How polyamine synthesis inhibitors and cinnamic acid affect tropane alkaloid production

Hairy roots of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to divert the carbon flux from competing pathways and thus enhance productivity, the polyamine biosynthesis inhibitors cyclohexylamine (CHA) and methylglyoxal-bis-guanylhy, drazone (MGBG) and the phenylalamine-ammonia-lyase inhibitor cinnamic acid were used. CHA decreased the specific productivity of both alkaloids but increased significantly the release of scopolamine (approx 500%) when it was added in the mid-exponential phase. However, when CHA was added for only 48 h during the exponential phase, the specific productivity of both alkaloids increased (approx 200%), favoring scopolamine. Treatment with MGBG was detrimental to growth but promoted release into the medium of both alkaloids. However, when it was added for 48 h during the exponential phase, MGBG increased the specific productivity (approx 200%) and release (250–1800%) of both alkaloids. Cinnamic acid alone also favored release but not specific productivity. When a combination of CHA or MGBG with cinnamic acid was used, the results obtained were approximately the same as with each polyamine biosynthesis inhibitor alone, although to a lesser extent. Regarding root morphology, CHA inhibited growth of primary roots and ramification. However, it had a positive effect on elongation of lateral roots.     

Application of metabolic engineering to the production of scopolamine

Scopolamine is an alkaloid widely used in medicine for its anticholinergic activity. The aim of this review is to show that metabolic engineering techniques constitute a suitable tool to improve the production of tropane alkaloids, focusing in particular on scopolamine. We present an overview of results obtained by various research groups, including our own, who have studied the overexpression of genes involved in the biosynthesis of scopolamine in different plant species that produce tropane alkaloids. Experiments carried out to improve production in hairy root cultures will also be described, as well as those attempting to biotransform hyoscyamine into scopolamine in roots and transgenic tobacco cells.     

Expression of tropane alkaloids in the hairy root culture of Atropa acuminata substantiated by DART mass spectrometric technique

Agrobacterium rhizogenes-mediated 'hairy root' cultures were established in Atropa acuminata. The chemical profiling of the hairy roots was carried out by a new mass spectrometric technique, direct analysis in real time (DART). The intact hairy roots were directly analyzed by holding them in the gap between the DART ion source and mass spectrometer. Two alkaloids, atropine and scopolamine, were characterized. The structural confirmation of the two alkaloids was made through their accurate molecular formula determinations. This is the first report of establishing hairy roots in A. acuminata as well as application of the DART technique for the chemical profiling of its hairy roots.     

Strategies for supercritical fluid extraction of hyoscyamine and scopolamine salts using basified modifiers

The supercritical fluid extraction behaviors of hyoscyamine and scopolamine were investigated and found to be highly dependent upon the chemical nature of the compounds. Free bases of hyoscyamine and scopolamine were freely soluble in supercritical CO2 with increasing temperature and pressure; however, the salts of these alkaloids were not soluble under any experimental conditions. It was found that alkaline modifiers such as methanol basified with diethylamine could enhance the solubilities and extraction yields of these alkaloids from plant matrices as compared to other modifiers.     

Alkaloids of the Genus Datura: Review of a Rich Resource for Natural Product Discovery

The genus Datura (Solanaceae) contains nine species of medicinal plants that have held both curative utility and cultural significance throughout history. This genus’ particular bioactivity results from the enormous diversity of alkaloids it contains, making it a valuable study organism for many disciplines. Although Datura contains mostly tropane alkaloids (such as hyoscyamine and scopolamine), indole, beta-carboline, and pyrrolidine alkaloids have also been identified. The tools available to explore specialized metabolism in plants have undergone remarkable advances over the past couple of decades and provide renewed opportunities for discoveries of new compounds and the genetic basis for their biosynthesis. This review provides a comprehensive overview of studies on the alkaloids of Datura that focuses on three questions: How do we find and identify alkaloids? Where do alkaloids come from? What factors affect their presence and abundance? We also address pitfalls and relevant questions applicable to natural products and metabolomics researchers. With both careful perspectives and new advances in instrumentation, the pace of alkaloid discovery—from not just Datura—has the potential to accelerate dramatically in the near future.     

