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Plants of the genus Hypericum, commonly known as “St. Johnʼs wort” (“spathohorto” or “valsamo” in Greek), have been used since antiquity for their therapeutic properties. Wild-harvested Hypericum plants are still popular today in herbal medicines, commercially exploited due to their bioactive compounds, hypericin and hyperforin, which have antidepressant, antimicrobial and antiviral activity. Species identification of commercial products is therefore important and DNA barcoding, a molecular method that uses small sequences of organismsʼ genome as barcodes, can be useful in this direction. In this study, we collected plants of the genus Hypericum that grow wild in North-Eastern Greece and explored the efficiency of matK, and trnH-psbA regions as DNA barcodes for their identification. We focused on 5 taxa, namely H. aucheri, H. montbretii, H. olympicum, H. perforatum subsp. perforatum, and H. thasium, the latter a rare Balkan endemic species collected for the first time from mainland Greece. matK (using the genus-specific primers designed herein), trnH-psbA, and their combination were effectively used for the identification of the 5 Hypericum taxa and the discrimination of different H. perforatum subsp. perforatum populations. These barcodes were also able to discriminate Greek populations of H. perforatum, H. aucheri, H. montbretii, and H. olympicum from populations of the same species growing in other countries.

Medicinal plants of the genus Hypericum are rich sources of bioactive naphthodianthrones, which are unique in the plant kingdom, but quite common in fungal endophytes. Cultivable endophytic fungi were isolated from 14 different Hypericum spp. originating from seeds grown under in vitro conditions and further acclimated to outdoor conditions. Among 37 fungal isolates yielded from the aerial and underground plant organs, 25 were identified at the species level by the fungal barcode marker internal transcribed spacer rDNA and protein-coding gene region of tef1α. Ten of them were isolated from Hypericum spp. for the first time. The axenic cultures of the isolated endophytes were screened for the production of extracellular enzymes, as well as bioactive naphthodianthrones and their putative precursors by Bornträgerʼs test and HPLC-HRMS. Traces of naphthodianthrones and their intermediates, emodin, emodin anthrone, skyrin, or pseudohypericin, were detected in the fungal mycelia of Acremonium sclerotigenum and Plectosphaerella cucumerina isolated from Hypericum perforatum and Hypericum maculatum, respectively. Traces of emodin, hypericin, and pseudohypericin were released in the broth by Scedosporium apiospermum, P. cucumerina, and Fusarium oxysporum during submerged fermentation. These endophytes were isolated from several hypericin-producing Hypericum spp. Taken together, our results reveal the biosynthetic potential of cultivable endophytic fungi harbored in Hypericum plants as well as evidence of the existence of remarkable plant-endophyte relationships in selected non-native ecological niches. A possible role of the extracellular enzymes in plant secondary metabolism is discussed.

Hypericin is a natural polycyclic quinone found in Hypericum perforatum. Although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably because hypericin is a highly lipophilic compound that is poorly soluble in a physiological medium. The major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the Caco-2 cell system under different experimental conditions. The permeation characteristics of hypericin (5 µM) in the absence or presence of the model flavonoid quercitrin (20 µM) were studied in the absorptive direction, without or with the addition of 10 % FBS to the transport buffer apically. Following the application of hypericin to the apical side of the monolayer, only negligible amounts of the compound were found in the basolateral compartment when the experiment was performed with a transport buffer. The amount of hypericin in the basolateral compartment increased in the presence of quercitrin (from 0 to 4 %). The majority of hypericin was found after cell extraction (44 % in the absence and 64 % in the presence of quercitrin). When 10 % FBS was added to the transport buffer in the apical compartment to improve the solubility of hypericin in the aqueous solution, around 68 % of hypericin was bound to the serum proteins. Under these experimental conditions, the amount of hypericin in the cells/cell membrane was only 13 % in the absence and 18 % in the presence of quercitrin. The low recovery and significant amounts of hypericin found after cell extraction and bound to the surface of the culture dish made a correct estimation of permeability constants impossible. Fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. The precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. However, our experiments demonstrated that regardless of the experimental conditions, the permeation characteristics of hypericin improved in the presence of the model flavonoid quercitrin.

