RESUMO
Ragworts like tansy ragwort (J. vulgaris Gaertn., syn. Senecio jacobaea L.) contain hepatotoxic and cancerogenic pyrrolizidine alkaloids (PA) and their corresponding pyrrolizidine alkaloid N-oxides (PANO). Due to increasing spread of ragworts (Jacobaea spp.) PA/PANO may pose a health risk to animals and humans consuming contaminated feed and food. Therefore, the aim of the present study was to investigate the transfer of individual PA/PANO originating from a well-defined PA/PANO extract into the milk of dairy cows. For this objective, 16 German Holstein cows were assigned to four treatment groups (n = 4) in a 28-day dose-response study. Administration into the reticulorumen was performed daily by gavage after the morning milking. Three groups received different amounts of the J. vulgaris extract resulting in a PA/PANO exposure of 0.47, 0.95, or 1.91 mg PA/PANO/kg body weight/day, respectively. Furthermore, a control group received molasses to account for the sugar content of the used PA/PANO extract. While the composition of the PA/PANO extract was more diverse, the PA/PANO pattern in milk was dominated by the PA in their free base form. It was shown that mainly PA considered stable in the rumen environment were transferred into the milk. The main compounds in milk were jacoline (74.3 ± 2.4% of the PA/PANO sum), jaconine (11.2 ± 1.3%), and jacobine (7.2 ± 0.6%) with concentrations up to 29.7, 4.65 µg/l, or in the highest exposed group, 3.44 µg/l. There was no dose-dependent effect on the total PA/PANO transfer rate into the milk. The average transfer rate was 0.064 ± 0.005% of the administered content.
Assuntos
Relação Dose-Resposta a Droga , Leite , Extratos Vegetais , Alcaloides de Pirrolizidina , Senécio , Animais , Alcaloides de Pirrolizidina/análise , Alcaloides de Pirrolizidina/química , Bovinos , Leite/química , Extratos Vegetais/química , Extratos Vegetais/administração & dosagem , Senécio/química , Feminino , Contaminação de Alimentos/análiseRESUMO
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.
Assuntos
Produtos Biológicos , Alcaloides de Pirrolizidina , Alcaloides de Pirrolizidina/análise , Humanos , Produtos Biológicos/análise , Produtos Biológicos/química , Plantas Medicinais/química , Espectrometria de Massas/métodos , Contaminação de Alimentos/análise , Toxinas Biológicas/análiseRESUMO
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.
Assuntos
Alcaloides de Pirrolizidina , Senécio , Tanacetum , Animais , Feminino , Bovinos , Senécio/química , Alcaloides de Pirrolizidina/toxicidade , Alcaloides de Pirrolizidina/análise , Leite/química , Plantas Tóxicas , Fígado , Aminoácidos/análiseRESUMO
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.
Assuntos
Mel , Alcaloides de Pirrolizidina , Gravidez , Animais , Criança , Feminino , Humanos , Alcaloides de Pirrolizidina/análise , Cromatografia Líquida , Espectrometria de Massas em Tandem , Mel/análise , Plantas/metabolismo , Chá , Contaminação de Alimentos/análiseRESUMO
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.
Assuntos
Alcaloides de Pirrolizidina , Humanos , Animais , Alcaloides de Pirrolizidina/análise , Cromatografia Líquida , Gana , Espectrometria de Massas em Tandem , Contaminação de Alimentos/análise , Suplementos Nutricionais/análiseRESUMO
Different Senecio species, especially S. inaequidens - a neophyte native to South Africa - have widely spread across Europe and now are found worldwide. The entire genus is known to contain toxic pyrrolizidine alkaloids (PAs), which renders them a possible health hazard to humans and livestock. As they can enter the food chain or occur as contaminants in herbal crops and phytopharmaceutical formulations (e.g. teas), efficient and straightforward assays for their qualitative and quantitative analysis are in high demand. Different techniques have been used for this purpose, most commonly HPLC or GC. As the analysis of PAs is a challenging task, alternative methodologies like ultra-high performance SFC (UHPSFC) may offer an additional benefit in terms of their separation efficiency and orthogonal selectivity. In this study an UHPSFC approach for the simultaneous determination of six PAs (free bases as well as N-oxides) is presented, which achieved the baseline separation of all standard compounds in seven min. Optimal separation was carried out in gradient mode on a Torus™ DEA column with 0.05% ammonia in methanol as modifier. The column temperature was 25 °C, ABPR 1900 psi and flow rate 1.1 mL/min, with a detection wavelength of 215 nm. The assay was validated and fulfilled all ICH criteria exhibiting good linearity (R2 ≥ 0.9994), precision (inter-day variance ≤ 3.67%, intra-day variance ≤ 3.92%) and recovery rates (96.3-104.1%), with detection limits typical for SFC-PDA (≤ 4.24 µg/mL). Furthermore, it could conveniently be coupled to MS-detection, which increased the sensitivity significantly. To confirm practical suitability of the method, different Senecio samples were analyzed, indicating a high qualitative as well as quantitative difference in their PA profile (e.g. total amounts of PA between 0.09 and 4.63 mg/g).