Seven New Monoterpenoid Indole Alkaloids from Gelsemium elegans

Seven new indole alkaloids were isolated from the roots of Gelsemium elegans Benth. and their structures were determined by spectroscopic analysis and chemical transformation from known alkaloids. Kounaminal ( 1 ) is a new koumine‐type alkaloid that contains an unusual aminal moiety. Humantenoxenine ( 2 ) and 15‐hydroxyhumantenoxenine ( 3 ) are humantenine‐type alkaloids that contain a novel β‐amino‐α,β‐unsaturated ketone residue. The other four novel alkaloids are two gelsedilam‐related and two gelsenicine‐related alkaloids.     

A Sequence of Aldol Condensations and a Michael Addition Giving a Cyclohexenone Ring System

 The reaction of 1,5-diphenylpentanetrione, 4-chlorobenzaldehyde, and acetone in the presence of methylamine resulted in 2,6-dibenzoyl-5-(4-chlorphenyl)-3-methyl-cyclohex-2-en-1-one, which was formed by a sequence of a Knoevenagel reaction, an aldol condensation, and a Michael addition in a one-pot reaction.     

Simultaneous determination of atropine,scopolamine, and anisodamine in Flos daturae by capillary electrophoresis using a capillary coated by graphene oxide

A novel CE method was developed for the separation and determination of three main tropane alkaloids in Flos daturae with a capillary coated by graphene oxide (GO). The GO‐coated capillary was characterized by SEM, energy dispersive X‐ray spectroscopy, and Raman spectroscopy, and the results indicated that the inner surface of the capillary was partially coated by GO. A phosphate solution (40 mM, pH7.0) containing 20% v/v methanol and 30% v/v acetonitrile was used as the running buffer for the analysis of the atropine, scopolamine, and anisodamine. The linear ranges of atropine, scopolamine, and anisodamine was 0.5–200 μg/mL with satisfactory correlation coefficients (R²) > 0.9987, and this novel method provided an efficient separation for three tropane alkaloids as well as a good reproducibility and stability. Finally, the method was successfully applied for the determination of these three tropane alkaloids in plant extracts.     

Two new indoloquinoline alkaloids from cryptolepis sanguinolenta: Cryptosanguinolentine and cryptotackieine

The roots of the indigenous West African shrub Cryptolepis sanguinolenta have proved to be a rich source of indoloquinoline alkaloids. To date, all of the alkaloids isolated have been analogs of indolo[3, 2-b]quinoline. We now wish to report examples of two new indoloquinoline alkaloids which differ in the fusion of the indole and quinoline rings. The first, cryptosanguinolentine, is an angular indolo[3, 2-c]quinoline. The second, cryptotackieine, is a linear indolo[2, 3-b]quinoline system. Both of these families of alkaloids are without precedent from C. sanguinolenta. The structures of both were established through the extensive use of inverse-detected micro nmr methods.     

A Sequence of Aldol Condensations and a Michael Addition Giving a Cyclohexenone Ring System

Summary.  The reaction of 1,5-diphenylpentanetrione, 4-chlorobenzaldehyde, and acetone in the presence of methylamine resulted in 2,6-dibenzoyl-5-(4-chlorphenyl)-3-methyl-cyclohex-2-en-1-one, which was formed by a sequence of a Knoevenagel reaction, an aldol condensation, and a Michael addition in a one-pot reaction. Received August 16, 2000. Accepted September 8, 2000     

Polyketide origin of 3-alkylpyridines in the marine mollusc Haminoea orbignyana

The paper reports the biosynthesis of the main alkylpyridine alkaloids, haminol-1 (1) and -2 (2), in the Mediterranean mollusc Haminoea orbignyana. Experiments were carried out by in vivo incorporation of [1,2- ¹³C₂ acetate]. Data give full account for a polyketide origin of haminols in the Mediterranean molluscs, showing the biosynthesis of these 3-alkylpyridine alkaloids by elongation with acetate of a starter unit of nicotinic acid.     

