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.

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.

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.

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.

High abundance of pyrrolizidine alkaloids in bee pollen collected in July 2019 from Southern Germany.

Pyrrolizidine alkaloids (PA) are secondary plant defense compounds and known pre-toxins when containing a 1,2-double bond. They are commonly produced by various plants and may thus be present in bee pollen which may be consumed by humans as food supplements. In this study, PA were determined in bee pollen samples from 57 locations in Southern Germany sampled by means of pollen traps in July 2019. Samples were analyzed by using palynological methodology and solid-phase extraction (SPE) followed by LC-MS/MS. In total, 52 pollen samples featured total pyrrolizidine alkaloids (ΣPA) with concentrations up to 48,000 ng/g bee pollen, while the N-oxides (NO) echinatine-NO and rinderine-NO clearly dominated. In contrast, the palynological analysis only detected 33 samples with pollen from PA-producing plants. Accordingly, the results showed that palynological analysis is not sufficient to determine PA in pollen. In addition, a risk assessment was followed to estimate the risk of the detected PA concentrations to humans.

Petasin is the main component responsible for the anti-adipogenic effect of Petasites japonicus.

Petasites japonicus is one of the most popular edible wild plants in Japan. Many biological effects of P. japonicus have been reported, including anti-allergy, anti-inflammation, and anticancer effects. Although its anti-obesity effect has been reported in several studies, the most important component responsible for this activity has not been fully elucidated. On screening the components that suppress adipocyte differentiation in 3T3-F442A cells, we found that the extract of the flower buds of P. japonicus has anti-adipogenic effect. Among the known major components of P. japonicus, petasin exhibited a potent anti-adipogenic effect at an IC50 value of 0.95 µM. Quantitative analysis revealed that the active component responsible for most of the anti-adipogenic effects of P. japonicus extract is petasin. Petasin suppressed the expression of markers of mature adipocytes (PPARγ, C/EBPα, and aP2). However, as isopetasin and petasol, analogs of petasin, did not exhibit these effects, it indicates that a double bond at the C11-C12 position and an angeloyl ester moiety were essential for the activity. Petasin affected the late stage of adipocyte differentiation and inhibited the expression of lipid synthesis factors (ACC1, FAS, and SCD1). Additionally, it was revealed that petasin could be efficiently extracted using hexane with minimal amount of pyrrolizidine alkaloids, the toxic components. These findings indicate that P. japonicus extract containing petasin could be a promising food material for the prevention of obesity.

Pyrrolizidine Alkaloid-Induced Hepatotoxicity Associated with the Formation of Reactive Metabolite-Derived Pyrrole-Protein Adducts.

Pyrrolizidine alkaloids (PAs) with 1,2-unsaturated necine base are hepatotoxic phytotoxins. Acute PA intoxication is initiated by the formation of adducts between PA-derived reactive pyrrolic metabolites with cellular proteins. The present study aimed to investigate the correlation between the formation of hepatic pyrrole-protein adducts and occurrence of PA-induced liver injury (PA-ILI), and to further explore the use of such adducts for rapidly screening the hepatotoxic potency of natural products which contain PAs. Aqueous extracts of Crotalaria sessiliflora (containing one PA: monocrotaline) and Gynura japonica (containing two PAs: senecionine and seneciphylline) were orally administered to rats at different doses for 24 h to investigate PA-ILI. Serum alanine aminotransferase (ALT) activity, hepatic glutathione (GSH) level, and liver histological changes of the treated rats were evaluated to assess the severity of PA-ILI. The levels of pyrrole-protein adducts formed in the rats' livers were determined by a well-established spectrophotometric method. The biological and histological results showed a dose-dependent hepatotoxicity with significantly different toxic severity among groups of rats treated with herbal extracts containing different PAs. Both serum ALT activity and the amount of hepatic pyrrole-protein adducts increased in a dose-dependent manner. Moreover, the elevation of ALT activity correlated well with the formation of hepatic pyrrole-protein adducts, regardless of the structures of different PAs. The findings revealed that the formation of hepatic pyrrole-protein adducts-which directly correlated with the elevation of serum ALT activity-was a common insult leading to PA-ILI, suggesting a potential for using pyrrole-protein adducts to screen hepatotoxicity and rank PA-containing natural products, which generally contain multiple PAs with different structures.

