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Cytochrome P450 (CYP)3A4 induction by drugs and pesticides plays a critical role in the enhancement of pyrrolizidine alkaloid (PA) toxicity as it leads to increased formation of hepatotoxic dehydro-PA metabolites. Addressing the need for a quantitative analysis of this interaction, we developed a physiologically-based toxicokinetic (PBTK) model. Specifically, the model describes the impact of the well-characterized CYP3A4 inducer rifampicin on the kinetics of retrorsine, which is a prototypic PA and contaminant in herbal teas. Based on consumption data, the kinetics after daily intake of retrorsine were simulated with concomitant rifampicin treatment. Strongest impact on retrorsine kinetics (plasma AUC 24 and C max reduced to 67% and 74% compared to the rifampicin-free reference) was predicted directly after withdrawal of rifampicin. At this time point, the competitive inhibitory effect of rifampicin stopped, while CYP3A4 induction was still near its maximum. Due to the impacted metabolism kinetics, the cumulative formation of intestinal retrorsine CYP3A4 metabolites increased to 254% (from 10 to 25 nmol), while the cumulative formation of hepatic CYP3A4 metabolites was not affected (57 nmol). Return to baseline PA toxicokinetics was predicted 14 days after stop of a 14-day rifampicin treatment. In conclusion, the PBTK model showed to be a promising tool to assess the dynamic interplay of enzyme induction and toxification pathways. [ABSTRACT FROM AUTHOR]

The market demand for herbal dietary supplements is rapidly growing and such products are becoming more common and accessible to consumers. However, the knowledge about their safety remains incomplete. Herbal dietary supplements are one of the food groups that can contribute significantly to human health concerns arising from chronic exposure to pyrrolizidine alkaloids and mycotoxins. This study aimed to simultaneously determine 79 natural contaminants, including mycotoxins, as well as pyrrolizidine and tropane alkaloids in herbal dietary supplements in one analytical run. Exposure assessment and human health risks were assessed for all compounds included in this study. The total concentration of naturally occurring contaminants in herbal dietary supplements reached 5.3 mg kg−1 and the most frequently detected mycotoxins were tentoxin and alternariol monomethyl ether. The latter was detected with the highest frequency, reaching concentrations up to 2.5 mg kg−1. The obtained results indicate a potential risk to public health related to herbal dietary supplement consumption. [ABSTRACT FROM AUTHOR]

1,2-Dehydro-pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO) and tropane alkaloids (TA), are toxic plant metabolites. If plant material, containing these toxins, is present in the feed of dairy cows these toxins can be transferred into milk. Here, milk was sampled directly from dairy farms in the German federal states of Bavaria and Schleswig-Holstein in 2020–2022 in order to investigate a possible contamination of milk at the production stage. In total, 228 milk samples were analysed for 54 PA/PANO and two TA by a sensitive LC–ESI-MS/MS method. In addition, a subset of milk samples (n = 85) was independently analysed for TA by a cooperating laboratory for verification. PA/PANO were found in 26 samples (11%) with a low median sum content of the contaminated samples of 0.024 µg/L. The highest level of contamination was 5.6 µg/L. Senecionine-, lycopsamine- and heliotrine-type PA/PANO were detected. In four samples (1.8%), atropine was determined up to 0.066 µg/L. The toxin levels in the milk samples hardly contributed to the total daily exposure. These data are first-time results on contamination rates and levels occurring in milk from individual dairy farms, based on a large sample number. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are specialized metabolites that are produced by various plant families that act as defense compounds against herbivores. On the other hand, certain lepidopteran insects uptake and utilize these PAs as defense compounds against their predators and as precursors of their sex pheromones. Adult males of Parantica sita, a danaine butterfly, convert PAs into their sex pheromones. In early summer, P. sita swarms over the flowers of Myosotis scorpioides, which belongs to the family Boraginaceae. M. scorpioides produces PAs, but the organs in which PAs are produced and whether P. sita utilizes PAs in M. scorpioides are largely unknown. In the present study, we clarified that M. scorpioides accumulates retronecine-core PAs in N-oxide form in all organs, including flowers. We also identified two M. scorpioides genes encoding homospermidine synthase (HSS), a key enzyme in the PA biosynthetic pathway, and clarified that these genes are expressed in all organs where PAs accumulate. Phylogenetic analysis suggested that these two HSS genes were originated from gene duplication of deoxyhypusine synthase gene like other HSS genes in PA-producing plants. These results suggest that PAs are synthesized and accumulated in the flower of M. scorpioides and provide a possibility for a PA-mediated interaction between P. sita and M. scorpioides. [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]

