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Plants synthesize a broad array of specialized chemical compounds that mediate their interactions with the surrounding environment. Some of this chemical diversity is functional and subject to natural selection, but the factors underlying chemical evolution at the intraspecific level remain largely unknown. Here, we combined chemical, environmental and genetic data to investigate the effect of aridity on the expression of chemotypes in the invasive shrub Senecio pterophorus. We studied the variation in pyrrolizidine alkaloids (PAs), a group of specialized metabolites widespread across the families Boraginaceae, Asteraceae and Fabaceae, from native populations spanning a cline of aridity and from three cross‐continental introductions, under natural and common garden conditions. We examined whether the relationship between chemistry and aridity was compatible with a process of adaptive differentiation using a method that partitions the variance and covariance by controlling for the population neutral genetic structure. We found a consistent shift from retrorsine‐like to seneciphylline‐like compounds under increasing aridity in both natural and controlled conditions in coherence with the biosynthetic pathways. This pattern was independent of the neutral genetic structure and occurred along the environmental gradient in the native range and in a convergent manner in all nonnative regions, which suggests adaptive differentiation in response to aridity. Our findings show that the diversity of PAs in S. pterophorus has been partially shaped by aridity. Investigating how abiotic factors influence chemical evolution is key to elucidating the plant responses in future climate scenarios and the cascading effects on other trophic levels. [ABSTRACT FROM AUTHOR]

Diatraea saccharalis Fabricius (Lepidoptera: Crambidae), known as sugarcane borer, is the most damaging pest of sugarcane (Saccharum officinarum L., Poaceae) in northwestern Argentina and it causes significant losses in sugar and ethanol production. Currently, chemical and biological controls are used, along with host plant resistance, but there is a clear trend toward adoption of integrated pest management strategies. Considering the potential environmental and health risks associated with using agrochemicals, our study focuses on utilizing bioactive natural products, specifically plant secondary metabolites. Senecio rudbeckiaefolius Meyen & Walp (Asteraceae) is a perennial shrub found in southern Peru, Bolivia, and northwestern Argentina, valued for its use in traditional medicine. The main compounds of the genus Senecio are sesquiterpenes, with some species containing pyrrolizidine alkaloids (PAs), known for their harmful effects on herbivorous insects and vertebrates. Plant extracts exhibit several modes of action, encompassing confusion, feeding deterrence, growth regulation, insecticidal, and repellent effects. Our goal was to analyze the chemical composition of an ethanolic extract (EE) of S. rudbeckiaefolius and evaluate its lethal and sublethal effects on D. saccharalis larvae. We also investigated the potential target sites affected by the ingestion of an alkaloidal fraction from the EE (AFEE) through a midgut histological study. The main secondary metabolites identified were the PAs and phenolic compounds, with the highest concentrations found in the flowers and leaves. The majority of the PAs identified were senecionine and integerrimine. Toxicity bioassays were carried out with larvae fed on a diet supplemented with an EE of S. rudbeckiaefolius. Concentration‐dependent mortality and a marked inhibition of larval growth were recorded. We described the midgut histological structure and compared it with that of AFEE‐fed larvae. Various morphological changes were observed in the intestinal epithelium, especially an increase in the number of goblet cells and pronounced acidophilic secretion in the lumen. These results suggest that the EE of S. rudbeckiaefolius could be promising for D. saccharalis control. [ABSTRACT FROM AUTHOR]

SUMMARY: Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey (Symphytum officinale, Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper‐containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, SoCuAO1 is expressed in young leaves, while SoCuAO5 is expressed in roots. CRISPR/Cas9‐mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA‐free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of SoCuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species‐specific, tight spatio‐temporal regulation, and independent evolutionary origins in multiple plant lineages. Significance Statement: Pyrrolizidine alkaloids are involved in the chemical defense of comfrey against herbivores and are synthesized in roots and young leaves. Using CRISPR/Cas9 genome editing and various biochemical experiments, we show that pyrrolizidine alkaloid biosynthesis in the two different organs of comfrey is realized by two different paralogs of homospermidine oxidase, revealing another aspect of biosynthetic complexity in plant specialized metabolism. [ABSTRACT FROM AUTHOR]

BACKGROUND: Symphytum (comfrey) genus, particularly Symphytum officinale, has been empirically used in folk medicine mainly for its potent anti‐inflammatory properties. In an attempt to shed light on the valorization of less known taxa, the current study evaluated the metabolite profile and antioxidant and enzyme inhibitory effects of nine Symphytum species. RESULTS: Phenolic acids, flavonoids and pyrrolizidine alkaloids were the most representative compounds in all comfrey samples. Hierarchical cluster analysis revealed that, within the roots, S. grandiflorum was slightly different from S. ibericum, S. caucasicum and the remaining species. Within the aerial parts, S. caucasicum and S. asperum differed from the other samples. All Symphytum species showed good antioxidant and enzyme inhibitory activities, as evaluated in DPPH (up to 50.17 mg Trolox equivalents (TE) g−1), ABTS (up to 49.92 mg TE g−1), cupric reducing antioxidant capacity (CUPRAC, up to 92.93 mg TE g−1), ferric reducing antioxidant power (FRAP, up to 53.63 mg TE g−1), acetylcholinesterase (AChE, up to 0.52 mg galanthamine equivalents (GALAE) g−1), butyrylcholinesterase (BChE, up to 0.96 mg GALAE g−1), tyrosinase (up to 13.58 mg kojic acid equivalents g−1) and glucosidase (up to 0.28 mmol acarbose equivalents g−1) tests. Pearson correlation analysis revealed potential links between danshensu and ABTS/FRAP/CUPRAC, quercetin‐O‐hexoside and DPPH/CUPRAC, or rabdosiin and anti‐BChE activity. CONCLUSIONS: By assessing for the first time in a comparative manner the phytochemical–biological profile of a considerably high number of Symphytum samples, this study unveils the potential use of less common comfrey species as novel phytopharmaceutical or agricultural raw materials. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Within the last decades, in the EU, there has been an increasing interest in toxic plant alkaloids as food contaminants, especially after the continuous and growing consumption of plant‐based foods compared with food of animal origin. In this regard, the once neglected presence of these tropane alkaloids (TAs) and pyrrolizidine alkaloids (PAs) has recently been reconsidered by the European Food Safety Authority, highlighting the lack of data and the need to develop risk assessment strategies. For this reason, the emphasis has been placed on detecting their occurrence in food through the development of accurate and sensitive analytical methods to achieve the determination of these compounds. The present study aims to elaborate and validate an analytical method based on QuEChERS sample preparation approach, exploiting the UHPLC coupled to the HRMS to simultaneously identify and quantify 21 PAs and two TAs in cereals and spices. For TAs, the obtained limit of detection (LOD) is 0.1 μg·kg−1 and the limit of quantification (LOQ) is 0.4 μg·kg−1, while for PAs, the LODs values ranging between 0.2 to 0.3 μg·kg−1 and the LOQ, between 0.4 and 0.8 μg·kg−1, ensuring compliance with the recently established European Regulations. Several commercial samples were analysed to further verify the applicability of this comprehensive analytical approach. [ABSTRACT FROM AUTHOR]

