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Four undescribed pyrrolizidine alkaloids (seneciojanine A-D), two enantiomeric pairs of unusual alkaloids (millingtojanine A-B) with a unique tricyclic core, and nine known pyrrolizidine alkaloids were isolated from whole plant extracts of Jacobaea vulgaris Gaertn. The structures of the undescribed compounds were established by extensive spectroscopic and spectrometric analyses and comparison of theoretical and experimental ECD data. Several of the structures were also confirmed by X-ray diffraction analysis. Seneciojanine A-D possess a rare natural necine moiety with an α,β-unsaturated carbonyl group located at C-3 and a hydroxyl substituent at C-8.

Apocynaceae are well-known for diverse specialized metabolites that are distributed in a phylogenetically informative manner. Pyrrolizidine alkaloids (PAs) have been reported sporadically in one lineage in the family, the APSA clade, but few species had been studied to date. We conduct the first systematic survey of Apocynaceae for retronecine-type PAs, sampling leaves from 231 species from 13 of 16 major lineages within the APSA clade using HPLC-MS/MS. We also follow up on preliminary evidence for infra-specific variation of PA detectability in Echites umbellatus Jacq. Four precursor ion scans (PREC) were developed for a high-throughput survey for chemicals containing a structural moiety common to many PAs, the retronecine core. We identified with high confidence PAs in 7 of 8 sampled genera of tribe Echiteae, but not in samples from the closely related Odontadenieae and Mesechiteae, confirming the utility of PAs as a taxonomic character in tribal delimitation. The presence of PAs in Malouetieae was confirmed, as we report with high confidence their presence in Galactophora schomburgkiana Woodson and Eucorymbia alba Stapf, but currently we have low confidence of their presence in Holarrena pubescens Wall. ex G. Don (the one Malouetieae species where they were previously reported), as well as in Kibatalia macrophylla (Pierre ex Hua) Woodson and in Holarrena curtisii King & Gamble. For the first time the presence of PAs in species of Wrightia R. Br. (Wrightieae) and Marsdenia R. Br. (Marsdenieae) was confirmed. Detectability of PAs was found to vary among samples of Echites umbellatus and intra-individual plasticity contributes to this variation. Of toxicological importance, novel potential sources of human exposure to pro-toxic PAs were identified in the medicinal plants, Wrightia tinctoria R.Br. and Marsdenia tinctoria R.Br., and the food plant, Echites panduratus A. DC., warranting immediate further research to elucidate the structures of the candidate PAs identified. Method development and limitations are discussed.

Tansy ragwort, Jacobaea vulgaris Gaertn. (syn. Senecio jacobaea L.), is a common Asteraceae in Europe and Asia and known to be an invasive pest in several regions in the world. Recently it is also spreading immensely in native regions like Northern Germany. Pyrrolizidine alkaloids (PAs), which are found in high amounts in Jacobaea vulgaris, are toxic for humans and potentially lethal for grazing animals. In this study we investigated 27 populations of tansy ragwort in Northern Germany for their PA concentration and composition using liquid chromatography coupled to high resolution mass spectrometry. Furthermore, we investigated the genetic structure of selected populations using amplified length polymorphism markers. We detected 98 different PAs in the samples and considerable differences of PA composition between populations. In contrast, PA content of populations did not differ significantly. Genetic (4%) differentiation among populations was low while average genetic diversity was high (0.35). There was no correlation between genetic and geographic distance. Neither genetic markers nor chemical composition revealed any connection to the geographic pattern. As we could not detect any pattern in genetic or chemical diversity, we suggest that the existence of this diversity is a result of a broad interaction with the environment rather than that of evolutionary constraints in the current selection process driving PA composition in J. vulgaris in certain chemotypes.

Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis.

