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Bu çalışmada Helleborus orientalis Lam. (Ranunculacaae) türünün çiçek morfolojisi, tohum ve meyve yapısı incelenmiştir. Helleborus L., düğün çiçeğigiller (Ranunculaceae) familyasından Orta, Güney ve Doğu Avrupa'dan Kafkasya sınırlarına kadar yayılmış yaklaşık 20 türü bulunan zehirli bir bitki cinsidir. Helleborus L. cinsi Türkiye’de iki tür ile temsil edilmektedir. Bunlar H. vesicarius Aucher ve H. orientalis Lam. türleridir. Ülkemizde “çöpleme” olarak bilinen bu tür, Türkiye’de doğal olarak yetişir. H. orientalis halk arasında bohça otu, kara çöpleme, siyah harbak, boynuz otu, danabağırtan, danakıran gibi yöresel isimlerle anılır. Çok yıllık otsu bir bitkidir. Gövdeleri yapraksız ve 60 cm uzunluğunda olup, taban yaprakları her daim yeşildir. Uzun yaprak saplı, yaprakları geniş, digitat biçiminde olup çiçek yapısı simozdur. Sepaller yeşilimsi beyaz, bazen morumsu, ovat, 20-30(-35) mm’ dir. Tek bir çiçeğin tamamen açıldıktan sonraki ortalama ömrü yaklaşık 6 gündür. Çiçek örtüsü iki sıra üzerine dizili, sepale ya da petale benzeyen dışta 5 geniş halkalı, meyvede kalıcı, içteki halkada bulunanlar küçük, tüpsü, 2-dudaklı parçalı (nektaryum) bulunur. Uzun tepecikte kısa anterler, nadiren küttür. Anterler en azından tabanda birleşmiş birkaç tohumlu folikül grubu meyve bulunmaktadır. Tohumlar genellikle 6’ dan daha fazladır. Çiçeklenme genellikle mart-mayıs arasındadır. H. orientalis türü genel olarak Karadeniz Bölgesi’nde yayılış göstermekte olup genel dağılımı Yunanistan ve Kafkasya bölgeleri üzerindedir. Genellikle orman açıklıkları ve orman altlarında, çalılık habitatlarda 1-2200 metre arasında bulunmaktadır. Türkiye’de A2, A3, A4, A5, A6, A7, A8, A9 ve B3 karelerinde yayılış göstermektedir. Çalışma materyali A3 karesi içinde yer alan Bilecik ilinin Çiçekli yayla mevkiinden 23.05.2020 tarihinde toplanarak herbaryum materyali haline getirilmiştir. Morfolojik çalışmalar canlı örnekler üzerinde ölçümler yapılarak incelenmiştir.

There are 176 endemic plants spread in southern Colchis, of which 45 can be used for some medical treatments. The bioecology and detailed phytochemical content of some medicinal plant populations have not been studied so far. The research objectiveis to study the phytochemical content of endemic species of Helleborus caucasicus, Helleborus abchasicus and Ficaria popovii spread in southern Colchis. The research method for the phytochemical content included separation analysis, whichwas performed byusing UPLC-MS (Waters Acquity QDa detector). Three Steroidal glycosideswere isolated from the MeOH extract of the plants of Helleborus caucasicus and Helleborus abchasicus: Hellebrigenin-D-glucose, 20 – Hydroxyecdysone and Hydroxyecdysone – 3 glucoside. Two saponins (Hederagenin 3-O -α-L-arabino pyranoside, Hederagenin28-O-[α-L-rhamno-pyranosyl(1→4)-β-D-glucopyranosyl-(1→6)]β-D-lucopyranoside) and four flavonoids (kaempferol 3-O-β-ᴅ- (6ʺ-α-ʟ-rhamnopyranosyl)-glucopyranoside (nicotiflorin), apigenin 8-C-β-ᴅ-glucopyranoside (vitexin), luteolin 8-C-β-ᴅ-glucopyranoside (orientin), quercetin 3-O-rutinoside) were isolated from the tubers and flowers of Ficaria Popovii. Three Steroidal glycosides and Hydroxyecdysone -3 glucoside were isolated from the MeOH extract of Helleborus caucasicus.In addition, two saponins and four flavonoids were isolated from the tubers and leaves of FicariaPopovii.

