Preliminary studies on selenium-containing proteins in Brassica juncea by size exclusion chromatography and fast protein liquid chromatography coupled to ICP-MS

An approach for screening and resolving selenium-containing plant proteins was developed based on the combination of sample preparation and multi-dimensional liquid chromatography coupled to ICP-MS. Different protein extraction protocols were investigated. A 24 h dodecylsulfate-mediated protein extraction in a sonication bath followed by acetone precipitation was found to be optimal. The use of different protein precipitate solubilizing agents (sodium dodecyl sulfate media and Tris-HCl buffer) demonstrates possible fractionation of the selenium-containing proteins. Selenium-containing protein screening and fractionation were carried out by means of SEC-ICP-MS. High molecular weight selenium containing proteins were solubilized with a sodium dodecyl sulfate-containing buffer, whereas the low molecular weight compounds were released into a Tris-HCl buffer. Size exclusion chromatography-fast protein liquid chromatography coupled to ICP-MS allowed separation and detection of several selenium-containing proteins in Se-supplemented wild type Brassica juncea plant, a fast growing selenium accumulator.     

Characterisation of selenium compounds in rye seedling biomass using <Superscript>75</Superscript>Se-labelling/SDS-PAGE separation/γ-scintillation counting,and HPLC-ICP-MS analysis of a range of enzymatic digests

In the present study, selenium-enriched plant biomass was investigated to evaluate the ability of rye seedlings to take up, and assimilate, inorganic selenium. Two different analytical approaches were used. Electrophoretic separation (SDS-PAGE) of proteins extracted from ⁷⁵Se-labelled biomass was used to investigate the biotransformation of selenite into organic forms of the element. Ion-pair chromatography coupled with ICP-MS detection was chosen for the analysis of selenium species, enzymatically extracted from the plant biomass. The results of three enzymatic hydrolysis procedures and three sequential enzymatic extractions procedures are compared. The most effective single extraction was proteolysis (using protease type XIV), giving an overall extraction efficiency of 48%. However, for combinations of enzymes, the most effective was cellulase (Trichoderma viride) followed by sequential extraction of the solid pellet using protease type XIV, giving an extraction efficiency of 70%. The complementary data from the electrophoretic fractionation of proteins, and the HPLC separation of Se-species in the proteolytic digests, reveal the existence of large number of selenium-containing compounds in the rye seedling plant biomass. The results showed the complete biotransformation of inorganic selenium into organic forms during germination of the rye seedlings. HPLC-ICP-MS analysis of extracts from the plant biomass did not show the presence of selenate or selenite. At the time of this study, the lack of suitable organic-MS facilities meant that it was not possible to characterise them fully. However, the data does show that a combination of different enzymes, rather than just the commonly-used protease, should be considered when developing an extraction strategy for selenium in different food types to those already reported in the literature.     

Insecticidal volatiles from the marigold plant (genustagetes). Effect of species and sample manipulation

Summary Volatiles from three species of the genustagetes, commonly called marigold have been isolated and characterized. Simultaneous steam distillation extractions (SSDE) produced consistently extracts of higher insecticidal activity than Soxhlet extractions. Methylene chloride was the best solvent. Volatiles isolated from theminutae species showed higher activity than those frompatula anderecta. Comparison of extracts from the flower, foliage and roots of the plant showed that most of the activity is located in the flower. The volatiles are highly effective toward both larvae and adult mosquitoes.     

Profiling of Alk(en)ylresorcinols in cereals by HPLC-DAD-APcI-MS n

5-Alk(en)ylresorcinols in rye, wheat, spelt, and barley have been characterized by high-performance liquid chromatography coupled to atmospheric pressure chemical ionization multistage mass spectrometry (HPLC-APcI-MS ⁿ ) for the first time. Among the 29 compounds analysed, several major and minor C₁₅, C₁₇, C₁₉, C₂₁, C₂₃, and C₂₅-substituted resorcinols with saturated, monoenoic, dienoic, and/or oxygenated side-chains were characterized by their specific fragmentation patterns in collision-induced dissociation experiments. Additionally, a C_27:0 homologue, which has probably been overlooked in previous studies based on HPLC alone, was detected in all cereals analysed. Furthermore, we provide tentative evidence for the occurrence of alkylresorcinols with triolefinic side-chains, which have, to our knowledge, so far not been reported in any cereal species.     

