Anthraquinones from Ophiorrhiza pumila tissue and cell cultures

We have succeeded in initiating and establishing systems of tissue and cell cultures of Ophiorrhiza pumila. Examination of the constituents of the methanol extract of the cultured calli revealed the presence of 11 anthraquinones including two new ones whose structures have been rigorously proved using advanced spectroscopic methods. These findings demonstrated a remarkable difference in the constituents between the wild plants and the callus tissue or cultured cells; the former is devoid of anthraquinones and contains a variety of camptothecin-related alkaloids whereas the latter contains a significant amount of anthraquinones and shows no indication of the presence of alkaloids after sever…

High-performance liquid chromatographic, capillary electrophoretic and capillary electrophoretic–electrospray ionisation mass spectrometric analysis of selected alkaloid groups

Systems for efficient separation of selected alkaloid groups by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE-ESI-MS) are described. The optimized HPLC system was applied for the separation of 23 standard indole alkaloids as well as for qualitative and quantitative analyses of crude alkaloid extracts of Rauvolfia serpentina X Rhazya stricta hybrid cell cultures. The developed conditions for CE analysis proved to be efficient for separation of mixtures of standard indole and beta-carboline alkaloids. The described buffer system is also applicable in the combination of CE w…

Glucosylation of isatin-3-oxime followed by 2D in situ NMR in plant cells at highest magnetic field without labelling.

The glucosylation of isatin-3-oxime (1) was monitored by in situ 2D 1H-13C inverse correlated gradient assisted NMR spectroscopy in plant cell suspension cultures of Rauvolfia serpentina without labelling. The applied high magnetic field of 800 MHz allowed measurements within 20 min at concentrations of 1 of 5.76 mM. Complete glucosylation of 1 occurs inside the cells within 72 hours. During this time isatin-3-oxime-glucoside (2) accumulates without further metabolism.

Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis - Electrospray Mass Spectrometry

Development of an efficient system for the separation of indole alkaloids by high performance liquid chromatography and its applications

An efficient system for the analysis of indole alkaloids by HPLC on a reversed-phase column using an ion pair technique is described. The optimised chromatographic conditions allowed the successful separation of 22 standard monoterpenoid indole alkaloids (including some isomers) and tryptamine. The described HPLC system was applied to the analysis of alkaloids in intergeneric somatic hybrid cell cultures of Rauvolfia serpentina x Rhazya stricta.

Structure-based engineering of strictosidine synthase: auxiliary for alkaloid libraries.

SummaryThe highly substrate-specific strictosidine synthase (EC 4.3.3.2) catalyzes the biological Pictet-Spengler condensation between tryptamine and secologanin, leading to the synthesis of about 2000 monoterpenoid indole alkaloids in higher plants. The crystal structure of Rauvolfia serpentina strictosidine synthase (STR1) in complex with strictosidine has been elucidated here, allowing the rational site-directed mutation of the active center of STR1 and resulting in modulation of its substrate acceptance. Here, we report on the rational redesign of STR1 by generation of a Val208Ala mutant, further describing the influence on substrate acceptance and the enzyme-catalyzed synthesis of 10-m…

General approach for the analysis of various alkaloid classes using capillary electrophoresis and capillary electrophoresis-mass spectrometry

Abstract The analysis of various alkaloid classes employing capillary electrophoresis (CE) and on-line combined CE-mass spectrometry (CE-MS) is described. A CE method is presented for the analysis of alkaloids without derivatisation or purification. The separation of four different groups of alkaloids consisting of monoterpenoid indole alkaloids, protoberberines/benzophenanthridines, β-carboline alkaloids, and isoquinolines from poppy by free zone capillary electrophoresis has been obtained using a 1:1 mixture of 100 mmol 1 −1 ammonium acetate (pH 3.1) and acetonitrile. The influence of alkaloid structure on the electrophoretic mobility is discussed. The CE-MS reconstructed total ion curren…

Analysis of Rauwolfia Alkaloids Employing Capillary Electrophoresis-Mass Spectrometry

Abstract Capillary electrophoresis-mass spectrometry (CE-MS) was applied to analyse extracts of roots and cell suspension cultures from Rauwolfia serpentina. Most of the alkaloids known to be present in the respective plant material have been resolved via CE and assigned according to their electrospray mass spectra.

Improved detection of alkaloids in crude extracts applying capillary electrophoresis with field amplified sample injection

Abstract A simple and effective method for the sensitive detection of alkaloids in crude plant extracts applying capillary electrophoresis with field amplified sample injection (FASI) is described. This method was compared with normal pressure injection for the determination of alkaloids in methanolic extracts from roots of Berberis vulgaris L. (Berberidaceae) and Hydrastis canadensis L. (Ranunculaceae) using a 1:1 mixture of 200 mM ammonium acetate at pH 3.1 and methanol. By introducing a short plug of 70% methanol (v/v) before electrokinetic injection with 16 kV for 8 s the concentration sensitivity was 1000-times higher compared to hydrodynamic injection for 1 s. No difference between bo…

Capillary zone electrophoresis of alkaloids

Abstract A comprehensive discussion of important aspects for the analysis of alkaloids by capillary zone electrophoresis (CZE) is given. The influence of structure on the electrophoretic mobility (EM) of indole alkaloids was investigated using a running buffer which is generally applicable to the CZE analysis of alkaloids. The EM, which at the applied conditions was mostly dependent on the size and shape of the solvated analyte ions, was additionally affected by the presence of hydrogen bonds or ion–dipole interactions between protonated and unprotonated alkaloids of the same species. This could be derived from the existence of alkaloidal dimer cluster ions [2M+H]+ when mass spectrometry wa…