Alkaloid patterns and biosynthetic capacity of root cultures from some pyrrolizidine alkaloid producing Senecio species

Root cultures of Senecio vulgaris, S. vernalis, S. erucifolius and S. squalidus were established. The patterns of pyrrolizidine alkaloids found in these root cultures were analyzed by high-resolution GC and GC-MS and compared with the alkaloids present in the respective plants. In vitro cultured roots produce alkaloid patterns and accumulate quantities which are comparable to those found in soil grown plants. With the exception of the otonecine derivative senkirkine all pyrrolizidines accumulate as N-oxides. Only senkirkine is partially released into the medium. The cultures incorporate biosynthetic precursors, e.g. ¹⁴C-labelled putrescine or spermidine with high efficiency into the alkaloids. Senecionine N-oxide was found to be the main product of biosynthesis. Evidence is presented that senecionine N-oxide is directly transformed into senkirkine, the main alkaloid of S. vernalis root cultures.Abbreviations GC Gas chromatography - MS Mass spectroscopy - PND Phosphorous-Nitrogen-Detector - FID Flame Ionization Detector - fr.wt Fresh weight     

Concomitant occurrence of pyrrolizidine and quinolizidine alkaloids in the hemiparasite Osyris alba L. (Santalaceae)

The investigation of the alkaloid extracts of the hemiparasitic plant Osyris alba, collected from three different localities in southern France, revealed the concomitant presence of both pyrrolizidine (PA) and quinolizidine (QA) alkaloids in the samples from two of these localities. The sample from the third locality contained only PAs. The eight QAs identified were sparteine, N-methylcytisine, cytisine, methyl-12-cytisine acetate, hydroxy-N-methylcytisine, N-acetylcytisine, lupanine, and anagyrine. Of the eleven detected PAs, eight were identified as chysin A, chysin B, 1-carboxypyrrolizidine-7-olide, senecionine, integerrimine, retrorsine, senecivernine and a new alkaloid janfestine (7R-hydroxychysin A or 1R-carbomethoxy-7R-hydroxypyrrolizidine). PAs were mainly present as their N-oxides This is, to our knowledge, the first report demonstrating the simultaneous presence of two classes of alkaloids, quinolizidine and pyrrolizidine alkaloids, in a single parasitic plant. As these alkaloids do not occur in the same host plant, the results indicate that Osyris must have tapped more than one host plant concomitantly. Since both quinolizidine and pyrrolizidine alkaloids serve as defence compounds against herbivores, affecting different molecular targets, the simultaneous acquisition of the two types of alkaloids by a single plant could provide a novel mode of defence of hemiparasites against herbivores.     

Metabolic profiling in Echium plantagineum: presence of bioactive pyrrolizidine alkaloids and napthoquinones from accessions across southeastern Australia

Geographically distinct populations of Paterson’s curse (Echium plantagineum L., Boragineacea), found near roadsides across New South Wales and Victoria, Australia were surveyed along 3 distinct longitudinal transects in spring of 2011 for presence of pyrrolizidine alkaloids and naphthoquinones in sampled plants. Composite samples of shoots and roots were collected from each of 45 sites; shoot extracts were subjected to solid phase extraction and LC-ESI/MS for determination of pyrrolizidine alkaloids (PAs) and related N-oxides (PANOs), while root periderm extracts were analysed for naphthoquinone content spectrophotometrically and by LC-ESI/MS. Metabolic profiling of 12 possible PAs and PANOs showed their consistent appearance in all shoot extracts, with lepthamine N-oxide, echimidine-N oxide and echumine N-oxide predominant. The three major PANOs were significantly higher in northern sampling locations than those further south. Root extracts contained shikonin and several related naphthoquinones, as well as two of the major PANOs found in the leaves. Naphthoquinones were highest in the northwest corner of the sampled region. The patterns of abundance of secondary metabolites in E. plantagienum suggest that climate change might result in greater production of defensive compounds by E. plantagineum, making this weed increasingly toxic to livestock.     

