Insight into pyrrolizidine alkaloids degradation and the chemical structures of their degradation products using ultra high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometry.

Pyrrolizidine alkaloids (PAs), released into the environment by donor plants, are absorbed by crops or transported by animals, posing a global food safety concern. Photolysis is an effective way to eliminate harmful substances in the environment or food. Photolysis happens as PAs move among plants, environment and crops. In this study, we first investigated the photolysis and hydrolysis of 15 PAs and identified their degradation products via ultra-high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometry. PAs were degraded under UV radiation but minimally affected by visible light from a xenon lamp, and solvent pH had little impact on their photolysis. PAs were stable in neutral and acidic solutions but degraded by 50% within 24 h in alkaline conditions. The degradation products of PAs were mainly PAs/PANOs isomers and some minor byproducts. Cytotoxicity and computational analysis revealed isomers had similar toxicity, with minor products being less toxic. This study is a precursor for revealing the potential PAs degradation dynamics in the environment and food products, providing a reference for systematic evaluations of potential health and ecological risks of their degradation products.

Pyrrolizidine Alkaloids in Foods, Herbal Drugs, and Food Supplements: Chemistry, Metabolism, Toxicological Significance, Analytical Methods, Occurrence, and Challenges for Future.

Consumers are increasingly seeking natural alternatives to chemical compounds, including the use of dried aromatic plants as seasonings instead of salt. However, the presence of pyrrolizidine alkaloids (PAs) in food supplements and dried plants has become a concern because of their link to liver diseases and their classification as carcinogenic by the International Agency for Research on Cancer (IARC). Despite European Union (EU) Regulation (EU) 2023/915, non-compliance issues persist, as indicated by alerts on the Rapid Alert System for Food and Feed (RASFF) portal. Analyzing PAs poses a challenge because of their diverse chemical structures and low concentrations in these products, necessitating highly sensitive analytical methods. Despite these challenges, ongoing advancements in analytical techniques coupled with effective sampling and extraction strategies offer the potential to enhance safety measures. These developments aim to minimize consumer exposure to PAs and safeguard their health while addressing the growing demand for natural alternatives in the marketplace.

The toxicokinetic and metabolism of structurally diverse pyrrolizidine alkaloids in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are a group of phytotoxins present in about 3% of flowering plants worldwide. Ingestion of PA-containing herbal products may lead to hepatotoxicity. Notably, the toxicokinetic (TK) behaviors, especially pyrrole-protein adducts (PPAs) having the same structure but generated from metabolic activation of different PAs, significantly affect the toxicity of structurally diverse PAs, therefore studying them in their pure form is preferable to extracts to stratify toxic potency of different PAs co-existing in herbal extracts. However, previous studies mainly focus on the establishment of TK profiles of the intact PAs, revealing less or no kinetic information on the main PA metabolites (PA N-oxides) and PPAs which mediate PA-induced hepatotoxicity. In this study, PPA was measured as the biomarker of PA exposure and PA-induced toxicity. AIM OF STUDY: This study aims to investigate the TK difference between structurally diverse PAs of retronecine-type PAs: retrorsine (RTS) and monocrotaline (MCT), and otonecine-type PA: clivorine (CLI), and their toxicity-related metabolite PPAs and PA N-oxides, the main metabolite of retronecine-type PAs, for the establishment of a more accurate risk assessment of PAs exposure. MATERIALS AND METHODS: The TK studies were conducted using rats through intravenous (i.v.) or oral (p.o.) administration of PAs at 20 mg/kg. The main TK parameters of PAs and PA N-oxides were determined from plasma concentration-time profiles, and the kinetic profiles of PPAs were assessed from both plasma and erythrocyte concentration-time profiles. RESULTS: MCT demonstrated the slowest but the highest extent of absorption among the three PAs, while RTS demonstrated a similar absorption rate with a lower extent than CLI. For elimination, MCT demonstrated a similar elimination rate as RTS but the lowest extent of elimination among the three PAs, and CLI exhibited significantly faster elimination than MCT and RTS. Moreover, the formation of PA N-oxide, which only occurs in retronecine-type PAs, was remarkably less in MCT-treated rats compared to RTS-treated ones. Of note, the retronecine-type RTS and MCT induced more PPAs via p.o. than i.v. administration route, whereas the otonecine-type CLI showed the opposite trend. CONCLUSION: Dramatic TK differences, including not only PAs but also PA N-oxides and the derived protein adduct PPAs, were found among structurally diverse PAs in rats, laying the basis for varied hepatotoxic potencies induced by different PA-containing herbal products. Notably, our findings for the first time uncovered that oral administration of retronecine-type PAs might cause severer toxicity compared with the intravenous route, which warrants further in-depth exploration.

