Chemical composition of Hypericum species from the Taeniocarpium and Drosanthe sections

The presence of several phytochemicals, namely naphthodianthrones hypericin and pseudohypericin, phloroglucinol derivatives hyperforin and adhyperforin, the phenolic acids as chlorogenic acid, neochlorogenic acid, caffeic acid and 2,4-dihydroxybenzoic acid, the flavonols, hyperoside, isoquercitrin, quercitrin, quercetin, avicularin, rutin, and flavanols (+)-catechin and (?)-epicatechin, as well as biflavonoid amentoflavone was investigated in seven Turkish species of Hypericum from Taeniocarpium and Drosanthe sections. Plants were harvested at flowering, dried at room temperature, dissected into different tissues and assayed for chemical contents by HPLC. All chemicals were detected at various levels depending on species and plant parts. Despite the observed quantitative variation in the chemical content of plant material, it was found that phytochemical profiles of the species from the same section were very similar. The present data could be helpful in selecting the future targets for phytochemical and biological studies as well as enriching our current chemical knowledge about Hypericum species. Such kind of data could also be useful for elucidation of the chemotaxonomical relationships among the sections of Hypericum genus.     

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

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

Altitudinal changes in the content of bioactive substances in Hypericum orientale and Hypericum pallens

Altitudinal changes in the content of hypericin, pseudohypericin, hyperforin, adhyperforin, chlorogenic acid, neochlorogenic acid, caffeic acid, 2,4-Dihydroxybenzoic acid, amentoflavone, hyperoside, isoquercitrin, quercitrin, quercetin, avicularin, rutin, (+)-catechin and (?)-epicatechin among Hypericum orientale L. and Hypericum pallens Banks and Sol. populations from Northern Turkey were investigated for the first time. Thirty flowering individuals were collected from five different altitudes (400, 950, 1,150, 1,620 and 2,150 m) for H. pallens and six different altitudes (500, 1,150, 1,650, 2,100, 2,720 and 3,250 m) for H. orientale. The plant materials were dried at room temperature and subsequently assayed for chemical contents by HPLC. All chemicals were detected in both species at various levels depending on altitude of growing sites except for caffeic acid which was absent in H. pallens. It was found that plants from higher altitudes produced significantly higher amount of the bioactive compounds tested. The results were discussed as a possible protective response of plants to the different abiotic stress factors as high ultraviolet (UV)-B radiation and low temperature which were prevalent in higher altitudes.     

Phenolic constituents and genetic profile of Hypericum perforatum L. from India

The content of hypericins (hypericin and pseudohypericin), hyperforin, and flavonoids (rutin, hyperoside, quercitrin, and quercetin) and genetic profiles of eight accessions of Hypericum perforatum L., collected from different locations in India, have been determined. The secondary metabolite content was determined using a highly selective LC/MS/MS method. Pearson and Spearman's correlation coefficient were used to investigate the relationships between the secondary metabolites and a significant positive correlation was found between hypericin and pseudohypericin contents. Genetic profiling was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) methods. Among the 49 random primers used for the initial screening, only nine yielded polymorphic RAPD profiles. The SSR analysis shows that seven out of the 11 primers were polymorphic. There exists only a partial correlation between the chemical content and genetic profiling data among the accessions under study.     

Altitudinal changes in secondary metabolite contents of Hypericum androsaemum and Hypericum polyphyllum

The genus Hypericum (Hypericaceae) has attracted scientific interest as its members have yielded many bioactive compounds. In the present study we investigated the content of hypericin, pseudohypericin, hyperforin, adhyperforin, chlorogenic acid, neochlorogenic acid, caffeic acid, 2,4-dihydroxybenzoic acid, 13,II8-biapigenin, hyperoside, isoquercitrin, quercitrin, quercetin, avicularin, rutin, (+)-catechin and (−)-epicatechin in aerial parts of plants from populations of H. androsaemum L. and H. polyphyllum Boiss. & Bal. from Turkey growing at different altitudes. The plant materials were dried and subsequently assayed for chemical content by HPLC. All the tested compounds were detected in both species at varying levels depending upon the altitude the plants were growing, except for hypercins and rutin which did not accumulate in H. androsaemum. It was observed that overall the compounds were more abundant in plants from higher altitudes. The differences in the levels of the compounds could contribute to the ability of the plants to deal with the abiotic stress of lower temperature and higher ultraviolet (UV)-B radiation which would be greater at higher altitudes compared to lower altitudes.     

