Anatomical and Phytochemical Characteristics of Different Parts of Hypericum scabrum L. Extracts, Essential Oils, and Their Antimicrobial Potential.

Hypericum (Hypericaceae) is a genus that comprises a high number of species around the world. In this study, the roots, aerial parts, flowers, fruits, and aerial parts with flowers from Hypericum scabrum were macerated separately by methanol and water and then fractionated by different solvents of, such as ethyl acetate, n-hexane, butanol, dichloromethane, aqueous residue sub-extracts, and ethnobotanical use. All the extracts, sub-extracts and essential oils of H. scabrum were investigated for the first time in detail for their antimicrobial, total phenolics, and antioxidant activities. Anatomical structures of the root, stem, leaf, upper and lower leaf surface, stamen, sepal, and petal of H. scabrum were examined. The biochemical layout of essential oils was determined by GC and GC/MS. The antioxidant activity was determined by free radical scavenging activity (by DPPH). Antimicrobial activity was applied against Candida albicans ATCC 10231, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 19659, and C. tropicalis ATCC 750 using microdilution methods. The essentials of the aerial parts, flower, and fruit are characterized by the presence of monoterpene hydrocarbons, whereas roots oil include alkanes. The GC-FID and GC-MS analysis showed that major components of roots, aerial parts, flowers, and fruits oils were undecane (66.1%); α-pinene (17.5%), γ-terpinene (17.4%), and α-thujene (16.9%); α-pinene (55.6%), α-thujene (10.9%), and γ-terpinene (7.7%); α-pinene (85.2%), respectively. The aerial part sub-extracts indicated a greater level of total phenolics and antioxidant potential. The n-hexane sub-extracts (from aerial part, flower, and aerial part with flower) showed the best activity against B. subtilis, with 39.06 µg/mL MIC value. The presented research work indicates that H. scabrum can be a novel promising resource of natural antioxidant and antimicrobial compounds.

Relation between hypericin content and morphometric leaf parameters in Hypericum spp.: A case of cubic degree polynomial function.

Higher plants often accumulate secondary metabolites in multicellular structures or in secretory reservoirs. Biotechnological production of such compounds by cell cultures lacking proper morphological structures is difficult, therefore possibilities for an efficient increase of their formation by organ cultures are being searched. The genus Hypericum comprises many species that store photoactive and phototoxic naphthodianthrones in the dark nodules on their above-ground parts. To date, the relation between the content of hypericins and their proto-forms accumulated in the nodules, and morphological characters of the plant parts containing these structures has not been sufficiently explained. The content of hypericins and leaf morphology characters were measured in 12 selected diploid seed-derived Hypericum species cultured in vitro. The leaf volume and the volume of the nodules per leaf were calculated. Based on these data, a cubic degree polynomial regression model with high reliability was constructed. The model enables an estimate of the biosynthetic capacity of the cultures, and may be useful in designing the experiments aimed at elicitation of these unique secondary metabolites in shoot cultures of Hypericum spp. An analogous model may be developed for interpretation of experimental results for other plant species which accumulate metabolites in specialized morphological structures.

Exodermis and endodermis are the sites of xanthone biosynthesis in Hypericum perforatum roots.

Xanthones are specialized metabolites with antimicrobial properties, which accumulate in roots of Hypericum perforatum. This medicinal plant provides widely taken remedies for depressive episodes and skin disorders. Owing to the array of pharmacological activities, xanthone derivatives attract attention for drug design. Little is known about the sites of biosynthesis and accumulation of xanthones in roots. Xanthone biosynthesis is localized at the transcript, protein, and product levels using in situ mRNA hybridization, indirect immunofluorescence detection, and high lateral and mass resolution mass spectrometry imaging (AP-SMALDI-FT-Orbitrap MSI), respectively. The carbon skeleton of xanthones is formed by benzophenone synthase (BPS), for which a cDNA was cloned from root cultures of H. perforatum var. angustifolium. Both the BPS protein and the BPS transcripts are localized to the exodermis and the endodermis of roots. The xanthone compounds as the BPS products are detected in the same tissues. The exodermis and the endodermis, which are the outermost and innermost cell layers of the root cortex, respectively, are not only highly specialized barriers for controlling the passage of water and solutes but also preformed lines of defence against soilborne pathogens and predators.

Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization.

Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.

Integrating fossils, phylogenies, and niche models into biogeography to reveal ancient evolutionary history: the case of Hypericum (hypericaceae).

In disciplines such as macroevolution that are not amenable to experimentation, scientists usually rely on current observations to test hypotheses about historical events, assuming that "the present is the key to the past." Biogeographers, for example, used this assumption to reconstruct ancestral ranges from the distribution of extant species. Yet, under scenarios of high extinction rates, the biodiversity we observe today might not be representative of the historical diversity and this could result in incorrect biogeographic reconstructions. Here, we introduce a new approach to incorporate into biogeographic inference the temporal, spatial, and environmental information provided by the fossil record, as a direct evidence of the extinct biodiversity fraction. First, inferences of ancestral ranges for those nodes in the phylogeny calibrated with the fossil record are constrained to include the geographic distribution of the fossil. Second, we use fossil distribution and past climate data to reconstruct the climatic preferences and potential distribution of ancestral lineages over time, and use this information to build a biogeographic model that takes into account "ecological connectivity" through time. To show the power of this approach, we reconstruct the biogeographic history of the large angiosperm genus Hypericum, which has a fossil record extending back to the Early Cenozoic. Unlike previous reconstructions based on extant species distributions, our results reveal that Hypericum stem lineages were already distributed in the Holarctic before diversification of its crown-group, and that the geographic distribution of the genus has been relatively stable throughout the climatic oscillations of the Cenozoic. Geographical movement was mediated by the existence of climatic corridors, like Beringia, whereas the equatorial tropical belt acted as a climatic barrier, preventing Hypericum lineages to reach the southern temperate regions. Our study shows that an integrative approach to historical biogeography-that combines sources of evidence as diverse as paleontology, ecology, and phylogenetics-could help us obtain more accurate reconstructions of ancient evolutionary history. It also reveals the confounding effect different rates of extinction across regions have in biogeography, sometimes leading to ancestral areas being erroneously inferred as recent colonization events.

Efficient decoding of 2D structured illumination with linear phase stepping in X-ray phase contrast and dark-field imaging.

The ability to map the phase distribution and lateral coherence of an x-ray wavefront offers the potential for imaging the human body through phase contrast, without the need to deposit significant radiation energy. The classic means to achieve this goal is structured illumination, in which a periodic intensity modulation is introduced into the image, and changes in the phase distribution of the wavefront are detected as distortions of the modulation pattern. Two-dimensional periodic patterns are needed to fully characterize a transverse wavefront. Traditionally, the information in a 2D pattern is retrieved at high resolution by acquiring multiple images while shifting the pattern over a 2D matrix of positions. Here we describe a method to decode 2D periodic patterns with single-axis phase stepping, without either a loss of information or increasing the number of sampling steps. The method is created to reduce the instrumentation complexity of high-resolution 2D wavefront sensing in general. It is demonstrated with motionless electromagnetic phase stepping and a flexible processing algorithm in x-ray dark-field and phase contrast imaging.

Evolution of cryptic gene pools in Hypericum perforatum: the influence of reproductive system and gene flow.

