Toxic Pyrrolizidine Alkaloids Research Articles

Damage to the behavior and physiological functions of Apis mellifera (Hymenoptera: Apidae) by monocrotaline via the modulation of tryptophan metabolism and the corazonin receptor

Monocrotaline (MCT) is a toxic pyrrolizidine alkaloid found in plants of the Crotalaria genus. As primary pollinators of Crotalaria plants, honeybees come into contact with this harmful substance. However, limited research has been conducted on the effects of MCT on Apis mellifera, particularly the risks of long-term exposure to sublethal concentrations. Through evaluating the proboscis extension reflex (PER) ability, analyzing the honeybee brain transcriptome, and analyzing the honeybee hemolymph metabolome, we discovered that sublethal concentrations of MCT impair the olfactory and memory capabilities of honeybees by affecting tryptophan (Trp) metabolism. Furthermore, MCT upregulates the expression of the corazonin receptor (CrzR) gene in the honeybee brain, which elevates reactive oxygen species (ROS) levels in the brain while reducing glucose levels in the hemolymph, consequently shortening the honeybees' lifespan. Our findings regarding the multifaceted impact of MCT on honeybees lay the foundation for exploring its toxicological pathways and management in honeybee populations.

Exploring the wound healing potential of Lobostemon fruticosus using in vitro and in vivo bioassays

Ethnopharmacological relevanceLobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. Aim of the studyAligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. Materials and methodsAn aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. ResultsSix major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 μg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 μg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. ConclusionsThe study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.

Pyrrolizidine Alkaloids as Hazardous Toxins in Natural Products: Current Analytical Methods and Latest Legal Regulations.

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.

Symphytum genus—from traditional medicine to modern uses: an update on phytochemistry, pharmacological activity, and safety

AbstractWith around 34 recognized species, Symphytum genus (comfrey) has a noteworthy position within the Boraginaceae family. Comfrey species have been empirically used since ancient times as wound-healing and skin-regenerating agents in ulcers, wounds, bone fractures, and rheumatic complaints. This review aims to provide a thorough examination of recent scientific advances and challenges within the Symphytum genus, covering data published between 2013 and 2023. It delivers an updated overview of the taxonomy, ethnopharmacological uses, chemical composition, and pharmacological activities of the genus. Special emphasis is put on molecular identification methods for species taxonomy, emerging extraction technologies for comfrey phytochemicals, metabolomics techniques for mapping chemical complexity, modern bioassay platforms revealing its poly-pharmacology, formulation strategies, and remediation approaches for removal of toxic pyrrolizidine alkaloids (PAs). For instance, recent metabolomic studies employing advanced spectro-chromatographic techniques have revealed a diverse chemical composition of comfrey plants, including polysaccharides, allantoin, benzoic and cinnamic acid derivatives, flavonoids, fatty acids, and unsaturated necine-structure-based PAs. The mechanisms underlying their anti-inflammatory, analgesic, wound-healing, anti-irritant, and osteo-regenerative properties were targeted in modern pharmacological setups. Thus, key compounds like allantoin, rosmarinic acid, globoidnans A and B, rabdosiin, and comfreyn A, have been identified as significant contributors to the anti-inflammatory and wound-healing effects of Symphytum-derived preparations. Despite their well-established clinical use, concerns about PAs-induced toxicity have prompted the development of novel PA remediation strategies, enabling the production of comfrey extracts with enhanced safety profiles that can meet the regulatory standards imposed by authorities.

Insight into pyrrolizidine alkaloids degradation and the chemical structures of their degradation products using ultra high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometry

Pyrrolizidine alkaloids (PAs), released into the environment by donor plants, are absorbed by crops or transported by animals, posing a global food safety concern. Photolysis is an effective way to eliminate harmful substances in the environment or food. Photolysis happens as PAs move among plants, environment and crops. In this study, we first investigated the photolysis and hydrolysis of 15 PAs and identified their degradation products via ultra-high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometry. PAs were degraded under UV radiation but minimally affected by visible light from a xenon lamp, and solvent pH had little impact on their photolysis. PAs were stable in neutral and acidic solutions but degraded by 50% within 24 h in alkaline conditions. The degradation products of PAs were mainly PAs/PANOs isomers and some minor byproducts. Cytotoxicity and computational analysis revealed isomers had similar toxicity, with minor products being less toxic. This study is a precursor for revealing the potential PAs degradation dynamics in the environment and food products, providing a reference for systematic evaluations of potential health and ecological risks of their degradation products.

