Pyrrolizidine Alkaloid Profiles Research Articles

Analysis of Pyrrolizidine Alkaloids in Stingless Bee Honey and Identification of a Botanical Source as Ageratum conyzoides.

Stingless bee honeys (SBHs) from Australian and Malaysian species were analysed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the presence of pyrrolizidine alkaloids (PAs) and the corresponding N-oxides (PANOs) due to the potential for such hepatotoxic alkaloids to contaminate honey as a result of bees foraging on plants containing these alkaloids. Low levels of alkaloids were found in these SBHs when assessed against certified PA standards in targeted analysis. However, certain isomers were identified using untargeted analysis in a subset of honeys of Heterotrigona itama which resulted in the identification of a PA weed species (Ageratum conyzoides) near the hives. The evaluation of this weed provided a PA profile matching that of the SBH of H. itama produced nearby, and included supinine, supinine N-oxide (or isomers) and acetylated derivatives. These PAs lacking a hydroxyl group at C7 are thought to be less hepatoxic. However, high levels were also observed in SBH (and in A. conyzoides) of a potentially more toxic diester PA corresponding to an echimidine isomer. Intermedine, the C7 hydroxy equivalent of supinine, was also observed. Species differences in nectar collection were evident as the same alkaloids were not identified in SBH of G. thoracica from the same location. This study highlights that not all PAs and PANOs are identified using available standards in targeted analyses and confirms the need for producers of all types of honey to be aware of nearby potential PA sources, particularly weeds.

Separation of pyrrolizidine alkaloids in different Senecio species using ultra-high performance supercritical fluid chromatography

Different Senecio species, especially S. inaequidens – a neophyte native to South Africa – have widely spread across Europe and now are found worldwide. The entire genus is known to contain toxic pyrrolizidine alkaloids (PAs), which renders them a possible health hazard to humans and livestock. As they can enter the food chain or occur as contaminants in herbal crops and phytopharmaceutical formulations (e.g. teas), efficient and straightforward assays for their qualitative and quantitative analysis are in high demand. Different techniques have been used for this purpose, most commonly HPLC or GC. As the analysis of PAs is a challenging task, alternative methodologies like ultra-high performance SFC (UHPSFC) may offer an additional benefit in terms of their separation efficiency and orthogonal selectivity. In this study an UHPSFC approach for the simultaneous determination of six PAs (free bases as well as N-oxides) is presented, which achieved the baseline separation of all standard compounds in seven min. Optimal separation was carried out in gradient mode on a Torus™ DEA column with 0.05% ammonia in methanol as modifier. The column temperature was 25 °C, ABPR 1900 psi and flow rate 1.1 mL/min, with a detection wavelength of 215 nm. The assay was validated and fulfilled all ICH criteria exhibiting good linearity (R2 ≥ 0.9994), precision (inter-day variance ≤ 3.67%, intra-day variance ≤ 3.92%) and recovery rates (96.3–104.1%), with detection limits typical for SFC-PDA (≤ 4.24 µg/mL). Furthermore, it could conveniently be coupled to MS-detection, which increased the sensitivity significantly. To confirm practical suitability of the method, different Senecio samples were analyzed, indicating a high qualitative as well as quantitative difference in their PA profile (e.g. total amounts of PA between 0.09 and 4.63 mg/g).

The Orphan Crop Crassocephalum crepidioides Accumulates the Pyrrolizidine Alkaloid Jacobine in Response to Nitrogen Starvation.

Crassocephalum crepidioides is an African orphan crop that is used as a leafy vegetable and medicinal plant. Although it is of high regional importance in Sub-Saharan Africa, the plant is still mainly collected from the wild and therefore efforts are made to promote its domestication. However, in addition to beneficial properties, there was first evidence that C. crepidioides can accumulate the highly toxic pyrrolizidine alkaloid (PA) jacobine and here it was investigated, how jacobine production is controlled. Using ecotypes from Africa and Asia that were characterized in terms of their PA profiles, it is shown that the tetraploid C. crepidioides forms jacobine, an ability that its diploid close relative Crassocephalum rubens appears to lack. Evidence is provided that nitrogen (N) deficiency strongly increases jacobine in the leaves of C. crepidioides, that this capacity depends more strongly on the shoot than the root system, and that homospermidine synthase (HSS) activity is not rate-limiting for this reaction. A characterization of HSS gene representation and transcription showed that C. crepidioides and C. rubens possess two functional versions, one of which is conserved, that the HSS transcript is mainly present in roots and that its abundance is not controlled by N deficiency. In summary, this work improves our understanding of how environmental cues impact PA biosynthesis in plants and provides a basis for the development of PA-free C. crepidioides cultivars, which will aid its domestication and safe use.

