ER body-resident myrosinases and tryptophan specialized metabolism modulate root microbiota assembly.

Endoplasmic reticulum (ER) bodies are ER-derived structures that contain a large amount of PYK10 myrosinase, which hydrolyzes tryptophan (Trp)-derived indole glucosinolates (IGs). Given the well-described role of IGs in root-microbe interactions, we hypothesized that ER bodies in roots are important for interaction with soil-borne microbes at the root-soil interface. We used mutants impaired in ER bodies (nai1), ER body-resident myrosinases (pyk10bglu21), IG biosynthesis (myb34/51/122), and Trp specialized metabolism (cyp79b2b3) to profile their root microbiota community in natural soil, evaluate the impact of axenically collected root exudates on soil or synthetic microbial communities, and test their response to fungal endophytes in a mono-association setup. Tested mutants exhibited altered bacterial and fungal communities in rhizoplane and endosphere, respectively. Natural soils and bacterial synthetic communities treated with mutant root exudates exhibited distinctive microbial profiles from those treated with wild-type (WT) exudates. Most tested endophytes severely restricted the growth of cyp79b2b3, a part of which also impaired the growth of pyk10bglu21. Our results suggest that root ER bodies and their resident myrosinases modulate the profile of root-secreted metabolites and thereby influence root-microbiota interactions.

Loss of MYB34 Transcription Factor Supports the Backward Evolution of Indole Glucosinolate Biosynthesis in a Subclade of the Camelineae Tribe and Releases the Feedback Loop in This Pathway in Arabidopsis.

Glucosinolates are specialized defensive metabolites characteristic of the Brassicales order. Among them, aliphatic and indolic glucosinolates (IGs) are usually highly abundant in species from the Brassicaceae family. The exceptions this trend are species representing a subclade of the Camelineae tribe, including Capsella and Camelina genera, which have reduced capacity to produce and metabolize IGs. Our study addresses the contribution of specific glucosinolate-related myeloblastosis (MYB) transcription factors to this unprecedented backward evolution of IG biosynthesis. To this end, we performed phylogenomic and functional studies of respective MYB proteins. The obtained results revealed weakened conservation of glucosinolate-related MYB transcription factors, including loss of functional MYB34 protein, in the investigated species. We showed that the introduction of functional MYB34 from Arabidopsis thaliana partially restores IG biosynthesis in Capsella rubella, indicating that the loss of this transcription factor contributes to the backward evolution of this metabolic pathway. Finally, we performed an analysis of the impact of particular myb mutations on the feedback loop in IG biosynthesis, which drives auxin overproduction, metabolic dysregulation and strong growth retardation caused by mutations in IG biosynthetic genes. This uncovered the unique function of MYB34 among IG-related MYBs in this feedback regulation and consequently in IG conservation in Brassicaceae plants.

Metabolomic Aspects of Conservative and Resistance-Related Elements of Response to Fusarium culmorum in the Grass Family.

BACKGROUND: Fusarium head blight (FHB) is a serious fungal disease affecting crop plants, causing substantial yield reductions and the production of mycotoxins in the infected grains. Achieving progress in the breeding of crops with increased resistance and maintaining a high yield is not possible without a thorough examination of the molecular basis of plant immunity responses. METHODS: LC-MS-based metabolomics approaches powered by three-way ANOVA and the selec-tion of differentially accumulated metabolites (DAMs) were used for studying plant immunity. A correlation network and functional enrichment analysis were conducted on grains of barley and wheat genotypes that were resistant or susceptible to FHB, as well as on the model grass Brachypodium distachyon (Bd), as this is still poorly understood at the metabolomic level. RESULTS: We selected common and genotype-specific DAMs in response to F. culmorum inoculation. The immunological reaction at the metabolomic level was strongly diversified between resistant and susceptible genotypes. DAMs that were common to all tested species from the porphyrin, flavonoid, and phenylpropanoid metabolic pathways were highly correlated, reflecting con-servativeness in the FHB response in the Poaceae family. Resistance-related DAMs belonged to different structural classes, including tryptophan-derived metabolites, pyrimidines, the amino acids proline and serine, as well as phenylpropanoids and flavonoids. The physiological re-sponse to F. culmorum of Bd was close to that of barley and wheat genotypes; however, metabo-lomic changes were strongly diversified. CONCLUSIONS: Combined targeted and untargeted metabolomics provides comprehensive knowledge about significant elements of plant immuni-ty that have the potential to be molecular biomarkers of enhanced resistance to FHB in the grass family. Thorough examination of the Bd metabolome in juxtaposition with diversified geno-types of barley and wheat facilitated its use as a model grass for plant-microbe interaction.