Photosensitizing properties of hypericin are well known, and the chicken chorioallantoic membrane has previously been used to test photodynamic effects of hypericin and other substances. In our study the photodynamic effect of hypericin in the ex ovo quail chorioallantoic membrane model was evaluated. Steady-state and time-resolved fluorescence spectroscopy of hypericin solution in PEG-400 and its mixture in PBS was performed to assess and characterize the process of aggregation and disaggregation of hypericin during the drug formulation preparation. A therapeutical formulation (2 µg/g of embryo weight) was topically applied on CAM into the silicone ring. Hypericin was excited by diode laser with wavelength 405 nm, fluence rate 140 mW/cm 2 , and fluence 16.8 J/cm 2 . Hypericin in 100 % PEG-400 exhibits typical fluorescence spectra with a maximum of about 600 nm, while hypericin 10% PEG-400 formulation exhibits almost no fluorescence. Time resolved spectra analysis showed fluorescence decay of hypericin in 100 % PEG-400 solution with a mean lifetime of 5.1 ns and in 10 % PEG 4.1 ns. Damage of quail chorioallantoic membrane vasculature after photodynamic therapy ranged from hemorrhage and vanishing of capillary vessels to thrombosis, lysis, and hemorrhage of larger vessels. The presented findings suggest that quail embryos can be used as a suitable model to test the effect of hypericin and other photodynamic compounds.

St. Johnʼs wort (Hypericum perforatum) has been intensively investigated for its antidepressive activity, but dermatological applications also have a long tradition. Topical St. Johnʼs wort preparations such as oils or tinctures are used for the treatment of minor wounds and burns, sunburns, abrasions, bruises, contusions, ulcers, myalgia, and many others. Pharmacological research supports the use in these fields. Of the constituents, naphthodianthrones (e.g., hypericin) and phloroglucinols (e.g., hyperforin) have interesting pharmacological profiles, including antioxidant, anti-inflammatory, anticancer, and antimicrobial activities. In addition, hyperforin stimulates growth and differentiation of keratinocytes, and hypericin is a photosensitizer which can be used for selective treatment of nonmelanoma skin cancer. However, clinical research in this field is still scarce. Recently, sporadic trials have been conducted in wound healing, atopic dermatitis, psoriasis, and herpes simplex infections, partly with purified single constituents and modern dermatological formulations. St. Johnʼs wort also has a potential for use in medical skin care. Composition and stability of pharmaceutical formulations vary greatly depending on origin of the plant material, production method, lipophilicity of solvents, and storage conditions, and this must be regarded with respect to practical as well as scientific purposes.

Environmental factors are known to influence strongly the accumulation of secondary metabo- lites in plant tissues. In a previous paper, we studied the contents of (pseudo)hypericin and its immediate precursors in wild populations of various Hypericum species on the island of Crete, Greece, in dependence on their developmental stage. In this study, we investigated the effect of the habitat altitude on the total hypericins con- tent of the plants, which is defined as the sum of protohypericin, hypericin, protopseudohypericin and pseudohypericin. Taking into account our previous finding that the highest accumulation is found during the flowering period in June, we collected the aerial parts of spontaneously grow- ing H. perforatum L., H. triquentrifolium Turra ,H. empetrifolium Willd. and H. perfoliatum L. during that time frame at elevations between 100 and 600 m above sea level, however, bearing in mind the time lag in development with increasing alti- tude. HPLC analysis of the plant material, separa- ted again into a flowers and a leaves/petioles fraction, revealed great differences in the total hypericin content in dependence on the altitude of the habitat. Specifically, a clear trend was re- vealed, showing an increase of the total hypericin content with increasing altitude. However, no changes could be observed in the ratio of hyperi- cin to protohypericin and in that of pseudohyper- icin to protopseudohypericin. The habitats of the employed plants were again randomly distrib- uted all over Crete. It is proposed that higher light intensities accompanied by enhanced UV-B radi- ation and lower air temperature might be re- sponsible for the increasing levels of total hyper- icins with increasing altitude.