Pyrrolizidine alkaloids (PAs) are found in many plants worldwide, including some in Canada. PAs have been linked to losses in livestock populations and the development of human PA diseases. Four PA-derived dehydrosupinidine (DHP) adducts formed at the exocyclic amino groups of DNA purines have been found in tumor tissue and flagged as potential biomarkers for tumor formation (denoted DHP–G/A–3 and DHP–G/A–4). Four additional adducts (DHP–G/A–1 and DHP–G/A–2) have also been identified, which differ in the stereochemistry at the DHP–nucleobase linker or DHP ring carbon containing the hydroxy group, as well as the length of the DHP–nucleobase linker. Since the impact of these distinct chemical features on adduct mutagenicity is currently unclear, the present work uses density functional theory calculations to uncover the structures and base-pairing properties of these experimentally-observed DHP-derived purine adducts. Adduct Watson–Crick–Franklin base pairs involving the canonical partner are energetically and structurally feasible. However, the G/A–3 and G/A–4 adducts are also highly susceptible to mispairing with G. Indeed, the longer and more flexible DHP–nucleobase linker in these adducts affords interactions between the DHP moiety and the pairing G that are not possible for the G/A–1 and G/A–2 counterparts. Our data thus rationalize the experimental identification of this subset of adducts in liver tumor tissue, and provide key insights to guide future experimental and computational studies that investigate the replication and broader biological outcomes of DHP-derived lesions. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are one type of phytotoxins distributed in various plants, including many medicinal herbs. Many organs might suffer injuries from the intake of PAs, and the liver is the most susceptible one. The diagnosis, toxicological mechanism, and detoxification of PAs-induced hepatotoxicity have been studied for several decades, which is of great significance for its prevention, diagnosis, and therapy. When the liver was exposed to PAs, liver sinusoidal endothelial cells (LSECs) loss, hemorrhage, liver parenchymal cells death, nodular regeneration, Kupffer cells activation, and fibrogenesis occurred. These pathological changes classified the PAs-induced liver injury as acute, sub-acute, and chronic type. PAs metabolic activation, mitochondria injury, glutathione (GSH) depletion, inflammation, and LSECs damage-induced activation of the coagulation system were well recognized to play critical roles in the pathological process of PAs-induced hepatotoxicity. A lot of natural compounds like glycyrrhizic acid, (-)-epicatechin, quercetin, baicalein, chlorogenic acid, and so on were demonstrated to be effective in alleviating PAs-induced liver injury, which rendered them huge potential to be developed into therapeutic drugs for PAs poisoning in clinics. This review presents updated information about the diagnosis, toxicological mechanism, and detoxification studies on PAs-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are one of the largest distributed classes of toxins in nature. They have a wide range of toxicity, such as hepatotoxicity, pulmonary toxicity, neuronal toxicity, and carcinogenesis. Yet, biological targets responsible for these effects are not well addressed. Using methods of computational biology for target identification, we tested more than 200 PAs. We used a machine-learning approach that applies structural similarity for target identification, ChemMapper, and SwissTargetPrediction. The predicted targets with high probabilities were muscarinic acetylcholine receptor M1. The predicted interactions between these two targets and PAs were further studied by molecular docking-based binding energies using AutoDock and VinaLC, which revealed good binding affinities. The PAs are bound to the same binding pocket as pirenzepine, a known M1 antagonist. These results were confirmed by in vitro assays showing that PAs increased the levels of intracellular calcium. We conclude that PAs are potential acetylcholine receptor M1 antagonists. This elucidates for the first time the serious neuro-oncological toxicities exerted by PA consumption. [ABSTRACT FROM AUTHOR]

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

Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors d-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity. [ABSTRACT FROM AUTHOR]

Heterocyclic organic compounds named pyrrolizidine alkaloids (PAs) belong to a group of alkaloids and are synthesized by either plants or microorganisms. Therefore, they are naturally occurring secondary metabolites. They are found in species applied in the pharmaceutical and food industries, thus a thorough knowledge of their pharmacological properties and toxicology to humans is of great importance for their further safe employment. This review is original because it synthesizes knowledge of plant and microbial PAs, which is unusual in the scientific literature. We have focused on the Boraginaceae family, which is unique due to the exceptional richness and diversity of its PAs in plant species. We have also presented the microbial sources of PAs, both from fungi and bacteria. The structure and metabolism of PAs have been discussed. Our main aim was to summarize the effects of PAs on humans, including both negative, toxic ones, mainly concerning hepatotoxicity and carcinogenicity, as well as potentially positive ones for pharmacological and medical applications. We have collected the results of studies on the anticancer activity of PAs from plant and microbial sources (mainly Streptomyces strains) and on the antimicrobial activity of PAs on different strains of microorganisms (bacteria and fungi). Finally, we have suggested potential applications and future perspectives. [ABSTRACT FROM AUTHOR]

A one-pot multi-component reaction was employed for the stereoselective synthesis of a novel set of dispiro-oxindolopyrrolizidines analogs incorporating a thiazolo[3,2-a]benzimidazole scaffold based on the [3 + 2] cycloaddition (32CA) reaction approach. The desired novel dispiro-oxindolopyrrolizidines 9a–d were achieved using the 32CA reaction of new ethylene derivatives based on thiazolo[3,2-a]benzimidazole moiety seven with thiazolidine derivatives eight and different substituted isatin compounds 5a–d (R = H, Cl, NO2, and Br). The final dispiro-oxindolopyrrolizidines cycloadducts were separated, purified, and fully characterized by means of a set of spectroscopic tools including IR, HNMR, CNMR, and MS. The Molecular Electron Density Theory (MEDT) was applied to explain the mechanism and stereoselectivity in the of the key 32CA reaction step. The reactive pseudo(mono)radical electronic structure of the in situ generated azomethine ylides and the high polar character of the corresponding 32CA reactions account for the low computed activation Gibbs free energies and total endo stereoselectivity of this kinetically controlled exergonic reaction. The computed relative Gibbs free activation energies of competitive reaction paths and regioisomers ratio distribution of 80:20 justify the major formation of 9a via the most favorable ortho/endo reaction path. [ABSTRACT FROM AUTHOR]

Gelsemium elegans (G.elegans) is a plant of the Loganiaceae family, known for its indole alkaloids, including gelsemine, koumine, and gelsenicine. Gelsemine and koumine are well-studied active alkaloids with low toxicity, valued for their anti-anxiety and analgesic properties. However, gelsenicine, another important alkaloid, remains underexplored due to its high toxicity. This study focuses on evaluating the analgesic properties of gelsenicine and comparing them with gelsemine and koumine. The results indicate that all three alkaloids exhibit robust analgesic properties, with gelsemine, koumine, and gelsenicine showing ED 50 values of 0.82 mg/kg, 0.60 mg/kg, and 8.43 μg/kg, respectively, as assessed by the hot plate method. Notably, the therapeutic dose of gelsenicine was significantly lower than its toxic dose (LD 50 = 0.185 mg/kg). The study also investigated the mechanism of action by analyzing the expression levels of GlyRα3 and Gephyrin. The PGE 2 model group showed decreased expression levels of GlyRα3 and Gephyrin, while groups treated with gelsemine, koumine, and gelsenicine were able to reverse this decrease. These results suggest that gelsenicine effectively alleviates PGE 2 -induced hyperalgesia by upregulating the expression of GlyRα3 and Gephyrin, which are key targets of the Gly receptor pathway. • This study explored the analgesic mechanism of gelsenicine for the first time. • Gelsenicine can significantly relieve the hypersensitivity pain caused by PGE 2. • Gelsenicine did not cause pathological damage to the mouse's spinal cord. • Gelsenicine has the best analgesic effect among the G.elegans alkaloids. [ABSTRACT FROM AUTHOR]

A concise and efficient enantiospecific total synthesis of (−)‐hyacinthacine A1 and (+)‐hyacinthacine A1 was achieved from commercially available starting material L‐pyroglutamic acid and D‐glutamic acid, respectively. For the synthesis of this trihydroxylated pyrrolizidine ring, we employed the nitrogen‐substituted donor‐acceptor cyclopropane as a key intermediate. The synthetic approach relies on two crucial steps, highly stereo‐ and regioselective intramolecular cyclopropanation with Rh2(OAc)4 and regioselective ring opening of a nitrogen‐substituted donor‐acceptor cyclopropane. [ABSTRACT FROM AUTHOR]

