Comparison of Metabolic Profiles of Fruits of Arctium lappa, Arctium minus, and Arctium tomentosum.

Metabolites of the edible and medicinal plant Arctium have been shown to possess beneficial activities. The phytochemical profile of Arctium lappa is well-explored and its fruits are known to contain mainly lignans, fatty acids, and sterols. But the fruits of other Arctium species have not been thoroughly investigated. Therefore, this study compares the metabolic profiles of the fruits of A. lappa, Arctium tomentosum, and Arctium minus. Targeted metabolomics led to the putative identification of 53 metabolites in the fruit extracts, the majority of these being lignans and fatty acids. Quantification of the major lignans showed that the year of collection had a significant effect on the lignan content. Furthermore, A. lappa fruits contained lesser amounts of arctigenin but greater amounts of arctigenin glycoside than A. minus fruits. Regarding the profile of fatty acids, A. minus fruits differed from the others in the presence of linolelaidic acid.

Changes in the fatty acid profiles and health indexes of bovine colostrum during the first days of lactation and their impact on human health.

Our objective was to analyze the changes in fatty acid (FA) profiles of bovine colostrum and immature milk during the first four days of lactation and assess their potential impact on human health. Colostrum and immature milk samples were collected from Czech Fleckvieh cows during their first to third lactation and the FA profiles were analyzed using multidimensional gas chromatography with a vacuum ultraviolet detector (GC×GC-VUV). The colostrum of primiparous cows contained lower levels of medium-chain and saturated fatty acids, and higher levels of mono- and unsaturated fatty acids compared to that of multiparous cows. The atherogenic and thrombogenicity indexes, as well as the hypocholesterolemic-to-hypercholesterolemic fatty acid ratio, were more favourable in primiparous cows. This makes colostrum fat an attractive product for human nutrition. To obtain the maximum health benefits, we recommend collecting and processing the colostrum of primiparous cows and immature milk at the end of the milk transition separately.

Origin of Wine Lignans.

Plant lignans possess several properties beneficial for human health and therefore, increasing their contents in foods and beverages is desirable. One of the lignan sources in human diet is wine. To elucidate the origin of lignans contained in wine, LC-MS was used to analyze resinol-related lignans in must, seeds, stems, and wine prepared using stainless steel tanks, oak barrels, and Qvevri (clay vessel). White wines aged in stainless steel tanks contained significantly lower amounts of lignan aglycones (20-60 µg/L) than red and Qvevri wines (300-500 µg/L). Generally, white wines aged in stainless steel tanks contained only low amounts of isolariciresinol and matairesinol. Qvevri wines and red wine aged in stainless steel tank contained up to five lignan compounds and in wine aged in oak barrel, six different lignans were identified. Consistently, only low concentration of isolariciresinol has been found in must, whereas more lignan compounds have been found in grape seeds (isolariciresinol, secoisolariciresinol, and pinoresinol) and stems (isolariciresinol and syringaresinol). Consequently, we conclude that lignan content in wine can be increased by maturation in contact with grape berries, seeds, or stems or with wood.

Production of Bovine Equol-Enriched Milk: A Review.

Milk and dairy products are important sources of nutrients in the human diet because they contain a number of essential substances and other biologically active components. Many of these substances can be modified, and thus offer opportunities to use milk and dairy products as functional food. Isoflavones are particularly important in human nutrition due to their diverse pharmacological and antioxidant properties. The clinical effectiveness of isoflavone-rich products is believed to be dependent on their ability to metabolize daidzein to equol, which may directly exert cancer preventive effects. However, only approximately 30-40% of humans are able to produce equol, while animals, in general, produce equol. Equol is the predominant product of bacterial metabolism of isoflavones and can be found in various amounts in some food of animal origin, especially in milk. Therefore, milk and dairy products can be considered to be sources of equol for humans who are not able to produce this metabolite. When the content of isoflavones in milk is to be modified, two groups of factors should be considered, i.e., dietary factors that include the source of isoflavones and the processing effects on feedstuffs and animal factors that include the intake of isoflavones, ruminal and postruminal changes, and the health and physiological status of animals. The approximate content of isoflavones in milk can be predicted using carry-over rates for different dietary sources or using a formula that describes the relationship between equol concentration in milk and formononetin intake. Processing and storage can affect the content and profile of isoflavones in milk and dairy products.

Health Beneficial Properties of Grapevine Seed Extract and Its Influence on Selected Biochemical Markers in the Blood, Liver and Kidneys of Rattus norvegicus.