Levels, Toxic Effects, and Risk Assessment of Pyrrolizidine Alkaloids in Foods: A Review.

Pyrrolizidine alkaloids (PAs) are naturally occurring secondary metabolites of plants. To date, more than 660 types of PAs have been identified from an estimated 6000 plants, and approximately 120 of these PAs are hepatotoxic. As a result of PAs being found in spices, herbal teas, honey, and milk, PAs are considered contaminants in foods, posing a potential risk to human health. Here, we summarize the chemical structure, toxic effects, levels, and regulation of PAs in different countries to provide a better understanding of their toxicity and risk assessment. With recent research on the risk assessment of PAs, this review also discusses the challenges facing this field, aiming to provide a scientific basis for PA toxicity research and safety assessment.

Assessment of Toxic Pyrrolizidine and Tropane Alkaloids in Herbal Teas and Culinary Herbs Using LC-Q-ToF/MS.

Pyrrolizidine alkaloids are secondary metabolites produced by plants as a defense against insects. These can cause acute or chronic toxicity in humans. Therefore, avoiding potential poisoning from the consumption of tea and culinary plants contaminated with pyrrolizidine alkaloids (PAs), pyrrolizidine alkaloids N-oxides (PANOs), and tropane alkaloids (TAs) is important for human health and food safety. Therefore, it is important to determine the levels of these substances with reliable and highly accurate methods. In this study, the PAs, PANOs, and TAs in herbal teas and culinary herbs sold in Turkish markets were identified and their levels were determined. Thus, the general profiles of herbal teas and culinary herbs in Turkey were revealed, and the compliance of the total amounts of PA and TA with the regulations was examined. The identification and quantification of 25 PAs and N-oxides and 2 TAs (atropine and scopolamine) in the samples was performed with a liquid chromatography-quadrupole time-of-flight tandem mass spectrometer (LC-Q-ToF/MS). At least a few of these substances were detected in all of the tested herbal teas and culinary herbs. The total contents of the black tea, green tea, mixed tea, flavored tea, chamomile tea, sage tea, linden tea, fennel tea, rosehip tea, peppermint, and thyme samples ranged from 4.6 ng g-1 to 1054.5 ng g-1. The results obtained shed light on the importance of analyzing the total dehydro PA, PANO, and TA amounts in plant-based products consumed in diets with sensitive and accurate methods, and they highlight the necessity of performing these analyses routinely in terms of food safety.

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.

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

ABSTRACTThe 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.

Hyperoside attenuates pyrrolizidine alkaloids-induced liver injury by ameliorating TFEB-mediated mitochondrial dysfunction.

Pyrrolizidine alkaloids (PAs) are potent hepatotoxins that can cause liver damage. Hyperoside (Hyp), a natural flavonoid, can be extracted from medicinal plants. Hyp displays hepatoprotective activity in various liver diseases. However, the potential effect and mechanism of action of Hyp in ameliorating PA-induced liver injury remain obscure. This study aimed to explore the protective effect of Hyp against PA-induced hepatotoxicity and its underlying mechanism. We established an in vitro model of PAs in mouse primary hepatocytes and developed a mouse model of acute PA toxicity to investigate the protective effect of Hyp. We found that Hyp notably attenuated PA-induced hepatotoxicity. RNA-sequencing showed that the beneficial effect of Hyp against PA-induced hepatotoxicity was associated with the transcription factor EB (TFEB)-peroxisome proliferator-activated receptor-γ coactivator-1-α (PGC1α) pathway. Our results confirmed that both the autophagy-lysosomal pathway and mitochondrial biogenesis were induced by Hyp through TFEB nuclear translocation in PA-induced liver injury. Furthermore, we demonstrated that activation of the mechanistic target of rapamycin complex 1 (mTORC1) by MHY 1485 decreased TFEB nuclear translocation and abrogated the protective effect of Hyp against PA-induced liver injury in mice. In contrast, inhibition of mTORC1 activity increased the level of TFEB and reduced hepatotoxicity induced by PAs in mouse livers. Likewise, Hyp-induced TFEB activation was validated in vitro. In conclusion, Hyp can activate the TFEB-mediated autophagy-lysosomal pathway and mitochondrial biogenesis through inhibition of mTORC1 activity, alleviating the liver injury induced by PAs, thus suggesting the potential value of Hyp in the treatment of PA-induced hepatotoxicity.

