Pyrrolizidine Alkaloids Concentration Research Articles

Variations in chemical defences and patterns of natural enemy attack between native and introduced populations of fireweed (Senecio madagascariensis): Implications for biological control

AbstractNative to South Africa, fireweed (Senecio madagascariensis Poiret; Asteraceae) is an annual or short‐lived perennial herb that is highly invasive in Australia, where it is a target for biological control. Preliminary research indicates that fireweed may be undergoing adaptive changes along its invasion gradient in Australia. Changes, such as a shift in plant chemical defence, may influence fireweed’s interaction with specialist insect herbivores, and hence the efficacy of a biocontrol program. As the testing of biocontrol candidates is a rigorous and lengthy process, confirmation of fireweed’s susceptibility to insect attack may assist in fast tracking candidate suitability, where changes in plant defensive chemistry are found. In a large experimental field study in fireweed’s native range, we compared the susceptibility of Australian and South African populations to insect natural enemy attack (in terms of plant biomass, insect richness and composition). Simultaneously, we assessed the variation in plant pyrrolizidine alkaloid concentrations and composition to rationalise any differences in natural enemy attack between populations. We found no significant differences in plant biomass across fireweed populations, despite significantly higher alkaloid concentrations in Australian populations. The composition of the endophagous (specialist) insect herbivore assemblage was related to alkaloid composition, but there were only minor differences in the assemblage between Australian and South African fireweed populations. Moreover, we found no significant differences in the abundance, richness or Shannon diversity indices of endophagous or ectophagous (generalist) insect herbivores between Australian and South African fireweed populations. Our results suggest that despite variations in chemical defence, Australian fireweed populations displayed neither reduced nor increased susceptibility to specialist insect herbivore attack, supporting the notion that if host‐specific biocontrol agents are released, their impact in Australia is unlikely to be influenced by any changes in plant chemical defence.

Cytotoxic effect of Senecio madagascariensis (Asteraceae) extracts on cancer derived cell lines

Species of the genus Senecio have been traditionally employed with medical purposes and it has been demonstrated that some of them have anti-proliferative activity on cancer-derived cell lines. In South America S. madagascariensis is one of the most important representatives of the genus. Almost all species contain pyrrolizidine alkaloids (PA), substances that can lead to hepatic damage. Thus, the aim of this work was to determine the cytotoxic activity of a low PAs extract of S. madagascariensis on a cancer-derived cell line. A chloroform extract from plant material was produced and it was demonstrated to have a PAs concentration lower than 200 μg/g (w/w). Two stock solutions were made with DMSO and acetone as solvents and were diluted in culture medium (DMEM). Cells from urinary bladder transitional cell carcinoma T24 were cultured in 96 well plates (104 cells/well) and incubated for 24 hours with dilutions of the extracts at the following concentrations, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL. Cell viability was evaluated by the MTT colorimetric technique. Both solutions of S. madagascariensis extracts (acetone and DMSO) showed cytotoxic activity. In both cases, the degree of cytotoxicity was dependent on the concentration of the solution.

Determination of pyrrolizidine alkaloids in tea and honey with automated SPE clean-up and ultra-performance liquid chromatography/tandem mass spectrometry

ABSTRACT For routine analytical purpose a method based on a combination of automated solid-phase extraction (SPE) clean-up and detection by Ultra-Performance Liquid Chromatography/Tandem Mass Spectrometry (UPLC-MS/MS) was developed for the determination of pyrrolizidine alkaloids (PA) in various food commodities. In this survey, honey, tea and herbal infusion samples from local retailers collected in 2012–2015 were obtained and analysed for their PA content. PA concentrations were found in 30% of the honey samples and in 42% of the tea and herbal infusion samples with levels up to 595 µg/kg. The survey included 17 individual PA, and their sum is also reported for each sample.