A non-aqueous solid phase extraction method for alkaloid enrichment and its application in the determination of hyoscyamine and scopolamine

A non-aqueous solid phase extraction (SPE) method utilizing silica based strong cation exchange (SCX) was developed and optimized for the enrichment of alkaloids. In this method, silica based SCX SPE columns were used for the elimination of non-alkaloid compounds and the preconcentration of alkaloids from the extracts. Mass spectrometry was employed to analyze the alkaloid-enriched fraction, and results showed that the SPE method developed in this study was effective for the removal of non-alkaloids. Then, this pretreatment method was combined with high performance liquid chromatography for the quantification of scopolamine and hyoscyamine from Scopolia tangutica Maxim. The recoveries of scopolamine and (-)-hyoscyamine were 98.51% and 91.12%, respectively. Relative standard deviation values were 1.4% for scopolamine and 1.6% for (-)-hyoscyamine. The linearity was good in the 0.01-0.8 mg mL(-1) range for hyoscyamine and 0.01-0.4 mg mL(-1) range for scopolamine.     

Rapid structural characterization of isomeric benzo[c]phenanthridine alkaloids from the roots of Zanthoxylum nitidium by liquid chromatography combined with electrospray ionization tandem mass spectrometry

The fragmentation mechanism of six alkaloids, namely: dihydronitidine, dihydrochelerythrine, 8-acetonyldihydronitidine, 8-acetonyldrochelerythrine, nitidine and 1,3-bis(8-dihydronitidinyl)acetone, was investigated by electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn). Tandem mass spectrometry experiments indicated that different substitution sites of the methoxyl groups at C-9 and C-10 or at C-10 and C-11 determined the different abundances of the MS2 fragmentation ions using the same collision energy. According to the different abundances of MS2 product ions, positional isomeric benzo[c]phenanthridine alkaloids can be differentiated. Moreover, ten constituents in the crude alkaloidol extract from the roots of Zanthoxylum nitidium were rapidly identified by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MSn), through comparing the retention times and ESI-MSn spectra with the authentic standards. This work demonstrates that not only the characteristic fragments but also the characteristic abundances of the fragment ions can be used for detailed structural characterization.     

Investigation of Tropane Alkaloids in Genetically Transformed <Emphasis Type="Italic">Atropa belladonna</Emphasis> L. Cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform – methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile – 30 mM phosphate buffer - methanol 12.2:79.7:8.1(v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K₅ (0,223 m/m%) and in hairy root clone #K₄ (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants.     

Simultaneous determination of atropine,scopolamine, and anisodamine from Hyoscyamus niger L. in rat plasma by high‐performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetics study

In order to investigate the pharmacokinetics of tropane alkaloids in Hyoscyamus niger L., a sensitive and specific high‐performance liquid chromatography with tandem mass spectrometry method for the simultaneous determination of atropine, scopolamine, and anisodamine in rat plasma is developed and fully validated, using homatropine as an internal standard. The separation of the four compounds was carried out on a BDS Hypersil? C₁₈ column using a mobile phase consisting of acetonitrile and water (containing 10 mmol ammonium acetate). Calibration curves were linear from 0.2 to 40 ng/mL for atropine, scopolamine, and from 0.08 to 20 ng/mL for anisodamine. The precision of three analytes was <5.89% and the accuracy was between ?1.04 to 2.94%. This method is successfully applied to rat pharmacokinetics analysis of the three tropane alkaloids after oral administration of H. niger extract. The maximum concentration of these three tropane alkaloids was reached within 15 min, and the maximum concentrations were 31.36 ± 7.35 ng/mL for atropine, 49.94 ± 2.67 ng/mL for scopolamine, and 2.83 ± 1.49 ng/mL for anisodamine. The pharmacokinetic parameters revealed areas under the curve of 22.76 ± 5.80, 16.80 ± 3.08, and 4.31 ± 1.21 ng/h mL and mean residence times of 2.08 ± 0.55, 1.19 ± 0.45, and 3.28 ± 0.78 h for atropine, scopolamine, and anisodamine, respectively.     

