Production of Polyphenolic Natural Products by Bract-Derived Tissue Cultures of Three Medicinal Tilia spp.: A Comparative Untargeted Metabolomics Study.

Medicinal plant tissue cultures are potential sources of bioactive compounds. In this study, we report the chemical characterization of the callus cultures of three medicinal Tilia spp. (Tilia cordata, Tilia vulgaris and Tilia tomentosa), along with the comparison to bracts and flowers of the same species. Our aim was to show that calli of Tilia spp. are good alternatives to the calli of T. americana for the production of polyphenols and are better sources of a subset of polyphenolic metabolites, compared to the original organs. Calli were initiated from young bracts and grown on woody plant medium containing 1 mg L-1 2,4-D and 0.1 mg L-1 BAP. For chemical characterization, a quality-controlled untargeted metabolomics approach and the quantification of several bioactive compounds was performed with the use of LC-ESI-MS/MS. While bracts and flowers contained flavonoid glycosides (astragalin, isoquercitrin) as major polyphenols, calli of all species contained catechins, coumarins (fraxin, esculin and scopoletin) and flavane aglyca. T. tomentosa calli contained 5397 µg g DW-1 catechin, 201 µg g DW-1 esculin, 218 µg g DW-1 taxifolin and 273 µg g DW-1 eriodictyol, while calli from other species contained lower amounts. T. cordata and T. tomentosa flowers were rich in isoquercitrin, containing 8134 and 6385 µg g DW-1, respectively. The currently tested species contained many of the bioactive metabolites described from T. americana. The production of catechin was shown to be comparable to the most efficient tissue cultures reported. Flowers and bracts contained flavonoid glycosides, including tiliroside, resembling bioactive fractions of T. americana. In addition, untargeted metabolomics has shown fingerprint-like differences among species, highlighting possible chemotaxonomic and quality control applications, especially for bracts.

Antifungal Activity of Glucosinolate-Derived Nitriles and Their Synergistic Activity with Glucosinolate-Derived Isothiocyanates Distinguishes Various Taxa of Brassicaceae Endophytes and Soil Fungi.

The glucosinolates of Brassicaceae plants are converted into bioactive isothiocyanates and other volatiles during a challenge by pathogens and other biotic stressors. However, the role of alternative downstream products with weaker potency (e.g., nitriles) is far from being fully understood. This study tested the possible synergistic antifungal interaction between various glucosinolate-derived nitriles and 2-phenylethyl isothiocyanate (PEITC) on 45 fungal strains, including endophytes from horseradish roots (Brassicaceae) and soil fungi, using an airtight system enabling the accurate study of extremely volatile antifungal agents. The median minimal inhibitory concentrations (MICs) were 1.28, 6.10, 27.00 and 49.72 mM for 1H-indole-3-acetonitrile (IAN), 3-phenylpropanenitrile (PPN), 4-(methylsulfanyl)-butanenitrile (MSBN) and 3-butenenitrile (BN, = allyl cyanide), respectively. Thus, nitriles were considerably weaker antifungal agents compared to PEITC with a median MIC of 0.04 mM. For the same nitriles, the median fractional inhibitory concentration indices (FICIs) of the combinations were 0.562, 0.531, 0.562 and 0.625, respectively. Altogether, 47.7%, 56.8%, 50.0% and 27.3% of tested fungal strains showed a synergistic antifungal activity (FICI ≤ 0.5) for the nitrile-isothiocyanate combinations, respectively. Hypocreales strains showed the least sensitivity towards the GSL decomposition products and their combinations. The mean MIC values for PEITC showed 0.0679 ± 0.0358, 0.0400 ± 0.0214, 0.0319 ± 0.0087 and 0.0178 ± 0.0171 mM for Hypocreales, Eurotiales, Glomerellales and Pleosporales, respectively. In addition, nitriles, especially IAN, also showed significant differences. For the same fungi, the median FICI values fell in the ranges of 0.61-0.67, 0.52-0.61, 0.40-0.50 and 0.48-0.67, respectively, depending on the nitrile. Our results suggest that glucosinolate-derived nitriles may enhance isothiocyanate antifungal activity and that they may play an active role in shaping the plant microbiome and contribute to the filtering of microbes by plants.

