Accumulation of furanocoumarins in Ruta graveolens L. shoot culture

Ruta graveolens L. shoots cultured in stationary liquid phase produced furanocoumarins: psoralen, bergapten, xanthotoxin, isopimpinellin and imperatorin at the amount totalling almost 1 g/100 g dry wt of the shoots. The dominating metabolites were therapeutically important compounds: xanthotoxin – 0.33 g/100 g dry wt and bergapten – 0.32 g/100 g dry wt. Maximum contents of the majority of the compounds were observed on 28th day of culture.     

Identification of Ruta graveolens L. metabolites accumulated in the presence of abiotic elicitors

The study aimed to elucidate the effects of benzothiadiazole (BTH) and saccharin on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, and furoquinolone alkaloids in shoots of R. graveolens cultivated in vitro. The biosynthesized metabolites were analyzed and identified by GC-MS and by comparison of Kovats indices. Eight coumarin metabolites were identified: bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin, and also four alkaloids: dictamnine, gamma-fagarine, skimmianine, and kokusaginine. Each of the tested BTH concentrations induced a significant production of furanocoumarins and furoquinolone alkaloids. The use of saccharin also increased the production of bergapten, isopimpinelin, pinnarin, psoralen, and xanthotoxin several times.     

Furanocoumarin metabolism in Papilio polyxenes: biochemistry,genetic variability,and ecological significance

The ubiquitous occurrence of series of biosynthetically related plant secondary compounds within individual species has given rise to the suggestion that such multiplicity is adaptive; one possible mechanism that would serve to maintain such within-plant diversity is analog synergism. In a series of experiments, we provide evidence that synergism may account for the presence of multiple structurally related furanocoumarins in apiaceous plants. The black swallowtail, Papilio polyxenes, feeds exclusively on plant species containing furanocoumarins. Growth of larvae fed parsley leaves treated with both xanthotoxin and angelicin, two furanocoumarins that co-occur widely in swallowtail hostplants, was significantly slower than that of larvae fed leaves with an equimolar concentration of either xanthotoxin or angelicin. A multivariate combination of growth, food consumption and frass excretion differed significantly between larvae fed leaves treated with both xanthotoxin and angelicin and larvae fed leaves treated with angelicin alone. In addition, we measured rates of in vitro cytochrome P450-mediated metabolism of three furanocoumarins — bergapten, xanthotoxin, and angelicin. While bergapten and xanthotoxin, both linear furanocoumarins, were metabolized at similar rates (8.07 and 9.86 nmoles/min/g fw caterpillar, respectively), angelicin, an angular furanocoumarin, was metabolized more slowly (2.76 nmoles/min/g fw caterpillar). When all three furanocoumarins were assayed together, overall rates of metabolism were significantly reduced, suggesting substrate inhibition. Thus, the pattern of growth of larvae is consistent with the pattern of in vitro metabolism and is evidence in support of analog synergism. In a separate experiment, metabolism of xanthotoxin and angelicin individually and together were compared in six maternal families. Again, angelicin was metabolized more slowly than xanthotoxin and each furanocoumarin inhibited metabolism of the other. That significant family effects were found for rates of metabolism and for the ratio of moles of angelicin metabolized for each mole of xanthotoxin metabolized raises the possibility that genetic variation exists for the rate and specificity of metabolism and suggests that insect herbivores may be able to adapt to analog synergism.     

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 1. Identification of induced products as coumarin derivatives

Dark-grown cell suspension cultures of parsley, Petroselinum hortense, produce furanocoumarins after treatment with elicitor preparations of either Phytophthora megasperma f.sp. glycinea (Pmg elicitor) or Alternaria carthami Chowdhury (Ac elicitor). The linear furanocoumarins, psoralen and xanthotoxin, and the benzodipyrandione, graveolone, are the major products synthesized in response to Pmg elicitor, besides small amounts of the furanocoumarin bergapten. Treatment with Ac elicitor induces predominantly the formation of bergapten and the furanocoumarin isopimpinellin, as well as small amounts of graveolone. While Pmg elicitor leads to cell death within a few days, cell mass increased for at least 6 days after treatment with Ac elicitor. Brefeldin A, a phytotoxin produced by A. carthami, inhibits growth of parsley cell suspension cultures considerably at a concentration of 0.01 mM and growth of the cells ceased at a concentration of 0.1 mM toxin. Concomitantly, furanocoumarin biosynthesis was suppressed in our system by a concentration of brefeldin A within 0.01-0.1 mM.     

