MALDI-HRMS Imaging Maps the Localization of Skyrin, the Precursor of Hypericin, and Pathway Intermediates in Leaves of Hypericum Species.

Hypericum perforatum and related species (Hypericaceae) are a reservoir of pharmacologically important secondary metabolites, including the well-known naphthodianthrone hypericin. However, the exact biosynthetic steps in the hypericin biosynthetic pathway, vis-à-vis the essential precursors and their localization in plants, remain unestablished. Recently, we proposed a novel biosynthetic pathway of hypericin, not through emodin and emodin anthrone, but skyrin. However, the localization of skyrin and its precursors in Hypericum plants, as well as the correlation between their spatial distribution with the hypericin pathway intermediates and the produced naphthodianthrones, are not known. Herein, we report the spatial distribution of skyrin and its precursors in leaves of five in vitro cultivated Hypericum plant species concomitant to hypericin, its analogs, as well as its previously proposed precursors emodin and emodin anthrone, using MALDI-HRMS imaging. Firstly, we employed HPLC-HRMS to confirm the presence of skyrin in all analyzed species, namely H. humifusum, H. bupleuroides, H. annulatum, H. tetrapterum, and H. rumeliacum. Thereafter, MALDI-HRMS imaging of the skyrin-containing leaves revealed a species-specific distribution and localization pattern of skyrin. Skyrin is localized in the dark glands in H. humifusum and H. tetrapterum leaves together with hypericin but remains scattered throughout the leaves in H. annulatum, H. bupleuroides, and H. rumeliacum. The distribution and localization of related compounds were also mapped and are discussed concomitant to the incidence of skyrin. Taken together, our study establishes and correlates for the first time, the high spatial distribution of skyrin and its precursors, as well as of hypericin, its analogs, and previously proposed precursors emodin and emodin anthrone in the leaves of Hypericum plants.

Ultrasound-assisted packed-bed extraction of hypericin from Hypericum perforatum L. and optimization by response surface methodology.

This paper presents the successful application of ultrasound-assisted packed-bed (UAE-PB) method for the extraction of hypericin from the Hypericum perfuratum L. The Soxhlet system was utilized for the determination of suitable solvent from ethanol, methanol or from the mixture of different proportions of ethanol-methanol. The mixture of 50:50 v/v ethanol-methanol was obtained to be the most suitable solvent since it led to the highest extraction amount of hypericin. The extraction amount of hypericin increased by 13.6% and 21.4% when the solvent changed from pure methanol to the mixture of 50:50 v/v ethanol-methanol for the extraction time of 3 and 8 h, respectively. Subsequently, the extraction was conducted through the UAE-PB, and the effects of temperature, time, and the ratio of solvent to the dried plant were studied. The response surface method (RSM) was used to investigate the effect of parameters on the extraction in the UAE-PB system. At the temperature of 60 °C, extraction time of 105 min, and the solvent to plant ratio of 15.3, the maximum extraction yield of hypericin was achieved. In the optimal conditions, the amount of extraction was 0.112 mg hypericin/g dried plant, which was in accordance with the optimized predicted value (0.111 mg hypericin/g dried plant) from Design-Expert software.

Chemometric evaluation of hypericin and related phytochemicals in 17 in vitro cultured Hypericum species, hairy root cultures and hairy root-derived transgenic plants.

OBJECTIVES: The objective of this study was to ascertain the presence and correlations among eight important secondary metabolites viz. hypericin, pseudohypericin, emodin, hyperforin, rutin, hyperoside, quercetin and quercitrin in different organs of 17 in vitro cultured Hypericum species, along with H. tomentosum and H. tetrapterum hairy root cultures, and hairy root-derived transgenic plants of H. tomentosum. METHODS: Samples were extracted and analysed by LC-MS. The LC-MS data were subjected to chemometric evaluations for metabolite profiling and correlating the phytochemical compositions in different samples. KEY FINDINGS: Hypericin, pseudohypericin and their proposed precursor emodin were detected in various levels in the leaves of eight Hypericum species. The highest content of hypericins and emodin was found in H. tetrapterum, which contains the studied secondary metabolites in all plant organs. A significant positive correlation between hypericins and emodin was observed both by principal component analysis (PCA) and multidimensional scaling (MDS), indicating the role of emodin as a possible precursor in the biosynthetic pathway of hypericins. Flavonoids were found in all tested plant organs except roots of H. pulchrum. The hairy roots lacked hypericin, pseudohypericin, emodin, hyperforin and rutin. However, the hairy root-derived transgenic plants showed a significant increase in flavonoids. CONCLUSIONS: This study broadens knowledge about the phytochemical composition of selected in vitro cultured Hypericum species, compared to that of hairy root cultures and hairy root-derived transgenic plants.

