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.     

Cannibalism Affects Core Metabolic Processes in Helicoverpa armigera Larvae—A 2D NMR Metabolomics Study

Cannibalism is known in many insect species, yet its impact on insect metabolism has not been investigated in detail. This study assessed the effects of cannibalism on the metabolism of fourth-instar larvae of the non-predatory insect Helicoverpa armigera (Lepidotera: Noctuidea). Two groups of larvae were analyzed: one group fed with fourth-instar larvae of H. armigera (cannibal), the other group fed with an artificial plant diet. Water-soluble small organic compounds present in the larvae were analyzed using two-dimensional nuclear magnetic resonance (NMR) and principal component analysis (PCA). Cannibalism negatively affected larval growth. PCA of NMR spectra showed that the metabolic profiles of cannibal and herbivore larvae were statistically different with monomeric sugars, fatty acid- and amino acid-related metabolites as the most variable compounds. Quantitation of ¹H-¹³C HSQC (Heteronuclear Single Quantum Coherence) signals revealed that the concentrations of glucose, glucono-1,5-lactone, glycerol phosphate, glutamine, glycine, leucine, isoleucine, lysine, ornithine, proline, threonine and valine were higher in the herbivore larvae.     

Laboratory Evaluation of <Emphasis Type="Italic">Artemisia annua</Emphasis> L. Extract and Artemisinin Activity against <Emphasis Type="Italic">Epilachna paenulata</Emphasis> and <Emphasis Type="Italic">Spodoptera eridania</Emphasis>

Ethanolic extract of aerial parts of Artemisia annua L. and artemisinin were evaluated as anti-insect products. In a feeding deterrence assay on Epilachna paenulata Germ (Coleoptera: Coccinellidae) larvae, complete feeding rejection was observed at an extract concentration of 1.5 mg/cm² on pumpkin leaf tissue. The same concentration produced a feeding inhibition of 87% in Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae). In a no-choice assay, both species ate less and gained less weight when fed on leaves treated with the extract. Complete mortality in E. paenulata and 50% mortality in S. eridania were observed with extract at 1.5 mg/cm². Artemisinin exhibited a moderate antifeedant effect on E. paenulata and S. eridania at 0.03–0.375 mg/cm². However, a strong effect on survival and body weight was observed when E. paenulata larvae were forced to feed on leaves treated at 0.03 and 0.075 mg/cm². Artemisia annua ethanolic extract of aerial parts at 1.5 mg/cm² showed no phytotoxic effect on pumpkin seedlings.     

Cucurbitacins of Cucumis prophetarum and Cucumis prophetarum

Cucurbitacin B, isocucurbitacin B, dihydrocucurbitacin B, cucurbitacin E, dihydrocucurbitacin E, isocucurbitacin D, dihydroisocucurbitacin D, cucurbitacin I, dihydrocucurbitacin I, cucurbitacin Q1, and dihydrocucurbitacin Q1 were identified for the first time as constituents of Cucumis prophetarum L.. Cucurbitacin B, cucurbitacin O, cucurbitacin P, cucurbitacin Q1, dihydrocucurbitacin Q1, isocucurbitacin E, and dihydroisocucurbitacin E also were identified as constituents of C. prophetarum Jusl. ssp. Dissectus. Isocucurbitacin E and dihydroisocucurbitacin E were isolated for the first time in nature. The chemical structures were determined with extensive spectroscopic analysis including 2D NMR, ¹H NMR, ¹³C NMR, correlated spectroscopy (COSY), heteronuclear chemical shift correlation (HETCOR), attached proton test (APT), and distortionless enhancement by polarization transfer (DEPT).     

An Oviposition Stimulant for Spicebush Swallowtail Butterfly, Papilio troilus, from Leaves of Sassafras albidum

Female butterflies of the spicebush swallowtail, Papilio troilus, are specialists, ovipositing on plants in the family Lauraceae. Column chromatography and HPLC were used to isolate an oviposition stimulant from the leaves of one of its hosts, Sassafras albidum. The stimulant was identified as 3-trans-caffeoyl-muco-quinic acid on the basis of FAB-MS and ¹H NMR spectra as compared to a compound previously isolated from another plant. It was not active alone, but it increased the oviposition activity of butterflies when combined with other stimulant(s) at a concentration of 7 ng/mm² leaf surface area. Other caffeoylquinic acid isomers tested did not have this effect. This is the first report of a swallowtail contact oviposition stimulant from a plant in the family Lauraceae.     