Furanocoumarin biosynthesis in Ruta graveolens cell cultures

The biosynthetic routes to four linear furanocoumarins—psoralen, xanthotoxin, bergapten. isopimpinellin-co-occurring in Ruta graveolens cell cultures have been investigated with six ¹⁴C-labelled compounds. Mevalonic acid was only poorly incorporated, in contrast to umbelliferone. In support of previous suggestions, 7-demethylsuberosin and (±)-marmesin were very good precursors of the linear furanocoumarins. 7-O-Prenylumbelliferone also was fairly well utilized, but this was probably owing to a prior ether cleavage yielding umbelliferone. Psoralen was well incorporated into bergapten and xanthotoxin, but not into the dimethoxylated isopimpinellin. Differences exist between the organized plant and its cell culture in terms of metabolic products and, by implication, precursor utilization. S(+)-Marmesin was obtained in small quantity from an acid-hydrolysable conjugate present in the culture medium. Syntheses of [2-¹⁴C]7-demethylsuberosin, [2-¹⁴C]osthenol, [2-¹⁴C]7-O-prenylumbelliferone, [3-¹⁴C] (±)-marmesin, and [3-¹⁴C]psoralen are described, as well as an improved method for separation of furanocoumarin mixtures by TLC and GLC.     

Histochemical localization in <Emphasis Type="Italic">Ruta graveolens</Emphasis> cell cultures: elucidating the relationship between cellular differentiation and furanocoumarin production

Cell cultures of Ruta graveolens L. were used as a model system to study the relationship between cellular organization and furanocoumarin production. Relative contributions of individual cells were traced using a combination of biochemical and localization techniques in three types of cell cultures: dispersed, aggregated, and organized. The proportion of relative furanocoumarins produced varied with the organization level in cultures. Productive population in dispersed cell culture was 10% which increased to 17% and to 35% in aggregated and organized cell cultures, respectively. Large cell clusters accumulating furanocoumarins were restricted to organized cell cultures. In these lines, sites for psoralen, bergapten, and xanthotoxin accumulation were spatially separated from each other, which has been reported for the first time. Variation in production was due to change in relative size of productive population in the three types of cultures studied. A model has been proposed for differential furanocoumarin producing ability of cells based on differentiation levels.     

Heritability estimates for octyl acetate and octyl butyrate in the mature fruit of the wild parsnip

The aliphatic esters octyl acetate and octyl butyrate occur as major components of essential oils in the vittae, or oil tubes, of the wild parsnip (Pastinaca sativa). We determined phenotypic variation and narrow-sense heritabilities of these octyl esters in wild parsnip fruits from 30 maternal families. The mean octyl acetate content was 1.56 microg/mg dry fruit (0.08-5.51 microg/mg dry fruit) and the mean octyl butyrate content was 4.28 microg/mg dry fruit (1.28-14.22 microg/ mg dry fruit). Narrow-sense heritabilities for each ester's content were calculated by analysis of half-sib families (HS) and parent-offspring regression (OP). Heritabilities were 0.389 (HS) and 0.654 (OP) for octyl acetate and 0.670 (HS) and 0.626 (OP) for octyl butyrate. The amounts of the esters were phenotypically correlated with each other and with the linear furanocoumarins bergapten and xanthotoxin, phototoxic compounds that co-occur in the vittae with the esters. Ester amounts were not genetically correlated, indicating that these compounds could respond independently to selection pressures. These octyl esters may serve as carrier solvents that enhance penetration of these furanocoumarins into herbivore integuments and gut walls.     

Chemical barriers to adaptation by a specialist herbivore

Summary Depressaria pastinacella, the parsnip webworm (Lepidoptera: Oecophoridae), feeds throughout eastern North America on Pastinaca sativa (wild parsnip) and few other species. The assumption that specialist herbivores such as the parsnip webworm are adapted to hostplant chemistry, and are therefore unaffected by chemical variation in hostplants, was tested. Flower buds from plants grown first in the greenhouse and then in the field were fed to ultimate instar webworms. Plant phenotype had a significant effect on virtually all webworm food utilization parameters. While nutritional factors (i.e., nitrogen content) were correlated with approximate digestibility, two constituents of the flowers — bergapten and xanthotoxin, both linear furanocoumarins — independently accounted for a significant amount of variation in food utilization indicies. The physiological effects of these furanocoumarins were confirmed in artificial diet experiments. Despite the fact that the two most important furanocoumarins in parsnip flowers relative to webworm feeding and growth are isomers, differing only in the positioning of a methoxy substituent, they have different physiological effects; while xanthotoxin in general has no effect on growth, bergapten decreases growth and digestibility of the diet. These results underscore the need in studies of plant-animal interactions to examine individual chemical components rather than classes of compounds.     

Furanocoumarins: Novel topoisomerase I inhibitors from Ruta graveolens L.

Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA–topoisomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxation of plasmid in enzyme–substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme–extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA–enzyme complex was observed after enzyme–extract preincubation. The activity could be assigned to furanocoumarins—psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors.     

Coumarins from Three Phebalium species

The twigs of Phebalium anceps yielded the common furocoumarins psoralen, bergapten and xanthotoxin whereas the leaves of P. coxii and P. ralstonii gare only 8-prenylated 7-methoxycoumarins. From R coxii murrangatin and murralongin were obtained whilst R ralstonii yielded both of these compounds and another coumarin, the novel 7-methoxy-8-(2-acetoxy-3-methyl-1oxobut-2-enyl)-coumarin. The chemotaxonomic implications of these results are discussed.     

Isolation and Identification of Xanthotoxin (8-Methoxypsoralen) and Bergapten (5-Methoxypsoralen) from Celery Infected with Sclerotinia sclerotiorum

Methods for the production, isolation, and identification of xanthotoxin and bergapten from celery diseased by Sclerotinia sclerotiorum (Libert) de Bary were investigated. The only conditions under which this mold was capable of producing xanthotoxin and bergapten occurred when the mold was actively growing on fresh (metabolizing) celery. Neither compound was found in uninfected celery, in the mold growing on nutrient media, on nutrient media fortified with 30% filter-sterilized celery juice, or on nonmetabolizing celery. Maximal xanthotoxin production of 320 mug per g of dry rotted celery occurred at 20 C, although mycelial growth increased until 30 C. Neither xanthotoxin nor bergapten was found when the mold grew on 11 agricultural commodities other than celery.     

Simultaneous and sensitive determination of xanthotoxin,psoralen, isoimpinellin and bergapten in rat plasma by liquid chromatography–electrospray ionization mass spectrometry

A sensitive, specific and rapid liquid chromatography–mass spectrometry (LC–MS) method has been developed and validated for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5,8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in rat plasma using pimpinellin as an internal standard (IS). The plasma samples were pretreated by protein precipitation with methanol and chromatographic separation was performed on a C₁₈ column with a mobile phase composed of 1 mmol ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ion-pairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for IS. The total run time was 6 min between injections. The calibration curves were linear over the investigated concentration range with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.21 ng/ml. The intra- and inter-day RSD were no more than 9.7% and the relative errors were within the range of ?8.1% to 4.5%. The average extraction recoveries for all compounds were between 90.7% and 106.2%. The proposed method was further applied to the determination of actual plasma samples from rats after oral administration of Radix Glehniae extract.     

Prenylated flavonoids, monoterpenoid furanocoumarins and other constituents from the twigs of Dorstenia elliptica (Moraceae)

A monoprenylated flavan and two monoterpenoid substituted furanocoumarins were isolated from the twigs of Dorstenia elliptica along with 3-(3,3-dimethylallyl)-4,2',4'-trihydroxylchalcone, psoralen, bergapten, O-[3-(2,2-dimethyl-3-oxo-2H-furan-5-yl)butyl]bergaptol, beta-sitosterol and its beta-D-glucopyranoside. The structure of the flavan was determined as 6(1,1-dimethylallyl)-7,4'-dihydroxylflavan and the monoterpenoid substituted furanocoumarins were assigned as O-[3-(2,2-dimethyl-3-oxo-2H-furan-5-yl)-3-hydroxybutyl]-bergaptol and O-[2-(5-hydroxy-2,6,6-trimethyl-3-oxo-2H-pyran-2-yl)ethyl]bergaptol, respectively, using spectroscopic analysis, especially, 2D NMR spectra.     

Linear furocoumarins and other constituents from thamnosma texana

Eight linear furocoumarins and three coumarins were isolated and identified from Thamnosma texana. They were xanthotoxin, imperatorin, bergapten, alloimperatorin methyl ether epoxide, heraclenin, isopimpinellin, psoralen, oxypeucedanin, and the coumarins herniarin, osthol and thamnosmin. The linear furocoumarins appear to be agents that account for the known photosensitizing properties of Thamnosma texana, and consequently its colloquial name, ‘blisterweed.’ This is the first report on the occurrence of imperatorin, heraclenin, oxypeucedanin, herniarin or osthol in any Thamnosma species.     

