Differential accumulation of hyperforin and secohyperforin in Hypericum perforatum tissue cultures

Hyperforin is a pharmacologically active constituent of Hypericum perforatum (St. John's wort). In vitro cultures of this medicinal plant were found to contain hyperforin and three related polyprenylated acylphloroglucinol derivatives. The accumulation of these compounds was coupled to shoot regeneration, with secohyperforin being the major constituent in morphogenic cultures. The structure of secohyperforin was elucidated online by LC-DAD, -MS, and -NMR. In multiple shoot cultures, the ratio of hyperforin to secohyperforin was strongly influenced by the phytohormones N6-benzylaminopurine (BAP) and naphthalene-1-acetic acid (NAA). While increasing concentrations of BAP stimulated the formation of hyperforin, increasing concentrations of NAA elevated the level of secohyperforin. No differential stimulation was observed after elicitor treatment. Hyperforin and secohyperforin are proposed to arise from a branch point in the biosynthetic pathway.     

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.     

Hyperforin depletes synaptic vesicles content and induces compartmental redistribution of nerve ending monoamines

Hyperforin, a phloroglucinol derivative found in Hypericum perforatum (St. John's wort) extracts has antidepressant properties in depressed patients. Hyperforin has a unique pharmacological profile and it inhibits uptake of biogenic monoamines as well as amino acid transmitters. We have recently showed that the monoamines uptake inhibition exerted by hyperforin is related to its ability to dissipate the pH gradient across the synaptic vesicle membrane thereby interfering with vesicular monoamines storage. In the present study we demonstrate that hyperforin induces dose-dependent efflux of preloaded [3H]5HT and [3H]DA from rat brain slices. Moreover, we show that hyperforin attenuates depolarization- dependent release of monoamines, while increasing monoamine release by amphetamine or fenfluramine. It is also demonstrated that preincubation of brain slices with reserpine is associated with dose- dependent blunting of efflux due to hyperforin. Our data indicate that hyperforin-induced efflux of [3H]5HT and [3H]DA reflect elevated cytoplasmic concentrations of the two monoamines secondary to the depletion of the synaptic vesicle content and the compartmental redistribution of nerve ending monoamines.     

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%.     

Biosynthesis of hyperforin and adhyperforin from amino acid precursors in shoot cultures of Hypericum perforatum

Hyperforin and adhyperforin contribute to the antidepressant effects of Hypericum perforatum. The involvement of branched-chain amino acids in the biosynthesis of hyperforin and adhyperforin was demonstrated in H. perforatum shoot cultures. L-[U-(13)C(5)]Valine and L-[U-(13)C(6)]isoleucine, upon administration to the shoot cultures, were incorporated into acyl side chain of hyperforin and adhyperforin, respectively. Feeding the shoot cultures with unlabelled L-isoleucine at a concentration of 2mM induced a 3.7-fold increase in the production of adhyperforin. The addition of 3mM L-threonine, a precursor of isoleucine, stimulated a 2.0-fold increase in the accumulation of adhyperforin. The administration of L-valine at concentrations of 0-5mM had no stimulating effect on the hyperforin production in H. perforatum shoot cultures.     

Biosynthesis of the hyperforin skeleton in Hypericum calycinum cell cultures

Hyperforin is an important antidepressant constituent of Hypericum perforatum (St. John's wort). Cell cultures of the related species H. calycinum were found to contain the homologue adhyperforin and to a low extent hyperforin, when grown in BDS medium in the dark. Adhyperforin formation paralleled cell culture growth. Cell-free extracts from the cell cultures contained isobutyrophenone synthase activity catalyzing the condensation of isobutyryl-CoA with three molecules of malonyl-CoA to give phlorisobutyrophenone, i.e. the hyperforin skeleton. The formation of the hyperforins during cell culture growth was preceded by an increase in isobutyrophenone synthase activity. The cell cultures also contained benzophenone synthase and chalcone synthase activities which are involved in xanthone and flavonoid biosyntheses, respectively. The three type III polyketide synthases were separated by anion exchange chromatography.     

