Linear Furanocoumarins Research Articles

Host plant, host plant chemistry and the polyembryonic parasitoidCopidosoma sosares: indirect effects in a tritrophic interaction

Host plant identity and host plant chemistry have often been shown to influence host finding and acceptance by natural enemies but comparatively less attention has been paid to the tritrophic effects of host plant and host plant chemistry on other natural enemy fitness correlates, such as survivorship, clutch size, body size, and sex ratio. Such studies are central to understanding both the selective impact of plants on natural enemies as well as the potential for reciprocal selective impact of natural enemies on plant traits. We examined the effects of host plant and host plant chemistry in a tritrophic system consisting of three apiaceous plants (Pastinaca sativa,Heracleum sphondyliumandH. mantegazzianum), the parsnip webworm (Depressaria pastinacella) and the polyembryonic parasitic waspCopidosoma sosares. All of these plants produce furanocoumarins, known resistance factors for parsnip webworms. Furanocoumarin concentrations were correlated neither with the presence nor the number of webworms on a given plant. Concentrations of two furanocoumarins were negatively associated withC. sosaresfitness correlates: isopimpinellin with the likelihood that a given webworm would be parasitized and xanthotoxin with both within‐brood survivorship (of all‐male and mixed‐sex broods) and clutch size. Brood sex ratio and body sizes of individual wasps were not correlated with furanocoumarin chemistry. Because additive genetic variation exists inP. sativafor furanocoumarin chemical traits, these are subject to selection by webworms through herbivory. Third trophic level selective impacts on furanocoumarin traits may include selection for reduced production of those chemicals that affect parasitoid survivorship yet do not influence host plant choice by the herbivore. That such might be the case is suggested by patterns of furanocoumarin production in populations ofP. sativawith different histories of infestation; in the Netherlands, where parasitism rates of webworms byC. sosaresare high, plants produce lower levels of all linear furanocoumarins and proportionately less isopimpinellin than do midwestern U.S. populations ofP. sativa, where natural enemies of the webworm are effectively absent.

Identification of variable amino acids in the SRS1 region of CYP6B1 modulating furanocoumarin metabolism

The homology model of Papilio polyxenes CYP6B1 places Ile115, one of two variable amino acids, in the SRS1 of various CYP6B subfamily proteins in close proximity to the heme and Ala113, another variable amino acid, in a more distal position. We have constructed mutant CYP6B1 proteins altered at either of these positions and homology models of each based on multiple alignments with crystallized P450 proteins. The homology models suggest the existence of significant structural diversity in the hydrogen bond network surrounding the heme as a result of single point mutations in SRS1. Mutagenesis of Ile115 or Ala113 to other residues present in the insect CYP6B subfamily indicates that these amino acids control the spin state of the heme and, as a result, the catalytic activity of this monooxygenase. In particular, the I115L mutation significantly increases the spin state of the heme coordinately with 2- to 4-fold increases in its turnover of linear furanocoumarins. Other A113V, A113L, A113Q, and A113E mutations display more variation in their effects but, in each case, strong correlations exist between furanocoumarin turnover and heme spin state. These data demonstrate that variable amino acids in SRS1 of the insect CYP6B subfamily exert dramatic effects on the range of furanocoumarins metabolized, even when they occur in positions potentially distal from the substrate. These effects are possibly mediated through rearrangement of the local hydrogen bond network.

Insect Antifeedant Furanocoumarins from Tetradium daniellii.

The dried fruits of Tetradium daniellii yielded a new linear furanocoumarin, 5-(6-hydroxy-3,7-dimethylocta-2,7-dienyloxy)psoralen, together with six other structurally related furanocoumarins. A similar chemical profile was recorded by HPLC analysis of a fragment of T. daniellii fruit obtained from an historic herbarium voucher specimen collected in September 1917 during an expedition to Yunnan province, China. Four of the compounds identified caused a potent feeding deterrent effect towards larvae of Spodoptera littoralis and Heliothis virescens.

Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase

One challenge in the heterologous expression of microsomal cytochrome P450 monooxygenases (P450s) is fulfilling their obligatory requirement for electrons transferred from NADPH P450 reductase. We have established co-expression parameters for Papilio polyxenes CYP6B1 and house fly P450 reductase in baculovirus-infected Sf9 cells that allow for efficient expression of both components and significantly enhance metabolic turnover of this insect P450’s substrates. These expression conditions have allowed us to reexamine the turnover capacities of CYP6B1 toward linear and angular furanocoumarins present in the host plants for the specialist caterpillar P. polyxenes. Coexpression of CYP6B1 and P450 reductase at equivalent viral concentrations [MOI (multiplicity of infection) ratio of 1] results in turnover rates for the linear furanocoumarins xanthotoxin and psoralen, which are increased 32–33 fold over the turnover rates obtained with CYP6B1 expressed alone. The turnover rate for the angular furanocoumarin angelicin is also significantly increased to 4.76 nmol/min/nmol P450 compared to its barely detectable level obtained with CYP6B1 expressed alone. Substrate binding analyses indicate that all three of these compounds elicit typical type I binding spectra but with varying magnitudes and affinities that are indicative of each substrate’s effectiveness at coordinating with the heme iron. The relative proportions of high spin state generated with these substrates are consistent with CYP6B1 metabolizing these furanocoumarins in the rank order xanthotoxin>psoralen>angelicin. These differential activities for CYP6B1 suggest that it may have been an ancient participant in the coevolutionary arms race between papilionid butterflies and their apiaceous host plants. Due to its ability to handle a range of furanocoumarin structures, CYP6B1 may have contributed to P. polyxenes’ early colonization of linear furanocoumarin-containing plants and to its subsequent colonization of angular furanocoumarin-containing plants.

Functional expression of cinnamate 4-hydroxylase from Ammi majus L.

Total RNA was isolated from dark-grown cell suspension cultures of Ammi majus L. that had been induced with fungal elicitor or treated with water for control and used as template with cytochrome P450-specific primers for DD-RT-PCR amplifications. A cDNA clone was generated from the elicited transcripts and assigned to cinnamate 4-monooxygenase based on sequence alignments and functional expression in yeast cells. Comparison of the translated polypeptide with database accessions of heterologous cytochrome P450 monooxygenases revealed a high degree of similarity (99.6%) with 98.6% identity to cinnamic acid 4-hydroxylase from parsley, documenting the close evolutionary relationship within the Apiaceae family. Maximal activity of the Ammi hydroxylase in yeast microsomes was determined at 25 °C and in the pH range of 6.5–7.0 reaching 2.5 pkat/mg on average. An apparent K m of 8.9 μM was determined for cinnamate. Preincubations with psoralen or 8-methoxypsoralen added up to 100 μM in the presence or absence of NADPH hardly affected the turnover rate. A. majus cell cultures accumulate sets of O-prenylated umbelliferones and linear furanocoumarins besides lignin-like compounds upon treatment with elicitor, and cinnamic acid 4-hydroxylase catalyzes the initial reaction leading from the general into the various phenylpropanoid branch pathways. Correspondingly, the hydroxylase transcript abundance was induced in the elicited cells.

Antibacterial coumarins from Angelica gigas roots.

Systematic fractionation of Angelica gigas roots led to the isolation of linear furano(pyrano)coumarins such as bergapten (1), decursinol angelate (2), decursin (3), nodakenetin (4) and nodakenin (5). The antibacterial activities of those compounds against pathogenic bacteria were investigated. Among the compounds tested, decursinol angelate (2) and decursin (3) exhibited significant antibacterial activity against Bacillus subtilis with the minimum inhibitory concentrations (MICs) of 50 and 12.5 microg/mL, respectively.

Insect antifeedant furanocoumarins from Tetradium daniellii

The dried fruits of Tetradium daniellii yielded a new linear furanocoumarin, 5-(6-hydroxy-3,7-dimethylocta-2,7-dienyloxy)psoralen, together with six other structurally related furanocoumarins. A similar chemical profile was recorded by HPLC analysis of a fragment of T. daniellii fruit obtained from an historic herbarium voucher specimen collected in September 1917 during an expedition to Yunnan province, China. Four of the compounds identified caused a potent feeding deterrent effect towards larvae of Spodoptera littoralis and Heliothis virescens.

