Phototoxicity in human lens epithelial cells promoted by St. John's Wort

St. John's Wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges and is phototoxic to skin. To determine if it also could be phototoxic to the eye, we exposed human lens epithelial cells to 0.1-10 microM hypericin and irradiated them with 4 J/cm2 UV-A or 0.9 J/cm2 visible light. Neither hypericin exposure alone nor light exposure alone reduced cell viability. In contrast, cells exposed to hypericin in combination with UV-A or visible light underwent necrosis and apoptosis. The ocular antioxidants lutein and N-acetyl cysteine did not prevent damage. Thus, ingested SJW is potentially phototoxic to the eye and could contribute to early cataractogenesis. Precautions should be taken to protect the eye from intense sunlight while taking SJW.     

Pseudohypericin and hypericin in St. John's wort extracts. Breakdown of pseudohypericin

Photodiode array detector-coupled HPLC analysis of extracts of St. John's wort (Hypericum perforatum) reveals two main compounds: pseudohypericin (PH) and hypericin (H) (peaks A and B respectively). When the pH of the extracts is raised, peak A irreversibly disappears whereas peak B does not change. This instability at high pH means that alkaline conditions should be avoided during extraction and raises a question concerning the bioavailability of pseudohypericin from St. John's wort extract-containing medicinal products designed for oral administration.     

Time-resolved microspectrofluorimetry and fluorescence lifetime imaging of hypericin in human retinal pigment epithelial cells

Hypericin is the active ingredient of the off-the-shelf antidepressant St. John's Wort. It is an effective phototoxic agent and its systemic administration at therapeutic doses could induce particular damage in the eye due to continuous light exposure. Hypercin is strongly fluorescent and its fluorescence properties can be monitored to investigate noninvasively its localization and interactions. To this aim, time-resolved microspectrofluorimetry and fluorescence lifetime imaging were used to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by retinal pigment epithelium (RPE) cells treated with Hyp at concentrations in the micromolar range (0.5-10 microM). In the presence of hypericin, the emission peaks at 600-605 nm and the fluorescence decay is best fitted with three lifetimes (5.5-7 ns, 1.9-2.5 ns and <0.8 ns). Spectral and temporal differences were observed between high (> or =5 microM) and low hypericin concentrations. In particular, upon increasing concentration, the emission spectrum of the slow component broadens and its lifetime shortens. The latter change is observed also when high concentrations are reached locally, due to more efficient localization within the cell.     

Photooxidation of lens alpha-crystallin by hypericin (active ingredient in St. John's Wort)

Hypericin is the active ingredient in the over the counter antidepressant medication St. John's Wort. Hypericin produces singlet oxygen and other excited state intermediates that indicate it should be a very efficient phototoxic agent in the eye. Furthermore it absorbs in the UV and visible range, which means it can potentially damage both the lens and the retina. Lens alpha-crystallin, isolated from calf lenses, was irradiated in the presence of hypericin (5 x 10(-5) M, 10 mM ammonium bicarbonate, pH 7.0) and in the presence and absence of light (> 300 nm, 24 mW/cm2). Hypericin-induced photosensitized photopolymerization as assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Further analysis of the oxidative changes occurring in alpha-crystallin using mass spectrometry showed specific oxidation of methionine, tryptophan and histidine residues, which increased with irradiation time. Hypericin did not damage the lens protein in the dark. Damage to alpha-crystallin could undermine the integrity of the lens directly by protein denaturation and indirectly by disturbing chaperone function. Therefore, in the presence of light, hypericin can induce changes in lens protein that could lead to the formation of cataracts. Appropriate precautions should be taken to protect the eye from intense sunlight while on this antidepressant medication.     

Liquid chromatographic determination of St. John's wort components in functional foods

A method was developed for determination of St. John's wort marker compounds hypericin, pseudohypericin, hyperforin, and adhyperforin in functional foods. Solid-phase extraction provided analyte extraction and significant sample cleanup prior to analysis using liquid chromatography (LC) with UV and fluorescence detection. In addition to quantification using LC-UV, confirmation was made with electrospray ionization LC mass spectrometry (LC/MS). Several commercially available tea and drink products claiming to contain St. John's wort were tested. Recoveries ranged from 51 to 98% for the liquid samples. Comparison of the concentrations in 4 St. John's wort teas showed a variation in analyte concentration (1044-10 ng/mL marker compounds in brewed tea) and composition. No marker compounds were found in the beverages, indicating possible decomposition of the marker compounds caused by low pH and/or exposure to light. A solvent extraction procedure was developed for analysis of the marker compounds from solid samples. Analytes were detected at low parts per million, with an average recovery of 75%. No St. John's wort components were found in the 2 solid functional food samples analyzed.     