Cadmium (Cd) is a heavy metal that occurs in all areas of the environment, including the food chain. In the body, it causes oxidative stress by producing free radicals that are harmful to the cells. Grape seed extract (GSE) contains a wide range of biologically active components that help to neutralize the adverse effects of free radicals. In this study, the effects of GSE prepared form semi-resistant grapevine cultivar Cerason, which is rich in phenolics, on biochemical markers of brown rats exposed to the effects of cadmium were monitored. GSE increased the plasma antioxidant activity and, in the kidneys and the liver, Cd content was significantly lowered by GSE co-administration. Accordingly, the increase in creatinine content and alanine aminotransferase activity and the decrease of catalase and superoxide dismutase activities caused by cadmium were slowed down by GSE co-administration. The results of this work reveal that grape seed extract offers a protective effect against the intake of heavy metals into the organism.

Feedborne Mycotoxins Beauvericin and Enniatins and Livestock Animals.

Mycotoxins are secondary metabolites produced by several species of fungi, including the Fusarium, Aspergillus, and Penicillium species. Currently, more than 300 structurally diverse mycotoxins are known, including a group called minor mycotoxins, namely enniatins, beauvericin, and fusaproliferin. Beauvericin and enniatins possess a variety of biological activities. Their antimicrobial, antibiotic, or ionoforic activities have been proven and according to various bioassays, they are believed to be toxic. They are mainly found in cereal grains and their products, but they have also been detected in forage feedstuff. Mycotoxins in feedstuffs of livestock animals are of dual concern. First one relates to the safety of animal-derived food. Based on the available data, the carry-over of minor mycotoxins from feed to edible animal tissues is possible. The second concern relates to detrimental effects of mycotoxins on animal health and performance. This review aims to summarize current knowledge on the relation of minor mycotoxins to livestock animals.

Production of Defense Phenolics in Tomato Leaves of Different Age.

Phenolics play an essential role in the defense reaction of crop plants against pathogens. However, the intensity of their production induced by infection may differ during the life of a plant. Here, we identified age-related differences in phenolic biosynthesis in the pathosystem Solanum lycopersicum cv. Amateur and Pseudomonas syringae pv. tomato DC3000. We analyzed concentrations of total phenolics, phenolic profiles, and concentrations of selected phenolic acids. The influence of bacterial infection, together with leaf and plant age, was assessed. The changes in concentrations of caffeic acid, 4-hydroxybenzoic acid, and salicylic acid glucoside caused by infection were found to be influenced by age. In concrete, the increases in the concentrations of these metabolites were all evident only in young plants.

In vitro metabolism of red clover isoflavones in rumen fluid.

The degradation of red clover isoflavones was studied in vitro using a rumen fluid buffer system. Various amounts of red clover extract (5-75 mg) together with hay or concentrate-rich diet were added to 40 ml of rumen fluid obtained from non-lactating and lactating dairy cows, respectively, and incubated for 0, 3, 6, 12 or 24 hr. Following incubation, concentrations of daidzein, genistein, formononetin, biochanin A and equol were determined in the samples. After 3 hr of incubation, isoflavone metabolism and equol production could be observed. The results obtained indicate that hay diet provides better conditions for isoflavone metabolism, as concentrations of daidzein, formononetin and biochanin A were higher in incubations based on the concentrate-rich diet and the production of equol was higher in incubations based on the hay diet. Furthermore, in incubations with higher amounts of added clover extract, a decrease in equol production was observed. Further studies are needed to clarify the role of adaptation of rumen microflora on isoflavone degradation kinetics and to clarify the interrelationship between various dietary factors, rumen microbiota and isoflavones. The knowledge of isoflavone metabolism kinetics in dependence on studied factors will be useful for the optimization of feeding dose.

Isoflavones.

Phytoestrogens are naturally occurring nonsteroidal phenolic plant compounds that, due to their molecular structure and size, resemble vertebrate steroids estrogens. This review is focused on plant flavonoids isoflavones, which are ranked among the most estrogenic compounds. The main dietary sources of isoflavones for humans are soybean and soybean products, which contain mainly daidzein and genistein. When they are consumed, they exert estrogenic and/or antiestrogenic effects. Isoflavones are considered chemoprotective and can be used as an alternative therapy for a wide range of hormonal disorders, including several cancer types, namely breast cancer and prostate cancer, cardiovascular diseases, osteoporosis, or menopausal symptoms. On the other hand, isoflavones may also be considered endocrine disruptors with possible negative influences on the state of health in a certain part of the population or on the environment. This review deals with isoflavone classification, structure, and occurrence, with their metabolism, biological, and health effects in humans and animals, and with their utilization and potential risks.

