Chemotaxonomy of the pantropical genus Merremia (Convolvulaceae) based on the distribution of tropane alkaloids

The occurrence and distribution of tropane and biogenetically related pyrrolidine alkaloids in 18 Merremia species of paleo-, neo-, and pantropical occurrence have been studied. The extensive GC-MS study included members of almost all sections of the genus and has been carried out with epigeal vegetative parts as well as with roots. It comprises altogether 74 tropanes and 13 pyrrolidines including nicotine. Along with datumetine known already from a solanaceous species, the study led to the isolation (from M. dissecta and M. guerichii, respectively) and structure elucidation (spectral data) of four novel 3alpha-acyloxytropanes, merresectines A-D: 3alpha-(4-methoxybenzoyloxy)nortropane (A), 3alpha-kurameroyloxytropane (B), 3alpha-nervogenoyloxytropane (C), 3alpha-[4-(beta-D-glucopyranosyloxy)-3-methoxy-5-(3-methyl-2-butenyl)benzoyloxy]tropane (beta-d-glucoside of D). Moreover, the novel 3alpha,6beta-di-(4-methoxybenzoyloxy)tropane (merredissine) has been isolated from M. dissecta and structurally elucidated. In addition the structures of datumetine and merresectine A could be confirmed by synthesis. Spectral data for two known 3alpha-acyloxytropanes (merresectine E beta-D-glucoside, 4'-dihydroconsabatine) and one known 3beta-acyloxytropane (concneorine) are documented for the first time. The structures of three further merresectines (F-H) have been determined by mass spectrometry. Furthermore, the linkage (2',3- and 2',4-, respectively) of two position isomer N-methylpyrrolidinylhygrines was proven by synthesis. The results of the study contribute to the solution of infrageneric taxonomic problems. Whereas all species yield pyrrolidine alkaloids without suitably differentiating results the diverging occurrence of tropane alkaloids leads to three groups of sections: (1) taxa free of tropanes, (2) taxa with simple tropanes, and (3) taxa with merresectines in addition to simple tropanes.     

Chemotaxonomy, variation and geographical distribution of the Empetraceae

Thirty-one flavonoids (and related phenolics) were found in leaves of the six species in the three genera of Empetraceae (90 populations studied) and five further pigments in fruits of Empetrum. Affinity with the Ericaceae is supported by the occurrence of the 3-galactosidesof gossypetin, malvidin, delphinidin, cyanidin and peonidin. The distribution of leaf flavonoids agrees with both the recognition of three genera and species-delimitation in Ceratiola and Corema. In Empetrum , the flavonoids differ modally in the three species usually accepted, thus confirming data on fruit colour and breeding systems, and suggesting that the genus comprises one species with about three subspecies at diploid and tetraploid levels. The black fruits of most Northern Hemisphere plants contain malvidin, petunidin and delphinidin, while the red fruits of almost all Southern Hemisphere populations ( E. rubrum ) contain cyanidin and peonidin and provide a rare example of the association of cyanidin (instead of delphinidin) with the wild-type allele.
In leaf flavonoid content the diploid E. nigrum is much closer to its presumed tetraploid derivative E. eamesii than to the diploid E. rubrum , which differs most from E. eamesii. Flavonoid patterns and breeding system distinguish most E. rubrum populations from those isolated in Tristan da Cunha-Gough Island. The phytogeographical importance of the phytOGhemical results is discussed.     

2,3-dimethylnonacosane and tropane alkaloids from Hyoscyamus albus

In addition to hyoscine and hyoscyamine, a new compound isolated from the leaves and stems of Hyoscyamus albus has been characterized as 2,3-dimethylnonacosane by spectral studies.     

GC-MS investigation of tropane alkaloids in Datura stramonium

Alkaloids, GS-MS, Datura stramonium The alkaloid spectrum in roots, leaves and seeds of Datura stramonium L. was investigated by GC-MS. Twenty-nine tropane alkaloids are detected. Twelve of them are new constituents for the species and the two tropane esters 3-(3'-acetoxytropoyloxy)tropane (21) and 3-(2'-hydroxytropoyloxy)tropane (26) are described for the first time.     