Chemical Constituents and Biological Properties of Genus Doronicum (Asteraceae).

The genus Doronicum, belonging to tribe Senecioneae (Fam. Asteraceae), is found mainly in the Asia, Europe and North Africa. This genus of plant has always been used in traditional medicinal treatments due to the many biological properties shown such as killing parasitic worms and for relieving constipation, as well as to improve heart health, to alleviate pain and inflammation, to treat insect bites, etc. According to the World Flora the genus Doronicum contains 39 subordinate taxa.[1-3] The purpose of this article, which covers data published from 1970 to 2021 with more than 110 articles, aims to carry out a complete and critical review of the Doronicum genus, examining traditional uses and reporting the antioxidant, antimicrobial, anti-inflammatory and antitumor activity shown from crude extracts or essential oils, and from single isolated compounds. Furthermore, critical considerations of the published data have been highlighted by comparing them with the results obtained from species of other genus belonging to the Asteraceae family.

Pyrrolizidine alkaloids in tea, herbal tea and iced tea beverages- survey and transfer rates.

The transfer rate of 37 pyrrolizidine alkaloids (PA) found in ten naturally contaminated teas and herbal teas to their brews was studied in detail. Mixed herbal, peppermint, red bush, senna, black tea and green tea infusions were prepared according to the ISO guide and vendor's instructions, respectively, and parameters like herb-to-water ratio, steeping time and multiple extractions studied. In general, a transfer rate of 38-100% (median 95%) for brews following vendor's instructions was determined. The total concentration range of PA in these ten samples was 154-2412 ng/g (median 422 ng/g) in the herb and for single analytes 0.1-170 ng/g. Seven of the 37 PA occurred unexpectedly; these were tentatively identified and quantified by liquid chromatography-high resolution mass spectrometry (LC-HR-MS), since their contributions to total PA-content matter. Additionally, 46 iced tea beverages were analysed for their PA-load, determined to be in the range 0-631 ng/L (median 40 ng/L). The applied solid-phase extraction (SPE) clean-up turned out to be capable of separating PA in the free base pyrrolizidine alkaloids (PAFB) and their N-oxides (PANO) in a two-step elution, which was a valuable tool to support identification of unexpected PA. Further, atropine was found in 50% of the ten tea herb samples (range: 1-4 ng/g) and in 13% of the iced tea beverage samples (range: 2-65 ng/L).

Determination of Toxic Pyrrolizidine Alkaloids in Traditional Chinese Herbal Medicines by UPLC-MS/MS and Accompanying Risk Assessment for Human Health.

Pyrrolizidine alkaloids (PAs) are a class of natural toxins with hepatotoxicity, genotoxicity and carcinogenicity. They are endogenous and adulterated toxic components widely found in food and herbal products. In this study, a sensitive and efficient ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was used to detect the PAs in 386 kinds of Chinese herbal medicines recorded in the Chinese Pharmacopoeia (2020). The estimated daily intake (EDI) of 0.007 µg/kg body weight (bw)/day was adopted as the safety baseline. The margin of exposure (MOE) approach was applied to evaluate the chronic exposure risk for the genotoxic and carcinogenic potential of PAs. Results showed that PAs was detected in 271 out of 386 samples with a content of 0.1-25,567.4 µg/kg, and there were 20 samples with EDI values above the baseline, 0.007 µg/kg bw/day. Beyond that, the MOE values for 10 out of 271 positive samples were below 10,000. Considering the actual situation, Haber's rule was used to assume two weeks exposure every year during lifetime, and still the MOE values for four out of 271 positive samples were under 10,000, indicating these products may have potential health risk. The developed method was successfully applied to detect the PAs-containing Chinese herbal medicines. This study provides convincing data that can support risk management actions in China and a meaningful reference for the rational and safe use of Chinese herbal medicines.

CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

Analysis and chemotaxonomic significance of pyrrolizidine alkaloids from two Boraginaceae species growing in Algeria.

In the current study the pyrrolizidine alkaloid profiles of the species Echium sabulicola ssp. decipiens (Pomel) Klotz and Solenanthus lanatus DC were studied in various extracts. In addition, a chemotaxonomic study within the genus and the family was carried out. The analysis was carried out by using gas chromatography coupled to mass spectrometry and by comparing the Kovats Indices and molecular and fragment ions with literature data. Twenty-three alkaloids were tentatively identified. The results showed the presence of already reported compounds as well as previously unreported ones leading both to a confirmation of the botanical classification of the two studied species and to a brand new path in the chemotaxonomy of Boraginaceae family. The presence of some pyrrolizidine alkaloids sets limits for the usage of these plants for medicinal purposes. The identified compounds confirm the botanical classification of the studied species as members of the Boraginaceae family and their presence advices against their use in the ethnopharmacological field.

Pyrrolizidine alkaloids and beehive products: A review.

Pyrrolizidine alkaloids (PA) are secondary metabolites of plants, which are mostly found in the genus Senecio, Echium, Crotalaria, and Eupatorium. The presence of 1,2-unsaturated PA in foods is a concern to food regulators around the world because these compounds have been associated to acute and chronic toxicity, mainly in the liver. The intake foods with PA/PANO usually occur through accidental ingestion of plants and their derivatives, besides to products of vegetal-animal origin, such as honey. PA/PANO are transferred to honey by their presence in nectar, honeydew, and pollen, which are collected from the flora by bees. In addition to honey, other beekeeping products, such as pollen, royal jelly, propolis, and beeswax, are also vulnerable to PA contamination. In this context, this review provides information about chemical characteristics, regulation, and toxicity, as well as summarizes and critically discusses scientific publications that evaluated PA in honeys, pollens, royal jelly, and propolis.

Tu-San-Qi (Gynura japonica): the culprit behind pyrrolizidine alkaloid-induced liver injury in China.

Herbs and dietary supplement-induced liver injury (HILI) is the leading cause of drug-induced liver injury in China. Among different hepatotoxic herbs, the pyrrolizidine alkaloid (PA)-producing herb Gynura japonica contributes significantly to HILI by inducing hepatic sinusoidal obstruction syndrome (HSOS), a liver disorder characterized by hepatomegaly, hyperbilirubinemia, and ascites. In China, G. japonica has been used as one of the plant species for Tu-San-Qi and is often misused with non-PA-producing Tu-San-Qi (Sedum aizoon) or even San-Qi (Panax notoginseng) for self-medication. It has been reported that over 50% of HSOS cases are caused by the intake of PA-producing G. japonica. In this review, we provide comprehensive information to distinguish these Tu-San-Qi-related herbal plant species in terms of plant/medicinal part morphologies, medicinal indications, and chemical profiles. Approximately 2156 Tu-San-Qi-associated HSOS cases reported in China from 1980 to 2019 are systematically reviewed in terms of their clinical manifestation, diagnostic workups, therapeutic interventions, and outcomes. In addition, based on the application of our developed mechanism-based biomarker of PA exposure, our clinical findings on the definitive diagnosis of 58 PA-producing Tu-San-Qi-induced HSOS patients are also elaborated. Therefore, this review article provides the first comprehensive report on 2214 PA-producing Tu-San-Qi (G. japonica)-induced HSOS cases in China, and the information presented will improve public awareness of the significant incidence of PA-producing Tu-San-Qi (G. japonica)-induced HSOS and facilitate future prevention and better clinical management of this severe HILI.

Genetic diversity, reproductive capacity and alkaloids content in three endemic Alkanna species.