Miniaturized Analytical Strategy Based on µ-SPEed for Monitoring the Occurrence of Pyrrolizidine and Tropane Alkaloids in Honey.

Currently, the analysis of trace-level contaminants in food must be addressed following green analytical chemistry principles and with a commitment to the sustainable development goals. Accordingly, a sustainable and ecofriendly microextraction procedure based on µ-SPEed followed by ultrahigh liquid chromatography coupled to ion-trap tandem mass spectrometry analysis was developed to determine the occurrence of pyrrolizidine and tropane alkaloids in honey samples. The µ-SPEed procedure took approximately 3 min per sample, using only 100 µL of organic solvent and 300 µL of diluted sample. The method was properly validated (overall recoveries 72-100% and precision RSD values ≤15%), and its greenness was scored at 0.61 out of 1. The method was applied to different honey samples, showing overall contamination levels from 32 to 177 µg/kg of these alkaloids. Atropine was found in all the samples, whereas retrorsine N-oxide, lasiocarpine, echimidine, and echimidine N-oxide were the main pyrrolizidine alkaloids in the samples analyzed.

Hepatotoxicity screening and ranking of structurally different pyrrolizidine alkaloids in zebrafish.

Pyrrolizidine alkaloids (PAs) are phytotoxins distributed in ∼6000 plant species. PA-contaminated/containing foodstuffs/herbs/supplements pose a potential threat to human health. Various regulatory authorities established different PA margins of exposure assuming an equal hepatotoxic potency of structurally diverse PAs, although they exhibit different toxic potencies. Therefore, understanding hepatotoxic potencies of different PAs would facilitate a more appropriate risk assessment of PA exposure. In this study, a zebrafish model, which mimics physiological processes of absorption, distribution, metabolism, and excretion, was selected to evaluate acute hepatotoxic potency of different PAs (7 PAs and 2 PA N-oxides) and explore possible physiological pathways involved in PA-induced hepatotoxicity. After 6 h oral administration, PAs caused distinct structure-dependent hepatotoxicity with a series of biochemical and histological changes in zebrafish. Based on the measured toxicological endpoints, the relative toxic potency order of different PAs was derived as lasiocarpine âˆ¼ retrorsine > monocrotaline > riddelliine > clivorine > heliotrine > retrorsine N-oxide âˆ¼ riddelliine N-oxide≫>platyphyline. Further, compared to control group, different upregulation/downregulation of mRNA expression in PA-treated groups indicated that inflammation, apoptosis, and steatosis were involved in PA-induced hepatotoxicity in zebrafish. These findings demonstrate that zebrafish model is useful for screening and ranking hepatotoxicity of PAs with diverse structures, which would facilitate the more accurate risk assessment of PA exposure.

Structural annotation, semi-quantification and toxicity prediction of pyrrolizidine alkaloids from functional food: In silico and molecular networking strategy.

Many traditional Chinese herbs contain pyrrolizidine alkaloids (PAs), which have been reported to be toxic to livestock and humans. However, the lack of PAs standards makes it difficult to effectively conduct a risk assessment in the varied components of traditional Chinese medicine. It is necessary to propose a suitable strategy to obtain the representative occurrence data of PAs in complex systems. A comprehensive approach for annotating the structures, concentration, and mutagenicity of PAs in three Chinese herbs has been proposed in this article. First, feature-based molecular networking (FBMN) combined with network annotation propagation (NAP) on the Global Natural Products Social Molecular Networking web platform speeds up the process of annotating PAs found in Chinese herbs. Second, a semi-quantitative prediction model based on the quantitative structure and ionization intensity relationship (QSIIR) is used to forecast the amounts of PAs in complex substrates. Finally, the T.E.S.T. was used to provide predictions regarding the mutagenicity of annotated PAs. The goal of this study was to develop a strategy for combining the results of several computer models for PA screening to conduct a comprehensive analysis of PAs, which is a crucial step in risk assessment of unknown PAs in traditional Chinese herbal preparations.

