Pyrrolizidine alkaloid profiles in Crotalaria species from Brazil: Chemotaxonomic significance

Gas chromatography–mass spectrometry analysis of seeds from 28 species of Crotalaria from Brazil (sections Calycinae, Crotalaria, Chrysocalycinae and Hedriocarpae) showed that pyrrolizidine alkaloids (PAs) are important as chemotaxonomic markers at the infrageneric level. The sections Calycinae and Crotalaria were characterized by 11-membered macrocyclic monocrotaline-type PAs. In the section Chrysocalycinae, a single species in the subsection Incanae, C. incana, showed integerrimine, a 12-membered macrocyclic senecionine-type, as main PA. The group close to the subsection Stipulosae (C. micans and C. maypurensis) showed distinctive PA patterns: C. micans presented mainly the 12-membered macrocyclic integerrimine, and C. maypurensis the unusual 7-hydroxy-1-methylene-8-pyrrolizidine. In the group close to the subsection Glaucae, the PAs with otonecine as the necine base were the main alkaloids, except in C. rufipila which showed an assamicadine-like PA (monocrotaline-type). The section Hedriocarpae showed main 12-membered macrocyclic senecionine-type PAs.     

Frequent gain and loss of pyrrolizidine alkaloids in the evolution of Senecio section Jacobaea (Asteraceae)

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.     

Acquisition, transformation and maintenance of plant pyrrolizidine alkaloids by the polyphagous arctiid Grammia geneura

The polyphagous arctiid Grammia geneura appears well adapted to utilize for its protection plant pyrrolizidine alkaloids of almost all known structural types. Plant-acquired alkaloids that are maintained through all life-stages include various classes of macrocyclic diesters (typically occurring in the Asteraceae tribe Senecioneae and Fabaceae), macrocyclic triesters (Apocynaceae) and open-chain esters of the lycopsamine type (Asteraceae tribe Eupatorieae, Boraginaceae and Apocynaceae). As in other arctiids, all sequestered and processed pyrrolizidine alkaloids are maintained as non-toxic N-oxides. The only type of pyrrolizidine alkaloids that is neither sequestered nor metabolized are the pro-toxic otonecine-derivatives, e.g. the senecionine analog senkirkine that cannot be detoxified by N-oxidation. In its sequestration behavior, G. geneura resembles the previously studied highly polyphagous Estigmene acrea. Both arctiids are adapted to exploit pyrrolizidine alkaloid-containing plants as "drug sources". However, unlike E. acrea, G. geneura is not known to synthesize the pyrrolizidine-derived male courtship pheromone, hydroxydanaidal, and differs distinctly in its metabolic processing of the plant-acquired alkaloids. Necine bases obtained from plant acquired pyrrolizidine alkaloids are re-esterified yielding two distinct classes of insect-specific ester alkaloids, the creatonotines, also present in E. acrea, and the callimorphines, missing in E. acrea. The creatonotines are preferentially found in pupae; in adults they are largely replaced by the callimorphines. Before eclosion the creatonotines are apparently converted into the callimorphines by trans-esterification. Open-chain ester alkaloids such as the platynecine ester sarracine and the orchid alkaloid phalaenopsine, that do not possess the unique necic acid moiety of the lycopsamine type, are sequestered by larvae but they need to be converted into the respective creatonotines and callimorphines by trans-esterification in order to be transferred to the adult stage. In the case of the orchid alkaloids, evidence is presented that during this processing the necine base (trachelanthamidine) is converted into its 7-(R)-hydroxy derivative (turneforcidine), indicating the ability of G. geneura to introduce a hydroxyl group at C-7 of a necine base. The creatonotines and callimorphines display a striking similarity to plant necine monoesters of the lycopsamine type to which G. geneura is well adapted. The possible function of insect-specific trans-esterification in the acquisition of necine bases derived from plant acquired alkaloids, especially from those that cannot be maintained through all life-stages, is discussed.     

Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species

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.     

