Determination of hypericin content in Hypericum triquetrifolium Turra (Hypericaceae) growing wild in Jordan

Hypericin content of methanolic extracts of dried flowers, leaves, stems, and roots of Hypericum triquetrifolium (Turra) were determined by HPLC. Conversion of protohypericin to hypericin was achieved by exposing samples to light for 30 min immediately before HPLC analysis. External standard calibration was used to quantify hypericin. Leaves showed the highest hypericin content of 0.36% w/w. Total aerial parts contained 0.43% w/w of hypericin. The relatively high hypericin content of the H. triquetrifolium in this study encourages the introduction, cultivation, and biological evaluation of hypericum specie in Jordan.     

Cytotoxic activity of antioxidant constituents from Hypericum triquetrifolium Turra

The Sulforodamine B (SRB) assay was used to test cytotoxicity against four human cancer cell lines and one normal cell line of antioxidant constituents isolated from Hypericum triquetrifolium Turra. Methanolic extract and pure compounds were tested against the large cell lung carcinoma cell line COR-L23, the hepatocellular carcinoma cell line HepG-2, renal cell adenocarcinoma ACHN, the amelanotic melanoma cell line C32 and normal human foetal lung MRC5. The results showed that I3-II8-biapigenin exhibited strong cytotoxic activity (IC50 = 5.73 micro g mL(-1)) showing a certain degree of selectivity against the different cell types.     

The essential oils of the aerial parts of two Hypericum taxa (Hypericum triquetrifolium and Hypericum aviculariifolium subsp. depilatum var. depilatum (Clusiaceae)) from Turkey

In this study, essential oil compositions of two Hypericum L. taxa (Hypericum triquetrifolium Turra. and Hypericum aviculariifolium Jaub. et Spach subsp. depilatum var. depilatum (Freyn et Bornm.) Robson) naturally grown in Turkey were determined using gas chromatography and gas chromatography-mass spectrometry systems. The essential oils were obtained by hydrodistillation from the aerial parts of plants. A total of 45 compounds were identified in the essential oils of H. triquetrifolium; 1-hexanal (18.8%), 3-methylnonane (12.5%) and α-pinene (12.3%). In this study, 41 components were identified in H. aviculariifolium subsp. depilatum var. depilatum oil; α-pinene (52.1%), germacrene D (8.5%) and β-pinene (3.6%) were the predominant constituents. The essential oil analysis showed that monoterpene concentrations were higher than that of the sesquiterpenes in both oils. The results were discussed in the meaning of the usefulness of these plants and their chemicals as natural products and potential usage in chemotaxonomy.     

Determination of hypericin and pseudohypericin from Hypericum perforatum in rat brain after oral administration

The popularity of St. John’s Wort (SJW) extracts for treating mild to moderate depression has increased over the last decades and great effort has been devoted to identify the active principle of SJW extract. Previous investigations suggest the contribution of at least three classes of compounds, the phloroglucinols, the quercetin flavonoids, and the phenanthroperylenequinones, to the clinical efficiency of SJW extracts. Up to now, a plausible molecular mechanism of action has been described only for the phloroglucinols. For the flavonoids and the phenanthroperylenequinones different targets were proposed on the basis of pharmacological studies. The vast majority of these targets are located in the CNS and therefore increasing interest in the question of the CNS availability of these substances arose. Recently, the ability of phloroglucinols and flavonoids to penetrate the blood brain barrier could be demonstrated. For the phenanthroperylenequinones an examination of CNS bioavailability is still missing. The aim of this work is to close this gap by developing and validating a HPLC method with electrochemical detection for the quantification of the phenanthroperylenequinones in brain tissue of rodents after oral application of SJW extract or pure hypericin. In our study, the phenanthroperylenequinone content in the CNS tissue was below the lower limit of detection of the analytical method and was thus lower than 16 pmol/g brain for hypericin and lower than 52 pmol/g brain for pseudohypericin after oral administration of 1600 mg/kg SJW extract or pure hypericin (5 mg/kg). Correspondence: Mario Wurglics, Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University/ZAFES, 60438 Frankfurt am Main, Germany.     