In vitro investigations on the hepatotoxicity and genotoxicity of food‐relevant pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PA) are secondary plant metabolites occurring in a great many plant species worldwide, known to exhibit hepatotoxic, genotoxic and carcinogenic properties after metabolic activation. In recent years, contamination of food, feed and herbal medicines with PA has become an increasing problem. The concept of interim relative potency factors (iREP) proposed by Merz and Schrenk in 2016 was a new approach for risk assessment of PA. While existing approaches of risk assessment assumed equivalent toxic potency for all PA congeners, the approach of Merz and Schrenk considered the structural features of individual PA congeners based on existing data from the literature. In order to generate further data on the structure‐specific toxicity of PA, congeners of different structural classes were investigated in different in vitro test systems.In vitro cytotoxicity was investigated in primary rat hepatocytes, HepG2 C9 cells (overespressing human CYP3A4) and naïve HepG2 cells. Overall, it could be observed that lasiocarpine and the cyclic di‐esters (except monocrotaline) showed much stronger cytotoxic effects in comparison to the tested mono‐esters in both, primary rat hepatocytes and in HepG2 C9 cells. Primary hepatocytes were the most sensitive cells investigating cytotoxicity of different PA congeners, followed by the HepG2 C9 cells. This is confirmed by markedly higher metabolism rates for all investigated PA in primary rat hepatocytes determined in the metabolism experiments. In naïve HepG2 cells no cytotoxic effects could be observed. The influence of cytochrome P450 (CYP) on the formation of toxic metabolites seem to play a crucial role. This assumption could be beared using ketoconazole as CYP inhibitor and testing various pre‐incubation times in primary rat hepatocytes. CYP activity was measured using 7‐ Benzoxyresorufin‐O‐Dealkylase (BROD) assay in primary rat hepatocytes and in HepG2 C9 cells. Glutahione (GSH) depletion using buthionine sulfoximine (BSO) showed slight stronger cytotoxic effects for several PA, but not for all tested.In contrast to the negative results of mutagenicity in ames fluctuation assay using Salmonella strains TA98 and TA100 with and without metabolic activation by S9 mix, all tested PA congeners showed micronuclei induction in the HepG2 C9 cell line. Again, laisocarpine and the cyclic di‐esters (except monocrotaline) were the most potent ones. In conclusion, the data from cytotoxicity and genototoxicity experiments from the tested PA congeners confirm published iREP factors with a few exceptions, in particular for monocrotaline or echimidine. Additionally, metabolism of six selected PA was studied in primary rat hepatocytes and HepG2 C9 cells. Genrally, it was found that almost all tested cyclic and open‐chained di‐esters (except retrorsine) showed much higher metabolism rates in both cell types, in comparison to the mono‐esters, for which only low metabolism rates could be measured. The same was observed for the quantified amounts of reactive metabolites in the supernatants of both cell types. In general, also these data bear the results from cytotoxicity and genotoxicity experiments and help to better understand the complex metabolism and the structure‐specific toxicity of different PA congeners.

