Pyrrolizidine Alkaloids In Plants Research Articles

Variations in chemical defences and patterns of natural enemy attack between native and introduced populations of fireweed (Senecio madagascariensis): Implications for biological control

AbstractNative to South Africa, fireweed (Senecio madagascariensis Poiret; Asteraceae) is an annual or short‐lived perennial herb that is highly invasive in Australia, where it is a target for biological control. Preliminary research indicates that fireweed may be undergoing adaptive changes along its invasion gradient in Australia. Changes, such as a shift in plant chemical defence, may influence fireweed’s interaction with specialist insect herbivores, and hence the efficacy of a biocontrol program. As the testing of biocontrol candidates is a rigorous and lengthy process, confirmation of fireweed’s susceptibility to insect attack may assist in fast tracking candidate suitability, where changes in plant defensive chemistry are found. In a large experimental field study in fireweed’s native range, we compared the susceptibility of Australian and South African populations to insect natural enemy attack (in terms of plant biomass, insect richness and composition). Simultaneously, we assessed the variation in plant pyrrolizidine alkaloid concentrations and composition to rationalise any differences in natural enemy attack between populations. We found no significant differences in plant biomass across fireweed populations, despite significantly higher alkaloid concentrations in Australian populations. The composition of the endophagous (specialist) insect herbivore assemblage was related to alkaloid composition, but there were only minor differences in the assemblage between Australian and South African fireweed populations. Moreover, we found no significant differences in the abundance, richness or Shannon diversity indices of endophagous or ectophagous (generalist) insect herbivores between Australian and South African fireweed populations. Our results suggest that despite variations in chemical defence, Australian fireweed populations displayed neither reduced nor increased susceptibility to specialist insect herbivore attack, supporting the notion that if host‐specific biocontrol agents are released, their impact in Australia is unlikely to be influenced by any changes in plant chemical defence.

Analysis of Pyrrolizidine Alkaloids in Queensland Honey: Using Low Temperature Chromatography to Resolve Stereoisomers and Identify Botanical Sources by UHPLC-MS/MS.

Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to food regulators in Australia and internationally. In this survey, honey samples sourced from markets and shops in Queensland (Australia), were analysed by high-resolution Orbitrap UHPLC-MS/MS for 30 common PAs. Relationships between the occurrence of pyrrolizidine alkaloids and the botanical origin of the honey are essential as pyrrolizidine alkaloid contamination at up to 3300 ng/g were detected. In this study, the predominant alkaloids detected were isomeric PAs, lycopsamine, indicine and intermedine, exhibiting identical MS/MS spectra, along with lesser amounts of each of their N-oxides. Crucially, chromatographic UHPLC conditions were optimised by operation at low temperature (5 °C) to resolve these key isomeric PAs. Such separation of these isomers by UHPLC, enabled the relative proportions of these PAs present in honey to be compared to alkaloid levels in suspect source plants. Overall plant pyrrolizidine alkaloid profiles were compared to those found in honey samples to help identify the most important plants responsible for honey contamination. The native Australian vines of Parsonsia spp. are proposed as a likely contributor to high levels of lycopsamine in many of the honeys surveyed. Botanical origin information such as this, gained via low temperature chromatographic resolution of isomeric PAs, will be very valuable in identifying region of origin for honey samples.

Analytical strategies for the determination of pyrrolizidine alkaloids in plant based food and examination of the transfer rate during the infusion process

Two sample preparation methods were developed (graphitised carbon and C18 solid phase extraction clean-up) and validated in house using liquid chromatography and tandem mass spectrometry (MS/MS) for the determination of 30 pyrrolizidine alkaloids (PAs) in salads, herbs, tea, herbals teas as well as tea infusion and ice-tea beverages. Total PAs concentration of samples purchased on the Belgian market varied greatly with matrix type ranging from <LOD to 187151 ng/g for dry samples, while for infusions and ice-tea beverages the highest PAs concentration was 2106 ng/mL. Surprisingly high PAs concentrations were detected in herbs and spices mixes. The infusion study indicated a transfer rate between 16 and 28% (except for monocrotaline) which highlights the overestimation of PAs concentrations in infusions when derived from a dilution factor (transfer rate of 100%) to the measured concentrations of the dry tea sample.