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

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

Enhanced production of perylenequinones in the endophytic fungus Shiraia sp. Slf14 by calcium/calmodulin signal transduction.

Perylenequinones (PQ) that notably produce reactive oxygen species upon exposure to visible light are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens such as Shiraia sp. The involvement of Ca2+/calmodulin (CaM) signalling in PQ biosynthesis was investigated by submerged culturing of Shiraia sp. Slf14, a species that produces hypocrellins HA and HB and elsinochromes EA, EB, and EC. Our results showed that the total content of PQ reached 1894.66 ± 21.93 mg/L under optimal conditions of Ca2+ addition, which represents a 5.8-fold improvement over controls. The addition of pharmacological Ca2+ sensor inhibitors strongly inhibited PQ production, which indicates that Ca2+/CaM signalling regulates PQ biosynthesis. The expression levels of Ca2+ sensor and PQ biosynthetic genes were downregulated following addition of inhibitors but were upregulated upon addition of Ca2+. Inhibition was partially released by external Ca2+ supplementation. Fluo-3/AM experiments revealed that similar cytosolic Ca2+ variation occurred under these conditions. These results demonstrated that Ca2+ signalling via the CaM transduction pathway plays a pivotal role in PQ biosynthesis.

Optimized and Validated HPLC Analysis of St. John's Wort Extract and Final Products by Simultaneous Determination of Major Ingredients.

Aim of this work was to develop a validated high performance liquid chromatography method for the analysis of extracts and final products of St. John's wort, according to international guidelines for bioanalytical method validation. Chromatographic separation was performed on a C18 column with a combination of gradient and isocratic steps; the mobile phase composed of ammonium acetate solution (pH 4.5; 10 mM), acetonitrile and methanol. Quantification and method validation was performed using extract spiked with external reference standards of chlorogenic acid, rutin, hyperoside, isoquercitrin, quercetin and hypericin. Validation study revealed that trans-chlorogenic acid is partially transformed into its cis-isomer during analysis. The method showed good linearity, precision and accuracy. Hyperforin was completely unstable. All other ingredients were stable at -18°C and after three freeze-thaw cycles, while stability of most ingredients was limited at room temperature and 4 - 8°C; quercetin was the most unstable one. The major ingredients of methanolic extracts, infusions and final products of Hypericum perforatum were completely resolved and quantified. Beyond its potential usefulness in the analysis of St. John's wort products, this study addresses the issue of validation from the perspective of the field of bioanalysis and reveals the wealth of critical information which can be derived.

Arbuscular mycorrhizal fungi altered the hypericin, pseudohypericin, and hyperforin content in flowers of Hypericum perforatum grown under contrasting P availability in a highly organic substrate.

St. John's Wort (Hypericum perforatum) is a perennial herb able to produce water-soluble active ingredients (a.i.), mostly in flowers, with a wide range of medicinal and biotechnological uses. However, information about the ability of arbuscular mycorrhizal fungi (AMF) to affect its biomass accumulation, flower production, and concentration of a.i. under contrasting nutrient availability is still scarce. In the present experiment, we evaluated the role of AMF on growth, flower production, and concentration of bioactive secondary metabolites (hypericin, pseudohypericin, and hyperforin) of H. perforatum under contrasting P availability. AMF stimulated the production of aboveground biomass under low P conditions and increased the production of root biomass. AMF almost halved the number of flowers per plant by means of a reduction of the number of flower-bearing stems per plant under high P availability and through a lower number of flowers per stem in the low-P treatment. Flower hyperforin concentration was 17.5% lower in mycorrhizal than in non-mycorrhizal plants. On the contrary, pseudohypericin and hypericin concentrations increased by 166.8 and 279.2%, respectively, with AMF under low P availability, whereas no effect of AMF was found under high P availability. These results have implications for modulating the secondary metabolite production of H. perforatum. However, further studies are needed to evaluate the competition for photosynthates between AMF and flowers at different nutrient availabilities for both plant and AM fungus.

