Chemical comparison and discrimination of two plant sources of Angelicae dahuricae Radix, Angelica dahurica and Angelica dahurica var. formosana, by HPLC-Q/TOF-MS and quantitative analysis of multiple components by a single marker.

INTRODUCTION: Angelica dahurica(BZ) and Angelica dahurica var. formosana(HBZ) are two plant sources of Angelicae dahuricae Radix. Although BZ and HBZ are commonly used herbal medicines with great medicinal and dietary values, study on their phytochemicals and bioactive compositions is limited. OBJECTIVE: To compare the chemical compositions of BZ and HBZ and find the chemical makers for discrimination and quality evaluation of the two botanical origins of Angelicae dahuricae Radix. METHODOLOGY: A high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method was established for chemical profiling of BZ and HBZ. Then, a quantitative analysis of multiple components by a single marker method was developed for simultaneous determination of nine bioactive coumarins (xanthotoxol, oxypeucedanin hydrate, byakangelicin, xanthotoxin, bergapten, oxypeucedanin, phellopterin, imperatorin and isoimperatorin). Moreover, chemometrics were performed to compare and discriminate BZ and HBZ samples. RESULTS: A total of 30 coumarins compounds were identified, and the chemical compositions in BZ and HBZ were quite similar. The quantitative analysis showed that there were significant differences in the contents of bioactive coumarins, and the chemometric analysis indicated five coumarins (xanthotoxol, xanthotoxin, bergapten, phellopterin and isoimperatorin) were responsible for the significant differences between BZ and HBZ, which could be used as chemical markers to distinguish the two original plant sources of Angelicae dahuricae Radix. CONCLUSION: The present work provided useful information for understanding the chemical differences between BZ and HBZ and also provided feasible methods for quality evaluation and discrimination of herbal medicines originating from multiple botanical sources.

Matrix-dependent absorption of 8-methoxypsoralen in extracorporeal photopheresis.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy for various diseases. Autologous leukocytes are collected, photoactivated with a photosensitizer (8-methoxypsoralen, 8-MOP) and UVA light, and subsequently reinfused back to the patient. Leukapheresis and UVA irradiation systems can be integrated into one device (inline) or handled by two separate devices (offline). ECP works via intercalation of 8-MOP into DNA helices and UVA-based interactions to inhibit DNA replication. 8-MOP is known to adhere to plastic materials, which might reduce its availability for intercalation. In the present study we examined the bioavailability of 8-MOP when different plastic materials and solvents are used as matrices. METHODS: Varying amounts of shredded ethylene vinyl acetate (EVA) and polyvinylchloride (PVC) from the MacoGenic irradiation bag (EVA1), UVA PIT irradiation bag (EVA2), UVA PIT recirculation bag (PVC A) and UVA PIT tubing (PVC B) by MacoPharma and PIT Medical Systems, respectively, were incubated with 200 ng mL-1 8-MOP dissolved in diisopropyl ether (DIPE) plus toluene 90/10 vol%, deionized water or plasma. After 2 h 8-MOP concentrations were determined by GC-MS. RESULTS: After incubation, 8-MOP concentrations varied depending on the amount and type of plastic (PVC > EVA) and solvent (water > plasma > DIPE/toluene). Absorption to 200 mg EVA1 or EVA2 resulted in 8-MOP concentrations of 57% or 32% in water, 91% or 80% in plasma, and 93% or 92% in DIPE/toluene, while 200 mg PVC A and PVC B yielded recovery rates of 26% and 10% in water, 76% and 75% in plasma, and 55% and 30% in DIPE/toluene, respectively. Remaining 8-MOP differed significantly between container materials (EVA vs. PVC; p < 0.022) but not manufacturers (MacoPharma vs. PIT Medical Systems). CONCLUSION: Absorption loss of 8-MOP depends on the type of plastic and solvent and is more pronounced with water than with plasma. As the DNA binding effect of 8-MOP is dose-dependent, ECP starting doses should be adjusted to ensure that a sufficient concentration of free bioavailable 8-MOP is present during UV irradiation.

Extracellular chromone derivatives in cell cultures of Pimpinella anisum. Influence of elicitation with methyl jasmonate and 2ß-methyl cyclodextrins.

