Determination of pyrrolizidine alkaloids in dietary sources using a spectrophotometric method

Pyrrolizidine alkaloids (PAs) are a class of toxic compounds found in the composition of more than 6000 plants. People can be exposed to PAs by consuming phytotherapeutic products, food from crops contaminated with seeds of some species with high content of PAs, and/ or contaminated animal products like bee products. For this reason we developed and validated a method for quantitative determination of PAs, from the most frequently contaminated food sources, honey and flour. Colorimetric Ehrlich reagent method was used with standard addition (1mg/kg senecionine). The extraction solvent was methanol 50% acidified with citric acid to pH 2-3, as this solvent can be used for alkaloids and N-oxides. We found that, in extracting the alkaloid only once from the dietary sources, the percent of recovery is low (52.5% for honey, and 45.75% for flour). Using successive extractions, three times with the same solvent, the senecionine retrieval percentage increased to 86.0% for honey and 76.0% for flour. The method was validated using the following parameters: selectivity, linearity (0,25- 20 mg/ mL senecionine), accuracy (average recovery 93.5 - 107.93%) and precision (RSD 3,26-4.55%.). The calculated limit of quantification (0.174 mg/ mL) makes this method applicable for determining Pas occurring at toxic levels for consumers

Soil-borne microorganisms and soil-type affect pyrrolizidine alkaloids in Jacobaea vulgaris

Secondary metabolites like pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. We studied the effects of soil-borne microorganisms and soil-type on pyrrolizidine alkaloids in roots and shoots of Jacobaea vulgaris. We used clones of two genotypes from a dune area (Meijendel), propagated by tissue culture and grown on two sterilized soils and sterilized soils inoculated with 5% of non-sterilized soil of either of the two soil-types. Soil-borne microorganisms and soil-type affected the composition of PAs. By changing the composition rather than the total concentration below and aboveground, plants have a more complex defense strategy than formerly thought. Interestingly, a stronger negative effect on plant growth was found in sterilized soils inoculated with their ‘own’ microbial community suggesting that pathogenic and/or other plant inhibiting microorganisms were adapted to their ‘own’ soil conditions

The Relationship between Structurally Different Pyrrolizidine Alkaloids and Western Flower Thrips Resistance in F2 Hybrids of Jacobaea vulgaros and Jacobaea aquatica

Segregating plant hybrids often have more ecological and molecular variability compared to parental species, and are therefore useful for studying relationships between different traits, and the adaptive significance of trait variation. Hybrid systems have been used to study the relationship between the expression of plant defense compounds and herbivore susceptibility. We conducted a western flower thrips (WFT) bioassay using a hybrid family and investigated the relationship between WFT resistance and pyrrolizidine alkaloid (PA) variation. The hybrid family consisted of two parental (Jacobaea vulgaris and Jacobaea aquatica) genotypes, two F1 genotypes, and 94 F2 hybrid lines. The J. aquatica genotype was more susceptible to thrips attack than the J. vulgaris genotype, the two F1 hybrids were as susceptible as J. aquatica, and susceptibility to WFT differed among F2 hybrid lines: 69 F2 lines were equally susceptible compared to J. aquatica, 10 F2 lines were more susceptible than J. aquatica and 15 F2 lines were as resistant as J. vulgaris or were intermediate to the two parental genotypes. Among 37 individual PAs that were derived from four structural groups (senecionine-, jacobine-, erucifoline- and otosenine-like PAs), the N-oxides of jacobine, jaconine, and jacoline were negatively correlated with feeding damage caused by WFT, and the tertiary amines of jacobine, jaconine, jacoline, and other PAs did not relate to feeding damage. Total PA concentration was negatively correlated with feeding damage. Among the four PA groups, only the total concentration of the jacobine-like PAs was negatively correlated with feeding damage. Multiple regression tests suggested that jacobine-like PAs play a greater role in WFT resistance than PAs from other structural groups. We found no evidence for synergistic effects of different PAs on WFT resistance. The relationship between PA variation and WFT feeding damage in the Jacobaea hybrids suggests a role for PAs in resistance to generalist insects

Detection and toxicity evaluation of pyrrolizidine alkaloids in medicinal plants Gynura bicolor and Gynura divaricata collected from different Chinese locations

Two edible plants in Southeast Asia, Gynura bicolor and G.divaricata, are not only known to be nutritive but also useful as medicinal herbs. Previous phytochemical investigation of Gynura species showed the presence of hepatotoxic pyrrolizidine alkaloids (PAs), indicating the toxic risk of using these two plants. The present study was designed to analyze the distribution of PA components and tried to evaluate the preliminary toxicity of these two Gynura species. Eight samples of G.bicolor and G.divaricata from five different Chinese locations were collected and their specific PAs were qualitatively characterized by applying an UPLC/MS/MS spectrometry method. Using a pre-column derivatization HPLC method, the total retronecine ester-type PAs in their alkaloids extracts were quantitatively estimated as well. Finally, their genotoxicity was investigated with an effective high-throughput screening method referred to as Vitotox test and their potential cytotoxicity was tested on HepG2 cells. It was found that different types of PAs were widely present in Gynura species collected from south of China. Among them, no significant genotoxic effects were detected with serial concentrations through the present invitro assay. However, the cytotoxicity assay of Gynura plants collected from Jiangsu displayed weak activity at the concentration of 100mg/ml. It is important to note that this research validates in part the indication that the use of Gynura species requires caution