Quality-controlled LC-ESI-MS food metabolomics of fenugreek (Trigonella foenum-graecum) sprouts: Insights into changes in primary and specialized metabolites.

Fenugreek (Trigonella foenum-graecum L.) is an important food and spice with bioactive compounds against diabetes. In this study, fenugreek seeds germinating in darkness for 72 h were studied using quantification of trigonelline and 4-hydroxyisoleucine and an LC-ESI-MS/MS-based metabolomic approach capable of accurately estimating 237 features from various primary and specialized compound classes. During germination, the concentrations of trigonelline and 4-hydroxyisoleucine rose by 33.5% and 33.3%, respectively. At the same time, untargeted metabolomics revealed 9 putative flavonoids increasing 1.19- to 2.77-fold compared to the dormant seeds. A set of 19 steroid saponins rose by 1.08- to 31.86-fold. Primary metabolites however showed much more variability: abundance changes in amino acid derivatives, peptides and saccharides fell in the 0.09- to 22.25-fold, 0.93- to 478.79-fold and 0.36- to 941.58-fold ranges, respectively. To increase biosynthesis of specialized metabolites during germination, sprouts were exposed to 1-100 mM methyl jasmonate (MeJA) and methyl salicylate (MeSA). The hormone treatments affected normal metabolism: 67.1-83.1 % and 64.1-83.5 % of compounds showed a reduction compared to the controls in 100 mM MeJA and MeSA treatments at different sampling time points. Contrary to expectations, the abundance of flavonoids decreased, compared to the control sprouts (0.75- and 0.68-fold change medians, respectively). The same was observed for most, but not all steroid saponins. The quality-controlled untargeted metabolomics approach proved to yield excellent insight into the metabolic changes during germination of fenugreek. The results suggest that although fenugreek germination causes major shifts in plant metabolism, there are no major qualitative changes in bioactive specialized metabolites during the first three days. This stability likely translates into good bioactivity that is similar to that of the seeds. Because the large changes in the primary metabolites likely alter the nutritive value of the seed, further studies are warranted.

Correlations Between the Metabolome and the Endophytic Fungal Metagenome Suggests Importance of Various Metabolite Classes in Community Assembly in Horseradish (Armoracia rusticana, Brassicaceae) Roots.

The plant microbiome is an increasingly intensive research area, with significance in agriculture, general plant health, and production of bioactive natural products. Correlations between the fungal endophytic communities and plant chemistry can provide insight into these interactions, and suggest key contributors on both the chemical and fungal side. In this study, roots of various horseradish (Armoracia rusticana) accessions grown under the same conditions were sampled in two consecutive years and chemically characterized using a quality controlled, untargeted metabolomics approach by LC-ESI-MS/MS. Sinigrin, gluconasturtiin, glucoiberin, and glucobrassicin were also quantified. Thereafter, a subset of roots from eight accessions (n = 64) with considerable chemical variability was assessed for their endophytic fungal community, using an ITS2 amplicon-based metagenomic approach using a custom primer with high coverage on fungi, but no amplification of host internal transcribed spacer (ITS). A set of 335 chemical features, including putatively identified flavonoids, phospholipids, peptides, amino acid derivatives, indolic phytoalexins, a glucosinolate, and a glucosinolate downstream product was detected. Major taxa in horseradish roots belonged to Cantharellales, Glomerellales, Hypocreales, Pleosporales, Saccharomycetales, and Sordariales. Most abundant genera included typical endophytes such as Plectosphaerella, Thanatephorus, Podospora, Monosporascus, Exophiala, and Setophoma. A surprising dominance of single taxa was observed for many samples. In summary, 35.23% of reads of the plant endophytic fungal microbiome correlated with changes in the plant metabolome. While the concentration of flavonoid kaempferol glycosides positively correlated with the abundance of many fungal strains, many compounds showed negative correlations with fungi including indolic phytoalexins, a putative glucosinolate but not major glucosinolates and a glutathione isothiocyanate adduct. The latter is likely an in vivo glucosinolate decomposition product important in fungal arrest. Our results show the potency of the untargeted metabolomics approach in deciphering plant-microbe interactions and depicts a complex array of various metabolite classes in shaping the endophytic fungal community.

