RFLP analysis of cpDNA in the genus <Emphasis Type="Italic">Hypericum</Emphasis>

The chloroplast DNA of 43 species including 16 sections from the genus Hypericum was studied by PCR-RFLP analysis. The PCR-amplified products of four cpDNA regions, trnC-trnD, psbC-trnS, trnL-trnF and rbcL were digested with four restriction endonucleases. A high level of interspecific variation was detected while intraspecific diversity was not observed. The resulting parsimony analysis indicated the monophyletic assemblage of the sections Androsaemum, Olympia, Drosocarpium and Trigynobrathys. Monophyly of Hypericum is weakly supported, but close relationships of H. perforatum and H. maculatum are indicated. The members of Ascyreia are weakly resolved, but clustering of H. kouytchense and H. oblongifolium is well supported, however, H. reptans is nested with Olympia. CpDNA profiles and the positions on the parsimony tree indicate that the chloroplast donor among the putative parents of the hybrid species H. ×inodorum is H. androsaemum.     

Essential oil composition of Hypericum L. species from Southeastern Serbia and their chemotaxonomy

The essential oils of the aerial parts of nine species of Hypericum (Hypericum barbatum, Hypericum hirsutum, Hypericum linarioides, Hypericum maculatum, Hypericum olympicum, Hypericum perforatum, Hypericum richeri, Hypericum rumeliacum and Hypericum tetrapterum), collected from different locations in Southeast Serbia, were obtained by steam distillation and analyzed by GC and GC–MS. The essential oils investigated were characterized by a high content of non-terpene compounds and a low content of monoterpenes. The contents of non-terpenes, monoterpenes and sesquiterpenes in oils of the species H. barbatum, H. richeri and H. rumeliacum (section Drosocaprium) were similar and these oils were characterized by high contents of fatty acids. The oils of H. hirsutum and H. linarioides (section Taeniocarpium) contained a high percentage of n-nonane. There were similarities in contents of non-terpenes and sesquiterpenes in oils of species that belong to the section Hypericum (H. maculatum, H. perforatum and H. tetrapterum). The oil of H. olympicum differed from others by higher terpene content. A comparison was also carried out of the chemical composition of the essential oils from flower, leaf and stem of H. perforatum and it revealed that the highest concentration of non-terpene compounds was found in the flower and stem oil, while a high concentration of sesquiterpenes was characteristic for leaf oil. There were significant differences in the concentrations of the same compounds in the essential oils of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location which could be explained by seasonal differences. All data were statistically processed with principal component analysis and cluster analysis. The main conclusion from the above data is that genetic and environmental factors both play a role in determining the composition of essential oils of the Hypericum species studied.     

Identification of anti-inflammatory constituents in Hypericum perforatum and Hypericum gentianoides extracts using RAW 264.7 mouse macrophages

Hypericum perforatum (St. John’s wort) is an herb widely used as supplement for mild to moderate depression. Our prior studies established synergistic anti-inflammatory activity associated with 4 bioactive compounds in a fraction of a H. perforatum ethanol extract. Whether these 4 compounds also contributed to the ethanol extract activity was addressed in the research reported here. Despite the popularity of H. perforatum, other Hypericum species with different phytochemical profiles could have their anti-inflammatory potentials attributed to these or other compounds. In the current study, ethanol extracts of different Hypericum species were compared for their inhibitory effect on LPS-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 mouse macrophages. Among these extracts, those made from H. perforatum and H. gentianoides demonstrated stronger overall efficacy. LC–MS analysis established the 4 compounds were present in the H. perforatum extract and pseudohypericin in all active fractions. The 4 compounds accounted for a significant part of the extract’s inhibitory activity on PGE2, NO, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in RAW 264.7 as well as peritoneal macrophages. Pseudohypericin was the most important contributor of the anti-inflammatory potential among the 4 compounds. The lipophilic fractions of H. gentianoides extract, which did not contain the previously identified active constituents, decreased PGE2 and NO potently. These fractions were rich in acylphloroglucinols, including uliginosin A that accounted for a proportion of the anti-inflammatory activity observed with the active fractions. Overall, the current study established that a different group of major anti-inflammatory constituents were present in H. gentianoides, while showing that the previously identified 4 compound combination was important for H. perforatum’s anti-inflammatory potential.     

