Anti-Epstein-Barr Viral Agents from the Medicinal Herb-Derived Fungus Alternaria alstroemeriae Km2286.

Chromatographic separation on the liquid-state fermented products produced by the fungal strain Alternaria alstroemeriae Km2286 isolated from the littoral medicinal herb Atriplex maximowicziana Makino resulted in the isolation of compounds 1-9. Structures were determined by spectroscopic analysis as four undescribed perylenequinones, altertromins A-D (1-4), along with altertoxin IV (5), altertoxin VIII (6), stemphyperylenol (7), tenuazonic acid (8), and allo-tenuazonic acid (9). Compounds 1-6 exhibited antiviral activities against Epstein-Barr virus (EBV) with EC50 values ranging from 0.17 ± 0.07 to 3.13 ± 0.31 µM and selectivity indices higher than 10. In an anti-neuroinflammatory assay, compounds 1-4, 6, and 7 showed inhibitory activity of nitric oxide production in lipopolysaccharide-induced microglial BV-2 cells, with IC50 values ranging from 0.33 ± 0.04 to 4.08 ± 0.53 µM without significant cytotoxicity. This is the first report to describe perylenequinone-type compounds with potent anti-EBV and anti-neuroinflammatory activities.

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.

Antidiabetic activity of perylenequinonoid-rich extract from Shiraia bambusicola in KK-Ay mice with spontaneous type 2 diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: Bitter and cold traditional Chinese medicines (TCMs) have been long used to treat diabetes mellitus (DM) based on unique medical theory system since ancient China. As one of bitter and cold TCMs, the stromatas of Shiraia bambusicola have been used for the treatment of DM and exerted clinical effects to a certain extent. However, the corresponding active principles and antidiabetic mechanism of the TCM still remain unknown. Therefore, the aim of the present investigation was to evaluate the potential antidiabetic effect of the active Shiraia bambusicola EtOAc extract (SB-EtOAc) in vitro and in vivo, and elucidate its probable antidiabetic mechanism. MATERIALS AND METHODS: A LC-PDA-ESIMS protocol was developed to determine the chemical principles of the active EtOAc extract rapidly and unambiguously. The effect of SB-EtOAc on the glucose transporter type 4 (GLUT4) translocation and glucose uptake in L6 cells was examined. SB-EtOAc was orally administration at the dose of 30, 60 and 120mg/kg/d in KK-Ay mice, for 21 days. Body weight, plasma glucose, oral glucose tolerance test, fasted blood glucose levels, oral glucose tolerance test and insulin tolerance test, serum insulin and blood-lipid indexes were measured. GLUT4 on L6 cells membrane and phosphorylation of the AMP-activated protein kinase (p-AMPK) expression in L6 cells were measured. The GLUT4 and p-AMPK expression in KK-Ay mice skeletal muscle were measured. Phosphorylation of the acetyl-CoA carboxylase (p-ACC) and p-AMPK were measured. RESULTS: In vitro, SB-EtOAc exhibited a strong effect of stimulation on GLUT4 translocation by 3.2 fold in L6 cells compared with basal group, however, the selective AMPK inhibitor compound C can completely inhibit the AMPK pathway and prevent the GLUT4 translocation caused by SB-EtOAc. The further western blotting experiments showed that SB-EtOAc can stimulate AMPK phosphorylation in L6 cells and improve the expression of GLUT4. In vivo, SB-EtOAc can improve the KK-Ay mice insulin resistant and oral glucose tolerance to a certain extent. And the body weight, blood glucose levels and the serum TC, TG, FFA, AST, ALT and LDL-C were significantly reduced and HDL-C were increased after 3 weeks treatment. Mechanistically, phosphorylation of the AMPK and ACC had been improved obviously and the levels of AMPK phosphorylation and GLUT4 had been also enhanced. CONCLUSION: In vitro, SB-EtOAc exhibited a strong effect of stimulation on GLUT4 translocation and improved significantly the glucose uptake. In vivo, SB-EtOAc significantly improved oral glucose tolerance and the insulin resistant as well as glucolipid metabolism. In this study, SB-EtOAc displayed promising positive antidiabetic activity in vitro and in vivo, partly by modulating AMPK-GLUT4 and AMPK-ACC signaling pathways.

[Pharmacognosy study of Verbascum species]. / A Verbascum sp. farmakognóziai vizsgálata.

