Search

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6β-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.

Datura inoxia belongs to the family of Solanaceae. This is a very common plant in New Caledonia that contains two main toxic alkaloids, l-atropine and l-scopolamine. In this study, we report the case of a 20-year-old male admitted to an Emergency Unit after consumption of 6 dried flowers in hot water for hallucinations, mydriasis, and agitation associated with tachycardia and increase of systolic blood pressure to 180. Full recovery was observed after one week. Three weeks later, a lock of about 80 hairs (200 mg) was collected from the subject in vertex posterior with scissors to be tested for both atropine and scopolamine. After decontamination with dichloromethane, a strand of hair was segmented into three parts, cut into small segments (< 1 mm), incubated overnight in 1 mL pH 8.4 phosphate buffer in the presence of 2.5 ng atropine-d(3), the internal standard, then extracted with 5 mL dichloromethane/isopropanol/n-heptane (50:17:33). The residue was reconstituted in 100 microL of methanol, from which 10 microL was injected into an XTerra MS C18 column (100 x 2.1 mm, 3.5 microm) eluted with a gradient of acetonitrile and formate buffer delivered at a flow rate of 0.2 mL/min. A Quattro Micro triple-quadrupole mass spectrometer (MS) was used for analyses. Ionization was achieved using electrospray in the positive ionization mode. For each compound, detection was related to two daughter ions (atropine: m/z 290.2 to 124.0 and 92.9; atropine-d(3): m/z 293.1 to 127.0 and 92.9; scopolamine: m/z 304.1 to 138.0 and 156.0). Although atropine was never detected (limit of detection = 2 pg/mg), scopolamine was identified in the three segments, in the range 14 to 48 pg/mg. The absence of atropine in hair is consistent with its very low dosage in the flower of Datura inoxia. Hair segmentation indicated that the subject was previously exposed on several occasions to the plant. Liquid chromatography-tandem MS appears to be a necessity for testing tropane alkaloids of the Datura group, given the low concentrations to be measured.

De-differentiation of transformed root cultures of Datura stramonium has previously been shown to cause a loss of tropane alkaloid synthetic capacity. This indicates a marked shift in physiological status, notably in the flux of primary metabolites into tropane alkaloids. Nitrogen metabolism in transformed root cultures of D. stramonium (an alkaloid-producing system) and de-differentiated suspension cultures derived therefrom (a non-producing system) has been compared using Nuclear Magnetic Resonance (NMR) spectroscopy. (15)N-Labelled precursors [((15)NH(4))(2)SO(4) and K(15)NO(3)] were fed and their incorporation into nitrogenous metabolites studied using Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy. In both cultures, the same amino acids were resolved in the HMBC spectra. However, marked differences were found in the intensity of labelling of a range of nitrogenous compounds. In differentiated root cultures, cross-peaks corresponding to secondary metabolites, such as tropine, were observed, whereas these were absent in the de-differentiated cultures. By contrast, N- acetylputrescine and gamma-aminobutyric acid (GABA) accumulated in the de-differentiated cultures to a much larger extent than in the root cultures. It can therefore be suggested that the loss of alkaloid biosynthesis was compensated by the diversion of putrescine metabolism away from the tropane pathway and toward the synthesis of GABA via N-acetylputrescine.

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.

Duboisia myoporoides R. Br. shoots were regenerated from non-organogenic and organogenic calli induced with nine different cytokinin/auxin combinations. Alkaloid colour reagents localized tropane alkaloids in the vascular regions which had large cells in the secondary xylem of the basal stem sections of the non-rooted shoots. Tropane alkaloids were localized in shoots regenerated from calli induced with two different cytokinin/auxin combinations. No alkaloids were localized in shoots regenerated from calli induced with other cytokinin/auxin combinations. However, only nicotine was detected by GC-MS in the non-rooted shoots regenerated from calli induced with three different cytokinin/auxin combinations. Tropane alkaloids were also localized in xylem cells of roots regenerated from calli induced with two different cytokinin and auxin combinations independently. The presence or absence of nicotine, hyoscyamine and scopolamine in different cultured plant materials was confirmed by GC-MS, indicating that although the root is the main site for alkaloid biosynthesis, with suitable cell differentiation, alkaloid biosynthesis may take place in cultured shoots without root initiation.