The floristic region of Adjara represents “Hotpoint” of Caucasian which is distinguished with the uniqueness of its it relict Colchis flora.It represents one of the most powerful refuge in western Eurasia , which is not touched by the chilling because has the special geographical location. There are 176 endemic plants spread in southern Colchis, of which 45 ones can be used for some medical treatments. The bioecology and detailed phytochemical content of some medicinal plant populations have not been studied so far. The research objective is to study the phytochemical content of endemic species of Helleborus caucasicus and Helleborus abchasicus spread in southern Colchis. The research method for the phytochemical content included the separation analysis, which was performed by using UPLC-MS (Waters Acquity QDa detector). Three Steroidal glycosides were isolated from the MeOH extract of the plants of Helleborus caucasicus and Helleborus abchasicus: Hellebrigenin-D-glucose, 20 – Hydroxyecdysone and Hydroxyecdysone – 3 glucoside. Three Steroidal glycosides and Hydroxyecdysone -3 glucoside have been isolated from the MeOH extract of Helleborus caucasicus.

The use of hellebore (Helleborus) species for medical purposes has a long-standing tradition. Our work aimed at providing a historical survey of their medicinal application in Europe, and data on current ethnobotanical use ofH. purpurascensWaldst. et Kit. in Transylvania (Romania), compared with earlier records of this region and other European countries. While the chemistry and pharmacology of hellebores have been researched extensively, little is known about their anatomical traits. Thus, we intended to provide a detailed histological analysis ofHelleborus odorusWaldst. et Kit.,H. purpurascens, andH. nigerL., based on transverse sections of aerial parts and root. Our survey revealed thatH. purpurascensis known for immunotherapy, wounds, and as antiemetic drug in ethnoveterinary medicine, but not in human therapy in the study area. Distinctive histological characters included diverse stele structure in the root; sclerenchymatous bundle caps around compound vascular bundles in the stem and the main leaf veins ofH. odorus; and amphistomatic leaves inH. purpurascens. Quantitative vegetative traits also revealed significant differences among species, but they may reflect environmental influences, too. In all three species the sepal was hypostomatic with mesomorphic stomata, while the modified petal comprised a proximal nectar-producing and a distal non-secretory part. Distinctive floral traits included shape of modified petal, presence of papillae and thickness of non-secretory part; as well as ornamentation of tricolpate pollen grains. Our findings suggest that the anatomy of various plant parts varies slightly with each species, including ethnomedicinally knownH. purpurascens, even though the basic structure is the same within the genus.

Plants are important antioxidant sources. In our study, total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) of endemic Helleborus vesicarius Aucher ex Boiss.were determined. The aerial parts of the plant samples were dried and extracted with ethanol (EtOH). TAS and TOS values of plant extract were determined using Rel Assay Diagnostics kits. As a result of the studies, the TAS value of H. vesicarius was determined 5.548±0.23, the TOS value was 13.778±0.119 and the OSI value was 0.249±0.009. In this context, the plant has been shown to have significant antioxidant potential.

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.

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.

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 IC

Ethnopharmacological relevance Ethnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece. Aim of the study A pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed. Materials and methods The roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling. Results The RME showed 80% inhibition of PTZ-induced locomotor activity (300 μg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 μM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 μM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35–37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids. Conclusions This study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction.

Lenten rose (Hellebores hybridus) is an herbaceous perennial grown in landscapes and valued for early spring flowers and high levels of deer resistance. An additional benefit as a landscape plant c...