OBJECTIVES: To explore the potentiality of undifferentiated Pimpinella anisum L. cell cultures for the production of secondary metabolites by means of elicitation. RESULTS: Two chromone compounds were secreted to the medium of undifferentiated cultures of P. anisum: 4-methoxyfuro[3,2-g]chromen-7-one, known as bergapten, which is constitutive to anise, and 5-hydroxy-7-methoxy-2-methylchromen-4-one, the rare chromone eugenin, not yet described in P. anisum. Caffeoyl quinic acid species were also identified in the biomass. Elicitation with methyl jasmonate enhanced chromone accumulation in the medium and stimulated phenolic acid metabolism in the biomass (11 mg caffeoyl quinic acids g-1 DW cells). The application of 2,6-dimethyl-ß-cyclodextrins to cultures led to an intense accumulation of chromones, with nearly 10 mg l-1 bergapten and 150 mg l-1 eugenin being accumulated extracellularly after optimal elicitation conditions. CONCLUSIONS: The significant amounts of eugenin obtained in the anise cultures and the stability of production over long periods of time can be of interest for its biotechnological production and for future studies on biosynthesis regulation.

A method for the quantification of 8-methoxypsoralen by mass spectrometry for offline extracorporeal photopheresis.

BACKGROUND: Extracorporeal photopheresis (ECP) is an efficient method to treat various autoimmune diseases, cutaneous T-cell lymphoma, and graft-versus-host disease. It is based on the ex vivo inactivation of lymphocytes by 8-methoxypsoralen (8-MOP)/UV light treatment. Despite the adhesive, lipophilic nature of 8-MOP, no quality control is established for the ECP procedure. METHODS: We developed a sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to monitor residual 8-MOP concentration after UVA irradiation in the whole blood supernatant after acetonitrile precipitation. RESULTS: The preanalytical stability of 8-MOP exceeded 7 days, allowing batch mode analysis. Linearity was determined with R2 above 0.99. The 8-MOP concentrations decreased exponentially after UV exposure, with decay constants of 0.0259 in plasma and 0.0528 in saline. The recovery of 8-MOP in photopheresates was about 68%, indicating binding to DNA as well as to plastic structures. UVA induced no 8-MOP fragmentation, but caused self-adducts under extreme conditions (10-fold UV dosage). CONCLUSIONS: Detection of 8-MOP proved to be feasible and demonstrated that the doses were in the pharmaceutically active range.

Investigations on the constituents of SagaPro tablets, a food supplement manufactured from Angelica archangelica leaf.

Saga Pro is a food supplement product manufactured in Iceland and marketed internationally. It is claimed to have anti-nocturia effect and the flavonoid isoquercitrin has been suggested to play a role in this assumed activity. The purpose of this study was to identify and quantify the main flavonoids and furanocoumarins in the SagaPro tablets and to evaluate the importance of their presence. Isoquercitrin was identified as a constituent in an amount of 158 µg/tablet. This is a p.o. dosage highly unlikely to have an effect on nocturia or any other pharmacologically significant effect in humans. The main furanocoumarins, xanthotoxin and imperatorin, were also identified and quantified to 280 and 2 µg/tablet, respectively.

Bioactivity-guided isolation of antimicrobial coumarins from Heracleum mantegazzianum Sommier & Levier (Apiaceae) fruits by high-performance counter-current chromatography.

An efficient strategy, based on bioassay-guided fractionation, high-performance liquid chromatography (HPLC), and high-performance counter-current chromatography (HPCCC), was established to purify and evaluate the bioactive compounds from the dichloromethane extract of the fruits of Heracleum mantegazzianum Sommier & Levier (Apiaceae). The quaternary solvent system n-heptane-ethyl acetate-methanol-water (6:5:6:5 v/v) was used in the reversed phase mode. Using this method, in a single run, seven fractions were isolated, among them three were the pure furanocoumarins: pimpinellin, imperatorin, and phellopterin. In order to purify xanthotoxin a more polar system (1:1:1:1 v/v) was further applied. The antimicrobial activity of extract, chromatographic fractions, and single compounds were in the range of MIC = 0.03-1 mg mL(-1). Xanthotoxin may have priority as a compound of further interest based on its antimicrobial activity. For the first time, an extensive antimicrobial study was performed for pimpinellin and phellopterin.