Summary: Two analytical methods for the quantification of pyrrolizidine (PAs) and tropane alkaloids (TAs) in honey bee‐collected pollen, teas and herbal infusions have been in‐house validated using the UHPLC‐MS/MS technique. These methods showed good linearity, good accuracy and robustness. The methods have been applied to a preliminary investigation of forty‐seven pollen samples and thirty‐three packs of tea‐bags in light of Commission Regulations (EU) 2020/2040 and 2021/1408 regarding maximum limits of PAs and TAs in foodstuffs, respectively. About half of the pollen samples contained searched PAs but in concentrations significantly lower than the maximum limit established; two samples were contaminated with TAs. One third of teas or herbal infusions contained PAs or TAs, one sample exceeded the EU limits for PAs and one for TAs. Despite the final consumer's health does not seem threatened, these results recommend accurate analytical methods and broader investigations for PAs and TAs in foods. [ABSTRACT FROM AUTHOR]

Plant‐based 1,2‐unsaturated Pyrrolizidine Alkaloids (PAs) are responsible for liver genotoxicity/carcinogenicity following metabolic activation, making them a relevant concern for safety assessment. Due to 21st century toxicology approaches, risk of PAs can be better discerned though an understanding of differing toxic potencies, but it is often mixtures of PAs that are found as contaminants in foods, for example, herbal teas and honey, food supplements and herbal medicines. Our study investigated whether genotoxicity potency of PAs dosed individually or in mixtures differed when measured using micronuclei formation in vitro in HepaRG human liver cells, which we and others have shown to be suitable for observing genotoxic potency differences across different PA structural classes. When equipotent concentrations of up to six different PAs representing a wide range of potencies in vitro were tested as mixtures, the observed genotoxic potency aligned favorably with results for single PAs. Similarly, when the BMD confidence intervals of these equipotent mixtures were compared with the confidence intervals of the individual PAs, only minimal variation was observed. These data support a conclusion that for this class of plant impurities, all acting via the same DNA‐reactive mode of action, genotoxic potency can be regarded as additive when assessing the risk of mixtures of PAs. [ABSTRACT FROM AUTHOR]

Metabolic oxidation of pyrrolizidine alkaloids (PAs) from herbal and dietary supplements by cytochrome P450 produces dehydro‐PAs (DHPs), which leads to toxicities. A highly reactive cation species generated from the active pyrrole ring of DHPs readily reacts with various cellular components, causing hepatotoxicity and cytotoxicity. Inspired by PA‐induced hepatic damage, we developed a therapeutic approach based on a cyclization precursor that can be transformed into a synthetic DHP under physiological conditions through gold‐catalyzed 5‐endo‐dig cyclization using a gold‐based artificial metalloenzyme (ArM) instead of through metabolic oxidation by cytochrome P450. In cell‐based assays, the synthesis of the DHP by a cancer‐targeting glycosylated gold‐based ArM substantially suppressed cell growth of the targeted cancer cells without causing cytotoxicity to untargeted cells, highlighting the potential of the strategy to be used therapeutically in vivo. [ABSTRACT FROM AUTHOR]

In plants, homospermidine synthase (HSS) is a pathway‐specific enzyme initiating the biosynthesis of pyrrolizidine alkaloids (PAs), which function as a chemical defense against herbivores. In PA‐producing Convolvulaceae ("morning glories"), HSS originated from deoxyhypusine synthase at least >50 to 75 million years ago via a gene duplication event and subsequent functional diversification. To study the recruitment of this ancient gene duplicate to PA biosynthesis, the presence of putative hss gene copies in 11 Convolvulaceae species was analyzed. Additionally, various plant parts from seven of these species were screened for the presence of PAs. Although all of these species possess a putative hss copy, PAs could only be detected in roots of Ipomoea neei (Spreng.) O'Donell and Distimake quinquefolius (L.) A.R.Simões & Staples in this study. A precursor of PAs was detected in roots of Ipomoea alba L. Thus, despite sharing high sequence identities, the presence of an hss gene copy does not correlate with PA accumulation in particular species of Convolvulaceae. In vitro activity assays of the encoded enzymes revealed a broad spectrum of enzyme activity, further emphasizing a functional diversity of the hss gene copies. A recently identified HSS specific amino acid motif seems to be important for the loss of the ancestral protein function—the activation of the eukaryotic initiation factor 5A (eIF5A). Thus, the motif might be indicative for a change of function but allows not to predict the new function. This emphasizes the challenges in annotating functions for duplicates, even for duplicates from closely related species. [ABSTRACT FROM AUTHOR]

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Comprehensive Summary: Five new pyrrolizidine alkaloids, bohemamines J—N (1—5), were isolated from Streptomyces sp. CPCC 200497. Their structures were assigned based on detailed spectroscopic analysis and semisynthesis. Bohemamine J (1) possesses a new chimeric skeleton derived from bohemamine A (6) and phenylacetaldehyde. Inspired by the nonenzymatic formation mechanism of the methylene‐bridged dimers isolated from this strain, we synthesized a series of chimeric derivatives (8, 9, and 12—23) through natural product chimera strategy. Compounds 13, 15, 19, and 21 showed significant antioxidant activity. [ABSTRACT FROM AUTHOR]

Native 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. [ABSTRACT FROM AUTHOR]

Kleptopharmacophagy: Milkweed butterflies scratch and imbibe from Apocynaceae-feeding caterpillars Here, we report novel observations from Sulawesi of adult male danaine butterflies scratching other danaine butterfly larvae and imbibing the exudates with their proboscis, presumably to steal their host-plant-derived alkaloids. Keywords: behavior; leaf-scratching; pharmacophagy; pyrrolizidine alkaloids EN behavior leaf-scratching pharmacophagy pyrrolizidine alkaloids 1 5 5 12/03/21 20211201 NES 211201 Milkweed butterflies (Lepidoptera: Nymphalidae: Danainae) are chemically defended and aposematic (Ackery and Vane-Wright 1984). Unique to the danaine butterflies is the unusual behavior known as "leaf-scratching" (Boppré 1983), in which adult butterflies damage plant parts with their sharp tarsal claws, liberating plant juices and sap to imbibe. [Extracted from the article]

Pyrrolizidine alkaloids are toxins having hepatotoxic and carcinogenic effects on human health. A ultra high performance liquid chromatography tandem mass spectrometry technique was developed for the first time for the simultaneous determination of eight pyrrolizidine alkaloids, including four diastereoisomers (intermedine, lycopsamine, rinderine, and echinatine) and their respective N‐oxide forms, in different parts of Eupatorium lindleyanum. The risk assessment method for pyrrolizidine alkaloids in Eupatorium lindleyanum was explored using the margin of exposure strategy for the first time based on a real‐life exposure scenario. Differences were found in all eight pyrrolizidine alkaloids in various parts of Eupatorium lindleyanum. Besides, the total levels of pyrrolizidine alkaloids in Eupatorium lindleyanum followed the order of root > flower > stem > leaf. Moreover, the risk assessment data revealed that the deleterious effects on human health were unlikely at exposure times of less than 200, 37, and 12 days during the lifetimes of Eupatorium lindleyanum leaves, stems, and flowers, respectively. This study reported both the contents of and risk associated with Eupatorium lindleyanum pyrrolizidine alkaloids. The comprehensive application of the novel ultra high performance liquid chromatography tandem mass spectrometry technique alongside the risk assessment approach provided a scientific basis for quality evaluation and rational utilization of toxic pyrrolizidine alkaloids in Eupatorium lindleyanum to improve public health safety. [ABSTRACT FROM AUTHOR]

