Structure-Dependent Toxicokinetics of Selected Pyrrolizidine Alkaloids In Vitro

Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo.     

Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.     

Pyrrolizidine alkaloid content in crude and processed borage oil from different processing stages

The pyrrolizidine alkaloid content of crude borage oil and borage oil from different processing stages was determined by GC-MS. The results showed that no pyrrolizidine alkaloids were present above a detection limit of 20 ppb. The reduction factors for pyrrolizidine alkaloids at various stages in the oil refining process were determined by means of spiking experiments using the commercially available pyrrolizidine alkaloid crotaline. It was shown that the pyrrolizidine content in crude borage oil was reduced overall by a factor of about 30,000 in the refining process.     

Differences in Effects of Pyrrolizidine Alkaloids on Five Generalist Insect Herbivore Species

The evolution of the diversity in plant secondary compounds is often thought to be driven by insect herbivores, although there is little empirical evidence for this assumption. To investigate whether generalist insect herbivores could play a role in the evolution of the diversity of related compounds, we examined if (1) related compounds differ in their effects on generalists, (2) there is a synergistic effect among compounds, and (3) effects of related compounds differed among insect species. The effects of pyrrolizidine alkaloids (PAs) were tested on five generalist insect herbivore species of several genera using artificial diets or neutral substrates to which PAs were added. We found evidence that structurally related PAs differed in their effects to the thrips Frankliniella occidentalis, the aphid Myzus persicae, and the locust Locusta migratoria. The individual PAs had no effect on Spodoptera exigua and Mamestra brassicae caterpillars. For S. exigua, we found indications for synergistic deterrent effects of PAs in PA mixtures. The relative effects of PAs differed between insect species. The PA senkirkine had the strongest effect on the thrips, but had no effect at all on the aphids. Our results show that generalist herbivores could potentially play a role in the evolution and maintenance of the diversity of PAs.     

Regio‐ and Stereoselective Biocatalytic Monoamination of a Triketone Enables Asymmetric Synthesis of Both Enantiomers of the Pyrrolizidine Alkaloid Xenovenine Employing Transaminases

The (+)‐ as well as the (−)‐enantiomer of the pyrrolizidine alkaloid xenovenine were prepared within five steps with 17 and 30% overall yields, respectively, in optically pure form, >99% ee as well as >99% de. In the asymmetric key step a transaminase performed a regio‐ and stereoselective monoamination of a triketone. By employing two enantiocomplementary transaminases from Arthrobacter sp. both enantiomers were accessible. The triketone was readily prepared via two steps starting from commercially available, achiral 2‐(n‐heptyl)furan. In the final catalytic hydrogenation step, the newly introduced chiral centre directed hydrogen addition to form preferentially the desired (5Z,8E)‐diastereomer. The regio‐ and stereoselective amination of a single ketone moiety out of three allowed the performance of the shortest and highest yielding total synthesis of the bicyclic showcase pyrrolizidine alkaloid without the need for protecting strategies.

     

Pyrrolizidine alkaloids from Senecio jacobaea affect fungal growth

We investigated the growth-reducing effects of pyrrolizidine alkaloids (PAs) from Senecio jacobaea on nine plant-associated fungi (five strains of Fusarium oxysporum, two of F. sambucinum, and two of Trichoderma sp). Fungal growth was monitored on water agar media containing different concentrations of monocrotaline, retrorsine, or a purified extract of PAs from S. jacobaea. The growth rate of six strains was inhibited by PAs at the highest test concentration (3.33 mM), with the magnitude of the inhibition (7–35%) being dependent upon the specific fungus-PA interaction. In general, the PA extract caused the largest inhibition. However, the fungi isolated from S. jacobaea were positively affected by the PA extract (7–9%). Retrorsine N oxide was as effective as retrorsine in its inhibition of mycelium growth.     

