Investigations on the Transfer of Quinolizidine Alkaloids from Lupinus angustifolius into the Milk of Dairy Cows.

Lupin varieties with a low content of quinolizidine alkaloids (QAs) like blue sweet lupin (BSL) have long been used as a protein source for dairy cows. A health concern for humans may arise from the transfer of acute toxic QAs from feed into cow's milk. This study is the first to quantify the transfer of QAs from BSL into cow's milk with experimental and modeling methods. Four lactating dairy cows were subjected to two 7 day feeding periods with 1 and 2 kg/d BSL, respectively, each followed by a depuration period. BSL contained 1774 mg/kg dry matter total QAs. Individual milk samples were taken twice daily and QA contents in feed and milk determined with liquid chromatography-tandem mass spectrometry. Transfer of QAs into the milk was already seen with the administration of 1 kg/d BSL, with differences in transfer rates (TRs) between individual QAs. A toxicokinetic model was derived to quantify and predict QA feed-to-food transfer. For the four most prominent QAs, our model shows an α-half-life of around 0.27 d. TRs were obtained for six QAs and were between 0.13 (sparteine) and 3.74% (multiflorine). A toxicological assessment of milk containing QAs as measured in this study indicated a potential health concern.

Bioconversion of alkaloids to high-value chemicals: Comparative analysis of newly isolated lupanine degrading strains.

This work explores the potential for development of a lupanine valorization process evaluating different isolated microorganisms for their capacity to metabolize the alkaloid. Ecotoxicological assessment demonstrated that lupanine is toxic for Vibrio fischeri and Daphnia magna exhibiting EC50 values of 89 mg L-1 and 47 mg L-1 respectively, while acting both as growth inhibitor for a monocotyledonous and as promoter for a dicotyledonous plant. Among the eight aerobic and anaerobic strains isolated and identified Rhodococcus rhodochrous LPK211 achieved 81% removal for 1.5 g L-1 lupanine, while no end-products were detected by NMR constituting a promising microorganism for lupanine biodegradation. Moreover, Rhodococcus ruber LPK111 and Rhodococcus sp. LPK311 exhibited 66% and 71% of removal respectively, including potential formation of lupanine N-oxide. Pseudomonas putida LPK411 reached 80% of lupanine removal and generated three fermentation products potentially comprising 17-oxolupanine and lupanine derivatives with open ring structures enabling the development of alkaloid valorization processes.

Microclimatic variation in UV perception and related disparity in tropane and quinolizidine alkaloid composition of Atropa acuminata, Lupinus polyphyllus and Hyoscyamus niger.

The aim of current research was to evaluate the physiological adjustment in three medicinal herbs viz., Atropa acuminata, Lupinus polyphyllus and Hyoscyamus niger to the winter period characterised by intense UV flux in Kashmir valley across the North Western Himalaya. Quinolizidine (QA) and tropane alkaloid (TA) concentrations were analysed in these herbs thriving at two different altitudes via GC-MS and correlated by PCA analysis. This study investigated the hypothesis that UV reflectance and absorbance at low temperatures are directly related to disparity in alkaloid accumulation. Among QAs in L. polyphyllus, ammodendrine and lupanine accumulated at higher concentration and exhibited significant variation of 186.36% and 95.91% in ammodendrine and lupanine respectively in both sites. Tetrahydrohombifoline displayed non-significant variation of about 9.60% irrespective of sites. Among tropane alkaloid (TA), hyoscyamine was recorded as the most abundant constituent irrespective of the plant and site while apotropine accumulated in lesser quantity in A. acuminata than H. niger. However, apotropine demonstrated significant variation of 175% among both sites. The final concentration of quinolizidine (QA) and tropane alkaloid (TA) reflects the interplay between reflectance and absorbance of UV radiation response field. These findings suggest that spectral response of UV light contributes directly to alkaloid biosynthesis.

Cascade iminium ion reactions for the facile synthesis of quinolizidines. Concise syntheses of (+/-)-epilupinine and (-)-epimyrtine.

Several novel cascade processes have been designed and developed that involve sequential reactions of imines and iminium ions to form substituted quinolizidine ring systems in a single step from simple and readily available starting materials. The utility and promise of these cascade reactions is evident from their application to extraordinarily concise syntheses of the representative quinolizidine alkaloids (+/-)-epilupinine and (-)-epimyrtine.