Pyrrolizidine Alkaloids of Blue Heliotrope (Heliotropium amplexicaule) and Their Presence in Australian Honey.

Blue heliotrope (Heliotropium amplexicaule) is an invasive environmental weed that is widely naturalized in eastern Australia and has been implicated as a source of pyrrolizidine alkaloid (PA) poisoning in livestock. Less well-documented is the potential of such carcinogenic alkaloids to contaminate honey from bees foraging on this plant species. In this study, the PA profile of H. amplexicaule plant material, determined by HRAM LC-MS/MS, revealed the presence of nine PAs and PA-N-oxides, including several PAs and PA-N-oxides of the indicine class, which have not previously been reported. The predominant alkaloid, indicine, represents 84% of the reduced PA content, with minor alkaloids identified as intermedine and the newly reported helioamplexine, constituting 7 and 9%, respectively. NMR analysis confirmed the identity of helioamplexine as a previously unreported indicine homologue. This is the first report of the isolation of intermedine, helioamplexine, and 3'-O-angelylindicine from H. amplexicaule. Also described is the identification of N-chloromethyl analogues of the major alkaloids as isolation-derived artifacts from reactions with dichloromethane. Analysis of regional-market honey samples revealed a number of honey samples with PA profiles analogous to that seen in H. amplexicaule, with measured PA contents of up to 2.0 μg of PAs per gram of honey. These results confirm the need for honey producers to be aware of H. amplexicaule as a potential PA source, most particularly in products where honey is sourced from a single location.

Heliotropium europaeum poisoning in cattle and analysis of its pyrrolizidine alkaloid profile.

Pyrrolizidine alkaloids (PAs) are carcinogenic and genotoxic phytochemicals found exclusively in angiosperms. The ingestion of PA-containing plants often results in acute and chronic toxicities in man and livestock, targeting mainly the liver. During February 2014, a herd of 15-18-month-old mixed-breed beef cattle (n = 73) from the Galilee region in Israel was accidently fed hay contaminated with 12% Heliotropium europaeum (average total PA intake was 33 mg PA/kg body weight/d). After 42 d of feed ingestion, sudden death occurred over a time period of 63 d with a mortality rate of 33%. Necropsy and histopathological examination revealed fibrotic livers and moderate ascites, as well as various degrees of hyperplasia and fibrosis of bile duct epithelial cells. Elevated γ-glutamyl-transferase and alkaline phosphatase levels were indicative of severe liver damage. Comprehensive PA profile determination of the contaminated hay and of native H. europaeum by LC-MS/MS revealed the presence of 30 PAs and PA-N-oxides, including several newly reported PAs and PA-N-oxides of the rinderine and heliosupine class. Heliotrine- and lasiocarpine-type PAs constituted 80% and 18% of the total PAs, respectively, with the N-oxides being the most abundant form (92%). The PA profile of the contaminated hay showed very strong resemblance to that of H. europaeum.

The chemical profile of pyrrolizidine alkaloids from selected greek endemic boraginaceae plants determined by gas chromatography/mass spectrometry.

Four Greek endemic Boraginaceae plants, Onosma erecta Sibth. & Sm., Onosma kaheirei Teppner, Onosma leptantha Heldr., and Cynoglossum columnae L. (aerial parts), were screened for their content of pyrrolizidine alkaloids (PAs). TLC with the Mattocks-Molyneux visualization reagent was used as a preliminary qualitative test for PA or PA N-oxide detection. The extracts of the species found to contain PAs and their N-oxides were further analyzed by GC/MS, so as to identify their structures by means of the mass spectra and retention index values of known PAs already published in the literature. Twenty-three PAs were identified. For additional peaks, recognized as possible PAs by their MS pattern, no exact structures were tentatively suggested, as a result of lack of matching literature data. Furthermore, a quantitative PA profile of the species was obtained.

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.

Generalist <i>versus</i> Specialist Herbivores on the Invasive <i>Senecio inaequidens</i> and a Native Related Species: What Makes the Difference?

We compared herbivory pressure in the native Jacobaea vulgaris (formely Senecio jacobaea) and the alien invasive S. inaequidens in sites where they co-occur in Belgium. We predicted that the alien species experiences relaxed herbivory pressure by specialist herbivores (enemy release hypothesis ERH) whereas it is still attacked by generalist herbivores. Impacts of two generalist (gastropods and rabbits) and one specialist (the caterpillar Tyria jacobaeae) herbivores were assessed with field observations and exclusion experiments. The generalist herbivores had a higher impact on the biomass and survival of the seedlings of the alien S. inaequidens than on the native J. vulgaris. On the contrary, the specialist Tyria jacobaeae attacked exclusively the adults of the native species, supporting one of the main predictions of the ERH. These results are discussed in relation to differences in pyrrolizidine alkaloid profiles between the two species.