Selective Sequestration of Pyrrolizidine Alkaloids from Diverse Host Plants by Longitarsus Flea Beetles

In 11 species of the flea beetle genus Longitarsus we investigated whether the insects sequester the pyrrolizidine alkaloids (PAs) present in their host plants of the families Asteraceae and Boraginaceae. In all cases where PAs could be detected in the leaves of the local host plant, they could also be detected in the corresponding beetles. In one host plant, Pulmonaria officinalis, no PAs could be detected in the leaves, yet were present in the beetles collected from them. We suggest this is due to uptake of PAs during the root-feeding larval stage. By comparing the GC-MS pattern of PAs found in the beetles with those of their hosts, we investigated the specificity of this sequestration. Furthermore, we compared the pattern of sequestered PAs across beetle species that had been feeding on the same plant, and across hosts in Longitarsus species that feed on different plants in the field. This allowed us to analyze to what extent the PA pattern in the insects is specific for the beetle species and depends on the local food plant. Our data indicate that the PAs found in the beetles are largely determined by the host plant, e.g., whether alkaloids typical of the Boraginaceae or Asteraceae are present. However, there are some indications for a selective uptake of PAs and apparently the beetles are able to metabolize them.     

Differential Utilization of Pyrrolizidine Alkaloids by Males of a Danaid Butterfly, <Emphasis Type="Italic">Parantica sita</Emphasis>, for the Production of Danaidone in the Alar Scent Organ

Males of the chestnut tiger butterfly, Parantica sita, secrete danaidone as a major component from the alar androconial organ (sex brand). Since danaidone has been postulated to be derived from various pyrrolizidine alkaloids (PAs), which males ingest as adults from PA-containing plants, we conducted oral administration tests of several PAs to examine their availability for danaidone production by P. sita males. Males fed with a mixture of intermedine (80%) and lycopsamine (20%) produced danaidone at an average of 25.7 g per individual, which was comparable to that found in field-caught males. In contrast, a smaller amount of danaidone (5.7 to 7.0 g/male) was formed when males ingested retronecine or heliotrine, and those fed with an HCl salt of monocrotaline or retrorsine produced only traces of danaidone (< 0.5 g/male). In addition, males showed a strong feeding response to intermedine/lycopsamine, whereas the other PAs elicited no positive feeding behavior. These results indicate that, unlike the arctiid moths, P. sita males can only successfully convert limited chemical types of PAs into danaidone, and further suggest that in the field, males selectively ingest particular PAs that are readily transformable into danaidone.     

Phenotypic Plasticity of Senecio vulgaris from Contrasting Habitat Types: Growth and Pyrrolizidine Alkaloid Formation

The growth–differentiation balance hypothesis (GDB), which postulates a physiological trade-off between growth and differentiation (morphological and chemical), has been tested almost exclusively for carbon-based secondary metabolites. Little attention has been paid to N-based compounds. In this study we aimed to test the predictions of the GDB hypothesis under field conditions for growth and pyrrolizidine alkaloid (PA) formation in Senecio vulgaris. We conducted a reciprocal transplant experiment at two sites differing widely in their nutrient supply. These included a conventionally managed vineyard (V) and a strip of local wild flowers between crop fields, which was established to promote species diversity in agroecosystems (C). No fertilizer or pesticides are allowed in such ecological compensation areas. In C, we expected lower growth but higher PA formation than in V. Due to differentiated selection regimes in the two habitat types with regard to nutrient (nitrogen) availability in the soil, we also expected different N-allocation patterns for the genotypes of the two collection sites. Plants of V produced more biomass and were taller than the plants of C. The relatively poor nitrogen conditions in C favored an earlier differentiation towards generative organs. In plants of C, higher concentrations of PAs were found than in plants of V. There exists a close negative correlation between growth and PA formation, indicating a trade-off. The origin of the plant material had only a little effect on PA formation. The observed phenotypic reaction of PA formation in S. vulgaris in the two habitats fits quite well the predictions of GDB theory. It is shown that this general response is overlaid by physiological factors leading to a pattern of PA accumulation, which is not readily predictable by nonmechanistic theories.     

