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Pyrrolizidine alkaloids (PAs) are toxic compounds that occur naturally in certain plants, however, there are many secondary pathways causing PA contamination of other plants, including medicinal herbs and plant-based food products, which pose a risk of human intoxication. It is proven that chronic exposure to PAs causes serious adverse health consequences resulting from their cytotoxicity and genotoxicity. This review briefly presents PA occurrence, structures, chemistry, and toxicity, as well as a set of analytical methods. Recently developed sensitive electrochemical and chromatographic methods for the determination of PAs in honey, teas, herbs, and spices were summarized. The main strategies for improving the analytical efficiency of PA determination are related to the use of mass spectrometric (MS) detection; therefore, this review focuses on advances in MS-based methods. Raising awareness of the potential health risks associated with the presence of PAs in food and herbal medicines requires ongoing research in this area, including the development of sensitive methods for PA determination and rigorous legal regulations of PA intake from herbal products. The maximum levels of PAs in certain products are regulated by the European Commission; however, the precise knowledge about which products contain trace but significant amounts of these alkaloids is still insufficient. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are toxic compounds that occur naturally in certain plants, however, there are many secondary pathways causing PA contamination of other plants, including medicinal herbs and plant-based food products, which pose a risk of human intoxication. It is proven that chronic exposure to PAs causes serious adverse health consequences resulting from their cytotoxicity and genotoxicity. This review briefly presents PA occurrence, structures, chemistry, and toxicity, as well as a set of analytical methods. Recently developed sensitive electrochemical and chromatographic methods for the determination of PAs in honey, teas, herbs, and spices were summarized. The main strategies for improving the analytical efficiency of PA determination are related to the use of mass spectrometric (MS) detection; therefore, this review focuses on advances in MS-based methods. Raising awareness of the potential health risks associated with the presence of PAs in food and herbal medicines requires ongoing research in this area, including the development of sensitive methods for PA determination and rigorous legal regulations of PA intake from herbal products. The maximum levels of PAs in certain products are regulated by the European Commission; however, the precise knowledge about which products contain trace but significant amounts of these alkaloids is still insufficient.

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]

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.

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.

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.

Pyrrolizidine alkaloids (PAs) are genotoxic carcinogenic phytotoxins mostly prevalent in the Boraginaceae, Asteraceae and Fabaceae families. Heliotropium species (Boraginaceae) are PA-producing weeds, widely distributed in the Mediterranean region, that have been implicated with lethal intoxications in livestock and humans. In Israel, H. europaeum, H. rotundifolium and H. suaveolens are the most prevalent species. The toxicity of PA-producing plants depends on the PA concentration and composition. PAs occur in plants as mixtures of dozens of various PA congeners. Hence, the risk arising from simultaneous exposure to different congeners has to be evaluated. The comparative risk evaluation of the three Heliotropium species was based on recently proposed interim relative potency (iREP) factors, which take into account certain structural features as well as in vitro and in vivo toxicity data obtained for several PAs of different classes. The aim of the present study was to determine the PA profile of the major organ parts of H. europaeum, H. rotundifolium and H. suaveolens in order to assess the plants’ relative toxic potential by utilizing the iREP concept. In total, 31 different PAs were found, among which 20 PAs were described for the first time for H. rotundifolium and H. suaveolens. The most prominent PAs were heliotrine-N-oxide, europine-N-oxide and lasiocarpine-N-oxide. Europine-N-oxide displayed significant differences among the three species. The PA levels ranged between 0.5 and 5% of the dry weight. The flowers of the three species were rich in PAs, while the PA content in the root and flowers of H. europaeum was higher than that of the other species. H. europaeum was found to pose a higher risk to mammals than H. rotundifolium, whereas no differences were found between H. europaeum and H. suaveolens as well as H. suaveolens and H. rotundifolium.