A rapid and sensitive ultra‐performance liquid chromatography‐tandem electrospray ionization mass spectrometry (UPLC‐ESI/MS) method was established and validated for simultaneous determination of seneciphylline and its main metabolite in rat plasma. The plasma sample was prepared by simple methanol‐mediated precipitation. Chromatographic separation was achieved within 3 min by gradient elution using acetonitrile and water containing 0.1% formic acid as mobile phase on a Waters ACQUITY BEH C18 column (100 × 2.1 mm, i.d. 1.7 μm). Quantitation was conducted in a positive multiple reaction monitoring mode. The linearity of the method was over the range of 1–1,000 ng/mL, with the lower limit of quantification of 1 ng/mL. The intra‐ and inter‐day precision and accuracy, extraction recovery, and matrix effect of analytes were within the acceptable limit. The analytes were stable during the process of sample collection, preparation, and analysis. The validated method was further applied to a pharmacokinetic study of seneciphylline in rats after oral and intravenous administration. The results revealed that seneciphylline was quickly absorbed into plasma (Tmax, 0.23–0.32 h) and reached the maximum concentration of 0.82–1.75 μg/mL after oral administration. Both seneciphylline and seneciphylline N‐oxide were eliminated from plasma quickly. The low system exposure (oral bioavailability, 5.43–10.31%) was related to the extensive metabolism in the liver and microflora. [ABSTRACT FROM AUTHOR]

Introduction: Symphytum officinale L. (comfrey, Boraginaceae) is a cultivated or spontaneously growing medicinal plant that is traditionally used for the treatment of bone fractures, hematomas, muscle pains and joint pains. A wide range of topical preparations and dried roots for ex tempore applications are marketed in European drug stores or pharmacies. Objective: The aim of this study was to perform the qualitative and quantitative analysis of pyrrolizidine alkaloids (PAs) and phenolic compounds in the hydroethanolic extracts of 16 commercial comfrey root batches purchased from 12 different European countries. Methods: Liquid chromatography hyphenated with high‐resolution tandem mass spectrometry (LC‐HRMS/MS) was used for the profiling of PAs and phenolic compounds, whereas LC‐MS/MS and liquid chromatography with diode array detection (LC‐DAD) were used for their quantification. Results: 20 PAs (i.e. intermedine, lycopsamine, acetylintermedine, acetyllycopsamine, symphytine, symphytine‐N‐oxide), 17 phenolic compounds (i.e. caffeic and rosmarinic acids, rabdosiin, globoidnan A, globoidnan B) and 9 nonphenolic compounds (sugars, organic and fatty acids) were fully or partly annotated in the analysed samples. In addition, the quantitative analyses revealed that globoidnan B, rabdosiin and globoidnan A are new phenolic markers that can be used together with rosmarinic acid and PAs for the quality control of commercial comfrey root batches. Conclusions: This study brings new insights into the phytochemical complexity of S. officinale, revealing not only numerous toxic PAs, but also a significant number of valuable phenolic compounds that could contribute to the bioactivities of comfrey‐based preparations. A total of 16 hydroethanolic extracts of commercial comfrey (Symphytum officinale L.) root batches purchased from 12 different European countries were analyzed by LC‐HRMS/MS, revealing 20 pyrrolizidine alkaloids, 17 phenolic compounds and 9 nonphenolic compounds (sugars, organic and fatty acids). In addition, the quantitative analysis showed that globoidnan B, rabdosiin and globoidnan A are new phenolic markers that can be used together with rosmarinic acid and pyrrolizidine alkaloids for the quality control of commercial comfrey root batches. [ABSTRACT FROM AUTHOR]

Background: The complex regulatory framework for the quality of herbal medicinal products, in particular with regard to potential contaminants, consists of legal provisions including Pharmacopoeia requirements, relating to limits as well as in most cases to methods for determination. Objective: This review intends to provide an overview on the legal requirements for testing potential contaminants in herbal drugs used for the production of herbal medicinal products. Methodology: The following issues are described and discussed: For the assessment of pesticide residues, Chapter 2.8.13 of the European Pharmacopoeia and Regulation (EC) 396/2005 applies, for heavy metals the general Pharmacopoeia monograph as well as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q3D guideline on elemental impurities. The Pharmacopoeia also includes limits and acceptance criteria for microbiological purity and aflatoxins, respectively. Polycyclic aromatic hydrocarbons are mentioned in Regulation (EC) 1881/2006 for food as well as in a Reflection Paper on herbal medicinal products. For several years, a potential contamination with pyrrolizidine alkaloids was discussed which led to measures being imposed on industry and health authorities. Results: Various examples demonstrate that the evaluation of data from daily analytical practice is extremely useful to gain an overview on the occurrence of residues and/or contaminants in herbal drugs and herbal preparations. Such evaluations might assist manufacturers in their individual risk assessment and the definition of their testing strategies and contribute to guarantee the quality and safety of the marketed products. Conclusion: Annual data evaluations demonstrate that a remarkable decrease of the pyrrolizidine alkaloid content burden in herbal drugs and extracts used for the production of herbal medicinal products has been achieved over the past few years. However, it has also become clear that this issue cannot be completely solved in the near future. This review summarizes the legal requirements for testing potential contaminants in herbal drugs used for the production of herbal medicinal products. Respective substances comprise e.g. pesticides, heavy metals, mycotoxins and pyrrolizidine alkaloids. Various examples of data evaluations from daily analytical practice are given which present an overview on the actual situation and might assist manufacturers in defining their testing strategies. [ABSTRACT FROM AUTHOR]

Hepatotoxic and genotoxic pyrrolizidine alkaloids have been involved in the acute poisoning of animals and humans. Crotalaria (Fabaceae) species contain these alkaloids. In this work, the diversity and distribution of pyrrolizidine alkaloids in roots, leaves, flowers, and seeds of Crotalaria pallida, Crotalaria maypurensis, Crotalaria retusa, Crotalaria spectabilis, Crotalaria incana, and Crotalaria nitens were studied. Matrix solid‐phase dispersion and ultra‐high‐performance liquid chromatography coupled with Orbitrap mass spectrometry were successfully employed in pyrrolizidine alkaloids extraction and analysis, respectively. Forty‐five pyrrolizidine alkaloids were detected and their identification was based on the mass spectrometry accurate mass measurement and fragmentation pattern analysis. The cyclic retronecine‐type diesters monocrotaline, crotaleschenine, integerrimine, usaramine, and their N‐oxides were predominantly present. Five novel alkaloids were identified for the first time in Crotalaria species, namely 14‐hydroxymonocrotaline, 12‐acetylcrotaleschenine, 12‐acetylmonocrotaline, 12‐acetylintegerrimine, and dihydrointegerrimine. Due to a lack of commercially available standards, the response factor of monocrotaline was used for quantification of pyrrolizidine alkaloids and their N‐oxides. Seeds and flowers possessed higher pyrrolizidine alkaloids amounts than roots and leaves. Due to their 1,2‐unsaturated pyrrolizidine alkaloids content, the ingestion of Crotalaria plant seeds or other parts through herbal products, infusions, or natural remedies is a serious health threat to humans and livestock. [ABSTRACT FROM AUTHOR]