UGT76F1 glycosylates an isomer of the C7-necic acid component of pyrrolizidine alkaloids in Arabidopsis thaliana.

Identification of unknown metabolites and their biosynthetic genes is an active research area in plant specialized metabolism. By following a gene-metabolite association from a genome-wide association study of Arabidopsis stem metabolites, we report a previously unknown metabolite, 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid glucoside, and demonstrated that UGT76F1 is responsible for its production in Arabidopsis. The chemical structure of the glucoside was determined by a series of analyses, including tandem MS, acid and base hydrolysis, and NMR spectrometry. T-DNA knockout mutants of UGT76F1 are devoid of the glucoside but accumulate increased levels of the aglycone. 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid is structurally related to the C7-necic acid component of lycopsamine-type pyrrolizidine alkaloids such as trachelantic acid and viridifloric acid. Feeding norvaline greatly enhances the accumulation of 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid glucoside in wild-type but not the UGT76F1 knockout mutant plants, providing evidence for an orthologous C7-necic acid biosynthetic pathway in Arabidopsis despite the apparent lack of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids in borage (Borago officinalis): Comprehensive profiling and development of a validated LC-MS/MS method for quantification.

Pyrrolizidine alkaloids (PA) from borage (Borago officinalis) consumed as herb and tea, may pose a food safety risk. Therefore, the European Union (EU) set maximum levels of PA in borage, among other foodstuffs, which are applicable since July 1st, 2022. Here, a comprehensive LC-MS/MS based profiling of PA and their N-oxides (PANO) in B. officinalis leaves is presented. Based on these results a targeted, quantitative LC-MS/MS method for the determination of individual PA/PANO present in borage was developed. Chromatographic separation was achieved for all PA/PANO detected in B. officinalis. An easy and fast extraction procedure was developed using a design of experiments approach (DOE). The most efficient extraction was achieved using 0.2% formic acid in 10% methanol at a temperature of 47.5 °C for 60 min. The final method was validated and showed good overall accuracy (recoveries 85-121%) and precision (RDS ≤11%). The method was applied to B. officinalis leave material, demonstrating its suitability for the intended purpose. In these borage samples, the acetylated forms, which are not regulated by EU, were among the quantitatively most relevant PA.

From Structural Alerts to Signature Fragment Alerts: A Case Study on Pyrrolizidine Alkaloids.

Even though modeling is considered a valid alternative to mutagenicity testing for substances with known structures, it can be applied for mixtures only if all of the single chemical structures are identified. Within the present work, we investigate a new avenue to exploit computational toxicology for mixtures, such as plant-based food ingredients. Indeed, considering that in the absence of toxicological information, an important early consideration is whether any substance may be genotoxic through the mutagenic mechanism of action, we tried to establish a correspondence between genotoxic structural alerts (SAs) and so-called signature fragment alerts (SFAs). Once this correspondence is established, chromatograms could be screened for chemical features associated with genotoxic alerts. Pyrrolizidine alkaloids (PAs), a large group of natural toxins (several of them known as genotoxic) were used as a case study because their early identification would bring significant benefits. The method was built using 56 PA pure standards, resulting in the characterization of signature fragment alerts. Finally, the approach was verified in real plant-based samples such as herbal tea and alfalfa, where the screening of signature fragment alerts allowed highlighting quickly the presence of genotoxic PAs in plant-based mixtures. Therefore, the SFA analysis can be used for risk prioritization of newly identified PAs and for their identification in mixtures, contributing to the unnecessary use of animal experimentation for genotoxicity testing.

Comment on Pyrrolizidine Alkaloids and Terpenes from Senecio (Asteraceae): Chemistry and Research Gaps in Africa.