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.     

Pseudohypericin and hyperforin in Hypericum perforatum from Northern Turkey: variation among populations, plant parts and phenological stages

Hypericum perforatum is a perennial medicinal plant known as "St. John's wort" in Western Europe and has been used in the treatment of several diseases for centuries. In the present study, morphologic, phenologic and population variability in pseudohypericin and hyperforin concentrations among H. perforatum populations from Northern Turkey was investigated for the first time. The aerial parts of H. perforatum plants representing a total of 30 individuals were collected at full flowering from 10 sites of Northern Turkey to search the regional variation in the secondary metabolits concentrations. For morphologic and phenologic sampling, plants from one site were gathered in five phenological stages vegetative,floral budding, full flowering, fresh fruiting and mature fruiting. The plant materials were air-dried at room temperature and subsequently assayed for chemical concentrations by high performance liquid chromatography. Secondary metabolite concentrations ranged from traces to 2.94mg/g dry weight (DW) for pseudohypedcin and traces -6.29mg/g DW for hyperforin. The differences in the secondary metabolite concentrations among populations of H. perforatum were found to be significant. The populations varied greatly in hyperforin concentrations, whereas they produced a similar amount of pseudohypericin. Concentrations of both secondary metabolites in all tissues increased with advancing of plant development and higher accumulation levels were reached at flowering. Among different tissues, full opened flowers were found to be superior to stems, leaves and the other reproductive parts with regard to pseudohypericin and hyperforin accumulations. The present findings might be useful to optimize the processing methodology of wild-harvested plant material and obtain Increased concentrations of these secondary metabolites.     

Comparison of light and dark nitrogenase activity in selected soil cyanobacteria

Frequency of heterocytes and nitrogenase activity (NA) under light and dark cultivation conditions was determined in 12 cyanobacterial strains isolated from various soil habitats. In spite of a high variability, significant differences in NA among the strains were found in response of light and dark cultivation. Relatively high NA (9.9-15.3 micromol/h C2H4 per g fresh mass) under light conditions and basal NA after 12 h of dark cultivation were detected in Anabaena, Nodularia, Tolypothrix, and 1 of Cylindrospermum strains. On the other hand, significantly lower NA (0.76-5.4 micromol/h C2H4 per g fresh mass) was found under light conditions in Trichormus, Nostoc and another Cylindrospermum strain; the activity completely disappeared after 12 h of dark cultivation. NA values were not directly related to the frequency of the heterocytes. The total NA of cyanobacterial colony was found to be probably independent of the number and/or position of heterocytes. Remarkable differences in NA between strains isolated from cultivated fields and strains originating from natural or non-cultivated soils were found.     

Hyperforin induces Ca-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A2 activation through select phospholipid interactions

Here, we investigated the modulation of cytosolic phospholipase A₂ (cPLA₂)-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10 µM) versus unstimulated cells, which was blocked by cPLA₂α-inhibition, and induced translocation of cPLA₂ to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca²⁺ by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA₂ at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA₂ phosphorylation. However, neither AA release nor translocation of cPLA₂ was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA₂ in the presence of Ca²⁺. However, when Ca²⁺ was omitted, hyperforin caused a prominent increase in cPLA₂ activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional ¹H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA₂ activation by enabling its access towards select lipid membranes independent of Ca²⁺ ions. Such Ca²⁺- and phosphorylation-independent mechanism of cPLA₂ activation may apply also to other membrane-interfering molecules.     

