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Stem bark, root bark, and leaf extracts of Erythroxylum pungens were subjected to phytochemical analysis. N,N-dimethyltryptamine (DMT) was isolated and characterized from E. pungens roots. This unprecedented result is remarkable since no indole alkaloid has been previously reported from Erythroxylaceae so far. Eleven known tropane alkaloids were identified by their mass spectra and 3-(2-methylbutyryloxy)tropan-6,7-diol as well as 3-(2-methylbutyryloxy)nortropan-6,7-diol were isolated and characterized based on mass spectrometry, 1H, 13C, COSY, and NOESY NMR analysis. The complete NMR data are reported for the first time. Inverse Structure-based and Ligand-Based virtual screening were carried out to identify possible targets for 3-(2-methylbutyryloxy)tropan-6,7-diol. The level of cytotoxicity of this tropane alkaloid aliphatic ester was discrete with potencies on the order of 0.3–1.0 mg/mL and better results against HeLa (50% cell viability reduction). Otherwise, atropine (0.3 mg/mL), a Solanaceae tropane alkaloid, and DMT (0.5 mg/mL) from E. pungens roots impaired at 50% the cell viability against HeLa, SiHa, PC3, and 786-0. This study stimulates scientific investigation of the impact of edaphoclimatic features in a semi-arid environment on tropane alkaloid biosynthesis.

Duboisia species, which belong to the family of Solanaceae, are commercially cultivated in large scale, as they are main source of the pharmaceutically-used active compound scopolamine. In this study, 1 H NMR-based metabolite profiling linking primary with secondary metabolism and additional quantification via HPCL-MS with special focus on the tropane alkaloids were applied to compare leaf and root extracts of three wild types and two hybrids of Duboisia myoporoides and D. leichhardtii at different developmental stages grown under controlled conditions in climate chambers and under agricultural field plantation. Based on the leaf extracts, a clear distinction between the Duboisia hybrids and the wild types Duboisia myoporoides and D. leichhardtii using principal component analysis of 1 H NMR data was observed. The average content in scopolamine in the hybrids of Duboisia cultivated in climate chambers increased significantly from month 3–6 after potting of the rooted cuttings, however not so for the examined wild types. The Duboisia hybrids grown in climate chambers showed higher growth and contained more sugars and amino acids than Duboisia hybrids grown in the field, which in contrast showed an enhanced flux towards tropane alkaloids as well as flavonoids. For a more detailed analysis of tropane alkaloids, an appropriate HPLC-MS method was developed and validated. The measurements revealed large differences in the alkaloid pattern within the different genotypes under investigation, especially regarding the last enzymatic step, the conversion from hyoscamine to scopolamine by the hyoscyamine 6β–hydroxylase. Scopolamine was found in highest concentrations in Duboisia hybrids (20.04 ± 4.05 and 17.82 ± 3.52 mg/g dry wt) followed by Duboisia myoporoides (12.71 ± 2.55 mg/g dry wt), both showing a high selectivity for scopolamine in contrast to Duboisia leichhardtii (3.38 ± 0.59 and 5.09 ± 1.24 mg/g dry wt) with hyoscyamine being the predominant alkaloid.

Complexation of alkaloids is an important strategy plants utilize to facilitate storage in vacuoles and avoid autotoxicity. Previous studies have implicated hydroxycinnamoyl quinate esters in the complexation of purine alkaloids in Coffea arabica. The goal of this study was to determine if Erythroxylum coca uses similar complexation agents to store abundant tropane alkaloids, such as cocaine and cinnamoyl cocaine. Metabolite analysis of various E. coca organs established a close correlation between levels of coca alkaloids and those of two hydroxycinnamoyl esters of quinic acid, chlorogenic acid and 4-coumaroyl quinate. The BAHD acyltransferase catalyzing the final step in hydroxycinnamoyl quinate biosynthesis was isolated and characterized, and its gene expression found to correlate with tropane alkaloid accumulation. A physical interaction between chlorogenic acid and cocaine was observed and quantified in vitro using UV and NMR spectroscopic methods yielding similar values to those reported for a caffeine chlorogenate complex in C. arabica. These results suggest that storage of cocaine and other coca alkaloids in large quantities in E. coca involves hydroxycinnamoyl quinate esters as complexation partners.

Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea.

A rapid in vitro propagation system leading to formation of shoots from callus, roots, and plantlets was developed for Schizanthus hookeri Gill. (Solanaceae), an endemic Chilean plant. The genus Schizanthus is of particular interest due to the presence of several tropane alkaloids. So far, in vitro propagation of species of this genus has not been reported. Propagation of S. hookeri consisted of two phases, the first one for callus initiation and shoot formation and the second for rhizogenesis and plantlet regeneration. From a single callus that rapidly increased in cell biomass (from approximately 50 mg to approximately 460 mg/culture tube [25 x 130 mm] in 60 days) in the presence of 2.69 microM NAA and 2.22 microM BA, more than 10 shoots/callus explant were formed. From the latter, approx. twenty plantlets formed after 90-110 days shoot subculture in medium devoid of growth regulators that favored root formation. Ten alkaloids ranging from simple pyrrolidine derivatives to tropane esters derived from angelic, tiglic, senecioic or methylmesaconic acids were obtained from in vitro regenerated plantlets. One of them, 3alpha-methylmesaconyloxytropane, was not previously described. The same growth conditions, as well as other growth regulator levels tested, were required to induce callus and root formation in S. grahamii Gill. Root organogenesis occurred despite a high level of BA vs. NAA used, (i.e., 4.44 microM BA and 0.54 microM NAA); however no shoot formation was achieved. In the case of S. tricolor Grau et Gronbach, only callus formation was obtained in the presence of various growth regulators.

Two stereospecific oxidoreductases constitute a branch point in tropane alkaloid metabolism. Products of tropane metabolism are the alkaloids hyoscyamine, scopolamine, cocaine, and polyhydroxylated nortropane alkaloids, the calystegines. Both tropinone reductases reduce the precursor tropinone to yield either tropine or pseudotropine. In Solanaceae, tropine is incorporated into hyoscyamine and scopolamine; pseudotropine is the first specific metabolite on the way to the calystegines. Isolation, cloning and heterologous expression of both tropinone reductases enabled kinetic characterisation, protein crystallisation, and structure elucidation. Stereospecificity of reduction is achieved by binding tropinone in the respective enzyme active centre in opposite orientation. Immunolocalisation of both enzyme proteins in cultured roots revealed a tissue-specific protein accumulation. Metabolite flux through both arms of the tropane alkaloid pathway appears to be regulated by the activity of both enzymes and by their access to the precursor tropinone. Both tropinone reductases are NADPH-dependent short-chain dehydrogenases with amino acid sequence similarity of more than 50% suggesting their descent from a common ancestor. Putative tropinone reductase sequences annotated in plant genomes other that Solanaceae await functional characterisation.

Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2–4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.

Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A. tumefaciens strain C58C1pRTGus104. The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages. Significant differences appeared between the alkaloid contents of the different clones. In particular, all the hairy root clones and the roots of the intact plant produced 1.5–3 and 4.5–9 times more littorine than scopolamine, respectively. The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine. The aerial parts of H. muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.

Hairy root cultures of Datura stramonium were treated with methyl jasmonate (MeJa), a cell wall preparation from baker's yeast and oligogalacturonides, respectively, and analysed for the accumulation of the tropane alkaloids, littorine, hyoscyamine and scopolamine, and their precursors, phenyllactate and tropine. The treatments increased alkaloid accumulation in the order MeJa>fungal elicitor>oligogalacturonide and, in all cases, this was associated with an increase in tropine but a decline in phenyllactate concentrations. Time–course studies following MeJa treatments confirmed that increased tropane alkaloid synthesis was due to the differential enhancement of tropine biosynthesis.