Absolute configuration of the creatonotines and callimorphines, two classes of arctiid-specific pyrrolizidine alkaloids

Arctiids which as larvae sequester pyrrolizidine alkaloids (PAs) from their food plants are known to synthesize insect-specific PAs by esterifying necine bases derived from plant PAs with necic acids of insect origin. There are two classes of insect PAs, the creatonotines and the callimorphines. The creatonotines contain as necic acids either 2-hydroxy-3-methylbutyric acid (creatonotine A) or 2-hydroxy-3-methylpentanoic acid (creatonotine B). The three known callimorphines contain 2-hydroxy-2-methylbutanoic acid whose hydroxyl group can be either free (deacetylcallimorphine) or acetylated (callimorphine) or propionylated (homocallimorphine). Insect PAs are assumed to play an important role in the recycling of plant derived necine bases and the processing by trans-esterification of PA monoesters that cannot be directly transmitted to the insect's pupal and adult life-stages. The absolute configuration of the insect-specific necic acids was elucidated in the context of the suggested role of the insect PAs as insect-made mimics of plant monoester PAs of the lycopsamine type. For this purpose all needed stereoisomers were synthesized and a gas chromatography-mass spectrometry (GC-MS) method was established that allows the enantioselective separation and assignment of the stereochemistry of all insect specific necic acids as their methyl esters. The method could also be applied to the GC-MS analysis of the intact alkaloids which were hydrolyzed during injection and converted into their methyl esters. Analysis of the creatonotines and callimorphines isolated from the polyphagous arctiids Estigmene acrea and Grammia geneura that were fed with pure PAs and defined PA mixtures revealed the following absolute configuration: the callimorphines and creatonotine A were present in 2'R configuration, whereas creatonotine B was found as mixture of (2'R, 3'S)- and (2'S, 3'S)-stereoisomers. The ratio of 2'S to 2'R was extremely variable ranging from 98% S to 94% R. The cause of the lack of stereospecificity is discussed particularly in respect of a possible epimerization of the hydroxyl group at C-2' in analogy to the known epimerization at C-3' of plant acquired PAs of the lycopsamine type.     

Tissue distribution and biosynthesis of 1,2-saturated pyrrolizidine alkaloids in Phalaenopsis hybrids (Orchidaceae)

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.     

Biochemical processing of plant acquired pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi

Leaf beetles of the genus Platyphora, feeding on plant species containing pyrrolizidine alkaloids of the lycopsamine type, not only sequester these alkaloids and concentrate them in their exocrine defensive secretions, but also specifically process the plant acquired alkaloids. Using P. boucardi as subject, three mechanisms were studied: (i). utilization of host plant alkaloids that are not sequestered per se; (ii). elucidation of the mechanism of the already documented C-7 epimerization of heliotridine O(9)-monoesters; (iii). the specificity of insect catalyzed necine base esterification. P. boucardi does not sequester the triester parsonsine, the principal alkaloid of its host plant Prestonia portobellensis (Apocynaceae). Beetles fed with a purified mixture of nor-derivatives of parsonsine, obtained from Parsonsia laevigata, did not sequester the triesters but transformed them by partial degradation into monoesters that are accumulated in the defensive secretions. The mechanism of the previously described transformation of rinderine into intermedine by C-7 epimerization was elucidated by feeding C-7 deuterated heliotrine (3'-methylrinderine). The transformation of heliotrine into epiheliotrine (3'-methylintermedine) catalyzed by P. boucardi is accompanied by complete loss of deuterium, indicating the same mechanism of an oxidation-reduction process via a ketone intermediate as recently demonstrated in a pyrrolizidine alkaloid sequestering lepidopteran. P. boucardi is able to form ester alkaloids from five different necine bases fed as radioactively labeled substrates. However, besides C-7 epimerization the beetles are not able to convert simple necine bases into retronecine. The functional importance of the various alkaloid transformations is discussed in comparison to striking parallels of analogous reactions known from pyrrolizidine alkaloid sequestering Lepidoptera.     