Exploring of bioactive compounds in essential oil acquired from the stem and root derivatives of Hypericum triquetrifolium callus cultures

The chemical profile of the essential oil of callus and cell suspension cultures derivatives from stem and root of Hypericum triquetrifolium were explored by ITEX/GC-MS. The major constituents for stem derivatives were undecane (78.44%) and 2,4,6-trimethyl-octane (9.74%) for fresh calli, 2,4-dimethyl-benzaldehyde (46.94%), 2,3-dimethyl-undecane (28.39%), 2,4-dimethyl-1-hexene (10.17%), 1,2-oxolinalool (3.64%) and limonene (3.55%) for dry calli and undecane (61.24%), octane, 2,4,6-trimethyl- (16.73%), nonane, 3-methyl-(3.74%), 2,5-diphenyl-benzoquinone (3.70%) and limonene (3.60%) for cell suspension. However, for root derivatives, the dominated components were: undecane (49.94%), eucalyptol (12.07%), limonene (9.98%), toluene (9.03%) and 3-methyl-nonane (4.29%) for fresh calli, 2,4-dimethyl-benzaldehyde (29.80%), 1,1-dimethylethyl-cyclohexane (14.99%), 3-methyl-pentanal (14.99%), undecane (10.04%), beta-terpinyl acetate (8.60%), 1,2-oxolinalool (6.27%) and 2-pentyl-furan (4.09%) for dry calli, undecane (52.38%), 2,4,6-trimethyl-octane (13.81%), 3-methyl-nonane (5.73%), toluene (4.82%) and limonene (4.57%) for cell suspension derivative in root. The attained outcomes indicated that the alkane, aldehyde and monoterpene fractions dominated the chemical composition of essential oils.     

Green synthesis of zinc oxide nanoflowers using Hypericum triquetrifolium extract: characterization,antibacterial activity and cytotoxicity against lung cancer A549 cells

Zinc oxide nanoparticles have attracted significant interest in recent years due to their unique multifunctional chemical and physical properties along with their biological activities. This study demonstrated for the first time the biogenetic synthesis of zinc oxide nanoparticles by utilization of the methanolic extract of Hypericum triquetrifolium (HT). The obtained nanoparticles (HT-ZnO) were characterized by ultraviolet–visible spectroscopy (UV–Vis), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). The shape of the resulted nanoparticles is fusiform nanoflowers with an average hydrodynamic size of 275.46 ± 0.20 nm and a zeta potential of −8.23 ± 0.26 mV. SEM micrographs revealed that HT-ZnO nanoflowers have a multi-process structure in which one of the processes is large and the others have similar smaller dimensions. The synthesized nanoflowers have an average length of 312.28 ± 78.93 nm and the tip of its processes has a width of 48.69 ± 9.71 nm. The antimicrobial activity of HT-ZnO nanoflowers was performed using microbroth dilution format. It showed a bactericidal mode of action against Gram-positive Staphylococcus aureus and Enterococcus faecalis with MIC/MBC values of 20 μg/mL and 5 μg/mL, respectively. MTT assay had revealed that HT-ZnO nanoflowers caused a dose-dependent decline in the viability of A549 adenocarcinomic human alveolar basal epithelial cells with an IC₅₀ value of 20.45 μg/mL. The effect of HT-ZnO nanoflowers on the migration and colony formation abilities against the same cells was evaluated as well. In conclusion, zinc oxide nanoflowers were successfully synthesized using methanolic extract of H. triquetrifolium. The resulting particles showed a bactericidal effect against Gram-positiveS. aureus and E. faecalis and a cytotoxic activity against A549 cells.     

In vitro study of the photocytotoxicity of some hypericin analogs on different cell lines

In the present study, hypericin analogs with an increased hydrophilic character were synthesized. As chemical modifications alter the lipophilicity/hydrophilicity balance together with the photophysical/chemical background of the molecule the influence of these structural changes on the cellular uptake, retention and subcellular localization in HeLa cells was investigated. Besides, their photocytotoxic effects using three cell lines (HeLa, MCF-7, A431), as well as their plasma protein binding were also assessed. To assess the relative hydrophilic/lipophilic character of hypericin and analogs their retention times were determined on a reversed phase high performance liquid chromatography (C-18) column. The retention time of all the hypericin analogs was < 46 min, except for dibenzyltetramethylhypericin (118 min), while the retention time of hypericin was > 200 min (solvent system: methanol/citrate buffer 30 mM pH 7; 70/30). Hypericin, hexa-, penta- and dibenzyltetramethylhypericin displayed a potent antiproliferative effect at the nanomolar range after photosensitization (3.6 J/cm2). On the contrary, photoactivated tetrasulfonhypericin and fringelite D had no antiproliferative effect on the three cell lines, whereas hypericin polyethylene glycol showed only an intermediate cytotoxic effect on A431 cells. In dark conditions no antiproliferative effect was observed for any photosensitizer. The antiproliferative photo-effect correlated well with the intracellular accumulation as measured using HeLa cells. In general, the photocytotoxic hypericin analogs concentrated to a large extent, while the noncytotoxic compounds were not taken up by the HeLa cells. Furthermore, confocal laser microscopy revealed that all photosensitizers mainly concentrated in the perinuclear region, probably corresponding with Golgi apparatus and the endoplasmic reticulum, except for tetrasulfonhypericin which located at the plasma membrane. In addition, the plasma protein binding studies illustrated that hypericin bind extensively to the low-density lipoproteins, while the other hypericin analogs were mainly bound to heavy proteins (mostly albumin) and to a small extent to low-density lipoproteins.     