TESTING PHARMACEUTICAL RELEASE OF ACTIVE SUBSTANCES FROM MEDICINAL PRODUCTS CONTAINING ST. JOHN'S WORT.

The aim of this study was to determine the content of hypericins and flavonoids in tablets and capsules containing the extract or powdered herb of St. John's wort, in herbs for infusion and herbal infusions and to release of these compounds from tablets and capsules. HPLC method was used to determine the assay of hypericins and flavonoids in all tested products. The hypericins content was between 0.35 mg and 1.44 mg per tablet or capsule. The release of hypericins from these products in the phosphate buffer of pH 6.8 is between 30 and 60% of the determined content. The degree of hypericins release from herbs into infusions was 15% on average, which corresponds to 0.64 mg of hypericins per infusion of 4 g of herbs. The flavonoids content was between 8.79 and 36.3 mg per tablet or capsule. The release of flavonoids in the phosphate buffer of pH 6.8 is between 63 and 85% of the determined content. The degree of flavonoids release was 76% on average, which corresponds to 77.0 mg per infusion of 4 g of herbs. The test results confirmed that infusions from the St. John's wort constitute are a rich source of flavonoids. At the same time, the universally accepted opinion that aqueous infusions contain only trace amounts of hypericins was not confirmed. Infusions from Herba hyperici may also be a source of hypericins in amounts comparable with the minimum dose recommended for the treatment of mild to moderate depressive episodes.

Shoot Tip Meristem Cryopreservation of Hypericum Species.

Based on our long-standing experience with in vitro culture of Hypericum perforatum, a clonal multiplication system and vitrification-based cryopreservation protocols have been applied to several Hypericum species: H. humifusum L., H. annulatum Moris, H. tomentosum L., H. tetrapterum Fries, H. pulchrum L., and H. rumeliacum Boiss. The shoot tips were cryopreserved using a uniform procedure that includes pretreatment with abscisic acid (ABA), PVS3 cryoprotection, and direct immersion into the liquid nitrogen (LN). The freezing-tolerant Hypericum species were pre-exposed to the cold acclimation conditions performed by a 7-day exposure to 4 °C. The content of naphtodianthrones (hypericins) including hypericin, pseudohypericin, and their protoforms was quantified by HPLC. Ploidy of plants was determined by both flow cytometry of leaf tissue and chromosome counts of root tip meristematic cells. We have shown that the post-thaw recovery rate of the shoot tips, pretreated with 0.076 µM ABA for 7 days at room temperature, led to the post-cryogenic survival from 5 % in H. tomentosum to 21 % in H. annulatum. As compared to the untreated (control) plants, the content of hypericins in plants regenerated after cryopreservation remained unchanged or decreased in H. perforatum, H. humifusum, H. annulatum, H. tomentosum, H. tetrapterum, and H. rumeliacum. However, the pre-exposition of the freezing-tolerant H. perforatum to cold acclimation prior to excision of the shoot tips has improved the post-thaw recovery to 45 % and resulted in threefold increase of the total hypericin content.

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

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

An innovative approach to prepare hypericin molecularly imprinted pearls using a "phyto-template".

In this paper, an innovative method that uses hypericin "phyto-template" molecules is being applied herein for the first time to produce molecularly imprinted polymer (MIP) pearls able to selectively retain hypericin from Hypericum Perforatum L primary extracts. For this purpose, the wet phase inversion method was preferred for preparing the hypericin-MIP pearls for several reasons referring to economical benefits but also due to the fact that hypericin "phyto-template" molecules can be generated along with the phase inversion of the copolymer. Practically, the precursor poly(acrylonitrile-co-methacrylic acid) solution was mixed with a purified and concentrated naphtodianthrone phyto-extract (consisting only of hypericin and pseudo-hypericin). In the subsequent phase inversion step hypericin was trapped in the copolymer droplets, as a result to its poor solubility in the inversion water bath, and further served as "phyto-template" in the imprinting step. This in situ repartition of hypericin and pseudo-hypericin was sustained by HPLC-DAD chromatograms which recorded only the presence of hypericin during the extraction stage of imprinted pearls. Batch rebinding measurements, all together, validated the efficiency of this innovative imprinting procedure. The hypericin rebinding of imprinted pearls was quantitative (up to 318 µg/L) and approximately 5 times more specific relative to the blank pearls. Competitive re-binding revealed a more selective behaviour of imprinted pearls for hypericin when the up-take was measured against pseudohypericin (selectivity coefficient above 4.50).