The growth and tropane content in transformed root cultures of Atropa belladonna (strain M8) grown in 300 ml flasks were studied. After one month in culture, the biomass of the transformed roots increased 15 times and reached 5 g dry wt l −1 ; the alkaloid contents were 0.7% for (−)-hyoscyamine, 0.1% for 6 β -hydroxyhyoscyamine, 0.02% for scopolamine and 0.02% for littorine. During the culture period, no alkaloid could be detected in the medium. Seven chemicals were individually added to 18-day-old cultures. Glutathione (5 mM), chitin (0.1%), chitosan (0.1%) or yeast extract (0.1%) had no effect on the release of alkaloids into the medium. However, treatment with 5 mM H 2 O 2 induced a transient release of tropane alkaloids from transformed roots and the maximum amount of alkaloids in the medium was ca 0.35 mg per flask (10% of alkaloid contained in tissues); tissue viability was still preserved. Cu 2 and Cd 2 (5 mM) also improved the excretion of tropane alkaloids into the medium. However, their addition led to lysis of transformed roots.

Two new dimeric tropane alkaloids, mooniines A and B were identified from the leaves of Erythroxylum moonii. Their structures were determined by mass spectrometry and multi-dimensional NMR spectroscopy.

Thirteen alkaloids were identified from the stem-bark of Erythroxylum lucidum by GC-MS. Seven of these have not been reported previously in the genus Erythroxylum. Most of them are precursors or intermediates in the biosynthetic pathway of tropane alkaloids. Additionally, one alkaloid characterized as 2,1'-dehydrohygrine, a key putative intermediate in the formation of tropinone, has been identified unambiguously.

Pentahydroxy- nor -tropane alkaloids, calystegines C 1 and C 2 , were isolated from the leaves of greenhouse-cultivated Duboisia leichhardtii , together with calystegines B 1 , B 2 and B 4 . Calystegine C 1 is a potent inhibitor of β-glucosidase and β-galactosidase, whereas calystegine C 2 exhibited no significant inhibitory activity against these enzymes. Only calystegine C 2 , the 2-epimer of calystegine C 1 , among calystegines isolated to date exhibited an inhibitory activity against all α-mannosidases tested.

Six new tropane alkaloids have been isolated from the leaves of the endemic Chilian plant Schizanthus litoralis (Solanaceae). The new structures include a new tropanol dimer of itaconic acid together with 6β-senecioyloxytropan-3α-methylmesaconate, 6β-cinnamoyloxytropan-3α-methylmesaconate, 6β-senecioyloxytropan-3α-ol and cis-trans N-(4-hydroxyphenethyl) ferulamides. Other known alkaloids were also isolated. Their structures were elucidated by spectroscopic methods.

Ester derivatives of labelled tropic acid (denoted tropoyl-A and -D) were isolated as metabolites of atropine from Duboisia myoporoides seedlings supplied with [carbonyl- 14 C]atropine sulphate and were then fed to D. leichhardtii root cultures to determine whether they were metabolic end-products or whether they could be metabolized further. Both tropoyl-A and tropoyl-D were incorporated into tropane alkaloids (atropine, 6-hydroxyatropine and scopolamine). Incorporation from tropoyl-D exceeded that from tropoyl-A. This result shows that both tropoyl-A and -D are precursors of tropane alkaloids and that the tropic acid moiety is possibly recycled in plants of Duboisia .

Twenty-six tropane alkaloids were identified from the root-bark of Erythroxylum monogynum by GC and GC-MS and twenty of these alkaloids had not previously been reported as constituents of the root-bark. 3α-(3′,4′,5′-Trimethoxybenzoyloxy) tropane was the main base and in addition, three new alkaloids were identified as 3α-isobutyryloxynortropane, 3α-(4-methylvaleroyloxy)tropane and tentatively, 3β-phenylacetoxytropane.