Pyrrolic and N-oxide metabolites formed from pyrrolizidine alkaloids by hepatic microsomes in vitro: Relevance to in vivo hepatotoxicity

An analytical method of improved sensitivity has enabled measurements to be made of N-oxide as well as pyrrolic metabolites formed from a range of unsaturated pyrrolizidine alkaloids in hepatic microsome preparations. Using microsomes from livers of phenobarbitone-pretreated male Fischer rats, all 13 alkaloids tested were metabolised to both N-oxides and pyrroles. The most lipophilic alkaloids gave enhanced rates of metabolism. No consistent relationship existed between rates of N-oxide and of pyrrole formation. The two pathways appeared to be independent. The ratio of N-oxide to pyrrolic metabolites varied, depending on the type of ester: it was highest for ‘open’ diester alkaloids, lowest for 12 membered macrocyclic diesters and for monoesters. Steric hindrance by the acid moiety could account for these differences, by affecting the balance between microsomal oxidation of the amino alcohol moiety at the nitrogen and C8 positions respectively and could explain the high pyrrole yields given by some macrocyclic diesters. The levels of pyrrolic metabolites bound to liver tissues and responsible for hepatotoxicity in rats given pyrrolizidine alkaloids, did not necessarily reflect the rates of formation of such metabolites measured in vitro. In the animal additional factors could influence the formation and tissue binding of pyrrolic metabolites, including the detoxication of alkaloids by hydrolysis and the chemical reactivity and stability of the toxic metabolites. A comparison of heliotridine esters with retronecine esters showed that the 7-hydroxyl or -ester configuration had a relatively small influence on the balance between formation of pyrrolic metabolites and detoxication by N-oxidation. The results did not support any hypothesis that heliotridine esters should generally be more hepatotoxic than analogous retronecine esters. The structure of the acid moiety was likely to have at least as much influence on toxicity as the base configuration.     

Correlation between Polyamines and Pyrrolidine Alkaloids in Developing Tobacco Callus

Since the diamine putrescine can be metabolized into the pyrrolidine ring of tobacco alkaloids as well as into the higher polyamines, we have investigated the quantitative relationship between putrescine and these metabolites in tobacco callus cultured in vitro. We measured levels of free and conjugated putrescine and spermidine, and pyrrolidine alkaloids, as well as activities of the putrescine-biosynthetic enzymes arginine and ornithine decarboxylase. In callus grown on high (11.5 micromolar) α-naphthalene acetic acid, suboptimal for alkaloid biosynthesis, putrescine and spermidine conjugates were the main putrescine derivatives, while in callus grown on low (1.5 micromolar) α-naphthalene acetic acid, optimal for alkaloid formation, nornicotine and nicotine were the main putrescine derivatives. During callus development, a significant negative correlation was found between levels of perchloric acid-soluble putrescine conjugates and pyrrolidine alkaloids. The results suggest that bound putrescine can act as a pool for pyrrolidine alkaloid formation in systems where alkaloid biosynthesis is active. In addition, changes in arginine decarboxylase activity corresponding to increased alkaloid levels suggest a role for this enzyme in the overall biosynthesis of pyrrolidine alkaloids.     

Biosynthesis of spermidine, a direct precursor of pyrrolizidine alkaloids in root cultures of Senecio vulgaris L.