[Effects of Browning Inhibitors on Suspension Cells Growth and Secondary Metabolites Production in Changium smyrnioides].

OBJECTIVE: To study the effects of browning inhibitors on Changium smyrnioides suspension cells growth and secondary metabolites production. METHODS: Different concentrations of V(C), AC, AHC, Na2S2O3 and PVP were added to the light brown suspension cells, and the contents of phenols, total coumarins, bergaptol and bergapten were determined by UV-Vis and HPLC. RESULTS: PVP with low concentration and V(C) improved the growth of the suspension cells in different degrees. It was showed that the content of phenols in the suspension cells was related to the kinds of browning inhibitors. The addition of V(C) in the medium increased the content of total coumarins significantly. After using 2 mg/mL of V(C), the gross increase rate of total coumarins was 51.53%, which was 4.8 times than that of the control group. The browning phenomenon caused by salicylic acid were inhibited by adding 2 mg/mL of V(C) into suspension culture system (with salicylic acid as the inducer). At the same time, the content of bergaptol and bergapten was increased 25.96% and 33.33%, respectively. CONCLUSION: V(C) is the best anti-browning agent in this study. It can inhibit browning, and promote cell growth and accumulation of secondary metabolites in Changium smyrnioides suspension cells.

Simultaneous determination of osthole, bergapten and isopimpinellin in rat plasma and tissues by liquid chromatography-tandem mass spectrometry.

A highly selective and sensitive method for simultaneous quantitation of osthole, bergapten and isopimpinellin in rat plasma and tissues was developed by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS). After liquid-liquid extraction of samples with methyl tert-butyl ether, the analytes and dextrorphan (internal standard, IS) were separated by a Hypersil GOLD AQ C18 column with gradient elution of acetonitrile and water containing 0.5‰ formic acid. Three determinands were detected using an electrospray ionization (ESI) tandem mass spectrometry in the multiple reaction monitoring (MRM) modes with positive electrospray ionization. Calibration curves were recovered over the concentration ranges of 1-200 ng/ml, 1-500 ng/ml, 0.25-200 ng/ml for osthole, bergapten and isopimpinellin in plasma; 1-100 ng/ml, 1-500 ng/ml, 0.5-100 ng/ml for osthole, bergapten and isopimpinellin in tissues, respectively. The intra-day precision (R.S.D.) was within 13.90% and the intra-day accuracy (R.E.) was within -6.27 to 6.84% in all biological matrixes. The inter-day precision (R.S.D.) was less than 13.66% and the inter-day accuracy (R.E.) was within -10.64 to 13.04%. Then the method was successfully applied to investigate plasma pharmacokinetic study and tissue distribution of osthole, bergapten and isopimpinellin in rats after oral administration of Fructus Cnidii extraction, especially for testis/uterus tissue distribution. The results demonstrated that osthole, bergapten and isopimpinellin were absorbed and eliminated rapidly with wide distributions in rats. Distribution data of these three bioactive components in testis/uterus tissues could offer useful information for the further preclinical and clinical studies of Fructus Cnidii in the treatment of genital system disease.

Absence of furanocoumarins in Advantra Z® (Citrus aurantium, bitter orange) extracts.

Grapefruit (Citrus paradisi) juice is known for its ability to alter drug metabolism through inhibition of the cytochrome P450-3A4 (CYP3A4) system, and result in drug-food interactions that may be life threatening. The primary active ingredients in grapefruit responsible for these effects are the furanocoumarins bergapten, bergamottin, and 6',7'-dihydroxybergamottin (DHB). Bergamottin and DHB appear to be the most important in terms of adverse drug interactions. Furanocoumarins are present in the juices and fruits of other Citrus species including C. aurantium (bitter oranges). Bergapten is the predominant furanocoumarin in bitter orange. Bitter orange extracts are widely used in products associated with weight loss, sports performance, and energy production. Questions have been raised about the potential of bitter orange extracts to cause drug interactions. This study examined the furanocoumarin content of four standardized bitter orange extracts (Advantra Z®) by liquid chromatography-mass spectroscopy. The results indicated that the total furanocoumarin content of each of the four extracts was less than 20 µg/g, amounts insufficient to exert significant effects on the metabolism of susceptible drugs in human subjects at the doses commonly used for these extracts.

