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Ulrich Matern - One of the best experts on this subject based on the ideXlab platform.
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Presentation_1_The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants.pptx
2018Co-Authors: Celia Krieger, Ulrich Matern, Bernd Schneider, Sandro Roselli, Sandra Kellner-thielmann, Gianni Galati, Jeremy Grosjean, Alexandre Olry, David Ritchie, Frederic BourgaudAbstract:The production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi Majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae.
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The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants
Frontiers in Plant Science, 2018Co-Authors: Celia Krieger, Ulrich Matern, Bernd Schneider, Sandro Roselli, Sandra Kellner-thielmann, Gianni Galati, Jeremy Grosjean, Alexandre Olry, David Ritchie, Frederic BourgaudAbstract:The production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi Majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae.
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Isolation and Functional Characterization of CYP71AJ4 Encoding for the First P450 Monooxygenase of Angular Furanocoumarin Biosynthesis
Journal of Biological Chemistry, 2008Co-Authors: Romain Larbat, Ulrich Matern, Alain Hehn, Joachim Hans, Sarah Schneider, Hélène Jugdé, Bernd Schneider, Frederic BourgaudAbstract:The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi Majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent K(m) of 2.1 +/- 0.4 microm for (+)-columbianetin and a k(cat) of 112 +/- 14 min(-1). Moreover, the use of 3'-deuterated (+)-columbianetin as substrate led to an almost complete "metabolic switch," resulting in the synthesis of anti-3'-hydroxy-3'-deuterated(+)-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3'. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution.
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Molecular Cloning and Functional Characterization of Psoralen Synthase, the First Committed Monooxygenase of Furanocoumarin Biosynthesis
Journal of Biological Chemistry, 2007Co-Authors: Romain Larbat, Silvia Specker, Frederic Bourgaud, Alain Hehn, Joachim Hans, Sandra Kellner, Eric Gontier, Ulrich MaternAbstract:Ammi Majus L. accumulates linear furanocoumarins by cyto-chrome P450 (CYP)-dependent conversion of 6-prenylumbellif-erone via ()-marmesin to psoralen. Relevant activities, i.e. pso-ralen synthase, are induced rapidly from negligible background levels upon elicitation of A. Majus cultures with transient maxima at 9 –10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocou-marin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for ()-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via ()-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, ()-columbianetin failed as sub-strate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate ()-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.
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Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis
2007Co-Authors: Sandra Kellner, Silvia Specker, Frederic Bourgaud, Alain Hehn, Joachim Hans, Eric Gontier, Ulrich MaternAbstract:Ammi Majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (؉)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. Majus cultures with transient maxima at 9 -10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (؉)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (؉)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (؉)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (؉)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution. Furanocoumarins are produced by many plants, mostly of the Apiaceae, Rutaceae, Moraceae, or the Coronilla and Psoralea genera of the Fabaceae (1-3). Multiple pharmacological effects have been ascribed to several of these metabolites (4 -6), which were included in clinical screenings but received attention also for their inhibitory effect on monooxygenases involved in drug metabolism (7-9) and potential toxicity (10). The (dihydro)furan-substituted 2H-1-benzopyran-2-one forms the characteristic core structure, and the annulation type distinguishes the linear furanocoumarins or psoralens from the angular furanocoumarin
Ewa łojkowska - One of the best experts on this subject based on the ideXlab platform.
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establishment of a co culture of Ammi Majus l and ruta graveolens l for the synthesis of furanocoumarins
Plant Science, 2003Co-Authors: Matylda Sidwagorycka, Frederic Bourgaud, Aleksandra Krolicka, Malgorzata Kozyra, Kazimierz Glowniak, Ewa łojkowskaAbstract:Abstract Ammi Majus and Ruta graveolens plants were chosen to establish a co-culture due to their abilities to produce secondary metabolites from the furanocoumarin (FC) family. The conditions for cultivating A. Majus hairy roots and R. graveolens shoots separately were established earlier. In continuation, the conditions of the co-culture of A. Majus and R. graveolens were determined in this work. Two systems of co-cultivation were tested: A. Majus hairy roots with R. graveolens cell suspension and A. Majus hairy roots with R. graveolens shoots. The second model was chosen for further experiments, namely to test different tissues inoculation ratios as well as the influence of the photoperiod and darkness conditions on A. Majus and R. graveolens tissues growth and the accumulation of secondary metabolites. The growth index ( t 25 / t 1 ) of R. graveolens shoots was higher in the co-cultures; moreover, 2.5 times higher concentration of xanthotoxin was observed in the co-culture in light conditions.