Process Development of Lappula squarrosa Oil Refinement: Monitoring of Pyrrolizidine Alkaloids in Boraginaceae Seed Oils

Boraginaceous plants are characterized by high levels of polyunsaturated fatty acids and show a high ratio of ω-3/ω-6 fatty acids. In addition, Lappula squarrosa (Boraginaceae) shows high levels of stearidonic acid content (ω-3; 18:4; 6,9,12,15-octadecatetraenoic acid) showing interesting medical and health promoting properties. On the other hand Boraginaceous plants contain genotoxic and carcinogenic pyrrolizidine alkaloids (PA). An HPLC–ESI–MS/MS sum parameter method was developed to monitor the total sum of 1,2-unsaturated PA in seed oil. The method was used to monitor different steps in oil refinement using lab model experiments and pilot scale refinement of L. squarrosa seed oil. A limit of detection and a limit of quantification (LOQ) of 0.02 μg retronecine equivalents (RE)/kg and 0.05 μg RE/kg were achieved, respectively. Multiple washing steps at pH 2.2 can significantly reduce the PA content to 0.07 % of the start value. In addition, combining washing with neutralization, bleaching and deodorization can reduce the PA content of L. squarrosa oil below the LOQ (0.05 μg RE/kg). The newly established method was further used to analyze the PA content of commercially available Boraginaceous seed oils (Echium spp., Borago officinalis). Three out of ten products were tested as PA positive (PA content ranging from not detected to 0.6 μg RE/kg product).     

Pyrrolizidine alkaloids as oviposition stimulants for the cinnabar moth,Tyria jacobaeae

In choice experiments with artificial leaves, we tested related pyrrolizidine alkaloids (PAs) for their stimulatory effects on the oviposition of the cinnabar moth, a specialist on the PA-containing plant Senecio jacobaea. The PAs from S. jacobaea that we tested stimulated oviposition. Monocrotaline also stimulated oviposition although this PA is not found in plants of the genus Senecio. The moths preferred ovipositing on filter paper with a PA mixture extracted from S. jacobaea to ovipositing on filter paper with single PAs. Senkirkine, heliotrine, and retrorsine did not stimulate oviposition. The nonactive retrorsine differs only in one OH group to the active senecionine, indicating that small structural differences alter the stimulatory activity of PAs. However, a PA mixture extracted from a nonhost plant, Senecio inaequidens, that consisted of 81% of the nonactive retrorsine did stimulate oviposition. Oviposition preferences between Senecio species seem to be determined by chemical compounds other than PAs.     

Direct Evidence for Membrane Transport of Host-Plant-Derived Pyrrolizidine Alkaloid N-Oxides in Two Leaf Beetle Genera

The chrysomelid leaf beetles Longitarsus jacobaeae, Oreina cacaliae, and O. speciosissima sequester pyrrolizidine alkaloids from their asteracean host plants and store them as nontoxic N-oxides. Previous analyses showed that Longitarsus is able to N-oxidize protoxic tertiary PAs, but did not resolve in which form N-oxides are taken up. For Oreina, beetles seem able to directly transmit the polar PA N-oxides from the gut into the hemolymph and prevent any reduction of them in the gut yielding protoxic free bases. Here, we confirm the predicted direct uptake of PAs as N-oxides by Oreina, and elucidate the situation for Longitarsus by applying double-labeled [14C]senecionine [18O]N-oxide as tracer. The beetles were fed with the tracer and subsequently senecionine N-oxide was recovered from the defensive secretions (Oreina) and beetle extracts (Longitarsus), purified by HPLC, and submitted to ESI-MS, GC-MS, and analysis of the specific radioactivity. The 18O-label is retained without any loss in the labeled senecionine N-oxide recovered from the two Oreina species. Analysis of the Longitarsus experiment was complicated by a contamination of the HPLC-purified senecionine N-oxide with a second compound, identified as a dihydrosenecionine N-oxide by high-resolution CID analysis. The dihydrosenecionine N-oxide, probably the 15,20-dihydro derivative, constitutes a major idiosyncratic senecionine metabolite present in the beetle. The recovered senecionine N-oxide retained 74% 18O-label. The remaining 25% is mostly due to loss of 18O by reduction and subsequent re-N-oxidation. The experiments confirm for both beetle genera a direct uptake of the polar nontoxic PA N-oxides, which requires specific membrane carriers. Accumulation of detrimental free base PA is prevented by glucosylation (Oreina) or N-oxidation (Longitarsus).     