The Risk of Pyrrolizidine Alkaloids in Human Food and Animal Feed.

Toxicity from naturally occurring plant pyrrolizidine alkaloids bears substantial risk to humans and livestock. Risk management strategies must focus on minimizing the likelihood of such alkaloids entering into the food chain. To address this challenge, we describe the role of a tiered risk characterization approach and specific farm management practices.

Structural screening by multiple reaction monitoring as a new approach for tandem mass spectrometry: presented for the determination of pyrrolizidine alkaloids in plants

In tandem mass spectrometry the multiple reaction monitoring (MRM) mode is normally used for targeted analysis but this mode also has the potential to screen for structural similarities of analytes. On the basis of the fact that in general similar molecular structures result in similar fragments or losses of neutrals, this approach was used for pyrrolizidine alkaloid (PA) screening but could also be easily adapted to screen for other compound classes. PA are plant toxins of which several hundred individual compounds have been identified. Our MRM screening approach uses the structural relation and similar core structure of all PA which results in a common and thus predictable mass spectrometric fragmentation behaviour. On this basis a method was developed which screens for PA structures by MRM transitions and allows the detection of each individual PA down to a low microgram per kilogram concentration range. The approach was applied to investigate plants from the families of Asteraceae (several species of Senecio and Eupatorium), Boraginaceae (Echium, Cynoglossum, Borago and Anchusa officinalis as well as Heliotropium europaeum) and Fabaceae (Crotalaria incana) for a complete qualitative and quantitative PA characterisation. All analytes that were detected as possible PA by MRM screening were further investigated by recording product ion spectra. Analytes which exhibited a typical PA fragmentation pattern were either confirmed as PA or otherwise deleted as false positive signals (false positive rate was below 10%). Sum formulas of confirmed PA were determined by additional measurements applying high resolution mass spectrometry. In that way 121 unknown PA were identified and for the first time complete PA profiles of different PA plants were delivered.

Preference for high concentrations of plant pyrrolizidine alkaloids in the specialist arctiid moth Utetheisa ornatrix depends on previous experience

Secondary metabolites are one the most pervasive defensive mechanisms in plants. Many specialist herbivores have evolved adaptations to overcome these defensive compounds. Some herbivores can even take advantage of these compounds by sequestering them for protection and/or mate attraction. One of the most studied specialist insects that sequesters secondary metabolites is the arctiid moth Utetheisa ornatrix. This species sequesters pyrrolizidine alkaloids (PAs) from its host plant, the legume Crotalaria spp. The sequestered PAs are used as a predator repellent and as a mating pheromone. We used this species to test larval preference for different concentrations of PAs. We purified PAs from plant material and added them at different concentrations to an artificial diet. Larvae of U. ornatrix previously feeding on low and high PA concentration artificial diets were allowed to choose between two new artificial diets with different PA concentrations. The amount of PAs sequestered and larval preference were dependent on their previous exposure to low or high PA content in the diet. Larvae that were pretreated with a low PA diet significantly consumed more diet with the high PA concentration, while larvae that were pretreated with a high PA diet showed no discrimination between future feeding of different PA concentration diets. We discuss our results using mechanistic and evolutionary approaches. Finally, we discuss how these results have important implications on the evolution of plant herbivore interactions and how specialist herbivores may decrease the levels of chemical defenses on plant populations.

A free lunch? No cost for acquiring defensive plant pyrrolizidine alkaloids in a specialist arctiid moth (Utetheisa ornatrix)

Many herbivorous insects sequester defensive chemicals from their host plants. We tested sequestration fitness costs in the specialist moth Utetheisa ornatrix (Lepidoptera: Arctiidae). We added pyrrolizidine alkaloids (PAs) to an artificial diet at different concentrations. Of all the larval and adult fitness components measured, only development time was negatively affected by PA concentration. These results were repeated under stressful laboratory conditions. On the other hand, the amount of PAs sequestered greatly increased with the diet PA concentration. Absence of a detectable negative effect does not necessarily imply a lack of costs if all individuals express the biochemical machinery of detoxification and sequestration constitutively. Therefore, we used qPCR to show that expression of the gene used to detoxify PAs, pyrrolizidine-alkaloid-N-oxygenase (pno), increased 41-fold in our highest PA treatment. Nevertheless, fitness components were affected only slightly or not at all, suggesting that sequestration in this species does not incur a strong cost. The apparent lack of costs has important implications for our understanding of the evolution of ecological interactions; for example, it implies that selection by specialist herbivores may decrease the levels of certain chemical defences in plant populations.