Introduction: Crotalaria spectabilis is an important species used as a pre‐plant cover for soybean crops to control the proliferation of endoparasitic nematodes. Species from the Crotalaria genus are known for presenting pyrrolizidine alkaloids (PAs) in their composition, however, C. spectabilis is still considered chemically under‐explored. Objective: The goal of this manuscript is the development and validation of a method for PAs and flavonoids identification and quantification of C. spectabilis seeds and leaves, a toxic plant used for nematode proliferation control in soil, especially in soybean crops. Materials and methods: Seeds and leaves extracts were analysed by high‐performance liquid chromatography coupled to tandem mass spectrometry (HPLC‐MS/MS) for the identification of the compounds. Results: PAs and phenolic compounds could be identified in both samples based on the MS/MS fragmentation pattern. Molecular formulas of the annotated compounds were confirmed by ultra‐high‐performace liquid chromatography‐quadrupole time‐of‐flight (UHPLC‐QToF), and monocrotaline could also be confirmed by standard comparison. The quantification of monocrotaline was performed by HPLC‐MS/MS, resulting in 123 times higher monocrotaline content in seeds than in the leaves, which could explain its efficiency in combating nematode proliferation in soil. Conclusion: This was the first report of phenolic compounds in C. spectabilis. The current study highlights the importance of C. spectabilis for nematode control due to the presence of toxic PAs, and the employment of analytical techniques for identification and quantification of compounds present in the extracts. Crotalariaspectabilis is an important species used as pre‐plant cover for soybean crops to control the proliferation of endoparasitic nematodes. A validated method for pyrrolizidine alkaloids (PAs) and flavonoids identification and quantification of Crotalaria spectabilis seeds and leaves was performed by HPLC‐MS/MS, resulting in about 120 times higher monocrotaline content in seeds than in the leaves, which could explain its efficiency in combating nematode proliferation in soil. [ABSTRACT FROM AUTHOR]

Herbal drug‐induced liver injury has been reported worldwide and gained global attention. Thousands of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of herbal medicines and preparations containing pyrrolizidine alkaloids (PAs), which are natural phytotoxins globally distributed. And herbal medicines, such as Gynura japonica, are the current leading cause of PA‐induced HSOS. The present study aimed to reveal the mechanism underlying the hepatotoxicity of seneciphylline (Seph), a main PA in G. japonica. Results showed that Seph induced severe liver injury through apoptosis in mice (70 mg/kg Seph, orally) and primary mouse and human hepatocytes (5–50 μM Seph). Further research uncovered that Seph induced apoptosis by disrupting mitochondrial homeostasis, inducing mitochondrial depolarization, mitochondrial membrane potential (MMP) loss, and cytochrome c (Cyt c) release and activating c‐Jun N‐terminal kinase (JNK). The Seph‐induced apoptosis in hepatocytes could be alleviated by Mdivi‐1 (50 μM, a dynamin‐related protein 1 inhibitor), as well as SP600125 (25 μM, a specific JNK inhibitor) and ZVAD‐fmk (50 μM, a general caspase inhibitor). Moreover, the Seph‐induced MMP loss in hepatocytes was also rescued by Mdivi‐1. In conclusion, Seph induced liver toxicity via activating mitochondrial‐mediated apoptosis in mice and primary hepatocytes. Our results provide further information on Seph detoxification and herbal medicines containing Seph such as G. japonica. Seneciphylline (Seph), a main toxic pyrrolizidine alkaloid in Gynura japonica, induced severe liver injury via activating mitochondrial‐mediated apoptosis in mice and primary hepatocytes. Seph induced apoptosis by disrupting mitochondrial homeostasis, inducing mitochondrial depolarization, MMP loss, Cyt c release etc. The Seph‐induced apoptosis and MMP loss in hepatocytes could be alleviated by Mdivi‐1, a dynamin‐related protein 1 inhibitor, providing further information on Seph detoxification and herbal medicines containing Seph such as G. japonica. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids are the most widely distributed natural toxins, and pyrrolizidine alkaloid‐containing herbal medicines are probably the most common poisonous plants affecting humans. We reported pyrrolizidine alkaloid‐molecularly imprinted polymer solid‐phase microextraction for the selective adsorption of toxic pyrrolizidine alkaloids from herbal medicine. A sulfonic compound, sodium allylsulfonate, was chosen as the functional monomer to interact with pyrrolizidine alkaloids through strong ionic interaction. To avoid template leakage and for the aim of cost saving, a relatively cheap dummy template was used for the fabrication of molecularly imprinted polymer‐solid‐phase microextraction fibers. The obtained fibers showed selective adsorption ability for four pyrrolizidine alkaloids, including europine, echimidine, lasiocarpine, and heliotrine. The extraction parameters, such as extraction time, extraction temperature, shaking speed, elution solvent and elution time, were optimized. Then ultra high performance liquid chromatography with mass spectrometry coupled with molecularly imprinted polymer‐solid‐phase microextraction method was developed for the fast and efficient analysis of four pyrrolizidine alkaloids from the model herbal plant Farfarae Flos. The established method was validated and exhibited satisfactory accuracy and precision. The present method provides an innovative and fast analytical strategy for the determination of trace toxic pyrrolizidine alkaloids in complicated samples. [ABSTRACT FROM AUTHOR]

Background: This study evaluated the degradation of pyrrolizidine alkaloids (PAs) from eastern groundsel (Senecio vernalis) in grass silage prepared with different inoculants. Silages were produced from ryegrass with 230 g kg−1 dry matter (DM) content and mixed with eastern groundsel (9:1; w/w fresh matter basis) containing 5.5 g kg−1 DM PA. Treatments were: CON (untreated control), LP (3.0 × 105 cfu g−1Lactobacillus plantarum DSMZ 8862/8866) or LBLC (7.3 × 104 cfu g−1Lactobacillus buchneri LN40177 / Lactobacillus casei LC32909), and each of the treatments in combination with 30 g kg−1 molasses. Silages were prepared in glass jars and opened after 3, 10, and 90 days. Fermentation characteristics were determined and the PAs analyzed. Results: Although the levels of fermentation acids differed between treatments, results indicated good quality of all silages during 90 days. Significant time (P < 0.001) and treatment (P < 0.001) effects were observed for PAs. Concentrations of senecionine and seneciphylline decreased with molasses, declined over time, and were negatively correlated with lactic, propionic, and butyric acid, or with lactic and butyric acid in case of seneciphylline. In all silages, seneciphylline and senecionine N‐oxides were undetectable after 3 days, whereas senkirkine, the most abundant PA, remained stable. Conclusions: Silage prepared from grass contaminated with eastern groundsel still contained high PA levels, and was hence a potential health hazard. Molasses supplementation reduced concentrations of senecionine and seneciphylline, while the bacterial inoculants had no effect. Other potentially toxic PA metabolites were not analyzed in the present study and further research is needed. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Jacobaea aquatica (=Senecio aquaticus, Asteraceae), marsh ragwort, grows regionally in low management intensity wet grasslands in various European countries. The plant can be regarded as a noxious weed as it contains pyrrolizidine alkaloids (PAs), which cause health problems in livestock. In the Waldviertel region of Northern Austria, marsh ragwort has established dense populations as the fertilizing and production intensity of the meadows changed during the last decades. Ragwort biomass production and alkaloid concentration in J. aquatica were recorded during three consecutive years at three sites to study the effects of two treatments: cutting twice a year either early in June plus September, or in July plus August, respectively. Six PAs (Z‐erucifoline > senecionine > integerrimine > seneciphylline > acetyl‐erucifoline and acetyl‐seneciphylline) were evaluated. The alkaloid contents were highest in July and August when 600–1,400 mg/kg dry matter (DM) total PAs occurred in most ragwort samples. Consequently, the forage can easily be contaminated with 40–80 mg/kg DM of PAs and may present a health risk for domestic animals and also affect human health as these alkaloids are transferred into milk. [ABSTRACT FROM AUTHOR]

