Determination of Aconitum alkaloids in blood and urine samples: II. Capillary liquid chromatographic–frit fast atom bombardment mass spectrometric analysis

Determination of fourteen alkaloids, toxic Aconitum alkaloids, aconitine, mesaconitine, jesaconitine, hypaconitine and deoxyaconitine, and their hydrolysis products, benzoylaconines and aconines, have been established using capillary liquid chromatography (LC) fast atom bombardment mass spectrometry (FAB-MS) with a frit interface. Protonated molecular ions were observed as base peaks in the FAB-MS for these fourteen alkaloids. All the alkaloids were simultaneously quantified with linear gradient LC elution by solvent mixture of acetonitrile and 0.3% trifluoroacetic acid using selected ion monitoring of the protonated molecular ions. The calibration curves of these alkaloids were linear in injection amounts ranging from 5 to 500 pg, and their detection limits were 1 pg per injection (S/N=3). Solid-phase extraction using Sep-Pak Plus PS-1 was also investigated to clean-up and concentrate alkaloids in blood and urine samples, and showed satisfactory recoveries. This capillary LC–frit-FAB-MS method enables determination of low levels of Aconitum alkaloids in blood and urine samples, coupled with solid-phase extraction.     

Determination of Aconitum alkaloids in blood and urine samples. I. High-performance liquid chromatographic separation, solid-phase extraction and mass spectrometric confirmation

Determination of four toxic Aconitum alkaloids, aconitine, mesaconitine, hypaconitine and jesaconitine, in blood and urine samples has been established using high-performance liquid chromatography (HPLC) combined with ultraviolet absorbance detection, solid-phase extraction and mass spectrometry (MS). These alkaloids were hydrolyzed rapidly in alkaline solution (half lives (t_1/2)<one day), were stable in solutions of acetonitrile, tetrahydrofuran and diluted hydrochloric acid (t_1/2>five months) and were unstable in solutions of methanol and ethanol (t_1/2<one month). These alkaloids were separated on an octadecylsilica column with isocratic elution using a solvent mixture of tetrahydrofuran and 0.2% trifluoroacetic acid (14:86, v/v), which was found to be the optimal solvent of the elution systems examined. Calibration curves with UV detection were linear on injection of amounts ranging from 2.5 to 500 ng, and the limit of detection was 1 ng (S/N = 3). These four alkaloids in aqueous solution were recovered almost totally by solid-phase extraction using the styrene polymer resin, Sep-Pak Plus PS-1, and were eluted using a mixture of acetonitrile and hydrochloric acid. These Aconitum alkaloids were confirmed by HPLC coupled with fast atom bombardment MS, giving their protonated molecular ions as base peaks. These alkaloids were detected by HPLC with UV detection from blood samples spiked with more than 50 ng ml⁻¹ of alkaloids, but were not detectable from urine samples spiked with 5 μg ml⁻¹ of alkaloids because of severe sample interference.     

Pharmacophagy in grasshoppers?. Zonocerus attracted to and ingesting pure pyrrolizidine alkaloids

Zonocerus elegans has been found to be attracted to pure pyrrolizidine alkaloids and to ingest them. This finding makes the species likely to be pharmacophagous; also, it might provide means of controlling Zonocerus, and it indicates the importance of olfaction for localizing and recognizing host-plants in grasshoppers.

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Zusammenfassung Zonocerus elegans wird von reinen Pyrrolizidin-Alkaloiden angelockt und nimmt sie auf. Dieser Befund macht es wahrscheinlich, daß die Art pharmacophag ist; ausserdem könnte er neue Möglichkeiten zur Kontrolle von Zonocerus veröffnen und gibt Hinweise auf die Bedeutung des geruchs für die Lokalisation und Erkennung von Wirtspflanzen bei Heuschrecken.