Active Metabolites in the Chronic Hepatotoxicity of Pyrrolizidine Alkaloids,including Otonecine Esters

WHEN she reported the chronic hepatotoxicity of the alkaloids senkirkine and hydroxysenkirkine, which are esters of the aminoalcohol otonecine, Schoental¹ suggested that 1,2-epoxides rather than pyrrolic derivatives^2,3 are the active metabolites of hepatotoxic pyrrolizidine alkaloids. We have results which confirm the hepatotoxicity of otonecine esters but argue against the suggested involvement of 1,2-epoxides.     

Novel secopyrrolizidine alkaloids from Crotalaria verrucosa

Crotaverrine and O-acetylcrotaverrine, isolated from the seeds of C. verrucosa Linn., have been shown by spectroscopy and chemical evidence to be the macrocyclic diesters of otonecine and diastereoisomeric integerrinecic acid. Hitherto, diastereoisomeric integerrinecic acid esters were not known to occur in nature.     

Estrogenic action of estriol fatty acid esters

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.     

Effect of fatty acid chain length on initial reaction rates and regioselectivity of lipase-catalysed esterification of disaccharides

In a reaction medium mixture of 9:11 t-BuOH and pyridine (v/v) the effect of fatty acid chain length (C-4-C-12) on C. antarctica lipase B (Novozym 435, EC 3.1.1.3) catalysed esterification was studied. alpha and beta maltose 6'-O-acyl esters in an anomeric molar ratio of 1.0:1.1 were synthesised independently of the chain length, but the initial specific reaction rate increased with decreasing chain length of the acyl donor. The product yield followed the same trend with a lauryl ester yield of 1.1% (mol/mol) and a butyl ester yield of 27.6% (mol/mol) after 24 h of reaction. With sucrose as the acyl acceptor the 6'-O-acyl and 6-O-acyl monoesters were formed with fatty acids of chain length C-4 and C-10 while the 6',6-O-acyl diester was formed only with butanoic acid (C-4:0) as acyl donor. The 6'-O-acyl and 6-O-acyl monoesters and the 6',6-O-acyl diester of butanoic acid were produced in a molar ratio of 1.0:0.5:0.2 and with decanoic acid (C-10:0) the 6'-O-acyl and 6-O-acyl monoesters were formed in the ratio of 1.0:0.3. The highest initial reaction rate and yield were obtained with the shortest chain length of the acyl donor. Initial reaction rates and ester yields were affected by the solubility of the disaccharide, with higher reaction rates and yields with maltose than with sucrose, while no formation of esters were observed with either cellobiose or lactose as acyl acceptors.     

Two-step enzymatic synthesis of maltooligosaccharide esters

Glucose and maltose esters were synthesised in organic media by employing a lipase (E.C. 3.1.1.3) from Candida antarctica. In a second reaction step, a transglycosylation catalysed by a cyclodextrin glycosyltransferase (E.C. 2.4.1.19) from either Paenibacillus sp. F8 or Bacillus sp. strain no. 169 (DSM 2518) extended the degree of polymerisation (DP) of the carbohydrate moieties of the carbohydrate esters. The donor substrates used were either a cyclodextrin, a maltooligosaccharide or starch. The highest rate of low DP maltooligosaccharide ester formation was obtained when starch was used as glycosyl donor and caproyl maltose as glycosyl acceptor. The structures of two of the products were identified by 1H and 13C NMR and MALDI-TOF MS as capronate monoesters of maltotriose and maltotetraose, with the ester bond at C-6 of the second glucose unit from the reducing end.     

Ipangulines and minalobines, chemotaxonomic markers of the infrageneric Ipomoea taxon subgenus Quamoclit, section Mina

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.     