BACKGROUND AND AIMS: Hypericum perforatum (St. John's wort) is a widespread Eurasian perennial plant species with remarkable variation in its morphology, ploidy and breeding system, which ranges from sex to apomixis. Here, hypotheses on the evolutionary origin of St. John's wort are tested and contrasted with the subsequent history of interspecific gene flow. METHODS: Extensive field collections were analysed for quantitative morphological variation, ploidy, chromosome numbers and genetic diversity using nuclear (amplified fragment length polymorphism) and plastid (trnL-trnF) markers. The mode of reproduction was analysed by FCSS (flow cytometric seed screen). KEY RESULTS: It is demonstrated that H. perforatum is not of hybrid origin, and for the first time wild diploid populations are documented. Pseudogamous facultative apomictic reproduction is prevalent in the polyploids, whereas diploids are predominantly sexual, a phenomenon which also characterizes its sister species H. maculatum. Both molecular markers characterize identical major gene pools, distinguishing H. perforatum from H. maculatum and two genetic groups in H. perforatum. All three gene pools are in close geographical contact. Extensive gene flow and hybridization throughout Europe within and between gene pools and species is exemplified by the molecular data and confirmed by morphometric analyses. CONCLUSIONS: Hypericum perforatum is of a single evolutionary origin and later split into two major gene pools. Subsequently, independent and recurrent polyploidization occurred in all lineages and was accompanied by substantial gene flow within and between H. perforatum and H. maculatum. These processes are highly influenced by the reproductive system in both species, with a switch to predominantly apomictic reproduction in polyploids, irrespective of their origin.

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum).

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. John's wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.

[Study on the growth, development and artificial propagation of Hypericum ascyron].

OBJECTIVE: To explore the morphological changes, growth conditions and artificial propagation of Hypericum ascyron. METHODS: The morphological changes were observed and recorded in the scene, the height and diameter of the plants were measured; the growth Verhaulst model was set up with the SPSS 17.0 software; the sexual reproduction and asexual reproduction were carried out in artificial cultivation. RESULTS: Hypericum ascyron started germinating in late April each year, branching in late May, flowering in late June, the period of full bearing was in early August, seeds were mature in early October. The Verhaulst models of the increase in the height (H), the quantity of leaf pairs (L) and the branching (B) were, H = 127.109/(1 + 23.744 x e(-0.062t)), L = 23.343/(1 + 11.303 x e(-0.062t)), B = 22.037/(1 + 73.068 x e(-0.068t)). The survival rate of whole graft and segmentation plant were 100% and 67.2% respectively on asexual reproduction; on the sexual reproduction, the seed germination rate was 15.2%, the survival rate of transplant seedlings was 36%. CONCLUSIONS: The period of growth and development of Hypericum ascyron is from April to October and it can be carried out artificial propagation.

Evidence for the evolution of reduced mycorrhizal dependence during plant invasion.

Introduced species inevitably experience novel selection pressures in their new environments as a result of changes in mutualist and antagonist relationships. While most previous work has examined how escape from specialist enemies has influenced herbivore or pathogen resistance of exotic species, post-introduction shifts in exotic dependence on mutualists have not been considered. In a common environment, we compared dependence on AM fungi of North American and European populations of Hypericum perforatum (St. John's Wort), a forb native to Europe. Introduced North American populations responded less to inoculation with AM fungi than did European populations. Root architecture was strongly correlated with mycorrhizal response, and introduced populations had finer root architecture than native populations. Finally, introduced populations exhibited decreased root and increased reproductive allocation relative to European populations, consistent with a transition to a weedier life history; however, biomass allocation patterns were uncorrelated with mycorrhizal response. These findings are the first demonstration of a genetically based reduction of mycorrhizal dependence and shift in root architecture in an introduced species.

Factors affecting polyphenol biosynthesis in wild and field grown St. John's Wort (Hypericum perforatum L. Hypericaceae/Guttiferae).