     

Hairy root induction of<Emphasis Type="Italic"> Papaver somniferum</Emphasis> var.<Emphasis Type="Italic"> album</Emphasis>, a difficult-to-transform plant,by<Emphasis Type="Italic"> A. rhizogenes</Emphasis> LBA 9402

Two strains of Agrobacterium rhizogenes (15834, LBA 9402) and one Agrobacterium tumefaciens strain [GV 3101 (PMP90RK, p35SGUS-2)] and four culture media were tested and compared for their ability to induce hairy root formation on wounded Papaver somniferum L. hypocotyls. Five weeks after the infection with A. rhizogenes LBA 9402, hairy roots appeared on 80% of the hypocotyls maintained in the hormone-free liquid medium. Six hairy-root cultures were established. Transformation was confirmed by polymerase chain reaction analysis. One clone was analysed for its alkaloid production. The total alkaloid content was higher in the transformed roots (0.46±0.06% DW) than in the untransformed roots (0.32±0.05% DW). The transformed roots accumulated three times more codeine (0.18±0.02% DW) than intact roots (0.05±0% DW). Moreover, morphine (0.255±0.03% DW) and sanguinarine (0.014±0% DW) were found in the liquid culture medium.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - LS Linsmaier and Skoog     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Four tetracyclic oxindole alkaloids and a taberpsychine derivative from a Malayan Tabernaemontana

Four tetracyclic oxindole alkaloids, 7(R)- and 7(S)-geissoschizol oxindole (1 and 2), 7(R),16(R)- and 7(S),16(R)–19(E)-isositsirikine oxindole (3 and 4), in addition to a taberpsychine derivative, N(4)-demethyltaberpsychine (5), were isolated from the Malayan Tabernaemontana corymbosa and the structures were established using NMR and MS analysis.     

西藏不同生长地砂生槐群体种子生物碱含量及其相关性分析

为了明确不同群体间和群体内砂生槐天然种子富含生物碱含量与组成的差异,该研究选取来自西藏“一江四河”流域、海拔2 900～4 100 m范围内10个群体的砂生槐种子作为研究材料,采用高效液相色谱法,测定10个群体300个单株种子重要生物碱(氧化苦参碱、苦参碱、槐果碱、槐定碱)含量,应用SPSS软件分析了地理分布特征与生物碱含量之间的相关性及群体遗传变异关系,以筛选生物碱含量较高的群体,为砂生槐药物开发利用提供依据。结果表明：(1)10个群体的砂生槐种子中均含有氧化苦参碱、苦参碱和槐果碱,含量依次为：氧化苦参碱(46.18～64.08 mg/g)>苦参碱(1.14～9.82 mg/g)>槐果碱(0.08～1.16 mg/g),其中氧化苦参碱占生物碱总量的90%以上,且群体4中含量最高(64.08±7.37 mg/g);其他微量生物碱在群体3中含量最高;群体间氧化苦参碱、苦参碱和槐果碱均差异极显著(P＜0.01)。(2)氧化苦参碱群体内和群体间变异相对较小,苦参碱和槐果碱群体内和群体间变异系数均较大,而且氧化苦参碱和槐果碱的群体间变异均大于群体内变异,苦参碱的群体间变异小于群体内变异。(3)氧化苦参碱与海拔呈显著正相关关系(r = 0.117^*),苦参碱与海拔、经度和纬度均呈极显著负相关关系(r ＜ -0.326^**),其他关系不显著。氧化苦参碱与苦参碱和槐果碱均呈显著负相关关系(r ＜-0.162^**),而苦参碱与槐果碱呈显著正相关关系(r =0.789^**)。(4)聚类分析将10个群体聚为4类,各类间的距离差异比较大,且大多与地理因素有关。     

Purine alkaloid formation and CO2 gas exchange in dependence of development and of environmental factors in leaves of Coffea arabica L.

In the leaves of Coffea arabica L., purine alkaloid formation was estimated by analyzing the theobromine and caffeine content and by measuring the methylation rate of [2-¹⁴C]theobromine to [2-¹⁴C]caffeine in short-term experiments (6–24 h). At the same time, growth (in terms of dry weight and area), net photosynthesis (NPS), and dark respiration were determined. During leaf development, which was considered to be terminated when NPS was at a maximum (60–80 mol g^-1 s^-1) and dark respiration at a minimum (5–7.5 mol g^-1 s^-1), the content of theobromine and the velocity of caffeine formation were both found to decrease by a factor of more than 100. The close correlation between the theobromine content and the methylation rate is suspended when purine alkaloid formation is influenced by factors other than leaf development. Among these factors, temperature is the most effective: the velocity of caffeine biosynthesis is increased by raising the temperature and vice versa. Although the plants were well irrigated, a drastic decrease of NPS in the afternoon was observed under all environmental conditions tested. Light saturation was reached between 170–360 mol m^-2 s^-1. The temperature optimum of NPS was shown to be very broad (24–33°C)m provided the adaptation time was sufficiently long.Abbreviations MR methylation rate - NPS net photosynthesis - RMC relative methylation coefficient Dedicated to Professor Hans Wanner, as promoter of these investigations, on occasion of his 65th birthday     

Sites of synthesis,translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L.