Effects of pyrrolizidine alkaloids through different trophic levels

Pyrrolizidine alkaloids (PAs), mainly those with a 1,2-double bond in the necine base moiety (=1,2-dehydropyrrolizidines), constitute a class of well studied compounds with respect to their flux through different trophic levels. Plants belonging to various clades (e.g. Echiteae, Eupatorieae and Senecioneae, Boraginaceae, and Crotalarieae) biosynthesize PAs as N-oxides, generally in the roots, and transport them through the phloem to stems, leaves, and reproductive structures, where they act as potent deterrents against non-specialist herbivores. On the other hand, PA specialist herbivores (mainly arctiid moths, danaine and ithomiine butterflies, and some leaf beetles) have become able to overcome this chemical barrier, and to sequester these alkaloids from their larval host plants or from sources visited by adults, such as flowers and dead or withered plants. Specialists use PAs for their own benefit as chemical defence against a vast array of predators (e.g. ants, lacewings, spiders, lizards, birds, and mammals), but some predators are able to feed on PA-insects, by avoiding or physiologically overcoming PAs present in tissues of the ingested prey. Parasitoids may be affected by PAs, depending on their degree of specialization in relation to PA-insects. Arctiidae, Danainae and Ithomiinae also use PAs as precursors of sexual pheromones. The effects of PAs on trophic interactions have been intensely studied over the last four decades, but some open questions remain, and are discussed, such as the underlying mechanisms that lead to PA diversification, activity of different PA structures, synergism among PAs and other so-called defensive substances in PA-plants, and the ability to overcome this chemical barrier by predators and parasitoids.     

Specific recognition, detoxification and metabolism of pyrrolizidine alkaloids by the polyphagous arctiid Estigmene acrea

Evidence is presented that the polyphagous arctiid Estigmene acrea is well adapted to sequester and specifically handle pyrrolizidine alkaloids of almost all known structural types representative of the major plant families with pyrrolizidine alkaloid-containing species, i.e. Asteraceae with the tribes Senecioneae and Eupatorieae, Boraginaceae, Fabaceae, Apocynaceae and Orchidaceae. The adaptation of E. acrea to pyrrolizidine alkaloids includes a number of specialized characters: (i) highly sensitive recognition of alkaloid sources by pyrrolizidine alkaloid-specific taste receptors; (ii) detoxification of pyrrolizidine alkaloids by N-oxidation catalyzed by a specific flavin-dependent monooxygenase; (iii) transfer and maintenance of all types of pyrrolizidine N-oxides through all developmental stages; (iv) conversion of the various structures into the male courtship pheromone hydroxydanaidal most probably through retronecine and insect specific retronecine esters (creatonotines) as common intermediates; (v) specific integration into mating behavior and defense strategies. Toxic otonecine derivatives, e.g. the senecionine analogue senkirkine, which often accompany the common retronecine derivatives and which cannot be detoxified by N-oxidation do not affect the development of E. acrea larvae. Senkirkine is not sequestered at all. Non-toxic 1,2-saturated platynecine derivatives that frequently occur together with toxic retronecine esters are sequestered and metabolized to hydroxydanaidal, indicating the ability of E. acrea to aromatize saturated pyrrolizidines. Although pyrrolizidine alkaloids, even if they are offered continuously at a high level (2%) in the larval diet, are non-toxic, E. acrea larvae are not able to develop exclusively on a pyrrolizidine alkaloid-containing plant like Crotalaria. Therefore, E. acrea appears to be specifically adapted to exploit pyrrolizidine alkaloid-containing plants as "drug source" but not as a food source.     

A facile one-step synthesis of delta 1,4-3-keto bile acid esters by iodoxybenzene and benzeneselenic anhydride

A facile one-step conversion of stereoisomeric methyl 3-hydroxycholanoates to 1,4-dien-3-one, by treatment in boiling toluene with iodoxybenzene catalyzed by benzeneselenic anhydride, is described. The direct oxidation-dehydrogenation at C-3 is applicable to other cholanoates (hyodeoxycholic, chenodeoxycholic, ursodeoxycholic, deoxycholic, and cholic) when the hydroxyl groups at C-6, C-7, or C-12 are protected. Without protection at C-6 and C-7, the products from hyodeoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic esters are complex mixtures, whereas, methyl deoxycholate yields the 1,4-dien-3,12-dione cleanly.     

