Difficulties in Differentiating Natural from Synthetic Alkaloids by Isotope Ratio Monitoring using 13C Nuclear Magnetic Resonance Spectrometry.

Within the food and pharmaceutical industries, there is an increasing legislative requirement for the accurate labeling of the product's origin. A key feature of this is to indicate whether the product is of natural or synthetic origin. With reference to this context, we have investigated three alkaloids commonly exploited for human use: nicotine, atropine, and caffeine. We have measured by 13C nuclear magnetic resonance spectrometry the position-specific distribution of 13C at natural abundance within several samples of each of these target molecules. This technique is well suited to distinguishing between origins, as the distribution of the 13C isotope reflects the primary source of the carbon atoms and the process by which the molecule was (bio)synthesized. Our findings indicate that labeling can be misleading, especially in relation to a supplied compound being labeled as "synthetic" even though its 13C profile indicates a natural origin.

Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) ß3 subunits in enhanced functional expression of mouse α6ß2ß3- and α6ß4ß3-nAChRs.

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where "*" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mß4hß3- (∼ 5-8-fold) or WT mα6mß4mß3-nAChRs (∼ 2-fold) yielded higher function than mα6mß4-nAChRs. Function was not detected when mα6 and mß2 subunits were expressed together or in the additional presence of hß3 or mß3 subunits. However, function emerged upon expression of mα6mß2mß3(V9'S)-nAChRs containing ß3 subunits having gain-of-function V9'S (valine to serine at the 9'-position) mutations in transmembrane domain II and was further elevated 9-fold when hß3(V9'S) subunits were substituted for mß3(V9'S) subunits. Studies involving WT or gain-of-function chimeric mouse/human ß3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hß3 subunits as templates for site-directed mutagenesis studies, substitution with mß3 subunit residues in extracellular N-terminal domain loops "C" (Glu(221) and Phe(223)), "E" (Ser(144) and Ser(148)), and "ß2-ß3" (Gln(94) and Glu(101)) increased function of mα6mß2*- (∼ 2-3-fold) or mα6mß4* (∼ 2-4-fold)-nAChRs. EC50 values for nicotine acting at mα6mß4*-nAChR were unaffected by ß3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops ß2-ß3 and E) interfaces of ß3 subunits are some of the molecular impediments for functional expression of mα6mß2ß3- or mα6mß4ß3-nAChRs.

[Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.

Biochemical, microbiological and phytochemical studies on aqueous-fermented extracts from Atropa belladonna L. Part 2--Phytochemistry.

Extraction methods of fresh plants into aqueous-fermented extracts according to German Homoeopathic Pharmacopoeia (HAB), regulation nos. 33 and 34 were evaluated. In the course of production, the extraction is accompanied by fermentation and the resulting preparation is stored for at least 6 months until further processing. In part 1 of the work, the biochemical reactions proceeding in aqueous-fermented extracts from the fresh flowering herb of Atropa belladonna var. belladonna (L.) were studied and the responsible microorganisms were identified. This second part aimed at shedding light on the phytochemical changes upon manufacture and storage. Additionally, questions were addressed at the robustness of the method by varying production conditions, its reproducibility and the comparison with ethanolic extraction. Studying 110 extracts produced from 2006 to 2009 as well as model experiments on isolated lactic acid bacteria in atropine solutions proved that the active substance atropine was stable under regular pH conditions. Interestingly, no difference between aqueous-fermented and ethanolic extracts could be found with respect to atropine concentration. In contrast, the amounts of scopoletin and kaempferol glycosides from Atropa belladonna differed depending on the extraction procedure.

Biochemical and structural characterization of recombinant hyoscyamine 6ß-hydroxylase from Datura metel L.

Hyoscyamine 6ß-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6ß-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6ß-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K(m) values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50µM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14M(-1) and 0.56M(-1), respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.

Responses of fungi to tropane alkaloids produced by a medicinal plant Hyoscyamus muticus (Egyptian henbane).

Antifungal activity of hyoscyamine (Hcy) and scopolamine (Sco) were determined by TLC-bioautography against fungi associated with H. muticus grown in Egypt, and those isolated from other plants grown in Japan. All 40 fungal strains were tolerant to Sco and sensitive to Hcy, exhibiting a growth inhibition zone around the Hcy spot on the bioautography plate. The strains were grouped into three types based on the appearance of the inhibition zone: (i) 17 strains exhibiting a clear inhibition zone, which remained clear at 8 d after incubation (type I); (ii) 22 strains exhibiting the inhibition zone with a brown circle surrounding the zone and regrowth within the inhibition zone (type II); (iii) 1 strain exhibiting the inhibition zone with no brown circle and regrowth within the inhibition zone (type III). In the type II and III strains, Hcy disappeared, and other alkaloids were found in the inhibition zones in its place. Hcy feeding experiments using Penicillium purpurogenum (type II) and Cunninghamella elegans (type III) revealed that these fungi may convert Hcy to a new alkaloid compound.

Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures.

Hyoscyamine-6beta-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation.

Immunolocalisation of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber sprouts.

Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.

Hairy root culture in a liquid-dispersed bioreactor: characterization of spatial heterogeneity.