Helleborus sp. are healing plants from the family Ranunculaceae. The aim of this paper is to consider ethnopharmacology and the toxicity of the hellebores which is focused on a detailed research of the value of phytochemicals, as pharmacological attributes of phytomedicine herbs. The data is based on active components. Also, this paper presents the chemical composition of the root in the overall effect of the drug, as well as the importance of the hellebores in pharmacy. It shows a wide range of pharmacological effects such as cardiotonic, immunostimulative, antibacterial, antitumor, diuretic, and emetic. Helleborus odorus Waldst. et Kit. is rich with secondary metabolites such as bufadenolides, flavonoids, phenolic heterozids. With this research it was concluded that these plants have great significance and great application in the formulation of pharmaceutical dosage forms. Keywords: Helleborus sp., phytomedicine, folk medicinal uses, cardiotonic, toxicity, hellebore

Helleborus caucasicus (Ranunculaceae) is an endemic plant of the Caucasian flora, widely distributed in West Georgia. Biological activities for the extracts of some Helleborus species including H. caucasicus have been reported. In this work we found that butanolic extract of the underground parts of H. caucasicus and isolated compounds decreased cell viability in vitro on cancer cell line of lung origin (Calu-1) in a concentration-dependent manner, compared to the normal cell line. In particular, we identified that furostanol derivative (25S)-22α,25-epoxyfurost-5-ene-3β,11β,26-triol 26-O-β-d-glucopyranoside (5), 20-hydroxyecdysone (6), and 3β,5β,14β-trihydroxy-19-oxo-bufa-20,22-dienolide 3-O-α-l-rhamnopyranoside, known as deglucohellebrin (7) exerted a strong cytotoxic effect on the same cells and on other cancer cell lines (HepG2 and Caco-2) reducing the S-phase entry (compound 6) and inducing cell apoptosis associated with activation of caspase-3 (compound 7). Moreover we demonstrated that 6 and 7 significantly decreased protein expression of GRP78, a general ER-stress marker, suggesting pro-apoptotic functions. These findings indicated that selected compounds from H. caucasicus are potential interesting agents in anti-cancer therapy.

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. Electronic supplementary material The online version of this article (10.1186/s12906-019-2517-5) contains supplementary material, which is available to authorized users.

Hellebore or Lenten rose (Helleborus × hybridus) is an evergreen, herbaceous perennial in the family Ranunculaceae. Hellebores are sold as decorative, potted plants and as shade-loving landscape plants favored for their attractive and prolonged blooms in late winter or early spring. In April of 2008, downy mildew-like growth was observed on the foliage of approximately 60 containerized plants of Helleborus ‘Blue Lady’, ‘Pink Lady’, ‘White Lady’, and ‘Royal Heritage’ grown outdoors in a retail nursery in coastal San Mateo County, California. Infected foliage had angular, vein-delimited, dark brown-to-black speckled lesions on adaxial leaf surfaces turning dry and necrotic with age. Young leaves were small and distorted. Affected flowers were spotted and brown. The abaxial sides of affected leaves had light brown-to-purplish downy mildew-like growth. Subhyaline conidia, globose to ellipsoid in shape, ranged from 25 to 31 × 17 to 24 μm (average 28 × 21 μm). Conidiophores ranged from 265 to 375 × 5 to 11.5 μm (average 333 × 8.9 μm), branching dichotomously four to five times in the upper half. Morphological measurements fell within the range previously described for Peronospora pulveracea and P. alpicola, which were reported on Helleborus spp. and Ranunculus aconitifolius, respectively (1,2). DNA sequence of the internal transcribed spacer region of rDNA of our isolate (Genbank Accession No. FJ384778) matched sequences of P. pulveracea (Genbank Accession No. AY198270) and P. alpicola (Genbank Accession No. AY198271) with 100% identity. These two organisms are taxonomically indistinguishable by rDNA sequences and are likely to be the same species (3). To our knowledge, this is the first report of P. pulveracea on Helleborus × hybridus in California and the United States. Lenten rose is commercially propagated by seed, which is a potential pathway for introduction of this pathogen. Mature plants are sold and shipped intra- and interstate as decorative flowering plants or nursery stock. The importance and economic impact of this disease is limited, but significant economic losses could occur during production. References: (1) E. A. Gäumann. Beitr. Kryptogamenflora Schweiz 5:113, 1923. (2) G. Hall. Mycopathologia 126:57, 1994. (3) H. Voglmayr. Mycol. Res. 107:1132, 2003.

