[Constitution of a new sapogenin from Helleborus odoros Waldtst. et Ket. and Helleborus niger L].
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In the Rumanian traditional medicine a transcutaneous implantation of the root of Helleborus purpurascens is used to provoke leucocytosis and neutrophils with the aim to activate chronic diseases for better healing. The skin is perforated with a thick needle (in cattle and horses in the area of the thorax, in sheep and pigs in the ear flap), the transplant is introduced and after 24 hours removed. Though the method is more than 100 years old, it is not reported in the literature. The way of action is unknown. The investigation are performed to show the efficacy of this method of Helleborus-implants in comparison with the effect of an placebo. It could be shown that Helleborus-implant increased leucocytes in horses, sheep and pigs and increased neutrophils in all animal species. In the same way the phagocytosis was increased. Only in horses an abscess was observed. As a loss of performance may occur and fever and inappetence is followed by the implantation the authors dissuade from the use of Helleborus-implants in cattle and horses. Pigs and sheep tolerate this procedure without any problems, so that the methods may be used in modern treatment.
Extracts of Helleborus species are used as phytopreparations with immunostimulatory properties in Romanian traditional medicine. In Germany, Helleborus niger is used in homeopathy and as an adjuvant therapy in the treatment of tumor patients in anthroposophical medicine. In vitro application of an aqueous extract from Helleborus niger resulted in a slight induction of sister chromatid exchanges (SCE) in cultured peripheral blood mononuclear cells (PBMC) from healthy individuals, an effect associated with a slight increase of the [3H]thymidine uptake in the DNA of isolated lymphocytes. Since the cytokines interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha were reported to increase the number of SCE, we measured the concentrations of these cytokines in the supernatants of cultured PBMC treated with the plant extract. Here, no significant changes were observed as compared with the controls, but a trend to higher supernatant concentrations of TNF-alpha in six out of ten individuals was noted. Compared with lymphocytes treated with the alkylating substance, cyclophosphamide, the increase of the SCE levels induced by the plant extract is weak. The relevance of this DNA destabilizing property remains to be clarified.
BACKGROUND AND AIMS: This study examined the effect of plant traits and environmental factors on pollinator visitation in the winter-flowering Helleborus foetidus (Ranunculaceae) in three distant regions in the Iberian Peninsula. METHODS: Geographical variation in floral visitor assemblage, plant traits and environmental factors were analysed during the flowering season. KEY RESULTS: Differences were found in all plant traits measured (number of open flowers, flower size, number of stamens per flower, and number of nectaries) both within and among regions, and differences among regions in all the environmental factors considered (air temperature, exposure to sunlight, canopy cover, and distance to the nearest neighbour). Differences were also found among regions in the probability that plants would be visited by pollinators. CONCLUSIONS: The results show that, although floral display (i.e. number of open flowers on a plant on a given day) consistently explained among-plant differences in visitation rate in all regions, visitation rate was not significantly affected by any other biological or environmental variable. In Helleborus foetidus, then, 'how' the plant is would seem to be more important than 'where' is it.
A new acylated flavonol glycoside, quercetin 3-O-(2-trans-caffeoyl)-alpha-L-arabinopyranosyl-(1-->2)-beta-D-glucopyranoside (1), together with the known 25R,26-[(beta-D-glucopyranosyl)oxy]-22alpha-hydroxy-5beta-furostan-3-beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-D-glucopyranoside (2), anemonin (3), beta-D-glucosyl-p-hydroxyphenylethyl alcohol (4), and 1-beta-O-caffeoyl-D-glucose (5) were isolated from Helleborus foetidus leaves and identified on the basis of detailed spectral analysis.