 The polyamine spermidine is an essential biosynthetic precursor of pyrrolizidine alkaloids. It provides its aminobutyl group which is transferred to putrescine yielding homospermidine, the specific building block of the necine base moiety of pyrrolizidine alkaloids. The enzymatic formation of spermidine was studied in relation to the unique role of this polyamine as an alkaloid precursor. S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and spermidine synthase (SPDS, EC 2.5.1.16) from root cultures of Senecio vulgaris were partially purified and characterized. The SAMDC-catalyzed reaction showed a pH optimum of 7.5, that of SPDS an optimum of 7.7. The K _m value of SAMDC for its substrate S-adenosylmethionine (SAM) was 15 μM, while the apparent K _m values of SPDS for its substrates decarboxylated SAM (dSAM) and putrescine were 4 μM and 21 μM, respectively. The relative molecular masses of the two enzymes, determined by gel filtration, were 29 000 (SAMDC) and 37 000 (SPDS). Studies with various potential inhibitors revealed, for most inhibitors, profiles that were similar to those established with the respective enzymes from other plant sources. However, putrescine which is not known to be an inhibitor of plant SAMDC, strongly inhibited the enzyme from S. vulgaris roots. Spermidine synthase was sensitive to inhibition by its product spermidine. In the presence of the stationary tissue concentrations of the two polyamines (ca. 0.1 mM each) the activities of SAMDC and SPDS would be inhibited by >80%. The results are discussed in relation to the role of spermidine in primary and secondary metabolism of alkaloid-producing S. vulgaris root cultures. Received: 15 September 1999 / Accepted 10 December 1999     

Alkaloids from Galanthus rizehensis

Two new Amaryllidaceae alkaloid N-oxides, incartine N-oxide (1) and lycorine N-oxide (2) together with one β-carboline alkaloid, 1-acetyl-β-carboline (3) and six known alkaloids namely, incartine (4), N-trans feruloyltyramine (5), lycorine (6), O-methylnorbelladine (7), vittatine (8) and 11-hydroxyvittatine (9) were isolated from Galanthus rizehensis Stern (Amaryllidaceae). The structures of the alkaloids were elucidated by spectroscopic analyses (UV, IR, MS, 1D and 2D NMR). Acetylcholinesterase inhibitory activity potentials of the compounds were also determined.     

Uptake and metabolism of pyrrolizidine alkaloids in<Emphasis Type="Italic"> Longitarsus</Emphasis> flea beetles (Coleoptera: Chrysomelidae) adapted and non-adapted to alkaloid-containing host plants

Several Longitarsus flea beetle species sequester pyrrolizidine alkaloids acquired from their Asteraceae and Boraginaceae host plants. We carried out feeding and injection experiments using radioactively labeled pyrrolizidine alkaloids to investigate the physiological mechanisms of uptake, metabolism and storage of alkaloids in adult beetles. We examined six Longitarsus species belonging to different phylogenetic clades in a comparative approach. All species that accepted pyrrolizidine alkaloids in a preceding food choice study showed the ability both to store pyrrolizidine alkaloid N-oxides and to metabolize tertiary pyrrolizidine alkaloids into their N-oxides. Regardless of whether the beetles' natural host plants contain pyrrolizidine alkaloids or not, these species were found to possess an oxidizing enzyme. This oxygenase appears to be specific to pyrrolizidine alkaloids: [³H]Atropine and [¹⁴C]nicotine, two alkaloids not related to pyrrolizidine alkaloids, were neither stored nor N-oxidized by any of the tested species. One species, L. australis, that strictly avoids pyrrolizidine alkaloids behaviorally, exhibited a lack of adaptations to pyrrolizidine alkaloids on a physiological level as well. After injection of tertiary [¹⁴C]senecionine, beetles of this species neither N-oxidized nor stored the compounds, in contrast to L. jacobaeae, an adapted species that underwent the same treatment. L. jacobaeae demonstrated the same efficiency in N-oxidation and storage when fed or injected with tertiary [¹⁴C]senecionine.Communicated by G. Heldmaier     

Comparative HILIC/ESI-QTOF-MS and HPTLC studies of pyrrolizidine alkaloids in flowers of Tussilago farfara and roots of Arnebia euchroma