The genus Senecio is one of the largest in Asteraceae. There are thousands of species across the globe, either confirmed or awaiting taxonomic delimitation. While the species are best known for the toxic pyrrolizidine alkaloids that contaminate honeys (as bees select pollen from the species) and teas via lateral transfer and accumulation from adjacent roots of Senecio in the rhizosphere, they are also associated with more serious cases leading to fatality of grazing ruminants or people by contamination or accidental harvesting for medicine. Surprisingly, there are significantly more sesquiterpenoid than pyrrolizidine alkaloid-containing species. The main chemical classes, aside from alkaloids, are flavonoids, cacalols, eremophilanes, and bisabolols, often in the form of furan derivatives or free acids. The chemistry of the species across the globe generally overlaps with the 469 confirmed species of Africa. A small number of species express multiple classes of compounds, meaning the presence of sesquiterpenes does not exclude alkaloids. It is possible that there are many species that express the pyrrolizidine alkaloids, in addition to the cacalols, eremophilanes, and bisabolols. The aim of the current communication is, thus, to identify the research gaps related to the chemistry of African species of Senecio and reveal the possible chemical groups in unexplored taxa by way of example, thereby creating a summary of references that could be used to guide chemical assignment in future studies.

The in vitro genotoxicity potency of mixtures of pyrrolizidine alkaloids can be explained by dose addition of the individual mixture components.

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.

Rumen Metabolism of Senecio Pyrrolizidine Alkaloids May Explain Why Cattle Tolerate Higher Doses Than Monogastric Species.

Rumen metabolism of Senecio pyrrolizidine alkaloids (PAs) and their N-oxide forms was studied by mass spectrometry in in vitro batch culture incubates and confirmed in in vivo samples. Most N-oxides were found to undergo rapid conversion to their corresponding free bases, followed by biotransformation to metabolites hydrogenated at both the necine base and the necic acid moiety. Therefore, rumen metabolism can be considered a detoxification step, as saturated necine base structures are known as the platyphylline type, which is regarded as less or nontoxic. Individual Senecio PAs, such as jacoline, are metabolized slowly during rumen fermentation. PAs that showed limited biotransformation in the rumen in this study also showed limited transformation and CYP-mediated bioactivation in the liver in other studies. This could not only explain why PAs that are comparatively metabolically stable can pass into milk but also suggest that such PAs might be considered compounds of lesser concern.

Structure-Dependent Toxicokinetics of Selected Pyrrolizidine Alkaloids In Vitro.

Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo.

Development and Validation of an UHPLC-MS/MS Method for the Determination of 32 Pyrrolizidine Alkaloids in Chinese Wild Honey.

BACKGROUND: Studies on pyrrolizidine alkaloid (PA) contamination in honey produced in China are scarce. Previously reported HPLC-MS/MS methods for the determination of PAs in honey often suffer from insufficient separation and uncertainties in PA isomers. OBJECTIVE: To develop and validate an Ultra-HPLC (UHPLC)-MS/MS method for baseline separation of PA isomers towards precise determination of 32 PAs in Chinese wild honey. METHODS: PAs were extracted from honey samples and separated on an ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 µm) column with (A) 0.1% formic acid aqueous solution containing 5 mM ammonium acetate and (B) methanol as mobile phase. The column temperature was maintained at 30°C, and flow rate was 0.3 mL/min. Detection was performed by tandem mass spectrometry. The total run time was reduced to 18 min. RESULTS: Thirty-one of 32 PAs were baseline separated efficiently within 18 min. The LOD and LOQ were 0.06-0.25 µg/kg and 0.22-0.82 µg/kg, respectively, except for that of clivorine, for which LOD and LOQ were 2.03 and 6.78 µg/kg, respectively. The average recoveries ranged between 66.3 and 95.1% and the average RSDs were 3.2 to 8%. The established method was used to analyze PAs in 22 types of Chinese wild honey, and the predominant PAs found in these honey samples were intermedine and lycopsamine. CONCLUSION: A high-throughput method for the determination of isomeric PAs in honey was developed and validated. Five of the 22 types of Chinese wild honey were contaminated with PAs concentrations of 2.2-207.0 µg/kg. HIGHLIGHTS: A new method capable of monitoring more PAs and providing better separation than previously reported protocols for the determination of multiclass PAs in honey is established.