The effect of pre-treatment and modified atmosphere packaging on contents of phenolic compounds and sensory and microbiological quality of shredded celeriac.

BACKGROUND: The aim of this study was to determine the effect of washing (4 °C, 120 s) or soaking (4 °C, 600 s) of shredded celeriac in tap water on changes in contents of phenolic compounds, including furanocoumarins, and sensory and microbiological quality during 12 days of storage. The product was packaged in air or modified atmosphere containing 2/10/88 kPa O2/CO2/N2. RESULTS: The applied pre-treatment consisting of washing or soaking of shredded celeriac in water resulted in decreases in 8-methoxypsoralen content by approximately 50 and 70% respectively and phenolic content by 30% compared with samples that were not subjected to pre-treatment. During storage of shredded celeriac, a further significant (P ≤ 0.05) reduction in phenolic compounds and an approximately 2.5-fold increase in the total content of furanocoumarins were found. The application of modified atmosphere packaging had a significant effect on the maintenance of good sensory and microbiological quality of the tested product. CONCLUSION: Modified atmosphere packaging of shredded celeriac not subjected to pre-treatment made it possible to obtain a product with good sensory and microbiological quality and the highest content of phenolic compounds. The level of furanocoumarins recorded in the tested product does not constitute a health hazard.

Contamination of deconjugation enzymes derived from Helix pomatia with the plant bioactive compounds 3,3'-diindolylmethane, 5-methoxypsoralen, and 8-methoxypsoralen.

Bioactive compounds from plant foods are intensely investigated for effects on disease prevention. ß-Glucuronidase/arylsulfatase from Helix pomatia (snail) is commonly used when quantifying exposure to metabolized dietary components. However, we describe here the contamination of multiple formulations of this enzyme preparation with 3,3'-diindolylmethane (DIM), 8-methoxypsoralen (8-MOP), and 5-methoxypsoralen (5-MOP), bioactives from cruciferous and apiaceous vegetables under investigation as putative cancer chemopreventive agents. We identified an Escherichia coli preparation of ß-glucuronidase as free from contamination with any of the compounds tested. These results demonstrate the importance of selecting appropriate enzyme preparations when quantifying naturally occurring, trace level compounds in biological fluids.

A quantitative mass spectrometry-based approach for assessing the repair of 8-methoxypsoralen-induced DNA interstrand cross-links and monoadducts in mammalian cells.

Interstrand cross-links (ICLs) are highly toxic DNA lesions that block transcription and replication by preventing strand separation. ICL-inducing agents were among the earliest and are still the most widely used forms of chemotherapeutic drugs. Because of the repair of DNA ICLs, the therapeutic efficacy of the DNA cross-linking agents is often reduced by the development of chemoresistance in patients. Thus, it is very important to understand how various DNA ICLs are repaired. Such studies are currently hampered by the lack of an analytical method for monitoring directly the repair of DNA ICLs in cells. Here we report a high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method, together with the isotope dilution technique, for assessing the repair of 8-methoxypsoralen (8-MOP)-induced DNA ICLs, as well as monoadducts (MAs), in cultured mammalian cells. We found that, while there were substantial decreases in the levels of ICL and MAs in repair-competent cells 24 h after 8-MOP/UVA treatment, there was little repair of 8-MOP-ICLs and -MAs in xeroderma pigmentosum, complementation group A-deficient human skin fibroblasts and excision repair cross-complementing rodent repair deficiency, complementation group 1-deficient Chinese hamster ovary cells over a 24 h period. This result provided unequivocal evidence supporting the notion that the 8-MOP photoadducts are substrates for nucleotide excision repair in mammalian cells. This is one of the first few reports about the application of LC-MS/MS for assessing the repair of DNA ICLs. The analytical method developed here, when combined with genetic manipulation, will also facilitate the assessment of the roles of other DNA repair pathways in removing these DNA lesions, and the method can also be generally applicable for investigating the repair of other types of DNA ICLs in mammalian cells.