The review summarizes the methods of synthesis of pyrrolizidine (hexahydro-1H-pyrrolizine) derivatives published in the literature over the past five years. The methods can be conditionally divided into two major groups: synthesis from pyrrolidine derivatives and cyclization of acyclic precursors. [ABSTRACT FROM AUTHOR]

Incidences of human exposure to pyrrolizidine alkaloids (PAs) and their adverse effects have previously been reported in three African countries. In view of the increasing intake of herbal remedies over the years, we highlight Africa's tropical and subtropical PA-containing plants commonly used in herbal preparations, and summarize their ethnomedicinal uses, contamination pathways and potential health risks. PA-containing plants and their honey-derived products were ascertained through searching peer-reviewed electronic databases, PubMed, Google Scholar, Science Direct, Web of Science, and Scopus databases from 2001 to 2023. Overall, 37 PA-containing plants were found to be used in herbal medicinal preparations in Africa, of which 25 belong to Asteraceae, Fabaceae (10), and Boraginaceae (2) plant families. Furthermore, three major paths of PA-contamination of herbal medicines were identified while PAs from the genera Chromolaena, Ageratum, Eupatorium, Crotalaria, Eupatorium, Echium , and Senecio were frequently detected in honey used in herbal preparations. It was also revealed that the adoption of the guidelines for Good Agriculture and Collection Practice could significantly reduce the risk of exposure to PAs. This review will be useful in facilitating the identification of PA-containing plants in the field so that they are not used in herbal preparations, whereas there is a need to minimize their health risks by monitoring and setting acceptable limits for PAs in herbal and food products. [Display omitted] • Pyrrolizidine alkaloid poisoning is endemic in some African countries. • Pyrrolizidine alkaloid plants are used in herbal medicinal preparations in Africa. • Pyrrolizidine alkaloids have been detected in honey used in herbal preparations. • African countries are yet to establish maximum limits for pyrrolizidine alkaloids. [ABSTRACT FROM AUTHOR]

The increasing spread of ragworts is observed with concern. Ragworts like tansy ragwort (Jacobaea vulgaris Gaertn.) or marsh ragwort (J. aquatica) contain pyrrolizidine alkaloids (PA) which may induce hepatotoxic effects. Grazing animals usually avoid ragworts if their pasture management is appropriate. Preserved feed prepared from ragworts contaminated meadows may, however, lead to a significant exposure to PA. Previous studies on toxicity of PA for dairy cows revealed inconsistent results due to feeding ragwort plant material which was associated with heterogeneous PA exposure and thus failed to conclusively deduce critical PA doses. Therefore, the aim of the present study was to expose dairy cows (n = 4 per group) in a short-term scenario for 28 days with increasing PA doses (PA1: 0.47 mg PA/kg body weight (BW)/day (d); PA2: 0.95 mg PA/kg BW/d; PA3: 1.91 mg PA/kg BW/d) via oral administration by gavage of a defined PA-extract. While group PA3 was dosed with the PA-extract alone, groups PA2 and PA1 received PA-extracts blended in similar volumes with molasses to provide comparable amounts of sugar. Additionally, two control groups were treated either with water (CONWater) or with molasses (CONMolasses) to assess the effects of sugar without PA interference. While clinical traits including dry matter intake, milking performance, rectal body temperature, ruminal activity and body condition score (BCS) were not influenced by PA exposure, activities of enzymes indicative for liver damages, such as gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH), increased significantly over time at an exposure of 1.91 mg total PA/kg BW/d. [ABSTRACT FROM AUTHOR]

Background: Plant-derived pyrrolizidine alkaloids (PAs) in feed cause metabolic disturbances in farm animals resulting in high economic losses worldwide. The molecular pathways affected by these PAs in cells and tissues are not yet fully understood. The objective of the study was to examine the dose-dependent effects of orally applied PAs derived from tansy ragwort in midlactation dairy cows. Methods: Twenty Holstein dairy cows were treated with target exposures of 0, 0.47, 0.95 and 1.91 mg of total PA/kg of body weight/d in control, PA1, PA2 and PA3, respectively, for 28 days. Liver tissue biopsy and plasma and milk samples were taken at day 28 of treatment to assess changes in metabolic pathways. A targeted metabolomics approach was performed to detect the metabolite profiles in all compartments. Results: The PA-affected metabolite profiling in liver tissue, plasma and milk revealed changes in three substrate classes: acylcarnitines (ACs), phosphatidylcholines (PCs) and sphingomyelins (SMs). In addition, in the plasma, amino acid concentrations were affected by PA exposure. Conclusions: PA exposure disturbed liver metabolism at many sites, especially devastating pathways related to energy metabolism and to amino acid utilization, most likely based on mitochondrial oxidative stress. The effects on the milk metabolite profile may have consequences for milk quality. [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]

Pyrrolizidine alkaloids (PAs) are noted for their hepatotoxic, genotoxic, and carcinogenic effects in animals and humans following metabolic activation in the liver. In this study, herbal supplements sold in Ghana for sexual improvement were analysed for the presence of 64 PAs using LC-MS/MS analysis. Up to 17 different PAs were identified in 19 out of the 37 samples analysed. The sum of PAs in samples ranged from 5 to 3204 μg kg−1. Since the PA content in the herbal medicinal preparations was generally lower than in honey samples, their presence was mainly attributed to cross-contamination. The observed levels would result in estimated daily intakes from 0.01 to 12 μg per day or 0.0002 to 0.2 μg kg−1 bw day−1 for a person weighing 70 kg. The margins of exposure ranged from 1200 to 1,400,000 with eight samples showing values below 10,000, thus indicating a health concern. [ABSTRACT FROM AUTHOR]