Pseudohypericin and hyperforin in two Turkish Hypericum species: Variation among plant parts and phenological stages

The genus Hypericum has received considerable interest from scientists, as it contains the variety of structurally diverse natural products which possess a wide array of biological properties, mainly hypericins and hyperforin. In the present study, variations of pseudohypericin and hyperforin were investigated in two Turkish species of Hypericum, namely Hypericum perfoliatum and Hypericum origanifolium. Wild growing plants were harvested at vegetative, floral budding, flowering, fresh fruiting and mature fruiting stages, and dissected into stem, leaf and reproductive tissues and assayed for chemical contents by high performance liquid chromatography method. Content of pseudohypericin and hyperforin in samples of the whole plant increased during the course of ontogenesis in both species. The highest levels of the chemicals were reached at full flowering (2.62 mg/g dry weight (DW) pseudohypericin and 1.84 mg/g DW hyperforin for H. perfoliatum; 0.93 mg/g DW pseudohypericin and 1.63 mg/g DW hyperforin for H. origanifolium). Among different reproductive parts, full opened flowers produced the highest amount of pseudohypericin (1.18 mg/g DW) and hyperforin (4.36 mg/g DW) in H. origanifolium. Similarly, the highest pseudohypericin accumulation was observed in full opened flowers in H. perfoliatum (7.41 mg/g DW) while floral buds of this species produced the highest amount of hyperforin (7.80 mg/g DW). These data can be useful when elucidating the medicinal properties of the species and the chemosystematic significance of hyperforin and pseudohypericin in the relationships among species of Hypericum.     

Inhibition of vesicular uptake of monoamines by hyperforin

Hyperforin is the major active ingredient of Hypericum perforatum (St John's Wort), a traditional antidepressant medication. This study evaluated its inhibitory effects on the synaptic uptake of monoamines in rat forebrain homogenates, comparing the nature of the inhibition at synaptic and vesicular monoamine transporters. A hyperforin-rich extract inhibited with equal potencies the sodium-dependent uptake of the monoamine neurotransmitters serotonin [5-HT], dopamine [DA] and norepinephrine [NE] into rat brain synaptosomes. Hyperforin inhibited the uptake of all three monoamines noncompetitively, in marked contrast with the competitive inhibition exerted by fluoxetine, GBR12909 or desipramine on the uptake of these monoamines. Hyperforin had no inhibitory effect on the binding of [3H]paroxetine, [3H]GBR12935 and [3H]nisoxetine to membrane presynaptic transporters for 5-HT, DA and NE, respectively. The apparent presynaptic inhibition of monoamine uptake could reflect a "reserpine-like mechanism" by which hyperforin induced release of neurotransmitters from synaptic vesicles into the cytoplasm. Thus, we assessed the effects of hyperforin on the vesicular monoamine transporter. Hyperforin inhibited with equal potencies the uptake of the three tritiated monoamines to rat brain synaptic vesicles. Similarly to the synaptosomal uptake, the vesicular uptake was also noncompetitively inhibited by hyperforin. Notably, hyperforin did not affect the direct binding on [3H]dihydrotetrabenazine, a selective vesicular monoamine transporter ligand, to rat forebrain membranes. Our results support the notion that hyperforin interferes with the storage of monoamines in synaptic vesicles, rather than being a selective inhibitor of either synaptic membrane or vesicular monoamine transporters.     

Biosynthesis, accumulation and degradation of theobromine in developing Theobroma cacao fruits

We have studied the purine alkaloid content and purine metabolism in Theobroma cacao fruits at differing growth stages: Stage A (young small fruit, fresh weight, ca. 2 g); stage B (medium size fruit, fresh weight, ca. 100 g) and stage C (large size, fresh weight, ca. 500 g). The major purine alkaloid in stage A fruits (mainly pericarp) was theobromine (0.7 micromol g(-1) fresh weight), followed by caffeine (0.09 micromol g(-1) fresh weight). The theobromine content of the pericarp decreased sharply with tissue age, and the caffeine content decreased gradually. A large amount of theobromine (22 micromol g(-1) fresh weight) had accumulated in seeds (mainly cotyledons) of stage C fruits. Theobromine was found also in the seed coat and placenta. Tracer experiments with [8-(14)C]adenine show that the major sites of theobromine synthesis are the young pericarp and cotyledons of T. cacao fruits. Limited amounts of purine alkaloids may be transported from the pericarp to seed tissue, but most purine alkaloids that accumulated in seeds appeared to be synthesised in cotyledons. Degradation of [8-(14)C]theobromine and [8-(14)C]caffeine to CO2 via 3-methylxanthine and ureides (allantoin and allantoic acid) was detected only in the pericarp of stage C fruits.     