Formulation, Characterization and Permeability Studies of Fenugreek (Trigonella foenum-graecum) Containing Self-Emulsifying Drug Delivery System (SEDDS).

Fenugreek is used as a spice and a traditional herbal medicine for a variety of purposes, given its antidiabetic and antioxidant effects. Self-emulsifying drug delivery systems (SEDDS) of herbal drugs are targets of extensive research aiming to increase bioavailability and stability. The study's objective was to formulate SEDDS containing Trigonella foenum-graecum extract to improve the stability of herbal extract and to increase their permeability through a Caco-2 monolayer. A characterized fenugreek dry extract was used for the formulations, while the SEDDS properties were examined by particle size analysis and zeta potential measurements. Permeability assays were carried out on Caco-2 cell monolayers, the integrity of which was monitored by follow-up trans-epithelial electric resistance measurements (TEER). Cytocompatibility was tested by the MTT method, and an indirect dissolution test was performed, using DPPH antioxidant reagent. Two different SEDDS compositions were formulated from a standardized fenugreek dry extract at either the micro- or the nanoemulsion scale with sufficient stability, enhanced bioavailability of the compounds, and sustained release from HPMC capsules. Based on our results, a modern, non-toxic, cytocompatible fenugreek SEDDS formulation with high antioxidant capacity was developed in order to improve the permeability and bioavailability of all components.

Interactions of fungi with non-isothiocyanate products of the plant glucosinolate pathway: A review on product formation, antifungal activity, mode of action and biotransformation.

The glucosinolate pathway, which is present in the order Brassicales, is one of the most researched defensive natural product biosynthesis pathways. Its core molecules, the glucosinolates are broken down upon pathogen challenge or tissue damage to yield an array of natural products that may help plants defend against the stressor. Though the most widely known glucosinolate decomposition products are the antimicrobial isothiocyanates, there is a wide range of other volatile and non-volatile natural products that arise from this biosynthetic pathway. This review summarizes our current knowledge on the interaction of these much less examined, non-isothiocyanate products with fungi. It deals with compounds including (1) glucosinolates and their biosynthesis precursors; (2) glucosinolate-derived nitriles (e.g. derivatives of 1H-indole-3-acetonitrile), thiocyanates, epithionitriles and oxazolidine-2-thiones; (3) putative isothiocyanate downstream products such as raphanusamic acid, 1H-indole-3-methanol (= indole-3-carbinol) and its oligomers, 1H-indol-3-ylmethanamine and ascorbigen; (4) 1H-indole-3-acetonitrile downstream products such as 1H-indole-3-carbaldehyde (indole-3-carboxaldehyde), 1H-indole-3-carboxylic acid and their derivatives; and (5) indole phytoalexins including brassinin, cyclobrassinin and brassilexin. Herein, a literature review on the following aspects is provided: their direct antifungal activity and the proposed mechanisms of antifungal action, increased biosynthesis after fungal challenge, as well as data on their biotransformation/detoxification by fungi, including but not limited to fungal myrosinase activity.

Horseradish Essential Oil as a Promising Anti-Algal Product for Prevention of Phytoplankton Proliferation and Biofouling.