High-resolution F and H NMR of a vinylidenefluoride telomer

The reaction products of vinylidenefluoride (VDF) with methanol as a telogen have been analysed in the solution state by ¹H and ¹⁹F nuclear magnetic resonance (NMR) spectroscopy. High-resolution ¹⁹F and ¹H NMR spectra were achieved using high-power ¹H and ¹⁹F decoupling, respectively, giving superior resolution and revealing previously unresolved signals of the vinylidenefluoride telomer (VDFT). ¹H and ¹⁹F homo- and hetero-nuclear scalar coupling constants are presented and the spectra of functional groups and reverse units (including the identification of short-chain structures) are discussed. Furthermore, the application of ¹⁹F or ¹H decoupling for the correct assessment of reverse-unit content and degree of polymerisation is demonstrated. This work highlights the need for high-resolution NMR spectroscopy to determine both the chemical structure and the composition of these important fluoropolymers.     

NMR Metabolomics of Thrips (<Emphasis Type="Italic">Frankliniella occidentalis</Emphasis>) Resistance in <Emphasis Type="Italic">Senecio</Emphasis> Hybrids

Western flower thrips (Frankliniella occidentalis) has become a key insect pest of agricultural and horticultural crops worldwide. Little is known about host plant resistance to thrips. In this study, we investigated thrips resistance in F ₂ hybrids of Senecio jacobaea and Senecio aquaticus. We identified thrips-resistant hybrids applying three different bioassays. Subsequently, we compared the metabolomic profiles of these hybrids applying nuclear magnetic resonance spectroscopy (NMR). The new developments of NMR facilitate a wide range coverage of the metabolome. This makes NMR especially suitable if there is no a priori knowledge of the compounds related to herbivore resistance and allows a holistic approach analyzing different chemical compounds simultaneously. We show that the metabolomes of thrips-resistant and -susceptible hybrids differed considerably. Thrips-resistant hybrids contained higher amounts of the pyrrolizidine alkaloids (PA), jacobine, and jaconine, especially in younger leaves. Also, a flavanoid, kaempferol glucoside, accumulated in the resistant plants. Both PAs and kaempferol are known for their inhibitory effect on herbivores. In resistant and susceptible F ₂ hybrids, young leaves showed less thrips damage than old leaves. Consistent with the optimal plant defense theory, young leaves contained increased levels of primary metabolites such as sucrose, raffinose, and stachyose, but also accumulated jacaranone as a secondary plant defense compound. Our results prove NMR as a promising tool to identify different metabolites involved in herbivore resistance. It constitutes a significant advance in the study of plant–insect relationships, providing key information on the implementation of herbivore resistance breeding strategies in plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of Trimethylolpropane‐Based Biolubricant

The oxidative stability index (OSI) of fatty acid methyl esters (FAME) and trimethylolpropane (TMP) esters or TMPE produced from five vegetable oils (Brassica rapa L., Linum usitatissimum L., Zea mays L., Brassica napus L., Camelina sativa L.) are compared. The highest stability is observed in vegetable oils while the processed products are less stable. The major causes in loss of OSI are attributed to excess FAME in the crude product and the loss of natural antioxidants due to refinement with silica and celite. The low‐temperature flow properties of TMPE produced from four different vegetable oils (B. juncea L., L. usitatissimum L., B. rapa L., and C. sativa L.) are investigated by proton nuclear magnetic resonance (¹H‐NMR). The T2 relaxations of different TMPE are measured to observe how the mobility of oil changed as temperature decreased. Increased oil mobility (represented by T2) is correlated with rising temperature. The Gaussian widths of the singlet in ¹H‐NMR spectra of each oil demonstrated increased molecular mobility as temperature increased. Extrapolation of the relation of T2 signals of these four oils indicates that T2 approached zero between 232 K and 239 K, suggesting the molecular motion leading to a T2 relaxation has largely ceased. Practical Applications: The OSI is determined for four vegetable oils as well as the product FAME and TMPE. The vegetable oils are more stable than their products. The loss of natural antioxidants during purification of FAME and TMPE contributes to the lower OSI compared to vegetable oil. The low‐temperature flow behavior of TMP‐based biolubricants is determined between 238 K and 298 K using T2 relaxation. As temperature decreases, a singlet resonance in ¹H‐NMR spectra attributed to TMP protons broadens until it disappears. The results suggest that the log of the spin‐spin relaxation time is linearly correlated with rising temperature and oil mobility.     