Rindera graeca is a Greek endemic plant of the Boraginaceae family which has never been studied before. Consequently, this study attempted to phytochemically examine the aerial parts of this species. Nine phenolic secondary metabolites were identified, consisting of seven caffeic acid derivatives and two flavonol glucosides, namely rutin and quercetin-3-rutinoside-7-rhamnoside. These flavonoids, together with rosmarinic acid, were isolated via column chromatography and structurally determined through spectral analysis. Quercetin-3-rutinoside-7-rhamnoside is an unusual triglycoside, which is identified for the first time in Rindera genus and among Boraginaceae plants. This metabolite was further examined with thermal analysis and its 3D structure was simulated, revealing some intriguing information on its interaction with biological membrane models, which might have potential applications in microcirculation-related conditions. R. graeca was also analyzed for its pyrrolizidine alkaloids content, and it was found to contain echinatine together with echinatine N-oxide and rinderine N-oxide. Additionally, the total phenolic and flavonoid contents of R. graeca methanol extract were determined, along with free radical inhibition assays. High total phenolic content and almost complete inhibition at experimental doses at the free radical assays indicate a potent antioxidant profile for this plant. Overall, through phytochemical analysis and biological activity assays, insight was gained on an endemic Greek species of the little-studied Rindera genus, while its potential for further applications has been assessed.

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.

Pyrrolizidine alkaloids (PAs) are natural toxins found in some genera of the family Asteraceae. However, it has not been reported whether PAs are present in the widely used Asteraceae plant Artemisia capillaris Thunb. (A. capillaris). The purpose of this study was to establish a sensitive and rapid UPLC-MS/MS method together with chemometrics analysis for simultaneous determination and risk assessment of PAs in A. capillaris. The developed UPLC-MS/MS method was validated and was confirmed to display desirable high selectivity, precision and accuracy. Risk assessment was conducted according to the European Medicines Agency (EMA) guideline. Chemometrics analysis was performed with hierarchical clustering analysis and principal component analysis to characterize the differences between PAs of A. capillaris. Finally, PAs were found in 29 out of 30 samples and at least two were detected in each sample, besides, more than half of the samples exceeded the EMA baseline. Nevertheless, the chemometrics results suggested that the PAs contents of A. capillaris from different sources varied significantly. The method was successfully applied to the detection and risk evaluation of PAs-containing A. capillaris for the first time. This study should provide a meaningful reference for the rational and safe use of A. capillaris.

The examination of the aerial parts, roots, and seeds of the endemic plant Rindera umbellata is reported in this paper for the first time. Phytochemical investigation of R. umbellata led to the isolation and characterization of ten pyrrolizidine alkaloids and eleven fatty acids in the form of triglycerides. Pyrrolizidine alkaloids 1–9 were found in the aerial parts, 7 and 8 in the roots, and 6–10, together with eleven fatty acids, in the seeds of this plant species. The structures of compounds 1–10 were established based on spectroscopic studies (1H- and 13C-NMR, 2D NMR, IR and CI-MS). After trans-esterification, methyl esters of the fatty acids were analyzed using GC-MS. The effect of lindelofine-N-oxide (7) on tubulin polymerization was determined.