The beneficial and potentially harmful bioactive components in the seeds and seed oil of Trichodesma indicum L. (Boraginaceae) were investigated in the present study. The T. indicum seeds were rich in oil (29.0%), phenolic compounds (PC, 1881.2 mg per 100 g), and pyrrolizidine alkaloids (PA, 2,702,338 ng g−1). Seven PC were identified in T. indicum seeds by liquid chromatography‐quadrupole‐time‐of‐flight mass spectrometry system (LC‐Q‐TOF‐MS). Rosmarinic acid (67%) and isomers of salvianolic acid B/E/L (26%) were the main phenolics, while melitric acid A and sebestenoid C/D constituted 6% and 1%, respectively. Only a minor part of the total PC and PA was transferred from the seeds into the oil fraction during the extraction procedure (<0.03%). The T. indicum seed oil was predominated by the following polyunsaturated fatty acids (PUFA):linoleic (23.2%), γ‐linolenic (6.0%), α‐linolenic (26.8%), and stearidonic (5.9%). High levels were also observed for oleic (26.7%) and palmitic (7.4%) acids. Additionally, notable amounts of γ‐tocopherol (92% of total tocochromanols) and β‐sitosterol (53% of total sterols) were found in T. indicum seed oil. The total content of tocochromanols, sterols, and carotenoids in T. indicum seed oil was 102.7, 236.0, and 0.6 mg per 100 g oil, respectively. Among 10 detected hepatotoxic PA in T. indicum seeds, intermedine/lycopsamine/indicine (90.9%), intermedine N‐oxide (4.9%), and lycopsamine N‐oxide (4.1%) consisted 99.9% of the total PA concentration. The T. indicum seeds should be used carefully due to the presence of PA. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Pro‐toxic dehydropyrrolizidine alkaloids are associated with liver disease in humans. The potential for long‐term, low‐level or intermittent exposures to cause or contribute to chronically‐developing diseases is of international concern. Eupatorium perfoliatum is a medicinal herb referred to as boneset. While the presence of dehydropyrrolizidine alkaloids in some Eupatorium species is well‐established, reports on Eupatorium perfoliatum are scant and contradictory. Objective: To investigate the presence of dehydropyrrolizidine alkaloids in a survey of boneset samples and related alcoholic tinctures, and hot water infusions and decoctions. Methods: Methanol, hot water or aqueous ethanol extracts of Eupatorium perfoliatum and three closely‐related species were subjected to HPLC‐ESI(+)MS and MS/MS analysis using three complementary column methods. Dehydropyrrolizidine alkaloids were identified from their MS data and comparison with standards. Results: Forty‐nine samples of Eupatorium perfoliatum were shown to contain dehydropyrrolizidine alkaloids (0.0002–0.07% w/w), the majority dominated by lycopsamine and intermedine, their N‐oxides and acetylated derivatives. Alcoholic tinctures and hot water infusions and decoctions had high concentrations of the alkaloids. Different chemotypes, hybridisation or contamination of some Eupatorium perfoliatum samples with related species were suggested by the co‐presence of retronecine‐ and heliotridine‐based alkaloids. Conclusions: Sampling issues, low and high alkaloid chemotypes of Eupatorium perfoliatum or interspecies hybridization could cause the wide variation in dehydropyrrolizidine alkaloid concentrations or the different profiles observed. Concerns associated with dehydropyrrolizidine alkaloids provide a compelling reason for preclusive caution until further research can better define the toxicity and carcinogenicity of the dehydropyrrolizidine alkaloid content of Eupatorium perfoliatum. [Correction added on 12 July 2018, after first online publication: The ‘Conclusions’ section in the abstract has been added.] [ABSTRACT FROM AUTHOR]

Abstract: Background and Aims: Hematopoietic stem cell transplantation related hepatic vein occlusive disease (HSCT‐HVOD) has been researched extensively; however, little is known about the clinical features and treatment of pyrrolizidine alkaloid‐induced HVOD (PA‐HVOD). This retrospective single centre study examined the clinical and laboratory characteristics of 108 patients with acute PA‐HVOD and explored the efficacy of anticoagulation and TIPS therapy. Methods: The study included 108 consecutive patients with PA‐HVOD between July 2008 and June 2016. The clinical manifestations and the results of laboratory and imaging tests were evaluated. The survival rates of patients treated with different approaches were recorded. Results: Serum total bilirubin was <34.2 μmol/L (2 mg/dL) in approximately 40% of patients. More than 90% of patients were presented with hepatomegaly, uneven liver perfusion in the balance phase, compressive stenosis of the hepatic segmental inferior vena cava and decreased peak velocity of portal vein blood flow. Severe portal hypertension was observed in all patients undergoing HVPG examination or TIPS operation. Anticoagulation therapy with low molecular weight heparin combined with warfarin was significantly more effective than liver protection and supportive therapy, and TIPS further improved the prognosis of patients who did not respond to anticoagulation therapy. The total effective rate of the anticoagulation‐TIPS ladder therapeutic strategy was 91%. Conclusions: Patients with PA‐HVOD had different characteristics than those with HSCT‐HVOD. Anticoagulation and TIPS treatment may be effective for patients with PA‐HVOD. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids are natural molecules playing important roles in different biochemical processes in nature and in humans. In this work, the electron ionization mass spectrum of retronecine, an alkaloid molecule found in plants, was investigated computationally. Its mass spectrum can be characterized by three main fragment ions having the following m/z ratios: 111, 94, and 80. In order to rationalize the mass spectrum, minima and transition state geometries were computed using density functional theory. It was showed that the dissociation process includes an aromatization of the originally five‐membered ring of retronecine converted into a six‐membered ring compound. A fragmentation pathway mechanism involving dissociation activation barriers that are easily overcome by the initial ionization energy was found. From the computed quantum chemical geometric, atomic charges, and energetic parameters, the abundance of each ion in the mass spectrum of retronecine was discussed. [ABSTRACT FROM AUTHOR]

The high‐resolution crystal structure of the flavin‐dependent monooxygenase (FMO) from the African locust Zonocerus variegatus is presented and the kinetics of structure‐based protein variants are discussed. Z. variegatus expresses three flavin‐dependent monooxygenase (ZvFMO) isoforms which contribute to a counterstrategy against pyrrolizidine alkaloids (PAs). PAs are protoxic compounds produced by some angiosperm lineages as a chemical defence against herbivores. N‐Oxygenation of PAs and the accumulation of PA N‐oxides within their haemolymph result in two evolutionary advantages for these insects: (i) they circumvent the defence mechanism of their food plants and (ii) they can use PA N‐oxides to protect themselves against predators, which cannot cope with the toxic PAs. Despite a high degree of sequence identity and a similar substrate spectrum, the three ZvFMO isoforms differ greatly in enzyme activity. Here, the crystal structure of the Z. variegatus PA N‐oxygenase (ZvPNO), the most active ZvFMO isoform, is reported at 1.6 Å resolution together with kinetic studies of a second isoform, ZvFMOa. This is the first available crystal structure of an FMO from class B (of six different FMO subclasses, A–F) within the family of flavin‐dependent monooxygenases that originates from a more highly developed organism than yeast. Despite the differences in sequence between family members, their overall structure is very similar. This indicates the need for high conservation of the three‐dimensional structure for this type of reaction throughout all kingdoms of life. Nevertheless, this structure provides the closest relative to the human enzyme that is currently available for modelling studies. Of note, the crystal structure of ZvPNO reveals a unique dimeric arrangement as well as small conformational changes within the active site that have not been observed before. A newly observed kink within helix α8 close to the substrate‐binding path might indicate a potential mechanism for product release. The data show that even single amino‐acid exchanges in the substrate‐entry path, rather than the binding site, have a significant impact on the specific enzyme activity of the isoforms. [ABSTRACT FROM AUTHOR]