A stereo‐, regio‐ and chemoselective synthesis of a series of structurally diverse novel oxobenzothiophene embedded spirooxindolopyrrolidine/pyrrolizidine hybrid heterocycles were achieved in excellent yields by multicomponent 1,3‐dipolar cycloaddition reaction process. The 1,3‐dipole component employed was generated in situ from isatin and N‐methylglycine/L‐proline while the dipolarophile, ethyl 2‐(3‐oxobenzo[b]thiophen‐2(3H)‐ylidene)acetate was prepared from thiophenol in two good yielding steps. The formation of spiroheterocyclic hybrids occurred through the formation of two C=C and one C−N bonds in a single synthetic transformation. The compounds formed consists of three adjacent stereocenters, out of which two are spirocarbons. The synthesized spiro compounds were assayed for their AChE/BChE inhibitory activity and most of the spirocompounds showed promising cholinesterase inhibitory activity. Among them, spiropyrrolidine that substituted with chlorine atom on the oxindole ring displayed significant activity (4.16±0.09 μM) with respect to standard drug, galantamine (2.09±0.11 μM). Molecular modelling simulation was explored for the most potent compound that revealed interesting binding templates to the active site of enzyme. [ABSTRACT FROM AUTHOR]

Comfrey (Symphytum officinale L.) has a long tradition of use in the treatment of musculoskeletal disorders. However, due to hepatotoxic pyrrolizidine alkaloids (PAs), the EMA restricts the use of comfrey root (CR) to external use only and for short periods of time. Recent studies indicate a low permeability of PAs across the skin, calling into question the safety of topical application of products containing comfrey preparations. The aim of our work was to develop and validate an HPLC method enabling the separation of isomeric PAs from comfrey and, on this basis, to assess the potential toxicity of CR and comfrey leaf (CL) obtained from various Polish sources. The qualitative and quantitative analysis of PAs via HPLC-MS/MS was performed in MRM mode. The results obtained confirmed a lower content of PAs in CL than in CR and showed a wide variation in the composition of PAs in CR, with a much more stable profile of PAs in CL. Factor analysis confirmed that CRs and CLs differ in PA content, which is influenced by the growth conditions and geographical origin. The determined concentrations of PAs prove that in some CRs available on the Polish herbal market, the content of PAs may exceed the daily dose considered safe. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by over 6000 plant species worldwide. PAs enter the food chain through accidental co-harvesting of PA-containing weeds and through soil transfer from the living plant to surrounding acceptor plants. In animal studies, 1,2-unsaturated PAs have proven to be genotoxic carcinogens. According to the scientific opinion expressed by the 2017 EFSA, the foods with the highest levels of PA contamination were honey, tea, herbal infusions, and food supplements. Following the EFSA's recommendations, data on the presence of PAs in relevant food were monitored and collected. On 1 July 2022, the Commission Regulation (EU) 2020/2040 came into force, repealed by Commission Regulation (EU) 2023/915, setting maximum levels for the sum of pyrrolizidine alkaloids in certain food. A total of 602 food samples were collected from the Italian market between 2019 and 2022 and were classified as honey, pollen, dried tea, dried herbal infusions, dried herbs, and fresh borage leaves. The food samples were analyzed for their PA content via an in-house LC-MS/MS method that can detect PAs according to Regulation 2023/915. Overall, 42% of the analyzed samples were PA-contaminated, 14% exceeded the EU limits, and the items most frequently contaminated included dried herbs and tea. In conclusion, the number of food items containing considerable amounts of PAs may cause concern because they may contribute to human exposure, especially considering vulnerable populations—most importantly, children and pregnant women. [ABSTRACT FROM AUTHOR]

Background and aims: Hepatic sinusoidal obstruction syndrome (HSOS) is caused by toxic injury, such as pyrrolizidine alkaloids, to the liver sinusoidal endothelial cells, and the gut microbiota may be involved. However, the specific role and underlying mechanism of gut microbiota in HSOS is unknown. Methods: HSOS model was established by gavage of monocrotaline (MCT) in rats. Fecal microbiota transplantation (FMT) with HSOS-derived or healthy gut flora was also conducted to validate the role of gut microflora in MCT-induced liver injury. The microbial 16 s rRNA analysis and untargeted metabolomics analysis in the faeces were performed to identify HSOS-related flora and metabolites. Finally, by supplementation with specific tryptophan metabolites, such as indole-3-acetaldehyde (IAAld) and indole acetic acid (IAA), we further confirmed the role of tryptophan metabolism in HSOS and the role of the AhR/Nrf2 pathway in MCT-induced liver injury. Results: MCT induced HSOS-like liver injury in rats with significantly altered gut microbiota. Particularly, some tryptophan-metabolizing bacteria reduced in MCT-treated rats, such as Bacteroides, Bifidobacterium, Lactobacillus and Clostridium, and accompanied by a decrease in microbial tryptophan metabolic activity and a series of tryptophan derivatives. Restoring the gut microbiota via FMT improved MCT-induced liver damage, while HSOS-derived gut microbiota aggravated the liver injury induced by MCT. Supplementation with microbial tryptophan derivatives (IAAld or IAA), or 6-formylindolo(3,2-b)carbazole (Ficz, an AhR agonist) could activate the AhR/Nrf2 signaling pathway, thereby attenuating the MCT-induced liver oxidative stress and liver sinusoidal endothelial cells injury. Conclusions: Gut microbiota plays a critical role in MCT-induced HSOS, with inadequate microbial tryptophan metabolism in the gut and consequently a lower activity of the AhR/Nrf2 signaling pathway in the liver, which should be a potential target for the management of HSOS. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: The present study reports a previously unknown metabolite and the function of UGT76F1 in Arabidopsis. This metabolite is structurally related to the C7‐necic acid moiety of certain pyrrolizidine alkaloids, suggesting that necic acid biosynthesis may have a wider taxonomic distribution in non‐alkaloid‐containing species. [ABSTRACT FROM AUTHOR]