Dutch survey pyrrolizidine alkaloids in animal forage

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites produced by a number of plants from the Asteraceae (Compositae), Boriginaceae and Fabaceae (Leguminosae) families. Many of these alkaloids have been shown to be highly toxic, causing hepatic veno-occlusive disease (VOD), liver cirrhosis and ultimately death. PAs may have also mutagenic and carcinogenic potential. Amongst livestock, cattle and horses are especially susceptible to the toxic effects of the PAs. Humans may also be at risk by the consumption of milk of livestock fed with PA-contaminated forage. At RIKILT - Institute of Food Safety a (semi)quantitative method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of PAs in animal feeds has been developed and validated. This method comprises 40 macrocyclic PAs (including tertiary amines and N-oxides) representative for ragwort species. The method has been used for the analysis of 147 forage samples collected in 2006-2008

Independent Recruitment of a Flavin-Dependent Monooxygenase for Safe Accumulation of Sequestered Pyrrolizidine Alkaloids in Grasshoppers and Moths

Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants

Chemical analysis and toxicity of pyrrolizidine alkaloids and assessment of the health risks posed by their occurrence in honey

Pyrrolizidine alkaloids (PA) are secondary plant metabolites. In certain types of honey in particular, increased levels of pyrrolizidine alkaloids can occur in dependence of the product’s origin. In the opinion of the BfR, efforts are required to reduce these levels. In addition, it is recommended that suitable measures are taken to avoid the intake of PA through food supplements, e.g. products that contain pollen of PA-containing plants. On the occasion of the contamination of a salad mix by blossoms and leaves of common groundsel (Senecio vulgaris L.), the BfR already recommended that special care be taken when harvesting and preparing lettuce, leafy vegetables and herbs. In total, more than 500 different pyrrolizidine alkaloids and their N-oxides are known which in turn can be contained in roughly 6,000 plant species. Given the large variety of individual substances, the available data on the toxicology, oral bioavailability and on the presence of pyrrolizidine alkaloids in food and feed must be seen as patchy. What is known, however, is that pyrrolizidine alkaloids have a detrimental effect in the liver in both humans and animals. They typically trigger a liver veno occlusive disease (VOD). However, experiments with animals also found pyrrolizidine alkaloids to be carcinogenic. On the basis of the existing data, the Federal Institute for Risk Assessment (BfR) has conducted a provisional assessment of the health risk posed by pyrrolizidine alkaloids in food and especially honey. The study took into account both the acute and chronic toxic effects. Taking into consideration all currently available data, the BfR comes to the conclusion that the overall exposure of the consumer to 1,2-unsaturated pyrrolizidine alkaloids with genotoxic and carcinogenic properties found in different foods is to be kept as low as possible. A daily intake of 0.007 micrograms (μg)1 of unsaturated pyrrolizidine alkaloids per kilogram of bodyweight should possibly not be exceeded

Pyrrolizidine alkaloids from seven wild-growing Senecio species in Serbia and Montenegro

The genus Senecio (family Asteraceae) is one of the largest in the world. It comprises about 1100 species which are the rich source of pyrrolizidine alkaloids. Plants containing pyrrolizidine alkaloids are among the most important sources of human and animal exposure to plant toxins and carcinogens. The pyrrolizidine alkaloids of seven Senecio species (S. erucifolius, S. othonnae, S. wagneri, S. subalpinus, S. carpathicus, S. paludosus and S. rupestris) were studied. Fourteen alkaloids were isolated and their structures determined from spectroscopic data (1H- and 13C-NMR, IR and MS). Five of them were identified in S. erucifolius, four in S. othonnae, two in S. wagneri, four in S. subalpinus, two in S. carpathicus, three in S. paludosus and three in S. rupestris. Seven pyrrolizidine alkaloids were found for the first time in particular species. The results have chemotaxonomic importance. The cytotoxic activity and antimicrobial activity of some alkaloids were also studied

Pyrrolizidine alkaloids from Echium glomeratum (Boraginaceae).

The methanolic extract of the whole plant of Echium glomeratum Poir. (Boraginaceae) has afforded five pyrrolizidine alkaloids, three that were (7S, 8R)-petranine (1), (7S, 8S)-petranine (2), and (7R, 8R)-petranine (3a) or (7R, 8S)-petranine (3b), comprising a tricyclic pyrrolizidine alkaloids subclass; and two that were known but to the species: 7-angeloylretronecine (4) and 9-angeloylretronecine (5). All compounds were tested against a human tumor panel for cytotoxicity; no activity was observed (EC50 values > 20 µg/ml)