We evaluated the pharmacokinetic interaction between a low-hyperforin St John's wort (SJW) extract and alprazolam, caffeine, tolbutamide, and digoxin. Previous reports on other SJW products had shown remarkably decreased plasma concentrations of certain co-medicated drugs, which was attributed to an inducing effect of SJW on cytochrome P-450 (CYP) and p-glycoprotein (p-gp) activity. Two randomised, placebo-controlled studies were performed with 28 healthy volunteers (age 18 - 55 years) in each study. In study A, single doses of alprazolam (1 mg; substrate of CYP3A4) and caffeine (100 mg; CYP1A2) were given on days 1 and 11. In study B, single doses of tolbutamide (500 mg, days 1 and 11; CYP2C9) and multiple doses of digoxin (0.75 mg on days -2 and -1, 0.25 mg/die on days 1 to 11; p-gp) were given. The participants received SJW (Esbericum capsules; 240 mg/die of extract, 3.5 mg hyperforin) or placebo on days 2 to 11. Blood for pharmacokinetic analysis was drawn on days 1 and 11. No statistically significant differences were found in the primary kinetic parameter, AUC0 - 24, of alprazolam, caffeine (AUC0 - 12), paraxanthine, tolbutamide, 4-hydroxytolbutamide, and digoxin between the placebo group and the SJW group at the end of the study. The SJW-induced change in AUCs was less than 12 % of the initial median AUC of the participants in studies A and B, thus clinically irrelevant. On day 11, trough concentrations were 2.0 (range 0.6 - 4.1) microg/L and 1.0 (0.2 - 3.9) microg/L for hypericin and pseudohypericin, respectively, whereas hyperforin concentrations were below the quantification limit (< 1 microg/L). Kinetics of investigated probe drugs were only marginally influenced by concomitant treatment with Esbericum capsules. This may be due in particular to the low hyperforin plasma concentration as this SJW component has been shown to activate the PXR receptor which regulates expression of CYP3A4 and p-gp. Our findings corroborate the view that reports about interactions of other SJW extracts seem not to be predictive for the product we studied.

While the interest in medicinal plants continues to grow, there is a lack of basic information with respect to efficient protocols for plant production. Recently, in vitro regeneration protocols have been developed to provide masses of sterile, consistent St. John's wort. The current study assessed the potential for acclimatization of in vitro grown St. John's wort plantlets to a nutrient film technique (NFT) hydroponic system in a controlled environment greenhouse. Quantitative analyses of hypericin, hyperforin and pseudohypericin in flower tissues were used as the parameters to assess the quality of the greenhouse-grown plants. The three bioactive compounds were found to be present in similar or higher amounts than previously reported values for field-grown plants. These data provide evidence that greenhouse hydroponic systems can be effectively used for the efficient production of St. John's wort and other medicinal plants.

NF-kappa B is a transcription factor involved in immune and inflammatory responses. Here we show that micromolar concentrations of hypericin inhibited the PMA- and TNF-alpha-induced activation of NF-kappa B in HeLa and TC10 cells, respectively. In contrast, NF-kappa B activated by H2O2 was not influenced by hypericin, indicating a pathway-specificity of hypericin. Hyperforin and a Hypericum perforatum extract standardised on 0.15% hypericin and 5% hyperforin were not active at pharmacologically relevant concentrations. The PMA/TNF-alpha-induced transcription of a reporter gene, which is under the control of the NF-kappa B-dependent IL-6 promoter, was strongly reduced by preincubation with hypericin.

It has been shown recently that the fraction Ilk of acrude extract of Hypericum per foratum, (St. Johns wort) thatcontained both hypericin (1) and pseudohypericin (2), was re-markably active in the rats forced swimming test (FST) afterPorsolt. However, neither of the naphthodianthrones isolatedfrom this fraction were sufficiently effective when administeredsuspended in water. The solubility of 1 and 2 is remarkably in-creased in the presence of a fraction containing procyanidins, especially procyanidin B2, which is present also in the active Hy- pericum fraction Ilic. The cooperative effect of procyanidins sig-nificantly increased the in vivo effects of 1 and 2, which exhib-ited inverted U-shaped dose response curves, in the FST. Theanti-immobility effect of solubilized 1 and 2 was antagonizedby the dopamine antagonist sulpiride. These data indicate thatnaphthodianthrones are antidepressant constituents of H. per-foratum and suggest that the dopaminergic system is involvedin their action.Phytomedicines based on extracts from the herb of Hypericum

