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Strahil Berkov - One of the best experts on this subject based on the ideXlab platform.
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nutrient medium optimization for hyoscyamine production in diploid and tetraploid Datura stramonium l hairy root cultures
World Journal of Microbiology & Biotechnology, 2009Co-Authors: Atanas Pavlov, Vasil Georgiev, Andrey S Marchev, Strahil BerkovAbstract:The common effect of NO3 −, KH2PO4 and sucrose on the biosynthesis of hyoscyamine by diploid and tetraploid Datura stramonium L. hairy root cultures were studied. Polynominal regression models were elaborated for the description of hyoscyamine biosynthesis as a consequence of variation of the investigated variables (NO3 − between 31 and 47 mM, and sucrose between 20.00 and 40.00 g/l for diploid and NO3 − between 31 and 47 mM; KH2PO4 between 0.50 and 1.25 mM, and sucrose between 50 and 70 g/l, for tetraploid hairy roots, respectively). Optimization procedures allowed us to establish the optimal concentrations of the investigated variables and to propose the modified MS nutrient media, with 1.10 g/l KNO3, 0.17 g/l KH2PO4, and 40 g/l sucrose, for diploid and with 1.10 g/l KNO3, 0.17 g/l KH2PO4, and 50 g/l sucrose, for tetraploid Datura stramonium L. hairy root cultures. The proposed modified MS media provided optimal hyoscyamine production yield for both investigated hairy root cultures.
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hyoscyamine biosynthesis in Datura stramonium hairy root in vitro systems with different ploidy levels
Applied Biochemistry and Biotechnology, 2009Co-Authors: Atanas Pavlov, Strahil Berkov, Jost Weber, Thomas BleyAbstract:Hyoscyamine biosynthesis in Datura stramonium hairy roots with different ploidy levels was investigated. For the first time, we report that hairy roots undergo endoreduplication and therefore consist mainly of cells with doupled sets of chromosomes of primary plant tissues, used for Agrobacterium transformation. The alkaloid profiles of hairy roots obtained from diploid and tetraploid plants were similar in terms of the major compounds, but they differed significantly with respect to the minor compounds (here defined as those that accounted for <1% of the total ion current of the alkaloid mixture in gas chromatography-mass spectrometric analyses). Significant differences in the effects of the main nutrients on the growth of the hairy roots obtained from diploid and tetraploid plants and their hyoscyamine contents were observed. The maximal yield of hyoscyamine (177 mg/L) was obtained when hairy roots from tetraploid plants were cultivated in Murashige-Skoog nutrient medium supplemented with 6% sucrose. Time courses of utilization of the main nutrients in the medium during cultivation of D. stramonium hairy root cultures are also presented.
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alkaloid patterns in some varieties of Datura stramonium
Fitoterapia, 2006Co-Authors: Strahil Berkov, Rawia Zayed, Tsvetelina DonchevaAbstract:A comparative GC-MS investigation of the alkaloid patterns of three varieties of Datura stramonium vars. stramonium, tatula and godronii, was carried out. Twenty-five tropane alkaloids were identified in the plant organs. Alkaloid patterns of the roots, leaves and seeds of the varieties grown at equal conditions in Bulgaria were very similar. In contrast, alkaloid pattern of D. stramonium var. stramonium, grown in Egypt, showed significant differences indicating that it is influenced more strongly by the environmental factors than genetic ones.
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ontogenetic variation of the tropane alkaloids in Datura stramonium
Biochemical Systematics and Ecology, 2005Co-Authors: Strahil Berkov, Tsvetelina Doncheva, Stefan Philipov, Kiril AlexandrovAbstract:Abstract 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).
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alkaloid spectrum in diploid and tetraploid hairy root cultures of Datura stramonium
Zeitschrift für Naturforschung C, 2003Co-Authors: Strahil Berkov, Atanas Pavlov, Petia Kovatcheva, Pepa Stanimirova, Stefan PhilipovAbstract:: 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.
David Ohagan - One of the best experts on this subject based on the ideXlab platform.
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elicitation of tropane alkaloid biosynthesis in transformed root cultures of Datura stramonium
Phytochemistry, 1999Co-Authors: Ioannis Zabetakis, Robert Edwards, David OhaganAbstract:Abstract 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.