Targeted and Untargeted Metabolomic Analyses Reveal Organ Specificity of Specialized Metabolites in the Model Grass Brachypodium distachyon.

Brachypodium distachyon, because of its fully sequenced genome, is frequently used as a model grass species. However, its metabolome, which constitutes an indispensable element of complex biological systems, remains poorly characterized. In this study, we conducted comprehensive, liquid chromatography-mass spectrometry (LC-MS)-based metabolomic examination of roots, leaves and spikes of Brachypodium Bd21 and Bd3-1 lines. Our pathway enrichment analysis emphasised the accumulation of specialized metabolites representing the flavonoid biosynthetic pathway in parallel with processes related to nucleotide, sugar and amino acid metabolism. Similarities in metabolite profiles between both lines were relatively high in roots and leaves while spikes showed higher metabolic variance within both accessions. In roots, differences between Bd21 and Bd3-1 lines were manifested primarily in diterpenoid metabolism, while differences within spikes and leaves concerned nucleotide metabolism and nitrogen management. Additionally, sulphate-containing metabolites differentiated Bd21 and Bd3-1 lines in spikes. Structural analysis based on MS fragmentation spectra enabled identification of 93 specialized metabolites. Among them phenylpropanoids and flavonoids derivatives were mainly determined. As compared with closely related barley and wheat species, metabolic profile of Brachypodium is characterized with presence of threonate derivatives of hydroxycinnamic acids.

Tryptophan metabolism and bacterial commensals prevent fungal dysbiosis in Arabidopsis roots.

In nature, roots of healthy plants are colonized by multikingdom microbial communities that include bacteria, fungi, and oomycetes. A key question is how plants control the assembly of these diverse microbes in roots to maintain host-microbe homeostasis and health. Using microbiota reconstitution experiments with a set of immunocompromised Arabidopsis thaliana mutants and a multikingdom synthetic microbial community (SynCom) representative of the natural A. thaliana root microbiota, we observed that microbiota-mediated plant growth promotion was abolished in most of the tested immunocompromised mutants. Notably, more than 40% of between-genotype variation in these microbiota-induced growth differences was explained by fungal but not bacterial or oomycete load in roots. Extensive fungal overgrowth in roots and altered plant growth was evident at both vegetative and reproductive stages for a mutant impaired in the production of tryptophan-derived, specialized metabolites (cyp79b2/b3). Microbiota manipulation experiments with single- and multikingdom microbial SynComs further demonstrated that 1) the presence of fungi in the multikingdom SynCom was the direct cause of the dysbiotic phenotype in the cyp79b2/b3 mutant and 2) bacterial commensals and host tryptophan metabolism are both necessary to control fungal load, thereby promoting A. thaliana growth and survival. Our results indicate that protective activities of bacterial root commensals are as critical as the host tryptophan metabolic pathway in preventing fungal dysbiosis in the A. thaliana root endosphere.

Separation of Chromatographic Co-Eluted Compounds by Clustering and by Functional Data Analysis.

Peak overlapping is a common problem in chromatography, mainly in the case of complex biological mixtures, i.e., metabolites. Due to the existence of the phenomenon of co-elution of different compounds with similar chromatographic properties, peak separation becomes challenging. In this paper, two computational methods of separating peaks, applied, for the first time, to large chromatographic datasets, are described, compared, and experimentally validated. The methods lead from raw observations to data that can form inputs for statistical analysis. First, in both methods, data are normalized by the mass of sample, the baseline is removed, retention time alignment is conducted, and detection of peaks is performed. Then, in the first method, clustering is used to separate overlapping peaks, whereas in the second method, functional principal component analysis (FPCA) is applied for the same purpose. Simulated data and experimental results are used as examples to present both methods and to compare them. Real data were obtained in a study of metabolomic changes in barley (Hordeum vulgare) leaves under drought stress. The results suggest that both methods are suitable for separation of overlapping peaks, but the additional advantage of the FPCA is the possibility to assess the variability of individual compounds present within the same peaks of different chromatograms.

Gene expression evolution in pattern-triggered immunity within Arabidopsis thaliana and across Brassicaceae species.