A liquid-dispersed reactor equipped with a vertical mesh cylinder for inoculum support was developed for culture of Atropa belladonna hairy roots. The working volume of the culture vessel was 4.4 L with an aspect ratio of 1.7. Medium was dispersed as a spray onto the top of the root bed, and the roots grew radially outward from the central mesh cylinder to the vessel wall. Significant benefits in terms of liquid drainage and reduced interstitial liquid holdup were obtained using a vertical rather than horizontal support structure for the biomass and by operating the reactor with cocurrent air and liquid flow. With root growth, a pattern of spatial heterogeneity developed in the vessel. Higher local biomass densities, lower volumes of interstitial liquid, lower sugar concentrations, and higher root atropine contents were found in the upper sections of the root bed compared with the lower sections, suggesting a greater level of metabolic activity toward the top of the reactor. Although gas-liquid oxygen transfer to the spray droplets was very rapid, there was evidence of significant oxygen limitations in the reactor. Substantial volumes of non-free-draining interstitial liquid accumulated in the root bed. Roots near the bottom of the vessel trapped up to 3-4 times their own weight in liquid, thus eliminating the advantages of improved contact with the gas phase offered by liquid-dispersed culture systems. Local nutrient and product concentrations in the non-free-draining liquid were significantly different from those in the bulk medium, indicating poor liquid mixing within the root bed. Oxygen enrichment of the gas phase improved neither growth nor atropine production, highlighting the greater importance of liquid-solid compared with gas-liquid oxygen transfer resistance. The absence of mechanical or pneumatic agitation and the tendency of the root bed to accumulate liquid and impede drainage were identified as the major limitations to reactor performance. Improved reactor operating strategies and selection or development of root lines offering minimal resistance to liquid flow and low liquid retention characteristics are possible solutions to these problems.

Sulphotransferase-dependent dehydration of atropine and scopolamine in guinea pig.

1. Enzymatic dehydration of atropine and scopolamine was studied in guinea pig. 2. The incubation of these alkaloids with guinea pig liver cytosol in the absence of cofactors gave no dehydrated metabolite. However, when atropine and scopolamine were incubated with cytosol supplemented with ATP and sodium sulphate, dehydrated metabolites, apoatropine and aposcopolamine were formed. The formation of these metabolites was confirmed by gas chromatography-mass spectrometry. 3. The reaction required ATP as well as cytosol as the obligatory factors. Deletion of sodium sulphate from the reaction mixture also resulted in a decrease of the activities, although this treatment showed limited effect when the low concentration of atropine was used. Furthermore, dehydroepiandrosterone, an excellent substrate for hydroxysteroid-sulphotransferase, effectively inhibited the in vitro activity of atropine dehydration. 4. Administration of dehydroepiandrosterone to guinea pig followed by atropine treatment caused decreased urinary excretion of apoatropine. 5. These results strongly suggested that the dehydration of atropine and scopolamine takes place via the sulphate conjugate intermediates produced from the sulphotransferase-catalysed reaction. The present finding is the first example of the sulphotransferase-dependent dehydration of a drug, and its generality in drug metabolism is discussed.

Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition.

The tropane alkaloid scopolamine is a medicinally important anticholinergic drug present in several solanaceous plants. Hyoscyamine 6 beta-hydroxylase (EC 1.14.11.11) catalyzes the oxidative reactions in the biosynthetic pathway leading from hyoscyamine to scopolamine. We introduced the hydroxylase gene from Hyoscyamus niger under the control of the cauliflower mosaic virus 35S promoter into hyoscyamine-rich Atropa belladonna by the use of an Agrobacterium-mediated transformation system. A transgenic plant that constitutively and strongly expressed the transgene was selected, first by screening for kanamycin resistance and then by immunoscreening leaf samples with an antibody specific for the hydroxylase. In the primary transformant and its selfed progeny that inherited the transgene, the alkaloid contents of the leaf and stem were almost exclusively scopolamine. Such metabolically engineered plants should prove useful as breeding materials for obtaining improved medicinal components.

Recent developments in the biosynthesis of the tropane alkaloids.

Recent work on the biosynthesis of the tropane moiety of cocaine, hyoscamine, scopolamine, and related alkaloids is reviewed. Revision of the generally accepted biosynthetic pathway to these alkaloids is now proposed in the light of new discoveries. New information on the biosynthesis of some of the acid moieties (benzoic, tiglic and tropic acid) of the tropane ester alkaloids is also discussed.

Cholinergic blockade, septal lesions, and DRL performance in the rat.

Four experiments describing the effects of cholinergic blockade produced by systemic injection of either atropine sulfate or atropine methyl nitrate on the differential reinforcement of low rate (DRL) responding of rats are reported. It was shown that atropine sulfate injected either chronically or at high dosage suppressed DRL responding. Injected acutely, atropine sulfate produced disinhibitory effects. When atropine was injected either chronically or acutely into animals with septal lesions, there was suppression of responding. It was suggested that the specific behavioral outcome resulting from cholinergic blockade depends on the balance resulting from the competing peripheral and central effects of such blockade.

Intracranial drug implants: an autoradiographic analysis of diffusion.

Labeled crystalline atropine, administered to the hypothlalamus of rats, remainted strictly localized in a sphere, 1.0 to 1.8 millimeters in diameter, ditring the first 3 minutes. A similar distribution obtained after 1 hour. At intermediate times, slightly elevated radioactivity, reflecting concentrations 2,000 to 10,000 times below behaviorally effective doses, was observed several millimeters from the implantation site.