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elicitation of secondary metabolites in in vitro cultures of Ammi Majus l
Enzyme and Microbial Technology, 2003Co-Authors: Izabela Staniszewska, Aleksandra Krolicka, Edmund Malinski, Ewa łojkowska, Janusz SzafranekAbstract:Abstract The present study was concentrated on the production of secondary metabolites in callus, cell suspension and hairy roots of Ammi Majus L. by exposing them to elicitors: benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BION®) and autoclaved lysate of cell suspension of bacteria—Enterobacter sakazaki. GC and GC–MS analysis of chloroform and methanol extracts indicated a higher accumulation of umbelliferone in the elicited tissues than in the control ones. Using GC–MS, two compounds not earlier found in A. Majus tissues were identified in callus cultures: scopoletin (7-hydroxy-6-metoxy-2H-1-benzopyron-2-one) and dehydrogeijerin (7-methoxy-6-(3-methyl-1-oxo-2-butenyl)-2H-1-benzopyran-2-one).
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hplc dad in identification and quantification of selected coumarins in crude extracts from plant cultures of Ammi Majus and ruta graveolens
IEEE Journal of Solid-state Circuits, 2003Co-Authors: Marian Kaminski, Aleksandra Krolicka, Ewa łojkowska, Matylda Sidwagorycka, Rafal Kartanowicz, Marcin Kaminski, Wojciech GorzenAbstract:This paper describes a method for the separation and determination of selected coumarins and furanocoumarins in the crude extracts from plant tissue cultures of Ammi Majus hairy roots and Ruta graveolens cell suspensions, cultured in vitro, separately or together as co-cultures. The usefulness of the three main components of the eluent used in reversed-phase high performance liquid chromatographic analysis, namely: methanol (MeOH), acetonitrile (ACN), and tetrahydrofuran (THF), and different elution programs, was assessed. In the optimal analytical method a Lichrospher® RP-18e 5-μm column, a THF-MeOH elution gradient, and a UV/VIS DAD detector were used. Due to the presence of many different compounds in the investigated plant extracts, the use of a UV/VIS DAD detector was essential. Coumarins were identified by comparison of their UV spectra with those of the analytical standards, and characterization of peak purity.
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establishment of hairy root cultures of Ammi Majus
Plant Science, 2001Co-Authors: Aleksandra Krolicka, Izabela Staniszewska, Krzysztof P Bielawski, Edmund Malinski, Janusz Szafranek, Ewa łojkowskaAbstract:Abstract Axenically grown Ammi Majus plantlets were inoculated with seven different Agrobacterium rhizogenes strains. Hairy root lines were established only after inoculation with the two agropine strains: A4 and LBA9402. The growth rate of hairy root cultures was about thirty times faster than that of callus and cell suspension cultures. Polymerase chain reaction with primers for the genes rolB and rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A. rhizogenes to the genome of hairy roots obtained after transformation by both Agrobacterium strains. The furanocoumarins (psoralen, xanthotoxine, bergapten and imperatorin) usually found in seeds of A. Majus were not detected in callus, cell suspension and hairy root cultures using Gas chromatography–mass spectrometry (GC–MS). However, umbelliferone, a precursor of furanocoumarins, was detected in callus, cell suspension and hairy root cultures. The umbelliferone content in extracts of hairy root cultures, obtained after transformation by A4, was similar to that determined in A. Majus seeds (19 μg/g DW) and higher than those obtained for cell suspension and callus cultures (2 and 9 μg/g DW, respectively).
Frederic Bourgaud - One of the best experts on this subject based on the ideXlab platform.
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The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants
Frontiers in Plant Science, 2018Co-Authors: Celia Krieger, Ulrich Matern, Bernd Schneider, Sandro Roselli, Sandra Kellner-thielmann, Gianni Galati, Jeremy Grosjean, Alexandre Olry, David Ritchie, Frederic BourgaudAbstract:The production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi Majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae.
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Presentation_1_The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants.pptx
2018Co-Authors: Celia Krieger, Ulrich Matern, Bernd Schneider, Sandro Roselli, Sandra Kellner-thielmann, Gianni Galati, Jeremy Grosjean, Alexandre Olry, David Ritchie, Frederic BourgaudAbstract:The production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi Majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae.
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Isolation and Functional Characterization of CYP71AJ4 Encoding for the First P450 Monooxygenase of Angular Furanocoumarin Biosynthesis
Journal of Biological Chemistry, 2008Co-Authors: Romain Larbat, Ulrich Matern, Alain Hehn, Joachim Hans, Sarah Schneider, Hélène Jugdé, Bernd Schneider, Frederic BourgaudAbstract:The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi Majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent K(m) of 2.1 +/- 0.4 microm for (+)-columbianetin and a k(cat) of 112 +/- 14 min(-1). Moreover, the use of 3'-deuterated (+)-columbianetin as substrate led to an almost complete "metabolic switch," resulting in the synthesis of anti-3'-hydroxy-3'-deuterated(+)-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3'. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution.
