Morphology and development of secretory structures in Hypericum perforatum and H. richeri

Secretory organs are a specialised anatomical feature of plants, these tissues function in the production and/or storage of specific chemical substances, which often have pharmacological properties. The genus Hypericum is characterised by the presence of specialised secretory structures such as black nodules, translucent glands and secretory canals. The presence of these structures is not homogeneous for all the species or between the various floral and vegetative parts of the plants. In this study, we have compared the distributions, morphology and development of secretory structures in the leaves of H. perforatum and H. richeri. Whilst, black nodules occurred in both species, translucent glands followed different development stages depending on the leaf age.     

Morphological and phytochemical features of secretory structures in Hypericum richeri (Clusiaceae)

Structural and complementary chemical studies were carried out on Hypericum richeri , a lesser known species amongst those reported for folk medical use. We found only one type of secretory glands consisting of black dots which are present even in early emerging leaves. In the fully expanded leaves the nodular structure appears to be composed by a cluster of cells. These become unfunctional and disassembled towards the end of their development, and are used only as reservoirs of secretion products. HPTLC analyses showed that flower buds and flowers are the plant parts richest in active compounds. However, the spectrum of active compounds accumulated by H. richeri was both quantitatively and qualitatively similar to those reported for the pharmaceutically utilized, H. perforatum , and thus could potentially represent a possible alternative to this species.     

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 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.     

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.     

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.     

Growth and rutin production in hairy root cultures of buckwheat (Fagopyrum esculentum M.)

Buckwheat (Fagopyrum esculentum Moench.) is a potentially important source of rutin, a natural flavonoid with antihyperglycemic, antihypertensive, and antioxidative properties. To examine in vitro production of rutin, we established a hairy root culture of buckwheat by infecting leaf explants with Agrobacterium rhizogenes R1000, and tested the growth conditions and rutin production rates of these cultures. Ten hairy root clones were established; their growth and rutin production rates ranged from 233 to 312 (mg dry wt per 30 mL flask, and 0.8 to 1.2 (mg/g dry wt), respectively. Clone H8, which had high growth and rutin production rates (312 mg dry wt per 30 mL flask and 1.2 mg/g dry wt, respectively), was selected for further experiments. H8 showed maximal growth and rutin content at 30 days in culture in MS medium. Of four tested culture media, half-strength MS medium was found to induce the highest levels of growth (378 mg dry wt per 30 mL flask) and rutin production (1.4 mg/g dry wt) by clone H8. In contrast, supplementation with auxins (0.1-1 mg/l IAA, IBA and NAA) increased the growth rate, but had no significant effect on rutin production by H8. Collectively, these findings indicate that hairy root cultures of buckwheat culture could be a valuable alternative approach for rutin production.     

Hairy roots of Hypericum perforatum L.: a promising system for xanthone production

Hypericum perforatum L. is a common perennial plant with a reputed medicinal value. Investigations have been made to develop an efficient protocol for the identification and quantification of secondary metabolites in hairy roots (HR) of Hypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR had an altered phenolic profile with respect to phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones comparing to control roots. Phenolics in control and HR cultures were observed to be qualitatively and quantitatively distinct. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots are capable of producing flavonol glycosides such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. The HPLC analysis of flavonoid aglycones in HR resulted in the identification of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. Among the twenty-eight detected xanthones, four of them were identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in HR. Altogether, these results indicated that H. perforatum HR represent a promising experimental system for enhanced production of xanthones.     

Role of flavonoids in controlling the phototoxicity of Hypericum perforatum extracts.

Hypericum perforatum extracts are used mainly as oral antidepressants. Depending on source the extracts contain various amounts of phenylpropanes, flavonol derivates, biflavones, proathocyanidines, xanthones, phloroglucinoles, some amino acids, naphtodianthrones (hypericines) and essential oil constituents. The therapeutic use of Hypericum perforatum extracts however is limited by their phototoxic potential. It was the aim of the present study to investigate the phototoxic potential of 3 Hypericum perforatum extracts from different sources as well as some of its main constituents. In order to systematically study the phototoxic potential we established a modified neutral red assay utilizing an immortalized human keratinocyte cell line (HaCaT cells) as substrate and UVA irradiation. This modified neutral red assay was found to be a simple and reliable method for detecting phototoxic effects of reference agents and plant extracts. The validity of this method was demonstrated with known phototoxic compounds like chloropromazine and psoralenes like 5-MOP. Hypericum perforatum extracts demonstrated cytotoxicity and photocytotoxicity in a dose and UVA-dose dependent manner. Hypericine itself also evoked severe phototoxic effects and was thus identified as the main phototoxic constituent. Among the tested flavonoids quercitrin was found to be cytotoxic, while rutin unexpectedly demonstrated phototoxicity whereas quercitrin was effective to control the phototoxic activity of Hypericum perforatum extracts.     

