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Birgit Dräger - One of the best experts on this subject based on the ideXlab platform.
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Brassicaceae contain norTropane alkaloids.
Phytochemistry, 2006Co-Authors: Andrea Brock, Tobias Herzfeld, Reinhard Paschke, Marcus A. Koch, Birgit DrägerAbstract: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.
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immunolocalisation of two tropinone reductases in potato solanum tuberosum l root stolon and tuber sprouts
Planta, 2006Co-Authors: Heike Kaiser, Ute Richter, Ronald Keiner, Anja Brabant, Bettina Hause, Birgit DrägerAbstract: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.
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tropinone reductases enzymes at the branch point of Tropane alkaloid metabolism
Phytochemistry, 2006Co-Authors: Birgit DrägerAbstract: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.
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overexpression of tropinone reductases alters alkaloid composition in atropa belladonna root cultures
Journal of Experimental Botany, 2005Co-Authors: Ute Richter, Grit Rothe, Annekatrin Fabian, Bettina Rahfeld, Birgit DrägerAbstract: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.
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Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N‐methyltransferase
Journal of Experimental Botany, 2003Co-Authors: Grit Rothe, Yasuyuki Yamada, Takashi Hashimoto, Akira Hachiya, Birgit DrägerAbstract: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.
Xinmiao Liang - One of the best experts on this subject based on the ideXlab platform.
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discovery of new muscarinic acetylcholine receptor antagonists from scopolia tangutica
Scientific Reports, 2017Co-Authors: Nana Du, Jixia Wang, Jian He, Jianqiang Zhao, Han Zhou, Xinmiao Liang, Xiuli ZhangAbstract:Scopolia tangutica (S. tangutica) is a traditional Chinese medicinal plant used for antispasmodics, anesthesia, analgesia and sedation. Its pharmacological activities are mostly associated with the antagonistic activity at muscarinic acetylcholine receptors (mAchRs) of several known alkaloids such as atropine and scopolamine. With our recent identification of four hydroxycinnamic acid amides from S. tangutica, we hypothesized that this plant may contain previously unidentified alkaloids that may also contribute to its in vivo effect. Herein, we used a bioassay-guided multi-dimension separation strategy to discover novel mAchR antagonists from S. tangutica. The core of this approach is to use label-free cell phenotypic assay to first identify active fractions, and then to guide purification of active ligands. Besides four Tropanes and six cinnamic acid amides that have been previously isolated from S. tangutica, we recently identified two new Tropanes, one new cinnamic acid amide, and nine other compounds. Six Tropane compounds purified from S. tangutica for the first time were confirmed to be competitive antagonists of muscarinic receptor 3 (M3), including the two new ones 8 and 12 with IC50 values of 1.97 μM and 4.47 μM, respectively. Furthermore, the cinnamic acid amide 17 displayed 15-fold selectivity for M1 over M3 receptors. These findings will be useful in designing lead compounds for mAchRs and elucidating mechanisms of action of S. tangutica.
Zhihua Liao - One of the best experts on this subject based on the ideXlab platform.
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enhancing Tropane alkaloid production based on the functional identification of tropine forming reductase in scopolia lurida a tibetan medicinal plant
Frontiers in Plant Science, 2017Co-Authors: Kaihui Zhao, Junlan Zeng, Tengfei Zhao, Haoxing Zhang, Chunxian Yang, Lingjiang Zeng, Min Chen, Zhihua LiaoAbstract:Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of Tropane alkaloids. However, the Tropane alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6β-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in Tropane alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of Tropane alkaloids. With the purified recombinant SlTRI from E. coli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the Tropane alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on Tropane alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were c. 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of Tropane alkaloid biosynthesis. It also provides a candidate gene for enhancing Tropane alkaloid biosynthesis in S. lurida via metabolic engineering.
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Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing Tropane alkaloids
Phytochemistry, 2016Co-Authors: Wei Qiang, Junlan Zeng, Chunxian Yang, Min Chen, Xiaoqiang Liu, Ke Xia, Qiaozhuo Zhang, Yuanshe Huang, Xiaozhong Lan, Zhihua LiaoAbstract: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.