Metabolites from an Endophytic Fungus <Emphasis Type="Italic">Sphaceloma</Emphasis> sp. LN-15 Isolated from the Leaves of <Emphasis Type="Italic">Melia azedarach</Emphasis>

Two new natural compounds, a symmetrical disulfide dimer didodecyl 3,3″-dithiodipropionate (1) and a pregnane steroid 5,16-pregnadien-3β-ol-20-one acetate (2), were isolated together with two known compounds, ergosta-4,6,8(14),22-tetraen-3-one (3) and ergosterol peroxide (4), from the ethyl acetate soluble extract of fermentation broth of an endophytic fungus, Sphaceloma sp. LN-15 isolated from the leaves of Melia azedarach L. and grown in pure culture. Their structures were determined on the basis of spectroscopic methods including 1D and 2D nuclear magnetic resonance spectroscopy (NMR) experiments and by mass spectrometric measurements (MS). These fungal metabolites were isolated for the first time from the genus Sphaceloma. The structure of 1 was also confirmed by chemical synthesis.     

Discrimination of Cod Liver Oil According to Wild/Farmed and Geographical Origins by GC and <Superscript>13</Superscript>C NMR

The objective of this study was to test the possibility of using lipid profiles obtained by gas chromatography (GC) and ¹³C nuclear magnetic resonance (NMR) in authentication of cod liver oils according to wild/farmed and geographical origin. GC and ¹³C-NMR data of cod liver oil from wild and farmed fish from different locations in Norway and Scotland were obtained, and analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) to test if it was possible to differentiate oil from wild and cultured cod (Gadus morhua L.), and to further elucidate differences between fish from the different farms/catch area. Cod liver oils of wild and farmed origin were clearly separated in the PCA score plot both from GC and NMR data. From NMR data it was also possible to observe groupings based on geographical origin (farm/catch area) of the different samples. Using LDA with cross validation the wild/farmed classification rates were 97% for GC data and 100% for NMR data. In the classification of cod liver oils according to geographical origin (38 samples from six different farms/catch area), the correct classification rate was 63% for GC data and 95% for NMR data.     

Identification and bioassay of kairomones forHelicoverpa zea

Hexane extracts of leaves of 307 accessions from 73 host plant species ofHelicoverpa zea were analyzed by gas chromatography (GC) and used forH. zea oviposition and neonate larvae orientation bioassays. The gas chromatographic (GC) retention times of compounds statistically associated with behavioral activity were identified by correlation of GC peak area with oviposition and larval orientation preferences. Although taxonomically diverse in their origin, compounds for study were purified from extracts of species of the genusLycopersicon, due to their relative abundance. The structures of eight long-chain alkanes associated with oviposition preference were assigned by mass spectrometry, and the structures of five similarly associated organic acids and a terpenoid alkene were identified by¹H and¹³C nuclear magnetic resonance spectroscopy. The structures of a number of other phytochemicals from the plant leaves were identified for comparative purposes, including a previously unknown terpene, 7-epizingiberene. Bioassays were performed on the isolated acids and on the alkane wax fractions of severalLycopersicon species, and significant differences were found in oviposition stimulation for both classes of compounds. Of the hundreds of compounds found in the extracts, none were observed to act as oviposition deterrents. The results of these bioassays may be useful in explaining the broad host range ofH. zea, as well as the process and evolution of host plant selection for oviposition.     