The genus Helleborus is represented in the Northwestern Balkans by 8 species. Some of them (H. atrorubens, H. foetidus (newcomer, introduced as well as escaped from culture), H. niger, and H. orientalis (newcomer, escaped from culture)) are rather easy for determination and discrimination. While other 4 species (H. dumetorum, H. multifidus, H. odorus, and H. viridis) are really difficult for determination because of their narrow morphological similarity, which also is realized a wide variety in leaf shape. The distribution areas of last 4 species overlap in the Northwestern Balkans, so that there are no really clear borders of their distribution. Dominating species in this region is H. multifidus, and especially common there is its subsp. istriacus. Other 3 species are rare and are therefore often overlooked and the herbarium specimens are frequently misidentified as H. multifidus. Moreover, all these 4 species produce number of hybrids, and as a result all kind of transistions between these taxa could be found. In particular, 11 hybrides could be confirmed for this region. The hybrid between H. dumetorum and H. multifidus is described here as Helleborus ×mucheri.

Three new steroidal saponins including two spirostanol glycosides (1–2) and one furostanol glycoside 1-sulphate (3) were isolated from the dried roots and rhizomes of Helleborus thibetanus. Structures of the compounds were determined on the basis of extensive use of 1-D and 2-D NMR experiments, together with HR–ESI–MS and IR measurements, as well as the results of acid hydrolysis. Compounds 1–2 represented steroidal saponins with an unusual substitution pattern, which possessed a double bond at C-25 and were glycosylated at 1-OH.

Helleborus trifolius subsp. corsicus Briq., Prodr. Fl. Corse 1: 582. 1910. Ξ Helleborus lividus subsp. corsicus (Briq.) P. Fourn. Lectotypus (désigné par YEO, 1986: 157): FRANCE. Corse: pentes du Pigno, à Bastia, 20.III.1865, Mabille 28 (G [G00441205]!; isolecto-: G-BU [G00185695]!). Syntypi: (G-BU [G00185693, G00185693, G00830131, G00830132, G00830133, G00830134, G00830135, G00830136]!)., Published as part of Jeanmonod, Daniel, 2018, Typification des noms de taxons corses de l'herbier Burnat (G-BU), pp. 61-74 in Candollea 73 (1) on page 67, DOI: 10.15553/c2018v731a6, http://zenodo.org/record/5722164, {"references":["YEO, P. F. (1986). The nomenclature of Helleborus lividus Aiton sens. lat. (Ranunculaceae). Taxon 35: 156 - 161."]}

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.

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.

In this study, we investigated cytohistochemistry, cycle progression, and relative DNA content of the female gametophyte cells of Helleborus bocconei Ten. before and after fertilization process. The early stages of embryo development were also investigated. H. bocconei possesses a monosporic seven-celled/eight-nucleate Polygonum type female gametophyte, characterized by a morpho-functional polarity. The cells of the embryo sac showed abundant reserves of polysaccharides, strongly increasing in the egg cell just before fertilization. With different timing in DNA replication during cell cycle progression, synergids, egg cells, and polar nuclei showed a haploid DNA content at the end of their differentiation, while antipodes underwent three DNA endoreduplication cycles. Programmed cell death symptoms were detectable in synergid and antipodal cells. After double fertilization, the central cell quickly underwent many mitotic cycles forming the endosperm, which exhibited a progressive increase in protein bodies and starch grains. Close to the developing embryo, the endosperm differentiated a well-defined region rich in a fibrillar carbohydrate matrix. The zygote, that does not start immediately to divide after double fertilization, developed in to an embryo that reached the heart stage at fruit maturation time. A weakly differentiated embryo at this time indicates a morpho-physiological dormancy of seeds, as a survival strategy imposed by the life cycle of this plant with seed dispersal in spring and their germination in the following winter.

