Benzophenanthridine Alkaloids Research Articles

Identification of benzophenanthridine alkaloids from Bocconia arborea by gas chromatography-mass spectrometry.

A methanol extract of the bark of Bocconia arborea was fractionated on silica gel and the fractions analysed using gas chromatography coupled with mass spectrometry (GC-MS). Several benzophenanthridine alkaloids were identified including dihydrosanguinarine, oxysanguinarine, 11-acetonyldihydrochelerythrine, dihydrochelerythrine, chelerythrine, chelerythridimerine and angoline as the principal constituents. The results show that the direct GC-MS analysis of these alkaloids is possible with a clear distinction between the compounds. The technique is shown to be a valuable tool and an alternative technique to classical phytochemical procedures permitting the fast analysis of alkaloids mixtures.

Antisense RNA-Mediated Suppression of Benzophenanthridine Alkaloid Biosynthesis in Transgenic Cell Cultures of California Poppy

California poppy (Eschscholzia californicaCham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3′-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisenseBBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a β-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional 1H-nuclear magnetic resonance and in vivo 15N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.

Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy.

California poppy (Eschscholzia californica Cham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3'-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisense BBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a beta-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional (1)H-nuclear magnetic resonance and in vivo (15)N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.

Inhibition of Human Blood Acetylcholinesterase and Butyrylcholinesterase by Some Alkaloids

A comparative study has been carried out on effects of berberine (diisoquinoline alkaloid) and sanguinarine and chelidonine (benzophenanthridine alkaloids( on erythrocyte acetylcholinesterase and serum butyrylcholinesterase from human blood. The studied alkaloids have been shown to be strong reversible inhibitors of the cholinesterase activity. Acetylcholinesterase is more sensitive to their action, than butyrylcholinesterase. The type of reversible inhibition was determined, and inhibitor constants were calculated. It is revealed that the character of inhibition is identical for the both cholinesterases. Berberine and sanguinarine are competitive-noncompetitive inhibitors, whereas chelidonine, a competitive inhibitor.

Structural studies of benzophenanthridine alkaloid free bases by NMR spectroscopy

AbstractThe free bases of eight quaternary benzo[c]phenanthridine alkaloids (chelerythrine, sanguilutine, chelirubine, chelilutine, sanguirubine, sanguinarine, nitidine and fagaronine) were investigated by 1H and 13C NMR spectroscopy. The pseudobases (the 6‐hydroxy‐5,6‐dihydro derivatives) were found to be the only products of the alkalization of these alkaloids using sodium carbonate in DMSO‐d6 solution. This observation differs from the measurements of preparative free bases in CDCl3 solution, where the structures of the bimolecular aminoacetals were unequivocally confirmed. According to the results obtained, in biological systems, in the presence of excess of water and ions, free bases of the investigated alkaloids are assumed to adopt the pseudobase (6‐hydroxy derivative) structure. In contrast, in less polar media (C6D6, CDCl3), the formation of the bimolecular aminoacetal structure is preferred. Copyright © 2001 John Wiley & Sons, Ltd.

The evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea corylifolia and Sanguinaria canadensis

Extracts from forty-three plant species were selected on account of reported traditional uses for the treatment of TB and/or leprosy. These were assayed for antimycobacterial activities. A simple in vitro screening assay was employed using two model species of mycobacteria, M. aurum and M. smegmatis. Crude methanolic extracts from three of the plants, C. mukul, P. corylifolia and S. canadensis, were found to have significant antimycobacterial activity against M. aurum only (MIC=62.5 μg/ml) . Bioassay guided fractionation led to the isolation of two known benzophenanthridine alkaloids, sanguinarine ( 1) and chelerythrine ( 2), from the roots S. canadensis and the known phenolic meroterpene, bakuchiol ( 3) from the seeds of P. corylifolia. The fractionation of the resin of C. mukul lead to a decrease in antimycobacterial activity and hence further work was not pursued. Compound ( 2) was the most active against M. aurum and M. smegmatis (IC 50=7.30 μg/ml [19.02 μM] and 29.0 μg/ml [75.56 μM], respectively). M. aurum was the most susceptible organism to all three compounds. No significant difference in antimycobacterial activity was observed when the two alkaloids were tested for activity in media of differing pH values. The activities of the pure compounds against M. aurum were comparable with those against M. bovis BCG with compound ( 2) being the most active ( M. bovis BCG, IC 50=14.3 μg/ml [37.3 μM]). These results support the use of these plants in traditional medicine.

