Pyrrolizidine Alkaloids Concentration Research Articles

Process Development of Lappula squarrosa Oil Refinement: Monitoring of Pyrrolizidine Alkaloids in Boraginaceae Seed Oils

AbstractBoraginaceous plants are characterized by high levels of polyunsaturated fatty acids and show a high ratio of ω‐3/ω‐6 fatty acids. In addition, Lappula squarrosa (Boraginaceae) shows high levels of stearidonic acid content (ω‐3; 18:4; 6,9,12,15‐octadecatetraenoic acid) showing interesting medical and health promoting properties. On the other hand Boraginaceous plants contain genotoxic and carcinogenic pyrrolizidine alkaloids (PA). An HPLC–ESI–MS/MS sum parameter method was developed to monitor the total sum of 1,2‐unsaturated PA in seed oil. The method was used to monitor different steps in oil refinement using lab model experiments and pilot scale refinement of L. squarrosa seed oil. A limit of detection and a limit of quantification (LOQ) of 0.02 μg retronecine equivalents (RE)/kg and 0.05 μg RE/kg were achieved, respectively. Multiple washing steps at pH 2.2 can significantly reduce the PA content to 0.07 % of the start value. In addition, combining washing with neutralization, bleaching and deodorization can reduce the PA content of L. squarrosa oil below the LOQ (0.05 μg RE/kg). The newly established method was further used to analyze the PA content of commercially available Boraginaceous seed oils (Echium spp., Borago officinalis). Three out of ten products were tested as PA positive (PA content ranging from not detected to 0.6 μg RE/kg product).

Analysis of Swiss honeys for pyrrolizidine alkaloids

SummaryVarious studies have shown that honey may contain pyrrolizidine alkaloids (PAs) and thus can pose a potential health risk for consumers. It seems that the level of contamination depends on the geographical and botanical origin of the honey. The geographically central location of Switzerland within Europe accounts for various different climate zones, and therefore can serve as a model to study PA contamination in honeys. We analysed 71 honeys between 2009 and 2011. Honeys from various botanical origins were collected from regions north and south of the Alps as well as the alpine regions. The PA concentration of the honeys was determined by target analysis using an HPLC-MS/MS-system, allowing the detection of 18 different PAs and PA-N-oxides found in the genera Echium, Eupatoriumand Senecio. 54% of the honeys contained PAs, while in 46% of the honeys PA concentrations were below the limit of quantitation (LOQ). The LOQs ranged from 1 μg/kg to 3 μg/kg, depending on the PA. The mean PA concentration of the positive samples was 6.7 μg/kg. The highest concentration of PAs (55 μg/kg) was found in a honey from Ticino, an area of the southern flank of the Alps. All the other positive honeys contained PAs at concentrations below 18 μg/kg. Therefore Swiss honey usually does not pose a risk for consumers. In the investigated honeys, honeys from the Swiss alpine regions (29 out of 37) tested more frequently positive for PAs compared to honeys from areas north of the Alps (9 out of 34). This probably reflects the different botanical settings of the central European and the alpine climate regions.

Pyrrolizidine Alkaloids in the Food Chain: Development, Validation, and Application of a New HPLC-ESI-MS/MS Sum Parameter Method

Contamination of food and feed with pyrrolizidine alkaloids is currently discussed as a potential health risk. Here, we report the development of a new HPLC-ESI-MS/MS sum parameter method to quantitate the pyrrolizidine alkaloid content in complex food matrices. The procedure was validated for honey and culinary herbs. Isotopically labeled 7-O-9-O-dibutyroyl-[9,9-(2)H2]-retronecine was synthesized and utilized as an internal standard for validation and quantitation. The total pyrrolizidine alkaloid content of a sample is expressed as a single sum parameter: retronecine equivalents (RE). Ld/Lq for honey was 0.1 μg RE/kg/0.3 μg RE/kg. For culinary herbs, 1.0 μg RE/kg/3.0 μg RE/kg (dry weight, dw) and 0.1 μg RE/kg/0.3 μg RE/kg (fresh weight, fw) were determined, respectively. The new method was applied to analyze 21 herbal convenience products. Fifteen products (71%) were pyrrolizidine alkaloid positive showing pyrrolizidine alkaloid concentrations ranging from 0.9 to 74 μg RE/kg fw.