Effect of drying temperature and blanching on the degradation of chlorophyll a and b in mint (Mentha spicata Huds.) and basil (Ocimum basilicum): Analysis by high performance liquid chromatography with photodiode array detection

Summary The effect of drying conditions on the preservation of chlorophyll pigments in mint and basil have been investigated in order to determine the effects of drying temperature and whether or not there was a prior blanching.Pigments extracted from fresh and dried samples were analyzed by reversed phase high performance liquid chromatography coupled to a photodiode array detector; isocratic separation was performed on a Zorbax ODS C18 column.The purity of the chromatographic peaks of chlorophylls and breakdown products was investigated. The visible spectra of standard samples of chlorophylls and pheophytins were compared, using least squares normalization with those of peaks from the extracts of fresh and dried mint and basil. The study has shown that chlorophylls were better preserved when drying was preceded by a short blanching; if samples were not blanched before drying, the degradation of chlorophylls a and b was best prevented by drying at low temperatures.     

Bioaccumulation study of acrylates in algae (<Emphasis Type="Italic">Chlorella kessleri</Emphasis>) by Py-GC and Py-GC/MS

Acrylate monomers methylmethacrylate (MMA) and cyclohexylmethacrylate (CHMA) bioaccumulation has been determined in aquatic organism, algae (Chlorella kessleri). Algae were collected in amount of 0.4 mg and directly injected to the pyrolytical cell. In algae bodies accumulated monomers were analysed by pyrolysis gas chromatography (Py-GC) and pyrolysis gas chromatography coupled with mass spectrometry (Py-GC/MS). Traces of the accumulated monomers in algae body can be determined after 1-, 2-, 3-weeks of incubation. Maximum content of MMA was determined after 3-week of experiment, contrariwise in the case of CHMA after 2-week exposition. Relationship with pyrolysis temperature has also been studied.     

Analysis of cysteine-containing proteins using precolumn derivatization with <Emphasis Type="Italic">N</Emphasis>-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry

N-(2-Ferroceneethyl)maleimide (FEM) is introduced as an electroactive derivatizing agent for thiol functionalities in proteins. Using appropriate reaction conditions, the derivatization is completed within five minutes and no unspecific labeling of free amino functions is observed. Liquid chromatography/electrochemistry/mass spectrometry was used to detect the reaction products. The reagent is a useful tool for determining the number of free thiol groups or the total number of free and disulfide-bound thiol groups in proteins. The electrochemical cell provides additional information, because the increase in mass spectrometric response upon electrochemical oxidation of the neutral ferrocene to the charged ferrocinium groups is monitored. The method was successfully applied to the analysis of native proteins and their tryptic digests. Dedicated to Prof. Werner Engewald on the occasion of his 70th birthday.     

Identification of selenium-containing proteins in selenium-rich yeast aqueous extract by 2D gel electrophoresis, nanoHPLC-ICP MS and nanoHPLC-ESI MS/MS

An approach based on the consecutive use of nanoHPLC-ICP collision cell MS and nanoHPLC-electrospray MS was proposed for the analysis of water-soluble selenium-containing proteins in selenium-rich yeast after their separation by 2D gel electrophoresis (GE). An ultrasonic probe was employed for fast protein extraction avoiding sample heating and thus reducing the risk of protein degradation. The efficiency of different extraction steps were critically evaluated by total selenium analysis and size-exclusion chromatography (SEC)-ICP MS. Prior to electrophoresis proteins were purified by acetone precipitation. The protein-containing spots from 2D GE were excised and digested with trypsin. The digests obtained were analyzed by nanoHPLC-ICP MS in order to check for the presence of selenium-containing peptides; this allowed the detection of target proteins for further analyses (two out of five spots). The subsequent analyses of the selected digests by nanoHPLC-ES MS/MS allowed the attribution of amino acid sequences to peaks detected by ICP MS revealing the presence of two selenium-containing proteins: SIP 18 and HSP 12.     

