Mycotoxins and pyrrolizidine alkaloids in herbal dietary supplements.

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.

Gynura segetum induces hepatic sinusoidal obstruction syndrome in a child: A case report.

RATIONALE: Hepatic sinusoidal obstruction syndrome (HSOS), which includes hepatic stasis and portal hypertension, is a rare vascular disorder of the liver. It is often associated with hematopoietic stem cell transplantation. It is also possible to treat this disease using Chinese herbal medicines that contain pyrrolizidine alkaloids (PAs). This disease is extremely rare in children and poses a serious threat to their health. To our knowledge, this is the first case of HSOS in a child with PAs. PATIENT CONCERNS: We report a 4-year-old boy suffering from abdominal pain, hepatomegaly, massive ascites, elevated liver enzyme level, and severe portal hypertension as a result of the consumption of Gynura segetum (also known as Tusanqi in Chinese, a traditional herbal medicine containing PAs). DIAGNOSES: The child was finally diagnosed with PA-HSOS based on pathological diagnosis and imaging examination. INTERVENTION: With active symptomatic and supportive care and sequential anticoagulation therapy, the abdominal distension and liver function improved in the patient. OUTCOMES: The patient was eventually recovered. The levels of liver enzymes, hemoglobin, and bilirubin were normal, and the international normalized ratio fluctuated between 2.0 and 3.0 during 1-year follow-up after discharge. LESSONS: This case report emphasizes the prevention of Chinese herb-induced liver injury in children and the importance of active long-term sequential anticoagulant therapy to reduce the progressive damage of PA-HSOS in the liver.

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.

Diagnosis, toxicological mechanism, and detoxification for hepatotoxicity induced by pyrrolizidine alkaloids from herbal medicines or other plants.

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.

Toxins in Botanical Drugs and Plant-derived Food and Feed - from Science to Regulation: A Workshop Review.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

Analysis of potential risks of clinical application of Yi Dian Hong and its proprietary Chinese medicines: A review.

Yi Dian Hong, belonging to the Asteraceae family, finds widespread use in traditional Chinese medicine for its effectiveness in clearing heat, detoxifying, promoting blood circulation, reducing swelling, and cooling the blood. Modern medical research has revealed that Yi Dian Hong and its proprietary Chinese medicines possess biological functions such as inhibiting tumor-specific angiogenesis and regulating immune-related molecules. However, studies have identified that the primary component of Yi Dian Hong contains pyrrolizidine alkaloids (PAs), a toxic substance with potential risks to the liver, lungs, genes, and a propensity for carcinogenicity. Many countries impose strict controls on the content of PAs in herbal medicines and products. Unfortunately, China currently lacks relevant content standards, thereby introducing greater clinical application risks. To ensure the safety of clinical use of Yi Dian Hong, this review will analyze the risk associated with Yi Dian Hong and its proprietary Chinese medicines in clinical applications based on the PAs content in these medicines and provide recommendations.

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.

Pyrrolizidine Alkaloids in Tea (Camellia sinensis L.) from Weeds through Weed-Soil-Tea Transfer and Risk Assessment of Tea Intake.

Pyrrolizidine alkaloids (PAs) have been detected in tea and can threaten human health. However, the specific source of PAs in tea is still unclear. Here, 88 dried tea products collected from six major tea-producing areas in Anhui Province, China, were analyzed. The detection frequency was 76%. The content of total PAs in dried tea was between 1.1 and 90.5 µg/kg, which was all below the MRL recommended by the European Union (150 µg/kg). In the Shexian tea garden, PAs in the weeds and weed rhizospheric soil around tea plants and the fresh tea leaves were analyzed. Intermedine (Im), intermedine-N-oxide (ImNO), and jacobine-N-oxide (JbNO) were transferred through the weed-to-soil-to-tea route into the fresh tea leaves; only Im and ImNO were detected in dried tea samples. Potential risk of the total PAs in the tea infusion was assessed according to the margin of exposure method, and it might be a low concern for public health.

Short-term exposure of dairy cows to pyrrolizidine alkaloids from tansy ragwort (Jacobaea vulgaris Gaertn.): effects on health and performance.

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.

OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects.

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.

Pyrrolizidine Alkaloids in Food on the Italian Market.

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.

Occurrence and associated health risks of pyrrolizidine alkaloids in supplements marketed in Ghana for improved sexual performance.

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.

Vacuum differential mobility spectrometry combined with column-switching liquid chromatography- mass spectrometry for the analysis of pyrrolizidine alkaloids in tea samples.