Chemical Composition and Antimicrobial Activity of the Essential Oils from Several Hypericum Taxa (Guttiferae) Growing in Central Italy (Appennino Umbro‐Marchigiano)

The chemical composition of the essential oils of nine taxa from seven sections of Hypericum L. (Guttiferae; H. perforatum subsp. perforatum, H. perforatum subsp. veronense, H. calycinum, H. montanum, H. richeri subsp. richeri, H. hyssopifolium, H. hirsutum, H. hircinum subsp. majus, and H. tetrapterum) occurring in central Italy (Appennino Umbro‐Marchigiano) was analyzed by GC/FID and GC/MS. A total of 186 compounds were identified in the different species and subspecies, accounting for 86.9–92.8% of the total oils. The major fraction of the oil was always represented by sesquiterpene hydrocarbons (30.3–77.2%), while quantitative differences occurred between the other classes of volatiles depending on the taxa considered. Chemical composition of the nine Hypericum entities with respect to the taxonomical classification was discussed. Essential oils obtained from six taxa, i.e., H. perforatum subsp. perforatum, H. perforatum subsp. veronense, H. calycinum, H. richeri subsp. richeri, H. hirsutum and H. tetrapterum, were also tested for their antimicrobial properties against five different microbial strains by the broth‐microdilution method, and they were found to have significant activity (expressed as MIC) on B. subtilis, moderate activity on C. albicans and S. aureus, and weak activity on E. coli and E. faecalis, the most active being those from H. hirsutum, H. richeri subsp. richeri, and H. tetrapterum.     

Rutin as Deoxyribonuclease I Inhibitor

DNase I inhibitory potential of water extract of nine Hypericum species (H. umbellatum, H. barbatum, H. rumeliacum, H. rochelii, H. perforatum, H. tetrapterum, H. olympicum, H. hirsutum, H. linarioides) and the most important Hypericum secondary metabolites (hypericin, hyperforin, quercetin, and rutin) was investigated. All examined Hypericum extracts inhibited DNase I with IC₅₀ below 800 μg/ml, whereby H. perforatum was the most potent (IC₅₀=391.26±68.40 μg/ml). Among the investigated Hypericum secondary metabolites, rutin inhibited bovine pancreatic DNase I in a non‐competitive manner with IC₅₀ value of 108.90±9.73 μm . DNase I inhibitory ability of rutin was further confirmed on DNase I in rat liver homogenate (IC₅₀=137.17±16.65 μm ). Due to the involvement of DNase I in apoptotic processes the results of this study indicate the importance of frequent rutin and H. perforatum consumption in daily human nutrition. Rutin is a dietary component that can contribute to male infertility prevention by showing dual mechanism of sperm DNA protection, DNase I inhibition and antioxidant activity.     

Variability of Hypericins and Hyperforin in Hypericum Species from the Sicilian Flora

Within Sicilian flora, the genus Hypericum (Guttiferae) includes 10 native species, the most popular of which is H. perforatum. Hypericum’s most investigated active compounds belong to naphtodianthrones (hypericin, pseudohypericin) and phloroglucinols (hyperforin, adhyperforin), and the commercial value of the drug is graded according to its total hypericin content. Ethnobotanical sources attribute the therapeutic properties recognized for H. perforatum, also to other Hypericum species. However, their smaller distribution inside the territory suggests that an industrial use of such species, when collected from the wild, would result in an unacceptable depletion of their natural stands. This study investigated about the potential pharmacological properties of 48 accessions from six native species of Hypericum, including H. perforatum and five ‘minor’ species, also comparing, when possible, wild and cultivated sources. The variability in the content of active metabolites was remarkably high, and the differences within the species were often comparable to the differences among species. No difference was enlightened between wild and cultivated plants. A carefully planned cultivation of Hypericum seems the best option to achieve high and steady biomass yields, but there is a need for phytochemical studies, aimed to identify for multiplication the genotypes with the highest content of the active metabolites.     