A chemical study of Bethencourtia hermosae, aerial parts and in vitro root cultures, transformed by Agrobacterium rhizogenes, afforded the hitherto unreported sesquiterpenes ceratopicanol angelate (1), 8β-hydroxy-african-4(5)-en-3-one tiglate (4), 8β-hydroxy-african-4(5)-en-3-one 3′-angeloxy-2′-methylbutanoate (5), 1α,8β-dihydroxy-african-4(5)-en-3-one 8β-angelate (7) and 6α,8β-dihydroxy-african-4(5)-en-3-one 8β-angelate (8). In addition, 8β-hydroxy-african-4(5)-en-3-one (6) was isolated for the first time from a natural source, along with the rare sesquiterpenoid senecrassidiol (10) and two jacaranone derivatives 14 and 16. Known pyrrolizidine alkaloids, together with previously unreported hermosine (23), have also been isolated from this plant. The insect antifeedant activities of the extracts and compounds were studied together with their cytotoxic effects against insect (Sf9) and mammalian (CHO) cell lines., This work has been supported by Grant CTQ2012-38219-C03-01, MINECO, Spain. We gratefully acknowledge A. Fernández, Director of the Garajonay National Park (Gomera Island) for his advice on plant collection and to N. Martín-Sánchez for technical assistance. L.J.A. is grateful to the Spanish Research Council (CSIC) and the European Social Foundation for an I3P fellowship. We thank S. Carlin and P. Agnew for language advice.

Three flavonoids, cropalliflavones A–C, including two homoisoflavonoids with rare skeletons; three previously undescribed alkaloids, usaramine-N-oxide and cropallins A-B; and sixteen known compounds, were isolated from the seeds of Crotalaria pallida Ait. The absolute configurations of cropalliflavone A and usaramine-N-oxide were established by an ECD calculation and X-ray crystallography, respectively. Additionally, cropalliflavone B showed anti-proliferative activity against the MCF-7 cell line with an IC 50 value of 6.77 μ M, and cropalliflavone C showed anti-inflammatory activity, with an IC 50 value of 16.07 μ M.

Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants.

Secondary metabolites such as pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. PAs can occur in plants in two forms: tertiary amine (free base) and N-oxide. PA extraction and detection are of great importance for the understanding of the role of PAs as plant defense compounds, as the tertiary PA form is known for its stronger influence on several generalist insects, whereas the N-oxide form is claimed to be less deterrent. We measured PA N-oxides and their reduced tertiary amines by liquid chromatography–tandem mass spectrometry (LC-MS/MS). We show that the occurrence of tertiary PAs is not an artifact of the extraction and detection method. We found up to 50% of tertiary PAs in shoots of Jacobine – chemotype plants of Jacobaea vulgaris. Jacobine and its derivatives (jacoline, jaconine, jacozine and dehydrojaconine) may occur for more than 20% in reduced form in the shoots and more than 10% in the roots. For 22 PAs detected in F2 hybrids (J. vulgaris × Jacobaea aquatica), we calculate the tertiary amine percentage (TA% = the tertiary amine concentration/(tertiary amine concentration + the corresponding N-oxide concentration) × 100). We found that the TA% for various PAs was genotype-dependent. Furthermore, TA% for the different PAs were correlated and the highest correlations occurred between PAs which share high structural similarity.

The system of pyrrolizidine alkaloids has proven to be a powerful system for studying the evolution of a biosynthetic pathway in plant secondary metabolism. Pyrrolizidine alkaloids are typical plant secondary products produced by the plant as a defense against herbivores. The first specific enzyme, homospermidine synthase, has been shown to have evolved by duplication of the gene encoding deoxyhypusine synthase, which is involved in primary metabolism. Despite the identical function of homospermidine synthase for pyrrolizidine alkaloid biosynthesis in the various plant lineages, this gene duplication has occurred several times independently during angiosperm evolution. After duplication, these gene copies diverged with respect to gene function and regulation. In the diverse plant lineages producing pyrrolizidine alkaloids, homospermidine synthase has been shown to be expressed in a variety of tissues, suggesting that the regulatory elements were recruited individually after the duplication of the structural gene. The molecular, kinetic, and expression data of this system are discussed with respect to current models of gene and pathway evolution.

