Separation of bufadienolides from Helleborus thibetanus Franch. by a combination approach involving macroporous resin column chromatography and gradient countercurrent chromatography.

In this study, a combination approach involving macroporous resin (MR) column chromatography and gradient countercurrent chromatography (CCC) was employed to enrich and purify bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch. Initially, a D101 MR-packed column chromatography was utilized for fractionation and enrichment of the bufadienolides, which were effectively eluted from the column using a 60% ethanol solution. CCC was subsequently introduced to separate the enriched product using the ethyl acetate/n-butanol/water (EBuWat, 4:1:5, v/v) and EBuWat (5:0:5, v/v) solvent systems in a gradient elution mode. As results, five bufadienolides, including 6.1 mg of hellebrigenin-3-O-ß-D-glucoside (1), 2.2 mg of tigencaoside A (2), 8.3 mg of deglucohellebrin (3), 3.5 mg of 14 ß-hydroxy-3ß-[ß-D-glucopyranosyl-(1→6)-(ß-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (4), and 3.0 mg of 14ß-hydroxy-3ß-[(ß-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (5), were effectively separated from 300 mg of the enriched product. The respective high-performance liquid chromatography purities were as follows: 95.2%, 75.8%, 85.7%, 82.3%, and 92.8%. This study provides valuable insights for the efficient enrichment and separation of bufadienolides from Helleborus thibetanus Franch.

[Chemical constituents of Helleborus thibetanus].

The chemical constituents of Helleborus thibetanus were isolated and purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative RP-HPLC, and the structures of all compounds were identified by modern spectrographic technology(MS, NMR). The MTT method was used to measure the cytotoxicity of compounds 1-8. Twelve compounds were isolated from the roots and rhizomes of H. thibetanus and were identified as(25R)-22ß,25-expoxy-26-[(O-ß-D-glucopyranosyl)oxy]-1ß,3ß-dihydroxyfurosta-5-en(1), ß-sitosterol myristate(2), ß-sitosterol lactate(3), ß-sitosterol 3-O-ß-D-glucopyrannoside(4), 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one(5), 1,3,5-trimethoxybenzene(6), 7,8-dimethylbenzo pteridine-2,4(1H,3H)-dione(7), 1H-indole-3-carboxylic acid(8), p-hydroxy cinnamic acid(9), lauric acid(10), n-butyl α-L-arabinofuranoside(11) and methyl-α-D-fructofuranoside(12), respectively. Among them, compound 1 is a new compound and named thibetanoside L; compounds 2, 5-8, 11 are first isolated from the family Ranunculaceae; compound 12 is isolated from the genus Helleborus for the first time. The results of MTT assay showed that the IC_(50) values of compounds 1-8 against HepG2 and HCT116 cells were greater than 100 µmol·L~(-1).

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene, homooxacalix[3]arene and homoazacalix[3]arene as extractant agents for amino acids from the aqueous extract.

The task of this work was to investigate the extraction capacity of various calixarenes for free and esterified amino acids from aqueous acid phases. Furthermore, this method was applied to aqueous extracts of Helleborus purpurascens. Generally, it is known that calixarenes can be used as extractants for ammonium compounds due to π-cation and lone pair cation interactions. As first, tert-Butyl-calix[6]arene and derivatives thereof were used. They had already proven their worth in previous investigations. In addition, tert-Butyl-hexahomooxa-calix[3]arene was used also, which can also enter into lone pair cation interactions. In addition to these well-known calixarenes, new calixarenes were produced and tested. Based on the tert-Butyl-hexahomooxa-calix[3]arene, a phosphor(III)bridged derivative was prepared, combining the three aromatic hydroxyl groups to a phosphite. As a seldom-described class of calixarenes, tert-Butyl-hexahomoaza-calix[3]arene derivatives were used. The nitrogen analogues of tert-Butyl-hexahomooxa-calix[3]arene could be produced as N-benzyl derivatives. The structure of the esterified carboxymethylated derivative of N,N',N″-Tribenzyl-tert-Butyl-hexahomoaza-calix[3]arene could be verified by X-ray structure analysis. It crystallized as a partial cone. The extraction capacity of the described calixarenes was investigated for amino acids from aqueous acidic solutions into an organic phase. For the testing were chosen asparagine, aspartic acid, tyrosine, tryptophane, phenylalanine and pipecolinic acid and their methyl esters. The amino acids and their methyl esters were dissolved in water at different pH values. The calixarenes were dissolved in dichloromethane (DCM) or chloroform. After this preparation, the aqueous acidic amino acid solutions were mixed with the solutions and shaken intensively. In addition, blank values were determined by extracting the aqueous stock solutions of the amino acids and their methyl esters with pure solvents. To determine the extraction rate, the phases were separated and each analysed using GC-FID, partially GC-MS(EI). The evaluation is performed in two ways. On the one hand the depletion in the aqueous phase and on the other hand the content in the organic phase was determined.