ChemInform Abstract: Pyrrolizidine Alkaloids in Plants Used in the Traditional Medicine of Madagascar and the Mascarene Islands

AbstractReview: 95 refs.

Attract and deter: a dual role for pyrrolizidine alkaloids in plant–insect interactions

Pyrrolizidine alkaloids (PAs) are the major defense compounds of plants in the Senecio genus. Here I will review the effects of PAs in Senecio on the preference and performance of specialist and generalist insect herbivores. Specialist herbivores have evolved adaptation to PAs in their host plant. They can use the alkaloids as cue to find their host plant and often they sequester PAs for their own defense against predators. Generalists, on the other hand, can be deterred by PAs. PAs can also affect survival of generalist herbivores. Usually generalist insects avoid feeding on young Senecio leaves, which contain a high concentration of alkaloids. Structurally related PAs can differ in their effects on insect herbivores, some are more toxic than others. The differences in effects of PAs on specialist and generalists could lead to opposing selection on PAs, which may maintain the genetic diversity in PA concentration and composition in Senecio species.

Update on analytical methods for toxic pyrrolizidine alkaloids

Methods for the determination of toxic pyrrolizidine alkaloids in plants and foods are described with emphasis on the important aspects of sample extraction and clean-up and the now preferred determination by liquid chromatography-mass spectrometry. The efficiencies of different extraction solvents and methods are described, as are the methods of reduction of N-oxides. Appropriate liquid chromatography-mass spectrometry conditions are tabulated. This concise review is intended to guide analysts towards adopting a more unified and reliable approach to the analysis of these important toxins.

LC-MS-MS Can Distinguish Between Toxic and Non-Toxic Pyrrolizidine Alkaloids in Plants

Several plants including medicinal plants and weeds that contaminate food crops contain pyrrolizidine alkaloids (PA). In the case of medicinal plants not used regularly, a low level of PA can be accommodated, but because food is consumed over a long time the allowable levels must be much lower [1]. Maize and soybean farmers in South Africa suffer economic losses due to the presence of Crotalaria sphaerocarpa seed pods in crops by the implementation of current regulations [1]. Because many Crotalaria species contain toxic PAs, there is a limit on the quantity of seeds that are allowed in the grain. C. sphaerocarpa undoubtedly contains pyrrolizidine alkaloids but toxicity to animals could not be established in several unpublished trials.

Quantitative analysis of total retronecine esters-type pyrrolizidine alkaloids in plant by high performance liquid chromatography

Pyrrolizidine alkaloids (PAs) are alkaloids which typically contain a necine (7-hydroxy-1-hydroxymethyl-6,7-dihydro-5H-pyrrolizidine) base unit, and they can be found in one third of the higher plants around the world. They are hepatotoxic, mutagenic and carcinogenic and pose a threat to human health and safety. A specific, quick and sensitive method is therefore needed to detect and quantify the PAs sometimes in trace amount in herbs, tea or food products. Based on high performance liquid chromatography with prior derivatization of the alkaloids using o-chloranil and Ehrlich's reagent, we report an improved method for quantitative analysis of the total amount of retronecine esters-type pyrrolizidine alkaloids (RET-PAs) in a plant extract. The total quantitation of RET-PAs is achieved because of a common colored retronecine marker, a 7-ethoxy-1-ethoxylmethyl retronecine derivative, is produced with all the different RET-PAs during the derivatization reaction. The chemical identity of the common retronecine marker was characterized on-line by positive mode electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. The limit of detection using the improved method is 0.26 nmol mL −1 and the limit of quantitation is 0.79 nmol mL −1. The advantages of this method are much enhanced sensitivity in detection and quantitation, and, no restriction on the choice of RET-PA as a calibration standard. Application of the developed method to the quantitation of total RET esters-type PAs in Senecio scandens from different regions of China is also reported.