Spatial chemo-profiling of hypericin and related phytochemicals in Hypericum species using MALDI-HRMS imaging.

Advanced analytical imaging techniques, including matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) imaging, can be used to visualize the distribution, localization, and dynamics of target compounds and their precursors with limited sample preparation. Herein we report an application of MALDI-HRMS imaging to map, in high spatial resolution, the accumulation of the medicinally important naphthodianthrone hypericin, its structural analogues and proposed precursors, and other crucial phytochemical constituents in the leaves of two hypericin-containing species, Hypericum perforatum and Hypericum olympicum. We also investigated Hypericum patulum, which does not contain hypericin or its protoforms. We focused on both the secretory (dark glands, translucent glands, secretory canals, laminar glands, and ventral glands) and the surrounding non-secretory tissues to clarify the site of biosynthesis and localization of hypericin, its possible precursors, and patterns of localization of other related compounds concomitant to the presence or absence of hypericin. Hypericin, pseudohypericin, and protohypericin accumulate in the dark glands. However, the precursor emodin not only accumulates in the dark glands but is also present outside the glands in both hypericin-containing species. In hypericin-lacking H. patulum, however, emodin typically accumulates only in the glands, thereby providing evidence that hypericin is possibly biosynthesized outside the dark glands and thereafter stored in them. The distribution and localization of related compounds were also evaluated and are discussed concomitant to the occurrence of hypericin. Our study provides the basis for further detailed investigation of hypericin biosynthesis by gene discovery and expression studies.

Hypericum perforatum Reduces Paracetamol-Induced Hepatotoxicity and Lethality in Mice by Modulating Inflammation and Oxidative Stress.

Hypericum perforatum is a medicinal plant with anti-inflammatory and antioxidant properties, which is commercially available for therapeutic use in Brazil. Herein the effect of H. perforatum extract on paracetamol (acetaminophen)-induced hepatotoxicity, lethality, inflammation, and oxidative stress in male swiss mice were investigated. HPLC analysis demonstrated the presence of rutin, quercetin, hypericin, pseudohypericin, and hyperforin in H. perforatum extract. Paracetamol (0.15-3.0 g/kg, p.o.) induced dose-dependent mortality. The sub-maximal lethal dose of paracetamol (1.5 g/kg, p.o.) was chosen for the experiments in the study. H. perforatum (30-300 mg/kg, i.p.) dose-dependently reduced paracetamol-induced lethality. Paracetamol-induced increase in plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations, and hepatic myeloperoxidase activity, IL-1ß, TNF-α, and IFN-γ concentrations as well as decreased reduced glutathione (GSH) concentrations and capacity to reduce 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate radical cation; ABTS˙(+) ) were inhibited by H. perforatum (300 mg/kg, i.p.) treatment. Therefore, H. perforatum protects mice against paracetamol-induced lethality and liver damage. This effect seems to be related to the reduction of paracetamol-induced cytokine production, neutrophil recruitment, and oxidative stress.

Effects of UV-B radiation on total phenolic, flavonoid and hypericin contents in Hypericum retusum Aucher grown under in vitro conditions.

This study was conducted to increase total phenolics, flavonoids and hypericin accumulation in in vitro cultures of Hypericum retusum Aucher to determine the appropriate time of UV radiations. Proliferation of plantlets on Murashige-Skoog medium containing 0.5 mg L(- 1)N-6-benzylaminopurine was achieved under in vitro conditions. Then, the plantlets were exposed to UV-B radiation for different periods (15, 30, 45 and 60 min). The highest total phenolics, flavonoids and hypericin accumulation (43.17 ± 0.8; 35.09 ± 0.8; 2.7 ± 0.05 mg g(- 1), respectively) was achieved at 45 minutes of exposure to UV-B radiation when compared with the contents of naturally growing plants (23.33 ± 0.9, 18.62 ± 0.3 and 1.6 ± 0.01 mg g(- 1), respectively) and control groups (control group was not subjected to UV-B radiation).

