Identification of isomeric tropane alkaloids from Schizanthus grahamii by HPLC-NMR with loop storage and HPLC-UV-MS/SPE-NMR using a cryogenic flow probe

Two fully automated HPLC-NMR methods are reported and compared for the structure elucidation of four isomeric tropane alkaloids from the stem-bark of an endemic Chilean plant, Schizanthus grahamii Gill. (Solanaceae). The first approach interfaced a conventional HPLC column to NMR by means of a loop storage unit. After elution with a mobile phase consisting of deuterated water and standard protonated organic solvents, the separated analytes were momentarily stored in a loop cassette and then transferred one-at-a-time to the NMR flow probe for measurements. The second strategy combined HPLC with parallel ion-trap MS detection and NMR spectroscopy using an integrated solid-phase extraction (SPE) unit for post-column analyte trapping. The SPE cartridges were dried under a gentle stream of nitrogen and analytes were sequentially eluted and directed to a cryogenically cooled flow-probe with an NMR-friendly solvent. The structures of the four isomeric alkaloids, 3alpha-senecioyloxy-7beta-hydroxytropane, 3alpha-hydroxy-7beta-angeloyloxytropane, 3alpha-hydroxy-7beta-tigloyloxytropane and 3alpha-hydroxy-7beta-senecioyloxytropane, were unambiguously determined by combining NMR assignments with MS data.     

Combinative application of pH-zone-refining and conventional high-speed counter-current chromatography for preparative separation of alkaloids from Stephania kwangsiensis

A method which involves the combination of pH-zone-refining counter-current chromatography (pH-zone-refining CCC) and conventional high-speed counter-current chromatography (HSCCC) was established for the preparative separation of alkaloids from the crude extracts of Stephania kwangsiensis. pH-zone-refining CCC was first performed with the solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v), where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 2.0 g of crude extract, 370 mg of sinoacutine and 600 mg of a mixture of three other alkaloids were obtained. Then, the mixture was further separated by conventional HSCCC with the solvent system composed of n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v), yielding 42 mg of (-)-crebanine, 50 mg of (-)-stephanine and 30 mg of l-romerine from 150 mg mixture of three other alkaloids, respectively. The purities of the four compounds were all over 98% as determined by HPLC, and the chemical structures of the four compounds were confirmed by positive ESI-MS and (1)H NMR data. Results of the present study successfully indicated that this method was efficient for the preparative separation of alkaloids from natural plants.     

Effects of different elicitors on yield of tropane alkaloids in hairy roots of Anisodus acutangulus

The four tropane alkaloids have played a pivotal role in controlling diseases such as the toxic and septic shock, the organophosphorus poison and the acute lung injury. Here, the elicitation effect of different elicitors on the production of tropane alkaloids and the molecular mechanism of enzyme genes in the pathway was firstly demonstrated in hairy roots of Anisodus acutangulus. The results showed ethanol, methyl jasmonate and Ag⁺ could improve the accumulation of tropane alkaloids up to 1.51, 1.13 and 1.08 times after 24 h treatment, respectively (P < 0.05), whereas salicylic acid decreased the average content of tropane alkaloids. Furthermore, expression profile analysis results revealed that up-regulation of hyoscyamine-6b-hydroxylase (AaH6H) and little regulation of tropinone reducase II (AaTR2) elicited by ethanol, increased expression of putrescine N-methyltransferase I (AaPMT1) elicited by Ag⁺, elevated expression of tropinone reducase I (AaTR1) elicited by methyl jasmonate, respectively, resulted in tropane alkaloids improvement. Our results showed that hairy root culture of A. acutangulus in combination with elicitors was a promising way for production of tropane alkaloids in the future.     