¹⁴C-Labelled alkaloid precursors (arginine, putrescine, spermidine) fed to Senecio vulgaris plants via the root system were rapidly taken up and efficiently incorporated into the pyrrolizidine alkaloid senecionine N-oxide (sen-Nox) with total incorporations of 3–6%. Considerable amounts of labelled sen-Nox were translocated into the shoot and were directed mainly into the inflorescences, the major sites of pyrrolizidine-alkaloid accumulation. Detached shoots of S. vulgaris were unable to synthesize pyrrolizidine alkaloids, indicating that the roots are the site of their biosynthesis. Further evidence was obtained from studies with in-vitro systems established from S. vulgaris: root cultures were found to synthesize pyrrolizidine alkaloids but not cell-suspension cultures, tumor cultures or shoot-like teratomas obtained by transformation with Agrobacterium tumefaciens. Studies on transport of [¹⁴C]sen-Nox, which was fed either to detached shoots or to the root system of intact plants, indicate that the alkaloid N-oxide does not simply follow the transpiration stream but is specifically channelled to the target tissues such as epidermal stem tissue and flower heads. Exogenously applied [¹⁴C]senecionine is rapidly N-oxidized. If the phloem path along the stem is blocked by a steam girdle translocation of labelled sen-Nox is blocked as well. Root-derived sen-Nox accumulated below the girdle and only trace amounts were found in the tissues above. It is most likely that the root-to-shoot transport of sen-Nox occurs mainly if not exclusively via the phloem. In accordance with previous studies the polar, salt-like N-oxides, which are often considered to be artifacts, were found to be the real products of pyrrolizidine-alkaloid biosynthesis as well as the physiological forms for long-distance transport, tissue-specific distribution and cellular accumulation.Abbreviations FW fresh weight - sen senecionine - sen-Nox senecionine N-oxide     

Indole alkaloids in clonal propagules of Rauwolfia serpentina benthe ex kurz

Plantlets were succesfully regenerated from shoot cultures of Rauwolfia serpentina initiated from auxillary meristems on medium containing BA (4.44 M) + NAA (0.54 M). Rooting was initiated in White's basal medium supplemented with NAA (0.54 M). Tissue culture derived piants of R. serpentina (RSTC) were similar to normal plants (RS) in their morphological characteristics and chemical consitution. The biomass of the RSTC plants was higher (47.11 gms) than the normal plant (18.23 gms) on a dry weight basis. Five RSTC plants were cloned and the cloned plants were similar in biomass and alkaloid content to the normal plant.Abbreviations BA benzyl adenine - NAA naphthalene acetic acid - MS Murashige and Skoog - TLC thin layer chromatography - HPLC high performance liquid chromatography - F.W. fresh weight - D.W. dry weight - RS normal field grown plant established from stem cuttings - RSTC tissue culture plants established from shoot cultures - R reserpine - Aj ajmalicine - A ajmaline - S serpentine     

Cerebroside and β-carboline alkaloids from the ascidian Ascidia longistriata

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Highlights? Ascidia longistriata was collected for secondary metabolites investigation. ? This is the first report of compounds 1-3 from ascidians of the genus Ascidia. ? Compound 2 might be derived from an associated organism.     

The exposure to trans-cinnamic acid of osmotically stressed Catharanthus roseus cells cultured in a 14-l bioreactor increases alkaloid accumulation

A Catharanthus roseus cell line was cultured in a 14-l bioreactor. Total alkaloid production decreased more than 80% while scaling up this cell line from 250 ml batch cultures to the bioreactor. However, the subsequent application of an osmotic stress and 1 mM trans-cinnamic acid, which inhibits the synthesis of phenolic compounds, restored the original alkaloid amounts.     

