Levbid
[A rapid determination method for scopolia extract in gastrointestinal drugs by capillary electrophoresis]

[Article in Japanese]

Ori K, Mikata H, Tsurumori T.

Healthcare Research Laboratories, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan.

A rapid and simple determination of tropane-alkaloids (hyoscyamine, scopolamine) in gastrointestinal drugs was investigated by capillary electrophoresis. Micellar electrokinetic capillary chromatography (MEKC) using a fused silica capillary (560 mm x 0.075 mm i.d.) in 0.1 M SDS 20 mM borate buffer (pH 10)/acetonitrile (97:3) gave complete separation of the two alkaloids within 40 min. A sample solution was introduced by pressure method (50 mbar, 3.7 s), separation conditions applied voltage 15 kV and on-column detection was performed at 210 nm. Calibration curves for hyoscyamine and scopolamine showed a good linearity in the range of 4-12 micrograms/ml (r = 0.9970) and 390-1150 ng/ml (r = 0.9976). The present method is applicable to the simple and rapid determination of hyoscyamine and scopolamine in commercial gastrointestinal drugs.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10590714&dopt=Abstract hyoscyamine Levbid SL



Levbid
Strategies for supercritical fluid extraction of hyoscyamine and scopolamine salts using basified modifiers.

Choi YH, Chin YW, Kim J, Jeon SH, Yoo KP.

College of Pharmacy, Seoul National University, South Korea.

The supercritical fluid extraction behaviors of hyoscyamine and scopolamine were investigated and found to be highly dependent upon the chemical nature of the compounds. Free bases of hyoscyamine and scopolamine were freely soluble in supercritical CO2 with increasing temperature and pressure; however, the salts of these alkaloids were not soluble under any experimental conditions. It was found that alkaline modifiers such as methanol basified with diethylamine could enhance the solubilities and extraction yields of these alkaloids from plant matrices as compared to other modifiers.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10591463&dopt=Abstract hyoscyamine Levbid SL



Levbid
Capillary electrophoresis-diode array detection--electrospray mass spectrometry for the analysis of selected tropane alkaloids in plant extracts.

Mateus L, Cherkaoui S, Christen P, Veuthey JL.

Laboratory of Pharmaceutical Analytical Chemistry, University of Geneva, Switzerland.

Capillary zone electrophoresis, coupled to UV and interfaced with electrospray ionization mass spectrometry (ESI-MS), is described for the simultaneous analysis of hyoscyamine and scopolamine. On-line UV detection occurred at 22 cm from the inlet of the capillary and ESI-MS monitoring was performed along the entire length of the capillary (85 cm). An alkaline solution of 40 mM ammonium acetate at pH 8.5 was suitable for the analysis of the alkaloids under consideration. Under the optimized conditions, including CE and ESI-MS parameters, the two alkaloids were resolved within a short time and with very high sensitivity. The differentiation of hyoscyamine and its positional isomer littorine, commonly encountered in plant material, is also presented using up-front collision-induced dissociation. Finally, the developed method was applied to the analysis of these alkaloids in Belladonna leaf extract and in Datura candida x D. aurea hairy root extract.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10608707&dopt=Abstract hyoscyamine Levbid SL



Levbid
Two tropinone reductases, that catalyze opposite stereospecific reductions in tropane alkaloid biosynthesis, are localized in plant root with different cell-specific patterns.

Nakajima K, Hashimoto T.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Japan.

In the plant species that produce tropane alkaloids, two tropinone reductases (TRs) catalyze the stereospecific reductions of the 3-carbonyl group of tropinone. This reduction is a key branch point that determines the metabolite flow into the separate alkaloid groups, each with different stereospecific configurations. In this study, a specific antibody was prepared for each of the TRs by immunizing mice with recombinant TR protein and subsequent immuno-affinity purification of the antiserum. Immunoblot analyses revealed that accumulation of both TRs was highest in the lateral roots of Hyoscyamus niger throughout its development. In cultured roots, TR proteins were accumulated in a basal region but not in root apex. These patterns were similar to that of hyoscyamine 6 beta-hydroxylase (H6H), an enzyme that catalyzes a downstream step in the same biosynthetic pathway. However, an immunohistochemical analysis revealed that the two TRs and H6H were accumulated with different cell-specific patterns in the cultured root, suggesting transportation of the alkaloid intermediate(s) across the different cell layers.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10635114&dopt=Abstract hyoscyamine Levbid SL



Levbid
The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida.

Pitta-Alvarez SI, Spollansky TC, Giulietti AM.

Catedra de Microbiologia Industrial y Biotecnologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, 6to piso, Buenos Aires, Argentina

Hairy root cultures of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to increase productivity, several biotic and abiotic elicitors were tested. Salicylic acid increased significantly the release of both alkaloids (2- to 12-fold) and it also acted positively on specific production without altering the production profile. AgNO(3) increased significantly scopolamine release (3-fold) and both alkaloid's accumulation (5- to 8-fold) in the roots, thus favoring the production of scopolamine (up to 2-fold). The inhibiting effects of AgNO(3) and salicylic acid on ethylene could be partly responsible for these responses. Yeast extract incremented the intracellular content of both alkaloids (ca. 3-fold), but particularly increased the release of scopolamine (7-fold). CaCl(2) had little effect on accumulation or release of either alkaloid. CdCl(2) acted positively on the release of both alkaloids (3- to 24-fold), but was highly detrimental to growth. Hairy roots of B. candida are therefore susceptible to elicitation by biotic and abiotic elicitors, with variations in the kinetics of induction and the extent of release of each metabolite, thereby also exerting different effects on the alkaloid profile.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10689085&dopt=Abstract hyoscyamine Levbid SL