Phytotoxicity Assessment of Certain Phytochemical Products Containing Pyrrolizidine Alkaloids

AbstractIntroduction: Tussilago farfara (coltsfoot), Petasites hybridus (common butterbur), Senecio vernalis (eastern groundsel) and Symphytum officinale (comfrey) are species traditionally used in phytotherapy that besides the therapeutic compounds contain toxic pyrrolizidine alkaloids (PAs). The aim of the paper is to determine the total PAs content and the phytotoxicity of the above species. Material and methods: The quantitative determination of pyrrolizidine alkaloids is based on the stoichiometric reaction of protonated alkaloids with methyl orange. In acidic conditions the dye is released from the complex and its color is assessed spectrophotometrically using a linear regression curve of senecionine as a standard. The phytotoxicity was assessed by Triticum bioassay that studies the effect of the extracts (0.001-5.00%, w/v) upon root elongation (inhibitory concentration - IC50) and on the karyokinetic film. Results: The highest amount of total PAs was found in Senecio vernalis (654.8 ± 35.96 μg/g dry plant) and the lowest in Petasites hybridus. The lowest IC50 was found for Tussilago farfara followed by Petasites hybridus, Senecio vernalis, and Symphytum officinale. The results were supported by microscopic examination. Conclusions: The results of the spectrophotometric assay are consistent with the ones found in the literature. All extracts inhibited the elongation of the main root of wheat caryopses, however, no correlation between phytotoxicity and the PAs concentration could be emphasized

A rapid and highly specific method to evaluate the presence of pyrrolizidine alkaloids in Borago officinalis seed oil

Pyrrolizidine alkaloids (PAs) are complex molecules, present in plants as free bases and N-oxides. They are known for their hepatotoxicity, and consequently there is a health risk associated with the use of medicinal herbs that contain PAs. Unfortunately, there is no international regulation of PAs in foods, unlike those for herbs and medicines: in particular, for herbal preparation or herbal extracts, the total PA content must not exceed 1 µg/kg or 1 µg/l, respectively. Borago officinalis seed oil is a source of γ-linolenic acid, and its use is increased in both pharmaceutical and health food industries. Even if studies based on gas chromatography and TLC methods showed that PAs are not co-extracted with oil, the development of a rapid and sensitive method able to evaluate the presence of PAs in commercially available products is surely of interest. The presence of PAs in a commercially available Borago officinalis seed oil was tested either in the oil sample diluted with tetrahydrofuran/methanol (MeOH)/H2 O (85/10/5 v:v:v) or after extraction with MeOH/H2 O (50/50 v:v) solution The samples were analysed by electrospray ionization in positive ion mode and in high mass resolution (60,000) conditions. In both cases to evaluate the effectiveness of the method, spiking experiments were performed adding known amount of two PA standards to the borage seed oil. A limit of detection in the order of 200 ppt was determined for these two compounds, strongly analogous to Borago officinalis seed oil PAs. Consequently, if present, PAs level in Borago officinalis seed oil must lower than 200 ppt.

Pyrrolizidine alkaloids: their occurrence in Spanish honey collected from purple viper’s bugloss (Echium spp.)

The incidence and concentration of pyrrolizidine alkaloids (PAs) from Echium spp. plant have been defined in 103 Spanish honey samples. Each sample was examined to determine total pollen content, the percentage of Echium spp. pollen, and simultaneous measurements of PAs and their N-oxides concentrations by the HPLC-ESI/MS method to identify the potential origin of PAs in honey. PAs were found in 94.2% of the raw honey samples analysed, in the range of 1–237 µg kg−1 (average concentration = 48 µg kg−1). The PA pattern was clearly dominated by echimidine, lycopsamine and their N-oxides, representing the 97.8% of total ∑PAs, and only echimidine and echimidine-N-oxide surpassed the 87% of total ∑PA content. Others PAs, seneciphylline and heliotrine-N-oxide, appear to be reported in a lower incidence and concentration (average of 3 and 1 µg kg−1, respectively). The Pearson Chi-squared test (p ≤ 0.01) confirms the non-correspondence between pollen plants and PA content. This study was also realised to generate a dataset in order to evaluate the potential risk of Spanish honeys containing PA plants belonging to the genera Echium.