Hyoscyamus albus is a solanaceous plant forming tropane alkaloids. The roots as the site of biosynthesis show a rich alkaloid spectrum of at least 26 compounds, whereas the alkaloid composition of the aerial parts is less complex. Some of the alkaloids, i.e. 3-propionyloxytropane, 3-isobutyryloxytropane, 3β-tigloyloxynortropane and 6-hydroxy-3-phenyl-acetoxytropane are described as components of H. albus for the first time. Three hitherto unknown compounds were tentatively identified as N-methylpyrrolidinyl-cuscohygrine, occurring in two isomeric forms, and as 3α-(p-hydroxyphenyl)lactoyloxytropane (4′-hydroxylittorine). Compared with plant roots, hairy roots show a rather similar but not identical alkaloid pattern. The low proportion of scopolamine in hairy roots is especially remarkable.

Labelled acetate (C 2 H 3 13 COONa) was fed to fast-growing hairy roots of Hyoscyamus albus transformed with Agrobacterium rhizogenes MA

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.

Adventitious and hairy root cultures of several solanaceous plant species were established. The highest content of tropane alkaloids was obtained in the adventitious roots of Hyoscyamus albus cultured in hormone-free 1/2 MS liquid medium. On the other hand the hairy roots of H. albus transformed with A. rhizogenes strain MAFF 03-01724 grew very fast and yielded over 5 mg/100 ml flask of hyoscyamine within 22 days of culture. The alkaloid content in the hairy roots of H. niger was higher than that of its adventitious roots. 7β-Hydroxyhyoscyamine, recently isolated, was produced in the hairy roots of Scopolia tangutica and Datura innoxia, while it could not be detected in their adventitious root cultures.

Hairy roots of Hyoscyamus albus were induced from the shoot cultures by transformation with A. rhizogenes strain MAFF 03-01724. The hairy root cultures were tested for growth and alkaloid production (5 tropane alkaloids simultaneously quantified by HPLC) in various media. Half-strength MS and Gamborg BS media containing 3% sucrose gave the highest alkaloid content (ca 2% dry wt), but only poor growth. On the other hand, fast growth was observed in Woody Plant medium containing 8% sucrose, while the production of tropane alkaloids was relatively low. The addition of KNO3 (1–50 mM) to Woody Plant medium (3% sucrose) enhanced the growth, and consequently the alkaloid yield in the hairy roots increased remarkably. The hairy roots cultured in the improved medium (Woody Plant medium containing 3% sucrose and 15 mM KNO3) for 28 days produced over 30 mg tropane alkaloids in one 100 ml flask (ca 0.8 mg 7β-hydroxyhyoscyamine, 1.7 mg littorine, 1.7 mg scopolamine, 7 mg 6β-hydroxyhyoscyamine and 19 mg hyoscyamine in 50 ml liquid medium).

A new γ-pyranotropane, darlingine N-oxide, was isolated from the bark and leaves of Darlingia darlingiana, along with the known compound, darlingine. 1D-NOESY NMR experiments indicated that the N-methyl groups of both alkaloids were orientated towards the pyran ring.

Key Word Index-Datum spp.; Hyoscyamus spp.; Scopolia spp.; Solanaceae; identification; accumulation; trans- formed roots; tropane alkaloids; scopolamine; hyoscyamine. - Abstract-Petioles from over 1000 individual plants of the genera Datura, Scopolia and Hyoscyamus were analysed to establish the variation in the levels and patterns of tropane alkaloids which occur between plants. Transformed root cultures were initiated from plants of which analyses suggested extremely high or low alkaloid biosynthetic capacities, or favourable alkaloid patterns. These root cultures were then studied in detail by HPLC, GC and GC-MS. Root cultures showed substantial differences in alkaloid patterns between different species, but the quantitative differences in tropoyl esters of cc-tropine (hyoscyamine plus scopolamine) observed between different lines were less than those seen between plants. This difference is interpreted as to some extent reflecting the influence of transport and storage effects, as well as biosynthetic capacity, in determining alkaloid levels in plant petioles. In addition, there is some indication that the full biosynthetic capacity may not always be expressed in culture. Despite these limitations, the identification of plants showing favourable characteristics still, however, proved a useful first step in the development of scientifically and potentially biotechnologically interesting root cultures.