Comparison of chemical composition of Hypericum perforatum and H. maculatum in Estonia

Of numerous species belonging to the medicinally important genus Hypericum, only H. perforatum L. and H. maculatum Crantz grow widely in Estonia. A comparative biochemical systematics study of hypericins, hyperforins and other phenolics within Hypericum spp. growing in Estonia was performed. For comprehensive metabolomic investigation, 42 samples of H. perforatum and 16 samples of aerial parts of H. maculatum were collected in two consecutive years from various locations; methanolic extracts were prepared from airdried leaves and flowers. The concentrations of a quinic acid derivative, caffeic acid glucoside, vanillic acid glucoside, neochlorogenic acid, chlorogenic acid, catechin, epicatechin, myricetin glucoside, hyperoside, isoquercitrin, rutin, quercetin pentoside, quercitrin, kaempferol glucoside, kaempferol rutinoside, quercetin, hyperforin, adhyperforin, protopseudohypericin, pseudohypericin, and hypericin were determined by LC-DAD-MS/MS. All the aforementioned compounds were detected in both species, although some at very different levels – H. maculatum contained rutin and hyperforins only in trace amounts and overall tended to contain more phenolic compounds. The level of total hypericins was the same for both species. These results constitute a further contribution to the systematic knowledge about the Hypericum spp. Results of principal component analysis (PCA) demonstrated distinct between-years and between-species diversity in the chemical composition of the plants studied. Between-years diversity in Hypericum spp. has not been addressed before.     

Lipid and Phenolic Constituents from Seeds of Hypericum perforatum L. and Hypericum tetrapterum Fr. and their Antioxidant Activity

Seeds of Hypericum perforatum and H. tetrapterum were extracted with dichloromethane and methanol and investigated by chromatographic and mass spectrometric methods. Both species yielded a fatty oil fraction amounting to 30.5% and 18.0% of the seed weight, respectively. Linoleic acid (C18:2n‐6) was shown to be the predominant fatty acid constituent. Moreover, xanthone derivatives, i.e. tetrahydroxyxanthones (THX), xanthone‐glycosides and xanthone‐sulfonates, were assigned in methanolic extracts. For structure elucidation, one representative xanthone, namely 1,3,6,7‐THX, was synthesized and analyzed via HPLC‐DAD/MSⁿ and GC/MS. Total THX contents were quantitated applying a validated HPLC‐DAD method, resulting in 1.25 g/kg (H. perforatum) and 0.27 g/kg (H. tetrapterum), respectively. Moreover, the free radical scavenging capacity of the methanol extracts was tested using the DPPH antioxidant assay. Both, H. perforatum (IC₅₀ = 8.7 mg/l) and 1,3,6,7‐THX (IC₅₀ = 3.0 mg/l), exhibited good DPPH free radical scavenging activity compared to Trolox (IC₅₀ = 6.6 mg/l).     

Interspecific variation in localization of hypericins and phloroglucinols in the genus Hypericum as revealed by desorption electrospray ionization mass spectrometry imaging

Plants of the genus Hypericum are widely known for their therapeutic properties. The most biologically active compounds of this genus are naphtodianthrones and phloroglucinols. Indirect desorption electrospray ionization mass spectrometry (DESI‐MS) imaging allows visualization and localization of secondary metabolites in different plant tissues. This study is focused on localization of major secondary compounds in the leaves of 17 different in vitro cultured Hypericum species classified in 11 sections. Generally, all identified naphtodianthrones, protohypericin, hypericin, protopseudohypericin and pseudohypericin were co‐localized in the dark glands of eight hypericin producing species at the site of their accumulation. The known phloroglucinols, hyperforin, adhyperforin, hyperfirin and some new phloroglucinols with m/z [M ? H]^? 495 and 569 were localized in the translucent and pale cavities within the leaf in the majority of studied species. The comparison of different Hypericum species revealed an interspecific variation in the distribution of the dark and translucent glands corresponding with the localization of hypericins and phloroglucinols. Moreover, similarities in the localization and composition of the phloroglucinols were observed in the species belonging to the same section. Adding to various quantitative studies focused on the detection of secondary metabolites, this work using indirect DESI‐MSI offers additional valuable information about localization of the above‐mentioned compounds.     