Increased proliferation of algae is a current problem in natural and artificial water bodies. Controlling nutrients is the most sustainable treatment of increased algal proliferation, however in certain cases, it is not sufficiently available, or it does not provide results fast enough. Chemicals derived from natural sources, which could be effective in low concentrations and are biodegradable, may have an advantage over conventional chemical treatments. The main aim of the present study was to investigate the anti-cyanobacterial and anti-algal properties of allyl-isothiocyanate-containing essential oil produced from horseradish roots with a complex approach of the topic: on laboratory strains of cyanobacteria and eukaryotic algae, on microcosms containing natural phytoplankton assemblages, and on semi-natural biofilms. The results show that acute treatment can significantly reduce the viability of all the tested cyanobacteria and eukaryotic algae. Results of microcosm experiments with natural phytoplankton assemblages show that horseradish essential oil from 7.1 × 10-6% (v/v) is applicable to push back phytoplankton proliferation even in natural assemblages. The individual number in the biofilm was dropped down to one-fifth of the original individual number, so 7.1 × 10-6% (v/v) and higher concentration of the essential oil can be considered as a successful treatment against biofouling.

Effects of Glucosinolate-Derived Isothiocyanates on Fungi: A Comprehensive Review on Direct Effects, Mechanisms, Structure-Activity Relationship Data and Possible Agricultural Applications.

Plants heavily rely on chemical defense systems against a variety of stressors. The glucosinolates in the Brassicaceae and some allies are the core molecules of one of the most researched such pathways. These natural products are enzymatically converted into isothiocyanates (ITCs) and occasionally other defensive volatile organic constituents (VOCs) upon fungal challenge or tissue disruption to protect the host against the stressor. The current review provides a comprehensive insight on the effects of the isothiocyanates on fungi, including, but not limited to mycorrhizal fungi and pathogens of Brassicaceae. In the review, our current knowledge on the following topics are summarized: direct antifungal activity and the proposed mechanisms of antifungal action, QSAR (quantitative structure-activity relationships), synergistic activity of ITCs with other agents, effects of ITCs on soil microbial composition and allelopathic activity. A detailed insight into the possible applications is also provided: the literature of biofumigation studies, inhibition of post-harvest pathogenesis and protection of various products including grains and fruits is also reviewed herein.

Formulation of Novel Liquid Crystal (LC) Formulations with Skin-Permeation-Enhancing Abilities of Plantago lanceolata (PL) Extract and Their Assessment on HaCaT Cells.

Exposure to reactive oxygen species can easily result in serious diseases, such as hyperproliferative skin disorders or skin cancer. Herbal extracts are widely used as antioxidant sources in different compositions. The importance of antioxidant therapy in inflammatory conditions has increased. Innovative formulations can be used to improve the effects of these phytopharmacons. The bioactive compounds of Plantago lanceolata (PL) possess different effects, such as anti-inflammatory, antioxidant, and bactericidal pharmacological effects. The objective of this study was to formulate novel liquid crystal (LC) compositions to protect Plantago lanceolata extract from hydrolysis and to improve its effect. Since safety is an important aspect of pharmaceutical formulations, the biological properties of applied excipients and blends were evaluated using assorted in vitro methods on HaCaT cells. According to the antecedent toxicity screening evaluation, three surfactants were selected (Gelucire 44/14, Labrasol, and Lauroglycol 90) for the formulation. The dissolution rate of PL from the PL-LC systems was evaluated using a Franz diffusion chamber apparatus. The antioxidant properties of the PL-LC systems were evaluated with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and malondialdehyde (MDA) assessments. Our results suggest that these compositions use a nontraditional, rapid-permeation pathway for the delivery of drugs, as the applied penetration enhancers reversibly alter the barrier properties of the outer stratum corneum. These excipients can be safe and highly tolerable thus, they could improve the patient's experience and promote adherence.

Volatile Organic Compounds (VOCs) of Endophytic Fungi Growing on Extracts of the Host, Horseradish (Armoracia rusticana).