Phytochemical analysis of the polar extracts of the leaves of Helleborus viridis (Ranunculaceae) resulted in the isolation of two new furostanol saponins (25R)-26-[(alpha-L-rhamnopyranosyl)oxy]-22alpha-methoxyfurost-5-en-3beta-yl O-beta-D-glucopyranosyl-(1-->3)-O-[6-acetyl-beta-D-glucopyranosyl-(1-->3)]-O-beta-D-glucopyranoside (1) and (25R)-26-[(alpha-L-rhamnopyranosyl)oxy]-22alpha-methoxyfurost-5-en-3beta-yl O-beta-D-glucopyranosyl-(1-->3)-O-beta-D-glucopyranosyl-(1-->3)-O-beta-D-glucopyranoside (2) and three new quercetin glycosides, quercetin 3-O-(2-E-caffeoyl)-alpha-L-arabinopyranosyl-(1-->2)-beta-D-galactopyranoside-7-O-beta-d-glucopyranoside (3), quercetin 3-O-(2-E-caffeoyl)-alpha-L-arabinopyranosyl-(1-->2)-beta-D-galactopyranoside (4), and quercetin 3-O-alpha-L-arabinopyranosyl-(1-->2)-beta-D-galactopyranoside (5). The structures of the new compounds were determined by spectroscopic analysis, including 2D NMR data and mass spectrometry.
Thionins are relatively small-sized multiple-cystine peptides that are probably involved in the plant defense against pathogens. As such, these peptides constitute promising candidates for engineered plant resistance in the agricultural industry. More recently, thionins have been proposed as potential immunotoxins in tumor therapy. In the search for pharmacologically active natural products, a new family of thionins was recently discovered in the roots of Helleborus purpurascens that accordingly were termed hellethionins. The structural characterization by NMR of one representative member of this family, i.e., of hellethionin D, clearly reveals that thionins from different sources share a highly conserved overall fold. In fact, the well-defined 3D structure of hellethionin D is very similar to those reported so far for viscotoxins, purothionins, or crambin, although distinct differences could be detected in the C-terminal portion, especially for loop 36-39. These differences may derive from the unusual distribution of charged residues in the C-terminal half of the peptide sequence compared to other thionins and from the uncommon occurrence of four contiguous threonine residues in loop 36-39. As expected, reduction of the disulfide bonds in hellethionin D leads to complete unfolding, but upon oxidative refolding by air oxygen in the presence of glutathione the correct isomer is recovered in high yields, confirming the very robust fold of this class of bioactive cystine peptides.
Reproductive development in the Christmas rose (Helleborus niger L.) differs from that in commonly investigated model plants in two important aspects: (i) the perianth develops a photosynthetic system, after fertilization, and persists until seed ripening; and (ii) the ripe seed contains an immature embryo which continues to mature off the mother plant. The possible roles of cytokinins in these processes are investigated here by analysing extracts of the perianth and the carpels/maturing fruit prepared during anthesis and four stages of post-floral development. trans-Zeatin, dihydrozeatin, N6-(Delta2-isopentenyl)adenine, and their ribosides were identified by tandem mass spectrometry. Single ion monitoring in the presence of deuterated internal standards demonstrated the additional presence of the corresponding riboside-5'-monophosphates, O-glucosides, and 9-glucosides, and afforded quantitative data on the whole set of endogenous cytokinins. Fruit cytokinins were mostly localized in the seeds. Their overall concentrations increased dramatically during early seed development and remained high for 6-8 weeks, until shortly before seed ripening (the last time point covered in this work). Overall cytokinin levels in the perianth did not change markedly in the period covered, but the level of N6-(Delta2-isopentenyl)adenine-type cytokinins appeared to increase slightly and transiently during the greening phase. The perianths of unpollinated or depistillated flowers, which survived, but did not pass through the complete greening process, contained significantly less cytokinins than observed in fruit-bearing flowers. This suggests that perianth greening requires defined cytokinin levels and supports the role of the developing fruit in their maintenance.
We examine the hierarchical geographic structure of the interaction between a plant, Helleborus foetidus, and its floral herbivores and pollinators (interactors). Six populations from three distant regions of the Iberian Peninsula were used to examine intra- and inter-regional variation in plant traits, interactors and plant fecundity, and to compare, through selection gradient and path analyses, which traits were under selection, and which interactors were responsible for differential selection. Geographic and temporal congruency in interactor-mediated selection was further tested using a recent analytical approach based on multi-group comparison in Structural Equation Models. Most plant traits, interactors and fecundity differed among regions but not between populations. Similarly, the identity of the traits under selection, the selection gradients (strength and/or the direction of the selection) and the path coefficients (identifying the ecological basis for selection) varied inter- but not intra-regionally. Results show a selection mosaic at the broad scale and, for some traits, a link of differential selection to trait differentiation.