The photodynamic action of hypericin (HYP) in vitro was evaluated using human leukemic HL-60 and lung carcinoma A549 cell lines. After illumination HYP (1 x 10 (-5) M) reduced the proliferation and/or survival of HL-60 and A549 cells vs. controls to almost to 0 % and 29 %, respectively. A lower concentration of HYP (1 x 10 (-6) M) decreased the proliferation and/or survival only in HL-60 cells. Non-cytotoxic concentrations of the carbonic anhydrase inhibitor acetazolamide (ACTZ) (1 x 10 (-3)-1 x 10 (-6) M) significantly potentiated these effects of HYP (1 x 10 (-6)M) in HL-60, but not in the A549 cell line. The highest concentration of ACTZ (1 x 10 (-3) M) also induced an increase of the subdiploid G 0 /G 1 population in HYP (1 x 10 (-6) M) treated HL-60 cells from 14 % to 24 %. The results indicate that the photogenerated pH drop may participate in the potentiation of the photodynamic action of HYP observed in leukemia cells.

Pseudohypericin (PH) and hypericin (H) are photosensitizing plant pigments from Hypericum perforatum. H displays cytotoxic and apoptosis-inducing effects in neoplastic cell lines. Here, we have assessed the phototoxic and apoptosis-inducing capacity of PH compared to H in a cell culture model with human leukemic lymphoma cells (Jurkat). Treatment with both photoactivated H and PH resulted in a dose-dependent inhibition of cell proliferation, whereas not photoactivated H and PH had no effect at the concentrations tested. The half-maximal inhibitory concentration (IC 50 ) of H was lower (100 ng/mL) as compared to PH (200 ng/mL) (p < 0.05). In an apoptosis assay we found a dose-dependent increase of DNA fragmentation after treatment with both photoactivated H and PH. The cytotoxic potential of PH should be taken into account during systemic therapy with Hypericum extracts, since PH is about two times more abundant than H in Hypericum perforatum.

Fagopyrin and its derivatives are dimerized anthraquinone polyphenolic substances from buckwheat. They act as photosensitizers upon excitation with visible light (540–610nm), which causes phototoxic effect after ingestion of large amounts of buckwheat called fagopyrism (1). According to known structures and transformations of hypericin derivatives from St. John's wort and their similarity to fagopyrin, several forms of fagopyrin derivatives have been postulated as pseudofagopyrin and protofagopyrin (2). It was shown that pre-forms of fagopyrin transform to fagopyrin in plant extract under daylight exposure (3). We optimized the extraction method of fagopyrins from plant material. We developed a HPLC method coupled with fluorescence detector (excitation wavelength 330nm, emission 590nm) for separation and detection of those compounds. The HPLC method yielded several chromatogram peaks with close retention times presenting different forms of fagopyrin. Various buckwheat products available on the market were analyzed using this method. The highest amount of fagopyrins was found in buckwheat herb samples and less in fruit samples, which contain most of the compounds in peels. Buckwheat herb was used for further observation of the nature of fagopyrins in plant extract. As described previously (4), we observed that the content of different forms of fagopyrin varied due to different extraction conditions (time, temperature, solvent). Since the differences were observed even if the conditions changed after the end of the extraction (removal of the herbal substance), we assume that this changes were due to transformations of fagopyrins and not only due to extraction efficacy. The transformations were at least partly reversible. References: 1. Chick H, Ellinger P (1941)J Physiol 212–230. 2. Brockmann H, Lackner H (1979) Tetrahedron Letters 18: 1575–1578. 3. Habermann B (2000) Arch Farm, Farm Med Chem 333, Suppl. 2. 4. Hinneburg, Neubert Reinhard HH (2005)J Agric Food Chem 53: 3–7.

