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).     

Tropane alkaloids of Datura innoxia from Morocco

Fifty three alkaloids were identified in the organs (roots, stems, leaves, flowers, and seeds) of Datura innoxia by GC/MS. Seventeen of them are reported for the first time for this species and one nor-derivative, 3-phenylacetoxynortropane (28), for the genus Datura. Furthermore, four new tropane esters were tentatively identified as 3-acetoxy-6,7-epoxytropane (acetylscopine) (10), 3-acetoxy-6-propionyloxy-7-hydroxytropane (15), 6,7-dehydro-3-phenylacetoxytropane (25), and 3-(2'-phenylpropionyloxy)-6,7-epoxynortropane (dihydroaponorscopolamine) (37) on the basis of their mass spectral data. Hyoscyamine (44) and scopolamine (48) figure as main alkaloids in the roots and aerial parts, respectively.     

Alkaloids of Erythroxylum zambesiacum root-bark

Six new alkaloids characterized from the root-bark ofErythroxylum 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.     

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.     

In vitro organogenesis and alkaloid accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.     

Isolation and identification of littorine from hairy roots of Atropa belladonna

A hairy root clone (M8) of Atropa belladonna, producing high levels of tropane alkaloids, was established by transformation with Agrobacterium rhizogenes (MAFF 03-01724). Littorine, an intermediate of tropane alkaloids, was detected by high-performance liquid chromatography and gas chromatography-mass spectrometry in the alkaloid fraction of the hairy roots and identified by nuclear magnetic resonance analysis. Littorine was also detected in the non-transformed root culture of A. belladonna. Received: 18 March 1998 / Revision received: 15 June 1998 / Accepted: 3 August 1998     

Distribution of alkaloids in Anthocercis littorea and A. viscosa

The distribution of tropane alkaloids in organs of Anthocercis littorea and A. viscosa is reported. The following alkaloids have been isolated: atropine (hyoscyamine), apoatropine, noratropine (norhyoscyamine), littorine, hyoscine, norhyoscine, meteloidine, 3α, 6β-ditigloyloxytropan-7β-ol, 6β-tigloyloxytropan-3α-ol, 3α-tigloyloxytropane, tigloidine, tropine, ψ-tropine, (?)-tropan-3α-6β-diol, cuscohygrine and unknown bases.     

Tropane alkaloids from Erythroxylum caatingae Plowman

Three tropane alkaloids, 1-3, were isolated from Erythroxylum caatingae, i.e., 6β-benzoyloxy-3α-[(4-hydroxy-3,5-dimethoxybenzoyl)oxy]tropane (1), a new tropane alkaloid, along with the known alkaloids 3α,6β-dibenzoyloxytropane (2) and 6β-benzoyloxy-3α-[(3,4,5-trimethoxybenzoyl)oxy]tropane (catuabine B; 3). Their structures were determined by 2D- ((1) H and (13) C) NMR. By LC/ESI-MS/MS analysis of the fractions of alkaloids 1-3, it was possible to detect five more alkaloids, 4-8, two of these, 4 and 8, possibly being new natural products. X-Ray crystallography of the chloride derivate of 1, i.e., 6β-benzoyloxy-3α-(4-hydroxy-3,5-dimethoxybenzoyloxy)tropane hydrochloride (1a) confirmed the structure of 1. Cytotoxicity was tested against the cell lines HEp-2, NCI-H292, and KB for the MeOH extract and alkaloid 3, and antitumor activity was tested against Sarcoma 180 only for the MeOH extract.     

Brassicaceae contain nortropane alkaloids

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.     

Induction of tropane alkaloid formation in transformed root cultures of Brugmansia suaveolens (Solanaceae)

Hairy root cultures of Brugmansia suaveolens were set up by infection of root tips with Agrobacterium rhizogenes. The successful transformation was confirmed by analysing rolC and virC genes using polymerase chain reaction (PCR). Hairy root cultures were employed to study the formation of tropane alkaloids, such as hyoscyamine. The transformed cultures were incubated with potential elicitors, such as methyljasmonate, quercetin and salicylic acid in order to stimulate the biosynthesis of tropane alkaloids. Profile and amounts of tropane alkaloids were analysed using capillary GLC-MS. At least 18 different tropane alkaloids could be identified. Treatment of the cultures with 200 microM methyljasmonate increased the alkaloid accumulation 25-fold up to a level of 1 mg/g fresh weight as compared to untreated controls. Quercetin enhanced the alkaloid production 10 fold (0.4 mg/g fresh weight) within 24 h. In contrast 100 microM salicylic acid decreased alkaloids to a level of 1 microg/g fresh weight.     