Isoquinoline-derived alkaloids from the bark of Guatteria olivacea (Annonaceae)

Phytochemical investigation of the bark of Guatteria olivacea R. E. Fries (Annonaceae) led to the isolation and identification of ten isoquinoline-derived alkaloids, including three phenanthrenes, atherosperminine, argentinine, and atherosperminine N-oxide; three aporphines, asimilobine, puterine, and discoguattine; two oxoaporphines, liriodenine and oxoputerine; and two tetrahydroprotoberberines, corypalmine and discretine. All these alkaloids are described for the first time in G. olivacea and their chemotaxonomic significance was discussed. The structure elucidation of these isolated alkaloids was established by extensive analyses of 1D and 2D NMR spectroscopy in combination with MS. The NMR data for atherosperminine, argentinine, and atherosperminine N-oxide were reviewed.     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Constituents from Chimonanthus praecox (wintersweet)

One new (1) and seven (2–8) known sesquiterpenoids, four dimeric tryptamine-related alkaloids (9–12) and six glycosides (13–18) were isolated from the fruits and leaves of Chimonanthus praecox (wintersweet). Based on its spectroscopic data, the new structure of compound 1, an oppositane-type sesquiterpenoid, was elucidated to be the C-4 epimer of bullatantriol (2). All isolated compounds were evaluated for their cytotoxicities against a small panel of human cancer cell lines, and only the chimonanthine-type alkaloids (10–12) were found to have cytotoxic effects against gastric carcinoma NUGC3 and hepatocarcinoma SNU739 cancer cells, with IC₅₀ values ranging from 10.3 to 19.7 μM.     

8-甲氧基二氢血根碱化感潜能的评估

以莴苣幼苗为材料,检测了分离自细果角茴香的生物碱对莴苣幼苗根生长及根毛发育的影响。结果表明:50~200μmol·L-1浓度范围内,8-甲氧基二氢血根碱对莴苣幼苗根长显著抑制;10、20和30μmol·L-18-甲氧基二氢血根碱对莴苣幼苗根毛的长度和数量有显著的抑制作用,且抑制作用均表现了浓度依赖性。通过对莴苣根尖细胞有丝分裂研究发现,50μmol·L-1的8-甲氧基二氢血根碱能显著抑制根尖细胞的有丝分裂,而且莴苣幼苗净增值率与根尖细胞的有丝分裂之间呈正相关,证明8-甲氧基二氢血根碱主要通过抑制根尖细胞有丝分裂对莴苣根的生长产生影响。     

Determination of ergot alkaloids in rye and rye flour

An effective and timesaving analytical method was developed for the determination of 12 ergot alkaloids (ergometrine, ergotamine, ergocristine, α-ergokryptine, ergosine, ergocornine, and their respective -inine isomers) in rye and rye flour. Samples were extracted with dichloromethane/ethyl acetate/methanol/aqueous ammonia (25%) (50/25/5/1, v/v/v/v), and extracts were purified using a basic alumina column. The eluate was dried in the nitrogen stream and redissolved in acetonitrile/ ammonia carbamate-buffer (0.2 g/1), (1/1, v/v), and injected into an HPLC-FLD system (λ_Ex 330 nm, λ_Em 415 nm), using the same mixture as mobile phase and a Phenyl-Hexyl column. Detection limits for the individual compounds ranged from 0.01 μg/kg to 0.5 μg/kg. In sample material spiked with a mixture of these compounds at two different levels (13 μg/kg and 27 μg/kg per compound), mean (n=5) recoveries were at 101% (s_r 6.4%) and 89% (s_r 3.1%), respectively. Presented at the 28th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Vindoline synthesis in in vitro shoot cultures of Catharanthus roseus

Vindoline, the major alkaloid in cultures of Catharanthus roseus shoots, reached 2 mg g(-1) dry wt after 27 d in culture. Maximal vindoline accumulation coincided with maximum activities of deacetoxyvindoline 4-hydroxylase, deacetylvindoline acetyl-CoA acetyl transferase and tryptophan decarboxylase. Shoot exposure to jasmonate shortened the time required for the maximal vindoline accumulation to 14 d.     

Primary effects of allelochemicals ofDatura stramonium L.

Summary Alkaloids leached from seed ofDatura stramonium were identified as scopolamine and hyoscyamine and levels leached from seed over a seven day period were quantified. Scopolamine is known to inhibit early seedling growth of some crop plants. The hypothesis that such inhibition results from interference with gibberellin-stimulated food reserve metabolism in germinating seedlings was tested, using barley and wheat in bioassays. Seedling growth of both cereals was inhibited by scopolamine but no evidence of interference with responses to gibberellins was obtained.