Necines of alkaloids in Heliotropium species from Mexico and the U.S.A.

The qualitative and quantitative compositions of necines in plants of 20 Heliotropium species collected in Mexico and the U.S.A. and one species from Spain are reported. Trachelanthamidine, supinidine and retronecine were found in all species after hydrolysis of their alkaloids; lindelofidine was detected in most species, whereas heliotridine only in four. Trachelanthamidine, lindelofidine, and supinidine were dominant in four, two and one species, respectively; retronecine was dominant in 15 species, whereas heliotridine only in one. The dominant necine in H. ternatum was either retronecine or lindelofidine depending on the collection locality. Qualitative as well as quantitative differences depending on the collection locality were found in H. curassavicum. Plants from Oaxaca, Mexico, contained lindelofidine and a pyrrolizidine-diol as major necines, trachelanthamidine as minor, and traces of retronecine. Plants originating from two other localities contained trachelanthamidine (dominant), retronecine, and supinidine. The necine patterns found in the examined species differ significantly from those previously reported for 21 species mainly collected in Asia, the Middle East and Australia.     

Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols.

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.     

A new pyrrolizidine alkaloid and other constituents from the roots of Ligularia achyrotricha (Diels) Ling

Ligularia achyrotricha (Diels) Ling has been used as a traditional Tibetan herbal medicine. Ligulachyroine A (1), a new twelve-membered macrocyclic pyrrolizidine alkaloid with a ketonic group located at C-3 and an hydroxy group linked at C-8, was isolated from the roots of L. achyrotricha. It is particularly noteworthy that the structure of 1 rarely appears in any other naturally occurring twelve-membered macrocyclic pyrrolizidine alkaloid reported to date. Three known lignans (2–4) and eight known coniferyl and sinapyl alcohol derivatives (5–12) were also obtained in this species. Their structures were elucidated through a careful analysis of their IR, MS, and NMR spectra. Compound 1 exhibited moderate activity against HL-60 and SMMC-7721 cells.     

Pyrrolizidine alkaloids: different acquisition and use patterns in Apocynaceae and Solanaceae feeding ithomiine butterflies (Lepidoptera: Nymphalidae)

Pyrrolizidine alkaloids (PAs) often serve as chemical mediators of plant-herbivore-predator interactions. Butterflies (Danainae and Ithomiinae) and moths (Arctiidae) usually acquire PAs from plant sources (larval host plants, flowers or withered leaves visited by adults—pharmacophagy) and thereby become chemically protected against predators; they also use PAs as pheromone precursors. Study by GC-MS of PAs in three species of Ithomiinae butterflies, their larval host plants and adult alkaloid sources showed three different acquisition patterns: (1) larvae of the primitive Tithorea harmonia sequester PAs from their food plant Prestonia acutifolia (Apocynaceae: Echitoideae), and adults may also acquire these alkaloids from plant sources; (2) larvae of the more derived Aeria olena feed on Prestonia coalita , in whose leaves no PAs were detected, but freshly emerged adults sometimes contain PAs and males intensively seek and sequester these alkaloids in plant sources; and (3) larvae of the still more advanced Mechanitis polymnia feed on several PA-free Solanum species, and adult males sequester the alkaloids from various plant sources. Males and females of all three species contain mostly two PAs, the diastereoisomeric retronecine monoesters lycopsamine and intermedine, stored in the N-oxide form. Larval host plants and adult plant sources showed a large array of PA structures, the most abundant and frequent being lycopsamine and its diastereoisomers intermedine, echinatine, rinderine and indicine, and the deoxy-analogues supinine and amabiline. Bioassays with wild caught and freshly emerged adults suggest that protection against predation by the orb weaving spider Nephila clavipes may be dependent on PA concentration and maybe some spider idiosyncrasies, but freshly emerged Aeria olena without PAs are also liberated by Nephila , suggesting other protective compounds. The role of this spider as a selective pressure for PA acquisition by ithomiines is not clear.