Novel Steroidal Glycosides from the Whole Plants of Helleborus foetidus.

Phytochemical analysis of the whole Helleborus foetidus plants identified 28 steroidal glycosides (1-28), including 20 novel spirostanol glycosides (1-20) and a novel furostanol glycoside (21). The structures of the newly identified compounds were elucidated by two-dimensional NMR spectroscopy and hydrolytic cleavage. Compounds 12, 13, and 15 were determined to be spirostanol trisdesmosides bearing sugar moieties at the C-1, -21, and -24 hydroxy groups of the aglycone unit. The isolated compounds were subsequently evaluated for cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung carcinoma cells. In particular, 7 showed cytotoxic activity against the HL-60 and A549 cells, with IC50 values of 5.9 and 6.6 µM, respectively, whereas 19 was selectively cytotoxic to A549 cells with an IC50 value of 5.5 µM.

[Chemical-toxicological diagnosis of hellebore (veratrum) poisoning]. / Khimiko-toksikologicheskaya diagnostika otravlenii chemeritsei.

The problem of laboratory diagnosis of acute and fatal poisoning by hellebore, which is possible when used in traditional medicine, the erroneous use of hellebore preparations orally or use of various types of this plant for food, remains relevant. Currently, in the practice of chemical-toxicological laboratories and the bureau of forensic medical examination there is no single approach to the laboratory diagnosis of such poisoning. The diagnosis is most often based on anamnesis. In this regard, the development and validation of a legally significant methodology for the determination of hellebore alkaloids in various biological objects seems relevant. The physicochemical and toxic properties of alkaloids of various types of hellebore are characterized. It was shown that for the identification of hellebore alkaloids, it is advisable to use HPLC-MS/MS as the most sensitive and specific instrumental method corresponding to the characteristics of hellebore alkaloids (high molecular weight, high thermal lability, high polarity).

Cardiac Glycoside Compound Isolated from Helleborus thibetanus Franch Displays Potent Toxicity against HeLa Cervical Carcinoma Cells through ROS-Independent Autophagy.

The current study aimed to examine the anticancer activity of HTF-1, a cardiac glycoside (CG) isolated from Helleborus thibetanus Franch, using a cell-based model and to discover the underlying mechanisms with specific focus on autophagy. We found that HTF-1 was able to potently decrease the viability of several cancer cell lines especially for HeLa cervical carcinoma cells. It was discovered that HTF-1 dose dependently induced overproduction of ROS in HeLa cells, and the cell viability can be rescued when adding ROS scavenger N-acetyl-l-cysteine (NAC). More, we found that HTF-1 induced ROS-independent autophagy in concentration- and time-dependent manners in HeLa cells. This can be collectively verified by LC3-II and p62 abundance and also eGFP-LC3 puncta assay, bafilomycin clamp experiment, and acidotropic dye fluorescent labeling experiment. Additionally, TEM examination showed more autophagic vacuoles for HTF-1-treated HeLa cells. In HeLa cells, pretreatment with wortmannin (an inhibitor of the initial stages of autophagy to block autophagosome formation, thus, it should weaken the autophagy induction effect of HTF-1) decreased the autophagic flux and partially antagonized cell death induced by HTF-1, indicating that autophagy induced by HTF-1 played a cancer-suppressing role. Furthermore, coadministration of BAF (as a distal inhibitor of autophagy) with HTF-1 demonstrated a synergistic anticancer effect against HeLa cells. We believe that our work will enrich the understanding of CGs and especially anticarcinoma activity, also, pave the way for natural-product-based anticancer drug development.