The mullein (Verbascum phlomoides L., V thapsus L., V. thapsiforme Schrad., V. speciosum L.) is a medicinal herb known and used for a long time, especially in traditional Turkish medicine. The aims of our study were to identify the species and study the plant's major active substances both qualitatively and quantitatively, comparing it to data found in scientific literature. The plants were identified as probable hybrids of V. phlomoides and V. thapsiforme. Microscopic analysis of the flowers showed no major difference between the specimens. The diameter of both stomata and pollen we observed was around 15-20 µm. Important flavonoids like rutin and quercetin were identified. Dosage resulted in a 0.135% total flavonoid aglycone content. (expressed as hypericin) and a 1.3% total flavonoid glycoside content (expressed as rutoside). Thin layer chromatography from saponines revealed two spots. A hemolytic index of 13095 was also determined. Repeating the dosage experiment a year later resulted in significantly lower flavonoid aglycone and glycoside content (0.006% and 0.95% respectively) as well as a hemolytic index of approximately 4000.

Hypericin biosynthesis in Hypericum hookerianum Wight and Arn: investigation on biochemical pathways using metabolite inhibitors and suppression subtractive hybridization.

The biochemical pathway to hypericin biosynthesis is presumed to be polyketide synthase (PKS) mediated, but it has not been experimentally validated, and no alternate route (chorismate/o-succinylbenzoate pathway) has been analyzed. We report here our earlier developed auxin inducible culture systems of Hypericum hookerianum as a model, to study the metabolic pathway to hypericin synthesis. Inhibitors of the alternate pathway at varying concentrations showed steady synthesis of total hypericins with means of 2.80±0.22, 18.75±0.01; 16.39±3.75, 29.60±1.90 (mevinolin) 2.53±0.10, 18.12±0.56; 0.14±0.01, 14.28±1.11 (fosmidomycin) and 2.7±0.35, 18.75±0.61; 0.14±0.01, 12.80±1.09 mg g(-1) DW (glyphosate) in the control and auxin-induced shoot and shoot-forming callus cultures, respectively. SSH analysis classified the differentially expressed sequences into protein synthesis (38%), modification (20%), electron transport (9%) and remaining as unclassified (11%) and unknown proteins (22%). Functional annotation of sequences indicates the presence of additional protein components besides PKS activity. Our results demonstrate direct biochemical and molecular evidence of PKS hypothesis of hypericin biosynthesis for the first time.

Hypericum perforatum: Influences of the habitat on chemical composition, photo-induced cytotoxicity, and antiradical activity.

CONTEXT: Hypericin, isolated from Hypericum perforatum L. and about another 300 Hypericum species (Guttiferae), is one of the most powerful photosensitizers found in nature. OBJECTIVE: The aim of this study was to assess the variability of chemical composition and biological activities of four H. perforatum samples, collected at different altitudes in the South Apennine of Italy. MATERIALS AND METHODS: MTT assay was used to evaluate the antiproliferative activity of different samples concentrations (0.6-100 µg/mL) after irradiation at 365 nm. The inhibition of nitric oxide production was evaluated after 24 h of incubation using the macrophage cell line RAW 264.7 and sample solutions ranging from 12.5 to 1000 µg/mL. Antioxidant activities were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and ß-carotene bleaching test (ranges were 12.5-1000 and 1-400 µg/mL, respectively). Chemical composition was evaluated through HPTLC, and different contents of hypericin and rutin have been observed. RESULTS: The most phototoxic sample was collected from Zumpano (no. 1 at 370 m), with IC50 values of 24.61 ± 0.02 µg/mL. Sample no. 1 showed also the best radical scavenging activity (IC50 value of 9.18 ± 0.03 µg/mL) and the best antioxidant activity (IC50 value of 10.04 ± 0.03 µg/mL after 30 min of incubation). Best activity of extract no. 1 was well in accordance with chemical data, including the phenolic total content and particular metabolome profile. DISCUSSION AND CONCLUSION: This paper confirms the usefulness in maintaining the exploration of H. perforatum activities, in order to confirm its potentiality as a multipurpose plant.

Antiproliferative activity of epi-cercosporin in human solid tumor cell lines.