Genetic transformation of <Emphasis Type="Italic">Ruta graveolens</Emphasis> L. by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: hairy root cultures a promising approach for production of coumarins and furanocoumarins

Ruta graveolens L. is a source of pharmacologically active compounds such as coumarins, furanocoumarins and furoquinolone alkaloids. Hypocotyls, callus and shoots of R. graveolens were inoculated with bacteria from two Agrobacterium rhizogenes strains. Hairy root cultures were established after inoculation of hypocotyls with wild A. rhizogenes strain LBA 9402. The transgenic nature of the regenerated tissue was confirmed by PCR amplification. Coumarins, furanocoumarins and alkaloids present in the hairy root tissue were identified by GC and GC-MS and compared with those present in in vitro shoot cultures. The level of pinnarin and rutacultin, bergapten, isopimpinelin and xanthotoxin was approximately twofold higher in hairy root than in shoot cultures. Two additional coumarins: osthole and osthenol, never been found in R. graveolens, were identified in hairy root tissue. Besides coumarins, alkaloids were identified: dictamnine, skimmianine, kokusaginine, rybalinine and an isomer of rybalinine. The levels of nearly all coumarins and alkaloids in hairy roots cultured in the darkness were higher than those accumulated under a photoperiod mode.     

广东省春季三叶草斑潜蝇寄主种类

对广东省冬后蔬菜调查结果表明,三叶草斑潜蝇Liriomyzatri folii(Burgess)的寄主主要有菊科茼蒿、非洲菊,伞形科西芹、香芹,豆科豆角,葫芦科冬瓜,茄科番茄等;其中,茼蒿、芹菜、番茄等受害较重。     

Furanocoumarins from Dorsteniafoetida

The linear furanocoumarins 5-(2,3-epoxy-3-methyl-butoxy)-chalepensin, 5-methoxy-3-(3-methyl-2,3-dihydroxybutyl)-psoralen-diacetate (7), 5-methoxy-3-[3-(β-d-glucopyranosyloxy)-2-acetyloxy-3-methyl-butyl]-psoralen and 5-(3-methyl-2,3-dihydroxybutyloxy)-3-[3-(β-d-glucopyranosyloxy)-2-hydroxy-3-methyl-butyl]-psoralen, and the coumarin derivative 7-hydroxy-5-methoxy-6-carboxymethyl-3-[3-(β-d-glucopyranosyloxy)-2-hydroxy-3-methyl-butyl]-coumarin were isolated from the leaves of Dorstenia foetida (Moraceae) along with the known compounds psoralen, bergapten, isopimpinellin, phellopterin, 5-methoxychalepensin and turbinatocoumarin. Further furanocoumarins were characterized by ESI-MS/MS investigations. The nonpolar extracts of D. foetida exhibit antifungal, antibacterial and cytotoxic activity, however, no anthelminthic activity.     

Epidemiological studies on Fasciola hepatica in Gafsa Oases (south west of Tunisia)

Epidemiological investigations on Fasciola hepatica fasciolasis were carried out from July 2004 to June 2005 in the Gafsa oases (Tunisia) after the detection of a human case. Three habitats were studied: one in El Gsar and two in Ain Soltan. The prevalence of human infection was 6.6%. The presence of the parasite was detected through serology in 14.3% of cattle, 35% of sheep and 68.4% of goats. The plants Apium nodiflorum, Oxalis cernua and Sonchus maritimus were suspected to be at the origin of animal contamination and Apium nodiflorum was incriminated in human infection. The prevalence of the infection of the intermediate host Galba truncatula (G. truncatula) was 19.2% from July 2004 to June 2005. Gafsa oases constitute a new location for the development of fasciolasis in the southern west of Tunisia.     

Root cultures of linum species section Syllinum as rich sources of 6-methoxypodophyllotoxin

Linum spp. from section Syllinum are promising for the production of aryltetralin lignans like podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (MPTOX). MPTOX is a PTOX congener that has cytotoxic activity comparable with PTOX. In this study root cultures of Linum Bungei from section Dasyllinum, L. strictum from section Linastrum, L. album, L. mucronatum ssp. mucronatum and L. nodiflorum from section Syllinum were established and their MPTOX levels were investigated in 1000 ml flasks. Root cultures of L. mucronatum ssp. mucronatum and L. nodiflorum were used to examine cell growth and production of MPTOX during a culture period of 36 days in 250 ml flasks. Considerable amounts of MPTOX in root cultures (1000 ml flasks) of L. album (6 mg/100 g DW), L. mucronatum ssp. mucronatum (770 mg/100 g DW) and L. nodiflorum (91 mg/100 g DW) were detected while it wasn't detected in root cultures of L. Bungei and L. strictum. In time course experiments, the maximum amount of MPTOX in L. nodiflorum root culture was at day 16 with 480 mg/ 100 g DW and the maximum amount of MPTOX in L. mucronatum ssp. mucronatum root culture was at day 12 with 130 mg/100 g DW. The results showed that root cultures of Linum species from section Syllinum are rich sources of MPTOX and since this lignan has remarkable cytotoxic activity, it can be used as a precursor for the production of antitumor agents.