In the plant species that produce tropane alkaloids, two tropinone reductases (TRs) catalyze the stereospecific reductions of the 3-carbonyl group of tropinone. This reduction is a key branch point that determines the metabolite flow into the separate alkaloid groups, each with different stereospecific configurations. In this study, a specific antibody was prepared for each of the TRs by immunizing mice with recombinant TR protein and subsequent immuno-affinity purification of the antiserum. Immunoblot analyses revealed that accumulation of both TRs was highest in the lateral roots of Hyoscyamus niger throughout its development. In cultured roots, TR proteins were accumulated in a basal region but not in root apex. These patterns were similar to that of hyoscyamine 6 beta-hydroxylase (H6H), an enzyme that catalyzes a downstream step in the same biosynthetic pathway. However, an immunohistochemical analysis revealed that the two TRs and H6H were accumulated with different cell-specific patterns in the cultured root, suggesting transportation of the alkaloid intermediate(s) across the different cell layers.

The cDNAs encoding putrescine N-methyltransferase (PMT), which catalyzes the S-adenosylmethionine-dependent N-methylation of putrescine at the first committed step in the biosynthetic pathways of tropane alkaloids, were isolated from Atropa belladonna and Hyoscyamus niger. These PMTs, however, lacked the N-terminal tandem repeat arrays previously found in Nicotiana PMTs. AbPMT1 RNA was much more abundant in the root of A. belladonna than was AbPMT2 RNA. The 5'-flanking region of the AbPMT1 gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to A. belladonna. Histochemical analysis showed that GUS is expressed specifically in root pericycle cells and that the 0.3-kb 5'-upstream region was sufficient for pericycle-specific expression. Treatment of A. belladonna roots with methyl jasmonate did not up-regulate the expression of GUS or endogenous AbPMT genes. The regulation of tropane alkaloid biosynthesis is discussed and compared with that of nicotine biosynthesis.

A tropane ester, three tropane ethers, atropine and mecamylamine were compared in mice for their anti-muscarinic and anti-nicotinic activity against arecoline-induced tremor and nicotine-induced convulsions, respectively. Their receptor-binding characteristics were studied in neuronal membranes prepared from rat cerebral cortex. The results showed that the tropane ester, 2 alpha R-tropanyl benzylate, was more potent than atropine in its anti-muscarinic activity, but the anti-muscarinic activity of the tropane ethers, 2 alpha-(2',2'-diphenyl-2'-hydroxy-ethoxy)tropane (alpha-DPT) and its two isomers (1R,2 alpha R- and 1S,2 alpha S-) were less potent than that of atropine. In contrast with their anti-muscarinic potency, 2 alpha R-tropanyl benzylate and the three tropane ethers were equipotent in their anti-nicotinic activities. The order of potencies of these compounds to displace the binding of [3H]quinuclidinyl benzylate to brain membranes was similar to that of their anti-muscarinic potencies. The binding of [3H]nicotine to nicotinic receptors from brain was not inhibited by these compounds. Analyses of structure-activity relationships of these compounds suggested that it is the ester groups that determine the anti-muscarinic potencies of 2 alpha R-tropanyl benzylate; their anti-nicotinic activities were independent of the structural changes and of the anti-muscarinic activities of these compounds.

Label from U- 14 C-arginine (Arg), -ornithine (Orn) and -phenylalanine (Phe) was incorporated into hyoscyamine and scopolamine by both dissected roots of intact plants and homogeneous or aggregating suspension cultures of Atropa belladonna L. The early biosynthetic compounds (hygrine, tropinone, tropanol) were found only in the roots, and alkaloid synthesis proceeded as far as to scopolamine there. In the synthesis of the tropane skeleton, Orn was used more efficiently than Arg. Phe was quickly metabolized both in roots and suspension cultures, and the label was incorporated into both ethanol-insoluble compounds, particularly proteins and different ethanol-soluble compounds, especially phenolics

Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids. Two stereospecifically distinct NADPH-dependent oxidoreductases, TR-I and TR-II, which, respectively, reduce tropinone to 3alpha-hydroxytropane (tropine) and 3beta-hydroxytropane (psi-tropine), were detected mainly in the root of tropane alkaloid-producing plants but not in nonproducing cultured root. Both reductases were purified to near homogeneity from cultured root of Hyoscyamus niger and characterized. The TR-I reaction was reversible, whereas the TR-II reaction was essentially irreversible, reduction of the ketone being highly favored over oxidation of the alcohol psi-tropine. Marked differences were found between the two reductase in their affinities for tropinone substrate and in the effects of amino acid modification reagents. Some differences in substrate specificity were apparent. For example, N-propyl-4-piperidone was reduced by TR-II but not by TR-I. Conversely, 3-quinuclidinone and 8-thiabicyclo[3,2,1]octane-3-one were accepted as substrates by TR-I but hardly at all by TR-II. Both enzymes were shown to be class B oxidoreductases, which transfer the pro-S hydrogen of NAD(P)H to their substrates. Possible roles of these tropinone reductases in alkaloid biosynthesis are discussed.