The absolute configurations (AC) of natural occurring 6-hydroxyeuryopsin (1), of its acetyl derivative 2, and of eremophilanolide 8 were confirmed by comparison of the experimental vibrational circular dichroism (VCD) spectra with theoretical curves generated from density functional theory (DFT) calculations. Initial analyses were carried out using a Monte Carlo searching with the MMFF94 molecular mechanics force field. All MMFF94 conformers were further optimized using DFT at the B3LYP/6-31G(d) level of theory, followed by calculations of their vibrational frequencies at the B3LYP/6-31G(d,p); the VCD spectra of 2 and 8 were also calculated at the B3PW91/DGDZVP level of theory. Good agreement between theoretical and experimental VCD curves unambiguously verified the 4S,5R,6S absolute configuration for 1 and 2, and the 1S,4S,5R,6S,8S,10S configuration for 8.

Based on direct infusion mass spectrometry we identified a novel alkaloid as a major component of perennial ryegrass (Lolium perenne). Initial mass spectral data suggested it to be a pyrrolizidine conjugate. As this class of alkaloids has not been described before from grasses, we isolated it to elucidate its structure. The isolated alkaloid proved to be a mixture of two stereoisomers. The structures of the two compounds as determined by 1D and 2D NMR spectroscopy, were E-thesinine-O-4′-α-rhamnoside (1) and Z-thesinine-O-4′-α-rhamnoside (2). These identifications were supported by the characterisation by GC-MS and optical rotation of (+)-isoretronecanol as the necine base released on alkaline hydrolysis of these alkaloids. 1 and 2 together with the aglycone and a hexoside were also detected in tall fescue (Festuca arundinacea). This is the first report of pyrrolizidine alkaloids produced by grasses (Poaceae).

Three species of the Boraginaceae were studied: greenhouse-grown plants of Heliotropium indicum and Agrobacterium rhizogenes transformed roots cultures (hairy roots) of Cynoglossum officinale and Symphytum officinale. The species-specific pyrrolizidine alkaloid (PA) profiles of the three systems were established by GC-MS. All PAs are genuinely present as N-oxides. In H. indicum the tissue-specific PA distribution revealed the presence of PAs in all tissues with the highest levels in the inflorescences which in a flowering plant may account for more than 70% of total plant alkaloid. The sites of PA biosynthesis vary among species. In H. indicum PAs are synthesized in the shoot but not roots whereas they are only made in shoots for C. officinale and in roots of S. officinale. Classical tracer studies with radioactively labelled precursor amines (e.g., putrescine, spermidine and homospermidine) and various necine bases (trachelanthamidine, supinidine, retronecine, heliotridine) and potential ester alkaloid intermediates (e.g., trachelanthamine, supinine) were performed to evaluate the biosynthetic sequences. It was relevant to perform these comparative studies since the key enzyme of the core pathway, homospermidine synthase, evolved independently in the Boraginaceae and, for instance, in the Asteraceae [Reimann, A., Nurhayati, N., Backenkohler, A., Ober, D., 2004. Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant Cell 16, 2772-2784.]. These studies showed that the core pathway for the formation of trachelanthamidine from putrescine and spermidine via homospermidine is common to the pathway in Senecio ssp. (Asteraceae). In both pathways homospermidine is further processed by a beta-hydroxyethylhydrazine sensitive diamine oxidase. Further steps of PA biosynthesis starting with trachelanthamidine as common precursor occur in two successive stages. Firstly, the necine bases are structurally modified and either before or after this modification are converted into their O(9)-esters by esterification with one of the stereoisomers of 2,3-dihydroxy-2-isopropylbutyric acid, the unique necic acid of PAs of the lycopsamine type. Secondly, the necine O(9)-esters may be further diversified by O(7)- and/or O(3')-acylation.