From cultures of Cercospora piaropi, a phytopathogenic fungus isolated from symptomatic leaves of water hyacinth was obtained a red compound, which, according to the spectroscopic data, was epi-cercosporin. It showed in vitro antiproliferative activity against the panel of human solid tumor cells HBL-100, HeLa, SW1573 and WiDr. Cell cycle studies revealed that epi-cercosporin induces accumulation of cells in G2/M phase.

[Chemical constituents of ethyl acetate fraction from Hypericum hengshanense].

OBJECTIVE: To study the constituents of ethyl acetate fraction form Hypericum hengshanense. METHODS: The constituents were isolated and purified by chromatography on silica gel and their structures were elucidated by MS and NMR spectral analysis. RESULTS: Ten compounds were isolated and identified as: hyperoside (1), hypericin (2), quercetin (3), quercitrin (4), sesamin (5), betulonic acid (6), rutin (7), kaempferol (8), beta-daucosterol (9), beta-sitosterol (10). CONCLUSION: All compounds are obtained from this plant for the first time.

Elicitation of secondary metabolism in Hypericum perforatum by rhizosphere bacteria and derived elicitors in seedlings and shoot cultures.

CONTEXT: Hypericum perforatum L. (Guttiferae) appears as an alternative treatment to mild and moderate depression and been traditionally used as a health enhancer based on the phytochemicals hyperforin and hypericin. However, field grown medicinal plants show variable levels of phytopharmaceuticals depending on environmental conditions. Elicitation is a good strategy to trigger secondary metabolism. OBJECTIVE: This study explored the ability of 6 rhizobacterial strains to trigger secondary metabolism in H. perforatum seedlings and molecular elicitors from the most effective strain N5.18 were tested in shoot cultures. MATERIALS AND METHODS: Hypericin and pseudohypericin were determined on seedlings and shoot cultures by HPLC. Three putative elicitors from bacterial culture media were assayed in three different concentrations. RESULTS: Strain N5.18 significantly increased hypericin up to 1.2 ppm and pseudohypericin up to 3.4 ppm, over controls (0.3 and 2.5 ppm, respectively) when delivered to seedlings. In shoot cultures, only pseudohypericin was detected (168.9 ppm) and significant increases were observed under the different elicitors, reaching values of 3164.8 ppm with small elicitors in the middle concentration. DISCUSSION AND CONCLUSION: Secondary metabolism in plants is highly inducible due to its role in plant communication and defense. Our findings demonstrate that some beneficial bacterial strains are able to trigger secondary metabolism in H. perforatum plants when delivered through the roots and bacterial determinants released to culture media are able to reproduce the effect in shoot cultures. Therefore, these elicitors have great potential to enhance phytopharmaceutical production.

[Rapid cloning and functional characterization of hypericin synthase gene].

Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, commonly known as St. John's wort. Hypericin has attracted a growing attention of the pharmaceutical industry because of its potential application to various therapies, including the treatment of depression and remarkable antiviral and photodynamic activities, hyp-1 gene encodes for phenolic coupling protein which catalyzes in vitro direct and specific conversion of emodin to hypericin which, however, has not formed common opinion so far. Six pairs of primers specific to hyp-1 gene were synthesized. The rapid cloning of hyp-1 gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pET32ahyp containing hyp-1 gene was constructed and was transformed into E. coli to induce heterologous expression. SDS-PAGE and Western blot results showed the recombinant Hyp-1 protein was expressed successfully in E. coli. The soluble fraction was used to test the function of the recombinant Hyp-1. Hypericin was detected by LC-MS/MS with emodin as a substrate under in vitro conditions. The above results corroborated the Hyp-1 function, a confusing question, which lay a material foundation for the synthesis of hypericin by synthetic biotechnology.

Secondary metabolites of Hypericum leptophyllum Hochst., an endemic Turkish species.

In the present study, the presence of the phloroglucinol derivative hyperforin, the naphthodianthrones hypericin and pseudohypericin, the phenylpropane chlorogenic acid and the flavonoids rutin, hyperoside, kaempferol, isoquercetine, quercitrine, and quercetine was investigated in Hypericum leptophyllum Hochst., an endemic Turkish species for the first time. The aerial parts representing a total of 30 individuals were collected at full flowering and dissected into floral, leaf, and stem tissues. After being dried at room temperature, the plant materials were assayed for secondary metabolite concentrations by HPLC. Aerial plant parts accumulated chlorogenic acid, hyperoside, isoquercetine, quercitrine, and quercetine, but they did not accumulate hyperforin, hypericin, pseudohypericin, rutin, and kaempferol. Accumulation levels of the detected compounds varied with plant tissues. Such kind of data could be useful for elucidation of the chemotaxonomical significance of the corresponding compounds and phytochemical evaluation of this endemic species.

