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Distribution of hyoscyamine and scopolamine in Datura stramonium.

Miraldi E, Masti A, Ferri S, Barni Comparini I.

Dipartimento di Scienze Ambientali, Sezione Biologia Farmaceutica, Universita di Siena, Via Mattioli 4, 53100 Siena, Italy. miraldi unisi.it

The production of hyoscyamine and scopolamine in Datura stramonium has been investigated in the different plant parts, at different stages of their life cycle. Maximum contents were found in the stems and leaves of young plants, hyoscyamine being always the predominant component.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11543961&dopt=Abstract hyoscyamine Levbid SL



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Molecular cloning, expression and characterization of tropinone reductase II, an enzyme of the SDR family in Solanum tuberosum (L.).

Keiner R, Kaiser H, Nakajima K, Hashimoto T, Drager B.

Institut fur Pharmazeutische Biologie, Martin-Luther-Universitat Halle-Wittenberg, Halle/Saale, Germany.

Calystegines are nortropane alkaloids that are found in Solanaceae containing the classical tropane alkaloids hyoscyamine and scopolamine, and in other Solanaceae such as potato, Solanum tuberosum (L.). Calystegines are assumed to derive from the classical tropane alkaloid pathway. We isolated a cDNA from S. tuberosum with high homology to the pseudotropine-forming tropinone reductase (TRII), which presents as the first putative metabolite specific to calystegines. The equivalent amino acid sequence shows typical motifs of a short-chain dehydrogenase (SDR). The recombinant TRII protein expressed in Escherichia coli catalyzes pseudotropine formation from tropinone with a Km value, a pH optimum, substrate and co-substrate preferences similar to those reported for the TRII enzymes from other Solanaceae species. The gene is expressed in roots, tubers and aerial parts of potato. The distribution of the TRII transcript in comparison with the calystegine concentrations in the tissues suggests transport of calystegines or their precursors between potato organs.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11855731&dopt=Abstract hyoscyamine Levbid SL



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Depolarization of membrane potential and the smooth muscle contraction by isothiocyanatobenzyl imidazoline in guinea-pig stomach circular muscle.

Jing L, Jagadeesh JM, De Los Angeles J, Miller DD, Patil PN.

The Ohio State University, College of Pharmacy, Division of Pharmacology, Columbus 43210, USA.

Isothiocyanatobenzyl imidazoline (IBI) produces characteristic slowly developing contraction of many smooth muscle preparations including the circular smooth muscle of the guinea-pig stomach. Changes in the membrane potential were recorded intracellularly, and the muscle contraction induced by IBI was investigated. IBI at 100 micromol/l slowly produced a sustained depolarization of the membrane with a maximum change of approximately 15 mV. This depolarization could not be blocked by 1-hyoscyamine, 100 nmol/l. An imidazoline analogue, oxymetazoline at 1 micromol/l, did not change the resting membrane potential as observed after IBI. Significant membrane depolarization after IBI still occurred in Ca2+-free medium. During IBI-induced depolarization, sudden reduction of Na+ to 30 mmol/l in the medium reduced the depolarization slightly. IBI-induced depolarization was additive with that produced by 20 mmol/l K+ in the medium. In the presence of tetraethylammonium chloride or levcromakalim or nifedipine, IBI continued to depolarize the membrane although functional pharmacological experiments showed that the contractile effects of IBI were significantly inhibited by 30 micromol/l levcromakalim and abolished by 100 nmol/l nifedipine. At 100 micromol/l phentolamine (reported by others as an inhibitor of ATP-sensitive potassium channels) completely blocked IBI-induced contraction. Phentolamine (30 micromol/l) blocked the contractile effects of IBI by 50%. On the other hand, S(-)-Bay K 8644, a voltage-dependent calcium channel activator, was additive with the contractile response of IBI. These results indicated that IBI produced membrane depolarization and contraction of the guinea-pig stomach circular muscle, by a mechanism not involving muscarinic receptors or alpha-adrenoceptors. Even though levcromakalim, an ATP-sensitive potassium channel opener, could not inhibit IBI-induced depolarization, the ATP-sensitive potassium channel and the voltage-dependent calcium channel may be intrinsically linked with the action of IBI.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10543437&dopt=Abstract hyoscyamine Levbid SL



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Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures.

Zhang L, Ding R, Chai Y, Bonfill M, Moyano E, Oksman-Caldentey KM, Xu T, Pi Y, Wang Z, Zhang H, Kai G, Liao Z, Sun X, Tang K.

State Key Laboratory of Genetic Engineering, Morgan-Tan International Center for Life Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China.

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 beta-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T(3)) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H(11) (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15084741&dopt=Abstract hyoscyamine Levbid SL



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Antinociceptive effect of R-(+)-hyoscyamine on the conjunctival reflex test in rabbits.

Ghelardini C, Galeotti N, Fantetti L, Gualtieri F, Scapecchi S, Bartolini A.

Department of Pharmacology, University of Florence, Italy.

R-(+)-Hyoscyamine (1-10 microg/kg, s.c.) dose-dependently increased the local anesthetic effect of procaine (50 microg/ml) and lidocaine (50 microg/ml) in the conjunctival reflex test in the rabbit. This potentiating effect is completely prevented by the M1 antagonist dicyclomine (10 mg/kg, s.c.). The intensity of R-(+)-hyoscyamine antinociception was comparable to that induced by morphine (2 mg/kg, s.c.) and minaprine (15 mg/kg, s.c.), used as analgesic reference drugs. In the same experimental conditions, the S-(-)-enantiomer of atropine (0.1-10 microg/kg, s.c.), was completely ineffective. The present results confirm the ability of R-(+)-hyoscyamine to produce a paradoxical antinociceptive effect mediated by a cholinergic mechanism not only in rodents but also in the rabbit.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10580368&dopt=Abstract hyoscyamine Levbid SL