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Birgit Dräger - One of the best experts on this subject based on the ideXlab platform.

  • Tropane alkaloid analysis by chromatographic and electrophoretic techniques: an update.
    Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2010
    Co-Authors: Elke Aehle, Birgit Dräger
    Abstract:

    Tropane alkaloids like aTropine are antidotes applied against organophosphorus intoxications. ATropine is toxic itself and should be closely monitored during treatment. Hence, simple, fast, and sensitive determination methods for tropane alkaloids in serum are desirable. Mostly adopted methods of analysis are gas chromatography (GC); high performance liquid chromatography (HPLC), and capillary electrophoresis (CE). Various liquid and solid capillary fillings used in micellar electrokinetic chromatography, microemulsion electrokinetic chromatography, capillary electrochromatography, and enantioseparation provide high versatility to CE applications. In HPLC, specialised columns enhance separation efficacy. Ultraviolet light detection is common practise, but recently sensitivity and analyte identification were enhanced by coupling GC, HPLC, and CE to mass spectrometry. Apart from medical treatment, tropane alkaloids, cocaine in particular, are abused with various intentions. Forensic analysis of tropane alkaloids and their metabolites comprises the additional difficulty of unequivocal drug identification. Because of severe legal consequences, sophisticated analytical methods were developed and may provide additional techniques for therapeutic drug monitoring. Examples from forensic cocaine analysis and from doping analysis are included in this review.

  • immunolocalisation of two tropinone reductases in potato solanum tuberosum l root stolon and tuber sprouts
    Planta, 2006
    Co-Authors: Heike Kaiser, Ute Richter, Ronald Keiner, Anja Brabant, Bettina Hause, Birgit Dräger
    Abstract:

    Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either Tropine for hyoscyamine and scopolamine formation or providing pseudoTropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudoTropine, the other formed Tropine and showed kinetic properties typical for Tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and Tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudoTropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.

  • tropinone reductases enzymes at the branch point of tropane alkaloid metabolism
    Phytochemistry, 2006
    Co-Authors: Birgit Dräger
    Abstract:

    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.

  • overexpression of tropinone reductases alters alkaloid composition in atropa belladonna root cultures
    Journal of Experimental Botany, 2005
    Co-Authors: Ute Richter, Grit Rothe, Bettina Rahfeld, Annekatrin Fabian, Birgit Dräger
    Abstract:

    The medicinally applied tropane alkaloids hyoscyamine and scopolamine are produced in Atropa belladonna L. and in a small number of other Solanaceae. Calystegines are nortropane alkaloids that derive from a branching point in the tropane alkaloid biosynthetic pathway. In A. belladonna root cultures, calystegine molar concentration is 2-fold higher than that of hyoscyamine and scopolamine. In this study, two tropinone reductases forming a branching point in the tropane alkaloid biosynthesis were overexpressed in A. belladonna. Root culture lines with strong overexpression of the transcripts contained more enzyme activity of the respective reductase and enhanced enzyme products, Tropine or pseudoTropine. High pseudoTropine led to an increased accumulation of calystegines in the roots. Strong expression of the Tropine-forming reductase was accompanied by 3-fold more hyoscyamine and 5-fold more scopolamine compared with control roots, and calystegine levels were decreased by 30-90% of control. In some of the transformed root cultures, an increase of total tropane alkaloids was observed. Thus, transformation with cDNA of tropinone reductases successfully altered the ratio of Tropine-derived alkaloids versus pseudoTropine-derived alkaloids.

  • alkaloids in plants and root cultures of atropa belladonna overexpressing putrescine n methyltransferase
    Journal of Experimental Botany, 2003
    Co-Authors: Grit Rothe, Akira Hachiya, Birgit Dräger
    Abstract:

    Putrescine N-methyltransferase (PMT) is the first alkaloid-specific enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, Tropine, pseudoTropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 microM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.

Antonia Garrido Frenich - One of the best experts on this subject based on the ideXlab platform.

  • effect of tea making and boiling processes on the degradation of tropane alkaloids in tea and pasta samples contaminated with solanaceae seeds and coca leaf
    Food Chemistry, 2019
    Co-Authors: Jesus Marinsaez, Roberto Romerogonzalez, Antonia Garrido Frenich
    Abstract:

    Abstract In this study, the degradation of tropane alkaloids in pasta under boiling (100 °C during 10 min) and tea making (100 °C and let cool 5 min) conditions has been evaluated for the first time. Pasta and green tea were contaminated with Datura Stramonium and Brugmansia Arborea seeds (pasta and green tea), whereas coca leaf tea was directly analysed. The compounds were extracted using solid-liquid extraction coupled to a preconcentration stage (only for the cooking water), and the compounds were analysed by liquid chromatography coupled to mass spectrometry (Exactive-Orbitrap analyser). Degradation studies indicate that concentration of tropane alkaloids decreases, and it depends on the compound, observing the highest degradation for tropinone, tropane, cuscohygrine and Tropine, as well as it was observed that compounds migrated to the aqueous phase during cooking step. Finally, post-targeted analysis was performed and other tropane alkaloids were found, as scopine, tigloidine or convolvine, showing a similar behaviour under cooking conditions.