Changes in plant defense chemistry (pyrrolizidine alkaloids) revealed through high-resolution spectroscopy

Plant toxic biochemicals play an important role in defense against natural enemies and often are toxic to humans and livestock. Hyperspectral reflectance is an established method for primary chemical detection and could be further used to determine plant toxicity in the field. In order to make a first step for pyrrolizidine alkaloids detection (toxic defense compound against mammals and many insects) we studied how such spectral data can estimate plant defense chemistry under controlled conditions.In a greenhouse, we grew three related plant species that defend against generalist herbivores through pyrrolizidine alkaloids: Jacobaea vulgaris, Jacobaea erucifolia and Senecio inaequidens, and analyzed the relation between spectral measurements and chemical concentrations using multivariate statistics.Nutrient addition enhanced tertiary-amine pyrrolizidine alkaloids contents of J. vulgaris and J. erucifolia and decreased N-oxide contents in S. inaequidens and J. vulgaris. Pyrrolizidine alkaloids could be predicted with a moderate accuracy. Pyrrolizidine alkaloid forms tertiary-amines and epoxides were predicted with 63% and 56% of the variation explained, respectively. The most relevant spectral regions selected for prediction were associated with electron transitions and CH, OH, and NH bonds in the 1530 and 2100nm regions.Given the relatively low concentration in pyrrolizidine alkaloids concentration (in the order of mgg−1) and resultant predictions, it is promising that pyrrolizidine alkaloids interact with incident light. Further studies should be considered to determine if such a non-destructive method may predict changes in PA concentration in relation to plant natural enemies. Spectroscopy may be used to study plant defenses in intact plant tissues, and may provide managers of toxic plants, food industry and multitrophic-interaction researchers with faster and larger monitoring possibilities.

Pyrrolizidine Alkaloid Composition Influences Cinnabar Moth Oviposition Preferences in Jacobaea Hybrids

Plants produce a variety of secondary metabolites (PSMs) that may be selective against herbivores. Yet, specialist herbivores may use PSMs as cues for host recognition, oviposition, and feeding stimulation, or for their own defense against parasites and predators. This summarizes a dual role of PSMs: deter generalists but attract specialists. It is not clear yet whether specialist herbivores are a selective force in the evolution of PSM diversity. A prerequisite for such a selective force would be that the preference and/or performance of specialists is influenced by PSMs. To investigate these questions, we conducted an oviposition experiment with cinnabar moths (Tyria jacobaeae) and plants from an artificial hybrid family of Jacobaea vulgaris and Jacobaea aquatica. The cinnabar moth is a specialist herbivore of J. vulgaris and is adapted to pyrrolizidine alkaloids (PAs), defensive PSMs of these plants. The number of eggs and egg batches oviposited by the moths were dependent on plant genotype and positively correlated to concentrations of tertiary amines of jacobine-like PAs and some otosenine-like PAs. The other PAs did not correlate with oviposition preference. Results suggest that host plant PAs influence cinnabar moth oviposition preference, and that this insect is a potential selective factor against a high concentration of some individual PAs, especially those that are also involved in resistance against generalist herbivores.

Above‐ and below‐ground herbivory effects on below‐ground plant–fungus interactions and plant–soil feedback responses