Within-plant heterogeneity in fecundity and herbivory induced by localized DNA hypomethylation in the perennial herb Helleborus foetidus.

PREMISE: Phenotypic heterogeneity of reiterated, homologous structures produced by individual plants has ecological consequences for plants and their animal consumers. This paper examines experimentally the epigenetic mosaicism hypothesis, which postulates that within-plant variation in traits of reiterated structures may partly arise from different parts of the same genetic individual differing in patterns or extent of genomic DNA methylation. METHODS: Leaves of paired ramets borne by field-growing Helleborus foetidus plants were infiltrated periodically over the entire flowering period with either a water solution of the demethylating agent zebularine or just water as the control. The effects of the zebularine treatment were assessed by quantifying genome-wide DNA cytosine methylation in leaves and monitoring inflorescence growth and flower production, number of ovules per flower, pollination success, fruit set, seed set, seed size, and distribution of sap-feeding insects. RESULTS: Genomic DNA from leaves in zebularine-treated ramets was significantly less methylated than DNA from leaves in control ones. Inflorescences in treated ramets grew smaller and produced fewer flowers, with fewer ovules and lower follicle and seed set, but did not differ from inflorescences in untreated ramets in pollination success or seed size. The zebularine treatment influenced the within-plant distribution of sap-feeding insects. CONCLUSIONS: Experimental manipulation of genomic DNA methylation level in leaves of wild-growing H. foetidus plants induced considerable within-plant heterogeneity in phenotypic (inflorescences, flowers, fecundity) and ecologically relevant traits (herbivore distribution), which supports the hypothesis that epigenetic mosaicism may partly account for within-plant variation.

Preclinical evaluation of safety and potential of black hellebore extracts for cancer treatment.

BACKGROUND: The therapeutic use of Helleborus niger L. is manifold due to its specific phytochemical composition. Two compound groups, the ranunculin derivates including protoanemonin and the steroidal saponins, are also associated with toxicity (genotoxicity, disintegration of membrane structures). Therefore, in vitro investigations were performed on safety aspects of a Helleborus niger aqueous fermented extract (HNE). In addition its therapeutic potential against various cancer cell lines was assessed to gain insight into the respective mechanisms of action. METHODS: To evaluate the safe use of HNE, Ames and hemolytic tests were carried out. Two angiogenesis assays in 2D and 3D design were conducted to assess the anti-angiogenetic potential, for which human umbilical vein endothelial cells (HUVEC) were chosen. A panel of tumor cell lines was used in 2D and 3D proliferation assays as well in the migration- and invasion-assay. All investigations were performed with HNE compared to reference substances. The 2D proliferation assay was additionally performed with isolated compounds of HNE (characteristic steroidal saponins). RESULTS: HNE did not exhibit any genotoxic potential. Concentrations up to 10 µl/ml were classified as non-hemolytic. HNE exerted anti-angiogenetic effects in HUVEC and anti-proliferative effects in five cancer cell lines, whereas hellebosaponin A and D as well macranthosid I did not show comparable effects neither singly nor in combination. Due to the inherent instability of protoanemonin in isolated form, parallel investigations with protoanemonin could not be performed. HNE (600-1000 µg/ml) inhibited the migration of certain cancer cells by > 80% such as Caki-2, DLD-1 and SK-N-SH. CONCLUSION: HNE exhibit neither genotoxic nor hemolytic potential. The present investigations verify the anti-angiogenetic effects on HUVEC, the anti-proliferative effects and migration-inhibiting properties on tumor cells. The lower effect of the relevant steroidal saponins compared to the whole extract underlines the fact that the latter is more effective than a blend of isolated pharmacologically active components.

Studies on the constituents of Helleborus purpurascens: analysis and biological activity of the aqueous and organic extracts.

