Sonja Sturm

Oxindole alkaloids from Uncaria tomentosa induce apoptosis in proliferating, G0/G1-arrested and bcl-2-expressing acute lymphoblastic leukaemia cells

Natural products are still an untapped source of promising lead compounds for the generation of antineoplastic drugs. Here, we investigated for the first time the antiproliferative and apoptotic effects of highly purified oxindole alkaloids, namely isopteropodine (A1), pteropodine (A2), isomitraphylline (A3), uncarine F (A4) and mitraphylline (A5) obtained from Uncaria tomentosa, a South American Rubiaceae, on human lymphoblastic leukaemia T cells (CCRF‐CEM‐C7H2). Four of the five tested alkaloids inhibited proliferation of acute lymphoblastic leukaemia cells. Furthermore, the antiproliferative effect of the most potent alkaloids pteropodine (A2) and uncarine F (A4) correlated with induction of apoptosis. After 48 h, 100 μmol/l A2 or A4 increased apoptotic cells by 57%. CEM‐C7H2 sublines with tetracycline‐regulated expression of bcl‐2, p16ink4A or constitutively expressing the cowpox virus protein crm‐A were used for further studies of the apoptosis‐inducing properties of these alkaloids. Neither overexpression of bcl‐2 or crm‐A nor cell‐cycle arrest in G0/G1 phase by tetracycline‐regulated expression of p16INK4A could prevent alkaloid‐induced apoptosis. Our results show the strong apoptotic effects of pteropodine and uncarine F on acute leukaemic lymphoblasts and recommend the alkaloids for further studies in xenograft models.

Mass spectrometry and NMR spectroscopy: modern high-end detectors for high resolution separation techniques--state of the art in natural product HPLC-MS, HPLC-NMR, and CE-MS hyphenations.

Current natural product research is unthinkable without the use of high resolution separation techniques as high performance liquid chromatography or capillary electrophoresis (HPLC or CE respectively) combined with mass spectrometers (MS) or nuclear magnetic resonance (NMR) spectrometers. These hyphenated instrumental analysis platforms (CE-MS, HPLC-MS or HPLC-NMR) are valuable tools for natural product de novo identification, as well as the authentication, distribution, and quantification of constituents in biogenic raw materials, natural medicines and biological materials obtained from model organisms, animals and humans. Moreover, metabolic profiling and metabolic fingerprinting applications can be addressed as well as pharmacodynamic and pharmacokinetic issues. This review provides an overview of latest technological developments, discusses the assets and drawbacks of the available hyphenation techniques, and describes typical analytical workflows.

Capillary electrophoretic analysis of oxindole alkaloids fromUncaria tomentosa

Abstract The main oxindole alkaloids from the root bark ofUncaria tomentosa were separated by capillary electrophoresis. The electrophoretic parameters for the separation of the six alkaloids were optimized by studying the impact of the buffer composition, the ionic strength and the pH and the applied electric field on the separation efficiency of the analytical system. The best separation was achieved with a fused-silica capillary tube and 20 mM phosphate running buffer (pH 5.6) at a constant voltage of 10 kV. The determination of oxindole alkaloids in a crude methanolic extract ofUncaria tomentosa demonstrates the applicability of this method.

Tyrolobibenzyls E and F from Scorzonera humilis and distribution of caffeic acid derivatives, lignans and tyrolobibenzyls in European taxa of the subtribe Scorzonerinae (Lactuceae, Asteraceae).

A chemosystematic study of the subtribe Scorzonerinae, a subtribe of the Lactuceae tribe of the Asteraceae family was performed, using the recently discovered tyrolobibenzyls as well as lignans and caffeic acid derivatives as diagnostic characters. In addition to the known compounds two new tyrolobibenzyls (E and F) were isolated and their structures were established by mass spectrometry and 1D and 2D NMR spectroscopy. Twenty four samples from rootstocks of seventeen different Scorzonerinae taxa, comprising members of three genera (Podospermum, Scorzonera, and Tragopogon), were analyzed. Tyrolobibenzyls A (1), B (2), C (5), D (3), E (6), and F (4) were identified in crude extracts by means of HPLC retention times, on-line UV spectra and on-line MS/MS spectra. Quantification of these compounds was performed by HPLC, using 2,2-bis-(4-hydroxyphenyl)-propane as an internal standard. Tyrolobibenzyls A-F were only detected in samples from Scorzonera humilis, while chlorogenic acid and 3,5-dicaffeoylquinic acid were detected in all samples investigated. In contrast, caffeoyl tartaric acid and cichoric acid were not detectable in any member of the subtribe Scorzonerinae.

Analysis of cucurbitacins in medicinal plants by high-pressure liquid chromatography-mass spectrometry.

HPLC–MS (electrospray ionization or atmospheric pressure chemical ionization) was applied to the analysis of cucurbitacins in medicinal plants. High molecular weight plant constituents were separated from cucurbitacins by gel chromatography of the crude methanolic extracts. HPLC was performed on a Zorbax SB-C-18 column using a 0.01% trifluoroacetic acid:acetonitrile gradient and diode array detection. The method described was sensitive, and its applicability was demonstrated through the determination of cucurbitacins in a number of medicinal plants such as Citrullus colocynthis (Cucurbitaceae), Bryonia cretica ssp. dioica (Cucurbitaceae), Gratiola officinalis (Scrophulariaceae), Picrorhiza kurroa (Scrophulariaceae) and Iberis umbellata (Brassicaceae). Copyright

Liquid chromatography–nuclear magnetic resonance coupling as alternative to liquid chromatography–mass spectrometry hyphenations: Curious option or powerful and complementary routine tool?