The administration of dl-[5-14C]ornithine to a root culture of Duboisia leichhardtii, afforded radioactive (S)-nicotine and scopolamine. Systematic degradations carried out on these labelled alkaloids established that the nicotine was labelled equally at its C-2′ and C-5′ positions, and the scopolamine was labelled equally at its C-1 and C-5 positions. These results are consistent with the hypothesis that the pyrrolidine ring of nicotine and that present in the tropane moiety of scopolamine have a common biosynthetic origin and that the ornithine is incorporated via a symmetrical intermediate. An efficient method for the conversion of scopolamine to hyoscyamine is described.

Relatively low levels of tropane alkaloid precursors (tropic acid, tropinone and tropanol) and alkaloids (hyoscyamine and scopolamine) were fed to suspension cultures of Atropa belladonna with repressed alkaloid synthesis to study the transport of these compounds into the cells, to detect possible limiting steps in the biosynthetic pathway and to find ways of stimulating alkaloid production. At the end of the experiments, the cell levels of tropic acid and tropinone had reached the initial nutrient medium concentrations, while cell levels of tropanol, hyoscyamine and scopolamine were high, suggesting an active uptake mechanism. Increased intracellular precursor levels did not forward the biosynthesis of the tropane alkaloids.

An investigation of the roots of Convolvulus siculus and C. sabatius ssp. mauritanicus afforded two new tropan-3 α -ol esters named consiculine and consabatine and the known base merresectine B (3). The acyl residues of the new alkaloids are formed by consiculic acid ( = 1 β ,2 α -dihydroxy-5-prenyl-cyclohex-5-en-4-one carboxylic acid) and its 2-deoxy derivative, respectively.

The report of cochlearine, the 3-hydroxybenzoate ester of tropine found in Cochlearia officinalis, Brassicaceae, initiated a screening for tropane alkaloids in Cochlearia species and for calystegines in further Brassicaceae. All ten Cochlearia species investigated contained cochlearine, tropine, and pseudotropine. Calystegines, nortropane alkaloids deriving from pseudotropine, were also identified in all Cochlearia species and accumulated up to 0.5% dry mass in leaves. Brassicaceae species of all major lineages of the family were analysed for calystegines. Of the 43 species included in the study, 18 accumulated calystegines of various structures. This is the first screening of Brassicaceae for products of the tropane alkaloid pathway, which is known as characteristic for plants of Solanaceae family. The identification of calystegines in all branches of the Brassicaceae family including Aethionema, a species at the basis of the family, suggests tropane alkaloids as secondary compound typical for Brassicaceae.

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.

The volatile compounds emitted from flowers of Brugmansia × candida Pers. were characterized to investigate reports that the floral scent may have intoxicating effects. The dominant components were trans -ocimene (38–52%) and 1,8-cineole (5–19%), while the minor components consisted of various other terpenoids, benzenoids and indole. The floral scent was typical of many white flowers. The volatile compounds emitted from the leaves were also examined and were found to be mainly terpenoids. Perillene (14–25%) was the major component and its analogous furanoid sesquiterpenoid, dendrolasin, was also present. Volatile tropane alkaloids could not be detected in either the floral or foliar odours, and none of the compounds emitted by the flowers are likely to cause intoxicating effects at low exposures.

Polyhydroxylated tropane alkaloids, not hitherto recorded from the potato ( Solanum tuberosum ), were detected in the leaves and tubers of this plant. They are potent inhibitors of glycosidases and may be responsible for neurological disorders in livestock. These alkaloids were also extracted from a sphingid moth and an ithomiine butterfly, the larvae of which feed on Solanum .