Pyrrolizidine alkaloid profile in a traditional Chinese herbal medicine Chuan Zi Wan (Ligulariae Radix et Rhizoma) by liquid chromatography/electrospray ionization ion trap mass spectrometry

Chuan Zi Wan (CZW) is a traditional Chinese herbal medicine derived from Ligularia species and used as an antitussive and expectorant folk remedy. Its toxicity was recently demonstrated in rats, for which the hepatotoxic pyrrolizidine alkaloids (PAs) were detected but not fully examined. In order to further assess its safety, the entire PA profile of CZW was investigated using high-performance liquid chromatography (HPLC) coupled to electrospray ionization ion trap mass spectrometry. Twenty-six toxic PAs (25 otonecine-type and one retronecine-type PAs) and one non-toxic PA of platynecine-type were detected and/or characterized based on diagnostic ions, mass fragmentation patterns, chromatographic behaviors and biosynthetic consideration. Except clivorine, ligularine, hodgsonine and ligularizine, all other PAs were described for the first time; moreover, most otonecine-type PAs were found to contain the acetyl group, an important substructure closely linked to their toxic effects in vivo. Clivorine was unequivocally identified as a major PA in the extract, with a content of 3.94 mg g−1 dried extractant determined by a HPLC/UV assay. The total content of the toxic PAs was estimated to be about 7.92 mg g−1 dried extract. These results indicated that this herbal product may have a potential health threat to humans in its Chinese medical practice.

The evolution of pyrrolizidine alkaloid biosynthesis and diversity in the Senecioneae

Pyrrolizidine alkaloids are characteristic secondary metabolites of the Asteraceae and some other plant families. They are especially numerous and diverse in the tribe Senecioneae and form a powerful defense mechanism against herbivores. Studies into the evolution of pyrrolizidine alkaloid biosynthesis using Senecio species have identified homospermidine synthase as the enzyme responsible for the synthesis of the first specific intermediate. These studies further indicated that the homospermidine synthase-encoding gene was recruited following gene duplication of deoxyhypusine synthase and that this occurred independently in several different angiosperm lineages. A review of published pyrrolizidine alkaloid data shows that the Senecioneae are characterized by a large qualitative and quantitative variation in pyrrolizidine alkaloid profiles and that these data demonstrate little phylogenetic signal. This suggests that although the first steps of this pathway are highly conserved, the diversification of secondarily derived pyrrolizidine alkaloids is extremely plastic.

Chemotaxonomic value of pyrrolizidine alkaloids in southern Brazil Senecio (Senecioneae: Asteraceae)

Pyrrolizidine alkaloids (PAs) were characterized by GC-MS in inflorescences of 14 Senecio species ( S. brasiliensis, S. conyzifolius, S. heterotrichius, S. oleosus, S. oxyphyllus, S. riograndensis, S. selloi, S. bonariensis, S. grossidens, S. icoglossus, S. juergensii, S. pulcher, S. ceratophylloides and S. crassiflorus) from southern Brazil, belonging to three taxonomic sections of the genus. The 1,2-saturated macrocycle diesters (such as platyphylline or rosmarinine), 1,2-saturated or unsaturated triangularine-like alkaloids such as senecioylplatinecine and senecioylretronecine were characteristic for the section Hualtatini. The section Corymbosi has, as characteristic PAs, 1,2-unsaturated 12-membered macrocyclic diesters (such as senecionine), while the section Suffrutecius is ambiguous in relation to the PA profile. The data indicate that the PA patterns are mostly in good agreement with morphological data for the sections Corymbosi and Hualtatini and that they can be useful chemotaxonomic markers at the infrageneric level.

Pyrrolizidine alkaloids in an overwintering population of monarch butterflies (Danaus plexippus) in California

California overwintering monarch butterflies contain both pyrrolizidine alkaloids (PAs) and theirN-oxides. Analysis of 76 individual monarchs by TLC, HPLC, GLC, and GC-MS has shown the presence of three types of PAs, the saturated diester sarracine, the saturated monoester 7-angelylplatynecine, and the unsaturated dialcohol retronecine. Monarchs arriving at the overwintering site in Santa Cruz, California, showed a wide variation in both the type and amount of PA present. Those sampled after a PA-containing plant (Senecio mikanioides) had bloomed at the site showed an altered PA profile. While the plant was found to contain sarracine and 7-angelylplatynecine, which are nontoxic to mammals, the monarchs showed an increase in retronecine levels, a toxic PA, after the plant bloom. Apparently monarchs utilize PA-containing plants both en route to their overwintering site and at the site, and potentially alter those PAs to forms toxic to mammals.