Both Hypericum perforatum and H. maculatum are common species throughout Estonia and neighbouring countries. The study used folklore texts from the HERBA [1] database collected between 1868-1994 on medicinal plants in Estonia. 117 texts describing 137 usage cases of plants bearing vernacular names related to naistepuna [Hypericum in Estonian, direct translation women's red] were selected and analyzed using textological methods. Almost 30% of the usage cases were related to gynecological problems (as the name implies), followed by 14% for stomach problems, 10% for bladder and kidney problems and 5% each for stomachache caused by overexertion, skin diseases and pain. Although altogether 8 species belonging to 7 genera or even families bore such vernacular names in different places, the most frequently used plants were two species of genus Hypericum. Those two externally very similar species were not distinguished by ordinary people and were used in folk medicine as one. The content of hypericin [2] and total flavonoids before [3] and after [4] hydrolysis of glucosides was determined spectrophotometrically in both Hypericum species. The standard deviations of these methods did not exceed 0.5%, 8.7% and 5.4%, respectively (n=4). The flowering tops (15cm) and flowers in three different blooming stages (at the beginning of blooming, in full bloom and at the end of blooming) of H. perforatum and H. maculatum, collected in 2008 from South-Estonia (Antsla parish), were used as plant material. H. maculatum contained about a 2.5 times more hypericin (141-228mg%) than H. perforatum (75-81mg%). The amount of hypericin detected was higher in the flowers and lower in the flowering tops of both analyzed Hypericum species, but the comparative analysis of H. perforatum ja H. maculatum showed no similar tendencies for flavonoid content. Both species contained practically the same amount of total flavonoids (5,6-6,2% and 4,6-6,0%, respectively), but H. perforatum contained somewhat more flavonoid aglycones (1,1-2,0%) than H. maculatum (0,7-1,5%). It is necessary to continue this work.

Hyperici Oleum (St. John's wort oil) used in wound healing contains no hypericin. By using the sunlight maceration method described in the supplement to DAB 6 (EB 6), lipophilic breakdown products of this compound are obtained which lend the oil its red colour. Hyperforin, which is responsible for the oil's therapeutic activity could, for the first time, be identified and quantitatively determined by TLC and HPLC after solid-phase extraction. The stability of hyperforin is limited; sufficient shelf-life could only be achieved by hot maceration of dried flowers with eutanol G and storage in the absence of air. By gradient HPLC further polar hyperforin analogues were detected in those St. John's wort oils in which hyperforin had decomposed. At the same time flavonoids and xanthones could be identified. A procedure for the quantitative determination of flavonoids in St. John's wort was validated. The action of light during preparation of the oil led to a rise in the content of flavonoids.

The 74 samples of six HYPERICUM species (H. PERFOROTUM, H. HIRSUTUM, H. MACULATUM, H. TETRAPTERUM, H. MONTANUM, AND H. HUMIFUSUM) were collected around Slovenia and analysed for the content of ten substances (rutin, hyperoside, isoquercetin, quercitrin, quercetin, I3,II8-biapigenin, amentoflavone, pseudohypericin, hypericin, and hyperforin). The flowers were analysed separately from the green parts of the plants (herbs). The highest content of most of the substances was found in the flowers of H. PERFOROTUM. Among the herbal samples (without flowers), H. MONTANUM and H. HIRSUTUM contained significantly higher levels of amentoflavone (average 3-fold and 2.5-fold higher, respectively), than the herbs of H. PERFOROTUM. In the herbal part of H. PERFOROTUM the contents of all constituents strongly correlate with the contents of the same compound in flowers, except for the content of amentoflavone, which is independent in these two parts. Rutin and hyperoside are in positive correlation, and quercitrin is in negative correlation with the altitude of the growing site.

The genus Hypericum (Hypericaceae) is represented by nearly 100 taxa grouped under 19 sections in Turkey. Among them, 45 species are endemic. All members of the genus may be referred to as St. John's wort in the world. The genus is known as “sari kantaron, kantaron, binbirdelik otu, mayasil otu” and most of them, especially H. perforatum, have been used for the treatment of burns, wounds, haemorroids, diarrhorea and ulcers in Turkish traditional medicine. The pharmacological studies showed that this species has several activities: anti-depressant, anti-inflammatory, anti-microbial, anti-viral, anti-nociceptive and wound healing. The chemical composition of the Hypericum species is composed of naphthodianthrones (especially hypericin and pseudohypericin), acylphloroglucinol derivatives (especially hyperforin and adhyperforin), flavonoids (especially quercetin, quercitrin, hyperoside and biapigenin), tannins and volatile oils. Investigations on the chemical composition and biological activities, as well as hypericin content of Turkish Hypericum species, have been carried out for about 25 years. The aim of this study was to review and summarise important studies about Turkish Hypericumspecies. Endemic species are indicated with (e) in the text. Key words: Hypericum species, Turkey, endemic, chemical composition, biological activity, agricultural study, traditional use.

