Pyrrolizidine alkaloids from Senecio longilobus and Senecio glabellus

TLC, capillary GC, packed column and capillary GC-MS, and¹H NMR were used to characterize pyrrolizidine alkaloids fromSenecio longilobus andS.glabellus.S.glabellus contained senecionine and integerrimine, andS.longilobus contained senecionine, integerrimine, seneciphylline and retrorsine, all present predominantly asN-oxides. Alkaloid content varied greatly in collections ofS.longilobus. This is the first report of integerrimine in these plants.     

Senecionine n-oxide,the primary product of pyrrolizidine alkaloid biosynthesis in root cultures of Senecio vulgaris

Root cultures of Senecio vulgaris synthesize pyrrolizidine alkaloids which are accumulated in the form of their N-oxides. The cultures incorporate biosynthetic precursors, such as arginine, ornithine, isoleucine, putrescine and spermidine, with high efficiency into the alkaloids. Senecionine N-oxide is found to be the primary product of biosynthesis. With putrescine and spermidine incorporation rates of 20–30% are obtained. The N-oxide synthesized does not appear to undergo significant turnover. Tertiary pyrrolizidine alkaloids, if found at all, occur in small amounts in old tissues only. They are derived from the corresponding oxides, and are easily formed spontaneously during alkaloid extraction. The suitability of N-oxides in alkaloid storage is discussed.     

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.     

1-Hydroxyplatyphyllide,a norsesquiterpene lactone from Senecio gilliesiano

A new norsesquiterpene lactone, 1-hydroxyplatyphyllide, was isolated from roots of Senecio gilliesiano together with the previously known platyphyllide and the common pyrrolizidine alkaloids, senecionine and retrorsine. The structures of the products were determinated by spectral and chemical means and by comparison with standards and previously reported data. The genetic pathway of fragmentation by electron impact induced fragmentation of platyphyllide and 1-hydroxyplatyphyllide have been studied with the aid of high resolution measurements and metastable descompositions by the use of linked scanning.     

Safety assessment of food and herbal products containing hepatotoxic pyrrolizidine alkaloids: interlaboratory consistency and the importance of N-oxide determination

Introduction  – Two recent mass spectrometry‐based reports concerning Senecio scandens yielded remarkably dissimilar pyrrolizidine alkaloid constituents. In both studies, and in a related analysis of Senecio scandens and Tussilago farfara using micellar electrokinetic chromatography, the presence of hazardous N‐oxides of the alkaloids was either not considered or was inadequately considered. This raises concerns about the effectiveness of the methodologies used in these, and similar, studies in assessing the pyrrolizidine alkaloid content and the safety of food, food supplements and medicines for human use. Objective  – To highlight essential analytical requirements for confident assessment of pyrrolizidine alkaloid‐related safety of food and herbal products for human use. Methodology  – Direct infusion‐ESI MS and HPLC‐ESI MS were used to analyse samples derived from liquid–liquid partitioning experiments and from strong cation exchange, solid‐phase extraction of pyrrolizidine alkaloids and their N‐oxides. Results  – A simple solvent partitioning experiment using pure senecionine and senecionine‐N‐oxide, two constituents reported in one of the mass spectrometry‐based studies of S. scandens, clearly demonstrated the inadequacy of the reported method to detect and quantitate hazardous pyrrolizidine alkaloid N‐oxide components. A preliminary LCMS analysis of commercially‐prepared extracts of comfrey roots (Symphytum officinale and S. uplandicum s. l.) was used as a model to highlight the analytical importance of N‐oxides in the safety assessment of pyrrolizidine alkaloid‐containing medicinal herbs. Conclusions  – This study highlighted significant differences in the reported identification of pyrrolizidine alkaloids from the same plant species, and clearly demonstrated the inadequacy of some procedures to include N‐oxides in the assessment of pyrrolizidine alkaloid‐related safety of food and herbal products. Copyright © 2008 John Wiley & Sons, Ltd.     

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.     

Plant-defensive sesquiterpenoids from Senecio species with biopesticide potential

Senecio species have been used in folk medicine for treatment of wounds, as antiemetic, anti-inflammatory and their crude extract or dry powder as crop protection agents. The toxicity exhibited to livestock by these plants has been attributed to their content in pyrrolizidine alkaloids and furanoeremophilane type sesquiterpenes. Sesquiterpenoids with eremophilane, cacalol, bisabolane, silphinene, caryophillane, humulane, germacrane and benzofurane skeletons have been isolated from this genus. Here we focus on bioactive sesquiterpenoids with plant defensive properties isolated from Senecio.     