Quantitative HPLC Determination and Stability Studies of Pyridostemin in Extracts and Water Dispersible Granule Formulations of Stemona curtisii

A reversed-phase high-performance liquid chromatographic method has been developed and validated for the determination of pyridostemin, the major pesticidal alkaloid found in Stemona curtisii. This methodology was applied to the investigation of plant extracts and water dispersible granule formulations. Stability indicating procedures have also been carried out. The chromatographic separation was on a C₁₈ column with a mixture of acetonitrile–water–triethylamine (30:70:0.12, v/v/v), using UV detection at 300 nm. Validation procedures showed that the method was specific, accurate and precise. The response was linear over a range of 5–25 μg mL⁻¹ with recoveries in the range of 98.28–102.85%. The RSD for intra- and inter-day precision were <0.72 and <1.29%, respectively. Extraction of plant material with dichloromethane gave a significantly higher pyridostemin content in the crude extracts when compared with extractions in methanol. Partial purification of the crude extracts by silica gel column chromatography was used to concentrate the mixture about fourfold. Degradation behavior of pyridostemin in the partially purified extracts followed first-order kinetics. The main pathways for its decomposition were base hydrolysis and oxidation.     

Determination of 20-hydroxyecdysone content by thin-layer chromatography and micellar electrokinetic chromatography

Summary Seasonal dependence of 20-hydroxyecdysone content ofSerratula tinctoria andSerratula wolffii (Asteraceae) was investigated by micellar electrokinetic chromatography (MEKC). Samples were collected each month through the vegetation period. The leaves were dried, milled and extracted with methanol. Clean-up of the extracts was by solid-phase extraction using a polyamide micro-column to remove flavonoids and other plant phenolics which can interfere with the analysis. This work deals with the separation of 20-hydroxyecdysone from polypodine B and the seasonal variation of 20-hydroxyecdysone concentration. Determinations have been performed by both thin-layer chromatography and capillary electrophoresis using micellar electrokinetic chromatography. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Characterization ofC-glycosylflavones fromDissotis rotundifolia by liquid chromatography — UV diode array detection — tandem mass spectrometry

Summary By high-performance liquid chromatography (HPLC) coupled with UV diode-array detection (DAD) and thermospray mass spectrometry (TSP-MS), four main constituents of a polar, whole plant extract fromDissotis rotundifolia T. were characterized. The fourC-glycosylflavones, isoorientin, orientin, vitexin and isovitexin were detected in the methanolic and hydroalcoholic extract of the plant as well as in the commercial drug preparation ‘Sirop de Dissotis’. Although the UV data and TSP mass spectra allowed rapid characterisation of all fourC-glycosylflavones, exact attribution of the peaks to their structures could not be achieved as neither the UV spectra nor the TSP mass spectra enabled differentiation of one position isomer from the other. Therefore a successful attempt was made to distinguish the 6-C from the 8-C-glycosylflavones by thermospray tandem mass spectrometry (TSP-MS-MS). The collision induced dissociation (CID) spectra of the particular ion [M+H-120]⁺ gave fragments which permitted differentiation of position isomers. To confirm the accuracy of on-line results, reference compounds were included in the HPLC study.     

An attempt to differentiate HPLC-ICP-MS selenium speciation in natural and selenised Agaricus mushrooms using different species extraction procedures

Total determination and speciation analysis of Se in commercial and selenised Agaricus mushrooms have been performed to investigate the Se species naturally occurring in non-enriched mushrooms as well as those present in specimens grown in a Se-enriched medium. Mushroom aqueous and enzymatic extracts have been analysed by three complementary chromatographic separation mechanisms (size-exclusion, anion-exchange and reversed-phase) coupled to an inductively coupled plasma mass spectrometer with an octopole reaction system. Post-column isotope dilution analysis has been used on-line with the separations for quantification of the Se species eluted. The ⁷⁸Se-to-⁷⁷Se isotope ratio was monitored after adequate corrections for both total determinations and Se species quantitative speciation. The results showed marked differences not only in total Se contents but also in Se species found in the two types of Agaricus mushrooms investigated. Selenomethionine was detected in both of them (free in commercial mushrooms and incorporated into proteins in selenised ones) together with a number of unknown selenocompounds.     