The effect of infusion time on Echium amoenum extract -induced hepatotoxicity in vitro

Echium amoenum is an annual herb native to the northern mountains of Iran which has medicinal application. Petals of Echium amoenum (Gole-Gavzaban) is one of the most valuable medicinal plants in Iranian folk medicine. The dry petals of E. amoenum have long been used as a sedative, tonic, anxiolytic and as a treatment for sore throat, cough and inflammation. Previous studies have shown that petals of E. amoenum contain four toxic pyrrolizidine alkaloids but conflicting results have been acquired in experimental studies investigating the hepatotoxicy of E. amoenum. However, the direct effect of E. amoenum on liver cells and the complete mechanisms of its possible cytotoxic effects toward these cells remain to be defined. The main aim of this study was to assay the mechanisms underlying the toxic effects of E. amoenum toward hepG2 cells. E. amoenum extract was obtained by infusion of dried petals in hot water (90 centigrade) for 15 or 30 min. Cell viability and mechanistic parameters were determined following 12 h incubation of hepG2 with E. amoenum extract that was obtained after 15 or 30 min infusion. The results indicated that E. amoenum extract exerts cytotoxic effects on hepG2 cells, probably through mitochondrial and lysosomal damage induced by glutathione depletion and oxidative stress.

Separation of pyrrolizidine alkaloids in different Senecio species using ultra-high performance supercritical fluid chromatography

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).

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.

Comment on Pyrrolizidine Alkaloids and Terpenes from Senecio (Asteraceae): Chemistry and Research Gaps in Africa.

The genus Senecio is one of the largest in Asteraceae. There are thousands of species across the globe, either confirmed or awaiting taxonomic delimitation. While the species are best known for the toxic pyrrolizidine alkaloids that contaminate honeys (as bees select pollen from the species) and teas via lateral transfer and accumulation from adjacent roots of Senecio in the rhizosphere, they are also associated with more serious cases leading to fatality of grazing ruminants or people by contamination or accidental harvesting for medicine. Surprisingly, there are significantly more sesquiterpenoid than pyrrolizidine alkaloid-containing species. The main chemical classes, aside from alkaloids, are flavonoids, cacalols, eremophilanes, and bisabolols, often in the form of furan derivatives or free acids. The chemistry of the species across the globe generally overlaps with the 469 confirmed species of Africa. A small number of species express multiple classes of compounds, meaning the presence of sesquiterpenes does not exclude alkaloids. It is possible that there are many species that express the pyrrolizidine alkaloids, in addition to the cacalols, eremophilanes, and bisabolols. The aim of the current communication is, thus, to identify the research gaps related to the chemistry of African species of Senecio and reveal the possible chemical groups in unexplored taxa by way of example, thereby creating a summary of references that could be used to guide chemical assignment in future studies.

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

Ethnopharmacological relevanceComfrey (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 studyIn this work, different PA-/mucilage-depleted/undepleted comfrey root extracts were subjected to detailed phytochemical characterization and biological evaluation. Materials and methodsThe 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-1β, IL-8, and TNF-α were determined (ELISA assay). Results12 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-1β, IL-8, and TNF-α) in LPS-stimulated neutrophils. ConclusionsIn light of their superior safety profiles, PA-depleted comfrey extracts can be utilized further in cosmetic and pharmaceutical products.

Seasonal Variability in Pyrrolizidine Alkaloids in Jacobaea alpina from the Trentino-Alto Adige Region (Northern Italy).

In recent years, increased attention has been given to plants containing toxic pyrrolizidine alkaloids (PAs). Jacobaea alpina (syn. Senecio cordatus) is a tall forb growing on mountain pastures and meadows containing such alkaloids and therefore, the plant is considered as a noxious weed in these environments. The repartition of toxic macrocyclic PAs in the plant and their evolution during the vegetation period has been studied in two populations. Eight PAs were found where senciphylline and senecionine accounted in most samples for more than 85 % of total alkaloids. Leaves in April and stems in May started with high PA concentrations (19-22 mg/g dry matter), then alkaloid levels declined. This decrease was more rapid in stems than in leaves. Depending on the population, fully developed inflorescences in June and July PA contents were higher or lower than in the respective leaves. Later, also in the inflorescences PA concentration decreased. Combined with growth data total alkaloid content in the whole plant as mg/plant was highest in midsummer and declined afterwards. Finally, new emerging leaves in September had high PA levels, which declined markedly towards the end of the season in November. In sum, over a large period PA concentration appeared to be high enough to present a health risk for grazing animals.