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Molecular Cloning and Functional Characterization of Psoralen Synthase, the First Committed Monooxygenase of Furanocoumarin Biosynthesis
Journal of Biological Chemistry, 2007Co-Authors: Romain Larbat, Silvia Specker, Frederic Bourgaud, Alain Hehn, Joachim Hans, Sandra Kellner, Eric Gontier, Ulrich MaternAbstract:Ammi Majus L. accumulates linear furanocoumarins by cyto-chrome P450 (CYP)-dependent conversion of 6-prenylumbellif-erone via ()-marmesin to psoralen. Relevant activities, i.e. pso-ralen synthase, are induced rapidly from negligible background levels upon elicitation of A. Majus cultures with transient maxima at 9 –10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocou-marin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for ()-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via ()-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, ()-columbianetin failed as sub-strate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate ()-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.
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Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis
2007Co-Authors: Sandra Kellner, Silvia Specker, Frederic Bourgaud, Alain Hehn, Joachim Hans, Eric Gontier, Ulrich MaternAbstract:Ammi Majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (؉)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. Majus cultures with transient maxima at 9 -10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (؉)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (؉)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (؉)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (؉)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution. Furanocoumarins are produced by many plants, mostly of the Apiaceae, Rutaceae, Moraceae, or the Coronilla and Psoralea genera of the Fabaceae (1-3). Multiple pharmacological effects have been ascribed to several of these metabolites (4 -6), which were included in clinical screenings but received attention also for their inhibitory effect on monooxygenases involved in drug metabolism (7-9) and potential toxicity (10). The (dihydro)furan-substituted 2H-1-benzopyran-2-one forms the characteristic core structure, and the annulation type distinguishes the linear furanocoumarins or psoralens from the angular furanocoumarin
Aleksandra Krolicka - One of the best experts on this subject based on the ideXlab platform.
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establishment of a co culture of Ammi Majus l and ruta graveolens l for the synthesis of furanocoumarins
Plant Science, 2003Co-Authors: Matylda Sidwagorycka, Frederic Bourgaud, Aleksandra Krolicka, Malgorzata Kozyra, Kazimierz Glowniak, Ewa łojkowskaAbstract:Abstract Ammi Majus and Ruta graveolens plants were chosen to establish a co-culture due to their abilities to produce secondary metabolites from the furanocoumarin (FC) family. The conditions for cultivating A. Majus hairy roots and R. graveolens shoots separately were established earlier. In continuation, the conditions of the co-culture of A. Majus and R. graveolens were determined in this work. Two systems of co-cultivation were tested: A. Majus hairy roots with R. graveolens cell suspension and A. Majus hairy roots with R. graveolens shoots. The second model was chosen for further experiments, namely to test different tissues inoculation ratios as well as the influence of the photoperiod and darkness conditions on A. Majus and R. graveolens tissues growth and the accumulation of secondary metabolites. The growth index ( t 25 / t 1 ) of R. graveolens shoots was higher in the co-cultures; moreover, 2.5 times higher concentration of xanthotoxin was observed in the co-culture in light conditions.
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elicitation of secondary metabolites in in vitro cultures of Ammi Majus l
Enzyme and Microbial Technology, 2003Co-Authors: Izabela Staniszewska, Aleksandra Krolicka, Edmund Malinski, Ewa łojkowska, Janusz SzafranekAbstract:Abstract The present study was concentrated on the production of secondary metabolites in callus, cell suspension and hairy roots of Ammi Majus L. by exposing them to elicitors: benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BION®) and autoclaved lysate of cell suspension of bacteria—Enterobacter sakazaki. GC and GC–MS analysis of chloroform and methanol extracts indicated a higher accumulation of umbelliferone in the elicited tissues than in the control ones. Using GC–MS, two compounds not earlier found in A. Majus tissues were identified in callus cultures: scopoletin (7-hydroxy-6-metoxy-2H-1-benzopyron-2-one) and dehydrogeijerin (7-methoxy-6-(3-methyl-1-oxo-2-butenyl)-2H-1-benzopyran-2-one).