• Presence of pyrrolizidine alkaloids (PAs) are of global concern in herbal teas. • First study to show direct transfer of PAs from weed residues via soil to rooibos plants. • Rooibos plants mainly accumulated the PA senecivirnine-N-oxide in field and pot trial. • Weeds with highest PAs in Niewoudtville rooibos fields were Spergula arvensis and Senecio species. • Rooibos plants in fields contained on average 20 ug kg−1 total PAs. Pyrrolizidine alkaloids (PAs) contamination of rooibos tea products has prompted global concern amongst distributors. Lateral transfer of PAs from weeds as a result of co-harvesting has been previously demonstrated. However, it is not currently clear whether there can be a direct transfer of PAs from decomposing weed material to rooibos tea via the soil. Furthermore, a large number of weed species occur in rooibos tea plantations in the Nieuwoudtville region, Northern Cape Province, South Africa and it is not fully known which species produce PAs. Therefore, the objectives of this study were, firstly, to quantify and identify PAs in rooibos tea and dominant weed species occurring in rooibos plantations in the Nieuwoudtville region using ultra performance liquid chromatography mass spectrometry, and secondly, to determine whether PAs from weed materials can be transferred to rooibos plants via the soil in a pot trial. amongst the 19 weed species identified in the plantations in spring, Spergula arvensis, Senecio abruptus, Senecio arenarius and Senecio burchellii were found to contain substantial contents of PAs (> 1000 µg kg−1). Rooibos tea at the sites was found to contain average total PA concentrations of 20 µg kg−1, dominated by senecivirnine-N-oxide (76%). In the pot trial, only rooibos plants where S. burchellii material was applied contained measurable PA levels in the form of senecivirnine-N-oxide (4.25 μg kg−1, SE 2.53 μg kg−1). However, the dominant PAs occurring in S. burchellii material applied in the pot trial were senecivirnine (30.1%), senecionine-N-oxide (25.4%), and retrorsine-N-oxide (23.5%). This indicates that senecivirnine-N-oxide was the most absorbable PA taken up the rooibos plants or was the one of the primary metabolic products from adsorbed PAs in rooibos. These findings also help to explain why rooibos plants at the field sites contained predominantly senecivirnine-N-oxide. This is the first study to demonstrate the direct transfer of PAs from PA-containing weed residues to rooibos tea by the soil. [ABSTRACT FROM AUTHOR]

A new prenylated indole alkaloid—Penicimutamide C N-oxide (1), a new alkaloid penicimutamine A (2), along with six known alkaloids were isolated from an endophytic fungus Pallidocercospora crystallina. A simple and accurate method was used to determine the N-O bond in the N-oxide group of 1. By using a β-cell ablation diabetic zebrafish model, compounds 1, 3, 5, 6 and 8 showed significantly hypoglycemic activities under the concentration of 10 μM. Further studies revealed that compounds 1 and 8 lowered the glucose level through promoting glucose uptake in zebrafish. In addition, all eight compounds showed no acute toxicity, teratogenicity, nor vascular toxicity in zebrafish under the concentrations range from 2.5 μΜ to 40 μM. Importantly, these results provide new lead compounds for the development of antidiabetes strategies. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) occur as contaminants in plant-based foods and herbal medicines. Following metabolic activation by cytochrome P450 (CYP) enzymes, PAs induce DNA damage, hepatotoxicity and can cause liver cancer in rodents. There is ample evidence that the chemical structure of PAs determines their toxicity. However, more quantitative genotoxicity data are required, particularly in primary human hepatocytes (PHH). Here, the genotoxicity of eleven structurally different PAs was investigated in human HepG2 liver cells with CYP3A4 overexpression and PHH using an in vitro test battery. Furthermore, the data were subject to benchmark dose (BMD) modeling to derive the genotoxic potency of individual PAs. The cytotoxicity was initially determined in HepG2-CYP3A4 cells, revealing a clear structure–toxicity relationship for the PAs. Importantly, experiments in PHH confirmed the structure-dependent toxicity and cytotoxic potency ranking of the tested PAs. The genotoxicity markers γH2AX and p53 as well as the alkaline Comet assay consistently demonstrated a structure-dependent genotoxicity of PAs in HepG2-CYP3A4 cells, correlating well with their cytotoxic potency. BMD modeling yielded BMD values in the range of 0.1–10 µM for most cyclic and open diesters, followed by the monoesters. While retrorsine showed the highest genotoxic potency, monocrotaline and lycopsamine displayed the lowest genotoxicity. Finally, experiments in PHH corroborated the genotoxic potency ranking, and revealed genotoxic effects even in the absence of detectable cytotoxicity. In conclusion, our findings strongly support the concept of grouping PAs into potency classes and help to pave the way for a broader acceptance of relative potency factors in risk assessment. [ABSTRACT FROM AUTHOR]

Retrorsine is a hepatotoxic pyrrolizidine alkaloid (PA) found in herbal supplements and medicines, food and livestock feed. Dose-response studies enabling the derivation of a point of departure including a benchmark dose for risk assessment of retrorsine in humans and animals are not available. Addressing this need, a physiologically based toxicokinetic (PBTK) model of retrorsine was developed for mouse and rat. Comprehensive characterization of retrorsine toxicokinetics revealed: both the fraction absorbed from the intestine (78%) and the fraction unbound in plasma (60%) are high, hepatic membrane permeation is dominated by active uptake and not by passive diffusion, liver metabolic clearance is 4-fold higher in rat compared to mouse and renal excretion contributes to 20% of the total clearance. The PBTK model was calibrated with kinetic data from available mouse and rat studies using maximum likelihood estimation. PBTK model evaluation showed convincing goodness-of-fit for hepatic retrorsine and retrorsine-derived DNA adducts. Furthermore, the developed model allowed to translate in vitro liver toxicity data of retrorsine to in vivo dose-response data. Resulting benchmark dose confidence intervals (mg/kg bodyweight) are 24.1–88.5 in mice and 79.9–104 in rats for acute liver toxicity after oral retrorsine intake. As the PBTK model was built to enable extrapolation to different species and other PA congeners, this integrative framework constitutes a flexible tool to address gaps in the risk assessment of PA. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PA) are phytochemicals that are known to act as human hepatotoxins and are also considered to be genotoxic carcinogens. Several plant-derived foods are frequently contaminated with PA, like teas and herbal infusions, spices and herbs or certain food supplements. With respect to the chronic toxicity of PA, the carcinogenic potential of PA is generally regarded as the critical toxicological effect. The risk assessment of the short-term toxicity of PA, however, is internationally less consistent. The characteristic pathological syndrome of acute PA toxicity is hepatic veno-occlusive disease. High PA exposure levels may lead to liver failure and even death as documented by several case reports. In the present report, we suggest a risk assessment approach for the derivation of an acute reference dose (ARfD) for PA of 1 µg/kg body weight per day based on a sub-acute animal toxicity study in rats after oral PA administration. The derived ARfD value is further supported by several case reports describing acute human poisoning following accidental PA intake. The here derived ARfD value may be used for PA risk assessment in cases where the short-term toxicity of PA is of interest in addition to the assessment of the long-term risks. [ABSTRACT FROM AUTHOR]