  • Screening of drugs and homeopathic products from Atropa belladonna seed extracts: Tropane alkaloids determination and untargeted analysis.
    Drug Testing and Analysis, 2018
    Co-Authors: Jesús Marín-sáez, Roberto Romero-gonzález, Antonia Garrido Frenich, Francisco J. Egea-gonzález
    Abstract:

    Homeopathic products are still a controversial issue in modern medicine, understood as complementary or alternative medicine (CAM). In this particular case, homeopathic products prepared from Atropa belladonna extracts may present specific problems due to the effects derived from its components. This article applies a simple, rapid, reliable method to the analysis of different homeopathic products obtained from Atropa belladonna; drugs containing high concentration of plant extracts; and Atropa belladonna seeds. The method was based on a simple solid-phase preconcentration method followed by ultra-high pressure liquid chromatography (UHPLC) coupled to high resolution mass spectrometry using Exactive-Orbitrap as an analyser. An in-house database was set and aTropine and scopolamine were the compounds detected at highest concentrations in homeopathic products from Atropa belladonna extracts (4.57 and 2.56 μg/kg, respectively), in Belladonna ointment (4007 and 1139 μg/kg, respectively) and Belladonna seeds (338 and 32.1 mg/kg, respectively). Other tropane alkaloids such as Tropine, apoaTropine, aposcopolamine, tropinone, homaTropine, and anisodamine were detected at lower concentrations (0.04-1.36 μg/kg). When untargeted analysis was performed, other tropane alkaloids were identified in the tested samples, such as ecgonine (0.003 μg/kg), benzoylecgonine (0.56 μg/kg), calystegines A (19.6 μg/kg), B (33.1 μg/kg), and C (1.01 μg/kg). Finally other compounds present in the homeopathic products, such as sugars (fructose, glucose, and lactose) or amino acids (valine, ornithine, leucine, and phenylalanine), were identified.

  • simultaneous analysis of tropane alkaloids in teas and herbal teas by liquid chromatography coupled to high resolution mass spectrometry orbitrap
    IEEE Journal of Solid-state Circuits, 2018
    Co-Authors: Ana Romeratorres, Roberto Romerogonzalez, Jose Luis Martinez Vidal, Antonia Garrido Frenich
    Abstract:

    A new method has been developed for the simultaneous determination of 13 tropane alkaloids in tea and herbal teas using high-performance liquid chromatography coupled to an Exactive-Orbitrap analyzer. A mixture of methanol, water, and formic acid was used for the extraction of the target compounds followed by a solid-phase extraction step. The validated method provided recoveries from 75 to 128% with intra- and interday precision lower than or equal to 24% (except for apoaTropine). Limits of quantification ranged from 5 to 20 μg/kg. Eleven tea and herbal tea samples and two contaminated samples with Datura stramonium seeds were analyzed. Tropane alkaloids were detected in six samples with concentrations from 5 (apoaTropine) to 4340 μg/kg (sum of physoperuvine, pseudoTropine, and Tropine), whereas concentrations from 5 (apoaTropine) to 1725 μg/kg (sum of physoperuvine, pseudoTropine, and Tropine) were found in the contaminated samples.

  • Multi-analysis determination of tropane alkaloids in cereals and solanaceaes seeds by liquid chromatography coupled to single stage Exactive-Orbitrap.
    Journal of Chromatography A, 2017
    Co-Authors: Jesús Marín-sáez, Roberto Romero-gonzález, Antonia Garrido Frenich
    Abstract:

    Tropane alkaloids are a wide group of substances that comprises more than 200 compounds occurring especially in the Solanaceae family. The main aim of this study is the development of a method for the analysis of the principal tropane alkaloids as aTropine, scopolamine, anisodamine, tropane, Tropine, littorine, homaTropine, apoaTropine, aposcopolamine, scopoline, tropinone, physoperuvine, pseudoTropine and cuscohygrine in cereals and related matrices. For that, a simple solid-liquid extraction was optimized and a liquid chromatographic method coupled to a single stage Exactive-Orbitrap was developed. The method was validated obtaining recoveries in the range of 60-109% (except for some compounds in soy), precision values (expressed as relative standard deviation) lower than 20% and detection and quantification limits equal to or lower than 2 and 3μg/kg respectively. Finally, the method was applied to the analysis of different types of samples as buckwheat, linseed, soy and millet, obtaining positives for anisodamine, scopolamine, aTropine, littorine and tropinone in a millet flour sample above the quantification limits, whereas aTropine and scopolamine were detected in a buckwheat sample, below the quantification limit. Contaminated samples with Solanaceaes seeds (Datura Stramonium and Brugmansia Arborea) were also analysed, detecting concentrations up to 693μg/kg (scopolamine) for contaminated samples with Brugmansia seeds and 1847μg/kg (aTropine) when samples were contaminated with Stramonium seeds.

Manel Hassine - One of the best experts on this subject based on the ideXlab platform.

Jan Jirschitzka - One of the best experts on this subject based on the ideXlab platform.

  • plant tropane alkaloid biosynthesis evolved independently in the solanaceae and erythroxylaceae
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Jan Jirschitzka, Gregor W Schmidt, Michael Reichelt, Bernd Schneider, Jonathan Gershenzon, John C Dauria
    Abstract:

    The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as aTropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-tropinone) stereospecifically to 2-carbomethoxy-3β-Tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.

  • Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Jan Jirschitzka, Gregor W Schmidt, Michael Reichelt, Bernd Schneider, Jonathan Gershenzon, John C. D’auria
    Abstract:

    The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as aTropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-tropinone) stereospecifically to 2-carbomethoxy-3β-Tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.

Milton Thomas William Hearn - One of the best experts on this subject based on the ideXlab platform.

  • studies on the oxidative n demethylation of aTropine thebaine and oxycodone using a feiii taml catalyst
    Green Chemistry, 2014
    Co-Authors: Duy Dinh Do Pham, Geoffrey F Kelso, Yuanzhong Yang, Milton Thomas William Hearn
    Abstract:

    The reaction pathway and selectivity of the oxidative N-demethylation of the alkaloid aTropine with H2O2 using a FeIII-TAML catalyst has been investigated. The conversion of aTropine in ethanol with aqueous H2O2 produces noraTropine as the main product and N-formyl-noraTropine and other aTropine derivatives involving carbon-hydroxylated tropane species as minor by-products. Comparative reactivity studies with noraTropine, N-formyl-noraTropine and aTropine N-oxide demonstrated that the FeIII-TAML catalyses the N-demethylation of aTropine by a biomimetic oxidation pathway involving the formation and then decomposition of a N-hydroxymethylnoraTropine intermediate. The reaction selectivity for aTropine N-demethylation versus N-methyl oxidation to N-formyl-noraTropine was found to be sensitive to the structure of the alcohol co-solvent, the rate of H2O2 addition and the concentration of water, whereas temperature mainly affected the aTropine conversion efficiency. The use of tert-butyl or cumene hydroperoxide as oxidants shifted the reaction selectivity toward N-methyl oxidation compared to aqueous H2O2. Various inorganic oxidants were found to be ineffective. The FeIII-TAML also catalysed the N-demethylation of the opiate alkaloids thebaine and oxycodone with aqueous H2O2 in higher conversion efficiencies compared to aTropine but with lower selectivity. These investigations thus document key mechanistic features of the FeIII-TAML-catalysed N-demethylation of these alkaloids and provide insight into how this benign catalytic system could find broader utilisation for N-demethylation in general.

  • one pot oxidative n demethylation of tropane alkaloids with hydrogen peroxide and a feiii taml catalyst
    Green Chemistry, 2012
    Co-Authors: Duy Dinh Do Pham, Geoffrey F Kelso, Yuanzhong Yang, Milton Thomas William Hearn
    Abstract:

    The oxidative N-demethylation of tropane alkaloids to their nortropane derivatives has been investigated using H2O2 and an iron(III) tetraamido macrocycle (FeIII-TAML) catalyst. The yields of the nortropanes were found to be dependent on the amount of H2O2 used in the reaction, the catalyst loading, the nature of the organic co-solvent and the type of Tropine substrate. N-Hydroxy-nortropane, N-formyl-nortropane and tropane-N-oxide derivatives were identified as by-products of the reaction. After screening various reaction conditions, the optimised conditions were applied to the N-demethylation of aTropine and scopolamine at preparative scales and the desired products, noraTropine and norscopolamine, obtained following one pot reactions in good yields and high purity without the need for any chromatographic purification steps.