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fluorinated tropane alkaloids generated by directed biosynthesis in transformed root cultures of Datura stramonium
Journal of The Chemical Society-perkin Transactions 1, 1999Co-Authors: David Ohagan, Richard J Robins, Marina Wilson, Chi W Wong, Marc Berry, Ioannis ZabetakisAbstract:2′-, 3′- and 4′-Fluorophenyl-(RS)-lactic acids were administered to transformed root cultures of Datura stramonium to determine their abilities as substrates for incorporation into the phenyllactoyl and tropoyl ester moieties of the tropane alkaloids, littorine and hyoscyamine respectively. In the event, all of the fluorinated phenyllactates generated the corresponding fluorinated littorine and hyoscyamine analogues. The efficiency of conversion for the isomerisation of the fluorinated littorines (F-lit) to fluorinated hyoscyamines was 3′-F-lit > 4′-F-lit 2′-F-lit.
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tropic acid ester biosynthesis in Datura stramonium and related species
Chemical Society Reviews, 1998Co-Authors: David OhaganAbstract:The origin of the tropic acid ester moiety, found in some of the tropane alkaloids, but particularly in hyoscyamine and scopolamine, has been a subject of discussion and investigation in biosynthesis for many years. Recently it has been shown in Datura stramonium root cultures that hyoscyamine arises by isomerisation of the tropane alkaloid littorine. The mechanism of this isomerisation process is not obvious and in this review we present our recent results and current thinking on this process.
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tropic acid biosynthesis the incorporation of rs phenyl 2 18o 2 2h lactate into littorine and hyoscyamine in Datura stramonium
Chemical Communications, 1998Co-Authors: Chi W Wong, David Ohagan, John T G HamiltonAbstract:The incorporation of oxygen-18 from (RS)-phenyl- [2-18O,2-2H]lactate into the tropane alkaloids littorine 1 and hyoscyamine 2 in Datura stramonium reveals that up to 29% of the oxygen-18 is lost during the transformation of 1 to 2.
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the biosynthesis of tropic acid the r d phenyllactyl moiety is processed by the mutase involved in hyoscyamine biosynthesis in Datura stramonium
Journal of The Chemical Society Chemical Communications, 1995Co-Authors: Nicola C J E Chesters, David OhaganAbstract:Incubations of (R)-D-phenyl[2-13C, 2H]lactic acid 5a and (S)-L-phenyl[2-13C, 2H]lactic acid 5b with transformed root cultures of Datura stramonium have revealed that the 13C–2H bond is retained, intact at C-3 of the tropate moiety of hyoscyamine, only in the case of the (R)-D enantiomer 5a( the deuterium is lost from the (S)-L-enantiomer 5b); therefore, it is the (R)-D-phenyllactate moiety of littorine which is the enantiomer processed by the mutase in hyoscyamine biosynthesis.
Richard J Robins - One of the best experts on this subject based on the ideXlab platform.
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use of heteronuclear multiple bond coherence nmr spectroscopy to monitor nitrogen metabolism in a transformed root culture of Datura stramonium
Congrès du GERM, 2001Co-Authors: Ophelie Fliniaux, Richard J Robins, Francois Mesnard, Sophie Raynaud, Sylvie Baltora, Marcandre FliniauxAbstract:le metabolisme azote dans des cultures de racines transformees de Datura stramonium. Dans le contexte d'une etude visant a definir les facteurs qui regulent les flux des metabolites primaires vers les alcaloides tropaniques, la resonance magnetique nucleaire (RMN) est utilisee pour suivre le metabolisme azote de racines transformees de Datura stramonium cultive en presence de ( 15 NH 4 ) 2 SO 4 et K 15 NO 3 . Cette etude est realisee en utilisant la sequence RMN HMBC. Cette technique, associant la RMN a deux dimensions et la detection inverse avec gradients, presente a la fois les avantages de la resolution bidimensionnelle et ceux de la sensibilite de la RMN du proton. De plus, la sequence HMBC permet d'observer par l'intermediaire de couplages 2 J et/ou 3 J les noyaux 15 N lies a des protons labiles, ce qui est approprie pour identifier des intermediaires de la voie de biosynthese des alcaloides tropaniques, particulierement les precurseurs du noyau tropane. Ainsi, dans des racines transformees de Datura stramonium, outre certains acides amines couramment observes en RMN monodimensionnelle de l'azote 15, d'autres acides amines sont aussi identifies par HMBC. Un pic correspondant a des composes N-acetyles peut egalement etre remarque. Le marquage d'une base pyrimidique, l'uridine, est aussi obtenu. Alors que ces metabolites primaires ont deja ete observes dans des cultures cellulaires de Nicotiana plumbaginifolia, des pics de correlation correspondant a des metabolites secondaires, tels que la tropine, sont egalement identifies. C'est la premiere fois que ce type de metabolites secondaires est mis en evidence par cette technique.