Assuntos
Cromatografia com Fluido Supercrítico , Alcaloides de Pirrolizidina , Senécio , Humanos , Alcaloides de Pirrolizidina/análise , Senécio/química , Cromatografia Líquida de Alta Pressão , Europa (Continente)RESUMO
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.
Assuntos
Alcaloides de Pirrolizidina , Animais , Humanos , Ratos , Alcaloides de Pirrolizidina/análise , Medição de Risco , Carcinógenos/toxicidade , Suplementos Nutricionais/análise , Contaminação de Alimentos/análiseRESUMO
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.
Assuntos
Boraginaceae , Confrei , Alcaloides de Pirrolizidina , Confrei/química , Alcaloides de Pirrolizidina/toxicidade , Alcaloides de Pirrolizidina/análise , Cromatografia Líquida , Antioxidantes/farmacologia , Monofenol Mono-Oxigenase , Interleucina-8 , Lipopolissacarídeos , Fator de Necrose Tumoral alfa , Espectrometria de Massas em Tandem , Boraginaceae/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Compostos Fitoquímicos/farmacologiaRESUMO
Pyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs.
Assuntos
Alcaloides de Pirrolizidina , Transcriptoma , Carcinogênese , Ciclo Celular , Células Clonais/química , Dano ao DNA , Células Hep G2 , Humanos , Monocrotalina , Alcaloides de Pirrolizidina/análise , Alcaloides de Pirrolizidina/toxicidade , Transcriptoma/genéticaRESUMO
BACKGROUND: Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with potential hepatic and carcinogenic diseases in humans and animals. The concern over PAs has increased as the consumption of herbal medicines has increased. OBJECTIVE: This study aimed to develop and validate a sensitive analytical method to determine 28 PAs in five herbal medicines using liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). Additionally, this study identified and quantified the amount of PAs in 10 samples of each herbal medicine. METHODS: The pretreatment in the proposed LC-MS/MS analysis comprised solvent extraction using 0.05M H2SO4 in 50% methanol and clean-up step using an mixed-mode cationic exchange (MCX)-solid-phase extraction (SPE) cartridge. The PA contents in herbal medicines were measured by using the developed method. RESULTS: The proposed method had recoveries ranging from 72.5-123.7% for the Atractylodis Rhizoma Alba, 70.6-151.7% for Alba Chrysanthmi Flos, 80.6-130.9% for Leonuri Herba, 70.3-122.9% for Gastrodiae Rhizoma, and 67.1-106.9% for Glycyrrhizae Radix. Even though a few samples showed recoveries in unsatisfactory values, the proposed method indicated entirely sufficient recoveries and precision in most samples. In monitoring results, only Leonuri Herba contained two PAs, which indicated Retrorsine (4/10) of 84.7-120.9 µg/kg and Senkirkine (10/10) of 60.9-170.7 µg/kg. CONCLUSION: The results obtained from this study demonstrate that the proposed method is fit for purpose to determine 28 PAs in herbal medicines. Therefore it could serve as a regulatory method capable of being used for controlling the risks of PAs in certain medicinal plants and dietary supplements. HIGHLIGHTS: An LC-MS/MS method for the determination of 28 pyrrolizidine alkaloids in herbal medicines was developed and validated through this study. The proposed method is considered as an useful method for monitoring pyroolizidine alkaloids in herbal medicines.