Ontogenetic variation of the tropane alkaloids in Datura stramonium

Alkaloid profile of Datura stramonium plant parts collected at different stages of development were investigated by GC–MS. Sixty-four tropane alkaloids were detected and 48 of them were determined. Two new tropane alkaloids, 3-phenylacetoxy-6,7-epoxynortropane and 7-hydroxyapoatropine were tentatively identified. The alkaloids scopoline, 3-(hydroxyacetoxy)tropane, 3-hydroxy-6-(2-methylbutyryloxy)tropane, 3β-tigloyloxy-6-hydroxytropane, 3,7-dihydroxy-6-tigloyloxytropane, 3-tigloyloxy-6-propionyloxytropane, 3-phenylacetoxy-6,7-epoxytropane, 3-phenylacetoxy-6-hydroxytropane, aponorscopolamine, 3β,6β-ditigloyloxytropane and 7-hydroxyhyoscyamine are reported for the first time for this species and 3-acetoxy-6-isobutyryloxytropane for the family Solanaceae. The number and the type of alkaloids in the different plant organs depend on the stages of development. The variability by types of alkaloids (monosubstituted, 3,6- and 3,7-disubstituted, 3,6,7-trisubstituted and 3-substituted-6,7-epoxytropanes) decreases from the roots of the plants in vegetative growth and immature fruits to the seeds of senile plants (mainly monosubstituted and 3-substituted-6,7-epoxytropanes).     

Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Tropane alkaloids from Erythroxylum caatingae Plowman

Three tropane alkaloids, 1-3, were isolated from Erythroxylum caatingae, i.e., 6β-benzoyloxy-3α-[(4-hydroxy-3,5-dimethoxybenzoyl)oxy]tropane (1), a new tropane alkaloid, along with the known alkaloids 3α,6β-dibenzoyloxytropane (2) and 6β-benzoyloxy-3α-[(3,4,5-trimethoxybenzoyl)oxy]tropane (catuabine B; 3). Their structures were determined by 2D- ((1) H and (13) C) NMR. By LC/ESI-MS/MS analysis of the fractions of alkaloids 1-3, it was possible to detect five more alkaloids, 4-8, two of these, 4 and 8, possibly being new natural products. X-Ray crystallography of the chloride derivate of 1, i.e., 6β-benzoyloxy-3α-(4-hydroxy-3,5-dimethoxybenzoyloxy)tropane hydrochloride (1a) confirmed the structure of 1. Cytotoxicity was tested against the cell lines HEp-2, NCI-H292, and KB for the MeOH extract and alkaloid 3, and antitumor activity was tested against Sarcoma 180 only for the MeOH extract.     

Preparative isolation and purification of chemical components from Aconitum coreanum by high-speed counter-current chromatography coupled with evaporative light scattering detection

Aconitum coreanum (Lèvl.) Rapaics (Guanbaifu in Chinese) is a widely used, centuries-old Chinese herb. A preparative high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) method was employed for isolation and purification of alkaloids from the crude extract of Aconitum coreanum (Lèvl.) Rapaics using ethyl acetate-n-butanol-methanol-0.2 m HCl (7:2:2:7, v/v) as a two-phase solvent system. Six alkaloids, including GFO, GFQ, GFZ, hetisinone, hetisine and GFAA, were obtained in one-step separation. The purity of these compounds was 97.6, 93.8, 91.8, 91.9, 96.2 and 91.1%, respectively.     

In vitro organogenesis and alkaloid accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.     

Comparative study of the alkaloids in tribe Datureae and their chemosystematic significance

Sixty-six tropane alkaloids from crude leaf and root alkaloid mixtures of 12 different species and their varieties and subspecies from tribe Datureae were determined by GC–MS. The alkaloids 3β-hydroxy-6β-acetoxytropane, 3-propionyloxy-6-hyroxytropane, 3β-hydroxy-6β-tigloyloxytropane, 3β-tigloyloxy-6β-acetoxytropane and 3-tigloyloxy-7-isobutyryloxytropane are new reported tropane alkaloids.     