Photocytotoxicity of hypericin in normoxic and hypoxic conditions

The normoxic and hypoxic photocytotoxicity of hypericin has been examined on A431 cells as assessed by the Neutral Red method, using cell-culture flasks made of polystyrene and glass, different hypericin concentrations and light fluences. Using polystyrene flasks, lower hypoxic photoactivities of hypericin than those in normoxic conditions are seen under low fluence. In these conditions the hypoxic photocytotoxic effect can be (partially) rescued by increasing the fluence. However, a completely different outcome is observed when using glass flasks, since most of the hypoxic photocytotoxicity is lost under these conditions. The differences can be explained in terms of efficiency of deoxygenation of the medium present in polystyrene or glass flasks. Polystyrene holds large amounts of oxygen that effuses very slowly. Glass, on the other hand, does not cause this inconvenience. Therefore the type of material of the container used to investigate the oxygen dependency of the photobiological activity of photosensitizers dramatically influences the outcome of the hypoxic experiments. Our results unequivocally prove that the cytotoxic effect induced by photoactivated hypericin is completely oxygen dependent. Hence hypericin does not differ from other phototherapeutics used in photodynamic therapy of cancer, since haematoporphyrin derivative and the second-generation photosensitizers used all seem to depend on the presence of oxygen for their antitumour activity.     

Between species diversity of Hypericum perforatum and H. maculatum by the content of bioactive compounds

The objective of present study was to establish and compare the contents of secondary metabolites of two Hypericum species, H. perforatum and H. maculatum, native to Lithuania, and to evaluate factors predetermining their variation with some practical implications for utilization and conservation. The HPLC analysis of the ethanolic extracts of the studied species showed some regularity in their composition. Both species contained chlorogenic acid, hyperoside, quercitrin, quercetin and hypericin. The presence of rutin and hyperforin was observed only in H. perforatum. The quantitative analysis showed higher content of quercitrin in H. perforatum, than in H. maculatum, whereas the differences in the contents of quercetin, hypericin and chlorogenic acid were not statistically significant between the species. H. maculatum contained a significantly higher content of hyperoside than H. perforatum. The data on phytochemical analysis suggest almost equivalent use of both H. perforatum and H. maculatum extracts in the food industry, cosmetics and pharmaceutics.     

Effects of St John's wort (Hypericum perforatum L.) extracts on epileptogenesis

The purpose of this study was to investigate the effects of treatment with water, n-butanol and ether extracts of Hypercom perforatum L. on epileptogenesis in rabbits. Animals from the control group received solvent-ethanol, and the kindling model of epilepsy was used. Epileptic focus was induced in Chinchilla rabbits by stimulation of the hippocampus. The following parameters were determined: the minimum current strength necessary to induce after-discharge (AD) - discharges appearing after cessation of stimulation; AD duration; the number of stimulations necessary to induce spontaneous kindling; and the latency time for the development of full kindling. The results obtained indicate that epileptogenesis is influenced by Hypericum perforatum L. extract treatment. Animals treated with an ether extract of Hypericum perforatum L. required significantly weaker minimum current strengths for the development of epileptogenic focus, and displayed longer AD times, while the number of electro-stimulations necessary for full kindling was less. In contrast, animals treated with water and n-butanol extracts required increased electro-stimulations for the development of epileptic discharge, and displayed shortened AD durations versus controls.     