Phenyl[1-14C]alanine which is a precursor of the acid moiety of tropane alkaloids and one of the possible intermediates—namely phenylpyruvic, phenyllactic and tropic acids—were simultaneously added to Duboisia leichhardtii root cultures in order to trap the labelled intermediates. When phenyllactic acid was added to 0.5–5 mm, incorporation of phenyl[1-14C]alanine into the organic acid fraction was much greater in comparison with the cases using phenylpyruvic acid, tropic acid or no intermediate at all. TLC-autoradiogram of the organic acid fraction obtained from phenyllactic acid feeding experiments showed the existence of a radioactive spot corresponding to phenyllactic acid. Trapping of labelled phenyllactic acid was confirmed by recrystallization, NMR and melting point.

The polyamine, cadaverine, was detected in transformed root cultures of Brugmansia candida (syn. Datura candida), a Solanaceae which produces the tropane alkaloids scopolamine and hyoscyamine. To the best of our knowledge, this is the first time that the existence of this uncommon polyamine has been detected in a Datura species. Cadaverine, however, could not be found in the whole plant. The occurrence of cadaverine in hairy roots could be a consequence of either the transformation or a response to stress. Also, cadaverine could be participating in other secondary pathways rather than to the tropane alkaloids. The common polyamines, putrescine, spermidine and spermine were also observed.

Isotopic labelling experiments have been carried out in Datura stramonium root cultures with the following isotopically labelled precursors; [ 2 H 3 ]-, [2- 13 C , 2 H 3 ]-, [1- 13 C , 18 O 2 ]-acetates, 2 H 2 O , [ 2 H 3 - methyl ]-methionine, [2- 13 C ]-phenyllactate, [3- 2 H ]-tropine and [2′- 13 C , 3- 2 H ]-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- 13 C , 18 O 2 ]-acetate. The intramolecular nature of the rearrangement of littorine 1 to hyoscyamine 2 is revealed again by a labelling study using [2′- 13 C , 3- 2 H ]-littorine, [2- 13 C ]-phenyllactate and [3- 2 H ]-tropine.

This review illustrates the distribution of tropane alkaloids within the families Solanaceae, Erythroxylaceae, Proteaceae, Euphorbiaceae, Rhizophoraceae, Convolvulaceae and Cruciferae. Whereas tropane alkaloids are characteristic of the genera Datura, Brugmansia (tree datura) and Duboisia of the Solanaceae, the distribution is more widespread with novel tropane derivatives in families not traditionally associated with these bases. The chemical nature of more recently discovered water-soluble calystegines and the di- and trimeric forms from the Convolvulaceae (e.g. schizanthines from Schizanthus spp.), truxillines from Bolivian coca leaves and moonines of Erythroxylum moonii are highlighted. Where possible and appropriate, links between the phytochemistry and taxonomy are discussed.

A family of novel polyhydroxy- nor -tropanes is described. Three of their structures were determined by mass spectrometry and 1 H and 13 C NMR spectroscopy. These calystegins occur in two species of the Convolvulaceae and in Atropa belladonna . Their potential ecological significance is discussed.

1-Methylpyrrolidine-2-acetic acid and related compounds were studied as precursors in the biosynthesis of the tropane alkaloids in Erythroxylum coca and Datura innoxia. (R,S)-[1′,2-13C2,2-14C,15N]-1-Methylpyrolidien-2-accetic acid, (R,S)-[1′,2′-13C2,1′-14C]-1-methylpyrrolidine-2-acetic acid, (R,S) ethyl [1′,2′-13C2,1′-14C]-1- methylpyrrolidine-2-acetate, and (R,S)-[2′-14C]-1-methylpyrrolidine-2-acetic acid N-acetylcysteamine thioester were synthesized and fed to intact plants by leaf-painting or hydroponic-feeding. Specific incorporation of these compounds into (−)-hyoscyamine, (−)-scopolamine, (−)-cocaine and the biosynthetically related cuscohygrine were very low. These results indicate that 1-methylpyrrolidine-2-acetic acid is not an efficient precursor of tropane alkaloids.