Calystegines in wild and cultivated Erythroxylum species

Calystegines were identified in the genus Erythroxylum for the first time. Erythroxylum novogranatense var. novogranatense, a species cultivated for cocaine production, contained 0.2% total calystegines in dry leaves. Forty six Erythroxylum herbarium species consisting mostly of leaf tissue were analysed for calystegines, and 38 were found positive. Calystegines were compared qualitatively and quantitatively between individual Erythroxylum species. Calystegines A(3) and B(2) were the major calystegines in most species. Total calystegine content reached up to 0.32% dry mass. The simultaneous occurrence of calystegines, cocaine, other alkaloids of a 3alpha-hydroxy- or 3beta-hydroxytropane structure together with nicotine supports the concept of common biosynthetic steps of these alkaloids in Erythroxylum. The present results are the basis for further investigations of the phylogenetic origin of tropane alkaloid biosynthesis in the taxonomically remote families Solanaceae and Erythroxylaceae.     

GC-MS investigation of tropane alkaloids in Datura stramonium

Alkaloids, GS-MS, Datura stramonium The alkaloid spectrum in roots, leaves and seeds of Datura stramonium L. was investigated by GC-MS. Twenty-nine tropane alkaloids are detected. Twelve of them are new constituents for the species and the two tropane esters 3-(3'-acetoxytropoyloxy)tropane (21) and 3-(2'-hydroxytropoyloxy)tropane (26) are described for the first time.     

Preparative isolation of alkaloids from Dactylicapnos scandens using pH-zone-refining counter-current chromatography by changing the length of the separation column

pH-Zone-refining counter-current chromatography was successfully applied for the preparative separation of alkaloids from Dactylicapnos scandens. The two-phase solvent system was composed of petroleum ether-ethyl acetate-methanol-water (3:7:1:9, v/v), where 20 mM of triethylamine (TEA) was added to the upper phase as a retainer and 5 mM of hydrochloric acid (HCl) to the aqueous phase as an eluter. In this experiment, the apparatus with an adjustable length of the separation column was used for the separation of alkaloids from D. scandens and the resolution of the compounds can be remarkably improved by increasing the length of the separation column. As a result, 70 mg protopin, 30 mg (+) corydine, 120 mg (+) isocorydine and 40 mg (+) glaucine were obtained from 1.0 g of the crude extracts and each with 99.2%, 96.5%, 99.3%, 99.5% purity as determined by HPLC. The chemical structures of these compounds were confirmed by positive ESI-MS and (1)H NMR.     

山莨菪植物体内4种莨菪烷类生物碱含量的变化

为了对山莨菪植物资源进行可持续利用,采用高效液相色谱（HPLC）技术对山莨菪植物体内4种莨菪烷类生物碱同时进行了定量测定,将4种生物碱很好地分离开来,大大缩短了出峰时间;并对这4种生物碱含量随着物候的变化进行了分析研究。研究结果表明：4种生物碱含量与物候的关系呈抛物线变化,樟柳碱在山莨菪植物体内含量最高,地上部分4种生物碱均在花盛期含量最高,地下部分不同的生物碱含量最高点则对应不同的物候期。     

Chemotaxonomy of the pantropical genus Merremia (Convolvulaceae) based on the distribution of tropane alkaloids

The occurrence and distribution of tropane and biogenetically related pyrrolidine alkaloids in 18 Merremia species of paleo-, neo-, and pantropical occurrence have been studied. The extensive GC-MS study included members of almost all sections of the genus and has been carried out with epigeal vegetative parts as well as with roots. It comprises altogether 74 tropanes and 13 pyrrolidines including nicotine. Along with datumetine known already from a solanaceous species, the study led to the isolation (from M. dissecta and M. guerichii, respectively) and structure elucidation (spectral data) of four novel 3alpha-acyloxytropanes, merresectines A-D: 3alpha-(4-methoxybenzoyloxy)nortropane (A), 3alpha-kurameroyloxytropane (B), 3alpha-nervogenoyloxytropane (C), 3alpha-[4-(beta-D-glucopyranosyloxy)-3-methoxy-5-(3-methyl-2-butenyl)benzoyloxy]tropane (beta-d-glucoside of D). Moreover, the novel 3alpha,6beta-di-(4-methoxybenzoyloxy)tropane (merredissine) has been isolated from M. dissecta and structurally elucidated. In addition the structures of datumetine and merresectine A could be confirmed by synthesis. Spectral data for two known 3alpha-acyloxytropanes (merresectine E beta-D-glucoside, 4'-dihydroconsabatine) and one known 3beta-acyloxytropane (concneorine) are documented for the first time. The structures of three further merresectines (F-H) have been determined by mass spectrometry. Furthermore, the linkage (2',3- and 2',4-, respectively) of two position isomer N-methylpyrrolidinylhygrines was proven by synthesis. The results of the study contribute to the solution of infrageneric taxonomic problems. Whereas all species yield pyrrolidine alkaloids without suitably differentiating results the diverging occurrence of tropane alkaloids leads to three groups of sections: (1) taxa free of tropanes, (2) taxa with simple tropanes, and (3) taxa with merresectines in addition to simple tropanes.     