Determination of St. John's wort components in dietary supplements and functional foods by liquid chromatography

St. John's wort (Hypericum perforatum L.) preparations, a top-selling botanical dietary supplement used primarily as an antidepressant, has recently been used as an ingredient in some food products sold as functional foods. A rapid extraction technique followed by a liquid chromatographic (LC) method was developed to determine 4 characteristic bioactive compounds (pseudohypericin, hypericin, hyperforin, and adhyperforin) from St. John's wort in dietary supplements and functional foods to which it was added. Solid samples, including dried leaf/flower mixture, dietary supplement capsules, tea bags, puff and snack bar, were extracted with methanol by sonication. Noncarbonated, fruit-flavored drinks were centrifuged and mixed with methanol. Compounds were then determined by isocratic, reversed-phase LC with UV detection at 2 wavelengths and further identified or confirmed by photodiode array spectra and LC/mass spectrometry. Within-laboratory method variations (% RSD) were satisfactory. Very low amounts, if any, of the 4 components were found in drink and puff samples, and none was found in the snack bar. The methods developed provide a useful means for the determination of St. John's wort components in dietary supplements and functional foods.     

Antioxidant properties of melanin in retinal pigment epithelial cells

The retinal pigment epithelium (RPE) is a monolayer of highly pigmented cells lining the inner aspect of Bruch's membrane. This pigmentation is due to eumelanin and a possible antioxidant role of melanin is reported here. The photo-oxidation of A2E, a constituent of RPE lipofuscin, leads to the sequential addition of up to nine oxygen atoms and/or the addition or loss of two hydrogen atoms. These photo-oxidations were investigated in the presence and absence of either calf or human RPE melanin in A2E-laden RPE cells. It was found that calf melanin was protective against the photo-oxidation of A2E, with an inhibition of oxidation of up to 50% in the case of the addition of two oxygen atoms. Calf melanin was also protective against blue light-induced damage to RPE cells. In addition this ability appears to decrease in humans as they grow older. With aging, a melanin-lipofuscin complex called melanolipofuscin forms. It is suggested that the oxidation or photo-oxidation of A2E in vivo may contribute to the age-related deterioration of the anti-oxidant role of RPE melanin and lead to various retinal disorders, such as age-related macular degeneration.     

Development and evaluation of methods for determination of naphthodianthrones and flavonoids in St. John's wort

Several major constituents in St. John's wort were determined for a homogenized plant sample. Three extraction techniques were evaluated: Soxhlet extraction, pressurized-fluid extraction (PFE), and sonication extraction. Levels of nine constituents (chlorogenic acid, rutin, hyperoside, isoquercitrin, quercitrin, quercetin, amentoflavone, pseudohypericin, and hypericin) were measured using liquid chromatography with ultraviolet/visible absorbance, mass spectrometric, and fluorescence detection. Levels of total naphthodianthrones determined by liquid chromatography (LC) with absorbance detection at 590 nm were compared with levels determined by direct spectrophotometry at the same wavelength. Additionally, the methods described in this paper were applied to several brands of St. John's wort finished products.     

Quantitative evaluation of some species of St. John's wort for their level of hypericins by the HPLC method

All-Union Institute of Medicinal Plants Scientific Production Combine, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 138, January–February, 1991.     

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum)

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. John's wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.     

Authenticity study of commercial samples of St. John's wort by paper spray ionization mass spectrometry and chemometric tools

Hypericum perforatum L. (St. John's wort) is one of the world's most consumed medicinal plants for treating depression and psychiatric disorders. Counterfeiting can occur in the medicinal plant trade, either due to the lack of active ingredients or the addition of substances not mentioned on the labels, often without therapeutic value or even harmful to health. Hence, 43 samples of St. John's wort commercially acquired in different Brazilian regions and other countries were analyzed by paper spray ionization mass spectrometry (PS-MS) and modeled by principal component analysis. Hence, samples (plants, capsules, and tablets) were extracted with ethanol in a solid–liquid extraction. For the first time, PS-MS analysis allowed the detection of counterfeit H. perforatum samples containing active principles typical of other plants, such as Ageratum conyzoides and Senna spectabilis. About 52.3% of the samples were considered adulterated for having at least one of these two species in their composition. Furthermore, out of 35 samples produced in Brazil, only 13 were deemed authentic, having only H. perforatum. Therefore, there is a clear need to improve these drugs' quality control in Brazil.     

