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Made available in DSpace on 2021-06-25T19:56:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-11 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) INCT BioNat Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Tropane alkaloids are specialized plant metabolites mostly found in the Erythroxylaceae and Solanaceae families. Although tropane alkaloids have a high degree of structural similarity because of the tropane ring, their pharmacological actions are quite distinct. Brazil is one of the main hotspots of Erythroxylum spp. diversity with 123 species (almost 66% of the species catalogued in tropical America). Erythroxylum pungens occurs in the Caatinga, a promising biome that provides bioactive compounds, including tropane alkaloids. As part of our efforts to investigate this species, 15 alkaloids in specimens harvested under different environmental conditions are presented herein. The occurrence of 3-(2-methylbutyryloxy)tropan-6,7-diol in the stem bark of plants growing in their natural habitat, greenhouse controlled conditions, and after a period of water restriction, suggests that it is a potential chemical marker for the species. This alkaloid was evaluated for several parameters in zebrafish ( Danio rerio ) as a model organism. Regarding toxicity, teratogenic effects were observed at 19.5 mu M and the lethal dose for embryos was 18.4 mu M. No mortality was observed in adults, but a behavioral screen showed psychostimulatory action at 116.7 mu M. Overall, the alkaloid was able to cause zebrafish behavioral changes, prompting further investigation of its potential as a new molecule in the treatment of depression-like symptoms. In silico , targets involved in antidepressant pathways were identified by docking. Univ Fed Rio Grande do Norte, Dept Pharm, Gal Gustavo Cordeiro Farias St S-N, BR-59012570 Natal, RN, Brazil Univ Fed Rio Grande do Norte, Dept Physiol & Behav, Natal, RN, Brazil Univ Fed Mato Grosso do Sul, Fac Pharmaceut Sci Food & Nutr, Campo Grande, MS, Brazil Univ Fed Rio Grande do Sul, Fac Pharm, Porto Alegre, RS, Brazil Univ Fed Rio Grande do Sul, Ctr Biotechnol & Dept Bot, Porto Alegre, RS, Brazil Sao Paulo State Univ, Chem Inst, Araraquara, SP, Brazil Sao Paulo State Univ, Chem Inst, Araraquara, SP, Brazil CNPq: 445149/2014-0 INCT BioNat: 465637/2014-0 CAPES: 001 FAPESP: 2014/50926-0

Cell suspensions initiated from Duboisia myoporoides–a shrub belonging to the Solanaceae family and being a rich source of tropane alkaloids–previously showed their ability to glycosylate scopoletin into scopolin, which represent coumarins showing health benefits. To investigate the time course of this glycosylation reaction, an in vivo NMR approach was developed using a perfusion system in an 8-mm NMR tube and 1H NMR with 1D and 2D (TOCSY and NOESY) experiments. The time course of metabolic changes could therefore be followed without any labeling.

Plant species of the Solanaceae family (nightshades) contain pharmacologically active anticholinergic tropane alkaloids, e.g., scopolamine and hyoscyamine. Tropane alkaloids are of special interest, either as active principles or as starting materials for semisynthetic production of other substances. For genetic evaluation, domestication, cultivation, harvest and post-harvest treatments, quantification of the individual active principles is necessary to monitor industrial processes and the resulting finished products. Up to now, frequently applied methods for quantification are based on high performance liquid chromatography and gas chromatography optionally combined with mass spectrometry. However, alternative analytical methods have the potential to replace the established standard methods partly. In this context, attenuated total reflection-Fourier transform infrared spectroscopy enabled chemotaxonomical classification of the Solanaceae Atropa belladonna, Datura stramonium, Hyoscyamus niger, Solanum dulcamara , and Duboisia in combination with cluster analysis. Also discrimination of genotypes within species was achieved to some extent. The most characteristic scopolamine bands could be identified in attenuated total reflection-Fourier transform infrared spectra of Solanaceae leaves, which allow a fast characterisation of plants with high scopolamine content. Applying a partial least square algorithm, very good calibration statistics were obtained for the prediction of the scopolamine content (residual prediction deviation = 7.67), and moderate prediction quality could be achieved for the hyoscyamine content (residual prediction deviation = 2.48).