Plants recognize surrounding microbes by sensing microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). Despite their significance for microbial control, the evolution of PTI responses remains largely uncharacterized. Here, by employing comparative transcriptomics of six Arabidopsis thaliana accessions and three additional Brassicaceae species to investigate PTI responses, we identified a set of genes that commonly respond to the MAMP flg22 and genes that exhibit species-specific expression signatures. Variation in flg22-triggered transcriptome responses across Brassicaceae species was incongruent with their phylogeny, while expression changes were strongly conserved within A. thaliana. We found the enrichment of WRKY transcription factor binding sites in the 5'-regulatory regions of conserved and species-specific responsive genes, linking the emergence of WRKY-binding sites with the evolution of gene expression patterns during PTI. Our findings advance our understanding of the evolution of the transcriptome during biotic stress.

Halogen Atoms in the Protein-Ligand System. Structural and Thermodynamic Studies of the Binding of Bromobenzotriazoles by the Catalytic Subunit of Human Protein Kinase CK2.

Binding of a family of brominated benzotriazoles to the catalytic subunit of human protein kinase CK2 (hCK2α) was used as a model system to assess the contribution of halogen bonding to protein-ligand interaction. CK2 is a constitutively active pleiotropic serine/threonine protein kinase that belongs to the CMGC group of eukaryotic protein kinases (EPKs). Due to the addiction of some cancer cells, CK2 is an attractive and well-characterized drug target. Halogenated benzotriazoles act as ATP-competitive inhibitors with unexpectedly good selectivity for CK2 over other EPKs. We have characterized the interaction of bromobenzotriazoles with hCK2α by X-ray crystallography, low-volume differential scanning fluorimetry, and isothermal titration calorimetry. Properties of free ligands in solution were additionally characterized by volumetric and RT-HPLC measurements. Thermodynamic data indicate that the affinity increases with bromo substitution, with greater contributions from 5- and 6-substituents than 4- and 7-substituents. Except for 4,7-disubstituted compounds, the bromobenzotriazoles adopt a canonical pose with the triazole close to lysine 68, which precludes halogen bonding. More highly substituted benzotriazoles adopt many additional noncanonical poses, presumably driven by a large hydrophobic contribution to binding. Some noncanonical ligand orientations allow the formation of halogen bonds with the hinge region. Consistent with a predominantly hydrophobic interaction, the isobaric heat capacity decreases upon ligand binding, the more so the higher the substitution.

Evolutionary changes in the glucosinolate biosynthetic capacity in species representing Capsella, Camelina and Neslia genera.

Glucosinolates are unique thioglucosides that evolved in the order Brassicales. These compounds function in plant adaptation to the environment, including combating plant pathogens, herbivore deterrence and abiotic stress tolerance. In line with their defensive functions glucosinolates usually accumulate constitutively in relatively high amounts in all tissues of Brassicaceae plants. Here we performed glucosinolate analysis in different organs of selected species representing Capsella, Camelina and Neslia genera, which similarly as the model plant Arabidopsis thaliana belong to the Camelineae tribe. We also identified orthologs of A. thaliana glucosinolate biosynthetic genes in the published genomes of some of the investigated species. Subsequent gene expression and phylogenetic analyses enabled us an insight into the evolutionary changes in the transcription of these genes and in the sequences of respective proteins that occurred within the Camelineae tribe. Our results indicated that glucosinolates are highly abundant in siliques and roots of the investigated species but hardly, if at all, produced in leaves. In addition to this unusual tissular distribution we revealed reduced structural diversity of methionine-derived aliphatic glucosinolates (AGs) with elevated accumulation of rare long chain AGs. This preference seems to correlate with evolutionary changes in genes encoding methylthioalkylmalate synthases that are responsible for the elongation of AG side chains. Finally, our results indicate that the biosynthetic pathway for tryptophan-derived indolic glucosinolates likely lost its main functions in immunity and resistance towards sucking insects and is on its evolutionary route to be shut off in the investigated species.

The Effect of Different Water Extracts from Platycodon grandiflorum on Selected Factors Associated with Pathogenesis of Chronic Bronchitis in Rats.