The examination of the aerial parts, roots, and seeds of the endemic plant Rindera umbellata is reported in this paper for the first time. Phytochemical investigation of R. umbellata led to the isolation and characterization of ten pyrrolizidine alkaloids and eleven fatty acids in the form of triglycerides. Pyrrolizidine alkaloids 1-9 were found in the aerial parts, 7 and 8 in the roots, and 6-10, together with eleven fatty acids, in the seeds of this plant species. The structures of compounds 1-10 were established based on spectroscopic studies (1H- and 13C-NMR, 2D NMR, IR and CI-MS). After trans-esterification, methyl esters of the fatty acids were analyzed using GC-MS. The effect of lindelofine-N-oxide (7) on tubulin polymerization was determined. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are genotoxic carcinogenic phytotoxins mostly prevalent in the Boraginaceae, Asteraceae and Fabaceae families. Heliotropium species (Boraginaceae) are PA-producing weeds, widely distributed in the Mediterranean region, that have been implicated with lethal intoxications in livestock and humans. In Israel, H. europaeum, H. rotundifolium and H. suaveolens are the most prevalent species. The toxicity of PA-producing plants depends on the PA concentration and composition. PAs occur in plants as mixtures of dozens of various PA congeners. Hence, the risk arising from simultaneous exposure to different congeners has to be evaluated. The comparative risk evaluation of the three Heliotropium species was based on recently proposed interim relative potency (iREP) factors, which take into account certain structural features as well as in vitro and in vivo toxicity data obtained for several PAs of different classes. The aim of the present study was to determine the PA profile of the major organ parts of H. europaeum, H. rotundifolium and H. suaveolens in order to assess the plants' relative toxic potential by utilizing the iREP concept. In total, 31 different PAs were found, among which 20 PAs were described for the first time for H. rotundifolium and H. suaveolens. The most prominent PAs were heliotrine-N-oxide, europine-N-oxide and lasiocarpine-N-oxide. Europine-N-oxide displayed significant differences among the three species. The PA levels ranged between 0.5 and 5% of the dry weight. The flowers of the three species were rich in PAs, while the PA content in the root and flowers of H. europaeum was higher than that of the other species. H. europaeum was found to pose a higher risk to mammals than H. rotundifolium, whereas no differences were found between H. europaeum and H. suaveolens as well as H. suaveolens and H. rotundifolium. [ABSTRACT FROM AUTHOR]

Rindera graeca is a Greek endemic plant of the Boraginaceae family which has never been studied before. Consequently, this study attempted to phytochemically examine the aerial parts of this species. Nine phenolic secondary metabolites were identified, consisting of seven caffeic acid derivatives and two flavonol glucosides, namely rutin and quercetin-3-rutinoside-7-rhamnoside. These flavonoids, together with rosmarinic acid, were isolated via column chromatography and structurally determined through spectral analysis. Quercetin-3-rutinoside-7-rhamnoside is an unusual triglycoside, which is identified for the first time in Rindera genus and among Boraginaceae plants. This metabolite was further examined with thermal analysis and its 3D structure was simulated, revealing some intriguing information on its interaction with biological membrane models, which might have potential applications in microcirculation-related conditions. R. graeca was also analyzed for its pyrrolizidine alkaloids content, and it was found to contain echinatine together with echinatine N-oxide and rinderine N-oxide. Additionally, the total phenolic and flavonoid contents of R. graeca methanol extract were determined, along with free radical inhibition assays. High total phenolic content and almost complete inhibition at experimental doses at the free radical assays indicate a potent antioxidant profile for this plant. Overall, through phytochemical analysis and biological activity assays, insight was gained on an endemic Greek species of the little-studied Rindera genus, while its potential for further applications has been assessed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are natural toxins found in some genera of the family Asteraceae. However, it has not been reported whether PAs are present in the widely used Asteraceae plant Artemisia capillaris Thunb. (A. capillaris). The purpose of this study was to establish a sensitive and rapid UPLC-MS/MS method together with chemometrics analysis for simultaneous determination and risk assessment of PAs in A. capillaris. The developed UPLC-MS/MS method was validated and was confirmed to display desirable high selectivity, precision and accuracy. Risk assessment was conducted according to the European Medicines Agency (EMA) guideline. Chemometrics analysis was performed with hierarchical clustering analysis and principal component analysis to characterize the differences between PAs of A. capillaris. Finally, PAs were found in 29 out of 30 samples and at least two were detected in each sample, besides, more than half of the samples exceeded the EMA baseline. Nevertheless, the chemometrics results suggested that the PAs contents of A. capillaris from different sources varied significantly. The method was successfully applied to the detection and risk evaluation of PAs-containing A. capillaris for the first time. This study should provide a meaningful reference for the rational and safe use of A. capillaris. [ABSTRACT FROM AUTHOR]