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.

Purification of hypocrellins from Shiraia bambusicola by coordinated high-speed countercurrent chromatography using cupric chloride as a complexing agent.

Hypocrellins are anthraquinone that can act as excellent photosensitizers for photodynamic therapy. In the present work, we found that high-speed countercurrent chromatography using cupric chloride as a complexing agent effectively separated hypocrellins from Shiraia bambusicola extract. The optimal two-phase solvent system consisted of petroleum ether/ethyl acetate/methanol/water (7:3:5.5:4.5, v/v/v/v), with 0.01 mol/L cupric chloride in the lower phase at pH of 2.45. This lower phase served as the mobile phase, whereas the upper phase acted as the stationary phase. Employing a continuous separation mode, three continuous injections were found to allow the purification of 1.2 g of crude extract in approximately 12 h. Hypocrellin B (10.8 mg), hypocrellin A (16.2 mg), and hypocrellin C (15.6 mg) were obtained from this process. Simulation of complexation of hypocrellin A with divalent copper ion by computational chemistry calculations indicated that three pairs of hydroxyl and carbonyl groups in hypocrellin A had similar binding energies, and demonstrated that hypocrellin A and B owned different metal-to-ligand ratios as compared to hypocrellin C. These factors could modify the partitioning of these compounds in two-phase solvent system, and resulting in a suitable separation factor. This method would also be used to purify other anthraquinones from natural products.

[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.

Determination of the absolute configuration of perylene quinone-derived mycotoxins by measurement and calculation of electronic circular dichroism spectra and specific rotations.

Altertoxins I-III, alterlosins I and II, alteichin (alterperylenol), stemphyltoxins I-IV, stemphyperylenol, stemphytriol, 7-epi-8-hydroxyaltertoxin I, and 6-epi-stemphytriol are mycotoxins derived from perylene quinone, for which the absolute configuration was not known. Electronic circular dichroism (ECD) spectra were calculated for these compounds and compared with measured spectra of altertoxins I-III, alteichin, and stemphyltoxin III and with reported Cotton effects. Specific rotations were calculated and compared with reported specific rotations. The absolute configuration of all the toxins, except for stemphyltoxin IV, could thus be determined. The validity of the assignment was high whenever reported ECD data were available for comparison, and the validity was lower when the assignment was based only on the comparison of calculated and reported specific rotations. ECD spectra are intrinsically different for toxins with a biphenyl substructure and for toxins derived from dihydroanthracene.

Altertoxins with potent anti-HIV activity from Alternaria tenuissima QUE1Se, a fungal endophyte of Quercus emoryi.

Screening of a small library of natural product extracts derived from endophytic fungi of the Sonoran desert plants in a cell-based anti-HIV assay involving T-cells infected with the HIV-1 virus identified the EtOAc extract of a fermentation broth of Alternaria tenuissima QUE1Se inhabiting the stem tissue of Quercus emoryi as a promising candidate for further investigation. Bioactivity-guided fractionation of this extract led to the isolation and identification of two new metabolites, altertoxins V (1) and VI (2) together with the known compounds, altertoxins I (3), II (4), and III (5). The structures of 1 and 2 were determined by detailed spectroscopic analysis and those of 3-5 were established by comparison with reported data. When tested in our cell-based assay at concentrations insignificantly toxic to T-cells, altertoxins V (1), I (3), II (4), and III (5) completely inhibited replication of the HIV-1 virus at concentrations of 0.50, 2.20, 0.30, and 1.50 µM, respectively. Our findings suggest that the epoxyperylene structural scaffold in altertoxins may be manipulated to produce potent anti-HIV therapeutics.

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.

[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.

Phaeosphaerins A-F, cytotoxic perylenequinones from an endolichenic fungus, Phaeosphaeria sp.

Six novel phototoxins, phaeosphaerins A-F, together with six known perylenequinones were isolated from an endolichenic fungus Phaeosphaeria sp. Their structures were determined unequivocally on the basis of comprehensive analysis of MS and NMR data as well as electronic circular dichroism calculations. These toxins kill cancer cells in vitro with accumulation in lysosomes, and the killing effects were potently intensified in the presence of light.

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.

[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.

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.

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.

[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.