Normal and androgenic diploid Datura innoxia plants were selfed and the progeny was analysed for its leaf alkaloid content. Since the androgenic lines had originally produced very different amounts of the tropane alkaloids, scopolamine and hyoscyamine, we were interested in determining whether this trait is transmitted by self-fertilization. The alkaloid content of the progeny was found to correlate well with that of the parental plants. Also, calli were initiated from leaf discs derived from plants with different capacities for alkaloid biosynthesis. These were further subcultured for 2 years. Again, the same correlations in hyoscyamine and scopolamine content were observed. This indicates that it is possible to initiate callus with a high alkaloid content starting from actively alkaloid-producing androgenic Datura innoxia plants.

The use of silver halide impregnated supports for high-pressure liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analysis of drug substances has been studied. Successful separations of xanthines and mixtures containing barbiturates, xanthines, ergot alkaloids, and tropane alkaloids have been achieved with isocratic conditions or by using simple gradients. The Ag-supports show a similar behavior as many chemically bonded stationary phases, such as modification fo adsorption sites on silica gel to give lower retention but better specificity; rapid reconditioning in connection with gradient elution (3-5 minutes). Reasonable cost, simplicity of preparation and usage, and good chemical and physical stability render the silver halide phases applicable in routine analysis and quite competitive with many commercially available chemically bonded stationary phases.

Hairy root clones of Scopolia japonica were established by selection of adventitious roots formed on the root segments inoculated with Agrobacterium rhizogenes strain 15834. Twenty-nine isolated hairy root clones displayed various phenotypes characterized by growth rate, opine production and tropane alkaloid production. Of these, two highly alkaloid productive clones SI and S22 were examined for their growth rate and alkaloid productivity under various cultural conditions. When the most scopolamine-productive clone SI was cultured for 4 weeks at 25°C in the dark, the weight of the root tissue was increased by 40 times and the content of scopolamine reached a level of 0.5% on a dry weight basis in each optimum medium. On culture of the most hyoscyamine-productive clone S22 under the same conditions as with S1, the weight was increased by 102 times and the content of hyoscyamine was 1.3% on a dry weight basis in each optimum medium.

A pseudotropine-forming tropinone reductase was extracted from root cultures of Hyoscyamus niger that produce the tropane alkaloids hyoscyamine and scopolamine. The enzyme stereospecifically reduces tropinone to pseudotropine, oxidizing NADPH. It has an approximate molecular weight of 84, 000 and a pH optimum between 5.8 and 6.25. The Km value for tropinone is 35.1 μmol/l and for NADPH 21.1 μmol/l. Substrate specificity was tested for NADPH and several tropinone analogues.

The 125I-radioimmunoassay (RIA) for benzylecgonine (cocaine metabolite) in urine was evaluated by comparison with gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) and the Enzyme-Multiplied Immunoassay Technique (EMIT). By RIA, a statistically significant concentration, 2 microng/liter, was observed for urinary benzoylecgonine. The coefficient of variation for the RIA was 2.58+/-0.38% inter-assay and 2.20+/-0.14% intraassay. There was cross-reactivity with cocaine (more reactive than benzoylecgonine) and other members of the tropane family of alkaloids. There was agreement between results by RIA and GLC in 95.5% of the samples, between RIA and TLC in 87.0%, and between RIA and EMIT in 84.5%. The percentage of true false-positives was 3.5% for the RIA in comparison to GLC, 8.8% in comparison to TLC, and 9.1% in comparison to EMIT. True false-negatives were insignificant (0 to 1.0%). GLC and RIA results correlated highly (phi=0.908). GLC, therefore, was the best comparison method for this evaluation study. RIA for benzoylecgonine is sensitive, reproducible, and reliable for the detection of cocaine in urine.

