Alkaloid concentration of the invasive plant species Ulex europaeus in relation to geographic origin and herbivory.

In the study of plant defense evolution, invasive plant species can be very insightful because they are often introduced without their enemies, and traits linked to defense can be released from selective pressures and evolve. Further, studying plant defense evolution in invasive species is important for biological control and use of these species. In this study, we investigated the evolution of the defensive chemicals quinolizidine alkaloids (QAs) in the invasive species gorse, Ulex europaeus. Using a common garden experiment, our goals were to characterize the role of QAs relative to specialist enemies of gorse and to investigate if QA concentration evolved in invaded regions, where gorse was introduced without these enemies. Our results showed that pod infestation rate by the seed predator Exapion ulicis and infestation by the rust pathogen Uromyces genistae-tinctoriae were negatively correlated to concentration of the QA lupanine. Quinolizidine alkaloid concentration was very variable between individuals, both within and among populations, but it was not different between native and invaded regions, suggesting that no evolution of decreased resistance occurred after gorse lost its enemies. Our study also suggests that QA concentrations are traits integrated into seed predation avoidance strategies of gorse, with plants that mass-fruit in spring but do not escape pod infestation in time being richer in QAs.

Alkaloid Profiling as an Approach to Differentiate Lupinus garfieldensis, Lupinus sabinianus and Lupinus sericeus.

INTRODUCTION: Many species in the Lupinus genus are poorly defined morphologically, potentially resulting in improper taxonomic identification. Lupine species may contain quinolizidine and/or piperidine alkaloids that can be acutely toxic and/or teratogenic, the latter resulting in crooked calf disease. OBJECTIVE: To identify characteristic alkaloid profiles of Lupinus sabinianus, L. garfieldensis and L. sericeus which would aid in discriminating these species from each other and from L. sulphureus. METHODS AND MATERIALS: Quinolizidine and piperidine alkaloids were extracted from herbarium specimens and recent field collections of L. sabinianus, L. garfieldensis and L. sericeus. The alkaloid composition of each species was defined using GC-FID and GC-MS and compared using multivariate statistics. RESULTS: Each of the three species investigated contained a diagnostic chemical fingerprint composed of quinolizidine and/or piperidine alkaloids. CONCLUSION: The alkaloid profiles of Lupinus sabinianus, L. garfieldensis and L. sericeus can be used as a tool to discriminate these species from each other and L. sulphureus as long as one considers locality of the collection in the case of L. sabinianus.

[Simultaneous determination of 7 alkaloid in herba Sophorae Alopecuroidis by HPLC].

OBJECTIVE: To establish a method for determination of 7 alkaloid in Herba Sophorae Alopecuroidis by HPLC. METHOD: X-Brige C18 (4.6 mm x 200 mm, 5 microm) column was used with acetonitriles-0.05 mol x L(-1) KH2PO4 solution (2.0 mL x L(-1) triethylamine) with gradient elution as the mobile phase and 1.0 mL x min(-1) as the flow rate. The detection wavelength was 205 nm. RESULT: Aloperin curve was linear in the range from 20.66 to 103.32 microg (r = 0.998 8) and the average recovery was 97.12% (RSD 7. 3%); sophoridine curve was linear in the range from 22.82 to 114.12 microg (r = 0.999 7) and the average recovery was 97.47% (RSD 3.0%); oxymatrine curve was linear in the range from 25.10 to 125.52 microg (r = 0.999 1) and the average recovery was 96.21% (RSD 4.5%); oxysophocarpine curve was linear in the range from 23.88 to 119.40 microg (r = 0.997 5) and the average recovery was 94. 64% (RSD 5.2%); matrine curve was linear in the range from 5.00 to 24.99 microg (r = 0.998 6) and the average recovery was 98.04% (RSD 5.4%); sophocarping curve was linear in the range from 4.69 to 23.46 microg (r = 0.999 6) and the average recovery was 96.24 (RSD 5.8%); lehmannine curve was linear in the range from 4.60 to 23.01 microg (r = 0.997 8) and the average recovery was 101.31% (RSD 4.3%). CONCLUSION: The method is accurate, simple and feasible. It can be used as a quality evaluation in Herba S. Alopecuroidis.