CONFORMATIONAL ANALYSIS OF 2-(DIPHENYLPHOSPHANYL)-N,N-DIMETHYL-1-BENZ AMIDE AND 2-(DIPHENYLPHOSPHANYL)-PHENYL-PYRROLIDIN-1-YL-METHANONE BY NMR SPECTROSCOPY

We have synthesized 2-(Diphenylphosphanyl)-N, N-dimethyl-1-benzamide (1) and 2-(Diphenylphosphanyl)- phenyl-pyrrolidin-1-yl-methanone (2), and examined their conformations on the basis of NMR spectral data. Conformational analysis of the compounds is useful in deducing the structure in which they are active as a catalyst. In the present NMR measurements, ¹H-X fg-JHMBC (field gradient J-Resolved Hetero-nuclear Multiple-Bond Correlation) spectroscopy was implemented as a tool for the determination of hetero-nuclear three bond, phosphorus and protons and carbon-protons coupling constants. By fitting a sine curve to the experimental data by the method of 3D J-resolved HMBC NMR measurements, accurate ⁿ J _HX coupling constants were obtained. From the coupling constants, the corresponding dihedral angles, H3-C2-C3-P, H12-C12-C11-P, H16-C16-C11-P, H18-C18-C17-P, and H22-C22-C17-P, of compounds (1) and (2) were determined. The optimized structures of the compounds were obtained by molecular orbital calculations in which the dihedral angles experimentally determined were used.     

Kairomones for the egg parasiteTrichogramma evanescens Westwood

In observation-cage experiments some new contact kairomones for the egg parasiteTrichogramma evanescens Westwood are demonstrated.T. evanescens females search significantly longer on cabbage leaves treated with the wing scales of two hosts,Pieris brassicae L. andP. rapae L. Further, egg washes ofP. brassicae containing an oviposition deterrent pheromone for the butterflies, were found to have a contact-kairomonal effect on the parasite.T. evanescens females search significantly longer on cabbage leaves sprayed with a methanol or water wash ofP. brassicae eggs than on leaves treated with the solvent only.Hymenoptera: Trichogrammatidae.Lepidoptera: Pieridae.     

Quantitative Isotopomer Rates in Real-Time Metabolism of Cells Determined by NMR Methods

Tracer-based metabolism is becoming increasingly important for studying metabolic mechanisms in cells. NMR spectroscopy offers several approaches to measure label incorporation in metabolites, including ¹³C- and ¹H-detected spectra. The latter are generally more sensitive, but quantification depends on the proton–carbon ¹J_CH coupling constant, which varies significantly between different metabolites. It is therefore not possible to have one experiment optimised for all metabolites, and quantification of ¹H-edited spectra such as HSQCs requires precise knowledge of coupling constants. Increasing interest in tracer-based and metabolic flux analysis requires robust analyses with reasonably small acquisition times. Herein, we compare ¹³C-filtered and ¹³C-edited methods for quantification and show the applicability of the methods for real-time NMR spectroscopy of cancer-cell metabolism, in which label incorporations are subject to constant flux. We find an approach using a double filter to be most suitable and sufficiently robust to reliably obtain ¹³C incorporations from difference spectra. This is demonstrated for JJN3 multiple myeloma cells processing glucose over 24 h. The proposed method is equally well suited for calculating the level of label incorporation in labelled cell extracts in the context of metabolic flux analysis.     

Identification and Synthesis of the Sex Pheromone of the Passionvine Mealybug, Planococcus minor (Maskell)

The sex pheromone of the mealybug, Planococcus minor was isolated by fractionation of crude pheromone extract obtained by aeration of virgin females. The pheromone was identified as the irregular terpenoid, 2-isopropyl-5-methyl-2,4-hexadienyl acetate, by mass spectrometry, microchemical tests, and ¹H NMR spectroscopy. The stereochemistry of the pheromone was assigned as (E) by comparison with synthetic standards of known geometry. The compound was highly attractive to males in laboratory bioassays, whereas the (Z)-isomer appeared to antagonize attraction.     