[The determination in cadaveric material of drugs used for the interruption of pregnancy]. / Opredelenie v trupnom materiale lekarstvennykh sredstv, primeniaemykh dlia preryvaniia beremennosti.

A method for detection and measurements in cadaveric material of some drugs used to induce abortions has been developed. The drugs were measured by spectrophotometry, photo-electro-colorimetry, and argentometry. Detection threshold by the said methods was 0.5 to 3 mg%.

Gas chromatography/mass spectrometry assays for the determination of debrisoquine and sparteine metabolites in microsomal fractions of rat liver.

Debrisoquine and sparteine are prototype substrates of a genetic deficiency in cytochrome P450-dependent drug metabolism. Sensitive assays of in vitro oxidation of sparteine and debrisoquine are required for evaluation of this polymorphism. The activities were measured by quantitative analysis of 2-dehydrosparteine and 4-hydroxydebrisoquine production, respectively, using capillary column gas chromatography coupled with mass selective ion detection. With a single extraction, separation of parent drug, metabolite, and a suitable internal standard was readily achievable. Time-dependent production of both metabolites could be detected from as little as 40 micrograms of microsomal protein. Both activities showed a maximal activity with a 240-min incubation period. The ability to simultaneously quantify the parent drug and its metabolite suggests it would also be useful for evaluation of in vivo metabolism.

Effective sample preparation method for extracting biologically active compounds from different matrices by a microwave technique.

A method is described for extracting lupin alkaloid (sparteine) and drug metabolites from different matrices (seeds and rat faeces) using microwave energy and for checking the homogeneity of the electric field in the microwave oven. The high-performance liquid chromatographic separation and determination of the extracted compounds showed that the microwave extraction method is more advantageous than other traditional extraction methods with regard to the yield of extraction and the time and cost of the procedure. The potential degradation of the extracted compounds may be considerably reduced.

Subnanogram GC/NPD method for the determination of sparteine in biological fluids.

A gas chromatographic method is presented for identification and quantification of sparteine in biological fluids using cyclizine as an internal standard. No derivation is necessary and after a single alkaline extraction, GC analysis of sparteine is achieved in less than 8 min. A subnanogram limit of detection is allowed by the use of a nitrogen phosphorous detector (NPD). This method is simple, reproducible, selective, and applicable in both clinical and pharmacokinetic studies.

A fatal ingestion of sparteine and meprobamate: medicolegal and toxicological data.

A case of suicidal overdose from the ingestion of Palpipax (meprobamate and sparteine) is presented. The drugs were quantified in biological fluids and tissues using gas chromatography. The blood concentrations of meprobamate and sparteine were found to be 88.2 and 40.4 micrograms/ml, respectively. Results are discussed in the light of the existing literature.

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver.

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).

(-)-alpha-Isosparteine from Lupinus argenteus var. stenophyllus.

Combined GLC-mass spectrometry revealed that an unidentified sparteine isomer was the major component of an alkaloid extract of the aboveground portions of Lupinus argenteus Pursh. var. stenophyllus (Rydb.) Davis (Leguminosae). After isolation, this alkaloid was characterized as the least common of the known sparteine isomers, (-)-alpha-isosparteine. A preliminary pharmacological study showed (-)-alpha-isosparteine to have a more rapid onset and a shorter duration of action when compared with (-)-sparteine on rat myocardium.

Analysis of alkaloid mixtures by charge-transfer complexation.

Binary mixtures of weak and strong UV-absorbing alkaloids were analyzed by a charge-transfer spectrophotometric method, utilizing iodine in ethylene dichloride as the acceptor. In the uncomplexed form, the strong absorbing alkaloid (papaverine, quinine, ergotamine, or reserpine) was measured at a wavelength where there was no interference from weak absorbers (at 335, 332, 315, or 300 nm, respectively). The weak absorbing alkaloid (ephedrine, codeine, atropine, or homatropine methylbromide) was determined by computing its contribution to the total charge-transfer band at 295 nm where absorbance was linearly additive for mixtures. The greater increase in the original epsilon-values of the weak absorbers upon complexation with iodine relative to the corresponding increase in the epsilon-values of the strong absorbers led to good recoveries even at the low dose ratios of the weakly absorbing, and often more potent, alkaloids.