The increasing diffusion of herbal products is posing new questions: why are products so often different in their composition and efficacy? Which approach is more suitable to increase the biochemical productivity of medicinal plants with large-scale, low-cost solutions? Can the phytochemical profile of a medicinal plant be modulated in order to increase the accumulation of its most valuable constituents? Will polyphenol-rich medicinal crops ever be traded as commodities? Providing a proactive answer to such questions is an extremely hard task, due to the large number of variables involved: intraspecific chemodiversity, plant breeding, ontogenetic stage, post-harvest handling, biotic and abiotic factors, to name but a few. An ideal path in this direction should include the definition of optimum pre-harvesting and post-harvesting conditions and the availability of specific Good Agricultural Practices centered on secondary metabolism enhancement. The first steps to be taken are undoubtedly the evaluation and the organization of scattered data regarding the diverse factors involved in the optimization of medicinal plant cultivation, in order to provide an interdisciplinary overview of main possibilities, weaknesses and drawbacks. This review is intended to be a synopsis of the knowledge on this regard focused on Hypericum perforatum L. (Hypericaceae/Guttiferae) secondary metabolites of phenolic origin, with the aim to provide a reference and suggest an evolution towards the maximization of St. John's Wort bioactive constituents. Factors considered emerged not only from in-field agronomic results, but also from physiological, genetical, biotic, abiotic and phytochemical data that could be scaled up to the application level. To increase quality for final beneficiaries, growers' profits and ultimately transform phenolic-rich medicinal crops into commodities, the emerging trend suggests an integrated and synergic approach. Agronomy and genetics will need to develop their breeding strategies taking account of the suggestions of phytochemistry, biochemistry, pharmacognosy and pharmacology, without losing sight of the economic balance of the production.

[Identification on Herba Hyperici Japonici and its adulterant].

OBJECTIVE: To study the identification of Herba Hyperici Japonici and its adulterant. METHODS: Herba Hyperici Japonici and Herba Hyperici graminei were identified by traditional and modern identification. RESULTS: Both of them showed difference in exterior character, microscope characteristic, TLC, UV and HPLC fingerprint graphics. CONCLUSION: The distinct characteristic features reveal this study can serve as evidence for the identification of Herba Hyperici Japonici.

[Morphological identification of 20 medicinal species in Hypericum].

OBJECTIVE: To provide anatomical evidences for the morphological and histological identification of 20 medicinal species in Hypericum. METHOD: Morphological and anatomical study on the organs of 20 medicinal species in Hypericum using tissue clearing, paraffin sectioning and thin sectioning. RESULT: According to their anatomical characteristics, the secretory structures can be divided into nodules, secretory cavities (canals) and tiny secretory tubes of 20 medicinal species in Hypericum. Hypericin was produced and stored in the nodules, while the volatile oil was produced and stored in the secretory cavities (canals) and tiny secretory tubes. The types differed markedly from each other in location, diameter and distributional density of leaf, and the anatomical structures differed from each other of stem, calyx, petal, anther and fruit among the 20 species in Hypericum. CONCLUSION: The secretory structures may be as anatomical evidences for the morphological and histological identification of 20 medicinal species in Hypericum.

Hyperforin.

Hyperforin is a polyprenylated acylphloroglucinol derivative from Hypericum perforatum (St. John's wort). It exhibits antidepressant activity by a novel mechanism of action, antibiotic activity against gram-positive bacteria, and antitumoral activity in vivo. However, it also produces drug-drug interactions by activation of the pregnan X receptor. No total synthesis has been described. Some natural and semisynthetic analogues are available to study structure-activity relationships. Enzymatically, the skeleton of hyperforin is formed by isobutyrophenone synthase from isobutyryl-CoA and three molecules of malonyl-CoA. The first prenylation step is catalyzed by a soluble and ion-dependent dimethylallyltransferase. Hyperforin mainly accumulates in pistils and fruits where it probably serves as defensive compound.

Morphological, histochemical and phytochemical investigation of the genus Hypericum of the Central Italy.

Eight entities of the genus Hypericum that spontaneously grow on the Central Italy (Appennino Umbro-Marchigiano) have been studied under the morphological, histochemical and phytochemical aspects. From the morphological standpoint, they differ in the shape and size of flowers and leaves and in the dimension and distribution of the secretory structures through the various parts of the plant. It has been possible, with the histochemical and phytochemical studies, to localize and identify some secondary metabolites inside the secretory structures.

[Species and distribution of medicinal plant Hypericum in Guizhou].

OBJECTIVE: To gain a clear idea on the resources of medicinal plant Hypericum in Guizhou. METHOD: Conducting field investigation and consulting related specimens and data. RESULT: The distribution, growing environment and medicinal parts of 18 species of Hypericun have been clarified, and a key for their identification is given. CONCLUSION: A scientific basis for further study of the medicinal plant Hypericum in Guizhou Province has been provided.