Tropane alkaloids from Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae)

Six tropane alkaloids were isolated from the Sri Lankan endemic plant Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae) and structurally elucidated by NMR and MS measurements. Three of them, erythrozeylanines A [1R,3R,5S,6R-6-acetoxy-3-(3',4',5'-trimethoxybenzoyloxy)tropane], B [cis-3 beta-(cinnamoyloxy)tropane], and C [cis-6 beta-acetoxy-3 alpha-(cinnamoyloxy)tropane] are new, whereas the others have already been found in other Erythroxylum species. For the first time, the absolute configuration of a tropane alkaloid (erythrozeylanine A) has been determined by quantum chemical CD calculations.     

Effect of tropane on the cholinoreceptors of the cerebral cortex

Spontaneous electric activity of single neurons of the sensorimotor cortex was recorded extracellularly in experiments on unanesthetized rabbits. During microiontophoretic application of tropane and acetylcholine to the neurons, the response to both the agents was the same. The cells excitable by acetylcholine are also excitable by tropane, while those inhibited by acetylcholine are also inhibited by tropane. The cells that do not respond to acetylcholine are also irresponsive to tropane. The excitatory response pattern to tropane is similar to that of acetylcholine. Under the same conditions of microiontophoretic application, tropane causes less excitation as compared with acetylcholine. Tropane preliminarily applied to the neuron reduces the excitatory effect of acetylcholine. The possible role of agonist-antagonist relations between tropane and acetylcholine in the mechanism of the pharmacological effects of tropane and its derivatives is discussed.     

Synthesis of tropane and nortropane analogues with phenyl substitutions as serotonin transporter ligands.

The effects of structural modifications of 2 beta-carbomethoxy-3 beta-phenyl tropane analogues were evaluated on in vitro affinity to the dopamine (DAT) and serotonin (5-HTT) transporters in rat brain tissue. The introduction of a large alkyl group at the 4'-position of the phenyl ring, affording 2 beta-carbomethoxy-3 beta-(4'-alkylphenyl) tropane, diminished the affinity for the DAT whereas moderate 5-HTT affinity was obtained. The introduction of an iodine at the 3'-position of the 4'-alkylphenyl, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) tropane, and N-demethylation, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) nortropane, improved affinity and specificity for the 5-HTT. It could be assumed from these results that the combination of these three modifications of tropane structure yielded highly selective compounds for the 5-HTT. Of the new compounds synthesized, the most selective cocaine derivative, 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-isopropylphenyl) nortropane (8d) labeled with iodine-123 or carbon-11, could be a potential ligand for exploration of the 5-HT transporter by SPET or PET.     

Alkaloid spectrum in diploid and tetraploid hairy root cultures of Datura stramonium

Hairy root cultures were obtained from diploid and induced tetraploid plants of Datura stramonium and analyzed by gas chromatography/mass spectrometry. Twenty alkaloids (19 for diploid and 9 for tetraploid hairy root cultures) were identified. A new tropane ester 3-tigloyloxy-6-propionyloxy-7-hydroxytropane was identified on the basis of mass spectral data. Hyoscyamine was the main alkaloid in both diploid and tetraploid cultures. In contrast to diploid hairy roots, the percentage contributions of the alkaloids, with exceptions for hyoscyamine and apoatropine, were higher in the total alkaloid mixture of tetraploid hairy roots.     

Some morphological and biochemical peculiarities of Datura innoxia callus- and regenerated cultures

Five nutrient media were tested for expression of morphological pathway in Datura innoxia callus cultures. Regenerants-rooted plants and non-rooted shoots obtained from three subsequent subcultures of callus cultures were assayed for tropane alkaloid content and composition. Although no wide variation of bio-chemical traits of regenerants was observed. For a given number of plants decrease of hyoscyamine in their roots was detected. Alkaloid content and composition of upper parts of plants having roots and shoots or lacking roots was studied-receiving an idea about biosynthesis of these organs.     

Tropane alkaloid production by Agrobacterium rhizogenes transformed hairy root cultures of Atropa baetica Willk. (Solanaceae)

Atropa baetica hairy root cultures were induced after infecting stem segments with Agrobacterium rhizogenes strain ATCC 15834. Accumulation of the tropane alkaloids atropine and scopolamine by hairy roots cultured in half- and full-strength Murashige and Skoog (MS) medium was high, although this was not growth associated. These alkaloids were also released into both liquid media. Higher tropane alkaloids present both in hairy roots and liquid medium occurred in half MS medium, showing a clear relationship between slow growth of cultures and higher product accumulation. The pH of both nutrient media varied as culture progressed, and seemed to be associated with the release of scopolamine. GC-MS analyses showed the presence of a new compound, namely tigloylpseudotropine; moreover, 3α-isobutyryloxytropane, formerly found only in plant leaf tissue, was also identified in the hairy roots. Received: 18 August 1997 / Revision received: 30 November 1997 / Accepted: 20 January 1998     

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.