元宝草化学成分的研究

目的　研究中药元宝草 (HypericumsampsoniiH .)的化学成分。 方法　用乙醇或甲醇回流提取 ,聚酰胺柱色谱和重结晶分离 ,FT -IR ,MS ,UV ,1HNMR等方法确定结构。结果　分得了 4个化学成分 ,分别为芦丁 ,金丝桃苷 ,槲皮素和金丝桃素。结论　 4种化合物均为首次从元宝草中分得和检出。     

Effects of cytokinins and elicitors on the production of hypericins and hyperforin metabolites in Hypericum sampsonii and Hypericum perforatum

Hypericum perforatum L. (St. John’s wort) and Hypericum sampsonii Hance are medicinal plants used in China in the treatment of viruses and other disorders. In the current study, we investigated the effects of cytokinins 6-benzylaminopurin (BA), zeatin (ZT) and thidiazuron (TDZ) on plant growth and production of hypericins (pseudohypericin and hypericin) and hyperforin. Our data suggested that culture of H. perforatum in modified MS (Murashige and Skoog) medium, with a 50% reduction in ammonium nitrate and potassium nitrate, and supplemented with BA (0.44 μM) and indolebutyric acid (IBA, 0.049 μM), resulted in increased production of hypericins. Similar results were noted with H. sampsonii with minor changes to the medium (0.46 μM ZT and 0.049 μM IBA). There were approximately 2.95-, 2.62-fold increases in H. perforatum pseudohypericin and hypericin production by TDZ (0.45 μM) induction compared to the controls. No enhancement of hypericins and hyperforin production was elicited by TDZ in H. sampsonii. The elicitor methyl jasmonate (MJA, 50 μM) and its analog, 2,3-dihydroxypropyl jasmonate (DHPJA, 50 μM), were also used in H. perforatum and H. sampsonii shoot culture to increase secondary metabolite production, eliciting an increase in the production of hypericins and hyperforin. While leaf senescence and biomass inhibition were observed in cultures induced by MJA, no such effects were observed with DHPJA.     

Antimicrobial activity of some Hypericum species

The crude methanolic extracts of six species of Hypericum [H. caprifoliatum Cham. & Schlecht., H. carinatum Griseb., H. connatum Lam., H. ternum A. St. Hil., H. myrianthum Cham. & Schlecht. and H. polyanthemum Klotzsch ex Reichardt] growing in southern Brazil were analyzed for antimicrobial activity against several microorganisms (bacteria and fungi). The most active plant was H. caprifoliatum, which showed activity against Staphylococcus aureus. Only H. polyanthemum and H. ternum extracts were active against Bacillus subtilis. None of the crude methanolic extracts showed activity against S. epidermidis, Escherichia coli or Saccharomyces cerevisiae. Extracts from these species were evaluated chemically and tannin, flavonoid and phenolic acids were the prominent compounds. The plants contained quercitrin, hyperoside (except H. connatum) and, less frequently, isoquercitrin and chlorogenic acid. In contrast to H. perforatum, which has high concentrations of rutin, these species do not produce this flavonoid or it appears as traces. The tannin concentration varied between 5.1 and 16.7% in H. myrianthum and H. ternum, respectively.     

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.     

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

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

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

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

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.     

Secondary metabolites of Hypericum orientale L. growing in Turkey: variation among populations and plant parts

The present study was conducted to determine the variation in the content of several plant chemicals, namely hyperforin, hypericin, pseudohypericin, chlorogenic acid, rutin, hyperoside, isoquercetine, kaempferol, quercitrine and quercetine among ten Hypericum orientale L. populations from Northern Turkey. The aerial parts representing a total of 30 individuals were collected at full flowering and dissected into floral, leaf and stem tissues. After dried at room temperature, the plant materials were assayed for chemical contents by HPLC. The populations varied significantly in chemical contents. Among different plant parts, the flowers were found to be the principle organ for hyperforin, hypericin, pseudohypericin and rutin accumulations while the rest of the chemicals were accumulated mainly in leaves in all growing localities. The chemical variation among the populations and plant parts is discussed as being possibly the result of different genetic, environmental and morphological factors.     

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

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

贯叶连翘挥发性化学成分研究

采用水蒸气蒸馏法提取中草药贯叶连翘(全草)挥发油。通过气相色谱/质谱联用技术测定了挥发油的化学成分,从中鉴定出75种化合物,其中百里香酚、桉油精、丁香油酚等酚类物质为首次检出。结果表明,贯叶连翘因产地不同、部位不同,其挥发油的成分有较大的差别。