Interaction between different extracts of Hypericum perforatum L. from Serbia and pentobarbital, diazepam and paracetamol.

Herb-drug interactions are an important safety concern and this study was conducted regarding the interaction between the natural top-selling antidepressant remedy Hypericum perforatum (Hypericaceae) and conventional drugs. This study examined the influence of acute pretreatment with different extracts of Hypericum perforatum from Serbia on pentobarbital-induced sleeping time, impairment of motor coordination caused by diazepam and paracetamol pharmacokinetics in mice. Ethanolic extract, aqueous extract, infusion, tablet and capsule of Hypericum perforatum were used in this experiment. The profile of Hypericum perforatum extracts as well as paracetamol plasma concentration was determined using RP-HPLC analysis. By quantitative HPLC analysis of active principles, it has been proven that Hypericum perforatum ethanolic extract has the largest content of naphtodianthrones: hypericin (57.77 µg/mL) and pseudohypericin (155.38 µg/mL). Pretreatment with ethanolic extract of Hypericum perforatum potentiated the hypnotic effect of pentobarbital and impairment of motor coordination caused by diazepam to the greatest extent and also increased paracetamol plasma concentration in comparison to the control group. These results were in correlation with naphtodianthrone concentrations. The obtained results have shown a considerable influence of Hypericum perforatum on pentobarbital and diazepam pharmacodynamics and paracetamol pharmacokinetics.

[Simultaneous determination of hypocrellin A, hypocrellin B, and hypocrellin C by HPLC].

OBJECTIVE: To develop a high-performance liquid chromatography (HPLC) method for simultaneous determination of hypocrellin A, hypocrellin B, and hypocrellin C. METHOD: The separation was carried out on a Kromasil C18 (4.6 mm x 250 mm, 5 micrm) colum eluted with in mobile phases of water containing 0.5% glacial acetic acid and acetonitrile. The column temperature was 35 degrees C, and the flow rate was 1.0 mL x min(-1). The detection wavelength was set at 265 nm. RESULT: The three compounds were well separated. Calibration curves of hypocrellin A, hypocrellin B, and hypocrellin C showed good linear relationship RSD > 2.0%. The average recoveries of the hypocrellin A, hypocrellin B, and hypocrellin C were 101.8%, 102.3%, 100.0%, respectively. CONCLUSION: The developed method is simple, accurate, and repeatable, and can be readily used as valid tool for the quality control of Hypocrella bambusae.

Hypericin and pseudohypericin concentrations of a valuable medicinal plant Hypericum perforatum L. are enhanced by arbuscular mycorrhizal fungi.

Hypericum perforatum L. (St. John's-wort, Hypericaceae) is a valuable medicinal plant species cultivated for pharmaceutical purposes. Although the chemical composition and pharmacological activities of H. perforatum have been well studied, no data are available concerning the influence of arbuscular mycorrhizal fungi (AMF) on this important herb. A laboratory experiment was therefore conducted in order to test three AMF inocula on H. perforatum with a view to show whether AMF could influence plant vitality (biomass and photosynthetic activity) and the production of the most valuable secondary metabolites, namely anthraquinone derivatives (hypericin and pseudohypericin) as well as the prenylated phloroglucinol-hyperforin. The following treatments were prepared: (1) control-sterile soil without AMF inoculation, (2) Rhizophagus intraradices (syn. Glomus intraradices), (3) Funneliformis mosseae (syn. Glomus mosseae), and (4) an AMF Mix which contained: Funneliformis constrictum (syn. Glomus constrictum), Funneliformis geosporum (syn. Glomus geosporum), F. mosseae, and R. intraradices. The application of R. intraradices inoculum resulted in the highest mycorrhizal colonization, whereas the lowest values of mycorrhizal parameters were detected in the AMF Mix. There were no statistically significant differences in H. perforatum shoot mass in any of the treatments. However, we found AMF species specificity in the stimulation of H. perforatum photosynthetic activity and the production of secondary metabolites. Inoculation with the AMF Mix resulted in higher photosynthetic performance index (PI(total)) values in comparison to all the other treatments. The plants inoculated with R. intraradices and the AMF Mix were characterized by a higher concentration of hypericin and pseudohypericin in the shoots. However, no differences in the content of these metabolites were detected after the application of F. mosseae. In the case of hyperforin, no significant differences were found between the control plants and those inoculated with any of the AMF applied. The enhanced content of anthraquinone derivatives and, at the same time, better plant vitality suggest that the improved production of these metabolites was a result of the positive effect of the applied AMF strains on H. perforatum. This could be due to improved mineral nutrition or to AMF-induced changes in the phytohormonal balance. Our results are promising from the biotechnological point of view, i.e. the future inoculation of H. perforatum with AMF in order to improve the quality of medicinal plant raw material obtained from cultivation.