The contents of (pseudo)hypericin and their immediate precursors were studied in wild populations of various Hypericum species on the island of Crete, Greece. Therefore, the aerial parts of wild grown H. perforatum, H. triquentrifolium, H. empetrifolium and H. perfoliatum shoots were collected throughout the island and the quantitative variations in (proto)hypericin and (proto)-pseudohypericin examined. The plant material was harvested at different stages of the life cycle of the species and the contents in the above-mentioned compounds analyzed discriminating between flowers/fruits and leaves/petioles. HPLC analysis ofhypericin, pseudohypericin and their immediate precursors, protohypericin and protopseudohypericin, revealed great differences in the contents of the compounds in dependence on the developmental stage of the plants. In all examined species the highest concentrations of hypericin were found during blossoming whereas the lowest concentrations were present during ripening of the fruits. H. perforatum and H. triquentrifolium show much higher hypericin levels in flowers/fruits compared to leaves/petioles, whereas the species H. empetrifolium and H. perfoliatum show similar concentrations of total hypericins in both flowers/ fruits and leaves/petioles. In the different species the levels of (proto)hypericin and (proto)pseudohypericin varied, but in almost all samples from flowers/fruits and leaves/petioles the ratio of (proto)hypericin to (proto)pseudohypericin was higher than one. When the total amount of hypericins per entire aerial part of a plant was calculated for all developmental stages, we found that H. perforatum contained the highest amount of hypericin. This in combination with the comparatively high concentration of hypericins in flowers/fruits and in leaves/petioles in this species, as well as the high ratio of (proto)hypericin to (proto)pseudohypericin, especially during the developmental stage of blossoming, encourages us to think about the possibility of cultivating Hypericum perforatum in Crete as a medicinal plant in the future.

Hypericum perforatum L. (St. John's wort) is a medicinal plant used for many pathologies, especially for the treatment of mild to moderate depression. In the present study we have investigated the cytotoxic activity of the locally collected (Epirus region) Hypericum perforatum L. against cultured T24 and NBT-II bladder cancer cell lines. The lipophilic extract of the herb, prepared using petroleum ether, induced apoptosis displaying LC(50) values at concentrations as low as 4 and 5 microg/mL. A fraction of this extract displayed 60 % cell growth inhibition at a concentration of 0.95 microg/mL. Evaluating the importance of various biologically active components of the extract, it was found that hypericins (hypericin, pseudohypericin, etc.) were identified only in the methanolic (lipophobic) extract of the herb, and not in the active lipophilic extract. In addition, hyperforin concentrations in the lipophilic extract and its most active fraction, were 0.94 microg/mL, and 0.17 microg/mL, respectively, while the active cytotoxic concentration of pure hyperforin appeared in the range of 1.8 microg/mL - 5.0 microg/mL. Therefore, pure hyperforin does not seem to contribute significantly to the cytotoxicity activity. Chlorophylls were identified in low, not significantly different, concentrations in all extracts and fractions and were not correlated to the biological activity. Owing to the combination of significant cytotoxic activity, natural abundance and low toxicity, the lipophilic extract of Hypericum perforatum holds the promise of being an interesting, new, antiproliferative agent against bladder cancer that deserves further investigation.

Suspension cultures were established from the shoots of sterile germinated seeds of various provenances of seven Hypericum (H.) species in a half strength modified Murashige and Skoog liquid medium. In most strains of H. perforatum (18 provenances) and all strains of H. maculatum (6 provenances) as well as in the cultures of H. tomentosum, H. bithynicum, H. glandulosum and H. balearicum, hypericin and pseudohypericin could be proven, however, in extremely varying amounts. In general, the pseudohypericin content was significantly higher than that of hypericin. The flavonoid patterns, comprising monomeric quercetin derivatives and dimeric apigenin derivatives, varied among strains over a wide range.

A common biological alteration in patients with major depression is the activation of the hypothalamic-pituitary-adrenal (HPA) axis, manifested as hypersecretion of adrenocorticotropic hormone (ACTH) and cortisol. The hyperactivity of the HPA axis in depressed patients can be corrected during clinically effective therapy with standard antidepressant drugs such as imipramine, indicating that the HPA axis may be an important target for antidepressant action. We previously showed that a methanolic extract of St. John's Wort (SJW) and hypericin, one of its active constituents, both have delayed effects on the expression of genes that are involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis , whereas the phloroglucinol derivative hyperforin was inactive in the same model . Since flavonoids of SJW are also discussed as active constituents it was of interest to determine whether these compounds can modulate HPA axis function. Imipramine (15 mg/kg), hypericin (0.2 mg/kg), hyperoside (0.6 mg/kg), isoquercitrin (0.6 mg/kg) and miquelianin (0.6 mg/kg) given daily by gavage for two weeks significantly down-regulated circulating plasma levels of ACTH and corticosterone by 40 - 70 %. However, none of the compounds tested had an effect on plasma ACTH and corticosterone levels after chronic treatment (daily gavage for 8 weeks). Our data suggest that besides hypericin, flavonoids of SJW play an important role in the modulation of HPA axis function. Furthermore, the results support the hypothesis that flavonoids are involved in the antidepressant effects of SJW.