Horses in a few, localized northern Colorado pastures exhibited weight loss and colic. At post mortem, intestinal fibrosis and vascular sclerosis of the small intestine was identified. The pastures where the affected horses grazed were overrun by field bindweed (Convolvulus arvensis). Bindweed from the pasture was found to contain the tropane alkaloids tropine, pseudotropine, and tropinone and the pyrrolidine alkaloids cuscohygrine and hygrine. Laboratory mice readily ate C. arvensis and exhibited a variety of abnormal clinical signs depending on the amount eaten. Similar alkaloids have been found in other Convolvulus species and cuscohygrine and calystegines (polyhydroxytropanes) have been previously reported from C. arvensis roots. This is the first report of simple tropane alkaloids in C. arvensis, a world wide problem weed. Pseudotropine, the major alkaloid, is known to affect motility and might represent a causative agent for the observed cases of equine intestinal fibrosis.

The development of sensitive chromatographic and spectroscopic techniques for the isolation and structure determination of natural products has greatly facilitated phytochemical investigations. Chemical investigations of herbarium material have resulted in the isolation of indole, quinoline and isoquinoline alkaloids from a wide number of plants. Examples of novel natural products from higher plants are given and include alkaloids, terpenoids, phenolics and quinones. Some plants investigated have not yielded the types of constituents which would have been predicted from them. Plant tissue cultures provide alternative sources of biologically active compounds and examples investigated include Cinchona, Ailanthus, Brucea and Artemisia for antiprotozoal compounds and Datura for tropane alkaloids. Biological tests are useful for bioassay-guided fractionation of plant extracts and examples of the isolation of a series of natural products with antiprotozoal and cytotoxic activities are given. Chemical and biological investigations into the traditional medicine Dragon's blood (Croton lechleri) from S. America and a Chinese prescription for the treatment of atopic eczema are described. The use of radio-ligand binding assays for the detection of a wide range of biological activities is discussed. Sensitivity of chemical and biological techniques has greatly improved prospects for finding new drug entities from plants and for investigating traditional medicines. Basic phytochemical investigations should continue to be encouraged especially in view of the rapid loss of plant species.

Besides butropine, valtropine, apoatropine and 6β-hydroxyhyoscyamine, a new alkaloid isolated from the leaves of Duboisia myoporoides has been characterized as tropine nonanoate by spectral and chemical methods.

The new piperidone alkaloid hyalbidone [2α,2′β-(1,2-ethanediyl)bis-( N -methyl-3-piperidone)] has been isolated from the hairy roots of Hyoscyamus albus transformed with Agrobacterium rhizogenes strain MAFF 03-01724. The alkaloid was characterized by its chemical and physical data. Together with the piperidone, eight tropane alkaloids were isolated and identified from their NMR data in comparison with those of authentic samples.

All 22 species of the tribe Anthocercideae examined contained tropane alkaloids. Anthocercis is characterized by a wide range of alkaloids. The taxonomically reinstated genus Cyphanthera shows phytochemical similarities to Duboisia in producing tobacco alkaloids (nicotine and anabasine) and tropane esters involving C3 and C4 acids. Anthotroche species all produce hyoscyamine and accumulate its apo- and nor-derivatives. In accordance with the new taxonomic revision of the tribe, the alkaloid spectra of Grammosolen and Crenidium resemble those of Cyphanthera species.

Three new tropanol dimers of mesaconic and itaconic acids and 3α-senecioyloxytropane, together with other known alkaloids, were isolated from Schizanthus grahamii . The structures of the new compounds were established by spectroscopic and chemical transformations including 2-D long-range 13 C- 1 H correlations.