Characterization of furanocoumarin metabolites in parsnip webworm, Depressaria pastinacella.

Although metabolites of furanocoumarins have been characterized in a wide range of organisms, to date they have been identified in only a single insect species, Papilio polyxenes. Depressaria pastinacella, the parsnip webworm, like P. polyxenes a specialist on Apiaceae, routinely consumes plant tissues higher in furanocoumarin content than does P. polyxenes and is capable of faster cytochrome P-450-mediated detoxification of these compounds. In this study, we characterized metabolites of xanthotoxin, a linear furanocoumarin, and sphondin, an angular furanocoumarin, in midguts and frass of parsnip webworms. Two metabolites were isolated and identified from webworms fed artificial diet containing xanthotoxin. LC-ESI-MS analysis resulted in the determination of a MW of 266 for the compound in the frass and one of the compounds in the midgut; 1H NMR confirmed its structure as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA). The second compound from the midgut had a MW of 252 and was identified by 1H NMR and 13C NMR analysis as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol) (HMCH). Whereas HCHA has been found in frass of Papilio polyxenes fed xanthotoxin, HMCH has not been reported previously in insects. Although the first step of metabolism of xanthotoxin in webworms as well as P. polyxenes is likely the formation of an epoxide on the furan ring, angular furanocoumarin metabolism in webworms appears to differ. The principal metabolite of sphondin was identified as demethylated sphondin (6-hydroxy-2H-furo[2,3-h]-1-benzopyran-2-one) by LC-ESI-MS and confirmed by 1H NMR and 13C NMR analyses. That webworms produce metabolites of xanthotoxin in common not only with other Lepidoptera (e.g., HCHA) but with other vertebrates (e.g., HMCH) suggests a remarkable conservatism in the metabolic capabilities of cytochrome P-450s and raises the possibility that insects may share other detoxification reactions with vertebrates with respect to toxins in foodplants.

One‐Step Dimerization of Linear Furanocoumarins.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Amino acids in SRS1 and SRS6 are critical for furanocoumarin metabolism by CYP6B1v1, a cytochrome P450 monooxygenase.

CYP6B1v1 is the principal cytochrome P450 monooxygenase (P450) that detoxifies dietary furanocoumarins in the guts of Papilio polyxenes, the black swallowtail caterpillar. Sequence alignments and structure comparisons of CYP6B1v1 with the mouse CYP2A5 and bacterial CYP102 proteins, which are also capable of metabolizing the linear furanocoumarin xanthotoxin (8-methoxypsoralen), suggested that Phe116, His117, Val368 and Phe484 might be active site residues. In a homology model developed for CYP6B1v1, the side chains of Phe116 and His117 located in the B'-C loop of SRS1 are predicted to be positioned above the haem plane, while the side chain of Phe484 located in SRS6 is predicted near the entrance of the catalytic pocket. Site-directed mutagenesis of residues Phe116, His117 and Phe484 indicated that these residues represent several of those that determine this protein's stability and substrate specificity. Whereas all aromatic mutants of Phe116 and Phe484 generated CO-difference spectra with maxima at 450 nm indicative of correctly configured monooxygenases, aromatic mutants of Phe116 exhibited reduced reactivities toward some furanocoumarins and aromatic mutants of Phe484 eliminated all reactivities toward furanocoumarins. All single and double aliphatic mutants of Phe116, His117 and Phe484 and aromatic mutants of His117 generated carbon monoxide (CO) difference spectra with maxima at 420 nm (P420) indicative of incorrectly configured monooxygenases. These studies define residues Phe116, His117 and Phe484 as determinants of this insect P450's catalytic site integrity and residues Phe116 and Phe484 as determinants of its substrate specificity. Conservation of Phe116 and His117 in an array of lepidopteran CYP6B proteins implies that these amino acids serve a similar function in other monooxygenases of the insect CYP6B subfamily.