In Southeast Europe, the ethnomedicinal use of Helleborus species has a very long tradition. Cardiac steroids (Hellebrin), cysteine-rich proteins (Hellethionins) and several steroidal saponins have been identified in these plants. Aim of the present work was to investigate the amino acid composition of native extracts from the root and rootstock of Helleborus purpurascens. The amino acids have been identified by the GC-MS technique on the previously derivatised (Phenomenex Faast Kit) extract samples by comparison with the mass spectra and retention-time of the standards. A remarkable finding was a relatively intensive peak attributed to the non-proteinogenic Pipecolic acid (Pic). A cyclisation of the derivatised glutamine was observed during the GC measurement and a mechanistic pathway is described. Samples of the extract and of some isolated fractions have also been tested on; altogether 12 cancer cell lines aimed to identify further potentially cytostatic components which should be less toxic than Hellebrin. The finding of one Hellebrin-free fraction (IC50 = 0.007 mg/L) with higher cytotoxicity than Hellebrin (IC50 = 0.008 mg/L) is remarkable.

Comparative spatial genetics and epigenetics of plant populations: heuristic value and a proof of concept.

Despite the recent upsurge of interest on natural epigenetic variation of nonmodel organisms, factors conditioning the spatial structure of epigenetic diversity in wild plant populations remain virtually unexplored. We propose that information on processes shaping natural epigenetic variation can be gained using the spatial structure of genetic diversity as null model. Departures of epigenetic isolation-by-distance (IBD) patterns from genetic IBD patterns for the same sample, particularly differences in slope of similarity-distance regressions, will reflect the action of factors that operate specifically on epigenetic variation, including imperfect transgenerational inheritance and responsiveness to environmental factors of epigenetic marks. As a proof of concept, we provide a comparative analysis of spatial genetic and epigenetic structure of 200 mapped individuals of the perennial herb Helleborus foetidus. Plants were fingerprinted using nuclear microsatellites, amplified fragment length polymorphisms (AFLP) and methylation-sensitive AFLP markers. Expectations from individual-level IBD patterns were tested by means of kinship-distance regressions. Both genetic and epigenetic similarity between H. foetidus individuals conformed to theoretical expectations under individual-level IBD models. Irrespective of marker type, there were significant negative linear relationships between the kinship coefficient for plant pairs and their spatial separation. Regression slopes were significantly steeper for epigenetic markers. Epigenetic similarity between individuals was much greater than genetic similarity at shortest distances, such epigenetic 'kinship excess' tending to decrease as plant separation increased. Results suggest that moderate-to-high heritability and responsiveness to local environments are major drivers of epigenetic spatial structure in H. foetidus, and illustrate the heuristic value of comparing genetic and epigenetic spatial structure for formulating and testing hypotheses on forces shaping epigenetic diversity in wild plant populations.

Continuous within-plant variation as a source of intraspecific functional diversity: Patterns, magnitude, and genetic correlates of leaf variability in Helleborus foetidus (Ranunculaceae).

PREMISE OF THE STUDY: Continuous within-plant variation in quantitative traits of reiterated, homologous structures is a component of intraspecific variation, but its contribution to functional diversity remains largely unexplored. For the perennial Helleborus foetidus, we measured functional leaf traits to quantify the contribution of within-plant variation to intraspecific functional variance and evaluate whether within-plant variability itself deserves separate consideration. METHODS: Within-individual variation in eight leaf traits was quantified for 138 plants sampled from 10 widely spaced locations in the Sierra de Cazorla, southeastern Spain. An amplified fragment length polymorphism (AFLP) technique was used to look for associations between within-plant variability and specific AFLP markers. KEY RESULTS: Leaflets from basal positions in ramets were longer, heavier, had greater surface area and larger stomata, and lower specific area, stomatal index, and stomatal density than those from distal positions. Continuous variation between leaves from the same ramet was the main source of population-wide variance for most traits. Within-plant variability differed among populations. Individuals differed in within-plant variability, which was largely independent of trait means and associated with genetic characteristics. Up to four AFLP markers were associated with the within-plant variability level of a given leaf trait. CONCLUSIONS: Subindividual variability in continuous leaf traits was independent of plant means and related to genetic features. The within-individual component generally exceeded the between-individual component of intraspecific variance. Within-plant variation may broaden the ecological breadth and enhance stability and persistence of plant populations and communities and may provide novel insights when incorporated in trait-based community ecology models.

