A comparison of alternative sample preparation procedures for the analysis of swainsonine using LC‐MS/MS

Introduction – Swainsonine, a polyhydroxy indolizidine alkaloid and known glycosidase inhibitor, is found in a number of different plants that cause a lysosomal storage disease known as locoism in the western USA. Most recently swainsonine has been analysed by LC‐MS/MS after sample extraction and preparation from ion‐exchange resins. Objective – To compare previously published sample preparation procedures with several new alternative procedures to provide methods using either commercially available solid‐phase extraction equipment or procedures which significantly reduce sample preparation time. Methodology – A previously reported and validated sample preparation method using ion‐exchange resin was compared with methods using a commercially available solid‐phase extraction cartridge, a solvent partitioning procedure or a single solvent extraction procedure using one of two solvents. Twenty different plant samples of varying swainsonine concentrations were prepared in triplicate and analysed by LC‐MS/MS. The measured concentration of swainsonine was then statistically compared between methods. Results – There were no statistically significant differences found between four of the five different sample preparation methods tested. Conclusion – A commercially available SPE cartridge can be used to replace the previously used ion‐exchange resin for swainsonine analysis. For very rapid analyses the SPE procedure can be eliminated and a simple, single solvent extraction step used for sample preparation. Published in 2010 by John Wiley & Sons, Ltd.     

Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.     

Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids.

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.     

甘肃棘豆草中苦马豆素的提取分离工艺比较研究

以甘肃棘豆草为试验材料,通过工业酒精热回流提取、水提取和酸水提取3种工艺提取其中的总生物碱,总生物碱经硅胶柱层析分离后减压升华得到,并比较了3种工艺对甘肃棘豆中总生物碱得率和苦马豆素得率的差异。结果表明,3种工艺总生物碱平均得率和苦马豆素平均得率依次分别为7.91、6.79、6.22 mg/g和18.61、12.22、6.23μg/g,由此确定工业酒精热回流为提取苦马豆素的最佳提取分离工艺。     

The uptake of swainsonine, a specific inhibitor of alpha-D-mannosidase, into normal human fibroblasts in culture

Swainsonine, an indolizidine alkaloid, found in plants of the genus Swainsona, has been shown to be a strong inhibitor in vitro of the alpha-D-mannosidase activity in normal human fibroblasts. Therefore, inhibition of alpha-D-mannosidase activity in extracts of harvested cells grown with swainsonine in the medium has been used to follow the association of the alkaloid with normal human fibroblasts in culture. Swainsonine that could not be removed by extensive washing became associated with the cells within 1 min, and it is concluded that the alkaloid is internalized rapidly by the cells. The amount of swainsonine taken up into the cells depended on the length of time in contact and the concentration of swainsonine in the medium, but at 37 degrees C a plateau of internalized swainsonine occurred after 2 hr with extracellular concentrations of swainsonine of 100 microM or greater. At lower concentrations of swainsonine the rate of uptake was found to be temperature-dependent, increasing greatly at 20 degrees C. The rapidity and temperature sensitivity of the uptake, together with the observation that mannose or mannose-6-phosphate did not prevent the association, suggest that swainsonine enters the cells by permeation rather than by endocytosis. When swainsonine is withdrawn from the culture medium, there is a decrease with time of cell-associated swainsonine. The kinetics of uptake and release of swainsonine and its slightly basic nature make it likely that swainsonine is concentrated initially in the lysosomes. This rapid, but reversible, concentration of swainsonine in lysosomes would be consistent with the observed effects of the toxin in vivo.     

Characterization of phosphatidylcholines in rabbit lung lavage fluid by positive and negative ion fast-atom bombardment mass spectrometry

The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M + H − 2H]⁺ ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods.     