Summary Feeding by insect herbivores can affect plant growth and the concentration of defense compounds in plant tissues. Since plants provide resources for soil organisms, herbivory can also influence the composition of the soil community via its effects on the plant. Soil organisms, in turn, are important for plant growth. We tested whether insect herbivores, via their effects on the soil microbial community, can influence plant‐soil feedbacks. We first examined the effects of above‐ground (AG) and below‐ground (B) insect herbivory on the composition of pyrrolizidine alkaloids (PAs) in roots and on soil fungi in roots and rhizosphere soil of ragwort (Jacobaea vulgaris). The composition of fungal communities in roots and rhizosphere soil was affected by both AG and BG herbivory, but fungal composition also differed considerably between roots and rhizosphere soil. The composition of PAs in roots was affected only by BG herbivory. Thirteen different fungal species were detected in roots and rhizosphere soil. The presence of the potentially pathogenic fungus Fusarium oxysporum decreased and that of Phoma exigua increased in presence of BG herbivory, but only in soil samples. We then grew new plants in the soils conditioned by plants exposed to the herbivore treatments and in unconditioned soil. A subset of the new plants was exposed to foliar insect herbivory. Plant‐soil feedback was strongly negative, but the feedback effect was least negative in soil conditioned by plants that had been exposed to BG herbivory. There was a negative direct effect of foliar herbivory on plant biomass during the feedback phase, but this effect was far less strong when the soil was conditioned by plants exposed to AG herbivory. AG herbivory during the conditioning phase also caused a soil feedback effect on the PA concentration in the foliage of ragwort. Synthesis. Our results illustrate how insect herbivory can affect interactions between plants and soil organisms, and via these effects how herbivory can alter the performance of late‐growing plants. Plant‐soil feedback is emerging as an important theme in ecology and these results highlight that plant‐soil feedback should be considered from a multitrophic AG and BG perspective.

Senecio brasiliensis e alcaloides pirrolizidínicos: toxicidade em animais e na saúde humana

No Brasil, as plantas tóxicas são responsáveis por grande parte dos prejuízos econômicos em animais de produção, principalmente em equinos e bovinos. Nesse contexto, no Rio Grande do Sul, o gênero Senecio, especialmente S. brasiliensis Lessing, merece maior destaque por ser a principal causa de mortes de bovinos adultos. A toxicidade causada por esse gênero vegetal ocorre devido à presença dos alcaloides pirrolizidínicos, os quais sofrem biotransformação no fígado, gerando metabólitos tóxicos: os pirrois. Esses compostos tóxicos podem também ser nocivos para o homem através do consumo de produtos comestíveis, de origem animal, contaminados ou pelo uso dessa planta na medicina popular. Assim, o presente artigo traz uma revisão, em particular de S. brasiliensis com ênfase nos seus princípios ativos tóxicos e no mecanismo pelo qual se desenvolve a patogenia, além de abordar outras plantas que contêm os mesmos princípios tóxicos e que causam efeitos nocivos à saúde humana.

Effects of Root Herbivory on Pyrrolizidine Alkaloid Content and Aboveground Plant-Herbivore-Parasitoid Interactions in Jacobaea Vulgaris

The importance of root herbivory is increasingly recognized in ecological studies, and the effects of root herbivory on plant growth, chemistry, and performance of aboveground herbivores have been relatively well studied. However, how belowground herbivory by root feeding insects affects aboveground parasitoid development is largely unknown. In this study, we examined the effects of root herbivory by wireworms (Agriotes lineatus) on the expression of primary and secondary compounds in the leaves and roots of ragwort (Jacobaea vulgaris). We also studied the effects of root herbivory on the performance of a generalist aboveground herbivore, Mamestra brassicae and its parasitoid Microplitis mediator. In contrast to what most other studies have reported, root herbivory in J. vulgaris had a strong negative effect on the total concentration of pyrrolizidine alkaloids (PAs) in shoot tissues. The composition of PAs in the shoots also changed after root herbivory. In particular, the concentration of less toxic N-oxide PAs decreased. There was no significant effect of root herbivory on PA composition and concentration in the roots. Although the concentration of PA in the leaves decreased, M. brassicae tended to grow slower on the plants exposed to root herbivory. Parasitoid performance was not affected by root herbivory, but parasitoids developed faster when the concentration of jacobine-type PAs in the foliage was higher. These results point at a putative role of individual PAs in multitrophic interactions and emphasize that generalizations about aboveground-belowground effects should be made with great caution.