Positive correlations between hypericin and putative precursors detected in the quantitative secondary metabolite spectrum of Hypericum

A spectrum of eight pharmacologically important secondary compounds, all putatively belonging to the polyketide pathway (hypericin, pseudohypericin, emodin, hyperforin, hyperoside, rutin, quercetin, and quercitrin) were analyzed in several hypericin-producing species of Hypericum by LC–MS/MS. Different organs such as leaves, stems and roots of wild-grown plants of Hypericum hirsutum L., Hypericum maculatum Crantz s. l., Hypericum montanum L., Hypericum tetrapterum Fr. collected in Slovakia and of Hypericum perforatum L. collected in India were examined individually. Highest contents of hypericin, pseudohypericin, and emodin were found in H. montanum, suggesting that there are alternative species to H. perforatum with high pharmaceutical value. Amounts of hyperforin and quercetin were highest in H. perforatum, whereas highest contents of hyperoside and quercitrin were found in H. maculatum. A significant positive correlation between hypericin and pseudohypericin as well as between hypericin and emodin was observed by Kruskal’s multidimensional scaling (MDS), indicating a parallel enhancement of emodin as a common precursor in the biosynthetic pathways of hypericin and pseudohypericin. Furthermore, MDS combined with principal component analysis (PCA) revealed strong correlations in the occurrence of pseudohypericin and emodin, pseudohypericin and quercitrin, hypericin and quercitrin, emodin and quercitrin, hyperoside and quercitrin, rutin and quercetin, and, hyperforin and quercetin. On the other hand, rutin showed a negative correlation with emodin as well as with quercitrin. Furthermore, hierarchical agglomerative cluster analysis (HACA) clustered hypericin and pseudohypericin, grouping emodin at equal distance from both. Considerable infraspecific variability in secondary compound spectrum and load of different populations of H. maculatum from Slovakia underscores the need for detailed studies of genotypic variation and environmental factors in relation to polyketide biosynthesis and accumulation.     

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.     

Structure Elucidation of the Main Tetrahydroxyxanthones of Hypericum Seeds and Investigations into the Testa Structure

Seeds from Hypericum species have recently been identified as an interesting source of xanthone derivatives. Extraction of seeds from H. perforatum with MeOH and subsequent concentration via polyamide adsorption yielded a fraction enriched in tetrahydroxyxanthones (THX), which were further semipurified by silica gel chromatography. Based on tentative structure assignment of the two main THX X1 and X2 by NMR a total synthesis was performed for both compounds (THX 1 and 2 , respectively), starting with an Ullmann ether synthesis. The synthesized 1 and 2 were characterized via 1D‐ and 2D‐NMR methods as well as by LC/HR‐MS analysis and proven to be 1,4,6,7‐THX ( 1 ) and 1,2,6,7‐THX ( 2 ). Final structure assignment of the natural Hypericum THX constituents was accomplished by comparing chromatographic and spectroscopic data (LC/MSⁿ and GC/MS) with those of 1 and 2 which were obtained by synthesis. Beyond, investigations into the seeds of H. perforatum and H. tetrapterum by scanning electron microscopy (SEM) provided insights of the structure of the testa (seed coat), which is established by two cell layers, with the lignified sclerenchyma presumably being the depository of the xanthones.     

Morphogenetic and diurnal variation of hypericin in some Hypericum species from Turkey during the course of ontogenesis