The utility of HPTLC and HILIC/ESI-QTOF-MS for the determination of pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) was compared in the selected plant species: Tussilago farfara L. (TF, flower) and Arnebia euchroma (Royle) I.M. Johnst. (AE, root). HPTLC confirmed the postulated presence of PAs (saturated and unsaturated) or PANOs in the tested extracts. In accordance with previous studies, HILIC/ESI-Q-TOF-MS confirmed the presence of the toxic PA senkirkine and the saturated otonecine-type PAs, tussilagine and isotussilagine in the TF extract and 7-angeloylretronecine and 9-angeloylretronecine in AE extract. Moreover, the following alkaloids were identified in AE root: intermedine, intermedine-N-oxide, leptanthine-N-oxide, echimidine-N-oxide (or their corresponding stereoisomers) and traces of 7-angeloylretronecine and 9-angeloylretronecine-N-oxide. The study demonstrates the HILIC/ESI-Q-TOF-MS method to be a very useful tool for monitoring PAs and PANOs in the test samples, even when not all of the necessary standards are available. Quantitative analysis of senkirkine in TF flower by HILIC/ESI-QTOF-MS featured high resolution, high precision, high mass accuracy, and very high sensitivity with limit-of-detection (LOD) of 27.50 fg/μL and limit-of-quantitation (LOQ) of 91.60 fg/μL. The results from both methods may be used for the development or rejection of European Pharmacopoeia (X) monographs of both investigated species.     

1H NMR Spectroscopy of pyrrolizidine alkaloids

The ¹H NMR spectra of three pyrrolizidine alkaloids of the macrocyclic diester type, retrorsine, seneciphylline and senecionine, plus their three N-oxides have been assigned. Previous ¹H NMR studies of these pyrrolizidine alkaloids have stressed the difficulties of spectral intrepretation. The results reported here will provide a useful resource for analysis of tertiary structure in these and related compounds.     

UPLC-MS based metabolomics study on Senecio scandens and S. vulgaris: an approach for the differentiation of two Senecio herbs with similar morphology but different toxicity

Pyrrolizidine alkaloids show significant hepatotoxicity as they can bind to DNA or proteins after being activated in liver. Senecio vulgaris L., like many Compositae herbs containing pyrrolizidine alkaloids, was reported to have great hepatotoxicity. However, Senicio scandens Buch.-Ham., from the same genus, which was also used as a herb and documented in China Pharmacopoeia published in 2010, hardly showed any side effects or relevant toxicity. In the present study, we conducted the metabolomics study using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to obtain the different metabolic profiles of the two Senecio herbs. In addition, principle component analysis (PCA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA) were introduced for the multivariate analysis, and MS/MS was applied to the identification of target alkaloid markers which contributed most to the established models. As a result, ten pyrrolizidine alkaloids, including adonifoline, senecionine, senecionine N-oxide, retrorsine, retrorsine N-oxide and seneciphylline, were selected and identified. Among them, adonifoline was found to be a specific marker for S. scandens while senecionine and its N-oxidative were characteristic markers for S. vulgaris. Furthermore, the hepatotoxicity studies in vivo and in vitro showed that senecionine had more potent toxicity (LD₅₀, 57.3?mg/kg; IC₅₀, 5.41???M) than that of adonifonine (LD₅₀, 163.3?mg/kg; IC₅₀, 49.91???M). Taken together, the present study provides not only better understanding of the different toxicity between the two Senecio herbs containing pyrrolizidine alkaloids but also a reference method, which can be applied to other genetically closed species with similar morphology but different toxicity.     

Sites of synthesis,translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L.

¹⁴C-Labelled alkaloid precursors (arginine, putrescine, spermidine) fed to Senecio vulgaris plants via the root system were rapidly taken up and efficiently incorporated into the pyrrolizidine alkaloid senecionine N-oxide (sen-Nox) with total incorporations of 3–6%. Considerable amounts of labelled sen-Nox were translocated into the shoot and were directed mainly into the inflorescences, the major sites of pyrrolizidine-alkaloid accumulation. Detached shoots of S. vulgaris were unable to synthesize pyrrolizidine alkaloids, indicating that the roots are the site of their biosynthesis. Further evidence was obtained from studies with in-vitro systems established from S. vulgaris: root cultures were found to synthesize pyrrolizidine alkaloids but not cell-suspension cultures, tumor cultures or shoot-like teratomas obtained by transformation with Agrobacterium tumefaciens. Studies on transport of [¹⁴C]sen-Nox, which was fed either to detached shoots or to the root system of intact plants, indicate that the alkaloid N-oxide does not simply follow the transpiration stream but is specifically channelled to the target tissues such as epidermal stem tissue and flower heads. Exogenously applied [¹⁴C]senecionine is rapidly N-oxidized. If the phloem path along the stem is blocked by a steam girdle translocation of labelled sen-Nox is blocked as well. Root-derived sen-Nox accumulated below the girdle and only trace amounts were found in the tissues above. It is most likely that the root-to-shoot transport of sen-Nox occurs mainly if not exclusively via the phloem. In accordance with previous studies the polar, salt-like N-oxides, which are often considered to be artifacts, were found to be the real products of pyrrolizidine-alkaloid biosynthesis as well as the physiological forms for long-distance transport, tissue-specific distribution and cellular accumulation.Abbreviations FW fresh weight - sen senecionine - sen-Nox senecionine N-oxide     