An Intramolecular Hydroaminomethylation-Based Approach to Pyrrolizidine Alkaloids under Microwave-Assisted Heating.

A general method for the synthesis of pyrrolizidine derivatives using an intramolecular hydroaminomethylation protocol (HAM) under microwave (MW) dielectric heating is reported. Starting from a 3,4-bis(benzyloxy)-2-[(benzyloxy)methyl]-5-vinylpyrrolidine, MW-assisted intramolecular HAM in the presence of gaseous H2 and CO gave the natural alkaloid hyacinthacine A2 protected as benzyl ether. The same approach gave a lentiginosine analogue starting from the corresponding vinyl N-hydroxypyrrolidine. The nature of the reaction products and the yields were strongly influenced by the relative stereochemistry of the starting pyrrolidines, as well as by the catalyst/ligand employed. The use of ethanol as a solvent provides environmentally friendly conditions, while the ligand/catalyst system can be recovered by separating the alkaloid product with an SCX column and recycling the ethanolic solution. HAM worked up to three times with the recycled catalyst solution without any significant impact on yield.

Electrochemiluminescence sensor for point-of-care detection of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and PA N-oxides are hepatotoxic natural products, produced by over 6000 plant species worldwide. However, an unmet need remains for confirmative measurement of PAs in routine clinical tests. Here, we develop a visual, easy-to-use, and economic mesoporous silica-electrochemiluminescence (MPS-ECL) sensor for point-of-care (POC) testing of PAs, utilizing MPS's amplification effect on positive ions. The relationship between PAs' different structures and corresponding Ru(bpy)32+ ECL activity shows that reaction mechanism, stability of intermediate, molecular geometry and alternative anodic reactivity significantly affect the ECL activity. The ECL intensity varies among different PAs: monocrotaline ˃ senecionine N-oxide ˃ retrorsine ˃ senkirkine. The POC sensors possess excellent linearity (0.9993 > R2 > 0.9944), low detection limits (0.02 µM-0.07 µM), and good recoveries (90.12%-105.93%), indicating good accuracy and practicability. The portable and low-cost sensor is user-friendly, which holds promise to be applied to POC testing of PAs in drugs, food products, and clinical samples, which is promising for initial assessments of PA-induced health risk.

Sulfonated halloysite nanotubes as a novel cation exchange material for solid phase extraction of toxic pyrrolizidine alkaloids.

Pyrrolizidine alkaloids are phytochemicals, which present a highly toxic class of compounds in multiple food resources and are therefore a late-breaking topic in food safety. This study describes the first use of modified halloysite nanotubes as a novel solid material for solid phase extraction. As a result of a fast one-pot sulfonation of the cheap and non-toxic halloysite nanotubes, an efficient cation exchange phase has been prepared. After optimization of the solid phase extraction protocol, high extraction efficiencies and overall recoveries were obtained for a mixture of four pyrrolizidine alkaloid structures through UHPLC-MS/MS analysis with caffeine as the internal standard. Furthermore, the novel solid phase was used for the selective binding of the toxic pyrrolizidine alkaloids in a real-life honey sample, which itself is often contaminated with these compounds. In-house validation showed great extraction efficiencies up to 99.9% for senecionine with a lower limit for lycopsamine with 59.3%, which indicated high selectivity even in the presence of potential interfering compounds. Subsequently, overall recoveries up to 91.5% could be obtained for senecionine while the lowest value was reached for lycopsamine with 55.1%. Comparison with a commercial strong cation exchange tube procedure showed the high competitiveness of the novel solid phase with respect to overall performance. Only slight disadvantages regarding precision and repeatability with values under 5.7% and 11.6% could be observed. Therefore, sulfonated halloysite nanotubes present themselves as an easy to prepare, cheap and highly efficient novel cation exchange material for the selective solid phase extraction of toxic pyrrolizidine alkaloids in frequently contaminated real-life samples like honey.

Quick and Green Microextraction of Pyrrolizidine Alkaloids from Infusions of Mallow, Calendula, and Hibiscus Flowers Using Ultrahigh-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry Analysis.