Inhibitory effects of sanguinarine on monoamine oxidase activity in mouse brain.

The effects of benzophenanthridine alkaloids, such as sanguinarine and chelidonine, on monoamine -oxidase (MAO) activity in mouse brain were investigated. Sanguinarine showed an inhibitory effect on MAO activity in a concentration dependent manner (53.4% inhibition at 25 microM). However, chelidonine did not inhibit MAO activity. The IC(50) value of sanguinarine was 24.5 microM. Sanguinarine inhibited non-competitively MAO activity using kynuramine as a substrate. The K(i) value for sanguinarine was 22.1 microM. These results suggest that sanguinarine partially contributes to the regulation of catecholamine content.

Elevational trends in defense chemistry, vegetation, and reproduction in Sanguinaria canadensis.

Evaluation of biotic interactions along geographic gradients reveals that pressure on plant populations by herbivores and pathogens increases as latitude decreases, and is accompanied by a parallel increase in the number and toxicity of alkaloid-bearing plants. We compared rhizome alkaloid content with plant reproductive and vegetative characters in Sanguinaria canadensis (Papaveraceae) along an elevational gradient over two growing seasons to ascertain 1) if alkaloid production in bloodroot varies among populations and systematically with elevation, and 2) if there exists a correlation between isoquinoline alkaloid, vegetative and reproductive production. In general, alkaloid content in bloodroot rhizomes declines with elevation, increases with rhizome water content, varies by site, and fluctuates seasonally with plant growth and reproduction. Alkaloid content was positively correlated with vegetative and reproductive effort with few exceptions. Analysis of total protopine and benzophenanthridine alkaloid concentrations revealed generally similar patterns as those of individual alkaloid concentrations, although significant differences did appear between individual alkaloid concentrations.

The effects of chelidonine on tubulin polymerisation, cell cycle progression and selected signal transmission pathways

Chelidonine is a tertiary benzophenanthridine alkaloid known to cause mitotic arrest and to interact weakly with tubulin. Our interest in chelidonine began when we found it to be a major contaminant of Ukrain, which is a compound reported to be selectively toxic to malignant cells. The effects of chelidonine in two normal (monkey kidney and Hs27), two transformed (Vero and Graham 293) and two malignant (WHCO5 and HeLa) cell lines, were examined. Chelidonine proved to be a weak inhibitor of cell growth, but no evidence for selective cytotoxicity was found in this study. It was confirmed that chelidonine inhibits tubulin polymerisation (IC50 = 24 microM), explaining its ability to disrupt microtubular structure in cells. A G2/M arrest results, which is characterised by abnormal metaphase morphology, increased levels of cyclin B1 and enhanced cdc2 kinase activity. Exposure of all cell lines examined to chelidonine leads to activation of the stress-activated protein kinase/jun kinase pathway (SAPK/JNK).

Agrobacterium-mediated genetic transformation of California poppy, Eschscholzia californica Cham., via somatic embryogenesis.

An efficient Agrobacterium-mediated protocol for the stable genetic transformation of Eschscholzia californica Cham. (California poppy) via somatic embryogenesis is reported. Excised cotyledons were co-cultivated with A. tumefaciens strain GV3101 carrying the pBI121 binary vector. Except for the co-cultivation medium, all formulations included 50 mg l-1 paromomycin as the selective agent and 200 mg l-1 timentin to eliminate the Agrobacterium. Four to five weeks after infection, paromomycin-resistant calli grew on 80% of explants in the presence of 2.0 mg l-1 1-naphthaleneacetic acid (NAA) and 0.1 mg l-1 6-benzylaminopurine (BAP). Calli were cultured on somatic embryogenesis induction medium containing 1.0 mg l-1 NAA and 0.5 mg l-1 BAP, and somatic embryos were visible on 30% of the paromomycin-resistant calli within 3-4 weeks. Three to four weeks after the somatic embryos were transferred to phytohormone-free plant regeneration medium, 32% converted to paromomycin-resistant plants. Detection of the neomycin phosphotransferase gene and high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all plant tissues confirmed the integrative transformation of the regenerated plants. The normal alkaloid profile of California poppy was unaffected by the transformation process; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regulation of the benzophenanthridine alkaloid pathway.