Brassicaceae contain nortropane alkaloids

The report of cochlearine, the 3-hydroxybenzoate ester of tropine found in Cochlearia officinalis, Brassicaceae, initiated a screening for tropane alkaloids in Cochlearia species and for calystegines in further Brassicaceae. All ten Cochlearia species investigated contained cochlearine, tropine, and pseudotropine. Calystegines, nortropane alkaloids deriving from pseudotropine, were also identified in all Cochlearia species and accumulated up to 0.5% dry mass in leaves. Brassicaceae species of all major lineages of the family were analysed for calystegines. Of the 43 species included in the study, 18 accumulated calystegines of various structures. This is the first screening of Brassicaceae for products of the tropane alkaloid pathway, which is known as characteristic for plants of Solanaceae family. The identification of calystegines in all branches of the Brassicaceae family including Aethionema, a species at the basis of the family, suggests tropane alkaloids as secondary compound typical for Brassicaceae.     

Effects of Oxygen on Nicotine and Tropane Alkaloid Production in Cultured Roots Duboisia myoporoides

The effects of oxygen on nicotine and tropane alkaloid production in root cultures of Duboisia myoporoides were investigated. Duboisia roots cultured in air produced both nicotine and tropane alkaloids equally. However, when roots were cultured in pure oxygen, the metabolic flux to tropane alkaloids increased, and that to nicotine alkaloids decreased. Intermediate product analysis by GC-MS showed an increase in tropine, but decreases in acetyl derivatives of tropane alkaloids and tropine esters with low-class fatty acids. Furthermore, hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11, the key enzyme in the pathway from hyosyamine to scopolamine) also increased. These results suggest that pure oxygen contributes to scopolamine production not only by activating the biosynthetic steps for scopolamine, but also by inactivating the biosynthetic steps for nicotine and other tropine derivatives.     

Tropane alkaloids of Datura innoxia from Morocco

Fifty three alkaloids were identified in the organs (roots, stems, leaves, flowers, and seeds) of Datura innoxia by GC/MS. Seventeen of them are reported for the first time for this species and one nor-derivative, 3-phenylacetoxynortropane (28), for the genus Datura. Furthermore, four new tropane esters were tentatively identified as 3-acetoxy-6,7-epoxytropane (acetylscopine) (10), 3-acetoxy-6-propionyloxy-7-hydroxytropane (15), 6,7-dehydro-3-phenylacetoxytropane (25), and 3-(2'-phenylpropionyloxy)-6,7-epoxynortropane (dihydroaponorscopolamine) (37) on the basis of their mass spectral data. Hyoscyamine (44) and scopolamine (48) figure as main alkaloids in the roots and aerial parts, respectively.     

Effect of electrolyte and solvent composition on capillary electrophoretic separation of some pharmaceuticals in non-aqueous media

Non-aqueous capillary electrophoresis was used to study the separation selectivity of positively charged drug substances and negatively charged diuretics. Study was made of the effects of organic solvent composition and the background electrolyte on the separation. The separation selectivity could be altered considerably by varying the methanol/acetonitrile composition. In addition, the migration order and the resolution of the pharmaceuticals could be altered merely by changing the electrolyte cation or the anion. The electrolytes tested were alkali metal acetates, ammonium acetate, ammonium chloride and ammonium bromide. As with aqueous background electrolyte solutions, the electroosmotic flow was decreased with increasing size of the alkali metal cation of the electrolyte in methanol/acetonitrile 50:50 (v/v).     

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.