Facilitation promotes changes in leaf economics traits of a perennial forb.

Optimal allocation of resources is crucial to maximize plant success. Plants modify their economic strategies by adjusting functional traits in response to shifts in environmental conditions. Facilitation has been recognized as a major biotic filter of trait distribution in communities, although the effect of facilitation on intraspecific variability has been scarcely explored. We evaluated intraspecific shifts in leaf functional traits of a perennial forb (Helleborus foetidus) in relation to the presence of a nurse plant (Juniperus sabina) in two sites with contrasting abiotic stress levels. The effects of abiotic (site) and biotic (juniper presence, microsite) environments on specific leaf area (SLA), leaf area (LA), lamina/petiole length ratio (LPR), intrinsic water use efficiency (iWUE) and leaf nutrient content (N, P and N:P) per mass were evaluated. Alleviation of drought stress associated with nurse plant presence was reflected in SLA, LA, LPR and iWUE at the high-stress site. Individuals growing in open areas showed more resource-conservative traits, supporting the argument that this strategy is advantageous in environments that limit opportunities for rapid carbon gain. Leaf nutrients were unrelated to other traits. The large amount of intraspecific variation in leaf functional traits related to facilitative processes highlights the importance of facilitation as a major source of plant trait variation. Both positive and negative biotic interactions, as well as intraspecific trait variability, should be considered in mechanistic models of plant communities' functional responses to environmental changes.

Helleborus purpurascens-Amino Acid and Peptide Analysis Linked to the Chemical and Antiproliferative Properties of the Extracted Compounds.

There is a strong drive worldwide to discover and exploit the therapeutic potential of a large variety of plants. In this work, an alcoholic extract of Helleborus purpurascens (family Ranunculaceae) was investigated for the identification of amino acids and peptides with putative antiproliferative effects. In our work, a separation strategy was developed using solvents of different polarity in order to obtain active compounds. Biochemical components were characterized through spectroscopic (mass spectroscopy) and chromatographic techniques (RP-HPLC and GC-MS). The biological activity of the obtained fractions was investigated in terms of their antiproliferative effects on HeLa cells. Through this study, we report an efficient separation of bioactive compounds (amino acids and peptides) from a plant extract dependent on solvent polarity, affording fractions with unaffected antiproliferative activities. Moreover, the two biologically tested fractions exerted a major antiproliferative effect, thereby suggesting potential anticancer therapeutic activity.

Variation in DNA methylation transmissibility, genetic heterogeneity and fecundity-related traits in natural populations of the perennial herb Helleborus foetidus.

Inferences about the role of epigenetics in plant ecology and evolution are mostly based on studies of cultivated or model plants conducted in artificial environments. Insights from natural populations, however, are essential to evaluate the possible consequences of epigenetic processes in biologically realistic scenarios with genetically and phenotypically heterogeneous populations. Here, we explore associations across individuals between DNA methylation transmissibility (proportion of methylation-sensitive loci whose methylation status persists unchanged after male gametogenesis), genetic characteristics (assessed with AFLP markers), seed size variability (within-plant seed mass variance), and realized maternal fecundity (number of recently recruited seedlings), in three populations of the perennial herb Helleborus foetidus along a natural ecological gradient in southeastern Spain. Plants (sporophytes) differed in the fidelity with which DNA methylation was transmitted to descendant pollen (gametophytes). This variation in methylation transmissibility was associated with genetic differences. Four AFLP loci were significantly associated with transmissibility and accounted collectively for ~40% of its sample-wide variance. Within-plant variance in seed mass was inversely related to individual transmissibility. The number of seedlings recruited by individual plants was significantly associated with transmissibility. The sign of the relationship varied between populations, which points to environment-specific, divergent phenotypic selection on epigenetic transmissibility. Results support the view that epigenetic transmissibility is itself a phenotypic trait whose evolution may be driven by natural selection, and suggest that in natural populations epigenetic and genetic variation are two intertwined, rather than independent, evolutionary factors.

