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Christopher W. W. Beecher - One of the best experts on this subject based on the ideXlab platform.
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involvement of protein kinase and g proteins in the signal transduction of benzophenanthridine alkaloid biosynthesis
Phytochemistry, 1998Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Abstract Benzophenanthridine alkaloid biosynthesis and biosynthetic enzyme activity were induced in suspension-cells of Sanguinaria canadensis L. by compounds that stimulate the activities of protein kinase and GTP-binding proteins. The results indicate that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis. We have previously reported that elicitor-induced benzophenanthridine alkaloid biosynthesis in suspension-cell cultures of Sanguinaria canadensis L. (SCP-GM) is mediated by a signal transduction system that involves calcium and possibly protein kinase(s). In this work, a number of exogenous agents were employed to further investigate the components of the signal transduction pathway involved in the induction of alkaloid biosynthesis by a fungal elicitor and abscisic acid (ABA). SCP-GM suspension-cells were treated with compounds that modify protein kinase activity, including phorbol esters, and 1-oleoyl-2-acetyl-rac-glycerol (OAG), a synthetic diacylglycerol analogue. Phorbol-12-myristate-13-acetate induced alkaloid accumulation by as much as 65-fold over control values, while the negative control, phorbol-13-monoacetate, had no effect. OAG also increased alkaloid production by approximately 25-fold as compared to controls. Likewise, pretreatment of the suspension-cell cultures with H-7 or staurosporine, significantly suppressed ABA- or fungal-induction of benzophenanthridine alkaloid biosynthesis. Modulators of GTP-binding protein activity were also active in this system. Treatment of the suspension-cells with cholera toxin (CHX) induced alkaloid accumulation by 25-fold, which increased to 34-fold when CHX was combined with a fungal elicitor derived from Penicillium expansum (PE), and 32-fold when CHX was combined with ABA. Treatment of SCP-GM cells with CHX also enhanced the activities of two N -methyltransferases in the benzophenanthridine biosynthetic pathway namely, tetrahydroberberine- N -methyltransferase and tetrahydrocoptisine- N -methyltransferase, by six and seven fold, respectively. Furthermore, benzophenanthridine alkaloid biosynthesis was induced by treating the suspension-cells with the G-protein activators, mastoparan, mas-7 or melittin, while the inactive homologue, mas-17, did not. Suppression of alkaloid accumulation occurred when the susoension-cells were treated with GDPsS or pertussis toxin prior to treatment of the SCP-GM cells with either PE or ABA. The results support the hypothesis that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis.
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Induction of benzo[c]phenanthridine alkaloid biosynthesis in suspension cell cultures of Sanguinaria canadensis by retinoic acid derivatives
Natural Product Letters, 1996Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Abstract Treatment of suspension-cell cultures of Sanguinaria canadensis L. (SCP-GM) with a series of natural and synthetic retinoic acid derivatives induced benzophenanthridine alkaloid biosynthesis in a dose-dependent manner. Suspension cells were treated with one of the following: retinoic acid (RA), retinol (ROH), retinal (RHO). 13- cis-retinoic acid (cis-RA), or retinyl acetate (ROAc) in concentrations ranging from 1 to 50 μM (24 h). Each of the RA derivatives tested increased the cellular alkaloid concentrations of sanguinarine and chelerythrine by 97 to 470% (approximately 0.045–0.23% dry wt) depending on the reinoid and the dosage employed. Control SCP-GM suspension-cells accumulated only trace amounts of sanguinarine and chelerythrine, approximately 0.005% dry wt and 0.004% dry wt, respectively. The activity of S-adenosyl methionine: tetrahydroberberine-.V-methyltransferase, a key branch point enzyme in the benzophenanthridine biosynthetic pathway, was also induced over 24 h after treatment of th...