(1) Background: Hepatic venous occlusion type of Budd–Chiari syndrome (BCS-HV) and pyrrolizidine alkaloid-induced hepatic sinusoidal obstructive syndrome (PA-HSOS), share similar clinical features, and imaging findings, leading to misdiagnoses; (2) Methods: We retrospectively analyzed 139 patients with BCS-HV and 257 with PA-HSOS admitted to six university-affiliated hospitals. We contrasted the two groups by clinical manifestations, laboratory tests, and imaging features for the most valuable distinguishing indicators.; (3) Results: The mean patient age in BCS-HV is younger than that in PA-HSOS (p < 0.05). In BCS-HV, the prevalence of hepatic vein collateral circulation of hepatic veins, enlarged caudate lobe of the liver, and early liver enhancement nodules were 73.90%, 47.70%, and 8.46%, respectively; none of the PA-HSOS patients exhibited these features (p < 0.05). DUS showed that 86.29% (107/124) of patients with BCS-HV showed occlusion of the hepatic vein, while CT or MRI showed that only 4.55%(5/110) patients had this manifestation (p < 0.001). Collateral circulation of hepatic veins was visible in 70.97% (88/124) of BCS-HV patients on DUS, while only 4.55% (5/110) were visible on CT or MRI (p < 0.001); (4) Conclusions: In addition to an established history of PA-containing plant exposure, local hepatic vein stenosis and the presence of collateral circulation of hepatic veins are the most important differential imaging features of these two diseases. However, these important imaging features may be missed by enhanced CT or MRI, leading to an incorrect diagnosis. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are widely distributed natural toxins and have been extensively studied for their hepatotoxicity. However, PA-induced pulmonary toxicity remains less studied regarding the initiating mechanism and treatment approaches. Our previous study demonstrated the formation of pyrrole–hemoglobin adducts after PA exposure in vivo, which is suspected to affect the oxygen-carrying capacity of erythrocytes [red blood cells (RBCs)] consequently. The present study aimed to investigate the effects of PAs on the oxygen-carrying capacity of RBCs and the potential of targeting RBC-mediated hypoxia to alleviate PA-induced lung injury. First, rats were treated with retrorsine (RTS) or monocrotaline (MCT) intravenously at 0.2 mmol/kg. The results of Raman spectrometry analysis on blood samples revealed both RTS and MCT significantly reduced the oxygen-carrying capacity of RBCs. Further, MCT (0.2 mmol/kg) was orally given to the rats with or without pretreatment with two doses of erythropoietin (Epo, 500 IU/kg/dose every other day), an RBC-stimulating agent. Biochemical and histological results showed pretreatment with Epo effectively reduced the cardiopulmonary toxicity induced by MCT. These findings provide the first evidence that adduction on hemoglobin, and the resulting RBC damage and impaired oxygen-carrying capacity, are the major initiating mechanism underlying PA-induced pulmonary arterial hypertension (PAH), while targeting the RBC damage is a potential therapeutic approach for PA-induced lung injury. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) can be transferred between plants via soil. Indicators of PAs in tea products are useful for tea garden management. In the present work a total of 37 weed species, 37 weed rhizospheric soils and 24 fresh tea leaf samples were collected from tea gardens, in which PAs were detected in 35 weeds species, 21 soil samples and 10 fresh tea leaves samples. In Shexian tea garden, 12.9 μg/kg of intermedine (Im) in one bud plus three leaves, 1.40 and 14.6 μg/kg of intermedine-N-oxide (ImNO) in one bud plus two leaves and one bud plus three leaves were detected, which were transferred from the PA-producing weeds via soil. However, no PAs were detected in fresh tea leaves collected from Langxi tea garden. The results indicated that synthesis of PAs in weeds and their transfer through the weed-soil-fresh tea leaf route varied with soil environments in different tea gardens. [ABSTRACT FROM AUTHOR]

Background and Aims: The definitive treatment for pyrrolizidine alkaloids (PAs)‐induced hepatic sinusoidal obstruction syndrome (HSOS) is not available. The effectiveness of anticoagulation therapy remains controversial. The efficacy of low molecular weight heparin (LMWH) should be investigated in patients and animal models, and the underlying mechanism should be explored. Methods: The prognosis of patients with PAs‐HSOS who received anticoagulation therapy was retrospectively analysed. The effect of enoxaparin on the liver injury was determined in animal models of monocrotaline (MCT)‐induced HSOS was determined, and the underlying mechanism was investigated using a murine model. Results: The cumulative survival rate of patients with PAs‐induced HSOS was 60.00% and 90.90% in the non‐anticoagulation group and anticoagulation group. Enoxaparin attenuated liver injury effectively in a rat model of MCT‐induced HSOS. Additionally, the improvement of severe liver injury was observed in MCT‐treated mice after the administration of enoxaparin (40 mg/kg). The alleviation of liver injury was observed in mice with hepatocyte‐specific deletion of oncostatin M (Osm△Hep). In MCT‐treated mice administrated with enoxaparin, no significant differences in liver injury were observed between Osm△Hep mice and Osmflox/flox mice. Additionally, adenovirus‐mediated overexpression of Osm resulted in severe liver injury in MCT‐induced mice after the administration of enoxaparin. Conclusions: LMWH attenuated severe liver injury in patients with PAs‐Induced HSOS and animal models of MCT‐induced HSOS, which provides a rationale for the application of anticoagulation therapy. [ABSTRACT FROM AUTHOR]

Three new compounds, the silphiperfolanol angelate ester umutagarananol (1), the macrocyclic pyrrolizidine alkaloids umutagarinine A and B (2–3), and five known secondary metabolites (4–8) were isolated from the CH2Cl2−MeOH (1 : 1) extract of the roots and the stem bark of Solanecio mannii (Hook. f.) (Asteraceae). The isolated compounds were characterized by NMR and IR spectroscopic, and mass spectrometric analyses, whereas the relative stereochemistry of 4 was established by NAMFIS‐based combined computational and solution NMR analysis. Synthetic modification of 5 provided two new compounds, 2‐angeloyloxy‐4,8‐epoxypresilphiperfolane (9) and 2‐angeloyloxy‐4,8‐epoxypresilphi‐perfolane (10). The crude extracts and the isolated constituents showed weak antibacterial activities (EC50 0.7–13.3 mM) against the Gram‐negative Escherichia coli and the Gram‐positive Bacillus subtilis. Compounds 2, 3 and 4 exhibited strong cytotoxicity against MCF‐7 human breast cancer cells, with EC50 values of 35.6, 21.7 and 12.5 μM, respectively. [ABSTRACT FROM AUTHOR]