The Structure of △"-Dehydroaloperine

A new alkaloid named ll-dehydroaloperine was isolated from the leaves and stalks of Sophora alopecuroides L. during flowering. Total alkaloid was obtained by diluted acid percolation and cation-exchange resin, then the new alkaloid was obtained via various polar solvent extraction under various pH value and Al2O3 column chromatography. The new alkaloid was proved to be analogous to aloperine through UV, IR, and mass spectrometry. Its structure was further identified by 1HNMR, 13CNMR, 1H-1H cosy spectrum, NOE as well as by some other chemical methods.     

Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis

Undifilum oxytropis (Phylum: Ascomycota; Family: Pleosporaceae) is a slow growing endophytic fungus that produces a toxic alkaloid, swainsonine. This endophyte resides in locoweeds, which are perennial flowering legumes. Consumption of this fungus by grazing animals induces a neurological disorder called locoism. The alkaloid swainsonine, an α-mannosidase inhibitor, is responsible for the field toxicity related to locoism. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi. Genetic manipulation of endophytic fungi is important to better understand biochemical pathways involved in alkaloid synthesis, but no transformation system has been available for studying such enzymes in Undifilum. In this study we report the development of protoplast and transformation system for U. oxytropis. Fungal mycelia required for generating protoplasts were grown in liquid culture, then harvested and processed with various enzymes. Protoplasts were transformed with a fungal specific vector driving the expression of Enhanced Green Florescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. In all cases, resistance to antibiotic hygromycin B was maintained. Such manipulation will open avenues for future research to decipher fungal metabolic pathways.     

Effect of swainsonine, an inhibitor of glycoprotein processing, on cultured mammalian cells

Swainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase II. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 μg/ml, swainsonine did not affect the growth rate of Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus-181 (SV-101), B-16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5-day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine-grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B-16 melanoma cells were grown for several days in the presence of swainsonine (100–500 ng/ml), these cells showed a 50–100% increase in their ability to bind [³H]concanavalin A, and a substantial decrease in the binding of [³H]wheat germ agglutinin. These alterations suggested an increase in high-mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B-886, a bacterium that binds to high-mannose glycoproteins, was also increased 1.5-to twofold, in cells grown in swainsonine. However, the binding of E. coli SS-142, another bacterial strain that does not bind to high-mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 μg/ml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [³H]leucine or [³H]proline into protein. There was, however, a significant inhibition in the incorporation of [³H]fucose, [³H]glucosamine, and [³H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, glucosamine by about 40 or 50%, and galactose by about 50%. In many cases (but not all), the incorporation of mannose was enhanced about 20–30% in cells grown in swainsonine.     

Mechanism of Inhibition of Jack Bean alpha-Mannosidase by Swainsonine

The indolizidine alkaloid, swainsonine, was previously shown to be a potent inhibitor of lysosomal and jack bean α-mannosidase (Dorling, Huxtable, Colegate 1980 Biochem J 191: 649-651). We examined the effects of various concentrations of this alkaloid on a number of commercially available glycosidases and found swainsonine to be quite specific for α-mannosidase (50% inhibition at 1-5 × 10⁻⁷ molar). Optimum inhibition was observed after a 2-minute preincubation of enzyme and inhibitor. Lineweaver-Burk plots of substrate concentration versus velocity in the presence of various amounts of swainsonine showed considerable curvature at high substrate concentrations, suggesting that swainsonine may be a competitive inhibitor that binds tightly to the enzyme and is only slowly removed. Periodate oxidation of swainsonine completely destroyed its inhibitory activity.     

A large-scale purification of paclitaxel from cell cultures of Taxus chinensis

A large-scale purification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. The complete method for mass production was a simple and efficient procedure, for the isolation and purification of paclitaxel from the biomass of Taxus chinensis, consisting of solvent extraction, synthetic adsorbent treatment, and two steps of precipitation, followed by two steps of high performance liquid chromatography (HPLC). The organic solvent extraction of biomass obtained crude extract containing paclitaxel. The use of synthetic adsorbent treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimise solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. The paclitaxel of over 99.5% purity can be simply obtained with high yield from crude paclitaxel by HPLC using reverse-phase separation on C18 as the first step and normal-phase separation on silica as the second step.     