Seasonal variation in defence compounds: A case study on pyrrolizidine alkaloids of clones of Jacobaea vulgaris, Jacobaea aquatica and their hybrids

Concentration of plant secondary metabolites (SMs) show seasonal variations. However, it is still not well understood how these abiotic and biotic factors influence the seasonal variations of SMs. In addition, it is of interest to know if and how SMs are reallocated to the different plant organs, in particular whether SMs are reallocated to the remaining tissues when biomass is lost, e.g., during winter. Here we used Jacobaea vulgaris, Jacobaea aquatica, two F1 and four F2 hybrids that differed in their pyrrolizidine alkaloids (PAs) bouquet as a study system. A series of clones of these genotypes were investigated during their vegetative stage spanning 14 months in a semi-natural environment. We found that the total PA concentration in roots and shoots showed a gradual increase until the spring of the second year, whereafter it dropped substantially in shoots. The variation in PA composition due to seasonal changes was significant but relatively small. Senecionine-like PAs were the dominant PAs in roots, while jacobine-/erucifoline-like PAs were dominant in shoots. The variation of PA concentration was significantly correlated with temperature, day length, and plant age. A correlation analysis showed that PAs were not reallocated when biomass was lost in winter. Overall, our study showed that PA composition of each genotype changed over seasons in a different manner but seasonal variation did not overrule the differences in PA composition among genotypes.

Pyrrolizidine alkaloid concentrations in local Italian and retail honeys of different origin: A scenario of human exposure

A validated method in liquid chromatography tandem mass spectrometry was applied to assess the contamination profile of 17 pyrrolizidine alkaloids and their N-oxide metabolites (PAs/PANOs) in 775 honey samples of different origins, including from local beekeepers (579, LB), large retail and online stores (Italian (LRI) and foreign (LRF) products). Fifty-five percent of the honey samples contained the 17 monitored compounds at below the limit of quantification. The LB samples were characterised by a total mean concentration of PAs/PANOs at 6.7 and 3.3 times lower than those of the LRF and LRI samples, respectively. The PA echimidine characterised mainly honey samples from the large retail stores (75.8 % and 72.5 % of LRI- and LRF-contaminated samples, respectively). The LB samples showed a senecionine-type contamination profile, providing beekeepers clues to the plants responsible for contamination. The results highlighted that the consumption of the local honey samples analysed does not represent a double risk for human health compared to the consumption of products from large retail stores. Careful attention must be paid to honey imported from areas where lycopsamine-type and senecionine-type PAs/PANOs-producing plants are invasive. A machine learning approach could predict the origin of the sample given its contaminant profile at an accuracy of 0.74, which is useful for detecting fraud in the declared honey origin.

Influence of the Post-Harvest Storage Time on the Multi-Biological Potential, Phenolic and Pyrrolizidine Alkaloid Content of Comfrey (Symphytum officinale L.) Roots Collected from Different European Regions.

Comfrey (Symphytum officinale L.) roots are well-known bioactive ingredients included in various cosmeceutical and pharmaceutical preparations. In this study, the influence of the post-harvest storage on the chemico-biological potential of roots collected from different European regions and stored for up to six months was investigated. Total phenolic content (TPC) and total phenolic acid content (TPAC) were spectrophotometrically estimated, whereas the levels of individual phenolic and pyrrolizidine alkaloidal markers were determined by HPLC-DAD and HPLC-MS/MS, respectively. The changes in the biological potential was tracked via antioxidant (DPPH, ABTS, CUPRAC, and FRAP) and anti-enzymatic (cholinesterase, tyrosinase, glucosidase, and amylase) assays. TPC and TPAC varied from 6.48–16.57 mg GAE/g d.w. root and from 2.67–9.03 mg CAE/g, respectively. The concentration of the four phenolics (rosmarinic acid, globoidnan A, globoidnan B, rabdosiin) and six pyrrolizidine alkaloids generally showed maximum values at 1–3 months, after which their levels significantly decreased. With respect to the bioassays, the samples showed a wide range of antioxidant and anti-enzymatic effects; however, a direct storage time–bioactivity relationship was not observed. Similar conclusions were also revealed by the multivariate and correlation analyses. Our study could improve the current knowledge of the shelf-life properties of comfrey-based products and enhance their industrial exploitation.