The Balkans endemic species Alkanna primuliflora Griseb., A. stribrnyi Velen., and A. graeca Boiss. & Spruner have limited distribution in the Balkan Peninsula and a large variation in the morphological characteristics. The populations of the three Alkanna species in the Bulgarian flora are small and fragmented. There are no previous reports on the chemical profile or on the embryology of these species. The hypothesis was that the limited distribution of A. primuliflora, A. stribrnyi, and A. graeca was due to their reproductive capacity and genetic diversity. Furthermore, we hypothesized that the three species will contain pyrrolizidine alkaloids (PAs), as other species of the genus Alkanna (Boraginaceae), but they would have differential alkaloids composition. The population genetic structure and differentiation showed a clear distinction between species and revealed average levels of genetic diversity among the natural populations of the three Alkanna species. The embryological investigation observed stability of the processes in the male and female generative spheres and high viability of mature pollen and embryo of the three species. The normal formation of male and female gametophytes without deviations or degenerative processes, and observed levels of genetic diversity between Alkanna individuals are important in maintaining the size and resilience of the Alkanna populations. Eight alkaloids were identified by GC-MS in A. primuliflora and A. graeca and six alkaloids in A. stribrnyi. The main pyrrolizidine alkaloids (PAs) in all investigated species was triangularine. A. primuliflora and A. graeca showed similar chemical composition that comprised 9-angeloylretronecine, 7-tigloylretronecine, 9-tigloylretronecine, triangularicine, dihydroxytriangularine, dihydroxytriangularicine, whereas, in A. stribrnyi 9-tigloylretronecine, triangularicine and dihydroxytriangularicine were not found. This is the first report on the presence of PAs in A. primuliflora, A. stribrnyi and A. graeca. Besides, this is the first report on the embryology of these endemic species. The results contribute to the knowledge of the three endemic Alkanna species and will facilitate policy-making and defining new strategies for their conservation.

Pyrrolizidine alkaloids in food and phytomedicine: Occurrence, exposure, toxicity, mechanisms, and risk assessment - A review.

Among naturally occurring plant constituents, the 1,2-unsaturated pyrrolizidine alkaloids (in the following termed 'PAs') play a distinct role because of the large number of congeners occurring in nature and the pronounced toxicity of some congeners. Several PAs are hepatotoxic in humans, experimental and farm animals and were shown to be potent hepatocarcinogens in laboratory rodents. Although the general mode of action leading to toxicity has been elucidated, i.e., being mediated by metabolic conversion of the parent molecule into a highly reactive electrophile capable of attacking cellular target molecules, major questions related to the risk assessment of PAs remain unresolved. It was the aim of a workshop held in September 2018 to shed more light on the occurrence, exposure, mode of action, toxicokinetics and -dynamics of PAs to improve the scientific basis for an advanced toxicological risk assessment. The contributions in nine chapters describe the scientific progress using advanced analytical methods, studies in subcellular fractions, cell culture, experimental animals and humans and the use of PBPK modeling and structure-activity relationship considerations aiming at a better understanding of PA toxicity and genotoxicity. Since PAs differ considerably in their toxic potencies and substantial species differences in sensitivity towards PA exposure exist, a special emphasis was placed on these issues.