Nana Du - One of the best experts on this subject based on the ideXlab platform.
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discovery of new muscarinic acetylcholine receptor antagonists from scopolia tangutica
Scientific Reports, 2017Co-Authors: Nana Du, Jixia Wang, Jian He, Jianqiang Zhao, Han Zhou, Xinmiao Liang, Xiuli ZhangAbstract:Scopolia tangutica (S. tangutica) is a traditional Chinese medicinal plant used for antispasmodics, anesthesia, analgesia and sedation. Its pharmacological activities are mostly associated with the antagonistic activity at muscarinic acetylcholine receptors (mAchRs) of several known alkaloids such as atropine and scopolamine. With our recent identification of four hydroxycinnamic acid amides from S. tangutica, we hypothesized that this plant may contain previously unidentified alkaloids that may also contribute to its in vivo effect. Herein, we used a bioassay-guided multi-dimension separation strategy to discover novel mAchR antagonists from S. tangutica. The core of this approach is to use label-free cell phenotypic assay to first identify active fractions, and then to guide purification of active ligands. Besides four Tropanes and six cinnamic acid amides that have been previously isolated from S. tangutica, we recently identified two new Tropanes, one new cinnamic acid amide, and nine other compounds. Six Tropane compounds purified from S. tangutica for the first time were confirmed to be competitive antagonists of muscarinic receptor 3 (M3), including the two new ones 8 and 12 with IC50 values of 1.97 μM and 4.47 μM, respectively. Furthermore, the cinnamic acid amide 17 displayed 15-fold selectivity for M1 over M3 receptors. These findings will be useful in designing lead compounds for mAchRs and elucidating mechanisms of action of S. tangutica.
Antonia Garrido Frenich - One of the best experts on this subject based on the ideXlab platform.
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Degradation of Tropane alkaloids in baked bread samples contaminated with Solanaceae seeds
Food research international (Ottawa Ont.), 2019Co-Authors: Jesús Marín-sáez, Roberto Romero-gonzález, Antonia Garrido FrenichAbstract:Abstract Solanaceae plant seeds, which contain high concentrations of Tropane alkaloids, have not been studied in real conditions of proofing and baking processes. In this work both lab vial trials and buckwheat and millet flour samples, contaminated with two species of Solanaceae plants, Datura stramonium and Brugmansia arborea, were undergone to proofing (37 °C) and baking (190 °C) processes. For the determination of Tropane alkaloids, a simple solid-liquid extraction with methanol:water 2:1 (v/v) containing 0.5% acetic acid was used to extract the targeted compounds, whereas a chromatographic method employing a Zorbax C18 column coupled to an Exactive-Orbitrap analyser was used for their determination. The results indicate that concentrations of Tropane alkaloids decrease under proofing conditions (degradation between 13 and 95%), while they are almost disappeared under baking conditions (degradation between 94 and 100%). Some degradation pathways have been clarified, showing that most of the compounds degrade into Tropane and tropine, and into tropine and tropinone under proofing and baking conditions respectively.
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Screening of drugs and homeopathic products from Atropa belladonna seed extracts: Tropane alkaloids determination and untargeted analysis.
Drug Testing and Analysis, 2018Co-Authors: Jesús Marín-sáez, Roberto Romero-gonzález, Antonia Garrido Frenich, Francisco J. Egea-gonzálezAbstract: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.
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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, 2018Co-Authors: Ana Romeratorres, Roberto Romerogonzalez, Jose Luis Martinez Vidal, Antonia Garrido FrenichAbstract: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.
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Multi-analysis determination of Tropane alkaloids in cereals and solanaceaes seeds by liquid chromatography coupled to single stage Exactive-Orbitrap.
Journal of Chromatography A, 2017Co-Authors: Jesús Marín-sáez, Roberto Romero-gonzález, Antonia Garrido FrenichAbstract: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.