The interaction between plant defensive metabolites and different plant-associated fungal species is of high interest to many disciplines. Volatile organic compounds (VOCs) are natural products that are easily evaporated under ambient conditions. They play a very important role in inter-species communication of microbes and their hosts. In this study, the VOCs produced by 43 different fungal isolates of endophytic and soil fungi during growth on horseradish root (Armoracia rusticana) extract or malt extract agar were examined, by using headspace-gas chromatography-mass spectrometry (headspace-GC-MS) and a high relative surface agar film as a medium. The proposed technique enabled sensitive detection of several typical VOCs (acetone, methyl acetate, methyl formate, ethyl acetate, methyl butanol isomers, styrene, beta-phellandrene), along with glucosinolate decomposition products, including allyl cyanide and allyl isothiocyanate and other sulfur-containing compounds-carbon disulfide, dimethyl sulfide. The VOC patterns of fungi belonging to Setophoma, Paraphoma, Plectosphaerella, Pyrenochaeta, Volutella, Cadophora, Notophoma, and Curvularia genera were described for the first time. The VOC pattern was significantly different among the isolates. The pattern was indicative of putative myrosinase activity for many tested isolates. On the other hand, endophytes and soil fungi as groups could not be separated by VOC pattern or intensity.

Special Issue: Plant Metabolomics.

The Special Issue is designed to provide an insight on the variability of the possible applications of the metabolomic approach within the field of plant sciences. The possibility to relatively quantify and putatively annotate hundreds to thousands of compounds has enabled scientists to obtain data on plant functions in previously unimaginable detail. Though the field is still evolving at a rapid pace, it is now clear that metabolomics has already become an efficient tool in the toolkit of colleagues studying secondary metabolism, plant physiology, plant-microbe interactions and several other fields, opening up new possibilities in agricultural, phytochemical and other sciences. The current Special Issue presents papers using various measurement techniques, plant genera and metabolite classes.

The cyanobacterial oligopeptides microginins induce DNA damage in the human hepatocellular carcinoma (HepG2) cell line.

Microginins (MGs) are bioactive metabolites mainly produced by Microcystis spp., (Cyanobacteria) commonly found in eutrophic environments. In this study, the cytotoxic and genotoxic activities of four MG congeners (MG FR3, MG GH787, cyanostatin B, MGL 402) and a well characterized cyanobacterial extract B-14-01 containing these metabolites were evaluated in the human hepatocellular carcinoma (HepG2) cell line. The cytotoxicity was measured with the MTT assay, while genotoxicity was studied with the comet, γH2AX and cytokinesis block (CBMN) micronucleus assays. The viability of cells after 24 h was significantly affected only by the extract, whereas after 72 h a concentration dependent decrease in cell proliferation was observed for the extract and tested microginins, with MGL 402 being the most potent and MG FR3 the least potent congener. The extract and all tested congeners induced DNA strand breaks after 4 and 24 h exposure. The most potent was the extract, which induced concentration and time dependent increase in DNA damage at concentrations ≥0.01 µg mL-1. Among microginins the most potent was MGL 402 (increase in DNA strand breaks at ≥ 0.01 µg mL-1) and MG FR3 was the least potent (increase in DNA strand breaks at ≥ 1 µg mL-1). However, no induction of DNA double strand breaks was observed after 24 and 72-h exposure to the cyanobacterial extract or MGs. Induction of genomic instability was observed in cells exposed to MG GH787, cyanostatin B and the extract B-14-01. This study is the first to provide the evidence that microginins exert genotoxic activity.

Variability of Bioactive Glucosinolates, Isothiocyanates and Enzyme Patterns in Horseradish Hairy Root Cultures Initiated from Different Organs.