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quercetin induced benzophenanthridine alkaloid production in suspension cell cultures of Sanguinaria canadensis
Planta Medica, 1994Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Addition of micromolar concentrations of quercetin or rutin to suspension cell cultures of Sanguinaria canadensis L. (bloodroot) induced the biosynthesis of sanguinarine and chelerythrine in a dose-dependent manner. In contrast, related compounds : baicalein, naringin, naringenin, catechin, caffeic acid and benzoic acid displayed very weak inductive activity. Off the two active flavonoids, quercetin was the most effective for inducing benzophenanthridine alkaloid biosynthesis, with dose of 100 μM increasing alkaloid production over 375% as compared to negative controls
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elicitor stimulated benzophenanthridine alkaloid biosynthesis in bloodroot suspension cultures is mediated by calcium
Phytochemistry, 1994Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Abstract Addition of an elicitor derived from the fungus Penicillium expansum Link (PE-elicitor) or the calcium ionophore A23187, to a suspension-cell culture of Sanguinaria canadensis induced the production of the benzophenanthridine alkaloids, sanguinarine and chelerythrine, in a dose-dependent manner. Pretreatment of the cells with the specific calcium chelatant EGTA (3 mM) or the calcium channel inhibitor verapamil (100 μM), for 1 hr prior to the addition of the PE-elicitor, decreased the accumulation of both sanguinarine and chelerythrine. Moreover, A23187-stimulated alkaloid accumulation was almost completely inhibited by pretreating the suspension cells with EGTA (3 mM) for 1 hr and this suppression was reversed by the readdition of calcium ions to the medium. Furthermore, addition of trifluoperazine (100 μM) to the suspension-cell cultures 1 hr before the PE-elicitor (35 μg Glc equ ml −1 ) treatment suppressed the accumulation of benzophenanthridine alkaloids by 51% as compared to suspension cells treated with PE-elicitor alone. These results demonstrate that an external source of calcium ions is required for elicitor-induced benzophenanthridine alkaloid accumulation and suggest that calcium and possibly calmodulin and/or protein kinase C may participate in a signal transduction system that mediates this process.
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Quercetin-induced benzophenanthridine alkaloid production in suspension cell cultures of Sanguinaria canadensis.
Planta medica, 1994Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Addition of micromolar concentrations of quercetin or rutin to suspension cell cultures of Sanguinaria canadensis L. (bloodroot) induced the biosynthesis of sanguinarine and chelerythrine in a dose-dependent manner. In contrast, related compounds: baicalein, naringin, naringenin, catechin, caffeic acid and benzoic acid displayed very weak inductive activity. Of the two active flavonoids, quercetin was the most effective for inducing benzophenanthridine alkaloid biosynthesis, with doses of 100 microM increasing alkaloid production over 375% as compared to negative controls. Quercetin's inductive effects were similar to that of an elicitor derived from fungus Penicillium expansum (PE-elicitor). Suppression of quercetin and PE-induced alkaloid biosynthesis by low doses of actinomycin D (5 micrograms/ml, alpha-amanitin (20 micrograms/ml), or cycloheximide (1 microgram/ml) demonstrate a requirement for both RNA and de novo cytoplasmic protein synthesis and suggest that alterations in gene expression are involved in the inductive mechanism. Furthermore, quercetin-induced alkaloid biosynthesis was significantly reduced by pretreatment of the cells with the calcium chelator, EGTA (3 mM), or the calcium channel inhibitor, verapamil (100 microM), suggesting that this process was calcium dependent.
Gail B Mahady - One of the best experts on this subject based on the ideXlab platform.
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In vitro susceptibility of Helicobacter pylori to isoquinoline alkaloids from Sanguinaria canadensis and Hydrastis canadensis
Phytotherapy research : PTR, 2003Co-Authors: Gail B Mahady, Susan L. Pendland, Adenia Stoia, Lucas R. ChadwickAbstract:Methanol extracts of the rhizomes of Sanguinaria canadensis, and the roots and rhizomes of Hydrastis canadensis, two plants used traditionally for the treatment of gastrointestinal ailments, were screened for in vitro antibacterial activity against 15 strains of Helicobacter pylori. The rhizome extracts, as well as a methanol extract of S. canadensis suspension-cell cultures inhibited the growth of H. pylori in vitro, with a MIC50 range of 12.5–50.0 µg/ml. Three isoquinoline alkaloids were identified in the active fraction. Sanguinarine and chelerythrine, two benzophenanthridine alkaloids, inhibited the growth of the bacterium, with an MIC50 of 50.0 and 100.0 µg/ml, respectively. Protopine, a protopine alkaloid, also inhibited the growth of the bacterium, with a MIC50 of 100 µg/ml. The crude methanol extract of H. canadensis rhizomes was very active, with an MIC50 of 12.5 µg/ml. Two isoquinoline alkaloids, berberine and β-hydrastine, were identified as the active constituents, and having an MIC50 of 12.5 and 100.0 µg/ml, respectively. Copyright © 2003 John Wiley & Sons, Ltd.