Premise of the Study: Understanding the phylogenetic distribution of defensive plant secondary metabolites is essential to the macroevolutionary study of chemically mediated plant–animal interactions. The chemical ecology of pyrrolizidine alkaloids (PAs) has been extensively studied in a number of plant–herbivore systems, including Apocynaceae (the milkweed and dogbane family) and Danainae (the milkweed and clearwing butterflies). A systematic survey is necessary to establish a detailed understanding of their occurrence across Apocynaceae. A survey of this species‐rich, mainly tropical and subtropical family will rely heavily on small tissue samples removed from herbarium specimens, some of which will be very old and/or preserved with alcohols or mercuric chloride. Methods: We optimized PA extraction methods from small leaf fragments of recently collected silica‐dried leaves of the PA‐positive Echites umbellatus, varying crushing and extraction time. We then applied our optimized method to leaf fragments from 70–167‐year‐old herbarium specimens of E. umbellatus. To simulate the effect of alcohol treatment on PA detectability in herbarium specimens, we incubated freshly collected leaves of the PA‐positive Parsonsia alboflavescens in three different alcohols before drying and compared PA recovery to freshly dried controls. PAs were quantified using high‐performance liquid chromatography–mass spectrometry analysis. X‐ray fluorescence was used to identify mercury‐containing specimens. Results: Fifteen seconds of leaf crushing followed by 24 h of extraction were optimal for PA free‐base and N‐oxide recovery. This method yielded ~50‐fold greater PA recovery than prior methods. Half of the herbarium specimens (13 of 23), including the oldest, tested positive for PAs; leaf age did not correlate with success in PA extraction. Treatment of fresh leaves with alcohol before drying did not diminish PA recovery; mercury was observed in both PA‐positive and PA‐negative specimens. Conclusions: PAs can be reliably detected in small tissue samples from herbarium specimens up to 167 years old, including specimens that had been treated with alcohol or mercury salts. The variability of PA presence among herbarium specimens of E. umbellatus indicates that multiple specimens will need to be tested before a particular species is determined to lack PAs. [ABSTRACT FROM AUTHOR]

Abstract: Farfarae Flos, the dried flower buds of Tussilago farfara L., is usually used to treat coughs, bronchitic and asthmatic conditions as an important traditional Chinese medicine. Tussilagone and methl butyric acid tussilagin ester are seen as representatives of two kinds of active substances. In addition, the pyrrolizidine alkaloids, mainly senkirkine and senecionine, present in the herb can be hepatoxic. In this study, a rapid and sensitive ultra‐high‐performance liquid chromatography coupled with hybrid triple quadrupole time‐of‐flight mass spectrometry method was successfully applied to identify the metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine. A total of 35, 37, 18 and nine metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine in rats were tentatively identified. Hydrolysis, oxidation, reduction and demethylation were the major metabolic reactions for tussilagone and methl butyric acid tussilagin ester. The main biotransformation routes of senkirkine and senecionine were identified as demethylation, N‐methylation, oxidation and reduction. This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways might provide further understanding of the metabolic fate of the chemical constituents after oral administration of Farfarae Flos extract in vivo. [ABSTRACT FROM AUTHOR]

Introduction Pyrrolizidine alkaloids (PAs) are secondary plant metabolites with considerable hepatoxic, tumorigenic and genotoxic potential. For separation, reversed phase chromatography is commonly used because of its excellent compatibility with detection by mass spectrometry. However, reversed phase chromatography has a low selectivity for PAs. Objective The objective of this work was to investigate the suitability of cation exchange chromatography for separation of PAs and to develop a rapid method for quantification of jacobine in Crassocephalum crepidioides that is suitable for analysis of huge sample numbers as required for mutant screening procedures. Results We demonstrate that cation exchange chromatography offers excellent selectivity for PAs allowing their separation from most other plant metabolites. Due to the high selectivity, plant extracts can be directly analysed after simple sample preparation. Detection with UV at 200 nm instead of mass spectrometry can be applied, which makes the method very simple and cost-effective. The recovery rate of the method exceeded 95%, the intra-day and inter-day standard deviations were below 7% and the limit of detection and quantification were 1 mg/kg and 3 mg/kg, respectively. Conclusion The developed method is sufficiently sensitive for reproducible detection of jacobine in C. crepidioides. Simple sample preparation and rapid separation allows for quantification of jacobine in plant material in a high-throughput manner. Thus, the method is suitable for genetic screenings and may be applicable for other plant species, for instance Jacobaea maritima. In addition, our results show that C. crepidioides cannot be considered safe for human consumption. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Introduction Crotalaria sessiliflora L. is a Chinese traditional herb for treatment of cutaneum carcinoma and cervical carcinoma. In addition to monocrotaline, coexisting pyrrolizidine alkaloids (PAs) also require further quantification for quality control and pharmaceutical uses of the herb. Objective To establish a UPLC-Q-Orbitrap/MS method of simultaneous determination of coexisting PAs with same parent structure for quality control and comprehensive researches of Crotalaria sessiliflora L. Methodology PAs in Crotalaria sessiliflora L. were analysed by UPLC-Q-Orbitrap/MS method. Coexisting PAs were identified by mass data of full MS-dd-MS2 based on the characteristic fragmentation pattern and necine-core structure. Moreover, quantification of PAs was conducted by parallel reaction monitoring (PRM) mode using m/ z 138, m/ z 120 and m/ z 94 from identical necine-core structure as quantitative ions with single monocrotaline standard for accurate calibration. Results Five PAs, named monocrotaline, retrorsine, senecionine, integerrimine, O-9-angeloylretronecine, were indentified and confirmed. Quantitative ions of m/ z 138, m/ z 120 and m/ z 94 were used for quantification of PAs containing the necine-core structure in Crotalaria sessiliflora L . The results demonstrated that contents, precision and recoveries of the five PAs mentioned earlier were respectively 3.307-30.35 μg/g, 1.1-4.5% and 88.91-92.33% while using m/ z 120 as the best quantitative ion. Conclusion The UPLC-Q-Orbitrap/MS method was established for simultaneous determination of five PAs in Crotalaria sessiliflora L. without all corresponding standards, and was proved that it was simple, convenient and effective for comprehensive quality control and pharmaceutical uses. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Echiteae (Apocynaceae) comprises about 200 species divided among 19 genera, 16 of which are restricted to the Neotropics, the other 3 to the Paleotropics. There are two large genera, Prestonia and Parsonsia, in the Neotropics and Paleotropics, respectively, whereas three-fourths of the genera contain only one to four species. In this study DNA of 82 species was extracted and amplified for four molecular markers: three plastid (trnL intron + trnL-trnF intergenic spacer, rpl16 intron, matK + 5'/ 3' trnK intron) and the nuclear ribosomal region ITS. The ingroup comprised 70 species from 17 genera in the tribe, and included representatives of all subtribes; the outgroup included 5 species each from the two putatively most closely related tribes, Odontadenieae and Mesechiteae, as well as 2 rooting species. Phylogenetic analyses were conducted using Bayesian inference and maximum likelihood approaches. Echiteae and four of its subtribes (Echitinae, Parsonsiinae, Peltastinae, Prestoniinae) as currently circumscribed were shown to be non-monophyletic. The fifth subtribe, Pentalinoninae, though monophyletic, does not belong to Echiteae; it was resolved as sister to a clade of Odontadenieae, where it fits better, since in both the characteristic secondary compounds are cardenolides. Prestonia and Temnadenia were resolved as polyphyletic, with P. riedelii grouped with Rhodocalyx and T. ornata nested within Prestonia. Fernaldia is nested within Echites and Peltastes is nested in Macropharynx. Fourteen genera are maintained. The new subtribe Laubertiinae is proposed, as are the combinations resulting from the merging of Fernaldia and Peltastes and the transfer of P. riedelii and T. ornata to Rhodocalyx and Prestonia, respectively. In the ancestors of Echiteae, we hypothesize that an evolutionary shift took place in which steroidal alkaloids and/or cardenolides, characteristic for apocynoids, were replaced by parsonsine-type pyrrolizidine alkaloids as the predominant defense compounds. [ABSTRACT FROM AUTHOR]