Assuntos
Plantas Medicinais , Alcaloides de Pirrolizidina , Cátions , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Humanos , Alcaloides de Pirrolizidina/análise , Extração em Fase Sólida , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Pyrrolizidine alkaloids are toxins having hepatotoxic and carcinogenic effects on human health. A ultra high performance liquid chromatography tandem mass spectrometry technique was developed for the first time for the simultaneous determination of eight pyrrolizidine alkaloids, including four diastereoisomers (intermedine, lycopsamine, rinderine, and echinatine) and their respective N-oxide forms, in different parts of Eupatorium lindleyanum. The risk assessment method for pyrrolizidine alkaloids in Eupatorium lindleyanum was explored using the margin of exposure strategy for the first time based on a real-life exposure scenario. Differences were found in all eight pyrrolizidine alkaloids in various parts of Eupatorium lindleyanum. Besides, the total levels of pyrrolizidine alkaloids in Eupatorium lindleyanum followed the order of root > flower > stem > leaf. Moreover, the risk assessment data revealed that the deleterious effects on human health were unlikely at exposure times of less than 200, 37, and 12 days during the lifetimes of Eupatorium lindleyanum leaves, stems, and flowers, respectively. This study reported both the contents of and risk associated with Eupatorium lindleyanum pyrrolizidine alkaloids. The comprehensive application of the novel ultra high performance liquid chromatography tandem mass spectrometry technique alongside the risk assessment approach provided a scientific basis for quality evaluation and rational utilization of toxic pyrrolizidine alkaloids in Eupatorium lindleyanum to improve public health safety.
Assuntos
Eupatorium/química , Componentes Aéreos da Planta/química , Alcaloides de Pirrolizidina/análise , Administração Oral , Cromatografia Líquida de Alta Pressão , Humanos , Conformação Molecular , Alcaloides de Pirrolizidina/administração & dosagem , Alcaloides de Pirrolizidina/efeitos adversos , Medição de Risco , Espectrometria de Massas em TandemRESUMO
To investigate the transfer of pyrrolizidine alkaloids (PAs) from feed to milk, rumen-cannulated dairy cows were intra-ruminally fed with 200 g/day of dried plant material of either ragwort (mixture of Jacobaea vulgaris and Senecio inaequidens), common groundsel (Senecio vulgaris) or viper's bugloss (Echium vulgare) for a period of 4 days. PA levels in the plant materials were 3767, 2792 and 1674 µg g-1 respectively. Feed intake, milk yield and several blood parameters indicative for liver function were not influenced by the treatment. When fed ragwort, increased levels of PAs were detected in the milk, in particular jacoline and an unidentified cyclic diester, possibly a hydroxylated metabolite from retrorsine. The latter was the most important PA in milk from cows fed common groundsel. For viper's bugloss, echimidine was the most abundant identified PA but in addition several hydroxylated PA metabolites were detected. For ragwort, the overall PA transfer was estimated at 0.05% and 1.4% for jacoline (N-oxide). Transfer rates were similar for viper's bugloss (0.05%) but lower for common groundsel (0.01%). Only a small portion of the administered PAs was quantified in milk, urine and faeces, with an overall balance of 4.5%, 2.9% and 5.8%, for ragwort, common groundsel and viper's bugloss, respectively. Samples taken from the rumen indicated that the N-oxides were converted into the free bases, which was confirmed by in vitro studies with the same plant species incubated with ruminal fluid. These results confirm that the transfer of PAs to milk is relatively low but may be of concern for human health regarding the genotoxic and carcinogenic properties of these compounds. The transfer rate depends on the type of PAs present in the weeds. The incomplete balance of input vs output stresses the need to further investigate the metabolism and the potential transfer of metabolites into edible products.