Spices, herbs, and tea have been used for dietary purposes since ancient times. Some of them may contain toxic natural plant toxins synthesized in plant secondary metabolism or because of co-harvesting or horizontal product transfer with other plants. This review highlights the main extraction and clean-up procedures developed for alkenylbenzenes, pyrrolizidine and tropane alkaloids from plant-derived food products published between 2010 and 2020. Analytical methods are discussed, paying attention to those based on chromatographic separation coupled with classical or mass spectrometry detectors. Finally, an overview of the occurrence of these compounds in the studied matrices is performed. [ABSTRACT FROM AUTHOR]

A method for the synthesis of isoindoline derivatives was developed based on a domino reaction involving a donor-acceptor cyclopropane containing a bromomethyl group in the ortho position of the aromatic substituent and structurally varied primary amines (anilines, benzylamines, cycloalkylamines). It was shown that the resulting N-benzyl-1,3-dihydroisoindole under hydrogenolysis conditions at room temperature underwent selective cleavage of the exocyclic N–CH2Ar bond followed by in situ lactamization to form benzo[b]pyrrolizidinone. The same product was obtained in a domino reaction starting with reduction of the azidomethyl group at the ortho position of the donor aromatic substituent. Similarly, the generation of the o-2-aminoethyl group was accompanied by two successive cyclization reactions leading to the formation of benzo[e]indolizidinone. [ABSTRACT FROM AUTHOR]

The persistent evolution of drug-resistant influenza strains represents a global concern. The innovation of new treatment approaches through drug screening strategies and investigating the antiviral potential of bioactive natural-based chemicals may address the issue. Herein, we screened the anti-influenza efficacy of some biologically active indole and β-carboline (βC) indole alkaloids against two different influenza A viruses (IAV) with varied host range ranges; seasonal influenza A/Egypt/NRC098/2019(H1N1) and avian influenza A/chicken/Egypt/N12640A/2016(H5N1). All compounds were first assessed for their half-maximal cytotoxic concentration (CC50) in MDCK cells and half-maximal inhibitory concentrations (IC50) against influenza A/H5N1. Intriguingly, Strychnine sulfate, Harmalol, Harmane, and Harmaline showed robust anti-H5N1 activities with IC50 values of 11.85, 0.02, 0.023, and 3.42 µg/ml, respectively, as compared to zanamivir and amantadine as control drugs (IC50 = 0.079 µg/ml and 17.59 µg/ml, respectively). The efficacy of the predefined phytochemicals was further confirmed against influenza A/H1N1 and they displayed potent anti-H1N1 activities compared to reference drugs. Based on SI values, the highly promising compounds were then evaluated for antiviral efficacy through plaque reduction assay and consistently they revealed high viral inhibition percentages at non-toxic concentrations. By studying the modes of antiviral action, Harmane and Harmalol could suppress viral infection via interfering mainly with the viral replication of the influenza A/H5N1 virus, whilst Harmaline exhibited a viricidal effect against the influenza A/H5N1 virus. Whereas, Strychnine sulfate elucidated its anti-influenza potency by interfering with viral adsorption into MDCK cells. Consistently, chemoinformatic studies showed that all studied phytochemicals illustrated HB formations with essential peptide cleft through the NH of indole moiety. Among active alkaloids, harmalol displayed the best lipophilicity metrics including ligand efficiency (LE) and ligand lipophilic efficiency (LLE) for both viruses. Compounds geometry and their ability to participate in HB formation are very crucial. [ABSTRACT FROM AUTHOR]

Retrosine (RTS) is a pyrrolozidine alkaloid and a known hepatotoxin that widely exist in nature. The mechanisms involved in toxic action of pyrrolizidine alkaloids need further investigation. The objective of the present study was to evaluate the correlation of RTS hepatotoxicity with hepatic RTS concentration and pyrrole-protein adduction. Mice were intragastrically treated with RTS alone or RTS and ketoconazole (KTZ) simultaneously. Sera and liver tissues were collected at various time points after administration, followed by the determination of changes in serum transaminase activity, hepatic RTS concentration and pyrrole-protein adduction. The correlation of RTS hepatotoxicity with hepatic RTS concentration and hepatic pyrrole-protein adduction were examined by use of Sigmoid-Emax PK/PD models. Dose-dependent hepatotoxicity, hepatic RTS concentration and pyrrole-protein adduction were observed in the animals, which could be modulated by co-treatment with KTZ. The fit parameters indicated pyrrole-protein adduction was more closely related with liver injury than hepatic RTS concentration. Similar correlation was observed in mice given low-dose of RTS for 4 consecutive days. RTS hepatotoxicity is correlated with hepatic pyrrole-protein adduction derived from RTS rather than hepatic RTS concentration. The observed protein modification would be a good indicator to predict the hepatoxicity of RTS at low dose. [Display omitted] • Hepatic protein covalent binding was found to be closely associated with pyrrolizidine alkaloid-induced hepatotoxicity. • A PK/PD model was established to predict the hepatotoxicity induced by retrorsine after low-dose continuous administration. • Pyrrole-protein adduction is a sensitive and suitable indicator to reflect the liver toxicity induced by retrorsine. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells. [ABSTRACT FROM AUTHOR]

Skin cancer has high rates of mortality and therapeutic failure. In this study, to develop a multi-agent strategy for skin cancer management, the selective cytotoxicity of several alkaloid fractions and pure alkaloids isolated from Amaryllidaceae species was evaluated in melanoma cells. In addition, UVB-stimulated keratinocytes (HaCaT) were exposed to seven alkaloid fractions characterized by GC-MS, and the production of intracellular reactive oxygen species (ROS) and IL-6, were measured to evaluate their photoprotection effects. The Eucharis caucana (bulb) alkaloid fraction (20 μg/ml) had a clear effect on the viability of melanoma cells, reducing it by 45.7% without affecting healthy keratinocytes. This alkaloid fraction and tazettine (both at 2.5 μg/ml) suppressed UVB-induced ROS production by 31.6% and 29.4%, respectively. The highest anti-inflammatory potential was shown by the Zephyranthes carinata (bulb) alkaloid fraction (10 μg/ml), which reduced IL-6 production by 90.8%. According to the chemometric analysis, lycoramine and tazettine had a photoprotective effect on the UVB-exposed HaCaT cells, attenuating the production of ROS and IL-6. These results suggest that Amaryllidaceae alkaloids have photoprotective and therapeutic potential in skin cancer management, especially at low concentrations. [ABSTRACT FROM AUTHOR]