The benefit of combining liquid chromatography (LC), supercritical fluid chromatography (SFC) and vacuum Differential Mobility Spectrometry - Mass Spectrometry (vDMS-MS) was investigated for the analysis of fourteen diastereomeric pyrrolizidine alkaloids (PA); intermedine, echinatine, lycopsamine, indicine, intermedine-N-oxide, echinatine-N-oxide, indicine-N-oxide, lycopsamine-N-oxide, senecivernine, senecionine, jacobine, senecivernine-N-oxide, senecionine-N-oxide, retrorsine. The mobile phase composition (15-100% MeOH and ACN), flow rate (8-100 µL/min), vDMS cell pressure, and F value showed an effect on the mobility behavior of the analytes. At 15% MeOH with a flow rate of 100 µL/min and 33 mbar vDMS pressure, 8 out 14 PA could be partially or totally separated by vDMS-MS. As well as providing an additional separation dimension vDMS improved the selectivity and a 5-minute assay method was developed for the quantification of 10 out of 14 single diastereomeric PA in tea samples, using a short LC column-switching and hyphenated to vDMS-MS in the selected ion monitoring mode. The performance of the method was found to be comparable with a 12-minute standard LC-MS/MS method using detection in the selected reaction monitoring mode. Additionally, the combination of vDMS and SFC-MS was investigated and suggests that the mixture of CO2/MeOH influences the CV shifting of the PA to more negative compensation voltage, and the signal-to-noise ratio is improved by a factor of three compared to SFC-MS without vDMS.

Target screening method for the quantitative determination of 118 pyrrolizidine alkaloids in food supplements, herbal infusions, honey and teas by liquid chromatography coupled to quadrupole orbitrap mass spectrometry.

An analytical procedure for the screening of 118 pyrrolizidine alkaloids (PAs) was successfully validated and applied to their quantitative determination in food supplements, herbal infusions, honey, and teas. It provides the reliable analyte identification by high-resolution tandem mass spectrometry (HRMS/MS), the accurate determination of 21 regulated PAs, and broad contamination profiles. 10% of 281 analyzed samples resulted contaminated at levels above the maximum levels (MLs) of European legislation. The contamination of herbal infusions of mixed plants can represent a possible health concern (23%; mean of PA sum above ML). A high number of PAs not included in the regulation was detected in honey and herbal food supplements, but their contribution was only relevant to the overall level in honey. The results indicate the need to continue collecting contamination data in food supplements and infusions of mixed herbs and to expand the PA-pool to be monitored in honey and related products.

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.

Risk assessment of short-term intake of pyrrolizidine alkaloids in food: derivation of an acute reference dose.

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.

Formation of DHP-DNA Adducts from Rat Liver Microsomal Metabolism of 1,2-Unsaturated Pyrrolizidine Alkaloid-Containing Plant Extracts and Dietary Supplements.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals. We previously determined that carcinogenic PAs and PA N-oxides commonly form a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, namely, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4. This set of DHP-DNA adducts has been implicated as a potential biomarker of PA-induced liver tumor initiation from metabolism of individual carcinogenic PAs. To date, it is not known whether this generality occurs from metabolism of PA-containing plant extracts. In this study, we investigate the rat liver microsomal metabolism of nine PA-containing plant extracts and two PA-containing dietary supplements in the presence of calf thymus DNA. The presence of carcinogenic PAs and PA N-oxides in plant extracts was first confirmed by LC-MS/MS analysis with selected reaction monitoring mode. Upon rat liver microsomal metabolism of these PA-containing plant extracts and dietary supplements, the formation of this set of DHP-DNA adducts was confirmed. Thus, these results indicate that metabolism of PA-containing plant extracts and dietary supplements can generate DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts, thereby potentially initiating liver tumor formation.

Profiling of pyrrolizidine alkaloids using a retronecine-based untargeted metabolomics approach coupled to the quantitation of the retronecine-core in medicinal plants using UHPLC-QTOF.