Assuntos
Contaminação de Alimentos/análise , Leite/química , Intoxicação por Plantas/veterinária , Alcaloides de Pirrolizidina/análise , Senécio/química , Ração Animal/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Echium/química , Fezes/química , Feminino , Humanos , Intoxicação por Plantas/metabolismo , Alcaloides de Pirrolizidina/metabolismo , Medição de Risco , Espectrometria de Massas em Tandem , Urina/químicaRESUMO
Pyrrolizidine alkaloids (PA) and PA N-oxides (PANO) are secondary plant metabolites exhibiting genotoxic and carcinogenic properties. Apart from the roots and leaves, PA/PANO are particularly present in pollen and nectar. Therefore, the spread of Jacobaea vulgaris in certain regions of northern Germany has an impact on the safety of honey produced in that region. In this study, raw honey samples (n = 437) were collected from usually three individual beehives per site (n = 73) in the district of Ostholstein and analyzed for 25 PA/PANO. The results reveal mean levels of 8.4, 1.5, and 72.6 µg/kg and maximum levels of 111, 59.4, and 3313 µg/kg, depending on the season (summer 2015 and spring/summer 2016, respectively). As far as individual data are concerned, sites near areas with J. vulgaris growth did not necessarily result in high PA/PANO values. Furthermore, intra-site investigations revealed remarkable differences in PA/PANO levels of raw honey collected by different bee colonies at the same site. Consumption of these regionally produced honeys entails an increased exposure to PA/PANO, especially in children and high consumers. Margin of exposure values of <10,000 and an exceedance of the health-based guidance value highlight that regionally produced and marketed honey must be considered with care for a proper risk assessment and risk management.
Assuntos
Asteraceae/metabolismo , Abelhas , Mel/análise , Óxidos/análise , Pólen/metabolismo , Alcaloides de Pirrolizidina/análise , Animais , Asteraceae/efeitos adversos , Qualidade de Produtos para o Consumidor , Alemanha , Pólen/efeitos adversos , Alcaloides de Pirrolizidina/efeitos adversos , Medição de Risco , Estações do Ano , Metabolismo Secundário , Fatores de TempoRESUMO
For a long time, honey has been recognized for its health-promoting properties and, consequently, has been used in traditional medicine worldwide. Apart from the beneficial bioactive compounds found in this food (e.g. polyphenols), molecules with potentially harmful effects may also be present, such as pyrrolizidine alkaloids. Aiming the quality assessment of honeys produced from Echium plantagineum L., a species known for its content in pyrrolizidine alkaloids, this work was focused in the search of these alkaloids and of polyphenols in one monofloral and two multifloral honeys, using chromatographic techniques. Additionally, their cytotoxicity and anti-inflammatory potential were assessed in cellular models. Several polyphenols were determined, but no pyrrolizidine alkaloid was detected in the analysed honey samples. Honey extracts exhibited capacity to decrease NO levels in lipopolysaccharide-stimulated murine macrophage-like cells (RAW 264.7) up to 40% at concentrations of 0.25 mg/mL. Therefore, this work highlights the health benefits of these honey samples.
Assuntos
Anti-Inflamatórios/farmacologia , Echium , Mel/análise , Polifenóis/análise , Alcaloides de Pirrolizidina/análise , Animais , Anti-Inflamatórios/química , Linhagem Celular Tumoral , Contaminação de Alimentos/análise , Qualidade dos Alimentos , Humanos , Camundongos , Óxido Nítrico/metabolismo , Pólen/química , Portugal , Células RAW 264.7RESUMO
Pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals, inducing liver tumors in experimental rodents. We previously determined that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), 7-glutathione-DHP, 7-cysteine-DHP, 7-N-acetylcysteine-DHP, and 1-CHO-DHP are DNA reactive pyrrolic metabolites potentially associated with PA-induced liver tumor initiation. In this study, we developed an LC/MS/MS multiple reaction monitoring (MRM) mode method to identify and quantify these metabolites formed from the metabolism of senecionine, a carcinogenic PA, by mouse, rat, and human liver microsomes, and primary rat hepatocytes. Together with the chemically prepared standards of these metabolites, this represents an accurate and convenient LC/MS/MS analytical method for quantifying these five reactive pyrrolic metabolites in biological systems.