Metabolic links between the biosynthesis of pyrrolizidine alkaloids and polyamines in root cultures of Senecio vulgaris

Isotope feeding and inhibitor experiments were performed in order to elucidate the pathway common to polyamine and alkaloid biosynthesis in root cultures of Senecio vulgaris L. -Difluoromethylarginine, a specific inhibitor of arginine decarboxylase, prevented completely the incorporation of radioactivity from [¹⁴C]arginine and [¹⁴C]ornithine into spermidine and the pyrrolizidine alkaloid senecionine N-oxide. In contrast, -difluoromethylornithine, a specific ornithine-decarboxylase inhibitor, had no effect on the flow of radioactivity from labelled ornithine and arginine into polyamines and alkaloids. Thus, putrescine, the common precursor of polyamines and pyrrolizidine alkaloids, is exclusively derived via the arginine-agmatine route. Ornithine is rapidly transformed into arginine. Recycling of the guanido moiety of agmatine back to ornithine can be excluded. Putrescine and spermidine were found to be reversibly interconvertable and to excist in a highly dynamic state. In contrast, senecionine N-oxide did not show any turnover but accumulated as a stable metabolic product. In-vivo evidence is presented that the carbon flow from arginine into the polyamine/alkaloid pathway may be controlled by spermidine. The possible importance of the metabolic coupling of pyrrolizidine-alkaloid biosynthesis to polyamine metabolism is discussed.Abbreviations DFMA D,l--difluoromethylarginine - DFMO D,l--difluoromethylornithine - FW fresh weight     

Experience influences gustatory responsiveness to pyrrolizidine alkaloids in the polyphagous caterpillar,Estigmene acrea

Electrophysiological recordings from taste sensilla of the caterpillar Estigmene acrea with the pyrrolizidine alkaloid, seneciphylline N-oxide, demonstrated that prior feeding on plants with pyrrolizidine alkaloids caused an increase in responsiveness of the PA-sensitive cells in two sensilla, relative to feeding on plants without such chemicals. Rearing on synthetic diet without pyrrolizidine alkaloids for up to seven generations caused a continuous decline in responsiveness, that could be reversed by experience with powdered Crotalaria pumila in the diet or by pure pyrrolizidine alkaloid, monocrotaline, in the diet. Response to the cardiac glycoside, ouabain, that stimulates one of the two pyrrolizidine alkaloid-sensitive cells, showed a similar decline. Pyrrolizidine alkaloids had no measurable effect on growth and development. Responses in all other taste cells were unaffected. The data are discussed in relation to the possible adaptive significance and the possible mechanisms involved.R.F. Chapman has died since this article was written     

Homospermidine in transgenic tobacco results in considerably reduced spermidine levels but is not converted to pyrrolizidine alkaloid precursors

Homospermidine synthase is the first specific enzyme in the biosynthesis of pyrrolizidine alkaloids. Whereas the substrates putrescine and spermidine are part of the highly dynamic polyamine pool of plants, the product homospermidine is incorporated exclusively into the necine base moiety of pyrrolizidine alkaloids. Recently, the gene encoding homospermidine synthase has been shown to have been recruited several times independently during angiosperm evolution by the duplication of the gene encoding deoxyhypusine synthase. To test whether high levels of homospermidine suffice for conversion, at least in traces, to precursors of pyrrolizidine alkaloids, transgenic tobacco plants were generated expressing homospermidine synthase. Analyses of the polyamine content revealed that, in the transgenic plants, about 80% of spermidine was replaced by homospermidine without any conspicuous modifications of the phenotype. Tracer-feeding experiments and gas chromatographic analyses suggested that these high levels of homospermidine were not sufficient to explain the formation of alkaloid precursors. These results are discussed with respect to current models of pathway evolution.     