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hplc dad in identification and quantification of selected coumarins in crude extracts from plant cultures of Ammi Majus and ruta graveolens
IEEE Journal of Solid-state Circuits, 2003Co-Authors: Marian Kaminski, Aleksandra Krolicka, Ewa łojkowska, Matylda Sidwagorycka, Rafal Kartanowicz, Marcin Kaminski, Wojciech GorzenAbstract:This paper describes a method for the separation and determination of selected coumarins and furanocoumarins in the crude extracts from plant tissue cultures of Ammi Majus hairy roots and Ruta graveolens cell suspensions, cultured in vitro, separately or together as co-cultures. The usefulness of the three main components of the eluent used in reversed-phase high performance liquid chromatographic analysis, namely: methanol (MeOH), acetonitrile (ACN), and tetrahydrofuran (THF), and different elution programs, was assessed. In the optimal analytical method a Lichrospher® RP-18e 5-μm column, a THF-MeOH elution gradient, and a UV/VIS DAD detector were used. Due to the presence of many different compounds in the investigated plant extracts, the use of a UV/VIS DAD detector was essential. Coumarins were identified by comparison of their UV spectra with those of the analytical standards, and characterization of peak purity.
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establishment of hairy root cultures of Ammi Majus
Plant Science, 2001Co-Authors: Aleksandra Krolicka, Izabela Staniszewska, Krzysztof P Bielawski, Edmund Malinski, Janusz Szafranek, Ewa łojkowskaAbstract:Abstract Axenically grown Ammi Majus plantlets were inoculated with seven different Agrobacterium rhizogenes strains. Hairy root lines were established only after inoculation with the two agropine strains: A4 and LBA9402. The growth rate of hairy root cultures was about thirty times faster than that of callus and cell suspension cultures. Polymerase chain reaction with primers for the genes rolB and rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A. rhizogenes to the genome of hairy roots obtained after transformation by both Agrobacterium strains. The furanocoumarins (psoralen, xanthotoxine, bergapten and imperatorin) usually found in seeds of A. Majus were not detected in callus, cell suspension and hairy root cultures using Gas chromatography–mass spectrometry (GC–MS). However, umbelliferone, a precursor of furanocoumarins, was detected in callus, cell suspension and hairy root cultures. The umbelliferone content in extracts of hairy root cultures, obtained after transformation by A4, was similar to that determined in A. Majus seeds (19 μg/g DW) and higher than those obtained for cell suspension and callus cultures (2 and 9 μg/g DW, respectively).
Matylda Sidwagorycka - One of the best experts on this subject based on the ideXlab platform.
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establishment of a co culture of Ammi Majus l and ruta graveolens l for the synthesis of furanocoumarins
Plant Science, 2003Co-Authors: Matylda Sidwagorycka, Frederic Bourgaud, Aleksandra Krolicka, Malgorzata Kozyra, Kazimierz Glowniak, Ewa łojkowskaAbstract:Abstract Ammi Majus and Ruta graveolens plants were chosen to establish a co-culture due to their abilities to produce secondary metabolites from the furanocoumarin (FC) family. The conditions for cultivating A. Majus hairy roots and R. graveolens shoots separately were established earlier. In continuation, the conditions of the co-culture of A. Majus and R. graveolens were determined in this work. Two systems of co-cultivation were tested: A. Majus hairy roots with R. graveolens cell suspension and A. Majus hairy roots with R. graveolens shoots. The second model was chosen for further experiments, namely to test different tissues inoculation ratios as well as the influence of the photoperiod and darkness conditions on A. Majus and R. graveolens tissues growth and the accumulation of secondary metabolites. The growth index ( t 25 / t 1 ) of R. graveolens shoots was higher in the co-cultures; moreover, 2.5 times higher concentration of xanthotoxin was observed in the co-culture in light conditions.
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hplc dad in identification and quantification of selected coumarins in crude extracts from plant cultures of Ammi Majus and ruta graveolens
IEEE Journal of Solid-state Circuits, 2003Co-Authors: Marian Kaminski, Aleksandra Krolicka, Ewa łojkowska, Matylda Sidwagorycka, Rafal Kartanowicz, Marcin Kaminski, Wojciech GorzenAbstract:This paper describes a method for the separation and determination of selected coumarins and furanocoumarins in the crude extracts from plant tissue cultures of Ammi Majus hairy roots and Ruta graveolens cell suspensions, cultured in vitro, separately or together as co-cultures. The usefulness of the three main components of the eluent used in reversed-phase high performance liquid chromatographic analysis, namely: methanol (MeOH), acetonitrile (ACN), and tetrahydrofuran (THF), and different elution programs, was assessed. In the optimal analytical method a Lichrospher® RP-18e 5-μm column, a THF-MeOH elution gradient, and a UV/VIS DAD detector were used. Due to the presence of many different compounds in the investigated plant extracts, the use of a UV/VIS DAD detector was essential. Coumarins were identified by comparison of their UV spectra with those of the analytical standards, and characterization of peak purity.