Effects of sodium orthovanadate on benzophenanthridine alkaloid formation and distribution in cell suspension cultures of Eschscholtzia californica

Cell suspension cultures of Eschscholtzia californica produce relatively large amounts of benzophenanthridine alkaloids upon elicitation. Sodium orthovanadate is used as an abiotic elicitor to induce alkaloid biosynthesis in cultures of E. californica. The response of the cell culture to this abiotic elicitor is very similar to that observed after elicitation with a biotic elicitor (a carbohydrate fraction from yeast extract). Treatment with orthovanadate leads to alkalinization of the growth medium, a 20-fold induction of the key enzyme tyrosine decarboxylase and increased alkaloid formation (up to 40 mg.L –1). Cells treated with the yeast elicitor excrete a large portion of alkaloids produced into the growth medium (up to 50 % of total alkaloids) while cells treated with orthovanadate release very small amounts of alkaloids into the medium (less than 10 % of total alkaloids). These results suggest that an active transport system, possibly specific for benzophenanthridine alkaloids, is present in the plasma membrane of E. californica cells. The nature of this putative vanadate-sensitive transporter is not known at present.

Activation of p38 and c-Jun N-terminal Kinase Pathways and Induction of Apoptosis by Chelerythrine Do Not Require Inhibition of Protein Kinase C

Chelerythrine, a natural benzophenanthridine alkaloid, has been reported to mediate a variety of biological activities, including inhibition of protein kinase C (PKC). Here we report that chelerythrine induced time- and dose-dependent activation of JNK1 and p38 in HeLa cells, which was mediated the upstream kinases, MEKK1 and MKK4. However, treatment with two other potent and selective PKC inhibitors, GF-109203X and Gö6983, or down-regulation of PKC activity by prolonged treatment with phorbol 12-myristate 13-acetate had no effect on JNK1 and p38 activities. Furthermore, under the conditions where JNK1 and p38 were activated, we did not observe any significant inhibitory effect of chelerythrine on the activities of PKC isozymes present in HeLa cells. Interestingly, pretreatment with the antioxidants, N-acetyl-L-cysteine, dithiothreitol, and glutathione, impaired chelerythrine-induced JNK1 and p38 activation. In addition, chelerythrine induced apoptosis that was blocked by the antioxidants and the dominant-negative mutants of MEKK1, MKK4, JNK1, and p38. Together, these results uncover a novel biochemical property of chelerythrine, i.e. activation of MEKK1- and MKK4-dependent JNK1 and p38 pathways through an oxidative stress mechanism, which mediate the induction of apoptosis, but are independent of PKC inhibition.

Topoisomerase I poisons and suppressors as anticancer drugs.

Inhibitors of topoisomerase I constitute a novel family of antitumor agents. The camptothecin derivatives topotecan and irinotecan represent new weapons in our arsenal for battling human cancer. These two drugs act specifically at the level of the topoisomerase I-DNA complex and stimulate DNA cleavage. This mechanism of action is not restricted to the camptothecins. Numerous topoisomerase I poisons including DNA minor groove binders such as Hoechst 33258 and DNA intercalators such as benzophenanthridine alkaloids and indolocarbazole derivatives have been discovered and developed. Another important group of topoisomerase I inhibitors contains drugs which prevent or reverse topoisomerase I-DNA complex formation. Many of these topoisomerase I suppressors are natural products (beta-lapachone, diospyrin, topostatin, topostin, flavonoids) which are believed to interact directly with the enzyme. This review is concerned with the different families of topoisomerase I poisons and suppressors. Their origin, chemical nature and mechanism of action are presented. The relationships between drug binding to DNA and topoisomerase I inhibition are discussed.