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.     

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.     

Variation in Hypericins from wild populations ofHypericum perforatum L. in the Pacific Northwest of the U.S.A.

Representatives from eight wild populations ofHypericum perforatum L. were collected from Montana and Northern California at flowering, and subsequently analyzed for hypericin and pseudohypericin using HPLC analysis. Total individual plant concentrations in these wild populations were from 0.0003–0.1250% dry weight (DW) hypericin and 0.0019–0.8458% DW pseudohypericin. In general, hypericin concentrations were highest in the plant’s reproductive (flower and bud) tissues, followed by leaf and stem tissues, respectively. Hypericin and pseudohypericin concentrations were positively correlated in all samples, although the relative ratio of hypericin to pseudohypericin varied with site location.     

Variation in the contents of pseudohypericin and hypericin in Hypericum perforatum from Lithuania

Hypericin and hypericin-like substances are considered the main active compounds in Hypericum perforatum L. (Hypericaceae). In this work pseudohypericin and hypericin of H. perforatum collected in Lithuania were quantified. Studies on accumulation dynamics and between-accession variation of the contents of these secondary metabolites were carried out by high performance liquid chromatography (HPLC). The data were statistically processed with ANOVA and PCA. Significant difference between pseudohypericin and hypericin content in floral budding and full flowering stages was detected. The highest amounts of the secondary metabolites were observed in the flowering stage. The study revealed evident within population variations in H. perforatum. Mean concentrations of pseudohypericin and hypericin among accessions varied from 3.45 to 6.82 mg/g and from 1.17 to 2.59 mg/g, respectively. Accessions of H. perforatum showed remarkable differences in chemical composition depending on the provenance of plants.     

Variation of bioactive substances and morphological traits in Hypericum perforatum populations from Northern Turkey

Hypericum perforatum L. has become one of the leading plant-based dietary supplements worldwide and its biological activities have been mainly attributed to hypericin and phenolic contents. The present study was conducted to determine chemical and morphological variability of H. perforatum sampled from different locations of Northern Turkey. The populations were investigated according to the hypericin, chlorogenic acid and the flavonoids, namely rutin, hyperoside, apigenin-7-O-glucoside, quercitrin and quercetin contents and morphological characters including leaf dark gland density, leaf area, leaf length/width ratio and plant height. Significant chemical and morphological variations were detected among the populations. Hypericin content among populations ranged between 0.44 and 2.82 mg/g dry weight, chlorogenic acid between 0.0 and 1.86 mg/g dry weight, rutin between 0.0 and 8.77 mg/g dry weight, hyperoside between 5.41 and 22.28 mg/g dry weight, quercitrin between 1.64 and 3.98 mg/g dry weight and quercetin between 1.01 and 1.76 mg/g dry weight. Hypericin content was found to be correlated positively with leaf dark gland density, however, negatively with leaf area and no correlation was detected between the other morphological traits and bioactive substances examined.     

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

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

Production of St. John's wort plants under controlled environment for maximizing biomass and secondary metabolites

Summary St. John's wort (Hypericum perforatum L.) is a medicinal plant used in the treatment of neurological disorders and has been recently shown to have anticancer potential. The principle medicinal components of St. John's wort are hypericin. pseudohypericin, and hyperforin. One of the problems associated with medicinal plant preparations including St. John's wort is the extreme variability in the phytochemical content, mostly due to environmental variability, and biotic and abiotic contamination during cropping. The current study was undertaken to transplant St. John's wort plants from in vitro bioreactors in a closed controlled environment system (CCES) with CO₂ enrichment for the optimized production of biomas and phytochemicals. The growth and levels of hypericin, pseudohypericin, and hyperforin in plants grown in CCES were compared with those of the greenhouse and in vitro-grown plants. The environmental parameters in the greenhouse were found to be variable whereas in the CCES these parameters were controlled. Generally, all the growth parameters and hypericin and psendohypericin levels were significantly higher in the plants grown in the CCES. These results provide the first indication that growing St. John's wort plants, under CO₂ enrichment in a closed environment system can enhance the biomass and medicinal contents. The adaptation of this growing system may be useful for the production of optimized products of St. John's wort and other medicinal species.     