HPLC micro-fractionation coupled to a cell-based assay for automated on-line primary screening of calcium antagonistic components in plant extracts

High performance liquid chromatography (HPLC) micro-fractionation was successfully coupled to an automated ⁴⁵Ca²⁺ uptake assay using GH₄C₁ cells for the separation of natural product extracts and for the primary detection of their calcium antagonistic components. The reliability of the procedure was first established with a reference solution consisting of pure compounds with a known effect on the Ca²⁺ uptake. No loss of activity was observed to occur after HPLC micro-fractionation. Extracts of Peucedanum palustre and Pinus sylvestris, showing high and no inhibition of Ca²⁺ uptake as total extracts, respectively, were analysed and the inhibitory activity of the P. palustre extract could be traced to two components, identified as columbianadin and isoimperatorin. As expected, no significant inhibition was observed with the micro-fractionated P. sylvestris samples. In summary, the procedure was found to be applicable for primary detection of calcium antagonistic components in complex matrices and to significantly reduce the time previously needed for bioactivity-guided isolation.     

Biotransformation of Valdecoxib by Plant Cell Cultures

Valdecoxib is a new anti-inflammatory drug that is highly selective for inhibition of the inducible form of cyclooxygenase (COX-2). In the present study, biotransformation of valdecoxib was investigated in cell cultures of five medicinal plants, viz., Catharanthus roseus, Azadirachta indica, Capsicum annuum, Ervatamia heyneana, and Nicotiana tabacum. Identification of the biotransformed products was carried out by using high-performance liquid chromatography coupled with diode array detection and liquid chromatography–tandem mass spectrometry analysis. All the cultures transformed valdecoxib into more polar compounds, and C. roseus also produced one unknown compound that is less polar than the substrate. The reactions performed by these plant cell cultures include hydroxylation, methylation, and demethylation. Optimization studies were performed to investigate the effect of the day of extraction and substrate concentration on biotransformation.     

Study of the influence of carboxymethylcellulose on the absorption of selenium (and selected metals) in a target plant

The aim of the present paper is the study of the influence of a polysaccharide (carboxymethylcellulose, CMC) on uptake by a target plant Lactuca sativa (LS) of selenium and some metals. LS was grown on a well characterized soil: such as, treated with 1.5 mg kg⁻¹ Se(IV) only and with two levels of CMC (3 and 30 mg kg⁻¹). Similar experiments were carried out by using Se(VI) instead of Se(IV). Uptake was evaluated through the quantification of total content of Se in dried leaves and roots by a suitable technique (graphite furnace atomic absorption spectrophotometry, instrumental neutron activation analysis and differential pulse cathodic stripping voltammetry). Results evidenced as the uptake of selenium was dependent on the form of selenium added to the soil: Se(VI) is accumulated much more then Se(IV) according to its lower toxicity and higher mobility. The simultaneous presence of CMC led to a lower selenium uptake in leaves, whereas no clear influence was evidenced in roots. Furthermore, the presence of CMC influenced also the mobility process (soil→plant) of several other metals: a lower content of them was detected in plants when CMC was present in the soil.     

Determination of Diterpenoids and Flavonoids in Isodon rubescens by LC-ESI-MS-MS

The diterpenoids and flavonoids in Isodon rubescens were analyzed simultaneously by high-performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) for the first time. The fragmentation pathway of rubescensin A (oridonin) and rubescensin B (ponicidin) in an electrospray ion trap mass spectrometer was also investigated. A total of 10 compounds, including five diterpenoids and five flavonoids, were identified or preliminarily characterized based on their mass spectra. Six of them were reported from Isodon species for the first time.     