Chemical Profile and Screening of Bioactive Metabolites of Rindera graeca (A. DC.) Bois. & Heldr. (Boraginaceae) In Vitro Cultures.

Rindera graeca is a rare endemic plant where in vitro culture has been used in order to investigate bioactive metabolites. Phytochemical study of the in vitro shoots and hairy roots led to the isolation of seven phenolic derivatives and the unusual furano-naphthoquinone rinderol. R. graeca was also analyzed for its pyrrolizidine alkaloids content by LC-MS, and it was found to contain echinatine together with echinatine and rinderine N-oxides. Rinderol, isolated only from in vitro hairy root culture for the first time in the genus, revealed promising bioactivities. It was evaluated in vitro against a panel of microorganisms, showing very strong activity specifically against Gram-positive bacteria (MIC values 0.98 × 10−2–1.18 µg/mL) as well as very interesting antiproliferative effect against the human non-small-cell bronchopulmonary carcinoma cell line NSCLC-N6-L16 and the epidermoid lung cancer cell line A549. These findings were compared with the chemical profile of the plant from nature, while this study is the first to report on the effects of R. graeca extracts obtained from in vitro culture, providing a valuable contribution to the scientific community towards this sustainable method of production of potential bioactive molecules.

Active Transport of Hepatotoxic Pyrrolizidine Alkaloids in HepaRG Cells.

1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites occurring as food contaminants that can cause severe liver damage upon metabolic activation in hepatocytes. However, it is yet unknown how these contaminants enter the cells. The role of hepatic transporters is only at the beginning of being recognized as a key determinant of PA toxicity. Therefore, this study concentrated on assessing the general mode of action of PA transport in the human hepatoma cell line HepaRG using seven structurally different PAs. Furthermore, several hepatic uptake and efflux transporters were targeted with pharmacological inhibitors to identify their role in the uptake of the PAs retrorsine and senecionine and in the disposition of their N-oxides (PANO). For this purpose, PA and PANO content was measured in the supernatant using LC-MS/MS. Also, PA-mediated cytotoxicity was analyzed after transport inhibition. It was found that PAs are taken up into HepaRG cells in a predominantly active and structure-dependent manner. This pattern correlates with other experimental endpoints such as cytotoxicity. Pharmacological inhibition of the influx transporters Na+/taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter 1 (SLC22A1) led to a reduced uptake of retrorsine and senecionine into HepaRG cells, emphasizing the relevance of these transporters for PA toxicokinetics.

Assessment of the diversity of comfrey (Symphytum officinale L. and S.\u2009\xd7\u2009uplandicum Nyman)

Comfrey Symphytum officinale L. (true comfrey) and S. × uplandicum Nyman (a hybrid between S. asperum Lepech × S. officinale L., Russian comfrey) are used externally for the treatment of pain, inflammation and swelling of muscles and joints in degenerative arthritis, in acute myalgia, sprains, contusions and strains after accidents. Besides plant secondary compounds associated with beneficial activities (e.g. rosmarinic acid and allantoin) comfrey forms also toxic pyrrolizidine alkaloids (PA). To improve further breeding and study the genetic relationships of a comfrey collection, a sample set of 219 S. officinale and 5 hybrid plants of S. × uplandicum were analysed with 34 SNP markers by KASP (Kompetitive Allele Specific PCR), developed from a next generation sequencing approach of three different individuals of S. officinale. In parallel, the plants were analysed for the polyphenol rosmarinic acid, the purine derivative allantoin, and the toxic pyrrolizidine alkaloids. Besides the two beneficial compounds, further 13 polyphenols and 10 purine derivatives were determined. The plants were grouped into six distinct genetic clusters. Rosmarinic acid was not linked to any of the clusters, while one cluster was distinctively different for some compounds, amongst them allantoin and globoidnan A. Also linked to a certain genetic cluster was a low content of PA, which could become a valuable gene pool for minimizing PA content by breeding. A subset of the samples was analysed in a second year again where rosmarinic acid and allantoin showed a medium stability, while globoidnan A was completely unstable.

