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.     

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.     

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     

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.     

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.     

The geographical distribution of rarity

Summary This paper asks the question: are most species that are censused as rare in particular localities rare throughout most of their geographic ranges, or are they common in substantial portions of their ranges elsewhere? The first alternative is labeled suffusive rarity and the second diffusive rarity. To answer this and similar questions, rarity can be measured as the fraction of censuses from some locality (e.g., a quadrat) in which a species occurs (occurrence rarity), or the relative or absolute abundance of the species averaged over all censuses from some locality (abundance rarity). The question was analyzed for occurrence-rarity data from Australian terrestrial birds distributed over 1° (10⁴-km²) quadrats. The great majority of species that are rare in a particular quadrat are not rare and are often common in a substantial number of other quadrats, i.e., these avian species are much closer to the diffusive than suffusive portion of the rarity continuum. The data also show that 1) the distribution of sizes of geographic ranges, whether breeding or total, is highly skewed, appearing exponential to more concave; 2) species are much rarer in their nonbreeding than breeding ranges; 3) more widespread species, whether breeding or total ranges are considered, tend to occur more rarely in a slightly but significantly greater fraction of their ranges; and 4) hawks and owls, typified by high abundance rarity, show occurrence rarity in a greater fraction of their ranges than the average nonraptorial species. Although continental birds may be especially predilected toward diffusive rarity, the present analysis points to identification of centers of abundance as major ways of preserving those species contributing most to recorded instances of rarity. Similar analyses with other kinds of organisms would be most welcome.     

Influence of inoculum morphology on growth of Atropa belladonna hairy roots and production of tropane alkaloids

Summary The specific growth rate of Atropa belladonna hairy roots measured in terms of root length increased by about 25% to 0.49 d^-1 when the inoculum consisted of a root with the primary root tip excised. The biomass dry weight produced after 14 d increased by 28%. In contrast, presence of laterals in the inoculum with or without lateral tips excised did not influence the growth rate. Although hyoscyamine was found to accumulate at higher concentrations in the more mature root tissues (2.1 ± 0.2 mg g^-1) than in the tips (1.1 ± 0.3 mg g^-1), hyoscyamine content in the harvested roots was independent of inoculum morphology.     

Absolute configuration of tropane alkaloids from Schizanthus species by vibrational circular dichroism

The absolute configuration (AC) of 6β-hydroxy-3α-senecioyloxytropane (1), 3α-hydroxy-6β-tigloyloxytropane (2), 3α-hydroxy-6β-senecioyloxytropane (3), and 3α-hydroxy-6β-angeloyloxytropane (4) was assigned as (1R,3R,5S,6R) using density functional theory (DFT) calculations at the B3LYP/DGDZVP level of theory in combination with experimental vibrational circular dichroism (VCD) measurements and comparison with the spectra of similar tropanes. The AC of 1 followed from a sample isolated from Schizanthus grahamii, while those of the mixture of 2 and 3, isolated from the same source, were determined by comparing the VCD measurement to a weighted calculation of the individual VCD spectra according to a 69:31 ratio of 2:3 determined by ¹H NMR signal integration. In turn, Schizanthus pinnatus provided a 7:3 mixture of 1:4 whose AC was determined using the experimental VCD absorptions in the 1150-950 cm⁻¹ spectral region which were compared with those observed for 1-3 and with those described for other 3α,6β-tropanediol derivatives.     

Chemotaxonomy of Anseriformes

The composition of the uropygial gland wax of 8 species of birds of the order Anseriformes was investigated. As was expected, the waxes are composed of more or less methyl-branched fatty acids and alcohols. The results obtained have been compared with those on other species of Anseriformes and discussed from a chemotaxonomical viewpoint. For this purpose the various parameters have been depicted on a 3-dimensional matrix.     

Production of tropane alkaloids by small-scale bubble column bioreactor cultures of Scopolia parviflora adventitious roots

The mass production of tropane alkaloids from adventitious root cultures of Scopolia parviflora, in small-scale bubble column bioreactor (BCB) was attempted. Adventitious roots of S. parviflora produced relatively enhanced levels of scopolamine and hyoscyamine in bioreactor compared to flask type cultures, and rapidly produced root clumps, with continuously increasing biomass throughout the culture period. The production of scopolamine and hyoscyamine in the top and bottom regions of root clumps were higher than in the core region. The adventitious root cultures of S. parviflora in the BCB required a relatively high level of aeration. The optimized conditions for the bioreactor culture growth and alkaloid production were found to be 3g of inoculum, on a fresh weight basis, a 15-day culture period and 0.4vvm of airflow. The elicitation by Staphylococus aureus increased the specific compound of scopolamine, while the production of hyoscyamine was slightly inhibited in BCB cultures.     

Effects of different elicitors on yield of tropane alkaloids in hairy roots of Anisodus acutangulus

The four tropane alkaloids have played a pivotal role in controlling diseases such as the toxic and septic shock, the organophosphorus poison and the acute lung injury. Here, the elicitation effect of different elicitors on the production of tropane alkaloids and the molecular mechanism of enzyme genes in the pathway was firstly demonstrated in hairy roots of Anisodus acutangulus. The results showed ethanol, methyl jasmonate and Ag⁺ could improve the accumulation of tropane alkaloids up to 1.51, 1.13 and 1.08 times after 24 h treatment, respectively (P < 0.05), whereas salicylic acid decreased the average content of tropane alkaloids. Furthermore, expression profile analysis results revealed that up-regulation of hyoscyamine-6b-hydroxylase (AaH6H) and little regulation of tropinone reducase II (AaTR2) elicited by ethanol, increased expression of putrescine N-methyltransferase I (AaPMT1) elicited by Ag⁺, elevated expression of tropinone reducase I (AaTR1) elicited by methyl jasmonate, respectively, resulted in tropane alkaloids improvement. Our results showed that hairy root culture of A. acutangulus in combination with elicitors was a promising way for production of tropane alkaloids in the future.     

Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures

Transformed root cultures of Datura stramonium, competent in tropane-alkaloid biosynthesis, have been treated with exogenous plant growth regulators. It was found that combinations of -naphthalene-acetic acid, kinetin (N⁶-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid induced de-differentiation, causing both the rooty phenotype and the hyoscyamine-biosynthetic capacity to be lost. Alkaloid biosynthesis disappeared rapidly and prior to the loss of morphological integrity. It was observed that the enzymes ornithine decarboxylase (EC 4.1.1.17), arginine decarboxylase (EC 4.1.1.19) and N-methylputrescine oxidase did not show the increase in level normally associated with subculturing the roots. The level of putrescine N-methyltransferase (EC 2.1.1.53) activity, the first enzyme fully committed to hyoscyamine biosynthesis, rapidly declined, about 80% being lost from the roots within 12h. This activity, although showing some temporary restoration, declined further after a few days, and was totally absent from fully dispersed cultures. N-Methylputrescine oxidase persisted at a low level. Following sub-culture of established de-differentiated lines to plant-growth-regulator-free medium, limited root regeneration occurred. The roots formed showed renewed competence in alkaloid biosynthesis and putrescine N-methyltransferase and N-methylputrescine oxidase activities were restored to their normal levels. The relationship between the morphological state and alkaloid-biosynthetic capacity of the cultures is discussed in relation to the overall control of alkaloid biosynthesis.Abbreviations ADC arginine decarboxylase - FW fresh weight - MPO N-methylputrescine oxidase - NAA -naphthalineacetic acid - ODC ornithine decarboxylase - pgr plant growth regulator - PMT putrescine N-methyltransferase We are most grateful to Abigael Peerless and Bridget Chapman for assistance with various part of this work.