Pyrrolizidine alkaloids in the antipodean genus Brachyglottis (Asteraceae)

The phylogeny of the genus Brachyglottis suggests that its constituent species should contain pyrrolizidine alkaloids. Consistent with this hypothesis, and the established occurrence of such alkaloids in Brachyglottis repanda, Brachyglottis kirkii, and Brachyglottis hectori, an investigation of Brachyglottis adamsii revealed the presence of senecionine and retrorsine; Brachyglottis huntii was found to contain senkirkine and retrorsine; 7-O-angelylheliotridine was the predominant alkaloid in Brachyglottis perdicioides, and the same alkaloid together with senecionine, senkirkine and intergerrimine was present in the Brachyglottis hectori × B. perdicioides “Alfred Atkinson” horticultural hybrid; Brachyglottis sciadophila contained clivorine and neopetasitenine (acetylfukinotoxin); the latter alkaloid was also present in B. kirkii together with the previously reported senkirkine and senkirkine 12-acetate.     

Gustatory responsiveness to pyrrolizidine alkaloids in the Senecio specialist, Tyria jacobaeae (Lepidoptera, Arctiidae)

Abstract.  Electrophysiological recordings from taste sensilla of the caterpillar Tyria jacobaeae with the pyrrolizidine alkaloids, characteristic compounds from their host plants, demonstrated sensitivity of a pyrrolizidine alkaloid-sensitive cell in the lateral galeal sensilla at concentrations as low as 1 × 10⁻¹¹ M. Another pyrrolizidine alkaloid-sensitive cell in the medial galeal sensilla responded at higher concentrations. Both pyrrolizidine alkaloid-cells were maximally sensitive to seneciphylline N -oxide and senecionine N -oxide. Seven other pyrrolizidine alkaloids were less stimulating. Monocrotaline N -oxide was the least stimulating. Observation experiments demonstrated that differences in sensitivity to different pyrrolizidine alkaloids at the electrophysiological level were correlated with differences in feeding behaviour; the first feeding bout was of longer duration on diet containing seneciphylline N -oxide than on diet containing monocrotaline N -oxide, and a plain diet was generally not accepted.     

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.     

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     

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     

Pyrrolizidine alkaloids in Senecio madagascariensis from Australia and Hawaii and assessment of possible livestock poisoning

The alkaloid content of Senecio madagascariensis collected from Australia and Hawaii was examined. Alkaloids were identified from the above ground whole plant (stems, leaves and flowers) by GC/MS analysis and included: senecivernine, senecionine, integerrimine, senkirkine, mucronatinine, retrorsine, usaramine, otosenine, acetylsenkirkine, desacetyldoronine, florosenine and doronine. Plant material collected from the Hawaiian Islands was found to be identical in pyrrolizidine alkaloid content to that from a single composite collection made from northern New South Wales, Australia. Overall, no appreciable differences in alkaloid content were found between locations, whereas variation among individual plants was evident. The average total pyrrolizidine alkaloid content varied from a low of 217 μg/g to a high of 1990 μg/g (dry weight basis) among the locations. Based on comparable alkaloid content and documented pyrrolizidine alkaloidosis cases from Australia, S. madagascariensis may pose a significant risk to livestock grazing heavily infested ranges on the Hawaiian Islands.     

Changes in Senecio grisebachii pyrrolizidine alkaloids abundances and profiles as response to soil quality

Abstract

Senecio grisebachii Baker is an invasive weed considered to be toxic due to the presence of pyrrolizidine alkaloids (PA) in its tissues. The PA production by S. grisebachii aerial parts was evaluated in samples grown in two Argentinean Rolling Pampa fields with the same kind of soil but differing in the length of their exploitation period by conventional tillage practices and, consequently, in their deterioration level.

We found significant differences in the relative concentrations of seven alkaloids between samples taken from the two fields. Seneciphylline was the most abundant alkaloid in inflorescences from less deteriorated soil (LD) while senecionine was the major one in those from highly deteriorated soil (D) being followed by seneciphylline, integerrimine, and minor amounts of spartiodine, jacobine, jacozine and retrorsine. A significant increase in total alkaloid content (TAC) was observed in inflorescences from samples growing in D soil (3.52±0.20 mg/g DW) when comparing with those from samples grown in LD one (3.23±0.26).     

Further pyrrolizidine alkaloids and furoeremophilanes from Senecio species

The investigation of 17 Senecio species afforded in addition to 14 known pyrrolizidine alkaloids 20 new ones, including some compounds of a novel type with an additional lactone moiety. Furthermore seven known and 11 new furoeremophilanes, two known and a new eremophilone, a further shikimic acid derivative and an acetylenic compound, most likely formed by intramolecular Diels-Alder reaction were isolated. The structures were elucidated by spectroscopic methods, especially high field ¹H and ¹³C NMR and NOE difference spectroscopy. The configuration of the seneremophilondiol esters has been corrected.     