The genus Hypericum has received considerable interest from scientists, as it is a source of a variety of biologically active compounds including the hypericins. The present study was conducted to determine ontogenetic, morphogenetic and diurnal variation of the total hypericins content in some species of Hypericum growing in Turkey namely, Hypericum aviculariifolium subsp. depilatum var. depilatum (endemic), Hypericum perforatum and Hypericum pruinatum. The Hypericum plants were harvested from wild populations at vegetative, floral budding, full flowering, fresh fruiting and mature fruiting stages four times a day. Plants were dissected into stem, leaf and reproductive tissues, which were dried separately, and subsequently assayed for total hypericin content. The density of dark glands on leaves at full flowering plants was determined for each species. Floral parts had the highest hypericin content in all species tested. But diurnal fluctuation in the hypericin content of whole plant during the course of ontogenesis varied among the species. It reached the highest level at floral budding and tended to increase at night in H. aviculariifolium subsp. depilatum var. depilatum and H. pruinatum, whereas in H. perforatum hypericin content was the highest at full flowering and no diurnal fluctuation was observed. In general, hypericin content of leaves and whole plant was higher in H. aviculariifolium subsp. depilatum var. depilatum whose leaves had more numerous dark glands than those of the two other species.     

Shoot anatomy and secretory structures in Hypericum species (Hypericaceae)

The anatomy and ultrastructure of internodes, leaves and petals were compared in Hypericum elegans, H. inodorum, H. olympicum, H. forrestii and two genotypes of H. perforatum. Internode anatomy was variable between species with respect to the structure of the cortical and pith parenchyma, including the presence of secretory reservoirs. Also, the secondary growth was more extensive in shrubs, i.e. H. inodorum and H. forrestii. In leaves, phloem secretory reservoirs were formed in all species, mesophyll secretory reservoirs were absent only in H. elegans and internal nodules were present only in H. elegans and H. perforatum. The petals differed between species in the mesophyll structure and the occurrence and location of secretory structures. The phloem secretory reservoirs lacked sheaths, whereas these were distinct in the mesophyll reservoirs. Other ultrastructural traits of the reservoirs were similar in all the species studied, with the exception of the leucoplast ultrastructure. In internal nodules, the inner cells vs. sheath cells differed in the number of vesicles and other membranous structures and plastid ultrastructure. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163 , 70–86.     

Computational identification of conserved microRNAs and their putative targets in the Hypericum perforatum L. flower transcriptome

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression in plants. Many miRNA families and their targets have been extensively studied in model species and major crops. We have characterized mature miRNAs along with their precursors and potential targets in Hypericum to generate a comprehensive list of conserved miRNA families and to investigate the regulatory role of selected miRNAs in biological processes that occur in the flower. St. John’s wort (Hypericum perforatum L., 2n = 4x = 32), a medicinal plant that produces pharmaceutically important metabolites with therapeutic activities, was chosen because it is regarded as an attractive model system for the study of apomixis. A computational in silico prediction of structure, in combination with an in vitro validation, allowed us to identify 7 pre-miRNAs, including miR156, miR166, miR390, miR394, miR396, and miR414. We demonstrated that H. perforatum flowers share highly conserved miRNAs and that these miRNAs potentially target dozens of genes with a wide range of molecular functions, including metabolism, response to stress, flower development, and plant reproduction. Our analysis paves the way toward identifying flower-specific miRNAs that may differentiate the sexual and apomictic reproductive pathways.     

Molecular phylogenetics and morphological evolution of St. John’s wort (Hypericum; Hypericaceae)

Phylogenetic hypotheses for the large cosmopolitan genus Hypericum (St. John’s wort) have previously been based on morphology, and molecular studies have thus far included only a few species. In this study, we used 360 sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) for 206 species representing Hypericum (incl. Triadenum and Thornea) and three other genera of Hypericaceae to generate an explicit phylogenetic hypothesis for the genus using parsimony and model-based methods. The results indicate that the small genus Triadenum is nested in a clade within Hypericum containing most of the New World species. Sister to Hypericum is Thornea from Central America. Within Hypericum, three large clades and two smaller grades were found; these are based on their general morphology, especially characters used previously in taxonomy of the genus. Relative to the most recent classification, around 60% of the sections of Hypericum were monophyletic. We used a Bayesian approach to reconstruct ancestral states of selected morphological characters, which resulted in recognition of characters that support major clades within the genus and a revised interpretation of morphological evolution in Hypericum. The shrubby habit represents the plesiomorphic state from which herbs evolved several times. Arborescent species have radiated convergently in high-elevation habitats in tropical Africa and South America.     

Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection

Background

Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated.

Results

Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material.

Conclusion

Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.     

Palynological investigations on some Hypericum taxa (Hypericaceae) growing naturally in Turkey

This study examined the detailed pollen morphological structures of sixteen Hypericum taxa (four endemic, twelve non endemic) including eight sections showing the natural distribution in Turkey: H. sect. Ascyreia (H. calycinum L.), H. sect. Heterophyllum (H. heterophyllum Vent.), H. Sect. Taeniocarpium (Hypericum confertum Choisy subsp. confertum, H. venustum Fenzl, H. linaroides Bosse), H. sect. Drosocarpium (H. montbretii Spach, H. bithynicum Boiss.), H. sect. Crossophyllum (H. adenotrichum Spach, H. orientale L.), H. sect. Olympia (H. olympicum L. subsp. olympicum), H. sect. Origanifolia (H. origanifolium Willd., H. avicularifolium Jaub and Spach subsp. depilatum (Freyn and Bornm.) Robson var. depilatum, H. avicularifolium Jaub. and Spach subsp. byzantinum) and H. sect. Hypericum (H. tetrapterum Fries, H. perforatum L., H. triquetrifolium Tura). These taxa were studied under light microscope and scanning electron microscope for the first time. Of the taxa studied, H. tetrapterum has the smallest pollen grains (on average 15.85–17.20 × 15.45–16.05 μm); and H. olympicum subsp. olympicum the largest grains (on average 22.90–23.10 × 21.40–22.10 μm). The pollen grains of 15 taxa are subprolate and one taxon is prolate-spheroidal. The regular pollen grains of all 16 taxa are 3-zonocolporate. Ornamentation is microreticulate; lumina regularly spaced in eight taxa, tectum perforatum; tectal perforations regularly spaced in seven taxa and tectum perforatum; and tectal perforations grouped together in one taxon. Endoaperture is cruciform porus, with short lateral and meridional extensions in ten taxa, cruciform porus, transversally elongated, with very small lateral extensions in one taxon and lalongate colpus in five taxa. Basic pollen types are ten taxa in type X, five taxa in type IV and one taxon in type II.     

Optimization of Protein Extraction from <Emphasis Type="Italic">Hypericum perforatum</Emphasis> Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development

Sample preparation is crucial for obtaining high-quality proteins for the purpose of electrophoretic separation and further analysis from tissues that contain high levels of interfering compounds. Hypericum perforatum is a medicinal plant that contains high amounts of phenolic compounds, of which hypericins, hyperforins, and flavonoids contribute to the antidepressant activities of the plant. This study focuses on obtaining optimized amounts of high-quality proteins from H. perforatum, which are suitable for electrophoretic analyses. From the tested protein extraction solutions, sodium borate buffers at pH 9 and 10 gave the best protein yields from mature H. perforatum leaves. With these buffers, relatively high protein yields could also be obtained from roots, stems, and flower buds. The protein extracts of all organs were well resolved in SDS-PAGE after an efficient removal of non-protein contaminants with PVPP, phenol extraction, and methanolic ammonium acetate precipitation. The method was suitable for high-quality protein extraction also from other tested species of genus Hypericum. The applicability of the protocol for immunoblotting was demonstrated by detecting Hyp-1 in H. perforatum leaves at different stages of development. Hyp-1, which has been suggested to attend to the biosynthesis of hypericin, accumulated in high amounts in H. perforatum leaves at mature stage.     

Measurement of hyperforin a constituent of St. John’s wort in plasma by high-performance liquid chromatography

Hyperforin is a constituent of Hypericum perforatum extracts (St. John’s wort, H. perforatum), which have antidepressant action. Hyperforin was extracted from plasma utilising a solid-phase extraction procedure. Chromatography was performed by isocratic reversed-phase high-performance liquid chromatography with UV end-point detection. The calibration curve was linear over the range 0.15–3 μg per ml of plasma. The sensitivity for hyperforin was 4.5 ng on-column. Mean inter- and intra-assay relative standard deviations over the range of the standard curve were less than 5%. The absolute recovery for hyperforin averaged 97.8%.