Human DNA Topoisomerase I: Quantitative Analysis of the Effects of Camptothecin Analogs and the Benzophenanthridine Alkaloids Nitidine and 6-Ethoxydihydronitidine on DNA Topoisomerase I-Induced DNA Strand Breakage

Human DNA topoisomerase I (topo I) has been purified from normal placenta and from a recombinant baculovirus expression system. A new radiolabeled plasmid DNA assay has been used to quantitate the activity of the purified enzymes and to compare the ability of several types of topo I-targeted drugs to induce topo I-mediated DNA strand breaks. The 100-kDa recombinant enzyme form isolated from the baculovirus expression system is able to relax 2564 ng of supercoiled M-13 mp19 plasmid per minute per nanogram of enzyme. The addition of camptothecin (1 μM) to the reaction lowers the rate to 1282 ng per minute per nanogram of enzyme. The 100-kDa topo I from human placenta is able to relax 1092 ng of supercoiled plasmid per minute per nanogram of enzyme and the 68-kDa topo I form from placenta is able to relax 2069 ng of supercoiled plasmid per minute per nanogram of enzyme. Camptothecin (1 μM) decreases the relaxation rate of the placental enzymes about 50%. In the presence of several different types of topo I-targeted drugs, both the recombinant and placental enzymes are induced to cleave plasmid DNA. Quantitative DNA cleavage assays with radioactive plasmid DNA and 9-aminocamptothecin, topotecan, SN-38, 10,11-methylenedioxycamptothecin, 7-ethyl-10,11-methylenedioxycamptothecin, 7-chloromethyl-10,11-methylenedioxycamptothecin, nitidine, and 6-ethoxy-5,6-dihydronitidine indicate that the order of potency in inducing topo I-mediated DNA breakage is methylenedioxycamptothecin analogs > SN-38 > 9-aminocamptothecin > topotecan and camptothecin > nitidine compounds. The order of potency correlates with the half-lives of the topo I-DNA drug complex determined with radiolabeled DNA in 0.45 M NaCl at 30°C. The half-life of the complex formed with 7-chloromethyl-10,11-methylenedioxycamptothecin is greater than 90 min whereas the half-life of the topo I–DNA complex with 6-ethoxy-5,6-dihydronitidine is less than 15 s. The other drugs tested were found to have drug complex half-lives which fall between these two extremes.

A Naturally Occurring Compound for Controlling Powdery Mildew of Greenhouse Roses

Quaternary benzophenanthridine alkaloids (QBAs) isolated from plants in the family Papaveraceae are effective for the control of some fungal diseases. Extracts from Macleaya cordata, a species rich in QBAs, were formulated at 150 mg·L–1 QBA for spray application to greenhouse roses (Rosa sp.) infected with Sphaerotheca pannosa var. rosae (powdery mildew). The QBA formulation was applied at 10-day intervals. For comparison, copper sulfate pentahydrate, piperalin, and fenarimol also were applied to mildewinfected plants within the same greenhouse at their respective labeled rates. One day after treatment, visible symptoms of mildew infection were reduced 60% by QBA, whereas fenarimol, copper sulfate pentahydrate, and piperalin reduced the symptoms of infection 50%, 75%, and 85%, respectively. Subsequent studies demonstrated that a tank mix of QBA and piperalin provided enhanced control of powdery mildew on rose. Results from this study indicate that QBAs have the potential to be developed as a biorational fungicide for greenhouse use with both fungicidal and fungistatic activity.

Two antimicrobial alkaloids from Bocconia arborea

Two alkaloid constituents of Bocconia arborea showed considerable antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans. Bioactivity-guided fractionation with thin-layer bioautography led to the isolation of the two known benzophenanthridine alkaloids, dihydrochelerythrine and dihydrosanguinarine. The minimal inhibitory concentration was determined for each compound using a twofold serial dilution assay. The structures of these compounds were determined by 1H and 13C NMR analyses.