Acquired and Partially De Novo Synthesized Pyrrolizidine Alkaloids in Two Polyphagous Arctiids and the Alkaloid Profiles of Their Larval Food-Plants

The profiles of pyrrolizidine alkaloids (PAs) in the two highly polyphagous arctiids Estigmene acrea and Grammia geneura and their potential PA sources in southeastern Arizona were compiled. One of four species of Boraginaceae, Plagiobothrys arizonicus, contained PAs; this is the first PA record for this plant species. The principle PA sources are Senecio longilobus (Asteraceae) and Crotalaria pumila (Fabaceae). The known PA pattern of S. longilobus was extended; the species was found to contain six closely related PAs of the senecionine type. Three novel PAs of the monocrotaline type, named pumilines A-C, were isolated and characterized from C. pumila, a species not studied before. The pumilines are the major PAs in the seeds, while in the vegetative organs they are accompanied by the simple necine derivatives supinidine and as the dominant compound subulacine (1beta,2beta-epoxytrachelanthamidine). In both plant species, the PAs are stored as N-oxides, except C. pumila seeds, which accumulate the free bases. Great variation in PA composition was observed between local populations of C. pumila. The PA profiles were established for larvae and adults of E. acrea that as larvae had fed on an artificial diet supplemented with crotalaria-powder and of G. geneura fed with S. longilobus. In both experiments, the larvae had a free choice between the respective PA source and diet or food plants free of PAs. The profiles compiled for the two species reflect the alkaloid profiles of their PA sources with one exception, subulacine could never be detected in E. acrea. Besides acquired PAs, insect PAs synthesized from acquired necine bases and necic acids of insect origin were detected in the two arctiid species. These insect PAs that do not occur in the larval food sources accounted for some 40-70% (E. acrea) and 17-37% (G. geneura) of total PAs extracted from the insects. A number of novel insect PAs were identified. Plant-acquired and insect PAs were found to accumulate as N-oxides. The results are discussed in relation to specific biochemical, electrophysiological, and behavioral mechanisms involved in PA sequestration by arctiids.     

Pyrrolizidine alkaloids in an overwintering population of monarch butterflies (Danaus plexippus) in California

California overwintering monarch butterflies contain both pyrrolizidine alkaloids (PAs) and theirN-oxides. Analysis of 76 individual monarchs by TLC, HPLC, GLC, and GC-MS has shown the presence of three types of PAs, the saturated diester sarracine, the saturated monoester 7-angelylplatynecine, and the unsaturated dialcohol retronecine. Monarchs arriving at the overwintering site in Santa Cruz, California, showed a wide variation in both the type and amount of PA present. Those sampled after a PA-containing plant (Senecio mikanioides) had bloomed at the site showed an altered PA profile. While the plant was found to contain sarracine and 7-angelylplatynecine, which are nontoxic to mammals, the monarchs showed an increase in retronecine levels, a toxic PA, after the plant bloom. Apparently monarchs utilize PA-containing plants both en route to their overwintering site and at the site, and potentially alter those PAs to forms toxic to mammals.     

