectrometry. To evaluate this methodology, Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress.

Elevated atmospheric carbon dioxide impairs the performance of root-feeding vine weevils by modifying root growth and secondary metabolites

Predicting how insect crop pests will respond to global climate change is an important part of increasing crop production for future food security, and will increasingly rely on empirically based evidence. The effects of atmospheric composition, especially elevated carbon dioxide (eCO(2)), on insect herbivores have been well studied, but this research has focussed almost exclusively on aboveground insects. However, responses of root-feeding insects to eCO(2) are unlikely to mirror these trends because of fundamental differences between aboveground and belowground habitats. Moreover, changes in secondary metabolites and defensive responses to insect attack under eCO(2) conditions are largely unexplored for root herbivore interactions. This study investigated how eCO(2) (700 mu mol mol-1) affected a root-feeding herbivore via changes to plant growth and concentrations of carbon (C), nitrogen (N) and phenolics. This study used the root-feeding vine weevil, Otiorhynchus sulcatus and the perennial crop, Ribes nigrum. Weevil populations decreased by 33% and body mass decreased by 23% (from 7.2 to 5.4 mg) in eCO(2). Root biomass decreased by 16% in eCO(2), which was strongly correlated with weevil performance. While root N concentrations fell by 8%, there were no significant effects of eCO(2) on root C and N concentrations. Weevils caused a sink in plants, resulting in 8-12% decreases in leaf C concentration following herbivory. There was an interactive effect of CO(2) and root herbivory on root phenolic concentrations, whereby weevils induced an increase at ambient CO(2), suggestive of defensive response, but caused a decrease under eCO(2). Contrary to predictions, there was a positive relationship between root phenolics and weevil performance. We conclude that impaired root-growth underpinned the negative effects of eCO(2) on vine weevils and speculate that the plant's failure to mount a defensive response at eCO(2) may have intensified these negative effects.

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.

Impacts of plant-based foods in ancestral hominin diets on the metabolism and function of gut microbiota in vitro

Ancestral human populations had diets containing more indigestible plant material than present-day diets in industrialized countries. One hypothesis for the rise in prevalence of obesity is that physiological mechanisms for controlling appetite evolved to match a diet with plant fiber content higher than that of present-day diets. We investigated how diet affects gut microbiota and colon cells by comparing human microbial communities with those from a primate that has an extreme plant-based diet, namely, the gelada baboon, which is a grazer. The effects of potato (high starch) versus grass (high lignin and cellulose) diets on human-derived versus gelada-derived fecal communities were compared in vitro. We especially focused on the production of short-chain fatty acids, which are hypothesized to be key metabolites influencing appetite regulation pathways. The results confirmed that diet has a major effect on bacterial numbers, short-chain fatty acid production, and the release of hormones involved in appetite suppression. The potato diet yielded greater production of short-chain fatty acids and hormone release than the grass diet, even in the gelada cultures, which we had expected should be better adapted to the grass diet. The strong effects of diet on hormone release could not be explained, however, solely by short-chain fatty acid concentrations. Nuclear magnetic resonance spectroscopy found changes in additional metabolites, including betaine and isoleucine, that might play key roles in inhibiting and stimulating appetite suppression pathways. Our study results indicate that a broader array of metabolites might be involved in triggering gut hormone release in humans than previously thought. IMPORTANCE: One theory for rising levels of obesity in western populations is that the body's mechanisms for controlling appetite evolved to match ancestral diets with more low-energy plant foods. We investigated this idea by comparing the effects of diet on appetite suppression pathways via the use of gut bacterial communities from humans and gelada baboons, which are modern-day primates with an extreme diet of low-energy plant food, namely, grass. We found that diet does play a major role in affecting gut bacteria and the production of a hormone that suppresses appetite but not in the direction predicted by the ancestral diet hypothesis. Also, bacterial products were correlated with hormone release that were different from those normally thought to play this role. By comparing microbiota and diets outside the natural range for modern humans, we found a relationship between diet and appetite pathways that was more complex than previously hypothesized on the basis of more-controlled studies of the effects of single compounds.