Distribution and speciation of selenium in Lecythis ollaria plant

Selenium has been determined in different parts of the Lecythis ollaria (LO) plant (Venezuela) and the determination of its speciation has been achieved in fruit seeds. The study has been performed using different analytical techniques including inductively coupled plasma atomic emission spectrometry (ICP-AES), differential pulse cathodic stripping voltammetry (DPCSV), instrumental neutron activation analysis (INAA), high performance liquid chromatography (HPLC) and mass spectrometry (MS). Different parts of the plant (leaves, bark, capsules and seeds) were examined as well as the soil where LO was growing. Among different considered parts, seeds show the highest content in selenium (5 g kg⁻¹) which is dependent on the maturation extent of the fruit. In seeds, about half of the total selenium content is soluble in water while the remaining is involved in protein structure. In the aqueous fraction, the prevailing form of selenium appears to be seleno-cystathionine with much lower amounts of Se(VI) and Se(IV).     

Asymmetric Synthesis of （R）- and （S）-Moprolol

A simple and effective procedure for the enantioselective synthesis of （R）- and （S）-moprolol was described. The key step was the asymmetric synthesis of enantiopure （R）- and （S）-guaifenesin, which were synthesized from enantioenriched （R）-3-chloro-l,2-propanediol and （S）-epichlorohydrin via kinetics of hydrolysis resolution of racemic epichlorohydrin by chiral Salen-Co^Ⅲ complex. The e.e. values of both the optical compounds were above 98%, and the chemical structures of the target compounds were confirmed by ^1H NMR, ^13C NMR, IR, and MS.     

Development and validation of a GC-MS method for rapid determination of galanthamine in Leucojum aestivum and Narcissus ssp.: a metabolomic approach

Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide salt for the treatment of Alzheimer's disease, is obtained from some Amaryllidaceae plants. A new method was developed and validated for its quantification by GC-MS in different plant sources: bulbs and leaves from Narcissus confusus; bulbs from N. pseudonarcissus cv. Carlton; and leaves and in vitro cultures from L. aestivum. Samples (50 mg) were extracted with methanol (1 mL) for 2 h, then aliquots of the extracts were silylated and analyzed by GC-MS. The calibration line was linear over a range of 15-800 μg galanthamine/sample, ensuring an analysis of samples with a content of 0.03-1.54% analyte referred to dry weight. The recovery was generally more than 95%. Good inter- and intra assay precision was observed (RSD < 3%). Principal component analysis of GC-MS chromatograms allowed discrimination of the plant raw material with respect to species, organs and geographical regions. The analytical method developed in this study proved to be simple, sensitive and far more informative than the routine analytical methods (GC, HPLC, CE and NMR), so it may be useful for quality control of plant raw materials in the pharmaceutical industry.     

Cytotoxic Activities of Extracts and Compounds from <Emphasis Type="Italic">Viscum coloratum</Emphasis> and its Transformation Products by <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The bioassay-oriented fractionation of mistletoe crude extracts (MCEE) using 75% ethanol and culture products of mistletoe transformed by Rhodobacter sphaeroides, a photosynthetic bacterium (PSBT), revealed that the high cytotoxic activities were due to the petroleum ether extracts (PEs) and the acid-precipitated proteins from the aqueous extracts (AQs) of MCEE and PSBT. The isolated triterpenes may account for the activities of the PEs of MCEE and PSBT, respectively. Extraction of MCEE using petroleum ether led to the isolation of 3-epi-betulinic acid (1), betulonic acid (2), oleanolic acid (3), and β-amyrin acetate (4), while petroleum ether extraction of PSBT led to the isolation of 1,3,4,betulinic acid (5), erythrodiol (6), and (3β)-olean-12-ene-3,23-diol (7). The PE of PSBT exerted higher cytotoxicity than the PE of MCEE, which was due to the different triterpene contents of these two extracts. The cytotoxic activities of all compounds were tested, and the results revealed that compounds 1, 2, 3, 5, 6, and 7 contributed significantly to the cytotoxicities of both PEs. The AQ of the PSBT exerted almost the same cytotoxic activity and lower toxicity compared to the AQ of the MCEE. These findings indicate that mistletoe products biotransformed by R. sphaeroides could be used to treat cancers, since they have lower toxicities and higher antitumor activities compared to standard treatments.