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.

[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.

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.

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.

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.

Psoralen phototoxicity: correlation with serum and epidermal 8-methoxypsoralen and 5-methoxypsoralen in the guinea pig.

Serum and epidermal concentrations of 8-methoxypsoralen and 5-methoxypsoralen 2 hours after oral administration to guinea pigs were determined by high-performance liquid chromatography. A linear relation was found between the serum and epidermal concentrations of 8-methoxypsoralen. In addition, a relation was found between serum concentrations of 5-methoxypsoralen and 8-methoxypsoralen and the appearance of phototoxicity. The lower phototoxicity of orally administered 5-methoxypsoralen as compared to 8-methoxypsoralen in the guinea pig appears to be due to its reduced concentrations in the epidermis, the primary site of the phototoxic events.

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.

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.

[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.

Variations in content of active ingredients causing drug interactions in grapefruit juice products sold in California.

The content of the active ingredients of grapefruit juice, naringin, naringenin and bergapten, was determined in 20 different commercial products of grapefruit juice sold in California. These included Minute Maid, Dole, Tropicana, Ocean Spray, Ralps, Albertson, Stater Bros, Vons, Langers, etc. The concentrations of naringin, naringenin and bergapten in grapefruit juice were assayed by specific HPLC methods. Naringin was found to be the most abundant flavonoid in grapefruit juice products, followed by naringenin and bergapten. The content of naringin varied among the products, ranging from 104 mg/l (Tropicana ruby red) to 628 mg/l (Ralphs white frozen concentrate). The mean contents of naringin in ruby red (158 +/- 66 [SD] mg/l) and pink (279 +/- 123 mg/l) grapefruit juice products were significantly lower than white (481 +/- 94 mg/l) (p <0.005) grapefruit juice products. Content of naringenin also varied from brand to brand and ranged from 3.9 mg/l (Vons white frozen concentrate) to 31.2 mg/l (Tree Sweet pink). Bergapten content was very low in grapefruit juice products ranging from 0 (not detectable) to 5.5 mg/l. There were no significant differences in naringenin and bergapten contents among the three types of grapefruit juice products. The information gained from this study would be useful in predicting the likelihood of grapefruit juice-drug interactions.

Flow cytometric analysis of 8-methoxypsoralen-DNA photoadducts in human keratinocytes.

Flow cytometric methods have been developed for the analysis of 8-methoxypsoralen-DNA adducts in SV40-transformed human keratinocytes (svk-14). Monoclonal antibodies which specifically recognize these adducts were used in conjunction with fluorescein isothiocyanate-labeled second antibodies and propidium iodide staining to simultaneously determine 8-methoxypsoralen-DNA adduct levels and DNA content, respectively. The sensitivity of the method was determined by quantitation of adduct levels in DNA isolated from treated cells by enzyme-linked immunosorbent assay. Adduct formation during various stages of the cell cycle was also investigated. Two separate cell populations with a DNA content indicative of cells in G1 but with different levels of fluorescein isothiocyanate labeling were observed. Pretreatment of cells with aphidicolin decreased the number of cells in S phase, and only one fluorescein isothiocyanate-staining G1 population was observed. There is an indication of greater adduct formation in S-phase cells when immunofluorescence is corrected for DNA content. These results suggest that adduct formation is not simply linear with respect to DNA content.

Immunological detection and visualization of 8-methoxypsoralen-DNA photoadducts.

Monoclonal antibodies specific for DNA damaged by 8-methoxypsoralen (8-MOP) plus ultraviolet A (UVA) light were used to study adduct formation in human keratinocytes and mouse and rat skin in vivo. This antibody does not cross-react with nonmodified DNA or free 8-MOP. Sensitive competitive enzyme-linked immunosorbent assays with color or fluorescence endpoints were used to quantitate adducts on DNA isolated from treated keratinocytes or skin samples. Localization of 8-MOP-DNA adducts was studied by indirect immunofluorescence with fluorescein-conjugated anti-mouse-IgG antibodies. When cultured keratinocytes were treated with 8-MOP and UVA, immunofluorescence was localized in the nucleus. There was no fluorescence in untreated control cells or treated cells incubated with nonspecific serum. Comparison of intensity of immunofluorescence staining with quantitation of adduct levels by enzyme-linked immunosorbent assay indicated that the limit of sensitivity of the immunofluorescence technique is 9.0 fmol adduct/micrograms DNA or 2.9 adducts/10(6) nucleotides.

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.

Quantitation of plasma levels of 8-methoxypsoralen by competitive enzyme-linked immunosorbent assay.

An immunologic method for the quantitation of 8-methoxypsoralen (8-MOP) levels in human plasma has been developed. A monoclonal antibody recognizing 8-MOP was prepared by immunizing mice with an 8-MOP derivative conjugated to bovine serum albumin with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide-HCl. The antibody was characterized by competitive enzyme-linked immunosorbent assay (ELISA) and recognizes 8-MOP (50% inhibition at 2 pmol) as well as structurally related psoralen derivatives including 4'-aminomethyl-4,5',8-trimethylpsoralen (50% inhibition at 50 pmol), 5-methoxypsoralen (50% inhibition at 150 pmole), and 6,4,4'-trimethylangelicin (50% inhibition at 360 pmol). The assay has a limit of sensitivity of 1 ng/ml plasma. For analysis of 8-MOP levels in plasma, samples were first extracted using SepPak C18 cartridges. The extracts were analyzed for 8-MOP levels both by ELISA and high-pressure liquid chromatography. There was a good correlation between the values determined by both methods (r = 0.92, p less than 0.0005). The development of immunologic methods should greatly facilitate the quantitation of 8-MOP levels in patient plasma.

The relationship between 8-methoxypsoralen skin and blood levels.

A method is described to determine the 8-methoxypsoralen (8-MOP) concentration in vivo in the skin by means of high-performance liquid chromatography (HPLC). Skin and blood samples were taken from 80 rats at specific intervals after oral administration of [3H]8-MOP. The pharmacokinetic results obtained for the skin levels were compared to the blood levels. In addition, liquid scintillation counting (LSC) was done on all the samples to compare the concentrations of 8-MOP plus metabolites to the concentrations of 8-MOP alone. There was a good correlation between the 8-MOP skin and blood levels. The values obtained with LSC were higher in function of time than the corresponding values obtained by HPLC, which indicates the presence of metabolites in both the skin and the blood. No statistically significant difference in the time of peaking was noted for the skin and blood levels. The blood levels seem to be a good parameter for the 8-MOP skin concentration.

Quantitative determination by bioassay of photoactive 8-methoxypsoralen in serum.

Photoactive 8-methoxypsoralen (8-MOP) levels in human and animal sera were determined by bioassay with Staphylcoccus aureus serving as the test organism. 8-MOP was extracted from sera with as 9:1 (V/V) ethyl acetate-n-hexane mixture and reconstituted in an aqueous medium following evaporation of the extractant. Bacterial suspensions containing extracted 8-MOP were irradiated for predetermined intervals with longwave ultraviolet light (UVA) at an intensity of 2.1 mw/cm2. Cultures containing known amounts of 8-MOP were used as standards and cytotoxicity (i.e., drug levels) determined by colony counts. The detection limit for 8-MOP was 5 ng/ml with an accuracy of +/- 10% above 10 ng/ml. Concomitant determinations of 8-MOP levels by high pressure liquid chromatography (HPLC) were in excellent agreement with the bioassay results.