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involvement of protein kinase and g proteins in the signal transduction of benzophenanthridine alkaloid biosynthesis
Phytochemistry, 1998Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Abstract Benzophenanthridine alkaloid biosynthesis and biosynthetic enzyme activity were induced in suspension-cells of Sanguinaria canadensis L. by compounds that stimulate the activities of protein kinase and GTP-binding proteins. The results indicate that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis. We have previously reported that elicitor-induced benzophenanthridine alkaloid biosynthesis in suspension-cell cultures of Sanguinaria canadensis L. (SCP-GM) is mediated by a signal transduction system that involves calcium and possibly protein kinase(s). In this work, a number of exogenous agents were employed to further investigate the components of the signal transduction pathway involved in the induction of alkaloid biosynthesis by a fungal elicitor and abscisic acid (ABA). SCP-GM suspension-cells were treated with compounds that modify protein kinase activity, including phorbol esters, and 1-oleoyl-2-acetyl-rac-glycerol (OAG), a synthetic diacylglycerol analogue. Phorbol-12-myristate-13-acetate induced alkaloid accumulation by as much as 65-fold over control values, while the negative control, phorbol-13-monoacetate, had no effect. OAG also increased alkaloid production by approximately 25-fold as compared to controls. Likewise, pretreatment of the suspension-cell cultures with H-7 or staurosporine, significantly suppressed ABA- or fungal-induction of benzophenanthridine alkaloid biosynthesis. Modulators of GTP-binding protein activity were also active in this system. Treatment of the suspension-cells with cholera toxin (CHX) induced alkaloid accumulation by 25-fold, which increased to 34-fold when CHX was combined with a fungal elicitor derived from Penicillium expansum (PE), and 32-fold when CHX was combined with ABA. Treatment of SCP-GM cells with CHX also enhanced the activities of two N -methyltransferases in the benzophenanthridine biosynthetic pathway namely, tetrahydroberberine- N -methyltransferase and tetrahydrocoptisine- N -methyltransferase, by six and seven fold, respectively. Furthermore, benzophenanthridine alkaloid biosynthesis was induced by treating the suspension-cells with the G-protein activators, mastoparan, mas-7 or melittin, while the inactive homologue, mas-17, did not. Suppression of alkaloid accumulation occurred when the susoension-cells were treated with GDPsS or pertussis toxin prior to treatment of the SCP-GM cells with either PE or ABA. The results support the hypothesis that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis.
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Induction of benzo[c]phenanthridine alkaloid biosynthesis in suspension cell cultures of Sanguinaria canadensis by retinoic acid derivatives
Natural Product Letters, 1996Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Abstract Treatment of suspension-cell cultures of Sanguinaria canadensis L. (SCP-GM) with a series of natural and synthetic retinoic acid derivatives induced benzophenanthridine alkaloid biosynthesis in a dose-dependent manner. Suspension cells were treated with one of the following: retinoic acid (RA), retinol (ROH), retinal (RHO). 13- cis-retinoic acid (cis-RA), or retinyl acetate (ROAc) in concentrations ranging from 1 to 50 μM (24 h). Each of the RA derivatives tested increased the cellular alkaloid concentrations of sanguinarine and chelerythrine by 97 to 470% (approximately 0.045–0.23% dry wt) depending on the reinoid and the dosage employed. Control SCP-GM suspension-cells accumulated only trace amounts of sanguinarine and chelerythrine, approximately 0.005% dry wt and 0.004% dry wt, respectively. The activity of S-adenosyl methionine: tetrahydroberberine-.V-methyltransferase, a key branch point enzyme in the benzophenanthridine biosynthetic pathway, was also induced over 24 h after treatment of th...