Caracterização dos efeitos tóxicos do 1, 2-dihidroxibenzeno em células originadas do sistema nervoso: investigação do efeito protetor de derivados de plantas / Characterization of toxic effects of 1, 2-dihydroxybenzene cells originating in the nervous system: investigating the protective effect of plant-derived

Catecóis são derivados do benzeno, podendo apresentar citotoxicidade, que pode constituir um modelo experimental útil para o desenvolvimento de novos fármacos. No bioma brasileiro inúmeras plantas produzem metabólitos com atividades diversas, como antioxidantes, ou inibidores do crescimento celular. No Brasil, as neoplasias são a segunda causa de óbito, especialmente aquelas derivadas do sistema nervoso, aumentando o interesse por novos antineoplásicos e agentes neuroprotetores. Este trabalho caracteriza efeitos citotóxicos do 1,2-dihidroxibenzeno (CAT) e discretamina (DSC) em células do sistema nervoso in vitro. Determinou-se a EC50 de CAT e DSC usando brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium (MTT), investigou-se sua auto-oxidação por espectrofotometria, avaliou-se mudanças morfológicas e condensação/fragmentação nuclear por microscopia. Avaliou-se a proteção de DSC e 8-metoxipsoraleno (8-MOP) contra a citotoxicidade do CAT. O padrão de morte celular foi analisado por citometria de fluxo. A espoliação de glutation reduzido (GSH) foi analisada usando monoclorobimano. A toxicidade do CAT para células SH-SY5Y e C6 depende da dose e associa-se à formação de quinonas. Houve mudanças morfológicas, condensação/fragmentação da cromatina e morte apoptótica, não relacionada à espoliação de GSH. DSC não foi tóxica para células SH-SY5Y, porém protegeu contra os efeitos do CAT em baixas concentrações. DSC mostrou-se citotóxica para células de glioma (GL-15 e C6) e potencializou o CAT. Pré-tratamento por 30 minutos com DSC protegeu contra a ação do CAT após 72 horas. 8-MOP potencializou os efeitos do CAT, não revertendo seus efeitos na viabilidade celular, morfologia celular, condensação/fragmentação nuclear, e espoliação de GSH. Esses resultados caracterizam um modelo de citotoxicidade que pode ser aplicado no desenvolvimento de novos agentes farmacológicos. Estudos complementares são necessários para elucidar a proteção da DSC.

Catechols are benzene derivatives, which may exhibit cytotoxic activity that can be employed to develop new drugs. Plants are important sources of metabolites with pharmacological activities such as antioxidants, or cell growth inhibitors. In Brazil, cancer is the second leading cause of death, especially those derived from the nervous system, which increase the interest for new antineoplastic and neuroprotective drugs. The cytotoxic effects promoted by 1,2-dihydroxybenzene (CAT) and discretamine (DSC) in nervous system cells were characterized in vitro. The protective effects of DSC and 8-methoxypsoralen (8-MOP) against CAT-induced cytotoxicity were also evaluated. CAT and DSC EC50 was determined by using 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT). CAT auto-oxidation was investigated by spectrophotometry. Morphological changes and nuclear condensation/ fragmentation were evaluated by microscopy. The pattern of cell death was obtained by flow cytometry. Reduced glutathione (GSH) depletion was analyzed by using monochlorobimane. CAT induced a dose-dependent toxicity to SH-SY5Y and C6 cells, associated with reactive quinones formation. It also induced morphological changes, nuclear condensation/fragmentation, and apoptotic death not caused by GSH depletion. DSC was not toxic to SH-SY5Y cells, but protected against CAT effects at low concentrations. DSC was be cytotoxic to glioma cells (GL-15 and C6) and potentiated CAT effects. However, pretreatment for 30 minutes with DSC protected them against CAT after 72 hours. 8-MOP also potentiated CAT effects instead to protect cells. These results characterize an experimental model useful for studies searching new pharmacological agents. However, further studies are needed to elucidate the DSC protective effects.

[High-performance liquid chromatography for determination of psoralene, bergapten and apigenin in Ficus hirta Vahl].