Epigenetic variation predicts regional and local intraspecific functional diversity in a perennial herb.

The ecological significance of epigenetic variation has been generally inferred from studies on model plants under artificial conditions, but the importance of epigenetic differences between individuals as a source of intraspecific diversity in natural plant populations remains essentially unknown. This study investigates the relationship between epigenetic variation and functional plant diversity by conducting epigenetic (methylation-sensitive amplified fragment length polymorphisms, MSAP) and genetic (amplified fragment length polymorphisms, AFLP) marker-trait association analyses for 20 whole-plant, leaf and regenerative functional traits in a large sample of wild-growing plants of the perennial herb Helleborus foetidus from ten sampling sites in south-eastern Spain. Plants differed widely in functional characteristics, and exhibited greater epigenetic than genetic diversity, as shown by per cent polymorphism of MSAP fragments (92%) or markers (69%) greatly exceeding that for AFLP ones (41%). After controlling for genetic structuring and possible cryptic relatedness, every functional trait considered exhibited a significant association with at least one AFLP or MSAP marker. A total of 27 MSAP (13.0% of total) and 12 AFLP (4.4%) markers were involved in significant associations, which explained on average 8.2% and 8.0% of trait variance, respectively. Individual MSAP markers were more likely to be associated with functional traits than AFLP markers. Between-site differences in multivariate functional diversity were directly related to variation in multilocus epigenetic diversity after multilocus genetic diversity was statistically accounted for. Results suggest that epigenetic variation can be an important source of intraspecific functional diversity in H. foetidus, possibly endowing this species with the capacity to exploit a broad range of ecological conditions despite its modest genetic diversity.

Rhizocola hellebori gen. nov., sp. nov., an actinomycete of the family Micromonosporaceae containing 3,4-dihydroxydiaminopimelic acid in the cell-wall peptidoglycan.

An actinomycete strain, K12-0602(T), was isolated from the root of a Helleborus orientalis plant in Japan. The 16S rRNA gene sequence of strain K12-0602(T) showed that it had a close relationship with members of the family Micromonosporaceae and the 16S rRNA gene sequence similarity values between strain K12-0602(T) and type strains of type species of 27 genera belonging to the family Micromonosporaceae were below 96.2%. MK-9 (H4) and MK-9 (H6) were detected as major menaquinones, and galactose, xylose, mannose and ribose were present in the whole-cell hydrolysate. The acyl type of the peptidoglycan was glycolyl. Major fatty acids were iso-C(15 : 0), iso-C(16 : 0), C(17 : 1)ω9c and anteiso-C(17 : 0). Phosphatidylethanolamine was detected as the phospholipid corresponding to phospholipid type II. The G+C content of the genomic DNA was 67 mol%. Analyses of the cell-wall peptidoglycan by TLC and LC/MS showed that it was composed of alanine, glycine, hydroxylglutamic acid and an unknown amino acid, which was subsequently determined to be 3,4-dihydroxydiaminopimelic acid using instrumental analyses, including NMR and mass spectrometry. On the basis of the phylogenetic analysis and chemotaxonomic characteristics, strain K12-0602(T) represents a novel species of a new genus in the family Micromonosporaceae, for which the name Rhizocola hellebori gen. nov., sp. nov. is proposed. The type strain of the type species is K12-0602(T) ( = NBRC 109834(T) = DSM 45988(T)). This is the first report, to our knowledge, of 3,4-dihydroxydiaminopimelic acid being found as a diamino acid in bacterial cell-wall peptidoglycan.

Individual variation in size and fecundity is correlated with differences in global DNA cytosine methylation in the perennial herb Helleborus foetidus (Ranunculaceae).