Putrescine N-methyltransferase (PMT, EC 2.1.1.53) catalyses the S-adenosyl- L-methionine (SAM or AdoMet)-dependent methylation of putrescine to N-methylputrescine within the biosynthetic pathways of calystegines, nicotine, and tropane alkaloids in medicinal plants and produces S-adenosyl- L-homocysteine (SAH or AdoHcy). Determination of PMT activity was time-consuming and hardly reproducible in the past because it required tedious separation steps after chemical derivatisation or radioactive labelling of N-methylputrescine. A convenient and accurate enzyme-coupled colorimetric assay is based on the conversion of SAH to homocysteine by 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN/SAHN, EC 3.2.2.9) and S-ribosylhomocysteine lyase (LuxS, EC 4.4.1.21). Homocysteine is quantified by 5,5'-dithiobis-2-nitrobenzoic acid. Putrescine was shown not to interfere with MTAN or LuxS. The colorimetric assay was validated by HPLC analysis. K(m) values determined by the assay, 108 microM for putrescine and 42 microM for SAM, are lower than the previously reported values, due to alleviation of PMT inhibition by SAH. DTNB:5,5'-dithiobis-2-nitrobenzoic acid LuxS: S-ribosylhomocysteine lyase MTAN:5'-methylthioadenosine nucleosidase PMT:putrescine N-methyltransferase SAH: S-adenosyl- L-homocysteine SAM: S-adenosyl- L-methionine TNB:2-nitro-5-thiobenzoic acid.

The effect of 34 alkaloids of the piperidine, pyridine, tropane, isoquinoline, indole, quinolizidine, quinoline, purine, and steroidal types on the growth of Trypanosoma brucei, T. congolense, and human HL-60 cells was investigated in vitro. Berbamine, berberine, cinchonidine, cinchonine, emetine, ergotamine, quinidine, quinine, and sanguinarine showed trypanocidal activities with ED(50) (50% effective dose) values below 10 microM. Berberine, emetine, and quinidine were the most active compounds found; their ED(50) values and minimum inhibitory concentrations were comparable to those of the antitrypanosomal drugs suramin and diminazene aceturate. However, most of these compounds were also cytotoxic. In the case of emetine, the ratio of cytotoxic/trypanocidal activity was only 3 while for quinidine it was 300 indicating that this alkaloid could be a candidate for further drug development. DNA intercalation in combination with protein biosynthesis inhibition, which is the major mode of action of the active alkaloids, could be responsible for the observed trypanocidal and cytotoxic effects.

Hairy root lines of Datura metel were established following infection of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium. The growth and production of hyoscyamine and scopolamine (mg/g dry wt.) of these root cultures was encouraged by using B5 liquid medium with half-strength salts. In these culture conditions, the capacity of the highest productive root line 25 to excrete scopolamine into the culture medium rose from 8.7 % to 70% when the permeabilizing agent Tween 20 was added for 24 h to the medium, after 2 and 4 weeks of culture. Using an airlift bioreactor (41) with modifications in order to increase root anchorage, the Tween 20 treatment encouraged both growth and alkaloid productivity of cultured root line 25. After 4 weeks their biomass yield was 2.3 mg/l/day and 0.84 mg/l/day of scopolamine was produced (70% extracellular). The scopolamine released into the culture medium was separated with an Amberlite XAD-2 column located in the media exit.

The tropane alkaloid anisodamine ( 2) is obtained by 6 beta-hydroxylation of hyoscyamine ( 1). The application of this alkaloid in medicine is gaining attention due to the wide range of therapeutic applications described in addition to its anticholinergic activity. In this work, the production of anisodamine ( 2) by IN VITRO cultures of BRUGMANSIA CANDIDA (Argentinean and Colombian samples) was studied. This alkaloid was estimated in different organs of IN VITRO-germinated seedlings as well as in hairy roots obtained from seedlings from both sources. Colombian roots exhibited the highest content of tropane alkaloids, with anisodamine ( 2) being the main alkaloid measured. In the leaves, the main alkaloid was scopolamine ( 3) and no significant differences were observed between Argentinean and Colombian leaves. The tropane alkaloid content in Argentinean hairy roots was significantly higher than in Colombian ones. Also, in the Argentinean samples the main alkaloid detected was anisodamine ( 2). Argentinean and Colombian B. CANDIDA seedlings and hairy roots appear to be a promising system for the production of anisodamine ( 2).