Yeasts frequently colonise floral nectar, where they can reach high densities. Recent investigations have further shown that yeast metabolism alters nectar properties by decreasing its total sugar content, modifying sugar composition, or raising nectar local temperature. However, the distribution patterns of nectar yeasts remain poorly investigated at multiple spatial and temporal scales. Here, we study natural variation of the nectar yeasts in a single host plant, Helleborus foetidus, in a mountainous region. We quantified spatio-temporal variation in the frequency and abundance of yeast species across six populations located along an altitudinal gradient. Variance partitioning techniques were used to estimate the relative magnitude of variation in yeast abundance between individual plants, flowers within plants, and nectaries within flowers. Although yeast frequency and abundance varied widely across sites and dates, the largest part of total variance occurred at the sub-individual level (i.e., flowers on the same plant). Pollinator composition and activity seemed the main factors explaining the observed patterns of yeast frequency and abundance across floral nectar samples

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

An actinomycete strain, K12-0602T, was isolated from the root of a Helleborus orientalis plant in Japan. The 16S rRNA gene sequence of strain K12-0602T showed that it had a close relationship with members of the family Micromonosporaceae and the 16S rRNA gene sequence similarity values between strain K12-0602T 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-C15 : 0, iso-C16 : 0, C17 : 1ω9c and anteiso-C17 : 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 K12-0602T 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 K12-0602T ( = NBRC 109834T = DSM 45988T). This is the first report, to our knowledge, of 3,4-dihydroxydiaminopimelic acid being found as a diamino acid in bacterial cell-wall peptidoglycan.

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. © 2014 John Wiley & Sons Ltd.

A non-sporulating fungus was isolated from different organs of Helleborus bocconei subsp. intermedius (Ranunculaceae) endemic to southern Italy and Sicily, known for the traditional use of dried roots in the treatment of lung diseases of cattle and horses. Molecular characterization of endophytic fungus based on the internal transcribed spacer (ITS) region of the rRNA gene sequences was done. The DNA sequence of full length ITS region of the studied fungus was a 100% match to that of Chaetomium strumarium strain dH 21642 (GenBank accession number JX280851.1). The morphological characters of colony and mycelium of this microfungus are reported here.

The paper discusses and supplements the knowledge of the phytosociological composition of Fagus sylvatica forests in the Rasa Valley, on the northeastern edge of the Karst, or more specifically, in the area between the plateau of Vrhe, the Senožece lowland and the Karst. They are classified into two associations, Seslerio autumnalis-Fagetum and Hacquetio-Fagetum . A new subassociation (- lamietosum orvalae ) and a new geographical subvariant (subvar. geogr. Helleborus istriacus ) were determined for the association Hacquetio-Fagetum. Although they occupy small areas these beech forests are an important part of the predominantly oak and hop hornbeam forest vegetation of the Karst, constituting a Natura 2000 habitat type Illyrian Fagus sylvatica forests ( Aremonio-Fagion ). They are threatened by climate change and excessive logging. Key words : phytosociology, synsystematics, Aremonio-Fagion, Hacquetio-Fagetum , Rasa, Kras, Slovenia Izvlecek Dopolnili smo vednost o fitocenoloski sestavi bukovih gozdov v dolini Rase, na severovzhodnem robu Krasa, natancneje med planoto Vrhe, Senožeskim podoljem in Krasom. Uvrscamo jih v dve asociaciji, Seslerio autumnalis-Fagetum in Hacquetio-Fagetum . Za slednjo smo ugotovili novo subasociacijo (- lamietosum orvalae ) in novo geografsko subvarianto (subvar. geogr. Helleborus istriacus ). Kljub majhnim povrsinam so ti bukovi gozdovi v zdajsnji prevladujoci hrastovi in crnogabrovi gozdni vegetaciji Krasa pomembni, saj sodijo v Natura 2000 habitatni tip Ilirski bukovi gozdovi ( Aremonio-Fagion ). Ogrožajo jih podnebne spremembe in premocne secnje. Kljucne besede : fitocenologija, sinsistematika, Aremonio-Fagion, Hacquetio-Fagetum , Rasa, Kras, Slovenija