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

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

Skuteczność i bezpieczeństwo stosowania korzenia Symphytum officinale L. – przegląd danych literaturowych

Comfrey root – Symphyti radix has been present in medicine since ancient times. Nowadays, European Medicine Agency (EMA) restricts its only for external use, in short therapies not exceeding 10 days. The last decade has been characterized by the progress in research on the chemical composition and metabolism of biologically active compounds present in the root and leaves. The result is the presence of herbal medicinal products containing comfrey root extracts with reduced content or free of toxic pyrrolizidine alkaloids on the European market of herbal products. Moreover, the medicinal properties of leaves from another Symphytum species, namely Russian comfrey, are also in the use. The results of clinical trials indicate the beneficial effects of plant raw materials obtained from comfreys in musculoskeletal system disorders – comfrey root and skin inflammations – leaves of Russian comfrey. The paper presents the history of use, chemical composition, therapeutic effect and toxicity of comfrey, as well as characterizes clinical trials on products containing comfrey extracts from root and leaves.

Retrospective HRMS Screening and Dedicated Target Analysis Reveal a Wide Exposure to Pyrrolizidine Alkaloids in Small Streams.

Pyrrolizidine alkaloids (PAs) are found to be toxic pollutants emitted into the environment by numerous plant species, resulting in contamination. In this article, we investigate the occurrence of PAs in the aquatic environment of small Swiss streams combining two different approaches. Pyrrolizidine alkaloids (PAs) are toxic secondary metabolites produced by numerous plant species. Although they were classified as persistent and mobile and found to be emitted into the environment, their occurrence in surface waters is largely unknown. Therefore, we performed a retrospective data analysis of two extensive HRMS campaigns each covering five small streams in Switzerland over the growing season. All sites were contaminated with up to 12 individual PAs and temporal detection frequencies between 36 and 87%. Individual PAs were in the low ng/L range, but rain-induced maximal total PA concentrations reached almost 100 ng/L in late spring and summer. Through PA patterns in water and plants, several species were tentatively identified as the source of contamination, with Senecio spp. and Echium vulgare being the most important. Additionally, two streams were monitored, and PAs were quantified with a newly developed, faster, and more sensitive LC-MS/MS method to distinguish different plant-based and indirect human PA sources. A distinctly different PA fingerprint in aqueous plant extracts pointed to invasive Senecio inaequidens as the main source of the surface water contamination at these sites. Results indicate that PA loads may increase if invasive species are sufficiently abundant.

The invasive butterbur contaminates stream and seepage water in groundwater wells with toxic pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) are persistent mutagenic and carcinogenic compounds produced by many common plant species. Health authorities recommend minimising human exposure via food and medicinal products to ensure consumer health and safety. However, there is little awareness that PAs can contaminate water resources. Therefore, no regulations exist to limit PAs in drinking water. This study measured a PA base concentration of ~ 70 ng/L in stream water adjacent to an invasive PA-producing plant Petasites hybridus (Asteraceae). After intense rain the PA concentration increased tenfold. In addition, PAs measured up to 230 ng/L in seepage water from groundwater wells. The dominant PAs in both water types corresponded to the most abundant PAs in the plants (senkirkine, senecionine, senecionine N-oxide). The study presents the first discovery of persistent plant toxins in well water and their associated risks. In addition, it for the first time reports monocrotaline and monocrotaline N-oxide in Petasites sp.