Evaporative light scattering detection of pyrrolizidine alkaloids

A reverse-phase high-performance liquid chromatography method utilizing evaporative light scattering detection (ELSD) has been developed for the simultaneous detection of hepatotoxic pyrrolizidine alkaloids with and without chromophores, namely, riddelliine, riddelliine N-oxide, senecionine, senecionine N-oxide, seneciphylline, retrorsine, integerrimine, lasiocarpine and heliotrine. Pyrrolizidine alkaloids were detected in five plant extracts (Senecio spartioides, S. douglasii var. longilobus, S. jacobaea, S. intergerrimus var. exaltatus and Symphytum officinale). The detection of heliotrine (which does not contain a chromophore) was much improved by ELSD compared with photodiode array detection.     

Alkaloid N-oxides as transport and vacuolar storage compounds of pyrrolizidine alkaloids in Senecio vulgaris L

Cell-suspension cultures of pyrrolizidinealkaloid-producing species selectively take up and accumulate senecionine (sen) and its N-oxide (sen-Nox). Cultures established from non-alkaloid-producing species are unable to accumulate the alkaloids. The uptake and accumulation of ¹⁴C-labelled alkaloids was studied using a Senecio vulgaris cell-suspension culture as well as protoplasts and vacuoles derived from it. The alkaloid uptake exhibits all characteristics of a carrier-mediated transport. The uptake of sen-Nox follows a multiphasic saturation kinetics. The K_m-values for sen Nox of 53 M and 310 M are evaluated. Senecionine competitively inhibits sen-Nox uptake, indicating that the tertiary alkaloid and its N-oxide share the same membrane carrier. The N-oxide of sen shows a pH optimum below 5.5, whereas sen is taken up over a range from pH 4 to 8. Activation energies of 90 and 53 kJ·mol^-1 are calculated for sen-Nox and sen transport, respectively. At concentrations of 10 to 100 M, sen-Nox is rapidly taken up by cells and protoplasts; within 2 h >90% of total N-oxide is within the cells. By contrast the uptake of sen is less efficient. Vacuoles isolated from protoplasts preloaded with sen-Nox totally retained the alkaloid N-oxide, whereas sen is rapidly lost during the procedure of vacuole preparation. N-oxidation converts the weak lipophilic tertiary base into a charged polar molecule which is excellently adapted to serve as the cellular transport and storage form of pyrrolizidine alkaloids.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP 2,4-dinitrophenol - sen senecionine - sen-Nox senecionine N-oxide     

Seasonal variability in pyrrolizidine alkaloids in Senecio inaequidens from the Val Venosta (Northern Italy)

Senecio inaequidens is a neophyte originating from South Africa that has managed to spread to Europe and colonize large areas. This plant has toxic pyrrolizidine alkaloids (PAs) that represent a health risk to humans and animals. The aim of this work was to measure the content of PAs of these plants in continuous intervals over the entire growing season. The plants were separated into their organs, such as sprouts, stems, leaves and inflorescences. PAs were extracted from the dried plants using an acidic methanol solution, purified and measured by GC/MS. The average PA content of plants over the growing season was 0.33% of dry weight. The highest PA levels were about 1% in the dry weight and were found in the young sprouts and flower heads. There were nine PAs present, of which six could be identified. The main alkaloid was retrorsine followed by senecivernine, senecionine, integerrimine, usaramine and seneciphylline. Due to the high PA content of the inflorescences, the long flowering period and the rapid invasion dynamics, this species presents a high health risk for humans and animals.     

Sandaracopimarene derivatives from Senecio subrubriflorus

Several new sandaracopimarene derivatives were isolated from Senecio subrubriflorus. A triphenyl acetate of shikimic acid and a 4, 7-oxide of bisabolene were also present. The structures were elucidated by high field ¹H NMR spectroscopy. The chemistry of this species differs considerably from that of other Senecio species.     

Novel N-oxide derivatives of benzyltetrahydroisoquinoline alkaloid from the tubers of Stephania viridiflavens H.S. Lo et M. Yang

An investigation on the phytochemistry of the medicinal plant Stephania viridiflavens H.S. Lo et M. Yang led to isolate two new naturally occurring benzyltetrahydroisoquinoline alkaloids, (+)-1S, 2R-laudanidine-N_β-oxide 2 and (+)-1S, 2S-laudanidine-N_α-oxide 3, along with four known benzyltetrahydroisoquinoline alkaloids: (+)-laudanidine 1, (+)-reticuline 4, (+)-1S, 2R-reticuline-N_β-oxide 5 and (+)-1S, 2S-reticuline-N_α-oxide 6. The structure and the stereochemistry of these compounds were determined on the basis of spectroscopic methods and also confirmed by partial synthesis. To examine putative acetycholinesterase (AChE) inhibitory or cytotoxic activities, various bioassays were performed, the N-oxide derivatives (5 and 6) demonstrated more potent cytotoxicity than the corresponding free base.