Variation in xanthotoxin content in Ammi majus L. cultures during in vitro flowering and fruiting

Ammi majus L. possess various linear furanocoumarins, in particular, xanthotoxin which is used in the treatment of leucoderma. We report a reproducible protocol for micropropagation through mature nodal segments and the variation in xanthotoxin content at various stages of in vitro flowering and fruiting. Direct organogenesis was achieved in 100% cultures on Murashige and Skoog's (MS)+indole-3-acetic acid (IAA) (0.5 mg l −1)+kinetin (Kn) (2.0 mg l −1)+casein hydrolysate (CH) (1000 mg l −1) and flowering and fruiting on MS+IAA (0.5 mg l −1)+Kn (5.0 mg l −1)+adenine (Ad) (40 mg l −1)+CH (500 mg l −1). Shoots were rooted on MS+indolebutyric acid (IBA) (0.2 mg l −1)+glutamine (Glu) (100 mg l −1). Rooted plantlets transferred to soil could be successfully established in pots. Xanthotoxin content of in vitro cultures bearing immature green fruit was 11.9 mg g −1 dry weight in contrast to 4.2 mg g −1 dry weight in natural populations. In vitro-raised plantlets transplanted to field showed 4.8 mg g −1 dry weight of xanthotoxin.

ONE STEP DIMERIZATION OF LINEAR FURANOCOUMARINS

ABSTRACT The one step annular dimerisation of naturally occuring linear furanocoumarins has been effected in 70–85% yield by PPA-SiO2.

Molecular analysis of multiple CYP6B genes from polyphagous Papilio species

Papilio glaucus (eastern tiger swallowtail) and Papilio. canadensis (Canadian tiger swallowtail) are two closely related species with broad but overlapping hostplant ranges. P. glaucus encounters toxic furanocoumarins occasionally in its diet in its rutaceous hostplants, whereas P. canadensis rarely if ever encounters these compounds. Analysis of their furanocoumarin-metabolic profiles indicates that these species induce cytochrome P450 monooxygenases (P450s) capable of metabolizing linear and angular furanocoumarins to varying degrees in response to dietary supplementation with xanthotoxin (a linear furanocoumarin). In P. glaucus, metabolism is induced to a significantly higher level than in P. canadensis. Cloning of multiple P450 genes from each species has revealed that both species contain and express two groups of P450s, designated CYP6B4 and CYP6B17, that are related to the P. glaucus CYP6B4v1 enzyme known to metabolize an array of furanocoumarins. Expression patterns of the CYP6B4 and CYP6B17 group transcripts differ in these species in both their basal and furanocoumarin-inducible levels. In P. glaucus, CYP6B4 transcripts, which are not detectable constitutively, are 311-fold induced by xanthotoxin and CYP6B17 transcripts, which are detectable constitutively, are 3-fold induced by xanthotoxin. In P. canadensis, CYP6B4 transcripts are only 8-fold induced and CYP6B17 transcripts are 13-fold induced. These findings are consistent with the postulated evolutionary history of these two species, according to which P. glaucus maintains its association with rutaceous hostplants and P. canadensis has differentiated to utilize hostplants in other families more extensively

Ruta graveolens L.: a promising species for the production of furanocoumarins

Psoralen and its derivatives (linear furanocoumarins) are used in dermatology, and are also very promising in the treatment of other diseases. These secondary metabolites occur naturally in plants. Ruta chalepensis, R. angustifolia, R. graveolens and R. montana (Rutaceae) have been studied to evaluate their potential for the production of furanocoumarins (psoralen, xanthotoxin, bergapten, isopimpinellin). The Ruta species contained from 4 to 17 mg g −1 dry weight (DW) of furanocoumarins (FCs). These concentrations were higher than those found in other families known to produce the same compounds (Moraceae, Apiacae and Fabaceae). Among the four species tested, R. graveolens exhibited high concentrations of FCs and the best biomass production. Sixteen different R. graveolens origins were cultivated in order to find the best candidate for FC production. Plants contained an average concentration of 10 mg g −1 DW of FCs, ranging from 2.8 to 17.5 mg g −1 DW, but there was no statistical difference due to origins. FCs allocation was studied within R. graveolens in different parts of the plant. Fruits had a greater FCs concentration, five–tenfold higher than roots, stems or leaves. Isopimpinellin, a minor FC in other organs, was highly concentrated in roots, where it reached 25% of the total FCs. Potential yield of FCs is the result of both biomass and secondary metabolite production. These parameters were affected by the phenological stage of plants, with greatest production from fruits, which had the highest concentrations, biomass and therefore yield of FCs. R. graveolens plants cultivated in field could possibly produce 12 kg of FCs per hectare, mainly in fruits and leaves.