Metabolic Activation of Herbal and Dietary Constituents and Its Clinical and Toxicological Implications: An Update

In recent years, there has been a globally increasing application of herbal medicines and dietary supplements to treat various chronic diseases and to promote health. However, there are increasing clinical reports on the organ toxicities associated with consumption of herbal medicines. This review updates the knowledge on metabolic activation of herbal components and its clinical and toxicological implications. Like many synthetic drugs undergoing metabolic activation to form reactive metabolites which are often associated with drug toxicity, it is recognized that some herbal components may also be converted to toxic, or even mutagenetic and carcinogenic metabolites by cytochrome P450s (CYPs) and less frequently by Phase II conjugating enzymes. This is exemplified by aristolochic acids (AAs) in Aristolochia spp, which undergo reduction of the nitro group by hepatic CYP1A1/2 or peroxidases in extrahepatic tissues to generate highly reactive cyclic nitrenium ions. The latter can react with macromolecules (DNA and protein), resulting in activation of H-ras oncogene and gene mutation in renal cells and finally carcinogenesis of the kidneys. Some naturally occurring flavonoids (e.g. quercetin) and alkenylbenzenes (e.g. safrole, methyleugenol and estragole) can undergo metabolic activation by sequential 1-hydroxylation and sulfation, resulting in reactive intermediates capable of forming DNA adducts and finally genotoxicity. Additional examples are pulegone present in essential oils from many mint species; and teucrin A, a diterpenoid found in germander (Teuchrium chamaedrys) used as an adjuvant to slimming dietary supplements but caused severe hepatotoxicity. Extensive pulegone metabolism generated p-cresol that was a glutathione depletory, whereas the furan ring of the diterpenoids in germander was oxidized by CYP3A4 to reactive epoxide which can inactivate hepatic CYP3A and epoxide hydrolase through covalent binding. The hepatotoxic and carcinogenic species of plant pyrrolizidine alkaloids (e.g. echimidine and jacobine), namely pyrrole-type metabolites, are generated by hepatic CYP2B6 and CYP3A4. Potential mechanisms underlying the hepatotoxicity of kava have been related to intracellular glutathione depletion and/or quinone formation. Some herbal constituents (e.g. capsaicin from chili peppers, glabridin from licorice root, oleuropein in olive oil, dially sulfone in garlic, and resveratrol found in red wine) behave as mechanism-based inhibitors of various CYPs. This may provide an explanation for some reported herb-drug interactions. In addition, the inhibition of CYPs by herbal constituents may decrease the formation of toxic metabolites and thus inhibit carcinogenesis, as CYPs play an important role in procarcinogen activation. Due to the wide use and easy availability of herbal medicines, further research should be conducted to ensure the safety and quality of herbal medicine.

Acquisition, transformation and maintenance of plant pyrrolizidine alkaloids by the polyphagous arctiid Grammia geneura