OBJECTIVE: To establish a high-performance liquid chromatography (HPLC)-based method for determining the contents of psoralene, bergapten and apigenin in Ficus hirta Vahl. METHODS: A Hypersil C18 column (250 mm × 4.6 mm, 5 µm) was used with the mobile phase of methanol-water (60:40), flow rate of 1.0 ml/min, detection wavelength of 268.7 nm and column temperature of 30 degrees celsius. RESULTS: The calibration curve was linear within the range of 10.0-30.0 µg/ml for psoralene (r=0.9998), 15.0-45.0 µg/ml for bergapten (r=0.9998) and 5.0-15.0 µg/ml for apigenin (r=0.9992). The average recovery of psoralene was 99.7% (RSD=1.99%), that of bergapten was 99.9% (RSD=1.71%) and that of apigenin was 100.3% (RSD=1.78%). CONCLUSION: The method is simple, economic and accurate with good reproducibility for the contents of psoralene, bergapten and apigenin.

In vitro photo-induced cytotoxic activity of Citrus bergamia and C. medica L. cv. Diamante peel essential oils and identified active coumarins.

CONTEXT: The search for innovative therapeutic approaches is gaining more interest in clinical oncology. OBJECTIVE: In the present investigation we reported the chemical profile and the photo-induced cytotoxic activity of two endemic Calabrian Citrus species (Rutaceae): Citrus bergamia Risso & Poit. and Citrus medica L. cv. Diamante. MATERIALS AND METHODS: Essential oils were obtained by hydrodistillation and analyzed by GC and GC/MS. In order to evaluate the cytotoxic activity two melanoma models, such as amelanotic melanoma C32 and malignant melanoma A375, were used. RESULTS: The essential oil of C. bergamia was characterized by limonene, linalyl acetate, gamma-terpinene, linalool and beta-pinene as major components. The most abundant compounds of C. medica cv. Diamante oil were limonene, gamma-terpinene, citral, geranial, beta-pinene and alpha-pinene. Two coumarins, bergapten and citropten, were also identified in C. bergamia and C. medica cv. Diamante, respectively and tested for biological activity. Both C. bergamia and C. medica cv. Diamante oils exhibited a selective interesting activity against the A375 cell line with IC(50) values of 79.3 and 89.1 microg/mL, respectively, after 100 min exposure to UV irradiation. The strong antiproliferative activity demonstrated with bergapten (IC(50) value of 71.3 microg/mL after 20 min of irradiation) was not found with citropten. DISCUSSION AND CONCLUSION: Our study suggested that UV irradiation is effective in activating essential oils and in particular bergapten. This phototoxicity may be considered as a treatment option in some cases of lentigo maligna or lentigo maligna melanoma.

[Contents of furanocoumarins in grapefruit juice and health foods].

Interactions between grapefruit juice and medications have long been recognized. In recent years, several furanocoumarins (FCs) that inhibit P450 activity in intestinal microsomes have been isolated from grapefruit juice. FCs, i.e., bergamottin (BG), bergapten (BP), bergaptol (BT) and 6',7'-dihydroxybergamottin (DHB), in samples was extracted with acetonitrile, and separated on a Phenyl column using 0.1% phosphoric acid-acetonitrile gradient as a mobile phase, with monitoring at 311 nm. The recoveries of BG, BP, BT and DHB from lemon juice spiked at the level of 5.0 microg/g were 103+/-0.7%, 99.5+/-0.2%, 96.5+/-0.2% and 90.1+/-0.2%, respectively. The quantification limits were 1.0 microg/g in samples. The contents of BG, BP and DHB in grapefruit juice (n=13), citrus fruit of 20 species and health food (n=16) were measured. The contents of BG were 0-16 microg/g, 0-16 microg/g and 0-5.6 microg/g, BT were 0-39 microg/g, 0-13 microg/g and 0-28 microg/g, DHB were 0-10 microg/g, 0-35 microg/g and 0-6.2 microg/g, respectively. BP was not detected. These results suggest that patients prescribed calcium antagonists or antiallergic agents should be cautions about their intake of FCs from grapefruit juice, citrus and health foods.

Isolation and identification of insecticidal components from Citrus aurantium fruit peel extract.