The aim of this study was to assess the activity of extracts from Platycodon grandiflorum A. DC (PG) in a model of chronic bronchitis in rats. The research was carried out on three water extracts: E1 - from roots of field cultivated PG; E2 - from biotransformed roots of PG; E3 - from callus of PG. The extracts differed in saponins and inulin levels-the highest was measured in E3 and the lowest in E1. Identification of secondary metabolites was performed using two complementary LC-MS systems. Chronic bronchitis was induced by sodium metabisulfite (a source of SO2). Animals were treated with extracts for three weeks (100 mg/kg, intragastrically) and endothelial growth factor (VEGF), transforming growth factors (TGF-ß1, -ß2, -ß3), and mucin 5AC (MUC5AC) levels were determined in bronchoalveolar lavage fluid, whereas C reactive protein (CRP) level was measured in serum. Moreover, mRNA expression were assessed in bronchi and lungs. In SO2-exposed rats, an elevation of the CRP, TGF-ß1, TGF-ß2, VEGF, and mucin was found, but the extracts' administration mostly reversed this phenomenon, leading to control values. The results showed a strong anti-inflammatory effect of the extracts from PG.

Phenolic Metabolites from Barley in Contribution to Phenome in soil Moisture Deficit.

Eight barley varieties from Europe and Asia were subjected to moisture deficit at various development stages. At the seedling stage and the flag leaf stage combined stress was applied. The experiment was designed for visualization of the correlation between the dynamics of changes in phenolic compound profiles and the external phenome. The most significant increase of compound content in water deficiency was observed for chrysoeriol and apigenin glycoconjugates acylated with methoxylated hydroxycinnamic acids that enhanced the UV-protection effectiveness. Moreover, other good antioxidants such as derivatives of luteolin and hordatines were also induced by moisture deficit. The structural diversity of metabolites of the contents changed in response to water deficiency in barley indicates their multipath activities under stress. Plants exposed to moisture deficit at the seedling stage mobilized twice as many metabolites as plants exposed to this stress at the flag leaf stage. Specific metabolites such as methoxyhydroxycinnamic acids participated in the long-term acclimation. In addition, differences in phenolome mobilization in response to moisture deficit applied at the vegetative and generative phases were correlated with the phenotypical consequences. Observations of plant yield and biomass gave us the possibility to discuss the developmentally related consequences of moisture deficit for plants' fitness.

Louse Flies of Charadrii and Scolopaci Shorebirds Migrating through Central Europe.

Louse flies are blood-sucking ectoparasites of birds and mammals of veterinary and medical importance. These flies damage host skin and serve as vectors for blood-associated pathogens. Little is known about their phenology, host selection, and prevalence in wild birds. Our study provided a broad-scale assessment of louse flies parasitizing Charadrii and Scolopaci shorebirds during their migration through Central Europe. We collected louse flies from 3,129 specimens in 22 species of shorebirds at a major migratory stopover site in central Poland in 2011-17. In total, 31 Ornithomya avicularia and 15 Ornithomya chloropus specimens occurred on five wader species: Common Snipe (Gallinago gallinago), Ruff (Calidris pugnax), Dunlin (Calidris alpina), Curlew Sandpiper (Calidris ferruginea), and Wood Sandpiper (Tringa glareola). Prevalences ranged from 0.23% to 4.62%. No individual birds were infested by both species of fly, and no flies were found on an additional 17 wader species. The rarer of the two flies, O. chloropus, occurred on all infested bird species, whereas O. avicularia occurred only on Common Snipe and Curlew Sandpiper. In comparison with the other four infested wader species, Common Snipe (n=1,194) was more frequently infested with O. avicularia than with O. chloropus. Our study shows that the prevalence of louse flies on shorebirds is much lower than on other avian taxa. Ecologic and evolutionary mechanisms leading to the relative rarity of louse flies on shorebirds remain to be understood.

Analytical Methods for Detection of Plant Metabolomes Changes in Response to Biotic and Abiotic Stresses.

Abiotic and biotic stresses are the main reasons of substantial crop yield losses worldwide. Research devoted to reveal mechanisms of plant reactions during their interactions with the environment are conducted on the level of genome, transcriptome, proteome, and metabolome. Data obtained during these studies would permit to define biochemical and physiological mechanisms of plant resistance or susceptibility to affecting factors/stresses. Metabolomics based on mass spectrometric techniques is an important part of research conducted in the direction of breeding new varieties of crop plants tolerant to the affecting stresses and possessing good agronomical features. Studies of this kind are carried out on model, crop and resurrection plants. Metabolites profiling yields large sets of data and due to this fact numerous advanced statistical and bioinformatic methods permitting to obtain qualitative and quantitative evaluation of the results have been developed. Moreover, advanced integration of metabolomics data with these obtained on other omics levels: genome, transcriptome and proteome should be carried out. Such a holistic approach would bring us closer to understanding biochemical and physiological processes of the cell and whole plant interacting with the environment and further apply these observations in successful breeding of stress tolerant or resistant crop plants.