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fluorinated tropane alkaloids generated by directed biosynthesis in transformed root cultures of Datura stramonium
Journal of The Chemical Society-perkin Transactions 1, 1999Co-Authors: David Ohagan, Richard J Robins, Marina Wilson, Chi W Wong, Marc Berry, Ioannis ZabetakisAbstract:2′-, 3′- and 4′-Fluorophenyl-(RS)-lactic acids were administered to transformed root cultures of Datura stramonium to determine their abilities as substrates for incorporation into the phenyllactoyl and tropoyl ester moieties of the tropane alkaloids, littorine and hyoscyamine respectively. In the event, all of the fluorinated phenyllactates generated the corresponding fluorinated littorine and hyoscyamine analogues. The efficiency of conversion for the isomerisation of the fluorinated littorines (F-lit) to fluorinated hyoscyamines was 3′-F-lit > 4′-F-lit 2′-F-lit.
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in vivo nmr analysis of tropane alkaloid metabolism in transformed root and de differentiated cultures of Datura stramonium
Phytochemistry, 1996Co-Authors: Yvesyannick Ford, George R Ratcliffe, Richard J RobinsAbstract:Abstract In vivo 15 N NMR spectroscopy has been used to study the metabolism of [ 15 N]tropinone in transformed root cultures of Datura stramonium . It was shown that, within only 2–4 hr of application of the labelled precursor, labelled metabolites can readily be detected. It was further demonstrated that de-differentiated cultures, derived from the roots by treatment with phytohormones, are capable of metabolising [ 15 N]tropinone to [ 15 N]tropine and simple [ 15 N]tropine esters but that these cultures cannot convert [ 15 N]tropinone to [ 15 N]hyoscyamine.
Miguel Ribo - One of the best experts on this subject based on the ideXlab platform.
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Influence of calcium ion-concentration in the medium on tropane alkaloid accumulation in Datura stramonium hairy roots
Plant Science, 1999Co-Authors: M. Teresa Piñol, R. M. Cusido, Javier Palazon, Miguel RiboAbstract:Hairy root cultures of Datura stramonium were established after the infection of stem sections with Agrobacterium rhizogenes A4, and cultured in phytohormone free B5 liquid medium containing different calcium concentrations in order to investigate the relationship between Ca2+- dependent peroxidase activity and hyoscyamine accumulation. Putrescine:SAM N- methyltransferase (PMT) activity and the levels of its mRNA were determined. Although calcium concentrations below that present in B5 medium (1.0 mM) did not affect hairy root line growth, they significantly reduced root peroxidase activity which is probably involved in the degradation of secondary products. Lower calcium concentrations also reduced the hyoscyamine synthesis ability of the hairy root lines. This loss of hyoscyamine production was concurrent with the loss of PMT activity, which is supported by Northern blot analysis performed on the total RNA fraction using cDNA corresponding to PMT of tobacco as probe.
R J Robins - One of the best experts on this subject based on the ideXlab platform.
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phytohormone induced gaba production in transformed root cultures of Datura stramonium an in vivo 15n nmr study
Journal of Experimental Botany, 1996Co-Authors: Yy Ford, R G Ratcllffe, R J RobinsAbstract:Primary nitrogen metabolism in transformed root cultures of Datura stramonium was observed by in vivo 15 N NMR. Treatment of the root cultures with the plant growth regulators α-naphthaleneacetic acid (NAA) and kinetin caused a de-differentiation of the root tissue, together with perturbation of primary and secondary nitrogen metabolism. The levels of newly-synthesized glutamine and glutamate during ammonium assimilation were depleted relative to control cultures, whereas GABA biosynthesis was enhanced. Although GABA production could be stimulated by a decrease in cytoplasmic pH (whether imposed artificially or induced by hypoxia), observation of the roots during phytohormone treatment by 31 P NMR showed that the cytoplasmic pH remained stable, indicating that the perturbation of nitrogen metabolism in the de-differentiated roots must be due to other causes.