The polyphagous arctiid Grammia geneura appears well adapted to utilize for its protection plant pyrrolizidine alkaloids of almost all known structural types. Plant-acquired alkaloids that are maintained through all life-stages include various classes of macrocyclic diesters (typically occurring in the Asteraceae tribe Senecioneae and Fabaceae), macrocyclic triesters (Apocynaceae) and open-chain esters of the lycopsamine type (Asteraceae tribe Eupatorieae, Boraginaceae and Apocynaceae). As in other arctiids, all sequestered and processed pyrrolizidine alkaloids are maintained as non-toxic N-oxides. The only type of pyrrolizidine alkaloids that is neither sequestered nor metabolized are the pro-toxic otonecine-derivatives, e.g. the senecionine analog senkirkine that cannot be detoxified by N-oxidation. In its sequestration behavior, G. geneura resembles the previously studied highly polyphagous Estigmene acrea. Both arctiids are adapted to exploit pyrrolizidine alkaloid-containing plants as “drug sources”. However, unlike E. acrea, G. geneura is not known to synthesize the pyrrolizidine-derived male courtship pheromone, hydroxydanaidal, and differs distinctly in its metabolic processing of the plant-acquired alkaloids. Necine bases obtained from plant acquired pyrrolizidine alkaloids are re-esterified yielding two distinct classes of insect-specific ester alkaloids, the creatonotines, also present in E. acrea, and the callimorphines, missing in E. acrea. The creatonotines are preferentially found in pupae; in adults they are largely replaced by the callimorphines. Before eclosion the creatonotines are apparently converted into the callimorphines by trans-esterification. Open-chain ester alkaloids such as the platynecine ester sarracine and the orchid alkaloid phalaenopsine, that do not possess the unique necic acid moiety of the lycopsamine type, are sequestered by larvae but they need to be converted into the respective creatonotines and callimorphines by trans-esterification in order to be transferred to the adult stage. In the case of the orchid alkaloids, evidence is presented that during this processing the necine base (trachelanthamidine) is converted into its 7-( R)-hydroxy derivative (turneforcidine), indicating the ability of G. geneura to introduce a hydroxyl group at C-7 of a necine base. The creatonotines and callimorphines display a striking similarity to plant necine monoesters of the lycopsamine type to which G. geneura is well adapted. The possible function of insect-specific trans-esterification in the acquisition of necine bases derived from plant acquired alkaloids, especially from those that cannot be maintained through all life-stages, is discussed.

Thesinine-4′- O-β- d-glucoside the first glycosylated plant pyrrolizidine alkaloid from Borago officinalis

The glycosylated pyrrolizidine alkaloid, thesinine-4′- O-β- d-glucoside, has been isolated from the aqueous methanolic extract of dried seeds of Borago officinalis (Boraginaceae). The structure was established by means of spectroscopic and chemical analyses.

Deoxyhypusine Synthase from Tobacco: cDNA ISOLATION, CHARACTERIZATION, AND BACTERIAL EXPRESSION OF AN ENZYME WITH EXTENDED SUBSTRATE SPECIFICITY

Deoxyhypusine synthase catalyzes the formation of a deoxyhypusine residue in the translation eukaryotic initiation factor 5A (eIF5A) precursor protein by transferring an aminobutyl moiety from spermidine onto a conserved lysine residue within the eIF5A polypeptide chain. This reaction commences the activation of the initiation factor in fungi and vertebrates. A mechanistically identical reaction is known in the biosynthetic pathway leading to pyrrolizidine alkaloids in plants. Deoxyhypusine synthase from tobacco was cloned and expressed in active form in Escherichia coli. It catalyzes the formation of a deoxyhypusine residue in the tobacco eIF5A substrate as shown by gas chromatography coupled with a mass spectrometer. The enzyme also accepts free putrescine as the aminobutyl acceptor, instead of lysine bound in the eIF5A polypeptide chain, yielding homospermidine. Conversely, it accepts homospermidine instead of spermidine as the aminobutyl donor, whereby the reactions with putrescine and homospermidine proceed at the same rate as those involving the authentic substrates. The conversion of deoxyhypusine synthase-catalyzed eIF5A deoxyhypusinylation pinpoints a function for spermidine in plant metabolism. Furthermore, and quite unexpectedly, the substrate spectrum of deoxyhypusine synthase hints at a biochemical basis behind the sparse and skew occurrence of both homospermidine and its pyrrolizidine derivatives across distantly related plant taxa.

Putative mechanisms of toxicity of 3-methylindole: from free radical to pneumotoxicosis.

Damage to lung tissue can result not only from exposure to chemicals by inhalation, but also from internal exposure to a variety of drugs or chemicals by ingestion. For example, the use of antineoplastic agents in the treatment of cancer (i.e. bleomycin, cyclophosphamide, carmustine, methotrexate), or the inadvertent ingestion of pesticide residues (i.e. paraquat, trialkylphos­ phoroth ioates) , or natural plant compounds (4-ipomeanol , plant pyrrolizidine alkaloids) are known to cause lung injury (1). In contrast to these ingested drugs or xenobiotics that producc critical lung disease, 3-methylindolc (3MI) is produced by resident microorganisms in the digestive system from tryptophan (TRP), an essential amino acid in the diet of humans and animals (2-4). Production of 3MI in the rumen in response to sudden changes in the feeding regimen can become detrimental to cattle by causing fatal pneumotoxicosis (5). 3M! is also produced in the colon of humans and animals, and the quantity varies with dietary composition, nutritional status, or disease (6). In addition to internal exposure to 3MI, humans can inhale 3Ml from cigarette smoke because it is a pyrolysis product of TRP in tobacco leaves (7). Investigation of the etiology of a naturally occurring lung disease in cattle,