Subcellular distributions and excited-state processes of hypericin in neurons

The photodynamic drug, hypericin, is studied in fetal rat neurons using fluorescence microscopy. Hypericin has an extremely high affinity for the cell membrane and is found to a smaller extent in the nucleus. Fluorescent excitation of hypericin is shown to cause irreversible damage to the cell membranes of living neurons. Fixed cells were used to make ultrafast time-resolved measurements to avoid the deleterious effects of long-term exposure to intense light and room temperatures. To our knowledge, these are the first ultrafast time-resolved measurements of the fluorescence lifetime of hypericin in a subcellular environment. Nonexponential fluorescence decay is observed in hypericin in the neurons. This nonexponential decay is discussed in terms of other examples where nonexponential decay is induced in hypericin upon its binding to biomolecules. The nonradiative processes giving rise to the nonexponential hypericin decay are attributed to excited-state electron transfer, excited-state proton transfer or both.     

Density and rheological properties of different nanofluids based on diesel oil at different mass concentrations

Journal of Thermal Analysis and Calorimetry - In this study, various nanofluids were synthesized and investigated in order to evaluate the density and viscosity of carbon nanoparticles...     

Toxic and essential element concentrations in different honey types

The physicochemical parameters (free acidity, moisture content, sucrose content, hydroxymethylfurfural, reducing sugars, electrical conductivity, diastase activity and water-insoluble content), elements (Cu, Fe, Zn, Mn, Cr, Co, Ni, Se, K, Na, Ca, Mg) and heavy metal/metalloid concentrations (Cd, Hg, Pb and As) of 84 honeys (linden, multifloral, honeydew and acacia) from Serbia, were analyzed using ICP-MS. Concerning the physicochemical parameters, all tested honeys were found to meet European Legislation (EC Directive 2001/110). Potassium was the major element, ranging between 943.9 ± 538.6 mg/kg and 2563 ± 1817 mg/kg. Statistical analysis showed significant differences in the concentrations of Zn, Mn, Co and Na between the honeys. Selenium and Hg levels were below the limits of detection. Positive correlations were found between electrical conductivity and most of the elements. The present study showed that Serbian honey examined were of good quality according to its physicochemical parameters and met safety criteria concerning the concentrations of Cd, Hg, Pb and As.     

Photosensitizing and radiosensitizing effects of hypericin on human renal carcinoma cells in vitro

The renal cell carcinoma (RCC) is extremely resistant to chemotherapy and radiotherapy. The prognosis of patients with metastatic RCC still remains poor, the median survival is less than 12 months. Therefore, new therapeutic options are desirable. The aim of this study was to investigate the photosensitizing and radiosensitizing effects of hypericin on human RCC cells in vitro. First the RCC-derived cell lines A498 and ACHN were incubated with different concentrations of hypericin. In vitro uptake and intracellular distribution of hypericin were confirmed by fluorescence microscopy. Subsequently cells were illuminated and irradiated with a dose of 2-8 Gy, respectively. Finally, metabolic activity, apoptosis and clonogenic survival were investigated. Uptake of hypericin was observed for almost all cells. Hypericin treatment combined with illumination led to a 94-97% decrease in metabolic activity and caused apoptosis in nearly 100% of RCC cells. Hypericin enhanced the radiosensitivity of A498 cells in vitro. The clonogenic survival after irradiation was significantly reduced by hypericin treatment. Taken together, the photosensitizing and radiosensitizing effects of hypericin on human RCC cells we found in this investigation could be of clinical relevance, e.g. for radiotherapy and intraoperative photodynamic therapy, respectively.     

Effects of different maturity stages on antioxidant content of Ivorian Gnagnan (Solanum indicum L.) berries

Gnagnan (Solanum indicum L.) is a spontaneous plant widely distributed in Ivory Coast. During ripening stages, Solanum indicum L. presents different colours (green, yellow and red) and is reported to contain several albeit poorly characterized antioxidant compounds. This paper describes in detail the antioxidant profile (ascorbic acid, carotenoids and polyphenols), antioxidant capacity (FRAP test and Folin-Ciocalteau assay) and the colour changes of Gnagnan berries at different ripening levels. Ascorbic acid content was similar in green and yellow berries, but significantly lower in red ones. Red berries showed a higher content of carotenoids compared to green and yellow ones. Regarding polyphenols, several phenolic acids and flavonoids were found in all berries. The content of caffeoylquinic acids, caffeic acid, flavonol glycosides and naringenin was higher in red berries, while the content of p-coumaric acid and feruloylquinic acids was similar among the three colours. The FRAP assay increased with the ripening process, whereas total polyphenols were similar among berries. Significant differences were found for the colorimetric indexes among products of different degrees of ripening. The present results show the important role of the ripening stage in increasing the antioxidant content of Gnagnan berries.     