From the leaves of Erythroxylum cuneatum, a new alkaloid (±)-3α,6β-dibenzoyloxytropane was isolated as principal alkaloid; four other benzoyl- and tigloyl-esters were obtained, together with nicotine. Three of these bases were also detected in the stem-bark. Tropacocaine was the principal alkaloid of E. ecarinatum leaves. Erythroxylum australe root-bark contained the new alkaloids 3α-(4-hydroxyphenylacetoxy)tropane and 3-cinnamoyloxytropane-6,7-diol, methylecgonidine as principal base, and other benzoyl- and cinnamoyl-esters.

Fourteen South American species of Erythroxylum representing four sections of the genus were examined for tropane and related alkaloids. The alkaloid content of the dried material ranged from 0.002 to 0.20 %. Commonly, the alkaloids involved were esters of various tropanols with benzoic and phenylacetic acids. A new alkaloid, nortropacocaine, was isolated from E. mamacoca. Mass spectrometry indicated the existence of other new bases, cuscohygrine and, in some species dihydrocuscohygrine. Chemotaxonomic implications are discussed.

Scopine, scopoline, pseudotropine as well as tropine supplied to the culture medium were converted into the corresponding acetates by cell cultures of Datura innoxia. Tropine was esterified preferentially with endogenous acetic acid, even if other organic acids in combination with tropine were supplied exogenously. When D. innoxia cell cultures were fed with tropine and tropic acid in the presence of different kinds of auxins, no alkaloidal metabolises but acetyltropine were detected in each treatment. Apart from Datura, tissue cultures induced from 15 other species belonging to 12 families were incapable of acetylating tropine.

Twelve new tropane alkaloids have been isolated from the leaves of the endemic New Caledonian plant Knightia strobilina Labill. (Proteaceae). The structure of seven of them, elucidated by spectroscopic methods, are described here.

Six new alkaloids characterized from the root-bark of Erythroxylum zambesiacum were: 3α-(3,4,5-trimethoxybenzoyloxy)nortropane, 3α-(3,4,5-trimethoxybenzoyloxy)tropan-6β-ol, 3α-(3,4,5-trimethoxybenzoyloxy)-nortropan-6β-ol, 6β-benzoyloxytropan-3α,-diol, 6β-benzoyloxy-3α-(3,4,5-trimethoxycinnamoyloxy)tropan-7β-ol, and 7β-acetoxy-6β-benzoyloxy-3a-(3,4,5-trimethoxycinnamoyloxy)tropane. Other bases identified included 3α-(3,4,5-trimethoxybenzoyloxy)tropane, 3α-(3,4,5-trimethoxycinnamoyloxy)tropane, 3a-phenylacetoxytropan-6β-ol, 3α-(3,4,5-trimethoxybenzoyloxy)tropan-6β,7β-diol, 6β-benzoyloxytropan-3α-ol, and 6β-benzoyloxy-3α-(3,4,5-trimethoxycinnamoyloxy)tropane; other bases could not be fully characterized. The chemotaxonomic implications of the esterifying acids are discussed.

Datura meteloides plants were fed via the roots with [1″,2′- 14 C]tigloyl hygroline and as a control, [2′- 14 C]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.

Cultured roots were obtained from calluses of Duboisia leichhardtii, D. myoporoides and D. hopwoodii. Cultured roots of all these species produced both tropane and pyridine-type alkaloids. The selected cultured roots of D. leichhardtii showed high contents of tropane alkaloids (hyoscyamine 0.53%, scopolamine 1.16%, on a dry weight basis).

The production of tropoylesters in suspension cultures of Datura innoxia stem callus was significantly stimulated by dl -tropic acid, phenylpyruvate or tropine, but was little affected by l -phenylalanine or l -ornithine. Analyses have shown that acetyltropine is synthesized in large quantities by cultured cells when tropine has been supplied to various cultures of D. innoxia and D. tatula . Acetyltropine has been isolated from either the culture medium or cells supplied with tropine. These results indicate that tropine absorbed by the cultured cells of Datura is esterified predominantly by acetic acid to form acetyltropine, instead of other tropane alkaloids.