Phytochemical analysis and genetic characterization of six Hypericum species from Serbia

The secondary metabolite contents and genetic profiles of six Hypericum species (H. barbatum Jacq., H. hirsutum L., H. linarioides Bosse, H. maculatum Crantz, H. rumeliacum Boiss. and H. tetrapterum Fries), collected from different locations in Serbia, have been analyzed. Methanol extracts of the aerial parts of the plants were obtained by accelerated solvent extraction (ASE) at 40 degrees C and 100 bar, and analyzed for five pharmacologically important standard constituents (hyperoside, quercitrin, pseudohypericin, hyperforin and hypericin) by LC-MS/MS. The highest content of hypericin and pseudohypericin was observed in the H. barbatum extract, while the highest content of hyperforin and quercitrin was found in the H. tetrapterum extract and the highest content of hyperoside in the H. maculatum extract. A literature survey shows that the above six Hypericum species, with the exception of H. maculatum, have not been previously genetically profiled. In order to correlate the chemical constituents of the species under investigation with their genetic factors, genetic profiling of these species was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) profiles of the above selected plants. Among the 52 random primers used for the initial screening, only 10 yielded polymorphic RAPD profiles. A total of 111 polymorphic markers were generated using these primers. The SSR analysis shows that 8 out of the 10 primers used were polymorphic. The correlation among the species under investigation using the two genetic markers was performed using Jaccuard's coefficients of similarity and a high correlation (r=0.99) was obtained. The main conclusion from the above data is that there exists a stronger correlation for secondary metabolite contents with RAPD data than with SSR data among the six Hypericum species from Serbia.     

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: A beneficial “side effect” of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic–phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.     

Simultaneous determination of total hypericin and hyperforin in St. John's wort extracts by HPLC with electrochemical detection

An analytical procedure was developed for the simultaneous determination of total hypericin (protopseudohypericin, pseudohypericin, protohypericin and hypericin) and hyperforin in Hypericum perforatum (St. John's wort) extracts and its preparations. The determination of total hypericin and hyperforin in one step was achieved by exposing the samples to artificial daylight in amber glass vials. This procedure allows both the photoconversion of the protoforms into the appropriate hypericins and the protection of the photosensitive hyperforin. For quantification, an HPLC method with electrochemical detection was applied. As an example of the application of the principle, two preparations containing St. John's wort were assayed.     

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: a beneficial "side effect" of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic-phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.     

Inter-population variability of secondary metabolites in Hypericum spp. (Hypericaceae) of the Northern Apennines, Italy

Hypericum spp. flowers were collected from different sites of Northern Apennines in Italy and were analysed by HPTLC technique to determine their contents in flavonoids (rutin, hyperosid, quercitrin, isoquercitrin and quercetin) and hypericins (pseudohypericin and hypericin). The H. perforatum and H. richeri composition was evaluated in order to investigate the influence of ecological conditions on secondary metabolites production. The species studied showed some differences in the amounts of investigated metabolites and high variability was observed in populations gathered in different places. No significant effects of environmental factors on active compound production were observed, except for the rutin content which is in positive correlation with the altitude of the growing site. Interesting informations are also given about the morphology of secretory structures found in Hypericum richeri.     

Protective effect of Hypericum calabricum Sprengel on oxidative damage and its inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophages

The present study shows for the first time the phenolic composition and the in vitro properties (antioxidant and inhibition of nitric oxide production) of Hypericum calabricum Sprengel collected in Italy. The content of hypericins (hypericin and pseudohypericin), hyperforin, flavonoids (rutin, hyperoside, isoquercetrin, quercitrin, quercetin and biapigenin) and chlorogenic acid of H. calabricum, have been determined. The ethyl acetate fraction from the aerial parts of H. calabricum exhibited activity against the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 value of 1.6 jig/ml. The test for inhibition of nitric oxide (NO) production was performed using the murine monocytic macrophage cell line RAW 264.7. The ethyl acetate fraction had significant activity with an IC50 value of 102 jig/ml and this might indicate that it would have an anti-inflammatory effect in vivo.     

The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L.

Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration post-elicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.