Biosynthetic studies on the tropane ring system of the tropane alkaloids from Datura stramonium

Isotopic labelling experiments have been carried out in Datura stramonium root cultures with the following isotopically labelled precursors; [2H3]- [2-13C, 2H3]-, [1-13C, 18O2]-acetates, 2H2O, [2H3-methyl]-methionine, [2-13C]-phenyllactate, [3-2H]-tropine and [2'-13C, 3-2H]-littorine. The study explored the incorporation of isotope into the tropane ring system of littorine 1 and hyoscyamine 2 and revealed that deuterium from acetate is incorporated only into C-6 and C-7, and not into C-2 and C-4 as previously reported. Oxygen-18 was not retained at a detectable level into the C(3)-O bond from [1-13C, 18O2]-acetate. The intramolecular nature of the rearrangement of littorine 1 to hyoscyamine 2 is revealed again by a labelling study using [2'-13C, 3-2H]-littorine, [2-13C]-phenyllactate and [3-2H]-tropine.     

Biosynthesis of tropane ester alkaloids in Datura

Datura meteloides plants were fed via the roots with [1″,2′-¹⁴C]tigloyl hygroline and as a control, [2′-¹⁴C]hygrine. After a week the alkaloids were isolated and degraded. Despite hydrolysis of the putative precursor it was possible, by label ratio, to show that esterification occurs after, and not before, the tropane ring has been synthesized. Hygroline is proposed as a possible intermediate.     

Chemotaxonomy of Portuguese Ulex: quinolizidine alkaloids as taxonomical markers

Six species of Portuguese Ulex L. in a total of nineteen populations were studied by GC-EIMS as to their content in quinolizidine alkaloids. Sparteine, beta-isosparteine, jussiaeiine A, N-methylcytisine, cytisine, 5,6-dehydrolupanine, rhombifoline, lupanine, jussiaeiine B, N-formylcytisine, N-acetylcytisine, anagyrine, jussiaeiine C, jussiaeiine D, pohakuline, baptifoline, and epibaptifoline were detected. Analysis of the chromatograms showed that the chemical profile of all species was mainly composed of N-methylcytisine, cytisine, anagyrine, and jussiaeiines A, B, C and D. Therefore a quantification study of these alkaloids in all the populations studied was done by GC. These data were then submitted to cluster analysis and principal component analysis, which allowed the definition of five chemotypes and the recognition of hybrids. N-methylcytisine, cytisine, and jussiaeiines A, C and D are recognized as markers of this genus in Portugal.     

Ontogenetic variation of the tropane alkaloids in Datura stramonium

Alkaloid profile of Datura stramonium plant parts collected at different stages of development were investigated by GC–MS. Sixty-four tropane alkaloids were detected and 48 of them were determined. Two new tropane alkaloids, 3-phenylacetoxy-6,7-epoxynortropane and 7-hydroxyapoatropine were tentatively identified. The alkaloids scopoline, 3-(hydroxyacetoxy)tropane, 3-hydroxy-6-(2-methylbutyryloxy)tropane, 3β-tigloyloxy-6-hydroxytropane, 3,7-dihydroxy-6-tigloyloxytropane, 3-tigloyloxy-6-propionyloxytropane, 3-phenylacetoxy-6,7-epoxytropane, 3-phenylacetoxy-6-hydroxytropane, aponorscopolamine, 3β,6β-ditigloyloxytropane and 7-hydroxyhyoscyamine are reported for the first time for this species and 3-acetoxy-6-isobutyryloxytropane for the family Solanaceae. The number and the type of alkaloids in the different plant organs depend on the stages of development. The variability by types of alkaloids (monosubstituted, 3,6- and 3,7-disubstituted, 3,6,7-trisubstituted and 3-substituted-6,7-epoxytropanes) decreases from the roots of the plants in vegetative growth and immature fruits to the seeds of senile plants (mainly monosubstituted and 3-substituted-6,7-epoxytropanes).     