The elicitin ß-cryptogein's activity in tomato is mediated by jasmonic acid and ethylene signalling pathways independently of elicitin-sterol interactions.

MAIN CONCLUSION: The level of resistance induced in different tomato genotypes after ß-CRY treatment correlated with the upregulation of defence genes, but not sterol binding and involved ethylene and jasmonic acid signalling. Elicitins, a family of small proteins secreted by Phytophthora and Pythium spp., are the most well-known microbe-associated molecular patterns of oomycetes, a lineage of fungus-like organisms that include many economically significant crop pathogens. The responses of tomato plants to elicitin INF1 produced by Phytophthora infestans have been studied extensively. Here, we present studies on the responses of three tomato genotypes to ß-cryptogein (ß-CRY), a potent elicitin secreted by Phytophthora cryptogea that induces hypersensitive response (HR) cell death in tobacco plants and confers greater resistance to oomycete infection than acidic elicitins like INF1. We also studied ß-CRY mutants impaired in sterol binding (Val84Phe) and interaction with the binding site on tobacco plasma membrane (Leu41Phe), because sterol binding was suggested to be important in INF1-induced resistance. Treatment with ß-CRY or the Val84Phe mutant induced resistance to powdery mildew caused by the pathogen Pseudoidium neolycopersici, but not the HR cell death observed in tobacco and potato plants. The level of resistance induced in different tomato genotypes correlated with the upregulation of defence genes including defensins, ß-1,3-glucanases, heveins, chitinases, osmotins, and PR1 proteins. Treatment with the Leu41Phe mutant did not induce this upregulation, suggesting similar elicitin recognition in tomato and tobacco. However, here ß-CRY activated ethylene and jasmonic acid signalling, but not salicylic acid signalling, demonstrating that elicitins activate different downstream signalling processes in different plant species. This could potentially be exploited to enhance the resistance of Phytophthora-susceptible crops.

Determination of in vitro isoflavone degradation in rumen fluid.

The aim of this study was to determine the degradation of dietary isoflavones in rumen fluid under 2 feeding regimens. The experiments were performed in vitro using a rumen fluid buffer system. The rumen fluid was taken from cows fed either a hay diet or a concentrate-rich diet (the diet consisted of 34.6% maize silage, 17.6% haylage, 12.8% alfalfa hay, and 35.0% supplemental mixture on a dry matter basis). As a source of isoflavones, 40% soybean extract (Biomedica, Prague, Czech Republic) at levels of 5, 25, 50, and 75 mg per 40 mL of rumen fluid was used. Samples of soybean extract were incubated in triplicate at 39°C for 0, 3.0, 6.0, 12.0, and 24.0 h in incubation solution. The metabolism of daidzein and genistein was faster under concentrate-rich diet conditions. In general, production of equol started after 3 to 6 h of incubation and reached the highest rate after approximately 12 h of incubation regardless of the type of diet or concentration of extract. In most of the experiments, production of equol continued after 24 h of incubation. Generally, equol production was greater under the hay diet conditions. Furthermore, experiments with higher amounts of added soybean extract revealed possible inhibitory effects of high levels of isoflavones on the rumen microflora.

Changes in equol and major soybean isoflavone contents during processing and storage of yogurts made from control or isoflavone-enriched bovine milk determined using LC-MS (TOF) analysis.

The effect of supplementing a basal diet for dairy cows with "Soybean extract 40" (Biomedica, Prague, Czech Republic), containing 40% soybean isoflavones, on the contents of daidzein, glycitein, genistein, and equol in milk as well as fresh and mature yogurts was estimated. To determine the contents of these isoflavonoids, an efficient analytical LC-MS (TOF) technique was used. The "Soybean extract 40" used in our study contained an especially high proportion of daidzein (307gkg-1). In both milk and yogurt samples, the amounts of daidzein and its metabolite equol were significantly higher in samples obtained from cows that received the isoflavone extract-supplemented diet than from those that received the basal diet, as the precursor daidzein contributed to the increased equol concentrations. Fermentation caused significant changes in the daidzein and glycitein concentrations. With maturation, the concentrations of daidzein and equol were unaffected, while the glycitein concentration decreased significantly.

MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins.

Diverse responses of wild and cultivated tomato to BABA, oligandrin and Oidium neolycopersici infection.