A sustainable microextraction of pyrrolizidine alkaloids (PAs) from edible flower infusions using the innovative µSPEed technique is proposed. Different sorbents and extraction conditions were tested, achieving the highest extraction efficiency with an octadecylsilane sorbent (4 mg). The extraction procedure just took 1 min per sample, and only 300 µL of methanol and 300 µL of the sample were used per extraction. Ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry was used for analysis. The method was properly validated, providing suitable linearity, selectivity, sensitivity (quantification limits 0.3-1 µg/L), overall recoveries (79-97%), and precision (≤17% relative standard deviation). Its application to the analysis of different infusions of mallow, calendula, and hibiscus flowers revealed similar total PA values (23-41 µg/L) and contamination profile among the mallow and hibiscus samples, with predominance of senecionine-type and heliotrine-type PAs, respectively. Conversely, calendula samples showed more variations (23-113 µg/L), highlighting the occurrence of intermedine N-oxide and europine N-oxide on them.

Isolation of Echimidine and Its C-7 Isomers from Echium plantagineum L. and Their Hepatotoxic Effect on Rat Hepatocytes.

Echimidine is the main pyrrolizidine alkaloid of Echium plantagineum L., a plant domesticated in many countries. Because of echimidine's toxicity, this alkaloid has become a target of the European Food Safety Authority regulations, especially in regard to honey contamination. In this study, we determined by NMR spectroscopy that the main HPLC peak purified from zinc reduced plant extract with an MS [M + H]+ signal at m/z 398 corresponding to echimidine (1), and in fact also represents an isomeric echihumiline (2). A third isomer present in the smallest amount and barely resolved by HPLC from co-eluting (1) and (2) was identified as hydroxymyoscorpine (3). Before the zinc reduction, alkaloids (1) and (2) were present mostly (90%) in the form of an N-oxide, which formed a single peak in HPLC. This is the first report of finding echihumiline and hydroxymyoscorpine in E. plantagineum. Retroanalysis of our samples of E. plantagineum collected in New Zealand, Argentina and the USA confirmed similar co-occurrence of the three isomeric alkaloids. In rat hepatocyte primary culture cells, the alkaloids at 3 to 300 µg/mL caused concentration-dependent inhibition of hepatocyte viability with mean IC50 values ranging from 9.26 to 14.14 µg/mL. Our discovery revealed that under standard HPLC acidic conditions, echimidine co-elutes with its isomers, echihumiline and to a lesser degree with hydroxymyoscorpine, obscuring real alkaloidal composition, which may have implications for human toxicity.

The chemical structure impairs the intensity of genotoxic effects promoted by 1,2-unsaturated pyrrolizidine alkaloids in vitro.

1,2-unsaturated pyrrolizidine alkaloids (PAs) represent a large group of secondary plant metabolites exhibiting hepatotoxic, genotoxic, and carcinogenic properties upon bioactivation. To examine how the degree of esterification affects the genotoxic profile of PA we investigated cytotoxicity, histone H2AX phosphorylation, DNA strand break induction, cell cycle perturbation, micronuclei formation, and aneugenic effects in different cell models. Analysis of cytotoxicity and phosphorylation of histone H2AX was structure- and concentration-dependent: diester-type PAs (except monocrotaline) showed more pronounced effects than monoester-type PAs. Cell cycle analysis identified that diester-type PAs induced a S-phase arrest and a decrease in the occurrence of cells in the G1-phase. The same structure-dependency was observed by flow-cytometric analysis of PA-induced micronuclei in CYP3A4-overexpressing V79 cells. Analysis of centromeres induced by lasiocarpine in the micronuclei by fluorescence in situ hybridization indicated an aneugenic effect in V79h3A4 cells. Comet assays revealed no significant induction of DNA strand breaks for all investigated PAs. Overall, diester-type PAs induced more pronounced effects than monoester-type PAs. Furthermore, our results indicate aneugenic effects upon exposure towards lasiocarpine in vitro. These data improve our understanding how structural features of PA influence the genotoxic profile. Especially, the monoester-type PAs seem to induce less severe effects than other PAs.