The physico-chemical background of alkaloid allocation within plants is outlined and discussed exemplarily for pyrrolizidine alkaloids ( PAs) and nicotine. The trigger for this discourse is the finding that, for example, PAs, which are taken up from the soil, are translocated in the xylem, whereas - when genuinely present in plants - they are allocated as N-oxides via phloem. Special emphasis is put on the impact of different pH values in certain compartments, as this entails significant changes in the relative lipophilic character of alkaloids: tertiary alkaloids diffuse readily through biomembranes, while the corresponding protonated alkaloids are retained in acidic compartments, i.e. vacuoles or xylem. Therefore, this phenomenon, well known as the 'ion trap mechanism', is also relevant for long-distance transport of alkaloids. Any efficient allocation of typical tertiary alkaloids within the phloem can thus be excluded. In contrast, due to their strongly increased hydrophilic properties, alkaloid- N-oxides or quarternary alkaloids cannot diffuse through biomembranes and, consequently, would be retained in the acidic xylem during translocation. The major aim of this paper is to sharpen the mind for the chemical peculiarities of alkaloids and to consider them adequately in forthcoming investigations on allocation of alkaloids. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids are the toxic components in Tussilago farfara L. Due to the lack of standard substances for quantitative analysis and traces of pyrrolizidine alkaloids in total alkaloids, the full quality control of Tussilago farfara L has been limited. In this study, we aimed to solve the difficulty of determination of pyrrolizidine alkaloids and identify more components in the total alkaloids. An on-line preconcentration method has been applied to improve determining sensitivity of pyrrolizidine alkaloids in Tussilago farfara L. in which included field-amplified sample stacking and sweeping in micellar electrokinetic capillary chromatography. The main parameters that affected separation and stacking efficiency were investigated in details. Under the optimal conditions, the sensitivity enhancement factors obtained by the developed method for the analytes were from 15- to 12-fold, the limits of detection of senkirkine and senecionine were 2∼5 μg/L. Senkirkine and senecionine have been detected in alkaloids ( c) of Tussilago farfara L, along ferulic acid methyl ester and methyl caffeate. The developed method was also applied to the analysis of acid extraction ( a) of Tussilago farfara L, and senkirkine could be detected directly. The results indicated that the developed method is feasible for the analysis of pyrrolizidine alkaloids in Tussilago farfara L with good recoveries. [ABSTRACT FROM AUTHOR]

Bacterial defense mechanisms have evolved to protect bacteria against predation by nematodes, predatory bacteria, or amoebae. We identified novel bacterial alkaloids (pyreudiones A-D) that protect the producer, Pseudomonas fluorescens HKI0770, against amoebal predation. Isolation, structure elucidation, total synthesis, and a proposed biosynthetic pathway for these structures are presented. The generation of P. fluorescens gene-deletion mutants unable to produce pyreudiones rendered the bacterium edible to a variety of soil-dwelling amoebae. [ABSTRACT FROM AUTHOR]

The (+)- as well as the (−)-enantiomer of the pyrrolizidine alkaloid xenovenine were prepared within five steps with 17 and 30% overall yields, respectively, in optically pure form, >99% ee as well as >99% de. In the asymmetric key step a transaminase performed a regio- and stereoselective monoamination of a triketone. By employing two enantiocomplementary transaminases from Arthrobacter sp. both enantiomers were accessible. The triketone was readily prepared via two steps starting from commercially available, achiral 2-( n-heptyl)furan. In the final catalytic hydrogenation step, the newly introduced chiral centre directed hydrogen addition to form preferentially the desired (5 Z,8 E)-diastereomer. The regio- and stereoselective amination of a single ketone moiety out of three allowed the performance of the shortest and highest yielding total synthesis of the bicyclic showcase pyrrolizidine alkaloid without the need for protecting strategies. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are toxins that are exclusively biosynthesized by plants and are commonly present in foods and herbs. PAs are usually associated with poisoning events in livestock and human beings. The aim of the present study was to evaluate the behavioral and neurochemical effects of prenatal exposure to PA integerrimine N-oxide of rats in adulthood. Pregnant Wistar rats received integerrimine N-oxide from the butanolic residue of Senecio brasiliensis by gavage on gestational days 6–20 at doses of 3, 6 and 9 mg/kg. During adulthood of the offspring, the following behavioral tests were performed: open-field, plus-maze, forced swimming, catalepsy and stereotypy. Histological analyses and monoamine levels were measured. Male offspring from dams that were exposed to 9 mg/kg showed an increase in locomotion in the open-field test, an increased frequency of entries and time spent in open arms in elevated plus-maze test, as well as decreased swimming time. In the female offspring from dams that were exposed to 9 mg/kg, there was an increased time of climbing in forced swimming and intensity of stereotyped behavior. The histological study indicates an increase in the number of multinucleated cells in the liver (6 and 9 mg/kg). In neurotransmitter analysis, specifically in the striatum, we observed change in dopamine and serotonin levels in the middle dose. Thus, our results indicate that prenatal exposure to integerrimine N-oxide changed behavior in adulthood and neurotransmitter levels in the striatum. Our results agree with previous studies, which showed that integerrimine N-oxide impaired physical and neurobehavioral development in childhood that can persist until adulthood. [ABSTRACT FROM AUTHOR]

In many countries, including the United States, herbal supplements, tisanes and vegetable products, including traditional Chinese medicines, are largely unregulated and their content is not registered, monitored or verified. Consequently, potent plant toxins including dehydropyrrolizidine alkaloids and other potential carcinogens can contaminate these products. As herbal and food supplement producers are left to their own means to determine the safety and purity of their products prior to marketing, disturbingly often good marketing practices currently in place are ignored and content is largely undocumented. Historical examples of poisoning and health issues relating to plant material containing dehydopyrrolizidine alkaloids and aristolochic acids were used as examples to demonstrate the risk and potential toxicity of herbal products, food supplements, or traditional medicines. More work is needed to educate consumers of the potential risk and require the industry to be more responsible to verify the content and insure the safety of their products. Published 2015. This article is a U.S. Government work and is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram-negative and Gram-positive bacteria indicates an important biological function in bacteria. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are a group of natural products with important biological activities. The discovery and characterization of the multifunctional FAD-dependent enzyme LgnC is now described. The enzyme is shown to convert indolizidine intermediates into pyrrolizidines through an unusual ring expansion/contraction mechanism, and catalyze the biosynthesis of new bacterial PAs, the so-called legonmycins. By genome-driven analysis, heterologous expression, and gene inactivation, the legonmycins were also shown to originate from non-ribosomal peptide synthetases (NRPSs). The biosynthetic origin of bacterial PAs has thus been disclosed for the first time. [ABSTRACT FROM AUTHOR]