Content of pyrrolizidine alkaloids in the leaves of coltsfoot (Tussilago farfara L.) in Poland

Coltsfoot (<em>Tussilago farfara</em> L.) is a common species, widely used in European and Chinese traditional medicine for the treatment of respiratory diseases. However, raw material from this plant contains hepatotoxic pyrrolizidine alkaloids (PAs). The aim of the study was to determine the variability of the level of PAs (senkirkine and senecionine) in leaves of coltsfoot originated from natural populations in Poland. In the phytochemical analysis, 20 samples of <em>T. farfara</em> were used. This plant material was obtained from the Garden of Medicinal Plants in Plewiska near Poznań and originated from different regions of Poland. Coltsfoot leaves were harvested in the middle of July of 2010 and then dried at room temperature. The alkaloid content was detected using the HPLC-DAD method. The amount of PAs in leaves of <em>T. farfara</em> changed in a wide range from 0.06 to 1.04 μg g<sup>−1</sup> of dry matter (DM). The content of senkirkine and senecionine was positively correlated (<em>r</em> = 0.68, <em>P</em> = 0.001). There was no statistically significant correlation between the amount of PAs as well as leaf weight and water content in leaves of <em>T. farfara</em>. Our results showed that a medium-sized leaf of coltsfoot (0.33 g DM) may contain from 0.02 to 0.34 μg of PAs (on average 0.14 μg). The level of PAs was not associated with the region of Poland, but phytochemical similarity of samples was usually visible at the local scale. Coltsfoot leaves are characterized by a high variability of the content of toxic PAs, much higher than in the case of the main active compounds, especially flavonoids and mucilage.<br /> This phytochemical variability is mainly genetically determined (samples came from a garden collection), and it can be increased by environmental factors. Our investigations indicate that Polish natural populations of <em>T. farfara</em> may provide raw material with a low level of toxic PAs.

Detection and Quantification of Pyrrolizidine Alkaloids in Antibacterial Medical Honeys

In recent years, there has been an increasing interest in antibacterial honey for wound care ranging from minor abrasions and burns to leg ulcers and surgical wounds. On the other hand, several recent studies demonstrated that honey for human consumption was contaminated with natural occurring, plant derived pyrrolizidine alkaloids.1,2-Unsaturated pyrrolizidine alkaloids are a group of secondary plant metabolites that show developmental, hepato-, and geno-toxicity as well as carcinogenic effects in animal models and in in vitro test systems. Hence, it was of particular interest to analyze the pyrrolizidine alkaloid content of medical honeys intended for wound care.19 different medical honey samples and/or batches were analyzed by applying a recently established pyrrolizidine alkaloid sum parameter method. 1,2-Unsaturated pyrrolizidine alkaloids were converted into the common necin backbone structures and were analyzed and quantified by GC-MS in the selected ion monitoring mode.All but one medical honey analyzed were pyrrolizidine alkaloid positive. The results ranged from 10.6 µg retronecine equivalents per kg to 494.5 µg retronecine equivalents/kg medical honey. The average pyrrolizidine alkaloid content of all positive samples was 83.6 µg retronecine equivalents/kg medical honey (average of all samples was 79.3 µg retronecine equivalents/kg medical honey). The limit of detection was 2.0 µg retronecine equivalents/kg medical honey, while the limit of quantification was 6.0 µg retronecine equivalents/kg medical honey (S/N > 7/1).Based on the data presented here and considering the fact that medical honeys can be applied to open wounds, it seems reasonable to discuss the monitoring of 1,2-unsaturated pyrrolizidine alkaloids in honey intended for wound treatment.

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.

Legacy effects of aboveground–belowground interactions

Root herbivory can greatly affect the performance of aboveground insects via changes in plant chemistry. These interactions have been studied extensively in experiments where aboveground and belowground insects were feeding on the same plant. However, little is known about how aboveground and belowground organisms interact when they feed on plant individuals that grow after each other in the same soil. We show that feeding by aboveground and belowground insect herbivores on ragwort (Jacobaea vulgaris) plants exert unique soil legacy effects, via herbivore-induced changes in the composition of soil fungi. These changes in the soil biota induced by aboveground and belowground herbivores of preceding plants greatly influenced the pyrrolizidine alkaloid content, biomass and aboveground multitrophic interactions of succeeding plants. We conclude that plant-mediated interactions between aboveground and belowground insects are also important when they do not feed simultaneously on the same plant.