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.

Fourteen commercial samples of the popular Brazilian aphrodisiac Catuaba specified as bark drugs of Anemopaegma, Erythroxylum and Trichilia species were examined for identity and purity. Only a minority of the examined Catuaba samples contained the crude drugs claimed on the labels. More than half of the products were adulterated with different crude drugs. The majority of the samples contained a bark originating from Trichilia catigua. The TLC fingerprints confirmed the heterogeneity, in 50% of the samples tropane alkaloids of various concentrations were detected. TLC and HPLC methods for separation and identification of the tropane alkaloids were developed and their analytical data (RF values, retention times, ESI-MS) given. The structure elucidation of the two main alkaloids, catuabine D and its hydroxymethyl derivative, is presented. The 1H- and 13C-NMR assignments of these alkaloids are discussed with regard to literature data. Neither aqueous nor methanolic extracts of the Trichilia catigua reference material nor alkaloid-enriched fractions of commercial samples showed any effect on the rabbit corpus cavernosum in an in vitro test.

The effects of tween 20 as permeabilizing agent on tropane alkaloids from Datura innoxia Mill, hairy roots have been studied. For various tween 20 concentrations both hyoscyamine and scopolamine accumulated in the culture medium. Plant material viability could be preserved after a 24 hours-2% tween 20 concentration treatment. The time-course study of alkaloid release showed that the maximum of excretion occurred after a 20 hour contact with tween 20. At that time, a concentration of hyoscyamine superior to 25 mg/l was detected in the medium.

Hairy root cultures of DATURA QUERCIFOLIA were established following infection with AGROBACTERIUM RHIZOGENES strain LBA 9402. Eight tropane alkaloids were identified in the hairy roots, hyoscyamine being the major constituent. The growth and the hyoscyamine content of transformed roots were investigated under various conditions. Gamborg B5, Murashige and Skoog, and Woody Plant media were tested. Gamborg B5 medium was the best for growth as well as for hyoscyamine accumulation. The influence of sucrose concentration was examined and a 5% concentration was found to be the most appropriate for growth and for alkaloid production. After 35 days of incubation in this medium, the hyoscyamine content of the roots was 1.24% based on dry weight. The influence of gibberellic acid and of Amberlite XAD-4 resin on hyoscyamine production was tested.

Increasingly, as a result of recent biochemical work, there exists a realistic possibility of taking a molecular genetic approach to the manipulation of alkaloid-producing pathways in plant tissue cultures. In the pathways forming indole alkaloids in CATHARANTHUS ROSEUS, tropane alkaloids in DATURA and HYOSCYAMUS species, and nicotine in NICOTIANA species, recent studies have identified a number of key enzymes and at least some of the factors that regulate their levels of activity. Such knowledge contributes the basis upon which it has become feasible to design a strategy by which the flux in these pathways may be enhanced at the genomic level. This review presents a summary of the state-of-the-art pertaining to these pathways and discusses the strategy to be adopted for a molecular approach to their manipulation, together with some of the pitfalls that may arise when trying to alter their natural regulation.

Anther culture of DATURA INNOXIA Mill, has permitted the obtention of spontaneous diploid androgenic plants which produced the tropane alkaloids. The source plants (zygotic diploid) showed no significant variations in the leaf alkaloid content. On the contrary, androgenic diploid plants obtained after the first cycle of androgenesis showed important quantitative and qualitative variations in the leaf alkaloid content. Thus, androgenesis was found to induce a large variation in the accumulation of these secondary metabolites in the leaves. It has also permitted the obtention of tropane alkaloid-overproductive plants, particularly rich in scopolamine. The analyses of zygotic plants obtained from seed germination of the first cycle androgenic plants have shown that this variability is transmissible by simple cross-pollination. The analyses of androgenic diploid plants obtained after the second cycle of androgenesis also showed variations in the leaf alkaloid content. IN VITRO androgenesis, therefore, clearly induced variability in the leaf alkaloid content of the androgenic plants. The role of IN VITRO androgenesis in inducing variability has been discussed.