Plant-soil feedback (PSF) effects on plant performance strongly depend on the plant species that conditioned the soil. Recent studies have shown that PSF can change above-ground plant-insect interactions via soil-mediated changes in plant quality, but whether these effects depend on species-specific soil conditioning is unknown. We examined how PSF effects of several plant species influence above-ground plant-aphid interactions., We grew ragwort ( Jacobaea vulgaris) in field soil conditioned specifically by 10 plant species, belonging to three functional groups (grasses, forbs and legumes), in a multispecies mixture of the conditioned soils and in control (unconditioned) field soil. We measured plant biomass, concentrations of primary (amino acids) and secondary (pyrrolizidine alkaloids) metabolites in phloem exudates, and performance of the generalist aphid Brachycaudus cardui and the specialist Aphis jacobaeae., We observed that plant species, via species-specific effects on soil fungal communities, exerted unique plant-soil effects on J. vulgaris biomass, amino acid concentrations in phloem exudates and aphid performance. The direction and magnitude of the species-specific PSF effects on aphid performance differed between both aphid species. PSF effects on soil fungal communities, plant biomass and A. jacobaeae performance also differed between grasses, forbs and legumes, with soil conditioning by forbs resulting in lowest plant biomass and aphid performance., Synthesis. Our study provides novel evidence that PSF effects on above-ground plant-insect interactions are highly species specific. Our results add a new dimension to the rapidly developing research fields of PSF and above-below-ground interactions, and highlights that these fields are tightly linked. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids are highly hepatotoxic natural chemicals that produce irreversible chronic and acute hepatotoxic effects on human beings. Purification of large amounts of pyrrolizidine alkaloids is necessary for toxicity studies. In this study, an efficient method for targeted analysis and purification of pyrrolizidine alkaloid cis/ trans isomers from herbal materials was developed for the first time. Targeted analysis of the hepatotoxic pyrrolizidine alkaloids was performed by liquid chromatography with tandem mass spectrometry (precursor ion scan and daughter ion scan), and the purification of pyrrolizidine alkaloids was achieved with a mass-directed auto purification system. The extraction and preparative liquid chromatography conditions were optimized. The developed method was applied to analysis of Gynura japonica (Thunb.) Juel., a herbal medicine traditionally used for detumescence and relieving pain but is potentially hepatotoxic as it contains pyrrolizidine alkaloids. Twelve pyrrolizidine alkaloids (six cis/ trans isomer pairs) were identified with reference compounds or characterized by liquid chromatography with tandem mass spectrometry, and five individual pyrrolizidine alkaloids, including ( E)-seneciphylline, seneciphylline, integerrimine, senecionine, and seneciphyllinine, were prepared from G. japonica roots with high efficiency. The results of this work provide a new technique for the preparation of large amounts of pyrrolizidine alkaloid reference substances, which will also benefit toxicological studies of pyrrolizidine alkaloids and treatments for pyrrolizidine alkaloid-induced toxicity. [ABSTRACT FROM AUTHOR]

Plants that contain pyrrolizidine alkaloids (PAs) have been reported as contaminants of pastures and food, as well as being used in herbal medicine. PAs are responsible for poisoning events in livestock and human beings. The aim of this present study was to evaluate effects of prenatal exposure to integerrimine N-oxide, the main PA found in the butanolic residue (BR) of Senecio brasiliensis , on both physical and behavioral parameters of Wistar rat offspring. The toxicity and maternal behavior were also evaluated. For this, pregnant Wistar rats received integerrimine N-oxide from the BR of Senecio brasiliensis , by gavage, on gestational days 6-20 (during organogenesis and fetal development period) at doses of 3, 6 and 9 mg/kg. During treatment, maternal body weight gain, and food and water intake were evaluated. After parturition, maternal behavior and aggressive maternal behavior were analyzed. In addition, physical development and behavioral assessments were observed in both male and female pups. Results showed that prenatal exposure to integerrimine N-oxide of S. brasiliensis induced maternal toxicity, impairment in maternal behavior and aggressive maternal behavior, mainly in the highest dose group. Between sexes comparison of pups showed loss of body weight, delayed physical development such as pinna detachment, hair growth, eruption of incisor teeth, eye and vaginal openings. These pups also showed a delay of palmar grasp, surface righting reflex, negative geotaxis and auditory startle reflexes. Thus, prenatal exposure to integerrimine N-oxide induces maternal toxicity, impairment of maternal care and delayed in physical and behavioral development of the offspring. [ABSTRACT FROM AUTHOR]

Boraginaceous 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). [ABSTRACT FROM AUTHOR]

In the last decades, cases of poisoning due to herbal medicines have occurred in many countries; Chinese herbal medicines (CHMs) are occasionally involved. The experience gained from traditional use is efficient to detect immediate or near-immediate relationship between administration and toxic effects but is quite unlikely to detect medium- to long-term toxicities; thorough investigations of herbal medicines (toxicity assessments, active pharmacovigilance) appear then essential for their safe use. Genotoxicity is an especially insidious toxicity that may result in carcinoma development years after exposure; it can arise from multiple compounds, with or without metabolic activation. The present work reviews traditional CHMs and phytochemicals that have been shown to present a genotoxic hazard. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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)/H2O (85/10/5 v:v:v) or after extraction with MeOH/H2O (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. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Recent studies revealed that plant-soil biotic interactions may cause changes in above-ground plant chemistry. It would be a new step in below-ground-above-ground interaction research if such above-ground chemistry changes could be efficiently detected. Here we test how hyperspectral reflectance may be used to study such plant-soil biotic interactions in a nondestructive and rapid way., The native plant species Jacobaea vulgaris and Jacobaea erucifolius, and the exotic invader Senecio inaequidens were grown in different soil biotic conditions. Biomass, chemical content and shoot reflectance between 400 and 2500 nm wavelengths were determined. The data were analysed with multivariate statistics., Exposing the plants to soil biota enhanced the content of defence compounds. The highest increase (400%) was observed for the exotic invader S. inaequidens. Chemical and spectral data enabled plant species to be classified with an accuracy > 85%. Plants grown in different soil conditions were classified with 50-60% correctness., Our data suggest that soil microorganisms can affect plant chemistry and spectral reflectance. Further studies should test the potential to study plant-soil biotic interactions in the field. Such techniques could help to monitor, among other things, where invasive exotic plant species develop biotic resistance or the development of hotspots of crop soil diseases. [ABSTRACT FROM AUTHOR]

The article presents a study on the protective effects of epidermal growth factor (EGF) on mitochondrial-mediated apoptosis in hepatocytes induced by clivorine. It mentions that the study investigated clivorine, an otonecine-type PA from a traditional Chinese medicine Ligularia hodgsonii Hook, to examine the EGF protection against its hepatoxicity. It incoporates the finding of previous related studies regarding the effects of reversed clivorine-induced cytotoxicity on human liver as observed through cell viability assay and cell morphology. The results show that the decrease in antiapoptotic Bcl-xL protein induced by clivorine is inhibited by EGF. Furthermore, it concludes that EGF protection is carried out by the modulation of mitochondrial apoptotic signal pathway.