The involvement of sodium and calcium ions in the release of amino acid neurotransmitters from mouse cortical slices elicited by hyperforin

Hyperforin is currently considered to be the major active antidepressant constituent of the medicinal herb St. John's wort (Hypericum perforatum L.). The mechanism of action however, is still largely unknown, although the involvement of sodium and calcium has been recently inferred. In the present study hyperforin (5 microM) significantly potentiated the release of endogenous aspartate and glutamate from mouse cortical slices when stimulated by veratridine or potassium. Hyperforin (5 microM) also stimulated the release of aspartate, glutamate, serine, glycine and GABA when perfused on its own. Perfusion of the sodium channel blocker, tetrodotoxin (TTX) inhibited the effect of hyperforin, whereas removal of extracellular calcium potentiated the effect. Our observations suggests that hyperforin increases the overflow of neurotransmitters from mouse cerebral cortex possibly through facilitating the entry of sodium into the neurone which leads to the release of calcium from intracellular stores.     

5 Alpha-reductase in the non-secretory cells of the rat ventral prostate epithelium

Kinetic constants for the 5 alpha-reductase were determined in freshly isolated epithelial cells from the rat ventral prostate. Studies were also performed on stromal tissue but not isolated stromal cells for comparison. Secretory and non-secretory epithelial cells were separated by centrifugation in a Percoll gradient. Both epithelial cell populations metabolized testosterone to predominantly 5 alpha-dihydrotestosterone (5 alpha-DHT), although when expressed per cell the capacity for conversion was 3-4-fold higher for secretory cells (7.4 pmol/min/10(6) cells) than for non-secretory cells (2.3 pmol/min/10(6) cells; P less than 0.01 in 4 separate studies). When compared per mg cytosol protein this difference became non-significant. Stromal tissue contained a 5 alpha-reductase Vmax (expressed) per mg protein) which was comparable to the non-secretory cell enzyme. Lineweaver-Burke plots revealed different Km values for the different cell populations (12.5, 5.9 and 4.7 microM for secretory, non-secretory and stromal cells, respectively) suggesting the presence of different isoforms of the enzyme, or differences in the intracellular concentrations of enzyme antagonists.     

Induction of phenolic compounds in wheat (Triticum aestivum L.) tissue cultures by streptomycin

The tissue cultures of wheat (Triticum aestivum L.) were induced from the mature embryos (explants) of the dry grains and grown on MS medium containing kinetin (0.1 mg/1) and 2,4 D (1.0 mg/l). The cultures were incubated for two weeks at (25+/-2) degrees C under a light/dark regime (16 h light daily). The formed calli were subcultured at the beginning of the stationary growth phase (15 days) with fresh MS medium containing 0, 5, 10, 25, 50, 100, 150 mg/l streptomycin elicitor and maintained for two weeks for three subcultures. A significant increase in phenylalanine ammonia lyase (PAL) activity coincided with the increase of the total phenolic compounds after elicitation with streptomycin. Maximum induction was recorded during the first two weeks, then gradually declined during the rest of the experimental period, but the values attained were still markedly higher than that of the control. The endogenous cinnamic acid content was also increased significantly with the increase in PAL activity making about 2-18% of the total phenolic acids. The growth and accumulation of phenolic compounds were inversely related. However, accumulation of phenolic compounds became limited for growth of wheat tissue culture especially during the long term cultivation.     