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quercetin induced benzophenanthridine alkaloid production in suspension cell cultures of Sanguinaria canadensis
Planta Medica, 1994Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Addition of micromolar concentrations of quercetin or rutin to suspension cell cultures of Sanguinaria canadensis L. (bloodroot) induced the biosynthesis of sanguinarine and chelerythrine in a dose-dependent manner. In contrast, related compounds : baicalein, naringin, naringenin, catechin, caffeic acid and benzoic acid displayed very weak inductive activity. Off the two active flavonoids, quercetin was the most effective for inducing benzophenanthridine alkaloid biosynthesis, with dose of 100 μM increasing alkaloid production over 375% as compared to negative controls
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elicitor stimulated benzophenanthridine alkaloid biosynthesis in bloodroot suspension cultures is mediated by calcium
Phytochemistry, 1994Co-Authors: Gail B Mahady, Christopher W. W. BeecherAbstract:Abstract Addition of an elicitor derived from the fungus Penicillium expansum Link (PE-elicitor) or the calcium ionophore A23187, to a suspension-cell culture of Sanguinaria canadensis induced the production of the benzophenanthridine alkaloids, sanguinarine and chelerythrine, in a dose-dependent manner. Pretreatment of the cells with the specific calcium chelatant EGTA (3 mM) or the calcium channel inhibitor verapamil (100 μM), for 1 hr prior to the addition of the PE-elicitor, decreased the accumulation of both sanguinarine and chelerythrine. Moreover, A23187-stimulated alkaloid accumulation was almost completely inhibited by pretreating the suspension cells with EGTA (3 mM) for 1 hr and this suppression was reversed by the readdition of calcium ions to the medium. Furthermore, addition of trifluoperazine (100 μM) to the suspension-cell cultures 1 hr before the PE-elicitor (35 μg Glc equ ml −1 ) treatment suppressed the accumulation of benzophenanthridine alkaloids by 51% as compared to suspension cells treated with PE-elicitor alone. These results demonstrate that an external source of calcium ions is required for elicitor-induced benzophenanthridine alkaloid accumulation and suggest that calcium and possibly calmodulin and/or protein kinase C may participate in a signal transduction system that mediates this process.
Naud Julie - One of the best experts on this subject based on the ideXlab platform.
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Espèces herbacées médicinales de sous-bois, cultivées en érablière sous différentes conditions de lumière et de sol, dans le sud du Québec (Canada)
Université Laval, 2009Co-Authors: Naud JulieAbstract:La majorité des plantes d’ombre d’intérêt médicinal sont récoltées en milieu naturel, menaçant ainsi les populations naturelles. Cette étude visait à évaluer l'impact des trouées et des caractéristiques chimiques du sol sur l’émergence, la croissance et la production de composés actifs de quatre plantes médicinales : Actaea racemosa, Sanguinaria canadensis, Caulophyllum thalictroides et Asarum canadense. Les boutures de rhizome ont été récoltées deux années après leur plantation en érablière. Les régressions multiples ont indiqué que la croissance de trois des espèces augmente avec l’irradiance et que toutes ont davantage répondu aux éléments reliés à l’acidité plutôt qu’à la richesse du sol. Les concentrations en composés actifs des rhizomes et des racines varient peu ce qui fait que leur contenu total augmente avec la biomasse. La création de trouées et le chaulage semble donc des interventions souhaitées pour augmenter le rendement en biomasse et en composés actifs chez ces espèces
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Espèces herbacées médicinales de sous-bois, cultivées en érablière sous différentes conditions de lumière et de sol, dans le sud du Québec (Canada).
Bibliotheque de l' Universite Laval, 2009Co-Authors: Naud JulieAbstract:La majorité des plantes d'ombre d'intérêt médicinal sont récoltées en milieu naturel, menaçant ainsi les populations naturelles. Cette étude visait à évaluer l'impact des trouées et des caractéristiques chimiques du sol sur l'émergence, la croissance et la production de composés actifs de quatre plantes médicinales : Actaea racemosa, Sanguinaria canadensis, Caulophyllum thalictroides et Asarum canadense. Les boutures de rhizome ont été récoltées deux années après leur plantation en érablière. Les régressions multiples ont indiqué que la croissance de trois des espèces augmente avec l'irradiance et que toutes ont davantage répondu aux éléments reliés à l'acidité plutôt qu'à la richesse du sol. Les concentrations en composés actifs des rhizomes et des racines varient peu ce qui fait que leur contenu total augmente avec la biomasse. La création de trouées et le chaulage semble donc des interventions souhaitées pour augmenter le rendement en biomasse et en composés actifs chez ces espèces.Agroforesteri
Jiiang-huei Jeng - One of the best experts on this subject based on the ideXlab platform.