Horseradish hairy root cultures are suitable plant tissue organs to study the glucosinolate-myrosinase-isothiocyanate system and also to produce the biologically active isothiocyanates and horseradish peroxidase, widely used in molecular biology. Fifty hairy root clones were isolated after Agrobacterium rhizogenes infection of surface sterilized Armoracia rusticana petioles and leaf blades, from which 21 were viable after antibiotic treatment. Biomass properties (e.g. dry weight %, daily growth index), glucosinolate content (analyzed by liquid chromatography-electronspray ionization-mass spectrometry (LC-ESI-MS/MS)), isothiocyanate and nitrile content (analyzed by gas chromatography-mass spectrometry (GC-MS)), myrosinase (on-gel detection) and horseradish peroxidase enzyme patterns (on-gel detection and spectrophotometry), and morphological features were examined with multi-variable statistical analysis. In addition to the several positive and negative correlations, the most outstanding phenomenon was many parameters of the hairy root clones showed dependence on the organ of origin. Among others, the daily growth index, sinigrin, glucobrassicin, 3-phenylpropionitrile, indole-3-acetonitrile and horseradish peroxidase values showed significantly higher levels in horseradish hairy root cultures initiated from leaf blades.

Comparative metabolomics of Tilia platyphyllos Scop. bracts during phenological development.

The medicinal plant drug "Tiliae flos" consists of the botanical flowers and bracts of Tilia sp., gathered almost exclusively during flowering. In this study, we examined the changes in the metabolome of specialized products in the bracts of Tilia platyphyllos from the appearance of the organ till the onset of senescence by LC-ESI-MS and data mining. A set of 504 natural products were detected, 241 of which showed significant seasonal variation (p < 9.92E-5). Seven compounds were quantified and an additional 45 were putatively identified. These included flavonoid glycosides, catechins, procyanidins, quinic acid derivatives (including chlorogenic acid) and coumarins. Compared to bracts during flowering, young tissues were characterized by a relatively high diversity of polyphenolic substances. Higher amounts of flavonol glycosides (quercetin, kaempferol), catechins and derivatives have been observed. Deoxyhexosides were almost exclusive to this phenological stage. Changes of about one order of magnitude were not uncommon. For some substances, 5-fold differences were observed (calibration with authentic standards). Some compounds (e.g. the coumarin fraxin) were more prominent at the late fruit growth stage. It was shown that bracts gathered before or after flowering could potentially be therapeutically useful. Changes are rapid during the early phase of bract development: three different groups of compounds presented their maxima during the first 32 days. Considering seasonal variation is of extreme importance during bioactivity tests and screening candidate sources for bioactive natural products. In the case of T. platyphyllos, young and old bracts can be of interest because of their high diversity of distinct specialized metabolites.

Microcystis Chemotype Diversity in the Alimentary Tract of Bigheaded Carp.

Most cyanobacterial organisms included in the genus Microcystis can produce a wide repertoire of secondary metabolites. In the mid-2010s, summer cyanobacterial blooms of Microcystis sp. occurred regularly in Lake Balaton. During this period, we investigated how the alimentary tract of filter-feeding bigheaded carps could deliver different chemotypes of viable cyanobacteria with specific peptide patterns. Twenty-five Microcystis strains were isolated from pelagic plankton samples (14 samples) and the hindguts of bigheaded carp (11 samples), and three bloom samples were collected from the scums of cyanobacterial blooms. An LC-MS/MS-based untargeted approach was used to analyze peptide patterns, which identified 36 anabaenopeptin, 17 microginin, and 13 microcystin variants. Heat map clustering visualization was used to compare the identified chemotypes. A lack of separation was observed in peptide patterns of Microcystis that originated from hindguts, water samples, and bloom-samples. Except for 13 peptides, all other congeners were detected from the viable and cultivated chemotypes of bigheaded carp. This finding suggests that the alimentary tract of bigheaded carps is not simply an extreme habitat, but may also supply the cyanobacterial strains that represent the pelagic chemotypes.

A Simple Method for On-Gel Detection of Myrosinase Activity.

Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4- is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031⁻0.25 U (pure Sinapis alba myrosinase, R² = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification.

Endophytic fungi from the roots of horseradish (Armoracia rusticana) and their interactions with the defensive metabolites of the glucosinolate - myrosinase - isothiocyanate system.

BACKGROUND: The health of plants is heavily influenced by the intensively researched plant microbiome. The microbiome has to cope with the plant's defensive secondary metabolites to survive and develop, but studies that describe this interaction are rare. In the current study, we describe interactions of endophytic fungi with a widely researched chemical defense system, the glucosinolate - myrosinase - isothiocyanate system. The antifungal isothiocyanates are also of special interest because of their beneficial effects on human consumers. RESULTS: Seven endophytic fungi were isolated from horseradish roots (Armoracia rusticana), from the genera Fusarium, Macrophomina, Setophoma, Paraphoma and Oidiodendron. LC-ESI-MS analysis of the horseradish extract incubated with these fungi showed that six of seven strains could decompose different classes of glucosinolates. Aliphatic, aromatic, thiomethylalkyl and indolic glucosinolates were decomposed by different strains at different rates. SPME-GC-MS measurements showed that two strains released significant amounts of allyl isothiocyanate into the surrounding air, but allyl nitrile was not detected. The LC-ESI-MS analysis of many strains' media showed the presence of allyl isothiocyanate - glutathione conjugate during the decomposition of sinigrin. Four endophytic strains also accepted sinigrin as the sole carbon source. Isothiocyanates inhibited the growth of fungi at various concentrations, phenylethyl isothiocyanate was more potent than allyl isothiocyanate (mean IC50 was 2.30-fold lower). As a control group, ten soil fungi from the same soil were used. They decomposed glucosinolates with lower overall efficiency: six of ten strains had insignificant or weak activities and only three could use sinigrin as a carbon source. The soil fungi also showed lower AITC tolerance in the growth inhibition assay: the median IC50 values were 0.1925 mM for endophytes and 0.0899 mM for soil fungi. CONCLUSIONS: The host's glucosinolates can be used by the tested endophytic fungi as nutrients or to gain competitive advantage over less tolerant species. These activities were much less apparent among the soil fungi. This suggests that the endophytes show adaptation to the host plant's secondary metabolites and that host metabolite specific activities are enriched in the root microbiome. The results present background mechanisms enabling an understanding of how plants shape their microbiome.

Allyl-Isothiocyanate and Microcystin-LR Reveal the Protein Phosphatase Mediated Regulation of Metaphase-Anaphase Transition in Vicia faba.

Horseradish allyl isothiocyanate (AITC, a volatile oil) and cyanobacterial microcystin-LR (MCY-LR, a cyclic heptapeptide) affect eukaryotic cell cycle. MCY-LR inhibits protein phosphatases PP1 and PP2A. We aimed to reveal the mechanisms of their cellular effects in a model eukaryote, Vicia faba. We have shown for the first time that AITC had minor effects on PP1 and PP2A activities in vitro, but it inhibited significantly PP1 in vivo. The combination of 10 µM AITC with 10 µM MCY-LR induced metaphase arrest after short-term (12 h) treatments. 10 µM AITC, 0.2-10 µM MCY-LR and their combinations induced histone H3 hyperphosphorylation, associated with the regulation of metaphase-anaphase transition. This hyperphosphorylation event occurred at any treatment which led to the inhibition of PP1 activity. 10 µM AITC + 10 µM MCY-LR increased the frequency of metaphase spindle anomalies, associated with metaphase arrest. We provide new insights into the mechanisms of metaphase-anaphase transition. Metaphase arrest is induced at the concomitant hyperphosphorylation of histone H3, alteration of metaphase spindle assembly and strong inhibition of PP1 + PP2A activity. Near-complete blocking of metaphase-anaphase transition by rapid protein phosphatase inhibition is shown here for the first time in plants, confirming a crucial role of serine-threonine phosphatases in this checkpoint of cell cycle regulation. Tissue-dependent differences in PP1 and PP2A activities induced by AITC and MCY-LR suggest that mainly regulatory subunits are affected. AITC is a potential tool for the study of protein phosphatase function and regulation. We raise the possibility that one of the biochemical events occurring during AITC release upon wounding is the modulation of protein phosphatase dependent signal transduction pathways during the plant defense response.