Our aim was to determine whether the precursor amino acids of the tropane skeleton affect nitrate reductase activity in Atropa belladonna and whether the simultaneous supply of NH 4 + ions leads to its repression. The interrelationship between nitrate reductase activity, growth and utilization of NO 3 − and NH 4 + were also studied

A reverse phase high pressure liquid chromatographic (HPLC) system utilizing ion-pairing, similar to that previously reported for methscopolamine bromide, was developed for the quantitation of atropine sulfate and scopolamine hydrobromide in tablets. This HPLC system is capable of differentiating the major tropane alkaloids, with the exception of the atropine- hyoscyamine optical isomer pair, by their k' values and separating a significant degradation product, tropic acid. Optimum sensitivity is obtained by using a variable wavelength ultraviolet detector at 230 nm resulting in a detection limit of < 0.5 μg alkaloid/injection. The response is linear in the range of 3–19 ---g atropine sulfate and scopolamine hydrobromide. The coefficient of variation for injection reproducibility is < 1 %. The method has been applied to composite and individual tablet assays of scopolamine hydrobromide and atropine sulfate. The HPLC results are comparable (within 1%) to those obtained using the USP procedure; the HPLC method requires less sample preparation.

The development of a second order derivative spectrophotometric assay of atropine, hyoscine and benztropine in formulations is described. The assay involves the extraction into 1,2-dichloroethane of the tetraphenylborate salt of the alkaloids precipitated from pH 6 solution by an excess of sodium tetraphenylboron and measurement of the amplitude near 273 nm to its shorter wavelength satellite in the second derivative spectrum. The second derivative amplitude gives a more precise determination of the alkaloids than absorbance. A two-point bracketting standardization of the amplitude is required owing to a small but significant intercept in the calibration data. The procedures are sufficiently sensitive and precise for the batch and unit-dose assay of tablets of atropine sulphate (0·6 mg) hyoscine hydrobromide (0·3 mg) and benztropine mesylate (2 mg), and for the assay of atropine sulphate injection (0·4 mg ml−1) and tincture of belladonna.

Summary The production of biosynthetic radioactive hyoscine and meteloidine in Datura ferox after injection of glycine-2-14C is described. The major part of the radioactive carbon is concentrated in the tropane residues.

The aerial parts of Scopolia lurida contain large quantities of tropane alkaloids and are a possible source of hyoscyamine and hyoscine. The leaves and stems are very similar to those of Atropa belladonna but they can be distinguished by certain microscopical characters; these include the presence of branched covering trichomes on the leaves and stems of Scopolia lurida and the absence of idioblasts containing microsphenoidal crystals of calcium oxalate from the leaf lamina.

Feeding and host-selection behavior of Lema trilineata daturaphila Kogan & Goeden were analyzed, with emphasis on the phases of host finding, recognition, acceptance, and suitability. Three sets of bioassays were developed to monitor orientation, biting, and continuous feeding activities. Whole-leaf powders of Datura meteloides de Candolle were fractionated and incorporated into a basic artificial medium. Complete larval development was achieved on an artificial medium containing whole-leaf powder. Bioassays compared larval responses obtained with different plant fractions incorporated into artificial media to those obtained with fresh leaf material. Artificial media containing certain tropane alkaloids and chlorogenic acid were used to test the role of these secondary metabolites found in host plants of L. t. daturaphila . A working hypothesis was formulated to explain host selection by insects associated with the Solanaceae based on a comparison of the host ranges of L. t. daturaphila , the Colorado potato beetle, Leptinotarsa decemlineata (Say), and the tobacco hornworm, Manduca sexta (Johannson), and on their reported tolerances to specific alkaloids of the Solanaceae. It is proposed that host selection is governed by feeding excitants that have a widespread occurrence among plants of this family. However, acceptance of certain plant species is limited by the distribution of particular alkaloids, namely tropane alkaloids that prevail in host plants of L. t. daturaphila , certain steroidal alkaloids occurring in preferred hosts of the Colorado potato beetle, and nicotine which is found in some hosts of the tobacco hornworm.

The results given by the hydrolytic and non-hydrolytic methods of determining the total alkaloidal content are in good agreement for belladonna, hyoscyamus and stramonium; the agreement is fair for Duboisia leichhardtii and Datura sanguinea, whilst for Duboisia myoporoides the non-hydrolytic method gives high values. An improved paper chromatographic system for the separation of the principal tropane alkaloids has been developed; the system was used to determine the individual alkaloidal content of the respective drugs and to provide the appropriate factor for converting titre to percentage of total alkaloid.