Phalaenopsis hybrids contain two 1,2-saturated pyrrolizidine monoesters, T-phalaenopsine (necine base trachelanthamidine) and its stereoisomer Is-phalaenopsine (necine base isoretronecanol). T-Phalaenopsine is the major alkaloid accounting for more than 90% of total alkaloid. About equal amounts of alkaloid were genuinely present as free base and its N-oxide. The structures were confirmed by GC-MS. The quantitative distribution of phalaenopsine in various organs and tissues of vegetative rosette plants and flowering plants revealed alkaloid in all tissues. The highest concentrations were found in young and developing tissues (e.g., root tips and young leaves), peripheral tissues (e.g., of flower stalks) and reproductive organs (flower buds and flowers). Within flowers, parts that usually attract insect visitors (e.g., labellum with colorful crests as well as column and pollinia) show the highest alkaloid levels. Tracer feeding experiments with (14)C-labeled putrecine revealed that in rosette plants the aerial roots were the sites of phalaenopsine biosynthesis. However active biosynthesis was only observed in roots still attached to the plant but not in excised roots. There is a slow but substantial translocation of newly synthesized alkaloid from the roots to other plant organs. A long-term tracer experiment revealed that phalaenopsine shows neither turnover nor degradation. The results are discussed in the context of a polyphyletic molecular origin of the biosynthetic pathways of pyrrolizidine alkaloids in various scattered angiosperm taxa. The ecological role of the so called non-toxic 1,2-saturated pyrrolizidine alkaloids is discussed in comparison to the pro-toxic 1,2-unsaturated pyrrolizidine alkaloids. Evidence from the plant-insect interphase is presented indicating a substantial role of the 1,2-saturated alkaloids in plant and insect defense.

We studied the variation in pyrrolizidine alkaloid (PA) patterns of lab-grown vegetative plants of 11 European Senecio jacobaea populations. Plants were classified as jacobine, erucifoline, mixed or senecionine chemotypes based on presence and absence of the PAs jacobine or erucifoline. Due to the presence of jacobine, total PA concentration in jacobine chemotypes was higher than in erucifoline chemotypes. Both relative and absolute concentrations of individual PAs differed between half-sib and clonal families, which showed that variation in PA patterns had a genetic basis. Within most populations relative abundance of PAs varied considerably between individual plants. Most populations consisted either of the jacobine chemotype or of the erucifoline chemotype, sometimes in combination with mixed or senecionine chemotypes.

This is the first study showing that alkaloids are present on the leaf surface of plants. A concentration of 30–230 pmol/cm 2 pyrrolizidine alkaloids (PA's) was detected in 8 different samples taken from Senecio jacobaea . PA concentration on the leaves was marginally correlated with PA concentration of the total leaf tissues. The PA spectrum on the leaf differed from the PA spectrum of the total leaf.

Six pyrrolizidine alkaloids were isolated from the whole herb of Liparis nervosa together with two previously known ones. Their structures were elucidated by extensive spectroscopic analyses and chemical reactions. The cytotoxicity of the isolates was evaluated against A549, HepG2, and MCF-7 human cancer cell lines; however, no significant growth inhibition was observed. All compounds were evaluated for the inhibition of LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, and most significantly inhibited NO production with IC50 values in the range of 2.16-38.25 μM.

Plants are attacked by many different herbivores. Some will consume whole leaves or roots, while others will attack specific types of tissue. Thus, insight into the metabolite profiles of different types of leaf tissues is necessary to understand plant resistance against herbivores. Jacobaea vulgaris, J. aquatica and three genotypes of their crossings were used to study the variation in metabolomic profiles between epidermis and mesophyll tissues. Extracts of epidermis and mesophyll tissues were obtained using carborundum abrasion (CA). Subsequently, (1)H nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses were applied to compare the metabolome profiles. Orthogonal partial least-squares-discriminant analysis (OPLS-DA) resulted in a clear separation of epidermis and mesophyll extracts. The epidermis contained significantly higher amounts of jacaranone and phenylpropanoids, specifically chlorogenic (5-O-CQA) and feruloyl quinic (FQA) acids compared to the mesophyll. In contrast, the mesophyll showed significantly higher concentrations of pyrrolizidine alkaloids (PAs), specifically jacobine and jaconine. The tissue specific distribution of these compounds was constant over all genotypes tested. Phenylpropanoids, 5-O-CQA and FQA, as well as PAs are known for their inhibitory effect on herbivores, especially against thrips. Thrips feeding commences with the penetration of the epidermis, followed by ingestion of sub-epidermal or mesophyll. Thrips thus may have to encounter phenylpropanoids in the epidermis as the first line of defence, before encountering the PAs as the ultimate defence in the mesophyll. The finding of tissue specific defense may have a major impact on studies of plant resistance. We cannot judge resistance using analyses of a whole roots, leafs or flowers. In such a whole-organism approach, the levels of potential defense compounds are far below the real ones encountered in tissues involved in the first line of defense. Instead, it is of great importance to study the defence compounds in the specific tissue to which the herbivore is confined.