Background and Aims: Current strategies for increased crop protection of susceptible tomato plants against pathogen infections include treatment with synthetic chemicals, application of natural pathogen-derived compounds or transfer of resistance genes from wild tomato species within breeding programmes. In this study, a series of 45 genes potentially involved in defence mechanisms was retrieved from the genome sequence of inbred reference tomato cultivar Solanum lycopersicum 'Heinz 1706'. The aim of the study was to analyse expression of these selected genes in wild and cultivated tomato plants contrasting in resistance to the biotrophic pathogen Oidium neolycopersici , the causative agent of powdery mildew. Plants were treated either solely with potential resistance inducers or by inducers together with the pathogen. Methods: The resistance against O. neolycopersici infection as well as RT-PCR-based analysis of gene expression in response to the oomycete elicitor oligandrin and chemical agent ß-aminobutyric acid (BABA) were investigated in the highly susceptible domesticated inbred genotype Solanum lycopersicum 'Amateur' and resistant wild genotype Solanum habrochaites . Key Results: Differences in basal expression levels of defensins, germins, ß-1,3-glucanases, heveins, chitinases, osmotins and PR1 proteins in non-infected and non-elicited plants were observed between the highly resistant and susceptible genotypes. Moreover, these defence genes showed an extensive up-regulation following O. neolycopersici infection in both genotypes. Application of BABA and elicitin induced expression of multiple defence-related transcripts and, through different mechanisms, enhanced resistance against powdery mildew in the susceptible tomato genotype. Conclusions: The results indicate that non-specific resistance in the resistant genotype S. habrochaites resulted from high basal levels of transcripts with proven roles in defence processes. In the susceptible genotype S. lycopersicum 'Amateur', oligandrin- and BABA-induced resistance involved different signalling pathways, with BABA-treated leaves displaying direct activation of the ethylene-dependent signalling pathway, in contrast to previously reported jasmonic acid-mediated signalling for elicitins.

Bacterial community dynamics in a rumen fluid bioreactor during in-vitro cultivation.

To study the various processes in the rumen the in vitro techniques are widely used to realize more controlled and reproducible conditions compared to in vivo experiments. Mostly, only the parameters like pH changes, volatile fatty acids content or metabolite production are monitored. In this study we examine the bacterial community dynamics of rumen fluid in course of ten day cultivation realize under standard conditions described in the literature. Whereas the pH values, total VFA content and A/P ratio in bioreactor were consistent with natural conditions in the rumen, the mean redox-potential values of -251 and -243mV were much more negative. For culture-independent assessment of bacterial community composition, the Illumina MiSeq results indicated that the community contained 292 bacterial genera. In course of ten days cultivation a significant changes in the microbial community were measured when Bacteroidetes to Firmicutes ratio changed from 3.2 to 1.2 and phyla Proteobacteria and Actinobacteria represented by genus Bifidobacterium and Olsenella significantly increased. The main responsible factor of these changes seems to be very low redox potential in bioreactor together with accumulation of simple carbohydrates in milieu as a result of limited excretion of fermented feed and absence of nutrient absorbing mechanisms.

Effects of Isoflavone-Enriched Feed on the Rumen Microbiota in Dairy Cows.

In this study, we compared the effects of two diets containing different isoflavone concentrations on the isoflavone transfer from feed into milk and on the rumen microbiota in lactating dairy cows. The on-farm experiment was conducted on twelve lactating Czech Fleckvieh x Holstein cows divided into two groups, each with similar mean milk yield. Twice daily, cows were individually fed a diet based on maize silage, meadow hay and supplemental mixture. Control group (CTRL) received the basal diet while the experimental group (EXP) received the basal diet supplemented with 40% soybean isoflavone extract. The average daily isoflavone intake in the EXP group (16 g/day) was twice as high as that in the CTRL group (8.4 g/day, P<0.001). Total isoflavone concentrations in milk from the CTRL and EXP groups were 96.89 and 276.07 µg/L, respectively (P<0.001). Equol concentrations in milk increased from 77.78 µg/L in the CTRL group to 186.30 µg/L in the EXP group (P<0.001). The V3-4 region of bacterial 16S rRNA genes was used for metagenomic analysis of the rumen microbiome. The experimental cows exhibited fewer OTUs at a distance level of 0.03 compared to control cows (P<0.05) and reduced microbial richness compared to control cows based on the calculated Inverse Simpson and Shannon indices. Non-metric multidimensional scaling analysis showed that the major contributor to separation between the experimental and control groups were changes in the representation of bacteria belonging to the phyla Bacteroidetes, Proteobacteria, Firmicutes, and Planctomycetes. Surprisingly, a statistically significant positive correlation was found only between isoflavones and the phyla Burkholderiales (r = 0.65, P<0.05) and unclassified Betaproteobacteria (r = 0.58, P<0.05). Previous mouse and human studies of isoflavone effects on the composition of gastrointestinal microbial populations generally report similar findings.