UNLABELLED: • PREMISE OF THE STUDY: Few studies have examined how epigenetic modifications of DNA may influence individual plant phenotypes and ecological processes in wild plant populations. We investigated natural variation in global DNA cytosine methylation and its phenotypic correlates in the perennial herb Helleborus foetidus.• METHODS: We focused specifically on individual differences in size- and fecundity-related traits and used HPLC to quantify percentage of total cytosines in the genome of young full-grown leaves that were methylated.• KEY RESULTS: About one third of all cytosines in H. foetidus genome were methylated. Methylation level differed significantly among individual plants (range = 26.4-36.6%; n = 60 plants), and this variation was significantly related to most size- and fecundity-related traits considered. Relatively hypomethylated plants bore more vegetative, reproductive, and total ramets, produced more flowers, larger inflorescences and more seed-bearing follicles, and their ramets remained vegetative for fewer years before reproducing sexually, than relatively hypermethylated ones. Taken together, results revealed that individual differences in size and reproductive output were inversely related to global cytosine methylation.• CONCLUSIONS: These results confirm, in a natural scenario, the association between DNA methylation and size- and fecundity-related traits that was previously found by experimental studies. Variations in global cytosine methylation were predictably related to individual differences in sexual reproduction through significant effects on flower and fruit production, which might ultimately influence patterns of selection and population dynamics in this species. This study provides novel insights on the potential ecological significance of epigenetic heterogeneity in wild plant populations.

Tandem mass spectrometric characterization of acetylated polyhydroxy hellebosaponins, the principal steroid saponins in Helleborus niger L. roots(#).

RATIONALE: Isolation and extensive nuclear magnetic resonance (NMR) analyses revealed polyhydroxy steroid saponins to be characteristic constituents in Helleborus niger L. roots. A comprehensive study including various multi-stage mass spectrometry (MS(n) ) experiments provided first solid chromatographic and mass spectrometric information facilitating future analysis and structural assessment of polyhydroxy saponins by LC/MS(n) techniques without isolation and NMR analyses. METHODS: The polyhydroxy saponins were analyzed by direct syringe injection or chromatographically separated on a capillary high-performance liquid chromatography (HPLC) system coupled to an electrospray ionization (ESI) source. MS(n) spectra were recorded on an ion trap mass spectrometer including up to four fragmentation stages (LC/ESI-MS/MS). Additionally, high-resolution mass spectra were recorded on an Orbitrap Fourier transform (FT) mass spectrometer equipped with a nanospray-ESI interface. RESULTS: The polyhydroxy hellebosaponins A and D were discovered to be significant constituents from H. niger roots. Extensive study of their MS(n) data revealed that they readily fragmented in the positive ion mode providing diagnostic fragments for elucidation of the steroidal character and number of OH groups. The negative ion mode yielded valuable information on the [M-H](-) ion, number and location of acetyl groups and sugar units. Additionally, fragmentation pathways for positive and negative ion modes were proposed. CONCLUSIONS: These results not only extend the knowledge about H. niger saponins, but also provide a facilitated approach to the analysis of polyhydroxy saponins by LC/MS(n) without prior isolation and extensive NMR identification. Additionally, proposed fragmentation pathways for positive and negative ionization modes provide a solid complementary database for further, more detailed MS(n) studies.

Evaluation of Geranium spp., Helleborus spp. and Hyssopus spp. polyphenolic extracts inhibitory activity against urease and α-chymotrypsin.

This study was meant to determine the inhibitory activity of tannins and flavonoid compounds from Geranium robertianum, Helleborus purpurascens and Hyssopus officinale plant polyphenol rich extracts against urease and α-chymotrypsin. The G. robertianum, H. purpurascens and H. officinale extracts were purified and concentrated by microfiltration and ultrafiltration. Phenolic compounds including flavonoids and tannins have been linked to many pharmacological activities. Thus, the polyphenolic content of the extracts was assessed by UV-Vis spectroscopy and HPLC. The concentrated extracts enriched in polyphenolic compounds (flavonoids, tannins and phenolic acids) showed a significant inhibition against urease from jack bean (over 90%), whereas in case of the α-chymotrypsin, they proved to have an inhibition below 54%. The results of this support the use of G. robertianum, H. purpurascens and H. officinale polyphenolic extracts as potential sources of urease inhibitors. Among the three plant extracts tested, H. officinale polyphenolic extracts exhibited a high inhibitory activity (92.67%) against urease and low inhibition (19.6%) against α-chymotrypsin and could be considered as possible remedy in ulcer treatment.