Combining the most powerful separation techniques, i.e. liquid chromatography (LC) or capillary electrophoresis (CE) with a information rich detection system - the mass spectrometer or the nuclear magnetic resonance (NMR) spectrometer - has been pursued for more than three decades. This compilation shall provide an overview of the advantages and limitations of the LC-NMR hyphenation in the light of its most valued application-the unequivocal analyte identification. Especially the post LC trapping of analytes with an in-line solid phase extraction (SPE) device prior to transferring the analyte of interest to the NMR spectrometer (LC-SPE-NMR) proved to be a robust installation allowing a significant cut-down of the amount of analyte needed for the generation of high quality heteronuclear NMR shift correlation data. Different available technical realizations will be discussed and typical application examples from natural product research and from industrial settings will be given.

HPLC analysis of the main oxindole alkaloids fromUncaria tomentosa

SummaryThe main oxindole alkaloids from the root bark ofUncaria tomentosa were separated by reversed phase HPLC with an acetonitrile/methanol/phosphate buffer solvent gradient. UV detection was carried out at 245 nm. The chromatographic parameters for the separation of the alkaloids were optimized by studying the impact of the pH value of the mobile phase and the column temperature on the separation efficiency of the analytical system. The best separation was achieved with a mobile phase pH of 6.6 and a column temperature of 15°C. The method developed is suitable for the qualitative characterisation and quantitative determination of oxindole alkaloids in crudeUncaria tomentosa extracts and phytopharmaceuticals.

Preparative isolation and purification of alkannin/shikonin derivatives from natural products by high-speed counter-current chromatography†

Alkannin and shikonin (A/S) and their derivatives have been found in the roots of several Boraginaceous species and are also produced through plant tissue cultures. The chiral compounds A/S are potent pharmaceutical substances with a wide spectrum of biological and pharmacological activities like wound healing, antimicrobial, anti-inflammatory, anticancer and antioxidant activity. High-speed counter-current chromatography (HSCCC) was applied for the first time to the separation, preparative isolation and purification of A/S and their esters from extracts of Alkanna tinctoria roots, as well as commercial samples. The constituents of HSCCC fractions and their purity were determined by high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS), since DAD cannot detect oligomeric A/S derivatives that are present in most of the samples containing the respective monomeric derivatives. The purity of HSCCC fractions was compared with the one of fractions isolated by column chromatography (CC) using as stationary phases silica gel and Sephadex LH-20. As shown, the purity of monomeric alkannin/shikonin was greater by HSCCC than CC separation of commercial A/S samples.

Analysis of Iridoid Glycosides from Picrorhiza kurroa by Capillary Electrophoresis and High Performance Liquid Chromatography - Mass Spectrometry

SummaryA micellar electrokinetic chromatography (MEKC) method was applied to the analysis of iridoid glycosides ofPicrorhiza kurroaRoyle ex Benth. Baseline separation was achieved within 16 min using a fused silica capillary and a borate buffer solution (100 mM, pH 8.60) containing 30 mM SDS and 1% acetonitrile. The applied voltage was 25 kV, the thermostating temperature was kept constant at 30°C. Injection was performed in the pressure mode for 2 s, the detection wavelength was 205 nm. For optimization of the CE method an iridoid-containing fraction of a methanolic extract ofP. kurroa was injected. The impact of the electrolyte composition, the pH value, the ionic strength, the concentration of SDS, the influence of organic additives, the voltage and the temperature on resolution of adjacent peaks was studied. The optimized method was used for quantitative determination of the main iridoids in crude methanolic extracts ofP. kurroa.Analysis was also performed by HPLC-MS using an electrospray ionization (ESI) interface. Conditions were optimized both for efficient HPLC and for the MS system. Mass spectra of all HPLC peaks showed one dominant signal corresponding to [M+Na]+. The good agreement of quantitative CE results with those obtained by LC clearly demonstrated the applicability of the presented methods.

Nonaqueous capillary electrophoresis-electrospray ionization-ion trap-mass spectrometry analysis of pyrrolo-and pyrido[1,2-a]azepine alkaloids in Stemona

Stemona alkaloids represent an outstanding class of natural compounds due to their pharmacological profile and their complex and unusual molecular structures. The aim of this study was the development of the first CE method for the separation, identification and quantification of these pyrrolo‐ and pyrido[1,2‐a]azepine derivatives in three Stemona species. The best results were obtained with a NACE‐ESI‐IT‐MS method, utilizing an electrolyte of 50 mM ammonium acetate, 1 M acetic acid and 10% methanol in ACN and a separation voltage of 30 kV. Samples were injected voltage‐assisted with 20 kV for 1 s. Isopropanol:water (1:1) was used as ESI sheath liquid at a flow rate of 3 µL/min. The assay was applied for the qualitative profiling of Stemona alkaloids in S. curtisii, S. collinsae and S. tuberosa. For unambiguous peak assignment of more than forty unidentified alkaloids MS/MS experiments were performed and fragmentation patterns studied. Subsequently the method was validated for the quantitative determination of four selected derivatives (RSD inter‐ and intraday <6%, LODs <7.5 µg/mL, LOQs <25.0 µg/mL, for all analytes, recovery rates >98.9%) in several Stemona sp. extracts.