Combination of integrated expanded bed adsorption chromatography and countercurrent chromatography for the direct extraction and purification of pseudohypericin and hypericin from St. John's wort (Hypericum perforatum L.)

St. John's wort has attracted particular attention because of its beneficial effects as an antidepressant, antiviral, and anticancer agent. A method for the combination of integrated expanded bed adsorption chromatography and countercurrent chromatography for the simultaneous extraction and purification of pseudohypericin and hypericin from the herb is presented in this paper. Firstly, the constituents were extracted and directly adsorbed by expanded bed adsorption chromatography under optimal conditions. The stepwise elution was then performed by expanded bed adsorption chromatography that enriched the targets with higher purities and recoveries compared to other methods. Secondly, the eluent fractions from expanded bed adsorption chromatography were further separated by two‐step high‐speed countercurrent chromatography. A two‐step high‐speed countercurrent chromatography method with a biphasic solvent system composed of n‐hexane/ethyl acetate/methanol/water with a volume ratio of 1:2:1:2 was performed by stepwise changing the flow rate of the mobile phase. Consequently, 5.6 mg of pseudohypericin and 2.2 mg of hypericin with purities of 95.5 and 95.0%, respectively, were successfully obtained from 40 mg of crude sample.     

Effects of sample preparation on methylmercury concentrations in Arctic organisms

The biogeochemical cycling of mercury (Hg) in the marine environment is an issue of global concern, as consumption of marine fish is a major route of human exposure to the toxic specie methylmercury (MeHg). The most widely utilised and accepted technique for preparing biological tissue samples for the analysis of MeHg involves an alkaline digestion of the sample. Recent studies suggest, however, that this technique is inadequate to produce satisfactory recoveries for certain biological samples, including fish, fur, feathers and other ‘indicator’ tissues which contain relatively high levels of MeHg. Thus an improved acidic extraction method has been proven to produce more satisfactory results for a wide range of biological tissues. The present study compares the two methods on real sample material from different organisms of an Arctic marine food chain, and shows how this could lead to misinterpretation of analytical results. Results show significantly (p < 0.05) lower concentrations for alkaline digestion for large parts of the food chain; especially in fish and birds. The mean differences in concentrations found between the two different methods were 28, 31 and 25% for fish (Polar and Atlantic cod), Little Auk and Kittiwake, respectively. For samples lower in the food chain (i.e. zooplankton and krill) no significant differences were found. This leads to a clear underestimation of the levels of MeHg found higher up in these food chains; the ratio of MeHg to Hg in biological samples; and thus potentially erroneous conclusions drawn from these results concerning the biological cycling of mercury species. We hypothesise that the main reasons for these differences are poor extraction efficiency and/or matrix effects on the ethylation step prior to analysis. This is the first study to examine the effects of these artefacts on real environmental samples covering a complete food chain.     

Analysis of selected constituents in methanolic extracts of Hypericum perforatum collected in different localities by capillary ITP-CZE

The on-line combination of CZE with capillary ITP (ITP-CZE) was used for the separation and quantification of selected flavonoids and phenolic acids in Hypericum perforatum leaves and flowers collected in six different localities in Slovakia. The leading electrolyte in the ITP preseparation step was 10 mM HCl with Tris as counterion (pH* 7.2). The terminating electrolyte was 50 mM boric acid of pH* 8.2 (adjusted with barium hydroxide). The BGE in the electrophoretic step contained 25 mM beta-hydroxy-4-morpholinopropanesulfonic acid (MOPSO), 50 mM Tris, 65 mM boric acid, pH* 8.3. The content of methanol in all electrolytes was 20% v/v. The total time of the analysis (including the preseparation step) was approximately 35 min. The rectilinear calibration ranges were between 0.125 and 5.0 microg/mL with kaempferol as internal standard. The correlation coefficients ranged between 0.9912 (for quercitrin and chlorogenic acid) and 0.9988 (for isoquercitrin). The RSD values are between 0.86 and 7.78% (n = 6) when determining rutin and quercetin (4 microg/mL). The optimized method was employed for the assay of flavonoids in medicinal plant extract of different collections of Hypericum perforatum haulm. The variability of the content of the active components depending on the place of collection was confirmed.