Identification of Toxic Pyrrolizidine Alkaloids and Their Common Hepatotoxicity Mechanism

Pyrrolizidine Alkaloids (PAs) are currently one of the most important botanical hepatotoxic ingredients. Glutathion (GSH) metabolism is the most reported pathway involved in hepatotoxicity mechanism of PAs. We speculate that, for different PAs, there should be a common mechanism underlying their hepatotoxicity in GSH metabolism. Computational methods were adopted to test our hypothesis in consideration of the limitations of current experimental approaches. Firstly, the potential targets of 22 PAs (from three major PA types) in GSH metabolism were identified by reverse docking; Secondly, glutathione S-transferase A1 (GSTA1) and glutathione peroxidase 1 (GPX1) targets pattern was found to be a special characteristic of toxic PAs with stepwise multiple linear regressions; Furthermore, the molecular mechanism underlying the interactions within toxic PAs and these two targets was demonstrated with the ligand-protein interaction analysis; Finally, GSTA1 and GPX1 were proved to be significant nodes in GSH metabolism. Overall, toxic PAs could be identified by GSTA1 and GPX1 targets pattern, which suggests their common hepatotoxicity mechanism: the interfering of detoxication in GSH metabolism. In addition, all the strategies developed here could be extended to studies on toxicity mechanism of other toxins.     

农药用生物碱的研究进展

综述了杀虫生物碱、灭鼠生物碱及灭菌生物碱的最新研究进展。苦参碱和烟碱在我国已进人工业化开发。黎芦碱、小蘖碱、雷公藤生物碱、甾醇生物碱均显示出良好的杀虫活性，番木鳖碱可用于灭鼠。而吡咯里西啶类生物碱则具有较强的杀菌活性。     

The Effects of Naringenin on miRNA-mRNA Profiles in HepaRG Cells

Naringenin, a natural flavonoid widely found in citrus fruits, has been reported to possess anti-oxidant, anti-inflammatory, and hepatoprotective properties as a natural dietary supplement. However, the regulatory mechanism of naringenin in human liver remains unclear. In the present study, messenger RNA sequencing (mRNA-seq), microRNA sequencing (miRNA-seq), and real-time qPCR were used to distinguish the expression differences between control and naringenin-treated HepaRG cells. We obtained 1037 differentially expressed mRNAs and 234 miRNAs. According to the target prediction and integration analysis in silico, we found 20 potential miRNA-mRNA pairs involved in liver metabolism. This study is the first to provide a perspective of miRNA–mRNA interactions in the regulation of naringenin via an integrated analysis of mRNA-seq and miRNA-seq in HepaRG cells, which further characterizes the nutraceutical value of naringenin as a food additive.     

刺桐属植物的生物碱成分及其药理作用研究进展

刺桐属（Erythrina）植物的主要成分有黄酮和生物碱。其中生物碱成分的结构类型主要为刺桐生物碱,药理作用主要有增强细胞毒性,抗焦虑,抗痉挛和DPPH自由基清除作用。本文对此属中分离得到的生物碱及其药理活性的研究现状进行综述,为今后对此属植物进一步研究提供参考。     

色谱法分析烟草生物碱及其代谢物的研究进展

介绍了近10多a来烟碱的分析测定情况,归纳整理了国内外主要杂志报道的有关烟草、卷烟及其烟气、头发、血液、尿等中烟草生物碱及其代谢物的色谱分析方法,并对烟草生物碱及其代谢物的色谱分析方法及其发展前景进行了评述。     

荠菜中总生物碱提取工艺研究

采用索氏提取法研究了荠菜中总生物碱的提取工艺条件。首先通过单因素试验考查了提取时间、提取剂用量和提取剂浓度等因素对荠菜总生物碱提取率的影响,然后利用正交试验确定了最适宜提取工艺条件为：原料荠菜粉20 g,用200 mL 90%乙醇提取9 h,总生物碱的提取率为0047%。