小果博落回的生物碱分离与鉴定

以D101大孔吸附树脂为吸附剂,将对小果博落回全草的酸水提取物分为50%乙醇洗脱部分和丙酮洗脱部分.对两部分洗脱物分别进行硅胶柱色谱分离,由50%乙醇洗脱部分得到5个生物碱:二氢血根碱(1),二氢白屈菜红碱(2),6-乙氧基血根碱(3),6-丙酮基血根碱(4)和Bocconarborine A (5).由丙酮洗脱部分得到原阿片碱(6),α-别隐品碱(7),β-别隐品碱(8)和小檗红碱(9).采用波谱分析对化合物进行了结构表征,结果表明,化合物3、4、5、9是首次从该植物中分离得到.     

Matrix solid‐phase dispersion with sand in chromatographic analysis of essential oils in herbs

Introduction – Matrix solid‐phase dispersion (MSPD) is a very simple, cheap and relatively quick sample preparation procedure which involves simultaneous disruption and extraction of various solid and semi‐solid samples due to the direct mechanical blending of the sample with a SPE sorbent, mainly C₁₈. Little is known about MSPD application as a sample preparation method for the analysis of essential oil components in herbs. Objective – To evaluate if C₁₈ sorbent, commonly used in MSPD process, can be substituted with sand in the procedure of essential oil analysis. Methodology – Essential oil extracts were obtained from mint, sage, chamomile, marjoram, savory and oregano using MSPD with C₁₈ sorbent or sand, pressurised liquid extraction and steam distillation. Their qualitative and quantitative compositions ware established by GC‐MS and GC‐FID. Results – The results prove that C₁₈ sorbent can be substituted with sand in the procedure of essential oil analysis in herbs. The recoveries of essential oil components estimated using MSPD/sand are almost equal to those using pressurised liquid extraction. Conclusion – The results presented in the paper reveal that MSPD with sand is suitable for the isolation of essential oil components from herbs. Its extraction efficiency is equivalent to pressurised liquid extraction, recognised as one of the most efficient extraction methods. The cost of MSPD procedure for essential oil analysis can be significantly diminished by substituting C₁₈ with sand. Copyright © 2010 John Wiley & Sons, Ltd.     

Real encoded genetic algorithm and response surface methodology to optimize production of an indolizidine alkaloid, swainsonine, from Metarhizium anisopliae

Response surface methodology (RSM) and artificial neural network-real encoded genetic algorithm (ANN-REGA) were employed to develop a process for fermentative swainsonine production from Metarhizium anisopliae (ARSEF 1724). The effect of finally screened process variables viz. inoculum size, oatmeal extract, glucose, and CaCl₂ were investigated through central composite design and were further utilized for training sets in ANN with training and test R values of 0.99 and 0.94, respectively. ANN-REGA was finally employed to simulate the predictive swainsonine production with best evolved media composition. ANN-REGA predicted a more precise fermentation model with 103 % (shake flask) increase in alkaloid production compared to 75.62 % (shake flask) obtained with RSM model upon validation.     

Analysis of swainsonine and its early metabolic precursors in cultures of Metarhizium anisopliae

The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 μg ml-1. Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition. This revised version was published online in November 2006 with corrections to the Cover Date.     

A DNA extraction method for RAPD analysis from plants rich in soluble polysaccharides

A simple procedure for the isolation of DNA from mature leaf tissue was developed. This procedure purified DNA using Sephacryl S-1000 column and PEG 8000 precipitation. Polysaccharide-like components were successfully removed from DNA samples from species in which polysaccharides were found to be difficult to remove by phenol/chloroform extraction. The DNA obtained by this method was suitable for PCR, RAPD, enzyme digestion, and Southern-blot analyses.     