Assuntos
Carcinógenos/análise , Alcaloides de Pirrolizidina/análise , Animais , Células Cultivadas , Cromatografia Líquida , DNA , Adutos de DNA , Hepatócitos/efeitos dos fármacos , Humanos , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Pirróis , Ratos , Espectrometria de Massas em TandemRESUMO
Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to food regulators in Australia and internationally. In this survey, honey samples sourced from markets and shops in Queensland (Australia), were analysed by high-resolution Orbitrap UHPLC-MS/MS for 30 common PAs. Relationships between the occurrence of pyrrolizidine alkaloids and the botanical origin of the honey are essential as pyrrolizidine alkaloid contamination at up to 3300 ng/g were detected. In this study, the predominant alkaloids detected were isomeric PAs, lycopsamine, indicine and intermedine, exhibiting identical MS/MS spectra, along with lesser amounts of each of their N-oxides. Crucially, chromatographic UHPLC conditions were optimised by operation at low temperature (5 °C) to resolve these key isomeric PAs. Such separation of these isomers by UHPLC, enabled the relative proportions of these PAs present in honey to be compared to alkaloid levels in suspect source plants. Overall plant pyrrolizidine alkaloid profiles were compared to those found in honey samples to help identify the most important plants responsible for honey contamination. The native Australian vines of Parsonsia spp. are proposed as a likely contributor to high levels of lycopsamine in many of the honeys surveyed. Botanical origin information such as this, gained via low temperature chromatographic resolution of isomeric PAs, will be very valuable in identifying region of origin for honey samples.
Assuntos
Contaminação de Alimentos/análise , Mel/análise , Alcaloides de Pirrolizidina/análise , Cromatografia Líquida de Alta Pressão , Plantas , Alcaloides de Pirrolizidina/química , Queensland , Estereoisomerismo , Espectrometria de Massas em Tandem , TemperaturaRESUMO
Blue heliotrope (Heliotropium amplexicaule) is an invasive environmental weed that is widely naturalized in eastern Australia and has been implicated as a source of pyrrolizidine alkaloid (PA) poisoning in livestock. Less well-documented is the potential of such carcinogenic alkaloids to contaminate honey from bees foraging on this plant species. In this study, the PA profile of H. amplexicaule plant material, determined by HRAM LC-MS/MS, revealed the presence of nine PAs and PA-N-oxides, including several PAs and PA-N-oxides of the indicine class, which have not previously been reported. The predominant alkaloid, indicine, represents 84% of the reduced PA content, with minor alkaloids identified as intermedine and the newly reported helioamplexine, constituting 7 and 9%, respectively. NMR analysis confirmed the identity of helioamplexine as a previously unreported indicine homologue. This is the first report of the isolation of intermedine, helioamplexine, and 3'-O-angelylindicine from H. amplexicaule. Also described is the identification of N-chloromethyl analogues of the major alkaloids as isolation-derived artifacts from reactions with dichloromethane. Analysis of regional-market honey samples revealed a number of honey samples with PA profiles analogous to that seen in H. amplexicaule, with measured PA contents of up to 2.0 µg of PAs per gram of honey. These results confirm the need for honey producers to be aware of H. amplexicaule as a potential PA source, most particularly in products where honey is sourced from a single location.
Assuntos
Heliotropium/química , Mel/análise , Alcaloides de Pirrolizidina/análise , Animais , Austrália , Abelhas/fisiologia , Cromatografia Líquida , Flores/química , Contaminação de Alimentos/análise , Espectrometria de Massas em TandemRESUMO
Pyrrolizidine alkaloids (PAs and corresponding N-oxides (PANOs)) are known to have adverse health effects. Their toxic effects on liver cells are especially well-documented. In addition, potential carcinogenic and mutagenic effects in chronic exposure via food and/or herbal medicines have been a subject of vivid discussion in the last decade. Liqueurs and elixirs are traditionally used alcoholic extracts made from parts of plants and herbs. PA cross-contamination of the final products seems likely. Hence, this study aims to detect and quantify the PAs in such products in the light of a possible PA-contamination. The PA content was determined in the form of a single sum parameter using HPLC-ESI-MS/MS and a stable isotope-labeled internal standard. Overall, 56 products available at German pharmacies, drugstores, or internet shops were analyzed, comprising in total 38 samples of liqueurs (mainly bitters), 12 samples of plant elixirs and six different herbal juices. The results showed that 9 out of 38 liqueurs were PA-positive (24%). The total amount of PAs ranged from non-detectable to 9.5⯵g/kg. Seven out of ten elixirs were PA-positive (70%) with a maximum PA-content of 3121⯵g/kg. Four out of six plant juices were PA-positive (67%) with an average of 4.4⯵g/kg (PA-positive samples only).The results and potential risks are discussed in the light of recommended portions for daily consumption or daily doses, in association with the detected PA amounts for individual products and product classes.