LC/MS study of the diversity and distribution of pyrrolizidine alkaloids in Crotalaria species growing in Colombia

Hepatotoxic and genotoxic pyrrolizidine alkaloids have been involved in the acute poisoning of animals and humans. Crotalaria (Fabaceae) species contain these alkaloids. In this work, the diversity and distribution of pyrrolizidine alkaloids in roots, leaves, flowers, and seeds of Crotalaria pallida, Crotalaria maypurensis, Crotalaria retusa, Crotalaria spectabilis, Crotalaria incana, and Crotalaria nitens were studied. Matrix solid-phase dispersion and ultra-high-performance liquid chromatography coupled with Orbitrap mass spectrometry were successfully employed in pyrrolizidine alkaloids extraction and analysis, respectively. Forty-five pyrrolizidine alkaloids were detected and their identification was based on the mass spectrometry accurate mass measurement and fragmentation pattern analysis. The cyclic retronecine-type diesters monocrotaline, crotaleschenine, integerrimine, usaramine, and their N-oxides were predominantly present. Five novel alkaloids were identified for the first time in Crotalaria species, namely 14-hydroxymonocrotaline, 12-acetylcrotaleschenine, 12-acetylmonocrotaline, 12-acetylintegerrimine, and dihydrointegerrimine. Due to a lack of commercially available standards, the response factor of monocrotaline was used for quantification of pyrrolizidine alkaloids and their N-oxides. Seeds and flowers possessed higher pyrrolizidine alkaloids amounts than roots and leaves. Due to their 1,2-unsaturated pyrrolizidine alkaloids content, the ingestion of Crotalaria plant seeds or other parts through herbal products, infusions, or natural remedies is a serious health threat to humans and livestock.

Status of content analysis of pyrrolizidine alkaloids in food and herbs

Pyrrolizidine alkaloids(PAs) are a group of naturally occurring alkaloids with a pyrrolizidine skeleton which can be found in about 3% of the world's flowering plants. It is notorious that PAs are cause the hepatoxic and genotoxic-carcinogenic effects by taking PA-containing herbs, food and dietary supplements. In order to control the poisoning caused by PAs, European Medicines Agency has set a limit of intake of PAs from herbal medicinal products at 0.007 μg of 1,2-unsaturated PAs/kg body weight. Nonetheless, a systematic overview of the amount of PAs in the herb has not been provided. Therefore, this paper is to systematically review the current status of PAs content analysis of herbal medicines and foods reported in the literature, and to provide theoretical and experimental support for the safety risk assessment and control of PAs in Chinese herbal medicines.

Occurrence of pyrrolizidine alkaloids in ragwort plants, soils and surface waters at the field scale in grassland

Pyrrolizidine alkaloids (PA)s are natural toxins produced by a variety of plants including ragwort. The PAs present a serious health risk to human and livestock. Although these compounds have been extensively studied in food and feed, little is known regarding their environmental fate. To fill this data gap, we investigated the occurrence of PAs in ragwort plants, soils and surface waters at three locations where ragwort was the dominant plant species to better understand their environmental distribution. The concentrations of PAs were quantified during the full growing season (April–November) and assessed in relation to rain events. PA concentrations ranged from 3.2–6.6 g/kg dry weight (dw) in plants, 0.8–4.0 mg/kg dw in soils, and 6.0–529 μg/L in surface waters. Maximum PA concentrations in the soil (4 mg/kg) and water (529 μg/L) were in mid-May just before flowering. The average distribution of PAs in water was approximately 5 g/10,000 L, compared to the average amounts present in ragwort (506 kg/ha), and soil (1.7 kg/ha). In general, concentrations of PAs increase in the soil and surface water following rain events.