In vitro assays for evaluating the ultraviolet B-induced damage in cultured human retinal pigment epithelial cells

The present study demonstrates broadband UV-B-induced damage of cultured human retinal pigment epithelial cells as an effort to develop an in vitro model that can be used, along with in vivo research and other in vitro efforts, to evaluate the need for retinal UV protection in humans after cataract removal. The human retinal pigment epithelial cell line, ARPE-19, was cultured in two groups: control and treated. Treated cells were irradiated with three broadband UVB radiations at energy levels of 0.05, 0.1 and 0.2J/cm(2). After irradiation, cells were incubated for 48h while cellular viability, morphology, and phagocytotic activity were analyzed using the Alamar blue assay, confocal microscopy, and fluorescent microspheres. Confocal analysis concentrated on the study of the cell nuclei and mitochondria. The Alamar blue assay of UV-B-exposed cells showed dose and time-dependent decreases in cellular viability in comparison to control cells. Loss of cell viability was measured at the two higher energy levels (0.2 and 0.1J/cm(2)), but the cell group exposed to 0.05J/cm(2) showed no significant viability change at 1-h time point. Morphological evaluation also showed dose and time-dependent degradation of mitochondria and nucleic acids. Cells exposed with 0.05J/cm(2) UVB did not show significant degradation of mitochondria and nucleic acids during the entire culture period. Phagocytotic activity assay data for UVB-exposed cells showed dose-dependent decreases in phagocytotic activity in comparison with the control cells. The control cells have significantly greater capacities for uptake than the 0.1 and 0.2J/cm(2) UV-B-exposed cells, while the 0.05J/cm(2) UV-B-exposed cell group showed no significant difference from the control cell group. The findings suggest that UVB radiation-induced cultured RPE cell damage can be evaluated by assays that probe cellular viability, morphological change, and phagocytotic activity, and that these assay methods together provide a valuable in vitro model for ultraviolet radiation-induced retinal toxicology research.     

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, Et₂O 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.     

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.     

Enzymatic degradation of A2E, a retinal pigment epithelial lipofuscin bisretinoid

Some forms of blinding macular disease are associated with excessive accumulation of bisretinoid lipofuscin in retinal pigment epithelial (RPE) cells of the eye. This material is refractory to lysosomal enzyme degradation. In addition to gene and drug-based therapies, treatments that reverse the accumulation of bisretinoid would be beneficial. Thus, we have examined the feasibility of degrading the bisretinoids by delivery of exogenous enzyme. As proof of principle we report that horseradish peroxidase (HRP) can cleave the RPE bisretinoid A2E. In both cell-free and cell-based assays, A2E levels were decreased in the presence of HRP. HRP-associated cleavage products were detected by ultraperformance liquid chromatography (UPLC) coupled to electrospray ionization mass spectrometry, and the structures of the aldehyde-bearing cleavage products were elucidated by 18O-labeling and 1H NMR spectroscopy and by recording UV?vis absorbance spectra. These findings indicate that RPE bisretinoids such as A2E can be degraded by appropriate enzyme activities.     

The role of A2E in prevention or enhancement of light damage in human retinal pigment epithelial cells

The process of sight (photostasis) produces, as a by-product, a chromophore called 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E, 5E-hexatrienyl]-pyridinium (A2E), whose function in the eye has not been defined as yet. In youth and adulthood, A2E is removed from human retinal pigment epithelial (h-RPE) cells as it is made, and so it is present in very low concentrations, but with advanced age, it accumulates to concentrations reaching 20 microM. In the present study we have used photophysical techniques and in vitro cellular measurements to explore the role of A2E in h-RPE cells. We have found that A2E has both pro- and antioxidant properties. It generated singlet oxygen (phiso = 0.004) much less efficiently than its precursor trans-retinal (phiso = 0.24). It also quenched singlet oxygen at a rate (10(8) M(-1) s(-1)) equivalent to two other endogenous quenchers of reactive oxygen species in the eye: alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C). The endogenous singlet oxygen quencher lutein, whose quenching rate is two orders of magnitude greater than that of A2E, completely prevented light damage in vitro, suggesting that singlet oxygen does indeed play a role in light-induced damage to aged human retinas. We have used multiphoton confocal microscopy and the comet assay to measure the toxic, phototoxic and protective capacity of A2E in h-RPE cells. At 1-5 microM, A2E protected these cells from UV-induced breaks in DNA; at 20 microM, A2E no longer exerted this protective effect. These results imply that the role of A2E is not simple and may change over the course of a lifetime. A2E itself may play a protective role in the young eye but a toxic role in older eyes.     