Background: Farfarae Flos (FF) is a frequently used traditional herbal medicine with outstanding antitussive actions. The adulteration of FF decoction pieces is common. Objective: This study aimed to study the effect of adulteration on the safety and quality of FF decoction pieces. Methods: The proportion of impurities was conducted by cone quartering method. A simple and accurate ultra-highperformance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was established to simultaneous determinate three pyrrolizidine alkaloids (PAs) as endogenous toxic compounds in FF. The traditional medicinal parts (flower bud), impurities (pedicel and rhizome) and unselected samples were determined respectively. The values of estimated daily intake (EDI) and margin of exposure (MOE) were used for risk assessment. Results: Twenty batches of samples were collected from different habitats, and the proportion of impurities ranged from 17.51% to 41.27%. Pedicel and rhizome were the main impurities, accounting for more than 87.40% of the total impurities. The content of PAs in impurities was significantly higher. The EDI value range was 5.34 to 16.59 lg/kg bw/day, which was much higher than the standard safety value of 7.00X10-3μg/kg bw/day. The MOE values ranges for life long time and shorter exposure were 14.29 to 44.37 and 371.53 to 1153.63, respectively, indicating that at least 80% of the samples had safety risks. Correlation analysis showed that the proportion of adulterated impurities had significant correlation with the values of EDI and MOE. Conclusions: Adulteration of non medicinal parts may significantly increase the risk of medications of FF decoction pieces. Highlights: This study provides an efficient methodology reference for the control of PAs and a basis for adulteration to affect the safety and quality of FF decoction pieces. [ABSTRACT FROM AUTHOR]

Background: Studies on pyrrolizidine alkaloid (PA) contamination in honey produced in China are scarce. Previously reported HPLC-MS/MSmethods 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 (2.1mm 100mm, 1.7 lm) column with (A) 0.1% formic acid aqueous solution containing 5mM 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 lg/kg and 0.22-0.82 lg/kg, respectively, except for that of clivorine, for which LOD and LOQ were 2.03 and 6.78 lg/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 lg/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. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to identify and determine the content of toxic components(diester‑type alkaloids) in Aconitum brachypodum, providing scientific support for the safe clinical use of this medicinal herb and offering a basis for quality control. Methods: Acidic water was used to extract the alkaloids from Aconitum brachypodum, which were then extracted with petroleum ether. Toxic components were separated and extracted with chloroform from the acid water, with the phenolic acidic fractions absorbed by alkaline oxidized alumina. The toxic components were identified by thin‑layer chromatography (TLC), and the content was measured by ultraviolet (UV)–visible spectrophotometry. Results: A method for special thin‑layer identification of toxic components and general determination of total alkaloid content in Aconitum brachypodum was established. The mean content of toxic diterpenoid alkaloids in Aconitum brachypodum cultivated in the Dongchuan district of Kunming was approximately 0.30%. Conclusion: Selective extraction of toxic alkaloids in Aconitum brachypodum by chemical methods, identification by TLC, and content determination by UV–visible spectrophotometry can control the toxicity of this herb, guide production, and ensure clinical safety. [ABSTRACT FROM AUTHOR]

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. [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]

Increasing studies have identified the potential of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in non-alcoholic fatty liver disease (NAFLD) treatment. Hence, we further focused on the potential of adipose-derived MSC (ADSC)-EVs in NAFLD by delivering miR-223-3p. The uptake of isolated ADSC-EVs by hepatocytes was assessed, and the expression of miR-223-3p in ADSC-EVs and hepatocytes was characterized. It was established that miR-223-3p, enriched in ADSC-EVs, could be delivered by ADSC-EVs into hepatocytes. Using co-culture system and gain-of-function approach, we evaluated the effect of ADSC-EVs carrying miR-223-3p on lipid accumulation and liver fibrosis in pyrrolizidine alkaloids (PA)-induced hepatocytes and a high-fat diet-induced NAFLD mouse model. Bioinformatics websites and dual-luciferase reporter gene assay were performed to determine the interactions between miR-223-3p and E2F1, which was further validated by rescue experiments. ADSC-EVs containing miR-223-3p displayed suppressive effects on lipid accumulation and liver fibrosis through E2F1 inhibition, since E2F1 was demonstrated as a target gene of miR-223-3p. The protective role of ADSC-EVs by delivering miR-223-3p was then confirmed in the mouse model. Collectively, this study elucidated that ADSC-EVs delayed the progression NAFLD through the delivery of anti-fibrotic miR-223-3p and subsequent E2F1 suppression, which may suggest miR-223-3p-loaded ADSC-EVs to be a potential therapeutic approach for NAFLD. [ABSTRACT FROM AUTHOR]

1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid–liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected. [ABSTRACT FROM AUTHOR]

In this paper, I review the effects of the diet of ladybirds on chemical defence in this group of beetles. The tendency to reflex bleed and the diversity of autogenously produced alkaloids in different taxa may be evolutionarily related to diet and the likelihood of food limitation. Within predatory species, both prey quantity and quality have been shown to affect autogenous alkaloid production. A few ladybird predators have been suggested to adaptively sequester toxins from their prey for their own defence. However, in most cases the evidence for this is limited, with questions remaining about the costs of accumulated toxins and their defensive value, especially over and above pre-existing autogenous defence. Only a single case (Hyperaspis trifurcata and carminic acid) is well supported. In the case of ladybird predators acquiring pyrrolizidine alkaloids from the ragwort aphid Aphis jacobaeae, I show that toxin accumulation is not very costly and the aphid is even an essential prey for some ladybirds. However, the defensive value of pyrrolizidine alkaloids to ladybirds is still not investigated. Intraspecific diversity in autogenous chemical defence could be reinforced further if chemical protection is conferred via sequestered chemicals. However, to understand better how ladybird diet and chemical defence interact, we need a clearer grasp of how the defensive chemicals of food are resisted or tolerated by ladybirds. [ABSTRACT FROM AUTHOR]