Specificity of MLL1 and TET3 CXXC domains towards naturally occurring cytosine modifications.

CXXC domains have traditionally been considered as CpG specific DNA binding domains that are repelled by cytosine modifications. This view has recently been challenged by the demonstration that CXXC domain of TET3 has relaxed sequence specificity and binds with the highest affinity to symmetric DNA duplex containing 5caCpG. Here, we present a comparative analysis of the MLL1-CXXC and TET3-CXXC sequence specificity and tolerance to cytosine modifications (5-methyl, 5-hydroxymethyl, 5-formyl, 5-carboxyl) in CpG and non-CpG context. For the first time, we take into consideration possible interference from cytosine bases elsewhere in the sequence. We show that despite similar overall structure, MLL1-CXXC has greater sequence and modification specificity than TET3-CXXC. MLL1-CXXC is specific only for CpG and does not tolerate any cytosine modifications. In contrast, TET3-CXXC does not require the CpG context of cytosine bases. Methyl-, formyl- and carboxyl-modifications are tolerated by TET3-CXXC, but only preceding G. Based on our and other data we propose a parsimonious model of MLL1-CXXC and TET3-CXXC DNA binding. This model explains why the binding of modified DNA duplexes by TET3-CXXC requires in some cases a register shift and is therefore context-dependent.

Comparison of bioactive compounds content in leaf extracts of Passiflora incarnata, P. caerulea and P. alata and in vitro cytotoxic potential on leukemia cell lines

ABSTRACT Passiflora caerulea L., P. alata Curtis and P. incarnata L. (synonym for P. edulis Sims), are the most popular representatives of the Passiflora genus in South America. In recent years, a growing attention is paid to the biological activity and phytochemical profiles of crude extracts from various species of Passiflora in worldwide. The aim of this study was to evaluate and to compare of anti-leukemic activity of the dry crude extracts from leaves of three Passiflora species from greenhouse of Poland in two human acute lymphoblastic leukemia cell lines: CCRF-CEM and its multidrug resistant variant. Two systems of liquid chromatography in order to assessment of phytochemical composition of extracts were applied. Extracts of P. alata and P. incarnata showed the potent inhibitory activity against human acute lymphoblastic leukemia CCRF-CEM, while P. caerulea not showed activity (or activity was poor). Despite similarities in quality phytochemical profile of extracts from P. caerulea and P. incarnata, differences in quantity of chemical compounds may determine their various pharmacological potency. For the activity of P. alata extract the highest content of terpenoids and a lack of flavones C-glycosides are believed to be crucial. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.

The Y. bercovieri Anbu crystal structure sheds light on the evolution of highly (pseudo)symmetric multimers.

Ancestral ß-subunit (Anbu) is homologous to HslV and 20S proteasomes. Based on its phylogenetic distribution and sequence clustering, Anbu has been proposed as the "ancestral" form of proteasomes. Here, we report biochemical data, small-angle X-ray scattering results, negative-stain electron microscopy micrographs and a crystal structure of the Anbu particle from Yersinia bercovieri (YbAnbu). All data are consistent with YbAnbu forming defined 12-14 subunit multimers that differ in shape from both HslV and 20S proteasomes. The crystal structure reveals that YbAnbu subunits form tight dimers, held together in part by the Anbu specific C-terminal helices. These dimers ("protomers") further assemble into a low-rise left-handed staircase. The lock-washer shape of YbAnbu is consistent with the presence of defined multimers, X-ray diffraction data in solution and negative-stain electron microscopy images. The presented structure suggests a possible evolutionary pathway from helical filaments to highly symmetric or pseudosymmetric multimer structures. YbAnbu subunits have the Ntn-hydrolase fold, a putative S1 pocket and conserved candidate catalytic residues Thr1, Asp17 and Lys32(33). Nevertheless, we did not detect any YbAnbu peptidase or amidase activity. However, we could document orthophosphate production from ATP catalyzed by the ATP-grasp protein encoded in the Y. bercovieri Anbu operon.

Effect of Salvia miltiorrhiza root extract on brain acetylcholinesterase and butyrylcholinesterase activities, their mRNA levels and memory evaluation in rats.