Effects of UV-B on secondary metabolites of St. John's Wort (Hypericum perforatum L.) grown in controlled environments.

The medicinal plant industry is under increasing scrutiny due to wide variance in active ingredient (AI) concentration from values claimed on labels. Reasons for this disparity include environmental and genotypic variation which influence AI concentration. St. John's wort (Hypericum perforatum) is a popular herbal remedy which also exhibits marked variance in AI concentration among products. This study evaluated concentration changes of three biologically active metabolites of H. perforatum after exposure to UV light while plants were still vegetative. Treatments were performed with 55-day-old plants grown under 400 µmol m(-2) s(-1) PAR for 16 h a day. Three UV light treatments were evaluated: a single dose, a daily dose and an increasing daily dose. Concentrations of hyperforin, pseudohypericin and hypericin were monitored for 7 days after each treatment. A daily dose and an increasing daily dose did not produce significantly greater increases in secondary metabolites compared to single dose treatments. These results suggest the small but significant transient metabolite concentration increases in H. perforatum can be induced by UV light exposure. Information from this study can be useful in optimizing total biomass and metabolite production in controlled environments.

Determination of total hypericins in St. John's wort and herbal medicinal products.

The work aimed to determine the levels of hypericins expressed as hypericin in the herbal substance of St. John's wort, in capsules and tablets containing the extract of St. John's wort, tablets containing powdered herb and in tincture and juice from fresh St. John's wort, by HPLC method with spectrophotometric detection. In addition, the amount of hypericins in the infusion prepared from St. John's wort was determined by HPLC and spectrophotometry methods. According to traditional indications aqueous infusions from St. John's wort containing mainly hydrophilic components are used in gastrointestinal diseases. On the other hand, ethanolic extracts containing hypericin and hyperforin affect the CNS and are indicated for the treatment of episodes of mild depressive disorders. The results obtained in the work indicate that the daily dose of hypericins taken by a patient as infusions is 0.328 mg on average for herbs in sachets and in bulk form. It can be compared to the daily dose of hypericins contained in tablets and capsules based on the alkoholic extract of St. John's wort and tablets containing powdered St. John's wort herb. For solid dosage forms, this dose ranges from 0.288 mg to 0.636 mg. The assays were performed using consistent analytical methods for all tested pharmaceutical products and consequently it was possible to compare doses taken by patients and their strength of action.

Lipid-mediated preferential localization of hypericin in lipid membranes.

Subcellular localization of a photosensitizer is critical to its therapeutic outcome during photodynamic therapy (PDT). We delineated the distribution of hypericin, a new generation photosensitizer, in model membrane systems to identify the operating principles of its subcellular accumulation. Results from fluorescence microscopy indicated preferential incorporation of hypericin in lipid of giant unilamellar vesicles. Monolayer fluorescence measurements further identified cholesterol as the key determinant for the observed selectivity of hypericin. The emission spectra of hypericin in lipid monolayers varied in a lipid-dependent manner and Stoke's shift behavior suggests that hypericin may form closely packed structure with cholesterol. Overall, our data lead to the conclusion that cholesterol is the major origin of the selectivity for hypericin in membrane systems. A hypothetical model depicting the intracellular and intravascular co-transport of hypericin and cholesterol because of their high affinity is presented.