Four pyrrolizidine alkaloids (PA) were isolated from Ageratum houstonianum and their structures elucidated by spectroscopical methods. Besides the already known lycopsamine three new PA were found. Their structures are the 2S-2-hydroxy-2,3-dimethylbutanoyl-O 9 as well as the O 7 esters of retronecine and the O 9 derivative of heliotridine. # 2001 Elsevier Science Ltd. All rights

In order to study the evolution of pathways of plant secondary metabolism, we use the biosynthesis of pyrrolizidine alkaloids (PAs) as a model system. PAs are regarded as part of the plant's constitutive defense against herbivores. Homospermidine synthase (HSS) is the first specific enzyme of PA biosynthesis. The gene encoding HSS has been recruited from the gene encoding deoxyhypusine synthase (DHS) from primary metabolism at least four times independently during angiosperm evolution. One of these recruitment occurred within the monocot lineage. We have used the PA-producing orchid Phalaenopsis to identify the cDNAs encoding HSS, DHS and the substrate protein for DHS, i.e., the precursor of the eukaryotic initiation factor 5A. A cDNA identified from maize was unequivocally characterized as DHS. From our study of Phalaenopsis, several pseudogenes emerged, of which one was shown to be a "processed pseudogene", and others to be transcribed. Sequence comparison of the HSS- and DHS-encoding sequences from this investigation with those of monocot species taken from the databases suggest that HSS and probably the ability to produce PAs is an old feature within the monocot lineage. This result is discussed with respect to the recent discovery of structural related PAs within grasses.

The methanolic extract of the whole plant of Echium glomeratum Poir. (Boraginaceae) has afforded five pyrrolizidine alkaloids, three that were (7S, 8R)-petranine (1), (7S, 8S)-petranine (2), and (7R, 8R)-petranine (3a) or (7R, 8S)-petranine (3b), comprising a tricyclic pyrrolizidine alkaloids subclass; and two that were known but to the species: 7-angeloylretronecine (4) and 9-angeloylretronecine (5). All compounds were tested against a human tumor panel for cytotoxicity; no activity was observed (EC50 values > 20 µg/ml).

The endemic Mexican genus Pittocaulon (subtribe Tussilagininae, tribe Senecioneae, Asteraceae) belongs to a monophyletic group of genera distributed in Mexico and North America. The five Pittocaulon species represent shrubs with broom-like succulent branches. All species were found to contain pyrrolizidine alkaloids (PAs). With one exception (i.e., stems of Pittocaulon velatum are devoid of PAs) PAs were found in all plant organs with the highest levels (up to 0.3% of dry weight) in the flower heads. Three structural types of PAs were found: (1) macrocyclic otonecine esters, e.g. senkirkine and acetylpetasitenine; (2) macrocyclic retronecine esters, e.g. senecionine, only found in roots, and (3) monoesters of 1,2-saturated necines with angelic acid. For an unambiguous assignment of the different stereoisomeric 1,2-saturated necine bases a GC-MS method was established that allows the separation and identification of the four stereoisomers as their diacetyl or trimethylsilyl derivatives. All otonecine esters that generally do not form N-oxides and the 1,2-saturated PAs were exclusively found as free bases, while the 1,2-unsaturated 7-angeloylheliotridine occurring in P. velatum was found only as its N-oxide. In a comparative study the 1H and 13C NMR spectra of the four stereoisomeric necine bases were completely assigned by the use of DEPT-135, H,H-COSY, H,C-HSQC and H,H-NOESY experiments and by iterative analysis of the 1H NMR spectra. Based on these methods the PA monoesters occurring in Pittocaulon praecox and P. velatum were assigned as 7-O-angeloyl ester respectively 9-O-angeloyl ester of dihydroxyheliotridane which could be identified for the first time as naturally occurring necine base. Unexpectedly, in the monoesters isolated from the three other Pittocaulon species dihydroxyheliotridane is replaced by the necine base turneforcidine with opposite configuration at C-1 and C-7. The species-specific and organ-typical PA profiles of the five Pittocaulon species are discussed in a biogenetic context.