Effective Solid Phase Extraction of Toxic Pyrrolizidine Alkaloids from Honey with Reusable Organosilyl-Sulfonated Halloysite Nanotubes

Pyrrolizidine alkaloids are plant secondary metabolites that have recently attracted attention as toxic contaminants in various foods and feeds as they are often harvested by accident. Furthermore, they prove themselves as hard to analyze due to their wide structural range and low concentration levels. However, even low concentrations show toxic behavior in the form of chronic liver diseases and possible carcinogenicity. Since sample preparation for this compound group is in need of more green and sustainable alternatives, modified halloysite nanotubes present an interesting approach. Based on the successful use of sulfonated halloysite nanotubes as inexpensive, easy-to-produce cation exchangers for solid phase extraction in our last work, this study deals with the further modification of the raw nanotubes and their performance in the solid phase extraction of pyrrolizidine alkaloids. Conducting already published syntheses of two organosilyl-sulfonated halloysite nanotubes, namely HNT-PhSO3H and HNT-MPTMS-SO3H, both materials were used as novel materials in solid phase extraction. After the optimization of the extraction protocol, extractions of aqueous pyrrolizidine alkaloid mixtures showed promising results with recoveries ranging from 78.3% to 101.3%. Therefore, spiked honey samples were extracted with an adjusted protocol. The mercaptopropyl-sulfonated halloysite nanotubes revealed satisfying loading efficiencies and recoveries. Validation was then performed, which displayed acceptable performance for the presented method. In addition, reusability studies using HNT-MPTMS-SO3H for solid phase extraction of an aqueous pyrrolizidine alkaloid mixture demonstrated excellent results over six cycles with no trend of recovery reduction or material depletion. Therefore, organosilyl-sulfonated halloysite nanotubes display a green, efficient and low-cost alternative to polymeric support in solid phase extraction of toxic pyrrolizidine alkaloids from complex honey matrix.

Source and Route of Pyrrolizidine Alkaloid Contamination in Tea Samples.

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in tea samples have remained unclear. In this work, an adsorbent method combined with UPLC-MS/MS was developed to determine 15 PAs in the weed Ageratum conyzoides L., A. conyzoides rhizospheric soil, fresh tea leaves, and dried tea samples. The average recoveries ranged from 78%-111%, with relative standard deviations of 0.33%-14.8%. Fifteen pairs of A. conyzoides and A. conyzoides rhizospheric soilsamples and 60 fresh tea leaf samples were collected from the Jinzhai tea garden in Anhui Province, China, and analyzed for the 15 PAs. Not all 15 PAs were detected in fresh tea leaves, except for intermedine-N-oxide (ImNO) and senecionine (Sn). The content of ImNO (34.7 µg/kg) was greater than that of Sn (9.69 µg/kg). In addition, both ImNO and Sn were concentrated in the young leaves of the tea plant, while their content was lower in the old leaves. The results indicated that the PAs in tea were transferred through the path of PA-producing weeds-soil-fresh tea leaves in tea gardens.

Comparative Pharmacokinetic Study of Two Pyrrolizidine Alkaloids Lasiocarpine and Heliotrine in Rats.

Lasiocarpine (LAS) and heliotrine (HEL) are two different ester types of toxic pyrrolizidine alkaloids (PAs): open-chain diester and monoester. However, the pharmacokinetics of these two types of PAs in rats have not been reported. In the present study, two LC-MS/MS methods for determining LAS and HEL were established and validated. The methods exhibited good linearity, accuracy, and precision and were then applied to a comparative pharmacokinetic study. After intravenous administration to male rats at 1 mg/kg, the AUC0-t values of LAS and HEL were 336 ± 26 ng/mL × h and 170 ± 5 ng/mL × h. After oral administration at 10 mg/kg, the AUC0-t of LAS was much lower than that of HEL (18.2 ± 3.8 ng/mL × h vs. 396 ± 18 ng/mL × h), while the Cmax of LAS was lower than that of HEL (51.7 ± 22.5 ng/mL × h vs. 320 ± 26 ng/mL × h). The absolute oral bioavailability of LAS was 0.5%, which was significantly lower than that of HEL (23.3%). The results revealed that the pharmacokinetic behaviors of LAS differed from that of HEL.