Absorption difference between hepatotoxic pyrrolizidine alkaloids and their N-oxides - Mechanism and its potential toxic impact.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are a group of phytotoxins widely present in about 3% of flowering plants. Many PA-containing herbal plants can cause liver injury. Our previous studies demonstrated that PA N-oxides are also hepatotoxic, with toxic potency much lower than the corresponding PAs, due to significant differences in their toxicokinetic fates. AIM OF STUDY: This study aimed to investigate the oral absorption of PAs and PA N-oxides for better understanding of their significant differences in toxicokinetics and toxic potency. MATERIALS AND METHODS: The oral absorption of PAs and PA N-oxides in rats and in rat in situ single pass intestine perfusion model was investigated. The intestinal permeability and absorption mechanisms of five pairs of PAs and PA N-oxides were evaluated by using Caco-2 monolayer model. RESULTS: The plasma concentrations of total PAs and PA N-oxides within 0-60 min were significantly lower in rats orally treated with a PA N-oxide-containing herbal alkaloid extract than with a PA-containing herbal alkaloid extract at the same dose, indicating that the absorption of PA N-oxides was lower than that of PAs. Using the rat in situ single pass intestine perfusion model, less cumulative amounts of retrorsine N-oxide in mesenteric blood were observed compared to that of retrorsine. In Caco-2 monolayer model, all five PAs showed absorption with Papp AtoB values [(1.43-16.26) × 10-6 cm/s] higher than those of corresponding N-oxides with Papp AtoB values lower than 1.35 × 10-6 cm/s. A further mechanistic study demonstrated that except for senecionine N-oxide, retrorsine N-oxide, and lycopsamine N-oxide, all PAs and PA N-oxides investigated were absorbed via passive diffusion. While, for these 3 PA N-oxides, in addition to passive diffusion as their primary transportation, efflux transporter-mediated active transportation was also involved but to a less extent with the efflux ratio of 2.31-3.41. Furthermore, a good correlation between lipophilicity and permeability of retronecine-type PAs and their N-oxides with absorption via passive diffusion was observed, demonstrating that PAs have a better oral absorbability than that of the corresponding PA N-oxides. CONCLUSION: We discovered that among many contributors, the lower intestinal absorption of PA N-oxides was the initiating contributor that caused differences in toxicokinetics and toxic potency between PAs and PA N-oxides.

Efficient Extraction of Pyrrolizidine Alkaloids from Plants by Pressurised Liquid Extraction - A Preliminary Study.

Pyrrolizidine alkaloids and their corresponding pyrrolizidine alkaloid-N-oxides are secondary plant constituents that became the subject of public concern due to their hepatotoxic, pneumotoxic, genotoxic, and cytotoxic effects. In contrast to the well-established analytical separation and detection methods, only a few studies have investigated the extraction of pyrrolizidine alkaloids/pyrrolizidine alkaloid-N-oxides from plant material. In this study, we have applied pressurized liquid extraction with the aim of evaluating the effect of various parameters on the recovery of pyrrolizidine alkaloids. The nature of the modifier (various acids, NH3) added to the aqueous extraction solvent, its concentration (1 or 5%), and the temperature (50 - 125 °C) were systematically varied. To analyse a wide range of structurally different pyrrolizidine alkaloids, Jacobaea vulgaris (syn. Senecio jacobaea), Tussilago farfara, and Symphytum officinale were included. Pyrrolizidine alkaloids were quantified by HPLC-MS/MS and the results obtained by pressurised liquid extraction were compared with the amount of pyrrolizidine alkaloids determined by an official reference method. Using this approach, increased rates of recovery were obtained for J. vulgaris (up to 174.4%), T. farfara (up to 156.5%), and S. officinale (up to 288.7%). Hence, pressurised liquid extraction was found to be a promising strategy for the complete and automated extraction of pyrrolizidine alkaloids, which could advantageously replace other time- and solvent-consuming extraction methods.

Pyrrolizidine Alkaloids: The Botanical Origin of Pollen Collected during the Flowering Period of Echium vulgare and the Stability of Pyrrolizidine Alkaloids in Bee Bread.

Previous studies have shown that pollen products sold as nutritional supplements and used in apitherapy may contain toxic pyrrolizidine alkaloids (PAs) if bees collect pollen from PA-containing plants, such as Echium vulgare. In this study, the botanical origin of pollen from two observation sites was studied. Despite a high PA content in pollen samples that bees collected during E. vulgare's flowering period, bees were found to collect relatively few Echium pollen loads. Thus, the monitoring of pollen loads collected at the apiaries is unviable to estimate the risk of PA contamination in pollen or bee bread. In a second step, the stability of PAs in bee bread samples containing PAs at concentrations of 2538 ng/g and 98 ng/g was assessed over a period of five or six months, respectively. No significant PA reduction was observed in bee bread stored at 15 °C, but there were overall PA reductions of 39% and 33% in bee bread stored at 30 °C, reflecting hive conditions. While PA N-oxides decreased over time, other types of PAs remained relatively stable. Monitoring PAs in pollen products remains important to ensure consumer safety and should include echivulgarine (and its N-oxide), the major PA type found in pollen from E. vulgare.