Indolizidine 239Q and quinolizidine 275I. Major alkaloids in two Argentinian bufonid toads (Melanophryniscus).

Alkaloid profiles in skin of poison frogs/toads (Dendrobatidae, Mantellidae, Bufonidae, and Myobatrachidae) are highly dependent on diet and hence on the nature of habitat. Extracts of the two species of toads (Melanophryniscus klappenbachi and Melanophryniscus cupreuscapularis) from similar habitats in the Corrientes/Chaco Provinces of Argentina have similar profiles of alkaloids, which differ considerably in profiles from other Melanophryniscus species from Brazil, Uruguay and Argentina. Structures of two major alkaloids 239Q (1) and 275I (2) were determined by mass, FTIR, and NMR spectral analysis as 5Z,9Z-3-(1-hydroxybutyl)-5-propylindolizidine and 6Z,10E-4,6-di(pent-4-enyl) quinolizidine, respectively. A third alkaloid, 249F (3), is postulated to be a homopumiliotoxin with an unprecedented conjugated exocyclic diene moiety.

Microemulsion electrokinetic chromatography coupling with field amplified sample injection and electroosmotic flow suppressant for analysis of some quinolizidine alkaloids.

A new rapid and reproducible method using microemulsion electrokinetic chromatography (MEEKC) combining field amplified sample injection and electroosmotic flow suppressant for the analysis of five quinolizidine alkaloids is developed in this paper. For the separation of five quinolizidine alkaloids, a running buffer composed of 1.2% (v/v) 1-butanol, 0.6% (v/v) ethyl acetate and 98.2% (v/v) 1 mM Na(2)B(4)O(7)-2 mM NaH(2)PO(4) buffer solution containing 21 mM sodium cholate (SC) (pH 6.5) was developed. The resolution of the analytes was improved significantly by adding a divalent cation (e.g., Mg(2+)) to the running buffer as an electroosmotic flow modification. In order to analyze trace quinolizidine alkaloids in traditional Chinese herbal medicines, field amplified sample injection (FASI) was applied to increase the detection sensitivity. The detection limits (defined as S/N=3) for the analytes could be as low as 0.0001 microg/mL. This method was applied for the determination of quinolizidine alkaloids in real samples with simple extraction procedures, and the assay results were satisfactory.

Quinolizidine alkaloids in seeds of lupin genotypes of different origins.

The intake of lupin-based foods could imply the exposure of consumers to quinolizidine alkaloids. The objectives of this study were to assess the genetic variation among and within 11 geographic regions of Lupinus albus ecotypes, verify the quinolizidine alkaloids amount of alkaloid-poor L. albus and Lupinus angustifolius varieties, and assess the effect of two climatically contrasting Italian environments on the alkaloid content. The quantitation was performed by GC-MS, and in all samples lupanine was the most abundant quinolizidine alkaloid, followed by albine and 13alpha-hydroxylupanine for L. albus and by 13alpha-hydroxylupanine and angustifoline for L. angustifolius. Some regions tended to have a high (Azores) or low (Egypt, Near East, Maghreb) total alkaloids content, but the variation among ecotypes within regions was larger than that among regions following the estimation of variance components. Alkaloid-poor varieties tended to have higher total alkaloid contents when grown in the subcontinental climate site, exceeding in some cases the limit of 0.200 mg/g.

Comparative analysis of quinolizidine alkaloids from different parts of Sophora alopecuroides seeds by UPLC-MS/MS.