Bioassay and UPLC-Q-Orbitrap-MS/MS guided isolation of polycyclic polyprenylated acylphloroglucinols from St. John's wort and their neuroprotective activity

St. John’s wort (Hypericum perforatum L.) is a popular dietary supplement ingredient used for the treatment of mild-to-moderate depression in the United States and Germany. Reported studies mainly focused on the biological evaluation and mechanism study of its crude extracts or two main components (namely, hypericin and hyperforin). However, it is unclear whether other phytochemicals including polycyclic polyprenylated acylphloroglucinols (PPAPs) contributed to the neuroprotective effects of H. perforatum. Here, bioassay and ultra performance liquid chromatography–quadrupole orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS/MS) guided isolation were applied to discover bioactive PPAPs from the crude extracts of H. perforatum. A new PPAP, named hyperforen A (2), along with nine known PPAPs (1 and 3–10), were identified from an ethyl acetate extract of H. perforatum. To the best of our knowledge, compound 2 represents the first PPAP with an unprecedented bicyclo[7.3.0]dodecane core. The chemical structures of the isolates were elucidated by extensive spectroscopic analysis and electronic circular dichroism calculations. Moreover, compounds 1–3, 6, and 10 (10 μM) exhibited significant neuroprotective effects against corticosterone-induced injury in PC12 cells. Fingings from the current study suggest that bioactive PPAPs from H. perforatum are promising compouds for the management of depression.     

The effect of telomerase expression on the escape from M2 crisis in virus-transformed human retinal pigment epithelial cells

Transformation with viral oncogene extends the lifespan of normal cells beyond replicative senescence called M1, but most of them eventually succumb to second crisis called M2 when telomeres become critically short. To acquire an infinite growth capacity, these cells have to overcome M2 crisis, which is known to follow telomerase activation. We have investigated if telomerase expression is required for virus-transformed pre-M2 cells to avert M2 crisis. Human retinal pigment epithelial (RPE) cells were transformed with simian virus 40 large T antigen and a VR3 clone in pre-M2 stage was obtained. Then, VR3 cells were transfected with a telomerase-containing vector and two cell lines that expressed telomerase temporarily or continuously were cloned and designated as ST1 and ST2, respectively. Normal RPE cells went into senescence after 36 population doublings. Although the lifespan was extended in the VR3 clone about 20 times more, it eventually underwent second crisis. The telomere length of VR3 decreased compared to that of normal RPE cells and the decrease continued during subculture. However, the ST1 and ST2 clones that expressed both T antigen and telomerase could avert this crisis. The initial telomere length of ST1 and ST2 was longer than that of normal cells. The ST1 underwent growth arrest again as telomerase expression faded out and elongated telomere was shortened, but the ST2 that maintained telomerase activity and telomere length proliferated continuously. In conclusion, telomerase activation is definitely required to overcome M2 crisis and acquire an infinite lifespan in human somatic epithelial cells and this mechanism is independent from M1 crisis escape in cell immortalization.     

Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells

Throughout the lifetime of an individual, light is focused onto the retina. The resulting photooxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration (AMD), the leading cause of legal blindness in the developed world, involves oxidative stress and death of the retinal pigment epithelium (RPE) followed by death of the overlying photoreceptors. Evidence suggests that damage due to exposure to light plays a role in AMD and other age-related eye diseases. In this work a system for light-induced damage and death of the RPE, based on the human ARPE-19 cell line, was used. Induction of mitochondria-derived reactive oxygen species (ROS) is shown to play a critical role in the death of cells exposed to short-wavelength blue light (425 +/- 20 nm). ROS and cell death are blocked either by inhibiting the mitochondrial electron transport chain or by mitochondria-specific antioxidants. These results show that mitochondria are an important source of toxic oxygen radicals in blue light-exposed RPE cells and may indicate new approaches for treating AMD using mitochondria-targeted antioxidants.     

Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells

The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon-carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E.