A new perylenequinone from a halotolerant fungus, Alternaria sp. M6.

AIM: To study the metabolites of a halotolerant fungus Alternaria sp. M6. METHODS: The metabolites were isolated and purified by various chromatographic techniques. Their structures were determined on the basis of physical properties and spectroscopic data. RESULTS: Nine compounds were isolated and identified as 8ß-chloro-3, 6aα, 7ß, 9ß, 10-pentahydroxy-9, 8, 7, 6a-tetrahydroperylen-4(6aH)-one (1), alterperylenol (2), dihydroalterperylenol (3), adenine (4), adenosine (5), deoxyadenosine (6), guanosine (7), tryptophan (8), and hexadecanoic acid (9). CONCLUSION: Compound 1 is a new perylenequinone.

[Study on the enzyme extraction process of hypericin by pectinase].

OBJECTIVE: To explore the best enzyme and optimal conditions for extracting Hypericin from Hypericum perforatum. METHODS: Chose the best enzyme from Pectinase, Xylanase, Glucanase, beta-Glucanase and Enzyme (SPE-007A). The effeet of solid-liquid ratio enzyme dosage, PH, temperature and the extraction time were investigated by L9 (3(4)) orthogonal design using extraction rate and the content of Hypericin as assessment index. RESULTS: The best enzyme was Pectinase and the optimum extraction process was as follows: PH 4.6, enzyme dosage 1.5%, temperature 50 degrees C, extraction time 5 h, liquid-solid ratio 15 times. CONCLUSION: This method is efficient and stable. It could be used in the future research of Hypericum perforatum.

Identification of anti-inflammatory constituents in Hypericum perforatum and Hypericum gentianoides extracts using RAW 264.7 mouse macrophages.

Hypericum perforatum (St. John's wort) is an herb widely used as supplement for mild to moderate depression. Our prior studies established synergistic anti-inflammatory activity associated with 4 bioactive compounds in a fraction of a H. perforatum ethanol extract. Whether these 4 compounds also contributed to the ethanol extract activity was addressed in the research reported here. Despite the popularity of H. perforatum, other Hypericum species with different phytochemical profiles could have their anti-inflammatory potentials attributed to these or other compounds. In the current study, ethanol extracts of different Hypericum species were compared for their inhibitory effect on LPS-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 mouse macrophages. Among these extracts, those made from H. perforatum and H. gentianoides demonstrated stronger overall efficacy. LC-MS analysis established the 4 compounds were present in the H. perforatum extract and pseudohypericin in all active fractions. The 4 compounds accounted for a significant part of the extract's inhibitory activity on PGE2, NO, tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in RAW 264.7 as well as peritoneal macrophages. Pseudohypericin was the most important contributor of the anti-inflammatory potential among the 4 compounds. The lipophilic fractions of H. gentianoides extract, which did not contain the previously identified active constituents, decreased PGE2 and NO potently. These fractions were rich in acylphloroglucinols, including uliginosin A that accounted for a proportion of the anti-inflammatory activity observed with the active fractions. Overall, the current study established that a different group of major anti-inflammatory constituents were present in H. gentianoides, while showing that the previously identified 4 compound combination was important for H. perforatum's anti-inflammatory potential.

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography.

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, ¹HNMR and ¹³CNMR.

Pharmacological studies on Hypericum perforatum fractions and constituents.