Helleborus (family Ranunculaceae) are well-known as ornamental plants, but less known for their therapeutic benefits. Over the past few years, Helleborus sp. has become a subject of interest for phytochemistry, pharmacology and other medical research areas. On the basis of their usefulness in traditional medicine, it was assumed that their biochemical profile could be a source of metabolites with the potential to overcome critical medical issues. There are studies involving natural extracts from these species which demonstrate that Helleborus plants are a valuable source of chemical compounds with great medical potential. Some phytochemicals produced by these species have been separated and identified a few decades ago: hellebrin, deglucohellebrin, 20-hydroxyecdysone and protoanemonin. Lately, many other active compounds have been reported and considered as promising remedies for severe diseases such as cancer, ulcer, diabetes and also for common medical problems such as toothache, eczema, low immunity and arthritis. This paper is an overview of the Helleborus genus focusing on some recentlydiscovered compounds and their potential for finding new drugs and useful biochemicals derived from these species.

The genus Helleborus is represented in the Northwestern Balkans by 8 species. Some of them (H. atrorubens, H. foetidus (newcomer, introduced as well as escaped from culture), H. niger, and H. orientalis (newcomer, escaped from culture)) are rather easy for determination and discrimination. While other 4 species (H. dumetorum, H. multifidus, H. odorus, and H. viridis) are really difficult for determination because of their narrow morphological similarity, which also is realized a wide variety in leaf shape. The distribution areas of last 4 species overlap in the Northwestern Balkans, so that there are no really clear borders of their distribution. Dominating species in this region is H. multifidus, and especially common there is its subsp. istriacus. Other 3 species are rare and are therefore often overlooked and the herbarium specimens are frequently misidentified as H. multifidus. Moreover, all these 4 species produce number of hybrids, and as a result all kind of transistions between these taxa could be found. In particular, 11 hybrides could be confirmed for this region. The hybrid between H. dumetorum and H. multifidus is described here as Helleborus ×mucheri.

Phytochemical reinvestigation of the dried roots and rhizomes of Helleborus thibetanus afforded four new minor spirostanol glycosides (1–4) and four known spirostanol glycosides (5–8). Their structures were determined on the basis of spectroscopic analyses, including 1D and 2D NMR experiments, together with HR-ESI-MS and IR measurements and the results of acid hydrolysis.

Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation-sensitive amplification polymorphism (MS-AFLP or MSAP) have been often used to assess methyl-cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome-wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome-wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl-cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context-specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation-based epigenetic processes in nonmodel plants.

Floral organogenesis in Helleborus thibetanus and Nigella damascena was examined and compared using scanning electron microscopy and light microscopy, and the putative relationships of Helleborus and Nigella were analysed. H. thibetanus and N. damascena share some features of floral phyllotaxis and development of the sepals, petals, stamens and carpels, which are also found in other members of Ranunculaceae. However, they differ strongly in the number and degree of fusion of the carpels: in H. thibetanus, the two carpels are slightly united at the base, whereas, in N. damascena, the gynoecium is syncarpous and the five carpels are united throughout the ovary. Differences are also noted in petal development. The blade of the young petal of H. thibetanus develops two bulges which become connate and then fuse with the blade at the sides, developing more quickly than the blade and forming a tubular petal. In N. damascena, a single ridge is formed on the petal blade which develops into the smaller adaxial labium of the bilabiate petal, whereas the blade itself develops into the larger abaxial labium bearing two pseudonectaries. The outermost stamens are delayed in development in Helleborus, but not in Nigella. Although the results from our investigation are preliminary, differences in floral development characters suggest that Helleborus and Nigella may not be closely related and possibly support placement into separate tribes. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 166, 431–443.