Several species of Lolium and other cool-season grasses (Poaceae subfamily Pooideae) tend to harbor symbiotic, seed-transmitted, fungi that enhance their fitness by various means. These fungal endophytes--species of Neotyphodium or Epichloe (Clavicipitaceae)--are known for production of antiherbivore metabolites such as the bioprotective loline alkaloids. Lolines are saturated pyrrolizidines with an exo-1-amine and an ether bridge between C-2 and C-7. The ether bridge is an unusual feature for a biogenic compound in that it links two bridgehead carbon atoms. Much of the loline-biosynthetic pathway has been elucidated by administering isotopically labeled precursors to fungal cultures and by comparisons of loline biosynthesis genes to known gene families. The first step appears to be an unusual gamma-substitution reaction involving an enzyme related to O-acetylhomoserine (thiol) lyase, but which uses the secondary amine of L-proline rather than a sulfhydryl group as the nucleophile. The strained ether bridge is added after formation of the pyrrolizidine rings. Lolines with dimethylated or acylated 1-amines have insect antifeedant and insecticidal activities comparable to nicotine, but little or no toxicity to mammals. Considering the surprising abundance of lolines in some grass-endophyte symbiota, possible additional effects on plant stress tolerance and physiology are worth future consideration. In this review, we discuss the history of loline discovery, methods of analysis, biological activities and distribution in nature, as well as progress on the genetics and biochemistry of their biosynthesis, and on the chemical synthesis of these alkaloids.

The pyrrolizidine alkaloid (PA) content of flowers, leaves, and roots of Anchusa strigosa (Boraginaceae) was analysed by ESI-LC-MS. Six PAs, including two new natural compounds, were detected, characterized by NMR spectroscopy, and quantified in each plant organ. The results indicated that the highest total concentration of PAs was in the leaves (23.63 mg/g of dried part), followed by the flowers (19.77 mg/g), and finally by the roots (1.80 mg/g). All PAs isolated were subjected to Spodoptera exigua and Pieris brassicae larvae. Feeding activity by both herbivore species using a bioassay was inhibited up to circa 75% depending on PA and applied concentration.

Pyrrolizidine alkaloids (PAs) of the macrocyclic senecionine type are secondary metabolites characteristic for most species of the genus Senecio (Asteraceae). These PAs are deterrent and toxic to most vertebrates and insects and provide plants with a chemical defense against herbivores. We studied the PA composition of 24 out of 26 species of Senecio section Jacobaea using GC-MS. The PA profiles of eight of these species have not been studied before and additional PAs were identified for most other species that were included in previous studies. With one exception (senecivernine) all 26 PAs identified in sect. Jacobaea can be regarded as derivatives of the biosynthetic backbone structure senecionine. Based on the PA profiles of the species of sect. Jacobaea and the results of previous tracer studies, we constructed two hypothetical biosynthetic scenarios of senecionine diversification. Both scenarios contain two major reactions: the conversion of the necine base moiety retronecine into the otonecine moiety and site-specific epoxidations within the necic acid moiety. Further reactions are site-specific hydroxylations, sometimes followed by O-acetylations, site-specific dehydrogenations, E, Z-isomerizations, and epoxide hydrolysis and chlorolysis. The GC-MS data and both biosynthetic scenarios were subsequently used to study the evolution of PA formation in sect. Jacobaea by reconstructing the evolutionary history of qualitative PA variation in this section. This was achieved by optimizing additive presence/absence data of PAs and types of enzymatic conversions on a maximum parsimony cladogram of section Jacobaea inferred from DNA sequence and morphological data. Besides showing large intra- and interspecific variation, PA distribution appears to be largely incidental within the whole clade. These results together with the finding that all but one of the PAs identified in sect. Jacobaea are also present in species of other sections of Senecio indicate that differences in PA profiles in Senecio can not be explained by the gain and loss of PA specific genes, but rather by a transient switch-off and switch-on of the expression of genes encoding PA pathway-specific enzymes.