Self-Nanoemulsifying Drug Delivery Systems Containing Plantago lanceolata-An Assessment of Their Antioxidant and Antiinflammatory Effects.

The most important components of Plantago lanceolata L. leaves are catalpol, aucubin, and acteoside (=verbascoside). These bioactive compounds possess different pharmacological effects: anti-inflammatory, antioxidant, antineoplastic, and hepatoprotective. The aim of this study was to protect Plantago lanceolata extract from hydrolysis and to improve its antioxidant effect using self-nano-emulsifying drug delivery systems (SNEDDS). Eight SNEDDS compositions were prepared, and their physical properties, in vitro cytotoxicity, and in vivo AST/ALT values were investigated. MTT cell viability assay was performed on Caco-2 cells. The well-diluted samples (200 to 1000-fold dilutions) proved to be non-cytotoxic. The acute administration of PL-SNEDDS compositions resulted in minor changes in hepatic markers (AST, ALT), except for compositions 4 and 8 due to their high Transcutol contents (80%). The non-toxic compositions showed a significant increase in free radical scavenger activity measured by the DPPH test compared to the blank SNEDDS. An indirect dissolution test was performed, based on the result of the DPPH antioxidant assay; the dissolution profiles of Plantago lancolata extract were statistically different from each SNEDDS. The anti-inflammatory effect of PL-SNEDDS compositions was confirmed by the ear inflammation test. For the complete examination period, all compositions decreased ear edema as compared to the positive (untreated) control. It can be concluded that PL-SNEDDS compositions could be used to deliver active natural compounds in a stable, efficient, and safe manner.

The Ratio of 2-AG to Its Isomer 1-AG as an Intrinsic Fine Tuning Mechanism of CB1 Receptor Activation.

Endocannabinoids are pleiotropic lipid messengers that play pro-homeostatic role in cellular physiology by strongly influencing intracellular Ca2+ concentration through the activation of cannabinoid receptors. One of the best-known endocannabinoid '2-AG' is chemically unstable in aqueous solutions, thus its molecular rearrangement, resulting in the formation of 1-AG, may influence 2-AG-mediated signaling depending on the relative concentration and potency of the two isomers. To predict whether this molecular rearrangement may be relevant in physiological processes and in experiments with 2-AG, here we studied if isomerization of 2-AG has an impact on 2-AG-induced, CB1-mediated Ca2+ signaling in vitro. We found that the isomerization-dependent drop in effective 2-AG concentration caused only a weak diminution of Ca2+ signaling in CB1 transfected COS7 cells. We also found that 1-AG induces Ca2+ transients through the activation of CB1, but its working concentration is threefold higher than that of 2-AG. Decreasing the concentration of 2-AG in parallel to the prevention of 1-AG formation by rapid preparation of 2-AG solutions, caused a significant diminution of Ca2+ signals. However, various mixtures of the two isomers in a fix total concentration - mimicking the process of isomerization over time - attenuated the drop in 2-AG potency, resulting in a minor decrease in CB1 mediated Ca2+ transients. Our results indicate that release of 2-AG into aqueous medium is accompanied by its isomerization, resulting in a drop of 2-AG concentration and simultaneous formation of the similarly bioactive isomer 1-AG. Thus, the relative concentration of the two isomers with different potency and efficacy may influence CB1 activation and the consequent biological responses. In addition, our results suggest that 1-AG may play role in stabilizing the strength of cannabinoid signal in case of prolonged 2-AG dependent cannabinoid mechanisms.