Expression of CYP6B1 and CYP6B3 cytochrome P450 monooxygenases and furanocoumarin metabolism in different tissues of Papilio polyxenes (Lepidoptera: Papilionidae)

The CYP6B1 and CYP6B3 cytochrome P450 monooxygenases in the midgut of the black swallowtail participate in the metabolism of toxic furanocoumarins present in its host plants. In this study, biochemical analyses indicate that the fat body metabolizes significant amounts of the linear furanocoumarins bergapten and xanthotoxin after larvae feed on xanthotoxin. Northern analyses of the combined CYP6B1/3 transcript expression patterns indicate that transcripts in this P450 subfamily are induced in the midgut and fat body by xanthotoxin. Semi-quantitative RT–PCR analyses of individual CYP6B1/CYP6B3 mRNAs indicate that CYP6B1 transcripts are induced by xanthotoxin in all tissues examined and that CYP6B3 transcripts are induced in the fat body only. These results indicate that the fat body participates in the P450-mediated metabolism of excess furanocoumarins unmetabolized by the midgut. Although transcripts of both genes were detected and CYP6B1 transcripts were induced by xanthotoxin in the integument, furanocoumarin metabolism was not detected. Comparison of these P450 promoters with the promoters of alcohol dehydrogenase genes expressed in the fat bodies of several Drosophila species suggest that the xanthotoxin inducibilities of these P450 genes in fat bodies are regulated by elements other than those modulating expression of Adh genes.

Oral administration of naturally occurring coumarins leads to altered phase I and II enzyme activities and reduced DNA adduct formation by polycyclic aromatic hydrocarbons in various tissues of SENCAR mice.

Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.

Furanocoumarins from the root of Angelica dahurica.

Five furanocoumarins including a new one were isolated from the root of Angelica dahurica by repeated silica gel column chromatography. Their chemical structures were determined to be isoimperatorin (1), oxypeucedanin hydrate-3"-butyl ether (2), imperatorin (3), knidilin (4), and oxypeucedanin hydrate (5). This represents the first study in which the compound 2 has been isolated and identified. The long-range coupling (5J) in the 1H-NMR spectrum observed in the linear furanocoumarin skeleton was also investigated in detail.

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.

A Bioassay Using Artemia salina for Detecting Phototoxicity of Plant Coumarins

Artemia salina (brine shrimp) has been successfully used for toxicity testing, and a screening test for phototoxicity has been developed based on this method. The ability of the method to test the phototoxic potential of seven known compounds was investigated. Athamantin (an angular furanocoumarin) and umbelliferone (a simple coumarin) showed no phototoxicity, while linear furanocoumarins exhibited phototoxic activity in the following order: psoralen > bergapten > peucedanin > xanthotoxin. The applicability of this method was also tested in screening the phototoxicity of plant material. Six plants from Apiaceae [Aegopodium podagraria L., Anethum graveolens L., Angelica archangelica L., Levisticum officinalis Koch, Petroselinum crispum (P. Mill) A. W. Hill., and Peucedanum palustre (L.) Moench] and one from Rutaceae (Ruta graveolens L.) were selected, all of them known to contain furanocoumarins. Extracts from leaves collected at different times during the growth period were used in the screening. Our results were in accordance with the furanocoumarin content of these plants and with the results of other phototoxicity tests. The Artemia salina method proved to be rapid, simple and inexpensive, and is therefore ideal in the initial biological screening of large numbers of samples for simultaneous detection of both toxicity and phototoxicity.

Simultaneous determination of phenolic acids and linear furanocoumarins in fruits of libanotis dolichostyla by solid-phase extraction and high performance liquid chromatography

Simultaneous determination of phenolic acids and linear furanocoumarins in fruits of libanotis dolichostyla by solid-phase extraction and high performance liquid chromatography