A comprehensive GC–MS analysis of 8 Ipomoea species belonging to the subgenus Quamoclit, section Mina revealed that the members of this taxon form combinations of two necine bases with rare necic acids resulting in unique pyrrolizidine alkaloids. The occurrence and diversity of these metabolites show remarkable variations: Some species, especially Ipomoea hederifolia and Ipomoea lobata , are able to synthesize a large number of alkaloids whereas others, especially Ipomoea coccinea and Ipomoea quamoclit , are poor synthesizers with only a few compounds. However, these metabolites are apparently chemotaxonomic markers of this infrageneric taxon in general. They represent either esters of (−)-platynecine (altogether 48 ipangulines and 4 further esters including results of a previous study) or esters of (−)-trachelanthamidine, an additional novel structural type called minalobines (altogether 21 alkaloids). Both types are characterized by section-specific rare necic acids, e.g., ipangulinic/isoipangulinic acid, phenylacetic acid. The alkaloids of Ipomoea cholulensis , I. coccinea , I. hederifolia , Ipomoea neei , and Ipomoea quamoclit were mono and diesters of platynecine. Minalobines turned out to be metabolites of I. lobata (Cerv.) Thell. (syn.: Mina lobata Cerv.) lacking ipangulines. The major alkaloid of this species, minalobine R, has been isolated and identified as 9- O -( threo -2-hydroxy-2-methyl-3-phenylacetoxy-butyryl)-(−)-trachelanthamidine on the basis of spectral data. Apparently only two of the species included in this study, Ipomoea cristulata and Ipomoea sloteri , are able to synthesize both, ipangulines as well as minalobines. Minalobine O could be isolated as a major alkaloid of I. cristulata , its structure has been established as 9- O -( erythro -2-hydroxy-2-methyl-3-tigloyloxy-butyryl)-(−)-trachelanthamidine on the basis of spectral data.

Deoxyhypusine synthase (DHS) is involved in the post-translational activation of the eukaryotic initiation factor 5A (eIF5A) and, as a side-reaction, catalyzes the formation of homospermidine if its substrate, the eIF5A precursor protein, is replaced by putrescine. Plant homospermidine synthase is assumed to be phylogenetically derived from DHS; it represents a DHS having lost its intrinsic activity. The enzyme is expressed in plants producing pyrrolizidine alkaloids where it catalyzes the formation of homospermidine the unique precursor of pyrrolizidine alkaloids. Here we show that 29 species randomly selected from 18 angiosperm families as well as a few other terrestrial plant species, all were able to produce small amounts of homospermidine. Basing on these results and in the context of literature on the occurrence of homospermidine in the organismic kingdoms, a universal occurrence of homospermidine is assumed and ubiquitous DHS is suggested to be responsible for its formation. The synthesis of homospermidine as an enzymatic by-product of an essential enzyme is discussed in respect to the evolutionary origin of homospermidine synthase and the biosynthetic pathway of pyrrolizidine alkaloids.

The glycosylated pyrrolizidine alkaloid, thesinine-4'-O-beta-D-glucoside, has been isolated from the aqueous methanol extract of dried, defatted seeds of Borago officinalis (Boraginaceae). The structure was established by means of spectroscopic and chemical analysis.