High anthocyanin accumulation in the dark by strawberry (Fragaria ananassa) callus

Fragaria ananassa (strawberry) callus, which produced high amounts of anthocyanin in the dark, was isolated from a cell line not producing anthocyanin. The isolated callus (FAR) was homogeneous and more than 90% of the cells were pigmented. The FAR callus accumulated more than 1000 g of anthocyanin per g fresh cell in the dark. Four different basal solid media were examined to maintain FAR callus: Though growth rate and anthocyanin concentration were different on each media, total anthocyanin production was about the same at 400 g anthocyanin/0.1 g fresh cell wt after 22 days. This FAR cell line could therefore be used for the industrial production of anthocyanin.     

Structural investigations of isomeric oxidised forms of hyperforin by HPLC-NMR and HPLC-MSn

The prenylated phloroglucinol hyperforin, thought to be an essential component for the anti-depressant activity of St. John's Wort (Hypericum perforatum), is unstable. The facile oxidative degradation of hyperforin poses serious problems for standardisation, and may also dramatically affect the pharmacological activity of the extracts. Hyperforin was dissolved in hexane and stored at room temperature for 3 days and yielded various closely related degradation products which, although difficult to isolate on the preparative scale, have been analysed by on-flow and stop-flow HPLC-NMR and HPLC-MS/MS. From on-line spectroscopic data, and with the aid of complementary in-mixture standard NMR two-dimensional correlation experiments, the different oxidised forms of hyperforin were found to be phloroglucinol derivatives in which a hydroxy-dihydrofuran ring is formed involving the enol OH at C-7 or C-9 (tautomeric form) and the prenyl chain at C-8 of the core nucleus of hyperforin. The strategy followed for the on-line identification of these constituents is discussed.     

In vitro antimalarial activity of hyperforin, a prenylated acylphloroglucinol. A structure-activity study

The antimalarial activity of hyperforin, a phenol-like compound that can be easily absorbed orally, and a series of derivatives variously modified on the cyclohexatrienone system was investigated. Hyperforin was active against Plasmodium falciparum with an IC(50) value in the micromolar range, and the activity was not critically dependent on either its phenol-like sensitivity to autooxidation or the presence of unsaturation on the prenyl residues. Related phloroglucinols like the hop beta-acids and the enantiomers of usnic acid showed only marginal activity, suggesting that hyperforin is a new antimalarial chemotype.     

Variation of Bioactive Compounds in Hypericum perforatum Growing in Turkey During Its Phenological Cycle

The present study was conducted to determine phenologic and morphogeneUc variation of hyperlcln, chlorogenlc acid and flavonoids, as rutin, hyperoside, apigenin-7-O-glucoside, quercitrin, quercetin content of Hypericum perforatum L. growing in Turkey. Wild growing plants were harvested at vegetative, floral budding, full flowering, fresh frulUng and mature fruiting stages and dissected into stem, leaf and reproductive tissues and assayed for bioacUve compounds by the High performance liquid chromatography （HPLC） method. Hypericin concentration ranged between 0 and 2.73 mg/g DW, chlorogenic acid 0.00-3.64 mg/g DW, rutin 0.00-3.36 mg/g DW, hyperoside 0.04- 22.42 mg/g DW, quercitrin 0.03-3.46 mg/g DW and quercetin 0.04-1.02 mg/g DW depending on ontogenetic and morphogenetic sampling. Leaves were found to be superior to stems and reproductive parts with regard to phenolic accumulation for all compounds tested while flowers accumulated the highest levels of hypericln. Quercltrln, quercetln and hypericin content in all tissues increased with advancing of developmental stages and reached their highest level during flower ontogenesis. Similarly, chlorogenic acid, hyperoside and apigenin-7-O-glucoside content in different plant parts increased during plant development, however, the highest level was observed at different stages of plant phenology for each tissue. Chlorogenic acid was not detected in stems, leaves and reproductive parts in several stages of plant phenology and its variation during plant growth showed inconsistent manner. In contrast to the other compounds examined, rutin content of stems and leaves decreased with advanc- ing of plant development and the highest level for both tissues was observed at the vegetative stage. However, content of the same compound in reproductive parts was the highest at mature fruiting. The present findings might be useful to obtain increased concentration of these natural compounds.