Calystegines as a new group of tropane alkaloids in Solanaceae

Calystegines are a new group of polyhydroxy alkaloids with a nortropane skeleton. They were detected in Atropa belladonna root cultures by chromatographic methods (TLC, GC) and identified by NMR and mass spectroscopy. Their occurrence was examined in several species of the Solanaceae. The biosynthesis of these compounds is suggested to proceed via the tropane alkaloid pathway, the first metabolite being pseudotropine. A pseudotropine-forming tropinone reductase was isolated and characterized from Atropa belladonna root cultures. Further evidence is given for the significance of tropinone and pseudotropine in calystegine formation by feeding experiments that increased calystegine formation. ¹⁵N-tropinone was shown to be incorporated into calystegines.Abbreviations GC gas chromatography - TBON 8-thiabicyclo[3.2.1]octan-3-one - TLC thin-layer chromatography     

Production of tropane alkaloids in cultured cells of Hyoscyamus niger

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS.Abbreviations NAA 1-Naphthaleneacetic acid - BA Benzyladenine - BSA N,O-Bis(trimethylsilyl) acetamide     

High-yield production of tropane alkaloids by hairy-root cultures of aDatura candida hybrid

Hairy root cultures were obtained following inoculation of the stems of sterile plantlets of aDatura candida hybrid withAgrobacterium rhizogenes. The scopolamine and hyoscyamine content was quantified by HPLC and compared with the non-transformed plants. The alkaloid yield (0.68% dry weight) obtained with the hairy roots was 1.6 and 2.6 times the amount found in the aerial parts and in the roots of the parent plants, respectively. Only a small proportion of alkaloids was released into the growth medium. Scopclamine was the principal alkaloid and the scopolamine/hyoscyamine ratio of ca. 5:1 makes these hairy roct cultures worthy of consideration as a source of scopolamine.     

Esterification reactions in the biosynthesis of tropane alkaloids in transformed root cultures

Transformed root cultures of Datura stramonium and of related species contain both aliphatic and aromatic tropane esters. It has been shown that these esters are produced by the action of several acyl transferases that transfer the acidic moiety to tropan-3-ol (tropine) or tropan-3-ol (pseudotropine) from various acyl-coenzyme A thioesters. The presence of these enzymes has been examined in a range of tropane-alkaloid-producing and non-tropane-alkaloid-producing species. Activities that esterify tropine appear to be confined to species that accumulate tropane alkaloids, whereas a number of species that do not accumulate tropane alkaloids possess some ability to esterify pseudotropine.The present state of knowledge of these enzymes is reviewed. One of these activities, tigloyl-Coenzyme A:pseudotropine acyl transferase, has been purified to near homogeneity and the properties of this enzyme are summarized.Abbreviations CoA coenzyme A - gc gas chromatography - ms mass spectrometry     

山莨菪植物体内4种莨菪烷类生物碱含量的变化

为了对山莨菪植物资源进行可持续利用,采用高效液相色谱（HPLC）技术对山莨菪植物体内4种莨菪烷类生物碱同时进行了定量测定,将4种生物碱很好地分离开来,大大缩短了出峰时间;并对这4种生物碱含量随着物候的变化进行了分析研究。研究结果表明：4种生物碱含量与物候的关系呈抛物线变化,樟柳碱在山莨菪植物体内含量最高,地上部分4种生物碱均在花盛期含量最高,地下部分不同的生物碱含量最高点则对应不同的物候期。     

Separation of four isomeric tropane alkaloids from Schizanthus grahamii by non-aqueous capillary electrophoresis