Binding of an oxindole alkaloid from Uncaria tomentosa to amyloid protein (Abeta1-40)

The primary aim of this work was to determine the interactions of an oxindole alkaloid (mitraphylline) isolated from Uncaria tomentosa with beta-amyloid 1-40 (Abeta1-40 protein) applying the capillary electrophoresis (CE) method. Specifically the Hummel-Dreyer method and Scatchard analysis were performed to study the binding of oxindole alkaloids with Abeta1-40 protein. Prior to these studies extraction of the alkaloid of interest was carried out. Identification of the isolated alkaloid was performed by the use of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) combined with electrospray ionization mass spectrometry (ESI-MS). The proposed approach was proved to be an efficient and accurate method for specific compound isolation and identification purposes. Moreover, analytical information from the CE approach can be considered as the valuable tool for binding constant determination. The binding constant of mitraphylline with Abeta1-40 protein determined by the Hummel-Dreyer method and Scatchard analysis equals K = 9.95 x 10(5) M(-1). The results obtained showed the significant binding of the tested compound with Abeta1-40 protein. These results are discussed and interpreted in the view of developing a strategy for identification of novel compounds of great importance in Alzheimer disease therapy.     

Inhibition of lysosomal alpha-mannosidase by swainsonine, an indolizidine alkaloid isolated from Swainsona canescens.

An indolizidine alkaloid (swainsonine) was isolated from the plant Swainsona canescens. Swainsonine is a specific and potent inhibitor of alpha-mannosidase (EC 3.2.1.24) and when administered to animals produces a phenocopy of the genetically based lysosomal storage disease, mannosidosis. Evidence is presented to suggest that swainsonine is a reversible active site-directed inhibitor of lysosomal alpha-mannosidase.     

Optimisation of supercritical fluid extraction of indole alkaloids from Catharanthus roseus using experimental design methodology--comparison with other extraction techniques

Response surface modelling, using MODDE 6 software for Design of Experiments and Optimisation, was applied to optimise supercritical fluid extraction (SFE) conditions for the extraction of indole alkaloids from the dried leaves of Catharanthus roseus. The effects of pressure (200-400 bar), temperature (40-80 degrees C), modifier concentration (2.2-6.6 vol%) and dynamic extraction time (20-60 min) on the yield of alkaloids were evaluated. The extracts were analysed by high-performance liquid chromatography and the analytes were identified using ion trap-electrospray ionisation-mass spectrometry. The method was linear for alkaloid concentration in the range 0.18-31 microg/mL. The limits of detection and quantification for catharanthine, vindoline, vinblastine and vincristine were 0.2, 0.15, 0.1 and 0.08 microg/mL and 2.7, 2.0, 1.3 and 1.1 microg/g, respectively. The dry weight content of major alkaloids in the plants were compared using different extraction methods, i.e. SFE, Soxhlet extraction, solid-liquid extraction with sonication and hot water extraction at various temperatures. The extraction techniques were also compared in terms of reproducibility, selectivity and analyte recoveries. Relative standard deviations for the major alkaloids varied from 4.1 to 17.5% in different extraction methods. The best recoveries (100%) for catharanthine were obtained by SFE at 250 bar and 80 degrees C using 6.6 vol% methanol as modifier for 40 min, for vindoline by Soxhlet extraction using dichloromethane in a reflux for 16 h, and for 3',4'-anhydrovinblastine by solid-liquid extraction using a solution of 0.5 m sulphuric acid and methanol (3:1 v/v) in an ultrasonic bath for 3 h.     

Bio-analysis of vinorelbine by high-performance liquid chromatography with fluorescence detection

A simple and selective procedure for the determination of vinorelbine, a new semi-synthetic vinca alkaloid, is presented. The method is based on ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection, combined with liquid—liquid extraction using diethyl ether for sample clean-up. The absence of endogenous interferences and the excellent chromatographic behaviour of vinca alkaloids provides accurate results even at low concentrations. The limit of determination in plasma is 1.5 μg/l (500-μl sample). Reproducible recoveries in urine were obtained if 10–50 μl of sample were processed supplemented with 500 μl of blank plasma.