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induction of necrosis and apoptosis to kb cancer cells by sanguinarine is associated with reactive oxygen species production and mitochondrial membrane depolarization
Toxicology and Applied Pharmacology, 2007Co-Authors: Meichi Chang, Chiupo Chan, Bor-ru Lin, Po Hsuen Lee, Ying Jan Wang, Lini Chen, Yiling Tsai, Yinlin Wang, Jiiang-huei JengAbstract:Abstract Sanguinarine is a benzopheanthridine alkaloid present in the root of Sanguinaria canadensis L. and Chellidonium majus L. In this study, sanguinarine (2 and 3 μM) exhibited cytotoxicity to KB cancer cells by decreasing MTT reduction to 83% and 52% of control after 24-h of exposure. Sanguinarine also inhibited the colony forming capacity (> 52–58%) and growth of KB cancer cells at concentrations higher than 0.5–1 μM. Short-term exposure to sanguinarine (> 0.5 μM) effectively suppressed the adhesion of KB cells to collagen and fibronectin (FN). Sanguinarine (2 and 3 μM) induced evident apoptosis as indicated by an increase in sub-G0/G1 populations, which was detected after 6-h of exposure. Only a slight increase in cells arresting in S-phase and G2/M was noted. Induction of KB cell apoptosis and necrosis by sanguinarine (2 and 3 μM) was further confirmed by Annexin V-PI dual staining flow cytometry and the presence of DNA fragmentation. The cytotoxicity by sanguinarine was accompanied by an increase in production of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential as indicated by single cell flow cytometric analysis of DCF and rhodamine fluorescence. NAC (1 and 3 mM) and catalase (2000 U/ml) prevented the sanguinarine-induced ROS production and cytotoxicity, whereas dimethylthiourea (DMT) showed no marked preventive effect. These results suggest that sanguinarine has anticarcinogenic properties with induction of ROS production and mitochondrial membrane depolarization, which mediate cancer cell death.
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Antiplatelet effect of sanguinarine is correlated to calcium mobilization, thromboxane and cAMP production.
Atherosclerosis, 2006Co-Authors: Jiiang-huei Jeng, Bor-ru Lin, Wan-hong Lan, Hsiao Hwa Chang, Po Hsuen Lee, Ying Jan Wang, Juo Song Wang, Yi-jane ChenAbstract:Sanguinarine is a plant alkaloid present in the root of Sanguinaria canadensis and Poppy fumaria species. Sanguinarine has been used as an antiseptic mouth rinse and a toothpaste additive to reduce dental plaque and gingival inflammation. In this study, we investigated the antiplatelet effects of sanguinarine, aiming to extend its potential pharmacological applications. Sanguinarine inhibited platelet aggregation induced by arachidonic acid (AA), collagen, U46619 and sub-threshold concentration of thrombin (0.05 U/ml) with IC(50) concentrations of 8.3, 7.7, 8.6 and 4.4 microM, respectively. Sanguinarine (5-10 microM) inhibited 10-31% of platelet TXB(2) production, but not platelet aggregation induced by higher concentration of thrombin (0.1 U/ml). SQ29548, a thromboxane receptor antagonist, inhibited the AA-induced platelet aggregation but not TXB(2) production. Sanguinarine suppressed cyclooxygenase-1 (COX-1) activity (IC(50)=28 microM), whereas its effect on COX-2 activity was minimal. Sanguinarine (8, 10 microM) further inhibited the AA-induced Ca(2+) mobilization by 27-62%. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the inhibitory effect of sanguinarine toward AA-induced platelet Ca(2+) mobilization and aggregation. These results suggest that sanguinarine is a potent antiplatelet agent, which activates adenylate cyclase, inhibits platelet Ca(2+) mobilization, TXB(2) production as well as suppresses COX-1 enzyme activity. Sanguinarine may have therapeutic potential for treatment of cardiovascular diseases related to platelet aggregation.
Carmine J Coscia - One of the best experts on this subject based on the ideXlab platform.
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Elicitation of dihydrobenzophenanthridine oxidase in Sanguinaria canadensis cell cultures.