The potential of non-aqueous capillary electrophoresis was investigated for the separation of four isomeric tropane alkaloids, namely 3alpha-senecioyloxy-7beta-hydroxytropane, 3alpha-hydroxy-7beta-senecioyloxytropane, 3alpha-hydroxy-7beta-angeloyloxytropane and 3alpha-hydroxy-7beta-tigloyloxytropane extracted from Schizanthus grahamii. The composition of the organic solvent and the nature of the electrolyte were of considerable importance with respect to selectivity. Different organic solvents (i.e. methanol, ethanol, acetonitrile, tetrahydrofuran) and mixtures thereof were investigated. Moreover, different electrolytes such as formate, acetate and trifluoroacetate were tested. After optimisation, an electrolyte consisting of 1 M trifluoroacetic acid and 25 mM ammonium trifluoroacetate in methanol:ethanol (40:60, v:v) was selected. It provided an efficient separation of the four positional isomers as well as a good repeatability of migration time (RSD < 0.2%). The method was successfully used with electrospray MS to confirm the molecular mass of the tropane alkaloids.     

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.     

High frequency plant regeneration and accumulation of tropane alkaloids in regenerated plants of Scopolia parviflora

An efficient in vitro plant regeneration system was established from callus culture of Scopolia parviflora. Callus was induced from adventitious roots on B5 medium with 0.45–9.04 μM 2,4-dichlorophenoxyacetic acid (2,4-D). In vitro plantlet regeneration was achieved on B5 medium supplemented with 44.38 μM benzyladenine (BA), 3% sucrose, and 0.38% gelrite. Plantlets were transplanted to artificial soil and grown to maturity successfully in a greenhouse. The tropane alkaloid contents in regenerated plants were analyzed using high-performance liquid chromatography (HPLC), and were found to be higher than those of adventitious roots, native growing plants, and acclimated plants. Regenerated plants from organogenic callus cultures produced a greater amount of tropane alkaloids.     

Selection on tropane alkaloids in native and non‐native populations of Datura stramonium

Theories of plant invasion based on enemy release in a new range assume that selection exerted by specialist herbivores on defence traits should be reduced, absent, or even selected against in the new environment. Here, we measured phenotypic selection on atropine and scopolamine concentration of Datura stramonium in eight native (Mexico) and 14 non‐native (Spain) populations. Native populations produced between 20 and 40 times more alkaloid than non‐native populations (atropine: 2.0171 vs. 0.0458 mg/g; scopolamine: 1.004 vs. 0.0488 mg/g, respectively). Selection on alkaloids was negative for atropine and positive for scopolamine concentration in both ranges. However, the effect sizes of selection gradients were only significant in the native range. Our results support the assumption that the reduction of plant defence in the absence of the plant's natural enemies in invasive ranges is driven by natural selection.     

缟蝇科昆虫生物学和地理分布分析

本文概述了缟蝇科昆虫的生活史、食性、访花习性、栖居环境等生物学特性,并对缟蝇科属、种的地理分布进行了分析。缟蝇科在世界动物地理区的分布统计结果表明,同脉缟蝇亚科为亚世界性分布,其属级阶元在东洋界丰富度最高,其次为澳洲界;其种级阶元在东洋界最为丰富,其次为澳洲界。缟蝇亚科为世界性分布,其属级阶元在新热带界丰富度最高,并且高度特化,其次是东洋界和澳洲界;其种级阶元在新热带界最为丰富,其次为古北界和澳洲界。缟蝇科在中国动物地理区和亚区的分布统计结果显示,其属、种级阶元在华南区最为丰富;同脉缟蝇亚科的种级阶元以海南岛亚区最为丰富,其次为台湾亚区;缟蝇亚科的种级阶元在台湾亚区最为丰富,西部山地高原亚区次之。     

Evolutionary ecology of the tropane alkaloids of Datura stramonium L. (Solanaceae)

Abstract.— Although insect herbivory is frequently assumed to be responsible for the maintenance of plant secondary metabolites such as alkaloids, the assumption is controversial and experimental evidence for this assumption is sparse. We examined natural selection on the two major alkaloids present in the leaves of Datura stramonium and found that both alkaloids came under active selection. We found negative directional selection for scopolamine (natural selection acting to reduce scopolamine levels) and stabilizing selection for hyoscyamine (natural selection acting to maintain an intermediate level of hyoscyamine). We also present evidence that insect herbivores act as the agents of selection on these alkaloids. Finally, we show that there were no trade-offs in resistance to different species of insects.