Three active components were identified by bioassay-guided fractionation of bitter orange ( Citrus aurantium L.) fruit peel petroleum ether extract. Silica gel fractionation of the extract yielded a fraction that inflicted up to 96% mortality to adults of the olive fruit fly Bactrocera oleae (Gmelin) three days post-treatment. Subsequent HPLC purification of the active fraction resulted in the isolation of three components, eluted in fractions F 222, F 224, and F 226, that induced adult mortality. Considering the data obtained from UV, FTIR, MS, and (1)H NMR spectra, they were identified as 7-methoxy-8-(3'-methyl-2'-butenyl)-2 H-1-benzopyran-2-one (osthol), 4-methoxy-7 H-furo[3,2- g]benzopyran-7-one (bergapten), and 4-(( E)-3'-methyl-5'-(3'',3''-dimethyloxiran-2''-yl)pent-2'-enyloxy)-7 H-furo[3,2- g][1]benzopyran-7-one (6',7'-epoxybergamottin). Our results are in concordance with those reported in the literature and were further verified by direct comparison to authentic components. 6',7'-Epoxybergamottin was toxic when tested individually, while bergapten and osthol were found to act synergistically to 6',7'-epoxybergamottin.

LC-MS/MS for the detection of DNA interstrand cross-links formed by 8-methoxypsoralen and UVA irradiation in human cells.

DNA interstrand cross-links (ICLs) are induced by many carcinogens and anitcancer drugs. ICL is a covalent linkage of both strands of DNA, preventing DNA strand separation during transcription and replication; thus, it is extremely cytotoxic in vivo. Psoralen and its derivatives are widely applied for the clinical treatment of several skin diseases and cutaneous T cell lymphoma, and they are also commonly used as model compounds for the study of ICL. Upon UVA photoactivation, 8-methoxypsoralen alkylates both strands of DNA at the 5,6-double bond of thymidines at the 5'-TpA-3' site, generating monoadducts and ICLs. Here we developed a method utilizing HPLC-tandem mass spectrometry, combined with nuclease P1 digestion, to assess the formation of ICL in DNA of human skin melanoma cells exposed to 500 ng/mL 8-methoxypsoralen and UVA irradiation. We were able to quantify ICL, in the form of tetranucleotide, at the level of 1 lesion/10(6) unmodified nucleobases using a low-microgram quantity of DNA. In addition, our results revealed that the formation of ICL increased linearly with the UVA dose. The yield of ICL increased by 15-fold from 4.5 to 76 lesions/10(6) nucleotides when the UV dose was increased from 0.5 to 5 J/cm2. This is the first report of an LC-MS assay for the quantification of DNA interstrand cross-links. The specificity and accuracy of this high-throughput approach are advantageous over other methods for the detection of ICLs formed in vitro and in vivo.

[Simultaneous determination of 5 active components in Fructus Cnidii by HPLC].

OBJECTIVE: To develop an RP-HPLC method for simultaneous determination of 5 constituents in Fructus Cnidii. METHOD: Analysis was performed on an Alltech C18 (4.6 mm x 250 mm, 5 microm) column. The mobile phases were acetonitrile water and acetic acid with gradient elution. The flow rate was 1 mL x min(-1). The monitoring wavelength was 325 nm and 245 nm. The column temperature was 40 degrees C. RESULT: The linear response ranges were 1-20 microg x mL(-1) (r = 0.999 9) for xanthotoxin, 1-20 microg x mL(-1) (r = 0.999 9) for isopimpinellin, 11-20 microg x mL(-1) (r = 0.999 8) for bergapten, 100-1 200 microg x mL(-1) (r = 0.999 7) for imperatorin, 100-2 000 microg x mL(-1) (r = 0.999 9) for osthole. The average recoveries were all above 95%. CONCLUSION: The method is simple, sensitive and accurate with good reproducibility.

Can dietary furanocoumarin ingestion enhance the erythemal response during high-dose UVA1 therapy?

As phototoxic skin reactions caused by psoralen are induced by wavelengths within the UVA1 spectrum, we assessed the potential of the small amount of psoralen in a normal diet to provoke phototoxicity in volunteers with skin types I and II. Threshold erythema was unaffected by ingestion of a 200-g portion of parsnip.