The biological evaluation of hypericin in various test models is hampered by its poor water solubility. In former studies we have shown that the water solubility of hypericin was remarkably enhanced in the presence of the procyanidins or flavonol glycosides of Hypericum extract. The present pharmacokinetic study was designed to find out whether the improved water solubility in the presence of procyanidin B2 or hyperoside is correlated to increased plasma levels of hypericin. Plasma levels of hypericin in rats in the presence and absence of procyanidin B2 or hyperoside were determined by reversed phase HPLC using fluorimetric detection. Both compounds increased the oral bioavailability of hypericin by ca. 58 % (B2) and 34 % (hyperoside). Procyanidin B2 and hyperoside had a different influence on the plasma kinetics of hypericin; median maximal plasma levels of hypericin were detected after 360 min (C max : 8.6 ng/mL) for B2, and after 150 min (C max : 8.8 ng/mL) for hyperoside. It can be speculated that, when administered together with these compounds, a significant accumulation of hypericin in rat plasma in the presence of both polyphenols might be responsible for the observed increased in vivo activity.

Hypericin, an active component of Hypericum perforatum, was evaluated for the regulation of interleukin-12 (IL-12) production in mouse macrophages. Hypericin significantly inhibited IL-12 production in lipopolysaccharide-activated macrophages in a dose-dependent manner (IC50 = 1.45 micrograms/ml). Furthermore, hypericin potently inhibited the activation of IL-12 gene promoter, suggesting that hypericin negatively regulated IL-12 production at the transcription level. These results may explain some known biological activities of hypericin including its anti-rheumatic effect.

In the present study, protohypericin was synthesised in order to compare its intrinsic photocytotoxicity with that of hypericin. The experimental work was performed in specific filtered light conditions that prevented both an unintended photoconversion of protohypericin and photosensitization of the cells. Assessing the photocytotoxicity as a function of irradiation time, it was found that the photocytotoxicity of both compounds converged after a long irradiation time (i.e., 15 min), while the difference between the photocytotoxicities was maximal after a short irradiation time (i.e., 1 min). Since this could not be accounted for by a redistribution of protohypericin during irradiation, and the different irradiation times corresponded to different degrees of photoconversion of protohypericin into hypericin, the results clearly suggest that protohypericin exhibits intrinsically a dramatically lower photoactivity as compared to hypericin.

Investigations of phytochemicals for antiviral activities are assuming greater importance; but little attention has been given to the influence of various reaction parameters. We found that the activities of several known antiviral phytochemicals were profoundly affected by the presence of serum components, but in different ways. Thus, the terthiophene, alpha-terthienyl (alpha-T), was inhibited in a concentration-dependent manner by serum. In the case of a carboxylic acid derivative of alpha-T, the compound appeared to have no antiviral activity at all in the presence of serum, yet in its absence this compound was as effective as alpha-T. In contrast the complex anthraquinone hypericin required a small amount of serum for maximal antiviral activity, although too much was inhibitory. The reactions were also strongly affected by the order of incubation of the components: virus, compound, serum, and light. The antiviral effects were not influenced significantly by temperature, in contrast to a report by other workers, provided the light exposures were controlled. These effects are significant because serum is commonly used in virus assays, and plant extracts often contain polypeptides. Furthermore, when phytochemicals are used in vivo, their effects could be modulated by components of tissues and body fluids.

Hypericin from HYPERICUM PERFORATUM was found to inhibit IN VITRO type A and B monoamine oxidase (MAO's) prepared by treating rat brain mitochondria with selective inhibitors. The inhibition of type A MAO by hypericin was higher than that of type B MAO. The inhibition was almost irreversible for both MAO types.