The evolution of pyrrolizidine alkaloid diversity among and withinJacobaeaspecies

Plants produce many secondary metabolites showing considerable inter- and intraspecific diversity of concentration and composition as a strategy to cope with environmental stresses. The evolution of plant defenses against herbivores and pathogens can be unraveled by understanding the mechanisms underlying chemical diversity. Pyrrolizidine alkaloids are a class of secondary metabolites with high diversity. We performed a qualitative and quantitative analysis of 80 pyrrolizidine alkaloids with liquid chromatography-tandem mass spectrometry of leaves from 17 Jacobaea species including one to three populations per species with 4–10 individuals per population grown under controlled conditions in a climate chamber. We observed large inter- and intraspecific variation in pyrrolizidine alkaloid concentration and composition, which were both species-specific. Furthermore, we sequenced 11 plastid and three nuclear regions to reconstruct the phylogeny of the 17 Jacobaea species. Ancestral state reconstruction at the species level showed mainly random distributions of individual pyrrolizidine alkaloids. We found little evidence for phylogenetic signals, as nine out of 80 pyrrolizidine alkaloids showed a significant phylogenetic signal for Pagel's λ statistics only, whereas no significance was detected for Blomberg's K measure. We speculate that this high pyrrolizidine alkaloid diversity is the result of the upregulation and downregulation of specific pyrrolizidine alkaloids depending on ecological needs rather than gains and losses of particular pyrrolizidine alkaloid biosynthesis genes during evolution.

Comprehensive investigation and risk study on pyrrolizidine alkaloid contamination in Chinese retail honey

Pyrrolizidine alkaloids (PAs) are common phytotoxins. We performed the first comprehensive investigation on PA contamination in Chinese honeys. LC-MS analysis revealed that 58% of 255 honey samples purchased from 17 regions across Mainland China and Taiwan contained PAs with total content ranging over 0.2–281.1 μg/kg. Monocrotaline (from Crotalaria spp), a PA never found in honey in other regions, together with echimidine (Echium plantagineum) and lycopsamine (from Senecio spp.), were three predominant PAs in PA-contaminated Chinese honeys. Further, PAs present in honeys were found to have geographically distinct pattern, indicating possible control of such contamination in future honey production. Moreover, we proposed a new risk estimation approach, which considered both content and toxic potency of individual PAs in honeys, and found that 12% of the PA-contaminated Chinese honeys tested might pose potential health risk. This study revealed a high prevalence and potential health risk of PA contamination in Chinese honeys.

Rindera graeca (Boraginaceae) Phytochemical Profile and Biological Activities.

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.

Extracting and Analyzing Pyrrolizidine Alkaloids in Medicinal Plants: A Review.

Pyrrolizidine alkaloids (PAs) are distributed in plant families of Asteraceae, Boraginaceae, and Fabaceae and serve in the chemical defense mechanism against herbivores. However, they became a matter of concern due to their toxicity associated with the high risk of intake within herbal preparations, e.g., phytopharmaceutical formulations, medicinal teas, or other plant-derived drug products. In 1992, the German Federal Ministry of Health established the first limits of PA content for fourteen medicinal plants. Because of the toxic effects of PAs, the Federal Institute of Risk Assessment (BfR) established more stringent limits in 2011, whereby a daily intake <0.007 µg/kg body weight was recommended and valid until 2018. A threefold higher limit was then advised by BfR. To address consumer safety, there is the need for more efficient extraction procedures along with robust, selective, and sensitive analytical methods to address these concerns. With the increased prevalence of, e.g., phytopharmaceutical formulations, this timely review comprehensively focuses on the most relevant extraction and analysis strategies for each of those fourteen plant genera. While a variety of extraction procedures has been reported, differences in PA content of up to 1110 ppm (0.11% (w/w)) were obtained dependent on the nature of the solvent and the applied extraction technique. It is evident that the efficient extraction of PAs requires further improvements or at least standardization of the extraction conditions. Comparing the various analytical techniques applied regarding selectivity and sensitivity, LC-MS methods appear most suited. This review shows that both standardized extraction and sensitive determination of PAs is required for achieving appropriate safety levels concerning public health in future.