Lupin alkaloids from sophora chrysophylla

A new lupin alkaloid, (?)-mamanine N-oxide, was isolated from Sophora chrysophylla together with 18 known alkaloids including some unusual lupin alkaloids such as kuraramine, lamprolobine, epilamprolobine, epilamprolobine N-oxide, (+)-mamanine and (?)-pohakuline. It was also shown that the alkaloid constituents of S. chrysophylla differed considerably in the leaves, stems and seeds.     

Selective uptake of pyrrolizidine N-oxides by cell suspension cultures from pyrrolizidine alkaloid producing plants

The N-oxides of pyrrolizidine alkaloids such as senecionine or monocrotaline are rapidly taken up and accumulated by cell suspension cultures obtained from plants known to produce pyrrolizidines, i.e. Senecio vernalis, vulgaris, viscosus (Asteraceae) and Symphytum officinale (Boraginaceae). The transport of the N-oxides into the cells is a specific and selective process. Other alkaloid N-oxides such as sparteine N-oxide are not taken up. Cell cultures from plant species which do not synthesize pyrrolizidine alkaloids are unable to accumulate pyrrolizidine N-oxides. The suitability of the pyrrolizidine N-oxides in alkaloid storage and accumulation is emphasized.     

13C NMR spectroscopy of Pyrrolizidine alkaloids

The ¹³C NMR spectra of nine pyrrolizidine alkaloids of the macrocyclic diester type, seven of the corresponding N-oxides and of the parent base retronecine have been recorded and the signals assigned. The 13C NMR signals were found to be sensitive to structural variation in both the diester moiety and the heterocyclic ring system, providing useful information for structural elucidation, particularly when the ¹H NMR spectra may be difficult to interpret.     

Some alkaloids of Alstonia undulata

Sixteen indole alkaloids have been identified in the root bark and in the leaves of Alstonia undulata from New Caledonia. They are vincamedine, tetrahydroalstonine, alstonidine, deplancheine, fluorocarpamine, pleiocarpamine, 11-methoxyakuammicine, cabucraline, desoxycabufiline, cabucraline-N(4)-oxide, 11-methoxyakuammicine N(4)-oxide, gentiacraline, plumocraline, vincorine, cathafoline and pericyclivine. Five bis indoles of unknown structure have been isolated. The root bark alkaloid mixture contains gentiacraline the first alkaloid made up of an indole and a pyridine alkaloid.     

Polyamines and leaf senescence in pyrrolizidine alkaloid-bearing Heliotropium plants

Putrescine, spermidine and spermine were found in leaves and inflorescences of H. angiospermum and H. indicum plants; the levels of these amines declined with leaf age. In addition, homospermidine was identified in the inflorescence axes and youngest leaves of H. indicum. The youngest tissues exhibited the highest levels of both putrescine and pyrrolizidine alkaloids. The detection of homospermidine in the plants supports the theory that the pyrrolizidine moiety is derived from two molecules of putrescine with homospermidine as an intermediate. In the youngest organs, the pyrrolizidines represented over 5% of the total nitrogen content. Their level was 50–100 fold higher than that of the polyamines, including putrescine. When detached and kept in the dark for 100–120 hr, mature older Heliotropium leaves, with a very low polyamine content, exhibited only a weak senescence syndrome. By contrast, in detached, darkened leaves of Avena sativa and Nicotiana alata having high polyamine levels, the chlorophyll and protein degradation and increases in free amino acids were very pronounced.