The seeds of Sophora alopecuroides L. have been utilized as a crude drug in China for thousands of years. Quinolizidine alkaloids are the main bioactive components of this plant. To determine the distribution and content of quinolizidine alkaloids in different seed organs (seed coat and cotyledon), a reliable method has been established using ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UPLC-MS/MS). Seven constituents, namely cytisine, oxymatrine, oxysophocarpine, sophoridine, sophoramine, matrine, and sophocarpine, were simultaneously determined in 10 min. The proposed method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. The analysis results showed there were remarkable differences in the distribution and contents of the chemical markers between seed coat and cotyledon. The established approach could be helpful for the quality control of S. alopecuroides seeds, and also for the determination of this type class of alkaloids in other medicinal herbs. The present study can provide necessary information for the rational utilization of S. alopecuroides resources.

Determination of quinolizidine alkaloids in different Lupinus species by NACE using UV and MS detection.

Lupin seeds are important for animal and human nutrition. However, they may contain toxic quinolizidine alkaloids (QA). Analytical methods for a reliable alkaloid determination are therefore of importance. Here the presented study reports on the first CE method for the analysis of QA in Lupinus species. A buffer system consisting of 100mM ammonium formate in methanol, acetonitrile, and small amounts of water and acetic acid enabled the baseline separation of sparteine, lupanine, angustifoline and 13alpha-hydroxylupanine in less than 10min. Applied voltage, temperature and detection wavelength were 25kV, 30 degrees C and 210nm, respectively. Additional compounds were identified in CE-MS experiments, in which all alkaloids could be assigned in positive ESI mode at corresponding [M+H](+) values. The CE method was validated for linearity, sensitivity, accuracy and precision, and then used to assess the seeds of seven different Lupinus species for their alkaloid content. Lupanine was present in all of them within a range from 0.02% (L. densiflorus, L. microcarpus) to 1.47% (L. albus). The highest percentage of an individual alkaloid was found in L. polyphyllus (3.28% of angustifoline), the content of total alkaloids ranged from 0.43% (L. microcarpus) to 5.13% in L. polyphyllus. The quantitative results were in good agreement with literature data.

Analysis of quinolizidine alkaloids in Sophora flavescens Ait. by capillary electrophoresis with tris(2,2'-bipyridyl) ruthenium (II)-based electrochemiluminescence detection.

A capillary electrophoresis method coupled with electrochemiluminescence detection for the analysis of quinolizidine alkaloids was established, especially, oxymatrine (OMT) which could not be measured by previous electrochemiluminescence methods was detected sensitively herein. Complete separation of sophoridine (SR), matrine (MT) and OMT was achieved within 13 min using a background electrolyte of 50mM phosphate buffer at pH 8.4 and a separation voltage of 15 kV. The calibration curves showed a linear range from 2.8 x 10(-8) to 4.4 x 10(-7) M for SR, 2.7 x 10(-8) to 4.4 x 10(-7) M for MT, and 2.5 x 10(-7) to 4.0 x 10(-6)M for OMT, respectively. The relative standard derivations for all analytes were below 3.1%. Good linear relationships were showed with correlation coefficients for all analytes exceeded 0.987. The detection limits were 1.0 nM for SR and MT, and 40 nM for OMT under the optimal conditions, respectively. The developed method was nearly harmless to the human and environment.

Evaluation of total quinolizidine alkaloids content in lupin flours, lupin-based ingredients, and foods.

Lupin proteins are gaining attention to replace animal proteins and other plants ingredients in several foods such as bakery products, imitation dairy and meat products, and beverages. One of the major safety issues of lupin-based foods is the presence of quinolizidine alkaloids (QAs), bitter compounds produced by lupin plants as a defense mechanism against predators. In mammals, QA intoxication is characterized by trembling, shaking, excitation, and convulsion. Lupanine and sparteine, the most common QAs, show acute oral toxicity due to neurological effects leading to the loss of motor co-ordination and muscular control. In this paper, 27 samples of lupin-based products, i. e., flours, protein isolates, and food (either model or commercially available ones), were analyzed for evaluating the QA content using a method based on GC/MS. All the analyzed samples were safe since they respect the maximum limit of 200 mg/kg fixed by the Health Authorities of Australia, New Zealand, Great Britain, and France, that have regulated this topic. The QA contents were particularly low in protein isolates and in foods containing these ingredients, indicating that their use is a very effective tool for keeping low the daily intake of QAs.