Delivering a hydrophobic anticancer drug for photodynamic therapy by amorphous formulation.

An amorphous formulation of hypocrellin A for photodynamic therapy is reported which can provide stable aqueous dispersion of such hydrophobic photosensitizers. In vitro studies have demonstrated the active uptake of amorphous formulation of hypocrellin A into the mitochondria of tumor cells. Compared with Tween-80 micelle embedded hypocrellin A, low dark-toxicity but similar light-toxicity of the amorphous one to drug impregnated tumor cells was observed. Thus, the potential of using amorphous formulation of hypocrellin A as drug delivery system for photodynamic therapy has been demonstrated.

Determination of enantiomerization barriers of hypericin and pseudohypericin by dynamic high-performance liquid chromatography on immobilized polysaccharide-type chiral stationary phases and off-column racemization experiments.

Direct enantiomer separation of hypericin, pseudohypericin, and protohypericin was accomplished by high-performance liquid chromatography (HPLC) using immobilized polysaccharide-type chiral stationary phases (CSPs). Enantioselectivities up to 1.30 were obtained in the polar-organic elution mode whereby for hypericin and pseudohypericin Chiralpak IC [chiral selector being cellulose tris(3,5-dichlorophenylcarbamate)] and for protohypericin Chiralpak IA (chiral selector being the 3,5-dimethylphenylcarbamate of amylose) gave favorable results. Enantiomers were distinguished by on-line electronic circular dichroism detection. Optimized enantioselective chromatographic conditions were the basis for determining stereodynamic parameters of the enantiomer interconversion process of hypericin and pseudohypericin. Rate constants delivered by computational simulation of dynamic HPLC elution profiles (stochastic model, consideration of peak tailing) were used to calculate averaged enantiomerization barriers (DeltaG(enant)(#)) of 97.6-99.6 kJ/mol for both compounds (investigated temperature range 25-45 degrees C). Complementary variable temperature off-column (i.e., in solution) racemization experiments delivered DeltaG(enant)(#) = 97.1-98.0 kJ/mol (27-45 degrees C) for hypericin and DeltaG(enant)(#) = 98.9-101.4 kJ/mol (25-55 degrees C) for pseudohypericin. An activation enthalpy of DeltaH(#) = 86.0 kJ/mol and an activation entropy of DeltaS(#) = -37.7 J/(K mol) were calculated from hypericin racemization kinetics in solution, whereas for pseudohypericin these figures amounted to 74.1 kJ/mol and -82.6 J/(K mol), respectively. Although the natural phenanthroperylene quinone pigments hypericin and pseudohypericin as well as their biological precursor protohypericin are chiral and can be separated by enantioselective HPLC low enantiomerization barriers seem to prevent the occurrence of an excess of one enantiomer under typical physiological conditions--at least as long as stereoselective intermolecular interactions with other chiral entities are absent.

Isolation, characterization, and production of red pigment from Cercospora piaropi a biocontrol agent for waterhyacinth.

A red pigment produced by a Mexican isolate of Cercospora piaropi (waterhyacinth pathogen) has been isolated and identified as cercosporin. The kinetic of cercosporin production in culture media during dark/light regimes was evaluated. When C. piaropi was cultivated in continuous light and potato dextrose broth culture, a maximum of cercosporin production was observed (72.59 mg/l). Despite other reports, C piaropi Mexican isolate produce cercosporin in dark conditions (25.70 mg/l). The results suggest that production of cercosporin in C. piaropi-waterhyacinth pathogenesis is an important factor to take into account in biocontrol strategies.

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.

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.

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.

[Studies on the chemical constituents of flowers of Prunus mume].

OBJECTIVE: To study the chemical constituents of flowers of Prunus mume. METHODS: Compounds were separated by silica gel. Their structures were identified and elucidated by spectral analysis and chemical methods. RESULTS: Eight compounds were obtained and identified as benzoic acid (I), isorhamnetin (II), quercetin (III), kaempferol-3-O-beta-D-galactopyranoside (IV), isorhamnetin-3-O-beta-D-glucopyranoside (V), isoquercitrin (VI), hypericin (VII) and rutin (VIII). CONCLUSION: Among them, II-VII are isolated from this plant for the first time.