CONTEXT: This study describes the antispasmodic, bronchodilator, and cardiovascular-modulatory activities of Hypericum perforatum Linn. (Hypericaceae) fractions and constituents. AIM OF STUDY: Pharmacological investigation of H. perforatum fractions and active principles. MATERIALS AND METHODS: H. perforatum extract fractions [petroleum spirit (HpPet), chloroform (HpCHCl(3)), ethyl acetate (HpEtAc), and aqueous (HpAq)] and its compounds (hyperforin, hypericin, and hyperoside) were studied in various isolated tissue preparations. RESULTS: In rabbit jejunum, HpCHCl(3), HpEtAc and HpAq, like papaverine, inhibited both spontaneous and K(+) (80 mM)-induced contractions at similar concentrations, whereas HpPet was relatively potent against K(+), as verapamil. All fractions caused rightward of Ca(2+) concentration-response curves (CRCs), similar to verapamil. HpCHCl(3), HpEtAc, and HpAq shifted isoprenaline-inhibitory CRCs to left, like papaverine, while HpPet was devoid of any such effect, as verapamil. In guinea-pig trachea, HpCHCl(3), HpEtAc, and HpAq equipotently relaxed carbachol and K(+)-induced contractions and shifted the isoprenaline-curves to the left, whereas HpPet was more effective against K(+), without potentiating isoprenaline effect. When tested in rabbit aorta, all fractions exhibited vasoconstrictor and vasodilator effects, except HpEtAc, which did not produce vasoconstriction. In guinea-pig atria HpCHCl(3), HpEtAc, and HpAq initially caused cardiac stimulation, followed by inhibition, similar to papaverine, whereas HpPet, like verapamil, caused only cardiac suppression. Hyperforin, hypericin, and hyperoside showed a similar pattern of spasmolytic effect to verapamil. DISCUSSION AND CONCLUSION: Thus, all tested fractions of H. perforatum exhibit a combination of Ca(2+) antagonist and phosphodiesterase-inhibition, except petroleum spirit which was devoid of later mechanism. The compounds tested showed only Ca(2+) channel blocking effect.

Chemical constituents and antidepressant activity of the new species Hypericum enshiense occurring in China.

Hypericum enshiense L. H. Wu et F. S. Wang is a new species of Hypericum occurring in China, which was first identified and denominated by our laboratory. No research has been reported on the antidepressant activity and chemical constituents of this new species. In this study, the qualitative and quantitative analyses of the chemical constituents in the hydroalcoholic extract of this species were performed using HPLC/DAD/ESI-MS online method. Hypericin, pseudohypericin and some flavonoids were identified or tentatively identified. Furthermore, H. enshiense had a high content of hypericins than H. perforatum. In addition, the antidepressant activity of the hydroalcoholic extract of the species was investigated using forced swimming test (FST) and tail suspension test (TST). The extract significantly shortened the immobility time in FST and TST, while did not alter the locomoter activity of mice. These results suggested for the first time that the hydroalcoholic extract of H. enshiense might possess potential antidepressant-like activity in the animal behavioral models, and this species might act as a new potential resource for developing antidepressants to treat depressive disorders.

LC-MS and LC-PDA analysis of Hypericum empetrifolium and Hypericum sinaicum.

Within the framework of our continuous efforts to explore Hypericum species from Jordan, we report the analysis of the major active metabolites, naphthodianthrones and phloroglucinols, in the methanolic extracts of two under-explored Hypericum species; H. empetrifolium Willd. and H. sinaicum Hochst. & Steud. ex Boiss., using LC-(+,-)-ESI-MS (TIC and SIM) and LC-UV/Vis spectroscopy. Based on their LC-UV/Vis profiles, retention times and (+,-)-ESI-MS (TIC and SIM) spectral data, hypericin, protohypericin and pseudohypericin were identified in both of the investigated species. In addition adhyperfirin was only detected in H. empetrifolium, while hyperforin and protopseudohypericin were only detected in H. sinaicum. This is the first report documenting the presence of hypericin, protohypericin, pseudohypericin, protopseudohypericin, and hyperforin in H. sinaicum, and adhyperfirin in H. empetrifolium.

Rapid and efficient purification of naphthodianthrones from St. John's wort extract by using liquid-liquid extraction and SEC.

Hypericin and pseudohypericin, the main naphthodianthrones present in Hypericum species are among the most promising natural products, but the research concerning their biological activities is hindered by their low content in the plant. In this paper a method for the rapid isolation of hypericin and pseudohypericin from Hypericum perforatum hydro-alcoholic dried extracts has been developed. Briefly, the method consists of a partition of the extract between organic and aqueous layers and further purification of the richest extract in naphthodianthrones with Sephadex LH-20 column chromatography. A final separation of hypericin from pseudohypericin was achieved using Sephadex LH-60 column chromatography. All partitions were carried out in triplicate and monitored by LC-MS and NMR analyses. The best results were obtained by successive extraction with n-hexane, Et(2)O and EtOAc. A three-step fractionation resulted in 98% content in total naphthodianthrones. To the best of our knowledge this is the first report on the separation of hypericin from pseudohypericin using size exclusion chromatography.