Assuntos
Bebidas Alcoólicas/análise , Contaminação de Alimentos/análise , Sucos de Frutas e Vegetais/análise , Preparações de Plantas/análise , Alcaloides de Pirrolizidina/análise , Cromatografia Líquida de Alta Pressão , Alemanha , Espectrometria de Massas em TandemRESUMO
In this study, a two-dimensional liquid chromatography tandem mass spectrometry method was developed and validated for the determination of pesticide residues and contaminants in whole wheat grains and oats. The samples were extracted with a mixture of acetonitrile and water and were injected into the two-dimensional LC-MS/MS system without any further clean-up or sample preparation. Samples were analyzed with four different matrix matched calibrations. Matrix effects were evaluated by comparing analyte signals in the respective matrix matched standard with the neat solvent standards. The final method was validated according to the current Eurachem validation guide and SANTE document. The number of successfully validated analytes throughout all three validation levels in oats and wheat, respectively, were as follows: 330 and 316 out of 370 pesticides, 6 and 13 out of 18 pyrrolizidine alkaloids and 7 out of 9 regulated mycotoxins. Moreover, both plant growth regulators mepiquat and chlormequat as well as the tropane alkaloids atropine and scopolamine met the validation criteria. The majority of pesticides showed limits of detection below 1 µg kg-1, pyrrolizidine alkaloids below 0.7 µg kg-1, tropane alkaloids below 0.2 µg kg-1, growth regulators below 0.7 µg kg-1 and mycotoxins below 8 µg kg-1 in both matrices.
Assuntos
Avena/química , Cromatografia Líquida/métodos , Contaminação de Alimentos/análise , Espectrometria de Massas em Tandem/métodos , Triticum/química , Clormequat/análise , Análise de Alimentos/métodos , Limite de Detecção , Micotoxinas/análise , Sistemas On-Line , Resíduos de Praguicidas/análise , Piperidinas/análise , Reguladores de Crescimento de Plantas/análise , Alcaloides de Pirrolizidina/análise , Reprodutibilidade dos Testes , Tropanos/análiseRESUMO
Pyrrolizidine alkaloids (PAs) are known hepatotoxins. Children have been reported to be particularly susceptible to PA-induced hepatotoxicity. To improve our understanding of the mechanisms of the age-dependent difference in susceptibility to PA hepatotoxicity, a comparative study of hepatotoxicities of retrorsine (RTS), a representative PA, was performed in newly weaned (3-4 weeks of age) and adult mice (8-12 weeks of age). Intraperitoneal administration of RTS at a dose of 50 mg/kg induced limited increases in serum ALT and AST activities in adult mice, while the same dosage of RTS caused intensified increases in serum ALT and AST activities in newly weaned mice. Toxicokinetic and metabolic activation studies in vitro and in vivo were performed to examine the factors responsible for the observed difference in toxicity susceptibility in mice of both ages. The values of AUC0-4h in plasma and liver of newly weaned mice are higher (all by 1.4-fold) than those in adult mice given the same dosage of RTS. As expected, more plasma pyrrolic ester-glutathione (GSH) conjugates (1.3-fold more) and pyrrolic ester-derived hepatic protein adduction (1.3-fold more) were found in newly weaned mice. Administration of RTS induced dramatic decreases in hepatic GSH levels (as little as 55% remained) of newly weaned mice, while the same dose of RTS did not reveal such GSH depletion in adult mice. The Vmax/ Km (CLint) for RTS metabolic activation in newly weaned mouse liver microsomes was found to be similar to that of adult mice. In conclusion, more internal exposure of RTS made newly weaned mice more susceptible to PA-induced hepatotoxicity.