Profiling of Alkaloids and Eremophilanes in Miracle Tea (Packera candidissima and P. bellidifolia) Products

Commercial preparations of the Mexican herbal drug known as "miracle tea" (Packera candidissima and P. bellidifolia) have been profiled qualitatively by HPLC and GC-MS. Eremophilanes (3-7) were the major components found in the hexane-soluble fraction, while pyrrolizidine alkaloids (PAs) were identified in the alkaloid extracts. The content of free PAs and their N-oxides was determined for a total of 22 samples, and the results showed that the amount of these hepatotoxic compounds (0.0005-0.94% free PAs; 0.0004-0.55% N-oxides), through the presence of retrorsine (1) and senesionine (2) as the main constituents, may reach toxic levels. Hexane-soluble extracts from commercial presentations (dried whole plants) of both species afforded neoadenostylone (3), 6-(2-methylbutanoyloxy)-9-oxo-1-(10)-furanoeremophilene (4), and epineoadenostylone (5), in addition to methyl-4-hydroxyphenylacetate (8) and methyl-2-(1-hydroxy-4-oxocyclohexyl)acetate (9). Also, epicacalone (6) and the new compound 2β-hydroxyneoadenostylone (7) were isolated from P. bellidifolia.

Differential tissue distribution of metabolites in Jacobaea vulgaris, Jacobaea aquatica and their crosses

Plants are attacked by many different herbivores. Some will consume whole leaves or roots, while others will attack specific types of tissue. Thus, insight into the metabolite profiles of different types of leaf tissues is necessary to understand plant resistance against herbivores. Jacobaea vulgaris, J. aquatica and three genotypes of their crossings were used to study the variation in metabolomic profiles between epidermis and mesophyll tissues. Extracts of epidermis and mesophyll tissues were obtained using carborundum abrasion (CA). Subsequently, 1H nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses were applied to compare the metabolome profiles. Orthogonal partial least-squares-discriminant analysis (OPLS-DA) resulted in a clear separation of epidermis and mesophyll extracts. The epidermis contained significantly higher amounts of jacaranone and phenylpropanoids, specifically chlorogenic (5-O-CQA) and feruloyl quinic (FQA) acids compared to the mesophyll. In contrast, the mesophyll showed significantly higher concentrations of pyrrolizidine alkaloids (PAs), specifically jacobine and jaconine. The tissue specific distribution of these compounds was constant over all genotypes tested. Phenylpropanoids, 5-O-CQA and FQA, as well as PAs are known for their inhibitory effect on herbivores, especially against thrips. Thrips feeding commences with the penetration of the epidermis, followed by ingestion of sub-epidermal or mesophyll. Thrips thus may have to encounter phenylpropanoids in the epidermis as the first line of defence, before encountering the PAs as the ultimate defence in the mesophyll. The finding of tissue specific defense may have a major impact on studies of plant resistance. We cannot judge resistance using analyses of a whole roots, leafs or flowers. In such a whole-organism approach, the levels of potential defense compounds are far below the real ones encountered in tissues involved in the first line of defense. Instead, it is of great importance to study the defence compounds in the specific tissue to which the herbivore is confined.

Detection of pyrrolizidine alkaloids in commercial honey using liquid chromatography–ion trap mass spectrometry

Pyrrolizidine alkaloids (PAs) are known secondary plant metabolites which can cause hepatotoxicity in both humans and livestock. PAs can be consumed through the use of plants for food, medicinal purposes and as contaminants of agricultural crops and food. PA contaminated grain has posed the largest health risk, although any PA contamination in our food chain should be recognised as a potential health threat. For this purpose, retail honeys were tested by LC–MS/MS. The method allows for specific identification of toxic retronecine and otonecine-type PAs by comparison to reference compounds via a spectral library. In total, 50 honey samples were matched to the reference spectra within a set of tolerance parameters. Accurate data analysis and quick detection of positive samples was possible. Positive samples contained an average PA concentration of 1260μgkg−1 of honey. Good linear calibrations were obtained (R2>0.991). LOD and LOQ ranged from 0.0134 to 0.0305 and 0.0446 to 0.1018μgmL−1, respectively.

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.