Pyrrolizidine alkaloids (PA) are secondary metabolites of high toxicological relevance. Several PA quantitative methodologies were developed based on a limited number of certified standards, including time consuming solid phase extraction (SPE) purification steps. Herein, we shed light on the variability of PA in herbal extracts and propose a quantification methodology based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) for the evaluation of the total PA content as retronecine-equivalents (RE) directly from crude matrices. Particularly in the focus of the investigation were Alkanna spp. (Boraginaceae), which possess a wide range of pharmaceutical properties. In addition, a comparative PA screening of crude and SPE enriched extracts was performed and PA-containing plants from Fabaceae and Compositae families were included to demonstrate universal applicability. In total, 105 PA were identified using HRMSe experiments, specific MS/MS fragmentation PA patterns, a customized in-house library and literature data. Among them, 18 glycosidic PA derivatives were reported for the first time in literature. Using a hierarchical clustering approach, PA distribution in herbal extracts was shown to be family-dependent and significantly different among species. This was further supported by the results of the total PA concentrations, obtained using a retronecine/heliotridine/internal standard-based targeted UHPLC-HRMS quantification method, which varied from 8.64 ± 0.08-3096.28 ± 273.72 µg RE/g extract dry weight in shoots extracts of Alkanna spp. and leaves extracts of Crotalaria retusa L. respectively. Worth mentioning is that the procedure allowed to quantify PA in Alkanna spp. If the procedure based on 35 specific PA recommended by European regulations had been used, results would have been equal to zero for the four species since none were observed in Alkanna spp. Finally, by combining the RE results with the corresponding dereplication results, a customized correction factor for each extract (ranging from 2.12 to 2.48) was assessed leading to a more accurate estimate of the PA content regardless of the molecular weight of each PA. The present methodology will facilitate PA quantification directly from crude extracts and avoid the underestimation the real PA content due to limited availabilty of authentic reference compounds in botanical extracts used in phytomedicines or food supplements/cosmetics.

An analytical platform for the screening and identification of pyrrolizidine alkaloids in food matrices with high risk of contamination.

An analytical platform for the detection of pyrrolizidine alkaloids (PAs) in honey, pollen, teas, herbal infusions, and dietary supplements is proposed; it includes a wide-scope suspect screening method, based on a diagnostic product ion filtering strategy for the characterization of PAs, and a target screening and identification method for the high-throughput detection of 118 PAs of a high-resolution mass spectral library. Salting-out assisted liquid-liquid extraction of aqueous extracts combined to ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry was employed. The limit of identification (0.6-30 µg kg-1) of 28 standards were fit-for-purpose in PA-monitoring applications, with a false negative rate <1.3 % at 4 µg L-1. The wide-scope suspect screening method allowed the tentative identification of 88 compounds. The screening of 282 commercial samples revealed a broad contamination of the studied matrices, demonstrating the effectiveness of the platform in detecting and identifying both target and untarget PAs.

Influence of pyrrolizidine alkaloids depletion upon the biological activity of Symphytum officinale L. extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Comfrey (Symphytum officinale L., Boraginaceae) root preparations are used as both traditional remedies and therapeutic agents in treating pain and inflammation associated with joint, bone, and muscle ailments. Even though numerous phytochemicals contribute to the beneficial effects of comfrey, the presence of toxic pyrrolizidine alkaloids (PAs) overshadows its uses. AIM OF THE STUDY: In this work, different PA-/mucilage-depleted/undepleted comfrey root extracts were subjected to detailed phytochemical characterization and biological evaluation. MATERIALS AND METHODS: The phytochemical profiling was performed by LC-HRMS/MS. The quantification of PAs and major phenolic compounds was carried out by LC-MS/MS and LC-DAD. Antioxidant and enzyme inhibitory activity was determined by in vitro free radical scavenging, ion reducing, metal chelating, cholinesterase, tyrosinase, amylase, and glucosidase assays. Using an ex vivo model of LPS-stimulated neutrophils, their viability (as measured by flow cytometry) and the release of IL-1ß, IL-8, and TNF-α were determined (ELISA assay). RESULTS: 12 phenolic acids, six PAs, three organic acids, two fatty acids, and two sugars were identified in the obtained comfrey extracts. The PA-depleted materials contained PAs levels below 2 ppm, whereas the removal of mucilage increased the content of rosmarinic acid, globoidnan A, globoidnan B, and rabdosiin. PA-depletion did not significantly affect the antioxidant potential. However, the radical scavenging and metal reducing properties were higher in the mucilage-depleted extracts. Neither PA-depletion nor mucilage-depletion had considerable effects on the in vitro inhibitory activity of cholinesterases, tyrosinase, amylase, and glucosidase or release of ex vivo pro-inflammatory cytokines (e.g., IL-1ß, IL-8, and TNF-α) in LPS-stimulated neutrophils. CONCLUSIONS: In light of their superior safety profiles, PA-depleted comfrey extracts can be utilized further in cosmetic and pharmaceutical products.