550 A Naturally Occurring Compound for Controlling Powdery Mildew of Greenhouse Roses

Quaternary benzophenanthridine alkaloids (QBAs) isolated from plants in the family Papaveraceae are effective for the control of some fungal diseases. Extracts from Macleaya cordata, a species rich in QBAs, were formulated at 150 mg·L–1 QBA for spray application to greenhouse roses infected with Sphaerotheca pannosa var. rosae (powdery mildew). The QBA formulation was applied at 10-day intervals. For comparison, copper sulfate pentahydrate, piperalin, and fenarimol also were applied to mildew-infected plants within the same greenhouse at their respective labeled rates. One day after treatment, visible symptoms of mildew infection were reduced 60% by QBA, whereas fenarimol, copper sulfate pentahydrate, and piperalin reduced the symptoms of infection 50%, 75%, and 85%, respectively. Subsequent studies demonstrated that a tank mix of QBA and piperalin provided enhanced control of powdery mildew on rose. Results from this study indicate that QBAs have the potential to be developed as a biorational fungicide for greenhouse use with both fungicidal and fungistatic activity.

Effects of alginate and immobilization by entrapment in alginate on benzophenanthridine alkaloid production in cell suspension cultures of Eschscholtzia californica

The production of benzophenanthridine alkaloids (sanguinarine, chelerythrine and macarpine) in cells of Eschscholtzia californica is enhanced by sodium alginate and by entrapment in Ca2+-alginate. Tyrosine decarboxylase, a key enzyme of alkaloid biosynthesis, is induced by the treatments. Alginate- entrapped cells are elicited over an extended period of time which leads to increased alkaloid biosynthesis (up to 800-fold enhancement). A major portion of alkaloids produced are released into the growth medium.

A redox-dependent, G-protein-coupled phospholipase A of the plasma membrane is involved in the elicitation of alkaloid biosynthesis in Eschscholtzia californica

In cultured cells of Californian poppy formation of benzophenanthridine alkaloids can be triggered by a yeast elicitor preparation independently of the hypersensitive reaction. A plasma membrane (PM) bound phospholipase A (PLA) is likely to play a role in the signalling process: PLA activity was detectable in individual cells, cell suspensions and PM vesicles with the fluorogenic phospholipid bis-BODIPY FL C 11-PC and was sensitive to known inhibitors of PLA 2. In microscopic assays, enzyme activity increased after elicitor contact of cells that were pretreated with non-saturating concentrations of PLA 2 inhibitors. In PM vesicles a PLA 2-like protein as well as G α- and G β-proteins were detected immunologically. Anti-G α or anti-G β antisera or mastoparan stimulated PLA activity thus indicating a G-protein-controlled enzyme. Elicitation of alkaloid production was sensitive to aristolochic acid and enhanced by PLA 2 products such as lysophosphatidylcholine and linolenic acid. Pretreatment of the cells with the artificial electron acceptors hexabromoiridate(V) or ferricyanide(III) reversibly abolished the effect of subsequent elicitation and reduced the activity of PLA both in intact cells and in PM vesicles. It appears, therefore, that PLA 2 is a point of interference of redox control with the signal path.

Shifts of intracellular pH distribution as a part of the signal mechanism leading to the elicitation of benzophenanthridine alkaloids . Phytoalexin biosynthesis in cultured cells of eschscholtzia californica

Cultured cells of Eschscholtzia californica (Californian poppy) respond to a yeast elicitor preparation or Penicillium cyclopium spores with the production of benzophenanthridine alkaloids, which are potent phytoalexins. Confocal pH mapping with the probe carboxy-seminaphthorhodafluor-1-acetoxymethylester revealed characteristic shifts of the pH distribution in challenged cells: within a few minutes after elicitor contact a transient acidification of cytoplasmic and nuclear areas occurred in parallel with an increase of the vacuolar pH. The change of proton concentration in the vacuole and in the extravacuolar area showed a nearly constant relation, indicating an efflux of vacuolar protons into the cytosol. A 10-min treatment with 2 mM butyric or pivalic acid caused a transient acidification of the cytoplasm comparable to that observed after elicitor contact and also induced alkaloid biosynthesis. Experimental depletion of the vacuolar proton pool reversibly prevented both the elicitor-triggered pH shifts and the induction of alkaloid biosynthesis. pH shifts and induction of alkaloid biosynthesis showed a similar dependence on the elicitor concentration. Net efflux of K+, alkalinization of the outer medium, and browning of the cells were evoked only at higher elicitor concentrations. We suggest that transient acidification of the cytoplasm via efflux of vacuolar protons is both a necessary and sufficient step in the signal path toward biosynthesis of benzophenanthridine alkaloids in Californian poppy cells.