Biomimetic synthesis of neodihydrothebaine and bractazonine from thebaine

Photochemical irradiation of the morphinan alkaloid thebaine 1 followed by reduction, affords the dibenz[d,f]azonine alkaloids neodihydrothebaine 2 and bractazonine 3, in a sequence paralleling the proposed biosynthesis of these alkaloids in Papaver bracteatum.     

Alkaloids from the Roots of Stemona tuberosa

Four new alkaloids, didehydrotuberostemonine A ( 1 ), stemoninone ( 2 ), tuberostemospiroline ( 3 ), and tuberostemonine L ( 4 ), together with seven known alkaloids, were isolated from the roots of Stemona tuberosa. Their structures were elucidated on the basis of spectroscopic analysis. The known alkaloids were identified as 2‐oxostenine ( 5 ), tuberostemonine ( 6 ), sessilifoliamide H ( 7 ), tuberostemonone ( 8 ), didehydrotuberostemonine ( 9 ), bisdehydrostemoninine ( 10 ), and tuberostemoamide ( 11 ).     

Functional genomic analysis of alkaloid biosynthesis in Hyoscyamus niger reveals a cytochrome P450 involved in littorine rearrangement

Tropane alkaloids are valuable pharmaceutical drugs derived from solanaceous plants such as Hyoscyamus niger (black henbane). The biosynthesis of these molecules, including the nature of the enigmatic rearrangement of (R)-littorine to (S)-hyoscyamine, is not completely understood. To test the hypothesis that a cytochrome P450 enzyme is involved in this rearrangement, we used virus-induced gene silencing to silence a cytochrome P450, CYP80F1, identified from H. niger roots by EST sequencing. Silencing CYP80F1 resulted in reduced hyoscyamine levels and the accumulation of littorine. Hyoscyamine was observed in CYP80F1-expressing tobacco hairy roots supplied with (R)-littorine. Expression in yeast confirmed that CYP80F1 catalyzes the oxidation of (R)-littorine with rearrangement to form hyoscyamine aldehyde, a putative precursor to hyoscyamine, and without rearrangement to form 3'-hydroxylittorine. Our data strongly support the involvement of CYP80F1 in the rearrangement of littorine to hyoscyamine.     

New nitro-benzo[c]phenanthridine and indolopyridoquinazoline alkaloids from Zanthoxylum atchoum

The first phytochemical investigation of the roots of Zanthoxylum atchoum has led to the isolation of two new nitro-benzo[c]phenanthridine alkaloids 6-nitronitidine (1) and 6-nitro-8-methoxy-7,8-dihydronitidine (2), two new salts of indolopyridoquinazoline alkaloids 3-hydroxy-8,13-dihydro-14-methyl-5-oxo-7H-indolo[2’,3’:3,4]pyrido[2,1-b]quinazolin-14-ium (3) and its zwitterionic form 3-phenolate-8,13-dihydro-14-methyl-5-oxo-7H-indolo[2’,3’:3,4]pyrido[2,1-b]quinazolin-14-ium (4) along with 18 (5–22) known compounds. Their chemical structures were elucidated by spectroscopic analysis including 1D and 2D NMR and MS techniques. This is the first report of the nitro group on the biosynthesis of the natural benzo[c]phenantridine alkaloids. Compound 2 exhibited potent antibacterial activity against Staphylococcus aureus of MIC₅₀ = 4 μg·mL⁻¹.     

Three new sesquiterpene alkaloids from the root of Tripterygium wilfordii

<正>Three new sesquiterpene alkaloids,1-desacetylwilforgine(1),1-desacetylwilforine(2),and 9′-hydroxy-2-nicotinoylwilforine (3) were isolated from the roots of Tripterygium wilfordii Hook f.,along with six known alkaloids.Their structures were established on the basis of spectral analysis.