Three bufadienolides, hellebortin A (5-[beta-D-glucopyranosyloxy]-10,14,16-trihydroxy-19-nor-[5beta,10beta,14beta,16beta]-bufa-3,20,22-trienolide [1]), hellebortin B (5-[beta-D-glucopyranosyloxy]-3,4-epoxy-14-hydroxy-19-oxo-bufa-20,22-dienolide [2]) and hellebortin C (5-[beta-D-glucopyranosyloxy]-3,4-epoxy-10,14-dihydroxy-19-nor-bufa-20,22-dienolide [3]), together with 20-hydroxyecdysone 3-O-beta-D-glucoside (4) and 20-hydroxyecdysone (5) have been isolated by bioassay- and RIA-directed HPLC analyses of a methanol extract of the seeds of Helleborus torquatus. The structure and relative stereochemistry of the novel bufadienolide hellebortin A (1) and the structures of hellebortin B (2) and hellebortin C (3) were determined unambiguously by comprehensive analyses of their 1D and 2D NMR data. These five compounds are isolated from Hellborus torquatus for the first time. The biological activities of compound 1, 4 and 5 as ecdysteroid agonists and antagonists have been assessed.
Numerical methods for the evaluation of the separation power of thirteen thin-layer chromatographic systems for splitting a methanolic extract of leaves of Helleborus atrorubens Waldst. et Kit. into 15 compounds (flavonoids and phenolic acids) have been investigated. For this purpose, the following mathematical approaches have been applied: calculation of the information content (I), determination of discriminating power (DP) and formation of clusters and dendrogram. The most suitable chromatographic system for the separation of investigated compounds is ethyl acetate-formic acid-water (65:15:20, v/v/v).
In fertilized flowers of Helleborus niger L., the sepals (the showy elements of the perianth at anthesis) grow, spread, and turn green, and the peduncles elongate. These processes did not proceed to completion when the pistils were removed at the bud stage, but could be restored by the application of plant growth regulators. Cytokinins and gibberellins stimulated the formation of well-developed chloroplasts in, and spreading of, the sepals; the gibberellin, GA3, and the auxin, 4-chloroindole-3-acetic acid, promoted peduncle elongation. In fruit-bearing flowers, on the other hand, paclobutrazol, an inhibitor of gibberellin biosynthesis, reduced chlorophyll formation in the sepals, reversed sepal spreading, and inhibited peduncle elongation. Of the endogenous growth regulators in developing fruit, the following cytokinins were identified: zeatin, dihydrozeatin, N6-(2-isopentenyl)adenine and their ribosides and 9-glucosides. Zeatin riboside drastically increased in abundance (about 200 times), shortly after fertilization, when chlorophyll accumulation in the sepals occurred at the fastest rate, and remained the most prominent identified cytokinin until seed ripening.
Autonomous self-pollination may be considered as a mechanism enhancing plant reproductive success when plant access to pollen sources may limit seed production. We have studied the relationship between geographical patterns of variation in pollinator service to Helleborus foetidus and self-pollination ability in three widely spaced regions in the Iberian Peninsula. As could be expected from its early flowering period, pollinator visitation rates to both plants and flowers of H. foetidus were very low at all sites. Pollinator composition remained consistent among regions, but there was significant variation among regions in pollinator service. Despite the low visitation rates, fruit set did not appear to be pollen limited in any of the study areas, which may be explained by the long duration of flowers (up to 20 d). When pollinators were excluded experimentally, fruit set decreased significantly, but substantial levels of self-pollination occurred at all regions. Autonomous self-pollination levels were lowest in the two regions with lowest pollinator service and highest in the region with highest pollinator service. This disagreement between our results and the expectations derived from the reproductive assurance hypothesis may reflect a nonequilibrium situation of the northern H. foetidus populations in relation to their current pollinating environment.
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