Systematic studies were carried out on two different strains (Gatersleben and Cairo) of Hyoscyamus muticus L. (Solananaceae) in order to analyse the variation in the contents of the two main tropa-alkaloids in individual plants and protoplast-derived cell culture clones. The hyoscyamine content was determined by radioimmunoassay, the scopolamine content by radio- and enzymeimmunoassay and the total tropane alkaloid content by quinuclidinyl benzilate assay. The development stage of the plant is important for alkaloid production. Clear maximal foliar scopolamine and hyoscyamine contents were reached at the onset of flowering and during the full blooming stage, respectively. In the roots the changes in the production of these alkaloids were not considerable. Hyoscyamine is a major alkaloid in the plants as well as in the cell culture clones. In a few exceptions the scopolamine content was greater than that of hyoscyamine, but this phenomenon was not inherited by the cultured clones derived from these plants. High and low tropane alkaloid production was inherited by the selfed F1, generation plants. Great variability in these alkaloids was observed among individual plants or clones in the same plant or clone population. There was a significant difference between the Cairo strain and the Gatersleben strain as regards their ability to produce tropane alkaloids. Haploid plants of both strains contained more hyoscyamine and scopolamine than the diploid ones. Hyoscyamine and scopolamine were the main alkaloids in the clones and in the plants. Via QNB-assay an interesting clone was found which contains remarkable amounts of unknown tropane-group alkaloids.

Atropine and scopolamine were assayed in leaf samples and cultured cells of ATROPA BELLADONNA L. by capillary gas chromatography. The samples were extracted and purified before the gas Chromatographic analysis by passage through disposable extraction columns of reversed phase type. Deviation among the analyses was 4.0% for atropine and 2.9% for scopolamine at 50 mg/l. The limit of detection was 5 mg/l for both alkaloids.

Antibodies were obtained from the immunization of rabbits with DL-tropic acid conjugated to bovine serum albumin. Their wide specificity enabled us to analyse simultaneously every main tropane alkaloid of Solanaceous plants: hyoscyamine, atropine ( DL-hyoscyamine), and scopolamine. The competitive ELISA assay used for this analysis was found to be a very rapid, sensitive method with good accuracy for the dosage of alkaloids in plant material: purified extracts, crude extracts, and dry powdered material. A tropine could be detected with a sensitivity of 0.1 ng. A racemization step was necessary to detect hyoscyamine and scopolamine with the same sensitivity.

Przewalskia tangutica Maxim. (Solanaceae), a plant of Tibetan traditional medicine, was investigated. Contents of tropine, cuscohygrine, (-)-6beta-hydroxyhyoscyamine, hyoscyamine, apoatropine, scopolamine and daturamine were determined (Table I). The roots of this plant showed high contents of both hyoscyamine (major constituent) and total alkaloids, amounting to 1.67-3.82% and 2.06-4.01%, respectively. This plant and the crude drugs prepared from it are noteworthy as new sources of tropane alkaloids. The occurrence of tropane alkaloids in solanaceous plants of China is discussed briefly (Table II).

Eight tropane alkaloids, anabasine and ursolic acid have been isolated from the newly-described species CRENIDIUM SPINESCENS. ANTHOCERCIS ILICIFOLIA possesses a similar alkaloid spectrum to that of A. LITTOREA to which it is taxonomically closely related. In alkaloid composition, A. GENISTOIDES is a variable species and the sample analysed here contained meteloidine as principal alkaloid. The alkaloids of CYPHANTHERA ODGERSII and C. TASMANICA are consistent with those of other species of the genus. At present, ursolic acid does not appear to be a useful characteristic for the classification of solanaceous taxa belonging to Anthocercideae.