Salvia miltiorrhiza (Lamiaceae), one of the most important and popular plants of traditional medicine of Asia, is used for the prevention and treatment of cardiovascular diseases and in central nervous system disturbances. The main aim of this study was to assess the influence of subchronic (28-fold) administration of Salvia miltiorrhiza root extract (SE, 200mg/kg, p.o.) on behavioural activity and memory of rats and to evaluate the activities of cholinesterases (AChE and BuChE) and gene expression levels of AChE and BuChE as well as of beta-secretase (BACE1) in the hippocampus and frontal cortex in vivo. Huperzine A (HU, 0.5mg/kg b.w., p.o.) served as a positive control substance, whereas scopolamine (0.5mg/kg, i.p.) injection was used as a well-known model of memory impairment. The results showed that subchronic administration of SE led to an improvement of long-term memory of rats. Strong inhibition of AChE and BuChE mRNA transcription in the frontal cortex of rats treated with SE or HU was observed. The BACE1 transcript level was significantly decreased. AChE activity was statistically significantly inhibited in the frontal cortex and the hippocampus by SE (47% and 55%, respectively). Similar effects were observed in the case of HU. In summary, activity of SE provides evidence that the plant can be a source of drugs used in the treatment of Alzheimer disease.

Drought-related secondary metabolites of barley (Hordeum vulgare L.) leaves and their metabolomic quantitative trait loci.

Determining the role of plant secondary metabolites in stress conditions is problematic due to the diversity of their structures and the complexity of their interdependence with different biological pathways. Correlation of metabolomic data with the genetic background provides essential information about the features of metabolites. LC-MS analysis of leaf metabolites from 100 barley recombinant inbred lines (RILs) revealed that 98 traits among 135 detected phenolic and terpenoid compounds significantly changed their level as a result of drought stress. Metabolites with similar patterns of change were grouped in modules, revealing differences among RILs and parental varieties at early and late stages of drought. The most significant changes in stress were observed for ferulic and sinapic acid derivatives as well as acylated glycosides of flavones. The tendency to accumulate methylated compounds was a major phenomenon in this set of samples. In addition, the polyamine derivatives hordatines as well as terpenoid blumenol C derivatives were observed to be drought related. The correlation of drought-related compounds with molecular marker polymorphisms resulted in the definition of metabolomic quantitative trait loci in the genomic regions of single-nucleotide polymorphism 3101-111 and simple sequence repeat Bmag0692 with multiple linkages to metabolites. The associations pointed to genes related to the defence response and response to cold, heat and oxidative stress, but not to genes related to biosynthesis of the compounds. We postulate that the significant metabolites have a role as antioxidants, regulators of gene expression and modulators of protein function in barley during drought.

THE USE OF EXTRACTS FROM PASSIFLORA SPP. IN HELPING THE TREATMENT OF ACANTHAMOEBIASIS.

Chronic progressive diseases of the central nervous system such as granulomatous amoebic encephalitis, amoebic keratitis, amoebic pneumonitis and also skin infections caused by free-living amoebae (Acanhamoeba spp.) are a significant challenge for pharmacotherapy. This is due to the lack of effective treatment because of encystation, which makes the amoebae highly resistant to anti-amoebic drugs. A very inter- esting and promising source of future drugs in this area are plant materials obtained not only from the habitat but also from plant in vitro culture as an alternative source of biomaterials. Alcoholic extracts from leaves of Passiflora incarnata, P. caerulea, P. alata (Passifloraceae) and from callus cultures were evaluated in vito for amoebicidal activity. Phytochemical analysis showed that all extracts contained phenolic compounds including flavonoids? Biological study revealed that all extracts showed amoebostatic and amoebicidal properties in concentrations from 4 to 12 mg/mL. Extracts of P. alata leaf and callus showed the most effective activities (IC5, 4.01 mg/mL, IC,5 7.29 mg/mL, respectively) after 48 h of exposure, which was correlated with the highest concentration of total phenolics and flavonoids in comparison with other extracts.

Structural Characterization of Flavonoid Glycoconjugates and Their Derivatives with Mass Spectrometric Techniques.

Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of plant secondary metabolite mixtures isolated from biological material including flavonoid glycoconjugates. Resolution of the applied mass spectrometers plays an important role in structural studies of mixtures of the target compounds isolated from biological material. High-resolution analyzers allow obtaining information about elemental composition of the analyzed compounds. Application of various mass spectrometric techniques, including different systems of ionization, analysis of both positive and negative ions of flavonoids, fragmentation of the protonated/deprotonated molecules and in some cases addition of metal ions to the studied compounds before ionization and fragmentation, may improve structural characterization of natural products. In our review we present different strategies allowing structural characterization of positional isomers and isobaric compounds existing in class of flavonoid glycoconjugates and their derivatives, which are synthetized in plants and are important components of the human food and drugs as well as animal feed.