Absolute configurational assignments for the1H-NMR spectrum of poly(vinyl alcohol) by use of two-dimensional NMR methods

Summary FOCSY two-dimensional J-resolved NMR spectroscopy is used to resolve¹H NMR resonances corresponding to triad and tetrad configurations of poly(vinyl alcohol). Two-dimensional auto correlation with ₁-scaling NMR spectroscopy is used to observe connectivities between triad and tetrad¹H NMR resonances. Comparison of these connectivities to the necessary compositional relationships at triad-tetrad level leads to absolute configurational assignments for the¹H NMR spectrum of poly(vinyl alcohol).     

Synthesis of dimer and oligomers from (R)-methyl hydnocarpate

We report synthesis and characterization of dimer and oligomer acids from chaulmoogra oil. (R)-Methyl hydnocarpate (methyl ester of the major fatty acid component of chaulmoogra oil) was brominated to give threo-2,3-dibromocyclopentane-1-methyl undecanoate. Formation of two diastereoisomers, viz., threo-2(R),3(R)-dibromocyclopentane-1(R)-methyl undecanoate and threo-2(S),3(S)-dibromocyclopentane-(R)-1-methyl undecanoate, was observed. Dehydrobromination of bromo derivatives using alcoholic KOH gave a cyclopentadiene derivative as intermediate, which underwent Diels-Alder reaction to give dimer and oligomer fatty acids. The products were characterized by ultraviolet, direct exposure probe-mass spectroscopy, ¹H nuclear magnetic resonance (NMR), and ¹³C NMR spectroscopic techniques.     

Solid-state nuclear magnetic resonance (NMR): Application to precursors of ceramics—Polycarbosilane

The chemical structures of oxidation- and electron irradiation-cured polycarbosilane fibers has been studied by IR and chemical analysis, but its structure has not been identified in detail. In this work, the chemical structure and curing mechanism was examined by solid-state high-resolution NMR spectroscopy. From the analysis of NMR spectra, it is explained that (i) in oxidation curing of PCS fibers, oxygen attacks first the SiC₃H bond and forms the SiC₃O bond and, next, the SiC₄ backbone bond and forms the SiC₂O₂ units; (ii) in electron irradiation curing, the signal intensity of SiC₃H units decreases with the increase in dose, the increase in the signal being due to the formation of SiC₄ units; (iii) solid-state²⁹Si high-resolution (CP/MAS) NMR spectroscopy is a powerful tool for investigating the chemical structure and curing mechanism of PCS fibers complementing infrared and solid-state¹³C high-resolution NMR spectroscopy; and (iv)¹H CRAMPS NMR spectroscopy is very useful for investigating the chemical structure and curing mechanism of PCS fibers.     

Lonicera Implexa Leaves Bearing Naturally Laid Eggs of the Specialist Herbivore Euphydryas Aurinia have Dramatically Greater Concentrations of Iridoid Glycosides than other Leaves

We tested in the field the hypothesis that the specialist butterfly Euphydryas aurinia (Lepidoptera: Nymphalidae, Melitaeinae) lays eggs on leaves of Lonicera implexa (Caprifoliaceae) plants with greater iridoid concentrations. We conducted our investigations in a Mediterranean site by analyzing leaves with and without naturally laid egg clusters. There were no significant differences in iridoid glycoside concentrations between leaves from plants that did not receive eggs and the unused leaves from plants receiving eggs, a fact that would seem to indicate that E. aurinia butterflies do not choose plants for oviposition by their iridoid content. However, the leaves of L. implexa that bore egg clusters had dramatically greater (over 15-fold) concentrations of iridoid glycosides than the directly opposite leaves on the same plant. These huge foliar concentrations of iridoids (15% leaf dry weight) may provide specialist herbivores with compounds that they either sequester for their own defense or use as a means of avoiding competition for food from generalist herbivores. Nevertheless, it may still be possible that these high concentrations are detrimental to the herbivore, even if the herbivore is a specialist feeder on the plant.