Phytochemistry, 1996Co-Authors: Atanas Ignatov, Steven D Cline, W.gregg Clark, Mikulas Psenak, Robert J. Krueger, Carmine J CosciaAbstract:Dihydrobenzophenanthridine (DHBP) oxidase catalyses the last step in the biogenesis of the benzo[c]phenanthridine alkaloid sanguinarine. Addition of autoclaved fungal preparations or putative plant defence signalling intermediates (jasmonic acid (JA), methyl jasmonate (MeJ), acetylsalicylic acid (ASA)) to Sanguinaria canadensis cell suspension cultures elicited an increase in the activity of DHBP oxidase. MeJ and ASA were better inducers of oxidase activity than were the fungal elicitor and JA. Enzyme-specific activity could be induced in a dose- and time-dependent manner up to 4- to 14-fold, respectively, when cells were treated with MeJ or with ASA. A change in total enzyme activity in cultured cells was observed only at the highest concentration of MeJ and not at any level of ASA tested. The results suggest that MeJ and ASA may play a role in the S. canadensis defence against pathogens by eliciting the enzymes involved in the synthesis of the phytoalexin benzophenanthridine alkaloids.
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differential enhancement of benzophenanthridine alkaloid content in cell suspension cultures of Sanguinaria canadensis under conditions of combined hormonal deprivation and fungal elicitation
Journal of Natural Products, 1993Co-Authors: Steven D Cline, Robert J Mchale, Carmine J CosciaAbstract:An elicitation protocol, resulting in the accumulation of sanguinarine in suspension cultures of Papaver bracteatum, was assessed for induction of the same alkaloid in Sanguinaria canadensis. Although only a trace constituent of P. bracteatum plants, sanguinarine is a major alkaloid (1-3% dry wt) of S. canadensis rhizomes. By combining hormonal deprivation for various intervals and a 3-day fungal (Verticillium dahliae) elicitation, benzophenanthridine alkaloid accumulation was induced in S. canadensis cell suspensions. Chelirubine content increased (0.1-1.3% dry wt) consistently in elicited cell cultures while chelerythrine (0.01-0.10% dry wt) and sanguinarine (0-0.02% dry wt) levels were considerably less. Alkaloid accumulation always occurred upon removal of hormone but only at certain time intervals in the log phase upon fungal elicitation. Levels of dopamine, a precursor of the alkaloids, fluctuated over the incubation period, but displayed a 2- to 6-fold increase in cell suspensions grown without hormone. In some experiments dopamine accumulated to levels > 20% dry wt, and these increases were enhanced by the addition of fungal elicitor. Although the same fungal elicitor induces benzophenanthridines in taxonomically related S. canadensis and P. bracteatum, it did not elicit the accumulation of the same alkaloid in the two different plant cultures.
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Purification and characterization of dihydrobenzophenanthridine oxidase from elicited Sanguinaria canadensis cell cultures
Archives of biochemistry and biophysics, 1992Co-Authors: Hirohumi Arakawa, Mikulas Psenak, W.gregg Clark, Carmine J CosciaAbstract:Abstract Upon treatment of Papaveracea cells with fungal elicitors, the biosynthesis of benzo[c]phenanthridine alkaloids is induced. Dihydrobenzophenanthridine oxidase, which catalyzes a later step in the biogenesis of these alkaloids, is one of the enzymes whose activity is elevated in the process. Here we report the 211-fold purification of the oxidase from elicited Sanguinaria canadensis by a combination of ammonium sulfate fractionation, DEAE-Sephadex, CM-Sephadex, Sephadex G-200, and either phenyl Superose or gel filtration chromatography. The purified enzyme utilized molecular oxygen to oxidize dihydrosanguinarine to sanguinarine with concomitant formation of hydrogen peroxide. A pH optimum of 7.0, Vmax of 27 nkat/mg protein, and apparent Km of 6.0 μ m for dihydrosanguinarine were determined. Dihydrochelerythrine was also found to be a substrate for the purified enzyme, displaying an apparent Km of 10 μ m . However, neither dihydronorsanguinarine nor the indole alkaloid ajmalicine was oxidized, indicating that the enzyme has some substrate specificity. Apparent molecular weight estimates by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis revealed that the most purified enzyme preparation obtained contained a major component of 77 kDa and two minor components between 59 and 67 kDa that can be associated with oxidase activity. Purified enzyme preparations possessed activity that was inhibited by sodium diethyldithiocarbamate, sodium azide, potassium cyanide, 1,4- dl -dithiothreitol, and mercaptoethanol.