S-4-3 - Rats with pulmonary hypertension induced by monocrotaline, a plant pyrrolizidine alkaloid.

S-4-3 - Rats with pulmonary hypertension induced by monocrotaline, a plant pyrrolizidine alkaloid.

Sequestration of plant pyrrolizidine alkaloids by chrysomelid beetles and selective transfer into the defensive secretions

Oreina cacaliae andO. speciosissima (Coleoptera, Chrysomelidae) sequester in their elytral and pronotal defensive secretions pyrrolizidine alkaloids (PAs) as Noxides (PA N-oxides). The PA N-oxide patterns found in the beetles and their host plants were evaluated qualitatively and quantitatively by capillary gas chromatography/mass spectrometry (GC-MS). Of the three host plantsAdenostyles alliariae (Asteraceae) is the exclusive source for PA N-oxide sequestration in the defensive secretions of the beetles. With the exception of O-acetylseneciphylline the N-oxides of all PAs ofA. alliariae, i.e. senecionine, seneciphylline, spartioidine, integerrimine, platyphylline and neoplatyphylline were identified in the secretion. PA N-oxides typical ofSenecio fuchsii (Asteraceae) were detected in the bodies of the beetles but not in their secretion. No PAs were found in the leaves of the third host plant,Petasites paradoxus (Asteraceae). The results suggest the existence of two distinctive storage compartments for PA N-oxides in the beetle: (1) the defensive secretion, containing specifically PA N-oxides acquired fromA. alliariae; (2) the body of the beetle, sequestering additionally but less selectively PA N-oxides from other sources,e.g. S. fuchsii or monocrotaline N-oxide fed in the laboratory. The concentration of PA N-oxides in the defensive secretion is in the range of 0.1 to 0.3 mol/1, which is more than 2.5 orders of magnitude higher than that found in the body of the beetle. No significant differences exist in the ability of the two species of beetles to sequester PA N-oxides fromA. alliariae, althoughO. speciosissima, but notO. cacaliae, produces autogenous cardenolides. A negative correlation seems to exist between the concentrations of plant-derived PA N-oxides andde novo synthesized cardenolides in the defensive secretion ofO. speciosissima.

Sequestration, N-Oxidation and Transformation of Plant Pyrrolizidine Alkaloids by the Arctiid Moth Tyria jacobaeae L

Larvae of the arctiid moth Tyria jacobaeae reared on Senecio jacobaea or S. vulgaris take up and store pyrrolizidine alkaloids (PAs) from their host plants. Individual PAs are taken up without preference. The PA patterns found in the insect bodies correspond to the PA composi­tion of their host plants. Like plants the insects store PAs as N-oxides, and larvae as well as pupae are specifically able to N -oxidize any tertiary PA. Callimorphine (O9-(2-methyl-2-acetoxybutanoyl)-retronecine), an insect PA well known from several arctiids, was found in pupae and imagines of Tyria which as larvae had been fed on S. jacobaea. It is accompanied by small amounts of its isomer O7-(2-methyl-2-acetoxybutanoyl)-retronecine named isocallimor-phine. The callimorphines may well account for 45% of total PAs found in the insect. Only small amounts of callimorphine were detected in pupae of Tyria which as larvae had been fed on S. vulgaris. [14C]Callimorphine N -oxide was isolated and identified from Tyria pupae which as larvae received [14C]retronecine. It is suggested that Tyria is able to esterify retronecine, derived from hydrolysis of ingested plant PAs with a necic acid produced by the insect. During metamorphosis the formation of callimorphine is restricted to the early stage of pupa­tion.