Soybean-Derived Isoflavone Determination in Rumen Fluid and Milk by LC-MS-(TOF).

Soybean-derived isoflavones belong to the family of biologically active phytoestrogens. The purpose of this study was to develop a sensitive method, which permits quantification of the soybean isoflavonoids and equol in bovine rumen fluid and milk using LC-MS-(TOF). The samples of rumen fluid and milk were obtained from 12 lactating dairy cows ingesting 7,500-9,500 mg of total isoflavones daily. The validation of the developed method showed the limits of quantification to be in the range of 0.9-5.0 ng/mL. The precision was determined as relative standard deviation, which was lower than 25% in all cases. The recoveries of the most isoflavonoids were satisfactory. Lower recoveries of daidzin and glycitin can be solved by adding an internal standard. The presented method will be useful for kinetic studies of isoflavone metabolism in ruminants due to simultaneous quantification of free aglycones and glycosides in the rumen fluid.

Elicitin-Induced Distal Systemic Resistance in Plants is Mediated Through the Protein-Protein Interactions Influenced by Selected Lysine Residues.

Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although α- and ß-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, ß-elicitins (possessing 6-7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the α-isoforms (with only 1-3 lysine residues). To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of ß-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein's charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins' movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

The effect of cryptogein with changed abilities to transfer sterols and altered charge distribution on extracellular alkalinization, ROS and NO generation, lipid peroxidation and LOX gene transcription in Nicotiana tabacum.

Cryptogein, a protein from oomycete Phytophthora cryptogea, induces a hypersensitive cell death in Nicotiana tabacum. We prepared a new series of cryptogein mutant proteins with altered abilities to bind sterols and with altered charge distribution in the proteins. The effect of the mutations on the cryptogein ability to induce plant defence mechanisms associated with hypersensitive cell death were examined. Our results with new mutants support the previous findings that the sterol binding does not influence synthesis of ROS, cytosol acidification and development of leaf necrosis as these events seem to be more likely affected by the charge distribution and the overall protein structure. This hypothesis was also applicable on other mechanisms involved in the execution of plant cell death such as the NO generation, the stimulation of lipid peroxidation (determination of malondialdehyde and hydroxy fatty acids levels) and LOX gene transcription. In addition, the ability to bind sterols was found to serve not only for pathogen utilisation in its own metabolism but also to have an important function for the destabilization of plant membrane facilitating the pathogen spread inside the plant tissue as well as intensively contributing to the development of plant cell death. Considering the insertion of charged amino acid residues in the protein structure, the change localized in the protein surface affected its biological activity more effectively than that change inside the protein cavity. Moreover, the insertion of negative charged amino acids influenced mainly the events involved in the early phase of defence reaction, while the positive residues affected especially the necrotic activity of cryptogein.

Preparation and evaluation of carriers for detection of cholinesterase inhibitors.

OBJECTIVES: The aim of the study was to use methods of pharmaceutical technology, and prepare carriers in the form of pellets suitable as a filling of detection tubes for enzymatic detection of cholinesterase inhibitors. The enzymatic detection was based on enzymatic hydrolysis of acetylthiocholine iodide and the subsequent colour reaction of its hydrolysis product with Ellman's reagent. The suitable carriers should be in the form of white, regular and sufficiently mechanically resistant particles of about 1 mm allowing it to capture the enzyme during the impregnation process and ensuring its high activity for enzymatic detection. METHODS: Carriers consisting of microcrystalline cellulose, lactose, povidone, and sodium carboxymethyl cellulose were prepared using extrusion-spheronization method under three different drying conditions in either a hot air oven or a microwave oven. Subsequently, the carriers were impregnated with acetylcholinesterase and their size, shape, mechanical resistance, bulk, tapped and pycnometric density, Hausner ratio, intraparticular and total tapped porosity, and activity were measured and recorded. RESULTS: In this procedure, carriers with different physical parameters and different acetylcholinesterase activity were evaluated. It was found that higher acetylcholinesterase activity was associated not only with a higher intraparticular porosity but also with more regular particles characterized by high sphericity and low total tapped porosity. CONCLUSION: This unique finding is important for the preparation of detection tubes based on enzymatic detection which is still irreplaceable especially in the field of detection and analysis of super-toxic cholinesterase inhibitors.